Levetiracetam synergises with common analgesics in producing antinociception in a mouse model of painful diabetic neuropathy

Pharmacological Research 97 (2015) 131–142

Contents lists available at ScienceDirect

Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs

Levetiracetam synergises with common analgesics in producing antinociception in a mouse model of painful diabetic neuropathy ´ Uroˇs Pecikoza, Nenad Ugreˇsic, ´ Radica Stepanovic-Petrovi ´ Ana Micov ∗ , Maja Tomic, c´ Department of Pharmacology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, POB 146, 11221 Belgrade, Serbia

a r t i c l e

i n f o

Article history: Received 23 February 2015 Received in revised form 21 April 2015 Accepted 26 April 2015 Available online 6 May 2015 Keywords: Levetiracetam Ibuprofen Aspirin Paracetamol Synergism Diabetic neuropathy Chemical compounds studied in this article: Levetiracetam (PubChem CID: 5284583) Ibuprofen (PubChem CID: 3672) Acetylsalicylic acid (PubChem CID: 2244) Paracetamol (PubChem CID: 1983) Streptozotocin (PubChem CID: 29327)

a b s t r a c t Painful diabetic neuropathy is difficult to treat. Single analgesics often have insufficient efficacy and poor tolerability. Combination therapy may therefore be of particular benefit, because it might provide optimal analgesia with fewer adverse effects. This study aimed to examine the type of interaction between levetiracetam, a novel anticonvulsant with analgesic properties, and commonly used analgesics (ibuprofen, aspirin and paracetamol) in a mouse model of painful diabetic neuropathy. Diabetes was induced in C57BL/6 mice with a single high dose of streptozotocin, applied intraperitoneally (150 mg/kg). Thermal (tail-flick test) and mechanical (electronic von Frey test) nociceptive thresholds were measured before and three weeks after diabetes induction. The antinociceptive effects of orally administered levetiracetam, analgesics, and their combinations were examined in diabetic mice that developed thermal/mechanical hypersensitivity. In combination experiments, the drugs were coadministered in fixed-dose fractions of single drug ED50 and the type of interaction was determined by isobolographic analysis. Levetiracetam (10–100 mg/kg), ibuprofen (2–50 mg/kg), aspirin (5–75 mg/kg), paracetamol (5–100 mg/kg), and levetiracetam-analgesic combinations produced significant, dosedependent antinociceptive effects in diabetic mice in both tests. In the tail-flick test, isobolographic analysis revealed 15-, and 19-fold reduction of doses of both drugs in the combination of levetiracetam with aspirin/ibuprofen, and paracetamol, respectively. In the von Frey test, approximately 7- and 9-fold reduction of doses of both drugs was detected in levetiracetam-ibuprofen and levetiracetamaspirin/levetiracetam-paracetamol combinations, respectively. These results show synergism between levetiracetam and ibuprofen/aspirin/paracetamol in a model of painful diabetic neuropathy and might provide a useful approach to the treatment of patients suffering from painful diabetic neuropathy. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Diabetic neuropathy is one of the most common complications of diabetes mellitus. It is estimated that 10–20% of patients with diabetic neuropathy feel pain [1]. The pathogenesis of painful diabetic neuropathy is complex and involves multiple neuropathic and inflammatory pain targets [2,3]. Painful diabetic neuropathy is difficult to treat. Conventional analgesics, nonsteroidal antiinflammatory drugs (NSAIDs) and opiates, often have limited

Abbreviations: AUC, area under the curve; COX, cyclooxygenase; ED50 add , theoretical additive ED50 for drug mixture; ED50 mix , experimental ED50 for drug mixture; %MPE, percentage of the maximal possible effect; NMDA, N-methyl-D-aspartate; NSAIDs, nonsteroidal anti-inflammatory drugs; TNF␣ , tumor necrosis factor alpha; ␥, interaction index. ∗ Corresponding author. Tel.: +381 11 39 51 374; fax: +381 11 39 70 840. E-mail addresses: [email protected] (A. Micov), ´ [email protected] (U. Pecikoza), [email protected] (M. Tomic), ´ [email protected] (R. Stepanovic-Petrovi ´ ´ [email protected] (N. Ugreˇsic), c). http://dx.doi.org/10.1016/j.phrs.2015.04.014 1043-6618/© 2015 Elsevier Ltd. All rights reserved.

efficacy. This has led to the use of antidepressants and anticonvulsants as the mainstay of treatment [1]. However, no single analgesic is fully effective in all patients and dose-related adverse effects in long-term use are also common. Therefore, combination therapy may be both necessary and justified [4,5]. Levetiracetam is a novel anticonvulsant with proven analgesic properties. Its antinociceptive/antihyperalgesic effects have been demonstrated in animal models of diabetic neuropathy [6,7], somatic and visceral inflammatory pain [8–11]. Several, small-scale, clinical studies have confirmed its efficacy in neuropathic pain treatment [12,13] and migraine prophylaxis [14]. Unusually favourable pharmacokinetic properties of levetiracetam (cytochrome P-450 independent metabolism, minimum plasma protein binding) [15,16], make it suitable for co-administration especially with drugs that are metabolized in the liver (e.g. ibuprofen, aspirin, paracetamol) and/or bound to plasma proteins (e.g. ibuprofen, aspirin). Levetiracetam exerted synergistic interactions with different analgesics (paracetamol, ibuprofen, celecoxib), caffeine and ceftriaxone in inflammatory pain models in rodents

132

A. Micov et al. / Pharmacological Research 97 (2015) 131–142

[10,11]. The interactions of levetiracetam with analgesics in neuropathic pain states have not been examined. Ibuprofen’s efficacy in treating inflammatory pain is wellestablished. Recent reports showed its efficacy in certain neuropathic pain models [17,18]. The long-term use of ibuprofen is limited by substantial gastrointestinal and cardiovascular (at higher doses) adverse effects [19]. Aspirin is a unique NSAID, which is widely used to treat mild to moderate pain, but also in prevention of cardiovascular events (at lower doses). Gastrointestinal adverse effects of aspirin limit its clinical usefulness. It was shown that aspirin in combination with minocycline prevents the development of experimental diabetic neuropathy [20]. Paracetamol is one of the most widely used analgesics with a favourable safety profile in therapeutic doses, while overdose could result in hepatotoxicity [21]. It has been extensively studied in combination with other analgesics. Synergistic interaction was obtained between paracetamol and tramadol in diabetic rats [22] and their combination analgesia was confirmed in patients with painful diabetic neuropathy [23,24]. Paracetamol combined with either morphine or gabapentin, resulted in synergistic efficacy in rats with spinal cord injury pain [25]. Combination of analgesics with different mechanisms of action might provide optimal analgesia with fewer adverse effects due to the reduced doses of single drugs. Thus, the purpose of this study was to examine the effects of two-drug combinations of levetiracetam with ibuprofen/aspirin/paracetamol in a mouse model of painful diabetic neuropathy and to determine the type of interaction between components (synergistic, additive or antagonistic). 2. Material and methods 2.1. Animals Experiments were performed on male C57BL/6 mice, weighing 20–30 g at the beginning of the study (Military Academy Breeding Farm, Belgrade, Serbia). The animals were housed under standard laboratory conditions: temperature of 22 ± 1 ◦ C, 60% relative humidity and maintained on a 12/12 h light/dark cycle with freely available food and water. The experimental groups consisted of 5–9 mice. All experiments were approved by the Institutional Animal Care and Use Committee of the Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia, and were carried out in compliance with the guidelines of European Communities Council Directive 2010/63/EU on the use of animals for scientific purposes. A total of 624 mice were used in the study. 2.2. Drugs and their administration Levetiracetam (Keppra; UCB Pharma AG, Brussels, Belgium), ibuprofen (Pharmagen GmbH, Frankfurt, Germany), aspirin (Aspirin; Bayer Bitterfeld GmbH, Bitterfeld-Wolfen, Germany), and paracetamol (Panadol; GlaxoSmithKline Dungarvan Ltd., Dungarvan, Ireland) were suspended in distilled water with the addition of 1 drop of Tween 80 and sonicated for 15 min for proper distribution. Drugs/drug combinations were administered to mice by oral gavage (p.o.), in a volume of 10 ml/kg body weight. 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 (10 ml/kg body weight). 2.3. Induction and assessment of diabetes Diabetes was induced by a single i.p. injection of streptozotocin (150 mg/kg) following an overnight fast [26]. Two weeks after the streptozotocin injection, the diabetes was confirmed by measuring

