Enantioselective N-Demethylation and Hydroxylation of Sibutramine in Human Liver Microsomes and Recombinant Cytochrome P-450 Isoforms

The enantioselective metabolism of sibutramine was examined using human liver microsomes (HLM) and recombinant cytochrome P-450 (CYP) isoforms. This drug is metabolized to N-mono-desmethyl- (M1) and N,N-di-desmethylsibutramine (M2), and subsequent hydroxylation results in hydroxyl M1 (HM1) and hydroxyl M2 (HM2). No significant difference was noted in formation of M1from sibutramine between R- and S-sibutramine in HLM. However, S-enantiomers of M1 and M2 were preferentially metabolized to M2, HM1, and HM2compared to R-enantiomers in HLM, and intrinsic clearance (Cl_int) ratios of S-enantiomers/R-enantiomers were 1.97, 4.83, and 9.94 for M2, HM1, and HM2, respectively. CYP3A4 and CYP3A5 were only involved in the formation of M1, whereas CYP2B6 and CYP2C19 were responsible for all metabolic reactions of sibutramine. CYP2C19 and CYP3A5 displayed catalytic preference for S-sibutramine to S-M1, whereas CYP2B6 and CYP3A4 showed little or no stereoselectivity in metabolism of sibutramine to M1. In the case of M2 formation, CYP2B6 metabolized S-M1 more rapidly than R-M1 with a Cl_int ratio of 2.14. However, CYP2C19 catalyzed less S-M1 than R-M1 and the Cl_int ratio of S-M1 to R-M1 was 0.65. The most significant enantioselectivity was observed in formation of HM1 from M1, and HM2 from M2. CYP2B6 and CYP2C19 exhibited preferential catalysis of formation of hydroxyl metabolites from S-enantiomers rather than R-enantiomers. These results indicate that S-sibutramine was more rapidly metabolized by CYP isoforms than R-sibutramine, and that enantioselective metabolism needs to be considered in drug interactions involving sibutramine and co-administered drugs.     

Cytochrome P450‐mediated metabolism of the synthetic cannabinoids UR‐144 and XLR‐11

In recent years, synthetic cannabinoids have emerged in the illicit drug market, in particular via the Internet, leading to abuse of these drugs. There is currently limited knowledge about the specific enzymes involved in the metabolism of these drugs. In this study, we investigated the cytochrome P450 (CYP) enzymes involved in the metabolism of the two synthetic cannabinoids (1‐pentyl‐1H‐indol‐3‐yl)‐(2,2,3,3‐tetramethylcyclopropyl)methanone (UR‐144) and [1‐(5‐fluoropentyl)‐1H‐indol‐3‐yl)](2,2,3,3‐tetramethylcyclopropyl)methanone (XLR‐11). This study extends previous studies by identifying the specific CYP enzymes involved in the metabolism of UR‐144 and XLR‐11 utilizing a panel of nine recombinant enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 3A4, and 2E1). This is followed by an investigation of the effect of specific inhibitors targeted against CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4 in human liver microsomes (HLM). Incubations of UR‐144 and XLR‐11 with recombinant CYP enzymes revealed that UR‐144 and XLR‐11 are extensively metabolized by CYP3A4 at the tetramethylcyclopropyl (TMCP) moiety, but also CYP1A2 and CYP2C19 showed activity. Inhibition of CYP3A4 in HLM attenuated the metabolism of UR‐144 and XLR‐11, while inhibition of the other CYP enzymes in HLM had only minor effects. Thus, CYP3A4 is the major contributor to the CYP mediated metabolism of UR‐144 and XLR‐11 with minor contributions from CYP1A2. Users of UR‐144 and XLR‐11 are thus subject to the influence of potential drug‐drug interactions, if they are concomitantly medicated with CYP3A4 inducers (e.g. some antiepileptics) or inhibitors (e.g. some antifungal drugs). Copyright © 2015 John Wiley & Sons, Ltd.     

