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Anesthesia & Analgesia:
doi: 10.1213/ANE.0b013e31826c7fc2
Pain and Analgesic Mechanisms

The Local Peripheral Antihyperalgesic Effect of Levetiracetam and Its Mechanism of Action in an Inflammatory Pain Model

Stepanović-Petrović, Radica M. PhD; Micov, Ana M. BPharm; Tomić, Maja A. PhD; Ugrešić, Nenad D. PhD

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From the Department of Pharmacology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia.

Accepted for publication July 25, 2012.

Published ahead of print November 9, 2012

Supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant 175045.

The authors declare no conflicts of interest.

This report was previously presented, in part, at the ECNP Congress.

Reprints will not be available from the authors.

Address correspondence to Radica M. Stepanovi`c-Petrovi`c, PhD, Department of Pharmacology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, P.O. Box 146, 11221, Belgrade, Serbia. Address e-mail to racabbr@eunet.rs

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Abstract

BACKGROUND: We have recently shown that levetiracetam, administered systemically, exerts an antihyperalgesic effect in a rat inflammatory pain model. In this study, we examined whether levetiracetam has local peripheral antihyperalgesic/anti-edematous effects in the same model of localized inflammation and whether opioidergic, adrenergic, purinergic, 5-HTergic, and GABAergic receptors are involved in its antihyperalgesic action.

METHODS: Rats were intraplantarly (IPL) injected with carrageenan. A paw pressure test was used to determine the effect/s of (a) levetiracetam when applied IPL, on carrageenan-induced hyperalgesia, and (b) naloxone (a nonselective opioid receptor antagonist), CTAP (a selective μ-opioid receptor antagonist); yohimbine (a selective α2-adrenoceptor antagonist), BRL 44408 (a selective α2A-adrenoceptor antagonist), MK-912 (a selective α2C-adrenoceptor antagonist); caffeine (a nonselective adenosine receptor antagonist), DPCPX (a selective adenosine A1 receptor antagonist); methysergide (a nonselective 5-HT receptor antagonist), GR 127935 (a selective 5-HT1B/1D receptor antagonist); and bicuculline (a selective GABAA receptor antagonist), all applied IPL, on the levetiracetam-induced antihyperalgesia. Moreover, levetiracetam’s influence on paw inflammatory edema was measured by plethysmometry.

RESULTS: Levetiracetam (200–1000 nmol/paw) produced a significant dose-dependent reduction of the paw inflammatory hyperalgesia and edema induced by carrageenan. Naloxone (75–300 nmol/paw), CTAP (1–5 nmol/paw); yohimbine (130–520 nmol/paw), BRL 44408 (50–200 nmol/paw), MK-912 (5–20 nmol/paw); caffeine (500–1500 nmol/paw), DPCPX (3–30 nmol/paw); methysergide (10–100 nmol/paw) and GR 127935 (50–200 nmol/paw); but not bicuculline (400 nmol/paw), significantly depressed the antihyperalgesic effects of levetiracetam (1000 nmol/paw). The effects of levetiracetam and antagonists were attributed to local peripheral effects because they were not observed after administration into the contralateral hind-paw.

CONCLUSIONS: Our results show that levetiracetam produces local peripheral antihyperalgesic and anti-edematous effects in a rat model of localized inflammation. Antihyperalgesia is at least in part mediated by peripheral μ-opioid, α2A,C-adrenergic, A1 adenosine, and 5-HT1B/1D receptors, but not by GABAA receptors. These findings could contribute toward a better understanding of the analgesic effects of levetiracetam, and improved treatments of inflammatory pain with a lower incidence of systemic side effects and drug interactions of levetiracetam.

