Design, Evaluation, and Comparison of Ghrelin ... - ACS Publications

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Design, Evaluation, and Comparison of Ghrelin Receptor Agonists and Inverse Agonists as Suitable Radiotracers for PET Imaging Constance Chollet,*,† Ralf Bergmann,‡ Jens Pietzsch,‡ and Annette G. Beck-Sickinger† †

Institute of Biochemistry, Universität Leipzig, Leipzig, Germany Institute of Radiopharmacy, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany

‡

ABSTRACT: Ghrelin agonist and inverse agonist radiotracers, suitable for positron emission tomography (PET), were developed to study the behavior of ghrelin receptor ligands in vivo and for further design of druggable peptides. The target peptides were synthesized on solid support and conjugated to the bifunctional chelator 1,4, 7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), which is known to form a stable complex with Ga3+. Complexation with 68Ga could be achieved under mild conditions and led to radiotracers with high radiochemical purity and specific activity. The biological activity of the radiotracers was evaluated in vitro by an inositol phosphate turnover assay. Pharmacokinetic profile and metabolic stability of the 68 Ga-NODAGA-radiotracers were investigated by small animal PET in rodent. Ghrelin derived agonists presented very high kidney accumulation, negligible tissue distribution, fast blood clearance, and poor stability in blood. Contrarily, the inverse agonist radiotracer exhibited very high stability in blood, large diffusion in tissues, reasonable kidney and liver metabolism, and slow blood clearance. This pharmacokinetic profile makes the ghrelin inverse agonist motif KwFwLL-CONH2 suitable for further development of radiotracers and a promising lead to design peptide-based therapeutics against obesity.

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INTRODUCTION Ghrelin is a peptide hormone acting as a predominant orexigenic signal. It triggers the preprandrial hunger and meal initiation and also acts as a positive adiposity signal favoring energy storage.1,2 Physiologically, ghrelin activates the endogenous ghrelin receptor (formerly called GHS-R1a), and stimulates the secretion of the orexigenic neurotransmitter NPY and agouti related peptides (AgRP) in the arcuate nucleus of hypothalamus.3−5 Ghrelin is a 28 amino acid peptide produced by the oxyntic mucosa of the stomach.4,6 Its sequence contains a unique n-octanoylation at serine 3, introduced post-translationally by ghrelin-O-acyltransferase (GOAT) and essential for binding the ghrelin receptor.7,8 Being the only known orexigenic signal from the periphery, the role of ghrelin in obesity has been widely studied. Indeed, although ghrelin levels are low in the obese, weight loss is correlated with an increase in ghrelin level and GHS-R1a expression.9 In addition, the ghrelin receptor possesses a naturally high constitutive activity thought to induce constant appetite and to trigger food intake between meals.10 Consequently, the ghrelin receptor has emerged as a new target to design drugs against not only obesity, but also cachexia and anorexia.11−13 However, ghrelin is an endogenous peptide and its short halflife and low stability in the organism limits its use as a drug. In addition, little is known about the peptide behavior in vivo. With the aim to develop peptide-based therapeutics targeting the ghrelin receptor, we developed radiotracers suitable for PET imaging. Indeed, the use of noninvasive imaging techniques, especially small animal PET, should provide better knowledge on © 2012 American Chemical Society

biodistribution, biokinetics, metabolic stability, and mode of action of ghrelin receptor ligands in vivo. To obtain peptidebased radiotracers, we used the bifunctional chelator (BFC) 1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid (NODAGA), that can be easily branched to different peptide cores and can chelate radionuclides in mild conditions. Hence, in this paper, we evaluate ghrelin agonist and inverse agonist peptides as radiotracers. [68Ga]-NODAGA-conjugates were synthesized using solid-phase peptide synthesis. The biological activity of the radiotracers was evaluated in vitro in a functional assay. Peptides behavior in vivo was investigated in rodents with biodistribution, biokinetics, and metabolism studies and with PET imaging.

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EXPERIMENTAL PROCEDURES Materials. Fmoc amino acid derivatives, Boc-L-Gly-OH, diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), piperidine, Wang resin, and Fmoc-L-Arg(Pmc)-Wang resin were purchased from Iris Biotech (Marktredwitz, Germany) or Novabiochem (Läufelfingen, Switzerland). If not specified, the side chain protecting groups are tBu for Glu, Ser, Thr, and Tyr; Boc for Lys and D-Trp; Trt for Gln; and His and Pbf for Arg. Fmoc-L-Lys(Dde)-OH, Fmoc-L-Ser(Trt)-OH, and Fmoc-L-Dpr(Mtt)-OH were also purchased from Iris Biotech Received: November 4, 2011 Revised: February 28, 2012 Published: February 28, 2012 771

