CSF-1R (IC50 = 3.2 nM); KIT (IC50 = 20 nM); FLT3 (IC50 = 190 nM)

体外活性：Edicotinib（原名 JNJ-40346527）是一种新型选择性口服生物可利用的集落刺激因子 1 (CSF-1) 受体激酶抑制剂。它正在接受临床研究，尽管接受疾病缓解抗风湿药物 (DMARD) 治疗，但仍能抑制活动性类风湿关节炎 (RA) 患者的巨噬细胞存活、增殖和分化。 JNJ-40346527 及其活性代谢物的药代动力学暴露高于药理活性所需的预计浓度，JNJ-40346527 治疗中 CSF-1 水平增加和 CD16+ 单核细胞减少证明了有效的靶点参与和活性证明，但没有接受安慰剂治疗的患者。 37 名 (58.7%) 接受 JNJ-40346527 治疗的患者和 16 名 (50.0%) 接受安慰剂治疗的患者报告≥ 1 次不良事件 (AE)； 1 名 (1.6%) 接受 JNJ-40346527 治疗的患者和 3 名 (9.4%) 接受安慰剂治疗的患者报告≥ 1 例严重 AE。激酶测定：Edicotinib（原名 JNJ-40346527）是一种新型选择性口服生物可利用的集落刺激因子 1 (CSF-1) 受体激酶抑制剂。

JNJ-40346527 及其活性代谢物的药代动力学暴露高于药理活性所需的预计浓度，JNJ-40346527 治疗中 CSF-1 水平增加和 CD16+ 单核细胞减少证明了有效的靶点参与和活性证明，但没有接受安慰剂治疗的患者。 37 名 (58.7%) 接受 JNJ-40346527 治疗的患者和 16 名 (50.0%) 接受安慰剂治疗的患者报告≥ 1 次不良事件 (AE)； 1 名 (1.6%) 接受 JNJ-40346527 治疗的患者和 3 名 (9.4%) 接受安慰剂治疗的患者报告≥ 1 例严重 AE。 Edicotinib（口服强饲；3、10、30 和 100 mg/kg；5 天）显着抑制 ME7 小鼠中的小胶质细胞增殖。仅在最高测试剂量 100 mg/kg 时，它才会减少小胶质细胞（CD45+CD11b+ 细胞总数）的数量，而 JNJ-527 在每个测试剂量（CD45+CD11bhighLy6C 中/低细胞）下会消耗高达 50% 的巡逻血单核细胞在 100 mg/kg 剂量下仅存在炎症单核细胞（Ly6C 高细胞）比例减少的趋势

CSF1R磷酸化的体外评估[1]
N13小鼠小胶质细胞系（Righi等人，1991）在添加了10%胎牛血清和50U/ml青霉素/链霉素的Dulbecco改良Eagle培养基（DMEM）中培养。细胞在T75培养瓶中保持在37°C、5%CO2加湿的环境中。将细胞以2×105个细胞/cm2的密度铺在6孔板中，并培养过夜以使其粘附。在刺激前将细胞在无血清培养基中保持4小时，然后在没有或有0.1、1、10、100或1000 nM的Edicotinib (JNJ-527；JNJ-40346527)的情况下孵育30分钟。将重组CSF1（100 ng/ml，R&D Systems）加入相应的孔中5分钟，之后立即在RIPA缓冲液中裂解细胞，补充蛋白酶和磷酸酶抑制剂鸡尾酒。根据制造商的说明，使用Microcon-10 kDa离心过滤器浓缩蛋白质裂解物，并使用Pierce BCA蛋白质检测试剂盒测定蛋白质浓度。为了估算IC50，使用GraphPad棱镜在非线性回归曲线中模拟CSF1R和ERK1/2磷酸化的值。

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发现了新型选择性口服生物可利用的集落刺激因子 1 (CSF-1) 受体激酶抑制剂艾迪克替尼（以前称为 JNJ-40346527）。

细胞系：N13 小胶质细胞
浓度：0.1 nM、1 nM、10 nM、100 nM、1000 nM
孵育时间：24 小时
结果：阻止 N13 小胶质细胞中的 CSF1R 和 ERK1/2 磷酸化

C57BL/6 J (Harlan) mice
3, 10, 30 and 100 mg/kg; 5 days
Oral gavage

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Pharmacological treatments [1]
For short-term treatments, Edicotinib (JNJ-527；JNJ-40346527) was dissolved in 0.9% Methocel™ and administered daily (morning) for five consecutive days by oral gavage at doses of 3, 10, 30 and 100 mg/kg. For long term treatments (4–8 weeks), Edicotinib (JNJ-527；JNJ-40346527) was incorporated into mouse chow as previously described by Olmos-Alonso et al. (2016), for a final dose of 30 mg/kg with an average daily ingestion of 5 g of food per mouse. Diet composition was identical in terms of fat, protein, etc. content, with the only addition of the compound. Mouse weight and food consumption were monitored in all experiments, and no differences were found between treated and untreated groups.

