P2X7 receptor: a critical regulator and potential target for breast cancer.

Breast cancer is currently the most common cancer and the leading cause of cancer death among women worldwide. Advanced breast cancer is prone to metastasis, and there is currently no drug to cure metastatic breast cancer. The purinergic ligand-gated ion channel 7 receptor is an ATP-gated nonselective cation channel receptor and is involved in signal transduction, growth regulation, cytokine secretion, and tumor cell development. Recent studies have shown that upregulation of the P2X7 receptor in breast cancer can mediate AKT signaling pathways, Ca2 þ-activated SK3 potassium channels, and EMT and regulate the secretion of small extracellular vesicles to promote breast cancer invasion and migration, which are affected by factors such as hypoxia and ATP. In addition, studies have shown that microRNAs can bind to the 3' untranslated region of the P2X7 receptor, which affects the occurrence and development of breast cancer by upregulating and downregulating P2X7 receptor expression. Studies have shown that new P2X7 receptor inhibitors, such as emodin and Uncaria tomentosa, can inhibit P2X7 receptor-mediated breast cancer invasion and are expected to be used clinically. This article reviews the research progress on the relationship between the P2X7 receptor and breast cancer to provide new ideas and a basis for clinical diagnosis and treatment.

Neuropsychopharmacology of JNJ-55308942: evaluation of a clinical candidate targeting P2X7 ion channels in animal models of neuroinflammation and anhedonia.

Emerging data continues to point towards a relationship between neuroinflammation and neuropsychiatric disorders. ATP-induced activation of P2X7 results in IL-1ß release causing neuroinflammation and microglial activation. This study describes the in-vitro and in-vivo neuropharmacology of a novel brain-penetrant P2X7 antagonist, JNJ-55308942, currently in clinical development. JNJ-55308942 is a high-affinity, selective, brain-penetrant (brain/plasma of 1) P2X7 functional antagonist. In human blood and in mouse blood and microglia, JNJ-55308942 attenuated IL-1ß release in a potent and concentration-dependent manner. After oral dosing, the compound exhibited both dose and concentration-dependent occupancy of rat brain P2X7 with an ED50 of 0.07 mg/kg. The P2X7 antagonist (3 mg/kg, oral) blocked Bz-ATP-induced brain IL-1ß release in conscious rats, demonstrating functional effects of target engagement in the brain. JNJ-55308942 (30 mg/kg, oral) attenuated LPS-induced microglial activation in mice, assessed at day 2 after a single systemic LPS injection (0.8 mg/kg, i.p.), suggesting a role for P2X7 in microglial activation. In a model of BCG-induced depression, JNJ-55308942 dosed orally (30 mg/kg), reversed the BCG-induced deficits of sucrose preference and social interaction, indicating for the first time a role of P2X7 in the BCG model of depression, probably due to the neuroinflammatory component induced by BCG inoculation. Finally, in a rat model of chronic stress induced sucrose intake deficit, JNJ-55308942 reversed the deficit with concurrent high P2X7 brain occupancy as measured by autoradiography. This body of data demonstrates that JNJ-55308942 is a potent P2X7 antagonist, engages the target in brain, modulates IL-1ß release and microglial activation leading to efficacy in two models of anhedonia in rodents.

The P2X7 receptor: A main player in inflammation.

Damage associated molecular patterns (DAMPs) are intracellular molecules released from infected or injured cells to activate inflammatory and reparatory responses. One of the most ancient and conserved DAMPs is extracellular ATP that exerts its phlogistic activity mainly through activation of the P2X7 receptor (P2X7R). The P2X7R is an ATP gated ion channel, expressed by most immune cells, including the monocyte-derived cell lineages, T and B lymphocytes and their precursors. Here we give an overview of recent and established literature on the role of P2X7R in septic and sterile inflammation. P2X7R ability in restraining intracellular bacteria and parasite infection by modulation of the immune response are described, with particular focus on Mycobacteria and Plasmodium. Emerging literature on the role of P2X7 in viral infections such as HIV-1 is also briefly covered. Finally, we describe the numerous intracellular pathways related to inflammation and activated by the P2X7R, including the NLRP3 inflammasome, NF-kB, NFAT, GSK3ß and VEGF, and discuss the involvement of P2X7R in chronic diseases. The possible therapeutic applications of P2X7R antagonists are also described.

The therapeutic potential of purinergic signalling.

This review is focused on the pathophysiology and therapeutic potential of purinergic signalling. A wide range of diseases are considered, including those of the central nervous system, skin, kidney, musculoskeletal, liver gut, lower urinary tract, cardiovascular, airways and reproductive systems, the special senses, infection, diabetes and obesity. Several purinergic drugs are already on the market, including P2Y12 receptor antagonists for stroke and thrombosis, P2Y2 receptor agonists for dry eye, and A1 receptor agonists for supraventricular tachycardia. Clinical trials are underway for the use of P2X3 receptor antagonists for the treatment of chronic cough, visceral pain and hypertension, and many more compounds are being explored for the treatment of other diseases. Most experiments are 'proof of concept' studies on animal or cellular models, which hopefully will lead to further clinical trials. The review summarises the topic, mostly referring to recent review articles.

