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)

In rat hepatic stellate cells (HSCs) and human HSC-derived TWNT-4 cells, facsudil hydrochloride semihydrate (100 μM) suppresses cell proliferation by blocking the production of stress fibers, cell spreading, and α-SMA expression[4]. Western blotting of rat HSCs and human HSC-derived TWNT-4 cells reveals that facudil hydrochloride semihydrate (50-100 μM; 24 hours) suppresses the phosphorylation of ERK1/2, JNK, and p38 caused by LPA (lysophoaphatidic acid)[4]. In human HSC-derived TWNT-4 cells, facsudil hydrochloride semihydrate (25–100 μM; 24 hours) promotes MMP-1 transcription while suppressing collagen and TIMP transcription[4].

Fasudil hydrochloride semihydrate (10 mg/kg; intravenous; one hour prior to surgery) has been shown to protect against cardiovascular disease, lessen JNK activation, and lessen AIF's mitochondrial-nuclear translocation during ischemia injury[5]. Fasudil hydrochloride semihydrate (50 mg/kg/d; ip) inhibits the proliferation of lymphocytes, results in downregulation of interleukin (IL)-17, and a significant decrease in the ratio of IFN-γ to IL-4. It also prevents acute and relapsing EAE (experimental autoimmune encephalomyelitis) caused by the proteolipid protein PLP p139–151[6]. Fasudil hydrochloride semihydrate (100 mg/kg/d; po) decreases inflammation, demyelination, axonal loss, and APP positive in the spinal cord cord of mice. It also significantly lowers the incidence and pathological examination score of experimental autoimmune encephalomyelitis (EAE) in SJL/J mice[6].

Cyclic AMP-dependent protein kinase activity is assayed in a reaction mixture containing, in a final volume of 0.2 mL, 50 mM Tris-HCl (pH 7.0), 10 mM magnesium acetate, 2 mM EGTA, 1 μM cyclic AMP or absence of cyclic AMP, 3.3 to 20 μM [r-32P] ATP (4×105 c.p.m.), 0.5 μg of the enzyme, 100 μg of histone H2B and compound. The mixture is incubated at 30°C for 5 min. The reaction is terminated by adding 1mL of ice-cold 20% trichloroacetic acid after adding 500 μg of bovine serum albumin as a carrier protein. The sample is centrifuged at 3000 r.p.m. for 15min, the pellet is resuspended in ice-cold 10% trichloro-acetic acid solution and the centrifugation-resuspension cycle is repeated three times. The final pellet is dissolved in 1 mL of 1 N NaOH and radioactivity is measured with a liquid scintillation counter.

Western Blot Analysis[4]
Cell Types: Rat HSCs and human HSC-derived TWNT-4 cells
Tested Concentrations: 50 μM; 100 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Suppressed the LPA-induced phosphorylation of ERK1/2, JNK and p38 MAPK by 60%, 70%,and 90%, respectively.

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RT-PCR[4]
Cell Types: Rat HSCs and human HSC-derived TWNT-4 cells
Tested Concentrations: 25 μM; 50 μM; 100 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: decreased the expression of type I collagen, a-SMA, and TIMP-1.

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.

Oral LD50 in rats: 335 mg/kg. Sensory organs and special senses: ptosis; Behavior: tremor; Behavior: seizures or effect on the epilepsy threshold. Yakuri to Chiryo. Pharmacology and Therapeutics, 20 (Supplement). Subcutaneous LD50 in rats: 123 mg/kg. Sensory organs and special senses: ptosis; Behavior: tremor; Behavior: seizures or effect on the epilepsy threshold. Pharmacology and Therapeutics, 20 (Supplement). Intravenous LD50 in rats: 59900 ug/kg. Sensory organs and special senses: ptosis; Behavior: seizures or effect on the epilepsy threshold; Gastrointestinal tract: changes in salivary gland structure or function. Pharmacology and Therapeutics, 20(Supplement)
Oral LD50 in mice: 274 mg/kg. Sensory organs and special senses: ptosis; Behavior: altered sleep duration (including altered righting reflex); Behavior: seizures or effect on epilepsy threshold. Yakuri to Chiryo. Pharmacology and Therapeutics., 20(Supplement)
Subcutaneous LD50 in mice: 124 mg/kg. Sensory organs and special senses: ptosis; Behavior: altered sleep duration (including altered righting reflex); Behavior: seizures or effect on epilepsy threshold. Yakuri to Chiryo. Pharmacology and Therapeutics, 20(Supplement)

