The P2X7 receptor in the HEK-hP2X7 cell line is blocked by the antagonist AZD9056 with an IC50 of 11.2 nM, demonstrating the antagonist's strong receptor selectivity. AZD9056, an antagonist of the P2X7 receptor, significantly inhibits murine microglial BV2 cells (IC50=1-3 μM) [1]. The BCRP inhibitor AZD9056 has an IC50 of 92 μM, which indicates a poor inhibition of BCRP-mediated methotrexate transport[2].
P2X7 receptor – selective antagonist; inhibits BCRP-mediated methotrexate transport with IC50 = 92 μM; inhibits BCRP-mediated estrone 3-sulphate transport with IC50 = 32 μM; does not inhibit OAT1 or OAT3 transporters [2]
P2X7 receptor antagonist that suppresses NF-κB pathway activation and reduces inflammatory cytokine expression in osteoarthritis [3]

The P2X7 receptor in the HEK-hP2X7 cell line is blocked by the antagonist AZD9056 with an IC50 of 11.2 nM, demonstrating the antagonist's strong receptor selectivity. AZD9056, an antagonist of the P2X7 receptor, significantly inhibits murine microglial BV2 cells (IC50=1-3 μM) [1]. The BCRP inhibitor AZD9056 has an IC50 of 92 μM, which indicates a poor inhibition of BCRP-mediated methotrexate transport[2].

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AZD9056 inhibited BzATP-induced inward currents in P2X7 receptor-expressing mouse microglia BV2 cells in patch-clamp experiments. The inhibition was concentration-dependent with an IC₅₀ of 1-3 μmol/L. [1]
AZD9056 (10 μmol/L) did not significantly inhibit Ca²⁺ influx in human astrocytoma 1321N1 cells overexpressing P2X1, P2X2, or P2X3 receptors upon ATP stimulation. It caused a 27% decrease in activity in P2X4-expressing cells and reduced the remaining activity to 9% in P2X7-expressing cells, demonstrating selectivity for the P2X7 receptor over other P2X receptor subtypes. [1]
AZD9056 inhibited ATP-induced hydrogen peroxide (H₂O₂) release from freshly isolated human mononuclear blood cells in a concentration-dependent manner, with an IC₅₀ of 3.0 μmol/L when cells were stimulated with 1 mmol/L ATP. [1]
AZD9056 (10 μmol/L) reversed the BzATP (50 μmol/L)-induced increases in cellular respiration, extracellular acidification (metabolic activity), and changes in cell impedance (morphology) in HEK293 cells overexpressing the human P2X7 receptor (HEK-hP2X7). The inhibition was reversible upon washout of the antagonist. No such effects were observed in parental HEK293 cells lacking P2X7. [1]
AZD9056 protected HEK-hP2X7 cells from ATP- or BzATP-induced cytotoxicity in a concentration-dependent manner. It antagonized ATP (2.5 mmol/L)-induced cytotoxicity with an IC₅₀ of 11.4 nmol/L and BzATP (0.25 mmol/L)-induced cytotoxicity with an IC₅₀ of 5.62 nmol/L. No protective effect was observed in parental HEK293 cells. [1]
AZD9056 (10 μmol/L) inhibited the ATP (250 μmol/L)-induced uptake of the fluorescent dye YoPro1 in HEK-hP2X7 cells, a marker for P2X7 receptor pore dilation, with an IC₅₀ of 11.2 nmol/L. [1]

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AZD9056 (0.3-100 μM) did not inhibit OAT1- or OAT3-mediated methotrexate uptake in oocyte assays. It inhibited BCRP-mediated methotrexate transport with an IC50 of 92 μM and BCRP-mediated estrone 3-sulphate transport with an IC50 of 32 μM. [2]
AZD9056 is a weakly basic secondary amine (pKa 9.77) with a hydrophobic adamantane moiety that is highly protein bound (97%). [2]

Treatment with AZD9056 has anti-inflammatory and analgesic properties. causes the expression of interleukin (IL)-1β, IL-6, matrix metalloproteinase-13 (MMP-13), substance P (SP), prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α) to be upregulated. AZD9056 can counteract the effects of MIA in cartilage tissue [3].

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In a rat model of monosodium iodoacetate (MIA)-induced osteoarthritis, AZD9056 (12.5 mg/kg, injected every 2 days for 7 days starting 2 weeks after MIA injection) significantly reduced weight-bearing asymmetry, increased paw withdrawal thresholds, and decreased knee edema size compared to MIA-only group (p < 0.05). [3]
AZD9056 reversed the MIA-induced upregulation of IL-1β, IL-6, and TNF-α in both serum and knee joint cartilage tissues of rats with osteoarthritis. [3]
AZD9056 significantly decreased the MIA-induced increase in P2X7 receptor, MMP-13, substance P (SP), and prostaglandin E2 (PGE2) expression in knee joint cartilage tissues. [3]
AZD9056 reversed the MIA-induced upregulation of IKKα, IKKβ, IκBα, NF-κB p65 and their phosphorylated forms in the knee cartilage tissues of rats with OA. [3]
In rheumatoid arthritis clinical studies, AZD9056 was evaluated for efficacy in patients with active disease despite treatment with methotrexate or sulphasalazine. [2]

