Effect Of 30-Day Administration Of Cellgevity® Supplement On Selected Rat Liver Cytochrome P450 Enzyme Activity And Supplement Interaction With Carbamazepine

There is considerable evidence that many patients concurrently take dietary supplements with conventional drugs, with a risk of potential drug-supplement interaction. The aim of this study was to determine the effect of Cellgevity® supplement on selected rat liver cytochrome P450 (CYP) enzymes and on the pharmacokinetics of carbamazepine. Methods Sprague-Dawley (SD) rats were put into 5 groups and modulation of CYP enzyme activity by Cellgevity® was determined by comparing the enzyme activity of Cellgevity-treated groups with the negative control group after 30 days of treatment. For the effect of Cellgevity® on the pharmacokinetics of carbamazepine, 12 SD rats were put into 2 groups; one group received an oral administration of carbamazepine plus Cellgevity®, and the other carbamazepine plus normal saline. Blood samples were collected at specific time points and analyzed for levels of carbamazepine.

With the increase in the incidence of non-communicable diseases (NCDs) such as diabetes, cardiomyopathies, cancers and epilepsy, which are often associated with oxidative stress, people usually resort to the use of dietary supplements (with antioxidant potential) to prevent these diseases (2). Interestingly, some individuals who use dietary supplements have the notion that these agents may enhance the effects of conventional drugs (3), possibly because some synergy between dietary supplements and conventional drugs have been reported (4). However, this notion may not always be true.
Available on the market currently are a number of dietary supplements known to replenish levels of reduced glutathione, a free radical scavenger in the cell. One such supplement, Cellgevity®, contains a glutathione precursor molecule, riboceine (D-ribose-L-cysteine). Riboceine is known to deliver cysteine into cells and enhance reduced glutathione level in the body (5,6). The other constituents of Cellgevity® are broccoli seed extract, turmeric root extract, resveratrol, grape seed extract, quercetin, curcumin, milk thistle, vitamin C, selenomethionine, cordyceps, black pepper and aloe extract. Some of these constituents are known as inducers and/or inhibitors of CYP enzymes (7,8,16). Max International, the marketer and distributor of Cellgevity®, has branches in 14 countries (United States, Nigeria, Cote d'Ivoire, New Zealand, Singapore, Costa Rica, Columbia, Philippines, El Salvador, Malaysia, Guatemala, Ghana and Hong Kong). Cellgevity® has gained popularity in these countries probably due to the knowledge that the supplement has a high antioxidant potential (9).
Reports suggest that there could be clinically important modulation of cytochrome P450 (CYP) enzymes by supplements and/or herbal products. This could result in adverse or sub-therapeutic effects of concurrently administered conventional drugs. For example, St. John's wort was reported to decrease the serum concentration of theophylline (a bronchodilator) as a result of CYP450 enzyme induction (10). This interaction between St John's wort and theophylline could lead to sub-therapeutic effect of normal doses of theophylline when there is co-administration. For this reason, patients are often advised not to take theophylline concomitantly with St. John's wort. In a previous study, we reported that Cellgevity® at 4 and 8 mg/kg significantly inhibited rat liver CYP2C9, CYP2B1/2B2 and CYP3A4 over a 7-day treatment period (11). Although there is evidence that food supplements could be effective at low dose in preventing some NCDs (COSMOS; NCT02422745), food supplements should also be tested at animal equivalent of the human dose (that is to mimic the human dose) when investigating their effectiveness in animal models. Therefore, since xenobiotics are known to modulate CYP enzymes depending on factors such as dose and treatment duration (12), the current study is follow-up to one we previously reported (11), but with Cellgevity® doses calculated after scaling from humans, and Cellgevity® administered over a longer period of time (30 days).
With reports suggesting concurrent administration of Cellgevity ® , known to inhibit rat liver CYP3A4 (11), and carbamazepine also known to be extensively metabolized by CYP3A4, there is the potential for interaction between these two agents. Carbamazepine is one of the most commonly prescribed drugs in the management of epilepsy. Due to the chronic nature of this disease, and the fact that patients have to take carbamazepine for a long time (lifetime in most cases), there is the potential for clinically significant interactions to occur between carbamazepine and co-administered agents like dietary supplements, herbal products and food (13). Therefore, the current study also sought to determine the effect of Cellgevity® on the pharmacokinetics of carbamazepine. 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.

