N-demethylation In Liver Microsomes Research Articles

The influence of methyl substitution on the N-demethylation and N-oxidation of normethadone in animal species.

Abstract N-Monodemethylation and N-oxidation were shown to be the major routes of metabolism of normethadone, (—)-methadone and (—)-isomethadone in vitro by hepatic microsomal preparations from rat, rabbit, guinea-pig, mouse and hamster. The rate of N-oxidation was decreased and the rate of N-demethylation was increased by the introduction of the methyl substituent into normethadone; the configuration of the methyl substituent influenced these processes. Km and Vmax values were determined for liver microsomal N-demethylation of normethadone, (—)-methadone and (—)-isomethadone by rat and guinea-pig and for the N-oxidation of normethadone by guinea-pig. The use of selective inhibitors showed that N-demethylation was not preceeded by N-oxidation in these compounds.

CYP3A5 Contributes Significantly to CYP3A-mediated Drug Oxidations in Liver Microsomes from Japanese Subjects

The purpose of this study was to evaluate a contribution of polymorphic cytochrome P450 (CYP) 3A5 to the oxidation of diltiazem, midazolam and testosterone by liver microsomes from Japanese subjects. Twenty-seven liver samples were classified into three groups according to the CYP3A5 genotypes; CYP3A5(*)1/(*)1 (n=3), (*)1/(*)3 (n=12) and (*)3/(*)3 (n=12). The results of genotyping and immunochemical quantitation of CYP3A5 protein showed a good accordance between the CYP3A5 genotype and CYP3A5 content but not CYP3A4 content in liver microsomes. The expression levels of hepatic CYP3A5 protein ranged from 20 to 60% of the sum of CYP3A4 and CYP3A5 contents in subjects with at least one wild type allele ((*)1). The CYP3A5 contents correlated well with liver microsomal activities of diltiazem N-demethylation, midazolam 1'- and 4-hydroxylations and testosterone 6beta-hydroxylation among subjects carrying at least one (*)1 allele. In addition, the correlation coefficients of CYP3A5 contents with the rates of diltiazem N-demethylation, midazolam 1'-hydroxylation and testosterone 6beta- hydroxylation were higher than those of CYP3A4, although the value of CYP3A5 with the midazolam 4-hydroxylation rate was similar to that of CYP3A4. Kinetic analyses revealed a biphasic diltiazem N-demethylation in liver microsomes from subjects carrying the (*)1 allele. The apparent V(max)/K(m) values for recombinant CYP3A5 indicated the greater contributions to diltiazem N-demethylation and midazolam 1'-hydroxylation as compared with CYP3A4. These results suggest that polymorphic CYP3A5 contributes markedly to the drug oxidations, particularly diltiazem N-demethylation, midazolam 1'- hydroxylation and testosterone 6beta-hydroxylation by liver microsomes from Japanese subjects.

Clinical implications of drug interactions with the cytochrome P-450 enzyme system associated with omeprazole

Interactions with the hepatic cytochrome P-450 microsomal enzyme system, as evidenced by statistically significant changes in pharmacokinetic parameters, have been described with some H2-receptor antagonists. Omeprazole is the first of a new class of antisecretory agents inhibiting gastric secretion by blocking hydrogen potassium ATPase. Omeprazole contains a benzimidazole moiety and thus has the potential to interact with the cytochrome P-450 enzyme group. In vitro, in vivo and human clinical studies have assessed whether such an interaction occurs, and the potential clinical consequences, in patients receiving omeprazole therapy. In vitro studies have demonstrated that omeprazole influences O-deethylation and N-demethylation in liver microsomes and the clearance and elimination half-life of antipyrine in isolated perfused liver preparations. Overall, the studies reviewed suggest that omeprazole has a differential affinity toward specific cytochrome P-450 isozymes. In vivo animal studies have demonstrated that omeprazole prolongs pentobarbital sleep times and half-life and decreases [14C]-aminopyrine elimination. Human clinical studies have not demonstrated the "all or none" effect of omeprazole on cytochrome P-450-mediated drug interactions, as is seen with cimetidine. These studies confirm in vitro findings that omeprazole is a differential inhibitor of drug metabolism: interactions have been demonstrated with the model drugs aminopyrine and antipyrine, and the therapeutic drugs diazepam, phenytoin, and warfarin but not with theophylline or propranolol. Although caution should be exercised when initiating omeprazole therapy in patients taking concomitant diazepam, warfarin, and phenytoin, clinically significant drug interactions appear unlikely.

