Lidocaine metabolism in human liver microsomes by cytochrome P450IIIA4

The metabolism of lidocaine to its major metabolite monoethylglycinexylidide (MEGX) was studied in human liver microsomes of 13 kidney transplant donors and of one patient with liver cirrhosis. Interindividual variation in metabolite formation was considerable. Biphasic kinetics indicated the involvement of at least two distinct enzymatic activities. With use of a series of antisera that recognize different human cytochrome P450 isozymes, we were able to identify an enzyme of the P450III gene family as one of two enzymes. By expressing human P450IIIA4 complementary deoxyribonucleic acid (cDNA) in HepG2 cells, we directly demonstrated lidocaine-deethylase activity for this P450 isozyme. These data suggest that P450IIIA4 is at least in part responsible for microsomal MEGX formation.     

NADPH-dependent metabolism of estrone by human liver microsomes

We characterized the NADPH-dependent metabolism of estrone (E1) by liver microsomes of 21 male and 12 female human subjects. The structures of 11 hydroxylated or keto metabolites of E1 formed by human liver microsomes were identified by chromatographic and mass spectrometric analyses. 2-Hydroxylation of E1 was the dominant metabolic pathway with all human liver microsomes tested. E1 is more prone to form catechol estrogens (particularly 4-OH-E1) than 17beta-estradiol (E2) and the average ratio of E1 4-hydroxylation to 2-hydroxylation (0.24) was slightly higher than the ratio of E2 4- to 2-hydroxylation (0.20, P < 0.001). An unidentified monohydroxylated E1 metabolite (y-OH-E1) was found to be one of the major metabolites formed by human liver microsomes of both genders. 6beta-OH-E1, 16alpha-OH-E1, and 16beta-OH-E1 were also formed in significant quantities. 16alpha-hydroxylation was not a major pathway for E1 metabolism. The overall profiles for the E1 metabolites formed by male and female human liver microsomes were similar, and their average rates were not significantly different. Hepatic CYP3A4/5 activity in both male and female liver microsomes correlated strongly with the rates of formation of several hydroxyestrogen metabolites. The dominant role of hepatic CYP3A4 and CYP3A5 in the formation of these hydroxyestrogen metabolites was further confirmed by incubations of human CYP3A4 or CYP3A5 with [3H]E1 and NADPH. Notably, human CYP3A5 has very high relative activity for E1 4-hydroxylation, exceeding its activity for E1 2-hydroxylation by approximately 100%. It will be of interest to determine the potential biological functions associated with any of the E1 metabolites identified in our present study.     

Chloramphenicol is a potent inhibitor of cytochrome P450 isoforms CYP2C19 and CYP3A4 in human liver microsomes

The inhibitory effect of chloramphenicol on human cytochrome P450 (CYP) isoforms was evaluated with human liver microsomes and cDNA-expressed CYPs. Chloramphenicol had a potent inhibitory effect on CYP2C19-catalyzed S-mephytoin 4′-hydroxylation and CYP3A4-catalyzed midazolam 1-hydroxylation, with apparent 50% inhibitory concentrations (inhibitory constant [K_i] values are shown in parentheses) of 32.0 (7.7) and 48.1 (10.6) μM, respectively. Chloramphenicol also weakly inhibited CYP2D6, with an apparent 50% inhibitory concentration (K_i) of 375.9 (75.8) μM. The mechanism of the drug interaction reported between chloramphenicol and phenytoin, which results in the elevation of plasma phenytoin concentrations, is clinically assumed to result from the inhibition of CYP2C9 by chloramphenicol. However, using human liver microsomes and cDNA-expressed CYPs, we showed this interaction arises from the inhibition of CYP2C19- not CYP2C9-catalyzed phenytoin metabolism. In conclusion, inhibition of CYP2C19 and CYP3A4 is the probable mechanism by which chloramphenicol decreases the clearance of coadministered drugs, which manifests as a drug interaction with chloramphenicol.     

