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Colloids and Surfaces B: Biointerfaces 101 (2013) 414423

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Colloids and Surfaces B: Biointerfaces

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Development of solid self-nanoemulsifying granules (SSNEGs) of ondansetron hydrochloride with enhanced bioavailability potential
Sarwar Beg a,c , Sidharth Sankar Jena a , Ch Niranjan Patra a , Mohammad Rizwan b, , Suryakanta Swain a , J. Sruti a , M.E. Bhanoji Rao a , Bhupinder Singh c
a b c

Department of Pharmaceutics, Roland Institute of Pharmaceutical Sciences, Khodasingi, Berhampur, Orissa, India Formulation Research, Wockhardt Research Center, Aurangabad, Maharashtra, India University Institute of Pharmaceutical Sciences (UGC Centre of Advanced Studies), Panjab University, Chandigarh, India

a r t i c l e

i n f o

a b s t r a c t
The current work aims to prepare the solid self-nanoemulsifying granules (SSNEGs) of ondansetron hydrochloride (ONH) to enhance its oral bioavailability by improving its aqueous solubility and facilitating its absorption though lymphatic pathways. Preformulation studies including screening of excipients for solubility and pseudoternary phase diagrams suggested the suitability of Capmul MCM as lipid, Labrasol as surfactant, and Tween 20 as cosurfactant for preparation of self-emulsifying formulations. Preliminary composition of the SNEDDS formulations were selected from the phase diagrams and subjected to thermodynamic stability studies and dispersibility tests. The prepared liquid SNEDDS formulations were characterized for viscosity, refractive index, droplet size and zeta potential. The TEM study conrmed the formation of nanoemulsion following dilution of liquid SNEDDS. The optimized liquid SNEDDS were transformed into free owing granules by adsorption on the porous carriers like Sylysia (350, 550, and 730) and NeusilinTM US2. Solid state characterization employing the FTIR, DSC and powder XRD studies indicated lack of any signicant interaction of drug with the lipidic and emulsifying excipients, and porous carriers. In vitro drug release studies indicated faster solubilization of the drug by optimized SSNEGs (over 80% within 30 min) vis--vis the pure drug (only 35% within 30 min). In vivo pharmacokinetic studies in Wistar rats observed signicant increase in Cmax (3.01-fold) and AUC (5.34-fold) using SSNEGs compared to pure drug, whereas no signicant difference (p > 0.1) was observed with the liquid SNEDDS. Thus, the present studies ratify the bioavailability enhancement potential of SSNEGs of ONH prepared using porous carriers. 2012 Elsevier B.V. All rights reserved.

Article history: Received 20 April 2012 Received in revised form 22 June 2012 Accepted 25 June 2012 Available online xxx Keywords: SNEDDS Hepatic rst-pass effect Porous carriers Self-nanoemulsifying Pharmacokinetics

1. Introduction Poor aqueous solubility of new drug entities is today considered as a formidable challenge for pharmaceutical scientist, which is now considered as an area of prime importance in the eld of biomedical research. Approximately one-half of the new molecular entities (NMEs) synthesized in pharmaceutical R&Ds employing with advanced techniques like combinatorial chemistry and computer-aided drug design (CADD) suffer from poor solubility and bioavailability [1,2]. To overcome such problems, various formulation approaches have been undertaken to improve oral bioavailability including surfactants [3], cyclodextrin complexes [4], solid dispersions [5], micronization and nanosizing [6], permeation enhancers [7], supercritical technology [8], gastroretentive

Corresponding author. Tel.: +91 8087766453. E-mail address: rizwan wck@yahoo.co.in (M. Rizwan). 0927-7765/$ see front matter 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.06.031

systems [9], nanosuspensions [10], dendrimers [11], carbon nanotubes [12] and lately, lipid-based formulations [13]. Ondansetron hydrochloride (ONH) is a 5-HT3 receptor antagonist, primarily used as rst-line drug for the management of nausea and vomiting associated with cancer chemotherapy, chronic medical illness, gastroenteritis and post-operative states [14]. The drug is poorly water soluble and possesses intermediate value of log P (i.e., 2.4) [15]. Ondansetron exhibits low (i.e., 45%) and inconsistent bioavailability, potentially due to high hepatic rst-pass metabolism and high P-gp efux, besides the inadequate aqueous solubility [16,17]. The myriad drug delivery systems of ONH developed so far have yielded limited fruition in oral bioavailability enhancement [18,19]. Self-nanoemulsifying drug delivery systems (SNEDDS) have recently gained wide acceptance due to robust formulation perspectives, practical enhancement of solubility and of oral bioavailability of drugs through lymphatic pathways [20]. These are pre-concentrates containing isotropic mixture of oils, surfactants and cosurfactants. They facilitate the dissolution of various

