Neuropharmacology Vol. 31, No. 10, pp. 1021-1026, 1992 Printed in Great Britain.All rights reserved
0028-3908/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd
ANTIPARKINSONIAN DRUGS MEMANTINE A N D TRIHEXYPHENIDYL POTENTIATE THE ANTICONVULSANT ACTIVITY OF VALPROATE AGAINST MAXIMAL ELECTROSHOCK-INDUCED SEIZURES E. URBAIqSKA,*M. DZIKI, S. J. CZUCZWAR,~"Z. KLEINROKand W. A. TURSKI Department of Pharmacology, Medical School, Jaczewskiego 8, 20-090 Lublin, Poland (Accepted 11 March 1992)
Summary--Memantine increased the threshold for electroconvulsions, when administered at 1.0--6.0mg/kg (i.p.) and given in subthreshold doses of 0.0156, 0.0625, 0.125 and 0.Smg/kg (i.p.) potentiated the protective efficacy of valproate, against maximal electroshock (50 mA)-induced seizures in mice, lowering the EDs0 from 235 to 197, 172, 164 and 130 mg/kg, respectively.Trihexyphenidyl,applied in doses of 30 and 50 mg/kg (i.p.), did not influence the electroconvulsivethreshold per se but when combined with valproate, strongly enhanced its anticonvulsant activity against maximal electroshockinduced seizures lowering the EDs0 from 206 to 103 and 46 mg/kg, respectively. The chimney test and retention testing in mice revealed that administration of memantine at 0.5 mg/kg (i.p.) or trihexyphenidyl at 30 mg/kg (i.p.) together with valproate in doses of 130 or 103 mg/kg (i.p.), respectively,resulted in motor impairment and caused impairment of long-term memory, similar to the effects of valproate alone, when applied at its EDso against maximal electroshock. Neither memantine nor trihexyphenidyl altered the total level of valproate in plasma. It may be concluded that the potentiation of the anticonvulsant activity of valproate, by memantine and trihexyphenidyl, is not associated with a pharmacokinetic interaction. Key words--valproate, memantine, trihexyphenidyl, seizures.
Both memantine and trihexyphenidyl are used clinically in the treatment of Parkinson's disease and other kinds of rigidity, due to their anticholinergic action (Bianchine, 1985; Masuo, Enomoto and Maeno, 1986). Memantine has been shown to act as an anticonvulsant drug in different models of electricallyand chemically-induced seizures in mice, raising the threshold for electroconvulsions and protecting against the tonic hind limb extension in pentylenetetrazol-, bicuculline-, 3-mercaptopropionic acidand picrotoxin-induced seizures (Maj, Sowifiska, Baran and Sarnek, 1974; Meldrum, Turski, Schwarz, Czuczwar and Sontag, 1986). Electrophysiological studies in cultures of spinal cord nerve cells from the mouse indicated that memantine is able to reduce the frequency of action potentials in neurones firing spontaneously and to shorten the duration of strychnine-elicited bursts (Netzer and Bigalke, 1990).
*Present address: Maryland Psychiatric Research Center, University of Maryland School of Medicine, P.O. Box 21247, Baltimore, MD 21228, U.S.A. tTo whom correspondence should be addressed at present address: Vanderbilt University, Department of Neurology, School of Medicine, 2100 Pierce Ave., Nashville, TN 37212, U.S.A. NP 31/10--E
Memantine and trihexyphenidyl were reported to inhibit the activity of N-methyl-D-aspartate (NMDA) receptors (Olney, Price, Labruyere, Salles, Friedrich, Mueller and Silverman, 1987; Bormann, 1989). Previous studies demonstrated that administration of dizocilpine (MK-801), the antagonist of the NMDA receptor channel (Clineschmidt, Martin, Bunting and Papp, 1982), together with valproate or phenobarbital, markedly potentiated the protective activity against maximal electroshock-induced seizures (Urbafiska, Dziki, Kleinrok, Czuczwar, Turski, 1991). The aim of the present study was to investigate the effects of memantine and trihexyphenidyl, when combined with valproate, on maximal electroshockinduced seizures. METHODS
Animals
The experiments were carried out on male Albino Swiss mice, weighing 20-25 g. The animals were housed in colony cages with free access to food (Murigran pellets) and tap water and maintained on a natural light--dark cycle. The experimental groups, consisting of 8-10 animals, were chosen by means of a randomized schedule. The convulsion and behavioural tests were performed between 10.00 and 13.00 hr and each mouse was used only once.
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Drugs Valproic acid (sodium salt) (Depakine, Labaz, Ambarez, France), at 20-300mg/kg, memantine hydrochloride (l.3-dimethyi- 5-aminoadamantane) (Merz, Frankfurt, Germany), at 0.0035-6.0 mg/kg and trihexyphenidyl hydrochloride (Sigma, St Louis, Missouri), at 10-50 mg/kg, were dissolved in sterile saline and administered intraperitoneally (i.p.) 30 min (valproate) and 60 min (the latter drugs) before the test. The control animals were given saline, at respective times prior to the test. The injection volume was always 0.05 ml/10 g body weight. Doses of drugs refer to their free forms.
Electroconvulsions The procedure was carried out according to Swinyard, Brown and Goodman (1952). Electroshock was applied by ear-clip electrodes and generated by a stimulator (COTM, Bialystok, Poland) which delivered an alternating current (50 Hz), the duration of the stimulus being 0.2 sec. The end-point was the tonic extension of the hind limbs. To evaluate the convulsion threshold, at least 4 groups of mice, consisting of 8-10 animals, were subjected to electroshocks of various intensities. An intensity-response curve was calculated from the percentage of mice showing the endpoint in each group. The convulsion threshold was evaluated as CS50, which was the strength of current (in mA), necessary to induce tonic hindlimb extension in 50% of the mice tested. The anticonvulsant potency of valproate was expressed as the EDs0, i.e. the dose of the drug (in mg/kg) required to protect 50% of the treated mice against maximal electroshock (50 mA)-induced tonic hindlimb extension. Both CS50 and EDs0 values were calculated according to the method of Litchfield and Wilcoxon (1949).
