INTRODUCTION:

Epilepsy a neurological cramp is a diverse set of neurological disorders characterized by seizures, which results from abnormal, excessive hyper synchronous neuronal activity^1,2. Despite the fact that experimental studies in recent decades have expanded the understanding of the pathogenesis of epilepsy, and have highlighted the influence of neurogenic inflammation on the development and progression of convulsive syndrome^3–5, the crucial role that pronounced as an anticonvulsant effect is under the control of transmembrane currents⁶.

This control can be both direct (the drug directly blocks sodium, calcium or potassium channels) and indirect, such as chlorine-induced hyperpolarization of neuronal membranes under the action of GABA mimetics or changes in membrane currents of calcium and sodium under the influence of modulators of glutamate receptors. All the newest antiepileptic drugs (AEDs), e.g. cenobamate, esogabine, perampanel, brivaracetam and eslicarbazepine acetate, which were approved by the FDA in the past decade, are known modulators of transmembrane ion currents^7–9. In addition, the results of experimental and clinical studies have shown pronounced anticonvulsant properties in known antiarrhythmic drugs like sodium channel blockers (lidocaine, propafenone and mexiletine), calcium channel blockers (verapamil, diltiazem, amlodipine, nifedipine and cinnarizine), potassium channel blocker (amiodarone), and blocker of If-channels of the sinus node (ivabradine)^10–18. A similar effect has been identified for herbal substances^19–24.

A promising target for influencing transmembrane ion currents is the basic enzyme that provides the membrane potential of neurons, Na+/K+ ATPase^25–30. However, information on the effect of known antiepileptic drugs (including the newest ones) on the Na+/K+ ATPase activity is extremely limited, in particular, there are only isolated studies indicating the stimulation of Na+/K+ ATPase in the brain microsomes of rats and cats under the influence of diphenylhydantoin medicines³¹. The anticonvulsant effect of the cardiac glycoside digoxin is probably associated with influence on the Na+/K+ ATPase activity^32,33. Digoxin has been shown to affect the membrane potential not only of cardiomyocytes, but also (due to its lipophilic properties and ability to overcome the blood-brain barrier) of neurons³⁴. In addition, cardiac glycoside is characterized by a pronounced dose dependence of action in cardiotherapeutic and high doses it blocks Na+/K+ ATPase, while in low (sub-cardiotonic) doses that do not affect the myocardium, digoxin increases the activity of the enzyme³².

Although the antiepileptic potential of digoxin monotherapy is not high enough, the effectiveness of epilepsy treatment has been verified by adding low doses of cardiac glycoside to classical anticonvulsant therapy regimens³⁵. Previously, a clear potentiating effect of digoxin has been shown at the sub-cardiotonic dose on the anticonvulsant effect of classical AEDs under pentylenetetrazole-induced seizures – a basic seizure model with a well-known GABA-negative mechanism³⁶. The issue of the influence of digoxin and its combinations with anticonvulsants on other mechanisms of seizure development remains open, as well as the search for effective combinations for the treatment of various types of epileptic seizures (including partial, generalized, etc.). The use of non-antiepileptic drugs (in particular digoxin), which have central neurotropic properties and mechanisms of action uncharacteristic of known AEDs, can significantly increase the effectiveness of pharmacotherapy of convulsive syndrome, which is the basis for improving the treatment of epilepsy, including multi-drug resistant forms of the disease.

The aim of the study was to determine the effects of low doses of cardiac glycoside digoxin on the anticonvulsant effect of five classical antiepileptic drugs – sodium valproate, topiramate, levetiracetam, clonazepam and phenobarbital – under experimental electro-induced seizures in mice.

MATERIALS AND METHODS:

Animals:

The experiments were conducted on 123 male random-breed albino mice weighing about 18-22gm. Animals were kept on a standard vivarium diet with free access to water, constant humidity and a temperature of 18-20°C on the basis of the Central Scientific and Research Laboratory of the Educational and Scientific Institute of Applied Pharmacy (ESIAP) of the National University of Pharmacy (Kharkiv, Ukraine).

Ethics approval:

All the experimental protocols were approved by the Bioethics Commission of the National University of Pharmacy (protocol No. 3, September 10, 2020). Experiments were conducted according with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.

