1521-0103/356/3/681–692$25.00 http://dx.doi.org/10.1124/jpet.115.229989THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 356:681–692, March 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Alternative Radioligands for Investigating the MolecularPharmacology of Melatonin Receptors

Céline Legros, Chantal Brasseur, Philippe Delagrange, Pierre Ducrot, Olivier Nosjean,and Jean A. BoutinPôle d’Expertise Biotechnologie, Chimie, Biologie (C.L., C.B., P.Du., O.N., J.A.B.), and Unité de Recherches et Découvertes enNeurosciences (P.De.), Institut de Recherches SERVIER, Croissy-sur-Seine, France

Received October 12, 2015; accepted January 11, 2016

ABSTRACTMelatonin exerts a variety of physiologic activities that are mainlyrelayed through the melatonin receptors MT1 and MT2 Lowexpressions of these receptors in tissues have led to widespreadexperimental use of the agonist 2-[125I]-iodomelatonin as asubstitute for melatonin. We describe three iodinated ligands:2-(2-[(2-iodo-4,5-dimethoxyphenyl)methyl]-4,5-dimethoxy phe-nyl) (DIV880) and (2-iodo-N-2-[5-methoxy-2-(naphthalen-1-yl)-1H-pyrrolo[3,2-b]pyridine-3-yl])acetamide (S70254), which arespecific ligands at MT2 receptors, and N-[2-(5-methoxy-1H-indol-3-yl)ethyl]iodoacetamide (SD6), an analog of 2-[125I]-iodomelatonin with slightly different characteristics. Here, wefurther characterized these new ligands with regards to theirmolecular pharmacology. We performed binding experiments,saturation assays, association/dissociation rate measurements,and autoradiography using sheep and rat tissues and recombinant

cell lines. Our results showed that [125I]-S70254 is receptor, andcan be used with both cells and tissue. This radioligand canbe used in autoradiography. Similarly, DIV880, a partial agonist[43% of melatonin on guanosine 59-3-O-(thio)triphosphatebinding assay], selective for MT2, can be used as a tool toselectively describe the pharmacology of this receptor in tissuesamples. The molecular pharmacology of both humanmelatoninreceptors MT1 and MT2, using a series of 24 ligands at thesereceptors and the new radioligands, did not lead to noticeablevariations in the profiles. For the first time, we described radio-labeled tools that are specific for one of the melatonin receptors(MT2). These tools are amenable to binding experiments and toautoradiography using sheep or rat tissues. These specific toolswill permit better understanding of the role and implication inphysiopathologic processes of the melatonin receptors.

IntroductionThe neurohormone melatonin (MLT) is produced by the

pineal gland during periods of darkness, particularly at night(Arendt and Broadway, 1987; Zawilska et al., 2009). Melato-nin pleiotropic effects include exerting control over biologicrhythms, such as circadian rhythms through the suprachias-matic nuclei (Coomans et al., 2015), and circannual rhythms,e.g., seasonal reproduction and molting (Lincoln et al., 2006).The melatonergic system has been implicated in depression,leading to development of the MLT receptor–targeting anti-depressive drug agomelatin (Valdoxan®) (Millan et al., 2003,2005; de Bodinat et al., 2010).Melatonin actions are exerted through its interactions

with three known targets: two of these targets are the

G-protein–coupledmelatonin receptorsMT1 andMT2 (Jockerset al., 2008), which aremainly coupled to Gi protein, leading toadenylate cyclase inhibition (Brydon et al., 1999; Barrett andBolborea, 2012); and the third melatonin target is quinonereductase 2 (formerly known as MT3) (Dubocovich, 1988b;Paul et al., 1999; Nosjean et al., 2000), which has raisedseveral questions regarding melatonin mechanisms of actionin some models, particularly at higher concentrations (Vellaet al., 2005; Jockers et al., 2008; Boutin, 2015). An interactionbetween the RAR-related orphan receptors andmelatonin hasbeen reported (Carlberg and Wiesenberg, 1995), but remainselusive to confirmatory studies.Prior to the cloning of these receptors in the late 1990s

(Reppert et al., 1994, 1995), it was very difficult to study MLTbinding on biologic membranes, since these receptors arenaturally expressed at very low levels and the availableradioligand [3H]-MLT had a poor specific activity. After thedx.doi.org/10.1124/jpet.115.229989.

ABBREVIATIONS: DIV880, 2-(2-[(2-iodo-4,5-dimethoxyphenyl)methyl]-4,5-dimethoxy phenyl); D600, methoxyverapamil; 5HT, 5-hydroxytrypta-mine; hMT, human melatonin receptors; MLT, melatonin; 2-[125I]-MLT, 2-[125I]-iodomelatonin; MT, melatonin receptors; oMT, ovine melatoninreceptors; 4P-PDOT, 4-phenyl-2-propionamidotetraline; SD1881, N-[2-(6-iodo-5-methoxy-1H-indol-3-yl)ethyl]acetamide; SD1882, N-[2-(4-iodo-5-methoxy-1H-indol-3-yl)ethyl]acetamide; SD1918, N-[2-(7-iodo-5-methoxy-1H-indol-3-yl)ethyl]acetamide; SD6, N-[2-(5-methoxy-1H-indol-3-yl)ethyl]iodoacetamide; 6-Cl-MLT, 6-chloromelatonin; S70254, (2-iodo-N-2-[5-methoxy-2-(naphthalen-1-yl)-1H-pyrrolo[3,2-b]pyridine-3-yl])acetamide;S73893, N-[3-methoxy-2-(7-methoxy-1-naphthyl)propyl]acetamide; S75436, 2-fluoro-N-[3-hydroxy-2-(7-methoxy-1-naphthyl)propyl]acetamide;S77834, N-[(8-methoxy-10,11-dihydro-5H-dibenzo[a,d][7]annulen-10-yl)methyl]acetamide; S77840, 1-[(8-methoxy-10,11-dihydro-5H-dibenzo[a,d]-[7]annulen-10-yl)methyl]urea; S20928, (N-[2-(1-naphthyl)ethyl]cyclobutanecarboxamide); S21278, N-[2-(6-methoxybenzimidazol-1-yl)ethyl]acet-amide; S22153, N-[2-(5-ethylbenzothiophen-3-yl)ethyl]acetamide; S27128-1, N-[2-(2-iodo-5-methoxy-6-nitro-1H-indol-3-yl)ethyl]acetamide;TESPA, 3-amino-propyl-ethoxy-silane; 2-Br-MLT, 2-bromomelatonin.

