P2X7 receptor – EC50 = 5.33 ± 0.05 (pEC50, human P2X7, calcium influx); EC50 = 5.01 ± 0.04 (pEC50, rat P2X7, calcium influx); EC50 = 3.99 ± 0.06 (pEC50, mouse BALB/c P2X7, calcium influx); EC50 = 4.02 ± 0.02 (pEC50, mouse C57BL/6 P2X7, calcium influx); also activates P2X1, P2X3, P2X2/3, P2X4 receptors with higher potency than P2X7 [1][2][3][4]
pEC50: 8.74 (P2X1), 5.26 (P2X2), 7.10 (P2X3), 6.19 (P2X2/3), 6.31 (P2X4), 5.33 (P2X7)[1] EC50 3.6 μM (rat P2X7); 285 μM (mouse P2X7)[2]

In Vitro: BzATP (30-100 μM) induced current in non-neuronal cells from rat dorsal root ganglia, which was significantly reduced by A-438079 (1 μM). [1]
BzATP (3 mM) induced IL-1β release from mouse peritoneal macrophages, which was dose-dependently inhibited by A-438079 (0.3-3 μM). At 3 μM, A-438079 inhibited IL-1β release by 75.6% (P < 0.01). [4]
BzATP (500 μM) induced a strong, sustained increase in intracellular Ca²⁺ concentration in primary mouse macrophages, which was concentration-dependently reduced by the P2X7 antagonist Brilliant Blue G (IC50 = 0.74 μM). BzATP also induced ethidium bromide uptake (pore formation) in macrophages, which was inhibited by BBG (IC50 = 1.57 μM). [4]
BzATP (10-100 μM) induced proliferation and migration of U87 and U251 human glioma cells in a concentration-dependent manner. Proliferation peaked at 100 μM BzATP. BzATP (100 μM) increased migration rate from 39.7 ± 2.3% (control) to 73.0 ± 2.1% (P < 0.05). BzATP also increased expression of ERK, p-ERK, and PCNA proteins in these cells. The P2X7 antagonist BBG blocked these effects. BzATP did not significantly increase apoptosis in glioma cells. [3]
BzATP (100 μM) upregulated P2X7 receptor protein expression in U87 and U251 glioma cells as shown by immunofluorescence and Western blot. [3]
BzATP (0.5-25 μM) activated P2X1, P2X3, and P2X2/3 receptors with high potency (pEC50 6.70-8.74), and was least active at P2X7 and P2X4 receptors. [1]
In 1321N1 cells expressing recombinant P2X7 receptors, BzATP mediated Ca²⁺ influx with EC50 values: human P2X7 pEC50 = 5.33, rat P2X7 pEC50 = 5.01, mouse BALB/c P2X7 pEC50 = 3.99, mouse C57BL/6 P2X7 pEC50 = 4.02. BzATP was a full agonist at all tested P2X7 receptors. [2]
BzATP stimulated Yo-Pro uptake (pore formation) in P2X7-expressing cells with pEC50 values: human 6.18, rat 5.20, mouse BALB/c 4.22, mouse C57BL/6 4.44. [2]
At P2X1 receptors, BzATP was the most potent agonist tested (pEC50 = 8.74). At P2X3 receptors (human), BzATP had pEC50 = 7.10 (90% efficacy). At P2X2/3 heteromeric receptors, BzATP had pEC50 = 7.50 (90% efficacy). [1]

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BzATP (10-1000 μM; 24 hours) triethylammonium promotes the proliferation and migration of U87 and U251 glioma cells [3]. BzATP (100 μM; 6-48 hours) triethylammonium induces P2X7R protein expression in human glioma cells.

In Vivo: In a mouse model of cyclophosphamide-induced hemorrhagic cystitis, BzATP (3 mM, applied to macrophages in vitro) induced IL-1β release. [4]
In a mouse model of sepsis (cecal ligation and puncture), BzATP (5 mg/kg, i.p., 24 h after CLP) increased P2X7R expression in intestines, increased serum IL-6 and TNF-α levels (P < 0.001 vs control), increased histological scores, MPO activity, intestinal permeability (serum FD-40), bacterial translocation, and intestinal epithelial apoptosis, and decreased tight junction protein expression (occludin, claudin-1, ZO-1). BzATP-treated septic mice had 91% mortality at 48 hours. [4]
In a mouse model of sepsis, BzATP (5 mg/kg, i.p.) significantly promoted P2X7R expression in intestines compared to sham (P < 0.001) and control (P < 0.001) groups. [4]
In a rat model of Parkinson's disease (6-OHDA), BzATP (not administered in vivo; used for in vitro macrophage stimulation). [1]

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BzATP (5 mg/kg) triethylammonium significantly promoted the expression of P2X7R in the intestine after cecal ligation and puncture (CLP) induction compared with the sham and control groups [4].

