K-115 enhances autophagic activity and attenuates TGF-β1-induced activation of human Tenon's fibroblasts / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To investigate the influence of K-115 on the proliferation and migration of human Tenon's fibroblasts(HTFs)and to access the possible mechanism. Furthermore, to provide new ideas for anti-scar treatment after glaucoma surgery.METHODS: The Tenon capsule tissues were collected from patients who underwent glaucoma surgery in Hebei General Hospital from September 2018 to September 2019. Primary culture of HTFs was performed by tissue block method. The transforming growth factor-β1(TGF-β1)was used to induce HTFs activation that can mimic glaucoma filtration surgery. The cells were treated with K-115 and divided into 4 groups: the control group was treated with dimethyl sulfoxide(DMSO); TGF-β1 group was treated with 10μg/L TGF-β1 for 24h; TGF-β1 +5 K-115 group was pretreated with 5μmol/L K-115 for 2h and then treated with 10μg/L TGF-β1 for 24h; TGF-β1+10 K-115 group was pretreated with 10μmol/L K-115 for 2h and then 10μg/L TGF-β1 was added for 24h. Cell proliferation was observed by cell proliferation experiment. The migration ability of cells was detected by scratch test. The formation of autophagosomes was observed by transmission electron microscopy. Apoptosis was visualized by Hoechst 33342/PI staining.RESULTS: Cell proliferation experiment revealed that K-115 could inhibit the proliferation of HTFs induced by TGF-β1. Scratch test suggested that K-115 could inhibit the migration of HTFs induced by TGF-β1. Transmission electron microscope results showed that K-115 could enhance autophagy of HTFs induced by TGF-β1. Hoechst 33342/PI staining suggested that K-115 did not induce apoptosis.CONCLUSIONS: K-115 may regulate the proliferation and migration of HTFs induced by TGF-β1 by increasing autophagy rather than inducing apoptosis.

The effects of ripasudil (K-115), a Rho kinase inhibitor, on activation of human conjunctival fibroblasts.

The most common cause of glaucoma surgery failure is scar formation induced by activation of wound-healing responses and resultant fibrosis at the surgical site. We investigated the effects of ripasudil, a Rho kinase inhibitor, on activation of human conjunctival fibroblasts (HConF). HConF were pretreated with different concentrations of ripasudil for 1 h before addition of transforming growth factor (TGF)-ß2, followed by incubation for 48 h. TGF-ß2-treated fibroblasts exhibited a significant increase in expression of α-smooth muscle actin (α-SMA), a marker of fibroblast-to-myofibroblast differentiation, and this increase was significantly suppressed, in a dose-dependent manner, by pretreatment with ripasudil. Ripasudil pretreatment also significantly attenuated TGF-ß2-induced fibronectin production and collagen gel contraction. TGF-ß2 increased both the number of viable cells and the number of cells in the G2/M phase of the cell cycle; these effects were attenuated by pretreatment with ripasudil. In addition, we explored the effects of ripasudil on stimulation of HConF by activated macrophages. Human monocytic cell line THP-1 cells were differentiated into M1 or M2 macrophage-like cells, and HConF were treated with conditioned media derived from these macrophages in the presence or absence of ripasudil. Conditioned medium from M2 macrophage-like cells induced a significant increase in α-SMA expression, viable cell numbers, and gel contraction, all of which were significantly suppressed by ripasudil. Thus, overall, ripasudil attenuated activation of human conjunctival fibroblasts. Ripasudil may be of therapeutic utility, preventing excessive scarring after glaucoma filtration surgery.

One-year clinical evaluation of 0.4% ripasudil (K-115) in patients with open-angle glaucoma and ocular hypertension.

PURPOSE: To investigate the intra-ocular pressure (IOP)-lowering effects and safety of 0.4% ripasudil (K-115), a Rho kinase inhibitor, twice daily for 52 weeks, in patients with open-angle glaucoma or ocular hypertension (OHT). METHODS: In this multicentre, prospective, open-label study, 388 patients with primary open-angle glaucoma, OHT or exfoliation glaucoma were enrolled and 354 of them were subdivided into four cohorts (monotherapy, 173; additive therapy to prostaglandin analogs, 62; ß-blockers, 60; or fixed combination drugs, 59). The IOP reduction at trough and peak from baseline and adverse events was investigated. RESULTS: Ripasudil showed IOP-lowering effects over 52 weeks in all the analyses of monotherapy, additive therapy and both subgroups (baseline IOP ≥21 mmHg and <21 mmHg) of monotherapy. The mean IOP reductions at trough and peak at week 52 were -2.6 and -3.7 mmHg for monotherapy, and -1.4 and -2.4, -2.2 and -3.0, and -1.7 and -1.7 mmHg, respectively, for additive therapy described above. The most frequently observed adverse events were conjunctival hyperaemia (n = 264, 74.6%), blepharitis (n = 73, 20.6%) and allergic conjunctivitis (n = 61, 17.2%). Most of the conjunctival hyperaemia findings were mild (97.0%), transient and resolved spontaneously (78.0%). Although 51 patients discontinued from the study due to blepharitis and/or allergic conjunctivitis (blepharitis, 28; allergic conjunctivitis, 17; both, 6), all the events resolved with or without treatment after the discontinuation of ripasudil administration. CONCLUSION: Fifty-two week administration of 0.4% ripasudil revealed IOP-lowering effects and an acceptable safety profile when administered as monotherapy or as additive therapy, in patients with open-angle glaucoma or OHT.