dx.doi.org/10.1021/bc2005889 | Bioconjugate Chem. 2012, 23, 771−784

Bioconjugate Chemistry

Article

was successively washed five times with DMF, DCM, MeOH, and Et2O, and dried in vacuo. The loaded resin was elongated automatically and manually as described above to yield peptide 4a on solid support. Automated Peptide Synthesis. To synthesize peptides 1−5a, amino acids were introduced automatically with a multiple peptide synthesis robot system, following the procedure recently described.14 For all peptides, the resin was first swollen for 15 min in DMF. Then, a 10-fold excess of the corresponding Fmoc-protected amino acid was coupled through an in situ activation with a 10-fold excess of both HOBt and DIC in 400 μL of DMF. Each amino acid was coupled twice for 40 min, followed by a washing step and the removal of the Fmoc group by adding 400 μL of 40% piperidine in DMF (v/v), twice, for 10 min. The last amino acid introduced was Fmoc-L-Gly-OH for peptide 1a, and Boc-L-Gly-OH for peptides 2−4a, and FmocL-Lys(BOC)-OH for peptide 5a. Manual Peptide Synthesis. Fmoc-L-Lys(Dde)-OH at position 16 of peptide 3a, Fmoc-L-Ser(Trt)-OH at position 3 of peptides 1−2a, Fmoc-L-Dpr(Mtt)-OH at position 3 of peptides 3−4a were introduced manually to be further modified. First, each peptide was swollen in 500 μL of DMF for 15 min. The respective Fmoc-amino acid (0.075 mmol, 5 equiv) and HOBt (0.075 mmol, 5 equiv) were then dissolved in 200 μL DMF and the solution was added to the resins with DIC (0.075 mmol, 5 equiv). The reaction mixtures were shaken for at least 3 h at room temperature. The peptides were then washed successively five times with DMF, DCM, MeOH, and Et2O, and dried in vacuo. The completion of the coupling reaction was checked with a Kaiser test. Selective Dde Cleavage at Lys16 of Peptides 2−4a. 500 μL of a hydrazine solution (2% v/v) was added to each peptide for 10 min. The cleavage was repeated 10 times. The peptides were then washed successively five times with DMF, DCM, MeOH, and Et2O, and dried in vacuo. NODAGA(tBu) 3 Coupling for Peptides 1−5a. NODAGA(tBu)3 was introduced at the N-terminus of peptides 1a and 5a and at the Nε of K16, after Dde cleavage, for peptides 2−4a. Each peptide was first swollen in 500 μL of DMF for 15 min. NODAGA(tBu)3 (0.03 mmol, 2 equiv) and HOBt (0.03 mmol, 2 equiv) were dissolved in 150 μL DMF and the solution was added to the resins with DIC (0.03 mmol, 2 equiv). The reaction mixtures were shaken overnight at room temperature. The peptides were then washed successively five times with DMF, DCM, MeOH, and Et2O, and dried in vacuo to yield peptides 1−5b. The completion of the coupling reaction was monitored with a Kaiser test. Selective Trt/Mtt Cleavage for Peptides 1−4b. Mtt at Ser3 of peptides 1−2b and Trt at Dpr3 of peptides 3−4b were selectively cleaved following the same procedure. The peptides were incubated at room temperature for 5 min with 500 μL of the cleavage solution: TFA 1%, triisopropylsilane 5%, DCM 94% (v/v). The cleavage was repeated 15 times. At the end of the cleavage reaction, the peptides were washed successively five times with DMF, DCM, MeOH, and Et2O, and dried in vacuo. Octanoic Acid Coupling at Ser3 of Peptides 1−2b. After Trityl cleavage, both peptides were swollen in 500 μL of NMP for 15 min. Octanoic acid (0.15 mmol, 10 equiv), DMAP (0.09 mmol, 6 equiv), and DCC (0.09 mmol, 6 equiv) were then dissolved in 200 μL NMP, and the solution was added to the resins. The reaction mixtures were shaken for 1 h at room temperature. The peptides were then washed twice with NMP

(Marktredwitz, Germany) and used for orthogonal modification. Thioanisole, p-thiocresol, ethandithiol, piperidine, hydrazine monohydrate, 4-dimethylaminopyridine (DMAP), triisopropylsilane (TIPS), and tert-butanol was purchased from Fluka (Taufkirchen, Germany). NODAGA(tBu)3 (4-(4,7-bis(2-tertbutoxy-2-oxoethyl)-1,4,7-triazonan-1-yl)-5-tert-butoxy-5-oxopentanoic acid) was obtained from CheMatec (Dijon, France). Dichloromethane (DCM) and N,N-dimethylformamide (DMF) were purchased from Biosolve (Valkenswaard, Netherlands). Trifluoroacetic acid (TFA), acetic anhydride, diisopropylethylamine (DIPEA), octanoic acid, and Ga(NO3)3 were obtained from Sigma-Aldrich (Taufkirchen, Germany). Gradient-grade high-performance liquid chromatography (HPLC) solvent acetonitrile (ACN) was from VWR (Darmstadt, Germany). All reagents and solvents were used without purification as provided from the commercial suppliers. Nonmodified ghrelin used as a control in biological assays was obtained from Polypeptide (Hillerød, Denmark). For cell culture and in vitro assays, Dulbecco’s phosphate buffered saline without calcium and magnesium (PBS), DMEM, high glucose (4.5 g·L−1) with L-glutamine, heat-inactivated fetal bovine serum (FBS), penicillin/ streptavidin, and trypsin/EDTA (1:250) were purchased from PAA (Pasching, Austria). Metafectene was purchased from Biontex (Martinsried, Germany). myo-[3H] Inositol (681 MBq.mmol−1; 25.0 Ci.mmol−1) was from GE Healthcare Europe GmbH (Braunschweig, Germany). Cell culture flasks (75 cm2) and 24-well plates were from TPP (Trasadingen, Switzerland). Instruments. Automated peptide synthesis was performed with a multiple peptide synthesizer MultiSynTech Syro II or MultiSynTech Syro XP (Bochum, Germany). Preparative and semipreparative HPLC were performed using a Merck-Hitachi system on a Shimadzu RP18-column (12.5 × 250 mm; 5 μm/ 300 Å). Analytical HPLC were performed using a MerckHitachi system with a Vydac RP18-column (4.6 × 250 mm; 5 μm/300 Å, Merck Hitachi, Darmstadt, Germany). All peptides were analyzed by matrix-assisted LASER desorption ionization−time-of-flight (MALDI-ToF) using a Bruker Daltonics Ultraflex III mass spectrometer. The 1.85-GBq (50-mCi) 68 Ge/68Ga generator was purchased from iThemba Laboratories (Somerset West, South Africa) with the 68Ge on a SnO2-cartrige and eluted according to the manufacturer’s recommendations using a remote-controlled module. Synthesis of Ghrelin Derivative Tracers. The peptides were synthesized on solid support, following a fluorenylmethoxycarbonyl/tert-butyl (Fmoc/tBu) strategy. Preloaded Fmoc-Arg(Pmc)-Wang was used for peptides 1−3a and Rink amide resin for peptide 5a. For peptide 4a, Wang resin was manually loaded with Fmoc-L-Lys(Dde)-OH. Manual Loading of Wang Resin with Fmoc-L-Lys(Dde)-OH for the Synthesis of Peptide 4a. The Wang resin (0.015 mmol, 0.64 mmol.g−1) was swollen in 500 μL of DMF for 15 min. Fmoc-L-Lys(Dde)-OH (0.075 mmol, 5 equiv) and HOBt (0.075 mmol, 5 equiv) were dissolved in DMF to a concentration of 0.5 M. The solution was added to the resin with DIC (0.075 mmol, 5 equiv), and the reaction mixture was shaken overnight at room temperature. The resin was successively washed five times with DMF, DCM, MeOH, and Et2O, and dried in vacuo. The coupling was repeated once, followed by an end-capping of the free hydroxyl residues of the Wang resin. For this purpose, the resin was swollen for 15 min in 500 μL of DCM. Acetic anhydride (50 μL) and DIPEA (50 μL) were then added to the resin with 400 μL DCM, and the mixture was incubated 15 min at room temperature. The resin 772



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Table 1. Analytical Data of Conjugates 1−4d, 5c, and Chelates 1−4e, 5d compound 1d 1e 2d 2e 3d 3e 4d 4e 5c 5d

