Clinical pain is not simply the consequence of “switching on the pain system” by a particular pathology in the periphery but instead reflects, to a considerable extent, the state of excitability of nociceptive pathways. Sensitization can be defined as an amplification of neural signaling within the nervous system and is the fundamental component of the pain hypersensitivity present in many pain states, including neuropathic (e.g., trigeminal neuralgia, diabetic neuropathy) and inflammatory pain (e.g., migraine, visceral pain hypersensitivity syndromes).1–4 Current therapies often are inadequate in effectively managing those pain states, and a possible approach to overcome this problem is the use of treatments that could normalize hyperexcitable neural activity.4
Antiepileptic drugs are used widely in the treatment of neuropathic pain, as well as in migraine prophylaxis.5,6 The efficacy of antiepileptic drugs in these indications is assumed to be the consequence of their ability to suppress neuron hyperexcitability.5,7
Eslicarbazepine acetate (ESL) is a novel antiepileptic drug that is structurally related to carbamazepine and oxcarbazepine but possesses a more favorable metabolic profile. Unlike carbamazepine, it is not metabolized to the toxic 10,11-epoxide and is not susceptible to enzyme induction. Unlike oxcarbazepine (which is a prodrug of both the S- and the R-enantiomers of licarbazepine), ESL is a prodrug of predominantly the S-enantiomer of licarbazepine (eslicarbazepine), which is the more pharmacologically active enantiomer.8 Recent studies also suggest that ESL may be more tolerable than carbamazepine/oxcarbazepine.9,10 Currently, there are no published preclinical data on the analgesic properties of ESL; however, clinical studies aimed at evaluating its efficacy in the treatment of migraine, postherpetic neuralgia, and painful diabetic neuropathy have begun.11
The aims of the present study were to establish the efficacy of ESL in models of trigeminal (orofacial formalin test), neuropathic (streptozotocin-induced diabetic neuropathy), and visceral pain (writhing test) and to determine whether serotonergic 5-HT1B/1D and cannabinoid CB1 and CB2 receptors are involved in the antinociceptive effect of ESL in the trigeminal pain model. The importance of serotonergic and cannabinoid pain-modulating systems, particularly in pain states that originate in trigeminal region, is well-known or has been recently described.12–16
All experiments were approved by the Institutional Animal Care and Use Committee of the Faculty of Pharmacy, University of Belgrade. The experiments were performed on male Swiss Webster (weighing 20–30 g) and C57BL/6 (initially weighing 20–30 g) mice obtained from the Military Academy Breeding Farm, Belgrade, Serbia. The animals were housed under standard laboratory conditions. Swiss Webster mice were used in the orofacial formalin, writhing, and rotarod tests. C57BL/6 mice were used for the streptozotocin-induced diabetic neuropathy model. The experiments were conducted in a blinded manner, between 8:00 AM and 4:00 PM, to avoid diurnal variations in behavioral tests. A total of 248 mice were used in the study.
Drugs and Their Administration
ESL (Zebinix, BIAL-Portela & Ca., S.A., Sao Mamede do Coronado, Portugal) was suspended in distilled water and applied by oral (p.o.) gavage to fasted animals in a volume of 10 mL/kg body weight.
The formalin solution was prepared from commercially available stock formalin (Reahem, Sremski Karlovci, Serbia) diluted in saline to the final concentration of 2%. It was applied subcutaneously into the perinasal area of mice in a volume of 20 μL by using a microliter syringe and a 26-gauge needle. Streptozotocin (Sigma-Aldrich Chemie GmbH, Munich, Germany) was dissolved in 0.03 mol/L citrate buffer (pH 4.5), immediately before intraperitoneal (i.p.) injection in mice (10 mL/kg body weight). Diluted acetic acid (0.75%; Zorka Pharma, Šabac, Serbia) was injected i.p. to mice in a volume of 10 mL/kg body weight.
Antagonist stock solutions were made by dissolving N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-1,1′-biphenyl-4-carboxamide hydrochloride hydrate (GR 127935; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) in distilled water, whereas 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM251) and 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630; both from Sigma-Aldrich Co., St. Louis, MO) were dissolved in dimethyl sulfoxide. All antagonist stock solutions were further diluted to appropriate concentrations in isotonic saline before application and administered i.p. to mice in a volume of 10 mL/kg body weight.