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poorly water-soluble drug candidates by in situ solubilization in lipidic components [2022]. They spontaneously emulsify when exposed to aqueous media or gastrointestinal (GI) uids to form a stable o/w emulsion with nanometric droplet size ranging between 20 and 200 nm. Further, they also provide distinct improvement in bioavailability for lipophilic drugs exhibiting dissolution rate limited absorption and poor permeation [23]. The conventional liquid SNEDDS, however, have limited acceptance owing to their major drawbacks, like precipitation of drug due to potential interaction of volatile cosolvents with soft gelatin capsule shells and GI irritation due to high level of surfactant in the formulations [19]. To overcome such problems, the solid SNEDDS are developed as the technological innovations, which incorporate liquid or semisolid ingredients into powders employing diverse solidication techniques like spray drying [24], melt granulation [25], extrusionspheronization [26], eutectic mixing and nanoparticle technology [27]. The solid SNEDDS are relatively more robust formulations with high stability, improved patient compliance and simple manufacturing [20,24,26]. Such solid SNEDDS can further be formulated into free owing powders, granules, pellets, tablets, solid dispersions, microspheres and nanoparticles [2830]. Besides, limited volume of literature is also available describing the use of porous carriers like cross-linked porous silicon dioxide (SylysiaTM 320, 350, 550, 750), magnesium aluminum silicate (NeusilinTM US2) and microporous calcium silicate (FloriteTM RE) for adsorption of liquid self-emulsifying formulations and transforming them into solid SNEDDS [3133]. Attempts, therefore, were made to prepare the SSNEGs of poorly water soluble drug, ONH, using porous carriers like SylysiaTM (350, 550, and 730) and NeusilinTM US2 to enhance its aqueous solubility, and oral bioavailability by possible avoidance of hepatic rst-pass effect, inhibition of P-gp efux and enhanced absorption through lymphatic pathways. 2. Materials and methods 2.1. Materials ONH was generously gifted by M/s Aurobindo Pharma, Hyderabad, India. Labrasol, Labrafac PG and Labral M were gifted by M/s Gattefosse, Saint-Priest Cedex, France. Captex 200P, Captex 355 and Capmul MCM were provided by M/s Abitec, Janesville, WI, USA. Cremophor RH40 was obtained from M/s BASF, Ludwigshafen, Germany. Different grades of Sylysia (350, 550 and 730), and NeusilinTM US2 were generously provided by M/s Fuji chemicals, Toyama, Japan, and by M/s Gangwal Chemicals, Mumbai, India, respectively. Deionized water was used for study obtained from Milli-Q-water purication system M/s Millipore, Massachusetts, USA. All other reagents and solvents used were of analytical reagent grade. 2.2. Methods 2.2.1. Solubility studies Solubility of ONH was determined in various excipients viz. natural oils (coconut oil, palmoline oil, olive oil, sesame oil, arachis oil, castor oil, and neem oil), medium chain triglycerides (Captex 200, Captex 355 and Capmul MCM), surfactants (Labrasol, Labral, Labrafac and Brij 35) and cosurfactants (Polypropylene glycol, Tween 20, Tween 80, PEG 200, PEG 400 and PEG 600). Excess amount of ONH was added to 2 mL of each excipient in sealed vials and vortex-mixed (Vortex mixer, Remi, Mumbai, India). The sealed vials were stirred in water bath (Julabo SW23, Allentown, PH, USA) at 37 0.5 C for 72 h to attain equilibrium. All samples were centrifuged at 3000 rpm (402.48 g) for 15 min using

laboratory centrifuge (Remi, Mumbai, India). The supernatant was ltered through 0.45 m membrane lter (Millipore, Mumbai, India), suitably diluted with methanol, and analyzed spectrophotometrically at a max of 310 nm using a double beam UV-VIS spectrophotometer (Shimadzu-1800, Japan). 2.2.2. Pseudo-ternary phase diagrams 2.2.2.1. Selection of surfactants and cosurfactants (Smix ) ratios. The excipients, viz. oil, surfactant and cosurfactant, selected from the solubility studies were used to construct the pseudo-ternary phase diagrams employing water titration method. The pseudo-ternary phase diagrams were prepared to identify the Smix ratios, where maximum nanoemulsion region forms. Various Smix ratios were prepared using different proportions of surfactant and cosurfactant to fulll HLB value requirement (1216) which favors nanoemulsion formation. 2.2.2.2. Construction of pseudo-ternary phase diagrams. Different ratios of oil to surfactant/cosurfactant mixture (Smix ) were selected, ranging between 1:9 and 9:1, to delineate the boundaries of nanoemulsion region. The homogenous mixture of oil and Smix was subjected to aqueous titration with the addition of 5 mL of water in each step, and was visually observed. The amount of water at which transparency-to-turbidity transition occurs was derived from the weight measurements. The generated sample, which was clear or slightly bluish in appearance, was taken as the nanoemulsion. To determine boundaries of nanoemulsion, the values of oil and Smix ratio were calculated from the weight measurements. Pseudoternary phase diagrams were constructed using PROSIM software (STRATEGE, Cedex, France). 2.2.3. Formulation of liquid SNEDDS Different liquid SNEDDS formulations were prepared by selecting the concentration of oil and Smix from pseudoternary phase diagrams, as presented in Table 1. The SNEDDS were prepared by simple admixture of drug with oil with mixtures of surfactant/cosurfactant using vortex mixer at an ambient temperature. 2.2.4. Characterization of liquid SNEDDS 2.2.4.1. Dispersibility test. One mL of liquid SNEDDS from each formulation was taken, and mixed with 500 mL of distilled water at 37 C with constant stirring at 50 rpm. The SNEDDS were observed for the formation of stable nanoemulsions. The nanoemulsions formed were visually observed for phase clarity, self-emulsication time (SEF time) and rate of emulsication. 2.2.4.2. Thermodynamic stability studies. The liquid SNEDDS formulations were subjected to heating-cooling test using six refrigerator cycles at 45 C and 4 C temperatures separately for 48 h in an incubator (Remi, Mumbai, India). Formulations, which were found to stable during heating cooling cycles, were further centrifuged at 3500 rpm (402.48 x g) for 30 min. The stable liquid SNEDDS were subjected again to freeze-thaw cycles (n = 3) at 21 C and 25 C, respectively, and stirred continuously. 2.2.4.3. Viscosity. Viscosity of the selected liquid SNEDDS were determined by placing 1 mL of undiluted formulation in the viscometer (R/S CPS Plus, Brookeld Engineering Lab. Inc., Middleboro, MA) using spindle #C 50-1 at 25 0.5 C. The spindle (50 mm diameter) was operated at a speed 70 rpm with shear stress of 413 rpm, keeping the wait time as 15 min. Finally, the developed shear rate was noted in terms of centipoise (cps).