Characterization of human cytochrome P450 isoenzymes involved in the metabolism of vinorelbine

Vinorelbine (VRL) (IV Navelbine) is a semi-synthetic vinca alkaloid, used in therapeutics for the treatment of non-small-cell lung cancer and advanced breast cancer. The aim of this study was to characterize the cytochrome P450 (CYP) isoenzymes involved in VRL metabolism. VRL was incubated at 1.28 x 10(-5) m for 90 min with human hepatic microsomes prepared from 14 donors (one woman and 13 men aged from 27 to 76 years old) and characterized for CYP1A2, CYP2D6, CYP2E1 and CYP3A4 activities. A four-combined-approach study was performed, including correlation between CYP activities and VRL metabolism among the 14 batches of microsomes, inhibition of VRL biotransformation by isoform-selective substrates and by specific inhibitory antibodies, and incubation with supersomes. Analysis of unchanged VRL and its metabolites was performed using an HPLC method coupled with both radioactive and UV detections. No correlation between CYP1A2 or CYP2E1 and VRL metabolism was observed in the 14 batches of microsomes used. A correlation was shown between VRL metabolism and CYP3A4 activity as determined with the dextromethorphan N-demethylase and nifedipine oxidase activities (r(2)=0.80 and 0.59, respectively). These results were strengthened by a correlation between the metabolism extent of VRL and CYP3A4 protein content determined by immunoblotting (r(2)=0.75). Furthermore, VRL biotransformation was inhibited by troleandomycine, the CYP3A4-specific inhibitor substrate (80% of inhibition) and by anti-CYP3A antibodies (36% of inhibition). On the contrary, a low correlation with CYP2D6 activity as determined by dextrometorphan O-demethylation (r(2)=0.31) was established. CYP2D6 supersomes did not metabolize the drug whereas 63.4% of VRL were metabolized by microsomes overexpressing CYP3A4 isoform. These data indicated that CYP3A4 is the main enzyme involved in the hepatic metabolism of VRL in human, whereas CYP2D6 is not involved.     

P-glycoprotein-mediated efflux of indinavir metabolites in Caco-2 cells expressing cytochrome P450 3A4.

The role of P-glycoprotein in secretion of indinavir metabolites produced by CYP3A4 was evaluated in Caco-2 cells expressing CYP3A4. Metabolism of indinavir by CYP3A4 expressing Caco-2 cells grown on filters resulted in the formation of N-dealkylation products (M5 and M6) and hydroxylation of indinavir, which were preferentially secreted into the apical compartment. Apical secretion of the metabolites was inhibited by cyclosporin A (CsA) with all three classes of metabolites showing similar sensitivity to CsA, suggesting that they are all secreted by the same pathway. M6 stimulated P-glycoprotein (Pgp)-ATPase activity in a concentration-dependent manner. This stimulation was inhibited by the Pgp-specific monoclonal antibody C219. A method was developed to specifically inhibit Pgp using the monoclonal antibody UIC2 to determine whether Pgp efflux accounts for a significant proportion of the apical secretion of indinavir metabolites. UIC2 recognizes an extracellular transient conformational epitope that is stabilized by some Pgp substrates or by ATP depletion. Incubation of Caco-2 cells with UIC2 in the presence of 1 microM CsA resulted in 50 to 80% inhibition of Pgp-mediated vinblastine efflux, with no significant inhibition observed by UIC2 or CsA alone. Inhibition of Pgp in CYP3A4-expressing Caco-2 cells by UIC2 and 1 microM CsA resulted in a significant decrease in the apical secretion of M6, M5, and OH-indinavir and an increase in the amount of the metabolites secreted in the basolateral compartment and retained in the cytosol. These results are consistent with a role of Pgp in elimination of CYP3A4-generated metabolites and indicate that even relatively polar metabolites may be secreted from the cell by Pgp.     

