P2X7 Receptor; P2X7 receptor – pIC50 = 7.97 (human P2X7, calcium flux), 7.81 (rat P2X7), 7.55 (mouse P2X7), 7.96 (macaque P2X7), 7.72 (dog P2X7); pK = 8.12 ± 0.08 (recombinant human P2X7), 8.5 ± 0.04 (recombinant rat P2X7), 8.2 ± 0.06 (rat cortex native P2X7); pIC50 = 7.68 ± 0.2 (human blood IL-1β release), 7.21 ± 0.1 (human PBMC IL-1β release) [1]
Selective over P2X1, P2X2, P2X3, P2X4, P2X2/3 (all human) at concentrations up to 10 μM; no significant activity in a panel of 50 ion channels, receptors, and transporters (tested at 10 μM) or kinase panel (tested at 1 μM) [1]

In Vitro: JNJ-55308942 is a high-affinity, selective, brain-penetrant P2X7 functional antagonist. It blocked calcium flux through P2X7 ion channels across multiple species including human, rat, mouse, macaque, and dog. [1]
In radioligand binding assays, JNJ-55308942 showed high affinity for recombinant human P2X7 (pK = 8.12 ± 0.08), recombinant rat P2X7 (pK = 8.5 ± 0.04), and native rat cortex P2X7 (pK = 8.2 ± 0.06). [1]
In human blood and human PBMCs, JNJ-55308942 potently blocked P2X7-dependent IL-1β release with pIC50 values of 7.68 ± 0.2 and 7.21 ± 0.1, respectively. [1]
In mouse primary microglia, JNJ-55308942 blocked IL-1β release in a concentration-dependent manner (LPS 100 ng/ml + Bz-ATP 380 μM) and attenuated Bz-ATP-induced microglial cell death. [1]
JNJ-55308942 did not block P2X1, P2X2, P2X3, P2X4, or P2X2/3 heteromers at concentrations up to 10 μM. No significant activity was detected in a panel of 50 ion channels, receptors, and transporters (tested at 10 μM) or a kinase panel (tested at 1 μM). [1]
In Caco-2 permeability assays, JNJ-55308942 showed high permeability with efflux ratio <2. [2]
Compound 35 (JNJ-55308942) had high solubility in aqueous media. Solubility at pH 2 and pH 7 was >400 μM. [2]
JNJ-55308942 showed no significant hERG channel inhibition up to 3 μM in an automated patch-clamp assay. [2]
In human liver microsomes, JNJ-55308942 showed no CYP3A4 time-dependent inhibition and no CYP1A1/2 induction, although it showed moderate activation of human PXR (66% of rifampicin control at 10 μM). [2]

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The pKi of the rat P2X7 channel and the recombinant human channel are 8.5 and 8.1, respectively, according to JNJ-55308942. In human blood, mouse blood, and microglia, JNJ-55308942 potently and concentration-dependently reduces the release of IL-1β [2].

In Vivo: After oral dosing in rats, JNJ-55308942 exhibited dose-dependent occupancy of rat brain P2X7 with ED50 = 0.07 mg/kg. The plasma and brain EC50 for occupancy were approximately 15 and 12 ng/ml, respectively. EC80 and EC90 plasma exposures were around 60 ng/ml and 135 ng/ml. [1]
At 10 mg/kg (p.o.), JNJ-55308942 rapidly occupied brain P2X7 (88% occupancy at 0.25 h), maintained >80% occupancy through 6 h, with 70% at 24 h, 14% at 48 h, and 0% at 72 h. Brain/plasma ratio was close to unity. [1]
JNJ-55308942 (3 mg/kg, oral) blocked Bz-ATP-induced brain IL-1β release in conscious rats as measured by microdialysis, demonstrating functional target engagement in the brain. [1]
JNJ-55308942 (30 mg/kg, oral, three doses at 0, 24, and 31 h) attenuated LPS-induced microglial activation in mice, reducing forward scatter (cell size), side scatter (cell complexity), CD45 and CD11b surface expression, and Ki-67-positive proliferating microglia. [1]
In the BCG model of neuroinflammatory depression in mice, JNJ-55308942 (30 mg/kg, oral, once daily) reversed BCG-induced deficits in sucrose preference (anhedonia-like behavior) and social interaction. [1]
In the chronic mild stress (CMS) rat model of depression, JNJ-55308942 (0.1 and 1 mg/kg, oral, once daily for 5 weeks) reversed stress-induced sucrose intake deficits. The 1 mg/kg dose increased sucrose intake after the first week of treatment and maintained efficacy. Efficacy correlated with high terminal brain P2X7 occupancy. [1]
In vivo microdialysis assay in freely moving rats, JNJ-55308942 (10 mg/kg, oral, 4 h prior to Bz-ATP infusion) significantly inhibited Bz-ATP-induced IL-1β release in the rat hippocampus at 5 and 6 h time points. [2]

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JNJ-55308942 (30 mg/kg; oral) reduces the activation of microglia in mice induced by LPS[2]. Oral JNJ-55308942 (30 mg/kg) treatment corrected BCG-induced sucrose preference and social interaction deficits in a model of depression generated by BCG. With an ED50 of 0.07 mg/kg, the chemical exhibited dose- and concentration-dependent occupancy of rat brain P2X7 following oral treatment. Target engagement has a functional influence on the brain of conscious rats, as demonstrated by the P2X7 antagonist (3 mg/kg, oral) which prevents the release of IL-1β in the brain caused by Bz-ATP[2]. F, Vss, CL, Cmax, and AUC24h values for JNJ-55308942 (5 mg/kg; po) are 81%, 1.7 L/kg, 3.7 mL min/kg, 1747 ng/mL, and 17549 (ng/mL) h, respectively[1].