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description of the strongly labeled super agonist 2-[125I]-iodomelatonin (2-[125I]-MLT) (Vakkuri et al., 1984, 1985), allexperiments were conducted with it.Few diverse tools have been developed for MLT molecular

pharmacology studies (Markl et al., 2011), such as subtype-specific agonists or antagonists, or biased agonists (Devavryet al., 2012b; Legros et al., 2014). We further screened ourcompounds (Yan et al., 2008) to find various types of ligandsthat are amenable to chemical conversion to iodinated andradioactive derivatives. Through this study, we identifiedan MT2-specific very partial agonist, 2-(2-[(2-iodo-4,5-dimethoxyphenyl)methyl]-4,5-dimethoxy phenyl) (DIV880),with a Ki value that is 2 logs less potent at MT1 thanat MT2. We synthesized the precursors of each ligand, makethem iodinated, and assessed their characteristics as bindersat the recombinant human MT1 and MT2 receptors. Thisapproach has been briefly described in our recent publication(Legros et al., 2013).In the present work, we described the behaviors of these new

ligands as compared with the standard ligands [3H]-MLT and2-[125I]-MLT. In particular, we investigated the ligand behav-iors at their respective cloned receptors, finding that two werespecific to the receptor isoform MT2. Furthermore, we estab-lished the molecular pharmacology of MT2 using those spe-cific ligands, and compared it with the profiles obtained withother, nonspecific ligands (i.e., 2-[125I]-MLT and [3H]-MLT).This study contributes to the knowledge in this field, to helpdevelop as many tools as possible to determine the charac-teristics of the purified receptors (Logez et al., 2014) as wellas specific ligand(s) amenable for autoradiography in nativetissues.

Materials and MethodsReagents and Ligands

We purchased [125I]-SD6 (N-[2-(5-methoxy-1H-indol-3-yl)ethyl]iodoacetamide; specific activity 2175 Ci·mmol21), [125I]-S70254 [(2-iodo-N-2-[5-methoxy-2-(naphthalen-1-yl)-1H-pyrrolo[3,2-b]pyridine-3-yl])acetamide; specific activity 2175 Ci·mmol21], and [125I]-DIV880(specific activity 2130 Ci·mmol21) from ANAWATrading SA (WangenZürich, Switzerland). We purchased 2-[125I]-MLT (specific activity2200 Ci·mmol21) from PerkinElmer (Boston, MA) and [3H]-MLT(specific activity 80–85 Ci·mmol21) from American RadiolabeledChemicals, Inc. (St. Louis, MO). We obtained MLT, 2-iodomelatonin,6-chloromelatonin, serotonin, andD600 (methoxy-verapamil) fromSigma-Aldrich (St. Louis,MO); 4P-PDOT (4-phenyl-2-propionamidotetraline) andluzindole (2-benzyl-N-acetyltryptamine) from Tocris (Bristol, UK); and2-bromomelatonin from Toronto Research Chemicals, Inc. (NorthYork, Canada). We also evaluated 15 analogs of MLT that stemmedfrom our product library (Legros et al., 2014). Compounds weredissolved in dimethylsulfoxide at a stock concentration of 10 mM,and stored at 220°C until use. All other reagents were obtainedfrom Sigma-Aldrich.

Cell Membrane Preparation

CHO-K1 cell lines stably expressing the MT1 or MT2 receptor(human, rat,mouse, or sheep: hMT, rMT,mMTand oMT, respectively)were grown to confluence, harvested in phosphate-buffered salinebuffer (GIBCO, Invitrogen, Cergy-Pontoise, France) containing 5 mMEDTA, and centrifuged for 20minutes at 1000� g (4°C). The resultingpellet was suspended in 5 mM Tris/HCl (pH 7.4) containing 2 mMEDTA, and homogenized using a Kinematica polytron (KinematicaAG, Luzern, Switzerland). The homogenate was then centrifuged for

30minutes at 20,000� g (4°C), and the resulting pellet was suspendedin 75 mM Tris/HCl (pH 7.4) containing 2 mM EDTA and 12.5 mMMgCl2. Protein content was determined using the Bradford method(Bradford, 1976) with a Bio-Rad kit (Bio-Rad SA, Ivry-sur-Seine,France). Membrane preparation aliquots were stored at 280°C inresuspension buffer containing 75 mM Tris/HCl (pH 7.40), 2 mMEDTA, and 12.5 mM MgCl2.

Cell Membrane Binding Assays. Cell membrane binding as-says were performed in 96-well plates at a final volume of 250 ml,with binding buffer containing 50 mM Tris/HCl (pH 7.4), 5 mMMgCl2, and 1 mM EDTA, plus 0.1% bovine serum albumin for [125I]-DIV880. For all radioactive compounds, the hMT1 and hMT2

membranes were used at a final concentration of 30 mg ml21. In allprotocols, the reaction was stopped by rapid filtration through GF/Bunifilters (filters were presoaked in 0.1% polyethyleneimine for [125I]-DIV880), followed by three successive washes with ice-cold 50 mMTris/HCl buffer (pH 7.4).

Kinetic parameters of [125I]-DIV880, [125I]-S70254, and [125I]-SD6were measured with hMT1 and hMT2 at 37°C and at room tempera-ture (Kon, Koff, and KDkinetics). Koff is the dissociation rate constant, Kon

is the association rate constant, while Kd is the equilibrium bindingconstant, computed as Koff/Kon. For association studies, membraneswere added to radioligands (0.1 nM [125I]-DIV880, 0.04 nM [125I]-S70254, and 0.08 nM [125I]-SD6), followed by incubation for increasingtime periods ranging from 5 to 240 minutes. For dissociation studies,membranes were incubated with the same radioligand concentrationsfor 20 minutes, 1 hour, or 2 hours, followed by addition of 10 mM coldMLT to initiate dissociation, and then incubation for increasing timeperiods from 0 to 240 minutes. To account for membrane variability,kinetic measurements were repeated at least twice on each pool ofmembranes. For displacement tests, the membranes in bindingbuffer were first incubated with compounds diluted in 10%dimethylsulfoxide, followed by the same duration of incubation withthe radioligands: [125I]-DIV880, 0.1 nM for hMT2, 1.5-hour incubations;[125I]-S70254, 0.04 nM for hMT2, 1-hour incubations; and [125I]-SD6,0.08 nM for both hMT1 and hMT2, 1-hour incubations for hMT1 and1.5-hour incubations for hMT2. The different incubation times wereselected based on the kinetics results to be in equilibrium in accordancewith the mass-action law. Ligand dilutions from 1025 to 10215 M werecreated using the MicroLab Starlet (Hamilton, Massy, France). Non-specific binding was defined using 10 mM MLT.