Levetiracetam (S-α-ethyl-2-oxo-1-pyrrolidine acetamide) was approved as an antiepileptic drug 10 years ago, but its antihyperalgesic properties were shown only recently in some animal models of neuropathic and inflammatory pain.1–4 It has been found that levetiracetam is effective in the treatment of neuropathic pain and migraine prevention in small uncontrolled studies and case series, as well as in one randomized, double-blind, placebo-controlled, crossover clinical study in the treatment of specific pain symptoms in multiple sclerosis.5–8 In a previous study, we showed that systemic administration of levetiracetam diminished the development of mechanical hyperalgesia in an inflamed rat paw. Also, we showed that this effect is at least in part mediated by opioid, α2-adrenergic, γ-aminobutyric acid (GABA)A, and 5-hydroxytryptamine (5-HT) (serotonin) receptors.4 However, the site(s) of levetiracetam’s antihyperalgesic action in inflammatory pain (central, peripheral, or both central and peripheral) remain(s) to be clarified.

Inflammatory pain depends to some degree on the peripheral activation of primary sensory afferent neurons. Peripheral nerve endings express a variety of inhibitory neuroreceptors such as opioid, α-adrenergic, adenosine, 5-HT, and GABAA receptors, and these receptors are potential targets for drugs.9 Given that peripheral antinociception can involve activation of multiple receptors on sensory neurons, the ability of a single agent to target several receptors may be of particular interest.

The localized peripheral administration of drugs can potentially optimize drug concentrations at the site of origin of the pain, with a lesser incidence of side effects/drug interactions caused by systemic absorption. In the present study, we examined (1) the antihyperalgesic and anti-edematous effects of levetiracetam administered peripherally by local injection in a rat model of localized inflammation, and (2) the potential role of opioid, adrenergic, adenosine, 5-HT, and GABA receptors and their subtypes, in levetiracetam’s local peripheral antihyperalgesic action.

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METHODS

Animals

Experiments were performed on 180–220 g male Wistar rats (Military Academy Breeding Farm, Belgrade, Serbia) and were approved by the Institutional Animal Care and Use Committee. The animals were housed in groups of 4 per cage (42.5 × 27 × 19 cm3) and maintained on a 12/12-hour light/dark cycle (lights were switched on at 06:00 hours) at 22°C ± 1°C and 60% relative humidity. Food and water were freely available, except during the experimental procedure. Before experimental manipulation, the animals were given at least 3 days to adapt to the laboratory. All experiments were performed at the same time of day, between 8:00 and 16:00 hours, to avoid diurnal variations in behavioral tests. Efforts were made to minimize the number of animals used. The total number of animals used in this study was 360.

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Model of Localized Inflammation

Inflammation was induced by an intraplantar (IPL) injection of carrageenan into the right hind-paw, which was followed by hyperalgesia and edema development.10

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Assessment of Antihyperalgesic Activity

The antihyperalgesic activity was assessed by a modified “paw pressure test,”11,12 using the apparatus for evaluating the force (in grams) exerted by a rat’s hind-paws to determine right/left differences (Hugo Sachs Elektronik, March-Hugstetten, Germany). The rat was placed with its hind-paws on 2 transducer platforms and pushed slowly and smoothly downward with the investigator’s hand, so that the force (pressure) was applied simultaneously to both paws. Pressure was applied until one of the paws exceeded the trigger level set at 100 g. At this point, an audible click by the apparatus was heard and the values of forces applied to right and left hind-paws at that moment were displayed on 2 digital panels. This trigger level was chosen to represent a mild nociceptive stimuli needed to detect the nociceptive hypersensitivity (hyperalgesia). The rat preferably used its noninflamed paw and spared the inflamed one, which resulted in a reduced application of force on the inflamed paw. The difference between forces (df) applied on the noninflamed and inflamed paw was determined after each paw pressure measurement by using following equation:

df = force (g) applied on the noninflamed paw − force (g) applied on the inflamed paw.12

Measurements were repeated 4 times at each time point and the average df for each rat was used for further calculations. The pretreatment df value was obtained before carrageenan or carrageenan + drug(s) IPL administration. The posttreatment df values were measured 60, 90, 120, 150, 180, 240, and 300 minutes after IPL injections.

Agents capable of reducing the df were recognized as those possessing antihyperalgesic activity. To express a reduction of the df value induced by antihyperalgesic treatment in test (carrageenan + drugs treated) groups in relation to control (carrageenan treated) group df value, the percent of antihyperalgesic activity (%AA) was calculated according to the following formula12:

%AA = [(control group average df − test group average df)/(control group average df)] × 100.

