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adverse effect ratio
European Journal of Pharmacology 848 (2019) 80–87
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
The combination of opioid and neurotensin receptor agonists improves their analgesic/adverse effect ratio
T
⁎
Emilie Eiselt, Jérôme Côté, Jean-Michel Longpré, Véronique Blais, Philippe Sarret , ⁎ Louis Gendron Département de pharmacologie-physiologie, Institut de pharmacologie de Sherbrooke, Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Isobologram Analgesia Constipation Formalin Morphine Neurotensin
Opioid and neurotensin (NT) receptors are expressed in both central and peripheral nervous systems where they modulate nociceptive responses. Nowadays, opioid analgesics like morphine remain the most prescribed drugs for the treatment of moderate to severe pain. However, despite their daily used, opioids can produce lifethreatening side effects, such as constipation or respiratory depression. Besides, NT analogs exert strong opioidindependent analgesia. Here, we thus hypothesized that the combined use of opioid and NT agonists would require lower doses to produce significant analgesic effects, hence decreasing opioid-induced adverse effects. We used isobologram analyses to determine if the combination of a NT brain-penetrant analog, An2-NT(8-13) with morphine results in an inhibitory, synergistic or additive analgesic response. We found that intravenous administration of An2-NT(8-13) reduced by 90% the nocifensive behaviors induced by formalin injection, at the dose of 0.018 mg/kg. Likewise, subcutaneous morphine reduced pain by 90% at 1.8 mg/kg. Importantly, isobologram analyses revealed that the co-injection of An2-NT(8-13) with morphine induced an additive analgesic response. We finally assessed the effects of morphine and An2-NT(8-13) on the gastrointestinal tract motility using the charcoal meal test. As opposed to morphine which significantly reduced the intestinal motility at the analgesic effective dose of 1.8 mg/kg, An2-NT(8-13) did not affect the charcoal meal intestinal transit at 0.018 mg/kg. Interestingly, at the dose providing 90% pain relief, the co-administration of morphine with An2NT(8-13) had a reduced effect on constipation. Altogether, these results suggest that combining NT agonists with morphine may improve its analgesic/adverse effect ratio.
1. Introduction Nowadays, opioids remain the most prescribed analgesics to treat moderate to severe pain (Gomes et al., 2014; Munzing, 2017). Morphine, the prototypical opioid, mediates its actions mainly through the Mu opioid receptor (MOP). In addition to analgesia, morphine also produces a number of unwanted and debilitating effects such as drowsiness, constipation, nausea, respiratory depression, and tolerance (Benyamin et al., 2008). Analgesic tolerance to morphine develops such that the dose needed to maintain a given level of analgesia has to be constantly increased. In order to limit the development of analgesic tolerance and concomitant unwanted effects, drug combination represents a promising alternative to currently used opioids. Indeed, combining an opioid with another analgesic drug might reduce the unwanted effects normally associated to each individual drug. This was
indeed found to be true when morphine was intrathecally injected with dexmedetomidine, a selective α2-adrenergic receptor agonist in a rat model of neuropathic pain (Kabalak et al., 2013). Similarly, the combined use of morphine and monoamine reuptake inhibitors was found to potentiate their respective analgesic effects in the rat formalin model (Shen et al., 2013). We and others have shown that neurotensin (NT) can produce significant pain relief in various animal models of acute and chronic pain (Boules et al., 2006; Demeule et al., 2014; Fanelli et al., 2015; Fantegrossi et al., 2005; Guillemette et al., 2012; Roussy et al., 2008; Tétreault et al., 2013). First identified for its hypotensive effects, NT is also know to participate in a wide variety of physiological functions, including modulation of body temperature, myocardial performance, gastrointestinal tract motility and secretion (Osadchii, 2015; Zhao and Pothoulakis, 2006). According to its wide distribution throughout the
⁎ Correspondence to: Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, Québec, Canada J1H 5N4. E-mail addresses: [email protected] (P. Sarret), [email protected] (L. Gendron).
https://doi.org/10.1016/j.ejphar.2019.01.048 Received 22 October 2018; Received in revised form 24 January 2019; Accepted 28 January 2019 Available online 29 January 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.
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contralateral, has little or no weight placed on it; 2, injected paw is elevated, not in contact with any surface; 3, injected paw is licked, bitten, or flinched. The behaviors believed to represent higher levels of pain intensity were given higher weighted scores. The weighted average pain intensity score (ranging 1–3) was then calculated by multiplying the time spent in each category by the category weight, summing these products, and dividing by the total time in a given interval of time. For a 180-second block, the pain score is thus be calculated as (1T1 + 2T2 + 3T3)/180, in which T1, T2, and T3 represent duration (in s) spent in behavioral categories 1, 2 and 3, respectively. Phase I and II values were calculated between 0–9 and 21–60 min, respectively. Effective doses (ED30, ED50, ED70 and ED90 values) for the inflammatory phase (phase II) of the formalin test were calculated from the dose-response curves generated for morphine sulfate (doses ranging from 0.3 to 10 mg/kg), An2-NT(8-13) (doses ranging from 0.005 to 0.15 mg/kg), and a combination of a constant dose of An2-NT(8-13) (0.009 mg/kg) and morphine sulfate (doses ranging from 0.1 to 10 mg/ kg).
central nervous system (Sarret and Beaudet, 2002), NT was also shown to exert brain functions, such as antipsychotic- and psychostimulantlike effects as well as regulation of food intake (Dobner, 2005; Dobner et al., 2003; Mazella et al., 2012). The physiological effects of NT are mediated by the activation of two closely related G protein-coupled receptors (GPCRs), namely NTS1 and NTS2 (Vincent et al., 1999). Importantly, NT-mediated analgesia was found to be independent of the opioid system. There is indeed compelling evidence demonstrating that opioid antagonists do not suppress NT-induced antinociception (Behbehani and Pert, 1984; Clineschmidt et al., 1982; Osbahr et al., 1981). Furthermore, NT also appears to be more potent than morphine in various pain tests (Nemeroff et al., 1979; al-Rodhan et al., 1991). In the present study, we hypothesized that combining opioid and NT peptides will present some advantages over pure opioid-based therapies. We thus undertook to determine whether systemically administered morphine combined to An2-NT(8-13), consisting of the brainpenetrant peptide Angiopep-2 (An2) conjugated to NT(8-13) (Demeule et al., 2014), can exert additive or synergistic antinociceptive interaction. We therefore evaluated the analgesic effects of combining morphine and An2-NT(8-13) in the rat model of formalin-induced persistent pain, and determined the extent of drug interaction using isobolograms. We further determined whether this drug combination can reduce opioid-induced constipation or NT-induced drop in blood pressure, thus leading to a better analgesic benefit/adverse effect ratio.
2.3. An2-NT(8-13) synthesis and conjugation Angiopep-2-NT(8-13) (An2-NT(8-13)) was synthesized by conjugation of the NT(8-13) peptide to the Angiopep-2-Cys (TFFYGGSRGKRNNFKTEEYC-NH2), which were both prepared by solid-phase peptide synthesis on a Symphony® peptide synthesizer (Protein Technologies Inc., Tucson, AZ, USA) using standard Fmoc chemistry with commercially available Fmoc amino acids (Chem-Impex International, Inc., Wood Dale, IL, USA). Analysis of purity for each peptide was performed by ACQUITY ultra-performance liquid chromatography (UPLC) (Waters Corporation, Milford, MA, USA) on a BEH Phenyl column at a flow rate of 0.5 ml min−1 and a constant temperature of 23 °C. Detection was performed at a wavelength of 229 nm, data acquisition and processing were done using the Compass™ suite of instrument control and data processing software (©Bruker Daltonik, Bremen, Germany). Peptide identity was confirmed on a micrOTOF ESI-TOF mass spectrometer (Bruker Daltronics, Inc., Billerica, MA, USA) with m/z ratios for the purified peptides of 817.01 Da for NT(8-13) (C38H64N12O8) and 2403.63 Da for the Angiopep-2-Cys (C107H155N31O31S). The conjugate An2-NT(8-13) was subsequently synthesized in phosphate buffered saline (PBS) by a two-step ligation method at room temperature (RT), monitored by RP-UPLC. The first conjugation step was performed in PBS 4 × , pH = 7.2 buffer, while the second step was in PBS 4 × , pH = 5.1. In the first step, a sulfo-EMCS (Molecular Biosciences Inc., Boulder, CO, USA) was used to incorporate a maleimidyl linker sitespecifically at arginine 8 of NT(8-13), resulting in [Arg(MHA)8]NT. In the second step, conjugation with the C-terminal Cys-modified An2 peptide was performed. Both reactions were stopped by the addition of 20% acetic acid. Purification was performed on a preparative HPLC column.
