CYP-selective Inhibitors Research Articles

Hydroxychloroquine is Metabolized by Cytochrome P450 2D6, 3A4, and 2C8, and Inhibits Cytochrome P450 2D6, while its Metabolites also Inhibit Cytochrome P450 3A in vitro.

This study aimed to explore the cytochrome P450 (CYP) metabolic and inhibitory profile of hydroxychloroquine (HCQ). Hydroxychloroquine metabolism was studied using human liver microsomes (HLMs) and recombinant CYP enzymes. The inhibitory effects of HCQ and its metabolites on nine CYPs were also determined in HLMs, using an automated substrate cocktail method. Our metabolism data indicated that CYP3A4, CYP2D6, and CYP2C8 are the key enzymes involved in HCQ metabolism. All three CYPs formed the primary metabolites desethylchloroquine (DCQ) and desethylhydroxychloroquine (DHCQ) to various degrees. Although the intrinsic clearance (CLint) value of HCQ depletion by recombinant CYP2D6 was > 10-fold higher than that by CYP3A4 (0.87 versus 0.075 µl/min/pmol), scaling of recombinant CYP CLint to HLM level resulted in almost equal HLM CLint values for CYP2D6 and CYP3A4 (11 and 14 µl/min/mg, respectively). The scaled HLM CLint of CYP2C8 was 5.7 µl/min/mg. Data from HLM experiments with CYP-selective inhibitors also suggested relatively equal roles for CYP2D6 and CYP3A4 in HCQ metabolism, with a smaller contribution by CYP2C8. In CYP inhibition experiments, HCQ, DCQ, DHCQ, and the secondary metabolite didesethylchloroquine were direct CYP2D6 inhibitors, with 50% inhibitory concentration (IC50) values between 18 and 135 µM. HCQ did not inhibit other CYPs. Furthermore, all metabolites were time-dependent CYP3A inhibitors (IC50 shift 2.2-3.4). To conclude, HCQ is metabolized by CYP3A4, CYP2D6, and CYP2C8 in vitro. HCQ and its metabolites are reversible CYP2D6 inhibitors, and HCQ metabolites are time-dependent CYP3A inhibitors. These data can be used to improve physiologically-based pharmacokinetic models and update drug-drug interaction risk estimations for HCQ. SIGNIFICANCE STATEMENT: While CYP2D6, CYP3A4, and CYP2C8 have been shown to mediate chloroquine biotransformation, it appears that the role of CYP enzymes in hydroxychloroquine (HCQ) metabolism has not been studied. In addition, little is known about the CYP inhibitory effects of HCQ. Here, we demonstrate that CYP2D6, CYP3A4, and CYP2C8 are the key enzymes involved in HCQ metabolism. Furthermore, our findings show that HCQ and its metabolites are inhibitors of CYP2D6, which likely explains the previously observed interaction between HCQ and metoprolol.

Metabolism of Benzalkonium Chloride by CYP‐Mediated ω‐Oxidation and C‐C Cleavage Followed by β‐Oxidation in Humans

