P2X7 receptor – IC50 = 321 ± 20 nM (rat P2X7, calcium influx assay); selective over other P2X and P2Y receptors (P2X1, P2X2a, P2X2b, P2X4, P2Y1, P2Y2) at concentrations up to 100 μM; weak or no activity at 75 different G-protein-coupled receptors, enzymes, transporters, and ion channels (ED50 > 5 μM) [1][2][3][4]

In Vitro: A-438079 blocked BzATP (10 μM)-evoked changes in intracellular calcium concentrations in 1321N1 cells stably expressing rat P2X7 receptors with an IC50 of 321 ± 20 nM. At concentrations up to 100 μM, it did not significantly reduce agonist-evoked changes in intracellular calcium concentrations mediated by a variety of other P2X and P2Y receptors (P2X1, P2X2a, P2X2b, P2X4, P2Y1, P2Y2). [1]
In whole-cell voltage clamp recordings on non-neuronal cells (likely satellite glial cells) from L4/L5 dorsal root ganglia of SNL rats, A-438079 (1 μM) significantly reduced the current produced by BzATP (30 μM). A-438079 (1 μM) showed a tendency to reduce current evoked by 100 μM BzATP, but this was not significant. [1]
In mouse peritoneal macrophages, A-438079 (0.3-3 μM) dose-dependently decreased the quantity of IL-1β released by BzATP (3 mM) challenge. At 3 μM, A-438079 inhibited IL-1β release by 75.6% compared to vehicle controls (P < 0.01). [4]

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A 438079 inhibits BzATP-10 μM-induced fluctuations in intracellular calcium concentration in 1321N1 cells that express the rat P2X7 receptor stable, with an IC50 of 321 nM. At doses up to 100 μM, 438079 is likewise selective for the P2X7 receptor [1].

In Vivo: In rat models of neuropathic pain (spinal nerve ligation, chronic constriction injury, vincristine-induced neuropathy), systemic administration of A-438079 (10-300 μmol/kg, i.p.) produced dose-dependent anti-allodynic effects. ED50 values were approximately 76 μmol/kg (i.p.) in SNL, 100 μmol/kg (i.p.) in CCI, and 200 μmol/kg (i.p.) in vincristine model. [1]
In the formalin model, A-438079 (100-300 μmol/kg, i.p.) significantly reduced nocifensive behaviors during the second phase with an approximate ED50 of 102 μmol/kg (i.p.). [1]
In vivo electrophysiology in SNL rats: A-438079 (80 μmol/kg, i.v.) reduced von Frey-evoked (10 g) activity in hmWDR and mWDR neurons, brush-evoked activity in LT neurons, pinch-evoked firing of NS, hmWDR, mWDR neurons, and heat-evoked (50°C) responses of NS and hmWDR neurons. A-438079 also significantly decreased spontaneous firing in all four classes of spinal neurons (hmWDR, mWDR, NS, LT) in SNL but not sham rats. Effects typically occurred by 5 min after injection and lasted for 35 min. [1]
In a neonatal status epilepticus model (P10 rats, intra-amygdala kainic acid), A-438079 (5 and 15 mg/kg, i.p., given 60 min after KA) reduced seizure severity (EEG amplitude, total power, spike count). A-438079 (5 mg/kg) reduced hippocampal neuronal damage (FJB staining by 80-90%, TUNEL staining) and IL-1β levels. Higher dose (50 mg/kg) was less effective (U-shaped dose response). [2]
In a 6-OHDA rat model of Parkinson's disease, A-438079 (30 mg/kg, i.p., given 60 min before and after 6-OHDA) partially but significantly prevented 6-OHDA-induced striatal DA depletion (median % DA on lesioned side: 31.3% vs 23.4% in saline group, P=0.016). However, it did not prevent DA nerve cell loss in the substantia nigra. [3]
In a mouse model of cyclophosphamide-induced hemorrhagic cystitis, A-438079 (50-200 μmol/kg, i.p.) dose-dependently reduced nociceptive behavior scores, oedema, haemorrhage, bladder wet weight, MPO activity, macrophage migration (F4/80 staining), and IL-1β and TNF-α levels. The 50 and 100 μmol/kg doses reduced haemorrhage scores, while 200 μmol/kg did not. A-438079 (100 μmol/kg) also reduced c-Fos expression in brain cortical areas and lumbar spinal cord. [4]

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In neuropathic rats, 438079 (80 μmol/kg, i.v.) decreases the evoked activity of many classes of spinal cord neurons, both painful and nonnoxious. In SNL and CCI models, the withdrawal threshold was dramatically enhanced by 438079 (100 and 300 μmol/kg, ip) [1]. Sixty minutes after convulsions began, an intraperitoneal dose of A 438079 (5 and 15 mg/kg) decreased intrahippocampal neuronal death and seizure severity. When compared to phenobarbital at the same dose (25 mg/kg), 438079 exhibits better neuroprotective properties [2]. The depletion of striatal DA reserves caused by 6-OHDA is somewhat but significantly prevented by 438079 [3]. In the HC model, a 438079 Pretreatment lowers nociceptive behavior scores [4].