Data were analyzed using the program PRISM (GraphPad SoftwareInc., San Diego, CA). For the saturation assay, the binding site density(Bmax) and the radioligand dissociation constant (KD) were calculatedfollowing the method of Scatchard (1949). Association kinetics data wereanalyzed by fitting specific binding data to the equation B 5 Bmax*[1 2exp(2k*t)], where B is binding at time t, and k is the observed associationrate constant. Dissociation kinetics data were analyzed by fitting specificbinding to the equation B 5 Bmax*exp

(2k1t) 1 plateau, where k is thedissociation constant rate. For displacement experiments, inhibitionconstants (Ki) were calculated using the Cheng-Prussof equation (ChengandPrusoff, 1973):Ki5 IC50/[11(L/KD)],where IC50 is the50% inhibitionconcentration, and L is the [3H]-MLT concentration. The extra sum-of-squares F-test was used for preferredmodel analysis, at one or two sites,for the saturation and kinetics experiments. The Pearson product-moment correlation coefficient was used for correlation analysis of theinhibition constant values, as their log: pKi.

Tissue Membrane Preparation. Lambs and young adult sheepwere obtained from the SODEM slaughterhouse in February, andwere killed during the morning between 7:00 and 12:00 (Le Vigeant,France). Retinas were collected, frozen in nitrogen liquid, and storedat 280°C, with a less than 10-minute interval between slaughter andfreezing. For analysis, the retinas were thawed on ice, crushed, andhomogenized with a Kinematica polytron (Kinematica AG) in 4°Cgrinding buffer comprising 5 mMTris/HCl and 2mMEDTA at pH 7.4.Homogenates were then centrifuged for 12 minutes at 1000 � g (4°C),and the supernatantwas ultracentrifuged for 35minutes at 20,000� g(4°C). The resulting pellets were suspended in conservation buffer

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comprising 50 mMTris/HCl, 6 mM ascorbic acid, 4 mMCaCl2, and 4%glycerol at pH 7.4. Protein content was determined following theBradford method (Bradford, 1976) using the Bio-Rad kit. Aliquots ofthe membrane preparation were stored at 280°C until use.

Tissue Membrane Saturation Assays. Tissue membrane satu-ration assays were performed in 96-well plates with a 250-ml finalvolume in binding buffer comprising 50 mM Tris/HCl, 6 mM ascorbicacid, and 4 mM CaCl2 at pH 7.4 (with 0.1% bovine serum albumin for[125I]-DIV880). For all radioactive compounds, the final membraneconcentrations were 600 mg·ml21. Membranes were incubated for2 hours at 37°C in the presence of 2-[125I]-MLT, [125I]-SD6, [125I]-S70254, or [125I]-DIV880 (0.1–2.5 nM). Nonspecific binding wasdefined using 10 mM MLT. Reactions were stopped by rapid filtrationthrough GF/B unifilters, followed by three successive washes with ice-cold 50 mM Tris/HCl buffer (pH 7.4).

Autoradiography of Rat and Sheep Brains. Autoradiographywas performed using brains from three rats and two sheep. Theanimals were sacrificed by decapitation, after which their brainswere quickly removed and frozen over liquid nitrogen. We used amicrotome-cryostat (Leitz Kryostat 1720) at220°C to generate frozenfrontal 15-mm sections throughout the rat brains, and from fourregions of interest (suprachiasmatic nuclei, pars tuberalis, hypothal-amus and hippocampus) from the sheep brains. These sections werecollected on TESPA (3-amino-propyl-ethoxy-silane; Sigma-Aldrich,Saint-Quentin Fallavier, France) gel-coated slides (Leica Microsys-temes, Nanterre, France), and stored at 280°C until incubation.

Sections were first washed with 50 mM Tris buffer at 4°C, thenincubated for 1 hour at room temperature with 100 ml of Tris buffercontaining 2-[125I]-MLT 280 pM (specific activity, 2200 Ci·mmol21) for

melatonergic binding sites (MT1/MT2) or [125I]-S70254 140 pM forMT2

binding sites. After this incubation, the sections were rinsed twice (for2 and 3 minutes) at 4°C with Tris buffer, fixed with 4% paraformal-dehyde for 10 minutes at 4°C, and dipped in water for 10 minutes. Toassess nonspecific 2-[125I]-MLT binding and [125I]-S70254 binding,respectively, adjacent sections were incubated with 20 and 10 nMMLT (Fluka; Sigma-Aldrich) (Malpaux et al., 1998).

Autoradiograms were generated by placing air-dried sections in X-raycassettes with Biomax MR hyperfilm (Amersham, Courtaboeuf, France).[125I] microscale standards were generated using seven concentrations of2-[125I]-MLT in solutionwith pure ethanol, dispersed on thin-layer chroma-tography silicate gel (Macherey-Nagel,Hoerdt, France). The exposure timewas 28 days for 2-[125I]-MLT binding and 7 days for [125I]-S70254 bindingat room temperature. To identify histologic structure, autoradiographysections were stained using the Klüver and Barrera methods (Kluverand Barrera, 1953) and compared with a rat brain atlas (PaxinosandWatson, 1997). Binding intensity was assessed using an imageanalysis system (Biocom Histo 500, Les Ulys, France). Nonspecificbinding levels were measured on the control section incubated withcold ligands. After removing these levels, the mean gray density wastransformed to counts per minute using the microscale standards,and data were converted to fmol·mgprotein

21 as previously described(Nazarali et al., 1989).