α

N -NODAGA-ghrelin(1−28) Nα-NODAGA(Ga)-ghrelin(1−28) [K16(NODAGA)]ghrelin(1−28) [K16(NODAGA(Ga))]ghrelin(1−28) [Dpr3;K16(NODAGA)]ghrelin(1−28) [Dpr3;K16(NODAGA(Ga))]ghrelin(1−28) [Dpr3;K16(NODAGA)]ghrelin(1−16) [Dpr3;K16(NODAGA(Ga))]ghrelin(1−16) Nα-NODAGA-KwFwLL-CONH2 Nα-NODAGA(Ga)-KwFwLL-CONH2

exact mass (g.mol‑1)

[M+H]+ found

purity (%)

yield (%)

elution (% ACN)

3726.0 3791.9 3726.2 3792.1 3725.0 3791.8 2365.3 2431.95 1247.67 1313.5

3727.0 3792.9 3727.2 3793.1 3726.0 3792.1 2366.3 2432.2 1248.7 1314.5

99 99 97 99 97 100 100 100 95 98

32 37 20 21 28 -

73 74 72 78 38 23 26 25 70 88

semipreparative HPLC to yield the following chelates: 1e, NαNODAGA(Ga)-ghrelin(1−28); 2e, [K16(NODAGA(Ga))]ghrelin(1−28); 3e, [Dpr 3 ;K 16 (NODAGA(Ga))]ghrelin (1−28); 4e, [Dpr3;K16(NODAGA(Ga))]ghrelin(1−16); 5d, Nα-NODAGA(Ga)-KwFwLL-CONH2. Identity and purity of the chelates 1−4e and 5d were confirmed by MALDI-ToF mass spectrometry and HPLC (see Table 1). TFA-HCl Exchange for Conjugates 2−4d and 5c for in Vivo Assays. Each conjugate (15 μmol) was dissolved in 4 mL of a 50 mM HCl solution in H2O and stirred at room temperature for 1 h. The conjugates were dried in a freeze−dryer and the procedure repeated two more times. Integrity and purity of the conjugates were controlled by MALDI-ToF mass spectrometry and HPLC. Signal Transduction Assay. Cell Culture. COS-7 cells (African green monkey, kidney) were cultured in DMEM containing 10% FCS (v/v), 100 units.mL−1 penicillin, and 100 μg.mL−1 streptomycin. Cells were grown as monolayers at 37 °C in a humidified atmosphere of 5% CO2. Measurement of Inositol Phosphate Accumulation. The functional assay to measure ghrelin receptor ligand potency was previously described in detail.14 Briefly, COS-7 cells were seeded into 24-well plates (8 × 105 cells/well) and transiently transfected with 0.3 μg of a vector GHS-R_EYFP_pVITRO2_mcs (containing hygromycin resistance), using 0.9 μL of Metafectene. At confluency, cells were then incubated with 4 μCi.mL−1 of myo-[3H] Inositol (25.0 Ci.mmol−1) about 16 h prior to stimulation. Cells were then washed once and stimulated in DMEM containing 10 mM LiCl and 0.1% BSA, in the absence or with increasing concentrations of ghrelin and chelates 1e−4e and 5d for 2 h at 37 °C. The stimulation was stopped by aspiration of medium and cell lysis with 0.1 M NaOH (150 μL/well). After adding 0.2 M formic acid (50 μL/ well) and sample dilution, intracellular inositol phosphate levels were determined by anion-exchange chromatography. Data were analyzed with GraphPad Prism 3.0 program (GraphPad Software, San Diego, USA). EC50 and Emax values were obtained from concentration−response curves. All signal transduction assays were performed in duplicate and repeated at least two times independently. Small Animal PET Assay. 68Ga-Production. 68Ga (T1/2 = 68 min, β+ = 89%, and EC = 11%) was available from two 68 Ge/68Ga-generator-systems (Cyclotron Co., Ltd., Obninsk, Russia (Eckert & Ziegler, Germany), iThemba, South Africa (IDB Holland bv, The Netherlands)) where 68Ge (T1/2 = 270.8 d) was attached to a column of an inorganic matrix based on titanium dioxide or tin oxide. The 68Ga was eluted with 1 M hydrochloric acid or water. The final HCl concentrations of the eluates of both generators were approximately 1 M

and the reaction repeated three more times. Subsequently, the peptides were successively washed five times with NMP, DMF, DCM, MeOH, and Et2O, and dried in vacuo to yield peptides 1−2c on solid support. The completion of the coupling reaction was monitored with a sample cleavage of the peptides from the resin. Octanoic Acid Coupling at Dpr3 of Peptides 3−4b. After Mtt cleavage, both peptides were swollen in 500 μL of DMF for 15 min. Octanoic acid (0.15 mmol, 10 equiv) and HOBt (0.15 mmol, 10 equiv) were dissolved in 200 μL DMF and the solution was added to the resin with DIC (0.15 mmol, 10 equiv). The reaction mixture was shaken for 4 h at room temperature. The peptides were then successively washed five times with DMF, DCM, MeOH, and Et2O, and dried in vacuo to yield peptides 3−4c on solid support. The completion of the coupling reaction was monitored with a Kaiser test. Cleavage from the Resin and Side-Chain Deprotection of Peptide 1−4c and 5b. Cleavage from the resin and side-chain deprotection were simultaneously achieved by incubating the resins for 3 h at room temperature with 1 mL of different cleavage mixtures: Peptides 1−4c were incubated with 90% of TFA, 5% of thioanisole, and 5% of thiocresol (v/v). Peptide 5c was incubated with 90% of TFA, 3% of thioanisole, and 7% of ethandithiol (v/v). After 3 h, filtration of the resins allows collection of the cleavage mixtures containing the peptides. Peptides were then precipitated in 10 mL of icecold diethyl ether for 20 min and collected by centrifugation. After removal of the supernatant, the precipitates were washed four times by resuspension in ice-cold ether and centrifugation to yield conjugates 1−4d and 5c as white powders. Identity of the following conjugates was confirmed by MALDI-ToF mass spectrometry (see Table 1): 1d, NαNODAGA-ghrelin(1−28); 2d, [K16(NODAGA)]ghrelin(1− 28); 3d, [Dpr 3 ;K 1 6 (NODAGA)]ghrelin(1−28); 4d, [Dpr3;K16(NODAGA)]ghrelin(1−16); 5c, Nα-NODAGAKwFwLL-CONH2. Purification of Conjugates 1−4d and 5c. All peptides were purified by preparative RP-HPLC using a linear gradient of 0.1% TFA in water (A) and 0.08% TFA in acetonitrile (B) at a flow rate of 10 mL.min−1 with different gradient systems. All conjugates showed a purity higher than 95% as determined by analytical RP-HPLC and identity of the conjugates was confirmed by MALDI-ToF mass spectrometry (see Table 1). Complexation of Conjugates 1−4d and 5c with Ga for in Vitro Assay. 0.5 μmol of each peptide was dissolved in 700 μL of an ammonium acetate solution (0.1 M in H2O). 300 μL of a Ga(NO3)3 solution (0.04 M in H2O) were added, and the pH adjusted to 4.5 with diluted HCl. The solutions were then incubated for 30 min at 37 °C and directly purified by 773