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416 Table 1 Composition of the liquid SNEDDS formulations. Components (% w/w) ONH Capmul MCM Labrasol Tween 20 PEG 600 Smix ratio F1 1 20 39.5 39.5 1:1 F2 1 25 37 37 1:1 F3 1 27 36 36 1:1 F4 1 30 34.5 34.5 1:1 F5 1 20 26.33 52.66 1:2 F6 1 25 24.66 49.33 1:2 F7 1 27 24 48 1:2 F8 1 30 23 46 1:2 F9 1 20 31.6 47.4 2:3 F10 1 25 29.6 44.4 2:3 F11 1 27 28.8 43.2 2:3 F12 1 30 27.6 41.4 2:3 S. Beg et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 414423

2.2.4.4. Refractive index. Refractive index was determined using Abbes refractrometer (Nirmal International, Mumbai, India) by placing a drop of undiluted liquid SNEDDS. 2.2.4.5. Droplet size analysis and zeta potential. The liquid SNEDDS were diluted with deionized water at 25 C under gentle agitation. The droplet size distribution and zeta potential were determined using dynamic light scattering principle (Malvern Zetasizer, Nano ZS-90, Worcestershire, UK). The values of mean droplet size and zeta potential were recorded. 2.2.4.6. Transmission electron microscopy (TEM). The TEM analysis of liquid SNEDDS was performed for morphological characterization and visualization of emulsion droplets. The liquid SNEDDS formulation was diluted with deionized water (1:25) and mixed by gentle shaking. A drop of sample obtained after dilution was placed on copper grids, stained with 1% phosphotungstic acid solution for 30 s, and nally kept under electron microscope (Philips Tecnai 12, Eindhoven, Netherlands) to visualize the particle morphology. 2.2.5. Formulation of SSNEGs The optimized liquid SNEDDS was transformed into free owing granules using various colloidal porous carriers as adsorbents like Sylysia (350, 550, 730) and NeusilinTM US2. The liquid SNEDDS formulation was poured onto porous carriers placed in a small china bowl, mixed well for 5 min to obtain a homogeneous mass. The mass was subsequently passed through a sieve (#BSS 22) and lled into the size 0 hard gelatin capsules (Capsugel, Mumbai, India). 2.2.6. Optimization and characterization of SSNEGs The SSNEGs prepared using different porous carriers were optimized based on their oil adsorption capacity, micromeritic properties, in vitro drug release and retention of self-emulsication property. Oil adsorption capacity was determined by slow addition of liquid SNEDDS formulations to adsorb onto the porous carriers until these solidied, and by estimating their drug content. Important micromeritic properties of granules like bulk density, tap density, angle of repose, Carrs index and Hausners ratio were determined using standard procedures. In vitro drug release from SSNEGs was determined by withdrawing periodic aliquots of samples during dissolution and analyzing the same spectrophotometrically at 310 nm. The samples were also observed under TEM for formation of nanoemulsion, if any. 2.2.7. Comparative in vitro drug release studies In vitro drug release studies were nally carried out for optimized SSNEGs and optimized liquid SNEDDS vis--vis marketed product (Zofran ODT, GlaxoSmithKline, India) and pure drug, each containing 8 mg of ONH. The studies were conducted out in 900 mL of simulated gastric uid (SGF) at 75 rpm and 37 0.5 C using USP II apparatus (Electrolab, Mumbai, India) [34]. At predetermined time intervals (5, 10, 15, 30, 45, 60, 90 and 120 min), aliquots of samples (5 mL) were collected and ltered through 0.45 m membrane lter, suitably diluted and analyzed spectrophotometrically at 310 nm. The in vitro dissolution data were analyzed using MSExcel spreadsheet and cumulative percentage drug release was

plotted against time (h). Further, the values of dissolution efciency at 15 min (DE15 min ) and mean dissolution time (MDT) were also determined for the test formulations using standard algorithms [23]. 2.2.8. Drug content estimation The liquid SNEDDS and SSNEGs containing ONH, each containing 8 mg of ONH, were suitably dispersed in 100 mL of SGF in a volumetric ask. The samples were mixed gently, sonicated (Remi, Mumbai, India) to extract ONH completely, and centrifuged at 3000 rpm (402.48 g) for 15 min to separate undissolved excipients. The supernatant was ltered through 0.45 m membrane lter (Millipore, Mumbai, India), suitably diluted and analyzed using UV-VIS spectrophotometer (Shimadzu 160A, Japan) at a max of 310 nm. The experimental studies were performed in triplicate. 2.2.9. In vivo pharmacokinetic studies The experiments were conducted as per the Committee for prevention, control and supervision of experimental animals (CPCSEA) guidelines. Wistar rats (200250 gm) of either sex were kept under standard laboratory conditions at 25 2 C temperature and 55 5% RH. The animals were housed in polypropylene cages, six per cage with free access to standard diet (Lipton feed, Mumbai, India) and water ad libitum. The pharmacokinetic studies were carried out in accordance with protocol approved by the Institutional Ethics Committee at Roland Institute of Pharmaceutical Sciences, Berhampur, Odisha, with protocol number RIPS/IAEC/46/2011. The rats were divided into ve groups with six animals in each group. Control group received distilled water, and other treatment groups received suspension of pure drug, marketed preparation (Zofran ODT), optimized liquid SNEDDS and SSNEGs, each containing 8 mg of ONH. Rats were fasted overnight before oral administration of different formulations using a 5 mL oral feeding needle attached with a cannula. Oral dose for rat was calculated after taking into consideration of surface area ratio of rat to that of human [35,36]. After administration of different test formulations, rats were anaesthetized using diethyl ether. Blood samples (0.5 mL each) were withdrawn from the tail vein in microcentrifuge tubes containing heparin as an anticoagulant. Blood samples collected were mixed with anticoagulant and centrifuged at 5000 rpm (1118 g) for 20 min. The supernatant plasma samples obtained were mixed with 0.5 mL of ethyl acetate and allowed to evaporate. The dried samples were reconstituted with chloroform:methanol mixture (9:1%, v/v) and stored frozen at 21 C in eppendorff tubes until analyzed. ONH was analyzed in plasma employing a modied RP-HPLC method [37]. The mobile phase was 0.7 M sodium perchlorate-acetonitrile (50:50, %v/v) and the ow-rate was 0.5 mL/min. The chromatographic separation was achieved using Capacel Pak C18 column (Shiseido, Tokyo, Japan) with 250 4.6 mm i.d. and 5 m particle size, coupled with UV-Vis detector at 210 nm, on a binary pump RPHPLC instrument (Shimadzu, Tokyo, Japan). The extracted plasma samples were injected by means of a Rheodyne injector tted with a 20 L loop and data acquisition was controlled by Shimadzu Class-VP 5.032 software. Pharmacokinetic parameters like Cmax , tmax , t1/2 , Ka , AUC0t , AUMC0t and MRT0t were determined from plasma concentrationtime prole for different test formulations.

