Novel P2X₇ receptor antagonists ease the pain

Abstract

In recent months, a series of chemically diverse antagonists has been identified for the ATP-gated P2X₇ receptor. In particular, two classes of highly-selective competitive P2X₇ antagonists have been developed by Michael Jarvis and his colleagues at Abbott Laboratories. These di-substituted tetrazole and cyanoguanidine derivatives are outstanding for a number of reasons (not least their stability, selectivity, potency and, of course, reversibility); most exciting is their near equal potency at human and rodent P2X₇ isoforms. Armed with drugs such as A740003 and newer A438079, Jarvis and colleagues have explored the role of P2X₇ receptors in the onset and persistence of chronic pain in animal models. Their findings - and applicability to the human condition - are reviewed in this current issue of British Journal of Pharmacology. This accompanying Commentary describes the progress made by Jarvis and others in developing novel P2X₇ antagonists for pain relief.

In equal measures, the P2X₇ receptor is viewed as the darling and devil of the adenosine triphosphate (ATP) receptor family. Scientists have been beguiled by the molecular complexity of this unique ATP-gated ion channel, including its rapid conversion from an ion-channel (8–11 Å diameter) to an enlarged pore (30–50 Å diameter), accompanied by the influx of permeants of large molecular weight (such as ethidium, propidium and Yo-Pro-1 ions) (see Egan et al., 2006), its ability to initiate morphological changes such as cell swelling and membrane is blebbing (see North, 2002) and its downstream activation of intracellular caspases and maturation of releasable interleukin-1β (IL-1β) (see Ferrari et al., 2006). Scientists have also been frustrated by the lack of pharmacological tools to control these functional properties and, hence, have been frustrated by an inability to study the role of P2X₇ receptors in inflammation and pain in a systematic way.

Since its isolation, the P2X₇ receptor has been considered to be the molecular embodiment of a cell-permeabilizing ATP receptor found first by UCL scientists in rat mast cells (Cockcroft and Gomperts, 1979). This ATP receptor was later classified as the P2Z receptor and was also identified as present in immune cells (Gordon, 1986). A gradual adjustment in thinking has led to the realization that at least two separate pathways (and accessory pores) can be linked molecularly to the activation of P2X₇ receptors (Pelegrin and Surprenant, 2007). The P2Z receptor appears to be much more than a simple homomeric P2X₇ receptor. This emerging viewpoint may explain why the permeant size pass for P2Z receptors differs between macrophages (<900 Da) and lymphocytes (<400 Da). The precise molecular nature of P2Z receptors still requires further evaluation, but the field is now much closer to resolving this question.

Early studies of the ATP⁴⁻-activated P2Z receptor showed that excess Mg²⁺ could reseal the membrane of rat mast cells (Di Virgilio and Gomperts, 1983). Mg²⁺-dependent reduction in permeability was also reported for murine macrophage P2Z receptors expressed in oocytes (Nuttle and Dubyak, 1994), human lymphocyte P2Z receptors expressed in oocytes (King and Wiley, unpublished data) and rat P2X₇ receptors expressed in HEK293 cells (Virginio et al., 1997). Excess Mg²⁺ is not an effective tool for studies in vivo and also has limited applicability to studies in vitro. However, it was the first available tool, and its insurmountable actions were indicative of more than a reduction of extracellular levels of free ATP⁴⁻ ions. Others have shown that inorganic cations (Co²⁺, Cu²⁺, Mn²⁺ and Zn²⁺, for example) can inhibit P2X₇ receptors, but only some of these exert an insurmountable antagonism (see Virginio et al., 1997). The most striking finding in recent months is the discovery of an oxide of the metal vanadium (decavanadate H₂V₁₀O₂₈⁴⁻) as a competitive antagonist at human, rat and mouse P2X₇ isoforms (Michel et al., 2006). The complex method of preparing decavanadate solutions and its potency at P2X₂ receptors may detract from its future and frequent use in vivo.

Turning to drugs other than modulatory inorganic ions, amiloride and its analogues were identified as antagonists of P2Z receptors (Wiley et al., 1990). However, amiloride derivatives are unsuitable for all studies except those on isolated immune cells, because of their high affinity for non-purinergic ion channels such as the epithelial sodium channel ENaC. Attention then turned to exploiting very low-efficacy ATP derivatives as weak antagonists, and those tested on P2Z receptors on rat mast cells revealed a range of weak partial agonists and one (2-MeSATP) with overt inhibitory activity (Tatham et al., 1988). Similar studies revealed that oxidized ATP (oATP) also possessed inhibitory activity at P2Z receptors (Murgia et al., 1993). However, 2-MeSATP and oATP ultimately showed little promise in the study of P2X₇ receptors in vivo, mainly due to their lack of selectivity. The same can also be said of general P2 receptor antagonists – such as suramin, Reactive Blue 2 and PPADS – that, in various studies, have been shown to exert inhibitory activity at P2Z receptors and recombinant P2X₇ receptors. Incidentally, Jarvis and colleagues have extensively studied the inhibitory actions of a wide range of established P2 receptor antagonists at human and rodent P2X₇ isoforms and this work was recently presented in poster form at a Society for Neuroscience meeting (Namovic et al., 2006).

