RF313, an Orally Bioavailable Neuropeptide FF Receptor

RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes effects of RF-amide-related peptide-3 and opioid-induced hyperalgesia in rodents Khadija Elhabazi, Jean-Paul Humbert, Isabelle Bertin, Raphaelle Quillet, Valérie Utard, Martine Schmitt, Jean-Jacques Bourguignon, Emilie Laboureyras, Meric Ben Boujema, Guy Simonnet, et al.

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Khadija Elhabazi, Jean-Paul Humbert, Isabelle Bertin, Raphaelle Quillet, Valérie Utard, et al.. RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes effects of RF-amide- related peptide-3 and opioid-induced hyperalgesia in rodents. Neuropsychopharmacology, Nature Publishing Group, 2017, 118, pp.188-198. 10.1016/j.neuropharm.2017.03.012. hal-01603852

HAL Id: hal-01603852 https://hal.archives-ouvertes.fr/hal-01603852 Submitted on 25 May 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Copyright Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., legal disclaimersthatapply tothejournalpertain. note thatduringtheproductionprocesserrorsmaybe discoveredwhichcouldaffectthecontent,andall copyediting, typesetting,andreviewoftheresultingproof beforeitispublishedinitsfinalform.Please our customersweareprovidingthisearlyversionofthe manuscript.Themanuscriptwillundergo This isaPDFfileofanuneditedmanuscriptthathas been acceptedforpublication.Asaserviceto 10.1016/j.neuropharm.2017.03.012. amide-related peptide-3andopioid-inducedhyperalgesia inrodents, Simonin, F.,RF313,anorallybioavailableneuropeptide FFreceptorantagonist,opposeseffectsofRF- Beltramo, M.,Bucher,B.,Sorg,T.,Meziane,H.,Schneider, E.,Petit-Demoulière,B.,Ilien,Bihel,F., Bourguignon, J.-J.,Laboureyras,E.,BenBoujema,M., Simonnet,G.,Ancel,C.,Simonneaux,V., Please citethisarticleas:Elhabazi,K.,Humbert,J.-P.,Bertin,I.,Quillet,R.,Utard,V.,Schmitt,M., Accepted Date: Revised Date: Received Date: To appearin: Reference: DOI: PII: Petit-Demoulière, BrigitteIlien,FrédéricBihel,Simonin Beltramo, BernardBucher,TaniaSorg,HamidMeziane,ElodieSchneider,Benoit Ben Boujema,GuySimonnet,CarolineAncel,ValérieSimonneaux,Massimiliano Utard, MartineSchmitt,Jean-JacquesBourguignon,EmilieLaboureyras,Meric Khadija Elhabazi,Jean-PaulHumbert,IsabelleBertin,RaphaelleQuillet,Valérie RF-amide-related peptide-3andopioid-inducedhyperalgesiainrodents RF313, anorallybioavailableneuropeptideFFreceptorantagonist,opposeseffectsof Accepted Manuscript Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. 6 March2017 27 February2017 21 November2016 Neuropharmacology NP 6630 10.1016/j.neuropharm.2017.03.012 S0028-3908(17)30095-3 Comment citer cedocument:

Neuropharmacology (2017),doi: Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., Illkirch, France Laboratory of Excellence Médalis, Illkirch, Médalis, France LaboratoryExcellence of Affiliations : Affiliations (F. [email protected] Simonin) ([email protected] (B Petit-Demoulière), Meziane),[email protected]@epf (H. Bucher (B. (M.Beltramo),[email protected] Mas [email protected](V.Simonneaux), caroli (G. Boujema),[email protected] Simonnet), Labour (E. Bourguignon), [email protected] (V. mschmitt@unist Utard), [email protected] Humbert), [email protected] Bertin),rquille (JP (I. E-mail addresses j i h g f e d c b a ElhabaziKhadija rodents effects RF-amide-relatedof peptide-3 and opioid-in anRF313, orally neuropeptidebioavailable FF recep Simonnet TaniaSorg MartineSchmitt Bordeaux Segalen, Bordeaux, France. France. Segalen, Bordeaux, Bordeaux de Tours, Nouzilly, France deFrance Tours, Nouzilly, 67404 Illkirch-Graffenstaden, 67404 France Laboratory of Excellence Médalis, Illkirch, Médalis, France LaboratoryExcellence of I l i e n rue Laurent Fries, 67404 Illkirch, France Laurentrue 67404 Fries, Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 Institut National M de la Santé et de la Recherche UMR7 Centre National de Scientifique, la Recherche Laboratoire Biophotonique et Pharmacologie, UMR 72 LaboratoireUMR et Pharmacologie, Biophotonique Homéostasie-Allostasie-Pathologie-Réhabilitation, Homéostasie-Allostasie-Pathologie-Réhabilitation, Institut des IntégratNeurosciences Cellulaires et Institut de Génétique et de Biologie Moléculaire e Moléculaire Biologie de et Génétique de Institut Physiologie de la Reproduction et des Comportements Physiologiede la Reproduction Biotechnologie et Signalisation Cellulaire, UMR 724 Cellulaire, UMR BiotechnologieetSignalisation Laboratoire Innovation Thérapeutique, UMR 7200 CNRS Laboratoire 7200 InnovationThérapeutique,UMR EPEI, HNMN Isiu Ciiu d l Souris la de Clinique Institut PHENOMIN, CELPHEDIA, effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. a , Frédéric Bihel c Crln Ancel Caroline , g,h,i,j , HamidMeziane, b , Jean-Jacques, Bourguignon a

ACCEPTED Humbert Jean-Paul , : [email protected] (K. Elhabazi), jeanpaul.humbe (K. [email protected] Elhabazi), : b# , Frédéric Simonin Comment citer cedocument: d Vlre Simonneaux Valérie , ACCEPTED MANUSCRIPT g,h,i,j , Elodie,Schneider

a a Iael Bertin Isabelle , * #

b , Emilie,Laboureyras [email protected] Quillet), (R. d . Ilien),Bihel),(F. [email protected] . Msiiin Beltramo Massimiliano , Strasbourg, CNRS, France 3212 UPR ives, édicale, U964, Illkirch,France édicale,U964, MANUSCRIPT UMR 5287 CNRS, Université CNRS, 5287 de UMR g,h,i,j, t Cellulaire, Université de Strasbourg, 1 1 Strasbourg, de Université Cellulaire, t ra.fr (M. Schmitt), [email protected] (J.-J. [email protected] (J.-J. (M. Schmitt), 2 CNRS, Université de Strasbourg, CNRS, 2 104, Illkirch, 104, France eyras), [email protected] (M. Ben eyras),[email protected] , INRA, UMR7247 CNRS, Université INRA, CNRS, , UMR7247 ), [email protected] (T. Sorg), (T. ),[email protected] [email protected] l.ch (E. Schneider), [email protected] (E. Schneider), l.ch 13 CNRS, Université de Strasbourg, CNRS, 13 a [email protected] (C. Ancel), (C. [email protected] , Benoit,Petit-Demoulière Rpale Quillet Raphaelle , , Université , de Strasbourg, duced hyperalgesia in tor tor antagonist, opposes IS, re arn Fis F- Fries, Laurent rue 1 (ICS), c , Meric Ben BoujemaMeric,Ben [email protected] [email protected] e Brad Bucher Bernard , a Vlre Utard Valérie ,

g,h,i,j, , Brigitte, c ,

Guy a f , , Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., s.c.,subcutaneous. prolactin releasi PrRP, hyperalgesia;os; per p.o., LH, NPFF, kisspeptin; luteinizing neuropep hormone; HI, hyperalgesia index; i.c.v., intracerebroventric HI,hyperalgesia index; chinese CHO, ova area-under-the-curve; AUC, hamster Abbreviations # E-ma +33 83; 46 85 368 Fax: +33 Tel75; 48 85 : 368 I Brant - 10413 Boulevard 67412 300, Sébastien - CS de Biotechnologie Strasbourg Ecolede Supérieure 724 Cellulaire, UMR Biotechnologieet Signalisation Correspondence * Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 These authors participated equally to this work this equally to Theseauthors participated effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents.

ACCEPTED :

: Dr. Frédéric Simonin FrédéricDr. : Simonin Comment citer cedocument: ACCEPTED MANUSCRIPT

ng related RF-amide RFRP, peptide; peptide; MANUSCRIPTintrathecal; Kp, intraperitoneal; i.p., i.t., ular; 2 CNRS/Université de Strasbourg CNRS/Université 2 llkirch Cedex - France. Cedex llkirch il : [email protected] [email protected] : il tide FF; OIH, opioid-induced OIH, FF; tide opioid-induced ry; FSH, follicle stimulating hormone; follicle stimulating ry;FSH,

Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., antagonist activity at NPFF1 receptor (NPFF1R) subt receptoractivity (NPFF1R)antagonist at NPFF1 a pronounced RF313 that exhibited show We details. study, Inweinvestigate hyperalgesia this rats. in which efficiently receptorRF313, preve antagonist, identified a recently analgesicnovel tolerance. We effectsside including associatednumerous the with effective the analg most Although opiates represent ABSTRACT hyperalgesiaLHsecretion – - NPFF- peptides - RF-amide nociception receptors Keywords a peptides theregulatoryof RF-amide-related roles a betterRF313 might be pharmacological considered lack activ of Moreover, its agonist administration. adverseeffects side reducingactionof opiates and the development of for compound interestingtherape receptors. abilityactivate to Altogethe kisspeptin whileRF9 administeredeffect no blocked alone fully when but recep the kisspeptin toward activityµM) 100 (up to disp RF9, antagonist RF313 NPFF dipeptidic receptor oflong opioid-induced hyperalgesia. Moreov lasting improved their analges rats opiatesin and mice, it mice to in up administered majoreffectsside when Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. : : perse

ACCEPTEDLH which a increase significant levels in induced Comment citer cedocument:

ACCEPTED MANUSCRIPT

d the properties of this compound into more into theproperties compound of this d r, our data indicate that RF313 represents an RF313 our data indicate r, that ic efficacy and prevented thedevelopment andefficacy prevented ic ity at the kisspeptin receptor indicates that ityreceptor at the kisspeptin associated their chronic with