Chimney test The effects of valproate, combined with memantine or trihexyphenidyl on motor impairment, were quantified by the chimney test of Boissier, Tardy and Diverres (1960), in which the animals have to climb up backwards in a plastic tube (3 cm inner diameter, 25 cm length). Motor impairment was indicated by the inability of the animals to climb up backwards in the tube within 60 sec and the results were shown as a percentage of animals which failed to perform the test.
Dark-avoidance acquisition and retention testing The mice were placed in an illuminated box (10 x 13 x 15cm), connected to a large dark box (25 x 20 x 15cm) which was equipped with an electric grid floor. Entrance into the dark box was punished by an electric footshock (0.6 mA for 2 sec; facilitation of acquisition). On the next day (24 hr later), the same mice were placed in the illuminated box. Mice avoiding the dark compartment for over
60 sec were considered as remembering the task. The retention was quantified as a percentage of animals avoiding the dark compartment. The step-through passive avoidance task may give the information about ability to acquire the task (learning) and to recall the task (retrieval) and may be regarded as a measure involving long-term memory (Venault, Chapouthier, de Carvalho, Simiand, Morre, Dodd and Rossier, 1986).
Determination of the level of valproate in plasma The mice were injected with either valproate and saline or valproate, in combination with memantine or trihexyphenidyl. The animals were killed by decapitation at respective times and samples of blood of approximately l ml were collected into Eppendorf tubes. Samples of blood were centrifuged at 10,000 rpm for 3 min and samples of plasma of 70 ,ul were transferred to Abbott System cartridges. The level of vaiproate in plasma was estimated by immunofluorescence, using an Abbott TDx analyzer (Abbott, Irving, Texas) and presented as the mean value + SD of at least 7 determinations.
Statistics Both CSs0 and EDs0 values and statistical analysis of the results, obtained in the electroconvulsive tests, were estimated by computer linear regression analysis, according to Litchfield and Wilcoxon (1949). Results from the passive avoidance task and chimney test were analysed statistically with the use of Fisher's exact probability test. Statistical significance was defined as P < 0.05. RESULTS
Effects of memantine and trihexyphenidyl upon the electroconvulsive threshold Memantine, administered in doses of 1.0, 2.0, 4.0 and 6.0mg/kg (i.p.), 60rain before the test, raised the electroconvulsive threshold from 12.7 to 14.0, 15.0, 16.4 and 16.7mA, respectively. Memantine, applied in doses of 0.5mg/kg (i.p.) and less, did not significantly influence the electroconvulsive threshold (Table 1). Trihexyphenidyl, up to 50 mg/kg injected (i.p.), 60 min prior to the test did not significantly alter the electroconvulsive threshold (Table 1).
Influence of memantine and trihexyphenidyl upon the protective efficacy of valproate Memantine, given at 0.0156, 0.0625, 0.125 and 0.5 mg/kg (i.p.), 60 rain before the test, when applied together with valproate (i.p. 30min prior to the test), significantly reduced the EDs0 value--from 235 to 187, 172, 164 and 130mg/kg, respectively. Trihexyphenidyl, administered (i.p.) 60min before the test, in doses of 30 and 50 mg/kg, when given together with valproate (i.p. 30 min prior to the test), potentiated the protective efficacy, decreasing the
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Valproate and antiparkinsonian drugs Table 1. Effects of memantine and trihexyphenidyl upon the electroconvulsive threshold
Treatment Saline Memantine'
Saline TrihexyphenidyP
Dose (in mg/kg)
Electroconvulsive threshold (CS50 in mA; 95% confidence limits)b
-0.5 1.0 2.0 4.0 6.0 -30.0 50.0
12.7 (I 1.9-13.6) 13.6 (I 2.5-14.8) 14.0 (13.3-14.7)* 15.0 (13.7-16.4)* 16.4 (15.5-17.4)* 16.7 (15.0-18.7)* 13.1 (I 1.8-14.5) 12.9 ( I 1.6-14.3) 13.9 (12.8-15.2)
*P < 0.05 vs compared to saline-treated group. 'Memantine and trihexyphenidyl were administered intraperitoncally, 60 min prior to the test. b'l'he CSs0 values (with 95% confidence limits) and statistical comparisons were calculated according to the method of Litchfield and Wilcoxon (1949).
