Studies on the metabolism of the novel, selective cyclooxygenase-2 inhibitor indomethacin phenethylamide in rat, mouse, and human liver microsomes: identification of active metabolites.

The metabolism of 2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]-N-phenethyl-acetamide (indomethacin phenethylamide, LM-4108), a highly selective cyclooxygenase-2 inhibitor, was studied in rat, mouse, and human liver microsomes. The primary site of oxidation in all species examined was on the methylene carbons of the phenethyl side chain to form the 1'- and 2'-hydroxy and 2'-oxo metabolites as determined by electrospray ionization liquid chromatography-tandem mass spectrometry. Half-lives for the disappearance of 10 microM LM-4108 in rat, human, and mouse liver microsomes (0.15 pmol P450/ml) were 11 min, 21 min, and 51 min, respectively. Indomethacin formation was not observed in incubations with rat, mouse, or human liver microsomes. Both the 2'-hydroxy-LM-4108 and 2'-oxo-LM-4108 metabolites were synthesized and found to be equipotent to the parent compound with regard to COX-2 inhibitory potency and selectivity [2'-hydroxy-LM-4108: IC(50)(COX-2) = 0.06 microM, IC(50)(COX-1) >66 microM; 2'-oxo-LM-4108: IC(50)(COX-2) = 0.05 microM, IC(50)(COX-1) >66 microM]. The formation of the metabolites was strongly inhibited by specific CYP3A4 inhibitors ketoconazole and troleandomycin but not by other isoform-selective inhibitors. These findings were confirmed by demonstrating that cloned, expressed CYP3A4 catalyzed side chain oxidation. O-Demethylation was a minor oxidative pathway in contrast to the metabolism of indomethacin and was catalyzed by CYP2D6. Upon intravenous administration of LM-4108 to Sprague-Dawley rats, oxidative metabolism on the phenethyl side chain constituted the rate-limiting steps in its clearance. The active metabolites, 2'-oxo- and 2'-hydroxy-LM-4108, as well as 1'-hydroxy-LM-4108, were all observed in rat plasma and thus may contribute to COX-2 inhibition in vivo. The glucuronides of 2'hydroxy-LM-4108 and O-desmethyl-2'-hydroxy-LM-4108 were also identified in rat bile.     

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.     

Identification of enzymes responsible for rifalazil metabolism in human liver microsomes

1. The major metabolites of rifalazil in human are 25-deacetyl-rifalazil and 32-hydroxy-rifalazil. Biotransformation to these metabolites in pooled human liver microsomes, cytosol and supernatant 9000g (S9) fractions was studied, and the enzymes responsible for rifalazil metabolism were identified using inhibitors of esterases and cytochromes P450 (CYP). 2. The 25-deacetylation and 32-hydroxylation of rifalazil occurred in incubations with microsomes or S9 but not with cytosol, indicating that both the enzymes responsible for rifalazil metabolism were microsomal. Km and Vmax of the rifalazil-25-deacetylation in microsomes were 6.5 microM and 11.9 pmol/min/mg with NADPH, and 2.6 microM and 6.0 pmol/min/mg without NADPH, indicating that, although rifalazil-25-deacetylation did not require NADPH, NADPH activated it. Rifalazil-32-hydroxylation was NADPH dependent, and its Km and Vmax were 3.3 microM and 11.0 pmol/min/mg respectively. 3. Rifalazil-25-deacetylation in microsomes was completely inhibited by diisopropyl fluorophosphate, diethyl p-nitrophenyl phosphate and eserine, but not by p-chloromercuribenzoate or 5,5'-dithio-bis(2-nitrobenzoic acid), indicating that the enzyme responsible for the rifalazil-25-deacetylation is a B-esterase. 4. Rifalazil-32-hydroxylation in microsomes was completely inhibited by CYP3A4-specific inhibitors (fluconazole, ketoconazole, miconazole, troleandomycin) and drugs metabolized by CYP3A4 such as cyclosporin A and clarithromycin, indicating that the enzyme responsible for the rifalazil-32-hydroxylation is CYP3A4.     

