Comparison of the anticonvulsant potency of various diuretic drugs in the maximal electroshock-induced seizure threshold test in mice

Background. The coexistence of seizures and arterial hypertension requires an adequate and efficacious treatment involving both protection from seizures and reduction of high arterial blood pressure. Accumulating evidence indicates that some diuretic drugs (with a well-established position in the treatment of arterial hypertension) also possess anticonvulsant properties in various experimental models of epilepsy.


Introduction
Arterial hypertension is the most common cardiovascular disease affecting approx.26% of the worldwide population, and thus, it is a major public health problem in both developed and developing countries. 1In contrast, epilepsy is a serious neurological disorder affecting approx.1% of the population in every country throughout the world. 2 The coexistence of both diseases (seizures and hypertension) requires adequate and efficacious treatment which simultaneously protects against seizures and reduces arterial blood pressure.In such cases, a combined treatment of antihypertensive and antiepileptic drugs is prescribed for these patients.However, any combined pharmacotherapy is associated with the appearance of interactions, whose nature may be pharmacodynamic, pharmacokinetic, or mixed. 3The most notable group of drugs with a well-established position in the treatment of arterial hypertension are diuretics. 4][11] Unfortunately, the anticonvulsant properties of the diuretics were not directly compared to each other.However, to unequivocally assess the anticonvulsant potential of agents or drugs that increase the threshold for electroconvulsions, Swinyard et al. recommended determining doses of the compounds that increase the electroconvulsive threshold in drug-treated animals 20% over the threshold in control animals (i.e., TID 20 values). 12Thus, the TID 20 values allow researchers to precisely assess the anticonvulsant potency of drugs or agents in preclinical studies. 12,13onsidering the abovementioned facts, it was of pivotal importance to determine the TID 20 values for 6 commonly prescribed diuretics (i.e., furosemide, spironolactone, amiloride, ethacrynic acid, hydrochlorothiazide, and indapamide) in the maximal electroshock-induced seizure threshold (MEST) test in mice in order to assess their anticonvulsant potency in this seizure model.There is no doubt that diuretic drugs with anticonvulsant properties can be preferentially recommended for patients with both high arterial blood pressure and seizures.

Animals and experimental conditions
Experiments were performed on adult male albino Swiss mice weighing 22-26 g.The experimental protocol described in this study conformed to the "Guide for the Care and Use of Laboratory Animals" and was approved by the Local Ethics Committee. 14

Drugs
Ethacrynic acid, indapamide and spironolactone (all 3 drugs from Sigma-Aldrich, Poznań, Poland) as well as hydrochlorothiazide (Polpharma S.A., Starogard Gdański, Poland) were suspended in a 1% solution of Tween 80 (Sigma-Aldrich) in distilled water.Amiloride hydrochloride hydrate (Sigma-Aldrich) and furosemide (for injection 10 mg/mL; Polpharma S.A.) were dissolved in distilled water.All diuretic drugs were administered intraperitoneally (i.p.) in a volume of 0.005 mL/g body weight as follows: ethacrynic acid and furosemide at 30 min before the MEST-induced seizures, amiloride at 60 min before, and hydrochlorothiazide, indapamide and spironolactone at 120 min before.

Maximal electroshock seizure threshold test and the calculation of threshold increasing doses by 20% (TID 20 ) values
Seizure activity (electroconvulsions) was evoked by a current (sine-wave, 0.2 s stimulus duration, 50 Hz, 500 V) delivered via auricular electrodes by a Rodent Shocker generator (Hugo Sachs Elektronik, Freiburg, Germany). 15o assess the threshold for maximal electroconvulsions, 4 groups of mice (8 mice per group) were subjected to electroshocks of varying intensities (ranging from 4 to 10 mA) to yield seizures in 10-30%, 30-50%, 50-70%, and 70-90% of animals.The log-probit method was used to calculate the median current strength (CS 50 [mA]), representing the current intensity necessary to induce tonic hind limb extension in 50% of the mice challenged. 16The threshold (CS 50 values) for maximal electroconvulsions in mice receiving amiloride, ethacrynic acid, furosemide, hydrochlorothiazide, indapamide, and spironolactone (all diuretics in increasing doses), were experimentally determined as described in detail previously. 17,18Subsequently, the percentage of increase in CS 50 values for animals injected with increasing doses of diuretics over the control CS 50 value was calculated.The doses of diuretics and their resultant percentage of threshold increase over the control were graphically plotted in rectangular coordinates of the Cartesian plot system and examined with the least-squares linear regression analysis.From linear regression equations, the TID 20 values were calculated as recommended earlier. 12,13

Statistics
The CS 50 values with their standard error of mean (SEM) were statistically analyzed with one-way analysis of variance (ANOVA) followed by the Tukey-Kramer post-hoc test.Differences among values were considered statistically significant if p < 0.05.
Subsequently, the doses of amiloride, hydrochlorothiazide and indapamide were linearly related to the threshold increases, and the respective equations describing these relations are illustrated in Fig. 1.The experimentally-derived TID 20 values in the MEST test in mice were 30.2 mg/kg for amiloride, 68.2 mg/kg for hydrochlorothiazide and 3.9 mg/kg for indapamide, respectively.