Metabolism of (‐)‐cis‐ and (‐)‐trans‐rose oxide by cytochrome P450 enzymes in human liver microsomes

The in vitro metabolism of (‐)‐cis‐ and (‐)‐trans‐rose oxide was investigated using human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes for the first time. Both isomers of rose oxide were incubated with human liver microsomes, and the formation of the respective 9‐oxidized metabolite were determined using gas chromatography‐mass spectrometry (GC‐MS). Of 11 different recombinant human P450 enzymes used, CYP2B6 and CYP2C19 were the primary enzymes catalysing the metabolism of (‐)‐cis‐ and (‐)‐trans‐rose oxide. CYP1A2 also efficiently oxidized (‐)‐cis‐rose oxide at the 9‐position but not (‐)‐trans‐rose oxide. α‐Naphthoflavone (a selective CYP1A2 inhibitor), thioTEPA (a CYP2B6 inhibitor) and anti‐CYP2B6 antibody inhibited (‐)‐cis‐rose oxide 9‐hydroxylation catalysed by human liver microsomes. On the other hand, the metabolism of (‐)‐trans‐rose oxide was suppressed by thioTEPA and anti‐CYP2B6 at a significant level in human liver microsomes. However, omeprazole (a CYP2C19 inhibitor) had no significant effects on the metabolism of both isomers of rose oxide. Using microsomal preparations from nine different human liver samples, (‐)‐9‐hydroxy‐cis‐ and (‐)‐9‐hydroxy‐trans‐rose oxide formations correlated with (S)‐mephenytoin N‐demethylase activity (CYP2B6 marker activity). These results suggest that CYP2B6 plays important roles in the metabolism of (‐)‐cis‐ and (‐)‐trans‐rose oxide in human liver microsomes. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Direct and metabolism‐dependent cytochrome P450 inhibition assays for evaluating drug–drug interactions

We developed methods for evaluating the ntial inhibition of human cytochrome P450 (CYP) enzymes, including CYP1A2, CYP2A6, CYP2B6, CYP2 C9, CYP2 C19, CYP2D6, CYP2E1 and CYP3A4, using pooled human liver microsomes (HLMs). The CYP inhibition assay used substrate cocktail sets [set A: phenacetin for CYP1A2, coumarin for CYP2A6, (S)‐(+)‐mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4; set B: bupropion for CYP2B6, tolbutamide for CYP2C9, chlorzoxazone for CYP2E1, and testosterone for CYP3A4] with quantitation by liquid chromatography–tandem mass spectrometry. A direct inhibition assay was performed with the substrate cocktails without β‐nicotinamide adenine dinucleotide phosphate (NADPH) pre‐incubation, and a metabolism‐dependent inhibition (MDI) assay was performed after 30 min of pre‐incubation with NADPH in HLMs. MDI was identified based on the half‐maximal inhibitory concentration (IC₅₀) shifts. The IC₅₀ values of the direct inhibitors determined using the probe substrate cocktails were in good agreement with previously reported values. Eight metabolism‐dependent inhibitors including furafylline, 8‐methoxypsoralen, tienilic acid, ticlopidine, fluoxetine, paroxetine, disulfiram and verapamil against CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, respectively, resulted in significant IC₅₀ shifts (≥2.5‐fold) after pre‐incubation. Thus, these CYP inhibition assays are considered to be useful tools for evaluating both direct inhibition and MDI at an early stage of the drug discovery and development process. Copyright © 2011 John Wiley & Sons, Ltd.     

