Automated screening with confirmation of mechanism-based inactivation of CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP1A2 in pooled human liver microsomes.

A strategy is proposed to profile compounds for mechanism-based inactivation of CYP3A4, CYP2C19, CYP2C9, CYP2D6, and CYP1A2 based on an apparent partition ratio screen. Potent positives from the screen are confirmed by time- and concentration-dependent inactivation assays. Quasi-irreversible inhibitions are then differentiated from irreversible inactivations by oxidation with potassium ferricyanide and/or dialysis. The three-step screening procedure has been validated with acceptable accuracy and precision for detection and confirmation of mechanism-based inactivators in drug discovery. We report here the apparent partition ratios for 19 mechanism-based inactivators and four quasi-irreversible inhibitors obtained under the same experimental conditions. The apparent partition ratio screen was automated to provide throughput for determining structure-mechanism-based inactivation relationships. Information about reversibility can be used to assess potential toxicity mediated by covalent adducts, as well as the potential for pharmacokinetic drug-drug interactions. Direct comparison of known mechanism-based inactivators and quasi-irreversible inhibitors, based on our screening of apparent partition ratios, has identified ritonavir, mibefradil, and azamulin as highly effective mechanism-based inactivators; e.g., 1 mol of CYP3A4 was inactivated on turnover of about 2 mol of compound. Other mechanism-based inactivators we identified include bergamottin (CYP1A2 besides previously reported CYP3A4), troglitazone (CYP3A4), rosiglitazone (CYP3A4), and pioglitazone (CYP3A4). Comparison of the apparent partition ratios and inactivation clearance data for the three glitazones suggests that the chromane moiety on troglitazone contributes to its greater potency for mechanism-based inactivation.     

CYP2C19基因型和CYP2C9对人肝微粒体中氟西汀N-去甲基代谢的影响(英文)

目的:本实验旨在研究CYP2C19基因型人肝微粒体中氟西汀N-去甲基代谢的酶促动力学特点并鉴定参与此代谢途径的细胞色素P-450酶。方法:测定基因型CYP2C19肝微粒体中去甲氟西汀形成的酶促动力学。鉴定氟西汀N-去甲基酶活性与细胞色素P-450 2C9,2C19,1A2和2D6酶活性的相关性,同时应用各种细胞色素P-450酶的选择性抑制剂和化学探针进行抑制实验,从而确定参与氟西汀N-去甲基代谢的细胞色素P-450酶。结果:去甲氟西汀生成的酶促动力学数据符合单酶模型,并具有Michaelis-Menten动力学特征。当底物浓度为氟西汀25μmol/L和100μmol/L时,去甲氟西汀(N-FLU)的生成率分别与甲磺丁脲3-羟化酶活性显著相关(r_1=0.821,P_1=0.001;r_2=0.668,P_2=0.013),当底物浓度为氟西汀100μmol/L时,N-FLU的生成率与S-美芬妥因4’-羟化酶活性显著相关(r=0.717,P=0.006)。PM肝微粒中磺胺苯吡唑和醋竹桃霉素对氟西汀N-去甲基代谢的抑制作用显著大于EM(73％vs 45％,P<0.01)。结论:在生理底物浓度下,CYP2C9是催化人肝微粒体中氟西汀N-去甲基代谢的主要CYP-450酶;而高底物浓度时,以CYP2C19的作用为主。     

Contribution of CYP3A4, CYP2B6, and CYP2C9 isoforms to N-demethylation of ketamine in human liver microsomes.

Ketamine is a widely used drug for its anesthetic and analgesic properties; it is also considered as a drug of abuse, as many cases of ketamine illegal consumption were reported. Ketamine is N-demethylated by liver microsomal cytochrome P450 into norketamine. The identification of the enzymes responsible for ketamine metabolism is of great importance in clinical practice. In the present study, we investigated the metabolism of ketamine in human liver microsomes at clinically relevant concentrations. Liver to plasma concentration ratio of ketamine was taken into consideration. Pooled human liver microsomes and human lymphoblast-expressed P450 isoforms were used. N-demethylation of ketamine was correlated with nifedipine oxidase activity (CYP3A4-specific marker reaction), and it was also correlated with S-mephenytoin N-demethylase activity (CYP2B6-specific marker reaction). Orphenadrine, a specific inhibitor to CYP2B6, and ketoconazole, a specific inhibitor to CYP3A4, inhibited the N-demethylation of ketamine in human liver microsomes. In human lymphoblast-expressed P450, the activities of CYP2B6 were higher than those of CYP3A4 and CYP2C9 at three concentrations of ketamine, 0.005, 0.05, and 0.5 mM. When these results were extrapolated using the average relative content of these P450 isoforms in human liver, CYP3A4 was the major enzyme involved in ketamine N-demethylation. The present study demonstrates that CYP3A4 is the principal enzyme responsible for ketamine N-demethylation in human liver microsomes and that CYP2B6 and CYP2C9 have a minor contribution to ketamine N-demethylation at therapeutic concentrations of the drug.     

