P2X7 receptor-mediated release of microglial prostanoids and miRNAs correlates with reversal of neuropathic hypersensitivity in rats.

BACKGROUND: P2X7 receptor antagonists have potential for treating various central nervous system (CNS) diseases, including neuropathic pain, although none have been approved for clinical use. Reasons may include insufficient understanding of P2X7 receptor signalling in pain, and the lack of a corresponding preclinical mechanistic biomarker. METHODS: Lu AF27139 is a highly selective and potent small molecule antagonist at rat, mouse and human forms of the P2X7 receptor, with excellent pharmacokinetic and CNS permeability properties. In the current experiments, we probed the utility of previously characterized and novel signalling cascades exposed to Lu AF27139 using cultured microglia combined with release assays. Subsequently, we assessed the biomarker potential of identified candidate molecules in the rat chronic constriction injury (CCI) model of neuropathic pain; study design limitations precluded their assessment in spared nerve injury (SNI) rats. RESULTS: Lu AF27139 blocked several pain-relevant pathways downstream of P2X7 receptors in vitro. At brain and spinal cord receptor occupancy levels capable of functionally blocking P2X7 receptors, it diminished neuropathic hypersensitivity in SNI rats, and less potently in CCI rats. Although tissue levels of numerous molecules previously linked to neuropathic pain and P2X7 receptor function (e.g. IL-6, IL-1ß, cathepsin-S, 2-AG) were unaffected by CCI, Lu AF27139-mediated regulation of spinal PGE2 and miRNA (e.g. rno-miR-93-5p) levels increased by CCI aligned with its ability to diminish neuropathic hypersensitivity. CONCLUSIONS: We have identified a pain-relevant P2X7 receptor-regulated mechanism in neuropathic rats, which could hold promise as a translatable biomarker and by association enhance the clinical progression of P2X7 receptor antagonists in neuropathic pain. SIGNIFICANCE: Sub-optimal translation of preclinical molecules has hindered the clinical development of novel mechanism of action analgesics. We have undertaken a comprehensive in vitro analysis of migroglial signalling mechanisms recruited upon P2X7 receptor activation, a number of which were shown to be modulated by a selective P2X7 receptor antagonist in a well characterized animal model of neuropathic pain. Subject to further confirmation in other neuropathic models, this opens up the possibility to investigate their clinical utility as potential pain biomarkers in patients.

Synthesis and Characterization of the Novel Rodent-Active and CNS-Penetrant P2X7 Receptor Antagonist Lu AF27139.

There remains an insufficient number of P2X7 receptor antagonists with adequate rodent potency, CNS permeability, and pharmacokinetic properties from which to evaluate CNS disease hypotheses preclinically. Herein, we describe the molecular pharmacology, safety, pharmacokinetics, and functional CNS target engagement of Lu AF27139, a novel rodent-active and CNS-penetrant P2X7 receptor antagonist. Lu AF27139 is highly selective and potent against rat, mouse, and human forms of the receptors. The rat pharmacokinetic profile is favorable with high oral bioavailability, modest clearance (0.79 L/(h kg)), and good CNS permeability. In vivo mouse CNS microdialysis studies of lipopolysaccharide (LPS)-primed and 2'(3')-O-(benzoylbenzoyl)adenosine-5'-triphosphate (BzATP)-induced IL-1ß release demonstrate functional CNS target engagement. Importantly, Lu AF27139 was without effect in standard in vitro and in vivo toxicity studies. Based on these properties, we believe Lu AF27139 will be a valuable tool for probing the role of the P2X7 receptor in rodent models of CNS diseases.

Quantitative analysis of lipids: a higher-throughput LC-MS/MS-based method and its comparison to ELISA.

