ROCK (IC50 = 3.6 nM); PKC (IC50 = 420 nM); CaMKII (IC50 = 810 nM)

Y-33075, also known as Y-39983, is a strong inhibitor of ROCK, with an IC50 of 3.6 nM. Furthermore, Y-33075 inhibits PKC and CaMKII more potently than Y-27632. Y-27632 and Y-33075 had IC50 values of 9.0 μM and 0.42 μM against PKC and 26 μM and 0.81 μM, respectively, against CaMKII. Y-27632 and Y-33075 had IC50s of 82 and 117 times that of ROCK against PKC, and 236 and 225 times that of ROCK against CaMKII, respectively [1]. In comparison to neurites in RGCs not receiving Y-39983 therapy, Y-33075 (Y-39983, 10 μM) lengthens them in RGCs [2]. Rabbit ciliary artery segments in a Ca2+-free solution are not able to contract due to histamine when exposed to Y-33075 (Y-39983, 1 μM). In solutions with high potassium (high K), Y-33075 (10 μM) does not affect the rise in [Ca2+]i [3].

Y-39983 (≥0.01%) dramatically reduces intraocular pressure (IOP) in rabbits two hours after topical application. IOP significantly decreases in monkeys' treated eyes with Y-39983 (0.05%) between two and seven hours after topical application. management[1]. Rat eyes with retinal ganglion cells (RGCs) have more regenerating axons when exposed to 100 μM Y-39983[2].

---

A biochemical assay showed that Y-39983 inhibited ROCK more potently than Y-27632. In rabbits, topical administration of Y-39983 significantly increased conventional outflow by 65.5%, followed by significant, dose-dependent reduction in IOP. Maximum IOP reduction was 13.2 +/- 0.6 mm Hg (mean +/- SE) at 0.1% Y-39983 in rabbits. In monkeys, at 3 hours after topical administration of 0.05% Y-39983, maximum reduction of IOP was 2.5 +/- 0.8 mm Hg. No serious side effects were observed in ocular tissues except sporadic punctate subconjunctival hemorrhage during long-term topical administration of Y-39983 four times a day (at 2-hour intervals) in rabbits or monkeys. However, punctate subconjunctival hemorrhage was not observed with administration twice daily (at a 6-hour interval) or three times a day (at 5-hour intervals).[1]

---

Topical administration of 0.05% Y-39983 solution significantly increased blood flow in ONH compared with the vehicle group in rabbits. Maximum increase in blood flow in the 0.05% Y-39983 group was 122.84 ± 5.98 % (Mean ± S.E.) at 90 minutes after administration compared with before administration. Neurites in rat RGCs treated with 10 μM Y-39983 were extended compared with those without Y-39983 treatment of RGCs in vitro. Y-39983 dose-dependently increased the number of RGCs with regenerating axons in vivo. The numbers of RGCs with regenerating axons in 10 and 100 μM Y-39983-treated rats were 99.3 ± 10.5 and 169.5 ± 43.3 cells/mm(2) (Mean ± S.D.), respectively, and significantly increased compared with those in saline-treated rats (43.3 ± 6.0 cells/mm(2)).[2]