IntroductionBenzalkonium chlorides (BACs) are widely used quaternary ammonium disinfectants. A recent study found that median levels of BACs in human blood have increased 174% during COVID from pre‐COVID levels, with BACs being detected in 98% of blood samples. Although the parent BACs are not readily excreted into urine, we have identified several BAC metabolites in anonymous human urine samples. Formation of these metabolites requires ω‐oxidation and oxidative C‐C cleavage of BAC alkyl chains followed by β‐oxidation to produce a series of chain‐shortened carboxylic acid (COOH) BAC metabolites.Here, we aim to elucidate the metabolic route(s) that can account for the observed urinary metabolites of BACs. Our approach includes LC‐MS‐based investigation of metabolite profiles in microsomes, hepatocytes, and recombinant CYPs in comparison with those observed in human urine. BACs of the alkyl chain lengths found in commercial mixtures (C12, C14, and C16) served as substrates. Authentic metabolite standards were synthesized and confirmed by NMR and LC‐MS/MS. Selective CYP inhibitors were employed to assign specific CYP isoform contributions to metabolite formation.ResultsAs an extension of our previous work, we confirmed that NADPH‐dependent ω‐oxidation in microsomes and recombinant CYPs produced ω‐OH and ω‐COOH metabolites, as well as ω‐1 oxidized products, of all BAC chain lengths. We further found that the responsible CYPs (2D6, 4F2, 4F11, and 4F12) are all capable of performing C‐C cleavage of BAC side chains’ terminal carbons. The resulting products were chain‐shortened ω‐COOH BACs having lost 1 or 2 carbons (‐CH2 or ‐C2H4). In hepatocytes, subsequent β‐oxidation of the ω‐COOH C10, C12, C14, and C16 BAC metabolites (having lost 0 or 2 carbons during CYP metabolism) produces a series of even‐carbon ω‐COOH products (C4, C6, and C8) given the successive 2 carbon removal (‐C2H4) from the even‐carbon starting chain lengths. Additionally, hepatocytes produce a series of odd‐carbon ω‐COOH products (C5, C7, and C9) that logically originate from β‐oxidation of ω‐COOH C11, C13, and C15 BAC metabolites (having lost 1 carbon during CYP metabolism). The ω‐COOH BAC metabolites produced in hepatocytes were the same as those detected in human urine samples, and the relative abundance among the various chain lengths of ω‐COOH products was similar between human hepatocytes and urine. In most urine samples, C8, C10, and C12 were the most abundant COOH metabolites of even‐carbon length, and C9 was most abundant among the odd‐carbon series. In hepatocytes, addition of HET0016 and quinidine to inhibit CYP4F/CYP2D6 abolished all metabolite formation, including the β‐oxidation products.ConclusionThe above data confirm that CYP4F and/or CYP2D6 are required to initiate BAC metabolism. The relative abundance of ω‐ and β‐oxidized products (ω‐COOH BACs C4 ‐ C16) observed in urine was consistent with the human cellular metabolism of BACs observed in hepatocytes. While parent BACs are commonly found in human blood, they are not readily excreted to urine. Thus, we propose to expand monitoring of human urine samples for chain‐shortened ω‐COOH BAC metabolites as a practical surrogate for assessing BAC exposure in the general population.

Phosmet bioactivation by isoform-specific cytochrome P450s in human hepatic and gut samples and metabolic interaction with chlorpyrifos

Data on the bioactivation of Phosmet (Pho), a phthalimide-derived organophosphate pesticide (OPT), to the neurotoxic metabolite Phosmet-oxon (PhOx) in human are not available. The characterization of the reaction in single human recombinant CYPs evidenced that the ranking of the intrinsic clearances was: 2C18>2C19>2B6>2C9>1A1>1A2>2D6>3A4>2A6. Considering the average human hepatic content, CYP2C19 contributed for the great majority (60%) at relevant exposure concentrations, while CYP2C9 (33%) and CYP3A4 (31%) were relevant at high substrate concentration. The dose-dependent role of the active isoforms was confirmed in human liver microsomes by using selective CYP inhibitors. This prominent role of CYP2C in oxon formation was not shared by other OPTs. The pre-systemic Pho bioactivation measured in human intestinal microsomes was relevant accounting for ¼ of that measured in the liver showing two reaction phases catalysed by CYP2C and CYP3A4. Phosmet efficiently inhibited CPF bioactivation and detoxication, with Ki values (≈30 μM) relevant to pesticide concentrations achievable in the human liver, while the opposite is unlikely (Ki ≈ 160 μM) at the actual exposure levels, depending on the peculiar isoform-specific Pho bioactivation. Kinetic information in humans can support the development of quantitative in vitro/in vivo extrapolation and in silico models for risk assessment refinement for single and multiple pesticides.

A Specific Probe Substrate for Evaluation of CYP4A11 Activity in Human Tissue Microsomes and a Highly Selective CYP4A11 Inhibitor: Luciferin-4A and Epalrestat.

The specificity of cytochrome P450 4A11 (CYP4A11) against luciferin-4A O-demethylation in human liver microsomes (HLMs) and human renal microsomes (HRMs) and selectivity of CYP4A11 inhibition by epalrestat were investigated. Kinetic analysis of luciferin-4A O-demethylation yielded Vmax and S50 values of 39.7 pmol/min per milligram protein and 43.2 μM for HLMs (Hill coefficient 1.24) and 39.4 pmol/min per milligram protein and 33.8 μM for HRMs (Hill coefficient 1.34), respectively. Among the selective CYP inhibitors tested, HET0016 (CYP4 inhibitor) exclusively inhibited luciferin-4A O-demethylation by HLMs and HRMs. Furthermore, anti-CYP4A11 antibody nearly abolished the activity of both tissue microsomes. Luciferin-4A O-demethylase activity of HLMs was significantly correlated with lauric acid ω-hydroxylase activity, a marker of CYP4A11 activity (r = 0.904, P < 0.0001). Next, effects of epalrestat on CYP-mediated drug oxidations were examined. Epalrestat showed the most potent inhibition against CYP4A11 (IC50 = 1.82 μM) among the 17 recombinant enzymes tested. The inhibitory effect of epalrestat on CYP4A11 was at least 10-fold stronger than those on CYP4F2, CYP4F3B, and CYP4F12. For known CYP4 inhibitors, in contrast, HET0016 inhibited the activities of CYP4A11 and CYP4F2 (IC50 = 0.0137-0.0182 μM); 17-octadecynoic acid reduced activities of CYP4A11, CYP4F2, CYP4F3B, and CYP4F12 to a similar extent (IC50 = 5.70-17.7 μM). Epalrestat selectively and effectively inhibited the CYP4A11 activity of HLMs (IC50 = 0.913 μM) and HRMs (IC50 = 0.659 μM). These results indicated that luciferin-4A O-demethylase activity is a good CYP4A11 marker of HLMs and HRMs, and that epalrestat is a more selective CYP4A11 inhibitor compared with known CYP4 inhibitors.

