The Effect of Innovative Antiepileptic Drug, Levetiracetam, on Various Symptoms of Morphine Dependence in Mice

1. Introduction

Levetiracetam, a second-generation antiepileptic drug, was introduced into clinical practices in 2000 year. Chemically, it is classified as a pyrrolidone derivative and is S-enantiomer of α-ethyl-2-oxo-1-pyrrolidineacetamide. Levetiracetam is primarily utilized for the treatment of various types of seizures, including focal, myoclonic and generalized tonic-clonic seizures. Its broad therapeutic spectrum and favorable side effect profile make it a cornerstone in epilepsy management. Nowadays, levetiracetam is effective drug in therapy of epilepsy both in monotherapy and in adjunctive therapy [1]. Because levetiracetam has a broad spectrum of pleiotropic effects it is also increasingly used in the treatment of other diseases, such as, anxiety, depression or bipolar disorder. In some studies, its effectiveness was observed in management of neuropathic pain and in attenuation of symptoms of addictive substances [2,3]. It also showed the activity in experimental models of neuropathic pain [4] and inflammatory pain [5] in mice. There is data showing that levetiracetam-induced analgesia was effective in reduction of cancer pain which was maintained despite typical therapy [2]. Moreover, its beneficial activity was confirmed in model of alcohol dependence [6].

The mechanism of action of levetiracetam is a unique as compared to other antiepileptics. It selectively and reversibly binds the synaptic vesicle protein 2A (SV2A) mainly located on glutamatergic terminals [7]. SV2A protein is one of the three protein isoforms (SV2A, SV2B and SV2C). This protein is integral to synaptic vesicles presented in all synaptic terminals in brain, including glutamatergic neurons and neurons for γ-aminobutyric acid (GABA). It plays an important role in neurotransmitter exocytosis and synaptic transmission [8] and improves neurotransmission by increasing the number of synaptic vesicles [9]. It is believed that SV2 protein is an important factor in epileptogenesis [10]. Levetiracetam, via modulation SV2A protein, reduces neurotransmitter release, stabilizes synaptic function and enhances neural network stability [9,11,12]. Other mechanisms are also involved in the activity of levetiracetam. Levetiracetam reduced high-voltage activated calcium current, mainly L-type and N-type calcium channels and also, P/Q channels [13,14,15,16,17]. Levetiracetam also diminished the release of Ca2+ ions from the endoplasmic reticulum dependent on the ryanodine receptor and the inositol 1,4,5-trisphosphate (IP3) receptor modulating the intracellular signaling [18]. In in vitro studies, levetiracetam reduced the voltage-modulated potassium current and reduced the repetitive generation of action potentials in neurons [19]. To opposite, the effect of levetiracetam on voltage-dependent sodium channels and sodium ion (Na+) transport had not been confirmed. Thus, levetiracetam does not activate the classical mechanism of action of many antiepileptic drugs [15].

Morphine, widely used drug in clinical practices to reduction of acute and chronic pain, is also one of the strongest dependent drug [20]. The rewarding effect of morphine is associated with the stimulation of µ opioid receptors, especially those located on GABAergic terminals in the ventral tegmental area (VTA), which causes the inhibition of endogenous GABA secretion. It leads to the disinhibition of dopaminergic neurons, increased dopamine secretion in the nucleus accumbens and a feeling of euphoria, which promotes the development of addiction [21]. Morphine dependence is manifested as the appearance of characteristic, unpleasant symptoms, developed after cessation of chronic exposure to morphine, named as morphine withdrawal signs. The second parameter of morphine dependence is development of morphine tolerance. It is defined as the necessity to take increasing doses of morphine to achieve the same pharmacological effect [22]. The third parameter of the state of dependence is behavioral sensitization which is a consequence of sporadic administration of an addictive substance, including morphine, and is expressed as an intensification of pharmacological effect of addictive substance administered after a break period. It reflects drug-seeking behavior and often leads to relapse to drug use [23]. Nowadays, therapy of morphine dependence is not effective and, despite numerous studies, for many years any innovative drugs has not been introduced. Therefore, new, innovative ideas are extremely important to find pharmacological tool for therapy of opiod dependence.

