Stereo‐Selective Metabolism of Methadone by Human Liver Microsomes and cDNA‐Expressed Cytochrome P450s: A Reconciliation

Abstract: In vitro metabolism of methadone was investigated in cytochrome P450 (CYP) supersomes and phenotyped human liver microsomes (HLMs) to reconcile past findings on CYP involvement in stereo‐selective metabolism of methadone. Racaemic methadone was used for incubations; (R)‐ and (S)‐methadone turnover and (R)‐ and (S)‐EDDP formation were determined using chiral liquid chromatography–tandem mass spectrometry. CYP supersome activity for methadone use and EDDP formation ranked CYP2B6 > 3A4 > 2C19 > 2D6 > 2C18, 3A7 > 2C8, 2C9, 3A5. After abundance scaling, CYP3A4, 2B6 and 2C19 accounted for 63–74, 12–32 and 1. 4–14% of respective activity. CYP2B6, 2D6 and 2C18 demonstrated a preference for (S)‐EDDP formation; CYP2C19, 3A7 and 2C8 for (R)‐EDDP; 3A4 none. Correlation analysis with 15 HLMs supported the involvement of CYP2B6 and 3A. The significant correlation of S/R ratio with CYP2B6 activity confirmed its stereo‐selectivity. CYP2C19 and 2D6 inhibitors and monoclonal antibody (mAb) did not inhibit EDDP formation in HLM. Chemical and mAb inhibition of CYP3A in high 3A activity HLM reduced EDDP formation by 60–85%; inhibition of CYP2B6 in 2B6 high‐activity HLM reduced (S)‐EDDP formation by 80% and (R)‐EDDP formation by 55%. Inhibition changed methadone metabolism in a stereo‐selective manner. When CYP3A was inhibited, 2B6 mediated (S)‐EDDP formation predominated; S/R stereo‐selectivity increased. When 2B6 was inhibited (S)‐EDDP formation fell and stereo‐selectivity decreased. The results confirmed the primary roles of CYPs 3A4 and 2B6 in methadone metabolism; CYP2C8 and 2C9 did not appear involved; 2C19 and 2D6 have minimal roles. CYP2B6 is the primary determinant of stereo‐selective metabolism; stereo‐selective inhibition might play a role in varied plasma concentrations of the two enantiomers.     

Stereoselective metabolism of rabeprazole-thioether to rabeprazole by human liver microsomes

Objective Rabeprazole is metabolized mainly non-enzymatically to rabeprazole-thioether. This in vitro study was designed to clarify the stereoselective oxidation mechanism and to identify the enzyme(s) involved in the metabolic breakdown of rabeprazole-thioether to rabeprazole. Methods Rabeprazole-thioether was incubated with human liver microsomes and several recombinant cytochrome P450 (CYP) enzymes (CYPs 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4). High-performance liquid chromatography was used for identification and quantification of each rabeprazole enantiomer. Results The K _m and V _max values for the formation of (R)-rabeprazole from rabeprazole-thioether in human liver microsomes were 6.6 μM and 92 pmol/min/mg protein, respectively, whereas those for the formation of (S)-rabeprazole were 5.1 μM and 21 pmol/min/mg protein, respectively. CYP3A4 was found to be the major enzyme responsible for (R)- and (S)-rabeprazole formation from rabeprazole-thioether. The intrinsic clearance (V _max /K _m ) for the oxidation by CYP3A4 of (R)-rabeprazole was 3.5-fold higher than that for the (S)-enantiomer (81 nl/min/pmol of P450 vs. 23 nl/min/pmol of P450). On the other hand, CYP2C19 and CYP2D6 were the main enzymes catalyzing the formation of desmethylrabeprazole-thioether from rabeprazole-thioether. The mean K _m and V _max values of desmethylrabeprazole-thioether formation for CYP2C19 were 5.1 μM and 600 pmol/min/nmol of P450, respectively, whereas those for CYP2D6 were 15.1 μM and 736 pmol/min/nmol of P450, respectively. Discussion Rabeprazole is reduced mainly non-enzymatically to rabeprazole-thioether, which is further stereoselectively re-oxidized by CYP3A4 mainly to (R)-rabeprazole. The difference in the enantioselective disposition of rabeprazole is determined by stereoselectivity in CYP3A4-mediated metabolic conversion from rabeprazole-thioether to rabeprazole.     