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TSPO autoradiography [1]
Mice were terminally anaesthetized with an overdose of sodium pentobarbital and transcardially perfused with 0.9% saline. Brains were harvested, frozen in isopentane at a temperature of −40°C and stored at −80°C. NBH (n = 7), ME7 (n = 8) and ME7 + Edicotinib (JNJ-527；JNJ-40346527) (n = 8) mouse brains were coronally cryosectioned at 20 μm and directly mounted onto glass slides. Slides were incubated at room temperature for 30 min in 100 mM Tris-HCl containing 1 nM [3H]PK11195 (specific activity 82.7 Ci per mmol), washed twice for 6 min in 100 mM Tris-HCl, rinsed dipping into dH2O and air dried.

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Pharmacokinetics: sample preparation and bioanalytical method [1]
Aliquots (10 µl) of plasma and brain homogenate (diluted 1:5 in phosphate buffer) were analysed for Edicotinib (JNJ-527；JNJ-40346527) concentrations using a method based on protein precipitation and HPLC-MS/MS analysis. To each sample, an internal standard (20 µl) and acetonitrile (150 µl) were added. Samples were mixed thoroughly (mechanical shaking for 10 min), and then centrifuged (5000g for 10 min at 4°C). An aliquot of supernatant (20 µl) was dispensed into a LCMS plate and 200 µl of 0.1% formic acid in methanol/water (50:50) were added. Analysis for Edicotinib (JNJ-527；JNJ-40346527) concentrations was performed using HPLC-MS/MS employing positive-ion electrospray ionization (Sciex API 4000) and a Zorbaz Eclipse Phenyl Hexyl, 3.5 μm (50 × 2.1 mm internal diameter) column. Elution was achieved at a flow rate of 0.5 ml/min with isocratic elution of 0.1% formic acid in methanol/water (85:15). The lower limit of quantification was 5–10 ng/ml for plasma and 10 ng/g for brain. The assay was linear up to 4000 ng/ml for plasma and 4000 ng/g for brain.

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In this randomized, double-blind, placebo-controlled, parallel group study, adults were randomized (2:1) to receive oral Edicotinib (JNJ-527；JNJ-40346527) 100 mg or placebo twice daily through Week 12. Patients with RA had disease activity [≥ 6 swollen/≥ 6 tender joints, C-reactive protein (CRP) ≥ 0.8 mg/dl] despite DMARD therapy for ≥ 6 months. The primary endpoint was change from baseline at Week 12 in the 28-joint Disease Activity Score with CRP (DAS28-CRP). Pharmacokinetic/pharmacodynamic analyses were also performed, and safety was assessed through Week 16.[2]

Researchers analysed the concentration of the compound in brain and plasma and found a linear dose dependent increase in JNJ-527 exposure, with an average brain to plasma ratio of 0.65 (Fig. 1E). We then assessed the impact of CSF1R blockade by JNJ-527 on microglial proliferation. Researchers found that JNJ-527 significantly inhibited microglial proliferation (Iba1+ BrdU+ cells) in the hippocampus of ME7 mice from 3 mg/kg, reaching a maximum effect of 80% inhibition at 30 mg/kg (Fig. 1F and G). Based on these data, we generated a sigmoid Emax pharmacokinetic/pharmacodynamics model for inhibition of microglial proliferation and determined that JNJ-527 EC50 was 196 ng/ml or 69 ng/g for plasma and brain exposures, respectively (Fig. 1H). Overall, Researchers demonstrated that JNJ-527 administered at 30 mg/kg significantly blocks microglial proliferation in ME7-prion mice, without altering the dynamics of the population in the healthy brain (Supplementary Fig. 2C and D). Therefore, Researchers used a dose of 30 mg/kg for all subsequent experiments. [1]

[1]. CSF1R inhibitor JNJ-40346527 attenuates microglial proliferation and neurodegeneration in P301S mice.Brain. 2019 Oct 1;142(10):3243-3264.

[2]. Results from a Phase IIA Parallel Group Study of JNJ-40346527, an Oral CSF-1R Inhibitor, in Patients with Active Rheumatoid Arthritis despite Disease-modifying Antirheumatic Drug Therapy.J Rheumatol. 2015 Oct;42(10):1752-60.

JNJ-40346527 has been used in trials studying the treatment of Health and Arthritis, Rheumatoid.
Edicotinib is a small molecule and orally available inhibitor of colony-stimulating factor-1 receptor (CSF1R; FMS) with potential antineoplastic activity. Edicotinib blocks the receptor-ligand interaction between FMS and its ligand CSF1, thereby preventing autophosphorylation of FMS. As a result, unphosphorylated FMS can not activate FMS-mediated signaling pathways, thus potentially inhibiting cell proliferation in FMS-overexpressed tumor cells. FMS, a tyrosine kinase receptor, is overexpressed in certain tumor cell types and plays an essential role in macrophage differentiation, recruitment, and activation as well as the regulation of cell proliferation.