Characterization of cytochrome P450 enzymes and P‐glycoprotein involved in the metabolism and transport of a new anti‐angiogenic agent KR‐31831

The in vitro metabolism and the transport of a novel anti‐angiogenic agent KR‐31831, (2R,3R,4S)‐6‐amino‐4‐[N‐(4‐chlorophenyl)‐N‐(1H‐imidazol‐2‐ylmethyl)amino]‐3‐hydroxy‐2‐dimethyoxymethyl‐3,4‐dihydro‐2‐methyl‐2H‐1‐benzopyran were investigated. Liquid chromatography‐mass spectrometry and tandem mass spectrometry were used for qualitative and quantitative analysis. The bidirectional transport studies of KR‐31831 using Caco‐2 cell monolayers showed the efflux to be significantly higher than influx (29.1 × 10^?6 compared to 11.5 × 10^?6 cm/s). P‐glycoprotein inhibitors significantly increased the influx of KR‐31831 and decreased the efflux of KR‐31831. These data indicate that KR‐313831 is a substrate for an efflux pump, P‐glycoprotein. The incubations of KR‐31831 with human liver microsomes produced three metabolites, M1, M2, and M3. M1 and M2 were identified as N‐(4‐chlorophenyl)‐N‐(1H‐imidazol‐2‐ylmethyl)amine and (2R,3R,4S)‐6‐amino‐4‐[N‐(4‐chlorophenyl)‐N‐(1H‐imidazol‐2‐ylmethyl)amino]‐3‐hydroxy‐2‐hydroxymethyl‐3,4‐dihydro‐2‐methyl‐2H‐1‐benzopyran by comparison with the authentic standards. M3 was tentatively characterized as hydroxy‐KR‐31831. CYP3A4 was identified as the major enzyme responsible for KR‐31831 metabolism to a major metabolite M1 using the combination of correlation analysis, immuno‐inhibition, chemical inhibition in human liver microsomes, and metabolism by cDNA expressed CYP enzymes. There is the possibility of drug–drug interactions when prescribing KR‐31831 concomitantly with known inhibitors or inducers of CYP3A4 and P‐glycoprotein. KR‐31831 was found to inhibit potently the metabolism of CYP2D6 substrate, suggesting that coadministration of KR‐31831 with CYP2D6 substrates may have significant effects on the pharmacokinetics of CYP2D6 substrates. Drug Dev. Res. 66:40–49. 2006. © 2006 Wiley‐Liss, Inc.     

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 of human cytochrome P450 enzymes involved in the hepatic and intestinal biotransformation of 20(S)‐protopanaxadiol

20(S)‐Protopanaxadiol (aPPD), a ginseng sapogenin, has been shown to be a promising anti‐cancer compound and anti‐depressant agent. Although the bacterial biotransformation of ginsenosides has been studied thoroughly, few have reported on the cytochrome P450 (P450) mediated metabolism of aPPD. Taken orally, aPPD must first undergo absorption and metabolism in the intestine before further metabolism in the liver. The present study investigated the comparative biotransformation profile of aPPD in human intestinal microsomes (HIM) and human liver microsomes (HLM) and characterized the human P450 enzymes involved in aPPD metabolism. Three major monooxygenated metabolites and five minor dioxygenated metabolites were identified as the predominant products in aPPD incubations with HIM and HLM using liquid chromatography–mass spectrometry. Reaction phenotyping studies were performed with a panel of specific P450 chemical inhibitors, antibody inhibition and human recombinant P450 enzymes. Ketoconazole, a CYP3A inhibitor, blocked the formation of oxygenated metabolites of aPPD in both HIM and HLM in a concentration dependent manner. Among the human recombinant P450 enzymes assayed, CYP3A4 exhibited the highest activity towards aPPD oxidative metabolite formation, followed by CYP3A5. In summary, the results have shown that aPPD is extensively metabolized by HIM and the metabolite profile following in vitro incubations is similar in HIM and HLM. CYP3A4 and CYP3A5 isoforms are the predominant enzymes responsible for oxygenation of aPPD in HIM and HLM. The characterization of aPPD as a CYP3A substrate may facilitate better prediction of drug–herb interactions when aPPD is taken concomitantly with other therapeutic agents. Copyright © 2013 John Wiley & Sons, Ltd.     