If the test group average df was greater than the control group average df, a value of 0 %AA was assigned.

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Assessment of Anti-Edematous Activity

The anti-edematous activity was assessed by quantification of the rat’s paw swelling by plethysmometry. The plethysmometer (Ugo Basile, Comerio-VA, Italy) consists of 2 vertical Perspex tubes that are interconnected and filled with electrolyte solution. The larger tube is used for dipping the paw, and the smaller one contains the transducer. When the hind-paw was dipped to the tibiotarsal joint,13 the fluid level in both tubes was changed and the transducer detected the volume of the displaced fluid that represents the volume of the rat’s paw. The paw volume value (in milliliters) is displayed on a digital panel.

Basal paw volume was obtained before treatment (carrageenan or carrageenan + drugs IPL administration). The posttreatment paw volume was measured 60, 90, 120, 150, 180, 240, and 300 minutes after IPL injections. After each posttreatment paw volume measurement, the difference (dv) between treated (inflamed) and a basal paw volume was calculated as:

dv = volume of the inflamed paw (mL) − basal volume of the same paw (mL).

Measurements were repeated 2 times at each time point, and the average dv for each rat was used for further calculations.

Agents capable of reducing the dv were recognized as those having anti-edematous activity. To express a reduction of the dv value induced by anti-edematous treatment in test (carrageenan + drugs treated) groups in relation to control (carrageenan treated) group dv value, the percent of anti-edematous activity (%AE) was calculated according to the following formula:

%AE = [(control group average dv − test group average dv)/(control group average dv)] × 100

If the test group average dv was greater than the control group average dv, a value of 0 %AE was assigned.

The values for %AA and %AE were calculated after each measurement of df and dv (60, 90, 120, 150, 180, 240, and 300 minutes after IPL injections) to establish the time of the peak effect. The ED50 value (the dose expected to result in 50% of the effect) with 95% confidence limits was estimated from the corresponding log dose-response curves.14

For measuring paw pressure and paw volume differences, the experimenter was blinded to the treatment received by the animals.

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Experimental Protocol

In this study, 6 series of experiments were performed (Fig. 1).First, to examine the local peripheral antihyperalgesic and anti-edematous effect of levetiracetam, the drug and carrageenan were coadministered IPL into the right hind-paw (Fig. 1A). Control animals received the same volume of carrageenan (IPL) (Fig. 1B). To exclude the possible systemic effect of levetiracetam (IPL), the highest tested dose of the drug was given contralaterally (into the left, noninflamed hind-paw) to a separate group of rats (Fig. 1C). Next, the influences of antagonists (naloxone, a nonselective opioid receptor antagonist; CTAP, a selective μ-opioid receptor antagonist; yohimbine, a selective α2-adrenoceptor antagonist; BRL 44408, a selective α2A-adrenoceptor antagonist; MK-912, a selective α2C-adrenoceptor antagonist; caffeine, a nonselective adenosine receptor antagonist; 1,3-dypropyl-8-cyclopentylxanthine, DPCPX, a selective adenosine A1 receptor antagonist; methysergide, a nonselective 5-HT receptor antagonist; GR 127935, a selective 5-HT1B/1D receptor antagonist; and bicuculline, a selective GABAA receptor antagonist) on the local peripheral antihyperalgesic effect of levetiracetam were tested. The antagonists were coadministered with levetiracetam and carrageenan (IPL), into the right hind-paw (Fig. 1D). The comparative group of animals received the same volume of carrageenan with levetiracetam (Fig. 1A). To exclude the possible systemic effect of IPL-injected antagonists, the highest tested dose of each antagonist was given contralaterally to separate groups of rats (Fig. 1E). Finally, the effects of the highest dose of each antagonist were evaluated by coadministration with carrageenan (Fig. 1F) and compared with the effect of carrageenan alone (Fig. 1B).

Figure 1
Figure 1
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The percent inhibition (%I) of the antihyperalgesic effect of levetiracetam by antagonists (naloxone, CTAP; yohimbine, BRL 44408, MK-912; caffeine, DPCPX; methysergide, GR 127935; and bicuculline) was calculated according to the following formula12:

%I = 100 − (%AA with antagonist/%AA without antagonist) × 100.

