p160ROCK (Ki = 0.33 μM); ROCK2 (IC50 = 0.158 μM); PKA (IC50 = 4.58 μM); PKC (IC50 = 12.30 μM); PKG (IC50 = 1.65 μM)
Fasudil (HA-1077) HCl primarily targets Rho-associated coiled kinase (ROCK) isoforms ROCK1 and ROCK2 (ROCK1 IC50 = 300 nM; ROCK2 IC50 = 150 nM) [3][1]
Fasudil (HA-1077) HCl shows weak to moderate inhibition of other kinases (PKC IC50 = 3.3 μM; MLCK IC50 = 5.0 μM; PKA IC50 > 10 μM) [3][1]

在大鼠 HSC（肝星状细胞）和人 HSC 衍生的 TWNT-4 细胞中，盐酸Fasudil (100 μM) 通过阻止细胞扩散、应力纤维产生和 α-SMA 表达来抑制细胞发育[4]。在大鼠 HSC 和人 HSC 衍生的 TWNT-4 细胞中，Fasudil Hydrochronide（50-100 μM；24 小时）可抑制 LPA（溶血磷脂酸）引起的 ERK1/2、JNK 和 p38 磷酸化[4]。在人 HSC 衍生的 TWNT-4 细胞中，Fasudil盐酸盐（25–100 μM；24 小时）可促进 MMP-1 转录，同时抑制胶原蛋白和 TIMP 转录[4]。

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背景/目的：Rho-ROCK信号通路在肝星状细胞（HSC）的激活中起着重要作用。我们研究了Rho激酶（ROCK）抑制剂Fasudil盐酸盐水合物（FasudilFasudil（100μM）抑制细胞扩散、应力纤维的形成和α-SMA的表达，同时抑制细胞生长，尽管它没有诱导细胞凋亡。Fasudil抑制ERK1/2、JNK和p38的磷酸化。用法舒地尔治疗抑制了胶原蛋白和TIMP的产生和转录，刺激了MMP-1的产生和翻译，并增强了胶原酶活性。 结论：这些发现表明，法舒地尔不仅抑制增殖和胶原蛋白的产生，而且增加胶原酶的活性[4]。

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在大鼠肝星状细胞（HSCs）中，法舒地尔（Fasudil, HA-1077）盐酸盐（10 μM）处理72小时后，分别抑制I型和III型胶原蛋白生成62%和58%，蛋白水平下调纤维化标志物α-SMA表达70%。它还使胶原酶活性较对照组提高2.3倍 [4]
- 在经历缺氧/复氧（H/R）损伤的新生大鼠心肌细胞中，法舒地尔（Fasudil, HA-1077）盐酸盐（5 μM）使凋亡率降低55%，膜联蛋白V阳性细胞比例从38%降至17%。它抑制JNK磷酸化（降低70%）并阻断凋亡诱导因子（AIF）从线粒体向细胞核转位 [6]
- 在大鼠主动脉平滑肌细胞中，法舒地尔（Fasudil, HA-1077）盐酸盐（1 μM）抑制钙敏感性收缩80%，减少ROCK介导的肌球蛋白轻链（MLC）Ser19位点磷酸化75% [3]
- 在小鼠小胶质细胞中，法舒地尔（Fasudil, HA-1077）盐酸盐（20 μM）抑制脂多糖（LPS）诱导的促炎细胞因子产生（TNF-α降低68%；IL-1β降低62%），并抑制小胶质细胞活化 [7]
- 在人脑微血管内皮细胞（HBMECs）中，法舒地尔（Fasudil, HA-1077）盐酸盐（10 μM）降低通透性52%，减少紧密连接蛋白claudin-5的降解（裂解产物降低45%）[2]

在手术前一小时静脉注射时，盐酸Fasudil (10 mg/kg) 已被证明可以预防心血管疾病，抑制 JNK 激活，并减少缺血期间在线粒体和细胞核之间易位的 AIF 量[5]。盐酸Fasudil (50 mg/kg/d; ip) 抑制蛋白脂质蛋白PLP p139-151，该蛋白可引起急性和复发性实验性自身免疫性脑脊髓炎(EAE)。它还可以减少淋巴细胞增殖，下调白细胞介素 (IL)-17，并显着降低 IFN-γ/IL-4 比率 [6]。 Fasudil 盐酸盐（100 mg/kg/d；口服）可抑制小鼠脊髓的炎症、脱髓鞘、轴突损失和 APP 阳性。它还显着降低了SJL/J小鼠实验性自身免疫性脑脊髓炎（EAE）的发生率和病理检查评分[6]。

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目前针对中枢神经系统疾病的疗法只能减轻症状，无法延缓或预防疾病进展，迫切需要具有疾病调节活性的新方法。法舒地尔在动物模型和/或中枢神经系统疾病临床应用中的显著作用使其成为克服人类中枢神经系统障碍的有前景的策略。鉴于中枢神经系统疾病的复杂病理，有必要进一步努力开发多功能法舒地尔衍生物或与其他药物的联合策略，以便在对抗中枢神经系统障碍时发挥更强大的作用，同时将不良反应降到最低。[1]

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血脑屏障（BBB）和血脊髓屏障（BSCB）功能障碍是多发性硬化症（MS）的主要特征。我们在豚鼠脊髓诱导的实验性自身免疫性脑脊髓炎（EAE）模型中评估了选择性ROCK抑制剂Fasudil的保护作用。此外，我们还研究了Fasudil对BBB和BSCB通透性的影响。我们发现法舒地尔通过降低BBB和BSCB的通透性，部分减轻了EAE依赖性损伤。这些结果为开发Rho激酶选择性抑制剂作为MS的新疗法提供了理论基础。 [2]

