Effects of Rho-Associated Kinase (Rock) Inhibitors (Alternative to Y-27632) on Primary Human Corneal Endothelial Cells

Abstract

(1) Rho-associated coiled-coil protein kinase (ROCK) signaling cascade impacts a wide array of cellular events. For cellular therapeutics, scalable expansion of primary human corneal endothelial cells (CECs) is crucial, and the inhibition of ROCK signaling using a well characterized ROCK inhibitor (ROCKi) Y-27632 had been shown to enhance overall endothelial cell yield. (2) In this study, we compared several classes of ROCK inhibitors to both ROCK-I and ROCK-II, using in silico binding simulation. We then evaluated nine ROCK inhibitors for their effects on primary CECs, before narrowing it down to the two most efficacious compounds—AR-13324 (Netarsudil) and its active metabolite, AR-13503—and assessed their impact on cellular proliferation in vitro. Finally, we evaluated the use of AR-13324 on the regenerative capacity of donor cornea with an ex vivo corneal wound closure model. Donor-matched control groups supplemented with Y-27632 were used for comparative analyses. (3) Our in silico simulation revealed that most of the compounds had stronger binding strength than Y-27632. Most of the nine ROCK inhibitors assessed worked within the concentrations of between 100 nM to 30 µM, with comparable adherence to that of Y-27632. Of note, both AR-13324 and AR-13503 showed better cellular adherence when compared to Y-27632. Similarly, the proliferation rates of CECs exposed to AR-13324 were comparable to those of Y-27632. Interestingly, CECs expanded in a medium supplemented with AR-13503 were significantly more proliferative in (i) untreated vs. AR-13503 (1 μM; * p < 0.05); (ii) untreated vs. AR-13503 (10 μM; *** p < 0.001); (iii) Y-27632 vs. AR-13503 (10 μM; ** p < 0.005); (iv) AR-13324 (1 μM) vs. AR-13503 (10 μM; ** p < 0.005); and (v) AR-13324 (0.1 μM) vs. AR-13503 (10 μM; * p < 0.05). Lastly, an ex vivo corneal wound healing study showed a comparable wound healing rate for the final healed area in corneas exposed to Y-27632 or AR-13324. (4) In conclusion, we were able to demonstrate that various classes of ROCKi compounds other than Y-27632 were able to exert positive effects on primary CECs, and systematic donor-match controlled comparisons revealed that the FDA-approved ROCK inhibitor, AR-13324, is a potential candidate for cellular therapeutics or as an adjunct drug in regenerative treatment for corneal endothelial diseases in humans.

ROCK Inhibitors in Corneal Diseases and Glaucoma—A Comprehensive Review of These Emerging Drugs

“…In addition to opening new therapeutic options in glaucoma, ROCK inhibitors have shown promise in the treatment of corneal endothelial diseases by promoting corneal endothelial cell regeneration and both functional and morphological recovery [4]. Laboratory studies have suggested that ROCK inhibitors improve cellular attachment and proliferation of cultured endothelial cells in vitro and improve wound healing on ex vivo corneas [5]. They facilitate cell cycle progression from the G1 to S phase and prevent actinomyosin contraction by inhibiting the Rho kinase signaling cascade [6].…”

“…Fasudil was originally approved in Japan in 1995 for the treatment of cerebral vasospasms caused by subarachnoid hemorrhage [8], and it is used in ophthalmology for the treatment of diabetic macular edema (DME) [9]. Finally, two ROCK inhibitors have not been approved yet: SNJ-1656, currently in phase II trial for controlling intraocular pressure (IOP) [10], and Y-27632, still in a pre-clinical phase for use in corneal endothelial diseases [5].…”