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

体外活性：法舒地尔 (1-10 μM) 和羟基法舒地尔 (0.3-10 μM) 显着预防内皮素诱导的心肌细胞肥大。 Hydroxyfasudil 显着减弱血清素 (IC) 诱导的 SA 血管收缩（-7 +/- 1% vs. 2 +/- 1%，p < 0.01）。 I/R 后，冠状动脉 I/R 显着损害了冠状血管对乙酰胆碱的舒张作用（SA，p < 0.05；A，与 I/R 之前相比，p < 0.01），L-NMMA 进一步减少了血管舒张，而羟基法舒地尔完全保留了反应。激酶测定：Hydroxyfasudil HCl（也称为 HA1100 HCl）是 Fasudil 的代谢物，是一种有效的 Rho 激酶抑制剂和血管扩张剂。它作为 ROCK 抑制剂，对 ROCK1 和 ROCK2 的 IC50 值分别为 0.73 和 0.72 μM。 Hydroxyfasudil 可防止缺氧条件下内皮型 NO 合酶 (eNOS) 的下调。 Hydroxyfasudil 以浓度依赖性方式增加 eNOS mRNA 和蛋白质表达，在 10 μmol/L 时分别增加 1.9 倍和 1.6 倍。这与 eNOS 活性和 NO 产量分别增加 1.5 倍和 2.3 倍相关。细胞测定：用浓度不断增加的羟基法舒地尔（0.1 至 100 μmol/L）处理人血管内皮细胞，并测量 eNOS 表达和活性。hr> 法舒地尔和羟基法舒地尔抑制蛋白激酶的选择性[1]
测定法舒地尔及其活性代谢产物羟基法舒地尔对丝氨酸/苏氨酸激酶的抑制活性。与所研究的其他激酶相比，法舒地尔和羟基法舒地尔对ROCK1和ROCK2的选择性相对较高，其中羟基法舒迪尔（ROCK1和ROK2的IC50值分别为0.73和0.72μmol/L）的选择性略高于法舒地尔（ROK1和ROCK2的IC50值分别为1.2和0.82μmol/L。与ROCK相比，法舒地尔和羟基法舒地尔对蛋白激酶A（PKA）的IC50值分别高出约5倍和50倍（法舒地尔的IC50值为5.3和37μmol/L）。其他激酶，包括蛋白激酶C（PKC）α，对法舒地尔和羟基法舒地尔的IC50值>100μmol/L（数据未显示）。这些发现表明，本研究中使用的法舒地尔和羟基法舒地尔的浓度对ROCK抑制具有相对的选择性。

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羟基法舒地尔对内皮型一氧化氮合酶mRNA和蛋白水平的影响[1]
羟基法舒地尔治疗使HAEC、HUVEC和HSVEC中的eNOS mRNA分别增加到160±10%、156±10%和156±20%（n=3，P<0.05）（图1a）。羟基法舒地尔以浓度依赖的方式增加eNOS mRNA水平，EC50值为0.8±0.3μmol/L（图1b）。因此，羟基法舒地尔对eNOS mRNA的EC50值与羟基法舒迪尔对ROCK抑制的IC50值相当。抑制其他蛋白激酶如PKC、PKA和MLCK不影响eNOS mRNA水平（数据未显示），而ROCK显性负突变体（DN-Rho-K）的过表达与对照LacZ相比增加了eNOS mRNA的水平（图1c）。这些结果表明，羟基法舒地尔对ROCK的抑制导致eNOS mRNA表达的增加。 用羟基法舒地尔治疗以浓度依赖的方式增加eNOS蛋白表达（图1d）。同样，另一种ROCK抑制剂Y-27632以及DN-Rho-K的过表达增加了eNOS蛋白水平（图1e；结果未显示）。

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羟基法舒地尔对内皮型一氧化氮合酶活性和一氧化氮生成的影响[1]
以浓度依赖的方式，羟基法舒地尔治疗增加了eNOS活性并刺激了NO的产生（图2a）。这些结果表明，eNOS蛋白水平的增加与eNOS活性和NO产生的增加相关。

