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Influence of antazoline and ketotifen on the anticonvulsant activity of conventional antiepileptics against maximal electroshock in mice
European Neuropsychopharmacology 14 (2004) 307 – 318 www.elsevier.com/locate/euroneuro
Influence of antazoline and ketotifen on the anticonvulsant activity of conventional antiepileptics against maximal electroshock in mice Mariusz S´wia˛der a, Marian Wielosz a, StanislCaw J. Czuczwar b,c,* a
Department of Pharmacology and Toxicology, Medical University, 20-090 Lublin, Jaczewskiego 8, Poland b Department of Pathophysiology, Medical University, 20-090 Lublin, Jaczewskiego 8, Poland c Isotope Laboratory, Institute of Agricultural Medicine, 20-950 Lublin, Jaczewskiego 2, Poland Received 25 March 2003; received in revised form 15 July 2003; accepted 16 September 2003
Abstract Experimental studies have indicated that the central histaminergic system plays an important role in the inhibition of seizures through the stimulation of histamine H1 receptors. H1 receptor antagonists, including classical antiallergic drugs, occasionally may induce convulsions in healthy children and patients with epilepsy. The purpose of this study was to investigate the effects of antazoline and ketotifen (two H1 receptor antagonists) on the anticonvulsant activity of antiepileptic drugs against maximal electroshock (MES)-induced convulsions in mice. The following antiepileptic drugs were used: valproate, carbamazepine, diphenylhydantoin and phenobarbital. In addition, the effects of antiepileptic drugs alone or in combination with antazoline or ketotifen were studied on long-term memory (tested in the passive avoidance task) and motor performance (evaluated in the chimney test), acutely and after 7-day treatment with these H1 receptor antagonists. The influence of antazoline and ketotifen on the free plasma and brain levels of the antiepileptics was also evaluated. Antazoline (at 0.5 mg/kg), given acutely and after 7-day treatment, significantly diminished the electroconvulsive threshold. Similarly, ketotifen, after acute and chronic doses of 8 mg/kg markedly reduced the threshold for electroconvulsions. In both cases, antazoline and ketotifen were without effect upon this parameter at lower doses. Antazoline (0.25 mg/kg) significantly raised the ED50 value of carbamazepine against MES (both, acutely and after 7-day treatment). Furthermore antazoline (0.25 mg/kg) also reduced the anticonvulsant activity of diphenylhydantoin, but only after repeated administration, without modifying the brain and free plasma level of this drug. Moreover, valproate and phenobarbital did not change their protective activity when combined with antazoline. Ketotifen (4 mg/kg) possessed a biphasic action, acutely it enhanced the anticonvulsant action of carbamazepine and phenobarbital while, following 7-day treatment, reduced the antiseizure activity of carbamazepine. Ketotifen did not affect the free plasma or brain levels of antiepileptics tested. Only acute antazoline (0.25 mg/kg) applied with valproate impaired the performance of mice evaluated in the chimney test. Ketotifen (4 mg/ kg) co-administered with conventional antiepileptic drugs impaired motor coordination in mice treated with valproate, phenobarbital or diphenylhydantoin. Acute and chronic antazoline (0.25 mg/kg) alone or in combination with antiepileptic drugs did not disturb long-term memory, tested in the passive avoidance task. Similarly, ketotifen (4 mg/kg) did not impair long-term memory, acutely and after 7-day treatment. However, valproate alone or in combination with chronic ketotifen (4 mg/kg) worsened long-term memory. The results of this study indicate that H1 receptor antagonists, crossing the blood brain barrier, should be used with caution in epileptic patients. This is because antazoline reduced the protective potential of diphenylhydantoin and carbamazepine. Also, ketotifen reduced the protection offered by carbamazepine and elevated the adverse activity of diphenylhydantoin, phenobarbital and valproate. D 2003 Elsevier B.V./ECNP. All rights reserved. Keywords: Antazoline; Ketotifen; Antiepileptic drugs; Electroshock maximal; Drug interactions; Seizures
1. Introduction Several lines of evidence suggest that central histaminergic neuron system plays an important role in inhibition * Corresponding author. Department of Pathophysiology, Medical University, 20-090 Lublin, Jaczewskiego 8, Poland. Tel.: +48-81-7425837; fax: +48-81-742-5828. E-mail address: [email protected] (S.J. Czuczwar). 0924-977X/$ - see front matter D 2003 Elsevier B.V./ECNP. All rights reserved. doi:10.1016/j.euroneuro.2003.09.005
of convulsions in mammals (Gerald and Richter, 1976; Onodera and Watanabe, 1995; Onodera et al., 1994; Scherkl et al., 1991a,b). This biogenic amine in the central nervous system acts via four subtypes of receptors: postsynaptic H1 and H2, presynaptic H3 and recently discovered H4 receptors. Tuomisto and Tacke (1986) have shown that metoprine, which raises brain histamine levels, inhibits hindlimb extension in maximal electroshock (MES) in rats, while Onodera et al. (1992) have found that it reduces
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audiogenic seizures in genetically epilepsy-prone rats. In mice, the anticonvulsant effects of L-histidine, a precursor of brain histamine, on clonic and convulsive coma phases are dependent on the brain histamine, and mediated through the central H1 receptors (Yokoyama et al., 1992). Also, thioperamide, H3 receptor antagonist, which elevated histamine levels in the brain, significantly and dose dependently inhibited electroconvulsions (Yokoyama et al., 1993a,b,c), amygdala-kindled seizures (Kakinoki et al., 1998) and pentylenetetrazole-induced convulsions (Vohora et al., 2000). Generally, the administration of first generation H1 receptor antagonists or H3 receptor agonists blocks the anticonvulsant effects of these pharmacological manipulations, which increased brain histamine (e.g. thioperamide, L -histidine). Moreover, dimethindene and promethazine diminish pentylenetetrazol seizure threshold (Scherkl et al., 1991b), while pyrilamine blocks the beneficial effects of L-histidine (Yokoyama et al., 1992). The intracerebroventricular administration of histamine and the selective H1 receptor agonist, 2-thizolylthylamine, inhibited electroshock- and pentylenetetrazol-induced seizures, whereas ketotifen and pyrilamine antagonized their effects (Yokoyama et al., 1994; for review see Sangalli, 1997). Furthermore, pyrilamine, ketotifen and D-chlorpheniramine intensified convulsions in 21- and 30-day-old mice but not in 42-day-old animals against electroconvulsions (Yokoyama et al., 1993b,c). In the same studies Yokoyama et al. (1993a); Yokoyama et al. (1993b); Yokoyama et al. (1993c) indicated that the second generation H1 receptor antagonists were ineffective in this test. In addition, clinical reports show that H1-antagonists occasionally could produce convulsions in epileptic patients (Churchull and Gammon, 1949) and younger children (Wyngarden and Seevers, 1951; see review Yokoyama and Iinuma, 1996). Moreover, it was reported that ketotifen might induce West syndrome or infantile spasm in neonatals, being treated for allergic diseases (Yasuhara et al., 1998). Also, administration of ketotifen for treatment of allergic rhinitis, in a child with secondary generalized epilepsy, resulted in an increase in seizure frequency (Yokoyama et al., 1993a). Drugs affecting H1 receptors can be divided in two generations; the first one, consists of older drugs easily penetrating through blood – brain barrier and characterized by several adverse effects, and the second generation of antihistaminic drugs, which mainly act peripherally because of poor penetration into the brain. Moreover, out of classical antihistamines one can distinguish antiallergic agents, such as ketotifen. It especially acts via blocking degranulation from basophils and enterochromaffin cells in peripheral tissues. However, ketotifen is also a potent H1 receptor inhibitor (much more than classical histamine receptor antagonist, such as D-chlorpheniramine) (Noguchi et al., 1992). It is noteworthy, that the first generation H1-antagonists influence other neurotransmitter receptors, and have also several pharmacological actions, including anticholinergic
and antidopaminergic effects (Goodman and Gilman, 1960; Tuomisto and Tuomisto, 1980). The present study deals with the effects of H1 receptor antagonists, antazoline and ketotifen, upon the anticonvulsant potential of conventional antiepileptic drugs against MES-induced seizures in mice. Antazoline and ketotifen were administered acutely or in repeated doses, once daily per week. In addition, the adverse effects were studied in the chimney test and passive avoidance task. A possibility of pharmacokinetic interactions between H1antagonists and antiepileptics was also evaluated. Part of this study was published in an abstract form (S´wia˛der et al., 1999, 2001).
