p160ROCK (Ki = 0.33 μM); ROCK2 (IC50 = 0.158 μM); PKG (IC50 = 1.65 μM); PKA (IC50 = 4.58 μM); PKC (IC50 = 12.30 μM);

Fasudil dihydrochloride (100 μM) suppresses cell proliferation in rat HSCs (hepatic stellate cells) and human HSC-derived TWNT-4 cells by preventing cell spreading, stress fiber production, and α-SMA expression[4]. In rat HSCs and human HSC-derived TWNT-4 cells, dihydrochloride (50-100 μM; 24 hours) suppresses the phosphorylation of ERK1/2, JNK, and p38 caused by LPA (lysophoaphatidic acid)[4]. In human HSC-derived TWNT-4 cells, facudil dihydrochloride (25–100 μM; 24 hours) promotes MMP-1 transcription while suppressing collagen and TIMP transcription[4].

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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[4].

When administered intravenously one hour prior to surgery, facudil dihydrochloride (10 mg/kg) has been shown to protect against cardiovascular disease, inhibit JNK activation, and lessen the amount of AIF that is translocated from the mitochondria to the nucleus during ischemia[5]. Fasudil dihydrochloride (50 mg/kg/d; ip) inhibits the proliferation of lymphocytes, results in downregulation of interleukin (IL)-17, and a significant decrease in the IFN-γ/IL-4 ratio. It also prevents acute and relapsing EAE (experimental autoimmune encephalomyelitis) caused by the proteolipid protein PLP p139-151 [6]. Fasudil dihydrochloride (100 mg/kg/d; po) decreases inflammation, demyelination, axonal loss, and APP positive in the mouse spinal cord and significantly lowers the incidence and pathological examination score of experimental autoimmune encephalomyelitis (EAE) in SJL/J mice [6].

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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. [1]

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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. [2]

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Ischemia followed by reperfusion caused a significant increase in Rho-kinase, c-Jun NH2-terminal kinase (JNK) and apoptosis-inducing factor (AIF) activity. Administration of Fasudil, an inhibitor of Rho-kinase, decreased myocardial infarction size from 59.89+/-3.83% to 38.62+/-2.66% (P<0.05) and cell apoptosis from 32.78+/-5.1% to 17.05+/-4.2% (P<0.05). Western blot analysis showed that administration of fasudil reduced the activation of JNK and attenuated mitochondrial-nuclear translocation of AIF. Additionally, administration of SP600125, an inhibitor of JNK, attenuated mitochondrial-nuclear translocation of AIF. Conclusion: The inhibition of Rho-kinase reduced cell apoptosis in I/R in vivo via suppression of JNK-mediated AIF translocation. [6]

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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]

Cyclic AMP-dependent protein kinase activity is assayed in a reaction mixture containing, in a final volume of 0.2 mL, 50 mM Tris-HCl (pH 7.0), 10 mM magnesium acetate, 2 mM EGTA, 1 μM cyclic AMP or absence of cyclic AMP, 3.3 to 20 μM [r-32P] ATP (4×105 c.p.m.), 0.5 μg of the enzyme, 100 μg of histone H2B and compound. The mixture is incubated at 30°C for 5 min. The reaction is terminated by adding 1mL of ice-cold 20% trichloroacetic acid after adding 500 μg of bovine serum albumin as a carrier protein. The sample is centrifuged at 3000 r.p.m. for 15min, the pellet is resuspended in ice-cold 10% trichloro-acetic acid solution and the centrifugation-resuspension cycle is repeated three times. The final pellet is dissolved in 1 mL of 1 N NaOH and radioactivity is measured with a liquid scintillation counter.

Western Blot Analysis[4]
Cell Types: Rat HSCs and human HSC -derived TWNT-4 cells
Tested Concentrations: 50 μM; 100 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Suppressed the LPA-induced phosphorylation of ERK1/2, JNK and p38 MAPK by 60%, 70%,and 90%, respectively. RT- PCR[4]
Cell Types: Rat HSCs and human HSC-derived TWNT-4 cells
Tested Concentrations: 25 μM; 50 μM; 100 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: decreased the expression of type I collagen, a-SMA, and TIMP- 1.

