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Gamma-aminobutyric acid and mefloquine-induced seizures in mice
Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 917 – 921 www.elsevier.com/locate/pnpbp
Gamma-aminobutyric acid and mefloquine-induced seizures in mice George J. Amabeoku*, Celeste C. Farmer Division of Pharmacology, School of Pharmacy, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa Accepted 18 April 2005 Available online 11 July 2005
Abstract The role of GABAergic mechanism in the convulsant effect of mefloquine was investigated in mice. Mefloquine dose dependently induced tonic seizures in mice. Aminooxyacetic acid, diaminobutyric acid and muscimol significantly protected mice against mefloquineinduced seizures by significantly delaying the onset and decreasing the incidence of the seizures. Bicuculline and picrotoxin significantly enhanced the seizure producing effect of mefloquine and also significantly antagonised the protective effect of muscimol against the seizures. Phenobarbitone and diazepam effectively protected mice against mefloquine-induced seizures. Phenytoin did not alter mefloquine-induced seizures. These data indicate that GABA mechanisms might be involved in seizures produced by mefloquine in mice. D 2005 Elsevier Inc. All rights reserved. Keywords: GABAergic mechanism; Mefloquine; Mice; Phenytoin; Tonic seizures
1. Introduction Prophylactic antimalarial drugs have been reported, at standard dosages, to produce convulsions in healthy subjects and more frequently in subjects with a history of epilepsy (Zaccara et al., 1990). Mefloquine, a 4-aminoquinoline methanol, is used for prophylaxis of chloroquineresistant Plasmodium falciparum malaria (Rang et al., 1999a; Rosenthal and Goldsmith, 2001). However, severe neurological and psychiatric adverse effects including acute psychosis, affective disorders, acute confusional states and seizures have been reported (Piening and Young, 1996; Laffitte, 1997; Heeringa et al., 1998; Rang et al., 1999a; Rosenthal and Goldsmith, 2001). The mechanism of mefloquine-elicited convulsions has not been reported in literature. Quinine, an antimalarial agent, which is chemically related to mefloquine has been shown to cause seizures by impairing gamma aminobutyric acid (GABA) neurotransmission (Amabeoku and Chikuni, 1992; Rosenthal and Abbreviations: AOAA, aminooxyacetic acid hemihydrochloride; DABA, dl-2,4-diamino-n-butyric acid hydrochloride; GABA, gamma aminobutyric acid; GABA-T, gamma aminobutyric acid transaminase. * Corresponding author. Tel.: +27 21 959 3439; fax: +27 21 959 3407. E-mail address: [email protected] (G.J. Amabeoku). 0278-5846/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2005.04.030
Goldsmith, 2001). GABA is a major inhibitory neurotransmitter and through its receptors, GABAA receptors, is widely implicated in epilepsy (Olsen, 1981; Homeida and Cooke, 1982; De Deyn et al., 1990; Gale, 1992). This study was, therefore, designed to investigate the role of GABA mechanism in mefloquine-induced seizures by studying the effects of aminooxyacetic acid hemihydrochloride (AOAA), dl-2,4-diamino-n-butyric acid hydrochloride (DABA), muscimol, bicuculline, picrotoxin, diazepam and phenobarbitone on the seizures in mice. The role of phenytoin, a standard anticonvulsant agent, known to exert its anticonvulsant effect by blocking sodium ion entry into brain cells (Porter and Meldrum, 2001), in mefloquine seizures was also investigated in mice.
2. Materials and methods 2.1. Animals Male albino mice bought from the University of Cape Town, South Africa, and weighing 20 –30 g were used throughout the study. The animals were housed in groups of eight per cage and maintained on tap water and food ad libitum. A daily 12-h light – dark cycle was maintained.
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G.J. Amabeoku, C.C. Farmer / Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 917 – 921
Eight mice per dose of drug or control vehicle were used in the study and each animal was used for one experiment only.
3. Results
2.2. Drugs
Mefloquine (100 – 137.5 mg/kg, i.p.) elicited tonic seizures in a dose-dependent manner. The onset and incidence of the seizures shortened and increased respectively with increase in the dose of mefloquine (Table 1). The control vehicles did not alter the gross behaviour of animals, such as rearing, sniffing, grooming, exploration patterns and so on, or the convulsant effect of mefloquine. Mefloquine seizures were characterised by wild running in all directions in the cage by the animals, followed by rapid shaking of their bodies and culminating in rigid extensor spasm of their hindlimbs.
Mefloquine hydrochloride (Roche), aminooxyacetic acid hemihydrochloride (AOAA, Sigma Chemical Co.), dl-2,4-diamino-n-butyric acid hydrochloride (DABA, Sigma Chemical Co.), muscimol (Sigma Chemical Co.), picrotoxin (Sigma Chemical Co.), phenobarbitone sodium (Gardenal sodium, Rhone-Poulenc) and 5,5-diphenylhydantoin sodium salt (Phenytoin, Sigma Chemical Co.) were all dissolved in physiological saline. Diazepam (Valium, Roche) was dissolved in a minimum amount of polyethylene glycol 400 (Fluka AG, Buchs) and adjusted to the appropriate volume with physiological saline. (+)Bicuculline (Sigma Chemical Co.) was suspended in 3% (v/v) Tween 80 and adjusted to the appropriate volume with physiological saline. All drugs were administered intraperitoneally (i.p.) in a volume of 1 ml per 100 g of animal. Control animals were given equal-volume injections of appropriate solvent vehicles including Tween 80 and polyethylene glycol 400 both made up to the appropriate volumes with physiological saline. Fresh drug solutions were prepared on each day of the experiment. The drug pretreatment times prior to the injection of mefloquine were AOAA (20 min), DABA (30 min), muscimol (1 h), picrotoxin (20 min), bicuculline (10 min), phenobarbitone (10 min), diazepam (20 min) and phenytoin (30 min). The pretreatment times and doses of all the drugs used were established from preliminary studies in our laboratory. 2.3. Assessment of convulsant activity The modified method of Vellucci and Webster (1984) was used to assess the convulsant activity of mefloquine. Eight mice per dose of drug were used. Each animal was kept in a cage (25 15 15 cm) for 30 min to acclimatize to its new environment before the administration of drugs. After treatment with mefloquine, the mice were observed for seizures for 30 min. The time taken for the onset of tonic seizures and the proportion of mice convulsing were recorded. Mice that did not convulse within the 30-min period of observation were regarded as not convulsing. The behaviour of animals before the onset of seizures was also noted. Both the control and test experiments were performed between 0830 h and 1730 h in a quite room with an ambient temperature of 23 T 2 -C. 2.4. Statistical analysis The data on the onset of tonic seizures were analysed using paired Student’s t-test while the number of animals that convulsed was analysed using Chi-squared test.
