Novel Chemically Modified Analogues of Neuropeptide Y for Tumor

Abbreviations used: NPY, neuropeptide Y; Ga, gallium metal; CF, 5(6)-carboxyfluorescein; GPCRs, G-protein coupled receptors; BFC, bifunctional chelato...
0 downloads 0 Views 404KB Size
Download PDF
1430

Bioconjugate Chem. 2008, 19, 1430–1438

Novel Chemically Modified Analogues of Neuropeptide Y for Tumor Targeting§ Denise Zwanziger,+,† Irfan Ullah Khan,+,† Ines Neundorf,† Stephanie Sieger,‡ Lutz Lehmann,‡ Matthias Friebe,‡ Ludger Dinkelborg,‡ and Annette G. Beck-Sickinger*,† Institute of Biochemistry, Leipzig University, Bru¨derstr. 34, 04103 Leipzig, Germany, and Bayer Schering Pharma, Global Drug Discovery, Berlin, Germany. Received November 21, 2007; Revised Manuscript Received April 20, 2008

The successful use of peptides as potential radiopharmaceuticals essentially requires the modification of the bioactive peptide hormones to introduce chelators for radiolabeling. In this study, four Y1/Y2 receptor-selective NPY analogues with different receptor subtype specificities have been investigated. For in Vitro studies, the cold metal surrogate was used. Gallium and indium complexes were introduced by using 1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid as bifunctional chelator. The peptides were synthesized by solid-phase peptide synthesis (SPPS), the chelator was coupled either at the N-terminus or at the Nε side chain of Lys4 of the resin-bound peptide, and the labeling was performed in solution after cleavage. Competitive binding assays showed high binding affinity of the receptor-selective analogues at NPY receptor expressing cells. To test internalization of the novel peptide analogues and the metabolic stability in human blood plasma, the corresponding 5(6)-carboxyfluorescein (CF) analogues were prepared and investigated. One of the most promising analogues, the Y1-receptor selective [Lys(DOTA)4, Phe7, Pro34]NPY was labeled with 111In and injected into nude mice that bear MCF-7 breast cancer xenografts, and biodistribution studies were performed. In Vitro and in ViVo studies suggest that receptor-selective analogues of NPY have promising characteristics for future applications in nuclear medicine for breast tumor diagnosis and therapy.

INTRODUCTION The use of radiolabeled receptor-mediated peptide analogues for the treatment of primary and metastatic tumors, namely, targeted radionuclide therapy, has been an important alternative to conventional therapeutic regimens. Receptor-avid peptides have been chosen as vehicles to selectively deliver cytotoxic radiation-emitting radionuclides to tumor cells, resulting in tumor cell death (1). Neuropeptide Y (NPY)1 is a 36 amino acid peptide amide of the pancreatic polypeptide family. It is expressed in the peripheral and central nervous system and is one of the most abundant neuropeptides in the brain (2). NPY acts peripherally as a vasoconstrictor and modulates the activity of further neurotransmitters. Several other physiological activities such as induction of food intake, inhibition of anxiety, increase in memory retention, etc., have also been attributed to NPY (3). Its receptors are produced in a number of neuroblastoma and cell lines derived thereof, which suggest them as optimal targets for tumor scintigraphy. So far, five receptor subtypes (Y1, Y2, Y4, Y5, y6) have been cloned that bind NPY with nanomolar affinity (4). They all belong to the large family of G-proteincoupled receptors (GPCRs) with their typical seven transmembrane helix structure (5). Previous studies showed that there is a molecular basis for a putative role of NPY in tumors, e.g., neuroendocrine tumors, glioblastomas, and breast cancers (6–9). The strong predomi§ Dedicated to Professor Dr. Hans-Dieter Jakubke on the occasion of his 75th birthday. * Correspondence to: Tel. +49-341-9736901; fax +49-341-9736909; Leipzig University, Bru¨derstr. 34, 04103 Leipzig; E-mail: [email protected]. + Contributed equally. † Leipzig University. ‡ Global Drug Discovery.

nance of Y1 receptors in breast carcinomas compared to Y2 in normal breast tissue suggests that neoplastic transformation can switch the NPY receptor expression from the Y2 to the Y1 receptor subtype. The high incidence of Y1 in in situ, invasive, and metastatic breast cancers therefore allows the possibility to target them for diagnosis and therapy with NPY Y1 receptorselective analogues by using an appropriate metalloradiopharmaceutical that contains, e.g., a metallic radionuclide (e.g., 67Cu, 90 Y, 99mTc, 111In, 177Lu, 186Re, or 212Bi). Additionally, the targeting biomolecule requires a bifunctional chelator (BFC), either directly or separated by a spacer molecule to coordinate the metallic radionuclide.

