P2X7 receptor – IC50 = 20 nM (human P2X7, calcium influx); IC50 = 42 nM (rat P2X7, calcium influx); IC50 = 150 nM (mouse P2X7, calcium influx); IC50 = 37 nM (BzATP-stimulated IL-1β release in differentiated human THP-1 cells); IC50 = 7 nM (BzATP-stimulated YO-PRO uptake in differentiated human THP-1 cells); pIC50 = 7.67 ± 0.04 (human), 7.36 ± 0.02 (rat), 6.83 ± 0.03 (mouse) [1]; pA2 = 8.1 (competitive antagonist) [1]
A-839977 is a selective P2X7 receptor antagonist. It potently blocks human (IC50 = 20 nM), rat (IC50 = 42 nM), and mouse (IC50 = 150 nM) P2X7 receptors. [1]
It also blocks BzATP-stimulated IL-1β release (IC50 = 37 nM) and YO-PRO uptake (IC50 = 7 nM) in differentiated human THP-1 cells. [1]

In Vitro: A-839977 potently blocked BzATP-evoked changes in intracellular calcium concentrations in 1321N1 cells stably expressing human (IC50 = 20 nM), rat (IC50 = 42 nM), or mouse (IC50 = 150 nM) P2X7 receptors. [1]
A-839977 potently blocked BzATP-stimulated IL-1β release (IC50 = 37 nM) and YO-PRO uptake (IC50 = 7 nM) in differentiated human THP-1 cells. [1]
A-839977 produced parallel rightward shifts in BzATP concentration-effect calcium influx curves with a pA2 value of 8.1, indicating competitive antagonism. [1]
In cultured rat optic nerve head astrocytes, A-839977 (50 nM) prevented the swelling-induced upregulation of IL-1β mRNA. [2]
In cultured mouse optic nerve head astrocytes, A-839977 (100 nM) reduced the swelling-triggered reduction in IκBα, indicating inhibition of NFκB activation. [2]

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A 839977 specifically prevents agonist-induced YO-PRO uptake and IL-1beta release in differentiated human THP-1 cells by inhibiting BzATP-induced calcium influx at the mammalian P2X7 receptor (IC50=20-150 nM). In animal studies, it has been demonstrated to lessen neuropathic pain and inflammation [1]. In optic astrocytes, 839977 (50 nM, 1 hour pretreatment) effectively inhibits the rise in IL-1β initiation that is produced by stress [2].

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In 1321N1 human astrocytoma cells stably expressing human, rat, or mouse P2X7 receptors, A-839977 potently blocked BzATP-evoked changes in intracellular calcium concentrations (human IC50 = 20 nM, rat IC50 = 42 nM, mouse IC50 = 150 nM). [1]
In differentiated human THP-1 cells, A-839977 potently blocked BzATP-stimulated IL-1β release (IC50 = 37 nM) and YO-PRO uptake (IC50 = 7 nM), which are functional consequences of P2X7 receptor activation. [1]
In primary rat optic nerve head astrocytes, A-839977 (50 nM) significantly prevented the mechanosensitive IL-1β priming (mRNA upregulation) induced by 4 hours of swelling in a 30% hypotonic solution. The rise in IL-1β was blocked by A-839977. [2]
In primary mouse optic nerve head astrocytes, the swelling-induced rise in IL-1β mRNA was significantly lower in cells from P2X7-/- mice compared to C57Bl/6J mice. The P2X7 receptor antagonist A-839977 (100 nM) also reduced the swelling-triggered reduction of IκB-α protein expression, an indicator of NFκB activation. [2]
In spinal cord samples from cancer-bearing rats, Western blot analysis showed no significant change in P2X7 receptor expression at days 3, 7, 10, and 14 post-surgery compared to sham or naive animals, although a tendency to increased expression was observed on day 10. [3]