Orofacial Formalin Test
The test was performed according to Luccarini et al.,17 with a few modifications. Mice were allowed to acclimate to Plexiglass observation chambers (30 × 25 × 25 cm3) for 30 minutes before we performed the test. After habituation, the formalin solution was subcutaneously injected into the right upper lip, just lateral to the nose, and the mice were returned immediately to the chambers. The formalin injection induces a biphasic nociceptive behavioral response (rubbing of the injected area with ipsilateral fore and/or hind paw), which was observed and quantified. In our study, the first phase lasted from 0 to 9 minutes (phase I), and the second phase lasted from 9 to 45 minutes (phase II) after formalin injection, respectively. The time that mice spent in nociceptive behavior was measured in 3-minute intervals and expressed as the total (cumulative) time spent in face rubbing, denoted as T (seconds), in each phase. ESL or vehicle was applied p.o. 60 minutes before the formalin injection.
The T values in each phase were converted to percentage of antinociceptive (%AN) activity, according to the following formula:
Streptozotocin-Induced Diabetic Neuropathy
Diabetes was induced by a single injection of streptozotocin (150 mg/kg body weight) after an overnight fast.18 Mice were considered diabetic when the fasting blood glucose level at 2 weeks after the streptozotocin injection was >250 mg/dL (GlucoSure Plus, Apex Biotechnology Corp, Hsinchu, Taiwan).
The nociceptive responses were evaluated by the radiant heat tail-flick test (cat. no. 7360; Hugo Sachs Elektronik, March-Hugstetten, Germany), as described previously.18 Tail-flick latencies (seconds) were measured before (baseline latency) and 3 weeks after the injection of streptozotocin (but before ESL administration—pretreatment latency) to assess the development of hyperalgesia. The heat intensity was adjusted to produce baseline latencies between 4 and 6 seconds. The cutoff time was set at 10 seconds to prevent tissue damage.
The percentage of hyperalgesia (%HA) was calculated for each animal using the following formula:
When diabetic neuropathy develops, the pretreatment latency is shorter than baseline latency, resulting in a negative value of %HA.
The antinociceptive effect of ESL was examined in diabetic mice who developed thermal hyperalgesia. To evaluate whether ESL effects are dependent on diabetes-induced neuropathic changes, additional experiments were performed on nondiabetic mice. ESL was applied p.o. immediately after the measurement of pretreatment latencies. The post-treatment latencies were recorded 30, 60, 90, 120, 180, and 240 minutes after the administration of ESL. Control diabetic and nondiabetic mice received the same volume of corresponding vehicle instead of ESL.
Antinociceptive effects were expressed as the percentage of the maximal possible effect (%MPE), calculated by using the following formula18:
The writhing test described previously19,20 was used. Mice received an i.p. injection of acetic acid solution. The number of writhes (N) was counted during a 15-minute period, starting 5 minutes after the administration of acetic acid. ESL (or vehicle in the control group) was administered 55 minutes before the acetic acid solution.
The N values were converted to %AN activity, according to the following formula:
Antagonist studies were conducted in the orofacial formalin test in mice. The influence of GR 127935 (selective 5-HT1B/1D serotonin receptor antagonist), AM251 (selective CB1 cannabinoid receptor antagonist), and AM630 (selective CB2 cannabinoid receptor antagonist) on the antinociceptive effect of a fixed, effective dose of ESL (60 mg/kg; p.o.) was evaluated. GR 127935, AM251, and AM630 were administered i.p., immediately after ESL (60 minutes before the formalin injection). The group of animals used for comparison received ESL (p.o.) and the same volume of vehicle (i.p.) instead of the antagonists. To exclude the possible intrinsic effects of antagonists, the influence of the greatest dose of each antagonist on formalin-induced nociceptive behavior was tested separately.
