Paclitaxel metabolism in rat and human liver microsomes is inhibited by phenolic antioxidants

Paclitaxel is an important, recently introduced anti-neoplastic drug. Paclitaxel metabolites are virtually inactive in comparison with the parent drug. The study investigated whether phenolic antioxidants could inhibit metabolic inactivation sufficiently to increase paclitaxel effects. Cytochrome P450 (CYP)-catalysed metabolism of paclitaxel was investigated in rat and human liver microsomes. In rat microsomes, paclitaxel was metabolised mainly to C3'-hydroxypaclitaxel (C3'-OHP), less to C2-hydroxypaclitaxel (C2-OHP), di-hydroxypaclitaxel (di-OHP) and another monohydroxylated paclitaxel. In human liver microsomes, 6-hydroxypaclitaxel (6-OHP), formed by CYP2C8, was the main metabolite, while C3'-OHP, C2-OHP and another product different from di-OHP were minor metabolites, formed by CYP3A4. In individual human livers 6-OHP was formed at 1.8-fold to 13-fold higher rates than C3'-OHP. Kinetic parameters (K_m and V_max) of production of various metabolites in rat and human liver microsomes revealed differences between species as well as human individual differences. Nine phenolic antioxidants ((+)-catechin, (-)-epicatechin, fisetin, gallic acid, morin, myricetin, naringenin, quercetin and resveratrol) were tested for inhibition of paclitaxel metabolism. In rat microsomes, resveratrol was more inhibitory than fisetin; the other phenolic antioxidants were without effect. In human microsomes, the inhibiting potency decreased in the order fisetin >quercetin >morin >resveratrol, while the other phenolic antioxidants were not inhibitory; the formation of 6-OHP (CYP2C8) was generally more inhibited than that of C3'-OHP. The inhibition was mostly mixed-type. The results suggest that oral administration of some phenolic substances might increase paclitaxel blood concentrations during chemotherapy.     

Metabolism of new-generation taxanes in human, pig, minipig and rat liver microsomes

The novel taxanes SB-T-1102, SB-T-1214 and SB-T-1216 are up to 1000-fold more cytotoxic for resistant tumour cells than clinically used paclitaxel and docetaxel, and the current study has examined the metabolism of these new taxanes in human, rat, pig and minipig liver microsomes. Metabolites were characterized by high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS/MS) analysis. Metabolic pathways derived from their structures were confirmed by investigating subsequent metabolism of purified metabolites. SB-T-1102, SB-T-1214 and SB-T-1216 were metabolized to 14, 10 and 11 products, respectively. In contrast to docetaxel, side-chain hydroxylation did not occur at their tert-butyl group, but on the isobutyl (SB-T-1102) or isobutenyl (SB-T-1214 and SB-T-1216) chains. Species differences in their metabolism were observed. For example, human and untreated rat microsomes hydroxylated SB-T-1216 preferentially at the side-chain, whereas pig and minipig microsomes preferentially metabolized more at the taxane core. The increased formation of secondary and tertiary metabolites in rat microsomes with high expression of CYP3A1/2 compared with uninduced rats confirmed the role of CYP3A in taxane metabolism. All major products were formed by human cDNA-expressed CYP3A4 and none by CYP1A2, 1B1, 2A6, 2C9 and 2E1, indicating the principal role of CYP3A orthologues in SB-T metabolism. The knowledge of metabolic pathways of the examined agents and of their rates of formation is important due to possible metabolic inactivation of these three novel drugs with a great potential for the therapy of taxane-resistant tumours. The relatively slow metabolism of SB-T-1102 could be favourable for its antitumour efficiency in vivo.     

In vitro biotransformation of sildenafil (Viagra) in the male rat: the role of CYP2C11.

