Sertraline N-demethylation is catalyzed by multiple isoforms of human cytochrome P-450 in vitro.

Sertraline, a new antidepressant of the selective serotonin reuptake inhibitor class, is extensively metabolized to desmethylsertraline in humans. We identified the cytochrome P-450 (CYP) isoforms involved in sertraline N-demethylation using pooled human liver microsomes and cDNA-expressed CYP isoforms. Eadie-Hofstee plots for the sertraline N-demethylation in human liver microsomes were monophasic. The estimated Michaelis-Menten kinetic parameters were: KM = 18.1 +/- 2.0 microM, Vmax = 0.45 +/- 0.03 nmol/min/mg of protein, and Vmax/KM = 25.2 +/- 4.3 microl/min/mg of protein. At the substrate concentration of 20 microM, which approximated the apparent KM value, sulfaphenazole (CYP2C9 inhibitor) and triazolam (CYP3A substrate) reduced the N-demethylation activities by 20 to 35% in human liver microsomes, whereas the inhibition induced by mephenytoin (CYP2C19 substrate) or quinidine (CYP2D6 inhibitor) was marginal. The anti-CYP2B6 antibody inhibited the sertraline N-demethylation activities by 35%. Sertraline N-demethylation activities were detected in all cDNA-expressed CYP isoforms studied. In particular, CYP2C19, CYP2B6, CYP2C9-Arg, CYP2D6-Val, and CYP3A4 all showed relatively high activity. When the contributions of CYP2D6, CYP2C9, CYP2B6, CYP2C19, and CYP3A4 were estimated from the Vmax/KM of cDNA-expressed CYP isoforms and from their contents in pooled human liver microsomes, the values were found to be 35, 29, 14, 13, and 9%, respectively. The results suggest that at least five isoforms of CYP (CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4) are involved in the sertraline N-demethylation in human liver microsomes and that the contribution of any individual isoform does not exceed 40% of overall metabolism. Therefore, concurrent administration of a drug that inhibits a specific CYP isoform is unlikely to cause a marked increase in the plasma concentration of sertraline.     

Silybin is metabolized by cytochrome P450 2C8 in vitro.

Silybin (a flavonolignan, the main component of silymarin, an extract from the seeds of Silybum marianum) has been used to date mostly as a hepatoprotectant. However, it also has other interesting activities, e.g., anticancer and hypocholesterolemic effects. It is also known that silybin can inhibit the activities of the cytochrome P450 (P450) enzymes. In this study, a weak interaction of silybin with human microsomal CYP2E1, 2A6, 2B6, 2C19, and 2D6 (IC(50) > or = 250 microM) was found; a moderate inhibition was observed for CYP1A2 and 2C8. The most prominent inhibition effect was found with CYP3A4 and CYP2C9 (IC(50) < or = 50 microM). Using mass spectometry detection, production of O-demethylated (the main metabolite) as well as hydroxylated derivatives of silybin formed by P450 enzymes was detected. The effect of different P450 inhibitors on the formation of O-demethylated product was also studied. In particular, a relatively specific inhibitor of CYP2C8 (quercetin) markedly inhibited the formation of this metabolite. With the help of recombinant enzymes (bactosomes), it was confirmed that the CYP2C8 enzyme is responsible for the reaction leading to O-demethylated silybin.     

Selegiline metabolism and cytochrome P450 enzymes: in vitro study in human liver microsomes