In Vivo: In a rat model of cancer-induced bone pain (intratibial MRMT-1 carcinoma cells), spinal application of A-839977 (0.4 and 1.2 mg/kg) significantly reduced dorsal horn neuronal responses to high-intensity mechanical stimulation (26g and 60g) and high-intensity thermal stimulation (48°C) in a dose-dependent manner. No effect was observed on responses to low-intensity stimuli or in sham/naïve animals. [3]
In the same model, systemic administration of A-839977 (40 mg/kg, i.p.) significantly increased mechanical withdrawal threshold (von Frey test) in cancer-bearing animals, with no effect in sham animals or with vehicle treatment. A higher dose (120 mg/kg) showed toxic effects. [3]
A-839977 (40 mg/kg, i.p.) significantly increased weight-bearing ratio (incapacitance test) and limb-use score in cancer-bearing animals at later stages of disease, with no effect of vehicle treatment. [3]
A-839977 (40 mg/kg, i.p.) did not affect motor coordination in naive rats as measured by rotarod test. [3]
In a mouse model of CFA-induced inflammatory pain, A-839977 dose-dependently reduced thermal hyperalgesia (ED50 = 40 μmol/kg, i.p. in mice; ED50 = 100 μmol/kg, i.p. in rats). The antihyperalgesic effect was absent in IL-1αβ knockout mice. [1]
In rats, A-839977 (30 mg/kg, i.p.) 30 min before testing attenuated CFA-induced thermal hyperalgesia. [1]
In a mouse model of retinal mechanical strain (controlled elevation of IOP), A-839977 (50 nM in vitro, or via P2X7 knockout) prevented the upregulation of IL-1β mRNA and protein. [2]

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In rats, A839977 (30 μmol/kg, 100 μmol/kg, 300 μmol/kg; 30 min preinjection) lowers thermal hyperalgesia in a dose-dependent manner when complete Freund's adjuvant (CFA) is injected plantarly [1]. The CFA model of inflammatory pain in wild-type mice was significantly affected by A839977 (10 μmol/kg, 30 μmol/kg, and 100 μmol/kg; pre-injection for 30 minutes), whereas IL-1alphabeta knockout mice were not significantly affected. Rats are ineffective [1]. In animals with cancer, A839977 reduces the responses of dorsal horn neurons [3].

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In a rat model of CFA-induced inflammatory pain, systemic administration of A-839977 (i.p.) dose-dependently reduced thermal hyperalgesia (ED50 = 100 μmol/kg), with a 65.2 ± 4.7% effect at the highest dose tested. It had no effect on the contralateral non-inflamed paw, indicating a specific antihyperalgesic effect. [1]
In a mouse model of CFA-induced inflammatory pain, A-839977 (i.p.) dose-dependently reduced thermal hyperalgesia in wild-type mice (ED50 = 40 μmol/kg, 68.4 ± 8.3% effect at the highest dose). However, this antihyperalgesic effect was completely absent in IL-1αβ knockout mice. [1]
In a rat model of cancer-induced bone pain (MRMT-1 cell inoculation), spinal application of A-839977 (0.4 and 1.2 mg/kg) dose-dependently reduced dorsal horn wide dynamic range (WDR) neuronal responses to high-intensity mechanical (26g and 60g) and thermal (48°C) stimulation. No effect was seen on responses to low-intensity or electrical stimulation. The antagonist had no effect on neuronal responses in sham or naive animals. [3]
In the same cancer-induced bone pain model, systemic administration of A-839977 (40 mg/kg, i.p.) significantly increased the mechanical withdrawal threshold (von Frey test) in cancer-bearing animals, indicating a reduction in mechanical hypersensitivity. It also significantly improved weight-bearing ratio and limb-use scores, indicating attenuation of movement-evoked and non-evoked pain. No effect was observed in sham or vehicle-treated animals. [3]

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. After washing, cells were incubated with A-839977 for 3 min before agonist addition. BzATP at EC70 concentrations (mouse: 150 μM, rat: 10 μM, human: 5 μM) was used as agonist. Fluorescence was measured for 3 min. IC50 values were calculated. [1]
YO-PRO uptake assay: Differentiated human THP-1 cells were plated and pre-incubated with A-839977 for 30 min. BzATP (90 mM) was added and YO-PRO uptake measured for 1 hour. [1]
IL-1β release assay: Differentiated human THP-1 cells were primed with LPS (25 ng/ml) and IFNγ (10 ng/ml) for 3 h. A-839977 was added 30 min before BzATP (1 mM) stimulation. IL-1β levels in supernatant were measured by ELISA. [1]

