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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.

Phenylcyclohexyldiethylamine (PCDE) is an analog of phencyclidine with low affinity for the N-methyl-d-aspartate receptor that is metabolized to an active monoethyl derivative, phenylcyclohexylethylamine (PCE). In a pharmacokinetic analysis of the ataxia response of rats to i.p. administered PCDE and PCE, ataxia intensity was determined together with plasma and cerebrospinal fluid concentrations of the drugs. The role of PCE as the active metabolite of PCDE was assessed quantitatively by correlating the response with both the plasma and cerebrospinal fluid drug levels. Increased PCE concentrations in the cerebrospinal fluid and plasma were associated with increased ataxia response when either PCDE or PCE was the administered drug. However, the concentration-response curves did not superimpose and the curve after PCDE was shifted to the left of that after PCE, suggesting that PCDE was contributing an effect not accountable by PCE concentration. This apparent potentiation must involve an interaction at sites other than the N-methyl-daspartate receptor. In the analysis of the behavior responses, PCDE was found to induce a greater backpedalling response which has been attributed to interaction with dopamine or serotonin systems, suggesting that other transmitter systems may contribute to the overall ataxia response.

The pharmacokinetics of three phencyclidine analogs--phenylcyclohexyl-diethylamine (PCDE), phenylcyclohexylethylamine (PCE), and phenylcyclohexylamine (PCA)--were determined in rats after intravenous administration of each drug. Because PCE and PCA are major metabolites of PCDE, their plasma levels were also measured after administration of PCDE. Similarly, PCA concentrations was determined after administration of PCE. The data were combined and analyzed by nonlinear regression procedures using compartmental and noncompartmental models to determine the kinetic parameters of PCDE metabolism. The object was to estimate the kinetic constants for the metabolic sequence, PCDE to PCE to PCA. A 6-compartment model (two pools for each analyte) that included saturable components for the conversion of PCDE to PCE and PCE to PCA gave the best fit to the combined data. Despite large uncertainties for some microparameters, useful estimates were obtained for clearances, distribution volumes, and fraction of PCDE or PCE converted to PCE and PCA in vivo under nonsaturating conditions. The estimated fraction of PCDE converted to PCA and the apparent Km value for the conversion of PCDE to PCE were comparable to values obtained in vitro with microsomal preparations, suggesting that metabolic studies in vitro provide reasonable predictors of the biotransformation process in vivo for this class of compounds.

1. Metabolites formed during incubation of methylenedioxybenzene (MDB) and methylenedioxymethamphetamine (MDMA) with rabbit liver microsomes were examined by h.p.l.c.-electrochemical detection and g.l.c.-mass spectrometry. 2. The trifluoroacetyl derivative of metabolite M-1, obtained from MDB, had a molecular ion at m/z 234 and was identified as 3,4-methylenedioxy-6-hydroxybenzene (sesamol) by comparison with authentic material. 3. The trifluoroacetyl derivative of metabolite M-2, obtained from MDMA, exhibited a molecular ion at m/z 401. Experiments with the deuterium substituted variants of MDMA indicated that the product was hydroxylated on the aromatic ring. 4. The formation of these hydroxylated metabolites required NADPH and was inhibited by carbon monoxide, indicating the possible participation of cytochrome P-450. Phenobarbital (PB) induction caused a marked enhancement of MDP hydroxylase activity whereas MDMA hydroxylation was not affected. 5. The aromatic hydroxylation of MDB and MDMA was also observed in a reconstituted system with cytochrome P-450 isozyme IIB4.