Enzyme Assay: Calcium influx FLIPR assay: 1321N1 cells stably expressing rat P2X7 receptors were plated in poly-D-lysine-coated black 96-well plates and loaded with Fluo-4 dye. Cells were washed with DPBS. A-438079 was tested at 11 half-log concentrations from 10⁻¹⁰ to 10⁻⁴ M. After agonist addition (BzATP at EC₇₀ concentration, 10 μM for rat P2X7), changes in intracellular Ca²⁺ concentrations were recorded for 3 min. For antagonist activity, A-438079 was added and fluorescence data collected for 3 min before agonist addition. pIC₅₀ values were derived from a single curve fit to mean data (n=17, in duplicate). [1]
In vitro electrophysiology on non-neuronal DRG cells: L4/L5 DRG from SNL rats were dissociated by enzyme digestion (0.1% collagenase, then collagenase/dispase). Whole-cell voltage clamp recordings were performed at room temperature within 48 h after dissociation. Pipette solution contained (mM): NaCl 154, EGTA 10, Hepes 5, pH 7.2; external solution contained (mM): NaCl 147, KCl 2, CaCl₂ 0.3, Hepes 10, glucose 12, pH 7.4. Electrode resistance was 2-4 MΩ. P2X currents were recorded at holding potential of -60 mV. A-438079 (1 μM) and BzATP (30-100 μM) were applied using a superfusion system. [1]
IL-1β release from macrophages: Resident resting macrophages were collected by peritoneal lavage, plated, and primed with LPS (3 μg/ml) for 2 h. A-438079 (0.3-3 μM) was applied followed 30 min later by BzATP (3 mM) challenge. Supernatants were collected 30 min after BzATP and analyzed for IL-1β by ELISA. [4]

Cell Assay: 1321N1 human astrocytoma cells stably expressing rat P2X7, human P2X4, P2X2a, P2X2b, P2X7, P2Y1, and P2Y2 receptors were maintained in DMEM with 10% FBS and appropriate selection antibiotics. For calcium influx assays, cells were plated in poly-D-lysine-coated black 96-well plates at 5 × 10⁶ cells per plate. [1]
Mouse peritoneal macrophages: Resident macrophages were collected by lavaging peritoneal cavities with RPMI 1640 containing 5% FCS. Cells were plated at 2 × 10⁶ cells/well, allowed to attach for 2 h, nonattached cells washed out, and cultured overnight. Cells were primed with LPS (3 μg/ml) for 2 h before A-438079 addition. [4]
Non-neuronal cells from rat DRG: L4/L5 DRG from SNL rats were dissociated by enzyme digestion and plated in PEI-treated 24-well plates in DMEM supplemented with 10% FBS, 50 mM NGF, 2 mM glutamine, 100 U/ml penicillin-streptomycin. Whole-cell voltage clamp recordings were performed within 48 h after dissociation. [1]

Animal Protocol: Neuropathic pain models (SNL, CCI, vincristine): Male Sprague-Dawley rats (250-400 g) underwent L5/L6 spinal nerve ligation, chronic constriction injury of the sciatic nerve, or vincristine infusion (30 μg/kg/day via osmotic pump). A-438079 (10-300 μmol/kg, i.p.) was administered, and tactile allodynia measured using von Frey filaments 2 weeks after surgery. [1]
Formalin model: Rats received formalin (5%, 50 μl) into the dorsal surface of the right hind paw. A-438079 (100-300 μmol/kg, i.p.) was injected, and nocifensive events recorded during the second phase. [1]
In vivo electrophysiology: SNL or sham rats were anesthetized with pentobarbital then maintained with propofol infusion (8-12 mg/kg/h, i.v.). A laminectomy was performed to remove vertebral segments T12-L3. Extracellular activity of spinal neurons was recorded using platinum-iridium microelectrodes. A-438079 (80 μmol/kg, i.v.) or vehicle was administered over 7 min. Stimuli included von Frey (10 g), brush, pinch (Bulldog Serrefine clamp), and heat (50°C water). [1]
Neonatal status epilepticus model: P10 rat pups underwent intra-amygdala kainic acid (2 μg in 0.2 μl) injection. A-438079 (0.5, 5, 15, 50 mg/kg, i.p.) was given 60 min after KA. EEG was recorded for 2 h. Rats were killed at 72 h for histology. [2]
6-OHDA Parkinson's model: Male Sprague-Dawley rats received unilateral intranigral 6-OHDA (0.2 μg/4 μl). A-438079 (30 mg/kg, i.p.) was given 60 min before and 60 min after 6-OHDA. Rats were killed 4 weeks later for striatal DA measurement by HPLC and TH immunohistochemistry. [3]
Cyclophosphamide-induced hemorrhagic cystitis model: Swiss or C57/BL6 mice received cyclophosphamide (300 mg/kg, i.p.). A-438079 (50, 100, 200 μmol/kg, i.p.) or Mesna (60 mg/kg) was given 30 min before and 4 h after cyclophosphamide (for behavioral experiments) or as a single dose 30 min before (for cytokine experiments). Bladders were collected at 6 h for gross evaluation, histology, immunohistochemistry (F4/80, P2X7R, c-Fos), MPO activity, and cytokine ELISA. [4]

