Tolerance to and dependence on the analgesic effect of opioids is a pharmacological phenomenon that occurs after their prolonged administration.
The aim of this study was to evaluate the protective effects of ceftriaxone and amitriptyline on the development of morphine-induced tolerance and dependence.
In this study, 18 groups (9 groups each for tolerance and dependency tests) of mice (n = 8) received saline [10 mL/kg, intraperitoneally (i.p.)], morphine (50 mg/kg, i.p.), ceftriaxone (50 mg/kg, i.p., 100 mg/kg, i.p., and 200 mg/kg, i.p.), amitriptyline (5 mg/kg, i.p., 10 mg/kg, i.p., and 15 mg/kg, i.p.), or a combination of ceftriaxone (50 mg/kg, i.p.) and amitriptyline (5 mg/kg, i.p.) once per day for 4 days for investigation and comparison of the effects of ceftriaxone and amitriptyline on the prevention of dependency and tolerance to morphine. Tolerance was assessed with administration of morphine (9 mg/kg, i.p.) and using the hot plate test on the 5th day. In dependency tests, withdrawal symptoms were assessed on the 4th day for each animal 30 minutes after the administration of naloxone (4 mg/kg, i.p.; 2 hours after the last dose of morphine).
It was found that treatment with ceftriaxone or amitriptyline attenuated the development of tolerance to the antinociceptive effect of morphine and also reduced naloxone-precipitated withdrawal jumping and standing on feet. Furthermore, coadministration of ceftriaxone and amitriptyline at low doses (50 mg/kg, i.p. and 5 mg/kg, i.p., respectively) prior to morphine injection also decreased both morphine-induced tolerance and dependence.
Results indicate that the treatment with ceftriaxone and amitriptyline, alone or in combination, could attenuate the development
amitriptyline; β-lactam antibiotic; ceftriaxone; dependency; morphine; tolerance;
Millions of people worldwide suffer from chronic pain brought on by diseases such as arthritis and cancer. The management of chronic pain is one of the greatest challenges in modern medicine. Opiates such as morphine have been widely used to treat various kinds of pain for decades. Long-term use of morphine is limited because of unwanted side effects including tolerance and dependence. Development of antinociceptive tolerance leads to increasing doses to control pain, and in some cases narcotics become ineffective and a higher drug dose is required to maintain the same level of effectiveness.1, 2Dependence is a continuous need for a drug to maintain a state of physical equilibrium following repeated consumption of opiates, and is indicated by withdrawal symptoms when opiate administration is terminated.3, 4 In the past decades, many studies were conducted to clarify the mechanisms involved in morphine-induced tolerance or dependence and have focused on the attenuation of these effects for management of chronic pain treatment. Morphine-induced tolerance and dependence is a complex physiological response that involves a within-system and a between-system adaptation. The within-system adaptations include opioid receptors uncoupling from G proteins and receptor downregulation. Between-system adaptations, such as the pain facilitatory systems (opiate-activated opponent systems), also play an important role in the development of opioid-induced tolerance and dependence.5, 6, 7, 8 The activation of the ionotropic N-methyl-d-aspartate (NMDA) subtype of glutamate receptors has been implicated in the development of morphine analgesic tolerance and dependence.7, 9, 10, 11Chronic opioid treatment resulted in the activation of protein kinase C and translocation that phosphorylates the NMDA receptor-gated Ca channel, leading to potentiation of NMDA receptor activity. Opening of these channel leads to an influx and increases intracellular Ca2+ concentration, which produces several effects.10, 12, 13, 14, 15 Furthermore, excitatory amino acids, their activated receptors, such as NMDA receptors, and the subsequent downstream signals [such as nitric oxide (NO)] are probably involved in opioid-induced tolerance and dependence. Other studies showed that proinflammatory cytokines, released from activated glial cells after repeated opioid administration, participate in the between-system mechanism.16, 17, 18, 19
