GLUCURONIDATION OF ENTACAPONE, NITECAPONE, TOLCAPONE, AND SOME OTHER NITROCATECHOLS BY RAT-LIVER MICROSOMES

Purpose. Nitrocatechol COMT inhibitors are a new class of bioactive co mpounds, for which glucuronidation is the most important metabolic pat hway. The objective was to characterize the enzyme kinetics of nitroca techol glucuronidation to improve the understanding and predicting of the pharmacokinetic behavior of this class of compounds. Methods. The glucuronidation kinetics of seven nitrocatechols and 4-nitrophenol, th e reference substrate for phenol UDP-glucuronosyltransferase activity, was measured in liver microsomes from creosote-treated rats and deter mined by non-linear fitting of the experimental data to the Michaelis- Menten equation. A new method that combined densitometric and radioact ivity measurement of the glucuronides separated by HPTLC was developed for the quantification. Results. Apparent K-m values for the nitrocat echols varied greatly depending on substitution pattern being comparab le with 4-nitrophenol (O.11 mM) only in the case of 4-nitrocatechol (0 .19 mM). Simple nitrocatechols showed two-fold V-max values compared w ith 4-nitrophenol (68.6 nmol min(-1) mg(-1)), while all disubstituted catechols exhibited much lower glucuronidation rate. V-max/K-m values were about 10 times higher for monosubstituted catechols compared to d isubstituted ones. The kinetic parameters for COMT inhibitors were in the following order: K-m nitecapone >> entacapone > tolcapone; V-max n itecapone > entacapone > tolcapone; V-max/K-m tolcapone > nitecapone > entacapone. Conclusions. Nitrocatechols can in principle be good subs trates of UGTs. However, substituents may have a remarkable effect on the enzyme kinetic parameters. The different behaviour of nitecapone c ompared to the other COMT inhibitors may be due to its hydrophilic 5-s ubstituent. The longer elimination half-life of tolcapone in vivo comp ared to entacapone could not be explained by glucuronidation kinetics in vitro.