Interactions between angiotensin AT1 receptor antagonists and second-generation antiepileptic drugs in the test of maximal electroshock

The anticonvulsant activity of angiotensin AT1 receptor antagonists, losartan (2‐n‐butyl‐4‐chloro‐5‐hydroxymethyl‐1‐[(2′(1H‐tetrazol‐5‐yl)‐biphenil‐4‐yl)methyl]imidazole) and telmisartan (49‐[(1,49‐dimethyl‐29‐propyl[2,69‐bi‐1H‐benzimidazo]‐19‐yl)methyl]‐[1,19‐biphenyl]‐2‐carboxylic acid), has been reported recently. It is suggested that AT1 receptor antagonists may affect the protective action of antiepileptic drugs. The aim of this study was to determine the influence of losartan and telmisartan on the anticonvulsant activity of some second‐generation antiepileptics (lamotrigine – LTG, oxcarbazepine – OXC, and topiramate – TPM). For this purpose, the maximal electroshock seizure (MES) test in mice was used. Additionally, the drug combinations were checked for adverse effects in the passive avoidance and chimney tests. In the MES test, losartan at the doses of 30 and 50 mg/kg, administered intraperitoneally (i.p.), potentiated the protective action of LTG (P < 0.01). This interaction was not accompanied by a significant change of LTG level either in plasma or in the brain. Telmisartan at the dose of 30 mg/kg i.p. enhanced the anticonvulsant action of TPM (P < 0.01). However, this interaction was pharmacokinetic in nature, as telmisartan significantly increased plasma and total brain concentrations of TPM (P < 0.001). The combinations of AT1 receptor antagonists with antiepileptic drugs did not affect retention in the passive avoidance test or motor coordination in the chimney test. The potentiation of the anticonvulsant action of LTG by losartan probably on account of pharmacodynamic interactions, make this combination important for further experimental and clinical studies. The combination of telmisartan and TPM is less beneficial due to pharmacokinetic interactions.

antiepileptic drugs; drug interactions; losartan; maximal electroshock; telmisartan

It is generally accepted that an independent renin–angiotensin system (RAS) exists in the brain [1] . The brain RAS is associated with the regulation of body water balance and thirst, blood pressure maintenance, vasopressin release, and influences on reproduction and sexual behaviors [2] . Additionally, this system has been implicated in the regulation of cerebral blood flow and cerebroprotection, stress, depression, alcohol consumption, memory consolidation, with possible roles in the etiology of Alzheimer's disease, Parkinson's disease, and diabetes [2] , [3] . There is also evidence that the brain RAS mediates seizure susceptibility. Angiotensin II (Ang II) administered intracerebroventricularly increased the threshold of pentylenetetrazol‐, bicuculline‐, and picrotoxin‐induced seizures and attenuated the intensity of clonic convulsions induced by pentylenetetrazol and 3‐mercaptopropionic acid in mice [4] . Using a rat model of epilepsy, it has been found that components of the RAS, angiotensin‐converting enzyme (ACE), and angiotensin AT1 receptor are up‐regulated in the brain following repetitive seizures [5] . In the same study, it has been demonstrated that losartan (2‐n‐butyl‐4‐chloro‐5‐hydroxymethyl‐1‐[(2′(1H‐tetrazol‐5‐yl)‐biphenil‐4‐yl)methyl]imidazole), an angiotensin AT1 receptor antagonist, suppressed seizures in Wistar audiogenic rats [5] . Furthermore, losartan and telmisartan (49‐[(1,49‐dimethyl‐29‐propyl[2,69‐bi‐1H‐benzimidazo]‐19‐yl)methyl]‐[1,19‐biphenyl]‐2‐carboxylic acid), another AT1 receptor antagonist, enhanced the anticonvulsant activity of valproate (VPA) when examined with classical antiepileptic drugs (AEDs) in the test of maximal electroshock (MES) in mice [6] . The MES test is thought to be a predictive model of human generalized tonic–clonic seizures [7] .

The above‐mentioned reports suggest that certain AT1 receptor antagonists may possess anticonvulsant‐like properties. Therefore, this work was undertaken to study the interaction between the AT1 receptor antagonists, losartan and telmisartan, and some second‐generation AEDs, lamotrigine (LTG), oxcarbazepine (OXC), and topiramate (TPM), against MES‐induced convulsions. Additionally, we took into consideration that losartan and telmisartan are commonly prescribed antihypertensive drugs in clinical practice [8] , [9] . Besides, losartan is indicated in patients with heart failure, and telmisartan is the only AT1 receptor antagonist indicated for cardiovascular prevention in a broad range of high‐risk patients with or without hypertension [8] . On the other hand, LTG and TPM are recommended to epileptic patients suffering from cardiovascular disease [10] . Thus, any pharmacological interactions between the AT1 receptor antagonists and the AEDs in this study, especially LTG and TPM, could have clinical implications.

The experiments were performed on male Swiss mice weighing 20–26 g. They were housed in colony cages under standardized laboratory conditions (room temperature of 21 ± 1 °C, humidity 50–60%) on a 12‐h light–dark cycle, with unlimited access to food and tap water. The experimental groups consisting of 8–16 animals were made up at random. Each mouse was used only once. All experimental procedures described in this manuscript were approved by the Local Ethics Committee for Animal Experiments and complied with the European Communities Council Directive of 24 November 1986 (86/609/EEC).

Angiotensin AT1 receptor antagonists, losartan (Xartan; Adamed, Pienkow, Poland) and telmisartan (Micardis; Boehringer Ingelheim, Ingelheim, Germany), and AEDs such as lamotrigine (Lamitrin; GlaxoSmithKline, Brentford, UK), oxcarbazepine (Trileptal; Novartis Pharma GmbH, Nurnberg, Germany), and topiramate (Topamax; Janssen‐Cilag International N.V., Beerse, Belgium) were used. All drugs were suspended in a 1% solution of Tween 80 (Sigma, St. Louis, MO, USA) in distilled water and were injected once intraperitoneally (i.p.) in a volume of 5 mL/kg body weight. Control animals received injections of the vehicle. The pretreatment times of the drugs before the tests were as follows: 120 min (losartan and telmisartan), 60 min (LTG and TPM), or 30 min (OXC). Losartan and telmisartan were administered at doses allowing sufficient amounts of the antagonists enter the brain and cause blockade of the brain renin–angiotensin system [11] , [12] , [13] . The pretreatment times of all drugs were based on previous reports on their biological activity [13] , [14] , [15] , [16] .

Electroconvulsions were produced according to Swinyard et al. [17] with ear‐clip electrodes and alternating current (50 Hz, 500 V) delivered by a Hugo Sachs generator (Rodent Shocker, Type 221; Freiburg, Germany). The stimulus duration was 0.2 s. In this test, a fixed current intensity of 25 mA was applied. The end point was the tonic extension of both hind limbs. The protective activities of AEDs were determined as their ability to protect 50% of mice against the MES‐induced tonic hindlimb extension and expressed as respective median effective doses (ED50 values in mg/kg). To calculate the respective ED50 values, at least three groups of mice were challenged with the MES‐induced seizures after receiving progressive doses of an AED. A dose–response curve for each AED was subsequently constructed on the basis of a percentage of animals protected against the convulsions.

On the first day, mice injected with drugs were placed separately in an illuminated box (12 × 20 × 15 cm) connected to a dark box (24 × 20 × 15 cm). A 4 × 7 cm doorway was located at floor level in the center of the connecting wall. The dark box was equipped with an electric grid floor. After the mouse entered the dark box, it was immediately punished by an electric foot shock (0.6 mA for 2 s). On the next day (24 h later), the retention test was conducted in which the same animals, without any treatment, were put into the illuminated box, and the latency (time) to enter the dark box was recorded. The mice that avoided the dark compartment for 180 s were considered remembering the task. The passive avoidance test may be regarded as a measure of long‐term memory [18] .

Motor performance was evaluated with the chimney test of Boissier et al. [19] . The animals had to climb backwards up a plastic tube (3 cm inner diameter and 25 cm in length). Impaired motor coordination was indicated as the inability of animals to climb backward up the tube within 60 s.

The measurement of the free plasma (nonprotein‐bound) and total brain concentrations of LTG and TPM was undertaken at doses of the AEDs corresponding to their ED50 values in combinations with losartan (50 mg/kg) and telmisartan (30 mg/kg), respectively, in the MES test. Mice injected with the drugs were killed by decapitation at times scheduled for MES and blood samples of approximately 1 mL were collected into heparinized Eppendorf tubes. Simultaneously, the brains of mice were removed from their skulls and placed into the deep freeze at −80 °C. Samples of blood were centrifuged at 5000 × g for 5 min, and plasma samples of 250 μL were stocked into the deep freeze. On the next day, the brains were weighed and homogenized using Abbott buffer (1 : 2 weight/volume) and the Ultra‐Turrax T8 homogenizer (IKA, Staufen, Germany). The homogenates were centrifuged at 10 000 × g for 10 min. Plasma and brain supernatant samples were checked for LTG or TPM concentrations using one of two methods depending on an AED.