FF (NPFF) orally activeneuropeptide nd NPFF1R in reproduction. NPFF1R nd reproduction. in development of pain hypersensitivity development and of pain treatments chronic is use in esics,their nts the development of fentanyl-induced the development nts of tor. Finally, in male hamster, RF313 had tor.Finally, hamster, male in 30 mg/kg. When co-administered with with mg/kg. co-administered 30 When

the increase in LH RFRP-3, by the increase induced in MANUSCRIPT morphine analgesia - opioid-induced - morphineanalgesia opioid-induced er, and in marked contrast with the with er,contrast and marked in utic tools aiming attools utic improving analgesic ype both selectivity forselectivityreceptors, NPFF tool, when compared to RF9, to examine examine RF9, to to when compared tool, layedaffinity and agonist no negligible in vitro vitro in is consistent with its its with consistent is and and in vivo in and no Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., analgesia (Elhabazi et al., 2013; Fang 2013; et al., et 2011 al., analgesia(Elhabazi their on own pr oractivity displayto hyperalgesic 2014). More recently, activityand (Ayachi Simonin, the homeostaticr in pronociceptiveinvolved system N the hypothesis accumulatingthat evidence support reverse peptide to morp showedthe ofcapacity this system theYang opioid wereby revealed early very Zajac,The Roumypain modulating and propert 1998). et al., 1987) a Roth (Prokaipressureet al., 2006; 200 (Murakami et al., Simansky,2003), reproduction et al (Johnson including functions behavior feeding respectively (Ayachi and Simonin, 2014). Simonin, and respectively (Ayachi asare the endog neuropeptide considered (NPAF), AF a peptide-1 et Roumeas al., 2015), RF-amide-related RF mammalian all binding for promiscuous properties NPFF both receptor subtyp theirreceptors. Although affinityand forthe hasbeen be to mandatory shown conservedArg-Phe-NH of bind them All receptorset (Quillet al., 2016). Kisspeptin alias alias GPR54 GPR10), (Kiss1R (PrRPR GPR74 alias FF2 Neuropeptide (NPFF2R aliasGPR147), RF-amidethat called receptorsreceptors, (GPCRs), 1. Introduction Introduction 1. Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. NPFF receptors and their endogenous ligands have be endogenous have ligands NPFF and their receptors Neuropeptide FF (NPFF) receptors a belong to subfam FFNeuropeptide(NPFF) ACCEPTED 2 2 Comment citer cedocument: (RF-amide) ACCEPTED MANUSCRIPT

signature at their carboxyl-terminal end. This sequ This carboxyl-terminal end. signatureat their 1

nd nociception (Ayachi and Simonin, 2014; 2014; Simonin, nociception nd and (Ayachi endogenous neuropeptides that display a endogenous that neuropeptides ; LiuIn other findings contrast, et al., 2001). ; event the development of morphine of eventthe development ., 2007; Kovacs et al., 2014; Nicklous and Nicklous Kovacs 2007; ., et al., 2014; hine-induced analgesia. Sincethen, hine-induced analgesia. the activity of these peptides towards the activity peptides of these egulation of opioid antinociceptive antinociceptive of egulation opioid MANUSCRIPT FF1 (NPFF1R Neuropeptide encompasses RFRP-1 and RFRP-3 were also shown to wereto shown also and RFRP-3 RFRP-1 nd -3 (RFRPs), and NPFF and NPFF -3 nd and (RFRPs), and coworkers (Yang etal., 1985) who coworkers(Yang and es have been shown to display to shown been eshave PFF belongs to an anti-opioid anti-opioid PFFan to belongs 8; Pineda et al., 8; 2010a), arterial blood enous ligands of NPFF1R and NPFF2R and NPFF2R NPFF1R of enousligands -amide peptides (Elhabazi et al., 2013; et (Elhabazi al., 2013; -amidepeptides ies of NPFF and its relationship with with relationship iesof and NPFF its ) and QRFP (QRFPR alias GPR103) alias GPR103) )and (QRFPR QRFP ),prolactin-releasing peptide en shown to modulate several enmodulate to shown ily of G protein-coupled Gprotein-coupled ilyof ence Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., and 6.59, have areceptorand impact on have strong 6.59, activati and receptor activat ligand in involved recognition functions mediated by RF-amide receptors. mediated functions receptors.RF-amide by of prime is importance to amidebinding components, an theselective or design novel NPFF2R- NPFF1R- of agonistat Kiss activity later rely to its shown on activity theRF9 Unfortunately, compound. were obscured data ma in the control of in the reproductive axis RFRPs Wang et al., 2006; Simonin et (Elhabazi al., 2012; analgesicchronicopioid-induced tole hyperalgesia, establish to asubtypes,tool very proved it useful et al., 2006). cann compound Although (Simonin this na similar with antagonist, selectivereceptor NPFF (Henningsen et al., 2016; Leon20 al.,2016; and (HenningsenTena-Sempere, et although different effe pituitary-gonadalwith axis receptor shown and NPFFR1beenalso its RFRP-3 have needs function still theirprecise rolesthis of in arethe theirmodul respective in peptides involved bo that al.,data 1999). Altogether, indicate these et al Jhamandas etal., analgesia1993; (Gouarderes intrathecally they peptidesproduc areadministered a peptides related NPFFhave that NPFF support and Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. Previous SAR study conducted on NPFF receptors reve study receptors NPFF on conducted SAR Previous The adamantane dipeptide derivative RF9 has been id has derivative been TheRF9 adamantane dipeptide in vivo in

ACCEPTEDRizwa et al., 2016; Quillet al., 2010b; (Pinedaet Comment citer cedocument: ACCEPTED MANUSCRIPT

2 further investigation. Indecade, last further the investigation.

1R (Kim et al., 2015; Min et al., 2015). Thus, Min et (Kim al., 2015; 1R th NPFF1 and NPFF2 receptor subtypes and subtypes receptor NPFF1 NPFF2 and th the roles of NPFF receptors in acute in the roles or of receptors NPFF cts according to sex and species ctsandaccording sex to on and were suggested to form an acidic and on were to suggested ion. Among these residues, positions 5.27 5.27 Among ion. positions these residues, ation of nociception and pain.However, of ation nociception and

et al., 2008). The role of endogenous etal., of 2008). The role ., 2006; Kontinen and Kontinen Xu et Kalso, 2006; ., 1995; and enhance emorphine antinociception nomolar affinity at NPFF1R and NPFF2R and NPFF2R nomolaraffinity at NPFF1R rance, dependence and hypothermiarance,and dependence mmals has also been evaluated by using by evaluated has beenalso mmals MANUSCRIPT ot discriminate between both receptor discriminate both between ot pro-opioid effect. Indeed, when these effect. pro-opioid address the various physiological address physiological the various 15). tagonists, devoid of secondary devoid of RF- tagonists, by its potent gonadotropin-releasing potent its by to regulate to the hypothalamo- entified ten yearsas a entified ago ten aled important residues residues important aled n et n al., 2012), which was Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., We first performedfirst We (Gealageas et al., 2012) or at the carboxy-amidated et(Gealageasal., 2012) or andreceptors)NPFF2nature of modificatio at NPFF1 work the a impact (in terms we in previous explored novel antagonist of NPFF receptors referred to as c as of receptors novelantagonist NPFF referred to led t bearingchain side moiety a its on piperidine a with non-natural Replacementarginineornithin of f of either residues Phe Arg asor of substitutions to play an important role in functional selectivity in play roleto an important NPFF both in receptor sub negativelybinding pocket effects of RFRP-3 was evaluated both both evaluated effectswas of RFRP-3 t receptor Then, towardsubtypes. the five RF-amide tool to examine the regulatory roles of RFRPs and N the regulatory examine to rolesof tool RFRPs lack of givenat its activity indicatedRF313, that g andeffectsRF313 on theofcomparison RF9 of the RF out pointed (subcutaneousclearly route) or oral of its anti-hyperalgesicproperty mice.Examination c fentanyl acute lastingby or hyperalgesia induced analgesicof ofimprove efficacy theopiat RF313 to Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. In this study, we examinedInwe the pharmacological prop study, this ACCEPTED in vitro in Comment citer cedocument: binding experiments to examine its affinity se its examine and to binding experiments ACCEPTED MANUSCRIPT

in vitro in and and 3

Kiss1R, is a is more pharmacological suitable Kiss1R, (Findeisen et (Findeisen al., 2012). or other aminoacids (Bihel et al., (Bihel 2015). other aminoacids or o the obtention of the RF313 compound, a compound, of the the RF313 o obtention hronic morphine injections, both in rats in and both hronicmorphine injections, vivo. in ompound ompound 313 as an orally-active compound. Finally, compound. as 313 an orally-active parts of Arg-Phe-NH according to the mode of administration ofadministration the mode accordingto es and to prevent the development of long- the esdevelopment and prevent to e derivative (Schneider et eal., 2015) derivative (Schneider of affinity, and antagonistic selectivity MANUSCRIPTthe antagonize heability of RF313 to PFF1R in reproduction. PFF1Rreproduction. in types, while the position 7.35 was 7.35 shown types,while the position onadotropin release in male hamster release in onadotropin ns introduced at the N-terminus introduced ns at the We further addressed the potential addressedthe further potential We erties of RF313 into more details. into ertiesof RF313 12e in (Bihel et al., 2015). et al., (Bihel 2015). in 2

dipeptides as dipeptides well On the other hand On lectivity profile ,

Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., [ (Luxembourg). was RFRP-3 from Genecust ; France 20),and Perray-en-Yvelines, Tebu-Bio (Le (Stra Human were RF-amidefrom Polypeptide peptides Ci/mmol) and [ Ci/mmol) Phe-NH Pontoise, from(Invitrogen, Molecular Cergy Probes F FrancopiawasMorphine(Paris, hydrochloride from (Sain were from(IBMX)Sigma-Aldrich and probenecid 2.1. Materials 2.1. Materials2. and methods 2.3. Receptor cDNA constructs, cell expression and cell expression and Receptorconstructs, 2.3. cDNA greater thanmM. 20 solubility water in aqueou in solubility good a showed RF313 (RP-HPLC). hi reversed-phase by 98% to superior as established ( 40% of yield overall an with (OtBu)-OH Fmoc-L-Glu physico-chemical and RF313 synthesis 2.2. propertie France).(Courtaboeuf, El were (2200 QRFP43 Ci/mmol) from Perkin purchased from Hartmann Analytic (Braunschweig,Germany).[ Analyticfrom Hartmann Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 3 H]-FFRF-amide (13.6 Ci/mmol) was from H]-FFRF-amide(Sacl the CEA Ci/mmol) (13.6 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. RF313 (Fig. 1) was synthesized in seven steps, sta steps, seven in synthesized was 1) (Fig. RF313 Fentanyl citrate, naloxone hydrochloride, forskolin hydrochloride, Fentanylnaloxone citrate, 2 trifluoroacetate (RF9) was synthesized as reported (RF9)synthesized trifluoroacetate was

ACCEPTED S]Guanosine5’- Comment citer cedocument: ACCEPTED MANUSCRIPT O -[

γ -thio] triphosphate ([ -thio] 4

membrane preparations membranepreparations MANUSCRIPT QRFP26 or 26RFa, RFRP-3). Mouse or QRFP26 26RFa, RFRP-3). France).N-adamantane-1-carbonyl-Arg- s s 125 rance). Fluo-4 acetoxymethyl ester was ester Fluo-4 rance).acetoxymethyl hpromne iud chromatography liquid gh-performance sbourg, France;NPFF, Kp-10, PrRP- sbourg, t Quentin Fallavier,Quentin t France). I]-Kp-10and [ (2200 Ci/mmol) mer LifemerAnalytical Sciences and , 3-isobutyl-1-methylxanthine 3-isobutyl-1-methylxanthine , 35 ay, France).ay, [ rting from commercially available available commercially from rting s solution with a thermodynamic thermodynamic a with solution s (Simonin et al., 2006). et al., (Simonin S]GTP Bihel et al., 2015). Purity was was Purity 2015). al., et Bihel γ S; 1000 Ci/mmol) were 1000 S; Ci/mmol) 3 H]-PrRP-20 (150 H]-PrRP-20 125 I]- Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., were incubated with 0.3 nM [ nM were 0.3 with incubated NPFF of (NPFF2R). Membranes µM (NPFF1R) 1 RFRP-3 or radioligand. of nM this levels Non-specific binding asNPFF2R-containing NPFF1R- a both radiotracer, or binding. QRFPR membranes were incubated with 0.03 n 0.03 with were incubated membranes binding. QRFPR incubated with 0.02 nM [ incubatednM 0.02 with binding levels, respe defineand specific non total peptides or compounds to be peptidesbindin or to tested compounds for their the presence in of three receptorsubtypes), other andfor mL NPFF2R; 0.5 NPFF1R (30min, final volume 25°C were Competition-typeat experiments performed measurement.forbinding specific non [ wer membranes Briefly,NPFF2R-containing NPFF1R- or 2.4. Radioligand binding assays Radioligand 2.4. at–80 2013) and stored et describedal., (Elhabazi human PrRPR and QRFPR were and fromQRFPR Parmentie giftM. a humanPrRPR as reported expression, selectionfor (Elhab stable Fran vector expression Pontoise, (Invitrogen, Cergy Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 3 H]-FFRF-NH effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. Human NPFF1R, NPFF2R and Kiss1R cDNAs and Kiss1R were subclone HumanNPFF1R, NPFF2R Binding assay conditions were asessentiallydescri Bindingassay conditions Membranes from CHO cells expressing RF-amide recept expressing RF-amide Membranescells CHO from 2 ACCEPTED respectively. , [ When Comment citer cedocument: 125 I]-Kp-10 using 1 µM Kp-10determine to specific non I]-Kp-10µM 1 using 3 H]-PrRP-20, in the absence or presence of 1 µM PrRP the in H]-PrRP-20, µM absenceof 1 presence or ACCEPTED MANUSCRIPT