Cytochrome P450 3A4 is the major enzyme involved in the metabolism of the substance P receptor antagonist aprepitant.

The contribution of human cytochrome P450 (P450) isoforms to the metabolism of aprepitant in humans was investigated using recombinant P450s and inhibition studies. In addition, aprepitant was evaluated as an inhibitor of human P450s. Metabolism of aprepitant by microsomes prepared from baculovirus-expressed human P450s was observed only when CYP1A2, CYP2C19, or CYP3A4 was present in the expression system. Incubation with CYP1A2 and CYP2C19 yielded only products of O-dealkylation, whereas CYP3A4 catalyzed both N- and O-dealkylation reactions. The metabolism of aprepitant by human liver microsomes was inhibited completely by ketoconazole or troleandomycin. No inhibition was observed with other P450 isoform-selective inhibitors. Aprepitant was evaluated also as a P450 inhibitor in human liver microsomes. No significant inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP2E1 was observed in experiments with isoform-specific substrates (IC50 > 70 microM). Aprepitant was a moderate inhibitor of CYP3A4, with Ki values of approximately 10 microM for the 1'- and 4-hydroxylation of midazolam, and the N-demethylation of diltiazem, respectively. Aprepitant was a very weak inhibitor of CYP2C9 and CYP2C19, with Ki values of 108 and 66 microM for the 7-hydroxylation of warfarin and the 4'-hydroxylation of S-mephenytoin, respectively. Collectively, these results indicated that aprepitant is both a substrate and a moderate inhibitor of CYP3A4.     

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。     

In vitro metabolism of indiplon and an assessment of its drug interaction potential

This study was designed to study the in vitro metabolism of indiplon, a novel hypnotic agent, and to assess its potential to cause drug interactions. In incubations with pooled human liver microsomes, indiplon was converted to two major, pharmacologically inactive metabolites, N-desmethyl-indiplon and N-desacetyl-indiplon. The N-deacetylation reaction did not require NADPH, and appeared to be catalyzed by organophosphate-sensitive microsomal carboxylesterases. The N-demethylation of indiplon was catalyzed by CYP3A4/5 based on the following observations: (1) the sample-to-sample variation in N-demethylation of indiplon ([S] = 100 microM) in a bank of human liver microsomes was strongly correlated with testosterone 6beta-hydroxylase (CYP3A4/5) activity (r(2) = 0.98), but not with any other CYP enzyme; (2) recombinant CYP1A1, CYP1A2, CYP3A4, CYP3A5 and CYP3A7 had the ability to catalyze this reaction; (3) the N-demethylation of indiplon was inhibited by CYP3A4/5 inhibitors (ketoconazole and troleandomycin), but not by a CYP1A2 inhibitor (furafylline). In pooled human liver microsomes, indiplon exhibited a weak capacity to inhibit CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4/5 and carboxylesterase (p-nitrophenylacetate hydrolysis) activities (IC50 >/= 20 microM). Clinical data available on indiplon support the conclusions of this paper that the in vitro metabolism of indiplon is catalyzed by multiple enzymes, and indiplon is a weak inhibitor of human CYP enzymes.     

Role of individual human cytochrome P450 enzymes in the in vitro metabolism of hydromorphone