2. Materials and methods 2.1. Animals Adult male Sprague-Dawley rats (225–250 g; Charles River Laboratories, St-Constant, QC, Canada) were maintained in a 12-h light/dark cycle and had ad libitum access to lab chow and water. Rats were acclimatized for 3 days to experimental room, manipulations and devices prior to the behavioral studies. All experiments were performed in a quiet room, different than the housing area, between 8:00 a.m. and 2:00 p.m. by the same experimenter. All experiments were approved by the animal care committee of the Université de Sherbrooke (Protocols #356-13/18 and 035-13/18) and all procedures conformed to the directives of the Canadian Council on Animal Care and guidelines of the International Association for the Study of Pain. All animal experiments were designed to minimize the number of animals used and their suffering. 2.2. Formalin test Antinociception was assessed using the formalin test as a model of tonic pain. For this purpose, male Sprague-Dawley rats were habituated for 30 min to the experimentation room. Thereafter, the rats received a 50 μl s.c. injection of 2% formaldehyde (i.e. 5% formalin, Fisher Scientific, Ottawa, ON, Canada) into the plantar surface of the right hind paw. Rats were then placed in clear plastic chambers (40 cm × 30 cm × 30 cm) equipped with a glass floor positioned over a mirror angled at 45° in order to allow an unobstructed view of the paws, and their behaviors were scored during 60 min. The intraplantar injection of formalin produces a typical biphasic nociceptive response (Sawynok and Liu, 2003). These two distinct phases of spontaneous pain behaviors that occur in rodents are proposed to reflect a direct effect of formalin on sensory nociceptors, corresponding to an acute phase (phase I), and a longer-lasting pain due to inflammation and central sensitization (phase II). These two phases are separated by a period of quiescence, called interphase, which is characterized by the active inhibition of the formalin-induced nociceptive behaviors. The nocifensive behaviors were assessed using a weighted score method (Coderre et al., 1993; Dubuisson and Dennis, 1977). After the injection of formalin into the right hind paw, the time spent was measured in each of three behavioral categories: 1, injected paw is comparable to
2.4. Dose-response studies with the formalin test 2.4.1. Morphine alone Five groups of rats were injected s.c. with various doses of morphine sulfate (0.3; 1; 1.5; 3 and 10 mg/kg) and saline i.v. five min. before the injection of a solution of 2% formaldehyde in the hind paw. Morphine sulfate was purchased from Medisca (St-Laurent, QC, Canada) and dissolved in 0.9% saline solution before use. 2.4.2. An2-NT(8-13) alone Four groups of rats were injected with various doses of An2-NT(813) (0.005; 0.015; 0.05 and 0.15 mg/kg i.v.) and saline s.c five min. before the injection of a solution of 2% formaldehyde in the hind paw. 2.4.3. Morphine plus An2-NT(8-13) Five groups of rats were injected with different doses of morphine 81
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sulfate (0.1; 0.3; 1; 3 and 10 mg/kg s.c.) and a fixed dose of An2-NT(813) (0.009 mg/kg i.v.) five min before the injection of a solution of 2% formaldehyde in the hind paw.
inflammatory phase (21–60 min). Indeed, at 3 mg/kg and 10 mg/kg, morphine completely inhibited the formalin-induced nociceptive behaviors during the inflammatory phase. Morphine also reduced the nocifensive behaviors during the acute phase (0–9 min), but only at the highest dose of 10 mg/kg. As shown in Fig. 1B, the administration of An2-NT(8-13) (0.005–0.15 mg/kg, i.v.) also produced dose-dependent antinociception in both phases of the formalin test. The potencies of morphine and An2-NT(8-13) were then determined from the area under the curves, calculated for each dose of the compounds. To this purpose, only the early inflammatory phase (21–40 min) was considered. We found that morphine inhibited formalin-induced pain with an ED30 of 1.25 mg/kg, ED50 of 1.39 mg/kg, ED70 of 1.53 mg/kg, and ED90 of 1.8 mg/kg (Fig. 1C; Table 1). Likewise, An2-NT(8-13) induced antinociception with an ED30 of 0.013 mg/kg, ED50 of 0.014 mg/kg, ED70 of 0.015 mg/kg, and ED90 of 0.018 mg/kg (Fig. 1D; Table 1). In order to evaluate if morphine and An2-NT(8-13) interacted in an additive or synergistic manner to produce antinociception, we first generated isobolograms from the doses required to produce 30%, 50%, 70% and 90% antinociception (Fig. 2) using the ED30, ED50, ED70 and ED90 values obtained for morphine and An2-NT(8-13) alone, as determined on the dose-response curves illustrated in Fig. 1C and D. To measure a possible interaction between morphine and An2-NT(8-13), the antinociceptive effect of increasing doses of morphine (0.1–3 mg/ kg) co-administered with a constant, sub-analgesic dose of An2-NT(813) (0.009 mg/kg i.v) was determined. As shown in Fig. 3A, the combination of morphine and An2-NT(8-13) produced a time- and dose (morphine)-dependent antinociceptive effect in the formalin test. When the antinociceptive effect of the drug combination is measured as the area under the curve (AUC; for the 21–40 min phase II), ED30, ED50, ED70 and ED90 values for morphine in the presence of An2-NT(8-13) can be determined. Graphical determination revealed that the ED30, ED50, ED70 and ED90 values of morphine, when co-administered with 0.009 mg/kg of An2-NT(8-13), are respectively 0.25 mg/kg, 0.39 mg/ kg, 0.6 mg/kg and 1.2 mg/kg (Fig. 3B; Table 1). Reporting these combinations on the corresponding isobolograms of Fig. 2 revealed an additive antinociceptive effect of morphine and An2-NT(8-13) combination as every combinations felt on the theoretical line of additivity for these drugs.
2.4.4. Isobolographic analysis The assessment of the additive effect of morphine and An2-NT(8-13) was done graphically using isobolograms (Gessner, 1995; Tallarida, 2006). The ED values for An2-NT(8-13) and morphine injected alone were respectively placed on the X and Y axes. The line connecting the ED value of each individual drug represents the theoretical line of additivity for these two drugs. On isobolograms, additivity is found when the level of antinociception of the drug combination falls on or in close proximity to the theoretical line of additivity. When the effect of the drug combination falls above or below the line of additivity, the interaction between drugs is respectively considered inhibitory and synergistic. 2.5. Charcoal meal test Constipation was assessed by measuring the gastrointestinal tract motility using the charcoal meal test. Briefly, food deprived (16 h) animals are injected (i.v. or s.c.) with saline, morphine sulfate (1.8 mg/kg s.c or 5 mg/kg s.c), An2-NT(8-13) (0.018 mg/kg i.v), or co-injected with morphine sulfate (0.9 mg/kg s.c) and An2-NT(8-13) (0.009 mg/kg i.v) or with morphine sulfate (5 mg/kg s.c) and An2-NT(8-13) (0.05 mg/kg i.v). Thirty min. after drug injection, 2 ml of a charcoal meal solution (5% arabic gum and 10% charcoal in water) is administered to the rats by gavage. The animals are euthanized 60 min after and the progression of the charcoal in the intestine is measured as a ratio of progression/ total length of the intestine. Results are presented as the percentage of progression of the charcoal meal in the intestine. 2.6. Blood pressure test Rats were anaesthetized with a mixture of ketamine/xylaxine (87 mg/kg: 13 mg/kg, i.m.) and placed in supine position on a heating pad. Mean arterial blood pressure was measured through a PE50 catheter filled with heparinized saline inserted in the right carotid artery and connected to a Micro-Med transducer (model TDX-300, Micromed LLC, Ampler, PA, USA) linked to a blood pressure Micro-Med analyzer (model BPA-100c, Micromed LLC). When basal pressure was stabilized, rats were first given a vehicle or An2-NT(8-13) intravenously (caudal vein), and vehicle or morphine subcutaneously 30 s later. In one group, morphine was administered 30 min prior to An2-NT(8-13). Only one paradigm of injections was performed prior to euthanasia. Changes in blood pressure from the baseline to maximal effect post-injection in individual animals were determined. Data represents the mean ± S.E.M. of at least six different experiments.
