Drug Metabolism Letters - Volume 11, Issue 2, 2017

Background: There has been an increase in the use of herbal products to complement conventional drugs in the treatment of various diseases especially in developing countries. This may be attributable to the potential cost-effectiveness and ease of accessibility of these products as well as the perception of their safety profiles. However, there are numerous literature reports on herbs altering the pharmacokinetics and pharmacodynamics of other co-administered drugs thereby modulating the therapeutic outcomes. The prevalence of diabetes is on a steady increase worldwide and it is now identified as one of the main threats to human health. Objective: It is important that knowledge on specific effects of antidiabetic herbs and their products on drug metabolizing enzymes are updated and documented so as to ensure optimization of their therapeutic utility. This review, therefore, aims to highlight herbal products with evidence-based antidiabetic effects, identify their bioactive phyto-constituents, and also focus on the important Cytochrome P450 and consequences of their inhibition or induction. Methods: An extensive literature search was undertaken and the information obtained were critically analyzed and discussed. Results and Conclusion: The literature abounds with reports on the utilization of herbal medications for the treatment of diabetes mellitus since time immemorial, but very few of these herbal products have undergone clinical trials. Also, studies on the herb-drug interactions were limited. Due to the complex phytochemical composition of the herbs, concomitant administration with conventional drugs resulted in alterations of pharmacological effects of some drugs. Evidences of beneficial interactions were identified for medical exploitation.

Background: Frequent recreational use of Anabolic Androgenic Steroids (AAS) is an instance of substance abuse which mimics the status of a natural hormone and upon prolonged exposure may lead to adverse drug reactions. These adverse drug reactions proceed in a manner so as to alter the normal metabolism of an enzyme mediated pathway such as the Cytochrome P450 (CYP) family of enzymes. Objective: The present study was conducted to investigate the impact of overuse of Nandrolone Decanoate (ND), an AAS, upon CYP enzyme activity and a CYP gene, belonging to CYP1 family. Methods: The study was carried out using normal and ND treated male albino mice. Genetic analysis was conducted using normalized and treated cDNA and reverse transcriptase polymerase chain reaction based assays. For enzyme assay, 0.1ml of 25 mg ND was administered to the animals twice a week for a period of 90 days. Genetic analysis was carried out with the same dose but administered for a period of 360 days. Results: CYP enzyme activity increased significantly (p<0.01) in the ND treated group of animals compared to that in the normal group. However, no noticeable alteration was observed at the molecular level. Conclusion: From the present study it could be inferred that, at elevated doses, ND has the potential to alter hepatic CYP enzyme activity without any modification in the CYP gene. This could be due to a possible adaptive response of the living system to such drugs.

Background: Tamoxifen is widely used in the therapy for breast cancer and has three major metabolites, N-desmethyltamoxifen, 4-hydroxytamoxifen, and endoxifen. Endoxifen has played a major role in the inhibition of tumor growth of breast cancer and the tumor growth is related to endoxifen concentration. Objectives: The aim of this study was to develop a pharmacokinetic-pharmacodynamic model to predict the distribution of tamoxifen and endoxifen quantitatively, and to discover the anti-tumor effect patterns of tamoxifen and endoxifen. Methods: The pharmacokinetic-pharmacodynamic model was established by integrating a four compartments pharmacokinetics model and a pharmacodynamic model, the first one include central compartment and peripheral compartment both of which contain tamoxifen and endoxifen. The parameters of the model were calculated by the values of plasma concentrations and the tumor growth data before and after the administration of tamoxifen. Results: The transport rate k42 (6.0003) of endoxifen from the peripheral compartment to the central compartment and the metabolism rate k34 (0.0031) from tamoxifen to endoxifen in the peripheral compartment were proven to be significant, which showed that tamoxifen and endoxifen are mainly distributed in the central compartment. The model provided reasonable predictions of tumor growth, which was inhibited after the administration and varies with the concentration of endoxifen. Conclusion: We established a PK-PD model of tamoxifen and endoxifen to predict the tumor growth. The parameters of the pharmacodynamic model, which characterized the tumor growth, revealed the patterns of tamoxifen's anti-tumor functions. The PK-PD model successfully provided illustration for the pharmacokinetics of tamoxifen and endoxifen, and predicted the inhibition effect of endoxifen on the tumor growth.