Treatment:

Mice were randomly divided into experimental groups like control group (animals with untreated seizures), mice with modeling seizures which received digoxin, and the remaining groups were mice with convulsions administered of sodium valproate, topiramate, levetiracetam, clonazepam, phenobarbital, and also combinations of these medicines with digoxin.

Classical antiepileptic drugs were administered 30 min before to seizure induction once intragastrically at conditionally effective (ED₅₀) and sub-effective (½ ED₅₀) doses³⁷ as below:

· Sodium valproate (Depakine, Sanofi Aventis, France) – at doses of 300 and 150 mg/kg body weight;

· Topiramate (Topamax, Janssen-Cilag S.p.A., Italy) – at doses of 300 and 150 mg/kg body weight;

· Levetiracetam (Keppra, UCB Pharma, Belgium) – at doses of 100 and 50 mg/kg body weight;

· Phenobarbital (Phenobarbital IC, Interkhim, Ukraine) – at doses of 20 and 10 mg/kg body weight;

· Clonazepam (Clonazepam IC, Interkhim, Ukraine) – at doses of 0.1 and 0.05 mg/kg body weight.

Digoxin (People’s Health, Ukraine) was administered once subcutaneously at a dose of 0.8 mg/kg body weight (¹/₁₀ LD₅₀)³³ 10-15 min before seizure induction.

Mice from control group received intragastrically purified water in an appropriate volume (0.1ml per 10 g body weight).

Experimental electro-induced seizures:

The influence of digoxin on the anticonvulsant effect of sodium valproate, topiramate, levetiracetam, clonazepam and phenobarbital has been studied in a baseline model of seizures caused by maximal electroshock (MES)^38,39.

Convulsions induced by MES were reproduced by passing an electric current with constant characteristics (strength – 50 mA, frequency – 50 Hz) for 0.2s through copper corneal electrodes. The anticonvulsant effect of drugs and their combinations was assessed by the following indicators: % of animals in the group separately with clonic and tonic seizures, seizure severity in points (3 points – clonic seizures without lateral position, 4 points – clonic-tonic seizures with lateral position, 5 points – tonic extension, 6 points – tonic extension, which led to the death of the animal), the duration of the convulsive period, recovery period – the time of exit from the lateral position (restoration of motor activity), time of death and lethality⁴⁰.

Statistical analysis:

Statistical analysis to the obtained results was made using the package, STATISTICA 12, with the calculation of mean, standard error of the mean, and confidence probability (p). Significance of differences between comparison groups in cases of normal distribution was evaluated by Student's parametric t-test, in case of its absence – by non-parametric Mann-Whitney U-test and when accounting for the results in an alternative form (lethality, % of mice with brain clonic and tonic convulsions) Fisher's angular transformation (φ*) was used.

RESULTS AND DISCUSSION:

Maximal electroshock allows to model generalized convulsions associated with the activation of transmembrane sodium currents and the induction of membrane potentials of neurons³⁹. Clinically, MES-induced seizures were initially detected by a very short phase of tonic flexion, followed by prolonged tonic extension of the hind limbs in the lateral position of the animal⁴¹. The rigidity of this model is confirmed by the high lethality for 8 mice out of 10 died without leaving the stage of tonic extension (Tables 1-5).

Table 1: Influence of sodium valproate, digoxin and their combination on seizures, induced by MES in mice

Experimental group	n	% of mice with convulsions		Severity, points	Duration, sec	Recovery time, sec	Death time, sec	Lethality, %
Experimental group	n	clonic	tonic	Severity, points	Duration, sec	Recovery time, sec	Death time, sec	Lethality, %
Control (untreated seizures)	10	100	100	5.80±0.13	11.50±1.71	19.50±3.50	13.88±0.81	80
Digoxin, 0.8 mg/kg	8	100	100	5.13±0.23*	5.00±1.96*	19.33±1.45	14.00	25**
Sodium valproate, 300 mg/kg	7	100	71**	3.86±0.26**^##	1.43±0.20**^#	9.00±2.42^#	–	0**^#
Sodium valproate, 150 mg/kg	7	100	86	5.00±0.38*^§	1.71±0.18**^#	10.20±2.76^#	16.00±1.00	29*^§
Sodium valproate, 150 mg/kg + Digoxin, 0.8 mg/kg	7	100	43**°	3.86±0.40**^#°	1.43±0.20**^#	8.86±1.70*^##	–	0**^#°