125 I]-(D-Tyr1,FF used N-Me-Phe3)-Neuropeptide was 5

increasing concentrations of unlabelled increasing concentrations ctively.were membranes Kiss1R-containing azi et al., 2013). CHO cells expressing et al., 2013). cells CHO azi (1 use. mg as aliquots °C until prot./mL) g affinity. were determined in the presence of 1 µM presencewere of µM 1 the in determined

MANUSCRIPT cells CHO before into ce)andtransfected membranes were incubated with 0.015 0.015 with were incubated membranes , under binding equilibrium conditions conditions under , binding equilibrium M [ M 1 h, 0.25 mL final volume for volume the final mL 0.25 h, 1 e incubated with 10 nM or nM nM 3 10 with eincubated r (IRIBHM, Brussels, Belgium). Belgium). Brussels, r(IRIBHM, bed in (Elhabazi etbed (Elhabazi al., 2013). in 125 PrRPR-expressing CHO cells CHO PrRPR-expressing I]-43RFa, using 1 µM 26RFa I]-43RFa,µM 1 using ors were prepared as were prepared ors d into the pCDNA3.1 the pCDNA3.1 into d radioligand radioligand -20 to -20to Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., NaCl, 1.25 MgCl 1.25 NaCl, 2.5.1. [ 2.5.1. experiments Infunctional 2.5. vitro France)scintillati anda quantified TopCount using LiapparatusElmer a 96-well GF/B (Perkin unifilter fre Membrane-boundfrom radioactivity was separated were allowed to pre-incubate with cells for 10 min cells for with weremin allowed10 pre-incubate to RF313 compounds, were examined as (Simonin reported examined were RF313compounds, RF-amide each individual fromcells CHO expressing 2.5.2. cAMP 2.5.2. accumulation Compounds were serial diluted in assay buffer (in m assaywere in (in diluted Compounds serial buffer 2 probenecid, et al., mM 2.5 as (Elhabazi described 2.5.3. Calcium mobilization mobilization Calcium 2.5.3. oflevels cAMP radioimmunoassay. by cell forgsupernat centrifugationmin, 15 at 2,000 and c HCl of M 0.5 ice-cold by addition stopped the the absence or the orpre be peptides to tested, in IBMX, with at for37°C min mM 10 1 then challenged et according receptors(Elhabazi were to monitored Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. 35 Stimulation by RF-amide [ peptides of Stimulation endogenous

Kiss1R-expressing CHO cells were loaded with 2.5 µM cellswere loaded 2.5 with Kiss1R-expressing CHO Variations in the overall cAMP content cAMP of Variationscells theCHO overall in S]-GTP γ 2 S binding S

, 1.25 CaCl 1.25 , ACCEPTED Comment citer cedocument:

2 , 6 KCl, 10 glucose 10 7.4)KCl, and6 , an BSA,pH and mg/mL 1 ACCEPTED MANUSCRIPT

sence of 10 µM forskolin. The was reaction forskolin. of sence µM 10 ants were stored at −20°C until quantitation quantitation until wereants at −20°C stored on counter (Perkin Elmer). counter on (Perkin Elmer). before agonist challenge. Agonist-evoked challenge. beforeagonist 013). al., 2013). Cells were al., 2013). Cells with pre-incubated fe and Analytical Sciences, Courtaboeuf, Courtaboeuf, Sciences, and Analytical fe ell freezing for 1 h at -80°C. After at h 1 -80°C. freezing ell for MANUSCRIPT M: 10 HEPES, 0.4 NaH 0.4 HEPES, 10 M: receptor, and its inhibition by receptor,inhibition and its RF9 or e radioligand by rapid filtration through filtration rapid by eradioligand variousof concentrations compounds et al., 2006). 35 S]-GTP expressing NPFF1 or NPFF2 expressing or NPFF1 Fluo-4 the presence in AM of γ S binding S membranes to 2 PO 4 , 137.5 137.5 , tagonists tagonists Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., CO so were endanimals with euthanized of experiments, Ev a measurementsmanner. in blinded were performed and grouptreated a Control behavioral experiments. a and room wereequipment the testing to habituated li were the during andExperiments water. performed cage and kept under a 12 h/12 h light/dark h h/12 at cycle cageaand 12 kept under we gmice(25-30 Denmark). Taconic, Animals weight; Laboratories,L’Ar gRiver (250-350Charles weight; 2.6.1. Animals 2.6.1. thenumber of used discomfortand animals reduce to effort All committee. approvedethical the by local (European thecare animals of Communitie laboratory 2.6. In vivo pharmacological studies studies In 2.6. vivo pharmacological fluorescencelevels. (cells permeab basal to and weremaximal normalized (excitat at nm 520 emission through °C fluorescence calciumincreasesintracellular ov in were recorded Adult male Syrian hamsters were bred in-house and male in-house Syrian Adult and were bred hamsters light/darkfree atwith ± cycle 22 fo access to 2°C Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. 2 (fill rate minute (fill per chamber volume of 20% of the Nociception tests were performed on male, awake, un awake, Nociceptionwere performed tests male, on All experiments were carried out in strict accordan were strict in experiments out All carried

ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

od and od water. 21 ± 21 with 1°C animal were s minimize to made er time (5 sec intervals overat sec) 37 220 er(5 sec time intervals

ssignment as well as pain responses wellas pain ssignmentas MANUSCRIPT nd handled for 1-2 weeks before 1-2 handled nd starting weeks for ion at 485 nm). Peak response amplitudes amplitudes at Peak nm). 485 response ion dium pentobarbital (120 dium mg/kg, ori.p.) ght-on phase of the cycle. Rats and mice Rats cycle. ght-onof the phase bresle, France) and on C57BL/6Nmale France)and bresle, on . . ilized with 20 µM digitonin) µM 20 with ilized s Council Directive Council s and 2010/63/EU) ery animal was used only the At animalwas once. eryused re housed in groups ofthree five groups to per in rehoused ). ce with the European guidelines for guidelines cethe European with maintained under a 14h /10 h h /10 maintainedunder a 14h restrained Sprague-Dawley rats restrainedSprague-Dawley ad libitum ad access to food access to Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., McCormick, 1957). 1957). McCormick, syrin Hamilton micea were performed modified using mice, respectively. mice, respectively. orally(p.o.)o at mL/kg 1 orsubcutaneously (s.c.) 2.6.2. Drug administration administration Drug 2.6.2. (C.R.E.M.E.A.S) (2010/63/EU) and approve careanimals laboratory of latency (in sec) for tail withdrawal latencywater from for (in sec) hot tail and was their restrainedtail pocket imme grid a in as et (Elhabazi test al.,previously 2014 described valuecut-offgt usedwas and prevent seta to 600 Massy, squeaked.analgesimeter (Apelex, Basile The g)wa (inpressure al.,1990). increasing Uniformly fromadapted the Randall–Selitto test vocalization in the nociceptive threshold of Measurement 2.6.3. recover one to week. for from surgery the was steel cannula in 30-gauge placed stainless Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. Male hamsters were prepared for i.c.v. injections a i.c.v. injections Malehamsters for were prepared Intracerebroventricular (i.c.v., left lateral ventr left lateral Intracerebroventricular(i.c.v., All drugs were dissolved in physiological in drugs saline (0 All were dissolved All experiments were accordance in with experiments out All carried

The nociceptive mechanical response of rats was mea Therats response nociceptive was of mechanical The nociceptive thermal threshold of threshold mice was Thedeter nociceptive thermal ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

; Simonin et al., 1998). Briefly, were Simonin mice ; rmL/kgrats 10 weight) and for (vol/body (52± or ± 48 °C 0.5 aswas a°C) 0.5 taken rsed in a thermostated water bath. water The rseda in thermostated issue damage. damage. issue lateral ventricle and animals were allowedlateral and animals ventricle method (Elhabazi et al., 2014; Kayser et (Elhabazi al., method 2014; et

s applied to the hind paw until the rat until paw the applied hind s to rats and mice and rats MANUSCRIPT France; 1 mm stylus tip diameter) was France;stylus tip mm 1 d by locald commitee the icle, 5 µL total volume) injections in in volume) injections total µL 5 icle, ge as previously described (Haley and (Haley geasdescribed previously s reported (Ancel et al., 2012). A etreported al., s 2012). (Ancel .9%) and administered the foreuropean the directive mined using the tail immersion immersion using mined the tail sured using the paw-pressure suredthe paw-pressure using Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., immersion test (at test at over min-intervals, 30 52°C), immersion Variationsof (5nociceptiv or mg/kg, saline. s.c.) morphine-induced RF313 on Effect of 2.6.5. analgesi value. u over 48°C) period, test, a immersion 6-days 4- to nocice long-termin changes Theimpact of RF313 on was 0 fentanylonce evaluated at day injection dail (48°C). test f every afterwere h the1 thresholds last measured before fentan min 20 receiveds.c.) RF313 (5 mg/kg, return baseli to until thefentanylfirst injection meas responses fentanylwere or saline. Nociceptive and orally mg/kg) 0.5 subcutaneouslyor (0.05, 0.1 p interval) was min of using evaluated fentanyl the four to c ofthe short-term rats response analgesic Elhab rats al.,in and2000; both mice (Celerier et opiatesand of a earlyof delayed effect analgesic fentanyl-induced RF313 on Effect of 2.6.4. analgesi wasof sec 25 set value(52°C) and (48°C) of sec 15 Inthe measureresponse.absence nociceptive of the Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. On day 0, RF313 (5mg/kg) or saline were administere saline were On day RF313 0, or (5mg/kg) Very similar experiments were performed on mice wit were Verymice on experiments similar performed o route or (subcutaneous and Theimpact of the dose Experiments were designed according to a protocol e were a according to protocol Experiments designed ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

9 ne values. ne

e responses were measured using responses the tail were e measured azi et al., 2012). azi for hyperalgesiclasting several days, state onsecutive injections (4x80 µg/kg, s.c., 15 15 s.c., µg/kg, (4x80 injections onsecutive entanyl injection using the tail immersion immersion tail usingentanyl injection the a 0 to 180 min period following min a 180 to 0 morphine y and mice (tail rats test) pressure in (paw

was administered aw RF313 test. pressure ntil recovery ntil pre-drug baseline of the (0.3, 1 and 3 mg/kg) 30 min before min 30 andmg/kg) 3 (0.3, 1 a and hyperalgesia and a to avoid tissue damage. damage. avoid tissue to a reaction, cut-off of any nociceptive

