Species differences in the metabolism and macromolecular binding of methapyrilene: a comparison of rat, mouse and hamster.

1. The metabolism of methapyrilene (MPH) by rat, hamster and mouse liver microsomes in vitro was investigated together with the binding of 14C-MPH to calf thymus DNA after metabolic activation. 2. Both quantitative and qualitative differences in MPH metabolism were observed in these three species. Mouse liver microsomes catalyse the formation of two novel isomers of hydroxypyrdylmethapyrilene (hydroxypyridyl-MPH) as determined by mass spectral analysis. N,N'-Didesmethylmethapyrilene (didesmethyl-MPH) was formed in detectable quantities only when hamster liver microsomes were used. 3. Incubation of liver microsomes from all three species catalysed the binding of 14C-MPH to exogenous DNA, which was quantitatively similar for all three species. The effect of the cytochrome P-450 inhibitor, 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), and methimazole, a flavin-dependent monooxygenase inhibitor, on binding differed significantly for the three species studied.

Testing for fluoxymesterone (Halotestin) administration to man: identification of urinary metabolites by gas chromatography-mass spectrometry.

Fluoxymesterone, an anabolic steroid, is metabolized in man primarily by 6 beta-hydroxylation, 4-ene-reduction, 3-keto-reduction, and 11-hydroxy-oxidation. These pathways of metabolism are suggested by the positive identification of 4 metabolites and the tentative identification of 3 other metabolites. Detection of the drug in urine is possible for at least 5 days after a single 10 mg oral dose to previously untreated adult males, by monitoring the presence of 2 metabolites, since the parent drug is not detectable more than 1 day after the dose.

A comparative in vitro metabolic study of methaphenilene and pyribenzamine.

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.

The in vivo metabolism of methapyrilene, a hepatocarcinogen, in the rat.

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.

In vitro metabolism of chlorpheniramine in the rabbit.

The in vitro metabolism of 3-(p-chlorophenyl)-3-(2-pyridyl)-N, N-dimethylpropylamine (chlorpheniramine, I) by rabbit liver microsomes was examined. The metabolites, tentatively identified by gas-liquid chromatography-mass spectrometry, included the mono- and didemethyl metabolites, the aldehyde that results from deamination, and further metabolites of this aldehyde including its intramolecular cyclization product, an indolizine, and its reduction product, the alcohol. Inhibition of metabolism of I by N2, CO, SKF-525A, 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), or deletion of NADPH implies some involvement of cytochrome P-450 in the metabolic reactions. Quantitation of metabolism in these studies accounted for only 69% of the dose, so that binding and/or other undetected metabolic pathways were operative.

Cytochrome P-450 dependent binding of methapyrilene to DNA in vitro.

Methapyrilene ([14C]MPH) was found to bind to calf thymus DNA only after activation by both rat liver microsomes and NADPH. The cytochrome P-450 inhibitors 2,4-dichloro-6-phenylphenoxyethylamine, 2-diethylaminoethyl-2,2-diphenylvalerate and metyrapone inhibited binding, but methimazole, a flavin-dependent monooxygenase inhibitor, had no effect. However, 1,2-epoxy-3,3,3-trichloropropane, an epoxide hydrolase inhibitor, decreased binding by 30%. Pre-treatment of rats with isosafrole, pregnenolone-16 alpha-carbonitrile or phenobarbital had little or no effect on binding while 3-methylcholanthrene pretreatment decreased binding by 37%. Incubations in the presence of either N-acetylcysteine, glutathione, catalase or glutathione-peroxidase decreased binding to DNA while superoxide dismutase had no effect. These data suggest that MPH is metabolically activated to a species which binds to DNA and that this activation may be mediated by cytochrome P-450 isozymes.

Species differences in the in vitro metabolism of methapyrilene.

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.

The effect of chronic methapyrilene treatment on methapyrilene metabolism in vitro.

Rats were fed 100 or 1000 p.p.m. methapyrilene (MPH) in their diet for 1, 2, 4, 8 or 16 weeks. Liver microsomes were prepared from both control and treated rats. After incubation with 1 mM MPH, eight metabolites were detected and six were quantitated. Five of the metabolites have been previously identified as 2-hydroxymethyl-thiophene (ThM), thiophene-2-carboxylic acid (ThCA), N-(2-thienylmethyl)-2-aminopyridine (TMAP), N-2-pyridyl-N',N'-dimethylethylenediamine (PMED), and 2-aminopyridine (AP). Three other metabolites have been tentatively identified based on their mass spectral fragmentation patterns as normethapyrilene (N-MPH), (5-hydroxypyridyl)methapyrilene (HP-MPH), and methapyrileneamide (MPH-A). The same metabolites were found in both control and treated animals, the most abundant being N-MPH and PMED. Pretreatment with MPH resulted in inhibition of both consumption of MPH and formation of some metabolites. However increases in the formation of all of the metabolites also occurred under different treatment conditions. In both control and treated tissue, the preliminary mass balance was less than 55%, except in incubations with tissue from rats treated with 1000 p.p.m. for 8 or 16 weeks where it was 92 and 89%, respectively. Dramatic increases in the fraction of TMAP, MPH-A, N-MPH, and HP-MPH relative to MPH consumed account for the increase in the mass balance after 8 weeks pretreatment with 1000 p.p.m. MPH, and increases in the amounts of PMED, HP-MPH and ThCA account for the higher mass balance after 16 weeks. The toxicological consequences of these complex metabolic changes may be important in the induction of cancer by MPH.