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hydrochloric acid. The pH of the fractionated [68Ga]-eluate (300 μL) was adjusted to pH 4.0−4.5 using 2 M NH4OAc. Radiolabeling. The solution of 68Ga(OAc)3 in acetate buffer was added to 20 nmol of each NODAGA-peptide 2−4d and 5c. The reaction mixture was shaken for 15 min at 37 °C to afford [68Ga]-labeled 2−4f and 5e in solution. The reaction mixture was analyzed by radio-HPLC. Analysis was performed on a Zorbax C18 300SB (250 × 9.4 mm; 4 μm) column with an eluent system C (water + 0.1% TFA) and D (acetonitrile +0.1% TFA) in a gradient 5 min 95% C, 10 min to 95% D, and 5 min at 95% D at a flow rate of 3 mL.min−1. Radiolabeled conjugates with radiochemical purity higher than 95% were used for radiopharmacological studies. Before formulation for the in vitro or in vivo application, the reaction mixture was filtrated (45 μm pore size, REZIST 13/0.45 PTFE, Schleicher & Schuell, Dassel, Germany). The filtrates were then diluted with electrolyte solution E-153 (140 mmol.L−1 Na+, 5 mmol.L−1 K+, 2.5 mmol.L−1 Ca2+, 1.5 mmol.L−1 Mg2+, 50 mmol.L−1 acetate, 103 mmol.L−1 Cl−, Serumwerk Bernburg, Germany) to reach concentration of about 80 MBq.mL mmol.L−1 and directly used for the radiopharmacological studies. Animals, Feeding, Husbandry, and Animal Preparation. The local animal research committee at the Landesdirektion Dresden approved the animal facilities and the experiments according to institutional guidelines and the German animal welfare regulations (permission number: 24−9168.21−4/ 2004−1 from 04/27/2004 valid up to 03/31/2014). The experimental procedure used conforms to the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS No. 123), to the Deutsches Tierschutzgesetz, and to the Guide for the Care and Use of Laboratory Animals published by the U. S. National Institutes of Health [DHEW Publication No. (NIH) 82−23, Revised 1996, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205]. Wistar rats (Wistar Unilever, HsdCpb:WU, Harlan Winkelmann, Borchen, Germany) were housed under standard conditions with free access to food (2016 Teklad Global 16% Protein Rodent Diet, Harlan Laboratories, Inc., Germany) and tap water. Rats were housed (5 rats per cage) in Uni Protect airflow cabinet (Ehret GmbH Labor and Pharmatechnik, Schönwalde, Germany) in polysulfone cages (380 × 200 × 590 mm3; floor area, 1815 cm2). The caging had a solid bottom and stainless steel wire lid. The cage floor was covered with chipped wood. The cages were changed weekly. Tap water was provided in polycarbonate bottles, changed twice weekly, and refilled once between the cage changes. The room temperature was 22 ± 1 °C and relative humidity was 53% ± 6%, but the temperature was 1−4 °C higher in the airflow cabinets. Artificial lighting was in 12 h light and dark cycles with fluorescent tubes (light color, warm white). Surgery. Male Wistar-Unilever rats weighing 183 ± 56 g (mean ± SD, n = 19) were anesthetized with desflurane. The guide value for breathing frequency was 65 breaths/min. Animals were put in the supine position and placed on a heating pad to maintain body temperature. The spontaneously breathing rats were treated with 100 units.kg−1 heparin (Heparin-Natrium 25.000-ratiopharm, Ratiopharm, Germany) by subcutaneous injection to prevent blood clotting on intravascular catheters. After local anesthesia by injection of Lignocaine 1% (Xylocitin loc, Mibe, Jena, Germany) into the right groin, catheter was introduced into the right femoral artery (0.8 mm Umbilical Vessel Catheter, Tyco Healthcare, Tullamore, Ireland) for blood samples for metabolite analysis,

for gas analysis, arterial blood pressure measurements and a second needle (35 G) catheter into one tail vein was used for administration of the 2−4f, 5e. Measurement of Original Compounds and Radioactive Metabolites. Metabolic stability studies were performed on male Wistar rats. Arterial blood samples were collected at 1, 3, 5, 10, 20, 30, and 60 min after injections and the activity (% ID.mL−1) was measured to give the arterial activity of 2−4f, 5e. Blood cells were separated by centrifugation (5 °C, 5 min, 8000 rpm), and plasma proteins were precipitated using 60% acetonitrile and subsequent centrifugation (5 °C, 5 min, 8000 rpm). The supernatant was analyzed by radio-HPLC. The radio-HPLC system (Agilent 1100 series) applied for metabolite analysis was equipped with UV detection (254 nm) and an external radiochemical detector (Ramona, Raytest GmbH, Straubenhardt, Germany). Analysis was performed on a Zorbax C18 300SB (250 × 9.4 mm; 4 μm) column with an eluent system C (water + 0.1% TFA) and D (acetonitrile + 0.1% TFA) in a gradient 5 min 95% C, 10 min to 95% D, and 5 min at 95% D at a flow rate of 3 mL.min−1. HPLC were performed on 2−4e, 5f added to a rat blood sample, and arterial blood samples from 1 to 60 min after injections and on urine sample from 60 min after injection. The metabolite fraction in blood was calculated as % of the parent compound in blood in HPLC. The extracts from other tissues were prepared by ultrasound homogenization as 10% solution in PBS, centrifugation (5 °C, 70 000 g × min) and subsequent precipitation by 60% acetonitrile. The supernatant was analyzed by the described radio-HPLC. Small Animal PET. Anesthetized, spontaneously breathing animals were allowed to stabilize for 10 min after preparation. A 10 min transmission scan was recorded during this time for each subject by using a rotating point source of 57Co. The transmission scan was used to correct the emission scan for γray attenuation caused by body tissues and supporting structures; it was also used to demarcate the body field for image registration. After the transmission scans, physiological data [mean arterial blood pressure (MAP), heart rate (HR), respiration rate (RR), and body temperature (T)] were measured and arterial blood samples were taken for pH, PaO2, PaCO2, hemoglobin (Hb), and hematocrit (Hct). The radioactivity of the injection solution was measured in a well counter (Isomed 2000, Dresden, Germany) cross-calibrated to the microPET P4 scanner (Siemens preclinical solutions, Knoxville, TN). The PET acquisition of 60 or 120 min emission scan was started and the infusion of the 68Ga-labeled compound was initiated with a delay of 30 s. 0.5 mL of [68Ga]-peptide solutions 2−4f and 5e, containing 90 ± 33 MBq was infused over 1 min (with a Harvard apparatus 44 syringe pump) into a tail vein. At the end of the experiment, the animals were deeply anesthetized and sacrificed by an intravenous injection of potassium chloride. Data Acquisition. Acquisition was performed in 3D list mode. Emission data were collected continuously. The list mode data were sorted into sinograms with 32 or 38 frames (15 × 10 s, 5 × 30 s, 5 × 60 s, 4 × 300 s, 3 × 600 s, or 9 × 600 s). The data were decay-, scatter-, and attenuation-corrected. The frames were reconstructed by Ordered Subset Expectation Maximization applied to 3D sinograms (OSEM3D) with 14 subsets, 15 OSEM3D iterations, 25 maximum a posteriori (MAP) iterations, and 1.8 mm resolution using the FastMAP algorithm (Siemens Preclinical Solutions, Knoxville, TN). The voxel size was 0.07 by 0.07 by 0.12 cm, and the resolution in the center of 774



