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Bosphorus

3rd BBBB
International Conference
on Pharmaceutical Sciences
October 26-28, 2009 - Antalya –TURKEY
With the Participation of Representatives of
University - Industry - Regulatory Scientists

Bosphorus
Bled
Balaton
Baltic
with the collaboration of the
Phage Societies (UK, USA, GE) and European Association of Faculties of Pharmacy (EAFP)

organized by
Turkish Pharmaceutical Technology Scientists’ Association (TÜFTAD)

www.bbbb-eufeps.org
PREFACE

W
ithin the last decade, very rapid changes have • Regulatory issues related  to
taken place in pharmaceutical sciences, phar- - disharmonization of BE of highly variable drugs,
maceutical industry activities, regulatory ap- - herbal medicinal products,
proaches and economical platforms. Among these, the im- - medical devices, and
pact of developments in computer sciences, technology and - pharmaceuticals
medical sciences have also added tremendous challenges to • Recent strategies for the oral delivery of poorly soluble
pharmaceutical practices in industry, regulatory body and drugs
academia. We all know what we are facing new challenges • Utilisation of bacteriophages  as  medicines in drug 
everyday in our mutual applications. What we have seen and delivery systems
experienced within this last decade explained us that we
• Bacterophages and lytic enzymes to combat new and
should transfer our experience, knowledge and guidance to
emerging bacteria
young scientists, and new generations.
• Self assembling nanoparticles as vaccine delivery ve-
The BBBB International Conferences have been initi- hicles for bacteriophages
ated under the auspices of EUFEPS by the joint proposal of • Bacteriophages in targeted medications
four founder countries’ associations and have been decided • Biotechnology, biological systems for delivery of drugs
to initiate organizing international conferences biennially on • New analytical techniques for enantiomeric purity of
the attractive, historical and well-known shore of a lake or on chiral drugs
the seacoast, e.g. in the region of Lake Balaton, the Baltic sea, • Microchips in drug analysis
Bled, and the Bosphorus in order to support young, bright • SCCE in pharmaceutical and bioanalysis
and future promising scientists of these countries and other • QSAR and prediction of  drug properties
participant countries as well.  Another important reason to • Alicylic beta aminoacids in drug research
emphasize was to open the doors in order to create close sci- • Further topics on medicinal chemistry and analytical
entific contacts between founding and participant countries’ techniques of drug development
scientists. Under the light of above mentioned approaches, • Disease-drug interactions
the 1st BBBB Balaton International Conference in Siofok,
• Cost effectiveness of drug therapy using pharmacoge-
Hungary was jointly organized by Hungary and Slovenia in
netic testings
2005. 2nd BBBB Baltic International Conference in Tartu
• Clinical pharmacy services in hospitals
was jointly organized by Estonia and Finland in 2007.
• Clinical pharmacy and pharmaceutical care
3
The 3rd BBBB-Bosphorus International Conference • PHARMINE or pharmacy education in Europe
on Pharmaceutical Sciences was organized from October
26 to 28, 2009 in Antalya-Turkey by Turkish Pharmaceutical
Technology Scientists’ Association (TÜFTAD). BBBB Bosphorus (Antalya-Turkey, 2009) International
Conference hosted around 200 participants from university,
Main target of the 3rd BBBB-Bosphorus International industry, and regulatory agencies from all over the world.
Conference on Pharmaceutical Sciences was to create a The contributions as oral and poster presentations were
pharmaceutical sciences platform in between the university,
published in this special issue. We hope these excellent pre-
industry and regulatory scientists for discussing the main
sentations serve as a guide to readers for new areas of phar-
topics of this symposium in a friendly atmosphere in histori-
cally and naturally attractive part of Turkey called “Turkish maceutical sciences.
Riviera”. BBBB Bosphorus (Antalya-Turkey, 2009) Internation-
As the guest editors of this issue, we wish to thank all the
al Conference consisted of six main themes:
authors for their scientific contributions and also to Elsevi-
• Regulatory Issues er giving us the chance of publishing this special issue. We
• Towards Innovative Novel Drug Delivery Systems would like to extend our thanks to the sponsoring compa-
• Biotechnology, Delivery of Biologics and Vaccination nies from the Turkish Pharmaceutical Industry.
• Progresses in Medicinal Chemistry and Analytical Tech-
September 26, 2009
niques of Drug Development
• Clinical Pharmacy - Pharmaceutical Care
• New Progresses in Pharmacy Education, Training and
Pharmacy Progression Guest Editors
However, under these six main themes, BBBB Bosphorus Prof. Dr. A. Atilla Hıncal
Conference brought the attendees a lot of hot topics and very İDE Information-Consultancy-Education, Inc., Ankara, Turkey
new area of interests for pharmaceutical sciences, such as:
Prof. Dr. Nevin Çelebi
• Modeling and simulation in modern pharmacy Gazi University, Faculty of Pharmacy, Ankara, Turkey
• HTS and solubility testing
• New approaches and techniques for drug delivery sys- Assoc. Prof. Dr. Nilüfer Yüksel
tems Ankara University, Faculty of Pharmacy, Ankara, Turkey

October 26 - 28, 2009 / Antalya - TURKEY


“Spelling mistakes and any other errors are under the responsibility
of the authors.”
ORAL PRESENTATIONS
ORAL PRESENTATIONS

O01
In vitro blood brain barrier (BBB) models
and their application in drug research
Christian R. Noe
University of Vienna, Austria

D
evelopment of new drugs acting on the central ner- BBB specific properties, such as tight junction formation and
vous system (CNS) is one of the major tasks in pharma- functionality. A comparative analysis of the models will be
ceutical sciences. In this context, CNS targeted drugs presented. In order to assess their reliability to predict in vivo
have to cross the BBB. The BBB maintains the homeostasis permeability of drugs, transport studies with sets of drugs of
of the brain microenvironment, which is crucial for neuronal different classes were performed. The broad applicability of
activity and function. Unlike peripheral endothelium, brain BBB models beyond transport studies will be presented as
microvascular endothelial cells are characterised by the well. The models may be used to study the impact of drugs
presence of tight intercellular junctions, minimal pinocytotic on the BBB system itself. There is significant evidence that
activity and the absence of fenestrations. dysfunction of the BBB might play a crucial role in neurode-
generative and other CNS diseases. BBB in vitro models are
The lecture will report about the side-by-side comparison an excellent toll to study options for specific therapeutic in-
of several BBB cell culture in vitro models. Transwell models tervention by design of drugs exerting their effects directly
and dynamic hollow-fiber models based on different cell at the BBB.
lines were established and characterised with regard to their

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

O02
AN ILLUSTRATION OF REGULATORY DISHARMONIZATION:
BIOEQUIVALENCE OF HIGHLY VARIABLE DRUGS
Laszlo Endrenyi and Laszlo Tothfalusi
University of Toronto and Semmelweis University of Medicine, Canada

H
armonization of regulations is a praiseworthy princi- Scaled average BE can be interpreted as an equivalence test
ple. In practice, however, various regulatory authori- for clinical effects sizes. It is also a special case of therapeutic
ties develop their expectations and procedures with switchability.
almost complete disregard of each other. As a reminder of
this divergence, the issue of individual bioequivalence (BE) FDA imposes also a secondary regulatory criterion. This
was strongly promoted for a decade in the United States but expects that the point estimates of the ratio of geometric
nowhere else. means of the pharmacokinetic parameters should be be-
tween 0.80 and 1.25. This criterion has only political but no
Another, currently timely topic is the determination of BE scientific background. It substantially complicates the eval-
for highly variable (HV) drugs and drug products which has uation of BE for HV drugs.
been, for a long time, a substantial problem. The usual reg-
ulatory requirement for satisfying BE is that the 90% confi- The FDA procedure, as stated currently, has some techni-
dence interval around the ratio of geometric means of phar- cal difficulties. Notably, the BE limits are discontinuous at a
macokinetic parameters (AUC and Cmax) for the contrasted within-subject variation of 30%. As a consequence, the con-
drug products be between 0.80 and 1.25. It is very difficult to sumer risk can be much higher than 5%, and the regulatory
satisfy this expectation, by utilizing the procedure of average uncertainty for making a decision about acceptance or rejec-
8 BE, when the within-subject variation of a drug is high unless tion is enhanced.
very many subjects are enrolled in a study.
EMEA in the European Union issued a Concept Paper in 2006
Various other approaches have been devised in order to re- on the problem of HV drugs. This was later withdrawn, and
duce this burden. Steady-state investigations often lower the EMEA resisted, until very recently, further consideration of
intrasubject variation. However, some regulatory authorities the topic. Currently, EMEA ponders the application of an ap-
have expressed reluctance to apply this approach. proach which parallels that of scaled average BE. Unscaled
average BE would be utilized in which the BE limits would ex-
In the past, regulatory disharmonization proceeded to an al- pand in proportion to the within-subject standard deviation.
most ironic extent. For instance, in Europe it was possible to However, the expansion would be limited to a range when
extend substantially, for a long time, the regulatory criterion the intrasubject coefficient of variation is between 30% and
for Cmax provided that a clinical justification was presented 50%. There is no scientific justification for this limitation.
in the protocol. FDA in the United States did not permit at
the time such exceptions. In the late 1990`s the roles re- Health Canada contemplates also unscaled average BE with
versed. In Europe, the tolerance was constricted whereas expanding limits. However, it is concerned that various sub-
FDA became, for a while, more relaxed. missions for the same drug could report differing within-
subject variations which could result in differing regulatory
Currently, FDA appears to prefer the procedure of scaled procedures. Therefore the BE limits would be determined on
average BE. Accordingly, the usual criterion for average BE the basis of all available information. It is contemplated that
is scaled by the within-subject standard deviation. FDA ex- the BE limits would expand stepwise by decades of within-
pects that the procedure be implemented in replicate-de- subject variation. The problem with this approach is that the
sign investigations. BE limits would be discontinuous again with the consequent
difficulties.
Computer-simulated studies demonstrate that by apply-
ing this approach, comparatively few subjects are needed The Medicines Control Council of South Africa considers accept-
to satisfy regulatory expectations. For instance, a few more able the use of alternative methods of evaluation, e.g. of scaled
than 36 subjects could be sufficient in a 3-period investiga- average BE, provided that this is stated a priori in the protocol.
tion, whereas only about 24 individuals could be needed in
a 4-period study. The producer risk is independent of the The possible presence of outliers is an issue related to the de-
within-subject variation. termination of BE for HV drugs and drug products. BE trials

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

often fail not because the drug is highly variable but because and sequential designs will be combined, for example, how
outliers could inflate the variability. Regulatory authorities a study will be assessed if a drug product is HV in one stage
propose different approaches to address this problem. FDA of the investigation but not in the other.
suggests a re-dosing strategy. In contrast, this approach is
missing from a recent CHMP draft. Both guidelines are very Thus, the various regulatory authorities make slow progress
negative on removing outliers merely for statistical reasons but separately from each other and without taking into ac-
whereas a recent Canadian proposal is more permissive. At count considerations and procedures developed by the
least, the application of sequential designs is a common other agencies. It is difficult to think of this as a harmonized
feature of the recent proposals on which consensus could process.
be reached. However, it is not clear at present, how scaling

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

O03
REGULATORY ISSUES for HERBAL MEDICINAL PRODUCTS
Bilge Şener
Gazi University Faculty of Pharmacy Department of Pharmacognosy 06330 Ankara-Turkey

H
erbal medicinal products have been playing an im- With the growing interest for alternative approaches in trea-
portant role in the primary health care of the people ting diseases, herbal medicinal products have also an impor-
around the world, specially in the developing coun- tant role for the development of new therapeutic agents.
tries. In order to keep pace with the modern medicines, the Herbal medicinal products named as “Phytomedicines” ex-
production of safe and effective herbal medicinal products hibit a variety of biological activities on human health. These
in a standardized way is essential. range from the control of regulatory processes by Health Au-
thorities is essential for human life. Therefore, herbal medici-
Assessment of the quality, safety and efficacy of herbal me- nal products are also subject to the same legislative controls
dicinal products are an important issue. Standardization of raw as other medicines.
materials, intermediates and final product of herbal medicines
are the main issue for the quality control of herbal medicinal The overview of the herbal medicinal products in the world-
products. All of the supporting evidence behind the use of phy- wide along with current registration guidelines and criteria
tomedicines has been on use of standardized extracts of the for the control and market situation of herbal medicinal
plant material to ensure reproducibility in the clinical setting. products will be highlighted.

10

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

O04
Development of paediatric medication – opportunity or hurdle?
Tom Sam
Director Global Regulatory Affairs CMC, Schering-Plough, The Netherlands

INTRODUCTION Children are a diverse patient population and their needs


The continued widespread practice of unverified extempora- should be addressed both in terms of delivering the dose
neous preparation and “off-label” use of adult medicines for of drug appropriate for their age and size and providing
children has led to the introduction of legislation in the EU the drug in a dosage form that will make it easy to admin-
and USA to stimulate the industrial development of paediatric ister and facilitate compliance. In order to create a situation
medicines. In Europe it has become a regulation both for new where substantially more children have access to safe and ef-
and existing patented products to include paediatric data in fective medication, an environment is needed that provides
the registration dossier in accordance with an agreed investi- the right balance between predictable and consistent regu-
gation plan and/or a waiver and/or a deferral. This hurdle of latory assessment and industrial feasibility.
extra work beyond developing an adult medicine is meant to
be counterbalanced by an incentive for industry of 6 months
extension of the Supplementary Patent Certificate.

11

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

O05
Medical Devices and Pharmaceuticals:
Differences and Regulatory Approach
Sema Çalış
Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, Sıhhiye, Ankara

M
edical devices have become an increasingly impor- are pure molecules, have toxicologic concerns and drug-
tant health care area in relation to their impact on interactions might be present, clinically studied and have a
health and health care expenditure. This sector cov- long market life. Medical devices are complex components,
ers numerous types of products ranging from simple ban- have biocompability concerns, device mulfunctions might
dages to hemodialysis machines, through life maintaining be possible. For production of drugs, Good Manufacturing
implantable devices, equipment to screen and diagnose dis- Practices are a requirement whereas Quality Managements
ease and health conditions to the most sophisticated diag- Systems are important for Medical devices. In devices ad-
nostic imaging and minimal invasive surgery equipment. verse effects are most often local in nature, they are intend-
ed for professional use.
According to the definition of The Global Harmonization
Task Force, a “Medical device” means any instrument, ap- According to the US FDA’s definition, “a combination device
paratus, implement, machine, appliance, implant, in vitro comprises two or more regulated components, i.e., drug/
reagent or calibrator, software, material or other similar or device, biologic/device, or drug/device/biologic, that are
related article, intended by the manufacturer to be used, physically, chemically, or otherwise combined or mixed and
alone or in combination, for human beings for one or more produced as a single entity: or two or more separate prod-
of the spesific purposes of: diagnosis, prevention, monitor- ucts packaged together in a single package or as a unit and
12 ing, treatment or alleviation of disease; diagnosis, monitor- comprised of drug and device products, device and biolgi-
ing, treatment, alleviation of or compensation for an injury; cal products, or biological and drug products”. Using locally
investigation, replacement, modification, or support of the controlled drug delivery, combination products have already
anatomy or of a physiological process; supporting or sustain- found applications in various areas of cardiovascular disease,
ing life; control of conception; disinfection of medical devic- diabetes, orthopedic, and cancer.
es; providing information for medical purposes by means of
in vitro examination. The regulatory aspects of medical devices differ from coun-
try to country. Manufacturers are obliged to establish the
Medical devices are distinguished from drugs and biologi- safety of their products before they are marketed. Medical
cal products on the basis of their intended modes of action, devices were addressed by three medical device directives,
in which, they are not metabolic, pharmacologic or immu- which were intended to ensure the safety and performance
nologic, whereas drugs primary mode of action are meta- of medical device in the EU:
bolic, pharmacologic or immunologic. For stating the main
differences between the drugs and medical devices; drugs 1. Active implantable medical devices directive –

Table 1. European medical devices classification


EU Class Device Type Example
Class I Low risk to the patient and, except for sterile products or measuring Stethoscopes, EEG and ECG electrodes, body liquid collection devices,
devices, such devices generally do not enter into contact or interact with wheel chair
the body.
Class IIA Medium risk, and are invasive in their interaction with the human body, Syringes and tubing intended for use with infusion pumps, blood
but that interact with the body only through natural body orifices. Also transfusion devices, non-medicated gauze dressing, urinary catheters
include therapeutic devices used in diagnosis or in wound management.
Class IIB Medium risk, and are either partially or totally implantable within the Dialysis apparatus, medicated gauze, dressing
human body, and may modify the biological or chemical composition of
body fluids
Class III High risk, and generally affect the functioning of vital organs and/or life Cardiac pacemarkers and defibrillators, drug-eluting stents, breast
support system. implants, knee impart

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

90/385/EEC 4: covers all powered implantable medical Class IIb or III (high risk).(Below please see European medi-
devices. cal devices classification up to the device type and related
2. Medical device directive – 93/42/EEC 5: regulates example)
most medical devices, e.g. bandages, condoms and di-
agnostic X-ray machines. REFERENCES
3. The in vitro diagnostic medical devices directive – 1. R. Swami, J. Singh, D. Kaushik, H.Dureja, Medical Devices: A
98/79/EC: this directive regulates products used to ex- Regulatory Overview, Latest Reviews, 2009 Vol. 7 Issue 1.
amine substance derived from human body. 2. J. Bright, European medical device regulatory law and
product liability, Journal of Hospital Infection, 1999, Vol.
Medical devices should be classified according to their in- 43, Suppl. 1, S169-S173.
tended use. There are 18 rules for classification of medical 3. www.fda.gov
devices. They helped the manufacturer to determine wheth- 4. www.who.int
er the device is Class I (low risk), Class II ( medium risk), or

13

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

O06
Cellular automata model for drug release sımulatıons
Timo Laaksonen1,Hannu Laaksonen2, Lasse Mutomäki3,4, Jouni Hirvonen1
1Division of the Pharmaceutical Technology, University of Helsinki, Finland
2Brain Research Unit, Low Tempreture Laboratory, Helsinki University of Technology, Finland
3Centre for Drug Research, University of Helsinki, Finland
4Laboratory of Physical Chemistry and Electrochemistry, Helsinki University of Technology, Finland

C
ontrolled release of drugs from polymeric tablets or abilities for various events such as moving to a neighboring cell
implants is a technologically and therapeutically im- or being converted into another type of a cell. The probability
portant field. In order to create and plan new dosage for its movement is set to 1 and all other probabilities are rela-
forms efficiently, it is important to be able to design the re- tive to this. Swelling and permeation have lower probabilities.
lease profile of the drug. These characteristics are influenced
by several factors, such as diffusion, geometry and solubility Table 1. Cell states considered in the model and the cellular
of the polymer used. There are four major release mecha- automata rule applied for each cell state in the simulations.
nisms which affect the release profile: diffusion, erosion, per- Adapted from ref. 2.
meation and swelling. Normally they are considered by solv-
Cell state & Cellular automata rule θ and γ values
ing differential equations, with relevant boundary and initial
symbol
conditions specific to each system. This can be quite time
consuming and must be done separately for every system. Water (W) No rules. θ = 0, γ = 0
Solid drug (D) When in contact with a W, p or O θ = θ0.
Here, a new approach for drug release modeling based on will convert into a mobile drug
a cellular automata model for virtually any geometry is pro- cell. Drug cells are not allowed to
posed1-2. It takes into account all four major drug release diffuse except through water or
14 mechanisms. Similar models have been used to model drug wet polymer.
release from eroding polymeric matrices3-7. In this model a
2D representation of the release device is divided into a grid Mobile drug Will randomly move to any If a transfer takes place,
space, where each cell represents a small part of the whole (dissolved) (d) neighboring water cell and con- the θ value of this cell is
system. Each cell is connected to 4 neighboring cells and vert it into a d cell. Can move into decreased by one and θ
can interact with them according to a simple set of rules de- a wet polymer cell with a prob- of the receiving cell is
signed to model the physics and chemistry of drug release. ability pp and form an O cell. increased by one.
For example, diffusion is modeled by allowing drug cells to Will be removed from the simu- Converts into a W cell
randomly move into adjacent water cells but not into other lation if it reaches the boundary when the value of θ is
drug cells. Diffusion inside permeable membranes is modeled of the simulation area. reduced to 0.
in the same manner, but with a lower diffusion coefficient. The
Polymer (P) Will become a p cell when in con- γ = γ0.
idea behind the model is to consider microscopic domains of
tact with water with a probabil-
the release device and treat them statistically instead of con-
ity pw. Polymer cells are assumed
sidering molecular interactions directly. For each physical or
to be passive unless they come
chemical reaction possible in the system, a probability for that
into contact with water.
occurrence is given. As this is the basis behind diffusion coeffi-
cients and kinetic rate constants, it is relatively straightforward Wet polymer Can swell with a probability ps if If swelling takes place,
to relate the probabilities to these constants. (p) γ > 1. the cell will increase
Has a probability pe to be eroded. the γ of a nearby cell
The model divides an assumed spherical drug release device The probability is higher if there by one and lower its
or tablet into a matrix of cells. The state of each cell represents are more water cells adjacent to own γ by one. Erosion
the physical contents of that part of the tablet. The states can it. decreases γ by one.
be either water, polymer, drug or a combination of the three. Converts into a W cell
Each cell represents a domain of the release device, not single when γ is reduced to 0.
molecules or even groups of molecules. During the simulations,
cells are allowed to interact with the 4 adjacent cells and change Wet polymer Obeys the rules of both d and p If θ falls to zero, this
their state according to a simple set of rules , shown in Table 1. with drug (O) cells. cell is converted into a
A dissolved drug filled domain is always assumed to be at the p cell. If γ falls to zero,
saturation concentration and concentration is represented by this cell is converted
the density of these cells. Each cell has a predefined set of prob- into a d cell.

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

As an example, swelling-controlled release is considered. In with limited calculation power required. It is therefore sug-
this case, water as well as drug diffusion and polymer swell- gested that it could prove to be useful for the design of swell-
ing has to be taken into account. Adding these rules to the ing drug formulations and other controlled release systems.
cellular automata model is quite straightforward as can be
seen from Table 1. An example of a swelling-controlled re- REFERENCES
lease simulation is shown in Fig 1. The typical near-zero or- 1. Laaksonen, T., Laaksonen, H., Hirvonen, J., Murtomäki,
der release profile is seen for most of the release time as the L., Cellular automata model for drug release from binary
thickness of the hydrogel layer stays constant throughout matrix and reservoir polymeric devices, Biomaterials,
the dissolution2. 2009, 30: 1978-1987.
2. Laaksonen, H., Hirvonen, J., Laaksonen, T., Cellular
automata model for swelling-controlled drug re-
lease, Int. J. Pharm., 2009, In press, doi:10.1016/j.ijp-
harm.2009.06.023
3. Göpferich, A., Langer, R., Modeling monomer release from
bioerodible polymers. J. Controlled Release, 1995, 33: 55-69.
4. Barat, A., Ruskin, H.J., Crane, M., Probabilistic methods
for drug dissolution. Part 2. Modelling a soluble binary
drug delivery system dissolving in vitro, Simul. Model.
Pract. Theory, 2006, 14: 857-873.
5. Barat, A., Ruskin, H.J. & Crane, M., Probabilistic models
for drug dissolution. Part 1. Review of Monte Carlo and
stochastic cellular automata approaches, Simul. Model.
Figure. 1: Illustration of a single simulation run for a swell- Pract. Theory, 2006, 14: 843-856.
ing-controlled drug release system. The status of the drug 6. Zygourakis, K., Development and temporal evolution of
release matrix in different time points during the simulation erosion fronts in bioerodible controlled release devices,
is shown above. The release curve and the location of the dif- Chem. Eng. Sci., 1990: 45: 2359-2366.
ferent fronts during the simulation are shown below. Black 7. Zygourakis, K., Markenscoff, P.A., Computer-aided de-
spots represent polymer, dark gray spots represent solid sign of bioerodible devices with optimal release char-
drug, gray spots represent wet polymer and light gray spots acteristics: a cellular automata approach, Biomaterials,
represent wet polymer with drug cells. Adapted from ref. 2. 1996, 17: 125-135.
8. Colombo, P., Santi, P., Bettini, R., Brazel, C.S., Peppas, N.A., 15
In conclusion, a cellular, extremely flexible model for con- Drug Release from Swelling-Controlled Systems. In:
trolled drug release simulations was presented. The model Wise, D.L. (ed), Handbook of Pharmaceutical Controlled
can take into account swelling and erosion of the polymer, Release Technology, Marcel Dekker, New York, 2000, pp.
drug and water permeations and solubility of the drug. The 183-209.
model was simple to implement and gave realistic results

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

O07
Modelling and simulation in modern pharmacy –bridging
regulatory bodies, academia and industry
Sebastian Polak
Unit of Pharmacoepidemiology and Pharmacoeconomics, Faculty of Pharmacy, Jagiellonian University Medical
College, Medyczna 9 Str., 30-688 Krakow, Poland
Simcyp Ltd, Blades Enterprise Centre, John Street,  S24SU Sheffield, UK

T
he main reasons for growing role of modelling and sim- on substance structure plays important role at the initial
ulation tools in pharmacy are widely known. Among stage of the drug discovery. Having possibility to use and
them dramatic cost increase of the drug discovery & compare both free (i.e. vcclab.org) and commercially avail-
development process, ethical reasons forcing scientist to re- able (i.e. Marvin, ACDlabs) software every substance can be
duce number of animals sacrificed during drug development characterized according to the BCS classification. Specialized
process could be listed, but the most important reason is a chemical software (MOE, Dragon) offers calculation of the
high reliability of currently used in silico methods. It is worth theoretical descriptors which can be further used for estima-
mention that modelling and simulation procedures are not tion of potential pharmacological and toxicological activity
only in common use at the universities during scientific re- (i.e. hERG channel inhibition, skin irritation, hepatotoxicity).
search studies and by pharmaceutical industry but also more At the development level using in vitro information obtained
and more widely accepted and even requested by the main in the research laboratories it is possible to simulate in vivo
regulatory bodies like FDA.  drug behaviour with use of a virtual population (Simcyp)
what enables considering inter-individual variability. Such a
M&S methods and tools are in common use beginning from facility can be directly used to plan Phase I clinical trials and
the very early discovery (physico-chemical parameters pre- reduce costs of unnecessary studies. It becomes extraordi-
diction), thorough qualitative forecasting and quantitative narily important for the paediatric population which can be
16 assessment of toxicity, pharmacokinetic characterization simulated with use of Simcyp Paediatric module. Concomi-
(including in vitro –  in vivo extrapolation and clinical trials tantly carried formulation studies can be optimized by drug
simulation), pharmacodynamic activity, biopharmacy, com- absorption prediction platforms (Simcyp) for various routes
puter aided drug formulation and Phase IV clinical trial con- of administration and specific, formulation focused (i.e. mi-
ducting (effect prediction for pharmacoeconomic studies).  croemulsions composition) systems.
Prediction of the basic physico-chemical parameters based

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

O08
Microtensiometry - novel surface tension based HTS method for
solubility testing of drug materials
Tiina Heikkilä, Leena Peltonen, Saila Taskinen, Timo Laaksonen and Jouni Hirvonen
Division of Pharmaceutical Technology, University of Helsinki, P.O. Box 56 (Viikinkaari 5E), 00014 University of
Helsinki, Finland

INTRODUCTION drug and diluting the next sample to half the concentration of the
High throughput screening (HTS) techniques are mostly based previous one. Last samples were blank. The presolvent solutions
on the determination of kinetic solubility. Active pharmaceuti- were then added to the medium in the 96-well plate where the
cal ingredient (API) is dissolved in dimethyl sulfoxide (DMSO) single well volume was 50 µl. The samples were let to stand for 10
and small volumes of the aqueous media are added until the minutes prior to the measurements without shaking5. Measure-
solubility limit is reached at the point where the API precipitates. ments were repeated at least 4 times at room temperature (appr.
In the existing 96-well plate methods, the kinetic solubility can 24 oC). Determined solubilities of ibuprofen and indomethacin var-
be determined by turbidity, nephelometry or UV absorption1-2. ied between 0.0005 – 10 mg/ml and 0.004 – 5 mg/ml, respectively.
Still, these methods have some weaknesses. For example, with
UV absorption, problems can arise from the low absorption of The surface tension measurements were done with a fully automat-
poorly soluble material. Also possible absorption by residues ed Delta-8 multichannel microtensiometer (Kibron inc., Finland).
or other materials, like buffering salts, in the same wavelength The method is based on Du Noüy maximum pull force technique
area as the studied compound can cause problems2. and the measurement of one 96-well plate lasted appr. 5 min.

In this study a microtensiometric method based on surface The shake-flask solubility tests were done in duplicate un-
tension measurements is introduced for drug solubility re- der the same conditions as the microtensiometric measure-
search purposes. Amphiphilic molecules decrease the surface ments according to a standard procedure. Samples were 17
tension (γ) within a concentration range limited by air/water analyzed after filtration by UV-spectrophotometry (λ= 264
partitioning at the lower concentration end and solubility/ nm for ibuprofen and 254 nm for indomethacin, Pharmacia
critical micelle concentration (CMC) at the higher end, which LKB, Ultrospec, Sweden).
is described by the classical Gibbs adsorption isotherm:
RESULTS AND DISCUSSION
Figure 1 shows an example of a measured Gibbs isotherm for
, ibuprofen in water. The solubility of ibuprofen can be seen from
the focal point between the slope and the level part of the iso-
where Γ is surface excess, R is gas constant, T is temperature therm. In this point, there is an equilibrium state between the
and c is concentration. solid and solvated phases. At higher concentrations, all material
If the compound is more likely to precipitate than to form mi- exceeding the solubility limit will be in the solid phase, which
celles, the value for solubility can be determined from these has no effect on the surface tension. Also the effect of changing
isotherms3 (Figure 1). In practice, almost all drug compounds pH is seen in Figure 1; lowering of the pH decreased the solubil-
have some surface activity and the measurement is possible. ity, which was seen as a shift in the adsorption curve.

The aim of this study was to utilize interfacial tension measure-


ments at the air/water interface for the kinetic solubility deter-
minations. Poorly water-soluble BCS class II drugs ibuprofen
and indomethacin were used as the model drugs. Different sol-
ubility areas were tested by changing the medium. Traditional
shake-flask measurements were used as a reference method.

MATERIALS AND METHODS


Solubilities of ibuprofen and indomethacin were studied in
buffer solutions pH 1.2 and pH 6.8 (4) and in water. Either
DMSO (10% v/v) or methanol (10 % v/v) was used as presol-
vent. Also the effects of NaCl and CaCl2 were studied. Figure 1: Gibbs isotherm for ibuprofen in water in two differ-
ent pHs. Surface tension is decreased with increasing drug
The studied drugs were first dissolved in presolvent. 12-22 samples concentration until a solubility limit is reached. Methanol
were prepared in each medium starting from ca. 100 mg/ml of the was used as a presolvent (n=12).

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

The correlation between shake-flask and microtensionmeter Figure 3: Solubilities of ibuprofen and indomethacin (marked
methods was good (Figures 2 and 3). As expected for weak in the figure) by the surface tension measurements (black
carboxylic acid, the solubility of ibuprofen decreased in the pillars, n=8-12) and shake-flask measurements (grey pillars,
shake-flask method from 0.09 mg/ml in pure water to 0.05 n=2).
mg/ml at pH 1.2, and increased to 3.1 mg/ml at pH 6.8. The
similar trends were obtained for indomethacin. NaCl and The developed surface tension based solubility testing meth-
CaCl2 concentrations of 0.9% and 2%, had only limited ef- od is fast, can be easily repeated and gives a large amount of
fects on the solubility of ibuprofen (Figure 3). Still, a linear qualitative data. As compared to other screening methods
dependence exists in all the media tested between the two for solubility testing, microtensiometric analysis does have
methods for both the model substances. some clear benefits. For example, in UV-based methods
problems arise if the absorption areas of different materi-
als overlap or the impurities with absorbance potential may
cause inaccuracy to the results or the material is poorly ab-
sorbing. One limitation for the technique is that the CMC and
solubility limits may not be differentiated. Though this is not
likely to cause problems for most of the drugs, very insoluble
drugs can, in some cases, not be analyzed since they might
not partition sufficiently to the water/air interface.

CONCLUSION
This study shows that the microtensiometric determination
of drug solubility is a promising new HTS method. The mea-
surements are fast to perform, can be easily repeated and
large amount of qualitative data with a minimum amount of
analyses required are achieved.

REFERENCES
Figure 2: Solubilities of ibuprofen from the surface tension 1. Bevan and Lloyd, Anal. Chem., 72(2000), 1781.
measurements (y-axis) as compared to the shake-flask mea- 2. Pan et al., J. Pharm. Sci., 90(2001), 521.
surements (x-axis). 3. Johans, www.kibron.com, 2004.
18 4. USP XXXI, 2007.
5. Heikkilä et al., Letters Drug Design Deliv., 5(2008), 471.

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

O09
THE POTENTIAL OF MAGNETIC RESONANCE IMAGING AS ANALYTICAL TOOL
FOR THE INVESTIGATION OF CONTROLLED RELEASE MATRIX TABLETS
S. Baumgartner1, J. Kristl1, U. Mikac2, A. Sepe2
1
University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana/Slovenia
2
Jožef Stefan Institute, Jamova 39, 1000 Ljubljana/Slovenia

INTRODUCTION The NMR spin-lattice (T1) and spin-spin relaxation (T2) times
Various hydrophilic polymers have been investigated as the were measured at 1H NMR frequency of 100MHz for hydro-
basis for gel-forming, controlled release tablets, of which gels with known XAN concentrations (Bruker, G, with a su-
HPMC currently appears to be among the most useful1. How- perconducting magnet Oxford Instruments Ltd., UK). The T1
ever, the polymers of the natural origin, like xanthan (XAN), was measured by standard inversion recovery sequence and
are becoming more and more important in the pharmaceu- T2 by CPMG pulse sequence.
tical research. The drug release from such tablets is a very
complex process and is to a great deal influenced by the gel MRI of XAN tablets during swelling
layer structures forming around the dry tablet core2. For T2-mapping multi-echo pulse sequence was used: TE =
6.2 ms, number of echoes N = 50, TR = 200 ms. The field of
The polymer swelling process has been studied by variety of view was 50 mm with in-plane resolution of 200 µm and slice
analytical techniques. Among them, magnetic resonance im- thickness of 3 mm. The concentration profiles were calculat-
aging (MRI) has been proven to be a powerful method for fol- ed from the signal intensity of the image with N = 1, i.e. TE =
lowing the formation of hydrogel because it is non-invasive, 6.2 ms. Sequential MR-images of matrix tablets during swell-
spatial selective and can provide quantitative information. ing in different media were taken and as a reference medium
Various approaches have been taken in using MRI for quanti- was used.
tative characterization of the swollen tablets3,4. In one of the 19
latest, benchtop MRI was introduced into this research5. RESULTS AND DISCUSSION
Drug release studies
The aim of this study was to investigate the influence of pH Release of PF from XAN tablets was prolonged by more than
and ionic strength of medium on dynamics of hydrogel for- 24 h in water and in HCl pH 3.0 medium. The slowing of re-
mation of the XAN matrix tablets. For resolving specific XAN lease was accompanied by evident swelling of the matrix
structural changes influencing drug release, measuring and tablet (Fig 1)
modeling tools of MRI were used.

MATERIALS AND METHODS


Materials
As polymer matrix xanthan (XAN, Sigma-Aldrich Chemie, G)
was used. As water soluble drug pentoxifylline (PF) was sup-
plied by Krka, d.d., SI. For drug release and MRI studies 6 dif-
ferent media were prepared: purified water pH 5.5, HCl pH
1.2 and pH 3.0. To increase ionic strength for 0.2M, NaCl was
added to each media.

Preparation of matrix tablets


For MRI, XAN was directly compressed (SP 300, Kilian&co.,
Cologne, G) to form tablets of hardness 100N±10 (VanKel
VK200, USA), m=500mg, 2r=12mm. For drug release 100 mg
of PF was incorporated into 400mg tablets. Figure 1: The release profile of pentoxifylline from XAN tab-
lets in different media
Drug release from XAN matrix tablets
Dissolution studies were performed in all 6 media on a dis- When tablets were placed in medium with increased ionic
solution apparatus, paddle method (Apparatus II, VK7000, strength or pH 1.2 the drug release was much faster. Since
USA), 900ml, 50rpm, 37°C±0.5. Samples were UV analyzed at the dynamics of swelling and arrangement of water mole-
274 nm (HP diode array UV spectrophotometer, 8453, G). cules within the formed gel structure is crucial for drug re-
lease, XAN tablets during swelling process were investigated
Measurements of NMR relaxation times by MRI.

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

MR imaging and T2-mapping


To be able to determine the polymer concentration pro-
file during swelling, the relaxation times T1 and T2 of gels at
known XAN concentrations were measured. From these re-
sults theoretical signal intensities (S) of gels were calculated
according to eq.

where k is the constant independent of polymer, ρ is the pro-


ton density, TE and TR are experimental parameters. Figure 3: XAN polymer concentration profiles across the tab-
The images at different times after hydration of tablets in all let and T2 profiles in HCl pH 1.2 + NaCl medium
6 media were taken (Fig 2). The dry tablet core is black and
the brightness of the newly formed gel Differently hydrated domains were proved as the conse-
layer depends on its concentration. quence of XAN conformational changes during swelling.
Furthermore, the drug release was faster, due to the pres-
ence of larger fraction of free water molecules and thinner
gel layer.

REFERENCES
1. Colombo P, Bettini R, et al. Swellable matrices for con-
trolled drug delivery: gel-layer behaviour, mechanisms
and optimal performance. Pharm. Sci. Technol. Today
2000; 3: 198-204.
2. Baumgartner S, Pavli M, Kristl J. Effect of calcium ions on
the gelling and drug release characteristics of xanthan
matrix tablets Eur. J. Pharm. Biopharm. 2008; 69: 698 –
Figure 2: MR images of XAN during swelling in HCl pH 1.2 707.
+ NaCl medium. Square on the 1st image denotes the area 3. Fyfe CA, Blazek-Welsh AI. Quantitative NMR imaging
20 that was chosen for evaluating the polymer concentration study of the mechanism of drug release from swelling
profile. White arrows indicate different domains within gel. HPMC tablets. J. Control. Release 2000; 68:313-333.
4. Baumgartner S, Lahajnar G, Sepe A, Kristl J. Quantita-
The polymer concentration profiles across swollen tablet are tive evaluation of polymer concentration profile during
much steeper at low pH and in presence of NaCl, proving swelling of hydrophilic matrix tablets using 1H NMR and
denser, but thinner gel network. Unusual gel formation at MRI methods. Eur. J. Pharm. Biopharm. 2005; 59: 299-
pH 1.2 was observed from MRI. For certain conditions during 306.
swelling, T2 can be quite long even at high XAN concentra- 5. Metz H, Mäder K. Benchtop-NMR and MRI–a new ana-
tion (Fig 3). lytical tool in drug delivery research, Int. J. Pharm. 2008;
364 170-175.

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

O10
USING MESOPOROUS INORGANIC MATERIALS FOR
DELIVERING POORLY SOLUBLE DRUGS
Vesa-Pekka Lehto
Department of Physics, University of Kuopio, FI-70211 Kuopio, Finland

INTRODUCTION
Loading of therapeutic compounds into the confined space
of mesoporous inorganic materials can radically change their
properties and fate when delivered in human body via various
administration routes. Not only the modified physico-chemical
properties obtained with the nanostructuring of the payloads,
but also the protection of the payloads against the harsh con-
ditions of the gastrointestinal tract, together with the possibil-
ity to cross biological barriers make inorganic porous nano/
microparticles attractive carriers for drug delivery purposes.
However, safety and biocompatibility are the basic require-
ments for any drug carrier. Biodegradability is also preferential
feature when concerning parenteral administration. Figure 1. A schematic drawing of drug loaded PSi particle
with functionalized surfaces.
It is estimated that over 95% of the new drug candidates suf-
fer from poor pharmaco-kinetic/ADME-properties when deliv- When aiming at oral delivery, microparticles are typically uti-
ered orally. The main reasons for the inadequate oral bioavail- lized; when aiming at systems for parenteral administration, 21
ability are poor thermodynamic solubility and/or dissolution nanoparticles are employed. However, utilizing nanoparticles
rate of the compound in the intestinal lumen, inadequate also in oral drug delivery may be advantageous as the residence
permeability properties of the drug across the gastrointesti- time in gastrointestinal (GI) tract can be prolonged by mucoad-
nal (GI) wall, as well as high intestinal and hepatic first pass hesion or the particles can even cross the GI epithelium.
metabolism. Thus, due to these problems in drug discovery,
delivery systems addressing issues regarding low solubility, The presentation focuses on the results obtained in a Finnish
enzymatic degradation and means to modulate drug perme- research consortium to manifest the feasibility of mesoporous
ability (absorption) have been emphasized as key methodolo- silicon as a drug carrier4-6. The encouraging results concern
gies. high payloads, stabilization of the payloads, improved disso-
lution, reduced pH dependence of dissolution, improved per-
Silicon based mesoporous materials have been reported to meation, biocompatibility/non-toxicity, bio-degradation and
have potential to overcome problems encountered in the de- sustained release. The aim has been in the development of PSi
velopment of effective drug delivery systems1,2. Mesoporous as a safe and effective platform for delivery of therapeutics.
silicon (PSi) is a top-down material produced typically from
silicon wafers via electrochemical etching3. PSi is a nano- MATERIALS AND METHODS
structured material with closely spaced 2 – 50 nm wide pores The PSi micro/nanoparticles were prepared by electrochemi-
throughout its volume. Silicon materials are biodegradable, cal etching as described previously3. The pore structure and
and they possess some beneficial properties regarding drug pore diameters were varied by changing the current density
delivery applications. The small size of the pores confines the in the etching or by a post-treatment of annealing. Particles
space of a drug and emphasizes the effects of surface inter- with different size fractions were produced by milling of the
actions of the drug molecules and the pore wall (Figure 1). PSi films and by a subsequent wet sieving. The surfaces of
The size of the pores and the surface chemistry of the pore the particles were modified according to the intended appli-
walls may be easily controlled. Depending on the size and cation as hydrogen terminated, oxidized, hydrocarbonized
the surface chemistry of the pores, increased or sustained re- or carbonized. The surfaces can also be grafted with carboxyl
lease of the loaded drug can be obtained. Drug loading from or amino groups to facilitate a further bioconjugation. Typi-
a solution at room temperature (or below) enables the use of cally the loading was performed by immersing the particles
PSi also with sensitive therapeutic compounds like peptides in an appropriate drug solution. Dry powders of drug loaded
and proteins. PSi microparticles were obtained after filtration and drying.
Regarding nanoparticles the material was used as suspen-

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

sion. The unloaded and loaded particles were studied with


various physical methods including FTIR, DSC and TG to ana-
lyze, e.g., the stability and the loading degree of the drug.
The drug loaded particles were characterized in vitro con-
cerning dissolution behaviour, permeation and cytotoxic-
ity (flow cytometric analysis, CellTiter-Glo Luminescent Cell
Viability assay and CellTiter-Fluor Cell Viability assay) with
Caco-2 cells. In vivo experiments were performed to charac-
terize the toxicity (cytokine release tests after subcutaneous
administration), biodegradation and biocompatibility of PSi
after s.c. administration in mice.

RESULTS AND DISCUSSION


The typical loading degrees (mdrug/mtotal) for small molecules
were ca. 30% ranging from 20% to 60%. The latter case con-
cerns material with high porosity and optimal loading. With
peptides the obtained loading degrees were around 20%. By
loading poorly water soluble drugs in PSi particles their dissolu-
tion behaviors was significantly improved. For example, with in-
domethacin a 28-fold improvement in the dissolution rate was
achieved (Figure 2). For furosemide, which dissolution behavior
is very pH dependent, the dependence was minimized when
loaded in PSi. The permeability across Caco-2 cell monolayer
was improved 1.5-4 fold without any adverse effects on the
monolayer integrity. The large PSi particles were well tolerated
even with high concentrations by Caco-2 cells in vitro, but small
particles (<25 µm) seemed to reduce the viability of the cells
(Figure 3). However, cytokine release tests showed normal cy-
tokine activity in mice in vivo after s.c. administration suggest-
ing the particles are not causing any inflammatory effects (Ta-
Table 1: Plasma concentrations of various cytokines after s.c. ad-
22 ble I). Microscopic examinations of the injection sites supported
ministration of thermally hydrocarbonised PSi microparticles.
this finding. Importantly, the particles were biodegradable.
CONCLUSION
Based on the recent results obtained in our consortium and
elsewhere mesoporous silicon seems to be a potential platform
for delivery of therapeutics. However, various administration
routes demand specific concern regarding the safety of the
mesoporous silicon material with different surface chemistries.

ACKNOWLEDGEMENTS
Jouni Hirvonen, Kristiina Järvinen, Karl-Heinz Herzig, Marjat-
ta Louhi-Kultanen, Ale Närvänen, Anu Airaksinen and Jarno
Figure 2: Release kinetics of indomethacin as free powder Salonen are thanked for fruitful collaboration. The financial
and loaded in thermally carbonized PSi microparticles. support from the Academy of Finland is acknowledged.

REFERENCES
1. Prestidge C, Barnes T, Lau CH, Barnett C, Loni A, Canham
L, Expert Opin. Drug Deliv. 2007; 4: 101-110.
2. Salonen J, Kaukonen AM, Hirvonen J, Lehto VP, J. Pharm.
Sci. 2008; 97, 632-653.
3. Salonen J, Lehto VP, Chem. Eng. J. 2008; 137: 162-172.
4. Salonen J, Laitinen L, Kaukonen AM, Tuura J, Björkqvist
M, Heikkilä T, Vähä-Heikkilä K, Hirvonen J, Lehto V-P, J.
Control. Release 2005; 108: 362-374.
5. Kaukonen AM, Laitinen L, Salonen J, Tuura J, Heikkilä T,
Limnell T, Hirvonen J, Lehto VP, Eur. J. Pharm. Biopharm.
2007; 66: 348-356.
Figure 3: Number of living cells (%) determined by flow cytom- 6. Kilpeläinen M, Riikonen J, Vlasova MA, Huotari A, Lehto
etry when Caco-2 cells were treated and incubated for 3 hours VP, Salonen J, Herzig KH, Järvinen K, J. Control. Release
with PSi microparticles with various surface chemistries. 2009; 137: 166-170.

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

O11
Carbon nanotubes as drug delivery systems
İsmail Tuncer Değim
Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06330, Etiler, Ankara, Turkey

C
arbon is the most common and versatile element in as carriers for other encapsulated molecules such as drugs or
the world. The type, strength, and number of bonds imaging reagents. Fullerene adducts or organic derivatives
of carbon in the molecule can form many different of fullerene molecules7 are the ideal candidates for applica-
elements with variety of properties. The diversity of bonds tion in peapod syntheses to create advanced materials. For
and their corresponding geometries enable the existence example, a C60 molecule could serve as a vector to enable a
of structural isomers, geometric isomers, and enantiomers. second, covalently conjugated molecule to be encapsulated
These are found in large, complex, and diverse structures inside a SWNT. This approach could facilitate the assembly of
and allow for an endless variety of organic and inorganic molecular “packages” wrapped in a protective SWNT for the
molecules. The properties of carbon are a direct conse- purpose of drug delivery. Unfortunately, fullerene adducts
quence of the arrangement of electrons around the nucleus typically decompose at relatively low temperatures, necessi-
of the atom. Carbon in the solid phase can exist in three allo- tating mild filling conditions using solutions8 or supercritical
tropic forms: graphite, diamond, and buckminsterfullerene. fluids. Although there are some reports and successful re-
Diamond has a crystalline structure where the different ge- sults, drug delivery attempts with CNTs rather concentrated
ometry of the chemical bonds makes graphite soft, slippery, to use of CNTs9.
opaque, and electrically conductive. Buckminsterfullerenes,
or fullerenes, are the third allotrope of carbon and consist An ideal cancer treatment would deliver a high dose of drugs
of a family of spheroidal or cylindrical molecules. The tubu- to tumor sites while minimizing side effects. Unfortunately, as 23
lar form of the fullerenes called nanotubes (CNTs), will be anticancer drugs are quickly cleared from the bloodstream,
the subjected to this presentation and their usage as drug large doses are usually needed to ensure that enough drugs
carrier system will be discussed. Although CNTs were first reach the tumor. And since these drugs can be absorbed by
discovered in 1952 by Radushkevich and Lukyanovich in normal cells, high doses cause unpleasant side effects. Now
Russia1 it was not announced through worldwide, therefore research from Stanford University10 has shown that carbon
it is generally accepted that at a carbon-carbon composites nanotubes loaded with anticancer drugs can target tumor
workshop, Rick Smalley proposed the existence of a tubular cells while steering clear of healthy tissue. The CNTs on av-
fullerene in 19902. Experimental evidence of the existence of erage 100 nanometers long and a few nanometers wide
CNTs came in 1991 when Iijima imaged multiwalled carbon pass easily through the leaky walls of tumor blood vessels
nanotubes (MWCNTs) and Iijima was first to recognize that but do not get into healthy blood vessels. So the researchers
nanotubes were concentrically rolled graphene sheets with realized that drugs attached to the nanotubes could be car-
a large number of potential helicities and chiralities rather ried inside tumors without harming normal tissue. To make
than a graphene sheet rolled up like a scroll as originally pro- working nano-drug transporters, the researchers coated the
posed by Bacon. Iijima initially observed only MWCNTs with nanotubes with a molecule called polyethylene glycol (PEG),
between two and twenty layers3, but in a subsequent publi- which has three branches on one end, then attached mol-
cation in 1993, he confirmed the existence of single-walled ecules of the anticancer drug paclitaxel to each branch10. The
carbon nanotubes (SWCNTs) and elucidated their structure4. drug-delivery technique was tested in breast cancer model
Since the discovery of CNTs, numerous ideas for applications in mice and they found that the tumors treated by nanotube
have arose in a wide variety of scientific disciplines, such as delivery were less than half the size of the tumors treated by
electronics and opto-electronics, sensors, batteries/fuel cells, the second most effective available conventional treatment
medicine/biology (fluorescent markers, labeling and drug with Taxol10. However, despite such a dramatic improve-
delivery carriers), catalysis and gas storage5. Because of ment, lingering concern over the potential toxicity of carbon
their intrinsic optical properties, nanotubes have been con- nanotubes means that they may be a long way from clinical
sidered potential candidates for drug delivery carriers. The use as a drug-delivery mechanism. That safety concerns will
capped ends of nanotubes may be opened up by oxidation, probably delay the approval of carbon nanotubes for medi-
allowing for the insertion of molecules of interest inside the cal purposes.
nanotube. Smith et al. observed peapods6, SWNTs filled with
C60, via high-resolution transmission electron microscopy, It has been also shown that single-walled carbon nanotubes
These peapods can form suggests that nanotubes may serve (SWNTs) and multiwalled carbon nanotubes (MWNTs) can

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

be internalized by living cells and pass across the biological ogy,1990, 6–7.
membranes in cell culture studies11. The internalization of 3. Iijima S, Helical microtubules of graphitic carbon, Na-
carbon nanotubes by corneo-cytes has been shown12 in the ture, 1991, 354: 56–58.
literature but their drug carrying properties through the skin 4. S. Iijima and T. Ichihashi. Single-shell carbon nanotubes
have not been fully evaluated. But in our previous study it of 1-nm diameter, Nature, 1993, 363: 603–605.
has been first shown that single-walled carbon nanotubes 5. O’Connell, MJ, Carbon Nanotubes Properties and Appli-
(SWNTs) and multiwalled carbon nanotubes (MWNTs) can cations, CRC Press, Taylor & Francis Group, FL, US, 2006.
be used to deliver drug molecules through deeper skin lay- 6. Smith BW, Monthioux M, and Luzzi DE, Encapuslated
ers. The application of iontophoresis using carbon nanotube C60 in carbon nanotubes, Nature, 1998, 396: 323–324.
electrode having adsorb drug molecules on their surface has 7. Kadish, K.M. and Ruoff, R.S., Eds., Fullerenes: Chemistry,
been shown and molecules successfully transferred through Physics and Technology, Wiley, New York, 2000.
deeper skin layers13. Indomethacin was selected as a hydro- 8. Yudasaka, M. et al., Nano-extraction and nano-conden-
phobic drug (Log Ko/w = 4.5) and it penetrates through full sation for C60 incorporation into single-wall carbon
thickness skin6. The penetration of indomethacin through nanotubes in liquid phases, Chem. Phys. Lett., 2003, 380:
fullthickness of rat skin was enhanced when indomethacin 42.
adsorbed CNTs were used (Flux values were 0.119±0.037 9. Britz DA. et al, Selective host–guest interaction of single-
μg/h for indomethacin alone; 0.330±0.052 μg/h for indo- walled carbon nanotubes with functionalised fullerenes,
methacin with MWCNTs and 0.347±0.106 indomethacin with Chem. Commun., 2004, 176.
double walled carbon nanotubes (DWCNTs, n=3,±SD) (14). 10. h t t p : / / w w w . t e c h n o l o g y r e v i e w . c o m /
The penetration enhancement was higher with DWCNTs Nanotech/21316/?a=f
and MWCNTs, however the mechanism was still unknown. 11. Lacerda L, Raffa V, Prato M, Bianco A, Kostarelos K, Cell-
The CNTs may act to facilitate presentation of the drug to penetrating carbon nanotubes in the delivery of thera-
the lipophillic membrane and/or they facilitate penetration peutics, Nano Today, 2007, 2: 38–43.
through the skin accompanying the drug into the dermal 12. Monteiro-Riviere NA, Inman AO, Wang YY, Nemanich RJ,
tissue. Similar results were obtained when pig ear skin was Surfactant effects on carbon nanotube interactions with
used. Penetration of indomethacin was found to be much human epidermal keratinocytes, Nanomedicine: Nano-
higher when indometahcin molecules were introduced to technology, Biology and Medicine, 2005.1: 293-299.
the skin surface with CNTs14. 13. Martínez-Pla JJ, Martín-Biosca Y, Sagrado S, Villanueva-
Camañas RM, Medina-Hernández MJ, Evaluation of the
All previous studies have shown that carbon nanotubes pH effect of formulations on the skin permeability of
24 can be used to deliver drug molecules to the active site. drugs by biopartitioning micellar chromatography, J.
Therefore it was aimed to determine adsorption of pacli- Chromatogr. A., 2004,1047: 255-262.
taxel, oxaliplatin and other non-steroidal anti-inflammatory 14. Degim IT, Verma S, Papadimitrakopoulos F, Burgess D,
drug naproxen. Recent studies indicated and showed that Carbon nanotubes (CNTs) for enhancing drug transport
cyclooxygenase (COX) can have a role in development of through skin, Controlled Release Society Annual Meet-
cancer and tumors. Nonsteroidal anti-inflammatory drugs ing & Exposition, July 12-16 2008, New York, U.S.A. (Oral
(NSAIDs) are therefore have noticeable effects on reducing presentation).
tumor mass and its development15-18. Naproxen sodium was 15. İlbasmış Tamer S, Yılmaz Ş, Degim IT, Nanoscience Nano-
selected as a model NSAID. The drug adsorption capacity of technology, Nanobiotechnology, Nanomedicine Con-
DWCNTs and MWCNTs were found to be 14.25 and 14.70% ference, June 11-14-2007. Bilkent, Ankara, Turkey (Oral
(weightCNTs/weightDrug) respectively19. presentation).
16. Xin B, Yokoyama Y, Shigeto T, Mizunuma H, Anti-tumor ef-
The adsorption properties of DWCNTs and MWCNTs were fect of non-steroidal anti-inflammatory drugs on human
also investigated for oxaliplatin and MWCNTs were found ovarian cancers, Pathology Oncology Research,2007,13:
to be adsorbed oxaliplatin higher19. Oxaliplatin and pacli- 365-369.
taxel were also adsorbed to the surface of fullerene and it 17. Piffara PM et al,, Naproxen, clenbuterol and insulin ad-
was found that in cell culture studies (MDCK cell lines) they ministration ameliorates cancer cachexia and reduce
can actively stop cell proliferation more than their free forms tumor growth in Walker 256 tumor-bearing rats, Cancer
(MTT tests)19. Letters, 2003, 201: 139–148.
18. Chen JH, Liu TY, Wu CW,. Chi C, Nonsteroidal anti-inflam-
As a conclusion CNTs were found to be suitable material matory drugs for treatment of advanced gastric cancer:
for drug transport having higher surface area and being an cyclooxygenase-2 is involved in hepatocyte growth
adsorptive material, CNTs can be useful materials for deliv- factor mediated tumor development and progression,
ery of drug molecules by simply adsorption and desorption Medical Hypotheses, 2001, 57: 503-505.
mechanism. 19. Ilbasmıs-Tamer S, DEGIM IT, Carbon nanotubes to de-
liver drug molecules, NanoTR5, , 5th National Congress
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1. http://en.wikipedia.org/wiki/Timeline_of_carbon_nan- Bilkent, Anadolu University, Eskişehir, Turkey (Poster pre-
otubes sentation).
2. Smalley RE, Formation and properties of C60 and the
fullerenes, National Institute of Standards and Technol-

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

O12
Colloidal Dispersions for Targeted Brain Drug Delivery
Paolo Blasi1, Aurélie Schoubben1, Stefano Giovagnoli1, C. Puglia2, L. Barberini3, C. Cirotto3, Carlo Rossi1,
Maurizio Ricci1
1
Dipartimento di Chimica e Tecnologia del Farmaco; Università degli Studi di Perugia, Italy
2
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Catania, Italy
3
Dipartimento di Biologia Cellulare e Ambientale, Università degli Studi di Perugia, Italy

INTRODUCTION gies was employed to optimize the fabrication procedure and


The concentration and clearance of endogenous and ex- to individuate the most favourable working conditions. The
ogenous molecules in the brain are strictly regulated by its obtained NPs were characterized using photon correlation
anatomic and physiologic features, such as the blood-brain spectroscopy (PCS), transmission electron microscopy (TEM),
barrier (BBB). Furthermore, the enormous efforts made for and differential scanning calorimetry (DSC). Lipid colloids
the individuation of new specific and powerful drug candi- were loaded with a fluorescent dye, nile red, and loperamide,
dates are often vain. For these reasons, different technologi- a model pharmacologically active compound used to investi-
cal strategies have been proposed and evaluated to solve gate the transport across the BBB. Method reproducibility and
the BBB issue. Among them, the use of nanoparticles (NPs) long term stability were investigated.
has been regarded as having great potential for the delivery
of drugs into the CNS1. In particular, polysorbate-coated NPs In vivo biocompatibility studies were performed on chick
have shown the capability to deliver active molecules within embryos using the chorioallantoic membrane (CAM) assay.
the brain parenchyma, probably thanks to a preferential apo- Fertilized eggs, windowed at day 4 and treated at day 7, were
lipoprotein adsorption and successive BBB transcytosis2,3. monitored for 1, 2 and 5 days after treatment.

In the scaring scenario of a continuous growing incidence of RESULTS AND DISCUSSION


CNS pathologies (in Europe ~35% of all illnesses is represented Hot high pressure homogenization was chosen as produc- 25
by CNS diseases), a fast translation from the benchside to the tion methodology due to avoidance of toxic solvents, high
bedside is highly desirable. Unfortunately, most of the nano- reproducibility and easy scalability.
technologies developed in the last two decades to deliver
drugs within the brain suffer from serious drawbacks, such as Even though both surfactant concentration and number
low biocompatibility of the employed material, limited carrier of homogenization cycles were seen to affect colloid mean
stability and difficult, if not impossible, industrial scale-up1. diameter (MD) and polydispersity, expressed as Gaussian
distribution width (GDW), a preponderant effect of P80 con-
Therefore, the aim of this study is to develop a highly stable centration on nanoparticle dimensions was found. ANOVA
and biocompatible colloidal dispersion using an easily scal- validation showed a high adequacy (P<0.0001) and non-
able method. significant lack-of-fit for both models (MD and GDW), con-
firmed also by the values of the variance (1.76; 5.70) and the
MATERIALS AND METHODS correlation coefficients (r2 0.9622; 0.7773). Check-point anal-
Materials ysis confirmed the good agreement between predicted and
Cetyl palmitate (CP) (m.p. ~ 55°C) was a kind gift of Gattefos- experimental values. Seven homogenization cycles and 2%
sé (Milan, Italy); polysorbate 80 (HX)™ of injectable grade was of P80 were individuated as optimal fabrication conditions.
a gift of NOF corporation (Tokyo, Japan). All other materials
were of analytical grade and used as provided. NPs were prepared in replicate of four to assess method repro-
ducibility. The prepared formulations showed a mean diameter
Colloidal dispersion preparation of 185 ± 8 nm, a method yield of ∼92 % and a good physical
CP (5g) was heated above its melting point and added to a stability at 4°C for 18 months. The encapsulation of the model
hot aqueous solution of surfactant (Polysorbate 80, P80) un- drug, loperamide, and of the fluorescent die, nile red, did not in-
der mixing with a high-speed stirrer (Ultra Turrax T25, Jahnke fluence significantly particle size and distribution. Loperamide
and Kunkel, Germany) for 1 minute at 8000 rpm to form a pre- was entrapped with an encapsulation efficiency of about 70%,
emulsion. Homogenization was carried out by using an Emul- corresponding to 2.3% of drug content. TEM analysis evidenced
siFlex-C5 homogenizer (Avestin, Ottawa, Canada) at the same a similar morphology for blank and loaded NPs.
temperatures used to prepare the pre-emulsion and at a pres-
sure of 1500 Bar. Ten homogenization cycles were performed. CAM assay showed high biocompatibility. In fact, neither in-
The hot dispersion was immediately cooled in an ice bath. A flammatory reaction nor angiogenesis was stimulated by
statistical approach exploiting response surface methodolo-

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

NPs, 2% P80 solution, and the raw material at 1, 2 and 5 days af- REFERENCES
ter treatment. On the contrary, carragenin and sodium dodecyl 1. P. Blasi, S. Giovagnoli, A. Schoubben, M. Ricci, C. Rossi,
sulphate, used as positive controls, showed a high level of in- Solid lipid nanoparticles for targeted brain drug deliv-
flammation and angiogenesis around the application site. ery. Adv. Drug Deliver. Rev. 59 (2007) 454-477.
2. J. Kreuter, Application of nanoparticles for the delivery
CP NPs possessed the characteristics needed for brain drug of drugs to the brain. Int. Congr. Ser. 1277 (2005) 85-94.
delivery and, for this reason, their capability to deliver drugs 3. A. Zensi, D. Begley, C. Pontikis, C. Legros, L. Mihoreanu,
to the brain is under evaluation. S. Wagner, C. Büchel, H. von Briesen, J. Kreuter, Albu-
min nanoparticles targeted with ApoE enter the CNS by
transcytosis and are delivered to neurones. J. Control.
Release 137 (2009) 78-86.

26

3rd BBBB International Conference on Pharmaceutical Sciences


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O13
Novel Lab-on-chip devices towards nanomedicine study at single
cell level
Giorgia Pastorin1 and Vincent Lee2
1 Department of Pharmacy, National University of Singapore
2 Department of Electrical and Computer Engineering, National University of Singapore

ABSTRACT macrophages (of about 21 μm). The basic separation princi-


Single-cell detection is a crucial aspect in several branches ple, analyzed with manysimulation designs, makes use of the
of medicine and biology,allowing the study of immune sys- inherent characteristic size differences between thedifferent
tem function, disease diagnosis and natural cell death. With- cell populations, to provide a quick and simple separation
regards to biology, an in-depth evaluation of the activities method. Coupled withmicromechanical filters and microflu-
associated with a particular cell, interms of its molecular up- idics designs, some cell prototypes (e.g. cancer cells) have-
take or production of unique features at its surface. Similarly, demonstrated to be effectively segregated from the majority
in the caseof medicine, the clear understanding of cells’ be- of other cells without the need foradditional active control
haviour, on the basis of their exposure toparticular stimuli or external forces. It has been shown, through fluidic simula-
and conditions, represents a fundamental requirement in tion, that cellsof larger radius (our target, of about 30-40 μm)
the development ofbioactive agents aimed to target a spe- are readily exposed to the central flow and carriedaway from
cific cell subtype without unwanted side effects. the blood stream. A row of pillars with certain separation
gap (of about 8 μm) islocated further in the downstream and
Several approaches have been recently developed to iden- arranged either parallel or oblique to the flow. This allowsthe
tify a limited number of cellswith good accuracy, and they smaller cells type to be efficiently removed from the main
have being divided into two main classes, namely Poly- channels. Hence, only the cells ofinterest are obtained at the
meraseChain Reaction(PCR)-based methods and cytometric end of the main channel. If confirmed with real samples, this 27
procedures. Both of them show advantagesand drawbacks, resultcould overcome the drawbacks of current diagnostic
so that no ideal technique is available. PCR in general shows approaches, since it enables an efficientidentification and
more sensitivity,since it is usually based on the design of spe- isolation of a few abnormal cells circulating into the blood-
cific sequences for the gene(s) of interest, but on theother stream, thus providingan early prognosis of more problem-
hand it might fail in distinguishing between intact units and atic diseases.
genetic material shed from dyingcells. On the other hand,
flow cytometry represents a valuable technique, dealing So far, research studies aimed to detect a few cell subtypes
with theintroduction of cells into an artificial flow and a “real- have often shownremarkable limitations in terms of rapid-
time” laser counting each cell during itspassage through a ity of sample processing, entrapment yields andinformation
single-file flow. Unfortunately, some cell loss is inevitable, regarding the mechanisms involved in cellular response.
and cell entrapmentis not feasible with this technique. In Taking into account all theseinadequacies, we have elabo-
other words, the development of techniques able not only rated a promising device that not only is able to surmount
toidentify cells, but also to isolate them from an entire cell thecurrent problems, but it also paves the way for interest-
population in order to carry out theirmorphological and mo- ing advances in the field ofNanomedicine. In fact our fluidic
lecular characterization, is extremely lacking and defective. simulations, together with preliminary estimations, seem
tosupport ambitious goals: first, our platform provides tools
Therefore, in this project we present a novel cytometric sys- for rapid screening of cell populations inorder to identify a
tem consisting of an advanceddevice that is able to isolate a few cell subtypes at very low concentrations. The main ad-
few cells among a heterogeneous mixture, and to enhance vantage relies on theuse of a disposable chip that leads the
theentrapment yields, which still represent the limiting as- evaluation of several cells at the same time, whilesignificant-
pect of this methodology. To that purpose,we will combine a ly raise up the flow rate, such that micro-litre sample will be
micromechanical fluidic approach with a detection mecha- examined in a range of fewtens of minutes to one hour. This
nism within a uniquecell-based lab-on-chip (LOC) device, in will be achieved through a high-pressure flow into the LOC
order to provide a fast and effective tool able to entrapcells witha concomitant increase of the flow rate. Second, it in-
mainly on the basis of their dimensions. The realization of corporates advanced technologies to entrapthese cells and
the LOC device required an initialphase of platform design, allow further investigations “in real-time”. Results obtained
in which we chose samples of blood as it is made up of het- from additional studiesat individual cell level are expected to
erogeneouscells and platelets, with dimensions ranging revolutionize cell diagnostics in vitro. Finally, it offersunique
from a few micrometers (e.g. erythrocytes, of 6-8 μm)to huge possibilities to enhance week signals through a read-out sys-

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

tem. As concerns this lastaspect, integrated signal enhancer suitable DDS and their direct cellular target. Thepresent
will enable us to characterize individual cells simultaneous- project is aimed to develop a novel, improved platform for
lywhen treated with nanomedicines. This additional strategy CNTs-based drug delivery andfor real-time monitoring of
should enable us to overcome the lackof sensitivity usually responses deriving from different living cells. It is worth
attributed to conventional detection approaches. mentioningthat, although CNTs represent suitable DDS, the
platform provided by the LOC device showshigh versatility:
A further logical development of this project is represented after the identification of the cancer cell subtype, CNTs are
by the combination of our LOCwith a drug delivery system introduced in thedevice through a branched channel, hence
(DDS), in order to study the effects of the administration of it theoretically enables other systems (such asliposomes or
bioactiveagents at the single cell level. Amidst the myriad polymeric structures) to be investigated as well. Using this
of systems currently employed for drug delivery,carbon platform, the effects ofDDS and DDS-drug conjugates on
nanotubes (CNTs) represent intriguing devices, due to their individual or assembled living cells could be analyzed in-
ability to carry bioactivederivatives and, at the same time, depth.
molecules that can increase cell penetration. Previous in vit-
rostudies on uptake of functionalized CNTs by cell lines, while On the whole, this project offers a high degree of innova-
providing useful insights on thedynamics of penetration, yet tion, in which state-of-the-artnanotechnology materials,
gave limited information regarding the mechanism and the novel biosensing techniques and micromechanical systems
distinctphysiological effects of various types of single cells or basedLOC are to be developed in order to elaborate a novel
tissues to CNTs. Due to the multiple cell-tocell interactions, detecting system for important biomedicalapplications. Due
reactions of groups of cells often impair understanding the to the advantageous aspects presented in our platform, to-
intracellularmechanisms underlying the observed respons- gether with the urgentneed and the precious role of these
es. Therefore, the evaluation of single cellsexposed to CNTs devices, it is reasonably envisaged that the system willde-
would be extremely beneficial in disclosing the real process termine a remarkable impact and a significant commercial
of interactionbetween the delivery of bioactive agents from value in relatively short time.

28

3rd BBBB International Conference on Pharmaceutical Sciences


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O14
NANOBIOTECHNOLOGY and NANOTOXICITY
Filiz Hıncal
Hacettepe University Faculty of Pharmacy Deparment of Toxicology, Ankara, 06100, TURKEY

N
anotechnology is defined as the design and manipu- database have fostered the perception that nanoparticles are
lation of materials at the atomic and molecular scale. likely to be more toxic than fine-sized particulates, and sur-
Exciting applications of nanotechnology in diverse face properties and free radical generation resulting from the
medical fields have recently provided significant health ben- interactions of particles with cells may play important roles
efits through the introduction of many intelligent nanomate- in nanoparticle toxicity. Emphasizing that there exist at pres-
rials such as biomarkers, molecular diagnostics, nanodevices, ent little understanding of the basics of the interactions of
new drug discoveries and drug delivery systems. Primary nanoparticles with living cells, organs and organisms, there
goals of nanotechnology in drug delivery are more specific are strong arguments that even if a certain nanoparticle does
and efficacious drug targeting and delivery, while minimiz- not appear toxic by itself, its interaction with other common
ing side effects to provide greater safety and biocompatibil- components in human body may cause problems to cell func-
ity and leading to better patient compliance. However, there tions. It is certain that further research and developments and
is a concern that certain nanomaterials could have negative improved knowledge of physiological effects and kinetics of
impacts on human health or the environment as a result of nanoparticles in the body, their long term effects, and persis-
their new properties. The diversity of nanomaterial is vast, tence and fate in the environment are needed as the integral
and their physical, chemical and biological properties differ part of the technological development processes.
from the bulk material. Results from the limited toxicological
29

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

O15
Poly(anhydride) nanoparticles as adjuvants for oral vaccination
Juan M. Irache
Department of Pharmaceutics and Pharmaceutical Technology, University of Navarra, 31008-Pamplona, Spain

I
n the last years, nanoparticles have been proposed as ad- Systemic and mucosal immune responses, reported after ad-
juvants for mucosal vaccination. However, conventional ministration of a single dose, indicated that both OVA-M-NP
nanoparticles usually display a low capability to target in and OVA-F-NP elicited higher and balanced systemic specific
larger extent specific sites within the gut (i.e. Peyer’s patch- antibody responses [IgG1 (Th2-response) and IgG2a (Th1-
es). Therefore, the immune response elicited with these response)] compared to non-coated ones. In addition, oral
antigen carriers is usually not as high as necessary to offer immunization using OVA-M-NP or OVAF-NP was able to elicit
the adequate degree of protection to the host. In order to higher levels of intestinal secretory IgA compared to subcu-
render nanoparticles more efficient as adjuvants for vacci- taneous administration. All of these findings may be directly
nation, one possible strategy can be their association with related with the high capability and tropism of both flagel-
compounds or molecules involved in the colonization pro- lin- and mannosylated-nanoparticles to interact with the
cess of microorganisms. This biomimetic approach may be of Peyer’s patches and other intestinal regions rich in antigen
interest to put in place more effective and safer vaccines. presenting cells.

