Engineering a long-acting relaxin agonist to treat liver fibrosis

项目摘要/摘要 松弛素是一种由53个氨基酸组成的异二聚体多肽激素，可诱导心血管顺应性和 妊娠和分娩期间生殖组织重塑。除了生殖器官，松弛 受体RXFP1也在肝、心、肺、肾、骨和皮肤中表达。这张宽大的纸巾 本地化使人们认识到松弛素是一种多功能激素，具有血管扩张、抗纤维化、组织 动物模型的重塑、抗细胞凋亡和抗炎特性。松弛素的疗效已被证实 在从急性和慢性心力衰竭到纤维性疾病的人体临床试验中进行测试 皮肤、肺和肝脏；然而，松弛素的短暂半衰期和持续静脉输注的需要有限 它的临床应用。尽管使用松弛素来治疗纤维化和心血管疾病的理由仍然很多， 必须开发更好的RXFP1激动剂，以显示更长的血清半衰期和可测量的 药效学读数，同时保持与慢性RXFP1激动剂相称的安全性 治疗。我们使用了斑马生物公司专有的蛋白质中蛋白(PIP)抗体技术来插入一个 免疫球蛋白G互补决定区(CDR)的单链松弛蛋白构建 我的脊梁。这项技术以前曾被用来设计半衰期为数天的蛋白质和多肽 而天然分子只需要几分钟的时间。一种松弛素-PIP，H2-PIP现在已经被设计成具有内在效力 在细胞检测中可与重组松弛素相媲美。这项提议的目的是澄清 H2-PIP的药代动力学和药效学性质。我们假设这种长效松弛素-PIP 与松弛素相比，激动剂分子将是一种更好的抗纤维化药物候选药物，允许概念验证 通过较少的皮下注射进行疗效和安全性评估，从而消除了对 持续静脉输液。 我们提交了第一阶段STTR赠款申请，以满足以下具体目标(SA)： SA1：H2-PIP的药代动力学-药效学(PK-PD)分析。SA1的目标是确定 H2-PIP的半衰期和最大活性血清浓度，并将血液暴露与已知的 急性CCl4毒性背景下RXFP1激活的药效学标志物。这种关联将允许 美国建立剂量和剂量频率的估计，以确定肝纤维化模型的疗效。 SA2：H2-PIP对小鼠肝纤维化模型的疗效。SA2的目标是确定疗效 H2-PIP在两种显示关键分子和组织病理学的慢性肝纤维化小鼠模型中的表达 人类肝纤维化的特点：1)接触CCl4和2)高脂饮食(HFD)治疗。 这些研究的成功完成将为一系列疾病的最终临床研究奠定基础。 组织纤维化和血流动力学病理表现明显。

Project Summary/Abstract Relaxin is a heterodimeric 53 amino acid peptide hormone that induces cardiovascular compliance and reproductive tissue remodeling during pregnancy and parturition. In addition to reproductive organs, the relaxin receptor, RXFP1, is also expressed in the liver, heart, lung, kidney, bone and skin. This broad tissue localization has led to the recognition that relaxin is a pleiotropic hormone with vasodilatory, antifibrotic, tissue remodeling, antiapoptotic, and anti-inflammatory properties in animal models. The efficacy of relaxin has been tested in human clinical trials in diseases ranging from acute and chronic heart failure, to fibrotic diseases of skin, lung, and liver; however, relaxin's short half-life and need for continual intravenous infusion have limited its clinical utility. While the rationale to treat fibrosis and cardiovascular diseases with relaxin remains high, better RXFP1 agonists must be developed that display longer serum half-lives and measurable pharmacodynamic readouts, while maintaining a safety profile commensurate with chronic RXFP1 agonist treatment. We have used Zebra Biologics' proprietary Protein-in-Protein (PiP) antibody technology to insert a single-chain relaxin construct into the complementarity-determining region (CDR) of an immunoglobulin G backbone. This technology has been used previously to engineer proteins and peptides with half-lives of days vs. minutes for the native molecules. A relaxin-PiP, H2-PiP, has now been engineered with an intrinsic potency comparable to recombinant relaxin in cellular assays. The objective of this proposal is to elucidate pharmacokinetic and pharmacodynamic properties of H2-PiP. We hypothesize that this long-acting relaxin-PiP agonist molecule will be a superior antifibrosis drug candidate compared to relaxin, allowing proof-of-concept efficacy and safety assessment with less frequent subcutaneous injections, thus obviating the need for continuous intravenous infusion. We submit a Phase 1 STTR grant application addressing the following Specific Aims (SA): SA1: Pharmacokinetic-pharmacodynamic (PK-PD) analysis of H2-PiP. The goal of SA1 is to determine the half-life and maximal active serum concentration of H2-PiP, and to correlate blood exposure with known pharmacodynamic markers of RXFP1 activation in the context of acute CCl4 toxicity. This correlation will allow us to establish estimates of dose and dose frequency for determining efficacy in models of liver fibrosis. SA2: Efficacy of H2-PiP in mouse models of hepatic fibrosis. The goal of SA2 is to determine the efficacy of H2-PiP in two mouse models of chronic liver fibrosis that display key molecular and histopathological features of human liver fibrosis: 1) CCl4 exposure and 2) high fat diet (HFD) treatment. Successful completion of these studies will set the stage for eventual clinical studies in a range of diseases where tissue fibrosis and hemodynamic pathologies are manifest.