Although being a drug therapeutically used for a long time, the enzymatic metabolism of selegiline has not been adequately studied. In the current work we have studied the cytochrome P450 (CYP)-catalyzed oxidative metabolism of selegiline to desmethylselegiline and 1-methamphetamine and the effects of selegiline, desmethylselegiline and 1-methamphetamine on hepatic CYP enzymes in human liver microsomes in vitro. The apparent Km values for desmethylselegiline and 1-methamphetamine formation were on an average 149 microM and 293 microM, and the apparent Vmax values, 243 pmol/min./mg and 1351 pmol/min./mg, respectively. Furafylline and ketoconazole, the known reference inhibitors for CYP1A2 and CYP3A4, respectively, inhibited the formation of desmethylselegiline with Ki value of 1.7 microM and 15 microM. Ketoconazole inhibited also the formation of 1-methamphetamine with Ki of 18 microM. Fluvoxamine, an inhibitor of CYP1A2, CYP2C19 and CYP3A4, inhibited the formation of desmethylselegiline and 1-methamphetamine with Ki values of 9 and 25 microM, respectively. On the basis of these results we suggest that CYP1A2 and CYP3A4 contribute to the formation of desmethylselegiline and that CYP3A4 participates in the formation of 1-methamphetamine. In studies with CYP-specific model activities, both selegiline and desmethylselegiline inhibited the CYP2C19-mediated S-mephenytoin 4'-hydroxylation with average IC50 values of 21 microM and 26 microM, respectively. The Ki for selegiline was determined to be around 7 microM. Selegiline inhibited CYP1A2-mediated ethoxyresorufin O-deethylation with a Ki value of 76 microM. Inhibitory potencies of selegiline, desmethylselegiline and 1-methamphetamine towards other CYP-model activities were much lower. On this basis, selegiline and desmethylselegiline were shown to have a relatively high affinity for CYP2C19, but no evidence about selegiline metabolism by CYP2C19 was obtained.     

In vitro metabolism of eupatilin by multiple cytochrome P450 and UDP-glucuronosyltransferase enzymes

Eupatilin, a pharmacologically active flavone derived from Artemisia plants, is extensively metabolized to eupatilin glucuronide, 4-O-desmethyleupatilin and 4-O-desmethyleupatilin glucuronide in human liver microsomes. This study characterized the human liver cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes responsible for the metabolism of eupatilin. The specific CYPs responsible for O-demethylation of eupatilin to the major metabolite, 4-O-desmethyleupatilin were identified using a combination of correlation analysis, immuno-inhibition, chemical inhibition in human liver microsomes and metabolism by human cDNA-expressed CYP enzymes. UGT enzymes involved in the eupatilin glucuronidation were identified using pooled human liver microsomes and human cDNA-expressed UGT enzymes. Eupatilin was predominantly metabolized by CYP1A2 and, to a lesser extent, CYP2C8 mediated O-demethylation of eupatilin to 4-O-desmethyleupatilin. Eupatilin glucuronidation was catalysed by UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT1A10.     

In vitro metabolism of eupatilin by multiple cytochrome P450 and UDP-glucuronosyltransferase enzymes

Eupatilin, a pharmacologically active flavone derived from Artemisia plants, is extensively metabolized to eupatilin glucuronide, 4-O-desmethyleupatilin and 4-O-desmethyleupatilin glucuronide in human liver microsomes. This study characterized the human liver cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes responsible for the metabolism of eupatilin. The specific CYPs responsible for O-demethylation of eupatilin to the major metabolite, 4-O-desmethyleupatilin were identified using a combination of correlation analysis, immuno-inhibition, chemical inhibition in human liver microsomes and metabolism by human cDNA-expressed CYP enzymes. UGT enzymes involved in the eupatilin glucuronidation were identified using pooled human liver microsomes and human cDNA-expressed UGT enzymes. Eupatilin was predominantly metabolized by CYP1A2 and, to a lesser extent, CYP2C8 mediated O-demethylation of eupatilin to 4-O-desmethyleupatilin. Eupatilin glucuronidation was catalysed by UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT1A10.     

Drug-drug interactions in the metabolism of imidafenacin: role of the human cytochrome P450 enzymes and UDP-glucuronic acid transferases, and potential of imidafenacin to inhibit human cytochrome P450 enzymes