Effects of individual ginsenosides, ginkgolides and flavonoids on CYP2C19 and CYP2D6 activity in human liver microsomes

1. The effects of four individual ginsenosides (Rb1, Rb2, Rc and Rd), two ginkgolides (A and B) and one flavonoid (quercetin) on CYP2C19-dependent S-mephenytoin 4 cent-hydroxylation and CYP2D6-mediated bufuralol 1 cent-hydroxylation were evaluated in human liver microsomes. 2. Increasing concentrations of each test compound were added to microsomal incubation mixtures containing a well-characterized marker substrate (S-mephenytoin for CYP2C19 or bufuralol for CYP2D6) to determine their IC(50) values (compound concentration yielding 50% inhibition of a marker enzyme activity), which were estimated by graphical inspection. 3. For CYP2C19, the IC(50) values were 46, 46 and 62 micromol/L for ginsenoside Rd, quercetin and ginsenoside Rb2, respectively, whereas only ginsenoside Rd had an IC(50) value of 57 micromol/L for CYP2D6. 4. The data suggest that the tested compounds are not likely to inhibit the metabolism of the concurrent use of a given drug whose primary route of elimination is through CYP2C19 or CYP2D6.     

CYP2C19 participates in tolbutamide hydroxylation by human liver microsomes.

Tolbutamide is a sulfonylurea-type oral hypoglycemic agent whose action is terminated by hydroxylation of the tolylsulfonyl methyl moiety catalyzed by cytochrome P-450 (CYP) enzymes of the human CYP2C subfamily. Although most studies have implicated CYP2C9 as the exclusive catalyst of hepatic tolbutamide hydroxylation in humans, there is evidence that other CYP2C enzymes (e.g., CYP2C19) may also participate. To that end, we used an immunochemical approach to assess the role of individual CYP2Cs in microsomal tolbutamide metabolism. Polyclonal antibodies were raised to CYP2C9 purified from human liver, and were then back-adsorbed against recombinant CYP2C19 coupled to a solid-phase support. Western blotting revealed that the absorbed anti-human CYP2C9 preparation reacted with only recombinant CYP2C9 and the corresponding native protein in hepatic microsomes, and no longer recognized CYP2C19 and CYP2C8. Monospecific anti-CYP2C9 not only retained the ability to inhibit CYP2C9-catalyzed reactions, as evidenced by its marked (90%) inhibition of diclofenac 4'-hydroxylation by purified CYP2C9 and by human liver microsomes, but also exhibited metabolic specificity, as indicated by its negligible (<15%) inhibitory effect on S-mephenytoin 4'-hydroxylation by purified CYP2C19 or hepatic microsomes containing CYP2C19. Monospecific anti-CYP2C9 was also found to inhibit rates of tolbutamide hydroxylation by 93 +/- 4 and 78 +/- 6% in CYP2C19-deficient and CYP2C19-containing human liver microsomes, respectively. Taken together, our results indicate that both CYP2C9 and CYP2C19 are involved in tolbutamide hydroxylation by human liver microsomes, and that CYP2C19 underlies at least 14 to 22% of tolbutamide metabolism. Although expression of CYP2C19 in human liver is less than that of CYP2C9, it may play an important role in tolbutamide disposition in subjects expressing either high levels of CYP2C19 or a catalytically deficient CYP2C9 enzyme.     