3.1. Convulsant effect of mefloquine
3.2. The effects of AOAA, DABA and muscimol on mefloquine-induced seizures AOAA (10 –20 mg/kg, i.p.), DABA (8 –16 mg/kg, i.p.) and muscimol (1 – 2 mg/kg, i.p.) effectively attenuated seizures elicited by mefloquine (137.5 mg/kg, i.p.). AOAA (10 – 20 mg/kg, i.p.) significantly reduced the number of animals convulsing. 20 mg/kg (i.p.) of AOAA significantly prolonged the onset of the seizures while 10 mg/kg (i.p.) did not significantly alter the onset of seizures. DABA (8– 16 mg/kg, i.p.) significantly delayed the onset of mefloquine (137.5 mg/kg, i.p.)-induced seizures. DABA (16 mg/kg, i.p.) also significantly reduced the incidence of the seizures whereas 8 mg/kg (i.p.) only caused 37.5% reduction in the incidence. Muscimol (1mg/kg, i.p.) completely protected mice against seizures induced by mefloquine (137.5 mg/kg, i.p.) while 2 mg/kg (i.p.) profoundly reduced the number of animals convulsing and significantly delayed the onset of the seizures (Table 2). All the doses of AOAA and DABA used did not alter the gross behaviour of mice but muscimol, in all the doses used, made the animals sedentary (results not shown). 3.3. The effects of bicuculline, picrotoxin and their interactions with muscimol on mefloquine-induced seizures Mefloquine (112.5 mg/kg, i.p.) elicited tonic seizures in 37.5% of mice used. Bicuculline (4 mg/kg, i.p.) and picrotoxin (1 mg/kg, i.p.) did not produce any convulsions when used alone but slightly enhanced the exploratory behaviour of the animals. Bicuculline (4 mg/kg, i.p.) given together with 112.5 mg/kg (i.p.) of mefloquine significantly Table 1 Convulsant effect of mefloquine in mice Mefloquine (mg/kg)
No. convulsed/No. used
Onset of tonic seizures (min) mean T S.E.M.
100 112.5 125 137.5
0/8 3/8 5/8 8/8
0 4.0 3.6 1.5
1.0 0.89 0.53
G.J. Amabeoku, C.C. Farmer / Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 917 – 921
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Table 2 The effects of aminooxyacetic acid (AOAA), diaminobutyric acid (DABA) and muscimol on mefloquine-induced seizures in mice Dose (mg/kg) mefloquine
AOAA
DABA
Muscimol
No. convulsed (min)/No. used
Onset of tonic seizures (min) mean T S.E.M.
137.5 137.5 137.5 137.5 137.5 137.5 137.5
– 10 20 – – – –
– – – 8 16 – –
– – – – – 1 2
8/8 2/8++ 3/8+ 5/8 2/8++ 0/8++++ 1/8+++
1.38 2.50 4.33** 4.20** 3.50* 0** 10.0**
0.18 0.25 0.20 0.52 2.12 0
*P < 0.01, **P < 0.001 versus mefloquine (137.5 mg/kg) control, Student’s t-test. + P < 0.05, ++P < 0.01, +++P < 0.005, ++++P < 0.001 versus mefloquine (137.5 mg/kg) control, Chi-square test.
increased the number of mice convulsing to 87.5% but did not significantly affect the onset of the seizures. Muscimol (1 mg/kg, i.p.) completely antagonised seizures produced by mefloquine (137.5 mg/kg, i.p.). Bicuculline (4 mg/kg, i.p.) significantly antagonised the protective effect of muscimol (1 mg/kg, i.p.) against mefloquine (137.5 mg/kg, i.p.)induced seizures by increasing the number of mice convulsing to 62.5%. Similarly, picrotoxin (1 mg/kg, i.p.) given together with mefloquine (112.5 mg/kg, i.p.) significantly increased the number of mice convulsing to 75% but did not significantly alter the onset of the seizures. Picrotoxin (1 mg/kg, i.p.) significantly antagonised the protective effect of muscimol (1 mg/kg, i.p.) against seizures induced by mefloquine (137.5 mg/kg, i.p.) by increasing the number of animals convulsing to 62.5% (Table 3). 3.4. The effects of diazepam, phenobarbitone and phenytoin on mefloquine-induced seizures Diazepam (0.5 mg/kg, i.p.) completely protected animals against mefloquine (137.5 mg/kg, i.p.)-induced seizures. Phenobarbitone (12.5 mg/kg, i.p.) significantly delayed the onset of mefloquine (137.5 mg/kg)-induced seizures and significantly reduced the number of mice convulsing. Phenytoin (32 mg/kg, i.p.), on the other hand, did not alter the onset or incidence of mefloquine (137.5 mg/kg, i.p.)induced seizures significantly (Table 4).