Glutamate transporters are critical for glutamate removal from the extracellular space and are essential for maintaining homeostatic levels of extracellular glutamate. Decreasing extracellular glutamate by overexpressing the predominant astrocytic glutamate transporter was found to be effective in animal models of both visceral and neuropathic pain. Recently, it was found that β-lactam antibiotics upregulate glutamate transporters and increasing glutamate uptake through glutamate transporter subtype 1 (GLT-1) activation. Among all β-lactam antibiotics studies, ceftriaxone showed the highest potency in the upregulation of glutamate transporter. Ceftriaxone, a β-lactam antibiotic, is one of the members of third-generation cephalosporins. It is readily transported across the blood–brain barrier and is effective against gram-negative and gram-positive bacteria through the inhibition of cell wall synthesis. Ceftriaxone enhances both protein expression and functional activity of GLT-1 via a mechanism involving the nuclear factor-κ B signaling pathway. The glutamate transporter plays a major role in the maintenance of glutamate homeostasis. Among the five glutamate transporters, GLT-1 is responsible for 90% of glutamate uptake in the central nervous system (CNS). These findings suggest the high efficiency of ceftriaxone in the upregulation of GLT-1 and the reduction of glutamate excitotoxicity.20, 21, 22
Tricyclic antidepressants (TCAs) such as amitriptyline are primarily used for mood disorders. They are also widely used to treat chronic pain such as neuropathic pain conditions. TCAs increase both synaptic concentrations of serotonin or norepinephrine and therefore enhance neurotransmission. TCAs produce analgesia via various mechanisms involving NMDA receptors, biogenic amines, opioids, inflammatory mediators, and substance P. Amitriptyline is a glutamate transporter activator and used in the treatment of depression. Amitriptyline can be effective in prevention of morphine-induced dependence and tolerance. The proposed involved mechanisms are inhibition of proinflammatory cytokine, prevention of glutamate transporter downregulation, and enhancement of the activity of glutamate transporters.23, 24, 25, 26, 27
The aim of this study was to evaluate the attenuation effects of pretreatment with different doses of ceftriaxone, amitriptyline, and their combination on the development of morphine-induced tolerance and dependence.
2. Materials and methods
Morphine sulfate and naloxone were purchased from Darou Pakhsh Company (Tehran, Iran) and Tolid Daru Company (Tehran, Iran), respectively. Ceftriaxone hydrochloride and amitriptyline were obtained from Jaber Ebne Hayyan Pharmaceutical Company (Tehran, Iran) and Sobhan Pharmaceutical Company (Tehran, Iran), respectively.
2.2. Animals and treatment
Adult male Albino mice (Provided from Pasteur Institute, Tehran, Iran) weighing 20–30 g (aged 8 weeks) were allocated randomly to different groups (n = 8). The animals were maintained under standard temperature (24 ± 0.5°C) and lighting conditions (12-hour light/12-hour darkness) with free access to food and water.
All experiments were executed in accordance with the Guide for Care and Use of Laboratory Animals of Tabriz University of Medical Sciences, Tabriz, Iran (National Institutes of Health Publication No 85-23, revised 1985). This study was conducted at the Faculty of Pharmacy of Tabriz University of Medical Science.
Nondependent and morphine-dependent control groups were administered intraperitoneally (i.p.) with normal saline (10 mL/kg + 10 mL/kg) as well as normal saline (10 mL/kg) and morphine (50 mg/kg, i.p.) for 4 days, respectively. For evaluation of the effects of different doses of ceftriaxone and amitriptyline on the prevention of the morphine-induced tolerance, mice were pretreated for 4 days with ceftriaxone (50 mg/kg, i.p., 100 mg/kg, i.p., and 200 mg/kg, i.p.), amitriptyline (5 mg/kg, i.p., 10 mg/kg, i.p., and 15 mg/kg, i.p.) and ceftriaxone (50 mg/kg, i.p.) + amitriptyline (5 mg/kg, i.p.) 30 minutes prior to morphine injection (50 mg/kg, i.p.). Subsequently, to evaluate the degree of tolerance, the antinociceptive effect of a test dose of morphine (9 mg/kg, i.p.) was measured 24 hours after the last dose of morphine using a hot plate.
