Antidepressants have been used for many years for the treatment of pain in humans.1 Classical tricyclic antidepressants have greater analgesic efficacy than selective amine reuptake inhibitors, and amitriptyline is the gold standard of analgesic antidepressants.2,3 On the other hand, selective serotonin reuptake inhibitors (SSRIs) are generally safer than classical tricyclic antidepressants and are more frequently prescribed to treat chronic pain despite weaker antinociceptive effects.4
The antinociceptive mechanisms of antidepressants are not completely clear. Increased availability of norepinephrine (NE) and serotonin (5-HT) at neuronal terminals produced by antidepressant drugs3,5 may activate multiple monoamine receptors, such as α1, α2A, 5-HT2, and 5-HT3 receptors.1,2,6 Moreover, the opioidergic system has been shown to be involved in the antinociceptive activity of antidepressants, including amitriptyline, paroxetine, and sibutramine.7 Similarly, activation of noradrenergic and serotonergic pathways occurs during opioid analgesia.8
The use of multiple compounds with different mechanisms of action, a practice termed “multimodal analgesia,” is frequently recommended for acute, moderate-to-severe pain treatment. Nonopioid and opioid analgesics can be combined to simultaneously address peripheral and central mechanisms of pain sensation, with improved opioid tolerability. Opioids, the most effective analgesics, are widely used via the intrathecal or epidural route, despite side effects such as nausea, sedation, pruritus, urinary retention, and respiratory depression.9
Intrathecal administration of monoamine reuptake inhibitors can enhance the antinociceptive effect of systemically injected opioids.8,10 However, there has been disagreement among clinical studies examining interactions between opioids and antidepressants. Depending on the drug and the type of pain, systemically administered antidepressants may enhance, have no effect on, or even decrease systemic opioid analgesia.11–13
Thus, the aim of this study was to compare the analgesic effects of intrathecal coadministration of morphine with the SSRI citalopram, the selective NE inhibitor maprotiline, and the nonselective antidepressant amitriptyline. The drug effects were examined in the thermal withdrawal test in rats using isobolographic analysis. We also tested the involvement of noradrenergic and opioidergic systems in the antinociceptive effect.
The Animal Care and Use Committee at Universidade Federal do Rio de Janeiro approved this study. Eighty-four male Wistar rats weighing 230–300 g were housed in a temperature- and humidity-controlled room with free access to food and water.
Amitriptyline hydrochloride, maprotiline hydrochloride, morphine sulfate, and citalopram hydrobromide were kindly donated by Cristália Produtos Químicos e Farmacêuticos Ltda (Itapira, SP, Brazil). Yohimbine hydrochloride and naloxone hydrochloride were purchased from Sigma (St. Louis, MO). All drugs were dissolved in saline, except yohimbine, which was dissolved in distilled water.
Intrathecal injection of drugs was performed as previously described.14 Briefly, under anesthesia with sevoflurane, rats were positioned for intrathecal injection of compounds at the L4–5 intervertebral space using a 30-gauge needle attached to a 300-μL syringe. The tail-flick reaction was used to confirm proper positioning of the needle in the intrathecal space.15 A 40-μL aliquot of drug solution was injected into each animal. Control animals received intrathecal saline only. Animals were allowed to recover from anesthesia for 10 min before starting the behavioral protocol.
The hind-paw thermal withdrawal test was used as previously described16 using an Analgesia Meter (IITC 336, Life Science Instruments, Woodland Hills, CA). Rats were individually placed in a box (20 cm × 20 cm × 20 cm) on heat-tempered glass. After 30-min of acclimatization period, a radiant heat source (12 V, 150 W halogen) with controlled bulb intensity was focused on the plantar surface of the hind-paw, and the paw withdrawal latency was recorded. The heat intensity was adjusted such that rats withdrew their paws within 2.5–3.5 s in the absence of a drug treatment. Both paws were tested and the average of their values was calculated. The cutoff time was 10 s to avoid tissue damage.
