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

描述(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种最常见的癌症。然而，由于严重的中性粒细胞减少和迟发性腹泻，CPT-11的生物激活和随后的代谢产生的副作用，其疗效和安全性受到了影响。CPT-11是一种前药，它被羧酸酯酶水解为具有活性的拓扑异构酶1抑制剂SN-38。失活和解毒主要通过UGT1A1催化的葡萄糖醛酸化生成SN-38葡萄糖醛酸苷(SN-38G)来实现。SN-38G通过胆管排泄到胃肠道(GI)，在那里它是由微生物群产生的细菌β-葡萄糖醛酸苷酶的底物。游离的SN-38通过被动扩散或主动转运被吸收到胃肠道。在2005年和2010年，FDA更新了CPT-11的标签，关于遗传UGT1A1*28等位基因并表现出UGT1A1表达降低的患者严重副作用的高风险。然而，最近的几项临床研究总结了UGT1A1*28等位基因纯合的个体存活率提高和肿瘤应答率显著提高的趋势。充分了解SN-38直接的葡萄糖醛酸化作用与CPT-11治疗引起的未决肠道组织损伤之间的关系是很重要的。最近，我们建立了一种针对肝组织(UGT1？HEP小鼠)UGT1基因座缺失的条件性基因敲除小鼠模型。令人惊讶的是，即使完全缺乏肝脏UGT1基因和所有UGT1A蛋白，UGT1DHep小鼠对CPT-11治疗和随之而来的肠道毒性仍具有相当的抵抗力。因此，我们假设UGT1A蛋白的肝外表达有助于SN-38的解毒，这一点在胃肠道UGT1基因缺失的小鼠(UGT1？GI小鼠)中得到进一步证实。重要的是，我们推测，胃肠道对小鼠UGT1基因的直接控制可以被用来直接检查该组织对SN-38引起的肠道损伤的贡献。为了研究小鼠UGT1基因的组织特异性表达在CPT-11引起的肠道损伤中的作用，我们将进行以下实验。具体目标1将用于确定器官特异性UGT1基因缺失在CPT-11迟发性腹泻中的影响。在这一目标下概述的研究将探索CPT-11在UGT1？HEP和UGT1？GI小鼠中引起的肠道毒性的差异。具体目标2将研究选择性诱导肠道UGT1A1表达的影响及其在保护肠道组织免受因SN-38浓度增加而产生的毒性损害方面的增强贡献。通过我们实验室最近开发的新的动物模型，这些研究将确定肝脏和肠道的葡萄糖醛酸化作用在CPT-11诱导的肠道毒性的病因中所起的作用。这些模型可以被用来确定将改善与CPT-11治疗相关的治疗指数和疗效的治疗方法。 公共卫生相关性：抗癌药物CPT-11被广泛用于治疗结直肠癌，但其严重副作用显著限制了其应用，主要是由于肠道上皮细胞暴露于活性代谢物SN-38。SN-38通过UGT1催化的葡萄糖醛酸化反应失活。我们将使用我们实验室最近开发的UGT1条件性基因敲除小鼠模型来研究SN-38指导的葡萄糖醛酸化作用对提高这种化疗化合物的系统耐受性的贡献。

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.