Pharmacology of P2X channels Gever, Joel R; Cockayne, Debra A; Dillon, Michael P ...
Pflügers Archiv,
08/2006, Letnik:
452, Številka:
5
Journal Article
Recenzirano
Significant progress in understanding the pharmacological characteristics and physiological importance of homomeric and heteromeric P2X channels has been achieved in recent years. P2X channels, gated ...by ATP and most likely trimerically assembled from seven known P2X subunits, are present in a broad distribution of tissues and are thought to play an important role in a variety of physiological functions, including peripheral and central neuronal transmission, smooth muscle contraction, and inflammation. The known homomeric and heteromeric P2X channels can be distinguished from each other on the basis of pharmacological differences when expressed recombinantly in cell lines, but whether this pharmacological classification holds true in native cells and in vivo is less well-established. Nevertheless, several potent and selective P2X antagonists have been discovered in recent years and shown to be efficacious in various animal models including those for visceral organ function, chronic inflammatory and neuropathic pain, and inflammation. The recent advancement of drug candidates targeting P2X channels into human trials, confirms the medicinal exploitability of this novel target family and provides hope that safe and effective medicines for the treatment of disorders involving P2X channels may be identified in the near future.
Lower urinary tract symptoms (LUTS) are present in many common urological syndromes. However, their current suboptimal management by muscarinic and α1‐adrenoceptor antagonists leaves a significant ...opportunity for the discovery and development of superior medicines. As potential targets for such therapeutics, purinoceptors have emerged over the last two decades from investigations that have established a prominent role for ATP in the regulation of urinary bladder function under normal and pathophysiological conditions. In particular, evidence suggests that ATP signaling via P2X1 receptors participates in the efferent control of detrusor smooth muscle excitability, and that this function may be heightened in disease and aging. ATP also appears to be involved in bladder sensation, via activation of P2X3 and P2X2/3 receptors on sensory afferent neurons, both within the bladder itself and possibly at central synapses. Such findings are based on results from classical pharmacological and localization studies in non‐human and human tissues, knockout mice, and studies using recently identified pharmacological antagonists – some of which possess attributes that offer the potential for optimization into candidate drug molecules. Based on recent advances in this field, it is clearly possible that the development of selective antagonists for these receptors will occur that could lead to therapies offering better relief of sensory and motor symptoms for patients, while minimizing the systemic side effects that limit current medicines.
British Journal of Pharmacology (2006) 147, S132–S143. doi:10.1038/sj.bjp.0706637
Background and Purpose
The P2X3 receptor is an ATP‐gated ion channel expressed by sensory afferent neurons and is used as a target to treat chronic sensitisation conditions. The first‐in‐class, ...selective P2X3 and P2X2/3 receptor antagonist, the diaminopyrimidine MK‐7264 (gefapixant), has progressed to Phase III trials for refractory or unexplained chronic cough. We used patch clamp to elucidate the pharmacology and kinetics of MK‐7264 and rat models of hypersensitivity and hyperalgesia to test its efficacy on these conditions.
Experimental Approach
Whole‐cell patch clamp of 1321N1 cells expressing human P2X3 and P2X2/3 receptors was used to determine mode of MK‐7264 action, potency, and kinetics. The analgesic efficacy was assessed using paw withdrawal threshold and limb weight distribution in rat models of inflammatory, osteoarthritic, and neuropathic sensitisation.
Key Results
MK‐7264 is a reversible allosteric antagonist at human P2X3 and P2X2/3 receptors. Experiments with the slowly desensitising P2X2/3 heteromer revealed concentration‐ and state‐dependency to wash‐on, with faster rates and greater inhibition when applied before agonist compared to during agonist application. The wash‐on rate (τ value) for MK‐7264 at maximal concentrations was much lower when applied before compared to during agonist application. In vivo, MK‐7264 displayed efficacy comparable to naproxen in inflammatory and osteoarthritic sensitisation models and gabapentin in neuropathic sensitisation models, increasing paw withdrawal threshold and decreasing weight‐bearing discomfort.
Conclusions and Implications
MK‐7264 is a reversible and selective P2X3 and P2X2/3 antagonist, exerting allosteric antagonism via preferential activity at closed channels. Its efficacy in rat models supports its clinical investigation for chronic sensitisation conditions.
