Suppression of Ocular Scarring - Controlled Delivery of an ALK-5 Inhibitor

描述(由申请人提供)： 青光眼在美国是导致失明的主要原因。青光眼的主要危险因素之一是眼压升高，青光眼治疗的目标是将眼压降低到安全水平。青光眼滤过手术(GFS)是青光眼患者经常进行的一种手术，据报道，它能产生最大和最持续的眼压下降。对于成功的眼压降低，保持滤过泡是关键，滤过泡由GFS后房水被排入的结膜下空间组成。眼部疤痕会留下疤痕并关闭滤过泡的结膜下空间，从而导致滤过泡和手术失败。作为结膜下抗瘢痕治疗，抗代谢药物通常是5-氟尿嘧啶(5-FU)和丝裂霉素C(MMC)，用于减少GFS术后瘢痕形成，提高GFS的远期疗效。虽然抗代谢药物的使用使许多患者受益，但它们会导致广泛的细胞死亡，并与严重的和潜在的致盲并发症有关，如迟发性感染、眼内炎。与其他眼科手术相比，GFS术后严重/致盲并发症的发生率极高，例如眼内炎，7.5%，例如白内障手术后0.028-0.15%(Wykoff等人，2010年；Freeman等人，2010年)。为了解决这些问题，还需要其他抗疤痕的方法。转化生长因子-2已成为创伤后纤维化反应的关键介质，并已被证明在青光眼滤过手术的并发症结膜下瘢痕形成中起重要作用。研究了抗转化生长因子-2或其受体的抗体和反义/siRNAs在玻璃体瘢痕形成中的作用。然而，在III期临床试验中，在预防青光眼初次小梁切除术失败方面，抗体和安慰剂之间没有区别。此外，反义/siRNAs的临床应用仍有疑问。相比之下，使用化合物是可行的，例如化学抑制剂，因为它们的结构稳定。受体和激酶通常也被认为是选择性地阻断各种生物系统中的信号通路的有效靶点。最近，激活素受体样激酶5(ALK-5，又称转化生长因子-2受体I)的抑制剂被认为是转化生长因子-2活性的有效抑制物。在我们之前的实验中，ALK-5抑制剂SB-505124在体外诱导转化生长因子-2下游蛋白显著减少。此外，在活体兔GFS模型中，SB-505124还显示延长了滤过泡的存活期。Sb-505124在玻璃纤维中的毒性明显低于丝裂原纤维。另一方面，尽管Sb-505124提高了滤泡的存活率，但在我们的数据中，添加Sb-505124的GFS的存活期仍然比丝裂原纤维短。据推测，在滤过泡中保持适当浓度的SB-505124是延长滤过泡存活期所必需的。因此，有必要对政府飞行服务队的受控药物运送进行调查。最近，利用凝胶作为储存库的受控给药系统已经被开发出来，它可以更容易地给药，并减少刺激/炎症反应。其中，热可逆凝胶具有生物相容性好、无刺激性、使用方便、释药时间长等显著优点。新型的聚乙二醇-聚己内酯-聚乳酸-聚己内酯-聚乙二醇五嵌段热可逆凝胶是我们实验室最近开发的一种新型热可逆凝胶，该凝胶已成功地应用于眼部缓释系统。五块热可逆凝胶似乎是包括GFS在内的眼部应用的最佳选择，因为与其他凝胶相比，它具有稳定和更长的药物释放性能。我们将在这个项目中使用这种新颖而有前途的药物释放系统--聚乙二醇-聚己内酯-聚乳酸-聚乙二醇热可逆凝胶，所获得的数据将对未来滤过泡的药物释放工作具有重要意义。我们研究的总体目标是在不使用任何抗代谢药物的情况下，在活体兔GFS模型中建立和研究抑制眼部瘢痕形成的作用。在兔GFS模型中，侵袭性瘢痕形成会导致早期滤过泡衰竭，通常在GFS后4至7天内，除非用抗代谢药物治疗，如MMC，使兔的滤过泡存活期延长至30天。为了实现我们的目标，我们的目标是使用激活素受体样激酶5(ALK-5)抑制剂SB-505124，它选择性地抑制转化生长因子-2(转化生长因子-2)途径。在目标1中，将在兔GFS模型中测定ALK-5抑制剂SB-505124在滤过泡中的体内治疗有效浓度。这一结果与建立和优化GFS受控给药系统的目标2直接相关。在目标2中，将建立和优化受控给药系统，该系统将保持滤过泡中SB-505124的治疗有效浓度，使滤过泡存活30天。ALK-5抑制剂SB-505124从热可逆凝胶中的控释将在体外和体内进行考察。含有该抑制剂的凝胶在活体兔GFS模型中的有效性和毒性将通过临床和桌面检查进行研究。体外和体内的结果将对青光眼治疗以及其他与瘢痕形成相关的眼部疾病的治疗有价值，如增殖性玻璃体视网膜病变。

DESCRIPTION (provided by applicant): Glaucoma is a leading cause of blindness in the United States. One of the major risk factors of glaucoma is an elevated intraocular pressure (IOP), and the goal of glaucoma therapy is to lower IOP to safe levels. Glaucoma filtration surgery (GFS) is a procedure frequently performed in glaucoma patients and has been reported to produce the largest and most sustained decrease in IOP. For successful IOP reduction, maintaining filtering blebs, which consist of the subconjunctival space where aqueous humor is drained into after GFS, is a key. Ocular scarring that scars and closes the subconjunctival space of filtering blebs leads to bleb and surgery failure. As subconjunctival anti-scarring treatments, antimetabolites, generally 5-fluorouracil (5-FU) and mitomycin-C (MMC), are commonly used to reduce post-operative scarring at the site of GFS and enhance the long-term success of GFS. While the use of antimetabolites benefits a number of patients, they do so by causing widespread cell death and are associated with severe and potentially blinding complications, such as late onset infection, endophthalmitis. The incidence of severe/blinding complications after GFS is extremely higher, e.g. endophthalmitis, 7.5% after GFS, than that after other ocular surgeries, e.g. 0.028-0.15% after cataract surgery (Wykoff et al, 2010; Freeman et al, 2010). In order to resolve these problems, other anti-scarring approaches are needed. TGF-2 has emerged as a key mediator of the fibrotic response to wounding and has been shown to be important in conditions in subconjunctival scarring, a complication of filtration surgery in glaucoma. Efficacy of antibody and antisense/siRNAs for TGF-2 or its receptors has been investigated in ocular scarring of GFS. However, in a phase III clinical trial, there was no difference between the antibody and a placebo in preventing the failure of primary trabeculectomy in human glaucoma