Metabolism of territrem B and C in liver microsomes from 14-wk-old Wistar rats is catalyzed by cytochrome P-450 3A

The metabolism of territrem B (TRB) and territrem C (TRC) in liver microsomes of 14-wk-old male and female Wistar rats was investigated. Metabolism of TRB to 4beta-hydroxylmethyl-4beta-demethylterritrem B (MB2), O-demethylation of the methoxy group of the aromatic moiety of TRB to form MB4 (same structure as TRC), and metabolism of TRC to 4beta-hydroxylmethyl-4beta-demethylterritrem C (MC) were observed in both genders. However, the amounts of MB2, MB4, and MC formed in females were much lower than in males. To investigate which cytochrome P-450 (CYP450) isoforms were involved in each step, four CYP450 isotype-specific inhibitors (furafylline, orphenadrine, cimetidine, and troleandomycin) and antibodies against CYP1A1, CYP2B1, CYP2C11, or CYP3A2 were used. Formation of MB2, MB4, and MC was markedly inhibited by cimetidine and troleandomycin, but less by furafylline and orphenadrine. Anti-CYP3A2 antibody completely inhibited MB, MB, and MC formation, while antibodies against CYP1A1, CYP2B1, or CYP2C11 produced no marked effect. Of the seven tested supersomes from baculovirus-transformed insect cells expressing rat CYP450 isoforms (1Al, 1A2, 2B1, 2C11, 2C12, 3A1, and 3A2), only those expressing CYP3A1 and CYP3A2 metabolized TRB and TRC. The amounts of MB2, MB4, and MC formed by male and female rat liver microsome preparations were related to the testosterone 6beta-hydroxylase activity and CYP3A1/2 protein content of the preparation. Immunoblotting showed that CYP3A1 was expressed in both genders, but at different levels, while CYP3A2 was only expressed in males. These results suggest that the formation of MB2, MB4, and MC in liver microsomes from 14-wk-old rats of either gender is mediated by CYP3A1 and CYP3A2.     

Role of human hepatic cytochrome P-450s in territrem A metabolism

The ability of human liver microsomal preparations (HLM1, 2, 3, and 5), microsomes from human lymphoblasts expressing different cytochrome P-450 (CYP450) isoforms, and CYP3A4 cDNA-transfected V79 Chinese hamster cells to metabolize territrem A (TRA) was studied. The only metabolite generated by any of these preparations was 6beta-hydroxymethyl-6beta-demethylterritrem A (MA(1)). MA(1) formation was observed with all four human liver microsomal samples. Of the eight microsomal preparations from human lymphoblasts expressing different cytochrome P-450 enzymes (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) examined, only those expressing CYP2C9, CYP2D6, or CYP3A4 metabolized TRA, with that expressing CYP3A4 being the most active. No TRA metabolites were formed by control V79MZ cells, but MA(1) was formed by CYP3A4 cDNA-transfected V79 Chinese hamster cells. In order to investigate which CYP450 isoforms were involved in MA(1) formation in the human liver microsomal preparations, the effects of six isoform-specific chemical inhibitors (furafylline, sulfaphenazole, omeprazole, quinidine, ketaconazole, and diethyldithiocarbamate) and anti-3A4, anti-2C9, and anti-2D6 antibodies on TRA metabolism by HLM2 and HLM5 were examined. MA(1) formation was markedly inhibited by ketaconazole, with quinidine and sulfaphenazole having less of an effect. Anti-CYP3A4 antibody markedly inhibited MA(1) formation, while antibodies against CYP2C9 or CYP2D6 had little effect. The amount of MA(1) formed using different HLM preparations was related to the 6beta-testosterone hydroxylase activity and CYP3A4 protein content of the preparations. These results suggest that CYP3A4 is the major enzyme involved in TRA metabolism by human liver microsomes, with CYP2C9 and CYP2D6 playing a minor role.     

Predicting the contribution of rat cytochrome P-450 3A1, 3A2 and human cytochrome P-450 3A4, 3A5 to territrem a 4beta-C hydroxylation using the relative activity factor

The relative activity factor (RAF) was used to predict the contribution of different cytochrome P-450 (CYP) 3A isoforms (3A1 and 3A2 in rat liver microsomes and 3A4 and 3A5 in human liver microsomes) to 4beta-C hydroxylation of territrem A (TRA). Seven recombinant rat and eight recombinant human CYP450 isoforms, five rat liver microsomes, and seven human liver microsomes were assessed. In liver microsomes from five male Wistar rats, TRA 4beta-C hydroxylation activity significantly correlated with CYP3A1/2 activity, while, in liver microsomes from seven humans, there was marked correlation with CYP3A4 activity. Immunoinhibition confirmed that CYP3A2 and CYP3A4 were responsible for the hepatic metabolism of TRA 4beta-C hydroxylation. Using RAF, the percent contributions of CYP3A1 and CYP3A2 to 4beta-C hydroxylation of TRA in rat liver microsomes were estimated as 5 to 6 and 94 to 96, respectively, and those of CYP3A4 and CYP3A5 in human liver microsomes as 70 to 72 and 28 to 30%, respectively. These results suggest that CYP3A2 and CYP3A4 are the main form involved in the 4beta-C hydroxylation of TRA in rat and human liver microsomes.     