1 Abbreviations used: NPY, neuropeptide Y; Ga, gallium metal; CF, 5(6)-carboxyfluorescein; GPCRs, G-protein coupled receptors; BFC, bifunctional chelator; His, histidine; Phe, phenylalanine; Lys, lysine; Pro, proline; Asp, aspartic acid; Glu, glumatic acid; Ser, serine; Thr, threonine; Asn, asparagine; Gln, glutamine; Arg, arginine; In, indium; Y, yttrium; DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; SPPS, solid phase peptide synthesis; Boc, tert-butyl-oxycarbonyl; Pmc, pentamethylchroman-6-sulfonyl; Dde, 4,4-dimethyl-2, 6-dioxocyclohex-1-ylidene-ethyl; DMF, N,N-dimethylformamide; DCM, dichloromethane; MeOH, methanol; Et2O, diethyl ether; HOBt, hydroxybenzotriazole; DIC, N,N′-diisopropylcarbodiimide; DIPEA, N,N-diisopropylethylamine; ACN, acetonitrile; MEM, minimum essential medium; DMEM, Dulbecco’s Modified Eagle’s Medium; FCS, fetal calf serum; BSA, bovine serum albumin; EDTA, ethylenediaminetetraacetic acid; MALDI-ToF, matrix-assisted laser desorption ionization-timeof-flight; HPLC, high performance liquid chromatography; TFA, trifluoroacetic acid; NSB, nonspecific binding; Ga(NO3)3, gallium nitrate; InCl3, indium chloride; 111InCl3, 111Indium chloride; PRRT, peptide receptor radiation therapy; PBS, phosphate buffered saline; Yy ) NPY receptor subtype Y (y ) 1, 2, or 5); PET, positron emission tomography; %ID/g, percent of injected dose per gram; HCl, hydrochloric acid; Bq, bequerel, CLSM, confocal laser scanning microscopy; BHK, baby hamster kidney.

10.1021/bc7004297 CCC: $40.75  2008 American Chemical Society Published on Web 06/24/2008

Chemically Modified Analogues of NPY for Tumor Targeting

We focused on the development of gallium- and indiumlabeled NPY receptor-selective analogues chelated by DOTA, since this chelator is capable of forming stable complexes with various 3+-charged radiometals, as demonstrated by the successful development of Ga-labeled somatostatin analogues (10). The effect on receptor binding properties was studied by labeling the DOTA-NPY analogues with nonradioactive Ga and In. All peptides were tested for receptor binding affinity at various NPY receptor expressing cells, e.g., human neuroepithelioma (SKN-MC), human breast adenocarcinoma (MCF-7), human neuroblastoma (SMS-KAN), and human endometrial carcinoma (HEC) cells. The SK-N-MC and MCF-7 cells selectively express NPY Y1 receptors, while SMS-KAN cells selectively express NPY Y2 receptors and HEC-1B-Y5 cells were stable transfected with Y5 receptors (11). All cell lines have frequently been used to investigate binding as well as signal transduction of NPY (12). Here, we report on the influence of the linker chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and conjugates of four different NPY analogues with respect to ligand binding. Interestingly, the position of the labeling of linker was of significant interest regarding the binding to the receptor. Moreover, we present the first series biodistribution data of one of the most potent Y1 receptor selective NPY analogues [Lys(DOTA)4, Phe7, Pro34]NPY. The peptide was successfully labeled with 111In and accordingly confirms the proof of the principle. The in ViVo characteristic of the peptide was studied in nude mice that bear MCF-7 breast cancer xenografts.