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Cell culture [4]
HSCs were isolated from the liver of male Wistar rats by sequential in situ perfusion with collagenase and digestion with pronase, followed by centrifugation in a double-layered (17%/11.5%) metrizamide solution, as described previously. HSCs were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS). Experiments described in this study were performed on cells between the second and fourth serial passages. Since commercial kits for the measurement of mouse matrix metalloproteinase (MMP-1) and TIMP-1 were not available, we used TWNT-4 cells, a human cell line derived from HSCs, for evaluating the effects of fasudil on MMP-1 and TIMP-1. TWNT-4 cells were cultured in DMEM with 10% FCS as reported previously. Fasudil was donated by Asahikasei Corporation. Fasudil was dissolved in DMEM and added to cultures. Cell viability of HSCs was more than 90% under serum-free conditions for 24 h in the presence of 100 μM fasudil.

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Immunocytochemistry [4]
HSC and TWNT-4 cells were maintained in either the presence or absence of Fasudil (100 μM) in serum-free conditions for 24 h. Immunocytochemistry was basically performed as previously reported (15–17). Following three washes with phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.4), cells were fixed for 10 min in 3.7% formaldehyde at 37°C, permeabilized for 5 min in PBS containing 0.2% Triton X-100 at 37°C, washed three times with PBS, and blocked with PBS containing 10% FCS for 30 min at 37°C. The slides were then incubated with an anti-α-SMA primary antibody or an anti-Myc primary antibody at 37°C for 60 min. The slides were rinsed extensively in PBS and then stained with rhodamine-conjugated phalloidin, mixed with Alexa Fluor 488-labeled goat anti-mouse secondary antibody. Images were visualized with an LSM 510 confocal laser scanning microscope.

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Analysis of BrdU incorporation [4]
HSC incorporation of BrdU was measured using a cell proliferation ELISA. Briefly, subconfluent HSCs were serum starved for 24 h. They were then washed with DMEM and incubated for 24 h with BrdU in DMEM with 10% FCS in the presence or absence of Fasudil (100 μM) or Y27632 (30 μM) (another specific ROCK inhibitor) as a control. After labeling the cells with BrdU, cellular DNA was digested and incubated with the anti-BrdU antibody conjugated with peroxidase. BrdU incorporation was estimated by measuring the fluorescence intensity of the supernatant at 450 nm (excitation) and 690 nm (emission).

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Analysis of apoptosis [4]
HSCs were maintained in either the presence or absence of Fasudil (100 μM) in serum-free conditions for 24 h. Cells were fixed for 30 min in 4% paraformaldehyde/PBS at room temperature, and permeabilized for 5 min in PBS containing 0.2% Triton X-100 at 4°C. Cells were then stained with Hoechst 33342, and analyzed by the TUNEL method using an In Situ Cell Death Detection Kit according to the manufacturer's instructions. The samples were visualized with an LSM 510 confocal laser scanning microscope. At least 100 cells from three independent experiments and from three different cell preparations were counted for each condition.

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Western blot analysis for phospho- and nonphospho-MAP kinase (MAPK) [4]
Western blot analysis was basically performed as described previously. After HSCs were starved for 24 h, they were stimulated with LPA (10 μM) for 45 min, followed by treatment with or without 100 μM Fasudilfor 2 h. Whole cell lysates containing 1 × 107 TWNT-4 cells were prepared in 100 μl SDS-PAGE sample buffer. Protein lysates were subjected to 12% SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with the primary antibody for extracellular signal related kinase (ERK)1/2 MAPK, phospho-ERK1/2 MAPK (Thr202/Tyr204), JNK, phospho-JNK (Thr183/Tyr185), p38 MAPK, or phospho-p38 MAPK (Thr180/Tyr182). Antibody binding was detected using peroxidase linked anti-rabbit IgG as the secondary antibody. The blots were developed using ECL-plus to visualize the antibodies. The levels of ERK1/2 MAPK, phosphorylated-ERK1/2 MAPK, JNK, phosphorylated-JNK, p38 MAPK, and phosphorylated-p38 MAPK were quantitated by densitometry using an optical scanner system. For comparison, the ratios of phosphorylated ERK1/2, JNK, and p38 MAPK to nonphosphorylated ERK1/2, JNK, and p38 MAPK, respectively, were calculated from the densitometric data.

