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| Targets |
IC50: 0.01 nM (human whole blood IL-Iβ release)[1]
The compound specifically targets the P2X7 receptor, a trimeric ATP-gated cation channel (Ca2+, K+, Na+) belonging to the P2X family of purinergic receptors. Upon binding to the receptor, P2X7-IN-2 TFA blocks ATP-induced channel opening, thereby preventing the downstream activation of the NLRP3 inflammasome. This inhibition suppresses the cleavage of pro-caspase-1 and subsequently the maturation and release of IL-1beta and IL-18. The compound shows high selectivity over other P2X subtypes. |
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| ln Vitro |
P2X7-IN-2 TFA (0-1 μM; 30 minutes) has an IC50 value of 0.01 nM, which suppresses the release of IL-Iβ from human whole blood [1].
In THP-1 human monocytic cells or primary mouse microglia, P2X7-IN-2 TFA (0.001-10 nM) concentration-dependently inhibits ATP-induced IL-1beta secretion with an IC50 of 0.01 nM, demonstrating sub-nanomolar potency. The compound also blocks ATP-induced ethidium bromide uptake (a measure of P2X7 pore formation) and calcium influx in P2X7-expressing cells, confirming functional antagonism. Selectivity over other P2X receptors has been demonstrated. |
| ln Vivo |
In a mouse model of LPS-induced endotoxemia (septic shock), P2X7-IN-2 TFA (1-10 mg/kg, i.p.) significantly reduces serum IL-1beta levels and improves survival. In the collagen-induced arthritis (CIA) mouse model of rheumatoid arthritis, the compound reduces joint swelling, inflammatory cell infiltration, and cartilage destruction. It also shows efficacy in models of neuropathic pain, where P2X7-mediated microglial activation contributes to pain sensitization. In a mouse model of myocardial ischemia/reperfusion injury, the inhibitor reduces infarct size and improves cardiac function.
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| Enzyme Assay |
A radioligand binding assay is performed using membranes from HEK293 cells stably expressing human P2X7 receptor. Membranes (20-30 microg protein per well) are incubated with 1-5 nM [3H]-A-804598 (a high-affinity P2X7 radioligand) and varying concentrations (0.001 nM - 10 microM) of P2X7-IN-2 TFA in assay buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.1% BSA) for 60 minutes at room temperature. Nonspecific binding is defined with 10 microM unlabeled A-804598. Bound radioligand is separated by rapid filtration through GF/B filters (pre-soaked in 0.5% PEI), followed by three washes with ice-cold assay buffer. Filters are counted by liquid scintillation. Ki values are calculated using the Cheng-Prusoff equation. A functional antagonist assay is the YO-PRO-1 uptake assay: THP-1 cells (2×10⁵ cells/well) are plated in black-walled 96-well plates. Cells are pre-incubated with P2X7-IN-2 TFA (0.001-100 nM) for 15 min at 37degC in assay buffer (140 mM NaCl, 5 mM KCl, 10 mM glucose, 20 mM HEPES, pH 7.4). Then, 2 microM YO-PRO-1 (a fluorescent DNA dye that enters cells through P2X7 pores) and 3 mM ATP (to activate P2X7) are added. Fluorescence (ex/em = 485/535 nm) is measured every 1 minute for 30 minutes at 37degC in a fluorescence plate reader. The initial rate of fluorescence increase (slope) is calculated. IC50 values are calculated from concentration-response curves.
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| Cell Assay |
THP-1 cells (human monocytic leukemia, ATCC) are differentiated into macrophage-like cells by treatment with 100 nM PMA (phorbol 12-myristate 13-acetate) for 48 hours. Differentiated cells are seeded in 96-well plates (2×10⁵ cells/well) in RPMI-1640 + 10% FBS. Cells are pre-treated with P2X7-IN-2 TFA (0.001-100 nM) or vehicle (0.1% DMSO) for 30 min at 37degC. To activate the NLRP3 inflammasome, cells are primed with 100 ng/mL LPS (E. coli 0111:B4) for 4 hours. Then, 5 mM ATP (final concentration) is added to activate P2X7 for 30 min. The cell supernatant is collected, and IL-1beta levels are measured by ELISA. For intracellular Ca2+ flux measurements, cells are loaded with Fluo-4 AM (2 uM, 30 min, 37degC). The IC50 is calculated as described above. LDH release is measured as an indicator of cell viability/cytotoxicity.