EXPERIMENTAL PROCEDURES Materials. The NR-Fmoc-protected amino acids were obtained from Alexis (La¨ufelfingen, Switzerland) and Novabiochem (La¨ufelfingen, Switzerland). The side chain protecting groups were tert-butyl for Asp, Glu, Ser, Thr, and Tyr; tertbutyloxycarbonyl (Boc) for Lys; trityl for Asn, Gln, and His; and 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) for Arg. The 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy (Rink Amide) resin was obtained from Novabiochem. N,N-Dimethylformamide, dichloromethane, methanol, and diethyl ether were purchased from Scharlau (La Jota, Barcelona, Spain). Physiological sodium chloride was obtained from Braun (Melsungen, Germany). 5(6)-Carboxyfluorescein (CF), 1-hydroxybenzotriazole (HOBt), thioanisole, p-thiocresol, piperidine, diisopropylethylamine, hydrazine monohydrate, and tert-butanol were purchased from Fluka (Buchs, Switzerland). N,N′-Diisopropylcarbodiimide (DIC) was purchased from Aldrich (Buchs, Switzerland). Acetonitrile (ACN) was obtained from Romil (Cambridge, England) and Merck (Darmstadt, Germany). Trifluoroacetic acid was obtained from Fluka (Buchs, Switzerland) and Merck. Ethanol (70%), water for chromatography, sodium acetate buffer, and saline solution were purchased from Merck. Dulbecco’s MEM/NutMix F12 medium (50:50, v/v), Minimum Essential Medium (MEM) with Earl’s salts, Dulbecco’s PBS, sodium pyruvate, phosphate buffered saline (NaCl/Pi), fetal calf serum, glutamine, nonessential amino acids, and trypan blue were purchased from Gibco (Life Technologies, Basel, Switzerland). Trypsin-EDTA was obtained from Gibco (Life Technologies, Basel, Switzerland) and Biochrom (Berlin, Germany). Isofluran Curamed was purchased from Cura MED Pharma (Karlsruhe, Germany) and MatrigelTM Matrix was obtained from Becton & Dickinson (Heidelberg, Germany). Bacitracin and BSA were from Sigma (Buchs, Switzerland), while EDTA, Pefabloc SC, and all other chemicals were from Fluka. [3H]propionyl-NPY and 111InCl3 were obtained from Amersham (Buckinghamshire, United Kingdom). Automatic Peptide Synthesis. The peptides were synthesized by fluorenylmethoxycarbonyl/tert-butyl (Fmoc/t-Bu) strategy

Bioconjugate Chem., Vol. 19, No. 7, 2008 1431

with an automated multiple peptide synthesis robot system (Syro, MultiSynTech, Bochum, Germany) using Rink amide resin (30 mg, 450 µmol). Each amino acid was coupled by a double coupling procedure (two times coupling with 40 min incubation) using a 10-fold excess of NR-Fmoc-protected amino acids and in situ activation with DIC and 500 µL HOBt (0.5 M in DMF) within 15 min incubation time at room temperature (13). The removal of the Fmoc-group was carried out with 40% piperidine in DMF (13). Cleavage of the peptide from the resin and of the side-chain protecting groups was accomplished in one step with 90% trifluoroacetic acid (TFA) in the presence of 10% scavengers (thioanisole/thiocresol 1:1) for 3 h. The peptides were precipitated from ice-cold diethyl ether and collected by centrifugation, resuspended in ether, and centrifuged again. This procedure was repeated four times. The identification of the products was confirmed by matrix-assisted LASER desorption ionization-time-of-flight (MALDI-ToF) mass spectrometry. The purification of the peptides was performed by a preparative RP-HPLC on a Shimadzu RP18-column (12.5 × 250 mm; 5 µm/300 Å). The pure products were characterized by analytical RP-HPLC on a Vydac RP18-column (4.6 × 250 mm; 5 µm/ 300 Å, Merck Hitachi, Darmstadt, Germany) by using a linear gradient of 0.1% TFA in water (A) and 0.08% TFA in acetonitrile (B) from 10% to 60% B in A over 30 min at a flow rate of 0.6 mL/min-1. The following peptides were synthesized: NPY, [Phe7, Pro34]NPY, [Ahx5-24]NPY, and [Ahx8-20]NPY. The analytical data of the peptides are summarized in Table 1. For the modification of the Nε side chain of Lys4, this residue was protected with 4,4-dimethyl-2,6-dioxocyclohex-1-ylideneethyl (Dde group) by using Fmoc-Lys(Dde), whereas the N-terminus was protected by the Boc-protecting group. After complete synthesis of the peptide sequence, the Dde protecting group was selectively removed from the resin by repeated washing steps with 2% hydrazine in DMF (14) followed by a 10 min incubation time at room temperature. The peptide remained still bound to the resin and all acid labile protecting groups remain fully protected under these conditions. After this, Lys (4) was labeled with the respective group (chelator or 5(6)carboxyfluorescein), followed by the cleavage of the labeled peptide from the resin as described above. Labeling with 5(6)-Carboxyfluorescein (CF). Prior to cleavage of the peptides, the peptides were labeled with 5(6)-carboxyfluorescein, following the procedure described by Weber et al. (15), in which a 10-fold excess of the CF group was used. HOBt and DIC were dissolved in DMF to give a 250 mM solution and an incubation period of 2 h was applied. Then, the resin was washed three times with 1 mL of DMF, DCM, MeOH, and Et2O each and dried in Vacuo. Cleavage of the peptides from the resin and identification of the CF-labeled peptides was performed as described above. The following peptides were produced: CF-NPY, [Lys(CF)4]NPY, CF-[Phe7, Pro34]NPY, [Lys(CF)4, Phe7, Pro34 ]NPY, CF-[Ahx5-24]NPY, [Lys(CF)4, Ahx5-24]NPY, CF[Ahx8-20]NPY, and [Lys(CF)4, Ahx8-20]NPY. Conjugation with DOTA. A protected DOTA analogue (DOTA-3tBu) was activated in a separate vial by 3 equiv HOBt and 3 equiv DIC in 250 µL DMF. It was transferred to the resinbound peptide and incubated at room temperature for 2 h. The resin was washed three times with 1 mL of DMF, DCM, DMF, MeOH, and Et2O each and dried in Vacuo. The following peptides were synthesized: DOTA-NPY, Lys(DOTA)4-NPY, DOTA-[Phe7, Pro34]NPY, Lys(DOTA)4-[Phe7, Pro34]NPY, DOTA-[Ahx5-24]NPY, Lys(DOTA)4-[Ahx5-24]NPY, DOTA[Ahx8-20]NPY, and Lys(DOTA)4-[Ahx8-20]NPY. Labeling of DOTA-NPY Analogues with Ga. A mixture of 500 µg (340 µmol) of DOTA-NPY in 500 µL of 0.4 M sodium acetate buffer (pH 5) was incubated with 102 µL of a