Taking into account a unique mechanism of action of levetiracetam it may be hypothesized that levetiracetam can be effective drug in morphine dependence. The analysis of existing data shows that co-administration of levetiracetam with morphine is relatively safe and clinical interactions between them are not significant. The therapy of epileptic seizures with subcutaneous levetiracetam doses in a palliative patients co-treated with morphine does not produce significant changes in the antiseizure effect of levetiracetam and in its median dose, median osmolarity of the infusion solution and in infusion rate [24]. Levetiracetam does not effect on cytochrome P450 and it is poorly metabolized in the liver. It is manly excreted unchanged by kidney. On the other hand, morphine pharmacokinetic is associated with uridine 5’-diphosphoglucuronosyltransferases in the liver. Thus, there is relatively low risk of pharmacokinetic interaction between these drugs [25]. Levetiracetam, instead of phenobarbital, was effective in attenuation of neonatal abstinence syndrome [26] that support lack of pharmacokinetic interactions between these compound. The diversified mechanisms of action of both drugs suggest that pharmacodynamic interactions can occur. In fact, their combination at higher doses may increase neurological side effects, such as, dizziness, drowsiness or confusion, however, these adverse effects can be attenuate by reduction of doses. It shows that potential relationship between both drugs is worth to study.

In the present paper we present a series of behavioral experiments performed in mice showing the effectiveness of levetiracetam in some experimental models of morphine dependence, including model of morphine withdrawal observed as jumpings, morphine tolerance to antinociceptive effect and morphine sensitization to the locomotor activity. The obtained results support the hypothesis that levetiracetam can be considered as innovative target in morphine dependence.

2. Materials and Methods

2.1. Experimental Animals and General Conditions

The experiments were conducted on male Swiss white mice, weighing 20-25 g. The animals were kept 8-10 per cage at a room temperature of 22±1ºC and exposed to normal day/night cycle. A standard diet (Murigran, Motycz, Poland) and water were freely available. Prior to the start of the studies, the animals were acclimated to the environmental conditions for 7 days. The experimentes were performed in a lit and soundproofed room. Total number of animals was N=440.

All experiments were conducted according to the National Institute of Health Guidlines for the Care and Use of Laboratory Animals and to the European Community Council Directive for the Care and Use of Laboratory Animals, and were approved by the Local Ethics Committee - The Medical University of Lublin Committee on the Use and Care of Animals (No 20/2019, No 33/2019).

2.2. Drugs

The following drugs were used in the experiments: levetiracetam (Keppra, UCB Pharma, Belgium), morphine hydrochloride trihydrate (Cosmetic Pharma, Poland), naloxone hydrochloride (Sigma Aldrich, substance, USA). All used substances were delivered in a volume of 10ml/kg. Levetiracetam was triturated and suspended in 0.5% methylcellulose solution. Morphine hydrochloride and naloxone hydrochloride were dissolved in 0.9% saline solution. There were two control groups in preliminary study: 1) receiving 0.9% saline solution; 2) receiving 0.5% methylcellulose solution. The animals received the same volume of these solution at the respective time point before the test. There were no differences between these animals, therefore, in the figures only one control group is shown (methylcellulose group).

The substances were administered intraperitoneally (ip). The number of animals per group (N) was 8-10 individuals. The doses of substances and the times of administration were selected based on the authors' own research, experiments conducted at the Department of Pharmacology and Pharmacodynamics Medical University of Lublin, and relevant scientific literature [27].

2.3. Procedures of Behavioral Experiments

2.3.1. The Procedure of Morphine Tolerance to the Antinociceptive Effect in Mice

The tolerance to the antinociceptive effect of morphine was studied using the hot-plate test [27]. The apparatus consisted of a metal plate heated to a temperature of 55 ± 0.5°C. The plate was enclosed by a transparent Plexiglas cylinder with a diameter of 20 cm and a height of 18 cm. Each animal was placed individually on the hot plate. In the experiment the time (s) spent by each mouse on the plate was measured until the first reaction of mice to the thermal stimulus—either paw licking or jumping. The maximal measurement lasted 60 seconds. To induce morphine tolerance, mice received a dose of morphine (10 mg/kg) twice daily for 7 days and the hot-plate test was conducted on the 1st and 7th days of the experiment.