4. Discussion 4.1. The effects of muscimol and bicuculline on mefloquineinduced seizures The role of GABA, a major inhibitory neurotransmitter, and its receptors, GABAA receptors, in epilepsy has been widely reported (Leidenheimer et al., 1991; Gale, 1992; Rang et al., 1999b). Blockade and stimulation of GABAA receptors in the brain have been shown to elicit and attenuate seizures respectively (Gale, 1992). The results of this study show that mefloquine in a dose-related manner induced tonic seizures in mice. Muscimol (1 mg/kg, i.p.) was shown to effectively protect mice against mefloquine (137.5 mg/kg, i.p.) seizures in this study. According to Vellucci (1989) and Rang et al. (1999b), muscimol, a selective GABA agonist, exerts its effects by stimulating GABAA receptors to mimic the effects of GABA. In the present study, bicuculline (4 mg/kg, i.p.) enhanced mefloquine (112.5 mg/kg, i.p.)-induced seizures and also antagonised the protective effect of muscimol (1 mg/kg, i.p.) against seizures produced by mefloquine (137.5 mg/kg, i.p.). Bicuculline is a potent GABAA receptor antagonist which exerts its convulsant effect by denying GABA access to its receptors (Forster, 1996; Rang et al., 1999b). These data suggest that GABAergic mechanisms may be involved in mefloquine-induced seizures. The data are also consistent
Table 3 The effects of bicuculline and picrotoxin and their interactions with muscimol on mefloquine-induced seizures in mice Dose mefloquine (mg/kg)
Bicuculline
Picrotoxin
Muscimol
No. convulsed/No. used
Onset of tonic seizures (min) mean T S.E.M.
137.5 112.5 – 112.5 137.5 137.5 – 112.5 135.7
– – 4 4 – 4 – – –
– – – – – – 1 1 1
– – – – 1 1 1 – 1
8/8 3/8 0/8 7/8‘‘ 0/8+ 5/8t 0/8 6/8‘ 5/8t
1.38 3.67 0 4.29 0r 8.0* 0 4.67 6.20*
r
p < 0.001 versus mefloquine (137.5 mg/kg) control, Student’s t-test. *p < 0.001 versus mefloquine (137.5 mg/kg) plus muscimol (1 mg/kg), Student’s t-test. + p < 0.001 versus mefloquine (137.5 mg/kg) control, Chi-square test. ‘ p < 0.05, ‘‘p < 0.01 versus mefloquine (112.5 mg/kg), Chi-square test. t p < 0.001 versus mefloquine (137.5 mg/kg) plus muscimol (1 mg/kg), Chi-square test.
0.18 0.74 0.70 0.79 1.28 0.39
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Table 4 The effects of diazepam, phenobarbitone and phenytoin on mefloquine-induced seizures in mice Dose (mg/kg) Mefloquine
Diazepam
Phenobarbitone
Phenytoin
No. convulsed/No. used
Onset of tonic seizures (min) mean T S.E.M.
137.5 137.5 137.5 137.5
– 0.5 – –
– – 12.5 –
– – – 32
8/8 0/8++ 3/8+ 8/8
2.25 0* 11.67* 2.75
0.16 0.63 0.25
*p < 0.001 versus mefloquine (137.5 mg/kg) control, Student’s t-test. + p < 0.05, ++p < 0.001 versus mefloquine (137.5 mg/kg), Chi-square test.
with the findings of Meldrum (1981) that muscimol antagonised sound-induced seizures in mice. 4.2. The effects of AOAA and DABA on mefloquine-induced seizures The present study shows that AOAA (10 – 20 mg/kg, i.p.) significantly attenuated mefloquine (137.5 mg/kg, i.p.)induced seizures. According to Wallach (1961) and Kerwin and Pycock (1978), AOAA is a potent inhibitor of GABA transaminase (GABA-T), an enzyme which metabolizes GABA. It accumulates GABA in the brain by preventing its breakdown. It is possible that the attenuation of mefloquine seizures by AOAA might be due to the increase in GABA levels in the brain. Iversen and Kelly (1975) and Horton (1989) reported that DABA is a potent and selective inhibitor of GABA reuptake which accumulates GABA in the brain. DABA (8 – 16 mg/kg, i.p.) was shown to antagonise mefloquine (137.5 mg/kg, i.p.)-induced seizures in this study. The antagonism of mefloquine seizures by DABA might be attributed to the increased level of GABA in the brain. These data further support the involvement of GABAergic mechanisms in mefloquine seizures. 4.3. The effect of picrotoxin on mefloquine-induced seizures Picrotoxin, a specific GABA antagonist (Nicoll, 2001), acts by blocking the GABAA-linked chloride ion channel which normally opens to allow increased chloride ion conductance when GABAA receptors are activated by GABA (Forster, 1996; Rang et al., 1999b). Interestingly, in the present study, picrotoxin (1 mg/kg, i.p.) effectively enhanced seizures elicited by mefloquine (112.5 mg/kg, i.p.) and also antagonised the protective effect of muscimol against mefloquine (137.5 mg/kg, i.p.)-induced seizures. These data further implicate GABAergic mechanisms in mefloquine seizures. 4.4. The effects of diazepam, phenobarbitone and phenytoin on mefloquine-induced seizures In the present study, diazepam (0.5 mg/kg., i.p.) and phenobarbitone (12.5 mg/kg, i.p.) effectively attenuated the tonic seizures elicited by mefloquine (137.5 mg/kg, i.p.). Post-synaptic GABAA receptors are functionally linked to barbiturate receptors, benzodiazepine receptors and chloride
channels to form the GABA-chloride ionophore complex which is involved in the modulation of GABAergic inhibitory transmission (Olsen, 1981; Porter and Meldrum, 2001). Barbiturates and benzodiazepines are thought to stimulate their respective receptors in the ionophore complex to increase chloride flux through chloride channels at GABAA receptors sites to enhance GABAergic functions (Olsen, 1981; Porter and Meldrum, 2001). It is significant, therefore, that phenobarbitone and diazepam, standard antiepileptic drugs, attenuated mefloquine-induced seizures in this study. Phenytoin, a standard antiepileptic drug, is thought to exert its antiepileptic effect by blocking sodium ion channels and inhibiting the generation of repetitive action potentials (Rang et al., 1999c; Porter and Meldrum, 2001). In the present study, phenytoin did not affect the mefloquine seizures in mice at all the doses used. The data obtained in this study indicate that mefloquine may be interfering with GABA mechanism in the central nervous system to produce seizures.
