ROCK2 (IC50 = 105 nM); ROCK1 (IC50 = 24 μM)

Belumosudil (SLx-2119; 40 µM) significantly reduces the levels of Tsp-1 and CTGF mRNA in PASMC. Five times more background is seen in the microarray hybridized with aRNA from HMVEC treated with belumosudil than in the other arrays[1].
Belumosudil (KD025, SLx-2119)  selectivity: Radiometric enzyme assays confirmed that SLx-2119 selectively inhibits activity of human ROCK2 (IC50 = 105 nM), while effects on human ROCK1 in this cell-free system were minimal (IC50 = 24 µM).

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This study was designed to compare gene expression profiles of atorvastatin with the newly developed ROCK2 inhibitor Belumosudil (KD025, SLx-2119)  in primary cultures of normal human endothelial cells, smooth muscle cells, and fibroblasts. Cells were treated with each compound for 24 h, after which total RNA was isolated and genome-wide gene-expression profiles were obtained with Illumina arrays. Because of the known effect of statins on the actin cytoskeleton and on connective tissue growth factor, a prominent growth factor involved in tissue fibrosis, the effects of SLx-2119 and atorvastatin on the actin cytoskeleton and connective tissue growth factor mRNA were also examined in cultures of smooth muscle cells with a fibrotic phenotype, isolated from biopsies of human intestine with radiation-induced fibrosis. Although SLx-2119 and atorvastatin affected expression of genes belonging to the same biological processes, individual genes were mostly different, consistent with synergistic or additive properties. Both SLx-2119 and atorvastatin reduced connective tissue growth factor mRNA and remodeled the actin cytoskeleton in fibrosis-derived smooth muscle cells, suggesting that both compounds have antifibrotic properties. These results form the basis for further studies to assess the possible therapeutic benefit of combined treatments [1].

Belumosudil (KD-025; 100, 200 or 300 mg/kg, i.p.) decreases the infarct volume dose-dependently following a brief middle cerebral artery blockage. Belumosudil works in aged, diabetic, or female mice just as well as it does in healthy adult male mice[2].

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Belumosudil (KD025, SLx-2119) dose-dependently reduced infarct volume after transient middle cerebral artery occlusion. The therapeutic window was at least 3 hours from stroke onset, and the efficacy was sustained for at least 4 weeks. KD025 was at least as efficacious in aged, diabetic or female mice, as in normal adult males. Concurrent treatment with atorvastatin was safe, but not additive or synergistic. KD025 was also safe in a permanent ischemia model, albeit with diminished efficacy. As one mechanism of protection, KD025 improved cortical perfusion in a distal middle cerebral artery occlusion model, implicating enhanced collateral flow. Unlike isoform-nonselective ROCK inhibitors, KD025 did not cause significant hypotension, a dose-limiting side effect in acute ischemic stroke. Interpretation: Altogether, these data show that KD025 is efficacious and safe in acute focal cerebral ischemia in mice, implicating ROCK2 as the relevant isoform in acute ischemic stroke. Data suggest that selective ROCK2 inhibition has a favorable safety profile to facilitate clinical translation[2].

Radiometric truncated enzyme ROCK1 and ROCK2 assays[1]
Cell-free enzyme assays were performed to determine the selective inhibition of ROCK1 and ROCK2 by SLx-2119. Reactions were performed on non-binding surface microplates. Four mU of human ROCK1 and ROCK2 were used to phosphorylate 30 µM of the synthetic ROCK peptide substrate S6 Long (sequence: KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK), prepared at American Peptide with the addition of 10 µM ATP, containing 33P-ATP in the presence of 10 mM Mg2+, 50 mM Tris, pH 7.5, 0.1 mM EGTA and 1 mM DTT at room temperature. One unit is the amount of kinase needed to catalyze the transfer of 1 nmol phosphate/min to the peptide. The reactions were allowed to proceed for 45 minutes and then stopped with 3% phosphoric acid to a final concentration of 1%. The reactions were captured on phospho cellulose filtration microplates and washed with 75 mM phosphoric acid and methanol using a vacuum manifold. Phosphorylation was measured on a Perkin-Elmer MicroBeta 1450.

