2D SMARTCyp reactivity-based site of metabolism prediction for major drug-metabolizing cytochrome P450 enzymes

Cytochrome P450 (CYP) 3A4, 2D6, 2C9, 2C19, and 1A2 are the most important drug-metabolizing enzymes in the human liver. Knowledge of which parts of a drug molecule are subject to metabolic reactions catalyzed by these enzymes is crucial for rational drug design to mitigate ADME/toxicity issues. SMARTCyp, a recently developed 2D ligand structure-based method, is able to predict site-specific metabolic reactivity of CYP3A4 and CYP2D6 substrates with an accuracy that rivals the best and more computationally demanding 3D structure-based methods. In this article, the SMARTCyp approach was extended to predict the metabolic hotspots for CYP2C9, CYP2C19, and CYP1A2 substrates. This was accomplished by taking into account the impact of a key substrate-receptor recognition feature of each enzyme as a correction term to the SMARTCyp reactivity. The corrected reactivity was then used to rank order the likely sites of CYP-mediated metabolic reactions. For 60 CYP1A2 substrates, the observed major sites of CYP1A2 catalyzed metabolic reactions were among the top-ranked 1, 2, and 3 positions in 67%, 80%, and 83% of the cases, respectively. The results were similar to those obtained by MetaSite and the reactivity + docking approach. For 70 CYP2C9 substrates, the observed sites of CYP2C9 metabolism were among the top-ranked 1, 2, and 3 positions in 66%, 86%, and 87% of the cases, respectively. These results were better than the corresponding results of StarDrop version 5.0, which were 61%, 73%, and 77%, respectively. For 36 compounds metabolized by CYP2C19, the observed sites of metabolism were found to be among the top-ranked 1, 2, and 3 sites in 78%, 89%, and 94% of the cases, respectively. The computational procedure was implemented as an extension to the program SMARTCyp 2.0. With the extension, the program can now predict the site of metabolism for all five major drug-metabolizing enzymes with an accuracy similar to or better than that achieved by the best 3D structure-based methods. Both the Java source code and the binary executable of the program are freely available to interested users.     

Liquid chromatography/tandem mass spectrometric determination of inhibition of human cytochrome P450 isozymes by resveratrol and resveratrol-3-sulfate

trans-Resveratrol, a phenolic phytoalexin occurring in grapes, wine, peanuts, and cranberries, has been reported to both have anticarcinogenic, antioxidative, phytoestrogenic, and cardioprotective activities, and to be a weak inhibitor of cytochrome P450 (CYP)3A4, which might have significance for drug-drug interactions. Since trans-resveratrol is rapidly converted in vivo to primarily trans-resveratrol-3-sulfate, a rapid, selective, and sensitive method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed to investigate human cytochrome P450 inhibition by trans-resveratrol-3-sulfate. Effects of trans-resveratrol and trans-resveratrol-3-sulfate on the metabolism of selective cytochrome P450 substrates (CYP1A2/ethoxyresorufin, CYP2C9/diclofenac, CYP2C19/(S)-mephenytoin, CYP2D6/bufuralol, CYP3A4/testosterone) were monitored using cDNA-expressed human recombinant isozymes. For method validation, LC/MS/MS was used to measure the inhibition of various cytochrome P450 isozymes by different concentrations (0-50 microM) of known selective inhibitors. IC(50) values of 3.2, 1.4, 8.9, 0.2, and 0.3 microM were obtained for the standard isozyme inhibitors CYP1A2/furafylline, CYP2C9/sulfaphenazole, CYP2C19/tranylcypromine, CYP2D6/quinidine, and CYP3A4/ketoconazole, respectively, which were in good agreement with literature values. trans-Resveratrol showed IC(50) values of 11.6 microM for CYP2C19 and 1.1 microM for CYP3A4, but the IC(50) values exceeded 50 microM for all the other CYP isozymes, which indicated no inhibition. No enzyme inhibition was observed for trans-resveratrol-3-sulfate. Our results indicate that trans-resveratrol is a marginal inhibitor of CYP3A4 and a weak inhibitor of CYP2C19, but its major metabolite trans-resveratrol-3-sulfate is not an inhibitor of any of the cytochrome P450 isozymes investigated.     

