114991-26-3

A sensitive and high-throughput liquid chromatography-tandem mass spectrometry system was developed and validated for the simultaneous determination of major human hepatic UDP-glucuronyltransferase forms in human liver microsomes. The analytes were detected using a triple-quadrupole mass spectrometer equipped with an electrospray ionization source in the negative ion and selected reaction monitoring modes. The method provided satisfactory linear concentration range, accuracy, precision, and stability. The developed method was successfully applied to the enzyme kinetic study of estradiol-3-O-glucuronidation, 4-methylumbelliferone-O-glucuronidation, propofol-O-glucuronidation, and 3-azido-3-deoxythymidine glucuronidation in human liver microsomes.

Icariside II (IC-II) is a well-known flavonoid glycoside with many bioactivities in Epimedium plants. However, its glucuronidation involving human UDP-glucuronosyltransferases (UGTs) remains unclear. In this study, IC-II was found to be metabolized to a glucuronide (IC-II-7-G) at C7-OH after incubation by human liver microsomes (HLM) and human intestine microsomes (HIM) with the intrinsic clearance (CLint) values of 2.14 and 1.22 μL/min/mg, respectively. In addition, reaction phenotyping and chemical inhibition assays indicated that UGT1A1, 1A7, 1A8, 1A9, and 1A10 were the main UGT isoforms for IC-II-7-O-glucuronidation. Furthermore, IC-II-7-O-glucuronidation was correlated with β-estradiol-3-O-glucuronidation (r = 0.772, p = 0.015) and propofol-O-glucuronidation (r = 0.675, p = 0.046) in a bank of individual HLMs (n = 9), respectively. Furthermore, based on the relative activity factor (RAF) approach, UGT1A1 and 1A9 contributed 25.9% and 16.0% for the IC-II-7-O-glucuronidation in HLM, respectively. Moreover, there were marked species difference (nearly 11-fold) between human and animals liver microsomes. Taken altogether, these results of combined approaches including reaction phenotyping, chemical inhibition assays, activity correlation analysis, and RAF analysis indicate that human UGT1A1, 1A7, 1A8, 1A9, and 1A10 are the main UGT contributors responsible for IC-II-7-O-glucuronidation. The results increase our knowledge about the metabolic fate of icariside II in vivo.

Purpose: Catalytic selectivity of human UGT1A9, an important membrane-bound enzyme catalyzing glucuronidation of xenobiotics, was determined experimentally using 145 phenolics and analyzed by 3D-QSAR methods. Methods: Catalytic efficiency of UGT1A9 was determined by kinetic profiling. Quantitative structure activity relationships were analyzed using CoMFA and CoMSIA techniques. Molecular alignment of substrate structures was made by superimposing the glucuronidation site and its adjacent aromatic ring to achieve maximal steric overlap. For a substrate with multiple active glucuronidation sites, each site was considered a separate substrate. Results: 3D-QSAR analyses produced statistically reliable models with good predictive power (CoMFA: q 2=0.548, r2=0.949, r pred 2 =0.775; CoMSIA: q2=0.579, r2=0.876, rpred2 =0.700). Contour coefficient maps were applied to elucidate structural features among substrates that are responsible for selectivity differences. Contour coefficient maps were overlaid in the catalytic pocket of a homology model of UGT1A9, enabling identification of the UGT1A9 catalytic pocket with a high degree of confidence. Conclusion: CoMFA/CoMSIA models can predict substrate selectivity and in vitro clearance of UGT1A9. Our findings also provide a possible molecular basis for understanding UGT1A9 functions and substrate selectivity.

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. Copyright

Chloramphenicol (CP), a broad spectrum antibiotic, is eliminated in humans by glucuronidation. The primary UGT enzymes responsible for CP O-glucuronidation remain unidentified. We have previously identified the 3-O-CP (major) and 1-O-CP (minor) glucuronides by β-glucuronidase hydrolysis, liquid chromatography-tandem mass spectrometry, and 1D/2D H NMR. Reaction phenotyping for the glucuronidation of CP with 12 expressed human liver UGT isoforms has identified UGT2B7 as having the highest activity for 3-O- and 1-O-CP glucuronidation with minor contributions from UGT1A6 and UGT1A9. The kinetics of CP 3-O-glucuronidation by pooled human liver microsomes (HLMs) exhibited biphasic Michaelis-Menten kinetics with the apparent high-affinity K m1 and low-affinity Km2 values of 46.0 and 1027 μM, whereas expressed UGT2B7 exhibited Michaelis-Menten kinetics with the apparent Km value of 109.1 μM. The formation of 1-O-CP glucuronide by pooled HLM and expressed UGT2B7 exhibited substrate inhibition kinetics with apparent Km values of 408.2 and 115.0 μM, respectively. Azidothymidine (AZT) and hyodeoxycholic acid (substrates of UGT2B7) inhibited 3-O- and 1-O-CP glucuronidation in pooled HLMs. In 10 donor HLM preparations, both CP 3-O- and CP 1-O-glucuronidation showed a significant correlation with AZT glucuronidation (UGT2B7) (rs = 0.85 and rs = 0.83, respectively) at 30 μMCP, whereas no significant correlation was observed between CP 3-O-glucuronidation and serotonin glucuronidation (UGT1A6) or propofol glucuronidation (UGT1A9) at this CP concentration. These results suggest that UGT2B7 is the primary human hepatic UDP-glucuronosyltransferase isoform catalyzing 3-O- and 1-O-CP glucuronidation with minor contributions from UGT1A6 and UGT1A9. Copyright