细胞色素P450酶系3A亚家族中CYP3A5的研究进展

细胞色素P450酶系(cytochrome P450 enzyme system,CYPs)是参与外源化合物解毒和内源化合物代谢的重要酶系,主要有CYP1、CYP2、CYP3三大家族。CYP3A5是CYP3A亚家族最主要的肝外分布形式,其在药物的相互作用中发挥至关重要的作用,并与肿瘤化疗耐药密切相关。对CYP3A5与耐药关系的研究,将为实施肿瘤个体化治疗提供理论基础,可能具一定的临床应用价值。本文从遗传、环境等多方面对目前的CYP3A5研究作一综述。     

Involvement of cytochrome P450 2C9, 2E1 and 3A4 in trimethadione N-demethylation in human microsomes

BACKGROUND AND OBJECTIVES: Trimethadione (TMO), an antiepileptic drug, may be used as a candidate for estimating hepatic drug-oxidizing activity. While TMO metabolism is mainly catalysed by CYP2C9, CYP2E1 and CYP3A4 the contribution of the different isoforms is unclear. In this study, we determined the percentage contribution of the three CYPs (CYP2C9, CYP2E1 and CYP3A4) to TMO N-demethylation. METHOD: We used human liver microsomes and human recombinant CYPs expressed in human B-lymphoblast cells and baculovirus-infected insect cells. RESULTS: The mean Km, Vmax and Vmax/Km values of TMO N-demethylation in human microsomes were 3.66 (mm), 503 (pmol/min/mg) and 2.61 (mL/h/mg), respectively. In the microsomes from human B-lymphoblast cells or baculovirus-infected insect cells, CYP 2C9, CYP 2E1 and CYP3A4 exhibited similar Km and higher Vmax in baculovirus-infected insect cells than B-lymphoblast cells. In baculovirus-infected insect cells, CYP2C9, CYP2E1 and CYP3A4 exhibited activities of 32, 286 and 77 pmol/min/pmol CYP, respectively. No CYP activity catalysed by CYP1A2 and 2D6 were detected in the two human cDNA expressed CYP isoforms. CONCLUSION: TMO is metabolized not only by CYP2E1 but also CYP3A4 and CYP2C9. The order of this metabolism is as follows: CYP2E1 > CYP3A4 > CYP2C9.     

Use of quinidine inhibition to define the role of the sparteine/debrisoquine cytochrome P450 in metoprolol oxidation by human liver microsomes

The oxidation of the beta adrenoceptor antagonist metoprolol exhibits genetic polymorphism of the sparteine/debrisoquine (SP/DB) type. The alpha-hydroxylation of metoprolol is absent in poor metabolizers, whereas metoprolol O-demethylation is only partially impaired, suggesting that an enzyme or enzymes other than cytochrome P450-SP/DB contribute to the latter reaction. Using inhibition by the quinidine/quinine isomer pair as a marker for the activity of cytochrome P450-SP/DB, the role of this enzyme in the in vitro oxidation of the enantiomers of metoprolol by human liver microsomes was examined. Unlike alpha-hydroxylation, only a portion of metoprolol O-demethylation showed the stereoselective inhibition by quinidine and quinine characteristic of in vitro reactions catalyzed by cytochrome P450-SP/DB. Furthermore, the kinetics of metoprolol O-demethylation were biphasic, the two components of O-demethylase activity being distinguishable by their enantioselectivity and sensitivity to inhibition by quinidine. Microsomes from one liver formed no detectable alpha-hydroxymetoprolol, and O-demethylation by these microsomes corresponded to the low affinity site observed in eight other livers. The rate of metoprolol O-demethylation by the quinidine-inhibitable high affinity component was directly proportional to the rate of alpha-hydroxylation. These findings support the hypothesis that cytochrome P450-SP/DB catalyzes the formation of alpha-hydroxymetoprolol, but is only partially responsible for metoprolol O-demethylation. Such a mechanism could explain the previously reported inability to detect polymorphism in the O-demethylation pathway in vivo.     

