Short communication
Ivabradine (a hyperpolarization activated cyclic nucleotide-gated channel blocker) elevates
the threshold for maximal electroshock-induced tonic seizures in mice
Jarogniew J. £uszczki1,2, Andrzej Prystupa3, Marta Andres-Mach1, Ewa Marzêda1, Magdalena Florek-£uszczki4
1Isobolographic Analysis Laboratory,4Department of Public Health, Institute of Rural Health, Jaczewskiego 2, PL 20-950 Lublin, Poland
2Department of Pathophysiology, Medical University, Jaczewskiego 8, PL 20-090 Lublin, Poland
3Department of Internal Medicine, Medical University, Staszica 11, PL 20-090 Lublin, Poland
Correspondence: Jarogniew J. £uszczki, e-mail: jarogniew.luszczki@gmail.com, jluszczki@yahoo.com
Abstract:
Background: The aim of this study was to determine the effect of ivabradine (a hyperpolarization activated cyclic nucleotide-gated channel (HCN) blocker) on the threshold for maximal electroshock (MEST)-induced tonic seizures in mice.
Methods: Electroconvulsions were produced in mice by means of a current (sine-wave, 50 Hz, maximum 500 V, strength from 3–10 mA, ear-clip electrodes, 0.2-s stimulus duration, tonic hindlimb extension taken as the endpoint).
Results: Ivabradine administered intraperitoneally (ip), 60 min before the MEST test, at doses of 5 and 10 mg/kg, did not alter the threshold for maximal electroconvulsions in mice. In contrast, ivabradine at doses of 15 and 20 mg/kg significantly elevated the threshold for maximal electroconvulsions in mice (p < 0.05 and p < 0.001, respectively). Linear regression analysis of ivabradine doses and their corresponding threshold increases allowed determination of the threshold increasing doses by 20 and 50% (TID20 and TID50values) that elevate the threshold in drug-treated animals over the threshold in control animals. The experimentally de- rived TID20and TID50values for ivabradine were 8.70 and 18.29 mg/kg, respectively.
Conclusions: Based on this preclinical study, one can ascertain that ivabradine dose-dependently increased the threshold for MEST-induced seizures, suggesting the antiseizure activity of the compound in this seizure model in mice.
Key words:
ivabradine, threshold for maximal electroshock-induced seizures, TID20, TID50, mice
Introduction
In various types of neurons a voltage-activated Na+/K+ current designated “Ih” has been identified. This cur- rent (Ih = hyperpolarization-activated depolarizing cur- rent) is activated by membrane hyperpolarization fa- cilitated by cAMP [1]. A current corresponding to Ih is
also present in cardiac pacemaker cells where it is named “funny current” (If). Many functional roles have been attributed to Ih/If, including the initiation and regulation of the heart beat (“pacemaker current”) [3]. An involvement of Ih in the control of rhythmic activity in neuronal circuits (e.g., in the thalamus) is also postulated. In addition, Ih was claimed to con-
Pharmacological Reports 2013, 65, 14071414 ISSN 1734-1140
Copyright © 2013 by Institute of Pharmacology Polish Academy of Sciences
by cloning a family of ionic channels known as HCN – hyperpolarization activated cyclic nucleotide-gated channels [12, 17, 33–35]. To date, four isoforms of HCN (1–4) have been identified in a variety of mam- malian tissues, including cardiac and neuronal tissues [1, 2, 7, 8, 31, 32].
HCN1 has been detected in the neocortex, hippo- campus, cerebellar cortex, and in the brainstem [24, 27, 34]. HCN2 is ubiquitously distributed in the cen- tral nervous system, and HCN3 is poorly expressed in the brain, except for the olfactory bulb, hypothala- mus, and retinal cones pedicles [3, 26, 32]. Only HCN4 channels are highly expressed in thalamic nu- clei, in the basal ganglia, and in the olfactory bulb [25, 27, 34]. In neurons, HCN channels contribute to a larger set of functions, such as working memory, motor learning, generation of rhythmic activity, con- trol of the membrane resting potential, regulation of cell excitability, dendritic integration, and synaptic transmission [3]. More detailed information on the properties and functions of Ih/HCN channels in the nervous system is presented elsewhere [3, 32, 35, 38].
Since among the functional roles of HCN channels in neurons, one of the most important is the control of cell excitability, it is highly likely that some dysfunc- tions in HCN channels might be involved in some forms of epileptic activity. For example, in a rat model of childhood febrile seizure, down-regulation of HCN1 and up-regulation of HCN2 have been ob- served in hippocampal CA1 pyramidal neurons after the epileptic event [5, 6, 9, 10, 33]. In HCN2-deficient mice, an almost complete abolishment of Ih in thala- mocortical and thalamic interneurons was associated with a large hyperpolarizing shift of the resting mem- brane potential, and thus a more pronounced burst- mode activity, leading to spike-and-wave discharges typical of absence epilepsy have been observed [16].