To assess the suitability of the male rat model for human studies on sildenafil metabolism, we examined the biotransformation of sildenafil in male rat liver microsomes and identified the role of specific cytochrome P450s (P450) using inhibitory antibodies and cDNA-expressed P450s. Rates of formation of the major circulating metabolite of sildenafil, UK-103,320, were 11-fold greater in the male rat than in human liver microsomes at 36 microM sildenafil, whereas substrate concentration corresponding to 50% V(max) (K(m) values) were 2.9-fold lower in the male rat. Although sildenafil is largely metabolized by CYP3A isoforms in humans, coincubation of rat liver microsomes with immunoinhibitory antibodies (CYP1A1/2, 2B1/2, 2C11, 2E1, and 3A1/2) revealed that metabolite formation was inhibited only by an antirat CYP2C11 antibody. Incubation of sildenafil with a cDNA-expressed CYP2C11 produced 10-fold higher levels of UK-103,320 than other P450s (CYP1A1, 1A2, 2B1, 2C6, 2C12, 2C13, 2E1, 3A1, and 3A2). Thus CYP2C11 contributes in a major way to the metabolism of sildenafil in the male rat. P450 isoforms mediating sildenafil biotransformation differ substantially between humans and the male rat, thereby limiting the applicability of this species as a model for sildenafil metabolism and drug interactions in humans.     

Metabolism of vanoxerine, 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine, by human cytochrome P450 enzymes.

Vanoxerine (1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine; GBR12909) is a promising agent for the treatment of cocaine dependence. Knowledge of the major pathway for GBR12909 metabolism is important for prediction of the likelihood of drug-drug interactions, which may affect the therapeutic clinical outcome, when this agent is used in cocaine-dependent individuals receiving multiple drug therapy. We studied biotransformation of GBR12909 in human liver microsomes (n = 4), human hepatocytes, and microsomes containing cDNA-expressed human P450 isoforms with GBR12909 concentrations within the range of steady-state plasma concentrations detected in healthy volunteers. A high-pressure liquid chromatography assay was used to measure parent GBR12909 and its primary metabolite. GBR12909 was metabolized by human liver microsomes, hepatocytes, and cDNA-expressed human P450s to a single metabolite. Ketoconazole, a selective inhibitor of CYP3A, reduced GBR12909 biotransformation in human liver microsomes and primary hepatocytes by 92 +/- 2 and 92.4 +/- 0.4%, respectively. Quercetin (an inhibitor of CYP2C8/3A4) was a less effective inhibitor producing 62 +/- 22% inhibition in human liver microsomes and 54 +/- 35% in hepatocytes. Other P450 selective inhibitors did not decrease GBR12909 biotransformation more than 29% in either human liver microsomes or hepatocytes with the exception of chlorzoxazone (CYP2E1), which inhibited GBR12909 biotransformation by 71.4 +/- 18.5% in primary human hepatocytes. Ciprofloxacin (CYP1A2), sulfaphenazole (CYP2C9), quinidine (CYP2D6), chlorzoxazone (CYP2E1), and mephenytoin (CYP2C19) did not demonstrate statistically significant inhibition (p > 0.05) of GBR12909 biotransformation in liver microsomes. cDNA-expressed P450 3A4 metabolized GBR12909 to a greater extent than 2C8 and 2E1. These data suggest the possibility that multiple P450 isoforms may be involved in human GBR12909 metabolism but that CYP3A appears to be the major enzyme responsible for human GBR12909 biotransformation.     

In vitro human metabolism and interactions of repellent N,N-diethyl-m-toluamide.

Oxidative metabolism of the insect repellent N,N-diethyl-m-toluamide (DEET) by pooled human liver microsomes (HLM), rat liver microsomes (RLM), and mouse liver microsomes (MLM) was investigated. DEET is metabolized by cytochromes P450 (P450s) leading to the production of a ring methyl oxidation product, N,N-diethyl-m-hydroxymethylbenzamide (BALC), and an N-deethylated product, N-ethyl-m-toluamide (ET). Both the affinities and intrinsic clearance of HLM for ring hydroxylation are greater than those for N-deethylation. Pooled HLM show significantly lower affinities (K(m)) than RLM for metabolism of DEET to either of the primary metabolites (BALC and ET). Among 15 cDNA-expressed P450 enzymes examined, CYP1A2, 2B6, 2D6*1 (Val(374)), and 2E1 metabolized DEET to the BALC metabolite, whereas CYP3A4, 3A5, 2A6, and 2C19 produced the ET metabolite. CYP2B6 is the principal cytochrome P450 involved in the metabolism of DEET to its major BALC metabolite, whereas CYP2C19 had the greatest activity for the formation of the ET metabolite. Use of phenotyped HLMs demonstrated that individuals with high levels of CYP2B6, 3A4, 2C19, and 2A6 have the greatest potential to metabolize DEET. Mice treated with DEET demonstrated induced levels of the CYP2B family, increased hydroxylation, and a 2.4-fold increase in the metabolism of chlorpyrifos to chlorpyrifos-oxon, a potent anticholinesterase. Preincubation of human CYP2B6 with chlorpyrifos completely inhibited the metabolism of DEET. Preincubation of human or rodent microsomes with chlorpyrifos, permethrin, and pyridostigmine bromide alone or in combination can lead to either stimulation or inhibition of DEET metabolism.     

