Development of Drug Detoxifying Bacteria for Chemotherapy Induced Gut Injury

项目摘要/摘要 基于伊立替康的化疗被广泛用于治疗各种类型的转移性癌症，然而， 严重的药物损伤或胃肠道(GI)毒性，尤其是严重的迟发性腹泻(SDOD) 由伊立替康的活性代谢物SN-38诱导，限制了其临床应用。伊立替康诱导 SDOD的发生导致住院导致的医疗费用增加，以及生活质量和 癌症患者的治疗结果。目前，大约有15%的人没有有效的治疗方法 接受伊立替康治疗的患者中，他们出现无法控制的SDOD症状，主要是由于他们无法 使结肠中的SN-38解毒。SN-38介导的肠道毒性在患者中被发现更严重 通过尿苷二磷酸糖基转移酶1A1(UGT1A1)基因的多态性，阐明了尿苷二磷酸糖基转移酶1A1的关键作用 结肠UGT1A1预防SN-38诱导的SDOD减少SN-38结肠癌的各种方法 暴露的尝试没有多少成功，SDOD仍然无法控制的剂量限制毒性 伊立替康在成人和儿童癌症患者中的应用。我们的学术研究合作者的初步研究 北得克萨斯大学和休斯顿大学发现了植物中的葡萄糖转移酶 能够高效代谢包括SN-38在内的典型人类UGT1A1底物的元宝紫花苜蓿 体外试验。这导致了生物工程共生菌E.Coli(EC)的新颖和创新概念 过量表达植物UGT71G1以产生一种可以解毒SN-38的药物解毒菌(DDB)。在本STTR中 项目中，我们建议生成概念验证的临床前证据，以支持开发安全、 DDB作为一种新型的活体生物治疗产品(LBP)，可有效地缓解和/或预防SN-B。 38介导的肠道毒性。在这里，我们将生物工程一种共生的大肠杆菌菌株，具有良好的人类肠道 定植潜能，过度表达UGT71G1(U71G1*n)最活跃的突变体，标记为绿色 荧光蛋白(GFP)，它将在保护胶囊中直接输送到结肠，以获得有效的 SN-38糖基化为SN-38-葡萄糖。为了实现这一目标，提出的具体目标是：1)发展 可追溯且更具活性的药物解毒菌EC_U71G1*n(活性突变体)对SN-38，2)进行解毒 开发一种可追踪和高活性冻干DDB的结肠优化胶囊递送，以及3)评估 2种结肠型DDBs对伊立替康所致SD大鼠模型的影响采用以下技术设计的活动分布式数据库 PUGT生物催化剂(用于SN-38的解毒)和GFP(用于示踪人体有效的肠道定植 用于疗效监测的粪便)将加速癌症患者从SDOD中恢复，并导致 伊立替康所致SDOD的临床有效管理。该项目的成功将为 Sanarentero拥有开发SN-38目标可追踪DDB所需的概念验证证据 第二阶段SBIR/STTR应用中的临床前和IND使能研究。小说中的LBP将扮演一个 治疗SDOD的补充疗法，应可改善生活质量，甚至改善治疗结果 对于转移性癌症患者。一旦这一努力取得成功，我们计划应用相同的方法来利用 不同的植物UGT用于灭活其他胃肠道毒性药物、异物、污染物或其有毒代谢物 结肠通过开发药物特异性DDB。

PROJECT SUMMARY/ ABSTRACT Irinotecan-based chemotherapy is widely used for the treatment of various types of metastatic cancers, however, severe drug-induced injury or gastrointestinal (GI) toxicity, especially severe delayed-onset diarrhea (SDOD) induced by SN-38 (an active metabolite of irinotecan), limits its clinical application of the drug. Irinotecan-induced SDOD incidences result in increased healthcare cost due to hospitalization, and poor quality of life and therapeutic outcomes for the cancer patients. Currently, there is no effective treatment available for about ~15% of patients receiving irinotecan therapy, who suffer unmanageable SDOD symptoms, mainly due to their inability to detoxify the SN-38 in the colon. SN-38 mediated intestinal toxicity was found to be more severe in patients with uridine diphosphate glycosyltransferases 1A1 (UGT1A1) polymorphism, delineating the critical role of colonic UGT1A1 in the prevention of SN-38 induced SDOD. Various approaches to reduce SN-38 colonic exposure has been tried without much success, and SDOD remains unmanageable dose-limiting toxicity of irinotecan in adult and pediatric cancer patients. Preliminary research of our academic research collaborators at the University of North Texas and the University of Houston discovered glucosyltransferases from plant Medicago truncatula that could efficiently metabolize typical human UGT1A1 substrates, including SN-38, in an in vitro assay. This led to the novel and innovative concept of bioengineering commensal bacteria E.coli (EC) for overexpressing plant UGT71G1 to create a drug detoxifying bacteria (DDB) that can detoxify SN-38. In this STTR project, we propose to generate proof-of-concept preclinical evidence in support of developing a safe, efficacious, and traceable DDB as a novel live biotherapeutic product (LBP) to alleviate and/or prevent the SN- 38-mediated intestinal toxicity. Here, we will bioengineer a commensal E.coli strain, with good human gut colonization potential, to overexpress the most active variant of UGT71G1 (U71G1*n) tagged with a green fluorescent protein (GFP), which will be delivered in a protective capsule directly to the colon for the effective glycosylation of SN-38 to SN-38-glucose. To achieve this goal, the proposed specific aims are 1) to develop traceable and more active drug detoxifying bacteria EC_U71G1*n (active variant) to detoxify SN-38, 2) to develop a colon-optimized capsule delivery of traceable and highly active lyophilized DDBs, and 3) to evaluate the efficacy of 2 colon-delivered DDBs in irinotecan induced SDOD rat model. An active DDB engineered with pUGT biocatalyst (for the detoxification of SN-38) and GFP (for tracing the effective gut colonization in human feces for therapeutic efficacy monitoring) will accelerate the recovery of cancer patients from SDOD, and lead to the effective management of irinotecan-induced SDOD in clinics. The success of this project will provide Sanarentero with the proof-of-concept evidence needed for developing an SN-38-targeted traceable DDB for preclinical and IND-enabling studies in a phase-II SBIR/STTR application. The novel LBP will act as a complementary therapy to treat SDOD, which should improve the quality of life and even therapeutic outcomes for metastatic cancer patients. Once successful in this endeavor, we plan to apply the same approach to utilize different plant UGTs for inactivating other GI-toxic drugs, xenobiotics, pollutants, or their toxic metabolites in the colon by developing drug-specific DDB.