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The pharmacokinetic proles of test formulations (liquid SNEDDS and SSNEGs) were compared with those of pure drug suspension and Marketed brand (Zofran ODT). Statistical analysis was carried out by paired t-test to determine the signicant difference between the test groups for any change in pharmacokinetic parameters. The relative bioavailability was calculated using Eq. (1): %F = AUC(test) Dose(std) AUC(std) Dose(test) (1)

3. Results 3.1. Solubility studies Amongst the various oils, surfactants and cosurfactants investigated for equilibrium solubility studies viz. Capmul MCM, Captex 200, Captex 355, olive oil, arachis oil, sesame oil, Labrasol, Labral, Labrafac, Tween 20, Tween 80, Polypropylene glycol, PEG 200, PEG 400 and PEG 600, the highest solubility of ONH was observed in Capmul MCM (32 3.2 mg/mL), Labrasol (31.1 1.8 mg/mL), PEG 600 (35.3 2.3 mg/mL) and Tween 20 (28.5 2.0 mg/mL) (data not shown). Hence, they were selected for construction of pseudoternary phase diagrams and further studies. 3.2. Pseudo-ternary phase diagrams The pseudo-ternary phase diagrams of surfactant, cosurfactant and oil were plotted, each of them represents an apex of the triangle [38]. The ternary phase diagrams with Smix ratios of 1:1, 1:2 and 2:3 for Labrasol:Tween 20 showed the maximum region for nanoemulsion(s) using Capmul MCM as the oil phase (Fig. 1). 3.3. Selection of liquid SNEDDS The prototype liquid SNEDDS formulations were prepared using the Smix ratios which exhibited higher self-emulsication efciency in water. A total of twelve formulations were prepared using the selected Smix ratio (1:1, 1:2, 2:3 for Labrasol:Tween 20, and 2:3 for Labrasol:PEG 600) and Capmul MCM as the oil in the percentage range of 2030% (w/w), which could solubilize single dose (8 mg) of ONH. These formulations were subjected to dispersibility test and thermodynamic stability studies to select the stable liquid SNEDDS formulations. The liquid SNEDDS F1, F2, F3, prepared with Smix ratio 1:1 for Labrasol, Tween 20 and Capmul MCM passed the thermodynamic stability and dispersibility studies. However, other selected liquid SNEDDS failed to clear the thermodynamic stability tests. Dispersibility tests showed that dilution of liquid SNEDDS (F1F3) with water formed slightly bluish colored clear and transparent solution within a minute. In contrast, other selected liquid SNEDDS did not produce transparent clear solution on dilution; either it took more than a minute or yielded a milky emulsion. 3.4. Characterization of liquid SNEDDS Dispersibility test showed that all the liquid SNEDDS formulations form ne bluish white nanoemulsion in less than 1 min. Table 2 represents the physiochemical properties of the selected liquid SNEDDS formulations (F1F3), such as viscocity, refractive index, droplet size and zeta potential. The liquid SNEDDS showed viscosity in the range of 27 and 38 cps, while the refractive index was in the range of 1.39 and 1.48. Droplet size analysis using dynamic light scattering was found to range between 118 and 250 nm, which indicated that emulsion droplets are in nanometric range. Zeta potential was found to range between 16.3 and 45.8 mV, conrming emulsion droplets as stable and wellseparated [29]. The TEM image of the optimized liquid SNEDDS appeared as dark globules with bright surrounding (Fig. 2). In vitro drug release prole of liquid SNEDDS indicated complete drug release within 30 min for all the formulations. 3.5. Optimization and characterization of SSNEGs

2.2.10. Fourier transformed infrared (FTIR) spectroscopy FTIR spectroscopic studies were employed to characterize the possible interactions, if any, between the drug and excipients. The FTIR spectra of samples of pure drug, physical mixture of drug with self-emulsifying excipients and SSNEGs were recorded using KBr disc using an FT-IR spectrophotometer (Shimadzu, Japan).