Plasma and brain levels of LTG were estimated by high‐performance liquid chromatography (HPLC). In this method, plasma and brain supernatant samples were prepared for analysis as follows: 200 μL of samples were pipetted into a 1.5‐mL plastic tube to which was added 200 μL of 0.08 m triethylammonium phosphate buffer solution, 400 μL of acetonitrile, and vortex‐mixed for 1 min. After centrifugation (10 000 × g for 10 min) in centrifugal filter devices (Millipore Corporation, Billerica, MA, USA), the organic layer was removed, and 20 μL of the aqueous phase was injected into HPLC system. The chromatograph (Dionex, Sunnyvale, CA, USA) was equipped with a gradient pump P580 LPG and a UV/VIS detector (UVD 340S) with a sensitivity setting of 0.1 absorbance units full scale (AUFS) and a time constant of 0.1 s. The Rheodyne 3601 injector valve with a 20‐μL sample loop was used for sample injection. For HPLC, a stainless steel HP ODS column (200 × 4.6 mm) was used at an ambient temperature. The mobile phase was 40 mm triethylammonium phosphate buffer : methanol : acetonitrile (660 : 80 : 160 vol/vol/vol; Fluka, HPLC grade). The mobile phase flow rate was 1.2 mL/min, and LTG absorbance was measured at 214 nm. The peak height for LTG was linearly related to its concentrations, which ranged from 0.16 to 5.0 g/mL. Plasma levels and total brain concentrations of LTG were expressed in μg/mL of plasma or brain supernatant as means ± SD (standard deviation of the mean) of eight determinations.

In the case of TPM, immunofluorescence estimation of plasma and brain levels of this AED was performed. Plasma samples of 250 μL were pipetted into a micropartitioning system, MPS‐1 (Amicon, Danvers, MA, USA), for the separation of free from protein‐bound microsolutes. The MPS‐1 tubes were centrifuged at 5000 × g for 10 min, and 60 μL filtrate samples were collected. Next, samples of 60 μL of filtrate or 75 μL of brain supernatant were analyzed for TPM content by fluorescence‐polarization immunoassay using a TDx analyzer (Abbott, Irving, TX, USA) and reagents (Seradyn Inc., Indianapolis, IN, USA). Plasma and total brain concentrations of TPM were expressed in μg/mL of plasma or brain supernatant as means ± SD of eight determinations.

ED50 values with their 95% confidence limits were calculated according to Litchfield and Wilcoxon method [20] . The 95% confidence limits obtained were transformed into standard errors of the mean (SEM), as described previously [21] . The anticonvulsant activities of AEDs alone or coadministered with losartan or telmisartan were analyzed using a one‐way analysis of variance (anova) followed by the post hoc Dunnett's test for multiple comparisons. A Kruskal–Wallis nonparametric anova followed by Dunn's multiple comparisons test was used to calculate data from the passive avoidance task. The results from the chimney test were compared using Fisher's exact probability test. Free (nonprotein‐bound) plasma and total brain concentrations of LTG and TPM were evaluated with the unpaired Student's t‐test. Group differences were considered statistically significant at P < 0.05.

It has been documented that AT1 receptor antagonists alone at doses applied in this study do not affect electroconvulsions [6] . At present, it has been found that losartan at the doses of 30 and 50 mg/kg i.p. potentiated the anticonvulsant action of LTG, decreasing its ED50 value from 5.1 to 3.6 and 3.8 mg/kg, respectively (P < 0.01). Losartan combined with OXC or TPM did not show such effect. Telmisartan at the dose of 30 mg/kg i.p. enhanced the protective action of TPM, reducing the ED50 value for TPM from 46.1 to 24.7 mg/kg (P < 0.01). The anticonvulsant activity of LTG or OXC was not significantly influenced by telmisartan in the MES test (Table [NaN] ).