Autoradiography of Rat and Sheep Retinas. Autoradiographywas performed using retinas from two rats and two sheep. Sheep weresacrificed by decapitation, the eyes were quickly removed, and then theretinas were collected by scraping the back of the eyes. The collectedretinas were frozen together over liquid nitrogen and then embedded inTissue-Tek (akura Finetek Europe B.V., AV Alphen aan den Rijn, The

TABLE 1Kinetic parameters of [3H]-MLT, 2-[125I]-MLT, [125I]-SD6, [125I]-S70254, and [125I]-DIV880 with the hMT1and hMT2 receptors at 37°C and at room temperature (RT)Kinetic parameters (Kon, Koff, and half-life) of [3H]-MLT, 2-[125I]-MLT, [125I]-SD6, [125I]-S70254, and [125I]-DIV880 weremeasured on membranes from CHO-K1 cells expressing either hMT1 or hMT2 receptors. pKd was calculated as pKd =2log(Koff/Kon). Results are given as the mean 6 S.E.M. for at least two experiments.

pKd(kinetics) Association Half-Life Dissociation Half-Life Kon Koff

37°C min min M21.s21 s21

[3H]-MLTa

hMT1 10.24 6 0.05 3.0 6 0.6 33.6 6 2.9 6.10 � 106 3.54 � 1024

hMT2 10.72 6 0.02 2.6 6 0.7 69.3 6 8.2 7.37 � 106 1.42 � 1024

2-[125I]-MLTa

hMT1 10.94 6 0.11 20.5 6 4.2 48.6 6 12.5 2.20 � 107 2.55 � 1024

hMT2 — 36.4 6 4.1 — 1.23 � 107 —

[125I]-SD6hMT1 10.96 6 0.02 5.18 6 3.43 32.25 6 5.35 3.48 � 107 3.79 � 1024

hMT2 10.36b 17.89 6 1.80 35.04 6 14.99 8.24 � 106 3.63 � 1024

[125I]-S70254hMT1 — — — — —

hMT2 10.97 6 0.07 12.72 6 0.39 47.42 6 7.14 2.37 � 107 2.56 � 1024

[125I]-DIV880hMT1 — — — — —

hMT2 10.26 6 0.13 14.59 6 1.49 25.30 6 4.40 8.85 � 106 5.00 � 1024

RT[3H]-MLTa

hMT1 10.30 6 0.05 2.7 6 0.7 35.0 6 2.1 6.75 � 106 3.37 � 1024

hMT2 — 6.1 6 3.8 — 3.78 � 106 —

2-[125I]-MLTa

hMT1 10.60 6 0.06 48.6 6 6.7 48.6 6 6.7 9.58 � 106 2.41 � 1024

hMT2 — 79.9 6 9.9 — 5.60 � 106 —

[125I]-SD6hMT1 10.38 6 0.01 13.63 6 9.25 21.84 6 3.23 1.34 � 107 5.56 � 1024

hMT2 9.41b 41.09 6 5.80 9.63 6 5.27 3.67 � 106 1.41 � 1023

[125I]-S70254hMT1 — — — — —

hMT2 10.45 6 0.08 18.42 6 0.68 21.37 6 3.42 1.63 � 107 5.87 � 1024

[125I]-DIV880hMT1 — — — — —

hMT2 10.06 6 0.29 16.87 6 7.23 24.02 6 7.50 7.90 � 106 7.64 � 1024

—, no binding.aData already published by Legros et al. (2014).bNo dissociation measurable at 20 min association time.

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Fig. 1. Time course of association and dissociation of [125I]-SD6, [125I]-S70254, and [125I]-DIV880 at hMT1 (top) and hMT2 (bottom). Time course ofassociation and dissociation of [125I]-SD6 (0.08 nM) binding to the hMT1 receptor at 37°C and at room temperature (RT). Time course of association anddissociation of [125I]-SD6 (0.08 nM), [125I]-S70254 (0.04 nM), and [125I]-DIV880 (0.1 nM) binding to the hMT2 receptor at 37°C and at room temperature.Dissociation was measured at different association times: 20 minutes, red curve; 60 minutes, green curve; and association curve in black. Data are themean (6 S.E.M.) of at least two experiments at the same time point.

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Netherlands) and used to generate 15-mm retina sections for analysis.Rats were sacrificed by decapitation, their eyes were collected, and thecrystalline was removed. The eyes were then embedded together on thelateral side in Tissue-Tek, frozen over liquid nitrogen, and usedto generate 15-mm sagittal sections. Binding in the rat and sheepretina tissue sections was assayed using the previously describedconditions.

ResultsCharacteristics of Human MT1 and MT2 Receptors

with Classic Ligands. Membrane preparations were firstcharacterized using the classic radioligands 2-[125I]-MLT and[3H]-MLT. As described earlier (Legros et al., 2013), averageaffinities and Bmax values are as follows. With 2-[125I]-MLT

Fig. 2. Correlation plots of binding affinities [expressed as pKi = 2log(Ki)] determined for [125I]-SD6, [125I]-S70254, [125I]-DIV880, and 2-[125I]-MLTbinding to the hMT1 and hMT2 receptors in membrane preparations.

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and hMT1, the average pKd value for the used preparationswas 10.696 0.07 nM, and Bmax was 6886 153 fmol·mgprotein

21.With 2-[125I]-MLT and hMT2, pKd was 10.16 6 0.03 nM, andBmax was 19986 318 fmol·mgprotein

21. The radioligands [125I]-S70254 and [125I]-DIV880 showed specific binding only tohMT2, with pKd values of 9.61 6 0.14 and 9.65 6 0.07,respectively, and Bmax values of 1778 6 87 and 2308 6539 fmol·mgprotein

21. [125I]-SD6 showed high affinity for bothreceptors, with a pKd of 10.85 6 0.01 and Bmax of 276 650 fmol·mgprotein

21 for hMT1, and a pKd of 10.186 0.11 and Bmax

of 19296 308 fmol·mgprotein21 for hMT2.Membraneswere also

characterized using [3H]-MLT, which showed biphasic curvesfor both receptors, with pKd values of 10.236 0.07 and 9.4660.01 and Bmax values of 5756 77 and 966 12 fmol·mgprotein

21

for hMT1, and pKd values of 9.87 6 0.05 and 9.26 6 0.05and Bmax values of 22206 178 and 4636 68 fmol·mgprotein