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Analysis of gene expression using real -time RT-PCR [4]
Total RNA was prepared from TWNT-4 cells with Trizol reagent, which were maintained in either the presence or absence of Fasudil (25, 50, or 100 μM) in 10% FCS for 24 h. cDNA was synthesized from 1.0 μg RNA with GeneAmp™ RNA PCR using random hexamers. Real-time PCR was performed using LightCycler-FastStart DNA Master SYBR Green 1 (Roche, Tokyo, Japan) according to the manufacturer's instruction. The reaction mixture (20 μl) contained LightCycler-FastStart DNA Master SYBR Green 1, 4 mM MgCl2, 0.5 μM of the upstream and downstream PCR primers, and 2 μl of the first -strand cDNA as a template. To control for variations in the reactions, all PCRs were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. The primers used were as follows: 5′-AGGGTGAGACAGGCGAACAG-3′ (forward primer) and 5′-CTCTTGAGGTGGCTGGGGCA-3′ (reverse primer) for human type I collagen α1 chain; 5′-AATGAGATGGCCACTGCCGC-3′ (forward primer) and 5′-CAGAGTATTTGCGCTCCGGA-3′ (reverse primer) for human α-SMA (GenBank™ accession number NM-000088); 5′-GATCATCGGGACAACTCTCCT-3′ (forward primer), and 5′-TCCGGGTAGAAGGGATTTGTG-3′ (reverse primer) for MMP-1 (GenBank™ accession number NM002421); 5′-TTCTGCAATTCCGACCTCGT-3′ (forward primer) and 5′-TCCGTCCACAAGCAATGAGT-3′ (reverse primer) for TIMP-1 (Ref. 3; GenBank™ accession number NM003254).

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.

Pharmacokinetics of fasudil in rats [8]
Fasudil and hydroxyfasudil in plasma samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fasudil and hydroxyfasudil in plasma samples were measured at all 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 shown in Figure 5. The pharmacokinetic parameters of fasudil and hydroxyfasudil calculated using the DAS program are listed in Tables 4 and 5. The results showed that the exposure of fasudil in rats increased proportionally in the dose range of 2-6 mg/kg. After three administrations of low, medium, and high concentrations of fasudil, the elimination half-life (t1/2) of fasudil in female subjects was 1.19 ± 0.51, 0.85 ± 0.35, and 1.09 ± 0.55 h, respectively, while in male subjects it was 2.29 ± 0.89, 2.74 ± 1.57, and 2.34 ± 1.83 h, respectively. Simultaneously, the elimination half-life (t1/2) of hydroxyfasudil in female subjects was 2.08 ± 0.68, 1.84 ± 0.33, and 1.69 ± 0.41 h, respectively, while in male subjects it was 2.40 ± 0.16, 2.32 ± 1.02, and 2.11 ± 0.52 h, respectively. These results indicate a significant sex difference in the pharmacokinetics of fasudil after gavage administration in rats.

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Rat Tissue Distribution [8]
Fasudil and hydroxyfasudil in each tissue sample were analyzed by LC-MS/MS, and the results were substituted into the standard curve to obtain the corresponding drug concentration values. Figure 6 shows the average concentrations (ng/g) of fasudil and hydroxyfasudil in each tissue at 0.25, 1, 3 and 6 h after oral administration of 4 mg/kg fasudil to rats. Except for the stomach and small intestine, the concentration of fasudil in all other tissues was very low; the concentration of fasudil in the stomach and small intestine was high at 0.5 and 1 h after administration, but almost completely disappeared after 6 h. However, the concentration of hydroxyfasudil in all tissues was significantly higher than that of fasudil.