Identification of CYP3A7 for glyburide metabolism in human fetal livers

Glyburide is commonly prescribed for the treatment of gestational diabetes mellitus; however, fetal exposure to glyburide is not well understood and may have short- and long-term consequences for the health of the child. Glyburide can cross the placenta; fetal concentrations at term are nearly comparable to maternal levels. Whether or not glyburide is metabolized in the fetus and by what mechanisms has yet to be determined. In this study, we determined the kinetic parameters for glyburide depletion by CYP3A isoenzymes; characterized glyburide metabolism by human fetal liver tissues collected during the first or early second trimester of pregnancy; and identified the major enzyme responsible for glyburide metabolism in human fetal livers. CYP3A4 had the highest metabolic capacity towards glyburide, followed by CYP3A7 and CYP3A5 (Cl_int,u = 37.1, 13.0, and 8.7 ml/min/nmol P450, respectively). M5 was the predominant metabolite generated by CYP3A7 and human fetal liver microsomes (HFLMs) with approximately 96% relative abundance. M5 was also the dominant metabolite generated by CYP3A4, CYP3A5, and adult liver microsomes; however, M1–M4 were also present, with up to 15% relative abundance. CYP3A7 protein levels in HFLMs were highly correlated with glyburide Cl_int, 16α-OH DHEA formation, and 4′-OH midazolam formation. Likewise, glyburide Cl_int was highly correlated with 16α-OH DHEA formation. Fetal demographics as well as CYP3A5 and CYP3A7 genotype did not alter CYP3A7 protein levels or glyburide Cl_int. These results indicate that human fetal livers metabolize glyburide predominantly to M5 and that CYP3A7 is the major enzyme responsible for glyburide metabolism in human fetal livers.     

Enantioselective pharmacokinetics of sibutramine in rat

Racemic sibutramine is widely used to treat obesity owing to its inhibition of serotonin and noradrenaline reuptake in synapses. Although the enantioselective effects of sibutramine and its two active desmethyl-metabolites, monodesmethylsibutramine (MDS) and didesmethylsibutramine (DDS), on anorexia and energy expenditure have been elucidated, the enantioselective pharmacokinetics of sibutramine are still unclear. Therefore, we aimed to characterize the enantioselective pharmacokinetics of sibutramine and its metabolites in plasma and urine following an intravenous and a single oral administration of sibutramine in rats. The absolute bioavailability of sibutramine was only about 7%. The pharmacologically less effective S-isomer of DDS was predominant in the plasma: the C _max and the AUC _inf were 28 and 30 times higher than those of the R-isomer, respectively (p<0.001). In the urine, the concentrations of the R-isomers of hydroxylated DDS and hydroxylated and carbamoylglucuronized MDS and DDS appeared to be 11.3-, 5.1-, and 5.3-times the concentrations of the respective S-isomers. Thus, regardless of increased potency than the S-enantiomers, the R-enantiomers of the sibutramine metabolites MDS and DDS were present at lower concentrations, owing to their rapid biotransformation to hydroxylated and/or carbamoylglucuronized forms and their faster excretion in the urine. The present study is the first to elucidate the enantioselective pharmacokinetics of sibutramine in rats.     

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

Abstract

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 K_i?=?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 K_m?=?6?±?2 versus 11?±?2?µM, P=0.04). CYP2C8*3/*3 (pooled HLM) also showed high binding affinity (K_m?=?4?µM) and single-enzyme weak autoinhibition (K_i?=?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.     

CYP3A4 mediates dextropropoxyphene N-demethylation to nordextropropoxyphene: human in vitro and in vivo studies and lack of CYP2D6 involvement

1.?The individual cytochrome P450 isoforms in dextropropoxyphene N-demethylation to nordextropropoxyphene were determined and the pharmacokinetics of dextropropoxyphene and nordextropropoxyphene in cytochrome P4502D6 (CYP2D6) extensive (EM) and poor (PM) subjects were characterized.

2.?Microsomes from six CYP2D6 extensive metabolizers and one CYP2D6 poor metabolizer were used with isoform specific chemical and antibody inhibitors and expressed recombinant CYP enzymes. Groups of three CYP2D6 EM and PM subjects received a single 65-mg oral dose of dextropropoxyphene, and blood and urine were collected for 168 and 96 h, respectively.