Evaluation of the Anticonvulsant Effect of Brilliant Blue G, a Selective P2X7 Receptor Antagonist, in the iv PTZ-, Maximal Electroshock-, and 6 Hz-Induced Seizure Tests in Mice.

Epilepsy is one of the most common neurological disorders which is diagnosed in around 65 million people worldwide. Clinically available antiepileptic drugs fail to control epileptic activity in about 30% of patients and they are merely symptomatic treatments and cannot cure or prevent epilepsy. There remains a need for searching new therapeutic strategies for epileptic disorders. The P2X7 receptor has been recently investigated as a new target in epilepsy treatment. Preclinical studies revealed that P2X7 receptor antagonists have anticonvulsant properties in some models of epilepsy. We aimed to investigate whether P2X7 receptor antagonist-brilliant blue G (BBG)-is able to change seizure threshold in three acute seizure models in mice, i.e., in the intravenous pentylenetetrazole seizure threshold, maximal electroshock seizure threshold and 6 Hz psychomotor seizure threshold tests. BBG was administered acutely (50-200 mg/kg, 30 min before the tests) and sub-chronically (25-100 mg/kg, once daily for seven consecutive days). Moreover, the chimney and grip strength tests were used to estimate the influence of BBG on the motor coordination and muscular strength in mice, respectively. Our results revealed only a week anticonvulsant potential of the studied P2X7 receptor antagonist because it showed anticonvulsant action only in the 6 Hz seizure test, both after acute and sub-chronic administration. BBG did not significantly influence seizure thresholds in the remaining tests. Motor coordination and muscular strength were not affected by the studied P2X7 receptor antagonist. In summary, BBG does not possess any remarkable anticonvulsant potential in acute seizure models in mice.

The P2X7 receptor antagonist, oxidized adenosine triphosphate, ameliorates renal ischemia-reperfusion injury by expansion of regulatory T cells.

Extracellular adenosine triphosphate (ATP) binds to purinergic receptors and, as a danger molecule, promotes inflammatory responses. Here we tested whether periodate-oxidized ATP (oATP), a P2X7 receptor (P2X7R) antagonist can attenuate renal ischemia-reperfusion injury and clarify the related cellular mechanisms. Treatment with oATP prior to ischemia-reperfusion injury decreased blood urea nitrogen, serum creatinine, the tubular injury score, and tubular epithelial cell apoptosis after injury. The infiltration of dendritic cells, neutrophils, macrophages, CD69+CD4+, and CD44+CD4+ T cells was attenuated, but renal Foxp3+CD4+ Treg infiltration was increased by oATP. The levels of IL-6 and CCL2 were reduced in the oATP group. Additionally, oATP treatment following injury improved renal function, decreased the infiltration of innate and adaptive effector cells, and increased the renal infiltration of Foxp3+CD4+ Tregs. Post-ischemia-reperfusion injury oATP treatment increased tubular cell proliferation and reduced renal fibrosis. oATP treatment attenuated renal functional deterioration after ischemia-reperfusion injury in RAG-1 knockout mice; however, Treg depletion using PC61 abrogated the beneficial effects of oATP in wild-type mice. Furthermore, oATP treatment after transfer of Tregs from wild-type mice improved the beneficial effects of Tregs on ischemia-reperfusion injury, but treatment after transfer of Tregs from P2X7R knockout mice did not. Renal ischemia-reperfusion injury was also attenuated in P2X7R knockout mice. Experiments using bone marrow chimeras established that P2X7R expression on hematopoietic cells rather than non-hematopoietic cells, such as tubular epithelial cells, plays a major role in ischemia-reperfusion injury. Thus, oATP attenuated acute renal damage and facilitated renal recovery in ischemia-reperfusion injury by expansion of Tregs.

Effect of artemisinin on neuropathic pain mediated by P2X4 receptor in dorsal root ganglia.

Neuropathic pain is a type of chronic pain caused by nervous system damage and dysfunction. The pathogenesis of chronic pain is complicated, and there are no effective therapies for neuropathic pain. Studies show that the P2X4 receptor expressed in the satellite glial cells (SGCs) of dorsal root ganglia (DRG) is related to neuropathic pain. Artemisinin is a monomeric component extracted from traditional Chinese medicine and has a variety of important pharmacological effects and potential applications. This study observed the effect of artemisinin on neuropathic pain and delineated its possible mechanism. The chronic constriction injury (CCI) rat model was used in this study. The results demonstrated that artemisinin relieved pain behaviors in the CCI rats, inhibited the expression of P2X4 receptor in the DRG, and decreased the ATP-activated currents in HEK293 cells transfected with P2X4 plasmid. Dual-labeling immunofluorescence showed that the coexpression of P2X4 receptor and glial fibrillary acidic protein (GFAP) in the DRG of CCI rats was increased compared to control rats. After CCI rats were treated with artemisinin, the coexpression of P2X4 receptor and GFAP in the DRG was significantly decreased compared to the CCI group. This finding suggested that artemisinin could inhibit the nociceptive transmission mediated by P2X4 receptor in the DRG SGCs and thus relieve pain behaviors in the CCI rats.