Additionally, it has been shown that lamotrigine and gabapentin, two antiepileptic drugs known to stimu- late Ih, act by increasing basal inhibition of CA1 hip- pocampal activity through the enhancement of Ih in inhibitory interneurons [29, 30].
Ivabradine (3-(3-{[((7S)-3,4-dimethoxybicyclo-[4,2,0]
octa-1,3,5-trien-7-yl)methyl]methyl-amino}propyl)- 1,3,4,5-tetrahydro-7,8-dimethoxy-2H-3-benzazepin-2- one, hydrochloride) is a pure heart rate lowering drug that specifically inhibits the If current involved in the
tassium channels [7]. Electrophysiological experi- ments revealed that ivabradine blocks mouse HCN1 and human HCN4 channels, being an open-channel blocker of hHCN4 and a closed-channel blocker of mHCN1 channels [8].
Anticonvulsant effects produced by drugs or agents are usually evaluated in experimental (preclinical) studies by displaying efficacy of these compounds in animal models of epilepsy [14, 18, 28, 36]. Of these models, the maximal electroshock-induced seizure threshold (MEST) test can determine whether or not the examined compounds affect the threshold for maximal electroconvulsions in animals. In order to unequivocally assess the anticonvulsant potential of agents or drugs in this test, doses increasing the threshold by 20 and 50% (TID20 and TID50 values) that elevate the threshold for electroconvulsions in drug-treated animals by 20 and 50% over the thresh- old in control animals can be denoted [14, 36]. The TID20 and TID50 values uniformly describe the anti- convulsant potency of drugs or agents in preclinical studies. To determine the TID20and TID50values, lin- ear regression analysis of drug doses and their corre- sponding threshold values was used [14, 36]. Assess- ment of dose-response relation with linear regression is a standard and common procedure in pharmacol- ogical studies, especially in those assessing the an- tielectroshock potential of drugs or agents [15].
Recently, we have reported that some second-generation antiepileptic drugs (i.e., gabapentin, levetiracetam, stiripen- tol, tiagabine, and vigabatrin) increased the threshold for electroconvulsions in mice that allowed the calculation of TID20and TID50values in mice [19–22, 24].
Considering the above-mentioned facts, it was of importance to determine the TID20and TID50 values for ivabradine (the HCN channel blocker) in the MEST test in mice.
Materials and Methods
Animals and experimental conditions
All experiments were performed on adult male Swiss mice weighing 22–26 g. The animals were purchased
from a licensed breeder (J. Ko³acz, Warszawa, Po- land). The mice were kept in colony cages with free access to food and tap water, under standardized housing conditions (natural light-dark cycle, tempera- ture 21 ± 1°C). After 7 days of adaptation to labora- tory conditions, the animals were randomly assigned to experimental groups consisting of 8 mice. Each mouse was used only once. All tests were performed between 9.00 a.m. and 2.00 p.m. Procedures involv- ing animals and their care were conducted within cur- rent European Community and Polish legislation on animal experimentation. Additionally, all efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data. The experimental protocols and proce- dures listed below conformed with the Guide for the Care and Use of Laboratory Animals and approved by the Second Local Ethics Committee at the Univer- sity of Life Sciences in Lublin (License No.:
77/2011). The total number of animals used in this study was 160.
Drug
Ivabradine (Procoralan®, Les Laboratoires Servier, Neuilly-sur-Seine, France) was suspended in a 1%
aqueous solution of Tween 80 (Sigma, St. Louis, MO, USA) and administered intraperitoneally (ip) in a vol- ume of 0.005 ml/g body weight at 60 min before MEST-induced seizures. The pretreatment time (60 min) before testing ivabradine was established in our pilot study as the time to peak of maximum anti- convulsant activity of the drug. The time to the peak of maximum anticonvulsant effects for ivabradine was used as the reference time in all behavioral tests.
