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, 8 (9), e74891
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Relative Contributions of Norepinephrine and Serotonin Transporters to Antinociceptive Synergy Between Monoamine Reuptake Inhibitors and Morphine in the Rat Formalin Model

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Relative Contributions of Norepinephrine and Serotonin Transporters to Antinociceptive Synergy Between Monoamine Reuptake Inhibitors and Morphine in the Rat Formalin Model

Fei Shen et al. PLoS One.

Abstract

Multimodal analgesia is designed to optimize pain relief by coadministering drugs with distinct mechanisms of action or by combining multiple pharmacologies within a single molecule. In clinical settings, combinations of monoamine reuptake inhibitors and opioid receptor agonists have been explored and one currently available analgesic, tapentadol, functions as both a µ-opioid receptor agonist and a norepinephrine transporter inhibitor. However, it is unclear whether the combination of selective norepinephrine reuptake inhibition and µ-receptor agonism achieves an optimal antinociceptive synergy. In this study, we assessed the pharmacodynamic interactions between morphine and monoamine reuptake inhibitors that possess different affinities and selectivities for norepinephrine and serotonin transporters. Using the rat formalin model, in conjunction with measurements of ex vivo transporter occupancy, we show that neither the norepinephrine-selective inhibitor, esreboxetine, nor the serotonin-selective reuptake inhibitor, fluoxetine, produce antinociceptive synergy with morphine. Atomoxetine, a monoamine reuptake inhibitor that achieves higher levels of norepinephrine than serotonin transporter occupancy, exhibited robust antinociceptive synergy with morphine. Similarly, a fixed-dose combination of esreboxetine and fluoxetine which achieves comparable levels of transporter occupancy potentiated the antinociceptive response to morphine. By contrast, duloxetine, a monoamine reuptake inhibitor that achieves higher serotonin than norepinephrine transporter occupancy, failed to potentiate the antinociceptive response to morphine. However, when duloxetine was coadministered with the 5-HT3 receptor antagonist, ondansetron, potentiation of the antinociceptive response to morphine was revealed. These results support the notion that inhibition of both serotonin and norepinephrine transporters is required for monoamine reuptake inhibitor and opioid-mediated antinociceptive synergy; yet, excess serotonin, acting via 5-HT3 receptors, may reduce the potential for synergistic interactions. Thus, in the rat formalin model, the balance between norepinephrine and serotonin transporter inhibition influences the degree of antinociceptive synergy observed between monoamine reuptake inhibitors and morphine.

Conflict of interest statement

Competing Interests: The authors are current employees of Theravance, Inc., a commercial company which has an interest in the development of novel monoamine reuptake inhibitors. Pending Patents: “Combination of a serotonin and norepinephrine reuptake inhibitor and an opioid agonist for the treatment of pain.” This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. All materials used in this publication are commercially available.

