Determination of drug glucuronidation and UDP-glucuronosyltransferase selectivity using a 96-well non-HPLC radiometric assay Separation of glucuronide products. Oasis TM HLB 30mg solid phase 96 well extraction

A rapid and sensitive radiometric assay for UDP-glucuronosyltransferase (UGT) is described. UGT substrates are incubated in 96-well plates with microsomes in the presence of [ 14 C]UDPGA, and [ 14 C]-labelled glucuronidation products are separated from the unreacted nucleotide sugar by solid phase extraction using 96-well extraction plates. The assay was validated with 15 structurally diverse UGT substrates containing acidic, phenolic and hydroxyl reacting groups. Glucuronidation velocities for these compounds were determined using human, rat, and dog liver microsomes, and reaction kinetics was studied with 1-naphthol and 4-methylumbelliferone. Results obtained with the new assay confirmed the previously reported rank order of glucuronidation velocity of several typical UGT substrates and the finding that glucuronidation of most of these compounds is significantly faster in dog than in human liver microsomes. UGT specificity of 5 compounds was determined using recombinant human UGTs. The major UGT isoforms identified were UGT1A6, UGT1A7, and UGT1A9 for 4-methylumbelliferone, UGT1A6 and UGT1A8 for 1-naphthol, UGT2B7 for naloxone, UGT1A3 and UGT2B7 for ketoprofen, and UGT1A4 for trifluoperazine. Identical results were obtained with a conventional HPLC method coupled to mass spectrometric detection. The new assay should prove to be valuable for rapidly benchmarking recombinant UGTs and microsomal preparations from different species and tissues, for identifying high turnover compounds during drug discovery, and for reaction phenotyping studies. drugs we describe a 96-well radiometric assay for the determination of UGT activity toward compounds containing carboxyl, phenol and hydroxyl moieties, based on the conjugation of test compounds with [ 14 C]UDPGA and separation of reaction products on 96-well SPE plates. The assay was validated with 15 structurally diverse substrates, used to compare reaction velocities in microsomes from 3 different species, and to determine the reaction kinetics and UGT specificity of typical substrates. Plates incubated 0 to and reactions terminated by the addition of 100 µl of 0.2 HCl. For zero time controls, HCl added to the reaction mixture prior of UDPGA. by centrifugation at 1750 x g for using a microplate rotor. Determination of substrate glucuronidation by recombinant UGTs. Compounds were incubated with recombinant UGT preparations containing 10 or 50 µ g of microsomal protein for 30 min, except for trifluoperazine, which was incubated for 120 min. Following addition of stopping solution and centrifugation, 150 µ l aliquots of deproteinized supernatants were subjected to SPE and processed as described above. Aliquots of 30 µ l aliquots were diluted with an equal volume of acetonitrile and analyzed by HPLC-MS/MS using an Agilent HP1100 liquid chromatograph equipped with a CTC Analytics PAL Autosampler. Chromatography was performed on an XTERRA MS C18 column (4.6 mm x 5 cm; 5µm; Waters) at a flow rate of 2 mL/min, using a linear gradient from water/ 0.1% formic acid (A) to 20% A, 80% acetonitrile/ 0.1% formic acid. The eluate was diverted to a Sciex API-3000 triple quadrupole mass with a Turbo Ionspray ionization source operated in the positive ion mode (glucuronides of ketoprofen, naloxone, trifluoperazine) or negative ion mode (glucuronides of 1NP and 4MU). concentrations (0.1 and 0.5 mg/ml) and stopped after 10, 20, and 40 min. Reactions were linear with time at one or both microsome concentrations. Reaction velocities were determined by linear regression analysis from the slope of the product formation time course. Results are mean values ± standard error of the fit, or mean values ± half-range of the velocities obtained at 2 microsome concentrations.