Imidafenacin (IM), 4-(2-methyl-1H-imidazol-1-yl)-2,2-diphenylbutanamide, is a newly synthesized antimuscarinic drug developed for the treatment of overactive bladder. To predict clinically relevant drug interactions in the metabolism of IM, the paper investigated: (1) the major enzymes responsible for the metabolism of IM, (2) the effects of concomitant drugs on the inhibition of metabolism of IM, and (3) the effects of IM and its metabolites on the inhibition of human cytochrome P450 (CYP). The elimination of IM and production of oxidative metabolites were mainly catalysed by recombinant CYP3A4, and the elimination of IM by human liver microsomes (HLM) was markedly inhibited by co-incubation with ketoconazole. The production of the N-glucuronide metabolite was only catalysed by recombinant UGT1A4. Clinically established CYP3A4 inhibitors including itraconazole, ketoconazole, erythromycin and clarithromycin inhibited the elimination of IM in HLM. IM and its major metabolites did not affect the activities of CYP enzymes in vitro. The results suggest that the major enzymes responsible for the metabolism of IM are CYP3A4 and UGT1A4, and oxidative metabolism of IM is reduced by concomitant administration of CYP3A4 inhibitors. In contrast, IM and its metabolites have no inhibitory effect on the CYP-mediated metabolism of concomitant drugs.     

Inhibition and induction of human cytochrome P450 enzymes in vitro by capsaicin

1. Widespread exposure to capsaicin occurs through food and topical medicines. To investigate potential food-drug or drug–drug interactions, capsaicin was evaluated in vitro against seven human drug-metabolizing cytochrome P450 (CYP) enzymes.

2. At concentrations occurring after ingestion of chili peppers or topical administration of a high-concentration patch, capsaicin did not cause direct inhibition of any CYP enzyme. Direct inhibition was only observed at much higher concentrations; the lowest IC₅₀ value was 2.0 μM. For CYP2E1, the IC₅₀ value was too high to calculate. With pre-incubation, inhibition decreased for CYP1A2, 2C9, 2C19 and 3A4/5, whereas inhibition of CYP2B6 increased and moderately increased for CYP2D6.

3. Induction of CYP activity was evaluated in microsomes from hepatocyte primary cultures. Capsaicin did not induce CYP1A2, 2B6, 2C9, 2C19, 2E1 or 3A4/5. 10 μM capsaicin caused a statistically significant increase in CYP1A2 activity (8.6% of the positive control).

4. Inhibition of drug metabolism by capsaicin should be minimal, as the ratio of [I]/K_i for direct inhibition is?<?0.1. Although pre-incubation did enhance the potency for CYP2B6 inhibition to 5.1 μM, given that exposure to capsaicin from either food or a topical medicine is very low (≤58?nM) and transient, effects on CYPs appear unlikely.

     

In vitro metabolism of naphthalene by human liver microsomal cytochrome P450 enzymes.

The polycyclic aromatic hydrocarbon naphthalene is an environmental pollutant, a component of jet fuel, and, since 2000, has been reclassified as a potential human carcinogen. Few studies of the in vitro human metabolism of naphthalene are available, and these focus primarily on lung metabolism. The current studies were performed to characterize naphthalene metabolism by human cytochromes P450. Naphthalene metabolites from pooled human liver microsomes (pHLMs) were trans-1,2-dihydro-1,2-naphthalenediol (dihydrodiol), 1-naphthol, and 2-naphthol. Metabolite production generated Km values of 23, 40, and 116 microM And Vmax values of 2860, 268, and 22 pmol/mg protein/min, respectively. P450 isoform screening of naphthalene metabolism identified CYP1A2 as the most efficient isoform for producing dihydrodiol and 1-naphthol, and CYP3A4 as the most effective for 2-naphthol production. Metabolism of the primary metabolites of naphthalene was also studied to identify secondary metabolites. Whereas 2-naphthol was readily metabolized by pHLMs to produce 2,6- and 1,7-dihydroxynaphthalene, dihydrodiol and 1-naphthol were inefficient substrates for pHLMs. A series of human p450 isoforms was used to further explore the metabolism of dihydrodiol and 1-naphthol. 1,4-Naphthoquinone and four minor unknown metabolites from 1-naphthol were observed, and CYP1A2 and 2D6*1 were identified as the most active isoforms for the production of 1,4-naphthoquinone. Dihydrodiol was metabolized by P450 isoforms to three minor unidentified metabolites with CYP3A4 and CYP2A6 having the greatest activity toward this substrate. The metabolism of dihydrodiol by P450 isoforms was lower than that of 1-naphthol. These studies identify primary and secondary metabolites of naphthalene produced by pHLMs and P450 isoforms.     

