PHARMACODYNAMIC AND PHARMACOKINETIC STUDIES

ON NOVEL VANADYL CHRYSIN COMPLEX

1 2 3
Bruno Stefan VELESCU , Valentina UIVAROI , Valentina ANUA ,
1 1
Anca BUZESCU , Simona NEGRE
1
Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy,
Carol Davila University of Medicine and Pharmacy, Bucharest, Romania
2
Department of Inorganic Chemistry, Faculty of Pharmacy,
Carol Davila University of Medicine and Pharmacy, Bucharest, Romania
3
Department of Physical chemistry, Faculty of Pharmacy,
Carol Davila University of Medicine and Pharmacy, Bucharest, Romania
ABSTRACT. A novel complex with general formula VO(chrysin) 4H O was studied in order to evaluate its
2 2
pharmacodynamic action, and its pharmacokinetic properties. The hypoglycemic activity of the complex was
evaluated in an alloxan diabetic rat model, and compared to chrysin and vanadyl sulphate. The pharmacokinetic
properties were studied following a single dose administration, in compartmental and non-compartmental
analyses. The complex has a low toxicity and a low hypoglycemic effect (p>0.05) correlated with the
pharmacokinetic parameters (volume of distribution, half time and micro-constants).
Keywords: vanadyl chrysin complex, pharmacodynamic action, pharmacokinetic properties,
hypoglycemic activity
INTRODUCTION entrapment of oxovanadium (IV) ion in complexes with
natural ligands such as the above mentioned, is a premise The use of inorganic compounds in therapy is an
for diminishing the major problems of toxicology of the increasing field of research motivated mainly by the
vanadium compounds (decreased GI absorption and GI
success of cisplatin in chemotherapy.
irritation) and increasing the oral absorption.
Such potential inorganic drugs are vanadium
The hypoglycemic activity of the complex
compounds used in the treatment of the type 1 (insulin-
VO(chrysin)24H2O (fig. 1) was assessed in an alloxan
dependent) and type 2 (non-insulin-dependent) diabetes.
induced diabetes rat model. The hypoglycemic activity
Early studies started with simple inorganic
was compared with vanadyl sulphate and chrysin as
vanadium(IV/V) salts which shown insulin-like effects,
reference substances. The pharmacokinetic parameters
but these vanadium salts are poorly absorbed from the
were assessed in a single dose experimental study with
gastro-intestinal tract, therefore the finding of orally
extra-vascular administration. Plasma levels of the VO-
active insulinomimetic compounds have to be directed
chrysin were determined with a validated HPLC method.
towards the improvement of bioavailability of such salts
Pharmacokinetic analysis was performed using TOPFIT
by complexation with less toxic low-molecular-weight
Ver2.0. software in Non Compartmental (NC) and
ligands. Complexes of oxovanadium (IV) cation and
Compartmental (C) methods.
flavonoid derivatives were recently developed. The
V
O
O
O H
O O
O
OH
O
O
Fig. 1 Structure of vanadyl-chrysin complex
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2012 Vasile Goldis University Press (www.studiauniversitatis.ro)
,
MATERIALS AND METHODS Venous glucose determinations were carried out using
Materials a commercial kit for glucose monitoring, BioLand G-423
Alloxan, chrysin, acetonitrile, trifluracetic acid, glucometer, BioLand Technology LTD; blood samples
dimethylsulfoxide, diethyl-ether, hexane were purchased from the tail vein of the rat were collected by puncture.
from Sigma Aldrich. The diabetic animals were randomly separated into 4
Vanadyl-chrysin complex was synthesized according groups of 6 rats:
to a method described in a previous paper. - diabetic rats (D) treated with water 1mL/100 g b.w. for 5
HPLC grade water was purified by a TKA-Genpure days;
UV system. Statistical analysis was performed using - diabetic rats treated with VO2+-chrysin complex (VO-
StatSoft, Inc. (2004). STATISTICA (data analysis ch) 0,8 mmol/kg b.w., aqueous suspension 0,8 mmol %
software system), version 7, www.statsoft.com. All for 5 days
results are expressed as mean standard deviation. - diabetic rats treated with chrysin (Ch) 0,16 mmol/kg
b.w., aqueous suspension 0,16 mmol % for 5 days
Experimental animals - diabetic rats treated with vanadyl sulphate (V) 0,8
Male Wistar and female rats weighing (129 10 g) mmol/kg b.w., aqueous solution 0,8 mmol % for 5 days
from the Cantacuzino Institute, Bucharest were used. The
rats were housed in plastic cages in an air-conditioned Pharmacokinetics
animal room and fed on granulated food with free access Study design
to water. 26 female Wistar rats (137 8 g) were randomly
The temperature and relative humidity were distributed in 5 groups of 5 animals 1 animal used as
continuously monitored using a thermohygrometer. The blank.
temperature was between 20C and 22C and the relative The animals received 100 mg/b.w. Vo-chrysin
humidity was generally maintained at 35-45%. complex, by intraperitoneal injection.
All procedures were carried out in accordance with Following the administration the animal groups were
the Directive 86/609/EEC of 24th November 1986, on the sacrificed at 30 min, 1h, 2h, 6h and 24h respectively. The
protection of animals used for experimental and other blood was collected on Na2EDTA and was centrifuged;
scientific purposes. plasma samples were stored at -22C for HPLC analyses.
Pharmacokinetic analysis was performed with TOPFIT
Diabetic animals Ver2.0. software.
Diabetes was induced by a single intraperitoneal
injection of alloxan monohydrate 13% (w/v) in a saline HPLC assay
solution at a dose of 130 mg/kg body weight, after a fasten The HPLC method used a Waters Chromatographic
period of 12 hours. Blood samples were collected 48 syst em (Wat ers, Mi l ford, MA, USA). The
hours later and glucose levels were determined to confirm chromatographic conditions are presented in table 1:
the development of diabetes.
