Click Chemistry for Immobilized Bone Morphogenetic Protein

描述（由申请人提供）：生长因子是有效的信号分子，启动分化、增殖和存活的基本细胞程序。直到最近，大分子如生长因子才被用于增强医学植入疗法的功效和特异性。特别地，需要天然组织与植入材料相互作用的程序受益于包含生长因子。在技术和生物学之间的界面上的成功合并通常可以预测随后的治疗结果。Affinergy Inc.，开发了双功能肽接头，称为界面生物材料（IFBM），有助于促进合成和生物之间关键界面的生物学。将一种旨在结合生长因子的肽连接到另一种旨在结合医疗设备的肽，提供了一种简单且靶向特异性的治疗策略。此外，我们的模块化方法允许肽半的可重复性，以产生潜在的双功能IFBM的组合阵列。目前，我们化学合成我们的肽作为一个连续的序列。这种方法提出了重大的挑战，因为固相肽合成的偶联效率随着链长接近30个残基而显著降低。我们提出了第一阶段的研究，探索使用[3 + 2]环加成或点击化学制备IFBM。该方法利用了我们的模块化方法，并通过偶联较短的肽（<25聚体）实现了更高的总产率。此外，在未保护的氨基酸侧链存在下的选择性偶联使得能够实现完整IFBM文库的高通量筛选能力。我们已经产生了两个IFBM结合可吸收的胶原海绵和骨形态发生蛋白（BMP），一个使用点击化学和一个使用传统的固相合成。在我们的I期研究中，我们发现用两种合成技术制备的IFBM的生物稳定性和体外活性几乎相同。这些目标的成功实现鼓励我们将IFBM技术扩展到其他生长因子和骨移植材料。我们在第二阶段的目标是优化IFBM制造的点击化学，检查原型IFBM的生物相容性，储存和灭菌，并测试其体内功效。Affinergy的IFBM技术旨在以三种潜在的方式改善生长因子治疗：1）促进BMP从现有载体中的持续释放; 2）减少提供所需治疗效果所需的重组生长因子的超生理量; 2）从细胞外环境捕获和浓缩内源性分子，潜在地减少或消除对重组蛋白的需求。项目叙述 骨移植物目前采取三种形式之一，包括自体移植物（通常从患者的髂嵴获取）、同种异体移植物（尸体骨）和合成材料（helistat海绵、羟基磷灰石或磷酸三钙）。自体移植物具有骨再生的所有内源性生物学能力，但代表了痛苦的额外手术步骤。同种异体移植物和合成物不需要这种二次手术，但具有有限的骨诱导能力或没有骨诱导能力。因此，我们的目标是通过在胶原基骨移植物中加入生长因子结合肽涂层，融合两种移植物的优点。我们设想这项技术作为一种减少昂贵的生长因子剂量的手段，同时消除与骨采集相关的疼痛和潜在并发症。该技术可能最适用于椎间融合和后外侧脊柱融合，手术频率越来越高，并且已知受益于骨诱导生长因子。每年进行超过45，000例脊柱融合手术，对改进技术、护理和成本的需求不太可能很快得到缓解。

DESCRIPTION (provided by applicant): Growth factors are potent signaling molecules, initiating essential cellular programs for differentiation, proliferation and survival. Only recently have large molecules like growth factors, been employed to enhance the efficacy and specificity of medical implant therapies. In particular, procedures that require native tissues to interact with an implanted material stand to benefit from inclusion of growth factors. A successful merger at the interface between technology and biology is generally predictive of subsequent therapeutic outcome. Affinergy Inc., has developed bifunctional peptide linkers, called interfacial biomaterials (IFBMs) that help promote biology at the critical interface between a synthetic and a biologic. Attaching one peptide designed to bind a growth factor, to another peptide designed to bind a medical device, offers a simple and target-specific therapeutic strategy. In addition, our modular approach allows interchangeability of peptide halves , to produce a combinatorial array of potential bifunctional IFBMs. Currently we chemically synthesize our peptides as one continuous sequence. This approach presents significant challenges, as coupling efficiency of solid-phase peptide synthesis decreases dramatically as chain length approaches 30 residues. We proposed a Phase I study, exploring the use of [3 + 2] cycloaddition or click chemistry to prepare IFBMs. This method takes advantage of our modular approach and allows for higher total yields through the coupling of shorter peptides (<25 mers). Additionally, selective coupling in the presence of unprotected amino acid side chains enables high-throughput screening capabilities of completed IFBM libraries. We have generated two IFBMs which bind a resorbable collagen sponge and a bone morphogenetic protein (BMP); one using click chemistry and one using traditional solid-phase synthesis. In our Phase I studies, we found nearly identical biostability and in vitro activity of IFBMs made with both synthesis techniques. Successful completion of these aims has encouraged us to expand our IFBM technology to other growth factors and bone graft materials. Our goal in Phase II is to optimize click chemistry for IFBM manufacturing, examine the biocompatibility, storage, and sterilization of prototype IFBMs, and test their efficacy in vivo. Affinergy's IFBM technology is designed to improve growth factor therapy in three potential ways: 1) promoting the sustained release of BMP from existing carriers; 2) reducing the supraphysiological amounts of recombinant growth factor required to provide the desired therapeutic effect and 2) capturing and concentrating endogenous molecules from the extracellular milieu, potential decreasing or eliminating the requirement for recombinant protein.Project Narrative Bone grafts currently take one of three forms, which include autograft (typically harvested from the patient's iliac crest) allograft (cadaveric bone) and synthetics (helistat sponge, hydroxyapatite or tricalcium phosphate). Autografts harbor all the endogenous biological capacity for bone regeneration, but represent a painful additional surgical step. Allograft and synthetics require no such secondary procedure, but have limited or no osteoinductive capabilities. Our goal is to therefore merge the benefits of both grafts, by adding a growth factor binding peptide coating to collagen-based bone grafts. We envision this technology as a means of reducing the dosage of expensive growth factors, while removing the pain and potential complications associated with bone harvesting. This technology is likely most applicable to intervertebral and posterior lateral spinal fusion, procedures growing in frequency and known to benefit from osteoinductive growth factors. With over 45,000 spinal fusion surgeries performed each year, the demand for improved technique, care and cost is unlikely to be soon assuaged.