Characterization of three diaminopyrimidines as potent and selective antagonists of P2X3 and P2X2/3 receptors with in vivo efficacy in a pain model.

BACKGROUND AND PURPOSE: P2X3 and P2X2/3 receptors are highly localized on the peripheral and central pathways of nociceptive signal transmission. The discovery of A-317491 allowed their validation as chronic inflammatory and neuropathic pain targets, but this molecule has a very limited oral bioavailability and CNS penetration. Recently, potent P2X3 and P2X2/3 blockers with a diaminopyrimidine core group and better bioavailability were synthesized and represent a new opportunity for the validation of P2X3-containing receptors as targets for pain. Here we present a characterization of three representative diaminopyrimidines. EXPERIMENTAL APPROACH: The activity of compounds was evaluated in intracellular calcium flux and electrophysiological recordings from P2X receptors expressed in mammalian cells and in a in vivo model of inflammatory pain (complete Freund's adjuvant (CFA) in rat paws). KEY RESULTS: Compound A potently blocked P2X3 (pIC(50)= 7.39) and P2X2/3 (pIC(50)=6.68) and showed no detectable activity at P2X1, P2X2, P2X4 and P2X7 receptors (pIC(50)< 4.7). Whole-cell voltage clamp electrophysiology confirmed these results. Compounds showed good selectivities when tested against a panel of different classes of target. In the CFA model, compound B showed significant anti-nociceptive effects (57% reversal at 3mg·kg(-1) ). CONCLUSIONS AND IMPLICATIONS: The diaminopyrimidines were potent and selective P2X3 and P2X2/3 receptor antagonists, showing efficacy in vivo and represent useful tools to validate these receptors as targets for inflammatory and neuropathic pain and provide promising progress in the identification of therapeutic tools for the treatment of pain-related disorders.

Translation of flecainide- and mexiletine-induced cardiac sodium channel inhibition and ventricular conduction slowing from nonclinical models to clinical.

INTRODUCTION: Nonclinical in vivo models used for cardiovascular safety testing have not previously been studied for their sensitivity for detection of conduction slowing resulting from cardiac sodium channel block. The goal of this study was to examine the sensitivity of in vivo models to cardiac sodium channel block, and translation of the effect from in vitro to in vivo models using sodium channel inhibitors flecainide and mexiletine; flecainide, but not mexiletine is commonly associated with QRS complex prolongation in humans. METHODS: Inhibition of cloned cardiac sodium channels (hNav1.5) was studied using the IonWorks platform. Conduction slowing was measured in vitro in the rabbit isolated ventricular wedge (RVW) and in vivo in the conscious telemetered rat and dog, and anaesthetised dog. RESULTS: Flecainide and mexiletine inhibited hNav1.5 channels with IC50 values of 10.7 and 67.2 µM respectively. In the RVW, QRS was increased by flecainide at 60 bpm, and at 120bpm, there was an increased effect of both drugs. In conscious rats, flecainide significantly increased QRS complex duration; mexiletine had no significant effect, but there was an increase at the highest dose in 4/6 animals. QRS complex was increased by flecainide and mexiletine in anaesthetised dogs but this was not statistically significant; in conscious dog, only flecainide produced a significant increase in QRS complex. DISCUSSION: When compared to clinical data, effects of flecainide and mexiletine in RVW and conscious dog compared well with effects in patients and healthy volunteers in terms of sensitivity. The anaesthetised dog was least sensitive for detection of changes in QRS. All assays showed some differentiation between the expected conduction slowing activity of flecainide and mexiletine. Based on these data, RVW and conscious dog were most predictive for effects of compounds on QRS complex and cardiac conduction.

Development of a functional reporter gene HTS assay for the identification of mGluR7 modulators.

For the identification of modulators of the metabotropic glutamate receptor mGluR7, a functional cell-based high throughput screening (HTS) assay was developed. This assay utilizes the signal transduction pathway of mGluR7, which is negatively coupled to adenylyl cyclase. A cAMP-responsive luciferase reporter gene and rat mGluR7 cDNA were cotransfected into CHO-K1 cells by electroporation. Stable recombinant cells were selected by resistance to the antibiotic G418. Functional selection was carried out by analyzing the effect of the agonist glutamate to reduce elevated cAMP levels after forskolin stimulation. Out of 83 G418-resistant cell clones, the clone with the best functional characteristics was selected. This clone displayed the strongest reduction of forskolin-stimulated cAMP levels. Glutamate (10 mM) decreased cAMP levels, as monitored by luciferase expression, by about 50%, and the more potent agonist L-2-amino-4-phosphonobutyrate resulted in nearly complete reduction, exhibiting an EC(50) of 0.9 mM. The functional response of the clone did not change during cell passages, indicating the stability of this novel recombinant cell line. The luciferase reporter gene assay, which allows easy nonradioactive luminescence detection of mGluR7 activity, was optimized for its application in automated HTS.