Effects of ethinylestradiol and testosterone implants on hepatic microsomal cytochrome P450 monooxygenases of birth gonadectomized male and female Dark Agouti rats

Monooxygenases in the cytochrome P450 IIIA subfamily are induced by a number of their xenobiotic substrates and by testosterone, an endobiotic substrate of importance in their regulation. 17α-Ethinylestradiol (EE) is also metabolized by these enzymes and in this study Dark Agouti rats were used to examine the effects of subcutaneous implantation of controlled release silastic capsules containing EE to determine if this steroid also induces these enzymes. Data were compared with results obtained from equivalent groups of animals implanted with capsules containing testosterone propionate (TP). Liver microsomes prepared from male and female rats were used to identify intrinsic gender differences in the monooxygenases studied and gender differences in the responses to the implanted steroids were also determined. Effects due to imprinting of growth hormone secretion patterns were controlled by using male and female birth gonadectomized animals. Results obtained from groups with blank implants showed there were no effects due to the silastic implant material itself on the monooxygenases studied. The specific activities of erythromycin N-demethylation in liver microsomes of both EE and TP implanted male and female birth gonadectomized animals were enhanced relative to corresponding blank implanted controls consistent with both steroids having an effect to induce activity attributable to cytochrome P450 IIIA isoforms. Immunoinhibition studies using microsomes from EE treated female rats with erythromycin as substrate provided further evidence for this steroid having this induction effect. The specific activity of ethylmorphine N-demethylation was however not increased in microsomes prepared from the EE implanted female animals and was decreased in the corresponding male preparations. These findings distinguished the response to this steroid from that to TP and suggested induction by this estrogen of an isoform(s) having a more limited range of substrates than has characteristically been found in this subfamily. EE treatment also caused an increase in diazepam C 3 hydroxylase consistent with an effect to induce P450 IIIA activity but this was found only in microsomes from birth gonadectomized female animals. This was in contrast to the effect of TP treatment which produced increases in this monooxygenase in both male and female animals. Another gender specific effect of EE was a striking decrease in morphine N-demethylase activity seen only in birth gonadectomized male rats. This again contrasted with the effect of TP which caused a marked increase in this activity in liver microsomes of both male and female birth gonadectomized animals consistent with the proposal that testosterone is important in the regulation of this activity. EE also caused decreases in diazepam N-demethylase specific activity in both male and female rats and spectral evidence indicated that mechanisms other than heme alkylation may be responsible for the decreased activities of these monooxygenases.

Simultaneous determination of aminopyrine hydroxylation and aminopyrine N-demethylation in liver microsomes by high-performance liquid chromatography

Aminopyrine and its metabolites, including 3-hydroxymethyl-2-methyl-4-dimethylamino-1-phenyl-3-pyrazoline-5-one which is a hydroxylated metabolite of aminopyrine, were separated on a reversed-phase (C 8) Radial-Pak column using a mobile phase of methanol—triethylamine—water (30:1:69) adjusted to pH 5.40 with acetic acid. Detection of the peak was performed by an ultraviolet detector at 254 nm. By the rapid and simple method, aminopyrine hydroxylation as well as aminopyrine N-demethylation in liver microsomes can be examined simultaneously.

Simultaneous determination of aminopyrine hydroxylation and N-demethylation in liver microsomes by HPLC and effects of inducers on these enzyme reactions

Aminopyrine(AM) has been used as a model substrate for investigation of drug metabolism. The major metabolic route is N-demethylation that was confirmed in liver microsomes. Another route, however, the oxidation of the 3-methyl group has not been confirmed in liver microsomes. AM and its metabolites including 3-hydroxymethyl-2-methyl-4-dimethylamino-1-phenyl-3-pyrazoline-5-one (AM-OH), which is a 3-hydroxylated metabolite, were separated on a reversed-phase(Cg) Radial-Pak column using a mobile phase of 30% methanol, 1% triethylamine and 69% water adjusted to pH 5.40 with acetic acid.By this rapid and simple method, AM hydroxylation was confirmed in liver microsomes of rats. The hydroxylation in control rat has two Km values (0.52 and 3.06 mM). Pretreatment of phénobarbital (PB) caused an induction of the hydroxylation activity and showed a low Km (0.51 mM) only. 3-Methylcholanthrene(3-MC) did not affect the activity and Km value of the hydroxylation. Similarly PB induced AM N-demethylation activity in agreement with former reports and changed the Km (low,0.80 and high,3.20 mM) into one Km (1.43 mM). Therefore AM hydroxylation as well as N-demethylation seems to be a PB-responding pathway.