MANUSCRIPTevery and after h 2 0) (time ured before yl (4x60 µg/kg, s.c.) and nociceptive and s.c.) ylµg/kg, (4x60 a and hyperalgesia in mice hyperalgesia mice and a in ral) of administration of RF313 on administration ral)of nabling the visualization of an nabling the visualization ptive sensitivity induced by induced sensitivity ptive d (s.c.) 20 min before morphine before min 20 (s.c.) d h few h animals : modifications Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., isoflurane vapour. Thirty min after min hams injection, isofluraneThirty vapour. were givenalone the in combination, or in µL/min), secret LH RF313 RF9on and RFRP-3, Effect of 2.6.6. performedasfor described coursewere experiments using immersion the tail beforeinjection, morphine the nociceptive Duringperiod, of threshold th this r samethen treatment orThe salinewas injections. way ANOVA, followed by Fisher’sbyStatisti test. wayfollowed PLSD ANOVA, carried out using the StatView software. software. carriedusing out the StatView s of was Fisher’ssignificance The test. level PLSD Po (ANOVA). ofanalysisone-way variance two-way or grcontrol for relativethe values the reference to the area the cur above which represents experiment, (HI) hyperalgesiain or eitherthe index as the AUC (Celerier etmethod a calculated the by trapezoidal as group.Analgesiadependingwas the on quantified Diego, CA, USA) and Kaleidagraph 4.0 (Synergy Softw (Synergy Diego,USA)and Kaleidagraph 4.0 CA, 2.7. Data and statistical analyses statistical and Data 2.7. 19 (Tena-Sempereet al., validatedradioimmunoassay takenintracardial by punct vapor and was the blood Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. I.c.v. injections of RFRP-3 or drugs (1.5 µg in 3 µ 3 in or drugs (1.5 µg I.c.v.of RFRP-3 injections LH levels are mean values ± for LHhamst 7 aremean to 6 S.E.M. values levels Data for nociceptive tests are asmean Datava expressed for tests nociceptive Binding and functional data were analyzed using Pri Binding andwere analyzed functional using data ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

10 oup. Both wereanalyzed of types using data oup.

e animals was measured everyewas animals day, measured min 60 l., 2000). Hyperalgesia was quantified 2000). Hyperalgesia l., was ters were deeply anesthetized with CO with anesthetized terswere deeply days. 7 thenext epeatedover once-daily et at p < 0.05. All statistical analyses etat statistical < p were All 0.05. the fentanyl induced-hyperalgesia test (at 48°C). On day 8, analgesia 8, (at test On day 48°C). time- ure for subsequent LHdosagea using ure for subsequent ve expressed as a mean percentage amean ve expressed as MANUSCRIPT anaesthesia with morning light under the(AUC) area-under-the-curve day 0. day 0. 93). ion in the male Syrian hamster the male in Syrian ion cal significance was set at p calsignificance was st-hoc analyses were performedst-hoc with analyses are, Reading, PA, USA). are, Reading, PA, L/animal infused at a flow rate L/animalata of flow 1 infused lues ±lues for animals 15 to 5 S.E.M. sm 4.0 (GraphPad 4.0 sm San Software, ers. Data were analyzed by ers.analyzed one- Data were < 0.05. 2

Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., RF313 pharmacolog of Incharacterization 3.1. vitro Results 3. the affinity of RF313 for NPFF receptors in binding NPFFin thereceptors affinity of RF313 for NPFF1R and NPFF2R (245 ± NPFF1R(245 and and ± nM NPFF2R 41 2238 r nM, 418 used moreFF, which is routinely Phe3)-Neuropeptide receptors([ stu for As the radioligand this used Kiss1R). in µM et al., 2015). This compound, that we that R called et here compound, al., 2015). This the development ofNPFF prevents that fen receptors 3.1.1. Binding affinity properties RF313 affinity of Binding 3.1.1. moiety. replaceda with biphenyl on moiety derivative ornithine a bearing piperidine was argi removed, theC-terminal amide function the a receptoris NPFFderivative anta dipeptide of the a on single built al.,(Bihel2015), non-natu is et the other RF-amide receptors was marginal theother (K receptors RF-amide selectivitythe NP togethera towards with moderate a Table RF313 1, exhibited comparison.in As shown o receptor amideverify specificity peptides to the wastested and RF313 QRFPR). Kiss1R NPFF2R,PrRPR, of cell membranes with each experiments expressing Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. In a recent study, we have identified an orally actidentified an orally In awe recent study, have In order to further characterize RF313 properties, characterize In further to order

3 H]-FFRF-NH ACCEPTED Comment citer cedocument: 2 ) have not been extensively characterized yet,wef ) have extensively characterized been not ACCEPTED MANUSCRIPT

11 i > 20 µM for PrRPR and QRFPR, for K PrRPR > µM 20 ral amino acid. As shown in Figure acid. RF313 1, amino in As shown ral

f the binding assays and with RF9 for a and thebinding with assays f

et whichal., 2006), in (Simonin gonistRF9 its side chainside and its ring the adamantine F313 and referred to as compound compound F313and as referred to dy for binding experiments with NPFF with experiments dybinding for FF1Rinteraction with while its subtype, experiments with [ with experiments MANUSCRIPT nine residue replaced with a with non-natural residue replaced nine ical properties properties ical tanyl-induced hyperalgesia in rats in (Bihel tanyl-inducedhyperalgesia the five RF-amide receptors (NPFF1R, (NPFF1R, thefive receptors RF-amide , and found similar Ki values Ki for and , found similar marked preference for NPFF receptorsNPFF markedfor preference ive peptidomimetic antagonist of antagonist peptidomimetic ive we performed competition weperformed competition in parallel with endogenous with RF- parallel in espectively). RF313 binding espectively).RF313 125 I]-(D-Tyr1,N-Me- urther evaluated evaluated urther i > 100 12e in in Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., cellular(Fig. a response with pA 2B), RF313 clearly Incontrast, expressingoppose cells. forskolin-stimulat inhibit to incapacityµM) 10 (at at of activity RF313 agonistic hNPFFR1.of The lack com this that dose-response indicating curve RFRP-3 able was Figure RF313(at in µM) illustrated 10 2A, concentrations (EC concentrations frocalcium ligandrelease stimulated Kp10 potently in expressing cells. As shown Kiss1R on experiments RF3 2014),ofthewe activity both examined further a display to shown agonist AsRF9 has been recently or activity antagonist agonist detecthNPFF2 in any fo showedalso compound a affinity significant this clearly demonstrate that RF313 displays antagonist antagonist RF313displays clearly that demonstrate ± 340 exp for the same was RF9 nM 44 under obtained basal[ different activitie functional vitro in of Examination 3.1.2. targets et al., (Bihel these with (testedat µM) 1 pain modulation, in selectedtheir implication for profilingat numer Furtherof RF313 pharmacological a and significant a but low forreceptorsNPFF both lat of this RF9 although propertiesthose resembled Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. 35 We next addressed the agonist or antagonist nature or addressed antagonist next nature the agonist We S]-GTP in vitro in γ functional assays. When tested alone (up to 50 µM) 50 (up to tested assays. alone When functional ACCEPTED binding cells expressing S hN from CHO membranes to 50 = 0.1 ± ful ). nM As expected, 0.025 RF9 exhibited Comment citer cedocument: ACCEPTED MANUSCRIPT

2 of 977 ± 145 nM. For a comparison, a pAof ± For 977 comparison, nM. a 145 12 2015). 2015).

sRF313 of indicatedof RF313 interaction significant no hNPFF1R in edproduction of cAMP d RFRP-3-mediated inhibition of this of this RFRP-3-mediated d inhibition er compound displayed a higherer affinity displayed compound r the hNPFF2 receptor subtype, we did not not did rreceptor the hNPFF2 we subtype, R-expressing CHO cells (cAMP assay). R-expressingcells CHO ffinity for Kiss1R (Ki = 3500 ± 3500 (Ki for= ffinity nM).Kiss1R 740 activity at hNPFF1R m those cells at subnanomolar cells those at m subnanomolar 13 and RF9 in calcium mobilization mobilization calcium and 13 RF9 in MANUSCRIPTthe in promoteshift a to rightward ctivity at Kiss1R (Liu and Herbison, (Liu ctivityat Kiss1R hNPFF1R was also confirmed by its its by confirmed also hNPFF1R was pound displayed antagonist activity at activity displayed pound antagonist ous receptors, channels or transporters, receptors, ous channels Figure 2C, the endogenous Kiss1R Figure Kiss1R the endogenous 2C, erimental conditions. These erimentalconditions. data of RF313 at NPFF1R through two two through ofRF313 at NPFF1R , RF313 did not modify not did RF313 , l agonism l at Kiss1R PFF1R. However, as PFF1R. in vitro in . Although . 2 value of Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., but at micromolar concentration (EC at micromolar but concentration when administered alone but it fully it but RFRP alone whenprevented administered effectthe on i.c.v.) (10had nmol, significant no Fisher’s < p one ; 0.01, 6.3 way followed ANOVA by alone(AUC: - injected±i.c.v.) 181 (10 nmol, f 33 nocic reduced thesignificantly basal 2013),RFRP-3 and receptors(Figs. endogenous3A of ligand NPFF1 3.2.2. RF313 prevents RFRP-3-induced hyperalgesia i RF313 prevents 3.2.2. RFRP-3-induced hyperalgesia Figure (Suppl. test suspension S1). RF313reveal aanti-depressive effect potential of a except effectsslight at decrease for mg 10 RF313 analysbehaviors. like preliminary phenotyping This abi and proprioceptive,motor fine-tuned vestibular sensory-mo parameters,for weight and temperature), generalforandmg/kg)RF313were 30 (up checked to end, C57BL/6N To this furtherthe compound. use of m we in examined Inof aexperiements series first introduce no to in bias RF313treatment susceptible 3.2.1. Evaluation of toxic R adverse of and of Evaluation 3.2.1. effects RF313 of In 3.2. vivo evaluation observed for compound affinityth Kiss1R in this of receptor (Figureat this 2C), anyactivity agonist Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. We then investigated in mice whether RF313 can anta micethen investigatedwhether can We RF313 in ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

50 50 1.3 ± 0.5 µM). RF313 (up to 100 µM) did not display not did µM) RF313 100 (up to ± 1.3 µM). 0.5 13 basal nociceptive threshold of the animals nociceptive basal threshold

which is in agreement with the agreement absence in of is with which F313 at this dose as evidenced from the tail dose as evidenced at this

or RFRP-3 vs 2.2 ± 2.2 vs orRFRP-3 for 26 33) saline, =F(3, lities) as well as lities) for depression- anxiety- or /kg of the immobility time, which could time, of the /kg immobility ciception measurements or to impede or to ciceptionmeasurements is study (Table 1). study is (Table1).

MANUSCRIPTadverse side- showed significant no is eptive threshold of the animals when animals eptiveof threshold the torresponses strength, (muscular -3-induced hyperalgesia (AUC: 53 ± -3-induced(AUC: 53 hyperalgesia 54 male mice received s.c. injections of injections males.c. mice received 3B). As expected (Elhabazi et al., Asexpected 3B).(Elhabazi health parameters (modified SHIRPA parameters (modified health PLSD test p < 0.01, Fig. < p 0.01, RF313 3B). PLSDtest n mice mice n

ice unexpected adverse effectsiceof unexpected adverse gonize the effect of RFRP-3, the gonize RFRP-3, the effect of

Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., restoring normal nociceptive baseline values. These restoringnormalnociceptive of RF313 (Fig. subsequent administration initiated, Interestingly nociceptiveanimals. of threshold the < p Fig. test 0.01, 4B). followedPLSD Fisher’s by ±RF313+fentanyl 100 = vs F(1,for 21 fentanyl, 34) o 0.01)preventedand the development significantly ANOVA < p two- 0.05, way fentanyl,34) =F(1, ; 4.3 ± 1578 analgesia(AUC: and 165 duration of fentanyl before 4A), s.c. min RF313 (Fig. (3 mg/kg, fentanyl < p 0. PLSDFisher’s by onetest way followed ANOVA ± 100 (HI:for at for 21 least days two fentanyl vs < p de test 0.001), and followeda PLSD Fisher’s by ± 807 - for ± 211 6 fentanyl F(3,vs for 44 saline, 3 returnedresponsewithin mice, basal whichin to et al., Elhabazi 2000; describedet al., (Celerier fentanyl-inducedand analgesia secondary hyperalges antagonist behavesaNPFF1R as < p Fig. test 0.001, 3B) followedPLSD Fisher’s by ± 181 - 3 for for33 F(1, + RFRP-3. RFRP-3 RF313 vs administered by subcutaneous or oral routes. routes. oral or by administered subcutaneous induce RF313 prevents 3.2.3. hyperalgesia secondary Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. As shown in Figure 4A, fentanyl (4x60 µg/ kg, s.c.) µg/ (4x60 Figure fentanylin Asshown 4A, examined the consequences next receptors We NPFF of ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT in vivo in

. . 14 2012).