After 30 min of acclimatization, control hind-paw withdrawal latencies were obtained. Rats were then anesthetized with sevoflurane, and the test substances were administered intrathecally. Citalopram, amitriptyline, maprotiline, and morphine were injected at doses of 144, 125, 1.25, and 2 μg, respectively. The dose of antidepressants chosen for this study was selected based on a previous study using amitriptyline,17 as well as potency and molar relationships with animal behavior in preliminary experiments (see Discussion). Paw withdrawal latency was measured at 15, 30, 45, and 60 min posttreatment and every hour until rats exhibited complete recovery from the analgesic effect. Antinociceptive effects were determined as a percentage of the maximum possible effect (MPE) according to the following formula:
For drug combination treatments, morphine was mixed with citalopram, amitriptyline, or maprotiline in the same syringe and intrathecally injected. Yohimbine (4 mg/kg) and/or naloxone (10 mg/kg) were given intraperitoneally 15 min before the intrathecal injection of morphine-maprotiline.
Isobolographic analysis was performed for the combination of morphine and maprotiline. Dose-response curves for morphine (0.3, 1, 2, and 4 μg) and maprotiline (0.625, 1.25, 2.5, and 5 μg) were obtained, and the ED50 (dose that yields a 50% maximum effect) was determined by linear regression analysis. A dose-response curve was then obtained by intrathecal coadministration of morphine with maprotiline in fixed ratio combinations of fractions of their respective ED50 values. Separate groups received (morphine ED50 + maprotiline ED50)/2, (morphine ED50 + maprotiline ED50)/4, and (morphine ED50 + maprotiline ED50)/8. ED50 values of the combinations were determined and used to generate the isobologram plot. The additive line was constructed by connecting the ED50 of morphine plotted on the ordinate with the ED50 of maprotiline plotted on the abscissa, and the theoretical additive combination dose was calculated as described by Tallarida.18 Experimental values that were on or near the additive line were considered to be additive interactions, those that lay below and to the left were considered to be synergistic, and those that lay above and to the right were considered to be subadditive or antagonistic interactions.
Statistical Analysis of Behavioral Data
Data are presented as mean ± sem. Statistical analysis was performed by one-way analysis of variance followed by Student–Newman–Keuls test or Dunnett test for intergroup comparisons, with significant differences considered at P < 0.05. The area under the curve (AUC) depicting %MPE versus time was calculated by the trapezoidal rule and is presented as mean ± sem for each group. Statistical differences between theoretical and experimental values obtained in the isobologram were assessed by Student's t-test, with P < 0.05 considered significant in all cases.
Single Dose Combinations of Morphine with Citalopram, Amitriptyline, and Maprotiline
A single intrathecal injection of morphine (2 μg) caused a time-dependent antinociceptive effect in rats. The maximal effect (51.6% ± 8.9% MPE) was achieved 15 min after administration and decreased to baseline within 120 min (Fig. 1). Citalopram (144 μg) produced an antinociceptive effect of 33.8% ± 5.2% MPE. Intrathecal injection of morphine with citalopram did not change the intensity or duration of morphine antinociception (40.6% ± 4.6% MPE).
The maximal antinociceptive effect of amitriptyline (125 μg) was 10.3% ± 3.2% MPE. However, simultaneous administration of amitriptyline with morphine significantly increased the effect to 91.3% ± 8.6% MPE (P < 0.001) and increased the antinociceptive duration to approximately 300 min (Fig. 2).
Intrathecal administration of maprotiline (1.25 μg) caused an antinociceptive effect of 48.5% ± 4.1% MPE, which returned to baseline in 240 min. Combining maprotiline with morphine increased the antinociceptive effect of morphine to 86.9% ± 9.2% MPE (P < 0.001) and the duration of the effect to 480 min (Fig. 3).
Mechanism of Interaction of Morphine with Maprotiline
Figure 4 shows the AUC calculated from the time course of the antinociceptive effect induced by morphine (2 μg), maprotiline (1.25 μg), or their combination. The AUC of maprotiline (4976 ± 523 U) was larger (P < 0.05) than that of morphine (2640 ± 581 U). Pretreatment of the animals with yohimbine (4 mg/kg) decreased the AUC of maprotiline to 318 ± 93 U (P < 0.05). The combination of morphine with maprotiline yielded an AUC of 18148 ± 1390 U, which was decreased to 2503 ± 566 U (P < 0.001) by pretreatment with yohimbine. Naloxone (10 mg/kg) further blocked this activity (P < 0.001).
