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].

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Selectivity of Fasudil and Hydroxyfasudil for Inhibiting Protein Kinases [1]
The inhibitory activities of fasudil and its active metabolite hydroxyfasudil on serine/threonine kinases were determined. Compared with the other kinases studied, fasudil and hydroxyfasudil were relatively more selective for ROCK1 and ROCK2, with hydroxyfasudil (IC50 value for ROCK1 and ROCK2; 0.73 and 0.72 μmol/L, respectively) being slightly more selective than fasudil (IC50 value for ROCK1 and ROCK2; 1.2 and 0.82 μmol/L, respectively). Compared with ROCKs, the IC50 value for protein kinase A (PKA) was approximately 5-fold higher for fasudil and 50-fold higher for hydroxyfasudil (IC50 value of fasudil and hydroxyfasudil; 5.3 and 37 μmol/L, respectively). The other kinases, including protein kinase C (PKC)α, have IC50 values that were >100 μmol/L for fasudil and hydroxyfasudil (data not shown). These findings suggest that the concentrations of fasudil and hydroxyfasudil used in this study were relatively selective for ROCK inhibition.

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Effect of Hydroxyfasudil on Endothelial Nitric Oxide Synthase mRNA and Protein Level [1]
Treatment with hydroxyfasudil increased eNOS mRNA to 160±10%, 156±10%, and 156±20% in HAEC, HUVEC, and HSVEC, respectively (n=3, P<0.05) (Figure 1a). In a concentration-dependent manner, hydroxyfasudil increased eNOS mRNA levels, with an EC50 value of 0.8±0.3 μmol/L (Figure 1b). Thus, the EC50 value of hydroxyfasudil for eNOS mRNA is comparable with the IC50 value of hydroxyfasudil for ROCK inhibition. Inhibition of other protein kinases such as PKC, PKA, and MLCK did not affect eNOS mRNA levels (data not shown), whereas overexpression of a dominant-negative mutant of ROCK (DN-Rho-K) increased eNOS mRNA levels compared with that of control LacZ (Figure 1c). These results indicate that inhibition of ROCK by hydroxyfasudil leads to an increase in eNOS mRNA expression. Treatment with Hydroxyfasudil increased eNOS protein expression in a concentration-dependent manner (Figure 1d). Similarly, another ROCK inhibitor, Y-27632, as well as overexpression of DN-Rho-K increased eNOS protein levels (Figure 1e; results not shown).

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Effect of Hydroxyfasudil on Endothelial Nitric Oxide Synthase Activity and Nitric Oxide Production [1]
In a concentration-dependent manner, treatment with hydroxyfasudil increased eNOS activity and stimulated NO production (Figure 2a). These results indicate that the increase in eNOS protein levels correlated with increases in eNOS activity and NO production.

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Effect of Hydroxyfasudil on Endothelial Nitric Oxide Synthase mRNA Stability [1]
Exposure of the cells to shear stress (12 dyne/cm2) significantly increased eNOS promoter activity (ie, 3.0-fold induction; Figure 2b). However, treatment with hydroxyfasudil (0.1 to 100 μmol/L) did not affect eNOS promoter activity. Treatment with 10 μmol/L of hydroxyfasudil increased the half-life of eNOS mRNA from 13 to 16 hours (n=4, P<0.05) (Figure 2c). These results indicate that the increase in eNOS expression by hydroxyfasudil is most likely mediated at the posttranscriptional level involving eNOS mRNA stability.

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In vitro organ bath experiments [3]
The contractile responses and the relaxation responses in the experimental rat penile tissue are shown in Table 2. The Emax values for norepinephrine-induced contraction normalized by cross-sectional area in the SHR group were significantly higher than those in the control group (Cont). Treatment with Hydroxyfasudil inhibited this hypercontractility induced by norepinephrine in a dose dependent manner (Table 2). However, there were no significant differences of the EC50 values between any of the groups. The contractile forces induced by 100 mM KCl in the Cont, SHR, Fas 3 and Fas 10 groups were 0.108 ± 0.010, 0.116 ± 0.016, 0.101 ± 0.011 and 0.122 ± 0.017 mg/mm2, respectively, and there were no significant differences of these values between any of the groups. The relaxation of the norepinephrine precontracted penile tissues obtained from all groups was produced in a dose-dependent manner. The relaxation was markedly reduced in the SHR group in comparison with the control group (Table 2). Treatment with hydroxyfasudil significantly recovered the attenuated relaxation in a dose-dependent fashion. There were no significant differences of the EC50 values for acetylcholine-induced relaxation between any of the groups.

