Pascal Tétreault
email: pascal.tétreault@usherbrooke.ca

Élie Besserer-Offroy

Rebecca L Brouillette
email: rebecca.brouillette@usherbrooke.ca

Adeline René
email: adeline.rene@gmail.com

Alexandre Murza
email: alexandre.murza@usherbrooke.ca

Roberto Fanelli
email: robyfanelli@hotmail.it

Karyn Kirby
email: karyn.kirby@usherbrooke.ca

Alexandre J Parent
email: alexandre.parent2@usherbrooke.ca

Isabelle Dubuc
email: isabelle.dubuc@univ-rouen.fr

Nicolas Beaudet
email: nicolas.beaudet@usherbrooke.ca

Jérôme Côté

Jean-Michel Longpré

Jean Martinez
email: jean.martinez@umontpellier.fr

Florine Cavelier
email: florine.cavelier@umontpellier.fr

Ph.D Philippe Florine Sarret
email: philippe.sarret@usherbrooke.ca

Ph.D Cavelier

Pain relief devoid of opioid side effects following central action of a silylated neurotensin analog

Keywords: unnatural aminoacid, antinociception, constipation, tolerance, formalin, neuropathic

Neurotensin (NT) exerts naloxone-insensitive antinociceptive action through its binding to both NTS 1 and NTS 2 receptors and NT analogs provide stronger pain relief than morphine on a molecular basis. Here, we examined the analgesic/adverse effect profile of a new NT(8-13) derivative denoted JMV2009, in which the Pro 10 residue was substituted by a silicon-containing unnatural amino acid silaproline. We first report the synthesis and in vitro characterization (receptor-binding affinity, functional activity and stability) of JMV2009. We next examined its analgesic activity in a battery of acute, tonic and chronic pain models. We finally evaluated its ability to induce adverse effects associated with chronic opioid use, such as constipation and analgesic tolerance or related to NTS 1 activation, like hypothermia. In in vitro assays, JMV2009 exhibited high binding affinity for both NTS 1 and NTS 2 , improved proteolytic resistance as well as agonistic activities similar to NT, inducing sustained activation of p42/p44 MAPK and receptor internalization. Intrathecal injection of JMV2009 produced dose-dependent antinociceptive responses in the tail-flick test and almost completely abolished the nociceptiverelated behaviors induced by chemical somatic and visceral noxious stimuli. Likewise, increasing doses of JMV2009 significantly reduced tactile allodynia and weight bearing deficits in nerveinjured rats. Importantly, repeated agonist treatment did not result in the development of analgesic tolerance. Furthermore, JMV2009 did not cause constipation and was ineffective in inducing hypothermia. These findings suggest that NT drugs can act as an effective opioid-free medication for the management of pain or can serve as adjuvant analgesics to reduce the opioid adverse effects.

Introduction

Today, opioids remain at the forefront of the pharmacological treatment of chronic pain (de [START_REF] De Leon-Casasola | Opioids for chronic pain: new evidence, new strategies, safe prescribing[END_REF][START_REF] Gomes | Trends in high-dose opioid prescribing in Canada[END_REF]. However, opioid therapy is associated with a wide range of side effects, such as constipation, nausea, drowsiness, dizziness, sleep disturbance, dry mouth, respiratory depression and tolerance that negatively affects patients' well-being and even discourages them for continuing their medications [START_REF] Benyamin | Opioid Complications and Side Effects[END_REF][START_REF] Bruneau | Management of opioid use disorders: a national clinical practice guideline[END_REF][START_REF] Devulder | Impact of long-term use of opioids on quality of life in patients with chronic, non-malignant pain[END_REF][START_REF] Mcnicol | Management of opioid side effects in cancer-related and chronic noncancer pain: a systematic review[END_REF]. In the ongoing opioid crisis, involving drug misuse, abuse, overdose and death [START_REF] Coyle | Opioid analgesic dose and the risk of misuse, overdose, and death: A narrative review[END_REF], there is therefore an urgent need for new, effective and safe non-opioid analgesics to improve the care of patients suffering from chronic pain [START_REF] Besserer-Offroy | Sending out Biased Signals: an Appropriate Proposition for Pain?[END_REF].

Among the alternative to opioids, neurotensin (NT) receptors recently emerged as attractive targets to develop new painkillers (Perez de [START_REF] Perez De Vega | Recent progress in non-opioid analgesic peptides[END_REF]. Through its binding with two G protein-coupled receptors (GPCRs), referred to as NTS 1 and NTS 2 , the tridecapeptide NT(1-13) was indeed found to induce a potent opioid-independent analgesia in a variety of pain paradigms following systemic or central administration [START_REF] Dobner | Neurotensin and pain modulation[END_REF][START_REF] Feng | The Roles of Neurotensin and its Analogues in Pain[END_REF][START_REF] Kleczkowska | Neurotensin and neurotensin receptors: characteristic, structure-activity relationship and pain modulation--a review[END_REF][START_REF] Sarret | Neurotensin and Its Receptors, Reference Module in Neuroscience and Biobehavioral Psychology[END_REF]. Accordingly, it was pharmacologically demonstrated that the antinociceptive responses induced by NT and its analogs, designed and synthesized based on the minimal biologically active C-terminal region (Arg 8 -Arg 9 -Pro 10 -Tyr 11 -Ile 12 -Leu 13 ), could not be reversed by the opioid antagonists, naloxone and naltrexone [START_REF] Behbehani | A mechanism for the analgesic effect of neurotensin as revealed by behavioral and electrophysiological techniques[END_REF][START_REF] Bredeloux | Interactions between NTS2 neurotensin and opioid receptors on two nociceptive responses assessed on the hot plate test in mice[END_REF][START_REF] Osbahr | Neurotensin-induced antinociception in mice: antagonism by thyrotropin-releasing hormone[END_REF][START_REF] Wustrow | Reduced amide bond neurotensin 8-13 mimetics with potent in vivo activity[END_REF]. Importantly, NT was also reported to provide stronger pain relief than morphine on a molecular basis [START_REF] Al-Rodhan | Structure-antinociceptive activity of neurotensin and some novel analogues in the periaqueductal gray region of the brainstem[END_REF][START_REF] Nemeroff | Alterations in nociception and body temperature after intracisternal administration of neurotensin, beta-endorphin, other endogenous peptides, and morphine[END_REF].

In the last two decades, peptide therapeutics have gained increased interest from the biomedical and pharmaceutical industry, filling the gap between the two main classes of available market drugs, small molecules and biologics [START_REF] Fosgerau | Peptide therapeutics: current status and future directions[END_REF][START_REF] Kaspar | Future directions for peptide therapeutics development[END_REF]. Peptides are definitely attractive drug candidates due to their high potency/efficacy, target specificity and safety/tolerability profile. Despite offering many advantages, their extensive use as therapeutics is, however, limited by their short half-life and rapid plasma clearance, which leads to low bioavailability [START_REF] Fosgerau | Peptide therapeutics: current status and future directions[END_REF][START_REF] Mcgonigle | Peptide therapeutics for CNS indications[END_REF]. Several strategies have therefore been developed to overcome the undesirable physicochemical properties of therapeutic peptides. These approaches successfully applied to the C-terminal NT(8-13) hexapeptide include backbone and side-chain modifications, such as Damino acid and non-natural amino acid substitutions, incorporation of reduced bonds, lactam bridged mimetics or peptide-peptoid hybrids, N-and C-terminal modifications as well as introduction of conformational constraints via cyclization [START_REF] Bittermann | Evaluation of lactam-bridged neurotensin analogues adjusting psi(Pro10) close to the experimentally derived bioactive conformation of NT(8-13)[END_REF][START_REF] Boules | NT79: A novel neurotensin analog with selective behavioral effects[END_REF][START_REF] Bredeloux | Synthesis and biological effects of c(Lys-Lys-Pro-Tyr-Ile-Leu-Lys-Lys-Pro-Tyr-Ile-Leu) (JMV2012), a new analogue of neurotensin that crosses the blood-brain barrier[END_REF][START_REF] Einsiedel | Discovery of highly potent and neurotensin receptor 2 selective neurotensin mimetics[END_REF][START_REF] Fanelli | Synthesis and Characterization in Vitro and in Vivo of (l)-(Trimethylsilyl)alanine Containing Neurotensin Analogues[END_REF][START_REF] Hughes | Identification and functional characterization of a stable, centrally active derivative of the neurotensin (8-13) fragment as a potential first-in-class analgesic[END_REF][START_REF] Labbé-Jullié | In vivo and in vitro structure-activity studies with peptide and pseudopeptide neurotensin analogs suggest the existence of distinct central neurotensin receptor subtypes[END_REF][START_REF] Sarret | Neurotensin and Its Receptors, Reference Module in Neuroscience and Biobehavioral Psychology[END_REF][START_REF] Sousbie | Structural Optimization and Characterization of Potent Analgesic Macrocyclic Analogues of Neurotensin (8-13)[END_REF][START_REF] Tyler-Mcmahon | Highly potent neurotensin analog that causes hypothermia and antinociception[END_REF][START_REF] Wustrow | Reduced amide bond neurotensin 8-13 mimetics with potent in vivo activity[END_REF].