AIM: Lipids such as prostaglandins, leukotrienes and thromboxanes are released as a result of an inflammatory episode in pain (central and peripheral). METHODOLOGY & RESULTS: To measure these lipids as potential mechanistic biomarkers in neuropathic pain models, we developed a higher-throughput LC-MS/MS-based method with simultaneous detection of PGE2, PGD2, PGF2α, LTB4, TXB2 and 2-arachidonoyl glycerol in brain and spinal cord tissues. We also demonstrate that the LC-MS/MS method was more sensitive and specific in differentiating PGE2 levels in CNS tissues compared with ELISA. CONCLUSION: The ability to modify the LC-MS/MS method to accommodate numerous other lipids in one analysis, demonstrates that the presented method offers a cost-effective and more sensitive alternative to ELISA method useful in drug discovery settings.

Inhibition of the potassium channel KCa3.1 by senicapoc reverses tactile allodynia in rats with peripheral nerve injury.

Neuropathic pain is a debilitating, chronic condition with a significant unmet need for effective treatment options. Recent studies have demonstrated that in addition to neurons, non-neuronal cells such as microglia contribute to the initiation and maintenance of allodynia in rodent models of neuropathic pain. The Ca2+- activated K+ channel, KCa3.1 is critical for the activation of immune cells, including the CNS-resident microglia. In order to evaluate the role of KCa3.1 in the maintenance of mechanical allodynia following peripheral nerve injury, we used senicapoc, a stable and highly potent KCa3.1 inhibitor. In primary cultured microglia, senicapoc inhibited microglial nitric oxide and IL-1ß release. In vivo, senicapoc showed high CNS penetrance and when administered to rats with peripheral nerve injury, it significantly reversed tactile allodynia similar to the standard of care, gabapentin. In contrast to gabapentin, senicapoc achieved efficacy without any overt impact on locomotor activity. Together, the data demonstrate that the KCa3.1 inhibitor senicapoc is effective at reducing mechanical hypersensitivity in a rodent model of peripheral nerve injury.

In vitro differentiation of dendritic cells in the presence of prostaglandin E2 alters the IL-12/IL-23 balance and promotes differentiation of Th17 cells.

PGE2, an endogenous lipid mediator released in inflammatory conditions, affects both dendritic cell (DC) differentiation and maturation. Whereas the effect of PGE2 on fully differentiated DC was studied extensively, little is known about its effects on DC differentiation. In this study, we show that bone marrow-derived DC generated in the presence of PGE2 (DCp) acquire a proinflammatory profile; produce higher levels of proinflammatory cytokines/chemokines; express higher levels of MHC class II, costimulatory molecules, and TLRs; and exhibit increased activation of the NF-kappaB-signaling pathway. In addition, DCp exhibit a different IL-12/IL-23 profile than DC generated in the absence of PGE2. The low IL-12 and high IL-23 production in LPS-stimulated DCp is associated with the down-regulation of p35 and the up-regulation of p19 expression, respectively. In agreement with the DCp proinflammatory phenotype and especially with the altered IL-12/IL-23 balance which strongly favors IL-23, DCp also affect T cell differentiation. In contrast to DC which favor Th1 differentiation, DCp promote Th17 and inhibit Th1/Th2 differentiation, in vitro and in vivo. Previous in vivo studies indicated that PGE2 had a proinflammatory effect, especially in models of autoimmune diseases. Our results suggest that the proinflammatory effects of PGE2 could be mediated, at least partially, through effects on differentiating DC and subsequent alterations in CD4+ T cell differentiation, resulting in the preferential development of pathogenic autoimmune Th17 cells.

PGE2-induced metalloproteinase-9 is essential for dendritic cell migration.

Following antigen acquisition and maturation, dendritic cells (DCs) disengage from the extracellular matrix, cross basement membranes, and travel to draining lymph nodes to activate T cells. CCR7 expression is necessary but not sufficient for the directional migration of DCs. Prostaglandin E2 (PGE2), present in inflammatory sites, induces DC migration, presumably by enacting a migration-permissive gene expression program. Since regulation of DC migration is highly important for their use in vaccination and therapy, we examined the PGE2-induced changes in the expression of metalloproteinases (MMPs). Our results indicate that PGE2 significantly up-regulates MMP-9 expression, induces both secreted and membrane-bound MMP-9, and that in turn, DC-derived MMP-9 is essential for DC chemotaxis in response to the CCR7 ligand CCL19, Matrigel migration, and in vivo migration in both wild-type and MMP-9-deficient hosts. We conclude that DCs matured within inflammatory sites require both CCR7 and PGE2-induced MMP-9 for their directional migration to draining lymph nodes.