3.2. Effect of morphine and An2-NT(8-13) on the gastrointestinal tract motility One of the main adverse effects of morphine is undoubtedly its propensity to produce constipation (Holzer, 2009). To see whether or not the combination of morphine with An2-NT(8-13) can advantageously be used to produce antinociception without inhibiting the gastrointestinal tract motility, the effect of morphine (1.8 mg/kg), An2NT(8-13) (0.018 mg/kg) and of a combination of morphine (0.9 mg/kg) and An2-NT(8-13) (0.009 mg/kg) were evaluated and compared. These doses were chosen because they all produced a 90% antinociceptive effect in the formalin test (cf. Fig. 1C, D, and Fig. 2D). As reported in Table 2, morphine (1.8 mg/kg s.c.) was found to produce a significant decrease of the charcoal meal progression in the intestine, when compared to saline (Ctrl). By contrast, An2-NT(8-13) did not induce any decrease of the gastrointestinal tract motility at the analgesic effective dose of 0.018 mg/kg. Interestingly, the combination of morphine (0.9 mg/kg) and An2-NT(8-13) (0.009 mg/kg), although equianalgesic to a 1.8 mg/kg dose of morphine, had no significant effect on the progression of the charcoal meal. To determine if the lack of effect of the drug combination on the gastro-intestinal tract motility can be due to an interference of An2-NT(8-13), we then tested whether or not a higher dose of An2-NT(8-13) can modify the effect of morphine. When injected at a dose of 5 mg/kg (s.c.), morphine induced a robust decrease of the charcoal meal intestinal transit (Table 2). The co-administration of 0.05 mg/kg of An2-NT(8-13) (i.v.) had no effect on morphine (5 mg/ kg; s.c.)-induced constipation, suggesting that An2-NT(8-13) does not
2.7. Statistical analysis Data of gastro-intestinal tract motility and blood pressure were compared using Kruskal-Wallis nonparametric test followed by Dunn's multiple comparison test. Statistical analyses were performed using GraphPad Prism (version 7.00; GraphPad Software Inc.). 3. Results 3.1. Antinociceptive effect of morphine and An2-NT(8-13) in the rat formalin test We first determined the analgesic potencies of morphine and An2NT(8-13) following separate administration. Our results demonstrate that the administration of morphine (0.3–10 mg/kg, s.c.) induced a dose-dependent antinociceptive effect in the rat formalin test (Fig. 1A). The most important effect of morphine was observed during the 82
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Fig. 1. Analgesic potency of morphine or An2-NT(8-13) in the formalin model of tonic pain. The antinociceptive effects of morphine (A) and An2-NT(8-13) (B) were measured following s.c. injection of formalin into the plantar surface of the right hind paw. A, B. Pain intensity were determined over time in the absence or presence of increasing doses of morphine, 0.3 mg/kg (n = 3), 1 mg/kg (n = 6), 1.5 mg/kg (n = 4), 3 mg/kg (n = 6) and 10 mg/kg (n = 3) (A) or An2-NT(8-13), 0.005 mg/kg (n = 6), 0.015 mg/kg (n = 5), 0.05 mg/kg (n = 5) and 0.15 mg/kg (n = 4) (B). Antinociceptive potencies of morphine (C) and An2-NT(8-13) (D) were determined graphically. The area under the curve (AUC) for the early inflammatory phase was calculated for each dose of morphine (C) and An2-NT(8-13) (D) and expressed graphically as a dose-response curve. For both, morphine and An2-NT(8-13), ED30, ED50, ED70 and ED90 were determined. The black dotted line represents the ED50 and the grey dotted lines represent the ED30, ED70 and ED90.
morphine s.c. + 0.009 mg/kg of An2-NT(8-13) i.v.), injected at the same time, triggered a rapid but transient drop of 20 mmHg in arterial blood pressure. However, as opposed to An2-NT(8-13) alone, the arterial blood pressure rapidly returned to baseline, without inducing a long-lasting pronounced hypotensive phase. As shown in Fig. 4B, when the total area under the curve (AUC) is analyzed, only the effect of An2NT(8-13) was found to be significantly different from the control group. Because the effect of s.c. morphine on blood pressure, if any, is likely to be delayed when compared to i.v. An2-NT(8-13), we also injected a group of rats with morphine 30 min prior to the injection of An2-NT(813). Under this condition, the effect of the drug combination was not different than when morphine and An2-NT(8-13) were injected at the same time.
Table 1 ED values of An2-NT(8-13), morphine or combination of morphine + An2NT(8-13) in the formalin test. Observed (mg/kg)
Predicted (mg/kg)
Drug
ED30
ED50
ED70
ED90
ED30
ED50
ED70
ED90
An2-NT(8-13) Morphine Morphine + An2NT(8-13)
0,013 1,25 0,25
0,014 1,39 0,39
0,015 1,53 0,6
0,018 1,8 1,2
0,38
0,5
0,61
0,86
impact morphine-induced inhibition of the charcoal meal progression. 3.3. Effect of morphine and An2-NT(8-13) on blood pressure
4. Discussion We and other have shown that NT, through activation of the NTS1 receptor, produces robust hypotensive effects (Demeule et al., 2014; Fantegrossi et al., 2005; Zogovic and Pilowsky, 2012). We therefore measured the effect of morphine, An2-NT(8-13), and a combination of the two drugs on the changes in blood pressure. As shown in Fig. 4A, i.v. injection of An2-NT(8-13) at 0.018 mg/kg induced a rapid (within the first 30 s) and robust drop (ΔMAP of 30 mmHg) in arterial blood pressure. The effect on blood pressure was biphasic, with a return to near baseline value by 80 s and a subsequent, more sustained decreased 2 min after An2-NT(8-13) injection. The effect of An2-NT(8-13) was found to persist for more than 15 min (not shown). Morphine injected alone at 1.8 mg/kg (s.c.) did not produce any change on the arterial blood pressure. The combination of the two drugs (0.9 mg/kg of
Despite its negative impact on the patient's health outcomes, chronic pain remains a highly prevalent, debilitating and costly medical condition that is frequently undertreated or inadequately managed. Indeed, the pharmacotherapy of chronic pain, which mainly depends on the use of opioids still shows limited analgesic efficacy and dose-limiting adverse effects in clinical practice (Bruneau et al., 2018). Although the recent research efforts have significantly improved our understanding of pain mechanisms or led to the identification of novel pain targets, little progress has been made in developing new, effective and safe analgesics (Kissin, 2010; Woolf, 2010). In light of the current limitations, there is therefore a need for alternative pain treatment strategies. Among them, drug combinations represent an attractive and 83
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Fig. 2. Isobolographic determination of the effect of combining morphine and An2-NT(813) in the formalin pain model. The ED30 (A), ED50 (B), ED70 (C) and ED90 (D) for the single administration of An2-NT(8-13) and morphine were respectively placed on the X and Y axes. The lines connecting each ED of single administration represent the theoretical line of additivity. The black square in the middle of each graph represents the ED30 (A), ED50 (B), ED70 (C) and ED90 (D) for the co-administration of An2-NT(8-13) and morphine. ED values and 95% confidence limits were determined using GraphPad Prism software.
opioid-NT combination regimen exerts complementary analgesic actions, producing an additive antinociceptive effect in the formalin test. Most interestingly, combining morphine with An2-NT(8-13) was found to inhibit the nocifensive behaviors induced by intraplantar formalin without producing the usual unwanted effects associated with the systemic administration of either morphine (constipation) or NT (hypotension). The antinociceptive effects of morphine have been extensively described in various models of pain, including the rat formalin model of persistent pain (Lötsch and Geisslinger, 2001; Sevostianova et al., 2003). Similarly, neurotensin receptor agonists have been found to produce antinociception in the formalin test through activation of both NTS1 and NTS2 receptors (Fanelli et al., 2015; Roussy et al., 2008, 2009). Admittedly, a major problem associated with the use of morphine or other opioid analgesics is the adverse effects observed at effective analgesic doses (de Boer et al., 2017). Likewise, both opioids and NT analogs were shown to induce significant unwanted effects in animal models of pain (Boules et al., 2006; DeWire et al., 2013). If one
beneficial treatment option for the management of chronic pain (Gilron et al., 2013). Indeed, as opposed to monotherapy the use of complementary medications may maximize the analgesic response by targeting multiple pain-related signaling pathways, while decreasing adverse drug reactions (van Hasselt and Iyengar, 2019). Although opioids have demonstrated their efficacy in relieving pain, their therapeutic use causes considerable adverse events to chronic pain patients, including constipation, respiratory depression, and nausea (Benyamin et al., 2008). Long-term opioid prescribing is further complicated by the potential for patients to develop tolerance or hyperalgesia (Chu et al., 2006). Importantly, the last decade has also seen an increase in abuse and misuse of opioids, thus leading to a drastic rise in drug overdoses (Coyle et al., 2018). In the present study, we investigated if the combination of morphine to the An2-NT(8-13) non-opioid analgesic may represent an alternative for the management of pain. Importantly, opioid and neurotensinergic systems target distinct receptors and signaling pathways, and exhibit non-overlapping adverse effect profiles. Here, we demonstrated that the
Fig. 3. Analgesic potency of morphine and An2-NT(8-13) in the formalin model of tonic pain. The antinociceptive effect of the co-administration of morphine and An2-NT(8-13) was measured in the rat model of formalin-induced tonic pain. A. Pain scores are shown over time in the absence or presence of increasing doses of morphine, 0.1 mg/kg (n = 6), 0.3 mg/kg (n = 5), 1 mg/kg (n = 6), 3 mg/kg (n = 6) and 10 mg/kg (n = 4) co-administered with the single dose 0.009 mg/kg of An2NT(8-13). Antinociceptive potencies of the co-administration of morphine and An2-NT(8-13) were determined graphically. B. The area under the curve (AUC) for the early inflammatory phase was calculated and expressed graphically as a dose-response curve. The ED30, ED50, ED70 and ED90 were determined. The black dotted line represents the ED50 and the grey dotted lines represent the ED30, ED70 and ED90. 84
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Table 2 Effect of morphine or/and An2-NT(8–13) on the gastro-intestinal tract motility. Vehicle (n = 13), morphine (1.8 mg/kg s.c n = 8 or 5 mg/kg s.c n = 8), An2NT(8–13) (0.018 mg/kg i.v) (n = 8) and the co-administration of morphine (0.9 mg/kg s.c.) and An2-NT(8–13) (0.009 mg/kg i.v.) (n = 8) or the co-administration of morphine (5 mg/kg s.c.) and An2-NT(8–13) (0.05 mg/kg i.v.) (n = 8) were injected 30 min before the force-feeding with a charcoal meal solution. The progression of the charcoal meal in the intestine was measured 1 h after the force-feeding. a P < 0.05, b P < 0.01 compared to control group; Kruskal-Wallis nonparametric test followed by Dunn multiple-comparison test. Drug Dose (mg/kg) Progression (%)
Saline
An2-NT(8–13) + Saline
– 87,9
0,018 88,1
Saline + Morphine 1,8 75 a
5 53,4
An2-NT(8–13) + Morphine
b
0,009 + 0,9 82,5
0,05 + 5 56,5 a
