Development of Drug Detoxifying Bacteria for Chemotherapy Induced Gut Injury

项目总结/摘要 基于伊立替康的化疗广泛用于治疗各种类型的转移性癌症，然而， 重度药物性损伤或胃肠道（GI）毒性，尤其是重度迟发性腹泻（SDOD） SN-38（伊立替康的活性代谢产物）诱导的，限制了其临床应用。伊立替康诱导 SDOD发病率导致住院治疗导致医疗保健成本增加，生活质量差， 癌症患者的治疗效果。目前，约15%的人没有有效的治疗方法 的接受伊立替康治疗的患者，他们患有难以控制的SDOD症状，主要是由于他们无法 来解毒结肠中的SN-38 SN-38介导的肠道毒性在患者中更严重 与尿苷二磷酸糖基转移酶1A 1（UGT 1A 1）多态性，描绘的关键作用， 结肠UGT 1A 1预防SN-38诱导的SDOD。减少SN-38结肠炎的各种方法 暴露已经尝试过，但没有太大的成功，SDOD仍然是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， 北德克萨斯大学和休斯顿大学从植物中发现了葡萄糖基转移酶， 蒺藜苜蓿，可以有效地代谢典型的人类UGT 1A 1底物，包括SN-38，在一个实验室中， 体外测定。这导致了生物工程大肠杆菌（EC）的新颖和创新概念， 过量表达植物UGT 71 G1以产生可使SN-38解毒的药物解毒细菌（DDB）。在本STTR中 项目中，我们建议生成概念验证临床前证据，以支持开发一种安全， DDB作为一种新型的活菌制剂（LBP），可缓解和/或预防SN- 38-介导的肠毒性。在这里，我们将生物工程一个大肠杆菌菌株，具有良好的人类肠道 定殖潜力，以过表达用绿色标记的UGT 71 G1（U 71 G1 *n）的最具活性的变体 荧光蛋白（GFP），它将在一个保护胶囊中直接输送到结肠， SN-38糖基化为SN-38-葡萄糖。为了实现这一目标，建议的具体目标是：1）发展 可追踪的和更有活性的药物解毒细菌EC_U71G1*n（活性变体），以解毒SN-38，2） 开发可追踪和高活性冻干DDBs的结肠优化胶囊递送，以及3）评估 在伊立替康诱导的SDOD大鼠模型中2种结肠递送的DDB的功效。主动DDB设计， pUGT生物催化剂（用于SN-38的解毒）和GFP（用于追踪人中的有效肠道定殖 粪便用于治疗效果监测）将加速癌症患者从SDOD的恢复，并导致 在临床中有效管理伊立替康诱导的SDOD。该项目的成功将提供 Sanarenectin提供了开发针对SN-38的可追踪DDB所需的概念验证证据， II期SBIR/STTR应用中的临床前和IND使能研究。新的LBP将作为 治疗SDOD的补充疗法，这应该改善生活质量，甚至改善治疗结果 治疗转移性癌症患者一旦成功，我们计划采用同样的方法， 不同的植物UGT用于灭活植物中的其他GI毒性药物、外源性物质、污染物或其毒性代谢物， 通过开发药物特异性DDB来治疗结肠。

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.