RT Journal Article
SR Electronic
T1 Optimized Assays for Human UDP-Glucuronosyltransferase (UGT) Activities: Altered Alamethicin Concentration and Utility to Screen for UGT Inhibitors
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 1051
OP 1065
DO 10.1124/dmd.111.043117
VO 40
IS 5
A1 Robert L. Walsky
A1 Jonathan N. Bauman
A1 Karine Bourcier
A1 Georgina Giddens
A1 Kimberly Lapham
A1 Andre Negahban
A1 Tim F. Ryder
A1 R. Scott Obach
A1 Ruth Hyland
A1 Theunis C. Goosen
YR 2012
UL http://dmd.aspetjournals.org/content/40/5/1051.abstract
AB 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: β-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 μg/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 enzyme-selective chemical inhibitors.