Effect of CYP3A5 expression on vincristine metabolism with human liver microsomes

Vincristine is preferentially metabolized to a secondary amine, M1, by CYP3A5 with a 9- to 14-fold higher intrinsic clearance than CYP3A4 using cDNA-expressed enzymes. The genetically polymorphic expression of CYP3A5 may contribute to interindividual variability in vincristine efficacy and toxicity. The current study quantifies the contribution of cytochromes P450 (P450s), including CYP3A4 and CYP3A5, to vincristine metabolism with a bank of human liver microsomes (HLMs). M1 was the major metabolite formed with HLMs, and selective chemical inhibition of P450s confirmed that CYP3A was the major metabolizing subfamily. The liver tissues were genotyped for low expression alleles, CYP3A5*3,*6, and *7, and the HLMs were phenotyped for CYP3A4 and CYP3A5 expression by Western blot. Testosterone 6beta-hydroxylation and itraconazole hydroxylation were used to quantify CYP3A4 activity in the HLMs. For each CYP3A5 high expresser (n=10), the rate of M1 formation from vincristine due to CYP3A5 was quantified by subtracting the CYP3A4 contribution as determined by linear regression with CYP3A5*3/*3 samples. For CYP3A5 high expressers, the contribution of CYP3A5 to the metabolism of vincristine was 54 to 95% of the total activity, and the rate of M1 formation mediated by CYP3A5 correlated with CYP3A5 protein content (r2=0.95). Selective inhibition of CYP3A4 demonstrated that the M1 formation rate with CYP3A5 high expressers was differentially inhibited based on CYP3A4 activity. Using median values, the estimated hepatic clearances were 5-fold higher for CYP3A5 high expressers than low expressers. We conclude that polymorphic expression of CYP3A5 may be a major determinant in the P450-mediated clearance of vincristine.     

Mechanism-based inactivation of cytochrome P450 3A4 by 17 alpha-ethynylestradiol: evidence for heme destruction and covalent binding to protein

17 alpha-Ethynylestradiol (EE), a major constituent of many oral contraceptives, inactivated the testosterone 6 beta-hydroxylation activity of purified P450 3A4 reconstituted with phospholipid and NADPH-cytochrome P450 reductase in a mechanism-based manner. The inactivation of P450 3A4 followed pseudo first order kinetics and was dependent on NADPH. The values for the K(I) and k(inact) were 18 microM and 0.04 min(-1), respectively, and the t(1/2) was 16 min. Incubation of 50 microM EE with P450 3A4 at 37 degrees C for 30 min resulted in a 67% loss of testosterone 6 beta-hydroxylation activity accompanied by a 35% loss of the spectral absorbance of the native protein at 415 nm and a 70% loss of the spectrally detectable P450-CO complex. The inactivation of P450 3A4 by EE was irreversible. Testosterone, an alternate substrate, was able to protect P450 3A4 from EE-dependent inactivation. The partition ratio was approximately 50. The stoichiometry of binding was approximately 1.3 nmol of an EE metabolite bound per nmol of P450 3A4 inactivated. SDS-polyacrylamide gel electrophoresis analysis demonstrated that [(3)H]EE was irreversibly bound to the P450 3A4 apoprotein. After extensive dialysis of the [(3)H]EE inactivated samples, high-pressure liquid chromatography (HPLC) analysis demonstrated that the inactivation resulting from EE metabolism led to the destruction of approximately half the heme with the concomitant generation of modified heme and EE-labeled heme fragments and produced covalently radiolabeled P450 3A4 apoprotein. Electrospray mass spectrometry demonstrated that the fraction corresponding to the major radiolabeled product of EE metabolism has a mass (M - H)(-) of 479 Da. HPLC and gas chromatography-mass spectometry analyses revealed that EE metabolism by P450 3A4 generated one major metabolite, 2-hydroxyethynylestradiol, and at least three additional metabolites. In conclusion, our results demonstrate that EE is an effective mechanism-based inactivator of P450 3A4 and that the mechanism of inactivation involves not only heme destruction, but also the irreversible modification of the apoprotein at the active site.     

Potent cytochrome P450 2C19 genotype-related interaction between voriconazole and the cytochrome P450 3A4 inhibitor ritonavir

