细胞色素P4502D6酶基因多态性与利培酮代谢的相关性研究

目的 研究利培酮(抗精神分裂症药)在人体代谢过程与CYP2D6*10基因多态性的关系.方法 不同CYP2D6*1/*1(n=5)、*1/*10(n=12)及*10/*10(n=6)基因型的健康志愿者,单次口服利培酮2 mg后,用LC-MS/MS方法测定血浆中利培酮及其活性代谢产物9-羟基利培酮浓度,研究利培酮药代动力学与CYP2D6*10基因型的相关性.结果 CYP2D6*10/*10和CYP2D6*1/*10基因型与CYP2D6*1/*1基因型的受试者相比,利培酮的AUC0-∞分别增加811%和212%,2者比较差异非常显著(P<0.001);利培酮/9-羟基利培酮的AUC比值在各CYP2D6*10基因型间存在显著性差异(P<0.001).结论 CYP2D6*10 C188T突变使CYP2D6对利培酮的代谢活性降低,体内利培酮暴露量增加.     

Stereoselective Metabolism of Bupropion by Cytochrome P4502B6 (CYP2B6) and Human Liver Microsomes

Purpose Hydroxylation of the antidepressant and smoking deterrent drug bupropion is a clinically important bioactivation and elimination pathway. Bupropion hydroxylation is catalyzed selectively by cytochrome P4502B6 (CYP2B6). CYP2B6-catalyzed bupropion hydroxylation has been used as an in vitro and in vivo phenotypic probe for CYP2B6 activity and CYP2B6 drug interactions. Bupropion is chiral, used clinically as a racemate, and disposition is stereoselective. Nevertheless, it is unknown whether CYP2B6-catalyzed bupropion hydroxylation is stereoselective. Methods Hydroxylation of racemic bupropion by recombinant CYP2B6 and human liver microsomes was evaluated using a stereoselective assay. Results At therapeutic concentrations, hydroxylation of (S)-bupropion was threefold and 1.5-greater than (R)-bupropion, respectively, by recombinant CYP2B6 and human liver microsomes. In vitro intrinsic clearances were likewise different for bupropion enantiomers. Conclusions Stereoselective bupropion hydroxylation may have implications for the therapeutic efficacy of bupropion as an antidepressant or smoking cessation therapy, and for the use of bupropion as an in vivo phenotypic probe for CYP2B6 activity.     

Involvement of CYP2B6 in the biotransformation of propofol by human liver microsomes

Objective To determine whether the cytochrome P4502B6(CYP2B6)is involved in the oxidation of propofol by human liver microsomes.Methods The change of propofol concentration in an incubation mixture with human liver microsomes was monitored by the high performance liquid chromatography(HPLC),in order to calculate the rate constants of metabolism of propofol.The correlation between the rate constants and the rate of metabolism of CYP2B6 selective substrate bupropion,and the effect of two different CYP2B6 specific inhibitors on the propofol metabolism were examined.Results The mean rate constant of propofol metabolism by liver microsomes obtained from twelve individuals was 3.9(95% confidence intervals 3.3,4.5)nmol·min-1·mg-1 protein.The rate constants of propofol metabolism by liver microsomes were significantly correlated with bupropion hydroxylation(r=0.888,P<0.001).Both selective chemical inhibitors of CYP2B6,orphenadrine and N,N',N″-triethylenethiophosphoramide(thioTEPA),reduced the rate constants of propofol metabolism by 37.5%(P<0.001)and 42.7%(P<0.001)in liver microsomes,respectively.Conclusions CYP2B6 is predominantly involved in the oxidation of propofol by human liver microsomes.     

人细胞色素P450 2B6在哺乳类细胞中的稳定表达

在诱变试验中广泛应用人羊膜FL细胞, 中国仓鼠CHL和V79细胞, 但由于它们不能表达内源性细胞色素P450(CYP)活化前致突变物, 而严重限制了它们的使用. 本工作构建了携有编码CYP2B6单胺氧化酶的cDNA重组表达体并分别导入上述三种细胞中表达, 经G418抗性筛选, 建立了FL-2B6, CHL-2B6和V79-2B6三种转基因细胞系, 用Northern印迹杂交证明它们都有CYP2B6的表达. 在CHL-2B6细胞中还测得较高水平的7-乙氧基-4-三氟甲基香豆素去乙基酶活性, 它对前致突变物甲基亚硝胺吡啶酮的致微核形成作用有很高敏感性, 并呈剂量效应关系, 但其母系细胞皆为阴性.     

