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DETERMINATION OF DRUG GLUCURONIDATION AND UDP-GLUCURONOSYLTRANSFERASE SELECTIVITY USING A 96-WELL RADIOMETRIC ASSAY

Department of Pharmacology, Istituto di Ricerche di Biologia Molecolare (IRBM) P. Angeletti, Merck Research Laboratories, Rome, Italy

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 [¹⁴C]UDP-glucuronic acid, and ¹⁴C-labeled 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 were 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 the glucuronidation of most of these compounds is significantly faster in dog than in human liver microsomes. UGT specificity of five 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 high-performance liquid chromatography method coupled to mass spectrometric detection. The new assay should prove valuable for rapidly benchmarking recombinant UGTs and microsomal preparations from different species and tissues, identifying high-turnover compounds during drug discovery, and reaction phenotyping studies.

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