2. Materials and methods 2.1. Animals Experiments were carried out on male Swiss mice weighing 20 –25 g. The animals were housed in colony cages with free access to food (chow pellets) and tap water. The experimental temperature was 21 F 1 jC and mice were on a natural light– dark cycle. After 7 days of adaptation to laboratory conditions, the animals were randomly assigned to experimental groups (consisting of 8 –12 animals). Each mouse was used only once. All experimental procedures were approved by a Lublin Bioethical Committee. 2.2. Drugs The following drugs were used throughout the study: antazoline (Phenazolinum, ICN Polfa Rzeszo´w, Poland) and ketotifen (Sigma, St. Louis, MO, USA) and four conventional antiepileptics: valproate magnesium (Dipromal, ICN Polfa Rzeszo´w, Poland), carbamazepine (Amizepin), diphenylhydantoin (Phenytoinum) and phenobarbital sodium (Luminalum Natrium, all three from Polfa, Warsaw, Poland). Antazoline, valproate and phenobarbital were dissolved in distilled water, while ketotifen, carbamazepine and diphenylhydantoin were suspended in a 1% solution of Tween 80 (Sigma). All drugs were administered intraperitoneally (i.p.) in a volume of 0.01 ml/g body weight. Antazoline, valproate magnesium and carbamazepine, 30 min; ketotifen and phenobarbital, 60 min; diphenylhydantoin, 120 min prior to the test. The doses of phenobarbital and valproate refer to their free forms. 2.3. Electroconvulsions Electroconvulsions were produced using ear-clip electrodes and alternating current delivered by a Hugo Sachs (Type 221, Freiburg, Germany) generator, the stimulus duration being 0.2 s. Tonic hindlimb extension was taken as the endpoint. The convulsive threshold was evaluated as CS50, which is the current strength (in mA) necessary
M. S´wia˛der et al. / European Neuropsychopharmacology 14 (2004) 307–318 Table 1 Influence of acute and chronic antazoline treatment on the electroconvulsive threshold Treatment (day)
Drugs (mg/kg)
Time before the test (min)
CS50 (with 95% confident limits; mA)
Acute treatment
Vehicle Antazoline Antazoline Antazoline Vehicle Antazoline Antazoline Antazoline Antazoline Vehicle Antazoline Antazoline Antazoline
30 30 30 30 60 60 60 60 60 30 30 30 30
6.2 5.5 5.7 5.1 6.2 6.0 6.1 5.7 5.8 6.6 6.1 5.5 5.2
Chronic treatment
(0.125) (0.25) (0.5) (0.125) (0.25) (0.5) (1.0) (0.125) (0.25) (0.5)
(5.6 – 7.1) (5.1 – 5.9) (5.0 – 6.4) (4.6 – 5.6)a (5.6 – 7.1) (5.6 – 6.4) (5.3 – 7.0) (5.1 – 6.4) (5.3 – 6.4) (5.7 – 7.5) (5.4 – 7.0) (5.1 – 6.1) (4.6 – 5.9)a
CS50 (in mA; 95% confidence limits in parentheses) is the current strength necessary to produce tonic convulsions in 50% of the animals tested. Antazoline was administered i.p. after acute (30 and 60 min before the test) and chronic treatment. Control groups received vehicle. The experimental groups consisted of at least eight animals. Statistic analysis was performed according to Litchfield and Wilcoxon (1949). a P < 0.05 vs. respective control group.
to produce tonic hindlimb extension in 50% of the animals tested. To estimate the convulsive threshold, at least four groups of mice (eight animals per group) were challenged with electroshocks of various intensities. Subsequently, an intensity – response curve was calculated on the basis of the percentage of mice convulsing. In order to evaluate the respective ED50 values of antiepileptic drugs (in mg/kg; a dose of an antiepileptic drug required to block the hindlimb tonic extension in 50% of the animal tested), mice pretreated with different doses of antazoline or ketotifen were challenged with MES (25 mA). At least four groups of mice, consisting of eight animals, were used to estimate each ED50 value. A dose – effect curve was constructed, based on the percentage of mice protected.
2.5. Passive avoidance acquisition and retention testing According to Venault et al. (1986) the step-through passive avoidance task may be applied as a measure of long-term memory acquisition. We used this test to compare the influence of antazoline, ketotifen, carbamazepine, valproate, phenobarbital and diphenylhydantoin alone or in combination (the H1 receptor antagonists combined with antiepileptics) on passive avoidance acquisition in mice. The pretreated animals were placed in an illuminated box (10 13 15 cm) connected to a larger (25 20 15 cm) dark compartment equipped with an electric grid floor. In this test, entry into the dark compartment was punished by an electric footshock (0.6 mA for 2 s; facilitation of acquisition). The mice that did not enter the dark compartment within 60 s were excluded from the experiment. On the following day (24 h later), the same animals, without treatment, were again placed in the illuminated box and those avoiding the dark compartment for longer than 180 s were regarded as remembering the task. Retention was expressed as medians with 25 and 75 percentiles. 2.6. Estimation of the free plasma levels and brain concentrations of antiepileptic drugs The plasma levels of antiepileptic drugs were measured according to Czuczwar et al. (1989). The animals were given either one of the studied antiepileptic drugs and saline (control group) or combinations of H1 receptor antagonists Table 2 Influence of acute and chronic ketotifen treatment on the electroconvulsive threshold Treatment (day)
Drugs (mg/kg)
Time prior to the test (min)
CS50 (with 95% confident limits; mA)
Acute treatment
Vehicle Ketotifen Ketotifen Ketotifen Vehicle Ketotifen Ketotifen Ketotifen Ketotifen Vehicle Ketotifen Ketotifen Ketotifen Vehicle Ketotifen Ketotifen Ketotifen
30 30 30 30 60 60 60 60 60 120 120 120 120 60 60 60 60
5.9 5.9 5.7 5.5 6.8 6.4 6.3 6.3 5.7 5.9 5.7 5.7 5.7 6.1 7.0 5.7 5.3
2.4. Chimney test The effects of antazoline or ketotifen on motor impairment were evaluated with the chimney test of Boissier et al. (1960). In this test, the animals had to climb backwards up a plastic tube (3 cm inner diameter, 25 cm length). Motor impairment was indicated by the inability of mice to climb backwards up the tube within 60 s. The animals were pretrained 24 h before treatment and those unable to perform the test were rejected from experimental groups. TD50 values (a dose of an antiepileptic required to produce motor impairment in 50% of the animals tested) for antazoline/ketotifen (administered at the maximal dose used in the experiment) alone or in combination with conventional antiepileptics were calculated according to the method of Litchfield and Wilcoxon (1949).
309
Chronic treatment
(2.0) (4.0) (8.0) (1.0) (2.0) (4.0) (8.0) (2.0) (4.0) (8.0) (2.0) (4.0) (8.0)
(5.1 – 6.8) (5.5 – 6.4) (5.1 – 6.3) (4.7 – 6.4) (6.1 – 7.5) (5.6 – 7.4) (6.0 – 6.7) (6.0 – 6.6) (5.2 – 6.3)a (5.1 – 6.8) (5.1 – 6.4) (5.1 – 6.5) (5.1 – 6.4) (5.6 – 6.7) (6.3 – 7.7) (5.3 – 6.2) (4.9 – 5.7)a
CS50 (in mA); 95% confidence limits in parentheses) is the current strength necessary to produce tonic convulsions in 50% of the animals tested. Ketotifen was administered i.p. after acute (30, 60 and 120 min before the test) and chronic treatment. Control groups received vehicle. The experimental groups consisted of at least eight animals. Statistic analysis was performed according to Litchfield and Wilcoxon (1949). a P < 0.05 vs. respective control group.
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with one of these drugs. Mice were decapitated at times scheduled for the convulsive test and blood samples of approximately 1 ml were collected into Eppendorf tubes. Whole brains were taken from the same animals immediately after decapitation at 4 jC. According to Borowicz
et al. (1999), brains of mice were homogenized in TDx buffer (Abbott, Irving, TX, USA). Samples of blood and brain were centrifuged and only plasma samples were pipetted into a micropartition system, MPS-1 (Amicon, Danvers, MA, USA). Then, the plasma samples were again
Fig. 1. Influence of antazoline, given acutely or for 7 days, upon the protective activity of antiepileptic drugs against MES-induced seizures in mice. Carbamazepine (CBZ) and valproate (VPA) were given i.p. 30 min, phenobarbital (PB), 60 min and diphenylhydantoin (DPH), 120 min before testing. (A) Antazoline in a single dose was given i.p. 30 min before testing. (B) Prolonged treatment of antazoline. The data are ED50 values of antiepileptic drugs (with 95% confidence limits), calculated and compared according to Litchfield and Wilcoxon (1949). a P < 0.05 vs. antiepileptic drug alone. c P < 0.001 vs. antiepileptic drug alone.
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centrifuged, and the free plasma levels and brain concentrations of antiepileptic drugs were determined by immunofluorescence, using an Abbott TDx analyzer (Abbott). The
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plasma or brain levels of antiepileptic drugs were expressed in Ag/ml of plasma or Ag/g of wet brain tissue as mean F S.D. of at least eight determinations.
Fig. 2. Influence of ketotifen, given acutely or for 7 days, upon the protective activity of antiepileptic drugs against MES-induced seizures in mice. Carbamazepine (CBZ) and valproate (VPA) were given i.p. 30 min, phenobarbital (PB), 60 min and diphenylhydantoin (DPH), 120 min before testing. (A) Ketotifen in a single dose was given i.p. 60 min before testing. (B) Prolonged treatment of ketotifen. The data are ED50 values of antiepileptic drugs (with 95% confidence limits), calculated and compared according to Litchfield and Wilcoxon (1949). a P < 0.05 vs. antiepileptic drug alone. b P < 0.01 vs. antiepileptic drug alone. c P < 0.001 vs. antiepileptic drug alone.
