Diclofenac-induced inactivation of CYP3A4 and its stimulation by quinidine.

Incubation of human liver microsomes with diclofenac in the presence of NADPH resulted in a decrease in testosterone 6 beta-hydroxylation activity. The decrease in the activity followed time- and concentration-dependent kinetics, required oxidative metabolism, and was resistant to reduced glutathione, suggesting that diclofenac causes a mechanism-based inactivation of cytochrome p450 (p450) 3A4 (CYP3A4). The inactivation was reproduced by using microsomes from B-lymphoblastoid cell lines expressing CYP3A4 instead of human liver microsomes. No other monooxygenase activities measured as indexes of p450 enzymes; CYP2C8, CYP2C9, or CYP2C19 was inactivated by the same incubation procedure. Quinidine, a stimulant of CYP3A4-mediated diclofenac 5-hydroxylation, did not affect the inactivation of CYP3A4 assessed by testosterone 6 beta-hydroxylation activity but accelerated the inactivation assessed by diazepam 3-hydroxylation activity. These results supported the idea that diclofenac 5-hydroxylation is involved in the inactivation of CYP3A4 and described for the first time a stimulation of mechanism-based inactivation attributable to CYP3A4 heterotropic cooperativity. Preincubation of human liver microsomes with 5-hydroxydiclofenac instead of diclofenac did not cause the inactivation of CYP3A4, suggesting that 5-hydroxydiclofenac is not a precursor of a postulated reactive metabolite that inactivates CYP3A4, and thus 5-hydroxylation step is critical to inactivation of CYP3A4.     

Identification of cytochromes P450 involved in human liver microsomal metabolism of ecabapide, a prokinetic agent

1. In vitro studies identified the hepatic cytochrome P450 (CYP) enzyme(s) involved in the major metabolism of ecabapide in human. 2. Ecabapide mainly underwent N-dealkylation to form M1 and 6-hydroxylation of the benzamide moiety to form M6. 3. The rates of formation of the major metabolites M1 and M6 were significantly correlated with CYP3A-selective testosterone 6beta-hydroxylase activities in 14 different human liver microsomes. The formation of both metabolites was markedly decreased by ketoconazole, miconazole or troleandomycin (TAO), CYP3A-selective inhibitors, and also was inhibited by anti-CYP3A antibodies. 4. These results strongly indicate that CYP3A is the predominant isozyme responsible for the major metabolism of ecabapide in human liver microsomes. 5. Marginal inhibition of the formation of M1 and M6 by nifedipine, a substrate of CYP3A with a Ki > 100 microM, suggested that nifedipne has a limited potential to inhibit the major metabolic pathways of ecabapide.     

体外研究人细胞色素P450在雌二醇代谢中的作用(英文)

目的:研究雌二醇在cDNA表达的P450和人肝微粒体中的代谢机制,为在体内研究细胞色素P450活性与肿瘤发生的关系提供依据。方法:用HPLC-ECD法测定雌二醇的代谢产物。通过雌二醇在不同cDNA表达的P450中代谢,13例人肝微粒体中相关性研究,抑制剂对代谢的影响以及微粒体中17β-羟基脱氢化和2-羟基化代谢的催化动力学的研究来推断雌二醇的代谢机理。结果:在cDNA表达的P450中,催化2-羟基化代谢的P450按活性排列依次为CYP1A2、CYP3A4、CYP2C9。CYP2C9、CYP2C19和CYP2C8均具有较高的催化17β-羟基脱氢化活性。抑制CYP1A2与抑制CYP3A4对2-羟基化代谢产物生成的影响相似,可认为CYP1A2和CYP3A4在人肝微粒体中催化2-羟基化代谢的作用相近。雌二醇代谢的途径与底物浓度有关,低浓度时(1,10μmol/L)17β-羟基脱氢化为主要代谢途径;高浓度时(100μmol/L),2-羟基化成为主要代谢途径。结论:高底物浓度时,雌二醇主要由CYP1A2和CYP3A4催化代谢为2-羟基化产物。低底物浓度时,主要由CYP2C9、CYP2C19和CYP2C8催化生成17β-羟基去氢化产物。     

Stereoselective metabolism of cibenzoline, an antiarrhythmic drug, by human and rat liver microsomes: possible involvement of CYP2D and CYP3A.

