CSF-1R (IC50 = 3.2 nM); KIT (IC50 = 20 nM); FLT3 (IC50 = 190 nM)
Colony-Stimulating Factor 1 Receptor (CSF1R); in vitro IC50 for inhibiting CSF1R phosphorylation in N13 cells: 18.6–22.5 nM; pharmacokinetic/pharmacodynamic (PK/PD)-derived EC50: 196 ng/mL (plasma) and 69 ng/g (brain tissue) [1]
- Colony-Stimulating Factor 1 Receptor (CSF1R); no specific IC50, Ki, or EC50 values provided; effective target engagement demonstrated by increased plasma CSF1 levels and decreased CD16+ monocytes in patients [2]

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 ME7 prion model mice: Mice were treated with Edicotinib at doses of 3, 10, 30, or 100 mg/kg for 5 days (with 4 daily BrdU injections to quantify proliferation). Edicotinib significantly reduced microglial proliferation (Iba-1+ BrdU+ cells/mm²) at all doses, with greater efficacy at 30 and 100 mg/kg; it also reduced hippocampal microglial number (PU.1+ cells), decreased IL1-β expression, and improved hyperactive locomotor activity and motor deficits (inverted screen, horizontal bar) [1] - In P301S tauopathy mice: Mice were treated with Edicotinib at 30 mg/kg for 8 weeks. Edicotinib reduced spinal cord CD11b+ CD45+ microglial cells, decreased IL1-β/TNFα mRNA and protein levels, inhibited cytoplasmic activation of JNK/p38, reduced tau phosphorylation (AT8) and aggregation, preserved L4–L5 spinal motor neurons (Nissl staining), and improved motor function (rotarod test) [1] - In patients with active rheumatoid arthritis (RA) (Phase IIA study): Patients received oral Edicotinib 100 mg twice daily for 12 weeks. Mean improvement in DAS28-CRP (28-joint Disease Activity Score with CRP) was 1.15 (vs. 1.42 in placebo, p=0.30, no statistical significance); however, effective target engagement was confirmed by increased plasma CSF1 levels and decreased CD16+ monocytes (vs. placebo) [2]

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.

---

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

---

CSF1R phosphorylation and ERK1/2 activation assay in N13 cells: N13 cells were seeded and pretreated with serial concentrations of Edicotinib for a set period, then stimulated with CSF1 to activate CSF1R signaling. Cells were lysed in buffer containing protease/phosphatase inhibitors, and total protein was extracted. Protein concentrations were quantified, and equal amounts of protein were separated by SDS-PAGE, transferred to membranes, and probed with primary antibodies against phosphorylated CSF1R, total CSF1R, phosphorylated ERK1/2, and total ERK1/2. Immunoreactive bands were visualized and quantified to assess inhibition efficacy [1]
- TSPO autoradiography assay: Brain tissues from ME7 mice (after Edicotinib treatment) were sectioned, and incubated with [3H]-PK11195 (a TSPO ligand) to detect microglial activation. Radioactivity was measured using autoradiography, and signal intensity was quantified to evaluate the effect of Edicotinib on neuroinflammation [1]

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

---

Microglial proliferation assay (Iba-1/BrdU double immunohistochemistry): Brain sections from ME7 mice (after Edicotinib and BrdU treatment) were deparaffinized, rehydrated, and subjected to antigen retrieval. Sections were incubated with primary antibodies against Iba-1 (microglial marker) and BrdU (proliferation marker), followed by species-matched secondary antibodies (DAB for Iba-1, alkaline phosphatase for BrdU). Proliferating microglia (Iba-1+ BrdU+ cells) were counted per mm² to quantify inhibition [1]
- Flow cytometry for microglial quantification: Spinal cord tissues from P301S mice were dissociated into single-cell suspensions, stained with fluorochrome-conjugated antibodies against CD11b and CD45 (microglial markers), and analyzed by flow cytometry. The number of CD11b+ CD45+ cells was quantified to assess Edicotinib-mediated microglial reduction [1]
- PCR for cytokine mRNA detection: Total RNA was extracted from spinal cord tissues of P301S mice (after Edicotinib treatment) using a phenol-chloroform method. Complementary DNA (cDNA) was synthesized from total RNA, and quantitative real-time PCR was performed with specific primers for IL1-β and TNFα (pro-inflammatory cytokines) and a reference gene. Relative mRNA expression levels were calculated to evaluate cytokine reduction [1]
- Western blot for tau phosphorylation and kinase activation: Spinal cord tissues from P301S mice were lysed, and protein extracts were subjected to SDS-PAGE. Membranes were probed with antibodies against phosphorylated tau (AT8 epitope), phosphorylated JNK, phosphorylated p38, and a loading control. Band intensities were quantified to assess Edicotinib’s effect on tau pathology and kinase activation [1]

C57BL/6 J (Harlan) mice
\n3, 10, 30 and 100 mg/kg; 5 days
\nOral gavage

---

\nPharmacological treatments [1]
\nFor 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.