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Fig. 3. Influence of antazoline, injected singly or following a 7-day treatment, upon motor impairment produced by conventional antiepileptic drugs. (A) Antazoline in a single dose. (B) Antazoline prolonged treatment. The data are TD50 values of antiepileptic drugs (with 95% confidence limits), calculated and compared according to Litchfield and Wilcoxon (1949). For abbreviations and treatment times see Fig. 1. a P < 0.05 vs. antiepileptic drug alone.
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Fig. 4. Influence of ketotifen, injected singly or following a 7-day treatment, upon motor impairment produced by conventional antiepileptic drugs. (A) Ketotifen in a single dose. (B) Ketotifen prolonged treatment. The data are TD50 values of antiepileptic drugs (with 95% confidence limits), calculated and compared according to Litchfield and Wilcoxon (1949). For abbreviations and treatment times see Fig. 2. a P < 0.05 vs. antiepileptic drug alone.
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2.7. Treatment protocol Experiments were carried out after acute or chronic treatment with antazoline or ketotifen. 1. Acute study: animals were injected with a single dose of H1 receptor antagonists and one of the antiepileptics at the time prior to the tests characterized above. Conventional antiepileptic drugs were tested at the time of their peak-anticonvulsant activity according to our previously published studies (Gasior et al., 1996; Wlaz´ et al., 1992, 1993), whilst the antazoline- and ketotifen-time of maximum activity were determined experimentally. 2. Chronic study: once a day (between 08:00 and 10:00 h) mice were injected as follows: group 1, saline for 6 days (control group); group 2, antazoline or ketotifen for 6 days (repeated doses). On the 7th day, mice from all groups received one of the conventional antiepileptic drugs (saline-treated group) or H1 receptor antagonists co-administered with one of the antiepileptics, at the same time, just like in the acute study. The CS50s of antazoline or ketotifen, ED50s and TD50s of antiepileptics alone or in combinations with H1-antagonists were calculated and statistically analyzed. Also, long-term memory and the free plasma or brain levels of antiepileptics were also evaluated in both experimental protocols.
by the Kruskal – Wallis test followed by Dunn’s test. Unpaired Student’s t-test was used for the statistical evaluation of the free plasma and brain levels of antiepileptic drugs.
3. Results 3.1. Effect of acute and chronic treatment with antazoline and ketotifen on the electroconvulsive threshold Antazoline, after acute and chronic treatment at the dose of 0.5 mg/kg, significantly reduced the electroconvulsive threshold. No effect was observed when antazoline was used at lower doses of 0.125 –0.25 mg/kg, 30 min before the test or up to 1 mg/kg, 60 min prior to electroconvulsions (Table 1). Ketotifen, given acutely or for 7 days, at the dose of 8 mg/kg markedly decreased the electroconvulsive threshold when given 60 min before the test. The CS50 value for ketotifen was 5.7 (5.2 –6.3) mA in comparison to 6.8 (6.1 – 7.5) mA of a control group. It was without effect upon this parameter at doses of 1– 4 mg/kg. When administered up to 8 mg/kg, 30 or 120 min before electroconvulsions, it did not affect the convulsive threshold (Table 2). 3.2. Influence of acute or repeated antazoline on the anticonvulsant activity of antiepileptic drugs against maximal electroshock-induced seizures in mice
2.8. Statistics CS50, ED50 and TD50 values and their statistical comparisons were calculated by computer probit analysis, according to Litchfield and Wilcoxon (1949). The results from the passive avoidance task were statistically verified
Antazoline (0.25 mg/kg), acutely and for 7 days, diminished the antiseizure activity of carbamazepine elevating its ED50 values from 13.7 (12.1 – 15.4) and 15.3 (13.4 – 17.6) mg/kg (control groups) to 31.9 (27.1 –37.6) and 24.6 (20.8 – 29.2) mg/kg, respectively (Fig. 1).
Table 3 Effects of ketotifen (after 1- or 7-day treatment) and antiepileptic drugs or their combinations on long-term memory in mice Drugs
Vehicle Ketotifen Carbamazepine Carbamazepine Carbamazepine Valproate Valproate Valproate Phenobarbital Phenobarbital Phenobarbital Diphenylhydantoin Diphenylhydantoin Diphenylhydantoin
Dosage (mg/kg)
Ketotifen
Acute
Chronic
– – 13.4 9.4 9.4 254 234 234 24.9 20.8 20.8 8.2 7.8 7.8
– – 11.0 14.9 14.9 256 260 260 23.2 27.8 27.8 10.0 10.4 10.4
– 4.0 – – 4.0 – – 4.0 – – 4.0 – – 4.0
Medians (25, 75 percentile) Acute
Chronic
180 180 180 180 180 28.5 63 89 128 180 153 180 180 180
180 (180, 180) 180 (170, 180) 180 (180, 180) 180 (180, 180) 180 (153, 180) 166 (66, 180) 115 (84.5, 163) 56 (46, 69.3)a,# 180 (91.5, 180) 180 (124, 180) 180 (147, 180) 180 (96, 180) 156 (104, 180) 180 (180, 180)
(180, 180) (175, 180) (118, 180) (166, 180) (146, 180) (14.3, 68.3)a (32.5, 180) (75.8, 180) (110, 175) (104, 180) (60.3, 180) (103, 180) (71, 180) (180, 180)
Presented values are the medians with 25 and 75 percentiles of 12 determinations. The retention was quantified as a time period (in seconds) the animals avoided the dark compartment. The results obtained from the passive avoidance task were statistically verified by the Kruskal – Wallis test followed by Dunn’s post-hoc test. For treatment times refer to Fig. 2. a P < 0.05 vs. control group. # P < 0.05 vs. valproate alone (260 mg/kg).
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Antazoline, in a single dose of 0.25 mg/kg did not affect the protective activity of diphenylhydantoin. It is noteworthy that the ED50 value of diphenylhydantoin was raised when it was co-administered with antazoline (0.25 mg/kg) in repeated doses, from 8.9 (6.8 – 11.6) to 12.6 (10.4 – 15.4) mg/kg (Fig. 1). However, the remaining antiepileptic drugs, co-administered with antazoline (0.25 mg/kg), after acute and chronic treatment, did not change their efficacy (Fig. 1). 3.3. Influence of ketotifen, after acute and chronic treatment, on the anticonvulsant activity of antiepileptic drugs against maximal electroshock-induced seizures in mice Ketotifen, injected acutely at the dose of 4 mg/kg, increased the anticonvulsant action of carbamazepine and phenobarbital, reflected by reductions of the respective ED50 values from 13.4 (11.7 –15.4) and 24.9 (22.5 –27.5) mg/kg to 7.9 (6.5 – 9.7) and 20.8 (18.9 – 22.9) mg/kg, respectively (Fig. 2). However, chronic ketotifen (4 mg/ kg) did not affect the protective action of phenobarbital. In contrast, ketotifen (4 mg/kg) given chronically diminished significantly the anticonvulsant action of carbamazepine, increasing its ED50 value from 11.0 (10.2 – 11.9) to 14.9 (13.3 –16.7) mg/kg. Neither ED50 of valproate nor that of diphenylhydantoin was changed when the drugs were coadministered with ketotifen (4 mg/kg), given acutely or for 7 days (Fig. 2). 3.4. Chimney test Acute antazoline (0.25 mg/kg) decreased the TD50 value of valproate from 375 (339 –414) to 329 (304 –355) while given chronically, it increased this value, from 376 (355 – 398) mg/kg to 409 (391 – 428) mg/kg. However, this H1 receptor antagonist did not intensify motor impairment after acute or chronic co-administration with carbamazepine, phenobarbital or diphenylhydantoin (Fig. 3). Ketotifen (4 mg/kg), administered in a single dose, further impaired motor coordination in mice treated with valproate, phenobarbital or diphenylhydantoin, which resulted in reductions of the TD50 values from 426 (398 – 456.3), 99.3 (86.8 – 113.5) and 79.1 (75.1 – 83.3) mg/kg to 384 (353 – 415), 76.7 (64.2 –91.5) and 72.7 (68.1 – 77.7) mg/kg, respectively. In contrast, chronic treatment with ketotifen (4 mg/kg) did not influence TD50 values of antiepileptic drugs (Fig. 4). 3.5. Passive avoidance task Acute antazoline (0.25 mg/kg) did not influence retention in the passive avoidance task. Carbamazepine, valproate, diphenylhydantoin and phenobarbital when applied at their ED50 values against MES-induced seizures or coadministered with antazoline, did not impair long-term memory in mice. Similiarly, antazoline (0.25 mg/kg), after 7-day treatment, alone or combined with the anticonvulsants produced no statistical worsening of long-term mem-
Fig. 5. Influence of antazoline, given acutely or for 7 days, upon the brain concentrations or free plasma levels of antiepileptic drugs. Presented values are the means (Ag/ml of plasma and Ag/g of brain wet tissue) of eight determinations F S.D. Unpaired Student’s t-test was used for statistical evaluation of the data. Blood or brain samples were taken at times scheduled for the convulsive test. For treatment times and abbreviations see also the legend of Fig. 1.