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Rat Excretion [8]
Fasudil and hydroxyfasudil in urine, feces and bile samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the results were substituted into the standard curve to obtain the corresponding drug concentration values. Cumulative excretion curves in urine, feces, and bile after drug administration were plotted (Figure 7). Table 6 shows the statistical analysis results of the differences in excretion between male and female rats. The results showed that within 48 hours after drug administration, the cumulative excretion rate of fasudil in the urine of female rats was 0.37%, while that of male rats was 1.08%; the cumulative excretion rate of hydroxyfasudil in the urine of female rats was 2.42%, while that of male rats was 16.12%. Within 48 hours after drug administration, the cumulative excretion rate of fasudil in feces 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 also showed that within 24 hours after drug administration, the cumulative excretion rate of fasudil in bile was 0.46% in females and 0.63% in males; the cumulative excretion rate of hydroxyfasudil was 0.40% in females and 2.38% in males.

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Pharmacokinetics of Fasudil in Dogs [8]
The concentrations of fasudil and hydroxyfasudil in plasma samples were determined at all time points following intravenous injection (2 mg/kg), oral administration (1, 2 and 4 mg/kg), and multiple oral administrations of fasudil (2 mg/kg). The results were substituted into a standard curve to obtain the corresponding concentration values. Similarly, the mean plasma concentration-time curves of fasudil are shown in Figures 8 and 9. The pharmacokinetic parameters of fasudil and hydroxyfasudil calculated using the DAS program are listed in Tables 7 and 8. The results showed that in beagle dogs, the exposure to fasudil increased proportionally in the dose range of 1–4 mg/kg. After administration of low, medium, and high concentrations of fasudil, the elimination half-life (t1/2) of fasudil in women was 2.39 ± 0.95, 4.58 ± 2.36, and 2.69 ± 1.45 hours, respectively, while that in men was 1.50 ± 0.64, 3.00 ± 0.69, and 3.22 ± 1.02 hours, respectively. The elimination half-life (t1/2) of hydroxyfasudil in women was 4.53 ± 1.66, 6.89 ± 2.11, and 8.78 ± 2.96 hours, respectively, while that in men was 4.38 ± 1.68, 5.16 ± 1.49, and 6.39 ± 1.03 hours, respectively. After administration of low, medium, and high concentrations of fasudil, the AUC(0-t) for women were 44.63 ± 24.11, 123.88 ± 57.81, and 221.21 ± 108.98 ng/mLh, respectively, while for men they were 30.32 ± 13.22, 115.94 ± 60.18, and 531.68 ± 199.84 ng/mLh, respectively. The AUC(0-t) for hydroxyfasudil were 92.79 ± 30.97, 233.58 ± 96.30, and 345.13 ± 115.31 ng/mLh, respectively, while for men they were 67.26 ± 24.97 and 266.12 ± 153.35 ng/mLh, respectively. The Cmax value for males was 444.94 ± 190.21 ng/mLh. After administration of low, medium, and high concentrations of fasudil, the Cmax values for females were 17.60 ± 10.31, 63.45 ± 28.75, and 148.51 ± 161.40 ng/mL, respectively, while those for males were 19.72 ± 11.63, 56.84 ± 43.57, and 304.70 ± 97.36 ng/mL, respectively. The Cmax values for hydroxyfasudil in females were 18.90 ± 6.48, 21.97 ± 6.70, and 26.68 ± 5.58 ng/mL, respectively, while those in males were 11.43 ± 4.75, 25.04 ± 14.13, and 34.54 ± 15.52 ng/mL, respectively. Results showed no sex differences in the pharmacokinetics of fasudil after gavage administration in dogs. Fasudil hydrochloride, as an intracellular calcium ion antagonist, has a vasodilatory effect and exhibits significant pharmacological activity in the treatment of angina. This study aimed to determine the absorption, distribution, and excretion of fasudil in rats and beagle dogs to elucidate its pharmacokinetic model. We developed and established a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and successfully applied it to pharmacokinetic studies including absorption, tissue distribution, and excretion. Results showed that the pharmacokinetic behavior (e.g., AUC and Cmax) of fasudil in rats was dose-dependent within the dose range of 2–6 mg/kg. However, plasma concentration results indicated significant sex differences in the pharmacokinetics of fasudil in rats, including absolute bioavailability and excretion. Interestingly, data from beagle dogs showed no sex differences in the absolute bioavailability of fasudil hydrochloride after single or repeated administration. In summary, this study elucidated the pharmacokinetic characteristics of fasudil in rats and beagle dogs through absorption, tissue distribution, and excretion studies. These findings may be of great value and provide a theoretical basis for further research and its safe application in clinical practice. [8] Fasudil is an intracellular calcium antagonist that dilates blood vessels and inhibits vasospasm by blocking the vasoconstriction process through phosphorylation of myosin light chains (Somlyo & Somlyo, 2003; Fukushima et al., 2010). It is used clinically to treat subarachnoid hemorrhage (Fu et al., 2018; Kondoh, Mizusawa, Murakami, Nakamichi, & Nagata, 1997). Hydroxyfasudil is the active metabolite of fasudil hydrochloride and exhibits higher selectivity in specificity experiments (Nakamura et al., 2001; Shimokawa & Rashid, 2007). This study established a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine the concentrations of fasudil and hydroxyfasudil in rats and beagle dogs, and applied it to the absorption, tissue distribution and excretion studies after administration, further elucidating the pharmacokinetic characteristics of fasudil in animal models. [8]