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Recombinant ROCK1 and ROCK2 assays[2]
Compound dilutions and reactions were performed in 96-well polystyrene low-binding plates. Filtration was done in 96-well filter plates containing hydrophilic phospho-cellulose cation exchanger membranes. Enzymatic activity of the recombinant ROCK1 and ROCK2 was measured radiometrically in 50 μL of reaction mixture containing assay buffer (50 mmol/L Tris, pH 7.5, 0.1 mmol/L ethyleneglycoltetraacetic acid, 10 mmol/L magnesium acetate and 1 mmol/L dithiothreitol). Long S6 peptide (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK, 30 μmol/L), ROCK (4 mU per reaction) and ATP (10 μmol/L, 1 μCi [γ-33P]ATP) and test compound were diluted to a final dimethylsulfoxide concentration of 1%. The reaction was incubated for 45 min at room temperature and stopped with 25 μL of 3% phosphoric acid. Phosphorylated long S6 peptide was separated from unreacted [γ-33P]ATP by filtration of the quenched reaction contents through a P30 phosphocellulose filter plate using the Millipore Multiscreen® vacuum manifold system. Each filter was washed three times with 75 μL of 75 mmol/L phosphoric acid and one time with 30 μL of 100% methanol. Filter plates were allowed to dry and 30 μL of OptiPhase ‘SuperMix’ scintillation fluid was added to each well. 33Phosphorous was quantified in an I450 MicroBeta scintillation counter and corrected by subtracting the radioactivity associated with the background samples. Data were analyzed and expressed as percent inhibition using the formula ((U − B)/(C − B)) × 100 where U is the unknown value, B is the average of staurosporine background wells, and C is the average of control wells. Curve fitting was performed by GraphPad Prism software using sigmoidal dose-response (variable slope) equation type analysis to generate IC50 values. Ki values were calculated from an equation of Ki = IC50/(1 + [S]/Km)), where [S] and Km are the concentration of ATP and the Km value of ATP, respectively.

The ROCK2-inhibitor, SLx-2119, was dissolved in DMSO to obtain a stock solution of 20 mM.[1] Human microvascular endothelial cells, PASMC, and NHDF at passage 7, and N-SMC and RE-SMC at passage 4, were plated in 6 cm culture dishes with 3 ml culture medium, at a density of 1×106 cells/dish. After 2 days (confluence 90%) the cells were incubated for 24 hours in 3 ml culture media containing vehicle (10 µl sterile PBS), 10 µM atorvastatin, a combination of 10 µM atorvastatin and 500 µM mevalonate, 10 µM SLx-2119, or 40 µM SLx-2119. Three independent experiments were performed, with 3 culture dishes in each treatment group. [1] RNA isolation[1] Twenty-four hours after treatment of HMVEC, PASMC and NHDF with vehicle, SLx-2119, atorvastatin, or atorvastatin combined with mevalonate, total RNA was isolated using Ultraspec RNA isolation reagent, according to the manufacturer’s instructions. Two µg of RNA was kept for microarray analysis (including quality control analysis) and 2 µg was used for real-time PCR.[1] Twenty-four hours after treatment of N-SMC and RE-SMC with vehicle, SLx-2119, atorvastatin, or atorvastatin combined with mevalonate, total RNA was isolated as described before. This RNA was used for real-time PCR.

Animals and drug treatments[2]
Young adult (C57BL/6, 2–3 months old, male 22–30 g, female 16–23 g), aged (C57BL/6, 12 months old, 33–52 g), or type 2 diabetic mice (db/db, B6.BKS(D)-Lepr db/J, 2–3 months old, male, 33–50 g) were used in all experiments. Only one animal was excluded due to technical failure (hemorrhage during filament middle cerebral artery occlusion [fMCAO] in db/db mouse assigned to the vehicle group). KD025 (formerly SLx-2119) was kindly provided by Kadmon Corporation (New York, NY). Vehicle (0.4% methylcellulose) or KD025 (100, 200 or 300 mg/kg) was administered every 12 h via orogastric gavage. The dosing paradigm was chosen based on the pharmacokinetic profile after oral administration in mice (see below). Atorvastatin (4 mg/mL) was dissolved in phosphate-buffered saline (pH 7.4) containing 45% 3-hydroxypropyl-B-cyclodextrin and 10% ethanol, and administered at a dose of 20 mg/kg per day as a single daily intraperitoneal injection for 2 weeks as previously described.

