Year : 2009 | Volume
: 20 | Issue : 4 | Page : 418--422
Evaluation of gastric tolerability, antinociceptive and antiinflammatory activity of combination NSAIDs in rats
Bhupinder Singh Kalra, Shalini, Sonali Chaturvedi, Vandana Tayal, Usha Gupta
Department of Pharmacology, Maulana Azad Medical College, Bahadur Shah Zafar Marg, New Delhi - 110 002, India
Bhupinder Singh Kalra
Department of Pharmacology, Maulana Azad Medical College, Bahadur Shah Zafar Marg, New Delhi - 110 002
Background : Non-steroidal antiinflammatory drugs (NSAIDs) are one of the most commonly prescribed drugs in clinical practice. Presently, several varieties of fixed dose combinations (FDCs) of NSAIDs are available over the counter and are being prescribed too. There is paucity of literature regarding comparative efficacy of these combinations against their individual component. Various clinical studies have documented increased incidence of gastric ulcerations with usage of more than one NSAID simultaneously.
Objectives : To study gastric tolerability, antinociceptive and antiinflammatory activity of combination NSAIDs in rats.
Materials and Methods : Gastric tolerability of different NSAIDs was observed after administration of drugs for 7 days orally. On 7 th day, 4 h after drug administration, rats were sacrificed and stomach mucosa was examined for ulcerations. Analgesic or antinociceptive activity of single and combination NSAIDs was evaluated using Writhing test model. For induction of writhing, 4% normal saline (hypertonic saline) was injected (0.1 ml/10 gm) intraperitoneally. Evaluation of antiinflammatory activity for FDCs of NSAIDs was done by using rat paw edema model with the aid of plethysmometer. Paw edema was induced by injecting 0.1 ml of 1% formalin in sub-planter region of hind paw.
Results : Analgesic activity was found to be enhanced or significant only in the group pretreated with combination of nimesulide with ibuprofen as compared to ibuprofen-alone group (P = 0.01). Decrease in mean paw edema (antiinflammatory activity) was not significant in rats pretreated with combination NSAIDs as compared to NSAID-alone group. Mean gastric ulcer index was significant in groups pretreated with diclofenac alone (P = 0.03) and in combination groups of nimesulide with diclofenac and ibuprofen with paracetamol as compared to control (P = 0.03, P = 0.007).
Conclusion : Addition of ibuprofen to paracetamol and combining diclofenac to nimesulide, significantly increased severity of gastric ulcerations. Fixed dose combination does not possess additional analgesic activity over their individual components, only exception being combination of nimesulide with ibuprofen, which has additional analgesic activity over ibuprofen alone, and this combination was not found to be ulcerogenic. Antiinflammatory activity of ibuprofen, paracetamol and nimesulide was significantly enhanced after addition of diclofenac.
|How to cite this article:|
Kalra BS, Shalini, Chaturvedi S, Tayal V, Gupta U. Evaluation of gastric tolerability, antinociceptive and antiinflammatory activity of combination NSAIDs in rats.Indian J Dent Res 2009;20:418-422
|How to cite this URL:|
Kalra BS, Shalini, Chaturvedi S, Tayal V, Gupta U. Evaluation of gastric tolerability, antinociceptive and antiinflammatory activity of combination NSAIDs in rats. Indian J Dent Res [serial online] 2009 [cited 2020 Feb 25 ];20:418-422
Available from: http://www.ijdr.in/text.asp?2009/20/4/418/59439
Nonsteroidal antiinflammatory drugs (NSAIDs) are among the most commonly prescribed drugs world over. They account for big market share in the pharmaceutical industry. Nonsteroidal antiinflammatory drugs are commonly used for treatment of pain and inflammation. They are prescribed in both acute and chronic conditions like dental pain, rheumatoid arthritis, osteoarthritis etc. Nonsteroidal antiinflammatory drugs use can range from one day to few months or even years depending upon disease condition.
Nonsteroidal antiinflammatory drugs use is frequently limited by gastrointestinal side effects, ranging from dyspepsia to life-threatening bleeding from ulceration.  It is believed that NSAIDs by inhibiting COX pathway causes inhibition of prostaglandins synthesis, which are responsible for maintaining gastric mucosal integrity.  Upper gastrointestinal endoscopy studies have shown a 15-30% prevalence of ulcers in the stomach of patients taking NSAIDs regularly.  According to prospective data from Arthritis, Rheumatism, and Aging Medical Information System (ARAMIS), 13 of every 1000 patients with rheumatoid arthritis, who take NSAIDs for one year have a serious gastrointestinal complication. ,
Fixed dose combination (FDC) of NSAIDs like nimesulide and diclofenac, ibuprofen and Paracetamol are being widely prescribed for painful conditions. These combinations are being extensively marketed by pharmaceutical industry claiming higher efficacy of combinations over single NSAID. Due to vigorous promotional activities, prescribers are driven towards prescribing such products in many clinical situations. Several other over-the-counter (OTC) combinations (e.g. nimesulide with paracetamol, diclofenac with paracetamol and ibuprofen with paracetamol) have also gained much wider acceptance and many people are increasingly using them after experiencing any sort of pain, fever or inflammation. In a qualitative review comparing effect of paracetamol, NSAIDs or their combinations in postoperative pain management, it was observed that addition of an NSAID to paracetamol may confer additional analgesic efficacy compared with paracetamol alone. 
