Is there a biochemical basis for purinergic P2X3 and P2X4 receptor antagonists to be considered as anti-seizure medications?

Patients with epilepsy require improved medications. Purinergic receptors were identified as late as 1976 and are slowly emerging as potential drug targets for the discovery of antiseizure medications. While compounds interacting with these receptors have been approved for use as medicines (e.g., gefapixant for cough) and continue to be explored for a number of diseases (e.g., pain, cancer), there have been no purinergic receptor antagonists that have been advanced for epilepsy. There are very few studies on the channel conducting receptors, P2X3 and P2X4, that suggest their possible role in seizure generation or control. However, the limited data available provides some compelling reasons to believe that they could be valuable antiseizure medication drug targets. The data implicating P2X3 and P2X4 receptors in epilepsy includes the role played by ATP in neuronal excitability and seizures, receptor localization, increased receptor expression in epileptic brain, the involvement of these receptors in seizure-associated inflammation, crosstalk between these purinergic receptors and neuronal processes involved in seizures (GABAergic and glutamatergic neurotransmission), and the significant attenuation of seizures and seizure-like activity with P2X receptor blockade. The discovery of new and selective antagonists for P2X3 and P2X4 receptors is ongoing, armed with new structural data to guide rational design. The availability of safe, brain-penetrant compounds will likely encourage the clinical exploration of epilepsy as a disease entity.

P2X4R Contributes to Central Disinhibition Via TNF-α/TNFR1/GABAaR Pathway in Post-stroke Pain Rats.

Central post-stroke pain (CPSP) is a disabling condition in stroke patients. It is a type of neuropathic pain for which the mechanism and relevant drug pathways remain unknown. Inflammatory response and central disinhibition have been suggested recently. Our previous research has shown targeting P2X4 receptors (P2X4R) may be effective in the treatment of CPSP, but the downstream pathway of the P2X4R has not been studied. In this study, we found the increase in tumor necrosis factor alpha (TNF-α) level and endocytosis of surface gamma-aminobutyric acid a receptors (GABAaR) in CPSP, and these effects were inhibited by blocking P2X4R. Furthermore, antagonizing TNF-α can increase surface GABAaR expression and mechanical pain threshold. Meanwhile, knocking down TNFR1 but not TNFR2 reversed the endocytosis of surface GABAaR and alleviated mechanical allodynia. Thus, the neuropathic pain was mediated, in part, through P2X4R/TNF-α/TNFR1/GABAaR signaling, which was induced after stroke. PERSPECTIVE: P2X4R regulates the pathophysiological mechanism of CPSP through central disinhibition mediated by TNF-α/TNFR1. Our results suggest that modulation of P2X4R-TNF-α/TNFR1-GABAaR signaling could provide a new therapeutic strategy to treat CPSP.

Cross-Talk between P2X and NMDA Receptors.

Purinergic P2X receptors (P2X) are ATP-gated ion channels widely expressed in the CNS. While the direct contribution of P2X to synaptic transmission is uncertain, P2X reportedly affect N-methyl-D-aspartate receptor (NMDAR) activity, which has given rise to competing theories on the role of P2X in the modulation of synapses. However, P2X have also been shown to participate in receptor cross-talk: an interaction where one receptor (e.g., P2X2) directly influences the activity of another (e.g., nicotinic, 5-HT3 or GABA receptors). In this study, we tested for interactions between P2X2 or P2X4 and NMDARs. Using two-electrode voltage-clamp electrophysiology experiments in Xenopus laevis oocytes, we demonstrate that both P2X2 and P2X4 interact with NMDARs in an inhibited manner. When investigating the molecular domains responsible for this phenomenon, we found that the P2X2 c-terminus (CT) could interfere with both P2X2 and P2X4 interactions with NMDARs. We also report that 11 distal CT residues on the P2X4 facilitate the P2X4-NMDAR interaction, and that a peptide consisting of these P2X4 CT residues (11C) can disrupt the interaction between NMDARs and P2X2 or P2X4. Collectively, these results provide new evidence for the modulatory nature of P2X2 and P2X4, suggesting they might play a more nuanced role in the CNS.