The cytochrome P450-mediated oxidative demethylenation of the benzo-1,3-dioxoles (methylenedioxyphenyl compounds, MDPs), methylenedioxybenzene (MDB), methylenedioxyamphetamine (MDA), and methylenedioxymethamphetamine (MDMA), by rabbit liver microsomes and cytochrome P450IIB4 (CYP2B4) was examined. Material balance studies indicated that demethylenation to catechol derivatives is a major metabolic pathway for MDB, MDA and MDMA. The reactions required NADPH and were inhibited by CO/O2 (4:1, v/v). Biphasic double-reciprocal plots of MDMA, MDA and MDB oxidation suggested participation of more than one isozyme of cytochrome P450 in the reaction. Phenobarbital (PB) induction was selective in that the Vmax values for MDB were increased but not those for MDA and MDMA. Exposure of liver microsomes from PB-pretreated animals to phencyclidine (PCP) markedly suppressed MDB oxidation but had little effect on MDA and MDMA demethylenation. Reconstitution experiments with CYP2B4 demonstrated that MDB is a good substrate for the isozyme; but the relative demethylenation activities for MDA and MDMA were 1 and 2% of that for MDB. These results indicate that the PB-inducible isozymes such as CYP2B4 appear to play an important role in MDB demethylenation, whereas MDA and MDMA oxidation is mediated mainly by constitutive isozymes.

The pharmacodynamics and pharmacokinetics of three phencyclidine analogs, differing from phencyclidine (PCP) only in the nature of the amine structure, were determined after intravenous doses of equimolar amounts to rats. The purpose of the study was to assess the role of pharmacokinetics in the in vivo potency of the compounds. The compounds examined were phenylcyclohexyl-pyrrolidine (PCPY), diethylamine (PCDE), ethylamine (PCE), and phencyclohexylamine (PCA). The behavior responses monitored included ataxia and others previously shown to be characteristic of PCP. In contrast to their relative affinities for the MK 801 binding site, the behavioral potencies of PCE, PCDE and PCPY were comparable to PCP. The major discrepancy occurred with PCDE, whose affinity for the NMDA receptor was 1/20th of PCP. The pharmacokinetic studies showed that the discrepancy between in vivo and in vitro activity of PCDE could be partially accounted for by its conversion to PCE, a relatively potent PCP-like agent.

The oxidative demethylenation reactions of (methylendioxy)phenyl compounds (MDPs), (methylenedioxy)benzene (MDB), (methylenedioxy)amphetamine (MDA), and (methylenedioxy)methamphetamine (MDMA), were evaluated by using two hydroxyl radical generating systems, the autoxidation of ascorbate in the presence of iron-EDTA and the iron-catalyzed Haber-Weiss reaction conducted by xanthine/xanthine oxidase with iron-EDTA. Reaction products generated when MDB, MDA, and MDMA were incubated with the ascorbate or xanthine oxidase system were catechol, dihydroxyamphetamine (DHA), and dihydroxymethamphetamine (DHMA), respectively. The reaction required the presence of either ascorbic acid or xanthine oxidase. Levels of each catechol increased in proportion to ferric ion concentration and were suppressed by desferrioxamine B methanesulfonate (desferal). Catalase (CAT) inhibited the oxidation by the ascorbate system whereas superoxide dismutase (SOD) had little effect. The addition of hydrogen peroxide to the reaction mixture stimulated the oxidation, but the reaction was not initiated by hydrogen peroxide alone, suggesting that hydrogen peroxide acts as a precursor of hydroxyl radical. SOD and CAT suppressed the demethylenation reactions in the xanthine oxidase system. Hydroxyl radical scavenging agents such as ethanol, benzoate, DMSO, and thiourea effectively inhibited the oxidation by both systems. Urea, which has little effect on hydroxyl radical, was without any effect. These results indicated that hydroxyl radical can effect the cleavage of methylenedioxy group on MDPs.

The metabolism of methapyrilene(I) in rat-liver 9000 g supernatant fraction produced four new metabolites positively identified by comparison of g.l.c. retention times and mass-spectral fragmentation patterns with those of authentic materials. These compounds are 2-thiophene-methanol(VI), 2-thiophenecarboxylic acid(VII), N-2-pyridyl-N'-dimethylethylenediamine(IX) and 2-aminopyridine(X). In addition, the previously known metabolite 2-[(2-thienylmethyl)amino]-pyridine(VIII) was also positively identified. Six other metabolites were tentatively identified by analysis of the mass-spectral fragmentation patterns of both the trimethylsilyl and the tertiary butyldimethylsilyl derivatives of each compound. These compounds are tentatively identified as: normethapyrilene(II), (hydroxypyridyl)-methapyrilene(XII), methapyrilenamide(XIV), (hydroxypyridyl)-normethapyrilene(XVI), (hydroxypyridyl)-desmethylmethapyrilenamide(XVII), and (hydroxypyridyl) methapyrilenamide(XVIII). Quantification of II, VI-X, XII and XIV account for approx. 65% of the metabolized methapyrilene.

