p160ROCK (Ki = 0.33 μM); ROCK2 (IC50 = 0.158 μM); PKG (IC50 = 1.65 μM); PKA (IC50 = 4.58 μM); PKC (IC50 = 12.30 μM)

在大鼠肝星状细胞 (HSC) 和人 HSC 衍生的 TWNT-4 细胞中，半水合物 facsudil 盐酸盐 (100 μM) 通过阻断应力纤维的产生、细胞扩散和 α-SMA 表达来抑制细胞增殖[4]。大鼠 HSC 和人 HSC 衍生的 TWNT-4 细胞的蛋白质印迹显示，facudil Hydrochloride semiHydrate（50-100 μM；24 小时）可抑制 LPA（溶血磷脂酸）引起的 ERK1/2、JNK 和 p38 磷酸化[4] 。在人 HSC 衍生的 TWNT-4 细胞中，facsudil HydroHClide semiHydrate（25–100 μM；24 小时）可促进 MMP-1 转录，同时抑制胶原蛋白和 TIMP 转录[4]。

盐酸法舒地尔半水合物（10 mg/kg；静脉注射；手术前一小时）已被证明可以预防心血管疾病，减少 JNK 激活，并减少缺血损伤期间 AIF 的线粒体核易位[5]。盐酸法舒地尔半水合物（50 mg/kg/d；腹腔注射）抑制淋巴细胞增殖，导致白细胞介素 (IL)-17 下调，并显着降低 IFN-γ 与 IL-4 的比率。它还可以预防由蛋白脂质蛋白 PLP p139-151 引起的急性和复发性 EAE（实验性自身免疫性脑脊髓炎）[6]。盐酸法舒地尔半水合物（100 mg/kg/d；口服）可减少小鼠脊髓的炎症、脱髓鞘、轴突损失和 APP 阳性。它还显着降低了SJL/J小鼠实验性自身免疫性脑脊髓炎（EAE）的发生率和病理检查评分[6]。

在最终体积为0.2 mL的反应混合物中，测定环AMP依赖性蛋白激酶的活性，该反应混合物中含有50 mM Tris-HCl (pH 7.0)， 10 mM醋酸镁，2 mM EGTA, 1 μM环AMP或不含环AMP, 3.3至20 μM [r-32P] ATP (4×105 c.p.m)， 0.5 μg酶，100 μg组蛋白H2B和化合物。在30℃下孵育5 min，加入500 μg牛血清白蛋白作为载体蛋白，加入20%三氯乙酸1mL，终止反应。样品在3000转/分离心15min后，在10%三氯乙酸冰冷溶液中重悬，重复离心-重悬循环3次。最后的颗粒溶解在1ml的1n NaOH中，用液体闪烁计数器测量放射性。

Western Blot 分析[4]
细胞类型：大鼠 HSC 和人 HSC 衍生的 TWNT-4 细胞
测试浓度： 50 μM； 100 μM
孵育时间： 24 小时
实验结果： 将 LPA 诱导的 ERK1/2、JNK 和 p38 MAPK 磷酸化抑制 60分别为 %、70% 和 90%。

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RT-PCR[4]
细胞类型：大鼠 HSC 和人 HSC 衍生的 TWNT-4 细胞
测试浓度： 25微米； 50μM； 100 μM
孵育时间：24 小时
实验结果：降低 I 型胶原蛋白、a-SMA 和 TIMP-1 的表达。

Animal/Disease Models: Myocardial ischemia and reperfusion in rat (250-300 g)[5]
Doses: 10 mg/kg
Route of Administration: intravenous (iv) injection; 1 h before operation
Experimental Results: Activated the Rho-kinase, JNK, and resulted AIF translocated to the nucleus. Inhibited Rho-kinase activity, and decreased myocardial infarct size and heart cell apoptosis.

