The present study was carried out using male adult Wistar rats, (weighing 180 to 230 g) and Swiss albino mice (weighing 25 to 30 g) between 09.00 AM and 2.00 PM. The animals were kept in a separate temperature-regulated room in cages (six animals in each cage) with air-cooling and 12 hours light and dark cycle. They had free access to water and standard laboratory diet. All experiments were conducted on separate groups of animals and each animal was used only once and only in a single test to avoid interference between tests.

Rats and mice were randomly divided into groups with six members in each group. Control groups: (Group I) were administered normal saline at the dose of 10 mL/kg and diclofenac 100 mg/kg (Group II). Ibuprofen was administered at dosage of 10 mg/kg (Group III), 30 mg/kg (Group IV) and the dosage for pregabalin was 6 mg/kg (Group V), 12 (Group VI) mg/kg while the dosage for combination of ibuprofen and pregabalin was 10/6 mg/kg (Group VII) and 30/12 mg/kg (Group VIII). The combination ratio of pregabalin and ibuprofen was determined considering the maximum recommended human dose of pregabalin and ibuprofen, respectively. Diclofenac is often used as reference drug to compare other analgesic drugs for inflammatory pain. All the drugs were administered through oral route after dissolving them in normal saline. All chemicals and solvents (pregabalin, Ibuprofen, λ-carrageenan and saline) used were at least of analytical grade and were obtained commercially.

The study was approved by the Institutional Animal Ethics Committee. All experiments and animal care were as per the recommendations of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and Good Laboratory Practice (GLP) Guidelines, between January 2018 and April 2018 and no animal was sacrificed at the end of the study.

The analgesic activity was evaluated in mice by injecting 0.6% acetic acid (10 mL/kg) intraperitoneally (ip) into the lower right quadrant of the abdominal cavity at an angle of 30° and a depth of 5 mm [8]. The test drugs were administered orally to the mice 30 minutes before acetic acid injection. Immediately after injection, the mice were observed for writhing reflex, which is characterized by abdominal muscle contractions associated with inward outstretching of the hind limbs, a hind paw reflex, or whole body extension. The number of writhing reflexes was observed over a period of twenty min for each animal after acetic acid injection and the results were expressed as the mean±SD. Analgesic effect was quantified as the percent reduction in the number of writhes produced by each drug dose. For a given dose, percentage inhibition was calculated using the following formula: % inhibition={(Wc-Wt)×100}/Wc where, Wc=No. of writhes in control group, Wt=No. of writhes in test group.

Mice were placed into individual restraining cages leaving the tail hanging out freely. The animals were allowed to adapt to the cages for 30 minutes before testing. The lower 5 cm portion of the tail was marked. This part of the tail was immersed in a cup of freshly filled water of exactly 55°C. The reaction time was recorded with the help of a stopwatch. After each determination the tail was carefully dried. The reaction time was determined before (0 hour) and thereafter at one and two hour following oral administration of the test substances. The cut off time of the immersion was 15 seconds. Tail immersion latency was measured as the time between tail immersion and tail withdrawal [9]. Change in tail immersion latency, Dt (s), was calculated for each animal according to the formula {Dt (s)=post drug latency-pre drug latency} [10]. For each animal, the % maximum possible effect (%MPE) was calculated using the following formula: {(Post drug latency-Pre drug latency)/(15-Pre drug latency)} X100.

The analgesic activity was also assessed using hot plate method of Eddy and Leimbach in rats [11]. In this experiment, the hot-plate apparatus was maintained at 55±0.1°C. Rats were placed in an acrylic cylinder (20 cm in diameter) on the heated surface, and the time between placement and licking of their hind paws or jumping was recorded as the response latency. A 20 seconds cut-off was used to prevent tissue damage. The response latency was recorded before (0 min) and thereafter at 1 and 2 hour following oral administration of drugs. Analgesia was defined as prolongation of latency without licking or flicking of hind limb or jumping. Animals presenting with latencies higher than 15 seconds at 0 minute were excluded. For each animal, the % maximum possible effect (%MPE) was calculated using the following formula: {(Post drug latency-Pre drug latency)/ (20-Pre drug latency)} X100.

We followed the method adopted by Winter CA et al., subsequently modified by Singh H and Ghosh MN, for evaluating the acute anti-inflammatory activity in rats. Paw oedema was induced in the right hind paw of each rat by intra-plantar injection of 100 μl of 1% (suspension in saline) lambda carrageenan [12,13]. The paw volume of rats was measured by traditional mercury plethysmometer, before the injection of carrageen, 2.5 hours after the injection of carrageen (just before the administration of the test drugs and then again one and two hours after test drug administration. The degree of oedema induced was assessed by the volume of the right hind paw before and after carrageenan treatment, respectively. Mean baseline paw volume ranged from 1.3 to 1.4 mL among the different treatment groups. When measured 2.5 hours after the injection of carrageenan, mean paw volume ranged from 2.4 to 2.7 mL among the different treatment groups. Drug effects were expressed as a difference score in which the paw volume measured 2 hours after administration of the drug was subtracted from that determined immediately before (i.e., 2.5 hours after carrageenan). Negative values, therefore, represent a reduction in inflammation.