Isobolographic analysis was used to determine the type of pharmacologic interaction between morphine and maprotiline. The maximum effects observed with 0.62, 1.25, 2.5, and 5 μg of maprotiline were 35.8% ± 3.1%, 48.5% ± 4.1%, 54.5% ± 5.8%, and 60.0% ± 3.4% MPE, respectively, with an ED50 of 1.82 μg. The antinociceptive effects produced by the intrathecal administration of morphine at 0.3, 1, 2, and 4 μg were 25.5% ± 0.8%, 40.0% ± 2.9%, 51.6% ± 8.9%, and 80.0% ± 4.5% MPE, respectively, with an ED50 of 1.35 μg.
Morphine and maprotiline were intrathecally coadministered at dose fractions (1/8, 1/4, and 1/2) of the ED50 of each drug. These dosages yielded antinociceptive effects of 40.5% ± 3.5%, 62.0% ± 9.1%, and 70.7% ± 11.8% MPE, respectively (Fig. 5), with an ED50 of 0.54 μg. This ED50 was significantly less than the theoretical additive line generated by each drug alone (P < 0.01; Fig. 6), indicating the occurrence of a synergistic interaction.
Animal and human studies show opioid,19 NE,20 and 5-HT21 involvement in spinal cord pain modulation, supporting the use of monoamine reuptake inhibitors for clinical pain control.22 Here, we compared the antinociceptive effects induced by a single intrathecal injection of citalopram, amitriptyline, or maprotiline in combination with morphine. Doses were selected based on a previous publication,17 potency, and molar relationships as well as on animal observations in preliminary experiments. Side effects such as sedation and muscle paralysis were observed after intrathecal injection of 250 μg amitriptyline but not after 125 μg; this dose only produced an antinociceptive effect of about 10%, and higher doses caused reduction of motor activity (sedation). The dose of citalopram (144 μg) was determined by the molar equivalent to amitriptyline test doses, from 72 to 216 μg, and showed an ED50 of 161 μg (not shown), with 70.8% ± 3.1% MPE. The 144-μg dose gave a 33% nociceptive effect and no side effects. The molar equivalent dose of maprotiline (125 μg) produced several side effects, whereas a 10-fold lower dose (12.5 μg) produced a 90% antinociceptive effect that was too high for morphine interaction studies. A 100-fold lower dose of maprotiline (1.25 μg) produced a 50% MPE and no side effects in an open field test even at 12.5 μg (not shown).
The three examined compounds produced different antinociceptive effects, with amitriptyline being the least active, in agreement with previous studies on amitriptyline.8,23 Citalopram and maprotiline had comparable antinociceptive effects to morphine, suggesting that both 5-HT and NE are important for pain regulation. Analgesia can be induced by an intrathecal injection of 5-HT24 as well as SSRIs25 in acute pain models via activation of 5-HT1 receptors in thermal and noxious electrical stimuli.26 Maprotiline is approximately 470-fold more selective for NE reuptake than for 5-HT uptake,27 whereas citalopram is selective for 5-HT uptake. Both 5-HT and NE can modulate pain responses. SSRIs are 37% effective, and NE reuptake inhibitors are 64% effective in animal pain models.28 Maprotiline increases NE reuptake and probably activates α2-adrenoceptors to mediate its antinociceptive effect; this putative mechanism agrees with findings that morphine activity is enhanced by the α2-adrenoceptor agonist, ST-91, NE, or clonidine.29–31
These three antidepressants had different effects on the intensity and duration of morphine analgesia. Citalopram did not affect morphine analgesia, whereas amitriptyline and maprotiline increased both its intensity (by 70% for amitriptyline) and duration, by 2.5- and 4-fold for amitriptyline and maprotiline, respectively. In absolute values, maprotiline increased the duration of morphine analgesia from 120 to 480 min, a larger increase than any other published drug combination with morphine. Systemic coadministration of morphine and amitriptyline synergistically inhibits cutaneous orofacial inflammatory pain in rats.32 The inactivity of citalopram suggests that amitriptyline works in this model through inhibiting NE reuptake, as the effects with maprotiline confirm.