ED50 value from 206 to 103 and 46 mg/kg, respectively (Table 2). Motor impairment Valproate caused marked motor impairment, as revealed by the chimney test, when applied in doses of 206 and 235 mg/kg (i.p.), 30 min before individual trials (i.e. at the dose resembling the EDs0 of vaiproate for each trial) (Table 3). Memantine, given in a dose of 0.5 mg/kg (i.p.), 60 min prior to the test, did not induce any motor impairment and trihexyphenidyl, administered in a dose of 30 mg/kg (i.p.), 60 min before the test did (Table 3). The combined treatment with memantine, at 0.5 mg/kg (i.p.) or trihexyphenidyl at 30mg/kg (i.p.) and valproate at 130 or 103 mg/kg (i.p.), respectively, resulted in motor impairment (Table 3). Dark-avoidance acquisition and retention testing Administration of valproate, in a dose equal to its EDs0 against maximal electroshock i.e. 206 or 235mg/kg (i.p.) (two independent trials), 30min Table 2. Influence of memantine and trihexyphenidyl upon the protective efficacy of valproate against maximal electrochockinduced seizures Treatment (in mg/kg) Valproate' + saline + memantine' 0.0035 0.0156 0.0625 0.125 0.5 Valproate a + saline + trihexyphenidyP 10 30 50
EDs0b of valproate (in mg/kg; 95% confidence limits) 235.4 (210.8-262.9) 237.4 (199.0-283.3) 187.6 ( 163.4-215.5)* 172.5 (141.2-210.0)* 164.2 (143.4-188.0)* 130.0 (108.6-155.5)* 206.3 (167.5-253.9) 194.7 (123.9-306.0) 103.3 (60.9-175.4)* 46.6 (25.5-85.3)*
*P < 0.05 vs compared to saline-treated group. 'Drugs were administered intraperitoneally: valproate 30min, memantine and trihexypbenidyl 60 min, before the test. bTbe data represent EDs0 values (in mg/kg; 95% confidence limits) for the protection against maximal electroshock. The EDso values and statistical comparisons were calculated according to the method of Litchfield and Wilcoxon (1949).
Table 3. Motor impairment after administration of valproate, memantine, trihexyphenidyl or combinations of valproate with one of the antiparkinsonian drugs Treatment (mg/kg)
Nb
% of animals showing motor impairmenff
Saline Valproatea (235) Valproate (130) Valproate (130) + memantine' (0.5) Memantine (0.5) Saline Valproate (206) Valproate (103) Valproate (103) + trihexypbenidyP (30) Trihexyphenidyl (30)
12 12 9
0 50* I 1.1
10 10 12 10 10
60* 10 0 50* 20
10 10
40* 50*
*P < 0.05 vs saline (Fisher's exact probability test). aDrugs were administered intraperitoneany: valproate 30min, memantine and trihexyphenidyl 60 min before the test. bNumber of animals. CEvaluated by the chimney test of Boissier et al. (1960). Mice had to climb up backwards in a plastic tube (3 era inner diameter, 25 cm length). Motor impairment was indicated when the animals were unable to climb up backwards in the tube, within 60 sec. The results concerning the motor impairment were expressed as a percentage of animals which failed to climb up during the 60 sec observation period.
before training, caused significant impairment of long-term memory, as revealed by the retention test 24 hr later (Table 4). Valproate, in a dose of 103 or 130 mg/kg (i.p.), equal to its EDs0 against maximal electroshock, when applied together with trihexyphenidyl (30 mg/kg i.p.) or memantine (0.5 mg/kg, i.p.), respectively, did not influence retention. Administration of memantine at 0.5 mg/kg (i.p.), 60 min before the training session had no effect on dark avoidance 24 hr later. Trihexyphenidyl, injected at the dose of 30 mg/kg (i.p.), 60 rain before training totally impaired retention (Table 4). The combination of memantine (0.5 mg/kg, i.p.) or trihexyphenidyl (30mg/kg, i.p.) with valproate (130 or 103 mg/kg, Table 4. Effects of valproate, memantine and trihexyphenidyl or combinations of valproate with one of the antiparkinsonian drugs on retention of a passive avoidance task in mice Treatment (mg/kg)
Nb
Retention (% of control)c
Saline Valproate' (235) Valproate (130) Valproate (130)+ memantine' (0.5) Memantine (0.5)
10 10 8 8 8
100 50* 62.5 37.5* 100
Saline Valproate (206) Valproate (103) Valproate (103)+tribexypbenidyl' (30) Trihexyphenidyl (30)
10 10 11 10 10
100 50* 90.9 0 *d 0 *a
*P < 0.05 vs saline-treated group; dp < 0.05 VS valproate (206 mg/kg)-treated group (Fisber's exact probability test). "Drugs were administered intraperitoneally: valproate 30min, memantine and trihexyphenidyl 60 min before training. bNumber of animals used. 'The retention was quantified as a percentage of mice avoiding the dark compartment. Entrance into the dark box was punished by an electric footshock (0.6 mA for 2 see). On the next day (24 hr later), the same mice were placed in the illuminated box. Mice avoiding the dark compartment for over 60 sec were considered as remembering the task.
