Antagonism of non-NMDA receptors inhibits handling-induced, strychnine-potentiated convulsions

European Journal of Pharmacology, 231 (1993)309-312

309

© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00

EJP 21177 Short communication

Antagonism of non-NMDA receptors inhibits handling-induced, strychnine-potentiated convulsions Kevin H. M c A l l i s t e r Sandoz Research Institute Berne Ltd., Monbijoustrasse 115, CH-3001 Berne, Switzerland

Received 24 November1992, accepted 1 December 1992

The effects of blockade of the a-amino-3-hydroxy-5-methyl-isoxazole-4-proprionate (AMPA) and kainate subtypes of excitatory amino acid receptor upon handling-induced, strychnine-potentiated convulsions in mice were investigated using NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) and compared to the effects of N-methyl-D-aspartate (NMDA) receptor blockade with D-CPPene (D-(E)-4-(3-phosphonoprop-2-enyl)-piperazine-2-carboxylic acid), a competitive NMDA antagonist. Both compounds exerted peak blocking activity 30 min after intraperitoneal administration. Using this pretreatment interval, a dose-response relationship for blocking handling-induced, strychnine-potentiated convulsions was generated for each compound. D-CPPene blocked seizures with an EDs0 of 0.72 (0.59-0.87) mg/kg and NBQX blocked seizures with an EDs0 of 68.0 (36.72-125.94) mg/kg. These results indicate that both NMDA and non-NMDA subtypes of excitatory amino acid receptors are activated in handling-induced, strychnine-potentiated convulsions. non-NMDA receptor antagonism; Spinal seizures; (Mouse)

1. Introduction

Studies of the effects of the excitatory amino acids glutamate and aspartate on neurons of the mammalian central nervous system have revealed that these substances exert neuronal excitation via specific receptors (Watkins and Evans, 1981). These receptors can be classified into at least five subtypes: N-methyl-Daspartate (NMDA), kainate, a-amino-3-hydroxy-5methyl-isoxazole-4-proprionate (AMPA), 2-amino-4phosphonobutyrate (AP4) and metabotropic receptors (Monaghan et al., 1989). Most attention has been focused upon the N M D A subtype of excitatory amino acid receptor resulting in the development of compounds blocking excitatory amino acid-mediated depolarisation via the N M D A receptor relatively selectively. These compounds are effective anticonvulsants in various animal models (Dingledine et al., 1990; Chapman et al., 1990) including one involving the precipitation of seizures by sensory stimulation in mice previously treated with a subconvulsive dose of strychnine. Handling-induced, strychnine-potentiated convulsions are sensitive to blockade by competitive N M D A receptor antagonists, uncompetitive ion channel blockers, as

well as antagonists acting via the strychnine-insensitive glycine, and polyamine modulatory sites of the N M D A receptor (McAllister, 1992). The quinoxaline NBQX exerts potent antagonism at non-NMDA excitatory amino acid receptors (Sheardown et al., 1990). It shows high affinity for the AMPA and kainate site with little activity at the N M D A and NMDA-associated strychnine-insensitive glycine binding sites. The compound has been demonstrated to exert anticonvulsant activity in animal models of epilepsy (Swedberg et al., 1990; Chapman et al., 1991; Smith et al., 1991). The present experiments investigated whether handling-induced, strychnine-potentiated convulsions could be blocked by antagonism of non-NMDA receptors and compared the activity of N B Q X (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) to that of D-CPPene (D-(E)-4-(3-phosphonoprop-2-enyl)-piperazine-2-carboxylic acid), a specific competitive antagonist of the N M D A receptor (Aebischer et al., 1989, Lowe et al., 1990).

2. Materials and methods 2.1. Experimental animals

Correspondence to: K.H. McAllister, Sandoz Research Institute Berne Ltd., Monbijoustrasse 115, CH-3001 Berne, Switzerland. Tel. 41 31 46 61 11, fax 41 31 46 69 46.