Effect of antifungal drugs on cytochrome P450 (CYP) 2C9, CYP2C19, and CYP3A4 activities in human liver microsomes

The effects of five antifungal drugs, fluconazole, itraconazole, micafungin, miconazole, and voriconazole, on cytochrome P450 (CYP) 2C9-mediated tolbutamide hydroxylation, CYP2C19-mediated S-mephenytoin 4'-hydroxylation, and CYP3A4-mediated nifedipine oxidation activities in human liver microsomes were compared. In addition, the effects of preincubation were estimated to investigate the mechanism-based inhibition. The IC50 value against tolbutamide hydroxylation was the lowest for miconazole (2.0 microM), followed by voriconazole (8.4 microM) and fluconazole (30.3 microM). Similarly, the IC50 value against S-mephenytoin 4'-hydroxylation was the lowest for miconazole (0.33 microM), followed by voriconazole (8.7 microM) and fluconazole (12.3 microM). On the other hand, micafungin at a concentration of 10 or 25 microM neither inhibited nor stimulated tolbutamide hydroxylation and S-mephenytoin 4'-hydroxylation, and the IC50 values for itraconazole against these were greater than 10 microM. These results suggest that miconazole is the strongest inhibitor of CYP2C9 and CYP2C19, followed by voriconazole and fluconazole, whereas micafungin would not cause clinically significant interactions with other drugs that are metabolized by CYP2C9 or CYP2C19 via the inhibition of metabolism. The IC50 value of voriconazole against nifedipine oxidation was comparable with that of fluconazole and micafungin and higher than that of itraconazole and miconazole. The stimulation of the inhibition of CYP2C9-, CYP2C19-, or CYP3A4-mediated reactions by 15-min preincubation was not observed for any of the antifungal drugs, suggesting that these drugs are not mechanism-based inhibitors.     

Contribution of CYP2C9, CYP2A6, and CYP2B6 to valproic acid metabolism in hepatic microsomes from individuals with the CYP2C9*1/*1 genotype.

The present study investigated the role of specific human cytochrome P450 (CYP) enzymes in the in vitro metabolism of valproic acid (VPA) by a complementary approach that used individual cDNA-expressed CYP enzymes, chemical inhibitors of specific CYP enzymes, CYP-specific inhibitory monoclonal antibodies (MAbs), individual human hepatic microsomes, and correlational analysis. cDNA-expressed CYP2C9*1, CYP2A6, and CYP2B6 were the most active catalysts of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation. The extent of 4-OH-VPA and 5-OH-VPA formation by CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C19, CYP2D6, CYP2E1, CYP4A11, CYP4F2, CYP4F3A, and CYP4F3B was only 1-8% of the levels by CYP2C9*1. CYP2A6 was the most active in catalyzing VPA 3-hydroxylation, whereas CYP1A1, CYP2B6, CYP4F2, and CYP4F3B were less active. Correlational analyses of VPA metabolism with CYP enzyme-selective activities suggested a potential role for hepatic microsomal CYP2A6 and CYP2C9. Chemical inhibition experiments with coumarin (CYP2A6 inhibitor), triethylenethiophosphoramide (CYP2B6 inhibitor), and sulfaphenazole (CYP2C9 inhibitor) and immunoinhibition experiments (including combinatorial analysis) with MAb-2A6, MAb-2B6, and MAb-2C9 indicated that the CYP2C9 inhibitors reduced the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by 75-80% in a panel of hepatic microsomes from donors with the CYP2C9*1/*1 genotype, whereas the CYP2A6 and CYP2B6 inhibitors had a small effect. Only the CYP2A6 inhibitors reduced VPA 3-hydroxylation (by approximately 50%). The extent of inhibition correlated with the catalytic capacity of these enzymes in each microsome sample. Overall, our novel findings indicate that in human hepatic microsomes, CYP2C9*1 is the predominant catalyst in the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA, whereas CYP2A6 contributes partially to 3-OH-VPA formation.     

Glucuronidation versus oxidation of the flavonoid galangin by human liver microsomes and hepatocytes.

In a previous study, we used human liver microsomes for the first time to study cytochrome P450 (P450)-mediated oxidation of the flavonoid galangin. The combination of CYP1A2 and CYP2C9 produced a V(max)/K(m) value of 13.6 +/- 1.1 microl/min/mg of protein. In the present extended study, we determined glucuronidation rates for galangin with the same microsomes. Two major and one minor glucuronide were identified by liquid chromatography/mass spectrometry. The V(max)/K(m) values for the two major glucuronides conjugated in the 7- and 3-positions were 155 +/- 30 and 427 +/- 26 microl/min/mg of protein, thus, exceeding that of oxidation by 11 and 31 times, respectively. This highly efficient glucuronidation appeared to be catalyzed mainly by the UDP-glucuronosyltransferase (UGT)1A9 isoform but also by UGT1A1 and UGT2B15. Sulfation of galangin by the human liver cytosol, mediated mainly but not exclusively by sulfotransferase (SULT) 1A1, also appeared to be efficient. These conclusions were strongly supported by experiments using the S9 fraction of the human liver, in which all three metabolic pathways could be directly compared. When galangin metabolism was examined in fresh plated hepatocytes from six donors, glucuronidation clearly predominated followed by sulfation. Oxidation occurred only to a minor extent in two of the donors. This study for the first time establishes that glucuronidation and sulfation of galangin, and maybe other flavonoids, are more efficient than P450-mediated oxidation, clearly being the metabolic pathways of choice in intact cells and therefore likely also in vivo.     

