Short Communication Close Association of UGT1A9 IVS1 399C>T with UGT1A1*28, *6, or *60 Haplotype and Its Apparent Influence on 7-Ethyl-10- hydroxycamptothecin (SN-38) Glucuronidation in Japanese

The anticancer prodrug, irinotecan, is converted to its active form 7-ethyl-10-hydroxycamptothecin (SN-38) by carboxylesterases, and SN-38 is inactivated by UDP-glucuronosyltransferase (UGT)1A1-mediated glucuronidation. UGT1A9 also mediates this reaction. In a recent study, it was reported that the UGT1A9 IVS1 399 (I399)C>T polymorphism is associated with increased SN-38 glucuronidation both in vitro and in vivo. However, its role in UGT1A9 expression levels and activity is controversial. Thus, we evaluated the role of I399C>T in SN-38 glucuronidation using 177 Japanese cancer patients administered irinotecan. I399C>T was detected at a 0.636 allele frequency. This polymorphism was in strong linkage disequilibrium (LD) with UGT1A9*1b ( 126_ 118T9>T10, D 0.99) and UGT1A1*6 (211G>A, 0.86), in moderate LD with UGT1A1*60 ( 3279T>G, 0.55), but weakly associated with UGT1A1*28 ( 54_ 39A(TA)6TAA>A(TA)7TAA, 0.25). Haplotype analysis showed that 98% of the I399C alleles were linked with low-activity haplotypes, either UGT1A1*6, *28, or *60. On the other hand, 85% of the T alleles were linked with the UGT1A1 wildtype haplotype *1. Although I399T-dependent increases in SN-38 glucuronide/SN-38 area under concentration-time curve (AUC) ratio (an in vivo marker for UGT1A activity) and decreases in SN-38 AUC/ dose were apparent (P < 0.0001), these effects were no longer observed after stratified patients by UGT1A1*6, *28, or *60 haplotype. Thus, at least in Japanese populations, influence of I399C>T on SN-38 glucuronidation is attributable to its close association with either UGT1A1*6, *28, or *60. Irinotecan is an important drug for treatment of various tumors including lung, colon, and gastric (Smith et al., 2006). The infused drug is metabolized to its active form 7-ethyl-10-hydroxycamptothecin (SN-38) by carboxylesterases, and SN-38 is inactivated by glucuronidation. At least four UDP-glucuronosyltransferase (UGT) isoforms, namely UGT1A1, UGT1A7, UGT1A9, and UGT1A10, are known to glucuronidate SN-38 (Gagné et al., 2002; Saito et al., 2007). The UGT1A gene complex consists of 9 active first exons including UGT1A10, 1A9, 1A7, and 1A1 (in this order) and common exons 2 to 5. One of the 9 first exons can be used in conjunction with the common exons (Tukey and Strassburg, 2000). The UGT1A N-terminal domains (encoded by the first exons) determine substrate-binding specificity, and the C-terminal domain (encoded by exons 2 to 5) is important for binding to UDP-glucuronic acid. The 5 or 3 -flanking region of each exon 1 is presumably involved in regulation of its expression. Substantial interindividual differences have been detected in mRNA and protein levels and enzymatic activity of the UGT1A isoforms (Fisher et al., 2000; Saito et al., 2007). SN-38 glucuronidation is thought to be mediated mainly by UGT1A1, and its genetic polymorphisms affecting irinotecan pharmacokinetics and adverse reactions have been already identified. The TA-repeat polymorphism, 54_ 39A(TA)6TAA A(TA)7TAA (UGT1A1*28 allele), is associated with lower promoter activity, resulting in reduced SN-38 glucuronidation (Beutler et al., 1998; Iyer et al., 1999). The single nucleotide polymorphism (SNP) 211G A (Gly71Arg, *6 allele), found mainly in East Asians, causes reduced protein expression levels and SN-38 glucuronidation activity (Gagné et al., 2002; Jinno et al., 2003). Another SNP in the enhancer region of UGT1A1, 3279T G (*60 allele), is also a causative factor for reduced expression (Sugatani et al., 2002). Allele frequencies have been reported for *28 (0.09–0.13), *6 (0.15–0.19), and *60 (0.26–0.32) in Japanese and Chinese populations and for *28 (0.30– 0.39), *6 ( 0), and *60 (0.44–0.55) in whites (Saito et al., 2007). In a previous study, in the Japanese population, we defined