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].
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].

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].

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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.

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.

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Pharmacokinetic profile [2]
To guide the dose and dose interval selection, we determined the pharmacokinetic profile of KD025 in mice. We administered the drug via orogastric gavage twice a day for 2 days and measured blood and brain tissue levels at predetermined time points starting immediately before the last dose at 48 h (time 0; Fig.2). We used both noncompartmental analysis, and zero and first-order kinetic absorption models for one-compartmental analysis (Table2). Plasma drug levels fitted better to the first-order absorption model (R2 = 0.98, Akaike Information Criterion [AIC] = 8.31), whereas the brain drug levels fitted better to zero order absorption model (R2 = 0.98, AIC = 6.52). Peak plasma and brain concentrations were reached within 2 h of dosing, and exceeded the in vitro IC50 by almost 10-fold. Brain exposure was ∼5% of plasma exposure based on brain/plasma area under the concentration (AUC) ratio. Half-life was shorter in the brain than plasma (2 vs. 5 h), presumably due to the higher elimination constant, distribution volume, and clearance rate for the brain. Observed mean residence time was 4 and 7 h for brain and plasma, respectively, suggesting that the compound did not accumulate in the body at the dosing interval selected in this study (accumulation factor [R] 1.15 and 1.02 for plasma and brain, respectively). Nevertheless, 200 mg/kg dose level provided sustained plasma and tissue concentrations for at least 12 h. Altogether, these data suggest that the selected dose levels and twice a day dosing paradigm were appropriate to test efficacy and safety in ischemia models.

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.

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Safety in combination with statins [2]
Statins inhibit ROCK signaling by reducing the synthesis of isoprenyl intermediates of cholesterol metabolism that are critical for Rho activation. This is believed to be responsible, at least in part, for the pleiotropic actions of statins. Therefore, Belumosudil (KD025, SLx-2119) may have additive or synergistic interactions with statins that may potentially be unsafe. We tested this in mice pretreated with atorvastatin (20 mg/kg per day) for 2 weeks. KD025 was safe in atorvastatin-pretreated mice, but did not show an additive or synergistic effect (Fig.8A).

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Safety in permanent ischemia [2]
Although most cerebral arterial occlusions eventually recanalize, it is impossible to predict whether an occlusion will remain permanent in the hyperacute stage. If the drug were not safe in the absence of reperfusion, this would preclude its hyperacute administration in the field, adding to the delay in treatment initiation until imaging demonstration of recanalization. We, therefore, tested the safety of Belumosudil (KD025, SLx-2119) in permanent fMCAO. Because the model carries a high mortality over time, we assessed the infarct volume at 24 h after ischemia onset to minimize excess losses. As expected, infarct volumes were larger in the permanent model (Fig.8B) compared to transient fMCAO (see Figs.3, 5). KD025 was safe but lost its efficacy in the presence of persistent arterial occlusion.

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Other safety endpoints [2]
Hemorrhagic transformation, weight loss, and mortality were recorded in all experiments. None of these safety endpoints was significantly altered by Belumosudil (KD025, SLx-2119) in any of the experimental groups, except for increased weight loss when it was combined with atorvastatin (Table3; Fig. S1). Because we did not have a sham group, it is unclear whether this increased weight loss is directly related to ischemia.

[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.

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In conclusion, the results of the current gene expression profiling study show that atorvastatin and the ROCK2-inhibitor SLx-2119 exhibit little overlap, but instead are mainly complimentary in their effects on gene expression in several primary human cell cultures. These data are consistent with a potential synergistic effect between statins and ROCK-inhibitors. It should also be noted that the ROCK pathway affects the function of several target proteins by posttranslation modification. Hence, more in vitro and in vivo studies are needed to further explore the possible therapeutic benefit of treatments in which ROCK-inhibitors and statins are combined.[1]

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Objective: Rho-associated kinase (ROCK) is a key regulator of numerous processes in multiple cell types relevant in stroke pathophysiology. ROCK inhibitors have improved outcome in experimental models of acute ischemic or hemorrhagic stroke. However, the relevant ROCK isoform (ROCK1 or ROCK2) in acute stroke is not known. Methods: We characterized the pharmacodynamic and pharmacokinetic profile, and tested the efficacy and safety of a novel selective ROCK2 inhibitor KD025 (formerly SLx-2119) in focal cerebral ischemia models in mice. Results: KD025 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]

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.)