According to recent structure-activity relationship (SAR) studies, structural modifications with unnatural amino acids were effective in producing NT(8-13) analogs with improved metabolic stability, receptor selectivity and in vivo activity [START_REF] Boules | NT79: A novel neurotensin analog with selective behavioral effects[END_REF][START_REF] Fanelli | Synthesis and Characterization in Vitro and in Vivo of (l)-(Trimethylsilyl)alanine Containing Neurotensin Analogues[END_REF][START_REF] Hapău | Stereoselective Synthesis of β-(5-Arylthiazolyl) α-Amino Acids and Use in Neurotensin Analogues[END_REF][START_REF] Hughes | Identification and functional characterization of a stable, centrally active derivative of the neurotensin (8-13) fragment as a potential first-in-class analgesic[END_REF][START_REF] Magafa | Novel stable analogues of the neurotensin C-terminal hexapeptide containing unnatural amino acids[END_REF][START_REF] Tokumura | Stability of a novel hexapeptide, (Me)Arg-Lys-Pro-Trp-tert-Leu-Leu-OEt, with neurotensin activity, in aqueous solution and in the solid state[END_REF][START_REF] Tyler-Mcmahon | Highly potent neurotensin analog that causes hypothermia and antinociception[END_REF]. In that respect, non-natural silylated amino acids are of great interest for their ability to modulate the physicochemical properties of bioactive peptides [START_REF] Cavelier | Biological activity of silylated amino acid containing substance P analogues[END_REF][START_REF] Cavelier | Influence of silaproline on peptide conformation and bioactivity[END_REF][START_REF] Dalkas | Study of a lipophilic captopril analogue binding to angiotensin I converting enzyme[END_REF][START_REF] Fanelli | Hydrophobic alpha,alpha-Disubstituted Disilylated TESDpg Induces Incipient 310-Helix in Short Tripeptide Sequence[END_REF][START_REF] Fanelli | Synthesis and Characterization in Vitro and in Vivo of (l)-(Trimethylsilyl)alanine Containing Neurotensin Analogues[END_REF][START_REF] Remond | Silicon-Containing Amino Acids: Synthetic Aspects, Conformational Studies, and Applications to Bioactive Peptides[END_REF]. Herein, we report the synthesis and in vitro/in vivo characterization of a new NT(8-13) analog, named JMV2009, which binds to both NTS 1 and NTS 2 receptors. In this compound, the Pro 10 residue was substituted by a silicon-containing unnatural amino acid proline surrogate, denoted silaproline (Sip), which has been shown to retain peptide conformation [START_REF] Cavelier | Conformational studies of proline-, thiaproline-and dimethylsilaproline-containing diketopiperazines[END_REF][START_REF] Cavelier | Influence of silaproline on peptide conformation and bioactivity[END_REF][START_REF] Remond | Silaproline, a Silicon-Containing Proline Surrogate[END_REF][START_REF] Vivet | Synthesis of silaproline, a new proline surrogate[END_REF]. This Sip-containing NT(8-13) analog was further screened in a battery of pain models to assess its putative analgesic properties and evaluate for its ability to induce adverse effects associated with chronic opioid therapy, such as constipation or analgesic tolerance.

Materials and methods

Materials

PD149163 was obtained by the U.S. National Institute of Mental Health, Chemical Synthesis and Drug Supply Program. Morphine sulfate was purchased from Sandoz (Boucherville, QC, Canada).

Synthesis and in vitro characterization of JMV2009

Synthesis, chemical characterization, and in vitro and in cellulo characterization (binding, signaling and plasma stability) of JMV2009 are reported into the accompanying Data in Brief article [START_REF] Besserer-Offroy | Data set describing the in vitro biological activity of JMV2009, a novel silylated neurotensin(8-13) analog[END_REF]

Animals

Experimental procedures were approved by the Animal Care and Ethical Committee of the Université de Sherbrooke, for experiments involving rats (animal use protocol number 035-18) and were in accordance with policies and directives of the Canadian Council on Animal Care.

Experiments involving mice were approved by the Animal Care and Ethical Committee of the Université de Rouen (Cenomax, Agreement of the French Ministry of Research number 54) and were in accordance with policies and directives of the French Council on Animal Care and European Community Council Directive of 24 November 1986 (86:609: EEC). Furthermore, all procedures involving animals followed the ARRIVE recommendations [START_REF] Kilkenny | Animal research: reporting in vivo experiments: the ARRIVE guidelines[END_REF].

Adult male Sprague-Dawley rats Charles River Laboratories,Quebec,Canada) or adult male Swiss albino mice (CD1, 22-26 g, Charles River, St-Aubin-les-Elbeuf, France) were both maintained on a 12:12 hrs light/dark cycle with access to food and water ad libitum. Animals were acclimated for four days to the animal facility and for two days to the manipulations and testing devices prior to behavioral studies. The total number of animals used in this study is 30 mice and 172 rats. Animals were independently and randomly assigned to a saline or treatment group prior any behavioral testing.

Behavioral studies involving mice

Intracerebroventricular injections

The intracerebroventricular injections (i.c.v.) were performed in conscious mice according to the method of [START_REF] Haley | Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse[END_REF] in a volume of 10 µl/mouse.

Visceral pain test

The intraperitoneal (i.p.) injection of a chemical irritant in mice induces a visceral noxious response characterized by a wave of constriction followed by elongation of the abdominal wall [START_REF] Collier | The Abdominal Constriction Response and Its Suppression by Analgesic Drugs in the Mouse[END_REF]. This tonic visceral nociceptive response is often described as the writhing test [START_REF] Koster | Acetic Acid for Analgesic Screening[END_REF]. Briefly, mice received an intracerebroventricular (i.c.v.) injection of saline or JMV2009 20 min before i.p. administration of an acetic acid solution (0.5%). The number of writhes produced for a 5-min period was then counted beginning 5 min after i.p. injection.

Behavioral studies involving rats

Intrathecal injections

Rats were lightly anesthetized with 2% isofluorane. Subsequently, a 25 µl volume of compound or vehicle was administered by injection into the subarachnoid space between lumbar vertebrae L5 and L6, using a 27 G 1/2 needle.

Acute pain test (Tail-Flick test)

Acute nociception was investigated using the tail-flick test. Thermal threshold latencies were determined at baseline and at 10, 20, 30, 40, 50, 60, and 90 min after intrathecal (i.t.) injection of JMV2009 or vehicle. Testing involved measuring the tail withdrawal latency (in s) after immersion in a 52˚C water bath. Six centimeters of the rat's tail were submerged in water and the latency to flick or curl the tail was recorded. Baseline responses (before drug administration) were typically ranging from 3 to 4 s. A cut-off was imposed at 10 s to avoid tissue damage.