interact with each other by a still unknown mechanism. This opposed to the apparent absence of synergistic interaction between morphine and An2-NT(8-13) found here. The reason why such a discrepancy was observed is unclear. The difference observed with NT79 may of course be due to the fact that it is a NTS2-selective agonist while An2-NT(8-13) can activate both NTS1 and NTS2 receptors. Indeed, despite the fact that the analgesic effects induced by NT have been shown to be independent of the opioidergic system (Behbehani and Pert, 1984; Clineschmidt et al., 1982; Osbahr et al., 1981), existence of complex interactions between neurotensinergic and opioidergic systems have nevertheless been described (Stiller et al., 1997). It was indeed reported that activation of the NTS2 receptor operates upstream of the mu opioid receptor (Bredeloux et al., 2006) while the NTS1 receptor rather acts downstream to the opioidergic system (Roussy et al., 2010). This organization along the pain pathways could thus be responsible for the difference observed between the NTS2-selective agonist NT79 and the non-selective An2-NT(8-13) when combined with morphine. However, we cannot apply these explanations to rationalize why An2-NT(8-13) and NT69L, which are both non-selective NT agonists exhibit distinct pharmacological profiles, producing respectively additive or synergistic interaction with morphine. In this case, the difference may arise from the fact that Boules and collaborators have used the acute thermal nociceptive hot plate test (Boules et al., 2009) while
of the most debilitating effects of morphine is constipation (Holzer, 2009), NT is known to significantly decrease arterial blood pressure (Boules et al., 2010; Tore and Tuncel, 2009) and body temperature (Bissette et al., 1976; Nemeroff et al., 1977) via the recruitment of the NTS1 receptor. Drug combinations thus represent an efficient strategy to reduce toxicity and adverse effects (Pemovska et al., 2018). Indeed, the combination of two different drugs has the potential to produce synergistic or additive effects. Here, we combined morphine to An2-NT(8-13) which was shown to cross the blood-brain barrier (Demeule et al., 2007, 2014) and found that they were, together, able to provide strong pain relief in formalininjected rats at lower doses than individual drugs. Isobologram representations revealed that combining subcutaneous morphine with intravenous An2-NT(8-13) produced an additive antinociceptive effect in the formalin test suggesting that the two drugs (and their respective receptors and downstream elements) do not directly interact. In other words, each drug neither masks nor increases the efficacy of the other drug. In support to this study, Boules and collaborators have previously reported that combining the non-selective agonist NT69L or the NTS2selective agonist NT79 with morphine results in analgesia at significantly lower doses (Boules et al., 2009, 2011). The combination of either NT69L or NT79 with morphine was however found to produce synergistic antinociceptive effects, suggesting that the two drugs
Fig. 4. Effects of morphine, An2-NT(8-13) and combination of both drugs on blood pressure. A. The Mean of Arterial blood Pressure (MAP) was determined for 300 s after injection of vehicle (n = 5), An2-NT(8-13) at 0.018 mg/kg (n = 5), morphine at 1.8 mg/kg (n = 4), the combination of morphine at 0.9 mg/kg and An2-NT(8-13) at 0.009 mg/kg (n = 7) or the injection of 0.9 mg/kg of morphine 30 min before injection of 0.009 mg/kg of An2-NT(813) (n = 5). Variation of the mean arterial blood pressure (ΔMAP) represents the difference in blood pressure between basal and treatment conditions. B. The area under the curve (AUC) was calculated and expressed graphically. *** P < 0.001 compared to the control group (saline iv + saline sc); KruskalWallis nonparametric test followed by Dunn multiple comparison test.
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the use of lower doses of each drug also gives rise to an improved analgesic benefit/adverse effect ratio.
we used the formalin persistent pain model. Admittedly, the formalin model and the hot plate test are very different behavioral tests involving distinct nociceptive mechanisms and descending/ascending pain circuits (Martin et al., 1999). Furthermore, the neuromodulators/neurotransmitters that mediate the nociceptive responses in each of these pain models can also differ (Basbaum, 1999; Dennis and Melzack, 1980; Dennis et al., 1980). In the formalin test, the nocifensive behaviors are first produced by the direct action of the formalin chemical stimulus on primary afferents (first phase), whereas the second phase, the one analyzed here, reflects the combined effects of afferent inputs, inflammation and central sensitization in the spinal dorsal horn (Hong and Abbott, 1995; Hunskaar and Hole, 1987). Importantly, the formalin test, which measures the licking, biting and shaking spontaneous nociceptive-related behaviors, is considered as a non-stimulus evoked behavioral test. Indeed, once injected, no additional external stimulus is required to evoke nociception. Additionally, the spontaneous behaviors can be quantified over a long period of time in freely moving animals. Unlike the formalin model, the hot plate test, which reflects supraspinal sensory integration, is a reflexive nociceptive test that measures pawlicking and jumping withdrawal responses induced by acute application of external heat stimuli to the hind paws (Le Bars et al., 2001). It is therefore possible that morphine and An2-NT(8-13) combination produce distinct effects (additive vs synergistic) depending on the behavioral nociceptive test used. At the molecular and cellular levels, it is known that NT can enhance glutamate signaling through NMDA receptors of mesencephalic and cortical neurons (Antonelli et al., 2007). Furthermore a link between NT and dopaminergic systems have been clearly established (Binder et al., 2001; Tyler-McMahon et al., 2000). The opioid system also appears to closely interact with both dopaminergic and glutamatergic systems, especially during addiction (Peters and De Vries, 2012). Although they both modulate the activity of the glutamatergic system, we learned from the present study that such interactions do not produce synergistic analgesic effects between NT and opioid systems. Morphine is commonly used in the treatment of both acute and chronic pain. However, the doses of morphine required to maintain analgesia often need to be increased due to the development of analgesic tolerance. If tolerance also develops for drowsiness and nausea, the effect on the gastrointestinal tract motility is amplified as the dose of morphine increases (Sizar and Gupta, 2018). The same effect is usually seen in preclinical models. Here, we found that for a similar level of antinociception, the combination of morphine with An2-NT(813) did not produce constipation in rats. As a comparison, when morphine was used alone, the higher dose required to produce the same level of antinociception also induced a reduction in the gastrointestinal tract motility. Similarly, when used alone, An2-NT(8-3) produced a robust hypotensive effect. Interestingly, the important drop in blood pressure observed when An2-NT(8-13) was injected alone was greatly reduced but also shortened when a lower dose was administered with morphine. It is interesting to note that morphine, by itself, had no effect on blood pressure. Indeed the injection of morphine thirty min before An2-NT(8-13) did not cause changes in blood pressure. Altogether, our results support the usefulness of combining analgesic drugs such as morphine and NT agonists to achieve effective analgesia with minimum adverse effects associated to individual drugs. These findings also support the idea that NT agonists may represent a new class of opioidsparing analgesic drugs. In summary, these results emphasized the need for expanded research on combination drug therapies to provide superior pain relief with reduced adverse effects to pain patients. Indeed, this study demonstrates the therapeutic advantage to combine two drugs that exert complementary analgesic actions through concurrent mechanisms of nociceptive transmission. Irrespective of their classification as additive or synergistic, these types of drug-drug interactions thus achieve adequate pain control at lower doses of each medication. In addition, as shown here, if the drugs display non-overlapping side-effect profiles,
Acknowledgements This work was supported by the Canadian Institute of Health Research (CIHR) (Grant nos. MOP-123399 and MOP-136871) awarded to L.G. and (Grant no. FDN-148413) awarded to PS. L.G. is the recipient of a Senior Salary support from the Fonds de la Recherche Québec – Santé. PS holds a Canada Research Chair in Neurophysiopharmacology of Chronic Pain. E.E. was the recipient of a PhD fellowship from the Institut de pharmacologie de Sherbrooke. E.E., P.S. and L.G. designed the study. E.E. and J.C. performed the experiments. E.E., J.C. P.S. and L.G. wrote the manuscript. J.-M.L and V.B. provided technical and professional assistance during the study and commented the manuscript. The authors declare no competing financial interests. References Antonelli, T., Fuxe, K., Tomasini, M.C., Mazzoni, E., Agnati, L.F., Tanganelli, S., Ferraro, L., 2007. Neurotensin receptor mechanisms and its modulation of glutamate transmission in the brain. Prog. Neurobiol. 83, 92–109. 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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
The combination of opioid and neurotensin receptor agonists improves their analgesic/adverse effect ratio
T
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Emilie Eiselt, Jérôme Côté, Jean-Michel Longpré, Véronique Blais, Philippe Sarret , ⁎ Louis Gendron Département de pharmacologie-physiologie, Institut de pharmacologie de Sherbrooke, Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Isobologram Analgesia Constipation Formalin Morphine Neurotensin
Opioid and neurotensin (NT) receptors are expressed in both central and peripheral nervous systems where they modulate nociceptive responses. Nowadays, opioid analgesics like morphine remain the most prescribed drugs for the treatment of moderate to severe pain. However, despite their daily used, opioids can produce lifethreatening side effects, such as constipation or respiratory depression. Besides, NT analogs exert strong opioidindependent analgesia. Here, we thus hypothesized that the combined use of opioid and NT agonists would require lower doses to produce significant analgesic effects, hence decreasing opioid-induced adverse effects. We used isobologram analyses to determine if the combination of a NT brain-penetrant analog, An2-NT(8-13) with morphine results in an inhibitory, synergistic or additive analgesic response. We found that intravenous administration of An2-NT(8-13) reduced by 90% the nocifensive behaviors induced by formalin injection, at the dose of 0.018 mg/kg. Likewise, subcutaneous morphine reduced pain by 90% at 1.8 mg/kg. Importantly, isobologram analyses revealed that the co-injection of An2-NT(8-13) with morphine induced an additive analgesic response. We finally assessed the effects of morphine and An2-NT(8-13) on the gastrointestinal tract motility using the charcoal meal test. As opposed to morphine which significantly reduced the intestinal motility at the analgesic effective dose of 1.8 mg/kg, An2-NT(8-13) did not affect the charcoal meal intestinal transit at 0.018 mg/kg. Interestingly, at the dose providing 90% pain relief, the co-administration of morphine with An2NT(8-13) had a reduced effect on constipation. Altogether, these results suggest that combining NT agonists with morphine may improve its analgesic/adverse effect ratio.
