Original papers Comparison of the anticonvulsant potency of various diuretic drugs in the maximal electroshock-induced seizure threshold test in mice Katarzyna Załuska1,B–D, Maria W. Kondrat-Wróbel1,B–D, Jarogniew J. Łuszczki1,2,A,C–F 1 Department of Pathophysiology, Medical University of Lublin, Poland 2 Isobolographic Analysis Laboratory, Institute of Rural Health, Lublin, Poland A – research concept and design; B – collection and/or assembly of data; C – data analysis and interpretation; D – writing the article; E – critical revision of the article; F – final approval of the article Advances in Clinical and Experimental Medicine, ISSN 1899-5276 (print), ISSN 2451-2680 (online) Adv Clin Exp Med. 2018;27(5):609–613 Address for correspondence Abstract Jarogniew J. Łuszczki E-mail: [email protected] 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. Accumulat- Funding sources None declared ing 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. Conflict of interest Objectives. The aim of this study was to assess the anticonvulsant potency of 6 commonly used diuretic Prof. J.J. Łuszczki has been involved in the design and development of new antiepileptics and CNS drugs (i.e., amiloride, ethacrynic acid, furosemide, hydrochlorothiazide, indapamide, and spironolactone) drugs. He has received, within the last 5 years, in the maximal electroshock-induced seizure threshold (MEST) test in mice. an unrestricted research grant from GlaxoSmith- Kline (Brentford, UK). The remaining authors have Material and methods. Doses of the studied diuretics and their corresponding threshold increases were no conflict of interest to disclose. linearly related, allowing for the determination of doses which increase the threshold for electroconvulsions Acknowledgements in drug-treated animals by 20% (TID20 values) over the threshold in control animals. This study was supported by grants from the Medi- Results. Amiloride, hydrochlorothiazide and indapamide administered systemically (intraperitoneally – i.p.) cal University of Lublin (DS 474/2012-2014 and DS 474/2015-2016). increased the threshold for maximal electroconvulsions in mice, and the experimentally-derived TID20 values in the maximal electroshock seizure threshold test were 30.2 mg/kg for amiloride, 68.2 mg/kg for hydro- chlorothiazide and 3.9 mg/kg for indapamide. In contrast, ethacrynic acid (up to 100 mg/kg), furosemide Received on June 14, 2016 (up to 100 mg/kg) and spironolactone (up to 50 mg/kg) administered i.p. had no significant impact on the Reviewed on October 1, 2016 threshold for electroconvulsions in mice. Accepted on January 27, 2017 Conclusions. The studied diuretics can be arranged with respect to their anticonvulsant potency in the MEST test as follows: indapamide > amiloride > hydrochlorothiazide. No anticonvulsant effects were observed for ethacrynic acid, furosemide or spironolactone in the MEST test in mice. Key words: diuretic drugs, threshold for electroconvulsions, TID20 values DOI 10.17219/acem/68694 Copyright © 2018 by Wroclaw Medical University This is an article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc-nd/4.0/) 610 K. Załuska, M. Kondrat-Wróbel, J. Łuszczki. Anticonvulsant effects of diuretic drugs Introduction Drugs Arterial hypertension is the most common cardio- Ethacrynic acid, indapamide and spironolactone (all vascular disease affecting approx. 26% of the worldwide 3 drugs from Sigma-Aldrich, Poznań, Poland) as well as population, and thus, it is a major public health problem hydrochlorothiazide (Polpharma S.A., Starogard Gdański, in both developed and developing countries.1 In contrast, Poland) were suspended in a 1% solution of Tween 80 (Sig- epilepsy is a serious neurological disorder affecting ap- ma-Aldrich) in distilled water. Amiloride hydrochloride prox. 1% of the population in every country throughout hydrate (Sigma-Aldrich) and furosemide (for injection the world.2 The coexistence of both diseases (seizures and 10 mg/mL; Polpharma S.A.) were dissolved in distilled hypertension) requires adequate and efficacious treatment water. All diuretic drugs were administered intraperito- which simultaneously protects against seizures and re- neally (i.p.) in a volume of 0.005 mL/g body weight as fol- duces arterial blood pressure. In such cases, a combined lows: ethacrynic acid and furosemide at 30 min before the treatment of antihypertensive and antiepileptic drugs MEST-induced seizures, amiloride at 60 min before, and is prescribed for these patients. However, any combined hydrochlorothiazide, indapamide and spironolactone at pharmacotherapy