The macrocyclic lactones ivermectin and moxidectin show differential effects on rotational behavior in the 6-hydroxydopamine mouse model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by motor and cognitive deficits, the result of dopamine (DA)-depletion within the basal ganglia. Currently, DA replacement therapy in the form of Sinemet (L-DOPA plus Carbidopa) provides symptomatic motor benefits and remains the "gold standard" for treatment. Several pharmacological approaches can enhance DA neurotransmission including the administration of DA receptor agonists, the inhibition of DA metabolism, and enhancing pre-synaptic DA release. DA neurotransmission is regulated by several receptor subtypes including signaling through the purinergic system. P2 × 4 receptors (P2 × 4Rs) are a class of cation-permeable ligand-gated ion channels activated by the synaptic release of extracellular adenosine 5'-triphosphate (ATP). P2 × 4Rs are expressed throughout the central nervous system including the dopaminergic circuitry of the substantia nigra, basal ganglia, and related reward networks. Previous studies have demonstrated that P2 × 4Rs can modulate several DA-dependent characteristics including motor, cognitive, and reward behaviors. Ivermectin (IVM) and moxidectin (MOX) are two macrocyclic lactones that can potentiate P2 × 4Rs. In this study, we sought to investigate the role of P2 × 4Rs in mediating DA neurotransmission by exploring their impact on DA-dependent behavior, specifically rotation frequency in the unilateral 6-hydroxydopamine-lesioned mouse model of DA-depletion. While we did not observe any differences in the degree of lesioning based on immunostaining for tyrosine hydroxylase between sexes, male mice displayed a greater number of rotations with L-DOPA compared to female mice. In contrast, we observed that IVM plus L-DOPA increased the number of rotations (per 10 min) in female, but not male mice. These findings highlight the potential role of pharmacologically targeting the purinergic receptor system in modulating DA neurotransmission as well as the importance of sex differences impacting outcome measures.

Dopamine Receptor Blockade Attenuates Purinergic P2X4 Receptor-Mediated Prepulse Inhibition Deficits and Underlying Molecular Mechanisms.

Sensorimotor gating refers to the ability to filter incoming sensory information in a stimulus-laden environment and disruption of this physiological process has been documented in psychiatric disorders characterized by cognitive aberrations. The effectiveness of current pharmacotherapies for treatment of sensorimotor gating deficits in the patient population still remains controversial. These challenges emphasize the need to better understand the biological underpinnings of sensorimotor gating which could lead to discovery of novel drug targets for therapeutic intervention. Notably, we recently reported a role for purinergic P2X4 receptors (P2X4Rs) in regulation of sensorimotor gating using prepulse inhibition (PPI) of acoustic startle reflex. P2X4Rs are ion channels gated by adenosine-5'-triphosphate (ATP). Ivermectin (IVM) induced PPI deficits in C57BL/6J mice in a P2X4R-specific manner. Furthermore, mice deficient in P2X4Rs [P2X4R knockout (KO)] exhibited PPI deficits that were alleviated by dopamine (DA) receptor antagonists demonstrating an interaction between P2X4Rs and DA receptors in PPI regulation. On the basis of these findings, we hypothesized that increased DA neurotransmission underlies IVM-mediated PPI deficits. To test this hypothesis, we measured the effects of D1 and D2 receptor antagonists, SCH 23390 and raclopride respectively and D1 agonist, SKF 82958 on IVM-mediated PPI deficits. To gain mechanistic insights, we investigated the interaction between IVM and dopaminergic drugs on signaling molecules linked to PPI regulation in the ventral striatum. SCH 23390 significantly attenuated the PPI disruptive effects of IVM to a much greater degree than that of raclopride. SKF 82958 failed to potentiate IVM-mediated PPI disruption. At the molecular level, modulation of D1 receptors altered IVM's effects on dopamine and cyclic-AMP regulated phosphoprotein of 32 kDa (DARPP-32) phosphorylation. Additionally, IVM interacted with the DA receptors antagonists and SKF 82958 in phosphorylation of Ca2+/calmodulin kinase IIα (CaMKIIα) and its downstream target, neuronal nitric oxide synthase (nNOS). Current findings suggest an involvement for D1 and D2 receptors in IVM-mediated PPI disruption via modulation of DARPP-32, CaMKIIα and nNOS. Taken together, the findings suggest that stimulation of P2X4Rs can lead to DA hyperactivity and disruption of information processing, implicating P2X4Rs as a novel drug target for treatment of psychiatric disorders characterized by sensorimotor gating deficits.