Fentanyl buccal soluble film (FBSF) has been developed as a treatment of breakthrough pain in opioid-tolerant patients with cancer. The objective of this study was to evaluate the efficacy of FBSF at ...doses of 200–1200 μg in the management of breakthrough pain in patients with cancer receiving ongoing opioid therapy.
This was a multicenter, randomized, double-blind, placebo-controlled, multiple-crossover study that included opioid-tolerant adult patients with chronic cancer pain who experienced one to four daily episodes of breakthrough pain. The primary efficacy assessment was the sum of pain intensity differences at 30 min (SPID30) postdose.
The intent-to-treat population consisted of 80 patients with ≥1 post-baseline efficacy assessment. The least-squares mean (LSM ± SEM) of the SPID30 was significantly greater for FBSF-treated episodes of breakthrough pain than for placebo-treated episodes (47.9±3.9 versus 38.1 ± 4.3; P = 0.004). There was statistical separation from placebo starting at 15 min up through 60 min (last time point assessed). There were no unexpected adverse events (AEs) or clinically significant safety findings.
FBSF is an effective option for control of breakthrough pain in patients receiving ongoing opioid therapy. In this study, FBSF was well tolerated in the oral cavity, with no reports of treatment-related oral AEs.
1
Many types of culture media contain a pH‐sensitive dye. One commonly occurring dye, Phenol red sodium (Na+) salt, was tested for blocking activity at rat P2X1−4 receptors (P2X1−4Rs) expressed in ...Xenopus oocytes.
2
Phenol red Na+‐salt antagonised adenosine 5′‐triphosphate (ATP) responses at P2X1R (IC50, 3 μM) and, at higher concentrations, also blocked P2X2R and P2X3R. Phenol red Na+‐salt, purified of lipophilic contaminants, blocked P2X1R and P2X3R by acting as an insurmountable antagonist.
3
Two lipophilic extracts of Phenol red antagonised ATP responses at P2XRs. Extract A was a potent antagonist at P2X1R (IC50, 1.4 μM), whereas extract B was a potent antagonist at P2X3R (IC50, 4.1 μM). A bisphenolic compound (RS151030) found in these extracts was a potent antagonist at P2X1R (IC50, 0.3 μM) and at P2X3R (IC50, 2.4 μM).
4
Phenolphthalein base was a potent irreversible antagonist at P2X1R (IC50, 1 μM), whereas Phenolphthalein K+‐salt was 25‐fold less potent here.
5
Phenolphthalein base was a reversible antagonist of ATP responses at rat P2X4R (IC50, 26 μM), whereas Phenolphthalein K+‐salt was inactive.
6
Dimethyl sulphoxide (DMSO), used to dissolve lipophilic extracts, showed pharmacological activity by itself at rat P2X1R and P2X4R.
7
Thus, Phenol red and related compounds are antagonists at rat P2X1R, but are also active at other rat P2XRs. Phenolphthalein base is a newly identified, low potency antagonist of ATP responses at P2X4R. Culture media containing these red dyes should be used cautiously in future pharmacological studies of P2XRs. Also, wherever possible, the solvent DMSO should be used with caution.
British Journal of Pharmacology (2005) 145, 313–322. doi:10.1038/sj.bjp.0706187
The microtubule-associated protein (MAP) tau has been implicated in the pathology of numerous neurodegenerative diseases. In the past decade, the hyperphosphorylated and aggregated states of tau ...protein have been important targets in the drug discovery field for the potential treatment of Alzheimer's disease. Although several compounds have been reported to reduce the hyperphosphorylated state of tau or impact the stabilization of tau, their therapeutic activities are remain to be validated. Recently, reduction of total cellular tau protein has emerged as an alternate intervention point for drug development and a potential treatment of tauopathies. We have developed and optimized homogenous assays, using the AlphaLISA and HTRF assay technologies, for the quantification of total cellular tau protein levels in the SH-SY5Y neuroblastoma cell line. The signal-to-basal ratios were 375 and 5.3, and the Z' factors were 0.67 and 0.60 for the AlphaLISA and HTRF tau assays, respectively. The clear advantages of these homogeneous tau assays over conventional total tau assays, such as ELISA and Western blot, are the elimination of plate wash steps and miniaturization of the assay into 1536-well plate format for the ultra-high-throughput screening of large compound libraries.