Patch-clamp electrophysiology: Mouse microglia BV2 cells, which express functional P2X7 receptors, were analyzed in the whole-cell patch-clamp configuration. The intracellular pipette solution contained KF, KCl, EGTA, and Hepes buffer. The extracellular bath solution contained NaCl, KCl, CaCl₂, Hepes, and glucose. Cells were held at a constant potential of -70 mV. Agonist-induced currents were recorded upon application of BzATP (100 μmol/L) in the presence or absence of different concentrations of AZD9056 via a computer-controlled perfusion system. Steady-state currents were measured, leak-corrected, and used to generate concentration-response curves for the antagonist. [1]
Calcium flux assay: Human astrocytoma 1321N1 cells overexpressing specific human P2X receptor subtypes (P2X1, P2X2, P2X3, P2X4, or P2X7) were loaded with the calcium-sensitive fluorescent dye Fluo-4 AM. After washing, cells were preincubated with 10 μmol/L AZD9056 for 2 minutes before stimulation with ATP (0.2-2 mmol/L). Fluorescence signals, indicating intracellular Ca²⁺ increase, were recorded after 15 minutes using a fluorescent plate reader to assess receptor activity and antagonist selectivity. [1]
YoPro1 uptake assay: This assay measures pore dilation of the P2X7 receptor. HEK-hP2X7 cells were seeded in plates. After removal of culture medium, assay buffer containing the membrane-impermeable dye YoPro1 was added along with ATP (250 μmol/L) and varying concentrations of AZD9056. Cells were incubated for 45 minutes, allowing dye entry through the dilated P2X7 pore. Fluorescence was measured using a plate reader (excitation 485 nm, emission 530 nm) to quantify dye uptake and antagonist inhibition. [1]

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Oocyte uptake assays: Pools of 10 OAT1- or OAT3-expressing or water-injected oocytes were washed with sodium uptake buffer (pH 7.4). Uptake solutions contained radiolabelled compound and appropriate concentrations of non-radiolabelled compound in sodium uptake buffer. After 1 h incubation at room temperature, oocytes were washed 5 times with ice-cold buffer, lysed with 10% SDS, and analyzed by liquid scintillation counting. [2]
Vesicle uptake assays: MRP2- and BCRP-expressing membrane vesicles (0.67 mg/mL) were incubated with radiolabelled substrates in uptake buffer (250 mM sucrose, 10 mM MgCl₂, 10 mM Tris/HCl, pH 7.4) with or without 5 mM ATP. After 4 min incubation at 37°C, reactions were terminated by filtration through GF/B filter plates. Radioactivity was measured by liquid scintillation counting. [2]

Hydrogen peroxide release assay: Freshly isolated human mononuclear blood cells were incubated in a buffer containing Amplex Red reagent and horseradish peroxidase. After adding ATP (1 mmol/L) or BzATP, the fluorescence resulting from H₂O₂-dependent conversion of Amplex Red to resorufin was quantified using a microplate reader (excitation 550 nm, emission/absorbance 600 nm). The effect of AZD9056 was tested by preincubating cells with the antagonist before agonist addition. [1]
Cytosensor real-time monitoring: HEK-hP2X7 or parental HEK293 cells were cultured on a sensor chip. A cytosensor system continuously recorded extracellular acidification (metabolism), oxygen consumption (respiration), and cellular impedance (morphology/adhesion) under a constant flow of assay medium. Cells were exposed to BzATP (0-100 μmol/L) in increasing concentrations during stop-flow cycles. For inhibition experiments, cells were pre-exposed to 10 μmol/L AZD9056 prior to stimulation with 50 μmol/L BzATP. Wash-out phases were included to assess reversibility. [1]
Cell viability/cytotoxicity assay: HEK-hP2X7 or parental HEK293 cells were seeded in plates and allowed to attach. For agonist toxicity, ATP or BzATP was added at varying concentrations. For antagonist protection, cells were preincubated with increasing concentrations of AZD9056 for 5 minutes before adding a fixed concentration of ATP (2.5 mmol/L) or BzATP (0.25 mmol/L). After a 30-minute incubation, a cell viability reagent was added, and fluorescence was measured after 1 hour (excitation 560 nm, emission 590 nm) to determine cell survival. [1]
Mitochondrial membrane potential assay: HEK-hP2X7 and parental HEK293 cells were seeded on coated slides and loaded with the JC-1 dye, which accumulates in mitochondria and exhibits red fluorescence in healthy cells with intact membrane potential. Cells were then treated with BzATP (31.3 or 250 μmol/L) for 1 hour. Depolarization of mitochondria leads to a shift in JC-1 fluorescence from red to green, which was monitored using confocal microscopy. Valinomycin served as a positive control for depolarization. This assay was used to demonstrate agonist-induced mitochondrial toxicity, but the direct effect of AZD9056 on mitochondrial potential was not described in this context. [1]