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ory (results not shown). Acute ketotifen (at 4 mg/kg), alone or combined with antiepileptic drugs at their ED50s against MES, did not affect the performance of mice in the passive
avoidance task. In contrast, valproate (254 mg/kg) administered alone at its ED50 against MES significantly disturbed this parameter. Moreover, chronic treatment with ketotifen (4 mg/kg) alone, as well as, co-administered with carbamazepine, diphenylhydantoin or phenobarbital did not affect long-term memory in mice. However, valproate (at doses providing a 50% protection against MES-induced seizures) combined with ketotifen (4 mg/kg), produced a significant impairment of the memory task (Table 3). This result was also significant when compared to valproate (260 mg/kg) alone. 3.6. Influence of antazoline and ketotifen on the free plasma and brain levels of antiepileptic drugs Antazoline (0.25 mg/kg), given acutely or chronically, did not affect either the free plasma levels or brain concentration of carbamazepine or diphenylhydantoin (Fig. 5). In addition, ketotifen (4 mg/kg), after acute or 7-day treatment, did not influence the free plasma or brain levels of carbamazepine and phenobarbital (Fig. 6).
4. Discussion
Fig. 6. Influence of ketotifen, given acutely or for 7 days, upon the brain concentrations or free plasma levels of antiepileptic drugs. Presented values are the means (Ag/ml of plasma and Ag/g of brain wet tissue) of eight determinations F S.D. Unpaired Student’s t-test was used for statistical evaluation of the data. Blood or brain samples were taken at times scheduled for the convulsive test. For treatment times and abbreviations see also the legend of Fig. 2.
The existing evidence indicates that there are about 30% of epileptic patients not satisfactorily controlled with existing antiepileptic drugs (Deckers et al., 2000). A possibility exists that at least some therapeutic failures in epileptic patients may result from undesired interactions between antiepileptics and other drugs, prescribed for reasons unrelated to epilepsy. Also, side effects of antiepileptic drugs may frequently be a cause of their withdrawal which is especially difficult in a situation of adequate seizure control (Deckers et al., 2000). Consequently, the present study was not only confined to the evaluation of drug interactions in terms of anticonvulsant efficacy but to adverse effects as well. In the present study, antazoline and ketotifen showed some proconvulsive activity. Both drugs reduced the electroconvulsive threshold, either after acute or chronic treatment. Moreover, antazoline (0.25 mg/kg) and ketotifen (4 mg/kg), at subeffective repeated doses, diminished the anticonvulsant activity of some antiepileptic drugs studied. Acute antazoline increased only the ED50 value of carbamazepine, while chronically injected it raised ED50s of carbamazepine and diphenylhydantoin. The interactions described between H1-antagonists and antiepileptic drugs were pharmacodynamic in nature. It is important to underline, that the ED50 value of carbamazepine when combined with acute or 7-day treatment antazoline (0.25 mg/kg) was about 2– 3-fold higher in comparison with control groups. The difference between diphenylhydantoin alone or coadministered with antazoline was about 50%. Ketotifen (4 mg/kg) administered acutely, enhanced the anticonvulsant action of carbamazepine and phenobarbital. In contrast, after
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chronic treatment with ketotifen (4 mg/kg) a reduction of the anticonvulsant activity of carbamazepine was observed. Antazoline (0.25 mg/kg) worsened motor coordination in mice when combined with valproate, only after acute treatment. Chronic treatment with antazoline resulted in improved motor performance in mice receiving valproate, which was reflected by the increased TD50 value of this antiepileptic drug. However, this H1 receptor antagonist did not influence passive avoidance task in mice. In contrast, acute ketotifen (4 mg/kg) co-administered with valproate, phenobarbital or diphenylhydantoin markedly raised their TD50 values, being without effect in this test after 7-day treatment. Further, valproate tested on long-term memory, after acute or 7-day treatment with ketotifen, impaired memory in mice when was administered alone or combined with this H1 receptor antagonist. Bearing in mind that H1 receptor antagonists affect different neurotransmitter systems, such as cholinergic, dopaminergic, serotoninergic and noradrenergic ones (Goodman and Gilman, 1960; Tuomisto and Tuomisto, 1980), it is difficult to consider all possible interactions. It is probable that an enhancement of the anticonvulsant activity of antiepileptics by acute ketotifen may result from an anticholinergic component. On the other hand, antazoline, after acute and chronic treatment, and ketotifen, administered repeatedly, diminished the antiseizure potency of antiepileptic drugs. As already mentioned, H1 receptor antagonists are proconvulsive in numerous experimental models of epilepsy such as MES (Yokoyama et al., 1993b,c), pentylenetetrazole-induced convulsions in mice (Gerald and Richter, 1976; Scherkl et al., 1991b) or amygdala-kindled seizures in rats (Chen et al., 2002; Kamei, 2001; Yokoyama et al., 1996). Moreover, metoprine and L-histidine, as well as, thioperamide, which elevate brain histamine levels, have demonstrated anticonvulsant activity against pentylenetetrazole-induced convulsions (Chen et al., 2000; Scherkl et al., 1991a,b; Vohora et al., 2001) or electroconvulsive seizures (Yokoyama et al., 1993b,c; Vohora et al., 2001). Furthermore, L-histidine decreases susceptibility to electrically induced convulsions in mice (Yokoyama et al., 1992) and also inhibits amygdala-kindled seizures in rats (Kamei, 2001; Kamei et al., 1998). Recently, Kamin´ski et al. (2003) have presented data, which indicates that L-histidine, but not the D-isomer of this amino acid, augmented the protective effects of carbamazepine or diphenylhydantoin against electroconvulsions. However, the roles of agents, elevating central histamine are controversial, since there are data in the literature about their ineffectiveness in amygdalakindled seizures (Borowicz et al., 2000; Yoshida et al., 2000). Generally, histamine acts in the central nervous system as an inhibitory neurotransmitter mostly via H1 receptors (Kamei et al., 2000). In conclusion, the present findings indicate that H1 receptor antagonists diminished the anticonvulsant activity of carbamazepine and diphenylhydantoin. It is important
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that the observed effects were evident after prolonged treatment with both antihistaminic drugs, and after acute treatment with antazoline. Considering thoroughly the results from the acute studies, it is clear that some antihistaminics co-applied with antiepileptic drugs may impair the seizure control in epileptic patients. Nevertheless, it seems evident that rather chronic co-administration of these drugs may give some adequate rationale for avoiding their combinations with antiepileptic drugs. For instance, the present results indicate that considering the anticonvulsant activity, negative interactions may be possible upon combinations of antazoline with carbamazepine and diphenylhydantoin or ketotifen with carbamazepine. Considering adverse effects, untoward interactions may accompany a co-administration of valproate with ketotifen.
Acknowledgements This study was supported by the grant No. P05A 044 18 from the State Committee for Scientific Research (Warszawa, Poland). The generous gift of valproate magnesium from ICN Polfa Rzeszo´w is appreciated.