This is the first isobolographic analysis of morphine and maprotiline coadministered intrathecally in the thermal withdrawal test. Maprotiline activity could be reversed by yohimbine, indicating that an α2-adrenoceptor mechanism was at work. Naloxone further enhanced this inhibition, indicating that opioid receptors were also involved. Similar results with yohimbine have been observed in the tail-flick test after intrathecal administration of subthreshold doses of desipramine and morphine.33
Isobolographic analysis demonstrated a synergistic interaction between intrathecal morphine and maprotiline. Analgesia induced by α2-agonists and opioids involves peripheral, spinal, and brain sites. Both α2-adrenoceptors and opioid receptors are Gi/Go-coupled receptors that decrease neuronal excitation by inhibiting adenylyl cyclase and consequently reducing the formation of cyclic adenosine monophosphate, inhibiting Ca2+ channels, and opening K+ channels.34,35 Unfortunately, the isobolographic study cannot explain the mechanisms of the synergistic interaction of morphine and maprotiline. The fact that yohimbine, naloxone, or their coadministration is able to block the effects of the morphine-maprotiline combination agrees with these observations.
In conclusion, the intensity and duration of morphine- mediated antinociceptive activity can be synergistically increased with an NE reuptake inhibitor but not a 5-HT reuptake inhibitor. Maprotiline increased the duration of morphine analgesia 4-fold via activation of α2-adrenoreceptors. Maprotiline is not neurotoxic after intrathecal administration, and future work should test the combination of morphine and maprotiline in clinical practice.
1. Ghelardini C, Galeotti N, Bartolini A. Antinociception induced by amitriptyline and imipramine is mediated by alpha2A-adrenoceptors. Jpn J Pharmacol 2000;82:130–7
2. Otsuka N, Kiuchi Y, Yokogawa F, Masuda Y, Oguchi K, Hosoyamada A. Antinociceptive efficacy of antidepressants: assessment of five antidepressants and four monoamine receptors in rats. J Anaesth 2001;15:154–8
3. Micó JA, Ardid D, Berrocoso E, Eschalier A. Antidepressants and pain. Trends Pharmacol Sci 2006;27:348–54
4. Schreiber S, Pick CG. From selective to highly selective SSRIs: a comparison of the antinociceptive properties of fluoxetine, fluvoxamine, citalopram and escitalopram. Eur Neuropsychopharmacol 2006;16:464–8
5. Obata H, Saito S, Koizuca S, Nishikawa K, Goto F. The monoamine-mediated antiallodynic effects of intrathecally administered milnacipran, a serotonin norepinephrine reuptake inhibitor, in a rat model of neuropathic pain. Anesth Analg 2005;100:1406–10
6. Yokogawa F, Kiuchi Y, Ishikawa Y, Otsuka N, Masuda Y, Oguchi K, Hosoyamada A. An investigation of monoamine receptors involved in antinociceptive effects of antidepressants. Anesth Analg 2002;95:163–8
7. Gray AM, Pache DM, Sewell RD. Do alpha2-adrenoceptors play an integral role in the antinociceptive mechanism of action of antidepressant compounds? Eur J Pharmacol 1999;378:161–8
8. Eisenach JC, Gebhart GF. Intrathecal amitriptyline: antinociceptive interactions with intravenous morphine and intrathecal clonidine, neostigmine, and carbamylcholine in rats. Anesthesiology 1995;583:1036–45
9. Solomon RE, Gebhart GF. Synergistic antinociceptive interaction among drugs administered to the spinal cord. Anesth Analg 1994;78:1164–72
10. Botney M, Fields HL. Amitriptyline potentiates morphine analgesia by a direct action on the central nervous system. Ann Neurol 1983;13:160–4
11. Coda BA, Hill HF, Schaffer RL, Luger TJ, Jacobson RC, Chapman CR. Enhancement of morphine analgesia by fenfluramine in subjects receiving tailored opioid infusions. Pain 1993;52:85–91
12. Eisenach JC, Hood DD, Curry R, Tong CY. Alfentanil but not amitriptyline reduces pain, hyperalgesia, and allodynia from intradermal injection of capsaicin in humans. Anesthesiology 1997;86:1279–87