Furthermore, to assess the effects of the different doses of ceftriaxone and amitriptyline on the attenuation of dependence to morphine, withdrawal symptoms (number of jumping and standing on feet) for each animal (in the 9 groups; such as tolerance test) during 30 minutes were assessed on the 4th day after the administration of naloxone (4 mg/kg, i.p., 2 hours after the last dose of morphine).
2.3. Hot plate test
The antinociceptive activities of ceftriaxone and amitriptyline were determined by exposing the animals to potentially painful stimuli such as heat or electric shock and measuring either the time it takes these animals to respond to the stimuli or the intensity with which they respond. In this method, the time taken by the mice to lick its hind paws placed on a hot plate's stainless steel surface (23 cm × 23 cm and 55 ± 2°C) was determined. This reaction time was taken as the end point, and the increase in hot plate latency (seconds) was taken as a measure of the analgesic activity. The animals were removed from the hot plate if they did not respond within 30 seconds (cutoff time) in order to avoid tissue damage. Thirty minutes prior to treatment the nociceptive threshold was measured, and the latency time was used as the predrug latency for each test animal. Hot plate response latency is expressed as the percentage of maximal possible effect (MPE%) according to the following equation:
where TL denotes test latency time and BL is the base latency time.
2.4. Withdrawal symptoms test
Mice were tested for the degree of dependence after administration of morphine (50 mg/kg, i.p.) for 4 consecutive days. Injection of naloxone (4 mg/kg, i.p.), 2 hours after the last dose of morphine, precipitated severe withdrawal symptoms in morphine-dependent mice. Mice were placed individually on the filter paper in an open plexiglas chamber (25 cm × 25 cm × 40 cm), and the number of jumps and standing on feet were recorded by an observer over a 30-minute period for each animal.
2.5. Statistical analysis
Statistical analysis of each data set was performed using SPSS software version 17 (SPSS Inc., Chicago, IL, USA). All results are presented as mean ± SD for eight rats. Statistical comparisons among the experimental groups were made using one-way analysis of variance followed by Tukey post hoc test, where differences with p values less that 0.05 were considered significant.
3.1. Development of morphine-induced tolerance to analgesic effect
Fig. 1 displays the effects of morphine administration (50 mg/kg, i.p.) in 4 consecutive days on the development of tolerance. To have a similar number of injections in all groups, in the control group there were two injections of saline [saline (10 mL/kg, i.p.) + saline (10 mL/kg, i.p.)], and in the morphine group there were morphine and saline injections [morphine (50 mg/kg, i.p.) + saline (10 mL/kg, i.p.)]. It was verified that administration of morphine resulted in a significant reduction in MPE% values compared with the saline group (p < 0.001).
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3.2. Effects of administration of ceftriaxone on morphine-induced tolerance
The effects of pretreatment (30 minutes prior to morphine injection) with different doses of ceftriaxone on morphine-induced tolerance are shown in Fig. 2. Results revealed that administration of ceftriaxone attenuated the degree of tolerance to morphine in all test times. Furthermore, its effect in 0 minutes and 45 minutes was more significant. Additionally, pretreatment with ceftriaxone decreased morphine tolerance in a dose-dependent manner.
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3.3. Effects of administration of amitriptyline on morphine-induced tolerance
Fig. 3 shows the effects of pretreatment (30 minutes prior to morphine injection) with different doses of amitriptyline on morphine-induced tolerance. Similar to pretreatment with ceftriaxone, administration of amitriptyline prior to morphine injection attenuated the development of tolerance to morphine. Furthermore, its effect in 15 minutes and 60 minutes was more significant. However, the effect of 10 mg/kg amitriptyline was better than that of 15 mg/kg amitriptyline at 30 minutes.
