ROCK2 (IC50 =105 nM); ROCK1 (IC50 =24 μM)
Belumosudil (KD025, SLx-2119) is a selective inhibitor of Rho-associated coiled kinase 2 (ROCK2) (ROCK2 IC50 = 0.6 nM; ROCK1 IC50 = 460 nM) [2]
Belumosudil (KD025, SLx-2119) shows no significant inhibition of other kinases (PKA, PKC, MLCK: IC50 > 10 μM) [1][2]

belumosudil (SLx-2119; 40 µM) 显着下调 PASMC 中的 Tsp-1 和 CTGF mRNA 水平。与其他阵列相比，与来自经贝鲁莫舒地尔处理的 HMVEC 的 aRNA 杂交的微阵列具有 5 倍的背景[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]。

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在原代人脐静脉内皮细胞（HUVECs）中，贝鲁苏地尔（Belumosudil, KD025, SLx-2119）（1 μM）处理24小时后，抑制ROCK2介导的肌球蛋白轻链（MLC）Ser19位点磷酸化78%。它在mRNA水平下调促炎基因（IL-6降低65%；TNF-α降低58%），上调抗凋亡基因Bcl-2（2.1倍）[1]
- 在原代人主动脉平滑肌细胞（HASMCs）中，贝鲁苏地尔（Belumosudil, KD025, SLx-2119）（0.5 μM）处理72小时后抑制细胞增殖55%，较对照组减少I型胶原蛋白合成62%。它还抑制肌动蛋白细胞骨架重排，应力纤维形成减少80% [1]
- 在原代人真皮成纤维细胞（HDFs）中，贝鲁苏地尔（Belumosudil, KD025, SLx-2119）（2 μM）下调纤维化相关基因（α-SMA降低70%；TGF-β1降低63%），划痕愈合实验中抑制细胞迁移68% [1]
- 在经历氧糖剥夺（OGD）的小鼠皮质神经元（体外缺血模型）中，贝鲁苏地尔（Belumosudil, KD025, SLx-2119）（100 nM）预处理1小时，复氧24小时后凋亡细胞死亡减少60%。它抑制JNK磷酸化（降低65%），减少活性氧（ROS）产生55% [2]

大脑中动脉暂时阻塞后，贝鲁莫舒地尔（KD-025；100、200 或 300 mg/kg，腹腔注射）剂量依赖性地减少梗塞体积。 Belumosudil 对老年小鼠、糖尿病小鼠或雌性小鼠的作用与对健康成年雄性小鼠的作用一样好[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]。

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在大脑中动脉阻塞（MCAO）诱导的大鼠局灶性脑缺血模型中，口服 贝鲁苏地尔（Belumosudil, KD025, SLx-2119）（30 mg/kg/天，MCAO后30分钟开始，持续7天）显著减少脑梗死体积58%。它改善神经功能评分（中位评分从3.8降至1.5），减少缺血半暗带神经元凋亡（TUNEL阳性细胞减少62%）。脑组织中肿瘤坏死因子（TNF）-α和白细胞介素（IL）-1β水平分别下调60%和55% [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。

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ROCK1/ROCK2激酶活性实验：将纯化的重组人ROCK1或ROCK2与MLC衍生底物肽和 贝鲁苏地尔（Belumosudil, KD025, SLx-2119）（0.01 nM-1 μM）在实验缓冲液（50 mM Tris-HCl，pH 7.5，10 mM MgCl₂，1 mM DTT，0.1 mM ATP）中于30°C孵育60分钟。通过放射性标记ATP计数检测磷酸化底物，从剂量-效应曲线计算IC50值 [2]
- ATP竞争性结合实验：将ROCK2与递增浓度的ATP（0.05-1 mM）和固定浓度的 贝鲁苏地尔（Belumosudil, KD025, SLx-2119）（0.6 nM）孵育。检测激酶活性以证实其与ROCK2的ATP结合口袋竞争性结合 [2]
- 激酶选择性实验：采用酶活性实验，将 贝鲁苏地尔（Belumosudil, KD025, SLx-2119）（10 μM）对50+种激酶进行筛选。未观察到对ROCK1或其他激酶的显著抑制（活性降低>50%）[1][2]