Profile of territrem metabolism and cytochrome P-450 3A expression in liver microsomes from Wistar rats of both genders as a function of age

This study determined territrem metabolites after incubation of territrem A, B, or C with NADPH and liver microsomes from Wistar rat of both genders aged 2 to 76 wk. The liver microsomal cytochrome P-450 content, NADPH-cytochrome P-450 reductase activity, and CYP3A1 and CYP3A2 protein and mRNA levels were also analyzed. Male rats had significantly higher liver microsomal cytochrome P-450 content and NADPH-cytochrome P-450 reductase activities than females at 14 to 26 wk. Microsomal cytochrome P-450 content was decreased in senescence in both genders compared with postpubertal and adulthood stages. The activity of 6beta-testosterone hydroxylase in male rats, which was significantly higher than those in females at all ages, decreased after 52 wk. After 26 wk, the levels of CYP3A1 protein markely declined in both genders, which resulted in a large gender difference (male greater than female). The protein levels and mRNA of CYP3A2 were constitutively expressed in 2- to 52-wk-old male rats, but they decreased after 76 wk, and decreased in females after 6 wk. The expression of CYP3A1 or CYP3A2 in males are generally higher than in females. The metabolites of territrems MA1, MAX, MA2, MB2, MB4, and MC were measured by high-performance chromatography (HPLC). Formation of MA1, MAX, and MA2 decreased after 52 wk in males, and MAX and MA2 were not formed after 6 wk in females. The amount of MB2 formed in females was less than in males, but the amount of MC (TRC metabolites) formed in females was higher than in males. The gender differences in metabolism of TRA were related to the protein and mRNA expression of CYP3A2. The protein levels and mRNA expression of CYP3A2 and efficiency of territrems metabolism were decreased after 76 wk. The results suggested that the effects of age and gender on territrem metabolism are due to differences in CYP3A1 and CYP3A2 expression in the liver microsomes.     

Biotransformation of territrems by S9 fraction from rat liver.

Male Wistar rats were pretreated with phenobarbital, 3-methyl-cholanthrene, or polychlorinated biphenyl. The S9 fraction was isolated from their livers. An amount of 40-microliters territrem (TRA, B, or C) (1 mg/ml methanol) was added to 5-ml reaction mixtures containing S9 (8 mg protein), NADP sodium salt (20 mumol), glucose-6-phosphate monosodium salt (25 mumol), MgCl2 (40 mumol), KCl (165 mumol), and sodium phosphate buffer, 0.1 M, pH 7.4, after preincubation for 5 min. Further incubation was carried out for 30 min by shaking (100 ocillations/min). The reaction was stopped by adding 2 ml acetone. The acetone was then removed by evaporation in a hood at room temperature. The residue was lyophilized and extracted with 2 ml methanol 3 times. When TRB was a substrate, at least four blue fluorescent products, designated as MB1, MB2, MB3, and MB4, were found in the methanol extract by TLC under view of long-wave UV light. MB2 was the major product. When TRA or TRC was a substrate, two products, MA1 (the major product) and MA2 from TRA, and one product, MC from TRC, were, respectively, detected in the methanol extract. The formation of the products was dependent on the presence of S9, NADP, glucose-6-phosphate, and territrem. The reaction was enhanced by pretreatment of rats with phenobarbital. It was demonstrated that MB2 and MA1 are hydroxylated products of the methyl group at the C4 position of TRB and TRA. MB4 was identified as TRC. MC was shown to be identical to MB1, which was the hydroxylated product at the methyl group of C4 position of TRC.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro study on the involvement of CYP1A2, CYP2D6 and CYP3A4 in the metabolism of haloperidol and reduced haloperidol