blood glucose levels with an automatic analyzer (GlucoSure Plus, Apex Biotechnology Corp, Hsinchu, Taiwan) using glucose oxidase peroxidase enzyme reagent stripes. Mice with a non-fasting serum glucose level above 250 mg/dl were considered diabetic and were used for the experiments [26]. 2.4. Assessment of nociceptive responses Thermal and mechanical hypersensitivity are typical manifestations of abnormal pain sensation in the early stage of experimental diabetes. Therefore, nociceptive responses in diabetic and nondiabetic mice to thermal and mechanical stimuli were evaluated. 2.4.1. Assessment of thermal nociceptive responses The thermal nociceptive responses in both diabetic and nondiabetic mice were evaluated by the radiant heat tail-flick test [26]. In brief, the tails of mice (about 4 cm from the tip of the tail) were exposed to an infrared radiant heat source of tail-flick apparatus (Hugo Sach Elektronik, Cat. No. 7360, March-Hugstetten, Germany). Tail-flick latency (s) was measured before (baseline thermal threshold) and 3 weeks after the injection of streptozotocin, but before corresponding drug treatment (pretreatment thermal threshold), to assess hyperalgesia development. The heat intensity was set up to provide a baseline latency time of 4–6 s. A cut-off time of 10 s was used to prevent tissue damage. 2.4.2. Assessment of mechanical nociceptive responses The mechanical nociceptive responses in diabetic/non-diabetic mice were assessed by measuring the paw withdrawal thresholds using an electronic Von Frey anesthesiometer (IITC Life Science, Woodland Hills, CA) [27]. Mice were individually placed in plastic boxes on a metallic mesh floor. The mechanical stimulus was delivered using a plastic, flexible filament coupled with a force transducer. The force was applied to the plantar surface of either hindpaw and the pressure was gradually increased until the animal withdrew the paw, which was automatically recorded. Paw withdrawal threshold (g) was measured before (baseline mechanical threshold) and 3 weeks after the injection of streptozotocin, but before corresponding drug treatment (pretreatment mechanical threshold), to assess allodynia development. A cut-off pressure of 6 g was used in the study. 2.4.3. Quantification of hypersensitivity and antinociceptive effects The percentage of hypersensitivity (thermal hyperalgesia or mechanical allodynia) was calculated for each animal using following formula: % hypersensitivity =

(baseline threshold-pretreatment threshold) baseline threshold × 100.

The antinociceptive effects of drugs/drug combinations were examined in diabetic mice that developed thermal/mechanical hypersensitivity. To evaluate whether their effects are dependent on diabetes-induced neuropathic changes, additional experiments on non-diabetic mice were performed. All treatments were applied p.o. immediately after the measurement of pretreatment thresholds. The post-treatment thresholds (thermal or mechanical) were recorded 30, 60, 90, 120, 180 and 240 min after drugs/drug combinations administration in both diabetic and non-diabetic mice. Control diabetic and non-diabetic mice received the same volume of corresponding vehicle (p.o.) instead of test compounds.

A. Micov et al. / Pharmacological Research 97 (2015) 131–142

Antinociceptive effects were expressed as the percentage of the maximal possible effect (%MPE), calculated for each animal using following formula: % MPE =

(post-treatment threshold-pretreatment threshold) (cut-off value-pretreatment threshold) × 100

If post-treatment threshold was shorter than pretreatment threshold, a value of 0%MPE was assigned, indicating the absence of antinociception. The values of ED50 (the dose expected to result in 50%MPE) with 95% confidence limits were estimated from corresponding log dose–response curves determined at the time of peak effect [28,29]. 2.5. Analysis of interactions between levetiracetam and analgesics The interactions between levetiracetam and the analgesics in diabetic mice were evaluated by isobolographic analysis at the ED50 level of the effect, as described previously [11,26,30]. Briefly, the ED50 value of each drug was obtained from the corresponding log dose–response curves. In combination experiments, levetiracetam and ibuprofen/aspirin/paracetamol were co-administered in fixeddose fractions of their ED50 . For drug mixture, experimental ED50 (ED50 mix ) was determined by linear regression analysis of the log dose–response curve and compared to a theoretical additive ED50 (ED50 add ). When the drug combination produced an ED50 mix that was significantly lower than the ED50 add , it was interpreted as there is a supra-additive (synergistic) interaction between the drugs. In addition, an interaction index () was used to describe the magnitude of interaction [11,26,31]. 2.6. Analysis of the duration of effect of drugs and drug combinations To compare the duration of the effect of the drug applied alone with the duration of the effect produced by the same drug applied in combination, the slopes of the %MPE-AUC regression lines were calculated [26]. The data were expressed as area under the curve (AUC): the area of a series of trapezoids in which the height was the difference in post-treatment and pretreatment thermal/mechanical thresholds (expressed in seconds (s) in the tail-flick test, and grams (g) in the von Frey test), and the base, the interval (min) between measurements [32]. AUC for each dose of drug/drug combination is expressed as a function of its peak %MPE (AUC = slope × %MPE + intercept). In %MPE-AUC regression line, the slope is the relative measure of the duration of the drug/drug combination effect; the treatment with a significantly greater slope exerts an effect of longer duration than that with a lesser slope [26,32]. Additionally, high correlation coefficient of the %MPE-AUC regression line indicates that the duration of the effect of the drug/drug combination treatment is dose-dependent [26,32]. 2.7. Assessment of rotarod performance Rotarod performance was assessed to evaluate the effects of levetiracetam and levetiracetam-analgesic combinations in diabetic mice on motor coordination or sedation as described previously [10,33] with minor modifications. The test was performed using a rotarod apparatus (Treadmill for mice 7600; Ugo Basile, Milano, Italy), consisting of a rod rotating at a constant speed of 15 rpm. Mice were trained to drive the rotarod for 3 days. Only those mice that could remain on the rod for 60 s on two consecutive trials (separated by 30 min pause between trials) were used in

