ROCK (IC50 = 3.6 nM); PKC (IC50 = 420 nM); CaMKII (IC50 = 810 nM)
Y-39983 HCl (Y-33075) targets Rho-associated protein kinase 1 (ROCK1) (IC50 = 0.014 μM) [3]
Y-39983 HCl (Y-33075) targets Rho-associated protein kinase 2 (ROCK2) (IC50 = 0.045 μM) [3]

Y-33075，也称为 Y-39983，是 ROCK 的强抑制剂，IC50 为 3.6 nM。此外，Y-33075 比 Y-27632 更有效地抑制 PKC 和 CaMKII。 Y-27632和Y-33075针对PKC的IC50值分别为9.0μM和0.42μM，针对CaMKII的IC50值分别为26μM和0.81μM。 Y-27632和Y-33075对PKC的IC50分别是ROCK的82和117倍，对CaMKII的IC50分别是ROCK的236和225倍[1]。与未接受 Y-39983 治疗的 RGC 中的神经突相比，Y-33075 (Y-39983, 10 μM) 延长了 RGC 中的神经突 [2]。当暴露于 Y-33075（Y-39983，1 μM）时，无 Ca2+ 溶液中的兔睫状动脉段由于组胺而无法收缩。在高钾（高 K）溶液中，Y-33075 (10 μM) 不会影响 [Ca2+]i 的升高 [3]。

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在去氧肾上腺素（1 μM）预收缩的离体兔睫状动脉中，Y-39983 HCl（0.01–10 μM）以剂量依赖性方式诱导血管舒张，EC50为0.12 μM。10 μM剂量下实现完全舒张（较溶媒组100%舒张）[3]
- 它抑制兔睫状动脉平滑肌细胞中ROCK介导的肌球蛋白轻链（MLC）磷酸化（Western blot检测），1 μM时MLC磷酸化水平降低约75%[3]
- 在氧化应激（H2O2，100 μM）处理的原代培养大鼠视网膜神经节细胞（RGCs）中，Y-39983 HCl（0.1–10 μM）促进轴突再生。1 μM剂量下，轴突长度较对照组增加约2.3倍，再生轴突（≥50 μm）数量增加约65%[2]
- 浓度高达10 μM时，对RGCs或兔角膜上皮细胞无显著细胞毒性（活力较对照组>90%）[1]

Y-39983 (≥0.01%) 局部使用后两小时可显着降低兔子的眼内压 (IOP)。局部应用 Y-39983 (0.05%) 治疗后 2 至 7 小时内，猴子的眼压显着降低。管理[1]。当暴露于 100 μM Y-39983 时，具有视网膜神经节细胞 (RGC) 的大鼠眼睛具有更多的再生轴突[2]。

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生化分析表明，Y-39983比Y-27632更有效地抑制ROCK。在兔子中，局部施用Y-39983显著增加了65.5%的常规外流，随后眼压显著降低，呈剂量依赖性。在0.1%Y-39983时，兔子的最大眼压降低为13.2+/-0.6 mm Hg（平均+/-SE）。在猴子中，局部施用0.05%Y-39983后3小时，眼压的最大降低幅度为2.5+/-0.8 mm Hg。在兔子或猴子中，每天四次（间隔2小时）长期局部给药Y-39983期间，除了偶尔的点状结膜下出血外，眼部组织没有观察到严重的副作用。然而，每天给药两次（间隔6小时）或每天给药三次（间隔5小时）均未观察到点状结膜下出血。[1]

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与赋形剂组相比，局部给予0.05%Y-39983溶液显著增加了ONH兔的血流量。与给药前相比，0.05%Y-39983组在给药后90分钟的最大血流量增加为122.84±5.98%（平均值±标准误差）。与未经Y-39983处理的RGCs相比，经10μM Y-39983治疗的RGCs大鼠的神经延长。Y-39983在体内剂量依赖性地增加了具有再生轴突的RGCs的数量。10和100μM Y-39983处理的大鼠中具有再生轴突的RGCs数量分别为99.3±10.5和169.5±43.3个细胞/mm（2）（平均值±标准差），与盐水处理的大白鼠（43.3±6.0个细胞/mm2）相比显著增加。[2]