2.2.11. Differential scanning calorimetry (DSC) DSC thermograms of pure drug, physical mixture (1:1) of drug with self-emulsifying excipients, porous carriers, optimized liquid SNEDDS and SSNEGs were carried out using Pyris-6-DSC Thermal analyzer (PerkinElmer, Tokyo, Japan). The liquid samples were sealed in the heat-resistant aluminium pans and lid was crimped on its surface by pressing under pellet press. The sample and reference pans were kept in the heating chamber, heated from 30 to 300 C with temperature rise at a rate of 10 C/min, and DSC spectra were recorded.

2.2.12. Powder XRD studies Powder XRD studies were carried out for solid state characterization including powder morphology and crystallographic structure of pure drug and SSNEGs. The diffraction pattern of ONH and SSNEGs were recorded by X-ray diffractometer (Philips PW 17291, Philips, The Netherlands) using Ni-ltered, Cu kV radiation, at a voltage of 40 kV and a 25 mA current. The scanning rate was kept between 10 and 40 angles with an increment of 1 min1 over 2 range.

2.2.13. Accelerated stability studies The optimized SSNEGs in size 0 capsules were sealed HDPE bottle and subjected to accelerated stability studies at 40 2 C/75% 5% RH upto three months. The SSNEGs capsules were evaluated for disintegration time, SEF time, droplet size after nanoemulsication, percentage drug release in 15 min (%DR15 min ), and MDT at specied time points (0, 1, 2 and 3 months). For estimation of shelf-life, the SSNEGs capsules in HDPE bottle were stored at 30 0.5 C, 40 0.5 C and 50 0.5 C temperature upto a period of three months. Samples were withdrawn after specied time intervals (0, 1, 2 and 3 months), concentration and log concentration of ONH remained was analyzed. Order of reaction in which drug degradation occur was estimated. The reaction rate constant (K) for the degradation was measured from slope of lines at each elevated temperature using Eq. (2), and an Arrhenius plot was constructed (i.e., plot of log K at various elevated temperatures against the reciprocal of absolute temperature). From the plot, K value at 25 C was determined and used for prediction of shelf-life by substituting in Eq. (3). K Slope = 2.303 t90 = 0.1052 K25 (2)

(3)

The optimized liquid SNEDDS (F1) was selected for the preparation of SSNEGs using different porous carriers. The composition of different SSNEGs prepared is presented in Table 3. The prepared SSNEGs were evaluated for oil absorption capacity, drug release

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Fig. 1. Pseudoternary phase diagram of systems containing 1:1, 1:2, 1:3, 2:3 Smix ratio Labrasol and Tween-20 using Capmul MCM as oil, and water as titrant.

Table 2 Characterization of the SNEDDS formulations. Formulation code F1 F2 F3 Mean viscosity SD (cps) After 120 s 27.5 1.01 32.4 0.99 38.0 0.91 Refractive Index SD 1.39 0.021 1.41 0.013 1.38 0.011 Zeta potential (mV) SD 45.8 0.02 23.5 0.11 16.3 0.13 Droplet size (nm) SD 118 0.12 224 0.24 250 0.30

Fig. 2. TEM images of the reconstituted nanoemulsions from (A) the optimized liquid SNEDDS F1 and (b) the optimized SSNEGs G1.

Table 3 Formulation composition of SSNEGs. Component SNEDDS (ml) Sylysia 350 (mg) Sylysia 550 (mg) Sylysia 730 (mg) Neusilin US2 (mg) G1 0.8 284 G2 0.8 488 912 260 100 G3 0.8 G4 0.8 G5 0.8 150

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Fig. 3. Comparative in vitro drug release prole of pure drug, conventional tablet (Zofran ODT), optimized liquid SNEDDS and SSNEGs formulation containing Sylysia 350. Data represented are cumulative %drug release versus time (min) in terms of mean SD (n = 3).

Fig. 4. Plasma concentrationtime prole of pure drug, conventional tablet (Zofran ODT), optimized liquid SNEDDS and SSNEGs containing 8 mg of ONH after oral administration in Wistar rats.

and micromeritic properties. All the batches of SSNEGs showed good ow characteristics with Carrs index range between 14 and 17%, Hausners ratio less than 1.25 and angle of repose ( ) < 25. The SSNEGs containing Sylysia 350 and Neusilin US2 (G1 and G5) showed the highest oil absorption tendency due to the highly porous nature and signicant voidage in the particles [39,40]. It has been observed that oil absorption capacity decreased with higher grades of Sylysia. The Sylysia 350 granules (G1) showed the highest oil absorption property due to high porosity and small particle size compared to the granules containing Sylysia 550 (G2) and Sylysia 730 (G3). The larger particle size of Sylysia shows lower porosity leading to lower oil absorption capacity [41]. The in vitro drug release studies revealed that all the SSNEGs formulations observed better drug release behavior with 50% drug release in 15 min and more than 80% drug release within 30 min. Among all the formulations, the formulation G1 and G5 containing Sylysia 350 and Neusilin US2 granules showed faster dissolution with drug release upto 91% in 30 min, ostensibly due to highest oil absorption capacity and faster solubilization in dissolution medium. 3.6. Comparative in vitro drug release studies The comparative dissolution proles of optimized SSNEGs formulation (G1) and optimized liquid SNEDDS (F1), marketed preparation (Zofran ODT) and pure drug as carried out in SGF (pH 1.2) are presented in Fig. 3. The dissolution prole shows that the optimized liquid SNEDDS and SSNEGs exhibited faster drug release (98.9% and 99.8% within 15 min) vis--vis pure drug and marketed preparation with maximum drug release in 30 min as 41% and 45.7%, respectively. No statistically signicant difference was observed between the drug release by SSNEGs with liquid SNEDDS (p > 0.1). The optimized SSNEGs (DE15 min = 73.6%) and optimized liquid SNEDDS (DE15 min = 81.4%) showed better dissolution performance vis--vis marketed preparation (DE15 min = 34.5%) and pure drug (DE15 min = 28.2%), owing to faster dissolution rate. Likewise, lower MDT values for optimized SSNEGs (MDT = 4.34 min) and optimized liquid SNEDDS (MDT = 5.49 min) also indicated higher dissolution rate and faster drug release compared to marketed preparation (MDT = 2.17 min) and pure drug (MDT = 2.43 min). Thus the optimized SSNEGs showed a 23-fold increase in dissolution rate vis--vis pure drug. 3.7. In vivo pharmacokinetic studies Fig. 4 depicts the mean plasma concentration prole as a function of time obtained during the in vivo pharmacokinetic studies carried out in rats on optimized SSNEGs (G1), optimized liquid SNEDDS (F1), marketed preparation (Zofran ) and pure drug. The noncompartmental model parameters used to evaluate various