21

for hMT2.Association and Dissociation Kinetics. Kinetics pa-

rameters were determined for each radioligand on hMT1 andhMT2. Table 1 presents all kinetics parameters (kon, koff,KDkinetics, and half-life) as the mean 6 S.E.M. from experi-ments with n of at least 2. The kinetics curves are presentedin Fig. 1. Table 1 recapitulates the results. It seems to usoutstanding that all the ligands presented low nanomolaraffinities at one or the other receptor, if not both. For the tworeference compounds, 2-[125I]-MLT and [3H]-MLT, no majordifferences were observed, as far as the affinities were

concerned, as already reported previously. To the contrary,there are huge differences between 2-[125I]-MLT and [3H]-MLT association half-life: 3 minutes for [3H]-MLT and 20–36minutes for 2-[125I]-MLT, at both receptor subtypes. [3H]-MLTis the only tested compound to show such short associationhalf-lives (ca. 3 minutes) among the five radiolabeled com-pounds tested, whereas their dissociation half-lives were notdramatically different. Only [125I]-SD6 was studied on hMT1

since the other radioligands did not bind to this receptor.Whatever the experimental temperature, nomajor differenceswere recorded with the ligands, as far as their affinities wereconcerned. As stated previously, it is almost impossible tomeasure an affinity at hMT1 for [125I]-S70254 and [125I]-DIV880. Of note is the fact that only data recorded with [3H]-MLT showed a second inflection point, leading to the possibilityof measuring a second pKd (9.91 6 0.02 at MT1) whatever theexperimental temperature. This feature is shared only by theDIV880 at MT2 with a pKd of 9.23 6 0.29. The meaning of thissecond binding site remains elusive.Pharmacology. It is important to evaluate the molecular

pharmacologyprofiles of the three investigatednewradioligandsin comparison with those of 2-[125I]-MLT and [3H]-MLT. Weassessed a set of 24 compounds that were previously describedin the literature [including 4-phenyl-2-propionamidotetraline(Dubocovich et al., 1997), luzindole (Dubocovich, 1988a), andramelteon (Uchikawa et al., 2002)] or that were issued fromour own medicinal chemistry programs (Depreux et al., 1994;

TABLE 2Binding affinities of reference ligands to the hMT1 receptor as measured with either [125I]-SD6, [3H]-MLT, or 2-[125I]-MLTExperiments were conducted using the recombinant hMT1 receptor expressed in CHO-K1 cells. Data are given as mean6S.E.M.

hMT1

pKi 6 S.E.M.a [3H]-MLT pKi 6 S.E.M.a 2-[125I]-MLT pKi 6 S.E.M. [125I]-SD6

MLT 10.15 6 0.12 9.65 6 0.02 9.85 6 0.012-I-MLT 12.12 6 0.20 10.71 6 0.08 10.49 6 0.114P-PDOT 7.56 6 0.16 6.85 6 0.04 7.07 6 0.14Luzindole 8.09 6 0.31 6.59 6 0.01 6.39 6 0.05Ramelteon 11.82 6 0.06 10.10 6 0.09 10.52 6 0.07SD6 11.33 6 0.34 9.94 6 0.01 10.45 6 0.116-Cl-MLT 9.25 6 0.07 8.73 6 0.03 9.08 6 0.192-Br-MLT 12.11 6 0.08 10.82 6 0.13 10.56 6 0.11S70254 7.32 6 0.31 7.03 6 0.09 6.36 6 0.28SD1881/6-I-MLT 6.83 6 0.24 8.84 6 0.01 8.87 6 0.10SD1882/4-I-MLT 7.95 6 0.07 7.76 6 0.12 7.66 6 0.07SD1918/7-I-MLT 7.88 6 0.10 7.34 6 0.15 7.65 6 0.16S22153 8.25 6 0.09 8.24 6 0.14 7.97 6 0.12S27128 9.03 6 0.12 8.92 6 0.01 8.96 6 0.05Agomelatine 10.17 6 0.25 9.92 6 0.01 9.93 6 0.02D600 (+/2) 7.76 6 0.15 7.04 6 0.02 7.19 6 0.01DIV880 7.44 6 0.12 6.10 6 0.04 5.89 6 0.565HT .5 .5 .5S20928 7.27 6 0.26 7.10 6 0.08 6.73 6 0.10S75436 8.53 6 0.06 7.88 6 0.01 8.31 6 0.12S21278 7.71 6 0.14 6.22 6 0.10 6.18 6 0.12S73893 8.60 6 0.06 8.36 6 0.16 7.98 6 0.06S77834 7.87 6 0.15 7.09 6 0.05 7.11 6 0.14S77840 8.16 6 0.11 6.15 6 0.08 6.50 6 0.11

6-Cl-MLT, 6-chloromelatonin; 2-Br-MLT, 2-bromomelatonin; D600, methoxyverapamil; 5HT, 5-hydroxytryptamine;S20928, (N-[2-(1-naphthyl)ethyl]cyclobutanecarboxamide); S21278, N-[2-(6-methoxybenzimidazol-1-yl)ethyl]acetamide;S22153, N-[2-(5-ethylbenzothiophen-3-yl)ethyl]acetamide; S27128-1, N-[2-(2-iodo-5-methoxy-6-nitro-1H-indol-3-yl)ethyl]acetamide; S73893, N-[3-methoxy-2-(7-methoxy-1-naphthyl)propyl]acetamide; S75436, 2-fluoro-N-[3-hydroxy-2-(7-methoxy-1-naphthyl)propyl]acetamide; S77834, N-[(8-methoxy-10,11-dihydro-5H-dibenzo[a,d][7]annulen-10-yl)methyl]acetamide; S77840, 1-[(8-methoxy-10,11-dihydro-5H-dibenzo[a,d][7]annulen-10-yl)methyl]urea; SD1881, N-[2-(6-iodo-5-methoxy-1H-indol-3-yl)ethyl]acetamide; SD1882, N-[2-(4-iodo-5-methoxy-1H-indol-3-yl)ethyl]acetamide; SD1918, N-[2-(7-iodo-5-methoxy-1H-indol-3-yl)ethyl]acetamide.

aData from Legros et al. (2014).