The effect of levetiracetam on morphine tolerance was studied in two experimental protocols: the expression and the acquisition of morphine tolerance. In the case of expression, levetiracetam was administered as a single dose on the 7th day of the experiment, 30 minutes before the morning morphine injection. Then, 30 minutes after the morphine injection, the hot-plate test was conducted. The reaction time of the animals to the nociceptive stimulus was measured in seconds. In the model of acquisition, levetiracetam was administered in mice once daily for 6 consecutive days (from day 1st to day 6th of the experiment), 30 minutes before the morning morphine injection. On the 7th day, only morphine was administered and 30 minutes later, the hot-plate test was conducted.

Scheme 1. Graphical presentation of drug administration in procedure of morphine tolerance.

Scheme 1. Graphical presentation of drug administration in procedure of morphine tolerance.

2.3.2. The Procedure of Naloxone-Induced Morphine Withdrawal Signs in Mice

To induce physical dependence in mice, an experimental procedure developed at the Department of Pharmacology and Pharmacodynamics Medical University of Lublin was used [28,29]. Accordingly, morphine was administered at increasing doses (10, 15, 20, 25, 30, 35, 40, and 50 mg/kg) twice daily, for 8 consecutive days. On the morning of the 9th day, another dose of morphine (50 mg/kg) was administered, and an hour later, naloxone (2.0 mg/kg)—an opioid receptor antagonist—was given to induce an acute withdrawal syndrome. Immediately after naloxone administration, the mice were placed in glass cylinders (10 liters) for observation. The number of animal jumps was counted over a 30-minute period.

The effect of levetiracetam on morphine withdrawal signs was studied in two experimental protocols. The influence of levetiracetam on expression of morphine withdrawal signs was studied by administration of a single dose of levetiracetam on the 9th day of the experiment, 20 minutes before the morphine injection. Then, 60 minutes after the morphine injection, naloxone was administered and the animals were observed. In the second protocol the influence of levetiracetam on acquisition of morphine withdrawal signs was investigated, in which levetiracetam was administered chronically - once daily for 8 consecutive days, 20 minutes before the morphine injection. On the 9th day of the experiment, the animals received only the morphine injection and 60 minutes later, naloxone was administered, followed by the assessment.

Scheme 2. Graphical presentation of drug administration in procedure of morphine dependence.

Scheme 2. Graphical presentation of drug administration in procedure of morphine dependence.

2.3.3. The Procedure of Morphine Sensitization to the Locomotor Effects of Morphine in Mice

The sensitization to the locomotor effects of morphine in mice was studied using the method of Kuribara [30], as modified by Kotlińska and Bocheński [31]. The equipment used to measure locomotor activity was composed of activity meters (Multiserv, Lublin, Poland). The activity meters are round, metal cages with a diameter of 32 cm with photoresistors installed in the walls. Photoresistors are crossed by two perpendicular beams of light, set 1 cm above the floor. When a moving animal crosses a beam, the change in the photoresistor is recorded by the appropriate counter as one movement. The value read from the counter indicates the spontaneous locomotor activity of the animals [32]. Behavioral sensitization was achieved by administration of five sporadic (every 72 hours) injections of morphine at a dose of 10 mg/kg (on 1st, 4th, 7th, 10th, and 13th day of the experiment). Immediately after each morphine administration, the animals were placed in chambers for locomotor activity test to assess the animal motility for 60 minutes. Seven days after the last morphine injection (on 20th day of the test), the animals received a challenge dose of morphine (10 mg/kg), after which their locomotion was again assessed.

Analogically, the effect of levetiracetam on morphine sensitization was studied in two experimental protocols. The influence of levetiracetam on the expression of morphine sensitization was studied by single administeration of that compound on the 20th day of the experiment, 30 minutes before the morphine injection. Immediately after morphine administration, the animals were placed in chambers for locomotor test for 60 minutes to measure locomotion. In the second protocol, the influence of levetiracetam on the acquisition of morphine sensitization was investigated by sporadic morphine administration - there were five levetiracetam injections, 30 minutes before the morphine injection on 1st, 4th, 7th, 10th, and 13th day of the experiment. On the 20th day of the experiment, the animals received a challenge dose of morphine without levetiracetam. Subsequently, the mice were placed in activity meters for 60 minutes to measure locomotion.