Purpose: Previous studies showed that lowering PrPC concomitantly reduced PrPSc in the brains of mice inoculated with prions. We aimed to develop assays that measure PrPC on the surface of human T98G ...glioblastoma and IMR32 neuroblastoma cells. Using these assays, we sought to identify chemical hits, confirmed hits, and scaffolds that potently lowered PrPC levels in human brains cells, without lethality, and that could achieve drug concentrations in the brain after oral or intraperitoneal dosing in mice. Methods: We utilized HTS ELISA assays to identify small molecules that lower PrPC levels by ⩾30% on the cell surface of human glioblastoma (T98G) and neuroblastoma (IMR32) cells. Results: From 44,578 diverse chemical compounds tested, 138 hits were identified by single point confirmation (SPC) representing 7 chemical scaffolds in T98G cells, and 114 SPC hits representing 6 scaffolds found in IMR32 cells. When the confirmed SPC hits were combined with structurally related analogs, >300 compounds (representing 6 distinct chemical scaffolds) were tested for dose–response (EC50) in both cell lines, only studies in T98G cells identified compounds that reduced PrPC without killing the cells. EC50 values from 32 hits ranged from 65nM to 4.1μM. Twenty-eight were evaluated in vivo in pharmacokinetic studies after a single 10mg/kg oral or intraperitoneal dose in mice. Our results showed brain concentrations as high as 16.2μM, but only after intraperitoneal dosing. Conclusions: Our studies identified leads for future studies to determine which compounds might lower PrPC levels in rodent brain, and provide the basis of a therapeutic for fatal disorders caused by PrP prions.
OBJECTIVE
To describe the preclinical pharmacology of Ro 115–1240, a peripherally acting selective α
1A/1L
‐adrenoceptor (AR) partial agonist, compared with the α
1A/1L
‐AR full agonist amidephrine, ...as AR agonists have some utility in the treatment of stress urinary incontinence (SUI) but are limited by undesirable cardiovascular and central nervous system side‐effects.
RESULTS
In radioligand‐binding studies Ro 115–1240 had greater affinity for α
1A
than for α
1B
and α
1D
subtypes. The potency and intrinsic activity of amidephrine and Ro 115–1240 relative to noradrenaline were determined in native and cell‐based assays using human recombinant α
1
‐ARs; they acted as selective α
1A/1L
‐AR full and partial agonists, respectively. In anaesthetized micropigs and rabbits, amidephrine and Ro 115–1240 produced non‐selective, dose‐dependent increases in intraurethral and arterial blood pressures but the magnitude of the pressure increases evoked by Ro 115–1240 were about a third of those with amidephrine. In conscious micropigs both agents produced dose‐dependent increases in urethral tension. Again, the magnitude of the urethral response to Ro 115–1240 was about a third of that with amidephrine. More importantly, only amidephrine produced dose‐dependent increases in blood pressure and decreases in heart rate. Ro 115–1240 produced a maximum increase in urethral tension with no effect on blood pressure or heart rate.
CONCLUSION
These results show that by combining selectivity for the α
1A/1L
‐AR subtype with a reduction in intrinsic agonist efficacy, Ro 115–1240 has reduced haemodynamic effects while retaining to some degree the contractile effects on urethral smooth muscle. These studies indicate that Ro 115–1240 may be useful as a novel treatment for SUI.
The only small-molecule compound demonstrated to substantially extend survival in prion-infected mice is a biaryl hydrazone termed "Compd B" ...(4-pyridinecarboxaldehyde,2-4-(5-oxazolyl)phenylhydrazone). However, the hydrazone moiety of Compd B results in toxic metabolites, making it a poor candidate for further drug development. We developed a pharmacophore model based on diverse antiprion compounds identified by high-throughput screening; based on this model, we generated biaryl amide analogs of Compd B. Medicinal chemistry optimization led to multiple compounds with increased potency, increased brain concentrations, and greater metabolic stability, indicating that they could be promising candidates for antiprion therapy. Replacing the pyridyl ring of Compd B with a phenyl group containing an electron-donating substituent increased potency, while adding an aryl group to the oxazole moiety increased metabolic stability. To test the efficacy of Compd B, we applied bioluminescence imaging (BLI), which was previously shown to detect prion disease onset in live mice earlier than clinical signs. In our studies, Compd B showed good efficacy in two lines of transgenic mice infected with the mouse-adapted Rocky Mountain Laboratory (RML) strain of prions, but not in transgenic mice infected with human prions. The BLI system successfully predicted the efficacies in all cases long before extension in survival could be observed. Our studies suggest that this BLI system has good potential to be applied in future antiprion drug efficacy studies.
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