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Scheme 1. Synthesis of Conjugate 1da

field of view was 1.8 mm. No correction for partial volume effects was applied. The image volume data were converted to Siemens ECAT7 format for further processing. The image files were then processed using the ROVER software (ABX GmbH, Radeberg, Germany). Masks for defining three-dimensional regions of interest (ROI) were set and the ROI’s were defined by thresholding and ROI time activity curves (TAC) were derived for the subsequent data analysis. The ROI data and TAC were further analyzed using R (R is available as Free Software under the terms of the Free Software Foundation’s GNU General Public License in source code form) and especially developed program packages (Jörg van den Hoff, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany). The standardized uptake values (SUV = (activity.mL−1 tissue)/ (injected activity/body weight), mL.g−1)) were calculated in the ROI. Biodistribution. The male Wistar rats (Wistar Unilever, HsdCpb:WU, Harlan Winkelmann GmbH, Borchen, Germany) were between 7 and 9 weeks of age. Two groups of four animals (mean body weight ± SD, 186 ± 11 g) for each time point were intravenously injected into a tail vein with approximately 0.3 MBq of the 0.5 mL of [68Ga]-tracer solutions 2−4f and 5e, Ga-68-labeled compound dissolved in 0.5 mL of electrolyte solution E-153. The specific activity ranged between 5 and 30 GBq.mmol−1 at the time of injection. Animals were sacrificed at 5 and 60 min post injection. Blood and the major organs were collected, weighed, and counted in a Wallac WIZARD automatic gamma counter (PerkinElmer, Germany). The radioactivity of the tissue samples was decay-corrected and calibrated by comparing the counts in tissue with the counts in aliquots of the injected tracer that had been measured in the gamma counter at the same time. The activity amount in the selected tissues and organs was expressed as percent of injected dose (% ID). The activity concentration in the biodistribution measurements were calculated as SUV (SUV = (activity/g tissue)/ (injected activity/body weight)) and expressed as means ± standard deviation (mean ± SD) for each group of four animals. Statistical Analysis. Values are expressed as mean ± SD or median and interquartile range (IQR). IQR is defined as the range of the values extending from the 25th percentile to the 75th percentile. Values were compared using an unpaired Student’s t-test with Welch’s correction and an F-test to compare the variances (GraphPad Prism 5.02 for Windows, GraphPad Software, San Diego, CA). The nonparametric Wilcoxon signed rank test and the D’Agostino-Pearson normality test were used for statistical evaluation for some of the data. A P value of 1000 0.72 ± 0.06 1.91 ± 0.40 1.41 ± 0.60 643.2 ± 9.1

1 >1000 1.3 3.5 2.6 n.c.*

112 ± 25 n.d. 122 ± 7 111 ± 11 150 ± 8 91 ± 1

4 3 3 3 3 2

agonist agonist agonist agonist agonist inverse agonist

EC50 values (mean ± SD) were obtained from concentration−response curves and showed the potency of the chelates. Efficacy values (ΔEfficacy) are the mean ± SD of |Efficacymax − Efficacymin|. *n.c.: noncalculated. a

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Figure 2. Biodistribution profiles of tracers 2−4f and 5e in male Wistar rats. Activity amount in selected organs were measured at 5 and 60 min after injection of radiotracers 2−4f and 5e. Values are given as mean ± SD for each group of four animals. Biodistribution profiles are expressed as percent of the injected dose (%ID) for 2f (A), 3f (C), 4f (E), and 5e (G). Biodistribution profiles are expressed as standard uptake values (SUV) for 2f (B), 3f (D), 4f (F) and 5e (H).

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However, the absolute retention of compound 5e in blood is higher than of 2−4f. The mean plasma retention times were consistent with the metabolic studies. The typical pattern of high blood activity of 5e is also visible in the PET image (Figure 5). The heart and the large vessels (jugular vein, vena cava) are clearly visible at midframe time of 5 min. At this time, the liver is prominent in the animal injected with 3f.

DISCUSSION

In this study, we developed both agonist and inverse agonist probes targeting the ghrelin receptor. Indeed, suitable probes with opposed behavior would be highly valuable for studying the hormone signalization in vivo. Radiotracers could be of major interest as diagnostic tools in hormonal dysfunctions and for further development of therapeutic peptides for diseases 779



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Table 4. Elimination Pathways: Comparison of the Renal Metabolism (Kidney) toward the Hepatobiliary Metabolism (Liver + Intestine) for Radiotracer 2−4f and 5ea renal metabolism

hepathobiliary metabolism

kidney %ID

5 min 26.6 33.2 27.7 4.4

2f 3f 4f 5e a

± ± ± ±

liver 60 min

11.8 2.2 2.9 0.2

63.3 65.3 52.3 4.4

± ± ± ±

intestine

5 min

25.9 2.2 5.9 0.6

11.1 22.6 6.2 11.6

± ± ± ±

60 min

0.9 1.6 1.3 1.4

8.1 22.3 3.4 9.4

± ± ± ±

5 min

0.8 2.4 0.2 0.7

3.2 2.3 2.8 9.1

± ± ± ±

60 min

0.3 0.2 0. 6 2.0

0.9 1.5 1.7 10.7

± ± ± ±

0.1 0.4 0.5 2.1

Values are given as mean ± SD for each group of four animals and are as expressed as percent of the injected dose (%ID).

