THE NOVEL UGT1A9 INTRONIC I399 POLYMORPHISM APPEARS AS A PREDICTOR OF 7-ETHYL-10-HYDROXYCAMPTOTHECIN GLUCURONIDATION LEVELS IN THE LIVER Hugo Girard, Lyne Villeneuve, Michael H. Court, Louis-Charles Fortier, Patrick Caron, Qin Hao, Lisa L. von Moltke, David J. Greenblatt, and Chantal Guillemette Laboratory of Pharmacogenomics, Oncology and Molecular Endocrinology Research Center, Centre Hospitalier de l’Universite ´ Laval (CHUL) Research Center and Faculty of Pharmacy, Laval University, Quebec, Canada (H.G., L.V., L.-C.F., P.C., C.G.); and Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, Massachusetts (M.H.C., Q.H., L.L.v.M., D.J.G.) Received February 18, 2006; accepted March 29, 2006 ABSTRACT: Polymorphisms in UGT1A9 were associated with reduced toxicity and increased response to irinotecan in cancer patients. UDP- glucuronosyltransferase (UGT) protein expression, glucuronida- tion activities for 7-ethyl-10-hydroxycamptothecin (SN-38), and probe substrates of the UGT1A9 and UGT1A1 were measured in 48 human livers to clarify the role of UGT1A9 variants on the in vitro glucuronidation of SN-38. Genotypes were assessed for UGT1A9 (2152C>T, 275T>A, and 118T 9>10 ), three novel UGT1A9 vari- ants (5366G>T, 4549T>C, and I399C>T), and UGT1A1 (53TA 6>7 , 3156G>A, and 3279T>G). Of all the variants, the UGT1A9 I399C>T was associated with the most dramatic change in SN-38-glucuronide (SN-38G) (2.64-fold; p  0.0007). Compared with UGT1A9 I399C/C, homozygous I399T/T presented elevated UGT1A1 and UGT1A9 proteins and higher glucuronidation of UGT1A9 and UGT1A1 substrates (p < 0.05). The very low linkage disequilibrium (r 2 < 0.19) between UGT1A9 I399 and all the other UGT1A1 and UGT1A9 variants suggests a direct effect or linkage to unknown functional variant(s) relevant to SN-38 glucuronidation. The UGT1A9 118T 9/10 was also linked to alteration of SN-38 glucuronidation profiles in the liver (p < 0.05) and was associated with higher UGT1A1 protein (p  0.03). However, UGT1A9 118T 10 appears to have low functional impact as a result of the lack of correlation with UGT1A9 protein levels and a modest 1.4-fold higher reporter gene expression associated with the 118T 10 allele in HepG2 cells (p  0.004). In contrast, the UGT1A9 5366T, 4549C, 2152T, and 275A, associated with higher UGT1A9 pro- tein (2-fold; p < 0.05), have no influence on SN-38G. Despite limi- tations resulting from sample size, results indicate that UGT1A9 I399 and 118T 9/10 may represent additional candidates in combi- nation with UGT1A1 promoter haplotypes for the prediction of SN-38 glucuronidation profile in vivo. Irinotecan (or CPT-11) is a topoisomerase inhibitor used to treat metastatic colorectal cancer (Douillard et al., 2000; Saltz et al., 2000). Its metabolism is complex and includes oxidation by cytochrome P450 and activation by carboxylesterase to form the active metabolite SN-38 (Kawato et al., 1991). Glucuronidation catalyzed by UDP- glucuronosyltransferases (UGTs) represents the major inactivation pathway for SN-38 (Gupta et al., 1994). Of the 16 functional human UGT enzymes, UGT1A1 and UGT1A9 were identified as the main hepatic enzymes involved in the inactivation of SN-38 to SN-38- glucuronide (SN-38G) (Hanioka et al., 2001; Gagne et al., 2002). UGT1A1 and UGT1A9 are encoded by a single gene by exon sharing of individual exons 1 with common exons 2 to 5, whereas at least six other UGT1A proteins originate from this gene (Gong et al., 2001). Several studies revealed the importance of UGT1A1 and UGT1A9 in the hepatic metabolism of SN-38 (Iyer et al., 1998; Gagne et al., 2002; Carlini et al., 2005). A number of polymorphisms in UGT1A1 and UGT1A9 affecting expression and protein function have been identified and could potentially modulate the metabolism of SN-38 in vivo (Zheng et al., 2001; Guillemette, 2003; Villeneuve et al., 2003; Girard et al., 2004; Yamanaka et al., 2004), as well as response to irinotecan-based chemotherapy (Ando et al., 2000; Innocenti et al., 2004; Marcuello et al., 2004; Rouits et al., 2004; Carlini et al., 2005). Among these genetic variations, patients with the UGT1A1*28 variant, associated with Gilbert’s syndrome (Bosma et al., 1995), show higher levels of SN-38 and experienced severe diarrhea and neutropenia (Ando et al., 2000; Innocenti et al., 2004; Marcuello et al., 2004; Rouits et al., 2004). Additional polymorphisms in the UGT1A1 gene have been reported and linked to a variable SN-38 glucuronida- tion, namely, the -3156GA and the -3279TG variations (Inno- centi et al., 2002; Sugatani et al., 2002). Genotyping of these varia- tions in the promoter region of UGT1A1, along with the -53 variant, has been suggested to improve the prediction of UGT1A1 status, and specific UGT1A1 haplotypes are associated with altered SN-38 pheno- types (Innocenti et al., 2002, 2005; Sai et al., 2004; Kitagawa et al., 2005). This work was supported by the Canadian Institutes of Health Research (CIHR; MOP-42392) and Canada Research Chair Program (C.G.), and by Grants GM- 61834, GM-74369, DA-05258, MH-58435, DA-13209, DK-58496, DA-13834, AG- 17880, and RR-00054 from the National Institutes of Health (M.H.C, D.J.G., L.L.v.M.). Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.106.009787. ABBREVIATIONS: SN-38, 7-ethyl-10-hydroxycamptothecin; UGT, UDP-glucuronosyltransferase; SN-38G, SN-38-glucuronide; PCR, polymerase chain reaction; HPLC, high-performance liquid chromatography; LD, linkage disequilibrium; MPA, mycophenolic acid. 0090-9556/06/3407-1220–1228$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 7 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 9787/3119193 DMD 34:1220–1228, 2006 Printed in U.S.A. 1220