The percentage inhibition (%I) of the antinociceptive effect of ESL produced by the antagonists was calculated according to the following formula21:
The rotarod test was used to evaluate the effects of ESL on motor coordination or sedation.19,20 The test was performed using a rotarod apparatus (Treadmill for mice 7600; Ugo Basile, Milano, Italy), rotating at a constant speed of 15 rpm. The animals were trained to drive the rotarod for 2 days. Only those mice that could remain on the rod for 60 seconds on 2 consecutive trials were used in the experiments. The post-treatment latency to remain on the rotating rod was recorded at 4 time points after ESL or vehicle administration, during 120 minutes. The cutoff time was 60 seconds.
The percentage of motor impairment was calculated according to the following formula22:
All pharmacologic computations were performed using Pharm Tools Pro (The McCary Group, Schnecksville, PA). The doses of ESL expected to produce 50% of the antinociceptive effect (ED50) or motor impairment (TD50) were estimated from the corresponding log dose-response curve by linear regression.23 The statistical analysis was conducted using SigmaPlot 11 (Systat Software Inc., Richmond, CA) and SPSS 18 for Windows (IBM SPSS Statistics, Chicago, IL). The sizes of the test groups were 7 to 9 mice and those of the control groups were 11 (for the orofacial formalin test) and 6 to 9 (for the writhing, tail-flick, and rotarod tests). The group sizes were chosen on the basis of our previous experience with those tests and are consistent with the literature data. The sample sizes for each group are indicated in the figures. The values from each group were graphed as means ± SEM.
The data were assessed for normality and equality of variance. Data from nociceptive tests were normally distributed (all P ≥ 0.064; Shapiro-Wilk test), whereas the results from the rotarod test were not (all P ≤ 0.047; Shapiro-Wilk test). Data from the orofacial formalin test, writhing test, and antagonist studies were analyzed by the Student t test or 1-way analysis of variance (ANOVA). The paired Student t test was used to compare the tail-flick latencies before and after diabetes induction. Time course data in the streptozotocin-induced diabetic neuropathy model were analyzed using 2-way repeated-measures ANOVA (type of treatment was the between-subject factor and the time after ESL/vehicle administration was the within-subject factor). In the orofacial formalin test (Fig. 1), the statistical assumption of equality of variances was violated (all P ≤ 0.011; Levene test) and the Games-Howell test was used for between-group comparisons in the post hoc analysis. The statistical assumption of equality of variances was not violated in the streptozotocin-induced diabetic neuropathy model (Fig. 2; all P ≥ 0.213; Mauchly test of sphericity) writhing test (Fig. 3; P = 0.469; Levene test) and antagonist studies (Fig. 4; all P ≥ 0.179; Levene test), and the Tukey honest significant difference (HSD) test was used for post hoc comparisons. In the streptozotocin-induced diabetic neuropathy model, there was a significant interaction between the 2 tested factors (P < 0.001), and we used 1-way ANOVA followed by the Tukey HSD test for every time point to determine which ESL-treated group was significantly different from the control (vehicle-treated) group. A P value of <0.05 was considered statistically significant in the post hoc analysis of data from nociceptive tests. Data from the rotarod test (Fig. 5) were analyzed by nonparametric Kruskal-Wallis 1-way ANOVA with Bonferroni correction for multiple comparisons in the post hoc analysis (exact Mann-Whitney U test). After the application of the Bonferroni correction, an exact P value of <0.017 was considered statistically significant in the Mann-Whitney U test. The corrected P value was obtained by lowering the critical significance probability to P < 0.05/3, where 3 equals the number of multiple comparisons in the rotarod test data analysis (i.e., for comparing 3 groups that received different doses of ESL to the control group). All reported P values for paired comparisons are post hoc–corrected P values, unless otherwise indicated.
The Effect of ESL in the Orofacial Formalin Test
The injection of 20 μL of 2% formalin into the perinasal area produced 2 characteristic phases of face-rubbing behavior (Fig. 1, A and B). Injection of an equivalent volume of saline did not produce any significant behavioral effect (not shown).
In the first phase of the test, only the highest tested dose of ESL (100 mg/kg) produced a significant antinociceptive effect of 41.9% (P = 0.003; Fig. 1, A and B). In the second phase of the test, ESL (15–100 mg/kg) caused a significant, dose-dependent antinociceptive effect in the range of 25.4% to 65.1% (P = 0.238 for ESL dose of 15 mg/kg and all P ≤ 0.011 for ESL doses 30–100 mg/kg; Fig. 1, A and B). The ED50 ± SEM for the second phase of the orofacial formalin test was 47.5 ± 2.8 mg/kg.