豆腐果苷对人肝微粒体CYP450酶体外抑制作用研究

目的:通过评价豆腐果苷在体外对人肝微粒体CYP450酶的7种亚型酶活性的影响,预测服用豆腐果苷可能出现的食物-药物及药物-药物代谢的影响。方法:将豆腐果苷与CYP450酶7种亚型的特异性探针底物咖啡因(CYP1A2)、右美沙芬(CYP2D6)、甲苯磺丁脲(CYP2C9)、S-美芬妥因(CYP2C19)、氯唑沙宗(CYP2E1)、香豆素(CYP2A6)及咪达唑仑(CYP3A4)与人肝微粒体进行孵育反应,采用HPLC和LC-MS/MS法测定对应的7种代谢产物(1,7-二甲基黄嘌呤、去甲右美沙芬、4-羟基甲苯磺丁脲、4-羟基美芬妥因、6-羟基氯唑沙宗、7-羟基香豆素和1-羟基咪达唑仑)的浓度,与对照组比较,确定豆腐果苷对以上7种亚酶活性的影响。结果:豆腐果苷在1～100μmol.L-1时对7种酶的抑制作用均无明显统计学意义(P>0.05)。结论:豆腐果苷可能不会引起有临床意义的CYP450酶抑制现象的发生。     

In vitro metabolism of mirtazapine enantiomers by human cytochrome P450 enzymes

The metabolism of mirtazapine enantiomers was investigated in vitro using human lymphoblast microsomes transfected with human cDNA to overexpress either CYP1A2, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 and assayed for mirtazapine enantiomers using a validated chiral method of high-performance liquid chromatography. (+)-Mirtazapine was extensively metabolised by CYP2D6 (K(m) = 9.3 +/- 3.3 &mgr;mol/l, V(max) = 40.9 +/- 7.9 &mgr;mol/h/mg, intrinsic clearance = 4.41 l/h/mg). CYP1A2 and CYP3A4 showed low metabolic activity towards (+)-mirtazapine and (-)-mirtazapine respectively. Neither CYP2C9 nor CYP2C19 appeared to be involved in the metabolism of the enantiomers of mirtazapine. Copyright 2001 John Wiley & Sons, Ltd.     

In vitro inhibition and induction of human hepatic cytochrome P450 enzymes by modafinil.

The ability of modafinil to affect human hepatic cytochrome P450 (CYP) activities was examined in vitro. The potential for inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5, and CYP4A9/11 by modafinil (5-250 microM) was evaluated with pooled human liver microsomes. Modafinil exhibited minimal capacity to inhibit any CYP enzyme, except CYP2C19. Modafinil inhibited the 4'-hydroxylation of S-mephenytoin, a marker substrate for CYP2C19, reversibly and competitively with a K(i) value of 39 microM, which approximates the steady-state C(max) value of modafinil in human plasma at a dosage of 400 mg/day. No irreversible inhibition of any CYP enzyme was observed, and there was no evidence of metabolism-dependent inhibition. The potential for induction of CYP activity was evaluated by exposing primary cultures of human hepatocytes to modafinil (10-300 microM). Microsomes were then prepared and assayed for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5 activities. The mean activities of microsomal CYP1A2, CYP2B6, and CYP3A4/5 from modafinil-treated hepatocytes were higher (up to 2-fold) than those in the solvent-treated controls but were less than those produced by reference inducers of these enzymes. At high concentrations of modafinil (>/=100 microM), the mean activity of CYP2C9 was decreased (up to 60%) relative to that in the solvent controls. Overall, modafinil was shown to have effects on human hepatic CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4/5 activities in vitro. Although effects obtained in vitro are not always predictive of effects in vivo, such results provide a rational basis for understanding drug-drug interactions that are observed clinically and for planning subsequent investigations.     