OBJECTIVES: Cytochrome P450 (CYP) 2C19 and CYP3A4 are the major enzymes responsible for voriconazole elimination. Because the activity of CYP2C19 is under genetic control, the extent of inhibition with a CYP3A4 inhibitor was expected to be modulated by the CYP2C19 metabolizer status. This study thus assessed the effect of the potent CYP3A4 inhibitor ritonavir after short-term administration on voriconazole pharmacokinetics in extensive metabolizers (EMs) and poor metabolizers (PMs) of CYP2C19. METHODS: In a randomized, placebo-controlled crossover study, 20 healthy participants who were stratified according to CYP2C19 genotype received oral ritonavir (300 mg twice daily) or placebo for 2 days. Together with the first ritonavir or placebo dose, a single oral dose of 400 mg voriconazole was administered. Voriconazole was determined in plasma and urine by liquid chromatography-mass spectrometry, and pharmacokinetic parameters were estimated by noncompartmental analysis. RESULTS: When given alone, the apparent oral clearance of voriconazole after single oral dosing was 26%+/-16% (P > .05) lower in CYP2C19*1/*2 individuals and 66%+/-14% (P < .01) lower in CYP2C19 PMs. The addition of ritonavir caused a major reduction in voriconazole apparent oral clearance (354+/-173 mL/min versus 202+/-139 mL/min, P = .0001). This reduction occurred in all CYP2C19 genotypes (463+/-168 mL/min versus 305+/-112 mL/min [P = .023] for *1/*1, 343+/-127 mL/min versus 190+/-93 mL/min [P = .008] for *1/*2, and 158+/-54 mL/min versus 22+/-11 mL/min for *2/*2) and is probably caused by inhibition of CYP3A4-mediated voriconazole metabolism. CONCLUSIONS: Coadministration of a potent CYP3A4 inhibitor leads to a higher and prolonged exposure with voriconazole that might increase the risk of the development of adverse drug reactions on a short-term basis, particularly in CYP2C19 PM patients.     

Differential expression and activities of cytochrome P450 3A in the rat brain microsomes and mitochondria

Midazolam (MDZ), a benzodiazepine derivative, is metabolized to 1′- and 4-hydroxylated metabolites (1′-OH-MDZ and 4-OH-MDZ, respectively) by cytochrome P450 3A (CYP3A). The purpose of this study was to investigate the CYP3A-mediated hydroxylation of MDZ in the rat brain mitochondria (MT). Brain microsomes (MC) and MT fractions were prepared from rats (n = 8) using differential and density gradient centrifugations, and the purity of the fractions was evaluated using VDAC1 and calreticulin as markers of MT and MC, respectively. The formation rates of 1′-OH-MDZ and 4-OH-MDZ in the rat brain MC and MT samples were determined using an LC–MS/MS method after validation. Subsequently, Michaelis–Menten kinetics of 1′- and 4-hydroxylation of MDZ were estimated. Western blot (WB) analysis was used to determine the protein expression of CYP3A in the rat brain MC and MT. The MC fractions had 5.93% ± 3.01% mitochondrial impurity, and the MT fractions had 19.3% ± 7.8% microsomal impurity (mean ± SD). The maximum velocity (V_max) values of the formation of the hydroxylated metabolites in the brain MT were 2.4–9-fold higher than those in MC. Further, the V_max values of 4-OH-MDZ in both MC and MT fractions were substantially higher than those of 1′-OH-MDZ. The WB analysis showed that the intensity of the CYP3A immunoreactive band in MT was more than twofold higher than that in MC. It is concluded that compared with MC, rat brain MT contains substantial CYP3A, which may affect the pharmacology or toxicology of centrally acting xenobiotic and endogenous substrates of this enzyme.     

Metabolism of rifabutin and its 25-desacetyl metabolite, LM565, by human liver microsomes and recombinant human cytochrome P-450 3A4: relevance to clinical interaction with fluconazole.