1. The aim was to identify the individual human cytochrome P450 (CYP) enzymes responsible for the in vitro N-demethylation of hydromorphone and to determine the potential effect of the inhibition of this metabolic pathway on the formation of other hydromorphone metabolites. 2. Hydromorphone was metabolized to norhydromorphone (apparent Km = 206 - 822 microM, Vmax = 104 - 834 pmol min(-1) mg(-1) protein) and dihydroisomorphine (apparent Km = 62 - 557 microM, Vmax = 17 - 122 pmol min(-1) mg(-1) protein) by human liver microsomes. 5. In pooled human liver microsomes, troleandomycin, ketoconazole and sulfaphenazole reduced norhydromorphone formation by an average of 45, 50 and 25%, respectively, whereas furafylline, quinidine and omeprazole had no effect. In an individual liver microsome sample with a high CYP3A protein content, troleandomycin and ketoconazole inhibited norhydromorphone formation by 80%. 5. The reduction in norhydromorphone formation by troleandomycin and ketoconazole was accompanied by a stimulation in dihydroisomorphine production.Recombinant CYP3A4, CYP3A5, CYP2C9 and CYP2D6, but not CYP1A2, catalysed norhydromorphone formation, whereas none of these enzymes was active in dihydroisomorphine formation. 6. In summary, CYP3A and, to a lesser extent, CYP2C9 catalysed hydromorphone N-demethylation in human liver microsomes. The inhibition of norhydromorphone formation by troleandomycin and ketoconazole resulted in a stimulation of microsomal dihydroisomorphine formation.     

Identification of cytochrome P4503A as the major subfamily responsible for the metabolism of roquinimex in man

1. Roquinimex, a novel immunomodulator, is metabolized in liver microsomes from mouse and rat via cytochrome P450s to four hydroxylated and two demethylated metabolites (R1-6). The study investigated which cytochrome P450 enzyme(s) is responsible for the metabolism of roquinimex in man. 2. Enzyme kinetic analysis demonstrated an apparent Km = 1.28-7.00 mM and Vmax = 50-159 pmol x mg(-1) microsomal protein x min(-1) for the primary metabolites in human liver microsomes. The sum of Cl(int) for the primary pathways was 0.167 microl x mg(-1) microsomal protein x min(-1). 3. A correlation between the formation rate of R1-6 and 6beta-hydroxylation of testosterone was obtained within a panel of liver microsomes from 11 individuals (r2 = 0.72-0.97). Furthermore, significant inhibition (>90%) of roquinimex primary metabolism was demonstrated by ketoconazole and troleandomycin, specific inhibitors of CYP3A4 as well as with anti-CYP3A4 antibodies. Moreover, a similar metabolite pattern was produced from roquinimex by incubation with cDNA-expressed CYP3A4 as by human liver microsomes. 4. In conclusion, these data indicate a major role for CYP3A4 in the formation of roquinimex primary metabolites in human liver microsomes.     

Model for the drug-drug interaction responsible for CYP3A enzyme inhibition. I: evaluation of cynomolgus monkeys as surrogates for humans

1. Anti-human cytochrome P450 (CYP) 3A4 antiserum completely inhibited midazolam metabolism in monkey liver microsomes, suggesting that midazolam was mainly metabolized by CYP3A enzyme(s) in monkey liver microsomes. 2. Midazolam metabolism was also inhibited in vitro by typical chemical inhibitors of CYP3A, such as ketoconazole, erythromycin and diltiazem, and the apparent K(i) values for ketoconazole, erythromycin and diltiazem were 0.127, 94.2 and 29.6 microM, respectively. 3. CYP3A inhibitors increased plasma midazolam concentrations when midazolam and CYP3A inhibitors were co-administered orally. However, the pharmacokinetic parameters of midazolam were not changed by treatment with CYP3A inhibitors when midazolam was given intravenously. This suggests that CYP3A inhibitors modified the first-pass metabolism in the liver and/or intestine, but not systemic metabolism. 4. The drug-drug interaction responsible for CYP3A enzyme(s) inhibition was observed when midazolam and inhibitors were co-administrated orally. Therefore, it was concluded that monkeys given midazolam orally could be useful models for predicting drug-drug interactions in man based on CYP3A enzyme inhibition.     

