Methadone has become one of the most widely used drugs for opiate dependency treatment. This drug is extensively metabolized by the cytochrome P450 hepatic enzyme family in man, yielding an N-demethylated metabolite that cyclizes spontaneously into 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine. The specific forms of cytochrome P450 involved in this oxidative N-demethylation were examined in a panel of 20 human liver microsomal preparations previously characterized with respect to their P450 enzyme contents. Methadone was demethylated with an apparent Km of 545 +/- 258 microM (n = 3). The metabolic rates were 745 +/- 574 pmol/(min.mg of protein). This metabolic pathway was strongly correlated with estradiol 2-hydroxylation, testosterone 6 beta-hydroxylation, nifedipine oxidation, erythromycin N-demethylation, and toremifene N-demethylation, all of these monooxygenase activities being supported by P450 3A4. Furthermore, the total P450 3A content of liver microsomal samples, determined by immuno-quantification using a monoclonal anti-human P450 3A4 antibody, was correlated with methadone demethylation (r = 0.72; p < 0.003). Methadone metabolism was 60-72% inhibited either by three mechanism-based inhibitors of P450 3A4 (gestodene, TAO, and erythralosamine) or by four reversible inhibitors of P450 3A (ketoconazole, dihydroergotamine, quercetin, and diazepam with an apparent Ki of 50 microM) and by two nonspecific inhibitors (metyrapone and SKF-525A). Conversely, quinidine (inhibitor of P450 2D6), 7,8-benzoflavone (inhibitor of P450 1A), or sulfaphenazole (inhibitor of P450 2C) did not significantly inhibit, and may even have activated, methadone metabolism. Four heterologously expressed P450 proteins were able to catalyze the N-demethylation of methadone, namely, P450 2C8, P450 2C18, P450 2D6, and P450 3A4. However, referring to their relative liver content, it can be asserted that P450 3A4 is the major enzyme involved in the N-demethylation of methadone on average. Accordingly, caution should be advised in the clinical use of methadone when other drugs are also administered that induce or inhibit P450 3A4, such as rifampicin or diazepam, respectively.

The capacity for the brain to produce acetaldehyde (AcHO) from ethanol was determined in rat brain homogenates. Rat brains were perfused with saline-heparin solution and homogenized in a phosphate buffer. Varying amounts of tissue were incubated with ethanol (0-100 mM) for periods of up to 60 min. The reaction was stopped by the addition of desferrioxamine and ice-cold perchloric acid. Supernatants were treated with dinitrophenylhydrazine reagent, extracted with isooctane in the presence of an internal standard, and the derivatives were separated by HPLC. The addition of 4-methyl pyrazole (an alcohol dehydrogenase inhibitor) or metyrapone (a cytochrome P450 inhibitor) had no effect on the amount of recovered AcHO. On the other hand, treatment with the catalase inhibitors sodium azide, cyanamide, or 3-amino-1,2,4-triazole blocked the production of AcHO while the addition of exogenous peroxide or a peroxide-generating system enhanced the production of AcHO. Overall, these results suggest that AcHO may be produced in the brain during alcohol intoxication, through the action of the enzyme catalase.

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