Thus poly(anhydride) nanoparticles (NP) were either coated In summary, oral immunization by biomimetic nanoparticles
with flagellin from Salmonella enteritidis (F) or mannosamine demonstrated strong long lasting systemic and mucosal im-
(M). The mucosal affinity of the resulting nanoparticles (F-NP mune responses than the respective non-conjugated vec-
30 and M-NP) was investigated by bioadhesion and fluores- tors.
cence microscopy studies. The immunoadjuvant proper-
ties when administered by the oral route were evaluated in
Balb/C mice using ovalbumin (OVA) as an antigen model.

3rd BBBB International Conference on Pharmaceutical Sciences


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O16
Different medicinal forms of bacteriophage preparations for
treatment of infectious diseases
Dr. Mzia Kutateladze
G. Eliava Institute of Bacteriophages, Microbiology and Virology, Tbilisi, Georgia

D
uring several years, phage therapy was included as a G. Eliava Institute of Bacteriophages, Microbiology and Vi-
powerful tool against infectious diseases of bacterial rology, Tbilisi, Georgia is the most famous institutions in the
etiology. Unfortunately, phage treatment was forgot- world focused on bacteriophage study, particularly isola-
ten in the Western World shortly after antibiotics became tion and selection of phage components, active to various
widely available. Although, nowadays rapid increase of multi- pathogenic and conditionally pathogenic bacterial strains.
drag resistant bacterial strains develop renewed interest to- The preparations developed in the Eliava Institute have been
wards phage therapy. studied through extensive preclinical and clinical trials. How-
ever, little of this information has ever been published and
Phage therapy remained as a standard part of healthcare even when details are available, the trial reports do not meet
system in Eastern Europe and USSR. Phage preparations internationally approved regulations and standards.
were used for therapy, prophylaxis and diagnostic of various
bacterial infections. Such preparations have been success- Depending of the source of infections to be cured, the phage
fully used against intestinal problems (dysentery, diarrhea, preparations have been produced at the Eliava Institute in
typhoid) and purulent-septic infections (wounds, infectious different forms: liquid (ampoules, vials), ointments, sup-
complications of burns, inflammation of organs, etc). positories, tablets, preparation for intravenous application.
The method for construction of phage-based product –
Bacteriophages have number of advantages in comparison phagebioderm has been developed at the Institute together 31
to antibiotics; they are ecologically safe - harmless to human, with the scientists from the Tbilisi Technical University. This
plant and animal; they are very specific and do not affect preparation includes original biodegradable polymer that
normal microflora; phage preparations are readily available is impregnated with phages against several pathogens. The
and easy to apply. There is no resistance appears to multi- original technology for preparing bioactive composite act-
component phage preparation. Today, the uncontrolled use ing in bacteriophages’ sustained/controlled release fashion
of antibiotics (among other factors) has limited the efficacy is developed at the Institute. The preparation is successfully
of their treatment and has contributed to the wide-spread used in infected wounds, burns, tropical ulcers etc.
evolution of multi-drug-resistant bacterial pathogens. Phage
therapy as an alternative approach for treatment of infec-
tions has become an evident and promising remedy.

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

O17
Utilization of Bacteriophages and lytic enzymes in Treatment and
Prevention of Staphylococcus aureus MRSA and other Infections
David Trudil1, Michał Bartoszcze2, Romuald Gryko2, Mzia Kutateladze3.
1Battelle/NHDe, 12616 Mt. Laurel Ct, Reisterstown, MD 21136 USA davidt@nhdetect.com
2Biological Threats Identification and Countermeasure Center of MIHIE, Pulawy, Poland
3G. Eliava Institute of Bacteriophages, Tbilisi, Georgia

B
acteriophages (phage), one of the most common or- to MRSA (Methicillin-resistant Staphylococcus aureus),
ganisms on earth, are viruses which infect bacteria. VRE (Vancomycin-resistant enterococcus), multi-resistant
The discovery of bacteriophages in the early 1900s had strains of Pseudomonas aeruginosa, or Escherichia coli which
primarily pointed to the possibilities of using them as natural produce lactamase of extended substrate spectrum (ESBL).
antibacterial agents; and led to numerous attempts to use These harbour in purulent infections (wounds, dermatologi-
them to treat bacterial infections in humans and animals. cal complications) as well as respiratory system and urinary
The discovery and subsequent practical application of anti- tract infections.
biotics (1940) halted both research work and clinical studies
in this area (particularly in the West) for many years; and it The G. Eliava Institute of Bacteriophages, Microbiology and
was only the growing number of bacterial strains resistant Virology in Tbilisi has developed a phage cocktail which has
to antibiotics that brought phage therapy into the limelight been proven to be effective against MRSA. The results of a
again. These studies concerning the possibilities offered by limited, one person challenge study of a chronically ill, MRSA
phage therapy, however, were centered mainly in the former infected patient treated with the Eliava phage cocktail will
Soviet Union and Poland. be provided. Additionally, the status of phage therapies, as
well as phage derived treatments and preventatives will also
Recent efforts focused on phages lytically active to a number be discussed.
32 of pathogens, including antibiotic-resistant strains, are gen-
erating the most interest. Worth noting is the lytic activity

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

O18
Bacteriophages as vaccine delivery vehicles
Jason Clark and John March
BigDNA Ltd., Wallace Building, Roslin Biocentre, Midlothian, EH25 9PP,UK.

B
acteriophages (or phages) are viruses which infect bac- We are currently designing an optimised lambda vector in
teria. Most phages are relatively simple and a number which the genetic material will be customised and produced
of systems are available where the genetic material or entirely synthetically. It will include several safety features
protein coat can be easily modified. This versatility makes and optimisations to improve yield. We are also developing
them ideal tools for nanotechnology and for biotechnology the means to grow these phages in fermentors at high yield
in general. In recent years phages have been used as alter- and the procedures to purify them to a level suitable for in-
natives to antibiotics, as biosensors, for targeting specific cell jection into humans.
types in anti-cancer therapies, as molecular scaffolds in bat-
tery production or as drug or vaccine delivery vehicles1,2. In May 2007, BigDNA ltd. was set up to commercialise the
phage vaccine technology. It has several patents including
One of the most well characterised phages is phage lambda, those for genetic and chimeric phage vaccines. We are cur-
which has a 50 nm head, is 200nm long and has a 50 kilobase rently planning and performing pre-clinical trials and aim to
genome (figure 1). We have used bacteriophage lambda as a start phase I clinical trials in 2011.
DNA vaccine delivery vehicle. In standard DNA vaccination,
genetic material containing a vaccine gene in a form that will
be expressed in mammalian cells is used as an immunogen.
Host cells near the site of injection take up the vaccine DNA, 33
the vaccine protein is expressed and an immune response is
raised. In bacteriophage vaccination, a DNA vaccine cassette
is cloned into the genome of the phage and whole phage
particles are used to inoculate the host. To the immune sys-
tem, phages structurally resemble a pathogenic virus and
they are rapidly cleared by the host’s immune system to
sites of antigen presentation (i.e. where the immune system Figure 1: Schematic representation of a bacteriophage lamb-
works most efficiently), where the vaccine component is ex-
da particle.
pressed. In effect, the host’s immune cells are turned into
vaccine producing factories. We have tested several vac-
cines in a number of animal species (mice3, rabbits4, sheep5
and fish). Example results using a vaccine against Hepatitis B
are shown in figure 2.

Phage vaccines have several advantages over traditional vac-


cines. They are relatively cheap to produce and purify, being
produced on E. coli in simple culture media. It is also therefore
very rapid to produce large quantities of vaccine. The phage
coat protects the DNA making the vaccines very stable, so
they are easy to store and transport6. No antibiotic resistance
is required on the vector and large inserts (up to 20kb) can be
used, so in theory multiple vaccines can be delivered in the
same vector. The versatility of the phage technology confers
a great deal of flexibility onto the platform. For example, it is Figure 2: Comparison of antibody responses in rabbits (5 per
possible to modify the phage coat to display fusion proteins, group) given a commercially available protein vaccine (A), a
so that the phages themselves can be targeted to specific cell bacteriophage vaccine based on the same antigen (B) and a
types. Alternatively a vaccine protein can be displayed on the control phage (C). Vaccinations were at weeks 0, 4 and 8. Re-
phage surface, so that protein and DNA vaccines are delivered sponses were measured by ELISA against the recombinant
on the same construct (a chimeric vaccine - figure 3). protein version of the antigen

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

2. Clark JR and March JB. (2006). Bacteriophages and Bio-


technology: Vaccines, Gene therapy and Antibacterials.
Trends Biotechnol. 24, 212-218.
3. March, J.B. et al. (2004) Genetic immunisation against
hepatitis B using whole bacteriophage lambda particles.
Vaccine 22, 1666-1671.
4. Clark, J.R. and March, J.B. (2004) Bacteriophage-medi-
ated nucleic acid immunisation. FEMS Immunol. Med.
Figure 3: Modifying bacteriophage lambda to display vac- Microbiol. 40, 21-26.
cine proteins, contain DNA vaccines or a combination of 5. Rocchi RM, Jepson CD, Clark JR, March JB, and McKeever
both. The whole phage particle is used as the vaccine. DJ. (2003). Efferent lymphatic cannulation for the evalu-
ation of immunogenicity of DNA vaccines. Immunology
REFERENCES 110 (Supplement 1), 121.
1. Clark JR, March JB. (2004). Bacterial viruses as human 6. Jepson, C.D. and March, J.B. (2004). Bacteriophage lamb-
vaccines? Expert Review of Vaccines 3, 463-474. da is a highly stable DNA vaccine delivery vehicle. Vac-
cine 22, 2413-2419.

34

3rd BBBB International Conference on Pharmaceutical Sciences


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O19
PHAGE-TARGETED MEDICATIONS
Valery A. Petrenko
Department of Pathobiology, Auburn University, AL 36849, U.S.A.,
Professor, office:334-844-2897; fax: 334-844-2652;

T
he more than 20-year evolution of phage display has tides fused to all copies of the major coat protein pVIII can be
dramatically affected the potential of this technique converted easily into gene-encapsulating particles or drug-
amid other bioengineering methods. The marriage of loaded vesicles that acquire the ability to recognize the same
combinatorial chemistry and biological selection has receptors, cells, tissues and organs that have been used for
been very powerful changing the methodology of biochemi- selection of the precisely targeted phage. The merge of
cal research by allowing selection simultaneously among bil- phage display technology with nanotechnology during the
lions of genetic species in one test tube. Genetically driven last several years is very promising and has already shown its
phage biotechnology has allowed development of libraries vitality and productivity contributing vigorously to different
of diverse nanostructures expressed on the phage surface areas of medicine and technology, such as medical diagnos-
providing a rich resource of diagnostic, detection and phar- tics and monitoring, molecular imaging, targeted drug and
maceutical probes. Phage engineering, which is based on gene delivery, vaccine development, as well as bone and tis-
natural mechanisms of selection, amplification and self-as- sue repair. This presentation focuses on the progress made
sembly, allows directed fabrication of bioselective materials in the development of these new materials and discusses
with possible applications in gene/drug-delivery, biosensors, the prospects of using phage as a bioselectable molecular
biosorbents, and other areas of medicine and technology. In recognition interface in medical and technical devices based
particular, landscape phage expressing tumor-specific pep- on the experience of the author in this area. 35

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

O20
Novel glutathion analogues and their effects in biological systems
Ursel Soomets
Department of Biochemistry, Faculty of Medicine, University of Tartu, The Centre of Excellence of Translational
Medicine, Ravila Str. 19, 50411 Tartu, Estonia

G
lutathione (GSH, γ-Glu-Cys-Gly) is the major intracel- tially pro-oxidative NAD(P)H oxidase were investigated. UPF
lular low molecular weight antioxidant. Its intracellu- peptides inhibited GPx activity only at high concentrations
lar concentration is up to 10 mM, whereas extracel- and this effect was more expressed for α-glutamyl moiety
lular concentrations are relatively low (5-50 µmol/l). Most of containing UPF17; did not affect the activity of GR but inhib-
the cellular reduced glutathione (GSH; 85-90%) is located in ited the human neutrophil NAD(P)H oxidase.
cytosol where it serves as non-enzymatic water-soluble an-
tioxidant. Additionally, GSH carries several other functions in Psoriasis is a chronic inflammatory T-cell mediated auto-
the human body concerning the metabolism of xenobiotics immune disease. Many different factors such as genetic
and eicosanoids, cellular signalling and thiol disulphide ex- features, immunological disorders, biochemical changes,
change reaction. High grade oxidative stress and decrease of environmental effects, trauma and stress contribute to the
GSH level is related to the development of several pathologi- pathogenesis of psoriasis. Recent studies have suggested
cal states including cardiovascular diseases, neurodegenera- that increased ROS production and deficient function of an-
tive diseases, cancer, HIV, inflammation, psoriasis, ischemia/ tioxidant systems activities play a role in the pathogenesis of
reperfusion injury, chronic obstructive pulmonary disease psoriasis. We found that the expression levels of GSH-linked
(COPD) etc. Compensatory administration of GSH is not ef- enzymes in the peripheral blood mononuclear cells (PBMC)
fective because of its rapid degradation, while more stable of psoriasis patients were lower compared to healthy sub-
36 and nontoxic glutathione analogues could be an effective jects. Statistically significant differences in “glutathione sys-
alternative. tem” enzymes expressions were established between the
study groups. Influence of antioxidant peptides (UPF1 and
The aim of our study was to create more stable and effective UPF17) to the expression of enzymes involved in glutathione
antioxidants than GSH, investigate their structure-activity re- metabolism was examined in the PBMCs. UPF peptides had
lationships and describe their biological activity in psoriasis different efficacy on control and patient cells, but in general,
and COPD patient blood cells. they were more effective in patient samples and normalized
the expression of studied enzymes to the same level as con-
By adding a fourth amino acid to the GSH molecule, we de- trols.
signed and synthesized library of tetrapeptidic glutathione Chronic obstructive pulmonary disease (COPD) is an increas-
analogues called UPF peptides. We showed that UPF pep- ing global health problem that is characterized by a specific
tides were far more active hydroxyl radical scavengers than pattern of chronic inflammation in small airways and lung pa-
glutathione (EC50 of GSH: 1231.0 mM; EC50 of UPF peptides: renchyma. Cigarette smoking is the major etiological factor
from 0.03 to 35 μM. GSH had slightly lower EC50 of DPPH radi- in this condition. COPD kills one person in every 10 seconds
cal scavenging than UPF peptides but comparing antiradi- worldwide. The effects of UPF analogues (UPF1 and UPF17)
cal efficiencies (reflects the speed of DPPH scavenging), UPF on the antioxidative and proinflammatory enzymes expres-
peptides exceeded GSH by antioxidative potential. In both sion levels in the blood mononuclear cells were studied. We
scavenging reactions the most effective structural modifica- found that these glutathione analogues affect expressions
tion was the substitution of the γ-Glu with the α-Glu moiety of studied enzymes by unknown mechanism.
in GSH/UPF backbone. UPF peptides did not affect the viabil-
ity and membrane integrity of human erythroleucemia K562 Our study confirmed that UPF peptides are promising leads
cells. The influences of selected UPF peptides (UPF1, UPF6, for design of powerful non-peptidic antioxidants that can be
UPF17, UPF19) on the activities of antioxidant enzymes (glu- used in treatment of different diseases involving oxidative
tathione peroxidase (GPx), -reductase (GR) etc.) and poten- stress.

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

O21
THE ROLE OF ABC MULTIDRUG TRANSPORTERS IN NORMAL
AND TUMOR STEM CELLS
András Váradi1, Gergely Szakács1, László Homolya2, Csilla Özvegy-Laczka2, and Balázs Sarkadi2
Institute of Enzymology, Hungarian Academy of Sciences, H-1113 Budapest, Hungary,
1

2
Membrane Research Group of the Hungarian Academy of Sciences, H-1113 Budapest, Hungary,

F
orty-eight genes coding ABC-transporters have been tion can be used during the process of drug discovery and
annotated in the human genome. Most of these pro- development. We provide a description of the relevant hu-
teins facilitate outward transport of small molecules man ABC drug transporters, and review the models and as-
from the cytosol, thus functioning as export pumps, while say systems that can be applied for the analysis of their ex-
some of them act as ion channels or ATP/ADP-sensors/recep- pected drug interactions. The use of the in vitro, in vivo, in
tors regulating ion channels. silico models, their combination, and the emerging clinical
information are evaluated with respect to their potential
The human ABC drug transporters play an important role in application in early drug screening. We also discuss the de-
cancer drug resistance, protection against xenobiotics, and tails of the chemoimmunity network hypothesis explaining
in general in the passage of drugs through cellular and tis- how a very few ABC transporters can be involved in a very
sue barriers. This review explores how human ATP-Binding wide substrate recognition thus providing a defense system
Cassette (ABC) transporters modulate the pharmacological against toxic xenobiotic and metabolites.
effects of various drugs, and how this predictable modula-

37

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

O22
Characterization of novel polimeric materıals from the point of
theır physical ageıng
Romána Zelkó and Virág Szente
University Pharmacy Department of Pharmacy Administration Semmelsweis UniversirtH-1092 Budapest, 7-9 Högyes
E. Street, Hungary

D
ifferent polymers are widely used in pharmaceutical cal ageing can be initiated by several factors (e.g. humidity)
technology for a diversity of purposes. They can be ap- and is manifested in macrostructural (enthalpy and volume)
plied as fillers, binders, matrix-forming and film-form- and microstructural (free volume distribution) alterations.
ing excipients in solid dosage forms, and most of the gelling These changes also occur during the processing and storage
agents of semisolid preparations and solutions are polymers, of pharmaceutical dosage forms containing such polymers,
as well. Furthermore, a wide variety of packaging materials is thus influencing their stability and bioavailability. Tracking
based on macromolecules. Although the chemical structure the ageing process of polymeric excipients is possible using
of the above mentioned polymers is rather heterogeneous, an array of techniques. Macrostructural changes can be fol-
their physical state is usually similar, as most of them are lowed by dilatometry and calorimetry, while microstructural
amorphous or partly amorphous. A well-known property of alterations can be monitored by means of positron annihila-
such materials is that they undergo physical ageing, which is tion lifetime spectroscopy, electron spin resonance spectros-
accompanied by volume and enthalpy relaxation and thus copy, fluorescence spectroscopy, small angle X-ray scattering
might result in severe structural changes in the polymer. The and scanning electron microscopy. Such investigations are of
enhanced molecular mobility, caused by the plasticization high importance because of the consequences considering
effect of absorbed water, has been proposed to be the ma- the stability of the excipients themselves and dosage forms
jor underlying factor in chemical and physical instability of prepared using these materials.
38 amorphous pharmaceutical materials. This so called physi-

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

O23
New analytical technologies and materials for determination of
enantiomeric purity of chiral drugs
Bezhan Chankvetadze
Department of Chemistry, School of Exact and Natural Sciences and Molecular Recognition and Separation Science
Laboratory, Tbilisi State University, Chavchavadze Ave 1, 0179 Tbilisi, Georgia

C
hirality represents increasingly important issue in drug higher. In addition, the less strict requirement to the value
development and use. The world-wide market for chi- of enantioselectivity allows to employ effectively a large
ral fine chemicals reached US dollar 6.63 billion in 2000 number of chiral selectors with relatively low chiral recogni-
and is expected to grow at 13.2% annually1. The drug indus- tion ability. In chromatography it is possible to obtain both
try is considered to be the engine that is driving this strong enantiomers in the optically pure from, even when the chiral
growth, accounting for 81.2% of the total, or US dollar 5.38 selector has an optical purity less than 100 %. Moreover, the
billion worth. These numbers become even more impressive absence of irreversible chemical reactions or phase transi-
when transferred to drug formulations. Thus, the world-wide tion during direct enantioseparations further favours the
market for dosage forms of single-enantiomer drugs was US application of this technique for analytical and preparative
dollar 123 billion in 2000, up 7.2% from US dollar 115 billion scale enantioseparations.
in 19991. The major current trend in enantioseparations is to
leave the classical column size and separation scale in two Recent developments in chiral chromatography (more uni-
opposite directions: On the one hand chromatography is versal, easily available, stable, tailor-designed chiral station-
becoming a valuable tool for obtaining of enantiomerically ary phasess) and technology (recycling, displacement and
pure drugs on preparative and production scale. On the oth- especially, simulated moving bed (SMB)) makes chromatog-
er hand, the techniques for analytical-scale enantiosepara- raphy a valuable alternative to the classical techniques for
tions are continuously miniaturized and microtechnologies, the analysis and preparation of pure enantiomers. 39
such as capillary liquid chromatography (CLC), capillary elec-
trophoresis (CE) and capillary electrochromatography (CEC) At present, high performance liquid chromatography (HPLC)
are gaining importance in this field. In addition, lab-on-chip with common size columns (250x4.6 mm) still dominates
technologies are establishing very rapidly. This presentation chromatographic enantiomeric analysis in industrial labo-
summarizes new developments for preparation of effective ratories. However, microanalytical techniques such as CLC,
chiral stationary phases for liquid phase enantioseparations CE and CEC are progressing very rapidly. It must be noted
as well as the innovations from the viewpoint of applied sep- that the acceptance of aforementioned microanalytical tech-
aration technologies. niques is not always the same by the conservative communi-
ty of separation scientists. Thus, despite the early remark by
Enantiomeric impurities, as well as other impurities in active Jorgenson that “The high theoretical plate counts make CE
pharmaceutical ingredients and drug formulations shall be particularly amenable to separating chiral molecules” [2] this
determined at the level below 0.1%. This applies very strict technique could not be established as the major technique
requirements to the analytical technology used for determi- for enantiomeric purity determination of chiral pharmaceu-
nation of enantiomeric purity of chioral drugs.Together with ticals. CLC relies certainly on the same separation principle as
separation techniques some other instrumental techniques HPLC but offers the advantages of miniaturisation compared
such as polarimetry and nuclear magnetic resonance (NMR) to the latter one. In particular, CLC requires less amount of
spectroscopy can be used for determination of enantiomeric the packing material, mobile phases and samples. It is cost-
purity of chiral drugs. However, chromatographic separa- effective and environmentally friendly technique and offers
tion techniques offer important conceptual advantages also the advantage of higher sensitivity when coupled with
compared to the “one step” processes such as polarimetry mass spectrometer.
and NMR spectroscopy. In chromatography as in a “multi-
step” separation method the overall resolution of the solute CE offers higher peak efficiency compared to HPLC. It is clear
originates from a large number of stereoselective “one-step” that even only at the expense of higher peak efficiency CE
adsorption-desorption cycles. The cumulative nature of the may allow to observe enantioseparations for certain chiral
chromatographic separation is the reason that a free energy analyte-selector pairs where the separation power of HPLC
difference in interactions of enantiomers as small as 0.025 kJ is insufficient for achieving this goal. In addition, chiral CE
mol-1 may in principle be sufficient for baseline chromato- offers almost unlimited possibility from the viewpoint of
graphic enantioseparations, whereas the free energy dif- adjustment of separation factor and the criticism “The effi-
ference required in “one-step” techniques is several orders ciency of CZE is high but, the flexibility of chromatography

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

in adjusting separation factor is lost” 3 does not apply at all to perfussive flow through the pores of the particles. As recent
chiral CE. Together with aforementioned conceptual advan- comparative studies on CLC and CEC indicated, the high
tages CE offers some favorable technical characteristics for plate numbers of CEC may allow observing the enantiosepa-
achieving high separation selectivity. Thus, chiral stationary rations in this technique under the conditions in which CLC
phases (CSPs) in HPLC contain commonly limited and pre- hardly provides any indication of chiral recognition5.
defined amounts of a chiral selector, whereas the concentra-
tion of a chiral selector is easily variable and just limited by In this presentation newly developed particulate and mono-
the solubility (for charged selectors also with Joule heating) lithic chiral stationary phases will be described for liquid-
of a chiral selector in a CE buffer. In addition, the combination phase enantioseparations 6-8.
of two or more (chiral) selectors is technically much easier
and not associated with instrumental difficulties in CE com- Thus, in the coming years separation science may turn from
pared to column-coupling in HPLC. Again, two columns are the workhorse HPLC with standard size chromatographic
coupled with given amounts of the chiral selectors in HPLC columns to microanalytical techniques (CLC, CE, CEC) for
whereas the ratio of chiral selectors in a combination can be enantiomeric purity determination of chiral drugs.
easily optimized in CE. Thus, chiral CE offers really enormous
flexibility from the viewpoint of the adjustment of the sepa- REFERENCES:
ration selectivity. This in combination with the inherently 1. S.C. Stinson, Chem. Eng. News, 2001, 79 (20), 45.
high separation efficiency makes chiral CE a very powerful 2. A. M. Rouhi, Chem. Eng. News, 2003, 81 (18) 56.
technique for enantioseparations4. 3. G. Guiochon, Int. Lab., 1999, January, 13C.
4. B. Chankvetadze, Capillary Electrophoresis in Chiral Anal-
Another electromigration technique, in particular CEC most ysis, 1997, Wiley&Sons, Chichester, UK.
likely does not offer a different separation mechanism for 5. B. Chankvetadze, J.Sep. Sci., 2001, 24, 691.
enantiomers compared to HPLC but higher peak efficiency. 6. S. Fanali, G. D’Orazio, K. Lomsadze, B. Chankvetadze, J.
Basically there are three different effects which may be har- Chromatogr. B, 2008, 875, 296-303.
nessed in order to obtain higher peak efficiency in CEC com- 7. K. Lomsadze, A. B. Martinez-Giron, M. Castro-Puyana, L.
pared to CLC: a) The plug-like flow profile of the electrically- Chankvetadze, A. L. Crego, A. Salgado, M. L. Marina, B.
driven flow; b) the potential of the application of smaller Chankvetadze, Electrophoresis, 2009, 30, 2803-2811.
particle size materials and longer separation beds and c) the 8. B. Chankvetadze, Electrophoresis, 2009, 30, S211-S221.

40

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

o024
Microchips in drug analysis
Risto Kostiainen
Division of Pharmaceutical Chemistry, P.O. Box 56, FI-00014 University of Helsinki, Finland

M
iniaturization of analytical instruments utilizing mi- Miniaturization of atmospheric pressure ionization tech-
cro-fabrication technology has been one of the hot- niques has gained rapidly enhanced interest in chip-based
test research topics in analytical chemistry over the analysis. Electrospray ionization (ESI) is currently the method
past decade. Driving force to this is an increasing demand of choice to connect a microchip with mass spectrometry
for low-cost instruments capable of rapidly analyzing very (MS). The flow rates used with microfluidic devices (nl-µl/
small amounts of samples with a high level of automation. min scale) are ideal for optimal sensitivity in ESI-MS. Differ-
A concept termed both “Miniaturized total analysis systems ent materials, such as silicon, glass, polymers have been used
(µ-TAS)” and “Lab-on-a-chip” aims to develop integrated mi- in fabrication of microchips. Recently SU-8 polymer has been
cro-analytical systems to perform complete analysis cycles shown to be highly suitable material for microfluidic separa-
(e.g. sample pre-treatment, chemical reactions, analytical tions and electrospray ionization.
separation, detection and data handling steps) on a single
micro-device. Even though ESI is an excellent method for polar and ionic
compounds, its sensitivity for neutral and non-polar com-
In the most micro-fluidic applications so far, on-chip detec- pounds may be poor. Atmospheric pressure chemical ioniza-
tion has relied on optical detection, and for sensitivity rea- tion (APCI) and especially atmospheric pressure photoion-
sons, fluorescence (FL) detection has been the most com- ization (APPI) offer alternative ionization techniques that is
monly utilized. Among the detection techniques alternative capable to ionize with high efficiency non-polar compounds. 41
to optical detection, mass spectrometry (MS) has gained rap- Recently we presented microchip APCI and APPI, which allow
idly enhanced interest in chip-based analysis, and during the flow rates down to 50 nl/min making it directly compatible
last few years great amount of reports have been published with microseparation systems. The chips provide excellent
in the field. At present, the main focus is in integrating ion- sensitivity, robust analysis, good reproducibility and cost ef-
ization methods to micro separation systems with MS. ficient manufacturing. The feasibility of the APCI and APPI
microchips in coupling of micro liquid chromatography (LC),
gas chromatography and microchip LC to mass spectrom-
etry is presented.

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

O25
Sensitive chiral capillary electrophoresis:
Application in bioanalysis
Éva Szökő, Tamás Tábi
Department of Pharmacodynamics, Semmelweis University, Budapest, Hungary

C
hirality has a great impact on the pharmacological methods. However, when simultaneous enantioseparation
properties of drug enantiomers, thus studying the of several sample components is required, even in case of
stereochemistry of the pharmacodynamic and phar- the suitable enantioselectivity, achieving the needed chemi-
macokinetic properties of a drug candidate in early phase cal (achiral) selectivity can be problematic. Separation pH,
of development has gained importance. Capillary electromi- applied electric field, capillary length and temperature are
gration methods are especially suitable for the simultaneous the main parameters to be optimized. Besides these, there
enantiomer separation of several compounds with similar are several other ways for influencing chemical selectivity
structures, such those formed during biotransformation pro- mainly through the improvement of separation efficiency
cesses. In contrast to chiral HPLC, capillary electrophoresis and/or widening the separation time window. We have used
(CE) methods have high efficiency and chemical selectivity buffer additives, such as organic modifier and chiral poly-
needed for the resolution of plethora of analytes in a sam- mer, or dual cyclodextrin system to improve achiral selectiv-
ple. The low reagent and sample requirement, as well as the ity when simultaneous enantiomeric separation of the chiral
quick and flexible method development provide a rapid and drug deprenyl, and several of its metabolites were to be per-
inexpensive way of analyzing enantiomers. These properties formed in metabolism studies.
are especial advantages, when screening is to be performed
or e.g. in in vitro metabolism studies. Drug quality control Major drawback of using CE techniques in bioanalysis is the
42 (chiral impurity testing), pharmacodynamic and pharma- poor concentration detection sensitivity by UV due to the
cokinetic studies, or forensic toxicological analysis are other small sample volume and short light path. The complex ma-
major fields of application of enantioselective CE. trix of biological samples further impairs separation efficien-
cy and the detection limits. However, during sample clean-
In capillary electrophoresis, the chiral selector is dissolved in up the composition of the sample matrix can be modified to
the background electrolyte and acts as a pseudo-stationary allow on-capillary sample stacking, and remove disturbing
phase that can interact with one of the enantiomers prefer- sample components. On-capillary sample stacking can be
ably. The enantioselectivity derives from the different sta- reached by decreasing the conductivity of the sample ma-
bility of diastereomer complexes the enantiomers form with trix, allowing higher sample injection volume without the
the chiral selector. This way, in chiral CE, the separation of the loss of separation efficiency. Stacking provides about ten
enantiomers is based on their distinct distribution between times increase in detection sensitivity. Liquid-liquid or solid
the separation buffer and the pseudo-stationary phase (chro- phase extractions are common sample clean-up methods
matographic principle), as well as on the different mobility for biological specimens. Their carefully designed use can
of the enantiomers under the separation conditions (electro- provide a sample matrix allowing the on-capillary sample-
migration principle). The combination of the two separation stacking and quantification limits in the submicromolar con-
measures provides the high peak capacity of the chiral CE centration range.

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

O26
QSAR IN PREDICTION OF TARGET AND ANTITARGET DRUG PROPERTIES
Milena Jadrijević - Mladar Takač
Department of Medicinal Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb/Croatia

INTRODUCTION 2007. The Lipinski’s rule-of-five analysis and parameters


Drug discovery has entered a new century with a wealth (miLogP, MW, TPSA, n atoms, nON, nOH NH, nrotab, volume)
of sophisticated technologies and information generation as well as druglikeness scores were computed using Mo-
platforms that theoretically will allow the more rapid devel- linspiration molecular processing engine, 2007.10. The
opment of medicines with improved selectivity and safety ‘n toxophoric score’ for each compound was calculated on
profiles. The most used, the quantitative structure activity the basis of presence one or more main toxophoric groups
relationship (QSAR) provides a great deal of information re- in molecule. All analyses were performed and data were
garding the nature of investigated NCEs and so far many ap- graphed using OriginPro 7.5 software (Origin Laboratories,
proaches were developed. In an extension of our earlier stud- Northampton, MA).
ies in attempting the role of the use of topological indices
(TIs) for QSAR studies in lead compound search among 1,4- (I) RESULTS AND DISCUSSION
and 3,1-benzoxazinones (II) ((n=62) we have undertaken the
present investigation in that we have used Wiener (W) and QSAR study
molecular connectivity (χ1, χMOD, CID) indices, molecular In present QSAR study W, χ1, χMOD and CID were correlated
descriptors (MDs), the lipophilicity parameter, log P (AlogP with AlogP, Sv and MICs (x10-3 moldm-3) for Staphilococcus
and miLogP) and van der Waals volume (Sv) in correlations aureus ATCCC 56511, Klebsiella pneumoniae ATCC 10031
with antimicrobial activity (MICx10-3moldm-3) obtained in and Candida monosa. The 2D correlations with similar sig- 43
tests with various microorganisms, including bacteria and nificancy between TIs (W, χ1, χMOD) and Sv versus MICx10-
fungi. The Lipinski’s rule-of-five study was also undertaken 3moldm-3 against S. aureus, were obtained and they are as
with the aim to explore, the potential oral bioavailability follows: i. MIC x 10-3 (mol dm-3) = 4.6115 – 0.00125 W (n =
and druglikeness properties of these compounds, in order 20, r = - 0.8627); ii. MIC x 10-3 (mol dm-3) = 7.35675 – 0.39864
to lead identification with the lowest potential toxophoric χ1 (n = 20, r = - 0.8952); iii. MIC x 10-3 (mol dm-3) = 7.35268
features. For this purpose the ‘n toxophoric scores’ were cal- – 0.06072 χMOD (n = 20, r = - 0.88495); and iv. MIC x 10-3
culated and used in correlations with data computed using (mol dm-3) = 6.87874 – 0.16685 Sv (n = 20, r = - 0.86517). The
Molinspiration software for druglikeness (GPCR ligand, ion lipophilicity parameter, AlogP were used in 2D and 3D cor-
channel modulator, kinase inhibitor, nuclear receptor li- relations. The 3D multiparameter correlations offered an ad-
gand), lipophylicity parameter, miLogP and molecular polar ditional insight in relationships among TIs, lipophilicity and
surface area (TPSA). MIC. QSAR results revealed that with an increasing of W, χ1
R1
R O R1
and AlogP, the MIC is decreasing for this set of compounds.
R
O The 2D correlations with the same TIs (W, χ1, χMOD and CID)
N O N O and Sv versus MIC against K. pneumoniae and C. monosa,
CH2N R2R3 CH2 NR2R3 showed comparatively less colinearities between investigat-
I II
F
ed parameters.
F
F
F F
Cl O
Cl
O
F Lipinski’s rule-of-five analysis
Lipinski’s rule-of-five predict poor absorption or permeation
NH O NH O
of orally administered compound if it meets the following
Efavirenz 1,4- isomer of efavirenz criteria: molecular mass greater than 500 Da, high lipophi-
licity (expressed as cLogP greater than 5), more than 5 hy-
Computed data for efavirenz and its 1,4- isomer were also drogen bond donors and more than 10 hydrogen bond ac-
included in this study. ceptors. Lipinski’s rule-of-five analysis was applied on a set
(n = 64) of I and II derivatives, including efavirenz and its 1,4-
MATERIAL AND METHODS isomer. Eight compounds out of 64 did not meet Lipinski’s
rule-of-five requirements (18, 19, 23, 27, 28, 30, 35 and 56),
All compounds I and II were previously synthesized and bio- mostly because of TPSA and number of H-bond acceptors in
logically tested. The TIs (W, χ1, χMOD and CID) and MDs (Sv, molecule.
Mv and AlogP) were computed using Dragon for Windows,

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

In addition to druglikeness data estimated using various mo- CONCLUSION


lecular properties, the ‘n toxophoric score’ were calculated on The results of QSAR study could be used in predicting more
the basis of presence one or more main toxophoric groups in active analogues of this series of compounds. The Lipinski’s
molecule. In correlations calculated n toxophoric scores with rules-of-five analysis revealed valuable information relevant
estimated druglikeness scores, the miLogP and TPSA, the 6 to potential drug candidate properties. The ‘n toxophoric
subgroups with relative and potential toxicity from 2 to 7 score’ have to be more sofisticated for the prediction of po-
were obtained. The ‘n toxophoric score’ have to be consid- tential toxicity of NCEs.
ered as relative value of indication for possible toxicity. By
this correlations only relative position of each compound in REFERENCES
particular subgroup and in relation to efavirenz for the par- 1. Höltje HD, Sippl W, Rognan D, Folkers G, Moelcular mod-
ticular druglikeness score, estimated miLogP and TPSA could eling: Basic principles and applications, Wiley-VCH Wein-
be evaluated. heim, DE, 2008.
6
Staphylococcus aureus ATCC 56511
2. Vaz RJ, Klabunde T, Antitargets: Prediction and preven-
Y = 4.6115 - 0.00125 X
r = -0.8627 tion of drug side effects, Vol. 38, Wiley-VCH, Weinheim,
n = 20

4
DE, 2008.
3. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ, Experi-
-3
MIC/10 mol dm

mental and computational approaches to estimate solu-


-3

2
bility and permeability in drug discovery and develop-
ment settings, Adv.Drug.Delivery Rev. 1997; 23: 4-25.
0
0 500 1000 1500 2000 2500 4. Jadrijević-Mladar Takač M, Kos I, Takač V, QSAR studies
W
on antimicrobial activity of benzoxazindione analogues,
68th International Congress of FIP. Abstracts Book. Basel,
CH, September 2008.

4
-3
MIC/10 mol dm
-3

0 3,0
500
2,5
2,0
1000 1,5
1500 1,0
W 2000 0,5 AlogP
2500 0,0

44
Figure 1: 2D and 3D correlations of W versus AlogP and MIC
(Staphylococcus aureus ATCC 56511)

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

O27
Alicyclic β-amino acids: new, valuable scaffolds in drug research
Ferenc Fülöp
Institute of Pharmaceutical Chemistry, University of Szeged, H-6720 Szeged, Eötvös utca 6, Hungary

A
lthough the drug discovery process seems to be an au- EtOOC COOEt EtOOC COOEt
tomatic and routine protocol in most pharmaceutical N N 1R
4R
COOEt + N N 1R
4S COOEt
N
companies, one of the most important key elements 2R 3R
N
2R 3R
for success is the availability of good hit molecules. In the past O
HO NHBoc HO NHBoc

10 years, fragment-based drug discovery (FBDD) become one NH


of the most accepted efficient and promising protocols.1 EtOOC COOEt EtOOC COOEt

N N 1R 4S COOEt N N 1R 4R COOEt
+ N
N
In our university project, we work on small, sometimes exotic, 2R 3S 2R 3S
HO NHBoc
well-designed molecules possessiny a great diversity of func- HO NHBoc

tionalities. The present lecture will demonstrate that cyclic The Scheme illustrates an example of the preparation of four
β-amino acids meet the above criteria. The alicyclic β-amino 1,2,3-triazole-substituted ethyl 2-amino-3-hydroxycyclopen-
acids have acquired great interest in recent years in view of tanecarboxylate diastereomers (3,4-disubstituted cispent-
their pharmacological potential.2 Cispentacin, an antifungal acins) with a cyclopentane skeleton in enantiomerically pure
antibiotic with a cyclopentane skeleton, is one of the most form from racemic β-lactam via enzymatic ring opening, ep-
important derivatives. (1R,2S)-2-Amino-4-methylenecyclo­ oxidation and selective ring opening of the oxirane ring with
pentane­carboxylic acid (Icofungipen) is a known antifungal sodium azide. The formation of the 1,2,3-triazole ring system
agent.2 Cyclic, conformationally rigid β-amino acids such involved click chemistry: 1,3-dipolar cycloaddition of the 45
as cis and trans-2-aminocyclo­pentane­carboxylic acid have corresponding 4-substituted azidocarboxylates with diethyl
been used as building blocks in the synthesis of peptides.2 acetylenedicarboxylate
The present lecture will point out different strategies that re-
sult in functionalized β-amino acids from 2-aminocycloalk- REFERENCES
enecarboxylic acids, and will also focus on different proto- 1. Keserű, M.G., Makara, G.M. Nature Rev. Drug Discovery
cols for the preparation of enantiomeric substances.3 2009, 8, 203.Congreve, M., Chessari, G., Tisi, D., Wood-
head, A.J. J. Med. Chem. 2008, 51, 3661.
2. Fülöp, F. Chem. Rev. 2001, 101, 2181. Fülöp, F., Martinek,
T.A., Tóth, G.K. Chem. Soc. Rev., 2006, 35, 323. Mándity,
I.M., Wéber, E., Martinek, T.A. Olajos G., Tóth, G.K., Vass, E.,
Fülöp, F., Angew. Chem. Int. Ed., 2009, 48, 2171.
3. Kiss, L., Forró, E., Sillanpää, R., Fülöp, F. Tetrahedron:
Asymmetry, 2008, 19, 2856. Kiss, L., Nonn, M., Forró, E.,
Sillanpää, R. Fülöp, F. Tetrahedron Lett., 2009, 50, 2605.

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

O28
HYDROXAMIC AND AMIDE DERIVATIVES OF NONSTEROIDAL
ANTIINFLAMMATORY DRUGS – SYNTHESIS AND BIOLOGICAL ACTIVITY
Zrinca Rajić, Branka Zorc
Department of Medicinal Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, 10000,
Zagreb/Croatia

NTRODUCTION amides 4a-i and 5a-f were obtained. Derivatives 6a-f were
Nonsteroidal antiinflammatory drugs (NSAIDs) are well- synthesized by the catalytic hydrogenation of compounds
known class of drugs with anagesic, antipyretic and antiin- 5a-f, by using heterogeneous Pd-ethylenediamine complex
flamatory activity based on their inhibition of cyclooxyge- catalyst. Benzotriazolides 3a-e,g were converted to NSAID
nase. Their numerous derivatives have been synthesized in hydroxamic acids 7a-x in the reaction with the correspond-
order to modify the pharmacokinetics, improve the selectiv- ing hydroxylamine (hydroxylamine, N-methylhydroxylam-
ity towards the enzymes or increase the variety of their ac- ine, O-methylhydroxylamine, O-ethylhydroxylamine or O-
tion1. benzylhydroxylamine) or by the catalytic hydrogenation.

Hydroxamic acids are well known to form strong complexes Biological Evaluation
with transition metals, and thus inhibit enzymes of human Compounds 4b,c,e,f, 5c,e,f and 6c,e,f were screened in vitro
and bacterial origin2. One of them is lipoxygenase, the cru- for their potential antiproliferative effects on a panel of 5 tu-
cial enzyme in the synthesis of leukotrienes. Many NSAIDs mor cell lines: HCT 116, SW620, MCF-7, MOLT-4, and H 460.
have been reported to act either as inhibitors of free radical The tested compounds showed very similar, moderate anti-
production or as radical scavengers. proliferative effect on the presented panel cell lines. NSAID
hydroxamic acid derivatives 7 were evaluated in vitro for the-
46 On the other hand, they are potential anticancer drugs ir cytostatic effects against the following malignant tumor
and effective chemopreventive agents3. In our previous cell lines: L1210, FM3A, Molt4/C8, MOLT-4, CEM, HeLa, MCF-
papers, potency of NSAID amides as cytostatic agents was 7, MiaPaCa-2, NCI H727, SW620, HCT 116, H 460 and SK-BR-3
screened4,5. It was shown that ketoprofen amidation signifi- and compared with their effects on the growth of human
cantly enhanced antiproliferative activity of the parent com- normal fibroblasts (WI 38). Indomethacin hydroxamic acid
pound and that lipophilicity of the compounds had a signifi- (7f) showed the strongest, but nonselective, antiproliferative
cant influence on their activity. activity on the tested tumor cell panel. In contrast, selective
and differential activity was exerted on MiaPaCa-2 by 7b (IC50
Here we report the synthesis of a new series of lipophilic = 2.2 μmol/L) and on HeLa by 7c (IC50 = 6.5 μmol/L).
ketoprofen amides and NSAID hydroxamic acids (deriva-
tives of ibuprofen, fenoprofen, ketoprofen, diclofenac and Antioxidant activity, soybean lipoxygenase inhibition and
indomethacin) and their O-alkyl and O-benzyl derivatives. inhibition of linoleic acid lipid peroxidation was tested for
Compounds were evaluated for their cytostatic, antioxidant compounds 4−7.
and antimicrobial activity, as well as their ability to inhibit Antioxidant activity was tested by the reaction with the sta-
soybean lipoxygenase (LO). ble free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the
results have shown that derivatives of the NSAID hydroxamic
RESULTS AND DISCUSSION acids 7 showed better activity than amide derivatives 4−6.
Chemistry The highest activity was exerted by compounds 7c and 7n.
Ketoprofen was the starting compound in the synthetic Inhibition of LO was tested by the UV absorbance based en-
pathway leading to reduced analogues 2a,b and their novel zyme assay. Ketoprofen derivatives 4−6 were better LO inhi-
amides 4a-i, 5a-f and 6a-f, while various NSAIDs (ibuprofen, bitors than NSAID hydroxamic acids derivatives 7. Perusal of
fenoprofen, diclofenac, indomethacin, ketoprofen, 2a,b) IC50 values showed that compound 6f was the most active
started the reaction path leading to the derivatives of NSAID by far (IC50 = 20.5 µmol/L).
hydroxamic acids 7a-x. Compounds 2a,b were synthesized
by the catalytic hydrogenation of ketoprofen under differ- Inhibition of linoleic acid lipid peroxidation (LP) was studied
ent reaction conditions. Further on, NSAIDs reacted with 1 by using 2,2’-azobis(2-amidinopropane) dihydrochloride
equivalent of 1-benzotriazole carboxylic acid chloride (1) af- (AAPH) as a free radical initiator to follow oxidative chan-
fording benzotriazolides 3a-g (6). In the reaction of benzot- ges of linoleic acid to conjugated diene hydroperoxide.
riazolides 3g,e with 1.1 equivalent of the appropriate amine The best LP inhibitors were 4d,e,f, 5c,d,e,f, and 7s,x,v,q,n
in the presence of 5 equivalents of triethylamine in toluene (92.2−99.9%).

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

Antimicrobial activity was tested for NSAID hydroxamic acid droxamic acids as pharmacological agents, Curr Med
derivatives 7. The results of the hole-plate diffusion method Chem. 2002; 9: 1631−1653.
showed that only NSAID hydroxamic acids with free and O- 3. Thun MJ, Henley SJ, Patrono C. Nonsteroidal anti-inflam-
ethyl substituted hydroxy groups possessed noticeable anti- matory drugs as anticancer agents: mechanistic, phar-
microbial activity at concentration 29.6 mg/mL. The minimal macologic, and clinical issues, J Nat Cancer Inst. 2002;
inhibitory (MIC) and the minimal microbicidal concentrations 94: 252−266.
(MMcC) were determined by the microdilution broth meth- 4. Wittine K, Benci K, Rajic Z, Zorc B, Kralj M et al. The novel
od. Compounds 7a and 7c showed the lowest MIC/MMcC phosphoramidate derivatives of NSAID 3-hydroxypro-
against B. subtilis and S. enterica subsp. enterica species. pylamides: Synthesis, cytostatic and antiviral activity
evaluations, Eur J Med Chem. 2009; 44: 143−151.
REFERENCES 5. Marjanovic M, Zorc B, Pejnovic L, Zovko M, Kralj M. Fe-
1. Bonina F, Puglia C, Santagati NA, Saija A, Tomaino A, Tita noprofen and ketoprofen amides as potential antitumor
B. Oligoethylene ester derivatives of ketoprofen, naprox- agents. Chem Biol Drug Des. 2007; 69: 222−226.
en and diclofenac as oral prodrugs: a pharmacological 6. Zorc B, Antolic S, Butula I. Macromolecular prodrugs. I.
evaluation, Pharmazie. 2002; 57: 552−555. Synthesis of some non-steroidal anti-inflammatory drug
2. Muri EMF, Nieto MJ, Sindelar RD, Williamson, JS. Hy- esters. Acta Pharm. 1993; 43: 127–133.

47

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

O29
Improving the effectiveness and cost effectiveness of drug therapy
using pharmacogenetic testing and comprehensive screening of
drug interactions
Lajos Botz
Pharmaceutical Institute, Faculty of Medicine, University of Pécs, Pécs, Szigeti u. 12, Hungary H-7624

R
ecently more and more controversial opinions surfaced viduals in the selected patients groups with increasing ex-
regarding the effectiveness of traditional pharmacot- posure to drug-drug interactions. The alleles selected were
herapy. These critics refer to the inadequate effective- CYP2D6 and CYP2C19. In Caucasians, the most common
ness of drugs, the high prevalence of adverse reactions and non-coding alleles are CYP2D6*4, CYP2D6*3, CYP2D6*5 and
the excessive use of medication. In many cases drug interac- CYP2D6*6. CYP2C19 has about 15 allele variants of which two
tions may be the root of the problem. The aims of the current (CYP2C19*2 and CYP2C19*3) play the most important role in
study were (1) to identify the most common drug interacti- the altered metabolism of antidepressant drugs. CYP2C19*3
ons in clinical practice; (2) to characterise of drug interactions is extremely rare in Caucasians, but accounts for approxi-
with computer analysis (3); to develop a software application mately 85% of all poor metabolisers. We used two methods
which utilises a drug interaction database based on the CYP to detect the patients’ genotype. The first method is based
450 metabolism; (4) to assess the influence of polymorphic on DNA melting point analysis. This method is less expensive
drug metabolism on the utilization of antidepressant drugs. but only validated for experimental use. The second method,
Many studies have demonstrated marked ethnogeographic which is based on a DNA chip analysis, is validated for clini-
variance in the distribution of CYP2D6 and CYP2C19 genoty- cal use and capable to detect 29 polymorphisms (33 alleles)
pes throughout the world. Many antidepressants are metab- of CYP2D6 and 2 polymorphisms (2 alleles) of CYP2C19. This
olized by the polymorphic cytochrome P450 enzymes. The method is very expensive and not approved by National
48 activities of CYP 2D6 and 2C19 enzymes in the metabolism Health Insurance System and health insurance companies.
of antidepressants are very different. The pharmacogenetic The patients for the experiment were selected from the Clin-
information may be beneficial in evaluating the drug-drug ics of Psychiatry and Psychotherapeutics at the University of
interactions. The combination of these two fields for pre- Pécs in Hungary. Non-responsiveness was defined as ineffec-
screening the drug interactions is useful for increasing the tive therapeutic response evaluated by the physician after a
efficiency of drug-drug interaction control software. We four-week treatment period with an adequate dosage. The
would also like to map and visualise the complex network type and number of adverse effects and therapeutic conse-
of interactions. This combined methodology may be used quences were recorded and reported at the end of the treat-
for optimising the drug therapy for patients suffering from ment with a particular antidepressant. The aims of the study
depression. Two drug interaction software applications have were to learn the drug interactions that occur in practice
been developed to detect drug interactions. The first soft- and to compare various aspects of the analysis. The results
ware application was developed by the Pharmacy Institute show that the most frequent drug-drug interactions were
of University of Pécs for community pharmacies. The interac- the acenocoumarol - acetylsalicylic acid and the naproxen -
tion analysis was based on three data sources: (1) the official enalapril. The overall rate of clinically significant interactions
summary of product characteristics for drugs, (2) relevant were 3.64% and 2.65%. The software application identifies
drug information databases and literature and (3) the effect and prevents the prescription of clinically dangerous drug
of the interactions on the cytochrome P450 enzyme system. interactions. The analysis of the drug interactions in patients
After having analysed the official Hungarian drug specifica- with depression also revealed startling relationships. Almost
tions and drug interactions, we developed the first software 8% of all drug administrations and more than 35 % of patients
application which filters out undesirable drug interactions. were using alprazolam in a combination. The Micromedex®
We only targeted drug interactions which are relevant in the drug interaction software application revealed 240 drug in-
clinical practice. The novel software application is based on teractions during the study period. Within 38% of all drug
the drug interaction database. The second software was an administration at least one drug interaction occurred. The
internationally used professional tool: Micromedex® Health- most important drugs were antidepressants and antiplate-
care series drug interaction system (Thomson Reuters). The let agents. The relationships between the frequency of ADRs
first data collection was 1997, when 709,000 prescriptions and genotype, the interactions in CYP enzyme-mediated
were analysed. The second systematic data collection was drug metabolism and ADRs were investigated. However, we
between 2001 -2004. This analysed 1,227,000 prescriptions could not find correlation between phenotype and the oc-
by a computer program. Our third survey included 401,000 currence of ADRs prior to this. We also analysed relationships
prescriptions in 2007. We determined the genotype of indi- between drug interactions of CYP enzyme mediated drug

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

metabolism and the presence of ADRs. Such analysis of drugs added to the existing one to characterize the relationship
may lead to a personalised medical therapy in the future. between active agent pairs. The possibility of analysis of the
Drug interactions via the inhibition or induction of a CYP en- complex network of interactions of the active ingredients of
zyme have been also analysed. These results show that there drugs helps to predict future interactions. This analysis can
are correlations between genetic background and therapy be facilitated by forming clusters of all used active ingredi-
outcome. Drug therapy decisions based on pharmacogenet- ent pairs. The relationship function resembles the independ-
ics are very complex and clear link cannot be confirmed yet. ent network models (logplot). This network analysis shows
Our software application contains three screening methods a map of all drug-drug interactions, and highlights every in-
regarding an active agent pair. To improve our software ap- teraction of a selected drug.. The analysis shows that severe
plication bioinformatics network matrix and a bioinformatics drug interactions can occur in 3-4% of patients and this may
software environment were introduced. A new database was cause 6-10% increase in medical costs.

49

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

O30
IMPLEMENTATION OF CLINICAL PHARMACY SERVICES IN HOSPITALS: WHERE TO
START?
Kutay Demirkan
Hacettepe University, School of Pharmacy, Ankara, Turkey

I
mplementation of clinical pharmacy requires an exten- macy. Clinical pharmacy education and practice have been
sive knowledge of therapeutics and medical terminology, developed by contribution from both pharmacy schools and
a good understanding of disease and drug monitoring university-based teaching hospitals.
processes, therapeutic planning skills, ability to assess and Since only few clinical pharmacists are available and clinical
interpret physical and laboratory findings and provision of pharmacy practices are not routinely performed in hospitals
information about medicines. in Turkey, most of the physicians are not familiar with clini-
cal pharmacy practices. Clinical pharmacy practices have
By means of its definition, responsibilities and duties of a been first implemented in nine-bed medical intensive care
clinical pharmacist include selection of the correct drug, unit (ICU) at the Hacettepe University Hospital in Türkiye.
proper dosage form, appropriate route of administration; The practicing clinical pharmacist was a part-time provider;
choice and timing of specimens for therapeutic drug moni- therefore clinical pharmacy service was limited to morning
toring; interpretation of pharmacokinetics data, detection visits of ICU throughout almost all week-days. Also, the clini-
and prevention of drug interactions, adverse drug effects cal pharmacist was available on call when needed. The rea-
and toxicity. son of “why ICU to start with” is very clear and simple: the
clinical pharmacist had a specialty in the ICU and the attend-
It is clear that clinical pharmacists have the potential to con- ing physician who also had an experience working with clini-
50 tribute in reduction of drug expenditures and improving cal pharmacists in USA. Implementation of clinical pharmacy
medication and patient safety. To achieve these outcomes, practice was also started in nutrition unit and neuro-ICU unit
clinical pharmacists should obtain medication histories, upon requests from physicians after the ICU activities at the
perform medication reviews, attend ward rounds, provide Hacettepe University Hospital. Other requests for implemen-
recommendations on drug selection and follow-up, and tation of clinical pharmacy services from other units such
provide counseling to patients and healthcare providers. as oncology, infectious diseases are not fulfilled yet due to
The clinical pharmacists should be willing to improve them- a lack of clinical pharmacist at the moment. Moreover, bed-
selves through ongoing learning activities about their dis- side teaching for pharmacy students at the hospital was ap-
cipline and should be able to keep up with current medical proved by the hospital administration office and chief phy-
literature. sicians, where a clinical pharmacist act as a mentor during
medication review process for undergraduate education.
Clinical pharmacy services are well developed in some coun-
tries (such as USA, Canada and the UK) and clinical pharma- The implementation of clinical pharmacy practice from a
cists are integral members of the health care team. However, pilot unit and thereafter an establishment of a clinical phar-
clinical pharmacy is a new arising issue in many countries macy service at the hospital might be easier to prove the
and therefore education and provision of clinical pharmacy efficacy and necessity of the service. It is better to choose
are not available yet. a pilot unit that patients are more complex and need more
attention, so pharmacists can have more active role and in-
Implementing a new clinical pharmacy service in a hospital terventions in short time period.
is challenging. Before the implementation of any service, the
barriers such as lack of time, lack of communication skills, al- Although implementing a new clinical pharmacy service in
location of new resources, lack of training and lack of clinical a hospital seems to be challenging; having qualified clinical
pharmacist, lack of reimbursement need to be identified and pharmacists, highly cooperative and enthusiastic healthcare
the plans to overcome for each barrier need to be consid- professionals along with supportive hospital administrative
ered. board, would be an initiative force to establish clinical phar-
macy services.
Schools of Pharmacy in most countries do not satisfy minimal
requirements for an appropriate education in clinical phar-

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

O31
THE ROLE OF THERAPEUTIC DRUG MONITORING IN OPTIMAZING
PHARMACOTHERAPY OF SELECTED ANTIEPILEPTICS
T. Vovk1, I. Grabnar1, M. Kerec Kos1, M.B. Jakovljević2, K. Vučičević3, A. Mrhar1
1
The Chair of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, University of Ljubljana, Ljubljana,
Slovenia
2
Pharmacology Department, Medical Faculty, University of Kragujevac, Kragujevac, Serbia
3
Department of Pharmacokinetics, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia

INTRODUCTION the TPR study volume of distribution (V/F) were estimated,


Clinical experience has demonstrated that individualized while the absorption rate constant was fixed. Effects of con-
dose adjustment by the aid of therapeutic drug monitoring tinuous and categorical covariates were tested against the
(TDM) can significantly improve treatment with antiepileptic base model. Alternative models were compared by the likeli-
drugs (AED). Development of a population pharmacokinetic hood ratio test (α = 0.05). Significant covariates were rank-
(PK) model is a logical extension of TDM, because it allows ordered and introduced into the full model. The final model
estimation of individual patients’ PK parameters based on was determined by backward elimination of covariates one
sparse concentration measurements and ultimately effec- by one from the full model to see if they should remain in
tive control of dosing. In addition to being a valuable tool in the model using the likelihood ratio test. Additional criterion
designing a safe and effective dosing regimen for patients for retention of a covariate in the model was reduction in the
with epilepsy, population PK models permit evaluation of unexplained interindividual variability. The model adequacy
various factors that can influence PK characteristics1. was evaluated by standard diagnostic plots, convergence of
minimization, number of significant digits more than 3, suc-
This paper presents the summary of carbamazepine2, valpro- cessful covariance step and gradients in the final iteration in
ic acid and topiramate studies aiming to develop population the range between 10-3 and 102.
PK models to evaluate the influences of demographic factors
and comedications on drugs PK. RESULTS AND DISCUSSION 51
Carbamazepine
METHODS Patient weight (WT), daily carbamazepine dose (DCBZ), daily
Patients dose of phenobarbitone (DPB) and valproic acid (VPA), when
For development of carbamazepine (CBZ) and valproic acid its daily dose exceeded 750 mg, significantly influenced CL/F,
(VPA) population pharmacokinetic models patients’ char- and were included in the final model:
acteristics and plasma drug concentrations were collected
retrospectively from patients’ charts as part of routine TDM.
Topiramate (TPR) study was prospective, and was not part of
routine TDM.

Blood sampling
Blood samples were drawn at the end of the dose interval,
before the morning dose, while in all cases of TPR study and
same cases in CBZ and VPR study a second blood sample was
drawn after the dosing approximately at the time of the peak
concentration. , where VPA is 1 if DVPA>750 mg or 0 otherwise. The rela-
tionship between CBZ CL/F and DCBZ is nonlinear, while that
Bioanalitical assay with DPB is linear. The effects of CBZ and PB on CZB CL/F are
In CBZ and VPR studies drugs were measured using homog- attributed to induction of CBZ metabolism. Moreover, VPA
enous enzyme immunoassay technique (EMIT; Cobas Mira, increases the CBZ CL/F probably by displacement of CBZ
Roche Diagnostics, Basel, Switzerland), while the TPR was de- from plasma proteins.
termined with HPLC fluorescent labelling method adapted
from literature. Valproic acid
The VPA CL/F was significantly influenced by patient’s WT,
Population modelling daily dose of VPA, and co-therapy with topiramate (TPR).
PK analysis was performed by a population PK modelling These covariates were included in the final pharmacokinetic
approach using NONMEM software. The structural model model which is described with the following equation:
used was a one-compartment PK model with first-order ab-
sorption and elimination. Apparent clearance (CL/F) and in

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

CONCLUSIONS
The proposed PK models can be used for Bayesian estima-
tion of drugs CL/F in individual patients based on sparse
concentration measurements and for selection of optimum
dosing regimen in routine patient care.
, where VPA is 1, if VPA dose is greater than 1000 mg/day, or 0
otherwise, and TPR equals 1, if patient is co-treated with TPR, REFERENCES
or 0, if not. This study confirms the interaction of VPA with 1. Johannessen SI, Landmark CJ. Value of therapeutic drug
TPR, which is presumably dependent on VPA dose. monitoring in epilepsy, Expert Rev Neurother. 2008; 8:
929-939.
2. Vučićević K, Miljković B, Veličković R, Pokrajac M, Mrhar
Topiramate A, et al. Population pharmacokinetic model of carbam-
The final model of TPR CL/F was significantly influenced by azepine derived from routine therapeutic drug monitor-
co-treatment with CBZ and patient’s age. While the TPR V/F ing data, Ther Drug Monit. 2007; 29: 781-788
was only dependent on patient’s weight. The final models
are presented by the following equations:

, where CBZ is 1 in patients co-treated with carbamazepine,


or 0 otherwise. CBZ most probably induces TPR metabolism
and therefore its clearance.

52

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

O32
IDENTIFYING TARGETS FOR THE TREATMENT OF DYSTONIA AND
PRECLINICALTESTING OF THE LEAD COMPOUNDS
Aygül Balcıoğlu
Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

A
common early-onset dystonia is a disease that is char- abnormalitiesin striatal DA release4 as well as function of
acterized by an abnormal rigidity of muscle, and pro- dopamine transporter (DAT) and amphetamine-induced-
longed, repetitive muscle contractions that may cause locomotion. We also described a deficit in motor learning5,
twisting or jerking movements of the body or abody part. which resembles that observed in humans.These animals do
Most cases of early onset torsin dystonia (DYT1) are caused not have overt dystonia, but they do not learn to stay on the
by a ΔGAG mutation in TOR1Agene resulting in the loss of a rotarod and they travelless distance under basal condition
glutamic acid in the carboxy terminal region of torsinA pro- as well as following amphetamine administration as a condi-
tein in theheterozygous state1. tion ofDYT1 dystonia.

There are 300,000 DYT1 patients in USA and over 3 mil- Using in vivo microdialysis in awake animals, we found the
lion dystonia patients in the worldwide waitingfor a treat- response to amphetamine to be markedlyattenuated in the
ment2. Therapy is primarily limited to either neurosurgery human mutant torsinA (hMT1) expressing mice compared
or chemodenervation3. Asmutant torsinA (ΔE) expression to their non-transgeniclittermates (253±71% vs. 561±132%,
results in decreased torsinA function, therapeutic strategies p<0.05, two-way ANOVA). Extracellular striatal tissue DA lev-
directedtoward enhancement of wildtype (WT) torsinA ac- elsmeasured using HPLC revealed no marked difference be-
tivity in patients, who are heterozygous for mutanttorsinA, tween hMT1 mice and their non-transgeniclittermates (48±9
may restore normal cellular functionality. One of many such vs. 34±6 nmol/mg, tissue respectively, n=6), suggesting no 53
efforts includes screening of thePrestwick C. elegans chemi- degeneration in striatum. Two targets involved in amphet-
cal library purchased from Prestwick Chemical Inc. (Illkirch, amine’s action are DAT and vesicular monoamine trans-
France). Thesecompounds are evaluated in nematodes (C. porter (VMAT2)are very important components of uptake,
elegans) to enhance WT torsinA activity in two separateas- storage and release of DA. Using the specific DAT inhibitor-
says. nomifensine (NOM), we demonstrated that the efficiency of
the inhibition was different at the sameconcentration (20
Transgenic nematode strains were utilized to quantitative- μM), with means of 50% for transgenics vs. 91% for non-
ly measure changes in protein misfolding as afunctional transgenics, but this did notreach statistical significance. This
readout in a screen for small molecule effectors of torsinA indicated to us that DAT is involved in amphetamine-induced
activity. We screened thePrestwick C. elegans library, a col- DArelease in transgenic mice, and the reduced DA release
lection of 240 chemically and therapeutically diverse drugs would be consistent with fewer or improperlyprocessed DAT
(prescreenedfor maximal dosage and toxicity to worms), on the cell surface of neurons in hMT1 mice. Consequently,
for compounds that either inhibited the dominanteffect of we studied the dynamics ofDAT in vivo using no-net flux
mutant torsinA (DE) or enhanced the normal activity of WT microdialysis6,7. The extraction fraction (indicates function
torsinA. This screen wasperformed in worms that presum- of DAT)for non-transgenic animals was 0.88±0.04 and for
ably best reflect the human DYT1 condition in that they ex- hMT1 transgenic mice 0.73±0.05 (p<0.05). Thisdifference in-
pressed boththe WT and DE gene products (WT/DE). At the dicates that DAT in hMT1 mice work less efficiently than in
third larval stage of C. elegans, a 60% increase inaggregates their controls, which couldresult from fewer or functionaly
was reproducibly observed in worms expressing WT/DE, compromised transporters on the cell surface. Additionally,
when compared to animalsexpressing only the WT human basal (datanot shown) and amphetamine-induced locomo-
protein. This difference represented an ideal developmental tion was much less in hMT1 transgenic mice compared to-
stage forreadily distinguishing molecules that significantly control littermates. The distance traveled following amphet-
altered torsinA chaperone function. amine administration was 48.5±6.7 metersfor transgenics vs.
73.7±9.8 meters for non-transgenics (p<0.05). These results
Work in our collaborative team has also focused on mouse support altered dynamicsof DAT in hMT1 transgenic mice
models of DYT1 dystonia, with the goal ofunderstanding consistent with behavioral consequences. These functional
the underlying mechanisms, which may be shared with analyses ofDAT support potential defects in processing of
other forms of dystonia. In atransgenic DYT1 model in which this protein by mutant torsinA, which is responsible from-
mice express human mutant torsinA, we have identified DYT1 dystonia.

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

We are currently working on the promising leading com- ish C, de Leon D, Brin M, Raymond D, CoreyDP, Fahn S,
pounds from Prestwick C. elegans library onimpaired am- Risch N, Buckler A, Gusella JF, Breakefield XO (1997) The
phetamine-induced DA release as well as basal and am- early-onset torsion dystoniagene (DYT1) encodes an
phetamine-induced locomotion in ananimal model of DYT1 ATP-binding protein. Nat Genet 17:40-48.
dsytonia. In a transgenic DYT1 model in which mice express 2. Defazio G (2007) in Handbook of Dystonia, ed Stacey ME
human mutanttorsinA, we have identified abnormalities in (Informa Healthcare USA,Inc. New York), pp 11-20.
A. Striatal amphetamine-induced DA release, B. Functionof 3. Jankovic J (2006) Treatment of dystonia. Lancet Neurol
dopamine transporter (DAT), and C. Amphetamine-induced 5: 864-872.
locomotion. These studies provide therationale that a com- 4. Balcioglu A, Kim MO, Sharma N, Cha JH, Breakefield XO,
promised DA system might be an important phenotype in Standaert DG. (2007) Dopamine release isimpaired in a
animal models of DYT1dystonia. Some compounds have mouse model of DYT1 dystonia. J Chem. 102: 783-788.
emerged as a promising candidate for DYT1 dystonia to test 5. Sharma N, Baxter MG, Petravicz J, Bragg CD, Schienda
itsefficacy in vivo before it (and/or similar derivatives) could A, Standaert DG, Breakefield XO (2005)Impaired motor
be given to DYT1 patients. Our workaccelerates determining learning in mice expressing torsinA with the DYT1 dys-
the efficacy and relevance to behavioral outcome of prom- tonia mutation. J Neurosci25: 5351-5355
ising compounds bytesting them on in vivo phenotype for 6. Jones SR, Gainetdinov RR, Jaber M, Giros B, Wightman
DYT1 dsytonia. It is critical to perform these translationalpre- RM, Caron MG (1998) Profound neuronalplasticity in
clinical tests to validate potential therapies under disease- response to inactivation of the dopamine transporter.
specific conditions in animal models beforethey can enter Proc Natl Acad Sci 95: 4029-4034
the development pipeline on their way to the bedside. 7. Chefer VI, Czyzyk T, Bolan EA, Moron J, Pintar JE, Ship-
penberg TS (2005) Endogenous kappa-opioidreceptor
REFERENCES systems regulate mesoaccumbal dopamine dynamics
1. Ozelius LJ, Hewett J, Page C, Bressman S, Kramer P, Shal- and vulnerability to cocaine. J Neurosci25: 5029-5037.v

54

3rd BBBB International Conference on Pharmaceutical Sciences


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O33
A EUROPEAN PERSPECTIVE ON THE FUTURE OF THE PHARMACY PROFESSION
Daisy Volmer
Department of Pharmacy, University of Tartu, 50411 Tartu, Estonia

INTRODUCTION assure unified professional competency of the pharmacists.


Historically the preparation of medicines has been considered
as one the core competencies of the pharmacy profession. Implementation of the professional competencies into practice
During the past fifty years described professional activity has Community pharmacy setting
been shifted from community and hospital setting to drug Within 15-20 years community pharmacy services have be-
industry and changed from preparation to manufacture of come more accessible and patient oriented. Patients are re-
medicinal products. The pharmacy profession, having a mo- ceiving more detailed drug information and pharmacists are
nopoly concerning preparation of medicines in the past, has regarded as professionals in the use of medicines2.
had to review its´ core values, practice roles and educational
methods. With a decrease in the preparation of medicines in Community pharmacies provide new services as patient
community and hospital pharmacies, provision of drug infor- medication review, taking blood pressure and other analysis,
mation has become an important service. To ensure the qua- monitoring the quality of therapy of patients with chronic
lity treatment of patients, new approaches including clinical conditions. Pharmacists are considered as advisors on
pharmacy and pharmaceutical care were initiated. healthcare issues and prescribers of medicines.