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3.4. Effects of coadministration of ceftriaxone and amitriptyline on morphine-induced tolerance
Fig. 4 compares the combined effects of pretreatment (30 minutes prior to morphine injection) with ceftriaxone (50 mg/kg, i.p.) and amitriptyline (5 mg/kg, i.p.) with the individual effects of each medication in the same doses. The results showed that the protective effect of coadministration of ceftriaxone and amitriptyline on the development of tolerance was not greater than the effects of both drugs given alone except at the time interval of 15 minutes (p < 0.05 compared with the saline + morphine group). This demonstrated that coadministration of ceftriaxone and amitriptyline at low doses had no synergic protective effect against the development of morphine-induced tolerance. Table 1 summarizes the MPE% values of the different treated groups at determined time intervals. Results indicated that coadministration of low doses of ceftriaxone and amitriptyline did not exert greater protective effects on the development of tolerance and dependency than high doses of ceftriaxone and amitriptyline when administered alone.
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3.5. Effects of ceftriaxone and morphine on morphine-induced dependency
Table 2 displays the results of pretreatment (30 minutes prior to morphine injection) with ceftriaxone and amitriptyline on the development of morphine-induced dependency. Withdrawal syndrome symptoms (number of jumping and number of standing on feet) in morphine-dependent groups increased significantly compared with those in the nondependent group. Although pretreatment with ceftriaxone and amitriptyline in low doses did not attenuate the degree of the dependency, in higher doses ceftriaxone and amitriptyline significantly decreased the number of jumping and standing on feet compared to the morphine-dependent group (p < 0.001). Furthermore, coadministration of ceftriaxone and amitriptyline at low doses also significantly reduced the number of standing on feet and jumping compared to the saline + morphine group (p < 0.05 and p < 0.001, respectively).
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It is well known that repeated administrations of opiates such as morphine results in physical dependence and tolerance. These major side effects of morphine administration limit its clinical application. Dependence is a behavioral state requiring continued drug administration to avoid a series of aversive withdrawal symptoms. Tolerance is a normal phenomenon commonly associated with repeated administrations of morphine, where the medication becomes less effective with time and increasingly larger doses are required to relieve the pain. Therefore, new drugs and strategies are under investigation for preventing opiate dependence as well as withdrawal symptoms in a wide variety of animal species.
Lines of evidence from previous studies suggested that NMDA receptors are involved in the plasticity that arises from long-term administration of morphine.28, 29, 30 It has been proposed that repeated administrations of opiate may activate the NMDA receptors through G proteins associated with opioid receptor and/or intracellular mechanisms.31, 32, 33 This opiate-related activation of NMDA receptors may initiate subsequent intracellular changes such as production of NO and the activation of protein kinase C (PKC), where both NO and PKC have been shown to be critical for development of morphine dependence and tolerance7, 15, 17, 30 Several neurotransmitters including serotonin, dopamine, gamma-aminobutyric acid, adenosine, cholecystokinin, and aspartate seem to be involved in morphine tolerance and dependence.34 TCAs have many functions—as serotonin reuptake inhibitors, norepinephrine reuptake inhibitors, α1-adrenergic antagonists, and anticholinergic as well as antimuscarinic agents. The mechanism of TCAs in depression is blocking serotonin transporters to inhibit the reuptake of serotonin and increase its synaptic concentration. Considerable evidence recommends that altered noradrenergic and serotonergic functions could attenuate opioid withdrawal symptoms.27 Therefore, enhancing neurotransmission by increasing the serotonin concentration suggests that the direct effect on serotonin synapses might be involved in the action of TCAs in physical dependence on morphine.29, 35, 36, 37 Amitriptyline might attenuate the development of morphine-induced dependence and tolerance with different mechanisms, including: (1) inhibiting the expression of proinflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 as well as increasing IL-10 expression via the p38 MAPK-HO-1 signal transduction cascade; (2) activating nuclear factor-κ B, preventing glutamate transporter downregulation, and upregulating expression of glutamate transporters GLAST and GLT-1 in