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The effects of cimetidine chronic treatment on conventional antiepileptic drugs in mice
Pharmacological Reports 68 (2016) 283–288
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The effects of cimetidine chronic treatment on conventional antiepileptic drugs in mice Mariusz J. S´wia˛der a,*, Bartłomiej Barczyn´ski a, Michał Tomaszewski a, Katarzyna S´wia˛der b, Stanisław J. Czuczwar c,d a
Department of Experimental and Clinical Pharmacology, Medical University, Lublin, Poland Department of Applied Pharmacy, The Medical University of Lublin, Lublin, Poland Department of Pathophysiology, Medical University of Lublin, Lublin, Poland d Department of Physiopathology, Institute of Agricultural Medicine, Lublin, Poland b c
A R T I C L E I N F O
Article history: Received 7 April 2015 Received in revised form 17 September 2015 Accepted 22 September 2015 Available online 9 October 2015 Keywords: Cimetidine Antiepileptic drugs Electroshock maximal Seizures Drug interactions
A B S T R A C T
Purpose: The aim of this study was to evaluate the effects of 1-day, 7-day and 14-day administrations of cimetidine on the anticonvulsant activity of conventional antiepileptic drugs (AEDs; valproate, carbamazepine, phenytoin and phenobarbital) against maximal electroshock (MES)-induced convulsions in mice. Methods: Electroconvulsions were evoked in Albino Swiss mice by a current delivered via ear-clip electrodes. In addition, the effects of cimetidine, AEDs alone and their combinations were studied on performance and long-term memory tests. Pharmacokinetic changes in plasma and brain concentrations of AEDs after cimetidine administration were evaluated with immunofluorescence. Results: Cimetidine (up to 100 mg/kg) after 1-day administration did not affect the electroconvulsive threshold in animals. Moreover, in the 14-day treatment, cimetidine administered at a dose of 40 mg/kg did not significantly change the electroconvulsive threshold in the MES-test, cimetidine administered 14-day (at 20 mg/kg) significantly increased the anticonvulsant activity of carbamazepine, staying without effects after a 1-day and 7-day studies. In contrast, both the 7-day and 14-day administrations of cimetidine resulted in significant reductions of protective efficacy of the phenobarbital. Only valproate and phenytoin were not affected by cimetidine (20 mg/kg) in all experimental period. Cimetidine administered 1-day, did not alter total brain concentrations and free plasma levels of all AEDs tested, whilst the 14-day study elevated carbamazepine plasma and brain concentration and reduced phenobarbital brain concentration. Cimetidine co-applied with AEDs did not impair performance of mice evaluated in the chimney test however, it worsened long-term memory in animals. Conclusions: Based on this preclinical study, a special caution is advised when treating epileptic patients with combinations of phenobarbital or carbamazepine with cimetidine. ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
Introduction Histamine is present in humans at different concentrations in the majority of human body organs (i.e., brain, lungs, stomach, intestine, uterus, and ureters). Since the mid-seventies of the XX century, when Garbarg et al. [1] had traced an ascending histamine neuronal pathway, numbers of research studies on the role of histaminergic neurons in the brain have been launched. The widespread distribution and magnitude of the human histamine system suggests that it must be functionally important [2]. For
* Corresponding author. E-mail address: [email protected] (M.J. S´wia˛der).
many years, three main histamine receptor subtypes have been distinguished as follows: H1, H2 and H3. Noticeably, all these histamine receptor subtypes are present in the brain [3]. Relatively recently, Tasaka et al. [4] have confirmed the existence of a novel histamine receptor subtype (H4). Previously, it has been found that H1 receptor antagonists provoked seizures in rodents, or even epilepsy attacks in humans (especially in very young children). Pro-seizure effects of H1 receptor antagonists may probably be caused by an insufficient concentration of histamine in the brain. First, enunciation of possible harmful adverse effects of histamine receptor antagonists have been described just after their approval for clinical practice [5,6]. Also, preclinical experiments on animals have indicated that the reduction in brain histamine concentration may lead to a
http://dx.doi.org/10.1016/j.pharep.2015.09.009 1734-1140/ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
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decrease in the convulsive threshold in various models of epilepsy [7–9]. In contrast, an increase in the histamine level in the CNS inhibits both maximal electroshock (MES)- [10] and pentylenetetrazole-induced seizures in rodents [11]. Cimetidine and other H2 receptor antagonists are widely used in clinical practice, as an antiulcer drug, although, clinical reports indicate various incidences of adverse effects from the CNS, including headache, drowsiness, disturbance of consciousness, mental confusion, hallucination or even convulsions [12,13]. Numerous experimental and clinical studies have reported that the administration of cimetidine or other H2 histamine receptor antagonists may be associated with a higher risk of the appearance of convulsive attacks [14–17]. Moreover, it has been found that seizures evoked by cimetidine or other H2 histamine receptor antagonists were blocked by muscimol – a GABAA receptor agonist [18]. On the other hand, brain endogenous histamine plays a protective role during the development of seizures in pentylenetetrazole-kindled rats [19]. However, the brain concentration of endogenous histamine seemed to be independent on the administration of H2 receptor antagonists [20]. Relatively recently, Cannon et al. [20] have suggested that some cimetidine-like drugs (i.e., famotidine, tiotidine, ranitidine and improgan) do not produce seizures via H2 and GABAA receptors, but through other, as of yet unknown mechanisms that might be involved in CNS adverse effects. Accumulating clinical data indicates that H2 receptor antagonists may also produce convulsions, which are difficult to be treated with standard antiepileptic drugs (AEDs), but they can be successfully blocked with physostigmine [21] or thiopental [15]. Experimental studies have shown that cimetidine and ranitidine (another H2 receptor antagonist) were able to induce seizures in mice after their intracerebral injection [16]. Moreover, it has been documented that muscimol, aminooxyacetic acid (AOAA) and diamino-n-butyric acid reversed tonic convulsions induced by intraperitoneal injection of cimetidine [14]. The present study was aimed at examining the effects of the H2 receptor antagonist cimetidine upon the anticonvulsant potential of conventional AEDs against MES-induced seizures in mice. Cimetidine was administered either acutely (1-day) or in repeated doses, once daily for 7 and 14 days. In addition, the adverse-effect profiles of combinations of cimetidine with conventional AEDs were studied in the chimney test (motor coordination) and passive avoidance task (long-term memory) following 1-day and 14-day administration of this H2 receptor antagonist. The existence of possible pharmacokinetic interactions between this H2 antagonist and conventional AEDs was also verified in the plasma or brain of experimental animals by using fluorescent polarization immunoassay.
described in this study were approved by the Local Ethics Committee at the Medial University of Lublin and complied with the European Communities Council Directive of November 24th, 1986 (86/609/ EEC). Drugs The following drugs were used in this study: cimetidine (Polfa, Warszawa, Poland), valproate (magnesium salt, Dipromal, ICN Polfa, Rzeszo´w, Poland), carbamazepine (Amizepin), phenytoin (Phenytoinum), and phenobarbital (sodium salt, Luminalum Natrium, all three drugs from Polfa Warszawa, Poland). Valproate, cimetidine and phenobarbital were dissolved in distilled water, whereas carbamazepine and phenytoin were suspended in a 1% solution of Tween 80 (Sigma, St. Louis, MO, USA) in distilled water. All AEDs were administered intraperitoneally (ip) in a volume of 5 ml/kg body weight. Pretreatment times for valproate and carbamazepine were 30 min, phenobarbital and cimetidine – 60 min, and phenytoin – 120 min prior to the electroconvulsive and behavioral tests. Electroconvulsions Electroconvulsions were induced by applying alternating current (50 Hz; 500 V) via ear-clip electrodes from a rodent shocker generator (type 221; Hugo Sachs Elektronik, Freiburg, Germany). Stimulus duration was 0.2 s. Tonic hind limb extension was used as the endpoint. This apparatus was used to induce seizures in 2 methodologically different experimental approaches: MES seizure threshold test and MES seizure test [22]. MES seizure threshold test The MES seizure threshold test was used to assess the anticonvulsant effects of cimetidine administered alone. The convulsive threshold was evaluated as median current strength (CS50 in mA), which is necessary to produce tonic hind limb extension in 50% of the animals tested. To estimate the convulsive threshold, at least four groups of mice (8 animals per group) were challenged with electroshocks of various intensities. Statistical analysis of data was performed with one-way ANOVA followed by the post hoc Tukey–Kramer test for multiple comparisons. Cimetidine at a dose of 40 mg/kg, which did not significantly affect the seizure threshold in the MES seizure threshold test, was selected for testing in combination with the four AEDs in the MES seizure test. This approach allowed us to rule out any contribution of the intrinsic anticonvulsant efficacy of cimetidine in the effects observed in combination with the AEDs in the MES seizure test.