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Pharmacokinetic studies[2]
We measured plasma and brain concentrations of KD025 in male mice. Animals received 100 or 200 mg/kg KD025 twice a day for a total of five doses via orogastric gavage. Blood and brain tissue were collected at different time points after the last dose. For each time point, a different group of mice was sacrificed (n = 5 each). Whole blood was collected via jugular vein into K3 ethylenediaminetetraacetic acid (EDTA) tubes, and centrifuged at 1000 g for 3 min at 4°C. Immediately following blood collection, mice were perfused with saline through the left ventricle to clear intravascular blood, and brains were harvested. All samples were stored at −80°C until analysis. Plasma and tissue KD025 concentrations were measured using high-resolution mass spectrometry. Pharmacokinetic parameters were calculated using PKSolver.22 A noncompartmental analysis was performed. The slope of the terminal log-linear part of the concentration versus time curve (λz) was calculated using the best-fit method. In addition, a one-compartmental analysis was performed for zero-or first-order kinetic models.

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Dissolved in 0.4% methylcellulose; 300 mg/kg; oral gavage
Type 2 diabetic mice

Absorption, Distribution and Excretion
Following oral administration, the mean bioavailability of berumosudil is 64%, with a median time to peak plasma concentration (Tmax) ranging from 1.26 to 2.53 hours. Compared to fasting administration, co-administration with a high-fat, high-calorie meal increased the peak plasma concentration (Cmax) and area under the curve (AUC) of berumosudil by 2.2-fold and 2-fold, respectively. Berumosudil is primarily excreted in feces. In healthy subjects, approximately 85% of the radioactive material is recovered in feces after oral administration of radiolabeled berumosudil, of which 30% is unmetabolized parent drug, and less than 5% is recovered in urine. The mean geometric volume of distribution after a single oral dose of berumosudil in healthy subjects is 184 L. The mean clearance of berumosudil is 9.83 L/h.

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Metabolism/Metabolites
Berumosudil is primarily metabolized via CYP3A4 in vitro, followed by CYP2C8, CYP2D6, and UGT1A9. The specific metabolites produced by berumosudil metabolism are not yet clear.

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Biological Half-Life
The mean elimination half-life of berumosudil after oral administration is 19 hours.

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Pharmacokinetic Characteristics[2]
To guide the selection of dosage and dosing interval, we determined the pharmacokinetic characteristics of KD025 in mice. We administered the drug twice daily via an oral tube for 2 consecutive days and measured the drug concentration in blood and brain tissue at predetermined time points, starting 48 hours before the last administration (time 0; Figure 2). We used non-compartmental model analysis as well as zero-order and first-order kinetic absorption models for single-compartmental model analysis (Table 2). Plasma drug concentrations conformed more closely to a first-order absorption model (R² = 0.98, Akaike Information Criterion [AIC] = 8.31), while brain tissue drug concentrations conformed more closely to a zero-order absorption model (R² = 0.98, AIC = 6.52). Peak drug concentrations in both plasma and brain tissue were reached within 2 hours of administration and were nearly 10 times higher than the in vitro IC50 values. Based on the area under the brain/plasma concentration-time curve (AUC) ratio, brain exposure was approximately 5% of plasma exposure. The brain half-life was shorter than that in plasma (2 hours vs. 5 hours), likely due to the higher elimination constant, volume of distribution, and clearance rate in the brain. The observed mean residence times in brain and plasma were 4 hours and 7 hours, respectively, indicating that the compound did not accumulate in vivo within the dosing interval selected in this study (accumulation factors [R] in plasma and brain were 1.15 and 1.02, respectively). Nevertheless, a dose level of 200 mg/kg maintained plasma and tissue concentrations for at least 12 hours. In summary, these data suggest that the selected dose level and twice-daily dosing regimen are suitable for testing the efficacy and safety of the drug in an ischemic model.

