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The Synergistic Interaction Between Morphine and Maprotiline After Intrathecal Injection in Rats

Pettersen, Vera L. A., MD*; Zapata-Sudo, Gisele, MD, PhD; Raimundo, Juliana M., PhD; Trachez, Margarete M., MD, PhD; Sudo, Roberto T., MD, PhD

doi: 10.1213/ane.0b013e3181b16ff5
Analgesia: Pain Mechanisms: Research Reports
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BACKGROUND: Antidepressant drugs act as potent inhibitors of norepinephrine and/or serotonin reuptake and are widely used with opioids for the treatment of chronic pain. The mechanism of this increased analgesic action is unclear. We compared the antinociceptive effects of the intrathecal administration of morphine with that of a nonselective (amitriptyline) or selective (maprotiline or citalopram) antidepressant drug using the thermal withdrawal test in rats. We also investigated the possible mechanisms involved in the interactions of these drugs.

METHODS: Male Wistar rats were anesthetized with sevoflurane and administered morphine and antidepressant drugs, or saline, through intrathecal injection. The antinociceptive effect was evaluated using the thermal withdrawal test before and after drug administration. The time for the withdrawal reaction was expressed as percentage of maximum possible effect (MPE). Animals were also pretreated with yohimbine (a nonselective alpha2-adrenergic antagonist) and naloxone (a nonselective opioid antagonist) for mechanism of action studies. Pharmacologic interaction was evaluated using isobolographic analysis of simultaneous administration of fixed proportions of maprotiline and morphine.

RESULTS: Single intrathecal administration of morphine (2 μg), amitriptiline (125 μg), citalopram (144 μg), and maprotiline (1.25 μg) produced 51.6% ± 8.9%, 10.3% ± 3.2%, 33.8% ± 5.2%, and 48.5% ± 9.2% MPE, respectively. The antinociceptive effect of morphine was increased when combined with amitriptyline (91.3% ± 4.6% MPE) and maprotiline (86.9% ± 9.2% MPE) but not with citalopram (40.6% ± 4.6% MPE). Coadministration of maprotiline increased the antinociceptive duration of morphine by 4-fold (from 120 to 480 min), which was reversed by pretreatment with the α-2 adrenoceptor inhibitor, yohimbine, and the mu-type opioid receptor antagonist, naloxone. Isobolographic analysis demonstrated a synergistic interaction between morphine and maprotiline.

CONCLUSIONS: Selective norepinephrine reuptake inhibitors can significantly increase the intensity and duration of morphine antinociceptive activity via both α2-adrenergic and opioid receptors. This interaction was not observed with the selective serotonin inhibitor, citalopram.

From the *Programa de Pós-Graduação em Cirurgia Geral da Universidade Federal do Rio de Janeiro; †Programa de Desenvolvimento de Fármacos do Instituto de Ciências Biomédicas da Universidade Federal do Rio de Janeiro; and ‡Serviço de Anestesiologia da Universidade Federal Fluminense, Rio de Janeiro, Brazil.

Accepted for publication May 15, 2009.

Supported by Coordenação de Aperfeiçoamento de Nível Superior (CAPES, BR), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, BR), Cristália Produtos Químicos e Farmacêuticos Ltda (BR), Fundação Universitária José Bonifácio (BR). Fellowships from CAPES (VLAP, JMR) and CNPq (GZS, RTS).

Address correspondence and reprint requests to Roberto Takashi Sudo, MD, PhD, Centro de Ciências da Saúde, Bloco J, Sala 14, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas 373, Cidade Universitária, Rio de Janeiro 21941-590, Brazil. Address e-mail to rtsudo@farmaco.ufrj.br.

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.

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METHODS

Animals

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.

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Drugs

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.

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Intrathecal Injection

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.

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Nociceptive Testing

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.

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Experimental Protocol

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.

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Isobolographic Analysis

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.

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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.

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RESULTS

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).

Figure 1.

Figure 1.

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).

Figure 2.

Figure 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).

Figure 3.

Figure 3.

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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).

Figure 4.

Figure 4.

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Isobolographic Analysis

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.

Figure 5.

Figure 5.

Figure 6.

Figure 6.

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DISCUSSION

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.

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