Document Type : Research Paper
Authors
Department of Pharmacology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
Abstract
Keywords
1. Introduction
It is known that continuous or long term use of opiate drugs may cause tolerance and dependence which limits the therapeutic efficacy of these drugs [1-3]. With repeated administration of these drugs, adaptive mechanisms are initiated. One such mechanism is the development of tolerance. Another results from development of counter adaptations such that once the drug is removed a sequence of rebound signs and symptoms are manifested. Recent work suggests that these adaptive processes at the cellular, synaptic, and network levels downstream from the receptor may hold the keys to understanding of addiction [4]. Perhaps the most important adaptations that develop as a result of chronic opioid administration occur in neural systems responsible for the transition from casual to compulsive drug use. At the cellular level, adaptive mechanisms that occur with repeated and/or continuous morphine treatment to mediate associative tolerance are probably mediated by separate mechanisms [4, 5]. Although compounds of extremely high potency have been produced, the problem of tolerance and dependence on these agonists persists.
Several studies have indicated that different neurotransmitters and receptors are involved in morphine tolerance [6, 7], and the influence of receptors and neurotransmitters on the morphine tolerance and other opioids are contradictory [6, 8]. One of the neurotransmit-ters involved in morphine tolerance is adenosine. Caffeine, a mild stimulant, is the most widely-used psychoactive drug in the world (Goodman). Caffeine is rapidly distributed throughout all tissues and easily crosses the placenta and brain barrier. Caffeine has intrinsic antinociceptive properties and is frequently used as an adjuvant analgesic drug [8, 9]. It is thought that caffeine analgesia is produced, at least in part, through adenosine receptor antagonism. The adenosine receptor family comprises four subtypes: A1, A2A, A2B, and A3 [10]. They are widely distributed in CNS and peripheral tissues. The A1 receptors are found in high density in the brain (cortex, cerebellum, and hippocampus), the dorsal horn of the spinal cord, at lower levels in other brain regions, and in peripheral tissues [10, 11]. Caffeine is an adenosine A1, A2A, and A2B receptor antagonist. In this study, we examined the role of caffeine on morphine tolerance and analgesia in mice. We demonstrated analgesic and anti-analgesic effects of caffeine in different doses in mice.
Figure 1. Analgesic effects of morphine on (○) tolerant and (●) non-tolerant mice. Animals received either saline (10 ml/kg,i.p.) or morphine (30 mg/kg, i.p.) for 4 days. Antinociception of a test dose of morphine (9 mg/kg, i.p.) was tested 24 h afterthe last dose of morphine (30 mg/kg, i.p.) in tolerant and non-tolerant mice. Each group had at least 9 mice. Results are expressed as Mean±SE.*Significantly different from tolerant group (p < 0.05), ** (p < 0.01).
Table 1. Analgesia induced by i.p. administration of caffeine.
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Latency Time(sec) |
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Drug/dose(mg/kg) |
Base line (sec) |
15 (min) |
30 (min) |
45 (min) |
60 (min) |
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Saline normal (10 ml/kg) |
11±0.72 |
10.78±0.87 |
10.89±0.63 |
10.68±0.68 |
9.88±0.61 |
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Caffeine (5 mg/kg) |
11.05±1.25 |
11.05±1.32 |
11.23±1.38 |
10.5±1.33 |
10.27±1.22 |
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Caffeine (10 mg/kg) |
11±1.15 |
11.5±0.99 |
11.93±1.19 |
10.69±1.06 |
10.62±0.78 |
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Caffeine (15 mg/kg) |
11.33±1.56 |
11.43±1.75 |
12±1.77 |
10.31±1.31 |
9.16±1.34 |
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Caffeine (25 mg/kg) |
11.44±0.76 |
12±0.78 |
12.38±0.89 |
10.77±1.02 |
10.22±1.05 |
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Caffeine (50 mg/kg) |
11.12±1.37 |
11.75±1.55 |
12.38±1.23 |
10.75±1.01 |
9.75±1.05 |
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Caffeine (75 mg/kg) |
11.5±0.77 |
11.98±0.63 |
13.06±0.43 |
12.12±0.31 |
11±0.57 |
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Caffeine (100 mg/kg) |
11.25±1.21 |
12.93±1.39 |
14.25±1.03* |
12.06±1.10 |
10.63±0.74 |
All administrations were i.p. Latency time was measured as explained under materials and methods by hot plate test; *Significantly different from Base line (p < 0.01).
2. Materials and methods
2.1. Animals
Male albino Swiss-Webster mice (20-30 g, Razi Institute, Tehran, Iran) were used throughout the study (9 mice for each experiment). Animals were housed under a 12/12-h light cycle (07:00–19:00), with food and water available ad libitum. The animals were randomly allocated to different experimental groups.