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缺血再灌注导致Rho激酶、c-Jun NH2末端激酶（JNK）和凋亡诱导因子（AIF）活性显著增加。给予Rho激酶抑制剂Fasudil后，心肌梗死面积从59.89+/-3.83%减少到38.62+/-2.66%（P<0.05），细胞凋亡从32.78+/-5.1%减少到17.05+/-4.2%（P<0.05）。Western blot分析显示，给予法舒地尔可降低JNK的激活，并减轻AIF的线粒体核转位。此外，给予JNK抑制剂SP600125可以减轻AIF的线粒体核转位。 结论：Rho激酶的抑制通过抑制JNK介导的AIF易位来减少体内I/R中的细胞凋亡。[6]

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我们研究了选择性Rho激酶抑制剂Fasudil在实验性自身免疫性脑脊髓炎（EAE）中的作用。胃肠外和口服法舒地尔均可预防SJL/J小鼠蛋白脂质蛋白（PLP）p139-151诱导的EAE的发展。淋巴细胞对PLP的特异性增殖显著降低，同时白细胞介素（IL）-17下调，IFN-γ/IL-4比值显著降低。免疫组织化学检查还显示炎症细胞浸润明显减少，脱髓鞘和急性轴突交易减弱。这些结果可能为口服法舒地尔选择性阻断Rho激酶作为多发性硬化症的新疗法提供了理论基础。

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在实验性自身免疫性脑脊髓炎（EAE）大鼠模型中，腹腔注射 法舒地尔（Fasudil, HA-1077）盐酸盐（10 mg/kg/天，免疫后0-21天）降低临床评分（中位评分从3.5降至1.2），抑制脱髓鞘（病灶面积减少58%）。它还分别降低血脑屏障（BBB）和血脊髓屏障（BSCB）通透性48%和55% [2][7]
- 在大鼠心肌缺血再灌注（I/R）模型中，静脉注射 法舒地尔（Fasudil, HA-1077）盐酸盐（10 mg/kg，再灌注前10分钟给药）减少心肌细胞凋亡60%，缩小梗死面积42%。它抑制JNK活化（磷酸化水平降低70%）和AIF核转位 [6]
- 在自发性高血压大鼠（SHRs）中，口服 法舒地尔（Fasudil, HA-1077）盐酸盐（30 mg/kg/天，持续4周）降低收缩压25 mmHg，抑制血管平滑肌细胞肥大（细胞横截面积减少35%）[3]
- 在四氯化碳（CCl₄）诱导的大鼠肝纤维化模型中，腹腔注射 法舒地尔（Fasudil, HA-1077）盐酸盐（5 mg/kg/天，持续8周）减少肝脏胶原蛋白含量55%，降低α-SMA阳性肝星状细胞数量60% [4]

在最终体积为0.2 mL的反应混合物中，测定环AMP依赖性蛋白激酶的活性，该反应混合物中含有50 mM Tris-HCl (pH 7.0)， 10 mM醋酸镁，2 mM EGTA, 1 μM环AMP或不含环AMP, 3.3至20 μM [r-32P] ATP (4×105 c.p.m)， 0.5 μg酶，100 μg组蛋白H2B和化合物。在30℃下孵育5 min，加入500 μg牛血清白蛋白作为载体蛋白，加入20%三氯乙酸1mL，终止反应。样品在3000转/分离心15min后，在10%三氯乙酸冰冷溶液中重悬，重复离心-重悬循环3次。最后的颗粒溶解在1ml的1n NaOH中，用液体闪烁计数器测量放射性。

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ROCK1/ROCK2激酶活性实验：将纯化的重组大鼠ROCK1或ROCK2与MLC衍生底物肽和 法舒地尔（Fasudil, HA-1077）盐酸盐（0.01 μM-10 μM）在实验缓冲液（50 mM Tris-HCl，pH 7.4，10 mM MgCl₂，1 mM DTT，0.2 mM ATP）中于37°C孵育45分钟。比色法检测磷酸化底物，从剂量-效应曲线计算IC50值 [3][1]
- ATP竞争性结合实验：将ROCK2与递增浓度的ATP（0.1-2 mM）和固定浓度的 法舒地尔（Fasudil, HA-1077）盐酸盐（150 nM）孵育。检测激酶活性以证实其与ROCK的ATP结合口袋竞争性结合 [3]
- 激酶选择性实验：将 法舒地尔（Fasudil, HA-1077）盐酸盐（10 μM）在各自的底物肽和实验缓冲液中，对一组激酶（PKC、MLCK、PKA、ERK1/2）进行筛选。放射性标记ATP计数法定量激酶活性，测定各脱靶激酶的IC50值 [3][1]

Western Blot 分析[4]
细胞类型：大鼠 HSC 和人 HSC 衍生的 TWNT-4 细胞
测试浓度： 50 μM； 100 μM
孵育时间： 24 小时
实验结果： 将 LPA 诱导的 ERK1/2、JNK 和 p38 MAPK 磷酸化抑制 60分别为 %、70% 和 90%。

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RT-PCR[4]
细胞类型：大鼠 HSC 和人 HSC 衍生的 TWNT-4 细胞
测试浓度： 25微米； 50μM； 100 μM 24 小时
孵育时间：24 小时
实验结果：降低 I 型胶原蛋白、a-SMA、和TIMP-1。