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Neuroinflammation and microglial activation are significant processes in Alzheimer's disease pathology. Recent genome-wide association studies have highlighted multiple immune-related genes in association with Alzheimer's disease, and experimental data have demonstrated microglial proliferation as a significant component of the neuropathology. In this study, we tested the efficacy of the selective CSF1R inhibitor JNJ-40346527 (JNJ-527) in the P301S mouse tauopathy model. We first demonstrated the anti-proliferative effects of JNJ-527 on microglia in the ME7 prion model, and its impact on the inflammatory profile, and provided potential CNS biomarkers for clinical investigation with the compound, including pharmacokinetic/pharmacodynamics and efficacy assessment by TSPO autoradiography and CSF proteomics. Then, we showed for the first time that blockade of microglial proliferation and modification of microglial phenotype leads to an attenuation of tau-induced neurodegeneration and results in functional improvement in P301S mice. Overall, this work strongly supports the potential for inhibition of CSF1R as a target for the treatment of Alzheimer's disease and other tau-mediated neurodegenerative diseases.[1]

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Objective: To assess the efficacy and safety of JNJ-40346527, a selective inhibitor of colony-stimulating factor-1 (CSF-1) receptor kinase that acts to inhibit macrophage survival, proliferation, and differentiation in patients with active rheumatoid arthritis (RA) despite disease-modifying antirheumatic drug (DMARD) therapy. Methods: In this randomized, double-blind, placebo-controlled, parallel group study, adults were randomized (2:1) to receive oral JNJ-40346527 100 mg or placebo twice daily through Week 12. Patients with RA had disease activity [≥ 6 swollen/≥ 6 tender joints, C-reactive protein (CRP) ≥ 0.8 mg/dl] despite DMARD therapy for ≥ 6 months. The primary endpoint was change from baseline at Week 12 in the 28-joint Disease Activity Score with CRP (DAS28-CRP). Pharmacokinetic/pharmacodynamic analyses were also performed, and safety was assessed through Week 16. Results: Ninety-five patients were treated (63 JNJ-40346527, 32 placebo); 8 patients discontinued treatment (6 JNJ-40346527, 2 placebo) through Week 12. Mean improvements in DAS28-CRP from baseline to Week 12 were 1.15 for the JNJ-40346527 group and 1.42 for the placebo group (p = 0.30); thus, a statistically significant difference was not observed for the primary endpoint. Pharmacokinetic exposure to JNJ-40346527 and its active metabolites was above the projected concentration needed for pharmacologic activity, and effective target engagement and proof of activity were demonstrated by increased levels of CSF-1 and decreased CD16+ monocytes in JNJ-40346527-treated, but not placebo-treated, patients. Thirty-seven (58.7%) JNJ-40346527-treated and 16 (50.0%) placebo-treated patients reported ≥ 1 adverse event (AE); 1 (1.6%) JNJ-40346527-treated and 3 (9.4%) placebo-treated patients reported ≥ 1 serious AE. Conclusion: Although adequate exposure and effective peripheral target engagement were evident, JNJ-40346527 efficacy was not observed in patients with DMARD-refractory active RA. ClinicalTrials.gov identifier: NCT01597739. EudraCT Number: 2011-004529-28.[2]

C27H35N5O2

461.61

461.279

C, 70.25; H, 7.64; N, 15.17; O, 6.93

1142363-52-7

1559069-92-9 (HCl);1142363-52-7;

25230468

White to off-white solid powder

1.2±0.1 g/cm3

1.591

5.16

104

2

5

4

34

838

0

O1C(C)(C)CC(C2C=CC(=C(C3=CCC(C)(C)CC3)N=2)NC(C2=NC=C(C#N)N2)=O)CC1(C)C

BNVPFDRNGHMRJS-UHFFFAOYSA-N

InChI=1S/C27H35N5O2/c1-25(2)11-9-17(10-12-25)22-21(32-24(33)23-29-16-19(15-28)30-23)8-7-20(31-22)18-13-26(3,4)34-27(5,6)14-18/h7-9,16,18H,10-14H2,1-6H3,(H,29,30)(H,32,33)

5-cyano-N-[2-(4,4-dimethylcyclohexen-1-yl)-6-(2,2,6,6-tetramethyloxan-4-yl)pyridin-3-yl]-1H-imidazole-2-carboxamide

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: ≥ 1.67 mg/mL (3.62 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 16.7 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 1.67 mg/mL (3.62 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 16.7 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 10 mg/mL (21.66 mM) in 17% Polyethylene glycol 12-hydroxystearate in 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。