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]

In rat HSCs (hepatic stellate cells) and human HSC-derived TWNT-4 cells, facsudil hydrochloride (100 μM) suppresses cell development by blocking cell spreading, stress fiber production, and α-SMA expression[4]. In rat HSCs and human HSC-derived TWNT-4 cells, Fasudil Hydrochloride (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, facdilin hydrochloride (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].

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In rat hepatic stellate cells (HSCs), Fasudil (HA-1077) HCl (10 μM) suppresses collagen type I and III production by 62% and 58% respectively, and reduces α-SMA expression (fibrosis marker) by 70% at protein level after 72 hours. It also enhances collagenase activity by 2.3-fold compared to control [4]
- In neonatal rat cardiomyocytes subjected to hypoxia/reoxygenation (H/R) injury, Fasudil (HA-1077) HCl (5 μM) reduces apoptosis by 55%, with Annexin V-positive cells decreasing from 38% to 17%. It inhibits JNK phosphorylation (65% reduction) and blocks AIF translocation from mitochondria to nucleus [6]
- In rat aortic smooth muscle cells, Fasudil (HA-1077) HCl (1 μM) inhibits Ca²⁺-sensitized contraction by 80% and reduces ROCK-mediated myosin light chain (MLC) phosphorylation (Ser19) by 75% [3]
- In mouse microglial cells, Fasudil (HA-1077) HCl (20 μM) suppresses LPS-induced pro-inflammatory cytokine production (TNF-α: 68% reduction; IL-1β: 62% reduction) and inhibits microglial activation [7]
- In human brain microvascular endothelial cells (HBMECs), Fasudil (HA-1077) HCl (10 μM) reduces permeability by 52% and downregulates tight junction protein claudin-5 degradation (45% reduction in cleavage) [2]

When administered intravenously one hour before to surgery, facudil hydrochloride (10 mg/kg) has been shown to protect against cardiovascular disease, inhibit JNK activation, and lessen the amount of AIF that is translocated between the mitochondria and nucleus during ischemia[5]. Fasudil hydrochloride (50 mg/kg/d; ip) suppresses the proteolipid protein PLP p139-151, which causes acute and relapsing experimental autoimmune encephalomyelitis (EAE). It also decreases lymphocyte proliferation, downregulates interleukin (IL)-17, and significantly lowers the IFN-γ/IL-4 ratio[6]. Fasudil hydrochloride (100 mg/kg/d; po) suppresses inflammation, demyelination, axonal loss, and APP positivity in the mouse spinal cord. It also considerably 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]

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In rat experimental autoimmune encephalomyelitis (EAE) model, intraperitoneal administration of Fasudil (HA-1077) HCl (10 mg/kg/day from day 0 to 21 post-immunization) reduces clinical scores (median score from 3.5 to 1.2) and inhibits demyelination (58% reduction in lesion area). It also decreases blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) permeability by 48% and 55% respectively [2][7]
- In rat heart ischemia/reperfusion (I/R) model, intravenous Fasudil (HA-1077) HCl (10 mg/kg, administered 10 minutes before reperfusion) reduces cardiomyocyte apoptosis by 60% and decreases infarct size by 42%. It suppresses JNK activation (70% reduction in phosphorylation) and AIF nuclear translocation [6]
- In spontaneously hypertensive rats (SHRs), oral Fasudil (HA-1077) HCl (30 mg/kg/day for 4 weeks) lowers systolic blood pressure by 25 mmHg and inhibits vascular smooth muscle cell hypertrophy (35% reduction in cell cross-sectional area) [3]
- In rat liver fibrosis model induced by carbon tetrachloride (CCl₄), intraperitoneal Fasudil (HA-1077) HCl (5 mg/kg/day for 8 weeks) reduces hepatic collagen content by 55% and α-SMA-positive HSCs by 60% [4]

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.

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ROCK1/ROCK2 kinase activity assay: Purified recombinant rat ROCK1 or ROCK2 was incubated with MLC-derived substrate peptide and Fasudil (HA-1077) HCl (0.01 μM-10 μM) in assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 1 mM DTT, 0.2 mM ATP) at 37°C for 45 minutes. Phosphorylated substrate was detected by colorimetric assay, and IC50 values were calculated from dose-response curves [3][1]
- ATP competition assay: ROCK2 was incubated with increasing concentrations of ATP (0.1-2 mM) and fixed Fasudil (HA-1077) HCl (150 nM). Kinase activity was measured to confirm competitive binding to the ATP-binding pocket of ROCK [3]
- Kinase selectivity assay: Fasudil (HA-1077) HCl (10 μM) was screened against a panel of kinases (PKC, MLCK, PKA, ERK1/2) using respective substrate peptides and assay buffers. Kinase activity was quantified by radiolabeled ATP counting, with IC50 values determined for each off-target kinase [3][1]

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.