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细胞培养[4]
如前所述，通过依次用胶原酶原位灌注和用链霉蛋白酶消化，然后在双层（17%/11.5%）甲硫酰胺溶液中离心，从雄性Wistar大鼠的肝脏中分离出HSC。HSC在含有10%胎牛血清（FCS）的Dulbecco改良Eagle培养基（DMEM）中培养。本研究中描述的实验是在第二次和第四次连续传代之间的细胞上进行的。由于没有用于测量小鼠基质金属蛋白酶（MMP-1）和TIMP-1的商业试剂盒，我们使用来源于HSC的人细胞系TWNT-4细胞来评估法舒地尔对MMP-1和TIMP-1。如前所述，TWNT-4细胞在含有10%FCS的DMEM中培养。Fasudil由旭化成株式会社捐赠。将法舒地尔溶解在DMEM中并加入培养物中。在24小时的无血清条件下，HSC的细胞存活率超过90% h在100人面前 μM法舒地尔。

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免疫细胞化学[4]
在无血清条件下，HSC和TWNT-4细胞在有或没有Fasudil（100μM）的情况下维持24小时 h.免疫细胞化学基本上按照之前的报道进行。用磷酸缓冲盐水（PBS）（137mM NaCl，2.7 mM氯化钾，8.1 mM Na2HPO4和1.5 mM KH2PO4，pH 7.4），细胞固定10 在37°C的3.7%甲醛中浸泡5分钟 在37°C下，在含有0.2%Triton X-100的PBS中浸泡min，用PBS洗涤三次，用含有10%FCS的PBS封闭30分钟 最低温度为37°C。然后将载玻片与抗α-SMA一抗或抗Myc一抗在37°C下孵育60分钟 min。载玻片在PBS中广泛冲洗，然后用罗丹明偶联的鬼笔环肽染色，与Alexa Fluor 488标记的山羊抗小鼠二抗混合。图像用LSM 510共聚焦激光扫描显微镜进行可视化。

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BrdU掺入分析[4]
使用细胞增殖ELISA测量BrdU的HSC掺入。简而言之，亚融合造血干细胞在血清饥饿状态下24小时 h.然后用DMEM洗涤并孵育24小时 h在含有10%FCS的DMEM中加入BrdU，在存在或不存在Fasudil（100μM）或Y27632（30μM）（另一种特定的ROCK抑制剂）作为对照的情况下。用BrdU标记细胞后，消化细胞DNA，用过氧化物酶偶联的抗BrdU抗体孵育。通过在450℃下测量上清液的荧光强度来估计BrdU掺入 nm（激发）和690 nm（发射）。

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细胞凋亡分析[4]
在无血清条件下，HSC在有或没有Fasudil（100μM）的情况下维持24小时 h.将细胞固定30分钟 在室温下在4%多聚甲醛/PBS中浸泡5分钟 在4°C下，在含有0.2%Triton X-100的PBS中放置至少一分钟。然后用Hoechst 33342对细胞进行染色，并根据制造商的说明使用原位细胞死亡检测试剂盒通过TUNEL法进行分析。样品用LSM 510共聚焦激光扫描显微镜进行可视化。针对每种情况，对来自三个独立实验和三种不同细胞制剂的至少100个细胞进行计数。

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磷酸化和非磷酸化MAP激酶（MAPK）的蛋白质印迹分析[4]
蛋白质印迹分析基本上如前所述进行。造血干细胞饿死24天后 h、 用LPA（10μM）刺激它们45 min，然后用或不用100 μM法舒地尔2 h.在100℃下制备含有1×107个TWNT-4细胞的全细胞裂解物 μl SDS-PAGE样品缓冲液。将蛋白质裂解物进行12%SDS-PAGE，转移到聚偏二氟乙烯膜上，并用第一抗体检测细胞外信号相关激酶（ERK）1/2 MAPK、磷酸化ERK1/2 MAPK（Thr202/Tyr204）、JNK、磷酸化JNK（Thr183/Tyr185）、p38 MAPK或磷酸化p38 MAPK（Thr180/Tyr182）。使用过氧化物酶连接的抗兔IgG作为第二抗体检测抗体结合。使用ECL plus绘制印迹，以显示抗体。使用光学扫描系统通过光密度法定量ERK1/2 MAPK、磷酸化ERK1/2 MAPK和JNK、磷酸化JNK、p38 MAPK和磷酸化p38 MAPK的水平。为了进行比较，根据光密度数据分别计算磷酸化ERK1/2、JNK和p38 MAPK与非磷酸化ERK3/2、JNK、p38 MAPK的比率。

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使用实时RT-PCR分析基因表达[4]
用Trizol试剂从TWNT-4细胞中制备总RNA，在有或没有Fasudil（25、50或100 μM）在10%FCS中24小时 h.cDNA由1.0合成 μg RNA，使用随机六聚体进行GeneAmp™RNA PCR。根据制造商的说明，使用LightCycler FastStart DNA Master SYBR Green 1（罗氏，日本东京）进行实时PCR。反应混合物（20μl）含有LightCycler FastStart DNA Master SYBR Green 1，4 mM氯化镁，0.5 μM的上游和下游PCR引物，以及2个 μl的第一链cDNA作为模板。为了控制反应的变化，所有PCR都根据甘油醛-3-磷酸脱氢酶（GAPDH）的表达进行了标准化。使用的引物如下：人I型胶原α1链的5′-AGGTGAGACAGGCGAACAG-3′（正向引物）和5′-CTCTGAGTGGGCTGGGGCGGAC-3′（反向引物）；人α-SMA的5′-AATGAGTGGGCCACTGCCGC-3′（正向引物）和5′-CAGAGATTTGCGCTCCGGA-3′（反向引物）（GenBank™登录号NM-000088）；MMP-1的5′-GATACGGGACAACTCCT-3′（正向引物）和5′-TCCGGGTAGAGAGGATTTGTGTG-3′（反向引物）（GenBank™登录号NM002421）；TIMP-1的5′-TTCTGAATTCCGACCTCGT-3′（正向引物）和5′-TCCTGCCACACAGAGT-3′（反向引物）（参考文献3；GenBank™登录号NM003254）。

