Physalin pool from Physalis angulata L. leaves and physalin D inhibit P2X7 receptor function in vitro and acute lung injury in vivo.

P2X7 receptor promotes inflammatory response and neuropathic pain. New drugs capable of impairing inflammation and pain-reducing adverse effects extracted from plant extracts have been studied. Physalis angulate L. possesses traditional uses and exhibits antiparasitic, anti-inflammatory, antimicrobial, antinociceptive, antimalarial, antileishmanial, immunosuppressive, antiasthmatic. diuretic, and antitumor activities. The most representative phytochemical constituents identified with medicinal importance are the physalins and withanolides. However, the mechanism of anti-inflammatory action is scarce. Although some physalins and withanolides subtypes have anti-inflammatory activity, only four physalins subtypes (B, D, F, and G) have further studies. Therefore, we evaluated the crude ethanolic extract enriched with physalins B, D, F, and G from P. angulata leaves, a pool containing the physalins B, D, F, G, and the physalins individually, as P2X7 receptor antagonists. For this purpose, we evaluated ATP-induced dye uptake, macroscopic currents, and interleukin 1-ß (IL-1ß) in vitro. The crude extract and pool dose-dependently inhibited P2X7 receptor function. Thus, physalin B, D, F, and G individually evaluated for 5'-triphosphate (ATP)-induced dye uptake assay, whole-cell patch-clamp, and cytokine release showed distinct antagonist levels. Physalin D displayed higher potency and efficacy than physalin B, F, and G for all these parameters. In vivo mice model as ATP-induced paw edema was potently inhibited for physalin D, in contrast to physalin B, F, and G. ATP and lipopolysaccharide (LPS)-induced pleurisy in mice were reversed for physalin D treatment. Molecular modeling and computational simulation predicted the intermolecular interactions between the P2X7 receptor and physalin derivatives. In silico results indicated physalin D and F as a potent allosteric P2X7 receptor antagonist. These data confirm physalin D as a promisor source for developing a new P2X7 receptor antagonist with anti-inflammatory action.

DT-0111: a novel drug-candidate for the treatment of COPD and chronic cough.

BACKGROUND: Extracellular adenosine 5'-triphosphate (ATP) plays important mechanistic roles in pulmonary disorders in general and chronic obstructive pulmonary disease (COPD) and cough in particular. The effects of ATP in the lungs are mediated to a large extent by P2X2/3 receptors (P2X2/3R) localized on vagal sensory nerve terminals (both C and Aδ fibers). The activation of these receptors by ATP triggers a pulmonary-pulmonary central reflex, which results in bronchoconstriction and cough, and is also proinflammatory due to the release of neuropeptides from these nerve terminals via the axon reflex. These actions of ATP in the lungs constitute a strong rationale for the development of a new class of drugs targeting P2X2/3R. DT-0111 is a novel, small, water-soluble molecule that acts as an antagonist at P2X2/3R sites. METHODS: Experiments using receptor-binding functional assays, rat nodose ganglionic cells, perfused innervated guinea pig lung preparation ex vivo, and anesthetized and conscious guinea pigs in vivo were performed. RESULTS: DT-0111 acted as a selective and effective antagonist at P2X2/3R, that is, it did not activate or block P2YR; markedly inhibited the activation by ATP of nodose pulmonary vagal afferents in vitro; and, given as an aerosol, inhibited aerosolized ATP-induced bronchoconstriction and cough in vivo. CONCLUSIONS: These results indicate that DT-0111 is an attractive drug-candidate for the treatment of COPD and chronic cough, both of which still constitute major unmet clinical needs. The reviews of this paper are available via the supplementary material section.

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.

Targeting P(2)X(7) receptor for the treatment of central post-stroke pain in a rodent model.