5. Conclusions The present study shows that AOAA, DABA, muscimol, diazepam and phenobarbitone, which enhance GABA neurotransmission, attenuated mefloquine seizures while those such as bicuculline and picrotoxin, which block GABA neurotransmission enhanced the seizures. Conversely, phenytoin, known to exert its antiepileptic effect by blocking sodium ion entry into the brain cells, did not alter mefloquine-induced seizures. These findings, therefore, suggest that GABA mechanism may be involved in mefloquine seizures in mice. Acknowledgements We wish to thank Roche Products (Pty) Ltd., South Africa, for their generous gift of mefloquine hydrochloride. We also thank Mr. B. Minnis for his technical assistance and for the welfare of the animals. References Amabeoku, G.J., Chikuni, O., 1992. Effects of some GABAergic agents on quinine-induced seizures in mice. Experientia 48, 659 – 662.
G.J. Amabeoku, C.C. Farmer / Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 917 – 921 De Deyn, P.P., Marescau, B., MacDonald, R.I., 1990. Epilepsy and the GABA-hypothesis, a brief review and some examples. Acta Neurol. Belg. 90, 65 – 81. Forster, R.W., 1996. Basic Pharmacology, Fourth edR Butterworth, Oxford. Chapter 5. Gale, K., 1992. GABA and epilepsy: basic concepts from preclinical research. Epilepsia 33 (Suppl. 5), S3 – S12. Heeringa, M., Kluster, J.A., Meyboom, R.H., Bouvy, M., 1998. Convulsions during prophylactic use of mefloquine. Ned. Tijdschr. Geneeskd. 145 (45), 2477 – 2480. Homeida, A.M., Cooke, R.G., 1982. Anticonvulsant activity of diazepam and clonidinme on metaldehyde-induced seizures in mice: effects on brain gamma-aminobutyric acid concentrations and monoamine oxidase activity. J. Vet. Pharmac. Ther. 5, 187 – 190. Horton, R.W., 1989. Amino acid neurotransmitter. In: Webster, R.A., Jordan, C.C. (Eds.), Neurotransmitters, Drugs and Diseases. Backwell, London, pp. 165 – 170. Iversen, L., Kelly, J.S., 1975. Uptake and metabolism of gammaaminobutyric acid by neurons and glial cells. Biochem. Pharmacol. 24, 933 – 938. Kerwin, R., Pycock, C., 1978. Baclofen (h-p-chlorophenyl-g-aminobutyric acid) enhances [3H] gamma-aminobutyric acid release from rat globus pallidus in vitro. J. Pharm. Pharmacol. 30, 622 – 627. Laffitte, E., 1997. Convulsions and mefloquine prophylaxis. Med. Trop. 53 (3), 307. Leidenheimer, N.J., Browning, M.D., Harris, R.A., 1991. GABAA receptor phosphorylation: multiple sites, actions and artifacts. Trends Pharmacol. Sci. 12, 84 – 87. Meldrum, B.S., 1981. GABA agonists as antiepileptic agents. Adv. Biochem. Psychopharmacol. 26, 207 – 217.
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Nicoll, R.A., 2001. Introduction to pharmacology of CNS drugs. In: Katzung, B.G. (Ed.), Basic and Clinical Pharmacology. McGraw-Hill and Lange, San Francisco, pp. 352 – 353. Olsen, B.W.J., 1981. GABA-benzodiazepine-barbiturate receptor interactions. J. Neurochem. 37, 1 – 3. Piening, R.B., Young, S.A., 1996. Mefloquine-induced psychosis. Ann. Emerg. Med. 27 (6), 792 – 793. Porter, R.J., Meldrum, B.S., 2001. Antiseizure drugs. In: Katzung, B.G. (Ed.), Basic and Clinical Pharmacology. McGraw-Hill and Lange, San Francisco, pp. 397 – 398. Rang, H.R., Dale, M.M., Ritter, J.M., 1999a. Pharmacology, Fourth edR Churchill Livingstone, Edinburgh. Chapter 46. Rang, H.R., Dale, M.M., Ritter, J.M., 1999b. Pharmacology, Fourth edR Churchill Livingstone, Edinburgh. Chapter 29. Rang, H.R., Dale, M.M., Ritter, J.M., 1999c. Pharmacology, Fourth edR Churchill Livingstone, Edinburgh. Chapter 36. Rosenthal, P.J., Goldsmith, R.S., 2001. Antiprotozoal drugs. In: Katzung, B.G. (Ed.), Basic and Clinical Pharmacology. McGraw-Hill and Lange, San Francisco, pp. 888 – 889. Vellucci, S.V., 1989. Anxiety. In: Webster, R.A., Jordan, C.C. (Eds.), Neurotransmitters, Drugs and Diseases. Blackwell, London, pp. 402 – 403. Vellucci, S.V., Webster, R.A., 1984. Antagonism of caffeine-induced seizures in mice by Ro 15-1788. Eur. J. Pharmacol. 97, 289 – 293. Wallach, D.P., 1961. Studies on the pathway: 1. The inhibition of gammaaminobutyric acid a-ketoglutamic acid transaminase in vitro and in vivo by 4-7524 (Aminooxyacetic acid). Biochem. Pharmacol. 5, 323 – 331. Zaccara, G., Muscas, G.C., Messon, A., 1990. Clinical features, pathogenesis and management of drug-induced seizures. Drug Safety 5 (2), 109 – 151.