The Effect of ESL on Streptozotocin-Induced Diabetic Neuropathy
In diabetic mice, tail-flick (pretreatment) latencies (3.16 ± 0.53 seconds; n = 35) were significantly lower than their basal latencies tested 3 weeks before (5.03 ± 0.85 seconds; n = 35; P < 0.001, paired Student t test), resulting in hyperalgesia with a value of −40 %HA. All animals demonstrated normal behavior during the study period.
ESL (60–240 mg/kg) produced a significant and dose-dependent antinociceptive effect in the tail-flick test in diabetic mice that developed thermal hyperalgesia (Fig. 2A). The 2-way repeated-measures ANOVA revealed a significant effect of both type of treatment and time after ESL/vehicle application on the %MPE determined in the tail-flick test (P < 0.001 for both factors). In addition, there was a significant interaction between the 2 tested factors (P < 0.001), so we performed 1-way ANOVA (followed by the Tukey HSD test) for every time point to determine which ESL-treated group was significantly different from the control group (it is normal that the effect of the drug changes over time). One-way ANOVA detected a significant antinociceptive effect of ESL in every time point (all P < 0.001), and the Tukey post hoc P values were ≤0.013 in all time points for most tested doses of ESL. The maximal antinociceptive effect of most tested doses was obtained 90 minutes after application and was from 32.2% to 65.8%. The ED50 ± SEM was 124.7 ± 4.2 mg/kg for the effect at 90 minutes after ESL administration. In nondiabetic mice, the greatest tested dose of ESL (240 mg/kg) did not produce a significant antinociceptive effect in the tail-flick test (P = 0.566 by 2-way repeated-measures ANOVA; Fig. 2B).
The Effects of ESL in the Writhing Test
In the writhing test, ESL (7.5–40 mg/kg) significantly reduced the number of writhes in a dose-dependent manner (P = 0.208 for ESL dose of 7.5 mg/kg and all P ≤ 0.003 for ESL doses of 15–40 mg/kg; Fig. 3). The antinociceptive effect was from 23.9% to 76.5% (not shown). The corresponding ED50 ± SEM was 19.3 ± 2.4 mg/kg.
The Influence of 5-HT1B/1D Serotonin Receptor Antagonist on the Antinociceptive Effect of ESL
In the second phase of the orofacial formalin test, GR 127935 (1 and 3 mg/kg) significantly reduced the antinociceptive effect of ESL (60 mg/kg) in a dose-related manner (all P ≤ 0.038; Fig. 4A). The values of inhibition of the antinociceptive effect of ESL produced by GR 127935 were 52.6% and 72.9% for doses of 1 and 3 mg/kg, respectively (not shown). The highest tested dose of GR 127935 (3 mg/kg) did not produce any effect in the second phase of the orofacial formalin test (P = 0.760, Student t test; not shown).
The Influence of CB1 and CB2 Cannabinoid Receptor Antagonists on the Antinociceptive Effect of ESL
AM251 (1 and 3 mg/kg) and AM630 (1 and 3 mg/kg) produced a significant and dose-related decrease in the antinociceptive effect of ESL (60 mg/kg) in the second phase of the orofacial formalin test (all P ≤ 0.013 for both AM251 and AM630; Fig. 4, B and C). The values of inhibition of the antinociceptive effect of ESL produced by AM251 were 51.9% and 70.5% for doses of 1 and 3 mg/kg, respectively (not shown). The inhibitory effects for AM630 doses of 1 and 3 mg/kg were 63.0% and 78.4%, respectively (not shown). The highest tested doses of AM251 (3 mg/kg) and AM630 (3 mg/kg) had no significant effect on nociceptive behavior in the second phase of the orofacial formalin test (P = 0.763 for AM251 and P = 0.835 for AM630, Student t test; not shown).