Materials and methods MES seizure test Animals and experimental conditions The 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 1 8C and the mice were kept in 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 and all tests were performed between 08:00 and 15:00 h. The procedures involving animals and their care were conducted in accordance with current European Community and Polish legislation on animal experimentation. Additionally, all efforts were made to minimize any animal suffering and to use only the number of animals necessary to produce reliable scientific data. The experimental protocols and procedures
In the MES seizure test, mice were challenged with a current of fixed intensity that was 4–5-fold higher than the CS50 value in controlled-treated mice [22]. To evaluate median effective doses (ED50, values, corresponding to the doses of AEDs, protecting 50% of the animals against tonic hind limb extension) for valproate, carbamazepine, phenytoin and phenobarbital, the animals pretreated with different doses of AEDs, alone or with cimetidine, were challenged with a MES (25 mA). At least four groups of mice, with 8 mice per group, were used to estimate each ED50 value (in mg/kg). A dose-effect curve was constructed, based on the percentage of mice protected against maximal electroconvulsions, and calculated according to the method of Litchfield and Wilcoxon [23] and one-way ANOVA followed by the post hoc Tukey–Kramer test for multiple comparisons.
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Chimney test Coordination impairment was evaluated with the chimney test of Boissier et al. [24]. In this test, animals had to climb backwards up a plastic tube (3 cm inner diameter, 25 cm in length). Coordination impairment was indicated by the inability of animals to climb backwards up the tube within 60 s. The mice were pretrained for 24 h before treatment and those unable to perform the test were rejected from the experimental groups. Median toxic doses (TD50 values) corresponding to the doses of AEDs required to produce coordination impairment in 50% of the animals tested, for conventional AEDs administered alone or in combination with cimetidine (at the highest dose tested in this study) were evaluated according to the method of Litchfield and Wilcoxon [23]. Passive avoidance acquisition and retention testing According to Venault et al. [25] the step-through passive avoidance task may be considered as a measure of long-term memory acquisition in rodents. We used this test to determine the effect of cimetidine and conventional AEDs administered alone or in combination on passive avoidance acquisition in mice. The animals were placed in an illuminated box (10 cm 13 cm 15 cm) connected to a larger (25 cm 20 cm 15 cm) dark compartment equipped with an electric grid floor. In this test, entry of the animals into the dark box was punished by an electric foot-shock (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 next day (24 h later), the same animals, without any 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 the medians with 25th and 75th percentiles.
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maximum anticonvulsant effect of corresponding AEDs. The time of administration of cimetidine was determined experimentally. Cimetidine was administered at the dose of 20 mg/kg, 60 min before the MES test. AEDs were injected as follows: phenytoin—120 min, phenobarbital—60 min, valproate and carbamazepine—30 min before electroconvulsions and behavioral tests. 2. Seven-day study: once a day (between 8.00 and 10.00 a.m.) the mice were injected with: group 1 – saline for 6 days (control group); group 2 – cimetidine (20 m/kg, ip) for 6 days. On the 7th day of experimentation, the mice from the first group received one of the AEDs + saline, while the mice from the second group received cimetidine (20 mg/kg) + one of the AEDs, at the same times as in the one-day study. 3. Fourteen-day study: once a day (between 8.00 and 10.00 a.m.) the mice were injected as follows: group 1 – was administered with saline for 13 days (control group); group 2 – cimetidine for 13 days. On the 14th day of experimentation, the mice from the first group received one of the AEDs + saline, while the mice from the second group received cimetidine (20 mg/kg, ip) + one of the AEDs, at the same times as in the one-day study. Statistics The CS50, ED50 and TD50 values (with 95% confidence limits) and their statistical comparisons were calculated by computer logprobity analysis according to Litchfield and Wilcoxon [23] and oneway ANOVA followed by the post hoc Tukey–Kramer test for multiple comparisons. The results from the passive avoidance task were statistically verified by the Kruskal–Wallis test followed by Dunn’s post hoc test. Unpaired Student’s t-test was used for the statistical evaluation of free plasma and brain concentrations of AEDs.
Measurement of free plasma levels and brain concentrations of AEDs Results Pharmacokinetic evaluation of free plasma levels and total brain AEDs concentrations were performed only for those combinations of cimetidine with AEDs for which the anticonvulsant effect in the MES seizure test was significantly greater than that for the control (an antiepileptic drug + vehicle-treated) animals. Thus, the measurement of free plasma levels and total brain concentrations of carbamazepine and phenobarbital were undertaken at the doses that corresponded to their ED50 values from the MES seizure test. Again, the animals were given either one of the studied AEDs + saline (control group) or the respective combinations of the cimetidine with an AED. Mice were decapitated at times scheduled for the MES and blood samples of approximately 1 ml were collected into heparinized Eppendorf tubes. The whole brains were removed from the skulls of the experimental animals, weighed, harvested and homogenized using Abbott buffer (1:2, w/v; Abbott Laboratories, North Chicago, IL, USA) in an Ultra-Turrax T8 homogenizer. Blood samples and brain homogenates were centrifuged at 10,000 g for 10 min. Next, plasma samples were pipetted into a micro-partition system, MPS-1 (Amicon, Danvers, MA, USA). Finally, free plasma samples or brain supernatants (at minimal values of 100 ml) were determined by immunofluorescence, using an Abbott TDx analyzer (Abbott) and reagents as described by the manufacturer. The free plasma and brain levels of AEDs were expressed in mg/ml of plasma and mg/g of wet brain tissue, respectively, as a means SD of at least eight determinations. Experimental design 1. One-day study: animals were injected with a single dose of cimetidine and one of the AEDs tested, at the times of peak
The effects of acute and chronic treatment with cimetidine in the electroconvulsive threshold Cimetidine (up to 100 mg/kg), administered acutely, 30 min prior to the test, did not affect the electroconvulsive threshold. No significant difference was observed in the electroconvulsive threshold of experimental animals when cimetidine was administered in the same doses, 60 and 120 min before the test (Table 1). Moreover, in the 14-day treatment, cimetidine administered at a dose of 40 mg/kg (60 min prior to the test) did not significantly change the electroconvulsive threshold (Table 1). Influences of acute and repeated administration of cimetidine on the anticonvulsant activity of AEDs against MES-induced seizures in mice Cimetidine administered acutely, at doses of 10 and 20 mg/kg, did not enhance the anticonvulsant action of carbamazepine, reducing its ED50 value from 14.5 (13.0–16.2) to 9.5 (8.1–11.3) and 8.9 (8.1–9.8) mg/kg, respectively (F[4;104] = 2.1078; p = 0.0763; Fig. 1A). In the 7-day studies, cimetidine (20 mg/kg) did not affect the protective action of carbamazepine. It is important to stress that the 14-day treatment with cimetidine (20 mg/kg) markedly potentiated the anticonvulsant activity of carbamazepine, which was associated with a reduction of its ED50 values from 13.4 (11.7–15.4) to 9.1 (8.2–10.1) mg/kg (p < 0.001; Fig. 1C). In contrast, the administration of cimetidine (20 mg/kg) during 7 or 14 days, diminished significantly the anticonvulsant action of phenobarbital, increasing its ED50 value from 24.3
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Table 1 Influence of 1-day and 14-day administration of cimetidine on the electroconvulsive threshold. Treatment (days)
Drugs (mg/kg)
Time before the test (min)
CS50 (mA)
1 day
Vehicle Cimetidine (20) Cimetidine (40) Cimetidine (100) F (3;100) = 1.040 Vehicle Cimetidine (20) Cimetidine (40) Cimetidine (100) F (3;76) = 0.3834 Vehicle Cimetidine (20) Cimetidine (40) Cimetidine (100) F (3;60) = 0.2246 Vehicle
30 30 30 30 p = 0.03783 60 60 60 60 p = 0.7652 120 120 120 120 p = 0.8789 60
7.1 6.4 7.2 7.6
(6.5–7.8) (5.9–6.9) (6.4–8.0) (7.2–8.1)
7.5 7.2 7.2 7.8
(6.5–8.6) (6.6–7.8) (6.7–7.8) (7.1–8.5)
7.1 6.8 7.0 6.6
(6.5–7.8) (6.3–7.3) (6.2–7.8) (6.2–7.1)
Cimetidine (20) Cimetidine (40) F (2;61) = 0.5991
60 60 p = 0.5525
5.4 (5.0–5.9) 5.1 (4.7–5.5)
14 days
5.8 (5.3–6.4)
Data are presented as median current strengths (CS50s with 95% confidence limits), necessary to produce tonic convulsions in 50% of the animals tested. Cimetidine was administered ip after single (one-day – 30, 60 and 120 min before the test) and 14-day treatments (60 min before the test on the 14th day administration). Control groups of animals received vehicle (1% of solution of Tween 80 in distilled water). Statistical analysis of data was performed with one-way ANOVA followed by the post hoc Tukey–Kramer test for multiple comparisons. F, statistics from one-way ANOVA; p, probability from one-way ANOVA.
