|Year : 2013 | Volume
| Issue : 3 | Page : 244-247
Evaluation of effect of allopurinol and febuxostat in behavioral model of depression in mice
Ashwini V Karve, Sagar S Jagtiani, Kunal A Chitnis
Department of Pharmacology, T.N. Medical College and B.Y.L. Nair Ch. Hospital, Mumbai, Maharashtra, India
|Date of Submission||26-Nov-2012|
|Date of Decision||18-Jan-2013|
|Date of Acceptance||26-Feb-2013|
|Date of Web Publication||15-May-2013|
Sagar S Jagtiani
Department of Pharmacology, T.N. Medical College and B.Y.L. Nair Ch. Hospital, Mumbai, Maharashtra
Source of Support: None, Conflict of Interest: None
Objective: To evaluate the effects of allopurinol and febuxostat on depression using Forced Swim Test (FST) in mice.
Materials and Methods: Allopurinol (39 mg/kg p. o) and febuxostat (15.6 mg/kg p. o) were administered once daily for 21 successive days to Swiss Albino mice. On the 21 st day, the effect of the drug on locomotion was tested using photo-actometer followed by the recording of immobility period in the FST and the results were compared with the standard drug fluoxetine (10 mg/kg p. o).
Results: Allopurinol and febuxostat expressed significant antidepressant like effect as indicated by reduction in the immobility period of mice in the FST as compared to control group. The effects of allopurinol and febuxostat were found to be comparable to that of fluoxetine.
Conclusion: The results of the present study indicate that allopurinol and febuxostat possess significant antidepressant like activity.
Keywords: Forced swim test, photo-actometer, serotonin, tryptophan
|How to cite this article:|
Karve AV, Jagtiani SS, Chitnis KA. Evaluation of effect of allopurinol and febuxostat in behavioral model of depression in mice. Indian J Pharmacol 2013;45:244-7
|How to cite this URL:|
Karve AV, Jagtiani SS, Chitnis KA. Evaluation of effect of allopurinol and febuxostat in behavioral model of depression in mice. Indian J Pharmacol [serial online] 2013 [cited 2021 Jan 25];45:244-7. Available from: https://www.ijp-online.com/text.asp?2013/45/3/244/111922
| » Introduction|| |
Depression is considered as an affective disorder characterized by a change of mood. It is a disorder of major public health importance, in terms of its prevalence, and the suffering, dysfunction, morbidity and economic burden. ,
The "monoamine hypothesis" has been the fundamental topic for research in the field of depression. It suggests that, a deficiency or imbalance in the monoamine neurotransmitters such as serotonin, dopamine and norepinephrine, can lead to depression. This hypothesis is also supported by the fact that the known antidepressants like monoamine oxidase inhibitors, tricyclic antidepressants and selective serotonin reuptake inhibitors have been known to boost monoamine function. 
The currently available antidepressants require many weeks and even months to start showing any effect. In addition, they do not have a very favorable adverse effect profile. They can cause a myriad of side effects such as epigastric distress, fatigue, sedation, palpitations, urinary retention, postural hypotension, sexual dysfunction, delirium, hypomanic, and manic states, etc. Furthermore, the metabolism of most antidepressants is greatly dependent on the activity of hepatic cytochrome enzymes sometimes producing clinically significant drug interactions. Moreover, there are reports in the medical literature of patients developing tolerance or physical dependence to the effect of these antidepressants. 
Tryptophan is the aromatic amino acid precursor of 5-hydroxytryptamine (Serotonin), which is a "monoamine" neurotransmitter in the brain. Tryptophan is metabolized with the help of the enzyme tryptophan pyrrolase. Xanthine oxidase has been identified as an endogenous activator of tryptophan pyrrolase enzyme,  which brings about increased degradation of tryptophan and decreases its level in the body.
Allopurinol and febuxostat are potent xanthine oxidase inhibitors, primarily used in the treatment of hyperuricemia and gout.  Due to their xanthine oxidase inhibiting property, they may cause a decreased activation of tryptophan pyrrolase enzyme, which ultimately might increase the level of tryptophan in the body. By increasing the tryptophan level, these drugs may be hypothesized to possess an anti-depressant effect.