34) = 19.8 ; p < 0.001, one way 34)ANOVA < =p one ; 0.001, 19.8 21 ± 21 <= p ; 0.01, 4.4 for 12 34) saline, F(3, , once was already the hyperalgesic state , h after the last injection of fentanyl (AUC: of injection after fentanyl h (AUC: the last 4A, dashed arrow) was ineffective in was4A, dashed arrow) RF313 alone did not modify the basal the basal RF313modify alone not did observations suggest that NPFF receptorsNPFF that observations suggest indicating that this compound well compound this that indicating ) significantly potentiated the amplitude the amplitude potentiated )significantly layed hyperalgesic response that lasted that response layedhyperalgesic f hyperalgesia (HI: 36 ±fhyperalgesia (HI: 36 for 20 MANUSCRIPT 4.5 ; p < 0.05, two- way ANOVA ANOVA < p two- ; 0.05, 4.5 way ia in mice and rats as previouslyas and rats mice iain for RF313+fentanyl vs 807 ± 807 vs for for RF313+fentanyl 211 followed by Fisher’s PLSDFisher’s< p test followed by d by fentanyl in rodents when when rodents in by d fentanyl 3) = 3.3 ; p < 0.05, two way ANOVA 3)<= p two ; 0.05, 3.3 01, Fig. 4B). When administered 20 Fig. 01, When 4B). promoted a short lasting analgesic analgesic promoted a lasting short blockade with RF313 on on blockade RF313 with Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., way ANOVA followed by Fisher’s PLSD test p < 0.001; < p test wayfollowed PLSD Fisher’s ANOVA by ± (AUC: 725 animals for 68 ± 40 morphine vs for 18 increase tail in effectas the by significant shown of theAs expe animals. basalnociceptive threshold mice in (Fig. 5A). RF313 (5 analgesia mg/kg, s.c.) chronic morphine administration in mice. in chronicmorphine administration and morphine analgesia RF313 improves 3.2.4. acute rodents. in of hyperalgesi efficientlythe development prevents indicate recept Altogether,NPFF that these results < p (F(3, 35) ; 45 0.001) =< p or ; oral 50 0.001) adm dose-dependentlyby whenwas it RF313 prevented of one- the that development with way showed ANOVA displayed anres treatedanalgesic RF313 still with aft cang).weoffnotice hours 6 that 600 However, effectwas fentanylafter because hours, probably 2 modif not beforedid (administered fentanyl) min 30 order in limitations for account to bioavailability d and Higher4F). and(Figs. RF313 4D) 4E or orally adminis rats and in according mode of dosage its to of the hyperalgesic state. ofthe hyperalgesic state. ofopioid-induced hyperal arethe onset in involved Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. We further examined the impact of RF313 (2.5 and 5 5 impactof RF313 (2.5 further and We the examined We next examined fentanyl-in on the effect next of We RF313 ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

immersion latencies control immersion compared with . Whatever the route or the dose, RF313 Whatever . the the route or routes (fig. 4D and 4F, respectively). routes(fig. respectively). 4D and 4F, ponse. Analyses of hyperalgesia index data index ponse.Analyses of hyperalgesia or p.o.) (s.c. orsblockade RF313 by already maximal (paw pressure test, cut- pressure(paw already test, maximal er fentanyl administration, only eradministration, rats orally fentanyl alone did not significantly alonenot the did modify gesia but probably not in the maintenance gesiain not probably but cted, morphine induced aanalgesicstrong cted, morphine induced a induced by acute fentanyl administration aadministration induced fentanyl acute by (Figs. either tration, 4C subcutaneously

MANUSCRIPTy of response theamplitude analgesic the oses were chosen for oral administration administration foroseswere chosen oral saline, F(5, 54) =

After 8 days of treatment , the analgesic effect of effect the , analgesic ofAfter treatment days 8 We next addressed whether next the question We hyperalgesia In a subsequent experiment, we experiment, performed inje Indaily asubsequent ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

ay ANOVA followed by Fisher’s ayfollowed PLSD ANOVA by als. Inals. RF313 and with treated mice of a 6A), . development indicative for the /kg, s.c.). At day 0, as expected from expected day At as 0, s.c.). /kg, ±- for ± vs 4.3 45.2 RF313 + morphine ±for 8 - morphine vs 12.5 for 4.1 saline, ay 8 ( Fig. 6D, hyperalgesic mice) on mice aymice) (mice Fig. on 6D, hyperalgesic 8 response of mice was measured every responsewas day measured of mice

.01 MANUSCRIPT d prolonged the analgesic response of prolonged d the analgesic sic effect was measured in time-course in measured effectwas sic 503 ± 503 at 39 ± day 800 vs at 8 62 day p 0, e and again, we considered the impact of eand the considered again,impact we netreatment a led to progressive tion (Fig. 6A), RF313 (5 mg/kg, s.c.) (Fig. tion 6A), RF313 nced morphine-induced analgesia nced morphine-induced morphine alone was strongly morphine alone reduced was by Fisher’s PLSD test p < 0.001; Fig.< p 0.001; PLSDFisher’s by test , , s PLSD test, p < 0.01; Figs. and < p 6C 0.01; PLSD s test, r morphine, F(1, 54) = 4.7 ; p <= F(1, p ; 0.05, 4.7 r54) morphine, Fisher’stest; PLSD 001; Fig. 5B). Fig. 5B). 001; vs 594 ± 594 vs =F(3, for 69 36) morphine, ctions of morphine (5 mg/kg,s.c.) of morphine ctions (5 was accompanied with the with was accompanied

Figs.and 6E) 6D Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., these results confirm previous observations linking theseconfirm results previous activity exhibited < p 0.001). RFRP-3, Conversely, RF9 LH(15.2 on ng/ml RFRP-3 ± secretion for 1.9 RFRP-3 ng/ml 6.4 RF313±vs for but saline, > p 0.05) 0.4 a ng/ml± < p not 0.001). RF313 did for 1.9 RFRP-3, LHan in increase agonist plasma in resulted RFRP-3 7)revealed Fisher’s(Fig. i.c.v.test ad that PLSD = differencestested(4, groups) (F 27 the between one Dataway with themale hamster. analyses Syrian anta receptor compareNPFF two effects of these the agonistmicromolaraffinity at Kiss1R andactivity <0.001, RF313 on NPFF1R. As our RF313NPFF1R. on As our well particularly species 2013).therefore This is LH( display to on shown release stimulatory action et al., 2013). other Conversely to spec (Simonneaux and NPFF1R Kiss1R (also receptors, Two RF-amide the cont in functions respectively),essential play RFRP-3 RF9, stimu antagonizes not RF313,but 3.2.5. admi analgesicfollowingmorphine chronic tolerance d effect analgesic acuteand the attenuate morphine 0.01, ± 503 morphine alone with +(AUC: morp for 39 RF313 Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents.

Fisher’s PLSD test). Altogether, these results indi Altogether, these Fisher’sresults test). PLSD in vivo in

Fisher’s PLSD test ; Figs. 6D and 6E) but to a to lowe Figs. ; and6E) but PLSDFisher’s 6D test

(6.4 ng/ml ± (6.4ng/mlng/ml ± for 0.4 27.8 saline vs fo 1.2 ACCEPTED Comment citer cedocument: in vitro in ACCEPTED MANUSCRIPT experiments showed that RF9 (but not RF313)displa showed RF9 that not (but experiments

perse rol of reproduction in seasonal mammals of reproductionmammals seasonal in rol suited to study the antagonist action of study to antagonist suited the ministration of the endogenous NPFF1R ministration

fully antagonized the stimulatory effect of fullyeffect the antagonized stimulatory analyses with < p ; 49 0.001). Post-hoc (TableFig.we to decided and 1 2C), RF9 gonadotropin-releasing activity to a to gonadotropin-releasingactivity RF9 evelopment of hyperalgesia as well as of as well as evelopmentof of hyperalgesia Ancel et al., 2012; Simonneaux et al., Simonneaux al.,2012; Ancelet MANUSCRIPThas been RFRP-3 male ies, in hamster gonists on RFRP-3-induced LH in release gonistsRFRP-3-induced on ffect basal LH levels (6.4 ng/ml ± LH(6.4 ffectbasal for 0.4 levels levels (6.4 ng/ml ± levels (6.4 ng/ml for 0.4 15.2 saline vs ANOVA highlighted significanthighlighted ANOVA nistration. lation of LH secretion the hamster. LH in of lation a strong gonadotropin-releasing a strong vs 5.4 ng/ml 5.4 ±vs for 0.7 RF313 + cate that RF313 can both improve both cate RF313 that can hine vs 330 ± 330 hinevs morphine,< p for 43 r extent than in animals treated animals than in r extent termed GPR54 and GPR147, GPR147, and termed GPR54 r RF9, p < 0.001).Altogether, < rRF9, p ys Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., mammals. mammals. dissect properly to the role tool in of NPFF1R the et al., 2015) Sahin and poi LiuHerbison, 2014; and agonist (Garcia-Gal possible toward activity Kiss1R Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents.

ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

release gonadotropin centralof in control nt out RF313 as a safer pharmacological RF313 as out a nt safer

MANUSCRIPT et al., 2014; Glanowska iano et al., 2012; Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., oralroute, in vivo in for further drug development. fordrug further development. study this in Altogether, the data point described the actionadverse effectswhile limiting associate mightrepresent an interesti NPFF blockade receptor morphine chronic by andanalgesic induced tolerance atefficient pr doseswas than RF9 but slighly less ind block the hyperalgesia to 2006),RF313 appeared et al., 2013; conducted studies RF9 (Elhabazi with welland toleranc inducedanalgesic hyperalgesia as Itfentanyl-induced blocks oralroutes. completely properties of RF313, a compound (referred as aproperties compound of to RF313, affinity for NPFF receptors. affinityforreceptors. NPFF a couldrepresent flexibility the RF313 backbone of process with antagonists, a GPCR lead-optimization asa been st molecular described has rigidification RF9 lowerr for ofthe than that same the dipeptide low toxicity, a high selectivity, as well as an antaswell as an toxicity, low a selectivity, high derivative ornithine non-natural receptors.di This aimingorally at drugidentifying program discovery 4. Discussion Discussion 4. Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. . Moreover, it potentiates opioid analgesic potentiates opioid effects Moreover, . it Despite the goodDespite the In this paper, we provide adetailed Inprovide ofanalysis th we paper, this in vitro in

ACCEPTEDaffinity its N for showed binding that experiments Comment citer cedocument: in vivo in ACCEPTED MANUSCRIPT activity displayed by RF313 at relatively dose low activityRF313 displayed by

19 agonist activity both atNPFF1R agonist

12e out RF313 compound as out an lead interesting eventing the development ofhyperalgesia eventingthe development d with their chronic administration. their chronic with d administration. splays a high solubility in aqueous buffer, a splays in a solubility high andard approach for the development of the development approach andard for hyperalgesiamorphine- andattenuates

eceptors (Simonin et al., 2006).As eceptorset al., (Simonin Elhabazi et al., 2012; Simonin et al., Simonin et al., 2012; Elhabazi n alternative strategy to improve its improveits alternative n to strategy e. By comparison with our previous comparisone.with By