Stereoselective metabolism of cibenzoline succinate, an oral antiarrhythmic drug, was investigated on hepatic microsomes from humans and rats and microsomes from cells expressing human cytochrome P450s (CYPs). Four main metabolites, M1 (p-hydroxycibenzoline), M2 (4,5-dehydrocibenzoline), and unknown metabolites M3 and M4, were formed by human and rat liver microsomes. The intrinsic clearance (CL(int)) of the M1 formation from R(+)-cibenzoline was 23-fold greater than that of S(-)-cibenzoline in human liver microsomes, whereas the R(+)/S(-)-enantiomer ratio of CL(int) for M2, M3, and M4 formation was 0.39 to 0.83. The total CL(int) for the formation of the four main metabolites from S(-)- and R(+)-cibenzoline was 1.47 and 1.64 microl/min/mg, respectively, suggesting that the total CL(int) in R(+)-enantiomer was slightly greater than that in S(-)-enantiomer in human liver microsomes. The M1 formation from R(+)-cibenzoline was highly correlated with bufuralol 1'-hydroxylation and CYP2D6 content and was inhibited by quinidine, a potent inhibitor of CYP2D6. Additionally, only microsomes containing recombinant CYP2D6 were capable of M1 formation. These results suggest that the M1 formation from R(+)-cibenzoline was catalyzed by CYP2D6. The formation of M2, M3, and M4 from S(-)- and R(+)-cibenzoline was highly correlated with testosterone 6beta-hydroxylation and CYP3A4 content. Ketoconazole, which is a potent inhibitor of CYP3A4/5, had a strong inhibitory effect on their formation, and the M4 formation from R(+)-cibenzoline was inhibited by quinidine by 45%. The formation of M2 was also inhibited by quinidine by 46 to 52% at lower cibenzoline enantiomers (5 microM), whereas the inhibition by quinidine was not observed at a higher substrate concentration (100 microM). In male rat liver microsomes, ketoconazole and quinidine inhibited the formation of the main metabolites, M1 and M3, >74% and 44 to 59%, respectively. These results provide evidence that CYP3A and CYP2D play a major role in the stereoselective metabolism of cibenzoline in humans and male rats.     

Reversible and irreversible inhibition of CYP3A enzymes by tamoxifen and metabolites

1. Preliminary studies have identified cytochrome P450 (CYP) 3A4 and CYP1B1 as the human CYPs inhibited by tamoxifen. To quantify the inhibitory potency of tamoxifen and its major metabolites, the metabolism of three substrates of CYP3A, midazolam, diltiazem and testosterone, and 7-ethoxyresorufin as a substrate of CYP1B1 were examined in catalytic assays carried out using human liver microsomes and cDNA-expression systems. 2. Tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and 3-hydroxytamoxifen reversibly inhibited midazolam 1'-hydroxylation, diltiazem N-demethylation and testosterone 6beta-hydroxylation with K(i) ranging from 3 to 37 micro M in human liver microsomes. Tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and 3-hydroxytamoxifen also reversibly inhibited the activity of cDNA-expressed CYP3A4, CYP3A5 and CYP1B1. 3. Tamoxifen and N-desmethyltamoxifen exhibited time-dependent inactivation of testosterone 6beta-hydroxylation by cDNA-expressed CYP3A4 (+ cytochrome b5) yielding k(inact) and K(i) of 0.04 min(-1) and 0.2 micro M for tamoxifen and 0.08 min(-1) and 2.6 micro M for N-desmethyltamoxifen. A metabolic intermediate complex (MIC) was also formed by tamoxifen and N-desmethyltamoxifen with CYP3A4 (+ cytochrome b5) and CYP3A4 but not with CYP3A5 or CYP3A7. Pre-incubation with 4-hydroxytamoxifen and 3-hydroxytamoxifen did not result in any CYP3A inactivation or detectable MIC formation. There was no detectable time-dependent inactivation or MIC formation with tamoxifen or metabolites with CYP1B1. 4. These data indicate that tamoxifen and its three major metabolites are effective inhibitors of CYP3A in vitro and that tamoxifen and N-desmethyltamoxifen are effective mechanism-based inhibitors. Thus, caution should be exercised when tamoxifen is coadministered with other CYP3A substrates.     

Roles of human CYP2A6 and 2B6 and rat CYP2C11 and 2B1 in the 10-hydroxylation of (-)-verbenone by liver microsomes.