Analytical chemistry at the Games of the XXIIIrd Olympiad in Los Angeles, 1984.

The equipment, methods, logistics, and results of doping-control analyses for the 1984 Los Angeles Olympic Games are discussed in this article. Within 15 days, 1510 different urine specimens underwent 9440 screening analyses by a combination of gas chromatography, gas chromatography-mass spectrometry, "high-performance" liquid chromatography, and radioimmunoassay. These tests covered more than 200 different drugs and metabolites, including psychomotor stimulants, sympathomimetic amines, central nervous system stimulants, narcotic analgesics, and anabolic steroids. The results are summarized by class of drug. Less than 2% of the samples were found to contain a banned drug.

Mutagenicity studies of selected antihistamines, their metabolites and products of nitrosation.

Methapyrilene, four structurally related antihistamines, three metabolites of methapyrilene and two products of the reaction of methapyrilene with nitrite were all tested for mutagenicity to Salmonella typhimurium. The two products of the methapyrilene-nitrite reaction have also been identified as metabolites of methapyrilene. None was mutagenic alone, either with or without rat liver S-9 activation. After reaction with sodium nitrite in acetic acid solution (nitrosation), the products of five of the ten compounds were mutagenic. These compounds were methaphenilene, 2-thiophenemethanol, 2-thiophenecarboxylic acid, N-(2-pyridyl)-N'N'-dimethylethylenediamine and N-(2-thenylmethyl)-2-aminopyridine.

Lidocaine metabolism by rabbit-liver homogenate and detection of a new metabolite.

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.

Metabolism of methapyrilene by rat-liver homogenate.

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.

Species differences in the in vitro metabolism of phencyclidine.

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.

Induction of phencyclidine metabolism by phencyclidine, ketamine, ethanol, phenobarbital and isosafrole.

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.

Phencyclidine metabolism in vitro. The formation of a carbinolamine and its metabolites by rabbit liver preparations.

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.

Cytochrome P-455-nm complex formation in the metabolism of phenylalkylamines. VI. Structure--activity relationships in metabolic intermediary complex formation with a series of alpha-substituted 2-phenylethylamines and corresponding N-hydroxylamines.

The formation of cytochrome P-450 metabolic intermediary (MI) complexes from a homologous series of alpha-substituted 2-phenylethylamines and corresponding N-hydroxylamines was investigated during NADPH-dependent metabolism in liver microsomes from phenobarbital-pretreated rats. The alpha-alkyl substituent consisted of branched and unbranched alkyl chains ranging from 0-4 carbons and the benzyl group. All the compounds but 2-phenylethylamine generated the complex and double-reciprocal plots of the highest observed rate of complex formation vs. substrate concentration gave linear relations over a defined substrate range. The Vmax(obs) values for complex formation by the amines increased markedly with increasing size of the alkyl group and a good correlation was obtained between log Vmax(obs) and the logarithm of the octanol/buffer partition coefficient of the substrates. With the N-hydroxy compounds, complex formation was a much less selective phenomenon and without exception the rates were quite high with Vmax(obs) values as much as 100 times greater than those of the amines. The disappearance of substrate amines was independent of structure and at an initial substrate concentration of 100 microM about a 50% decrease was noted during a 20-min period in all cases. The results substantiate the previous notion that N-oxidation is a prerequisite for MI-complex formation from primary amines. The results also suggest that C- and N-oxidation have different rate-limiting steps and the microsomal enzymes catalyzing the N-oxidation seem to be deeply submerged in the lipid matrix, as amines with a low distribution are inactive or poor substrates for generating the cytochrome P-450 ligand. Also, it is evident that the MI complex formation does not impair the overall metabolism of the amines.

Chemical transformations of xylamine (N-2'-chloroethyl-N-ethyl-2-methylbenzylamine) in solution. Pharmacological activity of the species derived from this irreversible norepinephrine uptake inhibitor.

Xylamine (N-2'-chloroethyl-N-ethyl-2-methylbenzylamine), a nitrogen mustard that irreversibly inhibits norepinephrine uptake, cyclizes in solution to form an aziridinium ion. The first-order rate constants for cyclization at 23 degrees and 37 degrees are 0.12 min-1 and 0.40 min-1, respectively. The aziridinium ion is relatively stable at 23 degrees but hydrolyzes at 37 degrees with a half-time of 70 min. A dimeric compound was indirectly shown to form at 1 mM xylamine through a reaction between the parent mustard and its aziridinium ion. A similar reaction between the 2-hydroxyethylamine and the aziridinium ion does not take place at pH 7.4. The aziridinium ion, its hydrolysis product, and the dimer were synthesized to evaluate directly their effects on norepinephrine uptake in rabbit thoracic aorta. The aziridinium ion was as potent as xylamine as an irreversible uptake inhibitor, and the effects of both compounds were sodium-dependent. The dimer was a weak competitive inhibitor of norepinephrine uptake, with an IC50 of about 10 microM. The 2-hydroxyethylamine, at 100 microM, competitively inhibited only 20% of control norepinephrine accumulation. These results demonstrate that the aziridinium ion is responsible for xylamine's uptake blocking activity and that the other xylamine derivatives do not influence this action.