3.?Nordextropropoxyphene formation in vitro was not different between the CYP2D6 extensive metabolizers (K_m = 179 ± 74 μM, Cl_int = 0.41 ± 0.26 ml mg^?1 h^?1) and the PM subject (K_m = 225 μM, Cl_int = 0.19 ml mg^?1 h^?1) and was catalysed predominantly by CYP3A4. There was no apparent difference in the pharmacokinetics of dextropropoxyphene and nordextropropoxyphene in CYP2D6 EM and PM subjects.

4.?CYP3A4 is the major CYP enzyme catalysing the major metabolic pathway of dextropropoxyphene metabolism. Hence variability in the pharmacodynamic effects of dextropropoxyphene are likely due to intersubject variability in hepatic CYP3A4 expression and/or drug–drug interactions. Reported CYP2D6 phenocopying is not due to dextropropoxyphene being a CYP2D6 substrate.     

甘草次酸在人细胞色素CYP450中体外代谢研究(英)

甘草根是中医临床常用解毒草药, 其活性成分甘草次酸主要是通过肝脏代谢。本文研究了人肝微粒体以及人源性CYP450s对甘草次酸的体外代谢影响, 以及甘草次酸对几种CYP450酶活性的影响。实验结果表明, 甘草次酸体外主要代谢酶为CYP3A4。体外药代动力学参数K_m, V_max和CL_int分别为18.6 μmol·L⁻¹, 4.4 nmol·mg⁻¹(protein)·min⁻¹和0.237 mL·mg⁻¹(protein)·min⁻¹。体外抑制试验显示, 50 μmol·L⁻¹甘草次酸可以抑制CYP2C19、CYP2C9、CYP3A4酶的活性, 其抑制率可高达50%以上。     

In-vitro metabolism of glycyrrhetinic acid by human and rat liver microsomes and its interactions with six CYP substrates

Objectives Glycyrrhetinic acid is the main metabolite of glycyrrhizin and the main active component of Licorice root. This study was designed to investigate the in‐vitro metabolism of glycyrrhetinic acid by liver microsomes and to examine possible metabolic interactions that glycyrrhetinic acid may have with other cytochrome P450 (CYP) substrates. Methods Glycyrrhetinic acid was incubated with rat liver microsomes (RLM) and human liver microsomes (HLM). Liquid chromatography tandem mass spectrometry was used for glycyrrhetinic acid or substrates identification and quantification. Key findings The K_m and V_max values for HLM are 33.41 µm and 2.23 nmol/mg protein/min, respectively; for RLM the K_m and V_max were 24.24 µm and 6.86 nmol/mg protein/min, respectively. CYP3A4 is likely to be the major enzyme responsible for glycyrrhetinic acid metabolism in HLM while CYP2C9 and CYP2C19 are considerably less active. Other human CYP isoforms have minimal or no activity toward glycyrrhetinic acid. The interactions of glycyrrhetinic acid and six CYP substrates, such as phenacetin, diclofenac, (S)‐mephenytoin, dextromethorphan, chlorzoxazone and midazolam were also investigated. The inhibitory action of glycyrrhetinic acid was observed in CYP2C9 for 4‐hydroxylation of diclofenac, CYP2C19 for 4′‐hydroxylation of (S)‐mephenytoin and CYP3A4 for 1′‐hydroxylation of midazolam with half maximal inhibitory concentration (IC50) values of 4.3‐fold, 3.8‐fold and 9.6‐fold higher than specific inhibitors in HLM, respectively. However, glycyrrhetinic acid showed relatively little inhibitory effect (IC50 > 400 µm ) on phenacetin O‐deethylation, dextromethorphan O‐demethylation and chlorzoxazone 6‐hydroxylation. Conclusions The study indicated that CYP3A4 is likely to be the major enzyme responsible for glycyrrhetinic acid metabolism in HLM while CYP2C9 and CYP2C19 are considerably less active. The results suggest that glycyrrhetinic acid has the potential to interact with a wide range of xenobiotics or endogenous chemicals that are CYP2C9, CYP2C19 and CYP3A4 substrates.     

The in vitro metabolism of bupropion revisited: concentration dependent involvement of cytochrome P450 2C19

1. The involvement of cytochrome P450 2B6 (CYP2B6) to the in vitro and in vivo metabolism of bupropion has been well studied. In these investigations we performed a detailed in vitro phenotyping study to characterize isoforms other than CYP2B6.