Cytochrome P450 reaction phenotyping and inhibition and induction studies of pinostrobin in human liver microsomes and hepatocytes

Pinostrobin (PI, 5‐hydroxy‐7‐methoxyflavanone) is a natural flavonoid known for its rich pharmacological activities. The objective of this study was to identify the human liver cytochrome P450 (CYP450) isoenzymes involved in the metabolism of PI. A single hydoxylated metabolite was obtained from PI after an incubation with pooled human liver microsomes (HLMs). The relative contributions of different CYP450s were evaluated using CYP450‐selective inhibitors in HLMs and recombinant human CYP450 enzymes, and the results revealed the major involvement of CYP1A2, CYP2C9 and CYP2E1 in PI metabolism. We also evaluated the ability of PI to inhibit and induce human cytochrome P450 enzymes in vitro . High‐performance liquid chromatography and liquid chromatography–tandem mass spectrometry analytical techniques were used to estimate the enzymatic activities of seven drug‐metabolizing CYP450 isozymes in vitro . In HLMs, PI did not inhibit CYP 1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 (IC₅₀ > 100 μm ). In the induction studies, PI had minimal effects on CYP1A2, CYP2B6and CYP3A4 activity. Based on these results, PI would not be expected to cause clinically significant CYP450 inhibition or induction.     

Direct inhibition of Re Du Ning Injection and its active compounds on human liver cytochrome P450 enzymes by a cocktail method

The aim of this study was to investigate the direct inhibitory effects of Re Du Ning Injection (RDN) and its active compounds on the major cytochrome P450 enzyme (CYP) isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) of human liver microsomes by ‘a cocktail method’. The activity of each CYP isform was represented as the formation rate of the specific metabolite from relevant substrate. Then a sensitive and specific ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed and validated to simultaneously analyze the seven metabolites. RDN (0.035–2.26 mg/mL) showed a strong inhibitiory effect on CYP2C8, followed by CYP2C9, CYP2B6, CYP2C19, CYP1A2 and CYP3A4. The IC₅₀ value for each enzyme was 0.19, 0.66, 0.72, 1.27, 1.66 and 2.13 mg/mL, respectively. RDN competitively inhibited the activities of CYP1A2 (K _i = 1.22 mg/mL), CYP2B6 (K _i = 0.65 mg/mL) and CYP3A4 (K _i = 0.88 mg/mL); it also exhibited mixed inhibition of CYP2C8, CYP2C9 and CYP2C19 with a K _i value of 0.26, 0.64 and 0.82 mg/mL, respectively. However, the activity of CYP2D6 was not significantly inhibited even by 2.26 mg/mL RDN. Moreover, the data of nine active compounds on the CYPs showed that cryptochlorogenin acid, sochlorogenic acid B and sochlorogenic acid C were the major contributors to the inhibitory effect of RDN on CYP2C8, while the inhibitory effect of RDN on CYP2C9 might be caused by sochlorogenic acid A and sochlorogenic acid C. Moreover, neochlorogenic acid might be the major contributor to the inhibitory effect on CYP2B6. All of the findings suggested that drug–drug interactions may occur and great caution should be taken when RDN is combined with drugs metabolized by these CYPs.     

Insights on cytochrome p450 enzymes and inhibitors obtained through QSAR studies

The cytochrome P450 (CYP) superfamily of heme enzymes play an important role in the metabolism of a large number of endogenous and exogenous compounds, including most of the drugs currently on the market. Inhibitors of CYP enzymes have important roles in the treatment of several disease conditions such as numerous cancers and fungal infections in addition to their critical role in drug-drug interactions. Structure activity relationships (SAR), and three-dimensional quantitative structure activity relationships (3D-QSAR) represent important tools in understanding the interactions of the inhibitors with the active sites of the CYP enzymes. A comprehensive account of the QSAR studies on the major human CYPs 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and a few other CYPs are detailed in this review which will provide us with an insight into the individual/common characteristics of the active sites of these enzymes and the enzyme-inhibitor interactions.     

