PHARMACODYNAMIC AND PHARMACOKINETIC STUDIES ON NOVEL VANADYL CHRYSIN COMPLEX

1 2 3Bruno Stefan VELESCU , Valentina UIVAROŞI , Valentina ANUŢA , 1 1Anca BUZESCU , Simona NEGREŞ

1Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy,

“Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania2Department of Inorganic Chemistry, Faculty of Pharmacy,

“Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania3Department 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)2·4H2O (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

OH

O O

O

OH

O

O

Fig. 1 Structure of vanadyl-chrysin complex

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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 daysresults 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 daysExperimental 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 daysfrom the Cantacuzino Institute, Bucharest were used. The rats were housed in plastic cages in an air-conditioned Pharmacokineticsanimal room and fed on granulated food with free access Study designto 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 20ºC and 22ºC 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 -22ºC 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 assaysolution 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 system (Waters , Milford, 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 assayTable 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 µg/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 µg/ml. Separate solutions were prepared stream, at 45ºC. The dry residue was reconstituted in 300 for the calibration curve samples and quality control. µl of mobile phase. An aliquot of 100 µl from the

resulting solution was injected into the chromatographic Sample preparation system.

To 0,5 mL of plasma sample, 250 µl of 0,2M

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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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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

μg/mL (r>0,9999); the lower limit of quantification significant substance loss during the study

(LLOQ) was 0,01 μ/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 methodTable 6

Mean plasma concentration of VO-chrysin complexTable 7

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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 analysisAkaike (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 modelsTable 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-chrysin complex to return in the central compartment REFERENCES(K21/K12 ratio is approx 5.5), and the fast elimination Akaike H (1974) A new look at the statistical model (t1/2 6.5 h), indicates that the intracellular distribution of identification. IEEE Trans Automat Control AC the complex is low; therefore the interaction with the antioxidant status in diabetic rat lenses. J Biosci cytosolic enzymes is diminished. 30(2):221–230

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