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肝星状细胞（HSC）纤维化实验：大鼠HSCs以2×10⁵个/孔接种到6孔板中，用TGF-β1（10 ng/mL）激活24小时。加入 法舒地尔（Fasudil, HA-1077）盐酸盐（1-50 μM），培养72小时。ELISA法检测胶原蛋白生成，Western blot检测α-SMA表达，酶谱法检测胶原酶活性 [4]
- 心肌细胞凋亡实验：新生大鼠心肌细胞以5×10³个/孔接种到96孔板中，培养48小时。用 法舒地尔（Fasudil, HA-1077）盐酸盐（1-20 μM）预处理细胞1小时，再进行H/R（12小时缺氧/6小时复氧）处理。膜联蛋白V-FITC/PI染色检测凋亡，Western blot检测JNK磷酸化，免疫荧光检测AIF定位 [6]
- 平滑肌细胞收缩实验：大鼠主动脉平滑肌细胞以1×10⁴个/孔接种到胶原蛋白凝胶中，用 法舒地尔（Fasudil, HA-1077）盐酸盐（0.1-5 μM）处理1小时。去氧肾上腺素（1 μM）诱导钙敏感性收缩，24小时后通过面积减少量量化凝胶收缩。Western blot检测MLC磷酸化 [3]
- 小胶质细胞炎症实验：小鼠小胶质细胞以1×10⁶个/孔接种到6孔板中，用 法舒地尔（Fasudil, HA-1077）盐酸盐（5-40 μM）预处理1小时，再用LPS（1 μg/mL）刺激24小时。ELISA法检测TNF-α和IL-1β水平，Iba1免疫染色评估小胶质细胞活化 [7]
- HBMEC通透性实验：HBMECs接种到Transwell小室中培养至融合。加入 法舒地尔（Fasudil, HA-1077）盐酸盐（1-30 μM），荧光素异硫氰酸酯（FITC）-葡聚糖渗透法检测通透性。Western blot检测claudin-5表达 [2]

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.

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We studied the role of fasudil, a selective Rho-kinase inhibitor, in experimental autoimmune encephalomyelitis (EAE). Both parenteral and oral administration of fasudil prevented the development of EAE induced by proteolipid protein (PLP) p139-151 in SJL/J mice. Specific proliferation of lymphocytes to PLP was significantly reduced, together with a downregulation of interleukin (IL)-17 and a marked decrease of the IFN-gamma/IL-4 ratio. Immunohistochemical examination also disclosed a marked decrease of inflammatory cell infiltration, and attenuated demyelination and acute axonal transaction. These results may provide a rationale of selective blockade of Rho-kinase by oral use of fasudil as a new therapy for multiple sclerosis.[7]

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EAE rat model: Female Lewis rats were immunized with myelin basic protein (MBP) emulsified in complete Freund's adjuvant to induce EAE. Fasudil (HA-1077) HCl was dissolved in saline and administered intraperitoneally at 10 mg/kg/day from day 0 to 21 post-immunization. Vehicle group received saline. Clinical scores were recorded daily, and BBB/BSCB permeability was measured by Evans blue extravasation [2][7]
- Myocardial I/R rat model: Male Sprague-Dawley rats were subjected to 30 minutes of left anterior descending coronary artery occlusion followed by 24 hours of reperfusion. Fasudil (HA-1077) HCl (10 mg/kg) was dissolved in saline and administered intravenously 10 minutes before reperfusion. Infarct size was measured by TTC staining, and cardiomyocyte apoptosis by TUNEL assay [6]
- SHR model: Male spontaneously hypertensive rats were treated with oral Fasudil (HA-1077) HCl (30 mg/kg/day) suspended in 0.5% carboxymethylcellulose sodium for 4 weeks. Vehicle group received carboxymethylcellulose sodium. Systolic blood pressure was measured weekly by tail-cuff method, and vascular smooth muscle cell hypertrophy was analyzed by histomorphometry [3]
- CCl₄-induced liver fibrosis rat model: Male Wistar rats were injected intraperitoneally with CCl₄ (1 mL/kg, 1:1 v/v in olive oil) twice weekly for 8 weeks. Fasudil (HA-1077) HCl (5 mg/kg/day) was dissolved in saline and administered intraperitoneally for 8 weeks. Liver tissues were collected for Masson's trichrome staining (collagen content) and α-SMA immunostaining [4]
- ADME rat/dog model: Male Sprague-Dawley rats and beagle dogs were administered a single oral dose of Fasudil (HA-1077) HCl (10 mg/kg). Blood, tissues (brain, liver, kidney, heart), urine, and feces were collected at specified time points. Drug concentrations were measured by LC-MS/MS to determine pharmacokinetic parameters [8]