1. The metabolism of methapyrilene (I), was examined in vivo by g.l.c. and g.l.c.-mass spectrometric analysis of rat urinary extracts. 2. Dosing the animals with tetradeuterium-labelled I helped identify 7 different metabolites of I in the urine, including (5-hydroxylpyridyl)-methapyrilene, which was identified by comparison with a synthetic reference standard. 3. After 4 weeks of treatment with I, rats also excrete detectable amounts of the 3- and (6-hydroxylpyridyl)-methapyrilene metabolites suggesting that pretreatment with I alters the metabolism of the pyridine ring. 4. Metabolic removal of the 2-thienylmethylene moiety is also facile, as large amounts of N'-(2-pyridyl)-N,N-dimethylethylenediamine and its metabolite N'-[2(5-hydroxylpyridyl)]-N,N-dimethylethylenediamine are excreted under all dosing regimens. 5. Urinary concn of both I and metabolites decline with time, despite continuous dosing, indicating a change in absorption, metabolism, and/or excretion of I on repeated dosing.

Four products of the in vitro oxidative metabolism of the piperidine ring of phencyclidine, 5-(1-phenylcyclohexylamino)valeraldehyde, V; N-(1-phenylcyclohexyl)-1,2,3,4-tetrahydropyridine, VIII; 5-(1-phenylcyclohexylamino)valeric acid, VII; and 1-phenylcyclohexylamine, IX, have been identified following derivatization, by GC/MS with stable isotope labeling and/or synthesis. The enamine, VIII, may be a work-up elimination product of a carbinolamine, alpha-hydroxy-N-(1-phenylcyclohexyl)piperidine, IV. The formation of all metabolites requires microsomal enzymes, but VII and the previously described N-(5-hydroxypentyl)-1-phenylcyclohexylamine, VI, also require soluble enzymes. Quantitative or semiquantitative data show that VI and VIII appear and disappear with time, whereas VII seems to be a terminal metabolite.

The metabolism of 1-(1-phenylcyclohexyl)-piperidine (phencyclidine or PCP) by liver preparations from cat, monkey, rabbit and rat has been studied. 4-Phenyl-4-piperidinocyclohexanol (I), 1-1-phenylcyclohexyl-4-hydroxy-piperidine (II), N-(5-hydroxypentyl)-1-phenylcyclohexylamine (IX) and 5-(1-phenylcyclohexylamino)-valeric acid (X) were found in all species, but liver preparations of rat and rabbit were much more active than those of cat or monkey in metabolizing PCP. Only rabbit produced 4-(4'-hydroxypiperidino)-4-phenylcyclohexanol (III) in amounts detectable by g.l.c. Mass balance calculations of PCP, I, II, III, IX and X in the cat, monkey and rat indicate that other metabolic pathways not measured in this study are operative.

1. In vitro metabolism of methaphenilene (MFN) and pyribenzamine (PBZ) was compared to that of methapyriline (MPH) in rat, because chronic treatment with MPH causes cancer in rats, whereas MFN and PBZ cause no cancer. 2. G.l.c. and mass spectrometry were used to identify 7 metabolites of MFN and 6 of PBZ in extracts of rat liver microsome incubations. 3. Quantification of the metabolic pathways revealed that N-oxide formation is considerably more important for both MFN and PBZ than for MPH, and only MPH forms an amide as a metabolic product. 4. Quantitative balance studies show that a lower recovery is apparent for metabolic experiments with MPH than for either MFN or PBZ under all conditions examined, indicating that significant metabolic pathways for MPH exist which are not being measured under these conditions.

