Engineering a long-acting relaxin agonist to treat liver fibrosis

项目概要/摘要 松弛素是一种异源二聚体的53个氨基酸的肽激素，其诱导心血管顺应性， 妊娠和分娩期间生殖组织重塑。除了生殖器官，松弛素 受体RXFP 1也在肝脏、心脏、肺、肾脏、骨骼和皮肤中表达。这个宽阔的组织 局部化导致认识到松弛素是一种多效激素，具有血管舒张、抗纤维化、组织 在动物模型中的重塑、抗凋亡和抗炎特性。松弛素的功效已经 在人类临床试验中测试了从急性和慢性心力衰竭到纤维化疾病的疾病， 皮肤、肺和肝脏;然而，松弛素的短半衰期和持续静脉输注的需要限制了 其临床效用。虽然用松弛素治疗纤维化和心血管疾病的理由仍然很充分， 必须开发更好的RXFP 1激动剂，其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RXFP 1激动剂相当的安全性特征 治疗我们使用Zebra Biologics专有的蛋白质中蛋白质（PiP）抗体技术， 单链松弛素构建体插入免疫球蛋白G的互补决定区（CDR） 骨干这项技术以前曾用于设计半衰期为几天的蛋白质和肽 vs.天然分子的时间。一种松弛素-PiP，H2-PiP，现在已经被设计成具有内在的效力， 在细胞测定中与重组松弛素相当。本提案的目的是阐明 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，激动剂分子将是上级抗纤维化候选药物， 有效性和安全性评估，皮下注射频率较低，因此无需 持续静脉输注。 我们提交了第一阶段STTR资助申请，解决以下具体目标（SA）： SA 1：H2-PiP的药代动力学-药效学（PK-PD）分析。SA 1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 在急性CCl 4毒性的背景下，RXFP 1激活的药效学标志物。这种关联将允许 我们建立剂量和给药频率的估计值，以确定在肝纤维化模型中的疗效。 SA 2：H2-PiP在肝纤维化小鼠模型中的功效。SA 2的目标是确定疗效 H2-PiP在两种慢性肝纤维化小鼠模型中的作用，显示关键的分子和组织病理学 人类肝纤维化的特征：1）CCl 4暴露和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 其中组织纤维化和血液动力学病理是明显的。

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.