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.

Pharmacological characterisation of the human small conductance calcium-activated potassium channel hSK3 reveals sensitivity to tricyclic antidepressants and antipsychotic phenothiazines.

A stable CHO-K1 cell line was developed which expresses the human small conductance calcium-activated potassium channel hSK3. Immunofluorescence microscopy using an anti-SK3 antibody and radioligand binding using [(125)I]-apamin demonstrated the presence of hSK3 channel in the recombinant cell line. This cell line was utilised in a fluorescence assay using the membrane potential-sensitive dye DiBAC(4)(3) to functionally analyse and pharmacologically characterise this potassium channel. The analysis of known blockers of calcium-activated potassium channels revealed the highest potency for apamin (IC(50)=13.2 nM). This result was confirmed by direct recordings of SK3 currents using the whole-cell patch-clamp technique. Tricyclic antidepressants such as desipramine, imipramine and nortriptyline as well as phenothiazines such as fluphenazine, promethazine, chlorpromazine and trifluoperazine blocked the hSK3 channel with micromolar potencies. These compounds also displaced [(125)I]-apamin binding to the hSK3 channel thus suggesting direct and competitive channel blocking activity. Since these compounds share a common three-ring molecular core structure, this feature seems to be important for channel blocking activity. The serine/threonine protein phosphatase inhibitors okadaic acid and calyculin A were able to abolish channel activation with nanomolar potencies, but did not displace [(125)I]-apamin binding. Thus, phosphorylation of hSK3 or an accessory channel subunit seems to be involved in its modulation.

Potent antihyperalgesic activity without tolerance produced by glycine site antagonist of N-methyl-D-aspartate receptor GV196771A.

Central sensitization is a condition of enhanced excitability of spinal cord neurons that contributes to the exaggerated pain sensation associated with chronic tissue or nerve injury. N-methyl-D-aspartate (NMDA) receptors are thought to play a key role in central sensitization. We have tested this hypothesis by characterizing in vitro and in vivo a novel antagonist of the NMDA receptor acting on its glycine site, GV196771A. GV196771A exhibited an elevated affinity for the NMDA glycine binding site in rat cerebral cortex membranes (pKi = 7.56). Moreover, GV196771A competitively and potently antagonized the activation of NMDA receptors produced by glycine in the presence of NMDA in primary cultures of cortical, spinal, and hippocampal neurons (pKB = 7.46, 8. 04, and 7.86, respectively). In isolated baby rat spinal cords, 10 microM GV196771A depressed wind-up, an electrical correlate of central sensitization. The antihyperalgesic properties of GV196771A were studied in a model of chronic constriction injury (CCI) of the rat sciatic nerve and in the mice formalin test. In the CCI model GV196771A (3 mg/kg twice a day p.o.), administered before and then for 10 days after nerve ligature, blocked the development of thermal hyperalgesia. Moreover, GV196771A (1-10 mg/kg p.o.) reversed the hyperalgesia when tested after the establishment of the CCI-induced hyperalgesia. In the formalin test GV196771A (0.1-10 mg/kg p.o.) dose-dependently reduced the duration of the licking time of the late phase. These antihyperalgesic properties were not accompanied by development of tolerance. These observations strengthen the view that NMDA receptors play a key role in the events underlying plastic phenomena, including hyperalgesia. Moreover, antagonists of the NMDA glycine site receptor could represent a new analgesic class, effective in conditions not sensitive to classical opioids.

A transient outward current dependent on external calcium in rat cerebellar granule cells.

The outward potassium current of rat cerebellar granule cells in culture was studied with the whole-cell patch-clamp method. Two voltage-dependent components were identified: a slow current, resembling the classical delayed rectifier current, and a fast component, similar to an IA-type current. The slow current was insensitive to 4-aminopyridine and independent of external Ca2+, but significantly inhibited by 3 mM tetraethylammonium. The fast current was depressed by external 4-aminopyridine, with an ED50 = 0.7 mM, and it was abolished by removal of divalent cations from the external medium. The sensitivity of the transient outward current to different divalent cations was investigated by equimolar substitution of Ca2+, Mn2+ and Mg2+. In 2.8 mM Mn2+, the transient potassium conductance was comparable to that in 2.8 mM Ca2+, while in 2.8 mM Mg2+ the transient component was drastically reduced, as in the absence of any divalent cations. However, when Ca2+ was present, Mg2+ up to 5 mM had no effect. The transient current increased with increasing concentrations of external Ca2+, [Ca2+]o, and the maximum conductance vs. [Ca2+]o curve could be approximated by a one-site model. In addition, the current recorded with 5.5 mM BAPTA in the intracellular solution was not different from that recorded in the absence of any Ca2+ buffer. These results suggest that divalent cations modulate the potassium channel interacting with a site on the external side of the cell membrane.

Voltage-dependent calcium currents in dissociated granule cells from rat cerebellum.

Voltage-dependent calcium currents were investigated by the patch-clamp technique in whole-cell recording configuration in cultures from 8-day-old rat cerebella, which contained greater than or equal to 90% granule cells. In solutions designed to minimize the sodium and potassium conductances and in 20 mM barium, an inward current activated around -25 mV, reached a peak amplitude at +20 mV and reversed around +80 mV. In 20 mM calcium, this current was approximately 50% of that in barium. From one to three days in vitro only 16% of the cells tested (n = 20) had a current exceeding 50 pA in maximum amplitude, while after four days in vitro the current reached 100 pA in all neurons tested (n greater than 70). Verapamil (50-100 microM) reversibly depressed this current. The dihydropyridine agonist Bay K 8644 (1 microM) enhanced the maximum conductance by 25 +/- 8% (n = 4), caused a negative shift in the activation of 21 +/- 5 mV and a prolongation of the deactivation time course as the voltage was stepped back from +20 to -80 mV. The GABAB agonist baclofen (50 microM) reversibly depressed the current by 27 +/- 8% in 80% of the cells. The effect was similar to that of GABA (10 microM), when the GABAA response (chloride current) was partially blocked by bicucculine. This current can be classified as a dihydropyridine-sensitive high-voltage-activated calcium current. The modulation by GABAB agonists is likely to be significant for presynaptic inhibition.

A transient voltage-dependent outward current in cultured cerebellar granules.

Granule cells were dissociated from rat cerebella with a procedure that yields a 98% pure cell population. Potassium currents in these cells were studied using the patch-clamp technique. Depolarizing pulses of 10 mV step and 100 ms duration from a holding potential of -80 mV elicited two different potassium outward currents: a transient, low-voltage activated component and a long lasting, high-voltage activated component. At +30 mV, the total current reached an amplitude of 2 nA (mean value of 15 experiments). The reversal potential of the transient current, estimated by measuring tail currents, was -77 mV, close to that predicted by the Nernst equation. The transient current was half inactivated with a holding potential of -78 mV and completely inactivated with -50 mV or more positive holding potentials. Finally, the current decay could be fitted by the sum of two exponentials with time constants of about 20 and 250 ms.