Predictive models for cytochrome p450 isozymes based on quantitative high throughput screening data

The human cytochrome P450 (CYP450) isozymes are the most important enzymes in the body to metabolize many endogenous and exogenous substances including environmental toxins and therapeutic drugs. Any unnecessary interactions between a small molecule and CYP450 isozymes may raise a potential to disarm the integrity of the protection. Accurately predicting the potential interactions between a small molecule and CYP450 isozymes is highly desirable for assessing the metabolic stability and toxicity of the molecule. The National Institutes of Health Chemical Genomics Center (NCGC) has screened a collection of over 17,000 compounds against the five major isozymes of CYP450 (1A2, 2C9, 2C19, 2D6, and 3A4) in a quantitative high throughput screening (qHTS) format. In this study, we developed support vector classification (SVC) models for these five isozymes using a set of customized generic atom types. The CYP450 data sets were randomly split into equal-sized training and test sets. The optimized SVC models exhibited high predictive power against the test sets for all five CYP450 isozymes with accuracies of 0.93, 0.89, 0.89, 0.85, and 0.87 for 1A2, 2C9, 2C19, 2D6, and 3A4, respectively, as measured by the area under the receiver operating characteristic (ROC) curves. The important atom types and features extracted from the five models are consistent with the structural preferences for different CYP450 substrates reported in the literature. We also identified novel features with significant discerning power to separate CYP450 actives from inactives. These models can be useful in prioritizing compounds in a drug discovery pipeline or recognizing the toxic potential of environmental chemicals.     

Simultaneous determination of bupropion,metroprolol, midazolam,phenacetin, omeprazole and tolbutamide in rat plasma by UPLC‐MS/MS and its application to cytochrome P450 activity study in rats

A specific ultra‐performance liquid chromatography tandem mass spectrometry method is described for the simultaneous determination of bupropion, metroprolol, midazolam, phenacetin, omeprazole and tolbutamide in rat plasma with diazepam as internal standard, which are the six probe drugs of the six cytochrome P450 isoforms CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19 and CYP2C9. Plasma samples were protein precipitated with acetonitrile. The chromatographic separation was achieved using a UPLC® BEH C₁₈ column (2.1 × 100 mm, 1.7 µm). The mobile phase consisted of acetonitrile and water (containing 0.1% formic acid) with gradient elution. The triple quadrupole mass spectrometric detection was operated by multiple reaction monitoring in positive electrospray ionization. The precisions were <13%, and the accuracy ranged from 93.3 to 110.4%. The extraction efficiency was >90.5%, and the matrix effects ranged from 84.3 to 114.2%. The calibration curves in plasma were linear in the range of 2–2000 ng/mL, with correlation coefficient (r²) >0.995. The method was successfully applied to pharmacokinetic studies of the six probe drugs of the six CYP450 isoforms and used to evaluate the effects of erlotinib on the activities of CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19 and CYP2C9 in rats. Erlotinib may inhibit the activity of CYP2B6 and CYP3A4, and may induce CYP2C9 of rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Prediction of cytochrome P450 3A4, 2D6, and 2C9 inhibitors and substrates by using support vector machines

Statistical learning methods have been used in developing filters for predicting inhibitors of two P450 isoenzymes, CYP3A4 and CYP2D6. This work explores the use of different statistical learning methods for predicting inhibitors of these enzymes and an additional P450 enzyme, CYP2C9, and the substrates of the three P450 isoenzymes. Two consensus support vector machine (CSVM) methods, "positive majority" (PM-CSVM) and "positive probability" (PP-CSVM), were used in this work. These methods were first tested for the prediction of inhibitors of CYP3A4 and CYP2D6 by using a significantly higher number of inhibitors and noninhibitors than that used in earlier studies. They were then applied to the prediction of inhibitors of CYP2C9 and substrates of the three enzymes. Both methods predict inhibitors of CYP3A4 and CYP2D6 at a similar level of accuracy as those of earlier studies. For classification of inhibitors of CYP2C9, the best CSVM method gives an accuracy of 88.9% for inhibitors and 96.3% for noninhibitors. The accuracies for classification of substrates and nonsubstrates of CYP3A4, CYP2D6, and CYP2C9 are 98.2 and 90.9%, 96.6 and 94.4%, and 85.7 and 98.8%, respectively. Both CSVM methods are potentially useful as filters for predicting inhibitors and substrates of P450 isoenzymes. These methods generally give better accuracies than single SVM classification systems, and the performance of the PP-CSVM method is slightly better than that of the PM-CSVM method.     