Effect of glimepiride and glibenclamide on S-warfarin 7-hydroxylation by human liver microsomes, recombinant human CYP2C9.1 and CYP2C9.3

The effect of glimepiride on metabolism of S-warfarin to 7-hydroxywarfarin was studied using human liver microsomes and recombinant cytochrome P450 2C9 microsomes (CYP2C9.1 and CYP2C9.3), and was compared with the results from the experiments using glibenclamide as an inhibitor. S-Warfarin 7-hydroxylation by recombinant CYP2C9.1 and CYP2C9.3 was inhibited by glimepiride competitively. The apparent K(i) value of glimepiride was lower at CYP2C9.3 than at CYP2C9.1. Glimepiride also inhibited 7-hydroxylation of S-warfarin in a competitive manner by microsomes from human liver which showed the genotypes of CYP2C9, as CYP2C9*1/*1 or CYP2C9*1/*3. The apparent K(i) value of glimepiride was lower than that of glibenclamide. These results may provide valuable information for optimizing the anticoagulant activity of warfarin when glimepiride is co-administered to patients.     

Inhibitory effects of angiotensin receptor blockers on CYP2C9 activity in human liver microsomes

We investigated the inhibitory effects of the angiotensin receptor blockers (ARBs), candesartan, irbesartan, losartan, losartan active metabolite (EXP-3174), olmesartan, telmisartan and valsartan (0.3-300 microM), on the CYP2C9 activity in human liver microsomes using (S)-(-)-warfarin as a typical CYP2C9 substrate. Except for olmesartan and valsartan, these ARBs inhibited the activity of 7-hydroxylation of (S)-(-)-warfarin with IC50 values of 39.5-116 microM. Of six synthetic derivatives of olmesartan, five compounds which possess either alkyl groups or a chloro group at the same position as that of the hydroxyisopropyl group in olmesartan inhibited CYP2C9 activity with IC50 values of 21.7-161 microM. Olmesartan and the olmesartan analogue, RNH-6272, both having a hydroxyisopropyl group, showed no inhibition, indicating that the hydrophilicity of this group greatly contributes to the lack of CYP2C9 inhibition by these two compounds. A three-dimensional model for docking between EXP-3174 and CYP2C9 indicated that the chloro group of EXP-3174 is oriented to a hydrophobic pocket in the CYP2C9 active site, indicating that the lipophilicity of the group present in ARBs at the position corresponding to that of the hydroxyisopropyl group in olmesartan is important in inhibiting CYP2C9 activity.     

Role of CYP3A4 and CYP2C19 in the stereoselective metabolism of lansoprazole by human liver microsomes