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The antagonist activity of A-839977 on P2X7 receptors was measured using a calcium influx assay. 1321N1 cells expressing recombinant human, rat, or mouse P2X7 receptors were loaded with a calcium-chelating dye. For antagonist activity measurement, the test compound was added to the cell plate, and fluorescence data were collected for 3 minutes before the addition of the agonist (BzATP, at its approximate EC70 concentration for each species). Fluorescence data were then collected for another 2 minutes after agonist addition. The concentration-response data were analyzed to derive pIC50 values. [1]
The effect of A-839977 on P2X7 receptor-mediated pore formation was assessed using a YO-PRO uptake assay. Cells (recombinant P2X7-1321N1 cells or differentiated THP-1 cells) were exposed to YO-PRO dye. After the addition of various concentrations of the antagonist, an EC70 concentration of BzATP was added to activate the receptor. YO-PRO dye uptake was observed by capturing the intensity of fluorescence over time (every 15 seconds for the first 10 minutes, then every 20 seconds for an additional 50 minutes). The percent maximal intensity was normalized to that induced by BzATP alone to calculate IC50 values. [1]
For the IL-1β release assay, differentiated THP-1 cells were incubated with A-839977 for 30 minutes at 37°C, followed by a challenge with 1 mM BzATP for an additional 30 minutes. Supernatants were collected and assayed for the presence of mature IL-1β by ELISA. Background IL-1β release from control cells was subtracted from the BzATP-induced release. [1]

Cell Assay: 1321N1 human astrocytoma cells stably expressing mouse, rat, or human P2X7 receptors were maintained in DMEM with 1% L-alanyl-L-glutamine, 1% antibiotic/antimycotic, 10% FBS, and 300 μg/ml geneticin. For calcium influx assays, cells were plated at 5 × 10⁶ cells per plate. [1]
THP-1 human monocytic cells were differentiated into macrophage phenotype with LPS (25 ng/ml) and IFNγ (10 ng/ml) for 3 h (IL-1β release) or overnight (pore formation). Cells were maintained in RPMI with 10% fetal calf serum. [1]
Rat optic nerve head astrocytes: Primary astrocytes were isolated from neonatal rat pups (PD3-5) and cultured in DMEM/F12 with 10% FBS, 1% penicillin/streptomycin, and 25 ng/ml EGF. Cells were >99% astrocytes by GFAP staining. For swelling experiments, cells were incubated in 30% hypotonic solution for 4 h. A-839977 (50 nM) was added 1 h before swelling. [2]
Mouse optic nerve head astrocytes: Isolated from 3-month-old C57BL/6J and P2X7-/- mice. Cultured similarly to rat astrocytes. A-839977 (100 nM) was used. [2]

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RT-PCR[2]
Cell Types: Optic astrocytes
Tested Concentrations: 50 nM
Incubation Duration: 1 hour (pre-treatment)
Experimental Results: Prevents IL-1β initiation in astrocytes

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For the calcium influx assay, 1321N1 human astrocytoma cells stably expressing mouse, rat, or human P2X7 receptors were plated onto poly-D-lysine coated black 96-well plates at a concentration of 5x10^6 cells per plate. Cells were loaded with a calcium-chelating dye for at least 1 hour but not more than 3 hours. After removing unincorporated dye, changes in intracellular Ca2+ concentrations upon agonist addition were recorded using a fluorescence imaging plate reader over a 3-minute period. For antagonist activity, the compound was added 3 minutes before the agonist (BzATP). [1]
For the YO-PRO uptake assay, cells (recombinant P2X7-1321N1 cells or differentiated THP-1 cells) were plated onto poly-D-lysine-coated black-walled 96-well plates at a density of 1x10^6 cells/plate. YO-PRO dye was diluted to a final concentration of 2 μM and placed on the cells immediately prior to agonist addition. Agonist-induced pore formation was assessed by capturing fluorescence intensity over time (every 15s for first 10 min, then every 20s for an additional 50 min) using a fluorescence imaging plate reader. [1]
For the IL-1β release assay, THP-1 cells were differentiated into a macrophage phenotype using LPS (25 ng/ml) and IFNγ (10 ng/ml) for 3 hours. Differentiated cells were then incubated with A-839977 for 30 minutes at 37°C, followed by challenge with 1 mM BzATP for an additional 30 minutes. Supernatants were collected and mature IL-1β was quantified by ELISA. [1]
For primary rat optic nerve head astrocyte experiments, cells were grown to confluence and then subjected to swelling by incubation in a 30% hypotonic solution for 4 hours, or to cyclical stretch (16% strain at 0.3 Hz for 4 hours) using a vacuum-operated tension system. RNA was extracted immediately after treatment, and qPCR was used to measure the expression of IL-1β and other inflammasome genes. Cells were pretreated with A-839977 (50 nM) for 1 hour before the application of test solutions. [2]
For immunoblotting, whole retinas or cultured astrocytes were lysed in RIPA buffer. Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. Blots were probed with primary antibodies against IL-1β, IκB-α, or β-actin, followed by HRP-conjugated secondary antibodies. Chemiluminescence was used for detection. [2]