Assurance of the professional competency in pharmacy New initiatives within the pharmacy profession relate more
Nowadays the pharmacy profession has been considered to providing effective drug therapy and resolving drug re- 55
as dynamic and multifaceted discipline. In the EU Directive lated problems. This is welcome, because a systematic ap-
2005/36/EC on the recognition of professional qualifications proach about how to avoid the problems connected with
pharmacists should be able to provide the following activi- use of medicines is missing.
ties and services:
• preparation and manufacturing of medicinal products; However, there are several factors serving as obstacles to
• testing the quality of medicinal products; the development of the community pharmacy practice. The
• storage, preservation and distribution of medicinal liberal pharmacy system with mostly private ownership of
products at the wholesale stage; community pharmacies not restricted by the pharmacy pro-
• preparation, testing, storage and supply of medicinal fession and operation of pharmacy chains has introduced
products at the retail sale stage; not healthcare but business oriented pharmacies. Shortage
• preparation, testing, storage and dispensing of medici- of professional staff and burn out syndrome of the pharma-
nal products at hospitals; cists decrease interest towards professional continuing edu-
• provision of information and advice on medicinal prod- cation initiatives.
ucts1).
It would be impossible to give one universal recipe for the
The question is how to provide the education to cover all afore- development of community pharmacy practice. For the ac-
mentioned aspects of the pharmaceutical activities. Directive tual steps, it is important to analyse existing situation, learn
2005/36/EC presents general info concerning length and con- the local health care traditions and develop guidelines, work
tent of the pharmacy studies at higher education institutions. out quality assurance instruments for provided or planned
During the last two decades there have been several discus- services, cooperate with different institutions, apply for the
sions and attempts to develop unified pharmacy education financial assistance from state and private organisations and
programme for Europe. In 2008 the PHARMINE programme has from EU funds.
been introduced by European Association of Faculties of Phar-
macy to map the pharmacy education in Europe and based on Hospital pharmacy setting
the received information to develop core curriculum for the In 1983 European Society of Clinical Pharmacy first pre-
pharmacy studies in Europe. In addition the extended curricula sented document with requirements and standards for the
in three main disciplines of pharmacy - community pharmacy, expertise and skills of clinical pharmacists. Following 10-15
clinical pharmacy and pharmaceutical industry will be devel- years several different organizations have been contributed
oped. The new curricula could serve as a standard for Europe to to modernization and presentation of the professional roles

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

of the clinical pharmacist3. However, nowadays there is con- - There could be mistrust towards new professional roles of
siderable variation in hospital pharmacy practice in Europe. the pharmacists by the patients and other health care profes-
The development of clinical pharmacy education and prac- sionals. Physicians may feel threatened by the pharmacists
tice has not been as fast as was expected. who perform the medical review or prescribe medicines.
In the countries where the clinical pharmacy is novel disci- - Trend towards business orientation may turn community
pline and/or the number of hospital pharmacies is small it pharmacies to the shops of medicinal products, where the
would be problematic to compose separate curriculum for pharmacist is only a seller with no obligation to give any
clinical pharmacy. Nevertheless, it is possible to introduce counselling to the patient.
some of the basic knowledge during the undergraduate
pharmacy studies followed by continuing educational cours- CONCLUSION
es abroad. Regardless of the reorientation of the core values, practice
and education of the pharmacy profession within the past
Possible challenges in implementation of the professional 20-30 years, the new initiatives and activities serve promis-
competencies ing opportunity for the future developments. Pharmacist
- Development of IT technologies has introduced new can consider themselves as a part of the system that discov-
powerful way to buy medicines – e-pharmacies. Online- ers, develops, produces, distributes and provides informa-
pharmacies obtain popularity due to ready availability and tion on medicines.
cost benefits of medicines. Currently each country in EU can
establish rules for opening of e-pharmacy (26). Despite on REFERENCES
national rules regulating the operation of Internet pharma-
cies, the nonexistent contact with the pharmacist, question- 1. EU Directive 2005/36/EC. Available at: http://eur-lex.eu-
able quality of the drug counselling and unclear conditions ropa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2005:255:0
to order prescription medicines may serve as the potential 022:0142:EN:PDF, visited July, 2009.
threat to the effective and safe medical treatment of the pa- 2. Leufkens H, Hekster Y, & Hudson S, (1997). Scenario anal-
tients. However, according to the recent developments the ysis of the future of clinical pharmacy. Pharm World and
e-pharmacies do not decrease the role of traditional com- Sci, 1997; 19 182–198.
munity pharmacies. It would be important to educate public 3. Volmer D, Bell JS, Janno R, Raal A, Hamilton DD, Airak-
to use this source of medicines properly. sinen MS, Change in public satisfaction with community
- Deregulation of the medicines distribution system at the pharmacy services in Tartu, Estonia, between 1993 and
end of 1990`s and on the beginning of 2000´s has ended 2005. Research in Social and Administrative Pharmacy,
56 pharmacy monopoly of the sale of OTC medicines in many in press.
European countries. Does the better accessibility to OTC-
medicines outweigh missing consultation of the pharma-
cist?

3rd BBBB International Conference on Pharmaceutical Sciences


ORAL PRESENTATIONS

O34
PHARMINE OR PHARMACEUTICAL EDUCATION IN EUROPE,
A CHALLENGE TO ALL PHARMACISTS
Bart Rombaut
(EAFP) Dept. Pharmaceutical Biotechnology and Molecular Biology, Vrije Universiteit Brussel,
Laarbeeklaan 103, 1090 Brussels, Belgium

I
n the XXI century EU pharmacists will play an increasingly The PHARMINE consortium consists of universities which
important role as partners in the efficient use of the health are members of the European Association of Faculties of
care resources of the EU (community and hospital phar- Pharmacy (EAFP) and EU partner associations representing
macists). They will also be major players in the development community, hospital of industrial pharmacy, together with
of the EU pharmaceutical industry (industrial pharmacists). the European Pharmacy Students’ Association and other in-
terested bodies. It will develop a bachelor/master/doctorate
Whilst abiding by the recommendations for the duration system for pharmacy education and training taking into ac-
and course content for EU pharmacy education and train- count the need for basic pharmaceutical competences (and
ing given in the directive 2005/36/EC on the recognition of mutual recognition of pharmacy qualifications) and the op-
professional qualifications, PHARMINE will examine the op- tion needed for pharmaceutical expertise in, for example,
portunities for the introduction of the principles of the Bolo- industry.
gna declaration into pharmacy education and training with
the aim of tuning the latter to the future needs in the three To do this, the consortium will survey existing EU pharmacy cur-
areas of pharmaceutical expertise: community, hospital and ricula and attempts to adapt these to the Bologna process.
industrial pharmacy.

57

October 26 - 28, 2009 / Antalya - TURKEY


ORAL PRESENTATIONS

O35
The PHARMINE survey of pharmacy education and training in Europe:
a databank of EU HEIs delivering pharmacy education and training
and a task force for the survey of competency curricula (work
programme 7 (WP7))
Jeffrey Atkinson and Bart Rombaut
*EAFP, c/o Dept. Pharmaceutical Biotechnology and Molecular Biology, Vrije Universiteit Brussel, Laarbeeklaan 103,
1090 Brussels, Belgium

Aims and objectives Performance indicators


The aims and objectives of WP7 are to produce a databank of The main performance indicator is the retrieval of full data
pharmacy education and training leading to basic pharma- from at least one HEI in each member state that delivers a
cist qualifications and to qualifications required for optional pharmacy diploma qualification. In the larger member states
activities, in pharmacy higher education institutes (HEIs) of an objective of two HEIs would be useful. As far as relation-
the EU. ships with other WPs and overall project it is obvious that the
success of the other WPs depends upon the quality of the
WP7 aims to survey to what extent the “Bologna” and the data obtained by this WP.
“Sectoral profession” (based on 2005/36/EC) models for
pharmacy education and training are compatible (if at all: Relationships with other WPs and overall project.
see figure). This WP will work together with other WPs regarding what
type of information is to be gathered, in which form it is to
Organisation be handed over to the other WPs.
The organisation of WP7 will be by electronic means and Although the main activity of WP7 will be over by month 12,
telephone conference. WP7 will collect data directly from its activities will continue (and be slowly turned over to the
pharmacy HEIs in the EU, from the EAFP archives (especially European Working Group on Pharmacy Education) with an
58 the previous TEMPUS programme, see abstract of Rombaut aim to ensuing that the information available in the data-
and Atkinson, this meeting), from PGEU, EAHP, EIPG and EPSA bank is kept up-to-date.
and from all other web and other sources available.
Anyone wishing to join WP7 should apply via the PHARMINE
Milestones website:
The principal milestone will be the delivery of the data re- www.pharmine.orgv
quired for the production of the white papers in WP2.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS
POSTER PRESENTATIONS

P001-Characterization and In- tablets exhibited good technological properties. Thus, this
Vitro Dissolution Studies of Solid study reveals the possibility of preparing SD tablets of GLC
with improved aqueous solubility and dissolution rate.
Dispersion Systems of Gliclazide with ACKNOWLEDGEMENT
Macrogols The authors would like to acknowledge Kuwait University for
funding this research project (grant No. PP01/07). Thanks is
Ibrahim Khattab1, Abel-Azim Zaghloul1, Aly Nada1 extended to Dr Farzana Bandarkar for excellent technical as-
sistance.
Department of Pharmaceutics, faculty of Pharmacy, Kuwait
University, Kuwait1 REFERENCES
1. L. Chengsheng, G. Kashappa. Characteristics of Rofecox-
INTRODUCTION ib-Polyethylene glycol 4000 Solid Dispersions and Tab-
Solid dispersion techniques are very useful in pharmaceuti- lets based on Solid Dispersions. Pharmaceutical devel-
cal sciences because of the increasing number of novel drug opment and Technology. 10; 467-477 (2005).
candidates that are poorly soluble, but there is still limited 2. D. Leonardi, M. G. Barrera, M. C. Lamas, and C. J. Salomón.
information available on their ability to be processed into Development of Prednisone:Polyethylene Glycol 6000
the final dosage forms 1,2. Many substances can be used as Fast-Release Tablets From Solid Dispersions: Solid-State
carriers to prepare solid dispersions among which, one of Characterization, Dissolution Behavior, and Formula-
the popular carriers used are polyethylene glycols (PEG)3,4 . tion Parameters. AAPS PharmSciTech. 8 (4); Article 108
(2007).
The objective of the present investigation was to improve 3. Y. Özkan, N. Doğanay, N. Dikmen, A. Işimer. Enhanced
the aqueous solubility and dissolution rate of Gliclazide release of solid dispersions of etodolac in polyethylene
(GLC), a hydrophobic anti-diabetic drug by solid dispersion glycol. Farmaco. 55; 433-438 (2000).
(SD) technique, using a water soluble carrier i.e. Macrogol 4. M. Franco, G. Trapani, A. Latrofa, C. Tullio, M.R. Proven-
(Polyethylene glycol). zano, M. Serra, M. Muggironi, G. Biggio, G. Liso. Dissolu-
tion properties and anticonvulsant activity of phenytoin
EXPERIMENTAL METHODS polyethylene glycol 6000 and -polyvinylpyrrolidone
Preliminary screening of various grades of PG’s (4000, 10000 K-30 solid dispersions. Int. J. Pharm. 225; 63-73 (2001).
and 20000) at different concentrations (0.5, 1, 2, 5 and 10%)
was done by phase solubility studies in order to analyze their
62 influence on solubility of GLC. SD’s of GLC and PG in differ-
ent ratios (1:1, 1:2, 1:5 and 1:10) were prepared by the fusion P002-PREPARATION AND EVALUATION
technique. The SD’s were compared with the original drug OF KETOPROFEN SOLID DISPERSIONS
as well as the physical mixtures (PM) in the same ratios for
in-vitro dissolution in distilled water and phosphate buffer Seda Rençber1, Mine Özyazıcı1, Tamer Güneri1, Gökhan
(pH 7.4) by the USP Type II method. DSC studies were per- Ertan1
formed to identify the physicochemical interaction between
the drug and the carrier. SD with satisfactory characteristics Ege University Faculty of Pharmacy Department of
was used for the formulation of tablets by wet granulation Pharmaceutical Technology1 , İzmir, Turkey
method and compared with a commercial brand.
INTRODUCTION
RESULTS AND DISCUSSION Ketoprofen [2-(3-benzoylphenyl)propionic acid] (KP) is an
It was evident from the phase solubility studies that the drug analgesic and non-steroidal anti-inflammatory (NSAI) drug,
solubility increased linearly with increasing PEG concentra- widely used in order to reduce pain, inflammation and stiff-
tions indicative of the AL type of phase diagram. Intrinsic ness caused by several condition such as osteorarthritis,
solubility of GLC in water was found to be only 6.144 µg/ rheumatoid arthritis, ankylosing spondylitis or abdominal
ml. Dissolution of GLC improved significantly in solid dis- cramps associated with menstruation. The mechanism of ac-
persions as compared with the original drug and physical tion of KP is mainly associated to the inhibition of the body’s
mixtures. SD’s of GLC with various grades of PEG in 1:2 w/w ability to synthesise prostaglandins (1, 2). KP has poor solu-
ratio showed almost 100% release in phosphate buffer (pH bility in water and acidic conditions and has short half-life,
7.4) in 30 mins, as compared to 23% dissolution of plain GLC. low bioavailability3.
Results of DSC studies demonstrated that enhanced dissolu-
tion of GLC from SD might be due to a decrease in crystallin- Solid dispersions (SDs) of many poorly water soluble drugs
ity of GLC in the molten state while its formulation by fusion with hydrophilic carrier matrix have been formulated for im-
method. SD tablets of GLC and PEG 20,000 in 1:2 w/w ratio proving drug dissolution rate, which can be further improved
exhibited a better dissolution profile (54.21%) than the com- if such polymers have surface active properties. Moreover,
mercial brand (30.26%). SDs may improve the bioavailability of poorly soluble drugs
by increasing the drug dissolution rate4,5.
CONCLUSION
The use of GLC:PEG 20,000 solid dispersion gave satisfy- Poloxamers are polyoxyethylene-polypropylene block co-
ing and reproducible drug dissolution. Also, the SD based polymer nonionic surfactants that have been widely used

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

as wetting and solubilizing agents, and surface adsorption


excipients. They have been employed to enhance the solu-
bility, dissolution and bioavailability of many poorly water
soluble drugs. Poloxamer 188 (P188) is selected to prepare
SDs because of its low melting point (about 56-57oC), sur-
factant properties and oral safety4.

The aim of the this study was to prepare KP–P188 solid dis-
persions in different ratios as drug-carrier system using melt-
ing method and to compare the in-vitro release of formula-
tion with KP and commercial product.

MATERIALS and METHODS Figure 2: The release rates of KP, SDs and CP in pH 4.5
Materials: Ketoprofen was a gift from Eczacibasi (Eczacıbaşı
İlaç S.T.A.Ş. Turkey). P188 was a gift from Basf.

Preparation of Formulation: KP and P188 1:1 (A1), 1:2 (A2),


1:3 (A3), 1:4 (A4), 2:1 (A5), 3:1 (A6) and 4:1 (A7) weight ratios
(w/w) were prepared by melting method. KP and P188 were
mixed at 1:1, 1:2, 1:3, 1:4, 2:1, 3:1 and 4:1 weight ratio and ob-
tained mixture was melted on water bath with stirring. After
10 -15 minutes, the mixture was cooled at fridge for 24 h. The
solidified solid dispersions were sieved and stored in a vial
at room temperature for further use. The formulations were
used containing equivalent to 150 mg KP.

In Vitro Release Studies: In vitro release studies of KP, SDs and


Figure 3: The release rates of KP, SDs and CP in pH 6.8
commercial product were performed using United Stetes
CONCLUSION
Pharmacopeia (USP) model digital tablet dissolution test ap-
Dissolution rate of KP were enhanced by preparing KP-P188
paratus at the paddle rotation speed of 50 rpm in 900 ml pH
(1:3) SDs in reletively easy, simple, quick, inexpensive and re-
1.2±0.1 (simulated gastric fluid), pH 4.5±0.1, pH 6.8±0.1 and
producible manner using low temperature melting method. 63
pH 7.5±0.1 (simulated intestinal fluid) at 37oC. The studies
Immediate release of free KP from A3 obtained rapid absop-
were tested and estimated at different time intervals spec-
tion and improved bioavailability compared to KP and CP.
trophotometrically at 259, 260, 261 and 260 nm, respectively
The solubility of KP was slightly increased from A3 formula-
(n=3).
tion in the pH 1.2, 4.5, 6.8 and 7.5.
RESULTS and DISCUSSION
REFERENCES
The in vitro release of KP, SDs and commercial product (CP)
1. Reynolds, J.E.F. (Ed.) Martindale The Extra Pharmacopeia,
in pH 1.2, 4.5, 6.8 and 7.5 were shown in Figure 1, Figure 2,
Vol.29, The Pharmaceutical Press, London, 1989; pp.25.
Figure 3 and Figure 4, respectively.
2. Bonina, F.P., Puglia, C., Barbuzzi, T., Caprariis, P., Palagiano,
F., Rimoli, M.G., Saija, A. In Vitro and In Vivo Evaluation
As seen in the Figure 1, 2, 3 and 4 the proporties of dissolu-
of Polyoxyethylene Esters as Dermal Prodrugs of Keto-
tion profiles of KP, SDs and CP were significantly different. Re-
profen, Naproxen and Diclofenac, Eur J Pharm Sci. 2001;
lease profile of A3 in pH 7.5 were obtained as 100% release,
14:123-134.
an the end of the 15 min.
3. Roda, A., Sabatini, L., Mirasoli, M., Baraldini, M., Roda,
E. Bioavailability of a New Ketoprofen Formulation
for Once-Daily Oral administration, Int J Pharm. 2002;
241:165-172.
4. Newa, M., Bhandari, K. H., Li, D. X., Kwon, T. H., Kim, J. A.,
Yoo, B. K., Woo, J. S., Lyoo, W. S., Yong, C. S., Choi, H. G.
Preparation, Characterization and In Vivo Evaluation of
Ibuprofen Binary Solid Dispersions with Poloxamer 188,
Int J Pharm. 2007; 343: 228-237.
5. Dordunoo, S. K., Ford, J. L., Rubinstein, M. H. Preformula-
tion Studies on Solid Dispersions Containing Triamterene
or Temazepam in Polyethylene Glycols or Gelucire 44/14
for Liquid Filling of Hard Gelatin Capsules, Drug Dev Ind
Pharm. 1991;17(12):1685-17.
Figure 1: The release rates of KP, SDs and CP in pH 1.2

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

Figure 4: The release rates of KP, SDs and CP in pH 7.5


Figure 1: Gelling profile of 10% P1670/water dispersion
P003-Study Of Gel-Forming
Properties Of Sucrose Esters For Contact angle measurements
Thermosensitive Drug Delivery The contact angle (Θ) of a solid was determined by the ses-
sile drop technique, using the OCA 20 Optical Contact Angle
Systems Measuring System (Dataphysics, Filderstadt, Germany), and
the method of Wu. The liquids used for contact angle mea-
Angela Szuts 1, Maria Budai Szucs1, Istvan Eros1, Naoya surement were bidistilled water and diiodomethane. The
Otomo2, Piroska Szabo Revesz1 polarity percentage was calculated from the γp and γ values:
(γp/γ )*100.
University of Szeged, Department of Pharmaceutical
Technology, Hungary 1 Dissolution studies
Mitsubishi Kagaku Foods Corporation, Tokyo, Japan2 For the dissolution tests, PAR-SE physical mixtures were filled
into hard gelatine capsules. The capsules contained 50 mg of
INTRODUCTION PAR and 50 mg of SE. The release of the drug was studied by
Sucrose esters (SEs) are biodegradable, non-toxic, non-ionic using Pharmatest equipment (Hainburg, Germany) at a pad-
64 surfactants. The aims of the present work were to study the dle speed of 100 rpm. 900 ml artificial gastric juice with a pH
gelling behaviour of SEs and the effects of this property on of 1.2 (±0.05) at 37 °C (±0.5 °C) was used. The drug contents
drug release. of the samples were measured spectrophotometrically (λ =
244 nm) (Unicam UV/Vis spectrophotometer).
MATERIALS AND METHODS
Materials Drug release kinetics
Sucrose palmitate P1670 (HLB=16), and sucrose stearate In order to describe the kinetics of the drug release from
S970 (HLB=9) were kindly provided by Syntapharm GmbH the formulations, the zero-order, first-order, Hixson-Crowell
(Germany). The model drug paracetamol (PAR) was supplied cube root law, Higuchi model and Korsmeyer–Peppas equa-
by Hungaropharma Ltd. (Hungary). tions were used.

Rheological measurements RESULTS AND DISCUSSION


The rheological properties were studied with a Physica MCR101 Rheological measurements
rheometer (Anton Paar, Austria). The measuring system was of The concentration dependences of the gelling temperatures
plate and plate type (diameter 50 mm, gap 0.1 mm). of the SE dispersions can be seen in Figure 2.

Dynamic frequency sweep tests were carried out at 37 °C,


at 1.0 Pa in the limit of the linear viscoelastic region. Stor-
age modulus (G’) and loss modulus (G”) were determined for
frequencies between 0.1 and 100 Hz. The gelling character-
istics were measured at a constant frequency of 1.0 Hz at a
constant strain of 1.5% (this strain was within the linear vis-
coelastic range of the SE gels). The heating rate was 2 °C/min.
The gelling temperature of an SE was defined as the tem-
perature where the storage modulus was half way between
G’ for the SE dispersion and G’ for the SE gel (Figure 1).

Figure 2: Concentration dependence of gelling tempera-


tures of SE dispersions

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

According to the frequency sweep tests, both dispersions Table 2 shows the correlation coefficients (r²) of the formula-
display gel characteristics, and S970 dispersions have more tions.
elastic behaviour (higher G’ values) and better gel character-
istics (lower slopes of the curves) (Figure 3). Table 2: Correlation coefficients of the linearization of drug
release plots
Sample Zero- First- Higuchi Hixson- Korsmeyer-
order order Crowell Peppas
PAR-P1670 0.8760 0.9703 0.9740 0.9444 0.9816
PAR-S970 0.9497 0.9604 0.9953 0.9570 0.9944

CONCLUSION
The results revealed that the gelling of the SEs is temperature-
and concentration-dependent. The examined sucrose stear-
ate (S970) has a stronger gel structure than that of sucrose
palmitate (P1670) and this behaviour has a great effect on the
drug release. The analysis of the dissolution kinetic data in this
study revealed that the dissolution follows the Korsmeyer-
Peppas (PAR-P1670) or Higuchi (PAR-S970) equations.
Figure 3: Frequency dependence of 5% aqueous dispersions
of P1670 and S970
P004-Preparation Of Famotidine Orally
Contact angle measurements Disintegrating Tablets
The results of contact angle measurements, which provide
information on the surface free energies and polarities of the Senem Sevtap Ölmez1, İmran Vural1
samples, are presented in Table 1.
Department of Pharmaceutical Technology, Faculty of
Table 1: Contact angles, surface free energies and polarities Pharmacy, Hacettepe University, 06100 Ankara/ Türkiye1
of the materials
Materials Θwater[°] Θdiiodomethane[°] Υ[mNm-1] Polarity INTRODUCTION
[%] The aim of this study was to formulate famotidine orally disinte- 65
P1670 18.49±0.85 58.76±0.72 70.10 60.96 grating tablets (ODTs) with sufficient mechanical integrity, and
to prepare ODTs by direct compression which has simple and
S970 46.79±1.76 62.99±1.10 55.25 53.85
low-cost manufacturing processes using materials with proven
PAR 39.32±1.77 28.72±2.42 67.96 40.52 safety as excipients. ODTs are solid dosage forms containing me-
PAR-P1670 23.11±1.83 47.13±1.46 70.55 53.61 dicinal substances that disintegrate rapidly in the mouth and re-
PAR-S970 37.19±1.13 54.74±1.81 62.37 53.05 quiring no chewing or additional water to facilitate swallowing.
ODTs are easy to use and convenient for the patient who cannot
swallow, such as the elderly, stroke victims, bedridden patients,
Dissolution studies patients affected by renal failure or dysphagia and patients who
Due to the swelling properties of the SEs, they sustained the refuse to swallow such as pediatric, geriatric and psychiatric pa-
drug release (Figure 4). The stronger gel structure (Figure 3) tients1. ODTs have convenience of administration and accurate
and lower polarity (Table 1) of S970 resulted in a slower drug dosing as compared to liquid dosage forms as well.
release.
MATERIALS AND METHODS
Materials
Famotidine was kindly provided by Mustafa Nevzat İlaç Sanayi,
İstanbul, Türkiye. Sodium stearyl fumarate (Pruv®) was obtained
as gift sample from JRS Pharma, Germany. Avicel PH102 (MCC),
low hydroxypropyl cellulose-11 (LHPC) and aspartame were
kindly provided by Abdi İbrahim İlaç Sanayi, İstanbul, Türkiye.

Preparation OF ODTs
MCC and LHPC were selected as the basic excipients due to
their proven safety and excellent compactibility. LHPC was
also used as a disintegrant. Pruv® was used as lubricant and
aspartame was used as sweetener. ODTs were prepared ac-
Figure 4: Dissolution curves of PAR and PAR-SE samples cording to the following procedure. Firstly, MCC and L-HPC
Drug release kinetics at 4:1 or 9:1 ratios were mixed for 5 min. Famotidine was
mixed well with the mixture of MCC and L-HPC and then, as-

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

partame (1%) was added and mixed. Lastly, sodium stearyl Friability was observed between % 0.33-0.38 which were
fumarate (2%) was added and mixed for 5 min. To prepare below 1% indicating the sufficient mechanical integrity and
200 mg tablets, the above-mentioned mixtures were com- strength of the prepared tablets. According to EP, orodis-
pressed by Erweka AR 400 Korsch/Germany. persible tablets disintegrate within 3 min using conventional
disintegration apparatus. The prepared tablets were disinte-
EVALUATION OF TABLET CHARACTERISTICS grated in 14.55 ± 4.21 and 10.03 ± 2.13 s for MCC: LHPC (4:1)
Weight Variation: Twenty tablets were randomly selected from and MCC: LHPC (9:1), respectively. The tablets containing
each formulation and weighed using a Shimadzu digital bal- lower concentration of L-HPC showed rapid disintegration
ance. The mean ± standard deviation (SD) were recorded. time and high crushing tolerance. The high concentration of
L-HPC in tablets results decreasing in the degree of wetting,
Measurement of Diameter and Thickness: Ten tablets from so increasing disintegration time. For in vitro dissolution test,
each formulation were taken randomly and their diameter the prepared tablets were complied the USP requirement for
and thickness were measured using a micrometer. The mean famotidine tablets (dissolved more than 75 % of the labeled
± SD values were calculated. amount of famotidine in 30 min) (Table 1). The dissolution
data is shown in Figure 1.
Hardness: Tablet hardness, which is defined as the force required
to break a tablet by radial compression, was measured with a
tablet hardness tester (n=10) (Schleuniger 2E/Sweetzerland).

Measurement of Friability: Friability of the tablets was deter-


mined using a friabilator (Smiths/England) at 25 rpm/min for 4
min. Pre-weighed tablets were placed in a plastic chambered
friabilator then, the tablets were de-dusted, reweighed and
percentage weight loss (friability) was calculated (n=20).

In vitro disintegration time: In vitro disintegration time of the


ODTs was determined using USP disintegration test appara-
tus without disk(Manesty tablet disintegration test unit MK4)
for six tablets. The 1000 mL of distilled water at 37 ± 1 °C was Figure 1: Dissolution profiles of prepared famotidine ODTs
used as test fluid at the rate of 30 ± 2 cycles / min. (: MCC: LHPC (4:1), º: MCC: LHPC (9:1)) (mean±SD, n=6)
66 CONCLUSION
In vitro dissolution test: It was performed according to USP In conclusion, famotidine tablets with sufficient crushing tol-
XXX using the paddle method at 50 rpm in pH 4.5, 0.1 M erance and short disintegration time can be prepared using
phosphate buffer utilizing a dissolution system (Sotax CH Avicel PH102 and LHPC-11.
4123). Samples (5 mL) were collected at predetermined time
intervals (2, 5, 10, 15, 20 and 30 min) and replaced with equal REFERENCES
volume of fresh medium and filtered. The detection of famo- 1- Sastry SV, Nyshadham JR, Fix JA. Recent technological ad-
tidine was performed spectrophotometrically at 266 nm. vances in oral drug delivery - A review, Pharm Sci Technolo
Today. 2000; 3: 138-145.
RESULTS AND DISCUSSION
The results of tablet characteristics of prepared ODT formu-
lations containing MCC and L-HPC at 4:1 or 9:1 ratios were
given in Table 1. P005-Effect Of Curing And Tableting
Table 1: Results of the tablet characteristics On Thermomechanical And Release
Formulation Properties Of Eudragit-Based
Tablet
MCC: LHPC (4:1) MCC: LHPC (9:1) Diclofenac Sodium Pellets
Characteristics
Mean ± SD Mean ± SD
Mean mass (mg) 200.89 ± 0.002 200.97 ± 0.003 Mohammadreza Abbaspour1, Masoud Alimorad2, Iman
Beygi1
Diameter (cm) 1.220 ± 0 1.220 ± 0
Thickness (cm) 0.15 ± 0 0.16 ± 0 School of pharmacy,Ahwaz Jundishapur University of Medical
Hardness (kp) 8.50 ± 0.67 8.57 ± 0.38 Sciences, Ahwaz, Iran1
Friability (%) 0.33 0.38 SOHA pharmaceutical co., Tehran, Iran2
Disintegration 14.6 ± 4.21 10.0 ± 2.13
time (s) INTRODUCTION
One of the most popular oral drug delivery systems is the
% dissolved after 94.62 ± 4.40 96.67 ± 10.05 multiparticulates such as pellets which their benefits over
30 min. single unit dosage forms have been widely studied. Such
pellets can be either filled in hard gelatin capsules or com-
pacted into multiple unit tablets. One challenge in the pro-

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

duction of such tablets is maintaining the desired drug re-


lease after compaction, as the applic-ation of compaction
force can damage the pellets and alter the drug release. It
has been shown that curing of eudragit based ibupr-ofen
pellets leads to decrease in drug release rate and improves
the mechanical strength of pellets to ensure maintaining pel-
lets integ-rity and their drug release rate after compact-tion1.
The aim of this study was to invest-igate the effect of curing
and tableting on thermo-mechanical and release properties
of diclofenac eudragit-based pellets and to design multipar-
ticulate tablets which upon oral ingestion rapidly disinte-
grate into comp-rising pellets and preserve primary release.

MATERIALS AND METHODS


Materials: Diclofenac Na (Hakim Pharmac-euticals, Iran), Avi-
cel PH101 (Razak Pharm-aceuticals, Iran), Eudragit RL PO and
RS PO (Rohm Pharma , Germany), PVP K30 and cross-linked
povidone (PVP xl) (Fluka, Switzerland), PEG 4000 and Mg-
stearate (Merck, Germany).

METHODS
Preparation of pellets: 9 formulations containing different
levels of diclofenac (10, 20, and 30%), different amounts of
Eudragit RS:RL (1:1) (20, 40 and 60%), 10% Avicel and 3% PVP
K30 were manufactured and studied based on 32 full facto-
rial design using extrusion spheronization. The obtained pel-
lets were dried at 40˚C for 10h. The pellets were cured at 60˚C
for 24h and then were allowed to cool at room temperature. Figure 1: Influence of amount of diclofenac and Eudragit on
MDT of: a) uncured and b) cured pellets
Mechanical tests: The crushing strength and elastic modu-
lus of 30 uncured and cured pellets were determined using a In this study all pellets had brittle behavior under the me- 67
material testing machine (Hounsfield, UK). The speed of the chanical tests and the curing process had no effect on their
upper mobile platen fitted with a 1 kN load cell was set at 1 strength and mechanical properties (data were not shown).
mm/min. DSC of diclofenac and eudragit physical mixtures showed
that diclofenac had no effect on Tg of polymers (Figure 2),
Tableting: Tablets containing 60% pellet and 40% filler blend but the peak showing melting point of drug at 300°C were
(59.5% Avicel, 10% PVP xl, 30% PEG 4000, 0.5% Mg-stearate) disappear probably due to electrostatic interactions be-
were prepared by manual filling 1000 mg of above mixture tween drug and polymers3.
in a 12 mm die and compressed using a single punch tablet-
ing machine (Erweka, Germany) Cured pellets containing 10% drug and 60% Eudragit which
had the maximum MDT was chose to study the effect of
Dissolution Test: The tests were carried out on pellets or tab- tableting on pellet properties. Tablets containing 60% pel-
lets containing 48 mg of drug (n=6) in USP apparatus I, at lets and 40% filler blend with acceptable hardness (60±8 N)
100 rpm, in phosphate buffer (pH 7.4), at 37˚C. The samples and disintegration time (5min) have been produced.
were taken at determined intervals and assayed by spectro-
photometer at 274 nm and converted to mean dissolution In vitro release profiles for compacted and un-compacted
time (MDT)2. pellets showed that no apparent damage to the pellets has
occurred as a result of the compaction process (Figure 3).
DSC: DSC analysis was performed on Eudragit RS or RL, di- Intact pellets were rapidly released from single unit dosage
clofenac and their physical mixtures using a DSC (Mettler form upon contact with dissolution medium.
Toledo DSC 822e, Switzerland).

RESULTS
The results showed that increase in amount of drug in both
uncured and cured pellets led to decrease in MDT, whereas
increasing amount of eudragit in uncured pellets had no
considerable effect on drug release but in cured pellets, spe-
cially at lower amounts of drug, led to increase in MDT or
decrease in drug release rate (Figure 1).

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

P006-Formulation Of Sustained Release


Oral Dosage Forms Of Hydralazine
Hydrochloride For The Treatment Of
Hypertension And Cancer

Melike Üner1, Benek Çelebi1

Istanbul University, Faculty of Pharmacy, Department of


Pharmaceutical Technology, 34116 Beyazit, Istanbul Turkey1

INTRODUCTION
Hydralazine hydrochloride (HH) has been used as a hypoten-
sive drug for years since it lowers the blood pressure by af-
fecting plain muscles of vessels1. In the last years, an increas-
, ing attention has been paid on this drug since it has the
Figure 2: DSC of physical mixture of diclofenac and eudragit RL demethylating effect on various suppressor genes. There-
fore, it can be used for various types of cancer in order to
support chemotherapy2.

Plasma half-life of HH is 3-4 hours. It is administered up to


200 mg daily as 3-4 divided doses by oral route1,4. The aim
of this study is to design oral dosage forms of HH release in
order to maintain drug in the systemic circulation for a lon-
ger time period. Therefore, its side effects are minimized by
lower dose administration, drug bioavailability is increased
and patient compliance is improved. For this aim, sutained
release (SR) tablets of HH were prepared by using various
polymers. Compression characterization of SR tablets and in
Figure 3: Dissolution profile for cured pellets and compacted vitro HH release were studied. This study is continued. It is
68 pellets already detailed according to the data obtained.

CONCLUSION MATERIALS AND METHODS


Curing has no effect on pellets hardness but decrease drug Materials
release rate. Multiparticulate tablets can be prepared with a Hydralazine hydrochloride (Novartis, Turkey), Methocel® K15
release profile similar to original pellets Premium EP (Colorcon, England), Carbopol® 971 PNF (No-
veon Inc., U.S.A.), Compritol® 888 ATO (Gattefossé, France),
REFERENCES cetyl alcohol (Cognis, Turkey), Avicel® PH-101 (Select-chemie
1. Abbaspour MR. et al., Design and study of ibuprofen dis- AG, Switzerland), magnesium stearate (Prever, Italy), Aerosil®
integrating sustained-release tablets comprising coated 200 (Degussa, Germany).
pellets, Eur. J. Pharm. Biopharm. 2008; 68: 747-759.
2. Costa FA. et al., Comparison of dissolution profiles of Preparation of SR Tablets
ibuprofen pellets, J. Control. Release 2003; 89: 199-212. Tablets were prepared by direct compression method (Table).
3. Sipos P. et al., An assessment of the interactions be- For this aim, a hydrolic press equipped with 10 mm diameter
tween diclofenac Na and ammonio methacrylate copo- flat-punch (Yeniyurt, Turkey) was used under 1500 psi force.
lymer using thermal analysis and Raman spectroscopy,
J. Pharm. Biomed. Anal. 2008; 46: 288-294. Physical characterization of tablets was tested in order to
determine weight variation, tablet diameter and thickness,
disintegration time, friability % and hardness5.

In Vitro Dissolution Studies


Release of HH from SR tablets was studied according to the USP
32 Method II (paddle) at 37°C ± 0.5 in 900 mL distilled water as
a dissolution medium at 50 rpm rotation speed. At predeter-
mined time intervals, 0.8 ml samples were taken, diluted to 10
ml and filtered through S&S5893 type blue ribbon filter paper.
The solution was assayed by UV spectrometry at 260 nm (Shi-
madzu UV-1700 Spectro- photometer, Japan) in water.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

Table 1: Composition (%) of tablets REFERENCES


Formula 1. Martindale. 31st Edition. Royal Pharmaceutical Society,
Tablet ingredients London, 1996.
A B C D
2. Song Y, Zhang C. Hydralazine inhibits human cervical
HH 28.57 28.57 28.57 28.57
cancer cell growth in vitro in association with APC dem-
Avicel 28.43 28.43 28.43 28.43 ethylation and re-expression. Cancer Chemo.Pharmacol.
Methocel 40 - - - 2009; 63: 605-613.
Carbopol - 40 - - 3. Estrada Flores L, Duenas Gonzalez A, Borbolla Garcia A,
Serna Martinez R, Rivera Hernandez A, Mohar Betan-
Compritol - - 40 -
court A. Pharmaceutical composition for the sustained
Cetyl alcohol - - - 40 release hydralazine and use thereof as a asupport for
Magnesium sterate 2 2 2 2 cancer treatment. 2007; EP 1 803 447 A1.
Aerosil 1 1 1 1 4. Kirsten R, Nelson K, Kirsten D, Heintz B. Clinical pharma-
cokinetics of vasodilators, Part II. Clin.Pharmacokinet.
RESULTS AND DISCUSSION 1998; 35: 9-36.
SR tablets were found to meet compendial requirements 5. Üner M, Altınkurt T. Evaluation of honey locust (Gleditsia
related to compression quality (weight variation, diameter, triacanthos Linn.) gum as sustaining material in tablet
thickness, friability % and hardness). dosage forms. Il Farmaco. 2004; 59: 567-573.

The highest release was observed with Formula D containing


cetyl alcohol (Figure). This formula released 100 % of drug P007-IMMOBILIZATION OF PANCREATIN
after 7 hours. Whereas, Formulas A, B and C released 79.05 USING ALGINATE BEADS
%, 61.24 % and 89.32 % of drugs from tablets after 8 hours,
respectively. When a system composed of hydrophylic poly- Nefise Özlen Şahin1, S. Ektiren2, S. Soylu3, A. Yüksel2
mers gets in contact with aqueous medium, medium passes
into the system and polymer at surface turns into a gel at Mersin University, Faculty of Pharmacy, 1Department
first. When medium transfer into the system continues, gel of Pharmaceutical Biotechnology, 2Department of
layer is improved to be thicker and the system swells. There- Pharmaceutical Technology, Yenisehir Campus, Mersin 33169,
fore, drug release occurs by passive diffusion. The system was Turkey.
waxy matrix because of containing cetyl alcohol in Formula 3
Mustafa Kemal University, Research Center, Hatay, Turkey.
D. The release was highest since cetyl alcohol concentration 69
was low in the formulation. INTRODUCTION
Impeded flow of pancreatic juice due to mechanical obstruc-
Drug release from all formulations were kinetically evaluated tion of the pancreatic duct in patients with cancer of the
and they were found to fit to Higuchi kinetic model. pancreatic head region results in exocrine pancreatic insuf-
ficiency with steatorrhoea and creatorrhoea. This may cause
profound weight loss that often occurs in these patients.
Weight loss in patients with pancreatic cancer may be cured
by pancreatic enzyme replacement therapy which will lead
to significant improvement in fat and protein absorption.
Various dosage forms of pancreatin including enteric-coated
tablets, pellets and microspheres are in the market.

Among the various drug delivery systems investigated to


achieve efficient and selective delivery of therapeutic agents
to the site of action, beads have gained more attention. Re-
cently, beads were prepared with natural polymers such as
alginate, chitosan, pectin become the focus of research in
this field. With all these in mind, we set our goal to prepare
Figure 1: Release profiles of HH from Formulas A, B, C and D pancreatin bearing beads for treatment of weight loss in the
in water. patients with pancreatic cancer. Alginate was used as bead
forming polymer to explore the temporary protection of
CONCLUSION pancreatin against acidic and enzymatic degradation during
Methocel, Carbopol and Compritol were determined to be gastric passage. In vitro dissolution results were compared
suitable polymers for preparation of SR tablets of HH in order to that of drug itself to ensure such protective effect.
to reduce dose intensity in the treatment of hipertension.
Additionally, they can be administrated as a supportive drug MATERIALS AND METHODS
in the cancer treatment since demethylating effect of HH will Materials:Both sodium alginate (medium viscosity) and pan-
also occure in a long time period. creatin were bought from Sigma Chem. Co, MO, USA. All
other reagents were analytical grade.

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

METHODS RESULTS & DISCUSSIONS


Preparation of gel beads: Alginate gel beads were prepared Using the method described above and electronic beads
by a slightly modified method of Yotsuyanagi et al. Pancre- generator, spherical and smooth surfaced beads with high
atin was added to 6 ml of 1.5-3.0 % (w/v) sodium alginate so- drug content (59.7 % entrapment efficiency) could be pro-
lution and dissolved completely. This solution was dropped duced. SEM photographs revealed that the surface of the
into an 85 ml 1-2% Barium chloride solution with mild agi- beads is porous. The bead size varied between 0.86-1.15 mm.
tation within a period of 17 mins. The beads were formed The drug loading capacity of the beads prepared with 1.5 %
instantaneously. Subsequently, they were transferred into alginate solution was %59.7. Drug loading capacity showed
falcon tubes and centrifuged at 900 rpm for 2 min. The su- an increasing trend as the polymer (3.0%) and initial drug
pernatant was removed by means of aspiration via vacuum. amount (%1) increased upto 76.4%.
Centrifugation process was repeated several times until no
barium chloride residue left on beads. Then, beads were In vitro drug release profiles of the beads are given in Fig-
dried at 50ºC for 3 h using an incubator. ure 1. Release was slower in a controlled manner over 6 h
for both formulations. The data indicated that in vitro drug
Determination of the size of the beads: The size distribution release was affected by alginate, drug and barium chloride
of the beads was determined by using sieve analysis (Reich, concentrations. An increase in alginate concentration from
Germany) (n =3). 1.5% to 3.00 % reduced the release of pancreatin from the
beads. Figure 2 indicates the effect of drug concentration on
Assessment of drug loading capacity: 100 mg of beads were the release profile. The release of the drug showed delayed
digested in 100 ml of pH 7.4 phosphate buffer. The solution characteristics as the drug concentration was increased.
was filtered and the absorbance of pancreatin was measured Lowering barium chloride concentration from 2% to 1% de-
spectrophotometrically at 660 nm. creased the drug release (Figure 3). Comparison of the re-
lease profiles of encapsulated drug and that of plain drug
Erosion studies: A certain amount of beads was placed in indicated that bead formation provides controlled release of
a 10 ml of screw-capped bottle containing 5 ml phosphate pancreatin.
buffer (pH 7.4) and agitated at 37ºC in a shaker bath. At vari-
ous time intervals, beads were removed, washed and dried.
Weight lose was determined gravimetrically (n=8).

Microscopic investigations: Shape and surface morphology


70 of the beads were investigated by both polarized and scan-
ning electron microscopes.

In vitro dissolution studies: Pancreatin bearing beads were


suspended in phosphate buffer (pH 7.4) contained in wide-
mouthed bottles, and stirred at 100 rpm in a shaker bath
at 37ºC. Samples were withdrawn at certain time intervals
(15, 30, 45, 60, 90, 120, 180, 240, 300, 360 min) and analyzed
spectrophotometrically at 660 nm.

Determination of swelling rate: Adequate amount of beads Figure 1: Effect of alginate concentration on the release pro-
were weighed and suspended in distilled water. 3 hours later, file of pancreatin (n=3).
they were filtered an the diameters were measured under the
dissection microscope. Diameters of the beads before and after
swelling process were compared for each bead. The differences
were calculated and converted into percentage swelling rate.

Table 1: Variables used for preparation of different alginate


bead formulations.
Variables Values Codes
Alginate concentration (% w/w) 1.5 F1A15*

3.0 F1A3
Drug concentration (% w/w) 0.5 F2P05*

1.0 F2P1
Barium chloride concentration (% w/w) 1.0 F3B1* Figure 2: Effect of drug concentration on the release profile
of pancreatin (n=3).
2.0 F3B2
*F1A15, F2P05 and F3B1 are the same formulations.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

P008-Evaluation Of Physical Stability


Of Piroxicam Using Gelucire 44/14,
Labrasol And Polyethylene Glycol
4000

Ayşegül Karataş1, Şerife Bekmezci2

Department of Pharmaceutical Technology, Faculty of


Pharmacy,Ankara University, 06100-Tandoğan, Ankara, Turkey1
Department of Pharmaceutical Technology, Faculty of
Pharmacy, Ankara University, 06100-Tandoğan, Ankara, Turkey2

Figure 3: Effect of Barium chloride concentration on the re-


INTRODUCTION
lease profile of pancreatin (n=3)
Piroxicam is non-steroidal anti-inflammatory drug, clasified
in the Biopharmaceutic Drug Clasification System (BCS) as a
Class II drug with low solubility and high permeability1. In our
Erosion profiles of alginate beads are plotted against time in
previous study2, semi-solid dispersion containing the mix-
Figure 4. It indicates that a considerable erosion occurs dur-
ture of 20 % of Gelucire 44/14 and 80 % of labrasol brought
ing the 180 min.
about the dissolution not less than 85 % of piroxicam within
30 min in each of the dissolution media (namely, acidic me-
dium, pH 1.2 SGF; pH 4.5 buffer; pH 6.8 buffer; and water). It
was probable that this formulation filled into the hard gelatin
capsules was thermosoftening at room temperature might
have tendency to turn into liquid form during storage. In this
study, Polyethylene glycol 4000 (PEG 4000) was added to Ge-
lucire and labrasol combination to solidify the formulation.
The modified semi-solid formulation containing gelucire
44/14, Labrasol and PEG 4000 were prepared in following ra-
tios: 20 %, 78 % and 2 %, respectively. Ratio of drug-excipient
was 1:20. Formulation was studied for in vitro dissolution be- 71
haviour and 12-month physical stability test at temperature
Figure 4: Erosion profiles of beads. of 25oC and relative humidity (RH) of 60%. Characterization
of the drug/carrier combination was conducted using Fou-
Swelling rate of pancreatin bearing alginate beads were rier –transformation- IR spectroscopy (FT-IR) and differential
found as 35.2%. scanning calorimetry (DSC).

CONCLUSIONS MATERIALS AND METHODS


Based on the data obtained in this study, we can suggest Materials: Piroxicam was obtained from dipharma (Turkey),
that alginate beads can serve as a convenient carrier for the Gelucire 44/14 and labrasol were supplied by Gattefosse
controlled release of pancreatin to be used in the treatment (Saint-Priest cedex, France). PEG 4000 was purchased from
of weight loss associated with pancreatic cancer. Merck (USA). Hard gelarine capsules were provided by Shion-
ogi (Qualicaps SA, Spain).
REFERENCES
1. Edelman ER, Mathiowitz E, Langer R, Klagsbrun M. Con- Preparation of semi-solid dispersion: Necessary amounts of
trolled and modulated release of basic fibroblast growth excipients heated to abouth 5 oC above the melting point
factor, Biomaterials 1991; 12: 619-625. of Gelucire 44/14 on the water bath. The calculated amount
2. Hermes RS, Narayani R. Polymeric alginate films and al- of drug was added to the molten vehicle with continuous
ginate beads for the controlled delivery of macromole- stirring. Then, mixture was poured into a plastic injector and
cules, Trends Biomater. Artif. Organs. 2002; 15(2), 54-56. volumetrically filled into hard gelatin capsules at the tem-
3. Arnesjö B, Ihse I, Odeberg H, Stahl E. Deficiency of lipoly- perature close to the solidification point of the material.
tic pancreatic enzymes in an adult man. Digestion, 1974;
11: 147-151. Dissolution testing: Dissolution studies were conducted us-
4. Bhopatkar D, Anal AK, Stevens WF. Ionotropic alginate ing USP Apparatus 1 (rotating basket method) (Sotax AT 7
beads for controlled intestinal protein delivery: effect of Smart, Swithzerland) with three replicates, according to the
chitosan and barium counter-ions on entrapment and USP monograph of the drug3. In all experiments, 5 mL of dis-
release. J Microencapsul. 2005; 22(1): 91-100. solution sample was withdrawn at 5, 10, 20, 30, 45, 60 min
and replaced with an equal volume of the fresh medium.
Samples were assayed by UV spectrophotometry.

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

FT-IR Spectroscopy: Drug–carrier interactions during storage


conditions were determined based on IR spectra measured
with a FT-IR spectroscope (Jasco, FT/IR 420) by the KBr meth-
od in the 4000-400 cm-1 region.

DSC: DSC analysis was carried out on a differential scanning


calorimeter (DSC 60 Shimatzu). The themograms were ob-
tained by heating the samples (5 mg) at a rate of 5 oC/min
from 25 oC to 250 oC under nitrogen purge.

Storage stability studies: Formulation was stored in closed glass Figure 1c


vials for a period of 12 months at 25°C ± 2°C/60% RH ± 5% RH. Dis-
solution testing was conducted at 3-6- and 12-month intervals to
assess changes in drug release characteristics. The dissolution pro-
cedure used was the same as described above. Changes in physical
state of the samples were also performed on the drug, excipients
and at 0-3-6- and 12-month intervals of formulation by FT-IR and
DSC analyses.

RESULTS AND DISCUSSION


Dissolution studies: It was show that the addition of PEG
4000 to the formulation afforded a solidification effect on
the formulation. In addition, the presence of the PEG poly- Figure 1d
mer in combination with Gelucire and labrasol showed simi- Figure 1.Dissolution profiles of formulation monitored initial
lar results in dissolution when compared to the original for- sample and samples stored for 3,6 and 12-month at 25°C ±
mulation. Namely, this formulation (initial) also provided at 2°C/60% RH ± 5% RH at different buffers (a) pH 1.2 (b) pH 4.5
least 85 % piroxicam dissolution within 30 min in each of the (c) pH 6.8 (d)water
media (Fig 1).
³²°°°°µµ°°° Storage stability studies
When dissolution of the stored samples (3-6- and 12-month
72 intervals) were compared with that of initial formulation,
samples at 6- and 12- month intervals showed a significant
decrease in dissolution (Fig 1).

FT-IR Spectroscopy
The presence of an interaction in formulation during storage
period could not be confirmed by FT-IR spectroscopy (Fig 2).

Figure 1a

Figure 1b Figure 2: FT-IR spectrograms

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

DSC possibilities in this field of pharmaceutical technology. Co-


The melting peak of formulation (Fig 3 b) was observed at micronization involving the use of spray-drying as a one-
around 38 oC showing amourphous state of the drug in step and non-conventional processing procedure presents
the dispersion. Melting peaks of stored samples were ob- new possibilities for the development of different carrier
served at 59.57 oC (at 6-month) and 113.1 and 120.67 oC (at systems and for investigations of how the physicochemical
12-month) (Fig 3 d,g). properties of the raw carrier can be modified. Moreover, the
addition of adequate stabilizers may prevent the agglomera-
tion of the resulting microparticles of the carrier. This study
demonstrates the effects of a sucrose ester (SE) on the habit
and polymorph modification of mannitol as compared with
Tween 80.

MATERIALS AND METHODS


Materials
Sucrose myristate M1695 (HLB=16) (SE) was kindly provided
by Syntapharm GmbH (Germany). β-D-Mannitol (M) and
Figure 3: DSC thermograms Tween 80 (polysorbate 80) (HLB=15) (T) were from Hungaro
Pharma Rt.
CONCLUSION
In terms of storage stability, formulation exhibited short- Preparation of co-micronized carrier systems
term stability of at least 3 months but beyond that seemed In the sample preparation, SE or T was chosen as surfactant
to decrease in dissolution at all pH’s to modify and stabilize the M crystals.

DSC results indicated the present of a chemical interaction The micronized carrier systems were prepared (Table 1) from
between the drug and excipients during storage period that 1 g of M and three different concentrations of SE or T and
results in a decrease in dissolution rates. ad 20 g of water, using an Ultraturrax (T-25 IKA-WERKE Ger-
many) and for spray-drying (SD) a Büchi Mini Dryer B-191
REFERENCES (Switzerland) was applied. A spray-dried mannitol (M-SD)
1. Amidon GL, Lennernas H, Shah VP, Crison JR. A theoreti- was also prepared, as a control sample without additives.
cal basis for a biopharmaceutics drug clasification: The
correlation of in vitro drug product dissolution and in Investigations 73
vivo bioavailability. Pharm Res. 1995; 12:423-420. The particle size was determined with a LEICA Q500MC Im-
2. Karataş A,Yüksel N, Baykara T. Improved solubility and age Processing and Analysis System (LEICA Cambridge Ltd.,
dissolution rate of piroxicam using Gelucire 44/14 and UK). The OCA Contact Angle System (Dataphysics OCA 20,
Labrasol. Il Farmaco. 2005; 60:777-782. Dataphysics Inc., GmbH, Germany) was used for studies of
3. US Pharmacopoeia 24, US pharmacopeial Convention the wettability. The morphology of the particles was exam-
Rockville, MD, 2000, pp.1342 ined with a scanning electron microscope (SEM) (Hitachi
S4700, Hitachi Scientific Ltd., Japan). The DSC measurements
were achieved with a Mettler Toledo DSC 821e thermal anal-
ysis system (Mettler Inc., Schwerzenbach, Switzerland). The
P009-Effects Of A Sucrose Ester On crystallinity index for mannitol (CIM) was calculated from the
Co-Micronized Mannitol-Based Drug heats of fusion. The physical state of M was evaluated by X-
Formulation ray powder diffraction (XRPD), using an X-ray Diffractometer
Miniflex II (Rigaku Co. Tokyo, Japan).
Rita Ambrus1, Anita Pomazi1, Peter Sipos1, Naoya Otomo2,
Piroska Szabo Revesz1 RESULTS AND DISCUSSION
The SEM pictures (Figure 1) and Table 2 demonstrate the dif-
University of Szeged Department of Pharmaceutical ferences in size and morphology of crystals produced.
Technology1
Market Development Department, Mitsubishi Kagaku Foods M has an irregular shape with a rough surface. After spray-
Corporation2 drying, we got significantly smaller particles were obtained
in both cases, but the resulting crystals were a spherical only
INTRODUCTION when SE was used, which is suitable for the 1-5 μm range of
The application of carrier-based drug formulation is strongly DPI.
dependent on the preparation factors and the physicochem-
ical properties of the carrier systems. Mannitol is a carrier The wettability study (Table 2) indicated that significantly
that is frequently used in the field of pharmaceutical appli- lower contact angles were measured with lower concentra-
cations, especially in dry powder inhalation (DPI) systems. In tions of additives. When T was applied, the polarity values
this case, the surfactant Tween 80 is generally used, as a sta- were nearly the same as for M. With SE however, especially at
bilizer. The sucrose esters, as wetting and solubilizing agents, lower concentration, the polarity increased.
and liberation and absorption-modifying agents, offer new

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

Table 3. lists the changes in the melting point and CIM of M. It Table 3. DSC analysis of products
may be seen that little change occured in the melting point. Samples Melting Point [°] CIM(%)
After spray-drying, the crystallinity of the products was near-
ly the same as that of M-SD, but at the highest concentration M Pure 166.62 100
of SE the recrystallization was blocked. M-SD 166.01 94.31
MP-SE1 166.01 94.45
The three crystal forms of M can easily be distinguished via MP-SE2 165.95 95.97
their XRPD patterns, as shown in Figure 2. The lower concen- MP-SE3 165.46 54.57
tration of M resulted in a turn from β to α, and the highest to MP-T1 166.58 91.15
δ form. But T presented only the α form of the carrier. MP-T2 165.58 85.32
MP-T3 166.30 96.46
CONCLUSION
Our study has demonstrated that the crystal habit and struc-
ture of mannitol can be modified by the use of additives. We
suggest a sucrose ester for co-micronized carrier-based drug
formulation, and especially DPI formulation.
/e-mail: arita@pharm.u-szeged.hu/

Table 1: Concentrations of the additives in micronized prod-


ucts (MPs)
SE-M1695 Tween 80
0.05 g (0.25%) MP-SE1 MP-T1
0.1 g (0.5%) MP-SE2 MP-T2
0.1 g (2.5%) MP-SE3 MP-T3
Figure 2: XRPD patterns of MP-SE1-3

P010-TESTING OF THE GASTRO-RESISTANCE


74 PROPERTIES OF OMEPRAZOLE CAPSULES AT
DIFFERENT PH VALUES

E. Vranić1, A. Uzunović2, O. Rahić1, A. Atilla Hıncal3

1
Department of Pharmaceutical Technology Faculty of
Pharmacy, University of Sarajevo, Čekaluša 90, 71000
Sarajevo, Bosnia and Herzegovina
2
Institute for Quality Control of Medicines, M. Tita 9, 71 000
Sarajevo, Bosnia and Herzegovina
3
Department of Pharmaceutical Technology, Faculty of
Figure 1: SEM pictures of pure carrier (A), product MP-T3 (B) Pharmacy, Hacettepe University, 06100 Sıhhiye-Ankara, Turkey
and product MP-SE3 (C)
INTRODUCTION
Table 2. Particle size and wettability of the samples Omeprazol, a substitued benzimidazole compound and
Main prototype antisecretory agent, is a potent non-reversible
Θwater Polarity inhibitor of the gastric proton pump H+/K+-ATPase which is
Materials particle Θdiiodomethane [°]
[°] (%) reversible for gastric acid secretion. It is weak base, so it is
size [μ]°
acid labile???, and could be rapidly degraded unless it may
M Pure 86.74 21.34 17.55 43.72
be protected against acid conditions1. Moreover, it decom-
M-SD 1.61 20.13 24.87 45.71
posing rapidly at pH< 5.0 and is sensitive to heat, moisture,
MP-SE1 2.33 8.40 62.15 64.35 organic solvents and to some degree, light. The degradation
MP-SE2 2.40 13.20 65.55 65.87 of omeprazole manifests itself in a loss of drug content and
MP-SE3 3.81 36.95 64.10 58.90 increasing amounts of degradation products2.
MP-T1 6.90 9.20 9.85 44.64
MP-T2 2.30 14.80 18.73 45.27 Currently the USP pharmacopoeial requirements and test
MP-T3 not measurable method for gastro-resistant capsules are that not more than
10% of the active ingredient is released within two hours in
0.1 mol/dm3 hydrochloric acid3.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

In this study, we have evaluated differences in the dissolu- Table 1.1: Fraction of dissolved omeprazole at pH value 1.1
tion characteristics of four omeprazole products (purchased pH = 1.1
from market) at different pH values.Our primary purpose in
F1 F2 F3 F4
this study was to determine amount of the active ingredient
released under dissolution test conditions at pH 4.5. 0.00 0.22 0.96 0.31

% dissolved
0.08 0.00 1.12 0.31
MATERIALS AND METHODS 0.12 0.26 2.11 0.34
Materials
The following reagents were used and provided by different 0.67 0.49 0.91 0.38
producers: omeprazole working standard (Ulkar Kimya), ac- 0.32 0.32 0.28 0.35
etonitrile-HPLC grade (Sigma Aldrich), phosphoric acid 85% 0.45 0.22 1.05 0.39
(Carlo Erba), sodium hydroxyde, hydrochloric acid (Merck),
X 0.27 0.25 1.07 0.35
sodium phosphate, disodium hydrogen-phosphate, potas-
sium hydrogen phosphate (Fluka). S.D. 0.25547 0.15930 0.590098 0.03386
R.S.D. 93.47 63.30 55.15 9.77
Dissolution Test Conditions
The dissolution tests of omeprazole gastro-resistant cap- Table 1.2: Fraction of dissolved omeprazole at pH value 6.8
sules (n=6) were performed using Apparatus 2 (n=6), Van
pH = 6.8
Kel VK 7010 dissolution tester, at stirring speed of 100 rpm.
The dissolution test was carried under the following condi- F1 F2 F3 F4
tions: -first stage: dissolution testing was performed for two 98.67 95.89 85.96 92.88

% dissolved
hours in 1000 ml of 0.1 mol/dm3 hydrochloric acid at pH=1.1; 101.78 90.74 81.77 93.74
-second stage: the same capsules were carried to dissolution
vessel, and 750 ml of phosphate buffer solution, pH=4.5, was 100.89 91.89 80.34 98.21
added. Samples in the amount of 10 ml were withdrawn after 95.43 89.06 86.98 95.44
45 minutes. Dissolution samples were collected for analysis 95.87 90.45 82.19 97.34
and replaced with an equal volume of fresh dissolution me-
92.57 94.39 8.04 98.51
dium to maintain a constant total volume; -final stage: within
the next five minutes 250 ml of phosphate buffer solution, X 3.53324 2.58691 2.62660 2.37205
pH=6.8, was added. The rotation speed was maintained at S.D. 97.54 92.07 83.21 96.02
100 rpm and continued to operate for next 45 minutes. R.S.D. 3.62 2.81 3.16 2.47 75
-the apparatus was maintained at 37oC throughout the
The results obtained in this study indicated problems in dis-
whole experiment
solution rate of F4 omeprazole formulation at pH 4.5 and
-all dissolution tests were performed in triplicate.
6.8.
HPLC Method
Additionally, excessive release at pH 4.5 is in contradiction
The following experiment examined the amount of the ac-
with insufficient dissolution rate in phosphate buffer at pH
tive ingredient released was carried out by high-performance
6.8. So, the results represent a sufficient reason for further
liquid chromatography (HPLC) method for omeprazole cap-
testing of the preparation in order to determine the reasons
sules in accordance with British Pharmacopoeia 2009 (4).
that would explain the results.
The system consisted of a pump, injection valve, autosam-
pler and variable wavelength detector (Shimadzu, Kyoto, Ja-
Table 2.1: Fraction of dissolved omeprazole at pH value 4.5
pan). The mobile phase consisted of phosphate buffer pH
7.6: water: acetonitrile = 250:350:400 v/v/v). The flow rate pH = 4.5
was 1.3 ml/min, the injection volume 50 µl, the column tem- F1 F2 F3 F4
perature 30oC and the detection wavelengths 302 nm. Zor- 0.00 0.08 1.06 27.73
% dissolved

bax Stable Bond C-18 column (250mm×4.6mm×5μm) was


used throughout the experiments. Optimization of original 0.04 0.05 1.4 28.02
isocratic HPLC method was performed using excel program 0.09 0.08 1.64 25.97
called ‘‘HPLC calculator v2.1’’5. 0.06 0.09 2.49 27.42
0.10 0.07 1.74 28.57
RESULTS AND DISCUSSION
The results of dissolution studies for Formulation (F) 1, 2, 0.00 0.06 1.14 27.60
3 and 4 are summarised in Table 1.1, 1.2, 2.1 and 2.2 which X 0.05 0.07 1.58 27.55
show fraction of the dissolved drug at different pH values, S.D. 0.04309 0.01472 0.52032 0.87287
1.1, 4.5 and 6.8., respectively.
R.S.D. 89.00 21.00 33.00 3.00

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

Table 2.2: Fraction of dissolved omeprazole at pH value 6.8 INTRODUCTION


pH = 6.8 Verapamil hydrochloride (VH) is a calcium-channel blocker
and is classified as a class IV anti-arrhythmic agent. It is used
F1 F2 F3 F4
in the control of supra ventricular tachyarrhythmia, and in
94.19 93.75 81.30 37.08 the management of classical and variant angina pectoris. It is
% dissolved

97.58 95.01 84.32 34.43 also used in the treatment of hypertension. These drugs are
91.87 97.42 80.63 32.49 available in sustained release and immediate release prepa-
rations. Overdose is associated with a high incidence of mor-
92.61 95.50 82.09 33.48 bidity and mortality. Calcium channel blockers accounted
96.62 98.01 81.82 33.81 for 16% of all cardiovascular drug exposures reported to the
98.29 98.50 85.12 37.39 American Association of Poison Control Centers, but they ac-
counted for 38% to 48% of deaths caused by cardiovascular
X 95.19 96.37 82.55 34.78
drug overdosage1.
S.D. 2.68499 1.88678 1.77376 2.00533
R.S.D. 3.00 2.00 2.00 6.00 Drug–excipient interactions have the potential to affect
many physiological processes and factors, such as: the pH of
CONCLUSION the microenvironment, protein binding, GI transit time, sta-
The results obtained in this study indicate that the existing bility in the GI tract, effects on gut flora, and so on. The po-
USP pharmacopeia request for gastro-resistance testing (2 tential outcome of all of these interactions might be to alter
hours in 0.1 mol/dm3 HCl (≤ 10% of declared content) is not the bioavailability of the drug2,3.
sufficient and that the additional criteria should be estab-
lished (such as gastro-resistance testing in buffer pH 4.5). Our primary purpose in this study was to investigate degree
of permeation of three different tablet formulations of VH
REFERENCES (purchased from the market) under various test conditions.
1. Espinosa Bosch M., Ruiz Sánchez A.J., Sánchez Rojas F.,
Bosch Ojeda C. Analytical methodologies for the deter- MATERIALS AND METHODS
mination of omeprazole: An overview. J. Pharm. Biomed. The used reagents were of analytical grade, unless otherwise
Anal. 2007; 44: 831–844 stated. Verapamil hydrochloride working standard was pro-
2. Murakami F. S., Lang K.L., Mendes C., Cruz A.P., Marco vided by Merck (Darmstadt, Germany). Phosphatidylcholine,
A.S., Filho C., Silva M.A.S. Physico-chemical solid-state cholesterol, chloroform, methanol were provided by Sigma
76 characterization of omeprazole sodium: Thermal, spec- Aldrich (Buchs, Switzerland).
troscopic and crystallinity studies. J. Pharm. Biomed.
Anal. 2009; 49: 72-80 Assessment of permeation of VH was performed using appro-
3. U.S. Pharmacopeia, 31st rev.; U. S. Pharmacopeial Con- priate in house prepared in vitro passive absorption model.
vention, Rockville, MD, 2007 In this model, four HPLC vials (acceptor compartment) were
4. British Pharmacopoea 2009, British Pharmacopoeia adjusted on paddle of dissolution tester and positioned ver-
Commission, Stationery Office, London, UK tically with our artificial membrane downside oriented into
5. Guillarme D., Nguyen D.T.T., Rudaz S., Veuthey J.L. Met- dissolution vessel (donor compartment).
hod transfer for fast liquid chromatography in pharma-
ceutical analysis.: Application to short columns packed Polytetrafluoroethylene (PTFE) filters, pore size 0.45μm (Sar-
wit small particle. Part I: Isocratic separation, Eur. J. torious GmbH, Goettingen, Germany), were impregnated
Pharm. Sci. 2007; 66: 475–482 with mixture of phosphatidylcholine and cholesterol (22:78
w/w). Chloroform/ methanol solution (70/30 v/ v) was used
as a solvent to prepare impregnation solution. Impregnation
procedure was completed after PTFE filters were submerged
p011-ASSESSMENT OF PERMEATION OF into phosphatidylcholine/ cholesterol solution during 30
THREE DIFFERENT TABLET FORMULATIONS minutes, on Sartorius Certomat MO II shaker (Sartorious
OF VERAPAMIL HYDROCHLORIDE BY IN VITRO GmbH, Goettingen, Germany), rotation speed 200 rpm. Un-
coated PTFE filters were used as blank during testing proce-
PASSIVE ABSORPTION MODEL
dure.
A. Uzunović1, E. Vranić2, O. Rahić2, A. Atilla Hıncal3
In vitro tests on VH (80 mg) tablets were performed on dis-
solution tester ERWEKA DT 800; USP apparatus 2 (n= 6); ro-
1
Institute for Quality Control of Medicines, M. Tita 9, 71 000
tating speed 50 rpm at 370C±0.50C; volume of dissolution
Sarajevo, Bosnia and Herzegovina
medium 900 ml.
2
Department of Pharmaceutical Technology, Faculty of Phar-
macy, University of Sarajevo, Čekaluša 90, 71000 Sarajevo,
In our experiments, three different media were used in both
Bosnia and Herzegovina
compartments: simulated gastric fluid at pH 1.2, simulated
3
Department of Pharmaceutical Technology, Faculty of Phar-
gastric fluid at pH 4.5 and phosphate buffer pH 7.4. Four
macy, Hacettepe University, 06100 Sıhhiye-Ankara,Turkey
HPLC vials were adjusted on each paddle of dissolution
tester. Three of the four vials were impregnated with phos-

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

pholipids; the fourth was blank and was used as a control to absorbed from the small intestine. Drugs that are weak acids
confirm that there is no significant permeation through non- are un-ionized in the stomach and hence might be absorbed
impregnated membrane. Samples were withdrawn from the well from there, whereas drugs that are weak bases might
vials, one by one, at the following intervals: 1, 2 and 4 hours. not be well absorbed from the stomach because they are
From the fourth vial (blank, non-impregnated PTFE filters) highly ionized. Also, most of the excipients are weak acids
the sample was withdrawn after 4 hours. or weak bases and possess various degree of ionisation in
different part of GI tract.
Quantification of VH was performed by UV/VIS spectropho-
tometric method at the absorption maximum at 277 nm (cal- A substance that increases the gastric pH would therefore
ibration curves: y = 11.69605x-0.00398, R2=0.99995 simulat- be expected to increase the absorption of weak bases and to
ed gastric fluid at pH 1.2; y= 12.04581x+0.01054, R2=0.99977 decrease the absorption of weak acids in the stomach. Also,
simulated gastric fluid at pH 4.5 and y= 11.95440x-0.00106, decrease or increase of pH of the GI tract change degree of
R2=1.00000 in phosphate buffer pH 7.4, respectively. The ionisation of excipients (weak acids/ weak bases) and change
precision of method was expressed by relative standard de- quality of drug-excipient interactions. A drug–excipient in-
viation (RSD) from 10 replications of samples, concentration teraction can be actively used to increase the bioavailabil-
range from 0.00125 mg/cm3 to 0.005 mg/ cm3. RSD was in ity of the drug (e.g. complexation with cyclodextrins). There
the range of 0.14 %-0.60 % are also many cases in which an interaction might not be
expected and might adversely affect the bioavailability of a
RESULTS AND DISCUSSION drug (e.g. where the mechanism of the interaction is surface
In donor cells, concentration of VH was identified by both adsorption).
weighing and spectrophotometric determination (not pre-
sented).Permeation was calculated as apparent permeability Table 2: Influence of different pH values of media and test
coefficient measured in cm min−1 (P) and results were pro- duration on VH in vitro permeation
vided in Figure 1. and Table 2.4,5
P x 104 [cm min−1] of VH

Test Tablet Tablet Tablet


pH of
duration formulation I formulation II formulation
media
(minutes) ± 2xSD ± 2xSD III ± 2xSD
60 315.64 ±41.3 213.69 ±35.1 125.64 ±12.6
1.2 ± 0.5 120 58.33 ±10.1 81.82 ±10.5 105.47 ±15.2 77
240 41.65 ±7.8 43.86 ±11.1 40.71 ±5.9
60 303.78 ±38.4 242.07 ±31.7 240.61 ±44.3
4.5 ± 0.5 120 137.16 ±9.9 102.40 ±16.3 72.40 ±21.3
240 51.99 ±10.0 37.50 ±8.4 33.89 ±6.8
60 248.47 ±42.3 156.36 ±24.1 89.95 ±19.4
7.4 ± 0.5 120 86.70 ±7.2 76.19 ±8.9 60.07 ±10.1
240 57.70 ±12.4 52.66 ±11.8 35.98 ±8.4

Apparent permeability coefficient achieved the highest val-


ue after the first hour of testing. This was caused by concen-
tration gradient between the acceptor/donor compartment
which value was decreased during further testing. In addi-
tion, as a result of decreased concentration gradient, two-
way permeation process equilibrium from one to another
compartment was established.