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羟基法舒地尔对内皮型一氧化氮合酶mRNA稳定性的影响[1]
细胞暴露于剪切应力（12达因/cm2）显著增加了eNOS启动子活性（即，诱导3.0倍；图2b）。然而，用羟基法舒地尔（0.1至100μmol/L）处理不影响eNOS启动子活性。用10μmol/L的羟基法舒地尔治疗后，eNOS mRNA的半衰期从13小时增加到16小时（n=4，P<0.05）（图2c）。这些结果表明，羟基法舒地尔增加eNOS表达很可能是在转录后水平介导的，涉及eNOS mRNA的稳定性。

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体外器官浴实验[3]
实验大鼠阴茎组织的收缩反应和松弛反应如表2所示。SHR组去甲肾上腺素诱导的收缩的Emax值（按横截面积归一化）明显高于对照组（续）。羟基法舒地尔治疗以剂量依赖的方式抑制了去甲肾上腺素诱导的这种高收缩性（表2）。然而，各组之间的EC50值没有显著差异。在Cont、SHR、Fas 3和Fas 10组中，100 mM KCl诱导的收缩力分别为0.108±0.010、0.116±0.016、0.101±0.011和0.122±0.017 mg/mm2，这些值在任何组之间都没有显著差异。从所有组获得的去甲肾上腺素预收缩阴茎组织的松弛以剂量依赖的方式产生。与对照组相比，SHR组的松弛明显减少（表2）。羟基法舒地尔治疗以剂量依赖的方式显著恢复了减弱的舒张作用。乙酰胆碱诱导的舒张的EC50值在任何一组之间都没有显著差异。

在控制条件下，冠状动脉内给予羟基法舒地尔（HF）会以剂量依赖性方式引起小动脉和小动脉显着的冠状血管舒张，从而导致CBF（冠状动脉血流量）增加。冠状动脉内羟基法舒地尔不会显着改变平均主动脉压或心率。用羟基法舒地尔预处理可显着减少缺血再灌注引起的心肌梗死面积，而羟基法舒地尔的这种有益作用会被 L-NMMA 显着减弱。 NO可能参与了羟基法舒地尔的心血管保护作用。羟基法舒地尔也可能有效治疗肺动脉高压。心力衰竭通过增加局部心肌血流量来保护心肌免受起搏引起的缺血。

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脑缺血对ROCK活性和内皮型一氧化氮合酶表达的影响[1]
为了确定ROCK抑制是否可以预防缺血性中风，给小鼠服用法舒地尔，在短暂MCA闭塞之前，法舒地尔在肝脏中代谢为活性代谢产物羟基法舒地尔。MCA闭塞后，脑缺血区的ROCK活性（通过肌球蛋白轻链磷酸酶肌球蛋白结合亚基（MYPT）的Thr696磷酸化测量）增加了2倍以上（图3a）。与赋形剂治疗相比，法舒地尔治疗使脑中ROCK活性降低了55%（P<0.05）。有趣的是，MCA闭塞与载体处理小鼠eNOS蛋白表达降低41%有关（图3b）。法舒地尔治疗的小鼠在MCA闭塞后的eNOS表达水平与对照组小鼠相同。

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腹腔注射羟基法舒地尔（10mg/kg）显著增加了平均和最大排尿量。羟基法舒地尔显著降低了最大逼尿肌压力，而收缩间期没有受到显著影响。给药0.1、0.3、1和3后 μM羟基法舒地尔，卡巴胆碱浓度反应曲线的最大收缩显著降低至74.5 ± 4.2%, 55.2 ± 5.6%, 29.4 ± 5.6%,21.6 ± 8.2% 分别表示控制值。 结论：  目前的研究结果表明，羟法舒地尔可能是CYP诱导的逼尿肌过度活动的一种新的治疗选择。[2]