Human hepatocytes and cytochrome P450-selective inhibitors predict variability in human drug exposure more accurately than human recombinant P450s.

Drugs metabolically eliminated by several enzymes are less vulnerable to variable compound exposure in patients due to drug-drug interactions (DDI) or if a polymorphic enzyme is involved in their elimination. Therefore, it is vital in drug discovery to accurately and efficiently estimate and optimize the metabolic elimination profile. CYP3A and/or CYP2D6 substrates with well described variability in vivo in humans due to CYP3A DDI and CYP2D6 polymorphism were selected for assessment of fraction metabolized by each enzyme (fmCYP ) in two in vitro systems: (i) human recombinant P450s (hrP450s) and (ii) human hepatocytes combined with selective P450 inhibitors. Increases in compound exposure in poor versus extensive CYP2D6 metabolizers and by the strong CYP3A inhibitor ketoconazole were mathematically modelled and predicted changes in exposure were compared with in vivo data. Predicted changes in exposure were within twofold of reported in vivo values using fmCYP estimated in human hepatocytes and there was a strong linear correlation between predicted and observed changes in exposure (r2 =0.83 for CYP3A, r2 =0.82 for CYP2D6). Predictions using fmCYP in hrP450s were not as accurate (r2 =0.55 for CYP3A, r2 =0.20 for CYP2D6). The results suggest that variability in human drug exposure due to DDI and enzyme polymorphism can be accurately predicted using fmCYP from human hepatocytes and CYP-selective inhibitors. This approach can be efficiently applied in drug discovery to aid optimization of candidate drugs with a favourable metabolic elimination profile and limited variability in patients.

Relative contribution of rat CYP isoforms responsible for stereoselective metabolism of carvedilol.

The relative contribution of cytochrome P450 (CYP) isoforms responsible for carvedilol (CAR) oxidation in rats were evaluated in order to compare with that of reported human CYPs responsible for the metabolism of CAR enantiomers. The depletion of CAR enantiomers by recombinant CYPs and the effects of CYP-selective inhibitors on the depletion catalyzed by rat liver microsomes (RLM) was determined. Quinine (rat CYP2D inhibitor) markedly inhibited the metabolism of both R- and S-CAR by RLM. The metabolism of S-CAR was inhibited more than that of R-CAR by furafylline, (a CYP1A2 inhibitor, 53.5% vs 11.3%), α-naphthoflavone (a CYP1A2 inhibitor, 64.5% vs 33.6%), and ketoconazole (a CYP3A inhibitor, 87.1% vs 51.2%). Among the CYPs examined, CYP2D2 showed the highest metabolic activities against both the enantiomers. R-CAR was mainly metabolized by CYP2D2 and CYP3A2. CYP2C11 and CYP3A1, in addition to CYP2D2 and CYP3A2 showed higher metabolic activities against S-CAR than that against R-CAR. These results suggest that CYP2D2 predominantly catalyzed R-CAR metabolism, whereas CYP2D2 and CYP3A1/2 catalyzed S-CAR metabolism in rats.

Variation in the Response of Clozapine Biotransformation Pathways in Human Hepatic Microsomes to CYP1A2- and CYP3A4-selective Inhibitors.

