IND-enabling development of Radioprotectin 1: a dual GI/HE radiation mitigator

广大美国民众，特别是军事人员和急救人员，面临着辐射的危险 由于核装置爆炸、核反应堆事故和辐射恐怖主义威胁而暴露。 FDA批准的放射医学对策(RCM)药物没有符合 胃肠道(GI)放射增强剂-一种缓解急性GI辐射综合征(GI-ARS)的药物 在暴露后给药。电离辐射杀死无法修复DNA的细胞，主要是通过 有丝分裂灾难和细胞凋亡性死亡。辐射后的遗传毒性应激和细胞损伤是一个悬而未决的问题 医学上的问题。阻碍RCM药物开发的一个关键障碍是，传统的人类 临床试验不是一种选择。因此，FDA对RCM的批准是根据动物规则进行的，该规则要求 详细了解其作用机制，在动物模型中展示其安全性和有效性 以及它在人体内的安全性。在这项过渡性研究建议中，我们建议进行研究，以充分满足 放射防护素-1(RP-1)--一种新的辐射减释剂--动物规程的作用机理 用以前的NIAID资金开发，并开发符合CONPOS的单剂缓释配方 RCM的要求。我们的总体目标是准备RP-1作为一流的合成药物获得监管部门的批准 胃肠道辐射缓释剂。RP-1是溶血磷脂酸(LPA)受体亚型2的第一个特异性激动剂 (LPA2)具有微克分子EC50，可降低辐射损伤诱导的小鼠死亡率。我们的中心假设 是由LPA2 G蛋白偶联受体介导的由RP-1激活的独特的长时间(16h)信号吗 (GPCR)，负责减轻遗传毒性应激和促进细胞存活。我们的假设预测 RP-1通过1)LPA2 GPCR的专职刺激，2)超分子的顺序募集来实现这一点 负责其长时间作用的信号相互作用，3)增强DNA修复和4) 提高LGR5肠道干细胞(ISC)的存活率。我们的目标是：1)详细确定独特的 RP-1如何通过LPA2募集克服遗传毒性所需相互作用的分子机制 应激，以及2)利用转基因小鼠在体内鉴定受RP-1保护的ISC的特定亚群 在带有ISC的LGR5标记中表达荧光蛋白，并开发单剂缓释 减轻GI-ARS的纳米颗粒配方。尽管这一信息对于推进RP-1是必要的 对于监管审批，我们将生成的知识体系也代表着重要的和以前的 关于辐射防护信号机制的未知信息。我们的预期结果将包括1) 建立依赖LPA2的IEX-1-TRIP6-ERK1/2-AKT互动组的招募是 减轻遗传毒性应激；2)确定增强DNA依赖蛋白激酶的RP-1效应的作用。 依赖的DNA修复和生存信号；3)证明RP-1是GI-ARS的有效缓解剂 通过保护小鼠的ISC；4)单剂缓释RP-1制剂。我们项目的影响将 直接影响我们在治疗辐射损伤患者时的第一反应选择。该项目的目标是： 目的1.验证Rp-1通过LPA2介导DNA修复和蛋白原长时间激活的假说。 LGR-5阳性的肠道干细胞中的生存信号。 目的2.研制治疗GI-ARS的纳米粒缓释RP-1制剂 老鼠和恒河猴。 所有获得的数据将用于RP-1药物主文件，并在IND前会议期间提交给FDA 在项目的最后一年，并与广大研究界分享。

The US population at large, and particularly military personnel and first responders, are at risk of radiation exposure due to the explosion of a nuclear device, a nuclear reactor accident, and the threat of radiation terrorism. There is no radiation medical countermeasure (RCM) drug approved by the FDA that meets the criterion of a gastrointestinal (GI) radiomitigator – an agent which mitigates the acute GI radiation syndrome (GI-ARS) when administered after the exposure. Ionizing radiation kills cells that are unable to repair their DNA, primarily via mitotic catastrophe and apoptotic cell death. Post-irradiation genotoxic stress and cell injury is an unsolved medical problem. A critical barrier to progress in development of RCM drugs is that a traditional human clinical trial is not an option. Therefore, FDA approval of a RCM is done under the Animal Rule that requires detailed understanding of its mechanism of action, demonstration of its safety, and efficacy in animal models, and its safety in humans. In this transitional research proposal, we propose studies to fully satisfy the mechanism of action requirement of the Animal rule for Radioprotectin-1 (RP-1) a new radiation mitigator we developed with previous NIAID funding and develop a single-dose extended release formulation that meets CONPOS requirements of a RCM. Our overall goal is to prepare RP-1 for regulatory approval as a first-in-class synthetic GI radiation mitigator. RP-1 is the first specific agonist of the lysophosphatidic acid (LPA) receptor subtype 2 (LPA2) with picomolar EC50, which reduces radiation injury-induced mortality in mice. Our central hypothesis is that RP-1–activated, uniquely long-lasting (> 16h) signaling mediated by the LPA2 G protein-coupled receptor (GPCR), is responsible for mitigation of genotoxic stress and promotion of cell survival. Our hypothesis predicts that RP-1 achieves this via 1) obligate stimulation of the LPA2 GPCR, 2) sequential recruitment of supramolecular signaling interactomes responsible for the long duration of its action, 3) augmentation of DNA repair and 4) enhanced survival of LGR5 intestinal stem cells (ISC). Our objectives are: 1) determine in detail the unique molecular mechanism of how RP-1 acts via LPA2 to recruit the interactomes required for overcoming genotoxic stress, and 2) identify the specific subpopulation of ISC that is protected by RP-1 in vivo using transgenic mice that express fluorescent protein in the LGR5 marker bearing ISC and 3) develop a single dose extended release nanoparticle formulation that mitigates the GI-ARS. Although this information is necessary to move RP-1 forward toward regulatory approval, the body of knowledge we will generate also represents significant and previously unknown information concerning radioprotective signaling mechanisms. Our expected outcomes will include 1) establishing that LPA2-dependent recruitment of the IEX-1–TRIP6–ERK1/2-AKT interactome is required for mitigation of genotoxic stress; 2) defining the role of RP-1 effects that enhance DNA-dependent Protein Kinase- dependent DNA repair and prosurvival signals; 3) demonstrating that RP-1 is an effective mitigator of GI-ARS by protecting ISC in mice; 4) a single-dose extended-release RP-1 formulation. The impact of our project will directly affect our first-response options in treating patients with radiation injury. The aims of the project are: Aim 1. Test the hypothesis that RP-1 via LPA2 mediates long-lasting activation of DNA repair and pro- survival signaling in LGR-5 positive intestinal stem cells. Aim 2. Develop a nanoparticle-based extended-release RP-1 formulation for the treatment of the GI-ARS of mice and Rhesus macaques. All data obtained will be used for a RP-1 Drug Master file and presented to the FDA during our PRE-IND meeting in the final year of the project and shared with the research community at large.