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Audinot et al., 2003, 2008; Mailliet et al., 2004; Devavry et al.,2012a,b; Ettaoussi et al., 2013; Legros et al., 2013, 2014).These compounds were tested against [125I]-SD6 on bothhMT1 and hMT2 membrane preparations, and against [125I]-S70254 and [125I]-DIV880 on hMT2 membrane preparationsonly. Under standard non-decoupling binding conditions,the binding data all consistently exhibited good correlationsamong the various data sets (Fig. 2; Tables 2 and 3). Themolecular pharmacology profiles of [125I]-SD6, [125I]-S70254,and [125I]-DIV880 mostly replicated that obtained with2-[125I]-MLT for both hMT1 and hMT2 (for all radioligands,r $ 0.95 and P , 0.0001, n $ 2).Tissue Membrane Saturation Assays. We next tested

binding of the [125I]-SD6, [125I]-S70254, and [125I]-DIV880radioligands on tissue membrane preparations. We chose touse sheep retina tissue because it is readily available in largeamounts, and because MT1 and MT2 mRNAs have beendetected in this tissue (Coge et al., 2009). As a positive control,membranes were also characterized using 2-[125I]-MLT.The saturation curves for 2-[125I]-MLT showed biphasic

curves and Scatchard regressions (Fig. 3) with a pKd1 (site 1)of 10.356 0.03, a pKd2 (site 2) of 9.256 0.13, a Bmax1 (site 1) of1.46 6 0.13 fmol·mgprotein

21, and a Bmax2 (site 2) of 2.32 60.98 fmol·mgprotein

21 (n 5 4). Similarly, [125I]-SD6 showedbiphasic curves and Scatchard regressions (Fig. 3) with apKd1 of 10.136 0.18, a pKd2 of 6.796 0.21, a Bmax1 of 0.6060.20 fmol·mgprotein

21, and aBmax25 2.116 1.13 fmol·mgprotein21

(n 5 3). Unfortunately, MT2 radioligands did not show anyproper saturation curves. Moreover, the signals of [125I]-S70254 were very low and highly variable, and it was notpossible to obtain a saturation curve better than that shown in

Fig. 3. We observed no specific binding of [125I]-DIV880 onretina membranes.Rat Brain Autoradiography. Rat brains were randomly

screened. We analyzed 150 sections from throughout thebrain, processed in groups of six. Compared with 2-[125I]-MLT, [125I]-S70254 showed higher specific binding in theperiacqueductal gray region (1.93 6 2.00 vs. 0.25 6 0.51fmol·mgprotein

21, respectively), in the interpeduncular nu-cleus (4.14 6 4.52 vs. 1.56 6 0.31 fmol·mgprotein

21), and inolfactory bulbs (4.88 vs. 2.24 fmol·mgprotein

21). In the sub-iculum and pontine nuclei, we observed only [125I]-S70254binding (3.05 6 3.17 and 3.10 6 2.33 fmol·mgprotein

21,respectively). In the suprachiasmatic nuclei and pars tuber-alis, we observed only 2-[125I]-MLT binding (4.07 and14.24 fmol·mgprotein

21, respectively). Representative imagesare shown in Fig. 4.Rat and sheep retina autoradiography. To complete

our characterization of these new radioligands, we alsoassessed binding of 2-[125I]-MLT and [125I]-S70254 in ratand sheep retinas (Fig. 5). The binding density of [125I]-S70254 was greater than that of 2-[125I]-MLT, with values of14.13 6 6.53 fmol·mgprotein

21 for [125I]-S70254 and 4.11 60.73 fmol·mgprotein

21 for 2-[125I]-MLT in the rat retina, andvalues of 4.00 6 0.24 fmol·mgprotein

21 for [125I]-S70254and 1.98 6 2.64 fmol·mgprotein

21 for 2-[125I]-MLT in thesheep retina.Radioligand Structure and Characteristics. Figure 6

presents a comparison of ligand electrostatic potentials, ascomputed using Molecular Operating Environment 2012.10(Chemical Computing Group, (www.chemcomp.com). Nota-bly, the iodine atom (either on position 2 or at the tip of the

TABLE 3Binding affinities of reference ligands to the hMT2 receptor as measured with [125I]-SD6, [125I]-S70254, [125I]-DIV880, [3H]-MLT, or 2-[125I]-MLTExperiments were conducted using the recombinant hMT2 receptor expressed in CHO-K1 cells. Data are given as mean 6 S.E.M.

hMT2

pKi 6 S.E.M.a[3H]-MLT pKi 6 S.E.M.a 2-[125I]-MLT pKi 6 S.E.M. [125I]-SD6 pKi 6 S.E.M. [125I]-S 70254 pKi 6 S.E.M. [125I]-DIV880

