RESUMO
In vitro studies were conducted to identify the hepatic cytochrome P450 (CYP) enzymes responsible for the oxidative metabolism of loxapine to 8-hydroxyloxapine, 7-hydroxyloxapine, N-desmethylloxapine (amoxapine) and loxapine N-oxide. These studies included use of cDNA-expressed enzymes, correlation analysis with 12 phenotyped human liver microsomal samples, and use of selective inhibitors of cytochrome P450s. The resultant data indicated that loxapine was mainly metabolized by human liver microsomes to (i) 8-hydroxyloxapine by CYP1A2, (ii) 7-hydroxyloxapine by CYP2D6, (iii) N-desmethyloxapine by CYP3A4 and (iv) loxapine N-oxide by CYP3A4. The involvement of flavin-containing monooxygenase (FMO) in the formation of loxapine N-oxide was also observed.
Assuntos
Antipsicóticos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Loxapina/metabolismo , Antipsicóticos/química , Sistema Enzimático do Citocromo P-450/química , Regulação Enzimológica da Expressão Gênica , Humanos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , OxirreduçãoRESUMO
Two of the abundant conjugates of human nicotine metabolism result from the N-glucuronidation of S-(-)-nicotine and S-(-)-cotinine, transformations we recently demonstrated in liver microsomes. We further studied these microsomal N-glucuronidation reactions with respect to human hepatic interindividual, human intertissue, and interspecies hepatic variation. The reactivities of microsomes from human liver (n = 12), various human tissues, and liver from eight species toward the N-glucuronidation of S-(-)-nicotine and S-(-)-cotinine, and also R-(+)-nicotine in human liver were examined. Assays with (14)C-labeled substrates involved radiometric high-performance liquid chromatography. For the human liver samples examined there were 13- to 17-fold variations in the catalytic activities observed toward S-(-)-nicotine, R-(+)-nicotine, and S-(-)-cotinine. Gender and smoking effects were studied, and after exclusion of an outlier a decrease in catalytic activity in females was observed. Significant correlations were observed between all three analytes, indicating that the same UDP-glucuronosyltransferase(s) enzyme is likely to be involved in these transformations. Catalytic activities were not observed for human gastrointestinal tract (colon, duodenum, ileum, jejunum, and stomach), kidney, or lung microsomes. For the seven animal species examined, activity was measurable only for monkey, guinea pig, and minipig, and only for S-(-)-nicotine N-glucuronidation and at rates 10- to 40-fold lower than humans. Activity was not measurable in the case of dog, mouse, rabbit, or rat, for the latter under five different treatment conditions for one of the strains. In conclusion, there are large hepatic interindividual variations in N-glucuronidation of S-(-)-nicotine and S-(-)-cotinine, in human extrahepatic metabolism seems limited, and none of the animal strains examined resembled human.
Assuntos
Cotinina/metabolismo , Variação Genética , Microssomos Hepáticos/metabolismo , Nicotina/metabolismo , Uridina Difosfato Ácido Glucurônico/metabolismo , Animais , Cotinina/química , Cães , Feminino , Variação Genética/genética , Cobaias , Humanos , Macaca fascicularis , Masculino , Camundongos , Nicotina/química , Coelhos , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Ratos Wistar , Fumar/metabolismo , Especificidade da Espécie , Porco MiniaturaRESUMO
Two predominant human glucuronide metabolites of nicotine result from pyridine nitrogen atom conjugation. The present objectives included determination of the kinetics of formation of S(-)-cotinine N1-glucuronide in pooled human liver microsomes and investigation of the UDP-glucuronosyltransferases (UGTs) involved in N-glucuronidation of nicotine isomers and S(-)-cotinine by use of recombinant enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15). Quantification was by radiochemical high-performance liquid chromatography with use of radiolabeled substrates. S(-)-Cotinine N1-glucuronide formation in human liver microsomes was proven by comparing the chromatographic behaviors and electrospray ionization-mass spectral characteristics of the metabolite with a synthetic reference standard. This glucuronide was formed by one-enzyme kinetics with K(m) and V(max) values of 5.4 mM and 696 pmol/min/mg, respectively, and the apparent intrinsic clearance value (V(max/Km)) was 9-fold less than that previously determined for S(-)-nicotine N1-glucuronide (0.13 versus 1.2 microl/min/mg) using the same pooled microsomes. This comparison of values is consistent with the observation that on smoking cigarettes, although the average S(-)-cotinine plasma levels usually far exceed S(-)-nicotine levels, the urinary recovery of S(-)-cotinine N1-glucuronide only averages 3-fold greater than for S(-)-nicotine N1-glucuronide. None of the UGTs examined catalyzed the N-glucuronidation of S(-)-nicotine, R(+)-nicotine, and S(-)-cotinine, including UGT1A3 and UGT1A4, the only isoforms known to catalyze many substrates at a tertiary amine. Also, neither S(-)-nicotine or S(-)-cotinine affected enzyme inhibition of trifluoperazine, a UGT1A4 substrate. It would appear that the same, as yet unexamined, UGT catalyzes the N-glucuronidation of both cotinine and nicotine.
Assuntos
Cotinina/análogos & derivados , Cotinina/metabolismo , Glucuronatos/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Nicotina/análogos & derivados , Nicotina/metabolismo , Catálise , Humanos , Técnicas In Vitro , Cinética , Microssomos Hepáticos/metabolismo , Proteínas Recombinantes/metabolismo , EstereoisomerismoRESUMO
N-Glucuronidation at an aromatic tertiary amine of 5-membered polyaza ring systems was investigated for a model series of eight 1-substituted imidazoles in liver microsomes from five species. The major objectives were to investigate substrate specificities of the series in human microsomes and interspecies variation for the prototype molecule, 1-phenylimidazole. The formed quaternary ammonium-linked metabolites were characterized by positive ion electrospray mass spectrometry. The incubation conditions for the N-glucuronidation of 1-substituted imidazoles were optimized; where for membrane disrupting agents, alamethicin was more effective than the detergents examined. The need to optimize alamethicin concentration was indicated by 4-fold interspecies variation in optimal concentration and by a change in effect from removal of glucuronidation latency to inhibition on increasing concentration. For the four species with quantifiable N-glucuronidation of 1-phenylimidazole, there were 8- and 18-fold variations in the determined apparent K(m) (range, 0.63 to 4.8 mM) and V(max) (range, 0.08 to 1.4 nmol/min/mg of protein) values, respectively. The apparent clearance values (V(max)/K(m)) were in the following order: human congruent with guinea pig congruent with rabbit > rat congruent with dog (no metabolite detected). Monophasic kinetics were observed for the N-glucuronidation of seven substrates by human liver microsomes, which suggests that one enzyme is involved in each metabolic catalysis. No N-glucuronidation was observed for the substrate containing the para-phenyl substituent with the largest electron withdrawing effect, 1-(4-nitrophenyl)imidazole. Linear correlation analyses between apparent microsomal kinetics and substrate physicochemical parameters revealed significant correlations between K(m) and lipophilicity (pi(para) or log P values) and between V(max)/K(m) and both electronic properties (sigma(para) value) and pKa.