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Dodd, R.H., Roissier, J., 1986. Benzodiazepines impair and beta-carbolines enhance performance in learning and memory tasks. Nature 321, 864 – 866. Vohora, D., Pal, S.N., Pillai, K.K., 2000. Thioperamide, a selective histamine H3 receptor antagonist, protects against PTZ-induced seizures in mice. Life Sci. 66, 297 – 301. Vohora, D., Pal, S.N., Pillai, K.K., 2001. Histamine and selective H3-receptor ligands: a possible role in the mechanism and management of epilepsy. Pharmacol. Biochem. Behav. 68, 735 – 741. Wlaz´, P., Rolinski, Z., Kleinrok, Z., Czuczwar, S.J., 1992. Anticonvulsant activity of carbamazepine and diphenylhydantoin against maximal electroshock in mice chronically treated with aminophylline. J. Neural Transm. 89, 41 – 48. Wlaz´, P., Rolinski, Z., Kleinrok, Z., Czuczwar, S.J., 1993. Influence of chronic aminophylline on the anticonvulsant efficacy of phenobarbital and valproate in mice. Epilepsia 34, 385 – 389. Wyngarden, J.B., Seevers, M.H., 1951. The toxic effects of antihistaminic drugs. J. Am. Med. Assoc. 145, 277 – 282. Yasuhara, A., Ochi, A., Harada, Y., Kobayashi, Y., 1998. Infantile spasms associated with a histamine H1 antagonist. Neuropediatrics 29, 320 – 321. Yokoyama, H., Iinuma, K., 1996. Histamine and seizures—implications for the treatment of epilepsy. CNS Drugs 5, 321 – 330. Yokoyama, H., Onodera, K., Maeyama, K., Yanai, K., Iinuma, K., Tuomisto, L., Watanabe, T., 1992. Histamine levels and clonic convulsions of electrically-induced seizure in mice: the effects of alpha-fluoromethylhistidine and metoprine. Naunyn-Schmiedeberg’s Arch. Pharmacol. 346, 40 – 45. Yokoyama, H., Iinuma, K., Yanai, K., Watanabe, T., Sakurai, E., Onodera, K., 1993a. Proconvulsant effect of ketotifen, a histamine H1 antagonist, confirmed by the use of D-chlorpheniramine with monitoring electroencephalography. Methods Find. Exp. Clin. Pharmacol. 15, 183 – 188. Yokoyama, H., Onodera, K., Iinuma, K., Watanabe, T., 1993b. Effect of thioperamide, a histamine H3 receptor antagonist, on electrically induced convulsions in mice. Eur. J. Pharmacol. 234, 129 – 133. Yokoyama, H., Onodera, K., Iinuma, K., Watanabe, T., 1993c. Proconvulsive effects of histamine H1-antagonists on electrically-induced seizure in developing mice. Psychopharmacology 112, 199 – 203. Yokoyama, H., Onodera, K., Iinuma, K., Watanabe, T., 1994. 2-Thiazolylethylamine, a selective histamine H1 agonist, decreases seizure susceptibility in mice. Pharmacol. Biochem. Behav. 47, 503 – 507. Yokoyama, H., Sato, M., Iinuma, K., Onodera, K., Watanabe, T., 1996. Centrally acting histamine H1-antagonists promote the development of amygdala kindling in rats. Neurosci. Lett. 217, 194 – 196. Yoshida, M., Noguchi, E., Tsuru, N., 2000. Lack of substantial effect of the H(3)-antagonist thioperamide and of the non-selective mixed H(3)-antagonist/H(1)-agonist betahistine on amygdaloid kindled seizures. Epilepsy Res. 40, 141 – 145.
Influence of antazoline and ketotifen on the anticonvulsant activity of conventional antiepileptics against maximal electroshock in mice Mariusz S´wia˛der a, Marian Wielosz a, StanislCaw J. Czuczwar b,c,* a
Department of Pharmacology and Toxicology, Medical University, 20-090 Lublin, Jaczewskiego 8, Poland b Department of Pathophysiology, Medical University, 20-090 Lublin, Jaczewskiego 8, Poland c Isotope Laboratory, Institute of Agricultural Medicine, 20-950 Lublin, Jaczewskiego 2, Poland Received 25 March 2003; received in revised form 15 July 2003; accepted 16 September 2003
Abstract Experimental studies have indicated that the central histaminergic system plays an important role in the inhibition of seizures through the stimulation of histamine H1 receptors. H1 receptor antagonists, including classical antiallergic drugs, occasionally may induce convulsions in healthy children and patients with epilepsy. The purpose of this study was to investigate the effects of antazoline and ketotifen (two H1 receptor antagonists) on the anticonvulsant activity of antiepileptic drugs against maximal electroshock (MES)-induced convulsions in mice. The following antiepileptic drugs were used: valproate, carbamazepine, diphenylhydantoin and phenobarbital. In addition, the effects of antiepileptic drugs alone or in combination with antazoline or ketotifen were studied on long-term memory (tested in the passive avoidance task) and motor performance (evaluated in the chimney test), acutely and after 7-day treatment with these H1 receptor antagonists. The influence of antazoline and ketotifen on the free plasma and brain levels of the antiepileptics was also evaluated. Antazoline (at 0.5 mg/kg), given acutely and after 7-day treatment, significantly diminished the electroconvulsive threshold. Similarly, ketotifen, after acute and chronic doses of 8 mg/kg markedly reduced the threshold for electroconvulsions. In both cases, antazoline and ketotifen were without effect upon this parameter at lower doses. Antazoline (0.25 mg/kg) significantly raised the ED50 value of carbamazepine against MES (both, acutely and after 7-day treatment). Furthermore antazoline (0.25 mg/kg) also reduced the anticonvulsant activity of diphenylhydantoin, but only after repeated administration, without modifying the brain and free plasma level of this drug. Moreover, valproate and phenobarbital did not change their protective activity when combined with antazoline. Ketotifen (4 mg/kg) possessed a biphasic action, acutely it enhanced the anticonvulsant action of carbamazepine and phenobarbital while, following 7-day treatment, reduced the antiseizure activity of carbamazepine. Ketotifen did not affect the free plasma or brain levels of antiepileptics tested. Only acute antazoline (0.25 mg/kg) applied with valproate impaired the performance of mice evaluated in the chimney test. Ketotifen (4 mg/ kg) co-administered with conventional antiepileptic drugs impaired motor coordination in mice treated with valproate, phenobarbital or diphenylhydantoin. Acute and chronic antazoline (0.25 mg/kg) alone or in combination with antiepileptic drugs did not disturb long-term memory, tested in the passive avoidance task. Similarly, ketotifen (4 mg/kg) did not impair long-term memory, acutely and after 7-day treatment. However, valproate alone or in combination with chronic ketotifen (4 mg/kg) worsened long-term memory. The results of this study indicate that H1 receptor antagonists, crossing the blood brain barrier, should be used with caution in epileptic patients. This is because antazoline reduced the protective potential of diphenylhydantoin and carbamazepine. Also, ketotifen reduced the protection offered by carbamazepine and elevated the adverse activity of diphenylhydantoin, phenobarbital and valproate. D 2003 Elsevier B.V./ECNP. All rights reserved. Keywords: Antazoline; Ketotifen; Antiepileptic drugs; Electroshock maximal; Drug interactions; Seizures
1. Introduction Several lines of evidence suggest that central histaminergic neuron system plays an important role in inhibition * Corresponding author. Department of Pathophysiology, Medical University, 20-090 Lublin, Jaczewskiego 8, Poland. Tel.: +48-81-7425837; fax: +48-81-742-5828. E-mail address: [email protected] (S.J. Czuczwar). 0924-977X/$ - see front matter D 2003 Elsevier B.V./ECNP. All rights reserved. doi:10.1016/j.euroneuro.2003.09.005
of convulsions in mammals (Gerald and Richter, 1976; Onodera and Watanabe, 1995; Onodera et al., 1994; Scherkl et al., 1991a,b). This biogenic amine in the central nervous system acts via four subtypes of receptors: postsynaptic H1 and H2, presynaptic H3 and recently discovered H4 receptors. Tuomisto and Tacke (1986) have shown that metoprine, which raises brain histamine levels, inhibits hindlimb extension in maximal electroshock (MES) in rats, while Onodera et al. (1992) have found that it reduces
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audiogenic seizures in genetically epilepsy-prone rats. In mice, the anticonvulsant effects of L-histidine, a precursor of brain histamine, on clonic and convulsive coma phases are dependent on the brain histamine, and mediated through the central H1 receptors (Yokoyama et al., 1992). Also, thioperamide, H3 receptor antagonist, which elevated histamine levels in the brain, significantly and dose dependently inhibited electroconvulsions (Yokoyama et al., 1993a,b,c), amygdala-kindled seizures (Kakinoki et al., 1998) and pentylenetetrazole-induced convulsions (Vohora et al., 2000). Generally, the administration of first generation H1 receptor antagonists or H3 receptor agonists blocks the anticonvulsant effects of these pharmacological manipulations, which increased brain histamine (e.g. thioperamide, L -histidine). Moreover, dimethindene and promethazine diminish pentylenetetrazol seizure threshold (Scherkl et al., 1991b), while pyrilamine blocks the beneficial effects of L-histidine (Yokoyama et al., 1992). The intracerebroventricular administration of histamine and the selective H1 receptor agonist, 2-thizolylthylamine, inhibited electroshock- and pentylenetetrazol-induced seizures, whereas ketotifen and pyrilamine antagonized their effects (Yokoyama et al., 1994; for review see Sangalli, 1997). Furthermore, pyrilamine, ketotifen and D-chlorpheniramine intensified convulsions in 21- and 30-day-old mice but not in 42-day-old animals against electroconvulsions (Yokoyama et al., 1993b,c). In the same studies Yokoyama et al. (1993a); Yokoyama et al. (1993b); Yokoyama et al. (1993c) indicated that the second generation H1 receptor antagonists were ineffective in this test. In addition, clinical reports show that H1-antagonists occasionally could produce convulsions in epileptic patients (Churchull and Gammon, 1949) and younger children (Wyngarden and Seevers, 1951; see review Yokoyama and Iinuma, 1996). Moreover, it was reported that ketotifen might induce West syndrome or infantile spasm in neonatals, being treated for allergic diseases (Yasuhara et al., 1998). Also, administration of ketotifen for treatment of allergic rhinitis, in a child with secondary generalized epilepsy, resulted in an increase in seizure frequency (Yokoyama et al., 1993a). Drugs affecting H1 receptors can be divided in two generations; the first one, consists of older drugs easily penetrating through blood – brain barrier and characterized by several adverse effects, and the second generation of antihistaminic drugs, which mainly act peripherally because of poor penetration into the brain. Moreover, out of classical antihistamines one can distinguish antiallergic agents, such as ketotifen. It especially acts via blocking degranulation from basophils and enterochromaffin cells in peripheral tissues. However, ketotifen is also a potent H1 receptor inhibitor (much more than classical histamine receptor antagonist, such as D-chlorpheniramine) (Noguchi et al., 1992). It is noteworthy, that the first generation H1-antagonists influence other neurotransmitter receptors, and have also several pharmacological actions, including anticholinergic
and antidopaminergic effects (Goodman and Gilman, 1960; Tuomisto and Tuomisto, 1980). The present study deals with the effects of H1 receptor antagonists, antazoline and ketotifen, upon the anticonvulsant potential of conventional antiepileptic drugs against MES-induced seizures in mice. Antazoline and ketotifen were administered acutely or in repeated doses, once daily per week. In addition, the adverse effects were studied in the chimney test and passive avoidance task. A possibility of pharmacokinetic interactions between H1antagonists and antiepileptics was also evaluated. Part of this study was published in an abstract form (S´wia˛der et al., 1999, 2001).
