Kentaro Konishi1, Daisuke Tenmizu2, Shin Takusagawa3. 1. Analysis and Pharmacokinetics Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki, 305-8585, Japan. kentaro.konishi@astellas.com. 2. Analysis and Pharmacokinetics Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki, 305-8585, Japan. 3. Clinical Pharmacology, Development, Astellas Pharma Inc., 2-5-1, Nihonbashi-Honcho, Chuo-ku, Tokyo, 103-8411, Japan.
Abstract
BACKGROUND AND OBJECTIVES: Mirabegron is cleared by multiple mechanisms, including drug-metabolizing enzymes. One of the most important clearance pathways is direct glucuronidation. In humans, M11 (O-glucuronide), M13 (carbamoyl-glucuronide), and M14 (N-glucuronide) have been identified, of which M11 is one of the major metabolites in human plasma. The objective of this study was to identify the uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) isoform responsible for the direct glucuronidation of mirabegron using human liver microsomes (HLMs) and recombinant human UGTs (rhUGTs). METHODS: Reaction mixtures contained 1-1000 μM mirabegron, 8 mM MgCl2, alamethicin (25 μg/mL), 50 mM Tris-HCl buffer (pH 7.5), human liver microsome (HLM) or rhUGT (1.0 mg protein/mL), and 2 mM UDP-glucuronic acid in a total volume of 200 μL for 120 min at 37 °C. HLMs from 16 individuals were used for the correlation study, and mefenamic acid and propofol were used for the inhibition study. RESULTS: Regarding M11 formation, rhUGT2B7 showed high activity among the rhUGTs tested (11.3 pmol/min/mg protein). This result was supported by the correlation between M11 formation activity and UGT2B7 marker enzyme activity (3-glucuronidation of morphine, r 2 = 0.330, p = 0.020) in individual HLMs; inhibition by mefenamic acid in pooled HLMs (IC50 = 22.8 μM); and relatively similar K m values between pooled HLMs and rhUGT2B7 (1260 vs. 486 μM). Regarding M13 and M14 formation, rhUGT1A3 and rhUGT1A8 showed high activity among the rhUGTs tested, respectively. CONCLUSIONS: UGT2B7 is the main catalyst of M11 formation in HLMs. Regarding M13 and M14 formation, UGT1A3 and UGT1A8 are strong candidates for glucuronidation, respectively.
BACKGROUND AND OBJECTIVES: Mirabegron is cleared by multiple mechanisms, including drug-metabolizing enzymes. One of the most important clearance pathways is direct glucuronidation. In humans, M11 (O-glucuronide), M13 (carbamoyl-glucuronide), and M14 (N-glucuronide) have been identified, of which M11 is one of the major metabolites in human plasma. The objective of this study was to identify the uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) isoform responsible for the direct glucuronidation of mirabegron using human liver microsomes (HLMs) and recombinant human UGTs (rhUGTs). METHODS: Reaction mixtures contained 1-1000 μM mirabegron, 8 mM MgCl2, alamethicin (25 μg/mL), 50 mM Tris-HCl buffer (pH 7.5), human liver microsome (HLM) or rhUGT (1.0 mg protein/mL), and 2 mM UDP-glucuronic acid in a total volume of 200 μL for 120 min at 37 °C. HLMs from 16 individuals were used for the correlation study, and mefenamic acid and propofol were used for the inhibition study. RESULTS: Regarding M11 formation, rhUGT2B7 showed high activity among the rhUGTs tested (11.3 pmol/min/mg protein). This result was supported by the correlation between M11 formation activity and UGT2B7 marker enzyme activity (3-glucuronidation of morphine, r 2 = 0.330, p = 0.020) in individual HLMs; inhibition by mefenamic acid in pooled HLMs (IC50 = 22.8 μM); and relatively similar K m values between pooled HLMs and rhUGT2B7 (1260 vs. 486 μM). Regarding M13 and M14 formation, rhUGT1A3 and rhUGT1A8 showed high activity among the rhUGTs tested, respectively. CONCLUSIONS:UGT2B7 is the main catalyst of M11 formation in HLMs. Regarding M13 and M14 formation, UGT1A3 and UGT1A8 are strong candidates for glucuronidation, respectively.
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