Enzyme Assay: Calcium flux FLIPR assay: 1321N1 cells expressing recombinant P2X7 channels were seeded at 25,000 cells/well in 96-well plates. Cells were loaded with Calcium-4 dye and incubated for 60 min. Test compounds were prepared in assay buffer. Cells were incubated with test compounds for 30 min. BzATP was added and fluorescence change measured for 180 seconds. pIC50 values were calculated. [1]
Radioligand binding assay: Cell membrane preparations from recombinant P2X7 1321N1 cells or rat cortex tissue were incubated with [³H]-JNJ-54232334 (10 nM) and test compounds in 50 mM Tris buffer with 0.1% BSA. Non-specific binding was determined with 10 μM A-740003. Incubation was for 1 min at room temperature followed by 4 × 5 min washes at 4°C. Quantitative autoradiography was performed using a β Imager. [1]
Ex vivo autoradiography for P2X7 occupancy: Rat brain sections (20 μm) were incubated with 10 nM [³H]-JNJ-54232334 in 50 mM Tris buffer with 0.1% BSA. Non-specific binding was assessed with 10 μM A-740003. Sections were incubated for 1 min at room temperature, washed 4 × 5 min at 4°C, then 2 dips in deionized water. Occupancy was quantified using β Imager and M3 Vision software. [1]
Brain IL-1β microdialysis: A guide cannula was surgically placed in rat hippocampus. After 4 days recovery, JNJ-55308942 was orally dosed. Bz-ATP (100 mM in aCSF with 0.15% BSA) was delivered via reverse dialysis over 2 h. Dialysate was collected and IL-1β levels measured by ELISA. [1]
CYP inhibition cocktail assay: Human liver microsomes were incubated with six CYP substrates and test compounds. Probe metabolite formation was analyzed by LC-MS/MS. [2]

Cell Assay: 1321N1 human astrocytoma cells stably expressing human, rat, mouse, macaque, or dog P2X7 channels were maintained in HyQ DME high glucose with 10% FBS and selection marker. Cells were seeded at 25,000 cells/well for calcium flux assays. [1]
Mouse primary microglia: Isolated from cortices of postnatal day 0-3 mouse pups. For IL-1β release, microglia were incubated with JNJ-55308942 for 1 h before 100 ng/ml LPS challenge for 22 h in the presence or absence of 380 μM Bz-ATP for an additional 2 h. IL-1β was quantified by ELISA. For cell death assay, microglia were pre-treated with JNJ-55308942 for 1 h before exposure to 380 μM Bz-ATP for 4.5 h. Cell viability was determined by CCK-8 kit. [1]
Human blood and PBMC assays: Whole blood or isolated PBMCs were stimulated with LPS (30 ng/ml) for 1 h, then test compounds added for 30 min, followed by Bz-ATP (0.5 mM for PBMC, 1 mM for blood) for 1.5 h. IL-1β in supernatant was measured by ELISA. [1]
Caco-2 permeability assay: Caco-2 cells were seeded onto 96-well plates and cultured for at least 21 days. Test compounds (10 μM) were applied to donor side and incubated at 37°C for 60 min (A→B) or 40 min (B→A). Apparent permeability coefficient (Papp) was calculated. [2]

Animal/Disease Models: Sixteen male C57/BL6J mice[2]
Doses: 30 mg/kg
Route of Administration: Po (after an ip injection of LPS (0.8 mg/kg, ip))
Experimental Results: Dramatically attenuated the effect of LPS on FSC, CD45 surface expression and CD11b surface expression.

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Animal/Disease Models: Rat[1]
Doses: Po (pharmacokinetic/PK Analysis)
Route of Administration: 5 mg/kg
Experimental Results: The F, Vss, CL, Cmax and AUC24h were 81%, 1.7 L/kg, 3.7 mL min /kg, 1747 ng/mL, and 17549 (ng/mL) h, respectively.