Stroke is a leading cause of death and disability in industrialized countries. Approximately 8-14% of stroke survivors suffer from central post-stroke pain (CPSP) when hemorrhagic stroke occurs in lateral thalamic regions, which severely affects their quality of life. Because the mechanisms of CPSP are not well understood, effective treatments have not been developed. In the present study, we tested the hypothesis that persistent CPSP is caused by P(2)X(7)receptor activation after brain tissue damage and subsequent elevations in inflammatory cytokines. A thalamic hemorrhagic rat model was used, characterized by thermal and mechanical allodynia that develops in the subacute to chronic phases upon CPSP onset. We found a significant increase in P(2)X(7) expression in reactive microglia/macrophages in thalamic peri-lesion tissues at 5 weeks post-hemorrhage. Thalamic P(2)X(7) receptors were directly involved in pain transmission and hypersensitivity. The systemic targeting of P(2)X(7) receptors during the acute stage of hemorrhage rescued abnormal pain behaviors and neuronal activity in the thalamocingulate pathway by reducing reactive microglia/macrophage aggregation and associated inflammatory cytokines. After CPSP onset, the targeting of interleukin-1ß reversed abnormal pain sensitivity. The aberrant spontaneous thalamocortical oscillations in rats with CPSP were modulated by blocking P(2)X(7) receptors. Taken together, our results suggest that targeting P(2)X(7) may be bi-effective in the treatment of CPSP, as both a pain blocker and immunosuppressant that inhibits inflammatory damage to brain tissue. P(2)X(7)receptors may serve as a potential target to prevent the occurrence of CPSP and may be beneficial for the recovery of patients from stroke.

Decrease of serum adenine nucleotide hydrolysis in an irritant contact dermatitis mice model: potential P2X7R involvement.

Extracellular adenosine 5'-triphosphate (ATP) has significant effects on a variety of pathological conditions and it is the main physiological agonist of P2X7 purinergic receptor (P2X7R). It is known that ATP acting via purinergic receptors plays a relevant role on skin inflammation, and P2X7R is required to neutrophil recruitment in a mice model of irritant contact dermatitis (ICD).The present study investigated the effects of chemical irritant croton oil (CrO) upon ATP, ADP, and AMP hydrolysis in mice blood serum, and the potential involvement of P2X7R. The topical application CrO induced a decrease on soluble ATP/ADPase activities (~50 %), and the treatment with the selective P2X7R antagonist, A438079, reversed these effects to control level. Furthermore, we showed that CrO decreased cellular viability (52.6 % ± 3.9) in relation to the control and caused necrosis in keratinocytes (PI positive cells). The necrosis induced by CrO was prevented by the pre-treatment with the selective P2X7R antagonist A438079. The results presented herein suggest that CrO exerts an inhibitory effect on the activity of ATPDase in mouse serum, reinforcing the idea that ICD has a pathogenic mechanism dependent of CD39. Furthermore, it is tempting to suggest that P2X7R may act as a controller of the extracellular levels of ATP.

A reassessment of P2X7 receptor inhibition as a neuroprotective strategy in rat models of contusion injury.

These experiments were completed as part of an NIH "Facilities of Research Excellence in Spinal Cord Injury" contract to support independent replication of published studies that could be considered for eventual clinical testing. Recent studies have reported that selective inhibition of the P2X7 receptor improves both the functional and histopathological consequences of a contusive spinal cord injury (SCI) in rats. We repeated two published studies reporting the beneficial effects of pyridoxal-5'-phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS) or Brilliant blue G (BBG) treatment after SCI (Wang et al., 2004 and Peng et al., 2009). Mild thoracic SCI was first produced in Experiment 1 by means of the MASCIS impactor at T10 (height 6.25 mm, weight 10 g) followed by intraspinal administration of a P2X7 antagonist (2 µl/10 mM) after injury. Treatment with PPADS or another highly selective P2X7R antagonist Brilliant Blue G (BBG) (2 µl/02 mM) did not improve locomotive (BBB rating scale) over a 7 week period compared to vehicle treated rats. Also, secondary histopathological changes in terms of overall lesion and cavity volume were not significantly different between the PPADS, BBG, and vehicle treated animals. In the second experiment, the systemic administration of BBG (10 or 50 mg/kg, iv) 15 min, 24 and 72 h after moderate (12.5 mm) SCI failed to significantly improve motor recovery or histopathological outcome over the 6 week observational period. Although we cannot conclude that there will be no long-term beneficial effects in other spinal cord injury models using selective P2X7 receptor antagonists at different doses or treatment durations, we caution researchers that this potentially exciting therapy requires further preclinical investigations before the implementation of clinical trials targeting severe SCI patients.

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.