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排尿行为监测[2]
如表所示 1,与对照组相比，单次腹腔注射羟基法舒地尔可显著增加平均和最大排尿量。两组之间的尿量或每日尿频没有差异。

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膀胱测压图[2]
典型的连续膀胱测量图如图所示 1. 服用羟基法舒地尔后，最大逼尿肌压力（Pdet max）降低。尿动力学结果如图所示 2 表明羟基法舒地尔显著降低了Pdet max，而在羟基法舒迪尔给药后，收缩间期（ICI）没有明显变化。

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羟基法舒地尔对卡巴胆碱CRC的影响[2]
羟基法舒地尔浓度范围为0.1至3 μM以浓度依赖的方式显著减弱了卡巴胆碱诱导的逼尿肌收缩（图。 3). 如表所示 2,服用0.1、0.3、1和3浓度的羟基法舒地尔后，Emax值显著降低 µM.在服用浓度为0.3、1和3的羟基法舒地尔后，pEC50值也显著降低 µM.

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高血压是勃起功能障碍的主要危险因素。尽管高血压性勃起功能障碍的病因是多因素的，目前尚不清楚，但Rho-Rho激酶途径是关键因素之一。为了研究给予Rho激酶抑制剂羟基法舒地尔是否可以预防自发性高血压大鼠（SHR）海绵体平滑肌中NO诱导的舒张功能障碍，12周龄的雄性SHR每天一次（3或10mg/kg，i.p.）治疗6周。Wistar大鼠和用赋形剂治疗的SHR用作年龄匹配的对照。测定阴茎cGMP浓度和Rho激酶活性，并通过去甲肾上腺素诱导的收缩和乙酰胆碱诱导的舒张的器官浴研究评估阴茎功能。分别通过定量实时PCR方法和免疫印迹分析研究eNOS的参与mRNA水平和eNOS和磷酸化eNOS的参加蛋白水平。SHR的阴茎组织中cGMP浓度显著降低，Rho激酶活性增加，去甲肾上腺素诱导的过度收缩和乙酰胆碱诱导的低松弛。羟基法舒地尔治疗以剂量依赖的方式显著改善了阴茎cGMP浓度降低、Rho激酶活性增加、去甲肾上腺素诱导的收缩增加和乙酰胆碱诱导的舒张减少。尽管各组之间eNOS的表达蛋白水平没有显著差异，但经羟基法舒地尔治疗后，eNOS mRNA的下调以及磷酸化的eNOS明显改善。我们的数据表明，羟基法舒地尔可能通过抑制SHR中Rho-Rho激酶通路和激活NO-eNOS通路来改善NO诱导的海绵体平滑肌舒张的高血压相关功能障碍。[3]

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实验大鼠的一般特征[3]
实验动物的一般特征如表1所示。与Wistar大鼠（对照组）相比，接受或不接受羟基法舒地尔治疗的SHR在18周龄时体重增加显著降低，阴茎重量显著降低。羟基法舒地尔治疗没有显著改变体重或阴茎重量。然而，除SHR和Fas 3组外，所有组的阴茎重量体重比相似。SHR、Fas 3和Fas 10组的心率明显低于对照组。SHR组的血压明显高于对照组。两种剂量的羟基法舒地尔治疗均能轻微但显著降低SHR的血压（表1）。

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大鼠阴茎cGMP浓度[3]
大鼠阴茎cGMP浓度如图1所示。SHR组的阴茎cGMP浓度明显低于对照组。羟基法舒地尔以剂量依赖的方式显著改善了阴茎cGMP含量的降低。

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海绵体Rho激酶活性的测定[3]
实验性海绵体中Rho激酶活性如表3所示。SHR组Rho激酶活性明显高于Wistar组。治疗羟基法舒地尔以剂量依赖的方式改善了SHR中Rho激酶活性的增加。低剂量羟基法舒地尔治疗略微但不显著降低了SHR海绵体中的Rho激酶活性（P=0.058）。对照组和Fas 10组海绵体Rho激酶活性没有显著差异。