Rifabutin and fluconazole are often given concomitantly as therapy to prevent opportunistic infections in individuals infected with the human immunodeficiency virus. Recent reports have shown increased levels of rifabutin and its 25-desacetyl metabolite, LM565, in plasma when rifabutin is administered with fluconazole. Since fluconazole is known to inhibit microsomal enzymes, this study was undertaken to determine if this rifabutin-fluconazole interaction was due to an inhibition of human hepatic enzymes. The metabolism of both rifabutin and LM565 was evaluated in human liver microsomes and recombinant human cytochrome P-450 (CYP) 3A4 in the presence of fluconazole and other probe drugs known to inhibit CYP groups 1A2, 2C9, 2D6, 2E1, and 3A. The concentrations of rifabutin (1 microg/ml), LM565 (1 microg/ml), and fluconazole (10 and 100 microg/ml) used were equal to those observed in plasma after the administration of rifabutin and fluconazole at clinically relevant doses. High-performance liquid chromatography was used to assess the metabolism of rifabutin and LM565. Rifabutin was readily metabolized to LM565 by human microsomes, but the reaction was independent of NADPH and was not affected by the P-450 inhibitors. No rifabutin metabolism by recombinant CYP 3A4 was found to occur. LM565 was also metabolized by human microsomes to two products, but metabolism was dependent on NADPH and was affected by certain P-450 inhibitors. In addition, LM565 was readily metabolized by the recombinant CYP 3A4 to the same two products found with its metabolism by human microsomes. Therefore, rifabutin is metabolized by human microsomes but not via cytochrome P-450 enzymes, whereas LM565 is metabolized by CYP 3A4.     

荧光多聚酶链反应测定细胞色素P450 3A5 1*3多态性

目的：建立荧光多聚酶链反应测定细胞色素P450 3A5 1＊3的多态性。方法：以TaqMan技术为基础,采用等位基因特异性引物,在引物的3′端第二位人为引入一个非配对碱基提高引物的分辨力。对PCR反应的Mg^2＋和探针浓度进行优化,同时对本方法的灵敏度和精密度进行了研究,应用直接DNA测序法对准确度进行评价。结果：优化后的Mg^2＋和探针浓度分别为3．5mmol／L和0．7μmol／L,批内（n=20）CV为1．41％,批间（n=20）CV为1．72％,在25μl反应体系中,样本DNA浓度5．0×10^2～5．0×10^6Pg范围内可获满意结果。本法确定的50份标本基因型与直接DNA测序法结果完全一致。结论：本法快速、灵敏、可靠,适用于细胞色素P450 3A5 1＊3多态性测定。     

Activation of human cytochrome P-450 3A4-catalyzed meloxicam 5'-methylhydroxylation by quinidine and hydroquinidine in vitro.

In humans, meloxicam is metabolized mainly by cytochrome P-450 (CYP)-dependent hydroxylation of the 5'-methyl group. The predominant P-450 enzyme involved in meloxicam metabolism is CYP 2C9, with a minor contribution of CYP 3A4. Quinidine, a CYP 3A4 substrate commonly used as a selective in vitro inhibitor of CYP 2D6, was found to markedly increase the rate of meloxicam hydroxylation during in vitro experiments with human liver microsomes. A similar activation was observed with other compounds that are structurally related to quinidine. Besides quinidine, quinine and hydroquinidine were the most potent activators of meloxicam hydroxylation. Using expressed cytochrome P-450 enzymes and selective chemical inhibitors of CYP 2C9 and CYP 3A4, it was found that quinidine markedly increased the rate of CYP 3A4-mediated meloxicam hydroxylation but was virtually without effect on CYP 2C9. Kinetic analysis was performed to obtain insight into the possible mechanism of activation of CYP 3A4 and into the mutual interaction of quinidine/hydroquinidine and meloxicam. Quinidine and hydroquinidine decreased Km and increased Vmax of meloxicam hydroxylation, which was consistent with a mixed-type nonessential activation. Meloxicam, in turn, decreased both Km and Vmax of quinidine metabolism by CYP 3A4, indicating an uncompetitive inhibition mechanism. These results support the assumption that CYP 3A4 possesses at least two different substrate-binding sites. A clinically relevant effect on meloxicam drug therapy is not expected, because the most likely outcome in practice is moderately decreased meloxicam plasma concentrations.     

Human cytochrome P450 metabolism of teniposide and etoposide.