Metabolism of new-generation taxanes in human,pig, minipig and rat liver microsomes

The novel taxanes SB-T-1102, SB-T-1214 and SB-T-1216 are up to 1000-fold more cytotoxic for resistant tumour cells than clinically used paclitaxel and docetaxel, and the current study has examined the metabolism of these new taxanes in human, rat, pig and minipig liver microsomes. Metabolites were characterized by high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS/MS) analysis. Metabolic pathways derived from their structures were confirmed by investigating subsequent metabolism of purified metabolites. SB-T-1102, SB-T-1214 and SB-T-1216 were metabolized to 14, 10 and 11 products, respectively. In contrast to docetaxel, side-chain hydroxylation did not occur at their tert-butyl group, but on the isobutyl (SB-T-1102) or isobutenyl (SB-T-1214 and SB-T-1216) chains. Species differences in their metabolism were observed. For example, human and untreated rat microsomes hydroxylated SB-T-1216 preferentially at the side-chain, whereas pig and minipig microsomes preferentially metabolized more at the taxane core. The increased formation of secondary and tertiary metabolites in rat microsomes with high expression of CYP3A1/2 compared with uninduced rats confirmed the role of CYP3A in taxane metabolism. All major products were formed by human cDNA-expressed CYP3A4 and none by CYP1A2, 1B1, 2A6, 2C9 and 2E1, indicating the principal role of CYP3A orthologues in SB-T metabolism. The knowledge of metabolic pathways of the examined agents and of their rates of formation is important due to possible metabolic inactivation of these three novel drugs with a great potential for the therapy of taxane-resistant tumours. The relatively slow metabolism of SB-T-1102 could be favourable for its antitumour efficiency in vivo.     

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

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

Involvement of CYP2A6 in the formation of a novel metabolite, 3-hydroxypilocarpine, from pilocarpine in human liver microsomes.

Pilocarpine is a cholinergic agonist that is metabolized to pilocarpic acid by serum esterase. In this study, we discovered a novel metabolite in human urine after the oral administration of pilocarpine hydrochloride, and we investigated the metabolic enzyme responsible for the metabolite formation. The structure of the metabolite was identified as 3-hydroxypilocarpine by liquid chromatography-tandem mass spectrometry and NMR analyses and by comparing to the authentic metabolite. To clarify the human cytochrome P450 (P450) responsible for the metabolite formation, in vitro experiments using P450 isoform-selective inhibitors, cDNA-expressed human P450s (Supersomes; CYP1A2, -2A6, -2B6, -2C9, -2C19, -2D6, -2E1, and -3A4), and liver microsomes from different donors were conducted. The formation of 3-hydroxypilocarpine in human liver microsomes was strongly inhibited (>90%) by 200 microM coumarin. Other selective inhibitors of CYP1A2 (furafylline and alpha-naphthoflavone), CYP2C9 (sulfaphenazole), CYP2C19 [(S)-mephenytoin], CYP2E1 (4-methylpyrazole), CYP2D6 (quinidine), and CYP3A4 (troleandomycin) had a weak inhibitory effect (<20%) on the formation. The highest formation activity was expressed by recombinant CYP2A6. The K(m) value for recombinant CYP2A6 was 3.1 microM, and this value is comparable with that of human liver microsomes (1.5 microM). The pilocarpine 3-hydroxylation activity was correlated with coumarin 7-hydroxylation activity in 16 human liver microsomes (r = 0.98). These data indicated that CYP2A6 is the main enzyme responsible for the 3-hydroxylation of pilocarpine. In conclusion, we identified a novel metabolite of pilocarpine, 3-hydroxypilocarpine, and we clarified the involvement of CYP2A6 in the formation of this molecule in human liver microsomes.     