Measurement of Inhibition of ROCK, Protein Kinase C, and Calmodulin-Dependent Protein Kinase II[1]
Recombinant ROCK (ROK α/ROCK II) and purified protein kinase C (PKC: mixture of α, β, γ isoforms) were purchased from Upstate Biotechnology. Recombinant calmodulin-dependent protein kinase II (CaMK II) was purchased from Daiichi Pure Chemical. ROCK (0.2 U/mL) was incubated with 1 μM [γ-32P] ATP and 10 μg/mL histone as substrates in the absence or presence of various concentrations of Y-27632, Y-39983, or staurosporine at room temperature for 20 minutes in 20 mM MOPS (3-(N-morpholino)propanesulfonic acid) buffer (pH 7.2) containing 0.1 mg/mL bovine serum albumin (BSA), 5 mM dithiothreitol [DTT], 10 mM β-glycerophosphate, 50 μM Na3VO4, and 10 mM MgCl2 in a total volume of 100 μL. PKC (10 ng/mL) was incubated with 1 μM [γ-32P] ATP and 20 μM PKC substrate in the absence or presence of various concentrations of Y-27632, Y-39983, or staurosporine at room temperature for 30 minutes in 20 mM MOPS buffer (pH 7.5) containing 0.1 mg/mL BSA, 10 mM DTT, 10 mM β-glycerophosphate, 50 μM Na3VO4, 2 mM CaCl2, 20 μg/mL phosphatidyl-l-serine, and 10 mM MgCl2 in a total volume of 100 μL. CaMK II (125 U/mL) was incubated with 1 μM [γ-32P] ATP, 10 μM calmodulin, and 20 μM CaMK II substrate, in the absence or presence of various concentrations of Y-27632, Y-39983, or staurosporine at room temperature for 30 minutes in 20 mM MOPS buffer (pH 7.5) containing 0.2 mg/mL BSA, 0.5 mM DTT, 0.1 mM β-glycerophosphate, 50 μM Na3VO4, 1 mM CaCl2, and 5 mM MgCl2 in a total volume of 100 μL. Incubation was terminated by the addition of 100 μL of 0.7% phosphoric acid. A 160 μL portion of the mixture was transferred to Multiscreen-PH plate (Millipore, MA). A positively charged phosphocellulose filter absorbed the substrate that bound 32P (Multiscreen-Vacuum manifold; Millipore). The filter was washed with 300 μL of 0.5% phosphoric acid and then twice with purified water and then dried. The radioactivity of the dried filter was measured with a liquid scintillation counter. Results are presented as 50% inhibitory concentrations and 95% confidence intervals (CIs).

Effects on Cultured Human Umbilical Venous Endothelial Cells[1]
Human umbilical venous endothelial cells (HUVECs) were purchased from Dainippon Pharmaceutical (Osaka, Japan). HUVECs were cultured in CS-C medium (Dainippon Pharmaceutical) and maintained in a 95% air-5% CO2 atmosphere at 37°C and passaged using the trypsin-EDTA method. HUVECs were seeded into 24-well plates. After seeding, HUVECs were incubated in medium containing 1 μM Y-39983 for 15 or 30 minutes and observed by phase-contrast microscopy. Medium was then removed, and HUVECs were incubated in medium without Y-39983 for 1 hour to evaluate recovery from the morphologic changes induced by Y-39983[1].

Y-39983 was compared with Y-27632 for selectivity of ROCK inhibition by biochemical assay. The IOP was monitored by pneumatonometer in albino rabbits and cynomolgus monkeys that were given topically administered Y-39983. The total outflow facility and uveoscleral outflow were measured by two-level constant-pressure perfusion and perfusion technique using fluorescein isothiocyanate-dextran, respectively, at 2 hours after topical administration of Y-39983 in albino rabbits. The ocular toxicologic effects of topical administration of Y-39983 were observed in albino rabbits and cynomolgus monkeys.[1]

---

Blood flow in ONH was measured by the laser speckle method after topical administration of 0.05% Y-39983 solution or its vehicle in rabbit eyes. To investigate the effects of Y-39983 on axonal regeneration of RGCs, RGCs purified from rat eyes were cultured with or without 10 μM Y-39983 and morphologically observed by phase-contrast microscopy. Moreover, the effects of intravitreal administration of Y-39983 were evaluated using an in vivo model of axotomized RGCs in peripheral nerve-grafted rats.[2]

---

Dissolved in DMSO, and diluted in saline (Rat); 0.9% NaCl (Mice); 30 mg/kg/day (Rat); 0-10 mg/kg (mice); Orally (Rat); i.p. (Mice)

Male Wistar rats with spontaneous or induced hypertension; Swiss albino mice with Ehrlich ascites carcinoma

[1]. Effects of Topical Administration of Y-39983, a Selective Rho-Associated Protein Kinase Inhibitor, on Ocular Tissues in Rabbits and Monkeys Invest. Ophthalmol. Vis. Sci. July 2007 vol. 48no. 7 3216-3222.

[2]. Effects of Y-39983, a selective Rho-associated protein kinase inhibitor, on blood flow in optic nerve head in rabbits and axonal regeneration of retinal ganglion cells in rats. Curr Eye Res. 2011 Oct;36(10):964-70.

