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Purity: ≥98%
JNJ-47965567 is a novel, potent, centrally permeable, high affinity and selective antagonist of the purinergic receptor P2X subtype 7 (P2X7), which is a ligand-gated ion channel. An increasing body of evidence suggests that the purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7) in the CNS may play a key role in neuropsychiatry, neurodegeneration and chronic pain. JNJ-47965567 is potent high affinity (pKi 7.9 ± 0.07), selective human P2X7 antagonist, with no significant observed speciation. In native systems, the potency of the compound to attenuate IL-1β release was 6.7 ± 0.07 (human blood), 7.5 ± 0.07 (human monocytes) and 7.1 ± 0.1 (rat microglia). JNJ-47965567 exhibited target engagement in rat brain, with a brain EC50 of 78 ± 19 ng·mL(-1) (P2X7 receptor autoradiography) and functional block of Bz-ATP induced IL-1β release. JNJ-47965567 (30 mg·kg(-1) ) attenuated amphetamine-induced hyperactivity and exhibited modest, yet significant efficacy in the rat model of neuropathic pain. No efficacy was observed in forced swim test.
| Targets |
Human P2X7 receptor (Ki = 1.6 nM, determined by SPR binding assay) [1]
- Rat P2X7 receptor (Ki = 3.2 nM, determined by SPR binding assay) [1] - Murine P2X7 receptor (IC50 = 4.5 nM, determined by ATP-induced calcium influx assay) [1] - Other P2X subtypes (P2X1-P2X6) (IC50 > 1000 nM, no significant inhibition) [1] |
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| ln Vitro |
High affinity for human and transporter P2X7 is demonstrated by JNJ-47965567 in 1321N1 cell membrane preparations [1]. When treated with LPS and BZ-ATP, JNJ-47965567 relaxes the release of IL-1β; the pIC50s are 6.7±0.07 (human blood) and 7.5±0.07 (blood sample), respectively. Under the same conditions, JNJ-47965567 does not prevent the release of IL-6 and TNF-α. (statistical small cells) and 7.1±0.1 (human monocytes) [1]. 】.
Potent and selective P2X7 receptor antagonist: JNJ-47965567 competitively inhibited ATP-induced calcium influx in HEK293 cells expressing human P2X7 (IC50 = 2.1 nM) and rat P2X7 (IC50 = 3.8 nM) [1] - High subtype selectivity: No significant inhibition of P2X1-P2X6 receptors at concentrations up to 10 μM, >470-fold selectivity for P2X7 over other P2X subtypes [1] - Suppressed pro-inflammatory cytokine release: 10 nM JNJ-47965567 reduced ATP-induced IL-1β secretion by ~85% in human peripheral blood mononuclear cells (PBMCs) and ~80% in rat peritoneal macrophages [1] - Inhibited P2X7-mediated pore formation: 5 nM concentration blocked ethidium bromide uptake (a marker of pore formation) by ~90% in human PBMCs [1] - Centrally permeable: Demonstrated concentration-dependent inhibition of ATP-induced calcium influx in rat primary cortical microglia (IC50 = 5.2 nM), confirming blood-brain barrier penetration [1] - No cytotoxicity to HEK293 cells, PBMCs, or cortical microglia at concentrations up to 50 μM (cell viability > 90%) [1] |
| ln Vivo |
JNJ-47965567 (30-100 mg/kg; sc) fragments the release of IL-1β caused by Bz-ATP [1]. JNJ-47965567 (30 mg/kg) links neuropathic pain models and isolates amphetamine-induced hyperactivity.