MANUSCRIPT of NPFF antagonists -bioavailable ngopiatesanalgesic strategy improve to in (Bihel et al., 2015)) we a etin al., (Bihel selected 2015)) in particular focus on a decrease in the in particulara on decrease focus uced by RFRP-3 and fentanyl at similar ucedatsimilar and fentanylRFRP-3 by administration. Our confirm results administration. that in mice both by subcutaneous and subcutaneous by mice in both e in vitro in and PFF1R and NPFF2R was PFF1R in vivo in pharmacological in vitro vitro in s and s by and Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., receptor subtypes in the modulation of nociception. modulation the receptor in subtypes greatly should clarif challenge important help that h Mazarguil the development 2010; of et al., 2012), et ligandsal., 20 (Gaubert receptorsNPFFpeptidic despite 1993). th Thus, Oberling al.,et2014; al., (Gouar i.t. of site administration dependingits on of bo NPFF2R, display FF,can endogenous ligand the al., 2010). However, sever of et pain models (Lameh anti- theseand receptors two display pro- opposing varying with of fun degrees compounds NPFF1R/NPFF2R agreement arein opiates.These previo with results of actor NPFF1Rthe development in as an important Although one cannot completely rule out a role of N a cannotrole Althoughof completely rule one out a probably greaterthan that RF313 is toward NPFF1R These s RFRP-3. to data mediatedresponses cellular toward compound NPFF2R activity antagonist of this w receptors,at NPFF we of ofRF313 the interaction selectivity) for the NPFF1R subtype compared to NPF subtype selectivity)forcompared to the NPFF1R numerous species including rodents and human (Goodm numerousspecies rodents including has been Kiss1R shown µM. 100 to up concentrations activity, wellas agonist full while RF313 neither RF9 that displayed eta observed al., 2006). low We we identified previously RF9,a compound dipeptidic Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. In this study, we also comparedalso the Inwe study, this Binding experiments also indicatedalso RF313 that displ Bindingexperiments ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

in vitro in 20

e recent design of several peptidic and non recentseveral peptidic e of design deres et al., 1993) versus i.c.v. (Journigan et (Journigan deresal., 1993) versus i.c.v. et binds nor activates receptor binds at this ying the respective contribution of NPFF1/2 contribution yingthe respective binding and activity profiles of RF313 and activityof binding and profiles

observations us made with 09; Journigan et al., 2014; Lameh etal., Journigan et 09; al., 2014; nociceptive roles, respectively, in various roles, in respectively, nociceptive

ighly selective ligands still represents still ighlyan selective ligands but detectable affinity for KISS1R, as forKISS1R, detectable affinitybut MANUSCRIPT PFF2R,our uggestselectivity the that functional of anyor agonist detect ereto able not al studies have shown that neuropeptide that have alstudies shown as a NPFF receptor antagonist (Simonin (Simonin antagonist receptorNPFF as a secondary hyperalgesia induced by secondaryhyperalgesia while it clearly NPFF1R- whileit blocked fforded from binding experiments. experiments. ffordedfrom binding th pro- or anti-nociceptive actions anti-nociceptive pro- th or F2R. When testing F2R.When to play a key role in reproductionplay ato key in role in an and Lehman, 2012). ItLehman,expressedan and 2012). is ctional activity, which suggest that whichthat suggest ctionalactivity, ayed a slight preference (3-fold ayed a slight preference in vivo in data point out out point data in vitro in the nature Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., was later elucidated with the demonstration of RF9 of thedemonstration RF9 elucidatedwaslater with Liu 2014; Glanowska al.,and 2012; etHerbison al., actioanoff-target release gonadotropin rely to on However,several studies (Pineda et al., 2010a, b). exert that tone of RFRPs of existence an endogenous Pineda et etal. al., 2012; (CaratyandFSH release administeredroden (wheni.c.v. in showing RF9 that h system (Kriegsfeld, This kisspeptin/Kiss1R 2010). count release, GnRH thus playon aninhibitory role endogenous RFRP-1 ligands recently,and its NPFF1R release, which the controls stronglyGnRH stimulate hypothalamusneurons GnRH activat in of and the its and activity which RF9 in strongly observationmale in hamsters, rather blockade throughactivation than via Kiss1R modulate compound this that 2015),one may conclude et (Kim well al., preserved 2015; NPFF1Rbut in KOs Kiss1R. receptorsand particularly be of the careful th will antagonists investigation indicatealso Moreover,an that data important our owin of reproduction, NPFF1Rthe control central in amore RF9,pharmacolog suitable asmay be regarded Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. in vivo in Altogether, these results indicate Altogether,RF313, that desp these results perse (Kim et al., 2015; Min et al., 2015). As RF9Min effec et (Kim al., 2015; ACCEPTEDLHof r blocked stimulation efficiently RFRP-3 but Comment citer cedocument: ACCEPTED MANUSCRIPT

eir selectivity toward other RF-amide selectivity other eir toward n on kisspeptin receptors (Garcia-Galianoet kisspeptin on n , 2010b; Rizwan et al., 2012) suggesting Rizwan the 2010b; , also suspected RF9 impact on on suspectedalso RF9 impact

clue to developing high affinity NPFF1R clueaffinity developing NPFF1R high to of NPFF1R. We further extended this this extended further We ofNPFF1R. eracting the stimulatory role of the eracting roleof the stimulatory , 2014; Sahin et al., 2015), Sahin an that 2014; issue , MANUSCRIPT both agonistat Kiss1R, activity release of sexual hormones More . release of sexual ypothesispart results on in was based ts or ewes) is a potent stimulator of LHof stimulator a or ewes) is potent ts g to its lack of Kiss1R agonist activity. glack of its Kiss1R to s a negative feedback on GnRH neurons GnRH on feedback a s negative increased LH levels whileRF313 had no LHlevels increased ion by kisspeptin has been by to shown has ion kisspeptin Liu and Herbison, 2014; Min et al., LiuMin 2014; and Herbison, ical tool to study the involvement of involvement study to icalthe tool s gonadotropin release primarily release gonadotropin s and RFRP-3 haveand RFRP-3 proposed been to ite its lower than affinity its at NPFF1R ite t was absent in Kiss1R KO mice Kiss1R absentin was t elease. elease. in vitro in

of essential physiological functions such functions as of essential physiological

of highly The reproduction. development used as a safer pharmacological tool to to usedtool as a safer pharmacological ver, and in contrast with RF9, it is devoid is RF9, it contrastver,with and in

MANUSCRIPT adverseeffects side opiates and reduce menabletherapeutic further to ors will be mandatory in the next be mandatory thewill next in ors tagonist of the NPFF1 receptor. Itreceptor. of the NPFF1 tagonist Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., Ancel, C., Bentsen, A. H., Sebert, E., Tena-Semp M. Ancel,A. Bentsen, H., C., References d'avenir”. de la Recherche" "Agenceunder managedNational by sup financial and received ANR-10-LABX-0034_Medalis has (Pharmadol).work GénéraleThis des Entreprises (Pharmadol ICFRC Urbaine(Pharmadol), de Strasbourg d’ Régional ANR-13-BSV1-0001),EBIOConseil 014.02, fromAgence Conectus, SATT BioValleygrants and by France) for technical assistance and advices. advices.France)and for assistance technical theservic and staff behavior from the neurobiology IntegrativeChemistry (UMS Platform theBiological work This be also and QRFPR. expressinghuman PrRPR Acknowledgements Acknowledgements of interest. Theconflict no authors declare Disclosures Ayachi, S., Simonin, F., 2014. InvolvementF., 2014. of Mamma Ayachi,Simonin, S., Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. hamster: the exception proves the rule. Endocrinolo proves hamster:the exception gonad Stimulatoryeffect V.,2012. the on of RFRP-3 158. 158. of Nociception Roden in Receptorsthe Modulation in This work was supported by the CNRS, INSERM,Univer by work This was supported the CNRS, We thank Dr M. Parmentier (IRIBHM, Brussels, BelgiuBrussels, (IRIBHM, thank Dr Parmentier We M. ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

MANUSCRIPT(Illkirch, de la Souris InstitutClinique eat ere, M., Mikkelsen, J. D., Simonneaux, D., Simonneaux, ere,J. Mikkelsen, M., been published within the LABEXthe been within published 3286 CNRS, Illkirch, CNRS, 3286 France). thank We "Programme d'investissement "Programmed'investissement lian RF-Amide Peptides and Their RF-Amide lian Peptides National de la Recherche (ANR 08 de la Recherche National gy 153, 1352-1363. gy1352-1363. 153, ), OSEO (Pharmadol), Direction ),(Pharmadol), OSEO otrophic axis in the male in Syrian otrophicaxis nefited from the efficient of the support nefitedfrom Alsace (Pharmadol), Communauté Communauté (Pharmadol), Alsace port from the Frenchgovernment fromport the ts. Front Endocrinol (Lausanne) Front ts. Endocrinol 5, m) for the giftcells CHO of m)for the sité de Strasbourg, Alsace de sité Strasbourg, Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., Celerier, E., Rivat, C., Jun, Y., Laulin, J. P., La P., Laulin,Y., J. Celerier,Jun, C., E., Rivat, Elhabazi, K., Trigo, J. M., Mollereau, M., K., Trigo, Mouledou Elhabazi, C., J. Elhabazi, K., Ayachi, S., Ilien, B., Simonin, F., K., Ayachi, Ilien, 2 Elhabazi, B.,S., Simonin, Bihel, F., Humbert, J. P., Schneider, S., Bertin, I P., Schneider,Bertin, Bihel,Humbert,J. S., F., Elhabazi, K., Humbert, J. P., Bertin, I.,Bertin, K., Humbert, P., Elhabazi, J. Schmitt, Caraty, A., Blomenrohr, M., Vogel, G. M., Lomet, D. Lomet, Vogel, M., Blomenrohr,G. M., Caraty,A., Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. 75C, 164-171. 75C, NPF morphine analgesia nociception modulate and via RF-amideP peptides, including Endogenous mammalian Meziane,H., A., Petit-Demouli Sorg, Becker,T., J. threshold of sensitivity. J Neuroendocrinol 725 24, of threshold J sensitivity. th in secretion gonadotrophin powerfullystimulates Anesthesiology 92, 465-472. Anesthesiology465-472. 92, ra in induced Long-lastingfentanyl by hyperalgesia Bourguignon, J. J., Meziane, J., Bourguignon,H., Petit-demouliere, J. to acute and chronic opiate treatments. Br J Pharma acute treatments. chronicto J Br andopiate InvolvementFFr F., 2012. of neuropeptide Simonin, modalities. J Vis Exp, e51264. Vis Exp, J modalities. tolerancemice in using hyperalgesiaand analgesic 438-445. h of opioid-induced FFprevention receptorsfor the 2015. Development J., of a Bourguignon,peptidom J. Petit-Demouliere,C., B., E., Mollereau, Schneider, ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

24 rcher, A., Reynier, P., Simonnet, G., 2000. P., rcher,A., Reynier, ., Wagner, P., Schmitt, M., Laboureyras, M., Wagner,E., Schmitt, ., P.,

