Animals care and safety
This research was approved by the College of Health Sciences Ethical and Protocol Review Committee (Protocol ID: CHS-Et/M.9 – P1.16/2017-2018) of the University of Ghana. All animal procedures and techniques used in this study were in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals.
Male Sprague-Dawley (SD) rats, weighing 150-200 g and 6-8 weeks old, were obtained from Center for Plant Medicine Research, Mampong, Eastern Region, Ghana. The animals were housed in stainless steel cages. Each rat occupied a minimum space of 2 cubic feet (61 cm x 31 cm x 31 cm) with softwood shavings as bedding for their comfort. They were fed with normal pellet diet (AGRIMAT, Kumasi, Ghana), given water ad libitum, and maintained under standard laboratory conditions (temperature ~25°C, relative humidity 60-70%, and 12 h light-dark cycle). The animals’ feeding and water troughs were cleaned regularly to prevent contamination. Animals were acclimatized under the above conditions for 7 days before the experiment was commenced.
Hepatic enzyme induction/inhibition studies
Animal grouping and treatment administration
In determining the influence of Cellgevity® on CYP enzymes, male SD rats were put into five groups (6 rats in each group). Group 1 was administered distilled water, the vehicle used in dissolving Cellgevity® purchased from Max International (Ghana), and that served as the negative control (N-C) group. Groups 2, 3 and 4 received daily a low dose (L-D) of 38.63 mg/kg, a medium dose (M-D) of 77.25 mg/kg and a high dose (H-D) of 154.50 mg/kg of Cellgevity®, respectively. The doses of Cellgevity® administered to SD rats were animal equivalent of the human dose, calculated as described by Nair AB and Jacob SA (14), the human dose being 12.46 mg/kg per os. The SD rats in Group 5 received an oral dose of phenobarbital (Kinapharma, Ghana) 15 mg/kg daily, and that served as positive control (P-C). All administrations were for 30 days. After the 30-day period, animals were then sacrificed by cervical dislocation. Livers were excised and washed in ice-cold saline solution and weighed. Livers were then stored at -80oC until use.
Livers were thawed and homogenized in potassium phosphate buffer (pH 7.4) using a mortar and pestle on ice. Homogenized samples were first centrifuged at 4,000 rpm for 20 min. The supernatant was taken-up and re-centrifuged (Beckman Avanti J-25, USA) at 25,000 rpm for 2 h. The pellets, which constituted the microsomes were collected and stored at -80oC until use.
CYP2C9 (Diclofenac Hydroxylation) and CYP2D6 (Dextromethorphan O-demethylation) Assays
The assay was performed as previously described (15), with some modification. A volume of 350 µL of 0.1M potassium phosphate buffer (pH 7.4), 50 µL of 1 mM substrate (diclofenac for CYP2C9 assay and dextromethorphan for CYP2D6 – both substrates purchased from Sigma-Aldrich, USA) and 50 µL of 2.5 mg/mL microsome (obtained from rat livers from respective groups) were mixed separately in Eppendorf tubes. The mixtures were pre-incubated at 37oC for 5 min. A volume of 50 µL of 1 mM nicotinamide adenine dinucleotide phosphate (NADPH) [Sigma-Aldrich, USA] was added, mixed and incubated at 37oC for 45 min. A 100 µL stopping solution (ZnSO4.7H2O) was added and the mixture centrifuged at 4000 rpm for 5 min. The supernatants were aliquoted into High-Performance Liquid Chromatography (HPLC) [Shimadzu, Japan] vials.
Samples were analyzed using HPLC. The chromatographic system consisted of a binary solvent delivery system (LC - 20AB), a degasser (DGU-20A3), an auto-sampler (SIL - 20ACHT), a column temperature controller (CTO - 10AS VP) and a photodiode array detector (SPD - M20A) for CYP2C9 metabolites and fluorescence detector (RF - 10A XL) for CYP2D6 metabolites. The following chromatographic conditions were used for the analysis of CYP2C9; column, C18 (Shimadzu, Japan), diameter 5 μm, length x width 150 mm × 4.6 mm; flow rate, 1 mL/min; column temperature, 40oC; injection volume, 20 μL; mobile phase, 20 mM potassium phosphate buffer (pH 7.4)/methanol/acetonitrile (60:22.5:17.5, v/v/v). The same chromatographic conditions were used for the analysis of the CYP2D6, with modification to the mobile phase (acetonitrile/distilled water/triethylamine; 24:75:1, v/v/v).
