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

Belumosudil (SLx-2119; 40 µM) 显着降低 PASMC 中 Tsp-1 和 CTGF mRNA 的水平。与来自经贝鲁莫舒地尔处理的 HMVEC 的 aRNA 杂交的微阵列中观察到的背景是其他阵列的五倍[1]。
Belumosudil（KD025，SLx-2119）选择性： 放射性酶测定证实，SLx-2119选择性抑制人ROCK2的活性（IC50=105 nM），而在这种无细胞系统中对人ROCK1的影响很小（IC50=24µM）。

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本研究旨在比较阿托伐他汀与新开发的ROCK2抑制剂Belumosudil（KD025，SLx-2119）在正常人内皮细胞、平滑肌细胞和成纤维细胞原代培养中的基因表达谱。用每种化合物处理细胞24小时，然后分离总RNA，用Illumina阵列获得全基因组基因表达谱。由于他汀类药物对肌动蛋白细胞骨架和结缔组织生长因子（一种参与组织纤维化的重要生长因子）的已知作用，在从患有辐射诱导纤维化的人肠道活检中分离出的具有纤维化表型的平滑肌细胞培养物中，也研究了SLx-2119和阿托伐他汀对肌动蛋白细胞架和结缔组织增长因子mRNA的影响。尽管SLx-2119和阿托伐他汀影响属于相同生物过程的基因的表达，但单个基因大多不同，具有协同或相加的特性。SLx-2119和阿托伐他汀都减少了结缔组织生长因子mRNA，并重塑了纤维化平滑肌细胞中的肌动蛋白细胞骨架，表明这两种化合物都具有抗纤维化特性。这些结果构成了进一步研究的基础，以评估联合治疗可能带来的治疗益处[1]。

Belumosudil（KD-025；100、200 或 300 mg/kg，腹腔注射）在短暂的大脑中动脉阻塞后可剂量依赖性地减少梗塞体积。 Belumosudil 对老年、糖尿病或雌性小鼠的作用与对健康成年雄性小鼠的作用一样[2]。

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Belumosudil（KD025，SLx-2119）在短暂性大脑中动脉闭塞后剂量依赖性地减少梗死体积。治疗窗口至少为中风发作后3小时，疗效持续至少4周。KD025对老年、糖尿病或雌性小鼠的疗效至少与正常成年雄性小鼠相同。与阿托伐他汀同时治疗是安全的，但不是相加或协同的。KD025在永久性缺血模型中也是安全的，尽管疗效降低。作为一种保护机制，KD025改善了大脑中动脉远端闭塞模型的皮质灌注，这意味着侧支血流增强。与同种型非选择性ROCK抑制剂不同，KD025不会引起严重的低血压，这是急性缺血性卒中的剂量限制性副作用。 解释：总的来说，这些数据表明KD025在小鼠急性局灶性脑缺血中是有效和安全的，这表明ROCK2是急性缺血性卒中的相关亚型。数据表明，选择性ROCK2抑制具有良好的安全性，有助于临床转化[2]。

放射性截短酶ROCK1和ROCK2测定[1]
进行无细胞酶测定，以确定SLx-2119对ROCK1和ROCK2的选择性抑制作用。反应在非结合表面微孔板上进行。使用4 mU的人ROCK1和ROCK2在室温下磷酸化30µM的合成ROCK肽底物S6 Long（序列：KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK），该底物在American peptide制备，加入10µM ATP，在10 mM Mg2+、50 mM Tris、pH 7.5、0.1 mM EGTA和1 mM DTT的存在下含有33P-ATP。一个单位是催化1 nmol磷酸盐/分钟转移到肽所需的激酶量。使反应进行45分钟，然后用3%磷酸停止至终浓度为1%。反应在磷酸纤维素过滤微孔板上捕获，并使用真空歧管用75mM磷酸和甲醇洗涤。在Perkin-Elmer MicroBeta 1450上测量磷酸化。

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重组ROCK1和ROCK2检测[2]
在96孔聚苯乙烯低结合板中进行化合物稀释和反应。在含有亲水性磷酸纤维素阳离子交换膜的96孔滤板中进行过滤。在含有测定缓冲液（50 mmol/L Tris，pH 7.5，0.1 mmol/L乙二醇四乙酸，10 mmol/L乙酸镁和1 mmol/L二硫苏糖醇）的50μL反应混合物中，用放射法测量重组ROCK1和ROCK2的酶活性。将长S6肽（KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK，30μmol/L）、ROCK（每个反应4 mU）和ATP（10μmol/L，1μCi[γ-33P]ATP）以及试验化合物稀释至二甲亚砜的最终浓度为1%。反应在室温下孵育45分钟，用25μL 3%磷酸停止。使用Millipore Multiscreen®真空歧管系统，通过P30磷酸纤维素滤板过滤淬灭的反应内容物，将磷酸化的长S6肽与未反应的[γ-33P]ATP分离。每个过滤器用75μL 75 mmol/L磷酸洗涤三次，用30μL 100%甲醇洗涤一次。让滤板干燥，并向每个孔中加入30μL OptiPhase“SuperMix”闪烁液。33磷在I450 MicroBeta闪烁计数器中定量，并通过减去与背景样品相关的放射性进行校正。使用公式（（U-B）/（C-B））×100对数据进行分析并表示为抑制百分比，其中U是未知值，B是星孢菌素背景孔的平均值，C是对照孔的平均数。通过GraphPad Prism软件使用S形剂量反应（可变斜率）方程类型分析进行曲线拟合，以生成IC50值。Ki值根据Ki=IC50/（1+[S]/Km）的方程计算，其中[S]和Km分别是ATP的浓度和ATP的Km值。

