Smart Polymer Based MK2 Inhibitor to Suppress Vascular Graft Intimal Hyperplasia

描述(申请人提供)：冠状动脉搭桥术(CABG)与自体管道有效地缓解了缺血性心脏病，但长期移植物通畅性是有问题的。移植失败的主要原因是内膜增生(IH)，血管平滑肌细胞(VSMCs)迁移、增殖和沉积过多的细胞外基质(ECM)导致新生内膜形成的过程。IH可以闭塞管腔，并容易迅速发展为晚期移植物疾病，从而抵消了手术的好处。我们已经确定MAPKAP激酶II(MK2)是预防血管移植物IH的潜在药物干预靶点。MK2通过p38丝裂原激活激酶(MAPK)通路被激活，该通路由移植物中的VSMCs在移植过程中经历的物理和生化应激触发。激活的MK2进而磷酸化热休克蛋白27(HSP27)，HSP27是IH病理性VSMC行为的已知下游介质。由于p38 MAPK的功能多样，p38抑制剂会导致非特异性的副作用。因此，MK2是抑制IH近端触发的一个合乎逻辑的靶点，该提议的中心假设是，有效的药物抑制MK2将防止IH。制药公司开发小分子MK2抑制剂的努力一直没有成功。多肽抑制剂有望成为一种特异性更强、毒性更低的替代品，但细胞内给药的障碍阻碍了它们的使用。为了验证我们的假设，我们提出了一种新型的、“智能”的聚合物载体，用于多肽MK2抑制剂(MK2i)的细胞内递送。初步数据显示，通过与细胞穿透肽(CPP-MK2i)融合而输送MK2i的人大隐静脉样本中IH减少。然而，我们的机制研究支持这一观点，即CPP介导的递送由于内化到细胞内小泡和隔离在细胞内小泡中而遭受细胞质生物活性的损害。这项建议的总体目标是克服这种细胞质传递障碍，并使用CPP内化的用于MK2i肽传递的“智能”聚合物(CISP-MK2i)来优化对MK2的抑制。被提议的“智能”聚合物载体将“感应”内体中的酸性环境，这将引发聚合物急剧转变为更疏水的膜破坏状态，从而将CISP-MK2i释放到细胞质中。此外，CISP-MK2i被设计成在MK2i和“智能”聚合物主干之间具有可减少的连接，当到达细胞质时，将发生“细胞要求的”MK2i释放，以确保载体不会在空间上阻碍MK2i的活性。在CISP-MK2i合成的提案和一系列体外、体外和体内试验中概述了三个目标，以比较通过Cisp-MK2i和CPP-MK2i传递MK2i的效果。

DESCRIPTION (provided by applicant): Coronary artery bypass grafting (CABG) with autologous conduits effectively alleviates ischemic heart disease, but long-term graft patency is problematic. Graft failure is primarily attributable to intimal hyperplasia (IH), the process by which vascular smooth muscle cells (VSMCs) migrate, proliferate, and deposit excessive extracellular matrix (ECM) resulting in neointima formation. IH can occlude the lumen and is prone to rapid development into advanced graft disease, negating the surgical benefit. We have identified MAPKAP kinase II (MK2) as a potential target for pharmacological intervention for preventing vascular graft IH. MK2 is activated through a p38 mitogen activated kinase (MAPK) pathway that is triggered by the physical and biochemical stresses that VSMCs in the graft experience during transplant. Activated MK2, in turn, phosphorylates heat shock protein 27 (HSP27), a known downstream mediator of pathological VSMC behavior in IH. Because p38 MAPK functions are diverse, p38 inhibitors lead to nonspecific side effects. Thus, MK2 is a logical target for inhibiting a proximal trigger of IH, and the central hypothesis of this proposal is that efficient pharmacological inhibition of MK2 will prevent IH. Pharmaceutical companies' efforts to develop a small molecule MK2 inhibitor have been unsuccessful. Peptide inhibitors hold promise as an alternative with greater specificity and reduced toxicity, but barriers against intracellular delivery hinder their use. To test our hypothesis, a novel, "smart" polymer vehicle for intracellular delivery of a peptidic MK2 inhibitor (MK2i) is proposed. Preliminary data have shown a reduction in IH in human saphenous vein samples delivered MK2i via fusion to a cell penetrating peptide (CPP-MK2i). However, our mechanistic studies support the notion that CPP-mediated delivery suffers from compromised cytoplasmic bioactivity due to internalization into and sequestration within intracellular vesicles. The overall goal of this proposal is to overcome this cytoplasmic delivery barrier and to optimize MK2 inhibition using a CPP-internalized "smart" polymer (CISP) for MK2i peptide delivery (CISP-MK2i). The proposed "smart" polymer carrier will "sense" the acidic environment in the endosomes, which will trigger a sharp polymer transition into a more hydrophobic, membrane disruptive state that releases CISP-MK2i into the cytoplasm. Furthermore, CISP-MK2i has been designed with reducible attachments between MK2i and the "smart" polymer backbone, and upon reaching the cytoplasm, "cell- demanded" MK2i release will occur to ensure that the carrier does not sterically hinder MK2i activity. Three aims are outlined in the proposal for CISP-MK2i synthesis and a series of in vitro, ex vivo, and in vivo tests to compare the efficacy of MK2i delivery via CISP-MK2i versus CPP-MK2i.