The mean latency at peak effect (40 min) was used for the determination of the analgesic efficacy and was converted to % of maximal possible effect (MPE) according to the formula: % maximum possible effect = [(test latency) -(baseline latency)]/[(cut-off) -(baseline latency)] × 100.

Formalin tonic pain test

Persistent pain was assessed using the formalin test. For this purpose, rats were placed for a 60-min habituation period in the experimentation room two days before the test. On the test day, rats received 50 μl subcutaneous injection of diluted 2% formaldehyde into the plantar surface of the right hind paw five min. after i.t. injection of JMV2009 or saline. Rats were then immediately placed in clear plastic chambers (40 × 30 × 30 cm) positioned over a mirror angled at 45°, to allow an unobstructed view of the 4 paws. Behaviors were recorded for the next 60 min.

An intra-plantar injection of formalin produces a biphasic nociceptive response that characterizes this tonic pain model [START_REF] Sawynok | The Formalin Test: Characteristics and Usefulness of the Model[END_REF]. The two distinct phases of spontaneous pain behaviors that occur in rats are suggested to reflect a direct effect of formalin on sensory receptors (phase 1) and a longer lasting pain due to inflammation and central sensitization (phase 2). The nocifensive behaviors were assessed using a weighed score, as described previously by [START_REF] Dubuisson | The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats[END_REF]. A nociceptive mean score was determined for each 3-min period by measuring the amount of time spent in each of four behavioral categories: 0, the injected paw is comparable to the contralateral paw; 1, the injected paw has little or no weight placed on it; 2, the injected paw is elevated and is not in contact with any surface; 3, the injected paw is licked, bitten, or shaken. The total AUC for the inflammatory phase (phase 2) was calculated between 21 to 60 min for each animal [START_REF] Gaumond | Sex differences in opioid-mediated pain inhibitory mechanisms during the interphase in the formalin test[END_REF].

Chronic Construction Injury (CCI) of the sciatic nerve

Surgical procedure

The surgical procedure of the neuropathic pain model was performed as previously described by [START_REF] Bennett | A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man[END_REF] with a modification in the suture used (5-0 Prolene, Ethicon, Inc. Somerville, NJ, USA) in order to minimize suture-induced inflammation. Briefly, animals were anesthetized using 3% isoflurane and the left sciatic nerve was loosely ligatured with 4 sutures distant by 1 mm upstream of the tibial, sural, and common peroneal nerve trifurcation. The muscle, conjunctive tissue and skin were closed with proper sutures and washed with 3% (v/v) hydrogen peroxide. Sham animals received the surgery, but no ligation of the sciatic nerve was performed. Rats were housed for 24 h in separate cages to recover from the surgery and were not given any medication to control post-surgical pain.

The pain-related behaviors were examined at day 0 (pre-surgery, considered the baseline [BL]) and at 3, 7, 14, 21, and 28 days after nerve injury. Animals were moved to the examination room at 8 a.m. and tested one h later. Dynamic weight bearing (DWB) measurements were conducted immediately after the mechanical allodynia assessment.

Evaluation of mechanical allodynia

To determine the presence of mechanical allodynia, rats were placed in enclosures with an elevated wire mesh floor. A dynamic plantar aesthesiometer (Ugo Basile, Stoelting, IL, USA), consisting of a metal probe (0.5 mm diameter), was directed against the hind paw pad exerting an upward force (3.33 g/s). The force required to elicit a withdrawal response was measured in grams and automatically registered when the paw was withdrawn, or the pre-set cut-off was reached (50 g). Five values were taken alternately on ipsi-and contra-lateral hind paw at 15 s intervals. The percentage of anti-allodynia was calculated with the AUC of every treatment for the time period comprised between 3 and 28 days, with the use of the following equation: % antiallodynia =100 × [(CCI + drug) -CCI control]/(sham -CCI control). From the latter formula, 0% represents no anti-allodynic effect of the compound, while 100% corresponds to a complete relief of mechanical hypersensitivity.

Dynamic weight bearing measurements

The DWB device (Bioseb, Boulogne, France) was used to evaluate the presence of nonevoked pain. This apparatus was characterized previously by our group [START_REF] Tétreault | Weight bearing evaluation in inflammatory, neuropathic and cancer chronic pain in freely moving rats[END_REF].

Briefly, rats are allowed to move freely within a Plexiglas enclosure (22 × 22 × 30 cm) with a floor sensor composed of 44 × 44 captors (10.89 mm 2 per captor). Pressure data and live recording are recorded and transmitted to a laptop computer. Raw pressure and visual data were colligated with the latest DWB software available at the time. To calculate weight recovery and rehabilitation, we used the same formula as the anti-allodynia, meaning that: % weight recovery (or rehabilitation) = 100 × [AUC(CCI + drug) -AUC(CCI control)]/[(AUC(sham) -AUC(CCI control)]. From this formula, 0% represents no weight recovery (or rehabilitation) induced by the compound, while 100% corresponds to complete weight recovery or total rehabilitation.

Monitoring of the adverse effects

Gastro-intestinal motility assessment

Constipation has been assessed by measuring the gastro-intestinal (GI) tract motility using the charcoal meal test. Food deprived (16 h) animals were injected s.c. with saline or the compounds to be tested. 30 min. after drug injection, 2-ml of a charcoal meal solution (5% arabic gum, 10% charcoal in water) was administered to the rats by gavage. Rats were euthanized exactly 60 min after, and the progression of the charcoal in the intestine was measured as a ratio of progression of the meal over the total length of the intestine. The results are presented as a percentage of progression of the charcoal meal in the intestine.

Tolerance to the acute thermal pain test

For tolerance experiments, rats were injected i.t. with 30 µg/kg/day of JMV2009 or with saline for five consecutive days. On the sixth day, tail-flick latencies were measured immediately before and over 90 min following i.t. injection of JMV2009 (90 µg/kg). The analgesic effect of this subchronic treatment was assessed using the tail-flick thermal sensitivity assay as described above.

Blood pressure monitoring

Male Sprague-Dawley rats were anesthetized with a mixture of ketamine/xylazine (87 mg/kg:13 mg/kg, i.m.) and placed in supine position on a heating pad. Mean, systolic and diastolic arterial blood pressure as well a heart rate were measured through a catheter (PE 50 filled with heparinized saline) inserted in the right carotid artery and connected to a Micro-Med transducer (model TDX-300, USA) linked to a blood pressure Micro-Med analyzer (model BPA-100c).

Another catheter was inserted in the left jugular vein for bolus injections (1 ml/kg, 5-10 s) of vehicle (isotonic saline), or JMV2009. For relative potency evaluation, changes in mean arterial blood pressure (ΔMABP) from baseline to post-injection in individual animals were determined.

Core body temperature

Body temperature was measured using a thermistor probe inserted into the rectum of adult Sprague-Dawley rats. Prior to testing, animals were individually acclimatized to manipulations and thermistor probe 5 min/day for three consecutive days. On the test day, temperature was measured before (baseline) and each 30 min for up to 90 min following i.t. injection of saline, JMV2009 (3, 30 and 90 µg/kg) or the NTS 1 -selective reference compound PD149163 [START_REF] Feifel | The acute and subchronic effects of a brain-penetrating, neurotensin-1 receptor agonist on feeding, body weight and temperature[END_REF]. Variations in body temperature (Δ body temp) were determined as changes from baseline for each individual animal.

Data analysis and statistical procedures

Data were plotted as mean ± standard error (S.E.M.) for all curves and bar graphs. Twoway ANOVAs followed by a Dunnett's post hoc test were performed for all curves. Kruskal-Wallis followed by a Dunn's correction for multiple comparisons tests were performed for all bar graphs except for the weight recovery and rehabilitation where a Mann-Whitney rank comparison test was used. Prism v7.0a software was used for all statistical analyses. A P value < 0.05 was considered significant and significant differences between groups were represented by * P < 0.05, ** P < 0.01, and *** P < 0.001. Dose-response curve was analyzed by using the log(agonist) vs. response (three parameters) non-linear regression fit of GraphPad Prism v7.0a.