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Drugs and Their Administration

Levetiracetam, carrageenan, naloxone hydrochloride, CTAP; yohimbine hydrochloride, BRL 44408 maleate, MK-912 hydrate; methysergide maleate, GR 127935 hydrochloride hydrate; bicuculline methiodide; DPCPX (all from Sigma-Aldrich Chemie, Munich, Germany); and caffeine (Galenika, Belgrade, Serbia) were dissolved or suspended in saline. Carrageenan was used in a fixed dose of 1 mg/paw. All substances were injected IPL in a final volume of 0.1 mL/paw, using a 1-mL syringe and 24-gauge (0.55 × 25 mm) needle. Through all experiments, the left hind-paw was injected immediately before the right one.

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Statistical Analysis

The results are presented as mean values ± SEM obtained from groups of 6 to 8 animals. All computations were done according to Tallarida and Murray14 and Tallarida15 using computer programs Pharm PCS and Pharm Tools Pro. To enable group comparisons, great efforts were made to maintain all groups comparable except for treatment: we used rats of the same gender, age, and of similar weight, the experiments were conducted by the same experimenter, on consecutive days, and always at the same time of day, in the relatively constant laboratory conditions (i.e., temperature, light, humidity, quietness). Differences between the corresponding means were verified by using Student t test or 1-way analysis of variance, followed by the Tukey HSD test. A P value of <0.05 was considered statistically significant.

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RESULTS

The Effect of IPL Levetiracetam on Carrageenan-Induced Hyperalgesia

The coadministration of levetiracetam (200–1000 nmol/paw; IPL) with carrageenan produced a significant dose-dependent reduction in the differences in forces exerted by the inflamed and the noninflamed rat’s hind-paw in a modified paw pressure test used (Fig. 2A). The peak effects of all of the tested doses occurred 120 minutes after IPL administration (Fig. 2B). The corresponding ED50 ± SEM (95% confidence limits) at the peak effect point was 562.2 ± 28.9 (241.3–1309.4) nmol/paw. The effect of levetiracetam was attributed to local effect, because it was not observed after injection of the highest dose into the contralateral hind-paw (Fig. 2, 2A and 2B).

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The Effect of IPL Levetiracetam on Carrageenan-Induced Edema

The coadministration of levetiracetam (200–1000 nmol/paw; IPL) with carrageenan produced a significant reduction of rat paw edema in a dose-dependent manner (Fig. 3A). The maximum anti-edematous effect values were 22% (200 nmol/paw; IPL), 40% (600 nmol/paw; IPL), and 46% (1000 nmol/paw; IPL) obtained at the 90-minute time point (Fig. 3B). The effect of levetiracetam was attributed to local effect, because it was not observed after injection of the highest dose into the contralateral hind-paw (Fig. 3, 3A and 3B).

Figure 3
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The Influence of IPL Opioid Receptor Antagonists on the Antihyperalgesic Effect of Levetiracetam

The coadministration of naloxone, a nonselective opioid receptor antagonist (75–300 nmol/paw; IPL), or CTAP, a selective μ-opioid receptor antagonist (1–5 nmol/paw; IPL), with levetiracetam (1000 nmol/paw; IPL) into the hind-paw significantly decreased the antihyperalgesic effect of levetiracetam in a dose-dependent manner (Fig. 4, 4A and 4B). The maximal inhibitory effects of naloxone on levetiracetam-induced antihyperalgesia were achieved 120 minutes after administration; the corresponding values were 76%, 83%, and 100% for the doses of 75, 150, and 300 nmol/paw (IPL), respectively (not shown). For CTAP, the maximal inhibition of levetiracetam-induced antihyperalgesia occurred 90 to 120 minutes after administration and the values were 51%, 76%, and 100% for the doses of 1, 3, and 5 nmol/paw (IPL), respectively (not shown). The effects of naloxone and CTAP were attributed to a local action, because they were not observed after injection of the highest doses into the contralateral hind-paw (Fig. 4, 4A and 4B). Peripheral coadministration of the highest tested doses of naloxone or CTAP with carrageenan failed to produce any significant effect on carrageenan-induced hyperalgesia (P > 0.05, Student t test, data not shown).