Angiotensin AT 1 receptor antagonists and antiepileptics in the MES test

1 LTG, lamotrigine; OXC, oxcarbazepine; TPM, topiramate.

• 2 Median effective doses (ED50s) with 95% confidence limits are expressed in mg/kg with SEM values. They were calculated on the basis of the method of Litchfield and Wilcoxon 20. n – the number of animals at doses of AEDs, for which anticonvulsant effects ranged between 4 and 6 probit (16 and 84%) according to Litchfield and Wilcoxon 20.
• 3 P < 0.01 vs. LTG + vehicle (anova and Dunnett's test).
• 4 P < 0.01 vs. TPM + vehicle (anova and Dunnett's test).

It has been previously demonstrated that losartan and telmisartan at doses used in this study did not cause an impairment of long‐term memory or motor coordination in these tests [6] . At present, combined treatment with losartan or telmisartan and AEDs did not impair memory retention in the passive avoidance task (Table [NaN] ). Similarly, AT1 receptor antagonists in combinations with AEDs did not affect motor performance in the chimney test (Table [NaN] ).

Combined treatment with angiotensin AT 1 receptor antagonists and antiepileptics in the passive avoidance test

• 5 n, the number of animals; LTG, lamotrigine; OXC, oxcarbazepine; TPM, topiramate.
• 6 Results are presented as the median values (in s) along with 25th and 75th percentiles.
• 7 Not significant vs. control groups (Kruskal–Wallis and Dunn's test).

Combined treatment with angiotensin AT 1 receptor antagonists and antiepileptics in the chimney test

• 8 n, the number of animals; LTG, lamotrigine; OXC, oxcarbazepine; TPM, topiramate.
• 9 Results are shown as the percentage of mice with impaired motor coordination in the chimney test.
• 10 Not significant vs. control groups (Fisher's exact probability test).

Losartan (50 mg/kg i.p.) did not significantly change either plasma or the brain level of LTG (Table [NaN] ). However, the combination of telmisartan (30 mg/kg i.p.) with TPM raised the free plasma level and the total brain concentration of this AED (P < 0.001) (Table [NaN] ).

Effect of losartan on plasma and total brain concentrations of lamotrigine

11 Data are expressed as the means ± SD of eight separate determinations. Not significant vs. control group (Student's t‐test).

Effect of telmisartan on plasma and total brain concentrations of topiramate

• 12 Data are presented as the means ± SD of eight separate determinations.
• 13 P < 0.001 vs. control group (Student's t‐test).

The obtained results indicate that losartan (30 and 50 mg/kg i.p.) enhanced the protective action of LTG in the MES test. It is remarkable that no pharmacokinetic interaction was found in this case. In turn, telmisartan (30 mg/kg i.p.) potentiated the anticonvulsant activity of TPM. However, this interaction was pharmacokinetic in nature as telmisartan considerably increased the brain and plasma concentrations of TPM.

At this time, we can only speculate about a potential mechanism responsible for the better anticonvulsant protection in mice injected with both losartan and LTG. Recently, it has been demonstrated that the functional inhibition of the RAS in the brain by losartan was able to significantly impair the triggering and maintenance of seizures in a rat audiogenic model of epilepsy [5] . It has been suggested that the blockade of Ang II action may indirectly interfere with the modulation of excitatory and inhibitory components of the central nervous system, and this has been proposed as a possible mechanism of action by which the inhibition of the RAS affects seizures [5] . In the current study, we used losartan and telmisartan at doses that are able to cross the blood–brain barrier and cause effective blockade of the brain RAS [11] , [12] , [13] . However, the positive interaction with LTG was only seen in the case of losartan. Therefore, other mechanisms not necessarily related to the blockade of the RAS may be involved in the observed phenomenon.