Although, there are a few studies suggesting additional analgesic activity with FDCs, none of these studies evaluated risk of upper gastrointestinal complications associated with fixed dose NSAID combinations. At the same time, it has been observed that users of multiple NSAIDs simultaneously have about ten times increased risk of developing upper gastrointestinal complications (UGIC) compared with non-users. 
There is no safety data available showing safety of combination analgesics when administered over a period of time. These combinations also burden the patient with extra cost. Keeping this in view, the present study was designed to study antiinflammatory and antinociceptive activity and gastric tolerability of NSAIDs alone and in combinations in albino rats.
Materials and Methods
This study was approved by institutional animal ethical committee and committee for prevention of Cruelty and Supervision of Experiment on Animals (CPCSEA).
Animals: Wistar rats weighing 150-200 gm of body weight were used. Animals were procured from central animal house and were housed in air-conditioned environment. A gap of one week was kept for acclimatization of animals. They were provided with normal rat food pellet diet with water ad libitum.
Rats were divided into 11 groups with 8 rats in each group [Table 1]. All the rats received drugs through oral route in the dosage as mentioned in [Table 1].
Antiinflammatory activity: Paw edema model
On the morning of experiment, rats were weighed and baseline paw volume was measured with the aid of plethysmometer.  To ensure uniformity, lateral malleolus of left hind limb was marked in all animals so that same length of paw is dipped in fluid each time. This was followed by oral administration of drugs. After 45 min of drugs administration, animals were injected with 0.1 ml of 1% formalin in sub-planter region for inducing inflammation.  Paw volume was again measured after 3 h of sub-planter injection of 1% formalin. The paw volume and percent decrease in paw edema was compared between control group and drug-treated groups.
Analgesic activity: Writhing test
Animals were fasted overnight. They were administered with drugs 45 min prior to injecting 4% normal saline (hypertonic saline) intraperitoneally. , They were then observed for number of wriths for next 90 s. Writhing movements were characterized by specific abdominal contractions accompanied by elongation of body with arching of back, belly touching the ground and dragging of hind limbs. Number of wriths observed over 90 s period was recorded and percent inhibition of writhing was also calculated and compared among control and drug-treated groups.
Animals were administered with drugs orally once daily for a period of 7 days. In the control group, rats received normal saline for 7 days. On day 7, 4 h after drug administration animals were sacrificed. Each rat was subjected to midline abdominal incision, abdomen was opened and stomach was removed after ligating both esophageal and pylorus end. Incision was given in the stomach along greater curvature; mucosal surface was exposed and washed with normal saline.  It was then stretched and pinned on cork board. Mucosal surface was then examined for erosions and ulcerations. Severity of lesions was recorded according to following scale. 
0 = normal gray-colored mucosal surface. 0.5 = pink to red coloration of mucosal surface. 1 = spot ulcer. 1.5 = hemorrhagic streak. 2 = number of ulcers less than five. 3 = number of ulcers more than five. 4 = ulcers with bleeding. Ulcer index was calculated by adding the total number of ulcers plus the severity score. Ulcer index were recorded and compared among drug-treated and control group.
Results of writhing test, paw edema model and ulcer index are expressed as mean ± standard error of mean. Mann-Whitney statistical test was used for analysis of writhing, paw edema and ulcer index outcomes. A probability value of less than 0.05 (P Analgesic activity: Writhing
Writhing was observed in all control animals with 4% normal saline. The mean count of wriths in control group was 6.37 ± 0.42. Pretreatment of rats with NSAIDs in all the groups showed statistically significant inhibition of wriths as compared to control [Table 2]. Decrease in mean count of wriths in nimesulide, diclofenac, ibuprofen and paracetamol pretreated groups when compared with their respective combination groups was not statistically significant, except in the group administered with combination of nimesulide and ibuprofen, where significant inhibition of writhing was observed as compared to ibuprofen alone group (3.75 ± 0.45 to 2.38 ± 0.26, P = 0.01) [Table 2]. In the group pretreated with combination of nimesulide and ibuprofen, 63% inhibition in writhing was observed, which was highest among all groups.
Antiinflammatory activity : Paw edema
Marked paw edema was produced in rats with sub-planter injection of 0.1 ml of 1% formalin. Mean paw edema in the control rats was 502.4 ± 131.5 cu. mm [Table 2]. Pretreatment of rats with diclofenac alone resulted in significant decrease in paw edema as compared to control (502.4 ± 131.5 to 175.84 ± 103.5, P = 0.03). However, in the groups treated with nimesulide, paracetamol and ibuprofen alone, no significant decrease in edema was observed as compared to control group [Table 2]. Pretreatment of rats with combinations of nimesulide with ibuprofen, nimesulide with diclofenac, diclofenac with paracetamol and ibuprofen with diclofenac significantly inhibited paw edema as compared to control group [Table 2]. Decrease in mean paw edema was not significant in rats pretreated with combination NSAIDs as compared to NSAID alone group. In the group administered with combination of nimesulide and diclofenac, 75% decrease in paw edema was observed as compared to control, which was highest among all groups [Table 2].