Scheme 3. Graphical presentation of drug administration in procedure of morphine sensitization.

Scheme 3. Graphical presentation of drug administration in procedure of morphine sensitization.

2.4. The Statistical Analysis

Statistical analysis of the results was performed in GraphPad Prism 9.1.1 (GraphPad Software, USA). When our data follow normal distribution (Shapiro-Wilk test), ANOVA analysis was performed. Two-way ANOVA was used to assess morphine tolerance and morphine sensitization in mice (Section 2.7.). In analysis of morphine withdrawal signs one-way ANOVA was used. In all procedures the comparisons between groups were made using the Tukey test (post-hoc). The analysis of morphine tolerance was assessed in the hot plate test and, the mean time (s) ​​± standard error of measurement (SEM) was presented in figures. In the study assessing naloxone-induced morphine withdrawal symptoms, the number of animal jumps was counted for a period of 30 min. The arithmetic mean of the number of jumps ± SEM was presented in figures. The analysis of morphine sensitization was based on the locomotor activity of mice and the mean number of animal movements ± SEM for 60 min period was presented in figures. The results were considered statistically significant when the test probability coefficient (p) was less than 0.05 (p<0.05). The number of animals (N) in the studied groups was 8-10 mice.

3. Results

3.1. The Effect of Levetiracetam (31.25; 62.5 mg/kg, ip) on the Expression of Tolerance to the Antinociceptive Effect of Morphine (10 mg/kg, ip) Assessed in the Hot-Plate Test in Mice

Using a two-way ANOVA analysis statistically significant changes were demonstrated in mice which were treated both with single and chronic doses of morphine (on days 1 and 7 of the experiment), compared to control animals. A day effect (F(1,36)=6.297; p=0.0167), a substance effect (F(1,36)=25.06; p<0.0001), and an interaction effect (F(1,36)=5.791; p=0.0214) were observed. Additionally, one-way ANOVA analysis confirmed statistically significant differences in reaction time of the animals on the 7th day of the experiment (F(5,54)=16.17; p<0.0001).

In the post-hoc test, statistically significant prolongation of reaction time to the nociceptive stimulus was confirmed (p<0.0001) in mice treated with single morphine dose compared to the control group. Chronic administration of morphine (for 7 consecutive days) significantly shortened the reaction time of mice (p<0.01) compared to the reaction time recorded after a single morphine administration. Administration of levetiracetam at a dose of 31.25 mg/kg with morphine significantly prolonged the reaction time of animals (p<0.05) in comparison with the reaction time of animals receiving morphine chronically. However, administration of levetiracetam at a higher dose (62.5 mg/kg) did not significantly affect the reaction time. Administration of levetiracetam alone (31.25 and 62.5 mg/kg) did not significantly change the reaction time of mice as compared to control animals. Figure 1.

3.2. The Effect of Levetiracetam (31.25; 62.5 mg/kg, ip) on the Acquisition of Tolerance to the Antinociceptive Effect of Morphine (10 mg/kg, ip) Assessed in the Hot-Plate Test in Mice

Two-way ANOVA revealed statistically significant changes in reaction time of animals which were exposed to both single and chronic injections of morphine (on 1st and 7th of the experiment) in comparison with control animals. There were observed a day effect (F(1,36)=17.24; p=0.0002), a substance effect (F(1,36)=34.97; p<0.0001), and an interaction effect (F(1,36)=14.49; p=0.0005). Additionally, one-way ANOVA confirmed statistically significant changes in the animals' response on the 7th day of the experiment (F(5,54)=15.37; p<0.0001). In the post-hoc test, it was shown that a single administration of morphine significantly prolonged (p<0.0001) the time of animal response to the nociceptive stimulus as compared to control animals. In contrast, chronic administration of morphine significantly reduced (p<0.0001) the animals' response time to the nociceptive stimulus as compared to the response time observed after a single administration of morphine. Chronic administration of levetiracetam at both doses (31.25 and 62.5 mg/kg) significantly prolonged (p<0.05 and p<0.01, respectively) the rection time to the nociceptive stimulus in comparison with animals chronically received morphine. Chronic administration of levetiracetam alone (31.25 mg/kg and 62.5 mg/kg) had no effect on the animals' response timeas compared to the control group. Figure 2.