All data were expressed as mean±SD. Data were evaluated by means of one-way analysis of variance (ANOVA), post-hoc comparisons were made using Dunnett’s t-test or Tukey HSD post-hoc test, wherever appropriate to establish the statistical difference between groups. The criterion for statistical significance was fixed at p<0.05. Statistical analyses were accomplished using Graph Pad Prism 5 (Graph Pad Software, San Diego, CAUSA) for Windows (Microsoft Corporation, USA).

There was a significant diminution in the amount of writhing in all the groups tested when compared to the control group (vehicle 10 mL/kg) as shown in [Table/Fig-1]. Although the combination of ibuprofen plus pregabalin (both lower and higher groups) were effective in reducing the number of writhing, there was no significant difference in the amount of analgesia between these groups (60.6 and 73.2%) as compared to the standard analgesic diclofenac (62.4%) indicating possibly an analgesic ceiling effect.

In the tail-immersion test, the treatment with ibuprofen and pregabalin increased the latency to thermal stimulation at one hour after the treatment when compared to the control group (vehicle 10 mL/kg) and was maintained until two hours after treatment [Table/Fig-2].

The results of the analgesic effect of the drugs using hot plate method are presented in [Table/Fig-3]. The results revealed that there was no significant difference on the thermal stimulus in rats treated with normal saline (control group) throughout the observation period. There was a statistically significant increase in reaction time at all time points compared to baseline values in all treatment groups and was greatest for combination treated group at 1 hour after treatment. The experimentally-derived value of the maximum possible antinociceptive effect for low and high dose combination at 1 hour were 81.5±6.8 and 83.1±6.1% while at 2 hours they were 71.9±6.1 and 76.6±4.3% respectively.

Mean baseline paw volume ranged from 1.28 to 1.35 mL among the different treatment groups [Table/Fig-4]. When measured 2.5 hours after the injection of carrageenan, mean paw volume ranged from 2.5 to 2.7 mL among the different treatment groups. Drug effects are expressed as a difference score in which the paw volume measured 1 and 2 hours after administration of the drug was subtracted from that determined immediately before (i.e., 2.5 hours after carrageenan). Negative values, therefore, represent a reduction in inflammation. The results of λ-carrageenan-induced paw oedema indicated that the diclofenac, ibuprofen and the low or high dose combination significantly inhibited the development of paw oedema after 1 and 2 hours of treatment. A notable observation was that the degree of inhibition of paw oedema was greater in the combination groups (37.7 and 37.8%; 39.5 and 42%) than ibuprofen alone (21.3 and 21.8%; 23.6 and 25.2%) or the standard anti-inflammatory drug diclofenac (23.4 and 28%) while pregabalin alone exhibited negligible anti-inflammatory activity.

Despite the vast amount of research, pain management still relies on the “one drug fits all” model. The ability to develop new analgesic drugs has been disappointing and the commonly used analgesics today are derivatives of drug classes known to be analgesics for centuries or decades-NSAIDs, opioids, cannabinoids, and tricyclics. The present study carried out to evaluate the analgesic and anti-inflammatory activity of ibuprofen and pregabalin when used either alone or in combination demonstrated a potential antinociceptive activity, independent of the animal models of nociception or the nociceptive stimulus in a dose dependent manner with significant anti-inflammatory activity.

Recent animal studies suggested that pregabalin was found to possess analgesic activity in the formalin test on paw licking/late phase-corresponding to inflammatory pain with a central sensitization component [14]. Since persistent and uncontrolled pain may transform into chronic or neuropathic pain where peripheral and sensitization components play a significant role, use of combination of drugs with pregabalin as in present study may aid in the prevention of sensitization component in acute pain settings.

Ibuprofen has been shown to have analgesic and anti-inflammatory properties through central and peripheral blockade of COX-1 and COX-2 isoenzymes and can also have an effect through cox-independent pathways [15]. An isobolographic approach to characterize the nature of the interaction of gabapentin or pregabalin with naproxen in an animal model of inflammatory pain indicated that pregabalin was found to interact in a synergistic manner with naproxen [16]. The results of present study indicate that the combination is effective in inhibiting nociception at different levels of the central nervous system, as the hot-plate test predominantly measures supraspinal response to a painful stimulus while the tail-flick test primarily measures the spinal response. It is likely that the enhanced analgesic effect of the combination may be due to the activation of different antinociceptive pathways that are inhibited by these drugs. Taken together, findings in the present study suggested that the combination appears to be a good candidate for multimodal coverage of a wider spectrum of pain.

The present study displayed prominent analgesic effect and serves as a proof-of-principle study for considering the combination of ibuprofen and pregabalin as a lead for the development of new dual-action analgesic drugs. Isobolographic analysis is warranted to delineate whether the enhanced analgesic efficacy observed in this study was due to synergistic or supra-additive interactions of ibuprofen and pregabalin and efforts are under way to conduct the same. Further, more pharmacological and chemical studies are necessary in order to characterize the mechanism(s) responsible for the antinociceptive and anti-inflammatory action of this combination.