Prostaglandin E2 exacerbates collagen-induced arthritis in mice through the inflammatory interleukin-23/interleukin-17 axis.

OBJECTIVE: Recently, Th17 cells, a new subset of CD4+ T cells, emerged as major players in inflammation/autoimmunity. Maintenance of the Th17 phenotype requires interleukin-23 (IL-23), whereas the Th1-promoting cytokine IL-12p70 exerts a negative effect on Th17 cell differentiation. The lipid mediator prostaglandin E(2) (PGE(2)) acts primarily as a proinflammatory agent in autoimmune conditions, through mechanisms that remain to be elucidated. The aim of this study was to investigate whether PGE(2) released in inflammatory foci activates resident dendritic cells (DCs) to express IL-23 (at the expense of IL-12) and IL-6, resulting in a shift toward Th17 cell responses. METHODS: The effect of PGE(2) on IL-23 production by DCs and subsequent induction of T cell-derived IL-17 was assessed in vitro and in vivo. The effect of the stable PGE analog misoprostol was evaluated in a murine model of rheumatoid arthritis, in conjunction with IL-23 and IL-17 expression in affected joints and draining lymph nodes. RESULTS: In vivo administration of PGE(2) induced IL-23-dependent IL-17 production. Administration of misoprostol exacerbated collagen-induced arthritis (CIA). CIA exacerbation was associated with increased levels of IL-23p19/p40 messenger RNA and reduced expression of IL-12p35, and with increased levels of the proinflammatory cytokines IL-17, IL-1beta, IL-6, and tumor necrosis factor in the affected joint. Following ex vivo restimulation, draining lymph node cells from misoprostol-treated mice secreted higher levels of IL-17 and lower levels of interferon-gamma. CONCLUSION: Our results indicate that PGE(2) enhances DC-derived IL-6 production and induces a shift in the IL-23/IL-12 balance in favor of IL-23, resulting in increased IL-17 production, presumably through the amplification of self-reactive Th17 cells.

The proinflammatory effect of prostaglandin E2 in experimental inflammatory bowel disease is mediated through the IL-23-->IL-17 axis.

Although Crohn's disease has been traditionally considered to be Th1-mediated, the newly identified Th17 cells emerged recently as crucial participants. Th1/Th17 differentiation is controlled primarily by the IL-12 family of cytokines secreted by activated dendritic cells (DCs) and macrophages. IL-23 and IL-12/IL-27 have opposite effects, supporting the Th17 and Th1 phenotypes, respectively. We found that PGE(2), a major lipid mediator released in inflammatory conditions, shifts the IL-12/IL-23 balance in DCs in favor of IL-23, and propose that high levels of PGE(2) exacerbate the inflammatory process in inflammatory bowel disease through the IL-23-->IL-17 axis. We assessed the effects of PGE(2) on IL-12, IL-27, and IL-23 and found that PGE(2) promotes IL-23, inhibits IL-12 and IL-27 expression and release from stimulated DCs, and subsequently induces IL-17 production in activated T cells. The effects of PGE(2) are mediated through the EP2/EP4 receptors on DCs. In vivo, we assessed the effects of PGE analogs in an experimental model for inflammatory bowel disease and found that the exacerbation of clinical symptoms and histopathology correlated with an increase in IL-23 and IL-17, a decrease in IL-12p35 expression in colon and mesenteric lymph nodes, and a substantial increase in the number of infiltrating neutrophils and of CD4(+)IL-17(+) T cells in the colonic tissue. These studies suggest that high levels of PGE(2) exacerbate the inflammatory process through the preferential expression and release of DC-derived IL-23 and the subsequent support of the autoreactive/inflammatory Th17 phenotype.