ROCK2抑制剂SLx-2119溶解在DMSO中，得到20 mM的储备溶液。[1] 将第7代的人微血管内皮细胞、PASMC和NHDF，以及第4代的N-SMC和RE-SMC，以1×10^6个细胞/皿的密度，接种在6 cm的培养皿中，培养基为3 ml。2天后（汇合90%），将细胞在3 ml培养基中孵育24小时，培养基中含有载体（10µl无菌PBS）、10µM阿托伐他汀、10µM阿托伐他汀和500µM甲羟戊酸的组合、10µmSLx-2119或40µM SLx-2119。进行了三个独立的实验，每个处理组有3个培养皿。 [1] RNA分离[1] 根据制造商的说明，在用赋形剂、SLx-2119、阿托伐他汀或阿托伐他汀与甲羟戊酸联合治疗HMVEC、PASMC和NHDF 24小时后，使用Ultraspec RNA分离试剂分离总RNA。保留2µg RNA用于微阵列分析（包括质量控制分析），2µg用于实时PCR。[1] 用赋形剂SLx-2119、阿托伐他汀或阿托伐他汀与甲羟戊酸联合治疗N-SMC和RE-SMC 24小时后，如前所述分离总RNA。该RNA用于实时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 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]. 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 with a maximum clinical trial phase of IV (across all indications) that was first approved in 2021 and is indicated for graft versus host disease and has 1 investigational indication. Belumosudil is used in the treatment of chronic graft-versus-host disease (GVHD) and has been investigated for the treatment of pulmonary arterial hypertension. It is an inhibitor of rho-associated coiled-coil-containing protein kinases (ROCK), with significantly more selectivity for ROCK2 as compared to ROCK1 (IC50 100 nM vs. 3 μM, respectively). In the treatment of GVHD, a condition in which donor T-cells begin to attack recipient tissues following allogeneic hematopoeitic stem cell transplantation (HSCT), belumosudil helps to resolve immune dysregulation by shifting the balance between Th17 cells and T-regulatory cells, thereby dampening the inflammatory cascade that can occasionally be fatal. Belumosudil was first approved by the FDA in July 2021, under the brand name Rezurock, for the treatment of chronic GVHD in patients who have tried and failed at least two prior lines of systemic therapy. In July 2022, Belumosudil was approved by Health Canada under the brand name RHOLISTIQ to treat the same condition in adult and pediatric patients 12 years or older.

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Belumosudil is an orally bioavailable inhibitor of Rho-associated coiled-coil kinase 2 (ROCK2; ROCK-II), with potential immunomodulating activity. Upon oral administration, belumosudil binds to and inhibits the serine/threonine kinase activity of ROCK2. This inhibits ROCK2-mediated signal transduction pathways and modulates various pro- and anti-inflammatory immune cell responses through the regulation of signal transducer and activator of transcription 3 and 5 (STAT3/STAT5) phosphorylation. This downregulates pro-inflammatory Th17 cells and increases regulatory T (Treg) cells. Belumosudil also inhibits ROCK2-mediated fibrotic processes, including stress fiber formation, myofibroblast activation and pro-fibrotic gene transcription. ROCK2 is upregulated in various diseases, including various fibrotic, neurodegenerative and autoimmune diseases.

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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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Absorption
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.
Route of Elimination
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.

Volume of Distribution
Following a single oral dose of belumosudil in healthy subjects, the mean geometric volume of distribution was 184 L.
Clearance
The mean clearance of belumosudil is 9.83 L/h.
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.
Biological Half-Life
The mean elimination half-life of belumosudil following oral administration is 19 hours.
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.
Mechanism of Action
Chronic graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation in which the transplanted donor T-cells recognize the recipient's tissues as foreign and mount an immune response. During the conditioning regimen prior to stem cell transplantation (e.g. involving irradiation or chemotherapy) the host tissues can become damaged which results in downstream inflammatory responses and the generation of inflammatory mediators like TNF-alpha and IL-1. These cytokines increase the expression of host major histocompatibility (MHC) antigens and adhesion molecules which enhances the ability of mature donor T-cells to recognize these molecules. The activation of these donor T-cells results in the activation of mononuclear phagocytes, whose effector functions are triggered by stimulatory molecules generated by the damage incurred during the conditioning phase of treatment. Activated macrophages and cytotoxic T-lymphocytes begin to directly lyse target cells and/or cause their apoptosis, which eventually leads to local tissue damage and further inflammatory responses. Belumosudil is an inhibitor of Rho-associated coiled-coil kinase 2 (ROCK2), a protein that plays a vital role in the pathogenesis of immune and fibrotic diseases. The inhibition of ROCK2 has been shown to resolve immune dysregulation by down-regulating pro-inflammatory Th17 cells and up-regulating regulatory T-cells by manipulating the phosphorylation of STAT3 and STAT5.

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Inhibitors of Rho kinase (ROCK) are a relatively new class of drugs with potential benefits in oncology, neurology, and fibrotic and cardiovascular diseases. ROCK inhibitors modulate many cellular functions, some of which are similar to the pleiotropic effects of statins, suggesting additive or synergistic properties. Studies to date have used compounds that inhibit both isoforms of ROCK, ROCK1 and ROCK2. This study was designed to compare gene expression profiles of atorvastatin with the newly developed ROCK2 inhibitor 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]

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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.[2]
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.[2]
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.[2]
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.

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

168 Ų

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

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。