Results

JMV2009 acts as a non-selective NT receptor agonist

We report here the synthesis and pharmacological evaluation of a NT(8-13) analog (JMV2009) carrying an unnatural silicon-containing proline surrogate, the 4-(dimethyl)silaproline (Sip) in replacement of the proline residue at position 10 (Fig. 12and Scheme 1 of the accompanying Data in Brief article) . We first found that this silaprolinecontaining peptide exhibited relatively high and similar binding affinities for both NTS 1 and NTS 2 receptors, with only slightly reduced affinity compared to the native NT peptide (Fig. 3 and Table 1 of the accompanying Data in Brief article). Our results further demonstrated that the presence of this silylated proline residue at position 10 can increase peptide resistance to proteolytic degradation. Accordingly, around 80 % of JMV2009 remained intact after a 5-min incubation with rat plasma, compared to 15 % for the native NT peptide, thus resulting in an improved peptide stability to proteases (Fig. 4

of the accompanying Data in Brief article).

We next examined if this NT peptide derivative was still able to induce receptor activation, by studying its ability to stimulate known intracellular signaling pathways activated by the NT receptor family. We first evaluated the ability of JMV2009 to trigger the activation of different G proteins (Gα q , Gα 13 , Gα i1 , and Gα oA ) and the recruitment of β-arrestins 1 and 2 as it was previously reported for NTS1 endogenous ligands [START_REF] Besserer-Offroy | The signaling signature of the neurotensin type 1 receptor with endogenous ligands[END_REF]. We found that JMV2009 was able to activate the four studied G proteins as well as to promote the recruitment of both β-arrestins 1 and 2 (Fig. 5

and Table 2 of the accompanying Data in Brief article).

We next evaluated the phosphorylation level of the mitogen-activated protein kinase ERK1/2 after NT or JMV2009 stimulation using cell lines stably expressing either NTS 1 or NTS 2 receptors. Our results revealed that JMV2009 was able to induce ERK1/2 activation in a similar manner as NT in both NTS 1 -and NTS 2 -expressing cells (Fig. 6 of the accompanying Data in Brief article). We then assessed the ability of JMV2009 to trigger NT receptor internalization by cell surface enzyme-linked immunosorbent assay (Fig. 7 and Table 3 of the accompanying Data in Brief article). We observed that both NT and JMV2009 promoted NTS 1 receptor internalization, resulting respectively in 59.4 ± 1.5% and 49.1 ± 1.4 % reduction in receptor cell surface expression within 1 h. Likewise, NT and JMV2009 induced NTS 2 -receptor internalization after 60 min of stimulation. Altogether, these results demonstrate that the substitution of Pro 10 by Sip leads to a functional NT agonist that might be associated with antinociceptive properties.

Central delivery of JMV2009 significantly reduces acute and tonic pain

We first used the tail-flick acute pain model to evaluate whether intrathecal (i.t.) delivery of JMV2009 attenuated the withdrawal responses to thermal nociceptive stimuli. We found that i.t. delivery of JMV2009 in rats elicited a dose-dependent antinociceptive response up to 60 min after injection, characterized by an increased tail-flick latency compared to saline-treated animals (Fig. 1A). Peak analgesic responses occurred 40 min after JMV2009 injection, tail-flick latencies returning to baseline by 90 min. Comparison of % MPE at 40 min following JMV2009 administration showed significant antinociceptive effects for each dose tested. Antinociception reached 35.2 ± 3.2 %, 43.4 ± 8.9 %, and 62.0 ±7.7 % at doses of 3, 30 and 90 µg/kg, respectively (Fig. 1B).

We then assessed the efficacy of JMV2009 to alleviate the nociceptive behaviors in two different persistent pain paradigms, using either intraplantar formalin or intraperitoneal injection of acetic acid as chemical somatic and visceral noxious stimuli, respectively. Intraplantar injection of formalin into the right hind paw of saline-pretreated rats induced a biphasic timedependent increase in pain score (Fig. 2). Importantly, i.t. pretreatment with JMV2009 significantly reduced the stereotypical nocifensive behaviors elicited by formalin during the tonic inflammatory phase (21 to 60 min), without affecting the early phase (1 to 9 min) (Fig. 2A).

Indeed, both doses tested markedly abolished the spontaneous persistent pain-related behaviors, reaching 49.7 % and 97 % of inhibition at 3 and 30 µg/kg, respectively (Fig. 2B). The antinociceptive effect of JMV2009 was further tested using the acetic acid-induced writhing model in mice (Fig. 2C). We found that i.c.v. injection of JMV2009 suppressed the visceral pain behaviors in a dose-dependent manner, reducing the number of writhes by 87 % at the highest dose tested and displaying a calculated ED 50 value of 2.33 ± 0.6 µg/kg (Fig. 2D).

JMV2009 attenuates neuropathy-induced mechanical hypersensitivity and improves the quality-of-life proxies

We next investigated the antinociceptive action of JMV2009 in a clinically relevant model of chronic neuropathic pain. Neuropathic pain was induced by chronic constriction injury (CCI) of the sciatic nerve and the mechanical hypersensitivity was determined by applying von frey filaments. As shown in Fig. 3, CCI rats developed tactile allodynia as soon as 3 days after surgery compared with preoperative values and persisted until at least 28 days. As expected, the paw withdrawal thresholds in response to tactile stimuli were not affected in sham-operated animals. Importantly, i.t. injection of JMV2009 was effective in reversing the allodynic state, even at the lowest dose tested (Fig. 3A). Indeed, JMV2009 produced robust anti-allodynic effects, achieving 51.4 ± 3.4 %, 69.2 ± 3.9 % and 66.2 ± 7.6 % of pain relief at the doses of 3, 30 and 90 µg/kg, respectively (Fig. 3B).

To date, the development of potential analgesic agents relies mainly on the measurement of withdrawal responses to acute application of external heat or mechanical stimuli (e.g., von frey filament). However, to improve the translation of analgesics to the clinic, it may be useful to monitor spontaneous innate behaviors indicative of animal well-being in the preclinical drug screening. Accordingly, we previously demonstrated in different chronic pain models that the dynamic weight bearing (DWB) method could be used to measure posture-locomotor functional impairment and rehabilitation as well as health and quality-of-life outcomes in freely moving rats [START_REF] Tetreault | Spinal NTS2 receptor activation reverses signs of neuropathic pain[END_REF][START_REF] Tétreault | Weight bearing evaluation in inflammatory, neuropathic and cancer chronic pain in freely moving rats[END_REF]. In addition to the reflexive mechanical nociceptive test, we thus used the DWB device to assess the effects of JMV2009 on weight load deficits and time-of-use of the injured paw. We first found that i.t. delivery of JMV2009 was effective to reverse the weight bearing deficit induced by the chronic constriction injury of the sciatic nerve (Fig. 4A). Indeed, a 30-µg/kg dose of JMV2009 significantly reduced the ipsilateral paw weight load deficits in non-restrained neuropathic rats, leading to a gain of 40.9 ± 6.4 % of weight recovery over the 28-day period of observation (Fig. 4A-B). Next, we evaluated the efficacy of JMV2009 to reverse the impaired use of the affected limb. As shown in Fig. 4C, JMV2009 improved the time spent by the animal on its injured paw. The drug effectiveness was also expressed as % of rehabilitation within the four-week period, where 100% rehabilitation corresponds to a complete recovery of the paw usage. This outcome parameter is considered clinically relevant and represents a good indicator of quality of life improvement [START_REF] Cobos | Inflammation-induced decrease in voluntary wheel running in mice: A nonreflexive test for evaluating inflammatory pain and analgesia[END_REF]. At the dose used, JMV2009 was found to improve the use of the affected limb to 52.8 ± 10.3% (Fig. 4D).