Maximal electroshock seizure threshold (MEST) test
Electroconvulsions were produced by means of an al- ternating current (sine-wave, 0.2 s stimulus duration, 50 Hz, maximum stimulation voltage of 500 V, cur- rent strength 3–10 mA) delivered via ear-clip elec- trodes by a rodent shocker generator (Type 221, Hugo Sachs Elektronik, Freiburg, Germany). The electrical system of the stimulator was self-adjustable so that changes in impedance did not result in alterations of current intensity (i.e., the system provided constant current stimulation). The criterion for the occurrence of seizure activity was the tonic hind limb extension
(i.e., the hind limbs of animals outstretched 180° to the plane of the body axis). To evaluate the threshold for maximal electroconvulsions, at least 4 groups of mice, consisting of 8 animals per group, were chal- lenged with electroshocks of various intensities to yield 10–30, 30–50, 50–70, and 70–90% of animals with seizures. Then, a current intensity-response rela- tionship curve was constructed, according to the log- probit method by Litchfield and Wilcoxon [13], from which a median current strength (CS50 in mA) was calculated. Each CS50 value represented the current intensity required to induce tonic hindlimb extension in 50% of the mice challenged. After administration of a single dose of ivabradine to 4 groups of animals, the mice were again subjected to electroconvulsions (each group with a constant current intensity). The threshold for maximal electroconvulsions was re- corded for 4 different doses of ivabradine: 5, 10, 15 and 20 mg/kg, which were empirically chosen for testing in the MEST test. Subsequently, the percent- age of increase in CS50 values for animals injected with increasing doses of ivabradine over the control (vehicle-treated animals) was calculated. The doses of ivabradine and their resultant percentage of threshold increase over the control (vehicle-treated animals) were graphically plotted in rectangular coordinates of the cartesian plot system, and examined with least- squares linear regression analysis [11]. From linear regression equation, the TID20and TID50values were determined, as recommended by Löscher et al. [14]
and Swinyard et al. [36]. The experimental procedure has been described in more detail in our earlier studies [19–22].
Step-through passive avoidance task
Each animal was administered ivabradine at a maxi- mally tested dose of 20 mg/kg on the first day before training. The time before commencement of the train- ing session (after drug administration) was identical to that for the MES test. Subsequently, the animals were placed in an illuminated box (10 × 13 × 15 cm) connected to a larger dark box (25 × 20 × 15 cm) equipped with an electric grid floor. Entrance of the animals to the dark box was punished by an adequate electric footshock (0.6 mA for 2 s). On the following day (24 h later), the pre-trained animals were placed again into the illuminated box and observed for up to 180 s. Mice that avoided the dark compartment for 180 s were considered as having remembered the
Ivabradine and threshold in the MEST test in mice
Jarogniew J. £uszczki et al.
about ability to acquire a task (learning) and to recall a task (retrieval).
Grip-strength test
The effect of ivabradine at a maximally tested dose of 20 mg/kg on skeletal muscular strength in mice was quantified by the grip-strength test. The grip-strength apparatus (BioSeb, Chaville, France) comprised of a wire grid (8 × 8 cm) connected to an isometric force transducer (dynamometer). The mice were lifted by the tails so that their forepaws could grasp the grid.
The mice were then gently pulled backward by the tail until the grid was released. The maximal force ex- erted by the mouse before losing grip was recorded.
The mean of 3 measurements for each animal was cal- culated, and subsequently, the mean maximal force of 8 animals per group was determined. The muscular strength in mice is expressed in N (newtons) as the means ± SE of at least 8 determinations.
Chimney test
The chimney test was used to quantify the adverse ef- fect potential of ivabradine administered at a maxi- mally tested dose of 20 mg/kg. In this test, the animals had to climb backwards up a plastic tube (3 cm inner diameter, 30 cm long) and impairment of motor per- formance was indicated by the inability of the mice to climb backward up the transparent tube within 60 s.
Statistical analysis
The CS50 values with their 95% confidence limits were calculated by computer-assisted log-probit analysis, according to Litchfield and Wilcoxon [13].
Subsequently, the 95% confidence limits were trans- formed to their standard errors (SE), according to
£uszczki et al. [23]. Statistical analysis of data was performed with one-way ANOVA followed by the Tukey/Kramer test for multiple comparisons, as docu- mented earlier [21, 22]. The results obtained in the step-through passive avoidance task were statistically evaluated using Mann-Whitney-Wilcoxon test. The
statistically significant if p < 0.05. All statistical tests were performed using commercially available Graph- Pad Prism version 4.0 for Windows (GraphPad Soft- ware, San Diego, CA, USA).