人细胞色素P4502B6在哺乳类细胞中的稳定表达

〗在诱变试验中广泛应用人羊膜ＦＬ细胞，中国仓鼠ＣＨＬ和Ｖ７９细胞，但由于它们不能表达内源性细胞色素Ｐ４５０（ＣＹＰ）活化前致突变物，而严重限制了它们的使用．本工作构建了携有编码ＣＹＰ２Ｂ６单胺氧化酶的ｃＤＮＡ重组表达体并分别导入上述三种细胞中表达，经Ｇ４１８抗性筛选，建立了ＦＬ－２Ｂ６，ＣＨＬ－２Ｂ６和Ｖ７９－２Ｂ６三种转基因细胞系，用Ｎｏｒｔｈｅｒｎ印迹杂交证明它们都有ＣＹＰ２Ｂ６的表达．在ＣＨＬ－２Ｂ６细胞中还测得较高水平的７－乙氧基－４－三氟甲基香豆素去乙基酶活性，它对前致突变物甲基亚硝胺吡啶酮的致微核形成作用有很高敏感性，并呈剂量效应关系，但其母系细胞皆为阴性．     

Identification of the human cytochrome P450 enzymes involved in the in vitro metabolism of artemisinin

AIMS: The study aimed to identify the specific human cytochrome P450 (CYP450) enzymes involved in the metabolism of artemisinin. METHODS: Microsomes from human B-lymphoblastoid cell lines transformed with individual CYP450 cDNAs were investigated for their capacity to metabolize artemisinin. The effect on artemisinin metabolism in human liver microsomes by chemical inhibitors selective for individual forms of CYP450 was investigated. The relative contribution of individual CYP450 isoenzymes to artemisinin metabolism in human liver microsomes was evaluated with a tree-based regression model of artemisinin disappearance rate and specific CYP450 activities. RESULTS: The involvement of CYP2B6 in artemisinin metabolism was demonstrated by metabolism of artemisinin by recombinant CYP2B6, inhibition of artemisinin disappearance in human liver microsomes by orphenadrine (76%) and primary inclusion of CYP2B6 in the tree-based regression model. Recombinant CYP3A4 was catalytically competent in metabolizing artemisinin, although the rate was 10% of that for recombinant CYP2B6. The tree-based regression model suggested CYP3A4 to be of importance in individuals with low CYP2B6 expression. Even though ketoconazole inhibited artemisinin metabolism in human liver microsomes by 46%, incubation with ketoconazole together with orphenadrine did not increase the inhibition of artemisinin metabolism compared to orphenadrine alone. Troleandomycin failed to inhibit artemisinin metabolism. The rate of artemisinin metabolism in recombinant CYP2A6 was 15% of that for recombinant CYP2B6. The inhibition of artemisinin metabolism in human liver microsomes by 8-methoxypsoralen (a CYP2A6 inhibitor) was 82% but CYP2A6 activity was not included in the regression tree. CONCLUSIONS: Artemisinin metabolism in human liver microsomes is mediated primarily by CYP2B6 with probable secondary contribution of CYP3A4 in individuals with low CYP2B6 expression. The contribution of CYP2A6 to artemisinin metabolism is likely of minor importance.     

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。     

Hydroxylation of tanshinone IIa in human liver microsomes is specifically catalysed by cytochrome P4502A6

1. Tanshinone IIa, the primary active component of a traditional Chinese medicine Salvia miltiorrhiza (Danshen), has a wide range of pharmacological activities. In the present study, the metabolism of tanshinone IIa (5?μM) by cytochrome P450s (CYPs) was investigated in human liver microsomes.

2. One mono-hydroxylated metabolite was detected in a reaction catalysed by human liver microsomes, and was identified as tanshinone IIb by comparing the tandem mass spectra and the chromatographic retention time with that of the standard compound.

3. The study with a chemical selective inhibitor, cDNA-expressed human cytochrome P450s, correlation assay, and kinetics study demonstrated that CYP2A6 was the specific isozyme responsible for the hydroxyl metabolism of tanshinone IIa (5?μM) in human liver microsomes.