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i.p., respectively), significantly impaired retention (Table 4). Influence of memantine or trihexyphenidyl on the level o f valproate in plasma
The total level of valproate, administered at 130 or 103 mg/kg (i.p.) (186.1 + 12.6, 150.9 + 30.1/zg/ml, respectively), in plasma was not altered in the presence of memantine, given at 0.5 mg/kg (i.p.) or trihexyphenidyl, at 30mg/kg (i.p.) (175.3+20.9, 135.5 + 10.7 ~g/ml, respectively). DISCUSSION The present studies demonstrated that memantine (but not trihexyphenidyl) exerted a distinct anticonvulsant action, elevating the threshold for electroconvulsions in mice, Moreover, both drugs when applied at doses which did not affect the electroconvulsive threshold per se, markedly potentiated the protective efficacy of valproate against maximal electroshock-induced seizures. The observed augmentation of the antiepileptic activity of valproate did not seem to be related to a pharmacokinetic interaction, in terms of total plasma levels, since the level of valproate in plasma remained unchanged in the presence of either memantine or trihexyphenidyl. Application of memantine or trihexyphenidyl, together with valproate, led to the impairment of motor activity and long-term memory, similar to the action of valproate alone when applied at the EDs0 dose against maximal electroshock, as revealed by the chimney test and retention testing in mice. The combined treatment with valproate and trihexyphenidyl worsened even the memory task, in comparison with valproate alone. Moreover trihexyphenidyl, given alone, strongly impaired long-term memory. Thus, memantine and trihexyphenidyl did not reverse the side-effects produced by valproate, as MK-801 did (Urbafiska et al., 1991). This might be explained by the fact that anticholinergic drugs are known to impair memory very potently (Izquierdo, 1989), as do NMDA antagonists like phencyclidine (DeNoble, Jones, Schaeffer and Bauerle, 1990; Parada-Turska and Turski, 1990). The mechanism of strong potentiation of protection, against maximal-electroshock-induced seizures in the case of valproate, administered together with memantine or trihexyphenidyl, might be associated with several processes. Both memantine and trihexyphenidyl are antiparkinsonian agents, displaying anticholinergic properties (Bianchine, 1985; Masuo et al., 1986). The involvement of cholinergic mechanisms in human and experimental epilepsy has been often demonstrated (see for review Turski, Ikonomidou, Turski, Bortolotto, Cavalheiro, 1989). Pilocarpine, a cholinomimetic drug, when administered systemically or intracerebrally in rodents, produced severe seizure activity (Turski, Czuczwar,
Kleinrok and Turski, 1983). Carbachol (a cholinergic agonist), injected into the amygdala, induced the development of kindled seizures (Wasterlain and Jonec, 1981). Atropine (a muscarinic antagonist) increased the latency to develop fully generalized seizures in electrical kindling (Albright, Burnham and Okazaki, 1979) and potentiated the anticonvulsant action of the NMDA receptor antagonist, 2-amino5-phosphono-pentanoic acid, against electroconvulsions (Czuczwar, Turski, Chmielewska, Turski and Kleinrok, 1985). The anticonvulsant benefits of the anticholinergic drugs mentioned above are mainly thought to be related to their interaction with the central cholinergic system. It may be suspected that potentiation of the antiepileptic properties of valproate by memantine and trihexyphenidyl, presented in this study, may result in part from their ability to block cholinergic transmission. However, based on the report indicating that antiparkinsonian agents, among them memantine and trihexyphenidyl, are phencyclidine agonists and NMDA antagonists (Olney et al., 1987), an alternative explanation may be proposed. The observed enhancement of the anticonvulsant activity of valproate might be related to their anti-NMDA properties. Recently, valproate was shown to suppress NMDA-induced, transient depolarizations in the neocortex of the rat in vitro (Zeise, Kasparow and Zieglgfinsberger, 1991), which may give a further explanation for the interaction of this antiepileptic drug with NMDA antagonists. Excitatory amino acid antagonists, especially those selective for the NMDA receptor, have potent anticonvulsant activity in a wide range of experimental seizure models. They include different types of chemically, electrically and sound induced seizures in rodents, photically-induced convulsions in baboons and myoclonus and seizures in the high-pressure neurological syndrome (see for review Chapman, 1988; Meldrum, 1991). Dizocilpine (MK-801), the most potent anticonvulsant drug among noncompetitive NMDA antagonists, has been already studied in clinical trials in epileptic patients (Troupin, Mendius, Cheng and Risinger, 1986). The present data are in agreement with the previous finding, showing that MK-801 strongly potentiated the protective efficacy of valproate and phenobarbital against maximal electroshock-induced seizures (Urbafiska et al., 1991). Consequently, the blockade of excitatory amino acid-mediated events may also be involved in the observed augmentation of the antiepileptic activity of valproate, when combined with memantine or trihexyphenidyl. Nevertheless, another possible mechanism of this interaction cannot be excluded. It was noticed that anticholinergic drugs, used in the treatment of Parkinson's disease, might improve the illness not only due to the impaired activity of the cholinergic system but also as a result of a change in the balance
Valproate and antiparkinsonian drugs between dopamine and acetylcholine, since the degeneration of the striatum in Parkinson patients leads to the deficiency of dopamine, within those structures of brain (Bianchine, 1985). Memantine was reported to induce locomotor hyperactivity in vivo and to increase the release of dopamine in vitro (Maj, 1982; Osborne, Beale, GolombiowskaNikitin and Sontag, 1982). Numerous investigations have revealed that precursors of dopamine and dopamine agonists exert anticonvulsant effects in different animal models of epilepsy (Anlezark, Marrosu and Meldrum, 1981; Kleinrok, Czuczwar, W6jcik, Przegalifiski, 1978, L6scher and Czuczwar, 1986). Therefore, the observed augmentation of the antiepileptic activity of valproate by memantine and trihexyphenidyl, might be caused by an interaction with more than one neurotransmitter system, excitatory amino acids, and/or acetylcholine and/or dopamine. The involvement of excitatory amino acid-mediated events seems to be the most probable, since MK-801 and new competitive N M D A antagonists also strongly enhanced the antiepileptic activity of valproate (Urbafiska et al., 1991; unpublished data). In conclusion, combined treatment with valproate and memantine or trihexyphenidyl, allowed a reduction of the EDs0 for valproate 2-4-fold, while the examined side-effects of this combination were comparable to those induced by valproate alone. The influence of trihexyphenidyl upon memory seemed to be exceptional but anticholinergics impair memory very potently, even at doses smaller than those used in these studies. More precise investigations of the mechanism leading to the enhanced antiepileptic activity of valproate, when administered together with memantine or trihexyphenidyl, may be a promising approach to find similar drugs, not exerting pronounced sideeffects. These data suggest that the ability to obtain drugs displaying weaker anticholinergic properties, while possessing stronger a n t i - N M D A activity may be of a clinical value. Drugs of this type might be useful in clinical practice as therapeutic agents potentiating the activity of common antiepileptics. Acknowledgement--The authors wish to thank Merz &