CYP1A1/1A2 - Ethoxyresorufin O-deethylase (EROD), CYP2B1/2B2 - Pentoxyresorufin O-depentylase (PROD) and CYP3A4 - Benzyloxyresorufin O-debenzylase (BROD) Assays
The assays were performed as previously described (16, 17), with some modification. In brief, microsomes (CYP enzymes) were tested in a total volume of 100 μL. Aliquots of 70 μL potassium phosphate buffer (pH 7.4) were placed into a 96-well black plate. This was followed by addition of 10 μL of 50 μM substrate concentration (resorufin ethyl ether for CYP1A1/2, pentoresorufin for CYP2B1/2 and resorufin benzyl ether for CYP3A4; all substrates purchased from Sigma-Aldrich, USA). The final substrate concentration in 100 μL total reaction volume was 5 μM with 0.25% (v/v) dimethyl sulfoxide (DMSO). It is noteworthy that CYP activities were not expected to be affected at the DMSO concentration used in this experiment (18). Aliquots of 10 μL enzyme (microsome from each rat liver from respective Groups) corresponding to 1 mg/mL protein concentration and the vehicle was added in triplicates. The mixtures were pre-incubated at 37oC for 5 min. A volume of 10 μL of NADPH was then added to each well and the setup was incubated for 10 min for CYP1A1/2, 20 min for CYP2B1/2 and 30 min for CYP3A4 assays, respectively. Aliquots of 40 μL of stopping solution (20% 0.5 M Tris: 80% acetonitrile) were added to each well and shaken gently. Fluorescence of wells was read at wavelengths of 530 nm excitation and 586 nm emission. Triplicate experiments were performed. The average absorbance of the blank was subtracted from the average absorbance of each sample.
Effect of Cellgevity® on the pharmacokinetics of carbamazepine
Animal grouping and treatment administration
Twelve male SD rats were obtained for this aspect of the study. The animals were put into 2 groups (Group 1 and Group 2) of 6. Group 1 was administered carbamazepine plus saline and Group 2, Cellgevity® plus carbamazepine. A dose of 77.25 mg/kg/day of Cellgevity® plus 80 mg/kg of carbamazepine, both scaled from humans (14), were administered orally to rats in Group 2. Rats in Group 1 received 80 mg/kg/day of carbamazepine plus normal saline (the same volume was calculated per rat for the Cellgevity® dose).
Blood sample collection
After administration of agents, every 24 h for 14 consecutive days to Groups 1 and 2, tail vein blood samples were taken following the dose administered on the 14th day. Samples were drawn after 0.5, 1, 4, 12 and 24 h. Blood was collected into microtainer gel tubes and centrifuged at 2000 rpm for 5 min to separate serum, and this was stored at -20ºC until analysis was done.
Assay for carbamazepine in serum
Due to low sample volumes, serum samples of rats from the same group (6 animals) at each time point were pooled together, such that, for instance, serum samples of Group 1 rats at time 4 h, were pooled together to obtain a single sample. Usually, challenges with low sample volume can be circumvented by the approach of sample-pooling (19). Analysis of carbamazepine in serum was done by Fluorescence Polarization Immunoassay (FPIA) [Cobas Integra® 400 Plus, Roche, Philippines].
CYP activity of treatment groups was expressed as a percentage relative to the negative control group. All values were expressed as mean ± standard deviation. Differences between groups were tested for significance using a One-Way Analysis of Variance (ANOVA). This was followed by post-hoc analysis using Bonferroni’s Multiple Comparison Tests. P-values < 0.05 were considered statistically significant.
Non-compartmental pharmacokinetic analysis was used to determine the various pharmacokinetic parameters of carbamazepine. The maximum serum drug concentration (Cmax) and its corresponding time (Tmax) were determined by visual inspection of the concentration-time curve. The linear trapezoidal rule was applied in extrapolating area under the concentration-time curves (AUCs) for the two groups. The elimination rate constant (Ke) was determined from the slope of the concentration-time curve, and this was then used to calculate the elimination half-life (t1/2).