pharmacokinetic parameters of ONH absorption, which are summarized in Table 4. Linear trapezoidal rule was used to calculate the area under curve (AUC0t ). Plasma level proles were significantly increased for SNEDDS and SSNEGs formulations compared to pure drug and marketed preparation. The Cmax of liquid SNEDDS and SSNEGs was about 3-fold higher than pure drug. The values of AUC0t of liquid SNEDDS and SSNEGs were 5.45.8-fold higher compared to pure drug, and so were the values of relative bioavailability (%F). Although the AUC of SSNEGs (638.36 ng h/mL) was slightly lower than that of liquid SNEDDS (693.31 ng h/mL), the difference was not found to be statistically signicant (p > 0.1) between the two formulations. Similarly, tmax also decreased for SNEDDS (1.73 h) and SSNEGs (1.86 h) compared to the pure drug (2.77 h) and the marketed preparation (1.96 h). The marginally delayed values of tmax of SSNEGs compared to liquid SNEDDS (1.86 h vs. 1.73 h) are consistent with their difference in dissolution performance. Similarly, other parameters like MRT0t and Ka are also found to be higher, while t1/2 was lower for SNEDDS and SSNEGs with respect to the pure drug and marketed preparation, indicating enhanced bioavailability. The results of all the pharmacokinetic parameters were found to be highly signicant (p < 0.05) for SNEDDS and SSNEGs formulations compared to the pure drug and marketed preparation. No signicant differences (p > 0.05), however, was observed in pharmacokinetic parameters between SNEDDS and SSNEGs. The above results corroborate that oral absorption of ONH was signicantly improved from SSNEGs and SNEDDS.

3.8. Fourier transformed infrared (FTIR) spectroscopy The FTIR spectra of drug with various excipients observed no specic physiochemical interaction. There was no signicant difference observed in wave number (cm1 ) or functional group of the drug in all spectra (Fig. 5).

3.9. Differential scanning calorimetry (DSC) The DSC thermogram of ONH exhibited a sharp melting endothermic peak at 185.5 C (Tfus ) and with onset at 179.2 C and recovery at 193.2 C, and total heat consumed was 793.9 J/cal ( H). DSC thermograms of different SSNEGs prepared by physical mixture of SNEDDS with porous carriers showed absence of a denite melting endothermic peak due to complete solubilization of the ONH in the vicinity of the lipidic excipients (Fig. 6). This has also indicated change in physical nature of the drug in SSNEGs from the erstwhile crystalline state to the amorphous one.

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Table 4 Pharmacokinetic parameters and relative bioavailability of liquid SNEDDS, SSNEGs, marketed tablet (Zofran ODT) and pure drug administered orally in rats (mean SD, n = 6). Parameters* tmax (h) Cmax (ng/ml) AUC0t (ng.h/ml) AUMC0t (ng.h/ml) Ka (h1 ) t1/2 (h) MRT0t (h) %F
*

Pure drug 2.77 52.5 119.5 368.9 3.04 0.228 3.091 0.32 5.21 45.88 102.3 0.24 0.71 1.17

Conventional tablets 1.96 55.7 118.8 379.1 4.32 0.160 3.19 99.40 0.43 6.33 37.21 121.6 0.62 0.51 1.44

Liquid SNEDDS 1.73 91.3 693.3 3602.0 7.34 0.094 5.20 580.06 0.67 3.15 51.67 143.7 0.38 0.63 1.76

SSNEG 1.86 89.5 638.3 3179.8 7.56 0.092 4.98 534.04 0.45 2.59 45.32 152.4 0.25 0.78 1.98

All the parameters were determined with statistical signicance set at P < 0.05.

Fig. 5. FTIR spectra of pure drug ONH (A), physical mixture of ONH with Capmul MCM (B), Labrasol (C), and Tween-20 (D), optimized liquid SNEDDS F1 (E), optimized SSNEGs containing Sylysia 350 (F).

Fig. 7. X-ray powder diffractometry of pure drug ONH (A), SSNEGs G1 (B), SSNEGs G2 (C), SSNEGs G3 (D), SSNEGs G4 (E), and SSNEGs G5 (F).

3.10. Powder XRD studies Fig. 7 depicts the X-ray diffraction patterns of pure drug and SSNEGs. X-ray diffractogram of pure drug showed sharp peaks at diffraction angle (2 ) such as 2 , 12 , 12.5 , 17 , 18.3 , 18.8 , 20.5 , 20.09 , 23.2 , 24.3 , 25.5 , 27.1 , 28 and 30 , respectively. However, X-ray diffractogram of SSNEGs showed diffused spectra without any characteristic peaks of ONH. 3.11. Accelerated stability studies During accelerated stability studies, no signicant change in SEF time, droplet size, %DR15 min , and disintegration time was observed upto the period of three months. Drug present in SSNEGs undergoes degradation by rst-order kinetics. The rst-order degradation rate constant K was measured from the slope of the lines at each elevated temperature (Table 5). From the Arrhenius plot (Fig. 8), K value at 25 C (K25 ) was determined as 0.00874 days1 , and was nally used to calculate the shelf-life of the optimized SSNEGs as 3.3 years.