1. Introduction Nowadays, opioids remain the most prescribed analgesics to treat moderate to severe pain (Gomes et al., 2014; Munzing, 2017). Morphine, the prototypical opioid, mediates its actions mainly through the Mu opioid receptor (MOP). In addition to analgesia, morphine also produces a number of unwanted and debilitating effects such as drowsiness, constipation, nausea, respiratory depression, and tolerance (Benyamin et al., 2008). Analgesic tolerance to morphine develops such that the dose needed to maintain a given level of analgesia has to be constantly increased. In order to limit the development of analgesic tolerance and concomitant unwanted effects, drug combination represents a promising alternative to currently used opioids. Indeed, combining an opioid with another analgesic drug might reduce the unwanted effects normally associated to each individual drug. This was
indeed found to be true when morphine was intrathecally injected with dexmedetomidine, a selective α2-adrenergic receptor agonist in a rat model of neuropathic pain (Kabalak et al., 2013). Similarly, the combined use of morphine and monoamine reuptake inhibitors was found to potentiate their respective analgesic effects in the rat formalin model (Shen et al., 2013). We and others have shown that neurotensin (NT) can produce significant pain relief in various animal models of acute and chronic pain (Boules et al., 2006; Demeule et al., 2014; Fanelli et al., 2015; Fantegrossi et al., 2005; Guillemette et al., 2012; Roussy et al., 2008; Tétreault et al., 2013). First identified for its hypotensive effects, NT is also know to participate in a wide variety of physiological functions, including modulation of body temperature, myocardial performance, gastrointestinal tract motility and secretion (Osadchii, 2015; Zhao and Pothoulakis, 2006). According to its wide distribution throughout the
⁎ Correspondence to: Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, Québec, Canada J1H 5N4. E-mail addresses: [email protected] (P. Sarret), [email protected] (L. Gendron).
https://doi.org/10.1016/j.ejphar.2019.01.048 Received 22 October 2018; Received in revised form 24 January 2019; Accepted 28 January 2019 Available online 29 January 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.
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contralateral, has little or no weight placed on it; 2, injected paw is elevated, not in contact with any surface; 3, injected paw is licked, bitten, or flinched. The behaviors believed to represent higher levels of pain intensity were given higher weighted scores. The weighted average pain intensity score (ranging 1–3) was then calculated by multiplying the time spent in each category by the category weight, summing these products, and dividing by the total time in a given interval of time. For a 180-second block, the pain score is thus be calculated as (1T1 + 2T2 + 3T3)/180, in which T1, T2, and T3 represent duration (in s) spent in behavioral categories 1, 2 and 3, respectively. Phase I and II values were calculated between 0–9 and 21–60 min, respectively. Effective doses (ED30, ED50, ED70 and ED90 values) for the inflammatory phase (phase II) of the formalin test were calculated from the dose-response curves generated for morphine sulfate (doses ranging from 0.3 to 10 mg/kg), An2-NT(8-13) (doses ranging from 0.005 to 0.15 mg/kg), and a combination of a constant dose of An2-NT(8-13) (0.009 mg/kg) and morphine sulfate (doses ranging from 0.1 to 10 mg/ kg).
central nervous system (Sarret and Beaudet, 2002), NT was also shown to exert brain functions, such as antipsychotic- and psychostimulantlike effects as well as regulation of food intake (Dobner, 2005; Dobner et al., 2003; Mazella et al., 2012). The physiological effects of NT are mediated by the activation of two closely related G protein-coupled receptors (GPCRs), namely NTS1 and NTS2 (Vincent et al., 1999). Importantly, NT-mediated analgesia was found to be independent of the opioid system. There is indeed compelling evidence demonstrating that opioid antagonists do not suppress NT-induced antinociception (Behbehani and Pert, 1984; Clineschmidt et al., 1982; Osbahr et al., 1981). Furthermore, NT also appears to be more potent than morphine in various pain tests (Nemeroff et al., 1979; al-Rodhan et al., 1991). In the present study, we hypothesized that combining opioid and NT peptides will present some advantages over pure opioid-based therapies. We thus undertook to determine whether systemically administered morphine combined to An2-NT(8-13), consisting of the brainpenetrant peptide Angiopep-2 (An2) conjugated to NT(8-13) (Demeule et al., 2014), can exert additive or synergistic antinociceptive interaction. We therefore evaluated the analgesic effects of combining morphine and An2-NT(8-13) in the rat model of formalin-induced persistent pain, and determined the extent of drug interaction using isobolograms. We further determined whether this drug combination can reduce opioid-induced constipation or NT-induced drop in blood pressure, thus leading to a better analgesic benefit/adverse effect ratio.
2.3. An2-NT(8-13) synthesis and conjugation Angiopep-2-NT(8-13) (An2-NT(8-13)) was synthesized by conjugation of the NT(8-13) peptide to the Angiopep-2-Cys (TFFYGGSRGKRNNFKTEEYC-NH2), which were both prepared by solid-phase peptide synthesis on a Symphony® peptide synthesizer (Protein Technologies Inc., Tucson, AZ, USA) using standard Fmoc chemistry with commercially available Fmoc amino acids (Chem-Impex International, Inc., Wood Dale, IL, USA). Analysis of purity for each peptide was performed by ACQUITY ultra-performance liquid chromatography (UPLC) (Waters Corporation, Milford, MA, USA) on a BEH Phenyl column at a flow rate of 0.5 ml min−1 and a constant temperature of 23 °C. Detection was performed at a wavelength of 229 nm, data acquisition and processing were done using the Compass™ suite of instrument control and data processing software (©Bruker Daltonik, Bremen, Germany). Peptide identity was confirmed on a micrOTOF ESI-TOF mass spectrometer (Bruker Daltronics, Inc., Billerica, MA, USA) with m/z ratios for the purified peptides of 817.01 Da for NT(8-13) (C38H64N12O8) and 2403.63 Da for the Angiopep-2-Cys (C107H155N31O31S). The conjugate An2-NT(8-13) was subsequently synthesized in phosphate buffered saline (PBS) by a two-step ligation method at room temperature (RT), monitored by RP-UPLC. The first conjugation step was performed in PBS 4 × , pH = 7.2 buffer, while the second step was in PBS 4 × , pH = 5.1. In the first step, a sulfo-EMCS (Molecular Biosciences Inc., Boulder, CO, USA) was used to incorporate a maleimidyl linker sitespecifically at arginine 8 of NT(8-13), resulting in [Arg(MHA)8]NT. In the second step, conjugation with the C-terminal Cys-modified An2 peptide was performed. Both reactions were stopped by the addition of 20% acetic acid. Purification was performed on a preparative HPLC column.