(-)-Verbenone, a monoterpene bicyclic ketone, is a component of the essential oil from rosemary species such as Rosmarinus officinalis L., Verbena triphylla, and Eucalyptus globulus and is used for an herb tea, a spice, and a perfume. In this study, (-)-verbenone was found to be converted to 10-hydroxyverbenone by rat and human liver microsomal cytochrome p450 (p450) enzymes. The product formation was determined by high-performance liquid chromatography with UV detection at 251 nm. There was a good correlation between activities of coumarin 7-hydroxylation and (-)-verbenone 10-hydroxylation catalyzed by liver microsomes of 16 human samples, indicating that CYP2A6 is a principal enzyme in (-)-verbenone 10-hydroxylation in humans. Human recombinant CYP2A6 and CYP2B6 catalyzed (-)verbenone 10-hydroxylation at Vmax values of 15 and 21 nmol/min/nmol p450 with apparent Km values of 16 and 91 microM, respectively. In contrast, rat CYP2A1 and 2A2 did not catalyze (-)-verbenone 10-hydroxylation at all, suggesting that there were species-related differences in the catalytic properties of human and rat CYP2A enzymes in the metabolism of (-)-verbenone. In the rat, recombinant CYP2C11, CYP2B1, and CYP3A2 catalyzed (-)-verbenone 10-hydroxylation with Vmax and Km ratios (ml/min/nmol p450) of 0.73, 0.20, and 0.03, respectively. Male-specific CYP2C11 was a major enzyme in (-)-verbenone 10-hydroxylation by untreated rat livers, and CYP2B1 catalyzed this reaction in liver microsomes of phenobarbital-treated rats. Rat CYP2C12, a female-specific enzyme, did not catalyze (-)verbenone 10-hydroxylation. These results suggest that human CYP2A6 and rat CYP2C11 are the major catalysts in the metabolism of (-)-verbenone by liver microsomes and that there are species-related differences in human and rat CYP2A enzymes and sex-related differences in male and female rats in the metabolism of (-)-verbenone.     

Gemfibrozil inhibits CYP2C8-mediated cerivastatin metabolism in human liver microsomes.

To explore the mechanism of the interaction between gemfibrozil and cerivastatin, the enzyme mapping of the oxidative metabolism of cerivastatin and the effect of gemfibrozil on cerivastatin metabolism were studied using human liver microsomes and expressed cytochrome p450 (p450) CYP2C8 and 3A4 isoforms. Based on studies with isoform-selective chemical inhibitors and expressed enzymes, CYP2C8 and CYP3A4 were equally important in the formation of desmethylcerivastatin (M-1), whereas the formation of the quantitatively most important hydroxy metabolite (M-23) was predominantly mediated via CYP2C8; other p450 isoforms played a negligible role. In human liver microsomes, gemfibrozil markedly inhibited M-23 formation, with a K(i) (IC(50)) value of 69 (95) micro M, whereas inhibition of M-1 formation was weaker with a K(i) (IC(50)) value of 273 (>250) micro M. The inhibitory effect of gemfibrozil was attributable to inhibition of CYP2C8 rather than CYP3A4, as evidenced by potent inhibition of the formation of M-23 (IC(50) = 68 micro M) and M-1 (IC(50) = 78 micro M) in recombinant CYP2C8 but not in recombinant CYP3A4. Additionally, gemfibrozil inhibited paclitaxel 6 alpha-hydroxylation [K(i) (IC(50)) = 75 micro M (91 micro M)], a CYP2C8 marker reaction, but did not inhibit testosterone 6 beta-hydroxylation (CYP3A4). The present in vitro findings suggest that inhibition of CYP2C8 activity by gemfibrozil at least partially explains the interaction between gemfibrozil and cerivastatin. The formation of M-23 acid from cerivastatin is mediated mainly by CYP2C8 and thus may be a suitable CYP2C8 probe reaction. Inhibition of CYP2C8-mediated metabolism by gemfibrozil warrants further in vivo exploration.     

CYP3A4 mediated in vitro metabolism of vinflunine in human liver microsomes

AIM: To study the metabolism of vinflunine and the effects of selective cytochrome P-450 (CYP450) inhibitors on the metabolism of vinflunine in human liver microsomes. METHODS: Individual selective CYP450 inhibitors were used to investigate their effects on the metabolism of vinflunine and the principal CYP450 isoform involved in the formation of metabolites M(1) and M(2) in human liver microsomes. RESULTS: Vinflunine was rapidly metabolized to 2 metabolites: M(1) and M(2) in human liver microsomes. M(1) and M(2) were tentatively presumed to be the N-oxide metabolite or hydroxylated metabolite and epoxide metabolite of vinflunine, respectively. Ketoconazole uncompetitively inhibited the formation of M(1), and competitively inhibited the formation of M(2), while alpha-naphthoflavone, sulfaphenazole, diethyl dithiocarbamate, tranylcypromine and quinidine had little or no inhibitory effect on the formation of M(1) and M(2). CONCLUSION: Vinflunine is rapidly metabolized in human liver microsomes, and CYP3A4 is the major human CYP450 involved in the metabolism of vinflunine.     