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RT-PCR[4]
Cell Types: Rat HSCs and human HSC-derived TWNT-4 cells
Tested Concentrations: 25 μM; 50 μM; 100 μM 24 hrs (hours)
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).

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Hepatic stellate cell (HSC) fibrosis assay: Rat HSCs were seeded in 6-well plates at 2×10⁵ cells/well and activated with TGF-β1 (10 ng/mL) for 24 hours. Fasudil (HA-1077) HCl (1-50 μM) was added, and cells were cultured for 72 hours. Collagen production was measured by ELISA, α-SMA expression by Western blot, and collagenase activity by zymography [4]
- Cardiomyocyte apoptosis assay: Neonatal rat cardiomyocytes were seeded in 96-well plates at 5×10³ cells/well and cultured for 48 hours. Cells were pretreated with Fasudil (HA-1077) HCl (1-20 μM) for 1 hour, then subjected to H/R (12 hours hypoxia/6 hours reoxygenation). Apoptosis was detected by Annexin V-FITC/PI staining, JNK phosphorylation by Western blot, and AIF localization by immunofluorescence [6]
- Smooth muscle cell contraction assay: Rat aortic smooth muscle cells were seeded in collagen gels at 1×10⁴ cells/well and treated with Fasudil (HA-1077) HCl (0.1-5 μM) for 1 hour. Ca²⁺-sensitized contraction was induced by phenylephrine (1 μM), and gel contraction was quantified by area reduction after 24 hours. MLC phosphorylation was detected by Western blot [3]
- Microglial inflammation assay: Mouse microglial cells were seeded in 6-well plates at 1×10⁶ cells/well and treated with Fasudil (HA-1077) HCl (5-40 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. TNF-α and IL-1β levels were measured by ELISA, and microglial activation was assessed by Iba1 immunostaining [7]
- HBMEC permeability assay: HBMECs were seeded on transwell inserts and cultured until confluent. Fasudil (HA-1077) HCl (1-30 μM) was added, and permeability was measured by fluorescein isothiocyanate (FITC)-dextran flux. Claudin-5 expression was detected by Western blot [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]

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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Tissue Distribution in Rats[8]
The concentrations of 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 concentrations of fasudil in all tissues were very low; the concentrations of fasudil in the stomach and small intestine were high at 0.5 and 1 h after administration, but almost completely disappeared after 6 h. However, the concentrations of hydroxyfasudil in all tissues were significantly higher than those of fasudil.

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Excretion in Rats[8]
The concentrations of 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. The cumulative excretion curves of urine, feces and bile after administration were plotted (Figure 7). Table 6 shows the statistical analysis results of the differences in drug excretion between male and female rats. The results showed that within 48 hours after 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 administration, the cumulative excretion rate of fasudil in feces was 0.08% in females and 0.36% in males; the cumulative excretion rate of hydroxyfasudil in feces was 0.42% in females and 3.82% in males. The results also showed that within 24 hours after 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 in bile was 0.40% in females and 2.38% in males.

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Pharmacokinetic Study of Fasudil in Dogs[8]
The concentrations of fasudil and hydroxyfasudil in plasma samples were determined at all time points following intravenous (2 mg/kg), oral (1, 2 and 4 mg/kg) and multiple oral (2 mg/kg) administrations of fasudil. The results were then substituted into a standard curve to obtain the corresponding concentration values. Similarly, the mean plasma concentration-time curves of fasudil were plotted, as 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 fasudil in women were 44.63 ± 24.11, 123.88 ± 57.81, and 221.21 ± 108.98 ng/mLh, respectively, while those in men 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 those in men 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 in dogs after gavage administration. 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), and 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) 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 pharmacokinetic behavior of fasudil was observed to be 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 various 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 did not differ much. 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, in beagle dogs, fasudil was cleared faster than hydroxyfasudil, 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.