CONCLUSION
Traditionally, excipients have been regarded as inert. How-
ever, there are many instances in which excipients have been
shown to have a significant effect on the biological availa-
Figure 1: Permeation profile of VH at different pH values of bility of the drug. Many of the drug–excipient interactions
media and test duration affected the process of dissolution/ membrane permeation.
In fact, an interaction between a drug and an excipient that
The pH of the GI tract varies greatly, from pH 1.0 –3.5 in the alters the dissolution or the membrane permeation of some
stomach to pH 7.0 –8.5 in the large intestines. Some drugs drugs has been shown to have a marked impact on the ab-
can be preferentially absorbed from a particular section of sorption and bioavailability of that drug.
the GI tract, although both weakly acidic and basic drugs are

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

Used in vitro model of passive absorption is discriminatory ting machine (Korsh-Erweka, Germany). The hardness of the
and suitable for testing the level of permeation for various tablets was kept constant (approximately 35 N) and was mea-
technological formulations (different preparation proce- sured with a hardness tester (Korsh-Erweka GmbH, Germany).
dures and different excipients). Future tests could be used
for the evaluation the nature of interactions with individual Table 1: Compositions of the tablets
excipients and active substances. T1 T2 T3 T4
Pioglitazone 13.6 13.6 13.6 13.6
REFERENCES
1. Verbrugge L.B., Harry B. van Wezel H.B. Pathophysiology HPMC K15M 25 - - -
of verapamil overdose: new insights in the role of insu- HPMC K4M - 25 - -
lin. J. Cardiothor. Vasc. An. 2007; 21(3):406-409. HPMC K100LV - - 25 -
2. Pifferi G., Restani P. The safety of pharmaceutical excipi-
ents. Il Farmaco. 2003; 58(8):541-550. HPMC E4M - - - 25
3. Jivraj M., Martini L.G., Thomson C.M. An overview of the Foam Powder 10 10 10 10
different excipients useful for the direct compression of Avicel pH200 50.9 50.9 50.9 50.9
tablets. Pharm. Sci. Technol. Today. 2000; 3(2):58-63.
Mg stearate 0.5 0.5 0.5 0.5
4. Corti G., Maestrelli F., Cirri M., et al. Development and
evaluation of an in vitro method for prediction of human
drug absorption I. Assessment of artificial membrane Table 2: Compositions of the tablets
composition. Eur. J. Pharm. Sci. 2006; 27(4):346-353. T5 T6 T7
5. Avdeef A. Absorption and drug development - Solubil- Pioglitazone 13.6 13.6 13.6
ity, permeability, and charge state. First edition, John
HPMC K15M 15 25 35
Wiley & Sons Inc., 2003.
Foam Powder 10 10 10
Avicel pH200 60.9 50.9 40.9
P012-Floating Matrix Tablets Based On Mg stearate 0.5 0.5 0.5
Low Density Foam Powder
Floating behaviour of the tablets
Sevgi Takka1, Didem Kuzu2 The in vitro floating behaviour of the tablets was studied by
placing them in 900 ml glass containers filled 500 ml pre-
78 heated 0.1 N HCl (pH 1.2, 37°C ± 2 ) for 8 hours. The float-
University of Gazi, Faculty of Pharmacy, Pharmaceutical
Technology Department, Etiler Ankara/Turkey1 ing lag times (time period between placing the tablet in the
Republic of Turkey Social Security Institution, Ataturk medium and tablet floating) and floating durations of the
Boulevard, Kavaklıdere Ankara/Turkey2 tablets were determined by visual.

INTRODUCTION Dissolution test


Several difficulties are faced in designing controlled release sys- Dissolution profiles of pioglitazone tablets were determined
tems for better absorption and enhanced bioavailability. One of using the USP 26 method II (Van Kel USA) with a paddle at 50
such difficulties is the inability to confine the dosage form in the rpm. Dissolution was performed in 500 mL 0.1 N HCl at 37°C
desired area of the gastrointestinal tract1,2. The aim of this study ± 2 temperature. The amount of drug released was detected
was to develop single unit, floating controlling drug delivery UV-spectrophotometrically at 269 nm for 8 hours.
systems and to study the effect of formulation parameters on
the floating and drug release behaviour of these systems. RESULTS AND DISCUSSIONS
The effect of the type and concentration of matrix polymer
MATERIALS AND METHODS (HPMC K4M, HPMC K100LV, HPMC E5 or HPMC K15M) used
Materials for the preparation of floating, low density tablets on the
Floating tablets were prepared by using low density foam resulting drug release is shown in Figs.1 and 2. The in vitro
powder (Aeorosil) (Evonik Degussa GmbH, Germany). Pio- floating behaviour of the tablets was shown in Table 3.
glitazone HCl and HPMC were a generous gift from Jubilan
Organosys, India and Colorcon, Orpington, UK, respectively.

Tablet preparation
Tablets were prepared by direct compression. All excipients
were screened through an 80-mesh sieve before mixing. The
respective powders (drug, foam powder and polymer) and the
additive (Avicel pH 200) were blended for 10 minutes. Then the
mixture was lubricated with magnesium stearate for another 3
minutes. The magnesium stearate level was fixed at 0.5 % for all
formulations (Tables 1 and 2). The mixture was compressed into Figure 1: Effect of type of matrix-forming polymer on piogli-
flat-faced tablets 10 mm in diameter using single punch tablet- tazone release in 0.1N HCl from floating matrix tablet

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

P013-In Vivo Investigations On Liquid


Crystal Coated Naproxen

Altan Yüksel1, Tadeusz Librowski2, Nefise Özlen Şahin1

Mersin University, Faculty of Pharmacy, Mersin, Turkey1


Jagellonian University, Faculty of Pharmacy, Krakow, Poland2

INTRODUCTION
Figure 2: Effect of concentration of matrix-forming polymer on Naproxen (NPX), a nonsteroidal anti-inflammatory drug, has
pioglitazone release in 0.1N HCl from floating matrix tablet been widely used for analgesic effect. It has low aqueous
solubility and hence, its absorption is low. The peak plasma
Table 3: Floating time specifications of formulations concentration is reached late. As NPX stays in the gastroin-
testinal track for a long time, it results in gastric irritation.
Formulation Floating Time Lag Time
T1 8 hours **** Preparing salts of water insoluble drug substances, forming
T2 8 hours **** water soluble complexes with various vehicles such as cy-
clodextrins have been used to improve the solubility of sub-
T3 6 hours 1.5 hours
stances with low aqueous solubility1. Coating insoluble drug
T4 **** **** powders with liquid crystals is another way of enhancing the
T5 **** **** aqueous solubility.
T6 8 hours 3 minutes
Liquid crystals are stable structures with various solvents
T7 8 hours **** (mostly water) of amphiphilic substances. However, they are
homogenous-looking structures behaving as if single phase
CONCLUSION unlike emulsions2.
A single unit, floating drug delivery system has been developed.
Incorporation of the highly porous foam powder in the matrix In this study it was aimed to improve the aqueous solubil-
tablets provides densities that are optional lower than the den- ity of naproxen upon coating with liquid crystal. In the first
sity of the release medium. The three HPMC types/grades dif- part of our study which was presented elsewhere, it was de-
fer in the type of substitution and/or molecular weight which termined that coating naproxen with liquid crystal formula-
79
can be correlated with the polymer viscosity. The viscosities of tions improved the solubility efficiently under in vitro condi-
2 % aqueous solutions of HPMC E5, K 100LV, K4M and K15M tions. In this part of the study, the analgesic effect of liquid
at 20°C are 5, 100, 4000 and 15000 cps, respectively. The drug crystal coated naproxen formulations was investigated to
release rate decreased in the rank order HPMC E5>HPMC test whether liquid crystal coating influenced the pharma-
K100LV>HPMC K4M>HPMC K15M. With increasing macromo- cological activity of the drug substance.
lecular weight, the degree of entanglement of the polymer
chains increases and this leads to decreased drug release rates. MATERIALS AND METHODS
With decreasing amounts of HPMC, the density of the swollen Materials
hydrogel network decreases, presenting less hindrance for drug Sodium lauryl sulfate and vaseline were purchased from
diffusion and consequently, the drug release increase. Merck, Co., Germany. Distilled water was obtained by Mili-
pore water purifying system. Naproxen was obtained from
REFERENCES Sigma (St. Louis, USA). All other chemicals were of analytical
1. Streubel A, Siepmann J, Bodmeier R. Gastroretentive quality.
drug delivery systems, Expert Opin. Drug Deliv. 2006;
3(2):217-233. Preparation of liquid crystal formulations for coating
2. Li S, L, Lin S, Daggy BP, Mirchandani HL and Chien YW. Formulations consisting of sodium lauryl sulfate-vaseline-
Effect of HPMC and Carbopol on the release and floating water were prepared at room temperature as described else-
properties of gastric floating drug delivery system using where3. Adequately weighed substances at various ratios
factorial design, Int J Pharm. 2003; 253: 13-22. were mixed in a scintillation vial using a simple agitation ap-
plied with a glass rod for 5 minutes. Liquid crystal formations
were confirmed upon examination under polarized light mi-
croscope.

Amount of naproxen was kept constant as 250 mg in each


formulation. Coating procedure for naproxen powder by
liquid crystal was carried out in a mortar. Liquid crystal was
added at various ratios (12.5; 25 and 37.5% w/w). It was mixed
with naproxen for 2 minutes without applying any pressure
on the material.

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

Analgesic activity of liquid crystal coated naproxen formulations increases with the amount of liquid crystal used in coating.
Albino male mice weighing approximately 25 g were used As expected, ED50 values decrease with increasing coating
in our study. Prior to the experiments, approval of ethical ratio. This is in good agreement with our hypothesis.
committee at Jagellonian University was obtained. Analge-
sic activity was determined with “Writhing Test”4. Eight mice CONCLUSION
(n= 8) were used in each group. Group I (control group): mice Liquid crystal coated naproxen formulations may be used to
without any drug treatment; Group II: mice treated with obtain high analgesic activity at low doses of application.
naproxen powder; Group III: mice treated with 12.5% liquid
crystal coated naproxen formulation; Group IV: mice treated REFERENCES
with 25% liquid crystal coated naproxen formulation; Group 1. Milic-Askrabic J, Rajic DS, Tasic L, Djuric S, Kasa P,
V: mice treated with 37.5% liquid crystal coated naproxen Pintye-Hodi K. Etodolac and solid dispersion with
formulation β-cyclodextrin, Drug development and industrial phar-
macy, 1997, 23(11): 1123-1129.
Initially, phenylquinone was applied intraperitonealy to in- 2. Friberg SE, Mandel L, Larsson M. Mesomorphous phases,
duce pain. Each formulation was dispersed in distilled water a factor of importance for the properties of emulsions, J.
and then, administered orally by means of a gavage at the Coll. Interface Sci., 1969; 29: 155-156.
doses of 7.5; 15; and 30 mg/kg to the groups II to V. One and 3. Uslu S, Ermis D, Yuksel A, Baykara T. Increasing of disso-
a half hour later, wriggling movements made in 10 minutes lution rates tablets containing naproxen using isotropic
were counted. Data obtained were evaluated statistically. liquid crystals, 5th International Congress of Pharma-
ceutical Sciences, 1997, June 24-27, Ankara, pp.6.
RESULTS AND DISCUSSION 4. Singh PP, Junnarkar AY, Rao CS, Varma RK, Shridhar DR.
Table 1: Analgesic activity and ED50 values of pure naproxen Acetic acid and phenylquinone writhing test: a critical
Analgesic study in mice, Methods Find Exp Clin Pharmacol., 1983,
Dose (mg/kg) ED50 (mg/kg) 5(9):601-606.
Activity (%)
30 90,51
15 86,75 6,49 P014-EVALUATION OF SOLUBILITY CLASS OF
7,5 53,61 WEAK ACIDIC DRUG: ETODOLAC

Table 2: Analgesic activity and ED50 values of liquid crystal Huriye Demir, Betül Arıca Yeğin, Levent Öner
80 coated naproxen formulations (12.5% of liquid crystal)
Analgesic Department of Pharmaceutical Technology, Faculty of
Dose (mg/kg) ED50 (mg/kg) Pharmacy, Hacettepe University, 06100, Ankara, Turkey
Activity (%)
30 86,16
INTRODUCTION
15 79,50 4,84 The BCS is a scientific framework for classifying drug sub-
7,5 59,04 stances based on their aqueous solubility and intestinal
permeability1. Etodolac was selected as a model drug which
Table 3: Analgesic activity and ED50 values of liquid crystal belongs to BCS Class II. BCS Class II drugs exhibit low solubil-
coated naproxen formulations (25% of liquid crystal) ity and high permeability. In this study, the total solubility
of a insoluble and ionizable weak acid etodolac (pKa=4.65)2
Analgesic
Dose (mg/kg) ED50 (mg/kg) in surfactant containing buffers was investigated. The aim of
Activity (%)
this study was determining pH-solubility profile and solubil-
30 88,57 ity class of etodolac with the presence of surfactant within
15 83,13 4,84 the intestinal pH range.
7,5 63,47 MATERIALS AND METHODS
Materials
Table 4: Analgesic activity and ED50 values of liquid crystal Etodolac was supplied by Dr. Reddy’s Laboratory. Sodium
coated naproxen formulations (37.5% of liquid crystal) Lauryl Sulfate (SLS) and disodium hydrogen phosphate were
Analgesic purchased from Merck Co., and citric acid was purchased
Dose (mg/kg) ED50 (mg/kg)
Activity (%) from Carlo Erba. All the other chemicals used were analytical
30 93,38 grade and used without further purification.
15 71,07 3,64 Preparation of media
7,5 73,47 Mcllvaine buffers at pH 4.4, 5.6 and 6.8 were prepared for
solubility studies.
Data indicated that analgesic activity of coated naproxen
formulation is independent of amount of coating at high Solubility Studies
doses (30 mg/kg). Unlikely, at low doses, analgesic activity For determining solubility of etodolac at pH 4.4, 5.6 and 6.8,

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

each medium containing 0.5% and 1% (w/v) surfactant or Solubility results were used to determine the solubility class
without surfactant, etodolac powder was weighed as 200 mg, of etodolac. The solubility class was determined by calculat-
400 mg and 600 mg, respectively. Etodolac powder were shak- ing the volume of an aqueous medium sufficient to dissolve
en in rubber-capped vials containing 10 mL buffer solutions at the highest dose strength of etodolac (400 mg) in the pH
37ºC in a horizontal shaker water bath (Memmert, Germany) range 4.4-6.8.
with 80 rpm. At the end of first hour and twenty fourth hours,
samples were collected and drug content were determined at Dose/solubility values for etodolac are presented in Table 2.
273 nm with UV spectrometer (Shimadzu, Japan). A drug substance is considered highly soluble when the
highest dose strength is soluble in 250 mL or less of aque-
RESULTS AND DISCUSSION ous media over the pH range of l-7.51. According to this, sol-
The equilibrium solubility of etodolac in different buffer so- ubility class evaluation for etodolac is shown at Table 3.
lutions containing 0.5 - 1% SLS is shown in Table 1.
Table 2: Dose/solubility values of etodolac at various buffer
Table 1: Equilibrium solubility of etodolac at various buffer solutions
solutions (n=6, x±SD). SLS Concentration Dose/solubility (mL)
SLS Solubility (mg.mL-1) (%) pH 4.4 pH 5.6 pH 6.8
Concentration 0 3216 521 62
pH 4.4 pH 5.6 pH 6.8
(%) 0.5 432 206 53
0 0.124±0.003 0.767±0.020 6..371±0.231 1 248 124 41
0.5 0.925±0.010 1.933 ±0.066 7.460 ±0.075
1 1.608±0.020 3.213±0.047 9.717±0.189 Table 3: Solubility classification of etodolac in various buffer
solutions.
The solubility results indicate that the total solubility of et-
odolac increases with pH and SLS concentration. At pH<pKa, SLS Concentration Solubility Classification (250 mL)
total solubility was low however when pH>pKa, solubility in- (%)
pH 4.4 pH 5.6 pH 6.8
creased. Solubility enhancement was found to be 5-50 folds 0 Low Low High
with pH and 1.5-12 folds with SLS concentration changes.
0.5 Low High High
Figures 1 and 2 illustrate surface plot and contour graphics
1 High High High
of solubility with Equation 1 as a function of pH and SLS con-
centration.
Stotal=38,940-16,127x1-2,696x2+1,664x12+0,776x1x2-0,7762x22
Figures 3 and 4 illustrate surface plot and contour graphics 81
1 of dose/Solubility values with Equation 2 as a function of pH
and SLS concentration.
D/S=15879,0x1- 4178,6x1-9889,3x2+271,9x12+1227,5x1x2+1886,2x2 2

Figure 1: Total solubility as function of pH and SLS concentration.


Figure 3: Dose/solubility of etodolac as function of pH and
SLS concentration.

Figure 2: Total solubility as function of pH and SLS concentra-


tion.

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

of chitosan coating and liposomal encapsulation on the per-


meability, cellular toxicity, and characterization parameters
of the drug delivery system was investigated.

MATERIALS AND METHODS


Materials
L-α-lecithin (PC, soybean phospholipid, 95.6% phosphatidyl-
choline) and cholesterol (Col) was obtained by Calbiochem
(USA) and Acros Organics (USA), respectively. Protosan UP
CL-113 (MW: 110 kDa, DD: 87%) was purchased from FMC
Biopolymers, Norway. Caco-2 cells were obtained from ATCC
(Rockville, MD, USA). All the other chemicals used were ana-
lytical grade and used without further purification.
Figure 4. Dose/solubility of etodolac as function of pH and
SLS concentration. Preparation of the Liposomes
Liposomes were prepared by the evaporation-sonication
CONCLUSION method and characterized in terms of particle size, zeta po-
Studies for combined effect of pH and surfactant on the tential, encapsulation efficiency (EE) and cell viability and
solubility showed that etodolac solubility is pH-dependent permeability on Caco-2 cells. PC and Col (10:1 molar ratio)
and enhancement of pH and SLS concentration increased were dissolved in chloroform. The lipid mixture purged with
its solubility. Dose/solubility values indicated at pH 6.8 and nitrogen and organic solvent was removed by rotary evapo-
pH 5.6 with SLS that etodolac is highly soluble. In summary, rator for forming the lipid film. The formed lipid film was de-
because of high solubility at pH range of absorption region hydrated with phosphate buffer (pH 7.4) and vortexed. Then
in the gastrointestinal tract, etodolac exhibits BCS class I be- it was ultrasonicated for 15 min (SONOPULS Ultrasonic ho-
havior. mogenizer HD 2200, Bandelin, Germany). For chitosan coat-
ing, 0.5 mL of the liposome solution was added drop wise
REFERENCE to 0.05%, 0.1%, 0.5% and 1% chitosan solutions. They incu-
1. CDER/FDA. (2000). Waiver of In Vivo Bioavailability and bated at room temperature under continuous stirring for 2
Bioequivalence Studies For Immediate-Release Solid hours. Furosemide encapsulated liposomes were prepared
Oral Dosage Forms Based on a Biopharmaceutics Clas- as described above but this time furosemide was dissolved
82 sification System. in methanol and added to the lipid mixture before evaporat-
2. Yazdanian, M., Briggs, K., Jankovsky, C.,Hawi, A. (2004) ing step. Prepared liposomes evaluated by the particle size
The “High Solubility” Definition of the Current FDA Guid- and zeta potential measurements (n=3)
ance on Biopharmaceutical Classification System May
Be Too Strict for Acidic Drugs. Pharmaceutical Research, Cytotoxicity of the Formulations
21 (2), 293-299. Cytotoxicity of formulations was evaluated by using Caco-2
cells and cell viability was assessed by using MTT assay.

Permeability Studies
P015-CHITOSAN COATED LIPOSOMES Furosemide permeation studies were carried out with
LOADED WITH FUROSEMIDE: Caco-2 cells. The cells were incubated in the flasks and they
CHARACTERIZATION AND CELLULAR trypsinized when near to confluency. 1.5 mL of medium was
INTERACTION transferred to the basolateral side of the wells. Cell suspen-
sions from 25 passages in 0.5 mL of medium were introduced
into the inserts’ apical side at 6.0x104 cells/well (THINCERTS,
Can Sarısözen1, Senem Sevtap Ölmez2, İmran Vural2
pore diameter 1µm, surface area 1.13cm2, Greiner Bio-one,
NC, USA). Medium was changed on every 2nd day. The cell
Department of Pharmaceutical Technology, Faculty of
monolayer had reached confluence after 20 days of seeding.
Pharmacy, Hacettepe University, Ankara, Turkey1
Wells with TEER value of >400 ohm.cm2 were selected for
Department of Pharmaceutical Technology, Faculty of
furosemide transport studies. Culture medium was replaced
Pharmacy, Hacettepe University , Ankara, Turkey 2
from each well by 0.5 mL and 1.5 mL HBSS in the apical and
basolateral side of the well. Free furosemide solution, loaded
INTRODUCTION uncoated liposomes and chitosan coated loaded liposomes
The aim of this study was to develop chitosan coated lipo- were added to the apical side of the monolayer. The wells
somal carrier systems for poorly soluble and permeable were then placed on a shaker at 30 rpm and 37°C for 2 hours.
drug Furosemide, which is a BCS Class IV compound and was After that the samples from the basolateral side was analyzed
used as a model drug1. Chitosan is a natural cationic poly- by validated HPLC method (r2 > 0.999). Permeability coeffi-
saccharide derived by deacetylation of chitin, which is, after cients (Papp, cm/s) were calculated using Eq. 1: Papp=(dQ/
cellulose, the most abundant polymer found in nature2. Chi- dt)(1/AC0) where dQ/dt is the flux across the monolayer, A
tosan was used as a coating material for liposomes due to is the surface area of the cell monolayer and C0 is the initial
its bioadhesive and permeability enhancer effects. The effect concentration on the apical side of the membrane.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

RESULTS AND DISCUSSION An 8 fold increase on Furosemide permeability was obtained


Liposomes coated with 0.5% chitosan was used due to its for chitosan coated liposomes when compared other formu-
zeta potential and particle size values. The results are given lations. The UFL and free drug showed similar permeation
below in Table 1. values of 1.96% and 1.77% respectively.

Table 1: Characterization parameters of the liposomes (n=3, CONCLUSION


mean ± SD) Furosemide which is a BCS Class IV drug was successfully en-
Particle Size Zeta Potential EE capsulated in the liposome formulations. According to the
Formulation cytotoxicity studies, chitosan coated liposome formulations
(nm) (mV) (%)
showed less cytotoxicity than the uncoated ones. The per-
UL 50.04 ± 0.78 2.00 ± 0.55 - meation enhancer effect observed for the chitosan coated
liposomes make them interesting candidates that could be
UFL 49.84 ± 0.85 -13.58 ± 0.56 42.6
effective to improve Class IV drugs bioavailability after oral
CL 68.38 ± 1.10 22.05 ± 2.66 - administration.

CFL 115.40 ± 2.86 32.42 ± 2.26 71.1 REFERENCES


1. Wu CY, Benet LZ. Predicting Drug Disposition via Ap-
Uncoated empty liposomes (UL) and uncoated furosemide plication of BCS: Transport/Absorption/Elimination In-
loaded liposomes (UFL) showed similar particle size. Chitosan terplay and Development of a Biopharmaceutics Drug
coating increased the average particle size for both unload- Disposition Classification System, Pharm Res. 2005; 22:
ed (CL) and loaded liposomes (CFL). For unloaded liposomes 11-23.
the zeta potential was nearly neutral, thus the mechanism 2. Kaş HS. Chitosan: Properties, preparations and applica-
of coating involved hydrogen bonding and hydrophobic tion to microparticulate systems, J Microencapsulation.
interactions. On the other hand when furosemide encapsu- 1997; 14: 689-711.
lated into the liposomes the zeta potential became negative
and the main interaction was electrostatic attraction. So the
drug loaded liposomes adsorbed more chitosan, and have P016-PEG-PE BASED MIXED MICELLES
increased particle sizes. Since chitosan carries high positive
LOADED WITH PACLITAXEL FOR
charge, the adsorption of chitosan increased the density of
positive charge made the zeta potentials positive. ANTITUMOR ACTIVITY: ENHANCED DRUG
SOLUBILIZATION 83
The encapsulation efficiencies of the formulations were giv-
en in Table 1. The results showed that the chitosan coating Can Sarısözen1, İmran Vural1, Vladimir P. Torchilin2, A. Atilla
increased the encapsulation of furosemide. Hıncal1

The cell viability results show that the chitosan coating in- Department of Pharmaceutical Technology, Faculty of
creased the Caco-2 cell viability both for furosemide loaded Pharmacy, Hacettepe University, 06100, Ankara, Turkey1
and unloaded liposomes. This observation supports the idea Department of Pharmaceutical Sciences and Center
that cell viability may be increased by chitosan coating. for Pharmaceutical Biotechnology and Nanomedicine,
Northeastern University, Boston, MA, USA2
The results of permeation studies were given in Figure 1. The
Papp values of the formulations were also indicated above INTRODUCTION
the bars in the figure. Paclitaxel is an anticancer drug derived from Taxus brevifo-
lia. It possesses its activity by stabilizing the microtubules.
In the recent years the researches showed that paclitaxel
has also induces apoptosis via the mitochondrial or intrin-
sic pathway. Even though paclitaxel is effective against vari-
ous cancer types like breast and ovarian cancers, it has very
low solubility. In the literature there are values for paclitaxel
solubility of 0.7 µg/ml1 to 30 µg/ml2. Due to its hydrophobic
properties, currently paclitaxel is formulated with alcohol
and Cremophor EL, which has severe side effects like neph-
rotoxicity, neurotoxicity and hypersensitivity. Also paclitaxel
itself represents relatively high toxicity. To overcome these
issues encapsulation of paclitaxel in different carriers sys-
tems have been developed extensively.

In the present work, the preparation and in vitro character-


Figure 1: Permeability study results of the formulations (n=3, ization of paclitaxel loaded PEG-PE/Vitamin E mixed micelles
mean±SD) have been described. Different molar rations of PEG2000-PE:

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

vitamin E and different paclitaxel amounts were used. The celles is formed by the hydrophobic diacyllipid part and the the
micellar drug solubilization effect and increased drug encap- corona is made of hydrophilic PEG chains. Thus the hydrophobic
sulation was evaluated. paclitaxel will be solubilized within in micelle core. Addition of
vitamin E is believed to increase the micelle size due to the hy-
MATERIALS AND METHODS drophobicity of the molecule. When vitamin E is incorporated
Materials into the micelle structures it increases the hydrophobic core vol-
1,2–distearoyl–sn–glycerol–3–phosphoethanolamine–N- ume of the micelle, thus increase the micelle size. This effect of
[methoxy(polyethylene glycol)-2000] (PEG2000-PE) was ob- the vitamin E can be clearly seen from the particle size data. The
tained from Avanti Plar Lipids (Alabaster, AL, USA). Vitamin increased vitamin E molar ratio resulted larger micelles (p<0.05).
E was purchased from Sigma (St. Louis, MO, USA). Paclitaxel
was from Applichem (Darmstadt, Germany). All the other As can be seen in the table, increased vitamin E ratio in-
chemicals used were analytical grade and used without fur- creased the encapsulation of paclitaxel. As explained be-
ther purification. fore, when vitamin E ratio was increased, core volume of the
mixed micelles becomes larger. The percent of paclitaxel in
Preparation of the paclitaxel loaded mixed micelles the micelles were found to be higher than 3% for both for-
The mixed micelles loaded with paclitaxel were prepared mulations. Also the amounts of the drug in 1 mL of micelle
with the following method. PEG2000-PE and vitamin E stock dispersion were about 1 mg. These results are consequent
solutions were prepared in chloroform. 85:15 and 90:10 mo- with literature3.
lar ratios of PEG2000-PE: vitamin E mixtures were prepared
and different amounts of paclitaxel solution (from the stock Table 1: Characterization parameters of the micelles (n=3,
solution of 1 mg/ml in methanol) was added to these mix- mean ± SD)
tures. Organic solvents were removed by rotavapor and mi- Formulation Particle Size Zeta Potential
celle material-paclitaxel film was formed. This film was freeze (PEG-PE: Vit. E) (mm) (mV)
dried to remove organic solvent residues. To form drug load- 85:15 (Empty) 23.9±2 -7.7±1.2
ed micelles, film was hydrated with PBS pH 7.4. The final con- 90:10 (Empty) 20.1±0.3 -9.0±0.4
centration of the micelle materials was 10 mM. 85:15 23.1±0.3 -24.6±0.2
90.19 21.4±0.2 -34.4±3
Particle size and zeta potential measurements
Particle size distribution and the zeta potentials of the micelles
Table 2: Incorporated drug values of the micelle formulations
were determined by laser diffractometry using a Malvern Na-
(mean ± SD)
84 noZS (Malvern Instruments Ltd., UK). The micelle samples were
diluted with MilliQ ultra pure water prior to measurements. All Drug Drug Drug amount
measurements have been made triplicate. Formulation incorporated weight in per mL of
(%) micelle (%) micelle (μg)
Drug solubilization and encapsulation efficiency 85:15 77.0±4.7 3.62±0.11 1095.7
The drug encapsulated in the micellar phase was measured 90:10 66.7±3.9 3.14±0.71 948.5
by a validated HPLC method. Mobile phase was methanol:
water (68:32, v/v), column was Develosil ODS-UG-5 C18 RP CONCLUSION
(4.6x150 mm, 5µm, Nomura Chemical, Seto city, Japan). The The results obtained showed that the PEG2000-PE/vitamin
injection volume was 10 µl and the flow rate was 1 ml/min. E mixed micelles can efficiently solubilized the paclitaxel, a
Paclitaxel was detected by UV detector at 230 nm. The ana- poorly soluble anticancer drug. The solubilization effect is
lytical method was validated by terms of specificity, linearity, significantly higher and 1 mg of paclitaxel can be incorpo-
accuracy, and precision. rated in 1 mL of mixed micelles at 10 mM concentration. The
sizes are around 20 nm, which is also very suitable for passive
The drug loaded micelle solutions was filtered through 0.22 targeting due to the EPR effect.
µm filter to remove non-incorporated and precipitated pa-
clitaxel crystals. The clear micelle suspensions then diluted REFERENCES
with methanol prior to injection. Micelle structure was dis- 1. Mathew AE, Mejillano MR, Nath JP, Himes RH, Stella VJ,
turbed by methanol and incorporated paclitaxel can be de- Synthesis and evaluation of some water-soluble prod-
termined. rugs and derivatives of taxol with antitumor activity,
Journal of Medicinal Chemistry, 1992; 35: 145-151
RESULTS AND DISCUSSION 2. Swindell CS, Krauss NE, Horwitz SB, Ringel I, Biologically
In our study the particle sizes and the zeta potentials of the active taxol analogs with deleted A-ring side chain sub-
micelle formulations are given in Table 1. The initial paclitaxel stituents and variable C-2’ configurations, Journal of Me-
amounts were added as the different percents of PEG-PE by dicinal Chemistry 1991; 34: 1176-1184
weight. The highest encapsulation was achieved by using 3. Sawant RR, Sawant RM, Torchilin VP, Mixed PEG-PE/vi-
the 5% paclitaxel of the PEG-PE weight. The results also indi- tamin E tumor targeted immunomicelles as carriers for
cate that micellar structure was intact. poorly soluble anti-cancer drugs: Improved drug solu-
bilization and enhanced in vitro cytotoxicity, European
The average size of both empty and loaded PEG2000-PE/Vitamin Journal of Pharmaceutics and Biopharmaceutics, 2008;
E mixed micelles was about 22 nm. The core of the PEG-PE mi- 70: 51-57

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

P017-FORMULATION AND IN VITRO and washed with distilled water. The washing step was re-
EVALUATION OF EUDRAGIT-L100/PLGA peated once before NP were resuspended in distilled water
and lyophilized overnight.
NANOPARTICLES CONTAINING SALMON
CALCITONIN Characterization of Nanoparticles
The morphological examination of the nanoparticles was
Meltem Çetin1, İmran Vural2, Alptuğ Atila3, Can Sarısözen4, performed using AFM (NanoMagnetics Instruments Ltd., UK).
Yücel Kadıoğlu5 The size (Z-average mean) and zeta potential of the nanopar-
ticles were analyzed by photon correlation spectroscopy and
Ataturk University, Faculty of Pharmacy, Dept. of laser doppler anemometry, respectively, in triplicate using a
Pharmaceutical Technology1 Zetasizer 3000HS (Malvern Instruments, UK). Size and zeta
Hacettepe University, Faculty of Pharmacy, Dept. of potential measurements were performed in triplicate fol-
Pharmaceutical Technology2 lowing a dilution of the nanoparticles suspension in distilled
Ataturk University, Faculty of Pharmacy, Dept. of Analytical water at 25 °C. Each measurement was done in triplicate. The
Chemistry3 entrapment efficiencies determined with high performance
Hacettepe University, Faculty of Pharmacy, Dept. of liquid chromatogram (HPLC).
Pharmaceutical Technology, Ankara4
Ataturk University, Faculty of Pharmacy, Dept. of Analytical RESULTS AND DISCUSSION
Chemistry, Erzurum, Turkey5 The nanoparticles were spherical and smooth on surface
as observed via AFM (Figure 1). Zeta potential of Eud/PLGA
INTRODUCTION and salmon calcitonin-loaded Eud/PLGA nanoparticles were
Salmon calcitonin (sCT) is an endogenous polypeptide hor- found to be -10,56 and -10,97 mV, respectively. There was no
mone composed of 32 amino acids (3,432 Da)1. sCT is current- significant differences were observed at the zeta potential
ly formulated as either a sterile solution for intramuscular or values (p> 0.05). Sizes of Eud/PLGA and salmon calcitonin-
subcutaneous injection or as a nasal spray2,3. This drug plays loaded Eud/PLGA nanoparticles were 218,5 and 234,1 nm
a crucial role in both calcium homeostasis and the treatment with a narrow polydispersity index of 0.068 and 0.051, re-
of bone disease such as osteoporosis1. Therefore, it is very spectively. The entrapment efficiency of salmon calcitonin
important to develop appropriate oral dosage forms of sCT was 68 %.
to increase patient compliance.

In the present study, we developed salmon calcitonin-load- 85


ed Eduragit®/PLGA nanoparticles by solvent evaporation
method. The formulations were characterized in vitro with
regard to encapsulation efficiency, surface morphology, size
and surface potential.

MATERIALS AND METHODS


Materials
Salmon calcitonin was purchased from PolyPeptide Lab.
(LimHamn, Sweden). Eudragit® L100 was supplied by Rohm
GmbH&Co. KG (Darmstadt, Germany). Acetone, polyvinyl
alcohol (Mw 30 000-70 000) were purchased from Sigma
(USA), poly (D, L-lactide-co-glycolide) (PLGA (50:50) Resomer
RG 502H) from Boehringer Ingelheim (Germany).

Preparation of Eud/PLGA-salmon calcitonin nanoparticles


The mixture of Eudragit® L100/PLGA polymers (Eud:PLGA)
was used for preparation of nanoparticles. Eudragit®/ L100
and PLGA were dissolved in anhydrous ethanol and acetone, Figure 1: AFM images of drug-loaded Eud/PLGA nanoparticles.
respectively. Then, the Eudragit® solution in ethanol was CONCLUSION
added slowly to the PLGA solution in acetone (1:2 v/v) with This study reports the preparation of salmon calcitonin-
a constant stirring rate of 500 rpm on magnetic stirrer at am- loaded Eudragit® L 100/PLGA nanoparticles and their in vitro
bient temperature. The organic phase was added into 6 ml characteristics. Eudragit® L 100/PLGA nanoparticles might
of PVA solutions (3 % w/v) containing salmon calcitonin in be well suited delivery systems for the oral delivery of salm-
the amber glass vial. The mixture was emulsified using 40 on calcitonin.
% power of ultrasonic probe (Sonoplus, HD 2070; Bandelin.
Electronics, Berlin, Germany) for 1,30 min. Finally, organic ACKNOWLEDGEMENTS
phases were evaporated under reduced pressure in a rota- This project was supported by the TUBITAK (Project no.
ry evaporator at 40 °C. After evaporation of the solvent, NP SBAG-108S275).
were recovered by centrifugation at 17,000 rpm for 30 min.

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

REFERENCES Table 1:Contents of tablet formulations


1. Na DH, Youn YS, Lee SD, Son MW, Kim WB et al. Moni-
toring of peptide acylation inside degrading PLGA mi- Magnesium Clopidogrel
Avicel HPMC
crospheres by capillary electrophoresis and MALDI-TOF Formulation Stearate Bisulphate
(mg) (mg
mass spectrometry, J Control Release. 2003; 92: 291– (mg) (mg)
299.
2. Azria M, The calcitonins, Physiology and pharmacology, 1 0 0 0.1 75
Karger, Basel, 1989. 2 0 25 0.1 75
3. Lee KC, Park MO, Na DH, Youn YS, Lee SD et al. Intranasal 3 0 50 0.1 75
delivery of PEGylated salmon calcitonins: hypocalcemic 4 25 0 0.1 75
effects in rats, Calcif Tissue
5 25 25 0.1 75
6 25 50 0.1 75
P018-DEVELOPMENT OF SUSTAINED RELEASE 7 50 0 0.1 75
8 50 25 0.1 75
FORMULATION OF ANTITHROMBOTIC DRUG
9 50 50 0.1 75
10 0 75 0.1 75
İsmail Tuncer Değim1, Celil Tan1
11 0 100 0.1 75
Gazi University, Faculty of Pharmacy, Department of 12 0 150 0.1 75
Pharmaceutical Technology, Ankara, Turkey1
Calculation of theoretical plasma profiles
The theoretical plasma profiles after virtually taking imme-
INTRODUCTION diate and sustained release formulations containing clopi-
A novel polymeric salt form of clopidogrel (clopidogrel resi- dogrel were calculated using known pharmacokinetic pa-
nate) has been also developed to for sustained release of ac- rameters and computer program (Stella v.6.0, Hanover, US).
tive compound1 and a patent application has been made to
prepare more effective and controlled release dosage forms In-vivo experiments
of clopidogrel2. The best commercially available tablets (fastest release), for-
mulation 3 and formulation 12 were subjected to animal ex-
Serious adverse drug reactions have been reported to be as- periments. Number of platelets (thrombocytes), prothrom-
86 sociated with clopidogrel therapy3. A controlled release dos- bine time, fibrinogen and thrombine time, active partial
age form of clopidogrel was developed to enhance the ben- thromboplastine time (APTT), collagen/ADP closer times
efit of the therapy and to lower incidence of side/adverse were determined.
effects. The design of the study was performed using Fuzzy
logic technique. Dissolution and release characteristics of RESULTS AND DISCUSSION
prepared tablets were determined. Commercially available Dissolution profiles of prepared tablets in 0.1 N HCl for first
tablets in Turkish market were also subjected to drug release two hours and then pH 6.8 were performed. Dissolution
study. The theoretical plasma profiles after virtually taking profiles of all formulations in 0.1 N HCl were also performed
immediate and sustained release formulations containing (Figure.1).
clopidogrel were calculated using known pharmacokinetic
parameters and computer program. Selected formulation
was given to rats and rabbits and blood samples were ana-
lyzed in terms of coagulation parameters.

MATERIALS AND METHODS


Clopidogrel bisulphate was obtained from Dr. Reddy’s, India.
Commercially available tablets were purchased from Turkish
market.

Methods
Preparation of tablet formulations
Formulations were designed using Fuzzy Tech 5.70b (IN-
FORM GmbH, Germany) computer program.

Dissolution and determination of drug release from tablets


Pharma Test-PTW S3C (Hainburg, Germany) dissolution tes- Figure 1: Dissolution profiles of all formulations in 0.1 N HCl
ter was used. Drug release tests were performed using pro- The theoretical blood profiles were calculated using a com-
cedure described in USP 28 as paddle method A. puter simulation program. Blood drug concentrations were
calculated after virtually given commercial tablets and after
virtually administration of formulation3 and.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

One commercially available formulation was seleceted as dissolving of the drug in the external aqueous phase during
a reference considering best release. All formulations were emulsification and hardening process, resulting with a low
given rabbits orally and their blood samples were analyzed. drug loading or encapsulation efficiency2.
The number of platelets in their blood were found to be sig-
nificantly increased after 4 hours for commercial tablets and In recent years, emulsion solvent diffusion method is pro-
formulation 12 (p<0.01).A significantly different results were posed and began to be used widely for microencapsula-
obtained after 4 hours with formulation 12 and commercial tion of various kinds of drugs. When the emulsion solvent
tablet (p<0.5) for prothrombin times.Percentage of fibrinojen diffusion method is compared with the solvent evaporation
coagulation results show that after first and 6 hours of tablets method, it is observed that the solidification of the liquid
administration, statistically significant results were obtained droplets realized much more faster in the former. Further-
for commercial tablets and formulation 3 (p<0.05) and sig- more, in the solvent evaporation process, it is known that, the
nificant difference between commercial tablets and formu- solvents used widely for preparation is classified in the “risky”
lation 12 and formulation 3 after 6 (p<0,01). When collagen/ groups whereas the heating stage to evaporate the solvents
ADP closer times were measured a significant difference were are avoided in emulsion solvent diffusion method1,2.
found between control and formulation 3 and 12 after 6 hours
(p<0.01). Dade Behring has performed a clinical study and the Tolmetin sodium (TS) is an antiinflammatory drug which is
reference value for collagen/ADP closer time was given with freely soluble in water. Therefore, in this study it is regarded
the value of 71-118 seconds. It is accepted with the general as a model drug for the realization of the microsphere for-
agreement that if this value is higher than 133 seconds it can mulation in the present study using solvent diffusion tech-
be said that this parameter is prolonged4. In our experiments nique with a high encapsulation efficiency.
this parameter was found to be 255 (210-300) and 180 (78-
274) seconds after 6 hours for formulation 3 and 12 respec- In the light of the above stated facts, in the present study,
tively. This indicates that our sustained release formulations the objective can be summarized as, to encapsulate the
were found to be potentially active after 6 hours and they can model hydrophilic drug tolmetin sodium within Eudragit®
be used as an alternative to conventional tablets. S100 polymer, so to increase the encapsulation efficiency of
the drug in microspheres with the addition of different salts
CONCLUSION (sodium chloride, potassium dihydrogen phosphate and
In conclusion, two sustained clopidogrel releasing formula- sodium bromide) into aqueous continuous phase to avoid
tions were developed and showed to be effective. dissolving of drug, that will end up with a low encapsulation
efficiency, also succeed the formulation of a nonsteroidal an-
REFERENCES tiinflammatory drug for oral application discarding the gas- 87
1. M.H. Ki, M.H. Choi, K.B. Ahn, B.S. Kim, D.S. Im, S.K. Ahn, trointestinal side effects3.
and H.J. Shin. The Efficacy and safety of clopidogrel resi-
nate as a novel polymeric salt form of clopidogrel. Arch MATERIALS AND METHODS
Pharm Res Vol. 31: 250-258 (2008). Materials
2. USPTO Patent Application #: 20070003628: Nanopar- Microsphere formulations were prepared by using Eudragit® S100 as
ticulate clopidogrel formulations polymer. Eudragit® S100(ES 100) was purchased from Röhm Pharma
3. http://en.wikipedia.org/wiki/Clopidogrel Polymers (Germany). Poly vinyl alcohol (PVA) was provided from Sig-
4. A.J. Grau, S. Reiners, C. Lincy, F. Buggle, and A. Ruf. Plate- ma (USA). Dichloromethane (DCM) and methanol were purchased
let function under aspirin, clopidogrel, and both after from Merck (Germany). Potassium dihydrogen phosphate (KH2PO4)
ischemic stroke. Stroke. April: 849-855 (2003). and sodium chloride (NaCl) were provided from Carlo Erba, (Italy),
sodium bromide (NaBr) was purchased from Merck (Germany) and
tolmetin sodium (TS) was provided from Santa Farma (Turkey).
P019-STUDIES ON THE IMPROVEMENT METHODS
OF ENCAPSULATION EFFICIENCY OF A Microspheres were prepared by emulsification diffusion
HYDROPHILIC DRUG technique by using 0,5 % PVA solution as the continuous
phase. The continuous phase contained NaBr, NaCl and
Göksu Yağ1, Betül Arıca Yeğin1, Sema Çalış1 KH2PO4 in different concentrations (see Table 1). For prepa-
ration process, first polymer was dissolved in methanol and
Department of Pharmaceutical Technology, Faculty of DCM mixture, then the drug was added in polymer solution
Pharmacy, Hacettepe University, Ankara, Turkey1 and stirred until it was dissolved. Following this step, this so-
lution was added into the continuous phase and emulsified
by stirring at 300 rpm for 1 hour with a propeller agitator.
INTRODUCTION Afterwards, the microspheres were collected by filtration.
Generally, the O/W method is one of the simplest techniques Tolmetin sodium, a hydrophilic model drug loaded micro-
to prepare microspheres and it provides many advantages sphere formulations were characterized for their particle
for efficient encapsulation of lipophilic drugs, such as high size, drug loading and in vitro release properties for evalu-
drug loading and the ability of preparing in a wide range of ation. For preparing the formulations, all of the formulation
particle sizes. Although these advantages, hydrophilic drugs parameters kept same except for the continuous phase. The
are poorly encapsulated by O/W method, which is due to codes of formulations are summarized Table 1 and the codes

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

were given according to the variability of the salts in the salts in the external phase were increased, the drug loading
outer phase. was increased. Also it was observed that the pH dependent
polymer provided a controlled release of the drug from mi-
Table 1: Formulation Codes crospheres. As a result, Eudragit® S100 was determined to be
suitable polymer for formulation of an oral microsphere for-
Codes Concentration of Salts
mulation of TS, thus emulsion diffusion technique in which
ES1 0,1 M NaCl various salts were included in the outer phase for satura-
tion, so reducing the dissolving of the drug appeared to be a
ES2 1 M NaCl proper technique for encapsulation of hydrophilic drugs.
ES3 0,1 M NaBr REFERENCES
1. Laleci, F., Çalış, S., Hıncal, L, A.A. Preparation and In Vit-
ES4 1 M NaBr ro Characterization of Tolmetin Sodium Loaded Oral
ES5 0,1 M KH2PO4 Eudragit Microspheres, Eur. J. Pharm. Sci. (Suppl. 1),
2005, 25:146-148.
ES6 1 M KH2PO4 2. Guinebretiere S., Briançon S., Lieto J. Study of the Emul-
sion Diffusion of Solvent Preparation and Characteriza-
RESULTS AND DISCUSSION tion of nanocapsules , Drug Dev. Research, 2002, 57:18-
The average production yields of microspheres and standard de- 33
viations were calculated, the results are as shown Table 2. 3. Maaieh Al A., R., Flanagan R., D. Salt and Cosalvent Ef-
The average encapsulation efficiency (% E.E.) and standard devia- fects on Ionic Drug Loading into Microspheres Using
tions were calculated, the results are as summarized in Table 3. an O/W Method, Journal of Controlled Release, 2001,
70:169-181
Table 2: Production yield (%) of microsphere formulations
ES1 ES2 ES3 ES4 ES5 ES6
P020-DEVELOPMENT OF LIPOSOME
Yield 62.3 69.7 70.1 72.1 55.8 60.7 FORMULATION OF RIVASTIGMINE AND
S.D. 0,54 0,34 0,78 0,87 1,12 0,67 INVESTIGATION OF TRANSPORT PROPERTIES
THROUGH MDCK AND CACO-2 CELL LINES
Table 3: Encapsulation efficiency of TS
88 Zelihagül Değim1, N. Başaran Mutlu1, Şükran Yılmaz2, Dinç
ES1 ES2 ES3 ES4 ES5 ES6 Eşsiz3
% E.E. 69.8 93.1 70.8 82.0 73.1 95.7
Gazi University,Department Of Pharmaceutical Technology,
S.D. 0.41 0.34 0.65 0.76 1.09 0.55 Faculty Of Pharmacy, Ankara, 063301
Food And Mouth Diseases Institute, Ankara, 065202
The mean particle size of TS loaded microspheres ranged Kafkas University, Department Of Pharmacology And
from 50μm - 81μm. The in vitro release profiles are shown Toxicology, Faculty Of Veterinary, Turkey3
in Figure 1.
INTRODUCTION
Alzheimer disease (AD) is one of the most common cause of
demantia which is characterised by decreased level of AChE
in brain. Rivastigmine is a carbamate derivative pseudo-irre-
versible cholinesterase inhibitor which can both inhibit AChE
and BuChE1, These drugs have been used for symptomatic
treatment of AD. MDCK (Madin Darby Canine Kidney) and
Caco-2 (Human Colon Carsinoma) cells are epithelial origi-
nated cells, which are widely used to test or determine oral
drug absorption. They are also increasingly used as an in
vitro blood-brain barrier (BBB) model in passive permeability
and membrane transport studies. This cell lines have similar
protein structures with BBB tight junctions and possess high
Figure 1. Release profiles of TS from microspheres transendothelial electrical resistance (TEER) values just like
brain cells2,3.
CONCLUSION
Finally, it was concluded that formulation parameters were In this study, liposome formulations of rivastigmine were
very important in achieving the high encapsulation efficien- developed. Cytotoxicity (MTT) tests were performed with
cy values espacially for hydrophilic drugs. When preparing different concentrations of the materials that were used in
microsphere formulations, addition of different salts into ex- the formulations. Transport studies were performed with
ternal phase, improve encapsulation efficiency of hydrophilic rivastigmine containing liposomes through Caco-2, MDCK
drugs. Moreover, it was observed that when the amount of cell cultures and also dialysis membrane. All results were

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

compared with rivastigmine solution. AChE inhibition effect was 35.4%. 1.5 mg/ml rivastigmine used for both Caco-2 and
of rivastigmine was also studied on mice and in vitro/in vivo MDCK transport studies according to MTT study results. Cu-
correlation studies were performed. mulative amount of rivastigmine penetrated through Caco-
2 and MDCK cells from apical to basolateral compartment
MATERIALS AND METHODS were found to be 60.9% and 54% respectively. Cumulative
Materials amount of rivastigmine released from liposomes and passed
Rivastigmine tartrate was obtained from Dr. Reddy’s (India) through Caco-2 and MDCK cells were found to be 3.6% and
and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium 15.2%(Figure 1). The apparent permeability coefficients (log.
bromide (MTT) were obtained from Sigma (Germany). Re- values) of rivastigmine solution and rivastigmine liposomes
agents which used in cell culture studies were obtained from were calculated as -1.96±0.04 cm/hour, -1.80±0.03 cm/hour,
Gibco Life Technologies (England). -1.03±0.29 cm/hour and -1.26±0.12 cm/hour for Caco-2 and
MDCK cell respectively. AChE inhibition values of rivastig-
Preperation of cell culture mine solution were calculated for both blood and brain in
MDCK cells were seeded on semipermiable polycarbonate mice treated with rivastigmine solution and liposomes (oral
filter inserts for 7 days (1.2 cm diameter, 0.4 µm pore size) and intraperitonally), and more effective inhibition was ob-
with 60000 cells/mL density. Caco-2 cells were cultured on served with rivastigmine liposomes.
polycarbonate membrane filter inserts for 21 days. Cells
were seeded on polycarbonate cell culture inserts (1.2 cm
diameter, 0.4 µm pore size) with a density of 80000 cells/ml.

MTT test
MTT test was performed for studying rivastigmine effect on
the viability of MDCK and Caco-2 cells. The serum effect on
cell viability was also investigated by using serum contain-
ing and serum free DMEM for 24 hours time period. Various
rivastigmine concentrations (1, 1.5, 3, 4.5, 6 µg/mL), were
studied in DMEM.

Transport Experiments
According to the MTT test results, the cell viability was
seemed to be higher with DMEM containing serum and it Figure 1: Cumulative amount of rivastigmine transported 89
was used for transport studies. Rivastigmin concentration through MDCK, Caco-2 cells and dialyse membrane.
was not exceeded to 1.5 mg/mL for 24 hours of transport
period. MDCK and Caco-2 monolayer containing membrane In conclusion more efficient and prolonged theraphy can be
was placed between donor and receptor compartments of possible for AD with rivastigmine liposomes after oral deliv-
vertical diffusion cells. 95% O2 and 5% CO2 were delivered ery and high in vivo/invitro correlation coefficients showed
to the system within the experiment period at 37ºC to main- that Caco-2 and MDCK cell lines are good tools for predicting
tain cell viability. Transport experiments were performed brain permeability of drugs.
from apical to basolateral compartment. Dialysis membrane
transport studies were also performed at 37ºC using pH 7.4 ACKNOWLEDGMENTS
phosphate buffer Samples were taken out at specific time This study was supported by grants from Gazi University
periods. Removed sample volume was always replenished (02/2006-17).
with fresh DMEM. The rivastigmine content of samples were
analysed by HPLC. REFERENCES
1. Williams BR, Nazarians A, Gill M. Clin.Ther. 2003;
AChE inhibiton experiments 25(6):1651-1663.
Ellman method was used for the determination AChE % in- 2. Waterbeemd HV, Gifford E. Drug Discovery, 2003;2:192-
hibition4. Rivastigmine solutions were applied to the mice 204.
both intraperitonally and orally. Blood samples were taken 3. Mahar Doan KM, et al. J Pharmcol. Exp. Ther.
at specific time periods. 20 µL of whole blood and 25 µL of 2002,303(3),1029-1037
DTNB were added to the 3 mL pH 8 0.1 M phosphate buffer 4. Ellman GL, et al. Biochem.Pharmacol. 1961; 7:88-95.
containing spectrophotometer cuvettes and autozeroed at
the wavelength of 412 nm. Then 20 µL of AChI was added
to the sample only and absorbans changes were recorded
during 6 minutes. AChE % inhibition values were then calcu-
lated considering the control group.

RESULTS AND DISCUSSION


Positive effect of serum on cell viability was shown by MTT
test results. Therefore rivastigmine concentration should
be below 1.5 mg/mL. Encapsulation efficiency of liposomes

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

P021-CNS DELIVERY OF THERAPEUTIC anticipated that these studies will lead to the developing a
PROTEINS IN LIVING CELLS new technology based on cell-mediated active delivery of
therapeutic polypeptides that attenuate neuroinflammation
Elena Batrakova1, Anna Brynskikh1, Yuling Zhao1, Shu Li1, R. and produce neuroprotection in patients with PD.
Lee Mosley2, Alexander Kabanov1, Howard Gendelman3

Center for Drug Delivery and Nanomedicine, Department


of Pharmaceutical Sciences, University of Nebraska Medical
Center1
Center for Neurovirology and Neurodegenerative Disorders,
University of Nebraska Medical Center2
Center for Drug Delivery and Nanomedicine, Center for
Neurovirology and Neurodegenerative Disorders, Department
of Pharmacology and Experimental Neuroscience, University
of Nebraska Medical Center3

INTRODUCTION
The need for delivery of therapeutic polypeptides to restore
brain tissues in PD cannot be overstated. Selective delivery
of antioxidants to the substantia nigra pars compacta (the
regions most affected in human disease) can potentially at-
tenuate oxidative stress and increase survival of dopaminer-
gic neurons in patients with PD. To this end, we developed ACKNOWLEDGMENTS
a bone-marrow derived monocyte (BMM) system to deliver We appreciate the support by the United States National In-
redox enzymes to PD affected brain regions in an animal stitute of Health grant RO1NS057748.
model (Scheme 1). To preclude BMM-mediated enzyme deg-
radation, catalase was packaged into a block ionomer com- REFERENCES
plex producing particles of nanoscale size, “nanozymes”. 1. Vinogradov S, Batrakova E, Li S, Kabanov A. Polyion com-
plex micelles with protein-modified corona for receptor-
MATERIALS AND METHODS mediated delivery of oligonucleotides into cells. Biocon-
90 Enzyme molecules were coupled with a cationic block co- jug Chem.1999; 10: 851-860.
polymer, polyethyleneimine-poly(ethylene glycol) (PEI-PEG), 2. Batrakova EV, Li S, Reynolds A, Thomas M, Bronich TK,
a synthetic polyelectrolyte of opposite charge, and cross- Kabanov AV, Gendelman HE. A macrophage-nanozyme
linked to obtain stable polyion complexes. Loading and re- delivery system for Parkinson’s Disease. Bioconjugate
lease of nanozyme from cell carriers were studied in vitro. A Chem. 2007; 18: 1498-1506.
neuro-immunological approach for the nanozyme delivery
was employed in MPTP mouse model of PD.
P022-DRUG RELEASE FROM PACLITAXEL
RESULTS AND DISCUSSION LOADED NIOSOMES AND EVALUATION
To build a protective nanocontainer for catalase, we synthe-
OF THEIR STABILITY AGAINST
sized a block copolymer by conjugation of PEI (2,000 Da) and
PEO (10,000 Da)1. The self-assembled catalase/PEI-PEG com- GASTROINTESTINAL ENZYMES
plexes, “nanozymes”, were rapidly taken up by BMM, retained
catalytic activity, and released in active form for greater than Zerrin Sezgin Bayındır1, Nilüfer Yüksel1
24 hours2. Significant amount of nanozyme was found in
MPTP-intoxicated brain in mice. Ankara Üniversitesi School of Pharmacy Department of
Pharmaceutical Technology, Ankara. Turkey1
Treatment of MPTP-intoxicated mice with catalase-loaded
BMM prevented neuroinflammation (microglial activation INTRODUCTION
and astrocytosis) at the level in healthy animals. Thus, MPTP Paclitaxel (PCT) is an antineoplastic agent isolated from
injections caused significant increase in GFAP staining par- the bark of Taxus brevifolia. Development of oral delivery
ticularly within the nigrostrial system. In contrast, treatment systems of PCT has attracted considerable attention1,2. In
of MPTP-injected mice with catalase-loaded monocytes pre- our previous study we developed several niosome formu-
vented neuroinflammation at the level in healthy animals. lations loaded with PCT. In this study in vitro drug release
Similar results were found for microglial activation. As a re- and release mechanisms of PCT from niosome formulations
sult, the neuroprotective effect of catalase-loaded mono- were investigated. The efficiency of niosomes to protect PCT
cytes in murine PD model was recorded. against gastrointestinal enzymes was researched for PCT
The obtained data suggest that “nanozyme”-loaded BMM mi- oral delivery.
grate across the BBB toward the site of pathology and release
the nanoparticles maintaining therapeutic concentrations of
the drug and providing prolonged antioxidative effect. It is

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

MATERIALS AND METHODS Table 2: PCT encapsulation efficiency of niosomes and % PCT
Film hydration method was used to incorporate PCT in nio- released at 24 hours.
somes. Briefly a thin film of surfactant (Table 1), dicetyl phos- Formula Code Entrapment efficiency % PCT released at 24 hours
phate, cholesterol and PCT was formed. After hydration of F1 96.6±0.48 23.3±1.96
the film with ultrapure water, unloaded drug was removed
by ultracentrifuge. F2 91.5±1.53 28.6±1.96
F3 86.4±2.04 19.9±0.87
The dialysis method was used to investigate PCT release from F4 20.3±0.62 32.1±1.39
niosomal formulations. Niosomal suspensions containing 60
F5 42.5±0.35 33.1±0.28
μg of PCT was placed into dialysis membrane bag and sunk
in the 50 mL of release medium, phosphate buffered saline F6 79.5±1.32 19.7±0.36
(PBS), pH 7.4 containing %0.1 (v/w) Tween 80 to maintain the F7 12.1±1.36 27.6±1.52
sink condition. The samples were placed in a water bath and
F8 81.1±0.12 23.8±0.88
shaken at 100 rpm 37 ºC. 1 mL of samples were taken from
the release medium at predetermined time intervals up to
24 hours and fresh medium was added to the dissolution Release data have been applied to various mathematical
medium (n=3). The collected samples were directly analyzed models and Higuchi model fits best to the release data. It is
by HPLC. concluded that drug is released from the niosomes by a dif-
fusion controlled mechanism.
The stability of formulations in gastrointestinal (GI) enzymes
trypsin, α-chymotrypsin and pepsin was investigated3. 500
μl of niosome containing 30 μg/mL PCT or free PCT in PBS
pH 7.4 was mixed with 500 μl of 5 IU trypsin solution in
phosphate buffer pH 7.8 or 16 IU α-chymotrypsin solution
in phosphate buffer pH 7.8 or pepsin solution in simulated
gastric fluid (SGF) pH 1.2. The mixtures were incubated at 37
ºC, 3 hours for trypsin and α-chymotrypsin and 1 hour for
pepsin. At the end of the incubation time 100 μl trifluoroa-
cetic acid (TFA) was added to 100 μl of sample to stop the en-
zymatic reaction. Samples were diluted with methanol and
PCT content was measured by HPLC (n=3). 91
Figure 1: PCT release from the niosome formulations made
Table 1: Chemical names and HLB values of the used non-
with different non-ionic surfactants.
ionic surfactants in the niosome formulations.
Code Surfactant Chemical Name HLB
Pure drug preserved 96.1 % of its structure without degrada-
Value
tion in SGF containing pepsin, while this percent decreased
F1 Span 40 Sorbitan monopalmitate 6.70 to 75.8 % and 27.8 % in trypsin and chymotrypsin containing
F2 Span 20 Sorbitan monolaruate 8.60 phosphate buffer pH 7.8 respectively. In general niosomes
F3 Span 60 Sorbitan monostearate 4.70 seem to protect PCT against chymotrypsin degradation.
F4 Tween 20 Polyoxyethylene (20) sorbitan monoaruate 16.7 F1 formulation showed the best protection against all en-
F5 Tween 60 Polyoxyethylene (20) sorbitan monostearate 15.0 zymes.
F6 Brij 76 Polyoxyethylene (10) stearyl ether 12.4
F7 Brij 78 Polyoxyethylene (20) stearyl ether 15.3
F8 Brij 72 Polyoxyethylene (2) stearyl ether 4.90

RESULTS AND DISCUSSION


A negative correlation was observed between encapsulation
efficiency and drug release (Table 2). The release of PCT from
Span niosomes (F1-F3) is increased while the HLB (see Table
1) of the surfactants increased.

When we consider all formulations, drug release from Tween


niosomes seems faster (Figure 1). This result can be contrib-
uted to structural differences on niosome assembly. The bi-
layer structure upon hydration of the film would be looser
and more flexible due to high aqueous solubility of Tweens,
leading an increased permeability to solutes. It has been
stated that more hydrophobic Span surfactants form more Figure 2: Stability of pure PCT and PCT loaded niosomes in
compact niosomes when hydrated in presence of choles- GI enzymes.
terol.

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

In vitro studies showed that F1 formulation might be more with stirring added, the solution was stirred for 15 min and was
beneficial for oral drug delivery among other formulations. left to cool down the room temperature. The resulted sediment
F1 will be further evaluated for their in vivo behaviors. was collected and kept in an oven at 40°C for 24 h. Finally, the
powder was kept in a desiccator at room temperature.
ACKNOWLEDGMENTS
This study has been supported by TUBITAK (The Scientific Crystal modification of lactose using QESD method
and Technological Research Council of Turkey) under grant 30 g lactose was dissolved in 50 mL of 80°C distilled water.
SBAG-3742. The solution was then cooled and reached to 40°C. 100 mL
ethanol was gradually and with stirring added to the slou-
REFERENCES tion and was left to cool down the room temperature. The
1. Peltier, S.; et al. Enhanced oral paclitaxel bioavailability resulted sediment was collected and kept in an oven at 40°C
after administration of paclitaxel-loaded lipid nanocap- for 24h. Finally, the powder was kept in a desiccator at room
sules. Pharm. Res. 2006; 23(6): 1243-1250. temperature.
2. Kruijtzer, C. M. F. et al. Weekly oral paclitaxel as first-line
treatment in patients with advanced gastric cancer. An- Powder flow
nals of Oncology 2003; 14: 197–204. Flow of 20 g of powders (untreated and modified) was re-
3. Pardakhty, A.; Varshosaz, J.; Rouholamini, A. In vitro study corded by use of Erweka flow meter (n=6).
of polyoxyethylene alkyl ether niosomes for delivery of
insulin. Int. J. Pharm. 2007; 328: 130-41. Angle of repose
The repose angle of 20 g of powders (untreated and modi-
fied) was measured using the static method (n=6).
P023-Optimization of flow and Densities and compressibility indices
compressibility characteristics Bulk and tapped density of powders were measured using
the tapping method by a 100 mL cylinder (n=6). Hausner ra-
of starch and lactose using quasi- tio and Carr’s index were calculated for each powder.
emulsion solvent diffusion method
aimed for using in direct compression Tablet preparation and evaluation
200 mg of each powder (untreated or modified) was fully
Abbas Akhgari1, Mohammad Ali Dabbagh1, Hasti Sadeghi1 mixed with 200 mg naproxen and the resulted powder was
compressed in a single punch tabletting machine (Korsch,
92 School of Pharmacy- Ahwaz Jundishapur University of Medical Germany) under the same pressure. The hardness of tablets
Sciences- Ahwaz- Iran1 was measured using Erweka hardness tester (n=10).

Data analysis
INTRODUCTION One-way analysis of variance was used to assess the sig-
Direct compression is one of the methods for tablet prepara- nificance of the differences among different groups. Tukey–
tion. Despite its advantages over granulation methods it has Kramer post-test was used to compare the means of differ-
not been completely generalized as the latter approaches ent treatment groups. Results with P < 0.05 were considered
which could be due to the limitation of compressibility and to be statistically significant.
flow of the usual excipients and drug powders. There are
some approaches to modify the excipient characteristics Results and discussion
such as flow and compressibility aimed for using in direct According to Fig. 1 untreated starch did not have a measur-
compression. Quasi-emulsion solvent diffusion (QESD) is a able flow. But modified starch had flow equal to 37.78±3.44
crystal modifying method by which the characteristics of ex- g/sec and it was even higher than free flow excipient, Flow-
cipients and drug substances could be changed1. Starch and lack®. Increase in flow due to crystal modification could be
lactose are two excipients generally used for tabletting but due to adhering of the particles to each other and producing
with poor flow. The aim of present study was to modify the the large and relatively round crystals2. Scanning electron
flow and compressibility characteristics of these excipients micrographs of the resulted powder (Fig. 2) confirmed this
using quasi-emulsion solvent diffusion (QESD) method. theory and showed that the modification method produced
the larger crystal particles compared to untreated starch. In
MATERIALS AND METHODS accordance with starch, modified lactose showed more flow
Materials ability (7.55±0.6 g/sec) than untreated powder and round-
Corn starch (Darusazi Hakim, Iran), lactose (Merck, Germa- ness of modified lactose particles (Fig. 2) was the related
ny), Flowlack® (Aburayhan, Iran), naproxen (Zambon, Spain), mechanism.
ethanol (Simintak, Iran), and hydrochloric acid (HCl) (Merck,
Germany) were prepared from indicated sources. Bulk and tapped density of lactose and starch increased
with the crystal modification (Table 1) which could be due to
Crystal modification of starch using QESD method change in morphology of particles, more uniformity in par-
Firstly, 200 mL HCl 0.1 M was heated until 80°C and 5 g starch ticle shape and the resulted better particle packing3. Also,
was gradually added to this solution. The solution was then Carr’s index and Hausner ratio of modified powders were de-
cooled and reached to 40°C. 200 mL ethanol was gradually and creased which confirms the above phenomenon.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

As shown in Fig. 3, tablets made of untreated starch had no


significant hardness. However, tablets containing modified
starch had suitable crushing strength. This phenomenon
could be explained by adhering smaller particles to each
other in the modified starch and more primary consolidation
of particles which could enhance by compression force and
conclude harder tablets. However, the increase in hardness
of lactose tablets was not very significant by crystal modifi-
cation (p>0.05).

Figure 3: Hardness of tablets made of different excipients

CONCLUSION
The results of this study demonstrated that quasi-emulsion
solvent diffusion (QESD) is a suitable method for modifying
tablet excipients aimed for use in direct compression. Change
in crystal morphology of starch and lactose enhanced their
flow characteristics. Also, by use of QESD method compress-
ibility of starch powder was increased and the resulted tab-
lets had good hardness and strength.

ACKNOWLEDGMENTS
The authors acknowledge for supporting this project by a
grant from Vice Chancellor for Research, Ahwaz University of
Medical Sciences.

REFERENCES
Figure 1: Flow of untreated and modified excipients
1. Katta J, Rasmuson A. Spherical crystallization of benzoic
acid. Int J Pharm. 2008; 348: 61-69.
2. Liu L, Marziano I, Bentham A, Litster J. Effect of particle
properties on the flowability of ibuprofen powders. Int J 93
Pharm. 2008; 362: 109-117.
3. Yadav V. Preparation and evaluation of polymeric car-
bamazepin spherical crystals by solvent diffusion tech-
nique. Asian J Pharm.2009; 3: 18-25.