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

Microsomal Preparation
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 -80 o C 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

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 37 o C 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.

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 14 th 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

Statistical analysis
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 (C max ) and its corresponding time (T max ) 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 (K e ) was determined from the slope of the concentrationtime curve, and this was then used to calculate the elimination half-life (t 1/2 ).

CYP2C9 activity after 30-day treatment
CYP2C9 enzyme activity in the treatment groups was estimated relative to the negative control (N-C) group. CYP2C9 enzyme activity was found to be elevated in the phenobarbital-and Cellgevity®treated groups in comparison with the negative control group. The phenobarbital-and Cellgevity®treated groups were found to differ significantly from the negative control. This increase in rat CYP2C9 enzyme activity by Cellgevity® was found to be dose-dependent. A representation of the effect of Cellgevity® on rat CYP2C9 enzyme is shown in Figure 1. CYP2D6 enzyme activity after the 30-day treatment is shown in Figure 2.

CYP1A1/2 activity after 30-day treatment
CYP1A1/2 enzyme activity in the treatment groups was estimated relative to the N-C group. CYP1A1/2 enzyme activity was found to be elevated in the phenobarbital-and Cellgevity®-treated groups in comparison with the N-C group. The Cellgevity®-treated L-D and M-D groups showed elevated CYP activity compared to N-C group, but these differences were not statistically significant. However, a significant difference was found between the H-D group and the N-C group. There was somewhat a dose-dependent increase in the effect of Cellgevity® on rat CYP1A1/2 enzyme activity. Levels of CYP1A1/2 enzyme activity after 30-day treatment are shown in Figure 3.

CYP2B1/2 activity after 30-day treatment
CYP2B1/2 enzyme activity in the treatment groups was estimated relative to the N-C group. CYP2B1/2 enzyme activity was found to be elevated in the phenobarbital-and Cellgevity®-treated groups in comparison with the N-C group. The Cellgevity®-treated groups showed elevated levels compared to N-C group, but the differences were not statistically significant. There was also no dose-dependent effect of Cellgevity® on rat CYP2B1/2 enzyme activity. Levels of CYP2B1/2 enzyme activity after 30day treatment are shown in Figure 4.

CYP3A4 activity after 30-day treatment
CYP3A4 enzyme activity in the treatment groups was estimated relative to the N-C group. CYP3A4 enzyme activity was found to be elevated in the phenobarbital-and Cellgevity®-treated groups in comparison with the N-C group. The Cellgevity®-treated groups showed elevated levels compared to the N-C group, but the differences were not statistically significant. There was also no dosedependent effect of Cellgevity® on rat CYP3A4 enzyme activity. Levels of CYP3A4 enzyme activity after 30-day treatment are shown in Figure 5.

Overall Effect of Cellgevity® on Rat CYP Enzyme Activity
When Cellgevity®-treated groups were compared to the N-C group, the activities of CYP3A4 and CYP2B1/2 did not differ significantly when compared to the N-C group. However, CYP1A1/2, CYP2C9 and CYP2D6 activity in rats treated with Cellgevity® were found to be significantly increased compared to the N-C group. Additionally, the increase in CYP2C9 activity was found to be dosedependent. The overall effect of Cellgevity® on selected CYP enzymes is shown in Table 1.

Effect of Cellgevity® on the pharmacokinetics of carbamazepine
The concentration-time curves of carbamazepine in rats administered carbamazepine with Cellgevity® and carbamazepine with normal saline is shown in Figure 6. From the concentration-time curve, rats administered carbamazepine with Cellgevity® had a higher peak at 4 h compared to the rats administered carbamazepine with normal saline.
The peak concentration for rats administered carbamazepine with Cellgevity® was 2-fold greater compared to carbamazepine with saline. Total drug exposure at the last sample time point (AUC 0→24 ) was also about 2-fold greater in rats administered carbamazepine with Cellgevity® compared to carbamazepine with saline. Pharmacokinetic parameters obtained from the concentration-time curves for the two groups are shown in Table 2.

