Received for publication March 31, 2008.
Revised May 5, 2008.
Accepted for publication May 7, 2008.

Regio- and stereospecific N-glucuronidation of medetomidine; the differences between UGT1A4 and UGT2B10 account for the complex kinetics of human liver microsomes

Abstract

Medetomidine is a chiral imidazole derivate whose dextro-enantiomer is pharmacologically active. The major metabolic pathway of dexmedetomidine 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 NMR. Studying medetomidine glucuronidation by human liver microsomes (HLM) 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. HLM catalyzed DG1 and DG2 formation, at a ratio of 3:1, with two-enzyme kinetics that contains both a high affinity component, K_m1s 6.6 and 8.7 µM, and a low affinity component, K_m2s >1 mM. The DG1:DG2 ratio in the case of UGT2B10 was lower, 1.4:1, while the substrate affinity for both reactions was high, K_m values 11 and 16 µM. 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 glucuronidation by HLM yielded mostly the N₃-glucuronide (LG1, structure determined by NMR), with monophasic kinetics and a K_m value of 14 µM. The activity of UGT1A4 towards levomedetomide was low and generated both LG1 and LG2, while UGT2B10 exhibited relatively high activity and sharp regioselectivity, yielding only LG1, with a K_m value of 7.4 µM. The results highlight the contribution of UGT2B10 to medetomidine glucuronidation, as well as its potential importance for other N-glucuronidation reactions within the human liver.

Key words: glucuronidation, phase II drug metabolism, UDP glucuronyltransferases