(21.9–27.0) and 23.9 (21.5–26.5) to 29.1 (27.2–31.0; p < 0.01) and 31.3 (29.2–33.4; p < 0.001) mg/kg respectively (Fig. 1B and C). On the other hand, the 1-day studies have shown no significant interaction between these drugs. The ED50s of valproate and phenytoin were not affected following the administration of cimetidine (20 mg/kg), either in 1-day, 7-day or 14-day studies (data not shown).
Chimney test Neither 1-day nor 14-day cimetidine administrations (20 mg/ kg) affected coordination impairment of animals co-administered with carbamazepine, valproate, phenobarbital or phenytoin (data not shown). Passive avoidance task Cimetidine (20 mg/kg, administered alone in the 1-day or 14day studies) did not affect the retention time in the passive avoidance task. However, cimetidine (at 20 mg/kg, given 1-day) combined with carbamazepine, valproate and phenobarbital, at doses corresponding to their ED50s against MES, worsened the long-term memory in animals. In Table 2, there are no significant differences in valproate 265 mg/kg and phenytoin 9.9 or 10.9 mg/ kg and phenobarbital 24.7 mg/kg. In the 14-day study, the combinations of valproate (257 mg/kg) or phenobarbital (31.3 mg/kg) with cimetidine (20 mg/kg) significantly impaired long-term memory in mice. It is worth stressing that phenobarbital and valproate administered alone at doses of 31 mg/kg or 274 mg/kg respectively, also impairs long-term memory. In contrast, 14-day administration of cimetidine (20 mg/kg) with carbamazepine and phenytoin had no impact on long-term memory in mice (Table 2). The effects of cimetidine on free plasma and brain concentrations of AEDs Cimetidine (20 mg/kg), given acutely, affected neither the free plasma nor brain concentrations of carbamazepine or phenobarbital (Table 3). On the other hand, cimetidine (20 mg/kg) in the 7-day study, significantly reduced brain concentration of phenobarbital, did not have an effect on the plasma levels of this AED. Also, in the 7-day study, cimetidine did not influence the free plasma and brain levels of carbamazepine (Table 3). Furthermore, a 14-day administration of cimetidine (20 mg/kg) significantly increased free plasma levels and brain concentrations of
Fig. 1. Influence of cimetidine, administered acutely (1 day) and chronically (for 7 and 14 days) upon the protective activity of antiepileptic drugs against maximal electroshock-induced seizures. Carbamazepine (CBZ) administered ip 30 min, phenobarbital (PB) and cimetidine – 60 min before testing. (A) Cimetidine in a single dose. (B) 7-day treatment of cimetidine. (C) 14-day treatment of cimetidine. Data are presented as ED50 values of antiepileptic drugs (with 95% confidence limits) calculated and compared according to log-probit method by Litchfield and Wilcoxon [23] or one-way ANOVA followed by Tukey–Kramer post hoc test for multiple comparisons. **p < 0.01, ***p < 0.001 vs. an antiepileptic drug alone.
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Table 2 Effects of cimetidine (after 1-day or 14-day treatment), antiepileptic drugs and their combinations on the retention time in the passive avoidance task. Drugs
Vehicle Cimetidine CBZ CBZ CBZ VPA VPA VPA PB PB PB PHT PHT PHT
Dosage (mg/kg)
Cimetidine
1-day
14-day
– – 14.5 8.9 8.9 288 265 265 24.7 22.2 22.2 9.9 10.9 10.9
– – 13.4 9.1 9.1 274 257 257 23.9 31.3 31.3 9.5 10.9 10.9
– 20 – – 20 – – 20 – – 20 – – 20
Medians (25, 75 percentile) 1-day
14-day
180 (180, 180) 180 (180, 180) 125 (40, 180) 180 (180, 180) 65 (30, 90)* 45 (25, 90)** 85 (27.5, 170) 50 (10, 125)** 127.5 (50, 180) 110 (45, 180) 28.5 (20, 140)** 180 (170, 180) 140 (90, 170) 180 (180, 180)
180 (168, 180) 180 (180, 180) 180 (163, 180) 180 (100, 180) 180 (158, 180) 85 (25, 154)* 110 (23.8, 165) 87.5 (52.5, 180)* 158 (100, 180) 100 (34, 160)* 65 (40, 113)** 180 (173, 180) 170 (65, 180) 180 (145, 180)
Values are presented as median retention times (with 25th and 75th percentiles) of 12 separate determinations. The retention was quantified as a time period (in seconds) the animals avoided entering the dark compartment. Carbamazepine (CBZ) and valproate (VPA) were given ip 30 min, phenobarbital (PB) and cimetidine – 60 min and phenytoin (PHT) – 120 min before testing. The results obtained from the passive avoidance task were statistically verified by the Kruskal–Wallis nonparametric ANOVA test followed by Dunn’s post hoc test. * p < 0.05. ** p < 0.01 vs. control group (Vehicle).