Although teniposide (VM26) and etoposide (VP16) are eliminated mostly by nonrenal mechanisms, their cytochrome P450 metabolism in humans has not been reported. Our objective was to determine the affinity and capacity of P450 O-demethylation of VM26 and VP16 in a variety of human livers. Formation of catechols of VM26 and VP16 was detected in 24 and 26 of 26 liver microsomal preparations, respectively, with wide variability in maximum catechol formation rates from VM26 (41-fold) and VP16 (39-fold range), even among normal livers. Maximal activity measurements at 500 microM substrate were lower for VM26 catechol formation (mean = 1.5 nmol/mg/hr) than for VP16 catechol (mean = 3.2 nmol/mg/hr) in 26 livers (P less than .001). Maximal activities for VP16 and VM26 O-demethylation, and ethoxycoumarin O-deethylation were significantly higher in normal than in diseased livers. No differences were found in activities related to age, sex or race of the liver donor. In all five livers tested over a range of substrate concentrations, Km (19.7, 23.2, 43.5, 30.1 and 22.0 microM) and Vmax values (1.0, 1.2, 4.4, 8.2 and 4.0 nmol/mg/hr) for VM26 were lower compared to values for VP16 (Km = 60.2, 115.1, 87.3, 42.1 and 81.9 microM; Vmax = 1.4, 3.3, 10.3, 27.5 and 10.1 nmol/mg/hr). Despite higher VP16 catechol formation, VM26 underwent greater overall reduced nicotinamide adenine dinucleotide phosphate-dependent metabolism than VP16, consistent with greater nonrenal clearance of VM26 in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genotype-phenotype associations of cytochrome P450 3A4 and 3A5 polymorphism with midazolam clearance in vivo

The molecular basis for the wide interindividual variability of cytochrome P450 (CYP) 3A metabolic activity was studied in vivo at a genetic level. A single oral dose of midazolam was administered to 26 healthy subjects. The variability in midazolam oral clearance was 11-fold. No differences in midazolam oral clearance related to gender or ethnicity were observed. Selective sequencing of CYP3A4 and CYP3A5 genes revealed 18 single nucleotide polymorphisms (SNPs), including 8 novel CYP3A4 SNPs. Thirteen novel CYP3A4 haplotypes, 2 novel CYP3A5 haplotypes, and 1 major novel multigene haplotype ( CYP3A4*VI - CYP3A5*3A ) were also identified. No significant genotype-phenotype or haplotype-phenotype associations were found for any of the SNPs or haplotypes studied, including CYP3A4*1B , CYP3A5*3 , and CYP3A5*6 , even when ethnicity was considered. The only exceptions were the haplotype CYP3A4*VI and the multigene haplotype CYP3A4*VI - CYP3A5*3A . The carriers of the haplotype CYP3A4*VI had a 1.8-fold higher clearance of midazolam in black subjects (ANOVA on ranks, P = .028) compared with other individuals, and the carriers of the multigene haplotype CYP3A4*VI - CYP3A5*3A had a 1.7-fold higher clearance in the entire population (ANOVA on ranks, P = .012). In conclusion, these results indicate that the genetic variants identified so far in the CYP3A4 and CYP3A5 genes have only a limited impact on CYP3A-mediated drug metabolism in vivo.     

Modulation of P450 CYP3A4-dependent metabolism by P-glycoprotein: implications for P450 phenotyping

Some compounds used for phenotyping of cytochrome P450s are substrates of P-glycoprotein (pgp). It is likely that in these cases, the level of pgp modulates the metabolism of in vivo probes. To address this important issue, we have analyzed the effects of pgp on CYP3A4-mediated reactions in two newly established cell lines (3A4/HR/MDR(-) and 3A4/HR/MDR(+)), which express CYP3A4 in the absence and presence of pgp, respectively. In cultured cells, the presence of pgp increased the apparent K(m) for the 6beta-hydroxylase activity of CYP3A4 toward testosterone and cortisol by a factor of 1.7 and 4, respectively. These steroids are poor and good substrates of pgp, respectively, and cortisol 6beta-hydroxylase has been frequently used as an in vivo probe for CYP3A4. Interestingly, we also found that pgp modulated the inhibition of CYP3A4-mediated metabolism by several compounds in intact cells. Although quinidine inhibited testosterone 6beta-hydroxylase activity in membranes or in intact cells that expressed recombinant CYP3A4 in the absence of pgp, low concentrations of this compound increased CYP3A4 activity in intact cells that expressed pgp. These results imply that pharmacokinetic drug-drug interactions involving CYP3A4 can be influenced by pgp.