Strong interest was expressed in Ca²⁺/calmodulin-dependent protein kinase II (CamKII) inhibitors, including calmodazolium and KN62, as potent inhibitors of P2X₇ receptors. Here, the first inklings surfaced for species- and use-dependent blocking activities for these inhibitors – with overt differences at human, rat and mouse P2X₇ isoforms. The molecular basis for such differences has yet to be resolved, and a recent review of the pharmacology of these compounds appears elsewhere (Gever et al., 2006). Similar problems surfaced for the polysulphonic dye, Coomassie Brilliant Blue G (BBG), which showed some selectivity for the rat P2X₇ receptor (Jiang et al., 2000). However, the lowered potency of BBG at the human P2X₇ receptor and similar potency at P2X₂ and P2X₄ receptor subtypes have limited its promise for use in vivo.

The involvement of Jarvis and colleagues in the search for selective P2X₇ receptors came at a relatively late stage. This group (and others based in pharmaceutical companies) had concentrated foremost on a role for native P2X₃ and P2X_2/3 receptors in the sensitization of spinal sensory neurons in experimental models of persistent pain. This work led to the discovery of 5-({(3-phenoxybenzyl)((1S)-1,2,3,4-tetrahydro-1-naphthalenyl)amino}carbonyl)-1,2,4-benzenetricarboxylic acid (A317491), a structurally novel and highly potent antagonist for P2X₃ and P2X_2/3 receptors (Jarvis et al., 2002). This antagonist was outstanding in terms of its novel chemistry, as it is structurally unrelated to any of the known P2 receptor antagonists, and also stood out in terms of its high potency and selectivity. Having cut their teeth on identifying and producing novel P2X₃ receptor antagonists, it was a natural step for Jarvis and colleagues to search for antagonists of other P2X receptor subtypes, including the hardest target, P2X₇.

A series of reports has commented on the presence of P2X₇-like immunoreactivity in nerve endings in central nuclei for second-order sensory neurons. There have been some problems with these findings, since positive immunoreactivity (albeit at reduced levels) still persists in gene-deleted P2X₇^−/− mice (see Anderson and Nedergaard, 2006). However, the presence of P2X₇ in microglia is widely acknowledged (as is the presence of microglia close to sensory neurons and axons), and P2X₇ receptor activation, in turn, does release IL-1β from microglia to initiate inflammation processes (Chakfe et al., 2002). In fact, functional P2X₇ receptors and COX-2 production are significantly upregulated in a number of human neurological disorders, including multiple sclerosis (see Yiangou et al., 2006). In this vein, Jarvis and colleagues identified P2X₇ receptors in non-neuronal cells in rat spinal ganglia (Zhang et al., 2005). With their chemistry, screening procedures and functional assays already fine-tuned, Jarvis and colleagues needed only a short period of time to produce the highly potent P2X₇ antagonist N-(1-{((cyanoimino)(5-quinolinylamino)methyl)amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide (A740003) (see Honore et al., 2006), which was followed thereafter by 3-((5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl) methyl pyridine (A438079) (Nelson et al., 2006).

In the accompanying review by Donnelly-Roberts and Jarvis (2007), the authors describe the pharmacological properties of their lead P2X₇ receptor antagonists and their utility in humans as potent analgesics of chronic pain. They provide a clear and balanced account of P2X₇ receptor pharmacology and also provide an unbiased comparison of their drugs with other lead P2X₇ receptor antagonists from industry rivals. The work summarized in this review represents a major step towards developing a new generation of analgesics for the relief of chronic pain. This review is not only timely, but also marks out a field that should be followed closely in the next few years.

Abbreviations

A317491

5-({(3-phenoxybenzyl)((1S)-1,2,3,4-tetrahydro-1-naphthalenyl)amino}carbonyl)-1,2,4-benzenetricarboxylic acid

A438079

3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl) methyl pyridine

A740003

N-(1-{((cyanoimino)(5-quinolinylamino)methyl)amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide