Short Communication INHIBITION OF UDP-GLUCURONOSYLTRANSFERASE 2B7-CATALYZED MORPHINE GLUCURONIDATION BY KETOCONAZOLE: DUAL MECHANISMS INVOLVING A NOVEL NONCOMPETITIVE MODE

Glucuronidation of morphine in humans is predominantly catalyzed by UDP-glucuronosyltransferase 2B7 (UGT2B7). Since our recent research suggested that cytochrome P450s (P450s) interact with UGT2B7 to affect its function [Takeda S et al. (2005) Mol Pharmacol 67:665–672], P450 inhibitors are expected to modulate UGT2B7catalyzed activity. To address this issue, we investigated the effects of P450 inhibitors (cimetidine, sulfaphenazole, erythromycin, nifedipine, and ketoconazole) on the UGT2B7-catalyzed formation of morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G). Among the inhibitors tested, ketoconazole was the most potent inhibitor of both M-3-G and M-6-G formation by human liver microsomes. The others were less effective except that nifedipine exhibited an inhibitory effect on M-6-G formation comparable to that by ketoconazole. Neither addition of NADPH nor solubilization of liver microsomes affected the ability of ketoconazole to inhibit morphine glucuronidation. In addition, ketoconazole had an ability to inhibit morphine UGT activity of recombinant UGT2B7 freed from P450. Kinetic analysis suggested that the ketoconazole-produced inhibition of morphine glucuronidation involves a mixedtype mechanism. Codeine potentiated inhibition of morphine glucuronidation by ketoconazole. In contrast, addition of another substrate, testosterone, showed no or a minor effect on ketoconazole-produced inhibition of morphine UGT. These results suggest that 1) metabolism of ketoconazole by P450 is not required for inhibition of UGT2B7-catalyzed morphine glucuronidation; and 2) this drug exerts its inhibitory effect on morphine UGT by novel mechanisms involving competitive and noncompetitive inhibition. A number of endogenous and exogenous compounds are biotransformed by phase I and phase II drug-metabolizing enzymes (Oguri et al., 1994; Radominska-Pandya et al., 1999; Ritter, 2000). The cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) families are two major enzyme groups responsible for phase I and II reactions. UGT-catalyzed glucuronidation is assumed to play a role in up to 35% of phase II reactions. UGT is anchored to the endoplasmic reticulum membrane by the transmembrane domain, and the main body of the enzyme, including the catalytic site, is believed to be present on the luminal side of the endoplasmic reticulum. This model of type I topology causes “latency,” a characteristic feature of UGT, whereby UGT activity is activated by disruption of the microsomal membranes with detergent. Among the UGTs identified in humans, UGT2B7 is of particular interest because it is a major isoform involved in morphine glucuronidation (Coffman et al., 1997). To date, UGT2B7 is the sole enzyme reported capable of forming morphine6-glucuronide (M-6-G) (Coffman et al., 1997), a metabolite having more potent analgesic activity than morphine. Although increasing evidence suggests that there are a number of factors that affect morphine UGT activity (Narayan et al., 1991; Ishii et al., 2001, 2004, 2005; Antonilli et al., 2003; Takeda et al., 2005a,b), little is known about inhibitors of UGT2B7. Experimental evidence in humans indicates that the glucuronidation of acetaminophen, codeine, zidovudine, carbamazepine, lorazepam, and propafenone is influenced by concomitantly administered drugs (Kiang et al., 2005). However, the glucuronidation of morphine is not affected by any of the drugs examined so far (Kiang et al., 2005). Although diclofenac is a potent inhibitor of codeine glucuronidation in vitro (Ki 7.9 M; Ammon et al., 2000), it has only a low ability to inhibit the glucuronidation of morphine by human liver microsomes (IC50 approximately 500 M; King et al., 2001). In contrast, using human liver microsomes, it has been reported that ketoconazole, a P450 inhibitor (Wrighton and Ring; 1994), competitively inhibits the glucuronidation of the UGT2B7 substrate zidovudine with a Ki of 80 M (Sampol et al., 1995). A recent study by Yong et al. (2005), also using human liver microsomes, showed that ketoconazole inhibited UGT1A-mediated glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38) and cDNA-expressed UGT1A isoforms. Since glucuronidation of zidovudine is catalyzed by human liver microsomal UGT (Sampol et al., 1995), it is conceivable that UGT2B7 is inhibited by ketoconazole. However, there are no data that demonstrate the direct inhibition of UGT2B7 by ketoconazole. Since P450s are suggested to interact with UGTs, including UGT2B7, to modulate its function (Taura et al., 2000; Fremont et al., 2005; Ishii et al., 2005; Takeda et al., 2005a,b), it is conceivable that P450 inhibitors modulate UGT2B7-catalyzed reactions. To examine this possibility, we carried out a comparative study to clarify whether This work was supported in part by a Grant-in-Aid for Scientific Research (C) (Research No. 17590128, recipient Y.I.) from the Japan Society for Promotion of