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0028-3908/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd
ANTIPARKINSONIAN DRUGS MEMANTINE A N D TRIHEXYPHENIDYL POTENTIATE THE ANTICONVULSANT ACTIVITY OF VALPROATE AGAINST MAXIMAL ELECTROSHOCK-INDUCED SEIZURES E. URBAIqSKA,*M. DZIKI, S. J. CZUCZWAR,~"Z. KLEINROKand W. A. TURSKI Department of Pharmacology, Medical School, Jaczewskiego 8, 20-090 Lublin, Poland (Accepted 11 March 1992)
Summary--Memantine increased the threshold for electroconvulsions, when administered at 1.0--6.0mg/kg (i.p.) and given in subthreshold doses of 0.0156, 0.0625, 0.125 and 0.Smg/kg (i.p.) potentiated the protective efficacy of valproate, against maximal electroshock (50 mA)-induced seizures in mice, lowering the EDs0 from 235 to 197, 172, 164 and 130 mg/kg, respectively.Trihexyphenidyl,applied in doses of 30 and 50 mg/kg (i.p.), did not influence the electroconvulsivethreshold per se but when combined with valproate, strongly enhanced its anticonvulsant activity against maximal electroshockinduced seizures lowering the EDs0 from 206 to 103 and 46 mg/kg, respectively. The chimney test and retention testing in mice revealed that administration of memantine at 0.5 mg/kg (i.p.) or trihexyphenidyl at 30 mg/kg (i.p.) together with valproate in doses of 130 or 103 mg/kg (i.p.), respectively,resulted in motor impairment and caused impairment of long-term memory, similar to the effects of valproate alone, when applied at its EDso against maximal electroshock. Neither memantine nor trihexyphenidyl altered the total level of valproate in plasma. It may be concluded that the potentiation of the anticonvulsant activity of valproate, by memantine and trihexyphenidyl, is not associated with a pharmacokinetic interaction. Key words--valproate, memantine, trihexyphenidyl, seizures.
Both memantine and trihexyphenidyl are used clinically in the treatment of Parkinson's disease and other kinds of rigidity, due to their anticholinergic action (Bianchine, 1985; Masuo, Enomoto and Maeno, 1986). Memantine has been shown to act as an anticonvulsant drug in different models of electricallyand chemically-induced seizures in mice, raising the threshold for electroconvulsions and protecting against the tonic hind limb extension in pentylenetetrazol-, bicuculline-, 3-mercaptopropionic acidand picrotoxin-induced seizures (Maj, Sowifiska, Baran and Sarnek, 1974; Meldrum, Turski, Schwarz, Czuczwar and Sontag, 1986). Electrophysiological studies in cultures of spinal cord nerve cells from the mouse indicated that memantine is able to reduce the frequency of action potentials in neurones firing spontaneously and to shorten the duration of strychnine-elicited bursts (Netzer and Bigalke, 1990).
*Present address: Maryland Psychiatric Research Center, University of Maryland School of Medicine, P.O. Box 21247, Baltimore, MD 21228, U.S.A. tTo whom correspondence should be addressed at present address: Vanderbilt University, Department of Neurology, School of Medicine, 2100 Pierce Ave., Nashville, TN 37212, U.S.A. NP 31/10--E
Memantine and trihexyphenidyl were reported to inhibit the activity of N-methyl-D-aspartate (NMDA) receptors (Olney, Price, Labruyere, Salles, Friedrich, Mueller and Silverman, 1987; Bormann, 1989). Previous studies demonstrated that administration of dizocilpine (MK-801), the antagonist of the NMDA receptor channel (Clineschmidt, Martin, Bunting and Papp, 1982), together with valproate or phenobarbital, markedly potentiated the protective activity against maximal electroshock-induced seizures (Urbafiska, Dziki, Kleinrok, Czuczwar, Turski, 1991). The aim of the present study was to investigate the effects of memantine and trihexyphenidyl, when combined with valproate, on maximal electroshockinduced seizures. METHODS
Animals
The experiments were carried out on male Albino Swiss mice, weighing 20-25 g. The animals were housed in colony cages with free access to food (Murigran pellets) and tap water and maintained on a natural light--dark cycle. The experimental groups, consisting of 8-10 animals, were chosen by means of a randomized schedule. The convulsion and behavioural tests were performed between 10.00 and 13.00 hr and each mouse was used only once.
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Drugs Valproic acid (sodium salt) (Depakine, Labaz, Ambarez, France), at 20-300mg/kg, memantine hydrochloride (l.3-dimethyi- 5-aminoadamantane) (Merz, Frankfurt, Germany), at 0.0035-6.0 mg/kg and trihexyphenidyl hydrochloride (Sigma, St Louis, Missouri), at 10-50 mg/kg, were dissolved in sterile saline and administered intraperitoneally (i.p.) 30 min (valproate) and 60 min (the latter drugs) before the test. The control animals were given saline, at respective times prior to the test. The injection volume was always 0.05 ml/10 g body weight. Doses of drugs refer to their free forms.
Electroconvulsions The procedure was carried out according to Swinyard, Brown and Goodman (1952). Electroshock was applied by ear-clip electrodes and generated by a stimulator (COTM, Bialystok, Poland) which delivered an alternating current (50 Hz), the duration of the stimulus being 0.2 sec. The end-point was the tonic extension of the hind limbs. To evaluate the convulsion threshold, at least 4 groups of mice, consisting of 8-10 animals, were subjected to electroshocks of various intensities. An intensity-response curve was calculated from the percentage of mice showing the endpoint in each group. The convulsion threshold was evaluated as CS50, which was the strength of current (in mA), necessary to induce tonic hindlimb extension in 50% of the mice tested. The anticonvulsant potency of valproate was expressed as the EDs0, i.e. the dose of the drug (in mg/kg) required to protect 50% of the treated mice against maximal electroshock (50 mA)-induced tonic hindlimb extension. Both CS50 and EDs0 values were calculated according to the method of Litchfield and Wilcoxon (1949).