OBJECTIVE: The aim of this investigation was to clarify the stereoselective properties in lansoprazole metabolism by monitoring the metabolic consumption for each enantiomer and the formation of the main metabolites, lansoprazole sulfone and 5-hydroxylansoprazole, in the presence of human liver microsomal enzymes. METHODS: Human liver microsomes or recombinant cytochrome P450 (CYP) enzymes were incubated with either (+/- )-, (+)-, or (-)-lansoprazole in the presence of reduced nicotinamide adenine dinucleotide phosphate. The metabolic consumption of lansoprazole enantiomers was estimated from the amounts of enantiomers consumed by microsomal enzymes after incubation at 37 degrees C for 60 min. Metabolites of lansoprazole, lansoprazole sulfone, and 5-hydroxylansoprazole were determined after incubation at 37 degrees C for 20 min, and kinetic parameters [Michaelis constant (Km) and maximum velocity (Vmax)] were obtained using Eadie-Hofstee plots. RESULTS: (-)-Lansoprazole was metabolized more preferentially than (+)-lansoprazole in human liver microsomes. Stereoselective sulfoxidation and hydroxylation [(+) > (-)] were observed in human liver microsomes. Strikingly, in sulfoxidation, a significantly higher intrinsic clearance (Vmax,l/Km,l) of (-)-lansoprazole (0.023 +/- 0.001 ml/min/mg) than (+)-lansoprazole (0.006 +/- 0.000 ml/min/mg) was observed. Consequently, sulfoxidation is likely to play an important role in the stereoselective metabolism of lansoprazole enantiomers. P450-isoform specificity for each enantiomer was evident. CYP3A4, which mainly catalyzed sulfoxidation, was more active toward (-)-lansoprazole in either a chiral or racemic drug as a substrate. CYP2C19, which catalyzed hydroxylation, preferentially metabolized (+)-lansoprazole. The consumption of (+)-lansoprazole was markedly inhibited by (-)-lansoprazole, indicating a metabolic enantiomer/enantiomer interaction. However, this alteration of recombinant CYP2C19 specificity for (+)-lansoprazole did not appear in metabolism in human liver microsomes. CONCLUSIONS: Stereoselective metabolism was observed in human liver microsomes, and this stereoselectivity was mainly based on CYP3A4 specificity for preferable metabolism of (-)-lansoprazole.     

Role of CYP2C19 in stereoselective hydroxylation of mephobarbital by human liver microsomes.

The 4-hydroxylation of mephobarbital enantiomers was investigated by using human liver microsomes from the extensive metabolizers (EM) and poor metabolizers of CYP2C19. The 4-hydroxylase activity of R-mephobarbital in the EM microsomes was >10 times higher than that of S-mephobarbital. In the poor metabolizer microsomes, the 4-hydroxylase activity of R-mephobarbital was much lower than that in the EM microsomes, and the ratio of 4-hydroxylase activity of R-mephobarbital to that of S-mephobarbital was also lower than that in the EM microsomes. Moreover, the 4-hydroxylase activity of R-mephobarbital showed a high correlation (r = 0.985, p<0.001) with the 4'-hydroxylase activity of S-mephenytoin in a panel of nine human liver microsomes. Anti-CYP2C antibody inhibited R-mephobarbital 4-hydroxylase activity by 85% of the control activity. R-Mephobarbital competitively inhibited S-mephenytoin 4'-hydroxylase activity (K(i) = 34 microM), while S-mephenytoin inhibited R-mephobarbital 4-hydroxylase activity (K(i) = 103 microM). Among the seven cDNA-expressed CYPs studied, only CYP2C19 catalyzed R-mephobarbital 4-hydroxylation. These findings suggest that the 4-hydroxylation of mephobarbital catalyzed by CYP2C19 is preferential for R-enantiomer in human liver microsomes.     

Metabolism of (+)- and (-)-limonenes to respective carveols and perillyl alcohols by CYP2C9 and CYP2C19 in human liver microsomes.

Limonene, a monocyclic monoterpene, is present in orange peel and other plants and has been shown to have chemopreventive activities. (+)- and (-)-Limonene enantiomers were incubated with human liver microsomes and the oxidative metabolites thus formed were analyzed using gas chromatography-mass spectrometry. Two kinds of metabolites, (+)- and (-)-trans-carveol (a product by 6-hydroxylation) and (+)- and (-)-perillyl alcohol (a product by 7-hydroxylation), were identified, and the latter metabolites were found to be formed more extensively, the former ones with liver microsomes prepared from different human samples. Sulfaphenazole, flavoxamine, and antibodies raised against purified liver cytochrome P450 (P450) 2C9 that inhibit both CYP2C9- and 2C19-dependent activities, significantly inhibited microsomal oxidations of (+)- and (-)-limonene enantiomers. The limonene oxidation activities correlated well with contents of CYP2C9 and activities of tolbutamide methyl hydroxylation in liver microsomes of 62 human samples, whereas these activities did not correlate with contents of CYP2C19 and activities of S-mephenytoin 4-hydroxylation. Of 11 recombinant human P450 enzymes (expressed in Trichoplusia ni cells) tested, CYP2C8, 2C9, 2C18, 2C19, and CYP3A4 catalyzed oxidations of (+)- and (-)-limonenes to respective carveols and perillyl alcohol. Interestingly, human CYP2B6 did not catalyze limonene oxidations, whereas rat CYP2B1 had high activities in catalyzing limonene oxidations. These results suggest that both (+)- and (-)-limonene enantiomers are oxidized at 6- and 7-positions by CYP2C9 and CYP2C19 in human liver microsomes. CYP2C9 may be more important than CYP2C19 in catalyzing limonene oxidations in human liver microsomes, since levels of the former protein are more abundant than CYP2C19 in these human samples. Species-related differences exist in the oxidations of limonenes in CYP2B subfamily in rats and humans.     