Therefore, the present study was undertaken to evaluate the effect of allopurinol and febuxostat on depression using Forced Swim Test (FST) in mice.
| » Materials and Methods|| |
The study was conducted after obtaining permission from the Institutional Animal Ethics Committee. Male Swiss Albino mice weighing between 20 g and 30 g were procured from Haffkine Institute, Mumbai. The mice were housed in polypropylene cages with husk paddy as the bedding and with stainless steel top grill having facilities for food and drinking water in glass bottles with stainless steel sipper tube. The animals were housed in air-conditioned rooms (23-30°C; Humidity 50-60%). The animals were maintained on a standard pellet diet and water ad libitum. A 12 h light and dark cycle was maintained.
Pure form of the drugs allopurinol, febuxostat and fluoxetine were obtained from Zydus Cadila.
The study was carried out in 24 male Swiss Albino mice weighing between 20 and 30 g. After an initial period of acclimatization of 7 days the animals were randomly divided into 4 groups of 6 mice each. The animals in group 1 were given 0.5 ml of the vehicle orally (0.5% carboxymethyl cellulose [CMC]). Animals in group 2 were administered fluoxetine (10 mg/kg p. o.), which is a known antidepressant and acted as the positive control.  Animals in group 3 and group 4 were administered allopurinol (39 mg/kg p. o.) and febuxostat (15.6 mg/kg p. o.) suspended in 0.5 ml of 0.5% CMC respectively. All the groups received the respective treatments for a period of 21 days. On the 21 st day, 1 h after administration of the drug, the effect of the drugs on locomotion was tested using photo-actometer followed by the recording of immobility period in the FST. During the test, the observer was blinded to the group of animals being tested to avoid bias.
Behavioral despair is proposed as a model to test for antidepressant activity by Porsolt et al. The animals are individually forced to swim inside a vertical Plexiglas cylinder (Height: 40 cm; diameter: 18 cm, containing 15 cm of water maintained at 25°C). After 2 min of vigorous activity, each animal assumes a typical immobile posture. A mouse will be considered to be immobile when it remains floating in the water without struggling making only minimum movements of its limbs necessary to keep its head above water. On the test day, each animal was placed in the water and forced to swim for 6 min. The total duration of immobility was recorded during the last 4 min of the total 6 min test. After removing the mice from the water they were allowed to dry for 15 min and then were replaced back into the cage. The water was changed prior to testing for each animal. 
The locomotor activity was recorded using a photo-actometer (INCO Ambala India).The animals are placed in the digital photo-actometer one by one, which consists of a cage, which is 30 cm long and 30 cm deep with a wire mesh at the bottom. A continuous beam of light from about six lights falls on the corresponding photoelectric cell; the photoelectric cell gets activated when an animal crosses the beam of light and thereby cuts-off the rays of light falling on it. These cut-offs were counted for a period of 10 min and the figure was taken as a measure of locomotor activity of the animal. Locomotor activity was expressed in terms of total photo beam counts for 10 min/animal. 
Results were represented as mean ± SD 0 Experimental data was analyzed using one way analysis of variance (ANOVA) followed by post hoc Tukey's test. P value of <0.05 was considered significant.
| » Results|| |
Effect on immobility in FST
In this study, the effect of allopurinol and febuxostat in depression was evaluated with the help of FST. The duration of immobility was recorded for both the drugs and was compared with that of control (0.5% CMC) and fluoxetine (positive control).
The mice in the control group who were given 0.5% CMC showed an immobility time of 178.5 ± 14.8 s. Duration of immobility of mice treated with fluoxetine was 114.7 ± 13.2 s. The duration of immobility of mice treated with allopurinol was 129.8 ± 10.5 s. Duration of immobility of mice with febuxostat was 125.0 ± 12.3 s [Table 1].
|Table 1: Effect of allopurinol and febuxostat on immobility period in forced swim test |
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Fluoxetine (10 mg/kg), which is the positive control, significantly reduced the immobility time in the FST as compared to control (P < 0.001) when administered for 21 successive days. Allopurinol (39 mg/kg) administered to mice for 21 successive days significantly reduced the immobility time in the FST as compared to the control group (P < 0.001). Furthermore, febuxostat (15.6 mg/kg) administered for 21 successive days significantly reduced the immobility time in the FST as compared to the control group (P < 0.001). The reduction in the immobility periods by allopurinol and febuxostat was also found to be comparable to fluoxetine, which is a known antidepressant. By observing the values, reduction of immobility time was more with febuxostat as compared to allopurinol.