Chimney test The effects of valproate, combined with memantine or trihexyphenidyl on motor impairment, were quantified by the chimney test of Boissier, Tardy and Diverres (1960), in which the animals have to climb up backwards in a plastic tube (3 cm inner diameter, 25 cm length). Motor impairment was indicated by the inability of the animals to climb up backwards in the tube within 60 sec and the results were shown as a percentage of animals which failed to perform the test.
Dark-avoidance acquisition and retention testing The mice were placed in an illuminated box (10 x 13 x 15cm), connected to a large dark box (25 x 20 x 15cm) which was equipped with an electric grid floor. Entrance into the dark box was punished by an electric footshock (0.6 mA for 2 sec; facilitation of acquisition). On the next day (24 hr later), the same mice were placed in the illuminated box. Mice avoiding the dark compartment for over
60 sec were considered as remembering the task. The retention was quantified as a percentage of animals avoiding the dark compartment. The step-through passive avoidance task may give the information about ability to acquire the task (learning) and to recall the task (retrieval) and may be regarded as a measure involving long-term memory (Venault, Chapouthier, de Carvalho, Simiand, Morre, Dodd and Rossier, 1986).
Determination of the level of valproate in plasma The mice were injected with either valproate and saline or valproate, in combination with memantine or trihexyphenidyl. The animals were killed by decapitation at respective times and samples of blood of approximately l ml were collected into Eppendorf tubes. Samples of blood were centrifuged at 10,000 rpm for 3 min and samples of plasma of 70 ,ul were transferred to Abbott System cartridges. The level of vaiproate in plasma was estimated by immunofluorescence, using an Abbott TDx analyzer (Abbott, Irving, Texas) and presented as the mean value + SD of at least 7 determinations.
Statistics Both CSs0 and EDs0 values and statistical analysis of the results, obtained in the electroconvulsive tests, were estimated by computer linear regression analysis, according to Litchfield and Wilcoxon (1949). Results from the passive avoidance task and chimney test were analysed statistically with the use of Fisher's exact probability test. Statistical significance was defined as P < 0.05. RESULTS
Effects of memantine and trihexyphenidyl upon the electroconvulsive threshold Memantine, administered in doses of 1.0, 2.0, 4.0 and 6.0mg/kg (i.p.), 60rain before the test, raised the electroconvulsive threshold from 12.7 to 14.0, 15.0, 16.4 and 16.7mA, respectively. Memantine, applied in doses of 0.5mg/kg (i.p.) and less, did not significantly influence the electroconvulsive threshold (Table 1). Trihexyphenidyl, up to 50 mg/kg injected (i.p.), 60 min prior to the test did not significantly alter the electroconvulsive threshold (Table 1).
Influence of memantine and trihexyphenidyl upon the protective efficacy of valproate Memantine, given at 0.0156, 0.0625, 0.125 and 0.5 mg/kg (i.p.), 60 rain before the test, when applied together with valproate (i.p. 30min prior to the test), significantly reduced the EDs0 value--from 235 to 187, 172, 164 and 130mg/kg, respectively. Trihexyphenidyl, administered (i.p.) 60min before the test, in doses of 30 and 50 mg/kg, when given together with valproate (i.p. 30 min prior to the test), potentiated the protective efficacy, decreasing the
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Valproate and antiparkinsonian drugs Table 1. Effects of memantine and trihexyphenidyl upon the electroconvulsive threshold
Treatment Saline Memantine'
Saline TrihexyphenidyP
Dose (in mg/kg)
Electroconvulsive threshold (CS50 in mA; 95% confidence limits)b
-0.5 1.0 2.0 4.0 6.0 -30.0 50.0
12.7 (I 1.9-13.6) 13.6 (I 2.5-14.8) 14.0 (13.3-14.7)* 15.0 (13.7-16.4)* 16.4 (15.5-17.4)* 16.7 (15.0-18.7)* 13.1 (I 1.8-14.5) 12.9 ( I 1.6-14.3) 13.9 (12.8-15.2)
*P < 0.05 vs compared to saline-treated group. 'Memantine and trihexyphenidyl were administered intraperitoncally, 60 min prior to the test. b'l'he CSs0 values (with 95% confidence limits) and statistical comparisons were calculated according to the method of Litchfield and Wilcoxon (1949).