2. A total of nine metabolites were identified (M1–M9) in the incubations with cDNA-expressed P450s (rhCYP) and human liver microsomes (HLM).

3. Incubations in rhCYP identified CYP2B6 as the isoform responsible for the formation of hydroxybupropion (M3). CYP2C19 was involved in bupropion metabolism primarily through alternate hydroxylation pathways (M4–M6) with higher activity at lower substrate concentrations, near 1 µM.

4. The results from HLM inhibition studies using CYP2B6 and CYP2C19 inhibitory antibodies indicated that CYP2B6 contributed to approximately 90% of M3 formation, and CYP2C19 contributed to approximately 70–90% of M4, M5, and M6 formation.

5. Studies using single donor HLM with varying degrees of CYP2B6 and CYP2C19 activities showed a good relationship between M3 formation and CYP2B6 activity and M4/M5 formation and CYP2C19 activity.

6. These results confirmed the principle role of CYP2B6 in hydroxybupropion formation, as a selective CYP2B6 probe. In addition, the new findings revealed that CYP2C19 also contributes to bupropion metabolism through alternate hydroxylation pathways.

     

Identification and relative contributions of human cytochrome P450 isoforms involved in the metabolism of glibenclamide and lansoprazole: evaluation of an approach based on the in vitro substrate disappearance rate

1.?The identification and relative contributions of human cytochrome P450 (CYP) enzymes involved in the metabolism of glibenclamide and lansoprazole in human liver microsomes were investigated using an approach based on the in vitro disappearance rate of unchanged drug.

2.?Recombinant CYP2C19 and CYP3A4 catalysed a significant disappearance of both drugs. When the contribution of CYPs to the intrinsic clearance (CL_int) of drugs in pooled human microsomes was estimated by relative activity factors, contributions of CYP2C19 and CYP3A4 were determined to be 4.6 and 96.4% for glibenclamide, and 75.1 and 35.6% for lansoprazole, respectively.

3.?CL_int of glibenclamide correlated very well with CYP3A4 marker activity, whereas the CL_int of lansoprazole significantly correlated with CYP2C19 and CYP3A4 marker activities in human liver microsomes from 12 separate individuals. Effects of CYP-specific inhibitors and anti-CYP3A serum on the CL_int of drugs in pooled human liver microsomes reflected the relative contributions of CYP2C19 and CYP3A4.

4.?The results suggest that glibenclamide is mainly metabolized by CYP3A4, whereas lansoprazole is metabolized by both CYP2C19 and CYP3A4 in human liver microsomes. This approach, based on the in vitro drug disappearance rate, is useful for estimating CYP identification and their contribution to drug discovery.     

Identification of cytochrome P450 enzymes involved in the metabolism of 4′-methoxy-α-pyrrolidinopropiophenone (MOPPP), a designer drug,in human liver microsomes

1. The metabolism of 4′-methoxy-α-pyrrolidinopropiophenone (MOPPP), a novel designer drug, to its demethylated major metabolite 4′-hydroxy-pyrrolidinopropio-phenone (HO-PPP) was studied in pooled human liver microsomes (HLM) and in cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes.

2. CYP2C19 catalysed the demethylation with apparent K_m and V_max values of 373.4 ± 45.1?μM and 6.0 ± 0.3?pmol?min^?1?pmol^?1 CYP, respectively (mean ± SD). Both CYP2D6 and HLM exhibited clear biphasic profiles with apparent K_m,1 values of 1.3 ± 0.4 and 22.0 ± 6.5?μM, respectively, and V_max,1 values of 1.1 ± 0.1 pmol?min^?1?pmol^?1 CYP and 169.1 ± 20.5?pmol?min^?1?mg^?1 protein, respectively.

3. Percentages of intrinsic clearances of MOPPP by particular CYPs were calculated using the relative activity factor (RAF) approach with (S)-mephenytoin-4′-hydroxylation or bufuralol-1′-hydroxylation as index reactions for CYP2C19 or CYP2D6, respectively.

4. MOPPP, HO-PPP and the standard 3′,4′-methylenedioxy-pyrrolidinopropio-phenone (MDPPP) were separated and analysed by liquid chromatography–mass spectrometry in the selected-ion monitoring (SIM) mode.

5. The CYP2D6 specific chemical inhibitor quinidine (3?μM) significantly (?p<0.0001) inhibited HO-PPP formation by 91.8 ± 0.5% (mean ± SEM) in incubation mixtures with HLM and 2?μM MOPPP.