Male OF-1 mice (Sandoz, Basle) weighing 18-25 g were used. The animals were maintained under con-

310 stant temperature (22°C) and humidity. Noise within the experimental room was kept to a minimum.

ensure a baseline of zero protection following vehicle administration.

2.2. Time course of blocking activity of D-CPPene and NBQX upon handling-induced, strychnine-potentiated convulsions

2.3. Dose-response of the inhibition of handling-induced, strychnine-potentiated convulsions by D-CPPene and NBQX

Separate groups of mice (n = 6-18 per group) were administered 1.0 m g / k g D-CPPene or 100.0 m g / k g NBQX intraperitoneally (i.p.) and subsequently tested at various time intervals after administration. These doses were chosen after initial experiments had revealed protection against handling-induced, strychnine-potentiated convulsions of greater than 50%. The procedure used to assess the inhibitory activity of compounds upon handling-induced, strychninepotentiated convulsions has been reported earlier (McAllister, 1992). Briefly, mice were injected with 0.42 m g / k g strychnine subcutaneously (s.c.). Ten minutes later the animals were lifted from the cage by the base of the tail and rotated along the body axis clockwise and anticlockwise alternatively three times. The mice were then rested on the laboratory bench. Typically, the mice show convulsions for periods up to 1 rain. The convulsions are characterised by a loss of righting, opistotonus, clonic movements of the limbs and rotation of the tail. The convulsive period is concluded with a short phase of inactivity and righting. The righting response is taken as the endpoint of the convulsion. If the animal did not convulse, the turning procedure was repeated. If the animal did not show a convulsion after three bouts of turning, then it was considered protected. That the majority of the control mice convulse upon the first bout of turning, and that all of the controls show convulsion, is employed as a criterion to

Using a pretreatment interval of 30 min, this being the optimal pretreatment time for both compounds (see Results), D-CPPene and NBQX were administered i.p. in a dose range of 0.1-3.0 m g / k g and 32.0100.0 m g / k g respectively to groups of 6-18 mice per dose for each compound. Difficulty was met in administering higher doses of NBQX.

2.4. Drugs All drug solutions were prepared on the day of the experiment. Strychnine nitrate dried overnight over silica gel was quickly weighed and dissolved in 0.9% saline. The strychnine solution was administered s.c. in a volume of 10.0 mg/kg. D-CPPene ((E)-4-(3-phosphonoprop-2-enyl)-piperazine-2-carboxylic acid; Aebischer et al., 1989) was dissolved in saline. NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline; Sheardown et al., 1990) was suspended in saline with Tween 80 and using ultrasonication.

2.5. Estimation of the ED5o for inhibition of handlinginduced, strychnine-potentiated convulsions The procedure described by Litchfield and Wilcoxon (1949) was used to calculate the dose that protected against seizures in 50% of the animals (EDs0) within 95% confidence limits

100"

PERCENTAGE INHIBITION OF HISP CONVULSIONS

0

I

I

I

30

60

90

I

120

i

150

•

180

TIME AFTER INJECTION (rnins)

Fig. 1. Percentage inhibition of handling-induced, strychnine-potentiatedconvulsions(ordinate) by 1.0 mg/kg D-CPPene (e) and 100.0 mg/kg NBQX (o) upon seperate groups of mice tested at various intervalsafter i.p. administration,n = 6-18 mice per time point for each compound.