Fig. 6. DSC thermograms of pure drug ONH (A), physical mixture of ONH and Sylysia 350 with Capmul MCM (B), Labrasol (C), Tween-20 (D) and optimized SSNEGs G2 (E).

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S. Beg et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 414423 Table 5 Degradation rate constant and the shelf-life of optimized SSNEG (G1). Temperature ( C) 25 30 40 50 Absolute temperature (K) 298.00 303.00 313.00 323.00 Slope 0.0004 0.0009 0.0011 0.0014 k 0.000874 0.002062 0.002533 0.003224 log k 2.9359 2.8236 2.5963 2.4015 1/t 1000 3.3557 3.3003 3.1944 3.0959 Shelf-life (t0.9 ) 421

3.3 years

4. Discussions The important factor considered during formulation of selfnanoemulsifying formulation is to avoid the precipitation of drug in vivo following the dilution in the gut lumen. Therefore, care was exercised to choose those oils in formulation, which have high solubilization capacity for the drug, ensuring complete solubilization of drug in the resultant dispersion [42]. During screening of excipients in the present study, Capmul MCM showed the highest solubility for ONH compared to other synthetic oils (Captex 200 and Captex 355) containing medium chain triglycerides (MCTs) and natural lipids containing long chain triglycerides (LCTs). Capmul MCM contains a mixture of C8 /C10 mono-/diglycerides which favors complete solubilization of drug in the vicinity of triglyceride chains due to shorter chain length. Captex 200/355 (C8 /C10 triglycerides), on the other hand, have longer chain length, which is invariably insufcient for complete solubilization of ONH [41,43]. Natural lipids were not selected due to low solubility of drug in these excipients, stability problems and biocompatibility issues. The lipids with higher number of medium chain glycerides provide higher surface area for drug solubilization due to shorter chain length compared to long chain triglycerides [38]. Also, the blends of Capmul MCM with various natural oils were investigated. But none of the combinations showed solubility more than Capmul MCM itself. Hence, Capmul MCM as lipid, Labrasol as surfactant, and Tween 20 and PEG 600 as cosurfactants were selected for the construction of pseudo-ternary phase diagrams. The efciency of emulsication was found to be promising, when the Smix concentration was more than 65% of SNEDDS formulation. However, emulsication was not efcient with less than 50% of surfactant ratio, because of inadequate concentration of surfactant causes poor emulsication [44]. It was observed that increasing the concentration of the surfactant increased the spontaneity of self-emulsication process, but decreased the extent of emulsication, possibly due to its lipophilic nature (Fig. 1). In a comparative study between two cosurfactants, Tween 20 showed larger self-emulsication region than PEG 600. This may be ascribed to the high HLB value which favors complete emulsication and dispersion of oil globules [1]. Thus, the Smix ratios of 1:1 and 2:3 for Labrasol:Tween 20, and of 2:3 for Labrasol:PEG 600 was selected for the preparation of SNEDDS. The SNEDDS formulations, prepared using selected Smix ratios and oils, were evaluated using dispersibility test and thermodynamic stability studies. It was observed that the SNEDDS

formulations with Smix ratio of 1:1 for Labrasol and Tween 20 passed all the preliminary screening tests. Hence, these were nally selected for preparing SNEDDS formulations. All the formulations were found to be clear and transparent. Refractive index of the SNEDDS formulation was found to range between 1.39 and 1.41, somewhat closer to 1, i.e., refractive index of water, thus conrming quite transparent nature of SNEDDS formulation. The viscocity evaluation showed that the prepared liquid SNEDDS exhibited a Newtonian type of ow behavior. The viscocity decreased with increasing amount of cosurfactant (i.e., Tween 20), ostensibly due to increase in lm exibility caused by hydrophilic nature of the cosurfactant [45,46]. The droplet size and zeta potential measurement revealed that increased in concentration of oil proportionately increased the emulsion droplet size. However, increase in surfactant concentration decreased the droplet size, plausibly owing to increase in net zeta potential [47]. The surfactant forms a thin lm at the interface and decreases the globule size and helps in stabilization of the emulsion. The cosurfactant, on the other hand, have limited role on droplet size and zeta potential rather on emulsication property due to its hydrophilic nature [35]. The TEM image revealed that nanoemulsion appeared as spherical globules after dilution with aqueous phase, attributable to reduction in surface tension due to surfactant and cosurfactant. The optimized liquid SNEDDS formulation (F1) which, prepared using a Smix ratio of 1:1 for Labrasol and Tween 20, constituted ONH (1%, w/w), Capmul MCM (20%, w/v), Labrasol (39.5%, w/v) and Tween 20 (39.5%, w/v). Complete drug release (99.8%) was observed in 30 min from liquid SNEDDS, whereas pure drug and marketed preparation showed maximum drug release in 30 min upto 41% and 45.7%, respectively. This indicated that free energy required to form an emulsion was quite low, allowing spontaneous emulsication of oil droplets at the oilwater interface resulting in immediate solubilization of drug in dissolution medium [48]. It has also been suggested that oil, surfactant and cosurfactant swell upon contact with water due to emulsication of oil droplets in the presence of surfactant [49]. This reduces the oil droplet size and eventually increases the release rate. Further, increase in concentration of oil decreased the drug release rate due to inadequate amount of surfactant, thus decreasing the emulsication rate. Among the various SSNEGs prepared from the corresponding SNEDDS using porous carriers, the granules G1 and G5 containing Sylysia 350 and Neusilin US2 showed superior oil adsorption tendency, good ow and faster drug release characteristics. Further, it was observed that oil adsorption capacity got decreased with higher grades of Sylysia, i.e., 350, 550 and 730. This may probably be due to increase in average particle size, which decreased the total pore volume and porosity [50,51]. In porous carriers, the drug is adsorbed to certain extent as a thin layer of oil surrounding the particles. During dissolution, the SSNEGs undergo faster hydration to produce o/w microemulsions. The high specic surface area of these particles contributes towards improved dissolution compared to pure drug. The in vitro dissolution studies from various SSNEGs revealed that drug release from porous carriers were marginally slower compared to liquid SNEDDS, though the difference was statistically insignicant (p > 0.05). This could be because of additional steps involve during dissolution such as disintegration of granules and desorption of liquid SNEDDS from the voids of