1. n – number of mice in the appropriate experimental group

2. * – p<0.05, in comparison with control (untreated seizures), ** – p<0.01, in comparison with control (untreated seizures)

3. ^# – p<0.05, in comparison with digoxin, ^## – p<0.01, in comparison with digoxin

4. ^§ – p<0.05, in comparison with sodium valproate at a dose of 300 mg/kg

5. ° – p<0.05, in comparison with sodium valproate at a dose of 150 mg/kg

Table 2: Influence of topiramate, digoxin and their combination on seizures, induced by MES in mice

Experimental group	n	% of mice with convulsions		Severity, points	Duration, sec	Recovery time, sec	Death time, sec	Lethality, %
Experimental group	n	clonic	tonic	Severity, points	Duration, sec	Recovery time, sec	Death time, sec	Lethality, %
Control (untreated seizures)	10	100	100	5.80±0.13	11.50±1.71	19.50±3.50	13.88±0.81	80
Digoxin, 0.8 mg/kg	8	100	100	5.13±0.23*	5.00±1.96*	19.33±1.45	14.00	25**
Topiramate, 300 mg/kg	7	100	86	4.00±0.22**^##	1.86±0.14**	21.71±4.14	–	0**^#
Topiramate, 150 mg/kg	7	100	100	4.71±0.36*	8.29±4.08	19.60±1.63	24.00±2.00	29*^§
Topiramate, 150 mg/kg + Digoxin, 0.8 mg/kg	7	100	100	4.14±0.14**^##	1.71±0.18**°	18.57±2.51	–	0**^#°

1. n – number of mice in the appropriate experimental group

2. * – p<0.05, in comparison with control (untreated seizures), ** – p<0.01, in comparison with control (untreated seizures)

3. ^# – p<0.05, in comparison with digoxin, ^## – p<0.01, in comparison with digoxin

4. ^§ – p<0.05, in comparison with topiramate at a dose of 300 mg/kg

5. ° – p<0.05, in comparison with topiramate at a dose of 150 mg/kg

Table 3: Influence of levetiracetam, digoxin and their combination on seizures, induced by MES in mice

Experimental group	n	% of mice with convulsions		Severity, points	Duration, sec	Recovery time, sec	Death time, sec	Lethality, %
Experimental group	n	clonic	tonic	Severity, points	Duration, sec	Recovery time, sec	Death time, sec	Lethality, %
Control (untreated seizures)	10	100	100	5.80±0.13	11.50±1.71	19.50±3.50	13.88±0.81	80
Digoxin, 0.8 mg/kg	8	100	100	5.13±0.23*	5.00±1.96*	19.33±1.45	14.00	25**
Levetiracetam, 100 mg/kg	7	100	100	5.14±0.40	9.00±2.93	18.33±9.35	14.75±2.06	57
Levetiracetam, 50 mg/kg	7	100	100	5.00±0.38	5.57±1.91*	17.00±5.72	10.67±1.67	43
Levetiracetam, 50 mg/kg + Digoxin, 0.8 mg/kg	7	100	86	4.29±0.29**^#	1.57±0.20**^#°	11.57±2.05^#	–	0**^#§§°°

1. n – number of mice in the appropriate experimental group

2. * – p<0.05, in comparison with control (untreated seizures), ** – p<0.01, in comparison with control (untreated seizures)

3. ^# – p<0.05, in comparison with digoxin

4. ^§ – p<0.05, in comparison with levetiracetam at a dose of 100 mg/kg, ^§§ – p<0.01, in comparison with levetiracetam at a dose of 100 mg/kg

5. ° – p<0.05, in comparison with levetiracetam at a dose of 50 mg/kg, °° – p<0.01, in comparison with levetiracetam at a dose of 50 mg/kg

Table 4: Influence of clonazepam, digoxin and their combination on seizures, induced by MES in mice

