Tadanobu Inaba

Cocaine metabolism: Cocaine and norcocaine hydrolysis by liver and serum esterases

The hydrolysis of cocaine and its N‐demethylated product, norcocaine, by esterases was examined in liver and serum. Both liver and serum enzymatically formed ecgonine methyl ester from cocaine. The liver enzyme had a much lower affinity for cocaine than that of serum, indicating that a different form of esterase was present in liver. The liver enzyme had a similar affinity for both norcocaine and cocaine. Likewise, the serum enzyme showed similar affinities for both substrates. The Vmax estimates, however, were consistently higher for norcocaine than cocaine in both liver and serum. Benzoyl ecgonine, a major metabolite of cocaine formed by hydrolysis, was not produced enzymatically in either serum or liver; the rate of spontaneous formation at physiological pH suggests that this metabolite may arise nonenzymatically in the body.

Metabolism of cocaine in man

Following ingestion of [N‐14CH3]cocaine (10 mg, 2.3 µCi) by 2 healthy subjects, breath, saliva, serum, and urine samples were collected serially. Labeled CO2 production was monitored as a measure of N‐demethylation of cocaine. The cumulative excretion of 14CO2 in 5 hr was 2.4% and 6.2% of the administered dose with half‐lives of 2.3 and 1.4 hr, respectively. The greater N‐demethylation was found in a subject with lower plasma cholinesterase activity. Radioactivity excreted in 0 to 28 hr urine reached 65% to 75% of the dose. Ecgonine methyl ester, a product of cocaine hydrolysis by plasma cholinesterase, was identified as a major metabolite in the urine of both subjects and accounted for 32% to 49% of the urinary metabolites.

Hydrolysis of cocaine in human plasma by cholinesterase

Abstract Hydrolysis of cocaine to ecgonine methyl ester in human plasma is mediated by cholinesterase. Cocaine hydrolysis by plasma is blocked by DFP and eserine and partially inhibited by fluoride. Highly purified cholinesterase from human plasma when diluted to the same benzoylcholine hydrolyzing activity as human plasma, shows the same rate of cocaine hydrolysis as human plasma. There was no detectable enzymatic conversion of cocaine to benzoyl ecgonine in plasma.

Competitive inhibition of sparteine oxidation in human liver by β-adrenoceptor antagonists and other cardiovascular drugs

The rate of oxidation of sparteine by the 9000 x g supernatant fraction of a human liver was measured in the presence of various drugs which exert cardiovascular effects. Hexamethonium, ouabain, caffeine and isoproterenol had no effect on this rate, while alprenolol, metoprolol, oxprenolol, propranolol, timolol, pindolol, lidocaine, mexiletine, 17-n-pentyl-sparteine, tolazoline, quinine, quinidine, cinchonine and cinchonidine inhibited the in vitro reaction competitively. Stereoselective inhibition was observed between quinine (Ki = 15 microM) and quinidine (Ki = 0.06 microM). Genetic evidence suggests that the primary metabolism of sparteine depends on a single species of cytochrome P450. In vitro competitive inhibition of sparteine oxidation by a drug indicates that this drug is capable of occupying the same enzymatic site as sparteine. This may mean that the competing drug is also metabolized at that site and thereby subject to the same genetic variation as sparteines oxidation; absence of inhibition excludes this possibility.

Interactions of amphetamine analogs with human liver CYP2D6

The interaction of fifteen amphetamine analogs with the genetically polymorphic enzyme CYP2D6 was examined. All fourteen phenylisopropylamines tested were competitive inhibitors of CYP2D6 in human liver microsomes. The presence of a methylenedioxy group in the 3,4-positions of both amphetamine (Ki = 26.5 microM) and methamphetamine (Ki = 25 microM) increased the affinity for CYP2D6 to 1.8 and 0.6 microM, respectively. Addition of a methoxy group to amphetamine in the 2-position also increased the affinity for CYP2D6 (Ki = 11.5 microM). The compound with the highest affinity for CYP2D6 was an amphetamine analog (MMDA-2) having both a methoxy group in the 2-position and a methylenedioxy group (Ki = 0.17 microM). Mescaline did not interact with CYP2D6. O-Demethylation of p-methoxyamphetamine (PMA) by CYP2D6 was characterized (Km = 59.2 +/- 22.4 microM, and Vmax = 29.3 +/- 16.6 nmol/mg/hr, N = 6 livers). This reaction was negligible in CYP2D6-deficient liver microsomes, was inhibited stereoselectively by the quinidine/quinine enantiomer pair, and was cosegregated with dextromethorphan O-demethylation (r = 0.975). The inhibitory effect of methylenedioxymethamphetamine (MDMA) was enhanced by preincubation with microsomes, suggesting that MDMA may produce a metabolite complex with CYP2D6. These findings suggest that phenylisopropylamines as a class interact with CYP2D6 as substrates and/or inhibitors. Their use may cause metabolic interactions with other drugs that are CYP2D6 substrates, and the potential for polymorphic oxidation via CYP2D6 may be a source of interindividual variation in their abuse liability and toxicity.