rat LD50 oral 335 mg/kg SENSE ORGANS AND SPECIAL SENSES: PTOSIS: EYE; BEHAVIORAL: TREMOR; BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD Yakuri to Chiryo. Pharmacology and Therapeutics., 20(Suppl
rat LD50 subcutaneous 123 mg/kg SENSE ORGANS AND SPECIAL SENSES: PTOSIS: EYE; BEHAVIORAL: TREMOR; BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD Yakuri to Chiryo. Pharmacology and Therapeutics., 20(Suppl
rat LD50 intravenous 59900 ug/kg SENSE ORGANS AND SPECIAL SENSES: PTOSIS: EYE; BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD; GASTROINTESTINAL: CHANGES IN STRUCTURE OR FUNCTION OF SALIVARY GLANDS Yakuri to Chiryo. Pharmacology and Therapeutics., 20(Suppl
mouse LD50 oral 274 mg/kg SENSE ORGANS AND SPECIAL SENSES: PTOSIS: EYE; BEHAVIORAL: ALTERED SLEEP TIME (INCLUDING CHANGE IN RIGHTING REFLEX); BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD Yakuri to Chiryo. Pharmacology and Therapeutics., 20(Suppl
mouse LD50 subcutaneous 124 mg/kg SENSE ORGANS AND SPECIAL SENSES: PTOSIS: EYE; BEHAVIORAL: ALTERED SLEEP TIME (INCLUDING CHANGE IN RIGHTING REFLEX); BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD Yakuri to Chiryo. Pharmacology and Therapeutics., 20(Suppl

[1]. Fasudil and its analogs: a new powerful weapon in the long war against central nervous system disorders? Expert Opin Investig Drugs. 2013 Apr;22(4):537-50.

[2]. The effects of fasudil on the permeability of the rat blood-brain barrier and blood-spinal cordbarrier following experimental autoimmune encephalomyelitis. J Neuroimmunol. 2011 Oct 28;239(1-2):61-7.

[3]. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature. 1997 Oct 30;389(6654):990-4.

[4]. Fasudil hydrochloride hydrate, a Rho-kinase (ROCK) inhibitor, suppresses collagen production and enhances collagenase activity in hepatic stellate cells. Liver Int. 2005 Aug;25(4):829-38.

[5]. Inhibition of the activity of Rho-kinase reduces cardiomyocyte apoptosis in heart ischemia/reperfusion via suppressing JNK-mediated AIF translocation. Clin Chim Acta. 2009 Mar;401(1-2):76-80.

[6]. The selective Rho-kinase inhibitor Fasudil is protective and therapeutic in experimental autoimmune encephalomyelitis. J Neuroimmunol. 2006 Nov;180(1-2):126-34. Epub 2006 Sep 22.

Fasudil hydrochloride hydrate is a hydrate that is the hemihydrate form of fasudil hydrochloride. It is a drug indicated for the prevention of cerebral vasospasm and ensuing cerebral ischemia following surgery for subarachnoid hemorrhage. It has a role as an antihypertensive agent, a calcium channel blocker, an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor, a neuroprotective agent, a nootropic agent and a vasodilator agent. It contains a fasudil hydrochloride.

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Fasudil is an isoquinoline substituted by a (1,4-diazepan-1-yl)sulfonyl group at position 5. It is a Rho-kinase inhibitor and its hydrochloride hydrate form is approved for the treatment of cerebral vasospasm and cerebral ischemia. It has a role as a geroprotector, an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor, a vasodilator agent, a nootropic agent, a neuroprotective agent, an antihypertensive agent and a calcium channel blocker. It is a N-sulfonyldiazepane and a member of isoquinolines. It is a conjugate base of a fasudil(1+).
Fasudil has been investigated in Carotid Stenosis.