2. Materials and methods 2.1. Animals Adult male Sprague-Dawley rats (225–250 g; Charles River Laboratories, St-Constant, QC, Canada) were maintained in a 12-h light/dark cycle and had ad libitum access to lab chow and water. Rats were acclimatized for 3 days to experimental room, manipulations and devices prior to the behavioral studies. All experiments were performed in a quiet room, different than the housing area, between 8:00 a.m. and 2:00 p.m. by the same experimenter. All experiments were approved by the animal care committee of the Université de Sherbrooke (Protocols #356-13/18 and 035-13/18) and all procedures conformed to the directives of the Canadian Council on Animal Care and guidelines of the International Association for the Study of Pain. All animal experiments were designed to minimize the number of animals used and their suffering. 2.2. Formalin test Antinociception was assessed using the formalin test as a model of tonic pain. For this purpose, male Sprague-Dawley rats were habituated for 30 min to the experimentation room. Thereafter, the rats received a 50 μl s.c. injection of 2% formaldehyde (i.e. 5% formalin, Fisher Scientific, Ottawa, ON, Canada) into the plantar surface of the right hind paw. Rats were then placed in clear plastic chambers (40 cm × 30 cm × 30 cm) equipped with a glass floor positioned over a mirror angled at 45° in order to allow an unobstructed view of the paws, and their behaviors were scored during 60 min. The intraplantar injection of formalin produces a typical biphasic nociceptive response (Sawynok and Liu, 2003). These two distinct phases of spontaneous pain behaviors that occur in rodents are proposed to reflect a direct effect of formalin on sensory nociceptors, corresponding to an acute phase (phase I), and a longer-lasting pain due to inflammation and central sensitization (phase II). These two phases are separated by a period of quiescence, called interphase, which is characterized by the active inhibition of the formalin-induced nociceptive behaviors. The nocifensive behaviors were assessed using a weighted score method (Coderre et al., 1993; Dubuisson and Dennis, 1977). After the injection of formalin into the right hind paw, the time spent was measured in each of three behavioral categories: 1, injected paw is comparable to
2.4. Dose-response studies with the formalin test 2.4.1. Morphine alone Five groups of rats were injected s.c. with various doses of morphine sulfate (0.3; 1; 1.5; 3 and 10 mg/kg) and saline i.v. five min. before the injection of a solution of 2% formaldehyde in the hind paw. Morphine sulfate was purchased from Medisca (St-Laurent, QC, Canada) and dissolved in 0.9% saline solution before use. 2.4.2. An2-NT(8-13) alone Four groups of rats were injected with various doses of An2-NT(813) (0.005; 0.015; 0.05 and 0.15 mg/kg i.v.) and saline s.c five min. before the injection of a solution of 2% formaldehyde in the hind paw. 2.4.3. Morphine plus An2-NT(8-13) Five groups of rats were injected with different doses of morphine 81
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sulfate (0.1; 0.3; 1; 3 and 10 mg/kg s.c.) and a fixed dose of An2-NT(813) (0.009 mg/kg i.v.) five min before the injection of a solution of 2% formaldehyde in the hind paw.
inflammatory phase (21–60 min). Indeed, at 3 mg/kg and 10 mg/kg, morphine completely inhibited the formalin-induced nociceptive behaviors during the inflammatory phase. Morphine also reduced the nocifensive behaviors during the acute phase (0–9 min), but only at the highest dose of 10 mg/kg. As shown in Fig. 1B, the administration of An2-NT(8-13) (0.005–0.15 mg/kg, i.v.) also produced dose-dependent antinociception in both phases of the formalin test. The potencies of morphine and An2-NT(8-13) were then determined from the area under the curves, calculated for each dose of the compounds. To this purpose, only the early inflammatory phase (21–40 min) was considered. We found that morphine inhibited formalin-induced pain with an ED30 of 1.25 mg/kg, ED50 of 1.39 mg/kg, ED70 of 1.53 mg/kg, and ED90 of 1.8 mg/kg (Fig. 1C; Table 1). Likewise, An2-NT(8-13) induced antinociception with an ED30 of 0.013 mg/kg, ED50 of 0.014 mg/kg, ED70 of 0.015 mg/kg, and ED90 of 0.018 mg/kg (Fig. 1D; Table 1). In order to evaluate if morphine and An2-NT(8-13) interacted in an additive or synergistic manner to produce antinociception, we first generated isobolograms from the doses required to produce 30%, 50%, 70% and 90% antinociception (Fig. 2) using the ED30, ED50, ED70 and ED90 values obtained for morphine and An2-NT(8-13) alone, as determined on the dose-response curves illustrated in Fig. 1C and D. To measure a possible interaction between morphine and An2-NT(8-13), the antinociceptive effect of increasing doses of morphine (0.1–3 mg/ kg) co-administered with a constant, sub-analgesic dose of An2-NT(813) (0.009 mg/kg i.v) was determined. As shown in Fig. 3A, the combination of morphine and An2-NT(8-13) produced a time- and dose (morphine)-dependent antinociceptive effect in the formalin test. When the antinociceptive effect of the drug combination is measured as the area under the curve (AUC; for the 21–40 min phase II), ED30, ED50, ED70 and ED90 values for morphine in the presence of An2-NT(8-13) can be determined. Graphical determination revealed that the ED30, ED50, ED70 and ED90 values of morphine, when co-administered with 0.009 mg/kg of An2-NT(8-13), are respectively 0.25 mg/kg, 0.39 mg/ kg, 0.6 mg/kg and 1.2 mg/kg (Fig. 3B; Table 1). Reporting these combinations on the corresponding isobolograms of Fig. 2 revealed an additive antinociceptive effect of morphine and An2-NT(8-13) combination as every combinations felt on the theoretical line of additivity for these drugs.
2.4.4. Isobolographic analysis The assessment of the additive effect of morphine and An2-NT(8-13) was done graphically using isobolograms (Gessner, 1995; Tallarida, 2006). The ED values for An2-NT(8-13) and morphine injected alone were respectively placed on the X and Y axes. The line connecting the ED value of each individual drug represents the theoretical line of additivity for these two drugs. On isobolograms, additivity is found when the level of antinociception of the drug combination falls on or in close proximity to the theoretical line of additivity. When the effect of the drug combination falls above or below the line of additivity, the interaction between drugs is respectively considered inhibitory and synergistic. 2.5. Charcoal meal test Constipation was assessed by measuring the gastrointestinal tract motility using the charcoal meal test. Briefly, food deprived (16 h) animals are injected (i.v. or s.c.) with saline, morphine sulfate (1.8 mg/kg s.c or 5 mg/kg s.c), An2-NT(8-13) (0.018 mg/kg i.v), or co-injected with morphine sulfate (0.9 mg/kg s.c) and An2-NT(8-13) (0.009 mg/kg i.v) or with morphine sulfate (5 mg/kg s.c) and An2-NT(8-13) (0.05 mg/kg i.v). Thirty min. after drug injection, 2 ml of a charcoal meal solution (5% arabic gum and 10% charcoal in water) is administered to the rats by gavage. The animals are euthanized 60 min after and the progression of the charcoal in the intestine is measured as a ratio of progression/ total length of the intestine. Results are presented as the percentage of progression of the charcoal meal in the intestine. 2.6. Blood pressure test Rats were anaesthetized with a mixture of ketamine/xylaxine (87 mg/kg: 13 mg/kg, i.m.) and placed in supine position on a heating pad. Mean arterial blood pressure was measured through a PE50 catheter filled with heparinized saline inserted in the right carotid artery and connected to a Micro-Med transducer (model TDX-300, Micromed LLC, Ampler, PA, USA) linked to a blood pressure Micro-Med analyzer (model BPA-100c, Micromed LLC). When basal pressure was stabilized, rats were first given a vehicle or An2-NT(8-13) intravenously (caudal vein), and vehicle or morphine subcutaneously 30 s later. In one group, morphine was administered 30 min prior to An2-NT(8-13). Only one paradigm of injections was performed prior to euthanasia. Changes in blood pressure from the baseline to maximal effect post-injection in individual animals were determined. Data represents the mean ± S.E.M. of at least six different experiments.
