|Year : 2015 | Volume
| Issue : 2 | Page : 106-110
Evaluation of analgesic activity of allopurinol and febuxostat in experimental analgesic models in mice
Promod D Shankpal, JH Hotwani, Kunal A Chitnis, Dnyanesh S Tadke, Vijay R Kokani
Department of Pharmacology, Topiwala National Medical College and Nair Hospital, Mumbai Central, Mumbai, Maharashtra, India
|Date of Web Publication||15-Apr-2015|
Dr. Promod D Shankpal
213, 2nd Floor College Building, Nair Hospital Campus, Nair Hospital, Mumbai Central, Mumbai - 400 008, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Allopurinol and febuxostat are xanthine oxidase inhibitors which are used in the treatment of hyperuricemia and gout. Pain is one of the important symptoms in gout patients. The present study was to evaluate the analgesic activity of allopurinol and febuxostat in two analgesic models in mice. Materials and Methods: The analgesic activity of allopurinol (39 mg/kg) and febuxostat (15.6 mg/kg) was evaluated using central analgesic model of Eddy's hot plate and peripheral analgesic model of acetic acid induced writhing. Both drugs were compared with the positive control, pentazocine for a hot plate method and aspirin for the writhing method. Furthermore, both allopurinol and febuxostat were compared with each other. Results: Both allopurinol and febuxostat showed significant increase in reaction time at various time periods in hot plate method and also showed significant delay in onset of writhing as well as decrease in number of writhes in writhing method. As compared to positive control result, allopurinol and febuxostat result were lower. Febuxostat shows better analgesic activity as compared to that of allopurinol. Conclusion: Allopurinol and febuxostat exhibited analgesic activity in both central and peripheral models of pain.
Keywords: Allopurinol, Eddy′s hot plate method, febuxostat, nonsteroidal anti-inflammatory drugs, reactive oxygen species, writhing method
|How to cite this article:|
Shankpal PD, Hotwani J H, Chitnis KA, Tadke DS, Kokani VR. Evaluation of analgesic activity of allopurinol and febuxostat in experimental analgesic models in mice. Indian J Pain 2015;29:106-10
|How to cite this URL:|
Shankpal PD, Hotwani J H, Chitnis KA, Tadke DS, Kokani VR. Evaluation of analgesic activity of allopurinol and febuxostat in experimental analgesic models in mice. Indian J Pain [serial online] 2015 [cited 2020 Feb 17];29:106-10. Available from: http://www.indianjpain.org/text.asp?2015/29/2/106/153589
| Introduction|| |
Pain is an unpleasant sensation but a protective mechanism of our body. Analgesics are defined as substances, which decrease pain sensation by increasing pain threshold to external stimuli without altering consciousness.  Gout is a painful, metabolic disease that most often affects middle-aged to elderly men and postmenopausal women. It results from an increased body pool of urate with hyperuricemia. It typically is characterized by episodic acute and chronic arthritis caused by deposition of monosodium urate crystals in joints and connective tissue tophi.  The current management for acute gout include anti-inflammatory drugs such as non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and for chronic gout include urate-lowering treatment such as allopurinol, febuxostat, and probenecid.  NSAIDs and corticosteroids have many noted serious adverse effects. , Therefore, analgesic drugs lacking the side effect as an alternative to NSAIDs and corticosteroids in the management of acute gout is on demand.
Allopurinol is a potent inhibitor of xanthine oxidase. It inhibits the transformation of hypoxanthine to xanthine and uric acid thereby reducing uric acid formation and purine degradation.  This leads to an increase in the concentration of hypoxanthine. Hypoxanthine is converted to inosine, inosine monophosphate (IMP), and consequently to adenosine via the purine salvage pathway.  Adenosine is a purine nucleoside that occurs naturally in all cells of the body. Its role in the modulation of pain has been confirmed by receptor-mediated action In spinal, supraspinal sites, and peripheral sites.  It may be related to the inhibition of neuronal conduction by: Increase in K + conductance; decrease in substance P and glutamate;  and attenuation of basal as well as N-methyl-D-aspartate (NMDA) induced nitric oxide production which is an excitatory neurotransmitter. 