133

the experiments. The post-treatment latencies to remain on the rotating rod were recorded at six time points, during 240 min. The cut-off time was 60 s. 2.8. Statistical analysis All computations were done according to Tallarida et al. [28–30] using computer programs Pharm PCS (Micro-Computer Specialists, Philadelphia, PA) and Pharm Tools Pro (The McCary Group, Schnecksville, PA). Statistical analysis was performed using SPSS 18 for Windows. The results are presented as mean values ± SEM. Differences between the corresponding means from the tail-flick and von Frey tests were verified by Student s t-test or one-way analysis of variance (One-Way ANOVA), followed by Tukey s HSD test. Data from the rotarod test were analyzed by Mann-Whitney U-test. Test for parallelism was used to compare the slopes of two regression lines [28]. The difference between theoretical ED50 (ED50 add ) and experimental ED50 (ED50 mix ) for drug combination was examined by Student’s t-test. A p value of less than 0.05 was considered statistically significant. 3. Results 3.1. Evaluation of streptozotocin-induced diabetes and nociceptive responses in diabetic mice Two weeks after streptozotocin injection (150 mg/kg; i.p.), the randomly chosen group of diabetic mice had significantly higher blood glucose levels (almost all above upper limit of detection of apparatus of 550.8 mg/dl; n = 20) compared to the basal blood glucose levels of the same group of mice obtained before induction of diabetes (100.8 mg/dl; n = 20; p < 0.01, paired Student’s t-test). Three weeks after diabetes induction, there was a significant decrease in the body mass in diabetic mice (21.82 ± 0.16 g; n = 382) compared to their body mass at the beginning of the study (24.21 ± 0.12 g; n = 382; p < 0.01, paired Student’s t-test). All animals demonstrated normal behaviour during the study period. Three weeks after streptozotocin-treatment, the nociceptive responses were substantially decreased in diabetic mice as compared with their basal values in both tests. In the tail-flick test, pretreatment latencies (3.96 ± 0.03 s; n = 208) were significantly lower than their basal latencies tested three weeks before (5.50 ± 0.03 s; n = 208; p < 0.01, paired Student’s t-test), resulting in thermal hyperalgesia with a value of 29%. In von Frey test, pretreatment withdrawal thresholds (2.40 ± 0.02 g; n = 174) were markedly lower than their basal thresholds tested three weeks before (4.18 ± 0.02 g; n = 174; p < 0.01, paired Student’s t-test), resulting in mechanical allodynia of 43%. 3.2. Antinociceptive effects of levetiracetam and analgesics alone In diabetic mice, levetiracetam (10–100 mg/kg; p.o.), ibuprofen (2–50 mg/kg; p.o.), aspirin (5–75 mg/kg; p.o.), and paracetamol (5–100 mg/kg; p.o.) caused significant, dose-dependent antinociceptive effects in the tail-flick and von Frey assays (Figs. 1, 2 and 5). Their ED50 values, calculated from the corresponding log dose–response curves (Fig. 5), are summarized in Table 1. In non-diabetic mice, levetiracetam (100 mg/kg; p.o.), ibuprofen (50 mg/kg; p.o.), and paracetamol (100 mg/kg; p.o.) failed to produce significant antinociception in both tests (p > 0.05, Student’s t-test) (not shown). Aspirin (75 mg/kg; p.o.) produced a weak antinociceptive effect (17%MPE, observed 60 min after administration) in non-diabetic mice in the tail-flick test (p < 0.05, Student’s t-test), and was without any efficacy in the von Frey test (p > 0.05, Student’s t-test) (not shown).

134

A. Micov et al. / Pharmacological Research 97 (2015) 131–142

Fig. 1. Time course of the antinociceptive effects of levetiracetam (LEV) (A), ibuprofen (IBU) (B), aspirin (ASA) (C), and paracetamol (PAR) (D) in diabetic mice, expressed as the percentage of maximal possible effect–MPE (%). Antinociceptive effect was measured in the tail-flick test. The drugs were administered orally (p.o.) (denoted by arrow). Each point represents the mean ± SEM of MPE (%). Statistical significance (* p < 0.05; ** p < 0.01; One-Way ANOVA, followed by Tukey s HSD test) was determined by comparison with the control group.

3.3. Antinociceptive effects of two-drug combinations of levetiracetam with analgesics and analysis of the type of interaction between components Two-drug combinations of levetiracetam with analgesics (ibuprofen, aspirin or paracetamol) administered in fixed-dose fractions of the ED50 (1/16, 1/8, 1/4 and 1/2) of each drug, produced significant and dose-dependent antinociceptive effects in the tailflick and von Frey tests in diabetic mice (Figs. 3 and 4). The ED50 mix values, calculated from the corresponding log dose–response curves (Fig. 5), are presented in Table 1 and in isobolograms (Fig. 6). In both tests, for all examined combinations, the ED50 mix was significantly lower than ED50 add and the interaction index was less than one (Fig. 6, Table 1), indicating a synergistic interaction.

According to the values of the interaction index, the rank of potencies in the tail-flick test was: levetiracetam-paracetamol > levetiracetam-ibuprofen = levetiracetam-aspirin (Table 1). In the von Frey test, the rank of potencies indicated by the interaction index values was: levetiracetam-paracetamol = levetiracetamaspirin > levetiracetam-ibuprofen (Table 1). In non-diabetic mice in both tests, levetiracetam-ibuprofen and levetiracetam-paracetamol combinations administered in the highest doses tested in diabetic mice (1/2 of the ED50 of each drug determined for diabetic mice), produced no significant antinociceptive effects (p > 0.05, Student’s t-test) (not shown). Levetiracetam-aspirin combination produced weak antinociceptive efficacy (17%MPE, observed 30 min after administration) in non-diabetic mice in the tail-flick test (p < 0.05, Student’s t-test),

A. Micov et al. / Pharmacological Research 97 (2015) 131–142

135

Fig. 2. Time course of the antinociceptive effects of levetiracetam (LEV) (A), ibuprofen (IBU) (B), aspirin (ASA) (C), and paracetamol (PAR) (D) in diabetic mice, expressed as the percentage of maximal possible effect–MPE (%). Antinociceptive effect was measured in the von Frey test. The drugs were administered orally (p.o.) (denoted by arrow). Each point represents the mean ± SEM of MPE (%). Statistical significance (* p < 0.05; ** p < 0.01; One-Way ANOVA, followed by Tukey s HSD test) was determined by comparison with the control group.

and was without effect in the von Frey test (p > 0.05, Student’s t-test) (not shown). 3.4. Analysis of the duration of antinociceptive effects of drugs applied alone and in combination The duration of the effects of levetiracetam, ibuprofen, aspirin, paracetamol, and levetiracetam-analgesic combinations were expressed as the slopes of the %MPE-AUC regression line. In both nociceptive tests, the slopes for all two-drug combinations (Table 2) were not different from the slopes for drugs used in corresponding combination when applied alone (p > 0.05, test for parallelism; Table 2), indicating a similar duration of the effects of drugs regardless of whether the drugs were administered alone or in combination.

The high correlation coefficients of the %MPE-AUC line for all the treatments indicated that the duration of the effects were dosedependent (Table 2). 3.5. Effects of levetiracetam and levetiracetam-analgesic combinations on the rotarod performance Levetiracetam (100 mg/kg; p.o.) and levetiracetam-analgesic combinations administered in the highest tested doses (1/2 of the ED50 of each drug determined in diabetic mice in the von Frey test), did not have significant influence on the rotarod performance (p > 0.05; Mann-Whitney U-test) (Fig. 7). However, there was a trend towards impaired rotarod performance (motor incoordination and/or sedation) for levetiracetam (p = 0.082; Mann-Whitney U-test) (Fig. 7).