311 D-CPPene (1.0 mg/kg) peaked 30 min after i.p. administration, with the amount of protection being less than 50% between 60 and 90 min post-administration. Seizure activity was blocked with an EDs0 of 0.72 mg/kg D-CPPene after i.p. administration 30 min prior to handling. These results parallel those of a previously reported study where an EDs0 of 0.6 mg/kg was estimated for D-CPPene under the same treatment regimen (McAllister, 1992). At a dose of 100.0 mg/kg i.p. and at the time points after administration tested in the present experiment, the inhibition of handling-induced, strychnine-potentiated convulsions by NBQX peaked 30 min post-administration. The amount of protection provided by NBQX fell below 50% between 30 and 60 min after administration. Using a pretreatment interval of 30 min, a dose-response relationship was generated for NBQX and an EDs0 of 68.0 mg/kg estimated. It would appear that the difference in potency between D-CPPene and NBQX is slightly greater in this model than that in inhibiting sound-induced seizures in DBA/2 mice. D-CPPene (given i.p.) blocks clonic seizures in D B A / 2 mice with an EDs0 of 0.39 mg/kg (Chapman et al., 1990). NBQX blocks seizures with an EDs0 of 9.5 mg/kg, i.p. 30 min post-administration (Chapman et al., 1991). The efficacy with which NBQX inhibits seizures in genetically-prone species is not limited to DBA/2 mice. Smith et al. (1981) found that NBQX blocks sound-induced seizures in GEPR-9 rats with an EDs0 of 13.68 mg/kg i.p., and photically induced myoclonus in photosensitive baboons within a dose range of 2.0-4.0 mg/kg i.v. The variance in the results between these models and that of handling-induced, strychnine-potentiated seizures may be due to the latter model reflecting seizure activity within the

3. Results

3.1. Time course of the blocking effects of D-CPPene and NBQX upon handling-induced, strychnine-potentiated convulsions Figure 1 shows the percentage inhibition of handling-induced, strychnine-potentiated convulsions provided by 1.0 mg/kg D-CPPene and 100.0 mg/kg NBQX as a function of time after administration. The blocking activity for D-CPPene reached a peak 30 min post-administration then declined over the next 120 min. NBQX also showed more blocking activity 30 min post-administration but also showed a high degree of activity after only 15 min post-administration.

3.2. Dose-response for blocking handling-induced, strychnine-potentiated convulsions by D-CPPene and NBQX It can be seen in fig. 2 that both D-CPPene and NBQX blocked handling-induced, strychnine-potentiated convulsions in a dose-dependent manner and that the dose-response curves are quite steep. The EDs0s for blocking the convulsions are 0.72 (0.59-0.87) mg/kg and 68.0 (36.72-125.94) mg/kg for D-CPPene and NBQX respectively.

4. Discussion

The results presented above confirm that handlinginduced, strychnine-potentiated convulsions are inhibited by antagonism of both NMDA and non-NMDA excitatory amino acid receptors. The blocking of handling-induced, strychnine-potentiated convulsions by

.100.

PE RC ENTAGE INHIBITION OF HISP 50CONVULSIONS

0

;

0.1

A

-

,

1.0

I/i 10.0

100.0

DOSE (rag Ikg) Fig. 2. Percentage inhibition of handling-induced,strychnine-potentiatedconvulsions(ordinate) as a functionof dose of D-CPPene (o) and NBQX(©) administeredi.p. 30 min before testing.The EDs0 for inhibitinghandling-induced,strychnine-potentiatedconvulsionsis 0.72 and 68.0 mg/kg for D-CPPene and NBQXrespectively,n = 6-18 miceper treatmentdose for each compound.

312 spinal cord. Strychnine blocks the chloride ion c o n d u c tance associated with the action of glycine at strychnine-sensitive inhibitory glycine receptors (Curtis et al., 1976), the resulting convulsions reflecting spinal seizure activity ( M e l d r u m , 1985). A l t h o u g h both N M D A a n d n o n - N M D A receptors are p r e s e n t in the spinal cord, the b e h a v i o u r a l c o n s e q u e n c e s u p o n s t i m u l a t i o n are different. F o r example, U r c a a n d Raigorodsky (1988) have d e m o n s t r a t e d qualitative a n d q u a n t i t a t i v e differences in the biting a n d scratching s y n d r o m e resulting from intrathecal a d m i n i s t r a t i o n of N M D A , quisqualate a n d kainate. Such differences may u n d e r l i e the discrepancy in efficacy with which N M D A a n d n o n N M D A receptor a n t a g o n i s m inhibits h a n d l i n g - i n duced, s t r y c h n i n e - p o t e n t i a t e d convulsions c o m p a r e d to o t h e r genetic seizure models.