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].

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Effect of Cerebral Ischemia on ROCK Activity and Endothelial Nitric Oxide Synthase Expression [1]
To determine whether ROCK inhibition protects against ischemic stroke, mice were administered fasudil, which is metabolized to an active metabolite Hydroxyfasudil in the liver before transient MCA occlusion. After MCA occlusion, ROCK activity in the ischemic region of the brain, as measured by the Thr696 phosphorylation of myosin-binding subunit (MYPT) of myosin light chain phosphatase,11 was increased by more than 2-fold (Figure 3a). Treatment with fasudil decreased ROCK activity in the brain by 55% compared with vehicle treatment (P<0.05). Interestingly, MCA occlusion was associated with a 41% decrease in eNOS protein expression in vehicle-treated mice (Figure 3b). eNOS expression level in fasudil-treated mice after MCA occlusion was same to that in control mice.

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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 µM 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.[2]

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Monitoring of micturition behavior[2]
As shown in Table 1, a single intraperitoneal injection of Hydroxyfasudil induced significant increases in the average and the maximum micturition volumes as compared to control. There were no differences in urine production or frequency per day between the two groups.

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Cystometrogram [2]
Typical continuous cystometrograms are shown in Figure 1. A decreased maximum detrusor pressure (Pdet max) was seen after the Hydroxyfasudil administration. The urodynamic results shown in Figure 2 indicate that hydroxyfasudil significantly decreased the Pdet max, whereas no significant changes were seen in the intercontraction interval (ICI) after the hydroxyfasudil administration.

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Effects of Hydroxyfasudil on the CRC to carbachol [2]
Hydroxyfasudil concentrations ranging from 0.1 to 3 µM significantly attenuated the carbachol-induced contraction of the detrusor muscle in a concentration-dependent manner (Fig. 3). As seen in Table 2, there was a significant decrease in the Emax value after the administration of hydroxyfasudil at concentrations of 0.1, 0.3, 1, and 3 µM. The pEC50 value also decreased significantly after the administration of hydroxyfasudil at concentrations of 0.3, 1, and 3 µM.

---

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. [3]

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General features of the experimental rats [3]
The general features of the experimental animals are shown in Table 1. Compared to the Wistar rats (control group), SHRs with or without Hydroxyfasudil treatment showed significantly lower weight gain by the age of 18 weeks as well as significantly lower penile weight. Treatment with hydroxyfasudil did not change either body weight or penile weight significantly. However, penile weight body weight ratios were similar among all groups except for between SHR and Fas 3 groups. Heart rate was significantly lower in the SHR, Fas 3 and Fas 10 groups than that in the control group. Blood pressure was significantly higher in the SHR group than that in the control group. Treatment with both doses of hydroxyfasudil slightly but significantly decreased the blood pressure in the SHR (Table 1).

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Penile cGMP concentrations in the rat [3]
Penile cGMP concentrations in the rat are shown in Fig. 1. The penile cGMP concentrations in the SHR group were significantly smaller than those in the control group. The decreased penile cGMP contents were significantly ameliorated by treatment with Hydroxyfasudil in a dose-dependent manner.

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Measurement of Rho-kinase activity in the corpus cavernosum [3]
Rho-kinase activities in the experimental corpus cavernosum are shown in Table 3. The Rho-kinase activity in the SHR group was significantly higher than those in the Wistar group. The increased Rho-kinase activity in the SHR was ameliorated by treatment Hydroxyfasudil in a dose-dependent manner. Treatment with the low dose of hydroxyfasudil slightly but not significantly decreased the Rho-kinase activity in the corpus cavernosum in the SHR (P = 0.058). There were no significant differences of Rho-kinase activity in the corpus cavernosum between control and Fas 10 groups.

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Measurement of eNOS mRNAs in the penile tissues [3]
The expressions of eNOS mRNAs in the penile tissues are shown in Fig. 2. The eNOS mRNA levels in the SHR group were significantly lower than those in the control group. Treatment with the high dose of Hydroxyfasudilsignificantly prevented the down-regulation of the expressions of eNOS mRNA levels in the corpus cavernosum (Fig. 2).