人肝细胞色素P450 2C金属酶与药物代谢*

由于人肝细胞色素P450 2C亚家族与临床药物代谢的密切关系,其研究已引起人们的广泛关注。本文综述了四种人肝细胞色素P450 2C,着重综述了其中的三种：CYP2C9,CYP2C8,CYP2C19的研究进展。评述了CYP2C9,CYP2C8和CYP2C19的某些氨基酸残基在催化过程中的作用,这三种酶的基因多态在不同人种中的分布及药物代谢的差异,以及它们与用药的特异性及某些疾病的易感性的联系,介绍了目前提出的CYP2C8的底物药效团模型,最后总结了CYP2C9,CYP2C8,CYP2C19,CYP2C18的主要特性。     

In silico modeling of p450 substrates, inhibitors, activators, and inducers

Cytochrome P450 enzymes are the predominant mediators of phase I metabolism of exogenous small molecules. As a result of their extensive role in metabolism of xenobiotics, drug compounds, and endogenous compounds, as well as their wide tissue distribution, significant drug discovery resources are spent to avoid interacting with this class of enzymes. Here we review historical and recent in silico modeling of 7 cytochrome P450 enzymes of particular interest, specifically CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. For each we provide a brief biological background including known inhibitors, substrates, and inducers, as well as details of computational modeling efforts and advances in structural biology. We also provide similar details for 3 nuclear receptors known to regulate gene expression of these enzyme families.     

Evaluation of the effects of Mitragyna speciosa alkaloid extract on cytochrome P450 enzymes using a high throughput assay

The extract from Mitragyna speciosa has been widely used as an opium substitute, mainly due to its morphine-like pharmacological effects. This study investigated the effects of M. speciosa alkaloid extract (MSE) on human recombinant cytochrome P450 (CYP) enzyme activities using a modified Crespi method. As compared with the liquid chromatography-mass spectrometry method, this method has shown to be a fast and cost-effective way to perform CYP inhibition studies. The results indicated that MSE has the most potent inhibitory effect on CYP3A4 and CYP2D6, with apparent half-maximal inhibitory concentration (IC(50)) values of 0.78 μg/mL and 0.636 μg/mL, respectively. In addition, moderate inhibition was observed for CYP1A2, with an IC(50) of 39 μg/mL, and weak inhibition was detected for CYP2C19. The IC(50) of CYP2C19 could not be determined, however, because inhibition was <50%. Competitive inhibition was found for the MSE-treated CYP2D6 inhibition assay, whereas non-competitive inhibition was shown in inhibition assays using CYP3A4, CYP1A2 and CYP2C19. Quinidine (CYP2D6), ketoconazole (CYP3A4), tranylcypromine (CYP2C19) and furafylline (CYP1A2) were ACCESSused as positive controls throughout the experiments. This study shows that MSE may contribute to an herb-drug interaction if administered concomitantly with drugs that are substrates for CYP3A4, CYP2D6 and CYP1A2.     

Inhibitory effects of Danhong Injection and its major constituents on human cytochrome P450 enzymes in vitro

Danhong Injection (DHI) as a Chinese patent medicine is mainly used to treat ischemic encephalopathy and coronary heart disease in combination with other chemotherapy. However, the information on DHI's potential drug interactions is limited. The goal of this work was to examine the potential P450‐mediated metabolism drug interaction arising from DHI and its active components. The results showed that DHI inhibited CYP2C19, CYP2D6, CYP3A4, CYP2E1 and CYP2C9 with IC₅₀ values of 1.26, 1.42, 1.63, 1.10 and 1.67% (v/v), respectively. Danshensu and rosmarinic acid inhibited CYP2E1 and CYP2C9 with IC₅₀ values of 36.63 and 75.76 μm , and 34.42 and 76.89 μm , respectively. Salvianolic acid A and B inhibited CYP2D6, CYP2E1 and CYP2C9 with IC₅₀ values of 33.79, 21.64 and 31.94 μm , and 45.47, 13.52 and 24.15 μm , respectively. The study provides some useful information for safe and effective use of DHI in clinical practice.     

Capillary electrophoresis to assess drug metabolism induced in vitro using single CYP450 enzymes (Supersomes): application to the chiral metabolism of mephenytoin and methadone