In rats, A-438079 (10 μmol/kg) showed moderate plasma clearance (CLp = 1.6 L/h/kg), moderate bioavailability (F = 19%, i.p.), and moderate plasma elimination half-life (T₁/₂ = 1.02 h, i.p.). Cmax and Tmax at 10 μmol/kg (i.p.) were 0.45 μg/ml and 0.25 h, respectively. Mean plasma and brain levels of A-438079 (10 μmol/kg) at 30 min were 0.2 ± 0.8 μg/ml and 0.56 ± 0.2 μg/g, respectively, giving a brain-to-plasma ratio of approximately 2:1. Plasma protein binding was 84% in rats. [1]
In P10 rat pups, A-438079 (5 mg/kg, i.p.) reached plasma levels of ~2.3 μg/ml at 10 min, declining rapidly thereafter. Brain levels followed the same profile, peaking at the earliest tested time (10 min) and declining rapidly. [2]

A-438079 did not affect rat rotarod performance up to the highest dose tested (300 μmol/kg, i.p.). [1]
In P10 rat pups, A-438079 alone (5 or 50 mg/kg) did not promote cell death in the brain. [2]
In Swiss mice, A-438079 (100 μmol/kg) did not produce significant changes in rearing, walking, or general exploring behavior in the open-field test. [4]

[1]. P2X7-related modulation of pathological nociception in rats. Neuroscience. 2007 Jun 8;146(4):1817-28.

[2]. P2X7 receptor inhibition interrupts the progression of seizures in immature rats and reduces hippocampal damage. CNS Neurosci Ther. 2014 Jun;20(6):556-64.

[3]. On the role of P2X(7) receptors in dopamine nerve cell degeneration in a rat model of Parkinson's disease: studies with the P2X(7) receptor antagonist A-438079. J Neural Transm (Vienna). 2010 Jun;117(6):681-7.

[4]. The role of P2X7 purinergic receptors in inflammatory and nociceptive changes accompanying cyclophosphamide-induced haemorrhagic cystitis in mice. Br J Pharmacol. 2012 Jan;165(1):183-96.

A-438079 (3-((5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methylpyridine; molecular weight 342.6) is a selective, competitive P2X7 receptor antagonist that readily enters the CNS after systemic delivery. It has been used to demonstrate the role of P2X7 receptors in neuropathic pain, inflammatory pain, status epilepticus, Parkinson's disease, and hemorrhagic cystitis. In electrophysiological studies, it reduced both evoked and spontaneous firing of spinal neurons in neuropathic rats. A-438079 has a U-shaped dose-response curve in some models (e.g., neonatal SE), with higher doses (50 mg/kg) being less effective than intermediate doses (5-15 mg/kg). [1][2][3][4]

C13H10CL3N5

342.6110

341

899507-36-9

A 438079 hydrochloride;899431-18-6

11673921

White to off-white solid powder

3.892

0

4

3

20

319

0

MBTJFFMIPPMRGR-UHFFFAOYSA-N

InChI=1S/C13H9Cl2N5.ClH/c14-11-5-1-4-10(12(11)15)13-17-18-19-20(13)8-9-3-2-6-16-7-9;/h1-7H,8H2;1H

3-[[5-(2,3-dichlorophenyl)tetrazol-1-yl]methyl]pyridine;hydrochloride

A-438079; A438079; A 438079; 3-{[5-(2,3-dichlorophenyl)-1H-1,2,3,4-tetrazol-1-yl]methyl}pyridine; 3-((5-(2,3-dichlorophenyl)-1H-1,2,3,4-tetrazol-1-yl)methyl)pyridine;

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 (~326.64 mM)
H2O : ~0.2 mg/mL (~0.65 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.17 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.17 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.)