The Effect of ESL on Rotarod Performance
ESL produced significant and dose-dependent motor impairment in mice (500–750 mg/kg; exact P = 0.014 for ESL dose of 625 mg/kg at 120 minutes; all exact P = 0.005 for ESL dose of 750 mg/kg at every time point; Fig. 5). The maximal effect on motor coordination was achieved 60 to 120 minutes after ESL application. The TD50 ± SEM was 736.5 ± 5.8 mg/kg (effect at 90 minutes after ESL administration).
In this study, we demonstrated that orally administered ESL produces dose-dependent antinociceptive effects in models of trigeminal, neuropathic, and visceral pain in mice. ESL exhibited similar efficacy across all models but had a different potency in producing antinociceptive effects. It was the most potent in the writhing test and the least potent in the streptozotocin-induced diabetic neuropathy model. Different doses required to produce antinociception could be because of different sensitivity of pain models/tests and/or mice strains to the action of ESL. After oral administration in mice, as in humans, ESL is rapidly and almost completely metabolized to eslicarbazepine.8,24,25 Thus, mice models are appropriate for exploring the effects and mechanisms of action of ESL.
In pain models used in this study, the sensitization of nociceptive pathways is an important component of nociceptive behavior in the second phase of the formalin test and in writhing test and plays a crucial role in the development of hyperalgesia in the diabetic neuropathy model. In the second phase of the formalin test and in the acetic acid writhing test, nociceptive behavior seems to be the consequence of both direct stimulation of nociceptors by a chemical agent (nociception) and peripheral inflammation that cause sensitization of pain pathways at peripheral and central sites.26–29 In the diabetic neuropathy model, thermal hyperalgesia is related to enhanced sensory functions of peripheral nociceptive fibers whose continuous discharge induces sensitization of spinal cord neurons.30,31 In all these models and phases of the tests, ESL exhibited highly effective dose-dependent antinociception. However, it had no significant effect in the tail-flick test in nondiabetic mice and exerted a dose-independent effect of lower degree in the first phase of the orofacial formalin test, which are considered to reflect “pure” nociception and are not associated with the sensitization of nociceptive pathways.29,32 These findings are not surprising because ESL, a voltage-gated sodium channel blocker,8,33 is capable of stabilizing the hyperexcitable neural activity. Our results might suggest that ESL exerts its antinociceptive effects mainly by reducing sensitization of nociceptive pathways and that it could be particularly effective in the treatment of clinical pain states that involve such sensitization, that is, different types of neuropathic and inflammatory pain.3,4 This hypothesis is consistent with a recent finding that oxcarbazepine, also a voltage-gated sodium channel blocker, is more effective for the relief of peripheral neuropathic pain in patients with the irritable nociceptor phenotype, that is, with signs of sensory hyperexcitability.34
The mechanisms that might be involved in the antinociceptive actions of ESL were examined in the orofacial formalin test in mice because it represents a trigeminal pain model and correlates, to a certain degree, with pain states that are treated clinically with carbamazepine/oxcarbazepine (i.e., trigeminal neuralgia) or other anticonvulsants (i.e., chronic migraine).5,35 We have demonstrated, in the second phase of the test, that selective antagonists of serotonergic 5-HT1B/1D and CB1/CB2 cannabinoid receptors exerted dose-related inhibition of the antinociceptive effect of ESL, which implicates an involvement of these receptors in the antinociceptive action of ESL, that is, eslicarbazepine.
The interaction of eslicarbazepine with serotonergic receptors might be either direct or indirect, via influence on endogenous serotonin (5-hydroxytryptamine [5-HT]). There are no data concerning the binding properties of eslicarbazepine to 5-HT receptors, and thus, a direct interaction could not be excluded. An indirect interaction seems, however, more possible because there is evidence that the monohydroxy metabolite of oxcarbazepine, structurally identical to eslicarbazepine (eslicarbazepine represents its S-enantiomer),8,33 promotes the release of 5-HT in neural tissue. Clinckers et al.36 showed a concentration-dependent elevation of extracellular 5-HT level in rats treated locally with monohydroxy metabolite of oxcarbazepine into hippocampus, which was closely related to its anticonvulsant action. If we suppose that eslicarbazepine could also promote 5-HT release in other regions of the nervous system, it seems likely that 5-HT could mediate its antinociceptive effects in the orofacial formalin test. It is well-known that 5-HT1B/1D receptor agonists can reduce pain in the trigeminal region. A 5-HT1B/1D receptor agonist (sumatriptan) induced dose-dependent antinociception in the orofacial formalin test.37 It was shown that the stimulation of 5-HT1B/1D receptors disrupts the communication between peripheral and central trigeminal neurons38 and activates central descending pathways to inhibit trigeminal nociceptive input.39 Taken together, it seems that eslicarbazepine could interact with serotonergic transmission, that is, serotonergic 5-HT1B/1D receptors and that this interaction probably takes place at central sites of pain transmission/modulation system.