Above metabolic effects of allopurinol have been demonstrated in the central nervous system and the periphery in animal studies.  Allopurinol was demonstrated to have anticonvulsant and anti-psychotic effects acting through above mechanism in animals and humans. , Schmidt et al. (2008)  found that acute inhibition of xanthine oxidase with allopurinol produced a modest adenosine A1 receptor-mediated anti-nociceptive effect in common tests of chemical and thermal nociception in mice. Xanthine oxidase is also generate reactive oxygen species (ROS) by catalyzing the oxidative hydroxylation of purine substrates,  this ROS may also mediate acute pain transmission.  Thus, allopurinol may have analgesic activity due to adenosine modulation and inhibition of generation of ROS or both.
Studies in rodents have demonstrated that spinal or systemic administration of adenosine and adenosine analogs inhibits pain behaviors in response to noxious stimuli in a variety of test system, including acute nociceptive pain tests, inflammatory pain tests, peripheral and central neuropathic pain tests. , Raising extracellular adenosine by inhibiting adenosine kinase (AK) in animal models induced an analgesic effect. , Investigations performed in patients with acute perioperative pain or chronic neuropathic pain treated with intravenous adenosine or an intrathecal adenosine, have shown analgesic activity with longer duration. ,
Febuxostat is a newer orally administered, nonpurine, selective inhibitor of both oxidized and reduced form of xanthine oxidase approved for the management of chronic hyperuricemia in patients with gout.  There are no data available if it been evaluated for the analgesic activity. It may have an analgesic activity through the aforementioned mechanism.
Therefore, our study was aimed at evaluating the analgesic activity of both allopurinol and febuxostat in experimental analgesic models in mice, thus establishing the class effects of xanthine oxidase inhibitors.
| Materials and Methods|| |
Swiss albino mice of either sex, weighing 15-20 g were used for the experiment. The mice were obtained from Haffkine Biopharma Corporation and were kept 3 mice/cage at room temperature of 25°C, humidity of 60%, and 12 h light and day cycle is maintained. Study was performed after obtaining the approval from the Institutional Animal Ethics Committee. Both allopurinol and febuxostat were obtained from Zydus Cadila, and their suspension was made in 1% carboxymethyl cellulose (CMC) in distilled water for oral administration. Aspirin in pure powder form manufactured by Qualigen while pentazocine parental preparation manufactured by Biochem Pharmaceutical, both are obtained from the hospital pharmacy. A suspension of 1% CMC in distilled water was used as a vehicle. 0.6% acetic acid solution was administered intraperitoneally to induce writhing in mice.
Eddy's hot plate method
After 7 days of acclimatization, 24 animals were divided into four groups containing six mice each [Table 1]. Temperature was maintained at constant temperature of 55-56°C. The time taken by the animal for either licking the paw or withdrawal of the paws or jumping off the surface, whichever observed first, was taken as the end point. Reaction time was measured by stopwatch in 1/100 s increments and was taken before (baseline) and 30, 60, 90, and 120 min after the drug or vehicle administration in each animal. 
After a washout period of 7 days, the animals were used for writhing method [Table 2]. Groups of mice received the drug or the vehicle. After 60 min interval, 0.1 ml of a 0.6% solution of acetic acid was injected intraperitoneally to the mice to induce writhes. A writhe was indicated by a constriction that travels along the abdominal wall. The time of onset of writhing and the number of writhing in the following 10 min were recorded by stopwatch in 1/100 increment.
One-way analysis of variance (ANOVA) followed by Tukeys multiple comparison test and repeated measures ANOVA followed by Dunnett test were used as a statistical method. P < 0.05 was considered significant.
| Results|| |
Hot plate results
Both test drugs, allopurinol and febuxostat and positive control showed significant analgesic activity as compared to compared to vehicle control (P < 0.001). Both drugs showed increase in reaction time at various time intervals in hot plate method [Table 3], [Figure 1] and delay in onset of writhing as well as decrease in numbers of writhes in writhing method [Table 4] and [Table 5], [Figure 2] and [Figure 3]. The analgesic activity seen with pentazocine was significantly better as compared to test drugs (P < 0.001). The analgesic activity seen with febuxostat was better as compared to allopurinol (P < 0.05).
|Figure 1: Comparison of reaction time between 1% carboxymethyl cellulose, pentazocine, allopurinol, and febuxostat|
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|Figure 2: Comparison of time of onset of writhing between 1% carboxymethyl cellulose, aspirin, allopurinol, febuxostat|
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|Figure 3: comparison of number of abdominal contraction between 1% carboxymethyl cellulose, aspirin, allopurinol, febuxostat|
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|Table 4: Comparison of time of onset of writhing between the groups in seconds|
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|Table 5: Comparison of number of writhes (abdominal contractions) and percentage of inhibition compared to that of control, between the groups|
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| Discussion|| |
Gout is a painful, chronic inflammatory condition of the joints. Its prevalence has been increasing in recent years and is currently one of the most common types of inflammatory arthritis.  If not treated it may lead to chronic gouty arthropathy and deposition of urate crystals in soft tissues, forming tophi. Current standards for first-line treatment of acute attacks of gout include NSAIDs, colchicines, corticosteroids. Urate lowering treatment such as allopurinol, febuxostat, and probenecid are usually recommended after the acute attack has resolved. 