P024-FORMULATION OF CHITOSAN COATED


SODIUM ALGINATE MICROCAPSULES
CONTAINING MELOXICAM

Hakan Eroğlu1, Levent Öner2


Figure 2: SEM of (a) untreated starch, (b) modified starch, (c)
untreated lactose and (d) modified lactose HAcettepe University Faculty of Pharmacy Department of Basic
Pharmaceutical Sciences1
Table 1: The results of factors related to flow and compress- Hacettepe University Faculty of Pharmacy Department of
ibility of powders (mean ± SD) Pharmaceutical Technology2

Bulk Tapped Carr’s Hauser INTRODUCTION


Samples
density density index ratio Meloxicam (MLX) is a well known member of non-steroidal
anti-inflammatory drugs (NSAIDs) with its analgesic and
untreated com starch 0.49±0.02 0.61±0.03 20±1 1.24±0.02 antipyretic effects. It is generally used for the treatment of
rheumatoid arthritis osteoarthritis in elderly patients. In this
modified com starch 0.71±0.01 0.85±0.00 17±1 1.20±0.02 study, we have formulated microcapsule formulations pre-
pared by the combination of two natural polymers chitosan
untreated lactose 0.37±0.01 0.49±0.02 25±1 1.34±0.02 and sodium alginate.

modified lactose 0.70±0.01 0.90±0.04 23±2 1.30±0.04 MATERIALS and METHODS


Materials
Flowlach 0.62±0.01 0.71±0.01 13±1 1.22±0.10
The model drug MLX was from Dr. Reddy’s Laboratories. The

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

polymers used in the preparation of microcapsule formula- dition of methanol, the total volume was completed up to
tions were chitosan and sodium alginate (Sigma Aldrich, 50 mL. The final solution was again mixed for 2 hours over a
USA). For the hardening agent, calcium chloride anhidr (Car- magnetic stirrer. The concentration of MLX has been deter-
lo Erba, Italy) was used in the preparation of microcapsule mined by the HPLC method.
formulations.
In-Vitro Release Studies
Preparation of Microcapsule Formulations The in-vitro release tests were carried out with USP- Apparatus
Microcapsule formulations were prepared by using the ori- 1. 15 mg of MLX were placed in the basket. The test was initially
fice-ionic gelation method1. As the matrix material, chitosan performed in 750 mL of 0.1 N HCl solution (pH=1.2) for 2 hours
(Medium M.Wt.) and sodium alginate (Medium M.Wt.) were at 75 rpm. After this period, 250 mL of 0.2 M tribasic sodium
used. MLX was homogeneously dispersed in the solution of phosphate solution that has been previously conditioned at
sodium alginate at concentrations varying from 1% up to 37±0.5 oC was added in order to maintain the pH of the me-
2% (w/v) in water with Ultra Turrax T-25 homogenizer. Sepa- dium at 6.8. At certain time intervals starting from the begin-
rately, the hardening agent CaCl2 was dissolved in 1000 mL ning of the experiment, samples of 5 mL were withdrawn and
of water and mixed with 25 mL of previously prepared chi- immediately replaced with fresh medium. The amount of MLX
tosan solution in diluted acetic acid. MLX dispersion in sodi- was quantified with the HPLC method. The release profiles of
um alginate solution was added drop wise into the solution two different microcapsule formulations were compared with
containing CaCl2 and chitosan. The resulting droplets were each other by using model independent approach
mixed for 12 hours over a magnetic stirrer and filtered. They
were washed with deionized water and dried at 30oC until RESULTS AND DISCUSSION
they reach a constant weight. Preparation Efficiency
The preparation efficiencies of the microcapsule formula-
Preparation Efficiency tions were calculated as 92.64 ± 2.57% and 95.12 ± 1.92 % for
The preparation efficiencies of the microcapsule formula- the formulations prepared by using 1% (w/v) and 2% (w/v)
tions were calculated with the formula: Yield=A/B * 100 sodium alginate, respectively.
where A is the total weight of the microcapsules and B is the
sum of the total weights of polymer and MLX used for prepa- Surface Morphology
ration of microcapsules. The microcapsules prepared by using sodium alginate at
different concentrations have similar surface properties.
Surface Morphology The particles neither had perfect circular shape nor smooth
94 The surface morphologies of the microcapsule formulations regular surface.
were investigated with Scanning Electron Microscopy (SEM).
The SEM investigations were further evaluated with JSM Particle Size Analysis
5600 device in 20 kV. The particle size distributions for microcapsule formulations
prepared with 1% (w/v) sodium alginate and 2% (w/v) sodi-
Particle Size Analysis um alginate polymer solutions were calculated as 505 ± 1.38
The particle size distribution of the microcapsule formula- µm and 740 ± 1.39µm, respectively.
tions containing MLX has been evaluated by using Ende-
cotts standard sieves having mesh aperture size in a range Assay of MLX
of 125-810 µm. The linearity of the calibration curve has been established
within the concentration range of 1-40 µg/mL for MLX
Assay of MLX (r2=0.9999) and the retention time for MLX was recorded as
The in-vitro quantification of MLX was carried out by using 2.62 min. The method was found to be accurate, precise, spe-
a HPLC equipped with an UV detector after the modification cific and sensitive for MLX.
of previously used method in literature2. The mobile phase
was composed of 50 mM phosphate buffer –acetonitrile- Determination of the MLX Content
methanol mixture at the ratio of 50:25:25 (v:v:v) The injec- MLX contents of the microcapsule formulations were deter-
tion volume and flow rate of the mobile phase was set as mines as 99.51 ± 1.38 % and 100.83 ± 2.46% for the formula-
15 µL and 1 mL/min, respectively. The UV detection of the tions prepared with sodium alginate polymer solution at the
samples was recorded at wavelength (λ) of 363.4 nm. ACE concentrations of 1% (w/v) and 2% (w/v), respectively.
C18 reverse phase column was used for separation (150 x 4.6
mm). For the validation of the in-vitro HPLC method, linear- In-Vitro Release Studies
ity, accuracy, precision, specificity and sensitivity parameters The release profiles of the formulations clearly indicate that
have been investigated. negligible release of MLX from microcapsule formulations
was determined within the first 2 hours of the experiment
Determination of the MLX Content where the pH of the release medium was 1.2. On the other
Microcapsules containing exactly 15 mg of MLX have been hand, as the pH was shifted to 6.8, release of MLX from micro-
accurately weighed and added into 25 mL of HCl solution capsule formulations starts depending on the increase in the
(0.1N). They were mixed for 2 hours and afterwards 8 mL solubility of chitosan coating. The increase in sodium alginate
of tribasic sodium phosphate was added. This solution was polymer concentrations from 1% (w/v) to 2% (w/v), governs
mixed for 4 hours and at the end of this period, by the ad- the release pattern of MLX from microcapsule formulations

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

as soon as the pH of the release medium is increased. with CTS containing different concentrations of CaCl2 (0.65,
1.25 and 2.5; samples 1, 2 and 3) were prepared and evalu-
CONCLUSION ated.
The results clearly indicate that release of MLX may be modi-
fied by using sodium alginate at varying concentrations. Also In vitro release studies
chitosan coating successfully prevented MLX release in gas- To compare the drug release from MPs under different pH
tric conditions. These formulations should further be used in conditions, the experiment were performed in buffer solu-
in-vitro/in-vivo correlation studies. tions at pH 2.0, 4.5, 6.8 and 7.4. The released 5-FU in different
time intervals was quantified spectrophotometrically (266
REFERENCES nm; Lambda 16, Perkin Elmer, USA).
1. Gonzales-Rodriguez M.L., Holgado M.A., Sanchez-Lafu-
ente C., et al. Alginate/chitosan particulate system for Ex vivo mucoadhesion studies and in vivo biodistribution
sodium diclofenac release. Int. J. Pharm. 2002, 232: 225- The muco/bioadhesive potential of the prepared MPs was de-
234. termined by modified everted sac method2. Unfasted Wistar
2. Nemutlu E. and Kır S. Method development and valida- rats, weighed ~400g were sacrificed, the colons were removed
tion for the analysis of meloxicam in tablets by CZE. Jour- and flushed with phosphate buffer saline pH 7.4 with 200 mg/
nal of Pharmaceutical and Biomedical Analysis 2003, 31: dl glucose (PBSG). Six cm segments were everted, filled with
393-400. 2ml of PBSG and introduced in conical tubes containing 40
mg of MPs, previously dispersed in 5ml PBSG. The sacs were
incubated at 37 °C on horizontal shaker (50 rpm, 30 min; Shak-
P025-MUCO/BIOADHESIVE PROPERTIES AND er Unitronic OR, Selecta, Spain). Afterwards, the sacs were re-
moved and the remaining MPs were freeze-dried (- 40 °C, 0.75
IN VIVO BIODISTRIBUTION OF CHITOSAN-CA- mBa, period 24h; Labconco, FreeZone 2.5L Feeze Dry System,
ALGINATE MICROPARTICLES LOADED WITH 5-FU USA). Colon segments in PBSG (blank sample) were used as
control. The muco/bioadhesive potential expressed as binding
Marija Glavas Dodov1, Sema Çalış2, Sonja Kuzmanovska3, efficiency (%) was calculated using the following equation:
Maja Simonoska Crcarevska1, Vesna Petrovska1, Nikola
Geskovski1, Katerina Goracinova1 binding efficiency (%) = MPs - (fdS-fdPBSG)/MPs x 100
Department of Pharmaceutical Technology, Faculty of
MPs - mass of the microparticles
Pharmacy, University Ss Cyril & Methodius, Skopje, Macedonia1
Pharmaceutical Technology Division, Faculty of Pharmacy,
fdS - mass of the sample after freeze-drying 95
fdPBSG - mass of blank sample after freeze-drying
Hacettepe University, Ankara, Turkey2
Department of Patophysiology, Medical faculty, University of
In order to determine the fate of prepared particles in vivo
Ss Cyril & methodius, Skopje, Macedonia3
upon oral administration, optimized formulation (sample 3)
INTRODUCTION was labeled with Technetium-99 (99mc-Tc). A dose of 400 µCi
The delivery of chemotherapeutic agents for the treatment of of 99mTc-labeled MPs dispersed in purified water was admin-
colon cancer using polymeric microparticles (MPs) has become istered orally to fasted Wistar rats. Imaging was performed at
one of the most popular areas of research because of the possi- different time intervals using an E-Cam Single Head gamma
bilities of reducing toxicity, enhancing controlled release activity camera (Siemens, Germany) in an order to determine the dis-
and also localizing the drug delivery. Working on this rationale, tribution of activity in GI tract.
chitosan-calcium-alginate MPs loaded with 5-FU were previously
prepared and characterized1. The aim of this contribution was to RESULTS AND DISCUSSION
investigate the binding efficiency of the prepared particles on rat Using one-step spray-drying process spherical particles with
intestinal mucosa and to test the biodistribution of this new mi- mean size of ~7 µm, narrow, unimodal particle size distribution,
croparticulated system after peroral administration in rats. high 5-FU loading efficiency, positive surface charge and pH
dependent swelling were prepared. The results from in vitro dis-
MATERIALS AND METHODS solution studies pointed that the rate and extent of drug release
Materials was a function of i). incorporation of acid-resistant particles of
5-FU was supplied as a gift sample from EBEWE Pharma, Ger- 5-FU into CTS-Ca-ALG matrix and ii). the degree of cross-linking
many. Chitosan (CTS) was obtained from Fluka, Switzerland and between both polymers; drug release decreased with increasing
sodium alginate (ALG) was kindly donated by FMC BioPolymer, CaCl2 concentration and. Formulation prepared with 2.5% CaCl2
Norway. Hydroxypropyl methylcellulose phthalate (HP-55) was (sample 3) has properties to minimize drug absorption in the
purchased from Shin Etsu Chemical Co. Ltd., Japan. Calcium upper part of GIT and at the same time could provide high drug
chloride (CaCl2) was obtained from Alkaloid, Macedonia. concentration over a prolonged period of time in colon region.

Preparation procedure All prepared formulation showed excessive adhesion to rat


Using one-step spray-drying process (Buchi 290, Mini Spray colon mucosa probably due to the design of the matrix and
Dryer, Swiss) cross-linked hydrogel MPs composed of acid- preserved flexibility of the polymer chains as well as posi-
resistant (HP-55 coated) particles of 5-FU in ALG core coated tive surface charge and significantswelling of the particles in
phosphate buffer at pH 7.4

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

cally. Theophylline is a hydrophobic drug and has a narrow


therapeutic index. Therefore, the dose must be monitored
and sustained release dosage forms have to be prepared. The
aim of this study was to load theophylline into PLGA micro-
spheres and to characterize them in vitro for the oral delivery
of the drug. In order to optimise the preparation process, two
formulation variables were investigated: 1) Concentration of
polymer solution by variation in the weight of PLGA polymer
and 2) The rate and duration of agitation during emulsifica-
tion. The prepared microspheres were tested for the particle
Figure 1: Interaction of MPs with rat colon segments size, zeta potential, encapsulation efficiency, in vitro release
studies and cell culture studies for cytotoxicity.
Also, the amount of interacted MPs with colon mucosa sur- MATERIALS AND METHODS
face decreased with increasing the concentration of CaCl2. Materials
Theophylline was kindly provided by Nobel İlaç Sanayi,
Our biodistribution data pointed that the MPs are traveling slow- İstanbul, Türkiye. Polyvinyl Alcohol (PVA) (Mw 30,000-70,000)
ly through GIT with higher concentration in the small intestine was purchased from Sigma (USA). 50:50 Poly(D,L-lactide-co-
after 5 hours and more than 65% of administered quantity pres- glycolide)–PLGA (Resomer RG503) was obtained from Boeh-
ent in the colon after 8 hours. 24 hours post administration ~55% ringer Ingelheim (Germany).
of the administered radioactivity was still present in the colon.
Several reasons might be involved in the pattern of MPs distribu- Preparation of PLGA Microspheres
tion, among them the particle size and surface properties of the PLGA microspheres were prepared using the oil-in-water
carrier as well as pH dependent swelling could probably lead to (o/w) emulsion/solvent evaporation method. PLGA was dis-
increased interaction with biological surfaces and muco/bioad- solved in dichloromethane and then injected using a syringe
hesion, thus providing the prolonged residence time of the car- into PVA solution. The prepared microspheres were then fil-
rier at the site of action and better therapeutic efficiency. tered under vacuum filtration using 0.45µm filter paper to
collect the microspheres and they were left to dry at room
CONCLUSION temperature for 48 hours.
Using an innovative microencapsulation technique, particles
with optimal physicochemical and biopharmaceutical prop- Particle Size Distribution and Zeta Potential
96 erties were prepared. Also, presented studies confirmed the Particle size distribution of microspheres and zeta potential
potential of CTS-Ca-ALG MPs for local colon delivery of 5-FU were determined using a Malvern Mastersizer (Malvern In-
and efficient treatment of colon cancer. struments Ltd, UK). The samples were analysed and average
particle size was expressed as the mean diameter and mean
REFERENCES zeta potential.
1. Glavas Dodov, M., et al., WGA-conjugated chitosan–Ca–
alginate microparticles for local colon delivery of 5-FU: Encapsulation Efficiency
Development and in vitro characterization. Int. J. Pharm. Drug loaded microspheres was dissolved in 1M NaOH, then
(2009), doi:10.1016/j.ijpharm.2009.06.037. vortexed for 1hour and kept overnight in dry, dark environ-
2. Santos, C. A., et al., Correlation of two bioadhesion as- ment. The following day, the solution was vortexed and 1M
says: the everted sac technique and the CAHN microbal- HCL was added. The solution was vortexed and the absor-
ance. J. Control. Release (1999), 61(1-2), 113-122. bance was measured using the UV spectrophotometer to
determine the concentration of theophylline in solution. The
blank microspheres were used as control.
p026-DEVELOPMENT AND
CHARACTERIZATION OF THEOPHYLLINE In Vitro Release Study
The in vitro release of theophylline from PLGA microspheres was
LOADED PLGA MICROSPHERES
determined in distilled water. Microspheres were suspended
in distilled water in centrifuge tube and placed into a shaking
Dania Shamil1, Can Sarısözen2, Sibel Bozdağ Pehlivan2, water bath at 37.5oC at 50cpm. At predetermined time inter-
Abdul Basit1, Selma Şahin2, İmran Vural2 vals, samples were centrifuged for 5 minutes and supernatant
removed for analysis in UV spectrophotometer. Fresh replace-
University of London, The School of Pharmacy, 29-39 ment media was added to re-suspend the microspheres.
Brunswick Square, London WC1N 1AX, United Kingdom1 Cytotoxicity Studies
Hacettepe University, Faculty of Pharmacy, Pharmaceutical Cytotoxicity of formulations was evaluated by using Caco-
Technology Department 06100 Ankara Türkiye2 2 cells (ATCC, Rockville, MD, USA). The cells were grown in
DMEM. Cell viability was assessed by using MTT assay. Caco-
INTRODUCTION 2 cells in culture medium were seeded in 96-well plates and
PLGA microspheres are biocompatible and biodegradable following 24 h incubation (for adherence) at 37oC in 5%
which show reproducible and slow-release characteristics in CO2, 20 µL of microsphere suspensions for each formulation
vivo. Therefore, they are widely used in research and clini- were added into each well. After incubation, 25 mL of MTT

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

solution (5 mg/mL) was added to each well and the plates


were incubated for further 4 h. The produced formazan was
solubilized and kept overnight at 37oC, the optical densities
at wavelength of 570 nm were measured using microplate
reader (Spectramax Plus, Molecular Devices).

RESULTS AND DISCUSSION


There was a general pattern of increase in particle size when
increasing the PLGA polymer ratio (weight of polymer to
drug) from 3:1 to 15:1. However, there was no apparent rela-
tionship between the stirring speed and particle size. When
increasing PLGA polymer amount, the encapsulation effi-
ciency also increased. When increasing the speed of stirring/
mixing from 3000rpm to 5000rpm, the encapsulation effi-
ciency decreased consecutively for all three concentrations
of PLGA polymer
(Table 1).
Figure 1. Cell viability values of formulations (mean ± SD,
n=3)
For all three concentration of PLGA, there was a similar trend
seen in the cumulative release profiles. Initial burst release
was observed followed by a constant release of theophylline.
For cytotoxicity studies, it was shown that, the PLGA micro- p027-ENHANCED ORAL BIOAVAILABILITY
spheres loaded with theophylline presented no cytotoxicity OF LORATADINE VIA A PH-INDEPENDENT
during the MTT assay (Figure 1). INCLUSION COMPLEX
CONCLUSION Agnes Nacsa1, Zoltan Aigner1, Peter Sipos1, Tamas Martinek2,
In conclusion, theophylline loaded PLGA microspheres have Gabor Blazso3, Agnes Balogh3, Piroska Szabo Revesz1
been prepared using an o/w single-emulsion solvent evapo-
ration method and characterized in vitro. This work can be
University of Szeged Department of Pharmaceutical
considered for further studies on evaluation of the in vivo
Technology1
efficacy of theophylline PLGA microspheres. 97
University of Szeged Institute of Pharmaceutical Chemistry2
University of Szeged Department of Pharmacodynamics and
Table 1: Particle characteristics of formulations.
Biopharmacy3
Polymer: Drug Ratio at
3000 rpm INTRODUCTION
One of the prerequisites for successful oral drug therapy is
adequate intestinal absorption. Loratadine (LOR), a second-
3:1 10:1 15:1
generation antihistamine, chosen for the current studies, is
a BCS II drug1, i.e. it has dissolution- or solubility-limited ab-
Mean Particle Size ┴ SD 3.57 4.93 7.44 sorption. In this work we studied the stoichiometry of inclu-
(μm) ┴0.90 ┴1.24 ┴0.74 sion complexes of LOR with dimethyl-β-cyclodextrin (DIMEB)
and their bioavailability.
-22.8 -21.28 -23.13
Zeta Potential ± SD MATERIALS AND METHODS
±0.69 ±0.57 ±0.56
Materials
Encapsulation Efficiency % 13.1% 21.50% 81.76% LOR was kindly provided by TEVA Ltd., Hungary. DIMEB was
purchased from Cyclolab Ltd., Hungary. Other chemical re-
Polymer: Drug Ratio at agents were of analytical grade purity.
3000 rpm
Preparation of products
3:1 10:1 15:1 The products were prepared in two molar ratios (LOR:DIMEB
= 1:1 and 1:2) by the kneading method (KP). The physical mix-
tures were suspended with the same mass of 50% ethanol,
Mean Particle Size ± SD 4.54 6.313 8.57
and the solvent was evaporated off at room temperature. Af-
(μm) ±0.75 ±2.61 ±0.97
ter drying, the products were ground and sieved (100 µm).

-18.6 -20.8 -21.9 Diffusion ordered spectroscopy (DOSY)


Zeta Potential ┴ SD
┴0.56 ┴0.42 ┴0.57 NMR measurements were performed on a Bruker Avance III
400 MHz spectrometer with a triple resonance 2.5-mm capil-
Encapsulation Efficiency % 2.02% 14.01% 25.25% lary probe with a z-gradient coil in D2O solution at 298.1 K.

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

The DOSY measurements were performed by using the stim- ESI-MS


ulated echo and longitudinal eddy current delay sequence.
Parameters: dephasing gradient pulse length: 2 ms; diffusion
delay: 250 ms; gradient strength: 5 to 95% of the maximum;
number of steps: 32. Each measurement was run with 256
scans and 2K time domain points.

Electron spray ionization mass spectrometry studies (ESI-MS)


The ESI-MSD-ion trap (AGILENT 1100 LC-MSD TRAP SL ion-
trap MS) was operated under positive ion and auto MS-MS
mode with previously described parameters2.

In vitro dissolution
The modified paddle method with the USP dissolution ap- Figure 2: The ESI of the products revealed that the complex stoi-
paratus (Pharmatest, Germany) was used to examine 200 mg chiometry is 1:1.
samples of pure LOR or products containing 200 mg of LOR In vitro dissolution
in 900 ml of simulated intestinal medium (pH = 7.0 ± 0.1, T =
37 ± 0.1). The rotation rate was 100 rpm. The LOR contents of
the samples were determined spectrophotometrically (λSIM
= 248 nm).

Study of the effect of pH on the solubility


Seven buffer solutions were prepared with different pH values
between 1.2 and 7.51. Products containing 10 mg of LOR were ex-
amined in 900 ml of dissolution media at 37 °C. The paddle was
rotated at 100 rpm. After 2 h, removed samples were filtered and
the LOR concentrations were measured spectrophotometrically.

In vivo experiments
In male rats of the Wistar strain weighing 150 ± 5 g, oedema Figure 3: The results of the in vitro dissolution.
98 was induced in the hind paw by the subplantar injection of
10 μg/0.1 ml compound 48/80. The extent of the oedema was Study of the effect of pH on the solubility
determined with a plethysmometer (Ugo Basile, Harvard Ap-
paratus, Germany) 30 min after the subplantar injection. The
animals were divided into five groups with 6 rats per group.
Group I served as a control and received the vehicle. The fol-
lowing materials were tested in this study: LOR, DIMEB, KP
1:1, KP 1:2. The materials were given orally in suspension in
0.25% methylcellulose (MC) at a LOR concentration of 10 mg/
kg 1 h before the subplantar injection of compound 48/80.
Evaluation was performed by one-way analysis of variance.

RESULTS AND DISCUSSION


DOSY

Figure 4: illustrates the pH-dependence of the solubility of


LOR and the products.

In vivo experiments

Figure 5: depicts the volume of the oedema in the control


and treated groups.
Figure 1: The DOSY spectra of KP 1:1

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

CONCLUSION was controlled in order to prevent extremely wetting of the


The stoichiometry of the complex is 1:1, but this amount of powder bed of sorbitol. The flask was rotated in the water
DIMEB is not sufficient for pH-independent dissolution to bath at 65-70°C under vacuum until sorbitol appeared to be
be achieved. Thus, there is a need for excess DIMEB, i.e. the dry, and then, another portion of stock solution was sprayed.
1:2 product is required. The KP 1:2 decreased the oedema by This process was repeated until all the stock solution had
more than 50% relative to LOR alone, in consequence of the been applied. After addition of the final aliquot, evapora-
higher blood concentration, through better and smoother tion was continued until the powder was completely dry.
bioavailability. The material was further dried in an oven (Selecta Vaciotem,
Australia) under vacuum at room temperature overnight to
ACKNOWLEDGEMENTS remove the residual chloroform (Formulation K1-spray, Table
This work was supported by a Sanofi-Aventis Fellowship. 1). Proniosomes were obtained as dry powder.

REFERENCES Slurry Method


1. Khan MZ et al. Classification of loratadine based on the Span 60, cholesterol and dicethylphosphate were dissolved
Biopharmaceutics Drug Classification concept and pos- in chloroform. The whole of this solution was introduced into
sible in Vitro - in Vivo Correlation, Biol. Pharm. Bull. 2004; the round-bottom flask containing required amount of sor-
27(10):1630–1635 bitol powder. After this process, a slurry was formed in the
2. alázsik K et al. A new rigid cinchona modified (α-IQ) plat- round-bottom flask. The flask was attached to a rotary evap-
inum catalyst for the enantioselective hydrogenation of orator to evaporate chloroform at a temperature of 45 + 2 °C.
activated ketones: Data to the origin of enantioselection, Evaporation was continued until the powder was completely
J. Molecular Catalysis A: Chemical 2007; 272: 265-274 dry. The material was further dried in owen under vacuum
at room temperature overnight (Formulation K1-Slurry,
Table 1). Proniosomes were obtained as dry powder. In this
p028-PRELIMINARY STUDIES ON method, different formulations were prepared (i)changing
the amount of chloroform (K2 formulation), (ii)using differ-
PRONIOSOME FORMULATIONS ent particle size range of sorbitol (K3 formulation), and (iii)
changing the ratio of surfactant mixture:sorbitol (K3-Medi-
Nilüfer Yüksel1, Murat Oral1 um, K4 and K5 formulations).

Ankara University School of Pharmacy Department of Table 1 :Composition of the proniosome formulations.
Pharmaceutical Technology Ankara-TURKEY1 Ingredients K1 K2 K3 K4 K5 99
Span 60 49.28 49.28 49.28 49.28 49.28
INTRODUCTION
Proniosomes are in the form of water soluble carrier par- Cholesterol 44.23 44.23 44.23 44.23 44.23
ticles coated by a film of non-ionic surfactant and choles- DCP 6.490 6.490 6.490 6.490 6.490
terol. Proniosomes can be hydrated easily prior to use and
Chloroform 10 5 10 10 10
converted to niosomal dispersions. In addition, they can be
prepared as per-oral dosage forms like tablets, capsules and Sorbitol 1000 1000 1000 2000 3000
beads. Because of being dry powders, proniosomes improve Particle size range of sorbitol used in the formulations (mm):
physical and chemical stability of niosomes. In this study, we K1: 0.25-0.75, K2: 0.25-0.75, K3-Large: 0.75-1.00, K3-Medium: 0.25-0.75,
aimed to evaluate two methods by which proniosomes are K3-Small: 0.25-0.75, K4: 0.25-0.75, and K5: 0.25-0.75
formed, and to investigate the effects of the formulation and
process variables on the characteristics of proniosomes. Determination of Niosome Size, Zeta Potential and Shape
The proniosomal powder were hydrated at 80°C and vor-
MATERIALS AND METHODS texed (Vortex mixer Heidolph D-91126, Germany) for 2 min.
Materials The effect of different vortexing times were evaluated on K4
Sorbitol (Merck, Germany) was used as the water soluble formulation. The size and zeta potential of the formed nio-
carrier. A non-ionic surfactant, Span 60 (Sigma Aldrich, USA) somes was measured using dynamic light scattering (DLS)
and cholesterol (Sigma Aldrich,USA) were used to form nio- and phase analysis light scattering (PALS) with a zetasizer
somes. Dicetyl phosphate (Sigma Aldrich,USA) is used as the (Malvern Zetasizer Nano ZS, UK). Leica Optical Microscope
negative charged agent for the stabilization of niosomal dis- (DM4000B, USA) was used to image the niosomes.
persion. All other chemicals were of analytical grade.
RESULTS AND DISCUSSION
Methods Spray and slurry methods were compared for particle size
Spray Method and shape of niosomes obtained by hydrating the pronio-
A 100 mL round bottomed flask containing required amount somes (K1-spray and K1-slurry).
of sorbitol powder was attached to a rotary evaporator (Ro-
tavapor, Buchi R-200,Germany). Span 60, cholesterol and The niosomes formed by the spray method are smaller than
dicethylphosphate were dissolved in chloroform. This stock those formed by the slurry method although the greater
solution was intermittently sprayed onto the surface of sor- amount of aggregated vesicles are seen in micrograph (Fig.
bitol powder. During spraying period the rate of application 1). Slurry method was chosen as the preparation method of

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

another proniosome formulations. The amount of chloroform Zeta potentials of all the proniosome-derived niosomes were
in K2 formulation was lowered (Table 1). Chloroform affected negative and between -32,2 to -68,8mV due to dicetylphos-
the consistency of the slurry during preparation resulting in phate (Table 2 and 3). These values indicates thermodynami-
bigger niosomes with a more heterogenous particle size dis- cally stabilized preparations.
tribution (Table 2). Optimum particle size of sorbitol used
as carrier for niosomes was also evaluated (K3 formulations, CONCLUSION
Table 2). The niosomes formed from proniosomes prepared According to the experimental findings, slurry method was
with medium particle size of sorbitol had monomodal par- found to be more convenient method for producing pronio-
ticle size distribution and average particle size was 580.6nm. somes by using lower amount of chloroform and sorbitol at
In the formulations of K3-Medium, K4, and K5, the ratio of the medium size. Niosomes could be formed without vortex
surfactant mixture:sorbitol was changed. Increasing amount mixing indicating the convenience of the proniosomes for
of sorbitol caused to increase of particle size of proniosome- per-oral administration as a solid dosage form (tablets and
derived niosomes (K4 and K5 formulations, Table 2). capsules) or a solution. In light of this observations, drug
loaded proniosomes will be prepared with the different type
of surfactants in an attempt to enhance drug solubility.

REFERENCES
1. Chengjiu H, Rhodes D, Proniosomes: A novel drug car-
rier preparation, Int. Journal of Pharm. 185 (1999) 23-35.
2. Solanki B, Parikh R, Formulation and optimization of
proxicam proniosomes by 3-factor 3-level Box Behnken
design, AAPS PharmSciTech 2007, 8 (4) Article 86.
3. Varshosaz et al, Sorbitan monopalmitate based pronio-
Figure 1: Optic micrographs of the niosomes obtained from somes for transdermal delivery of clorpheniramine
K1 formulations prepared by two different methods. maleate, Drug Delivery 12:2, 75-82

Table 2 : Zeta potentials and particle sizes of the niosomes


obtained from proniosomes. P029-OPTIMISATION STUDIES FOR MAKING
Peaks (nm) (% area of peak
Zeta potential FREE-FLOWING COMPACTABLE POWDER AND
indicating the volume of particle GRANULE CONTAINING RAMIPRIL
100 Formulation (mV±S.E.)
population)
1st peak 2nd peak Buket Aksu1, Ayça Yıldız2, Özgen Özer3, Tamer Güneri3
K1-spray 117.9 (100) - -32.2±0.482
K1-slurry 1156 (97.7) 125.3 (2.3) -35.7±0.99 Ege University, Pharmacy Faculty, Department of
Pharmaceutical Technology, Bornova-İzmir-Turkey Santafarma
K2 1326 (94.3) 314.9 (5.7) -46.7±0.698
Pharmaceuticals, 34382, Istanbul-Turkey1
K3-Large 1771 (89.6) 154.9 (10.4) -44.6±1.12 Istanbul University, Pharmacy Faculty, Department of
K3-Medium 580.6 (100) - -44.9±1.13 Pharmaceutical Technology, 34116, Istanbul-Turkey2
K3-Small 882.2 (62.9) 171.8 (14.6) -39.9±1.55 Ege University, Pharmacy Faculty, Department of
Pharmaceutical Technology, Bornova-İzmir-Turkey3
K4 1189 (100) - -67.1±3.04
K5 1178 (98.5) 158 (1.5) -57.0±1.68 INTRODUCTION
Granulation is a process by which fine powders are agglom-
The time of vortex mixing was changed to see its effect on erated into larger particles using a liquid binder. Flow prop-
particle size of niosomes after hydrating proniosomes. Par- erties of granules are of critical importance in fabrication of
ticle size of the niosomes was not considerably changed de- reproducible tablet formulations1. An optimum flow of gran-
pending on the vortex process indicating that niosomes can ules should be achieved to ensure uniform feed from funnel
also be formed without vortex mixing (Table 3). into dies to obtain tablet formulations with acceptable con-
tent uniformity, weight variation and physical consistency2.
Tablo 3 : The effect of vortex mixing time on particle size of In our study, therefore, with an eye to developing optimum
niosomes derived from proniosomes (K4). tablet formulations of ramipril which is an angiotensin-con-
Formulation Peaks (nm) (% area Zeta potential verting enzyme inhibitor, granule formulations were fabri-
of peak) (mV±S.E.) cated by wet granulation technique and the effect of some
parameters such as granule size, type and amount of lubri-
K4-without vortex 1046 (100) -68.8 ± 50
cants on their flow characteristics were investigated.
K4-2 min. vortex 1189 (100) -67.1 ± 3.01
K4-10 min. vortex 1227 (100) -33.3 ± 0.946 MATERIAL AND METHODS
Materials
Ramipril was purchased from Santa Farma Drug Company.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

Hydroxypropyl methylcellulose (HPMC, Collorcon), Magne- ing amounts of both lubricants. Granules which were sieved
sium stearate (Faci/Italy), Sodium stearyl fumarate (JRS Phar- through the 1.20 mm screen and lubricated with sodium
ma). All the other excipients were of pharmaceutical grade. stearyl fumarate showed less caking characteristics than
others due to their lowest caking strength (Figure 2).
Fabrication Granule Formulations
Ramipril (5 mg) and other additives (Lactose monohydrate,
pregelatinised starch, Ac-Di-Sol, sodium hydrogen carbon-
ate, ferrum oxide) were granulated in “High shear granulator”
(ProCept, Belgium) HPMC solution and passed through an
oscillating granulator fitted with 0,250 mm screen, and dried
in a hot-air oven at 60ºC. After re-sieving using 0.80 mm and
1.25 mm screens, the granules were blended with different
amount of two distinct lubricants (magnesium stearate and
sodium stearyl fumarate) for 5 min (Table 1).

Granule Flow Characterisation Figure 1: Effect of granule size and different amount of (a)
Granule characterization was performed on unlubricated Magnesium stearate, (b) Sodium stearyl fumarate on cohe-
and lubricated granules using Powder reometer (TA-XTPlus, siveness of granule formulations
Stable Micro Systems, UK). The instrument was calibrated for
force and distance measurements. A cylindrical vessel was
partly filled (140 mL) with each granule formulation and
set on the platform of the apparatus. A helical blade which
moves into the granule bed was used to probe and to mea-
sure a) the cohesion (Cohesion test), b) the caking (Caking
test) and c) the changes on granule flow properties due to
the increasing and decreasing flow speeds (Powder flow
speed dependence test).

Table 1. Formulation parameters of ramipril granule formula-


tions
Amount of Lubricant (%) 101
Formulations Sleve mesh size Magnesium Sodium stearyl
starate fumerate
F1 0.8 - -
Figure 2: Effect of lubricant type and amount on cake strength
F2 0.8 0.75 - of granule formulations
F3 0.8 1.00 -
F4 0.8 - 0.60 Granule flow properties may change with increasing and de-
creasing flow speeds. Powders/granules may become more
F5 0.8 - 1.20 resistant to flow as it is forced to flow faster or indeed it may
F6 1.2 - - become more free flowing as the flow speed increases. While
F7 1.2 0.75 - marginal or no change of the compaction coefficient with
flow speed would show that the granule is flow speed in-
F8 1.2 1.00 -
dependent, an increase or decrease in the compaction coef-
F9 1.2 - 0.60 ficient as the test speed dependence may result inadequate
F10 1.2 - 1.20 feed from bulk container into dies during tablet preparation.
Flow stability also gives important information about the
RESULTS AND DISCUSSION flow resistance of the granules. Compaction coefficients of
Cohesion is tendency for particles of granules to cling to- the lubricated granules were not significantly change with
gether. Cohesion test results showed that the addition of the increasing of tip speed (Figure 3) and their flow stability
increasing amounts of both lubricants decreased the co- values were found to be very close to 1.00 (Figure 4). On the
hesiveness of granule formulations. Whereas the screen other hand, granules which were sieved through the 1.2 mm
size (0.8 mm and 1.2 mm) did not significantly change the screen and lubricated with 0.6 % sodium stearyl fumarate
cohesiveness of the granules, sodium stearyl fumarate as a had lowest compaction coefficient.
lubricant was found to be more effective than magnesium
stearate (Figure 1).

Caking is the tendency of granules to form larger agglom-


erates during storage and transportation. Caking test indi-
cated that the caking strength decreased with the increas-

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

P030-ENHANCEMENT OF THE RELEASE OF


IBUPROFEN FROM SOLID DOSAGE FORM

Andres Lust1, Urve Paaver1

University of Tartu, Department of Pharmacy, Tartu, Estonia 1

INTRODUCTION
Release of poorly soluble drugs from oral solid dosage forms
in organism is guaranteed by endogenous surface-active
agents. For checking in vitro quality of pharmaceutical prep-
arations, bile acids are used for imitating in vivo conditions.
Bile acids are expensive and their usage for in vitro investiga-
tions is hardly reproducible because these are poorly soluble
and inadequately stable in various buffer solutions. Bile acids
are substituted with non-ionic surfactants that improve the
wettability of poorly soluble active pharmaceutical ingredi-
ent (API). The results of in vitro release should be in corre-
lation with pharmacokinetic characteristics and as a result
Figure 3: Powder flow speed dependence test results. Effect make possible to assess based on in vitro results the quality
of tip speed on compaction coefficent of granules of conventional solid oral pharmaceutical preparations con-
taining poorly soluble API.
Aims of the study
The aim of the study was
- to determine the effect of non-ionic surfactants with differ-
ent HLB value to the in vitro release of ibuprofen at different
pH conditions

MATERIALS AND METHODS


102 Formulation studied: Ibumetin® 200 (Nycomed) (ibuprofen -
II class by BCS, D0 = 80, solubility 0.01 mg/ml).

Surfactants: Polysorbate 60 – HLB 14.9, Polysorbate 80 – HLB


Figure 4: Flow stability values of unlubricated and lubricated
15.0, Polysorbate 40 – HLB 15.6, Polysorbate 20 – HLB 16.7,
granule formulations.
Triton X-100 HLB 13.4, Triton X-102 – HLB 14.4, Triton X-165 –
HLB 15.5, Triton X-305 – HLB 17.3 (Sigma-AldrichTM)
CONCLUSION
As a conclusion, sodium stearyl fumarate was found to be
The dissolution apparatus: Thermostat – Sotax AT7; Peristal-
the most effective lubricant for ramipril granule formulations
tic pump – Watson-Marlow 202 U/AA; Flow-through spec-
due to its suitable properties.
trophotometer – Ultrospec III (LKB Biochrom Ltd.); Software
– TDS (LKB Biochrom Ltd.).
REFERENCES
1. Kani T, Suzuki T, Tsukada M, Kamiya H. Influence of sur-
Testing conditions: Rotating paddle method. Speed of rota-
face-adhered nanoparticles and nanoporous structure
tion (r.p.m.) – 50. Dissolution medium – pH 1.2 and pH 4.5.
on bulk flowability of silica Powder Technology, 176,
Volume of dissolution medium – 900 ml Wavelength – 221
108-113, 2007.
nm. Temperature of dissolution medium - 37±0.5° C.
2. Navaneethan CV, Missaghi S, Fassihi R. Application of
Powder Rheometer to Determine Powder Flow Proper-
RESULTS AND DISCUSSION
ties and Lubrication Efficiency of Pharmaceutical Par-
Ibuprofen is used mainly for relief of acute pain. Ibuprofen
ticulate Systems. AAPS PharmSciTech, 6(3), Article 49,
is rapidly and completely absorbed after oral administration
2006.
through-out the gastrointestinal tract. In studies with solu-
tions for oral administration, peak plasma concentrations
were achieved 0.5 to 0.7 hours after administration [1]. For
conventional tablets tmax values of 1.3 to 2.7 h have been
reported2.

In the present study, in vitro release of ibuprofen at pH 1.2 and


4.5 was very low, 10.3±0.7% and 19.8±2.1% respectively.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

Figure 1: Ibuprofen release at pH 1.2 and 4.5 during 1 hour in Figure 3: Ibuprofen release at pH 1.2 during 1 h in the pres-
the presence of various concentrations of Tritons ence of various concentrations of Polysorbates.

Extent of dissolution increased in the presence of surfac- In case of Polysorbates, PS 80 (HLB 15.0) should be preferred.
tants. Tritons increased the release of ibuprofen more at pH In case of Tritons, the best results at low concentrations were
1.2 than at pH 4.5. Except for Triton X-100, that acted contrary obtained with Triton X-100 (HLB 13.4 – 0.035%) and Triton
(Figure 1). At pH 4.5 low concentrations (0.1%) of Polysor- X-165 (HLB 15.5 – 0.05%).
bates increase the release of ibuprofen on the average from
50% to 57.7% but not differentiate by HLB value. CONCLUSION
For enhancement the release of ibuprofen from solid dosage
It can be seen from Figure 2 and 3 that at pH 1.2 that to ob- forms at pH 1.2 Triton X-100, Triton -165, Polysorbate 80 and
tain the same release of active ingredient (AI), the amount of Polysorbate 60 were at most suitable.
Tritons is 5 to 10 times smaller than in case of Polysorbates. At pH 4.5 among the surfactants studied Triton X-100 in low
Also the presumption that a surfactant with higher HLB val- concentrations (below 0.1%) guaranteed the best result.
ue facilitates the release of API was not true.
ACKNOWLEDGMENT
This study was supported by the research fund of Estonian 103
Academical Society of Pharmacy.

REFERENCES
1. W.R. Gillespie, A.R. DiSanto, R.E. Monovich and K.S. Albert, Rel-
ative bioavailability of commercially available ibuprofen oral
dosage forms in humans, J. Pharm. Sci. 1982; 71: 1034–1038.
2. Bramlage P, Goldis A, Bioequivalence study of three ibu-
profen formulations after single dose administration in
healthy volunteers, BMC Pharmacology 2008; 8:18.

p031-VITAMIN E TPGS COATED


BIODEGRADABLE POLYMERIC
Figure 2: Ibuprofen release at pH 1.2 during 1 h in the pres-
ence of various concentrations of Tritons
NANOPARTICLES OF MELOXICAM FOR
COLON-SPECIFIC DELIVERY
The surfactants have mainly a wetting activity. The experi-
ments performed at pH 1.2 revealed that the increase of the Ceyda Tuba Şengel Türk1, Canan Hasçiçek1, Nurşin Gönül1
hydrophilicity of the amphiphilic surfactive polysorbates
does not correlate with the amounts of AI released. Addi- Ankara University Faculty of Pharmacy Department of
tion of the 0.5% and 1% Polysorbate 60 (HLB 14.9) into the Pharmaceutical Technology, Ankara, Turkey1
dissolution medium increased the ibuprofen release during
one hour from 67.5±2.9% up to 91.2±1.1%. For Polysorbate INTRODUCTION
80 (HLB 15.0) was concentration 0.5% enough for releasing Colon-specific drug delivery has been widely investigated
87.2±4.2% of drug. However, the higher HLB value Polysor- for the treatment of local colonic diseases such as Crohn’s
bate 40 (HLB 15.6) and Polysorbate 20 (HLB 16.7) addition diseases, ulcerative colitis, inflammatory bowel disease and
increased the release of the ibuprofen only by 65.6±1.1% colorectal polyps and cancer. The effect of non-steroidal anti-
and 84.8±1.7 %. inflammatory drugs (NSAIDs) on colorectal polyps and can-
cer arises interests in recent years1. Meloxicam, an enolic ac-

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

id-type NSAID, is used in the treatment of arthritis and other Table 2: Characteristics of the NPs.
joint diseases. Recent investigations about relationship be- Code EE (%) Sign (nm) PDI Zeta potential (mV)
tween meloxicam and colorectal cancer have reported that F1 80.17 178.30 0.146 -21.23
meloxicam has been regarded as a potential drug for the V1 80.63 178.60 0.149 -18.20
prevention and treatment of colorectal polyps and cancer2. V2 80.08 205.20 0.133 -23.30
Therefore, the development of a particulate carrier system
63.41
such as nanoparticles (NPs) which are designed to localize V3 79.96 0.279 -23.50
231.80
drugs at the target site for the therapy of colonic cancer is
valuable. The aim of this research is to develop a polymeric
The diameter of the NPs coated with higher than 2.5% coat-
nanoparticulate drug delivery system of Meloxicam for colon
ing ratio was found to be bigger than uncoated nanoparti-
cancer and investigate the effects of the coating process on
cles (p<0.05). This incerase in size was expected and attrib-
the physicochemical properties of the NPs. NP formulations
uted to the thickness of the coating. The size distribution of
which based on poly(D,L-lactide-co-glycolide) (PLGA) as a
the NP formulations was narrow (PDI: 0.133-0.149) except V3
biodegradable polymer were prepared by a combined tech-
coded NPs (PDI: 0.279). Zeta potential value is an important
nique that is based on salting-out and emulsion-evaporation
particle characteristics for the stability of the NP suspension.
steps and they were coated with Vitamin E TPGS which is a
All NPs were found to be stable in dispersion state, having
new water soluble derivative of natural Vitamin E by using
negative surface charges.
surface adsorpsion technique3.
The morphology of the prepared NPs was investigated by
MATERIALS AND METHODS
using transmission electron microscopy (TEM). As shown
Materials
in Figure 1, NPs have exhibited spherical shapes within the
Meloxicam (Ulkar Chem. Co., Turkey), PLGA (Mw: 40-75 kDa,
nanometer size.
copolimer ratio 50:50) and PVA (Mw: 9-10 kDa) (Sigma-Al-
drich, Germany), Vitamin E TPGS (Eastman Chemical, USA)
Dissolution rate studies on NPs were performed with static
were supplied and used as received.
method and resultant drug release profiles shown in Figure
Preparation of Nanoparticles
2, were found to be significantly different depending on the
PLGA and Meloxicam were dissolved in an organic mixture of
variation of the coating percentage for the formulation in-
acetone and methylene chloride. This solution was poured
vestigated (p<0.05). With an increase in the % coating ratio,
into a 25 ml aqueous phase containing PVA with magnesium
the drug release rate significantly decreased as a result of the
chloride and emulsified by ultrasonication in an ice bath for 3
thickness of the coating.
104 min at 80% output energy. Additional aqueous PVA was then
added under gentle stirring and stirring was continued for 24
h to insure complete organic solvents evaporation. NPs were
purified using ultracentrifugation and then freeze-dried for
48 h. For NPs coating, various ratios of Vitamin E TPGS was
dissolved in water and an adequate amount of lyophilized
NPs were suspended in this solution by ultrasonication for 30
sec. The suspension of NPs were then freeze-dried for 48 h3.

Characterization of Nanoparticles
Uncoated and Vitamin E TPGS coated PLGA NPs were evalu-
ated for their encapsulation efficiency (EE), zeta potential, Figure 1: TEM images of NPs coded F1.
particle size and polydispersity index, morphology and in-
vitro dissolution rate. Formulations, codes and the coating
percentages are given in Table 1.

Table 1: Formulation codes and the coating percentages.


Formulations Codes Coating percentage (%, w/w)
Uncoated NPs F1 0
Coated NPs V1 2.5
Coated NPs V2 5.0
Coated NPs V3 10.0

RESULTS AND DISCUSSION


The physicochemical NPs are summarized in Table 2. EE of
meloxicam within coated and uncoated NPs was between
79.96-80.63% and was not affected significantly by coating
process and percentages (p>0.5). Figure 2. In-vitro drug release profiles of the NPs.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

CONCLUSION MATERIALS AND METHODS


In this present research, it is evident that PLGA NPs were suc- Materials
cessfully coated with Vitamin E TPGS for the colonic delivery Triamcinolone acetonide (TA) was kindly provided by I.E.
of meloxicam. The physicochemical properties and the re- Ulagay Drug Company (Turkey). Carbopol 934® (C934) (No-
lease behaviour of the uncoated NPs were modified by coat- veon-USA), high molecular weight chitosan (HMW - viscosity
ing with various of Vitamin E TPGS at different percentages. > 400mPa), medium molecular weight chitosan (MMW - vis-
cosity 200mPa–400mPa) (Fluka Biochemika-Japan), HPMC-
ACKNOWLEDGMENTS Methocel E 4M (Colorcon LTD-USA) were used as bioadhesive
This research has been supported by Management of Scien- polymers. Poloxamer 407® (P407) (BASF-Germany) were used
tific Research Projects of Ankara University (project number for it’s good gelling property. Transcutol (TC) (Fluka Bioche-
is 07B3336003). mika, Japan) and propylene glycol (PG) (Sigma-Aldrich, USA)
REFERENCES were selected as penetration enhancer. As the commercial
1. Makhlof A, Tozuka Y, Takeuchi, pH-sensitive nanospheres product contained 0.1% TA (Kenacort–A orabase®) was used.
for colon-specific drug delivery in experimentally in- All other reagents were analytical grade.
duced colitis rat model, Eur. J. Pharm. and Biopharm.
2009; 72: 1-8. Preparation of the gel systems
2. Goldman AP, Williams CS, Sheng H, Lamps LW, Williams Bioadhesive gels containing P407 – C934 were prepared by
VP, et al. Meloxicam inhibits the growth of colorectal cold method2. Other mucoadhesive formulations were pre-
cancer cells, Carcinogenesis 1998; 19: 2195-2199. pared by dissolving chitosan (MMW or HMW) and HPMC
3. Şengel-Türk CT, Hasçiçek C, Gönül N, Development in diluted lactic acid solution. Compositions of the gels are
of PLGA nanoparticles surface modified with didode- seen in Table1.
cyldimethyl ammonium bromide: preparation and in-
vitro characterization, 36th Annual Metting and Exposi- Table 1: Compositions of the formulations
tion of the Controlled Release Society 2009; PO 898. P407 C934 TC PG TA
(w/w) (w/w) (w/w) (w/w) (w/w)
F3 15 2 - 12 0.1
p032-A COMPARISION OF MECHANICAL F4 20 1.5 - 12 0.1
PROPERTIES OF BUCCAL GEL FORMULATIONS HPMC chitosan TC PG TA
BASED ON DIFFERENT BIOADHESIVE (w/w) (w/w) (w/w) (w/w) (w/w)
POLYMERS CM-3 - 3(MMV) 10 12 0.1
105
CH-3 - 3(HMV) 10 12 0.1
Gülin Amasya1, Sinem Y. Karavana2, Tangül Şen1, Esra X1 1 3(MMV) 10 12 0.1
Baloğlu2, Nilüfer Tarımcı1 X2 2 3 (MMV)
10 12 0.1

Ankara University, Faculty of Pharmacy, Department of Viscosity measurements


Pharmaceutical Technology, 06100 Ankara, TURKEY1 The viscosity measurements were carried on using by digital
Ege University, Faculty of Pharmacy, Department of viscometer (Brookfield DV II) spindle no: T96 at 20 rpm and
Pharmaceutical Technology, 35100 İzmir, TURKEY2 at 25±1°C.

INTRODUCTION Texture profile analysis of TA gels


Bioadhesive drug delivery formulations are very beneficial, The mechanical properties of the gels were determined us-
since they provide controlled drug release overtime and ing software-controlled penetrometer, TA-XT Plus texture
localize the drug to a specific site of the body. They should analyzer (Stable Micro System, UK). From the resultant force-
exhibit suitable mechanical properties including acceptable time plot, mechanical parameters such as compressibility,
viscosity, easy of expression from the container, easy of ap- hardness, cohesiveness and elasticity were defined3.
plication, good spreadability, appropriate hardness and pro-
longed residence time in the oral cavity. These mechanical Mucoadhesion testing
properties may affect the performance of the formulations The mucoadhesive properties of formulations were evaluated
and their acceptance by patients1. with using TA-XT Plus texture analyzer (Stable Micro Systems,
UK). Freshly excised bovine buccal tissue was employed and
The aim of this study was to investigate the mechanical and three replicates were performed for each sample.
adhesive properties of buccal gel formulations containing
0.1% triamcinolone acetonide prepared with different poly- RESULTS AND DISCUSSION
mers. The results were also compared with the commercial pH and viscosity results are shown in Table 2. For chitosan
product contained the same amount of the triamcinolone based formulations, the pH values are lower than commer-
acetonide. cial product because of used lactic acid. The pH values of
P407 - C934 combinations were more applicable to the buc-
cal mucosa than chitosan based formulations and the com-
mercial product. Enhanced viscosity could suitable for easily
spread on the lesions as a thin film layer, the viscosity values

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

of developed formulations except CM-3 were also more fa- The cohesiveness parameter increases the performance of
vorable for topical application than commercial product. the product at the application site. The high value of cohe-
siveness provides full structural recovery following gel appli-
Table 2: pH, viscosity and bioadhesion work values of the for- cation. Elasticity which defines the rate of deformed sample
mulations and commercial product returns to its undeformed condition should be high. Accord-
Work of ing to the TPA results elasticity decreased when bioadhesive
Viscosity (Pa.s) polymer concentration increased for C934 based formula-
pH mucoadhesion
25 ± 1⁰C tions. Also negative correlation was obtained between the
(mJ/cm2)
F3 6.17 ± 0.01 175.56 ± 3.88 0.102 ± 0.02 molecular weight of the chitosan and elasticity values.
F4 6.43 ± 0.02 231.78 ± 2.05 0.082 ± 0.00
CM-3 4.53 ± 0.00 6.28 ± 0.36 0.004 ± 0.03 CONCLUSION
The results suggest that chitosan, Carbopol 934® and HPMC
CH-3 4.44 ± 0.01 29.00 ± 0.83 0.029 ± 0.06
can be used as vehicle for active substance to the oral cavity
X1 4.72 ± 0.01 10.06 ± 0.68 0.107 ± 0.00 because of their good textural properties. Also patients’ ac-
X2 4.17 ± 0.01 26.89 ± 1.24 0.115 ± 0.01 ceptance can be increased when compare with the commer-
Kenacort-A Orabase® 5.13 ± 0.01 200.00 ± 1.63 0.010 ± 0.03 cial product. In addition, different amounts of HPMC (1%-
2%) were improved bioadhesiveness of the chitosan based
Decreasing concentration of C934 from 2% to 1.5% (w/w), formulations without affecting the mechanical properties.
decreased work of mucoadhesion as seen Table 2. Adhe-
siveness of chitosan based gels was affected by polymer REFERENCES
molecular weight. According to these studies, the adhesive 1. Jones D S, Woolfson A D, Brown A F. Textural, viscoelas-
properties of the formulations increased as a function of tic and mucoadhesive properties of pharmaceutical gels
bioadhesive polymer concentration and molecular weight composed of cellulose polymers, Int J Pharm. 1997; 151:
of chitosan. The highest work of mucoadhesion was deter- 223-233.
mined for the formulation coded X2 (0.115±0.01 mJ/cm²). It 2. Scmolka I V. Artificial skin I. Preparation and properties
was also noted that, the mucoadhesive performance of the of Pluronic F-127 gels for treatment of burns, J Biomed.
chitosan based formulations was improved with HPMC. The Mater. Res. 6: 571-582.
textural profiles results are given in Table 3. Hardness and 3. Cevher E, Taha M A, Orlu M, Araman A. Evaluation of me-
compressibility values should be low for taking the formula- chanical and mucoadhesive properties of clomiphene
tions from the container and applying easily to the mucosa. citrate gel formulations containing carbomers and their
106 Increasing the hardness and compressibility values of the thiolated derivatives, Drug Deliv. 2008; 15: 57–67.
formulations depended on the viscosity. Commercial prod-
uct had the highest hardness and compressibility values
when compared with all the developed formulations. Also
HPMC (1% - 2%) were improved the viscosity without affect-
P033-PREPARATION OF A CHITOSAN-BASED
ing the hardness and compressibility. FORMULATION FOR THE TREATMENT OF
ORAL MUCOSAL DISORDERS
Table 3: Mechanical properties of gel formulations
Compressibility Ayşe Özdemir1, Gülçin Akça2, Z. Gülşen Öner3, Merve
Codes Hardness (N) ± SD Günbeyaz3, Sevda Şenel3
(N.mm) ± SD
F3 0.26 ± 0.03 0.90 ± 0.11
University of London, The School of Pharmacy, London, United
F4 0.59 ± 0.06 1.84 ± 0.14 Kingdom1
CM-3 0.02 ± 0.00 0.09 ± 0.00 Gazi University, Faculty of Dentistry, Department of Basic
Medical Sciences, Microbiology Laboratory, Ankara, Turkey2
CH-3 0.04 ± 0.00 0.12 ± 0.01
Hacettepe University, Faculty of Pharmacy, Department of
X1 0.02 ± 0.00 0.09 ± 0.01 Pharmaceutical Technology, 06100- Ankara, Turkey3
X2 0.03 ± 0.00 0.10 ± 0.00
INTRODUCTION
Kenakort-A Orabase® 0.87 ± 0.03 1.92 ± 0.13 The aim of this study was to develop a chitosan based for-
Cohesiveness ± SD Elasticity ± SD mulation for the treatment of oral mucosal infections. In ad-
F3 0.86 ± 0.03 0.91 ± 0.01 dition to its favorable properties such as biodegradability,
biocompatibility, and bioadhesivity, chitosan itself exhibits
F4 0.93 ± 0.03 0.96 ± 0.01 antimicrobial activity as well1. Gel formulations were pre-
CM-3 0.94 ± 0.02 1.05 ± 0.10 pared using different types of chitosan. An antimicrobial
agent, chlorhexidine gluconate (Chx) was incorporated into
CH-3 0.95 ± 0.01 0.98 ± 0.01
the formulations at 0.1 and 0.2% w/v concentrations. For-
X1 0.93 ± 0.03 1.02 ± 0.02 mulations were investigated in vitro for rheological, release
X2 0.96 ± 0.01 1.00 ± 0.02 properties, and antimicrobial activities.
Kenakort-A Orabase® 0.63 ± 0.12 0.96 ±0.06

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

MATERIALS AND METHODS


Preparation of chitosan gels
Various types of chitosan with different molecular weight and
solubility were used to prepare gel formulations (Table 1).
Table 1: Gel formulations used in the study
Formualation Chitosan Concn of
Chx DD** chitosan Solvent
Code Type MV* (kDa)
(%w/w) (%) (%w/w)
S - Chitopharm 1% (v/v)
S-01 0.1 S (Cognis, 50 - 1000 >70 3 aqueous
S-02 0.2 Germany) lactic acid
M - Chitopharm 1% (v/v) Figure 1: Flow curves of chitosan gels
M-01 0.1 M (Cognis, 300 - 2000 >70 2 aqueous
M-02 0.2 Germany) lactic acid
L - Chitopharm 1% (v/v)
L-01 0.1 L (Cognis, 500 -5000 >70 1 aqueous
L-02 0.2 Germany) lactic acid
Sig - Chitosan
1% (v/v)
Sig-01 0.1 medium
190 - 310 75 - 85 2 aqueous
(Sigma
Sig-02 0.2 lactic acid
adrich, USA)
Pro - Protasan
Pro-01 0.1 UP CI 213 Distilled
150 - 400 75 - 76 3
(Novomatrix, water
Pro-02 0.2 Figure 2: Release profiles of Chx from gels in distilled water
Norway)
at 37°C
Viscosity measurements
Antimicrobial activity
Viscosity measurements of gels were performed on a Brook-
Inhibition zone diameters obtained for chitosan gels are
field digital viscometer (Model DV-II+) at 25°C.
given in Fig. 3a-c. 107
In vitro release studies
Chitosan gels alone were shown to exert antimicrobial activity.
Release from gels was studied using Franz diffusion cells. The
Water soluble chitosan was found to be less effective against
samples were assayed for Chx at 253 nm using UV 1800 Shi-
all the strains investigated when compared to basic chitosans.
madzu Spectrophotometer.
Highest antimicrobial activity was obtained with the high mo-
lecular weight chitosan in absence of Chx whereas in presence
Antimicrobial activity studies
of Chx the activity seemed to be influenced by the viscosity.
In vitro antimicrobial activity and growth inhibition of for-
Increasing the concentration of Chx in the gels did not signifi-
mulations against various microorganisms were evaluated
cantly enhance the antimicrobial activity except for the Sig-02
using well diffusion method on their specific agar media
formulation. Chitosan has a synergic effect which offers an ad-
according to the measurement of the inhibition zones. Can-
vantage in delivery of antimicrobial agents at lower concen-
dida albicans (ATCC#90028) and an oral isolate of Candida al-
trations avoiding undesired side effects.
bicans strains were used as fungal pathogens, and standard
strains of Agregatibacter actinomycetemcomitans (A.a) and
Porphyromonas gingivalis (ATCC#33277) and were used as
bacterial pathogens which can be found in oral cavity.

RESULTS AND DISCUSSION


Viscosity measurements
Viscosity was found to increase significantly with the increas-
ing chitosan concentration as well as increasing molecular
weight (Fig. 1). Chitosan gels exhibited pseudoplastic behav-
ior which indicates a gel structure.

In vitro release studies


The release of Chx was found to be higher from the gels
prepared with low molecular weight chitosans. Release was
found to be drug concentration dependent(Fig. 2).

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

The main factor, which contributes in its instability, is hydrolysis


of the ester linkage3. This study demonstrates the rate degra-
dation of artesunate in different buffer solutions at a range of
pH values (2-10.5) and with different IV fluids. The temperature
dependency was also investigated at 5, 25 and 37 °C.

MATERIALS AND METHODS


Materials
Artesunate powder was provided by Apin chemical LTD
Company. All other solvents used were HPLC grade and wa-
ter was purified through a Milli-Q apparatus

Methods
The high performance liquid chromatography instrument
consisted of a high pressure pump (Waters 501, HPLC pump,
Millipore USA), a Apollo C18 (5 µm) column of 150 mm length
and ID of 4.6 mm, a Rheodyne Model 7125 syringe loading
sample injector with 20 µL sample loop, a ultraviolet detector
(Waters 484, Tunable Absorbance Detector, Millipore, USA).
Mobile phase used was phosphate buffer- acetonitrile (70:30)
50 mM at pH 7. The monitoring wavelength was 210 nm.

Artesunate powder was dissolved in buffer solutions sepa-


rately to make a concentration of 0.6 mg ml-1. Buffer solu-
tions (phosphate at pH 2.00, 2.50, 5.50, 6.50 and 7.50, citrate
at pH 3.50, acetate at pH 4.00 and 5.00, Bis Tris at pH 8.50 and
carbonate at pH 9.50 and 10.50) were employed to evalu-
ate the degradation of artesunate. These buffer solutions of
varying concentrations were prepared to evaluate the rate of
Figure 3: Inhibition zone diameters for gels a) C. albicans, b)
degradation of artesunate at a constant pH, temperature (37
P. gingivalis and c) A.a.
108 °C), and ionic strength of 0.5 mol.L-1 (NaCl). Concentrations
of residual drug were determined by HPLC. Degradation
CONCLUSION
rate of artesunate in 0.6 mg mL-1 in Hartman, glucose and
Chitosan gels can be suggested as promising systems not
normal saline fluids was investigated at 37 °C. Temperature
only for delivery but also for treatment of the oral mucosal
dependence was investigated by preparing a concentration
infections due to its antibacterial/ antifungal activity and
of 0.6 mg mL-1 artesunate in buffer solutions of pH 1.20, 6.00
also due to its bioadhesive properties which would provide
and 10.50. Each selected pH of artesunate was separately in-
occlusion on the applied site.
vestigated at three temperatures (278, 295 and 303K).
REFERENCES
1. İkinci G, Şenel S, Akıncıbay H, Kas S, Ercis S, Wilson CG, A plot of log percent drug concentration versus time was
plotted to determine the rate of degradation and to find the
Hıncal AA. Effect of chitosan on a periodontal pathogen P.
rate constant of each reaction. Rate constant were reproduc-
gingivalis, Int J. Pharm. 2002; 235: 121– 127.
ible to within ± 4%
RESULTS AND DISCUSSION
p034-The degradation of artesunate in A plot of log percent remaining concentration of artesunate
aqueous solutions in different type of buffer solution versus time showed a lin-
ear relationship over than ≥ 2 t1/2 of reaction which indi-
Muder Al Haydar1, Bruce Sunderland2 cated first order of degradation of artesunate.

Pharmacy School , Curtin Unniversity, WA Australia1 The catalytic effect of the buffer systems used in the kinetic stud-
Pharmacy School, Curtin University, WA Australia2 ies was determined by experiments at constant pH, temperature,
ionic strength and drug concentration, the only variation being
INTRODUCTION was in the overall buffer concentration at a given pH. This was ex-
Artesunate, dihydroarteminsinin -10α – hemisuccinate, is a amined at 0.6 mg mL-1 of artesunate, 37˚C, µ of 0.5 mol L-1 and
semi-synthetic derivative of artemisinin which is used for the at selected pH values. The plots of log residual artesunate con-
treatment of both uncomplicated and severe malaria1. It is for- centrations versus time indicated linear relationship which also
mulated for oral, parenteral (intramuscular and intravenous) indicated a first-order rate for artesunate degradation.The rate
and rectal administration and used clinically worldwide2. Al- constant values did not show significantly increased catalysis
though it is frequently employed as a water soluble drug, it is with increased of buffer concentration as shown in Table (1). This
not sufficiently stable to allow for long term storage in solution. indicates that the buffer system does not have a marked catalytic
effect on the degradation rate of artesunate.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

Table 1: The observed rate values of artesunate in different


pH values.
Rate constant (hr-1) at the following buffer
concentration
pH 0.1 mol L-1 0.15 mol L-1 0.2 mol L-1
2 1.57 1.6 1.64
2.5 0.297 0.331 0.337
3.5 0.226 0.243 0.268
4 0.15 0.162 0.165
5 0.086 0.091 0.094
5.5 0.083 0.882 0.893
6.5 0.083 0.088 0.089
7.5 0.06 0.073 0.074 Figure 2: log k’- pH profile of artesunate degradation in aque-
8.5 0.07 0.071 0.072 ous solution at 37˚C and µ of 0.5 mol L-1
9.5 0.138 0.179 0.19
10.5 0.34 0.34 0.345 CONCLUSION
The degradation rate of artesunate in the buffer systems ki-
The rate constant values at zero buffer concentration, k΄, were ob- netically obeyed a pseudo-first order reaction and with gen-
tained from the plots of pseudo first-order rate constants versus eral acid-base catalysis, however; it was not a major factor in
the total buffer concentration Figure (1). At zero buffer concentra- artesunate degradation. Maximum stability of artesunate in
tion only specific acid-base catalysis (H+ / OH¯) or a spontaneous aqueous solution was between pH 5.50 and 7.50. Stability
water reaction occurs. The logarithms of these rate constants are was markedly temperature dependent.
plotted versus pH in the pH range of 2.00 – 10.50. The pH-rate
profile Figure (2) has a U-shape which is characteristic of reaction ACKNOWLEDGMENT
susceptible to specific acid-base catalysis. The pH of maximum I would sincerely thank Prof. Bruce Sunderland for his contin-
stability for artesunate in buffer free conditions is over the pH ual support and encouragement in his roles as supervisor.
range of 5.50 to 7.50. Hence below pH 5.50 the rate of reaction
increases with decrease in pH whereas above 7.50 the rate of re- REFERENCES
action again increases with increased pH. There were three im- 1. Lalloo DG, Shingadia D, Pasvol G, Chiodini PL, Whitty
portant pH regions. One where the acid catalysed reaction domi- CJ, Beeching NJ, et al. UK malaria treatment guidelines. 109
nated, which was below pH 5.50, the pH-independent region Journal of Infection. 2007; 54(2):111-121.
over the pH range of 5.50 to 7.50 and the third one where the 2. Dondorp AM, Day NPJ. The treatment of severe malaria.
hydroxide ion catalysed reaction took place, which was above pH Transactions of the Royal Society of Tropical Medicine
7.50. The shelf lives of artesunate at pH 5.5, 6.50 and 7.50 and 37 and Hygiene. 2007; 101(7):633-634.
°C were estimated to be 1.3, 2.2 and 1.5 hr respectively. 3. Batty KT, Ilett KF, Davis T, Davis ME. Chemical stability
of artesunate injection and proposal for its administra-
The temperature dependent studies on artesunate indicated tion by intravenous infusion. J Pharm Pharmacol. 1996;
that the degradation of artesunate was increased with in- 48(1):22-6.
creased temperature. The Arrhenius plots were obtained and
the apparent activation energy values were calculated at pH p035-VALIDATION OF AN HPLC METHOD
1.2, 6.5 and 10.5 to be 100.38, 100.96 and 95 kJ mol-1 respec-
FOR THE DETERMINATION OF NAPROXEN
tively. These indicate a significant temperature dependence.
SODIUM IN POLY(LACTIDE-CO-GLYCOLIDE)
CORNEAL SCAFFOLDS

Seren Kayıran1, Sibel Bozdağ Pehlivan1, Mustafa Çelebier2,


Nurşen Ünlü1

Hacettepe University Faculty of Pharmacy Department of


Pharmaceutical Technology1
Hacettepe University Faculty of Pharmacy Department of
Analytical Chemistry2

INTRODUCTION
The aim of this study was to develop a new specific HPLC
method for the determination of released naproxen sodium
(NS) from poly-(lactide-co-glycolide) (PLGA) (50:50) scaffolds
Figure 1: Catalytic effect of the buffer solutions on 0.6mg and to assess the related validation parameters for the ana-
mL-1 artesunate at 37 °C and µ of 0.5 mol L-1 lytical method. We intend to formulate PLGA (50:50) scaffolds

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

containing corneal epithelial cells and NS to treat severe cor- Stability


neal injuries by the replacement of epithelial cells with NS The standard solutions of NS were stored at +4 °C for 24 hour
into the damaged, inflammatory area1. The total amount of and protected from daylight. During this period, it was ana-
NS in scaffolds and the released amount of NS from the for- lyzed periodically at 6, 12, 24 h.
mulations will be determined by developed HPLC method.
RESULTS AND DISCUSSION
MATERIALS AND METHODS Specificity
Materials The chromatogram obtained from NS containing PLGA(50:50)
The HPLC system consisted of Waters 2690 Separations scaffold was identical with that obtained from the blank scaf-
Module equipped with a Waters 2695 Fluorescence Detec- fold containing PLGA(50:50). The representative chromato-
tor (Waters, USA) was used for the validation of the method. grams (Figure 1) show no other peaks on the retention time
NS was supplied from Abdi İbrahim (Turkey). PLGA (50:50) of NS. Accordingly, the proposed method can be considered
was commercially available as Resomer RG 503 and was pur- selective.
chased from Boehringer-Ingelheim (Germany). HPLC grade
methanol and acetic acid were purchased from Sigma Al- Linearity Range
drich (Germany). High purity water was prepared by using The peak areas of NS were plotted against the correspond-
Millipore Milli-Q plus water purification system. ing nominal concentration to obtain the calibration graph.
Thus, the method was evaluated linear in the range of 0.08
HPLC Method to 75 μg.mL-1 for NS.
An HPLC method with fluorescence detection was selected
for the method of analyses. The reversed-phase procedure The r2 for the regression line is 0.9996 with a slope of
utilized a Waters Spherisorb S10 ODS2 column (C18; 200 x 24239118.360 and a y-intercept of 230293899.733. These re-
4.6mm). The mobile phase comprised of methanol and ac- sults were considered acceptable.
etate buffer (pH 5.1) (55:45 v/v). Analyses were run at a flow-
rate of 1 mL.min-1 at an ambient temperature. The fluoro- Sensitivity
metric detector was set at 254 nm ex/ 352 nm em. At this The values of LOD and LOQ for NS were 0.03 μg.mL-1 and
wavelength, peak areas were measured and used for the 0.08 μg.mL-1.
quantitative evaluations. Precision and Accuracy
The relative standard deviation (RSD) and the Bias of intra-
Standard solution of NS was prepared in simulated lacrimal and inter-day studies were within acceptable range indi-
110 fluid (SLF). SLF was selected as the solvent because of its cating that the precision and the accuracy of the method
property as a release medium for ophthalmic formulations. were satisfactory. The intra- and inter-day precision relative
standard deviation was 3.71 % or less, and the accuracy was
Analytical Method Validation within 2.96 % deviation of the nominal concentration.

Specificity Stability
Specificity was examined by analyzing release medium and During 24 h, no unexpected peak appeared and the peak
the blank PLGA (50:50) scaffold in the release medium. areas did not changed significantly (p > 0.05) for the com-
pound when it was compared with freshly prepared one.

Linearity Range
The linearity of an analytical procedure is its ability to ob-
tain test results which are directly proportional to the con-
centration of analyte in the sample2. Seven concentrations
between 0.08-75 μg.mL-1 of NS solutions were prepared to
show the linearity of the method.

Sensitivity
The sensitivity of the analytical method was evaluated by de-
termining limits of detection (LOD) and quantitation (LOQ).
The signal-to-noise ratios of 3:1 and 10:1 were taken as LOD
and LOQ, respectively2.
Figure 1: The chromatogram obtained in optimum condi-
Precision and Accuracy tions. a: blank solution; b: PLGA(50:50) standart solution (1
Three different concentrations of standard NS were analyzed μg.mL1); c: NS standard solution
three consecutive days (inter-day precision) and within the
same day (intra-day precision). The relative standard devia-
tion (RSD) an the Bias of intra- and inter-day studies were
calculated.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

HPMC were evaluated for gelling capacity in order to identify


the compositions suitable for use as in-situ gelling systems. In-
situ gel formulations were prepared in a citrophosphate buffer
(pH 6). Buffer salts were dissolved in purified water, HPMC was
added and allowed to hydrate. Carbopol was sprinkled over
this solution and allowed to hydrate. CPH or inclusion com-
plex was dissolved in NaOH solution and benzalkonium chlo-
ride was added. The drug solution was added to the carbopol/
HPMC solution under constant stirring. Then the volume was
made up to 100 ml with purified water (Table 1).