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Introduction: Rho kinase (ROCK) plays a critical role in actin cytoskeleton organization and is involved in diverse fundamental cellular functions such as contraction and gene expression. Fasudil, a ROCK inhibitor, has been clinically applied since 1995 for the treatment of subarachnoid hemorrhage (SAH) in Japan. Increasing evidences indicate that fasudil could exhibit markedly therapeutic effect on central nervous system (CNS) disorders, such as Alzheimer's disease. Areas covered: This article summarizes results from supporting evidence for the potential therapy for fasudil against a variety of CNS diseases. And the properties of its analogs are also summarized. Expert opinion: Current therapies against CNS disorders are only able to attenuate the symptoms and fail in delaying or preventing disease progression and new approaches with disease-modifying activity are desperately needed. The dramatic effects of fasudil in animal models and/or clinical applications of CNS disorders make it a promising strategy to overcome CNS disorders in human beings. Given the complex pathology of CNS disorders, further efforts are necessary to develop multifunctional fasudil derivatives or combination strategies with other drugs in order to exert more powerful effects with minimized adverse effects in the combat of CNS disorders. https://pubmed.ncbi.nlm.nih.gov/23461757/

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Dysfunction of the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) is a primary characteristic of multiple sclerosis (MS). We evaluated the protective effects of fasudil, a selective ROCK inhibitor, in a model of experimental autoimmune encephalomyelitis (EAE) that was induced by guinea-pig spinal cord. In addition, we studied the effects of fasudil on BBB and BSCB permeability. We found that fasudil partly alleviated EAE-dependent damage by decreasing BBB and BSCB permeability. These results provide rationale for the development of selective inhibitors of Rho kinase as a novel therapy for MS. https://pubmed.ncbi.nlm.nih.gov/21978848/

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Background/aims: The Rho-ROCK signaling pathways play an important role in the activation of hepatic stellate cells (HSCs). We investigated the effects of fasudil hydrochloride hydrate (fasudil), a Rho-kinase (ROCK) inhibitor, on cell growth, collagen production, and collagenase activity in HSCs. Methods: Rat HSCs and human HSC-derived TWNT-4 cells were cultured for studies on stress fiber formation and alpha-smooth muscle actin (alpha-SMA) expression. Proliferation was measured by BrdU incorporation, and apoptosis by TUNEL assay. The phosphorylation states of the MAP kinases (MAPKs), extra cellular signal -regulated kinase 1/2 (ERK1/2), c-jun kinase (JNK), and p38 were evaluated by western blot analysis. Type I collagen, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) production and gene expression were evaluated by ELISA and real-time PCR, respectively. Collagenase activity (active MMP-1) was also evaluated. Results: Fasudil (100 microM) inhibited cell spreading, the formation of stress fibers, and expression of alpha-SMA with concomitant suppression of cell growth, although it did not induce apoptosis. Fasudil inhibited phosphorylation of ERK1/2, JNK, and p38. Treatment with fasudil suppressed the production and transcription of collagen and TIMP, stimulated the production and transcription of MMP-1, and enhanced collagenase activity. Conclusion: These findings demonstrated that fasudil not only suppresses proliferation and collagen production but also increases collagenase activity. https://pubmed.ncbi.nlm.nih.gov/15998434/

2[C14H17N3O2S].2[HCL].H2O

673.67452

672.172

C, 49.92; H, 5.69; Cl, 10.52; N, 12.48; O, 11.87; S, 9.52

186694-02-0

Fasudil;103745-39-7;Fasudil dihydrochloride;203911-27-7

23724856

Typically exists as solid at room temperature

6.672

150.59

5

11

4

43

421

0

O.Cl.Cl.C1NCCN(S(C2=CC=CC3C=NC=CC2=3)(=O)=O)CC1.C1NCCN(S(C2=CC=CC3C=NC=CC2=3)(=O)=O)CC1

AACOJGPCMIDLEY-UHFFFAOYSA-N

InChI=1S/2C14H17N3O2S.2ClH.H2O/c2*18-20(19,17-9-2-6-15-8-10-17)14-4-1-3-12-11-16-7-5-13(12)14;;;/h2*1,3-5,7,11,15H,2,6,8-10H2;2*1H;1H2

5-(1,4-diazepan-1-ylsulfonyl)isoquinoline;hydrate;dihydrochloride

Fasudil hydrochloride hydrate; 186694-02-0; Fasudil hydrochloride hemihydrate; Eril-S; fasudil HCl semihydrate; 5-((1,4-Diazepan-1-yl)sulfonyl)isoquinoline hydrochloride hemihydrate; Fasudil hydrochloride hydrate [JAN]; LI4L0R5Y7T;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。