136

A. Micov et al. / Pharmacological Research 97 (2015) 131–142

Table 1 Parameters of isobolographic analysis for levetiracetam-analgesic combinations in a mouse model of painful diabetic neuropathy. Drugs

ED50 a ± SEM (confidence limits) Tail-flick test ± ± ± ±

Von Frey test

Levetiracetam Ibuprofen Aspirin Paracetamol

25.33 7.82 17.83 10.83

Drug combinations

ED50 a ± SEM (confidence limits)

Levetiracetam + ibuprofen Levetiracetam + aspirin Levetiracetam + paracetamol

1.52 (19.28–34.50) 1.27 (3.89–15.72) 2.09 (10.73–29.61) 1.74 (5.41–21.68)

28.23 12.91 27.59 17.28

± ± ± ±

1.29 (23.18–34.39) 2.99 (4.77–34.96) 5.99 (10.83–70.29) 2.82 (8.55–34.83)

ED50 add c

ED50 mix d

b

ED50 add c

ED50 mix d

b

16.57 ± 0.99 (12.75–19.30) 21.58 ± 1.30 (18.35–24.64) 18.07 ± 1.16 (13.86–20.27)

2.25 ± 0.45 (1.07–4.39)* 3.08 ± 0.41 (1.75–5.43)* 1.95 ± 0.69 (0.42–9.03)*

0.14 0.14 0.11

20.57 ± 1.63 (15.96–25.94) 27.91 ± 3.06 (21.52–34.43) 22.75 ± 1.55 (19.19–26.75)

6.36 ± 0.97 (3.30–12.28)* 6.34 ± 0.66 (4.04–9.95)* 5.15 ± 0.26 (4.16–6.38)*

0.31 0.23 0.23

a

ED50 = Effective dose required to produce 50% antinociceptive activity.  = ED5 LEVETIRACETAM COMBINED WITH ANALGESIC /ED50 LEVETIRACETAM GIVEN ALONE + ED50 ANALGESIC COMBINED WITH LEVETIRACETAM /ED50 ANALGESIC GIVEN ALONE . Values near 1 indicate an additive interaction, values more than 1 imply an antagonistic interaction and values less than 1 indicate a synergistic interaction [31]. c ED50 add = Theoretical additive ED50 for drug mixture. d ED50 mix = Experimental ED50 for drug mixture. * p < 0.05 between ED50 add and ED50 mix (t-test), indicates a synergistic interaction [30]. b

Table 2 The parameters of duration of antinociceptive action of levetiracetam, analgesics and levetiracetam-analgesic combinations in a mouse model of painful diabetic neuropathy. Drugs/drug combinations

Tail-Flick test Slopea ± SEM

Levetiracetam Ibuprofen Aspirin Paracetamol Levetiracetam + ibuprofen Levetiracetam + aspirin Levetiracetam + paracetamol

8.93 11.18 10.08 11.44 8.45 10.11 5.74

± ± ± ± ± ± ±

0.64 0.66 1.06 1.74 2.51 0.49 2.26

Von Frey test rb

Slopea ± SEM

1 1 0.99 0.98 0.92 1 0.87

5.47 8.68 6.34 5.47 7.37 5.50 6.96

± ± ± ± ± ± ±

0.42 0.46 0.68 0.32 0.90 1.29 0.23

rb 0.99 1 0.99 0.99 0.99 0.95 1

a Slope of the %MPE-AUC regression lines is a relative measure for the duration of the effect of drug/drug combination; treatment with significantly greater slope has a longer duration of antinociceptive effect [26,32]. b r = Correlation coefficients of the %MPE-AUC regression lines; values near 1 indicate that the duration of the effect of drug/drug combination treatment is dose-dependent [26,32].

4. Discussion The major finding of this study is that the anticonvulsant levetiracetam exerts synergistic interaction with ibuprofen/aspirin/paracetamol in a mouse model of painful diabetic neuropathy. Anticonvulsants and nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in monotherapy of painful diabetic neuropathy, and here we show for the first time their combination analgesia. Considering that insufficient efficacy and/or poor tolerability may limit their clinical use in monotherapy, the presented data may offer a novel therapeutic option for the treatment of painful diabetic neuropathy. Behavioural responses to mechanical and thermal stimuli are the most widely used assays in animal studies of painful diabetic neuropathy. Consistent with the literature data, we observed that streptozotocin-induced diabetic mice developed thermal hyperalgesia and mechanical allodynia in the early stage of experimental diabetes. Both endpoints have certain clinical relevance. Thermal hyperalgesia has been registered in some patients with mild painful diabetic neuropathy [34], and mechanical sensitization of afferent fibers were found in patients with early development of diabetic neuropathy [35]. We observed that levetiracetam, ibuprofen, paracetamol, levetiracetam-ibuprofen, and levetiracetam-paracetamol combinations produced significant antinociception in diabetic, but not in non-diabetic mice in the tail-flick and von Frey assays. Aspirin and levetiracetam-aspirin combination produced high antinociceptive efficacy in diabetic mice in both tests compared to weak

efficacy (tail-flick test), and no effect (von Frey test) in nondiabetic mice. The general lack of antinociception of individually applied drugs in non-diabetic mice is in line with previous studies which showed no effect of levetiracetam [6,7], ibuprofen [36], aspirin [37,38] and paracetamol [39] in healthy mice/rats in thermal/mechanical pain tests. Therefore, we could hypothesize that the determined antinociceptive effects of levetiracetam, analgesics and levetiracetam-analgesic combinations in diabetic mice are dependent on neuropathic changes caused by diabetes. The current study demonstrates that ibuprofen (2–50 mg/kg; p.o.) produced a significant, dose-dependent reduction of thermal hyperalgesia and mechanical allodynia in diabetic mice. To our knowledge, there is no evidence about ibuprofen’s efficacy in animal models of peripheral diabetic neuropathy. However, recent reports demonstrate analgesic effects of ibuprofen in other models of peripheral neuropathy. Ibuprofen was effective in decreasing thermal hyperalgesia and mechanical allodynia induced by peripheral nerve lesions after systemic (per os) [18] and local (intraplantar) [17] administration. Our finding that aspirin (5–75 mg/kg; p.o.) increased thermal and mechanical nociceptive thresholds in diabetic mice was not consistent with the observation of Courteix et al. [37] who showed that aspirin (100 mg/kg; intravenous) did not affect vocalization thresholds in diabetic rats in the paw pressure test. The use of different nociceptive endpoints, animal species and route of aspirin administration may contribute to this discrepancy. Our results are consistent with recent findings of Wang et al. [40] who showed that aspirin administered in the form of its endogenous mediator

A. Micov et al. / Pharmacological Research 97 (2015) 131–142

137

Fig. 3. Time course of the antinociceptive effects of levetiracetam-ibuprofen (LEV + IBU) (A), levetiracetam-aspirin (LEV + ASA) (B), and levetiracetam-paracetamol (LEV + PAR) (C) combinations in diabetic mice, expressed as the percentage of maximal possible effect–MPE (%). Antinociceptive effect was measured in the tail-flick test. Drugs were administered orally (denoted by arrow), in a fixed-dose fraction of their ED50 (1/16 = 0.0625, 1/8 = 0.125, 1/4 = 0.25, and 1/2 = 0.5). Each point represents the mean ± SEM of MPE (%). Statistical significance (** p < 0.01; One-Way ANOVA, followed by Tukey s HSD test) was determined by comparison with the control group.