2. Materials and methods 2.1. Animals Experiments were carried out on male Swiss mice weighing 20 –25 g. The animals were housed in colony cages with free access to food (chow pellets) and tap water. The experimental temperature was 21 F 1 jC and mice were on a natural light– dark cycle. After 7 days of adaptation to laboratory conditions, the animals were randomly assigned to experimental groups (consisting of 8 –12 animals). Each mouse was used only once. All experimental procedures were approved by a Lublin Bioethical Committee. 2.2. Drugs The following drugs were used throughout the study: antazoline (Phenazolinum, ICN Polfa Rzeszo´w, Poland) and ketotifen (Sigma, St. Louis, MO, USA) and four conventional antiepileptics: valproate magnesium (Dipromal, ICN Polfa Rzeszo´w, Poland), carbamazepine (Amizepin), diphenylhydantoin (Phenytoinum) and phenobarbital sodium (Luminalum Natrium, all three from Polfa, Warsaw, Poland). Antazoline, valproate and phenobarbital were dissolved in distilled water, while ketotifen, carbamazepine and diphenylhydantoin were suspended in a 1% solution of Tween 80 (Sigma). All drugs were administered intraperitoneally (i.p.) in a volume of 0.01 ml/g body weight. Antazoline, valproate magnesium and carbamazepine, 30 min; ketotifen and phenobarbital, 60 min; diphenylhydantoin, 120 min prior to the test. The doses of phenobarbital and valproate refer to their free forms. 2.3. Electroconvulsions Electroconvulsions were produced using ear-clip electrodes and alternating current delivered by a Hugo Sachs (Type 221, Freiburg, Germany) generator, the stimulus duration being 0.2 s. Tonic hindlimb extension was taken as the endpoint. The convulsive threshold was evaluated as CS50, which is the current strength (in mA) necessary
M. S´wia˛der et al. / European Neuropsychopharmacology 14 (2004) 307–318 Table 1 Influence of acute and chronic antazoline treatment on the electroconvulsive threshold Treatment (day)
Drugs (mg/kg)
Time before the test (min)
CS50 (with 95% confident limits; mA)
Acute treatment
Vehicle Antazoline Antazoline Antazoline Vehicle Antazoline Antazoline Antazoline Antazoline Vehicle Antazoline Antazoline Antazoline
30 30 30 30 60 60 60 60 60 30 30 30 30
6.2 5.5 5.7 5.1 6.2 6.0 6.1 5.7 5.8 6.6 6.1 5.5 5.2
Chronic treatment
(0.125) (0.25) (0.5) (0.125) (0.25) (0.5) (1.0) (0.125) (0.25) (0.5)
(5.6 – 7.1) (5.1 – 5.9) (5.0 – 6.4) (4.6 – 5.6)a (5.6 – 7.1) (5.6 – 6.4) (5.3 – 7.0) (5.1 – 6.4) (5.3 – 6.4) (5.7 – 7.5) (5.4 – 7.0) (5.1 – 6.1) (4.6 – 5.9)a
CS50 (in mA; 95% confidence limits in parentheses) is the current strength necessary to produce tonic convulsions in 50% of the animals tested. Antazoline was administered i.p. after acute (30 and 60 min before the test) and chronic treatment. Control groups received vehicle. The experimental groups consisted of at least eight animals. Statistic analysis was performed according to Litchfield and Wilcoxon (1949). a P < 0.05 vs. respective control group.
to produce tonic hindlimb extension in 50% of the animals tested. To estimate the convulsive threshold, at least four groups of mice (eight animals per group) were challenged with electroshocks of various intensities. Subsequently, an intensity – response curve was calculated on the basis of the percentage of mice convulsing. In order to evaluate the respective ED50 values of antiepileptic drugs (in mg/kg; a dose of an antiepileptic drug required to block the hindlimb tonic extension in 50% of the animal tested), mice pretreated with different doses of antazoline or ketotifen were challenged with MES (25 mA). At least four groups of mice, consisting of eight animals, were used to estimate each ED50 value. A dose – effect curve was constructed, based on the percentage of mice protected.
2.5. Passive avoidance acquisition and retention testing According to Venault et al. (1986) the step-through passive avoidance task may be applied as a measure of long-term memory acquisition. We used this test to compare the influence of antazoline, ketotifen, carbamazepine, valproate, phenobarbital and diphenylhydantoin alone or in combination (the H1 receptor antagonists combined with antiepileptics) on passive avoidance acquisition in mice. The pretreated animals were placed in an illuminated box (10 13 15 cm) connected to a larger (25 20 15 cm) dark compartment equipped with an electric grid floor. In this test, entry into the dark compartment was punished by an electric footshock (0.6 mA for 2 s; facilitation of acquisition). The mice that did not enter the dark compartment within 60 s were excluded from the experiment. On the following day (24 h later), the same animals, without treatment, were again placed in the illuminated box and those avoiding the dark compartment for longer than 180 s were regarded as remembering the task. Retention was expressed as medians with 25 and 75 percentiles. 2.6. Estimation of the free plasma levels and brain concentrations of antiepileptic drugs The plasma levels of antiepileptic drugs were measured according to Czuczwar et al. (1989). The animals were given either one of the studied antiepileptic drugs and saline (control group) or combinations of H1 receptor antagonists Table 2 Influence of acute and chronic ketotifen treatment on the electroconvulsive threshold Treatment (day)
Drugs (mg/kg)
Time prior to the test (min)
CS50 (with 95% confident limits; mA)
Acute treatment
Vehicle Ketotifen Ketotifen Ketotifen Vehicle Ketotifen Ketotifen Ketotifen Ketotifen Vehicle Ketotifen Ketotifen Ketotifen Vehicle Ketotifen Ketotifen Ketotifen
30 30 30 30 60 60 60 60 60 120 120 120 120 60 60 60 60
5.9 5.9 5.7 5.5 6.8 6.4 6.3 6.3 5.7 5.9 5.7 5.7 5.7 6.1 7.0 5.7 5.3
2.4. Chimney test The effects of antazoline or ketotifen on motor impairment were evaluated with the chimney test of Boissier et al. (1960). In this test, the animals had to climb backwards up a plastic tube (3 cm inner diameter, 25 cm length). Motor impairment was indicated by the inability of mice to climb backwards up the tube within 60 s. The animals were pretrained 24 h before treatment and those unable to perform the test were rejected from experimental groups. TD50 values (a dose of an antiepileptic required to produce motor impairment in 50% of the animals tested) for antazoline/ketotifen (administered at the maximal dose used in the experiment) alone or in combination with conventional antiepileptics were calculated according to the method of Litchfield and Wilcoxon (1949).
309
Chronic treatment
(2.0) (4.0) (8.0) (1.0) (2.0) (4.0) (8.0) (2.0) (4.0) (8.0) (2.0) (4.0) (8.0)
(5.1 – 6.8) (5.5 – 6.4) (5.1 – 6.3) (4.7 – 6.4) (6.1 – 7.5) (5.6 – 7.4) (6.0 – 6.7) (6.0 – 6.6) (5.2 – 6.3)a (5.1 – 6.8) (5.1 – 6.4) (5.1 – 6.5) (5.1 – 6.4) (5.6 – 6.7) (6.3 – 7.7) (5.3 – 6.2) (4.9 – 5.7)a
CS50 (in mA); 95% confidence limits in parentheses) is the current strength necessary to produce tonic convulsions in 50% of the animals tested. Ketotifen was administered i.p. after acute (30, 60 and 120 min before the test) and chronic treatment. Control groups received vehicle. The experimental groups consisted of at least eight animals. Statistic analysis was performed according to Litchfield and Wilcoxon (1949). a P < 0.05 vs. respective control group.
