Identification of non-amidine inhibitors of acid-sensing ion channel-3 (ASIC3).

A series of benzothiophene methyl amines were examined in an effort to identify non-amidine chemotypes with reduced polypharmacology from existing leads with the goal of finding potent ASIC3 channel blockers to advance the therapeutic evaluation of ASIC3 inhibition.

High concentration electrophysiology-based fragment screen: discovery of novel acid-sensing ion channel 3 (ASIC3) inhibitors.

The Merck Fragment Library was screened versus acid-sensing ion channel 3 (ASIC3), a novel target for the treatment of pain. Fragment hits were optimized using two strategies, and potency was improved from 0.7 mM to 3 µM with retention of good ligand efficiency and incorporation of reasonable physical properties, off-target profile, and rat pharmacokinetics.

Amiloride derived inhibitors of acid-sensing ion channel-3 (ASIC3).

A series of amiloride derivatives modified at the 5-position of the pyrazine ring were evaluated as inhibitors of acid-sensing ion channel-3 (ASIC3), a novel target for the treatment of chronic pain.

Amidine derived inhibitors of acid-sensing ion channel-3 (ASIC3).

A series of indole amidines modified at the 2-position of the indole ring were evaluated as inhibitors of Acid-Sensing Ion Channel-3 (ASIC3), a novel target for the treatment of chronic pain.

Botulinum toxin type A inhibits sensory neuropeptide release in rat bladder models of acute injury and chronic inflammation.

OBJECTIVE: To determine the effect of botulinum toxin type A (BTX-A) on the release of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from isolated bladder preparations after acute injury with HCl and the induction of cyclophosphamide (CYP)-induced cystitis, as neurogenic inflammation has been increasingly identified in urological disorders such as interstitial cystitis. MATERIALS AND METHODS: Adult rats had either an intraperitoneal injection with CYP or saline over a 10-day period to induce chronic bladder inflammation, after which the bladder was harvested, or normal bladder explants were injured acutely with incubation (20 s) in HCl (0.4 m). To measure the effect of BTX-A on the release of neurotransmitters, harvested bladders were incubated in an organ bath containing BTX-A (10 U) or vehicle. Bladders were transferred to a subsequent bath (physiological saline) and incubated for 15 min, and the bathing medium analysed to measure neurotransmitter release, as determined by radioimmunoassay. Bladder specimens from sham treatment, controls and experimental rats were compared histologically. RESULTS: Acute injury with HCl caused a significantly greater release of both CGRP and SP release (1235 and 1655 pg/g, respectively) than in controls (183 and 449 pg/g, respectively; P < 0.001). This increase in neurotransmitter release was partly inhibited by exposure to BTX-A (870 and 1033 pg/g (P < 0.05 and <0.01). CYP-induced chronic inflammation caused significantly greater release of SP than in the controls (1060 and 605 pg/g, respectively; P < 0.005). Exposure to BTX-A partly inhibited the release of SP after CYP-induced cystitis (709 pg/g, P < 0.05). CONCLUSIONS: The application of BTX-A inhibits the release of sensory neurotransmitters from isolated bladder preparations in rat bladder models of both acute injury and chronic inflammation, suggesting a potential clinical benefit of BTX-A in the treatment of neurogenic inflammation.

Use-dependent inhibition of P2X3 receptors by nanomolar agonist.

P2X3 receptors desensitize within 100 ms of channel activation, yet recovery from desensitization requires several minutes. The molecular basis for this slow rate of recovery is unknown. We designed experiments to test the hypothesis that this slow recovery is attributable to the high affinity (< 1 nM) of desensitized P2X3 receptors for agonist. We found that agonist binding to the desensitized state provided a mechanism for potent inhibition of P2X3 current. Sustained applications of 0.5 nM ATP inhibited > 50% of current to repetitive applications of P2X3 agonist. Inhibition occurred at 1000-fold lower agonist concentrations than required for channel activation and showed strong use dependence. No inhibition occurred without previous activation and desensitization. Our data are consistent with a model whereby inhibition of P2X3 by nanomolar [agonist] occurs by the rebinding of agonist to desensitized channels before recovery from desensitization. For several ATP analogs, the concentration required to inhibit P2X3 current inversely correlated with the rate of recovery from desensitization. This indicates that the affinity of the desensitized state and recovery rate primarily depend on the rate of agonist unbinding. Consistent with this hypothesis, unbinding of [32P]ATP from desensitized P2X3 receptors mirrored the rate of recovery from desensitization. As expected, disruption of agonist binding by site-directed mutagenesis increased the IC50 for inhibition and increased the rate of recovery.

Reversal of acid-induced and inflammatory pain by the selective ASIC3 inhibitor, APETx2.

