Organ-specific Ugt1 profiling in detoxifying the anticancer drug CPT-11

描述（由申请人提供）：伊立替康（CPT-11）已被用作治疗结直肠癌的一线药物，结直肠癌是世界上第三常见的癌症。然而，由于严重的中性粒细胞减少症和晚期腹泻，CPT-11生物活化和随后的代谢引起的副作用，其有效性和安全性受到损害。CPT-11是一种前药，由羧酸酯酶水解成活性拓扑异构酶1抑制剂SN-38。失活和解毒主要通过UGT1A1催化葡萄糖醛酸化生成SN-38葡萄糖醛酸（SN-38G）发生。SN-38G通过胆管排泄到胃肠道，在那里它作为细菌的底物。微生物产生的-葡萄糖醛酸酶。游离的SN-38通过被动扩散或主动运输被消化道吸收。在2005年和2010年，FDA更新了CPT-11的标签，考虑到遗传UGT1A1*28等位基因并表现出UGT1A1表达降低的患者严重副作用的风险增加。然而，最近的一些临床研究总结了UGT1A1*28等位基因纯合子个体的生存率和统计学意义上更高的肿瘤反应率的趋势。充分了解SN-38定向葡萄糖醛酸化与CPT-11治疗引起的肠道组织损伤之间的关系是很重要的。最近，我们建立了一个Ugt1条件敲除小鼠模型，专门针对肝组织中Ugt1位点的缺失(Ugt1？消息灵通的老鼠)。令人惊讶的是，即使完全缺乏肝脏Ugt1位点和所有UGT1A蛋白，Ugt1DHep小鼠对CPT-11治疗和随后的肠道毒性具有相当的抗性。因此，我们假设UGT1A蛋白的肝外表达有助于SN-38的解毒，这可以在胃肠道缺失Ugt1位点(Ugt1？胃肠道老鼠)。重要的是，我们推测在小鼠中Ugt1基因座的胃肠道定向控制可以用来直接检测该组织对SN-38引起的肠道损伤的贡献。为了研究Ugt1基因座在小鼠中组织特异性表达对CPT-11引发的肠道损伤的贡献，我们将进行以下实验。特异性目的1将用于确定器官特异性缺失Ugt1位点在CPT-11延迟性腹泻中的影响。在此目的下概述的研究将探讨CPT-11诱导的Ugt1？Hep和Ugt1？胃肠道老鼠。特异性目的2将研究诱导选择性肠道UGT1A1表达的影响及其对保护肠道组织免受SN-38浓度增加所产生的毒性损伤的增强作用。利用我们实验室最近开发的新型动物模型，这些研究将确定肝脏和肠道葡萄糖醛酸化在CPT-11诱导肠道毒性的病因学中的作用。这些模型可以用来确定治疗方法，以提高与CPT-11治疗相关的治疗指数和疗效。

DESCRIPTION (provided by applicant): Irinotecan (CPT-11) has been used as a first line drug in the treatment of colorectal cancer, the third most commonly diagnosed cancer in the world. However, its efficacy and safety is compromised because of severe neutropenia and late diarrhea, the side-effects resulting from CPT-11 bioactivation and subsequent metabolism. CPT-11 is a prodrug that is hydrolyzed by carboxylesterase to the active topoisomerase 1 inhibitor, SN-38. Inactivation and detoxification occurs primarily by UGT1A1 catalyzed glucuronidation to form SN-38 glucuronide (SN-38G). SN-38G is excreted via the biliary ducts into the gastrointestinal (GI) tract, where it serves as a substrate for bacterial ?-glucuronidase enzymes produced by microflora. Free SN-38 is absorbed into the GI tract through passive diffusion or active transport. In 2005 and 2010, the FDA updated the label for CPT-11 regarding the heightened risk of serious side effects for patients that inherit the UGT1A1*28 allele and exhibit reduced expression of UGT1A1. However, several recent clinical studies summarize a trend of improved survival and statistically significant higher tumor response rates among individuals that are homozygous for the UGT1A1*28 allele. It is important to fully understand the association between SN-38 directed glucuronidation and the pending intestinal tissue damage resulting from CPT-11 therapy. Recently, we have generated an Ugt1 conditional knockout mouse model targeting deletion of the Ugt1 locus specifically in liver tissue (Ugt1?Hep mice). Surprisingly, even with the complete absence of the hepatic Ugt1 locus and all UGT1A proteins, Ugt1DHep mice are quite resistant towards CPT-11 treatment and the ensuing intestinal tract toxicity. Thus, we hypothesize that extrahepatic expression of the UGT1A proteins contributes to the detoxification of SN-38, which can be further confirmed in mice following gastrointestinal tract deletion of the Ugt1 locus (Ugt1?GI mice). Importantly, we speculate that GI tract directed control of the Ugt1 locus in mice can be leveraged to directly examine the contribution of this tissue towards SN-38 elicited intestinal damage. To examine the contribution of tissue specific expression of the Ugt1 locus in mice towards CPT-11 initiated intestinal damage, we will perform the following experiments. Specific Aim 1 will be directed to determine the impact of organ specific deletion of the Ugt1 locus in CPT-11 delayed diarrhea. Studies outlined under this aim will explore differences in CPT-11 induced intestinal toxicity in both Ugt1?Hep and Ugt1?GI mice. Specific Aim 2 will examine the impact of inducing selectively intestinal UGT1A1 expression and its enhanced contribution towards protecting intestinal tissue from the toxic insult generated from increasing concentrations of SN-38. With novel animal models recently developed in our laboratory, these studies will identify the role of hepatic and intestinal glucuronidation towards the etiology of CPT-11 induced intestinal toxicity. These models can be exploited to identify therapeutics that will improve the therapeutic index and efficacy associated with CPT-11 treatment. PUBLIC HEALTH RELEVANCE: Anticancer drug CPT-11 is used widely to treat colorectal cancer, but its application is markedly limited by its severe side effects, resulting primarily fro the exposure of intestinal epithelial cells to the active metabolite SN-38. SN-38 is deactivated through UGT1-catalyzed glucuronidation. We will employ Ugt1 conditional knockout mouse models that have been recently developed in our laboratory to investigate the contribution of SN-38 directed glucuronidation to improve the systemic tolerance of this chemotherapeutic compound.