Objective: To investigate in vitro which CYP isoforms (CYP1A2, CYP2D6 and CYP3A4) are involved in the biotransformation of haloperidol (HAL) and reduced haloperidol (RHAL). Methods: The biotransformation of HAL and RHAL is evaluated by measuring HAL and RHAL remaining after incubation with human liver microsomes and with supersomes from human baculovirus-infected cells expressing human P ₄₅₀ isoforms. The influence of chemical- and immuno-inhibition of specific isoforms on the disappearance of HAL and RHAL was also studied. Results: After 60-min incubation of 2 μM and 20 μM HAL or RHAL with human liver microsomes, for HAL, 58% and 64%, respectively, remained in the incubation mixture, for RHAL, 53% and 66%, respectively. Ketoconazole had the most pronounced inhibitory effect on the biotransformation of both substrates, while for quinidine and furafylline there was only a weak or no influence. Anti-CYP3A4 antibodies inhibited strongly the biotransformation of HAL and RHAL, while the influence of anti-CYP2D6 antibodies was much less pronounced. After incubation with supersomes of recombinant CYP3A4, HAL and RHAL disappeared rapidly; disappearance was slow after incubation with CYP2D6 supersomes, and negligible with CYP1A2 supersomes. Conclusion: The results show that CYP3A4 is the most important CYP isoenzyme involved in the biotransformation of HAL and RHAL, and that the metabolism by CYP2D6 is only a minor pathway; CYP1A2 has no or only a negligible influence. Received: 12 April 1999 / Accepted in revised form: 2 August 1999     

Involvement of CYP1A2 and CYP3A4 in lidocaine N-deethylation and 3-hydroxylation in humans.

The roles of cytochrome P-450 (CYP) enzymes in the N-deethylation, i.e., formation of monoethylglycinexylidide (MEGX), and 3-hydroxylation of lidocaine were studied with human liver microsomes and recombinant human CYP isoforms. Both CYP1A2 and CYP3A4 were found to be capable of catalyzing the formation of MEGX and 3-OH-lidocaine. Lidocaine N-deethylation by liver microsomes was strongly inhibited by furafylline (by about 60%) and anti-CYP1A1/2 antibodies (>75%) at 5 microM lidocaine, suggesting that CYP1A2 was the major isoform catalyzing lidocaine N-deethylation at low (therapeutically relevant) lidocaine concentrations. Troleandomycin inhibited the N-deethylation of lidocaine by about 50% at 800 microM lidocaine, suggesting that the role of CYP3A4 may be more important than that of CYP1A2 at high lidocaine concentrations. Chemical inhibition and immunoinhibition studies also indicated that 3-OH-lidocaine formation was catalyzed almost exclusively by CYP1A2, CYP3A4 playing only a minor role. Although the CYP2C9 inhibitor sulfaphenazole (100 microM) inhibited MEGX formation by about 30%, recombinant human CYP2C9 showed very low catalytic activity, suggesting a negligible role for this enzyme in lidocaine N-deethylation. Chemical inhibition studies indicated that CYP2C19, CYP2D6, and CYP2E1 did not play significant roles in the metabolism of lidocaine in vitro. Taken together, these results demonstrate that CYP1A2 and CYP3A4 enzymes are the major CYP isoforms involved in lidocaine N-deethylation. Therefore, the MEGX test (formation of MEGX from lidocaine) is not a suitable marker of hepatic CYP3A4 activity in vivo.     

The influence of CYP2B6, CYP2C9 and CYP2D6 genotypes on the formation of the potent antioestrogen Z-4-hydroxy-tamoxifen in human liver

AIMS: To investigate in a large panel of 50 human liver samples the contribution of CYP2C9, CYP2D6, and CYP3A4 to the overall formation of the potent antioestrogen Z-4-hydroxy-tamoxifen, and how various genotypes affect its formation from tamoxifen. METHODS: The formation of Z-4-hydroxy-tamoxifen from 10 microm tamoxifen was studied in human liver microsomes (n=50), characterized for CYP2B6, CYP2C9, CYP2D6 and CYP3A4 expression, and CYP2B6, CYP2C9 and CYP2D6 genotype. The effect of chemical and monoclonal antibody inhibitors, and the formation in supersomes expressing recombinant CYP isoforms was also investigated. Z-4-hydroxy-tamoxifen was quantified using LC-MS analysis. RESULTS: Z-4-hydroxy-tamoxifen was formed by supersomes expressing CYP2B6, CYP2C9, CYP2C19 and CYP2D6, but not CYP3A4. In agreement with these data, the mean formation of Z-4-hydroxy-tamoxifen was inhibited 49% by sulphaphenazole (P=0.001), 38% by quinidine (P<0.05) and 13% by monoclonal antibody against CYP2B6 (MAB-2B6, P<0.05). Furthermore, Z-4-hydroxy-tamoxifen formation significantly correlated with both CYP2C9 expression (r(s)=0.256, P<0.05) and CYP2D6 expression (r(s)=0.309, P<0.05). Genotypes of CYP2D6, CYP2B6 and CYP2C9 had an effect on metabolite formation in such a way that samples with two nonfunctional CYP2D6, or two variant CYP2C9 or CYP2B6 alleles, showed lower enzyme activity compared with those with two functional or wild-type alleles, (5.0 vs 9.9 pmol mg(-1) protein min(-1), P=0.046, 5.1 vs 9.9 pmol mg(-1) protein min(-1), P=0.053, and 6.8 vs 9.4 pmol mg(-1) protein min(-1), P=0.054, respectively). CYP2D6 and CYP2C9 contribute on average 45 and 46%, respectively, to the overall formation of Z-4-hydroxy-tamoxifen. CONCLUSIONS: CYP2B6, CYP2C9 and CYP2D6 genotypes all affected Z-4-hydroxy-tamoxifen formation and can predict individual ability to catalyse this reaction.     