Inhibitory effects of H1-antihistamines on CYP2D6- and CYP2C9-mediated drug metabolic reactions in human liver microsomes

OBJECTIVE: To screen the inhibitory effects of H1-antihistamines on hepatic bufuralol 1'-hydroxylation and on tolbutamide 4-methylhydroxylation in human liver microsomes. METHODS: Bufuralol 1'-hydroxylation and tolbutamide 4-methylhydroxylation were used as index reactions for CYP2D6 and CYP2C9, respectively. The metabolites of both reactions were measured using high-performance liquid chromatography and were used as indicators of whether CYP2D6 or CYP2C9 activities were inhibited or unaffected by the agents. RESULTS: All five H1-antihistamines studied showed a concentration-dependent inhibition of CYP2D6-mediated bufuralol 1'-hydroxylation with 50% inhibitory concentration (IC50) values of 32-109 microM. Cyclizine and promethazine showed inhibitory effects on tolbutamide 4-methylhydroxylation with IC20 values of 85 microM and 88 microM, respectively. Tripelennamine, chlorpheniramine, and diphenhydramine showed no inhibitory effects on CYP2C9. CONCLUSION: All five H1-antihistamines studied inhibited CYP2D6 markedly, but only cyclizine and promethazine inhibited CYP2C9 at concentrations above that usually seen in plasma. Promethazine and chlorpheniramine inhibited CYP2D6 at concentrations that are very close to their therapeutic plasma concentrations. Further studies in humans, especially in poor metabolizers of CYP2D6, will be required to confirm these findings.     

Evidence for involvement of polymorphic CYP2C19 and 2C9 in the N-demethylation of sertraline in human liver microsomes

AIMS: The present study was designed to define the kinetic behaviour of sertraline N-demethylation in human liver microsomes and to identify the isoforms of cytochrome P450 involved in this metabolic pathway. METHODS: The kinetics of the formation of N-demethylsertraline were determined in human liver microsomes from six genotyped CYP2C19 extensive (EM) and three poor metabolisers (PM). Selective inhibitors of and specific monoclonal antibodies to various cytochrome P450 isoforms were also employed. RESULTS: The kinetics of N-demethylsertraline formation in all EM liver microsomes were fitted by a two-enzyme Michaelis-Menten equation, whereas the kinetics in all PM liver microsomes were best described by a single-enzyme Michaelis-Menten equation similar to the low-affinity component found in EM microsomes. Mean apparent Km values for the high-and low-affinity components were 1.9 and 88 microm and V max values were 33 and 554 pmol min-1 mg-1 protein, respectively, in the EM liver microsomes. Omeprazole (a CYP2C19 substrate) at high concentrations and sulphaphenazole (a selective inhibitor of CYP2C9) substantially inhibited N-demethylsertraline formation. Of five monoclonal antibodies to various cytochrome P450 forms tested, only anti-CYP2C8/9/19 had any inhibitory effect on this reaction. The inhibition of sertraline N-demethylation by anti-CYP2C8/9/19 was greater in EM livers than in PM livers at both low and high substrate concentrations. However, anti-CYP2C8/9/19 did not abolish the formation of N-demethylsertraline in the microsomes from any of the livers. CONCLUSIONS: The polymorphic enzyme CYP2C19 catalyses the high-affinity N-demethylation of sertraline, while CYP2C9 is one of the low-affinity components of this metabolic pathway.     

CYP2B6, CYP3A4, and CYP2C19 are responsible for the in vitro N-demethylation of meperidine in human liver microsomes.