Osteoarthritis rat model: Male Wistar rats received a single intra-articular injection of monosodium iodoacetate (MIA, 5 mg/kg) in sterile 0.9% saline into the left hind knee through the infrapatellar ligament using a 26G needle. AZD9056 (12.5 mg/kg) was injected every 2 days for 7 days starting 2 weeks after MIA injection. Behavioral tests (paw withdrawal threshold using Von Frey monofilaments, hindlimb weight-bearing asymmetry using Incapacitance Tester, and knee edema size using digital calipers) were evaluated at days 3, 7, 10, 14, 15, 17, 19, and 21 after MIA injection. Rats were sacrificed on days 7, 14, and 21 for tissue collection. [3]

The therapeutic dose of AZD9056 is predicted to be 400 mg, giving a predicted total maximum plasma concentration of 2 μM. The theoretical intestinal concentration is calculated to be 3.5 mM. Unbound AZD9056 plasma concentration is 0.06 μM. The compound is highly protein bound (97%). [2]

No specific toxicity data (LD50, hepatotoxicity, nephrotoxicity, drug-drug interactions) were described for AZD9056 in these papers. The compound was evaluated as a potential cause of drug-drug interaction with methotrexate via transporter inhibition. The [I₁]/IC₅₀ ratio for AZD9056 inhibition of BCRP-mediated estrone 3-sulphate transport was 0.06 (total concentration) or 0.002 (unbound concentration), and for BCRP-mediated methotrexate transport was 0.02 (total) or 0.0007 (unbound), indicating low risk of systemic DDI. The intestinal [I₂]/IC₅₀ ratio was 109, suggesting potential for intestinal DDI with BCRP substrates. [2]

[1]. ATP-induced cellular stress and mitochondrial toxicity in cells expressing purinergic P2X7 receptor. Pharmacol Res Perspect. 2015 Mar;3(2):e00123.

[2]. In vitro risk assessment of AZD9056 perpetrating a transporter-mediated drug-drug interaction with methotrexate. Eur J Pharm Sci. 2011 May 18;43(1-2):41-9.

[3]. Blocking of the P2X7 receptor inhibits the activation of the MMP-13 and NF-κB pathways in the cartilage tissue of rats with osteoarthritis. Int J Mol Med. 2016 Dec;38(6):1922-1932.

AZD9056 is described as a selective antagonist of the purinergic P2X7 receptor. [1]
As mentioned in the introduction, its efficacy in rheumatoid arthritis has been evaluated in a phase II clinical trial. [1]
In this study, AZD9056 was primarily used as a pharmacological tool to demonstrate that various cellular effects induced by ATP or its analogue BzATP (ion flow, dye uptake, metabolic changes, oxidative stress, cytotoxicity) are specifically mediated by activation of the P2X7 receptor. [1]
In cell sensor experiments, the antagonist exhibited reversible inhibitory effects. [1]

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AZD9056 is a purinergic P2X7 receptor antagonist that was being developed for the oral treatment of rheumatoid arthritis. It is a weakly basic secondary amine (pKa 9.77) with a hydrophobic adamantane moiety. In vitro studies showed that AZD9056 is neither an inhibitor of OAT1 nor OAT3 and is unlikely to cause a systemic transporter-mediated DDI with methotrexate. However, as a BCRP inhibitor, it has potential to perpetrate an intestinal DDI with co-administered BCRP substrate drugs. In an osteoarthritis rat model, AZD9056 exerted anti-inflammatory and pain-relieving effects by inhibiting P2X7R and the NF-κB signaling pathway, reducing the expression of IL-1β, IL-6, TNF-α, MMP-13, SP, and PGE2. The compound was evaluated in clinical trials for rheumatoid arthritis but did not meet efficacy endpoints. [2][3]

C24H36CL2N2O2

455.460844993591

454.215

C, 63.29; H, 7.97; Cl, 15.57; N, 6.15; O, 7.03

345303-91-5

345304-65-6

10161380

White to off-white solid powder

5.774

4

3

10

30

514

0

C1C2CC3CC1CC(C2)(C3)CNC(=O)C4=C(C=CC(=C4)CCCNCCCO)Cl.Cl

LZBBHFRRZLDHLE-UHFFFAOYSA-N

InChI=1S/C24H35ClN2O2.ClH/c25-22-5-4-17(3-1-6-26-7-2-8-28)12-21(22)23(29)27-16-24-13-18-9-19(14-24)11-20(10-18)15-24;/h4-5,12,18-20,26,28H,1-3,6-11,13-16H2,(H,27,29);1H

Benzamide, 2-chloro-5-(3-((3-hydroxypropyl)amino)propyl)-N-(tricyclo(3.3.1.13,7)dec-1-ylmethyl)- HCl

AZD-9056 HCl; AZD9056 hydrochloride; 345303-91-5; AZD-9056 hydrochloride; UNII-0CZ6S167ZM; 0CZ6S167ZM; AZD 9056 HCl; AZD9056.

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, avoid exposure to moisture.

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

DMSO : ~50 mg/mL (~109.78 mM)
H2O : ~1.67 mg/mL (~3.67 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.49 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 (5.49 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 3: ≥ 2.5 mg/mL (5.49 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.)