6-Chloro-5-[4-(1-Hydroxycyclobutyl)Phenyl]-1H-Indole-3-Carboxylic Acid is a Highly Selective Substrate for Glucuronidation by UGT1A1, Relative toβ-Estradiol

6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic acid (PF-06409577) is a direct activator of the human β1-containing adenosine monophosphate-activated protein kinase (ΑMPK) isoforms. The clearance mechanism of PF-06409577 in animals and humans involves uridine diphosphoglucuronosyl transferase (UGT)–mediated glucuronidation to an acyl glucuronide metabolite of PF-06409577 [(2S,3S,4S,5R,6S)-6-((6-chloro-5-(4-(1-hydroxycyclobutyl)phenyl)-1H-indole-3-carbonyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (M1)], which retains selective activation of human β1-containing AMPK isoforms. This paper describes a detailed characterization of the human UGT isoform(s) responsible for glucuronidation of PF-06409577 to M1. Studies using a panel of 13 human recombinant UGT (hrUGT) enzymes indicated that PF-06409577 was converted to M1 in a highly selective fashion by UGT1A1, which was further verified in human liver microsomes treated with specific chemical inhibitors, and in different UGT1A1 expressers. Conversion of PF-06409577 to M1 by UGT1A1 occurred in a relatively selective fashion, compared with β-estradiol (ES), a conventional probe substrate of UGT1A1. The Michaelis-Menten constant (K_M) and V_max values describing the formation of M1 from PF-06409577 in hrUGT1A1 and microsomal preparations from human intestine, liver, and kidney ranged from 131 to 212 μM (K_M) and 107–3834 pmol/min per milligram (V_max) in the presence of 2% bovine serum albumin. Relative activity factors (RAF) were determined for UGT1A1 using PF-06409577 and ES to enable estimation of intrinsic clearance from various tissues. RAF values from PF-06409577 and ES were generally comparable with the exception of intestinal microsomes, where ES overestimated the RAF of UGT1A1 due to glucuronidation by intestinal UGT1A8 and UGT1A10. Our results suggest the potential utility of PF-06409477 as a selective probe UGT1A1 substrate for UGT reaction phenotyping and inhibition studies in preclinical discovery/development.