carbamazepine, being simultaneously without any effect on the plasma and brain levels of phenobarbital (Table 3). Discussion Unsuccessfully controlled epilepsy is reported in about 30% of patients [26]. A part of the clinical failure may be caused by the use of medications for other reasons than epilepsy. For example, methylxanthine derivatives (especially, theophylline, pentoxifylline or caffeine) have been documented to sharply decrease the protective potential of conventional AEDs against MES-induced convulsions in mice [27,28]. Interestingly, chronic caffeine was far more potent in this respect than the acute administration of the methylxanthines [27,28]. Similarly, prolonged administration of histamine H1 receptor antagonists strongly reduced the anticonvulsant activity of conventional AEDs against MES-induced convulsions in mice [7,8]. Table 3 Influence of cimetidine, administered for 1 day, 7 days or 14 days upon the free plasma and brain concentrations of carbamazepine and phenobarbital. Treatment (days)
Drugs (mg/kg)
Brain concentration (mg/g)
Free plasma concentration (mg/ml)
1 day
CBZ (8.9) CBZ (8.9) + Cimetidine PB (22.2) PB (22.2) + Cimetidine
1.56 0.28 1.57 0.15 4.02 0.77 4.56 0.47
0.82 0.05 0.87 0.04 16.82 0.9 17.47 0.4
7 days
CBZ (12.5) CBZ (12.5) + Cimetidine PB (29.1) PB (29.1) + Cimetidine
4.67 0.83 4.70 0.46 8.8 0.36 8.0 0.44
1.45 0.14 1.65 0.33 23.65 1.51 25.04 0.88
14 days
CBZ (9.1) CBZ (9.1) + Cimetidine PB (31.3) PB (31.3) + Cimetidine
2.16 0.40 3.88 0.88*** 9.27 0.65 9.44 0.82
2.50 0.45 3.58 0.55*** 23.19 1.04 22.53 0.69*
Carbamazepine (CBZ) administered ip 30 mins, and phenobarbital (PB) – 60 mins before the plasma and brain sampling. Cimetidine in the day of experimentation administered ip, 60 mins before testing at the dose of 20 mg/kg. Presented values are the means (mg/ml of plasma or mg/g of brain wet tissue) SD of at least eight determinations. Unpaired Student’s t-test was used for statistical evaluation of data. Blood or brain samples were taken at times scheduled for the convulsive test. * p < 0.05. *** p < 0.001 vs. an antiepileptic drug alone.
The present study indicates that cimetidine (10 and 20 mg/kg) given singularly did not affect the anticonvulsant activity of carbamazepine. In addition, 7-day and 14-day applications of cimetidine (at a dose of 20 mg/kg), significantly diminished the anticonvulsant activity of phenobarbital. It is worth mentioning that the 14-day administration of cimetidine significantly raised the anticonvulsant activity of carbamazepine. Our previous study [29] showed that acute administration of cimetidine (at a dose of 20 mg/kg) enhanced the anticonvulsant activity of ethosuximide but did not affect the anticonvulsant activity of ethosuximide, valproate, clonazepam and phenobarbital when administered chronically against PTZ-induced convulsions. Moreover, a recent study indicated that single and repeated intraperitoneal administration of famotidine, another H2 histamine receptor antagonist, combined with valproate and phenytoin significantly enhanced the anticonvulsant activity of AEDs, but did not change valproate and phenytoin free plasma levels simultaneously elevated brain concentration of the first drug [30]. The 1-day and 14-day administrations of cimetidine (at 20 mg/ kg), both, alone and in combinations with conventional AEDs were devoid of any significant acute adverse effects evaluating in the chimney test with respect to coordination. On the other hand, cimetidine (in the 1-day and 14-day studies) impaired significantly long-term memory in the passive avoidance task when applied together with some AEDs tested. In this case, cimetidine shortened the retention time when co-administered with phenobarbital, valproate or carbamazepine. On the other hand, valproate and phenobarbital when administered alone also produced the impairment of coordination per se in mice. This fact indicates evidently that the memory impairment in animals cannot be entirely ascribed to cimetidine, since the effects of these AEDs alone or combined with this H2 receptor antagonist did not differ statistically. The results presented in analogy to earlier studies suggest that long-term memory impairment was observed after the administration of PB [7] and VPA [8,31] given alone in their dosages resulting from the ED50 values. These results may also confirm our previous publications, where H1 and H2 antagonists studied did not affect the memory in mice [8,30]. However, Taati et al. reported that cimetidine reversed the exercise-induced improvement in learning and memory in rats [32]. The experimental evidence indicates that central histamine plays a main role in cognitive function and it was shown to
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enhance memory in both the passive [33] and active avoidance tasks [34,35]. However, Eidi et al. [36] have shown that pyrilamine (a histamine H1 receptor antagonist) and cimetidine (a histamine H2 receptor antagonist) decreased memory retention in animals. Noticeably, cimetidine is a well-know inhibitor of numerous isoforms of hepatic enzymes (cytochrome P450), and thus, it may increase plasma levels of co-administered AEDs [37]. In the present study, we observed a decrease in brain phenobarbital concentration following the 14-day administration of cimetidine. It should be stressed that pharmacokinetic interaction observed in this study reflected the brain concentration of AED, not plasma levels in mice. This discrepancy probably results from the changed blood– brain barrier permeability mediated by cimetidine. Previously, it has been found experimentally that some H1 and H2 receptor antagonists changed the permeability of blood–brain barrier and thereby, they influenced the distribution of histamine in the histaminergic neurons [38]. Hence, it is possible that the 14-day administration of cimetidine could decrease the penetration of PB through the blood–brain barrier, leading finally to a reduced brain phenobarbital concentration. In light of this fact, it is worth noting that the brain concentration of carbamazepine was not affected by the 7-day administration of cimetidine, whereas the 14-day treatment with cimetidine significantly elevated both free plasma and total brain concentrations of carbamazepine. This may be related to the relatively long periods of therapy (when comparing the opposite effects observed for a 7-day administration of cimetidine). This observed discrepancy may result from pharmacokinetic interactions related to the inhibition of hepatic cytochrome P450 enzymes. However, the inhibitory effect of cimetidine on CYP3A4 is not substantial, and the interaction with CBZ is of a modest clinical significance [37]. Summing up, the present study provides evidence that the chronic 14 day treatment with cimetidine may result in a decrease of the electroconvulsive threshold in animals. Moreover, the coadministration of conventional AEDs with cimetidine affected the anticonvulsant effects of carbamazepine and phenobarbital, but not those of valproate and phenytoin. In such cases, special caution is advised when combining cimetidine with conventional AEDs. Conflict of interest The authors declare no conflict of interests. Acknowledgements This study was supported by grants from the State Committee for Scientific Research (No. P05A 044 18, Warsaw, Poland) and Medical University of Lublin. References [1] Garbarg M, Barbin G, Feger J, Schwartz JC. Histaminergic pathway in rat brain evidenced by lesions of the medial forebrain bundle. Science 1974;186:833–5. [2] Panula P, Airaksinen MS, Pirvola U, Kotilainen E. A histamine-containing neuronal system in human brain. Neuroscience 1990;34:127–32. [3] Hill SJ. Distribution properties, and functional characteristics of three classes of histamine receptor. Pharmacol Rev 1990;42:45–83. [4] Tasaka K, Mio M, Akagi M, Saito T. Histamine release induced by histone and related morphological changes in mast cells. Agents Actions 1990;30:114–7. [5] Churchull JA, Gammon GD. The effect of antihistaminic drugs on convulsive seizures. J Am Med Assoc 1949;141:18–21. [6] Wynagaarden JB, Seevers MH. The toxic effects of antihistaminic drugs. J Am Med Assoc 1951;145:277–82. [7] S´wia˛der M, Wielosz M, Czuczwar SJ. Interaction of astemizole, an H1 receptor antagonist, with conventional antiepileptic drugs in mice. Pharmacol Biochem Behav 2003;76:169–78.