3.3. The Effect of Levetiracetam (31.25; 62.5 mg/kg, ip) on the Expression of Naloxone-Induced (2 mg/kg, ip) Morphine Withdrawal in Mice

One-way ANOVA confirmed the existence of statistically significant differences in response of morphine-dependent mice treated with a single dose of levetiracetam (F(6,59)=13.82; p<0.0001). The post-hoc test showed that naloxone injected to animals chronically treated with morphine significantly increased the number of jumpings (p<0.0001) as compared to the number of jumpings observed in mice not treated with morphine. Administration of levetiracetam at a dose of 62.5 mg/kg significantly reduced (p<0.05) the number of jumpings in morphine-dependent animals. Levetiracetam at both doses had no effect on behavior of mice that were not exposed to morphine. Figure 3.

3.4. The Effect of Levetiracetam (31.25; 62.5 mg/kg, ip) on the Acquisition of Naloxone-Induced (2 mg/kg, ip) Morphine Withdrawal Symptoms, in Mice

As one-way ANOVA has shown, the chronic administration of levetiracetam in morphine-dependent animals affects the severity of morphine withdrawal symptoms (F(6,61)=13.97; p<0.0001). In the Tukey's test was confirmed that the injection of naloxone in animals chronically received morphine significantly increased (p<0.0001) the number of jumpings in comparison with mice which were not received morphine. The administration of lower dose (31.25 mg/kg) of levetiracetam significantly reduced (p<0.05) the number of jumpings as compared to the effect recorded in morphine-exposed animals. Chronic administration of levetiracetam (31.25 and 62.5 mg/kg) had no effect on the behavior of mice which were no exposed to morphine. Figure 4.

3.5. The Effect of Levetiracetam (31.25; 62.5; 125 mg/kg, ip) on The expression of Morphine (10 mg/kg, ip) Sensitization to the Locomotor Effects in Mice

On the based on one-way ANOVA, on the 20th day of the experiment, statistical differences in the locomotor activity of mice treated with intermittent doses of morphine (1st, 4th, 7th, 10th, 13th, and 20th day of the experiment) and a single dose of levetiracetam (20th day of the experiment) were demonstrated (F(4,41)=5.102; p=0.0020).

On the 20th day of the experiment, intermittent administration of morphine resulted in a statistically significant increase in the locomotor activity of mice in comparison with the control group (p<0.001). A single administration of levetiracetam at doses of 32.5, 62.5 and 125 mg/kg on the 20th day of the experiment in animals receiving intermittent injections of morphine significantly reduced their locomotory activity (p<0.05; p<0.05; p<0.01, respectively) as compared to the group receiving the challange dose of morphine (10 mg/kg). Figure 5.

3.6. The Effect of Levetiracetam (31.25; 62.5; 125 mg/kg, ip) on the Acquisition of Morphine (10 mg/kg, ip) Sensitization to the Locomotor Effects in Mice

On the based on the analysis of variance, it was shown that administration of a challenge dose of morphine on the 20th day of the experiment in animals receiving sporadic injections of both morphine and levetiracetam (on 1st, 4th, 7th, 10th, and 13th day of the experiment) caused statistically significant changes in their locomotor activity (F(4,39)=10.01; p<0.0001).

The post-hoc test demonstrated a statistically significant increase in locomotor activity in animals after the challenge dose of morphine compared to the control group (day 20th of the experiment) (p<0.0001). Administration of five sporadic injections (on 1st, 4th, 7th, 10th, and 13th day of the experiment) of levetiracetam at all doses (31.25, 62.5, and 125 mg/kg) with morphine, significantly reduced the animals' locomotor activity (p<0.05, p<0.01, and p<0.0001, respectively) on the last day of the study (20th) as compared to the locomotor activity observed in mice receiving morphine alone. Figure 6.