Cytochrome P-4503A1 catalyzes the formation of MA1 from territrem a in liver microsomes of 7-week-old female Wistar rats

Liver microsomal territrem A (TRA) metabolism was studied in 7-wk-old female Wistar rats. Pretreatment with phenobarbital (PB) or dexamethasone (DEX) resulted in a significant increase in 4 beta-hydroxymethyl-4 beta-demethylterritrem A (MA1) production. SKF 525A (0.025 and 0.05 mM), a general cytochrome P-450 (CYP450) inhibitor, blocked MA1 formation in liver microsomes from PB-pretreated female rats. Anti-CYP2B antibody had no marked effect on MA1 formation, although orphenadrine (0.5 mM), which inhibits CYP2B, blocked MA1 formation in liver microsomes from PB-treated female rats. An immunoinhibition study showed that anti-CYP3A2 antibody reduced MA1 formation to nondetectable levels in liver microsomes from PB-treated female rats. Furthermore, immunoblotting showed that CYP3A1 protein was expressed in 7-wk-old female rat and only MA1 was formed from TRA using supersomes from CYP3A1-expressing baculovirus-infected insect cells. Further, Western blot analysis indicated that CYP3A2 protein was expressed in 2-wk-old rats of both sexes and 7-wk-old male rats, and 3 metabolites of TRA, such as MA1, MAX, and MA2, were formed using supersomes from CYP3A2-expressing baculovirus-infected insect cells. These results suggest that MA1 formation in liver microsomes of 7-wk-old female Wistar rats is mediated by CYP3A1.     

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.     

The role of CYP3A4 in amiodarone-associated toxicity on HepG2 cells

Amiodarone is a class III antiarrhythmic drug with potentially life-threatening hepatotoxicity. Recent in vitro investigations suggested that the mono-N-desethyl (MDEA) and di-N-desethyl (DDEA) metabolites may cause amiodarone's hepatotoxicity. Since cytochrome P450 (CYP) 3A4 is responsible for amiodarone N-deethylation, CYP3A4 induction may represent a risk factor. Our aim was therefore to investigate the role of CYP3A4 in amiodarone-associated hepatotoxicity. First, we showed that 50 μM amiodarone is more toxic to primary human hepatocytes after CYP induction with rifampicin. Second, we overexpressed human CYP3A4 in HepG2 cells (HepG2 cells/CYP3A4) for studying the interaction between CYP3A4 and amiodarone in more detail. We also used HepG2 wild type cells (HepG2 cells/wt) co-incubated with human CYP3A4 supersomes for amiodarone activation (HepG2 cells/CYP3A4 supersomes). Amiodarone (10–50 μM) was cytotoxic for HepG2 cells/CYP3A4 or HepG2 cells/CYP3A4 supersomes, but not for HepG2 cells/wt or less toxic for HepG2 cells/wt incubated with control supersomes without CYP3A4. Co-incubation with ketoconazole, attenuated cytotoxicity of amiodarone incubated with HepG2 cells/CYP3A4 or HepG2 cells/CYP3A4 supersomes. MDEA and DDEA were formed only in incubations containing HepG2 cells/CYP3A4 or HepG2 cells/CYP3A4 supersomes but not by HepG2 cells/wt or HepG2 cells/wt with control supersomes. Metabolized amiodarone triggered the production of reactive oxygen species, induced mitochondrial damage and cytochrome c release, and promoted apoptosis/necrosis in HepG2 cells/CYP3A4, but not HepG2 cells/wt. This study supports the hypothesis that a high CYP3A4 activity is a risk factor for amiodarone's hepatotoxicity. Since CYP3A4 inducers are used frequently and amiodarone-associated hepatotoxicity can be fatal, our observations may be clinically relevant.     