Table 5. Concentrations of the Radiotracers 2−4f and 5e in Blood, Kidney, Liver, and Brain of Male Wistar Rat at 5 and 60 min after the Injectiona blood (mean ± SD) SUV 2f 3f 4f 5e

5 min 1.82 1.19 1.30 3.78

± ± ± ±

0.19 0.12 0.23 0.43

kidney (mean ± SD)

60 min 0.18 0.15 0.26 1.53

± ± ± ±

0.04 0.05 0.09 0.25

5 min 36.58 37.02 30.93 4.88

± ± ± ±

4.02 3.95 3.15 0.48

liver (mean ± SD)

60 min 85.70 70.39 56.31 5.09

± ± ± ±

6.2 6.51 7.99 0.58

5 min 2.13 4.77 1.36 1.96

± ± ± ±

0.11 0.52 0.34 0.33

brain (mean ± SD)

60 min 1.53 5.31 0.83 1.75

± ± ± ±

0.15 0.45 0.12 0.43

5 min 0.06 0.04 0.05 0.11

± ± ± ±

0.01 0.00 0.01 0.02

60 min 0.01 0.01 0.01 0.05

± ± ± ±

0.00 0.00 0.00 0.01

a Values are given as mean ± SD for each group of four animals and are as expressed in standard uptake value SUV = (activity.g−1 tissue)/(injected activity/body weight).

internalization and thus increase receptor concentration on membranes. Moreover, the hexapeptide is a current lead for development of antiobesity peptides, and in vivo data for 5e could guide further SAR. In vivo studies highlighted a very distinct behavior between agonists 2−4f and the inverse agonist 5e. Agonist tracers 2f, 3f, and 4f were mainly found in the kidney, and the high increase in concentration reflected an accumulation in this organ over time. The renal metabolism and the hepatobiliary metabolism were also compared. Although the renal metabolism was predominant, accumulation in the kidney is a high bias to measure the renal elimination and longer biodistribution studies (i.e., with radionuclide having longer half-life) need to be performed to get an exact picture of metabolism pathways. However, agonists 2f and 4f presented a low hepatobiliary metabolism, whereas accumulation of 3f could be observed in the liver. On the other hand, the inverse agonist 5e was mainly excreted via the hepatobiliary system, and accumulation was visible neither in the kidney nor in the liver. In addition, very low doses of agonists 2−4f were found in blood and in all other tissues, whereas inverse agonist 5e presented a significant perfusion in all tissues, and especially a high concentration in blood even after 1 h. Metabolic stability studies gave us extremely valuable insight for further development of radiotracers. Compounds 2−4f all presented a fast blood clearance but showed differences in stability. The N-octanoylated ghrelin analogue 3f appeared stable within 5 min after injection, but was degraded after 1 h. The O-octanoylated ghrelin tracer 2f showed a spontaneous conversion into a main metabolite, detected before injection in a low proportion and increasing in blood over the time. This metabolite is probably the desoctanoylated form of tracer 2f as desoctanoylation of ghrelin occurs very fast under physiological conditions. Interestingly, 4f was completely metabolized after 5 min and a unique metabolite was found in blood and urine. 4f is a N-octanoylated and C-terminally truncated ghrelin analogue. The C-terminal (17−28) segment, although not essential for ghrelin receptor recognition, appeared to stabilize

associated with ghrelin dysregulation like obesity, cachexia, or anorexia nervosa. The probes consist of a ghrelin agonist or inverse agonist peptides bound to the bifunctional chelator NODAGA and further labeled with the positron emitting radionuclide 68Ga to allow in vivo imaging of the radiolabeled peptides. NODAGA is derived from the NOTA ring (1,4,7triazacyclononane-1,4,7-triacetic acid), and the protected precursor NODAGA(tBu)3 was suitable for solid-phase peptide synthesis. Peptide synthesis and coupling of NODAGA were straightforward, as they could both be achieved on solid support. Complexation of cold gallium for in vitro studies was easily achieved under mild conditions and supported the existing reports on NODAGA chemistry and stability.17,18 Moreover, all [Ga]NODAGA−peptide complexes were stable and could easily be purified. Potency of the chelates was evaluated at the ghrelin receptor by an in vitro inositol phosphate turnover assay, and results were consistent with previous ghrelin SAR studies.19 Tracers 1−4f are agonists derived from the ghrelin motif. The agonist activity was maintained when the bifunctional chelator was introduced at position 16 of the ghrelin sequence (2−4e), whereas modification at the N-terminus was not tolerated (1e). Introduction of an amide bond instead of the native ester bond at position 3 (3e) or truncation of the C-terminal segment (17−28) (4e) were both tolerated and did not affect the agonist activity at the receptor. Compound 5e is an inverse agonist tracer derived from the short inverse agonist KwFwLLCONH2, developed in our group.20 Previous SAR showed that Lys1 was essential for the inverse agonist activity of the hexapeptide. Hence, modification at Lys1 side chain was excluded. Introduction of NODAGA was restricted to the peptide Nterminus and led to an important loss of activity compared with the native peptide (KwFwLL-CONH2). Nevertheless, 5e was included in the in vivo studies to gain information on the peptide pharmacokinetic and nonspecific biodistribution of the native peptide. Indeed, the development of an inverse agonist tracer could be very interesting for a dynamic receptor mapping, as inverse agonists are less likely to mediate receptor 780



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Figure 3. Blood clearance, metabolite fractions after injection of tracers 2−4f and 5e in rats and HPLC analysis. (A) Blood clearance was measured for the total radioactivity related to each tracer. (B) Blood clearance of original compounds and (C) metabolite fractions corresponding to each tracer were calculated according to HPLC analysis. (D) Chromatogram of tracer 2f. (E) Chromatogram of tracer 3f. (F) Chromatogram of tracer 4f. (G) Chromatogram of tracer 5e. For each chromatogram, pick intensities are normalized over the pick intensity of the major compound and the baselines are shifted to avoid overlapping.

the peptide sequence. Indeed, a recent study showed that Cterminal truncation of (8−28) and (21−28) segments dramatically decreased ghrelin half-life in rats.21 It was also reported that ghrelin interaction with plasma lipoprotein via both its N- and C-terminus may confer protection against esterases.22 Consequently, C-terminal truncation is not suitable for the development of stable ghrelin agonist motifs. On the other hand, 5e exhibited a slow blood clearance and appeared impressively stable in blood and urine after 60 min. Indeed, the peptidomimetic structure of 5e with two D-amino acids and an amidated C-terminus certainly protects the tracer from peptidases. Moreover, the peptide hydrophobicity, normally correlated with an elevated binding to plasma protein, can be the reason for a slow blood clearance.