Gamma-aminobutyric acid and mefloquine-induced seizures in mice George J. Amabeoku*, Celeste C. Farmer Division of Pharmacology, School of Pharmacy, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa Accepted 18 April 2005 Available online 11 July 2005
Abstract The role of GABAergic mechanism in the convulsant effect of mefloquine was investigated in mice. Mefloquine dose dependently induced tonic seizures in mice. Aminooxyacetic acid, diaminobutyric acid and muscimol significantly protected mice against mefloquineinduced seizures by significantly delaying the onset and decreasing the incidence of the seizures. Bicuculline and picrotoxin significantly enhanced the seizure producing effect of mefloquine and also significantly antagonised the protective effect of muscimol against the seizures. Phenobarbitone and diazepam effectively protected mice against mefloquine-induced seizures. Phenytoin did not alter mefloquine-induced seizures. These data indicate that GABA mechanisms might be involved in seizures produced by mefloquine in mice. D 2005 Elsevier Inc. All rights reserved. Keywords: GABAergic mechanism; Mefloquine; Mice; Phenytoin; Tonic seizures
1. Introduction Prophylactic antimalarial drugs have been reported, at standard dosages, to produce convulsions in healthy subjects and more frequently in subjects with a history of epilepsy (Zaccara et al., 1990). Mefloquine, a 4-aminoquinoline methanol, is used for prophylaxis of chloroquineresistant Plasmodium falciparum malaria (Rang et al., 1999a; Rosenthal and Goldsmith, 2001). However, severe neurological and psychiatric adverse effects including acute psychosis, affective disorders, acute confusional states and seizures have been reported (Piening and Young, 1996; Laffitte, 1997; Heeringa et al., 1998; Rang et al., 1999a; Rosenthal and Goldsmith, 2001). The mechanism of mefloquine-elicited convulsions has not been reported in literature. Quinine, an antimalarial agent, which is chemically related to mefloquine has been shown to cause seizures by impairing gamma aminobutyric acid (GABA) neurotransmission (Amabeoku and Chikuni, 1992; Rosenthal and Abbreviations: AOAA, aminooxyacetic acid hemihydrochloride; DABA, dl-2,4-diamino-n-butyric acid hydrochloride; GABA, gamma aminobutyric acid; GABA-T, gamma aminobutyric acid transaminase. * Corresponding author. Tel.: +27 21 959 3439; fax: +27 21 959 3407. E-mail address: [email protected] (G.J. Amabeoku). 0278-5846/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2005.04.030
Goldsmith, 2001). GABA is a major inhibitory neurotransmitter and through its receptors, GABAA receptors, is widely implicated in epilepsy (Olsen, 1981; Homeida and Cooke, 1982; De Deyn et al., 1990; Gale, 1992). This study was, therefore, designed to investigate the role of GABA mechanism in mefloquine-induced seizures by studying the effects of aminooxyacetic acid hemihydrochloride (AOAA), dl-2,4-diamino-n-butyric acid hydrochloride (DABA), muscimol, bicuculline, picrotoxin, diazepam and phenobarbitone on the seizures in mice. The role of phenytoin, a standard anticonvulsant agent, known to exert its anticonvulsant effect by blocking sodium ion entry into brain cells (Porter and Meldrum, 2001), in mefloquine seizures was also investigated in mice.
2. Materials and methods 2.1. Animals Male albino mice bought from the University of Cape Town, South Africa, and weighing 20 –30 g were used throughout the study. The animals were housed in groups of eight per cage and maintained on tap water and food ad libitum. A daily 12-h light – dark cycle was maintained.
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G.J. Amabeoku, C.C. Farmer / Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 917 – 921
Eight mice per dose of drug or control vehicle were used in the study and each animal was used for one experiment only.
3. Results
2.2. Drugs
Mefloquine (100 – 137.5 mg/kg, i.p.) elicited tonic seizures in a dose-dependent manner. The onset and incidence of the seizures shortened and increased respectively with increase in the dose of mefloquine (Table 1). The control vehicles did not alter the gross behaviour of animals, such as rearing, sniffing, grooming, exploration patterns and so on, or the convulsant effect of mefloquine. Mefloquine seizures were characterised by wild running in all directions in the cage by the animals, followed by rapid shaking of their bodies and culminating in rigid extensor spasm of their hindlimbs.
Mefloquine hydrochloride (Roche), aminooxyacetic acid hemihydrochloride (AOAA, Sigma Chemical Co.), dl-2,4-diamino-n-butyric acid hydrochloride (DABA, Sigma Chemical Co.), muscimol (Sigma Chemical Co.), picrotoxin (Sigma Chemical Co.), phenobarbitone sodium (Gardenal sodium, Rhone-Poulenc) and 5,5-diphenylhydantoin sodium salt (Phenytoin, Sigma Chemical Co.) were all dissolved in physiological saline. Diazepam (Valium, Roche) was dissolved in a minimum amount of polyethylene glycol 400 (Fluka AG, Buchs) and adjusted to the appropriate volume with physiological saline. (+)Bicuculline (Sigma Chemical Co.) was suspended in 3% (v/v) Tween 80 and adjusted to the appropriate volume with physiological saline. All drugs were administered intraperitoneally (i.p.) in a volume of 1 ml per 100 g of animal. Control animals were given equal-volume injections of appropriate solvent vehicles including Tween 80 and polyethylene glycol 400 both made up to the appropriate volumes with physiological saline. Fresh drug solutions were prepared on each day of the experiment. The drug pretreatment times prior to the injection of mefloquine were AOAA (20 min), DABA (30 min), muscimol (1 h), picrotoxin (20 min), bicuculline (10 min), phenobarbitone (10 min), diazepam (20 min) and phenytoin (30 min). The pretreatment times and doses of all the drugs used were established from preliminary studies in our laboratory. 2.3. Assessment of convulsant activity The modified method of Vellucci and Webster (1984) was used to assess the convulsant activity of mefloquine. Eight mice per dose of drug were used. Each animal was kept in a cage (25 15 15 cm) for 30 min to acclimatize to its new environment before the administration of drugs. After treatment with mefloquine, the mice were observed for seizures for 30 min. The time taken for the onset of tonic seizures and the proportion of mice convulsing were recorded. Mice that did not convulse within the 30-min period of observation were regarded as not convulsing. The behaviour of animals before the onset of seizures was also noted. Both the control and test experiments were performed between 0830 h and 1730 h in a quite room with an ambient temperature of 23 T 2 -C. 2.4. Statistical analysis The data on the onset of tonic seizures were analysed using paired Student’s t-test while the number of animals that convulsed was analysed using Chi-squared test.