Hepatotoxicity
In open-label premarketing clinical trials of berumoshudil for the treatment of patients with refractory chronic graft-versus-host disease (GvHD), up to 7% of subjects experienced elevated serum transaminases. These elevations were generally mild and transient, with only 1% to 2% of patients experiencing elevations exceeding 5 times the upper limit of normal (ULN). These elevations sometimes led to premature discontinuation of treatment, but in most cases, they resolved spontaneously without dose adjustment. No clinically significant liver injury associated with berumoshudil was observed in the premarketing studies. Since the approval and widespread use of berumoshudil, no reports of hepatotoxicity related to its use have been published. Probability Score: E (Unlikely to cause clinically significant liver injury). Protein Binding Berumoshudil appears to bind extensively to a variety of proteins in plasma—in vitro studies showed binding rates of 99.9% to serum albumin and 98.6% to α1-acid glycoprotein, respectively.

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Safety of use in combination with statins[2]
Statins inhibit the ROCK signaling pathway by reducing the synthesis of isoprene intermediates in cholesterol metabolism, which are essential for Rho activation. This is thought to at least partially explain the pleiotropic effects of statins. Therefore, Belumosudil (KD025, SLx-2119) may have an additive or synergistic effect with statins, which may pose a potential safety risk. We tested it in mice pretreated with atorvastatin (20 mg/kg/day) for 2 weeks. KD025 was safe in mice pretreated with atorvastatin but did not show an additive or synergistic effect (Figure 8A).

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Safety in permanent ischemia[2]
Although most cerebral artery occlusions eventually recanalize, it is impossible to predict whether the occlusion will persist in the hyperacute phase. If the drug is unsafe in the absence of reperfusion, this would hinder its administration in the field during the hyperacute phase, further delaying the start of treatment until imaging confirms recanalization. Therefore, we tested the safety of Belumosudil (KD025, SLx-2119) in a permanent fMCAO model. Given the high mortality rate over time in this model, we assessed the infarct volume 24 hours after ischemia to minimize additional loss. As expected, the permanent model (Fig. 8B) had a larger infarct volume compared to the transient fMCAO model (see Figs. 3, 5). KD025 was safe but lost its efficacy in the case of persistent arterial occlusion.

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Other safety endpoints[2]
Hemorrhagic transformation, weight loss, and mortality were recorded in all trials. Except for increased weight loss when Belumosudil (KD025, SLx-2119) was used in combination with atorvastatin (Table 3; Fig. S1), Belumosudil (KD025, SLx-2119) did not significantly alter these safety endpoints in any trial group. Since we did not have a sham surgery group, it is unclear whether this weight loss was directly related to ischemia.

[1]. Blood Coagul Fibrinolysis . 2008 Oct;19(7):709-18.

[2]. Ann Clin Transl Neurol . 2014 Jan 1;1(1):2-14.

Belumosudil mesylate is a small molecule drug that has completed the most Phase IV clinical trials (covering all indications) and was first approved in 2021 for the treatment of graft-versus-host disease (GVHD), with one other indication under investigation. Belumosudil is used to treat chronic GVHD and is also being studied for the treatment of pulmonary hypertension. It is a Rho-associated coiled-coil protein kinase (ROCK) inhibitor with significantly higher selectivity for ROCK2 than ROCK1 (IC50 values of 100 nM and 3 μM, respectively). In the treatment of GVHD, donor T cells begin attacking recipient tissues after allogeneic hematopoietic stem cell transplantation (HSCT), and belumosudil helps resolve immune dysregulation by modulating the balance between Th17 cells and regulatory T cells, thereby suppressing the sometimes fatal inflammatory cascade. Berumoshudil was first approved by the U.S. Food and Drug Administration (FDA) in July 2021 under the brand name Rezurock for the treatment of patients with chronic GVHD who have failed at least two prior systemic therapies. In July 2022, berumoshudil was approved by Health Canada under the brand name RHOLISTIQ for the treatment of the same disease in adults and children aged 12 years and older. Berumoshudil is an orally bioavailable Rho-associated coiled-coil kinase 2 (ROCK2; ROCK-II) inhibitor with potential immunomodulatory activity. After oral administration, berumoshudil binds to and inhibits the serine/threonine kinase activity of ROCK2. This inhibits the ROCK2-mediated signal transduction pathway and modulates various pro-inflammatory and anti-inflammatory immune cellular responses by regulating the phosphorylation of signal transduction and transcription activator factors 3 and 5 (STAT3/STAT5). This downregulates pro-inflammatory Th17 cells and increases the number of regulatory T cells (Tregs). Berumosudil also inhibits ROCK2-mediated fibrotic processes, including stress fiber formation, myofibroblast activation, and transcription of pro-fibrotic genes. ROCK2 is upregulated in a variety of diseases, including various fibrotic diseases, neurodegenerative diseases, and autoimmune diseases.