Evidence for involvement of human CYP3A in the 3-hydroxylation of quinine

Aims Our previous studies using in vitro hepatic microsomal preparations suggested that the hepatic metabolism of quinine to form the major metabolite 3-hydroxyquinine is most likely catalysed by human P450 3A (CYP3A). The present study was carried out to investigate the kinetics and to identify and further characterise the human liver CYP isoforms involved in the metabolism of quinine.
Methods In vitro human microsomal techniques were employed.
Results The mean apparent K _m value for 3-hydroxyquinine formation was 83±19 (s.d.)  μm, ranging from 57  μm to 123  μm in microsomes from ten human livers. There was a 6.7-fold variation in V _max values (mean 547±416  pmol min⁻¹  mg⁻¹ ). Quinine 3-hydroxylation was inhibited by the specific CYP3A inhibitors, troleandomycin, midazolam and erythromycin. Inhibitors selective for CYP1A1/2, CYP2D6, CYP2E1, CYP2C9/10 or CYP2C19 had little or no effect on quinine 3-hydroxylation. Using microsomes from a panel of livers, significant correlations were found only between 3-hydroxyquinine activity and other CYP3A activities (caffeine 8-oxidation, omeprazole sulphoxidation, midazolam 1'-hydroxylation and midazolam 4-hydroxylation) and immunoreactive CYP3A content. There were no statistically significant correlations with activities selective for CYP1A2, CYP2C9 and CYP2E1. Competitive inhibition of quinine 3-hydroxylation was observed with a substrate known to be specifically metabolized by human CYP3A, i.e. midazolam, with an apparent K _i value of 11.0  μm.
Conclusions The present results strongly indicate that the conversion of quinine to 3-hydroxyquinine is the major metabolic pathway in human liver in vitro and that the reaction is catalysed by CYP3A isoforms.     

Relative contributions of CYP2C9 and 2C19 to phenytoin 4-hydroxylation in vitro: inhibition by sulfaphenazole, omeprazole, and ticlopidine

OBJECTIVES: To determine the relative contribution of cytochromes P450 (CYP) 2C9 and 2C19 to the formation of 5-(-4-hydroxyphenyl)-5-phenylhydantion (HPPH) from phenytoin (PPH). DESIGN: Hydroxylation of PPH to form HPPH was studied in vitro using human liver microsomes and microsomes from cDNA-transfected human lymphoblastoid cells. RESULTS: Formation of HPPH from PPH in liver microsomes had a mean (+/- SEM) apparent Km [substrate concentration corresponding to 50% of maximal reaction velocity (Vmax)] of 23.6 +/- 1.8 mumol/l. Coincubation with the CYP2C9 inhibitor, sulfaphenazole (SPA), at 5 mumol/l reduced reaction velocity to less than 15% of control values. The mean inhibitor concentration at which 50% inhibition is achieved (IC50 value) for SPA versus PPH hydroxylation (0.49 microM) was similar to the SPA IC50 versus flurbiprofen hydroxylation (0.46 microM) and tolbutamide hydroxylation (0.7-1.5 microM). In contrast, the CYP2C19 inhibitor omeprazole (OME) at 10 mumol/l produced only a small degree of inhibition. Incubation of PPH with microsomes from cDNA-transfected human lymphoblastoid cells containing CYP1A2, 2A6, 2B6, 2C8, 2D6, 2E1, or 3A4 yielded no detectable formation of HPPH. Only CYP2C9 and 2C19 had PPH hydroxylation activity, with apparent Km values for the high-affinity component of 14.6 mumol/l and 24.1 mumol/l, respectively. Based on Vmax values in liver microsomes, the Vmax and Km values in expressed CYPs and the relative abundance of the two isoforms in human liver, CYP2C9, and 2C19 were estimated to have relative contributions of 90% and 10%, respectively, to net intrinsic clearance. CONCLUSIONS: Formation of HPPH from PPH is mediated exclusively by CYP2C9 and 2C19, with CYP2C9 playing the major role.     

Metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin by human hepatic CYP isoforms: evidence for selectivity towards CYP3A4