[3]. Effects of Rho-associated protein kinase inhibitors Y-27632 and Y-39983 on isolated rabbit ciliary arteries.Jpn J Ophthalmol. 2011 Jul;55(4):411-7. Epub 2011 Jun 11.

Purpose: To elucidate the intraocular pressure (IOP)-lowering effects and associated characteristics of Y-39983, a selective Rho-associated coiled coil-forming protein kinase (ROCK) inhibitor derived from Y-27632, in animal eyes. Conclusions: Y-39983 causes increased outflow facility followed by IOP reduction. Y-39983 ophthalmic solution may be a candidate drug for lowering of IOP, since it increases conventional outflow and produces relatively few side effects.[1]

---

Purpose: To investigate the effects of Y-39983, a selective Rho-associated coiled coil-forming protein kinase inhibitor, on blood flow in the optic nerve head (ONH) in rabbits and axonal regeneration of retinal ganglion cells (RGCs) in rats. Conclusion: Y-39983 may be a candidate drug not only for lowering of IOP but also for increasing of blood flow in ONH in the treatment of glaucoma. Moreover, Y-39983 may have therapeutic potential for axonal regeneration of RGCs in the treatment of diseases with degenerating axons of RGCs including glaucoma, although improvements of formulation or route of administration are needed in order to reach an effective concentration in retina.[2]

---

Purpose: In normotensive eyes, reduced ocular blood flow can lead to glaucoma pathogenesis. Drugs that reduce intraocular pressure (IOP) often cause vasodilation of the ciliary arteries and improve blood flow to the eye. A novel class of drugs called Rho-associated coiled coil-forming protein kinase (ROCK) inhibitors can lower IOP. Therefore, we tested the ability of two ROCK inhibitors, Y-27632 and Y39983, to relax rabbit ciliary arteries.[3]
Methods: We measured in vitro ciliary artery smooth muscle contractions by isometric tension recordings and changes of intracellular free calcium concentration ([Ca(2+)](i)) by fluorescence photometry.[3]
Results: Both Y-27632 and Y-39983 induced a concentration-dependent relaxation in rabbit ciliary arteries precontracted with a high-potassium (high-K) solution. The amplitude of relaxation induced by Y-27632 and Y-39983 was not affected by either 100 μM N (G)-nitro-L: -arginine methyl ester (L: -NAME) or 10 μM indomethacin. In Ca(2+)-free solution, Y-27632 and Y-39983 significantly inhibited the transient contraction of ciliary arteries induced by 10 μM histamine. However, neither Y-27632 nor Y-39983 affected the elevation of [Ca(2+)](i) induced by high-K solution and histamine.[3]
Conclusions: We concluded that Y-27632 and Y-39983 relaxed isolated rabbit ciliary artery segments in vitro. The mechanism of relaxation was not dependent on endothelial-derived factors such as nitric oxide (NO) or prostacyclin, nor was it dependent on changes in intracellular Ca(2+) concentration.[3]

C16H18CL2N4O

353.25

352.086

C, 54.40; H, 5.14; Cl, 20.07; N, 15.86; O, 4.53

173897-44-4

Y-33075;199433-58-4;Y-33075 hydrochloride;471843-75-1

20601328

White to gray solid powder

5.212

5

3

3

23

367

1

C[C@H](C1=CC=C(C=C1)C(=O)NC2=C3C=CNC3=NC=C2)N.Cl.Cl

CKFHAVRPVZNMGT-YQFADDPSSA-N

InChI=1S/C16H16N4O.2ClH/c1-10(17)11-2-4-12(5-3-11)16(21)20-14-7-9-19-15-13(14)6-8-18-15;;/h2-10H,17H2,1H3,(H2,18,19,20,21);2*1H/t10-;;/m1../s1

4-[(1R)-1-aminoethyl]-N-(1H-pyrrolo[2,3-b]pyridin-4-yl)benzamide;dihydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.08 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (7.08 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (7.08 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: Saline: 30 mg/mL

---

Solubility in Formulation 5: 100 mg/mL (283.09 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

 (Please use freshly prepared in vivo formulations for optimal results.)