Rho-associated protein kinase (ROCK) inhibitors allow for causative glaucoma therapy. Unfortunately, topically applied ROCK inhibitors suffer from high incidence of hyperemia and low intraocular bioavailability. Therefore, we propose the use of poly (lactide-co-glycolide) (PLGA) microspheres as a depot formulation for intravitreal injection to supply outflow tissues with the ROCK inhibitor fasudil over a prolonged time. Fasudil-loaded microspheres were prepared by double emulsion solvent evaporation technique. The chemical integrity of released fasudil was confirmed by mass spectrometry. The biological activity was measured in cell-based assays using trabecular meshwork cells (TM cells), Schlemm’s canal cells (SC cells), fibroblasts and adult retinal pigment epithelium cells (ARPE-19). Cellular response to fasudil after its diffusion through vitreous humor was investigated by electric cell-substrate impedance sensing. Microspheres ranged in size from 3 to 67 µm. The release of fasudil from microspheres was controllable and sustained for up to 45 days. Released fasudil reduced actin stress fibers in TM cells, SC cells and fibroblasts. Decreased collagen gel contraction provoked by fasudil was detected in TM cells (~2.4-fold), SC cells (~1.4-fold) and fibroblasts (~1.3-fold). In addition, fasudil readily diffused through vitreous humor reaching its target compartment and eliciting effects on TM cells. No negative effects on ARPE-19 cells were observed. Since fasudil readily diffuses through the vitreous humor, we suggest that an intravitreal drug depot of ROCK inhibitors could significantly improve current glaucoma therapy particularly for patients with comorbid retinal diseases.

中文翻译：

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法舒地尔负载 PLGA 微球作为青光眼治疗的潜在玻璃体内贮库制剂。

Rho 相关蛋白激酶 (ROCK) 抑制剂可用于治疗青光眼。不幸的是，局部应用的 ROCK 抑制剂存在充血发生率高和眼内生物利用度低的问题。因此，我们建议使用聚（丙交酯-共-乙交酯）（PLGA）微球作为玻璃体内注射的长效制剂，以在较长时间内为流出组织提供 ROCK 抑制剂法舒地尔。通过双乳液溶剂蒸发技术制备负载法舒地尔的微球。释放的法舒地尔的化学完整性通过质谱确认。使用小梁网细胞 (TM 细胞)、施累姆氏管细胞 (SC 细胞)、成纤维细胞和成人视网膜色素上皮细胞 (ARPE-19) 在基于细胞的测定中测量生物活性。通过细胞-基质阻抗传感研究了通过玻璃体液扩散后对法舒地尔的细胞反应。微球的尺寸范围为 3 至 67 µm。法舒地尔从微球中的释放是可控的，可持续长达 45 天。释放的 fasudil 减少了 TM 细胞、SC 细胞和成纤维细胞中的肌动蛋白应力纤维。在 TM 细胞（~2.4 倍）、SC 细胞（~1.4 倍）和成纤维细胞（~1.3 倍）中检测到由 fasudil 引起的胶原凝胶收缩减少。此外，法舒地尔很容易通过玻璃体液扩散到达其目标隔室并引发对 TM 细胞的影响。没有观察到对 ARPE-19 细胞的负面影响。由于法舒地尔很容易通过玻璃体液扩散，