Jinno Hideto, Saeki Mayumi, Saito Yoshiro, Tanaka-Kagawa Toshiko, Hanioka Nobumitsu, Sai Kimie, Kaniwa Nahoko, Ando Masanori, Shirao Kuniaki, Minami Hironobu, Ohtsu Atsushi, Yoshida Teruhiko, Saijo Nagahiro, Ozawa Shogo, Sawada Jun-ichi
Division of Environmental Chemistry, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan.
J Pharmacol Exp Ther. 2003 Aug;306(2):688-93. doi: 10.1124/jpet.103.051250. Epub 2003 May 1.
SN-38 (7-ethyl-10-hydroxycamptothecin), an active metabolite of the antitumor prodrug irinotecan, is conjugated and detoxified to SN-38 10-O-beta-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 766G>A 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 Km values were 19.3 and 44.4 microM, and the Vmax 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 Vmax/Km) 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.
SN-38（7-乙基-10-羟基喜树碱）是抗肿瘤前药伊立替康的活性代谢产物，通过肝脏尿苷二磷酸葡萄糖醛酸基转移酶（UGT）1A1与葡萄糖醛酸结合并解毒生成SN-38 10-O-β-D-葡萄糖醛酸。最近的研究表明，其他UGT1A同工型，即UGT1A7和UGT1A9，也参与SN-38的葡萄糖醛酸化。虽然已报道了影响SN-38葡萄糖醛酸化活性的UGT1A1和UGT1A7的几种基因多态性，但尚未鉴定出UGT1A9的此类多态性。在本研究中，对61例均接受伊立替康治疗的日本癌症患者的UGT1A9外显子1及其侧翼区域进行了测序。在UGT1A9外显子1中鉴定出一种新的非同义单核苷酸多态性，杂合子766G>A导致氨基酸D256N替换。野生型和D256N UGT1A9在COS-1细胞中以相似的蛋白质水平瞬时表达，并对其膜组分进行体外SN-38葡萄糖醛酸化活性表征。野生型和D256N变体的表观Km值分别为19.3和44.4 microM，Vmax值分别为2.94和0.24 pmol/min/mg膜蛋白。D256N的SN-38葡萄糖醛酸化效率（标准化Vmax/Km）不到野生型UGT1A9的5%。这些结果清楚地表明，D256N变体在SN-38葡萄糖醛酸化方面基本上无功能。这些发现突出了进一步研究UGT1A9 D256N变体对体内伊立替康代谢潜在影响的重要性。