Effect on locomotor activity in photo-actometer
The locomotor activity of animals subjected to FST was tested using photo-actometer in all groups. This test was conducted 15 min before subjecting animals to FST.
Allopurinol (197.0 ± 17.04) and febuxostat (195.0 ± 14.25) did not produce any significant change in locomotor activity after 21 days successive administration as compared to control (175.7 ± 10.78) and fluoxetine (196.0 ± 11.71) (P = 0.6494). There was no statistically significant difference between test groups for the total photo beam counts measured suggesting that the reduction in duration of immobility with test drugs was due to antidepressant effect and was not a false positive [Table 2].
|Table 2: Effect of allopurinol and febuxostat on locomotor activity in photo-actometer |
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| » Discussion|| |
The FST is the most commonly used behavioral model of depression in animals. This test is quite sensitive and relatively specific to all major classes of anti-depressant drugs. It is widely employed in rodents to predict anti-depressant potential of drugs.  In the test, the immobility time of the animal in water is ascertained and a drug having anti-depressant like effect decreases the immobility time. This immobility, referred to as behavioral despair in animals, is claimed to reproduce a condition similar to human depression.  In the present study, allopurinol, and febuxostat administered to mice for 21 successive days, showed significant anti-depressant like activity in the FST. The efficacy of allopurinol and febuxostat was found to be comparable to that of fluoxetine, a known anti-depressant.
The immobility time in FST, is also reduced by CNS stimulants, but they tend to increase the locomotor activity  in the animals as opposed to the anti-depressants, which bring about no change in locomotion.  In the present study, allopurinol and febuxostat administered for 21 successive days, did not show any significant change in locomotor activity of mice, as compared to the control group, which helps to confirm the anti-depressant like activity.
Depression is a wide-spread mood disorder that affects an individual's life, disturbing his mood, thoughts, thinking, behavior, feelings, etc. Reduced monoaminergic signalling has long been thought to underlie depressive disorders. 
Tryptophan is the only aromatic amino acid precursor of 5-hydroxytryptamine (Serotonin), which is a "monoamine" neurotransmitter in the brain. After entering the brain, tryptophan is converted into serotonin in a two-step synthesis process. L-tryptophan is first converted into 5-hydroxytryptophan by the enzyme tryptophan hydroxylase (Tph). 5-hydroxytryptophan is then decarboxylated by another enzyme, aromatic amino acid decarboxylase (5-hydroxy-l-tryptophan decarboxylase) forming serotonin. Tph is the rate-limiting enzyme in the synthesis of serotonin. 
Serotonin has strongly been linked in the pathophysiology of depressive syndromes and in the mechanism of antidepressant drug action. The rate of 5-Hydroxytryptamine (5-HT) synthesis is dependent on plasma tryptophan, and on how much of it crosses the Blood Brain Barrier (BBB), hence, depletion of plasma tryptophan may modulate brain activity and mood.  Tryptophan is degraded to amphibolic intermediates via the kynurenine-anthranilate pathway. Tryptophan pyrrolase is the enzyme that oxidizes tryptophan. 
The antidepressant action of tryptophan has been demonstrated to be greater than that as compared to placebo. One hundred and fifteen patients participated in a 12-week, double-blind study comparing L-tryptophan, amitriptyline, L-tryptophan-amitriptyline combination and placebo in the treatment of depression. Analysis of total score on the Hamilton Depression Scale and a global rating of depression showed that all 3 active treatments were more effective than placebo. 
Furthermore, the anti-depressive effect of tryptophan has been compared with electroconvulsive therapy (E.C.T) in patients with severe depression. The results of this study suggest that, tryptophan was as effective as E.C.T. in treating depressive illness. 
A double-blind study administered tryptophan-depleted and taste-matched placebo challenge drinks to individuals in order to investigate the effect of acute tryptophan depletion (ATD) on positive affect and depressed mood. ATD has shown to cause an increase in depressed mood and overall depressive symptoms in patients, especially in those with a family history and in those who have undergone remission. 