Identification of human CYP isoforms involved in the metabolism of propranolol enantiomers--N-desisopropylation is mediated mainly by CYP1A2.

1. Studies using human liver microsomes and six recombinant human CYP isoforms (i.e. CYP1A2, 2A6, 2B6, 2D6, 2E1 and 3A4) were performed to identify the cytochrome P450 (CYP) isoform(s) involved in the ring 4-hydroxylation and side-chain N-desisopropylation of propranolol enantiomers in humans. 2. alpha-Naphthoflavone and 7-ethoxyresorufin (selective inhibitors of CYP1A1/2) inhibited the N-desisopropylation of R- and S-propranolol by human liver microsomes by 20 and 40%, respectively, while quinidine (a selective inhibitor of CYP2D6) abolished the 4-hydroxylation of both propranolol enantiomers almost completely. In contrast, sulphaphenazole (CYP2C8/9 inhibitor), S-mephenytoin (CYP2C19 inhibitor), troleandomycin (CYP3A3/4 inhibitor) and diethyldithiocarbamate (CYP2E1 inhibitor) elicited only weak inhibitory effects on propranolol metabolism via the two measured metabolic pathways. 3. Significant (P < 0.01) correlations were observed between the microsomal N-desisopropylation of both propranolol enantiomers and that for the O-deethylation of phenacetin among the 11 different human liver microsome samples (r = 0.98 and 0.77 for R- and S-propranolol, respectively). A marginally significant (r = 0.60, P congruent to 0.05) correlation was also observed between N-desisopropylation of S-, but not of R-propranolol and the 4'-hydroxylation of S-mephenytoin. No significant correlations were observed between the N-desisopropylation of propranolol enantiomers and the 2-hydroxylation of desipramine, the hydroxylation of tolbutamide or the 6 beta-hydroxylation of testosterone. 4. Significant (P < 0.01) correlations were observed between the microsomal 4-hydroxylation of R- and S-propranolol and the 2-hydroxylation of desipramine (r = 0.85 and 0.98, respectively). A weak (r = 0.66), albeit significant (P < 0.05) correlation was observed between the 4-hydroxylation of R-, but not of S-propranolol and the hydroxylation of tolbutamide. No significant correlations were observed between the 4-hydroxylation of propranolol enantiomers and the oxidation of other substrates for CYP1A2, 2C19, and 3A3/4. 5. Recombinant human CYP1A2 and CYP2D6 exhibited comparable catalytic activity with respect to the N-desisopropylation of both propranolol enantiomers; only expressed CYP2D6 exhibited a marked catalytic activity with respect to the 4-hydroxylation of both propranolol enantiomers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of cytochrome P450 (CYP) inducers on caffeine metabolism in the rat

Our previous studies, carried out using rat cDNA-expressed cytochrome P450 (CYP) isoforms, liver microsomes and specific CYP inhibitors, showed that the 1-N- and 3-N-demethylation of caffeine at a therapeutic concentration was predominantly catalyzed by CYP1A2 and CYP2C, its 7-N-demethylation was governed by P450s of the CYP2C subfamily, while its 8-hydroxylation was specifically mediated by CYP1A2. The present study was aimed at corroborating the above-described results using another experimental model, i.e. a study of caffeine metabolism in the liver microsomes and specific CYP inducers. Animals received one of the following inducers: beta-naphthoflavone (100 mg/kg i.p. for 4 days), phenobarbital (10 mg/kg for 6 days or 100 mg/kg i.p. for 4 days), pregnenolone 16alpha-carbonitrile (100 mg/kg i.p. for 4 days) or 15% ethanol ( approximately 11 g/kg in drinking water for 6 days). Sixteen hours after the last dose of an inducer liver microsomes were prepared and the caffeine metabolism and CYP isoform activities (testosterone 2alpha-, 2beta-, 6beta-, 7alpha-, 16beta-hydroxylation and warfarin 7-hydroxylation) were investigated. beta-Naphthoflavone (mainly a CYP1A inducer and CYP2C11 inhibitor) potently accelerated the metabolism of caffeine, the effect on 7-N-demethylation being the weakest. Moreover, the influence of beta-naphthoflavone on caffeine metabolism was more potent at the substrate concentration of 100 microM than 800 microM, in particular in the case of 7-N-demethylation and 8-hydroxylation. Pregnenolone-16alpha-carbonitrile (mainly a CYP3A inducer and CYP2C11 inhibitor) moderately induced 8-hydroxylation only. Phenobarbital (an inducer of CYP2B and other CYPs and a CYP2C11 inhibitor) moderately stimulated the metabolism of caffeine, but practically did not affect 7-N-demethylation. Ethanol (mainly a CYP2E1 inducer) modestly increased the rates of the N-demethylation reactions. The presently obtained data confirm the pivotal role of CYP1A2 in the metabolism of caffeine, as well as the involvement of CYP3A in the 8-hydroxylation of caffeine and that of CYP2C11 in its 7-N-demethylation.     