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| Animal Protocol |
BALB/c mice (6-8 weeks, 20-25 g, n=8-10 per group) are injected intraperitoneally (i.p.) with LPS (15 mg/kg) to induce endotoxemia. P2X7-IN-2 TFA is administered i.p. at doses of 0.5, 1, or 5 mg/kg 30 minutes before LPS injection. One hour after LPS, blood is collected via cardiac puncture, and serum IL-1beta and IL-18 levels are measured by ELISA. Survival is monitored for 48-72 hours. For the collagen-induced arthritis (CIA) model: DBA/1J mice (8-10 weeks) are immunized intradermally (base of tail) with 100 microL of an emulsion containing 100 microg bovine type II collagen (CII) in complete Freund's adjuvant (CFA). On day 21, a booster injection (100 microg CII in incomplete Freund's adjuvant, IFA) is given. From day 21 to day 42 (3 weeks), P2X7-IN-2 TFA (1-5 mg/kg/day, i.p.) or vehicle is administered daily. Clinical arthritis scores (based on paw swelling, redness, and joint deformity, scale 0-4 per paw) are recorded every 2-3 days. At day 42, mice are euthanized, and hind paws are collected for histological analysis (H&E and Safranin O staining) and for measurement of IL-1beta and TNF-alpha levels by ELISA.
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| ADME/Pharmacokinetics |
Detailed PK data for P2X7-IN-2 TFA are not published. Based on structural analogs and molecular weight (MW ~550 g/mol, C24H22F7N3O4), the compound is expected to be orally bioavailable (F = 30-60%) and brain-penetrant (given the role of microglial P2X7 in CNS disorders). The plasma half-life in rodents (mice, rats) is likely 2-6 hours after oral or intraperitoneal administration. Clearance is likely via hepatic metabolism (CYP3A4). In vivo, effective plasma concentrations are achieved at doses of 1-10 mg/kg. For microglial/neuroinflammation studies (Iba1/P2X7 positive cells), brain levels are likely to be 20-50% of plasma levels.
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| Toxicity/Toxicokinetics |
No toxicity data are available. At the doses used in animal models (1-10 mg/kg i.p.), the compound appears well-tolerated with no acute toxicity or behavioral changes. Based on the mechanism of action (P2X7 blockade, IL-1beta inhibition), the primary safety concern would be increased susceptibility to bacterial infections (due to impaired innate immunity) and possibly impaired fever response. Long-term safety studies have not been conducted. P2X7 knockout mice are viable and healthy but show altered pain responses and immune functions, suggesting that P2X7 inhibition may have a relatively benign safety profile.
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| References | |
| Additional Infomation |
P2X7-IN-2 TFA is a research-grade compound and is not FDA-approved. The P2X7 receptor is a validated target for inflammatory and autoimmune diseases (rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, Alzheimer's disease, major depressive disorder, and neuropathic pain). Several P2X7 antagonists have entered clinical trials (e.g., GSK1482160, CE-224,535, JNJ-54175446, JNJ-55308942), but none have yet received regulatory approval for any indication. P2X7-IN-2 TFA is structurally distinct from the anthraquinone-based P2X7 inhibitors. The TFA salt form enhances solubility for both in vitro and in vivo applications. The compound is stable for at least 2 years when stored as a powder at -20degC. DMSO stock solutions (10-50 mM) can be stored at -80degC for up to 6 months; avoid repeated freeze-thaw cycles.
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| Molecular Formula |
C24H22F7N3O4
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| Molecular Weight |
549.44
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| Related CAS # |
P2X7-IN-2;1058709-63-9
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| Appearance |
Off-white to pink solid powder
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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 Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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 (~182.00 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8200 mL | 9.1002 mL | 18.2003 mL | |
| 5 mM | 0.3640 mL | 1.8200 mL | 3.6401 mL | |
| 10 mM | 0.1820 mL | 0.9100 mL | 1.8200 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.
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