Fluvoxamine is a more potent inhibitor of lidocaine metabolism than ketoconazole and erythromycin in vitro

CYP3A4 is generally believed to be the major CYP enzyme involved in the biotransformation of lidocaine in man; however, recent in vivo studies suggest that this may not be the case. We have examined the effects of the CYP3A4 inhibitors erythromycin and ketoconazole and the CYP1A2 inhibitor fluvoxamine on the N-deethylation, i.e. formation of monoethylglycinexylidide (MEGX), and 3-hydroxylation of lidocaine by human liver microsomes. The experiments were carried out at lidocaine concentrations of 5 microM (clinically relevant concentration) and 800 microM. The formation of both MEGX and 3-hydroxylidocaine was best described by a two-enzyme model. At 5 microM of lidocaine, fluvoxamine was a potent inhibitor of the formation of MEGX (IC50 1.2 microM). Ketoconazole and erythromycin also showed an inhibitory effect on MEGX formation, but ketoconazole (IC50 8.5 microM) was a much more potent inhibitor than erythromycin (IC50 200 microM). At 800 microM of lidocaine, fluvoxamine (IC50 20.7 microM) and ketoconazole (IC50 20.4 microM) displayed a modest inhibitory effect on MEGX formation, whereas erythromycin was a weak inhibitor (IC50 >250 microM). The 3-hydroxylation of lidocaine was potently inhibited by fluvoxamine at both lidocaine concentrations (IC50 0.16 microM at 5 microM and 1.8 microM at 800 microM). Erythromycin and ketoconazole showed a clear inhibitory effect on the 3-hydroxylation of lidocaine at 5 microM of lidocaine (IC50 9.9 microM and 13.9 microM, respectively), but did not show a consistent effect at 800 microM of lidocaine (IC50 >250 microM and 75.0 microM, respectively). Although further studies are needed to elucidate the role of distinct CYP enzymes in the biotransformation of lidocaine in humans, the findings of this study suggest that while both CYP1A2 and CYP3A4 are involved in the metabolism of lidocaine by human liver microsomes, CYP1A2 is the more important isoform at clinically relevant lidocaine concentrations.     

CYP3A4-mediated hepatic metabolism of the HIV-1 protease inhibitor saquinavir in vitro

1. The aim was to identify the major metabolites of saquinavir (SQV) from human hepatic microsomal incubations and the CYP isoform(s) responsible. 2. Ten fractions containing various metabolites were separated by isocratic reversed-phase HPLC and characterized by HPLC, mass spectrometry and NMR. 3. Metabolites were either mono- or di-hydroxylated derivatives of SQV. Fast-atom bombardment and electrospray MS showed that hydroxylation was predominantly situated on the decahydroisoquinoline ring. A major metabolite (M4) was rigorously identified as 6-equatorial-hydroxy SQV. 4. Metabolism of saquinavir to all metabolites was inhibited by the CYP3A4-selective inhibitor ketoconazole (IC50 = 0.55 +/- 0.12 microM). Other isoform-selective inhibitors were non-inhibitory. The protease inhibitors ritonavir, indinavir and nelfinavir potently inhibited SQV metabolism in hepatic microsomes with IC50 = 0.025 +/- 0.004, 0.82 +/- 0.26 and 0. 58 +/- 0.14 microM, respectively. 5. Saquinavir metabolism correlated with immunochemically determined CYP3A4 levels and testosterone 6beta-hydroxylation, but it failed to correlate with either immunochemically determined CYPIA2 levels or marker activities for CYP1A2, 2C9 or 2E1. 6. Heterologously expressed CYP3A4 metabolized saquinavir with a similar metabolic profile to that of human liver microsomes. 7. Km, and Vmax for total SQV metabolism were 0.61 +/- 0.19 microM and 1.82 +/- 1.13 nmol mg(-1) min(-1), respectively. 8. The extensive involvement of hepatic CYP3A4 in the metabolism of saquinavir predicts high intrinsic clearance of saquinavir. Inhibitors of CYP3A4 such as other protease inhibitors will substantially increase the bioavailability of saquinavir.     

Species difference in stereoselective involvement of CYP3A in the mono-N-dealkylation of disopyramide.