haplotype *28 as the haplotype harboring the *28 allele, haplotype *6 as that harboring the*6 allele, and haplotype *60 as that harboring the *60 allele (and without the *28 or *6 allele) (Sai et al., 2004; Saeki et al., 2006). Note that most of the *28 haplotypes concurrently harbored the *60 alleles, and that the *28 and *6 alleles were exclusively present on the different chromosomes (Sai et al., 2004; Saeki et al., 2006). We have also revealed that the haplotype *28, *6, or *60 was associated with reduced SN-38 glucuronide (SN-38G)/SN-38 area under concentration-time curve (AUC) ratios, an in vivo parameter for UGT1A activity (Minami et al., 2007). In a recent study, an intronic SNP of UGT1A9, IVS1 399 (I399)C T, has been shown to be associated with increased UGT1A9 protein levels and glucuronidation activities toward SN-38 and the UGT1A9 probe drug propofol (Girard et al., 2006). Elevation of This study was supported in part by the program for the Promotion of Fundamental Studies in Health Sciences from the National Institute of Biomedical Innovation [Grant 05-25]; and a Health and Labour Sciences Research grant from the Ministry of Health, Labour and Welfare in Japan [Grant KHB1008]. Y.S. and K.S. contributed equally to this work. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.108.024208. ABBREVIATIONS: SN-38, 7-ethyl-10-hydroxycamptothecin; UGT, UDP-glucuronosyltransferase; SNP, single nucleotide polymorphism; SN-38G, SN-38 glucuronide; AUC, area under concentration-time curve; I399, UGT1A9 IVS1 399; LD, linkage disequilibrium. 0090-9556/09/3702-272–276$20.00 DRUG METABOLISM AND DISPOSITION Vol. 37, No. 2 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 24208/3426314 DMD 37:272–276, 2009 Printed in U.S.A. 272 at A PE T Jornals on A ril 0, 2017 dm d.aspurnals.org D ow nladed from SN-38 glucuronidation activity by this SNP is significant among subjects without UGT1A1*28. Sandanaraj et al. (2008) have also reported that I399C/C patients showed higher SN-38 AUC than C/T and T/T patients. With the same UGT1A1 diplotypes, patients with I399T/T (and UGT1A9 126_ 118T10/T10) have shown higher SN38G Cmax than I399C/T (and T9/T10) patients. UGT1A9*1b (UGT1A9 126_ 118T9 T10) has been shown to have no affect on UGT1A9 expression levels (Girard et al., 2006; Ramírez et al., 2007; Sandanaraj et al., 2008). Thus, two groups did suggest that I399T allele was associated with higher glucuronidation activity. However, using human liver microsomes, Ramírez et al. (2007) showed that I399C T had no significant effect on both UGT1A9 mRNA levels and glucuronidation activities for two UGT1A9 substrates. Therefore, the roles of I399C T in UGT1A9 activities as well as SN-38 glucuronidation remain inconclusive. In the present report, we reveal the linkage of I399C T with UGT1A1, UGT1A7, and UGT1A9 polymorphisms and analyze its association with the SN-38G/SN-38 AUC ratio and SN-38 AUC/dose (per dose) to clarify its role in SN-38 glucuronidation. Materials and Methods Patients. One hundred and seventy-seven patients (81 lung, 63 colon, 19 stomach, and 14 other cancer patients) administered irinotecan at the National Cancer Center were enrolled in this study as described previously (Minami et al., 2007). This study was approved by the ethics committees of the National Cancer Center and the National Institute of Health Sciences, and written informed consent was obtained from all participants. Eligibility criteria, patient profiles, and irinotecan regimens are summarized in our previous report (Minami et al., 2007). In brief, patients consisted of 135 males and 42 females with a mean age of 60.5 (26–78 years old), and their performance status was 0 (84 patients), 1 (89 patients), or 2 (4 patients). Irinotecan administrations were conducted according to the standard protocols in Japan as follows: i.v. 90-min infusion at a dose of 100 mg/m weekly or 150 mg/m biweekly in irinotecan monotherapy; and 60 mg/m weekly with cisplatin in most combi-