Potentiation of antinociceptive effect of NSAIDs by a specific lipooxygenase inhibitor, acetyl 11-keto-beta boswellic acid

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1 Indian Journal of Experimental Biology Vol. 44, February 2006, pp Potentiation of antinociceptive effect of NSAIDs by a specific lipooxygenase inhibitor, acetyl 11-keto-beta boswellic acid Mahendra Bishnoi, Chandrashekar S Patil, Anil Kumar & Shrinivas K Kulkarni* Pharmacology Division, University Institute of Pharmaceutical Sciences, Chandigarh , India Received 10 January 2005; revised 10 October 2005 The present study was aimed to assess the combined effects of cyclooxygenase and 5-lipoxygenase (COX/5-LOX) inhibitors in different animal models of nociception. nimesulide and rofecoxib are well-established antinociceptive agents acting via COX inhibition. AKBA (acetyl-keto-beta-boswellic acid) is a 5-LOX inhibitor. AKBA ( mg/kg) produced a dose dependent and significant antinociceptive effect in different animal models of nociception. Based on the earlier reports from our laboratory, sub effective doses of all the three COX Inhibitors were selected and they were administered (naproxen, 5 mg/kg; nimesulide, 1 mg/kg; and rofecoxib, 1 mg/kg) with AKBA (100 mg/kg). This produced a more significant antinociceptive effect as compared to per se effect observed in all the three models of nociception. However, the effect of combination of nimesulide with AKBA was more pronounced as compared to naproxen and rofecoxib and their combination with AKBA. The present finding provided an evidence for the potentiation of antinociceptive effect of NSAIDs with AKBA. Such a combination may help to reduce the therapeutic doses of conventional NSAIDs and also reduce side effects (gastric, cardiac and renal) that are popularly associated with the NSAIDs. Keywords: Antinociception, Boswellic acid, Nociception, NSAID Oxidized arachidonic acid derivatives, collectively termed as eicosanoids (prostaglandins and leukotrienes) have important modulatory role in inflammation, blood clotting, control of vascular tone, renal functions, reproductive functions as well as pain 1. Role of prostaglandins (PGs) in pain is long and extensively studied. PGs act directly on the peripheral terminals of the nociceptors through G protein coupled receptors to modulate (via camp/pka second messenger) the tetrodotoxin resistant sodium channel and to produce pain. Besides this, leukotrienes, especially LTB 4, result into increase in vascular permeability and chemotaxis of polymorphonuclear leukocytes as well as release of chemicals from leukocytes, which produce the sensitization of nociceptors 2,3. The lipoxygenase products of arachidonic acid metabolism have also been implicated directly in activating the capsaicin sensitive vanilloid receptors. 5-lipooxygenase products are also involved in hyperalgesia 4,5. Acetyl-11-keto-beta-boswellic acid (AKBA) is one of the four major pentacyclic triterpenic acids present in the acidic extract of the Boswellia serrata gum resin, which is used for a variety of inflammatory *Correspondent author skpu@yahoo.com <mailto:skpu@yahoo.com> disorders, such as rheumatoid arthritis, osteoarthritis, and cervical spondolytis 6. AKBA is a novel and highly specific inhibitor of 5-lipooxygenase, the key enzyme in leukotriene biosynthesis. AKBA inhibits 5- LOX either directly interacting with the enzyme itself or interacting with 5-lipooxygenase activating proteins (FLAP) 6,7. Based on the possible role of the leukotrienes in the sensitization and provocation of nociceptors and the already established role of cyclooxygenase metabolites, we studied the effect of different COX inhibitors (naproxen, nimesulide and rofecoxib) and LOX inhibitor, acetyl-11-keto-beta-boswellic acid (AKBA), and their combination. The study was undertaken to determine whether the combined inhibition showed better analgesic activity than single inhibition in different models of pain. Materials and Methods Animals Laca mice (20-30 g) of either sex, bred in central animal house of Panjab University, Chandigarh maintained at 12 hr light and dark cycle were used in the study. Animals were housed under standard laboratory conditions, with free access to food and water. All experiments were carried out between 0900 and 1700 hrs and the experimental protocol was approved by IAEC of the university.