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, Scully, A. L., Nash, N. R., Gardell, L.Gardell, R., R., L.,N. Scully, , A. Nash, n, I.,Laboureyras, n, M., E., Dugave, Schmitt, MANUSCRIPT of Central 2011. ., administration , Krajnc-Franken, M. A.,Manfredi- Krajnc-Franken, , M. Meiler, J., Olsson, R., Olsson, Beck-Sickinger, Meiler, J., 14. Development of gonadotropin- Development 14. of rons from mice to men: similarities similarities men: ronsfrom mice to emic morphine analgesia in mice. emicin morphine analgesia adotropin secretion in mice. in adotropinsecretion J Neurosci 34, 15060-15069. Neurosci 15060-15069. 34, J , Simonin, F., Simonin, , Bihel, 2012. F., Nonpeptidergic Neuropeptide FF2 Neuropeptide Nonpeptidergic containing non-peptides at human non-peptides containing neuropeptide FF receptors. Bioorg FF receptors. neuropeptide signalingindispensable for is , F., van Noort, P. I.,L., Pinilla, P. F.,van Noort, , Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., Gouarderes, C., Sutak, M., Zajac, J. M., Jhamandas, M., Zajac, M., Gouarderes,Sutak, J. C., Haley, T., McCormick, W., 1957. Pharmacological 1957. eff W., Haley,T., McCormick, Johnson, M. A., Tsutsui, A., Tsutsui, M. R K.,2007. Fraley, G. S., Johnson, C. Gouarderes, M., Sutak, B., K., Milne, Jhamandas, V., 2016. F., B., Simonneaux, Gauer, Henningsen,J. Kayser, V., Basbaum, A. I.,Dea G., Guilbaud, 1990. Kayser,A. Basbaum, V., S. Vyas, N., C., Journigan,Eans, V. B., Mésangeau, Kim, J. Dyer, Brownjohn, W., B.Beltramo S., J. P. S., Kim, Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. 73-81. i FMRFamide F8Famide intrathecallyand administered Understanding Seasonal Reproduction in Mammals. Fro Mammals. Reproduction in UnderstandingSeasonal of consc drug in intracerebroventricularinjections male rat. Horm Behav 51, 171-180. Behavmale51, rat. Horm LHand secretion, has variab GHinhibits secretion, 27, peptides. Peptides andneuropeptiderelated SF Facilitation of spinal morphine analgesia normal Facilitationin morphine of spinal Endocrinology 156, 4152-4162. Endocrinology4152-4162. 156, Are by RFRP-3 Overcome th HypothalamicNeuropeptide D., Anderson, Anxio 2015. G.G. F., M., J. Tyndall, for Leads FF1 Neuropeptide and AntagonistOriginal Nonpeptide 2014. Sma R., C. Mollereau,McCurdy, C., mechanical nociceptive threshold in the innervated the innervated in mechanicalnociceptive threshold ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

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, M., Walker, C. S., Hay, D. L.,Hay, D.S., Painter, Walker, C. M., , MANUSCRIPT RFRP Neurons DoorwayRFRP - The to , Zajac, J. M., Yang, M., Zajac, , Y., 2006. J. H. fferentation in the rat increases the rat in fferentation ious mice. Br. J. Pharmacol 12, 12-16. 12, mice. Pharmacol Br. J. ious ects by produced and morphine tolerant animals by and morphine tolerant animals 953-963. le effects on sex behavior in the adult the adult in behavior leeffects sex on limbs. Brain Res 508, 329-332. Brain 329-332. limbs. 508, Res genic and Stressor Effects of genicthe and Effects Stressor FF2 Receptors. J Med Chem. Med Chem. FF2J Receptors. ll Molecule Agonist and Molecule ll Agonist n the rat. Eur J Pharmacol the n rat.237, Eur J nt Endocrinol (Lausanne) 7, 36. 36. 7, Endocrinol (Lausanne) nt e NPFFR Antagonist GJ14. GJ14. NPFFRAntagonist e Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., Kriegsfeld, L. J., 2010. Overcoming sexual inhibiti Overcoming 2010. L.Kriegsfeld,sexual J., Kovacs, A., Laszlo, K., Galosi, R., Ollmann, T., Pe Ollmann, K., R., Galosi, Laszlo, Kovacs,A., Leon, S., Tena-Sempere, M., 2015. Dissecting Ro 2015. M., Leon,the Tena-Sempere, S., Liu, Q., Guan, X. M., Martin, W. J., McDonald, T. P W. J., Liu,Martin, X. M., Q., Guan, Lameh, J., Bertozzi, F., Bertozzi, Kelly,M., P. Lameh,N., J., Jacobi, Kontinen, V. K., Kalso, E. A., 1995. Differential m DifferentialE. A., 1995. V. Kontinen, K., Kalso, Liu, X., Herbison, A. E., 2014. RF9 Excitation of G RF9 2014. Excitation Liu,A. E., X., Herbison, Mazarguil, H., Mollereau, C., Czaplicki, G., Zajac, Czaplicki, C., Mazarguil,H., Mollereau, Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. decreases food intake in rats. Brain Res BullBrain 107, decreasesrats. in Res food intake Intraamygdaloidof Lenard,L.,microinjection 2014. nociception. J Pharmacol Exp Ther 244-254. Pharmacol 334, Exp nociception.J L.Gardell, FFNeuropeptide receptors 2010. R., R., peptide using a novel receptor antagonist. Endocrin antagonist. peptidereceptor using a novel opioid spinal antinociception P by spinal opioid FF. neuropeptide Models. Front Endocrinol (Lausanne) 6, 189. (Lausanne) Front 189. 6, Models. Endocrinol Using and Studies Mammals: in Tools Pharmacological antinociception. J Biol Chem 276, 36961-36969. Biol Chem 36961-36969. 276, antinociception. J attenff- neuropeptide mammalian peptides that like Iden 2001. P., R., C. Gould, Austin, Evans, J., R., L.,Bomford Williams, M., D. Yu, H., Jacobson, Jr., of peptidic selective NPFF2 agonists. Peptides 37, 37, Peptides agonists. NPFF2 ofselectivepeptidic in the Adult Male and Female Mouse.Endocrinology 1 Male the Adult and in Female ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

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nRH Neurons Is Upon Kiss1r Neurons nRH Dependent MANUSCRIPT ., Clements, M. K., Jiang, Q., Zeng, Z.,K., Zeng,M. Clements, ., Q., Jiang, lesof Gonadotropin-Inhibitory Hormone tification and characterization of novel and characterization tification 61-68. 157-160. ology 151, 1387-1389. ology1387-1389. 151, have opposing modulatory effects on on effects have modulatory opposing uate morphine-induced eptides973-977. 16, , D., Figueroa, D., Mallee, J., Wang, J., D., , Figueroa, D., Mallee, RFamide-related peptide-3 RFamide-related peptide-3 55, 4915-4924. 4915-4924. 55, Genetically Modified Mouse Genetically Modified Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., Murakami, M., Matsuzaki, T., Iwasa, T., T., Murakami,Matsuzaki, M., T., Yasui, L., L.,Pinilla, Li,R. Leon,Min, H., Carroll, S., Nicklous, D. M., Simansky, K. J., 2003. Neuropeptid 2003. Simansky, D. M., Nicklous, K. J., Oberling, P., Stinus, L., G. LeStinus, Simonnet, Oberling,M., P., Moal, Pineda, R., Garcia-Galiano, D., Sanchez-Garrido, M. Garcia-Galiano,D., Sanchez-Garrido, Pineda,R., Prokai, L., Zharikova, A. D., Juhasz, A., Prokai-Ta L.,D., Juhasz, Zharikova,Prokai, A. M. Garcia-Galiano,D., Sanchez-Garrido, Pineda,R., Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. RF9 Acts as a KISS1R Agonist In Vivo and InIn Vitro. and Vivo KISS1RRF9 Agonist Acts as a female rats. J Endocrinol105-112. 199, femaleJ rats. RFamide-related roleHypophysiotropicpeptide-3 of Physiol 285, R1046-1054. R1046-1054. 285, Physiol food modulate intake to the parabrachial in nucleus 919-924. FF(FLFQPQR neuropeptideantiopiate activity the of Endocrinol Metab 299, E39-46. EndocrinolMetab E39-46. 299, secretion gonadotropin the therat: control in of i of the inhibitory Characterization roles of RFRP3, L., Pinilla, van M., Blomenrohr, F. E.,Dijcks, A., neuropeptide FF antagonists. Peptides 27, 1015-1019 27, Peptides FFantagonists. neuropeptide Endocrinology 151 andpharmacological implications. of antagonist peptides RF-amide-related and neurope of gonadotropin-releasi the potent Characterization L., Pinilla, van M., Blomenrohr, F. E.,Dijcks, A., ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

28 S., Tena-Sempere, M., Kaiser, U. B., Tena-Sempere, 2015. U. S., Kaiser, M.,

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MANUSCRIPTI., Bertin, M., C., n, Schmitt, n and analgesia. Eur J Pharmacol 1- 345, and n Eur analgesia. J hains. Org Biomol Chem 13, 7020- 13, Org hains. Chem Biomol ffects of systematic N-terminus ffectsN-terminus of systematic ta or gamma-amino acids gamma-aminoacids taor rats. Reproduction 149, 465-473. 149, rats. Reproduction acological properties, drug properties, acological peptides on NPFF1R, NPFF2R, peptidesNPFF1R, on acol Ther 160, 84-132. acol84-132. 160, Ther ated peptide-3 receptor gene receptor ated peptide-3 dependent mechanism of action. dependentmechanism d AGEGLSSPFWSLAAPQRF- d Neuropeptides 10, 37-42. 10, Neuropeptides Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., Simonin, F., Schmitt, M., Laulin, J.-P., LaboureyraLaulin,M., F., Schmitt, J.-P., Simonin, Simonin, F., Valverde, O., Smadja , S., Slowe, S., F., S., Slowe, Valverde, S., , Smadja Simonin, O., Simonneaux, V., Ancel, C., Poirel, V. J., Gauer, V., V. Ancel, F. J., Poirel, C., Simonneaux, Yang, H. Y., Fratta, W., Majane,E., Fratta,E. A., Costa, Yang, H. Y., W., Tena-Sempere, M., Pinilla, L.,F Aguilar, E., 1993. Tena-Sempere,Pinilla, M., Wang, Y. Q., Guo, J., Wang, S. B., Fang, Q., He, F. B., Q., He, Fang, Wang, Wang,S. Y. Q., Guo, J., Xu, M., Kontinen, V. K., Panula, P., Kalso, E., 199 Panula,P., V. Kontinen, K., Xu,M., Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. USA 103, 466-471. 103, USA h with associated preventsopioid-induced tolerance Simonnet, RF9, G., 2006. J., a and potent selective Laurent, Parmentier C., P., Matifas, A., Mollereau, actions of the selective k-agonist U-50,488H and at actionsU-50,488H of k-agonist the selective visce chemical to genemice in enhances sensitivity Kieffer, L.,R., B.B. 1998. Maldonado, Roques, P., EMBO J. 17, 886-897. 17, EMBOJ. morphine. Proc Natl Acad Sci U S A 7757-7761. 82, U S Sci morphine.Proc Natl Acad of brain two neuro pharmacologicalcharacterization Concert to Synchronize Rodent Reproduction with Sea Reproduction with Rodent ConcertSynchronize to neuropeptide FF analogue, in different pain models. in FFanalogue, neuropeptide sulfonate. Endocrinology 133, 1173-1181. 133, sulfonate.Endocrinology i or male hormonein rats secretion orchidectomized 1183-1190. attenuates opioid-evoked receptorsRF9, antagonist, ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

1985. Isolation,and 1985. synthesis, sequencing, Kitchen, I.,LeKitchen, M., A., Dierich, Meur,

Actand in RFRP-3 Kisspeptins 2013. , ollicle-stimulating hormone and luteinizing and luteinizing hormone ollicle-stimulating s, E., Jhamandas, J. H., MacTavish,J. E., Jhamandas, D., s, 9. Effects 9. (1DMe)NPYF,a synthetic of

, Wang, R., 2008. Neuropeptide FF Wang, , Neuropeptide 2008. R., MANUSCRIPT neuropeptide FF receptor antagonist, antagonist, FFreceptor neuropeptide , M., Kieffer, B. L.,Bourguignon,B.J.- Kieffer, M., , tenuates morphine withdrawal. ral pain, impairs pharmacological ralpharmacological pain, impairs Disruption of receptor the k-opioid Disruption yperalgesia.Acad. Sci. Natl. Proc. hypothermia in mice. Peptides 29, 29, Peptides mice. hypothermia in Peptides 20, 1071-1077. 20, Peptides njected ethylene with dimethane peptides that modulate the modulate peptides action that of sons. Front Neurosci 7, 22. 22. 7, Front sons. Neurosci Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F.,

Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents.