The atypical antipsychotic agent clozapine (CLZ) is effective in many patients who are resistant to conventional antipsychotic drugs. Cytochromes P450 (CYPs) 1A2 and 3A4 oxidize CLZ to norCLZ and CLZ N-oxide in human liver. Concurrent treatment with inducers and inhibitors of CYP1A2 modulates CLZ elimination that disrupts therapy. Drug-drug interactions involving CYP3A4 are also significant but less predictable. To further characterize the factors underlying these interactions, we used samples from a cohort of human livers to assess variation in CLZ oxidation pathways in relation to intrinsic CYP3A4 and CYP1A2 activities and the effects of the corresponding selective inhibitors ketoconazole (0.2 and 2 μM) and fluvoxamine (1 and 10 μM). The CYP3A4-selective inhibitor ketoconazole (2 μM) impaired CLZ N-oxide formation in all 14 of the livers used in inhibition studies (≥50% inhibition) while the CYP1A2-selective inhibitor fluvoxamine (10 μM) decreased norCLZ formation in nine. Ketoconazole effectively inhibited CLZ metabolism in five of seven livers that catalysed CYP3A4-dependent testosterone 6β-hydroxylation at or above the median rate and in four other livers with lower intrinsic CYP3A4 activity. Similarly, fluvoxamine (10 μM) readily inhibited CLZ oxidation in seven livers with high CYP1A2-mediated 7-ethoxyresorufin O-deethylation activity (at or above the median) and three livers with lower intrinsic CYP1A2 activity. In three livers, CLZ biotransformation was impaired by both ketoconazole and fluvoxamine, consistent with a major role for both CYPs. These findings suggest that the intrinsic activities of CYPs 1A2 and 3A4 are unrelated to the response to CYP-selective inhibitors and that assessment of the activities in vivo may not assist the prediction of drug-drug interactions.

The role of biotransformation and oxidative stress in 3,5-dichloroaniline (3,5-DCA) induced nephrotoxicity in isolated renal cortical cells from male Fischer 344 rats.

Among the mono- and dichloroanilines, 3,5-dichloroaniline (3,5-DCA) is the most potent nephrotoxicant in vivo and in vitro. However, the role of renal biotransformation in 3,5-DCA induced nephrotoxicity is unknown. The current study was designed to determine the in vitro nephrotoxic potential of 3,5-DCA in isolated renal cortical cells (IRCC) obtained from male Fischer 344 rats, and the role of renal bioactivation and oxidative stress in 3,5-DCA nephrotoxicity. IRCC (∼ 4 million cells/ml) from male rats were exposed to 3,5-DCA (0-1.0mM) for up to 120 min. In IRCC, 3,5-DCA was cytotoxic at 1.0mM by 60 min as evidenced by the increased release of lactate dehydrogenase (LDH), but 120 min was required for 3,5-DCA 0.5mM to increase LDH release. In subsequent studies, IRCC were exposed to a pretreatment (antioxidant or enzyme inhibitor) prior to exposure to 3,5-DCA (1.0mM) for 90 min. Cytotoxicity induced by 3,5-DCA was attenuated by pretreatment with inhibitors of flavin-containing monooxygenase (FMO; methimazole, N-octylamine), cytochrome P450 (CYP; piperonyl butoxide, metyrapone), or peroxidase (indomethacin, mercaptosuccinate) enzymes. Use of more selective CYP inhibitors suggested that the CYP 2C family contributed to 3,5-DCA bioactivation. Antioxidants (glutathione, N-acetyl-l-cysteine, α-tocopherol, ascorbate, pyruvate) also attenuated 3,5-DCA nephrotoxicity, but oxidized glutathione levels and the oxidized/reduced glutathione ratios were not increased. These results indicate that 3,5-DCA may be activated via several renal enzyme systems to toxic metabolites, and that free radicals, but not oxidative stress, contribute to 3,5-DCA induced nephrotoxicity in vitro.

Impact of CYP2C8*3 polymorphism on in vitro metabolism of imatinib to N-desmethyl imatinib

1. Imatinib is metabolized to N-desmethyl imatinib by CYPs 3A4 and 2C8. The effect of CYP2C8*3 genotype on N-desmethyl imatinib formation was unknown.2. We examined imatinib N-demethylation in human liver microsomes (HLMs) genotyped for CYP2C8*3, in CYP2C8*3/*3 pooled HLMs and in recombinant CYP2C8 and CYP3A4 enzymes. Effects of CYP-selective inhibitors on N-demethylation were also determined.3. A single-enzyme Michaelis–Menten model with autoinhibition best fitted CYP2C8*1/*1 HLM (n = 5) and recombinant CYP2C8 kinetic data (median ± SD Ki = 139 ± 61 µM and 149 µM, respectively). Recombinant CYP3A4 showed two-site enzyme kinetics with no autoinhibition. Three of four CYP2C8*1/*3 HLMs showed single-enzyme kinetics with no autoinhibition. Binding affinity was higher in CYP2C8*1/*3 than CYP2C8*1/*1 HLM (median ± SD Km = 6 ± 2 versus 11 ± 2 µM, P=0.04). CYP2C8*3/*3 (pooled HLM) also showed high binding affinity (Km = 4 µM) and single-enzyme weak autoinhibition (Ki = 449 µM) kinetics. CYP2C8 inhibitors reduced HLM N-demethylation by 47–75%, compared to 0–30% for CYP3A4 inhibitors.4. In conclusion, CYP2C8*3 is a gain-of-function polymorphism for imatinib N-demethylation, which appears to be mainly mediated by CYP2C8 and not CYP3A4 in vitro in HLM.