6. It can be concluded from the data obtained from kinetic and inhibition studies that polymorphically expressed CYP2D6 is the enzyme mainly responsible for MOPPP demethylation.     

Identification of cytochrome P450 enzymes involved in the metabolism of 3′,4′-methylenedioxy-α-pyrrolidinopropiophenone (MDPPP), a designer drug,in human liver microsomes

The metabolism of 3′,4′-methylenedioxy-α-pyrrolidinopropiophenone (MDPPP), a novel designer drug, to its demethylenated major metabolite 3′,4′-dihydroxy-pyrrolidinopropiophenone (di-HO-PPP) was studied in pooled human liver microsomes (HLM) and in cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes. CYP2C19 catalysed the demethylenation with apparent K_m and V_max values of 120.0?±?13.4?µM and 3.2?±?0.1?pmol/min/pmol?CYP, respectively (mean?±?standard deviation). CYP2D6 catalysed the demethylenation with apparent K_m and V_max values of 13.5?±?1.5?µM and 1.3?±?0.1 pmol/min/pmol?CYP, respectively. HLM exhibited a clear biphasic profile with an apparent K_m,1 value of 7.6?±?9.0 and a V_max,1 value of 11.1?±?3.6?pmol/min/mg?protein, respectively. Percentages of intrinsic clearances of MDPPP by specific CYPs were calculated using the relative activity factor (RAF) approach with (S)-mephenytoin-4′-hydroxylation or bufuralol-1′-hydroxylation as index reactions for CYP2C19 or CYP2D6, respectively. MDPPP, di-HO-PPP and the standard 4′-methyl-pyrrolidinohexanophenone (MPHP) were separated and analysed by liquid chromatography-mass spectrometry in the selected-ion monitoring (SIM) mode. The CYP2D6-specific chemical inhibitor quinidine (3?µM) significantly (p?相似文献   

CYP3A4 and CYP3A5 catalyse the conversion of the N-methyl-D-aspartate (NMDA) antagonist CJ-036878 to two novel dimers

CJ-036878, N-(3-phenethoxybenzyl)-4-hydroxybenzamide, was developed as an antagonist of the N-methyl-D-aspartate receptor NR2B subunit. Two dimeric metabolites, CJ-047710 and CJ-047713, were identified from the incubation mixture with CJ-036878 in human liver microsomes (HLM). The identification of the enzymes involved in the formation of these dimeric metabolites was investigated in the current study. Inhibition of the formation of CJ-047710 and CJ-047713 in pooled HLM by 1-aminobenztriazole, SKF-525A, and ketoconazole were observed. Ketoconazole played a significant role in inhibiting formation of these two metabolites in a concentration-dependent manner. Recombinant CYP3A4 and CYP3A5 exhibited a markedly high activity toward the formation of CJ-047710 and CJ-047713 from CJ-036878, but the contribution of other CYP enzymes to these formations was at a very low level or negligible. The formation of CJ-047710 and CJ-047713 in pooled HLM, CYP3A4, and CYP3A5 showed sigmoid characteristics. S₅₀ values for CJ-047710 and CJ-047713 formation in HLM were almost equivalent with those for CYP3A4 and CYP3A5. For the CYP3A enzymes, maximal clearance due to auto-activation values for CJ-047710 and CJ-047713 formation catalysed by CYP3A5 were 3.6- and 3.1-fold higher than those catalysed by CYP3A4. This is the first report that shows both CYP3A4 and CYP3A5 simultaneously contribute to dimerization through oxidative C-C and C-O coupling reactions.     

Pharmacokinetic differences between the enantiomers of lansoprazole and its metabolite, 5-hydroxylansoprazole,in relation to CYP2C19 genotypes

