Smart Polymer Based MK2 Inhibitor to Suppress Vascular Graft Intimal Hyperplasia

描述（申请人提供）：自体导管冠状动脉旁路移植术（CABG）可有效缓解缺血性心脏病，但移植物长期通畅存在问题。移植失败主要是由于内膜增生（IH），即血管平滑肌细胞（VSMCs）迁移、增殖和沉积过多的细胞外基质（ECM）导致新内膜形成的过程。IH可以阻塞管腔，容易迅速发展为晚期移植物疾病，使手术效果失效。我们已经确定MAPKAP激酶II （MK2）作为预防血管移植IH的药物干预的潜在靶点。MK2通过p38丝裂原活化激酶（MAPK）途径被激活，该途径是由移植物中的VSMCs在移植过程中经历的物理和生化应激触发的。活化的MK2反过来磷酸化热休克蛋白27 (HSP27)， HSP27是IH病理VSMC行为的已知下游介质。由于p38 MAPK的功能多种多样，p38抑制剂会导致非特异性副作用。因此，MK2是抑制IH近端触发因子的逻辑靶点，该建议的中心假设是MK2的有效药理抑制将预防IH。制药公司开发小分子MK2抑制剂的努力一直没有成功。肽抑制剂具有更大的特异性和更低的毒性，但对细胞内递送的障碍阻碍了它们的使用。为了验证我们的假设，提出了一种新的“智能”聚合物载体，用于细胞内递送肽型MK2抑制剂（MK2i）。初步数据显示，通过融合细胞穿透肽（pcp -MK2i）递送MK2i的人隐静脉样品中的IH降低。然而，我们的机制研究支持这样一种观点，即cpp介导的递送由于内在化和隔离在细胞内囊泡内而受到细胞质生物活性的损害。本提案的总体目标是克服这种细胞质递送障碍，并使用cpp内化的“智能”聚合物（CISP）优化MK2抑制，用于MK2i肽递送（CISP-MK2i）。提出的“智能”聚合物载体将“感知”内体中的酸性环境，这将触发聚合物急剧转变为更疏水的膜破坏状态，释放CISP-MK2i到细胞质中。此外，CISP-MK2i被设计为在MK2i和“智能”聚合物主链之间具有可还原的附着，并且在到达细胞质时，“细胞要求”的MK2i释放将发生，以确保载体不会立体阻碍MK2i的活性。该提案概述了CISP-MK2i合成和一系列体外、离体和体内试验的三个目标，以比较CISP-MK2i与CPP-MK2i递送MK2i的功效。

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.