The interaction of eslicarbazepine with cannabinoid receptors could not be easily explained. In the absence of data on binding properties, the direct stimulation of cannabinoid receptors cannot be excluded, but seems less possible, because there is no structural similarity between eslicarbazepine and cannabinoid receptors ligands.8,40 The other possibility is the stimulation of endocannabinoid (eCB) release. Agents capable of increasing eCB levels exerted antinociceptive effects in various models of inflammatory and neuropathic pain, and these effects have been shown to be mediated via CB1 and/or CB2 receptors.40,41 The link between eslicarbazepine and the eCB system in producing antinociception in the orofacial formalin test could be the aforementioned increased serotonergic transmission. Burattini et al.42 have demonstrated that 5-HT can provoke the production and release of eCB in the brain. Consistent with this finding, Akerman et al.12 proposed that the antimigraine action of triptans, 5-HT1B/1D receptor agonists may, in part, be mediated via eCB containing neurons. Thus, it seems possible that the stimulation of 5-HT1B/1D receptors could lead to eCB release. Existing data suggest that both CB1 and CB2 receptors could be involved in the antinociception produced by cannabinoid receptors agonists in trigeminal pain models.13,14,16 With all this in mind, we can propose that eslicarbazepine might interact with cannabinoid receptors indirectly, by releasing 5-HT, and subsequently eCB, to produce antinociception in the orofacial formalin test. Further experiments are needed to clarify the nature and place(s) of eslicarbazepine’s interaction with serotonergic and cannabinoid systems in reducing pain.
As there is a great structural similarity between eslicarbazepine and carbamazepine/oxcarbazepine, these interesting findings may point to some new insights in the mechanisms involved in the antinociceptive action of carbamazepine/oxcarbazepine. Both drugs could also elevate 5-HT in the brain in a concentration-dependent manner36,43; thus, all the proposed subsequent steps may be involved in their antinociceptive actions as well and should be explored.
The doses of ESL that exhibited antinociceptive effects in our study were well below those required to produce significant motor impairment in the rotarod test. Our results show that the ED50 values were about 6-fold (in the streptozotocin-induced diabetic neuropathy model), 15-fold (in the second phase of the orofacial formalin test), and 38-fold (in the writhing test) lower than the TD50 values. By comparison, in a previous study,19 we demonstrated on the same mice strain that the ED50 values for carbamazepine and oxcarbazepine in the writhing test were 2- and 15-fold lower than the TD50 values determined in the rotarod test. These findings may suggest that ESL might have better tolerability than carbamazepine and oxcarbazepine when used as an analgesic.
In conclusion, ESL exhibited efficacy in models of trigeminal, neuropathic, and visceral pain. The antinociceptive effect of ESL in the trigeminal model is, at least in part, mediated by 5-HT1B/1D serotonergic and CB1 and CB2 cannabinoid receptors. This study indicates that ESL could be potentially useful in the clinical treatment of inflammatory and neuropathic pain states.
Name: Maja A. Tomić, PhD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Maja A. Tomić has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Uroš B. Pecikoza, BPharm.
Contribution: This author helped design the study, conduct the study, and analyze the data.
Attestation: Uroš B. Pecikoza has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Ana M. Micov, PhD.
Contribution: This author helped conduct the study and analyze the data.
Attestation: Ana M. Micov has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Radica M. Stepanović-Petrović, PhD.
Contribution: This author helped design the study and analyze the data.
Attestation: Radica M. Stepanović-Petrović has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Jianren Mao, MD, PhD.
The authors thank Kristina Veljković, MSc (from the Department of Physics and Mathematics, Faculty of Pharmacy, University of Belgrade), for her help with the statistical analysis of data.
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