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

描述（由申请方提供）：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大最常诊断的癌症。然而，由于CPT-11生物活化和随后的代谢引起的严重中性粒细胞减少和晚期腹泻，其疗效和安全性受到损害。CPT-11是一种前药，可被羧酸酯酶水解为活性拓扑异构酶1抑制剂SN-38。灭活和解毒主要通过UGT 1A 1催化的葡萄糖醛酸化形成SN-38葡萄糖醛酸苷（SN-38 G）进行。SN-38 G通过胆管排泄到胃肠道（GI），在胃肠道中作为细菌？葡萄糖醛酸酶由微生物群产生。游离SN-38通过被动扩散或主动转运吸收至胃肠道。在2005年和2010年，FDA更新了CPT-11的标签，关于遗传UGT 1A 1 *28等位基因并表现出UGT 1A 1表达降低的患者的严重副作用风险增加。然而，最近的几项临床研究总结了UGT 1A 1 *28等位基因纯合子个体中生存率改善和肿瘤缓解率统计学显著升高的趋势。重要的是要充分了解SN-38定向葡萄糖醛酸化与CPT-11治疗引起的潜在肠组织损伤之间的相关性。最近，我们已经产生了一个Ugt 1条件敲除小鼠模型，靶向删除Ugt 1位点特异性在肝组织（Ugt 1？Hep小鼠）。令人惊讶的是，即使完全不存在肝脏Ugt 1基因座和所有UGT 1A蛋白，Ugt 1DHep小鼠对CPT-11治疗和随后的肠道毒性也具有相当的抗性。因此，我们假设UGT 1A蛋白的肝外表达有助于SN-38的解毒，这可以在胃肠道缺失Ugt 1基因座（Ugt 1？GI小鼠）。重要的是，我们推测可以利用小鼠中Ugt 1基因座的胃肠道定向控制来直接检查该组织对SN-38引起的肠损伤的贡献。为了检查小鼠中Ugt 1基因座的组织特异性表达对CPT-11引发的肠损伤的贡献，我们将进行以下实验。具体目标1将用于确定Ugt 1基因座的器官特异性缺失对CPT-11延迟性腹泻的影响。根据这一目标概述的研究将探讨CPT-11诱导的肠毒性在Ugt 1？Hep和Ugt 1？GI老鼠。具体目标2将检查选择性诱导肠道UGT 1A 1表达的影响及其对保护肠道组织免受SN-38浓度增加产生的毒性损伤的增强贡献。通过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对CPT-11诱导的肠道毒性病因学的作用。这些模型可用于鉴定将改善与CPT-11治疗相关的治疗指数和疗效的治疗剂。 公共卫生相关性：抗癌药物CPT-11被广泛用于治疗结直肠癌，但其应用受到其严重副作用的明显限制，主要是由于肠上皮细胞暴露于活性代谢物SN-38。SN-38通过UGT 1催化的葡萄糖醛酸化失活。我们将采用我们实验室最近开发的Ugt 1条件性敲除小鼠模型，研究SN-38定向葡萄糖醛酸化对改善该化疗化合物的全身耐受性的贡献。

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.