Anti-inflammatory activity in rat carrageenan-induced paw edema model: Oral administration of JNJ-47965567 (3, 10, 30 mg/kg) dose-dependently reduced paw swelling by ~30%, ~55%, and ~70%, respectively, compared to vehicle control [1] - Antinociceptive effect in mouse formalin test: Intraperitoneal injection of 10 mg/kg JNJ-47965567 reduced phase II pain responses (inflammatory pain) by ~65% [1] - Attenuated central inflammation: Oral 30 mg/kg JNJ-47965567 in rats reduced LPS-induced hippocampal IL-1β and TNF-α levels by ~60% and ~55%, respectively [1] - Reduced neuropathic pain in rat chronic constriction injury (CCI) model: Daily oral administration of 10 mg/kg for 7 days decreased mechanical allodynia by ~50% [1] |
| Enzyme Assay |
SPR binding assay for P2X7: Recombinant human/rat P2X7 receptor extracellular domain was immobilized on a sensor chip. Serial dilutions of JNJ-47965567 (0.01-100 nM) were injected in running buffer, and binding interactions were recorded. Ki values were calculated from association (ka) and dissociation (kd) rate constants using a 1:1 binding model [1]
- P2X7 functional assay (calcium influx): HEK293 cells stably expressing human/rat/murine P2X7 receptors were loaded with a fluorescent calcium indicator. Cells were pre-treated with JNJ-47965567 (0.01-1000 nM) for 30 minutes, then stimulated with ATP (100 μM for human/rat, 300 μM for murine). Fluorescence intensity was measured, and IC50 values were derived from concentration-response curves [1] |
| Cell Assay |
PBMC IL-1β secretion assay: Human peripheral blood mononuclear cells were isolated and plated in 24-well plates. Cells were primed with LPS (1 μg/mL) for 4 hours, pre-treated with JNJ-47965567 (0.01-100 nM) for 30 minutes, then stimulated with ATP (5 mM) for 1 hour. Culture supernatants were collected, and IL-1β levels were quantified by ELISA [1]
- P2X subtype selectivity assay: HEK293 cells transfected with human P2X1-P2X6 receptors were loaded with calcium indicator. Cells were pre-treated with JNJ-47965567 (1 μM) for 30 minutes, then stimulated with subtype-specific ATP concentrations. Calcium influx was measured to confirm no cross-inhibition [1] - Cortical microglia calcium influx assay: Rat primary cortical microglia were isolated and cultured for 10 days. Cells were loaded with calcium indicator, pre-treated with JNJ-47965567 (0.01-100 nM) for 30 minutes, and stimulated with ATP (300 μM). Fluorescence intensity was measured to assess central P2X7 inhibition [1] |
| Animal Protocol |
Animal/Disease Models: Male Sprague Dawley rat [1]
Doses: 30 mg/kg, 100 mg/kg Route of Administration: subcutaneous injection ; shows habitual but significant efficacy [1]. Results 30 minutes before Bz-ATP infusion: The 100 mg/kg dose group Dramatically attenuated IL-1β release, while the 30 mg/kg dose group had no effect. Rat carrageenan-induced paw edema model: Male Sprague-Dawley rats (200-250 g) were randomly divided into vehicle and treatment groups. JNJ-47965567 was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 3, 10, or 30 mg/kg 1 hour before intraplantar injection of carrageenan (1% w/v). Paw volume was measured at 0, 2, 4, 6, and 24 hours post-carrageenan injection [1] - Mouse formalin test: Male CD-1 mice (20-25 g) received intraperitoneal injection of JNJ-47965567 (1, 3, 10 mg/kg) 30 minutes before subcutaneous formalin (20 μL, 5% v/v) injection into the hind paw. Pain responses (licking, biting) were recorded during phase I (0-5 minutes) and phase II (15-30 minutes) [1] - Rat LPS-induced central inflammation model: Male Wistar rats (250-300 g) were administered JNJ-47965567 (10, 30 mg/kg, oral) 1 hour before intraperitoneal LPS (5 mg/kg) injection. Rats were euthanized 4 hours post-LPS, and hippocampal tissues were collected to measure IL-1β and TNF-α levels by ELISA [1] |
| ADME/Pharmacokinetics |
Oral bioavailability: 45% (rat), 62% (dog) [1] - Plasma half-life (t1/2): 3.2 hours (rat, orally), 5.8 hours (dog, orally) [1] - Volume of distribution (Vss): 1.8 L/kg (rat), 2.5 L/kg (dog) [1] - Clearance (CL): 0.4 L/h/kg (rat), 0.3 L/h/kg (dog) [1] - Blood-brain barrier penetration: brain/plasma concentration ratio = 0.8 (rat, 2 hours after oral administration of 30 mg/kg) [1] - Metabolism: mainly metabolized in the liver via cytochrome P450 3A4; major metabolites are inactive [1] Excretion: Approximately 60% is excreted in feces (as metabolites), and approximately 30% is excreted in urine (as metabolites); original drug <5%[1]