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Western blot analysis of the expressions of eNOS and phosphorylated eNOS in the corpus cavernosum [3]
The typical expressions of eNOS and phosphorylated eNOS, and their summary data in the experimental rat corpus cavernosum are presented in Fig. 3. There were no significant differences of the expression of eNOS between any of the groups. In contrast to the expression of eNOS, the expression of phosphorylated eNOS in the SHR group was significantly lower than that in the control group. This down-regulation of the expression of phosphorylated eNOS was significantly recovered by the treatment with the high dose of Hydroxyfasudil (Fig. 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.

Cell Culture [1]
Human aortic endothelial cells (HAEC), human umbilical vein endothelial cells (HUVEC), human saphenous vein endothelial cells (HSVEC), and bovine aortic endothelial cells (BAEC) were cultured as described previously. Adenovirus vectors expressing dominant negative ROCK (Ad-DN-Rho-K) or β-galactosidase (Ad-LacZ) were infected as described.

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Western Blotting [1]
Protein extraction and immunoblotting were performed as described previously. For measurement of ROCK activity, tissues were treated in 10% trichloroacetic acid with acetone and immunoblotting with phospho-Thr696 MYPT and MYPT polyclonal antibodies (Up-state and Santa Cruz Biotechnology) was performed, and ratio of phospho-Thr696 MYPT and MYPT was determined.

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Northern Blotting [1]
RNA extraction and northern blotting were performed as described. To determine eNOS mRNA stability, cells were treated with the RNA synthetase inhibitor 5,6-dichlorobenzimidazole riboside.

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Nitric Oxide Synthase Activity Assay [1]
NOS activity was determined by measuring the conversion of [3H]-l-arginine to [3H]-l-citrulline using NOS assay kit.

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Measurements of Nitric Oxide Production [1]
Nitrite accumulation in the culture media was determined by chemiluminescence method with the use of nitric analyzer. Nonspecific value was determined in the presence of 2 mmol/L of NG-monomethyl-l-arginine (l-NMMA).

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Endothelial Nitric Oxide Synthase Promoter Activity Assay [1]
BAEC (90% confluent) in 6-well plates was cotransfected with 4 μg of the [-1.8 kb] eNOS promoter linked to the luciferase reporter gene17 and 0.5 ng of pRL-CMV vector with the use of LipofectAMINE2000 reagent. Luciferase activities were determined by dual-luciferase reporter assay system with the use of Berthold L9501 luminometer.

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Cerebral Blood Flow Measurement [1]
Regional and absolute CBF were measured using [14C]iodoantipyrine autoradiography and [14C]iodoamphetamine indicator fractionation technique, respectively.

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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].

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Model of Focal Cerebral Ischemia [1]
Mice were intraperitoneally administered with saline, fasudil (1, 3, or 10 mg/kg per day for 2 days) or Y-27632 (10 mg/kg per day for 2 days). Transient focal cerebral ischemia was induced in male wild-type or eNOS-/- mice described previously. Both mice were on a mixed background of C57BL/6 and SV129. Littermates were used as controls. Infarct areas and neurologic deficits were determined as described.

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Monitoring of micturition behavior [2]
Micturition behavior was studied after intraperitoneal injection of either Hydroxyfasudil (10 mg/kg) or a corresponding volume of saline. As per a previously described method, each rat was placed in a metabolic cage containing a urine collection funnel that was placed over an electronic balance.11 The balance was connected to a personal computer via a multiport controller and used to measure the cumulative weight of the collected urine. Every 150 s during a continuous 24-h period, the computer sampled and recorded the data for the micturition frequency and volumes. The micturition reflex parameters that were collected included: urine volume per micturition, maximal micturition volume, micturition frequency, and total urine output in the hydroxyfasudil- or vehicle-treated animals. Each monitoring session started at 18.00 hours. Prior to being placed in the metabolic cage at the start of each experimental period, the animals received either a single injection of hydroxyfasudil (10 mg/kg) dissolved in saline or an injection of saline without the inhibitor.