In vitro metabolism of a novel antithrombotic compound,S002-333, and its enantiomers: quantitative cytochrome P450 phenotyping,metabolic profiling and enzyme kinetic studies

Abstract

1.?S002-333, (2-(4′-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-pyrido (3,4-b) indole-3-carboxylic acid amide) is a novel potent antithrombotic molecule currently under development phase. It is the racemic mixture of two enantiomers, namely S004-1032 (R-form) and S007-1558 (S-form).

2.?The contribution of five major isoenzymes, namely CYP2B6, 2C9, 2C19, 2D6 and 3A4 was quantified using recombinant P450s in the phase-I metabolism through relative activity factor approach. CYP2C19 was found to be the major contributor for S002-333 and S007-1558, while CYP3A4 showed greater involvement in S004-1032 metabolism. Chemical inhibition and immunoinhibition studies reconfirmed the results in human liver microsomes (HLM).

3.?Four major phase-I metabolites of S002-333; M-1 and M-3 (oxidative), M-2 (O-demethylated) and M-4 (dehydrogenated) were characterized in HLM. These metabolites constituted 11.2, 11.3 and 21.5% of the parent in comparison with the net phase-I metabolism of 29.9, 31.4 and 38.3% of S002-333, S004-1032 and S007-1558, respectively.

4.?Among CYP2C9, 2C19 and 3A4, the relative contribution of CYP2C9 was found to be maximum during M-1 through M-4 formation. Enzyme kinetic analysis for detected metabolites indicated that M-1 to M-3 followed classical hyperbolic kinetics, whereas M-4 showed evidence of autoactivation. In conclusion, the results suggest prominent role of CYP2C9, 2C19 and 3A4 isoforms for enantioselective disposition of S002-333 in vitro.     

An in vitro approach to potential methadone metabolic-inhibition interactions

Objective The aim of this study was to assess the drug interaction potential of psychotropic medication on methadone N-demethylation using cDNA-expressed cytochrome P450 CYP enzymes. Methods Methadone was incubated with various drugs (n = 10) and cDNA-expressed CYP3A4, CYP2D6, CYP2B6, CYP2C19 and CYP1A2 enzymes to screen for their inhibition potency. The nature of enzyme selective activity for inhibition was further investigated for potent inhibitors. To test for a mechanism-based component in inhibition, all substances were tested with preincubation and without. 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) concentration was determined by liquid chromatography/tandem mass spectrometry following liquid/liquid extraction. Results Formation of EDDP was catalysed by CYP3A4, CYP2D6 and CYP2C19. The N-demethylation of methadone was preferentially inhibited by amitriptyline, buprenorphine, methylenedioxymethamphetamine (MDMA) and zolpidem. Both amitriptyline and buprenorphine were strong, reversible inhibitors of CYP3A4. Similarly, amitriptyline and MDMA were identified as inhibitors of CYP2D6. Zolpidem revealed a mechanism-based inhibition of CYP3A4. Conclusion Amitriptyline, MDMA and zolpidem are likely to slow down conversion of methadone and to increase its area under the curve (AUC). A consideration of the in vitro evidence of drug-methadone interactions should help to improve patient care during methadone maintenance treatment.     

Identification of cytochromes<Emphasis Type="Italic"> P</Emphasis><Subscript>450</Subscript> 2C9 and 3A4 as the major catalysts of phenprocoumon hydroxylation in vitro