Meperidine is an opioid analgesic metabolized in the liver by N-demethylation to normeperidine, a potent stimulant of the central nervous system. The purpose of this study was to identify the human cytochrome P450 (P450) enzymes involved in normeperidine formation. Our in vitro studies included 1) screening 16 expressed P450s for normeperidine formation, 2) kinetic experiments on human liver microsomes and candidate P450s, and 3) correlation and inhibition experiments using human hepatic microsomes. After normalization by its relative abundance in human liver microsomes, CYP2B6, CYP3A4, and CYP2C19 accounted for 57, 28, and 15% of the total intrinsic clearance of meperidine. CYP3A5 and CYP2D6 contributed to < 1%. Formation of normeperidine significantly correlated with CYP2B6-selective S-mephenytoin N-demethylation (r = 0.88, p < 0.0001 at 75 > microM meperidine, and r = 0.89, p < 0.0001 at 350 microM meperidine, n = 21) and CYP3A4-selective midazolam 1'-hydroxylation (r = 0.59, p < 0.01 at 75 microM meperidine, and r = 0.55, p < 0.01 at 350 microM meperidine, n = 23). No significant correlation was observed with CYP2C19-selective S-mephenytoin 4'-hydroxylation (r = 0.36, p = 0.2 at 75 microM meperidine, and r = 0.02, p = 0.9 at 350 microM meperidine, n = 13). An anti-CYP2B6 antibody inhibited normeperidine formation by 46%. In contrast, antibodies inhibitory to CYP3A4 and CYP2C8/9/18/19 had little effect (<14% inhibition). Experiments with thiotepa and ketoconazole suggested inhibition of microsomal CYP2B6 and CYP3A4 activity, whereas studies with fluvoxamine (a substrate of CYP2C19) were inconclusive due to lack of specificity. We conclude that normeperidine formation in human liver microsomes is mainly catalyzed by CYP2B6 and CYP3A4, with a minor contribution from CYP2C19.     

Roles of human CYP2A6 and rat CYP2B1 in the oxidation of (+)-fenchol by liver microsomes

The metabolism of (+)-fenchol was investigated in vitro using liver microsomes of rats and humans and recombinant cytochrome P450 (P450 or CYP) enzymes in insect cells in which human/rat P450 and NADPH-P450 reductase cDNAs had been introduced. The biotransformation of (+)-fenchol was investigated by gas chromatography-mass spectrometry (GC-MS). (+)-Fenchol was oxidized to fenchone by human liver microsomal P450 enzymes. The formation of metabolites was determined by the relative abundance of mass fragments and retention times on GC. Several lines of evidence suggested that CYP2A6 is a major enzyme involved in the oxidation of (+)-fenchol by human liver microsomes. (+)-Fenchol oxidation activities by liver microsomes were very significantly inhibited by (+)-menthofuran, a CYP2A6 inhibitor, and anti-CYP2A6. There was a good correlation between CYP2A6 contents and (+)-fenchol oxidation activities in liver microsomes of ten human samples. Kinetic analysis showed that the V_max/K_m values for (+)-fenchol catalysed by liver microsomes of human sample HG03 were 7.25?nM^?1?min^?1. Human recombinant CYP2A6-catalyzed (+)-fenchol oxidation with a V_max value of 6.96?nmol?min^?1?nmol^?1 P450 and apparent K_m value of 0.09?mM. In contrast, rat CYP2A1 did not catalyse (+)-fenchol oxidation. In the rat (+)-fenchol was oxidized to fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol by liver microsomes of phenobarbital-treated rats. Recombinant rat CYP2B1 catalysed (+)-fenchol oxidation. Kinetic analysis showed that the K_m values for the formation of fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol in rats treated with phenobarbital were 0.06, 0.03 and 0.03?mM, and V_max values were 2.94, 6.1 and 13.8?nmol?min^?1?nmol^?1 P450, respectively. Taken collectively, the results suggest that human CYP2A6 and rat CYP2B1 are the major enzymes involved in the metabolism of (+)-fenchol by liver microsomes and that there are species-related differences in the human and rat CYP2A enzymes.     

Roles of human CYP2A6 and rat CYP2B1 in the oxidation of (+)-fenchol by liver microsomes