Background: Timolol is clinically administered topically (ocular) to reduce intraocular pressure and treat open-angle glaucoma. Ocular administration of timolol in low doses (0.5% w/v in the form of eye drops) has led to challenges for in vivo metabolite identification. An understanding of drug metabolism in the eye is important for clinical ocular therapeutics and potential drug candidates. Methods: We aimed to investigate the metabolism of timolol in rat ocular and liver S9 fractions, as well as rat ocular tissue and plasma following a 0.5% topical (ocular) dose of timolol. We explored the potential in vitro metabolic bioactivation in the eye/liver by conducting trapping studies for putative aldehyde and iminium ion intermediates that may arise from the morpholine functionality. Results: Oxidative metabolism of timolol to its major metabolite (M4) in ocular S9 and recombinant rat cytochrome P450 (CYP) isoforms supports the possible role of rat ocular CYP2D2, 2D4, and/or 2D18. Observation of N-acetyl-timolol (M5) is suggestive that the ocular N-acetyltransferases may also play a larger role in ocular disposition of timolol, a previously unreported finding. This research is the first comprehensive report of in vitro ocular metabolism of timolol in rat. Conclusion: This study also indicates that in vitro hepatic metabolism is over-predictive of ocular metabolism following topically ocular dosed timolol. The research, herein, highlights the eye as an organ capable of first pass metabolism for topical drugs. Thus, new ophthalmologic considerations for studying and designing long term topical therapies in preclinical species are needed in drug discovery.

Background: Non-selective chemical inhibitors of phase I and phase II enzymes are commonly used in in vitro metabolic studies to elucidate the biotransformation pathways of drugs. However, the inhibition of the inhibitors on efflux and uptake transporters is not well investigated, potentially leading to unexpected and ambiguous results in these studies. Objective: The commonly used metabolizing enzyme inhibitors, 1-aminobenzotriazole (ABT), SKF- 525A, pargyline, allopurinol, menadione, methimazole, piperine and raloxifene, were examined for their potential inhibition of the major hepatic ABC (ATP binding cassette) and SLC (solute carrier) transporters. Methods: Different concentrations of the metabolizing enzyme inhibitors were used to study their effects on ABC and SLC transporters expressed in MDR1-MDCKI, Bcrp1-MDCKII, OATP1B1-HEK, OATP1B3-HEK, OCT1-HEK, OCT3-HEK cells and MRP2 vesicles. Results: ABT, allopurinol and methimazole had no inhibitory effects on MDR1, Bcrp1, MRP2 or on OATP1B1, OATP1B3, OCT1 or OCT3. Pargyline did not inhibit OATP1B1 or OATP1B3, but weakly inhibited OCT1 and OCT3. In contrast, SKF-525A showed inhibition of not only MDR1, Bcrp1 and MRP2 but also OATP1B1, OATP1B3 and OCT1. Menadione and raloxifene weakly inhibited Bcrp1, but the inhibition of raloxifene on MDR1 was as potent as on the xanthine oxidase pterin oxidation. Piperine showed inhibition of MDR1, Bcrp1, OATP1B1, OCT1 and OCT3. Conclusion: ABT, pargyline, allopurinol and methimazole have no inhibitory effects on the studied ABC and SLC transporters, suggesting the inhibitors are unlikely to cause confounding inhibition of transporters when used in metabolism studies. However, SKF525A, menadione, raloxifene and piperine can inhibit the activities of ABC and/or SLC transporters.

Background: Epidermal Growth Factor (EGF) is a well-known mitogen that has importance in cell proliferation and differentiation. This property has led to the common use of EGF as an additive to some cell culture media. EGF has been previously shown to modulate constitutive Cytochrome P450 (CYP) expression in vitro. Objectives: To assess the influence of EGF on the basal and induced expression of CYP3A4, CYP1A2 and CYP2B6 in plated human hepatocytes. Methods: Human hepatocytes were treated with EGF with and without in the presence of positive control inducers. After treatment, CYP isoform mRNA expression and enzyme activity were measured. Results: EGF at concentrations ranging from 0.001-500 ng/mL resulted in a concentration-dependent decrease in basal CYP3A4 catalytic activity by up to 92%. In contrast, rifampicin (RIF)-induced activity was decreased only slightly (up to 23%). CYP3A4 mRNA also decreased in an EGF concentrationdependent manner. In contrast to CYP3A4, CYP1A2 and CYP2B6 activity and mRNA were either not suppressed or suppressed to a lower extent. The preferential effect with CYP3A4 was confirmed in 4 additional donors using a single concentration of EGF (10 ng/mL) and time-dependence experiments revealed that suppression appeared after only 24h of treatment. Conclusion: Because of the larger effect on the basal CYP3A4 compared to the induced response, EGF as a media additive enables a higher dynamic range in a CYP3A4 induction assay, potentially expanding the range of donor hepatocytes suitable for use in induction studies. These findings also suggest that EGF may be an important regulator of CYP3A4 expression in vivo.

