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

Patients treated with JNJ-40346527, but not placebo, showed increased levels of CSF-1 and decreased CD16+ monocytes, indicating that the pharmacokinetic exposure to the compound and its active metabolites was above the expected concentration required for pharmacologic activity. Thirty-seven (58.7%) Patients treated with JNJ-40346527 and 16 (50.0%) receiving a placebo reported at least one adverse event (AE); 1 (1.6%) reported more than one. Three patients (9.4%) receiving placebo and those receiving JNJ-40346527 reported at least one serious adverse event. In ME7 mice, edicoxinib (oral gavage; 3, 10, 30, and 100 mg/kg; 5 days) significantly reduces the proliferation of microglia. JNJ-527 reduces up to 50% of patrolling blood monocytes at every dose tested (CD45+CD11bhighLy6Cintermediate/low cells), with only a tendency for a reduction in the proportion of inflammatory monocytes (Ly6C high cells) at 100 mg/kg.It only reduces the number of microglia (total CD45+CD11b+ cells) at the highest dose tested of 100 mg/kg.

In vitro assessment of CSF1R phosphorylation [1]
The N13 murine microglia cell line (Righi et al., 1991) was cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% foetal bovine serum and 50 U/ml penicillin/streptomycin. Cells were maintained in T75 flasks at 37°C in a 5% CO2 humidified atmosphere. Cells were plated at a density of 2 × 105 cells/cm2 in 6-well plates and cultured overnight to allow adherence. Cells were kept in serum-free medium for 4 h prior to stimulation and then incubated without or with 0.1, 1, 10, 100 or 1000 nM of Edicotinib (JNJ-527；JNJ-40346527) for 30 min. Recombinant CSF1 (100 ng/ml, R&D Systems) was added to respective wells for 5 min, after which cells were immediately lysed in RIPA buffer, supplemented with protease and phosphatase inhibitor cocktails. Protein lysates were concentrated using Microcon-10 kDa Centrifugal Filter Units, according to manufacturer’s instructions and protein concentration was determined using the Pierce BCA Protein Assay Kit. For estimation of IC50, values for CSF1R and ERK1/2 phosphorylation were modelled in a non-linear regression curve using GraphPad prism.

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The novel selective and orally bioavailable inhibitor of colony-stimulating factor-1 (CSF-1) receptor kinase, edicotinib (previously known as JNJ-40346527), was discovered.

Cell Line: N13 microglial cells
Concentration: 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM
Incubation Time: 24 hours
Result: Prevented CSF1R and ERK1/2 phosphorylation in N13 microglial cells

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

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)

Solubility in Formulation 1: ≥ 1.67 mg/mL (3.62 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 16.7 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: ≥ 1.67 mg/mL (3.62 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 10 mg/mL (21.66 mM) in 17% Polyethylene glycol 12-hydroxystearate in 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.)