ED50 value from 206 to 103 and 46 mg/kg, respectively (Table 2). Motor impairment Valproate caused marked motor impairment, as revealed by the chimney test, when applied in doses of 206 and 235 mg/kg (i.p.), 30 min before individual trials (i.e. at the dose resembling the EDs0 of vaiproate for each trial) (Table 3). Memantine, given in a dose of 0.5 mg/kg (i.p.), 60 min prior to the test, did not induce any motor impairment and trihexyphenidyl, administered in a dose of 30 mg/kg (i.p.), 60 min before the test did (Table 3). The combined treatment with memantine, at 0.5 mg/kg (i.p.) or trihexyphenidyl at 30mg/kg (i.p.) and valproate at 130 or 103 mg/kg (i.p.), respectively, resulted in motor impairment (Table 3). Dark-avoidance acquisition and retention testing Administration of valproate, in a dose equal to its EDs0 against maximal electroshock i.e. 206 or 235mg/kg (i.p.) (two independent trials), 30min Table 2. Influence of memantine and trihexyphenidyl upon the protective efficacy of valproate against maximal electrochockinduced seizures Treatment (in mg/kg) Valproate' + saline + memantine' 0.0035 0.0156 0.0625 0.125 0.5 Valproate a + saline + trihexyphenidyP 10 30 50
EDs0b of valproate (in mg/kg; 95% confidence limits) 235.4 (210.8-262.9) 237.4 (199.0-283.3) 187.6 ( 163.4-215.5)* 172.5 (141.2-210.0)* 164.2 (143.4-188.0)* 130.0 (108.6-155.5)* 206.3 (167.5-253.9) 194.7 (123.9-306.0) 103.3 (60.9-175.4)* 46.6 (25.5-85.3)*
*P < 0.05 vs compared to saline-treated group. 'Drugs were administered intraperitoneally: valproate 30min, memantine and trihexypbenidyl 60 min, before the test. bTbe data represent EDs0 values (in mg/kg; 95% confidence limits) for the protection against maximal electroshock. The EDso values and statistical comparisons were calculated according to the method of Litchfield and Wilcoxon (1949).
Table 3. Motor impairment after administration of valproate, memantine, trihexyphenidyl or combinations of valproate with one of the antiparkinsonian drugs Treatment (mg/kg)
Nb
% of animals showing motor impairmenff
Saline Valproatea (235) Valproate (130) Valproate (130) + memantine' (0.5) Memantine (0.5) Saline Valproate (206) Valproate (103) Valproate (103) + trihexypbenidyP (30) Trihexyphenidyl (30)
12 12 9
0 50* I 1.1
10 10 12 10 10
60* 10 0 50* 20
10 10
40* 50*
*P < 0.05 vs saline (Fisher's exact probability test). aDrugs were administered intraperitoneany: valproate 30min, memantine and trihexyphenidyl 60 min before the test. bNumber of animals. CEvaluated by the chimney test of Boissier et al. (1960). Mice had to climb up backwards in a plastic tube (3 era inner diameter, 25 cm length). Motor impairment was indicated when the animals were unable to climb up backwards in the tube, within 60 sec. The results concerning the motor impairment were expressed as a percentage of animals which failed to climb up during the 60 sec observation period.
before training, caused significant impairment of long-term memory, as revealed by the retention test 24 hr later (Table 4). Valproate, in a dose of 103 or 130 mg/kg (i.p.), equal to its EDs0 against maximal electroshock, when applied together with trihexyphenidyl (30 mg/kg i.p.) or memantine (0.5 mg/kg, i.p.), respectively, did not influence retention. Administration of memantine at 0.5 mg/kg (i.p.), 60 min before the training session had no effect on dark avoidance 24 hr later. Trihexyphenidyl, injected at the dose of 30 mg/kg (i.p.), 60 rain before training totally impaired retention (Table 4). The combination of memantine (0.5 mg/kg, i.p.) or trihexyphenidyl (30mg/kg, i.p.) with valproate (130 or 103 mg/kg, Table 4. Effects of valproate, memantine and trihexyphenidyl or combinations of valproate with one of the antiparkinsonian drugs on retention of a passive avoidance task in mice Treatment (mg/kg)
Nb
Retention (% of control)c
Saline Valproate' (235) Valproate (130) Valproate (130)+ memantine' (0.5) Memantine (0.5)
10 10 8 8 8
100 50* 62.5 37.5* 100
Saline Valproate (206) Valproate (103) Valproate (103)+tribexypbenidyl' (30) Trihexyphenidyl (30)
10 10 11 10 10
100 50* 90.9 0 *d 0 *a
*P < 0.05 vs saline-treated group; dp < 0.05 VS valproate (206 mg/kg)-treated group (Fisber's exact probability test). "Drugs were administered intraperitoneally: valproate 30min, memantine and trihexyphenidyl 60 min before training. bNumber of animals used. 'The retention was quantified as a percentage of mice avoiding the dark compartment. Entrance into the dark box was punished by an electric footshock (0.6 mA for 2 see). On the next day (24 hr later), the same mice were placed in the illuminated box. Mice avoiding the dark compartment for over 60 sec were considered as remembering the task.