Figure 2: The regression line for NS (0.08-75 μg.mL-1). Table 1: Ingredients of the developed formulations
Code
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10
CONCLUSION w/v %
In this study, a simple, very specific, and reliable HPLC meth- CPH 0.35 0.35 0.35 0.35 0.35 0.35 0.35 - - -
od using fluorometric detection has been developed and Inclusion
- - - - - - - 1.675 1.675 1.675
validated for the assay of NS from PLGA (50:50) scaffolds. complex
C940 0.5 0.5 0.3 0.5 - - - - - -
REFERENCES C934 - - - - 0.5 0.3 0.5 0.5 0.3 0.5
1. Nishida K. Tissue engineering of the cornea, Cornea HPMC 1.5 1.5 1.5 1 1.5 1.5 1 1.5 1.5 1
2003; 22(7):28-34. Tween 20 - 1 - - - - - - - -
2. ICH Guideline for Industry, Text on Validation of Analyti-
Benzalkonium
cal Procedures Q2A ,1995. 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02
Chloride

Drug Content Uniformity and pH


P036-PH INDUCED IN SITU OPHTHALMIC The CPH concentration of formulations was determined at
GELLING SYSTEM FOR CIPROFLOXACIN / 271 nm. pH of the formulations were measured with SenTix
82 pH electrode.
HP-Β-CD COMPLEX
Viscosity Measurements
Berrin Başaran1, Asuman Bozkır1 111
Viscosity of formulations were measured with a Brookfield
RVTDV-II viscometer at 25°C, pH 6 and at 34°C, pH 7,4.
Ankara University Faculty of Pharmacy Department of
Pharmaceutical Technology, Ankara, Turkey1 In Vitro Release Studies
The in vitro release of CPH from the formulations was stud-
INTRODUCTION ied using a dialysis membrane in a circulating water bath at
In this study, a pH induced in-situ gel for Ciprofloxacin HCl (CPH) 80 rpm and 34±1°C. The diffusion medium was 50 ml of pH
was developed and different carbopol types were studied as 7,4 phosphate buffer.
the gelling agent in combination with hydroxypropylmethyl-
cellulose (HPMC) which acted as a viscosity enhancing agent. Antimicrobial Efficacy Studies
CPH was complexed with hydroxypropyl-beta-cyclodextrin Antimicrobial efficacy of formulations were determined by
(HP-β-CD) and this complex was also used in the formulations. agar diffusion test employing the cup plate technique2.
Viscosities of formulations were measured. In vitro release and
antimicrobial efficacy of selected formulations were evaluated.
MATERIALS AND METHODS RESULTS AND DISCUSSION
Materials Gelling Capacity
Ciprofloxacin HCl (Bayer, Germany), Carbopol 934 C980 containing formulations showed the weakest gelation.
(C934),Carbopol 940 (C940) (BF Goodrich,USA), Carbopol Formulations prepared with 0,5% C934 or C940 with 1 or
980 (C980) (Noveon,USA), HPMC (Fluka,Switzerland) All oth- 1,5% HPMC and 0,3% C934 or C940 with 1,5% HPMC were
er reagents were of analytical grade. shown to have the best gelling capacities.

Preparation and Characterization of CPH: HP-β-CD Inclusion Drug Content and pH


Complex The drug content and pH of the formulations were satisfac-
The inclusion complex of CPH with HP-β-CD in a 1:1 molar tory. Standard deviations were found to be below 2 %.
ratio was prepared by freeze-drying technique. Formation of
the complex was confirmed using FTIR Spectrophotometry, Rheological Studies
DSC analysis and 1H NMR spectroscopy (Data not shown)1. Decrease in viscosity was observed with increase in angular
velocity (pseudoplastic rheology)3. C934 containing formula-
Preparation of In Situ Gelling System tions exhibited lower viscosity than formulations containing
Solutions of varying concentrations of carbopol types and C940 at 25°C and pH 6. C7 and C10 formulations containing

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

0,5% C934 and 1% HPMC exhibited lower viscosity values. Antimicrobial Efficacy Studies
After gelation of C7 and C10 formulations, viscosity values The zone of inhibition values against P. aeruginosa were
increased 3 fold and 7 fold, respectively (Table 2). So, C7 and higher than that against S. aureus (Table 3). This study indi-
C10 formulations were selected in terms of both instilling cates that CPH retained its antimicrobial efficacy when incor-
convenience and viscosity increase after gelation. porated in an in situ gel system.

Table 2: Viscosity of formulations (10 rpm) (n=3) Table 3. Antimicrobial efficacy of formulations
Viscosity (cp) Zone of inhibition (mm) (% efficiency)
Formulations Viscosity (cp) (250C)
(340C, pH 7.4) Concentration
C1 14333 ± 1443 13000 ± 0 Std C7 C10
(μg/ml)
C3 11000 ± 2500 13500 ± 1250
100 42 28 (66.67) 28 (66.67)
C4 7333 ± 1443 15000 ± 2500 S.aureus
500 45 32 (71.11) 33 (73.33)
C5 10000 ± 0 13333 ± 1443
C6 9500 ± 1250 11670 ± 1443 P.aeruginosa 500 37 33 (89.19) 35 (94.60)
C7 3500 ± 1250 9670 ± 1443
C8 9500 ± 1250 21000 ± 2500 CONCLUSION
C9 8500 ±1250 11333 ± 1443 C7 and C10 formulations are an alternative to eye drops
because of instillation easiness and decreased frequency
C10 3000 ± 0 20333 ± 1443
of administration for better patient acceptance. Inclusion
complex was used for increasing the stability of CPH. These
In Vitro Release Studies
formulations seem promising opthalmic delivery system for
Increasing carbopol concentration decreased the drug re-
CPH.
lease for C5, C6, C7 formulations. While carbopol concentra-
tion was pegged, increase in HPMC concentration decreased
REFERENCES
the drug release. The most slowly drug release was observed
1. Basaran B, Bozkır A. Thermosensitive, prolonged release
with C5 and C8 formulations, but these formulations were
ophthalmic gel for ciprofloxacin/HP-β-CD complex. 2nd
found to have higher viscosity before gelation and instilla-
PharmSciFair, Nice-France, 2009.
tion wasn’t easy. C7 and C10 formulations showed the slower
2. Jain SP, Shah SP, Rajadhyaksha NS. In situ ophthalmic gel
drug release than C6 and C9 formulations and their viscosity
of ciprofloxacin hydrochloride for once a day sustained
values were convenience for instillation, so C7 and C10 for-
delivery. Drug Dev. Ind. Pharm. 2008; 34: 445-452.
112 mulations were selected (Figures 1 and 2).
3. Basaran B, Bozkır A. Preparation and evaluation of chito-
san-based in-situ gelling ophthalmic formulations con-
taining ciprofloxacin. Advances in Chitin Science 2007;
X: 343-348.

P037-SURFACE ANALYSIS AND STRUCTURE


CHARACTERIZATION OF A DRY POWDER
CONTAINING CHITOSAN-HYALURONIC ACID
NANOPARTICLES

S. Al-Qadi1, C. Remuñán-López2

Figure 1: In vitro release of C5, C6 and C7 formulations Department of Pharmaceutical technology, Faculty of
Pharmacy, University Santiago de Compostela,Spain1
Department of Pharmaceutical technology, Faculty of
Pharmacy, University Santiago de Compostela,Spain2

INTRODUCTION
Recently, we proposed the micro-encapsulation of nano-
particles in dry powders for pulmonary administration of
therapeutic macromolecules such as proteins and DNA1. It is
known that particle surface chemistry interfaces with biolog-
ical environments, and this governs its biological behaviour2.
The aim of this study was to characterize the structure and
surface composition of chitosan/hyaluronic acid/ tripoly-
phosphate nanoparticles (CS NPs) encapsulated in mannitol
Figure 2: In vitro release of C8, C9 and C10 formulations
microspheres (M-NPs). Confocal laser scanning microscopy
(CLSM) was used to investigate the spatial distribution of NPs

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

in the microcarrier. Besides, the surface-sensitive analytic


techniques X-ray spectroscopy (XPS) and time of flight sec-
ondary ion mass spectrometry (TOF-SIMS) were employed
for further characterization.

MATERIALS AND METHODS


Materials
Ultrapure chitosan (CS) HCl salt (Protasan UP CL 113) was
purchased from FMC Biopolymers [Norway]; hyaluronic acid
was provided by Bioiberica; fluorescein sodium salt, penta-
sodium tripolyphosphate (TPP) and D-mannitol were ob-
tained from Sigma-Aldrich [Spain]; Bodipy® 630/650-X was
provided by Molecular Probes [Netherlands].
Figure1b: SEM micrograph of M-NPs
Preparation and characterization of microencapsulated
The CLSM micrographs depicted in Fig. 2 show the labelled
nanoparticles
mannitol forming a continuous matrix in which NPs are ho-
CS was labelled by fluorescein (FITC) following the method
mogeneously distributed.
described by De Campos et al3. Morphology visualization
of NPs was conducted by transmission electron microscopy
(TEM) [CM 12 Philips, Eindhoven, Netherlands]. To prepare
the NPs-loaded M microspheres (M-NPs), mannitol was la-
belled with the flourophore Bodipy® and then co-spray dried
with the NPs suspension using a laboratory-scale spray-dryer
[Büchi mini spray dryer, B-290, Switzerland].The resultant mi-
crospheres were observed using a scanning electron micro-
scope [SEM, Leo 435VP, U. K.] and their particle size charac-
terized with an Aerosizer®-Aerodisperser® [Amherst Process
Instruments, Inc., Amherst, MA, USA].

Structural Characterization using CLSM


Fig.2. CLSM images of microspheres: (a) mannitol stained by
The internal structure of the microspheres was observed by 113
CLSM, using a TCS-SP2 vertical microscope [Leica GmbH, Ger-
many]. Laser excitation λ of 488 nm and 633 nm were used to
scan the powder; and fluorescent emissions from FITC (emis-
sion λ) (492-550 nm) and Bodipy® (emission λ) (650-725 nm)
were collected using separate channels.
Analysis Using XPS and TOF-SIMS
Surface analysis of microencapsulated NPs was performed
using XPS [VG Escalab 250 iXL ESCA, VG Scientific, U. K.] and
TOF-SIMS [TOF-SIMS IV, Ion-TOF GmbH, Germany]. Mannitol
microspheres and isolated NPs were used as controls.

RESULTS AND DISCUSSION (b) NPs labelled by FITC (green channel)


NPs displayed a regular shape and M-NPs showed a spherical
morphology (Fig. 1).

and (c) overlapping of both channelsBodipy® (red channels)

Analysis using XPS and TOF-SIMS


As shown in Table 1, the ratio C/O and the absence of the
Figure 1a: TEM micrograph of CS/HA/TPP NPs signal of P and Na in the case of mannitol and M-NPs indicate
that NPs are embedded inside the mannitol microspheres.

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

Table 1: Surface composition (atomic percentage) following P038-SURFACE-MODIFIED PLGA


XPS analysis of CS/HA/TPP NPs, mannitol microspheres (M), MICROPARTICLES AS PULMONARY DELIVERY
and microencapsulated nanoparticles (M-NPs). C: carbon,O:
oxygen,N:nitrogen,P:phosphor,Na:sodium.
VEHICLES FOR RHIL-2

Element CS/HA/TPP NPs M M-NPs Burcu Devrim1, Asuman Bozkır1, Kandemir Canefe1
C 51,7 50.6 43.3
Department of Pharmaceutical Technology, Faculty of
O 37.1 43.0 37.0
Pharmacy, Ankara University, 06100- Tandoğan, Ankara/
N 3.4 0.5 1.0 Turkey1
P 0.6 0 0
Na 1.7 0 0 INTRODUCTION
C/O 1.39 1.176 1.170 Interleukin-2 (IL-2), a lymphokine produced by activated T
cells, has a wide variety of actions and plays a central role
The mass spectra obtained by TOF-SIMS proves that the char- in immune regulation. Because of this biological function,
acteristic mass of mannitol m/z 183 (molecular ion + H+) is recombinant human IL-2 (rhIL-2) has been tested in clinical
present in the case of mannitol and M-NPs and absent in the trials for treatment of tumors1. Since the protein has a short
case of the isolated NPs (Fig.3). Also, the mass of phosphate half-life, frequent administration of rhIL-2 is required to ob-
fragment (PO2, PO3), attributed to TPP, appears in the case tain a therapeutic effect, which can result in severe toxic side
of NPs but not in the spectrum of mannitol microspheres (M) effects. Controlled-release technology lends itself naturally
and M-NPs, suggesting that the NPs are totally covered with to this type of medical problem. Systems can be designed
mannitol (data not shown). that provide release of molecules over days to weeks, in such
a manner that a systemic effect is achieved without toxic-
ity. In addition to this, modifying of microparticles with mu-
coadhesive polymers may help to increase the residence
time and increase efficacy when administered by the nasal
or pulmonary routes2.

In this study, surface-modified PLGA microparticles was


prepared by using w/o/w double emulsion solvent extrac-
114 tion method. And the physicochemical properties of rhIL-2
loaded PLGA microparticles was determined. Chitosan (CS)
chloride and Carbopol 971P was used as surface modifying
agents.

MATERIALS AND METHODS


Materials
Microparticle formulations containing rhIL-2 were prepared
Figure 3: TOF-SIMS mass spectrum of NPs, mannitol and M- by PLGA with a lactide/glycolide ratio of 50:50 (Resomer RG
NPs. 502) (Bohringer Ilgelheim, Germany). CS chloride (Protosan
UP CL 113) was obtained from Novomatrix (ABD). Carbopol
CONCLUSION 971P was obtained from Noveon (ABD). rhIL-2 was a gift from
CLSM, XPS and TOF-SIMS analysis prove that NPs are efficiently Novartis (Novartis, Turkey) and all the other chemicals used
encapsulated and homogeneously distributed within M-NPs. were analytical grade.

ACKNOWLEDGEMENTS Preparation of Surface-Modified PLGA Microparticles


MAEC-AECID fellowship of the Spanish Agency to S. Al-Qadi PLGA microparticles containing rhIL-2 were prepared by a
and the Spanish Government (CICYT, SAF2002-03314) are w/o/w double emulsion solvent extraction method3. In brief,
highly appreciated. C. Serra (CACTI, Vigo) is acknowledged aqueous solution of rhIL-2 was poured into a PLGA solution
for her assistance. in dicholoromethane. The primary emulsion was generated
by a high-speed homogenizer (Ultraturrax T-25 Homogeniz-
REFERENCES er, Germany). This water-in-oil emulsion, was added to exter-
1. Grenha A, Seijo B, Remuñán-López C. Micro-encapsulat- nal aqueous solution of polyvinyl alcohol (PVA) and stirred
ed chitosan nanoparticles for lung protein delivery, Eur. to produce the secondary emulsion. To modify the micropar-
J. Pharm. Sci. 2005; 25:427-437. ticle surfaces, 0.5% (w/v) CS chloride or Carbopol 971P was
2. Mc Arthur S. Applications of XPS in bioengineering, Surf. added to PVA solution. The w/o/w emulsion was poured into
Interface Anal. 2006; 38: 1380-1385. additional aqueous PVA solution and stirred with a mechani-
3. De Campos A M, Diebold Y, Carvalho ELS et al. Chitosan cal stirrer for 3 h. Finally, the microparticles were collected by
nanoparticles as new ocular drug delivery systems: in centrifugation, washed with bidistilled water and lyophilized
vitro stability, in vivo fate, and cellular toxicity, Pharm. to obtain a dry powder.
Res. 2004; 21: 803–810.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

Characterization of Microparticles modification with mucoadhesive polymers. CS chloride


To determining morphology of the microparticles, samples coating led to an increase in the particle size of micropar-
were coated with a thin layer of gold and viewed using a ticles. In contrast to the CS chloride, surface modification
JEOL-6400 scanning electron microscopy(SEM)(JEOL, Ja- with Carbopol 971P was resulted decrease in particle size of
pan). microparticles (Table 1).

The particle size distributions of the microparticles were deter- Table 1: Characterization of unmodified and surface modi-
mined by laser diffraction (Sympatec laser diffraction particle fied microparticles
sizer, Germany) after suspending the microparticles in water. Zeta
Three determinations were carried out for each formulation. Formulation Coating Particle size E.E
potantial
Code material (μm) ± S.D. (%) ± S.D.
(mV) ± SD.
Estimation of rhIL-2 Content in PLGA Microparticles F1 - 4.03 ± 0.0058 99.22 ± 1.20 -8.31 ± 1.24
The encapsulation efficiency for biodegradable PLGA micropar- F2 Chitosan HCl 5.68 ± 0.026 97.57 ± 2.36 +19.30 ± 0.30
ticles containing rhIL-2 was determined by dissolving micropar- Carbopol
ticles in 2% (w/v) SDS in 0.1 M sodium hydroxide solution. After F3 1.37 ± 0.010 98.12 ± 1.53 -28.60 ± 0.44
971P
the solution was neutralized with 0.1 M HCl, the amount of rhIL-2
was determined by micro bicinchoninic acid (microBCA) assay.
Placebo microparticles (without protein) were used as control.
The rhIL-2 encapsulation efficiency of PLGA microparticles
Measurement of Zeta Potantial
was found in range of 97.54-99.20% determined by microB-
Surface charge of freeze-dried microparticles was deter-
CA assay.
mined by zeta potential measurement on a Malvern Zeta-
sizer (Malvern, UK).
The modification of the surfaces of PLGA microparticles with
CS chloride or Carbopol 971P was confirmed by resultant
In vitro Release of rhIL-2 from Microparticles
changes in zeta potential. The surface charge of the unmodi-
The release of rhIL-2 from the PLGA microparticles was de-
fied PLGA microparticles was negative, whereas the zeta po-
termined by suspending microparticles in pH 7.2 phosphate
tantial was recorded to be positive when the microparticles
buffer (PB) containing 0.1% SDS and 0.02% sodium azide.
were modified with CS chloride. Conversely, the zeta poten-
The samples were incubated at 37 °C in a shaking water bath.
tial of Carbopol 971P coated microparticles became more
A vial was withdrawn at each time-point, the contents of vial
negatively charged compared with un-coated microparticles
were centrifuged and the supernatant containing released
(Table 1).
rhIL-2 was collected. After each sample collection, the sam- 115
ple PB was replaced with fresh buffer. The measurement of
In vitro release results indicate that coating the micropar-
protein concentration was performed by microBCA.
ticles with mucoadhesive polymers reduced the initial burst
of drug release.
RESULTS AND DISCUSSION
The surface-modified PLGA microparticles prepared by dou-
ble emulsion method were spherical in shape with smooth
surface (Figure 1).

Figure 2: Cumulative release of rhIL-2 from unmodified and


surface modified PLGA microparticles.

CONCLUSION
PLGA microparticles coated with mucoadhesive polymers
were successfully prepared by double emulsion solvent ex-
traction method. The surface coating of microparticles with
polymer was confirmed by the change in zeta potential.
Figure 1: SEM micrographs of PLGA microparticles. a)Unmod- Additionally, surface modification with mucoadhesive poly-
ified microparticles (F1) b)chitoasan hydrochloride coated mers reduced the initial burst of drug release.
microparticles (F2) c)Carbopol 971
REFERENCES
The particle size of microparticles was affected on surface 1. Skubitz KM, Anderson PM. Inhalational interleukin-2 li-

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

posomes for pulmonary metastases: a phase I clinical samples were obtained at a scanning rate of 10 °C/min with
trial, Anti-Cancer Drugs 2000, 11:555-563. a temperature range of 25–140 °C. Melting point of surfactin
2. Alpar HO, Somavarapu S, Atuah KN, Bramwell VW. Biode- was also determined by a capillary melting point apparatus
gradable mucoadhesive particulates for nasal and pul- (Thomas Hoover, USA).
monary antigen and DNA delivery, Adv. Drug Del. Rev.
2005, 57:411-430. HPLC Analysis
3. Devrim B, Bozkır A, Canefe K. Biodegradable PLGA mi- An HPLC system (Waters) equipped with a photodiode array
croparticles as a delivery system for protein: effect of detector (DAD) was utilized for the quantitative analysis of
molecular weight of PLGA on the microparticle charac- surfactin. The mobile phase A was composed of 0.5% trifluo-
teristics, Pharm. J. Slovenia 2008; 59:107-108. roacetic acid in water; B was 100% acetonitrile at a flow rate
of 0.7 ml/min. The injection volume was 40 μl. A reversed-
phase C18 column (4.6x250 mm, 5μm) kept at 20°C was
p039-FORMULATION AND CHARACTERIZATION used. The detection wavelength was 205 nm.
OF SURFACTIN-CONTAINING SELF-
RESULTS AND DISCUSSION
EMULSIFYING DRUG DELIVERY SYSTEMS Preparation of Formulations
Several compositions of oils and surfactants were prepared
F. Hande Kural1, R. Neslihan Gürsoy1 using ternary phase diagrams as part of preformulation
studies. Of the excipients tested, the composition of Gelucire
Hacettepe University, Faculty of Pharmacy, Department of 44/14, Labrasol, PEG 3000 and Vitamin E gave the most sta-
Pharmaceutical Technology, Ankara, Turkey1 ble SEDDS (Figure 1). Stable microemulsions were obtained
as shown in region A. In region B, unstable microemulsions
INTRODUCTION which became cloudy as a function of time, were obtained.
Surfactin, one of the most powerful biosurfactants, is a cyclic In region C, coarse emulsions were obtained.
lipopeptide that is produced by Bacillus subtilis. It has been
shown to exhibit antiviral, antifungal, antimicrobial and anti-
tumor effects1. In this study, we have designed and character-
ized surfactin-containing self-emulsifying drug delivery sys-
tems (SEDDS) for future therapeutic applications of surfactin.

116 MATERIALS AND METHODS


Materials
Surfactin (≥ 98% purity) was purchased from Sigma. Gelucire
44/14 and Labrasol were purchased from Gattefossé. Vitamin
E was a kind gift from Roche Pharmaceuticals (İstanbul, Tur-
key). Poly ethylene glycol 3000 (PEG 3000) was purchased
from Fluka. Nanopure water was used throughout the ex-
periments.

Preparation of Formulations
In the preformulation studies, optimum surfactant, co-sur-
factant and oil combinations were determined with ternary
phase diagrams. After mixing the excipients on a water bath
at 40°C, the formulations were diluted to 10 ml with phos-
phate buffer (pH 7).

Microscopic Examination
The formulations were examined utilizing a polarizing light
microscope (Leica DM EP, Germany) to observe the appear-
ance of possible liquid crystal structures due to the presence
of high amounts of surfactants in the formulations.

Droplet Size and Zeta Potential


Droplet size and zeta potential measurements were carried
out by Malvern Nanosizer ZS 2000 (United Kingdom). Each
sample was analyzed in triplicate.

DSC Analysis
The thermal analyses of the excipients, surfactin and formu-
lations were performed with a differential scanning calorim-
eter (TA Instruments Q100, USA). The thermograms of the

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

Figure 1: Ternary phase diagram of SEDDS formulations


The SEDDS formulations in region A exhibited Newtonian
type rhelogical behavior upon aqueous dilution.

Microscopic Examination
Due to the nano size range of the SEDDS formulations, no
image was observed under the polarizing light microscope,
Figure 2: Chromatogram of surfactin solution (100 μg/ml)
as expected. Moreover, no liquid crystal structures were ob-
served, which are known to exhibit characteristic images un-
CONCLUSION
der the polarizing light microscope.
Stable SEDDS formulations were designed and character-
ized, and the bioactive surfactin compound was successfully
Droplet Size and Zeta Potential
incorporated into these formulations. The characterization
A monomodal droplet size distribution with a narrow range
studies of surfactin-containing SEDDS formulations indicat-
was obtained with stable SEDDS formulations. The average
ed that the addition of surfactin caused no changes in the
particle size of the SEDDS formulations were in the 15–21
stability and physicochemical properties of the formulations.
nm range. The average droplet size of the SEDDS formula-
To the best of our knowledge, this is the first study showing
tion containing surfactin was found to be 19.41±0.42 nm.
the successful incorporation of surfactin into a drug delivery
The average zeta potential values were in the range of -9.5–
system such as SEDDS.
5.0 mV. Since only nonionic surfactants were used in these
formulations, zeta potential values close to neutrality were
ACKNOWLEDGEMENT
obtained. The average zeta potential of the surfactin-con-
This project was supported by State Planning Office (DPT) of
taining formulation was -1.53±0.88 mV.
the Republic of Turkey.
DSC Analysis
REFERENCES
One of the most stable formulations from region A was test-
1. Seydlova G, Svobodova J, Review of Surfactin chemical
ed for DSC experiments.
properties and the potential biomedical applications,
Cent Eur J Med, 2008;3(2):123-133.
Table 1: DSC data for the excipients and the SEDDS formula-
2. Oka K, et al., Satisfactory separation and MS-MS spec-
tions (n=3)
trometry of six surfactins isolated from Bacillus subtilis
Component Tm(0C) ∆H (J/g) 117
natto,1993;41(5):1000-1002.
Gelucire 44/14 47.88 ± 2.09 60 ± 7.12 3. Abdel-Mawgoud AM et al., Characterization of surfactin
PEG3000 61.97 ± 0.85 194.68 ± 2.04 produced by Bacillus Subtilis isolate BS5, appl Biochem
SEDDS before dilution 39.48 ± 0.79 12.81 ± 4.31 Biotechnol, 2008;150:289-303.
SEDDS after dilution 70.50 ± 3.51 205.94 ± 149.13t

The melting temperature of surfactin was found to be 131.54 p040-THE EFFECTS OF PULMONARY
°C. This data is in accordance with the results obtained by the ADMINISTRATION OF INTERLEUKIN-2
capillary melting point method (132 °C) and the literature2. LIPOSOMES ON IMMUNE SYSTEM IN RATS
Table 1 illustrates the melting temperature (Tm) and the en-
thalpy of transition (ΔH) for Gelucire 44/14, PEG 3000 and the
Nevin Çelebi1, Ayşe Çilek1, Ayşegül Atak2, Gonca Akbulut3
formulations.

It can be inferred from the DSC data that excipients forming Gazi University Faculty of Pharmacy Department of
the SEDDS behave differently before and after dilution. It ap- Pharmaceutical Technology, Ankara, Turkey1
pears that following aqueous dilution, the molecules exhib- Gazi University Faculty of Medicine Department of
ited structural rearrangements so only the Gelucire melting Immunology, Ankara, Turkey2
endotherm appeared before dilution (39.48 °C) and only the Gazi University Faculty of Medicine Department of Physiology,
PEG 3000 melting endotherm (70.50 °C) following dilution, Ankara, Turkey3
both of them exhibiting a shift from their actual values. This
may be due to the hydrophilicity of PEG 3000 which prob- INTRODUCTION
ably leads to more interaction with the water molecules. The Interleukin-2 (IL-2) is a cytokine which is produced by acti-
melting endotherm of surfactin was not observed in the ther- vated hepler T lymphocytes and is an important mediator of
mogram of the surfactin-containing SEDDS formulation. the immune response. Because of this biological function,
recombinant human IL-2 (rhIL-2) has been tested in clinical
HPLC Analysis trials for treatment of tumors1. rhIL-2 is a relatively hydro-
Figure 2 shows that surfactin isoforms eluted in 20 minutes. phobic protein with limited aqueous solubility.Since it has a
The chromatographic profile of surfactin was comparable to short-half life, frequent administration of rhIL-2 is required to
those obtained by other researchers utilizing different meth- obtain a therapeutic effect, which to can result in toxic side
ods3. effects. In order to overcome these problems, drug delivery

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

systems (e.g liposomes and polymatrices) have been investi-


gated to increase the therapeutic efficacy and minimize the
side effects of rhIL-22. Recently, pulmonary administration of
IL-2 have been gained importance. In the previous study, we
have reported that the effects of pulmonary administration
of IL-2 liposome formulation on pharmacokinetics param-
eters in rats3. The objective of in this part of our study was to
investigate the effects of pulmonary administration of IL-2
liposomes on immunological parameters in serum and lung
homogenisate and to compare with pulmonary administra-
tion of IL-2 solution and intravenous (i.v) administration.

MATERIALS AND METHODS


Preparation of Liposomes
Liposomes were prepared by using the film method as re-
ported previously3. Liposomes were characterized by par-
ticle size and zeta potantial measurements. Encapsulation
efficiency of IL-2 in the liposomes were determined3.

In vivo Studies
Wistar male rats were used throughout the study. In vivo
experiments were conducted in accordance with the eth-
ics guidelines upon obtaining the approval Gazi University
laboratory animal care committee. The rats were divided into
three major groups including 8 rats each. Pulmonary admin-
istration of IL-2 formulations (liposomes and solution) to rats
were performed using PennCentury device. IL-2 was admin-
istered as 107IU/mL and 2.106 dose levels via pulmonary
and intravenous, respectively. IL-2 containing formulations
was administered at the beginnig and on the seventh day in
118 order to generate secondary immunity. The immunological
parameters (IgA, IgM, IgG, TNFα, IFNγ and subgroups IgG1,
IgG2a) in serum and lung homogenizate were measured by
ELISA kits (Biosource).The same immunolgical parameters
were once again measured on the fourteenth day. On the
other hand, the blood samples were taken from hearts at
the end of the seventh and on the fourteenth days. Then, the
animals were sacrificed. The lung homogenisate was pre-
pared as the following procedures:The lung was removed
and washed with saline solution. Traceal parts of lung have
been cleaned and was divided into small parts. After then, Figure 1: The IgM Levels in Serum
the parts have been homogenized with saline solution and
centrifuged at 4000 rpm for 20 minutes. The supernatant
was separated and the above-mentioned immunological
parameters were measured.

RESULTS AND DISCUSSION


The IgM levels were significantly reduced with pulmonary ad-
ministration of IL-2 liposome on the fourteenth day (p<0.05)
(Figure 1). When IgG levels in lung were evaluated, the best
result was obtained with liposome formulation since sup-
pressed the immune system at most ( Figure 2).

According to the experimental findings, pulmonary IL-2 lipo-


some formulation caused long-time reduction in serum IgM
and IgA levels. It was shown that intravenous IL-2 solution
increased the TNFα and IFNγ levels more than the other ad- Figure 2: The IgM Levels in Lung Homogenisate
ministration methods did. Reduction of IgG, IgG2 and IgG2a
levels by the pulmonary IL-2 liposome was shown on the CONCLUSION
fourteenth day. The results indicate that the prepared IL-2 liposome for pul-
monary administration can be effective on immune system.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

ACKNOWLEDGEMENTS Methods
This study was supported by grants from Turkish Scientific SLN were produced by modified microemulsion dilution
and Technical Research Council (TUBITAK, 105S084). technique using stearic acid as matrix lipid, Tween 80 as sur-
factant and ethanol as co-surfactant, distilled water as dis-
REFERENCES pertant and either EQ1 (N,N-di-(b-steaorylethyl)-N,N-dime-
1. Cadee JA, Gnoot CJ,Jiskoot W, et al.Release of recombi- thylammonium chloride) or stearylamine as charge carrier.
nant human interleukin-2 from dextran-based hydro- The lipids were melted with a mixture of water, co-surfactant
gels, J Control Rel. 2002; 78:1-13. and the surfactant at 80ºC. A transparent, thermodynami-
2. Kanaoka E,Takahashi K, Yoshikawa T, et al. A significant cally stable system was formed when the compounds were
enhancement of therapeutic effect against hepatic me- mixed at certain ratios as seen in Table 1. This microemulsion
tastases of M5076 in mice by a liposomal interleukin-2 was then dispersed in a cold aqueous medium (+2ºC ±1) un-
(mixture),J Control Rel. 2002; 82:183-187. der mild mechanical stirring at a ratio of 1:10 microemulsion/
3. Çelebi N, Çilek A, Evaluation of pharmacokinetics of in- aqueous medium3.
terleukin- liposomes for pulmonary delivery, 2nd Pharm-
SciFair, June 8-12 2009. Table 1: SLN formulations

Distilled water (% w/w)


Stearylamine (% w/w)
Stearic Acid (% w/w)

Esterquat1 (% w/w)

Tween 80 (% w/w)

Ethanol (% w/w)
p041-PREPARATION AND

*Formulations
CHARACTERISATION OF CATIONIC SOLID
LIPID NANOPARTICLES AS A POTENTIAL DNA
DELIVERY SYSTEM
Gülten Kantarcı1, Devrim Demir Dora1, Hasan Akbaba1
1 2 2.5 2.5 15 12 66
2 2 2 2 15 12 67
Ege University Faculty Of Pharmacy- Pharmaceutical
3 2 2 2 15 12 68
Biotechnology Department, İzmir, Turkey 1
4 2 1 1 15 11 70
INTRODUCTION 5 2 3 3 15 12 65
In recent years, solid lipid nanoparticles (SLN) have been *In all formulations, 1ml microemulsion was dispersed in 10
developed as potential carriers for a number of drugs. SLN ml of dispertion medium. 119
usually consists of physiologically well-tolerated ingredients
already approved for pharmaceutical applications in hu-
mans. SLN can readily be produced in large scale, have good Droplet Size and Zeta Potential Determinations
storage capabilities including freeze-drying, can be sterilised The particle size distribution and average droplet size of
and show low cytotoxicity, when injected intravenously. In microemulsions were determined using Zetasizer Nano ZS
addition, the advantage of SLN is that the charge of the par- (Malvern Instruments Ltd., UK).
ticles can be modulated via the composition, thus allowing
binding of oppositely charged molecules via electrostatic in- pH Measurements
teractions1. SLN can be produced in nano-scale size (10–200 The observed pH values of the samples were measured by
nm), wherefore the particles are sufficiently small to traverse pH meter (Inolab Level 1, WTW Wissenschaftlich-Techniche
the microvascular system and prevent macrophage uptake Werkstätten GmbH, Weilheim, Germany), at 20±2ºC.
and are therefore particularly suitable for systemic delivery2.
Conductivity Measurements
The purpose of this study is to develop a novel cationic solid The electrical conductivity was measured using a conducto-
lipid nanoparticle formulations for targeting plasmid DNA meter (Jenway 4071, UK), at 20±2ºC.
for non-viral gene therapy.
Stability of SLN Formulations
MATERIALS AND METHODS The particle size and the polydispersity index of cationic SLN
Materials formulations were measured after two weeks storage in dis-
Tween 80....................... Merck-Co. (Hohenbrunn, Germany), persion medium at 4-8 ºC.
Stearic Acid................... Merck-Co. (Hohenbrunn, Germany),
Ethanol........................... Merck-Co. (Hohenbrunn, Germany), Plazmid DNA Purification
Stearylamine................ Sigma-Aldrich Co. (St. Louis, MO, USA). The Green Fluorescent Protein (GFP) expression plasmid
Esterquat 1(EQ1.......... Gerbu Biochemicals GmbH (Gaiberg, (pcDNA3.1/NT-GFP, 6160 bp) purchased from Invitrogen
Germany) (USA) was transformed into Escherichia coli JM109 cells. Plas-
Distilled Water............. pH 7.4 (adjusted with sodium hydrox- mid DNA purification was done by MiniPrep Plasmid DNA
ide) Isolation Kit (Fermentas, Germany), from the overnight liquid
50 µS (adjusted with sodium chloride) culture of transformed E. coli cells in Luria-Bertani medium
at an Ampicillin concentration of 80 µg/mL. The DNA con-
centration was determined by using Qubit fluorometer with

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

Quant-iT™ ds DNA BR Assay Kit (Invitrogen). The purity of the


isolated plasmid DNA was checked by 0.8 % (w/v) agarose
gel electrophoresis and contamination with RNA or genomic
DNA was not detected.

Preparation of SLN-pDNA Complexes


SLN-pDNA complexes were prepared by incubating 0.1 µg/
mL pDNA solution with a cationic SLN suspension for 30 min-
utes at 25ºC, at a DNA/EQ1 ratio of 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:12,
1:16 and 1:20 w/w. The complex formation was then checked
by agarose gel electrophoresis (Figure 1) (4). Figure 1. Agarose gel electrophoresis photograph of SLN-
pDNA complexes at different DNA/EQ1 ratios. Lanes from
Agarose Gel Electrophoresis left: moleculer weight marker (GeneRuler™ 1kb DNA Ladder,
SLN-pDNA complexes were diluted in Ultra-Pure distilled wa- Fermentas), naked pDNA, DNA/EQ1 mixtures w/w ratios of
ter to a final concentration of 0.1 µg DNA/µL and subjected 1:0,5, 1:1, 1:2, 1:4, 1:8, 1:16. 1:20, 0:16 respectively.
to electrophoresis on an 0.8 % (w/v) agarose gel (0.5 % (w/w)
ethidium bromide included for visualization) for 40 min at The cationic SLN was developed as DNA delivery system and
120 V. The bands were visualized by a UV transilluminator it was planned to determine its transfection efficiency in hu-
(Vilber-Lourmat) and images were captured using a digital man cancer cell lines.
camera.
ACKNOWLEDGMENTS
RESULTS AND DISCUSSION We would like to thank to Sevil Zencir for her kind help.
Table 2 summarizes particle size, zeta potential and polydis-
persity index of SLN composed by different amounts of EQ1 REFERENCES
and Stearylamine. Formulation 2 was selected for further 1. Müller, R.H., Mader, K. and Gohla, S. Solid lipid nanopar-
studies because of its appropriate zeta potential (41,3±3,11 ticles (SLN) for controlled drug delivery - a review of the
mV) for DNA binding. state of the art, European Journal of Pharmaceutics and
Biopharmaceutics, 2000; 50: 161-177.
Table 2: Particle Size, PDI and Zeta Potential of the formula- 2. Pedersen, N., Hansen, S., Heydenreich, A. V., Kristensen,
tions H.G. and Poulsen, H.S. Solid lipid nanoparticles can effec-
120 Particle Size Zeta Potential tively bind DNA, streptavidin and biotinylated ligands,
Formulations PDI European Journal of Pharmaceutics and Biopharmaceu-
(nm) (mV)
1 34,93 (±0,6431 0,235 (±0,006) 51,7 (±0,681) tics, 2006; 62: 155-162.
3. M.R. Gasco., Method for producing solid lipid micro-
2 33,14 (±0,4954) 0,237 (±0,007) 41,3 (±3,11
spheres having a narrow size distribution, US Patent
3 30,85 (±0,3848) 0,285 (±0,006) 30,9 (±3,88) 5250236, 1993.
4 42,83 (±0,5327) 0,271 (±0,001) 29,7 (±2,44) 4. Heydenreich, A.V., Westmeier, R., Pedersen, N., Poulsen,
5 35,18 (±0,2552) 0,217 (±0,005) 27,1 (±1,61) H.S., Kristensen, H.G. Preparation and purification of
cationic solid lipid nanospheres-effects on particle size,
We observed that the particle size and the polydispersity physical stability and cell toxicity, International Journal
index of cationic SLN formulations were increased after two of Pharmaceutics, 2003; 254: 83–87.
weeks storage in dispersion medium at 4-8 ºC. The results of 5. Vighi, E., Rouzi, B., Montanari, M., Batini R. and Leo, E.
the measurements showed that the particle size of all formu- Re-dispersible cationic solid lipid nanoparticles (SLNs)
lations increased after 15 days of storage in distilled water freeze-dried without cryoprotectors: Characterization
(50µS, pH 7.4). Therefore, samples lyophilized at -45ºC and and ability to bind the pEGFP-plasmid, European Jour-
0,07 mbar for 48h (Christ Alpha 1-2 LD Plus freze-dryer, İzmir) nal of Pharmaceutics and Biopharmaceutics, 2007; 67:
for the stability. Lyophilized samples were reconstituted in 320–328.
distilled water (adjusted to pH 7,4 and 50 µS) by mixing with
vortex then the resulting formulations filtered at 0,22 µm
millipore filter5. p042-Long-term biocompatibility of a
Binding of the cationic SLN to pDNA was checked by the new liquid polyurethane embolization
analysis of the electrophoretic mobility of the pDNA within material
an agarose gel. As shown in Figure 1, the cationic SLN formu-
lation 2 was immobilised detectable amounts of DNA at 1:8 Zsuzsa Tóvölgyi1, István Hudák2, Tamás Dóczi2, Lajos Botz1
DNA/EQ1 (w/w) ratios (Lane 6).
Pharmaceutical Institute, Faculty of Medicine, University of
Pécs, Pécs, Szigeti u. 12, Hungary H-76241
Department of Neurosurgery, Faculty of Medicine, University of
Pécs, Pécs, Rét u. 2, Hungary H-76232

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

INTRODUCTION cellulose acetate. . The polyurethane was dissolved in a ster-


Endovascular embolization offers a novel and minimally in- ile solution of DMSO and ethanol. As soon as an aqueous so-
vasive treatment to several vascular pathologies in the pe- lution, e.g. blood, or a water-containing surface comes into
riphery and central nervous system - including occlusion of contact with the liquid polyurethane, solvents rapidly diffuse
arteriovenous malformations (AVMs), arteriovenous fistulas, out from the mixture, precipitation process starts and is fin-
saccular aneurysms and even hypervascularized tumours. ished within minutes. A solid, elastic intravascular “implant”
The most commonly used liquid embolic materials are the without adhesion to the vascular wall is the result. Sterile, mi-
tissue adhesive acrylics, such as isobutyl cyanoacrylate cronized tantalum powder (BALT Extrusion, France) was sus-
(IBCA, Bucrylate) and n-butyl cyanoacrylate (nBCA, TRUFILL® pended in the solution to obtain appropriate radioopacity
by Cordis Neurovascular Inc. US) and non-adhesives, such during endovascular occlusion of vessels. Forty-eight adult
as ethylene vinyl alcohol copolymer (EVOH, ONYX® by ev3 male and female CD rats weighing 250-350 g from Charles
Inc. US) and cellulose acetate. However, these devices do River (Hungary) were employed. Under general anaesthesia
not meet all requirements of the endovascular surgeon and and aseptic conditions the femoral arteries of the animals
therapeutical problems often arise after embolisation mainly were disclosed carefully. The right femoral arteries of 24 rats
due to cellular reaction in the vessel wall and in surrounding were embolized with the liquid polyurethane embolization
tissue. Mutagenic and carcinogenic potential of IBCA was re- material. In the control group (n=24) animals received physi-
ported in animal experiments, therefore, the use of IBCA was ologic NaCl injection into the right femoral artery. Animals
ceased very early1. The cyanoacrylate glues induce a signifi- were sacrificed at 1 day (n=6), 1 week (n=6), 8 weeks (n=6),
cant acute and chronic inflammatory response due to toxic 24 weeks (n=6) after vessel occlusion to explant vessels and
reaction products and the exothermic polymerization2. After surrounding tissue. The removed arterial segments were
successful embolization of aneurysms and AVMs by means immediately immersed and fixed in 4% (w/w) neutral buff-
of cellulose acetate polymer solution only mild inflamma- ered (pH=7.4) formalin at 4°C. The embedded tissue sample
tory reactions with no chronic granulomatous reaction was blocks were frozen cut 10 μm thick sections, stained with
reported, however, recanalisation was also seen3. Natarajan haematoxylin and eosin (HE) and periodic acid-Schiff (PAS)
et al. 4 observed inflammation in equal frequency (90-91%), to evaluate cellular and collagen states in the artery and the
with both EVOH and nBCA embolization groups. Angione- surrounding tissue.The sections were also immunostained
crosis occurred in higher rate in patients treated with EVOH with an antibody (CD31) to identify the presence of endothe-
(59%) than with nBCA (40%). Chronic foreign body giant lium in vessel walls within the embolizing material.
cells and recanalisation was observed after EVOH but not
after nBCA embolization. The maximum exposure time of RESULTS AND DISCUSSION
specimens embolized with nBCA was less than 24 days in this All sections were examined under light microscope for pres- 121
study, this time span had been probably not long enough for ence of occlusion, recanalisation and inflammation and also
the development of foreign body giant cells and recanalisa- to evaluate vessel wall and tissue integrity and regeneration.
tion (4). Studies on technical feasibility and histotoxicity of No inflammation in the perivascular tissues and no evidence
EVOH pointed out that the carrier solvent, DMSO (0,5 to 1,0 of necrosis was observed. Only mild intravascular inflam-
ml) caused severe acute vasospasm and toxic effects, such as mation response with scattered foreign body-type reaction
angionecrosis, endothelial denudation, subarachnoid hae- with giant cells containing embolisation material were de-
morrhage, cerebral infarction and death, but angitoxicity of tected and minor changes to the vessel wall were seen. As a
DMSO may be dose-dependent5. prove to good biocompatibility of the agent examined, it has
not induced severe inflammation or toxic destruction of ves-
An ideal transcatheter embolization device must have low sel wall, accordingly no contraindication has been found to
toxicity, should cause minimal inflammation and produce its application for endovascular procedures. As vessel occlu-
reliable and permanent occlusion without extravasation and sions have remained stable for up to 6 months the suitability
recanalisation. for embolization vascular disorders, such as arteriovenous
malformations and aneurysms has also been proved.
The authors examined the properties of a potential emboli-
sation polyurethane material. The purpose of this study is to REFERENCES
determine the long-term biocompatibility of polyurethane 1. Vinters HV, Lundie MJ, Kaufmann JC: Long-term patho-
a new embolisation material in a preclinical animal studies. logical follow-up of cerebral arteriovenous malforma-
Gross and microscopic changes were studied in the embo- tions treated by embolization with bucrylate. N Engl J
lised artery and the surrounding tissue, presence of angion- Med 1986;314:477-483.
ecrosis, arterial revascularization, intra- and perivascular in- 2. Vinters HV, Galil KA, Lundie MJ, Kaufmann JCE: The histo-
flammatory reactions were searched for in rats in a 6 months toxicity of cyanoacrylates. Neuroradiology 1985;27:279–
experiment. 291.
3. Sugiu K, Kinugasa K, Mandai S, Tokunaga K, Ohmoto T.:
MATERIALS AND METHODS Direct thrombosis of experimental aneurysms with cel-
Ingredients of the polyurethane are 4,4’-methylene-bisphe- lulose acetate polymer (CAP): technical aspects, angio-
nyl-isocyanate (MDI), 1,4- butandiol (BD) and polytetrahy- graphic follow up, and histological study. J Neurosurg.
drofurane (PTHF). The chemical structure of polyurethane is 1995;83(3):531-538.
linear. This property offers numerous advantages compared 4. Natarajan SK, Born D, Ghodke B, Britz GW, Sekhar LN.:
to the other monomers or polymers like nBCA, EVOH and Histopathological changes in brain arteriovenous mal-

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

formations after embolization using Onyx or N-butyl cy- aprotinin into pre-weighted water phase, and then adding
anoacrylate. J Neurosurg. 2009;111(1):105-13. into the oil, surfactants and co-surfactants mixture.
5. Chaloupka JC, Viñuela F, Vinters HV, Robert J: Technical
feasibility and histopathologic studies of ethylene vinyl Determination of phase inversion temperatures (P.I.T.) of the
copolymer (EVAL) using a swine endovascular emboliza- microemulsions :
tion model. Am J Neuroradiol 1994;15:1107-1115. Blank microemulsions were placed in a beaker and the bea-
ker was heated in a water bath. The temperature of the bath
was increased at 1°C/min steadily. The microemulsions were
p043-EVALUATION OF MICROEMULSION agitated with a stirrer and the change in the conductivity
FORMULATIONS FOR PARENTERAL DRUG was recorded. The measurements were taken with a conduc-
tivity meter (Conductivity meter 4071, Jenway) (4).
DELIVERY OF APROTININ: FORMULATION,
DESIGN AND STABILITY STUDIES Stability of microemulsions
Stability of the different microemulsion formulations was
H. Yeşim Karasulu1, Neslihan Üstündağ1, Zeynep Ay tested at 5±3°C, 25±2°C and 40±2°C. Microemulsions were
Şenyiğit1, Tamer Güneri1 evaluated with their pH, viscosity, droplet size, electrical
conductivity, density and refractive index changes during 6
Ege University, Faculty of Pharmacy, Department of months.
Pharmaceutical Technology, İzmir, Turkey1
Determination of the osmolality of water phase of the mi-
INTRODUCTION croemulsions
Microemulsions are thermodynamically- stable, isotropic, Measurement of the osmalality of the water phase with or
single-phase dispersions of oil and water stabilized by one or without aprotinin was based on the freezing-point method.
more surface active agents Microemulsion formulations have After calibration of the osmometer (Knauer Semi-Micro Os-
distinct advantages over macroemulsion systems when de- mometer) with reference standarts (distilled water and 400
livered parenterally; because of the fine particle microemul- miliosmol NaCl solution), the osmolality was measured with
sion is cleared more slowly than the coarse particle emulsion 0.15 mL of sample5.
and therefore, has longer residence time in the body1.
Characterization of aprotinin-loaded microemulsion
Aprotinin is a monomeric globular polypeptide derived from formulations
122 bovine lung tissue; it has a molecular weight of 6512 and The characteristic properties of drug-loaded microemulsions
consists of 16 different amino acids arranged in a chain of 58 such as pH, viscosity, refractive index, electrical conductivity,
amino acid residues. It is a Kunitz protease inhibitor and has droplet size and zeta potential were evaluated.
a wide action with particular against trypsin, chymotrypsin
and kallikrein, making it theorically attractive in ameliorating Determination of zeta potential
the effects of acute pancreatitis2. Zeta potential measurements were performed at 25 oC us-
ing a zetasizer (Malvern Zetasizer Nano ZS, UK).
The aim of this study is to design and evaluate novel micro-
emulsion formulations for parenteral aprotinin administra- RESULTS AND DISCUSSION
tion. The optimum formulations according to phase diagrams are
shown in Table 1.
MATERIALS AND METHODS
Materials Table 1: Composition of the microemulsion formulations
Aprotinin (Sigma, Germany), oleic acid (Merck, Germany), M1 M2 M3
isopropyl myristat (Sigma, Germany), Labrasol (Gattefosse, Isopropyl
Oil phase Oleic acid 33.92% Oleic acid 9.34%
France), Cremophor EL (Sigma, Germany), ethanol (J.T.Baker, myristate 10.18%
Holland) and isopropyl alcohol (Merck, Germany) were used Cremophor EL /
Surfactants/co- Cremophor EL/ Labrasol/Isopropyl
for microemulsion formulation. All other chemicals were Isopropyl alcohol
surfactants Ethanol 51.62% alcohol 66.58%
used as analytical grade. 54%

Preparation of microemulsion formulations 0.9% NaCl 0.9% NaCl 0.9% NaCl


Water phase
The microemulsion systems were formulated with various solution 14.46% solution 36.66% solution 23.25%
compositions of oil, surfactants and co-surfactants. The HLB value 12.5 12.5 14
pseudo-ternary phase diagram was constructed by titration The phase inversion temperatures of M1, M2 and M3 were
of homogenous liquid mixtures of oil, surfactant and co-sur- found 48.00±0, 50.666±0.577, 47.666±0.015 respectively.
factant with 0.9% NaCl solution as water phase at ambient Also, the results were shown in Fig. 1, Fig. 2 and Fig. 3.
temperature. After the identification of microemulsion area
in the phase diagrams, optimum formulations were selected Microemulsion formulations didn’t show significant changes
according to the gravity centers of these diagrams3. Drug- and were determined stabil at 5±3°C, 25±2°C and 40±2°C
loaded microemulsions were prepared by dissolving 2 mg of during 6 mounts.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

The water phase of the parenteral microemulsions should be Table 2: Characterization of the aprotinin-loaded microemul-
augmented by the incorporation of ionic or osmotic agents sion formulations
because emulsified oil exerts no osmotic effect. Therefore,
M1 M2 M3
the osmolaltity of water phase (0.9% NaCl solution) and 2
mg aprotinin incorporarted water phases of the formulatons pH 4.073±0.012 4.5±0.010 4.693±0.030
were tested. The osmolality these water phases were found
in range of 300-320 milliosmol/kg.
Viscosity (cP) 19.71±0.043 17.00±0.017 11.223±0.025
Characterization of the aprotinin-loaded microemulsion for-
mulations were listed in Table 2.

Zeta potential of the blank M1, M2 and M3 microemulsion Electrical 118.7±0.100 0.43±0.020 mS/ 0.155±0.007
formulations were found +0.113±0.153, +0.0197±0.0834, Conductivity µS/cm cm mS/cm
-0.0472±0.228, respectively. In addition, zeta potential
of aprotinin loaded M1, M2 and M3 formulations were
14.066±1.250, -0.038±0.033, 0.199±0.021, respectively.
Refractive index
1.4325±0.0006 1.3942±0.0059 1.4149±0.0016
(nm)

Droplet size (nm) 1.023±0.074 6.411±0.509 2.902±0.044

Polydispersity
0.288±0.042 0.254±0.078 0.493±0.161
Index

CONCLUSION
The results of characterization and stability studies, physi-
cochemical property tests showed that all microemulsion
Figure 1: Change of conductivity of M1 with various tem- formulations studied may be appropriate vehicles for paren-
perature. teral uses. In conclusion, microemulsion formulations con- 123
taining aprotinin may be appropriate vehicles for aprotinin
parenteral application.

ACKNOWLEDGEMENTS
This study was supported by The Scientific and Technologi-
cal Research Council of Turkey (Tubitak-108 S 083).

REFERENCES
1. Jadhav KR, Shaikh IM, Ambade KW, Kadam VJ. Applica-
tions of Microemulsion Based Drug Delivery System,
Curr Drug Deliv. 2006; 3:267-273.
2. Smith M, Kocher H, Hunt BJ. Aprotinin in severe acute
Figure 2: Change of conductivity of M2 with various tem- pancreatitits, The Int J Clin Pract. 2009; 1-9
perature. 3. Karasulu HY, Üstündağ N, Ay Şenyiğit Z, Güneri T. A
Novel Aprotinin Microemulsion For Pancreatit: Design,
Optimization And Physichemical Characterization, Drug
Reserach and Development Abstract Book, Ankara, Tur-
key, 4-7 May 2009.
4. Yue Y, San-ming L, Li-min Y, Pan D, Da-fang Z. Physico-
chemical properties and evaluation of microemulsion
systems for transdermal delivery of meloxicam, Chem
Res Chinese U. 2007; 23(1):81-86.
5. Ganta S, Paxton JW, Baguley BC, Garg S. Pharmacokinet-
ics and pharmacodynamics of chlorambucil delivered in
parenteral emulsion, Int J Pharm. 2008; 360:115–121.

Figure 3: Change of conductivity of M3 with various tem-


perature.

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

p044-DEVELOPMENT OF A PARENTERAL Sterility tests


SOLVENT SYSTEM FOR THE SOLUBILIZATION The stability of the solvent system was examined on auto-
claving and on filtration. Stability of the solvent system was
OF AZOLE TYPE ANTIFUNGALS confirmed on autoclaving on 120°C for 20 minutes.

Kristóf Kovács1, György Stampf1, István Antal1, Imre Static filter tests were conducted on 0.2µm pore size cellu-
Klebovich1, Krisztina Ludányi1 lose acetate, cellulose nitrate and polyamide membrane fil-
ters. Integrity of the membrane was examined prior to and
Semmelweis University, Department of Pharmaceutics, Hogyes following testing and the ability of the membrane to bond
Endre street 7, H-1092 Budapest, Hungary1 miconazole was also examined.

INTRODUCTION Stability test


Poor water solubility of active pharmaceutical ingredients is Accelerated stability test of the solvent system was per-
a great issue in pharmaceutical development. Unfortunately formed according to Guideline on stability testing: stability
a wide range of chemicals exhibit inadequate solubility1,2. testing of existing active substances and related finished
products.
Generally systemic mycoses are chronic and develop slowly.
A group of active pharmaceutical ingredients, the azoles RESULTS AND DISCUSSION
have been shown to be effective agents against various Solubility studies
fungi and can be used in the treatment of systemic fungal in- The solubility tests were systematically built up in order to achieve
fections. Although these compounds are potent molecules, adequate solubility of miconazole. First aqueous solutions of the
most of them show poor water solubility, thus formulation solubilizing excipients were examined, then binary and ternary
of a composition, which comprises therapeutically effective solvent systems were tested. The solubilizing effect of one of the
quantities of active agent, is difficult3. It has been shown that solvent systems from each group is shown in figure 1.
the solubility of azoles can be advantageously altered by the
use of cyclodextrins and other solubilizers, but all of them
have certain disadvantages.

In this study we developed a solvent system for azoles, which


is a novel combination of well established excipients show-
124 ing synergetic solubilizing effect.

MATERIALS AND METHODS


Materials
Ketoconazole (MW: 531.44) and miconazole (MW: 416.1) of
minimum 98% purity were obtained from Bosche Scien-
tific, New Brunswick, NJ, USA. Acetic acid, ammonium ac-
etate, ethanol 96%, calcium gluconate, glycerol, macrogol
400, phosphoric acid, polysorbate 20, 60 and 80, potassium
dihydrogen phosphate, propylene glycol and sodium tau-
rocholeate were used as solubilizers and were all pharma-
copoeial grade (Molar Chemicals Ltd., Budapest, Hungary). Figure 1: Solubilizing effect of various solvent systems
Ammonium acetate, acetic acid, methanol and water were
all of HPLC gradient grade (Sigma-Aldrich Ltd., Budapest, As seen in the table the a ternary solvent system compris-
Hungary). ing 5% polysorbate 80, 25% ethanol and 0.05M ammonium
acetate (pH 3.1) is able of solubilizing more than 40 mg/mL
Methods of miconazole. Furthermore, the same composition is able of
Solubility studies dissolving more than 150 mg/mL of ketoconazole.
Solubility tests were conducted using pH adjusters, co-sol-
vents, surfactants and their combinations. Solvent systems Optimization of the solvent system
were prepared and an excess of drug was added to the sol- Optimization of the solvent system was performed and re-
vents. After reaching equilibrium the excess active ingre- sulted in a solvent system containing less excipients, but
dient was removed and an HPLC method was used for the with a similarly substantial solubilizing capacity.
quantitative determination of miconazole.
Sterility tests
Optimization of the solvent system The final composition was autoclaved and was found to be
After ensuring adequate solubility of miconazole, the com- stable upon heat sterilization.
position of the ternary solvent system was optimized by
reducing the amount of polysorbate and ethanol based on The tested membrane filters did not lose their integrity
solubility and dilution tests. Dilution tests were performed during the static filter tests, but the cellulose nitrate mem-
using 5% dextrose and 0.9% sodium chloride infusions. brane filter absorbed substantial miconazole during the test.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

Therefore it was concluded that aside filtration with cellulose Construction of phase diagrams
nitrate membrane filters, all of the tested methods of steril- Phase diagrams were constructed at 5,57 and 8,40 HLB values
ization may be applied for the novel solvent system. with S85 and T80. Oil phase was mixed with the surfactant
phase at the ratios of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40,
Stability test 70:30, 80:20, 90:10. These mixtures were slowly titrated with
Accelerated stability tests performed over a period of 12 distilled water until the turbidity was observed. Microemulsion
months prove the long term stability of the solvent system. areas (MA) were detected with the aid of phase diagrams drawn
using a computer program developed in the computer center,
CONCLUSION Faculty of Pharmacy, University of Ege [4]. Four microemulsion
Based on the results it can be concluded that a novel solvent formulations were chosen for further studies from the gravity
system was worked out, which showed a very substantial solu- centers of microemulsion formation areas (Table 1).
bilizing power. After optimizing the composition of the solvent
system it was shown that the solvent system is readily capable Table 1: The contents of the microemulsion formulations.
of being sterilized by both heat sterilization and filtration. The % of ingredients
accelerated stability tests show long term stability of the sol- Oils Formations HLB
Oil Surf Water
vent system. Based on these results a concentrate for infusion OM1 5.57 31.0 66.5 2.5
containing miconazole or ketoconazole was formulated. Olive
OM2 8.40 31.6 64.4 4.0
SM1 5.57 32.1 65.4 2.5
REFERENCES Soybean
1. Strickley RG. Solubilizing excipients in oral and inject- SM2 8.40 21.0 75.0 4.0
able formulations, Pharm Res. 2004; 21(2): 201-230.
2. Yalkowsky SH, Solubility and Solubilization in Aqueous Physichochemical characterization
Media, New York, 1999. pH values were measured using pH meter (Inolab Level 1,
3. Richardson MD, Jones BL, Therapeutic Guidelines in Sys- WTW Wissenschaftlich-Techniche Werkstätten GmbH, Weil-
temic Fungal Infections, 3rd Ed., England, 2004. heim, Germany), at 25±2°C. Viscosity and flow characeris-
tics were determined by Programmable DV-III+ Rheometer
(Brookfield Engineering Labs. Inc., Middleboro, USA) (Spin-
dle: SC4-21) at 25.0±0.1°C. Electrical conductivity was mea-
P045-PREPARATION OF W/O sured using a conductometer (Jenway 4071, UK), at 25±2°C.
MCIROEMULSIONS WITH DIFFERENT Droplet size distribution and PDI were determined by Zeta-
VEGETABLE OILS FOR DRUG DELIVERY sizer Nano ZS (Malvern Instruments Ltd., UK) at 25±0,1°C. 125
Gülten Kantarcı1, Mustafa Kotmakchiev1, Buket Aksu2, Tamer Stability of microemulsion formulations
Güneri3 The centrifuge method (5000 rpm, 15 min), and particle size
measurements are used for determining the physical stability
Ege University, Faculty of Pharmacy, Department of of microemulsions. The storage stability of microemulsions
Pharmaceutical Biotechnology1, İzmir, Turkey were checked after 6 months storage at room temparature.
Ege Universiry, Faculty of Pharmacy, Department of
Pharmaceutikal Technology2, İzmir, Turkey RESULTS
Ege Universiry, Faculty of Pharmacy, Department of As shown in Table 1, W/O microemulsion formulations were de-
Pharmaceutical Technology3 , İzmir, Turkey veloped with soybean oil and olive oil at different HLB values.
The clear transparent W/O microemulsion areas are plotted
INTRODUCTION on pseudoternary phase diagram for the systems oil/T80:S85/
Microemulsions are thermodynamically stable, isotropic so- water. At 8,40 HLB, w/o microemulsion areas were larger than
lutions of water and oil stabilized by appropriate surfactants those at lower HLB with both oils. The MA’s with 5.57 HLB were
and co-surfactants. Several plant oils, in combination with so similar for SM1 (166.90) and for OM1(166.31). At 8.40 HLB,
different surfactants or surfactant/co-surfactant mixtures the area of OM2 (267.49) was larger than area of SM2 (231.69).
have been studied in order to construct biologically suitable This may be related to the difference between the triglyceride
microemulsions1,2,3. compositions of olive oil and soybean oil1,5. Olive oil contains
78 percent monounsaturated, 8 percent poly unsaturated and
The purpose of this study was to investigate the effects of 12 percent saturated fatty acids, while soybean oil contains 25,
different vegetable oils on microemulsion formulation. 62 and 11.5 percent, respectively. The two oils showed similar
microemulsion forming areas which are narrow. These results
MATERIALS AND METHODS agree with the results of Parris et al. who concluded that trig-
Materials lyceride oils could hardly be microemulsified5.
Soybean oil and Olive oil (as oil phase) from Küçükbay Yağ
Sanayi A.Ş. (Izmir, Turkey), Tween® 80 (T80) (as surfactant) The viscosity was decreased after storage for all formula-
from Merck-Co. (Hohenbrunn, Germany), Span® 85 (S85) tions. As seen in table 2, the change of viscosity was so small
(as surfactant) from Sigma-Aldrich Co. (St. Louis, MO, USA). for OM2. The formulations showed newtonian flow before
Distilled water was used in all experiments as the aqueous and after the storage (Figure2, only before storage). On pH
phase. values, little changes were observed.

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

Table 2: Physichochemical properties of microemulsions. DS- bility after OM2. They also exhibited good stability in their
droplet size; PDI- polydispersity index. droplet size after 6 months storage, and may be appropriate
Before storage vehicles for drug delivery.
Formulations Viscosity
pH DS (mm) PDI REFERENCES
(cP)
OM1 214.3 6.84 58.21±2.480 0.096±0.017 1. Do LD., Withayyapayanon A., Harwell JH. and Sabatini
OM2 220.0 7.11 60.48±2.151 0.266±0.038 DA. Environmentally Friendly Vegetable Oil Microemul-
sions Using Extended surfactants as Linkers, J Surfact
SM1 177.3 6.80 73.08±0.850 0.086±0.050
Deterg. 2009; 12: 91-99.
SM2 328.7 7.24 74.09±12.457 0.181±0.007 2. Gupta S.; Sanyal S.K.; Datta S.; Moulik S.P. Preparation of
After storage prospective plant oil derived micro-emulsion vehicles
OM1 134.4 6.90 57.10±1.412 0.243±0.006 for drug delivery, Indian J Biochem Biophys. 2006; 43:
OM2 21..2 7.24 58.83±1.403 0.320±0.026 254-257.
SM1 118.9 7.05 72.53±1.105 0.090±0.042 3. Kantarci G.; Özgüney I.; Karasulu H.Y.; Arzık S.; Güneri
SM2 242.7 7.12 66.10±0.746 0.228±0.023 T. Comparison of Different Water/Oil Microemulsions
Containing Diclofenac Sodium: Preparation, Character-
The conductivity results are not included in Table 2, since all ization, Release Rate, and Skin Irritation Studies, AAPS
formulations exhibited non-conducting behavior at prepa- PharmSciTech, 2007; 8(4): Article 91
ration and after the storage period. 4. Ege MA, Karasulu HY, Guneri T. Triangle phase diyagram
analysis software, The 4th International Postgraduate
All formulations were proven stable by the centrifuge meth- Research Symposium on Pharmaceutics, Istanbul ,2004.
od. Droplet sizes of the formulations showed a little bit de- 5. Parris N., Joubran RF. and Lu DP. Triglyceride microemul-
crease after storage, except SM2. sions: Effect of nonionic surfactants and the nature of
the oil, J Agric Food Chem. 1994; 42: 1295-1299.

p046-NANOEMULSION FORMULATION OF A
NEW Candida albicans CYP51 INHIBITOR

Aurélie Schoubben1, Elio Cenci2, Fausto Schiaffella1, Paolo


126 Blasi1, Lara Milanese1, Carlo Rossi1, Anna Vecchiarelli2,
Maurizio Ricci1, Renata Fringuelli1

Department of Drug Chemistry and Technology, University of


Perugia1
Department of Experimental Medicine and Biochemical
Sciences, University of Perugia2
Figure 1: Pseudoternary phase diagrams of W/O type micro-
emulsions. INTRODUCTION
In a program aimed at the design and synthesis of novel azole
inhibitors of C. albicans CYP51 (CA-CYP51), a series of azole
1,4-benzothiazines and 1,4-benzoxazines were recently syn-
thesized. Among them a novel derivative, 2-butyl-6-{1-[(4-
chlorobenzyl)oxy]-2-(1H-1-imidazoyl)ethyl}-4-methyl-3,4-di-
hydro-2H-1,4-benzothiazin-3-one (Fig.1), characterized by a
very good MIC against C. albicans CA-6, showed a low water
solubility that probably limits its in vivo activity1.

The water solubility, determined at 25°C, was of about 4 µg/


mL that, according to the European Pharmacopoeia, corre-
sponds to a “practically insoluble” compound2.

Therefore, the aim of the study was to assess the feasibility


of using nanoemulsion technology to overcome the com-
Figure 2: Flow behavior of w/o type microemulsions before pound solubility issues.
storage.
MATERIALS AND METHODS
CONCLUSION Materials and animals
Among all tested formulations OM2 showed minimal chang- PEG 400 was purchased from Sigma (Milan, Italy). Flucon-
es on physicochemical properties. SM1 has also good sta- azole was obtained from Pfizer (Latina, Italy).

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

For experimental systemic candidiasis, mice were inoculated


with the highly virulent C. albicans strain CA-6 isolated from clini-
cal specimens in the laboratories of the Microbiology Section,
University of Perugia3. Female, 8-10 weeks old, outbred CD1 mice
were obtained from Harlan Nossan Laboratories (Milan, Italy) and
housed at the Animal Facilities of the University of Perugia, Italy.

Methods
To prepare the nanoemulsion, the compound was first solu-
bilized in PEG 400 and then added dropwise to deionised
water under stirring. The obtained emulsion was character-
ized in terms of dimensions and stability using photocorrela-
tion spectroscopy.
Figure 2: Volume diameter distribution of the nanoemul-
In vitro anti-Candida activity was determined according to sion.
the NCCLS guidelines4.
REFERENCES
For systemic infection, mice were infected intravenously on 1. Schiaffella F, Macchiarulo A, Milanese L, Vecchiarelli A,
day 0 with 2.5x105 blastospores of the highly virulent C. al- Costantino G, Pietrella D, Fringuelli R. Design, Synthesis,
bicans CA-6 strain and untreated or treated 2 h before chal- and Microbiological Evaluation of New Candida albicans
lenge and once a day for 6 consecutive days after challenge CYP51 Inhibitors. J Med Chem. 2005; 48: 7658-7666.
with the synthetic compound, nanoemulsion, fluconazole 2. European Pharmacopoeia. 6th Edition; 1: 5.
(all at 10 mg/kg body weight, i.p.). PEG 400 (0.2 mL, i.p.) was 3. Bistoni F, Vecchiarelli A, Cenci E, Puccetti P, Marconi P,
used as a negative control. Infected animals were monitored Cassone A. Infect Immun. 1986; 51: 668.
for survival and organ clearance. 4. Reference Method for Broth Dilution Antifungal Suscep-
tibility Testing of Yeast; Approved Standard NCCLS Doc-
RESULTS AND DISCUSSION ument M27-A; National Committee for Clinical Labora-
The nanoemulsion was characterized by dimensions of tory Standards:Waye, PA, 1997.
189±55 nm and the formulation showed to be stable for at
least 7 months even when diluted (Fig. 2).
p047-EFFECTS OF STERILIZATION 127
The nanometric compound droplets should accelerate TECHNIQUES ON INJECTABLE STEALTH BONE
its in vivo solubilization with respect to the drug solution
TARGETED NANOPARTICLES
(DMSO:H2O, 1:4). In fact, it can be hypothesized that once
injected, DMSO dilution or absorption may lead to drug
phase separation and low effective surface available for dis- İpek Özcan1, Freimar Segura Sanchez2, Kawthar Bouchemal2,
solution. In addition to the improved solubility properties, Özlem Abacı3, Özgen Özer1, Tamer Güneri1, Gilles Ponchel2
this formulation allowed to eliminate the use of DMSO that
should be preferably avoided in vivo. Ege University, Faculty of Pharmacy, Department of
Pharmaceutical Technology, 35100 Izmir, Turkey1
The preliminary results of in vivo experiments showed that Paris-South University, UMR CNRS 8612, Faculty of Pharmacy,
the use nanoemulsion turned out to be a valuable strategy Physicochimie Pharmacotechnie Biopharmacie, 92290
to overcome solubility problems encountered during early Chatenay-Malabry, France2
discovery and at the formulation stage as well. Ege University, Faculty of Science, Department of Biology, Basic
& Industrial Microbiology, 35100 Izmir, Turkey3

INTRODUCTION
Synthetic biodegradable polymers are well-known materi-
als for the preparation of nanoparticles. Poly(γ-benzyl-L-
glutamate) PBLG, a synthetic degradable polypeptide and
various chemical moieties could be quite easily introduced in
the structure of PBLG to form various copolymers1. Nanopar-
ticles exhibit different properties such as targeting, stealth
properties, efficient drug loading and controlled drug re-
lease.