1. The metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BFBFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomes and in cDNA-expressed human liver CYP isoforms. For purposes of comparison, some limited studies were also performed with 7-benzyloxyquinoline (7BQ). 2. Initial interactive docking studies with a homology model of human CYP3A4 indicated that BFBFC was likely to be a selective substrate for CYP3A4 with a relatively high binding affinity, due to the presence of several key hydrogen bonds with active site amino acid residues. 3. Kinetic analysis of NADPH-dependent BFBFC metabolism to HFC in three preparations of pooled human liver microsomes revealed mean (+/- TSEM) Km and Vmax = 4.6 +/- 0.3 microM and 20.0 +/- 3.8 pmol/min/mg protein, respectively. 4. The metabolism of BFBFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing a BFBFC substrate concentration of lO microM (i.e. around twice Km). Good correlations (r2 = 0.736-0.904) were observed between BFBFC metabolism and markers of CYP3A isoforms. 5. While 10O microM BFBFC was metabolized to HFC by cDNA-expressed CYP3A4, little or no metabolism was observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 6. The metabolism of 10 microM BFBFC in human liver microsomes was markedly inhibited by 5-50 microM troleandomycin and 0.2-5 microM ketoconazole, but stimulated by 0.2-10 microM alpha-naphthoflavone. The metabolism of 10 microM BFBFC in human liver microsomes was also markedly inhibited by an antibody to CYP3A4. 7. Kinetic analysis of NADPH-dependent 7BQ metabolism to 7-hydroxyquinoline (7HQ) in human liver microsomes revealed Km and Vmax = 70 microM and 3.39 nmol/min/mg protein, respectively. 8. While 80 microM 7BQ was metabolized to 7HQ by cDNA-expressed CYP3A4, only low rates of metabolism were observed with cDNA-expressed CYPIA2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 9. In summary, by correlation analysis, the use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFBFC metabolism in human liver microsomes appears to be primarily catalysed by CYP3A4. BFBFC may be a useful fluorescent probe substrate for human hepatic CYP3A4, but compared with 7BQ has only a low rate of metabolism in human liver microsomes.     

Comparison of (S)-mephenytoin and proguanil oxidation in vitro: contribution of several CYP isoforms

AIMS: To compare the oxidative metabolism of (S)-mephenytoin and proguanil in vitro and to determine the involvement of various cytochrome P450 isoforms. METHODS: The kinetics of the formation of 4'-hydroxymephenytoin and cycloguanil in human liver microsomes from 10 liver samples were determined, and inhibition of formation was studied using specific chemical inhibitors and monoclonal antibodies directed towards specific CYP450 isoforms. Expressed CYP450 enzymes were used to characterize further CYP isoform contribution in vitro. Livers were genotyped for CYP2C19 using PCR amplification of genomic DNA followed by restriction endonuclease digestion. RESULTS: All livers were wildtype with respect to CYP2C19, except HLS#5 whose genotype was CYP2C19*1/CYP2C19*2. The Km, Vmax and CLint values for the formation of 4'-hydroxymephenytoin from (S)-mephenytoin and the formation of cycloguanil from proguanil ranged from 50.8 to 51.6 and 43-380 microm, 1.0-13.9 and 0.5-2.5 nmol mg-1 h-1, and 20.2-273.8 and 2.7-38.9 microl h-1 mg-1, respectively. There was a significant association between the Vmax values of cycloguanil and 4'-hydroxymephenytoin formation (rs=0.95, P=0.0004). Cycloguanil formation was inhibited significantly by omeprazole (CYP2C19/3A), troleandomycin (CYP3A), diethyldithiocarbamate (CYP2E1/3A), furafylline (CYP1A2), and (S)-mephenytoin. 4'-Hydroxymephenytoin formation was inhibited significantly by omeprazole, diethyldithiocarbamate, proguanil, furafylline, diazepam, troleandomycin, and sulphaphenazole (CYP2C9). Human CYP2E1 and CYP3A4 monoclonal antibodies did not inhibit the formation of cycloguanil or 4'-hydroxymephenytoin, and cycloguanil was formed by expressed CYP3A4 and CYP2C19 supersomes. However, only expressed CYP2C19 and CYP2C19 supersomes formed 4'-hydroxymephenytoin. CONCLUSIONS: The oxidative metabolism of (S)-mephenytoin and proguanil in vitro is catalysed by CYPs 2C19 and 1A2, with the significant association between Vmax values suggesting that the predominant enzymes involved in both reactions are similar. However the degree of selectively of both drugs for CYP isoforms needs further investigation, particularly the involvement of CYP3A4 in the metabolism of proguanil. We assert that proguanil may not be a suitable alternative to (S)-mephenytoin as a probe drug for the CYP2C19 genetic polymorphism.     