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The Influence of IPL α-Adrenoceptor Antagonists on the Antihyperalgesic Effect of Levetiracetam

The coadministration of yohimbine, a selective α2-adrenoceptor antagonist (130–520 nmol/paw; IPL), BRL 44408, a selective α2A-adrenoceptor antagonist (50–200 nmol/paw; IPL), or MK-912, a selective α2C-adrenoceptor antagonist (5–20 nmol/paw; IPL), with levetiracetam (1000 nmol/paw; IPL) into the hind-paw significantly and dose-dependently decreased the antihyperalgesic effect of levetiracetam (Fig. 5, A–C). The maximum inhibitory effects of yohimbine on levetiracetam-induced antihyperalgesia were achieved 90 minutes after administration; the corresponding values were 51%, 80%, and 100% for the doses of 130, 260, and 520 nmol/paw (IPL), respectively (not shown). BRL 44408 exerted the maximal inhibitory effects 90 to 120 minutes after administration, and the corresponding values were 54%, 79%, and 100% for the doses 50, 100, and 200 nmol/paw (IPL), respectively (not shown). The maximum inhibitory effects of MK-912 on the antihyperalgesic effect of levetiracetam were achieved 90 to 150 minutes after administration. The corresponding values were 53%, 80%, and 100% after administration of 5, 10, and 20 nmol/paw (IPL) of MK-912, respectively (not shown). When 520 nmol/paw yohimbine, 200 nmol/paw BRL 44408, or 20 nmol/paw MK-912 was administered contralaterally, they did not produce a significant effect on levetiracetam-induced antihyperalgesia (Fig. 5, A–C). Peripheral coadministration of the same doses of α-adrenoceptor antagonists and carrageenan failed to produce any significant effect on carrageenan-induced hyperalgesia (P > 0.05, Student t test, data not shown).

Figure 5
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The Influence of IPL Adenosine Receptor Antagonists on the Antihyperalgesic Effect of Levetiracetam

Caffeine, a nonselective adenosine receptor antagonist (500–1500 nmol/paw; IPL), as well as DPCPX, a selective adenosine A1 receptor antagonist (3–30 nmol/paw; IPL), coadministered with levetiracetam (1000 nmol/paw; IPL) into the hind-paw, significantly decreased the local peripheral antihyperalgesic effect of levetiracetam in a dose-dependent manner (Fig. 6, A and B). The maximal inhibitory effects of caffeine on levetiracetam-induced antihyperalgesia were achieved 90 to 120 minutes after administration, and the corresponding values were 73%, 86%, and 100% for the doses of 500, 1000, and 1500 nmol/paw (IPL), respectively (not shown). The maximal inhibitory effects of DPCPX on levetiracetam-induced antihyperalgesia occurred 90 minutes after administration. The values of maximal inhibition were 45%, 78%, and 100% for the doses of 3, 10, and 30 nmol/paw (IPL), respectively (not shown). The effects of caffeine and DPCPX were attributed to the local action, because they were not observed after injection of the highest dose into the contralateral hind-paw (Fig. 6, A and B). Local peripheral coadministration of the highest tested doses of caffeine or DPCPX with carrageenan did not produce a significant effect on carrageenan-induced hyperalgesia (P > 0.05, Student t test, data not shown).

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The Influence of IPL 5-HT Receptor Antagonists on the Antihyperalgesic Effect of Levetiracetam