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Interaction of topiramate with conventional antiepileptic drugs in mice. Eur J Pharmacol 2000;399:35–41. [32] Taati M, Moghaddasi M, Esmaeili M, Pourkhodadad S, Nayebzadeh H. The role of the central histaminergic receptors in the exercise-induced improvements of the spatial learning and memory in rats. Brain Res 2014;1587:112–8. [33] de Almeida MA, Izquierdo I. Memory facilitation by histamine. Arch Int Pharmacodyn Ther 1986;283:193–8. [34] Kamei C, Okumura Y, Tasaka K. Influence of histamine depletion on learning and memory recollection in rats. Psychopharmacology (Berl) 1993;111: 376–82. [35] Miyazaki S, Imaizumi M, Onodera K. Effects of thioperamide on the cholinergic system and the step-through passive avoidance test in mice. Methods Find Exp Clin Pharmacol 1995;17:653–8. [36] Eidi M, Zarrindast MR, Eidi A, Oryan S, Parivar K. Effects of histamine and cholinergic systems on memory retention of passive avoidance learning in rats. Eur J Pharmacol 2003;465:91–6. 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The effects of cimetidine chronic treatment on conventional antiepileptic drugs in mice Mariusz J. S´wia˛der a,*, Bartłomiej Barczyn´ski a, Michał Tomaszewski a, Katarzyna S´wia˛der b, Stanisław J. Czuczwar c,d a
Department of Experimental and Clinical Pharmacology, Medical University, Lublin, Poland Department of Applied Pharmacy, The Medical University of Lublin, Lublin, Poland Department of Pathophysiology, Medical University of Lublin, Lublin, Poland d Department of Physiopathology, Institute of Agricultural Medicine, Lublin, Poland b c
A R T I C L E I N F O
Article history: Received 7 April 2015 Received in revised form 17 September 2015 Accepted 22 September 2015 Available online 9 October 2015 Keywords: Cimetidine Antiepileptic drugs Electroshock maximal Seizures Drug interactions
A B S T R A C T
Purpose: The aim of this study was to evaluate the effects of 1-day, 7-day and 14-day administrations of cimetidine on the anticonvulsant activity of conventional antiepileptic drugs (AEDs; valproate, carbamazepine, phenytoin and phenobarbital) against maximal electroshock (MES)-induced convulsions in mice. Methods: Electroconvulsions were evoked in Albino Swiss mice by a current delivered via ear-clip electrodes. In addition, the effects of cimetidine, AEDs alone and their combinations were studied on performance and long-term memory tests. Pharmacokinetic changes in plasma and brain concentrations of AEDs after cimetidine administration were evaluated with immunofluorescence. Results: Cimetidine (up to 100 mg/kg) after 1-day administration did not affect the electroconvulsive threshold in animals. Moreover, in the 14-day treatment, cimetidine administered at a dose of 40 mg/kg did not significantly change the electroconvulsive threshold in the MES-test, cimetidine administered 14-day (at 20 mg/kg) significantly increased the anticonvulsant activity of carbamazepine, staying without effects after a 1-day and 7-day studies. In contrast, both the 7-day and 14-day administrations of cimetidine resulted in significant reductions of protective efficacy of the phenobarbital. Only valproate and phenytoin were not affected by cimetidine (20 mg/kg) in all experimental period. Cimetidine administered 1-day, did not alter total brain concentrations and free plasma levels of all AEDs tested, whilst the 14-day study elevated carbamazepine plasma and brain concentration and reduced phenobarbital brain concentration. Cimetidine co-applied with AEDs did not impair performance of mice evaluated in the chimney test however, it worsened long-term memory in animals. Conclusions: Based on this preclinical study, a special caution is advised when treating epileptic patients with combinations of phenobarbital or carbamazepine with cimetidine. ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
Introduction Histamine is present in humans at different concentrations in the majority of human body organs (i.e., brain, lungs, stomach, intestine, uterus, and ureters). Since the mid-seventies of the XX century, when Garbarg et al. [1] had traced an ascending histamine neuronal pathway, numbers of research studies on the role of histaminergic neurons in the brain have been launched. The widespread distribution and magnitude of the human histamine system suggests that it must be functionally important [2]. For
* Corresponding author. E-mail address: [email protected] (M.J. S´wia˛der).
many years, three main histamine receptor subtypes have been distinguished as follows: H1, H2 and H3. Noticeably, all these histamine receptor subtypes are present in the brain [3]. Relatively recently, Tasaka et al. [4] have confirmed the existence of a novel histamine receptor subtype (H4). Previously, it has been found that H1 receptor antagonists provoked seizures in rodents, or even epilepsy attacks in humans (especially in very young children). Pro-seizure effects of H1 receptor antagonists may probably be caused by an insufficient concentration of histamine in the brain. First, enunciation of possible harmful adverse effects of histamine receptor antagonists have been described just after their approval for clinical practice [5,6]. Also, preclinical experiments on animals have indicated that the reduction in brain histamine concentration may lead to a
http://dx.doi.org/10.1016/j.pharep.2015.09.009 1734-1140/ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
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decrease in the convulsive threshold in various models of epilepsy [7–9]. In contrast, an increase in the histamine level in the CNS inhibits both maximal electroshock (MES)- [10] and pentylenetetrazole-induced seizures in rodents [11]. Cimetidine and other H2 receptor antagonists are widely used in clinical practice, as an antiulcer drug, although, clinical reports indicate various incidences of adverse effects from the CNS, including headache, drowsiness, disturbance of consciousness, mental confusion, hallucination or even convulsions [12,13]. Numerous experimental and clinical studies have reported that the administration of cimetidine or other H2 histamine receptor antagonists may be associated with a higher risk of the appearance of convulsive attacks [14–17]. Moreover, it has been found that seizures evoked by cimetidine or other H2 histamine receptor antagonists were blocked by muscimol – a GABAA receptor agonist [18]. On the other hand, brain endogenous histamine plays a protective role during the development of seizures in pentylenetetrazole-kindled rats [19]. However, the brain concentration of endogenous histamine seemed to be independent on the administration of H2 receptor antagonists [20]. Relatively recently, Cannon et al. [20] have suggested that some cimetidine-like drugs (i.e., famotidine, tiotidine, ranitidine and improgan) do not produce seizures via H2 and GABAA receptors, but through other, as of yet unknown mechanisms that might be involved in CNS adverse effects. Accumulating clinical data indicates that H2 receptor antagonists may also produce convulsions, which are difficult to be treated with standard antiepileptic drugs (AEDs), but they can be successfully blocked with physostigmine [21] or thiopental [15]. Experimental studies have shown that cimetidine and ranitidine (another H2 receptor antagonist) were able to induce seizures in mice after their intracerebral injection [16]. Moreover, it has been documented that muscimol, aminooxyacetic acid (AOAA) and diamino-n-butyric acid reversed tonic convulsions induced by intraperitoneal injection of cimetidine [14]. The present study was aimed at examining the effects of the H2 receptor antagonist cimetidine upon the anticonvulsant potential of conventional AEDs against MES-induced seizures in mice. Cimetidine was administered either acutely (1-day) or in repeated doses, once daily for 7 and 14 days. In addition, the adverse-effect profiles of combinations of cimetidine with conventional AEDs were studied in the chimney test (motor coordination) and passive avoidance task (long-term memory) following 1-day and 14-day administration of this H2 receptor antagonist. The existence of possible pharmacokinetic interactions between this H2 antagonist and conventional AEDs was also verified in the plasma or brain of experimental animals by using fluorescent polarization immunoassay.