Roles of cytochromes P450 1A2, 2A6, and 2C8 in 5-fluorouracil formation from tegafur, an anticancer prodrug, in human liver microsomes.

Tegafur, an anticancer prodrug, is bioactivated to 5-fluorouracil (5-FU) mainly by cytochrome P450 (P450) enzymes. The conversion from tegafur into 5-FU catalyzed by human liver microsomal P450 enzymes was investigated. In fourteen cDNA-expressed human P450 enzymes having measurable activities, CYP1A2, CYP2A6, CYP2E1, and CYP3A5 were highly active in catalyzing 5-FU formation at a tegafur concentration of 100 microM. Kinetic analysis revealed that CYP1A2 had the highest V(max)/K(m) value and that the V(max) value of CYP2A6 was high in 5-FU formation. In human liver microsomes, the activities of 5-FU formation from 10 microM, 100 microM, and 1 mM tegafur were significantly correlated with both coumarin 7-hydroxylation (r = 0.83, 0.86, and 0.74) and paclitaxel 6 alpha-hydroxylation (r = 0.77, 0.62, and 0.85) activities, respectively. Coumarin efficiently inhibited the 5-FU formation activities from 100 microM and 1 mM tegafur catalyzed by human liver microsomes that had high coumarin 7-hydroxylation activity. On the other hand, furafylline, fluvoxamine, and quercetin, as well as coumarin, showed inhibitory effects in liver microsomes that had high catalytic activities of 5-FU formation. The other P450 inhibitors examined showed weak or no inhibition in human liver microsomes. Polyclonal anti-CYP1A2 antibody, monoclonal anti-CYP2A6, and anti-CYP2C8 antibodies inhibited 5-FU formation activities to different extents in those two microsomal samples. These results suggest that CYP1A2, CYP2A6, and CYP2C8 have important roles in human liver microsomal 5-FU formation and that the involvement of these three P450 forms differs among individual humans.     

Oxidation of troglitazone to a quinone-type metabolite catalyzed by cytochrome P-450 2C8 and P-450 3A4 in human liver microsomes.

Troglitazone, a new oral antidiabetic drug, is reported to be mostly metabolized to its conjugates and not to be oxidized by cytochrome P-450 (P-450) enzymes. Of fourteen cDNA-expressed human P-450 enzymes examined, CYP1A1, CYP2C8, CYP2C19, and CYP3A4 were active in catalyzing formation of a quinone-type metabolite at a concentration of 10 microM troglitazone, whereas CYP3A4 had the highest catalytic activity at 100 microM substrate. In human liver microsomes, rates of the quinone-type metabolite formation (at 100 microM) were correlated well with rates of testosterone 6beta-hydroxylation (r = 0.98), but those at 10 microM troglitazone were not correlated with any of several marker activities of P-450 enzymes. Quercetin efficiently inhibited quinone-type metabolite formation (at 10 microM troglitazone) in human samples that contained relatively high levels of CYP2C, whereas ketoconazole affected these activities in liver microsomes in which CYP3A4 levels were relatively high. Anti-CYP2C antibodies strongly inhibited quinone-type metabolite formation (at 10 microM troglitazone) in CYP2C-rich human liver microsomes (by approximately 85%); the intensity of this effect depended on the human samples and their P-450 status. The results suggest that in human liver both CYP2C8 and CYP3A4 have major roles in quinone-type metabolite formation and that the hepatic contents of these two P-450 forms determine which P-450 enzymes play major roles in individual humans. CYP3A4 may be expected to play a role in formation of quinone-type metabolite from troglitazone even at a low concentration in humans.     

Stereo- and regioselectivity account for the diversity of dehydroepiandrosterone (DHEA) metabolites produced by liver microsomal cytochromes P450.