MLT 9.67 6 0.26 9.27 6 0.02 9.16 6 0.06 9.10 6 0.13 9.34 6 0.152-I-MLT 11.40 6 0.18 9.83 6 0.03 9.58 6 0.06 9.49 6 0.06 9.60 6 0.064P-P-DOT 9.07 6 0.51 8.97 6 0.05 8.99 6 0.05 9.14 6 0.10 9.27 6 0.02Luzindole 7.80 6 0.17 7.57 6 0.01 7.49 6 0.33 7.51 6 0.08 7.45 6 0.06Ramelteon 11.52 6 0.14 10.30 6 0.19 9.60 6 0.02 9.56 6 0.07 9.59 6 0.00SD6 11.33 6 0.13 9.89 6 0.22 9.48 6 0.13 9.50 6 0.01 9.60 6 0.026-Cl-MLT 10.09 6 0.24 9.56 6 0.12 9.23 6 0.12 9.28 6 0.07 9.42 6 0.012-Br-MLT 11.47 6 0.23 9.94 6 0.12 9.59 6 0.12 9.51 6 0.05 9.64 6 0.02S 70254 8.31 6 0.50 9.04 6 0.08 8.39 6 0.06 8.35 6 0.04 8.38 6 0.06SD1881 / 6-I-MLT 8.64 6 0.14 8.61 6 0.04 8.27 6 0.09 8.42 6 0.12 8.49 6 0.06SD1882 / 4-I-MLT 8.04 6 0.14 7.99 6 0.14 7.63 6 0.05 8.10 6 0.55 8.27 6 0.69SD1918 / 7-I-MLT 7.53 6 0.52 7.32 6 0.15 7.14 6 0.11 7.44 6 0.20 7.45 6 0.07S 22153 8.47 6 0.44 8.01 6 0.09 7.96 6 0.03 8.12 6 0.20 8.31 6 0.10S 27128 9.40 6 0.26 9.17 6 0.06 8.57 6 0.28 9.17 6 0.17 9.31 6 0.13Agomelatine 11.21 6 0.17 9.93 6 0.06 9.56 6 0.06 9.53 6 0.09 9.59 6 0.03D600 (+/2) .5 .5 10.42 6 1.27 .5 .5DIV00880 8.08 6 0.34 8.04 6 0.06 7.77 6 0.16 8.05 6 0.18 7.77 6 0.025HT .5 .5 .5 .5 .5S 20928 7.65 6 0.28 7.05 6 0.25 6.86 6 0.15 7.01 6 0.24 7.08 6 0.03S 75436 9.68 6 0.11 8.87 6 0.15 8.85 6 0.10 9.12 6 0.22 9.20 6 0.07S 21278 7.78 6 0.22 6.22 6 0.03 6.08 6 0.07 6.13 6 0.05 6.20 6 0.06S 73893 8.90 6 0.09 8.11 6 0.23 8.22 6 0.01 ND 8.41 6 0.08S 77834 8.51 6 0.19 8.53 6 0.06 8.34 6 0.39 ND 8.08 6 0.10S 77840 7.98 6 0.05 7.71 6 0.15 7.14 6 0.05 ND 7.27 6 0.15

6-Cl-MLT, 6-chloromelatonin; 2-Br-MLT, 2-bromomelatonin; D600, methoxyverapamil; 5HT, 5-hydroxytryptamine; S20928, (N-[2-(1-naphthyl)ethyl]cyclobutanecarbox-amide); S21278, N-[2-(6-methoxybenzimidazol-1-yl)ethyl]acetamide; S22153, N-[2-(5-ethylbenzothiophen-3-yl)ethyl]acetamide; S27128-1, N-[2-(2-iodo-5-methoxy-6-nitro-1H-indol-3-yl)ethyl]acetamide; S73893, N-[3-methoxy-2-(7-methoxy-1-naphthyl)propyl]acetamide; S75436, 2-fluoro-N-[3-hydroxy-2-(7-methoxy-1-naphthyl)propyl]acetamide;S77834, N-[(8-methoxy-10,11-dihydro-5H-dibenzo[a,d][7]annulen-10-yl)methyl]acetamide; S77840, 1-[(8-methoxy-10,11-dihydro-5H-dibenzo[a,d][7]annulen-10-yl)methyl]urea; SD1881, N-[2-(6-iodo-5-methoxy-1H-indol-3-yl)ethyl]acetamide; SD1882, N-[2-(4-iodo-5-methoxy-1H-indol-3-yl)ethyl]acetamide; SD1918, N-[2-(7-iodo-5-methoxy-1H-indol-3-yl)ethyl]acetamide. aData from Legros et al. (2014).

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side chain of MLT) does not affect the hydrogen bondingpotential with the 5-methoxy oxygen atom and the amidegroup on the side chain, as previously described (Pala et al.,

2013). The iodine and the naphthyl (S70254) on the indole C2position have a similar hydrophobic shape in the vicinityof the indole ring. Furthermore, the second aromatic of

Fig. 3. Saturation of 2-[125I]-melatonin, [125I]-SD6, and [125I]-S70254 on sheep retina membranes, and Scatchard regression with incubation for 2 hoursat 37°C. Closed circles, total binding; open circles, specific binding; closed triangles, nonspecific binding. Graphs are representative of all experimentswith each setup.

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the naphthyl group likely confers the MT2 selectivity toS70254. On the side chain of compounds S70254 and SD6, theiodine atom does not prevent the amide group from makinghydrogen bonds to the receptor, suggesting a hydrophobicpocket in that region.

DiscussionOne main factor limiting studies of the melatonin receptors

MT1 and MT2 is the lack of antagonist and discriminantradioligands. The two presently known radioligands, [3H]-MLT and 2-[125I]-MLT, are pure agonists, and exhibit thesame affinity and almost identical pharmacological parame-ters for both receptors (Browning et al., 2000; Masana andDubocovich, 2001; Legros et al., 2013, 2014). In the presentwork, we described three new radioligands that can be usedfor MT1 and MT2 receptor characterization. Our resultsindicated that each radioligand has specific properties thatcan be exploited for different kinds of pharmacologicalstudies. In particular, we found that [125I]-S70254 isspecific for the MT2 receptor, and can be used with cellsand tissue.The three newly investigated radioligands could each bind

hMT1 and/or hMT2 receptors. Specifically, [125I]-SD6 showedaffinities (pKd6 S.E.M.) of 10.856 0.01 for hMT1 and 10.1860.11 for hMT2, and [125I]-DIV880 and [125I]-S70254 bound

hMT2 with affinities of 9.61 6 0.14 and 9.65 6 0.07, re-spectively. Despite its slow dissociation from the hMT2 re-ceptor, [125I]-SD6 showed affinities for MT receptors andkinetics parameters (including pKd and half-life) that werevery similar to those of 2-[125I]-MLT. The set of compoundstested in our dose-response experiments also showed affin-ities for receptors that were similar to those of 2-[125I]-MLT.These findings suggest that the position of the iodine (whichconstitutes 50% of the size of MLT, i.e., 125 vs. 232 Da) doesnot influence the binding, at least when it is located atposition 2 on the indolic moiety or at the extremity of thelateral chain.Although both [125I]-S70254 and [125I]-DIV880 showed low

affinities for hMT1 [pKi of 6.25 and 6.08, respectively (Legroset al., 2013)], neither showed specific binding for this receptor.Compared with 2-[125I]-MLT, [125I]-S70254 and [125I]-DIV880each showed a slightly lower affinity (half-log lower) for thehMT2 receptor, although this difference disappeared whenusing the kinetics pKd. The lower affinity could be explainedby the difficulty reaching a saturation plateau in the Scatch-ard assay. Apart from the lack of affinity for hMT1, [

125I]-S70254 and [125I]-DIV880 mainly differ from 2-[125I]-MLT inthat they show fast and total dissociation at 37°C and partialdissociation at room temperature, whereas 2-[125I]-MLT dis-sociates partially at 37°C and does not dissociate at roomtemperature.