Objective This in-vitro study aimed at an identification of cytochrome P₄₅₀ (CYP) enzymes catalysing the (S)- and (R)-hydroxylation of the widely used anticoagulant phenprocoumon (PPC) to its major, inactive metabolites.Methods Relevant catalysts were identified by kinetic, correlation and inhibition experiments using human liver microsomes and recombinant enzymes.Results Kinetics revealed (S)-7-hydroxylation as quantitatively most important. Biphasic Eadie-Hofstee plots indicated more than one catalyst for the 4-, 6- and 7-hydroxylation of both enantiomers with mean K_m1 and K_m2 of 144.5±34.9 and 10.0±6.49 µM, respectively. PPC hydroxylation rates were significantly correlated with CYP2C9 and CYP3A4 activity and expression analysing 11 different CYP-specific probes. Complete inhibition of PPC hydroxylation was achieved by combined addition of the CYP3A4-specific inhibitor triacetyloleandomycin (TAO) and a monoclonal, inhibitory antibody (mAb) directed against CYP2C8, 9, 18 and 19, except for the (R)-4-hydroxylation that was, however, inhibited by ~80% using TAO alone. (S)-PPC hydroxylation was reduced by ~2/3 and ~1/3 using mAb2C8–9-18–19 and TAO, respectively, but (R)-6- and 7-hydroxylation by ~50% each. Experiments with mAbs directed against single CYP2C enzymes clearly indicated CYP2C9 as a major catalyst of the 6- and 7-hydroxylation for both enantiomers. However, CYP2C8 was equally important regarding the (S)-4-hydroxylation. Recombinant CYP2C8 and CYP2C9 were high-affinity catalysts (K_m <5 µM), whereas CYP3A4 operated with low affinity (K_m >100 µM).Conclusion CYP2C9 and CYP3A4 are major catalysts of (S)- and (R)-PPC hydroxylation, while CYP2C8 partly catalysed the (S)-4-hydroxylation. Increased vigilance is warranted when PPC treatment is combined with substrates, inhibitors, or inducers of these enzymes.Part of this work was presented at the 6th Congress of the European Association for Clinical Pharmacology and Therapeutics, Istanbul, June 2003.     

Inhibitory effects of memantine on human cytochrome P450 activities: prediction of in vivo drug interactions

Objective: The aim of the present study was to predict the drug interaction potential of memantine by elucidation of its inhibitory effects on cytochrome P450 enzymes using pooled human liver microsomes (HLM) and recombinant P450s. Methods: The inhibitory potency of memantine on CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 activities was examined with specific probe drugs in HLM and recombinant P450s. The in vivo drug interactions of memantine were predicted in vitro using the [I ]/([I ] + K_I) values. Results: In HLM, memantine inhibited CYP2B6 and CYP2D6 activities, with K_I (IC₅₀) values of 76.7 (279.7) and 94.9 (368.7) M, respectively. Both inhibitions were competitive. In addition, cDNA-expressed P450s were used to confirm these results. Memantine strongly inhibited recombinant CYP2B6 activity with IC₅₀ (K_I) value of 1.12 (0.51) M and activity of recombinant CYP2D6 with IC₅₀ (K_I) value of 242.4 (84.4) M. With concentrations up to 1,000 M, memantine showed no appreciable effect on CYP1A2, CYP2E1, CYP2C9, or CYP3A4 activities and a slight decrease of CYP2A6 and CYP2C19 activities. Based on [I ]/([I ] + K_I) values calculated using peak total plasma concentration (or enzyme-available concentration in the liver) of memantine and the K_I obtained in HLM, 1.3 (13.5), and 1.0% (11.2%), inhibition of the clearance of CYP2B6 and CYP2D6 substrates could be expected, respectively. Nevertheless, when considering K_I values obtained from cDNA-expressed CYP2B6, as generally recommended, even 66.2% (95.9%) decrease in metabolism of coadministered CYP2B6 substrates could be anticipated. Conclusion: Memantine exerts selective inhibition of CYP2B6 activity at clinically relevant concentrations, suggesting the potential for clinically significant drug interactions. Inhibition of other CYPs during memantine therapy is unlikely. Moreover, memantine represents a new, potent, selective inhibitor of recombinant CYP2B6, which may prove useful for screening purposes during early phases of in vitro drug metabolism studies with new chemical entities.     