EFFECTS OF MORPHINE TREATMENT AND STARVATION ON THE SUBSTRATE INTERACTION WITH P-450 IN THE OXIDATION OF DRUGS BY LIVER MICROSOMES

In previous papers (1, 2), it has been reported that in male rats the administration of morphine and also starvation caused a marked decrease in the activities of drug-metabolizing enzymes of liver microsomes for hexobarbital hydroxylation and aminopyrine N-demethylation, but this was not the case in female rats. On the other hand, the activities of drugmetabolizing enzymes for aniline hydroxylation and zoxazolamine hydroxylation were not significantly altered in both sexes or even increased by the same treatments. The castration of male rats markedly decreased the activities of hexobarbital hydroxylation and aminopyrine N-demethylation, while the activities were not altered by the castration of female rats (1, 2). The administration of methyltestosterone to the castrated male and female rats increased the activities of hexobarbital hydroxylation and aminopyrine N-demethylation in liver microsomes and these activities again decreased by the administration of morphine and by starvation in the androgen-treated rats (1, 2). These results indicate that both morphine and starvation decrease the activities of drug-metabolizing enzymes which are stimulated by androgens, probably through the impairment of an androgen-dependent stimulating mechanism (1-4). Recent studies have established that an unique hemoprotein clled P-450 (5) is involved as the oxygen activating component in a number of NADPH-dependent monooxygenase reactions, such as hydroxylations of drugs and steroid hormones (6-10). Imai and Sato (11) and Schenkman et al. (12) have recently reported that a number of drugs, substrates of hepatic microsomal hydroxylases, react with the microsomal cytochrome to give two characteristic types of spectral change. These results suggest that the spectral changes observed are indicative of substrate interaction with cytochrome P-450 presumably representing the primary binding of substrate for enzymic hydroxylation (13, 14). Moreover, Schenkman et al. (15) demonstrated that the magnitude of the hexobarbital-induced spectral change was greater in liver microsomes isolated from male rats than in those from female rats and castration of male rats abolished this sex difference. These observations suggested that the sex difference in the hydroxylation of hexobarbital may be due to the difference in the substrate interaction with P-450. The purpose of the present communication is to investigate whether the decrease in the hexobarbital hydroxylation and aminopyrine N-demethylation in liver microsomes from morphine-treated and fasted male rats is related to an impairment of androgeninduced stimulation of the substrate-P-450 interaction.

Effect of Food Additives and Colors on Microsomal Drug-metabolizing Enzymes of Rat Liver

Effects of short term administration of food additives and colors on microsomal drug-metabolizing enzyme in rats liver were investigated.1. Oil yellow OB, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, dibutylhydroxytoluene, propyl gallate and sodium dehydroacetate caused liver enlargement in rats.2. A significant decrease in the activity of serum cholinesterase was observed in the rats treated with oil yellow OB or naphthol yellow S.3. The activities of aminopyrine N-demethylation, hexobarbital hydroxylation and butynamine N-demethylation in liver microsomes were significantly increased in dibutylhydroxytoluene treated rats.4. The duration of pentobarbital narcosis was significantly decreased in dibutylhydroxytoluene treated rats and it appeared to be decreased in dehydroacetic acid treated rats.It is, therefore, supposed that the relatively high lipid soluble compounds which are metabolized in liver microsomes, such as dibutylhydroxytoluene, increased the activities of drug-metabolizing enzymes in liver microsomes.

Die oxydative N-demethylierung von N-monomethyl-p-nitranilin

1. A method is described for the direct measurement of oxidative N-demethylation in liver microsomes. It is based on the spectral difference of the substrate N-monomethyl-p-nitroaniline and its demethylation product p-nitro-aniline. The kinetics of the disappearance of N-monomethyl-p-nitroaniline in suspensions of washed rat or mouse liver microsomes can be determined spectrophotometrically at 420 nm. The pyridine-nucleotide cofactor requirement of this reaction has been studied with the result that only NADPH is active, while NADH has no effect. The reaction can be inhibited by the microsome inhibitor SKF 525-A.