3.1. Convulsant effect of mefloquine
3.2. The effects of AOAA, DABA and muscimol on mefloquine-induced seizures AOAA (10 –20 mg/kg, i.p.), DABA (8 –16 mg/kg, i.p.) and muscimol (1 – 2 mg/kg, i.p.) effectively attenuated seizures elicited by mefloquine (137.5 mg/kg, i.p.). AOAA (10 – 20 mg/kg, i.p.) significantly reduced the number of animals convulsing. 20 mg/kg (i.p.) of AOAA significantly prolonged the onset of the seizures while 10 mg/kg (i.p.) did not significantly alter the onset of seizures. DABA (8– 16 mg/kg, i.p.) significantly delayed the onset of mefloquine (137.5 mg/kg, i.p.)-induced seizures. DABA (16 mg/kg, i.p.) also significantly reduced the incidence of the seizures whereas 8 mg/kg (i.p.) only caused 37.5% reduction in the incidence. Muscimol (1mg/kg, i.p.) completely protected mice against seizures induced by mefloquine (137.5 mg/kg, i.p.) while 2 mg/kg (i.p.) profoundly reduced the number of animals convulsing and significantly delayed the onset of the seizures (Table 2). All the doses of AOAA and DABA used did not alter the gross behaviour of mice but muscimol, in all the doses used, made the animals sedentary (results not shown). 3.3. The effects of bicuculline, picrotoxin and their interactions with muscimol on mefloquine-induced seizures Mefloquine (112.5 mg/kg, i.p.) elicited tonic seizures in 37.5% of mice used. Bicuculline (4 mg/kg, i.p.) and picrotoxin (1 mg/kg, i.p.) did not produce any convulsions when used alone but slightly enhanced the exploratory behaviour of the animals. Bicuculline (4 mg/kg, i.p.) given together with 112.5 mg/kg (i.p.) of mefloquine significantly Table 1 Convulsant effect of mefloquine in mice Mefloquine (mg/kg)
No. convulsed/No. used
Onset of tonic seizures (min) mean T S.E.M.
100 112.5 125 137.5
0/8 3/8 5/8 8/8
0 4.0 3.6 1.5
1.0 0.89 0.53
G.J. Amabeoku, C.C. Farmer / Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 917 – 921
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Table 2 The effects of aminooxyacetic acid (AOAA), diaminobutyric acid (DABA) and muscimol on mefloquine-induced seizures in mice Dose (mg/kg) mefloquine
AOAA
DABA
Muscimol
No. convulsed (min)/No. used
Onset of tonic seizures (min) mean T S.E.M.
137.5 137.5 137.5 137.5 137.5 137.5 137.5
– 10 20 – – – –
– – – 8 16 – –
– – – – – 1 2
8/8 2/8++ 3/8+ 5/8 2/8++ 0/8++++ 1/8+++
1.38 2.50 4.33** 4.20** 3.50* 0** 10.0**
0.18 0.25 0.20 0.52 2.12 0
*P < 0.01, **P < 0.001 versus mefloquine (137.5 mg/kg) control, Student’s t-test. + P < 0.05, ++P < 0.01, +++P < 0.005, ++++P < 0.001 versus mefloquine (137.5 mg/kg) control, Chi-square test.
increased the number of mice convulsing to 87.5% but did not significantly affect the onset of the seizures. Muscimol (1 mg/kg, i.p.) completely antagonised seizures produced by mefloquine (137.5 mg/kg, i.p.). Bicuculline (4 mg/kg, i.p.) significantly antagonised the protective effect of muscimol (1 mg/kg, i.p.) against mefloquine (137.5 mg/kg, i.p.)induced seizures by increasing the number of mice convulsing to 62.5%. Similarly, picrotoxin (1 mg/kg, i.p.) given together with mefloquine (112.5 mg/kg, i.p.) significantly increased the number of mice convulsing to 75% but did not significantly alter the onset of the seizures. Picrotoxin (1 mg/kg, i.p.) significantly antagonised the protective effect of muscimol (1 mg/kg, i.p.) against seizures induced by mefloquine (137.5 mg/kg, i.p.) by increasing the number of animals convulsing to 62.5% (Table 3). 3.4. The effects of diazepam, phenobarbitone and phenytoin on mefloquine-induced seizures Diazepam (0.5 mg/kg, i.p.) completely protected animals against mefloquine (137.5 mg/kg, i.p.)-induced seizures. Phenobarbitone (12.5 mg/kg, i.p.) significantly delayed the onset of mefloquine (137.5 mg/kg)-induced seizures and significantly reduced the number of mice convulsing. Phenytoin (32 mg/kg, i.p.), on the other hand, did not alter the onset or incidence of mefloquine (137.5 mg/kg, i.p.)induced seizures significantly (Table 4).