The differences in peptide structures and metabolic stability can explain the divergence in biodistribution and metabolism. Indeed, peptides 2−4f are hydrophilic biomolecules, structurally derived from the endogenous ghrelin. They present a molecular weight of, respectively, 3792, 3791, and 2431 Da and a high charge ratio. Thus, the three tracers 2−4f present high renal elimination and poor tissue perfusion, characteristic for hydrophilic macromolecules. In addition, 3f is more stable than 2f and 4f toward metabolism. This can explain the higher hepatobiliary elimination of the original compound 3f, whereas renal elimination is favored for metabolites of 2f and 4f. It is important to mention that the renal accumulation of the tracers 2−4f could also be attributed to the bifunctional chelator (BFC) increasing the charge ratio of the peptides. Indeed, renal 781



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as tracer 5e in the present study and other NOTA or NODAGA based tracers in earlier reports did not display any renal accumulation.18,24 Nevertheless, a high renal uptake can lead to renal toxicity, and is a serious drawback for PET imaging. Hence, other bifunctional chelator and different agonist moieties should be investigated in the future to overcome this issue. On the other hand, the inverse agonist tracer 5e is a short peptidomimetic (MW = 1315 Da). It presents hydrophobic properties that can directly be correlated with (a) a favored hepatobiliary elimination; (b) a high perfusion in tissues due to unspecific and passive membrane diffusion; and (c) a high concentration in blood due to a high affinity for plasma protein. Importantly, none of the tracers were detected in the brain. The concentration gradient between the blood plasma and the brain tissue seems too small to transfer significant amounts of radiotracers through the blood-brain barrier (BBB) for all tracers 2−4f and 5e. As a consequence, we could not detect any active transport into the brain at the investigated tracer concentrations. However, ghrelin response is thought to be mediated via the arcuate nucleus of hypothalamus where ghrelin receptors are mainly expressed.5,25,26 Interestingly, a previous study reported the accumulation in hypothalamus of a [11C]quinazolinone ligand of the ghrelin receptor, at tracer concentration.27 Low concentrations of the tracer could also be detected in the brain region with low ghrelin receptor density (cortex, cerebellum, and hippocampus). Therefore, in the current study, the lack of tracers 2−4f in the brain may be due to their hydrophilic nature or an excessively high charge ratio. In addition, the higher hydrophobicity and lower charge ratio of 5e made it more likely to cross the BBB although no trace of the tracer could be detected in the brain. Accordingly, the moderate in vitro activity of 5e could limit the tracer detection in the brain if the uptake was specific. Importantly, as the NODAGA motif highly modifies the physicochemical properties of the compounds, no conclusion can be drawn concerning the blood-brain barrier passage of the native peptide KwFwLL-CONH2. Therefore, extensive SAR should be conducted on 5e to identify a radiotracer with higher binding affinity at the receptor. To conclude, the in vitro and in vivo evaluation of four [68Ga]Ga-NODAGA-peptide conjugates provides an interesting insight for the development of imaging probes and peptidebased therapeutics targeting the ghrelin receptor. On one hand, despite a very good affinity for the receptor, ghrelin derived agonists 2−4f presented very high kidney accumulation, negligible tissue distribution, fast blood clearance, and poor stability in blood. Consequently, the ghrelin motif does not

accumulation of BFC-functionalized peptides has often been reported.23 However, it cannot be considered an intrinsic property of the NOTA motif (1,4,7-triazacyclononane-1,4,7-triacetic acid),

Figure 4. Radioactive metabolites in rat liver 5 min after injection of 3f. For each chromatogram, pick intensities are normalized over the pick intensity of the major compound and the baseline are shifted to avoid overlapping.

Figure 5. Maximum intensity projection of PET studies of Ga68 labeled compounds 2−4f and 5e at 5 min midframe time (duration of measurement 6 min). The images were scaled to the maximum activity (Bq.cm−3) in each image.

Figure 6. Time−activity curves of 68Ga-labeled compounds 2f−4f and 5e for three regions of interest (ROI) in rats. (A) kidneys, (B) liver, and (C) heart. Values are given as mean ± SD for each group of four animals and are expressed in standard uptake values (SUV). 782



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receptor in pituitary and hypothalamus that functions in growth hormone release. Science 273 (5277), 974−7. (4) Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., and Kangawa, K. (1999) Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402 (6762), 656−60. (5) Ferrini, F., Salio, C., Lossi, L., and Merighi, A. (2009) Ghrelin in central neurons. Curr. Neuropharmacol. 7 (1), 37−49. (6) Kojima, M., and Kangawa, K. (2005) Ghrelin: structure and function. Physiol. Rev. 85 (2), 495−522. (7) Gutierrez, J. A., Solenberg, P. J., Perkins, D. R., Willency, J. A., Knierman, M. D., Jin, Z., Witcher, D. R., Luo, S., Onyia, J. E., and Hale, J. E. (2008) Ghrelin octanoylation mediated by an orphan lipid transferase. Proc. Natl. Acad. Sci. U. S. A. 105 (17), 6320−5. (8) Yang, J., Brown, M. S., Liang, G., Grishin, N. V., and Goldstein, J. L. (2008) Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell 132 (3), 387−96. (9) Cummings, D. E., Weigle, D. S., Frayo, R. S., Breen, P. A., Ma, M. K., Dellinger, E. P., and Purnell, J. Q. (2002) Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N. Engl. J. Med. 346 (21), 1623−30. (10) Holst, B., and Schwartz, T. W. (2006) Ghrelin receptor mutations--too little height and too much hunger. J. Clin. Invest. 116 (3), 637−41. (11) Leite-Moreira, A. F., and Soares, J. B. (2007) Physiological, pathological and potential therapeutic roles of ghrelin. Drug Discovery Today 12 (7−8), 276−88. (12) Chollet, C., Meyer, K., and Beck-Sickinger, A. G. (2009) Ghrelin--a novel generation of anti-obesity drug: design, pharmacomodulation and biological activity of ghrelin analogues. J. Pept. Sci. 15 (11), 711−30. (13) Depoortere, I. (2009) Targeting the ghrelin receptor to regulate food intake. Regul. Pept. 156 (1−3), 13−23. (14) Els, S., Beck-Sickinger, A. G., and Chollet, C. (2010) Ghrelin receptor: high constitutive activity and methods for developing inverse agonists. Methods Enzymol. 485, 103−21. (15) Eisenwiener, K. P., Prata, M. I., Buschmann, I., Zhang, H. W., Santos, A. C., Wenger, S., Reubi, J. C., and Macke, H. R. (2002) NODAGATOC, a new chelator-coupled somatostatin analogue labeled with [67/68Ga] and [111In] for SPECT, PET, and targeted therapeutic applications of somatostatin receptor (hsst2) expressing tumors. Bioconjugate Chem. 13 (3), 530−41. (16) Velikyan, I., Maecke, H., and Langstrom, B. (2008) Convenient preparation of 68Ga-based PET-radiopharmaceuticals at room temperature. Bioconjugate Chem. 19 (2), 569−73. (17) de Sa, A., Matias, A. A., Prata, M. I., Geraldes, C. F., Ferreira, P. M., and Andre, J. P. (2010) Gallium labeled NOTA-based conjugates for peptide receptor-mediated medical imaging. Bioorg. Med. Chem. Lett. 20 (24), 7345−8. (18) Knetsch, P. A., Petrik, M., Griessinger, C. M., Rangger, C., Fani, M., Kesenheimer, C., von Guggenberg, E., Pichler, B. J., Virgolini, I., Decristoforo, C., and Haubner, R. (2011) [(68)Ga]NODAGA-RGD for imaging alpha(v)beta (3) integrin expression. Eur. J. Nucl. Med. Mol. Imaging 38 (7), 1303−12. (19) Bednarek, M. A., Feighner, S. D., Pong, S. S., McKee, K. K., Hreniuk, D. L., Silva, M. V., Warren, V. A., Howard, A. D., Van Der Ploeg, L. H., and Heck, J. V. (2000) Structure-function studies on the new growth hormone-releasing peptide, ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a. J. Med. Chem. 43 (23), 4370−6. (20) Holst, B., Lang, M., Brandt, E., Bach, A., Howard, A., Frimurer, T. M., Beck-Sickinger, A., and Schwartz, T. W. (2006) Ghrelin receptor inverse agonists: identification of an active peptide core and its interaction epitopes on the receptor. Mol. Pharmacol. 70 (3), 936− 46. (21) Morozumi, N., Hanada, T., Habara, H., Yamaki, A., Furuya, M., Nakatsuka, T., Inomata, N., Minamitake, Y., Ohsuye, K., and Kangawa, K. (2011) The role of C-terminal part of ghrelin in pharmacokinetic profile and biological activity in rats. Peptides 32 (5), 1001−7.