is associated with the appearance of in- 120 min before. teractions, whose nature may be pharmacodynamic, phar- macokinetic, or mixed.3 The most notable group of drugs Maximal electroshock seizure threshold with a well-established position in the treatment of arterial test and the calculation of threshold hypertension are diuretics.4 increasing doses by 20% (TID20) values Accumulating experimental evidence indicates that some diuretic drugs also possess anticonvulsant properties Seizure activity (electroconvulsions) was evoked by a cur- in various experimental models of epilepsy.5–8 Previously, rent (sine-wave, 0.2 s stimulus duration, 50 Hz, 500 V) it was discovered that amiloride, hydrochlorothiazide and delivered via auricular electrodes by a Rodent Shocker indapamide (3 classical diuretics) elevated the threshold for generator (Hugo Sachs Elektronik, Freiburg, Germany).15 maximal electroconvulsions in experimental animals.9–11 To assess the threshold for maximal electroconvulsions, Unfortunately, the anticonvulsant properties of the di- 4 groups of mice (8 mice per group) were subjected to elec- uretics were not directly compared to each other. How- troshocks of varying intensities (ranging from 4 to 10 mA) ever, to unequivocally assess the anticonvulsant potential to yield seizures in 10–30%, 30–50%, 50–70%, and 70–90% of agents or drugs that increase the threshold for electro- of animals. The log-probit method was used to calculate convulsions, Swinyard et al. recommended determining the median current strength (CS50 [mA]), representing doses of the compounds that increase the electroconvulsive the current intensity necessary to induce tonic hind limb threshold in drug-treated animals 20% over the threshold extension in 50% of the mice challenged.16 The threshold 12 in control animals (i.e., TID20 values). Thus, the TID20 val- (CS50 values) for maximal electroconvulsions in mice re- ues allow researchers to precisely assess the anticonvulsant ceiving amiloride, ethacrynic acid, furosemide, hydrochlo- potency of drugs or agents in preclinical studies.12,13 rothiazide, indapamide, and spironolactone (all diuretics Considering the abovementioned facts, it was of pivotal in increasing doses), were experimentally determined as 17,18 importance to determine the TID20 values for 6 common- described in detail previously. Subsequently, the per- ly prescribed diuretics (i.e., furosemide, spironolactone, centage of increase in CS50 values for animals injected amiloride, ethacrynic acid, hydrochlorothiazide, and in- with increasing doses of diuretics over the control CS50 dapamide) in the maximal electroshock-induced seizure value was calculated. The doses of diuretics and their re- threshold (MEST) test in mice in order to assess their an- sultant percentage of threshold increase over the control ticonvulsant potency in this seizure model. There is no were graphically plotted in rectangular coordinates of the doubt that diuretic drugs with anticonvulsant properties Cartesian plot system and examined with the least-squares can be preferentially recommended for patients with both linear regression analysis. From linear regression equa- high arterial blood pressure and seizures. tions, the TID20 values were calculated as recommended earlier.12,13 Material and methods Statistics Animals and experimental conditions The CS50 values with their standard error of mean (SEM) were statistically analyzed with one-way analysis of vari- Experiments were performed on adult male albino Swiss ance (ANOVA) followed by the Tukey-Kramer post-hoc mice weighing 22–26 g. The experimental protocol de- test. Differences among values were considered statistically scribed in this study conformed to the “Guide for the Care significant if p < 0.05. and Use of Laboratory Animals” and was approved by the Local Ethics Committee.14 Adv Clin Exp Med. 2018;27(5):609–613 611 Results threshold in mice (f (3; 76) = 2.535; p = 0.0631) (Table 1). Moreover, indapamide administered i.p. at doses of 1.5, Effects of various diuretic drugs 3 and 6 mg/kg raised the threshold for MEST-induced on the threshold for maximal seizures by 2.2%, 12.1% and 37.4%, respectively (Table 1). electroshock-induced seizures In this case, indapamide (6 mg/kg) significantly increased (p < 0.05) the threshold for electroconvulsions in mice Amiloride administered systemically (i.p.) at doses of 5, (f (3; 60) = 3.718; p = 0.0161) (Table 1). In the case of ethac- 10, 50, 75, and 100 mg/kg elevated the threshold for MEST- rynic acid, the drug at a maximally tested dose of 100 mg/kg induced seizures