In humans, ingestion of tryptophan-free amino acid mixture has been shown to produce a 70-90% reduction in plasma tryptophan and 80-90% reduced Cerebrospinal Fluid (CSF) tryptophan after 5-7 and 7-10 h respectively. Tryptophan depletion was also found to diminish the rate of serotonin synthesis by 90% of baseline value. 
Allopurinol and febuxostat are potent inhibitors of the enzyme xanthine oxidase and are primarily used in the treatment of hyperuricemia and gout.  Xanthine oxidase has been identified as an endogenous activator of the enzyme trytophan pyrrolase.
Allopurinol has been shown to inhibit the activity of rat and mice liver tryptophan pyrrolase.  It is suggested that allopurinol inhibits liver tryptophan pyrrolase activity by preventing the conjugation of the apoenzyme with its haem activator.  As a result of this decreased degradation, there is an increase in the tryptophan levels in the body.
Pituitary-adrenal activation,  or exogenous hydrocortisone administration,  have been demonstrated to increase the synthesis of tryptophan pyrrolase. This may lead to increased degradation of tryptophan, and in turn, reduction in 5-HT levels, which may lead to depression. This is further supported in a study, where intraperitoneal injection of hydrocortisone was associated with a reduction in the rat brain 5-HT levels.  It is also demonstrated that the rise of tryptophan pyrrolase activity, mediated by hydrocortisone, was prevented by the concurrent administration of the xanthine oxidase inhibitor, allopurinol. 
Allopurinol is also known to increase the volume of distribution of tryptophan. Allopurinol increased the level of tryptophan in the brain when a load of tryptophan was administered to a group of allopurinol pretreated rats as compared to control. 
Allopurinol is a time tested drug and has been in the market for many decades now. It is tolerated well by most patients. The most common adverse effects are hypersensitivity reactions that may occur after months or years of medication. Fever, malaise, myalgias, hepatomegaly are other rare side-effects. Furthermore, the drug interactions seen with allopurinol are less than those observed with the currently available antidepressants. 
Febuxostat is non-purine xanthine oxidase inhibitor. It is more potent and has a better adverse effect profile as compared to allopurinol.  The most frequent treatment-related adverse events are liver function abnormalities, diarrhea, headache, and nausea. Furthermore, febuxostat appears to be well tolerated in patients with a history of allopurinol intolerance.  To our knowledge, activity of febuxostat on tryptophan levels and in depression has not been assessed till date.
Hence, in summary, we can conclude that allopurinol and febuxostat possess anti-depressant like activity, which needs to be confirmed in other models of depression and in human trials. If developed, these drugs can form important tools in the arsenal of anti-depressant drugs.
| » References|| |
|1.||Shalam MD, Shantakumar SM, Laxmi Narasu M. Pharmacological and biochemical evidence for the antidepressant effect of the herbal preparation Trans-01. Indian J Pharmacol 2007;39:231-4. |
|2.|| Grover S, Dutt A, Avasthi A. An overview of Indian research in depression. Indian J Psychiatry 2010;52:S178-88. |
|3.|| Lee S, Jeong J, Kwak Y, Park SK. Depression research: Where are we now? Mol Brain 2010;3:8. |
|4.||O'Donnell JM, Shelton RC. Drug Therapy of Depression and Anxiety Disorders. In: Brunton L, Chabner B, Knollman B, editors. Goodman and Gilman's the Pharmacological Basis of Therapeutics. 12 th ed. New York: McGraw Hill; 2011. p. 397-415. |
|5.||Becking GC, Johnson WJ. The inhibition of tryptophan pyrrolase by allopurinol, an inhibitor of xanthine oxidase. Can J Biochem 1967;45:1667-72. |
