RESUMO
Metabolic disposition of drugs and other xenobiotics includes glucuronidation reactions that are catalyzed by the uridine diphosphate glucuronosyltransferases (UGTs). The most common glucuronidation reactions are O- and N-glucuronidation and in this review, we discuss both, while the emphasis is on N-glucuronidation. Interspecies difference in glucuronidation is another central issue in this review due to its importance in drug development. Accordingly, the available data on glucuronidation in different animals comes mainly from the species that are used in preclinical studies to assess the safety of drugs under development. Both O- and N-glucuronidation reactions are chemically diverse. Different O-glucuronidation reactions are described and discussed, and many drugs that undergo such reactions are indicated. The compounds that undergo N-glucuronidation include primary aromatic amines, hydroxylamines, amides, tertiary aliphatic amines, and aromatic N-heterocycles. The interspecies variability in N-glucuronidation is particularly high, above all when it comes to aliphatic tertiary amines and aromatic N-heterocycles. The N-glucuronidation rates in humans are typically much higher than in animals, largely due to the activity of two enzymes, the extensively studied UGT1A4, and the more recently identified as a main player in N-glucuronidation, UGT2B10. We discuss both enzymes and review the findings that revealed the role of UGT2B10 in N-glucuronidation.
Assuntos
Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Catálise , Humanos , Difosfato de Uridina/metabolismo , Xenobióticos/metabolismoRESUMO
Medetomidine is a chiral imidazole derivate whose dextroenantiomer is pharmacologically active. The major metabolic pathway of dexmedetomidine [(+)-4-(S)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole] in humans is N-glucuronidation at the imidazolate nitrogens. We have purified the N3- and N1-glucuronides of dexmedetomidine, termed DG1 and DG2, respectively, according to their elution order in liquid chromatography and determined their structure by 1H nuclear magnetic resonance (NMR). Studying medetomidine glucuronidation by human liver microsomes (HLMs) and recombinant UDP glucuronosyltransferase (UGT) 1A4 indicated that another human UGT plays a major role in these activities. We now demonstrate that this enzyme is UGT2B10. HLMs catalyzed DG1 and DG2 formation, at a ratio of 3:1, with two-enzyme kinetics that contain both a high-affinity component, K(m1) values of 6.6 and 8.7 microM, and a low-affinity component, K(m2) values > 1 mM. The DG1/DG2 ratio in the case of UGT2B10 was lower, 1.4:1, whereas the substrate affinity for both reactions was high, K(m) values of 11 and 16 microM. UGT1A4 produced mainly DG1 (DG1/DG2 ratio of 6.6:1) at low substrate affinities, K(m) values above 0.6 mM, but superior expression-normalized V(max) values. Levomedetomidine [(-)-4-(R)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole] glucuronidation by HLMs yielded mostly the N3-glucuronide (LG1, structure determined by NMR), with monophasic kinetics and a K(m) value of 14 microM. The activity of UGT1A4 toward levomedetomide was low and generated both LG1 and LG2, whereas UGT2B10 exhibited relatively high activity and sharp regioselectivity, yielding only LG1, with a K(m) value of 7.4 microM. The results highlight the contribution of UGT2B10 to medetomidine glucuronidation and its potential importance for other N-glucuronidation reactions within the human liver.