[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 the hemihydrate form of fasudil hydrochloride. It is a drug used to prevent cerebral vasospasm and secondary cerebral ischemia following subarachnoid hemorrhage surgery. It has multiple effects, including antihypertensive, calcium channel blocking, EC 2.7.11.1 (nonspecific serine/threonine protein kinase) inhibitor, neuroprotective, nootropic, and vasodilator effects. It contains fasudil hydrochloride. Fasudil is an isoquinoline compound with a (1,4-diazacycloheptan-1-yl)sulfonyl group substituted at the 5-position. It is a Rho kinase inhibitor, and its hydrochloride hydrate form is approved for the treatment of cerebral vasospasm and cerebral ischemia. It has multiple effects, including anti-aging, EC 2.7.11.1 (nonspecific serine/threonine protein kinase) inhibition, vasodilation, nootropic, neuroprotective, antihypertensive, and calcium channel blocking. It is an N-sulfonyldiazacycloheptanane compound, belonging to the isoquinoline class. It is the conjugate base of fasudil (1+). Fasudil has been investigated in the treatment of carotid artery stenosis. Introduction: Rho kinase (ROCK) plays a crucial role in the organization of the actin cytoskeleton and is involved in a variety of essential cellular functions, such as contraction and gene expression. Fasudil is a ROCK inhibitor that has been used in Japan since 1995 to treat subarachnoid hemorrhage (SAH). Increasing evidence suggests that fasudil may have significant therapeutic effects on central nervous system (CNS) diseases, such as Alzheimer's disease. This article summarizes the evidence supporting the potential efficacy of fasudil in treating various CNS diseases and outlines the characteristics of its analogues. Expert Opinion: Current therapies for CNS diseases only alleviate symptoms and cannot slow or stop disease progression; therefore, there is an urgent need for new approaches with disease-modifying effects. The significant efficacy of fasudil in animal models and/or clinical applications for CNS diseases makes it a promising strategy for treating human CNS diseases. Given the complex pathological mechanisms of central nervous system diseases, it is necessary to further develop multifunctional fasudil derivatives or use them in combination with other drugs to achieve stronger efficacy and minimize adverse reactions in combating central nervous system diseases. https://pubmed.ncbi.nlm.nih.gov/23461757/

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Blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) dysfunction is a major feature of multiple sclerosis (MS). We evaluated the protective effect of the selective ROCK inhibitor fasudil in a guinea pig spinal cord-induced experimental autoimmune encephalomyelitis (EAE) model. Furthermore, we investigated the effect of fasudil on BBB and BSCB permeability. We found that fasudil partially alleviated EAE-induced damage by reducing BBB and BSCB permeability. These results provide a theoretical basis for developing selective Rho kinase inhibitors as novel therapies for MS. https://pubmed.ncbi.nlm.nih.gov/21978848/

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Background/Objective: The Rho-ROCK signaling pathway plays a crucial role in the activation of hepatic stellate cells (HSCs). We investigated the effects of the Rho kinase (ROCK) inhibitor fasudil hydrochloride hydrate (fasudil) on HSC cell growth, collagen production, and collagenase activity. Methods: Rat HSCs and human HSC-derived TWNT-4 cells were cultured to study stress fiber formation and α-smooth muscle actin (α-SMA) expression. Cell proliferation was detected using the BrdU incorporation assay, and apoptosis was detected using the TUNEL assay. The phosphorylation status of MAP kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun kinase (JNK), and p38 was detected using Western blotting. The production and gene expression of type I collagen, matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinases-1 (TIMP-1) were detected by enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR, respectively. Collagenase activity (active MMP-1) was also measured. Results: Fasudil (100 μM) inhibited cell spreading, stress fiber formation, and α-SMA expression, accompanied by cell growth inhibition, but did not induce apoptosis. Fasudil inhibited the phosphorylation of ERK1/2, JNK, and p38. Fasudil treatment inhibited the production and transcription of collagen and TIMP, stimulated the production and transcription of MMP-1, and enhanced collagenase activity. Conclusion: These findings indicate that fasudil not only inhibits cell 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

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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