Acknowledgements The author would like to thank Dr. T. Honor6 (Novo Nordisk A/S, Denmark) for generously supplying NBQX. The author would also like to thank Drs. A.K. Dixon and D.A. Lowe for comments on the manuscript, and Mrs. V. Haussener for technical assistance.

References Aebischer, B., P. Frey, H.-P. Haerter, P.L. Herrling, W. Mueller, H.J. Olverman and J.C. Watkins, 1989, Synthesis and NMDA antagonistic properties of the enantiomers of 4-(3-pbosphonopropyl)piperazine-2-carboxylic acid (CPP) and of the unsaturated analogue (E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid (CPP-ene), Helv. Chim. Acta 72, 1043. Chapman, A.G., J. Graham and B.S. Meldrum, 1990, Potent oral anticonvuisant action of CPP and CPPene in DBA/2 mice, Eur. J. Pharmacol. 178, 97.

Chapman, A.G., S.E. Smith and B.S. Meldrum, 1991, The anticonvulsant effect of the non-NMDA antagonists, NBQX and GYKI 52466, in mice, Epilepsy Res. 9, 92. Curtis, D.R., A.W. Duggan and G.A.R. Johnston, 1976, The specificity of strychnine as a glycine antagonist in the mammalian spinal cord, Exp. Brain Res. 12, 541. Dingledine, R., C.J. McBain and J.O. McNamara, 1990, Excitatory amino receptors in epilepsy, Trends Pharmacol. Sci. 11, 334. Litchfield, J.T. and F. Wilcoxon, 1949, A simplified method of evaluating dose-effect experiments, J. Pharmacol. Exp. Ther. 96, 99. Lowe, D.A., H.C. Neijt and B. Aebischer, 1990, D-CPPene (SDZ EAA 494), a potent and competitive N-methyl-D-aspartate (NMDA) antagonist: effect on spontaneous activity and NMDAinduced depolarisations in the rat neocortical slice preparation, compared with other CPP derivatives and MK-801, Neurosci. Lett. 113, 315. Meldrum, B.S., 1985, GABA and other amino acids, in: Antiepileptic Drugs, ed. H.-H. Frey and D. Janz (Springer-Verlag, Berlin) p. 153. McAllister, K.H., 1992, NMDA receptor antagonists and channel blockers have different effects upon a spinal seizure model in mice, Eur. J. Pharmacol. 211, 105. Monaghan, D.T., R.J. Bridges and C.W. Cotman, 1989, The excitatory amino acid receptors: Their classes, pharmacology, and distinct properties in the function of the central nervous system, Ann. Rev. Pharmacol. Toxicol. 29, 365. Sheardown, M.J., E.O. Nielsen, A.J. Hansen, P. Jacobsen and T. Honor6, 1990, 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline: A neuroprotectant for cerebral ischemia, Science 247, 571. Smith, S.E., N. Diirmiiller and B.S. Meldrum, 1991, The non-Nmethyl-D-aspartate receptor antagonists, GYKI 52466 and NBQX are anticonvulsant in two animal models of reflex epilepsy, Eur. J. Pharmacol. 201, 179. Swedberg, M.D.B., P. Jacobsen and T. Honor6, 1990, NBQX (2,3-dihydroxy-6-nitro-sulfamoyl-benzo(F)quinoxaline): Anticonvulsant effects in mice, Soc. Neurosci. Abstr. 16, 489.8. Urca, G. and G. Raidorodsky, 1988, Behavioral classification of excitatory amino acid receptors in mouse spinal cord, Eur. J. Pharmacol. 153, 211. Watkins, J.C. and R.H. Evans, 1981, Excitatory amino acid transmitters, Ann. Rev. Pharmacol. Toxicol. 21, 165.