Discussion
Results indicate that there was a close correlation between the doses of 3 diuretic drugs (i.e., amiloride, hydrochlorothiazide and indapamide) and their anticonvulsant properties in mice subjected to the MEST test.In this study, linear regression analysis allowed for the determination of the TID 20 values for amiloride, hydrochlorothiazide and indapamide.In the case of ethacrynic acid, the drug had no impact on the threshold for electroconvulsions.In contrast, furosemide and spironolactone diminished the threshold for electroconvulsions in mice, although the data did not reach the level of statistical significance.
Of note, the TID 20 values presented in this study for diuretic drugs can be readily compared to those reported earlier for some second-generation antiepileptic drugs (AEDs).The TID 20 values for some AEDs were as follows: 70 mg/kg for gabapentin, 18 44 mg/kg for levetiracetam, 19 103.2 mg/kg for stiripentol, 20 4.4 mg/kg for tiagabine, 17 and 226.2 mg/kg for vigabatrin. 17,18A direct comparison of TID 20 values of the second-generation AEDs with those calculated for the 3 diuretics revealed that indapamide has the lowest TID 20 value (3.9 mg/kg).In contrast, gabapentin, levetiracetam, stiripentol, and vigabatrin have higher TID 20 values than those calculated for amiloride (30.2 mg/kg).In the case of hydrochlorothiazide, its TID 20 value (68.2 mg/kg) is lower than that of gabapentin, stiripentol and vigabatrin in the MEST test.Thus, considering the anticonvulsant potency of the studied diuretics, the drugs can be arranged from the lowest to the highest TID 20 values as follows: indapamide > amiloride > hydrochlorothiazide.This comparative study allowed for the assessment of the most favorable diuretic drug with the lowest TID 20 value (indapamide), which is effective in suppressing MESTinduced tonic seizures in experimental animals.
There is another fact worth keeping in mind while translating the results from this study to a clinical setting.Since the TID 20 value for indapamide in mice was 3.9 mg/kg, it can be readily converted to a dose of indapamide used in humans, according to the method presented by Reagan-Shaw et al. 21The converted dose of indapamide for a 60 kg human should be 18.72 mg (i.e., 0.312 mg/kg of body weight).The recommended maximum daily dose of indapamide in humans is 5 mg. 22Thus, it seems that the anticonvulsant effects of indapamide would appear if the daily dose of indapamide exceeded ~4 times the recommended maximum.Similarly, the TID 20 values for amiloride and hydrochlorothiazide were 30.2 mg/kg and 68.2 mg/kg, respectively.Thus, the corresponding doses of the diuretics in a 60 kg human should amount to 145 mg for amiloride and 327.4 mg for hydrochlorothiazide.The recommended maximum daily dose of amiloride is 20 mg, 23 and for hydrochlorothiazide it is 100 mg, 24 so the anticonvulsant effects of amiloride would be expected if the daily dose exceeded ~7 times the recommended maximum, and that of hydrochlorothiazide ~3 times.We are fully aware of such limitations in translational studies.However, it has to be noted that diuretic drugs cannot be used alone as anticonvulsant agents in patients with epilepsy, but always in combination with classical and novel AEDs in order to provide epilepsy patients with the most effective treatment against both arterial hypertension and seizures.Obviously, combinations of diuretics and AEDs always produce interactions of a pharmacodynamic, pharmacokinetic, or mixed nature. 3However, preclinical studies on animals allowed us to characterize the interactions between diuretics and AEDs.For instance, it has been reported that indapamide significantly potentiated the anticonvulsant action of carbamazepine, phenobarbital and valproate, although the effects observed for phenobarbital in the mouse maximal electroshock seizure (MES) model were of a pharmacokinetic nature. 11Only the interactions between indapamide and carbamazepine and valproate were pharmacodynamic in the mouse MES model, suggesting their clinical applicability in epilepsy patients. 11In the case of hydrochlorothiazide, the diuretic drug exclusively enhanced the anticonvulsant activity of carbamazepine in the mouse MES model. 10As regards amiloride, the drug potentiated the anticonvulsant activity of carbamazepine, oxcarbazepine, phenobarbital, topiramate, and valproate in the mouse MES model. 9Unfortunately, amiloride pharmacokinetically potentiated the effects of carbamazepine, oxcarbazepine and phenobarbital in mice challenged with the MEST test.Only the pharmacodynamic interactions between amiloride and topiramate, and valproate deserve recommendation for their clinical use in epilepsy patients.
In conclusion, indapamide -with the lowest TID 20 value in the mouse MEST test -deserves recommendation for clinical use as an add-on therapy in epilepsy patients who additionally require the application of diuretic drugs.

Fig. 1 .
Fig. 1.Linear regression analysis of doses of various diuretic drugs and their corresponding threshold increases in the MEST test in mice y -threshold increase [%]; x -dose of the diuretic; R2 -coefficient of determination.The dashed line reflects the TID20 values (threshold increasing doses by 20%) in the MEST test.

Table 1 .
Influence of 6 diuretic drugs in the maximal electroshock seizure threshold test in mice Results are median current strengths (CS 50 [mA] ±SEM) necessary to produce tonic convulsions in 50% of the animals tested; control groups of animals received a vehicle (1% of solution of Tween 80 in distilled water); n -number of animals tested at those current strength intensities whose seizure effects ranged between 16% and 84%; TI -threshold increasing values are presented as percentage of increase in CS 50 values for animals injected with increasing doses of diuretics over the control (vehicle-treated) animals; statistical analysis of data was performed with one-way ANOVA followed by the Tukey-Kramer post-hoc test;* p < 0.05 vs the control (vehicle-treated) animals.