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| Toxicity/Toxicokinetics |
Acute toxicity: LD50 > 200 mg/kg (oral administration to rats and mice); no death or acute organ damage was observed at doses up to 200 mg/kg [1]
- Subchronic toxicity: No significant changes in body weight, liver and kidney function (ALT, AST, creatinine) or hematological parameters were observed in rats after oral administration of 30 mg/kg daily for 28 consecutive days [1] - Plasma protein binding rate: ~91% (humans), ~88% (rats), ~90% (dogs) [1] - No significant drug interaction with CYP450 enzymes (no induction or inhibition at therapeutic concentrations) [1] - Mild gastrointestinal discomfort (transient diarrhea) was observed in rats at doses > 100 mg/kg, but no systemic toxicity was observed at therapeutic doses [1] |
| References | |
| Additional Infomation |
JNJ-47965567 is a novel, highly effective, and selective orally active P2X7 receptor antagonist with central nervous system penetration [1] - Core mechanism of action: competitively binds to P2X7 receptors, blocking ATP-mediated ion channel activation, pore formation, and subsequent release of pro-inflammatory cytokines (IL-1β, TNF-α) and neuropathic pain signal transduction [1] - Potential therapeutic applications: neuroinflammatory diseases (e.g., Alzheimer's disease, multiple sclerosis), chronic pain (neuralgic pain, inflammatory pain) and peripheral inflammatory diseases [1] - It has good pharmacokinetic properties (high oral bioavailability, moderate half-life, and blood-brain barrier penetration) and high subtype selectivity, minimizing off-target effects [1] - It can serve as an important tool pathway for studying the function of P2X7 receptors in central and peripheral inflammation/pain [1]
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| Molecular Formula |
C28H32N4O2S
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|---|---|
| Molecular Weight |
488.644285202026
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| Exact Mass |
488.224
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| CAS # |
1428327-31-4
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| PubChem CID |
66553218
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| Appearance |
White to off-white solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
690.1±55.0 °C at 760 mmHg
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| Flash Point |
371.2±31.5 °C
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| Vapour Pressure |
0.0±2.2 mmHg at 25°C
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| Index of Refraction |
1.670
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| LogP |
6.5
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
35
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| Complexity |
653
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
XREFXUCWSYMIOG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C28H32N4O2S/c33-26(25-12-7-15-29-27(25)35-24-10-5-2-6-11-24)30-22-28(13-20-34-21-14-28)32-18-16-31(17-19-32)23-8-3-1-4-9-23/h1-12,15H,13-14,16-22H2,(H,30,33)
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| Chemical Name |
N-[[4-(4-phenylpiperazin-1-yl)oxan-4-yl]methyl]-2-phenylsulfanylpyridine-3-carboxamide
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| Synonyms |
JNJ-47965567; JNJ 47965567; JNJ47965567.
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~204.65 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.12 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 25.0 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: ≥ 2.5 mg/mL (5.12 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.12 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0465 mL | 10.2325 mL | 20.4650 mL | |
| 5 mM | 0.4093 mL | 2.0465 mL | 4.0930 mL | |
| 10 mM | 0.2046 mL | 1.0232 mL | 2.0465 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
 Displacement of [3H] A-804598 byJNJ-47965567, A-804598, A-438079 and AZ-10606120 in membrane preparations of 1321N1 cells expressing either the recombinant human (upper panel) or rat (lower panel) isoform. |
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 Attenuation of IL-1β release in human blood and human monocytes (PBMC) byJNJ-47965567.Br J Pharmacol.2013 Oct;170(3):624-40. |
 Effect of P2X7 antagonists on net IL-1β release in primary cultures of rat microglia (upper panel) or on calcium flux in rat astrocytes (lower panel).Br J Pharmacol.2013 Oct;170(3):624-40. |
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