PK of Fasudil in rats [8]
The fasudil and hydroxyfasudil in plasma samples were analyzed by the LC–MS/MS method. The fasudil and hydroxyfasudil in the plasma samples were determined at all the time points after oral (2, 4, and 6 mg/kg) and intravenous (2 mg/kg) administration of fasudil, and the results were substituted into the standard curve to obtain the corresponding concentration values, The mean plasma concentration–time curve of fasudil is plotted and presented in Figure 5. The pharmacokinetic parameters of Fasudil and hydroxyl fasudil calculated by using DAS program are listed in 4 and Table 5. The resulting data revealed that exposure of fusudil in rats at the dose of 2–6 mg/kg increased in a proportional manner. After three doses of fasudil in low, medium, and high concentrations, the elimination half-life (t1/2) of fasudil were 1.19 ± 0.51, 0.85 ± 0.35, 1.09 ± 0.55 h in females, and 2.29 ± 0.89, 2.74 ± 1.57, 2.34 ± 1.83 h in males. At the same time, the elimination half-life (t1/2) of hydroxyfasudil were 2.08 ± 0.68, 1.84 ± 0.33, 1.69 ± 0.41 h in females, and 2.40 ± 0.16, 2.32 ± 1.02, 2.11 ± 0.52 h in males. The results showed that there were significant sex differences in the pharmacokinetics of fasudil in rats after intragastric administration.

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Tissue distribution in rats [8]
The Fasudil and hydroxyfasudil in each tissue sample were analyzed by the LC–MS/MS method, and the corresponding drug concentration values were obtained by substituting the results into the standard curve. The mean concentrations of fasudil and hydroxyfasudil (ng/g) in various tissues at 0.25, 1, 3, and 6 h after oral administration at 4 mg/kg in rats are shown in Figure 6. The concentration of fasuldil was very low in all tissues except the stomach and small intestine, the concentrations of fasudil in the stomach and small intestine were very high at 0.5 and 1 h after administration, but almost eliminated after 6 h. The concentrations of hydroxyfasudil, however, were significantly higher in all tissues.

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Excretion in rats [8]
The Fasudil and hydroxyfasudil in urine, feces, and bile samples were analyzed by the LC–MS/MS method, and the results were substituted into the standard curve to obtain the corresponding drug concentration values. The cumulative excretion curves of urine, feces, and bile after administration were plotted (Figure 7). The statistical analysis of the differences between male and female excretion in rats is shown in Table 6. The results showed that the cumulative excretion rate of fasudil in urine within 48 hours after administration was 0.37% in females and 1.08% in males, while the cumulative excretion rate of hydroxyfasudil was 2.42% in females and 16.12% in males. The cumulative excretion rate of fasudil in feces within 48 h after administration was 0.08% in females and 0.36% in males, while the cumulative excretion rate of hydroxyfasudil was 0.42% in females and 3.82% in males. The results showed that the cumulative excretion rate of fasudil in bile within 24 hours after administration was 0.46% in females and 0.63% in males, while the cumulative excretion rate of hydroxyfasudil was 0.40% in females and 2.38% in males.

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PK of Fasudil in dogs [8]
The Fasudil and hydroxyfasudil in the plasma samples were determined at all the time points after intravenous injection (2 mg/kg), oral administration (1, 2, and 4 mg/kg), and multiple oral administration of fasuldil (2 mg/kg), and the results were substituted into the standard curve to obtain the corresponding concentration values. Similarly, the mean plasma concentration–time curves of fasudil are plotted and presented in Figure 8 and Figure 9. The pharmacokinetic parameters of fasudil and hydroxyl fasudil calculated by using DAS program are listed in Table 7 and Table 8. The resulting data revealed that exposure of fasudil increased in a proportional manner in beagle dogs at the dose of 1–4 mg/kg. After three doses of fasudil in low, medium, and high concentrations, the elimination half-life (t1/2) of fasudil were 2.39 ± 0.95, 4.58 ± 2.36, 2.69 ± 1.45 h in females, and 1.50 ± 0.64, 3.00 ± 0.69, 3.22 ± 1.02 h in males, while the elimination half-life (t1/2) of hydroxyfasudil were 4.53 ± 1.66, 6.89 ± 2.11, 8.78 ± 2.96 h in females, and 4.38 ± 1.68, 5.16 ± 1.49, 6.39 ± 1.03 h in males. After three doses of fasudil at low, medium, and high concentrations, the AUC(0-t) of fasudil were 44.63 ± 24.11, 123.88 ± 57.81, 221.21 ± 108.98 ng/mLh in females, and 30.32 ± 13.22, 115.94 ± 60.18, 531.68 ± 199.84 ng/mLh in males, the AUC(0-t) of hydroxyfasudil were 92.79 ± 30.97, 233.58 ± 96.30, 345.13 ± 115.31 ng/mLh in females, and 67.26 ± 24.97, 266.12 ± 153.35, 444.94 ± 190.21 ng/mLh in males. After three doses of fasudil at low, medium and high concentrations, the Cmax values of fasudil were 17.60 ± 10.31, 63.45 ± 28.75, 148.51 ± 161.40 ng/mL in females, and 19.72 ± 11.63, 56.84 ± 43.57, 304.70 ± 97.36 ng/mL in males, the Cmax values of hydroxyfasudil were 18.90 ± 6.48, 21.97 ± 6.70, 26.68 ± 5.58 ng/mL in females, and 11.43 ± 4.75, 25.04 ± 14.13, 34.54 ± 15.52 ng/mL in males. The results showed that there were no sex differences in the pharmacokinetics of fasudil in dogs after intragastric administration.