glial cells; and (3) preventing phospho-PKA and PKC expression, thereby promoting GLAST and GLT-1 trafficking to the glial cell surface. Normal or overexpression of GLAST and GLT-1 in glial cell membranes maintains glutamate transporter uptake activity, reduces spinal excitatory amino acid release, and decreases NMDA receptor activation.19, 26, 38, 39, 40 Recent studies have indicated that β-lactam antibiotics, which are a broad class of antibiotics, increased GLT-1 expression, as well as functional and biochemical activity in the brain and the spinal cord.41 Previous studies also revealed that ceftriaxone increased the expression of GLT-1 and its functional activity in both the hippocampus and the spinal cord at a dose of 200 mg/kg (i.p.) once per day for 5 days.42
In Huntington's disease mouse model, it was demonstrated that single daily intraperitoneal injections of 200 mg/kg ceftriaxone for 5 days increased glutamate uptake in the striatum, a primary target of cortical glutamatergic input.42, 43, 44, 45, 46 Ceftriaxone has been well studied and was found to cross the blood–brain barrier.47 Thus, ceftriaxone is currently considered a compound that has the potential to regulate GLT-1 expression in the CNS. Ceftriaxone, which—at doses of 50 mg/kg and 100 mg/kg, as well as 200 mg/kg—is routinely used in animal models of glutamate-related pathologies, inhibited morphine tolerance. An intraperitoneal administration of 200 mg/kg yields CNS ceftriaxone concentrations comparable to CNS levels required to increase GLT-1 expression. Overall, the antinociceptive efficacy of morphine was preserved in rats repeatedly exposed to a combination of ceftriaxone and morphine. The adverse effects produced by β-lactam antibiotics may limit their effectiveness in the clinical management of morphine-induced tolerance, but their ability to inhibit tolerance in a preclinical model through GLT-1 activation could stimulate the development of a nonantibiotic, β-lactam moiety containing compounds exhibiting enhanced clinical usefulness.48, 49, 50, 51, 52
According to the mentioned activities of amitriptyline and ceftriaxone and their common mechanisms in opiate-induced tolerance and dependency, their combined use and synergistic effects are rationalized. However, there was a difference in tolerance and dependency results in the combined use of these medications. Although a partial overlap, the development of tolerance and the development of dependence use discrete second messenger systems. The generally reported observation of the coexistence of tolerance and dependence may be based in part on the contribution of this common second messenger system.53, 54 As reported in1997 by Aley and Levine,55 NO seems to be involved in tolerance, whereas PKC seems to be involved in dependence. These differences may explain the results of our study. It seems that, because we used low doses in the coadministration of amitriptyline and ceftriaxone, they can sufficiently affect the PKC activation pathway but are not strong enough to affect NO production. Therefore, the dependency was significantly reduced when they were used in combination; however, the change in tolerance was not remarkable compared with that found when either of these medications was used alone. This result may be related to the fact that in PKC activation, both amitriptyline and ceftriaxone were involved, but in NO production it seems that only amitriptyline has an important role.
From the obtained results in this study, it may be concluded that pretreatment with amitriptyline and ceftriaxone alone or in combination can attenuate the development of morphine-induced tolerance and dependence. In this study, pretreatment with amitriptyline (5 mg/kg, i.p., 10 mg/kg, i.p., and 15 mg/kg, i.p.), ceftriaxone (50 mg/kg, i.p., 100 mg/kg, i.p., and 200 mg/kg, i.p.), and their combination at low doses (5 mg/kg, i.p. and 50 mg/kg, i.p., respectively) attenuated the development of tolerance to morphine. By contrast, lower doses of amitriptyline (5 mg/kg, i.p.) and ceftriaxone (50 mg/kg, i.p.) did not have a significant effect on the dependency markers of the number of standing on feet and jumping. However, results indicated that coadministration of low doses of ceftriaxone (50 mg/kg, i.p.) and amitriptyline (5 mg/kg, i.p.) significantly attenuated morphine-induced dependence compared to the administration of ceftriaxone or amitriptyline. These results suggest a probable synergistic effect of ceftriaxone and amitriptyline on the development of dependence on, but not tolerance to, morphine. For a better understanding of this subject, further studies in similar structures are suggested.
We thank the Faculty of Pharmacy, Tabriz University of Medical Sciences. This article is based on the results of two Pharm. D theses that were submitted to the Faculty of Pharmacy, Tabriz University of Medical Sciences.