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The 4-hydroxylation of S(+)- and R(-)-methamphetamine by rat liver microsomes was examined in Sprague-Dawley and Dark Agouti strains to determine the role of cytochrome P4502D (CYP2D) subfamily isozymes in catalyzing the reaction. In the study, anti-P450-BTL IgG, bufuralol, and quinine, a substrate and inhibitors of CYP2D isozymes, respectively, were found to block approximately 90% of the reaction as catalyzed by microsomes from Sprague-Dawley rats. Reconstituted systems of CYP2D isozymes purified from rat liver microsomes also mediated the reaction. These observations and the minimal activity found in microsomes from Dark Agouti rats support the notion that methamphetamine, like other phenylisopropylamine compounds, is oxidized on the 4-position of the aromatic ring by CYP2D isozymes.

The purpose of this study was to determine whether phencyclidine (PCP) inhibits constitutive hepatic cytochrome P450 (CYP) isozymes when administered to naive adult male Sprague-Dawley rats. Animals were pretreated with PCP (25 mg/kg/day for 2 days), killed 3 and 16 hr after the last dose, and liver microsomes prepared. The washed microsomes were then assayed for benzphetamine, methamphetamine (MA), and methylenedioxymethamphetamine (MDMA) N-demethylation together with MDMA demethylenation and MA 4-hydroxylation activities. MDMA demethylenation (low substrate concentration), MA 4-hydroxylation, and metoprolol alpha-hydroxylation reactions, which are catalyzed by CYP2D isozymes, were reduced > 74% 3 hr after the last PCP dose and were only partially restored 13 hr later. Benzphetamine and (-)-MDMA N-demethylation activities were restored to control values 16 hr after the last dose. These results indicate that PCP suppresses constitutive isozymes, including CYP2C11 and members of the CYP2D subfamily. The suppression of cytochromes P450 activity by PCP in vivo is consistent with its in vitro actions found in this and other studies, and demonstrates that alteration of CYP activity is another pharmacological effect of this compound.

The metabolism of methylenedioxymethamphetamine (MDMA, "ecstasy") was examined in a microsomal preparation of the yeast Saccharomyces cerevisiae expressing human debrisoquine hydroxylase, CYP2D6. Only one product, dihydroxymethylamphetamine (DHMA), was detected in the incubation mixture, and this product accounted for all of the substrate consumption at low concentration (10 microM). Mean +/- SD values of apparent Km(microM) and Vmax (nmol/min per nmol P450) for the demethylenation of (+) and (-)-MDMA at low concentrations (1-100 microM) were 1.72, 0.12 and 6.45, 0.10 and 2.90, 0.10 and 7.61, 0.06, respectively. At high concentrations (> 1000 microM) substrate inhibition was noted, with Ki values of 14.2 and 28.2 mM, respectively, for the (+) and (-) enantiomers. Incubation of MDMA isomers with human liver microsomes indicated that their demethylenation is deficient in the poor metabolizer phenotype. Thus, MDMA is converted to the catecholamine DHMA by CYP2D6, and this may give rise to genetically-determined differences in toxicity.

The cytochrome P450 isozymes in rat liver microsomes that catalyze the demethylenation of methylenedioxymethamphetamine enantiomers to the corresponding dihydroxymethamphetamine were characterized. Dihydroxymethamphetamine formation in liver microsomes from male Sprague-Dawley rats exhibited multienzyme kinetics, with Km values in the micromolar/millimolar range. The stereoselectivity [(+)-isomer versus (-)-isomer] varied from 0.78 to 1.94 after pretreatment of the rats with phenobarbital, 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile, or pyrazole, suggesting that different isozymes participate in the reaction. The low-Km demethylenation was not induced by these compounds and was not inhibited by antibodies raised against CYP2C11. Liver microsomes from female Dark-Agouti rats, a strain genetically deficient in CYP2D1, exhibited demethylenation activities that were 9% of those in microsomes from male Sprague-Dawley rats. The low-Km demethylenation was also inhibited by CYP2D substrates such as sparteine, bufuralol, or desipramine and was almost completely inhibited by antibodies against P450 BTL, which belongs to the CYP2D family. The higg-Km demethylation activity was induced by phenobarbital and pregnenolone-16 alpha-carbonitrile and the activity in both untreated and phenobarbital-induced microsomes was suppressed by anti-CYP2B1 IgG. Experiments with IgG raised against cytochrome b5 suggested that the hemoprotein contributed to the low-Km activity but not the high-Km activity. These results indicate that cytochrome P450 isozymes belonging to the CYP2D subfamily catalyze demethylenation with low Km values and that the reaction occurring with high Km values is likely to be mediated by members of the CYP2B family, but with the possible participation of other phenobarbital-inducible isoforms.

The cytochrome P450 isozymes catalyzing the oxidation of the methylenedioxyphenyl compounds methylenedioxybenzene (MDB) and methylenedioxyamphetamine (MDA) have been investigated in rabbit liver preparations. The aromatic ring in MDB undergoes both demethylenation to catechol and aromatic hydroxylation to sesamol, whereas that in MDA undergoes only demethylenation to dihydroxyamphetamine. Formation of catechol and sesamol from MDB in microsomal incubation mixtures was enhanced about 5- and 3-fold, respectively, by pretreatment of the rabbits with phenobarbital, which induced CYP2B4 and CYP4B1. The cytochrome P450 isozyme responsible for aromatic hydroxylation of MDB was induced by beta-naphthoflavone and was inhibited by alpha-naphthoflavone. Microsomal demethylenation of MDA was minimally sensitive to pretreatment of the rabbits with phenobarbital, beta-naphthoflavone, pyrazole, or rifampicin. However, MDA competitively inhibited the N-demethylation of erythromycin. Antibodies against CYP2B4, but not those against CYP4B1, caused a marked inhibition of the demethylenation and aromatic hydroxylation of MDB. Antibodies against CYP2C3 did not inhibit the demethylenation of MDA, nor did substrates or inhibitors of the CYP2D family except for bufuralol. MDB and MDA were both capable of forming metabolic intermediate complexes, and the rate of complex formation was accelerated by phenobarbital induction. Reconstitution experiments with CYP2B4 suggested that phenobarbital-inducible complex formation from MDA was not due to the carbene pathway involving the methylenedioxy group but was due to oxidation of the amino group. These results indicate that CYP2B4 oxidizes different regions of methylenedioxyphenyl compounds depending on their structure. MDB undergoes oxidation at the methylenedioxy group (major) and the benzene ring (minor). MDA is oxidized at the alkylamino side chain at the nitrogen and alpha-carbon. The results suggested that one or more constitutive isoforms (probably unknown) of cytochrome P450 present in rabbit liver microsomes are primarily responsible for MDA demethylenation but that CYP3A6 contributes slightly.