Discussion
This study was a follow-up on an earlier one that sought to investigate the potential of Cellgevity® to modulate CYP enzymes in rats. In the earlier study, low doses of Cellgevity® (4 mg/kg and 8 mg/kg) were used over a period of 7 days (11). In that study, Cellgevity® was found to inhibit rat liver CYP3A4, CYP2C9 and CYP1A2 after the 7-day treatment period (11). In the current study, animal equivalent doses of Cellgevity® per serving in humans (12.46 mg/kg) were used in SD rats. Since xenobiotics are known to modulate CYP enzymes depending on factors such as dose and treatment duration (12), this study, therefore, sought to investigate the effect of Cellgevity® on rat liver CYP enzymes using 3 doses of Cellgevity® calculated after scaling from humans, and administering Cellgevity® over a period of 30 days.
In the present study (after the 30-day treatment period) Cellgevity® was found to have increased the activity of CYP1A1/2, CYP2C9 and CYP2D6 significantly. These results are contrary to what was reported by N'guessan et al., (11), where Cellgevity® significantly inhibited rat CYP2B1/2B2, CYP3A4, and CYP2C9 after a 7-day treatment period. Horn, Reichert (12) showed that CYP activity is both dose and treatment duration dependent. On the other hand, Pichard-Garcia, Hyland (20) reported that higher concentrations of eletriptan induced CYP3A in the culture medium. However, lower doses of eletriptan did not cause CYP3A induction. Organisms after exposure to xenobiotics or foreign chemicals often develop adaptive mechanisms where they increase metabolism in an attempt to get rid of the insulting agent.
Indeed, it may not be entirely prudent to extrapolate animal studies to humans, but these data give credence to the fact that dietary supplements could modulate CYP enzymes in humans. If this increase in enzyme activity observed for CYP1A1/2, CYP2C9 and CYP2D6 (dose-dependent in the case of CYP2C9) are clinically relevant, then emphasis should be made on maximum therapeutic daily doses of Cellgevity® in humans.
There are reports of potential interaction between dietary supplements/herbal products and conventional drugs. The commonest of these interactions appear to occur at the level of drug metabolism; especially with liver microsomal enzymes. The current study, therefore, also sought to determine the effect of Cellgevity® on the pharmacokinetics of carbamazepine in SD rats. The total carbamazepine exposure (AUC) for rats administered carbamazepine with normal saline was 347 µmol.h/L, as against 170 µmol.h/L for rats administered carbamazepine with Cellgevity®. With elimination rates being 0.28 h -1 for carbamazepine with Cellgevity® and 0.41 h -1 for carbamazepine with normal saline, this meant that there was a slower clearance of carbamazepine in rats administered carbamazepine with Cellgevity®. This ultimately led to a longer half-life (2.3 h) among rats administered carbamazepine with Cellgevity®. It can, therefore, be inferred from the current study that Cellgevity® had some level of interaction with carbamazepine, possibly through inhibition of CYP3A, the enzyme that is known to metabolize carbamazepine.
Anecdotal reports that suggested that epileptic patients were taking diosmin (a widely used flavonoid in the treatment of varicose veins and haemorrhoids) along with carbamazepine, raised concerns that led to a study to ascertain possible interaction between these two agents in an animal model (21). In that study, diosmin significantly enhanced C max , AUC, and t 1/2 of carbamazepine as compared to control rats. Diosmin also significantly decreased k e and apparent oral clearance (CL/F) of carbamazepine as compared to control rats. This, therefore, corroborates findings from the current study, that there is a potential for herbal medicines, dietary supplements, and food to interact with conventional drugs in vivo (22), and that studies of this nature ought to be conducted to ascertain such interactions.

Conclusion
In the current study, Cellgevity® was found to cause an appreciable increase in the activities of rat liver CYP1A1/2, CYP2C9 and CYP2D6 after a 30-day treatment period. Additionally, Cellgevity® was found to alter the pharmacokinetics of carbamazepine in Sprague-Dawley rats. Although this study was conducted in an animal model, this finding is noteworthy, as this may serve as a basis for future studies in humans.

Availability of data and materials
The data used to support the findings of this study are available from the corresponding author upon request.

Competing interests
The authors declare that they have no competing interests.

Consent for publication
Not applicable.