     

In vitro metabolism of nobiletin,a polymethoxy-flavonoid,by human liver microsomes and cytochrome P450

1. Cytochrome P450 enzymes (CYPs) in the liver metabolize drugs prior to excretion, with different enzymes acting at different molecular motifs. At present, the human CYPs responsible for the metabolism of the flavonoid, nobiletin (NBL), are unidentified. We investigated which enzymes were involved using human liver microsomes and 12 cDNA-expressed human CYPs.

2. Human liver microsomes metabolized NBL to three mono-demethylated metabolites (4′-OH-, 7-OH- and 6-OH-NBL) with a relative ratio of 1:4.1:0.5, respectively, by aerobic incubation with nicotinamide adenine dinucleotide phosphate (NADPH). Of 12 human CYPs, CYP1A1, CYP1A2 and CYP1B1 showed high activity for the formation of 4′-OH-NBL. CYP3A4 catalyzed the formation of 7-OH-NBL with the highest activity and of 6-OH-NBL with lower activity. CYP3A5 also catalyzed the formation of both metabolites but considerably more slowly than CYP3A4. In contrast, seven CYPs (CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) were inactive for NBL.

3. Both ketoconazole and troleandomycin (CYP3A inhibitors) almost completely inhibited the formation of 7-OH- and 6-OH-NBL. Similarly, α-naphthoflavone (CYP1A1 inhibitor) and furafylline (CYP1A2 inhibitor) significantly decreased the formation of 4′-OH-NBL.

4. These results suggest that CYP1A2 and CYP3A4 are the key enzymes in human liver mediating the oxidative demethylation of NBL in the B-ring and A-ring, respectively.

     

Inhibition of cytochrome P450 2E1 by propofol in human and porcine liver microsomes

While almost anesthetics are metabolized by the cytochrome P450 (CYP) 3A4, some major volatile ones such as halothane and sevoflurane are metabolized by CYP2E1 in humans. To determine whether 2,6-diisopropylphenol (propofol), a widely used intravenous anesthetic agent, known to inhibit CYP3A4 and CYP1A2, also inhibits CYP2E1, 6-OH hydroxylation of chlorzoxazone, a prototypical CYP2E1 substrate, was estimated using two pools of human microsomes and one pool of porcine microsomes from seven livers. Basal human enzyme activities were characterized by a V(max) of 1426+/-230 and 288+/-29 pmol min(-1)mg(-1) protein and a K(m) of 122+/-47 and 149+/-42 microM, while the corresponding porcine activities were associated with a V(max) of 352+/-42 pmol min(-1)mg(-1) protein and a K(m) of 167+/-38 microM. A competitive inhibition of CYP2E1 by propofol was observed with low inhibition constants in the therapeutic range in both porcine (19 microM) and human (48 microM) liver microsomes. These in vitro results suggest that propofol could have a protective effect on toxic metabolite activation of compounds catalyzed by CYP2E1.     

Regioselectivity and Enantioselectivity of Metoprolol Oxidation by Two Variants of cDNA-Expressed P4502D6

Purpose. The oxidative metabolism of metoprolol was investigated in two human lymphoblastoma cell-lines transfected with variants of cDNA for cytochrome P4502D6. Methods. The regioselective and enantioselective features of the oxidations of deuterium-labeled pseudoracemic metoprolol were characterized by GC/MS analysis of the substrate and products. Results. There were significant differences between the two P4502D6 variants in the formation kinetics of O-demethylmetoprolol and -hydroxymetoprolol. The h2D6-Val microsomes highly favored the formation of the O-demethylmetoprolol regioisomer 6.3:1 and 2.8:1, respectively from (R)-metoprolol-d₀ and (S)-metoprolol-d₂, while the corresponding ratios for h2D6v2 microsomes were much lower. For both variants, O-demethylmetoprolol formation favored the (R)-substrate 1.5 to 2-fold, while -hydroxymetoprolol formation was non-enantioselective. Similar Km values of metoprolol oxidation, 10-20 µM, were observed for the two microsomal preparations. Conclusions. The regioselectivity, enantioselectivity, and Km values for the h2D6-Val microsomes resemble those observed for the native P4502D6 in human liver microsomes, whereas the h2D6v2 microsomes deviated remarkably in regioselectivity.     