Fig. 4. Autoradiography of 15-mm sections of rat brain using 2-[125I]-MLT (0.28 nM) and [125I]-S70254 (0.14 nM), with a 1-hour incubation at roomtemperature. Nonspecific binding was assessed with 10 and 20 nM melatonin for [125I]-S70254 and 2-[125I]-MLT, respectively. C, cortex; CG, centralgray; DG, dentate gyrus; Hipp, hippocampus; IP, interpeduncular nuclei; PAG, periaqueductal nucleus; PT, pars tuberalis; S, subiculum; SCN,suprachiasmatic nuclei.

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Our kinetics experiments illustrated the ability of theradiocompound to associate and dissociate from the receptor.After 1 hour of incubation at room temperature, 2-[125I]-MLTdid not reach an equilibrium plateau, and only a proportionwas bound to a receptor (3/4 with hMT1 and 1/2 with hMT2).

The 2-[125I]-MLT radioligand has been widely used to studymelatonin receptors in the brain and peripheral organs byautoradiography (Morgan et al., 1994; Thomas et al., 2002;Sallinen et al., 2005). Usually an incubation time of about1 hour is used, which allows a balance between radioligand

Fig. 5. Rat and sheep retina autoradiography with 2-[125I]-melatonin (0.28 nM) and [125I]-S70254 (0.14 nM). For rat tissue, 15-mm sagittal sections ofeyes without crystalline from two different animals were embedded together in Tissue-Tek. For sheep tissue, we used 15-mm sections of retina alone,collected by scraping the back of the eye. Nonspecific binding was assessed with 10 and 20 nMmelatonin for [125I]-S70254 and 2-[125I]-MLT, respectively.Representative pictures are shown for both animals.

Fig. 6. Graphical representation of the ligands. Compounds are shown with the electrostatic potential mapped on the molecular surface. Red and blueregions depict negative and positive electrostatic potential, respectively. (A) Melatonin; (B) 2-iodo melatonin; (C) S70254; (D) SD6; (E) DIV880. Theserepresentations were created using Molecular Operating Environment software 2012.10 (Chemical Computing Group, www.chemcomp.com). On theindividual figures, the iodine is shown by a red circle.

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binding and degradation of the tissue slice, which is not fixedin this kind of protocol. These data suggest that the realdensities of receptors and binding sites are underestimatedwith the use of 2-[125I]-MLT. Some prior studies may havedetected no melatonin receptors because there exists only alow density of binding sites, and because these sites are onlypartially detected due to the use of an incubation time that istoo short—for example, in the finding of no 2-[125I]-MLTbinding on the suprachiasmatic nuclei in sheep brain tissue(de Reviers et al., 1991).The use of alternative radioligands could potentially help

with this problem of partial binding site labeling. In thepresent work, we showed that such autoradiography experi-ments could be performed, and could lead to new specificinformation about MT2 localization. The ligand 2-[125I]-MLTshows similar affinities for both receptors as well as similarkinetics, making it a poor selection for autoradiography. Onthe other hand, [3H]-MLT has the advantage of fast associa-tion kinetics but also shows a very low specific activity thatalso makes it a poor candidate for autoradiography. We foundthat [125I]-S70254 labeled the MT2 receptor in autoradiogra-phy. However, as with 2-[125I]-MLT, we must consider theassociation time that results in partial labeling of the MT2

receptor (about 2/3 or 3/4). Our present results did not show[125I]-S70254 binding on the suprachiasmatic nuclei. Thiscould have been due to the random screening without target-ing of structures and the small size of the rat suprachiasmaticnuclei (2–4 mm2) (Paxinos and Watson, 1997), or it couldhave been because the rat MT2 expression was too low andthe reinforcement of this problem by the partial labelingof the receptors by the radioligand with an incubation timeof 1 hour.In our assays using the retina membrane preparation, we

observed specific binding with 2-[125I]-MLT and [125I]-SD6.Sheep retina tissue expresses both the ovine MT1 and MT2

receptors (Coge et al., 2009). In comparison with the Kd valueon transfected cell membrane preparations, the first highestaffinity binding site could be oMT1 or oMT2 in sheep retinatissue (Mailliet et al., 2004). The second binding site in thesaturation experiment remains to be identified. The binding of[125I]-S70254 was highly variable, and the saturation curvewas difficult to obtain. This may have been due to the lowdensity of the oMT2 receptor, which could potentially beremedied by increasing the protein concentration only if wealso changed the filtration protocol, as filter obstruction wouldoccur with the presently used protocol. Another possible causeis the affinity of the radioligand for the ovine form of thereceptor (Kiovine 5 7.67 6 0.07, unpublished data). This couldbe investigated by performing tests with tissue membranepreparations from different species.It is unfortunately clear that, as of today, the use of [125I]-SD6

would not determine other information than that determined by2-[125I]-MLT. It remains to be seen if further characterization ofthe agonistic nature of the compound, using as many differentsignaling pathways, will reveal a different view of these agonists(C. Legros and J.A. Boutin, manuscript in preparation).Overall, our present data contribute information and

potential new tools that will be useful in further studies ofMLT molecular pharmacology. Greater availability of suchtools would help pharmacologists to better understand thenature of involvement of MLT receptors in the many patho-logic processes in which MLT is active. However, further

structure-based modeling will be required to design newagonist compounds and to better understand the structuremechanism of the various action relationships (agonist,partial agonist, and antagonist) that can, incidentally, serveas radioligands after iodination. Continued investigations ofthis field and the development of new techniques could leadto novel therapies targeting the melatonergic system.

Authorship Contributions

Participated in research design: Legros, Nosjean, Delagrange,Boutin.

Conducted experiments: Legros, Brasseur, Ducrot.Wrote or contributed to the writing of the manuscript: Legros,

Boutin, Ducrot.

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Address correspondence to: Jean A. Boutin, PEX BCB, Institut deRecherches SERVIER, 125 chemin de Ronde, 78290 Croissy-sur-Seine, France.E-mail: jean.boutin@servier.com

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