JMV2009 displays a safer profile compared to opioids or other NT derivatives

To achieve good analgesia, patients' compliance to a prescribed pain treatment is of high importance. Among the various reasons of noncompliance, the presence of mild to severe side effects seriously affects medication adherence in patients with chronic pain [START_REF] Benyamin | Opioid Complications and Side Effects[END_REF]. For instance, constipation, nausea, respiratory depression and tolerance are common clinical concerns that may lead to discontinuation of long-term opioid therapy. Here, we therefore assessed whether JMV2009 could induce constipation after systemic administration, as it is reported that NT and NT derivatives could have a direct effect on smooth muscles of the intestine in isolated ileum organ bath [START_REF] Fanelli | Synthesis and Characterization in Vitro and in Vivo of (l)-(Trimethylsilyl)alanine Containing Neurotensin Analogues[END_REF]. To this end, we measured the effects of JMV2009 and morphine on the gastrointestinal tract motility using the charcoal meal test in starved rats (Fig. 5A). Morphine (10 mg kg -1 s.c.) was found to reduce by 45% the progression of the charcoal meal in the intestine, when compared to saline-treated animals. By contrast, both NT and JMV2009 subcutaneously injected at 10 mg kg -1 did not affect the charcoal meal intestinal transit. Rats treated with JMV2009 even showed an almost significant increase of the gastrointestinal tract motility, since the charcoal meal travelled 98.7 ± 0.8 % of the intestine (P = 0.064) compared to 82.2 ± 2.4 % and 87.4 ± 2.5 % when rats were treated with either NT or saline, respectively (Fig. 5A).

We next determined whether repeated injection of JMV2009 induced the development of tolerance to its analgesic effect. For this purpose, rats were subjected to i.t. injections of JMV2009 (30 µg/kg) daily for five consecutive days, and tail-flick tests were conducted over 90 min on the last day to evaluate changes of JMV2009-induced antinociception. In this experimental paradigm, repeated JMV2009 exposure did not produce tolerance, as demonstrated by the absence of changes in tail-flick latency, when compared to saline pre-treated animals (Fig. 5B). Next, since NT administered centrally also causes hypothermia through an NTS 1 -dependent mechanism [START_REF] Bissette | Hypothermia and intolerance to cold induced by intracisternal administration of the hypothalamic peptide neurotensin[END_REF][START_REF] Feifel | The acute and subchronic effects of a brain-penetrating, neurotensin-1 receptor agonist on feeding, body weight and temperature[END_REF], we measured the ability of JMV2009 to induce changes in body temperature after central administration. We found here that JMV2009 administered i.t. at effective analgesic doses did not induce hypothermia. In sharp contrast, the NTS 1 -selective agonist PD149163, used here as a reference compound induced a pronounced time-dependent drop in body temperature (Fig. 5C). Finally, we investigated the ability of JMV2009 to induce changes in blood pressure after systemic injection. We found here that a dose of 0.1 mg kg -1 of JMV2009 injected i.v. was able to induce a drop of blood pressure similar to the one induced by the same dose of NT. Changes in mean arterial blood pressure (MABP) were found to be of -29.2 ± 5.7 mmHg for rats receiving JMV2009 compared to -25.0 ± 2.5 mmHg for NT-injected animals (Fig. 5D).

Discussion

G-protein coupled receptors are the target of more than 25% of the FDA-approved drugs [START_REF] Overington | How many drug targets are there?[END_REF] and represent one of the largest classes of therapeutic targets in pain medicine [START_REF] Stone | In search of analgesia: emerging roles of GPCRs in pain[END_REF]. Nowadays, validation of new molecular targets and development of new effective, non-addicting pain medications are critical, but still remain extremely slow [START_REF] Kissin | The development of new analgesics over the past 50 years: a lack of real breakthrough drugs[END_REF][START_REF] Yaksh | Development of New Analgesics: An Answer to Opioid Epidemic[END_REF]. In the past decades, both NTS 1 and NTS 2 have generated a growing interest as potential targets for pain treatment [START_REF] Dobner | Neurotensin and pain modulation[END_REF][START_REF] Feng | The Roles of Neurotensin and its Analogues in Pain[END_REF][START_REF] Kleczkowska | Neurotensin and neurotensin receptors: characteristic, structure-activity relationship and pain modulation--a review[END_REF][START_REF] Sarret | Neurotensin and Its Receptors, Reference Module in Neuroscience and Biobehavioral Psychology[END_REF]. This increasing attention is probably driven in part by the fact that NT drugs act as effective opioid-free medications for pain management, producing significant pain relief in various animal models of acute and chronic pain, including neuropathic pain for which opioids have shown their limitations [START_REF] Guillemette | Intrathecal administration of NTS1 agonists reverses nociceptive behaviors in a rat model of neuropathic pain[END_REF][START_REF] Perez De Vega | Recent progress in non-opioid analgesic peptides[END_REF][START_REF] Tetreault | Spinal NTS2 receptor activation reverses signs of neuropathic pain[END_REF]. Importantly, the combination of NT analogs to opioids has also the potential to exert complementary (synergistic or additive) analgesic actions, thereby minimizing the adverse effects related to chronic opioid use [START_REF] Boules | Analgesic synergy of neurotensin receptor subtype 2 agonist NT79 and morphine[END_REF][START_REF] Boules | NT69L, a novel analgesic, shows synergy with morphine[END_REF][START_REF] Eiselt | The combination of opioid and neurotensin receptor agonists improves their analgesic/adverse effect ratio[END_REF].