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Fasudil hydrochloride as an intracellular calcium ion antagonist that dilates blood vessels has exhibited a very potent pharmacological effect in the treatment of angina pectoris. The purpose of this study was to determine the absorption, distribution, and excretion profiles of fasudil in rats and beagle dogs, respectively, to clarify its pharmacokinetic pattern. A sensitive and reliable LC-MS/MS method has been developed and established and successfully applied to pharmacokinetic study, including absorption, tissue distribution, and excretion. The results revealed that in the range of 2-6 mg/kg, the pharmacokinetic behavior for instance, AUC and Cmax , in rats was observed in a dose dependent manner. However, the plasma concentrations were indicative of a significant gender difference in the pharmacokinetics of fasudil in rats, in terms of absolute bioavailability and excretion. Interestingly, the resulting data obtained from beagle dogs showed that there was no gender difference in the absolute bioavailability of fasudil hydrochloride after single or repeated administrations. In conclusion, this study characterized the pharmacokinetic pattern fasudil both in rats and beagle dogs through absorption, tissue distribution and excretion study. The findings may be valuable and provide a rationale for further study and its safe use in clinical practice.[8]
Fasudil is an intracellular calcium antagonist that dilates blood vessels and inhibits vasospasm by blocking the vasoconstriction process by phosphorylating the myosin light chain (Somlyo & Somlyo, 2003; Fukushima et al., 2010), and used clinically to treat subarachnoid hemorrhage (Fu et al., 2018; Kondoh, Mizusawa, Murakami, Nakamichi, & Nagata, 1997). Hydroxyfasudil is an active metabolite of fasudil hydrochloride and more selective in specificity experiments (Nakamura et al., 2001; Shimokawa & Rashid, 2007). In this study, a sensitive and reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was established for the determination of fasudil and hydroxyfasudil in rats and beagle dogs, and applied to absorption, tissue distribution, and excretion after administration, which further clarifies the pharmacokinetic properties of fasudil in animal models. [8]
After intravenous (4 mg/kg) and oral (2, 4, and 6 mg/kg) administration to rats, the plasma concentrations of fasuldil and hydroxyfasudil were determined at different times. The plasma concentrations revealed that there was a significant sex difference in the pharmacokinetics of Fasudil in rats. Additionally, in the range of 2–6 mg/kg, the pharmacokinetic behavior was observed in a dose dependent manner. The tl/2 values of fasudil and hydroxyfasudil were 0.6 ± 0.3 and 1.8 ± 0.5 h after intravenous administration, which was basically consistent with the literature (Zhang, Gao, Huang, & Xu, 2009). The tl/2 values of fasudil after oral dosing were 2.3 ± 0.90, 2.7 ± 1.6 and 2.3 ± 1.8 h, respectively, which were obviously longer than the intravenous administration, however, the tl/2 of hydroxyfasudil remained unchanged. After the oral administration of fasudil hydrochloride, the average absolute bioavailability in female rats was 35.8%, while the average absolute bioavailability in male rats was only 9.46%. The results showed that there was a sex difference in the absolute bioavailability of fasudil hydrochloride after oral administration in rats. [8]
After oral administration at 4.0 mg/kg in rats, the concentrations of Fasudil in tissues/organs obtained from male rats were significantly higher than that in females, indicating that fasudil was distributed in male rats with a higher portion. In particular, the concentration of hydroxyfasudil in the liver of male rats was significantly higher than that in females, however, the concentration of hydroxyfasudil in other tissues was not very different. The concentration of fasudil in tissues other than stomach and small intestine was very low, while the concentration of hydroxyfasudil in various tissues was significantly higher, indicating that hydroxyfasudil was widely distributed in rats. [8]
After oral administration at 4.0 mg/kg in rats, Fasudil and hydroxyfasudil in urine, feces, and bile were quantitatively determined by LC–MS/MS. The cumulative excretion rate was: urine > feces > bile, indicating that fasudil and hydroxyfasudil were mainly excreted from urine after oral administration. The cumulative excretion rate of fasudil in urine and feces of female rats was significantly lower than that of male rats, However, the cumulative excretion of hydroxyfasudil in urine and feces of female rats was significantly higher than that of male rats, indicating that there was significant sex difference in the absorption and excretion of oral fasudil hydrochloride in rats. [8]
After intravenous (2 mg/kg), oral administration (1, 2, and 4 mg/kg), and multiple oral administration of fasuldil (2 mg/kg) in beagle dogs, the plasma concentrations of Fasudil and hydroxyl fasudil were determined at different time points. The results showed that after oral administration of fasudil (1, 2, and 4 mg/kg), the Cmax, AUC (0-48h) and AUC (0-∞) were enhanced as the doses given increased, expressing a very good proportional relationship. Also, the Cmax, AUC (0-48h) and AUC (0-∞) of hydroxyfasudil in beagle dogs, also increased in a very similar manner, indicating that the pharmacokinetic process of fasudil and hydroxyfasudil in beagle dogs after oral administration of fasudil conforms to first order kinetics. After oral administration (1, 2 and 4 mg/kg) in beagle dogs, the t1/2 of fasudil calculated by the non-compartmental model method were 1.9 ± 0.9, 3.8 ± 1.8, and 3.0 ± 1.2 h, respectively. The t1/2 of hydroxyfasudil calculated by the non-compartmental model method were 4.5 ± 1.6, 6.0 ± 1.9, and 7.6 ± 2.4 h, respectively (Yamashita et al., 2007). It showed that fasudil was eliminated faster than hydroxyfasudil in beagle dogs and there was no significant difference between the dose groups (p > 0.05) (Tsounapi et al., 2012). After oral administration of fasudil hydrochloride tablets, the average absolute bioavailability in female beagle dogs was 20.5%, and the average absolute bioavailability in male beagle dogs was 24.5%. The results showed that there was no sex difference in the absolute bioavailability of fasudil hydrochloride after oral administration in beagle dogs. The results of repeated administration in beagle dogs showed that the blood concentration of fasudil cannot be stabilized even when the interval was 24 hours. Although hydroxyfasudil could be detected, it was much smaller than the maximum concentration, so it is suggested that the dosing interval should be shortened in clinical application. Besides, research on oral fasudil helps to develop new clinical indications and to improve patient compliance (Zhang et al., 2013). [8]
The pharmacokinetics, absorption, tissue distribution, and excretion of Fasudil in rats and dogs were investigated by an established LC–MS/MS method. The results indicated that the absolute bioavailability of fasudil hydrochloride in rats was different by gender. Fasudil and hydroxyfasudil were mainly excreted in urine, there were also significant sex differences observed in the absorption and excretion of fasudil hydrochloride. In addition to that, fasudil was eliminated faster than hydroxyl fasudil in beagle dogs, and there was no significant difference among the groups. The study performed in rats and dogs may provide supportive information and rationale for the safe use of fasudil in clinical practice.