Experimental group	n	% of mice with convulsions		Severity, points	Duration, sec	Recovery time, sec	Death time, sec	Lethality, %
Experimental group	n	clonic	tonic	Severity, points	Duration, sec	Recovery time, sec	Death time, sec	Lethality, %
Control (untreated seizures)	10	100	100	5.80±0.13	11.50±1.71	19.50±3.50	13.88±0.81	80
Digoxin, 0.8 mg/kg	8	100	100	5.13±0.23*	5.00±1.96*	19.33±1.45	14.00	25**
Clonazepam, 0.1 mg/kg	7	100	100	5.29±0.36	12.14±3.99	14.67±5.78	20.00±2.80	57
Clonazepam, 0.05 mg/kg	7	100	100	5.86±0.14^#	13.29±2.29	11.00	15.17±1.54	86^##
Clonazepam, 0.05 mg/kg + Digoxin, 0.8 mg/kg	7	100	100	4.43±0.20**^#°°	1.86±0.14**°°	16.00±2.73	–	0**^#§§°°

1. n – number of mice in the appropriate experimental group

2. * – p<0.05, in comparison with control (untreated seizures), ** – p<0.01, in comparison with control (untreated seizures)

3. ^# – p<0.05, in comparison with digoxin, ^## – p<0.01, in comparison with digoxin

4. ^§ – p<0.05, in comparison with clonazepam at a dose of 0.1 mg/kg, ^§§ – p<0.01, in comparison with clonazepam at a dose of 0.1 mg/kg

5. ° – p<0.05, in comparison with clonazepam at a dose of 0.05 mg/kg, °° – p<0.01, in comparison with clonazepam at a dose of 0.05 mg/kg

Table 5: Influence of phenobarbital, digoxin and their combination on seizures, induced by MES in mice

Experimental group	n	% of mice with convulsions		Severity, points	Duration, sec	Recovery time, sec	Death time, sec	Lethality, %
Experimental group	n	clonic	tonic	Severity, points	Duration, sec	Recovery time, sec	Death time, sec	Lethality, %
Control (untreated seizures)	10	100	100	5.80±0.13	11.50±1.71	19.50±3.50	13.88±0.81	80
Digoxin, 0.8 mg/kg	8	100	100	5.13±0.23*	5.00±1.96*	19.33±1.45	14.00	25**
Phenobarbital, 20 mg/kg	7	100	71**	4.00±0.31**^#	1.29±0.18**^##	12.57±2.18^#	–	0**^#
Phenobarbital, 10 mg/kg	7	100	100^§	5.57±0.20^§§	10.43±2.86^§§	11.33±2.73^#	15.75±2.39	57^§§
Phenobarbital, 10 mg/kg + Digoxin, 0.8 mg/kg	7	100	86	4.86±0.40*	8.14±3.20^§	10.40±1.33^##	19.50±0.50	29*^§

1. n – number of mice in the appropriate experimental group

2. * – p<0.05, in comparison with control (untreated seizures), ** – p<0.01, in comparison with control (untreated seizures)

3. ^# – p<0.05, in comparison with digoxin, ^## – p<0.01, in comparison with digoxin

4. ^§ – p<0.05, in comparison with phenobarbital at a dose of 20 mg/kg, ^§§ – p<0.01, in comparison with phenobarbital at a dose of 20 mg/kg

The anticonvulsant activity of phenobarbital, however, is dose-dependent: at a ½ ED₅₀ (10 mg/kg) the drug does not have a significant effect on the pathogenesis of electro-induced paroxysms, statistically significantly inferior to the effects of phenobarbital at an ED₅₀.

When adding digoxin to phenobarbital at a ½ ED₅₀, this classic AED has anticonvulsant properties: against the background of a combination statistically significant lowering the number of dead animals (51% against control indicator, p<0.05) and the severity of seizures decreasing (in 1.2 times compared with control, p<0.05) are noted. However, the severity of the anticonvulsant effect of the combination of phenobarbital at a ½ ED₅₀ and digoxin is significantly inferior to the effects of phenobarbital at an ED₅₀, which provides a full protective effect on the integrated indicator – lethality.