In recent years, changes in the pharmaceutical industry have led to renewed interest in the use of peptides as therapeutics [START_REF] Fosgerau | Peptide therapeutics: current status and future directions[END_REF][START_REF] Kaspar | Future directions for peptide therapeutics development[END_REF]. The current pipeline of peptide therapeutics includes over 60 approved drugs and 150 in active clinical development, with 40% of these peptide drug candidates targeting GPCRs [START_REF] Lau | Therapeutic peptides: Historical perspectives, current development trends, and future directions[END_REF]. The development of peptide drugs with high therapeutic potency remains however challenging and requires the implementation of chemical strategies and/or computational structural prediction to synthesize modified peptides with improved stability, pharmacokinetics and in vivo activity profiles. Among the chemical approaches used to optimize the properties of the NT(8-13) peptide, incorporation of unnatural amino acids, such as N-methylarginine, Dornithine, D-α-naphthylalanine, tert-leucine or L-(trimethylsilyl)alanine respectively at positions 8, 9, 11, 12 and 13, has been shown to be effective, increasing significantly NTS 1 and NTS 2 receptor binding affinity or selectivity as well as peptide stability and in vivo biological activity [START_REF] Boules | NT79: A novel neurotensin analog with selective behavioral effects[END_REF][START_REF] Fanelli | Synthesis and Characterization in Vitro and in Vivo of (l)-(Trimethylsilyl)alanine Containing Neurotensin Analogues[END_REF][START_REF] Hughes | Identification and functional characterization of a stable, centrally active derivative of the neurotensin (8-13) fragment as a potential first-in-class analgesic[END_REF][START_REF] Rossi | Systemically and topically active antinociceptive neurotensin compounds[END_REF][START_REF] Tokumura | Stability of a novel hexapeptide, (Me)Arg-Lys-Pro-Trp-tert-Leu-Leu-OEt, with neurotensin activity, in aqueous solution and in the solid state[END_REF][START_REF] Tyler-Mcmahon | Highly potent neurotensin analog that causes hypothermia and antinociception[END_REF]. To date, only few studies have investigated the impact of Pro 10 substitutions on NT receptor binding and to our knowledge, none of them have evaluated their efficacy in vivo. The proline residue in position 10 plays a crucial role for peptide conformation and SAR studies reveal that substituted proline analogs that promote a reverse turn are more tolerated than those inducing an extended backbone conformation [START_REF] Bittermann | Evaluation of lactam-bridged neurotensin analogues adjusting psi(Pro10) close to the experimentally derived bioactive conformation of NT(8-13)[END_REF][START_REF] Held | Impact of the proline residue on ligand binding of neurotensin receptor 2 (NTS2)-selective peptide-peptoid hybrids[END_REF][START_REF] Heyl | Structure-activity and conformational studies of a series of modified C-terminal hexapeptide neurotensin analogues[END_REF]. Indeed, replacement of Pro 10 by thioproline, hydroxyproline, its 4 and 6 member ring counterparts azetidine carboxylic acid and pipecolic acid, or Tic and Aic cyclic aromatic derivatives all induces conformational changes resulting in a significant loss of NT receptor binding affinity [START_REF] Bittermann | Evaluation of lactam-bridged neurotensin analogues adjusting psi(Pro10) close to the experimentally derived bioactive conformation of NT(8-13)[END_REF][START_REF] Heyl | Structure-activity and conformational studies of a series of modified C-terminal hexapeptide neurotensin analogues[END_REF]. These results are consistent with the crystal structure of the NTS 1 -NT(8-13) complex in its activatedlike conformation, which shows a tight oriented hydrophobic binding site for the pyrrolidine ring of Pro 10 as well as extensive van der Waals interactions with the residue W339 in ECL3 [START_REF] Krumm | Structure and dynamics of a constitutively active neurotensin receptor[END_REF][START_REF] White | Structure of the agonist-bound neurotensin receptor[END_REF]. Here, we have therefore replaced Pro 10 by a silylated proline surrogate that exhibits very similar conformational properties that the proline residue in peptides [START_REF] Remond | Silicon-Containing Amino Acids: Synthetic Aspects, Conformational Studies, and Applications to Bioactive Peptides[END_REF][START_REF] Remond | Silaproline, a Silicon-Containing Proline Surrogate[END_REF]. The substitution of the γ-carbon by a dimethylsilyl group was found to slightly decrease the affinity of JMV2009 for NTS 1 while this chemical substitution seemed to be adequate for NTS 2 binding. Accordingly, proline isostere bearing a thiazolidine unit instead of the pyrrolidine ring, as well as fluoro-substituted proline analogs on C γ have already been reported to be well tolerated when incorporated into NTS 2 -selective peptoids [START_REF] Held | Impact of the proline residue on ligand binding of neurotensin receptor 2 (NTS2)-selective peptide-peptoid hybrids[END_REF]. This Sip-containing NT(8-13) peptide also exhibited an improved resistance to proteolytic degradation, probably to the endopeptidases 24.11 and 24.16 responsible for inactivating NT by cleaving at the Pro 10 -Tyr 11 bond [START_REF] Labbé-Jullié | In vivo and in vitro structure-activity studies with peptide and pseudopeptide neurotensin analogs suggest the existence of distinct central neurotensin receptor subtypes[END_REF][START_REF] Myers | Cancer, chemistry, and the cell: molecules that interact with the neurotensin receptors[END_REF]. The steric hindrance of the dimethyl group borne by the silicon atom could indeed induce a misrecognition of the Sip 10 -Tyr 11 peptide bond by these metallo-endopeptidases. To further increase the plasma stability of this NT derivative, it would be interesting to combine the substitution of Pro 10 by Sip to N-terminal protection through the incorporation of blocked amino group (i.e. N αmethylarginine) at position 8 or reduced amide bond between positions 8 and 9 as well as to replace Ile 12 by a tert-leucine residue to avoid enzymatic cleavage at the Tyr 11 -Ile 12 peptide bond [START_REF] Dubuc | JMV 449: a pseudopeptide analogue of neurotensin-(8-13) with highly potent and long-lasting hypothermic and analgesic effects in the mouse[END_REF][START_REF] Fanelli | Synthesis and Characterization in Vitro and in Vivo of (l)-(Trimethylsilyl)alanine Containing Neurotensin Analogues[END_REF][START_REF] Tokumura | Stability of a novel hexapeptide, (Me)Arg-Lys-Pro-Trp-tert-Leu-Leu-OEt, with neurotensin activity, in aqueous solution and in the solid state[END_REF]. Importantly, this Sip-containing NT(8-13) analog was effective in triggering intracellular signaling responses at both NTS 1 and NTS 2 , as shown by the activation of G proteins, recruitment of β-arrestins, ERK1/2 activation and receptor internalization [START_REF] Besserer-Offroy | The signaling signature of the neurotensin type 1 receptor with endogenous ligands[END_REF][START_REF] Besserer-Offroy | Data set describing the in vitro biological activity of JMV2009, a novel silylated neurotensin(8-13) analog[END_REF][START_REF] Gendron | Lowaffinity neurotensin receptor (NTS2) signaling: internalization-dependent activation of extracellular signal-regulated kinases 1/2[END_REF].

We then reported the analgesic efficacy of this silylated NT(8-13) analog in different experimental pain models. We first found that JMV2009 was able to significantly reduce acute thermal pain, even at the lower dose of 3 µg/kg. This result is consistent with previous studies demonstrating that both NTS 1 and NTS 2 agonists decrease the tail and foot withdrawal nociceptive responses to noxious heat stimuli [START_REF] Boules | NT69L, a novel analgesic, shows synergy with morphine[END_REF][START_REF] Hughes | Identification and functional characterization of a stable, centrally active derivative of the neurotensin (8-13) fragment as a potential first-in-class analgesic[END_REF][START_REF] Rossi | Systemically and topically active antinociceptive neurotensin compounds[END_REF][START_REF] Sarret | Potent spinal analgesia elicited through stimulation of NTS2 neurotensin receptors[END_REF]. In persistent pain paradigms, JMV2009 was also able to totally abolish the nociceptive behaviors induced by either intraplantar injection of formalin or visceral noxious stimulation. Interestingly, JMV2009 displays more potent analgesic effects in the formalin pain model than the other non-selective NT analogs, NT69L and JMV2007 previously described [START_REF] Fanelli | Synthesis and Characterization in Vitro and in Vivo of (l)-(Trimethylsilyl)alanine Containing Neurotensin Analogues[END_REF][START_REF] Roussy | Spinal NTS1 receptors regulate nociceptive signaling in a rat formalin tonic pain model[END_REF]. At equimolar dose, it is also more potent than the metabolically stable NTS 2 -selective analog JMV431 in reducing the formalin-induced nocifensive behaviors [START_REF] Roussy | Evidence for a role of NTS2 receptors in the modulation of tonic pain sensitivity[END_REF]. In the peripheral neuropathic pain model, JMV2009 induced relief of CCI-induced evoked hypersensitivity and ongoing pain. Indeed, we first observed that acute i.t. administration of JMV2009 was effective in reversing the development of tactile allodynia in nerve-injured rats. These data thus reinforce previous findings demonstrating that central or systemic delivery of either NTS 1 or NTS 2 agonists reduces the mechanical hypersensitivity in preclinical models of neuropathic pain [START_REF] Demeule | Conjugation of a brainpenetrant peptide with neurotensin provides antinociceptive properties[END_REF][START_REF] Guillemette | Intrathecal administration of NTS1 agonists reverses nociceptive behaviors in a rat model of neuropathic pain[END_REF][START_REF] Hughes | Identification and functional characterization of a stable, centrally active derivative of the neurotensin (8-13) fragment as a potential first-in-class analgesic[END_REF][START_REF] Tetreault | Spinal NTS2 receptor activation reverses signs of neuropathic pain[END_REF]. Finally, and perhaps most importantly, the spinal delivery of JMV2009 was also able to reverse significantly the weight bearing deficits as well as to improve the rehabilitation outcomes. In freely moving rats, we indeed found that JMV2009 induced partial recovery of the weight load on the injury leg and increased the time-ofuse of the affected limb. The monitoring of these spontaneous nociceptive-related behaviors is now considered critical for evaluating the effectiveness of new drugs [START_REF] Deuis | Methods Used to Evaluate Pain Behaviors in Rodents[END_REF][START_REF] Mogil | Animal models of pain: progress and challenges[END_REF][START_REF] Tappe-Theodor | Studying ongoing and spontaneous pain in rodents-challenges and opportunities[END_REF]. Most behavioral tests currently used rely only on the measurement of stimuli-evoked pain behaviors, such as mechanical hyperalgesia and allodynia. However, neuropathic pain patients predominantly suffer from non-evoked ongoing and spontaneous pain, and not from stimulus-evoked hypersensitivity. This type of pain is the one having the most negative impact, seriously affecting the patient's daily activities and quality of life, and leading to multiple comorbid conditions, such as mood and anxiety disorders, sleep perturbation, depression or attention deficits. Compared to the previously reported analog, JMV431, JMV2009 produces stronger anti-allodynic effects in CCI-treated animals as well as substantial improvement in the management of the health and quality-of-life outcomes [START_REF] Tetreault | Spinal NTS2 receptor activation reverses signs of neuropathic pain[END_REF]. Finally, when compared to typical first-and second-line medications for neuropathic pain, JMV2009 appeared as potent as the tricyclic antidepressant amitriptyline and more effective than morphine, pregabalin, gabapentin and ibuprofen in relieving spontaneous pain behaviors [START_REF] Angeby-Moller | Using the CatWalk method to assess weight-bearing and pain behaviour in walking rats with ankle joint monoarthritis induced by carrageenan: effects of morphine and rofecoxib[END_REF][START_REF] Deseure | Differential drug effects on spontaneous and evoked pain behavior in a model of trigeminal neuropathic pain[END_REF][START_REF] Nakazato-Imasato | Pharmacological characteristics of the hind paw weight bearing difference induced by chronic constriction injury of the sciatic nerve in rats[END_REF][START_REF] Tetreault | Spinal NTS2 receptor activation reverses signs of neuropathic pain[END_REF].