described in this study were approved by the Local Ethics Committee at the Medial University of Lublin and complied with the European Communities Council Directive of November 24th, 1986 (86/609/ EEC). Drugs The following drugs were used in this study: cimetidine (Polfa, Warszawa, Poland), valproate (magnesium salt, Dipromal, ICN Polfa, Rzeszo´w, Poland), carbamazepine (Amizepin), phenytoin (Phenytoinum), and phenobarbital (sodium salt, Luminalum Natrium, all three drugs from Polfa Warszawa, Poland). Valproate, cimetidine and phenobarbital were dissolved in distilled water, whereas carbamazepine and phenytoin were suspended in a 1% solution of Tween 80 (Sigma, St. Louis, MO, USA) in distilled water. All AEDs were administered intraperitoneally (ip) in a volume of 5 ml/kg body weight. Pretreatment times for valproate and carbamazepine were 30 min, phenobarbital and cimetidine – 60 min, and phenytoin – 120 min prior to the electroconvulsive and behavioral tests. Electroconvulsions Electroconvulsions were induced by applying alternating current (50 Hz; 500 V) via ear-clip electrodes from a rodent shocker generator (type 221; Hugo Sachs Elektronik, Freiburg, Germany). Stimulus duration was 0.2 s. Tonic hind limb extension was used as the endpoint. This apparatus was used to induce seizures in 2 methodologically different experimental approaches: MES seizure threshold test and MES seizure test [22]. MES seizure threshold test The MES seizure threshold test was used to assess the anticonvulsant effects of cimetidine administered alone. The convulsive threshold was evaluated as median current strength (CS50 in mA), which is necessary to produce tonic hind limb extension in 50% of the animals tested. To estimate the convulsive threshold, at least four groups of mice (8 animals per group) were challenged with electroshocks of various intensities. Statistical analysis of data was performed with one-way ANOVA followed by the post hoc Tukey–Kramer test for multiple comparisons. Cimetidine at a dose of 40 mg/kg, which did not significantly affect the seizure threshold in the MES seizure threshold test, was selected for testing in combination with the four AEDs in the MES seizure test. This approach allowed us to rule out any contribution of the intrinsic anticonvulsant efficacy of cimetidine in the effects observed in combination with the AEDs in the MES seizure test.
Materials and methods MES seizure test Animals and experimental conditions The 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 1 8C and the mice were kept in 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 and all tests were performed between 08:00 and 15:00 h. The procedures involving animals and their care were conducted in accordance with current European Community and Polish legislation on animal experimentation. Additionally, all efforts were made to minimize any animal suffering and to use only the number of animals necessary to produce reliable scientific data. The experimental protocols and procedures
In the MES seizure test, mice were challenged with a current of fixed intensity that was 4–5-fold higher than the CS50 value in controlled-treated mice [22]. To evaluate median effective doses (ED50, values, corresponding to the doses of AEDs, protecting 50% of the animals against tonic hind limb extension) for valproate, carbamazepine, phenytoin and phenobarbital, the animals pretreated with different doses of AEDs, alone or with cimetidine, were challenged with a MES (25 mA). At least four groups of mice, with 8 mice per group, were used to estimate each ED50 value (in mg/kg). A dose-effect curve was constructed, based on the percentage of mice protected against maximal electroconvulsions, and calculated according to the method of Litchfield and Wilcoxon [23] and one-way ANOVA followed by the post hoc Tukey–Kramer test for multiple comparisons.
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Chimney test Coordination impairment was evaluated with the chimney test of Boissier et al. [24]. In this test, animals had to climb backwards up a plastic tube (3 cm inner diameter, 25 cm in length). Coordination impairment was indicated by the inability of animals to climb backwards up the tube within 60 s. The mice were pretrained for 24 h before treatment and those unable to perform the test were rejected from the experimental groups. Median toxic doses (TD50 values) corresponding to the doses of AEDs required to produce coordination impairment in 50% of the animals tested, for conventional AEDs administered alone or in combination with cimetidine (at the highest dose tested in this study) were evaluated according to the method of Litchfield and Wilcoxon [23]. Passive avoidance acquisition and retention testing According to Venault et al. [25] the step-through passive avoidance task may be considered as a measure of long-term memory acquisition in rodents. We used this test to determine the effect of cimetidine and conventional AEDs administered alone or in combination on passive avoidance acquisition in mice. The animals were placed in an illuminated box (10 cm 13 cm 15 cm) connected to a larger (25 cm 20 cm 15 cm) dark compartment equipped with an electric grid floor. In this test, entry of the animals into the dark box was punished by an electric foot-shock (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 next day (24 h later), the same animals, without any 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 the medians with 25th and 75th percentiles.
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maximum anticonvulsant effect of corresponding AEDs. The time of administration of cimetidine was determined experimentally. Cimetidine was administered at the dose of 20 mg/kg, 60 min before the MES test. AEDs were injected as follows: phenytoin—120 min, phenobarbital—60 min, valproate and carbamazepine—30 min before electroconvulsions and behavioral tests. 2. Seven-day study: once a day (between 8.00 and 10.00 a.m.) the mice were injected with: group 1 – saline for 6 days (control group); group 2 – cimetidine (20 m/kg, ip) for 6 days. On the 7th day of experimentation, the mice from the first group received one of the AEDs + saline, while the mice from the second group received cimetidine (20 mg/kg) + one of the AEDs, at the same times as in the one-day study. 3. Fourteen-day study: once a day (between 8.00 and 10.00 a.m.) the mice were injected as follows: group 1 – was administered with saline for 13 days (control group); group 2 – cimetidine for 13 days. On the 14th day of experimentation, the mice from the first group received one of the AEDs + saline, while the mice from the second group received cimetidine (20 mg/kg, ip) + one of the AEDs, at the same times as in the one-day study. Statistics The CS50, ED50 and TD50 values (with 95% confidence limits) and their statistical comparisons were calculated by computer logprobity analysis according to Litchfield and Wilcoxon [23] and oneway ANOVA followed by the post hoc Tukey–Kramer test for multiple comparisons. The results from the passive avoidance task were statistically verified by the Kruskal–Wallis test followed by Dunn’s post hoc test. Unpaired Student’s t-test was used for the statistical evaluation of free plasma and brain concentrations of AEDs.