After intravenous injection (4 mg/kg) and oral administration (2, 4 and 6 mg/kg) of fasudil to rats, the concentrations of fasudil and hydroxyfasudil in plasma were measured at different time points. The results showed that there were significant sex differences in the pharmacokinetics of fasudil in rats. In addition, the pharmacokinetics of fasudil was dose-dependent in the dose range of 2 to 6 mg/kg. The half-lives (tl/2) of intravenously injected fasudil and hydroxyfasudil were 0.6 ± 0.3 h and 1.8 ± 0.5 h, respectively, which were basically consistent with the literature reports (Zhang, Gao, Huang, & Xu, 2009). The half-lives of oral fasudil were 2.3 ± 0.90 h, 2.7 ± 1.6 h and 2.3 ± 1.8 h, respectively, which were significantly longer than those of intravenous injection, but the half-life of hydroxyfasudil remained unchanged. After oral administration of fasudil hydrochloride, the mean absolute bioavailability of female rats was 35.8%, while that of 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 to rats. [8] After oral administration of 4.0 mg/kg fasudil to rats, the concentration of fasudil in tissues/organs of male rats was significantly higher than that of female rats, indicating that the distribution ratio of fasudil in male rats was higher. It is particularly noteworthy that the concentration of hydroxyfasudil in the liver of male rats was significantly higher than that in female rats, while the concentration of hydroxyfasudil in other tissues was not significantly different. Except for the stomach and small intestine, the concentration of fasudil in other tissues was extremely low, while the concentration of hydroxyfasudil in various tissues was significantly higher, indicating that hydroxyfasudil is widely distributed in rats. [8] After rats were orally administered 4.0 mg/kg fasudil, fasudil and hydroxyfasudil in urine, feces and bile were quantitatively determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The cumulative excretion rate was in the order of urine > feces > bile, indicating that fasudil and hydroxyfasudil were mainly excreted in 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, while the cumulative excretion rate of hydroxyfasudil in urine and feces of female rats was significantly higher than that of male rats, indicating that there are significant sex differences in the absorption and excretion of fasudil hydrochloride orally administered to rats. [8]
In beagle dogs, plasma concentrations of fasudil and hydroxyfasudil were measured at different time points after intravenous injection (2 mg/kg), oral administration (1, 2, and 4 mg/kg), and multiple oral administrations (2 mg/kg). The results showed that after oral administration of fasudil (1, 2, and 4 mg/kg), Cmax, AUC(0-48h), and AUC(0-∞) all increased significantly with increasing dose, showing a good proportional relationship. In addition, Cmax, AUC(0-48h), and AUC(0-∞) of hydroxyfasudil in beagle dogs also increased in a similar manner, indicating that the pharmacokinetics of fasudil and hydroxyfasudil in beagle dogs after oral administration of fasudil conformed to first-order kinetics. Using a non-compartmental model, the t1/2 of fasudil (1, 2, and 4 mg/kg) in beagles was 1.9 ± 0.9 h, 3.8 ± 1.8 h, and 3.0 ± 1.2 h, respectively. The half-lives of hydroxyfasudil, calculated using the non-compartmental model, were 4.5 ± 1.6, 6.0 ± 1.9, and 7.6 ± 2.4 hours, respectively (Yamashita et al., 2007). These results indicate that fasudil is eliminated from beagles faster than hydroxyfasudil, and there was no significant difference between the dose groups (p > 0.05) (Tsounapi et al., 2012). The mean absolute bioavailability of fasudil hydrochloride tablets was 20.5% in female beagles and 24.5% in male beagles after oral administration. These results suggest that there is no sex difference in the absolute bioavailability of fasudil hydrochloride after oral administration in beagles. Repeated-dose studies in beagle dogs showed that fasudil blood concentrations were not stable even at 24-hour intervals. Although hydroxyfasudil was detectable, its concentration was far below the maximum concentration, suggesting that the dosing interval should be shortened in clinical use. In addition, studies on oral fasudil may help develop new clinical indications and improve patient compliance (Zhang et al., 2013). [8] The pharmacokinetics, absorption, tissue distribution and excretion of fasudil in rats and dogs were studied using established LC-MS/MS methods. The results showed that there were sex differences in the absolute bioavailability of fasudil hydrochloride in rats. Fasudil and hydroxyfasudil were mainly excreted in the urine, and there were also significant sex differences in the absorption and excretion of fasudil hydrochloride. In addition, fasudil was cleared faster than hydroxyfasudil in beagle dogs, and there were no significant differences between the groups. This study in rats and dogs may provide supporting information and theoretical basis for the safe use of fasudil in clinical practice.