Metabolism of lidocaine in rabbit liver 9000 g supernatant fraction was examined. A capillary g.l.c. assay was developed to separate seven known metabolites of lidocaine, and all seven metabolites were identified in extracts of incubations of lidocaine with rabbit-liver fractions. These metabolites were monoethylglycinexylidide(I), glycinexylidide(II), 3-hydroxymonoethylglycinexylidide(III), 3-hydroxylidocaine(IV), 4-hydroxylidocaine(V), xylidine(VI) and 4-hydroxyxylidine(VII). A new metabolite, 2-amino-3-methylbenzoic acid(VIII), was identified in extracts of incubations of lidocaine with rabbit-liver fractions, by comparison of the mass-spectral fragmentation patterns and g.l.c. retention time with those of the authentic compound. The formation of VIII is dependent on protein, NADPH, time, O2, and the presence of soluble enzymes. Quantitative analysis of metabolites I-VIII after a two hour incubation accounts for 89% of the metabolized lidocaine.

The in vitro metabolism of phencyclidine by rabbit liver 9000g supernatant fraction produces primarily three known hydroxylated metabolites-namely, 4-phenyl-4-piperidinocyclohexanol, 4-(4'-hydroxypiperdino)-4phenylcyclohexanol, and 1-(1-phenylcyclohexyl)-4-hydroxypiperidine-plus a new metabolite formed by oxidative scission of the piperidine ring yielding an aminoalcohol, and much smaller amounts of five unidentified metabolites. Incubation with 50% deuterium-labeled PCP indicates that these compounds are metabolites by monitoring the resulting doublets found in their mass spectra. The time, cofactor, and protein-dependent formation of these compounds confirms that they are indeed, metabolites. DPEA inhibits the production of the four quantitated metabolites with maximal I50 values of approximately 50 microM, implying the involvement of cytochrome P-450 in these reactions.

The in vitro metabolism of phencyclidine (PCP) was investigated in 9000 g supernatant fractions of both control and PCP-, ketamine-, ethanol-, phenobarbital- or isosafrole-pretreated rats. Levels of PCP, trans-4-phenyl-4-piperidinocyclohexanol (I), 1-(1-phenylcyclohexyl)-4-hydroxypiperidine (II), N-(5-hydroxypentyl)-1-phenylcyclohexylamine (IX), and 5-(1-phenylcyclohexylamino)-valeric acid (X) were monitored by gas chromatographic analysis in all cases. The inhibition of metabolism by N2, CO, SKF-525A or 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), or deletion of NADPH or protein, implied the involvement of cytochrome P-450 in the reactions. The various inducing agents affected the metabolism of PCP in different ways, implying that at least several isozymes of cytochrome P-450 were involved in the total metabolism. The majority of the consumed PCP was not accounted for by the measured metabolites so that some other metabolic pathways of major quantitative importance must be operative.

1. The metabolism of N,N-dimethyl-N'-(2-pyridyl)-N'-(2-thienylmethyl)-1,2- ethanediamine(methapyrilene, I) by liver microsomes from rat, guinea pig, and rabbit has been examined. 2. Methapyrilene-N-oxide, (III), normethapyrilene, (II), 2-thiophene methanol, (VI), 2-thiophene carboxylic acid, (VII), N-(2-pyridyl)-N',N'-dimethylethylenediamine, (IX), and methapyrilene amide, (XIV) were found in all species. 3. N-(2-Thienylmethyl)-2-amino pyridine, (VIII), 2-aminopyridine, (X), and (5-hydroxypridyl)-methapyrilene, (XII), were detected in rat and rabbit only. 4. N-Hydroxynormethapyrilene, (XXI), was tentatively identified by mass spectral fragmentation patterns only in rabbit liver microsomes incubations; however, it was found in 9000 g supernatant fraction incubations of rabbit, rat and guinea pig. 5. The formation of IX and XII was quantitatively more important in the rat than in either rabbit or guinea pig.