3.2. Effect of morphine and An2-NT(8-13) on the gastrointestinal tract motility One of the main adverse effects of morphine is undoubtedly its propensity to produce constipation (Holzer, 2009). To see whether or not the combination of morphine with An2-NT(8-13) can advantageously be used to produce antinociception without inhibiting the gastrointestinal tract motility, the effect of morphine (1.8 mg/kg), An2NT(8-13) (0.018 mg/kg) and of a combination of morphine (0.9 mg/kg) and An2-NT(8-13) (0.009 mg/kg) were evaluated and compared. These doses were chosen because they all produced a 90% antinociceptive effect in the formalin test (cf. Fig. 1C, D, and Fig. 2D). As reported in Table 2, morphine (1.8 mg/kg s.c.) was found to produce a significant decrease of the charcoal meal progression in the intestine, when compared to saline (Ctrl). By contrast, An2-NT(8-13) did not induce any decrease of the gastrointestinal tract motility at the analgesic effective dose of 0.018 mg/kg. Interestingly, the combination of morphine (0.9 mg/kg) and An2-NT(8-13) (0.009 mg/kg), although equianalgesic to a 1.8 mg/kg dose of morphine, had no significant effect on the progression of the charcoal meal. To determine if the lack of effect of the drug combination on the gastro-intestinal tract motility can be due to an interference of An2-NT(8-13), we then tested whether or not a higher dose of An2-NT(8-13) can modify the effect of morphine. When injected at a dose of 5 mg/kg (s.c.), morphine induced a robust decrease of the charcoal meal intestinal transit (Table 2). The co-administration of 0.05 mg/kg of An2-NT(8-13) (i.v.) had no effect on morphine (5 mg/ kg; s.c.)-induced constipation, suggesting that An2-NT(8-13) does not
2.7. Statistical analysis Data of gastro-intestinal tract motility and blood pressure were compared using Kruskal-Wallis nonparametric test followed by Dunn's multiple comparison test. Statistical analyses were performed using GraphPad Prism (version 7.00; GraphPad Software Inc.). 3. Results 3.1. Antinociceptive effect of morphine and An2-NT(8-13) in the rat formalin test We first determined the analgesic potencies of morphine and An2NT(8-13) following separate administration. Our results demonstrate that the administration of morphine (0.3–10 mg/kg, s.c.) induced a dose-dependent antinociceptive effect in the rat formalin test (Fig. 1A). The most important effect of morphine was observed during the 82
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Fig. 1. Analgesic potency of morphine or An2-NT(8-13) in the formalin model of tonic pain. The antinociceptive effects of morphine (A) and An2-NT(8-13) (B) were measured following s.c. injection of formalin into the plantar surface of the right hind paw. A, B. Pain intensity were determined over time in the absence or presence of increasing doses of morphine, 0.3 mg/kg (n = 3), 1 mg/kg (n = 6), 1.5 mg/kg (n = 4), 3 mg/kg (n = 6) and 10 mg/kg (n = 3) (A) or An2-NT(8-13), 0.005 mg/kg (n = 6), 0.015 mg/kg (n = 5), 0.05 mg/kg (n = 5) and 0.15 mg/kg (n = 4) (B). Antinociceptive potencies of morphine (C) and An2-NT(8-13) (D) were determined graphically. The area under the curve (AUC) for the early inflammatory phase was calculated for each dose of morphine (C) and An2-NT(8-13) (D) and expressed graphically as a dose-response curve. For both, morphine and An2-NT(8-13), ED30, ED50, ED70 and ED90 were determined. The black dotted line represents the ED50 and the grey dotted lines represent the ED30, ED70 and ED90.
morphine s.c. + 0.009 mg/kg of An2-NT(8-13) i.v.), injected at the same time, triggered a rapid but transient drop of 20 mmHg in arterial blood pressure. However, as opposed to An2-NT(8-13) alone, the arterial blood pressure rapidly returned to baseline, without inducing a long-lasting pronounced hypotensive phase. As shown in Fig. 4B, when the total area under the curve (AUC) is analyzed, only the effect of An2NT(8-13) was found to be significantly different from the control group. Because the effect of s.c. morphine on blood pressure, if any, is likely to be delayed when compared to i.v. An2-NT(8-13), we also injected a group of rats with morphine 30 min prior to the injection of An2-NT(813). Under this condition, the effect of the drug combination was not different than when morphine and An2-NT(8-13) were injected at the same time.
Table 1 ED values of An2-NT(8-13), morphine or combination of morphine + An2NT(8-13) in the formalin test. Observed (mg/kg)
Predicted (mg/kg)
Drug
ED30
ED50
ED70
ED90
ED30
ED50
ED70
ED90
An2-NT(8-13) Morphine Morphine + An2NT(8-13)
0,013 1,25 0,25
0,014 1,39 0,39
0,015 1,53 0,6
0,018 1,8 1,2
0,38
0,5
0,61
0,86
impact morphine-induced inhibition of the charcoal meal progression. 3.3. Effect of morphine and An2-NT(8-13) on blood pressure
4. Discussion We and other have shown that NT, through activation of the NTS1 receptor, produces robust hypotensive effects (Demeule et al., 2014; Fantegrossi et al., 2005; Zogovic and Pilowsky, 2012). We therefore measured the effect of morphine, An2-NT(8-13), and a combination of the two drugs on the changes in blood pressure. As shown in Fig. 4A, i.v. injection of An2-NT(8-13) at 0.018 mg/kg induced a rapid (within the first 30 s) and robust drop (ΔMAP of 30 mmHg) in arterial blood pressure. The effect on blood pressure was biphasic, with a return to near baseline value by 80 s and a subsequent, more sustained decreased 2 min after An2-NT(8-13) injection. The effect of An2-NT(8-13) was found to persist for more than 15 min (not shown). Morphine injected alone at 1.8 mg/kg (s.c.) did not produce any change on the arterial blood pressure. The combination of the two drugs (0.9 mg/kg of
Despite its negative impact on the patient's health outcomes, chronic pain remains a highly prevalent, debilitating and costly medical condition that is frequently undertreated or inadequately managed. Indeed, the pharmacotherapy of chronic pain, which mainly depends on the use of opioids still shows limited analgesic efficacy and dose-limiting adverse effects in clinical practice (Bruneau et al., 2018). Although the recent research efforts have significantly improved our understanding of pain mechanisms or led to the identification of novel pain targets, little progress has been made in developing new, effective and safe analgesics (Kissin, 2010; Woolf, 2010). In light of the current limitations, there is therefore a need for alternative pain treatment strategies. Among them, drug combinations represent an attractive and 83
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Fig. 2. Isobolographic determination of the effect of combining morphine and An2-NT(813) in the formalin pain model. The ED30 (A), ED50 (B), ED70 (C) and ED90 (D) for the single administration of An2-NT(8-13) and morphine were respectively placed on the X and Y axes. The lines connecting each ED of single administration represent the theoretical line of additivity. The black square in the middle of each graph represents the ED30 (A), ED50 (B), ED70 (C) and ED90 (D) for the co-administration of An2-NT(8-13) and morphine. ED values and 95% confidence limits were determined using GraphPad Prism software.