Assuntos
Agonistas alfa-Adrenérgicos/farmacocinética , Analgésicos não Narcóticos/farmacocinética , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Isoenzimas/metabolismo , Medetomidina/farmacocinética , Microssomos Hepáticos/enzimologia , Cromatografia Líquida , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Proteínas Recombinantes/metabolismo , Espectrofotometria UltravioletaRESUMO
Nicotine biotransformation affects the smoking habits of addicted individuals and therefore their health risk. Using an improved analytical method, we have discovered that the human UDP-glucuronosyltransferase (UGT) 2B10, a liver enzyme previously unknown to conjugate nicotine or exhibit considerable activity toward any compound, plays a major role in nicotine inactivation by direct conjugation with glucuronic acid at the aromatic nitrogen atom. The K(m) value of recombinant UGT2B10 for nicotine (0.29 mM) was similar to that determined for human liver microsomes (0.33 mM), whereas the K(m) value of UGT1A4 for nicotine was almost 10-fold greater (2.4 mM). UGT2B10 was also more active than UGT1A4 in N-glucuronidation of cotinine (oxidative nicotine metabolite), whereas UGT2B7 exhibited only low nicotine glucuronidation activity and was essentially inactive toward cotinine. UGT1A9 did not glucuronidate nicotine or cotinine. Quantitative reverse transcription-polymerase chain reaction showed that UGT2B10 mRNA was exclusively expressed in human liver, whereas UGTs 1A4 and 2B7 were expressed at comparable, although somewhat lower, levels in liver and several other extrahepatic tissues, including kidney and intestine. These findings for UGT2B10 (but not for UGT1A4 and UGT2B7) were mirrored by human tissue activities because nicotine and cotinine glucuronidation rates in intestine microsomes were less than 0.1% that of human liver microsomes. These novel findings solve two seemingly separate questions: which UGT is primarily responsible for nicotine glucuronidation in human liver, and what conjugation reactions are catalyzed by UGT2B10.
Assuntos
Glucuronídeos/análise , Glucuronosiltransferase/metabolismo , Nicotina/metabolismo , Sequência de Aminoácidos , Biotransformação , Sequência Conservada , Cotinina/química , Cotinina/metabolismo , Feminino , Glucuronídeos/química , Glucuronídeos/metabolismo , Glucuronosiltransferase/química , Humanos , Intestinos/química , Intestinos/enzimologia , Isoenzimas/análise , Isoenzimas/metabolismo , Cinética , Masculino , Microssomos/química , Microssomos/enzimologia , Microssomos Hepáticos/química , Microssomos Hepáticos/enzimologia , Dados de Sequência Molecular , Estrutura Molecular , Nicotina/química , Sinais Direcionadores de Proteínas , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , EstereoisomerismoRESUMO
N-Glucuronidation in vitro of six 4-arylalkyl-1H-imidazoles (both enantiomers of medetomidine, detomidine, atipamezole, and two other closely related compounds) by rat, dog, and human liver microsomes and by four expressed human UDP-glucuronosyltransferase isoenzymes was studied. Human liver microsomes formed N-glucuronides of 4-arylalkyl-1H-imidazoles with high activity, with apparent V(max) values ranging from 0.59 to 1.89 nmol/min/mg of protein. In comparison, apparent V(max) values for two model compounds forming the N-glucuronides 4-aminobiphenyl and amitriptyline were 5.07 and 0.56 nmol/min/mg of protein, respectively. Atipamezole showed an exceptionally low apparent K(m) value of 4.0 microM and a high specificity constant (V(max)/K(m)) of 256 compared with 4-aminobiphenyl (K(m), 265 microM; V(max)/K(m), 19) and amitriptyline (K(m), 728 microM; V(max)/K(m), 0.8). N-Glucuronidation of medetomidine was highly enantioselective in human liver microsomes; levomedetomidine exhibited a 60-fold V(max)/K(m) value compared with dexmedetomidine. Furthermore, two isomeric imidazole N-glucuronides were formed from dexmedetomidine, but only one was formed from levomedetomidine. Dog liver microsomes formed N-glucuronides of 4-arylalkyl-1H-imidazoles at a low rate and affinity, with apparent V(max) values ranging from 0.29 to 0.73 nmol/min/mg of protein and apparent K(m) values from 279 to 1640 microM. Rat liver microsomes glucuronidated these compounds at a barely detectable rate. Four expressed human UDP-glucuronosyltransferase isoenzymes (UGT1A3, UGT1A4, UGT1A6, and UGT1A9) were studied for 4-arylalkyl-1H-imidazole-conjugating activity. Only UGT1A4 glucuronidated these compounds at an activity of about 5% of that measured for 4-aminobiphenyl. The observed activity of UGT1A4 does not explain the high efficiency of glucuronidation of 4-arylalkyl-1H-imidazoles in human liver microsomes.