Development of a Rapid and Cost Effective Assay for the Screening of Reversible Cytochrome P450 Inhibition in Parallel with Cyp3a4 Metabolism-Dependent Inhibition Using Recombinant Proteins

In the present study we have developed a high quality, rapid and cost effective CYP450 inhibition assay that does have the ability to detect both reversible and CYP3A4 metabolism-dependent inhibition (MDI), using recombinantly expressed P450 isoforms and fluorogenic P450 substrates. CYP3A4 isoform is screened with diethoxyflourescein (DEF) as probe substrate. The IC50 values can then be calculated for test compounds against the CYP3A4 isoform, based on the rate of metabolism of the probe substrate, measured for 10 minutes. In addition, the CYP3A4 metabolism-dependent inhibitory potential of test compounds is determined by extending for 30 minutes the determination of the rate of metabolism of diethoxyflourescein and calculating IC50 values every 5 minutes of the incubation period. An estimate of the CYP3A4 metabolism-dependent inhibitory potential of the test compounds can be determined comparing IC50 values, measured following 10 and 30 minutes incubation. The incubation was performed using the selective CYP inhibitors miconazole, for direct P450 inhibition, and troleandromycin, for metabolism-dependent inhibition, as positive controls. The entire screening process was fully-automated in 96-well plate format with the use of Hamilton liquid-handling robot technology coupled with two fluorimeters (Tecan) and a custom laboratory-information management. This assay is currently applied to screen compounds early in the lead optimization process and identify those compounds that cause reversible and/or metabolism-based CYP450 inhibition and therefore progress those molecules or chemical series with the lowest DDI potential possible. The high number of data generated through this assay can also be used to build an informative database and improve predictive models.

The cytochrome P450-catalyzed metabolism of levomepromazine: a phenothiazine neuroleptic with a wide spectrum of clinical application

The aim of the present study was to identify cytochrome P450 isoenzymes (CYPs) involved in the 5-sulfoxidation and N-demethylation of the aliphatic-type phenothiazine neuroleptic levomepromazine in human liver. Experiments were performed in vitro using cDNA-expressed human CYP isoforms (Supersomes 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4), liver microsomes from different donors and CYP-selective inhibitors. The obtained results indicate that CYP3A4 is the main isoform responsible for levomepromazine 5-sulfoxidation (72%) and N-demethylation (78%) at a therapeutic concentration of the drug (10μM). CYP1A2 contributes to a lesser degree to levomepromazine 5-sulfoxidation (20%). The role of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 in catalyzing the above-mentioned reactions is negligible (0.1–8%). Moreover, at a higher, toxicological concentration of the neuroleptic (100μM), the relative contribution of CYP1A2 to levomepromazine metabolism visibly increases (from 20% to 28% for 5-sufoxidation, and from 8% to 32% for N-demethylation), while the role of CYP3A4 significantly decreases (from 72% to 59% for 5-sulfoxidation, and from 78% to 47% for N-demethylation). The obtained results indicate that the catalysis of levomepromazine 5-sulfoxidation and N-demethylation in humans shows a strict CYP3A4 preference, especially at a therapeutic drug concentration. Hence pharmacokinetic interactions involving levomepromazine and CYP3A4 substrates (e.g. tricyclic antidepressants, calcium channel antagonists, macrolide antibiotics, testosterone), inhibitors (e.g. ketoconazole, erythromycin, SSRIs) or inducers (e.g. rifampicin, carbamazepine) are likely to occur.

Enantioselectivity in the cytochrome P450-dependent conversion of tegafur to 5-fluorouracil in human liver microsomes.