Chromatographic conditions of the HPLC assay
Table 1
Plasma standards preparation phosphate buffer pH 5,4 were added. The samples were
Standard plasma samples: Stock solution containing vortex-mixed for homogenization, and 3 ml
250 g/ml of VO-chrysin were made by dissolving 10,0 diethylether:hexane mixture (7:3 v/v) was added. The
mg of VO-chrysin in dimethylsulfoxide in a 25 ml samples were vortexed horizontally for 20 minutes at 120
volumetric flask. Further dilutions were made with blank rpm, and 2,5 ml of the organic layer was transferred into a
plasma, in order to obtain standard plasma samples in the conical tube, and evaporated to dryness under nitrogen
range 0,01-50 g/ml. Separate solutions were prepared stream, at 45C. The dry residue was reconstituted in 300
for the calibration curve samples and quality control. l of mobile phase. An aliquot of 100 l from the
resulting solution was injected into the chromatographic
Sample preparation system.
To 0,5 mL of plasma sample, 250 l of 0,2M
Studia Universitatis Vasile Goldi, Seria tiinele Vieii
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2012 Vasile Goldis University Press (www.studiauniversitatis.ro)
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Velescu B. S. et al.
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RESULTS AND DISCUSSIONS Daily blood level values were measured for the treated
Hypoglycemic activity animals.
Of the collectivity receiving alloxan, 72,42% were The mean blood glucose evolution is presented in the
found to be diabetic (blood glucose > 200 mg/mL). tables 2-4.
Mean blood glucose evolution vs. basal and day 1 of treatment
(24 hours after alloxan administration) for D group (%)
Table 2
Mean blood glucose evolution vs. basal and day 1 of treatment
(24 hours after alloxan administration) for VO-ch group (%)
Table 3
Mean blood glucose evolution vs. basal and day 1 of treatment
(24 hours after alloxan administration) for V group (%)
Table 4
Fig. 3 Calibration plot of the method
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2012 Vasile Goldis University Press (www.studiauniversitatis.ro)
pp. 525-530
Pharmacodynamic and pharmacokinetic studies
on novel vanadyl chrysin complex
527
Mean blood glucose evolution vs. basal and day 1 of treatment
(24 hours after alloxan administration) for Ch group(%)
Table 5
At the end of the experiment, by analyzing the data For LLOQ concentration, the precision (characterized
values, it can observed the glycemia decreased statistically by the relative standard deviation) was 2,13%, and
significant compared to D group (ANOVA p<0.05), only accuracy (defined as the deviation between the true and the
for the animals within the group receiving vanadyl measured value expressed in percent) was -0,77%. The
sulphate. intra-assay precision and accuracy was estimated by
analysing the quality control samples (low, medium and
HPLC validation high concentration) five times in the same analytical run.
Validation of the analytical method: Both intra-assay accuracy and precision were within the
For the plasma quantification of VO-chrysin, a accepted limits (Table 6). The precision was better than
chromatographic method was developed and validated. A 5% and the bias did not exceed 4 % at all concentration
representative chromatogram is presented in fig 2. The levels tested. The recovery from plasma was influenced by
method was validated in accordance with international the protein plasma binding of Vo-chrysin (77 % mean
regulations. recovery); nevertheless the value of the recovery is
appropriate for the determination of plasma level
concentrations of the analyte.
The concentration changes related to the nominal
The calibration curve was linear in the range 0,01-50 concentration were less than 15%, indicating no
g/mL (r>0,9999); the lower limit of quantification significant substance loss during the study
(LLOQ) was 0,01 /mL (N = 6) (Fig. 3) Plasma concentrations of VO-chrysin complex, at the
sampling moments, are shown in table 7 and figure 4:
Fig. 2 Representative chromatogram for the
plasma analyses of VO-chrysin
Fig. 3 Calibration plot of the method
Intra-assay accuracy and precision of the method
Table 6
Mean plasma concentration of VO-chrysin complex
Table 7
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2012 Vasile Goldis University Press (www.studiauniversitatis.ro)
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Velescu B. S. et al.
528
Fig. 4 Plasma concentration time profile
for VO-chrysin complex
The proposed pharmacokinetic model for VO-chrysin
complex is described in fig. 5.
Pharmacokinetics
NC analysis
The pharmacokinetic parameters calculated by the NC
methods are presented in table 6.
The calculated micro- and macro- constants are
presented in table 8.
C analysis
Akaike (AIC) criteria were used for accepting the
pharmacokinetic model described by the complex. The
model with the lowest AIC value is accepted.
VO-chrysin complex evolution in the rat organism is The pharmacokinetic parameters of the novel VO-
described by a bicompartmental model (table 7). chrysin complex are presented in table 9.
Pharmacokinetic parameters calculated
by the NC methods
Table 6
AIC values for the compartmental models
Table 7
Fig. 5 Proposed pharmacokinetic model
for VO-chrysin complex
Calculated micro- and macro- constants
for the bicompartmental model
Table 8
CONCLUSIONS As a consequence, the inappropriate pharmacokinetic
The calculated micro- and macro- constants for the profile can lead to a low pharmacodynamic effect .
VO-chrysin showed the complex tendency to a fast
elimination. AKNOWLEDGEMENTS
In order to manifest its biologic activity, vanadium This work was supported by the PNCDI II grant
species have to interfere with different cytosolic enzymes. 61042/2007 of the Romanian Ministry of Education and
Targeting these intracellular enzymes is ensured by a Research.
proper pharmacokinetic profile. The tendency of VO-
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