2.2. Drugs
Morphine sulfate (Darupakhsh, Iran), powder of anhydrous caffeine (Hunan Pharmaceutical Factory), atropine (Darupakhsh, Iran) were used.
Figure 2.. Effects of different doses of caffeine (75, 100 mg/kg, i.p.) on tolerance determined by hot-plate test in morphinetolerantmice. Each group had at least 9 mice. Results are expressed as Mean±SE. *(p < 0.05), **(p < 0.01),***(p < 0.001) Significantly different from the control group [morphine (30 mg/kg)].
2.3. Method
Hot-plate test: each animal was placed on a surface (23×23 cm) maintained at 55±2 ºC surrounded by a Plexiglas wall 20 cm high. Licking of hands was used at the end point for determination of response latencies. Failure to respond by 45 seconds was a marker for termination of the test (cut off).
2.4. Induction of tolerance
In order to induce tolerance, groups of 9 mice were chosen randomly. Mice were treated intraperitonealy (i.p.) by morphine (30 mg/kg) in combination with either caffeine or saline or both once a day for four days. To evaluate the degree of tolerance, the antinociceptive effect of a test dose of morphine (9 mg/kg) was measured 24 h after the last dose of morphine in combination with caffeine or saline or both.
2.5. Evaluation of analgesia
Analgesic effects of caffeine (10, 15, 25, 50, 75, 100 mg/kg, i.p.) were evaluated alone or in combination with different doses of morphine (3, 6, 9 mg/kg, IP).
2.6. Statistical analysis
The results are expressed as the Mean±SE. Differences between the individual mean values in different groups were analyzed by one-way analysis of variance (ANOVA). Differences with a p < 0.05 were considered significant.
Figure 3. Effect of atropine (5 mg/kg, s.c.) on tolerance induction by morphine + caffeine co -administration. Each group had at least 9 mice. Results are expressed as Mean ± SE.
3. Results
3.1. Development of tolerance to morphine antinociception
As shown in Figure 1, animals received morphine (30 mg/kg, i.p.) once a day for four days, and in each mice antinociceptive response to a test dose of morphine (9 mg/kg, i.p.) was assayed 24 h after the last dose of morphine. Animals that became tolerant to effects of morphine exhibited only a small antinociceptive effect.
3.2. Analgesia induced by administration of caffeine
Table 1 shows the response of various doses of administration of caffeine (i.p.) in hot-plate test. Animals received saline (10 ml/kg, i.p.) or different doses of caffeine (5, 10, 15, 25, 50, 75, 100 mg/kg, i.p.). As shown in Table 1, only caffeine with a dose of 100 mg/kg, produced a significant (p < 0.05) antinociceptive effect as compared to the saline in hot-plate test.
Figure 4. Figure 4. Effects of caffeine (25 mg/kg, i.p.) + morphine (3 mg/kg, i.p.) on analgesia determined by hot-plate test. Results expressed as Mean±SE of 9 mice.; ** (p < 0.01) Significantly different from the control group [morphine (3 mg/kg)].
Figure 5. Effects of caffeine (15 mg/kg, i.p.) + morphine (6 mg/kg, i.p.) administration on analgesia determined by hot-platetest. Results expressed as Mean±SE of 9 mice.*(p < 0.05, ***(p < 0.001) Significantly different from the control group [morphine (6 mg/kg)].
3.3. Effect of caffeine on tolerance to chronic morphine therapy
As shown in Figure 2, caffeine injection (75, 100 mg/kg, i.p.), 30 min before daily morphine administration, significantly decreased tolerance to the analgesic effects of morphine (p < 0.01).
3.4. Effect of atropine in tolerance induced by co-administration of morphine + caffeine
Figure 3 shows that pretreatment with atropine (5 mg/kg, s.c.), 30 min. before daily morphine and caffeine co-administration, changed morphine tolerance, significantly.
Figure 6. Effects of caffeine (10, 50 mg/kg, i.p.) + morphine (9 mg/kg, i.p.) administration on analgesia determined by hotplate test. Results expressed as Mean±SE of 9 mice. *(p < 0.05), **(p < 0.01), ***(p < 0.001) Significantly different from the control group [morphine (9 mg/kg)].
Figure 7. Effects of caffeine (50, 75, 100 mg/kg, i.p.) + morphine (3 mg/kg, i.p.) administration on analgesia determined by hot-plate test. Results expressed as Mean±SE of 9 mice. **(p < 0.01), ***(p < 0.001) Significantly different from the control group [morphine (3 mg/kg)].