1. To determine which CYP isoenzyme is involved in the N-dealkylation of disopyramide (DP) metabolism in human and dog, and to determine the stereoselectivity of DP metabolism with human CYP and dog CYP isoenzymes, the following in vitro metabolism studies of DP were conducted: correlation between human CYP isoenzyme activities and DP metabolism with human liver microsomes; inhibition of DP metabolism in human and dog liver microsomes with chemical inhibitors of CYP isoenzymes; inhibition of DP metabolism in human microsomes with human CYP antibodies; inhibition of DP metabolism in dog liver microsomes with human and dog CYP antibodies; metabolism of DP with human (CYP3A4) and dog (CYP3A12) cDNA-expressed isoenzymes; determination of Km and Vmax of DP enantiomers by using cDNA-expressed CYP3A4 and CYP3A12. 2. In human liver microsomes, the formation of the mono-N-dealkylated disopyramide (MNDP) metabolite was best correlated with CYP3A4 activities. DP metabolism was substantially inhibited by ketoconazole, troleandomycin (TA) and human CYP3A4 antibody. DP was metabolized by cDNA-expressed CYP3A isoenzymes. In dog liver microsomes, DP metabolism was inhibited by ketoconazole, TA and dog anti-CYP3A12. DP was also metabolized by cDNA-expressed CYP3A12. 3. CYP3A4 and CYP3A12 are the principal isoenzymes involved in DP metabolism in human and dog respectively. There was no stereoselectivity in N-dealkylation of DP by human CYP3A4. However, there was notable stereoselectivity in the N-dealkylation by dog CYP3A12.     

Enzyme kinetics for the formation of 3-hydroxyquinine and three new metabolites of quinine in vitro; 3-hydroxylation by CYP3A4 is indeed the major metabolic pathway.

The formation kinetics of 3-hydroxyquinine, 2'-quininone, (10S)-11-dihydroxydihydroquinine, and (10R)-11-dihydroxydihydroquinine were investigated in human liver microsomes and in human recombinant-expressed CYP3A4. The inhibition profile was studied by the use of different concentrations of ketoconazole, troleandomycin, and fluvoxamine. In addition, formation rates of the metabolites were correlated to different enzyme probe activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in microsomes from 20 human livers. Formation of 3-hydroxyquinine had the highest intrinsic clearance in human liver microsomes (mean +/- S.D.) of 11.0 +/- 4.6 micro l/min/mg. A markedly lower intrinsic clearance, 1.4 +/- 0.7, 0.5 +/- 0.1, and 1.1 +/- 0.2 micro l/min/mg was measured for 2'-quininone, (10R)-11-dihydroxydihydroquinine and (10S)-11-dihydroxydihydroquinine, respectively. Incubation with human recombinant CYP3A4 resulted in a 20-fold higher intrinsic clearance for 3-hydroxyquinine compared with 2'-quininone formation whereas no other metabolites were detected. The formation rate of 3-hydroxyquinine was completely inhibited by ketoconazole (1 micro M) and troleandomycin (80 micro M). Strong inhibition was observed on the formation of 2'-quininone whereas the formation of (10S)-11-dihydroxydihydroquinine was partly inhibited by these two inhibitors. No inhibition on the formation of (10R)-11-dihydroxydihydroquinine was observed. There was a significant correlation between the formation rates of quinine metabolites and activities of the CYP3A4 selected marker probes. This in vitro study demonstrates that 3-hydroxyquinine is the principal metabolite of quinine and CYP3A4 is the major enzyme involved in this metabolic pathway.     

An investigation of the interaction between halofantrine, CYP2D6 and CYP3A4: studies with human liver microsomes and heterologous enzyme expression systems.

1. We have assessed the interaction of the antimalarial halofantrine with cytochrome P450 (CYP) enzymes in vitro, with the use of microsomes from human liver and recombinant cell lines. 2. Rac-halofantrine was a potent inhibitor (IC50 = 1.06 microM, Ki = 4.3 microM) of the 1-hydroxylation of bufuralol, a marker for CYP2D6 activity. Of a group of structurally related antimalarials tested, only quinidine (IC50 = 0.04 microM) was more potent. 3. Microsomes prepared from recombinant CYP2D6 and CYP3A4 cell lines were shown to catalyse halofantrine N-debutylation. 4. The metabolism of halofantrine to its N-desbutyl metabolite by human liver microsomes showed no correlation with CYP2D6 genotypic or phenotypic status and there was no consistent inhibition by quinidine. 5. The rate of halofantrine metabolism showed a significant correlation with both CYP3A4 protein levels (r = 0.88, P = 0.01) and the rate of felodipine metabolism (r = 0.86, P = 0.013), a marker substrate for CYP3A4 activity. Inhibition studies showed that ketoconazole is a potent inhibitor of halofantrine metabolism (IC50 = 1.57 microM). 6. In conclusion, we have demonstrated that halofantrine is a potent inhibitor of CYP2D6 in vitro and can also be metabolised by the enzyme. However, in human liver microsomes it appears to be metabolised largely by CYP3A4.     