The purpose of this study was to quantify the oxidative metabolism of dehydroepiandrosterone (3beta-hydroxy-androst-5-ene-17-one; DHEA) by liver microsomal fractions from various species and identify the cytochrome P450 (P450) enzymes responsible for production of individual hydroxylated DHEA metabolites. A gas chromatography-mass spectrometry method was developed for identification and quantification of DHEA metabolites. 7alpha-Hydroxy-DHEA was the major oxidative metabolite formed by rat (4.6 nmol/min/mg), hamster (7.4 nmol/min/mg), and pig (0.70 nmol/min/mg) liver microsomal fractions. 16alpha-Hydroxy-DHEA was the next most prevalent metabolite formed by rat (2.6 nmol/min/mg), hamster (0.26 nmol/min/mg), and pig (0.16 nmol/min/mg). Several unidentified metabolites were formed by hamster liver microsomes, and androstenedione was produced only by pig microsomes. Liver microsomal fractions from one human demonstrated that DHEA was oxidatively metabolized at a total rate of 7.8 nmol/min/mg, forming 7alpha-hydroxy-DHEA, 16alpha-hydroxy-DHEA, and a previously unidentified hydroxylated metabolite, 7beta-hydroxy-DHEA. Other human microsomal fractions exhibited much lower rates of metabolism, but with similar metabolite profiles. Recombinant P450s were used to identify the cytochrome P450s responsible for DHEA metabolism in the rat and human. CYP3A4 and CYP3A5 were the cytochromes P450 responsible for production of 7alpha-hydroxy-DHEA, 7beta-hydroxy-DHEA, and 16alpha-hydroxy-DHEA in adult liver microsomes, whereas the fetal/neonatal form CYP3A7 produced 16alpha-hydroxy and 7beta-hydroxy-DHEA. CYP3A23 uniquely formed 7alpha-hydroxy-DHEA, whereas other P450s, CYP2B1, CYP2C11, and CYP2D1, were responsible for 16alpha-hydroxy-DHEA metabolite production in rat liver microsomal fractions. These results indicate that the stereo- and regioselectivity of hydroxylation by different P450s account for the diverse DHEA metabolites formed among various species.     

Comparison of the formation of N-alkylprotoporphyrin IX after interaction of porphyrinogenic xenobiotics with single cDNA-expressed human P450 enzymes in microsomes prepared from baculovirus-infected insect cells and human lymphoblastoid cell lines.

In a previous study using microsomes from human lymphoblastoid cell lines (HLCL) containing single cDNA-expressed human cytochrome P450 (P450) enzymes, human P450 enzymes were identified that are susceptible to mechanism-based inactivation by the porphyrinogenic xenobiotics, 3-[(arylthio)ethyl]sydnone (TTMS), 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethylDDC) and allylisopropylacetamide (AIA). In this study, we tested the hypothesis that N-alkylprotoporphyrin IX (N-alkylPP) formation following interaction of porphyrinogenic xenobiotics with single cDNA-expressed human P450 enzymes in microsomes from HLCL would occur only with P450 enzymes that had undergone mechanism-based inactivation. In a previous study, when 4-ethylDDC and NADPH interacted with human liver microsomes possessing elevated levels of CYP1A2 and 2C9, N-ethylprotoporphyrin IX (N-ethylPP) was not formed despite the fact that it was formed in microsomes from baculovirus-infected insect cell lines (BIICL) containing either CYP1A2 or 2C9. In this study, we tested the hypothesis that 4-ethylDDC underwent biotransformation by CYP3A4 present in human liver microsomes, diverting the xenobiotic from CYP1A2 and 2C9. Fluorometry was used to measure N-alkylPP formation following interaction of porphyrinogenic xenobiotics and NADPH with cDNA-expressed human P450 enzymes in microsomes from HLCL or BIICL. With TTMS and 4-ethylDDC but not with AIA, N-alkylPP formation was observed only with human P450 enzymes CYP2D6, 1A2, 3A4, or 2C9 in microsomes from HLCL, which had undergone mechanism-based inactivation. Microsomes from BIICL containing CYP3A4 were added to a mixture of NADPH, 4-ethylDDC, and microsomes from BIICL containing CYP1A2 and 2C9. The addition of CYP3A4 to CYP1A2 and 2C9 did not decrease N-ethylPP formation, providing no support for the hypothesis.     