Deoxyshikonin reversibly inhibits cytochrome P450 2B6

Deoxyshikonin, a natural shikonin derivative, is the major component of Lithospermum erythrorhizon and exhibits various pharmacological effects such as lymphangiogenetic, antibacterial, wound healing, and anticancer effects. To investigate the herb–drug interaction potential associated with deoxyshikonin, the inhibitory effects of deoxyshikonin on eight major cytochrome P450 (CYP) enzymes were examined using cocktail substrate-incubated human liver microsomes. Deoxyshikonin strongly inhibited CYP2B6-catalyzed bupropion hydroxylation, with a K_i value of 3.5 μM, and the inhibition was confirmed using purified human CYP2B6. The inhibition was reversible because the inhibitory effect of deoxyshikonin was not dependent on the preincubation time. The results indicated that deoxyshikonin-induced drug–drug interaction should be considered when any herb containing deoxyshikonin is used for conventional medications.     

Marmoset cytochrome P450 2B6, a propofol hydroxylase expressed in liver

1. The common marmoset (Callithrix jacchus) is a useful experimental animal to evaluate the pharmacokinetics of drug candidates. Cytochrome P450 (P450) 2B enzyme in marmoset livers has been identified; however, only limited information on the enzymatic properties and distribution has been available.

2. Marmoset P450 2B6 amino acids showed high sequence identities (>86%) with those of primates including humans and cynomolgus monkeys. Phylogenetic analysis using amino acid sequences indicated that marmoset P450 2B6 was closer to human and cynomolgus monkey P450 2B6 than to P450 2B orthologs of other species, including pigs, dogs, rabbits and rodents.

3. Quantitative polymerase chain reaction analysis using specific primers showed P450 2B6 mRNA predominantly expressed in livers among the five marmoset tissues, similar to those of humans and cynomolgus monkeys.

4. Marmoset P450 2B6 heterologously expressed in Escherichia coli membranes oxidized 7-ethoxycoumarin, pentoxyresorufin, propofol and testosterone, at roughly similar rates to those of humans and/or cynomolgus monkeys. A high capacity of marmoset P450 2B6 with propofol 4-hydroxylation (at low ionic strength conditions) with a low K_m value was relatively comparable to that for marmoset livers.

5. These results collectively indicated a high propofol 4-hydroxylation activity of P450 2B6 expressed in marmoset livers.

     

肝细胞微粒体的制备和细胞色素P450氧化酶活性测定

目的：为测定人肝细胞微粒体细胞色素Ｐ４５０氧化酶的活性。方法：用差速离心法制备３例人肝细胞微粒体。结果：细胞色素Ｐ４５０的含量为０．５２３±０．００５ｎｍｏｌ·ｍｇ－１；细胞色素ｂ５为０．２８５±０．０２５ｎｍｏｌ·ｍｇ－１；氨基比林Ｎ－脱甲基酶的活力为０．５±０．６ｎｍｏｌ·ｍｇ－１；乙基吗啡Ｎ－脱甲基酶活力为０．９８±０．０８ｎｍｏｌ·ｍｇ－１。结论：Ｐ４５０酶活性影响因素较多，个体差异大。临床用药时应考虑患者的个体情况。     

Involvement of multiple human cytochromes P450 in the liver microsomal metabolism of astemizole and a comparison with terfenadine

AIMS: The aims of the present study were to investigate the metabolism of astemizole in human liver microsomes, to assess possible pharmacokinetic drug-interactions with astemizole and to compare its metabolism with terfenadine, a typical H1 receptor antagonist known to be metabolized predominantly by CYP3A4. METHODS: Astemizole or terfenadine were incubated with human liver microsomes or recombinant cytochromes P450 in the absence or presence of chemical inhibitors and antibodies. RESULTS: Troleandomycin, a CYP3A4 inhibitor, markedly reduced the oxidation of terfenadine (26% of controls) in human liver microsomes, but showed only a marginal inhibition on the oxidation of astemizole (81% of controls). Three metabolites of astemizole were detected in a liver microsomal system, i.e. desmethylastemizole (DES-AST), 6-hydroxyastemizole (6OH-AST) and norastemizole (NOR-AST) at the ratio of 7.4 : 2.8 : 1. Experiments with recombinant P450s and antibodies indicate a negligible role for CYP3A4 on the main metabolic route of astemizole, i.e. formation of DES-AST, although CYP3A4 may mediate the relatively minor metabolic routes to 6OH-AST and NOR-AST. Recombinant CYP2D6 catalysed the formation of 6OH-AST and DES-AST. Studies with human liver microsomes, however, suggest a major role for a mono P450 in DES-AST formation. CONCLUSIONS: In contrast to terfenadine, a minor role for CYP3A4 and involvement of multiple P450 isozymes are suggested in the metabolism of astemizole. These differences in P450 isozymes involved in the metabolism of astemizole and terfenadine may associate with distinct pharmacokinetic influences observed with coadministration of drugs metabolized by CYP3A4.     