Sterilization of injectable nanoparticles is a major challenge


in the designing of appropriate drug delivery systems. Mem-
Figure 1: Chemical structure of 2-butyl-6-{1-[(4-chloroben-
brane filtration is a safe technique based on physical removal
zyl)oxy]-2-(1H-1-imidazoyl)ethyl}-4-methyl-3,4-dihydro-2H-
of present microorganisms that does not require excessive
1,4-benzothiazin-3-one
heat or radiation causing irreversible effects on the nano-

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

particles. However, it is very much limited to the size of the RESULTS AND DISCUSSION
particles. Heat sterilization by autoclaving is a highly effec- Characterizations of nanoparticles were evaluated on sterile
tive technique involving high temperatures, which may in- and non-sterile samples. Table 1 and 2 summarize the mean
fluence decomposition or degradation of active ingredient diameter, polydispersity index and zeta potential values of
as well as the nanoparticle material2,3. non-sterile and sterile nanoparticles, respectively.

In the present work, effect of sterilization technique on pre- Table 1: Physicochemical characteristics of non-steril nano-
viously designed stealth bone targeted nanoparticles prop- particles
erties such as particle size, zeta potential and polydispersity
index were investigated. Formulation Mean diameter Zeta potential Polydispersity
code (nm) (mV) index
MATERIALS AND METHODS
Preparation of Nanoparticles
The surfaces of the nanoparticles were modified with alen- F1 50±14 -25.6±0.77 0.137±0.03
dronate (ALD) (Chemos GmbH) as targeting agent, polyeth-
ylene glycol (PEG) (Shearwater Corporation) as hydrophilic
F2 49±16 -25.5±0.81 0.118±0.05
agent and fluorescein isothiocyanate (FITC) (Sigma Aldrich)
as imaging agent. F1 and F2 coded nanoparticles were pre-
pared by nanoprecipitation technique using composite Table 2: Physicochemical characteristics of nanoparticles af-
polymers (PBLG-ALD/PBLG-PEG/PBLG-FITC and PBLG-PEG- ter different sterilization techniques
ALD/PBLG-PEG/PBLG-FITC) at a ratio of 2:2:1, respectively).
Mean diameter Zeta potential Polydispersity
Sterilization of Nanoparticles Formulation code (nm) (mV) index
Membrane Filtration
The formulations were filtered through a Milipore Express™ Mean brane filtration
membrane filter system (Millipore®, Volketswil, Switzerland). Af-
terwards, sterile suspension was poured into sterile glass vials. F1 52±10 27.4±0.85 0.132±0.05

F2 48±14 -23.9±0.48 0.122±0.04


Autoclaving (Heat Sterilization)
Samples were divided into two groups of equal volume af-
Autoclaving
128 ter preparation. They were put into glass vials, which were
sealed with rubber stoppers and aluminum caps. One group 100±11 -20.2±0.55 0.355±0.15
F1
was sterilized at 121 °C for 20 min while the other group (ref-
erence) was kept at 8 °C for comparative evaluation of physi- F2 115±16 -22.9±0.32 0.272±0.26
cochemical properties with their sterilized analogue.

Characterizations of Nanoparticles As seen in tables, membrane filtration does not seem to have
The mean diameter and particle size distribution was deter- any effect on particle size and polydispersity index. Also,
mined before and after sterilization by dynamic laser light slight changes was seen in zeta potential of nanoparticles.
scattering (Nanosizer Coulter N4 Plus®) and also their obser- The clogging doesn’t observed because of the particle size
vation in transmission electron microscopy (TEM-Philips EM is significantly smaller than the membrane pore size. How-
208). Zeta potential measurements were carried out using a ever, alternative technique, autoclaving, has a direct impact
Zetasizer 4, Malvern Instrument (n=6). on nanoparticle size. Nanoparticles tend to aggregate at
high temperature employed in autoclaving which leads to
Microbial Sterility Testing of Formulations increased particle size and polydispersity index.
The sterility testing was performed on the nanoparticles,
following USP XXIII guidelines. Thioglycollate resazurine From TEM results it could be seen that nanoparticles exhib-
broth (BioMerieux®, Marcy, France) was used as anaerobic ited elipsoidal geometries. After the sterile filtration, there
medium for the detection of bacteria and tripcase soy broth was no change in particle shape (Figure 1a,b).
was used as medium for the detection of yeasts and fungi.
Mediums were sterilized at 121 °C/ 1.1 atm for 15 min. Then,
steril nanoparticles were immersed in tubes containing ap-
propriate media. The tubes were incubated for 14 days at 37
°C (thioglycollate resazurine medium) or at 25 °C (tripcase
medium). Non-sterile nanoparticles were used as positive
controls. The turbidity of the media was then observed over
a basic period of 14 days in comparison to positive controls.
The experiment was done three times.

Figure 1: TEM micrographs of (a) non-steril nanoparticles; (b)


steril nanoparticles

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

In order to ensure the sterility of the final nanoparticles (i.e. p048-TUMOR TARGETED NANOSIZED
free of all forms of viable microorganisms), the sterility test- LIPOSOME-BASED DIAGNOSTIC AGENTS FOR
ing was performed according pharmacopoeial requirements.
Sterility was assessed by the observation of the media during
HYBRID IMAGING SYSTEMS
the incubation period. All tested steril nanoparticles showed
no detectable visible growth of microorganisms at the end Mine Silindir1, Suna Erdoğan1, A. Yekta Özer1, A. Lale Doğan2,
of the 14 days for three different medium (Figure 2a,b). Con- Murat Tuncel 3, Vladimir P. Torchilin4
trarily to positive controls for which a substantial increase of
turbidity was systematically observed. Department of Radiopharmacy, Faculty of Pharmacy,
Hacettepe University, 06100, Ankara, Turkey1
Department of Oncology, Faculty of Medicine, Hacettepe
University, 06100, Ankara, Turkey 2
Department of Nuclear Medicine, Faculty of Medicine,
Hacettepe University, 06100, Ankara, Turkey3
Department of Pharmaceutical Sciences, Northeastern
University, 02129, Boston, M A, U S A4

INTRODUCTION
Among the currently used cancer imaging methods; Nuclear
Medicine modalities provide metabolic wheras the radiolog-
ical modalities provide anotomical information. In the evalu-
ation of the patients, the data obtained from both of these
modalities were used collaboratively. However the different
Figure 2: Photos of incubation medium (a) 1. day; (b) 14.day acquisition times, on separate machines, decrease the speci-
ficity and accuracy of both modalities. To solve this problem
CONCLUSION hybrid imaging modalities like SPECT/CT which opened a
According to the experimental findings, the sterilization of new era in the field of cancer imaging were developed.
nanoparticle formulations by autoclaving is not advisable. It
was shown that membrane filtration was a suitable method After the wide spread usage of the hybrid imaging modali-
for the sterilization of nanoparticles without measurable ties there is a need for a single contrast agent which can be
physical properties loss. used for both imaging modality. Nanoparticle carrier sys- 129
tems which can carry multiple contrast group are very prom-
REFERENCES ising. Among these systems; liposomes are the most popular
1. Barbosa EM, Montembault V, Cammas-Marion S, Ponchel ones.
G, Fontaine L. Synthesis and characterization of novel
poly(γ -benzyl-L-glutamate) derivatives tailored for the Herein, we studied the preparation, characterization and
preparation of nanoparticles of pharmaceutical interest, in vitro cell binding of plain and tumor targeted PEGylated
Polym Int. 2007; 56: 317-324. liposomes contains iopromide as a contrast agent in liquid
2. Memisoglu-Bilensoy E, Hincal AA. Sterile, injectable cy- compartment and radionuclide (Tc-99m)on the surface.
clodextrin nanoparticles: Effects of gamma irradiation
and autoclaving, Int J Pharm. 2006; 311: 203-208. MATERIAL AND METHODS
3. Konan YN, Gurny R, Alleman E. Preparation and charac- Synthesis of DTPA-PE
terization of sterile and freeze-dried sub-200 nm nano- DTPA-phosphatidylethanolanime was synthesized as de-
particles, Int J Pharm. 2002; 233: 239-252. scribed before1.
4. Ozcan I, Segura-Sánchez F, Bouchemal K, Ozer O, Guneri Synthesis of pNP-PEG-PE
T, Ponchel G. Design of nanoparticles composed of poly pNP-PEG-PE was synthesized as described before2.
(benzyl glutamate) derivatives for targeted drug deliv-
ery to bone, The 7th Central European Symposium on Preparation of plain liposomes
Pharmaceutical Technology and Biodelivery Systems, Lipid mixtures (200 mmol/L) of DPPC, PEG2000-PE, choles-
Ljubljana, Slovenia, September 2008. terol and DTPA-PE were dissolved in ethanol at 70°C. The
lipid- ethanol solution was then hydrated with Iopromide
(185 mg/mL of iodine). The initial ethanol content was 10%
vol. The resulting multilamellar vesicles were then extruded
5 times with 2 stacked polycarbonate membranes of 0.2 µm
and 0.1 µm pore size at 70°C. Then, liposomes were dialyzed
overnight against 20 mM Tris buffer (pH 7.4) (3). For liposome
formulation consist of PL 90G as phospholipid, all procedures
were carried out at 35°C.

Preparation of immunoliposomes
To prepare antibody (mAb 2C5) conjugates with PEG3400-

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

PE, solution of pNP-PEG-PE in citrat buffer saline (pH 5.0)


was added to an equal volume of a 1 mg/mL solution of a
antibody in Tris-buffered saline (pH 8.5). The mixtures were
incubated overnight at 4°C.

Then, 2C5(PEG-PE) conjugate were co-incubated with Iopro-


mide-loaded plain liposomes for 1 day at 4°C. Following the
incubation, free mAb 2C5 were removed by dialysis using
cellulose ester dialysis tubes with a cutoff size of 350 kDa.

Mean particle size Figure 1: In vitro binding of Tc-99m labeled Iopromide con-
The mean particle size of liposomes was measured by dynam- taining plain and immunoliposomes to different tumor cell
ic light scattering (DLS) at 25°C using Malvern Zeta Sizer. lines

Entrapment efficiency Table 1: Compositions of liposomes


After removing free iopromide by dialysis, vesicles were ly- Formulations Codes
sed by 20 mM Tris (pH 7.4) buffer containing 10% (w/v) Triton
X-100. Encapsulated iopromide amount was determined by DPPC:PEG2000:Chol:DTPA-PE A
spectrophotometrically at 297nm.
PL90G:PEG2000:Chol:DTPA-PE B
Radiolabeling of liposomes with Tc-99m mAb 2C5- DPPC:PEG2000:Chol:DTPA-PE C
Liposomes were labeled with Tc-99m by the tin reduction
method. Briefly, 0.5 mL SnCl2.2H2O (1 mg.ml-1) and 1.5 mCi mAb 2C5- PL90G:PEG2000:Chol:DTPA-PE D
Tc-99m were mixed with liposome dispersions and were left
to react for 30 min. After incubation samples were dialyzed Table 2: Characterization results of liposome formulations
against to 20mM Tris Buffer (pH 7.4) to remove free Tc-99m.
Formulations Particle Size (nm) Entrapment Efficiency (%)
In vitro cell culture experiments A 94 (0,13*) 10,3±0,02
The cells (MCF-7 and EL4) were seeded at (800.000 cells/
well) into six-well tissue culture plates and allowed to reat- B 99 (0,15*) 8,59±0,01
130 tach overnight at 37°C, 5% CO2 in a humidified atmosphere.
After 24h incubation, the cells were treated with 1% BSA in C 110 (0.14*) 16,1±0,02
a serum free media for 1h to prevent a nonspecific binding. D 115 (0,16*) 13,59±0,01
Then, the medium was replaced with plain and mAb 2C5
modified Iopromide-loaded liposomes for 2h at 37°C. The * PI: Polydispersity index
cells were washed with serum free media, trypsinised, and
resuspended in 1 mL serum-free media. Their radioactivities ACKNOWLEDGEMENTS
were measured by gamma counter. This work is supported by TUBITAK-SBAG (Project No:
108S058).
RESULTS AND DISCUSSION The authors would like to thank to Schering-Turkey for gen-
The objective of this study was to formulate liposomes contain- erous gift of Iopromide.
ing iopromide in their aqueous phase and DTPA-PE on their sur-
face to evaluate the feasibility of these vesicles for hybrid imag- REFERENCES
ing systems. All formulations are described in Table 1. 1. Grant CWM, Karlık S, Florio E. Magnetic Resonance in
Table 2 illustrated the results of mean particle size and en- Medicine, A liposomal MRI contrast agent: Phosphoti-
trapment efficiency for the plain and antibody modified li- dylethanolamine-DTPA, 1989; 11: 236-243.
posomes. It was seen that tumor specific antibody modified 2. Torchilin VP, Levchenko TS, Whiteman KR et al. p-nitro-
liposomes have bigger particle size and higher entrapment phenylcarbonyl-PEG-PE-liposomes: fast and simple
efficiency than plain liposomes. attachment of speci¢c ligands, including monoclonal
antibodies, to distal ends of PEG chains via p-nitrophe-
The results of in vitro cell binding experiments were shown in nylcarbonyl groupsBiomaterials 2001; 22: 3035-44.
Figure 1. The data presented in Fig. 1 clearly demonstrate that 3. Zheng J, Perkins G, Kirilova A et al. Multimodal contrast
Iopromide containing PEGylated 2C5-immunoliposomes ca- agent for combined computed tomography and mag-
pable of specific binding to various cancer cells than control netic resonance imaging applications, Investigative Ra-
non-specific liposomes of the same composition diology, 2006; 41: 339-348.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

p049-EFFECT OF SOD AND KET RELEASE The in vitro SOD and KET release was determined in 0.1M
FROM BIODEGRADABLE MICROSPHERES ON pH 7.4 phosphate buffer, at 37°C. Samples were analyzed by
Micro-BCA protein assay and UV.
THE IN VIVO RESPONSE TO IMPLANTED ALG-
PLO-ALG MICROCAPSULES Alginate analysis and MC fabrication and characterization
The purified ALG was assayed by the Bradford protein assay,
Stefano Giovagnoli1, Paolo Blasi1, Giovanni Luca2, Francesca the LAL (limulus amebocyte lysate) test at both the Universi-
Fallarino3, Mario Calvitti3, Francesca Mancuso3, Aurélie ty of Perugia (Italy) and Living Cell Technologies laboratories
Schoubben1, Maurizio Ricci1, Giuseppe Basta4, Ennio (SOP Q209, Belgium) and for metals at Sereco Biotest s.p.a.
Becchetti5, Carlo Rossi1, Riccardo Calafiore4 (Perugia, Italy). MS inclusion into ALG/PLO/ALG MC was car-
ried out according to a previously optimized method. SOD
Dipartimento di Chimica e Tecnologia del Farmaco, Faculty of and KET release from ALG/PLO/ALG MC was performed in
Pharmacy, University of Perugia1 0.1M pH 7.4 phosphate buffer at 37°C. PLO leakage from the
Dipartimento di Medicina Interna (Di.M.I.), Sezione di Medicina MC was also investigated by Micro-BCA analysis.
Interna e Scienze Metaboliche ed Endocrine, University of
Perugia, School of Medicine, 2 MC transplant and cell overgrowth analysis
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, The experiment was performed according to a protocol pre-
University of Perugia,3 viously approved by the Ethic Committee of the University
Dipartimento di Medicina Interna (Di.M.I.), Sezione di Medicina of Perugia (p.n. 19382 PRE 805/2003). CD1 mice (average
Interna e Scienze Metaboliche ed Endocrine, University of weight 25 g, Charles River Laboratories, Wilmington, MA),
Perugia, School of Medicine4 divided into (n=9) one control with conventional MC, one
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, multicompartmental MC with SOD and one with KET, were
University of Perugia5 terminated at day 10, 20, and 30.

INTRODUCTION The recovered MC were examined by optical microscopy (Le-


Two major obstacles affect Pancreatic islet transplantation: ica, Italy) for cell overgrowth and cells were detached from
the restricted availability of human donors and the need for the MC and treated with rat anti-CD16/32 (2.4G2) and as-
life-long recipient’s general immunosuppression1. sayed by EPICS flow cytometer (Beckman Coulter). Rat anti-
F4/80 and anti-CD8 or anti-CD11b, anti-CD25 and anti-CD69
These drawbacks could be solved by using neonatal por- were applied.
cine islets for human substitutes2 and by entrapping cells 131
into multicompartmental alginate-poly L-ornithine- alginate RESULTS AND DISCUSSION
(ALG-PLO-ALG) microcapsules (MC) supplied with systems Protein loading afforded 90-92% efficiency and 9% drug con-
enabling delivery of vitamins and enzymes, and growth fac- tent. The mean volume diameter ranged between 13 and 18
tors [3]. Sustained release of these actives could be used to µm. Span was 2.46. SOD activity within MS was successfully
treat post-transplant adverse events as well4. retained. KET loaded PLA MS showed the same characteris-
tics previously recorded, with mean size around 5 µm and
Therefore, this work was aimed to determine the in vivo ef- 99% of the population < 20 μm [5]. Drug content and encap-
fect of SOD and KET controlled release from poly(D,L-lac- sulation efficiency were 17% (w/w) and 75%, respectively.
tide-co-glycolide) (PLGA) and poly(D,L-lactide) (PLA) micro-
spheres (MS) on unexplained random in vivo host’s response SEM microphotographs highlighted a rather smooth surface
to intraperitoneal grafting of ALG-PLO-ALG MC prepared and fairly regular shape for both MS formulations (Fig. 1).
with purified ALG.
Optical microscopy showed MC of rather regular and uni-
MATERIALS AND METHODS form shape and size (Fig. 2). Both conventional (Panel 2A)
MS Preparation and characterization and multicompartmental (Panel 2B, 2C) had the same fea-
SOD loaded PLGA (Bidachem, Italy) MS and KET loaded PLA tures. All MC showed similar size around 450 µm. The MS dis-
(Sigma, Milan, Italy) MS were prepared by a W/O/W double tribution within the MC was homogeneous and no adhesion
emulsion and an O/W emulsion solvent extraction/evaporation to the double coated ALG membrane was observed.
methods respectively. The protein content was determined by
Micro-BCA protein assay (Sigma, Milan, Italy) by using a 8453 SOD had a sustained release from PLGA MS with just 25% ini-
Agilent UV/VIS spectrophotometer (Agilent Technologies, tial burst and the release reached 90% after 64 days in an al-
Germany). KET content was determined by UV/VIS after dis- most linear manner (r2=0.993) (Fig. 3). SOD release from multi-
solving the MS in methylene chloride. MS size distribution was compartmental MC barely approached 40% at 30 days with a
determined by an Accusizer TM 770 Optical Particle Sizer (PSS slow progression over time and stopped afterwards (Fig. 3).
Inc., Santa Barbara, CA, USA). Morphology was investigated by
scanning electron microscopy (SEM) using a Philips XL30 mi- KET release reached 90% after 7 days from PLA MS, while it
croscope (Philips, the Netherlands). The entrapped SOD activ- dropped down to 70% from inside the MC. PLO was not re-
ity retention was evaluated in vitro according to the pyrogallol leased from the MC as no significant amounts (2-3% w/w)
(Sigma, Milan, Italy) autoxidation inhibition assay. were found in the release medium up to 10-12 days.

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

Although the purified ALG was considered biocompatible,


the conventional MC collected at day 10, 20 and 30 were
completely coated by cells (Fig. 4A). In turn, the MC contain-
ing KET became completely covered by cells only at day 20
(Fig. 4B), while the MC with SOD did not show significant cell
growth over the entire post-transplant follow-up (Fig. 4C).

The profile of cell phenotypes recovered from the collected


MC is summarized in Table 1. The control group showed, at
day 10, 50% of F4/80+ peritoneal macrophages, which de-
creased to about 40% at day 30. 30% of the macrophages
expressed a constant percentage of CD11b+ cells over time.
The KET group presented 92% CD11b+ cells since day 10,
which at day 30 decreased down to 51%, while the F4/80+
cells were 61%. The SOD group displayed, at day 20, 61% of
F4/80+ cells, among which 48% expressed also for CD11b+,
while, at day 30, a bare 8% of F4/80+ cells and no CD11b+
phenotype.

T-cell expression at day 30 was about 19% CD8+ in the con-


trol, 6% in the KET and barely 4% in the SOD group. In the
control group, 50% of CD8+ expressed an activated pheno-
type.

132

Figure 2 - Optical observations of the obtained MC: A) conven-


tional and multicompartmental MC with B) SOD and C) KET.

Figure 1 - SEM microphotographs of A) SOD loaded PLGA MS


and B) KET loaded PLA MS.

Figure 3 - In vitro release profiles of SOD loaded PLGA MS


alone and from inside the MC.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

REFERENCES
1. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, et al.
Islet transplantation in seven patients with type 1 diabe-
tes mellitus using glucocorticoid-free immunosuppres-
sive regimen, N. Engl. J. Med. 2000; 343: 230-238.
2. Korbutt GS, Elliott JF, Ao Z, Smith DK, Warnock GL, et al.
Large scale isolation, growth, and function of porcine
eonatal islet cells, J. Clin. Invest. 1996; 97: 2119-2129.
3. Calafiore R Alginate microcapsules for pancreatic islet
cell graft immunoprotection: struggle and progress to-
wards the final cure for type1 diabetes mellitus, Expert.
Opin. Biol. Ther. 2003; 3: 201–205.
4. Ricci M, Blasi P, Giovagnoli S, Rossi C, Macchiarulo G, et
al. Ketoprofen controlled release from composite mi-
crocapsules for cell encapsulation: effect on post-trans-
plant acute inflammation, J. Control. Release 2005; 107:
Figure 4 - Microscopy observations of MC overgrowth at 395–407.
each time point for A) conventional MC and multicompart- 5. de Vos P, Faas MM, Strand B, Calafiore R Alginate-based
mental MC with B) KET and C) SOD. microcapsules for immunoisolation of pancreatic islets,
Biomaterials 2006; 27: 5603–5617.
Table 1: Cell expression on the recovered MC
*F4/80+ (CD11b+) CD8+
Timepoints (days)
Control P050-EVALUATION OF VAGINAL TABLETS
10 50% (30%) - CONTAINING POLYCARBOPHIL AND
20 *** - HYDROXYPROPYLMETHYLCELLULOSE AS
30 40% (30%) 19% MUCOADHESIVE POLYMERS
KET
Aslıhan Hilal Kurtoğlu, Tamer Baykara
10 100% (92%) -
20 *** - 133
Department of Pharmaceutical Technology, Faculty of
30 61% (51%) 6% Pharmacy, Ankara University, 06100 Tandoğan, Ankara/Turkey
SOD
10 n.a.** - INTRODUCTION
Mucoadhesive vaginal drug delivery systems extend the res-
20 61% (48%)" -
idence time in the vagina, so that reduce dosing frequency
30 8% n.d.† 4% and quantity of drug administered. Thus the therapeutic ef-
*Total activated cells = F4/80+, CD11b is a fraction of the ficacy of locally acting drugs may be improved due to the
F4/80+ phenotype. The number of the recovered cell was increased availability of the vaginal epitelium through mu-
qualitatively estimated being in the order: Control group > coadhesion1. The purpose of this work was to develop mu-
KET group >> 500 group >>> SOD group of all timepoints. coadhesive tablets able to prolong the permanence of the
**not applicable. The mass of cells collected at day 10 for the drug in the vaginal cavity. As a model drug we chose a board
SOD group was not sufficient to allow flow cytometry analy- spectrum antifungal agent Miconazole nitrate which is used
sis. As alternative data at day 20 were used. to treat topical fungal and yeast infections. The matrix tablets
***Day 20 for the control and KET groups were not used as a were designed using a combination of bioadhesive polymers
slight contamination was observed. polycarbophil (Noveon® AA1) and hydroxypropyl methylcel-

not detected lulose (Methocel K15M) in different ratios. The swelling, in
vitro drug release and ex vivo mucoadhesion properties of
CONCLUSIONS the tablets were evaluated.
In spite of ALG of tested clinical-grade, host’s reactions to the mate-
rial employed cannot be excluded. Therefore, the use of a combina- MATERIALS AND METHODS
tion of KET and SOD releasing MS is recommended to preserve the Materials
efficiency of grafts in the immediate and longer post-transplant fol- Miconazole nitrate (MCZ) (Selectchemie AG, Switzerland),
low-up and to warrant success of cell transplantation and survival. Polycarbophil (PCP) (Noveon® AA1), hydroxypropyl methyl-
cellulose (Methocel K15M) (Dow Chemical Company), Man-
ACKNOWLEDGMENTS nitol (Partect® M100). All other chemicals used were analyti-
This work was supported by the “Consorzio Interuniversitario cal grade.
per i Trapianti d’Organo”, Rome, Italy. The authors wish to thank
Dr Luca Poletti from Sereco Biotest s.p.a. for his support to ALG Preparation of mucoadhesive tablets
metal content analysis. Mucoadhesive tablets were basically formulated PCP alone

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

and in combination with HPMC. Each tablet contained a RESULTS AND DISCUSSION
constant amount of miconazole nitrate (MCZ), mannitol and Swelling studies of tablets
magnesium stearate (Table 1). The extension in swelling time is favorable for mucoadhe-
sion3 and this time is prolonged by, through increasing
Table 1: The composition of tablet formulations (mg) HPMC concentration in our tablets (Figure 1).
HPMC Magnesium
Formulations MCZ PCP Mannitol
(K15M) stearate
F1 100 100 - 300 5
F2 100 80 20 300 5
F3 100 60 40 300 5
F4 100 50 50 300 5

Figure 1: Swelling index (%) of the mucoadhesive tablets


Tablets were prepared by direct compression of powder
mixture using a hydraulic press (Ayaşlı, Üçler, Ankara, Turkey) In vitro drug release studies
equipped with flat-faced punches of 16 mm in diameter for Increasing HPMC concentration in the tablets forms a viscous
swelling and in vitro drug release studies and 8 mm in diam- gel layer and slows down the release rate of drug by provid-
eter for ex vivo mucoadhesion studies. ing a control4. HPMC also modifies the release profiles of the
drug (F2-F4) as well as regulates the hydration (Figure 2).
Swelling studies of tablets
Swelling properties of the tablets were evaluated by gravi-
metric method. Each tablet was accurately weighed (W1)
and immersed in a petri dish containing pH 4.5 phosphate
buffer solution at 37°C. The swollen tablets were withdrawn
from the medium at the end of predetermined time intervals
and weighed (W2) after removal of excess surface water with
a filter paper (n=3). The swelling index (%) was determined
by Eq.1
134
Figure 2: In vitro release profiles of MCZ from the mucoad-
SI (%) = (W2-W1) / (W1) x 100 Eq.1
hesive tablets
In vitro drug release studies
Ex vivo mucoadhesion studies
Phosphate buffer (pH 4.5) with 35% of 1,4-dioxane was used
Mucoadhesive performance was gradually decreased with
as release medium because of the low solubility of micon-
the increase in HPMC amount in the tablets (Table 2).
azole nitrate. Drug release test from mucoadhesive tablets
was carried out in a shaking water bath at 37°C. Each tablet
Table 2: The work of adhesion of the 8 mm tablets
was put separately in bakers containing 150 ml of release
Formulations Work of adhesion N.mm(mJ)±SE
medium at 37°C. The bakers were shaken horizontally at 50
rpm in the water bath maintaining 37±1°C. Samples of 1 ml F1 0.263±0.017
were withdrawn for 12 hours and replaced with equal vol- F2 0.141±0.017
ume of release medium equilibrated 37°C (n=3). F3 0.090±0.014
Ex vivo mucoadhesion studies F4 0.065±0.023
Mucoadhesion testing of the tablets was carried out using
a texture analyzer2 (TA.XT plus, Stable Micro Systems, UK). Even so PCP has a better mucoadhesion alone, it was pre-
8 mm tablet was attached to the cylindrical probe with dicted from the retardation of the hydration for F2-F4, HPMC
double-sided adhesive tape. Cow vaginal tissue (20x20 mm, tablets could stay on mucosa for an adequate time during
stored at -30°C) was equilibrated for 15 min at 37oC before the treatment.
placing onto the holder stage of mucoadhesive holder and
maintained at 37°C during the test in 700 ml of the medium CONCLUSION
(pH 4.5). A volume of 0.1 ml buffer (pH 4.5) was slowly added Our overall results indicate that, HPMC retards drug release
using Hamilton syringe onto the tablet for pre-hydration and appropriately and has an adequate mucoadhesion prop-
waited for five minutes before the probe was placed. The in- erty when it is combined with PCP. Consequently, mixtures
strumental parameters and test conditions were constant of PCP/HPMC polymers could be preferred for the develop-
during the study. The total amount of the forces involved in ment of mucoadhesive vaginal tablet formulations.
during the probe withdrawal from the tissue (work of adhe-
sion, Wad) was calculated (n=3). ACKNOWLEDGEMENTS
We wish to thank to Ege University, Faculty of Pharmacy,
Department of Pharmaceutical Technology for the permis-

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

sion of using the texture analyzer TA.XT plus, Stable Micro The aim of this study is to prepare the mucoadhesive vagi-
Systems. nal tablet formulations of econazole nitrate with thiomers
for the treatment of vaginal candidiasis and to evaluate their
REFERENCES properties in vitro conditions.
1. Ndesendo VMK, Pillay V, Choonara YE, Khan RA, Meyer L, Keywords: Thiomers, econazole nitrate, PAA-Cys, vaginal
Buchmann E, Rosin U. In vitro and ex vivo bioadhesivity drug delivery, mucoadhesion
analysis of polymeric intravaginal caplets using physico-
mechanics and computational structural modeling Int J MATERIALS AND METHODS
Pharm. 2009; 370:151-159 Materials
2. Thirawong N, Nunthanid J, Puttipipatkhachorn,S, Sri- Econazole nitrate (EN) and poly(acrylic acid) (PAA) (450 kDa)
amornsak, P. Mucoadhesive properties of various pec- were purchased from Sigma-Aldrich (Germany). All other
tins on gastrointestinal mucosa: An in vitro evaluation chemicals were of analytical grade.
using texture analyzer, Eur J of Pharm Biopharm. 2007;
67:132–140 Synthesis of poly(acrylic acid)–cysteine conjugates (PAA–Cys)
3. Baloğlu E, Özyazıcı M., Hızarcıoğlu SY, Şenyiğit T, Özturt The PAA–Cys were synthesized according to a method de-
D, Pekçetin Ç. Bioadhesive controlled release systems of scribed previously5.
ornidazole of vaginal delivery. Pharm Dev Technology,
2006;11: 477-484 Determination of the thiol group content
4. Ceschel GC, Bergamante V, Calabrese V, Biserni S. Design The amount of free thiol groups immobilized on polymer
and evaluation in vitro of controlled release mucoad- backbones was determined photometrically with Ellman’s
hesive tablets containing chlorhexidine, Drug Dev Ind reagent. In addition, the oxidized thiol moieties available in
Pharm. 2006; 32:53-61 form of disulphide bonds were quantified after reduction
with NaBH4 and addition of Ellman’s reagent.

p051-STUDIES ON MUCOADHESIVE Particle Size Determination of EN


Particle size of EN was determined using a laser particle size
VAGINAL TABLETS OF ECONAZOLE NITRATE analyzer (FRITSCH, Germany). The size of the particles was
FORMULATED WITH THIOMERS determined in water as a non-dissolving medium.

Esra Baloğlu1, Zeynep Şenyiğit1, Anna Vetter2, Sinem Yaprak Preparation of Tablets
Karavana1, Tamer Güneri1, Andreas Bernkop- Schnuerch2 For each tablet, 40 mg of the PAA-Cys conjugate and PAA 135
were hydrated in dematerialized water. 5 mg of EN was add-
University of Ege, Faculty of Pharmacy, İzmir, Turkey1 ed for each tablet, homogenized and lyophilized. The 45 mg
University of Innsbruck, Institute of Pharmacy, Inssbruck, Austria2 of polymer-drug lyophilisates were compressed into 5.0 mm
diameter flat faced tablets. The compaction pressure was
INTRODUCTION kept constant during the preparation of all tablets.
The increasing incidence of Candida vaginitis has highlighted
the importance of establishing therapeutic strategies aimed After the preparation of the tablets, the quality control stud-
at ensuring successful eradication of the infectious agent, ies such as weight variation, friability, hardness and diam-
short-term treatment, achievement of high drug amount at eter/thickness ratio were performed according to USP XXX.
the target site and safety. From such a perspective, a ratio-
nal choice for management of vaginitis is topical treatment. In vitro disintegration studies
Traditional vaginal delivery systems might not ensure suffi- Disintegration behavior of the tablets examined using a dis-
cient therapeutic efficacy, most likely short residence time integration test apparatus according to European Pharma-
at the site of administration1. Therefore, topical treatment of copeia. Tablets were exposured in simulated vaginal fluid at
vaginal infections is focused on developing suitable drug de- 37 °C and the oscillation frequency was set to 0.5 s−1.
livery systems to enable a prolonged intravaginal residence
time for administered drug. Evaluation of the swelling behavior
The swelling capacity of the tablets was determined by a gravi-
Mucoadhesive vaginal tablets are able to adhere on the mucus metric method. Tablets were fixed to a needle and immersed in
layer of vagina and prolong the residence time of the dosage a beaker containing simulated vaginal fluid at 37°C. At sched-
form and allows a sustained-release in order to maximize the uled time intervals, the swollen tablets were taken out of the in-
therapeutic effect2. A new generation of mucoadhesive poly- cubation medium, excess water was removed, and the amount
mers, called ‘thiolated polymers’ are promising hydrophilic of water uptake was determined gravimetrically. The swelling
polymers derivatised with thiol groups on their side chains. ratio was then calculated according to the following equation:
Due to the formation of inter- and/or intrachain disulphide Swelling ratio =Wut/Wo
bonds, these conjugates show strongly improved cohesive Wut: weight of uptaken water at time t; Wo: initial weight of
and excellent mucoadhesive properties3. Econazole nitrate is the dry tablet
an imidazole derivative which plays an important role in the
treatment of diseases caused by different fungal infections 4. Mucoadhesion studies via rotating cylinder
Time of adhesion of compressed discs to the bovine vaginal

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

mucosa was established by the rotating cylinder as follows. The results of tablet quality control studies are shown in
Tablets were attached to freshly excised mucosa which was pro- Table 2.
vided by the local slaughterhouse and fixed on a stainless-steel
cylinder using a cyanoacrylate adhesive. Thereafter the cylinder Table 2: Results of tablet quality control studies
was placed into the dissolution apparatus and completely im- Weight
mersed into the simulated vaginal fluid at 37°C. The cylinder Friability Hardness Diameter/
Formulations variation
was rotated at a speed of 100 rpm. Every 30 min the changes in (%) (kg) Thickness (mm)
(mg)
the test system were observed visually and registered until all
4.963±0.016/
of the discs were either disintegrated or detached from the mu- EN+PAA 48.744±2.362 0.0676 > 16
1.855±0.052
cosa. This test was performed at least five times for each tablet.
4.987±0.008/
EN+PAA-Cys 46.850±2.076 0.426 > 16
Release studies 1.834±0.0533
The release rate of EN from tablets was determined in vitro us-
ing a perfusor as it was shown in Figure 1. Tablets were placed Tablets prepared with unmodified PAA showed a complete
into tubes and kept at 37±0.5°C with a heater. A perfusor (Per- disintegration and erosion within 5.4±1.140 hours. On the
fusor Fm, Braun) was used to give 6 mL of vaginal fluid to tab- other hand, thiolated tablets showed no disintegration or
lets in 24 hours because the daily production of vaginal fluid erosion within 72 hours.
is approximately 6 mL/day and 0.5-0.75 mL continually present
in the vagina6. The same amount of samples was collected con- The swelling ratio of the tablets in simulated vaginal fluid at
currently. The samples were assayed for EN content by HPLC. 37°C is shown in Figure 3.

136
Figure 1: Schematic drawing of the in vitro release studies

RESULTS AND DISCUSSION Figure 3: Swelling behaviour of EN+PAA tablets () and
The amount of free thiol groups and disulfide bonds of the EN+PAA-Cys tablets () simulated vaginal fluid at 37°C. Indi-
polymer are listed in Table 1. cated values are the me

Table 1: The amount of thiol groups and disulfide bonds im- The swelling state of the polymer contributes to its mucoadhe-
mobilized on PAA sive behavior. The formulation prepared with PAA-Cys showed
longer mucoadhesion time than PAA formulation (Figure 4).
-SH [µmol/g polymer ±S. D.] -S-S [µmol/g polymer ±S. D.] It was thought that the presence of thiol groups allowed the
formation of covalent bonds with cysteine- rich sub domains
PAA-Cys 172.80 ± 30.33 377.93 ± 85.10
of the mucus gel layer, leading to increased residence time.
The mean size of econazole nitrate in distilled water was
determined to be 34.245 µm. Size distribution results are
shown in Figure 2.

Figure 4: Comparison of the mucoadhesion time of tablets


on the rotating cylinder. Indicated values are means ±SD of
at least three experiments.
Figure 2: Size distribution of econazole nitrate

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

The presence of disulphide bonds may significantly alter the P052-Liposomal Gels for Vaginal Drug
mechanism of drug release from the delivery system due to Delivery of Cidofovir
increased rigidity and cross linking7. Figure 5 showed the sig-
nificant difference between the release profiles of EN from
Fatmanur Tuğcu Demiröz1, Fusun Acartürk1
the formulation containing thiomer or not.
Department of Pharmaceutical Technology, Faculty of
Pharmacy, Gazi University, Ankara, Turkey1

INTRODUCTION
Vaginal delivery of local and systemically acting drugs has in-
creased over the past decade presumably reflecting greater
attention to women’s health care1. Liposomes can provide
a controlled release of an incorporated drug, phospholipid
vesicles have a potential as vaginal delivery system. The hy-
drogels prepared from Carbopol resins as a vehicle for incor-
poration of liposomes destined for vaginal delivery2.
Figure 5: Release rate of EN+PAA tablets () and EN+PAA- Cervical cancer which is mainly associated with Human Papil-
Cys tablets () simulated vaginal fluid at 37°C. Indicated val- loma Virus (HPV) infection is the second most common cause
ues are the means of of cancer death among women worldwide affecting approx-
imately 1% of all female population3. The therapy regimens
CONCLUSION for HPV-related genital diseases include the administration
The overall results obtained during this study suggested that of antiviral agents such as cidofovir (CV). CV is a prototype of
thiolated EN tablet formulation can be inferred as a prom- antiviral compounds, which is active against HPV, as well as
ising candidate for topical treatment of Candida vaginitis. against a large number of other DNA viruses4.
It was planned that the formulation would be evaluated in
vivo. Here, we report the development of a liposomal delivery
system for vaginal administration of CV, able to provide con-
ACKNOWLEDGEMENTS trolled release of drug. CV was entrapped in liposomes pre-
This work was financed by The Scientific and Technological pared by the polyol dilution method (PD), whereby different
Research Council of Turkey (106/S/196). phospholipid compositions were used.
137

REFERENCES MATERIALS AND METHODS


1. Knuth K, Amiji M, Robinso JR. Hydrogel delivery system Materials
for vaginal and oral applications, Adv Drug Delivery Rev. L-alpha-Dipalmitoyl phosphatidylcholine (DPPC) and 1-2 Dipalm-
1993; 11:137-167. itoyl-sn-glycero-3- phosphatidylcholine (DGPC) were purchased
2. Vermani K, Garg S. The scope and potential of vaginal from Acros Organics, USA and Carbopol 974P NF from Noveon,
delivery, Pharm Sci Tech Today. 2000; 3:1-9. USA. Stearylamine (SA) was also provided from Sigma, USA. Cido-
3. Leitner VM, Walker GF, Bernkop-Schnürch A. Thiolated fovir was generous gift from Gilead Sciences, Inc., USA.
polymers: evidence for the formation of disulphide
bonds with mucus glycoproteins, Eur J Pharm Biopharm. Preparation of Liposomes
2003; 56:207-214. Liposomes of CV were prepared according to the PD method5.
4. Czeizel AE, Vargha P. A population-based case–control After preparation, the liposome suspension was extruded
teratological study of vaginal econazole treatment dur- through polycarbonate filter (pore size, 400 nm) by using a Mini
ing pregnancy, Eur J Obstet Gynecol Reprod Biol. 2003; Extruder (Avanti Polar Lipids). PS2 formulation was prepared by
111:135–140. the freeze-thawing (FT) method5. For this purpose, preparations
5. Bernkop-Schnürch A, Schwarz V, Steininger S. Polymers were rapidly frozen in liquid nitrogen and then brought to 37°C
with thiol groups: a new generation of mucoadhesive to thaw completely. The procedure was performed five times.
polymers?, Pharm Res. 1999; 16:876–881. The composition of the liposomes are given in Table 1.
6. Bernkop-Schnürch A, Steininger S. Synthesis and Char-
acterisation of mucoadhesive thiolated polymers, Int J Particle Size and Zeta Potential of the Liposomes
Pharm. 2000; 22;1861-1867. The particle size and ζ-potential of liposomes were deter-
7. Andrews GP, Laverty TP, Jones DS. Mucoadhesive poly- mined using Zetasizer Nano Series (Nono-ZS, Malvern Inst.,
meric platforms for controlled drug delivery. Eur J Pharm UK). The analysis was performed at a temperature of 25°C,
Biopharm. 2009; 71:505–518. using samples appropriately diluted with distilled water.

Encapsulation Efficiency Determination


The encapsulation efficiency (EE) was calculated indirectly
by determining the remaining concentration of CV in the
supernatant after centrifugation. Samples were ultracentri-

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

fuged at 44,803 g for 1 h. The supernatant containing the dis- leased. The release of CV from the P2 formulation prepared with
solved free CV was used to determine the concentration. The DPPC and DGPC was observed to be high. Drug release varied
EE was calculated using the following equation: with the lipid content in the formulations. The PS2 formulation
exhibited a drug release of 88.1±2.1 % at the end of 48 hours.
EE = [(CVtot−CVsup)/ CVtot]x100 Thus, considering the release of liposomes during vaginal ad-
ministration, as well as controlled release of CV, hydrogels made
where, CVtot is the total amount of CV and CVsup is the of Carbopol 974P would be the proper choice of vehicle.
amount of CV in supernatant after centrifugation.
Table 1: Composition and preparation method of the lipo-
Preparation of Carbopol Gel Formulations some formulations
Carbopol 974P NF (1 g) was dispersed in demineralised water Liquid Preparation
(88 g) by stirring at 800 rpm for 60 min. Then propylene glycol Code CV (Molar Ratio) Molar Ratio
Composition Method
(10 g) was added and the mixture was neutralised by drop-wise P1 - DPPC: DPPC 9:1 PD
addition of 10% NaOH. Mixing was continued until a transpar- P2 0.5 DPPC: DPPC 9:1 PD
ent gel appeared, whereas the amount of base was adjusted to PS1 0.5 DPPC:S4 9:3 PD
achieve a hydrogel with pH 4.5. Carbopol 974P gel was kept at
PS2 DPPC:S4 9:3 PD-FT
+4°C overnight before application, in order to remove air bub-
bles. The PS2 liposomes added to the gel in the ratio of 10:90 to
Table 2: Characterization of liposomes
prepare the liposomal gel formulation PS2KR.
Code of liposomal Mean Zeta Potential Encapsulation
Drug Diffusion Studies of the Liposomes and Liposomal Gels gel formulation Diameter (nm) (mV) efficiency (EE)(%)
All glass Franz type diffusion cells were used. The diffusional P1 286.2±0.8 -0.522±0.171 -
cross sectional area was 1 cm2. Dialysis membrane (Sigma®, P2 298.7±0.9 -0.488±0.354 26.4
USA) having a pore size of 12,000 Da was used. The stirred recep-
PS1 326±9 2.54±0.405 15.53
tor phase (2.5 mL) containing citrate phosphate buffer (pH 4.5)
was thermostatted at 37°C. The donor compartment contained PS2 315±5 30.83±2.79 41.09
liposomes or liposomal gel formulation. Samples were taken
periodically from the receptor phase. Permeant concentrations
were determined by spectrophotometrically at 278.4 nm.

138 RESULTS AND DISCUSSION


Characterization of liposomes
Particle size of empty liposome formulation P1 was found
to be 286.2±0.8 nm. As shown in Table 2, liposome formu-
lations P1 and P2 had statistically significant difference be-
tween mean particle diameters (p>0.05). The modification of
liposomes with SA increased the particle size, depending on
its molar ratio (PS1 and PS2 formulations).
Figure 1: Diffusion profiles of CV from liposomes and lipo-
The zeta potential values of P1 and P2 formulations were somal gel formulations through dialysis membrane (n=6;
negative. The addition of SA caused a higher positive surface bars represent SD)
charge. Incorporation of DPPC or SA into liposome mem-
brane resulted negative zeta potential values for DPPC/DGPC CONCLUSION
liposomes or positive values for DPPC/SA liposomes respec- The polyol dilution method has been proven to be simple, repro-
tively. Regarding the encapsulation of CV, P2 liposomes en- ducible and appropriate for encapsulation of CV. Negatively and
capsulated 26.4% of the amount of drug. However, positively positively charged liposomes yielded different encapsulation ef-
charged PS2 liposomes prepared by the FT method encap- ficiencies. Incorporation of liposomes in to bioadhesive Carbopol
sulated a higher amount of the drug (41%). This results have 974P gel (PS2KR formulation) further confirmed for the applicabil-
shown that preparation method can affect the encapsula- ity of liposomes as a novel vaginal delivery system for controlled
tion of CV and the zeta potential of liposomes. release of encapsulated CV for the local treatment of genital HPV.

Drug Diffusion the Liposomes and Liposomal Gels ACKNOWLEDGMENTS


The drug diffusion profiles of CV from liposome formula- This study was supported by a research grant (SBAG 2807-
tions are shown in Figure 1. P2 formulation released 99.5±2 104S324) from the Scientific and Technical Research Council
% of the drug at the end of 48 h. PS1 and PS2 formulations of TURKEY (TÜBİTAK) and Research Foundation of Gazi Uni-
containing SA released 96.4±1.8 and 88.1±2.1 % of the drug versity (02/2006-08). We would like to thank Gilead Sciences,
throughout the diffusion studies, respectively (Fig. 1). Inc., USA for the gift sample of Cidofovir.

It was observed that, the liposomal gel formulations with Car- REFERENCES
bopol 974P released about 40.4 % of CV at 48 h. This showed 1. Acartürk F, Robinson JR, Pharm. Res. 1996; 13: 950-951.
that, liposomal gels almost prevented the drug from being re-

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

2. Pavelić Ž, Škalko N, Jalšenjak I, Eur. J. Pharm. Sci. 1999; 8: 345–351. ethyl alcohol-PBS (3:7) mixture thermostatted at 37°C, was about
3. Bilensoy E, Çırpanlı Y, Şen M, Doğan L, Çalış S, J Incl Phe- 4 ml. Infinite dose regimen was applied in all experiments.
nom Macrocycl Chem. 2007; 57: 363-370.
4. Gross G, Int. J. Antimic. Agents. 1999; 12: 1-3. At the end of the 6 h permeation experiments, the excess amount
5. Pavelić Ž, Škalko-Basnet N, Schubert R, J. Control. Rel. of the formulation was removed and SC and epidermis were sep-
2001; 219: 139-149. arated from dermis with heat application. The drug was extracted
using acetonitrile:water (60:40) mixture. The amount of CP or MF
in the permeation and accumulation samples was determined by
p053-THE INFLUENCE OF CHEMICAL HPLC using Luna C18 (2) 150 mm x 3 mm column (Phenomenex,
ENHANCERS ON SKIN ACCUMULATION OF USA) and a mobile phase composed of acetonitrile/water (55:45)
at 1 ml/min. UV detection at 240 nm was employed.
TOPICAL CORTICOSTEROIDS FROM GEL
FORMULATIONS Statistical Analysis
All experiments were replicated six times. Statistical differ-
Taner Şenyiğit1, Cristina Padula2, Özgen Özer1, Patrizia Santi2 ences were determined using ANOVA followed by Dunnet
multiple comparison test.
Ege University, İzmir, Turkey1
Universita degli Studi di Parma, Dipartimento Farmaceutico, RESULTS AND DISCUSSION
Parma, Italy2 In vitro permeation and accumulation of CP and MF in pig
ear skin layers after passive diffusion was studied using chi-
INTRODUCTION tosan gel containing different penetration enhancers as do-
Topical glucocorticoids (TG) are the most frequently prescribed nor reservoir. During the permeation studies, CP or MF was
drugs by dermatologists. Despite their benefit in the therapy of never found in the receptor medium at the end of 6 h.
inflammatory diseases, TG are associated number of side effects
that limit their use1. In this study, two TG were selected as model CP or MF skin concentration was calculated by normalizing the
active agents: clobetasol-17-propionate (CP) and mometasone amount of drug recovered in the SC + epidermis and dermis by the
furoate (MF). One of the approaches to reduce the systemic weight of tissues and expressed as μg of drug per mg of tissue.
adverse effects of TG is to enhance their permeability so as to
reduce the topically applied dose2. The use of chemical penetra- Figure 1 and Figure 2 present the effect of chemical enhanc-
tion enhancers is the most widely used approach to increase ers on the accumulation of CP and MF in the epidermis and
topical and transdermal delivery3. Two terpene derivatives (D- dermis, respectively. 139
limonene and nerolidol) and Transcutol® P were evaluated as
chemical penetration enhancers in this study. The aim of this
work was to investigate the effect of chemical enhancers on the
skin accumulation and permeation of CP and MF from chitosan
gels. The goal was to increase skin retention without increasing
penetration, to reduce the possible side effects of these drugs.

MATERIALS AND METHODS


Materials
CP and MF were kind gifts from GlaxoSmithKline (Turkey)
and Orva Drug Company (Turkey), respectively. Medium
molecular weight chitosan from Sigma (Germany). All other
chemicals were of analytical grade.
Figure 1: Effect of enhancers on the accumulation of CP (dark
Preparation of Chitosan Gels bars) and MF (light bars) in the epidermis. *significantly dif-
For the preparation of chitosan gel, 2 % chitosan was dis- ferent from chitosan gel.
solved in 1.5 % (w/v) acetic acid solution. 0.05 % CP or 0.1 %
MF was dissolved in 10 % PEG-400 and added to the chitosan
solution with continuous stirring until uniformity.

The concentration of terpenes and Transcutol P in chitosan


gels was 2 and 20 %, respectively.

In Vitro Permeation Experiments


The in vitro permeation experiments were performed using
Franz-type diffusion cells across pig ear skin. Previous studies
demonstrated that pig skin is a reasonable model for the human
barrier in passive condition4. The available diffusion area was 0.6
cm2 and the volume of the receptor compartment, containing

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

Figure 2: Effect of enhancers on the accumulation of CP (dark but the clinical use is often limited because of their potential
bars) and MF (light bars) in the dermis. *significantly differ- to cause adverse effects such as irritation and ulceration of
ent from commercial cream the gastro-intestinal mucosa. Administration of these agents
via dermal route can bypass these disadvantages of the oral
In the case of CP, nerolidol provided a significant enhance- route and may maintain relatively consistent plasma levels
ment of both epidermis and dermis retention (p < 0.01 and for long term therapy from a single dose1. Microemulsions
p < 0.05, respectively). Besides, D-limonene improved only have several advantages such as enhanced drug solubility,
dermis accumulation (p < 0.01) while, surprisingly, Transcu- good thermodynamic stability, enhancing effect on trans-
tol had only a modest, non significant, effect on epidermis dermal ability over conventional formulations2. The aim of
retention. this study is to develop new naproxen microemulsion formu-
lations as drug carrier for topical application and to compare
When the effect of enhancers on MF accumulation data is the ex vivo and in vivo permeation rate of naproxen from
considered, only nerolidol produced a statistically signifi- these formulations with the commercial formulation (C).
cant increase at the epidermis level (p < 0.05). No significant
increment was observed in the dermis accumulation of MF MATERIALS AND METHODS
with three penetration enhancers tested. Materials
Naproxen (Deva Holding, Turkey (gift)), Isopropyl myristate
CONCLUSION (IPM) (Sigma, Germany), Span 80 (Merck, Germany),Labrafil-
According to the experimental findings of this study, it was M (Gattefosse, France), Labrasol (Gattefosse, France), Ethanol
showed that Nerolidol resulted to be the best chemical pen- (J.T.Baker, Holland), Cremophor-EL (Sigma, Germany), Isopro-
etration enhancer among the three enhancers tested, with pyl alcohol (IPA) (Merck, Germany), Methanol (Merck, Hohen-
significantly higher accumulation data especially in the epi- brunn, Germany), Acetonitrile (Sigma, Aldrich, Germany). All
dermis of both active agents. D-Limonene and Transcutol the other chemicals and reagents used were of HPLC or ana-
did not increase the skin accumulation of active agents. lytical grade samples.

REFERENCES Preparation of w/o and o/w microemulsion formulations


1. Wiedersberg S, Leopold CS, Guy RH. Bioavailability and Microemulsions were prepared using isopropyl myristate
bioequivalence of topical glucocorticoids, Eur J Pharm (IPM) as oil phase, Span 80, Labrafil-M, Labrasol, Cremophor-
Biopharm. 2008; 68:453–466. EL as surfactants, ethanol, isopropyl alcohol as co-surfac-
2. Fang JY, Fang CL, Sung KC, Chen HY. Effect of low fre- tants and 0.5 N NaOH solution as aqueous phase. M1 and M2
140 quency ultrasound on the in vitro percutaneous ab- formulations were loaded with 10% naproxen, M3 and M4
sorption of clobetasol 17-propionate, Int J Pharm. 1999; formulations are blank.
191:33-42.
3. Moster K, Kriwet K, Naik A, Kalia YN, Guy RH. Passive skin Characterization of microemulsions
penetration enhancement and its quantification in vitro, The physicochemical properties of microemulsions were
Eur J Pharm Biopharm. 2001; 52: 103-112. measured.
4. Sekkat N, Kalia YN, Guy RH. Biophysical study of porcine
ear skin in vitro and its comparison to human skin in Ex-Vivo permeation studies
vivo, J Pharm Sci. 2002; 91(11): 2376-2381. Diffusion cell was used in the permeability studies of naprox-
en. The apparatus consisted of clamped preconditioned male
rat abdominal skin on to glass diffusion cell between donor
p054-Evaluation of transdermal and receptor compartments. Phosphate buffer pH 7.4 (10
absorption and anti-inflammatory mL) (600 rpm, 37oC) was used in the receptor compartment.
The samples were withdrawn at predetermined time inter-
and analgesic activities of naproxen vals then, immediately analyzed HPLC system consisting of a
through rat skin: ex vivo and in vivo UV spectrometric detector and C18 column (270 nm), direct-
ly. The mobil phase contained methanol/acetonitrile/ puri-
H. Yeşim Karasulu1, Neslihan Üstündağ1, N.Ülkü Karabay fied water (20/28/52 v/v/v) and 0.4mL triethylamine . Three
Yavaşoğlu2, Şebnem Apaydın3 replicates of each experiment were performed.

Department of Pharmaceutical Technology, Faculty of Assessment of anti-inflammatory activity


Pharmacy, University of Ege1 The anti-inflammatory activity was evaluated by the carra-
Department of Biology, Faculty of Science, Ege University2 geenan-induced paw edema test in the rat. Male Wistar rats
Center for Drug R&D and Pharmacokinetic Applications, Ege were deprived of food overnight and treated by dermal route
University3 with M1, M2, M3, M4, C and serum physiologic (SP), 60 min-
ute before 0.1 mL 1% carrageenan in isotonic saline was in-
INTRODUCTION jected subplantarly into left hind paw. The contralateral paw
Naproxen is a non-steroidal anti-inflammatory drug (NSAID) was injected with 0.1 mL saline and used as a control. Paw
compound with analgesic and antipyretic effects, used for volume was measured by water plethysmometer (n = 7).
treatment of rheumatoid arthritis, osteoarthritis and trau-
matic contusions. Oral therapy of NSAIDs is very effective, Assessment of Analgesic activity

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

Tail flick test Permeation rate from microemulsion formulations and com-
The tail flick test was evoked by a score of radiant heat, which mercial formulation were given Figure 1.
was focused on the dorsal surface of the tail. Rats were ex-
amined for latency (seconds) to withdraw their tails from a
noxious thermal stimulus using a tail-flick meter (MAY-TF
0703, Turkey). Each rat was then tested before and 30, 45, 60,
75, 90, 105, 120 and 180 minute after the topical administra-
tion of formulations (n = 7) (4).

Hot plate test


Rats were placed on aluminium hot plate (MAY-AHP 0603,
Turkey) kept at a temperature of 62±0.5 oC for a maximum
Figure 1: Permeation rate from microemulsion formulations
time of 30 s. Reaction time was determined (when animals
and commercial formulation (n=3)
licked their fore and hind paws and jumped) before and 15,
30, 45, 60, 90, 120 and 180 min after the topical application
Statistical comparison of flux values obtained from ex vivo
of formulations (n = 7) (4).
studies throughout 24 h showed that most of the microe-
mulsions provided fluxes (P<0.05) higher than the commer-
Statistical data analysis
cial formulation.
The statistical analysis of values, were performed using a one
way anaysis of variance (ANOVA) to test the difference be-
The intraplantar injection of carrageenan caused a time-
tween the means of microemulsion formulations and com-
dependent paw edema in the rat, although saline injection
mercial formulation permeation experiments. Data with P <
caused no swelling. Dermal application of formulations (M1,
0.05 are considered statistically significant.
M2, C) inhibited paw swelling. However, M3, M4 and SF for-
mulations did not inhibit paw swelling (Figure 2).
RESULTS AND DISCUSSION
Composition of the microemulsion formulations were given
in Table 1. M1, M2 formulations contain naproxen, however
M3, M4 are blank microemulsion formulations.
Table 1: Composition of the Microemulsion Formulations
Formulation(%) M1(w/o) M2(o/w) M3(w/o) M4 (o/w)
IPM 26.22 8.53 26.22 8.53 141
Labrafil-M - 3.964 - 3.964
Labrasol 1.38 - 1.38 -
Span 80 6.91 - 6.91 -
Cremophor EL - 11.893 - 11.893
Figure 2: Volume increase % during 6 hours (n=7) for M1,M2,C,
Ethanol 58.10 - 58.10 - 1,2,3 h after the injection, p<0.05; according to SP. For M1,
IPA - 47.572 - 47.572 M2, 4,5,6 h after the inje
Distilled water 7.38 28.04 7.38 28.04
Naproxen 10 10 - - C, M1 and M2 administration increased the tail-flick and hot
HLB value 5.916 10.375 5.916 10.375 plate latencies when compared with control rats (SP, M3 and
The physicochemical parameters of the microemulsions M4 administration on rats) (Figure 3 and Table 3).
were listed in Table 2. In the skin irritation study, no formulation was found to be
irritant.
Table 2: Characterization of microemulsion formulations
Formulation /
M1 M2 M3 M4
Characterization
pH 6.39 ±0.025 6.62± 0.020 7.12 ± 0.01 8.3± 0.025
15.33± 10.33± 15.33± 10.33±
Viscosity (cP)
0.577 0.577 0.577 0.577
1.405 ± 5.617 ±
Droplet size (nm) 1.007±0.011 4.583±0.560
0.040 0.199
141.5±0.1 0.51±0.01 149.5 ± 0.1 0.59±0.02
Conductivity
(µs/cm) (ms/cm) (µs/cm) (ms/cm)
0.922 ± 0.875 ± 0.925 ±
Density (g/mL) 0.881±0.001
0.025 0.010 0.025
1.405 1.413 ± 1.452 ± Figure 3: Tail flick results, *p<0.05 according to SP (n=7)
Refractive index 1.461±0.002
±0.002 0.001 0.002
PIT (oC) 41 ± 0.006 61 ± 0.020 41 ± 0.006 61 ± 0.020

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

Table 3: Hot plate results (n=7) results can be found on the effectiveness of antifungal treat-
0 15 30 45 60 90 120 180 ment. Only three of the papers were based on double-blind,
Group
(min.) (min.) (min.) (min.) (min.) (min.) (min.) (min.) placebo-controlled studies and of these three only one was
4.85± 4.75± 4.90± 4.85± 4.70± 4.40± 4.57± 4.34± multicentric, making the interpretation of the results rather
SP (sn)
0.15 0.22 0.24 0.18 0.22 0.15 0.14 0.14 difficult. The treatment period ranged between 4 and 80
4.71± 5.08± 8.41± 9.28± 8.87± 7.41± 6.52± 5.34± weeks, and the form of drug/placebo application also varied.
M1 (sn)
0.28 0.23 0.72 *# 0.44 *# 0.43 *# 0.35 *# 0.28 *# 0.22 * The concentration and the daily amount of amphotericin B
M2 (sn)
4.55± 5.02± 8.31± 9.55± 9.54± 8.28± 6.91± 5.81± recommended also differed. The conditions of drug storage
0.31 0.29 0.70 *x 0.45 *x 0.42 * 0.37 *x 0.32 *x 0.39 *x and checking the stability of the amphotericin B solutions
4.41± 8.35± 9.38± 8.88± 9.02± 8.54± 5.62± 4.87± was completely ignored in most cases. Due to the fact that
C (sn)
0.24 0.85 * 0.38 * 0.70 * 0.59 * 0.73 * 0.25 * 0.19 the patient compliance was examined in only one study it
4.85± 4.71± 4.68± 4.75± 4.51± 4.51± 4.48± 4.52± is extremely difficult to draw conclusions concerning the ef-
M3 (sn)
0.42 0.32 0.40 0.37 0.37 0.41 0.39 0.42 fectiveness or ineffectiveness of amphotericin B treatment.
4.54± 4.34± 4.28± 4.34± 4.35± 3.94± 3.94± 3.92± Thus during the preparation phase of the pilot study at Pécs
M4 (sn)
0.25 0.21 0.29 0.17 0.22 0.22 0.24 0.20 we had to face several pharmaceutically important questions
* SP; p<0.05, # M3; p<0.05, x M4; p<0.05
such as the preparation of study samples; optimal dosage
form; proper concentration of the active ingredient; optimal
CONCLUSION storage conditions, stability and analysis of samples and the
According to the ex vivo and in vivo studies, it can be con- compliance of the included patients. The significance and
cluded that when naproxen was incorporated into the brief summary of these topics is introduced and the outcome
microemulsion, its permeation rate from the formulation of the pilot clinical study is presented by the authors.
increases. Furthermore, microemulsion formulation contain-
ing naproxen may be an appropriate vehicle for the topical MATERIALS AND METHODS
application of naproxen. As the current work is a summary of different projects in di-
verse pharmaceutical sciences, the detailed description of
REFERENCES the materials and methods can be found in recently pub-
1. Beetge E, Plessis E, Müler J, Goosen DG, Rensburg FJ. The lished papers.1, 2, 3, 4
influence of the physicochemical characteristics and
pharmacokinetic properties of selected NSAID’s on their RESULTS AND DISCUSSION
transdermal absorption, Int. J. Pharm. 2000; 193: 261- In a double-blind, randomized, placebo-controlled study,
264.
142 33 patients received amphotericin B or placebo nasal sprays
2. Chen H, Chang X, Yang X. Microemulsion-based hydro- for 12 months after endoscopic polypectomy. Our aim was
gel formulation of ibuprofen for topical delivery. Int. J. to determine whether any difference could be observed
Pharm. 2006; 315: 52-58. between the two groups in the rates of recurrence of nasal
3. Guneli E., Yavaşoğlu-Karabay N.Ü., Apaydın, Ş., Uyar M., polyposis, in the symptoms, in the quality of life or in the end-
Uyar M. Analysis of the Antinociceptive Effect of System- socopic findings. The protocol of the study was approved by
ic Administration of Tramadol and Dexmedetomidine the Regional Ethics Committee of the University of Pécs. Our
Combination on Rats Models of Acute and Neuropatic patients displayed marked improvements in the sinus com-
Pain, Pharmacology, Biochemistry and Behavior. 2007; plaints by the end of the 1-year course of treatment, though
88: 9-17. without a significant difference in the extent of change be-
tween the two groups in the primary (computer tomogra-
phy evaluation) and secondary (sinonasal symphtoms and
p055-CLINICAL AND ANALYTICAL ASPECTS quality of life) outcome measures. Our clinical study does
OF THE IMPROVEMENT OF A LOCAL not justify the need for the administration of amphoteri-
ANTIFUNGAL PREPARATION FOR THE cin B nasal spray for 12 months following endoscopic nasal
TREATMENT OF NASAL POLYPOSIS polypectomy.

Andras Fittler1, Lajos Botz1 The knowledge of patient adherence (the extent to which
the patient’s actual history of drug administration corre-
Department of Pharmaceutics and Central Clinical Pharmacy1 sponds to the prescribed regimen) is important both in
Pharmaceutical Institute, Faculty of Medicine, University of medical research and in clinical practice. The results of clini-
Pécs, Pécs, Szigeti u. 12, Hungary H-76242 cal trials cannot be interpreted realistically without adher-
ence information. Two indirect methods were used for the
INTRODUCTION measurement of adherence: (1) recording of the amount of
In 2006 a double blind, randomized pilot clinical study was medication self-administered and (2) self-reporting by the
launched to assess the efficacy of long term intranasal anti- patient via the Hungarian translated version of the standard-
fungal treatment in patients suffering from chronic rhinosi- ized BMQ revised in 2003. Assessments were made of how
nusitis with nasal polyposis, because it has been hypnotized the adherence changed during the 12-month period, which
that fungal cells in the sinuses exacerbate adverse immune factors influenced the attitudes of the patients and what
response that result in the formation of nasal polyps. Several adverse events were reported. It can be stated that patient
studies have been published on the issue and controversial adherence was relatively good; only a moderate decline was

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

seen during the 12 months. At the end of the study, the over- view of the antifungal therapy, Eur Arch Otorhinolaryn-
all adherence was 94,7 ± 11,5 %. During the last month of gol. 2009; 266: 847-55.
treatment, 55,6 % of the patients were totally adherent, in 2. Gerlinger I, Fittler A, In reference to The Effect of Topi-
37 % sporadic non-adherence was measured, and only 2 pa- cal Amphotericin B on Inflammatory Markers in Patients
tients (7,4 %) were categorized as non-adherent. with Chronic Rhinosinusitis: A Multicenter Randomized
Controlled Study, Laryngoscope. 2009; 119:401-408
Stability testing requires the measurement of the active 3. Fittler A, Mayer A, Kocsis B, Gerlinger I, Fónay F, Botz L,
ingredient. Analysis and quantitaive determination of poly- Stability testing of amphotericin B nasal spray solutions
enes can be carried out with different methods such as with chemical and biological analysis, Acta Pharm Hung.
chemical and microbiological analysis. The two types of 2007; 77: 159-64.
analytical methods differ fundamentally from each other. 4. Fittler A, Matus Z, Kocsis B, Botz L, Chemical and micro-
With the aid of chemical methods, those parameters (polar- biological aspects of the quantitative analysis of ampho-
ity, light absorption) are studied which are associated to the tericin B, Acta Pharm Hung. 2008; 78:95-102.
molecular structure of the molecule. On the other hand with
the aid of biological assays directly the biologic effect can
be measured. In our three month stability test we investi- P056-CO-ENZYME Q10 LOADED SOLID LIPID
gated the stability of 5mg/ml amphotericin B solutions (Fun- NANOPARTICLES INCORPORATED INTO HPMC
gizone®) with chemical (spectrophotometry) and biological
(bioassay) detection. The effect of storage temperature and GELS: IN VITRO ANTIOXIDANT ACTIVITY
the addition of 5% glucose was evaluated on the stability of
the solutions for three months. The two detection methods Evren Homan Gökçe1, Emrah Korkmaz1, Sibel Konyalıoğlu2,
showed strikingly different results. According to the chemi- Özgen Özer1
cal analysis the samples are considered relatively stable un-
der all observed conditions. As opposed to chemical analysis, Department of Pharmaceutical Technology, Faculty of
bioassay showed complete loss of antifungal activity after 35 Pharmacy, University of Ege, 35100, Izmir, Turkey 1
days of storage, at room temperature and notable decrease Department of Biochemistry, Faculty of Pharmacy, University
was observed at 4°C. These results drew our attention to of Ege, 35100, Izmir, Turkey 2
the major differences that can be detected with the two dif-
ferent methods. Further analytical studies were performed INTRODUCTION
with high performance liquid chromatography (HPLC) and Solid Lipid Nanoparticles (SLNs) are lipidic colloidal systems
with an inner structure based on pure solid lipids. Advantag-
agar diffusion (bioassay) to get better understanding of the 143
changes in the amphotericin B solutions during storage. es of SLNs for dermal and transdermal applications include
skin occlusion, modulation of drug/cosmetic release, incre-
We have optimized previously used HPLC eluent systems ment in skin hydration and elasticity, UV blocking effects,
and with the aid of a gradient program, four heptaene com- drug targeting and enhancement of stability1. Coenzyme
ponents could be detected besides AmB main component Q10 (Q10) is known as an endogenous cellular antioxidant.
at 407 nm. The amount of these constituents is about one In terms of dermal/cosmetic applications, this active has
hundredth of the main AmB. Only traces of tetraenes were shown the ability to reduce photoaging in vivo with a cor-
detected in the samples at the wavelength of 305 nm. responding decrease in wrinkle depth. Due to its structure,
the aqueous solubility is very low, causing difficulties in for-
In our bioassay study we evaluated five commonly used as- mulation steps2.
say media and two test microorganisms for the quantitative
determination of amphotericin B in a water base solutions. HPMC hydrogels possess good rheological properties result-
Our result showed, that Mueller Hinton agar supplemented ing in long residue times at the site of administration3.
with 2 % glucose and 0,5 µg/ml methylene blue, inoculated
with C. albicans (ATCC 90028) proved to be the most suitable The aims of the present work were to prepare Q10 loaded
bioassay parameters for the measurement of amphotericin SLNs incorporated into HPMC hydrogels and to investigate
B during a stability test, where a relatively wide concentra- their antioxidant properties in vitro.
tion change is to be measured. The linear segment of the log
dose-response curve was between 1,54-60,0 μg/ml and the EXPERIMENTAL METHODS
inhibition zones could be read easily and accurately. Materials
Compritol 888 ATO (C) was a kind gift from Gattefossèe.
CONCLUSION Poloxamer 188 (Plu) (Pluronic® F 68, Basf ) and Tween 80
Our poster illustrates the complex view of hospital pharma- (T80) (Fluka) were used as surfactants. Q10 was purchased
cists which can be highly beneficial during the preparations from Fluka. HPMC (Methocel K100M) were obtained from
for and execution of clinical studies. Colorcon.

REFERENCES MATERIALS AND METHODS


1. Gerlinger I, Fittler A, Fónai F, Patzkó A, Mayer A, Botz L, SLN Preparation
Postoperative application of amphotericin B nasal spray SLNs were prepared using high shear homogenization and
in chronic rhinosinusitis with nasal polyposis, with a re- ultrasound method (4). C and Q10 (24%) as the lipid phase

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

(100 mg) was melt. The aqueous phase was based on Plu (50 Hardness and compressibility values of gels should be low to
mg) and T80 (25 mg) dissolved in 12.5 ml of bidistilled water take the prepared gel from the container. The highest hard-
and heated to 80°C. Aqueous phase was poured into the lip- ness value was obtained with gels containing Q10 loaded SLNs.
id phase under homogenization at 24000 rpm for 5 min with There was a significant difference between HPMC gels and Q10
Ultra-Turrax (T25). Subsequently particles were dispersed in and SLN incorporated gel formulations (p<0.05). Nothing incor-
12.5 ml bidistilled water at 4°C and kept at 4°C for one day. porated HPMC gel showed the highest compressibility.

SLN Characterization Table 2: The mechanical properties of gel formulations.