eyes. In addition, clinical usage of antisense/siRNAs is still questionable. By contrast, it is practical to use chemical compounds, such as chemical inhibitors, because of their structural stability. Receptors and kinases are also generally believed to be effective targets for selectively blocking signaling pathways in a variety of biological systems. Recently, inhibitors of activin receptor-like kinase 5 (ALK-5, also called TGF-2 receptor type I) have been described to be effective suppressors of TGF-2 activity. In our previous experiments, the ALK-5 inhibitor SB-505124 induced a marked reduction in downstream proteins of TGF-2 in vitro. In addition, in an in vivo rabbit GFS model, it was shown that SB-505124 extended the period of filtering bleb survival. Toxicity of SB-505124 in GFS was shown to be substantially lower than that of MMC. On the other hand, while the bleb survival was improved by SB-505124, the survival period of GFS with SB-505124 was still shorter compared to that of MMC in our data. It is speculated that maintaining an appropriate concentration of SB-505124 in the filtering bleb is required for extending the period of bleb survival. Therefore, investigation of a controlled drug delivery for GFS needs to be conducted. Recently, controlled drug delivery systems using gel as a depot have been developed, which allow for easier administration and reduced irritation/inflammatory response. Among them, the thermoreversible gel has significant advantages, which include being biocompatible, non-irritable, easy for application and having a longer period of drug release. Novel PEG-PCL-PLA-PCL-PEG pentablock thermoreversible gel was recently developed in the laboratory of our Co-Investigator, and the gel has been successfully applied as an ocular sustained release system. The pentablock thermoreversible gel seems the best choice for ocular application including GFS because of its stable and longer drug release properties compared to other gels. We will use this novel and promising drug delivery system, PEG-PCL-PLA-PCL-PEG thermoreversible gel, in this project, and the data obtained in this proposed project will be significant for the future works of drug delivery in filtering blebs. The overall goal of our research is to establish and investigate the suppression of ocular scarring in an in vivo rabbit GFS model without using any antimetabolites. In a rabbit GFS model, aggressive scarring produces early bleb failure, generally within 4 to 7 days after GFS, unless the blebs are treated with antimetabolites, such as MMC, with which the period of bleb survival in a rabbit is extended to 30 days. To achieve our goal, we aim to use an activin receptor-like kinase 5 (ALK-5) inhibitor, SB-505124, which selectively inhibits the transforming growth factor-2 (TGF-2) pathway. In aim 1, the in vivo therapeutically effective concentration of ALK-5 inhibitor SB-505124 in filtering bleb will be determined in a rabbit GFS model. The result is directly relevant to aim 2 to establish and optimize controlled drug delivery system for GFS. In aim 2, the controlled drug delivery system, which maintains the therapeutically effective concentration of SB-505124 in the filtering bleb to allow bleb survival for 30 days, will be established and optimized. The controlled release of the ALK-5 inhibitor SB-505124 from thermoreversible gel will be examined both in vitro and in vivo. The efficacy and toxicity of the gel containing the inhibitor in the in vivo rabbit GFS model will be investigated by both clinical and bench-top examinations. The in vitro and in vivo results will be valuable for glaucoma therapy as well as in treatments for other ocular disorders related to scarring, such as proliferative vitreoretinopathy.