Characterization of human cytochrome P450 isoforms involved in the metabolism of 7-epi-paclitaxel

1. The C-7 chiral centre in paclitaxel is subject to epimerization under physiological conditions, thus making 7-epi-paclitaxel as the principal degradant. This study was designed to characterize the cytochrome P450 (CYP) enzymes involved in 7-epi-paclitaxel metabolism, and to examine possible metabolic interactions that this C-7 epimer may have with paclitaxel.

2. In human liver microsomes, 7-epi-paclitaxel was oxidized to two monohydroxylated metabolites while the metabolic sites occurred at the C-13 side-chain for M-1 and taxane core ring for M-2. A combination of correlation analysis, chemical inhibition studies, assays with recombinant CYPs, and enzyme kinetics indicated that M-1 was generated predominantly by CYP3A4 and M-2 by CYP2C8. Co-incubation of 7-epi-paclitaxel with paclitaxel in human liver microsomes resulted in potent inhibition of 6α-hydroxypaclitaxel formation (IC₅₀?=?2.1?±?0.2 μM), thus decreasing the metabolic elimination of paclitaxel.

3. In conclusion, both CYP3A4 and CYP2C8 play a major role in biotransformation of 7-epi-paclitaxel in human liver microsomes. The existence of epimeric interactions between paclitaxel and its degradant might be a noteworthy factor resulting in the complex pharmacokinetic profile of paclitaxel.

     

Cytochrome P4502C9: an enzyme of major importance in human drug metabolism

Accumulating evidence indicates that CYP2C9 ranks amongst the most important drug metabolizing enzymes in humans. Substrates for CYP2C9 include fluoxetine, losartan, phenytoin, tolbutamide, torsemide, S-warfarin, and numerous NSAIDs. CYP2C9 activity in vivo is inducible by rifampicin. Evidence suggests that CYP2C9 substrates may also be induced variably by carbamazepine, ethanol and phenobarbitone. Apart from the mutual competitive inhibition which may occur between alternate substrates, numerous other drugs have been shown to inhibit CYP2C9 activity in vivo and/or in vitro . Clinically significant inhibition may occur with coadministration of amiodarone, fluconazole, phenylbutazone, sulphinpyrazone, sulphaphenazole and certain other sulphonamides. Polymorphisms in the coding region of the CYP2C9 gene produce variants at amino acid residues 144 (Arg144Cys) and 359 (Ile359Leu) of the CYP2C9 protein. Individuals homozygous for Leu359 have markedly diminished metabolic capacities for most CYP2C9 substrates, although the frequency of this allele is relatively low. Consistent with the modulation of enzyme activity by genetic and other factors, wide interindividual variability occurs in the elimination and/or dosage requirements of prototypic CYP2C9 substrates. Individualisation of dose is essential for those CYP2C9 substrates with a narrow therapeutic index.     

Characterization of the metabolism of fenretinide by human liver microsomes, cytochrome P450 enzymes and UDP-glucuronosyltransferases

BACKGROUND AND PURPOSE

Fenretinide (4-HPR) is a retinoic acid analogue, currently used in clinical trials in oncology. Metabolism of 4-HPR is of particular interest due to production of the active metabolite 4′-oxo 4-HPR and the clinical challenge of obtaining consistent 4-HPR plasma concentrations in patients. Here, we assessed the enzymes involved in various 4-HPR metabolic pathways.

EXPERIMENTAL APPROACH

Enzymes involved in 4-HPR metabolism were characterized using human liver microsomes (HLM), supersomes over-expressing individual human cytochrome P450s (CYPs), uridine 5′-diphospho-glucoronosyl transferases (UGTs) and CYP2C8 variants expressed in Escherichia coli. Samples were analysed by high-performance liquid chromatography and liquid chromatography/mass spectrometry assays and kinetic parameters for metabolite formation determined. Incubations were also carried out with inhibitors of CYPs and methylation enzymes.