Oral LD50 in rats: 335 mg/kg. Sensory organs and special senses: ptosis; Behavior: tremor; Behavior: seizures or effect on the epilepsy threshold. Yakuri to Chiryo. Pharmacology and Therapeutics, 20 (Supplement). Subcutaneous LD50 in rats: 123 mg/kg. Sensory organs and special senses: ptosis; Behavior: tremor; Behavior: seizures or effect on the epilepsy threshold. Pharmacology and Therapeutics, 20 (Supplement). Intravenous LD50 in rats: 59900 ug/kg. Sensory organs and special senses: ptosis; Behavior: seizures or effect on the epilepsy threshold; Gastrointestinal tract: changes in salivary gland structure or function. Pharmacology and Therapeutics, 20(Supplement)
Oral LD50 in mice: 274 mg/kg. Sensory organs and special senses: ptosis; Behavior: altered sleep duration (including altered righting reflex); Behavior: seizures or effect on epilepsy threshold. Yakuri to Chiryo. Pharmacology and Therapeutics., 20(Supplement)
Subcutaneous LD50 in mice: 124 mg/kg. Sensory organs and special senses: ptosis; Behavior: altered sleep duration (including altered righting reflex); Behavior: seizures or effect on epilepsy threshold. Yakuri to Chiryo. Pharmacology and Therapeutics, 20(Supplement)