Rhein inhibits ATP-triggered inflammatory responses in rheumatoid rat fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disorder, which may lead to joint disabilities. So far the pathogenesis of RA remains largely undetermined, and there are still no potent drugs for clinical treatment. Rhein, a natural bioactive anthraquinone derivative, exhibited significant anti-inflammatory activities demonstrated by previous studies. Here we aimed to investigate the effects of rhein on ATP-induced inflammation responses in fibroblast-like synoviocytes isolated from a rat model of collagen induced arthritis (CIA). Our results showed that ATP triggered rapid cytosolic calcium concentration ([Ca2+]c) increase depending on extracellular Ca2+ entry. Given the major P2 subtypes expressed in rat synoviocytes were P2X4 and P2Y2 receptors, ATP-elicited calcium entry should be mainly resulted from activating P2X4. Interestingly, rhein could effectively block the ATP-induced [Ca2+]c increases in a dose-dependent manner. Besides, rhein also suppressed the production of intracellular reactive oxygen species (ROS) induced by ATP in synoviocytes that was resulted from P2X4-mediated Ca2+ entry. Brilliant blue G (BBG), which can block P2X4 receptor at high concentration, showed similar suppressive effects on above responses. Furthermore, in lipopolysaccharide-primed cells, application of ATP synergistically promoted the gene expression of cyclooxygenase-2, interleukin-6 and matrix metalloproteinase-9. Both rhein and BBG attenuated these inflammatory gene expressions enhanced by ATP. Above data together suggested a potential anti-arthritic role of rhein by inhibiting ATP-induced [Ca2+]c increase, ROS production and inflammatory gene expression targeting P2X4 in CIA rat synoviocytes, which will provide a novel insight in the therapy of RA.

‬The expression of purinergic P2X4 and P2X7 receptors in selected mesolimbic structures during morphine withdrawal in rats.

Morphine is one of the most potent analgesics used in medicine and it's long-term use is associated with the risk of the state of dependence. The cessation of chronic morphine administration leads to withdrawal signs which are associated with neurotransmitter dysregulations within mesolimbic system. Adenosine 5'-triphosphate (ATP) and purinergic system play an important role in the activity of central nervous system (CNS). Purinergic receptors are widely distributed in neurons and glial cells throughout the CNS taking part in integration of functional activity between neurons, glial and vascular cells. In the present study the mRNA and protein expression of purinergic P2X4 and P2X7 receptors in selected mesolimbic structures (striatum, hippocampus and prefrontal cortex) during morphine withdrawal in rats was investigated by RT-PCR and Western Blot analysis. Two experimental models of morphine withdrawal were studied: single and repeated morphine withdrawal. We demonstrated that expression of P2X4 and P2X7 receptors was altered during morphine withdrawal period in rats. These alterations were varied in particular mesolimbic areas depending on the scheme of morphine administration. Our results extend the current knowledge on morphine withdrawal and for the first time high-light interactions between purinergic system and morphine withdrawal. It seems, the purinergic system may be a new, valuable tool in searching for a new strategy of management of opioid dependence.

Preclinical evaluation of avermectins as novel therapeutic agents for alcohol use disorders.