Results
Effect of ivabradine on the threshold for maximal electroshock-induced tonic seizures Ivabradine administered ip, 60 min before electrocon- vulsions, increased in a dose-dependent manner the threshold for MEST-induced seizures in mice. The experimentally-derived CS50 value for ivabradine at a dose of 20 mg/kg was 6.95 mA and significantly dif- fered from that of control animals, which was 4.50 mA (p < 0.001; Tab. 1). Similarly, the CS50value for ivabradine at a dose of 15 mg/kg was 6.42 mA and significantly differed from that of control animals (p <
0.05; Tab. 1). Ivabradine at lower doses of 5 and
Tab. 1. Effect of ivabradine on electroconvulsive threshold in mice
Treatment (mg/kg) CS50(mA) n TI (%)
Control (vehicle) 4.50 ± 0.51 24 –
Ivabradine (5) 4.93 ± 0.44 8 9.6
Ivabradine (10) 5.44 ± 0.44 16 20.9
Ivabradine (15) 6.42 ± 0.45* 16 42.7
Ivabradine (20) 6.95 ± 0.43*** 24 54.4 F (4, 83) = 5.083; p = 0.0010
Values are presented as median current strengths (CS50± SE) nec- essary to evoke seizure activity (tonic hindlimb extension) in 50% of animals tested. Each experimental group consisted of 8 animals. To determine each CS50value, four groups of animals (8 mice per group) were used. Statistical evaluation of data was performed with one-way ANOVA, followed by the post-hoc Tukey/Kramer test. Addi- tionally, the threshold for control animals was considered as a base- line (reference) value, allowing for the subsequent calculation of a percentage of threshold increase (TI) in animals after ivabradine administration. n – number of animals tested at current strengths with effect ranging between 4 and 6 probits. However, log-probit method considered only those groups of animals whose seizure effects ranged between 4 and 6 probits. This is why, number of animals (n) is different for various CS50 values. F – F-statistics from one-way ANOVA; p – probability from one-way ANOVA. * p < 0.05 and *** p <
0.001 vs. control (vehicle-treated) animals
10 mg/kg also elevated the threshold for MEST- induced clonic seizures by 9.6 and 20.9%, respec- tively; however, the results did not attain statistical significance with Tukey/Kramer post-hoc test (Tab. 1).
The equation for dose-threshold increase relationship for ivabradine was, as follows:
y = 3.129 x – 7.222 (r2= 0.983)
where y is the threshold increase in %, x is the drug dose, and r2is the coefficient of determination. The experimentally derived TID20and TID50values for iv- abradine were 8.70 and 18.29 mg/kg, respectively, in the MEST test in mice (Fig. 1).
Effects of ivabradine on motor performance, long-term memory and skeletal muscular strength of animals in the chimney, step-through passive avoidance and grip-strength tests
Ivabradine administered alone at a dose of 20 mg/kg did not impair long-term memory in animals chal- lenged with the step-through passive avoidance task (Tab. 2). Similarly, ivabradine at 20 mg/kg neither af- fected muscular strength in mice in the grip-strength test, nor impaired motor coordination in mice sub- jected to the chimney test (Tab. 2).
Discussion
The objective of this study was to determine the TID20
and TID50 values for ivabradine in the MEST test.
Linear regression analysis of ivabradine doses and their corresponding threshold increasing values over the threshold for control animals in the MEST test re- vealed that there was a close relationship between the doses of ivabradine and their biological effects in terms of seizure suppression in mice (Fig. 1). There is no doubt that the MEST test allows selection of agents or drugs having the anticonvulsant properties in both, preclinical studies on animals and clinical set- tings in patients with epilepsy.
In the presented study, it was found for the first time that ivabradine as a HCN channel blocker ele- vated the threshold for electroconvulsions in mice and thus possesses anticonvulsant potency against electro- shock-induced tonic seizures in mice. It can be sup- posed that ivabradine, due to its HCN channel blocker properties, is able to support the ionic homeostasis in the brain, and therefore, the drug elevated the thresh- old for electroconvulsions in mice.
It is important to note that linear regression analy- sis was used in this study to denote the doses of iv- abradine increasing the threshold for electroconvul-
Ivabradine and threshold in the MEST test in mice
Jarogniew J. £uszczki et al.
Fig. 1. Dose-threshold increase relationship for ivabradine in maximal electroshock seizure threshold (MEST) test in mice. Points placed on the graph represent threshold increasing doses of ivabradine, experimentally denoted in the MEST test in mice. Linear regression analysis allowed determination of the equation for dose-threshold increase relationship for ivabradine, as follows: y = 3.129 x – 7.222 (r2= 0.983); where y is the threshold increase in %, x is the drug dose, and r2is the coefficient of determination [11]. From this equation one denotes the TID20and TID50 (threshold increasing doses by 20 and 50%) for the MEST test. In this study, these values were 8.70 and 18.29 mg/kg, respectively. Ivabradine was administered ip, 60 min before the threshold evaluation
sions in animals by a fixed, previously established, percentage (20 and 50%), as mentioned earlier
by drugs tested at various doses. Therefore, to denote other effects, more experimental groups are required.