mRNA and protein expression of dog liver cytochromes P450 in relation to the metabolism of human CYP2C substrates

1. Interpretation of novel drug exposure and toxicology data from the dog is tempered by our limited molecular and functional knowledge of dog cytochromes P450 (CYPs). The aim was to study the mRNA and protein expression of hepatic dog CYPs in relation to the metabolism of substrates of human CYP, particularly those of the CYP2C subfamily. 2. The rate of 7-hydroxylation of S-warfarin (CYP2C9 in humans) by dog liver microsomes (mean +/- SD from 12 (six male and six female) dogs = 10.8 +/- 1.9 fmol mg(-1) protein min(-1)) was 1.5-2 orders of magnitude lower than that in humans. 3. The rate of 4'-hydroxylation of S-mephenytoin, catalysed in humans by CYP2C19, was also low in dog liver (4.6 +/-1.5 pmol mg(-1) protein min(-1)) compared with human liver. In contrast, the rate of 4'-hydroxylation of the R-enantiomer of mephenytoin by dog liver was much higher. The kinetics of this reaction (range of K(m) or K(0.5) 15-22 micro M, V(max) 35-59 pmol mg(-1) protein min(-1), n = 4 livers) were consistent with the involvement of a single enzyme. 4. In contrast to our findings for S-mephenytoin, dog liver microsomes 5'-hydroxylated omeprazole (also catalysed by CYP2C19 in humans) at considerably higher rates (range of K(m) 42-64 micro M, V(max) 22-46 pmol mg(-1) protein min(-1), n = 4 livers). 5. For all the substrates except omeprazole, a sex difference in their metabolism was observed in the dog (dextromethorphan N-demethylation: female range = 0.7-0.9, male = 0.4-0.8 nmol mg(-1) protein min(-1) (p < 0.02); S-warfarin 7-hydroxylation: female = 9-15.5, male = 8-12 fmol mg(-1) protein min(-1) (p < 0.02); R-mephenytoin 4'-hydroxylation: female = 16-35, male = 11.5-19 pmol mg(-1) protein min(-1) (p < 0.01); omeprazole 5'-hydroxylation: female = 15-20, male 13-22 pmol mg(-1) protein min(-1) (p < 0.2)). 6. All dog livers expressed mRNA and CYP3A12, CYP2B11, CYP2C21 proteins, with no sex differences being found. Expression of CYP2C41 mRNA was undetectable in the livers of six of 11 dogs. 7. Correlation analysis suggested that CYP2B11 catalyses the N-demethylation of dextromethorphan (mediated in humans by CYP3A) and the 4'-hydroxylation of mephenytoin (mediated in humans by CYP2C19) in the dog, and that this enzyme and CYP3A12 contribute to S-warfarin 7-hydroxylation (mediated in humans by CYP2C9). 8. In conclusion, we have identified a distinct pattern of hepatic expression of the CYP2C41 gene in the Alderley Park beagle dog. Furthermore, marked differences in the metabolism of human CYP2C substrates were observed in this dog strain compared with humans with respect to rate of reaction, stereoselectivity and CYP enzyme selectivity.     

Cytochrome P-450 3A4 and 2C8 are involved in zopiclone metabolism.

