Functional and molecular characterization of UDP-glucuronosyltransferase 2 family in cynomolgus macaques.

UDP-glucuronosyltransferases (UGTs) are essential enzymes metabolizing endogenous and exogenous chemicals. However, characteristics of UGTs have not been fully investigated in molecular levels of cynomolgus macaques, one of non-human primates widely used in preclinical drug metabolism studies. In this study, three UGT2A cDNAs (UGT2A1, 2A2, and 2A3) were isolated and characterized along with seven UGT2Bs previously identified in cynomolgus macaques. Several transcript variants were found in cynomolgus UGT2A1 and UGT2A2, like human orthologs. Cynomolgus UGT2A and UGT2B amino acid sequences were highly identical (87-96%) to their human counterparts. By phylogenetic analysis, all these cynomolgus UGT2s were more closely clustered with their human homologs than with dog, rat, or mouse UGT2s. Especially, UGT2As showed orthologous relationships between humans and cynomolgus macaques. All the cynomolgus UGT2 mRNAs were expressed in livers, jejunum, and/or kidneys abundantly, except that UGT2A1 and UGT2A2 mRNAs were predominantly expressed in nasal mucosa, like human UGT2s. UGT2A and UGT2B genes together form a gene cluster in the cynomolgus and human genome. Among the seven cynomolgus UGT2Bs heterologously expressed in yeast, UGT2B9 and UGT2B30 showed activities in estradiol 17-O-glucuronidation and morphine 3-O-glucuronidation but did not show activities in estradiol 3-O-glucuronidation, similar to human UGT2Bs. In liver microsomes, cynomolgus macaques showed higher estradiol 17-O-glucuronidase and morphine 3-O-glucuronidase activities than humans, suggesting functional activities of the responsible UGT2B enzymes in cynomolgus macaques. Therefore, cynomolgus UGT2s had overall molecular similarities to human UGT2s, but also showed some differences in UGT2B enzyme properties.

Suitable albumin concentrations for enhanced drug oxidation activities mediated by human liver microsomal cytochrome P450 2C9 and other forms predicted with unbound fractions and partition/distribution coefficients of model substrates.

Albumin has reportedly enhanced cytochrome P450 (P450)-mediated drug oxidation rates in human liver microsomes. Consequently, measurements of clearances and fractions metabolized could vary depending on the experimental albumin concentrations used. In this study, the oxidation rates of diclofenac and warfarin by human liver microsomes were significantly enhanced in the presence of 0.10% (w/v) bovine serum albumin, whereas those of tolbutamide and phenytoin required 1.0% and 2.0% of albumin for significant enhancement. Values of the fractions metabolized by P450 2C9 for four substrates did not markedly change in the presence of albumin at the above-mentioned concentrations. The oxidation rates of bupropion, omeprazole, chlorzoxazone and phenacetin in human liver microsomes were reportedly enhanced by 0.5%, 1%, 2% and 2% of albumin, respectively. Analysis of reported intrinsic clearance values and suitable albumin concentrations for the currently analyzed substrates and the reported substrates revealed an inverse correlation, with warfarin as an outlier. Suitable albumin concentrations were multivariately correlated with physicochemical properties, that is, the plasma unbound fractions, octanol-water partition coefficient and acid dissociation constant (r = 0.98, p<.0001, n = 10). Therefore, multiple physicochemical properties may be determinants of suitable albumin concentrations for substrate oxidations in human liver microsomes.