Crossover Randomized Study of Pharmacologic Effects of Ripasudil-Brimonidine Fixed-Dose Combination Versus Ripasudil or Brimonidine.

INTRODUCTION: Multidrug regimens for glaucoma treatment often result in adherence issues due to inconvenience; these issues may be improved with fixed-dose combination drugs. The ophthalmic solution of ripasudil-brimonidine fixed-dose combination (RBFC; K-232) is the first treatment combining a Rho kinase inhibitor and an α2-adrenoceptor agonist, and has demonstrated ability to lower intraocular pressure (IOP) and have various effects on conjunctival hyperemia and corneal endothelial cell morphology. This study evaluates the pharmacologic effects of RBFC treatment versus its separate components-ripasudil or brimonidine. METHODS: This single-center, prospective, randomized, open-label, blinded endpoint study with 3 × 3 crossover design randomly assigned healthy adult men to three groups (1:1:1) to undergo consecutive 8-day administration phases (with drug-free intervals of at least 5 days). Subjects received twice-daily instillation of RBFC → ripasudil → brimonidine (group A), ripasudil → brimonidine → RBFC (group B), or brimonidine → RBFC → ripasudil (group C). Endpoints included change in IOP, severity of conjunctival hyperemia, corneal endothelial cell morphology, pupil diameter, and pharmacokinetics. RESULTS: Eighteen subjects were assigned in total (six to each group). RBFC significantly reduced IOP from baseline at 1 h post-instillation on days 1 and 8 (12.7 vs. 9.1 and 9.0 mmHg, respectively; both P < 0.001), and provided significantly greater IOP reductions than ripasudil or brimonidine at several time points. The most common adverse drug reaction with all three treatments was mild conjunctival hyperemia, which transiently increased in severity with RBFC or ripasudil, peaking at 15 min post-instillation. In post hoc analyses, conjunctival hyperemia scores were lower with RBFC than with ripasudil at several time points. Transient morphologic changes in corneal endothelial cells occurred for up to several hours with RBFC or ripasudil, but not with brimonidine. Pupil diameter did not change with RBFC. CONCLUSION: RBFC significantly reduced IOP compared with each agent alone. A combination of each agent's pharmacologic profile was observed in that of RBFC. TRIAL REGISTRATION: Japan Registry of Clinical Trials; Registration No. jRCT2080225220.

Additive Intraocular Pressure-Lowering Effects of Ripasudil with Glaucoma Therapeutic Agents in Rabbits and Monkeys.

Ripasudil hydrochloride hydrate (K-115), a specific Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, is developed for the treatment of glaucoma and ocular hypertension. Topical administration of ripasudil decreases intraocular pressure (IOP) by increasing conventional outflow through the trabeculae to Schlemm's canal, which is different from existing agents that suppress aqueous humor production or promote uveoscleral outflow. In this study, we demonstrated that ripasudil significantly lowered IOP in combined regimens with other glaucoma therapeutic agents in rabbits and monkeys. Ripasudil showed additional effects on maximum IOP lowering or prolonged the duration of IOP-lowering effects with combined administration of timolol, nipradilol, brimonidine, brinzolamide, latanoprost, latanoprost/timolol fixed combination, and dorzolamide/timolol fixed combination. These results indicate that facilitation of conventional outflow by ripasudil provides additive IOP-lowering effect with other classes of antiglaucoma agents. Ripasudil is expected to have substantial utility in combined regimens with existing agents for glaucoma treatment.

Ocular Penetration and Pharmacokinetics of Ripasudil Following Topical Administration to Rabbits.

PURPOSE: We evaluated the ocular pharmacokinetics of ripasudil (K-115), a selective Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, following topical administration to rabbits. METHODS: We determined the ocular distribution of [(14)C]ripasudil by whole-head autoradiography and the radioactivity of each ocular tissue after single and multiple instillation of [(14)C]ripasudil to pigmented rabbits. We also measured the aqueous humor concentrations after concomitant instillation of ripasudil and a combination agent (0.005% latanoprost and 0.5% timolol) to pigmented rabbits as well as the tear fluid concentrations after instillation into rabbits, dogs, and monkeys using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Membrane permeability was evaluated using an in vitro parallel artificial membrane permeability assay system and Ussing chamber with rabbit cornea and conjunctiva. RESULTS: [(14)C]Ripasudil was rapidly absorbed into the cornea and distributed throughout the eye after topical instillation. The melanin-containing ocular tissues, such as the iris-ciliary body and retina-choroid, showed much higher concentrations of radioactivity than the other nonpigmented tissues. Concomitant instillation showed minor effects on the aqueous humor concentrations of each compound in rabbits. Membrane permeability of ripasudil was higher than other glaucoma drugs in vitro and ex vivo. The aqueous humor concentrations of ripasudil in rabbits were higher than those in dogs and monkeys in the early period after instillation and associated with tear turnover rate. CONCLUSIONS: These results indicate favorable intraocular penetration characteristics of ripasudil following topical administration.

Vascular Normalization by ROCK Inhibitor: Therapeutic Potential of Ripasudil (K-115) Eye Drop in Retinal Angiogenesis and Hypoxia.

