Tailoring Novel Therapeutics for Emerging Drug-Resistant C. Difficile Colitis

描述(由申请人提供)： 直到最近，艰难梭菌相关性腹泻(CDAD)作为一种机会性感染出现在使用抗生素的老年住院患者中。目前，CDAD已变得更加令人担忧，因为由于产生高毒素、产生二元毒素、对喹诺酮类药物耐药(Bl)菌株的出现，CDAD在美国和加拿大的频率和严重性普遍增加。此外，威胁生命的CDAD现在正在影响社区中健康的年轻人，即使最近没有使用抗生素。更糟糕的是，目前由更多抗生素组成的治疗正在失败，往往会延长脱落或复发的时间。因此，我们迫切需要新的方法来阻断毒素诱导的结肠炎，以防止艰难梭菌(S毒素)引起的结肠炎的进展，而不进一步破坏保护性菌群。弗吉尼亚大学的这项申请将我们经验丰富的肠学实验室团队与毒素发病机制和药物开发方面的基础科学同事以及一个药物合作伙伴(腺苷治疗公司，LLC)聚集在一起。我们将用一种创新的抗毒方法来解决新出现的对喹诺酮类药物耐药的艰难梭菌结肠炎，该方法得到了试验数据的支持，证明了相当有希望。我们建议单独和联合研究三种类型的抗炎药：阻断白细胞激活的腺苷A2a受体激动剂；阻断上皮细胞和肥大细胞产生细胞因子的A2B拮抗剂；以及促进吸收损伤修复的丙氨酰谷氨酰胺。前三个密切相关的特定目标旨在确定这些新疗法如何调节肠道细胞靶点，以阻止毒素诱导的细胞凋亡、炎症和分泌。我们计划：1)使用新的人肠上皮细胞类器官和C57BL/6小鼠模型来定义纯化的毒素A和B以及B变体和新的B1菌株上清液的靶点；2)使用腺苷受体敲除和Cre/loxP靶向细胞AAA受体缺失的C57BL/6小鼠来定义相关的腺苷靶点，以抑制毒素效应；以及3)使用AIMS 1和2中获得的信息来测试A2A激动剂、A2B拮抗剂和丙氨酰-谷氨酰胺的组合，以便为这些针对人类日益严重的CDAD的新药疗法的临床试验做准备。我们的第四个目标涉及开发更高吸收的AAR激动剂的发现工作，以及合成支持这项赠款所需的拟议A2A、激动剂和A2B拮抗剂。

DESCRIPTION (provided by applicant): Until recently, C. difficile associated diarrhea (CDAD) occurred as an opportunistic infection in older hospitalized patients on antibiotics. Currently, CDAD has become much more worrisome because of widespread increases in its frequency and severity throughout the US and Canada due to the emergence of hypertoxinogenic, binary toxin-producing, quinolone-resistant (Bl) strains. Furthermore, life threatening CDAD is now affecting healthy younger people in communities, even without recent antibiotic use. Even worse, current treatment consisting of more antibiotics is failing and often prolongs shedding or relapses. Hence we urgently need novel approaches that block toxin-induced colitis to prevent the progression of colitis produced by C. difficile toxin(s)) without further disruption of protective flora. This application from the University of Virginia brings together our experienced enterics laboratory team with basic science colleagues in toxin pathogenesis and in drug development, and a pharmaceutical partner (Adenosine Therapeutics, LLC). We shall address the emerging quinolone-resistant C. difficile colitis with an innovative antitoxic approach that is supported by pilot data demonstrating considerable promise. We propose to investigate three types of anti-inflammatory agents alone and in combination: adenosine A2A receptor agonists that block leukocyte activation; A2B antagonists that block cytokine production by epithelial and mast cells, and the pro-absorptive injury repairing agent, alanyl-glutamine. The first three closely interrelated specific aims are designed to determine how these novel therapeutics regulate intestinal cell targets to block toxin-induced apoptosis, inflammation, and secretion. We plan to: 1) use new human intestinal epithelial cell organoid and C57BL/6 murine models to define the targets of purified toxins A and B as well as B-variant and new Bl strain supernatants; 2) use C57BL/6 mice with adenosine receptor knockouts and Cre/loxp targeted cellular AaA receptor deletions to define the relevant adenosine targets to inhibition of toxin effects; and 3) use the information gained in aims 1 and 2 to test combinations of A2A agonists, A2B antagonists and alanyl-glutamine, alone and in combination, in animal models in order to prepare for clinical trials of these novel drug therapies for increasingly serious CDAD in humans. Our fourth aim involves discovery efforts towards developing more highly absorbed AaR agonists and the synthesis of the proposed A2A, agonists and A2B antagonists necessary to support this grant.