Tegafur (FT) is a prodrug of 5-fluorouracil (5-FU) used in cancer chemotherapy, and the bioactivation of FT to 5-FU is mainly catalyzed by cytochrome P450 (CYP) in hepatic microsomes. FT has a chiral center and is a racemate consisting of the enantiomers, R- and S-FT. In the present study, we clarified the enantioselectivity in the conversion of FT to 5-FU and identified human CYP isoforms involved in the metabolism of its enantiomers using human hepatic preparations and recombinant CYP isoforms. Although 5-FU was generated from both FT enantiomers, R-FT was a preferred substrate than S-FT, because of the considerably higher intrinsic clearance for 5-FU formation from R-FT in liver. Eadie–Hofstee plots in microsomes showed that the conversions of R- and S-FT to 5-FU followed biphasic and monophasic kinetics, respectively. Based on the evaluation using cDNA-expressed enzymes, CYP2A6 showed the highest activity for 5-FU formation from R-FT with the Km value similar to that of the high-affinity component in microsomes. Also, CYP2A6 was the most effective catalyst for S-FT. Inhibition studies using CYP-selective inhibitors and anti-CYP antibodies demonstrated that CYP2A6 mainly contributed to the enantioselective metabolism of FT, and were almost in accordance with the relative percentage contribution of each CYP isoform to the metabolism of FT estimated using relative activity factor methods. These results suggest that the enantioselectivity in the bioactivation of FT to 5-FU in humans is mainly due to the large difference of the catalytic activity of CYP2A6 between R- and S-FT.

Effects of cytochrome P450 inhibitors and inducers on the metabolism and pharmacokinetics of ospemifene

The objectives were to determine the cytochrome P450 (CYP) enzymes involved in the metabolism of ospemifene and its main hydroxylated metabolites and to examine the effects of CYP inhibitors and inducers on ospemifene pharmacokinetics. In vitro metabolism studies were conducted using human liver microsomes; CYP-selective inhibitors and CYP-specific substrates were used to determine the roles of nine CYP isoforms in ospemifene metabolism. Two Phase 1 clinical trials were conducted in healthy postmenopausal women; crossover designs examined the effects of pretreatment with the CYP modulators rifampicin, ketoconazole, fluconazole and omeprazole on ospemifene pharmacokinetics. Although several CYP inhibitors decreased the in vitro formation of ospemifene metabolites, none of them completely blocked metabolism. Roles for CYP3A4, CYP2C9, CYP2C19 and CYP2B6 in the metabolism of ospemifene and its two main metabolites, 4--hydroxyospemifene and 4'-hydroxyospemifene, were confirmed. The in vivo experiments demonstrated that ospemifene serum concentrations were decreased by rifampicin pretreatment, increased by ketoconazole or fluconazole pretreatment, and minimally affected by omeprazole pretreatment. The clinical pharmacokinetic findings and in vitro data suggest that CYP3A4 is important for ospemifene metabolism, but other CYP isoforms and metabolic pathways also contribute. Strong CYP3A or CYP2C9 inducers (e.g. rifampicin) would be expected to decrease the exposure to ospemifene. Ospemifene should be used with caution when coadministered with the modest CYP3A inhibitor ketoconazole and should not be coadministered with the potent CYP3A/CYP2C9/CYP2C19 inhibitor fluconazole. The potent CYP2C19 inhibitor omeprazole is unlikely to cause clinically significant changes in ospemifene pharmacokinetics.

TheCYP2B6*6Allele Significantly Alters theN-Demethylation of Ketamine Enantiomers In Vitro

Ketamine is primarily metabolized to norketamine by hepatic CYP2B6 and CYP3A4-mediated N-demethylation. However, the relative contribution from each enzyme remains controversial. The CYP2B6*6 allele is associated with reduced enzyme expression and activity that may lead to interindividual variability in ketamine metabolism. We examined the N-demethylation of individual ketamine enantiomers using human liver microsomes (HLMs) genotyped for the CYP2B6*6 allele, insect cell-expressed recombinant CYP2B6 and CYP3A4 enzymes, and COS-1 cell-expressed recombinant CYP2B6.1 and CYP2B6.6 protein variant. Effects of CYP-selective inhibitors on norketamine formation were also determined in HLMs. The two-enzyme Michaelis-Menten model best fitted the HLM kinetic data. The Michaelis-Menten constants (K(m)) for the high-affinity enzyme and the low-affinity enzyme were similar to those for the expressed CYP2B6 and CYP3A4, respectively. The intrinsic clearance for both ketamine enantiomers by the high-affinity enzyme in HLMs with CYP2B6*1/*1 genotype were at least 2-fold and 6-fold higher, respectively, than those for CYP2B6*1/*6 genotype and CYP2B6*6/*6 genotype. The V(max) and K(m) values for CYP2B6.1 were approximately 160 and 70% of those for CYP2B6.6, respectively. N,N'N'-triethylenethiophosphoramide (thioTEPA) (CYP2B6 inhibitor, 25 μM) and the monoclonal antibody against CYP2B6 but not troleandomycin (CYP3A4 inhibitor, 25 μM) or the monoclonal antibody against CYP3A4 inhibited ketamine N-demethylation at clinically relevant concentrations. The degree of inhibition was significantly reduced in HLMs with the CYP2B6*6 allele (gene-dose P < 0.05). These results indicate a major role of CYP2B6 in ketamine N-demethylation in vitro and a significant impact of the CYP2B6*6 allele on enzyme-ketamine binding and catalytic activity.