BACKGROUND AND PURPOSE: Inflammatory pain is triggered by activation of pathways leading to the release of mediators such as bradykinin, prostaglandins, interleukins, ATP, growth factors and protons that sensitize peripheral nociceptors. The activation of acid-sensitive ion channels (ASICs) may have particular relevance in the development and maintenance of inflammatory pain. ASIC3 is of particular interest due to its restricted tissue distribution in the nociceptive primary afferent fibres and its high sensitivity to protons. EXPERIMENTAL APPROACH: To examine the contribution of ASIC3 to the development and maintenance of muscle pain and inflammatory pain, we studied the in vivo efficacy of a selective ASIC3 inhibitor, APETx2, in rats. KEY RESULTS: Administration of APETx2 into the gastrocnemius muscle prior to the administration of low pH saline prevented the development of mechanical hypersensitivity, whereas APETx2 administration following low-pH saline was ineffective in reversing hypersensitivity. The prevention of mechanical hypersensitivity produced by acid administration was observed whether APETx2 was applied via i.m. or i.t. routes. In the complete Freund's adjuvant (CFA) inflammatory pain model, local administration of APETx2 resulted in a potent and complete reversal of established mechanical hypersensitivity, whereas i.t. application of APETx2 was ineffective. CONCLUSIONS AND IMPLICATIONS: ASIC3 contributed to the development of mechanical hypersensitivity in the acid-induced muscle pain model, whereas ASIC3 contributed to the maintenance of mechanical hypersensitivity in the CFA inflammatory pain model. The contribution of ASIC3 to established hypersensitivity associated with inflammation suggests that this channel may be an effective analgesic target for inflammatory pain states.

Synthesis, structure-activity relationship, and pharmacological profile of analogs of the ASIC-3 inhibitor A-317567.

The synthesis, structure-activity relationship (SAR), and pharmacological evaluation of analogs of the acid-sensing ion channel (ASIC) inhibitor A-317567 are reported. It was found that the compound with an acetylenic linkage was the most potent ASIC-3 channel blocker. This compound reversed mechanical hypersensitivity in the rat iodoacetate model of osteoarthritis pain, although sedation was noted. Sedation was also observed in ASIC-3 knockout mice, questioning whether sedation and antinociception are mediated via a non-ASIC-3 specific mechanism.

Botulinum toxin type a inhibits calcitonin gene-related peptide release from isolated rat bladder.

PURPOSE: Increasing evidence suggests that sensory nerve dysfunction may underlie several urological disorders, including interstitial cystitis and sensory urgency. We determined the effect of botulinum toxin type A (Allergan, Irvine, California) on baseline and chemically evoked release of the sensory neuropeptide, calcitonin gene-related peptide in an isolated bladder preparation. MATERIALS AND METHODS: Whole rat bladders were incubated in a series of tissue baths containing physiological salt solution. Following bladder equilibration in PSS sequential incubation was performed and this sample was used to measure baseline CGRP release. To evoke CGRP release tissue was subsequently incubated in PSS containing capsaicin (30 nM) and adenosine triphosphate (10 microM). To measure the effect of BTX-A on baseline and evoked CGRP release bladders were incubated for 6 hours in an organ bath containing BTX-A (50 microM) or vehicle prior to bladder equilibration. CGRP release was determined by radioimmunoassay. RESULTS: Mean baseline release of CGRP +/- SEM was 346 +/- 44 pg/gm. Adenosine triphosphate/capsaicin application increased CGRP release by 75% over baseline (606 +/- 98 pg/gm, p < 0.005). BTX-A application resulted in a 19% decrease in baseline release of CGRP, although this difference did not achieve statistical significance. BTX-A application significantly decreased evoked CGRP by 62% vs control (606 +/- 98 vs 229 +/- 21 pg/gm, p < 0.005). CONCLUSIONS: BTX-A application inhibits the evoked release of CGRP from afferent nerve terminals in isolated rat bladder. This finding suggests a potential clinical benefit of BTX-A for the treatment of interstitial cystitis or sensory urgency.

A role for the P2X receptor in urinary tract physiology and in the pathophysiology of urinary dysfunction.

OBJECTIVE: We provide a historical perspective of the P2X receptor class in bladder physiology and the pathophysiology of urinary dysfunction. METHODS: A literature search was performed using the MEDLINE database. RESULTS: Evidence suggests that P2X receptors serve a combined function in sensory and motor activity of human bladder. P2X receptors mediate excitation of sensory neurons and evoke muscle contraction in response to ATP release. Anatomical and functional defects in the P2X receptor signaling are associated with a variety of urologic diseases. CONCLUSION: Current research underscores the importance of P2X receptors in urologic physiology. Potential applications exist in relation to the diagnosis and treatment of urinary dysfunction. However, the detailed mechanism of P2X receptor function in bladder physiology and in urinary tract disease remains unknown and warrants further investigation.

ATP and UTP excite sensory neurons and induce CREB phosphorylation through the metabotropic receptor, P2Y2.

Extracellular ATP rapidly excites nociceptive sensory neurons by opening ATP-gated ion channels (P2X receptors). Here, we describe two actions of both ATP and UTP on rat sensory neurons that are relatively slow and sustained: phosphorylation of the transcription factor CREB and delayed action potential firing that persists for tens of seconds after removal of the ligand. The pharmacology of these responses indicates that they are mediated by the metabotropic receptor P2Y2, and not by P2X receptors. CREB phosphorylation occurred in a subset of small peripherin-positive neurons likely to be unmyelinated nociceptors. In situ hybridization analysis revealed widespread expression of P2Y2 mRNA in sensory neurons. CREB phosphorylation is mediated by both action-potential-evoked calcium influx and calcium release from intracellular stores. These findings suggest that P2Y2 contributes to the transduction of ATP-mediated sensory signalling, and may be involved in the activity-dependent regulation of nociceptor phenotype.