The pathogenesis of the MES model is qualitatively different from other chemo-induced experimental seizures. MES is based on the activation of sodium currents, dysregulation of membrane potentials and generalization of excitation. MES is thought to induce tonic-clonic seizures with a tonic component dominance. Under the conditions of electro-induced seizures, anticonvulsant properties are inherent in blockers of sodium voltage-gated channels – phenytoin, carbamazepine, oxcarbazepine, lamotrigine. At the same time, antiepileptic drugs with GABAergic properties (such as benzodiazepines, barbiturates, valproates, vigabatrin, levetiracetam, topiramate) either show much less activity in the MES seizure model, or are ineffective at all⁴⁴.

The potentiation of the anticonvulsant action of all, without exception, the studied classical AEDs at a ½ ED₅₀ with low doses of digoxin can be explained primarily by the influence on various pathways of experimental seizures development. Cardiac glycoside acts by a mechanism that is not typical for most known anticonvulsants – it activates neuronal Na⁺/K⁺ ATPase, which realize the main mechanism of occurrence and conduction of excitation^25,28,29. Although drug pharmacokinetic interactions, including increased concentration of AEDs in the brain caused by digoxin, cannot be completely ruled out, there is currently no reliable information on the ability of cardiac glycosides (including digoxin) to affect the permeability of the blood-brain barrier to other drugs. Digoxin also does not show the properties of either an inhibitor or an inducer of glycoprotein-P, which prevents the penetration of xenobiotics into the CNS^45–47.

Although all cardiac glycosides belong to drugs with a narrow therapeutic index⁴⁸, the peculiarities of pharmacodynamics – "saltatory" development of cardiac effects with increasing dose and, consequently, the lack of impact on the myocardium at low doses of digoxin³⁵ – predict the safety of its use as an anticonvulsant medicine.

Therefore, in the model of MES seizures in mice, it was established that digoxin in low doses enhances the effect of classical antiepileptic drugs, supplying a pronounced pharmacological effect of their sub-effective doses. This shows that digoxin can be used as an adjuvant agent in complex treatment of epilepsy, as it allows to reduce the dose of widely used AEDs without decrease in the effectiveness of therapy. Digoxin modulates the activity of anticonvulsants with inappropriate mechanisms of action in relation to the used convulsive model (including levetiracetam and clonazepam) and significantly increases the therapeutic potential of classical anticonvulsants even in a situation of incomplete seizure control, which is the basis for the use of low doses of cardiac glycoside at multidrug-resistant epilepsy.

The obtained results create preconditions for further investigation of the influence of digoxin on the anticonvulsant action profile of widely used AEDs, molecular mechanisms of its action, as well as potential risks of long-term combined use of AEDs with digoxin.

CONCLUSION:

The low-dose influence of digoxin on the antiepileptic potential of sodium valproate, levetiracetam, topiramate, clonazepam and phenobarbital in electro-induced seizures in mice has been studied. Digoxin has been shown to potentiate the effects of classical AEDs sodium valproate, topiramate and phenobarbital, supplying a pronounced pharmacological effect of their sub-effective doses. In addition, low-dose digoxin has been shown to modulate the effects of levetiracetam and clonazepam, increasing their therapeutic potential even under incomplete seizure control, the equivalent of drug-resistant epilepsy. The obtained results substantiate the expediency of further study of digoxin as an anticonvulsant drug in the adjuvant therapy of epilepsy and other seizures conditions.

CONFLICT OF INTEREST:

The authors have no conflict of interest to declare.

FUNDING:

The research was conducted as a part of a fundamental scientific study of the Ministry of Health of Ukraine No. 0120U102460 "Rationale for improving the treatment of multidrug-resistant epilepsy through the combined use of classical anticonvulsant medicines with other drugs" at the expense of the State Budget of Ukraine.

ACKNOWLEDGMENTS:

Authors express their highest esteem and thanks to Deputy Director for Science of the ESIAP of the National University of Pharmacy, researcher Tatiana Yudkevich for her help in hosting the research.

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Received on 11.12.2021 Modified on 13.04.2022

Research J. Pharm. and Tech 2022; 15(9):4241-4247.

DOI: 10.52711/0974-360X.2022.00713