Elizabeth A. Kowaluk

A-317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat

P2X3 and P2X2/3 receptors are highly localized on peripheral and central processes of sensory afferent nerves, and activation of these channels contributes to the pronociceptive effects of ATP. A-317491 is a novel non-nucleotide antagonist of P2X3 and P2X2/3 receptor activation. A-317491 potently blocked recombinant human and rat P2X3 and P2X2/3 receptor-mediated calcium flux (Ki = 22–92 nM) and was highly selective (IC50 >10 μM) over other P2 receptors and other neurotransmitter receptors, ion channels, and enzymes. A-317491 also blocked native P2X3 and P2X2/3 receptors in rat dorsal root ganglion neurons. Blockade of P2X3 containing channels was stereospecific because the R-enantiomer (A-317344) of A-317491 was significantly less active at P2X3 and P2X2/3 receptors. A-317491 dose-dependently (ED50 = 30 μmol/kg s.c.) reduced complete Freunds adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED50 = 10–15 μmol/kg s.c.) in attenuating both thermal hyperalgesia and mechanical allodynia after chronic nerve constriction injury. The R-enantiomer, A-317344, was inactive in these chronic pain models. Although active in chronic pain models, A-317491 was ineffective (ED50 >100 μmol/kg s.c.) in reducing nociception in animal models of acute pain, postoperative pain, and visceral pain. The present data indicate that a potent and selective antagonist of P2X3 and P2X2/3 receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X3 and P2X2/3 receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.

Inhibition of adenosine kinase during oxygen-glucose deprivation in rat cortical neuronal cultures

Adenosine kinase (AK) inhibitors potentiate the actions of endogenous adenosine (ADO) and ameliorate cerebral ischemic damage in animal models. The present study examined the effects of the AK inhibitor, 5-iodotubercidin (5-IT) in an in vitro model of neuronal ischemia, specifically, combined oxygen-glucose deprivation of rat cortical mixed neuronal-glial cultures. Oxygen-glucose deprivation caused extensive neuronal loss which was accompanied by a marked increase in ADO release into the extracellular medium, was ameliorated by exogenous ADO (10 microM(-1) mM), and was exacerbated by a high concentration of the selective A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 10 microM). 5-IT (1 microM) had no effect on extracellular ADO levels nor on neuronal loss. However, AK activity in these cultures was markedly suppressed during oxygen-glucose deprivation. Taken together, these data demonstrate a marked down-regulation of AK activity during oxygen-glucose deprivation in this in vitro model, providing an endogenous mechanism contributing to the accumulation of extracellular ADO, which exerts neuroprotective effects by activating the ADO A1 receptor.

Characterization of the effects of adenosine kinase inhibitors on acute thermal nociception in mice.

Adenosine (ADO) is an inhibitory neuromodulator that can increase the nociceptive threshold in animals exposed to a variety of noxious stimuli. Inhibition of the ADO-metabolizing enzyme, ADO kinase (AK), provides a means of locally enhancing extracellular ADO concentrations. In the present study, the AK inhibitors 5-amino,5-deoxy-ADO (NH2dADO), 5-iodotubercidin (5-IT), and 5-deoxy,5-iodotubercidin (5d-5IT) were examined for their analgesic efficacy in the hot-plate model of acute somatic nociception. Control and drug-treated adult male mice were placed on a 55 degrees C hot plate and the latency to the 10th jump was recorded via a computer driven infrared-beam photosensor. All three AK inhibitors were found to significantly increase jump latencies in a dose-dependent fashion. 5d-5IT was the most potent AK inhibitor (approx. ED50 value = 1 micromol/kg, IP), followed by 5-IT (ED50 value = 10 micromol/kg, IP), and NH2dADO (ED50 value = 100 micromol/kg, IP). 5d-5IT was found to be more potent and equally efficacious to morphine (ED50 value = 5.2 micromol/kg, IP) in this assay. In a model of persistent chemical pain, the phenyl-p-quinone-induced abdominal constriction assay, 5d-5IT (ED50 value = 1.5 micromol/kg, SC) and morphine (ED50 value = 3.0 micromol/kg, SC) dose dependently reduced nociception. Pretreatment of mice with either the nonselective ADO receptor antagonist, theophylline (56 micromol/kg, IP), but not the peripherally acting antagonist, 8-(p-sulfophenyl)-theophylline (8-PST, 200 micromol/kg, IP) significantly attenuated the antinociceptive effects of 5d-5IT in the hot-plate assay. Furthermore, the antinociceptive effects of 5d-5IT were completely blocked by an ADO A1 receptor selective antagonist, DPCPX, while an ADO A2A receptor selective antagonist, ZM 241385, showed markedly less antagonist activity. The analgesic effects of 5d-5IT were not blocked by the opioid receptor antagonist naloxone; however, 5d-5IT could produce additive analgesic effects with morphine when both compounds were administered in combination. The apparent efficacy of 2.5 micromol/kg, IP, of 5d-5IT was not significantly altered following the repeated administration of this dose twice daily for 4 days. The present data provide evidence for an antinociceptive action of AK inhibitors in the hot-plate test, which, at least for 5d-5IT, is mediated by an enhancement of ADOs actions at the ADO A1 receptor subtype, is nonopioid in nature, and which does not exhibit tolerance following repeated administration.

