Harnessing transthyretin to extend the in vivo half-life of therapeutic peptides

描述（由申请人提供）：利用甲状腺素运载蛋白延长治疗性肽的体内半衰期项目概述：肽的巨大治疗潜力尚未实现，并且迄今为止失败的潜在肽疗法远远超过成功的肽疗法。阻碍肽作为治疗剂的更广泛使用的主要挑战是其差的药代动力学（PK）特征，这是由于血清蛋白酶灭活和肾快速消除导致的短体内半衰期。因此，延长肽的体内半衰期显然是期望的，以便实现其治疗潜力，而不需要高剂量和/或频繁施用。 肽与大分子（例如聚乙二醇或血清蛋白）的共价缀合一直是用于增强肽的体内半衰期的主要方法。然而，这些大的大分子的空间位阻通常损害肽与其细胞外受体的结合，这损害了肽的药效学（PD）特性。该提案的总体目标是开发一种全新的方法，用于在不影响其生物活性的情况下提高肽的体内半衰期。该提议的主要假设是测试肽与选择性甲状腺素运载蛋白（TTR，一种血清蛋白）配体的缀合是否允许这些肽缀合物可逆地结合血清中的TTR。这将导致增加肽的体内半衰期，但最重要的是，将保持肽对其受体的结合亲和力。一个主要的重点将是我们最近发现的最有效的和选择性的可逆TTR配体的日期，AG 10。我们已经通过合成第一个AG 10-肽缀合物（Conj 1;以GnRH作为模型肽）取得了重大进展，我们对Conj 1的体外初步结果与这一假设一致。在这个提议中，我们将在体内评估Conj 1，并研究最佳的接头系统和对TTR的亲和力， 提供这些AG 10-GnRH缀合物的PK和PD之间的最佳平衡。本论文的主要目标有三：第一，设计合成一系列AG 10-GnRH偶联物，确定最佳的连接体长度和偶联位点（对AG 10和GnRH），其将保持缀合物对TTR和GnRH受体两者的最大结合亲和力和血清选择性;目标2，评价体外半-结合物在血清中的寿命，并确定最佳接头系统，该系统允许GnRH对血清蛋白酶的保护和对TTR的结合亲和力之间的最佳平衡和GnRH受体;和目的3，评价缀合物的药代动力学性质以确定与TTR的选择性结合是否将增加体内半衰期，同时维持缀合物在大鼠中的功效。如果我们成功地实现了这一提议的目标，我们的方法将代表一种创新技术，该技术可能适用于提高蛋白质、磷脂、寡糖、病毒样颗粒、成像剂和其他小分子药物的体内半衰期。

DESCRIPTION (provided by applicant): Harnessing transthyretin to extend the in vivo half-life of therapeutic peptides PROJECT SUMMARY: The tremendous therapeutic potential of peptides has not been fulfilled and potential peptide therapies that have failed far outnumber the successes so far. A major challenge impeding the more widespread use of peptides as therapeutics is their poor pharmacokinetic (PK) profile, due to short in vivo half-life resulting from inactivation by serum proteases and rapid elimination by kidney. Therefore, extending the in vivo half-life of peptides is clearly desirable in order for their therapeutic potential to be realized, without the need for high doses and/or frequent administration. Covalent conjugation of peptides to macromolecules (e.g. polyethylene glycol or serum proteins) has been the mainstay approach for enhancing the in vivo half-life of peptides. However, the steric hindrance of these large macromolecules often harms the binding of the peptide to its extra-cellular receptor, which compromises the pharmacodynamic (PD) properties of the peptide. The overall goal of this proposal is to develop a fundamentally new approach for enhancing the in vivo half-life of peptides without affecting its biological activity. The main hypothesis of this proposal isto test whether conjugation of peptides to a selective transthyretin (TTR, a serum protein) ligand would allow these peptide conjugates to reversibly bind to TTR in serum. This will result in enhancing the peptide's in vivo half-life, but most importantly, will maintain the binding affinityof the peptide to its receptor. A major emphasis will be on our recent discovery of the most potent and selective reversible TTR ligand to date, AG10. We have already made significant progress by synthesizing the first AG10-peptide conjugate (Conj1; with GnRH as a model peptide) and our in vitro preliminary results for Conj1 are consistent with this hypothesis. In this proposal, w will evaluate Conj1 in vivo and investigate the optimal linker system and affinity to TTR that will provide best balance between PK and PD of these AG10-GnRH conjugates. Three specific aims will be aggressively pursued: aim 1, design and synthesize a series of AG10-GnRH conjugates and determine the optimal linkers length and conjugation sites (on AG10 and GnRH) that will preserve the maximum binding affinity and serum selectivity of conjugates to both TTR and GnRH receptor; aim 2, evaluate the in vitro half-life of conjugates in serum and determine the optimal linker system that would allow for best balance between protection of GnRH against serum proteases and binding affinity to TTR and GnRH receptor; and aim 3, evaluate the pharmacokinetic properties of conjugates to determine if selective binding to TTR will increase the in vivo half-life, while maintaining the efficacy of conjugates in rats. If we successfully accomplished the goal of this proposal, our approach would represent an innovative technology that could potentially be applicable to enhancing the in vivo half-life of proteins, oligonucleotids, oligosaccharides, virus like particle, imaging agents, and other small molecule drugs.