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with one of these drugs. Mice were decapitated at times scheduled for the convulsive test and blood samples of approximately 1 ml were collected into Eppendorf tubes. Whole brains were taken from the same animals immediately after decapitation at 4 jC. According to Borowicz
et al. (1999), brains of mice were homogenized in TDx buffer (Abbott, Irving, TX, USA). Samples of blood and brain were centrifuged and only plasma samples were pipetted into a micropartition system, MPS-1 (Amicon, Danvers, MA, USA). Then, the plasma samples were again
Fig. 1. Influence of antazoline, given acutely or for 7 days, upon the protective activity of antiepileptic drugs against MES-induced seizures in mice. Carbamazepine (CBZ) and valproate (VPA) were given i.p. 30 min, phenobarbital (PB), 60 min and diphenylhydantoin (DPH), 120 min before testing. (A) Antazoline in a single dose was given i.p. 30 min before testing. (B) Prolonged treatment of antazoline. The data are ED50 values of antiepileptic drugs (with 95% confidence limits), calculated and compared according to Litchfield and Wilcoxon (1949). a P < 0.05 vs. antiepileptic drug alone. c P < 0.001 vs. antiepileptic drug alone.
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centrifuged, and the free plasma levels and brain concentrations of antiepileptic drugs were determined by immunofluorescence, using an Abbott TDx analyzer (Abbott). The
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plasma or brain levels of antiepileptic drugs were expressed in Ag/ml of plasma or Ag/g of wet brain tissue as mean F S.D. of at least eight determinations.
Fig. 2. Influence of ketotifen, given acutely or for 7 days, upon the protective activity of antiepileptic drugs against MES-induced seizures in mice. Carbamazepine (CBZ) and valproate (VPA) were given i.p. 30 min, phenobarbital (PB), 60 min and diphenylhydantoin (DPH), 120 min before testing. (A) Ketotifen in a single dose was given i.p. 60 min before testing. (B) Prolonged treatment of ketotifen. The data are ED50 values of antiepileptic drugs (with 95% confidence limits), calculated and compared according to Litchfield and Wilcoxon (1949). a P < 0.05 vs. antiepileptic drug alone. b P < 0.01 vs. antiepileptic drug alone. c P < 0.001 vs. antiepileptic drug alone.
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Fig. 3. Influence of antazoline, injected singly or following a 7-day treatment, upon motor impairment produced by conventional antiepileptic drugs. (A) Antazoline in a single dose. (B) Antazoline prolonged treatment. The data are TD50 values of antiepileptic drugs (with 95% confidence limits), calculated and compared according to Litchfield and Wilcoxon (1949). For abbreviations and treatment times see Fig. 1. a P < 0.05 vs. antiepileptic drug alone.
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Fig. 4. Influence of ketotifen, injected singly or following a 7-day treatment, upon motor impairment produced by conventional antiepileptic drugs. (A) Ketotifen in a single dose. (B) Ketotifen prolonged treatment. The data are TD50 values of antiepileptic drugs (with 95% confidence limits), calculated and compared according to Litchfield and Wilcoxon (1949). For abbreviations and treatment times see Fig. 2. a P < 0.05 vs. antiepileptic drug alone.
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2.7. Treatment protocol Experiments were carried out after acute or chronic treatment with antazoline or ketotifen. 1. Acute study: animals were injected with a single dose of H1 receptor antagonists and one of the antiepileptics at the time prior to the tests characterized above. Conventional antiepileptic drugs were tested at the time of their peak-anticonvulsant activity according to our previously published studies (Gasior et al., 1996; Wlaz´ et al., 1992, 1993), whilst the antazoline- and ketotifen-time of maximum activity were determined experimentally. 2. Chronic study: once a day (between 08:00 and 10:00 h) mice were injected as follows: group 1, saline for 6 days (control group); group 2, antazoline or ketotifen for 6 days (repeated doses). On the 7th day, mice from all groups received one of the conventional antiepileptic drugs (saline-treated group) or H1 receptor antagonists co-administered with one of the antiepileptics, at the same time, just like in the acute study. The CS50s of antazoline or ketotifen, ED50s and TD50s of antiepileptics alone or in combinations with H1-antagonists were calculated and statistically analyzed. Also, long-term memory and the free plasma or brain levels of antiepileptics were also evaluated in both experimental protocols.
by the Kruskal – Wallis test followed by Dunn’s test. Unpaired Student’s t-test was used for the statistical evaluation of the free plasma and brain levels of antiepileptic drugs.
3. Results 3.1. Effect of acute and chronic treatment with antazoline and ketotifen on the electroconvulsive threshold Antazoline, after acute and chronic treatment at the dose of 0.5 mg/kg, significantly reduced the electroconvulsive threshold. No effect was observed when antazoline was used at lower doses of 0.125 –0.25 mg/kg, 30 min before the test or up to 1 mg/kg, 60 min prior to electroconvulsions (Table 1). Ketotifen, given acutely or for 7 days, at the dose of 8 mg/kg markedly decreased the electroconvulsive threshold when given 60 min before the test. The CS50 value for ketotifen was 5.7 (5.2 –6.3) mA in comparison to 6.8 (6.1 – 7.5) mA of a control group. It was without effect upon this parameter at doses of 1– 4 mg/kg. When administered up to 8 mg/kg, 30 or 120 min before electroconvulsions, it did not affect the convulsive threshold (Table 2). 3.2. Influence of acute or repeated antazoline on the anticonvulsant activity of antiepileptic drugs against maximal electroshock-induced seizures in mice
2.8. Statistics CS50, ED50 and TD50 values and their statistical comparisons were calculated by computer probit analysis, according to Litchfield and Wilcoxon (1949). The results from the passive avoidance task were statistically verified
Antazoline (0.25 mg/kg), acutely and for 7 days, diminished the antiseizure activity of carbamazepine elevating its ED50 values from 13.7 (12.1 – 15.4) and 15.3 (13.4 – 17.6) mg/kg (control groups) to 31.9 (27.1 –37.6) and 24.6 (20.8 – 29.2) mg/kg, respectively (Fig. 1).
Table 3 Effects of ketotifen (after 1- or 7-day treatment) and antiepileptic drugs or their combinations on long-term memory in mice Drugs
Vehicle Ketotifen Carbamazepine Carbamazepine Carbamazepine Valproate Valproate Valproate Phenobarbital Phenobarbital Phenobarbital Diphenylhydantoin Diphenylhydantoin Diphenylhydantoin
Dosage (mg/kg)
Ketotifen
Acute
Chronic
– – 13.4 9.4 9.4 254 234 234 24.9 20.8 20.8 8.2 7.8 7.8
– – 11.0 14.9 14.9 256 260 260 23.2 27.8 27.8 10.0 10.4 10.4
– 4.0 – – 4.0 – – 4.0 – – 4.0 – – 4.0
Medians (25, 75 percentile) Acute
Chronic
180 180 180 180 180 28.5 63 89 128 180 153 180 180 180
180 (180, 180) 180 (170, 180) 180 (180, 180) 180 (180, 180) 180 (153, 180) 166 (66, 180) 115 (84.5, 163) 56 (46, 69.3)a,# 180 (91.5, 180) 180 (124, 180) 180 (147, 180) 180 (96, 180) 156 (104, 180) 180 (180, 180)
(180, 180) (175, 180) (118, 180) (166, 180) (146, 180) (14.3, 68.3)a (32.5, 180) (75.8, 180) (110, 175) (104, 180) (60.3, 180) (103, 180) (71, 180) (180, 180)
Presented values are the medians with 25 and 75 percentiles of 12 determinations. The retention was quantified as a time period (in seconds) the animals avoided the dark compartment. The results obtained from the passive avoidance task were statistically verified by the Kruskal – Wallis test followed by Dunn’s post-hoc test. For treatment times refer to Fig. 2. a P < 0.05 vs. control group. # P < 0.05 vs. valproate alone (260 mg/kg).