2 BISHNOI et al.: POTENTIATION OF ANTINOCICEPTIVE EFFECT OF NSAID 129 Drug, dose, route The drugs, nimesulide (1 and 2 mg/kg), naproxen (5 and 10 mg/kg), rofecoxib (1 and 2 mg/kg) and test substance AKBA (50 and 100 mg/kg) were prepared in 0.5% carboxy-methylcellulose (CMC) and administered orally (po) half an hour before subjecting to nociceptive stimulus in the different models of nociception. They were administered simultaneously whenever a combination was attempted. All the substances were obtained from M/s Panacea Biotec, Ltd., Lalru, India. Assessment of anti-nociceptive activity The anti-nociceptive activity of the given drugs and their combination was assessed using different models of nociception as follows: Acetic acid-induced writhing (abdominal contractions) Abdominal contractions were induced by 1% acetic acid solution (0.1 ml/10 g, ip) in mice. The number of abdominal writhes was scored over 20 min after the injection of acetic acid 8. Tail-flick test The antinociceptive effect was also determined by using the tail flick test. The response was recorded at 30, 60 and 120 min after drug administration 8. Tail-immersion test The distal end (2 cm) of the mouse-tail was marked and this part of the tail was immersed in a water bath containing warm water at 55ºC. Withdrawal of the tail from the water was taken as the endpoint. The reaction time was determined before and at various time intervals (30,60,120 min) after different treatments. A cut off time of 10 sec was observed to avoid injury to the tail 8. Results were expressed in % maximum possible effect (% MPE) % MPE= (Post-treatment latency pre-treatment latency) 100 Pre-treatment latency Statistical analysis The data was expressed as mean±sem and was analyzed using Students t test. P<0.05 was considered as significant. Results Acetic acid induced-writhing (abdominal contractions) Animals from the control group showed 61±1.58 abdominal writhes during an observation period of 20 min after acetic acid injection. All the 4 test drugs studied nimesulide (1, 2 mg/kg), naproxen (5,10 mg/kg), rofecoxib (1, 2 mg/kg) and AKBA (50,100 mg/kg) showed significant and dosedependent decrease in the number of abdominal constrictions as compared to the control group. Nimesulide (2 mg/kg, po) showed the maximum effect. When the sub-effective dose of COX inhibitors (nimesulide, 1 mg/kg; naproxen, 5 mg/kg; rofecoxib, 1 mg/kg) was combined with 5-LOX inhibitor AKBA (100 mg/kg) the analgesic effect of all the three drugs was potentiated significantly as compared to control as well as their per se effect. Effect of combination of nimesulide with AKBA was more prominent than other two COX inhibitors (Table 1). Tail- flick test All the 4 test drugs, nimesulide (1, 2 mg/kg), naproxen (5, 10 mg/kg), rofecoxib (1, 2 mg/kg) and AKBA (50, 100 mg/kg) showed significant and dose-dependent increase in % MPE as compared to control. The effect was maximum at 60 min in all the 4 drugs and after that the effect started decreasing and at 120 min it was minimum. Combination of AKBA with naproxen or rofecoxib Table 1 Effect of COX and 5-LOX inhibitors and their combination at different doses on acetic acid induced writhing in mice Number of writhes % Inhibition Control 61.0± ±2.48 a ±2.15 a ±4.31 a ±5.11 a ±1.24 a ±1.24 a ±1.80 a ±4.31 a ac 30.2± abd 22.6± ae 30.6± a P< 0.05 as compared to control values, b as compared to AKBA (100 mg/kg) values, c as compared to naproxen (5mg/kg) values, d as compared to nimesulide (1 mg/kg) values, e as compared to rofecoxib (1 mg/kg) values. Drugs were administered orally.

3 130 INDIAN J EXP BIOL, FEBRUARY 2006 appeared to potentiate the effect of these NSAIDs at 30 and 60 min, though the maximum effect was lesser than that observed for AKBA alone. However, the observation at 120 min revealed a clear potentiation in terms of duration of action as well as efficacy when compared to their individual effects. Combination of AKBA with nimesulide clearly exhibited a potentiation at all periods of observation when compared to their individual effects. Effect of combination of nimesulide with AKBA was more prominent than other two COX inhibitors (Table 2). Tail immersion test All the 4 test drugs (nimesulide, 1 and 2 mg/kg; naproxen 5 and 10 mg/kg; rofecoxib, 1 and 2 mg/kg; and AKBA 50, 100 mg/kg) showed significant and dose-dependent increase in % MPE as compared to the control. The effect was maximum at 60 min in all the drug treated groups and the effect started decreasing thereafter. Nimesulide (2 mg/kg, po) showed the maximum effect. When sub-effective dose of COX inhibitors (nimesulide, 1 mg/kg; naproxen, 5 mg/kg; and rofecoxib, 1 mg/kg) were combined with 5-LOX inhibitor AKBA (100 mg/kg) the analgesic effect of all the three drugs was potentiated significantly as compared to control as well as their per se effect at 60 min. Although the effect was potentiated at 30 and 120 min, it was not significant. The effect of combination was also not increased significantly as compared to AKBA per se. The effect of the combination of nimesulide with AKBA was more prominent than the other two COX inhibitors (Table 3). Discussion Evidences clearly indicate that prostanoids, especially PGE2 are involved in the hyperalgesia and act synergistically with other mediators to produce inflammatory pain. They also sensitize the receptors on the afferent nerve endings and produce synergistic effect to the actions of bradykinin and histamine 1. There are also evidences supporting the role of leukotrienes in acute pain. LTB4 and HETE are potent chemo-tactic factors for the polymorphs, which in turn lower the firing threshold of the pain fibers and thus stimulate the nociceptor directly 3. Capsaicin receptors (VR-1) that are cloned recently are reported to be activated by noxious heat and acid suggesting their role in thermal and chemical pain 9. Several reports had confirmed that the metabolic products of the lipoxygenase enzyme are the able candidates for the endogenous activation of capsaicin receptors 10. Besides this, the inhibition of epinephrine induced hyperalgesia (which act directly on primary afferent nociceptors) by different selective and non-selective 5 Table 2 Effect of COX and 5-LOX inhibitors and their combination at different doses on tail-flick latency in tail-flick test in mice 30 min 60 min 120 min Control 15.23± ± ± ± ± ± ±5.61 a 86.42±6.98 a 48.34±3.98 a ± ± ± ±3.52 a ±5.4 a 58.34±9.62 a ± ± ± ±9.35 a 98.24±8.21 a 60.04±6.65 a ± ± ± ±6.58 a 97.28±4.89 a 29.86±3.56 a ±1.25 ac ±6.98 ac 72.25±6.42 abc ±5.21 ad ±9.32 abd 84.56±9.98 abd ±3.25 ae ±8.25 ae 82.49±7.21 abe a P< 0.05 as compared to control values, b as compared to AKBA (100 mg/kg) values, c as compared to naproxen (5 mg/kg) values, d as compared to nimesulide (1 mg/kg) values, e as compared to rofecoxib (1 mg/kg) values. Drugs were administered orally.

4 BISHNOI et al.: POTENTIATION OF ANTINOCICEPTIVE EFFECT OF NSAID 131 Table 3 Effect of COX and 5-LOX inhibitors and their combination at different doses on tail-withdrawal latency in tail-immersion test in mice 30 min 60 min 120 min Control 4± ± ± ± ± ± ±1.56 a 92.20±7.52 a 52.64±5.54 a ± ± ± ±4.54 a ±8.54 a 70.45±6.58 a ± ± ± ±4.58 a ±9.87 a 103.2±10.4 a ± ± ± ±6.88 a 115.5±10.21 a 46.11±5.65 a ±3.65 ac ±9.25 ac 62.10±3.69 abc ±5.16 ad ±8.97 abd 62.58±5.64 abd ±3.98 a 124.5±9.94 ae 60.0±6.04 ab a P< 0.05 as compared to control values, b as compared to AKBA (100 mg/kg) values, c as compared to naproxen (5mg/kg) values, d as compared to nimesulide (1mg/kg) values, e as compared to rofecoxib (1 mg/kg) values. Drugs were administered orally. and 12 lipoxygenase inhibitors also supports the suggestion that lipooxygenase products might be involved in the nociceptor sensitization lipoxygenase products of arachidonic acid metabolism also act as the second messenger, downstream to protein kinase A and protein kinase C and modulate the action of these kinases in primary afferent nociceptors to mediate their sensitization to mechanical and chemical noxious stimuli. 5 and 12 lipooxygenase products act synergistically with PGE2 to produce the mechanical hyperalgesia 12,13. Acetyl-11-keto-beta-boswellic acid (AKBA) is one of the four major pentacyclic triterpenic acids present in the acidic extract of Boswellia serrata gum resin 14. Earlier studies indicate that the primary mode of action involves the inhibition of the 5-lipoxygenase, the key enzyme responsible in the formation of inflammatory compounds, leukotrienes 15. The mechanism of inhibition is poorly understood as it might inhibit 5-LOX either directly interacting with the enzyme itself or interacting with 5-lipooxygenase activating proteins (FLAP) 6. Other studies report that AKBA is devoid of any significant side effect and toxicity 7. Hence, the role of prostaglandin and leukotrienes were supposed to act synergistically to produce mechanical and chemically induced inflammatory pain. Therefore, the combination of their inhibitors might have pronounced effect than their individual effect. Results obtained from different experimental proofs suggest that dual inhibitors of COX and 5- LOX isoenzymes are more pronounced analgesic agents than individual COX inhibitors, whether selective and nonselective 16. In the present study, there was dose-dependent increase in the analgesic activity of all the given drugs, nimesulide, 1, 2 mg/kg; naproxen, 5, 10 mg/kg; rofecoxib, 1, 2 mg/kg; and 50,100 mg/kg in all the three nociceptive models. Effect of all the drugs was maximum after 60 min of noxious stimuli, which started to decline thereafter. Results of above experiments provided evidence that metabolites of COX isoenzymes as well as metabolites of the other pathway of the arachidonic acid metabolism namely, LOX pathway had a role in the acute pain. Further, combination of sub-maximal dose of COX inhibitors nimesulide (1 mg/kg), naproxen (5 mg/kg), rofecoxib (1 mg/kg) with 5-LOX inhibitor AKBA (100 mg/kg) not only synergistically increased the analgesic effect of submaximal doses of COX inhibitors, but also increased the duration of their action. The analgesic activity and the duration of action of combination were equivalent or even better than individual maximal dose treatment of different COX inhibitors.