Tizanidine is mainly metabolized by cytochrome p450 1A2 in vitro

AIMS: To identify the cytochrome p450 (CYP) enzyme(s) that catalyze the metabolism of tizanidine in vitro. METHODS: The effect of CYP isoform inhibitors on the elimination of tizanidine was studied using pooled human liver microsomes. The metabolism of the drug by a range of human recombinant CYP isoforms was then investigated. RESULTS: Incubation of tizanidine (80 nm) with human liver microsomes resulted in time- and NADPH-dependent substrate consumption with a half-life of 50 min, initial reaction velocity of 1.1 pmol x min-1 x mg-1 protein and intrinsic clearance of 17 ml x min-1 x kg-1. The predicted in vivo hepatic clearance (CLh) of tizanidine using the well-stirred and parallel-tube model was close (68% and 82%, respectively) to its estimated in vivo CLh. Fluvoxamine and furafylline strongly inhibited tizanidine metabolism. Inhibitors specific to isoforms other than CYP1A2 had no substantial effect. Recombinant CYP1A2 metabolized tizanidine to a substantial degree (35% in 45 min), but other recombinant CYPs had little metabolic capacity for the drug. CONCLUSIONS: CYP1A2 is primarily responsible for the metabolism of tizanidine. CYP1A2 inhibitors may inhibit its metabolism also in vivo.     

Stereoselective metabolism of omeprazole by human cytochrome P450 enzymes.

This study demonstrates the stereoselective metabolism of the optical isomers of omeprazole in human liver microsomes. The intrinsic clearance (CL(int)) of the formation of the hydroxy metabolite from S-omeprazole was 10-fold lower than that from R-omeprazole. However, the CL(int) value for the sulfone and 5-O-desmethyl metabolites from S-omeprazole was higher than that from R-omeprazole. The sum of the CL(int) of the formation of all three metabolites was 14.6 and 42.5 microl/min/mg protein for S- and R-omeprazole, respectively. This indicates that S-omeprazole is cleared more slowly than R-omeprazole in vivo. The stereoselective metabolism of the optical isomers is mediated primarily by cytochrome P450 (CYP) 2C19, as indicated by studies using cDNA-expressed enzymes. This is the result of a considerably higher CL(int) of the 5-hydroxy metabolite formation for R- than for S-omeprazole. For S-omeprazole, CYP2C19 is more important for 5-O-desmethyl formation than for 5-hydroxylation. Predictions of the CL(int) using data from cDNA-expressed enzymes suggest that CYP2C19 is responsible for 40 and 87% of the total CL(int) of S- and R-omeprazole, respectively, in human liver microsomes. According to experiments using cDNA-expressed enzymes, the sulfoxidation of both optical isomers is metabolized by a single isoform, CYP3A4. The CL(int) of the sulfone formation by CYP3A4 is 10-fold higher for S-omeprazole than for R-omeprazole, which may contribute to their stereoselective disposition. The results of this study show that both CYP2C19 and CYP3A4 exhibit a stereoselective metabolism of omeprazole. CYP2C19 favors 5-hydroxylation of the pyridine group of R-omeprazole, whereas the same enzyme mainly 5-O-demethylates S-omeprazole in the benzimidazole group. Sulfoxidation mediated by CYP3A4 highly favors the S-form.     

Polymorphic human cytochrome P450 enzymes: an opportunity for individualized drug treatment.

Approximately 40% of human P450-dependent drug metabolism is carried out by polymorphic enzymes, which can cause abolished, quantitatively or qualitatively altered or enhanced drug metabolism. The latter situation is due to stable duplication, multiduplication or amplification of active genes, most likely in response to dietary components that have resulted in a selection of alleles with multiple non-inducible genes. Several examples exist where subjects carrying certain alleles suffer from a lack of drug efficacy due to ultrarapid metabolism or, alternatively, adverse effects from the drug treatment due to the presence of defective alleles. Knowledge in this field has grown rapidly and can now be applied to both drug development and clinical practice. This is facilitated by the recent development of high-throughput methods for mutation detection and oligonucleotide chips array technology for the identification of a multitude of mutations in the genes encoding drug-metabolizing enzymes. The outcome will allow for safer and more efficient drug therapies.     