(aspirin-triggered Lipoxin A4) reversed mechanical allodynia in other model of peripheral neuropathy (chronic constriction injury in rats). We observed that paracetamol (5–100 mg/kg; p.o.) exhibited dose-dependent antinociception in diabetic mice in the tail-flick and von Frey assays. Our results are in accordance with that of Gong et al. [22] who showed that paracetamol, after systemic application (26–208 mg/kg; i.p.), reversed thermal and mechanical hyperalgesia in a dose-dependent manner in diabetic rats. In our study, levetiracetam (10–100 mg/kg; p.o.) produced dose-dependent antinociception in the tail-flick and von Frey tests in streptozotocin-induced painful diabetic neuropathy. As

levetiracetam did not produce a significant impairment of rotarod performance in diabetic mice at the highest used dose (100 mg/kg; p.o.), it would appear that its antinociception was not due to motor impairment or sedation. In the same model, Ozcan et al. [7] showed dose-dependent antinociception of levetiracetam (20–200 mg/kg; i.p.) in diabetic mice using the hot plate test, and Ardid et al.[6] observed significant antihyperalgesic effect of levetiracetam (17–120 mg/kg; i.p.) in diabetic rats in the paw pressure test. So, our data confirmed and extended previous findings as we provided information about levetiracetam’s antinociception in painful diabetic neuropathy after the clinically preferred oral route in a dose range that is not associated with either motor disturbance

Fig. 4. Time course of the antinociceptive effects of levetiracetam-ibuprofen (LEV + IBU) (A), levetiracetam-aspirin (LEV + ASA) (B), and levetiracetam-paracetamol (LEV + PAR) (C) combinations in diabetic mice, expressed as the percentage of maximal possible effect–MPE (%). Antinociceptive effect was measured in the von Frey test. Drugs were administered orally (denoted by arrow), in a fixed-dose fraction of their ED50 (1/16 = 0.0625, 1/8 = 0.125, 1/4 = 0.25, and 1/2 = 0.5). Each point represents the mean ± SEM of MPE (%). Statistical significance (** p < 0.01; One-Way ANOVA, followed by Tukey s HSD test) was determined by comparison with the control group.

138

A. Micov et al. / Pharmacological Research 97 (2015) 131–142

Fig. 5. Log dose–response curves for levetiracetam (LEV), ibuprofen (IBU), aspirin (ASA), paracetamol (PAR), as well as levetiracetam-ibuprofen (LEV + IBU), levetiracetamaspirin (LEV + ASA), and levetiracetam-paracetamol (LEV + PAR) combinations for antinociception at the time of peak effects in diabetic mice in the tail-flick (A, C, and E) and von Frey (B, D, and F) tests. Data are expressed as the percentage of maximal possible effect–MPE (%). Each point represents the mean ± SEM of MPE (%) obtained in 5–9 animals.

A. Micov et al. / Pharmacological Research 97 (2015) 131–142

139

Fig. 6. Isobolograms for the levetiracetam-ibuprofen (LEV + IBU) (A, B), levetiracetam-aspirin (LEV + ASA) (C, D), and levetiracetam-paracetamol (LEV + PAR) (E, F) combinations in diabetic mice in the tail-flick (A, C, and E) and von Frey (B, D, and F) tests. The ED50 values for each drug (obtained at the time of peak effects) are plotted at the axes. The straight line connecting the each ED50 value is the theoretical additive line, and the point in this line is the ED50 add (theoretical additive ED50 ). There is a significant difference (p < 0.05; t-test) between the ED50 add and the ED50 mix (experimental ED50 for drug mixture), indicating a synergistic drug interaction for the combination tested.

140

A. Micov et al. / Pharmacological Research 97 (2015) 131–142

Fig. 7. The effects of levetiracetam (LEV), levetiracetam-ibuprofen (LEV + IBU), levetiracetam-aspirin (LEV + ASA), and levetiracetam-paracetamol (LEV + PAR) combinations on the rotarod performance of diabetic mice expressed as the time spent on the rotarod (s). Pretreatment time spent on the rotarod was obtained immediately before peroral administration of levetiracetam (100 mg/kg) or levetiracetam-analgesic combinations (1/2 of the ED50 for antinociception in the von Frey test of each drug) or vehicle. Each column represents the mean ± SEM of the time spent on the rotarod. Statistical significance (Mann–Whitney test U-test) was determined by comparison with the control group.

or sedation. Despite encouraging data on levetiracetam’s antinociception from animal models of painful diabetic neuropathy, no clinically relevant effect of levetiracetam on painful polyneuropathy caused by diabetes was observed in small clinical trial [41]. Levetiracetam’s combinations with analgesics could be of greater clinical value. We demonstrate that levetiracetam interacts synergistically with ibuprofen/aspirin/paracetamol in producing antinociception in diabetic mice. Levetiracetam-analgesic combinations did not influence the rotarod performance in diabetic mice and their effects in the tail-flick and von Frey tests were antinociceptive. In the tail-flick test, isobolographic analysis revealed 15-, and 19-fold reduction of doses of both drugs in the combination of levetiracetam with aspirin/ibuprofen, and paracetamol, respectively. In the von Frey test, approximately 7- and 9-fold reduction of doses of both drugs was detected in levetiracetamibuprofen and levetiracetam-aspirin/levetiracetam-paracetamol combinations, respectively. All compared to equianalgesic doses of individual drugs. Our research group has demonstrated synergistic interactions between levetiracetam and ibuprofen/paracetamol in somatic inflammatory model of pain, recently [11]. Drug–drug interaction can be due to various mechanisms and levels of drugs action (pharmacodynamic interaction) and/or to alteration in their kinetics (pharmacokinetic interaction). In order to assess the possibility of pharmacodynamic interactions, the sites and mechanisms of action of individual drugs have to be discussed. Little is known about the mechanism of action of NSAIDs/ paracetamol in neuropathic pain states. Further, the pathogenesis of painful diabetic neuropathy is not well understood. Evidence suggests that up-regulation of cyclooxygenase-2 (COX-2) in the peripheral nerves and dorsal root ganglia neurons, as well as, raised serum tumor necrosis factor alpha (TNF␣ ) play an important role in the pathogenesis of painful diabetic neuropathy [18,42–44]. Therefore, the antinociceptive effects of ibuprofen [18,45], aspirin [40,46], and paracetamol [21,47,48] in this type of pain could be explained by their inhibition of COX-2 and ability to decrease TNF␣ levels. It has been recently shown that ibuprofen [49], and aspirin [50] inhibit microglial activity, which could also explain their