The coadministration of methysergide, a nonselective 5-HT receptor antagonist (10–100 nmol/paw; IPL), and GR 127935, a selective 5-HT1B/1D receptor antagonist (50–200 nmol/paw; IPL), with levetiracetam (1000 nmol/paw; IPL) into the hind-paw caused a significant and dose-dependent reduction of levetiracetam-induced antihyperalgesia (Fig. 7, 7A and 7B). The maximal inhibitory effects of methysergide on levetiracetam-induced antihyperalgesia were achieved 90 minutes after administration, and the corresponding values were 46%, 61%, and 89% for doses of 10, 50, and 100 nmol/paw (IPL), respectively (not shown). For GR 127935, the maximal inhibition of levetiracetam antihyperalgesia occurred 90 to 120 minutes after administration and the values were 49%, 77%, and 100% for the doses of 50, 100, and 200 nmol/paw (IPL), respectively (not shown). The effects of methysergide and GR 127935 were attributed to a local action, because they were not observed after injection of the highest tested doses into the contralateral hind-paw (Fig. 7, 7A and 7B). Peripheral coadministration of the highest tested doses of 5-HT antagonists with carrageenan did not produce a significant effect on carrageenan-induced hyperalgesia (P > 0.05, Student t test, data not shown).

Figure 7
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The Influence of IPL GABAA Antagonist on the Antihyperalgesic Effect of Levetiracetam

The coadministration of bicuculline, a selective GABAA receptor antagonist (400 nmol/paw; IPL), with levetiracetam (1000 nmol/paw; IPL) did not produce a statistically significant reduction of its peripheral antihyperalgesic effect (Fig. 8). Bicuculline itself (400 nmol/paw; IPL) did not exhibit an intrinsic effect in this experimental model (P > 0.05, Student t test, data not shown).

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DISCUSSION

The Antihyperalgesic and Anti-Edematous Effects of Levetiracetam

The present study demonstrates the efficacy of levetiracetam as a local peripheral antinociceptive drug in an experimental model of inflammatory pain. The local nature of this action was verified by the lack of levetiracetam’s effect after being injected into the contralateral hind-paw. Other antiepileptics such as phenytoin, gabapentin, ethosuximide, carbamazepine, oxcarbazepine, and topiramate also produced local peripheral antinociceptive/antihyperalgesic effects in thermal and inflammatory animal pain models.16–19 In all these studies, local peripheral doses of the antiepileptics were many times lower than the lowest effective systemic dose. Likewise, in the present study, significant local effects of levetiracetam (200–1000 nmol/paw = 0.034–0.17 mg/rat, corresponds to 0.17–0.85 mg/kg) were attainable with much lower doses (up to 59 times lower) than the lowest effective systemic dose that was determined in our previous study (10 mg/kg). This could suggest that levetiracetam given systemically is able to achieve effective concentrations at the periphery and that there is considerable contribution of the peripheral antinociceptive effect to the net effect of systemically administered levetiracetam. Moreover, locally administered levetiracetam may induce an analgesic effect with fewer potentially serious adverse effects/drug interactions seen after systemic administration.

Our study also shows that levetiracetam is able to inhibit in a dose-dependent manner the development of edema produced by carrageenan. Therefore, although levetiracetam is effective in reducing hyperalgesia associated with inflammation, it is also effective in reducing other manifestations of inflammation, such as edema. This finding is similar to the results of Bianchi et al.13 and our unpublished data, which have demonstrated an anti-edematous effect of other antiepileptics, carbamazepine and topiramate, also in a rat model of localized inflammation. Further experiments should be conducted to elucidate the possible mechanism(s) of local peripheral anti-edematous activity of levetiracetam.

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Involvement of the Opioid System in Antihyperalgesia by Levetiracetam

