Ripasudil hydrochloride dihydrate ROCK inhibitor

In monkey trabecular meshwork (TM) cells, Ripasudil induces retraction and rounding of cell bodies as well as disruption of actin bundles. In Schlemm's canal endothelial (SCE) cells, Ripasudil significantly decreases transendothelial electrical resistance (TEER), increases the transendothelial flux of FITC-dextran, and disrupts cellular localization of ZO-1 expression. [3]

Kinase Inhibition Assay

ROCK 1 (0.75 ng/μL) and ROCK 2 (0.5 ng/μL) are incubated with various concentrations of K-115, Y-27632, or HA-1077 at 25 °C for 90 min in 50 mmol/L Tris-HCl buffer (pH 7.5) containing 100 mmol/L KCl, 10 mmol/L MgCl₂, 0.1 mmol/L EGTA, 30 μmol/L Long S6 Kinase Substrate peptide, and 1 μmol/L ATP in a total volume of 40 μL. PKACα, PKC, and CaMKIIα are also incubated with various concentrations of K-115, Y-27632, or HA-1077. PKACα (0.0625 ng/μL) is incubated at 25 °C for 30 min in 40 mmol/L Tris-HCl buffer (pH 7.5) containing 20 mmol/L MgCl₂, 1 mg/mL BSA, 5 μmol/L Kemptide peptide substrate, and 1 μmol/L ATP in a total volume of 40 μL. PKC (0.025 ng/μL) is incubated at 25 °C for 80 min in 20 mmol/L Tris-HCl buffer (pH 7.5) containing 20 mmol/L MgCl₂, 0.4 mmol/L CaCl₂, 0.1 mg/mL BSA, 0.25 mmol/L EGTA, 25 ng/mL phosphatidylserine, 2.5 ng/mL diacylglycerol, 0.0075% Triton-X-100, 25 μmol/L DTT, 10 μmol/L Neurogranin (28–43) peptide substrate, and 1 μmol/L ATP in a total volume of 40 μL. CaMKIIα (0.025 ng/μL) is incubated at 25 °C for 90 min in 50 mmol/L Tris-HCl buffer (pH 7.5) containing 10 mmol/L MgCl₂, 2 mmol/L CaCl₂, 0.04 mg/mL BSA, 16 μg/mL purified calmodulin from bovine testis, 500 μmol/L DTT, 50 μmol/L Autocamitide 2, and 1 μmol/L ATP in a total volume of 40 μL. After incubation, 40 μL of Kinase-Glo Luminescent Kinase Assay solution is added, and allowed to remain at 25 °C for 10 min, and Relative Light Units (RLU) are measured using a luminometer. The RLU without test compound is set as 100% (Control value), and that without enzyme and compound is set as 0% (Normal value). The reaction rate (% of control) is then calculated from the RLU with addition of each concentration of test compounds, and the 50% inhibitory concentrations (IC50) are determined by logistic regression analysis using SAS.

In albino rabbits and monkeys, topical instillation of Ripasudil significantly reduces intraocular pressure (IOP) with maximum IOP reduction of 8.55 mmHg and 4.36 mmHg at 0.5 % and 0.4%, respectively. [1]

Ripasudil (1 mg/kg daily, p.o.) exerts a neuroprotective effect on retinal ganglion cell (RGC) after optic nerve crush (NC) by suppressing oxidative stress through pathways involving the Nox family in a mouse model. [2]