N-Glucuronidation of Drugs and Other Xenobiotics

UDP-glucuronosyltransferases (UGTs) are a family of metabolic enzymes responsible for the detoxification of a wide range of endoand xenobiotics, including drugs. UGTmediated metabolism is a major determinant of the pharmacokinetic behavior of many drugs in the human body, contributing to parameters such as bioavailability and elimination. UGTs catalyze the transfer of glucuronic acid from UDP-glucuronic acid (UDPGA) to the aglycone substrates, producing water-soluble glucuronide conjugates that are mostly devoid of pharmacological activity. Some of the 19 human UGTs have the ability to conjugate different nitrogen-containing compounds, thus forming Nglucuronides. N-Glucuronidation exhibits marked differences across species. As an example, the ability to form quaternary ammonium glucuronides from tertiary amines is a reaction largely, but not completely, restricted to humans. Among the human UGTs, UGT1A4 has been considered the enzyme “specializing” in N-glucuronidation. The goal of this study was to characterize species differences related to Nglucuronidation and to elucidate whether human UGT enzymes other than the previously reported UGT1A4 catalyze this reaction. The nitrogen-containing substrates investigated were firstly a set of 4-arylalkyl-1H-imidazoles, including the sedative drug dexmedetomidine [(+)-4-(S)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole], and secondly nicotine, the addictive agent in tobacco products, along with its major metabolite cotinine. The study was performed using different in vitro systems, such as human and animal liver microsomes and recombinant human UGT enzymes produced in baculovirus-infected insect cells. The 4-arylalkyl-1H-imidazole substrates were incubated with a radiolabeled cofactor, C-UDPGA, and the glucuronide products were analyzed by liquid chromatography using ultraviolet detection combined with a flow scintillation analyzer. Nicotine and cotinine glucuronides were quantified using liquid chromatography – mass spectrometry. Analyses of liver microsome incubates indicated that N-glucuronidation of medetomidine was efficient in humans, while the glucuronidation rates measured in rat, mouse, guinea-pig, rabbit, dog, mini-pig, and monkey liver microsomes were rather low. Studies with recombinant human UGTs revealed that the “orphan enzyme” UGT2B10, which has previously demonstrated no or only very limited activity when screened against a large variety of substrates, plays an important role in the N-glucuronidation of the compounds investigated. More specifically, we discovered that UGT2B10 is the enzyme mainly responsible for nicotine N-glucuronidation in the human liver, and, furthermore, this enzyme is a major contributor to the N-glucuronidation of medetomidine. Nuclear magnetic resonance (NMR) analyses revealed that the N-glucuronidation of medetomidine by human liver microsomes was highly regioselective, and that N3 was the preferred site of glucuronidation. Moreover, this chiral drug was N-glucuronidated stereoselectively. Regioand stereospecific N-glucuronidation of medetomidine in human liver microsomes was explained by complex kinetics involving two enzymes, UGT1A4 and UGT2B10. UGT2B10 was found to be a high-affinity (low Km) enzyme towards medetomidine, while the affinity of UGT1A4 was considerably lower. Levomedetomidine [(–)-4-(R)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole], in particular, turned out be a high-affinity, specific substrate of UGT2B10. The results emphasize the species differences of N-glucuronidation and the importance of in vitro studies utilizing human-derived material, along with animal studies, at the early stages of drug development. Furthermore, this study highlights the contribution of UGT2B10, along with UGT1A4, to the N-glucuronidation of drugs and other xenobiotics in the human liver.