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Berumosudil appears to inhibit multiple pro-fibrotic and pro-inflammatory processes, thereby preventing and treating damage caused by graft-versus-host disease. Given its mechanism of action and animal studies, berumosudil is considered to have embryo-fetal toxicity, and exposure to this drug in pregnant women may cause serious harm to the developing fetus.

Female patients of childbearing potential or male patients whose partners are women of childbearing potential should be advised to use effective contraception during treatment with berumosudil and for one week after the last dose.

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Absorption
After oral administration, the mean bioavailability of berumosudil is 64%, and the median time to peak plasma concentration (Tmax) is 1.26 to 2.53 hours. Compared with fasting administration, co-administration with a high-fat, high-calorie meal increases the peak plasma concentration (Cmax) and area under the curve (AUC) of berumosudil by 2.2-fold and 2-fold, respectively.
Excretion
Berumosudil is primarily excreted in feces. After oral administration of radiolabeled berumosudil to healthy subjects, approximately 85% of the radioactive material is recovered in feces, of which 30% is unmetabolized parent drug, and less than 5% is recovered in urine.
Volume of Distribution
The mean geometric volume of distribution after a single oral dose of berumosudil in healthy subjects was 184 L.
Clearance
The mean clearance of berumosudil was 9.83 L/h.
Metabolism/Metabolites
Berumosudil is primarily metabolized via CYP3A4 in vitro, followed by CYP2C8, CYP2D6, and UGT1A9. The specific metabolites produced by berumosudil metabolism are not yet clear.
Biological Half-Life
The mean elimination half-life after oral administration of berumosudil was 19 hours.
Protein Binding
Berumosudil appears to bind extensively to a variety of proteins in plasma—in vitro studies showed protein binding rates of 99.9% to serum albumin and 98.6% to α1-acid glycoprotein, respectively. Mechanism of Action
Chronic graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation, in which transplanted donor T cells recognize recipient tissue as foreign and trigger an immune response. During pre-transplant conditioning protocols (e.g., including radiation or chemotherapy), host tissue may be damaged, leading to downstream inflammatory responses and the production of inflammatory mediators such as TNF-α and IL-1. These cytokines can increase the expression of host major histocompatibility complex (MHC) antigens and adhesion molecules, thereby enhancing the ability of mature donor T cells to recognize these molecules. Activation of these donor T cells leads to activation of mononuclear phagocytes, whose effector functions are triggered by stimulating molecules generated by damage during the treatment conditioning phase. Activated macrophages and cytotoxic T lymphocytes begin to directly lyse target cells and/or induce their apoptosis, ultimately leading to local tissue damage and further inflammatory responses. Berumosudil is an inhibitor of Rho-associated coiled-coil kinase 2 (ROCK2), a protein that plays a key role in the pathogenesis of immune and fibrotic diseases. Studies have shown that ROCK2 inhibitors can address immune dysregulation by regulating the phosphorylation of STAT3 and STAT5, downregulating pro-inflammatory Th17 cells and upregulating regulatory T cells. Rho kinase (ROCK) inhibitors are a relatively new class of drugs with potential therapeutic value in oncology, neurology, fibrosis, and cardiovascular diseases. ROCK inhibitors can modulate multiple cellular functions, some of which are similar to the pleiotropic effects of statins, suggesting a possible additive or synergistic effect. Current research mainly uses compounds that can simultaneously inhibit both ROCK1 and ROCK2 subtypes. This study aimed to compare the gene expression profiles of atorvastatin and the newly developed ROCK2 inhibitor SLx-2119 in primary cultures of normal human endothelial cells, smooth muscle cells, and fibroblasts. After treating cells with each compound for 24 hours, total RNA was extracted, and whole-genome gene expression profiles were obtained using Illumina microarrays. Since statins are known to affect the actin cytoskeleton and connective tissue growth factor (an important growth factor involved in tissue fibrosis), this study also examined the effects of SLx-2119 and atorvastatin on the actin cytoskeleton and connective tissue growth factor mRNA. These smooth muscle cells had a fibrotic phenotype and were isolated from human intestinal biopsy tissues with radiation-induced fibrosis. Although SLx-2119 and atorvastatin affected the expression of genes belonging to the same biological processes, the expression of individual genes was mostly different, which is consistent with synergistic or additive effects. Both SLx-2119 and atorvastatin reduced the expression of connective tissue growth factor mRNA and remodeled the actin cytoskeleton in fibrotic smooth muscle cells, indicating that both compounds have anti-fibrotic properties. These results lay the foundation for further research on the potential efficacy of combination therapy. [1]