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Antazoline, in a single dose of 0.25 mg/kg did not affect the protective activity of diphenylhydantoin. It is noteworthy that the ED50 value of diphenylhydantoin was raised when it was co-administered with antazoline (0.25 mg/kg) in repeated doses, from 8.9 (6.8 – 11.6) to 12.6 (10.4 – 15.4) mg/kg (Fig. 1). However, the remaining antiepileptic drugs, co-administered with antazoline (0.25 mg/kg), after acute and chronic treatment, did not change their efficacy (Fig. 1). 3.3. Influence of ketotifen, after acute and chronic treatment, on the anticonvulsant activity of antiepileptic drugs against maximal electroshock-induced seizures in mice Ketotifen, injected acutely at the dose of 4 mg/kg, increased the anticonvulsant action of carbamazepine and phenobarbital, reflected by reductions of the respective ED50 values from 13.4 (11.7 –15.4) and 24.9 (22.5 –27.5) mg/kg to 7.9 (6.5 – 9.7) and 20.8 (18.9 – 22.9) mg/kg, respectively (Fig. 2). However, chronic ketotifen (4 mg/ kg) did not affect the protective action of phenobarbital. In contrast, ketotifen (4 mg/kg) given chronically diminished significantly the anticonvulsant action of carbamazepine, increasing its ED50 value from 11.0 (10.2 – 11.9) to 14.9 (13.3 –16.7) mg/kg. Neither ED50 of valproate nor that of diphenylhydantoin was changed when the drugs were coadministered with ketotifen (4 mg/kg), given acutely or for 7 days (Fig. 2). 3.4. Chimney test Acute antazoline (0.25 mg/kg) decreased the TD50 value of valproate from 375 (339 –414) to 329 (304 –355) while given chronically, it increased this value, from 376 (355 – 398) mg/kg to 409 (391 – 428) mg/kg. However, this H1 receptor antagonist did not intensify motor impairment after acute or chronic co-administration with carbamazepine, phenobarbital or diphenylhydantoin (Fig. 3). Ketotifen (4 mg/kg), administered in a single dose, further impaired motor coordination in mice treated with valproate, phenobarbital or diphenylhydantoin, which resulted in reductions of the TD50 values from 426 (398 – 456.3), 99.3 (86.8 – 113.5) and 79.1 (75.1 – 83.3) mg/kg to 384 (353 – 415), 76.7 (64.2 –91.5) and 72.7 (68.1 – 77.7) mg/kg, respectively. In contrast, chronic treatment with ketotifen (4 mg/kg) did not influence TD50 values of antiepileptic drugs (Fig. 4). 3.5. Passive avoidance task Acute antazoline (0.25 mg/kg) did not influence retention in the passive avoidance task. Carbamazepine, valproate, diphenylhydantoin and phenobarbital when applied at their ED50 values against MES-induced seizures or coadministered with antazoline, did not impair long-term memory in mice. Similiarly, antazoline (0.25 mg/kg), after 7-day treatment, alone or combined with the anticonvulsants produced no statistical worsening of long-term mem-
Fig. 5. Influence of antazoline, given acutely or for 7 days, upon the brain concentrations or free plasma levels of antiepileptic drugs. Presented values are the means (Ag/ml of plasma and Ag/g of brain wet tissue) of eight determinations F S.D. Unpaired Student’s t-test was used for statistical evaluation of the data. Blood or brain samples were taken at times scheduled for the convulsive test. For treatment times and abbreviations see also the legend of Fig. 1.
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ory (results not shown). Acute ketotifen (at 4 mg/kg), alone or combined with antiepileptic drugs at their ED50s against MES, did not affect the performance of mice in the passive
avoidance task. In contrast, valproate (254 mg/kg) administered alone at its ED50 against MES significantly disturbed this parameter. Moreover, chronic treatment with ketotifen (4 mg/kg) alone, as well as, co-administered with carbamazepine, diphenylhydantoin or phenobarbital did not affect long-term memory in mice. However, valproate (at doses providing a 50% protection against MES-induced seizures) combined with ketotifen (4 mg/kg), produced a significant impairment of the memory task (Table 3). This result was also significant when compared to valproate (260 mg/kg) alone. 3.6. Influence of antazoline and ketotifen on the free plasma and brain levels of antiepileptic drugs Antazoline (0.25 mg/kg), given acutely or chronically, did not affect either the free plasma levels or brain concentration of carbamazepine or diphenylhydantoin (Fig. 5). In addition, ketotifen (4 mg/kg), after acute or 7-day treatment, did not influence the free plasma or brain levels of carbamazepine and phenobarbital (Fig. 6).
4. Discussion
Fig. 6. Influence of ketotifen, given acutely or for 7 days, upon the brain concentrations or free plasma levels of antiepileptic drugs. Presented values are the means (Ag/ml of plasma and Ag/g of brain wet tissue) of eight determinations F S.D. Unpaired Student’s t-test was used for statistical evaluation of the data. Blood or brain samples were taken at times scheduled for the convulsive test. For treatment times and abbreviations see also the legend of Fig. 2.
The existing evidence indicates that there are about 30% of epileptic patients not satisfactorily controlled with existing antiepileptic drugs (Deckers et al., 2000). A possibility exists that at least some therapeutic failures in epileptic patients may result from undesired interactions between antiepileptics and other drugs, prescribed for reasons unrelated to epilepsy. Also, side effects of antiepileptic drugs may frequently be a cause of their withdrawal which is especially difficult in a situation of adequate seizure control (Deckers et al., 2000). Consequently, the present study was not only confined to the evaluation of drug interactions in terms of anticonvulsant efficacy but to adverse effects as well. In the present study, antazoline and ketotifen showed some proconvulsive activity. Both drugs reduced the electroconvulsive threshold, either after acute or chronic treatment. Moreover, antazoline (0.25 mg/kg) and ketotifen (4 mg/kg), at subeffective repeated doses, diminished the anticonvulsant activity of some antiepileptic drugs studied. Acute antazoline increased only the ED50 value of carbamazepine, while chronically injected it raised ED50s of carbamazepine and diphenylhydantoin. The interactions described between H1-antagonists and antiepileptic drugs were pharmacodynamic in nature. It is important to underline, that the ED50 value of carbamazepine when combined with acute or 7-day treatment antazoline (0.25 mg/kg) was about 2– 3-fold higher in comparison with control groups. The difference between diphenylhydantoin alone or coadministered with antazoline was about 50%. Ketotifen (4 mg/kg) administered acutely, enhanced the anticonvulsant action of carbamazepine and phenobarbital. In contrast, after
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chronic treatment with ketotifen (4 mg/kg) a reduction of the anticonvulsant activity of carbamazepine was observed. Antazoline (0.25 mg/kg) worsened motor coordination in mice when combined with valproate, only after acute treatment. Chronic treatment with antazoline resulted in improved motor performance in mice receiving valproate, which was reflected by the increased TD50 value of this antiepileptic drug. However, this H1 receptor antagonist did not influence passive avoidance task in mice. In contrast, acute ketotifen (4 mg/kg) co-administered with valproate, phenobarbital or diphenylhydantoin markedly raised their TD50 values, being without effect in this test after 7-day treatment. Further, valproate tested on long-term memory, after acute or 7-day treatment with ketotifen, impaired memory in mice when was administered alone or combined with this H1 receptor antagonist. Bearing in mind that H1 receptor antagonists affect different neurotransmitter systems, such as cholinergic, dopaminergic, serotoninergic and noradrenergic ones (Goodman and Gilman, 1960; Tuomisto and Tuomisto, 1980), it is difficult to consider all possible interactions. It is probable that an enhancement of the anticonvulsant activity of antiepileptics by acute ketotifen may result from an anticholinergic component. On the other hand, antazoline, after acute and chronic treatment, and ketotifen, administered repeatedly, diminished the antiseizure potency of antiepileptic drugs. As already mentioned, H1 receptor antagonists are proconvulsive in numerous experimental models of epilepsy such as MES (Yokoyama et al., 1993b,c), pentylenetetrazole-induced convulsions in mice (Gerald and Richter, 1976; Scherkl et al., 1991b) or amygdala-kindled seizures in rats (Chen et al., 2002; Kamei, 2001; Yokoyama et al., 1996). Moreover, metoprine and L-histidine, as well as, thioperamide, which elevate brain histamine levels, have demonstrated anticonvulsant activity against pentylenetetrazole-induced convulsions (Chen et al., 2000; Scherkl et al., 1991a,b; Vohora et al., 2001) or electroconvulsive seizures (Yokoyama et al., 1993b,c; Vohora et al., 2001). Furthermore, L-histidine decreases susceptibility to electrically induced convulsions in mice (Yokoyama et al., 1992) and also inhibits amygdala-kindled seizures in rats (Kamei, 2001; Kamei et al., 1998). Recently, Kamin´ski et al. (2003) have presented data, which indicates that L-histidine, but not the D-isomer of this amino acid, augmented the protective effects of carbamazepine or diphenylhydantoin against electroconvulsions. However, the roles of agents, elevating central histamine are controversial, since there are data in the literature about their ineffectiveness in amygdalakindled seizures (Borowicz et al., 2000; Yoshida et al., 2000). Generally, histamine acts in the central nervous system as an inhibitory neurotransmitter mostly via H1 receptors (Kamei et al., 2000). In conclusion, the present findings indicate that H1 receptor antagonists diminished the anticonvulsant activity of carbamazepine and diphenylhydantoin. It is important
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that the observed effects were evident after prolonged treatment with both antihistaminic drugs, and after acute treatment with antazoline. Considering thoroughly the results from the acute studies, it is clear that some antihistaminics co-applied with antiepileptic drugs may impair the seizure control in epileptic patients. Nevertheless, it seems evident that rather chronic co-administration of these drugs may give some adequate rationale for avoiding their combinations with antiepileptic drugs. For instance, the present results indicate that considering the anticonvulsant activity, negative interactions may be possible upon combinations of antazoline with carbamazepine and diphenylhydantoin or ketotifen with carbamazepine. Considering adverse effects, untoward interactions may accompany a co-administration of valproate with ketotifen.
Acknowledgements This study was supported by the grant No. P05A 044 18 from the State Committee for Scientific Research (Warszawa, Poland). The generous gift of valproate magnesium from ICN Polfa Rzeszo´w is appreciated.
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