Efficient synthesis of 13C‐labelled erythromycin biosynthetic intermediate. 1: S‐2‐acetylaminoethyl (2R,3R,4R,5R)‐3,5‐diacetoxy‐2,4‐dimethyl‐4‐([13C]methoxy)‐heptanethioate

An efficient ¹³C‐labelling synthesis of the putative erythromycin biosynthetic intermediate, S‐2‐acetylaminoethyl (2R,3R,4R,5R)‐3,5‐diacetoxy‐2,4‐dimethyl‐4‐([¹³C]methoxy)heptanethioate, which would be useful for the investigation of the chain elongation mechanism in erythromycin biosynthesis, was achieved by utilizing iodo[¹³C]methane and (2S,3R,4R,5R)‐4‐hydroxy‐3,5‐O‐isopropylidene‐2,4‐dimethylheptanol, obtained in our previous studies on erythromycin A synthesis. Copyright © 2008 John Wiley & Sons, Ltd.     

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.     

Synthesis of [2‐13C, 4‐13C]‐(2R,3S)‐catechin and [2‐13C, 4‐13C]‐(2R,3R)‐epicatechin

The first synthesis of doubly labeled, [2‐¹³C, 4‐¹³C]‐(2R,3S)‐catechin 15 and [2‐¹³C, 4‐¹³C]‐(2R,3R)‐epicatechin 18 starting from labeled 2‐hydroxy‐4, 6‐bis(benzyloxy)acetophenone 3 and labeled 3, 4‐bis(benzyloxy)‐benzaldehyde 7 are described. Copyright © 2010 John Wiley & Sons, Ltd.     

Methadone N-demethylation in human liver microsomes: lack of stereoselectivity and involvement of CYP3A4

AIMS: To investigate the kinetics of CYP-mediated N-demethylation of methadone in human liver microsomes, and examine the role of stereoselectivity and CYP isoforms involved. METHODS: The kinetics of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) formation via N-demethylation of rac-, (R)- and (S)-methadone in human liver microsomes prepared from six liver samples were determined by h.p.l.c., and inhibition of metabolic function was studied using isoform-specific chemical inhibitors and monoclonal antibodies. Microsomes containing expressed CYP3A4, CYP2D6 and CYP2C19 were also used to examine the formation of EDDP. RESULTS: The V max, Km, and CLint values for the formation of EDDP from rac-, (R)- and (S)-methadone were in the ranges of 20-77 nmol mg-1 protein h-1, 125-252 microm, and 91-494 ml h-1 g-1 protein. Km and CLint values for (R)- and (S)-methadone were not statistically significantly different (P >0.05), while V max values for (S)-methadone were 15% (P=0.045) lower than for (R)-methadone. Expressed CYP3A4 and CYP2C19 showed similar reaction rates for both (R)- and (S)-methadone, while CYP2D6 did not catalyse this reaction. Selective chemical inhibitors of CYP3A (troleandomycin, ketoconazole) and monoclonal human CYP3A4 antibodies significantly inhibited (P<0.05) the formation of EDDP in a concentration dependent manner by up to 80%. Sulphaphenazole (CYP2C9) also significantly inhibited (P<0.05) EDDP formation (range 14-25%). There were no statistically significant differences in the inhibition observed between the three substrates. Selective inhibitors of CYP1A2 (furafylline), CYP2A6 (coumarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (quinidine) and CYP2E1 (diethyldithiocarbamic acid sodium salt and monoclonal human CYP2E1 antibodies) had no significant (P >0.05) effect. CONCLUSIONS: The N-demethylation of methadone in human liver microsomes is not markedly stereoselective, and is mediated mainly by CYP3A4 with the possible involvement of CYP2C9 and CYP2C19. Thus, the large interindividual variation reported for methadone pharmacokinetics may be due to variability in the expression of these CYP isoforms, and the reported stereoselectivity in the systemic clearance of methadone in vivo is not due to stereoselectivity in N-demethylation.     

