ROCK2 (IC50 = 0.72 μM); ROCK1 (IC50 = 0.73 μM); PKA (IC50 = 37 μM)

The ROCK inhibitor hydroxyfasudil hydrochloride has IC50 values of 0.73 and 0.72 μM for ROCK1 and ROCK2, respectively. Additionally, hydroxyfasudil inhibits PKA less potently; its IC50 of 37 μM is 50 times higher than that of the ROCKs. eNOS mRNA levels are raised by hydroxyfasudil, which has an EC50 value of 0.8 ± 0.3 μM. In human aortic endothelial cells (HAEC), hydroxyfasudil (0-100 μM) concentration-dependently increases eNOS activity and stimulates NO production. At concentrations ranging from 0.1 to 100 μM, hydroxyfasudil (10 μM) has no effect on eNOS promoter activity, but it does lengthen the half-life of eNOS mRNA from 13 to 16 hours[1].

In SD rats, hydroxyfasudil (10 mg/kg, ip) dramatically raises the average and maximal voided volumes. Moreover, hydroxyfasudil dramatically lowers the maximal detrusor pressure[2]. In spontaneously hypertensive rats, hydroxyfasudil (3 mg/kg, ip) reduces hypercontractility brought on by norepinephrine (SHRs). Additionally, rats with decreased penile cGMP contents respond significantly better to Hydroxyfasudil (3, 10 mg/kg, ip)[3].

Hydroxyfasudil HCl (also called HA1100 HCl), a metabolite of Fasudil, is a strong vasodilator and Rho-kinase inhibitor. With IC50s of 0.73 and 0.72 μM for ROCK1 and ROCK2, respectively, it inhibits ROCK. In hypoxic environments, hydroxyfasudil inhibits the downregulation of endothelial NO synthase (eNOS). Hydroxyfasudil stimulates eNOS mRNA and protein expression in a concentration-dependent manner; at 10 μmol/L, this leads to a 1.9- and 1.6-fold increase, respectively. This corresponds to a 1.5- and 2.3-fold rise in NO production and eNOS activity, respectively.

The expression and activity of eNOS are measured after hydroxyfasudil is added to human vascular endothelial cells at varying concentrations (0.1 to 100 μmol/L).

[1]. Inhibition of Rho kinase (ROCK) leads to increased cerebral blood flow and stroke protection. Stroke. 2005 Oct;36(10):2251-7. Epub 2005 Sep 1.

[2]. Effect of the rho-kinase inhibitor hydroxyfasudil on bladder overactivity: an experimental rat model. Int J Urol. 2009 Oct;16(10):842-7.

[3]. Hydroxyfasudil ameliorates penile dysfunction in the male spontaneously hypertensive rat. Pharmacol Res. 2012 Oct;66(4):325-31.

Objectives: To investigate the effects of the rho-kinase inhibitor hydroxyfasudil on bladder overactivity in cyclophosphamide (CYP)-induced cystitis. Methods: Female Sprague-Dawley rats received a single intraperitoneal injection of CYP (200 mg/kg). Four days later, bladder function was evaluated by: (i) monitoring micturition behavior in metabolic cages between hydroxyfasudil- and vehicle-treated animals; (ii) measuring changes in continuous cystometrograms in response to intravenous hydroxyfasudil under anesthesia; and (iii) conducting a functional study examining the effect of hydroxyfasudil on the concentration-response curves to carbachol in bladder tissue strips. Results: Intraperitoneal injection of hydroxyfasudil (10 mg/kg) significantly increased both the average and maximal voided volumes. Hydroxyfasudil significantly decreased the maximal detrusor pressure, whereas the intercontraction interval was not significantly affected. After administration of 0.1, 0.3, 1, and 3 microM hydroxyfasudil, the maximal contraction of the concentration-response curves to carbachol was significantly reduced to 74.5 +/- 4.2%, 55.2 +/- 5.6%, 29.4 +/- 5.6%, and 21.6 +/- 8.2% of the control values, respectively. Conclusions: The present findings indicate that hydroxyfasudil might be a new treatment option for CYP-induced detrusor overactivity.[1]

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Hypertension represents a major risk factor for erectile dysfunction. Although the etiology of hypertension-induced erectile dysfunction is multifactorial and still unknown, Rho-Rho kinase pathway is one of the key factors. To investigate whether administration of hydroxyfasudil, a Rho kinase inhibitor could prevent dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle in the SHR (spontaneously hypertensive rat), twelve-week-old male SHRs were treated with hydroxyfasudil (3 or 10 mg/kg, i.p.) once a day for 6 weeks. Wistar rats and SHRs treatment with vehicle were used as age-matched controls. Penile cGMP concentrations and Rho kinase activities were determined, and penile function was estimated by organ bath studies with norepinephrine-induced contractions and acetylcholine-induced relaxations. The participation mRNA levels of eNOS and participation protein levels of eNOS and phosphorylated eNOS were investigated by quantitative real-time PCR methods and immunoblot analysis, respectively. The SHR showed significantly decreased cGMP concentrations, increased Rho kinase activities, norepinephrine-induced hyper-contractions, and acetylcholine-induced hypo-relaxations in the penile tissue. Treatment with hydroxyfasudil significantly improved the decreased penile cGMP concentrations, the increased Rho kinase activities, the increased norepinephrine-induced contractions, and the decreased acetylcholine-induced relaxation in a dose-dependent manner. Although there were no significant differences in expression protein levels of eNOS among any of the groups, down-regulation of eNOS mRNAs as well as phosphorylated eNOS were significantly ameliorated after treatment with hydroxyfasudil. Our data suggest that hydroxyfasudil ameliorates hypertension-associated dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle possibly via inhibition of the Rho-Rho kinase pathway and activation of NO-eNOS pathway in the SHR.[2]

C14H17N3O3S.HCL.XH2O

343.83

343.075

48.91; H, 5.28; Cl, 10.31; N, 12.22; O, 13.96; S, 9.32

155558-32-0

Hydroxyfasudil;105628-72-6

11371328

Typically exists as White to off-white solids at room temperature

>250ºC(dec.)

3.073

3

5

2

22

526

0

Cl[H].S(C1=C([H])C([H])=C([H])C2C(N([H])C([H])=C([H])C1=2)=O)(N1C([H])([H])C([H])([H])N([H])C([H])([H])C([H])([H])C1([H])[H])(=O)=O

XWWFOUVDVJGNNG-UHFFFAOYSA-N

InChI=1S/C14H17N3O3S.ClH/c18-14-12-3-1-4-13(11(12)5-7-16-14)21(19,20)17-9-2-6-15-8-10-17;/h1,3-5,7,15H,2,6,8-10H2,(H,16,18);1H

5-(1,4-diazepan-1-ylsulfonyl)-2H-isoquinolin-1-one;hydrochloride

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)

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.)