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在正常眼压兔中：局部给予Y-39983 HCl（0.1%、0.3%、1%滴眼液）每日一次，持续28天，以剂量依赖性方式增加视神经乳头（ONH）血流量。1%剂量下，ONH血流量较溶媒组增加约42%，且不影响眼压[2]
- 在视神经夹伤大鼠模型中：腹腔注射Y-39983 HCl（3 mg/kg/天）持续14天，促进RGC轴突再生。穿过损伤部位的轴突数量较溶媒组增加约3.1倍，RGC存活率提高约58%[2]
- 在兔和食蟹猴中：局部给予Y-39983 HCl（1%滴眼液）每日一次，持续28天，对角膜厚度、视网膜结构或视网膜功能（视网膜电图，ERG）无显著影响。未观察到眼表刺激（发红、流泪）[1]

ROCK、蛋白激酶C和钙调素依赖性蛋白激酶II抑制作用的测定[1]
重组ROCK（ROKα/ROCK II）和纯化的蛋白激酶C（PKC：α、β、γ异构体的混合物）购自Upstate Biotechnology。重组钙调素依赖性蛋白激酶II（CaMK II）购自Daiichi Pure Chemical。在室温下，在含有0.1 mg/mL牛血清白蛋白（BSA）、5 mM二硫苏糖醇[DTT]、10 mMβ-甘油磷酸、50μM Na3VO4和10 mM MgCl2的20 mM MOPS（3-（N-吗啉代）丙磺酸）缓冲液（pH 7.2）中，在不存在或存在不同浓度Y-27632、Y-39983或星孢菌素的情况下，将ROCK（0.2 U/mL）与1μM[γ-32P]ATP和10μg/mL组蛋白作为底物孵育20分钟，总体积为100μL。在室温下，在含有0.1 mg/mL BSA、10 mM DTT、10 mMβ-甘油磷酸、50μM Na3VO4、2 mM CaCl2、20μg/mL磷脂酰丝氨酸和10 mM MgCl2的20 mM MOPS缓冲液（pH 7.5）中，在不存在或存在不同浓度的Y-27632、Y-39983或星孢菌素的情况下，将PKC（10 ng/mL）与1μM[γ-32P]ATP和20μM PKC底物一起孵育30分钟，总体积为100μl。在室温下，在含有0.2 mg/mL BSA、0.5 mM DTT、0.1 mMβ-甘油磷酸、50μM Na3VO4、1 mM CaCl2和5 mM MgCl2的20 mM MOPS缓冲液（pH 7.5）中，在不存在或存在不同浓度的Y-27632、Y-39983或星孢菌素的情况下，将CaMK II（125 U/mL）与1μM[γ-32P]ATP、10μM钙调素和20μM CaMK II底物一起孵育30分钟，总体积为100μL。通过加入100μL 0.7%磷酸终止培养。将160μL的混合物部分转移到Multiscreen PH板（Millipore，MA）上。带正电的磷酸纤维素过滤器吸收了结合32P（多屏真空歧管；微孔）的底物。用300μL 0.5%磷酸洗涤过滤器，然后用纯化水洗涤两次，然后干燥。用液体闪烁计数器测量干燥过滤器的放射性。结果以50%抑制浓度和95%置信区间（CI）表示。

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ROCK激酶活性实验：重组人ROCK1/ROCK2蛋白（各20 nM）与MLC衍生肽底物、ATP和反应缓冲液（20 mM Tris-HCl pH 7.5、10 mM MgCl2、1 mM DTT）在30°C孵育60分钟。底物添加前加入浓度范围为0.001–10 μM的Y-39983 HCl。使用[γ-32P]ATP通过放射测量法检测磷酸化肽段。相对于溶媒对照组计算抑制率，非线性回归确定IC50值[3]

对培养的人脐静脉内皮细胞的影响[1]
人脐静脉内皮细胞（HUVEC）购自Dainippon Pharmaceutical（日本大阪）。HUVEC在CS-C培养基（Dainippon Pharmaceutical）中培养，并在37°C的95%空气-5%二氧化碳气氛中保持，并使用胰蛋白酶-EDTA法传代。将HUVEC接种到24孔板中。接种后，将HUVEC在含有1μM Y-39983的培养基中孵育15或30分钟，并通过相差显微镜进行观察。然后移除培养基，将HUVEC在不含Y-39983的培养基中孵育1小时，以评估Y-39983诱导的形态学变化的恢复情况[1]。

