Ripasudil hydrochloride dihydrate, Inhibitor

Ripasudil hydrochloride dihydrate

Cat. Num. AAA5764566

• Purity: 100.00%
• Synonyms: Ripasudil hydrochloride dihydrate; K-115; inhibitor

• Purity/Purification: 100.00%
• Form/Format: Solid. Powder

• Solubility: Water: 45mg/mL (113.67mM); (<1mg/ml refers to the product slightly soluble or insoluble)
• Formula: C15H23ClFN3O4S
• SMILES: O.O.Cl.C[C@H]1CNCCCN1S(=O)(=O)c1cccc2cncc(F)c12
• CAS Number: 887375-67-9
• Cell Research: Trabecular meshwork (TM) cells are plated on 6 well plates at a density of 1x10^4 cells per well in DMEM containing 10% FBS. Following overnight culture, when cells have reached semiconfluence, 1 or 10uM of Ripasudil, 10uM of Y-27632, or 10uM of fasudil are added to culture wells. PBS is used as a control vehicle. After 60min, drug solutions are removed and replaced with DMEM containing 10% FBS. Cells are observed by phase-contrast microscopy and photographed 60min after drug application and 2h after drug removal. For immunohistochemistry, TM cells are plated on gelatin-coated 8 well chamber slides at a density of 1x10^4 cells per well in DMEM containing 10% FBS. After overnight culture, when cells reach semiconfluence, cells are incubated in Ripasudil at 1 or 10uM, Y-27632 at 10uM, or fasudil at 10uM for 60minutes. PBS is used as a control vehicle. Drug solutions are removed and replaced with DMEM containing 10% FBS after 2hours. Cells are fixed with 4% paraformaldehyde in PBS for 15min then washed with cytoskeletal buffer (10mM MES, 150mM NaCl, 5mM EGTA, 5mM MgCl2, 5mM glucose, pH 6.1) and serum buffer (10% FBS in PBS). Cells are permeabilized with 0.5% Triton X-100 in PBS for 12minutes at room temperature and blocked with serum buffer for at least 2h at 4 degree C. Filamentous actin (F-actin) is labeled with 0.05mg/mL Phalloidin-TRITC for 1h at room temperature. After washing with PBS, cells are mounted with a commercial mounting medium containing DAPI and observed using a fluorescence microscope. The exposure to take images for F-actin and DAPI are 0.1 and 0.05sec, respectively [2].
• Kinase Assay: ROCK 1 (0.75ng/mL) and ROCK 2 (0.5ng/mL) are incubated with various concentrations of Ripasudil, Y-27632, or HA-1077 at 25 degree C for 90 min in 50mM Tris-HCl buffer (pH 7.5) containing 100mM KCl, 10mM MgCl2, 0.1mM EGTA, 30mM Long S6 Kinase Substrate peptide, and 1mM ATP in a total volume of 40mL. PKACa, PKC, and CaMKIIa are also incubated with various concentrations of Ripasudil, Y-27632, or HA-1077. PKACa (0.0625ng/mL) is incubated at 25 degree C for 30 min in 40mM Tris-HCl buffer (pH 7.5) containing 20mM MgCl2, 1mg/mL BSA, 5mM Kemptide peptide substrate, and 1mM ATP in a total volume of 40mL. PKC (0.025ng/mL) is incubated at 25 degree C for 80 min in 20mM Tris-HCl buffer (pH 7.5) containing 20mM MgCl2, 0.4mM CaCl2, 0.1mg/mL BSA, 0.25mM EGTA, 25ng/mL phosphatidylserine, 2.5ng/mL diacylglycerol, 0.0075% Triton-X-100, 25mM DTT, 10mM Neurogranin (28-43) peptide substrate, and 1mM ATP in a total volume of 40mL. CaMKIIa (0.025ng/mL) is incubated at 25 degree C for 90 min in 50mM Tris-HCl buffer (pH 7.5) containing 10mM MgCl2, 2mM CaCl2, 0.04mg/mL BSA, 16mg/mL purified calmodulin from bovine testis, 500mM DTT, 50mM Autocamitide 2, and 1mM ATP in a total volume of 40mL. After incubation, 40mL of KinaseGlo Luminescent Kinase Assay solution is added, and allowed to remain at 25 degree 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 [1].
• In Vitro: Ripasudil reduces intraocular pressure in a concentration-dependent manner at concentrations between 0.1% and 0.4% in monkey eyes and 0.0625% to 0.5% in rabbit eyes, respectively [1]. Ripasudil (1mg/kg, p.o. daily) shows a neuroprotective effect on retinal ganglion cells (RGCs) after nerve crush (NC). Ripasudil also inhibits the oxidative stress induced by axonal injury in mice. Ripasudil suppresses the time-dependent production of ROS in RGCs after NC injury [3].
• Preparation and Storage: Store at -20 degree C for 3 years powder; -80 degree C for 2 years in solvent

Structure

Related Product Information for Ripasudil hydrochloride dihydrate, inhibitor

Ripasudil (K-115) hydrochloride dihydrate is a specific ROCK inhibitor (IC50s: 51/19nM for ROCK1/ROCK2).

Product Categories/Family for Ripasudil hydrochloride dihydrate, inhibitor

Cell Cycle/Checkpoint; ROCK; Stem Cells

References

Isobe T, et al. Effects of K-115, a rho-kinase inhibitor, on aqueous humor dynamics in rabbits. Curr Eye Res. 2014 Aug;39(8):813-22.
Isobe T, et al. Effects of K-115, a rho-kinase inhibitor, on aqueous humor dynamics in rabbits. Curr Eye Res. 2014 Aug;39(8):813-22.
Kaneko Y, et al. Effects of K-115 (Ripasudil), a novel ROCK inhibitor, on trabecular meshwork and Schlemm's canal endothelial cells. Sci Rep. 2016 Jan 19;6:19640.
Kaneko Y, et al. Effects of K-115 (Ripasudil), a novel ROCK inhibitor, on trabecular meshwork and Schlemm's canal endothelial cells. Sci Rep. 2016 Jan 19;6:19640.
Yamamoto K, et al. The novel Rho kinase (ROCK) inhibitor K-115: a new candidate drug for neuroprotective treatment,in glaucoma. Invest Ophthalmol Vis Sci. 2014 Oct 2;55(11):7126-36.
Yamamoto K, et al. The novel Rho kinase (ROCK) inhibitor K-115: a new candidate drug for neuroprotective treatment,in glaucoma. Invest Ophthalmol Vis Sci. 2014 Oct 2;55(11):7126-36.