Fig. 8. Arrhenius plot for the optimized SSNEGs (G1).

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porous carriers [31,52]. The SSNEGs when exposed to dissolution medium, leads to desorption of the liquid SNEDDS from the silica surface due to stronger interaction between silica and dissolution medium than those between silica and liquid SNEDDS [37]. Drug release from SSNEGs was initially slower due to increase in diffusion path length for adsorbed liquid formulation in the matrix of porous carriers [53]. Also, the capillary forces and wicking properties exhibited by the liquid-lled porous carriers, upon contact with dissolution uid, could also be responsible for slower drug release. Literature reports that drug release from the liquid SNEDDS and SSNEGs tends to follow nearly zero-order kinetics followed by non-Fickian kinetics, owing to interplay of diffusion and convection mechanisms [30,54,55]. In comparative drug release study the SNEDDS and SSNEGs showed quite superior drug release compared to pure drug and marketed preparation, indicating 2.45-fold increase in extent of dissolution at 30 min. On the analogous heels, in vivo pharmacokinetic studies indicated higher Cmax , AUC0t , AUMC0t , MRT0t and shorter tmax for both the liquid SNEDDS and SSNEGs. Enhanced bioavailability of self-nanoemulsifying formulations may be due to higher solubilization of drug in the gastric milieu and lymphatic transport through intestinal transcellular pathways. The synthetic oil (Capmul MCM) containing MCTs promote lipoprotein synthesis and subsequent lymphatic absorption. Further, the surfactants present in self-emulsifying formulation helps in bioavailability enhancement by augmenting oral absorption through disruption of intestinal lipid bilayers [41]. On the basis of in vitro dissolution and in vivo pharmacokinetic studies, it has been conrmed that self-nanoemulsifying formulations can better enhance the bioavailability of ONH. Use of porous carriers in development of SSNEGs can be a better formulation alternative, as these tend to preserve the physiochemical and biopharmaceutical integrity of SNEDDS. No signicant change in the characteristic peak of the drug in the FTIR spectra indicated compatibility of drug with excipients. Similarly, DSC spectra of drug excipient mixture of SNEDDS, SSNEGs exhibited specic change in the fusion temperature of the drug and total heat consumption, due to loss of crystallinity of drug in SNEDDS formulation is known to occur [56]. Transition from the crystalline to amorphous state is known to occur in the selfnanoemulsifying formulations, thus augmenting the free energy of the molecules in the system, lowering the drug melting point, and eventually improving solubility and dissolution rate [57]. The compatibility study between the drug-excipient mixtures of SSNEGs as per DSC indicated no possibility of interactions, in agreement with the results from FTIR spectra. The powder XRD study demonstrated the crystalline nature of pure drug. In case of the SSNEGs, however, absence of any prominent peak indicated the amorphous nature of the drug present in the solubilized state in the proximity of lipidic excipients [44]. Stability studies ratied that the optimized SSNEGs was robust under the accelerated temperature and humidity conditions. High duration of the predicted shelf-life (i.e., 3.3 years) at room temperature, calculated using Arrhenius plot also corroborates the stability of the SSNEGs.

to be suitable in transforming the SNEDDS into SSNEGs, ostensibly due to their oil adsorption property. The optimized SSNEGs formulation exhibited 3.01-fold augmentation in oral bioavailability of ONH in rats as compared to pure drug and marketed preparation. Besides faster drug dissolution in the GI tract, transport through lymphatic pathways could be the plausible mechanism playing stellar role in enhanced drug absorption. The promising outcomes of present studies on SSNEGs formulated using porous carriers can also be extrapolated for successfully augmenting the oral bioavailability of other BCS class II compounds undergoing extensive hepatic rst-pass effect. Conicts of interest Authors have no conicts of interest. Acknowledgements The authors are thankful to M/s Aurobindo Pharma, Hyderabad, India, for providing the gift sample of ondansetron hydrochloride. The authors are also thankful to the Institute of Life Sciences, Bhubaneswar, India, for providing the necessary facilities for particle size analysis, and to the Panjab University, Chandigarh, India, for carrying out the powder XRD studies. References
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5. Conclusions The present studies successfully embarked upon formulation of the SSNEGs of ONH, a highly promising antiemetic drug, and their subsequent applications in enhancement of oral bioavailability by improving its dissolution rate and lymphatic absorption of drug. The pseudo-ternary phase studies successfully lead to the development of optimized SNEDDS formulations rational blends of lipid (i.e., Capmul MCM), surfactant (i.e., Labrasol) and cosurfactant (i.e., Tween 20). The porous carriers (Sylysia 350, 550, 730) were found

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