In contrast, the linear regression analysis allows pre- diction of the doses increasing the threshold for elec- troconvulsions by the respective percentage, without testing additional groups of experimental animals [19–22]. Moreover, with the linear regression analy- sis, one can readily assess the same effect for various agents and drugs, contributing to the evaluation of their anticonvulsive potency by comparing their TID20and TID50values.
The results presented in this study can be compared to those reported earlier for some classical and second-generation antiepileptic drugs (Tab. 3). It has been documented that carbamazepine, clonazepam, diazepam, gabapentin, levetiracetam, phenobarbital, phenytoin, stiripentol, tiagabine, valproate, vigabatrin and WIN 55,212-2 mesylate (a non-selective cannabi- noid CB1 and CB2 receptor agonist) increased the threshold for electroconvulsions in mice [14, 19–22, 24]. The direct comparison of TID50 values of the classical and second-generation antiepileptic drugs with those calculated for ivabradine, revealed that car- bamazepine, clonazepam, diazepam, phenobarbital, phenytoin, tiagabine and WIN 55,212-2 mesylate had TID50values lower than this of ivabradine (Tab. 3). In contrast, the TID50 values denoted for gabapentin, levetiracetam, stiripentol, valproate and vigabatrin were considerably higher than this denoted for iv- abradine in the presented study (Tab. 3). Thus, one can ascertain that ivabradine possesses anticonvulsant potency in the MEST model in mice.
Regarding the acute adverse effects produced by ivabradine, it was found that ivabradine administered alone at a maximal dose of 20 mg/kg neither affected long-term memory in mice challenged with the step- through passive avoidance task, nor changed skeletal muscular strength in mice subjected to the grip- strength test. In the chimney test, it was documented that ivabradine administered singly at a dose of 20 mg/kg had no significant impact on motor coordi- nation in mice.
Finally, based on this preclinical study, it can be as- certained that ivabradine, in a dose dependent manner, suppressed MEST-induced tonic seizures in mice.
Tab. 3. Characteristics of various drugs in maximal electroshock sei- zure threshold (MEST) test in mice
Drug TID20 TID50 References
Carbamazepine N.D. 1.5 [14]
Clonazepam N.D. 0.65 [14]
Diazepam N.D. 2.7 [14]
Gabapentin 70.0 112.7 [24]
Ivabradine 8.70 18.29 [present study]
Levetiracetam 44.0 150.0 [19]
Phenobarbital N.D. 4.0 [14]
Phenytoin N.D. 5.4 [14]
Stiripentol 103.2 195.8 [21]
Tiagabine 4.4 8.0 [20]
Valproate N.D. 69 [14]
Vigabatrin 226.2 N.D. [20, 24]
WIN 55,212-2 6.3 17.2 [22]
Results are presented as doses of drugs increasing the threshold for electroconvulsions by 20 and 50% (TID20and TID50in mg/kg), ex- perimentally denoted in the MEST test in mice. N.D. – not determined
(mg/kg) (s) (N) coordination
impairment (%)
Vehicle 180 (180; 180) 0.915 ± 0.051 0 Ivabradine
(20)
180 (175.5; 180) 0.898 ± 0.057 0
Results are presented as: 1) median retention times (in seconds, with 25thand 75thpercentiles in parentheses) from the passive avoidance task, assessing long-term memory in mice; 2) mean grip-strengths (in newtons ± SE) from the grip-strength test, assessing muscular strength in mice; and 3) percentage of animals showing motor coor- dination impairment in the chimney test in mice. Each experimental group consisted of 8 animals, and 3 different groups of animals were used to study behavioral effects of ivabradine in the grip-strength, chimney, and step-through passive avoidance tests in mice. Statisti- cal analysis of data from the passive avoidance task was performed with Mann-Whitney-Wilcoxon test; data from the grip-strength test were analyzed with Student’s t-test. Fisher’s exact probability test was used to analyze the results from the chimney test. Ivabradine was administered ip at time scheduled from the MES test
Disclosure of conflicts of interest:
The authors have no disclosures to declare.
Acknowledgments:
This study was supported by a grant from the Institute of Rural Health in Lublin, Poland. Professor J.J. £uszczki is a Member of the Academy of Young Scholars of the Polish Academy of Sciences in Warszawa, Poland.
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Received: September 16, 2012; in the revised form: April 4, 2013;
accepted: May 13, 2013.