1432 Bioconjugate Chem., Vol. 19, No. 7, 2008

Zwanziger et al.

Table 1. Analytical and Binding Data of the Modified/Labelled NPY Analogues pKi no.

peptide

MWcalc. [D]

MWexpt. [D]

RTa [min]

SK-N-MC

MCF-7

SMS-KAN

HEC-1B-Y5

1. 2. 3. 4. 1a. 1b. 2a. 2b. 3a. 3b. 4a. 4b. 2e. 2f. 2g. 1c. 1d. 2c. 2d. 3c. 3d. 4c. 4d.

NPY [Phe7, Pro34]NPY [Ahx5-24]NPY [Ahx8-20]NPY DOTA-NPY [Lys(DOTA)4]NPY DOTA-[Phe7, Pro34]NPY [Lys(DOTA)4, Phe7, Pro34]NPY DOTA-[Ahx5-24]NPY [Lys(DOTA)4, Ahx5-24]NPY DOTA-[Ahx8-20]NPY [Lys(DOTA)4, Ahx8-20]NPY [Lys(Ga-DOTA)4, Phe7, Pro34]NPY [Lys(In-DOTA)4, Phe7, Pro34]NPY [Lys(111In-DOTA)4, Phe7, Pro34]NPY CF-NPY [Lys(CF)4]NPY CF-[Phe7, Pro34]NPY [Lys(CF)4, Phe7, Pro34]NPY CF-[Ahx5-24]NPY [Lys(CF)4, Ahx5-24]NPY CF-[Ahx8-20]NPY [Lys(CF)4, Ahx8-20]NPY

4254.0 4255.1 2220.2 2981.0 4640.7 4640.7 4642.0 4642.0 2607.6 2607.6 3368.5 3368.5 4708.6 4753.7 4753.7 4612.0 4612.0 4614.1 4614.1 2578.9 2578.9 3339.8 3339.8

4254.8 4255.4 2221.3 2982.4 4641.5 4641.9 4643.5 4643.0 2608.5 2608.8 3369.6 3369.3 4709.7 4754.7 n.d. 4613.1 4613.4 4615.1 4614.5 2580.6 2580.3 3340.8 3340.8

21.8 22.1 14.9 19.9 22.3 21.4 23.5 22.8 17.4 17.8 20.5 21.1 23.6 21.6 20.9 24.8 24.7 25.3 25.1 17.9 18.1 13.3 22.8

9.5 ( 0.1 10.3 ( 0.2 6.8 ( 0.1 8.0 ( 0.1 n.d. n.d. 8.6 ( 0.2 9.5 ( 0.2 n.d. n.d. n.d. n.d. 8.8 ( 0.1 8.8 ( 0.1 n.d. 8.2 ( 0.1 7.1 ( 0.1 8.9 ( 0.2 8.1 ( 0.1 n.d. n.d. 6.8 ( 0.1 6.8 ( 0.1