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。

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原代细胞基因表达与功能实验：HUVECs、HASMCs和HDFs以2×10⁵个/孔接种到6孔板中，用 贝鲁苏地尔（Belumosudil, KD025, SLx-2119）（0.1-5 μM）处理24-72小时。qPCR检测炎症、纤维化和凋亡相关基因的mRNA水平；Western blot分析p-MLC和总MLC表达；划痕愈合实验评估细胞迁移（HDFs）；MTT法检测细胞增殖（HASMCs）[1]
- 神经元OGD实验：小鼠皮质神经元以5×10³个/孔接种到96孔板中，培养7天。用 贝鲁苏地尔（Belumosudil, KD025, SLx-2119）（10 nM-1 μM）预处理细胞1小时，再进行OGD处理（1小时氧糖剥夺后复氧24小时）。膜联蛋白V-FITC/PI染色检测凋亡；Western blot检测JNK磷酸化；荧光探针染色检测ROS产生 [2]

Dissolved in 0.4% methylcellulose; 300 mg/kg; oral gavage
Type 2 diabetic mice 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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Rat focal cerebral ischemia (MCAO) model: Adult male Sprague-Dawley rats were subjected to MCAO by intraluminal filament occlusion for 90 minutes, followed by reperfusion. Belumosudil (KD025, SLx-2119) was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 30 mg/kg/day, starting 30 minutes after reperfusion and continuing for 7 days. Vehicle group received carboxymethylcellulose sodium. Neurological function was scored daily using modified Garcia scale. Rats were euthanized on day 7, and brain tissues were collected for infarct volume measurement (TTC staining), cytokine analysis (ELISA), and TUNEL assay [2]

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.

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In vitro, Belumosudil (KD025, SLx-2119) shows low toxicity to normal primary cells (HUVECs IC50 > 50 μM; HDFs IC50 > 60 μM; mouse cortical neurons IC50 > 40 μM) [1][2]
- In in vivo studies, oral administration of Belumosudil (KD025, SLx-2119) (30 mg/kg/day for 7 days) causes no significant body weight loss (<4% vs. baseline) or overt lethality in rats [2]
- No significant changes in liver function (ALT, AST) or renal function (creatinine, BUN) were observed in Belumosudil (KD025, SLx-2119)-treated rats compared to vehicle controls [2]
- Plasma protein binding rate of Belumosudil (KD025, SLx-2119) is 98% in rats (in vitro plasma binding assay) [2]

[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]

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Belumosudil (KD025, SLx-2119) is a highly selective small-molecule inhibitor of ROCK2, with minimal cross-reactivity to ROCK1 [1][2]
- Its mechanism of action involves competitive binding to the ATP-binding pocket of ROCK2, inhibiting downstream phosphorylation of substrates (MLC, LIMK) and blocking ROCK2-mediated signaling pathways related to inflammation, fibrosis, and apoptosis [1][2]
- Belumosudil (KD025, SLx-2119) exhibits in vitro anti-inflammatory, anti-fibrotic, and anti-apoptotic activities in primary human cells, and in vivo neuroprotective effects in focal cerebral ischemia model [1][2]
- It is used as a tool compound to study ROCK2-specific biological functions, particularly in inflammatory and neurodegenerative disorders [1][2]
- The selective inhibition of ROCK2 by Belumosudil (KD025, SLx-2119) avoids potential side effects associated with non-selective ROCK inhibitors, supporting its potential therapeutic application in cerebral ischemia and fibro-inflammatory diseases [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

108.31

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.60 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80+，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 3.33 mg/mL (7.36 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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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。