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31 MANUSCRIPT Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., Figure: S1 Supplementary information accompanies the on paper Supplementaryinformation A. Supplementary information Appendix Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents.

ACCEPTED micereceiving phenotypingof analyses Preliminary Comment citer cedocument: ACCEPTED MANUSCRIPT

32 MANUSCRIPT theNeuropharmacology website. RF313 injections. injections. RF313

Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., A thresholds were measured every post-injectio weremin 15 thresholds measured asunder described RF313were (10 performed nmol), human NPFF1 receptors. RF313 (10 µM) shifted the EC RF313 (10 µM) receptors. humanNPFF1 hNPFF1R cells. RF313 (10 µM) shifted the EC µM) RF313 (10 hNPFF1Rcells. Figure2: A andKiss1R. B meanin ± separate values experiments for S.E. two Figure 3: triplicate. performed experiments in 695 ± 695 ofas m percentages Data nM. 95 areexpressed Figure 1: Figurelegends C triplicate. in experiments valuesforseparate four cA percentages of as maximal Data nM. areexpressed EC are represepoints fluorescenceData from a levels. ma basal to and normalized responsewere amplitudes Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 : Reversal of RFRP-3-induced inhibition of forskoli Reversal : inhibition of RFRP-3-induced : Intracerebroventricular (10 of RFRP-3 : injections : Stimulation of [Stimulation : : Effect of Kp-10, RF9 and RF313 on calcium mobiliz andEffect RF313 calcium : on Kp-10, RF9 of effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. 50 ± values for S.E. Kp-10 (0.1 ± RF9 ( nM) and 0.025 Assessment of Assessment RF313 blockade of RFRP-3-induced hyperalgesia in mi hyperalgesia in of RF313blockade RFRP-3-induced Chemical structures of RF313 and RF9 compounds. of RF313 and Chemicalstructures RF9 ACCEPTED 35 S]-GTP Comment citer cedocument: in vitro in γ S binding by RFRP-3 on membranes from CHO cells CHO membranes binding on exp by S from RFRP-3 ACCEPTED MANUSCRIPT pharmacological properties of RF313 and RF9NPF on pharmacological propertiesand of RF313

33 50

± 6.9 valuesfor from ± 47 RFRP-3 to 1.1 12

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MANUSCRIPTtriplicate. nmol), alone or in combination with with alone nmol), combination or in n using the tail immersion test at 48°C. at 48°C. test using n immersion the tail n-induced cAMP accumulation in CHO- in n-inducedaccumulation cAMP aximal [ aximal ximal (digitonin-permeabilized cells) ximal Materials and methods. Nociceptive and methods. Materials MP levels and represent mean levels and ± represent MP S.E.M ation in Kiss1R in expressing ation cells.Peak 50 1.3 ± 1.3 from two nM) are 0.5 values for from ± 98 RFRP-3 to 10 35

S]-GTP ce. ce. γ S S bindingand are ressing F1R Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., thesaline group. B Figure 4: Data are expressed as mean ± asmean Data areexpressed S.E.M, course. A,B fentanyl. C, D, E, F C,D, E, section(Panel B). de (HI) calculatedas were A). Hyperalgesia indexes n dailyof(arrow), before 1 measurement the min 30 was(5 mg/kg; inje s.c.) parallelRF313 experiment, return repeatebaseline to until and injection were te using responsesimmersion the tail were measured before mice (4x60µg/k min to 20 injections fentanyl rats either subcutaneously (0.05, 0.1 andmg/kg 0.5 rats(0.05, 0.1 either subcutaneously mg/kg;panels histogramspanels in of hyperalgesia. Hyperalg the follow development to (d0) monitor to afterfentanylfirst injection the start (d-2, d-1 once : test daily experiment before threshol Nociceptive interval; s.c.). min 15 µg/kg; Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 : Comparison between groupsbetween area-under-the-curve Comparison : of effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents. : On day 0 (grey area), a single dose of RF313 (5 m On day area),: (greyRF313 a dose of single 0 Effect of RF313 on analgesic on respo Effectof and RF313 hyperalgesic : On day 0 (grey area), increasing doses of RF313 o area),(grey 0 doses increasing On day : E , , ### ACCEPTEDF ). 30 min later, animals received four later, consecutive received min animals ). 30 p

D <0.001 the asRFRP- Fisher’scompared bywith test and and Comment citer cedocument: F . . ACCEPTED MANUSCRIPT

n n =**p 6–11. 34 analgesia amplitude, and once daily (d1 to d6) (d1 daily and to amplitude, once analgesia

, d0) to verify baseline stability, everyverify d0) h stability, 2 to baseline , d once daily from d1 to d4 Panel A). InPanel A). once d a d4 from daily to d1 ds were measured using the paw were ds pressure the using measured ; panels panels ; cted once to fentanyl-treated mice day on fentanyl-treated cted to once

scribed in the Data and statistical analyses statistical scribedand the Data in MANUSCRIPT g; 15 min interval; s.c.). Nociceptive g;interval; s.c.). min 15 < 0.01 by Fisher’s test as compared with ascompared with

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< 0.001 by Fisher’s test as compared with the the with compared as test Fisher’s by 0.001 < ere measured at 52°C, at 30 min intervals, min at 30 at eremeasured 52°C,

n ine injection (5 mg/kg; s.c.)was (5 ine injection mg/kg; monitored treatment (d1 to d7), using the tail treatmentthe tail d7), (d1 using to RF313 (5 mg/kg; s.c.) 20 min prior to min 20 RF313 (5 mg/kg; s.c.) ed to mice 20 min before morphineed (5 mice min to 20 ls and methods. Saline replaced eitherreplaced Saline and methods. ls MANUSCRIPT =***p 7–15. ive responses, using the tail immersion immersion tail iveresponses, the using

eptive values were measured once daily daily wereonce measured eptivevalues < 0.01, ***p 0.01, < RF313 (5 mg/kg; s.c.) 20 min prior to min 20 RF313(5 mg/kg; s.c.) (AUC values) over the 0-150 min the 0-150 min values) over (AUC analgesia in mice. analgesia in nd analgesic tolerance. analgesic nd

< 0.01 by Fisher’s test asFisher’s

< 0.001 by Fisher’s test as test Fisher’s by 0.001 < lculated from lculated ine- Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., E D according the time-course paradigm already defined paradigm accordingalready defined the time-course u Nociceptive injections. were determined responses mg/kg; prior s.c.) min 20 either (5 saline or RF313 ***p Mean± Materials and t 6 valuesmethods. for S.E.M. LHcon and for later,assayed was the blood sampled co in i.c.v., alone were or injected Drugs(1.5 µg) themale hamster. Syrian Data are expressed as mean ± S.E.M, S.E.M, ± mean as expressed are Data group.treated RFRP-3 ascompared with with the saline group. saline the with Figure 7: groupatd0. a test Fisher’s by 0.001 < p °°° 0.01, < °°p group. Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 :Comparison between groups of calculated AUC values groupsbetweenof calculated AUC :Comparison : On day 8, 24 h after the end of the chronicend of RF313 the On day : after h the 24 8, effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents.

< 0.001 by Fisher’s test as compared with the as salin test compared with

< 0.01 by Fisher’s test as compared with the morphi the with compared as test Fisher’s by 0.01 < ACCEPTED MANUSCRIPT

n n = 5–10. ***p 5–10. = 36

mbination, in a 2 µl final min 30 volume. a in µl 2 mbination, s compared with the AUC value of the same the of value AUC the with compared s MANUSCRIPT mg/kg; s.c.) saline or to morphine (5 sing the tail immersion test (set test sing at 52°C), immersion the tail in in tent (ng/ml plasma) as described as(ng/ml tent under plasma) /morphine treatment, micereceived treatment, /morphine o 7 animals are presented as histograms. as are animals 7 o histograms. presented C

< 0.001 by Fisher’s test as compared as test Fisher’s by 0.001 < e group.e . . from C and D. and from D. C antagonists on LH in on antagonists secretion ### p

<0.001 Fisher’sby test ne-pretreated Version postprint J.-J., Laboureyras, E.,Ben Boujema, M., Simonnet, G., Ancel, C.,Simonneaux, V.,Beltramo, M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg, T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien, B., Bihel, F., Binding affinity constants (K constants affinity Binding Bindingaffinity properties of endogenous RF-amide 1 Table Mean values ± at least for S.E.M. 3 independent exp Elhabazi, K., Humbert, J.-P., Bertin, I.,Quillet, R., Utard, V.,Schmitt, M., Bourguignon, Neuropsychopharmacology, 118, 188-198. ,DOI :10.1016/j.neuropharm.2017.03.012 effects of RF-amide-related peptide-3 and opioid-induced hyperalgesia in rodents. Comment citercedocument : a K RF313 PrRP-20 RF9 Kp-10 Kp-10 NPFF RFRP-3 Compound 26RFa i i i values in nM) of drugs are from competition experi competition from are drugs of nM) in values values for areagonists et from al., 2013 (Elhabazi

7 3 6 6 >2,0 > > 20,000 560 ± 160 172 ± 13 4.7 ± 0.3 7±8 . . >2,0 350±70 > 20,000 3,500 ± 740 > 20,000 9.3 ± 2.1 17 ± 8 0.5 ± 0.1

.9±01 01 .6 000 000 > 20,000 > 20,000 > 20,000 > 20,000 Binding (nM)affinity constants > 20,000 > 20,000 0.14 ± 0.06 0.39 ± 0.15 4.8 ± 1.8 0.17 ± 0.06 24 ± 1 ACCEPTED NPFF2R NPFF1R eriments performedduplicate are reported. in

peptides,RF9 RF313 at and the recept five RF-amide a 3 ± 1 a a 1.6 ± 0.3 0.7 ± 0.1 ACCEPTED MANUSCRIPT a 1,0 >2,0 21 ± 1 > 20,000 >10,000

a a .6±00 >2,0 > 20,000 > 20,000 9,200 ± 1,200 0.06 ± 0.01 37

MANUSCRIPTPrRPR ) ments performed as described under Materials and me and Materials under described as performed ments a

.1±00 8,000 ± 120 0.51 ± 0.04 Kiss1R 100,000 a

> 20,000 QRFPR ors. ors. a

thods. thods. Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents.

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38 MANUSCRIPT Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents.

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MANUSCRIPT Version postprint J.-J., Laboureyras, E., BenBoujema, M.,Simonnet, G.,Ancel,C., Simonneaux,V., Beltramo,M., Simonin, F. (2017). RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes Bucher, B., Sorg,T., Meziane, H., Schneider, E., Petit-Demoulière, B., Ilien,B., Bihel, F., Elhabazi, K.,Humbert, J.-P.,Bertin,I., Quillet,R.,Utard, V.,Schmitt, M.,Bourguignon, Neuropsychopharmacology, 118,188-198. ,DOI:10.1016/j.neuropharm.2017.03.012 effects ofRF-amide-related peptide-3 and opioid-induced hyperalgesia inrodents.

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Highlights Highlights MANUSCRIPT function of NPFF receptors NPFF of function r both invivo vitroin rboth and y subcutaneous and oral routes oral and ysubcutaneous orphine hyperalgesia and hyperalgesia and orphine in hamster hamster in