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大最常见的癌症。然而，由于严重的中性粒细胞减少症和晚期腹泻、CPT-11生物激活和随后的代谢产生的副作用，其功效和安全性受到影响。 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。失活和解毒主要通过 UGT1A1 催化葡萄糖醛酸化形成 SN-38 葡萄糖醛酸苷 (SN-38G) 发生。 SN-38G 通过胆管排泄到胃肠道 (GI)，在那里它充当微生物群产生的细菌 β-葡萄糖醛酸酶的底物。游离的 SN-38 通过被动扩散或主动转运被吸收到胃肠道中。 2005年和2010年，FDA更新了CPT-11的标签，认为对于继承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，最近的几项临床研究总结了 UGT1A1*28 等位基因纯合个体的生存率提高和肿瘤反应率统计显着升高的趋势。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联非常重要。最近，我们建立了一个 Ugt1 条件敲除小鼠模型，其目标是特异性删除肝组织中的 Ugt1 位点（Ugt1？Hep 小鼠）。令人惊讶的是，即使肝脏 Ugt1 基因座和所有 UGT1A 蛋白完全缺失，Ugt1DHep 小鼠对 CPT-11 治疗和随之而来的肠道毒性也具有相当的抵抗力。因此，我们假设UGT1A蛋白的肝外表达有助于SN-38的解毒，这可以在胃肠道删除Ugt1基因座的小鼠（Ugt1？GI小鼠）中得到进一步证实。重要的是，我们推测可以利用小鼠中 Ugt1 基因座的胃肠道定向控制来直接检查该组织对 SN-38 引起的肠道损伤的贡献。为了检查小鼠中 Ugt1 基因座的组织特异性表达对 CPT-11 引发的肠道损伤的贡献，我们将进行以下实验。具体目标 1 将旨在确定 Ugt1 基因座的器官特异性缺失对 CPT-11 迟发性腹泻的影响。在此目的下概述的研究将探讨 CPT-11 在 Ugt1?Hep 和 Ugt1?GI 小鼠中诱导肠道毒性的差异。具体目标 2 将检查选择性诱导肠道 UGT1A1 表达的影响及其对保护肠道组织免受因 SN-38 浓度增加而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 诱导肠道毒性病因的作用。这些模型可用于识别可提高与 CPT-11 治疗相关的治疗指数和疗效的治疗方法。 公共健康相关性：抗癌药物 CPT-11 广泛用于治疗结直肠癌，但其应用明显受到其严重副作用的限制，这些副作用主要是由于肠上皮细胞暴露于活性代谢物 SN-38 造成的。 SN-38 通过 UGT1 催化的葡萄糖醛酸化作用失活。我们将采用我们实验室最近开发的 Ugt1 条件敲除小鼠模型来研究 SN-38 定向葡萄糖醛酸化对改善这种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.