Role of human CYP2B6 in S-mephobarbital N-demethylation.

The role of cytochrome P-450s (CYPs) in S-mephobarbital N-demethylation was investigated by using human liver microsomes and cDNA-expressed CYPs. Among the 10 cDNA-expressed CYPs studied (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), only CYP2B6 could catalyze S-mephobarbital N-demethylation. The apparent K(m) values of human liver microsomes for S-mephobarbital N-demethylation were close to that of cDNA-expressed CYP2B6 (about 250 microM). The N-demethylase activity of S-mephobarbital in 10 human liver microsomes was strongly correlated with immunodetectable CYP2B6 levels (r = 0.920, p<.001). Orphenadrine (300 microM), a CYP2B6 inhibitor, inhibited the N-demethylase activity of S-mephobarbital in human liver microsomes to 29% of control activity. Therefore, it appears that CYP2B6 mainly catalyzes S-mephobarbital N-demethylation in human liver microsomes.     

Antitumor activity of methoxymorpholinyl doxorubicin: potentiation by cytochrome P450 3A metabolism

Methoxymorpholinyl doxorubicin (MMDX) is a novel liver cytochrome P450 (P450)-activated anticancer prodrug whose toxicity toward cultured tumor cells can be potentiated up to 100-fold by incubation with liver microsomes and NADPH. In the present study, a panel of human liver microsomes activated MMDX with potentiation ratios directly correlated to the CYP3A-dependent testosterone 6beta-hydroxylase activity of each liver sample. Microsome-activated MMDX exhibited nanomolar IC(50) values in growth-inhibition assays of human tumor cell lines representing multiple tissues of origin: lung (A549 cells), brain (U251 cells), colon (LS180 cells), and breast (MCF-7 cells). Analysis of individual cDNA-expressed CYP3A enzymes revealed that rat CYP3A1 and human CYP3A4 activated MMDX more efficiently than rat CYP3A2 and that human P450s 3A5 and 3A7 displayed little or no activity. MMDX cytotoxicity was substantially increased in Chinese hamster ovary cells after stable expression of CYP3A4 in combination with P450 reductase. CYP3A activation of MMDX abolished the parent drug's residual cross-resistance in a doxorubicin-resistant MCF-7 cell line that overexpresses P-glycoprotein. CYP3A-activated MMDX displayed a comparatively high intrinsic stability, with a t(1/2) of approximately 5.5 h at 37 degrees C. MMDX was rapidly activated by CYP3A at low ( approximately 1-5 nM) prodrug concentrations, with 100% tumor cell kill obtained after as short as a 2-h exposure to the activated metabolite. These findings demonstrate that MMDX can be activated by CYP3A metabolism to a potent, long-lived, and cell-permeable cytotoxic metabolite and suggest that this anthracycline prodrug may be used in combination with CYP3A4 in a P450 prodrug activation-based gene therapy for cancer treatment.     

Identification of the human liver cytochrome P450 isoenzymes responsible for the 5-methylhydroxylation of the novel anti-fibrotic drug AKF-PD

Identification of cytochrome P450 isoforms (CYPs) involved in flourofenidone (5-methyl-1-(3-fluorophenyl)-2-[1H]-pyridone, AKF-PD) 5-methylhydroxylation was carried out using human liver microsomes and cDNA-expressed human CYPs (supersomes). The experiments were performed in the following in vitro models: (A) a study of AKF-PD metabolism in liver microsomes: (a) correlations study between the rate of AKF-PD 5-methylhydroxylation and activity of CYPs; (b) the effect of specific CYPs inhibitors on the rate of AKF-PD 5-methylhydroxylation; (B) AKF-PD biotransformation by cDNA-expressed human CYPs (1A2, 2D6, 2C9, 2C19, 2E1, 3A4). In human liver microsomes, the formation of AKF-PD 5-methylhydroxylation metabolite significantly correlated with the caffeine N3-demethylase (CYP1A2), chlorzoxazone 6-hydroxylase (CYP2E1), midazolam 1'- hydroxylase (CYP3A4), tolbutamide 4-hydroxylase (CYP2C9), and debrisoquin 4-hydroxylase (CYP2D6) activities. The production of AKF-PD 5-methylhydroxylation metabolite was completely inhibited by a-naphthoflavone (a CYP1A2 inhibitor) with the IC50 value of 0.12 μM in human liver microsomes. The cDNA-expressed human CYPs generated different amounts of AKF-PD 5-methylhydroxylation metabolites, but the preference of CYP isoforms to catalyze AKF-PD metabolism was as follows: 2D6?>?2C19?>?1A2?>?2E1?>?2C9?>?3A4. The results demonstrated that CYP1A2 is the main isoform catalyzing AKF-PD 5-methylhydroxylation while CYP3A4, CYP2C9, CYP2E1, CYP2C19, and CYP2D6 are engaged to a lesser degree. Potential drug-drug interactions involving CYP1A2 may be noticed when AKF-PD is used combined with CYP1A2 inducers or inhibitors.     