Animal/Disease Models: Male SD (SD (Sprague-Dawley)), balb/c (Bagg ALBino) mouse and IL-1 (−/−) mice, for CFA-induced chronic inflammation Doses: 30 μmol/kg, 100 μmol/kg, 300 μmol/kg (rat); 10 μmol/kg, 30 μmol/kg, 100 μmol/kg (mouse)
Route of Administration: injection; 30-minute pre-injection
Experimental Results: Attenuated CFA-induced thermal hyperalgesia in a dose-related manner in rats and mice, However, it had no effect on IL-1 (−/−) mice.

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In the rat CFA-induced thermal hyperalgesia model, unilateral inflammation was induced by injecting 150 μl of a 50% CFA solution into the plantar surface of the right hind paw. A-839977 was dissolved in 30% NMP, 30% PEG400, and 40% hydroxypropyl-β-cyclodextrin and administered intraperitoneally (i.p.) 30 minutes before testing, which was conducted 48 hours after CFA injection. [1]
In the mouse CFA model, unilateral inflammation was induced by injecting 25 μl of a 50% CFA solution into the plantar surface of the right hind paw. A-839977 was prepared in the same vehicle and administered i.p. 30 minutes before testing, 48 hours post-CFA. [1]
For the rat cancer-induced bone pain model (electrophysiology), male Sprague-Dawley rats were inoculated with 3x10^3 MRMT-1 carcinoma cells into the intramedullary cavity of the tibia. Electrophysiological recordings were made 12-16 days after inoculation. A-839977 was dissolved daily in 10% DMSO, 10% ChromEL, and sterile saline and administered directly onto the spinal cord at doses of 0.2, 0.4, or 1.2 mg/kg. [3]
For the behavioral experiments in the cancer-induced bone pain model, rats were injected with 5x10^3 MRMT-1 cells. Mechanical hypersensitivity was assessed using von Frey filaments (up-down method). Weight-bearing was measured using an incapacitance tester, and limb use was scored on a scale from 0 to 3. A-839977 was dissolved in 30% NMP, 30% PEG400, and 40% hydroxypropyl-β-cyclodextrin and administered i.p. at 40 mg/kg (or 120 mg/kg, which showed clear toxic effects). Behavioral tests were performed 10-15 minutes post-administration. The rotarod test (accelerating from 3.5 to 35 rpm over 5 min) was used to assess motor coordination in naive animals following saline, vehicle, or drug administration. [3]
For the controlled elevation of IOP (CEI) model in rats and mice, IOP was unilaterally elevated to 50-60 mmHg for 4 hours. In some rat experiments, the P2X7 antagonist A-839977 was not used in vivo in this model, but the related antagonist BBG was used. In vitro, A-839977 (50 nM) was used on isolated astrocytes. [2]

A-839977 is CNS-penetrating with a brain/spinal cord to plasma ratio of 0.15-0.25. [1][3]
For in vivo electrophysiology, A-839977 was dissolved daily in 10% DMSO, 10% ChromEL, and sterile saline. [3]
For behavioral studies, A-839977 was dissolved in 30% NMP, 30% PEG400, and 40% hydroxypropyl-β-cyclodextrin. [3]

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A-839977 is able to penetrate into the central nervous system (CNS). It has a brain/spinal cord to plasma ratio of 0.15-0.25. [1]
A-839977 is CNS penetrating with a brain/spinal cord to plasma ratio of 0.15-0.25. [3]

Toxicity/Toxicokinetics: In cancer-induced bone pain model, a dose of 120 mg/kg A-839977 (i.p.) demonstrated clear toxic effects and was not tested further. The 40 mg/kg dose was well tolerated with no observed motor coordination deficits. [3]
In the CFA inflammatory pain model, A-839977 did not produce any overt signs of behavioral disruption at effective doses. [1]

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[1]. The antihyperalgesic activity of a selective P2X7 receptor antagonist, A-839977, is lost in IL-1alphabeta knockout mice. Behav Brain Res. 2009 Dec 1;204(1):77-81.