In rats (0.5/2.5 mg/kg iv/po): CL = 12 mL/min/kg, Vss = 2.5 L/kg, t1/2 = 2.5 h, F = 90%. [2]
In dogs (0.125/0.625 mg/kg iv/po): CL = 3.7 mL/min/kg, Vss = 2.6 L/kg, t1/2 = 8.8 h, F = 81%. [2]
In monkeys (0.125/0.625 mg/kg iv/po): CL = 2.6 mL/min/kg, Vss = 1.4 L/kg, t1/2 = 0.9 h, F = 77%. [2]
JNJ-55308942 showed high oral bioavailability across species (77-100%). Brain/plasma ratio was approximately 1. [1][2]
Human PK predictions: CL 0.8 mL/min/kg, Vss 1.1 L/kg, t1/2 16 h. For a 6 mg once daily dose, predicted Cmax 105 ng/mL, AUC 1786 (ng/mL)·h, maintaining trough levels above EC50 (45 ng/mL) over 24 h. For EC80 (225 ng/mL), estimated dose 30 mg once daily with Cmax 525 ng/mL and AUC 8930 (ng/mL)·h. [2]

In rat 4-day toleration study, JNJ-55308942 at 250 mg/kg/day was associated with mild increase in cholesterol (125 and 250 mg/kg) and slight increase in total protein and albumin (250 mg/kg). Mean day 4 Cmax at 250 mg/kg was 9260 ng/mL, AUC0-24h was 108,000 ng·h/mL. [2]
In dog 5-day toleration study at 300 mg/kg/day, there were no clinical pathology, macroscopic, or microscopic abnormalities. Day 5 Cmax at 300 mg/kg was 5330 ng/mL, AUC0-24h was 78,350 ng·h/mL. [2]
For compound 35 (JNJ-55308942), in rat 5-day study at 125 mg/kg/day, mean day 5 Cmax was 26,000 ng/mL, AUC0-24h was 291,000 ng·h/mL. Doses of 250 and 500 mg/kg/day were associated with adverse effects (body weight, weight gain, body temperature, neurobehavioral integrity). In dog 5-day study at 10 mg/kg/day, Cmax was 10,680 ng/mL, AUC0-24h was 167,000 ng·h/mL. Doses of 50 and 250 mg/kg/day resulted in body weight loss, lower food consumption, emesis, salivation, and lymphoid depletion of spleen and/or Peyer's patches. [2]
Cardiovascular safety studies in anesthetized guinea pig and dog showed no notable electrophysiological effects up to highest doses tested (plasma levels 15,500 and 12,150 ng/mL). NOAEL for blood pressure effects was 1280 ng/mL in dog and 1850 ng/mL in guinea pig. [2]

[1]. Neuropsychopharmacology of JNJ-55308942: evaluation of a clinical candidate targeting P2X7 ion channels in animal models of neuroinflammation and anhedonia. Neuropsychopharmacology. 2018;43(13):2586-2596.

[2]. A Dipolar Cycloaddition Reaction To Access 6-Methyl-4,5,6,7-tetrahydro-1H-[1,2,3]triazolo[4,5-c]pyridines Enables the Discovery Synthesis and Preclinical Profiling of a P2X7 Antagonist Clinical Candidate. J Med Chem. 2018;61(1):207-223.

JNJ-55308942 is a P2X7 antagonist currently in clinical development (NCT03151486). It was discovered through a dipolar cycloaddition reaction/Cope elimination sequence enabling synthesis of 6S-methyl-1,4,6,7-tetrahydro-5H-[1,2,3]triazolo[4,5-c]pyridine cores. The compound is orally bioavailable, binds to brain P2X7, and blocks IL-1β release from adult rodent brain. It reversed stress-induced anhedonia in two rodent models (BCG and chronic mild stress) and attenuated LPS-induced microglial activation. The compound has high solubility (>400 μM at pH 2 and 7) and is a BCS class I compound. It was chosen as a backup clinical candidate to JNJ-54175446. [1][2]

C17H12F5N7O

425.315499305725

425.102

C, 48.01; H, 2.84; F, 22.33; N, 23.05; O, 3.76

2166558-11-6

90408860

White to light yellow solid powder

1.5

0

11

2

30

632

1

FC1C(C(F)(F)F)=NC=CC=1C(N1CC2=C(C[C@@H]1C)N(C1N=CC(=CN=1)F)N=N2)=O

LMDWZBQISRTEBH-QMMMGPOBSA-N

InChI=1S/C17H12F5N7O/c1-8-4-12-11(26-27-29(12)16-24-5-9(18)6-25-16)7-28(8)15(30)10-2-3-23-14(13(10)19)17(20,21)22/h2-3,5-6,8H,4,7H2,1H3/t8-/m0/s1

[(6S)-1-(5-fluoropyrimidin-2-yl)-6-methyl-6,7-dihydro-4H-triazolo[4,5-c]pyridin-5-yl]-[3-fluoro-2-(trifluoromethyl)pyridin-4-yl]methanone

JNJ-55308942; JNJ55308942; Zanvipixant; JNJ 55308942; B7YN3CQ7S7;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 100 mg/mL (235.12 mM)

Solubility in Formulation 1: ≥ 4 mg/mL (9.40 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 40.0 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.

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Solubility in Formulation 2: ≥ 4 mg/mL (9.40 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 40.0 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.

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Solubility in Formulation 3: ≥ 4 mg/mL (9.40 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 40.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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