Haixia Yu

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.

Modulation of BzATP and formalin induced nociception: attenuation by the P2X receptor antagonist, TNP‐ATP and enhancement by the P2X3 allosteric modulator, cibacron blue

Exogenous ATP produces acute and localized pain in humans, and P2X receptor agonists elicit acute nociceptive behaviours in rodents following intradermal administration to the hindpaw. The predominant localization of P2X3 mRNA in sensory neurones has led to the hypothesis that activation of P2X3 and/or P2X2/3 receptors contributes to nociception. The local administration of the P2X receptor agonist, BzATP (100 – 1000 nmol paw−1, s.c.) into the rat hindpaw produced an acute (<15 min) paw flinching response that was similar to that observed in the acute phase of the formalin (5%) test. The co‐administration of the potent P2X receptor antagonist, TNP‐ATP (30 – 300 nmol paw−1), but not an inactive analogue, TNP‐AMP, with BzATP into the rat hindpaw attenuated BzATP‐induced nociception. Similarly, co‐administration of TNP‐ATP, but not TNP‐AMP, with 5% formalin reduced both acute and persistent nociception in this test. Co‐administration of cibacron blue (30 and 100 nmol paw−1), a selective allosteric enhancer of P2X3 and P2X2/3 receptor activation, with BzATP (30 and 100 nmol paw−1) into the rat hindpaw produced significantly greater nociception as compared to the algogenic effects of BzATP alone. Intradermal co‐administration of cibacron blue (30 and 100 nmol paw−1) with formalin (1 and 2.5%) into the rat hindpaw also produced significantly greater nociceptive behaviour as compared to formalin alone. The ability of TNP‐ATP and cibacron blue to respectively attenuate and enhance nociceptive responses elicited by exogenous BzATP and formalin provide further support for the hypothesis that activation of peripheral P2X3 containing channels contributes specifically to both acute and persistent nociception in the rat.

Analgesic and anti-inflammatory effects of A-286501, a novel orally active adenosine kinase inhibitor

&NA; Adenosine (ADO) is an inhibitory neuromodulator that can increase nociceptive thresholds in response to noxious stimulation. Inhibition of the ADO‐metabolizing enzyme, adenosine kinase (AK) increases extracellular ADO concentrations at sites of tissue trauma and AK inhibitors may have therapeutic potential as analgesic and anti‐inflammatory agents. N7‐((1′R,2′S,3′R,4′S)‐2′,3′‐dihydroxy‐4′‐amino‐cyclopentyl)‐4‐amino‐5‐bromo‐pyrrolo[2,3‐a]pyrimidine (A‐286501) is a novel and potent (IC50=0.47 nM) carbocyclic nucleoside AK inhibitor that has no significant activity (IC50>100 &mgr;M) at other sites of ADO interaction (A1, A2A, A3 receptors, ADO transporter, and ADO deaminase) or other (IC50 values>10 &mgr;M) neurotransmitter and peptide receptors, ion channel proteins, neurotransmitter reuptake sites and enzymes, including cyclooxygenases‐1 and ‐2. A‐286501 showed equivalent potency to inhibit AK from several mammalian species and kinetic studies revealed that A‐286501 was a reversible and competitive inhibitor with respect to ADO and non‐competitive with respect to MgATP2−. A‐286501 was orally effective to reduce nociception in animal models of acute (thermal), inflammatory (formalin and carrageenan), and neuropathic (L5/L6 nerve ligation and streptozotocin‐induced diabetic) pain. A‐286501 was particularly potent (ED50=1 &mgr;mol/kg, p.o.) to reduce carrageenan‐induced inflammatory thermal hyperalgesia as compared to its analgesic actions in other pain models (acute and neuropathic) and its ability to alter hemodynamic function and motor performance. A‐286501 was also effective to reduce carrageenan‐induced paw edema and myeloperoxidase activity, a measure of neutrophil influx (ED50=10 &mgr;mol/kg, p.o.), in the injured paw. The anti‐nociceptive effects of A‐286501 in the L5/L6 nerve injury model of neuropathic pain (ED50=20 &mgr;mol/kg, p.o.) were not blocked by the opioid antagonist naloxone, but were blocked by the ADO receptor antagonist, theophylline. Following repeated administration, A‐286501 showed less potential to produce tolerance as compared to morphine. Thus, A‐286501 is a structurally novel AK inhibitor that effectively attenuates nociception by a non‐opioid, non‐non‐steroidal anti‐inflammatory drug ADO, receptor mediated mechanism.

Structure-activity studies of 5-substituted pyridopyrimidines as adenosine kinase inhibitors.

The synthesis and SAR of a novel series of non-nucleoside pyridopyrimidine inhibitors of the enzyme adenosine kinase (AK) are described. It was found that pyridopyrimidines with a broad range of medium and large non-polar substituents at the 5-position potently inhibited AK activity. A narrower range of analogues was capable of potently inhibiting adenosine phosphorylation in intact cells indicating an enhanced ability of these analogues to penetrate cell membranes. Potent AK inhibitors were found to effectively reduce nociception in animal models of thermal hyperalgesia and persistent pain.

Adenosine kinase inhibitors: polar 7-substitutent of pyridopyrimidine derivatives improving their locomotor selectivity.

We have discovered that polar 7-substituents of pyridopyrimidine derivatives affect not only whole cell AK inhibitory potency, but also selectivity in causing locomotor side effects in vivo animal models. We have identified compound, 1o, which has potent whole cell AK inhibitory potency, analgesic activity and minimal reduction of locomotor activity.

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.