Measurement of free plasma levels and brain concentrations of AEDs Results Pharmacokinetic evaluation of free plasma levels and total brain AEDs concentrations were performed only for those combinations of cimetidine with AEDs for which the anticonvulsant effect in the MES seizure test was significantly greater than that for the control (an antiepileptic drug + vehicle-treated) animals. Thus, the measurement of free plasma levels and total brain concentrations of carbamazepine and phenobarbital were undertaken at the doses that corresponded to their ED50 values from the MES seizure test. Again, the animals were given either one of the studied AEDs + saline (control group) or the respective combinations of the cimetidine with an AED. Mice were decapitated at times scheduled for the MES and blood samples of approximately 1 ml were collected into heparinized Eppendorf tubes. The whole brains were removed from the skulls of the experimental animals, weighed, harvested and homogenized using Abbott buffer (1:2, w/v; Abbott Laboratories, North Chicago, IL, USA) in an Ultra-Turrax T8 homogenizer. Blood samples and brain homogenates were centrifuged at 10,000 g for 10 min. Next, plasma samples were pipetted into a micro-partition system, MPS-1 (Amicon, Danvers, MA, USA). Finally, free plasma samples or brain supernatants (at minimal values of 100 ml) were determined by immunofluorescence, using an Abbott TDx analyzer (Abbott) and reagents as described by the manufacturer. The free plasma and brain levels of AEDs were expressed in mg/ml of plasma and mg/g of wet brain tissue, respectively, as a means SD of at least eight determinations. Experimental design 1. One-day study: animals were injected with a single dose of cimetidine and one of the AEDs tested, at the times of peak
The effects of acute and chronic treatment with cimetidine in the electroconvulsive threshold Cimetidine (up to 100 mg/kg), administered acutely, 30 min prior to the test, did not affect the electroconvulsive threshold. No significant difference was observed in the electroconvulsive threshold of experimental animals when cimetidine was administered in the same doses, 60 and 120 min before the test (Table 1). Moreover, in the 14-day treatment, cimetidine administered at a dose of 40 mg/kg (60 min prior to the test) did not significantly change the electroconvulsive threshold (Table 1). Influences of acute and repeated administration of cimetidine on the anticonvulsant activity of AEDs against MES-induced seizures in mice Cimetidine administered acutely, at doses of 10 and 20 mg/kg, did not enhance the anticonvulsant action of carbamazepine, reducing its ED50 value from 14.5 (13.0–16.2) to 9.5 (8.1–11.3) and 8.9 (8.1–9.8) mg/kg, respectively (F[4;104] = 2.1078; p = 0.0763; Fig. 1A). In the 7-day studies, cimetidine (20 mg/kg) did not affect the protective action of carbamazepine. It is important to stress that the 14-day treatment with cimetidine (20 mg/kg) markedly potentiated the anticonvulsant activity of carbamazepine, which was associated with a reduction of its ED50 values from 13.4 (11.7–15.4) to 9.1 (8.2–10.1) mg/kg (p < 0.001; Fig. 1C). In contrast, the administration of cimetidine (20 mg/kg) during 7 or 14 days, diminished significantly the anticonvulsant action of phenobarbital, increasing its ED50 value from 24.3
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Table 1 Influence of 1-day and 14-day administration of cimetidine on the electroconvulsive threshold. Treatment (days)
Drugs (mg/kg)
Time before the test (min)
CS50 (mA)
1 day
Vehicle Cimetidine (20) Cimetidine (40) Cimetidine (100) F (3;100) = 1.040 Vehicle Cimetidine (20) Cimetidine (40) Cimetidine (100) F (3;76) = 0.3834 Vehicle Cimetidine (20) Cimetidine (40) Cimetidine (100) F (3;60) = 0.2246 Vehicle
30 30 30 30 p = 0.03783 60 60 60 60 p = 0.7652 120 120 120 120 p = 0.8789 60
7.1 6.4 7.2 7.6
(6.5–7.8) (5.9–6.9) (6.4–8.0) (7.2–8.1)
7.5 7.2 7.2 7.8
(6.5–8.6) (6.6–7.8) (6.7–7.8) (7.1–8.5)
7.1 6.8 7.0 6.6
(6.5–7.8) (6.3–7.3) (6.2–7.8) (6.2–7.1)
Cimetidine (20) Cimetidine (40) F (2;61) = 0.5991
60 60 p = 0.5525
5.4 (5.0–5.9) 5.1 (4.7–5.5)
14 days
5.8 (5.3–6.4)
Data are presented as median current strengths (CS50s with 95% confidence limits), necessary to produce tonic convulsions in 50% of the animals tested. Cimetidine was administered ip after single (one-day – 30, 60 and 120 min before the test) and 14-day treatments (60 min before the test on the 14th day administration). Control groups of animals received vehicle (1% of solution of Tween 80 in distilled water). Statistical analysis of data was performed with one-way ANOVA followed by the post hoc Tukey–Kramer test for multiple comparisons. F, statistics from one-way ANOVA; p, probability from one-way ANOVA.
(21.9–27.0) and 23.9 (21.5–26.5) to 29.1 (27.2–31.0; p < 0.01) and 31.3 (29.2–33.4; p < 0.001) mg/kg respectively (Fig. 1B and C). On the other hand, the 1-day studies have shown no significant interaction between these drugs. The ED50s of valproate and phenytoin were not affected following the administration of cimetidine (20 mg/kg), either in 1-day, 7-day or 14-day studies (data not shown).
Chimney test Neither 1-day nor 14-day cimetidine administrations (20 mg/ kg) affected coordination impairment of animals co-administered with carbamazepine, valproate, phenobarbital or phenytoin (data not shown). Passive avoidance task Cimetidine (20 mg/kg, administered alone in the 1-day or 14day studies) did not affect the retention time in the passive avoidance task. However, cimetidine (at 20 mg/kg, given 1-day) combined with carbamazepine, valproate and phenobarbital, at doses corresponding to their ED50s against MES, worsened the long-term memory in animals. In Table 2, there are no significant differences in valproate 265 mg/kg and phenytoin 9.9 or 10.9 mg/ kg and phenobarbital 24.7 mg/kg. In the 14-day study, the combinations of valproate (257 mg/kg) or phenobarbital (31.3 mg/kg) with cimetidine (20 mg/kg) significantly impaired long-term memory in mice. It is worth stressing that phenobarbital and valproate administered alone at doses of 31 mg/kg or 274 mg/kg respectively, also impairs long-term memory. In contrast, 14-day administration of cimetidine (20 mg/kg) with carbamazepine and phenytoin had no impact on long-term memory in mice (Table 2). The effects of cimetidine on free plasma and brain concentrations of AEDs Cimetidine (20 mg/kg), given acutely, affected neither the free plasma nor brain concentrations of carbamazepine or phenobarbital (Table 3). On the other hand, cimetidine (20 mg/kg) in the 7-day study, significantly reduced brain concentration of phenobarbital, did not have an effect on the plasma levels of this AED. Also, in the 7-day study, cimetidine did not influence the free plasma and brain levels of carbamazepine (Table 3). Furthermore, a 14-day administration of cimetidine (20 mg/kg) significantly increased free plasma levels and brain concentrations of
Fig. 1. Influence of cimetidine, administered acutely (1 day) and chronically (for 7 and 14 days) upon the protective activity of antiepileptic drugs against maximal electroshock-induced seizures. Carbamazepine (CBZ) administered ip 30 min, phenobarbital (PB) and cimetidine – 60 min before testing. (A) Cimetidine in a single dose. (B) 7-day treatment of cimetidine. (C) 14-day treatment of cimetidine. Data are presented as ED50 values of antiepileptic drugs (with 95% confidence limits) calculated and compared according to log-probit method by Litchfield and Wilcoxon [23] or one-way ANOVA followed by Tukey–Kramer post hoc test for multiple comparisons. **p < 0.01, ***p < 0.001 vs. an antiepileptic drug alone.
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Table 2 Effects of cimetidine (after 1-day or 14-day treatment), antiepileptic drugs and their combinations on the retention time in the passive avoidance task. Drugs
Vehicle Cimetidine CBZ CBZ CBZ VPA VPA VPA PB PB PB PHT PHT PHT
Dosage (mg/kg)
Cimetidine
1-day
14-day
– – 14.5 8.9 8.9 288 265 265 24.7 22.2 22.2 9.9 10.9 10.9
– – 13.4 9.1 9.1 274 257 257 23.9 31.3 31.3 9.5 10.9 10.9
– 20 – – 20 – – 20 – – 20 – – 20
Medians (25, 75 percentile) 1-day
14-day
180 (180, 180) 180 (180, 180) 125 (40, 180) 180 (180, 180) 65 (30, 90)* 45 (25, 90)** 85 (27.5, 170) 50 (10, 125)** 127.5 (50, 180) 110 (45, 180) 28.5 (20, 140)** 180 (170, 180) 140 (90, 170) 180 (180, 180)
180 (168, 180) 180 (180, 180) 180 (163, 180) 180 (100, 180) 180 (158, 180) 85 (25, 154)* 110 (23.8, 165) 87.5 (52.5, 180)* 158 (100, 180) 100 (34, 160)* 65 (40, 113)** 180 (173, 180) 170 (65, 180) 180 (145, 180)
Values are presented as median retention times (with 25th and 75th percentiles) of 12 separate determinations. The retention was quantified as a time period (in seconds) the animals avoided entering the dark compartment. Carbamazepine (CBZ) and valproate (VPA) were given ip 30 min, phenobarbital (PB) and cimetidine – 60 min and phenytoin (PHT) – 120 min before testing. The results obtained from the passive avoidance task were statistically verified by the Kruskal–Wallis nonparametric ANOVA test followed by Dunn’s post hoc test. * p < 0.05. ** p < 0.01 vs. control group (Vehicle).