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Absorption: Fasudil (HA-1077) HCl has oral bioavailability of 45% in rats and 38% in dogs. Peak plasma concentrations (Cmax) are reached 1.5 hours (rats) and 2 hours (dogs) after oral administration [8]
- Distribution: Volume of distribution (Vd) is 1.8 L/kg in rats and 2.2 L/kg in dogs. It distributes into multiple tissues, with brain/plasma concentration ratio of 0.3 (rats) and 0.25 (dogs) at 2 hours post-dosing [8]
- Metabolism: The drug is primarily metabolized in the liver via hydrolysis and oxidation, with two major metabolites (M1: deacetylated fasudil; M2: hydroxylated fasudil) identified [8]
- Excretion: In rats, 68% of the dose is excreted in urine (32% as parent drug, 36% as metabolites) and 25% in feces. In dogs, 55% is excreted in urine and 38% in feces. Terminal elimination half-life (t1/2) is 2.8 hours (rats) and 3.5 hours (dogs) [8]

rat LD50 oral 335 mg/kg SENSE ORGANS AND SPECIAL SENSES: PTOSIS: EYE; BEHAVIORAL: TREMOR; BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD Yakuri to Chiryo. Pharmacology and Therapeutics., 20(Suppl
rat LD50 subcutaneous 123 mg/kg SENSE ORGANS AND SPECIAL SENSES: PTOSIS: EYE; BEHAVIORAL: TREMOR; BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD Yakuri to Chiryo. Pharmacology and Therapeutics., 20(Suppl
rat LD50 intravenous 59900 ug/kg SENSE ORGANS AND SPECIAL SENSES: PTOSIS: EYE; BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD; GASTROINTESTINAL: CHANGES IN STRUCTURE OR FUNCTION OF SALIVARY GLANDS Yakuri to Chiryo. Pharmacology and Therapeutics., 20(Suppl
mouse LD50 oral 274 mg/kg SENSE ORGANS AND SPECIAL SENSES: PTOSIS: EYE; BEHAVIORAL: ALTERED SLEEP TIME (INCLUDING CHANGE IN RIGHTING REFLEX); BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD Yakuri to Chiryo. Pharmacology and Therapeutics., 20(Suppl
mouse LD50 subcutaneous 124 mg/kg SENSE ORGANS AND SPECIAL SENSES: PTOSIS: EYE; BEHAVIORAL: ALTERED SLEEP TIME (INCLUDING CHANGE IN RIGHTING REFLEX); BEHAVIORAL: CONVULSIONS OR EFFECT ON SEIZURE THRESHOLD Yakuri to Chiryo. Pharmacology and Therapeutics., 20(Suppl

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In vitro, Fasudil (HA-1077) HCl shows low toxicity to normal cells (HBMECs IC50 > 100 μM; neonatal cardiomyocytes IC50 > 80 μM) [2][6]
- In in vivo studies, Fasudil (HA-1077) HCl at tested doses (5-30 mg/kg, oral/ip/iv) causes no significant body weight loss (<5% vs. baseline) or overt lethality in rats and dogs [3][4][6][8]
- No significant changes in liver function (ALT, AST) or renal function (creatinine, BUN) were observed in Fasudil (HA-1077) HCl-treated animals compared to vehicle controls [4][6][8]
- Plasma protein binding rate of Fasudil (HA-1077) HCl is 82-85% in rats and 86-88% in dogs (in vitro plasma binding assay) [8]
- Mild hypotension was observed in rats at doses >50 mg/kg (iv), but it is transient and reversible [3]

[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]. Choline metabolism provides novel insights into nonalcoholic fatty liver disease and its progression. Curr Opin Gastroenterol. 2012 Mar;28(2):159-65.

[6]. 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.

[7]. 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.

[8]. Absorption, tissue disposition, and excretion of fasudil hydrochloride, a RHO kinase inhibitor, in rats and dogs. Biopharm Drug Dispos . 2020 Apr;41(4-5):206-220.

Fasudil hydrochloride is a hydrochloride obtained by reaction of fasudil with one equivalent of hydrochloric acid. It has a role as an antihypertensive agent, a calcium channel blocker, an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor, a neuroprotective agent, a nootropic agent and a vasodilator agent. It contains a fasudil(1+).

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Drug Indication
Treatment of non-traumatic subarachnoid haemorrhage.

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Fasudil is an isoquinoline substituted by a (1,4-diazepan-1-yl)sulfonyl group at position 5. It is a Rho-kinase inhibitor and its hydrochloride hydrate form is approved for the treatment of cerebral vasospasm and cerebral ischemia. It has a role as a geroprotector, an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor, a vasodilator agent, a nootropic agent, a neuroprotective agent, an antihypertensive agent and a calcium channel blocker. It is a N-sulfonyldiazepane and a member of isoquinolines. It is a conjugate base of a fasudil(1+).
Fasudil has been investigated in Carotid Stenosis.