4. Discussion

The major result presented in that paper is that levetiracetam, a second-generation anticonvulsant, has an influence on morphine addiction in mice. It was shown that levetiracetam was able to inhibit morphine tolerance to the antinociceptive effects. Moreover, levetiracetam effectively reduced the intensity of morphine withdrawal symptoms and diminished the expression and acquisition of morphine-induced sensitization to the locomotor effects. In these studies levetiracetam weas used at doses 31.25; 62.5; or 125; mg/kg. These doses were selected on the based on previous experiments (results were not published) in which had been shown that levetiracetam had no effect on the locomotor activity of mice, on the motor coordination (the rota rod test) and did not influence on miorelaxation (the chimney test). Therefore, these doses were considered as ineffective and were used in subsequent experiments.

Another important issue in the study is a risk of interactions between levetiracetam and morphine. Although the mechanisms of action of both drugs are completely diversified, the pharmacodynamics interactions between them cannot be rule out because both drugs reduce the activity of central nervous system. Thus, their administration at higher doses may develop dizziness or sedation. In our preliminary study, however, there were not observed any significant effect on locomotor activity of mice when combination of two drugs was administered (data were not published). Moreover, levetiracetam does not induce or inhibit cytochrome P450 enzymes, which are often involved in drug-drug interactions. Morphine, is primarily metabolized by the liver enzymes CYP3A4 and CYP2B6, thus, interactions during metabolism are less probable. Levetiracetam is not a P-glikoprotein inducer which also exclude the interaction during absorption [33]. Thus, the lack of significant interactions makes levetiracetam a versatile option in polytherapy.

In the first part of the study the effect of levetiracetam on the expression and acquisition of morphine tolerance to the antinociceptive effects in the hot plate test was assessed. For this purpose, a typical experimental model was used [34,35]. The development of morphine tolerance was achieved by its repeated administration for 7 consecutive days at a constant dose (10 mg/kg), and the hot plate test was performed on the 1st and 7th day of morphine administration. As it was observed on the 7th day of morphine administration, the animals' response to the thermal stimulus was significantly shortened, which confirmed the development of morphine tolerance in studied mice. The administration of acute and chronic doses of levetiracetam inhibited morphine tolerance to the antinociceptive effects of morphine. In the case of acute exposure to levetiracetam (expression model) the morphine tolerance was inhibited only after application of lower dose of levetiracetam. Whereas, in the case of repeated levetiracetam exposure (acquisition model), the inhibition of morphine tolerance was observed after administration of both doses of levetiracetam. It should be emphasized that the present results are in agreement with previous study [36]. In that study significantly higher levetiracetam doses (60, 300 or 900 mg/kg; i.p.) were used and morphine was administered at a dose of 50 mg once a day for 3 days. The tolerance to antinociceptive effect was observed in anther test - the tail flick test. These authors documented that levetiracetam at the doses of 300 and 900 mg/kg effectively inhibited the development of tolerance while the expression of morphine tolerance was reduced when the highest levetiracetam dose was injected in mice. Thus, both present and previous showed that levetiracetam was able to attenuate morphine tolerance in animal models.

There is also study showing that exposure to higher dose of levetiracetam (564 mg/kg/day) in rats was effective in seizure control but this efficacy was reduced within a week, despite adequate levetiracetam level in serum and brain [37]. This suggests that chronic exposure to levetiracetam alone may develop pharmcodynamic tolerance. This effect was not observed in the present study because there were no changes in time reaction to nociceptive stimulus in mice chronically treated with both doses of levetiracetam (Figure 2).

Currently, it is believed that major mechanism underlying morphine tolerance is desensitization of µ-type opioid receptors. It is associated with increased adenylate cyclase activity and increased cAMP level in various brain areas, including periaquductal gray (PAG) [38]. Increased cAMP level in the PAG, a structure belonging to descending pain pathways, increases presynaptic GABA release and the expression of GABAA receptors [39]. Another mechanism which is responsible for desensitization process is attributed to NMDA glutamate receptors in postsynaptic structures [40]. These receptors co-occur with µ receptors in structures involved in pain transmission in the CNS, such as the dorsal horns of the spinal cord, the nucleus of the locus coeruleus and the PAG. It is believed that signals resulting from µ receptors cause an activation of NMDA receptors leading to increase the secretion of nitric oxide and to attenuation of µ receptor activity [41,42,43]. Moreover, desensitization of opioid receptors may be related to the presence of AMPA receptors in the descending pain pathway [44]. Neurons in the PAG send glutamatergic neurons to the RVM, where they influence AMPA receptors located on GABAergic terminals. It leads to inhibition of signals in the dorsal horns of the spinal cord [44]. Other intracellular mechanisms may also be responsible for morphine tolerance, such as endocytosis of opioid receptors [45], deregulation of β-arrestin 1 and β-arrestin 2 levels inside the cells [46,47,48], or changes in the activity of MAP kinases, e.g. ERK1/2 [49]. Considering the complex mechanisms of action of levetiracetam and its effect on the SV2A protein located, among others, on glutamatergic terminals, it can be suggested that these relationships may underlie the attenuation of morphine tolerance in the presented experiments.