Lack of specificity of [35S]-ATPγS and [35S]-ADPβS as radioligands for ionotropic and metabotropic P2 receptor binding

Adenosine‐5′‐O‐3‐thio[35S]triphosphate ([35S]‐ATPγS) has been reported to specifically bind several P2X receptor subtypes, including P2X1, P2X2, P2X3, and P2X4. Similarly, adenosine‐5′‐O‐2‐thio[35S]diphosphate ([35S]‐ADPβS) has been reported to label putative P2Y receptors. To address whether these radioligands selectively label P2 receptors, the functional activity of various P2 ligands was compared with their ability to compete for [35S]‐ATPγS and [35S]‐ADPβS binding to cell membrane preparations from rat brain, HEK293 cells, and to native and P2X4 transfected 1321N1 astrocytoma cells. [35S]‐ATPγS (0.2 nM) and [35S]‐ADPβS (0.1 nM) displayed a high percentage of specific binding to membranes prepared from 1321N1 human astrocytoma cells, which were found to be devoid of detectable P2X and P2Y functional activity. [35S]‐ATPγS and [35S]‐ADPβS also exhibited equivalent high percentages of specific binding to HEK293 cell membranes, which endogenously express the P2Y1 and P2Y2 receptor subtypes, to 1321N1 cells stably transfected with the human P2X4 receptor, and to rat brain membranes, which have previously been shown to contain both P2X and P2Y receptor subtypes. The potency order of P2 agonists to compete for radioligand binding to these cell membrane preparations was significantly different from the functional rank order potencies determined in HEK293 cells and 1321N1 cells expressing the P2X4 receptor, as measured by cytosolic calcium flux. These data indicate that [35S]‐ATPγS and [35S]‐ADPβS appear to bind sites that do not correspond to known functional P2 receptor subtypes. The apparent lack of specificity of these radioligands for labeling P2 receptors is similar to that reported for other radiolabeled nucleotides and illustrates the need for caution in interpreting the apparent pharmacology of native P2 receptors on the basis of binding data alone. Drug Dev. Res. 48:84–93, 1999.

Synthesis of phosphorus-containing amino acid analogs as inhibitors of nitric oxide synthase

Abstract Two series of α-amino phosphonic and α-amino phosphinic analogs of arginine were prepared and tested as inhibitors of nitric oxide synthase (NOS). Two of the analogs were found to possess inhibitory activity for the neuronal isoform of NOS.

Opportunities in pain therapy:beyond the opioids and NSAIDs

Pain remains a major factor in patients seeking physician assistance. Neither the NSAIDs (non-steroidal, anti-inflammatory drugs) nor the opioids represent a complete answer to pain control, either alone or in combination. Recent research advances using molecular biological techniques have established that pain is a complex process involving multiple neurotransmitter/neuromodulator targets in the spinal cord, in ascending and descending spinal pathways and in the central nervous system as well as receptors and enzymes that are induced as part of the pain process. These advances, coupled with the considerable unmet medical need in the area of analgesics, are anticipated to lead to novel, non-opioid, non-NSAID approaches to pain relief and control.