Capillary electrophoresis (CE) with multiwavelength absorbance detection is demonstrated to be an effective tool for the assessment of in vitro drug metabolism studies using microsomes containing single human cytochrome P450 enzymes (CYPs) expressed in baculovirus-infected insect cells (Supersomes). Mephenytoin (MEPH), dextromethorphan, diclofenac, caffeine, and methadone (MET) were successfully applied as test substrates for CYP2C19, CYP2D6*1, CYP2C9*1, CYP1A2, and CYP3A4, respectively. For each system, the CE-based assay could be shown to permit the simultaneous analysis of the parent drug and its targeted metabolite. Using a chiral micellar electrokinetic capillary chromatography assay, the aromatic hydroxylation of MEPH catalyzed by CYP2C19 could thereby be confirmed to be highly stereoselective, an aspect that is in agreement with data obtained via urinary analysis after intake of racemic MEPH by extensive metabolizer phenotypes. The MET to 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) conversion was investigated with a chiral zone electrophoresis assay. Incubation of racemic and nonracemic MET with CYP3A4 revealed no stereoselectivity for the transformation to EDDP, whereas no EDDP formation was observed with CYP1A2. CYP2C9 and CYP2C19 provided enhanced formation of R-EDDP and CYP2D6 incubation resulted in the preferential conversion to S-EDDP. Investigations using racemic MET and human liver microsomes revealed a modest stereoselectivity with an R/S EDDP ratio < 1 which is similar to the in vivo findings in urine.     

Coexpression of CPR from Various Origins Enhances Biotransformation Activity of Human CYPs in S. pombe

Cytochrome P450 enzymes (CYPs or P450s) are the most important enzymes involved in the phase I metabolism of drugs (and other xenobiotics) in humans, and the corresponding drug metabolites are needed as reference substances for their structural confirmation and for pharmacological or toxicological characterization. We have previously shown that biotechnological synthesis of such metabolites is feasible by whole-cell biotransformation with human CYPs recombinantly expressed in the fission yeast Schizosaccharomyces pombe. It was the aim of this study to compare the activity of seven human microsomal CYPs (CYP2C9, CYP2D6, CYP3A4, CYP3A5, CYP3A7, CYP17, and CYP21) upon coexpression with NADPH-cytochrome P450 oxidoreductases (CPRs) from various origins, namely, human CPR (hCPR) and its homologues from fission yeast (ccr1) and the bishop’s weed Ammi majus (AmCPR), respectively. For this purpose, 28 recombinant strains were needed, with five of them having been constructed previously and 23 strains being newly constructed. Bioconversion experiments showed that coexpression of a CPR does not only influence the reaction rate but, in some cases, also exerts an influence on the metabolite pattern. For CYP3A enzymes, coexpression of hCPR yielded the best results, while for another two, hCPR was equally helpful as ccr1 (both CYP17 and CYP21) or AmCPR (CYP17 only), respectively. Interestingly, CYP2D6 displayed its highest activity when coexpressed with ccr1 and CYP2C9 with AmCPR. These results corroborate the view of CPR as a well-suited bio-brick in synthetic biology for the construction of artificial enzyme complexes.     

Comparison of metabolic soft spot predictions of CYP3A4, CYP2C9 and CYP2D6 substrates using MetaSite and StarDrop

Metabolite identification study plays an important role in determining the sites of metabolic liability of new chemical entities (NCEs) in drug discovery for lead optimization. Here we compare the two predictive software, MetaSite and StarDrop, available for this purpose. They work very differently but are used to predict the site of oxidation by major human cytochrome P450 (CYP) isoforms. Neither software can predict non-CYP catalyzed metabolism nor the rates of metabolism. For the purpose of comparing the two software packages, we tested known probe substrate for these enzymes, which included 12 substrates of CYP3A4 and 18 substrates of CYP2C9 and CYP2D6 were analyzed by each software and the results were compared. It is possible that these known substrates were part of the training set but we are not aware of it. To assess the performance of each software we assigned a point system for each correct prediction. The total points assigned for each CYP isoform experimentally were compared as a percentage of the total points assigned theoretically for the first choice prediction for all substrates for each isoform. Our results show that MetaSite and StarDrop are similar in predicting the correct site of metabolism by CYP3A4 (78% vs 83%, respectively). StarDrop appears to do slightly better in predicting the correct site of metabolism by CYP2C9 and CYP2D6 metabolism (89% and 93%, respectively) compared to MetaSite (63% and 70%, respectively). The sites of metabolism (SOM) from 34 in-house NCEs incubated in human liver microsomes or human hepatocytes were also evaluated using two prediction software packages and the results showed comparable SOM predictions. What makes this comparison challenging is that the contribution of each isoform to the intrinsic clearance (Clint) is not known. Overall the software were comparable except for MetaSite performing better for CYP2D6 and that MetaSite has a liver model that is absent in StarDrop that predicted with 82% accuracy.     