KEY RESULTS

HLM were found to predominantly produce 4′-oxo 4-HPR, with an additional polar metabolite, 4′-hydroxy 4-HPR (4′-OH 4-HPR), produced by individual CYPs. CYPs 2C8, 3A4 and 3A5 were found to metabolize 4-HPR, with metabolite formation prevented by inhibitors of CYP3A4 and CYP2C8. Differences in metabolism to 4′-OH 4-HPR were observed with 2C8 variants, CYP2C8*4 exhibited a significantly lower V_max value compared with *1. Conversely, a significantly higher V_max value for CYP2C8*4 versus *1 was observed in terms of 4′-oxo formation. In terms of 4-HPR glucuronidation, UGTs 1A1, 1A3 and 1A6 produced the 4-HPR glucuronide metabolite.

CONCLUSIONS AND IMPLICATIONS

The enzymes involved in 4-HPR metabolism have been characterized. The CYP2C8 isoform was found to have a significant effect on oxidative metabolism and may be of clinical relevance.     

酮康唑对大鼠肝脏CYP450酶系的影响*

目的:观察酮康唑对大鼠肝细胞色素P450及其主要亚型的影响。方法:Sprague-Dawley大鼠用140,280,420μmol.kg-1.d-1酮康唑连续灌胃7 d,测定肝脏微粒体中总CYP450含量和CYP1A1,1A2,1B1,2B1/2,2E1和3A亚型活性。结果:不同剂量酮康唑给药后大鼠肝脏脏器系数、CYP1A1和1B1亚型活性明显增高(P<0.05,P<0.01);总CYP450含量和CYP3A活性显著降低(P<0.01);低剂量的酮康唑抑制CYP1A2和CYP2B1/2亚型的活性,高剂量却出现了诱导作用(P<0.05,P<0.01)。各剂量组对CYP2E1均无明显影响。结论:酮康唑对大鼠肝脏CYP450及主要药物代谢亚型CYP1A1,1A2,1B1,2B1/2和3A有影响,临床长期用药或与经肝脏CYP450代谢的药物联合应用时要注意监测血药浓度和肝脏功能,防止药物代谢减缓出现蓄积中毒或药物代谢加快而降低药效。     

The xenobiotic inhibitor profile of cytochrome P4502C8

AIMS: To investigate inhibition of recombinant CYP2C8 by: (i) prototypic CYP isoform selective inhibitors (ii) imidazole/triazole antifungal agents (known inhibitors of CYP), and (iii) certain CYP3A substrates (given the apparent overlapping substrate specificity of CYP2C8 and CYP3A). METHODS: CYP2C8 and NADPH-cytochrome P450 oxidoreductase were coexpressed in Spodoptera frugiperda (Sf21) cells using the baculovirus expression system. CYP isoform selective inhibitors, imidazole/triazole antifungal agents and CYP3A substrates were screened for their inhibitory effects on CYP2C8-catalysed torsemide tolylmethylhydroxylation and, where appropriate, the kinetics of inhibition were characterized. The conversion of torsemide to its tolylmethylhydroxy metabolite was measured using an h.p.l.c. procedure. RESULTS: At concentrations of the CYP inhibitor 'probes' employed for isoform selectivity, only diethyldithiocarbamate and ketoconazole inhibited CYP2C8 by > 10%. Ketoconazole, at an added concentration of 10 microM, inhibited CYP2C8 by 89%. Another imidazole, clotrimazole, also potently inhibited CYP2C8. Ketoconazole and clotrimazole were both noncompetitive inhibitors of CYP2C8 with apparent Ki values of 2.5 microM. The CYP3A substrates amitriptyline, quinine, terfenadine and triazolam caused near complete inhibition (82-91% of control activity) of CYP2C8 at concentrations five-fold higher than the known CYP3A Km. Kinetic studies with selected CYP3A substrates demonstrated that most inhibited CYP2C8 noncompetitively. Apparent Ki values for midazolam, quinine, terfenadine and triazolam ranged from 5 to 25 microM. CONCLUSIONS: Inhibition of CYP2C8 occurred at concentrations of ketoconazole and diethyldithiocarbamate normally employed for selective inhibition of CYP3A and CYP2E1, respectively. Some CYP3A substrates have the capacity to inhibit CYP2C8 activity and this may have implications for inhibitory drug interactions in vivo.