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i.p., respectively), significantly impaired retention (Table 4). Influence of memantine or trihexyphenidyl on the level o f valproate in plasma
The total level of valproate, administered at 130 or 103 mg/kg (i.p.) (186.1 + 12.6, 150.9 + 30.1/zg/ml, respectively), in plasma was not altered in the presence of memantine, given at 0.5 mg/kg (i.p.) or trihexyphenidyl, at 30mg/kg (i.p.) (175.3+20.9, 135.5 + 10.7 ~g/ml, respectively). DISCUSSION The present studies demonstrated that memantine (but not trihexyphenidyl) exerted a distinct anticonvulsant action, elevating the threshold for electroconvulsions in mice, Moreover, both drugs when applied at doses which did not affect the electroconvulsive threshold per se, markedly potentiated the protective efficacy of valproate against maximal electroshock-induced seizures. The observed augmentation of the antiepileptic activity of valproate did not seem to be related to a pharmacokinetic interaction, in terms of total plasma levels, since the level of valproate in plasma remained unchanged in the presence of either memantine or trihexyphenidyl. Application of memantine or trihexyphenidyl, together with valproate, led to the impairment of motor activity and long-term memory, similar to the action of valproate alone when applied at the EDs0 dose against maximal electroshock, as revealed by the chimney test and retention testing in mice. The combined treatment with valproate and trihexyphenidyl worsened even the memory task, in comparison with valproate alone. Moreover trihexyphenidyl, given alone, strongly impaired long-term memory. Thus, memantine and trihexyphenidyl did not reverse the side-effects produced by valproate, as MK-801 did (Urbafiska et al., 1991). This might be explained by the fact that anticholinergic drugs are known to impair memory very potently (Izquierdo, 1989), as do NMDA antagonists like phencyclidine (DeNoble, Jones, Schaeffer and Bauerle, 1990; Parada-Turska and Turski, 1990). The mechanism of strong potentiation of protection, against maximal-electroshock-induced seizures in the case of valproate, administered together with memantine or trihexyphenidyl, might be associated with several processes. Both memantine and trihexyphenidyl are antiparkinsonian agents, displaying anticholinergic properties (Bianchine, 1985; Masuo et al., 1986). The involvement of cholinergic mechanisms in human and experimental epilepsy has been often demonstrated (see for review Turski, Ikonomidou, Turski, Bortolotto, Cavalheiro, 1989). Pilocarpine, a cholinomimetic drug, when administered systemically or intracerebrally in rodents, produced severe seizure activity (Turski, Czuczwar,
Kleinrok and Turski, 1983). Carbachol (a cholinergic agonist), injected into the amygdala, induced the development of kindled seizures (Wasterlain and Jonec, 1981). Atropine (a muscarinic antagonist) increased the latency to develop fully generalized seizures in electrical kindling (Albright, Burnham and Okazaki, 1979) and potentiated the anticonvulsant action of the NMDA receptor antagonist, 2-amino5-phosphono-pentanoic acid, against electroconvulsions (Czuczwar, Turski, Chmielewska, Turski and Kleinrok, 1985). The anticonvulsant benefits of the anticholinergic drugs mentioned above are mainly thought to be related to their interaction with the central cholinergic system. It may be suspected that potentiation of the antiepileptic properties of valproate by memantine and trihexyphenidyl, presented in this study, may result in part from their ability to block cholinergic transmission. However, based on the report indicating that antiparkinsonian agents, among them memantine and trihexyphenidyl, are phencyclidine agonists and NMDA antagonists (Olney et al., 1987), an alternative explanation may be proposed. The observed enhancement of the anticonvulsant activity of valproate might be related to their anti-NMDA properties. Recently, valproate was shown to suppress NMDA-induced, transient depolarizations in the neocortex of the rat in vitro (Zeise, Kasparow and Zieglgfinsberger, 1991), which may give a further explanation for the interaction of this antiepileptic drug with NMDA antagonists. Excitatory amino acid antagonists, especially those selective for the NMDA receptor, have potent anticonvulsant activity in a wide range of experimental seizure models. They include different types of chemically, electrically and sound induced seizures in rodents, photically-induced convulsions in baboons and myoclonus and seizures in the high-pressure neurological syndrome (see for review Chapman, 1988; Meldrum, 1991). Dizocilpine (MK-801), the most potent anticonvulsant drug among noncompetitive NMDA antagonists, has been already studied in clinical trials in epileptic patients (Troupin, Mendius, Cheng and Risinger, 1986). The present data are in agreement with the previous finding, showing that MK-801 strongly potentiated the protective efficacy of valproate and phenobarbital against maximal electroshock-induced seizures (Urbafiska et al., 1991). Consequently, the blockade of excitatory amino acid-mediated events may also be involved in the observed augmentation of the antiepileptic activity of valproate, when combined with memantine or trihexyphenidyl. Nevertheless, another possible mechanism of this interaction cannot be excluded. It was noticed that anticholinergic drugs, used in the treatment of Parkinson's disease, might improve the illness not only due to the impaired activity of the cholinergic system but also as a result of a change in the balance
Valproate and antiparkinsonian drugs between dopamine and acetylcholine, since the degeneration of the striatum in Parkinson patients leads to the deficiency of dopamine, within those structures of brain (Bianchine, 1985). Memantine was reported to induce locomotor hyperactivity in vivo and to increase the release of dopamine in vitro (Maj, 1982; Osborne, Beale, GolombiowskaNikitin and Sontag, 1982). Numerous investigations have revealed that precursors of dopamine and dopamine agonists exert anticonvulsant effects in different animal models of epilepsy (Anlezark, Marrosu and Meldrum, 1981; Kleinrok, Czuczwar, W6jcik, Przegalifiski, 1978, L6scher and Czuczwar, 1986). Therefore, the observed augmentation of the antiepileptic activity of valproate by memantine and trihexyphenidyl, might be caused by an interaction with more than one neurotransmitter system, excitatory amino acids, and/or acetylcholine and/or dopamine. The involvement of excitatory amino acid-mediated events seems to be the most probable, since MK-801 and new competitive N M D A antagonists also strongly enhanced the antiepileptic activity of valproate (Urbafiska et al., 1991; unpublished data). In conclusion, combined treatment with valproate and memantine or trihexyphenidyl, allowed a reduction of the EDs0 for valproate 2-4-fold, while the examined side-effects of this combination were comparable to those induced by valproate alone. The influence of trihexyphenidyl upon memory seemed to be exceptional but anticholinergics impair memory very potently, even at doses smaller than those used in these studies. More precise investigations of the mechanism leading to the enhanced antiepileptic activity of valproate, when administered together with memantine or trihexyphenidyl, may be a promising approach to find similar drugs, not exerting pronounced sideeffects. These data suggest that the ability to obtain drugs displaying weaker anticholinergic properties, while possessing stronger a n t i - N M D A activity may be of a clinical value. Drugs of this type might be useful in clinical practice as therapeutic agents potentiating the activity of common antiepileptics. Acknowledgement--The authors wish to thank Merz &
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