Background: Nuclear Receptors (NRs), including PXR and CAR, are presumed to be ligand-dependent transcription factors, but ligand binding is not an absolute requirement for activation. Indeed, many compounds activate PXR and CAR by indirect mechanisms. Detecting these indirect activators of specific nuclear receptors in vitro has been difficult. As NR activation of either or both PXR and CAR can lead to drug-drug interactions and adverse drug effects, false negatives obtained with screening tools incapable of detecting indirect activators could present liabilities. Objective: The aim of this study was to establish assays that identify indirect activators of human PXR and CAR. Methods: Commercially available human PXR and CAR transactivation assays were used for analyses. Results: We show that transactivation assays containing full-length nuclear receptors with native promoters can identify indirect activators of human CAR and PXRwhen compared to those of commercially available assays containing only the LBD of PXR and CAR. Of these two assay systems, only human PXR and CAR1 assays with full-length receptors and native promoters are capable of detecting indirect and ligand activators. With this capability, several kinase inhibitors were identified that activate PXR and CAR by indirect mechanisms. Furthermore by using both the LBD and full-length receptors, phenobarbital and midostaurin were found to be direct and indirect activators of PXR while human CAR activation by phenobarbital occurs by indirect mechanisms only. Conclusion: Cell based transactivation assays employing the full-length receptors and native promoters identify both direct and indirect activators of either or both human PXR and CAR.

Background: The nuclear hormone receptor, Farnesoid X Receptor (FXR) regulates the transcription of genes associated with bile acid metabolism and disposition. Objective: This study investigates possible changes in the expression of target genes responsible for amino acid conjugation, i.e., Bile Acid-CoA Synthetase (BACS) and bile acid-CoA: amino acid Nacetyltransferase (BAT). These genes have been shown to be inducible by FXR agonists in rat models, however, to date no studies have been conducted in a human hepatocyte model. Results: In human hepatocytes, treatment with the FXR agonists GW4064 (1.0 μM) and WAY362450 (0.1 μM) did not significantly induce the mRNA expression of BACS and BAT genes. However, other target genes associated with FXR activation, such as Bile Salt Export Pump (BSEP), Short Heterodimer Partner (SHP), Multidrug Resistance-associated Protein 2 (MRP2) and Multidrug Resistance Protein 3 (MDR3), were upregulated. Interestingly, a follow up study conducted in rat hepatocytes indicated that GW4064 induced the BACS gene while WAY362450 induced the BAT gene, confirming literature results that these genes can be induced in rat. Conclusion: In conclusion, there appears to be some species differences in the activation of FXR target genes.

Background: 4-(piperazin-1-yl)-8-(trifluoromethyl)pyrido[2,3-e][1,2,4]triazolo[4,3-a]pyrazine (1) is a small-molecule which demonstrated a sub-nM inhibitory potency toward the histamine H4 receptor (H4R). However, it was found to be mutagenic in an in vitro Ames assay. Metabolic bioactivation of 1 could potentially arise from the piperazine moiety by forming reactive intermediates such as glyoxal, aldehyde-imine and/or iminium ion, which could all lead to genotoxicity. The aim of this study was to investigate bioactivation of 1 to determine the potential causes of the genotoxicity and mitigate liabilities in this scaffold. Methods: 1 was investigated for its genotoxicity in phenobarbital and β-naphthoflavone induced Sprague Dawley rat liver S9 fractions. Trapping agents such as o-phenylenediamine was used postincubation. Results: Following metabolic profiling of 1, two oxidative metabolites were observed and identified in phenobarbital- and β -naphthoflavone induced Sprague Dawley rat liver S9 fractions. Metabolic pathway of 1 was primarily mediated by the metabolism of the piperazine moiety. The trapped glyoxal was identified by using high resolution LC-MS instrument. Structural characterization of the trapped glyoxal was determined by comparison of retention time, accurate mass measurement and Collision Induced Dissociation (CID) spectra to authentic standard. Conclusion: In the present investigation, a novel method was developed to trap glyoxal, which may potentially be liberated from piperazine moiety. These findings led to modifications on the piperazine ring to mitigate the bioactivation pathways leading to mutagenicity. Subsequently, the next generation compounds with modified piperazine moiety, retained H4R inhibitory potency in vitro and were not genotoxic in the Ames mutagenicity assay.