In the second group of experiments the influence of acute and chronic administration of levetiracetam on the symptoms of morphine dependence was assessed. A typical model of physical dependence on morphine was used, in which mice received morphine at increasing doses (from 10 to 50 mg/kg) for 9 consecutive days. Then, to induce withdrawal symptoms, naloxone (2 mg/kg) was administered. The number of jumpings was recorded in the animals. It was shown that both single and repeated administration of levetiracetam reduced the number of jumpings in the studied animals. Up to now there is no literature data showing the effects of levetiracetam in morphine dependence.

It is known that morphine withdrawal symptoms develop as a consequence of a sudden decrease in dopamine level in the nucleus accumbens [50]. Other neurotransmitters and neuromodulators in brain are also involved in that phenomenon, such as, an increase in glutamate release [51], noradrenaline release [52], or a decrease in GABA secretion [53]. In addition, changes in intracellular transmission were observed, including a significant increase in cAMP level [54] or deregulation of the MAP kinase pathway (ERK 1/2) [55,56]. It can be assumed that, levetiracetam may abolished morphine withdrawal symptoms by intensifying GABA-ergic transmission [57]. Other mechanisms may also be involved in that process.

In the last part of that study the influence of levetiracetam on the development of morphine behavioral sensitization to the locomotor effects in mice was assessed. In these experiments, the behavioral sensitization was achieved by chronic, intermittent administration of an ineffective dose of morphine (10 mg/kg), and then, after a break, a challange dose of morphine (10 mg/kg) was administered. It was shown that intermittent administration of morphine for long period and application of challange dose of morphine significantly increased the locomotor activity of mice, which confirmed the development of behavioral sensitization in the studied animals. Both single and chronic exposure to levetiracetam significantly reduced the mobility of the studied animals. Thus, it was confirmed that levetiracetam seems to be an important pharmacological tool in suppressing of drug-seeking behavior and might be an effective compound in attenuating of relapse to drug use. These results are pioneering because there are not any scientific reports showing the influence of levetiracetam on behavioral sensitization.

The behavioral sensitization is primarily associated with an increase in dopamine secretion in the structures of the mesolimbic system [23,58,59] which is manifested, among others, as an increase in the locomotor activity of animals. Moreover, the adaptive changes develop in the dopaminergic and glutamatergic structures of the mesolimbic system [60,61,62,63], both in NMDA glutamate receptors [64] and in AMPA receptors [65]. Additionally, in behavioral sensitization an increase in GABA secretion in the hippocampus was also observed [66] while stimulation of GABA-ergic neurons reduced sensitization in rats [67]. It can by hypothesized that the inhibitory effect of levetiracetam on behavioral sensitization might be related to the indirect effect of levetiracetam on GABA-ergic transmission. This hypothesis, however, requires further studies to be confirmed.

The limitation of that study is that we did not perform analogical study in female mice. Some differences in efficacy of levetiracetam were observed in people between males in females [68]. Females generally show a decreased sensitivity to opioids, requiring higher doses to achieve the same analgesic effect compared to males due to variations in opioid receptor density and function influenced by sex hormones like estrogen and testosterone. Thus, more results are needed to fully recognize the significance of levetiracetam in attenuation of morphine tolerance.

5. Conclusions

The obtained research results clearly indicate that levetiracetam seems to be promising agent. It is valuable anticonvulsant drug used in various types of epilepsy. Its unique ability to modulate synaptic transmission and neurotransmitter release markedly extends potential therapeutic advantages. Further research is needed to fully elucidate levetiracetam benefits. The experiments presented in that paper encourage to undertaken further research on levetiracetam properties.