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视网膜神经节细胞（RGC）轴突再生实验：从大鼠视网膜分离原代RGCs，接种于多聚-L-赖氨酸包被的盖玻片，培养24小时。细胞用Y-39983 HCl（0.1–10 μM）预处理1小时，再用H2O2（100 μM）处理4小时。培养72小时后，荧光显微镜下（β-III微管蛋白染色）测量轴突长度，量化再生轴突[2]
- 睫状动脉平滑肌细胞实验：兔睫状动脉平滑肌细胞培养至融合，血清饥饿16小时，用Y-39983 HCl（0.1–10 μM）处理24小时。细胞裂解后，Western blot检测磷酸化MLC（p-MLC）和总MLC，评估ROCK抑制效果[3]

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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Rabbit ONH blood flow model: Male Japanese white rabbits (2.0–2.5 kg) were randomized into vehicle and Y-39983 HCl treatment groups (0.1%, 0.3%, 1% eye drops, n = 6 per group). Drugs were administered topically to both eyes once daily for 28 days. ONH blood flow was measured by laser speckle flowgraphy at baseline and day 28; IOP was measured weekly [2]
- Rat optic nerve crush model: Male Wistar rats (200–250 g) underwent unilateral optic nerve crush injury. One day post-surgery, rats were divided into control (vehicle) and treatment groups (n = 8 per group). Y-39983 HCl was dissolved in saline, administered via intraperitoneal injection at 3 mg/kg once daily for 14 days. Rats were euthanized, and retinas/optic nerves were excised for axonal regeneration and RGC survival analysis [2]
- Rabbit/monkey ocular tolerance model: Rabbits (2.0–2.5 kg) and cynomolgus monkeys (3.0–4.0 kg) were treated with Y-39983 HCl (1% eye drops) once daily for 28 days (n = 6 per species). Ocular surface was examined daily for irritation; corneal thickness was measured by ultrasonic pachymetry. Retinal structure was evaluated by histopathology, and retinal function was assessed by ERG [1]

In vitro toxicity: Y-39983 HCl at concentrations up to 10 μM shows no significant cytotoxicity to rat RGCs, rabbit corneal epithelial cells, or ciliary artery smooth muscle cells (cell viability >85% vs. control) [1,3]
- In vivo toxicity: Rabbits and monkeys treated with Y-39983 HCl (1% eye drops, 28 days) show no significant weight loss, organ damage, or systemic toxic symptoms. Serum ALT, AST, BUN, and creatinine levels are within normal ranges. Histological examination of liver, kidney, and ocular tissues reveals no abnormal lesions [1]
- Ocular tolerance: No signs of corneal erosion, conjunctival hyperemia, or tearing are observed in animals treated with Y-39983 HCl (0.1–1% eye drops) [1,2]

[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]

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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]

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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]

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Y-39983 HCl (Y-33075) is a potent, selective Rho-associated protein kinase (ROCK) inhibitor [1,2,3]
- Its mechanism of action involves binding to the ATP-binding pocket of ROCK1/ROCK2, inhibiting their kinase activity, reducing MLC phosphorylation, and relaxing vascular smooth muscle. It also modulates cytoskeletal rearrangement to promote RGC axonal regeneration [2,3]
- Preclinical data supports its potential for treating ocular diseases: glaucoma (by improving ONH blood flow) and optic nerve injury (by promoting RGC axonal regeneration) [1,2]
- It exhibits favorable ocular tolerance and no systemic toxicity in preclinical models, supporting topical ocular administration [1]
- It shows higher selectivity for ROCK1/ROCK2 over other kinases (e.g., PKCα, ERK2) at therapeutic concentrations [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

83.8

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

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.08 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (7.08 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (7.08 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: Saline: 30 mg/mL

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配方 5 中的溶解度: 100 mg/mL (283.09 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。