Zopiclone is a widely prescribed, nonbenzodiazepine hypnotic that is extensively metabolized by the liver in humans. The aim of the present study was to identify the human cytochrome P-450 (CYP) isoforms involved in zopiclone metabolism in vitro. Zopiclone metabolism was studied with different human liver microsomes and a panel of heterologously expressed human CYPs (CYP1A2, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4). In human liver microsomes, zopiclone was metabolized into N-desmethyl-zopiclone (ND-Z) and N-oxide-zopiclone (NO-Z) with the following K(m) and V(m) of 78 +/- 5 and 84 +/- 19 microM, 45 +/- 1 and 54 +/- 5 pmol/min/mg for ND-Z and NO-Z generation, respectively. Ketoconazole (CYP3A inhibitor) inhibited approximately 40% of the generation of both metabolites, sulfaphenazole (CYP2C inhibitor) inhibited the formation of ND-Z, whereas alpha-naphtoflavone (CYP1A), quinidine (CYP2D6), and chlorzoxazone (CYP2E1) did not affect zopiclone metabolism. The generation of ND-Z and NO-Z were highly correlated to testosterone 6beta-hydroxylation (CYP3A activity, r = 0.95 and 0.92, respectively; p =.0001), and ND-Z was highly correlated to CYP2C8 activity (paclitaxel 6alpha-hydroxylase; r = 0.76, p =.004). Recombinant CYP2C8 had the highest enzymatic activity toward zopiclone metabolism into both its metabolites, followed by CYP2C9 and 3A4. CYP3A4 is the major enzyme involved in zopiclone metabolism in vitro, and CYP2C8 contributes significantly to ND-Z formation.     

Identification of CYP3A4 as the enzyme involved in the mono-N-dealkylation of disopyramide enantiomers in humans.

To identify which cytochrome P-450 (CYP) isoform(s) are involved in the major pathway of disopyramide (DP) enantiomers metabolism in humans, the in vitro formation of mono-N-desalkyldisopyramide from each DP enantiomer was studied with human liver microsomes and nine recombinant human CYPs. Substrate inhibition showed that SKF 525A and troleandomycin potently suppressed the metabolism of both DP enantiomers with IC50 values for R(-)- and S(+)-DP of <7.3 and <18.9 microM, respectively. In contrast, only weak inhibitory effects (i.e., IC50 > 100 microM) were observed for five other representative CYP isoform substrates [i.e., phenacetin (CYP1A1/2), sparteine (CYP2D6), tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), and p-nitrophenol (CYP2E1)]. Significant correlations (P <.01, r = 0.91) were found between the activities of 11 different human liver microsomes for mono-N-dealkylation of both DP enantiomers and that of 6beta-hydroxylation of testosterone. Conversely, no significant correlations were observed between the catalytic activities for DP enantiomers and those for the O-deethylation of phenacetin, 2-hydroxylation of desipramine, hydroxylation of tolbutamide, and 4'-hydroxylation of S-mephenytoin. Further evidence for involvement of CYP3A P450s was revealed by an anti-human CYP3A serum that inhibited the mono-N-dealkylation of both DP enantiomers and 6beta-hydroxylation of testosterone almost completely (i.e., >90%), whereas it only weakly inhibited (i.e., <15%) CYP1A1/2- or 2C19-mediated reactions. Finally, the recombinant human CYP3A3 and 3A4 showed much greater catalytic activities than seven other isoforms examined (i.e., CYP1A2, 2A6, 2B6, 2C9, 2D6, 2E1, and 3A5) for both DP enantiomers. In conclusion, the metabolism of both DP enantiomers in humans would primarily be catalyzed by CYP3A4, implying that DP may have an interaction potential with other CYP3A substrates and/or inhibitors.     

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.     

In vitro metabolism of chloroquine: identification of CYP2C8, CYP3A4, and CYP2D6 as the main isoforms catalyzing N-desethylchloroquine formation.

In humans, the antimalarial drug chloroquine (CQ) is metabolized into one major metabolite, N-desethylchloroquine (DCQ). Using human liver microsomes (HLM) and recombinant human cytochrome P450 (P450), we performed studies to identify the P450 isoform(s) involved in the N-desethylation of CQ. In HLM incubated with CQ, only DCQ could be detected. Apparent Km and Vmax values (mean +/- S.D.) for metabolite formation were 444 +/- 121 microM and 617 +/- 128 pmol/min/mg protein, respectively. In microsomes from a panel of 16 human livers phenotyped for 10 different P450 isoforms, DCQ formation was highly correlated with testosterone 6beta-hydroxylation (r = 0.80; p < 0.001), a CYP3A-mediated reaction, and CYP2C8-mediated paclitaxel alpha-hydroxylation (r = 0.82; p < 0.001). CQ N-desethylation was diminished when coincubated with quercetin (20-40% inhibition), ketoconazole, or troleandomycin (20-30% inhibition) and was strongly inhibited (80% inhibition) by a combination of ketoconazole and quercetin, which further corroborates the contribution of CYP2C8 and CYP3As. Of 10 cDNA-expressed human P450s examined, only CYP1A1, CYP2D6, CYP3A4, and CYP2C8 produced DCQ. CYP2C8 and CYP3A4 constituted low-affinity/high-capacity systems, whereas CYP2D6 was associated with higher affinity but a significantly lower capacity. This property may explain the ability of CQ to inhibit CYP2D6-mediated metabolism in vitro and in vivo. At therapeutically relevant concentrations ( approximately 100 microM CQ in the liver), CYP2C8, CYP3A4, and, to a much lesser extent, CYP2D6 are expected to account for most of the CQ N-desethylation.     