Nefiracetam metabolism by human liver microsomes: role of cytochrome P450 3A4 and cytochrome P450 1A2 in 5-hydroxynefiracetam formation.

An in-vitro study was conducted to investigate the metabolism of nefiracetam in human liver microsomes and to identify the enzymes responsible for the metabolism. Nefiracetam was hydroxylated by human liver microsomes to 5-hydroxynefiracetam (5-OHN). Eadie-Hofstee plots for the formation of 5-OHN suggested substrate activation. The kinetic parameters, apparent Km, Vmax, and Hill coefficient, for the formation of 5-OHN by pooled human liver microsomes were 4012 microM, 2.66 nmol min(-1) (mg protein)(-1), and 1.65, respectively. The formation of 5-OHN was significantly correlated with cytochrome P450 (CYP)3A4-mediated testosterone 6beta-hydroxylase activity and dextromethorphan N-demethylase activity. The 5-OHN formation was inhibited (94%) by antibody to human CYP3A4/5. The 5-OHN formation was also inhibited by the CYP3A4 inhibitors ketoconazole and troleandomycin, but not significantly inhibited by several other P450 inhibitors. The microsomes containing cDNA-expressed CYP3A4 formed 5-OHN with sigmoidal kinetics. CYP3A5-containing microsomes did not form 5-OHN. These results indicated that CYP3A, most likely CYP3A4, was the major isozyme responsible for the formation of 5-OHN in human liver microsomes. CYP1A2 and CYP2C19 microsomes were also capable of forming 5-OHN. However, the contribution of CYP1A2 was considered to be relatively minor compared with that of CYP3A4, and the contribution of CYP2C19 was assumed to be negligible, based on the result of the immunoinhibition study and taking into account both the turnover rate by each isozyme and the relative abundance of each isozyme in human liver. We conclude that on average the formation of 5-OHN, the major metabolite of nefiracetam, is principally mediated by CYP3A4 with a relatively minor contribution by CYP1A2.     

Characterization of the cytochrome P-450 gene family responsible for the N-dealkylation of the ergot alkaloid CQA 206-291 in humans.

The ergot alkaloid CQA 206-291 (CQA) was converted by human liver microsomes (n = 16) almost exclusively to the N-deethylated metabolite (I), as identified by the on-line coupling of liquid chromatography and mass spectroscopy. Metabolite I formation exhibited monophasic and linear enzyme kinetics (2.9-300 microM), and a 5.6-fold interindividual variability (7.2-40.2 nmol/mg/hr). Chemical inhibition experiments revealed that imidazole antimycotic agents (ketoconazole, miconazole, and clotrimazole) were potent inhibitors of this N-deethylation. Polymorphically metabolized substrates (sparteine and phenytoin), well-established cytochrome P-450 probe substrates (antipyrine and tolbutamide), and steroid hormones (estradiol and testosterone) were noninhibitory, indicating that their metabolism is catalyzed by forms of cytochrome P-450 that do not catalyze this route of CQA biotransformation. The ergot alkaloids--dihydroergotamine, bromocriptine, and SDZ 208-911--were competitive inhibitors of metabolite I formation, suggesting that these compounds are metabolized by similar enzymes. Cyclosporine A was a potent competitive inhibitor of CQA metabolism, providing initial evidence that formation of metabolite I was catalyzed by proteins of the CYP3 gene family. This was substantiated by the finding that CQA metabolism was completely inhibited by a polyclonal antibody directed against a pregnenolone 16 alpha-carbonitrile-inducible cytochrome P-450 of rat liver. The rate of CQA metabolism correlated significantly to the level of CYP3A4 expression, the rate of cyclosporine A metabolism to each of the primary metabolites (M-1, M-17, and M-21), and the rate of midazolam 4-hydroxylation. COS 1 cells transfected with human CYP3A4 and CYP3A5 provided direct evidence that these enzymes catalyze the metabolism of CQA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of human drug-metabolizing enzymes involved in the metabolism of SNI-2011.