PURPOSE: In this study, we investigated the therapeutic potential of a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor ripasudil (K-115) eye drop on retinal neovascularization and hypoxia. METHODS: In vitro, human retinal microvascular endothelial cells (HRMECs) were pretreated with ripasudil and then stimulated with VEGF. ROCK activity was evaluated by phosphorylation of myosin phosphatase target protein (MYPT)-1. Endothelial migration and cell viability were assessed by cell migration and MTT assay, respectively. The concentration of ripasudil in the retina was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In vivo, normal saline, 0.4%, or 0.8% ripasudil were administered three times a day to mice with oxygen-induced retinopathy (OIR). The areas of neovascularization and avascular retina were also quantified with retinal flat-mounts at postnatal day (P) 15, P17, or P21. The retinal hypoxic area was evaluated using hypoxia-sensitive drug pimonidazole by immunohistochemistry at P17. The vascular normalization was also evaluated by immunohistochemistry at P17. RESULTS: Ripasudil but not fasudil significantly reduced VEGF-induced MYPT-1 phosphorylation in HRMECs at 30 µmol/L. Ripasudil significantly inhibited VEGF-induced HRMECs migration and proliferation. The concentration of ripasudil in the retina was 3.8 to 10.4 µmol/L and 6.8 to 14.8 µmol/L after 0.4% and 0.8% ripasudil treatment, respectively. In the 0.4% and 0.8% ripasudil treated OIR mice, the areas of neovascularization as well as avascular area in the retina was significantly reduced compared with those of saline-treated mice at P17 and P21. Pimonidazole staining revealed that treatment with 0.4% and 0.8% ripasudil significantly inhibited the increase in the hypoxic area compared with saline. 0.8% ripasudil could cause intraretinal vascular sprouting and increase retinal vascular perfusion. CONCLUSIONS: Novel ROCK inhibitor ripasudil eye drop has therapeutic potential in the treatment of retinal hypoxic neovascular diseases via antiangiogenic effects as well as vascular normalization.

Effects of K-115 (Ripasudil), a novel ROCK inhibitor, on trabecular meshwork and Schlemm's canal endothelial cells.

Ripasudil hydrochloride hydrate (K-115), a specific Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, was the first ophthalmic solution developed for the treatment of glaucoma and ocular hypertension in Japan. Topical administration of K-115 decreased intraocular pressure (IOP) and increased outflow facility in rabbits. This study evaluated the effect of K-115 on monkey trabecular meshwork (TM) cells and Schlemm's canal endothelial (SCE) cells. K-115 induced retraction and rounding of cell bodies as well as disruption of actin bundles in TM cells. In SCE-cell monolayer permeability studies, K-115 significantly decreased transendothelial electrical resistance (TEER) and increased the transendothelial flux of FITC-dextran. Further, K-115 disrupted cellular localization of ZO-1 expression in SCE-cell monolayers. These results indicate that K-115 decreases IOP by increasing outflow facility in association with the modulation of TM cell behavior and SCE cell permeability in association with disruption of tight junction.

Species differences in metabolism of ripasudil (K-115) are attributed to aldehyde oxidase.

1. We examined the metabolism of ripasudil (K-115), a selective and potent Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor, by in vitro and in vivo studies. 2. First, we identified metabolites and metabolic enzymes involved in ripasudil metabolism. Species differences were observed in metabolic clearance and profiles of metabolites in liver S9 fraction and hepatocytes. In addition, ripasudil was metabolised in humans and monkey S9 without nicotinamide adenine dinucleotide phosphate (NADPH). Studies using specific inhibitors and human recombinant enzyme systems showed that M1 (main metabolite in humans) formation is mediated by aldehyde oxidase (AO). 3. Therefore, we developed ripasudil as an ophthalmic agent. First, we compared the pharmacokinetic profiles of ripasudil in humans and rats. The results indicated rapid disappearance of ripasudil from the circulation after instillation in humans and its level remained relatively high only in M1. In contrast, we found six metabolites from M1 to M6 in plasma after oral administration to rats. 4. Analysis of enzyme kinetics using S9 showed that the formation of M1 is the major metabolic pathway of ripasudil in humans even though CYP3A4/3A5 and CYP2C8/3A4/3A5 were associated with the formation of M2 and M4, respectively. In conclusion, AO causes differences in ripasudil metabolism between species.

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.

Function and regulation of taurine transport at the inner blood-retinal barrier.

In the retina, taurine exerts a number of neuroprotective functions as an osmolyte and antioxidant. The purpose of the present study was to elucidate the taurine transport system(s) at the inner blood-retinal barrier (BRB). [(3)H]Taurine transport at the inner BRB was characterized using in vivo integration plot analysis and a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). The expression of the taurine transporter (TauT) was demonstrated by RT-PCR and immunoblot analyses. The apparent influx permeability clearance of [(3)H]taurine in the rat retina was found to be 259 muL/(ming retina), supporting carrier-mediated influx transport of taurine at the BRB. [(3)H]Taurine uptake by TR-iBRB2 cells was Na(+)-, Cl(-)- and concentration-dependent with a K(m) of 22.2 muM and inhibited by TauT inhibitors, such as beta-alanine and hypotaurine. RT-PCR and immunoblot analyses demonstrated that TauT is expressed in TR-iBRB2 and primary cultured human retinal endothelial cells. The uptake of [(3)H]taurine and the expression of TauT mRNA in TR-iBRB2 cells increased under hypertonic conditions but decreased following pretreatment with excess taurine. In conclusion, TauT most likely mediates taurine transport and regulate taurine transport at the inner BRB.