Effect of intravitreal Rho kinase inhibitor ripasudil (K-115) on feline retinal microcirculation.

Ripasudil (K-115) is a novel Rho kinase inhibitor with a potent intraocular pressure-lowering effect. However, it is unclear whether ripasudil affects the retinal blood flow (RBF). We investigated the effect of ripasudil on feline retinal microcirculation. Ripasudil (5 µM, 50 µM or 5 mM; n = 5 each concentration) or vehicle (PBS; n = 5) was injected intravitreally. The vessel diameter (D) and blood velocity (V) were measured by laser Doppler velocimetry simultaneously in the first-order retinal arterioles and the RBF was calculated. The measurements started 5 min before the injection and were performed every 10 min for 120 min. After the intravitreal injection, the retinal circulatory parameters did not change significantly in PBS or 5 µM of ripasudil. The blood V and RBF increased significantly compared to baseline, whereas the vessel D did not change significantly in 50 µM and 5 mM of ripasudil. The V in 50 µM, and the V and RBF in 5 mM of ripasudil significantly increased compared to those in PBS. Intravitreal administration of ripasudil increased the blood V and RBF in cats, suggesting that ripasudil has the potential to improve the retinal blood flow.

Intra-ocular pressure-lowering effects of a Rho kinase inhibitor, ripasudil (K-115), over 24 hours in primary open-angle glaucoma and ocular hypertension: a randomized, open-label, crossover study.

PURPOSE: To investigate the intra-ocular pressure (IOP)-lowering effects of a selective Rho kinase inhibitor, ripasudil (K-115), over 24 hr in patients with primary open-angle glaucoma (POAG) or ocular hypertension (OHT). METHODS: In this multicenter, prospective, randomized, open-label, 3-period, Latin-square crossover clinical study, 28 patients with POAG or OHT whose IOP level was 21 mmHg or higher were subdivided into three groups. Each patient was treated with placebo and ripasudil in concentrations of 0.2 and 0.4%, at 9:00 and 21:00 on day 1 through a total of 3 periods separated by washout periods. IOP was measured at 9:00, 10:00, 11:00, 13:00, 16:00, 19:00, 21:00, 22:00 and 23:00 on day 1, and 1:00, 4:00, 7:00 and 9:00 on day 2 in sitting position using Goldmann applanation tonometer. Main outcome measure was the IOP reduction of placebo and ripasudil from baseline. RESULTS: The mean IOP reduction was -5.2 mmHg for 0.2%, -6.4 mmHg for 0.4% and -2.0 mmHg for placebo at 2 hr after the first instillation. Also, the corresponding values were -6.8 mmHg for 0.2%, -7.3 mmHg for 0.4% and -4.1 mmHg for placebo at 2 hr after the second instillation. Statistically significant IOP reduction, compared with placebo, was found for both 0.2 and 0.4% from 1 through 7 hr after each instillation. In safety, conjunctival hyperaemia was observed in 22 patients (79%) for 0.2%, 27 patients (96%) for 0.4% and three patients (11%) for placebo. CONCLUSION: Ripasudil is a promising new topical medication to lower IOP for at least 7 hr after instillations in patients with POAG or OHT.

Effects of K-115, a rho-kinase inhibitor, on aqueous humor dynamics in rabbits.

PURPOSE: To evaluate the topical instillation of K-115, a selective Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor, on intraocular pressure (IOP), ocular distribution, and aqueous humor dynamics in experimental animals. METHODS: Kinase inhibition by K-115 was measured by biochemical assay. IOP was monitored using a pneumatonometer in albino rabbits and monkeys after topical instillation of K-115. The ocular distribution of [(14)C]K-115 was determined by whole-head autoradiography. The aqueous flow rate was determined by fluorophotometry. The total outflow facility and uveoscleral outflow were measured by two-level constant pressure perfusion and perfusion technique using fluorescein isothiocyanate-dextran, respectively. RESULTS: Biochemical assay showed that K-115 had selective and potent inhibitory effects on ROCKs. In rabbits, topical instillation of K-115 significantly reduced IOP in a dose-dependent manner. Maximum IOP reduction was observed 1 h after topical instillation, which was 8.55 ± 1.09 mmHg (mean ± SE) from the baseline IOP at 0.5%. In monkeys, maximum IOP reduction was observed 2 h after topical instillation, which was 4.36 ± 0.32 mmHg from the baseline IOP at 0.4%, and was significantly stronger than that of 0.005% latanoprost. Whole-head autoradiography showed that the radioactivity level was maximum at 15 min after instillation of [(14)C]K-115 in the ipsilateral eye. Single instillation of 0.4% K-115 showed no effect on aqueous flow rate or uveoscleral outflow, but significantly increased conventional outflow facility by 2.2-fold compared to vehicle-treated eyes in rabbits. CONCLUSIONS: These results indicated that K-115 ophthalmic solution, a selective and potent ROCK inhibitor, is a novel and potent antiglaucoma agent.