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Objective: Rho-associated kinase (ROCK) is a key regulator of many processes in various cell types that are closely related to the pathophysiology of stroke. ROCK inhibitors have improved outcomes in experimental models of acute ischemic or hemorrhagic stroke. However, the ROCK subtypes (ROCK1 or ROCK2) associated with acute stroke remain unclear. [2]
Methods: We characterized the pharmacodynamics and pharmacokinetics of a novel selective ROCK2 inhibitor, KD025 (formerly SLx-2119), and tested its efficacy and safety in a mouse model of focal cerebral ischemia. [2]
Results: KD025 reduced infarct volume in a dose-dependent manner following transient middle cerebral artery occlusion. The treatment window was at least 3 hours after stroke onset and the efficacy lasted at least 4 weeks. KD025 was at least as effective as normal adult male mice in aged, diabetic, or female mice. Combination therapy with atorvastatin was safe, but no additive or synergistic effects were observed. KD025 was also safe in a model of permanent ischemia, although efficacy was reduced. As a protective mechanism, KD025 improved cortical perfusion in a distal middle cerebral artery occlusion model, suggesting enhanced collateral circulation. Unlike non-selective ROCK inhibitors, KD025 did not cause significant hypotension, a common dose-limiting side effect in acute ischemic stroke. [2] Conclusion: In summary, these data indicate that KD025 is effective and safe in a mouse model of acute focal cerebral ischemia, suggesting that ROCK2 is a relevant subtype of acute ischemic stroke. The data suggest that selective ROCK2 inhibitors have good safety profiles and are beneficial for clinical translation.

C27H28N6O5S

548.6134

548.184189

C, 59.11; H, 5.14; N, 15.32; O, 14.58; S, 5.84

2109704-99-4

Belumosudil;911417-87-3

146681181

Light yellow to yellow solid powder

4

9

7

39

770

0

CC(C)NC(=O)COC1=CC=CC(=C1)C2=NC3=CC=CC=C3C(=N2)NC4=CC5=C(C=C4)NN=C5.CS(=O)(=O)O

ILQJXEIRBCHLOM-UHFFFAOYSA-N

InChI=1S/C26H24N6O2.CH4O3S/c1-16(2)28-24(33)15-34-20-7-5-6-17(13-20)25-30-23-9-4-3-8-21(23)26(31-25)29-19-10-11-22-18(12-19)14-27-32-22;1-5(2,3)4/h3-14,16H,15H2,1-2H3,(H,27,32)(H,28,33)(H,29,30,31);1H3,(H,2,3,4)

2-(3-(4-(1H-indazol-5-ylamino)quinazolin-2-yl)phenoxy)-N-(propan-2-yl)acetamide

Belumosudil mesylate; KD-025; Belumosudil mesylate (KD025 mesylate); Belumosudil (mesylate); Rezurock; Belumosudil/KD-025; CHEMBL4802130;KD 025; KD025; WHO-11343; WHO 11343; WHO11343; SLx2119; SLx-2119; SLx 2119

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)

DMSO: ~12.5 mg/mL (~22.8 mM)

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.)