appear suitable to develop radiotracer for imaging. Moreover, the ghrelin motif as such is not an appropriate lead for drug development. However, we demonstrated that structural modifications on the ghrelin sequence influence the peptide behavior in vivo. Indeed, the N-octanoylated ghrelin derivatives were more stable than the O-octanoylated ghrelin derivatives toward metabolism in blood, and the C-terminal (17−28) segment appeared essential for ghrelin metabolic stability. On the other hand, tracer 5e exhibited very high stability in blood, large diffusion in tissues, reasonable kidney and liver metabolism without accumulation, and slow blood clearance. Consequently, the unspecific diffusion of the inverse agonist limits its use as a radiotracer for understanding ghrelin receptor signaling. Nevertheless, the pharmacokinetic profile of 5e is excellent for further development of druggable peptides targeting the ghrelin receptor. Hence, the ghrelin inverse agonist sequence KwFwLL-CONH2 emerges as a promising lead to design peptidebased therapeutics against obesity.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49 341 97 36904. Fax: +49 341 97 36909. E-mail: [email protected]. Notes

The authors declare no competing financial interest. If not specified, amino acids are L and written in the standard one or three letter codes.

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ACKNOWLEDGMENTS This project was financially supported by the EU (Gastrointestinal Peptides in Obesity, GIPIO, Grant Agreement Number 223057) and the Alexander von Humboldt foundation. Regina ReppichSacher and Kristin Löbner are kindly acknowledged for technical assistance during the synthesis and in vitro experiments. The authors also thank Regina Herrlich, Andrea Suhr, Natalie Thieme, Mareike Barth, and Catharina Heinig for contributing to the in vivo experiments.

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ABBREVIATIONS BBB, blood-brain barrier; BFC, bifunctional chelator; Boc, tertbutyloxycarbonyl; Dde, 1-(4,4-dimethyl-2,6-dioxocyclohex1-ylidene)ethyl; Dpr, diaminopropionic acid; Fmoc, 9-fluorenylmethyloxycarbonyl; Mtt, 4-methyltrityl; NODAGA(tBu)3, 4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazacyclononan1-yl)-5-(tert-butoxy)-5-oxopentanoic acid; NOTA, 1,4,7-triazacyclononane-1,4,7-triacetic acid; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl; Pmc, 2,2,5,7,8-pentamethylchroman; SAR, structure−activity relashionship; tBu, tert-butyl; Trt, trityl

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REFERENCES

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(22) De Vriese, C., Hacquebard, M., Gregoire, F., Carpentier, Y., and Delporte, C. (2007) Ghrelin interacts with human plasma lipoproteins. Endocrinology 148 (5), 2355−62. (23) Tornesello, A. L., Aurilio, M., Accardo, A., Tarallo, L., Barbieri, A., Arra, C., Tesauro, D., Morelli, G., and Aloj, L. (2011) Gastrin and cholecystokinin peptide-based radiopharmaceuticals: an in vivo and in vitro comparison. J. Pept. Sci. 17 (5), 405−12. (24) Fani, M., Del Pozzo, L., Abiraj, K., Mansi, R., Tamma, M. L., Cescato, R., Waser, B., Weber, W. A., Reubi, J. C., and Maecke, H. R. (2011) PET of Somatostatin Receptor-Positive Tumors Using 64Cuand 68Ga-Somatostatin Antagonists: The Chelate Makes the Difference. J. Nucl. Med. 52 (7), 1110−8. (25) Gnanapavan, S., Kola, B., Bustin, S. A., Morris, D. G., McGee, P., Fairclough, P., Bhattacharya, S., Carpenter, R., Grossman, A. B., and Korbonits, M. (2002) The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J. Clin. Endocrinol. Metab. No. 6, 2988. (26) Zigman, J. M., Jones, J. E., Lee, C. E., Saper, C. B., and Elmquist, J. K. (2006) Expression of ghrelin receptor mRNA in the rat and the mouse brain. J. Comp. Neurol. 494 (3), 528−48. (27) Potter, R., Horti, A. G., Ravert, H. T., Holt, D. P., Finley, P., Scheffel, U., Dannals, R. F., and Wahl, R. L. (2011) Synthesis and in vivo evaluation of (S)-6-(4-fluorophenoxy)-3-((1-[11C]methylpiperidin-3-yl)methyl)-2-o-tolylquinazolin-4(3H)-one, a potential PET tracer for growth hormone secretagogue receptor (GHSR). Bioorg. Med. Chem. 19 (7), 2368−72.

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