In vitro studies were conducted to identify human drug-metabolizing enzymes involved in the metabolism of SNI-2011 ((+/-)-cis-2-methylspiro [1,3-oxathiolane-5,3'-quinuclidine] monohydrochloride hemihydrate, cevimeline hydrochloride hydrate). When 14C-SNI-2011 was incubated with human liver microsomes, SNI-2011 trans-sulfoxide and cis-sulfoxide were detected as major metabolites. These oxidations required NADPH, and were markedly inhibited by SKF-525A, indicating that cytochrome P450 (CYP) was involved. In a chemical inhibition study, metabolism of SNI-2011 in liver microsomes was inhibited (35-65%) by CYP3A4 inhibitors (ketoconazole and troleandomycin) and CYP2D6 inhibitors (quinidine and chlorpromazine). Furthermore, using microsomes containing cDNA-expressed CYPs, it was found that high rates of sulfoxidation activities were observed with CYP2D6 and CYP3A4. On the other hand, when 14C-SNI-2011 was incubated with human kidney microsomes, SNI-2011 N-oxide was identified as a major metabolite. This N-oxidation required NADPH, and was completely inhibited by thiourea, indicating that flavin-containing monooxygenase (FMO) was involved. In addition, microsomes containing cDNA-expressed FMO1, a major isoform in human kidney, mainly catalyzed N-oxidation of SNI-2011, but microsomes containing FMO3, a major isoform in adult human liver, did not. These results suggest that SNI-2011 is mainly catalyzed to sulfoxides and N-oxide by CYP2D6/3A4 in liver and FMOI in kidney, respectively.     

Examination of 209 drugs for inhibition of cytochrome P450 2C8

Cytochrome P450 2C8 is involved in the metabolism of drugs such as paclitaxel, repaglinide, rosiglitazone, and cerivastatin, among others. An in vitro assessment of 209 frequently prescribed drugs and related xenobiotics was carried out to examine their potential to inhibit CYP2C8. A validated sensitive, moderate-throughput high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay was used to detect N-desethylamodiaquine, the CYP2C8-derived major metabolite of amodiaquine metabolism, using heterologously expressed recombinant CYP2C8 (rhCYP2C8) and pooled human liver microsomes. The 209 drugs were first tested at 30 muM for their ability to inhibit rhCYP2C8. Forty-eight compounds exhibited greater than 50% inhibition and were further evaluated for measurement of IC50. The six most potent inhibitors (IC50 <1 microM) from this set were measured for IC50 in pooled human liver microsomes, and the most potent inhibitor identified was the leukotriene receptor antagonist, montelukast (IC50 = 19.6 nM). Inhibitors of CYP2C8 were identified from a wide variety of therapeutic classes, with no single class predominating. Other potent inhibitors included candesartan cilexetil (cyclohexylcarbonate ester prodrug of candesartan), zafirlukast, clotrimazole, felodipine, and mometasone furoate. Seventeen moderate inhibitors of rhCYP2C8 (1 < IC50 < 10 microM) included salmeterol, raloxifene, fenofibrate, ritonavir, levothyroxine, tamoxifen, loratadine, quercetin, oxybutynin, medroxyprogesterone, simvastatin, ketoconazole, ethinyl estradiol, spironolactone, lovastatin, nifedipine, and irbesartan. These in vitro data were used along with clinical pharmacokinetic information in predicting potential drug-drug interactions that could occur by inhibition of CYP2C8. Although almost all drugs tested are not expected to cause drug interactions via inhibition of CYP2C8, montelukast was identified as being of concern as a potential inhibitor of clinical relevance. These findings are discussed in context to potential drug interactions that could be observed between these agents and drugs for which CYP2C8 is involved in metabolism and warrant investigation of the possibility of clinical drug interactions mediated by inhibition of this enzyme.     