3.5. Analgesia induced by administration of morphine alone or plus caffeine
Table 1 shows the effects of different doses of caffeine (5, 10, 15, 25, 50, 75, 100 mg/kg, i.p.) on morphine-induced antinociception in hot-plate test.
Figures 4 to 7 show the results of co-administration of caffeine (5, 10, 15, 25, 50, 75, 100 mg/kg, i.p.) with morphine (3, 6, 9 mg/kg, i.p.). It was found that the combination of caffeine (10, 15, 25, 50 mg/kg, i.p.) with morphine (3, 6, 9 mg/kg, i.p.), decreased the analgesic effect of morphine, significantly (p < 0.01). But high doses of caffeine (100 mg/kg) increased the analgesic effect of morphine, significantly (p < 0.01).
3.6. Effect of atropine (5 mg/kg, SC) on the caffeine + morphine analgesia induction
Figure 8 shows the effect of atropine (5 mg/kg, s.c.) on analgesia induced by the administration of caffeine+morphine in hot-plate test. Animals which received caffeine (100 mg/kg, i.p.) with morphine (3 /kg, i.p.), showed increased analgesic effect of morphine (p < 0.05). This effect was inhibited by atropine (5 mg/kg, s.c.) pretreatment (15 min).
4. Discussion
In this study, we evaluated the effects of systemic administration of caffeine (Adenosine A1 , A2A, and A2B receptor antagonist) on morphine tolerance and analgesia in mice. Results indicated that injection of caffeine, 30 min. before daily administration of morphine, decreased tolerance to the analgesic effects of morphine, significantly. On the other hand, administration of atropine, before daily co-administration of morphine and caffeine changed in morphine tolerance, significantly.
In several studies [12, 13] repeated injections of morphine, cocaine, and amphetamine showed an increase in presynaptic inhibition caused by endogenous adenosine. Originally done in brain slices from guinea pigs treated with either morphine or cocaine, the presynaptic regulation of the GABAB IPSP measured in dopaminergic cells of the VTA was changed in drug-treated animals, significantly. This work showed that the activation of adenylyl cyclase by either D1 receptors or forskolin had two opposing actions. One was to augment GABA release through the activation of PKA, and the second was to inhibit GABA release by activation of a presynaptic A1 adenosine receptor. In slices from drug-treated animals, the enhancement of inhibition by adenosine was so great that the effect of D1 receptor activation reversed direction and inhibited, rather than augmented, GABAB IPSPs. This inhibition was blocked by adenosine receptor antagonists and agents that blocked the transport (Probenecid) or metabolism (a phosphodiesterase inhibitor) of cAMP. The opposing actions of forskolin have been reported in several brain areas [14-18]. In general, the persistent increase in extra-cellular adenosine is occurred in repeated injections of morphine. Then, effects of caffeine (morphine tolerance inhibition) are believed to occur by means of competitive antagonism at adenosine receptors. On the other hand, this effect was inhibited by atropine pretreatment (15 min.). Thus, the mechanism of caffeine on morphine tolerance seems to be dependent on cholinergic activation.
Figure 8. Effects of atropine (5 mg/kg, s.c.) on analgesia induced by combination of caffeine (100 mg/kg, i.p.) + morphine (3 mg/kg, i.p.) administration, determined by hot-plate test. Results expressed as Mean±SE of 9 mice.*(p < 0.05) Significantly different from the control group.
The second part of this study examined analgesia induced by treatment with caffeine. In different doses of caffeine, as shown in Table 1, only caffeine with a dose of 100 mg/kg, produced a significant antinociceptive effect as compared to the saline in hot-plate test. Combination of caffeine with morphine decreased morphine analgesic effect, but high doses of caffeine (100 mg/kg) increased the analgesic effect of morphine, significantly.Adenosine has dual activity on nociception.It acts centrally within the spinal cord to suppress nociceptive signaling [19], presumably through the activation of A1 and A2 adenosine receptors [20]. In the periphery, adenosine has algogenic activity, which is probably mediated by A2 receptors [19, 21].
5. Conclusion
Caffeine, a virtually nonselective A1-, A2A, and A2B-adenosine receptor antagonist,exhibits antinociceptive effects in high doses. Thus, caffeine acts centrally in high doses within the spinal cord to suppress nociceptive signaling. Other activities of caffeine (alteration of catecholamine or acetylcholine release and turnover, inhibition of phospho-diesterase, influence on intracellular calcium concentrations, and interaction with GABAA receptors) may contribute to its antinocicep-tive effects [19, 22]. Of course for clarifying the exact effects, further studies are required.
Acknowledgements
Special thanks, and gratitude goes to those who helped us in carrying out the study successfully, especially the Pharmacology Department of Tabriz University of Medical Sciences for their helpful supports.
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