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阴茎组织中eNOS mRNA的测定[3]
eNOS mRNA在阴茎组织中的表达如图2所示。SHR组eNOS mRNA水平明显低于对照组。高剂量羟基法舒地尔治疗显著阻止了海绵体eNOS mRNA水平表达的下调（图2）。

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海绵体eNOS和磷酸化eNOS表达的Western blot分析[3]
eNOS和磷酸化eNOS在实验大鼠海绵体中的典型表达及其总结数据如图3所示。各组eNOS的表达无显著差异。与eNOS表达相反，SHR组磷酸化eNOS的表达明显低于对照组。用高剂量羟基法舒地尔治疗后，磷酸化eNOS表达的下调得到了显著恢复（图3）。

Hydroxyfasudil HCl（也称为 HA1100 HCl）是 Fasudil 的代谢物，是一种强血管扩张剂和 Rho 激酶抑制剂。它抑制 ROCK，ROCK1 和 ROCK2 的 IC50 分别为 0.73 和 0.72 μM。在缺氧环境中，羟基法舒地尔抑制内皮一氧化氮合酶（eNOS）的下调。 Hydroxyfasudil 以浓度依赖性方式刺激 eNOS mRNA 和蛋白质表达；在 10 μmol/L 时，这分别导致 1.9 倍和 1.6 倍的增加。这相当于 NO 产生和 eNOS 活性分别增加 1.5 倍和 2.3 倍。

细胞培养[1]
如前所述培养人主动脉内皮细胞（HAEC）、人脐静脉内皮细胞（HUVEC）、人隐静脉内皮细胞和牛主动脉内皮细胞。如所述感染表达显性阴性ROCK（Ad-DN-Rho-K）或β-半乳糖苷酶（Ad-LacZ）的腺病毒载体。

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蛋白印迹[1]
如前所述进行蛋白质提取和免疫印迹。为了测量ROCK活性，组织在10%三氯乙酸中用丙酮处理，并用磷酸化Thr696 MYPT和MYPT多克隆抗体（Up-state和Santa Cruz Biotechnology）进行免疫印迹，并测定磷酸化Thr 696 MYPT/MYPT的比例。

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北方印迹[1]
如所述进行RNA提取和northern印迹。为了确定eNOS mRNA的稳定性，用RNA合成酶抑制剂5,6-二氯苯并咪唑核苷处理细胞。

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一氧化氮合酶活性测定[1]
通过使用NOS测定试剂盒测量[3H]-l-精氨酸转化为[3H]-1-瓜氨酸来测定NOS活性。

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一氧化氮生成的测量[1]
用硝酸分析仪通过化学发光法测定培养基中亚硝酸盐的积累。在2 mmol/L的NG-单甲基-L-精氨酸（L-NMMA）存在下测定非特异性值。

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内皮型一氧化氮合酶启动子活性测定[1]
使用LipofectAMINE2000试剂，将6孔板中的BAEC（90%融合）与4μg与萤光素酶报告基因17连接的[-1.8kb]eNOS启动子和0.5ng pRL-CMV载体共转染。采用Berthold L9501光度计，通过双荧光素酶报告系统测定萤光素酶活性。

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脑血流测量[1]
分别使用[14C]碘安替比林放射自显影和[14C]碘伏指示剂分级技术测量区域和绝对CBF。

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将羟基法舒地尔以不同浓度（0.1至100μmol/L）添加至人血管内皮细胞后，测量eNOS的表达和活性。

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

90.65

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)

配方 1 中的溶解度: ≥ 0.5 mg/mL (1.63 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 5.0 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 0.5 mg/mL (1.63 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 5.0 mg/mL 澄清 DMSO 储备液加入 900 μL 20% SBE-β-CD 生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 0.5 mg/mL (1.63 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 5.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。