carbamazepine, being simultaneously without any effect on the plasma and brain levels of phenobarbital (Table 3). Discussion Unsuccessfully controlled epilepsy is reported in about 30% of patients [26]. A part of the clinical failure may be caused by the use of medications for other reasons than epilepsy. For example, methylxanthine derivatives (especially, theophylline, pentoxifylline or caffeine) have been documented to sharply decrease the protective potential of conventional AEDs against MES-induced convulsions in mice [27,28]. Interestingly, chronic caffeine was far more potent in this respect than the acute administration of the methylxanthines [27,28]. Similarly, prolonged administration of histamine H1 receptor antagonists strongly reduced the anticonvulsant activity of conventional AEDs against MES-induced convulsions in mice [7,8]. Table 3 Influence of cimetidine, administered for 1 day, 7 days or 14 days upon the free plasma and brain concentrations of carbamazepine and phenobarbital. Treatment (days)
Drugs (mg/kg)
Brain concentration (mg/g)
Free plasma concentration (mg/ml)
1 day
CBZ (8.9) CBZ (8.9) + Cimetidine PB (22.2) PB (22.2) + Cimetidine
1.56 0.28 1.57 0.15 4.02 0.77 4.56 0.47
0.82 0.05 0.87 0.04 16.82 0.9 17.47 0.4
7 days
CBZ (12.5) CBZ (12.5) + Cimetidine PB (29.1) PB (29.1) + Cimetidine
4.67 0.83 4.70 0.46 8.8 0.36 8.0 0.44
1.45 0.14 1.65 0.33 23.65 1.51 25.04 0.88
14 days
CBZ (9.1) CBZ (9.1) + Cimetidine PB (31.3) PB (31.3) + Cimetidine
2.16 0.40 3.88 0.88*** 9.27 0.65 9.44 0.82
2.50 0.45 3.58 0.55*** 23.19 1.04 22.53 0.69*
Carbamazepine (CBZ) administered ip 30 mins, and phenobarbital (PB) – 60 mins before the plasma and brain sampling. Cimetidine in the day of experimentation administered ip, 60 mins before testing at the dose of 20 mg/kg. Presented values are the means (mg/ml of plasma or mg/g of brain wet tissue) SD of at least eight determinations. Unpaired Student’s t-test was used for statistical evaluation of data. Blood or brain samples were taken at times scheduled for the convulsive test. * p < 0.05. *** p < 0.001 vs. an antiepileptic drug alone.
The present study indicates that cimetidine (10 and 20 mg/kg) given singularly did not affect the anticonvulsant activity of carbamazepine. In addition, 7-day and 14-day applications of cimetidine (at a dose of 20 mg/kg), significantly diminished the anticonvulsant activity of phenobarbital. It is worth mentioning that the 14-day administration of cimetidine significantly raised the anticonvulsant activity of carbamazepine. Our previous study [29] showed that acute administration of cimetidine (at a dose of 20 mg/kg) enhanced the anticonvulsant activity of ethosuximide but did not affect the anticonvulsant activity of ethosuximide, valproate, clonazepam and phenobarbital when administered chronically against PTZ-induced convulsions. Moreover, a recent study indicated that single and repeated intraperitoneal administration of famotidine, another H2 histamine receptor antagonist, combined with valproate and phenytoin significantly enhanced the anticonvulsant activity of AEDs, but did not change valproate and phenytoin free plasma levels simultaneously elevated brain concentration of the first drug [30]. The 1-day and 14-day administrations of cimetidine (at 20 mg/ kg), both, alone and in combinations with conventional AEDs were devoid of any significant acute adverse effects evaluating in the chimney test with respect to coordination. On the other hand, cimetidine (in the 1-day and 14-day studies) impaired significantly long-term memory in the passive avoidance task when applied together with some AEDs tested. In this case, cimetidine shortened the retention time when co-administered with phenobarbital, valproate or carbamazepine. On the other hand, valproate and phenobarbital when administered alone also produced the impairment of coordination per se in mice. This fact indicates evidently that the memory impairment in animals cannot be entirely ascribed to cimetidine, since the effects of these AEDs alone or combined with this H2 receptor antagonist did not differ statistically. The results presented in analogy to earlier studies suggest that long-term memory impairment was observed after the administration of PB [7] and VPA [8,31] given alone in their dosages resulting from the ED50 values. These results may also confirm our previous publications, where H1 and H2 antagonists studied did not affect the memory in mice [8,30]. However, Taati et al. reported that cimetidine reversed the exercise-induced improvement in learning and memory in rats [32]. The experimental evidence indicates that central histamine plays a main role in cognitive function and it was shown to
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enhance memory in both the passive [33] and active avoidance tasks [34,35]. However, Eidi et al. [36] have shown that pyrilamine (a histamine H1 receptor antagonist) and cimetidine (a histamine H2 receptor antagonist) decreased memory retention in animals. Noticeably, cimetidine is a well-know inhibitor of numerous isoforms of hepatic enzymes (cytochrome P450), and thus, it may increase plasma levels of co-administered AEDs [37]. In the present study, we observed a decrease in brain phenobarbital concentration following the 14-day administration of cimetidine. It should be stressed that pharmacokinetic interaction observed in this study reflected the brain concentration of AED, not plasma levels in mice. This discrepancy probably results from the changed blood– brain barrier permeability mediated by cimetidine. Previously, it has been found experimentally that some H1 and H2 receptor antagonists changed the permeability of blood–brain barrier and thereby, they influenced the distribution of histamine in the histaminergic neurons [38]. Hence, it is possible that the 14-day administration of cimetidine could decrease the penetration of PB through the blood–brain barrier, leading finally to a reduced brain phenobarbital concentration. In light of this fact, it is worth noting that the brain concentration of carbamazepine was not affected by the 7-day administration of cimetidine, whereas the 14-day treatment with cimetidine significantly elevated both free plasma and total brain concentrations of carbamazepine. This may be related to the relatively long periods of therapy (when comparing the opposite effects observed for a 7-day administration of cimetidine). This observed discrepancy may result from pharmacokinetic interactions related to the inhibition of hepatic cytochrome P450 enzymes. However, the inhibitory effect of cimetidine on CYP3A4 is not substantial, and the interaction with CBZ is of a modest clinical significance [37]. Summing up, the present study provides evidence that the chronic 14 day treatment with cimetidine may result in a decrease of the electroconvulsive threshold in animals. Moreover, the coadministration of conventional AEDs with cimetidine affected the anticonvulsant effects of carbamazepine and phenobarbital, but not those of valproate and phenytoin. In such cases, special caution is advised when combining cimetidine with conventional AEDs. Conflict of interest The authors declare no conflict of interests. Acknowledgements This study was supported by grants from the State Committee for Scientific Research (No. P05A 044 18, Warsaw, Poland) and Medical University of Lublin. References [1] Garbarg M, Barbin G, Feger J, Schwartz JC. Histaminergic pathway in rat brain evidenced by lesions of the medial forebrain bundle. Science 1974;186:833–5. [2] Panula P, Airaksinen MS, Pirvola U, Kotilainen E. A histamine-containing neuronal system in human brain. Neuroscience 1990;34:127–32. [3] Hill SJ. Distribution properties, and functional characteristics of three classes of histamine receptor. Pharmacol Rev 1990;42:45–83. [4] Tasaka K, Mio M, Akagi M, Saito T. Histamine release induced by histone and related morphological changes in mast cells. Agents Actions 1990;30:114–7. [5] Churchull JA, Gammon GD. The effect of antihistaminic drugs on convulsive seizures. J Am Med Assoc 1949;141:18–21. [6] Wynagaarden JB, Seevers MH. The toxic effects of antihistaminic drugs. J Am Med Assoc 1951;145:277–82. [7] S´wia˛der M, Wielosz M, Czuczwar SJ. Interaction of astemizole, an H1 receptor antagonist, with conventional antiepileptic drugs in mice. Pharmacol Biochem Behav 2003;76:169–78.
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