4. Discussion 4.1. The effects of muscimol and bicuculline on mefloquineinduced seizures The role of GABA, a major inhibitory neurotransmitter, and its receptors, GABAA receptors, in epilepsy has been widely reported (Leidenheimer et al., 1991; Gale, 1992; Rang et al., 1999b). Blockade and stimulation of GABAA receptors in the brain have been shown to elicit and attenuate seizures respectively (Gale, 1992). The results of this study show that mefloquine in a dose-related manner induced tonic seizures in mice. Muscimol (1 mg/kg, i.p.) was shown to effectively protect mice against mefloquine (137.5 mg/kg, i.p.) seizures in this study. According to Vellucci (1989) and Rang et al. (1999b), muscimol, a selective GABA agonist, exerts its effects by stimulating GABAA receptors to mimic the effects of GABA. In the present study, bicuculline (4 mg/kg, i.p.) enhanced mefloquine (112.5 mg/kg, i.p.)-induced seizures and also antagonised the protective effect of muscimol (1 mg/kg, i.p.) against seizures produced by mefloquine (137.5 mg/kg, i.p.). Bicuculline is a potent GABAA receptor antagonist which exerts its convulsant effect by denying GABA access to its receptors (Forster, 1996; Rang et al., 1999b). These data suggest that GABAergic mechanisms may be involved in mefloquine-induced seizures. The data are also consistent
Table 3 The effects of bicuculline and picrotoxin and their interactions with muscimol on mefloquine-induced seizures in mice Dose mefloquine (mg/kg)
Bicuculline
Picrotoxin
Muscimol
No. convulsed/No. used
Onset of tonic seizures (min) mean T S.E.M.
137.5 112.5 – 112.5 137.5 137.5 – 112.5 135.7
– – 4 4 – 4 – – –
– – – – – – 1 1 1
– – – – 1 1 1 – 1
8/8 3/8 0/8 7/8‘‘ 0/8+ 5/8t 0/8 6/8‘ 5/8t
1.38 3.67 0 4.29 0r 8.0* 0 4.67 6.20*
r
p < 0.001 versus mefloquine (137.5 mg/kg) control, Student’s t-test. *p < 0.001 versus mefloquine (137.5 mg/kg) plus muscimol (1 mg/kg), Student’s t-test. + p < 0.001 versus mefloquine (137.5 mg/kg) control, Chi-square test. ‘ p < 0.05, ‘‘p < 0.01 versus mefloquine (112.5 mg/kg), Chi-square test. t p < 0.001 versus mefloquine (137.5 mg/kg) plus muscimol (1 mg/kg), Chi-square test.
0.18 0.74 0.70 0.79 1.28 0.39
920
G.J. Amabeoku, C.C. Farmer / Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 917 – 921
Table 4 The effects of diazepam, phenobarbitone and phenytoin on mefloquine-induced seizures in mice Dose (mg/kg) Mefloquine
Diazepam
Phenobarbitone
Phenytoin
No. convulsed/No. used
Onset of tonic seizures (min) mean T S.E.M.
137.5 137.5 137.5 137.5
– 0.5 – –
– – 12.5 –
– – – 32
8/8 0/8++ 3/8+ 8/8
2.25 0* 11.67* 2.75
0.16 0.63 0.25
*p < 0.001 versus mefloquine (137.5 mg/kg) control, Student’s t-test. + p < 0.05, ++p < 0.001 versus mefloquine (137.5 mg/kg), Chi-square test.
with the findings of Meldrum (1981) that muscimol antagonised sound-induced seizures in mice. 4.2. The effects of AOAA and DABA on mefloquine-induced seizures The present study shows that AOAA (10 – 20 mg/kg, i.p.) significantly attenuated mefloquine (137.5 mg/kg, i.p.)induced seizures. According to Wallach (1961) and Kerwin and Pycock (1978), AOAA is a potent inhibitor of GABA transaminase (GABA-T), an enzyme which metabolizes GABA. It accumulates GABA in the brain by preventing its breakdown. It is possible that the attenuation of mefloquine seizures by AOAA might be due to the increase in GABA levels in the brain. Iversen and Kelly (1975) and Horton (1989) reported that DABA is a potent and selective inhibitor of GABA reuptake which accumulates GABA in the brain. DABA (8 – 16 mg/kg, i.p.) was shown to antagonise mefloquine (137.5 mg/kg, i.p.)-induced seizures in this study. The antagonism of mefloquine seizures by DABA might be attributed to the increased level of GABA in the brain. These data further support the involvement of GABAergic mechanisms in mefloquine seizures. 4.3. The effect of picrotoxin on mefloquine-induced seizures Picrotoxin, a specific GABA antagonist (Nicoll, 2001), acts by blocking the GABAA-linked chloride ion channel which normally opens to allow increased chloride ion conductance when GABAA receptors are activated by GABA (Forster, 1996; Rang et al., 1999b). Interestingly, in the present study, picrotoxin (1 mg/kg, i.p.) effectively enhanced seizures elicited by mefloquine (112.5 mg/kg, i.p.) and also antagonised the protective effect of muscimol against mefloquine (137.5 mg/kg, i.p.)-induced seizures. These data further implicate GABAergic mechanisms in mefloquine seizures. 4.4. The effects of diazepam, phenobarbitone and phenytoin on mefloquine-induced seizures In the present study, diazepam (0.5 mg/kg., i.p.) and phenobarbitone (12.5 mg/kg, i.p.) effectively attenuated the tonic seizures elicited by mefloquine (137.5 mg/kg, i.p.). Post-synaptic GABAA receptors are functionally linked to barbiturate receptors, benzodiazepine receptors and chloride
channels to form the GABA-chloride ionophore complex which is involved in the modulation of GABAergic inhibitory transmission (Olsen, 1981; Porter and Meldrum, 2001). Barbiturates and benzodiazepines are thought to stimulate their respective receptors in the ionophore complex to increase chloride flux through chloride channels at GABAA receptors sites to enhance GABAergic functions (Olsen, 1981; Porter and Meldrum, 2001). It is significant, therefore, that phenobarbitone and diazepam, standard antiepileptic drugs, attenuated mefloquine-induced seizures in this study. Phenytoin, a standard antiepileptic drug, is thought to exert its antiepileptic effect by blocking sodium ion channels and inhibiting the generation of repetitive action potentials (Rang et al., 1999c; Porter and Meldrum, 2001). In the present study, phenytoin did not affect the mefloquine seizures in mice at all the doses used. The data obtained in this study indicate that mefloquine may be interfering with GABA mechanism in the central nervous system to produce seizures.
