ACEA-1031

Inhibition of spinal glutamate receptors induces antinociceptive effects in numerous animal models of pain. The present study compares the effects of intrathecally administered N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor antagonists on nociceptive responses in the tail flick test. Potency of antagonists at NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors was first measured by electrical assays in Xenopus oocytes expressing rat cerebral cortex poly(A)+ RNA. Subsequently, Swiss Webster mice were injected intrathecally with the antagonists and tested for antinociception. The drugs tested were: NBQX and GYKI-52466, selective AMPA receptor antagonists, ketamine, MK-801, R(+) HA-966 and ACEA-0762, selective NMDA receptor antagonists, and ACEA-1031, ACEA-1328 and ACEA-0593, NMDA receptor antagonists that also show inhibition of non-NMDA receptors. Selective NMDA receptor antagonists induced essentially no antinociceptive effects in the tail flick test. Antinociceptive activity generally correlated with inhibition of AMPA receptors. The exception was the non-competitive AMPA receptor antagonist GYKI-52466, which was unexpectedly weak. This may be due to inadequate dosing, because the compound has limited solubility, or may be due to differences in the non-NMDA receptor subtype-selectivity profile of GYKI-52466 as compared to competitive antagonists such as NBQX. Overall, our results suggest that inhibition of spinal non-NMDA receptors is the primary, and necessary, mechanism of antinociception by these drugs in the tail flick test in mice.

N-methyl-D-aspartate (NMDA) receptor antagonists show therapeutic potential as neuroprotectants, analgesics, and anticonvulsants. In this context, we used electrical recording techniques to study the in vitro pharmacology of two novel quinoxalinediones, i.e., ACEA-1021 and ACEA-1031 (5-nitro-6,7- dichloro- and 5-nitro-6,7-dibromo-1,4-dihydro-2,3-quinoxalinedione, respectively). Assays with NMDA receptors expressed by rat brain poly(A)+ RNA in Xenopus oocytes and with NMDA receptors in cultured rat cortical neurons indicated that ACEA-1021 and ACEA-1031 are potent competitive antagonists at NMDA receptor glycine sites. Apparent dissociation constants (Kb values) for ACEA-1021 and ACEA-1031 ranged between 6 and 8 nM for oocyte assays and between 5 and 7 nM for neuronal assays. Cloned NMDA receptors expressed in oocytes showed up to 50-fold variation in sensitivity, depending upon subunit composition. For example, using fixed agonist concentrations (10 microM glycine and 100 microM glutamate) IC50 values for ACEA-1021 with four binary combinations were as follows: NMDA receptor (NR)1A/2A, 29 nM; NR1A/2B, 300 nM; NR1A/2C, 120 nM; NR1A/2D, 1500 nM. Measurement of EC50 for glycine and calculation of Kb for the inhibitors indicated that differences in IC50 values are due to subunit-dependent variations in glycine affinity (EC50 ranged between approximately 0.1 and 1 microM) combined with variations in affinity of the antagonists themselves (Kb of approximately 2-13 nM). In addition to the strong antagonism of NMDA receptors, ACEA-1021 and ACEA-1031 were also moderately potent competitive inhibitors of non-NMDA receptors activated either by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid or by kainate. Antagonist affinities were similar whether measured with receptors expressed by rat brain poly(A)+ RNA in oocytes (Kb of 1-2 microM) or with cultured neurons (Kb of 1.5-3.3 microM). Our results suggest that the in vivo neuro-protective actions of ACEA-1021 and ACEA-1031 are predominantly due to inhibition at NMDA receptor glycine sites, although additional inhibition at non-NMDA receptors may play an ancillary role.