Identification of UDP-Glucuronosyltransferases Responsible for the Glucuronidation of Darexaban, an Oral Factor Xa Inhibitor, in Human Liver and Intestine

Abstract

ABSTRACT:Darexaban maleate is a novel oral direct factor Xa inhibitor, which is under development for the prevention of venous thromboembolism. Darexaban glucuronide was the major component in plasma after oral administration of darexaban to humans and is the pharmacologically active metabolite. In this study, we identified UDPglucuronosyltransferases (UGTs) responsible for darexaban glucuronidation in human liver microsomes (HLM) and human intestinal microsomes (HIM). In HLM, the K m value for darexaban glucuronidation was >250 M. In HIM, the reaction followed substrate inhibition kinetics, with a K m value of 27.3 M. Among recombinant human UGTs, UGT1A9 showed the highest intrinsic clearance for darexaban glucuronidation, followed by UGT1A8, -1A10, and -1A7. All other UGT isoforms were inactive toward darexaban. The K m value of recombinant UGT1A10 for darexaban glucuronidation (34.2 M) was comparable to that of HIM. Inhibition studies using typical UGT substrates suggested that darexaban glucuronidation in both HLM and HIM was mainly catalyzed by UGT1A8, -1A9, and -1A10. Fatty acid-free bovine serum albumin (2%) decreased the unbound K m for darexaban glucuronidation from 216 to 17.6 M in HLM and from 35.5 to 18.3 M in recombinant UGT1A9. Recent studies indicated that the mRNA expression level of UGT1A9 is extremely high among UGT1A7, -1A8, -1A9, and -1A10 in human liver, whereas that of UGT1A10 is highest in the intestine. Thus, the present results strongly suggest that darexaban glucuronidation is mainly catalyzed by UGT1A9 and UGT1A10 in human liver and intestine, respectively. In addition, UGT1A7, -1A8, and -1A9 play a minor role in human intestine.

Reaction Phenotyping: Advances in the Experimental Strategies Used to Characterize the Contribution of Drug-Metabolizing Enzymes

“…In a similar experiment, Shiraga and colleagues showed kinetic benefit from low levels of BSA with UGT1A9 when evaluating darexaban. At 0.1% BSA, kinetic parameters similar to those obtained at 2% BSA were observed when corrected for the free fraction (Shiraga et al, 2012); however, the effects were most noticeable on the unbound K m and not the V max . …”

Identification of Enzymes Responsible for the N-Oxidation of Darexaban Glucuronide, the Pharmacologically Active Metabolite of Darexaban, and the Glucuronidation of Darexaban N-Oxides in Human Liver Microsomes

“…Extrapolation of kinetic constants (CL int ) generated in the presence of bovine serum albumin or fatty acid-free human serum albumin has been reported to improve the prediction of in vivo hepatic clearance by glucuronidation for substrates of UGT1A9 and UGT2B7 via a decrease in K m values, with little or no effect on V max values. [24][25][26] In the case of darexaban glucuronidation by HLM, 5) 2% bovine serum albumin decreased the unbound K m value from 216 to 17.6 μM, and increased the unbound CL int value approximately 10-fold. The unbound K m is closely similar to that of recombinant human UGT1A9 (18.3 μM) in the presence of 2% BSA.…”

“…These findings on UGT isoforms are consistent with the results of darexaban glucuronidation. 5) Human UGT isoforms are expressed in a tissue-specific manner. 20,21) Recent reports indicated that among UGT1A7, UGT1A8, UGT1A9, and UGT1A10, the mRNA expression level of UGT1A9 was highest and those of the other isoforms were extremely low or not detected in human liver.…”

“…4) In humans, darexaban glucuronidation is mainly catalyzed by uridine 5′-diphosphate (UDP)-glucuronosyltransferase (UGT) 1A9 and UGT1A10 in liver and intestine, respectively, after oral administration. 5) Additionally, UGT1A7, UGT1A8, and UGT1A9 play a minor role in darexaban glucuronidation in human intestine. 5) In addition to YM-222714, its N-oxides (YM-222714 Noxides, a 1 : 1 mixture of two diastereomers, Fig.…”

“…5) Additionally, UGT1A7, UGT1A8, and UGT1A9 play a minor role in darexaban glucuronidation in human intestine. 5) In addition to YM-222714, its N-oxides (YM-222714 Noxides, a 1 : 1 mixture of two diastereomers, Fig. 1) were also detected as minor metabolites of darexaban in human plasma (unpublished data).…”