The deleterious effects of alcohol use disorders (AUDs) on human health have been documented worldwide. The enormous socioeconomic burden coupled with lack of efficacious pharmacotherapies underlies the need for improved treatment strategies. At present, there is a growing body of preclinical evidence that demonstrates the potential of avermectins [ivermectin (IVM), selamectin (SEL), abamectin (ABM), and moxidectin (MOX)] in treatment of AUDs. Avermectins are derived by fermentation of soil micro-organism, Streptomyces avermitilis, and have been extensively used for treatment of parasitic infections. From the mechanistic standpoint, avermectins are positive modulators of purinergic P2X4 receptors (P2X4Rs). P2X4Rs belong to P2X superfamily of cation-permeable ion channels gated by adenosine 5'-triphosphate (ATP). Building evidence has implicated a role for P2X4Rs in regulation of ethanol intake and that ethanol can inhibit ATP-gated currents in P2X4Rs. Investigations using recombinant cell models and animal models of alcohol drinking have reported that IVM, ABM, and MOX, but not SEL, were able to antagonize the inhibitory effects of ethanol on P2X4Rs in vitro and reduce ethanol intake in vivo. Furthermore, IVM was shown to reduce ethanol consumption via P2X4R potentiation in vivo, supporting the involvement of P2X4Rs in IVM's anti-alcohol effects and that P2X4Rs can be used as a platform for developing novel anti-alcohol compounds. Taken together, these findings support the utility of avermectins as a novel class of drug candidates for treatment of AUDs.

Reduced expression of purinergic P2X4 receptors increases voluntary ethanol intake in C57BL/6J mice.

Purinergic P2X4 receptors (P2X4Rs) belong to the P2X superfamily of ionotropic receptors that are gated by adenosine 5'-triphosphate (ATP). Accumulating evidence indicates that P2X4Rs play an important role in regulation of ethanol intake. At the molecular level, ethanol's inhibitory effects on P2X4Rs are antagonized by ivermectin (IVM), in part, via action on P2X4Rs. Behaviorally, male mice deficient in the p2rx4 gene (P2X4R knockout [KO]) have been shown to exhibit a transient increase in ethanol intake over a period of 4 days, as demonstrated by social and binge drinking paradigms. Furthermore, IVM reduced ethanol consumption in male and female rodents, whereas male P2X4R KO mice were less sensitive to the anti-alcohol effects of IVM, compared to wildtype (WT) mice, further supporting a role for P2X4Rs as targets of IVM's action. The current investigation extends testing the hypothesis that P2X4Rs play a role in regulation of ethanol intake. First, we tested the response of P2X4R KO mice to ethanol for a period of 5 weeks. Second, to gain insights into the changes in ethanol intake, we employed a lentivirus-shRNA (LV-shRNA) methodology to selectively knockdown P2X4R expression in the nucleus accumbens (NAc) core in male C57BL/6J mice. In agreement with our previous study, male P2X4R KO mice exhibited higher ethanol intake than WT mice. Additionally, reduced expression of P2X4Rs in the NAc core significantly increased ethanol intake and preference. Collectively, the findings support the hypothesis that P2X4Rs play a role in regulation of ethanol intake and that P2X4Rs represent a novel drug target for treatment of alcohol use disorder.

Toll-like receptor-4/p38 MAPK signaling in the dorsal horn contributes to P2X4 receptor activation and BDNF over-secretion in cancer induced bone pain.

Our previous research suggested that the P2X4 receptor (P2X4R) expression in microglia was involved in the activation of toll-like receptor-4 (TLR4) in the dorsal horn in the rat model of cancer induced bone pain (CIBP). In this study, we focused on whether TLR4- mitogen-activated protein kinases, p38 (p38 MAPK) contributes to P2X4R activation and brain-derived neurotrophic factor (BDNF) over-secretion in CIBP. In in vitro experiment, the results showed that BDNF expression evoked by ATP stimulation was dependent on TLR4-p38. In in vivo experiment, the results demonstrated that an intrathecal injection of TLR4 siRNA alleviated nociception induced by lipopolysaccharide (LPS) plus ATP or CIBP with decreased expression of P2X4R, TLR4, BDNF, interleukin-6 (IL-6) and phosphorylated-p38 MAPK (p-p38 MAPK). Moreover, injection with p38MAPK inhibitor SB203580 resulted in an identical pattern compared with treatment with TLR4 siRNA. Our results demonstrate that the activation of TLR4-p38MAPK-P2X4R signaling in microglial possibility plays an important role in the process of nociceptive transmission in CIBP, suggesting new mechanism and potential therapeutic targets for CIBP.