Adherence to long-term opioid therapies represents a very clinical issue. Indeed, although opioids, such as morphine have been shown to be effective in relieving pain, the development of bothersome side effects may outweigh the benefits of opioid treatment and even lead to prompt discontinuation of the drug treatment [START_REF] Benyamin | Opioid Complications and Side Effects[END_REF][START_REF] Besserer-Offroy | Sending out Biased Signals: an Appropriate Proposition for Pain?[END_REF].

Constipation is particularly difficult to manage since patients don't usually develop tolerance upon repeated morphine administration [START_REF] Bowers | The Evolving Role of Long-Term Pharmacotherapy for Opioid-Induced Constipation in Patients Being Treated for Noncancer Pain[END_REF]. Likewise, tolerance to the respiratory depressant effects of opioids develops only slowly and incompletely, putting patients at risk for respiratory depression with dose escalation [START_REF] Algera | Opioid-induced respiratory depression in humans: a review of pharmacokinetic-pharmacodynamic modelling of reversal[END_REF]. Furthermore, the long-term use of opioids can lead to analgesic tolerance and opioid-induced hyperalgesia [START_REF] Chu | Opioid tolerance and hyperalgesia in chronic pain patients after one month of oral morphine therapy: a preliminary prospective study[END_REF]. Unlike morphine, we found here that both NT and JMV2009 did not cause constipation. On the contrary, JMV2009 seemed to induce better gastrointestinal transit compared to NT-injected rats. These results are consistent with previous studies demonstrating that NT stimulates colonic motility in rats, cats and humans [START_REF] Hellstrom | Effects of neurotensin, substance P and methionineenkephalin on colonic motility[END_REF][START_REF] Pellissier | Peripheral neurotensin participates in the modulation of pre-and postprandial intestinal motility in rats[END_REF][START_REF] Thor | Neurotensin increases colonic motility[END_REF]. Supporting a role for NT in propulsive colonic motility is also the finding that i.v. injection of NT in human subjects stimulates defecation and may improve the intestinal movements in colons of patients with slow transit constipation [START_REF] Calam | Neurotensin stimulates defaecation[END_REF][START_REF] Tomita | The effects of neurotensin in the colon of patients with slow transit constipation[END_REF]. We further demonstrated that JMV2009 did not produce analgesic tolerance following repeated agonist administration. Accordingly, various NT analogs, such as [D-Trp 11 ]-NT, ABS212 and β-lactotensin do not induce the development of antinociceptive tolerance [START_REF] Dubuc | Tolerance to the hypothermic but not to the analgesic effect of [D-Trp11]neurotensin during the semichronic intracerebroventricular infusion of the peptide in rats[END_REF][START_REF] Hughes | Identification and functional characterization of a stable, centrally active derivative of the neurotensin (8-13) fragment as a potential first-in-class analgesic[END_REF][START_REF] Yamauchi | Antinociception induced by beta-lactotensin, a neurotensin agonist peptide derived from beta-lactoglobulin, is mediated by NT2 and D1 receptors[END_REF]. We also showed that JMV2009 was ineffective for inducing hypothermia, as opposed to the NT(8-13) agonist PD149163 that triggered a sustained decrease in body temperature at equimolar dose. This result is quite surprising since most NT agonists acting at NTS 1 can produce a significant drop in body temperature [START_REF] Bissette | Hypothermia and intolerance to cold induced by intracisternal administration of the hypothalamic peptide neurotensin[END_REF][START_REF] Fantegrossi | Antinociceptive, hypothermic, hypotensive, and reinforcing effects of a novel neurotensin receptor agonist, NT69L, in rhesus monkeys[END_REF][START_REF] Feifel | The acute and subchronic effects of a brain-penetrating, neurotensin-1 receptor agonist on feeding, body weight and temperature[END_REF]. Nevertheless, few NT analogs acting at NTS 1 were previously reported to be able to distinguish between hypothermia and analgesia, such as the NT(8-13) analogs, NT27 and NT77L [START_REF] Boules | Neurotensin analog selective for hypothermia over antinociception and exhibiting atypical neuroleptic-like properties[END_REF][START_REF] Tyler | Evidence for additional neurotensin receptor subtypes: neurotensin analogs that distinguish between neurotensin-mediated hypothermia and antinociception[END_REF]. The concept of ligand biased signaling provides support to the idea that NT analogs acting at NTS 1 may have the ability to part between analgesia and hypothermia by recruiting different receptor downstream signaling pathways [START_REF] Kenakin | Biased Receptor Signaling in Drug Discovery[END_REF]. Finally, we showed that JMV2009, when injected systemically, was able to induce a drop in blood pressure which was comparable to the drop induced by NT. This effect, mediated by NTS 1 , can be triggered after an i.v. injection of a non-selective neurotensinergic compound but also after i.t. delivery at higher doses [START_REF] Zogovic | Intrathecal neurotensin is hypotensive, sympathoinhibitory and enhances the baroreflex in anaesthetized rat[END_REF]. Indeed, as shown by Zogovic and Pilowsky, administration of at least 75 µM of NT is required to lower blood pressure after spinal delivery [START_REF] Zogovic | Intrathecal neurotensin is hypotensive, sympathoinhibitory and enhances the baroreflex in anaesthetized rat[END_REF]. For comparison purposes, the analgesic dose of 30 µg/kg of JMV2009 corresponds to an injection of less than 500 nM (for a rat weighting 300 g). Finally, NT-and opioid-related adverse effects could be mitigated by the use of combining neurotensinergic and opioidergic ligands either bi-topic ligands or combination therapy, as recently demonstrated using systemic injections of morphine combined to a brain-penetrant NT agonist [START_REF] Eiselt | The combination of opioid and neurotensin receptor agonists improves their analgesic/adverse effect ratio[END_REF][START_REF] Kleczkowska | PK20, a new opioid-neurotensin hybrid peptide that exhibits central and peripheral antinociceptive effects[END_REF].

Conclusion

In conclusion, we demonstrate here for the first time that the NT(8-13) peptide can accommodate the substitution of the proline residue to give rise to a powerful analgesic peptide.