opioid-NT combination regimen exerts complementary analgesic actions, producing an additive antinociceptive effect in the formalin test. Most interestingly, combining morphine with An2-NT(8-13) was found to inhibit the nocifensive behaviors induced by intraplantar formalin without producing the usual unwanted effects associated with the systemic administration of either morphine (constipation) or NT (hypotension). The antinociceptive effects of morphine have been extensively described in various models of pain, including the rat formalin model of persistent pain (Lötsch and Geisslinger, 2001; Sevostianova et al., 2003). Similarly, neurotensin receptor agonists have been found to produce antinociception in the formalin test through activation of both NTS1 and NTS2 receptors (Fanelli et al., 2015; Roussy et al., 2008, 2009). Admittedly, a major problem associated with the use of morphine or other opioid analgesics is the adverse effects observed at effective analgesic doses (de Boer et al., 2017). Likewise, both opioids and NT analogs were shown to induce significant unwanted effects in animal models of pain (Boules et al., 2006; DeWire et al., 2013). If one
beneficial treatment option for the management of chronic pain (Gilron et al., 2013). Indeed, as opposed to monotherapy the use of complementary medications may maximize the analgesic response by targeting multiple pain-related signaling pathways, while decreasing adverse drug reactions (van Hasselt and Iyengar, 2019). Although opioids have demonstrated their efficacy in relieving pain, their therapeutic use causes considerable adverse events to chronic pain patients, including constipation, respiratory depression, and nausea (Benyamin et al., 2008). Long-term opioid prescribing is further complicated by the potential for patients to develop tolerance or hyperalgesia (Chu et al., 2006). Importantly, the last decade has also seen an increase in abuse and misuse of opioids, thus leading to a drastic rise in drug overdoses (Coyle et al., 2018). In the present study, we investigated if the combination of morphine to the An2-NT(8-13) non-opioid analgesic may represent an alternative for the management of pain. Importantly, opioid and neurotensinergic systems target distinct receptors and signaling pathways, and exhibit non-overlapping adverse effect profiles. Here, we demonstrated that the
Fig. 3. Analgesic potency of morphine and An2-NT(8-13) in the formalin model of tonic pain. The antinociceptive effect of the co-administration of morphine and An2-NT(8-13) was measured in the rat model of formalin-induced tonic pain. A. Pain scores are shown over time in the absence or presence of increasing doses of morphine, 0.1 mg/kg (n = 6), 0.3 mg/kg (n = 5), 1 mg/kg (n = 6), 3 mg/kg (n = 6) and 10 mg/kg (n = 4) co-administered with the single dose 0.009 mg/kg of An2NT(8-13). Antinociceptive potencies of the co-administration of morphine and An2-NT(8-13) were determined graphically. B. The area under the curve (AUC) for the early inflammatory phase was calculated and expressed graphically as a dose-response curve. The ED30, ED50, ED70 and ED90 were determined. The black dotted line represents the ED50 and the grey dotted lines represent the ED30, ED70 and ED90. 84
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Table 2 Effect of morphine or/and An2-NT(8–13) on the gastro-intestinal tract motility. Vehicle (n = 13), morphine (1.8 mg/kg s.c n = 8 or 5 mg/kg s.c n = 8), An2NT(8–13) (0.018 mg/kg i.v) (n = 8) and the co-administration of morphine (0.9 mg/kg s.c.) and An2-NT(8–13) (0.009 mg/kg i.v.) (n = 8) or the co-administration of morphine (5 mg/kg s.c.) and An2-NT(8–13) (0.05 mg/kg i.v.) (n = 8) were injected 30 min before the force-feeding with a charcoal meal solution. The progression of the charcoal meal in the intestine was measured 1 h after the force-feeding. a P < 0.05, b P < 0.01 compared to control group; Kruskal-Wallis nonparametric test followed by Dunn multiple-comparison test. Drug Dose (mg/kg) Progression (%)
Saline
An2-NT(8–13) + Saline
– 87,9
0,018 88,1
Saline + Morphine 1,8 75 a
5 53,4
An2-NT(8–13) + Morphine
b
0,009 + 0,9 82,5
0,05 + 5 56,5 a
interact with each other by a still unknown mechanism. This opposed to the apparent absence of synergistic interaction between morphine and An2-NT(8-13) found here. The reason why such a discrepancy was observed is unclear. The difference observed with NT79 may of course be due to the fact that it is a NTS2-selective agonist while An2-NT(8-13) can activate both NTS1 and NTS2 receptors. Indeed, despite the fact that the analgesic effects induced by NT have been shown to be independent of the opioidergic system (Behbehani and Pert, 1984; Clineschmidt et al., 1982; Osbahr et al., 1981), existence of complex interactions between neurotensinergic and opioidergic systems have nevertheless been described (Stiller et al., 1997). It was indeed reported that activation of the NTS2 receptor operates upstream of the mu opioid receptor (Bredeloux et al., 2006) while the NTS1 receptor rather acts downstream to the opioidergic system (Roussy et al., 2010). This organization along the pain pathways could thus be responsible for the difference observed between the NTS2-selective agonist NT79 and the non-selective An2-NT(8-13) when combined with morphine. However, we cannot apply these explanations to rationalize why An2-NT(8-13) and NT69L, which are both non-selective NT agonists exhibit distinct pharmacological profiles, producing respectively additive or synergistic interaction with morphine. In this case, the difference may arise from the fact that Boules and collaborators have used the acute thermal nociceptive hot plate test (Boules et al., 2009) while
of the most debilitating effects of morphine is constipation (Holzer, 2009), NT is known to significantly decrease arterial blood pressure (Boules et al., 2010; Tore and Tuncel, 2009) and body temperature (Bissette et al., 1976; Nemeroff et al., 1977) via the recruitment of the NTS1 receptor. Drug combinations thus represent an efficient strategy to reduce toxicity and adverse effects (Pemovska et al., 2018). Indeed, the combination of two different drugs has the potential to produce synergistic or additive effects. Here, we combined morphine to An2-NT(8-13) which was shown to cross the blood-brain barrier (Demeule et al., 2007, 2014) and found that they were, together, able to provide strong pain relief in formalininjected rats at lower doses than individual drugs. Isobologram representations revealed that combining subcutaneous morphine with intravenous An2-NT(8-13) produced an additive antinociceptive effect in the formalin test suggesting that the two drugs (and their respective receptors and downstream elements) do not directly interact. In other words, each drug neither masks nor increases the efficacy of the other drug. In support to this study, Boules and collaborators have previously reported that combining the non-selective agonist NT69L or the NTS2selective agonist NT79 with morphine results in analgesia at significantly lower doses (Boules et al., 2009, 2011). The combination of either NT69L or NT79 with morphine was however found to produce synergistic antinociceptive effects, suggesting that the two drugs
Fig. 4. Effects of morphine, An2-NT(8-13) and combination of both drugs on blood pressure. A. The Mean of Arterial blood Pressure (MAP) was determined for 300 s after injection of vehicle (n = 5), An2-NT(8-13) at 0.018 mg/kg (n = 5), morphine at 1.8 mg/kg (n = 4), the combination of morphine at 0.9 mg/kg and An2-NT(8-13) at 0.009 mg/kg (n = 7) or the injection of 0.9 mg/kg of morphine 30 min before injection of 0.009 mg/kg of An2-NT(813) (n = 5). Variation of the mean arterial blood pressure (ΔMAP) represents the difference in blood pressure between basal and treatment conditions. B. The area under the curve (AUC) was calculated and expressed graphically. *** P < 0.001 compared to the control group (saline iv + saline sc); KruskalWallis nonparametric test followed by Dunn multiple comparison test.
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the use of lower doses of each drug also gives rise to an improved analgesic benefit/adverse effect ratio.
we used the formalin persistent pain model. Admittedly, the formalin model and the hot plate test are very different behavioral tests involving distinct nociceptive mechanisms and descending/ascending pain circuits (Martin et al., 1999). Furthermore, the neuromodulators/neurotransmitters that mediate the nociceptive responses in each of these pain models can also differ (Basbaum, 1999; Dennis and Melzack, 1980; Dennis et al., 1980). In the formalin test, the nocifensive behaviors are first produced by the direct action of the formalin chemical stimulus on primary afferents (first phase), whereas the second phase, the one analyzed here, reflects the combined effects of afferent inputs, inflammation and central sensitization in the spinal dorsal horn (Hong and Abbott, 1995; Hunskaar and Hole, 1987). Importantly, the formalin test, which measures the licking, biting and shaking spontaneous nociceptive-related behaviors, is considered as a non-stimulus evoked behavioral test. Indeed, once injected, no additional external stimulus is required to evoke nociception. Additionally, the spontaneous behaviors can be quantified over a long period of time in freely moving animals. Unlike the formalin model, the hot plate test, which reflects supraspinal sensory integration, is a reflexive nociceptive test that measures pawlicking and jumping withdrawal responses induced by acute application of external heat stimuli to the hind paws (Le Bars et al., 2001). It is therefore possible that morphine and An2-NT(8-13) combination produce distinct effects (additive vs synergistic) depending on the behavioral nociceptive test used. At the molecular and cellular levels, it is known that NT can enhance glutamate signaling through NMDA receptors of mesencephalic and cortical neurons (Antonelli et al., 2007). Furthermore a link between NT and dopaminergic systems have been clearly established (Binder et al., 2001; Tyler-McMahon et al., 2000). The opioid system also appears to closely interact with both dopaminergic and glutamatergic systems, especially during addiction (Peters and De Vries, 2012). Although they both modulate the activity of the glutamatergic system, we learned from the present study that such interactions do not produce synergistic analgesic effects between NT and opioid systems. Morphine is commonly used in the treatment of both acute and chronic pain. However, the doses of morphine required to maintain analgesia often need to be increased due to the development of analgesic tolerance. If tolerance also develops for drowsiness and nausea, the effect on the gastrointestinal tract motility is amplified as the dose of morphine increases (Sizar and Gupta, 2018). The same effect is usually seen in preclinical models. Here, we found that for a similar level of antinociception, the combination of morphine with An2-NT(813) did not produce constipation in rats. As a comparison, when morphine was used alone, the higher dose required to produce the same level of antinociception also induced a reduction in the gastrointestinal tract motility. Similarly, when used alone, An2-NT(8-3) produced a robust hypotensive effect. Interestingly, the important drop in blood pressure observed when An2-NT(8-13) was injected alone was greatly reduced but also shortened when a lower dose was administered with morphine. It is interesting to note that morphine, by itself, had no effect on blood pressure. Indeed the injection of morphine thirty min before An2-NT(8-13) did not cause changes in blood pressure. Altogether, our results support the usefulness of combining analgesic drugs such as morphine and NT agonists to achieve effective analgesia with minimum adverse effects associated to individual drugs. These findings also support the idea that NT agonists may represent a new class of opioidsparing analgesic drugs. In summary, these results emphasized the need for expanded research on combination drug therapies to provide superior pain relief with reduced adverse effects to pain patients. Indeed, this study demonstrates the therapeutic advantage to combine two drugs that exert complementary analgesic actions through concurrent mechanisms of nociceptive transmission. Irrespective of their classification as additive or synergistic, these types of drug-drug interactions thus achieve adequate pain control at lower doses of each medication. In addition, as shown here, if the drugs display non-overlapping side-effect profiles,
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