In our study, we have evaluated analgesic activity of allopurinol and febuxostat in experimental analgesic models in mice. Eddy's hot plate for central analgesic activity and acetic acid induced writhing test for peripheral analgesic activity, were used for evaluation of analgesic activity of allopurinol and febuxostat in our study. Pentazocine, an opioid agonist-antagonist was used as standard positive control for central hot plate test and aspirin was used as standard positive control for peripheral writhing test.
Our study showed that allopurinol and febuxostat have analgesic activity in both central analgesic model of the hot plate and peripheral analgesic model of acetic acid induced writhing. The analgesic activity seen with febuxostat is higher as compared to that of allopurinol in both the pain models. Schmidt et al. (2008) is also evaluated analgesic activity of allopurinol.
Allopurinol and febuxostat are an inhibitor of the enzyme xanthine oxidase, the final step in purine metabolism, converting hypoxanthine and xanthine into uric acid. The inhibition of xanthine oxidase leads to accumulation of hypoxanthine and xanthine. This favors the action of the enzyme hypoxanthine-guanine-phosphoribosyltransferase, which is responsible for purine salvage. This leads to a decrease in the systemic concentration of uric acid and an increase in the concentration of the precursors, hypoxanthine and xanthine. The hypoxanthine is converted to inosine, IMP and consequently to adenosine.  Xanthine oxidase is also responsible for generation of ROS by catalyzing the oxidative hydroxylation of purine substrates,  ROS have been proposed to mediate acute pain transmission.  The proposed mechanism of allopurinol and febuxostat as an analgesic is the modulation of adenosine levels and inhibition of production of ROS.
Adenosine exerts analgesic activity at peripheral and central sites which may be related to the inhibition of neuronal conduction by increase in K + conductance; decrease in release of substance P and glutamate;  and attenuation of basal a well as NMDA-induced nitric oxide production which is an excitatory neurotransmitter.  Agents inhibiting a major adenosine metabolizing enzyme, AK, are very effective anti-nociceptive agents in a wide range of pain models. 
Non-steroidal anti-inflammatory drugs are used as an analgesic, which are associated with adverse effects such as gastritis, stomach ulcers, and gastric bleeding, bleeding disorder. On prolonged use of NSAIDs, it will cause renal dysfunction, hepatic dysfunction, asthma, vasomotor rhinitis, angioneurotic edema, urticaria, laryngeal edema, and even cardiovascular collapse. Cardiovascular risks of NSAIDs, especially COX-2 inhibitors have become a major focus of attention over the past several years.  They may be contraindicated in some patients due to coexistent medical problems, especially in the elderly, as the majority of these patients have preexisting conditions such as diabetes, renal, cardiovascular or gastrointestinal diseases.  Thus, the use of xanthine oxidase inhibitors such as allopurinol and febuxostat over that of uricosuric agents such as probenecid and benzbromarone in long-term treatment of gout, may provide an added advantage of substitution or lowering the dose of NSAIDs, thus, avoiding the side effects of long-term administration of NSAIDs. They may be useful to treat pain syndromes in humans.
Furthermore, xanthine oxidase inhibitors, allopurinol and febuxostat may be combined with other analgesics which act predominantly on nonadenosine systems in the management of gout. They may also provide useful adjunct therapies in chronic diseases such as sickle cell anemia and diabetic neuropathy, where their possible dual actions on ROS and adenosine levels may be particularly helpful in improving vascular endothelial function and limiting pain. 
However, further animal studies using other pain models should be done. Human studies should also be carried to throw further light on the effect of xanthine oxidase inhibitors on pain. In our study, we did not measure the cerebrospinal fluid levels of adenosine and the levels of ROS, which would have confirmed the mechanism of action for the analgesic activity of xanthine oxidase inhibitors.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]