Gastric tolerability: Ulcer index
Mean ulcer index in control rats was 0.18 ± 0.09 [Table 3] and [Figure 1]. Rats pretreated with diclofenac alone showed significant increase in mean ulcer index as compared to control (0.18 ± 0.09 to 0.75 ± 0.32, P = 0.03) [Figure 2], whereas rats pretreated with nimesulide, ibuprofen and paracetamol did not show significant change in mean ulcer index in comparison to the control [Table 3]. Mean change in ulcer index was significant in rats pretreated with combination of nimesulide and diclofenac as compared to control 0.18 ± 0.09 to 0.75 ± 0.32, P = 0.03, [Figure 2]. Also, in the group administered with ibuprofen and paracetamol, mean increase in ulcer index was highly significant as compared to control (0.18 ± 0.09 to 1.18 ± 0.45, P = 0.007) [Table 3]. No significant change in mean ulcer index was observed when single NSAID groups were compared with their respective combination groups except in rats pretreated with combination of ibuprofen and paracetamol, where significant increase in mean ulcer index was seen as compared to paracetamol alone group (0.25 ± 0.09 to 1.18 ± 0.45, P = 0.01). In the groups administered with combination of ibuprofen with diclofenac and also animals pretreated with ibuprofen alone showed raised ulcer index, but it was not statistically significant [Table 3].
It has been argued that FDCs should only be used if each component is necessary for the desired effect and if the advantages outweigh the added risks of using two or more drugs. Fixed dose combination of NSAIDs are being aggressively promoted by pharmaceutical industry, which do lead to excessive prescribing too. There is a lack of evidence regarding superior efficacy of FDC over each of its individual component NSAID. Also, FDCs are more likely to be useful when the incidence of adverse reactions is not increased or remains leveled to that of its individual component.
Very few clinical studies have demonstrated that combination of NSAIDs increases analgesic activity. ,,,, In our study, we observed increase in analgesic activity; however, this increase was not statistically significant in comparison to their individual component NSAID. Out of all FDC studied, only combination of nimesulide and ibuprofen showed significant increase in analgesic activity over ibuprofen alone.
In our study, diclofenac showed significant inhibition of paw edema (65%) in rat paw edema model, corroborating the findings of earlier studies and diclofenac happens to be most potent antiinflammatory drug among all NSAIDs that we studied. We observed that, when nimesulide was co-administered with diclofenac, antiinflammatory activity of diclofenac further increased by 10% (65 to 75%), but this increase was not significant in comparison to diclofenac alone. We found that combinations of nimesulide with paracetamol and ibuprofen with paracetamol lack significant antiinflammatory activity in rat paw edema model. On comparing the antiinflammatory activity of single NSAIDs with their respective combination, we observed no significant increase in antiinflammatory activity indicating no additional advantage of combining two NSAIDs.
It has been documented that patients receiving more than one NSAID are more likely to develop gastric ulcerations.  We also observed similar findings in our study that rats administered with diclofenac for 7 days had significant increase in gastric ulcer index. Rats pretreated with nimesulide, ibuprofen and paracetamol did not show increase in incidence of gastric ulcerations indicating diclofenac to be most ulcerogenic among NSAIDs studied. Among the combinations, nimesulide with diclofenac and ibuprofen with paracetamol were observed to be ulcerogenic. We also observed that addition of ibuprofen to paracetamol is ulcerogenic but paracetamol or ibuprofen in isolation is not ulcerogenic. Fixed dose combination of nimesulide and ibuprofen, nimesulide and paracetamol, diclofenac and ibuprofen, diclofenac and paracetamol were not found to be ulcerogenic.
As NSAIDs are associated with severe drug-induced gastric toxicity, and other adverse effects, the use of these drugs in combinations would pose greater risk to the patient particularly the elderly who in most cases suffer from chronic painful and inflammatory conditions and often require prolonged administration of analgesic and antiinflammatory agents.  Moreover, over the counter availability and non-prescription use of FDC further increases its consumption. The evidence on efficacy, which have so far been gathered has not yet justified their usefulness.
We observed that FDC does not possess additional analgesic activity over their individual components, only exception being combination of nimesulide with ibuprofen, which has additional analgesic activity over ibuprofen alone and this combination was not found to be ulcerogenic. We also observed that by combining diclofenac with ibuprofen, paracetamol and nimesulide led to significant increase in their antiinflammatory activity. More evidence and trials are needed for evaluation of additional analgesic antiinflammatory efficacy of NSAIDs combinations along with monitoring of adverse reactions to justify their utility.
We are thankful to Win Medicare, Delhi for providing diclofenac powder in pure form, Indoco Remedies Ltd for paracetamol and ibuprofen powder, Panacea Biotec Ltd for providing us gift sample of nimesulide powder. We are thankful to Ridhi Gupta for helping us out with research.
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