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

Tsp-1 and CTGF mRNA levels in PASMC are significantly down-regulated by belumosudil (SLx-2119; 40 µM). Compared to the other arrays, the microarray hybridized with aRNA from HMVEC treated with belumosudil has a 5-times greater background[1].

After temporary middle cerebral artery blockage, belumosudil (KD-025; 100, 200, or 300 mg/kg, ip) dose-dependently decreases the infarct volume. Belumosudil works just as well in old, diabetic, or female mice as it does in healthy adult male mice[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.

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 belumosudil is 64% and the median Tmax at steady-state is 1.26 to 2.53 hours. As compared to administration in a fasted state, belumosudil Cmax and AUC increased by 2.2 and 2 times, respectively, when administered with a high-fat, high-calorie meal.
Belumosudil is eliminated primarily in the feces. Following the administration of a radiolabeled oral dose of belumosudil in healthy subjects, approximately 85% of the radioactivity was recovered in the feces, 30% of which was unchanged parent drug, with less than 5% recovered in the urine.
Following a single oral dose of belumosudil in healthy subjects, the mean geometric volume of distribution was 184 L.
The mean clearance of belumosudil is 9.83 L/h.

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Metabolism / Metabolites
The _in vitro_ metabolism of belumosudil occurs primarily via CYP3A4 and to a lesser extent by CYP2C8, CYP2D6, and UGT1A9. The specific metabolites generated by belumosudil metabolism remain unclear.

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

Hepatotoxicity
In the open label prelicensure clinical trials of belumosudil in patients with refractory chronic GvHD, serum aminotransferase elevations occurred in up to 7% of treated subjects. The elevations were typically mild and transient and values above 5 times the upper limit of normal (ULN) occurred in only 1% to 2% of patients. The elevations occasionally led to early discontinuations, but more often resolved even without dose adjustment. In prelicensure studies, there were no instances of clinically apparent liver injury attributed to belumosudil. Since approval and more widescale availability of belumosudil, there have been no published reports of hepatotoxicity associated with its use.
Likelihood score: E (unlikely to be a cause of clinically apparent liver injury).

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Protein Binding
Belumosudil appears to be extensively protein-bound in plasma - _in vitro_ protein binding to serum albumin and alpha-1-acid glycoprotein was found to be 99.9% and 98.6%, respectively.

[1]. Comparative gene expression profiling in three primary human cell lines after treatment with a novel inhibitor of Rho kinase. Blood Coagul Fibrinolysis. 2008 Oct;19(7):709-18.

[2]. Selective ROCK2 Inhibition In Focal Cerebral Ischemia. Ann Clin Transl Neurol. 2014 Jan 1;1(1):2-14.

Pharmacodynamics
Belumosudil appears to inhibit several pro-fibrotic and pro-inflammatory processes in order to prevent and treat the damage incurred by graft-versus-host disease. Given its mechanism of action and findings in animal trials, belumosudil is considered to carry embryo-fetal toxicity and may cause significant harm to a developing fetus should a pregnant mother be exposed. Female patients of reproductive potential, or male patients with female partners of reproductive potential, should be advised to use effective contraception during treatment with belumosudil and for one week after the last dose.

C26H24N6O2

452.51

452.196

C, 69.01; H, 5.35; N, 18.57; O, 7.07

911417-87-3

Belumosudil mesylate;2109704-99-4

11950170

Typically exists as Off-white to light brown solids at room temperature

1.3±0.1 g/cm3

682.6±55.0 °C at 760 mmHg

366.6±31.5 °C

0.0±2.1 mmHg at 25°C

1.705

3.61

3

6

7

34

678

0

O=C(COC1C=C(C2N=C(NC3C=C4C(NN=C4)=CC=3)C3C(=CC=CC=3)N=2)C=CC=1)NC(C)C

GKHIVNAUVKXIIY-UHFFFAOYSA-N

InChI=1S/C26H24N6O2/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/h3-14,16H,15H2,1-2H3,(H,27,32)(H,28,33)(H,29,30,31)

2-[3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy]-N-(1-methylethyl)-acetamide

Belumosudil; KD-025; SLx-2119; KD025; SLx2119; KD 025; SLx 2119; SLx-2119; 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide; SLx 2119; UNII-834YJF89WO; ROCK inhibitor;

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.60 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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: 3.33 mg/mL (7.36 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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