Optimized Assays for Human UDP-Glucuronosyltransferase (UGT) Activities: Altered Alamethicin Concentration and Utility to Screen for UGT Inhibitors

Abstract

ABSTRACT:The measurement of the effect of new chemical entities on human UDP-glucuronosyltransferase (UGT) marker activities using in vitro experimentation represents an important experimental approach in drug development to guide clinical drug-interaction study designs or support claims that no in vivo interaction will occur. Selective high-performance liquid chromatography-tandem mass spectrometry functional assays of authentic glucuronides for five major hepatic UGT probe substrates were developed: ␤-estradiol-3-glucuronide (UGT1A1), trifluoperazine-N-glucuronide (UGT1A4), 5-hydroxytryptophol-O-glucuronide (UGT1A6), propofol-O-glucuronide (UGT1A9), and zidovudine-5-glucuronide (UGT2B7). High analytical sensitivity permitted characterization of enzyme kinetic parameters at low human liver microsomal and recombinant UGT protein concentration (0.025 mg/ml), which led to a new recommended optimal universal alamethicin activation concentration of 10 g/ml for microsomes. Alamethicin was not required for recombinant UGT incubations. Apparent enzyme kinetic parameters, particularly for UGT1A1 and UGT1A4, were affected by nonspecific binding. Unbound intrinsic clearance for UGT1A9 and UGT2B7 increased significantly after addition of 2% bovine serum albumin, with minimal changes for UGT1A1, UGT1A4, and UGT1A6. Eleven potential UGT and cytochrome P450 inhibitors were evaluated as UGT inhibitors, resulting in observation of nonselective UGT inhibition by chrysin, mefenamic acid, silibinin, tangeretin, ketoconazole, itraconazole, ritonavir, and verapamil. The pan-cytochrome P450 inhibitor, 1-aminobenzotriazole, minimally inhibited UGT activities and may be useful in reaction phenotyping of mixed UGT and cytochrome P450 substrates. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of human UGT activities and the identification of UGT enzymeselective chemical inhibitors.

The Letrozole Phase 1 Metabolite Carbinol as a Novel Probe Drug for UGT2B7

“…For both supersomes and HLM data, K m values were not affected by the presence or absence of alamethicin, which is consistent with alamethicin's pore-forming function not influencing UGT enzymes. Our results are in line with previously reported data in HLMs that indicated a 2-to 3-fold increased activity for various substrates in the presence of alamethicin (Fisher et al, 2000;Walsky et al, 2012).…”

Human UDP-Glucuronosyltransferase (UGT) 2B10: Validation of Cotinine as a Selective Probe Substrate, Inhibition by UGT Enzyme-Selective Inhibitors and Antidepressant and Antipsychotic Drugs, and Structural Determinants of Enzyme Inhibition

“…The mean K m for human liver microsomal cotinine N-glucuronidation was marginally higher (P = 0.06) than that for UGT2B10, whereas the V max with HLM as the enzyme source was 6.8-fold higher compared with UGT2B10. The activities of numerous UGTs, particularly with HLM as the enzyme source, are known to be increased in the presence of BSA (0.5-2% w/v) due to sequestration of inhibitory membrane long-chain unsaturated fatty acids released during the course of an incubation (Rowland et al, 2007(Rowland et al, , 2008Kilford et al, 2009;Manevski et al, 2011;Walsky et al, 2012). Addition of BSA (1% w/v) to incubations of HLM resulted in a 45% reduction in K m and a small (11%) but statistically significant increase in V max .…”

“…The addition of BSA (0.5-2% w/v) to incubations has been reported to decrease the K m values (with occasional effects on V max ) for substrates of several hepatically expressed UGT enzymes, particularly UGT 1A9, 2B4, 2B7, and 2B15 (Rowland et al, 2007(Rowland et al, , 2008Kilford et al, 2009;Manevski et al, 2011Manevski et al, , 2013Walsky et al, 2012). The albumin effect appears to arise from sequestration of inhibitory membrane fatty acids released during the course of an incubation.…”

Key to Opening Kidney for In Vitro–In Vivo Extrapolation Entrance in Health and Disease: Part I: In Vitro Systems and Physiological Data

“…Analogous to human liver microsomes, investigation of renal glucuronidation requires inclusion of alamethicin. This pore-forming peptide disrupts microsomal membranes to overcome reaction latency associated with UGTs due to localisation of the enzyme active site facing the lumen of the endoplasmic reticulum (7,17). Furthermore, the addition of albumin has been implemented in the renal glucuronidation in vitro assays (7) in order to account for the inhibitory effect of fatty acids released during microsomal incubations on UGTs.…”