Objective The purpose of this study was to elucidate the pharmacokinetics of each enantiomer of lansoprazole and 5-hydroxylansoprazole in three different CYP2C19 genotype groups of Japanese subjects.Methods Healthy subjects (n=18), of whom 6 were homozygous extensive metabolizers (homEMs), 6 were heterozygous extensive metabolizers (hetEMs) and 6 were poor metabolizers (PMs), participated in the study. After a single oral dose of 60 mg of racemic lansoprazole, the plasma concentrations of the lansoprazole enantiomers, 5-hydroxylansoprazole enantiomers and lansoprazole sulfone were measured for 24 h post-dose.Results The plasma concentrations of (R)-lansoprazole were remarkably higher in all three CYP2C19 genotype groups than those of the corresponding (S)-enantiomer. The mean maximum plasma concentration (C _max) of (S)-lansoprazole differed significantly among the three groups, whereas there was no difference for the (R)-enantiomer. The relative area under the plasma concentration (AUC) ratios of (R)- and (S)-lansoprazole in the homEMs, hetEMs, and PMs were 1:1.5:4.0 and 1:1.8:7.4, respectively. Yet, the relative AUC ratios of 5-hydroxylansoprazole to lansoprazole for the (R)- and (S)-enantiomers in the homEMs, hetEMs, and PMs were almost the same (1:0.73:0.12 and 1:0.77:0.13, respectively). However, the AUC ratios of the (S)-enantiomer were 13-fold greater for the three CYP2C19 genotypes than those of the corresponding (R)-enantiomer.Conclusions The magnitude of the contribution of CYP2C19 to the 5-hydroxylation of (S)-lansoprazole was greater than that of the (R)-enantiomer. The R/S ratios for the AUC of lansoprazole for the homEMs, hetEMs and PMs were 12.7, 8.5 and 5.8, respectively, suggesting a significant effect of CYP2C19 polymorphisms on the stereoselective disposition of lansoprazole.     

Stereoselectivity in metabolism of ifosfamide by CYP3A4 and CYP2B6

The aim was to identify the hepatic cytochromes P450 (CYPs) responsible for the enantioselective metabolism of ifosfamide (IFA). The 4-hydroxylation, N²- and N³-dechloroethylation of IFA enantiomers were monitored simultaneously in the same metabolic systems using GC/MS and pseudoracemate techniques. In human and rat liver microsomes, (R)-IFA was preferentially metabolized via 4-hydroxylation, whereas its antipode was biotransformed in favour of N-dechloroethylation. CYP3A4 was the major enzyme responsible for metabolism of IFA enantiomers in human liver. The study also revealed that CYP3A (human CYP3A4/5 and rat CYP3A1/2) and CYP2B (human CYP2B6 and rat CYP2B1/2) enantioselectively mediated the 4-hydroxylation, N²- and N³-dechloroethylation of IFA. CYP3A preferentially supported the formation of (R)-4-hydroxyIFA (HOIF), (R)-N²-dechloroethylIFA (N2D) and (R)-N³-dechloroethylIFA (N3D), whereas CYP2B preferentially mediated the generation of (S)-HOIF, (S)-N2D and (S)-N3D. The enantioselective metabolism of IFA by CYP3A4 and CYP2B1 was confirmed in cDNA transfected V79 cells.     

Functional Characterization of CYP2C8.13 and CYP2C8.14: Catalytic Activities toward Paclitaxel

Abstract: Cytochrome P450 2C8 (CYP2C8) plays important roles in the metabolism of various drugs, including the anti‐cancer drug, paclitaxel. We recently identified two novel CYP2C8 alleles (CYP2C8*13 and CYP2C8*14; wild‐type, CYP2C8*1A) with non‐synonymous single nucleotide polymorphisms in a Japanese population. To precisely investigate the effect of amino acid substitutions (CYP2C8*13, Ile223Met; CYP2C8*14, Ala238Pro) on CYP2C8 function, CYP2C8 proteins of the wild‐type (CYP2C8.1) and variants (CYP2C8.13 and CYP2C8.14) were heterologously expressed in yeast cells, and their paclitaxel 6α‐hydroxylation activities were determined. The K_m, V_max and CL_int values for paclitaxel 6α‐hydroxylation of CYP2C8.1 were 2.3 μM, 4.1 pmol/min./pmol CYP and 1.7 μl/min./pmol CYP, respectively. The K_m value of CYP2C8.14 was significantly higher (2.9‐fold) than that of CYP2C8.1. The V_max value of CYP2C8.14 was comparable to that of CYP2C8.1 and the CL_int value was reduced to 46% of CYP2C8.1. In contrast, the K_m, V_max and CL_int values of CYP2C8.13 were similar to those of CYP2C8.1. These results suggest that Ala238Pro substitution in CYP2C8.14 decreases the affinity toward paclitaxel of the CYP2C8 enzyme, and that the genetic polymorphism of the CYP2C8*14 allele may influence the clinical response to drugs metabolized mainly by CYP2C8.     