It can be suggested that a specific losartan mechanism of action is involved in the enhancement of the anticonvulsant activity of LTG. It has been reported that losartan inhibits voltage‐dependent Na+ channels in the rat adrenal medulla as in the presence of losartan, veratridine‐evoked (an activator of Na+ channels) catecholamine secretion was greatly inhibited [22] . On the other hand, telmisartan delays the inactivation of voltage‐gated Na+ channels in cardiomyocytes [23] . Although these activities of AT1 receptor antagonists occurred in tissues distinct from the central nervous system and concern specific Na+ channels, we cannot exclude that a different effect of the AT1 receptor antagonists on voltage‐dependent Na+ channels in the brain may have a role in the phenomenon. The primary anticonvulsant mechanism of action of LTG, similarly to classical antiepileptic drugs such as carbamazepine (CBZ) and phenytoin (PHT), is by blockade of voltage‐dependent Na+ channels [24] . Actually, LTG inhibits sustained repetitive firing of action potentials by blocking Na+ channels in a voltage‐ and use‐dependent manner [24] . In microdialysis studies, LTG reduced the basal and veratridine‐evoked extracellular concentration of glutamate in the hippocampus of freely moving rats [25] . This excitatory neurotransmitter is suggested to play an important role in the pathogenesis of epilepsy [26] . In turn, the hippocampus, particularly hippocampal dentate gyrus which is a gateway that regulates seizure activity in this structure [27] , belongs to brain structures strongly associated with the expression of MES‐induced generalized tonic seizures when identified using the induction of the c‐fos protein (Fos) as a marker of neuronal activity [28] . It is worth noting that pretreatment with losartan i.p. prevented the impairment of retention of an inhibitory avoidance response by angiotensin II administration to the dentate gyrus [29] . It is suggested that a potential inhibitory effect of losartan on glutamate concentrations in the hippocampus, caused by blockade of voltage‐dependent Na+ channels, may have contributed to the enhanced protective action of LTG in the MES test. However, this hypothesis is speculative and needs to be confirmed in advanced neurochemical studies.

In contrast to the combination with LTG, losartan did not enhance the anticonvulsant activity of other inhibitors of Na+ channels, CBZ and PHT, in the MES test [6] . In this study, it did not potentiate the protective action of OXC, a keto analog of CBZ, whose the primary anticonvulsant action is also by blockade of voltage‐dependent Na+ channels [24] . These different results could be related to markedly different effects of LTG, CBZ, or PHT on the neuroactive amino acids such as aspartate, glutamate, taurine, and γ‐aminobutyric acid (GABA) reported by some authors [25] . For example, in contrast to LTG, CBZ and PHT did not affect the basal and veratridine‐evoked extracellular concentration of glutamate in the hippocampus [25] . Further, TPM was another AED studied with AT1 receptor antagonists, and its anticonvulsant activity was not enhanced by losartan either. This antiepileptic drug is believed to possess multiple mechanisms of action, which includes the inhibition of voltage‐dependent Na+ channels [30] . However, in opposition to LTG, the evidence does not indicate that this is the primary mechanism responsible for the anticonvulsant activity of TPM [31] . It has been found that TPM at the highest concentration only inhibited the veratridine‐evoked release of glutamate by around 20% in hippocampal nerve endings [31] .

On the contrary, telmisartan potentiated the protective action of TPM in the MES test, probably by increasing the brain concentration of TPM. In general, pharmacokinetic interactions can occur during absorption, distribution, metabolism, or elimination of drugs [32] . It is rather impossible to find out from this study, at which stage of disposition of the drugs the interaction occurred. The most important pharmacokinetic interactions with AEDs are those involving cytochrome P450 (CYP) isoenzymes in hepatic metabolism [33] . Telmisartan is extensively metabolized by conjugation to glucuronic acid in the liver [34] , but it has been also reported to have inhibitory effects on CYP2C19 isoenzyme activity in vitro[35] . This suggests that telmisartan may decrease the clearance of CYP2C19 substrates. It has been documented that telmisartan showed inhibitory effects on indapamide metabolism, a substrate for CYP2C19, due to inhibiting this isoenzyme [36] . On the other hand, like telmisartan, TPM is an inhibitor of CYP2C19 [37] . Although an inhibitor of a given enzyme can be a substrate for the same enzyme [38] , [39] , such the substrate activity of TPM for CYP2C19, which may influence the interaction with telmisartan needs to be confirmed. The same apply for losartan which also shows some inhibitory potency on CYP2C19 [40] ; however, it did not affect the anticonvulsant action of TPM. Therefore, further studies on the interaction potential of telmisartan and TPM are required.

In conclusion, the current study showed that losartan enhanced the anticonvulsant action of LTG in mice. Taking into consideration, a widely clinical use of losartan as an antihypertensive drug and that this interaction was pharmacodynamic in nature, make the combination of losartan and LTG important for further experimental and clinical studies. The combination of telmisartan and TPM seems less beneficial due to pharmacokinetic interactions.

This study was supported by grant No. 2 P05D 095 30 from the Ministry of Science and Higher Education, Warsaw, Poland.