In vitro metabolism of eupatilin by multiple cytochrome P450 and UDP-glucuronosyltransferase enzymes

Eupatilin, a pharmacologically active flavone derived from Artemisia plants, is extensively metabolized to eupatilin glucuronide, 4-O-desmethyleupatilin and 4-O-desmethyleupatilin glucuronide in human liver microsomes. This study characterized the human liver cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes responsible for the metabolism of eupatilin. The specific CYPs responsible for O-demethylation of eupatilin to the major metabolite, 4-O-desmethyleupatilin were identified using a combination of correlation analysis, immuno-inhibition, chemical inhibition in human liver microsomes and metabolism by human cDNA-expressed CYP enzymes. UGT enzymes involved in the eupatilin glucuronidation were identified using pooled human liver microsomes and human cDNA-expressed UGT enzymes. Eupatilin was predominantly metabolized by CYP1A2 and, to a lesser extent, CYP2C8 mediated O-demethylation of eupatilin to 4-O-desmethyleupatilin. Eupatilin glucuronidation was catalysed by UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT1A10.     

Identification of cytochrome P450 enzymes responsible for N -dealkylation of a new oral erectogenic, mirodenafil

The purpose of this paper is to characterize the cytochrome P450 (CYP) enzymes involved in the metabolism of a new oral erectogenic, mirodenafil, to a major circulating active metabolite, N-dehydroxyethyl-mirodenafil, and to investigate the inhibitory potential of mirodenafil on seven CYP enzymes in human liver microsomes. CYP3A4 was identified as the major enzyme and CYP2C8 as a minor enzyme responsible for mirodenafil N-dealkylation based on correlation analysis, inhibition studies, and cDNA-expressed CYP enzyme activities. Plasma concentrations of mirodenafil and its N-dealkylated metabolite could therefore change with co-administration of known CYP3A4 inducers or inhibitors. Mirodenafil inhibited CYP3A4, CYP2C19 and CYP2D6 activities with IC50 values of 15.6, 38.2 and 77.0 microM, respectively, in human liver microsomes. However, it is very unlikely that mirodenafil will significantly alter the clearance of other compounds metabolized by CYPs 1A2, 2A6, 2C8, 2C9, 2C19, 2D6 and 3A4 because the maximum plasma concentration of mirodenafil is 0.55 microM after oral dosing of mirodenafil (100 mg) in male volunteers.     

Identification of human cytochrome P450 isoforms involved in the metabolism of S-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid.

