4-Substituted 2,6-dimethyl-3,5-bis(alkoxycarbonyl)-1,4-dihydropyridines are important because of their roles as calcium channel blockers. The mixed-function oxidation of 14 4-aryl- and four 4-alkyl-substituted derivatives by human liver microsomes was examined. The major product of enzymatic oxidation of all the 4-aryl compounds was the pyridine derivative containing the 4-aryl group. The 4-alkyl compounds, in contrast, formed a pyridine derivative in which a hydrogen atom was present at the 4-position and the alkyl group was lost; these compounds also inactivated cytochrome P-450 and caused the loss of nifedipine oxidase activity after enzymatic oxidation. All of these reactions were extensively inhibited by an antibody raised to purified human liver nifedipine oxidase cytochrome P-450 (P-450NF), indicating a major role for this enzyme in the oxidation of these compounds. Oxidation of the 4-alkyl compounds led not only to the loss of P-450NF but also to decreases in catalytic activities of cytochrome P-450 isozymes catalyzing other reactions (phenacetin O-deethylation and hexobarbital 3'-hydroxylation). The results indicate that P-450NF (or closely related enzyme forms) is responsible for the oxidation of these nifedipine-related compounds in human liver microsomes and that metabolism is highly dependent upon 4-substitution; with alkyl substituents, radicals are postulated to leave P-450NF to attack other proteins.

Silibinin, the main constituent of silymarin, a flavonoid drug from silybum marianum used in liver disease, was tested for inhibition of human cytochrome P-450 enzymes. Metabolic activities were determined in liver microsomes from two donors using selective substrates. With each substrate, incubations were carried out with and without silibinin (concentrations 3.7-300 mM) at 37ae in 0.1 M KH 2 PO 4 buffer containing up to 3% DMSO. Metabolite concentrations were determined by HPLC or direct spectroscopy. First, silibinin IC 50 values were determined for each substrate at respective K M concentrations. Silibinin had little effect (IC 50 Ͼ200 mM) on the metabolism of erythromycin (CYP3A4), chlorzoxazone (CYP2E1), S(π)-mephenytoin (CYP2C19), caffeine (CYP1A2) or coumarin (CYP2A6). A moderate effect was observed for high affinity dextromethorphan metabolism (CYP2D6) in one of the microsomes samples tested only (IC 50 Ω173 mM). Clear inhibition was found for denitronifedipine oxidation (CYP3A4; IC 50 Ω29 mM and 46 mM) and S(ª)-warfarin 7-hydroxylation (CYP2C9; IC 50 Ω43 mM and 45 mM). When additional substrate concentrations were tested to assess enzyme kinetics, silibinin was a potent competitive inhibitor of dextromethorphan metabolism at the low affinity site, which is not CYP2D6 (K i,c Ω2.3 mM and 2.4 mM). Inhibition was competitive for S(ª)-warfarin 7-hydroxylation (K i,c Ω18 mM and 19 mM) and mainly non-competitive for denitronifedipine oxidation (K i,n Ω9 mM and 12 mM). With therapeutic silibinin peak plasma concentrations of 0.6 mM and biliary concentrations up to 200 mM, metabolic interactions with xenobiotics metabolised by CYP3A4 or CYP2C9 cannot be excluded.

No abstract

Liver microsomes are a frequently used probe to investigate the phase I metabolism of xenobiotics in vitro. Structures containing nucleophilic hetero-atoms are possible substrates for cytochrome P450 enzymes (P450) and flavin-containing monooxygenases (FMO). Both enzymes are located in the endoplasmatic reticulum of hepatocytes and both need oxygen and NADPH as cofactors. The common method to distinguish between the two enzyme systems is to use the thermal inactivation of FMO and to inhibit P450 completely with carbon monoxide, N-octylamine or N-benzylimidazole. In the literature no indication could be found that the heat inactivation of FMO does not affect any of the human P450 enzymes or that the overall P450 inhibitors inhibit the different human P450 enzymes sufficiently and do not affect the FMO. The effect of N-benzylimidazole and heat inactivation was tested on specific activities of seven P450 enzymes in human liver microsomes, 1A2, 2A6, 2C9, 2C19, 2D6, 3A4/5, and 2E1, using methoxyresorufin O-demethylation, coumarin 7-hydroxylation, (S)-warfarin 4-hydroxylation, (S)-(+)-mephenytoin 4-hydroxylation, dextrometorphan O-demethylation, oxidation of denitronifedipine, and chlorzoxazone 6-hydroxylation respectively. The sulfoxidation of methimazole (MMI) was used as a specific probe for the determination of FMO activity. Methimazole sulfoxidation was compared with the well known assay for FMO metabolism, the formation of N,N-dimethylaniline (DMA) N-oxide, to be confirmed as an exclusively FMO mediated reaction. The participation of P450 and FMO in the sulfoxidation of four sulfur containing peptides, ametryne; terbutryne, prometryne and methiocarb was investigated using human liver microsomes. All four reactions were demonstrated to be catalysed predominantly by cytochrome P450.

Compounds of the s-triazine family are among the most heavily used herbicides over the last 30 years. Some of these derivatives are suspected to be carcinogens. In this study the identity of specific phase-I enzymes involved in the metabolism of s-triazine derivatives (atrazine, terbuthylazine, ametryne, and terbutryne) by human liver microsomes was determined. Kinetic studies demonstrated biphasic kinetics for all pathways examined (S-oxidation, N-dealkylation, and side-chain C-oxidation). Low K(m) values were in a range of about 1-20 microM, whereas high K(m) values were up to 2 orders of magnitude higher. For a correlation study, 30 human liver microsomal preparations were screened for seven specific P450 activities, and these were compared to activities for the metabolites derived from these s-triazines. A highly significant correlation in the high-affinity concentration range was seen with cytochrome P450 1A2 activities. Chemical inhibition was most effective with alpha-naphthoflavone and furafylline at low s-triazine concentrations and additionally with ketoconazole and gestodene at high substrate concentrations. Studies with 10 heterologously expressed P450 forms demonstrated that several P450 enzymes are capable of oxidizing these s-triazines, with different affinities and regioselectivities. P450 1A2 was confirmed to be the low-K(m) P450 enzyme involved in the metabolism of these s-triazines. A potential participation of flavin-containing monooxygenases (FMOs) in sulfoxidation reactions of the thiomethyl derivatives ametryne and terbutryne in human liver was also evaluated. Sulfoxide formation in human liver microsomes as a function of pH, heat, and chemical inhibition indicated no significant involvement of FMOs. Finally, purified recombinant FMO3, the major FMO in human liver, exhibited no significant activity (< 0.1 nmol (nmol of FMO3)-1 min-1) in the formation of the parent sulfoxides of ametryne and terbutryne. Therefore, P450 1A2 alone is likely to be responsible for the hepatic oxidative phase-I metabolism of the s-triazine derivatives in exposed humans.