Identification of novel glutathione adducts of benzbromarone in human liver microsomes

Benzbromarone (BBR) is a potent uricosuric drug that can cause serious liver injury. Our recent study suggested that 1′-hydroxy BBR, one of major metabolites of BBR, is metabolized to a cytotoxic metabolite that could be detoxified by glutathione (GSH). The aim of this study was to clarify whether GSH adducts are formed from 1′-hydroxy BBR in human liver microsomes (HLM). Incubation of 1′-hydroxy BBR with GSH in HLM did not result in the formation of GSH adducts, but 1′,6-dihydroxy BBR was formed. In addition, incubation of 1′,6-dihydroxy BBR with GSH in HLM resulted in the formation of three novel GSH adducts (M1, M2 and M3). The structures of M1 and M2 were estimated to be GSH adducts in which the 1-hydroxyethyl group at the C-2 position and the hydroxyl group at the C-1′ position of 1′,6-dihydroxy BBR were substituted by GSH, respectively. We also found that the 6-hydroxylation of 1′-hydroxy BBR is mainly catalyzed by CYP2C9 and that several CYPs and/or non-enzymatic reaction are involved in the formation of GSH adducts from 1′,6-dihydroxy BBR. The results indicate that 1′-hydroxy BBR is metabolized to reactive metabolites via 1′,6-dihydroxy BBR formation, suggesting that these reactive metabolites are responsible for BBR-induced liver injury.     

The role of human cytochrome P450 enzymes in metabolism of acrylamide in vitro

Abstract

Objective: Acrylamide (AA), a probable human carcinogen, is present in fried and baked starch-rich food. In vivo, the substance is partly biotransformed to glycidamide (GA), which may account for carcinogenic effects. Existing data suggest an important but not exclusive contribution of CYP2E1 to GA formation. The aim of this project was to derive respective enzyme kinetic parameters for CYP2E1 and to assess a possible role of other important human CYPs for this reaction in vitro.

Methods: AA (0.2–20?mM) was incubated with human liver microsomes (HLM) and human cytochrome P450 enzymes (supersomes?). GA was quantified by a specific LC-MS/MS method. Enzyme kinetic parameters were estimated assuming a single binding site. Furthermore, inhibition experiments were performed with diethyldithiocarbamate (DDC), a potent inhibitor of CYP2E1.

Results: The mean?±?SD maximum formation rate (V_max) and Michaelis–Menten constant (K_m) for GA formation in HLM were 199?±?36?pmol GA/mg protein/min and 3.3?±?0.5?mM, respectively. In AA incubations with supersomes?, only for CYP2E1 measurable GA formation was detected in all tested AA concentrations (V_max and K_m were 5.4?nmol GA/nmol CYP2E1/min and 1.3?mM, respectively). Inhibition constant (IC₅₀) of DDC was 3.1?±?0.5?µM for HLM and 1.2?±?0.2?µM for CYP2E1 supersomes?. Therefore, relevant participation of CYPs other than CYP2E1 in the metabolism of AA to GA in humans does not seem likely.

Conclusion: Our results confirm the major role of CYP2E1 in GA formation from AA, albeit with low affinity and low capacity. Further studies are needed to identify other pathways of GA formation.     

Relative roles of CYP2C19 and CYP3A4/5 in midazolam 1′-hydroxylation

1. During the characterization of recombinant CYP2C19, it was observed that this enzyme metabolized midazolam, which is generally regarded as CYP3A4/5 substrate, and we therefore decided to pursue this observation further.

2. CYP2C19 showed a Michaelis-Menten pattern for midazolam 1′-hydroxylation and was inhibited by (+)-N-3-benzylnirvanol and S-mephenytoin, which are a standard potent inhibitor and a substrate of CYP2C19, respectively.

3. The inhibitory potency by CYP3A4/5 inhibitor on the midazolam 1′-hydroxylation in human liver microsomes (HLM) was correlated with the CYP3A4/5 specific catalytic activity, but such correlation was not observed in CYP2C19 enzyme. The in vitro intrinsic clearance value for midazolam 1′-hydroxylation was not changed by the addition of (+)-N-3-benzylnirvanol in four individual HLM preparations.

4. These results indicated that although CYP2C19 is capable of catalyzing midazolam 1′-hydroxylation, CYP3A4/5 play a more important role.