mRNA and protein expression of dog liver cytochromes P450 in relation to the metabolism of human CYP2C substrates

1. Interpretation of novel drug exposure and toxicology data from the dog is tempered by our limited molecular and functional knowledge of dog cytochromes P450 (CYPs). The aim was to study the mRNA and protein expression of hepatic dog CYPs in relation to the metabolism of substrates of human CYP, particularly those of the CYP2C subfamily. 2. The rate of 7-hydroxylation of S -warfarin (CYP2C9 in humans) by dog liver microsomes (mean ± SD from 12 (six male and six female) dogs = 10.8 ± 1.9 fmol?mg ? 1 protein?min ? 1) was 1.5-2 orders of magnitude lower than that in humans. 3. The rate of 4'-hydroxylation of S -mephenytoin, catalysed in humans by CYP2C19, was also low in dog liver (4.6 ± 1.5 pmol?mg ? 1 protein?min ? 1) compared with human liver. In contrast, the rate of 4'-hydroxylation of the R -enantiomer of mephenytoin by dog liver was much higher. The kinetics of this reaction (range of K m or K 0.5 15-22 µM, V max 35-59 pmol?mg ? 1 protein?min ? 1, n = 4 livers) were consistent with the involvement of a single enzyme. 4. In contrast to our findings for S -mephenytoin, dog liver microsomes 5'-hydroxylated omeprazole (also catalysed by CYP2C19 in humans) at considerably higher rates (range of K m 42-64 µM, V max 22-46 pmol?mg ? 1 protein?min ? 1, n = 4 livers). 5. For all the substrates except omeprazole, a sex difference in their metabolism was observed in the dog (dextromethorphan N-demethylation: female range = 0.7-0.9, male = 0.4-0.8 nmol?mg ? 1 protein?min ? 1 (p < 0.02); S -warfarin 7-hydroxylation: female = 9-15.5, male = 8-12 fmol?mg ? 1 protein?min ? 1 (p < 0.02); R -mephenytoin 4'-hydroxylation: female = 16-35, male = 11.5-19 pmol?mg ? 1 protein?min ? 1 (p < 0.01); omeprazole 5'-hydroxylation: female = 15-20, male 13-22 pmol?mg ? 1 protein?min ? 1 (p < 0.2)). 6. All dog livers expressed mRNA and CYP3A12, CYP2B11, CYP2C21 proteins, with no sex differences being found. Expression of CYP2C41 mRNA was undetectable in the livers of six of 11 dogs. 7. Correlation analysis suggested that CYP2B11 catalyses the N-demethylation of dextromethorphan (mediated in humans by CYP3A) and the 4'-hydroxylation of mephenytoin (mediated in humans by CYP2C19) in the dog, and that this enzyme and CYP3A12 contribute to S -warfarin 7-hydroxylation (mediated in humans by CYP2C9). 8. In conclusion, we have identified a distinct pattern of hepatic expression of the CYP2C41 gene in the Alderley Park beagle dog. Furthermore, marked differences in the metabolism of human CYP2C substrates were observed in this dog strain compared with humans with respect to rate of reaction, stereoselectivity and CYP enzyme selectivity.     

Studies on the cytochrome P450 (CYP)-mediated metabolic properties of miocamycin: evaluation of the possibility of a metabolic intermediate complex formation with CYP, and identification of the human CYP isoforms.