Assessment of the stereoselective metabolism of methaqualone in man by capillary electrophoresis

Methaqualone (MQ) and its hydroxylated metabolites are quinazoline derivatives that exhibit atropisomerism. As a continuation of our previous work with these compounds (Electrophoresis 2001, 22, 3270-3280), chiral capillary zone electrophoresis with hydroxypropyl-beta-cyclodextrin as buffer additive and multiwavelength absorbance detection is shown to be an effective tool to provide insight into the stereoselectivity of the MQ metabolism. The five major monohydroxy MQ metabolites formed during biotransformation do not show enantiomerization at temperatures up to 85 degrees C. Enzymatic and acidic hydrolysis of urines that were collected after concomitant administration of 250 mg of MQ and 25 mg diphenhydramine (DH) chloride are both shown to provide stereoselective metabolic patterns with 4'-hydroxymethaqualone, the major urinary metabolite, being excreted almost exclusively as a single enantiomer. A stereoselectivity in the formation of 2'-hydroxymethaqualone and 2-hydroxymethaqualone was also observed in vitro using human liver microsomes and preparations containing the cytochrome P450 enzyme (CYP) CYP3A4 only. The presence of DH during incubation with human liver microsomes did not reveal a difference in the metabolic pattern obtained. Furthermore, CYP2D6 and CYP2C19 do not significantly contribute to the metabolism of MQ. This was independently observed in vitro and via analysis of urines of individuals that are either efficient metabolizer phenotypes or poor metabolizer phenotypes for the two polymorphic enzymes. Although interindividual differences in the monitored metabolic patterns were noted, no marked difference could be related to a CYP2D6 or CYP2C19 polymorphism.     

Biochemical and analytical development of the CIME cocktail for drug fate assessment in humans

Phenotyping based on drug metabolism activity appears to be informative regarding mechanism‐based interactions during drug development. We report here the first steps of the development of the innovative CIME cocktail. This cocktail is designed not only for the major cytochrome P450, with caffeine, amodiaquine, tolbutamide, omeprazole, dextromethorphan and midazolam as substrates of CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A, respectively, but also phase II enzymes UGT 1A1/6/9 with acetaminophen, P‐gp and OATP1B1 with digoxin and rosuvastatin, and renal function with memantine. An assay combining ultra‐performance liquid chromatography using a 1.7 µm particle size column with tandem mass spectrometry (UPLC/MS/MS) was set up for the simultaneous quantification of the 20 substrates and metabolites after extraction from human plasma using solid‐phase extraction. The method was validated in the spirit of the FDA guidelines. Mean accuracy ranged from 87.7 to 115%, the coefficient of variance (CV%) of intra‐ and inter‐run from 1.7 to 16.4% and from 1.6 to 14.9%, respectively, and for the limit of quantification (LOQ) with ten lots of plasma, accuracy ranged from 84 to 115% and CV% precision was <16%. Short‐term stability was evaluated in eluate (4 h, room temperature), plasma (24 h, room temperature), the autosampler (24 h, 4°C) and in three freeze/thaw cycles in plasma. All except three analytes were stable under these conditions. For the three others a specific process can be followed. This robust, fast and sensitive assay in human plasma provides an analytical tool for ten‐probe drugs of the CIME cocktail. Clinical samples will be assayed in the near future using this new assay method. Copyright © 2010 John Wiley & Sons, Ltd.     

Convenient Gram-Scale Metabolite Synthesis by Engineered Fission Yeast Strains Expressing Functional Human P450 Systems

The growing need for the characterization of cytochrome P450 (P450) metabolites often necessitates their synthesis up to Gram-scale. This task may in principle be achieved by using various techniques including chemical synthesis, the use of laboratory animals, in vitro P450 systems or microbial biotransformation. However, these approaches are in many instances unfavorable due to low yields, laborious purification, costs of cofactors, or the formation of non-physiologic metabolites. The fission yeast Schizosaccharomyces pombe has previously been shown by others and us to be very well suited for the heterologous expression of human P450s. In this study, we demonstrate whole-cell biotransformation reactions carried out with fission yeast strains that coexpress human cytochrome P450 reductase (CPR) and one of the following P450 isoforms: CYP2B6, CYP2C9, CYP2C19, CYP2D6, or CYP3A4, respectively. These strains could successfully convert their respective standard substrates but showed different responses with respect to incubation pH, the presence of glucose, and temperature, respectively. In addition, the preparative of synthesis of 2.8?g of 4'-hydroxydiclofenac was achieved by whole-cell biotransformation of diclofenac using a CPR-CYP2C9 coexpressing fission yeast strain.