Enzyme Assay: Calcium influx FLIPR assay: 1321N1 cells stably expressing P2X7 receptors were plated in poly-D-lysine-coated black 96-well plates and loaded with Fluo-4 dye. BzATP was tested at 11 half-log concentrations from 10⁻¹⁰ to 10⁻⁴ M. After agonist addition, changes in intracellular Ca²⁺ concentrations were recorded for 3 min. Concentration-effect curves were analyzed using a four-parameter logistic Hill equation. [1][2]
Yo-Pro uptake assay: 1321N1 cells expressing P2X7 receptors were plated in poly-D-lysine-coated black-walled 96-well plates. Cells were rinsed twice with PBS without Mg²⁺ or Ca²⁺ ions. Yo-Pro iodide dye (2 μM final) was added immediately prior to agonist addition, and dye uptake was measured for 1 hour. [2]
IL-1β release from macrophages: Resident resting macrophages were collected by peritoneal lavage, plated, and primed with LPS (3 μg/ml) for 2 h. BzATP (3 mM) was then added, and supernatants were collected 30 min later for IL-1β ELISA. [4]
Intracellular Ca²⁺ measurement in macrophages: Primary mouse macrophages were loaded with Fluo-4, and BzATP (500 μM)-induced changes in [Ca²⁺]c were measured. BBG was used as antagonist. [4]
Pore formation assay in macrophages: Macrophages were treated with BzATP (500 μM) in the presence of ethidium bromide (25 μM). EB uptake was measured fluorometrically. BBG was used as antagonist. [4]

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Activation of rat P2X3 and P2X2/3 receptors in vitro [https://pubmed.ncbi.nlm.nih.gov/11156585/]
The recombinant rat P2X3 and rat P2X2/3 receptor cDNAs were identical to the previously published sequences used in the in vitro characterization of the pharmacology of the rat homomeric and heteromeric P2X3 receptors (Bianchi et al., 1999). 1321N1 human astrocytoma cells stably expressing rat P2X3, or rat P2X2/3 receptors were constructed using standard lipid-mediated transfection methods. All cell lines were maintained in D-MEM containing 10% FBS and antibiotics as follows: 300 μg ml−1 G418 for rat P2X3 containing cells; and 75 μg ml−1 hygromycin and 150 μg ml−1 G418 for rat P2X2/3 containing cells. Cells were grown at 37°C in a humidified atmosphere containing 5% CO2. P2X receptor function was determined on the basis of agonist-mediated increases in cytosolic Ca2+ concentration as previously described (Bianchi et al., 1999). BzATP (10 μM) and α,β-meATP (10 μM) were used to activate rat P2X3 and P2X2/3 receptors, respectively. Briefly, a fluorescent Ca2+ chelating dye (Fluo-4) was used as an indicator of the relative levels of intracellular Ca2+ in a 96-well format using a Fluorescence Imaging Plate Reader (FLIPR). Cells were grown to confluence in 96-well black-walled tissue culture plates and loaded with the acetoxymethylester (AM) form of Fluo-4 (1 μM) in D-PBS for 1 – 2 h at 23°C. Fluorescence data was collected at 1 – 5 s intervals throughout each experimental run. Concentration response data were analysed using a four-parameter logistic Hill equation in GraphPad Prism.https://pubmed.ncbi.nlm.nih.gov/11156585/

Cell Assay: 1321N1 human astrocytoma cells stably expressing recombinant P2X1, P2X2, P2X3, P2X4, P2X7, P2Y1, P2Y2 receptors were maintained in DMEM with 10% FBS and appropriate selection antibiotics. Cells were plated in 96-well plates for calcium influx assays. [1][2]
Primary mouse peritoneal macrophages: Collected by lavage, plated, primed with LPS (3 μg/ml, 2 h), then treated with BzATP (3 mM) for IL-1β release. For Ca²⁺ and pore formation assays, macrophages were treated with BzATP (500 μM). [4]
U87 and U251 human glioma cells: Cultured in DMEM with 10% FBS. Cells were treated with BzATP (5-1000 μM) for MTT proliferation assays (optimal at 100 μM, 24 h). Migration was assessed by scratch wound assay. Western blot and immunofluorescence were used to detect P2X7R, ERK, p-ERK, PCNA expression. Apoptosis was assessed by TUNEL staining. [3]
Mouse lamina propria macrophages: Isolated from intestinal mucosa of septic mice, cultured in RPMI-1640 with 10% FBS. Treated with BzATP (500 μM) or BBG (10 μM) for 30 min. [4]

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Cell Proliferation Assay[3]
Cell Types: U87 and U251 glioma
Tested Concentrations: 5, 10, 50, 100, 500 and 1000 μM
Incubation Duration: 2, 6, 12, 24, 48 and 72 hours
Experimental Results: The proliferation of U87 and U251 glioma cell lines was significantly increased in the presence of 10-1000 uM and 100-1000 μM, respectively. The peak of cell proliferation of both U87 and U251 cell lines was at 100 μM. The optimal incubation time is 24 hours in both U87 and U251 cells lines.