Figures

Figure 1
Figure 1. Neither esreboxetine nor fluoxetine exhibited antinociceptive synergy with morphine in the rat formalin model.
(A) The selective NET inhibitor esreboxetine alone (Esrbx, IP, 10 mg/kg), failed to shift the morphine (Mor) dose-response curve (n = 6). Morphine alone: ED50 = 2.0 mg/kg (95% CI: 1.8–2.5); morphine+ esreboxetine (IP, 10 mg/kg): ED50 = 1.6 mg/kg (95% CI: 1.1–2.4). All data points are shown as mean ± SEM for each group and are expressed as percentage of controls. Inset (A) Esreboxetine (IP, 10 mg/kg) was associated with 82±5% NET and 3±5% SERT occupancy measured ex vivo at 75 min post-dose. All occupancy data represent mean (± SEM) for each group. (B) The selective SERT inhibitor fluoxetine alone (Flx, IP, 10 mg/kg), failed to shift the morphine dose-response curve (n = 6). Morphine+fluoxetine (IP, 10 mg/kg): ED50 = 1.6 mg/kg (95% CI: 1.2–2.2). Inset (B) Fluoxetine (IP, 10 mg/kg) was associated with 6±12% NET and 89±5% SERT occupancy measured ex vivo at 75 min post-dose.
Figure 2
Figure 2. Atomoxetine exhibited antinociceptive synergy with morphine using a fixed-dose design in the rat formalin model.
(A) Both 3 and 10 mg/kg atomoxetine (Atx, IP) shifted the morphine (Mor) dose-response curve leftward in the rat formalin model (n = 6–16). Morphine alone: ED50 = 2.3 mg/kg (95% CI: 2.0–2.5); morphine+atomoxetine (IP, 3 mg/kg): ED50 = 1.1 mg/kg (95% CI: 0.8–1.6); and morphine+atomoxetine (IP, 10 mg/kg): ED50 = 0.6 mg/kg (95% CI: 0.4–0.8). All data points are shown as mean ± SEM for each group and are expressed as percentage of controls. Inset (A) Atomoxetine (IP) at 3 and 10 mg/kg was associated with 67±10% and 84±3% for NET and 35±9% and 64±5% for SERT occupancy measured ex vivo at 75 min post-dose, respectively. All occupancy data represent mean (± SEM) for each group. (B) A subefficacious dose of morphine 1 mg/kg (SC) left-shifted the atomoxetine dose-response curve (n = 6–16). Atomoxetine alone: ED50 = 27.8 mg/kg (95% CI: 22–36); and atomoxetine+morphine (SC, 1 mg/kg): ED50 = 2.5 mg/kg (95% CI: 1.3–4.7). (C) A fixed combination of NET selective inhibitor esreboxetine (Esrbx, IP, 10 mg/kg) and SERT selective inhibitor fluoxetine (Flx, IP, 1 mg/kg) left-shifted the morphine dose-response curve (n = 6–12). Morphine alone: ED50 = 2.3 mg/kg (95% CI: 2.0–2.5); morphine+esreboxetine (IP, 10 mg/kg)+fluoxetine (IP, 1 mg/kg): ED50 = 0.3 mg/kg (95% CI: 0.2–0.7).
Figure 3
Figure 3. Atomoxetine exhibited antinociceptive synergy with morphine using a fixed-ratio design in the rat formalin model.
(A) The dose-response curve of a fixed-ratio of 3 parts atomoxetine (Atx, IP) to 1 part morphine (Mor, SC) leftward shifted relative to the atomoxetine dose-response curve alone (n = 6–12). All data points are shown as mean ± SEM for each group and are expressed as percentage of controls. (B) An isobologram for the combined effects of atomoxetine and morphine in a fixed ratio combination 3∶1. The ED50 value for morphine is plotted on the abscissa, and the ED50 value for atomoxetine is plotted on the ordinate. The solid line represents the line of additivity and the isobol point (observed ED50 value) is located to the left and below the theoretical additive ED50 value (with non-overlapping 95% CI). (C) The dose-response curve of a fixed-ratio of concomitant administration of 10 part atomoxetine (IP) to 1 part morphine (SC) leftward shifted relative to the atomoxetine dose-response curve alone (n = 6–16). All data points are shown as mean ± SEM for each group and are expressed as percentage of controls. (D) An isobologram for the combined effects of atomoxetine and morphine in a fixed ratio combination 10∶1. The isobol point (observed ED50 value) is located to the left and below the theoretical additive ED50 value (without overlapping 95% CI).
Figure 4
Figure 4. The antinociceptive activity of atomoxetine in the rat formalin model was independent of µ-opioid receptor activation.
The µ-opioid receptor antagonist naloxone (Nal, IP, 5 mg/kg), at a dose which effectively blocked morphine (Mor)-induced analgesia in the rat formalin model, did not inhibit atomoxetine (Atx)-induced antinociception (n = 5–7). All values are shown as mean ± SEM for each group and are expressed as percentage of controls. Student’s t test, t (10) = 7.668, ***p<0.001.
Figure 5
Figure 5. Antinociceptive synergy between atomoxetine and morphine did not reflect impaired motor coordination.
The white bars represent the % reduction in the flinching behavior compared to vehicle-treatment in the rat formalin model (n = 10–22), and the grey bars represent the change in latency for rats to fall from an accelerating rotating rod compared to vehicle treatment in the rat RotaRod test (n = 8). All data points are shown as mean ± SEM for each group and are expressed as percentage of controls. Data from one-way ANOVA are as follows: rat formalin model: F (4, 53) = 36.12, p<0.0001; RotaRod: F (4, 34) = 4.604, p = 0.004. Data from the post hoc Dunnett’s test follows: **p<0.01, q = 3.265; ***p<0.001, q = 9.258–9.370, compared to vehicle treatment.
Figure 6
Figure 6. Duloxetine failed to exhibit antinociceptive synergy with morphine in the rat formalin model.
(A) Duloxetine (Dlx) at 5 mg/kg failed to shift the morphine (Mor) dose-response curve leftward. Morphine alone: ED50 = 2.3 mg/kg (95% CI: 2.0–2.5); morphine+duloxetine (IP, 5 mg/kg): ED50 = 2.0 mg/kg (95% CI: 1.3–3.0). All data points are shown as mean ± SEM for each group and are expressed as percentage of controls. Inset (A) Duloxetine (IP) at 5 mg/kg was associated with 62±5% for NET and 92±3% for SERT occupancy measured ex vivo at 75 min post-dose. All occupancy data represent mean (± SEM) for each group. (B) A subefficacious dose of morphine 1 mg/kg (SC) failed to left-shift the duloxetine dose-response curve (n = 6–12). Duloxetine alone: ED50 = 10.9 mg/kg (95% CI: 8–15); and duloxetine+morphine (SC, 1 mg/kg): ED50 = 7.7 mg/kg (95% CI: 4–16).
Figure 7
Figure 7. Coadministration of ondansetron potentiates the antinociceptive response to duloxetine and morphine in the rat formalin model.
(A) Co-administration of the 5-HT3 receptor selective antagonist ondansetron (Ond, IP, 3 mg/kg) potentiates the antinociceptive response to duloxetine (Dlx, IP, 5 mg/kg) and morphine (Mor, SC, 1 mg/kg). Ondansetron alone, or in combination with either morphine or duloxetine, did not exhibit antinociceptive activity (n = 6–19). All data points are shown as mean ± SEM for each group and are expressed as percentage of controls. One-way ANOVA: F (7, 75) = 7.447, p<0.0001. Data from the post hoc Newman-Keuls test follows: ***p<0.001, q = 4.956–9.764 for duloxetine+morphine+ondansetron versus the other groups. (B) Co-administration of ondansetron (IP, 3 mg/kg) did not reveal antinociceptive synergy between the SERT selective reuptake inhibitor fluoxetine (Flx, IP, 10 mg/kg) and morphine (SC, 1 mg/kg). Ondansetron alone, or in combination with either morphine or fluoxetine, did not exhibit antinociceptive activity (n = 7). One-way ANOVA followed by post hoc Newman-Keuls: p>0.05 morphine versus fluoxetine+morphine; p>0.05 fluoxetine+morphine versus fluoxetine+morphine+ondansetron.

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