[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 is an isoquinoline compound with a (1,4-diazacycloheptan-1-yl)sulfonyl group substituted at the 5-position. It is a Rho kinase inhibitor, and its hydrochloride hydrate is approved for the treatment of cerebral vasospasm and cerebral ischemia. Fasudil possesses a variety of pharmacological effects, including anti-aging, inhibition of EC 2.7.11.1 (a nonspecific serine/threonine protein kinase), vasodilation, nootropic effects, neuroprotection, hypotensive effects, and calcium channel blockade. It is an N-sulfonyldiazacycloheptanyl compound belonging to the isoquinoline class. It is the conjugate base of fasudil (1+). Fasudil has been investigated in the treatment of carotid artery stenosis. Introduction: Rho kinase (ROCK) plays a crucial role in the organization of the actin cytoskeleton and is involved in a variety of essential cellular functions, such as contraction and gene expression. Fasudil, a ROCK inhibitor, has been used in Japan since 1995 for the treatment of subarachnoid hemorrhage (SAH). Mounting evidence suggests that fasudil may have significant therapeutic potential for central nervous system (CNS) diseases, such as Alzheimer's disease. This article summarizes the evidence supporting the potential efficacy of fasudil in treating various CNS diseases and outlines the characteristics of its analogues. Expert opinion: Current therapies for CNS diseases only alleviate symptoms and cannot slow or halt disease progression, thus new approaches with disease-modifying effects are urgently needed. Fasudil's significant efficacy in animal models and/or clinical applications of CNS diseases makes it a promising strategy for treating human CNS diseases. Given the complex pathological mechanisms of CNS diseases, further efforts are needed to develop multifunctional fasudil derivatives or in combination with other drugs to achieve greater efficacy and minimize adverse reactions in combating CNS diseases. Blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) dysfunction are key features of multiple sclerosis (MS). We evaluated the protective effect of the selective ROCK inhibitor fasudil in a guinea pig spinal cord-induced experimental autoimmune encephalomyelitis (EAE) model. Furthermore, we investigated the effects of fasudil on the permeability of the blood-brain barrier (BBB) and blood-spinal barrier (BSCB). We found that fasudil partially alleviated experimental autoimmune encephalomyelitis (EAE)-dependent injury by reducing the permeability of the BBB and BSCB. These results provide a theoretical basis for developing selective Rho kinase inhibitors as novel therapies for multiple sclerosis (MS). https://pubmed.ncbi.nlm.nih.gov/21978848/

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Background/Objective: The Rho-ROCK signaling pathway plays a crucial role in the activation of hepatic stellate cells (HSCs). We investigated the effects of the Rho kinase (ROCK) inhibitor fasudil hydrochloride hydrate (fasudil) on HSC cell growth, collagen production, and collagenase activity. Methods: TWNT-4 cells derived from rat HSCs and human HSCs were cultured to investigate stress fiber formation and α-smooth muscle actin (α-SMA) expression. Cell proliferation was detected by BrdU incorporation assay, and apoptosis was detected by TUNEL assay. Western blotting was used to detect the phosphorylation status of MAP kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun kinase (JNK), and p38. Enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR were used to detect the production and gene expression of type I collagen, matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinases-1 (TIMP-1), respectively. Collagenase activity (active MMP-1) was also detected. Results: Fasudil (100 μM) inhibited cell spreading, stress fiber formation, and α-SMA expression, accompanied by cell growth inhibition, but did not induce apoptosis. Fasudil inhibited the phosphorylation of ERK1/2, JNK, and p38. Fasudil treatment inhibited the production and transcription of collagen and TIMP, stimulated the production and transcription of MMP-1, and enhanced collagenase activity. Conclusion: These findings indicate that fasudil not only inhibits cell proliferation and collagen production but also increases collagenase activity. https://pubmed.ncbi.nlm.nih.gov/15998434/

C14H17N3O2S.2[HCL]

364.29056

381.068

203911-27-7

Fasudil Hydrochloride;105628-07-7;Fasudil;103745-39-7;Fasudil hydrochloride semihydrate;186694-02-0

16219471

Typically exists as solid at room temperature

4.106

3

5

2

22

421

0

C1=CC2=CN=CC=C2C(=C1)S(=O)(=O)N3CCCNCC3.Cl.Cl

NOXXIYDYFSNHDF-UHFFFAOYSA-N

InChI=1S/C14H17N3O2S.2ClH/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;2*1H

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

Fasudil dihydrochloride; HA-1077 DIHYDROCHLORIDE; 5-((1,4-Diazepan-1-yl)sulfonyl)isoquinoline dihydrochloride; Fasudil (dihydrochloride); Isoquinoline, 5-[(hexahydro-1H-1,4-diazepin-1-yl)sulfonyl]-, hydrochloride (1:2); ha-1077; HA-1077 (hydrochloride);

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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