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Western Blot Analysis[3]
Cell Types: U87 and U251 glioma
Tested Concentrations: 100 μM
Incubation Duration: 6-48 hours
Experimental Results: Induced the upregulation of P2X7R.

Animal/Disease Models: Male 2-month-old C57BL/6 mice (each weighing between 20 and 25 g)[4]
Doses: 5 mg/kg
Route of Administration: Injected through the intraperitoneal route
Experimental Results: At 48 hours, mice in the treated group and control group exhibited mortalities of 91% and 86%, respectively.

In a mouse sepsis model, BzATP (5 mg/kg, i.p., 24 h after CLP) increased mortality to 91% at 48 hours (compared to 86% in control and 73% in A740003-treated group). BzATP increased histological damage scores, MPO activity, intestinal permeability, and epithelial apoptosis in septic mice. [4]
In U87 and U251 glioma cells, BzATP (100 μM, 24 h) did not significantly increase apoptosis as measured by TUNEL staining. [3]

[1]. Pharmacological characterization of recombinant human and rat P2X receptor subtypes. Eur J Pharmacol. 1999 Jul 2;376(1-2):127-38.

[2]. Amino acid residues in the P2X7 receptor that mediate differential sensitivity to ATP and BzATP. Mol Pharmacol. 2007 Jan;71(1):92-100.

[3]. Involvement of P2X 7 Receptor in Proliferation and Migration of Human Glioma Cells. Biomed Res Int. 2018 Jan 9;2018:8591397.

[4]. Systemic blockade of P2X7 receptor protects against sepsis-induced intestinal barrier disruption. Sci Rep. 2017 Jun 29;7(1):4364.

BzATP (2',3'-O-(4-benzoylbenzoyl)ATP) is a synthetic ATP analog widely used as a P2X7 receptor agonist. However, it is not selective for P2X7; it is more potent at P2X1 and P2X3 receptors. BzATP is a partial agonist at rat P2X7 receptors (90% efficacy) and at human P2X7 receptors (98% efficacy). It is a full agonist at mouse P2X7 receptors. BzATP is also an agonist at P2X4 receptors (80% efficacy at human, 81% at rat). In glioma cells, BzATP promotes proliferation and migration via ERK pathway activation. In sepsis, BzATP exacerbates inflammation and intestinal barrier dysfunction. BzATP is used as a tool to study P2X7 receptor function in various disease models. [1][2][3][4]