The novel antifungal agent PLD-118 is neither metabolized by liver microsomes nor inhibits cytochrome P450 in vitro

PLD-118 is a novel, oral antifungal drug, under development for the treatment of Candida infections. Possible metabolism of PLD-118 by rat, dog and human S9 liver homogenates and inhibition of human cytochrome P450 (CYP) enzymes were investigated. PLD-118 (10 and 100 microM) incubated for 0-60 min with S9 fractions and NADPH was determined by HPLC, using the Waters AccQ.Tag method after derivatization of amino acids to stable, fluorescent derivatives. CYP assays were performed using pooled human liver microsomes with substrates, selective towards human CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, incubated at concentrations around the Km. Incubation mixtures were preincubated with PLD-118 (0.1-100 microM) or control inhibitor for 5 min. No metabolism of PLD-118 was detected with rat and dog S9 fractions. A small (8%) decrease in PLD-118 at 100 microM (not detected at 10 microM) with human microsomes was considered to be biologically irrelevant. PLD-118 did not inhibit any of the tested CYPs. PLD-118, at concentrations up to 100 microM, is not metabolized by rat, dog or human liver S9 homogenates and does not inhibit human CYPs in vitro, suggesting little likelihood for interaction of PLD-118 with drugs metabolized by these enzymes.     

Involvement of multiple cytochrome P450 and UDP-glucuronosyltransferase enzymes in the in vitro metabolism of muraglitazar.

Muraglitazar (Pargluva), a dual alpha/gamma peroxisome proliferator-activated receptor activator, has both glucose- and lipid-lowering effects in animal models and in patients with diabetes. The human major primary metabolic pathways of muraglitazar include acylglucuronidation, aliphatic/aryl hydroxylation, and O-demethylation. This study describes the identification of human cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) enzymes involved in the in vitro metabolism of muraglitazar. [(14)C]Muraglitazar was metabolized by cDNA-expressed CYP2C8, 2C9, 2C19, 2D6, and 3A4, but to a very minimal extent by CYP1A2, 2A6, 2B6, 2C18, 2E1, and 3A5. Inhibition of the in vitro metabolism of muraglitazar in human liver microsomes, at a clinically efficacious concentration, by chemical inhibitors and monoclonal antibodies further supported involvement of CYP2C8, 2C9, 2C19, 2D6, and 3A4 in its oxidation. A combination of intrinsic clearance (V(max)/K(m)) and relative concentrations of each P450 enzyme in the human liver was used to predict the contribution of CYP2C8, 2C9, 2C19, 2D6, and 3A4 to the formation of each primary oxidative metabolite and to the overall oxidative metabolism of muraglitazar. Glucuronidation of [(14)C]muraglitazar was catalyzed by cDNA-expressed UGT1A1, 1A3, and 1A9, but not by UGT1A6, 1A8, 1A10, 2B4, 2B7, and 2B15. The K(m) values for muraglitazar glucuronidation by the three active UGT enzymes were similar (2-4 muM). In summary, muraglitazar was metabolized by multiple P450 and UGT enzymes to form multiple metabolites. This characteristic predicts a low potential for the alteration of the pharmacokinetic parameters of muraglitazar via polymorphic drug metabolism enzymes responsible for clearance of the compound or by coadministration of drugs that inhibit or induce relevant metabolic enzymes.     

Stereoselective metabolism of lansoprazole by human liver cytochrome P450 enzymes.