In vitro proguanil activation to cycloguanil is mediated by CYP2C19 and CYP3A4 in adult Chinese liver microsomes

AIM: To identify the cytochrome P450 isoforms involved in proguanil (PG) activation to cycloguanil (CG) in Chinese liver microsomes. METHODS: The kinetics of the CG formation from PG was determined in the liver microsomes of 6 Chinese subjects. Selective chemical inhibitors to various cytochrome P450 isoforms were employed to conduct inhibition experiments. The relationship between the CG formation and S-mephenytoin 4'-hydroxylation was analyzed. RESULTS: The kinetic behaviors of CG formation were described well by a single-enzyme Michaelis-Menten equation in five livers. The apparent Km and Vmax were (82 +/- 47) mumol.L-1 and (8 +/- 6) pmol.min-1.mg-1 protein, respectively. However, the remaining one displayed a two-enzyme kinetic behavior. Inhibition experiments showed that troleandomycin (100 mumol.L-1) and diethyldithiocarbamate (100 mumol.L-1), as potent CYP3A4 and CYP2E1 inhibitors, respectively, reduced the formation rate of CG by 81.1% and 47.23%, while quinidine (10 mumol.L-1), furafylline (20 mumol.L-1), and sulfaphenazole (10 mumol.L-1), which were inhibitors towards CYP2D6, 1A2 and 2C9/10, respectively, did not display significant inhibition. At a low PG concentration of 5 mumol.L-1, the CG formation correlated well with S-mephenytoin 4'-hydroxylation (r = 0.805, P < 0.05). Nevertheless, when a high substrate concentration (500 mumol.L-1) was used, the correlation coefficient decreased (r = 0.581, P < 0.05). CONCLUSION: The present study indicates that CYP3A4 and CYP2C19 are involved in PG activation to CG in adult Chinese liver microsomes. CYP2C19 played an important role in the clearance of PG at a substrate concentration close to in vivo therapeutic concentrations, while CYP3A4 gradually made a dominant contribution with the increase of PG concentration.     

Role of human liver microsomal CYP3A4 and CYP2B6 in catalyzing N-dechloroethylation of cyclophosphamide and ifosfamide

The anticancer alkylating agents cyclophosphamide (CPA) and ifosfamide (IFA) are prodrugs that undergo extensive P450-catalyzed metabolism to yield both active (4-hydroxylated) and therapeutically inactive but neurotoxic (N-dechloroethylated) metabolites. Whereas the human liver microsomal P450 catalysts of CPA and IFA 4-hydroxylation are well characterized, the P450 enzyme catalysts of the alternative N-dechloroethylation pathway are poorly defined. Analysis of a panel of fifteen human P450 cDNAs in the baculovirus expression system ('Supersomes') demonstrated that CYP3A4 exhibited the highest N-dechloroethylation activity toward both CPA and IFA, whereas CYP2B6 displayed high N-dechloroethylation activity toward IFA, but not CPA. The contributions of each human P450 to overall liver microsomal N-dechloroethylation were calculated using a recently described relative substrate-activity factor method, and were found to be in excellent agreement with the results of inhibition studies using the CYP3A inhibitor troleandomycin and an inhibitory monoclonal antibody to CYP2B6. With CPA as substrate, CYP3A4 was shown to catalyze >/=95% of liver microsomal N-dechloroethylation, whereas with IFA as substrate, CYP3A4 catalyzed an average of approximately 70% of liver microsomal N-dechloroethylation (range = 40-90%), with the balance of this activity catalyzed by CYP2B6 (range = 10-70%, dependent on the CYP2B6 content of the liver). Because CYP2B6 can make a significant contribution to human liver microsomal IFA N-dechloroethylation, but only a minor contribution to IFA 4-hydroxylation, the selective inhibition of hepatic CYP2B6 activity in individuals with a high hepatic CYP2B6 content may provide a useful approach to minimize the formation of therapeutically inactive but toxic N-dechloroethylated IFA metabolites.     

Contribution of CYP3A4, CYP2B6, and CYP2C9 isoforms to N-demethylation of ketamine in human liver microsomes.

Ketamine is a widely used drug for its anesthetic and analgesic properties; it is also considered as a drug of abuse, as many cases of ketamine illegal consumption were reported. Ketamine is N-demethylated by liver microsomal cytochrome P450 into norketamine. The identification of the enzymes responsible for ketamine metabolism is of great importance in clinical practice. In the present study, we investigated the metabolism of ketamine in human liver microsomes at clinically relevant concentrations. Liver to plasma concentration ratio of ketamine was taken into consideration. Pooled human liver microsomes and human lymphoblast-expressed P450 isoforms were used. N-demethylation of ketamine was correlated with nifedipine oxidase activity (CYP3A4-specific marker reaction), and it was also correlated with S-mephenytoin N-demethylase activity (CYP2B6-specific marker reaction). Orphenadrine, a specific inhibitor to CYP2B6, and ketoconazole, a specific inhibitor to CYP3A4, inhibited the N-demethylation of ketamine in human liver microsomes. In human lymphoblast-expressed P450, the activities of CYP2B6 were higher than those of CYP3A4 and CYP2C9 at three concentrations of ketamine, 0.005, 0.05, and 0.5 mM. When these results were extrapolated using the average relative content of these P450 isoforms in human liver, CYP3A4 was the major enzyme involved in ketamine N-demethylation. The present study demonstrates that CYP3A4 is the principal enzyme responsible for ketamine N-demethylation in human liver microsomes and that CYP2B6 and CYP2C9 have a minor contribution to ketamine N-demethylation at therapeutic concentrations of the drug.     