We have also shown that when naloxone (75–300 nmol/paw; IPL), a nonselective opioid receptor antagonist, was administered peripherally, it significantly decreased the antihyperalgesic effect of levetiracetam in a dose-dependent manner. However, of all 3 subtypes of opioid receptors, the μ-opioid receptor agonists are generally the most potent at producing peripheral analgesia.9 Naloxone does not discriminate between opioidergic receptor subtypes, and experiments in which the selective μ-opioid receptor antagonist CTAP (1–5 nmol/paw; IPL) suppressed levetiracetam antihyperalgesia showed that the μ-opioid receptors have a significant role in the mechanism of the antinociception action of levetiracetam. Because these effects were not observed after the administration of the highest doses of the antagonists into the contralateral (noninflamed) hind-paw, the systemic effects of the antagonists due to absorption from the site of injection are excluded. The finding that the peripheral μ-opioid receptors are involved in levetiracetam’s antihyperalgesia could be interpreted that levetiracetam may act on μ-opioid receptors either directly or indirectly by influencing the endogenous opioidergic system to produce antinociception. Opioid peptides are endogenous ligands of opioid receptors. They are produced by the immune cells migrating to the site of inflammation.20,21 Antinociception by μ-opioid agonists in inflammation results predominantly from actions on sensory nerves.9 There are no available data describing the binding properties of levetiracetam to μ-opioid receptors on peripheral endings of sensory neurons. However, several in vitro studies have revealed that levetiracetam selectively inhibits the N-type Ca2+ channels and reduces Ca2+ conductance.22,23 μ-Opioid receptor agonists also inhibit N-type Ca2+ channels in a voltage-dependent manner.24 In vitro studies have shown that peripheral sensory neurons are rich in N-type Ca2+ channels.25 Peripheral local administration of N-type Ca2+ channel antagonist produced an antinociceptive effect in rat nerve injury–induced hyperalgesia, but not in normal rats, perhaps because sensory neurons become sensitized after nerve injury. This is likely to involve alterations in ion conductance, which results from a change in ion channel function and expression, or from modulation of ion conductance by hyperalgesic factors that are released from either neuronal or nonneuronal cells.26,27 Therefore, by influencing N-type Ca2+ channels in peripheral sensory neurons, levetiracetam might incorporate μ-opioid receptor-induced antinociception in its effect.

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Involvement of the Adrenergic System in Antihyperalgesia by Levetiracetam

Our experiments have also revealed that the local peripheral administration of yohimbine, a selective α2-adrenoceptor antagonist (130–520 nmol/paw; IPL), significantly decreased the antihyperalgesic effects of levetiracetam, in a dose-dependent manner. The α2-adrenoceptors are located on primary afferent terminals, and could mediate analgesia or hyperalgesia, depending on the subtype of α2-adrenoceptors involved.9,19,28,29 Both α2A- and α2C-adrenoceptors that are activated by noradrenaline from postganglionic sympathetic nerve fibers mediate antinociception.30–33 The reversal of the antihyperalgesic effect of levetiracetam by a selective α2A-adrenoceptor antagonist BRL 44408 (50–200 nmol/paw; IPL), and by a selective α2C-adrenoceptor antagonist MK-912 (5–20 nmol/paw; IPL), revealed that both α2A- and α2C-adrenoceptors have a significant role in the mechanism of the antinociceptive effect of levetiracetam. Because the same effects were not observed when yohimbine, BRL 44408, and MK-912 were administered to the contralateral hind-paw, we concluded that these antagonists exerted only local effects. There are no available data on the binding properties of levetiracetam to α2-adrenoceptors. Thus, the suppression of the antihyperalgesic effect of levetiracetam by peripheral α2-adrenoceptor antagonists is most likely explained by its indirect interaction with the peripheral α2-adrenoceptors, in particular with α2A- and α2C-adrenoceptors.

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Involvement of the Adenosine System in Antihyperalgesia by Levetiracetam

The purine nucleoside, adenosine, has an important role in pain modulation. Adenosine A1 receptors have been visualized in sensory afferent neurons using immunohistochemistry.34 Peripheral administration of A1 receptor agonists leads to antinociception against mechanical35 and thermal hyperalgesia after nerve injury,36 whereas adenosine A2 and A3 receptors mediate nociception peripherally.37 Inhibition of the local peripheral antihyperalgesic action of levetiracetam by caffeine (500–1500 nmol/paw; IPL), a nonselective adenosine receptor antagonist, does not determine which subtypes of adenosine receptors are involved in this action. However, because DPCPX (3–30 nmol/paw; IPL), a selective A1 receptor antagonist, also suppressed the antihyperalgesic effect of levetiracetam, levetiracetam probably exerted its effect through the activation of adenosine A1 receptors. Because the same effects were not observed when caffeine or DPCPX was administered to the contralateral hind-paw, it was concluded that these antagonists only exerted local effects. This is in agreement with our previous findings that were obtained in the same experimental model. We showed that the antiepileptics carbamazepine and oxcarbazepine produced their peripheral antinociceptive effects through A1 receptors.17,18 Because there are no data showing that levetiracetam binds to adenosine A1 receptors, we speculate that the local peripheral antihyperalgesic effect of levetiracetam is most likely mediated through indirect activation of peripheral adenosine A1 receptors.