5. Conclusions The present study shows that AOAA, DABA, muscimol, diazepam and phenobarbitone, which enhance GABA neurotransmission, attenuated mefloquine seizures while those such as bicuculline and picrotoxin, which block GABA neurotransmission enhanced the seizures. Conversely, phenytoin, known to exert its antiepileptic effect by blocking sodium ion entry into the brain cells, did not alter mefloquine-induced seizures. These findings, therefore, suggest that GABA mechanism may be involved in mefloquine seizures in mice. Acknowledgements We wish to thank Roche Products (Pty) Ltd., South Africa, for their generous gift of mefloquine hydrochloride. We also thank Mr. B. Minnis for his technical assistance and for the welfare of the animals. References Amabeoku, G.J., Chikuni, O., 1992. Effects of some GABAergic agents on quinine-induced seizures in mice. Experientia 48, 659 – 662.
G.J. Amabeoku, C.C. Farmer / Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 917 – 921 De Deyn, P.P., Marescau, B., MacDonald, R.I., 1990. Epilepsy and the GABA-hypothesis, a brief review and some examples. Acta Neurol. Belg. 90, 65 – 81. Forster, R.W., 1996. Basic Pharmacology, Fourth edR Butterworth, Oxford. Chapter 5. Gale, K., 1992. GABA and epilepsy: basic concepts from preclinical research. Epilepsia 33 (Suppl. 5), S3 – S12. Heeringa, M., Kluster, J.A., Meyboom, R.H., Bouvy, M., 1998. Convulsions during prophylactic use of mefloquine. Ned. Tijdschr. Geneeskd. 145 (45), 2477 – 2480. Homeida, A.M., Cooke, R.G., 1982. Anticonvulsant activity of diazepam and clonidinme on metaldehyde-induced seizures in mice: effects on brain gamma-aminobutyric acid concentrations and monoamine oxidase activity. J. Vet. Pharmac. Ther. 5, 187 – 190. Horton, R.W., 1989. Amino acid neurotransmitter. In: Webster, R.A., Jordan, C.C. (Eds.), Neurotransmitters, Drugs and Diseases. Backwell, London, pp. 165 – 170. Iversen, L., Kelly, J.S., 1975. Uptake and metabolism of gammaaminobutyric acid by neurons and glial cells. Biochem. Pharmacol. 24, 933 – 938. Kerwin, R., Pycock, C., 1978. Baclofen (h-p-chlorophenyl-g-aminobutyric acid) enhances [3H] gamma-aminobutyric acid release from rat globus pallidus in vitro. J. Pharm. Pharmacol. 30, 622 – 627. Laffitte, E., 1997. Convulsions and mefloquine prophylaxis. Med. Trop. 53 (3), 307. Leidenheimer, N.J., Browning, M.D., Harris, R.A., 1991. GABAA receptor phosphorylation: multiple sites, actions and artifacts. Trends Pharmacol. Sci. 12, 84 – 87. Meldrum, B.S., 1981. GABA agonists as antiepileptic agents. Adv. Biochem. Psychopharmacol. 26, 207 – 217.
921
Nicoll, R.A., 2001. Introduction to pharmacology of CNS drugs. In: Katzung, B.G. (Ed.), Basic and Clinical Pharmacology. McGraw-Hill and Lange, San Francisco, pp. 352 – 353. Olsen, B.W.J., 1981. GABA-benzodiazepine-barbiturate receptor interactions. J. Neurochem. 37, 1 – 3. Piening, R.B., Young, S.A., 1996. Mefloquine-induced psychosis. Ann. Emerg. Med. 27 (6), 792 – 793. Porter, R.J., Meldrum, B.S., 2001. Antiseizure drugs. In: Katzung, B.G. (Ed.), Basic and Clinical Pharmacology. McGraw-Hill and Lange, San Francisco, pp. 397 – 398. Rang, H.R., Dale, M.M., Ritter, J.M., 1999a. Pharmacology, Fourth edR Churchill Livingstone, Edinburgh. Chapter 46. Rang, H.R., Dale, M.M., Ritter, J.M., 1999b. Pharmacology, Fourth edR Churchill Livingstone, Edinburgh. Chapter 29. Rang, H.R., Dale, M.M., Ritter, J.M., 1999c. Pharmacology, Fourth edR Churchill Livingstone, Edinburgh. Chapter 36. Rosenthal, P.J., Goldsmith, R.S., 2001. Antiprotozoal drugs. In: Katzung, B.G. (Ed.), Basic and Clinical Pharmacology. McGraw-Hill and Lange, San Francisco, pp. 888 – 889. Vellucci, S.V., 1989. Anxiety. In: Webster, R.A., Jordan, C.C. (Eds.), Neurotransmitters, Drugs and Diseases. Blackwell, London, pp. 402 – 403. Vellucci, S.V., Webster, R.A., 1984. Antagonism of caffeine-induced seizures in mice by Ro 15-1788. Eur. J. Pharmacol. 97, 289 – 293. Wallach, D.P., 1961. Studies on the pathway: 1. The inhibition of gammaaminobutyric acid a-ketoglutamic acid transaminase in vitro and in vivo by 4-7524 (Aminooxyacetic acid). Biochem. Pharmacol. 5, 323 – 331. Zaccara, G., Muscas, G.C., Messon, A., 1990. Clinical features, pathogenesis and management of drug-induced seizures. Drug Safety 5 (2), 109 – 151.