Evaluation of Antinociceptive Effect of Pregabalin in Mice and Its Combination with Tramadol Using Tail Flick Test

Iranian Journal of Pharmaceutical Research (2013), 12 (3): 483-493 Copyright © 2013 by School of Pharmacy Received: April 2012 Shaheed Beheshti University of Medical Sciences and Health Services Accepted: December 2012 Original Article Evaluation of Antinociceptive Effect of Pregabalin in Mice and its Combination with Tramadol using Tail Flick Test Fariborz Keyhanfara, Manzumeh Shamsi Meymandia,b*, Gholamreza Sepehrib, Ramin Rastegaryanzadehb and Gioia Heravib aPharmacology Department., Iran University of Medical Sciences, Tehran, Iran. bKerman Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran. Abstract The development of combination therapy is a coherent approach in severe pain treatment. The present study investigated the antinociceptive effect of pregabalin alone and in combination with tramadol in acute pain modeling. Therefore, three groups of male mice received either pregabalin (1 to 400 mg/Kg), tramadol (10 to 80 mg/Kg) or their combination intraperitoneally. Then latency time, maximum possible effect (%MPE) and area under curve (AUC) were calculated in tail flick test. The antinociceptive indexes were significantly increasedin10, 100 and 200 mg/kg ofpregabalin while tramadol showed dose-dependentantinociception (effective dose 50% was 54 to 79 mg/Kg). The antinociceptive effect of 100 mg/Kg of pregabalin (%MPE = 35±4%) was similar to that of 50 mg/Kg of tramadol. The combination of non- analgesic doses (10 mg/Kg) of tramadol and pregabalin did not increase %MPE and AUC, but the co-administration of 30 mg/Kg of tramadol with pregabalin (10 mg/Kg) increased all antinociceptive indexes significantly compared to the controls and with each drug alone. In conclusion, pregabalin showed a comparable antinociceptive effect to tramadol. The increase in analgesic effect was observed after the combination of low analgesic doses of tramadol with pregabalin, while the combination of non-analgesic doses of each drug reversed the interaction to antagonism. Therefore to increase the analgesic effect in pain management, more attention should be paid to respecting right proportion of drug combination. Further studies that specify the mechanism(s) and statement of interaction are needed to expand these findings to clinical applications. Keywords: Pregabalin; Tramadol; Combination; Antinociception; Tail flick. Introduction Pregabalin known as CI-1008 or S(+)-3- isobutylgaba is a novel anticonvulsant of second The development of effective analgesics with generation of gabapentinoids that exerts analgesic fewer side effects and appropriate combination effect ofits own or as adjuvant in different models therapy is one of the most important objectives of pain (1-3). Pregabalin has been proposed of drug researches. Therefore, new drug classes in particular for pain pharmacotherapy of and combination therapy have been proposed in difficult treating syndromes (4-5) and in clinical pain treatment. neuropathic pain treatment (1,4-6). Pregabalin as an adjuvant interacts synergistically with * Corresponding author: naproxen to reverse hyperalgesia (7); it increases E-mail: [email protected] the efficacy of celecoxib in the treatment of Keyhanfar F et al. / IJPR (2013), 12 (3): 483-493 low back pain (8) and reduces pain and opioid was conformed to the guidelines on the study consumption after surgery (1,9-10).Tramadol of pain in conscious animals established by the as an atypical opioid is another analgesic used institutional guidelines. Ethical approval for this widely in the treatment of moderate to severe study (N° 90/141) was provided by local Ethical pain. Unlike typical opioids, tramadol does Committee of Kerman University of Medical not produce significant respiratory depression, Sciences according to the Guide for the Use of constipation and sedation. Moreover, it has low Laboratory Animals. potential for dependency, tolerance and drug abuse. However, tramadol may cause nausea, Drugs vomiting and tiredness (11-12). Pregabalin, The drugs used were: Pregabalin (Hetero despite of its high efficacy in neuropathic pain, Drugs Limited, India) and Tramadol (Kamud shows minor side effects such as constipation, Drugs, India). All drugs were freshly dissolved in transient dizziness, visual disturbance and normal saline and were injected at volume of 0.1 euphoria (4-6, 10). mL/10g body weight of mice intraperitoneally Furthermore, owing to the favorable (IP). pharmacokinetic parameters, combination of tramadol and pregabalin can be considered as Antinociception measurement desirable in pain treatment. These drugs have Tail flick test as an acute model of pain was common characteristics; both are orally active, used to assess the antinociceptive effect of the hold adequate volume of distribution and cross drugs by measuring the latency of response. blood brain barriers (10, 13-14). Tramadol protein Radiant heat was applied to the tail at 5-8 binding is 20%, while pregabalin does not bind cm from the tip using a tail flick apparatus to plasma proteins (10, 14). These parameters (PANLAB 7160, Spain). Tail flick latency time assign pregabalin as a drug holding minimal was measured as the time from the onset of the probability for drug interactions (10, 14).Thus it heat exposure to the withdrawal of the tail. The can be added to tramadol in cases of inadequate intensity of radiant heat was adjusted to yield the pain relief. Moreover, the other advantage of this baseline latencies of 2-4 sec. The heat stimulus combination is beneficial anticonvulsive effect was discontinued after 10 sec to avoid tissue of pregabalin that can cover a less frequent side damages (Cut off point =10 sec). effect of tramadol which occurred also with The animals were allowed to habituate to recommended doses (15). However, the efficacy laboratory surroundings before tail flick test and of this combination in acute pain treatment has the investigator was blind to the drug injected. not been reported yet. For each animal, baseline latency was obtained Thus, the current study was designed to as the mean of three measurements before establish antinociceptive effect of pregabalin the administration of each drug. The animals alone and in combination with tramadol in acute showing values of baseline latency time less model of pain using tail flick test in mice. than 2 or more than 4 sec were excluded from the study. The remnant mice were randomly Experimental assigned to experimental groups of eight mice. The latency times were determined in 15 min Animals intervals for 90 min from the time of drug or A total of 150 adult male NMRI mice weighing normal saline injection. 25 - 35 g were used. Animals were housed in 4 or Time course of antinociceptive response 5 mice per cage at a controlled temperature (22 ± of individual drug was constructed by plotting 2°C) and 12 h light-dark cycle with free access to the mean of latency times as a function of time. food and water. The experiments were carried out Antinociception was quantified as either tail during light cycle between 12:00 to 16:00 PM. flick latency time or percentage of maximal All animals were used only for one procedure possible effect (%MPE) or the area under curve and were humanely sacrificed under anesthesia (AUC) which includes both maximum effects after the completion of experiment. Animal care and duration of action (16). The %MPE was 484 Evaluation of Antinociceptive Effect of Pregabalin in Mice calculated as [(T1 - T0)/(T2 - T0)]×100. T0 and T1 Statistics were the tail flick latencies before and after drug Results were expressed as mean ± SEM of administration, and T2 was the cut off time. eight mice except for the control group (n = 12) in The AUC was calculated considering Tail combinational comparisons. Two way repeated Flick Latency time (TFL) from 15 to 90 post- measures of Analysis of Variance (ANOVA) was injection based on Trapezoid rules (16-17) used to assess the effect of dose, time and their as follows: AUC=15×TFL[(MIN 15)+(MIN interaction in time- response curve of each drug 30)+(MIN 45)+(MIN 60)… +(MIN 90)/2]. during seven consecutive (0, 15th, 30th, 45th, 60th ,…) measurements of latency times (time course). Procedure In this model, the dependent variable was latency In order to determine the antinociceptive effect time or %MPE and latency time before the of tramadol and pregabalin, the treated groups of injection was considered as covariate. One way eight mice received tramadol at the doses of 10, analysis of variance determined the differences 20, 40 and 80 mg/Kg and pregabalin at doses of of %MPE or AUC among independent treated 1, 10, 100, 200 and 400 mg/Kg (IP) respectively, groups and the post-hoc used was Tukey test. while control group received normal saline. The ED50 values with 95% confidence interval were chosen doses were aimed to cover the maximum calculated by linear regression using GraphPad possible range of antinociceptive effect. The dose Prism software version 5 (GraphPad Software range of tramadol was selected based on previous Inc., San Diego, CA, USA). Statistical analysis studies (18-20), while for pregabalin as a new was done using SPSS software version 11.5 compound in tail flick procedure,we referred to (Chicago, Illinois, USA). A value of p < 0.05 other animal models of pain assessment (21-22). was considered significant. However, the doses of more than 200 mg/Kg were never used before. Then based on latency Results times, the AUC and %MPE were calculated for each group. The %MPE at the 60th and 75th Antinociceptive effect of tramadol alone minutes were the highest ones in the course of The antinociceptive effect of tramadol study, which were used for further comparisons. showed increasing trend at all doses (10, 20, 40 To determine the ED50 (estimated dose to and 80 mg/Kg) and maximum antinociception produce 50% of antinociception), %MPE value was observed after injection of 80 mg/Kg at the th at the point of time when greatest antinociception 75 min (%MPE75= 88.4±4.8%). was observed were used to built dose-response Based on the results of repeated ANOVA curve.

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