Functional Characterization of Human UDP-Glucuronosyltransferase 1A9 Variant, D256N, Found in Japanese Cancer Patients

Abstract

, an active metabolite of the antitumor prodrug irinotecan, is conjugated and detoxified to SN-38 10-O-␤-D-glucuronide by hepatic UDP-glucuronosyltransferase (UGT) 1A1. Recent studies have revealed that other UGT1A isoforms, UGT1A7 and UGT1A9, also participate in SN-38 glucuronidation. Although several genetic polymorphisms are reported for UGT1A1 and UGT1A7 that affect the SN-38 glucuronidation activities, no such polymorphisms have been identified for UGT1A9. In the present study, UGT1A9 exon 1 and its flanking regions were sequenced from 61 Japanese cancer patients who were all treated with irinotecan. A novel nonsynonymous single nucleotide polymorphism was identified in UGT1A9 exon 1, heterozygous 766GϾA resulting in the amino acid substitution of D256N. The wild-type and D256N UGT1A9s were transiently expressed at similar protein levels in COS-1 cells, and their membrane fractions were characterized in vitro for the glucuronidation activities toward SN-38. The apparent K m values were 19.3 and 44.4 M, and the V max values were 2.94 and 0.24 pmol/min/mg of membrane protein for the wild-type and D256N variant, respectively. The SN-38 glucuronidation efficiency (normalized V max /K m ) of D256N was less than 5% that of wild-type UGT1A9. These results clearly indicate that the D256N variant is essentially nonfunctional with regard to SN-38 glucuronidation. These findings highlight the importance of further studies into the potential influence of UGT1A9 D256N variant to irinotecan metabolism in vivo.Irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin; Fig. 1) is an antitumor prodrug of a potent topoisomerase I inhibitor, SN-38 (7-ethyl-10-hydroxycamptothecin), which is now applied clinically with or without other chemotherapeutic agents such as 5-fluorouracil and cisplatin to a variety of malignant tumors including gastrointestinal and lung cancers (Rothenberg, 2001). Irinotecan is hydrolyzed by carboxylesterases in the human liver, intestinal mucosa, and plasma to liberate SN-38 (Rivory et al., 1996;Ahmed et al., 1999;Kehrer et al., 2000), which undergoes subsequent glucuronidation by UDP-glucuronosyltransferases (UGTs) into an inactive 10-O-␤-D-glucuronide (SN-38G; Fig. 1) and biliary excretion (Gupta et al., 1994;Iyer et al., 1998). Although the precise mechanism remains to be elucidated, the hepatic SN-38 glucuronidation capacity is thought to be an important determinant factor of severe diarrhea, the major dose-limiting side effect of irinotecan (Ratain, 2002). Therefore, genetic polymorphisms in UGT1A could influence the incidence of the irinotecan-induced diarrhea.The UGT1A family is known to include 9 functional isoforms (UGT1A1, UGT1A3 to UGT1A10). UGT1A2P, UGT1A11P, UGT1A12P, and UGT1A13P are thought to be pseudogenes based on their frame-shift mutations. The UGT1A family members have common carboxyl terminal sequences (245 amino acids in length) derived from four This study was supported in part by the Program for Promotion of Fundamental Studies in Health Sciences...

Prevalence of UGT1A9 and UGT2B7 nonsynonymous single nucleotide polymorphisms in West African, Papua New Guinean, and North American populations

“…Asian-Americans carrying 8A (n=2), 98C (n=1), or 766A (n=1) alleles were different individuals. Our results, by LDR-FMA, validate previous findings [14,15]that UGT1A9 nonsynonymous SNPs are relatively uncommon.…”

“…UGT1A9 enzyme variant M33T, heterologously expressed in HEK293 cells, showed 1.7-fold reduced intrinsic clearance (V max /K m ) for mycophenolic acid 7-O-glucuronide (mycophenolic acid is the active metabolite of a standard immunosuppressive drug mycophenolate mofetil) [40], and 26-fold reduced intrinsic clearance for SN-38 glucuronide (SN-38 is the active metabolite of an anticancer drug irinotecan) [14]. The other UGT1A9 enzyme variant D256N, heterologously expressed in COS-1 cells, showed 22-fold reduced intrinsic clearance for SN-38 glucuronide [15]. In addition to M33T and D256N, UGT1A9 promoter polymorphisms −275T>A and −2152C>T, associated with 2.3-fold higher protein expression and 2.1-fold higher glucuronidation activity in human liver samples [41], may influence the pharmacokinetics of mycophenolate mofetil in transplant recipients [42].…”

“…Using HEK293 cells expressing UGT allelic variants, this polymorphism was not found to alter glucuronidation activity for anticancer drugs [14]. The reported frequencies of UGT1A9 polymorphisms are: 8G>A (2.5%, African-Americans) [14], 98T > C (2.2-3.6%, Caucasians) [14], 766G>A (<1%, Japanese) [15], and 726T>G (0.5%, Japanese) [16].…”

“…Recently, pharmaco-vigilance of artemisinin-based combination therapy in Africa has been suggested in order to assess the safety of these drugs when used widely [23]. Unfortunately, only limited information is available regarding UGT1A9 and UGT2B7 pharmacogenetic variations in various populations; the information pertains mainly to Caucasian and Japanese populations [14,15,[18][19][20][21][22]. Here, for the first time, we present a comprehensive account of UGT1A9 and UGT2B7 nonsynonymous SNPs in populations from two different malaria-endemic regions, West Africa (Ghana, Guinea, Ivory Coast, Sierra Leone, and Senegal) and PNG, and in four major North American ethnic groups (Caucasian, African, Asian, and Hispanic).…”

Integrating Field Analyses with Laboratory Exposures to Assess Ecosystems Health

“…UGT1 contains five exons and has a unique gene structure in which there are 13 individual promoters/first exons, from UGT1A1 to UGT1A13P, and exons 2-5 are used in common in all mRNAs (Guillemette et al, 2000b;Gong et al, 2001;Jinno et al, 2003). Since all members of this family (UGT1A1-UGT1A13) share exons 2-5, the resulting isoforms are identical in their last 245 amino acids.…”