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

Hydroxyfasudil is a ROCK inhibitor, with IC50 values for ROCK1 and ROCK2 of 0.73 and 0.72 μM, respectively. Moreover, hydroxyfasudil inhibits PKA less potently than ROCKs, with an IC50 of 37 μM, which is 50 times higher. The eNOS mRNA levels are increased by hydroxyfasudil, which has an EC50 value of 0.8 ± 0.3 μM. Human aortic endothelial cells (HAEC) produce more NO and exhibit an increase in eNOS activity in response to hydroxyfasudil (0-100 μM) concentration. At concentrations of 0.1 to 100 μM, hydroxyfasudil (10 μM) has no effect on eNOS promoter activity, but it lengthens the half-life of eNOS mRNA from 13 to 16 hours[1].

Hydroxyfasudil (10 mg/kg, i.p.) amatically raises the average and maximal voided volumes in SD rats. Moreover, the maximal detrusor pressure is significantly reduced by hydroxyfasudil[2]. Among spontaneously hypertensive rats (SHRs), hydroxyfasudil (3 mg/kg, i.p.) suppresses norepinephrine-induced hypercontractility. Furthermore, Hydroxyfasudil (3, 10 mg/kg, i.p) significantly restores rats' lower penile cGMP contents[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).

The behavior of micturition is examined following an intraperitoneal injection of saline or Hydroxyfasudil (10 mg/kg). Every rat has its own metabolic cage with a urine collection funnel set above an electronic balance. The cumulative weight of the pee collected is measured by the balance, which is connected to a PC via a multiport controller. The computer takes a sample and stores the data for the micturition frequency and volumes every 150 s over the course of a 24-hour period. The micturition reflex parameters that are measured in animals treated with Hydroxyfasudil or a vehicle include total urine output, frequency of micturitions, maximal micturition volume, and urine volume per micturition. Every monitoring session began at eighteen minutes to the hour. The animals receive either an injection of saline without the inhibitor or a single injection of Hydroxyfasudil (10 mg/kg) dissolved in saline prior to being placed in the metabolic cage at the beginning of each experimental period[2].

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

[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

307.37

307.099

C, 54.71; H, 5.58; N, 13.67; O, 15.62; S, 10.43

105628-72-6

Hydroxyfasudil hydrochloride;155558-32-0

3064778

White to off-white solid powder

1.329g/cm3

613.7ºC at 760mmHg

325ºC

9.04E-16mmHg at 25°C

1.859

2

5

2

21

526

0

O=C1NC=CC2=C1C=CC=C2S(=O)(N3CCNCCC3)=O

ZAVGJDAFCZAWSZ-UHFFFAOYSA-N

InChI=1S/C14H17N3O3S/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)

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

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: ≥ 0.5 mg/mL (1.63 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 5.0 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: ≥ 0.5 mg/mL (1.63 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 5.0 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: ≥ 0.5 mg/mL (1.63 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 5.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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