effectiveness in diabetic neuropathy, as activated microglia may be involved in the development of diabetic neuropathy [51,52]. Recent studies have suggested that the antinociception of paracetamol in neuropathic pain is mediated by cannabinoid receptors [25,53]. As it was shown that activation of 5-HT7 receptor attenuates thermal hyperalgesia in streptozocin-induced diabetic mice [54], paracetamol might achieve analgesia in this pain state through activation of descending serotonergic pathways and spinal 5-HT7 receptors [55]. Paracetamol blocks spinal hyperalgesia induced by glutamate receptors agonist N-methyl-D-aspartate (NMDA) [56], which could also contribute to its antinociceptive effect since glutamate excitotoxicity has been implicated in diabetic neuropathic pain [34,57]. The mechanisms of analgesic action of levetiracetam in neuropathic pain have not been evaluated yet. In order to evaluate the molecular basis of levetiracetam’s antiepileptic action, it has been shown that levetiracetam selectively inhibits N-type Ca2+ -channels [58]. Bearing in mind that N-type Ca2+ -channels have an essential role in neuropathic pain development [59] and that blockers of these channels achieve analgesia in an experimental model of diabetic neuropathy [60], it could be suggested that levetiracetam exerts its antinociceptive effects through blockade of N-type Ca2+ channels. Levetiracetam’s antinociception could also be related to its ability to hyperpolarize membrane potential via K+ -channels activation and inhibition of Ca2+ entry [61]. Beside altered channels function, many neurotransmitter systems are also affected in diabetic neuropathy [62]. Thus, reduced spinal inhibition through ␥-aminobutyric acid (GABA) and decreased noradrenaline (via ␣2 -adrenoceptors) as well 5-hydroxytryptamine (5-HT) (via various 5-HT receptors) descending inhibition from brainstem to the spinal cord contribute to central sensitization and pain chronification [3,62]. ␮-Opioid receptors are integral part of this circuity too [62]. In our previous studies, we have shown that levetiracetam’s antihyperalgesia in an inflammatory pain model involves indirect activation of central and/or peripheral GABAA , ␣2 -adrenergic, 5-HT-ergic and ␮-opioidergic receptors [8,9]. Since inflammatory component contributes to the pathogenesis and/or maintenance of painful diabetic neuropathy [2,3,62] and both

A. Micov et al. / Pharmacological Research 97 (2015) 131–142

central and peripheral changes accompany neuropathy, it could be assumed that these mechanisms of levetiracetam-induced analgesia in an inflammatory pain model may also be relevant for its antinociception in diabetic neuropathic pain. As mentioned above, the synergism between levetiracetam and ibuprofen/aspirin/paracetamol is most probably due to pharmacodynamic interactions and could be explained by the involvement of multiple different targets in their antinociception. Combination analgesics that impact multiple targets are of particular benefit in treating complex pain conditions involving multiple causes, such as painful diabetic neuropathy. The pharmacokinetic interactions between levetiracetam and ibuprofen/aspirin/paracetamol were not within the scope of our study, but we assume that they are unlikely to occur for two reasons: (1) levetiracetam is almost devoid of pharmacokinetic interactions [15,16], and (2) we observed an unchanged duration of the antinociceptive effects when drugs are administered in combinations (prolongation of the effects of drug combinations is likely to be expected in the case of pharmacokinetic interactions which would result in enhancement of the pharmacological effect). Taken together with literature data, our findings most probably rule out pharmacokinetic interactions between levetiracetam and ibuprofen/aspirin/paracetamol. The current approach to management of painful diabetic neuropathy proposes monotherapy as a first therapeutic option [3,62,63]. Our results indicate that levetiracetam as well as ibuprofen/aspirin/paracetamol might be efficacious in some patients suffering from painful diabetic neuropathy. Evidence from clinical trials indicates that monotherapy with first-line drugs for treating painful diabetic neuropathy (pregabalin and duloxetine; given at standard dose), provide substantial clinical pain relief in only about 40% of patients [63]. Therefore, combination therapy could be useful for patients who have inadequate pain control with monotherapy. In clinical practice most analgesic combinations are used empirically in treating painful symptoms in diabetic neuropathy. Results from preclinical studies showing efficacy of certain combinations of analgesics may serve as a basis for their potential clinical use. In this context, our results suggest that levetiracetam used in combination with ibuprofen or aspirin or paracetamol might achieve optimal analgesia. Additionally, multifold reduction of doses of single drugs could lower incidence and/or intensity of adverse effects of levetiracetam (somnolence, weakness, dizziness), aspirin/ibuprofen (gastrointestinal disturbances, bleeding), as well as, diminish the possibility of paracetamol overdose. Preclinical examination of the adverse effects of levetiracetam-analgesic combinations may have less predictive value than analgesic effects anticipation, but referring to the rotarod results it could be assumed that motor impairment and sedation are less likely to occur. Therefore, the examined combinations might have better efficacy, but also a better safety profile than the corresponding monotherapies. An additional advantage of levetiracetam-aspirin combination might be its usefulness for both relieving neuropathic pain and preventing cardiovascular events in diabetic patients, given that the doses of aspirin (used in combinations with levetiracetam) that provided significant analgesia in neuropathic animals (1–14 mg/kg) correlate with aspirin doses used in humans (75 mg daily dosage in humans corresponds to 13 mg/kg aspirin in mice) [64] for cardiovascular events prevention.

5. Conclusions Our study shows that anticonvulsant levetiracetam exerts synergistic interaction with ibuprofen/aspirin/paracetamol in a mouse model of painful diabetic neuropathy, with multifold reduction of