5 132 INDIAN J EXP BIOL, FEBRUARY 2006 The study also indicated that the combination of AKBA (5-LOX inhibitor) with nimesulide (selective COX-2 inhibitor) produced a more pronounced effect than its combination with naproxen (nonselective COX inhibitor) or rofecoxib (selective COX-2 inhibitor). In conclusion, the present finding provided evidence that combination of a safe 5-LOX inhibitor like AKBA might reduce the therapeutic doses of conventional NSAIDs and also spared the different side effects (gastric, renal, cardiac), which are associated with the conventional NSAIDs therapy. It will be more advantageous to identify molecules that inhibit both the pathways of AA metabolism namely COX and LOX for better antinociceptive effect. Acknowledgement Authors are grateful to the Panacea Biotech, Lalru, Punjab, India for providing drug samples for this work. References 1 Reichling D B & Levine J D, The primary afferent nociceptor as the pattern generator, Pain, 6 (1999) S Piomelli D, The ligand that comes from within, Trends Pharmaco Sci, 22 (2001) Boden S E, Schweizer S, Dufer M, Drews G & Safayhi H, Stimulation of leukotrienes synthesis in intact polymorph nuclear cells by 5-lipoxygenase inhibitor 3-oxo-tirucallic acid, J Neurosci, 60 (2002) Aley K O & Messing R O, Mochly-Rosen D & Levine J D, Chronic hypersensitivity for inflammatory nociceptor sensitization mediated by the epsilon isozyme of protein kinase C, J Neurosci, 20 (2000) Cunha F Q, Teixeira M M & Ferriera S H, Pharmacological modulation of secondary mediator systems-cyclic AMP and cyclic GMP on inflammatory hyperalgesia, Br J Pharmacol, 127 (1999) Safayhi H, Sailor E R & Ammon H P T, 5-lipooxygenase inhibition by Acetyl-11-keto-beta-boswellic acid (AKBA) by a novel mechanism, Phytomedicine 3(1996) Singh G B, Singh S & Bani S, Anti-inflammatory actions of boswellic acids, Phytomedicine, 3 (1996) Jain N K, Singh A & Kulkarni S K, Analgesic, antiinflammatory and ulcerogenic activity of zinc naproxen complex in mice and rats, Pharm Pharmacol Commun, 5 (1999) Hwang S W, Cho H, Kwak S Y & Oh U, Direct activation of capsaicin receptors by products of lipooxygenases: Endogenous capsaicin like substances, Proc Natl Acad Sci USA, 97 (2000) Craib S J, Ellington H C, Pertwee R Q & Ross R A, A possible role of lipooxygenase in the activation of vanilloid receptors by anandamide in the guinea pig bronchous, Br J Pharmacol, 134 (2001) Khasar S G, Mccarter G & Levine J D. Epinephrine produces a beta-adrenergic receptor mediated mechanical hyperalgesia and in vitro sensitization of rat nociceptors, J neurophysiol, 81 (1999) Aley K O, Martin A, Mcmohan T, Mok J & Levine J D, Nociceptor sensitization by extracellular signal regulated kinases, J Neurosci, 21 (2001) Aley K O & Levine J D, Contribution of 5 and 12 lipooxygenase products to mechanical hyperalgesia induced by prostaglandin E2 and epinephrine in the rats, Exp Brain Res, 148 (2003) Ammon H P T, Safayhi H & Mack T, Mechanism of antiinflammatory actions of curcumin and boswellic acids, J Ethanopharmacol, 38 (1993) Singh G B & Atal C K, Pharmacology of an extract of salai guggal, Boswellia serrata, a new non-steroidal anti inflammatory agent, Agents Action, 18 (1986) Rioja I, Terencio C, Ubeda A, Molina P & Alcaraz M J, A pyrroloquinazoline derivative with anti-inflammatory and analgesic activity by dual inhibition of cyclooxygenase-2 and 5-lipoxygenase, Euro J Pharmacol, 434 (2002) 177.