Ripasudil hydrochloride dihydrate

Catalog No.S7995 Batch:S799501

Technical Data

Formula

C₁₅H₁₈FN₃O₂S.Cl H.2 H₂ O

Molecular Weight

395.88

CAS No.

887375-67-9

Solubility (25°C)*

In vitro

Water

79 mg/mL (199.55 mM)

DMSO

26 mg/mL (65.67 mM)

Ethanol

5 mg/mL (12.63 mM)

In vivo (Add solvents to the product individually and in order)

	5%DMSO 1 40%PEG300 2 5%Tween80 3 50%ddH2O	1.32mg/ml
	5% DMSO 1 95% Corn oil	0.65mg/ml

* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
* Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

Preparing Stock Solutions

Biological Activity

Description

Ripasudil hydrochloride dihydrate is a potent ROCK inhibitor with IC50 of 51 nM and 19 nM for ROCK1 and ROCK2, respectively, used for the treatment of glaucoma and ocular hypertension.

Targets

ROCK2 ^[1] (Cell-free assay)	ROCK1 ^[1] (Cell-free assay)
19 nM	51 nM

In vitro

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]

In vivo

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]

Protocol (from reference)

Kinase Assay:^{^[1]}	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.
Animal Study:^{^[2]}	Animal Models Male C57BL/6 mice Dosages 1 mg/kg Administration p.o.

Kinase Assay:^{^[1]}

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.

Animal Study:^{^[2]}

Animal Models
Male C57BL/6 mice
Dosages
1 mg/kg
Administration
p.o.

References

Selleck's Ripasudil hydrochloride dihydrate has been cited by 9 publications

Inhibitors of Rho kinases (ROCK) induce multiple mitotic defects and synthetic lethality in BRCA2-deficient cells [ Elife, 2023, 12e80254]	PubMed: 37073955
Characterization of mitotic phenotypes associated with a MYC synthetic lethal compound [ bioRxiv, 2023, 10.1101/2023.04.03.535438]	PubMed: None
Inhibition of Rho-associated protein kinase activity enhances oxidative phosphorylation to support corneal endothelial cell migration [ FASEB J, 2022, 36(7):e22397]	PubMed: 35661268
Lowering the Intraocular Pressure in Rats and Rabbits by Cordyceps cicadae Extract and Its Active Compounds [ Molecules, 2022, 27(3)707]	PubMed: 35163975
In situ immunogenic clearance induced by a combination of photodynamic therapy and rho-kinase inhibition sensitizes immune checkpoint blockade response to elicit systemic antitumor immunity against intraocular melanoma and its metastasis [ J Immunother Cancer, 2021, 9(1)e001481]	PubMed: 33479026
The Global Phosphorylation Landscape of SARS-CoV-2 Infection [ Cell, 2020, 182(3):685-712.e19]	PubMed: 32645325
The Global Phosphorylation Landscape of SARS-CoV-2 Infection [ Cell, 2020, 182(3):685-712.e19]	PubMed: 32645325
Drug and siRNA screens identify ROCK2 as a therapeutic target for ciliopathies [ bioRxiv, 2020, 10.1101/2020.11.26.393801]	PubMed: N/A
Deep Learning Enhancing Kinome-Wide Polypharmacology Profiling: Model Construction and Experiment Validation. [ J Med Chem, 2019, 10.1021/acs.jmedchem.9b00855]	PubMed: 31364850

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