[2]. Albalawi F et.al, The P2X7 Receptor Primes IL-1β and the NLRP3 Inflammasome in Astrocytes Exposed to Mechanical Strain. Front Cell Neurosci. 2017 Aug 8;11:227.

[3]. P2X7 receptor-mediated analgesia in cancer-induced bone pain. Neuroscience. 2015 Apr 16; 291:93-105.

A-839977 (1-(2,3-dichlorophenyl)-N-(2-(pyridin-2-yloxy)benzyl)-1H-tetrazol-5-amine) is a potent, selective, competitive P2X7 receptor antagonist derived from a series of tetrazole-based compounds. It is CNS-penetrant and has been used to demonstrate that P2X7 receptor antagonism produces antinociception in inflammatory and cancer-induced bone pain models. The antihyperalgesic effect of A-839977 is mediated through blockade of IL-1β release, as it is lost in IL-1αβ knockout mice. A-839977 has also been used to study P2X7 receptor involvement in inflammasome priming in astrocytes following mechanical strain. [1][2][3]

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The antihyperalgesic effects of A-839977 in an inflammatory pain model in mice are mediated by blocking the release of IL-1β, as its effect was lost in IL-1αβ knockout mice. [1]
A-839977 is a structurally novel P2X7 antagonist derived from a series of potent, selective, and competitive P2X7 receptor antagonists. It produced parallel rightward shifts in BzATP concentration-effect calcium influx curves with a pA2 value of 8.1. [1]
The P2X7 receptor is a key player in IL-1 processing and release, and its activation by ATP stimulates the rapid maturation and release of IL-1β from macrophages and microglial cells. A-839977 blocks this process. [1]
In a model of mechanical strain (elevated intraocular pressure), the P2X7 receptor is implicated in the mechanosensitive priming of the NLRP3 inflammasome, leading to increased IL-1β expression. A-839977 is used as a tool to block this pathway. [2]
The analgesic effect of A-839977 in cancer-induced bone pain is state-dependent, as it reduced nociceptive responses in cancer-bearing animals but had no effect in sham or naive animals. This suggests the P2X7 receptor might be a more disease-specific target compared to traditional analgesics. [3]

C19H14CL2N6O

413.260060787201

412.061

C, 55.22; H, 3.41; Cl, 17.16; N, 20.34; O, 3.87

870061-27-1

53325875

White to off-white solid powder

4.19

1

6

6

28

489

0

ClC1C(Cl)=C(N2C(NCC3C(OC4C=CC=CN=4)=CC=CC=3)=NN=N2)C=CC=1

GMVNBKZQJFRFAR-UHFFFAOYSA-N

InChI=1S/C19H14Cl2N6O/c20-14-7-5-8-15(18(14)21)27-19(24-25-26-27)23-12-13-6-1-2-9-16(13)28-17-10-3-4-11-22-17/h1-11H,12H2,(H,23,24,26)

1-(2,3-dichlorophenyl)-N-{[2-(pyridin-2-yloxy)phenyl]methyl}-1H-1,2,3,4-tetrazol-5-amine

A839977; A-839977; A-839,977; A839,977; 1-(2,3-dichlorophenyl)-N-{[2-(pyridin-2-yloxy)phenyl]methyl}-1H-1,2,3,4-tetrazol-5-amine; 1-(2,3-dichlorophenyl)-N-((2-(pyridin-2-yloxy)phenyl)methyl)-1H-1,2,3,4-tetrazol-5-amine; 870061-27-1; A 839977

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)

DMSO : ~100 mg/mL (~241.98 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.05 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.

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Solubility in Formulation 2: 2.5 mg/mL (6.05 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.05 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 25.0 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.)