Click Chemistry for Immobilized Bone Morphogenetic Protein

描述(申请人提供)：生长因子是强有力的信号分子，启动分化、增殖和生存所必需的细胞程序。直到最近，像生长因子这样的大分子才被用于提高医学植入疗法的有效性和特异性。特别是，需要天然组织与植入材料相互作用的程序可以从纳入生长因子中受益。技术和生物学之间的成功融合通常预示着随后的治疗结果。AffinEnergy Inc.开发了双功能多肽连接物，称为界面生物材料(IFBMs)，有助于在合成和生物之间的关键界面促进生物学。将一种旨在结合生长因子的多肽与另一种旨在结合医疗设备的多肽结合起来，提供了一种简单且靶向特异的治疗策略。此外，我们的模块化方法允许肽半部分的互换，以产生潜在的双功能IFBM的组合阵列。目前，我们通过化学方法将我们的多肽合成为一个连续序列。这种方法带来了巨大的挑战，因为当链长接近30个残基时，固相多肽合成的偶联效率急剧下降。我们提出了一项第一阶段的研究，探索使用[3，2]环加成或点击化学来制备IFBM。这种方法利用了我们的模块化方法，并允许通过偶联较短的多肽(&lt；25 MERS)获得更高的总产率。此外，在不受保护的氨基酸侧链存在的情况下进行选择性偶联，能够高通量筛选已完成的IFBM文库。我们已经产生了两种结合可吸收胶原海绵和骨形态发生蛋白(BMP)的IFBM；一种使用点击化学，另一种使用传统的固相合成。在我们的第一阶段研究中，我们发现用这两种合成技术制备的IFBMs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的生物兼容性、储存和灭菌，并在体内测试它们的效果。AffinEnergy的IFBM技术旨在通过三种可能的方式改进生长因子治疗：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学数量；2)捕获和浓缩细胞外环境中的内源分子，潜在地减少或消除对重组蛋白的需求。 骨移植目前采取三种形式之一，包括自体移植(通常取自患者的髂骨)、同种异体移植(身体骨)和合成材料(Helistat海绵、羟基磷灰石或磷酸三钙)。自体移植具有骨再生的所有内源性生物学能力，但代表着痛苦的额外外科步骤。同种异体骨和人造骨不需要这样的二次手术，但具有有限的或没有诱导成骨的能力。因此，我们的目标是通过在胶原基骨移植物中添加生长因子结合肽涂层来融合两种移植物的优点。我们设想这项技术可以减少昂贵的生长因子的用量，同时消除与骨采集相关的疼痛和潜在的并发症。这项技术可能最适用于椎间融合术和脊柱后外侧融合术，这些融合术的频率越来越高，并已知受益于骨诱导生长因子。由于每年进行的脊柱融合手术超过4.5万例，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.