We further provide evidence that NT agonists do not induce the common adverse effects associated with the use of opioids (i.e. constipation and tolerance), thus offering a non-opioid option for the treatment of pain. Finally, these findings suggest that NT agonists used combined with opioid therapy might contribute to adequate pain relief, while reducing the severity of opioid-and neurotensin-driven adverse events. Writhes were counted over a 5-min period after i.p. injection of 0.5% acetic acid. ** P < 0.01 and *** P < 0.001 (vs. Saline), ## P < 0.01 ( JMV2009 40 vs. JMV2009, 0.4 µg/kg) in a Kruskal-Wallis test ( 2 (4)= 31.06, P < 0.0001) followed by a Dunn's correction for multiple comparisons.

Figure legends

The number of indicated writhes is the mean ± S.E.M. from groups of 6 mice. (D) Dose-response curve of JMV2009-induced antinociception in the acetic acid in the writhing model in mice.

JMV2009 displayed an ED 50 of 2.33 ± 0.6 µg/kg, as determined by non-linear regression fit in GraphPad Prism (R 2 = 0.61).

Fig. 1 .

 1 Fig. 1. Antinociceptive response to i.t. injection of JMV2009 in the tail-flick test. (A) Doseand time-dependent antinociceptive effect of JMV2009 (3, 30, and 90 µg/kg) on tail-flick latencies in rats. Baseline latencies were taken three times before acute i.t. injection of JMV2009. Latencies were determined every 10 min for up to 60 min and a final measure was taken at 90 min following drug administration. ** P < 0.01 (JMV2009, 3 µg/kg vs. Saline); ## P < 0.01 (JMV2009, 30 µg/kg vs. Saline); † P < 0.05, and † † † P < 0.001 (JMV2009, 90 µg/kg vs. Saline) in a two-way ANOVA (F(7, 393) = 25.40) followed by a Dunnett's post hoc test. (B) Percentage of maximum possible effect (MPE) ± S.E.M. determined at the time of peak anti-nociceptive response (40 min) for each dose (3, 30, and 90 µg/kg). * P < 0.05 and *** P < 0.001 (JMV2009 vs. Saline) in a Kruskal-Wallis test ( 2 (3)= 19.48, P = 0.0002) followed by a Dunn's correction for multiple comparisons. Each data point represents the mean ± S.E.M. n = 8 rats per group.

Fig. 2 .

 2 Fig. 2. Effects of central administration of JMV2009 on tonic and visceral pain. (A)Reduction of the nocifensive behaviors after i.t. injection of JMV2009 (3 and 30 µg/kg) in the formalin-induced tonic pain model. * P < 0.05 (JMV2009, 3 µg/kg vs. Saline) and ### P < 0.01 (JMV2009, 30 µg/kg vs. Saline) in a two-way ANOVA (F(2, 455) = 168.7) followed by a Dunnett's post hoc test. (B) The cumulative nociceptive response, expressed as mean area under the curve (AUC) ± S.E.M., is measured during the inflammatory phase of the formalin test (21 -60 min). *** P < 0.001 (JMV2009 vs. Saline) in a Kruskal-Wallis test ( 2 (2)= 17.82, P = 0.0001) followed by a Dunn's correction for multiple comparisons. Each symbol represents the mean ± S.E.M. of determinations made in 6 rats. (C) Dose-dependent antinociceptive effects of JMV2009 (0.4, 4, 12 and 40 µg/kg, i.c.v.) in the acetic acid-induced writhing model in mice.

Fig. 3 .

 3 Fig. 3. Effects of JMV2009 on mechanical allodynia induced by the chronic constriction injury (CCI) of the sciatic nerve in rats. (A) Paw withdrawal thresholds were assessed with an automated von Frey hair during the 4-week period after CCI surgery. Baseline (BL) withdrawal thresholds were determined for all CCI-and sham-operated rats prior to surgery. In neuropathic rats, acute i.t. injection of JMV2009 (3, 30, and 90 µg/kg) at day 3, 7, 14, 21, and 28 post-surgery effectively reduces the mechanical hypersensitivity. ** P < 0.01, *** P < 0.001 (JMV2009, 3 µg/kg vs. CCI); ### P < 0.001 (JMV2009, 30 µg/kg vs. CCI); † † † P < 0.001 (JMV2009, 90 µg/kg vs. CCI) in a two-way ANOVA (F(4, 182) = 236.8) followed by a Dunnett's post hoc test. (B) Percentage of anti-allodynia calculated over the 3-28 day period. All tested doses of JMV2009 were effective to attenuate the development of mechanical allodynia. *** P < 0.001 (JMV2009 vs. Saline) in a Kruskal-Wallis test ( 2 (3)= 26.59, P < 0.0001) followed by a Dunn's correction for multiple comparisons. Vertical bars represent S.E.M. (n = 6 per group).

Fig. 4 .FiguresFig. 1

 41 Fig. 4. Effects of JMV2009 on neuropathy-induced weight-bearing deficits and decrease in time-of-use of the injured limb. (A) Weight bearing impairment was measured using the dynamic weight bearing (DWB) after acute i.t. injection of JMV2009 (30 µg/kg) at day 3, 7, 14, 21, and 28 post-surgery. Baseline values (BL) were assessed before CCI surgery. * P < 0.05 and ** P < 0.01 (JMV2009, 30 µg/kg vs. CCI) in a two-way ANOVA (F(2, 30) = 61.79) followed by a Dunnett's post hoc test. (B) Treatment effectiveness was determined by measuring the percentage of weight recovery over the 3-28 day period, with 100% corresponding to full recovery in the weight borne on the injured limb. *** P < 0.001 in a two-tailed Mann-Whitney test. (C) Percentage of time spent on the injured limb was determined using the DWB device over the 28-day period following CCI surgery. * P < 0.05 (JMV2009, 30 µg/kg vs. CCI) in a two-way ANOVA (F(2, 30) = 19.19) followed by a Dunnett's post hoc test. (D) Determination of the percentage of functional rehabilitation was examined over the 28-day period after acute administration of JMV2009. 100% rehabilitation corresponds to a full recovery in the use of the injured limb. * P < 0.05 in a two-tailed Mann-Whitney test. Each symbol represents the mean ± S.E.M. of determinations made in 6 rats.

  Fig.2

Acknowledgements

PT is the recipient of a post-doctoral fellowship from the Canadian Institutes for Health Research (CIHR, grant MFE-135472). ÉBO is the recipient of a Fond de recherche du Québec -Santé 29 (FRQ-S, grant 255989) and a CIHR (grant MFE-164740) research fellowships. RLB has been awarded an FRQ-S and a Faculty of Medicine and Health Sciences of the Université de Sherbrooke Ph.D. scholarships. PS is the recipient of a Tier 1 Canada Research Chair in Neurophysiopharmacology of Chronic Pain and a member of the FRQ-S-funded Québec Pain Research Network (QPRN). The National Institute of Mental Health (NIMH) is acknowledged for providing us with the NTS 1 -selective compound PD149163.

Élie Besserer-Offroy: Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Writing -Original Draft, Writing -Review & Editing, Visualization. Rebecca L Brouillette: Investigation. Adeline René: Ressources. Alexandre Murza: Investigation. Roberto Fanelli: Ressources. Karyn Kirby: Investigation. Alexandre J Parent: Investigation. Isabelle Dubuc: Investigation. Nicolas Beaudet: Conceptualization, Validation, Formal Analysis. Jérôme Côté: Investigation. Jean-Michel Longpré: Conceptualization, Validation, Formal Analysis. Jean Martinez: Ressources, Supervision. Florine Cavelier: Ressources, Writing -Review & Editing, Supervision, Funding Acquisition. Philippe Sarret: Conceptualization, Validation, Formal Analysis, Supervision, Funding Acquisition.

All the authors approved the version of the manuscript to be published.

Funding sources

This research was supported by grants from the Canadian Institutes for Health Research [grant numbers MOP-74618, FDN-148413]

Authors contributions

Pascal Tétreault: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing -Original Draft, Writing -Review & Editing, Visualization.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article. Each data point represents the mean ± S.E.M. n = 6 rats per group.