The stereoselective metabolism of lansoprazole enantiomers was evaluated by incubation of human liver microsomes and cDNA-expressed cytochrome p450 (p450) enzymes to understand and predict their stereoselective disposition in humans in vivo. The intrinsic clearances (Clint) of the formation of both hydroxy and sulfone metabolites from S-lansoprazole were 4.9- and 2.4-fold higher than those from the R-form, respectively. The sums of formation Clint of both metabolites were 13.5 and 57.3 microl/min/mg protein for R- and S-lansoprazole, respectively, suggesting that S-lansoprazole would be cleared more rapidly than the R-form. The p450 isoform selective inhibition study in liver microsomes, and the incubation study of cDNA-expressed enzymes, demonstrated that the stereoselective sulfoxidation is mediated by CYP3A4 and that the hydroxylation is mediated by CYP2C9 and CYP3A4 as well as by CYP2C19. Total Clint values of hydroxy and sulfone metabolite formation catalyzed by all these p450 enzymes were consistently higher for S-lansoprazole than for the R-form. The CYP3A4 produced the greatest difference of Clint between S- and R-enantiomers, mainly due to a difference of sulfoxidation metabolism (Clint 76.5 versus 10.8 microl/min/nmol of p450, respectively), whereas CYP2C19-catalyzed hydroxylation resulted in a minor difference of Clint between S- and R-enantiomers (179.6 versus 143.3 microl/min/nmol of p450, respectively). However, the affinity of CYP2C19 on hydroxylation was 5.7-fold higher for S-enantiomer than for the R-form (Km 2.3 versus 13.1 microM), suggesting that the role of CYP2C19 on stereoselective hydroxylation would be more prominent at concentrations around the usual therapeutic level. These findings suggest that both CYP2C19 and CYP3A4 are major enzymes contributing to the stereoselective disposition of lansoprazole, but stereoselective hydroxylation of lansoprazole enantiomers is mainly influenced by CYP2C19, especially at the usual therapeutic doses.     

Metabolism of nomifensine to a dihydroisoquinolinium ion metabolite by human myeloperoxidase, hemoglobin, monoamine oxidase A, and cytochrome P450 enzymes.

Nomifensine is an antidepressant agent that was removed from use because of a high incidence of hemolytic anemia. It contains an N-methyl-8-aminotetrahydroisoquinoline ring which has the potential to be oxidized to quaternary dihydroisoquinolinium and isoquinolinium ions, albeit such a transformation had not been previously observed. In this report, we demonstrate the conversion of nomifensine to a dihydroisoquinolinium ion metabolite by several human enzymes. Human liver microsomes supplemented with NADPH generated the dihydroisoquinolinium ion metabolite along with other hydroxylated metabolites, whereas when supplemented with t-butyl peroxide, only the dihydroisoquinolinium ion metabolite was observed. Monoamine oxidase A, but not monoamine oxidase B, catalyzed this reaction, as well as human hemoglobin supplemented with H2O2. Human myeloperoxidase catalyzed this reaction in the presence of H2O2, and activation of the reaction was observed when incubations were conducted in the presence of acetaminophen at concentrations relevant to those measured in humans. The reaction was also observed in human whole blood. The equilibrium between the dihydroisoquinolinium ion and carbinolamine was shown to have a pK of about 11.7. The dihydroisoquinolinium ion was shown to react with cyanide and borohydride, but not glutathione. These findings suggest that the electrophilic nomifensine dihydroisoquinolinium metabolite, which can be generated by several enzymes, could be behind toxic responses to nomifensine such as hemolytic anemia and hepatotoxicity.     

Inhibitory effects of gypenosides on seven human cytochrome P450 enzymes in vitro

Among the various possible causes for drug interactions, pharmacokinetic factors such as inhibition of drug-metabolizing enzymes, especially cytochrome P450 (CYP) enzymes, are regarded as the most frequent and clinically important. Gypenosides is widely used as functional food and over-the-counter drug in East Asia. In this study, the in vitro inhibitory effects of gypenosides on the major human CYP enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) activities in human liver microsomes were examined using liquid chromatography–tandem mass spectrometry. Gypenosides showed the strongest inhibition of CYP2D6, followed by CYP2C8, CYP3A4 and CYP2C9. The IC₅₀ values were 1.61 μg/mL, 20.06 μg/mL, 34.76 μg/mL (CYP3A4/midazolam), 46.73 μg/mL (CYP3A4/testosterone), and 54.52 μg/mL, respectively. Gypenosides exhibited competitive inhibition of CYP2D6 (Ki = 1.18). In conclusion, Gypenosides might cause herb–drug interactions via inhibition of CYP2D6. An in vivo study is needed to examine this further.