Effects of Some Antihypertensive Drugs on the Metabolism and Pharmacokinetics of Indapamide in Rats

Indapamide, a non-thiazide antihypertensive diuretic agent, has been widely coadministered with other classes of antihypertensive agents to reach target systolic blood pressure. Indapamide is extensively metabolized by cytochromes P450. Interaction of indapamide and other antihypertensive drugs are unknown. We investigated the effects of other antihypertensive drugs on the metabolism and pharmacokinetics of indapamide in vitro and in vivo. Indapamide metabolism was studies in vitro using human liver microsomes pretreated with or without different concentrations of CYP-selective inhibitors and seven major antihypertensive drugs, felodipine, nifedipine, nitrendipine, telmisartan, irbesartan, valsartan and puerarin. Furthermore, the pharmacokinetics of indapamide was determined by HPLC-MS/MS to evaluate the effects of felodipine coadministered on the bioavailability of indapamide in rats in vivo. The Km and Vmax of indapamide metabolism were 114.35 ± 3.47 μM and 23.13 ± 6.61 μmol/g/min. The metabolites of indapamide, hydroxyl-indapamide and dehydrogen-indapamide, were followed. CYP3A4 and CYP2C19 were involved in indapamide metabolism in human live microsomes. In addition, felodipine, nifedipine and nitrendipine significantly inhibited indapamide metabolism with the maximum inhibitory rates of 82.6%, 72% and 95%, respectively. Felodipine significantly elevated indapamide plasma concentration and prolonged its half-life. Combination therapy of indapamide and felodipine might lead to the alteration of indapamide metabolism and pharmacokinetics. The consequence of such an interaction that may include increased effectiveness and side effect needs to be tudeis in human.

In vitro inhibition of cytochrome P450-mediated reactions by gemfibrozil, erythromycin, ciprofloxacin and fluoxetine in fish liver microsomes

Inhibition of mammalian cytochrome P450 enzymes (CYPs) is well characterized; major hepatic CYPs can be inhibited by drugs and other environmental contaminants. CYP function and inhibition has not yet been well established in fish yet these studies are important for several reasons. First, such studies will provide functional information for non-mammalian CYPs. Second, specific inhibitors can be used as a diagnostic tool for studying CYP-mediated reactions. Lastly, pharmaceutical mixtures are found in the aquatic environment and adverse effects associated with drug–drug interactions, including CYP inhibition by pharmaceuticals may be of concern. Using liver microsomes from untreated and β-naphthoflavone (BNF)-treated rainbow trout, eight fluorescent CYP-mediated catalytic assays were used to assess in vitro CYP inhibition by four pharmaceuticals: fluoxetine, ciprofloxacin, gemfibrozil and erythromycin. Expressed zebrafish CYP1 proteins (CYP1A, CYP1B1, CYP1C1 and CYP1C2) were assessed for inhibition with selected substrates. All pharmaceuticals decreased the metabolism of a number of substrates. Fluoxetine was the strongest and most broad inhibitor of CYP-mediated reactions in liver microsomes. Zebrafish CYP1s were strongly inhibited by erythromycin and fluoxetine. Although the pharmaceuticals are selective CYP inhibitors in mammals, inhibition across a number of substrates suggests they are broad inhibitors in fish. These data demonstrate that in vitro hepatic CYP inhibition by pharmaceuticals is possible in fish and the patterns seen here are different than what would be expected based on CYP inhibition in mammals.

Metabolism of dl-praeruptorin a in rat liver microsomes using HPLC-electrospray ionization tandem mass spectrometry

dl-Praeruptorin A (Pd-Ia) is the major active constituent of the traditional Chinese medicine Peucedanum praeruptorum Dunn. Recently it has been identified as a novel agent in the treatment and prevention of cardiovascular diseases. Accordingly, we investigated the metabolism of Pd-Ia in rat liver microsomes. The involvement of cytochrome P450 (CYP) and CYP isoforms were identified using a CYP-specific inhibitor (SKF-525A), CYP-selective inhibitors (α-naphthoflavone, metyrapone, fluvastatin, quinidine, disulfiram, ketoconazole and ticlopidine) and CYP-selective inducers (phenobarbital, dexamethasone and β-naphthoflavone). Residual concentrations of the substrate and metabolites were determined by HPLC, and further identified by their mass spectra and chromatographic behavior. These experiments showed that CYP450 is involved in Pd-Ia metabolism, and that the major CYP isoform responsible is CYP3A1/2, which acts in a concentration-dependent manner. Four Pd-Ia metabolites (M1, M2, M3, and M4) were detected after incubation with rat liver microsomes. Hydroxylation was the primary metabolic pathway of Pd-Ia, and possible chemical structures of the metabolites were identified. Further research is now needed to link the metabolism of Pd-Ia to its drug-drug interactions.