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ATP, a rapid neurotransmitter, exerts its effects by specifically activating a class of ligand-gated ion channels called P2X receptors. In this study, six distinct recombinant P2X receptor subtypes were pharmacologically characterized in a heterologous expression system lacking endogenous P2X receptor activity. cDNAs encoding four human P2X receptor subtypes (hP2X1, hP2X3, hP2X4, and hP2X7) and two rat P2X receptor subtypes (rP2X2 and rP2X3) were stably expressed in 1321N1 human astrocytes. Furthermore, the rP2X2 and rP2X3 receptor subtypes were co-expressed in these cells, forming heteromeric receptors. The pharmacological characteristics of each receptor subtype were determined based on the assumed P2 ligand-stimulated Ca2+ influx activity. The potency and kinetics of each observed response were receptor subtype specific and correlated with their respective electrophysiological properties. Each receptor subtype exhibits unique pharmacological characteristics based on its sensitivity to nucleotide analogs, diadenosine polyphosphate, and putative P2 receptor antagonists. αβ-methylene ATP (αβ-meATP) is a putative P2X receptor selective agonist, and studies have shown it to exhibit potent agonist activity only on the hP2X1, hP2X3, and rP2X3 receptor subtypes. Benzoylbenzoic acid ATP (BzATP, a mixed 2' and 3' isomer) has been reported as a P2X7 receptor selective agonist, exhibiting minimal activity on rat and human P2X7 receptors, but is a potent (nM-level) agonist on hP2X1, rP2X3, and hP2X3 receptors. These data systematically investigate the functional pharmacology of P2X receptor activation. [1] The agonist properties of the P2X7 receptor (P2X7R) differ significantly from other P2X receptors in two main aspects: activation of the receptor requires high concentrations of ATP (>100 μM), and the ATP analog 2',3'-O-(4-benzoyl-benzoyl)ATP (BzATP) is more potent than ATP and induces a higher maximal current. However, these properties vary significantly across species. We attempted to exploit the large differences in the responses of rat and mouse P2X7R to ATP and BzATP to identify regions or specific residues on P2X7R that may enable these agonists to play unique roles. We measured the membrane current responses of wild-type rat and mouse P2X7R, chimeric P2X7R, and mouse P2X7R with point mutations to ATP and BzATP. Wild-type rat P2X7R was 10 times more sensitive to ATP and 100 times more sensitive to BzATP than wild-type mouse P2X7R. We found that the EC50 value of the agonist was entirely determined by the extracellular domain of P2X7R. When the two segments (amino acid residues 115-136 and 282-288) were interchanged, the sensitivity of mice was converted to that of rats. Point mutation analysis of these regions revealed that one amino acid residue in rat P2X7R—asparagine 284—fully explained the 10-fold difference in ATP sensitivity, while the 100-fold difference in Bz ATP sensitivity required the transfer of lysine 127 and asparagine 284 from rats to mice. Thus, a single amino acid difference between species can lead to a significant change in agonist potency and can distinguish between two widely used P2X7 receptor agonists. [2] Previous studies have shown that activation of the P2X7 receptor (P2X7R) can promote the proliferation and migration of certain types of tumors. This study aimed to investigate whether and how activated P2X7R promotes the proliferation and migration of human glioma cells. The results showed that the number of P2X7R positive cells increased with increasing tumor grade. In both U87 and U251 human glioma cell lines, P2X7R was expressed, and its expression could be enhanced by the P2X7R agonist 3'-O-(4-benzoylbenzoyl)ATP (BzATP) and siRNA. Our results also showed that 10 μM BzATP was sufficient to significantly induce glioma cell proliferation, while 100 μM BzATP caused cell proliferation to peak. In addition, BzATP treatment significantly enhanced the migration ability of U87 and U251 cells. However, BzATP treatment did not significantly change the number of apoptotic cells in U87 and U251 cells. In addition, the expression of ERK, p-ERK and proliferating cell nuclear antigen (PCNA) proteins was increased in BzATP-treated U87 and U251 glioma cells. The MEK/ERK pathway inhibitor PD98059 blocked the proliferation and migration of BzATP-activated glioma cells. These results indicate that the ERK pathway is involved in P2X7R activation-induced glioma cell proliferation and migration. [3]

C30H39N6O15P3

816.58

816.168625

112898-15-4

71308559

White to off-white solids at room temperature

3.396

C1=CC=C(C=C1)C(=O)C2=CC=C(C=C2)C(=O)O[C@@H]3[C@@H](COP(=O)(O)OP(=O)(O)OP(=O)(O)O)O[C@H]([C@@H]3O)N4C=NC5=C(N)N=CN=C54.CCN(CC)CC

HVOVBTNCGADRTH-WBLDMZOZSA-N

InChI=1S/C24H24N5O15P3.C6H15N/c25-21-17-22(27-11-26-21)29(12-28-17)23-19(31)20(16(41-23)10-40-46(36,37)44-47(38,39)43-45(33,34)35)42-24(32)15-8-6-14(7-9-15)18(30)13-4-2-1-3-5-13;1-4-7(5-2)6-3/h1-9,11-12,16,19-20,23,31H,10H2,(H,36,37)(H,38,39)(H2,25,26,27)(H2,33,34,35);4-6H2,1-3H3/t16-,19-,20-,23-;/m1./s1

[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-4-hydroxy-2-[[hydroxy-[hydroxy(phosphonooxy)phosphoryl]oxyphosphoryl]oxymethyl]oxolan-3-yl] 4-benzoylbenzoate;N,N-diethylethanamine

Benzoylbenzoyl-ATP triethylammonium; BzATP triethylammonium salt; 112898-15-4; benzoylbenzoyl-ATP; [(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-4-hydroxy-2-[[hydroxy-[hydroxy(phosphonooxy)phosphoryl]oxyphosphoryl]oxymethyl]oxolan-3-yl] 4-benzoylbenzoate;N,N-diethylethanamine; 2/'- AND 3/'-O-(4-BENZOYLBENZOYL)-ADENOSINE 5/'-TRIPHOSPHATE TRIETHYLAMMONIUM SALT; bbATP triethylammonium salt; Benzoylbenzoic adenosine 5'-triphosphate; CHEMBL4226675;

2934.99.9001

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.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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