13. Gordon NC, Heller PH, Gear RW, Levine JD. Interactions between fluoxetine and opiate analgesia for postoperative dental pain. Pain 1994;58:85–8
14. Papir-Kricheli D, Frey J, Laufer R, Gilon C, Chorev M, Selinger Z, Devor M. Behavioural effects of receptor-specific substance P agonists. Pain 1987;31:263–76
15. Storkson RV, Kjorsvik A, Tjolsen A, Hole K. Lumbar catheterization of the spinal subarachnoid space in the rat. J Neurosci Methods 1996;65:167–72
16. Meller ST, Cummings CP, Traub RJ, Gebhar GF. The role of nitric oxide in the development and maintenance of the hyperalgesia produced by intraplantar injection of carrageenan in the rat. Neuroscience 1994;60:367–74
17. Gerner P, Haderer AE, Mujtaba M, Sudoh Y, Narang S, Abdi S, S
rinivasa V, Pertl C, Wang GK. Assessment of differential blockade by amitriptyline and its N
-methyl derivative in different species by different routes. Anesthesiology 2003;98:1484–90
18. Tallarida RJ. Statistical analysis of drug combinations for synergism. Pain 1992;49:93–7
19. Watanabe C, Okuda K, Sakurada C, Ando R, Sakurada T, Sakurada S. Evidence that nitric oxide-glutamate cascade modulates spinal antinociceptive effect of morphine: a behavioural and microdialysis study in rats. Brain Res 2003;990:77–86
20. Reddy SV, Yaksh TL. Spinal noradrenergic terminal system mediates antinociception. Brain Res 1980;189:391–401
21. Wang JK. Antinociceptive effect of intrathecally administered serotonin. Anesthesiology 1977;47:269–71
22. Hwang AS, Wilcox GL. Analgesic properties of intrathecally administered heterocyclic antidepressants. Pain 1987;28:343–55
23. Dirksen R, Van Diejen D, Van Luijtelaar EL, Booij LH. Site- and test-dependent antinociceptive efficacy of amitriptyline in rats. Pharmacol Biochem Behav 1994;47:21–6
24. Bardin L, Lavarenne J, Eschalier A. Serotonin receptor subtypes involved in the spinal antinociceptive effect of 5-HT in rats. Pain 2000;86:11–8
25. Schreiber S, Backer MM, Yannai J, Pick CG. The antinociceptive effect of fluvoxamine. Eur Neuropsychopharmacol 1996;6:281–4
26. Nadeson R, Goodchild CS. Antinociceptive role of 5-HT1A receptors in rat spinal cord. Br J Anaesth 2002;88:679–84
27. Richelson E. Pharmacology of antidepressants. Psychopathology 1987;1:1–12
28. Eschalier A, Mestre C, Dubray C, Ardid D. Why are antidepressants effective as pain relief? CNS Drugs 1994;2:261–7
29. Monasky MS, Zinsmeister AR, Stevens CW, Yaksh TL. Interaction of intrathecal morphine and ST-91 on antinociception in the rat: dose-response analysis, antagonism and clearance. J Pharmacol Exp Ther 1990;254:383–92
30. Plummer JL, Cmielewski PL, Gourlay GK, Owen H, Cousins MJ. Antinociceptive and motor effects of intrathecal morphine combined with intrathecal clonidine, norepinephrine, carbachol or midazolam in rats. Pain 1992;49:145–52
31. Wilcox GL, Carlsson KH, Jochim A, Jurna I. Mutual potentiation of antinociceptive effects of morphine and clonidine on motor and sensory responses in rat spinal cord. Brain Res 1987;405:84–93
32. Luccarini P, Perrier L, Dégoulange C, Gaydier AM, Dallel R. Synergistic antinociceptive effect of amitriptyline and morphine in the rat orofacial formalin test. Anesthesiology 2004;100:690–6
33. Reimann W, Schlütz H, Selve N. The antinociceptive effects of morphine, desipramine, and serotonin and their combinations after intrathecal injection in the rat. Anesth Analg 1999;88:141–5
34. Stone LS, MacMillan LB, Kitto KF, Limbird, LE, Wilcox GL. The alpha2a adrenergic receptor subtype mediates spinal analgesia evoked by alpha2 agonists and is necessary for spinal adrenergic-opioid synergy. J Neurosci 1997;17:7157–65
© 2009 International Anesthesia Research Society
35. Law PY, Wong YH, Loh HH. Molecular mechanisms and regulation of opioid receptor signaling. Annu Rev Pharmacol Toxicol 2000;40:389–430