---

\nTSPO autoradiography [1]
\nMice 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.

---

\nPharmacokinetics: sample preparation and bioanalytical method [1]
\nAliquots (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.

---

\nIn 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]

---

\nME7 prion model mouse experiment: Mice were divided into groups: NBH (control), NBH + Edicotinib 30 mg/kg, ME7 (disease control), ME7 + Edicotinib (3, 10, 30, 100 mg/kg; n=6–14 per group). Edicotinib was administered for 5 days, and BrdU was injected 4 times daily during treatment to label proliferating cells. At the end of treatment, plasma and brain tissues were collected to measure Edicotinib concentrations; brain sections were analyzed by immunohistochemistry to quantify microglial proliferation [1]
\n- P301S tauopathy model mouse experiment: P301S transgenic mice and wild-type mice were used; P301S mice were treated with Edicotinib 30 mg/kg for 8 weeks. During treatment, motor function was assessed using the rotarod test. At the end of treatment, spinal cord tissues were collected for flow cytometry (microglial count), PCR (cytokine mRNA), western blot (tau pathology), and Nissl staining (motor neuron count) [1]

Researchers analyzed the concentrations of this compound in brain tissue and plasma, finding that JNJ-527 exposure increased linearly in a dose-dependent manner, with a mean brain-to-plasma concentration ratio of 0.65 (Figure 1E). Subsequently, we evaluated the effect of JNJ-527's CSF1R blocking effect on microglia proliferation. We found that JNJ-527 significantly inhibited the proliferation of microglia (Iba1+ BrdU+ cells) in the hippocampus of ME7 mice starting at a dose of 3 mg/kg, reaching a maximum inhibition rate of 80% at a dose of 30 mg/kg (Figures 1F and G). Based on these data, we constructed an S-shaped Emax pharmacokinetic/pharmacodynamic model for inhibiting microglia proliferation and determined the EC50 values of JNJ-527 in plasma and brain tissue to be 196 ng/ml and 69 ng/g, respectively (Figure 1H). Overall, the researchers demonstrated that JNJ-527 administered at a dose of 30 mg/kg significantly inhibited microglial proliferation in ME7 prion mice without altering the dynamic changes of microglial cells in healthy brain tissue (Supplementary Figures 2C and D). Therefore, the researchers used a dose of 30 mg/kg in all subsequent experiments. [1] In ME7 mice, the concentrations of Edicotinib in plasma and brain tissue were linearly correlated with the administered dose (3, 10, 30, 100 mg/kg), with tissue/plasma (T/P) ratios of 0.5–1. [1] In patients with rheumatoid arthritis (RA) (phase IIA study), pharmacokinetic exposures of Edicotinib and its active metabolites were demonstrated to be higher than the concentrations required for the expected pharmacological activity. [2]

In patients with rheumatoid arthritis (RA) (phase IIA study): 58.7% of patients treated with Edicotinib (n=63) reported ≥1 adverse event (AE), compared to 50.0% in the placebo group (n=32); 1.6% of patients treated with Edicotinib reported ≥1 serious adverse event (SAE), compared to 9.4% in the placebo group. No significant changes in hematological parameters, liver function, or kidney function were reported (data not provided) [2] - No significant toxicities (e.g., weight loss, organ damage) were observed at the test doses (3–100 mg/kg) in ME7 and P301S mouse models [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 clinical trials investigating its use in treating healthy individuals and rheumatoid arthritis. Edicotinib is a small-molecule, orally administered colony-stimulating factor-1 receptor (CSF1R; FMS) inhibitor with potential anti-tumor activity. Edicotinib blocks the receptor-ligand interaction between FMS and its ligand CSF1, thereby preventing FMS autophosphorylation. Therefore, unphosphorylated FMS cannot activate FMS-mediated signaling pathways, potentially inhibiting the proliferation of FMS-overexpressing tumor cells. FMS is a tyrosine kinase receptor overexpressed in certain tumor cell types and plays a crucial role in the regulation of macrophage differentiation, recruitment, activation, and cell proliferation. Neuroinflammation and microglial activation are important processes in the pathology of Alzheimer's disease. Recent genome-wide association studies have identified several immune-related genes closely associated with Alzheimer's disease, and experimental data have also confirmed that microglial proliferation is an important component of the neuropathology of this disease. This study tested the efficacy of the selective CSF1R inhibitor JNJ-40346527 (JNJ-527) in a P301S mouse tau protein disease model. We first demonstrated the antiproliferative effect of JNJ-527 on microglia and its influence on the inflammatory spectrum in an ME7 prion model, and provided potential central nervous system biomarkers for clinical research, including pharmacokinetic/pharmacodynamic and efficacy assessments via TSPO autoradiography and cerebrospinal fluid proteomics. Subsequently, we demonstrated for the first time that inhibiting microglia proliferation and altering microglia phenotypes alleviated tau protein-induced neurodegeneration and improved function in P301S mice. Overall, this study strongly supports the potential of CSF1R inhibition as a therapeutic target for Alzheimer's disease and other tau protein-mediated neurodegenerative diseases. [1]