141

doses of single drugs. Therefore, the examined combinations might have a better efficacy and safety profile than the corresponding monotherapies. The present findings indicate that levetiracetamibuprofen, levetiracetam-aspirin, and levetiracetam-paracetamol combinations could be useful in the treatment of painful diabetic neuropathy and should be explored further in clinical trials. Conflicts of interest None of the authors have professional or financial relationships that could result in conflicts of interest related to work described in this manuscript. Acknowledgement Funding sources: This work was supported by the Serbian Ministry of Education, Science and Technological Development (24 Nemanjina St., Belgrade, Serbia), Grant 175045. References [1] N. Rudroju, D. Bansal, S.T. Talakokkula, K. Gudala, D. Hota, A. Bhansali, et al., Comparative efficacy and safety of six antidepressants and anticonvulsants in painful diabetic neuropathy: a network meta-analysis, Pain Phys. 16 (2013) E705–E714. [2] K.L. Farmer, C. Li, R.T. Dobrowsky, Diabetic peripheral neuropathy: should a chaperone accompany our therapeutic approach, Pharmacol. Rev. 64 (2012) 880–900. [3] S. Tesfaye, D. Selvarajah, Advances in the epidemiology, pathogenesis and management of diabetic peripheral neuropathy, Diabetes Metab. Res. Rev. 28 (Suppl. 1) (2012) 8–14. [4] I. Gilron, T.S. Jensen, A.H. Dickenson, Combination pharmacotherapy for management of chronic pain: from bench to bedside, Lancet Neurol. 12 (2013) 1084–1095. [5] D. Ziegler, Current concepts in the management of diabetic polyneuropathy, Curr. Diabetes Rev. 7 (2011) 208–220. [6] D. Ardid, Y. Lamberty, A. Alloui, M.A. Coudore-Civiale, H. Klitgaard, A. Eschalier, Antihyperalgesic effect of levetiracetam in neuropathic pain models in rats, Eur. J. Pharmacol. 473 (2003) 27–33. [7] M. Ozcan, A. Ayar, S. Canpolat, S. Kutlu, Antinociceptive efficacy of levetiracetam in a mice model for painful diabetic neuropathy, Acta Anaesthesiol. Scand. 52 (2008) 926–930. ´ B. Popovic, ´ R. Stepanovic-Petrovi ´ ´ The antihyperalgesic [8] A. Micov, M. Tomic, c, effect of levetiracetam in an inflammatory model of pain in rats: mechanism of action, Br. J. Pharmacol. 161 (2010) 384–392. ´ ´ A.M. Micov, M.A. Tomic, ´ N.D. Ugreˇsic, ´ The local [9] R.M. Stepanovic-Petrovi c, peripheral antihyperalgesic effect of levetiracetam and its mechanism of action in an inflammatory pain model, Anesth. Analg. 115 (2012) 1457–1466. ´ ´ A.M. Micov, M.A. Tomic, ´ J.M. Kovaˇcevic, ´ B.D. Boˇskovic, ´ [10] R.M. Stepanovic-Petrovi c, Antihyperalgesic/antinociceptive effects of ceftriaxone and its synergistic interactions with different analgesics in inflammatory pain in rodents, Anesthesiology 120 (2014) 737–750. ´ A.M. Micov, R.M. Stepanovic-Petrovi ´ ´ Levetiracetam interacts [11] M.A. Tomic, c, synergistically with nonsteroidal analgesics and caffeine to produce antihyperalgesia in rats, J. Pain 14 (2013) 1371–1382. [12] M. Falah, C. Madsen, J.V. Holbech, S.H. Sindrup, A randomized, placebocontrolled trial of levetiracetam in central pain in multiple sclerosis, Eur. J. Pain 16 (2012) 860–869. [13] S. Rossi, G. Mataluni, C. Codecà, S. Fiore, F. Buttari, A. Musella, et al., Effects of levetiracetam on chronic pain in multiple sclerosis: results of a pilot, randomized, placebo-controlled study, Eur. J. Neurol. 16 (2009) 360–366. [14] F. Brighina, A. Palermo, A. Aloisio, M. Francolini, G. Giglia, B. Fierro, Levetiracetam in the prophylaxis of migraine with aura: a 6-month open-label study, Clin. Neuropharmacol. 29 (2006) 338–342. [15] T. De Smedt, R. Raedt, K. Vonck, P. Boon, Levetiracetam: the profile of a novel anticonvulsant drug-part I: preclinical data, CNS Drug Rev. 13 (2007) 43–56. [16] P.N. Patsalos, Clinical pharmacokinetics of levetiracetam, Clin. Pharmacokinet. 43 (2004) 707–724. [17] J. Guindon, P. Beaulieu, Antihyperalgesic effects of local injections of anandamide, ibuprofen, rofecoxib and their combinations in a model of neuropathic pain, Neuropharmacology 50 (2006) 814–823. [18] Y. Wang, X. Zhang, Q.L. Guo, W.Y. Zou, C.S. Huang, J.Q. Yan, Cyclooxygenase inhibitors suppress the expression of P2X(3) receptors in the DRG and attenuate hyperalgesia following chronic constriction injury in rats, Neurosci. Lett. 478 (2010) 77–81. [19] P. McGettigan, D. Henry, Cardiovascular risk with non-steroidal antiinflammatory drugs: systematic review of population-based controlled observational studies, PLoS Med. 8 (2011) e1001098.

142

A. Micov et al. / Pharmacological Research 97 (2015) 131–142

[20] L.K. Bhatt, A. Veeranjaneyulu, Minocycline with aspirin: a therapeutic approach in the treatment of diabetic neuropathy, Neurol. Sci. 31 (2010) 705–716. [21] C. Mallet, A. Eschalier, Pharmacology and mechanism of action of acetaminophen, In: P. Beaulieu, D. Lussier, F. Porreca, A.H. Dickenson (Eds.), Pharmacology of Pain, IASP Press, Seattle, 2010, pp. 65–85. [22] Y.H. Gong, X.R. Yu, H.L. Liu, N. Yang, P.P. Zuo, Y.G. Huang, Antinociceptive effects of combination of tramadol and acetaminophen on painful diabetic neuropathy in streptozotocin-induced diabetic rats, Acta Anaesthesiol. Taiwan 49 (2011) 16–20. [23] R. Freeman, P. Raskin, D.J. Hewitt, G.J. Vorsanger, D.M. Jordan, J. Xiang et al., CAPSS-237 Study Group, Randomized study of tramadol/acetaminophen versus placebo in painful diabetic peripheral neuropathy, Curr. Med. Res. Opin. 23 (2007) 147–161. [24] S.H. Ko, H.S. Kwon, J.M. Yu, S.H. Baik, I.B. Park, J.H. Lee, et al., Comparison of the efficacy and safety of tramadol/acetaminophen combination therapy and gabapentin in the treatment of painful diabetic neuropathy, Diabet. Med. 27 (2010) 1033–1040. [25] A.T. Hama, J. Sagen, Cannabinoid receptor-mediated antinociception with acetaminophen drug combinations in rats with neuropathic spinal cord injury pain, Neuropharmacology 58 (2010) 758–766. ´ S.M. Vuˇckovic, ´ R.M. Stepanovic-Petrovi ´ ´ A.M. Micov, N.D. [26] M.A. Tomic, c, ˇ Prostran, et al., Analysis of the antinociceptive interactions in ´ M.S. Ugreˇsic, two-drug combinations of gabapentin, oxcarbazepine and amitriptyline in streptozotocin-induced diabetic mice, Eur. J. Pharmacol. 628 (2010) 75–82. [27] M. Ohsawa, M. Aasato, S.S. Hayashi, J. Kamei, RhoA/Rho kinase pathway contributes to the pathogenesis of thermal hyperalgesia in diabetic mice, Pain 152 (2011) 114–122. [28] R.J. Tallarida, R.B. Murray, Manual of Pharmacologic Calculations with Computer Programs, Springer Verlag, New York, Berlin, Heidelberg, London, Paris, Tokyo, 1986. [29] R.J. Tallarida, Drug Synergism and Dose-Effect Data Analysis, Chapman & Hall/CRC, Boca Raton, London, New York, Washington, 2000. [30] R.J. Tallarida, D.J. Stone Jr, R.B. Raffa, Efficient designs for studying synergistic drug combinations, Life Sci. 61 (1997) PL 417–425. [31] R.J. Tallarida, The interaction index: a measure of drug synergism, Pain 98 (2002) 163–168. [32] T.L. Yaksh, R.Y. Noueihed, P.A. Durant, Studies of the pharmacology and pathology of intrathecally administered 4-anilinopiperidine analogues and morphine in the rat and cat, Anesthesiology 64 (1986) 54–66. [33] C. Gelegen, T.C. Gent, V. Ferretti, Z. Zhang, R. Yustos, F. Lan, et al., Staying awake—a genetic region that hinders ␣2 adrenergic receptor agonist-induced sleep, Eur. J. Neurosci. 40 (2014) 2311–2319. [34] I.G. Obrosova, Diabetic painful and insensate neuropathy: pathogenesis and potential treatments, Neurotherapeutics 6 (2009) 638–647. ˇ [35] K. Rrstavik, B. Namer, R. Schmidt, M. Schmelz, M. Hilliges, C. Weidner, et al., Abnormal function of C-fibers in patients with diabetic neuropathy, J. Neurosci. 26 (2006) 11287–11294. [36] Y.A. Kolesnikov, R.S. Wilson, G.W. Pasternak, The synergistic analgesic interactions between hydrocodone and ibuprofen, Anesth. Analg. 97 (2003) 1721–1723. [37] C. Courteix, M. Bardin, C. Chantelauze, J. Lavarenne, A. Eschalier, Study of the sensitivity of the diabetes-induced pain model in rats to a range of analgesics, Pain 57 (1994) 153–160. [38] S. Zelcer, Y. Kolesnikov, I. Kovalyshyn, D.A. Pasternak, G.W. Pasternak, Selective potentiation of opioid analgesia by nonsteroidal anti-inflammatory drugs, Brain 1040 (2005) 151–156. [39] A.A. Assi, The influence of divalent cations on the analgesic effect of opioid and non-opioid drugs, Pharmacol. Res. 43 (2001) 521–529. [40] Z.F. Wang, Q. Li, S.B. Liu, W.L. Mi, S. Hu, J. Zhao, et al., Aspirin-triggered Lipoxin A4 attenuates mechanical allodynia in association with inhibiting spinal JAK2/STAT3 signaling in neuropathic pain in rats, Neuroscience 273 (2014) 65–78. [41] J.V. Holbech, M. Otto, F.W. Bach, T.S. Jensen, S.H. Sindrup, The anticonvulsant levetiracetam for the treatment of pain in polyneuropathy: a randomized, placebo-controlled, cross-over trial, Eur. J. Pain 15 (2011) 608–614. [42] G. Hussain, S.A. Rizvi, S. Singhal, M. Zubair, J. Ahmad, Serum levels of TNF-␣ in peripheral neuropathy patients and its correlation with nerve conduction velocity in type 2 diabetes mellitus, Diabetes Metab. Syndr. 7 (2013) 238–242.