Enhancement of Muscle T Regulatory Cells and Improvement of Muscular Dystrophic Process in mdx Mice by Blockade of Extracellular ATP/P2X Axis.

Infiltration of immune cells and chronic inflammation substantially affect skeletal and cardiac muscle degeneration in Duchenne muscular dystrophy. In the immune system, extracellular adenosine triphosphate (ATP) released by dying cells is sensed as a danger associated molecular pattern through P2 purinergic receptors. Specifically, the P2X7 subtype has a prominent role in regulating immune system physiology and contributes to inflammasome activation also in muscle cells. Here, we show that in vivo blockade of the extracellular ATP/P2X purinergic signaling pathway by periodate-oxidized ATP delayed the progression of the dystrophic phenotype and dampened the local inflammatory response in mdx mice, a spontaneous mouse model of dystrophin deficiency. Reduced infiltration of leukocytes and macrophages and decreased expression of IL-6 were revealed in the muscles of periodate-oxidized ATP-treated mdx mice. Concomitantly, an increase in Foxp3(+) immunosuppressive regulatory T cells was observed and correlated with enhanced myofiber regeneration. Moreover, we detected reduced concentrations of profibrotic cytokines, including transforming growth factor-ß and connective tissue growth factor, in muscles of periodate-oxidized ATP-treated mdx mice. The improvement of inflammatory features was associated with increased strength and reduced necrosis, thus suggesting that pharmacologic purinergic antagonism altering the adaptive immune component in the muscle infiltrates might represent a promising therapeutic approach in Duchenne muscular dystrophy.

Pharmacologic P2X purinergic receptor antagonism in the treatment of collagen-induced arthritis.

OBJECTIVE: To assess the therapeutic potential of a P2X purinergic receptor antagonist, namely, periodate oxidized ATP, in collagen-induced arthritis (CIA). METHODS: Arthritis was induced in male DBA/1J mice by immunization with type II collagen (CII). Animals showing digit inflammation and paw swelling were treated intraperitoneally with 100 µl of 3 mM oxidized ATP daily for 10 days. At the end of the treatment period, animals were killed and paws were removed for histologic analysis and evaluation of T cell infiltration. Humoral response to CII was analyzed, and specific serum autoantibody levels were correlated with the clinical scores observed in the different treatment groups. RESULTS: Treatment with oxidized ATP resulted in a sustained reduction in disease activity, which was associated with a significant decrease in CD3+ T cell infiltration in arthritic lesions and a significant amelioration of cartilage erosion. Peripheral Treg cells were significantly increased upon P2X blockade in mouse lymph nodes. Moreover, a marked reduction in circulating autoantibodies directed against mouse CII was detected. There was a significant correlation between serum autoantibody levels and the clinical efficacy of oxidized ATP. CONCLUSION: Our findings indicate that P2X receptor antagonism has important therapeutic potential in chronic inflammatory rheumatic disorders. Taken together, our results underscore the value of the P2X receptor signaling pathway as a potential pharmacologic target for the modulation of adaptive immunity in CIA.

Characterization of three diaminopyrimidines as potent and selective antagonists of P2X3 and P2X2/3 receptors with in vivo efficacy in a pain model.

BACKGROUND AND PURPOSE: P2X3 and P2X2/3 receptors are highly localized on the peripheral and central pathways of nociceptive signal transmission. The discovery of A-317491 allowed their validation as chronic inflammatory and neuropathic pain targets, but this molecule has a very limited oral bioavailability and CNS penetration. Recently, potent P2X3 and P2X2/3 blockers with a diaminopyrimidine core group and better bioavailability were synthesized and represent a new opportunity for the validation of P2X3-containing receptors as targets for pain. Here we present a characterization of three representative diaminopyrimidines. EXPERIMENTAL APPROACH: The activity of compounds was evaluated in intracellular calcium flux and electrophysiological recordings from P2X receptors expressed in mammalian cells and in a in vivo model of inflammatory pain (complete Freund's adjuvant (CFA) in rat paws). KEY RESULTS: Compound A potently blocked P2X3 (pIC(50)= 7.39) and P2X2/3 (pIC(50)=6.68) and showed no detectable activity at P2X1, P2X2, P2X4 and P2X7 receptors (pIC(50)< 4.7). Whole-cell voltage clamp electrophysiology confirmed these results. Compounds showed good selectivities when tested against a panel of different classes of target. In the CFA model, compound B showed significant anti-nociceptive effects (57% reversal at 3mg·kg(-1) ). CONCLUSIONS AND IMPLICATIONS: The diaminopyrimidines were potent and selective P2X3 and P2X2/3 receptor antagonists, showing efficacy in vivo and represent useful tools to validate these receptors as targets for inflammatory and neuropathic pain and provide promising progress in the identification of therapeutic tools for the treatment of pain-related disorders.