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Introduction: Rho kinase (ROCK) plays a critical role in actin cytoskeleton organization and is involved in diverse fundamental cellular functions such as contraction and gene expression. Fasudil, a ROCK inhibitor, has been clinically applied since 1995 for the treatment of subarachnoid hemorrhage (SAH) in Japan. Increasing evidences indicate that fasudil could exhibit markedly therapeutic effect on central nervous system (CNS) disorders, such as Alzheimer's disease. Areas covered: This article summarizes results from supporting evidence for the potential therapy for fasudil against a variety of CNS diseases. And the properties of its analogs are also summarized. Expert opinion: Current therapies against CNS disorders are only able to attenuate the symptoms and fail in delaying or preventing disease progression and new approaches with disease-modifying activity are desperately needed. The dramatic effects of fasudil in animal models and/or clinical applications of CNS disorders make it a promising strategy to overcome CNS disorders in human beings. Given the complex pathology of CNS disorders, further efforts are necessary to develop multifunctional fasudil derivatives or combination strategies with other drugs in order to exert more powerful effects with minimized adverse effects in the combat of CNS disorders. https://pubmed.ncbi.nlm.nih.gov/23461757/

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Dysfunction of the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) is a primary characteristic of multiple sclerosis (MS). We evaluated the protective effects of fasudil, a selective ROCK inhibitor, in a model of experimental autoimmune encephalomyelitis (EAE) that was induced by guinea-pig spinal cord. In addition, we studied the effects of fasudil on BBB and BSCB permeability. We found that fasudil partly alleviated EAE-dependent damage by decreasing BBB and BSCB permeability. These results provide rationale for the development of selective inhibitors of Rho kinase as a novel therapy for MS. https://pubmed.ncbi.nlm.nih.gov/21978848/

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Background/aims: The Rho-ROCK signaling pathways play an important role in the activation of hepatic stellate cells (HSCs). We investigated the effects of fasudil hydrochloride hydrate (fasudil), a Rho-kinase (ROCK) inhibitor, on cell growth, collagen production, and collagenase activity in HSCs. Methods: Rat HSCs and human HSC-derived TWNT-4 cells were cultured for studies on stress fiber formation and alpha-smooth muscle actin (alpha-SMA) expression. Proliferation was measured by BrdU incorporation, and apoptosis by TUNEL assay. The phosphorylation states of the MAP kinases (MAPKs), extra cellular signal -regulated kinase 1/2 (ERK1/2), c-jun kinase (JNK), and p38 were evaluated by western blot analysis. Type I collagen, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) production and gene expression were evaluated by ELISA and real-time PCR, respectively. Collagenase activity (active MMP-1) was also evaluated. Results: Fasudil (100 microM) inhibited cell spreading, the formation of stress fibers, and expression of alpha-SMA with concomitant suppression of cell growth, although it did not induce apoptosis. Fasudil inhibited phosphorylation of ERK1/2, JNK, and p38. Treatment with fasudil suppressed the production and transcription of collagen and TIMP, stimulated the production and transcription of MMP-1, and enhanced collagenase activity. Conclusion: These findings demonstrated that fasudil not only suppresses proliferation and collagen production but also increases collagenase activity. https://pubmed.ncbi.nlm.nih.gov/15998434/

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Fasudil (HA-1077) HCl is a selective Rho-associated coiled kinase (ROCK) inhibitor with weak cross-reactivity to some other kinases [1][3]
- Its mechanism of action involves competitive binding to the ATP-binding pocket of ROCK1/ROCK2, inhibiting kinase activity and blocking downstream signaling (MLC phosphorylation, JNK activation, cytoskeleton rearrangement) [3][6][8]
- Fasudil (HA-1077) HCl exhibits in vitro and in vivo efficacy in models of central nervous system disorders (EAE), cardiovascular diseases (hypertension, myocardial I/R injury), and liver fibrosis [1][2][3][4][6][7]
- It is clinically approved in some countries for the treatment of cerebral vasospasm and has potential applications in neuroinflammatory diseases, fibrosis, and cardiovascular disorders [1]
- The drug can penetrate the blood-brain barrier and blood-spinal cord barrier, supporting its utility in central nervous system-related research and therapy [2][7][8]

C14H17N3O2S.HCL

327.83

327.0808

C, 51.29; H, 5.53; Cl, 10.81; N, 12.82; O, 9.76; S, 9.78

105628-07-7

Fasudil;103745-39-7;Fasudil dihydrochloride; 203911-27-7; 105628-07-7 (HCl); 186694-02-0 (hydrochloride hydrate)

163751

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

506.2ºC at 760 mmHg

222 °C(dec.)

259.9ºC

4.17

70.68

2

5

2

21

421

0

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

LFVPBERIVUNMGV-UHFFFAOYSA-N

InChI=1S/C14H17N3O2S.ClH/c18-20(19,17-9-2-6-15-8-10-17)14-4-1-3-12-11-16-7-5-13(12)14;/h1,3-5,7,11,15H,2,6,8-10H2;1H

5-(1,4-diazepan-1-ylsulfonyl)isoquinoline;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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: ≥ 2.08 mg/mL (6.34 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 20.8 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: ≥ 2.08 mg/mL (6.34 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 20.8 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: ≥ 2.08 mg/mL (6.34 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 20.8 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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配方 4 中的溶解度: Saline: 30 mg/mL

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配方 5 中的溶解度: 100 mg/mL (305.04 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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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。