---

Objective: To evaluate the efficacy and safety of JNJ-40346527. JNJ-40346527 is a selective colony-stimulating factor-1 (CSF-1) receptor kinase inhibitor that inhibits macrophage survival, proliferation, and differentiation in patients with active rheumatoid arthritis (RA) despite receiving disease-modifying antirheumatic drugs (DMARDs). Methods: In this randomized, double-blind, placebo-controlled, parallel-group study, adult patients were randomized 2:1 to receive either oral JNJ-40346527 100 mg or placebo twice daily for 12 weeks. Disease activity in RA patients was defined as [≥ 6 swollen joints/≥ 6 tender joints, C-reactive protein (CRP) ≥ 100 mg]. CRP levels remained at 0.8 mg/dL despite ≥ 6 months of DMARD treatment. The primary endpoint was the change in the 28-joint disease activity score (DAS28-CRP) from baseline at week 12. Pharmacokinetic/pharmacodynamic analyses were performed, and safety was assessed up to week 16. Results: A total of 95 patients received treatment (63 received JNJ-40346527 and 32 received placebo); by week 12, 8 patients discontinued treatment (6 received JNJ-40346527 and 2 received placebo). From baseline to week 12, the mean improvement in DAS28-CRP was 1.15 in the JNJ-40346527 group and 1.42 in the placebo group (p = 0.30); therefore, no statistically significant difference was observed in the primary endpoint. Pharmacokinetic exposures to JNJ-40346527 and its active metabolites were higher than the concentrations required for the expected pharmacological activity, and the increased CSF-1 levels and decreased CD16+ monocytes in patients treated with JNJ-40346527 confirmed the effective targeting and activity of the drug, which was not observed in the placebo group. Of the 37 patients (58.7%) treated with JNJ-40346527 and 16 patients (50.0%) treated with placebo, ≥1 adverse event (AE) was reported; of the 1 patient (1.6%) treated with JNJ-40346527 and 3 patients (9.4%) treated with placebo, ≥1 serious adverse event (SAE) was reported. Conclusion: Despite adequate drug exposure and effective binding to the peripheral target, no efficacy of JNJ-40346527 was observed in patients with DMARD-refractory active rheumatoid arthritis (RA). ClinicalTrials.gov Identifier: NCT01597739. EudraCT Number: 2011-004529-28. [2]

---

Edicotinib (JNJ-40346527) is a selective CSF1R inhibitor that works by inhibiting the survival, proliferation and differentiation of microglia and reducing the production of pro-inflammatory cytokines. [1][2]
- In preclinical studies, Edicotinib alleviated tau protein-induced neurodegeneration and improved functional prognosis in P301S tau protein disease mice, supporting its potential for treating Alzheimer's disease and other tau protein-mediated neurodegenerative diseases. [1]
- In a phase IIA study of active rheumatoid arthritis (despite treatment with DMARDs), Edicotinib showed effective peripheral target binding but no statistically significant efficacy in improving DAS28-CRP (the primary endpoint). ClinicalTrials.gov Registration No.: NCT01597739; EudraCT No.: 2011-004529-28 [2] - In ME7 prion model mice, Edicotinib reduced microglial proliferation and restored behavioral changes (motor activity, motor dysfunction), and identified potential central nervous system biomarkers (TSPO autoradiography, cerebrospinal fluid proteomics) for clinical research [1]

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.

---

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.

---

View More

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.

---

 (Please use freshly prepared in vivo formulations for optimal results.)