1. To identify the cytochrome P450 (CYP) isoenzymes responsible for the major metabolic pathways of S-2-[4-(3-methyl-2-thienyl)phenyl] propionic acid (S-MTPPA) in man, the metabolism of S-MTPPA was examined using human liver microsomes and microsomes containing cDNA-expressed CYP isozymes (CYP1A2, 2A6, 2B6, 2C9-Arg, 2C9-Cys, 2C19, 2D6-Val, 2E1 and 3A4). 2. S-MTPPA was mainly oxidized to the 5-hydroxylated metabolite of the thiophene ring (MA6) with human liver microsomes in the presence of NADPH. The formation of MA6 was inhibited by SKF 525-A, suggesting that CYP plays role in the formation of MA6. 3. Eadie-Hofstee plots for the 5-hydroxylation of S-MTPPA in the range 5-100 microM were linear for all samples studied, suggesting that the formation of MA6 by human liver microsomes follows simple Michaelis-Menten kinetics. Apparent Vmax = 1.42+/-0.64 nmol/min/mg protein; Km = 12+/-5 microM. 4. Among the CYP inhibitors examined (alpha-naphthoflavone, sulphaphenazole, omeprazole, quinidine and troleandomycin), sulphaphenazole (a CYP2C9 inhibitor) showed the most potent inhibitory effect on the 5-hydroxylation of S-MTPPA by human liver microsomes. 5. When incubated with microsomes containing cDNA-expressed CYP isozymes, S-MTPPA was substantially oxidized to MA6 only by CYP2C9. 6. These results suggest that formation of the major metabolite of S-MTPPA, MA6, in human liver microsomes is catalysed predominantly by a single CYP isoenzyme, namely CYP2C9.     

Metabolism of tauromustine in liver and lung microsomes from various species

1. The cytochrome P450 (CYP)-mediated metabolism of tauromustine has been evaluated in liver and lung microsomes from various species. Liver microsomes from rat pretreated with typical CYP inducers, human liver microsomes and cDNA-expressed human CYP enzymes were used to study the enzymatic basis of the metabolism. The further metabolism of the monodemethylated product of tauromustine and that of the denitrosated product were also investigated. 2. The major routes of tauromustine metabolism were demethylation to the alkylating active compound, R2, and denitrosation to the inactive metabolite, M3. The extent of metabolism and the activity of demethylation versus denitrosation varied among the species. The highest metabolism was found in mouse (BDF strain) followed by dog, rat and the human liver. Tauromustine was also metabolized to a low extent in lung microsomes from these species. 3. The further metabolism of R2 and M3 was ~100 times lower in activity than that of tauromustine. Both the demethylation and the denitrosation of tauromustine were increased 3-fold in liver microsomes from rat pretreated with phenobarbital, whereas treatment with cyanopregnenolone enhanced the denitrosation 11-fold, indicating the involvement of CYP3A. 4. Metabolism across a panel of 10 human liver microsomal samples demonstrated a correlation with testosterone 6beta-hydroxylation of demethylation (r2= 0.86) and denitrosation of tauromustine (r2=0.79). Among the human cDNA expressed CYP enzymes, not only was tauromustine determined to be catalysed predominantly by CYP3A4, but also to some extent by CYP2C19 and CYP2D6. 5. In conclusion, the present results indicate a major role of CYP3A enzymes in the metabolism of tauromustine.     

Metabolism of tauromustine in liver and lung microsomes from various species.

1. The cytochrome P450 (CYP)-mediated metabolism of tauromustine has been evaluated in liver and lung microsomes from various species. Liver microsomes from rat pretreated with typical CYP inducers, human liver microsomes and cDNA-expressed human CYP enzymes were used to study the enzymatic basis of the metabolism. The further metabolism of the monodemethylated product of tauromustine and that of the denitrosated product were also investigated. 2. The major routes of tauromustine metabolism were demethylation to the alkylating active compound, R2, and denitrosation to the inactive metabolite, M3. The extent of metabolism and the activity of demethylation versus denitrosation varied among the species. The highest metabolism was found in mouse (BDF strain) followed by dog, rat and the human liver. Tauromustine was also metabolized to a low extent in lung microsomes from these species. 3. The further metabolism of R2 and M3 was approximately 100 times lower in activity than that of tauromustine. Both the demethylation and the denitrosation of tauromustine were increased 3-fold in liver microsomes from rat pretreated with phenobarbital, whereas treatment with cyanopregnenolone enhanced the denitrosation 11-fold, indicating the involvement of CYP3A. 4. Metabolism across a panel of 10 human liver microsomal samples demonstrated a correlation with testosterone 6beta-hydroxylation of demethylation (r2 = 0.86) and denitrosation of tauromustine (r2 = 0.79). Among the human cDNA expressed CYP enzymes, not only was tauromustine determined to be catalysed predominantly by CYP3A4, but also to some extent by CYP2C19 and CYP2D6. 5. In conclusion, the present results indicate a major role of CYP3A enzymes in the metabolism of tauromustine.