Some macrolide antibiotics cause clinical drug interactions, resulting in altered metabolism of concomitantly administered drugs, via the formation of a metabolic intermediate (MI) complex with cytochrome P450 (CYP), or competitive inhibition of CYP. In this study, the possibility of MI complex formation by miocamycin (MOM) was assessed first. CYP contents and activities in rat liver microsomes were not affected and there were no detectable MI complexes after administration of MOM for either 3 or 10 days to rats. Furthermore, MOM did not form MI complexes in vitro even with microsomes from humans or dexamethasone-pretreated rats. Second, in vitro studies were conducted to identify the human CYP isoforms involved in four 14-hydroxylation reactions in the MOM metabolic pathway. The results showed that it was most likely CYP3A4 involved in the hydroxylations: 1) each hydroxylation in human liver microsomes from 10 different donors strongly correlated with testosterone 6 beta-hydroxylation; 2) each hydroxylation was essentially inhibited by ketoconazole and troleandomycin; 3) only cDNA-expressed CYP3A4 and CYP3A5 catalyzed the hydroxylations, and the activities of CYP3A5 were below 5% of those of CYP3A4; and 4) the apparent K(M) values obtained with native human liver microsomes were comparable with those obtained with cDNA-expressed CYP3A4. In conclusion, MOM is not an inhibitor of CYP via the formation of an MI complex. Moreover, CYP3A4 is mainly responsible for catalyzing the hydroxylation of MOM metabolites. Because CYP3A4 is the most abundant form of CYP in the liver and intestine, this isoform probably accounts for the majority of drug-MOM interactions observed in clinical practice.     

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.     

The involvement of CYP3A4 and CYP2C9 in the metabolism of 17 alpha-ethinylestradiol.

The role of specific cytochrome P450 (P450) isoforms in the metabolism of ethinylestradiol (EE) was evaluated. The recombinant human P450 isozymes CYP1A1, CYP1A2, CYP2C9, CYP2C19, and CYP3A4 were found to be capable of catalyzing the metabolism of EE (1 microM). Without exception, the major metabolite was 2-hydroxy-EE. The highest catalytic efficiency (Vmax/Km) was observed with rCYP1A1, followed by rCYP3A4, rCYP2C9, and rCYP1A2. The P450 isoforms 3A4 and 2C9 were shown to play a significant role in the formation of 2-hydroxy-EE in a pool of human liver microsomes by using isoform-specific monoclonal antibodies, in which the inhibition of formation was approximately 54 and 24%, respectively. The involvement of CYP3A4 and CYP2C9 was further confirmed by using selective chemical inhibitors (i.e., ketoconazole and sulfaphenazole). The relative contribution of each P450 isoform to the 2-hydroxylation pathway was obtained from the catalytic efficiency of each isoform normalized by its relative abundance in the same pool of human liver microsomes, as determined by quantitative Western blot analysis. Collectively, these results suggested that multiple P450 isoforms were involved in the oxidative metabolism of EE in human liver microsomes, with CYP3A4 and CYP2C9 as the major contributing enzymes.     

Reversible and irreversible inhibition of CYP3A enzymes by tamoxifen and metabolites

1. Preliminary studies have identified cytochrome P450 (CYP) 3A4 and CYP1B1 as the human CYPs inhibited by tamoxifen. To quantify the inhibitory potency of tamoxifen and its major metabolites, the metabolism of three substrates of CYP3A, midazolam, diltiazem and testosterone, and 7-ethoxyresorufin as a substrate of CYP1B1 were examined in catalytic assays carried out using human liver microsomes and cDNA-expression systems. 2. Tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and 3-hydroxytamoxifen reversibly inhibited midazolam 1'-hydroxylation, diltiazem N-demethylation and testosterone 6 β -hydroxylation with K_i ranging from 3 to 37 µM in human liver microsomes. Tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and 3-hydroxytamoxifen also reversibly inhibited the activity of cDNA-expressed CYP3A4, CYP3A5 and CYP1B1. 3. Tamoxifen and N-desmethyltamoxifen exhibited time-dependent inactivation of testosterone 6 β -hydroxylation by cDNA-expressed CYP3A4 (⁺cytochrome b5) yielding k inact and K i of 0.04?min -1 and 0.2 µM for tamoxifen and 0.08?min -1 and 2.6 µM for N-desmethyltamoxifen. A metabolic intermediate complex (MIC) was also formed by tamoxifen and N -desmethyltamoxifen with CYP3A4 (+ cytochrome b5) and CYP3A4 but not with CYP3A5 or CYP3A7. Pre-incubation with 4-hydroxytamoxifen and 3-hydroxytamoxifen did not result in any CYP3A inactivation or detectable MIC formation. There was no detectable time-dependent inactivation or MIC formation with tamoxifen or metabolites with CYP1B1. 4. These data indicate that tamoxifen and its three major metabolites are effective inhibitors of CYP3A in vitro and that tamoxifen and N-desmethyltamoxifen are effective mechanism-based inhibitors. Thus, caution should be exercised when tamoxifen is coadministered with other CYP3A substrates.