Quantitative contribution of CYP2D6 and CYP3A to oxycodone metabolism in human liver and intestinal microsomes.

Oxycodone undergoes N-demethylation to noroxycodone and O-demethylation to oxymorphone. The cytochrome P450 (P450) isoforms capable of mediating the oxidation of oxycodone to oxymorphone and noroxycodone were identified using a panel of recombinant human P450s. CYP3A4 and CYP3A5 displayed the highest activity for oxycodone N-demethylation; intrinsic clearance for CYP3A5 was slightly higher than that for CYP3A4. CYP2D6 had the highest activity for O-demethylation. Multienzyme, Michaelis-Menten kinetics were observed for both oxidative reactions in microsomes prepared from five human livers. Inhibition with ketoconazole showed that CYP3A is the high affinity enzyme for oxycodone N-demethylation; ketoconazole inhibited >90% of noroxycodone formation at low substrate concentrations. CYP3A-mediated noroxycodone formation exhibited a mean K(m) of 600 +/- 119 microM and a V(max) that ranged from 716 to 14523 pmol/mg/min. Contribution from the low affinity enzyme(s) did not exceed 8% of total intrinsic clearance for N-demethylation. Quinidine inhibition showed that CYP2D6 is the high affinity enzyme for O-demethylation with a mean K(m) of 130 +/- 33 microM and a V(max) that ranged from 89 to 356 pmol/mg/min. Activity of the low affinity enzyme(s) accounted for 10 to 26% of total intrinsic clearance for O-demethylation. On average, the total intrinsic clearance for noroxycodone formation was 8 times greater than that for oxymorphone formation across the five liver microsomal preparations (10.5 microl/min/mg versus 1.5 microl/min/mg). Experiments with human intestinal mucosal microsomes indicated lower N-demethylation activity (20-50%) compared with liver microsomes and negligible O-demethylation activity, which predict a minimal contribution of intestinal mucosa in the first-pass oxidative metabolism of oxycodone.     

Multiple Human Cytochromes Contribute to Biotransformation of Dextromethorphan In-vitro: Role of CYP2C9, CYP2C19, CYP2D6, and CYP3A

Cytochromes mediating the biotransformation of dextromethorphan to dextrorphan and 3-methoxymorphinan, its principal metabolites in man, have been studied by use of liver microsomes and microsomes containing individual cytochromes expressed by cDNA-transfected human lymphoblastoid cells. In-vitro formation of dextrorphan from dextromethorphan by liver microsomes was mediated principally by a high-affinity enzyme (K_m (substrate concentration producing maximum reaction velocity) 3–13 μM). Formation of dextrorphan from 25 μM dextromethorphan was strongly inhibited by quinidine (IC50 (concentration resulting in 50% inhibition) = 0.37 μm); inhibition by sulphaphenazole was approximately 18% and omeprazole and ketoconazole had minimal effect. Dextrorphan was formed from dextromethorphan by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, ?2C19, and ?2D6 but not by those expressing CYP1A2, ?2E1 or ?3A4. Despite the low in-vivo abundance of CYP2D6, this cytochrome was identified as the dominant enzyme mediating dextrorphan formation at substrate concentrations below 10 μM. Formation of 3-methoxy-morphinan from dextromethorphan in liver microsomes proceeded with a mean K_m of 259 μM. For formation of 3-methoxymorphinan from 25 μM dextromethorphan the IC50 for ketoconazole was 1.15 μM; sulphaphenazole, omeprazole and quinidine had little effect. 3-Methoxymorphinan was formed by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, ?2C19, ?2D6, and ?3A4, but not by those expressing CYP1A2 or ?2E1. CYP2C19 had the highest affinity (K_m = 49 μM) whereas CYP3A4 had the lowest (K_m = 1155 μM). Relative abundances of the four cytochromes were determined in liver microsomes by use of the relative activity factor approach. After adjustment for relative abundance, CYP3A4 was identified as the dominant enzyme mediating 3-methoxymorphinan formation from dextromethorphan, although CYP2C9 and ?2C19 were estimated to contribute to 3-methoxymorphinan formation, particularly at low substrate concentrations. Although formation of dextrorphan from dextromethorphan appears to be sufficiently specific to be used as an in-vitro or in-vivo index reaction for profiling of CYP2D6 activity, the findings raise questions about the specificity of 3-methoxymorphinan formation as an index of CYP3A activity.