Possible involvement of Cyp3a enzymes in the metabolism of tetrahydrocannabinols by mouse brain microsomes

Δ8-Tetrahydrocannabinol (THC) and Δ9-THC were mainly oxidized on the pentyl side chain at the 4′-position by mouse brain microsomes. 5′-Hydroxy-THCs were also formed as minor metabolites. However, 11-hydroxy metabolites of THCs, which are major metabolites of the cannabinoids produced by mouse hepatic microsomes, were not detectable as the metabolites formed by mouse brain microsomes. Cytochrome P450 (CYP) enzymes involved in the brain microsomal metabolism of Δ8-THC and Δ9-THC were identified by using CYP-selective inhibitors. The 4′- and 5′-hydroxylations of THCs were strongly inhibited by ketoconazole and troleandomycin, the known inhibitors of CYP3A enzymes, but not by other isozyme-selective inhibitors, such as 7,8-benzoflavone (CYP1A), quinidine (CYP2D), and sulfaphenazole (CYP2C). These results indicate that Cyp3a enzymes are responsible at least in part for the metabolism of Δ8-THC and Δ9-THC by mouse brain microsomes. Our present results using mouse brain seem to support the idea that the mode of THC metabolism by CYP enzymes in human brain is different from that in human liver.

CYP1A2-mediated biotransformation of cardioactive 2-thienylidene-3,4-methylenedioxybenzoylhydrazine (LASSBio-294) by rat liver microsomes and human recombinant CYP enzymes

We describe herein the metabolic fate of cardioactive 1,3-benzodioxolyl N-acylhydrazone prototype LASSBio-294 (4) and the structural identification of its major phase I metabolite from rat liver microsomal assays. Our results confirmed the hard-metabolic character of N-acylhydrazone (NAH) framework of LASSBio-294 (4). The development of a reproducible analytical methodology for the major metabolite by using HPLC–MS and the comparison with an authentic synthetic sample, allowed us to identify 2-thienylidene 3,4-dihydroxybenzoylhydrazine derivative (7), formed by oxidative scission of methylenedioxy bridge of LASSBio-294, as the main metabolite formed by action of CYP1A2 isoform. The identification of this isoform in the LASSBio-294 in the clearance of LASSBio-294 (4) oxidation was performed by the use of selective CYP inhibitors or human recombinant CYP enzymes.

Evaluation of Cytochrome P450 Inhibition Assays Using Human Liver Microsomes by a Cassette Analysis /LC-MS/MS

In vitro inhibition assays are used to screen new chemical entities (NCEs) for inhibition studies by using human liver microsomes. High-throughput inhibition assays using pooling methods have been developed to keep pace with screening requirements at the lead ADME stage. This method can determine IC(50) NCEs using microsomes from various organs from any species. A cassette analysis inhibition assay for five of the major CYP enzymes (phenacetin for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, bufurarol for CYP2D6 and midazolam, nifedipine and atorvastatin for CYP3A4) in pooled human liver microsomes using ultraperformance liquid chromatography tandem mass spectrometry (LC/MS/MS) were developed. The major metabolites of seven CYP-specific probe substrates for the five P450 isoforms were monitored and quantified to determine IC(50) values. Human liver microsomal incubation samples at each test compound concentration were combined and analyzed simultaneously by the LC/MS/MS method. The incubation was performed using the selective CYP inhibitors for each isoform: fluvoxamine (CYP1A2), sulphaphenazole (CYP2C9), ticlopidine (CYP2C19), quinidine (CYP2D6), and Ketoconazole (CYP3A4). Similar IC(50) results were obtained using the cassette analysis and discrete analysis method. The IC(50) values determined for typical CYP inhibitors were reproducible and consistent with those reported in the literature. The assay was fully automated in 96 well plate formats using Microlab 4000 series (Hamilton) coupled with two termomixer comfort (Eppendorf). An overall 70% time savings was achieved by pooling four isoforms samples (1A2, 2C9, 2C19 and 2D6) into one sample and also by pooling three CYP 3A4 substrate samples (MDZ, ATR, NIF) into one sample. Cassette analysis minimized the number of injections on LC/MS/MS analysis which results in a decrease in the LC/MS/MS analysis time.