[43] A.P. Kellogg, H.T. Cheng, R. Pop-Busui, Cyclooxygenase-2 pathway as a potential therapeutic target in diabetic peripheral neuropathy, Curr. Drug Targets 9 (2008) 68–76. [44] J. Satoh, S. Yagihashi, T. Toyota, The possible role of tumor necrosis factor-alpha in diabetic polyneuropathy, Exp. Diabesity Res. 4 (2003) 65–71. [45] S. Rai, P.K. Kamat, C. Nath, R. Shukla, Glial activation and post-synaptic neurotoxicity: the key events in Streptozotocin (ICV) induced memory impairment in rats, Pharmacol. Biochem. Behav. 117 (2014) 104–117. [46] P. Rao, E.E. Knaus, Evolution of nonsteroidal anti-inflammatory drugs (NSAIDs): cyclooxygenase (COX) inhibition and beyond, J. Pharm. Pharm. Sci. 11 (2008) 81s–110s. [47] B. Hinz, O. Cheremina, K. Brune, Acetaminophen (paracetamol) is a selective cyclooxygenase-2 inhibitor in man, FASEB J. 22 (2008) 383–390. [48] B. Kis, J.A. Snipes, S.A. Simandle, D.W. Busija, Acetaminophen-sensitive prostaglandin production in rat cerebral endothelial cells, Am. J. Physiol. Regul. Integr. Comp. Physiol. 288 (2005) R897–R902. [49] E. Redondo-Castro, X. Navarro, Chronic ibuprofen administration reduces neuropathic pain but does not exert neuroprotection after spinal cord injury in adult rats, Exp. Neurol. 252 (2014) 95–103. [50] F. Wang, H. Zhai, L. Huang, H. Li, Y. Xu, X. Qiao, et al., Aspirin protects dopaminergic neurons against lipopolysaccharide-induced neurotoxicity in primary midbrain cultures, J. Mol. Neurosci. 46 (2012) 153–161. [51] M.B. Graeber, M.J. Christie, Multiple mechanisms of microglia: a gatekeeper’s contribution to pain states, Exp. Neurol. 234 (2012) 255–261. [52] S.H. Kim, J.K. Kwon, Y.B. Kwon, Pain modality and spinal glia expression by streptozotocin induced diabetic peripheral neuropathy in rats, Lab. Anim. Res. 28 (2012) 131–136. [53] M. Dani, J. Guindon, C. Lambert, P. Beaulieu, The local antinociceptive effects of paracetamol in neuropathic pain are mediated by cannabinoid receptors, Eur. J. Pharmacol. 573 (2007) 214–215. [54] A. Ulugol, C. Oltulu, O. Gunduz, C. Citak, R. Carrara, M.R. Shaqaqi, et al., 5-HT7 receptor activation attenuates thermal hyperalgesia in streptozocin-induced diabetic mice, Pharmacol. Biochem. Behav. 102 (2012) 344–348. [55] A. Dogrul, M. Seyrek, E.O. Akgul, T. Cayci, S. Kahraman, H. Bolay, Systemic paracetamol-induced analgesic and antihyperalgesic effects through activation of descending serotonergic pathways involving spinal 5-HT7 , receptors, Eur. J. Pharmacol. 677 (2012) 93–101. [56] R. Björkman, K.M. Hallman, J. Hedner, T. Hedner, M. Henning, Acetaminophen blocks spinal hyperalgesia induced by NMDA and substance P, Pain 57 (1994) 259–264. [57] W. Zhang, Y. Murakawa, K.M. Wozniak, B. Slusher, A.A. Sima, The preventive and therapeutic effects of GCPII (NAALADase) inhibition on painful and sensory diabetic neuropathy, J. Neurol. Sci. 247 (2006) 217–223. [58] E.A. Lukyanetz, V.M. Shkryl, P.G. Kostyuk, Selective blockade of N-type calcium channels by levetiracetam, Epilepsia 43 (2002) 9–18. [59] H. Saegusa, T. Kurihara, S. Zong, A. Kazuno, Y. Matsuda, T. Nonaka, et al., Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N-type Ca2+ channel, EMBO J. 20 (2001) 2349–2356. [60] A. Kolosov, C.S. Goodchild, I.C.N.S.B004 Cooke, (Leconotide) causes antihyperalgesia without side effects when given intravenously: a comparison with ziconotide in a rat model of diabetic neuropathic pain, Pain Med. 11 (2010) 262–273. [61] M. Ozcan, A. Ayar, Modulation of action potential and calcium signaling by levetiracetam in rat sensory neurons, J. Recept. Signal. Transduct. Res. 32 (2012) 156–162. [62] S. Tesfaye, A.J. Boulton, A.H. Dickenson, Mechanisms and management of diabetic painful distal symmetrical polyneuropathy, Diab. Care 36 (2013) 2456–2465. [63] S. Tesfaye, S. Wilhelm, A. Lledo, A. Schacht, T. Tölle, D. Bouhassira, et al., Duloxetine and pregabalin: high-dose monotherapy or their combination? The COMBO-DN study–a multinational, randomized, double-blind, parallel-group study in patients with diabetic peripheral neuropathic pain, Pain 154 (2013) 2616–2625. [64] S. Reagan-Shaw, M. Nihal, N. Ahmad, Dose translation from animal to human studies revisited, FASEB J. 22 (2008) 659–661.