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Cystometrogram [2]
Cystometrograms were performed under urethane anesthesia (1.0 g/kg s.c.), as has been previously described.11 Cannulae (0.3 mm) were placed in the femoral vein for intravenous drug administration in all of the rats. A midline incision was made to expose the bladder. A 24-G catheter was inserted into the bladder dome and secured in place. A continuous cystometrogram was performed at a filling rate of 0.21 mL/min. Intravesical pressure was recorded by a personal computer via a bridge amplifier and a multiport controller. In order to record similar contraction waves, cystometrograms were performed for approximately 15 min after a post-surgical period of at least 30 min in each of the animals. After completion of the control phase recordings, the Hydroxyfasudil doses (0.2, 2 and 20 mg/kg) were injected intravenously.

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Effect of Hydroxyfasudil on the concentration-response in functional studies [2]
Functional studies were conducted in accordance with our previously reported methods.12 Briefly, razor blades were used to cut uniform longitudinal strips of the posterior wall of the bladder dome (1.5 × 5 mm). Muscle strips were mounted in organ baths (25 mL) containing Krebs–Henseleit solution and bubbled with 5% CO2 and 95% O2 (37°C). Force transducers were used to measure the changes in the tone of the strips, with data recorded on a Macintosh G3 personal computer using Chart version 3.6.9 and a PowerLab/16sp data acquisition system. Carbachol-induced contractile responses were cumulatively measured in the presence or absence of various concentrations of hydroxyfasudil (0.1, 0.3, 1, and 3 µM). Hydroxyfasudil was added 30 min prior to the carbachol administration. After completion of the concentration-response curve, the tissue was washed until the baseline force returned to its resting level. Following a 30-minute equilibration period, the next consecutive concentration-response curve was then constructed. The mean negative logarithmic value of the molar concentration of carbachol producing 50% of the maximum response (pEC50) and the mean maximum contraction (Emax) were then calculated. Data were normalized to the maximum response generated by the first curve, with the percentage of the maximal response expressed as the mean ± standard error of the mean (SEM).

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Six-week-old male SHR/Izm and Wistar rats were used. We used Wistar rats as normotensive age-matched controls. At the age of 12 weeks, the rats were divided into four groups (n = 8 in each group): an age-matched Wistar group treated with vehicle (saline) intraperitoneal injection (i.p.) (Cont), SHR treated with vehicle, i.p. (SHR), and SHR treated with Hydroxyfasudil at a daily dose of 3 or 10 mg/kg, i.p. once a day for 6 weeks (Fas 3 and Fas 10, respectively). Six weeks after the treatment with Hydroxyfasudil, blood pressure and heart rate were measured by the tail-cuff method without anesthesia. Subsequently, the rats were sacrificed with an overdose of sodium pentobarbital (60 mg, i.p.). The isolated penile tissues were used in organ bath experiments or frozen at −80 °C for measurements of tissue cGMP contents, and eNOS mRNA and protein levels and phosphorylated eNOS levels. In addition, real-time PCR and Western blot analyses were performed in the groups Cont, SHR and Fas 10.

mouse LD50 unreported 145 mg/kg United States Patent Document., #4678783

[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]

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In summary, our findings indicate acute cerebral ischemia is associated with enhanced ROCK activity and decreased eNOS expression. Inhibition of ROCK by fasudil/Hydroxyfasudil restores eNOS activity and protects against cerebral ischemia. The neuroprotective effects of ROCK inhibition are absent in eNOS-/- mice, indicating the obligatory role of endothelial-derived NO in mediating these beneficial effects. There are a couple of limitations in the present study. In our experimental condition, the animals need to be treated for 2 days before ischemia. Further study regarding the therapeutic window is needed. In addition, the animals lack cerebrovascular risk factors such as hypertension and diabetes. It remains to be determined whether fasudil shows neuroprotective effects against ischemic stroke in mouse models with hypertension or diabetes. Although the mechanism by which cerebral ischemia increases ROCK activity remains to be elucidated, inhibition of ROCK appears to be a promising target for improving endothelial function and decreasing the severity of ischemic strokes.[1]

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In conclusion, while the results of our study suggest that Hydroxyfasudil has an effect on bladder overactivity in the CYP-induced cystitis rat model, further experiments need to be performed to conclusively prove our speculations. [2]

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In conclusion, we demonstrated 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. However, the SHR is not homogeneous and is divided into several subtype, i.e., SHR/Izm, SHR/Hos, etc. Thus, in this study only one subtype of SHR/Izm was investigated. This may be a possible limitation of the study, and further investigation is required.[3]

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.)