Novel detection assay by PCR-RFLP and frequency of the CYP3A5 SNPs, CYP3A5*3 and *6, in a Japanese population.

In this study, we established useful and reliable methods for the direct detection of the variants of CYP3A5 gene by polymerase chain reaction (PCR) and DdeI restriction analysis. The frequency of CYP3A5 related SNPs in 200 healthy Japanese male subjects was determined. The homozygous wild-type (*1/*1) frequency was 7.0% (14/200), the heterozygous (*1/*3) frequency was 32.5% (65/200) and the homozygous mutant-type (*3/*3) frequency was 60.5% (121/200). The *6 allele was not detected in any of the Japanese individuals. This result suggests that an estimated 40% of the Japanese express relatively high levels of metabolically active CYP3A5 protein. The proposed detection assays are useful for screening the CYP3A5 related SNPs in pharmacogenetic research.

Antipyrine metabolism in the rat by three hepatic monooxygenases

Abstract The relative contribution of 3 oxidative reactions of antipyrine to its metabolism in vivo were assessed by comparing male and female rats, and by studying the effects of phenobarbital (PB) and 3-methylcholanthrene (MC). After [N 14 CH 3 ] antipyrine, 11–15% of the dose was excreted as 14 CO 2 in both sexes as a consequence of N-demethylation. PB pretreatment had no effect but MC doubled 14 CO 2 excretion. The other metabolites, 4-hydroxyantipyrine (4OHA) and 3-hydroxymethylantipyrine (3HMA), in urine were determined by thin-layer chromatography. Of the total activity in the urine, 4OHA represented 30% in male and 40% in female rats; 3HMA represented 35% in male and 20% in female rats. The ratio of 4OHA to 3HMA in both sexes increased after PB and MC, the effect being more pronounced with the latter. The results show that the 3 major oxidative pathways of antipyrine metabolism are mediated by different enzymes, almost certainly different forms of cytochrome P450.

Sparteine metabolism in Canadian Caucasians.

The capacity for sparteine (SP) metabolism was determined in 48 Caucasian subjects by measuring amounts of drug and dehydrogenated metabolites in urine after an oral dose of SP sulfate. Three phenotypes were recognized and were assumed to represent individuals homozygous for poor SP oxidation (group III) and those heterozygous (group II) and homozygous (group I) for extensive SP oxidation. Separation of groups I and II, although incomplete, was improved by alterations in the published analytic procedure. The pattern of deviations from the normal distribution was similar for both dehydrosparteine metabolites. This supports the hypothesis of a common intermediate, the formation of which is monogenically controlled. Correlation analysis of the two metabolites indicates the possibility of further metabolism of 5‐dehydrosparteine.

Deficient metabolism of debrisoquine and sparteine

Genetic deficiencies of alicyclic hydroxylation of debrisoquine and of sparteine oxidation are independently discovered entities, each of clinical significance in its sphere. This paper reports evidence to indicate that these 2 deficiencies have the same cause. Previous investigation of one of the affected subjects had revealed normal oxidative metabolism of amobarbital and antipyrine in terms of both metabolic rates and urinary metabolite patterns. Thus the genetic defect in the metabolism of sparteine and debrisoquine is not a generalized deficiency of drug oxidation or of the cytochrome P450 system.

Inhibition of sparteine oxidation in human liver by tricyclic antidepressants and other drugs.

Testing for competitive inhibition of sparteine oxidation in the 9000 x g supernatant fraction from human liver provides an in vitro means to identify drugs which can bind to the same form of cytochrome P450 which oxidizes sparteine. There has so far been only two outcomes of this test: either the drug examined competed with sparteine for a common binding site, or it did not inhibit the reaction. The results of such in vitro testing implicated the involvement of guanoxan, nortriptyline, desipramine, imipramine, amitriptyline and chlorpromazine with this enzyme. Amobarbital, tolbutamide and guanethidine in therapeutic concentrations did not interfere with sparteine oxidation by this preparation.