|6.||Rundles RW. The development of allopurinol. Arch Intern Med 1985;145:1492-503. |
|7.||Devadoss T, Pandey DK, Mahesh R, Yadav SK. Effect of acute and chronic treatment with QCF-3 (4-benzylpiperazin-1-yl) (quinoxalin-2-yl) methanone, a novel 5-HT(3) receptor antagonist, in animal models of depression. Pharmacol Rep 2010;62:245-57. |
|8.||Dhingra D, Sharma A. Evaluation of antidepressant like activity of glycyrrhizin in mice. Indian J Pharmacol 2005;37:390-4. |
|9.||Badhe SR, Badhe RV, Ghaisas MM, Chopade VV, Deshpande AD. Evaluation of antidepressant activity of Anacyclus pyrethrum root extract. Int J Green Pharm 2010;4:79-82. |
|10.||Dhingra D, Kumar V. Evidences for the involvement of monoaminergic and GABAergic systems in antidepressant-like activity of garlic extract in mice. Indian J Pharmacol 2008;40:175-9. |
|11.||Tabassum I, Siddiqui ZN, Rizvi SJ. Effects of Ocimum sanctum and Camellia sinensis on stress-induced anxiety and depression in male albino Rattus norvegicus. Indian J Pharmacol 2010;42:283-8. |
|12.||Porsolt RD, Anton G, Blavet N, Jalfre M. Behavioural despair in rats: A new model sensitive to antidepressant treatments. Eur J Pharmacol 1978;47:379-91. |
|13.||Toker L, Amar S, Bersudsky Y, Benjamin J, Klein E. The biology of tryptophan depletion and mood disorders. Isr J Psychiatry Relat Sci 2010;47:46-55. |
|14.|| Rodwell VW. Catabolism of carbon skeleton of amino acids. In: Murray RK, Granner DK, Mayes PA, Rodwell VW, editors. Harper's Illustrated Biochemistry. 26 th ed. New York: Lange Medical Books; 2006. p. 249-63. |
|15.||Thomson J, Rankin H, Ashcroft GW, Yates CM, McQueen JK, Cummings SW. The treatment of depression in general practice: A comparison of L-tryptophan, amitriptyline, and a combination of L-tryptophan and amitriptyline with placebo. Psychol Med 1982;12:741-51. |
|16.||Coppen A, Shaw DM, Herzberg B, Maggs R. Tryptophan in the treatment of depression. Lancet 1967;2:1178-80. |
|17.||Altman SE, Shankman SA, Spring B. Effect of acute tryptophan depletion on emotions in individuals with personal and family history of depression following a mood induction. Neuropsychobiology 2010;62:171-6. |
|18.|| Badawy AA, Evans M. The mechanism of inhibition of rat liver tryptophan pyrrolase activity by 4-hydroxypyrazolo(3,4-d)pyrimidine (Allopurinol). Biochem J 1973;133:585-91. |
|19.||Knox WE, Auerbach VH. The hormonal control of tryptophan peroxidase in the rat. J Biol Chem 1955;214:307-13. |
|20.||Julian JA, Chytil F. A two-step mechanism for the regulation of tryptophan pyrrolase. Biochem Biophys Res Commun 1969;35:734-40. |
|21.||Curzon G, Green AR. Effect of hydrocortisone on rat brain 5-hydroxytryptamine. Life Sci 1968;7:657-63. |
|22.||Green AR, Aronson JK, Curzon G, Woods HF. Metabolism of an oral tryptophan load. II: Effect of pretreatment with the putative tryptophan pyrrolase inhibitors nicotinamide or allopurinol. Br J Clin Pharmacol 1980;10:611-5. |
|23.||Grosser T, Smyth E, FitzGerald GA. Anti-Inflammatory, antipyretic and analgesic agents; pharmacotherapy of gout. In: Brunton L, Chabner B, Knollman B, editors. Goodman and Gilman's the Pharmacological Basis of Therapeutics. 12 th ed. New York: McGraw Hill; 2011. p. 959-1004. |
|24.||Becker MA, Schumacher HR, Espinoza LR, Wells AF, MacDonald P, Lloyd E, et al. The urate-lowering efficacy and safety of febuxostat in the treatment of the hyperuricemia of gout: The CONFIRMS trial. Arthritis Res Ther 2010;12:R63. |
|25.||Furst DE, Ulrich RW, Altamirano CV. Nonsteroidal anti-inflammatory drugs, disease-modifying antirheumatic drugs, nonopioid analgesics, & drugs used in gout. In: Katzung BG, Masters SB, Trevor AJ, editors. Basic and Clinical Pharmacology. 11 th ed. New Delhi: Tata McGraw Hill; 2009. p. 621-42. |
[Table 1], [Table 2]
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