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].

---

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].

---

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]

---

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]

---

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]

---

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.

---

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.

---

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.

---

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).

---

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.

---

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.

---

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.

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.

---

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.

---

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.

---

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/mL*h in females, and 30.32 ± 13.22, 115.94 ± 60.18, 531.68 ± 199.84 ng/mL*h in males, the AUC(0-t) of hydroxyfasudil were 92.79 ± 30.97, 233.58 ± 96.30, 345.13 ± 115.31 ng/mL*h in females, and 67.26 ± 24.97, 266.12 ± 153.35, 444.94 ± 190.21 ng/mL*h 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.

---

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.

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

[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 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.

---

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/

---

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/

---

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/

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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