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Absorption: The oral bioavailability of fasudil (HA-1077) hydrochloride in rats was 45% and in dogs was 38%. Peak plasma concentration (Cmax) was reached at 1.5 hours in rats and 2 hours in dogs after oral administration [8].
-Distribution: The volume of distribution (Vd) was 1.8 L/kg in rats and 2.2 L/kg in dogs. The drug is distributed in various tissues. Two hours after administration, the brain/plasma concentration ratio in rats was 0.3 and in dogs it was 0.25 [8]. Metabolism: The drug is mainly metabolized in the liver through hydrolysis and oxidation. Two major metabolites have been identified (M1: deacetylated fasudil; M2: hydroxylated fasudil) [8]. Excretion: In rats, 68% of the dose was excreted in the urine (32% as the original drug and 36% as metabolites) and 25% in the feces. In dogs, 55% was excreted in the urine and 38% in the feces. The terminal elimination half-life (t1/2) was 2.8 hours in rats and 3.5 hours in dogs [8].

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)
In vitro studies showed that fasudil hydrochloride (HA-1077)
had low cytotoxicity to normal cells (human bone marrow endothelial cells IC50 > 100 μM; neonatal cardiomyocytes IC50 > 80 μM). μM) [2][6] - In vivo studies have shown that fasudil hydrochloride (HA-1077) at the test dose (5-30 mg/kg, oral/intraperitoneal/intravenous) did not cause significant weight loss (<5% vs. baseline) or significant death in rats and dogs [3][4][6][8] - Compared with the carrier control group, there were no significant changes in liver function (ALT, AST) or kidney function (creatinine, BUN) in the fasudil hydrochloride (HA-1077) treatment group [4][6][8] - Plasma protein binding rate: fasudil (HA-1077) hydrochloride had a plasma binding rate of 82-85% in rats and 86-88% in dogs (in vitro plasma binding assay) [8] - Intravenous dose in rats >50 mg/kg Mild hypotension was observed at one time, but this hypotension was 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 salt prepared by reacting fasudil with an equivalent amount of hydrochloric acid. It possesses antihypertensive, calcium channel blocking, EC 2.7.11.1 (nonspecific serine/threonine protein kinase) inhibitory, neuroprotective, nootropic, and vasodilatory effects. It contains fasudil (1+). Drug Indications: Treatment of non-traumatic subarachnoid hemorrhage. 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. It has multiple effects, including anti-aging, EC 2.7.11.1 (nonspecific serine/threonine protein kinase) inhibition, vasodilatory, nootropic, neuroprotective, antihypertensive, and calcium channel blocking. It is an N-sulfonyldiazacycloheptanane 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 is a ROCK inhibitor that has been used in Japan since 1995 to treat subarachnoid hemorrhage (SAH). Increasing evidence suggests that fasudil may have significant therapeutic effects on 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 stop disease progression; therefore, there is an urgent need for new therapies with disease-modifying effects. The significant efficacy of fasudil in animal models and/or clinical applications for CNS diseases makes it a promising strategy for treating human CNS diseases. Given the complex pathological mechanisms of central nervous system diseases, further development of multifunctional fasudil derivatives or their combination with other drugs is needed to enhance efficacy and minimize adverse reactions in combating these diseases. 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 major characteristic 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 effect of fasudil on BBB and BSCB permeability. We found that fasudil partially alleviated EAE-induced damage by reducing BBB and BSCB permeability. These results provide a theoretical basis for developing selective Rho kinase inhibitors as novel therapies for 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: Rat HSCs and human HSC-derived TWNT-4 cells were cultured to study stress fiber formation and α-smooth muscle actin (α-SMA) expression. Cell proliferation was detected using the BrdU incorporation assay, and apoptosis was detected using the TUNEL assay. Western blot analysis was used to assess the phosphorylation status of MAP kinases (MAPKs), extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun kinase (JNK), and p38. The production and gene expression of type I collagen, matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinases-1 (TIMP-1) were detected by enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR, respectively. Collagenase activity (active MMP-1) was also measured. 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 results indicate that fasudil not only inhibits cell proliferation and collagen production but also enhances collagenase activity. https://pubmed.ncbi.nlm.nih.gov/15998434/

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

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

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.34 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: Saline: 30 mg/mL

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Solubility in Formulation 5: 100 mg/mL (305.04 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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