SLNs were characterized for particle size (PS), polydispersity Hardness Compressibility
index (PI) and zeta potential (ZP) by means of PCS (Malvern Elasticity ± SD
(N) ± SD (Nnum)± SD
Zetasizer Nano ZS). HPMC2.5 0.145 ± 0.01 0.991 ± 0.001 17.804 ± 1.36

Preparation of SLN HPMC Hydrogels HPMC2.5Q10 7.631 ± 0.12 0.994 ± 0.001 10.267 ± 0.43
HPMC gels were prepared at a concentration of 2.5% un- HPMC2.5SLN 6.608 ±0.19 0.993 ± 0.001 8.768 ± 0.24
der magnetic stirring with (HPMC2.5Q10) and without Q10 HPMC2.5Q10SLN 10.84 ±0.36 0.991 ± 0.001 14.131 ± 0.32
(HPMC2.5). HPMC were added to the nano-suspension after
the preparation of blank (HPMC2.5SLN) and loaded SLNs The high value of cohesiveness provides full structural recov-
(HPMC2.5SLNQ10). The resulting mixture was stirred at room ery following gel application. When we compared our formula-
temperature with a magnetic stirrer. The formulations were tions, the cohesiveness was significantly different from HPMC
put in an ultrasonicator (Transsonic 660/h) to avoid air bub- gels (Table 3). Incorporated gels showed similar cohesiveness.
bles before TPA analysis. The most adhesive formulation was HPMC gel (Fig.1).

Texture Profile Analysis (TPA) Table 3: The mechanical properties of gel formulations.
Evaluation of the mechanical properties of the preparations
Adhesiveness ± SD Coheiveness ± SD
was performed using an STS Stable Micro Systems texture
analyzer (Model TA-XT. plus) in the TPA mode. Gel formula- HPMC2.5 22.576 ± 1.78 0.975 ± 0.01
tions were transferred into a 10 mL beaker and packed to a HPMC2.5Q10 14.118 ± 0.35 1.017 ± 0.02
fixed height, taking care to avoid the introduction of air into HPMC2.5SLN 10.632 ±0.29 1.014 ± 0.02
the samples. The analytical probe (10 mm diameter) was
HPMC2.5Q10SLN 17.540 ±0.48 1.011 ± 0.01
compressed twice into each sample to a depth of 15 mm at
144 a rate of 8.0 mm s-I. A delay period of 15 s was allowed be-
tween the end of the first and the beginning of the second
compression. All tests were performed at least in quadrupli-
cate on samples at ambient temperature.

In vitro Antioxidant Capacity


The trolox equivalent antioxidant capacity (TEAC) assay was
employed for the assessment of antioxidant capacity. 7mM
of ABTS [2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic
acid)] was converted to its radical cation by the addition of
potasium persulfate and keeping at 30°C for 12-16 h in a dark
incubator.
The formulations were heated to 80°C in 5mM phosphate
Figure 1: Texture profile analysis graphs of the gel formula-
buffer and 100 μl of these mixtures were added to the ABTS
tions
radical cation. The reaction was monitored spectrophoto-
metrically at 746 nm for 6 min. The difference between the
TEAC assay showed that the antioxidant capacity of Q10
absorbance was evaluated5.
loaded SLN incorporated HPMC gels was the highest in com-
parison to other formulations. The percentage of inhibition
RESULTS AND DISCUSSION
was detected as 8.7% which is equivalent to a trolox concen-
The determined PS of formulations was approximately 220
tration as 1.76 mmol/L.
nm and PI indicated a uniform system. ZP was found nega-
tive for both blank and loaded formulations. The charge of
CONCLUSION
the particles seemed to decrease after the incorporation of
It can be stated that Q10 loaded SLN incorporated HPMC
Q10 in the system (Table 1).
(2.5% w/v) gel formulations performed suitable mechanic
Table 1: PS, PI and Ç of SLNs (n=6)
properties with a significant antioxidant capacity. These sys-
PS (nm)±SD PI ±SD ZP ±SD tems might be evaluated as promising delivery systems for
Blank 143.8 ±23.60 0.317 ±0.03 -23.8 ±3.23 dermal/cosmetic applications of Q10.
Loaded 136.5 ±11.14 0.253 ±0.01 -18.7 ±2.96
Blank SLNs without Q10 REFERENCES
Loaded SLNs loaded with Q10 1. V. Teeranachaideekul et al. Eur. J. Pharm. Biopharm.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

2007;67:141-148. RESULTS AND DISCUSSION


2. C. Hsu et al. AAPS PharmSciTech 2003 ;4(3) Article 32. Figure 1 shows the results of the thiobarbituric acid assay of
3. S.Y. Karavana et al. Pharm. Dev. Technol. (2009) the simple stratum corneum lipid model system after the ad-
doi:10.1080/10837450902882351 dition of different drugs as test substances. The figure shows
4. E.H. Gokce et al. Int. J. Pharm. 2008;364:76-86. surprising effects for ascorbic acid and amantadine. Whereas
5. C.C. Wang et al. Clinical Chem. 2004;50:952-954. the latter drug acted antioxidatively, for the samples includ-
ing ascorbic acid the secondary lipid peroxidation products
were increased significantly.
p057-FURTHER SCREENING FOR NEW
ANTIOXIDATIVE COMPOUNDS FOR TOPICAL
ADMINISTRATION USING LIPID MODEL
SYSTEMS

Trommer Hagen1, Neubert Reinhard1

Martin Luther University Halle Wittenberg, School of


Pharmacy, Institute of Pharmaceutics and Biopharmaceutics,
Halle, Germany1

INTRODUCTION
Ultraviolet radiation is able to damage all important biomol-
ecules, such as lipids, DNA, carbohydrates and proteins se-
verely via the initiation of free radical reactions1. The human
skin is of particular interest, because it is constantly exposed
to UV radiation, oxygen and other noxious environmental Figure 1: Concentration of the TBA reaction products (TBA-
influences2. RP Conc.) and influence of UV irradiation and different test
drugs (100 µM each) in the LLA
In this study, lipid model systems of different complexity
were used as in vitro counterparts of the intercellular lipid Among the four flavonoids tested in this study only the St.
matrix of the stratum corneum. The effects of forty seven Mary`s thistle ingredient silibinin and the flavone quercetin 145
different substances (drugs, plant extracts, plant ingredients showed a lipid protecting potency when added to the sam-
and polysaccharides) on UV irradiation induced lipid peroxi- ples before UV irradiation treatment (Figure 2). Hesperetin
dation were investigated3. and rutin did not act antioxidatively in this setting. All the
plant extracts used in the screening showed protective ef-
MATERIALS AND METHODS fects by decreasing the amount of UV irradiation induced
Sample Preparation lipid peroxidation. Figure 2 illustrates this for St. John`s Wort,
Two lipid systems were used as in vitro screening models. The melissa and sage.
simple system was an oil in water dispersion of alpha-Linolenic
acid. The complex system consists of liposomes prepared by use
of the thin layer method. The lipids used for liposome prepara-
tion were: alpha-Linolenic acid (LLA), L-alpha-Dipalmitoylphos-
phatidylcholine (DPPC) and Cholesterol (CHOL). Ferrous sulfate
(10 µM) was added to the samples as an electron donor to initi-
ate oxygen radical generation via a Fenton type reaction4.

Ultraviolet Irradiation
UV-B irradiation experiments were carried out using a UV ir-
radiation chamber (Dr. Gröbel UV-Elektronik, Ettlingen, Ger-
many) enabling a selective exposure to UV-B. The samples
were treated with an UV-B dose of 0.25 J/cm².

Thiobarbituric Acid Assay


The thiobarbituric acid (TBA) test was used to determine ma-
londialdehyde (MDA) as a classic lipid peroxidation second-
ary product. Two millilitres of a stock TBA reagent were add-
ed to 1.0 ml of the UV-B treated sample. During the reaction, Figure 2: Concentration of the TBA reaction products (TBA-
a red TBA:MDA complex (2:1) is formed which is detected by RP Conc.) and influence of UV irradiation, flavonoids (100 µM
fluorescence measurement. HPLC was used to quantify the each) and plant extracts (0.2%)
pigment.

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

The buckwheat extract effects on the lipids is demonstrated P058-THE EFFECT OF HOMOGENIZATION
by showing the results of both simple and complex screen- SPEED ON THE IN VITRO CHARACTERISTICS
ing system (Figure 3). The flavonoid fraction of Fagopyrum
esculentum contains several flavonoids which showed an
OF THE PLGA MICROPARTICLES PREPARED
antioxidative effect already when tested as a single com- USING O/W EMULSION – SOLVENT
pound such as the aglycon quercetin. EVAPORATION METHOD

F. Ulya (Numanoğlu) Badıllı1, Tangül Şen1, Nilüfer Tarımcı1

Ankara University Faculty Of Pharmacy Department Of


Pharmaceutical Technology, Ankara, Turkey 1

INTRODUCTION
Psoriasis is a chronic, genetically influenced and immuno-
logically based inflammatory skin disease1. Clobetasol pro-
pionate (CP), which is a superpotent topical corticosteroid,
is commonly used for the topical treatment of psoriasis.
However, the local and systemic side effects of topical cor-
ticosteroids have limited their use2. In this study, clobetasol
propionate loaded PLGA microparticles were prepared as a
topical drug delivery system for psoriasis treatment. By this
way, a reduction in the side effects of the drug was intended.
The effect of the homogenization speed on the character-
Figure 3: Concentration of the TBA reaction products (TBA-RP istics of the microparticles were investigated. Also, in vitro
Conc.) and influence of UV irradiation and buckwheat extract (0.1 drug release studies of the particles prepared with two dif-
%, 0.2 %, 0.5 %) in the simple and complex screening system ferent drug : polymer ratios were carried out.
The screening for antioxidative compounds for topical ad-
ministration resulted in new promising findings. In the drug MATERIALS AND METHODS
testings amantadine was found to act antioxidatively where- Materials
as for ascorbic acid prooxidative effects were determined. Clobetasol propionate (CP) was kindly provided by Sandoz
146 Buckwheat extract significantly reduced the level of irradia- (Gebze-Kocaeli, Turkey). Poly(D,L-lactic-co-glycolic acid)
tion induced lipid peroxidation as well as the extracts of St. copolymer (PLGA 50:50, Mw=40000-75000) and Poly(vinyl
John`s Wort, melissa and sage. alcohol) (PVA) (Mw=30000-70000) were purchased from
Sigma-Aldrich (Steinheim, Germany). All of the solvents and
CONCLUSIONS reagents were analytical grade.
Human skin has constant exposure to UV light and oxygen.
Therefore, the administration of protectors in cosmetic formu- Preparation of PLGA Microparticles
lations or sunscreens, as found in this study, may be helpful for PLGA microparticles containing CP were prepared by oil-in-
skin protection against UV induced damage. Our results show water (O/W) emulsion – solvent evaporation technique as de-
new ways of skin protection by topical applicable substances scribed in our previous studies (3,4). Briefly, CP was dissolved
with antioxidative potency. In vivo experiments with the pro- in the solution of polymer in DCM. The organic phase obtained
tector substances should follow as well as preformulation stud- was added to aqueous PVA solution (0.5%) and homogenized
ies to test the stability of the potential antioxidants in several using UltraTurrax T25 Homogenizer®. This mixture was stirred
formulations and examinations of the penetration behaviour. at 25 °C to evaporate the organic solvent. Microparticles were
collected by centrifugation, washed three times with ultra
REFERENCES pure water and liyophilized for 48 hours. The codes of the mi-
1. Krutmann J. New developments in photoprotection of croparticle formulations are shown in Table 1.
human skin, Skin Pharmacol Appl Skin Physiol. 2001;
14(6):401-407. Table 1: The codes of the microparticle formulations
2. Thiele JJ. Oxidative targets in the stratum corneum. A Formul Code Drug: Ploymer Ratio Homogenization speed (rpm)
new basis for antioxidative strategies, Skin Pharmacol F1 1:10 8000
Appl Skin Physiol. 2001; 14(Suppl 1):87-91.
3. Trommer H, Neubert RHH. Screening for new antioxida- F2 1:10 9500
tive compounds for topical administration using skin F3 1:5 8000
lipid model systems, J Pharm Pharm Sci. 2005; 8(3):494- F4 1:5 9500
506.
4. Trommer H, Wagner J, Graener H, Neubert RHH. The ex- In vitro Characterization of Microparticles
amination of skin lipid model systems stressed by ultra- The production yield, encapsulation efficiency and particle
violet irradiation in the presence of transition metal ions, size of the microparticle formulations were determined. The
Eur J Pharm Biopharm. 2001; 51(3):207-214. shape and surface morphology of the F1 and F3 were ex-

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

amined using scanning electron microscope. DSC and XRD


analysis of CP, polymer, F1, F3 and blank microparticles were
performed. In vitro drug release studies of the microparticles
were carried out using modified Franz diffusion cells during
24 hours at 37 °C. The CP content was analyzed by HPLC at a
wavelength of 242 nm.

RESULTS AND DISCUSSION


Encapsulation efficiencies for all microparticle formulations
were found to be higher than 90% (Table 2).

Table 2: Production yield, encapsulation efficiency and par- Figure 2. In vitro release profiles of F3 and F4
ticle size of the microparticles
Encapsultaion CONCLUSION
Production PArticle Size PLGA microparticles containing CP were succesfully pre-
Formul code Efficiency (%)±
Yield (%) (μ+SD) pared using O/W emulsion – solvent evaporation method
SD
and the effect of the homogenization speed was investi-
F1 59.66 93.73 ± 0.22 14.61 ±1.45
gated. In conclusion, the particle size decreased when the
F2 77.56 95.09 ± 0.88 6.39 ±1.63 homogenization speed was increased.
F3 73.25 97.06+ 0.63 8.54 ±1.66
F4 77.285 08.34 ±0ç45 4.57 ±2.47 ACKNOWLEDGMENTS
This study is supported by Management of Scientific Re-
The particle size of the microparticles prepared with 1:10
search Projects of Ankara University (Project number:
drug : polymer ratio at 9500 rpm (F2) was significantly lower
07B3336002).
than the microparticles prepared at 8000 rpm (F1) (p<0.05).
Similar results were obtained for the microparticle formu-
REFERENCES
lations prepared with 1:5 drug : polymer ratio at different
1. Linden KG, Weinstein GD. Psoriasis: current perspectives with
speed values. The results of the particle analysis showed that
an emphasis on treatment, American Journal of Medicine.
the particle size significantly decreased when the homog-
1999; 107: 595-605.
enization speed was increased from 8000 rpm to 9500 rpm.
2. Lebwohl M, Ali S. Treatment of psoriasis. part 1. topical ther- 147
apy and phototherapy, J Am Acad Dermatol. 2001; 45: 487-
Scanning electron micrographs showed that the microparticles
498.
were spherical in shape and no drug crystals were observed
3. Numanoglu U, Şen T, Tarimci N. Preparation and in vitro eval-
on the surface of the microparticles. It was also observed that,
uation of clobetasol propionate loaded PLGA microparticles
F3 coded microparticles had highly porous surfaces whereas
as topical drug delivery system for treatment of psoriasis vul-
the surface of the F1 coded microparticles was non-porous.
garis, FIP World Congress. 2008, Basel-Switzerland; 182.
4. Badilli (Numanoğlu) FU, Şen T, Tarimci N. Clobetasol propi-
DSC and XRD results showed that CP was totally entrapped
onate loaded PLGA microparticles for topical drug delivery:
in the polymer and the drug was present in an amorphous
the effect of the emulsifier ratio, 36th CRS. 2009, Cophenha-
phase in the microparticles.
gen-Denmark; Poster No: 1024.
The results of the in vitro release studies showed that, the release
rate of the drug from the F2 coded microparticles was significant-
ly higher than the F1 microparticles (Figure 1). This was attributed P059-ATR-FTIR SPECTROSCOPIC ANALYSIS
to the smaller particle size of the F2 coded microparticles. Unex- OF THE EFFECT OF TERPENE/CO-SOLVENT
pectedly, the amount of the drug released from F4 microparticles MIXTURES ON PIG SKIN
was lower than that of F3 coded microparticles (Figure 2). It was
thought that, the release of the drug increased due to the highly M. Sedef Erdal1, Deniz Özdin1, Sevgi Güngör1
porous surface of the F3 coded microparticles.
Istanbul University Faculty of Pharmacy Department of
Pharmaceutical Technology, Istanbul, Turkey1

INTRODUCTION
Terpene penetration enhancers are widely used in combina-
tion with ethanol and propylene glycol (PG). It has been pro-
posed that the interaction of terpenes with SC in presence of
different solvents may not be similar1.

Fourier Transformed Infrared (FTIR) spectroscopic imaging in


Figure 1: In vitro release profiles of F1 and F2 Attenuated Total Reflection (ATR) mode is a useful tool for

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

studying biomedical samples. It provides considerable in- at 2851.63±0.10 and 2920.39±0.14 cm-1, respectively. In
formation about the interactions of penetration enhancers the presence of EU and LM, SSV shifted by ~1.9 cm-1 to
with the lipids and proteins of the intercellular matrix of SC2. 2853.53±0.29 cm-1 which indicates the introduction of
gauche conformers in the carbon skeleton of lipid alkyl
The aim of our study was to investigate the interaction of chains.
three different terpenes: nerolidol (amphiphilic sesquiter-
pene), dl-limonene (cyclic terpene) and eucalyptol (oxygen As both SSV and ASSV collapse in the presence of NR, we can
containing monoterpene) with intact pig SC in two co-sol- suggest that the inter-chain interactions of epidermal lipids
vent systems, propylene glycol (PG) or Transcutol (TC) with are affected (Fig 1).
the goal of comparing these two co-solvents and the effect
of TC for the first time. Treatment of skin with pure PG or pure TC revealed no signif-
icant shift in lipid peak frequency, however, the dramatically
ATR-FTIR spectroscopy was employed to understand the ef- decrease in area of C-H stretching peaks is indicative of lipid
fect of terpene/co-solvent systems on molecular organiza- extraction from SC (Table 2).
tion of intact pig SC.
Table 1: Composition of terpene/co-solvent systems
MATERIALS AND METHODS Formulation Code Treatment (terpene 3%)
Materials LM dl-im onene
Nerolidol, Dl-limonene, Eucalyptol and Propylene Glycol LMPG dl-lim onene +PG
were purchased from Sigma. Transcutol® was kindly provid- LMTC dl-lim onene + TC
ed by Gattefossè. NR Nerolidod
NRTG Nerolidod +PG
Preparation of terpene/co-solvent systems NRTC Nerolidod + TC
Terpenes were added in PG or TC at 3% concentration (Table EU Eucalyptol
1). After 3 min vortexing, mixtures were equilibrated for 30 EUPG Eucalyptol + PG
min without stirring. EUTC Eucalyptol TC
PG Propylene Gylcol
Skin samples
The subcutaneous fat tissue of excised pig skin was removed TC Transcutol
with a scalpel and the hairs were clipped with hair clippers.
148 The skin was stored frozen at -25 oC (wrapped in Parafilm
and packed in ZipLock bags) for not longer than 2 months
prior to use.
Before collection of ATR-FTIR spectra, samples were de-
frosted 8 h at 4 oC. After defrosting, skin surface was wiped
2 times with wet cotton swab and excess water was gently
dried with paper tissue. 100 µl of terpene, co-solvent or ter-
pene/co-solvent system was applied on the surface and kept
for 3h at 25 oC. After the treatment period, excess of formu-
lation was gently blotted away with paper tissue from the
surface of the skin.
Figure 1: The IR spectra of intact pig SC treated with NR, EU
IR Spectroscopy and LM.
All spectra were collected with a spectral resolution of 4cm-1
in the 4000-650 cm-1 range using a Perkin Elmer Spectrum Table 2: Peak areas of SSV and ASSV of intact pig SC treated
100 FT-IR spectrometer (UK) equipped with a ZnSe ATR crys- with terpenes, co-solvents and terpene/co-solvent systems
tal. Each spectrum was an average of 4 scans. Skin sample,
Treatment Peak Area ASSV Peak Area of (SSV)
cutted to dimensions 2x2 cm, was placed SC side down onto
the ATR crystal. To ensure reproducible contact between the CNT 4.49 ± 0.22 2.25 ± 0.11
sample and the crystal, always the same pressure on top of PG 0.32 ± 0.25 n.a*
samples was applied (force gauge 100 N). TC 0.36 ± 0.32 n.a*
LM 3.60 ±0.11 1.56 ± ±0.17
For each skin sample peak area was measured after enhancer NR n.a* n.a*
treatment. Attention was focused on characterizing the oc- EU 2.01 ±1.21 1.42 ±0.39
currence of peaks near 2850 and 2920 cm-1 which were due LMPG 1.23 ±0.26 0.06 ±0.034
to the symmetric (SSV) and asymmetric (ASSV) C-H stretching LMTC 1.23 ±0.26 n.a*
vibrations, respectively. A non treated skin sample served as NRPG 1.81 ±0.05 0.05 ±0.04
control (CNT). Perkin Elmer Spectrum Version 6.0.2 software
NRTC 0.17 ±0.01 n.a*
was used for calculations.
RESULTS AND DISCUSSION EUPG 1.12 ±0.17 0.3 ±0.30
The SSV and ASSV of control skin were observed to be EUTC 1.17 ±0.25 0.26 ±0..26

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

CONCLUSION buffered saline (PBS) ingredients were supplied from Sigma


The synergistic effect of TC in this study can be explained (USA). All solvents used in this study were High Performance
by improved partitioning of terpenes into the SC in the Liquid Chromatography (HPLC) grade. All chemicals were of
presence of TC. Our investigation will provide a basis of us- the purest grade available.
ing a suitable terpene/co-solvent system in a transdermal
formulation. Ex vivo permeation studies will be carried out Methods
using hydrophilic and lipophilic model drugs to investigate Preparation of Liposomes Containing OMC
the relationship between the observed flux enhancements The multilamellar liposomes (MLVs) containing OMC were
and the effect of terpene/co-solvent systems on skin barrier prepared by fusion method and o/w emulsion was prepared
property. as FDA standard sunscreen method (3, 4).

REFERENCES Morphology and Size Analysis of Liposomes


1. Babita K., Kumar V., Rana V., Jain S, Tiwary A.K. Thermo- Optical microscope (OLYMPUS, Germany) was used for study-
tropic and spectroscopic behavior of skin : relationship ing the morphological features of MLV liposomes containing
with precutaneous permeation enhancement, Curr Drug OMC. The mean diameter and particle size distribution of li-
Del. 2006, 3 : 95-113 posomes were determined by a particle size analyzer (PSA;
2. Kazarian S.G., Chan K.L.A. Applications of ATR-FTIR spec- Klotz, Germany)1,3.
troscopic imaging to biomedical samples, Biochim Bio-
phys Acta. 2006,1758 : 858-867. SPF determination of Homosalate reference, COLIPA stan-
dard, o/w emulsion and liposomes containing OMC by in
vivo method
p060-DETERMINATION OF SPF AND
MOISTURIZING EFFECTS OF LIPOSOMAL AND The SPFs of the formulations were determined by in vivo
method according to Australian standard. The exposure area
CONVENTIONAL FORMULATIONS OF OMC AS was the back of 10 volunteers. Subsites of the backs were
A SUNSCREEN exposed to solar simulator. The Minimum erythemal dose for
unprotected skin MED (US) was observed in the next day. (Fig
Shiva Golmohammadzadeh1, Mahmoud Reza Jaafari2, 1) The sunscreen was spread (2mg/cm2) over the area. Each
Noman Khalili3 test subsite in a series was exposed to controlled amounts of
simulated sunlight by a constant ratio. In the third day the
Pharmaceutical Research Center and School of Pharmacy, MEDs of the formulations were observed. The SPF was deter- 149
Mashhad University of Medical Sciences, Mashhad, Iran1 mined by the ratio between the MED while using sunscreen
Pharmaceutical Research Center and Biotechnology Research and MED (US)4.
Center, School of Pharmacy, Mashhad University of Medical
Sciences2 Moisture Content of the Skin Following Application of o/w
School of Pharmacy, Mashhad University of Medical Sciences, Emulsion, Liposomes Containing OMC and NaCl 3% in Euc-
Mashhad, Iran3 erin Using Corneometer

INTRODUCTION The moisture content of the skin was determined before and
It has been known for decades that sunscreens are capable after 30 min, 2, 3, 6 and 10 h post application of the formu-
of protecting man from harmful effects of solar radiation1, 2 lations using Corneometer by measuring electrical capaci-
Ramon et al. showed that liposomes could be regarded as al- tance5.
ternatives to conventional oil/water emulsions in the formu-
lations of lipidic sun filters. When liposomes with a composi- RESULTS AND DISCUSSION
tion and structural organization similar to that of the stratum The liposomes made by fusion method were morphologi-
corneum lipids are used, the skin penetration is retarded1. cally homogenous multilamellar vesicles, as observed under
optical microscope. Mean diameters of MLVs liposomes con-
The objectives of this study were to determine and compare taining OMC determined by PSA were 2.21 ± 0.06 μm (n = 3)
the SPF (Sun Protection Factor) and moisturizing effects of the (Fig 2). Small unilamellar vesicles (SUV) were with a size of
liposomal and conventional formulations containing octyl 62.41 ± 21.2 μm (n = 300) (Fig 2).
methoxycinnamte (OMC) as a sunscreen by in vivo methods. The SPF of the liposomes containing OMC was greater than
lotion at the same concentration of OMC. However this was
MATERIALS AND METHODS considered not quite significant by unpaired t test (Table 1).
Materials It was observed that in each formulation there is significant
OMC, cholesterol and vitamin E were purchased from Merck increase in moisture content after 30 minutes. After 2 hours
(Darmstadt, Germany). Lanolin, white petrolatum, stearic the moisture contents were decreased in all of the formu-
acid, propylparaben, methylparaben disodium EDTA, pro- lations. At 30 minutes, the highest water content was ob-
pylene glycol and triethanolamine were purchased from served for OMC lotion. However, there were no significant
Sigma (USA). Soya Phosphatidylcholine was obtained from differences between NaCl 3% in eucerin as control and OMC
the Avanti Polar Lipids (Alabaster, Alabama, USA). Phosphate liposomes.

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

P061-EVALUATION OF LIPOSOMAL AND


CONVENTIONAL FORMULATIONS ON THE
HUMAN PERCUTANEOUS ABSORPTION OF
OMC AS A SUNSCREEN
Figure 1; The MED observed 20 hours after UV
Shiva Golmohammadzadeh1, Mahmoud Reza Jaafari2,
Table 1:The SPF values of COLIPA, OMC lotion and OMC-MLV Noman Khalili3
liposomes by in vivo method in ten subjects (S) with differ-
ent skin types and MEDs. Pharmaceutical Research Center and School of Pharmacy,
Mashhad University of Medical Sciences, Mashhad, Iran1
SPS Pharmaceutical Research Center and Biotechnology Research
Sunscreen S1 S2 S3 S4 S5 S6 S7 S8 S9 S10
(Men±SD) Center, School of Pharmacy, Mashhad University of Medical
Sciences, Mashhad, Iran2
Homosalate
reference
4 4.46 4.56 4.90 4 4.02 3.62 4.44 5.63 4.05 4.35±0.64 School of Pharmacy, Mashhad University of Medical Sciences,
Mashhad, Iran3
COLIPA 12.4 1.5 19.4 14.5 17.1 12.4 15.5 15.5 12.4 19.4 15.2±3.4
INTRODUCTION
OMC lotion 14.2 15.5 12.4 12.3 1.5 17.1 12.3 12.4 15.5 12.3 13.95±1.83
It has been known for decades that sunscreens are capable of
OMC liposomes 17.1 19.3 19.4 12.4 15.5 14.5 19.4 17.1 14.5 15.5 16.85±1.40 protecting man from harmful effects of solar radiation. Many
factors manage the delivery of the drugs and cosmetics into
the skin from topically applied formulations1,2. The objective
of this study was to determine the influence of vehicle on
the penetration of octyl methoxycinnamate (OMC), as a UV
absorber, to the skin layers by stripping method. The formu-
lations were a conventional o/w emulsion, multilamellar and
small unilamellar liposomes (MLVs and SUVs).
Figure 2: Optical microscopic picture of liposomes prepared
by fusion method containing OMC (magnification of image MATERIALS AND METHODS
is × 1000). Materials
150 OMC, cholesterol and vitamin E were purchased from Merck
CONCLUSION (Darmstadt, Germany). Lanolin, white petrolatum, stearic
The results of this study indicate that the SPFs obtained from acid, propylparaben, methylparaben disodium EDTA, pro-
our in vivo results is almost the same as published SPFs for pylene glycol and triethanolamine were purchased from
Homosalate reference and MLVs prepared by fusion method Sigma (USA). Soya Phosphatidylcholine was obtained from
is a good vehicle for OMC as a sunscreen since it provides the Avanti Polar Lipids (Alabaster, Alabama, USA). Phosphate
proper SPF and increase the moisture content of the skin. buffered saline (PBS) ingredients were supplied from Sigma
(USA). All solvents used in this study were High Performance
ACKNOWLEDGMENTS Liquid Chromatography (HPLC) grade. All chemicals were of
This study was supported financially by the Pharmaceuti- the purest grade available.
cal Research Center and Biotechnology Research Center,
Shool of Pharmacy, Mashhad University of Medical Sciences Methods
(MUMS). Preparation of liposomes containing OMC
MLVs containing OMC were prepared by fusion method and
REFERENCES then converted to SUVs by probe sonication3.
1. Ramon E, Alonso C, Coderch L et al. Liposomes as alter-
native vehicles for sun filter formulations. Drug Deliv. Determination of percutaneus absorption of OMC using
2005; 12: 2, 83-88. stripping method
2. Chatelain E, Gabard B, Surber C. Skin penetration and Formulations were applied at the level of the forearm of six
sun protection factor of five uv filters: effect of the ve- volunteers in a dose of 2 mg/cm2. Afterwards stripping was
hicle. Skin Pharmacol Appl Skin Phys. 2003: 16, 28-35. performed by 22 stripping and divided to different groups.
3. Foldvari M. 1998, Biphasic Liposomes. In US Patent. No. The sunscreen agent was assessed by HPLC4,5.
5,853,755.
4. Australian/New Zealand Standard. Sunscreen products. Morphology and Size Analysis of Liposomes
Evaluation and classification. AS/NZS 2604:1998, 1-32. Optical microscope (OLYMPUS, Germany) was used for study-
5. Alanen E, Nuutinen J, Niclen K et al. Measurement of hy- ing the morphological features of MLV liposomes containing
dration in the stratum corneum with the MoistureMeter OMC. The mean diameter and particle size distribution of li-
and comparison with the Corneometer. Skin Res Tech- posomes were determined by a particle size analyzer (PSA;
nol. 2004; 10: 32-37. Klotz, Germany)2.

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

RESULTS AND DISCUSSION Research Center and Biotechnology Research Center, Mash-
The liposomes made by fusion method were morphologi- had University of Medical Sciences (MUMS).
cally homogenous multilamellar vesicles, as observed under
optical microscope. Mean diameters of MLVs liposomes con- REFERENCES
taining OMC determined by PSA were 2.21 ± 0.06 μm (n = 3) 1. Sarveiya V, Risk S, Benson HAE. Liquid Chromatographic
(Fig 1). Small unilamellar vesicles (SUV) were with a size of assay for common sunscreen agents: application to in
62.41 ± 21.2 μm (n = 300) (Fig 2). vivo assessment of skin penetration and systemic ab-
sorption in human volunteers. J Chromatogr B 2004;
803: 225-231.
2. Verma D, Fahr A. Particle size of liposomes influences
dermal delivery of substances into skin, Int J Pharm.
Figure 1: Optical microscopic picture of liposomes prepared 2003; 258: 141-151.
by fusion method containing OMC (magnification of image 3. Foldvari M. 1998, Biphasic Liposomes. In US Patent. No.
is × 1000). 5,853,755.
4. Chatelain E, Gabard B, Surber C. Skin penetration and
sun protection factor of five uv filters: effect of the ve-
hicle. Skin Pharmacol Appl Skin Phys. 2003: 16, 28-35.
5. Periolia L, Ambrogi V, Bertini B, Ricci M, Nocchetti M et al.
Anionic clays for sunscreen agent safe use: Photoprotec-
Figure 2: Transmission electron micrographs of SUV lipo- tion, photostability and prevention of their skin penetra-
somes containing OMC by negative staining using phospho- tion. Eur J Pharm Biopharm. 2006; 62: 185–193.
tungstate and viewed in an electron microsc

The results of this study indicate that skin accumulation of P062-THE KNOWLEDGE OF PHARMACISTS
OMC in MLVs was significantly greater than o/w emulsion
and SUVs. The penetration of SUVs into the deeper skin lay-
IN REGARDE TO THE APPLICATION OF
er was significantly more than MLVs and conventional o/w SUNSCREENS AND MOISTURIZERS
emulsion. Higher amounts of OMC were recovered from
the upper layers of stratum corneum than deeper layers in Shiva Golmohammadzadeh1, Jebraeel Movaffagh2, Yousef
all formulations. The conventional lotion may remain on the Setayesh3
surface without penetration to the skin compared with lipo- 151
somes (Fig 3, 4). Pharmaceutical Research Center and School of Pharmacy,
Mashhad University of Medical Sciences, Mashhad, Iran1
School of Pharmacy, Mashhad University of Medical Sciences,
Mashhad, Iran2
School of Health, Mashhad University of Medical Sciences,
Mashhad, Iran 3

INTRODUCTION
Figure 3: Amount of OMC in the group of strips, after 3 h of Sunscreen usage is a widely accepted method of primary
application, (mean ± SD; ns p >0.05, **p <0.01, ***p <0.001 prevention against deleterious effects of ultraviolet radiation
vs. MLV), Group 1: strips 1-2; G (UVR); skin cancer, sunburn, freckles and photoaging. How-
ever, studies have shown that rates of regular sunscreen use
are very low, despite evidence of the efficacy of sunscreen
application1. Also it has been known for decades that mois-
turizers are useful for skin health care. Pharmacists are piv-
otal to prescribe the drugs and cosmetics. A pharmacist has
different functions; the pharmacist as a dispenser of drugs;
the pharmacist as a drug consultant and the pharmacist as a
Figure 4: Amount of OMC in the group of strips after 7 h of
‘substitute doctor’2.
application, (mean ± SD; ns p >0.05, **p <0.01, ***p <0.001
vs. MLV), Group 1: strips 1-2; Gr
The aim of this study was to assess the knowledge of phar-
macists in regard to the application of sunscreens and mois-
CONCLUSION
turizers in Mashad, Iran.
The result of this study indicate that MLVs prepared by fusion
method are better vehicle for OMC as a sun screen since it
MATERIAL AND METHODS
remains more in the stratum corneum and its penetration to
In May to July 2007, 81 pharmacists completed surveys by
the deeper layers is less.
filling the questionnaire. In this descriptive study, 25 ques-
tions addressed to determine the demographic character-
ACKNOWLEDGMENT
istics plus the knowledge of the pharmacists towards sun-
This study was supported financially by the Pharmaceutical

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

screens and moisturizers application. The factors influencing ACKNOWLEDGMENTS


the knowledge regarding sunscreens and moisturizers appli- Grant support was provided by Mashad University of Medi-
cation were evaluated3. cal Science (No. 86054). Mohammad Naser Shafiee, Faculty
member of MUMS is gratefully acknowledged.
RESULTS AND DISCUSSION
This is the first report which compares the knowledge and beliefs of REFERENCES
pharmacists on sunscreens and moisturizers use in Iran and other 1. Ling TC, Faulkner C, Rhodes LE. A questionnaire survey
countries. Our study includes a sample of 81 Pharmacists from the of attitudes to and usage of sunscreens in northwest
pharmacies in Mashhad including 41 (50.6%) men with 31.6±14.4 England. Photodermatol Photoimmunol Photomed
age and 39 (48.1%) women with 36.6±12.3 age. with 67% between 2003;19(2):98-101.
the ages 26 and 35. The median period of practicing as a pharma- 2. van Grootheest K, Olsson S, Couper M, de Jong-van den
cist since graduation was 6 (5–14) years (Table 1). From the overall Berg L. Pharmacists’ role in reporting adverse drug reac-
respondents 56.8% had used sunscreens and 12.3% had used the tions in an international perspective. , Pharmacoepide-
sunscreens in the correct time. 45.7% knew the correct applica- miol Drug Saf 2004; 13(7): 457–464.
tion of sunscreens, 34.6% didn’t know and 19.8% gave no answer. 3. Ling TC, Faulkner C, Rhodes LE. A questionnaire survey
In the case of the moisturizers, 65.4% had used moisturizers. The of attitudes to and usage of sunscreens in northwest
respondents in this study generally performed very poorly on the England. Photodermatol Photoimmunol Photomed
knowledge test. The mean score received, 39.33±16.77 out of 100 2003; 19(2):98-101.
points, which is less than 50%. Sex was not significantly associated
with the knowledge. In the overall sample the results showed that
higher knowledge scores were associated with younger age. Also P063-THE CONTENT OF TOTAL
higher knowledge scores were associated with less work history. POLYPHENOLS, TANNINS AND FLAVONOIDS
The mean score of the knowledge in formulations received was
9.25±7.59 out of 100 points (figure 1). IN DIFFERENT PARTS OF EPILOBIUM
ANGUSTIFOLIUM L.
Table 1: Percentage distribution of selected characteristics,
in pharmacists in Mashad, Iran, 2007(n=81) Ain Raal1, Siiri Jürgenson1
Characteristics Percentage
Use of sunscreens 56.8 University of Tartu, Tartu, Estonia1
Use of sunscreens in correct time 12.3
152 Use of sunscreens in all seasons 34.6 INTRODUCTION
Epilobium species (Onagraceae) are commonly used herbal
SBF definiton correctly answered 46.9
remedies in traditional, adjuvant therapy of benignus pros-
Accurate application of sunscreens 45.7 tate hyperplasia. Willow herb (Epilobium anguistifolium L.)
Use of moisturizers 65.4 products usually consist of mixtures from various species,
Knowledge regarding to sunscreens and moisturizers 39.33±16.77 with different phenoloid content, often only partially identi-
Knowledge regarding to conscistuence ofsunscreens and 9.25±7.59 fied. The main constituent, oenothein B belong to the group
moisturizers of tannins, but also lot of tannins, flavonoids and other vari-
ous polyphenols were detected in the willow herb1,2,3.

The aim of the study was to investigate the dynamics of total


polyphenols, tannins and flavonoids in different parts of wil-
low herb growing in Estonia.

MATERIALS AND METHODS


The plant material - 15 plants of willage herb were collected
from May to November 2006 in Unipiha village (Tartu county,
Estonia). The plants were cut at the height of 20 cm to avoid
stems without leaves. As soon as possible, the plants were
separated into different organs (roots, stems, leaves, flowers
Figure 1: Histogram curve of knowledge distribution in phar-
and fruits), which were dried during a week at the room tem-
macists regarding to sunscreen sand moisturizers in Mashad
perature. For receiving the herb, plant material was collected
city in 2007.
from the same site in July 2007.
CONCLUSION
The herb cut at 20 cm is henceforth called whole herb. Herb
This study showed that higher knowledge scores in formula-
without main stems was obtained with separating main stalk
tions were associated with more knowledge. In this study a
from the whole herb. The dried and cut herb was sieved (diam-
significant proportion of pharmacists do not have adequate
eter 1 mm) before extraction of biologically active substances.
knowledge concerning the sunscreens and moisturizers. These
Total polyphenols
results indicate that more education in cosmetic and toiletries
The plant material was extracted with 40% ethanol during
field is necessary for pharmacists, specially the older ones.
30 minutes at 60 degrees in thermostat. Folin-Denise re-

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

agent and sodium carbonate saturated solution were added CONCLUSION


to the obtained extract. After an hour the optical density of Based on the content of flavonoids and tannins, it seems to
the solutions was detected spectrophotometrically at the be optimum to use herb without main stalk collected while
730 nm. Results were calculated according to the absorption flowering.
coefficient of 1% chlorogenic acid (123, 5).
REFERENCES
Total tannins 1. Kosalec I, Zovko M, Sankovic K, Kremer D, Pepeljnjak S.
Plant material was extracted while boiling in distilled water Antioxidant and antimicrobial activity of willow herb
for 30 minutes on electric cooker. 1% solution of potassium- (Epilobium angustifolium L.). Planta Med. 2008; 74(9):
ferricyane and 1% solution of ferri(III)chloride were added to 948-948.
the filtered solution. After shaking and after 5 minutes, opti- 2. Shikov AN, Pozharitskaya ON, Ivanova SA, Makarov VG,
cal density at the 720 nm was detected spectrophotometri- Tikhonov VP, Galambosi B. Variation in concentration of
cally. The results were calculated based on the optical den- oenothein B in different samples of cultivated Epilobium
sity of tannin solutions. The standard chart was composed angustifolium L. Planta Med. 2008; 74(9): 1120-1120.
based on the optical density of tannin solutions with differ- 3. Hevesi Tóth B, Blazics B, Kéry A. Polyphenol composition
ent concentration and the two solutions mentioned above and antioxidant capacity of Epilobium species, J Pharm
were added in advance. Biomed Anal. 2009; 49(1): 26-31.

Total flavonoids
Plant material was extracted while boiling with 70% ethanol p064-EFFECT OF CATECHIN DERIVATIVES
for 30 minutes under cooled reflux condenser. 10% sulphuric ON SERUM ALBUMIN CONFORMATION
acid was added to the extract and it was boiled under cooled
reflux condenser for 30 minutes for the hydrolysis of gluco- AND ITS GLYCATION BY REACTIVE ALPHA-
sides. Alcohol was steamed on the water bath and the ex- DICARBONYL COMPOUNDS
tract was filtered. Sediment on the filter paper was dissolved
in 95% ethanol warmed up to 50 degrees. The solution of Jaroslav Dršata1, Lucie Trnková2, Iva Bousová1
aglycones was diluted and the optical density was detected
at the 370 nm spectrophotometrically. Charles University Faculty of Pharmacy, Czech Republic1
University of Hradec Králové, Czech Republic 2
RESULTS AND DISCUSSION
During the vegetation period, the most polyphenols can be 153
INTRODUCTION
found in the herb with main stalks collected during full flow-
Flavonoids are natural polyphenolic compounds that oc-
ering. It can also be found in significant amounts in roots and
cur ubiquitously in plants. Multiple biological effects of fla-
stems, but not so much in leaves, fruits and flowers. In roots,
vonoids including antioxidant, antiviral, antibacterial, anti-
stems and leaves the tannins can be found the most in May
inflammatory, and vasodilatory activity have been reported.
(14.9-41.4%). In herbs collected in July the content is about
Besides positive acting, flavonoids exert also pro oxidative
50% lower (11.9-18.7%). The least flavonoids can be found
effects in vitro and are able to bind with proteins via hydro-
in stems and the most in leaves. Significant amounts of fla-
phobic interactions and hydrogen bonds. Such interactions
vonoids can be also found in flowers and fruits (Table 1).
with proteins may on one side influence relative antioxidant
potency of these compounds as well as physiological role of
Table 1: The content of polyphenols, tannins and flavonoids
affected protein. Catechins (flavan 3 ols), a group of naturally
in different parts of Epilobium angustifolium L. from May to
occurring flavonoids, can be found predominantly in green
November 2006
tea. Trapping of reactive alpha-dicarbonyl species (e.g. meth-
Total of ylglyoxal) by epigallocatechin, epicatechin 3 gallate (ECG),
Total of Total of and epigallocatechin 3-gallate in vitro has been described.
Organs flavonoids,
polyphenols, % tannins, %
mg% MATERIALS AND METHODS
Roots 4.0-8.5 7.7-16.2 4.7-19.3 The aim of this work was to evaluate both direct impact of
eight catechin derivatives on conformation of human serum
Stems 5.5-8.3 2.7-14.9 5.7-20.8 albumin (HSA) and their possible anti-glycation activity in
Leaves 2.4-6.3 16.0-41.4 113.1-235.7 the model containing HSA, methylglyoxal (MGO), and cat-
echin derivative (0-50 µM) using fluorescence spectroscopy.
Flowers 3.4* 36.1* 154.7*
Fruits 3.5-4.6** 17.5-20.2** 68.1-121.7** RESULTS AND DISCUSSION
Whole herb 10.2*** 11.9*** 30.4*** For catechin-albumin interaction, catechins proved to be
reactive compounds, which were able to cause changes in
Herb without protein conformation. Their binding affinity decreased in fol-
6.5*** 18.7*** 345.1*** lowing order: ECG > catechin gallate > epigallocatechin gal-
main stalk
*Collected in July 2006, ** collected in July, August and Sep- late > gallocatechin gallate > epicatechin > catechin > gallo-
tember 2006, *** collected in July 2007 catechin ≥ epigallocatechin. Furthermore, these compounds

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

were able to decrease catalytical activity of glutathione concentration (MIC) and minimal bactericidal concentration
S-transferase in vitro. For anti-glycation study, fluorescence (MBC) values of extracts were recorded4.
of advanced glycation end products (AGEs) was measured
(370/440 nm) and % of inhibition of AGEs for individual cat- RESULTS AND DISCUSSION
echin derivatives were determined. The inhibitory activity of Among all obtained extracts, ethanol extracts of both spe-
these compounds was compared to effect of aminoguani- cies especially that of Malva sylvestris, showed the best ac-
dine (1 mM) and Trolox (2.5 mM). The most effective anti-gly- tivity against bacteria, followed by aqueous extracts. The
cation agent, even better than aminoguanidine, appeared to best of antibacterial MIC values was 0.4 g/ml obtained with
be ECG. Its inhibitory effect as well as effects of other cat- S. pyogenes cultures. All extracts were active against S. au-
echin derivatives was concentration-dependent. This study reus, P. aeruginosa, P. vulgaris. Aqueous and chloroform ex-
could help to elucidate role of catechin derivatives in the tracts had better antifungal activity. The best of antifungal
process of glycation and also contribute to assessment of MIC values was 0.6 g/ml for Malva sylvestris aqueous extract
their interaction with protein molecules. against A. niger cultures. All extracts had activity against A.
niger, A. fumigatus and C. albicans.
ACKNOWLEDGMENTS
Supported by the Development program of the Czech Minis- The prevention and treatment of wound infections espe-
try of Education No. 14/87/2009. cially after contaminated burn and surgical events is a criti-
cal issue. The proper interpretation of cultures taken from
wounds is uncertain. Multiple organisms are invariably iso-
p065-EFFICACY OF CHLOROFORM, ETHANOL lated from non-sterile wounds including indigenous aerobic
AND WATER EXTRACTS OF MEDICINAL and anaerobic flora, aerobic gram-negative rods, and fungi.
Antibiotic trials with routine antimicrobial agents are losing
PLANTS, MALVA SYLVESTRIS AND MALVA efficacy due to continual development of antibiotic resis-
NEGLECTA ON SOME BACTERIAL AND tances in the causative microorganisms. On the other hand,
FUNGAL CONTAMINANTS OF WOUND the antibacterial spectrum of an agent used for burn and
INFECTIONS surgical would treatment and prophylaxis should include
coverage for a wide range of microorganisms including
Payman Zare1, AmirBabak Sioofi 1, Saeed Kolahian 2 pathogenic agents and normal flora, especially strains of an-
tibiotic multi-resistant bacteria, S. aureus and P. aeruginosa
with different and varying sensitivity patterns. Presence of
Department of Pathobiology, Faculty of Veterinary Medicine,
154 fungal agents including C. albicans and A. niger adds to this
University of Tabriz, Tabriz, Iran.1 coverage problem. The inflammation and healing process of
Department of Basic Sciences, Faculty of Veterinary Medicine, wounds is an additional issue. An ideal treatment may also
University of Tabriz, Tabriz, Iran.2 accelerate the healing process. Natural medicinal plant ex-
tracts have an ancient background in this aspect and mod-
INTRODUCTION ern medicine gains valuable benefits with them. The present
Valuable gains have been documented on the bactericidal, study shows the acceptable efficacy of chloroform, water
bacteriostatic, antifungal and immuno-modulatory proper- and ethanol extracts of medicinal plants Malva sylvestris and
ties of Malva sylvestris and Malva neglecta extracts. The pres- Malva neglecta on some bacterial and fungal contaminants
ent study investigates the efficacy of chloroform, ethanol of wound infections. Previous studies have got to valuable
and water extracts of these medicinal plants on some bacte- gains on the bactericidal, antifungal and immune-modulato-
rial and fungal contaminants of wound infections. ry properties of these plant extracts (1, 2, 3). Our results show
that all extracts are active against S. aureus, P. aeruginosa, P.
MATERIALS AND METHODS vulgaris which have been reported to be troublesome bacte-
Whole plants of wild Malva sylvestris and Malva neglecta ria in wound infections, especially in the aspect of antibiotic
were collected freshly from free pastures in mountain areas resistance as multi-resistant microorganisms. The ethanol
of East Azerbaijan, Iran. Dried ground powder from various extracts had the highest antibacterial activity than all other
parts of plants were extracted with water (aqueous extracts), solvents. The results suggest that essential oils as non-polar
ethanol, chloroform (Soxhlet extracts). All of the extracts organic compounds could be the main active compounds in
were filtered through 0.45 mm filters under sterile condi- these plants. Anthocyanin of Malva sylvestris has approved
tions. Different concentrations of these extracts were added bacteriostatic activity1. This water soluble pigment can be
to fresh cultures of test microorganisms in MHB broth media responsible for acceptable antibacterial effects of aqueous
and as previously described by NCCLS/ NCLI Documents4,5. extracts of both plants. aqueous extracts of Malva sylvestris
Test microorganisms included Staphylococcus aureus, have shown great antifungal activity against stored products
Pseudomonas aeruginosa, Streptococcus pyogenes, Proteus fungi including A. niger3. A. niger is also one of the most im-
vulgaris, Aspergillus niger, Aspergillus fumigatus and Can- portant fungal contaminants of wound infections. Efficacy of
dida albicans. Broth dilution assay was performed to evalu- all extracts especially aqueous extracts against A. niger, A. fu-
ate antimicrobial activity of the extracts according to NCCLS/ migatus and C. albicans can be a promising result of the pres-
NCLI guidelines4,5. After shaker incubation at 35°C for 24 and ent study. The polysaccharide ingredients of aqueous extract
48 h, test cultures as well as control media were evaluated of Malva sylvestris has approved immunomodulatory effects
spectrophotometrically at 570 nm3,5, and Minimal inhibitory in in vitro studies; it has shown to switch off IL-4 transcription

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

and to activate macrophages and T helper-1 lymphocytes2. the flowering stage from Shush (latitude 32°11’21’’ N, lon-
Ongoing studies of the authors of the present study on the gitude 48°15’28’’ E, Khuzestan Province, Iran) in September
in vivo application of a combination of extracts especially 2006.
aqueous and ethanol extracts on infectious and non infec-
tious wounds will be complementary to these results. Air-dried aerial parts were subjected to hydrodistillation us-
ing a Clevenger-type apparatus for 3 h to yield 0.08% v/w of
CONCLUSION yellowish oil. The obtained oil with a distinct odor was col-
Our results add more reasons to the clinical application of lected in pentane (1 mL), dried over anhydrous sodium sul-
these extracts in the prophylaxis and treatment of wound in- phate and was stored under N2 atmosphere in amber vials
fections. The wide range of efficacy on different bacterial and at 4°C until analysis. The oil was analyzed within 24 h from its
fungal agents and possible healing acceleration obtained by production.The identification of the chemical constituents
these extracts makes them acceptable candidates for the was based on comparisons of their retention times and mass
promotion of healing in wound infections. spectra with those obtained from authentic samples and/or
the NIST/NBS, Wiley libraries’ spectra as well with literature
REFERENCES data2.
1. Cheng Cui-lin and Wang Zhen-yu. Bacteriostatic activ-
ity of anthocyanin of Malva sylvestris J. of Forestry Res. Susceptibility tests
2006; 17(1):83-85. Teen strains of C. albicans (isolated from different patients)
2. Ghaoui WB, Ghanem EB, Chedid LA and Abdelnoor AM. were studied. Anti fungal activity of D. graveolens essential
The effects of Alcea rosea L., Malva sylvestris L. and Sal- oil was investigated by agar well diffusion method3.
via libanotica L. water extracts on the production of An-
ti-egg albumin antibodies, interleukin-4, gamma inter- RESULTS AND DISCUSSION
feron and interleukin-12 in BALB/c mice. Phytother Res. A total of twenty two components added up to 98.69% of
2008 Dec; 22(12):1599-604. the oil was identified. The main constituents were 1,8 Cineol
3. Magro A, Carolino M, Bastos M and Mexia L. Efficacy (54.89%), P-Cymen (16.2%), [beta]-pinene (6.94%) and Bor-
of plant extracts against stored products fungi. Rev neol (5.44%).
Iberoam micol 2006; 3:176-178.
4. NCCLS. 2002. Reference method for broth dilution an- The oil components can be classified into monoterpene hy-
tifungal susceptibility testing of filamentous fungi. Ap- drocarbons (28%), oxygenated monoterpenes (66.70%) and
proved standard M38-A. NCCLS, Wayne, Pa. sesquiterpene hydrocarbons (3.99%).
5. NCCLS. 2003. Methods for dilution antimicrobial sus- 155
ceptibility testing of bacteria that grow aerobically; ap- In the present study the anti Candida activities of the essen-
proved standard – sixth edition. Document M7-A6. NC- tial oil of D. graveolens were evaluated against teen isolates
CLS. Wayne PA. of C. albicans by agar well diffusion method. Several concen-
trations of the essential oil of D. graveolens were applied on
isolates. The results showed remarkable activities against
p066-Volatile constituents and anti tested isolates. As shown the lowest MIC for teen isolates of
candida activity of the aerial parts C. albicans was 28.84 mg/ml. Totally the MIC for 10 isolates of
C. albicans was 30.675mg/ml. In the present study the MIC of
essential oil of Dittrichia graveolens hyphal forms of C. albicans was 15-16 mg/ml.
(L.) Greuter grown in Iran
Numerous essential oils have been tested for in vivo and in
Nasrin Aghel1, Ali Zarei Mahmoudabadi1 vitro antimycotic activity and some demonstrated to be po-
tential antifungal agents. Their mechanism of action appears
Joundishapour Medical Sciences University, Ahvaz, Iran1 to be predominantly on the fungal cell membrane, disrupt-
ing its structure causing leakage and cell death; blocking the
INTRODUCTION membrane synthesis; inhibition of the spore germination,
Because of the antimicrobial properties showed by essential fungal proliferation and cellular respiration1. Because of high
oils, the aromatherapy has been used for treatment of seri- volatility and lipophilicity of the essential oils, they are read-
ous skin diseases, in special, superficial mycoses1. ily attached to penetrate into the cell membrane to exert
their biological effect4.
The aim of present study was to identify the chemical com-
positions of the aerial parts essential oil of Dittrichia gravol-
ens grown in Khuzestan, south-west of Iran. Also, in order to REFERENCES
estimate the possibility of using it as an antifungal agent for 1. Harris R. Progress with superficial mycoses using essen-
skin diseases (cutaneous candidiasis and dermatomycosis), tial oils. International Journal of Aromatherapy, 2002; 12:
the antifungal activity of this oil against Candida albicans 83-91.
was studied. 2. Adams RP. Identification of Essential Oil Components by
Gas Chromatography/Quadropole Mass Spectroscopy.
Plant Material 3rd ed. Illinois: Allured Publlishing Corporation; 2001.
Aerial parts of Dittrichia graveolens were collected during 3. Shahidi Bonjar GH, Aghighi S, Karimi Nik A. Antibacte-

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

rial and Anti fungal Survey in Plants used in Indigenous MATERIALS AND METHODS
Herbal-Medicine of South East Regions of Iran. J Biol Sci. In vitro HDAC Inhibition Activity Screening
2005; 4: 405-412 Among pan-HDAC enzymes, HDAC8 was the first human
4. Inouye S. Laboratory evaluation of gaseous essential oils HDAC whose three-dimensional and X-ray crystal structure
(part 1). International Journal of Aromatherapy, 2003; was described. Therefore pan-HDAC and HDAC8 inhibition
13: 95-107. screening studies are important to develop new inhibitor
candidates.

P067-HISTON DEACETYLASE INHIBITION Pan-HDAC and HDAC8 inhibition activity screening of the
ACTIVITIES OF ROSMARINIC ACID AND compounds were determined in vitro by using a fluoromet-
ric HDAC inhibitor drug screening kit (BioVision/Biomol) ac-
QUERCETIN cording to the manufacturer’s protocol. Briefly, HeLa nuclear
extract/HDAC8 enzyme and a HDAC fluorometric substrate,
Deniz Ulutürk1, G. Bora Tatar2, D. Dayangaç Erden2, A. S. which comprises an acetylated lysine side chain, were incu-
Demir3, S. Dalkara4, H. Erdem Yurter2 bated with/without one of the compounds at 50 and 500 µM
concentrations for 37°C at 30 min. Deacetylation sensitized
Hacettepe University1 the substrate and treatment the with lysine developer (37°C
Department of Medical Biology, Faculty of Medicine, Hacettepe at 30 min) produced the fluorophore. Fluorescence was
University, Ankara, Turkey2 measured with fluorescence plate reader (Molecular Devices
Department of Chemistry, Faculty of Arts and Science, Middle Spectramax M2) at excitation 350 nm and emmision 440 nm.
East Technical University, Ankara, Turkey3 TSA was used as negative control according to the protocol
Department of Pharmaceutical Chemistry, Faculty of and NaBA was used as reference compound in all tests as
Pharmacy, Hacettepe University, Ankara, Turkey4 well. Compounds were screened in triplicate.

INTRODUCTION Data Analysis


The acetylation and deacetylation of histones have been shown HDAC inhibition activity was analyzed according to the con-
to play an important role in transcription regulation and are centration against arbitrary fluorescence unit (AFU). HDAC
regulated by histone deacetylases (HDACs) and histone acetyl- inhibition activity was assessed according to the decrease in
transferases (HATs). HATs add acetyl groups to lysine residues fluorescence signal. Decreasing fluorescence signal showed
of amino terminal of histone proteins, whereas HDACs remove HDAC activity.
156 the acetyl groups. HDACs can act as transcription repressors
due to histone deacetylation that causes chromatin condensa- RESULTS AND DISCUSSION
tion. HDACs have emerged as attractive drug targets and sev- In both pan-HDAC and HDAC8 assays, HDAC inhibition ac-
eral HDAC inhibitors have been developed as drug candidates. tivities of rosmarinic acid and quercetin were analyzed (Figs
According to the accumulated preclinical data, HDAC inhibitors 1. and 2). We found that both of them have more potent
may be used for the treatment of numerous neurodegenerative HDAC inhibition activities than NaBA which is a well-known
disorders such as spinal muscular atrophy1,2. carboxylate inhibitor.

During the past 15 years, a number of structurally diverse HDAC


inhibitors have been identified including carboxylic acids3. In
our previous studies we found that cinnamic acid-like pheno-
lic compounds (caffeic acid derivatives) are HDAC inhibitors
with at least equal or better activity compared to conventional
carboxylates such as sodium butyrate (NaBA) and valproic acid
(VPA). Based on these data, two of the well-known natural caf-
feic acid derivatives, chlorogenic acid and curcumin showed
high activities5. These results prompted us to investigate HDAC
inhibitory activity of rosmarinic acid and quercetin, which are
dimer and cyclic ester analogs of caffeic acid, respectively:

Figure 1: Inhibition of pan-HDAC activity by rosmarinic acid


and quercetin

Formulas

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

P068-IDENTIFICATION OF
PHARMACOPHORES IN ANTIOXIDATIVE UPF
PEPTIDES.

Kalle Kilk1, Riina Mahlapuu1, Säde Viirlaid2, Jaak Järv2, Ursel


Soomets1

Institute of Biochemistry, Medical Faculty, University of Tartu,


The Centre of Excellence for Translational Medicine, 19 Ravila
St, 50411 Tartu, Estonia1
Institute of Chemistry, University of Tartu, 2 Jakobi St, 51014
Tartu, Estonia2

Figure 2: Inhibition of HDAC8 activity by rosmarinic acid and INTRODUCTION


quercetin Oxidative stress and its control by antioxidants is an issue
related to many pathological conditions. Peptides UPF1 and
CONCLUSION UPF17 are analogues of one of the most important natural
Rosmarinic acid and quercetin showed a higher HDAC inhi- antioxidant glutathione (GSH). In addition to glycyl, cysteinyl
bition activity than NaBA. This result is in accordance with and glutamyl residues they contain an extra N-terminal O-
our previous data and support our proposal that caffeic acid methyl-tyrosine (Fig 1). These peptides have demonstrated
is a promising lead structure as HDAC inhibitor (4,5). There- excellent free radical scavenging properties exceeding GSH1.
fore, cell culture studies of these two caffeic acid derivatives We have aimed to determine the main pharmacophores in
is considered for future to conclude that these compounds these novel antioxidants.
may be potential candidate molecules for treatment of can-
cer, neurodegenerative, hereditary and inflammatory dis-
eases.

ACKNOWLEDGEMENT
This project was supported by The Scientific and Technologi-
cal Council of Turkey (TÜBİTAK, Project number: 105G014) 157

REFERENCES
Figure 1: Structures of UPF1 (A) and UPF17 (B)
1. Hahnen E, Hauke J, Tränkle C, Eyüpoglu IY, Wirth B,
Blümcke I. Histone deacetylase inhibitors: possible im-
MATERIALS AND METHODS
plications for neurodegenerative disorders, Expert Opin
Liquid chromatography – mass spectrometry
Investig Drugs. 2008; 17(2): 169-84.
For peptide oxidation studies 10µM CuCl2 in 200 µl PBS and
2. Balasubramanian S, Verner E, Buggy JJ. Isoform-specific
10mM H2O2 were mixed in order to generate OH• in a Fen-
histone deacetylase inhibitors: the next step?, Cancer
ton like reaction. Peptide was added to final concentration
Lett. 2009; 8;280(2): 211-21.
100µM.
3. Miller TA, Witter DJ, Belvedere, S. Histon Deacetylase In-
hibitors, J Med Chem. 2003; 46 (24): 5097-5116.
LC-MS was carried out on a Shimadzu Prominence - Applied
4. Bora-Tatar G, Dayangaç-Erden D, Demir A.S, Dalkara S,
Biosystems QTrap 3200 tandem system. C18 column was
Yelekçi K, , Erdem-Yurter H. Molecular modifications on
used to separate the buffer salts, peptides and oxidation
carboxylic acid derivatives as potent histone deacety-
products from each other. Flow rate was 0.2 ml/min. Step-
lase inhibitors: Activity and docking studies. Bioorg Med
wise gradient started with 10 min isocratic flow of 99.9%
Chem. 2009 Jul 15;17(14):5219-28.
H2O 0.01% HCOOH, after which acetonitrile concentration
5. Dayangaç-Erden D, Bora G, Ayhan P, Kocaefe C, Dalkara
rose to 50% within 10 min. Finally, 5 min elution at 50% ac-
S, Yelekçi K, Demir AS, Erdem-Yurter H. Histone deacety-
etonitrile was done. Mass spectrometry detection range was
lase inhibition activity and molecular docking of (e)-
50-1500 Da. Ions were generated in negative mode, -4500
resveratrol: its therapeutic potential in spinal muscular
volts, 150 ºC.
atrophy, Chem Biol Drug Des. 2009; 73(3): 355-64.
Determination of pKa
The principe of thiol and thiolate ratio measurement was
their different absorbance at 240nm. 1 ml 50 μM solution of
peptide in PBS was titrated with 10 or 5 μl volumes of 1 M
NaOH. pH and absorbance changes were determined after
each addition. Absorbance was recorded on PerkinElmer
Lambda 25 spectrometer.

October 26 - 28, 2009 / Antalya - TURKEY


POSTER PRESENTATIONS

Kinetic measurements Figure 3: Kinetic differences in DPPH radical scavenging by


Scavenging of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) alpha and gamma glutamyl containing peptides.
was monitored spectrophotometrically. Briefly, 0.25 ml of
peptide solution in citric acid–sodium citrate buffer (20 mM, The linker between the thiol and aromatic ring, glutamyl
pH 3.9) was added to 0.25 ml of DPPH solution in 95% etha- residue, was found to have an impact as well. The glutamyl
nol. Changes in the absorbance at 517 nm, occurring due to residue can be attached to cysteine either via its alpha or
scavenging the free radical, were monitored in thermostated gamma carboxylic group. Compounds with alpha glutamyl
quartz cuvette at 25 °C (UV–VIS spectrophotometer Unicam residue showed significatly faster reaction rates compared
UV300, ThermoSpectronic, USA). Data analysis was done by to respective gamma-glutamyl analogues (Fig. 3). Thus, ei-
Graphpad Prism 4.0 software. ther the length of this linker or position of the free carbocylic
group is a determinant radical scavenging kinetics.
RESULTS AND DISCUSSION
By liquid chromatography – mass spectrometry we deter- CONCLUSION
mined that under used conditions aromaticity containing In summary, we have identified the major structural features
UPF peptides oxidize further than non-aromatic controls that determine the biological activity of antioxidative UPF
(Fig. 2). Oxidation products beyond expected disulphide peptides (Fig. 4).
were identified as sulfinic (-SO2H) and sulfonic (-SO3H) acids.
No alteration in the aromatic ring was found and analogues
without the thiol group remained unreactive. Thus, the cen-
tre of the oxidative defense is the thiol group.

158
Figure 4: Main pharmacophores of UPF peptides.

ACKNOWLEDGEMENT:
This study was financed by Estonian Science Foundation
grants 6503, 7494, 7856, Estonian Ministry of Education and
Research (Grant SF0180064s08), and by the European Re-
Figure 2: Mass spectra of oxidation of (A) GSH and (B) UPF17 gional Development Fund.
by OH• at the beginning of the reaction and in 5 h.
REFERENCES
By titration assays it was found that additional N-terminal 1. Ehrlich K, Viirlaid S, Mahlapuu R, Saar K, Kullisaar T,
amino acids do not lower the pKa of the thiol, but rather Zilmer M, Langel U, Soomets U. Design, synthesis and
increase it slightly (8.97±0.3 vs 9.22±0.1). Hence the reason properties of novel powerful antioxidants, glutathione
why aromatic UPF peptides oxidize more readily than non- analogues. Free Radic Res. 2007 Jul;41(7):779-87
aromatic analogues must lie in pi-electron cloud – free radi-
cal interaction. This interaction could prolong the time a free
radical spends near the thiol and thereby “catalyze” the reac- p069-Wfs1 KO AND WILD TYPE MICE DIFFER
tion. IN THEIR STRIATAL DOPAMINE OUTPUT
IN RESPONSE TO K+ BUT NOT MORPHINE
CHALLANGE

Vallo Matto1, Anton Terasmaa2, Sulev Kõks2

Department of Pharmacy, University of Tartu, Estonia1


Department of Physiology and Centre of Excellence for
Translational Medicine, University of Tartu , Estonia 2

INTRODUCTION
Wolfram syndrome is a very rare inherited disorder caused
by the loss-of-function mutations of in the Wfs1 gene and

3rd BBBB International Conference on Pharmaceutical Sciences


POSTER PRESENTATIONS

characterized by a complex of endocrinological symptoms RESULTS AND DISCUSSION


and/or neuropsychological problems. In mutant mice mod- The concentrations of DA of the baseline samples were 0.8
els with Wfs1 gene knocked out (Wfs1 KO) evidence has been nM to 1.4 nm for the wild type animals and 0.6 to 1.3 nM
collected that the wfs1 protein is an important link between for the Wfs1 KO mice. Repeated measures ANOVA revealed
the endocrinological and CNS systems. The Wfs1 KO mice a significant time (F(1,11)= 18.9, p<0.001) and KO animal
are physically retarded, they have serious carbohydrate me- (F(1,11)= 14.6, p<0.01) effect, as well as an interaction be-
tabolism dysregulation, and they are also emotionally devi- tween these factors (F(1,11)= 10.6, p<0.01). 10 mg/kg mor-
ated from wild type conspecifics1. Dopamine (DA) is a major phine injection increased the striatal DA output in all tested
neuromediator involved in the mediation of the emotional, wild type animals (up to 160% from baseline) while in the
motivational, and locomotor components of behavior. The KO animals only a minor increase (up to 105% from baseline,
present set of experiments was intended to explore the pos- with a large in-group variation) was found. Time-point-by-
sible differences in extracellular DA concentrations in wild time-point post hoc analysis (20 – 120 min) considered this
type and Wfs1 KO mice in response to acute morphine treat- difference as statistically insignificant. Expectedly, the appli-
ment and high concentration K+ challenge. cation of the [K+]-rich modified Ringer solution dramatically
increased the striatal DA output in wild type animals (up to
MATERIALS AND METHODS 3000% for the last time point measured) but the increase of
Animals and microdialysis surgery the striatal DA output of Wfs1 KO mice was less pronounced
The mice (male 3–5 months old F2 hybrids) were anesthe- yielding only up to 450% from the baseline values (Figure
tized with Fentanyl-Fluanizone-Midazolam 30 min before 1). There was a statistically significant difference between
the surgery. The mice were attached from outer ear canals to these two mice groups (p<0.01 and p<0.001 for the time
the stereotaxic apparatus equipped with mouse nose hold- points 160 and 180 min, respectively). Our results show that
er. The coordinates for the guide cannulae (CMA/7, CMA/Mi- the regulation of the DA output in striatum of the Wfs1 KO
crodialysis) were aimed at the point above the striatum (AP = mice is impaired. This can be due to the impaired regulatory
+0.6 mm, L = +2.0 mm, DV = -2.3 mm, relative to bregma). pathways or alternatively, the Wfs1 KO mice have limited DA
intraneuronal storage capacity. Further studies are needed
Microdialysis experiments to assess the exact mechanisms of these findings and the
The microdialysis experiments were carried out using the possible causal relationship between the deviated behavior
technique described by Airavaara et al., 2, with some modi- and impaired striatal DA output of Wfs1 KO mice.
fications. A microdialysis probe was inserted into the guide
cannula, and the probe was infused with a modified Ringer
solution (147 mM NaCl, 1.2 mM CaCl2, 2.7 mM KCl, 1.0 mM 159
MgCl2, and 0.04 mM ascorbic acid) at the flow rate of 1.3 mi-
crol/min. The collection of the actual microdialysis samples
(every 20 min, 26 microl/sample) was started after two hours
of stabilization period. The DA concentrations were deter-
mined by an Agilent (HP 1100 Series) high performance liq-
uid chromatography (HPLC) system additionally equipped
with an electrochemical detector (Coulochem II, ESA), a
model 5011 analytical cell, an Agilent 35900E interface, and a
Rheodyne 7725i manual injector. The column (Zobirax SB-C-
18, 4×140 mm, 3.5 micron ODS) was kept at room tempera-
ture (20±1 °C). The mobile phase used (flow rate 1.0 ml/min)
consisted of 0.1 M NaH2PO4 buffer, pH 4.2 (adjusted with 1.0
mM citric acid), 0.6–0.8 mM octanesulphonic acid, 12–15%
methanol, and 1.2 mM EDTA.
Figure 1:Striatal DA output in wild type and Wfs1 KO mice.
20 microliters of dialysate per sample was injected into the
column. DA was reduced with an amperometric detector ACKNOWLEDGEMENTS
(-100 mV) after being oxidized with a coulometric detector This study was financed by a University of Tartu grant No
(+600 mV). The DA concentrations are presented as relative PARFA 08902.
to the baseline (± SEM, the average concentration of three
consecutive stable samples were defined as 100%). The ani- REFERENCES
mals received after one hour an i.p. injection of 10 mg/kg 1. Kõks S, Vasar E (Eds.), Wfs1 protein (Wolframin): Emerg-
morphine, one hour before the completion of the experi- ing link between the emotional brain and endocrine
ment the modified Ringer solution was replaced with the pancreas, Research Signpost, Kerala, India, 2008.
[K+]-rich modified Ringer solution (corresponds to 100 mM 2. Airavaara M, Mijatovic J, Vihavainen T, Piepponen TP,
[K+]). Saarma M, Ahtee L. In heterozygous GDNF knockout