In vitro biotransformation of sildenafil (Viagra): identification of human cytochromes and potential drug interactions.

The in vitro biotransformation of sildenafil to its major circulating metabolite, UK-103,320, was studied in human liver microsomes and in microsomes containing heterologously expressed human cytochromes. In human liver microsomes, the mean K(m) (+/-S.E. ) was 14.4 +/- 2.0 microM. A screen of the chemical inhibitors omeprazole (10 microM), quinidine (10 microM), sulfaphenazole (10 microM), and ketoconazole (2.5 microM) only revealed detectable inhibition with ketoconazole. Sildenafil biotransformation (36 microM) was inhibited by increasing concentrations of ketoconazole and ritonavir (IC(50) values less than 0.02 microM), which are established cytochrome P450 (CYP) 3A4 inhibitors. Using microsomes containing cDNA-expressed cytochromes, UK-103,320 formation was found to be mediated by four cytochromes: CYP3A4, -2C9, -2C19, and -2D6. Estimated relative contributions to net intrinsic clearance were 79% for CYP3A4 and 20% for CYP2C9; for CYP2C19 and -2D6, estimated contributions were less than 2%. These results demonstrate that CYP3A4 is the primary cytochrome mediating UK-103,320 formation and that drugs that inhibit CYP3A4 are likely to impair sildenafil biotransformation.     

Metabolic interactions between mibefradil and HMG-CoA reductase inhibitors: an in vitro investigation with human liver preparations

AIMS: To determine the effects of mibefradil on the nletabolism in human liver microsomal preparations of the HMG-CoA reductase inhibitors simvastatin, lovastatin, atorvastatin, cerivastatin and fluvastatin. METHODS: Metabolism of the above five statins (0.5, 5 or 10 microM), as well as of specific CYP3A4/5 and CYP2C8/9 marker substrates, was examined in human liver microsomal preparations in the presence and absence of mibefradil (0.1-50 microM). RESULTS: Mibefradil inhibited, in a concentration-dependent fashion, the metabolism of the four statins (simvastatin, lovastatin, atorvastatin and cerivastatin) known to be substrates for CYP3A. The potency of inhibition was such that the IC50 values (<1 microM) for inhibition of all of the CYP3A substrates fell within the therapeutic plasma concentrations of mibefradil, and was comparable with that of ketoconazole. However, the inhibition by mibefradil, unlike that of ketoconazole, was at least in part mechanism-based. Based on the kinetics of its inhibition of hepatic testosterone 6beta-hydroxylase activity, mibefradil was judged to be a powerful mechanism-based inhibitor of CYP3A4/5, with values for Kinactivation, Ki and partition ratio (moles of mibefradil metabolized per moles of enzyme inactivated) of 0.4 min(-1), 2.3 microM and 1.7, respectively. In contrast to the results with substrates of CYP3A, metabolism of fluvastatin, a substrate of CYP2C8/9, and the hydroxylation of tolbutamide, a functional probe for CYP2C8/9, were not inhibited by mibefradil. CONCLUSION: Mibefradil, at therapeutically relevant concentrations, strongly suppressed the metabolism in human liver microsomes of simvastatin, lovastatin, atorvastatin and cerivastatin through its inhibitory effects on CYP3A4/5, while the effects of mibefradil on fluvastatin, a substrate for CYP2C8/9, were minimal in this system. Since mibefradil is a potent mechanism-based inhibitor of CYP3A4/5, it is anticipated that clinically significant drug-drug interactions will likely ensue when mibefradil is coadministered with agents which are cleared primarily by CYP3A-mediated pathways.