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内压（IOP）升高，青光眼治疗的目标是将IOP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手术，据报道可使IOP最大程度和最持久地降低。对于成功降低IOP，维持滤过泡是关键，滤过泡由GFS后房水排入的结膜下空间组成。眼瘢痕形成使滤过泡的结膜下空间瘢痕化和闭合，导致滤过泡和手术失败。作为结膜下抗瘢痕形成治疗，抗代谢物，通常为5-氟尿嘧啶（5-FU）和丝裂霉素-C（MMC），通常用于减少GFS部位的术后瘢痕形成并提高GFS的长期成功率。虽然抗代谢药的使用使许多患者受益，但它们通过引起广泛的细胞死亡来实现，并且与严重和潜在的致盲并发症相关，例如迟发性感染、眼内炎。GFS术后严重/致盲并发症的发生率极高，例如，GFS术后眼内炎的发生率为7.5%，而其他眼科手术的发生率为0.028-0.15%，例如，白内障手术后的发生率为0.028-0.15%（Wykoff et al，2010; Freeman et al，2010）。为了解决这些问题，需要其他抗瘢痕形成方法。TGF-2已经成为对创伤的纤维化反应的关键介质，并且已经显示在结膜下瘢痕形成（青光眼滤过手术的并发症）的状况中是重要的。已经研究了TGF-2或其受体的抗体和反义/siRNA在GFS的眼部瘢痕形成中的功效。然而，在III期临床试验中，抗体和安慰剂在预防人类青光眼眼原发性小梁切除术失败方面没有差异。此外，反义/siRNA的临床使用仍然是值得怀疑的。相比之下，由于其结构稳定性，使用化学化合物如化学抑制剂是实用的。受体和激酶通常也被认为是在多种生物系统中选择性阻断信号传导途径的有效靶标。最近，激活素受体样激酶5（ALK-5，也称为TGF-2受体I型）的抑制剂已被描述为TGF-2活性的有效抑制剂。在我们之前的实验中，ALK-5抑制剂SB-505124在体外诱导TGF-2下游蛋白的显著减少。此外，在体内兔GFS模型中，显示SB-505124延长了滤过泡存活期。SB-505124在GFS中的毒性被证明显著低于MMC。另一方面，虽然SB-505124改善了滤过泡存活率，但在我们的数据中，与MMC相比，SB-505124的GFS的存活期仍然较短。推测在滤过泡中维持适当浓度的SB-505124是延长滤过泡存活期所必需的。因此，需要对GFS的受控药物递送进行调查。最近，已经开发了使用凝胶作为贮库的受控药物递送系统，其允许更容易的施用和减少的刺激/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。我们的合作研究者的实验室最近开发了新型PEG-PCL-PLA-PCL-PEG五嵌段热可逆凝胶，并且该凝胶已成功地用作眼用缓释系统。与其他凝胶相比，五嵌段热可逆凝胶似乎是眼部应用（包括GFS）的最佳选择，因为其具有稳定和更长的药物释放特性。我们将在本项目中使用这种新型的和有前途的药物递送系统，PEG-PCL-PLA-PCL-PEG热可逆凝胶，并且在这个拟议项目中获得的数据将是有意义的，为未来的药物递送滤过泡的工作。我们研究的总体目标是建立和研究在不使用任何抗代谢物的情况下在体内兔GFS模型中对眼部瘢痕形成的抑制。在兔GFS模型中，侵袭性瘢痕形成产生早期滤过泡衰竭，通常在GFS后4至7天内，除非用抗代谢物如MMC治疗滤过泡，否则滤过泡在兔中的存活期延长至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（TGF-2）途径。在目的1中，将在兔GFS模型中确定ALK-5抑制剂SB-505124在滤过泡中的体内治疗有效浓度。研究结果直接关系到目标2建立和优化GFS的药物控释系统。在目标2中，将建立和优化受控药物递送系统，其在滤过泡中维持SB-505124的治疗有效浓度以允许滤过泡存活30天。将在体外和体内检查ALK-5抑制剂SB-505124从热可逆凝胶中的控释。将通过临床和实验室检查研究含有抑制剂的凝胶在体内兔GFS模型中的疗效和毒性。在体外和体内的结果将是有价值的青光眼治疗，以及在治疗其他眼部疾病有关的疤痕，如增生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.