ROCK (IC50 = 3.6 nM); PKC (IC50 = 420 nM); CaMKII (IC50 = 810 nM)
Y‑39983 targets ROCK (both ROCK1 and ROCK2) as a potent inhibitor; it also inhibits PKC and CaMKII at higher concentrations. Its action on ROCK reduces actin cytoskeleton contraction, promotes neurite outgrowth, and decreases intraocular pressure by relaxing trabecular meshwork and ciliary muscle.

At 3.6 nM for IC50, Y-33075, also known as Y-39983, is a strong inhibitor of ROCK. More potently than Y-27632, Y-33075 inhibits PKC and CaMKII. In contrast, Y-27632 and Y-33075 have IC50s of 26 μM and 0.81 μM against CaMKII and 9.0 μM and 0.42 μM, respectively, against PKC. Comparing Y-27632 and Y-33075 against PKC, their IC50s are 82 and 117 times greater than ROCK, respectively, but against CaMKII, they are 236 and 225 times greater than ROCK, 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].

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In vitro, Y‑39983 (10 μM) extends neurites in retinal ganglion cells compared to untreated controls. It inhibits histamine‑induced contraction of rabbit ciliary artery at 1 μM in Ca²⁺‑free conditions, without affecting high‑K⁺‑induced calcium influx at 10 μM, confirming ROCK‑mediated vasodilation.

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].

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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]

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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]

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In vivo, topical Y‑39983 (≥0.01%) lowers intraocular pressure in rabbits and monkeys within 2 hours; it promotes regeneration of crushed optic nerve axons in adult cats and attenuates experimental autoimmune encephalomyelitis by inhibiting demyelination, indicating potential for glaucoma, nerve repair, and autoimmune diseases.

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).[1]

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ROCK inhibitory activity is determined in cell‑free kinase assays using recombinant ROCK enzyme, a peptide substrate, and ATP; phosphorylation is measured by scintillation or fluorescence. IC50 is derived from dose‑response curves. Similar assays are used for PKC and CaMKII to assess selectivity.

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].

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Cell‑based assays use primary retinal ganglion cell cultures; neurite outgrowth is quantified after treatment with 10 μM Y‑39983. Ciliary artery segments are used to measure contraction ex vivo, and intracellular calcium is monitored with fluorescent dyes in cells treated with the compound.

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 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]

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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]

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In vivo studies: topical ocular administration in rabbits and monkeys for IOP measurement; optic nerve crush model in cats for axonal regeneration; EAE model in mice for demyelination assessment. Doses and routes vary; outcomes include IOP reduction, nerve regeneration score, and clinical EAE severity.

Y‑39983 (MW 280.32, C₁₆H₁₆N₄O) is soluble in DMSO. Detailed PK parameters (half‑life, bioavailability, metabolism) are not fully reported, but its topical ocular efficacy suggests sufficient corneal penetration. Systemic PK would need further characterisation for systemic indications.

No detailed toxicology data are available; however, in vivo studies at effective doses did not report overt toxicity. As a ROCK inhibitor, potential side effects include hypotension and altered vascular tone, but topical application minimises systemic exposure. Further safety studies are required.

[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.

4-[(1R)-1-aminoethyl]-N-(1H-pyrrolo[2,3-b]pyridin-4-yl)benzamide is a pyrrolopyridine.
See also: Y-39983 (note moved to).

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Y‑39983 is an investigational compound not approved; studied for glaucoma, optic nerve regeneration, and multiple sclerosis. Its ROCK inhibition promotes neuronal repair and lowers IOP, making it a promising multi‑target candidate. Clinical translation requires further efficacy and safety validation.

C16H16N4O

280.33

280.132

C, 68.55; H, 5.75; N, 19.99; O, 5.71

199433-58-4

Y-33075 dihydrochloride;173897-44-4;Y-33075 hydrochloride;471843-75-1

9810884

White to off-white solid powder

1.32

444.644ºC at 760 mmHg

222.713ºC

3.608

3

3

3

21

367

1

O=C(NC1=C2C(NC=C2)=NC=C1)C3=CC=C(C=C3)[C@H](N)C

JTVBXQAYBIJXRP-SNVBAGLBSA-N

InChI=1S/C16H16N4O/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)/t10-/m1/s1

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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 (8.92 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.92 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.92 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.

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Solubility in Formulation 4: Saline: 30 mg/mL

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