Received for publication September 14, 2005.
Revised December 18, 2005.
Accepted for publication December 19, 2005.

Selectivity of substrate (trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbuatzone, quinidine, quinine and sulfinpyrazone) 'probes' for human UDP-glucuronosyltransferases

Abstract

Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucuronosyltransferases (UGT). This work investigated the selectivity of trifluoperazine (TFP), as a substrate, and amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine and sulfinpyrazone, as inhibitors, for human UGTs. Selectivity was assessed using UGT 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7 and 2B15 expressed in HEK293 cells. TFP was confirmed as a highly selective substrate for UGT1A4. However, TFP bound extensively to both HEK293 lysate and to human liver microsomes in a concentration-dependent manner (fu_inc 0.20 - 0.59). When corrected for non-specific binding, K_m values for TFP glucuronidation were similar for both UGT1A4 (4.1 µM) and human liver microsomes (6.1 ± 1.2 µM) as the enzyme sources. Of the compounds screened as inhibitors, hecogenin alone was selective; significant inhibition was observed only for UGT1A4 (IC₅₀ 1.5 µM). Using phenylbutazone and quinine as 'models', inhibition kinetics were variously described by competitive and noncompetitive mechanisms. Inhibition of UGT2B7 by quinidine was also investigated further since the effects of this compound on morphine pharmacokinetics (a known UGT2B7 substrate) have been ascribed to inhibition of P-glycoprotein. Quinidine inhibited human liver microsomal and recombinant UGT2B7 with respective K_i values of 335 ± 128 µM and 186 µM. In conclusion, TFP and hecogenin represent selective substrate and inhibitor probes for UGT1A4, although the extensive non-selective binding of the former should be taken into account in kinetic studies. Amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine and sulfinpyrazone are non-selective UGT inhibitors.

Key words: glucuronidation, in vitro-in vivo prediction, microsomes, phase II drug metabolism, recombinant proteins

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