The metabolism of (+)-fenchol was investigated in vitro using liver microsomes of rats and humans and recombinant cytochrome P450 (P450 or CYP) enzymes in insect cells in which human/rat P450 and NADPH-P450 reductase cDNAs had been introduced. The biotransformation of (+)-fenchol was investigated by gas chromatography-mass spectrometry (GC-MS). (+)-Fenchol was oxidized to fenchone by human liver microsomal P450 enzymes. The formation of metabolites was determined by the relative abundance of mass fragments and retention times on GC. Several lines of evidence suggested that CYP2A6 is a major enzyme involved in the oxidation of (+)-fenchol by human liver microsomes. (+)-Fenchol oxidation activities by liver microsomes were very significantly inhibited by (+)-menthofuran, a CYP2A6 inhibitor, and anti-CYP2A6. There was a good correlation between CYP2A6 contents and (+)-fenchol oxidation activities in liver microsomes of ten human samples. Kinetic analysis showed that the Vmax/Km values for (+)-fenchol catalysed by liver microsomes of human sample HG03 were 7.25 nM-1 min-1. Human recombinant CYP2A6-catalyzed (+)-fenchol oxidation with a Vmax value of 6.96 nmol min-1 nmol-1 P450 and apparent Km value of 0.09 mM. In contrast, rat CYP2A1 did not catalyse (+)-fenchol oxidation. In the rat (+)-fenchol was oxidized to fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol by liver microsomes of phenobarbital-treated rats. Recombinant rat CYP2B1 catalysed (+)-fenchol oxidation. Kinetic analysis showed that the Km values for the formation of fenchone, 6-exo- hydroxyfenchol and 10-hydroxyfenchol in rats treated with phenobarbital were 0.06, 0.03 and 0.03 mM, and Vmax values were 2.94, 6.1 and 13.8 nmol min-1 nmol-1 P450, respectively. Taken collectively, the results suggest that human CYP2A6 and rat CYP2B1 are the major enzymes involved in the metabolism of (+)-fenchol by liver microsomes and that there are species-related differences in the human and rat CYP2A enzymes.     

Cocktail-substrate assay system for mechanism-based inhibition of CYP2C9, CYP2D6, and CYP3A using human liver microsomes at an early stage of drug development

1. We established a mechanism-based inhibition cocktail-substrate assay system using human liver microsomes and drug–probe substrates that enabled simultaneous estimation of the inactivation of main cytochrome P450 (CYP) enzymes, CYP2C9, CYP2D6, and CYP3A, in drug metabolism.

2. The inactivation kinetic parameters of typical mechanism-based inhibitors, tienilic acid, paroxetine, and erythromycin, for each enzyme in the cocktail-substrate assay were almost in agreement with the values obtained in the single-substrate assay.

3. Using this system, we confirmed that multiple CYP inactivation caused by mechanism-based inhibitors such as isoniazid and amiodarone could be detected simultaneously.

4. Mechanism-based inhibition potency can be estimated by the determination of the observed inactivation rate constants (k_obs) at a single concentration of test compounds because the k_obs of eleven CYP3A inactivators at 10?μM in the assay system nearly corresponded to k_inact/K_I values, an indicator of a compound’s propensity to alter the activity of a CYP in vivo (R²?=?0.97).

5. Therefore, this cocktail-substrate assay is considered to be a powerful tool for evaluating mechanism-based inhibition at an early stage of drug development.

     

Effects of acid and lactone forms of statins on S-warfarin 7-hydroxylation catalyzed by human liver microsomes and recombinant CYP2C9 variants (CYP2C9.1 and CYP2C9.3)

The inhibition of CYP2C9-mediated warfarin metabolism by acid or lactone forms of statin converted in the body and effects of CYP2C9 genetic variants on their inhibition are not fully understood. Here, the effects of acid and lactone forms of statins on S-warfarin 7-hydroxylation were investigated in vitro. S-Warfarin 7-hydroxylase activities of human liver microsomes (HLMs), recombinant CYP2C9.1 (rCYP2C9.1), and rCYP2C9.3 (Ile359Leu variant) in the presence of statins were determined by high-performance liquid chromatography. Lactone forms of atorvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin inhibited the activity of HLMs more potently than the corresponding acid forms, whereas fluvastatin acid showed stronger inhibition than fluvastatin lactone. When the effects of statins on rCYP2C9 variants were examined, inhibition profiles of acid versus lactone forms of statins except for fluvastatin were similar between rCYP2C9.1 and rCYP2C9.3. However, the degrees of inhibition by atorvastatin lactone, fluvastatin acid, fluvastatin lactone, lovastatin lactone, and pitavastatin lactone (K_i values) were significantly different between these variants. These results indicated that lactone forms of statins other than fluvastatin showed more potent inhibition of CYP2C9-catalyzed S-warfarin 7-hydroxylation than the corresponding acid forms. Furthermore, our results indicated that Ile359Leu substitution in CYP2C9 affected the inhibitory potencies of statins.