Synthesis of (1R,2R)‐ and (1S,2R)‐1,2‐Epoxy‐3‐hydroxypropylphosphonates as Analogues of Fosfomycin

Cyclohexylammonium (1R,2R)‐1,2‐epoxy‐3‐hydroxypropylphosphonate was conveniently synthesized from dibenzyl (1S,2R)‐2,3‐O‐cyclohexylidene‐1,2,3‐trihydroxypropylphosphonate by a reaction sequence including mesylation, hydrolysis of acetal, intramolecular Williamson reaction, and hydrogenation in the presence of cyclohexylamine. For dibenzyl (1S,2R)‐2,3‐O‐cyclohexylidene‐1,2,3‐trihydroxypropylphosphonates the same approach was not successful, since prior the epoxide‐ring closure tritylation of HO–C3 in dibenzyl (1R,2R)‐2,3‐dihydroxy‐1‐mesyloxypropylphosphonate was necessary and the hydrogenolysis of dibenzyl (1S,2R)‐1,2‐epoxy‐3‐trityloxypropylphosphonate yielded a complex reaction mixture.     

Effects of CYP2C19 variants on methadone metabolism in vitro

CYP2C19 is an important member of the cytochrome P450 (CYP450) enzyme super family and is responsible for clearing approximately 10% of commonly used clinical drugs that undergo phase I metabolism. Genetic polymorphisms of CYP2C19 significantly influence the efficacy and safety of some drugs, which might cause undesirable adverse effects or cure failure at standard dosages. The aim of this study was to clarify the catalytic activities of 31 CYP2C19 alleles on the oxidative in vitro metabolism of methadone. Insect microsomes expressing the CYP2C19 alleles were incubated with 50–2000 μM methadone for 30 min at 37 °C and terminated by cooling to ‐80 °C immediately. Methadone and its metabolite EDDP were analyzed by an ultra performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) system. Of the 31 tested CYP2C19 allelies variants, CYP2C19*1 is the wild‐type. Compared with CYP2C19*1, two CYP2C19 variants (CYP2C19*3 and *35FS) had no detectable enzyme activity, one variant L16F exhibited slightly increased intrinsic clearance values, and one variant N277K showed no significant difference. In addition, 26 variants exhibited significantly decreased values (from 1.48% to 80.40%). These findings suggest that more attention should be paid in clinical administration of methadone to individuals carrying these CYP2C19 alleles. Copyright © 2016 John Wiley & Sons, Ltd.     

Synthesis of (S)‐cyano(3‐phenoxyphenyl)methyl (1R,3R)‐ 3‐[(1Z)‐2‐chloro‐3,3,3‐trifluoro‐1‐propenyl]‐2,2‐dimethylcyclopropanecarboxylate‐1‐14C

γ‐Cyhalothrin ( 1a ), (S)‐cyano(3‐phenoxyphenyl)methyl (1R,3R)‐3‐[(1Z)‐2‐chloro‐3,3,3‐trifluoro‐1‐propenyl]‐2,2‐dimethylcyclopropanecarboxylate, is a single‐isomer, synthetic pyrethroid insecticide marketed by Pytech Chemicals GmbH, a joint venture between Dow AgroSciences and Cheminova A/S. As a part of the registration process there was a need to incorporate a carbon‐14 label into the cyclopropyl ring of this molecule. A high yielding radiochemical synthesis of γ‐cyhalothrin was developed from readily available carbon‐14 labeled N‐t‐Boc protected glycine. This seven step synthesis, followed by a preparative normal phase HPLC separation of diastereomers, provided 21.8 mCi of γ‐cyhalothrin‐1‐¹⁴C ( 1b ) with >98% radiochemical purity. Copyright © 2007 John Wiley & Sons, Ltd.