Taken together, it is possible that peripheral antinociception induced by levetiracetam requires the physical presence of multiple μ-opioid, α2-adrenergic, and A1 adenosine receptors on the primary afferents, according the finding of Aley and Levine32 who found that despite the antinociception that is produced by the activation of μ, α2, or A1 receptors, it is possible that these receptors may not act independently, but rather require to be associated with a complex to produce antinociception.

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Involvement of the Serotonergic System in Antihyperalgesia by Levetiracetam

5-HT involvement in pain processing is complex; 5-HT can inhibit or facilitate nociceptive transmission depending on the nature of the nociceptive stimuli, the site of action, and the receptor subtype it acts on.28 In the present study, methysergide (10–100 nmol/paw, IPL), a broad-spectrum antagonist of 5-HT receptors (5-HT1, 5-HT2, and 5-HT5–7), significantly reduced the local peripheral antihyperalgesic effect of levetiracetam. It has been shown that 5-HT1B/1D receptors are involved in reducing inflammatory pain.38,39 Thus, it was not surprising that GR 127935, a selective 5-HT1B/1D receptor antagonist, at doses that ranged from 50 to 200 nmol/paw, IPL, significantly attenuated the antihyperalgesic effect of levetiracetam. Moreover, these antagonistic effects were not attributed to a systemic action because their contralateral administration was ineffective. To our knowledge, there are no available data describing the binding properties of levetiracetam to 5-HT1 receptors, nor its ability to influence 5-HT release in nociceptive transmission. At the periphery, triptans (5-HT1B/1D receptor agonists) prevent or block neurogenic inflammation, at least in part, by inhibiting the release of substance P and calcitonin gene-related peptide from peptidergic afferents.40 Our results indicate that 5-HT receptors, probably 5-HT 1B/1D, are involved indirectly in the peripheral antinociceptive effect of levetiracetam.

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Involvement of the GABAergic System in Antihyperalgesia by Levetiracetam

GABA modulates pain signaling processes at central and peripheral levels.9,41 Endogenous peripheral GABA could arise from primary afferent fibers and acts at GABAA receptors that are present on some unmyelinated afferent axons.9,42 The finding that bicuculline did not change the peripheral antihyperalgesia of levetiracetam implies that the peripheral GABAA receptors were not involved in its action. This is in accordance with evidence that GABA, via GABAA receptors, assumes a role in modulating the antihyperalgesia of carbamazepine, oxcarbazepine, and topiramate in a model of inflammatory pain, at central, but not at peripheral sites.19,43 Bearing in mind the results presented herein and our previous finding that bicuculline administered systemically significantly decreased the antihyperalgesic effect of levetiracetam,4 it can be concluded that GABAA receptors are involved in the antihyperalgesic effect of levetiracetam most likely only at the central level.

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CONCLUSIONS

Our data show that levetiracetam produces local peripheral antihyperalgesic and anti-edematous effects in an experimental model of localized inflammation. Its antihyperalgesic effect is mediated, at least in part, through peripheral μ-opioidergic, α2A,C-adrenergic, A1 adenosine, and 5-HT1B/1D receptors but not through GABAA receptors. These findings could contribute toward a better understanding of the mechanism of the peripheral antihyperalgesic effect of levetiracetam, and improved treatments of inflammatory pain with a lower incidence of systemic side effects and drug interactions.

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DISCLOSURES

Name: Radica M. Stepanovic`-Petrovic`, PhD.

Contribution: This author helped design the study and write the manuscript.

Attestation: Radica M. Stepanovic`-Petrovic` 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, BPharm.

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: Maja A. Tomic`, PhD.

Contribution: This author helped analyze the data.

Attestation: Maja A. Tomic` has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Nenad D. Ugrešic`, PhD.

Contribution: This author helped analyze the data.

Attestation: Nenad D. Ugrešic` has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

This manuscript was handled by: Steven L. Shafer, MD.

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