9.5 ( 0.1 9.1 ( 0.1 n.d.b 7.7 ( 0.1 n.d. n.d. 8.9 ( 0.1 9.1 ( 0.1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. 9.3 ( 0.2 7.2 ( 0.1 9.5 ( 0.2 7.9 ( 0.1 n.d. n.d. 7.6 ( 0.1 7.1 ( 0.1

8.8 ( 0.1 7.6 ( 0.1 9.2 ( 0.3 8.2 ( 0.1 n.d. n.d. 6.5 ( 0.1 7.2 ( 0.1 8.8 ( 0.1 8.5 ( 0.1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 8.2 ( 0.1 8.0 ( 0.1 8.8 ( 0.1 7.7 ( 0.1

8.2 ( 0.1 8.6 ( 0.5 n.d. 6.9 ( 0.1 n.d. n.d. 7.1 ( 0.3 7.5 ( 0.1 7.0 ( 0.3 6.8 ( 0.2 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

b

n.d. not determined. a Retention time.

Figure 1. Sequences and modifications of the peptides used in this study: (a) modification with DOTA at the N-terminus, (b) modification with DOTA at the Nε side chain of Lys4, (e,f) modification with Ga/In, (g) modification with 111In, (c) modification with CF at the N-terminus, (d) modification with CF at the Nε side chain of Lys4.

0.01 M aqueous solution of nonradioactive Ga(NO3)3 for 30 min at 37 °C and cooled to room temperature. Lys(DOTA)4[Phe7, Pro34]NPY was labeled with Ga to produce [Lys(GaDOTA)4, Phe7, Pro34]NPY. Labeling of DOTA-NPY Analogues with In. A mixture of 500 µg (340 µmol) of DOTA-NPY in 500 µL of 0.4 M ammonium acetate buffer (pH 5) was incubated with 100 µL of a 0.01 M aqueous solution of nonradioactive InCl3 for 30 min at 37 °C and cooled to room temperature. Lys(DOTA)4[Phe7, Pro34]NPY was labeled with In to produce [Lys(InDOTA)4, Phe7, Pro34]NPY. Labeling of DOTA-NPY Analogues with 111In. A mixture of 25 µg (1 mg/0.5 mL saline solution (2.5 M)) of NPY analogue in 12.5 µL of saline solution (2.5 M) was incubated with 100 µL of 111InCl3 (41 MBq) in 0.04 M HCl and 20 µL of a 2.5 M sodium acetate buffer (pH 5.6) for 15 min at 75 °C and cooled for 10 min (16). The product was analyzed by radio-HPLC.

Analysis was performed on the HPLC system Agilent (Luna column 5 µm, C18, 250 × 4.6 mm), by using a linear gradient of 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (ACN) (B) from 25% to 50% B in A in 30 min with a flow of 1.0 mL/min. Lys(DOTA)4-[Phe7, Pro34]NPY was labeled with 111In to produce [Lys(111In-DOTA)4, Phe7, Pro34]NPY. Cell Culture. Cells were grown in 75 cm2, 175 cm2, and 225 cm2 culture flasks at 37 °C, 5% CO2, in a humidified atmosphere. SK-N-MC cells were cultivated in MEM with Earl’s salts supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 4 mM L-glutamine, 0.2 mM nonessential amino acids, and 1 mM sodium pyruvate. SMS-KAN cells were grown in DMEM/nutrient mix Ham’s F12 (1:1) with 15% v/v FCS, 4 mM L-glutamine, and 0.2 mM nonessential amino acids. MCF-7 cells were cultivated in DMEM/nutrient mix Ham’s F12 (1:1) with 10% (v/v) FCS and 2 mM L-glutamine. All cells were grown to confluence before use.

Chemically Modified Analogues of NPY for Tumor Targeting

Bioconjugate Chem., Vol. 19, No. 7, 2008 1433

Figure 2. Typical analytical data of compound 2f [Lys(In-DOTA)4, Phe7, Pro34]NPY (A) HPLC chromatogram from 10% to 60% ACN in 30 min, (B) MALDI spectrum of the compound.

Receptor Binding Affinity Assays. Cells were resuspended in incubation buffer (minimal essential medium with Earl’s salts containing 0.1% bacitracin, 50 mM Pefabloc SC, and 1% bovine serum albumin). A total of 200 µL of the cell suspension containing approximately 60 000 cells was incubated with 25 µL of a 10 nM solution of 3H-propionyl-NPY and 25 µL of NPY or analogue in a concentration range of 10 µM to 10 pM in 1% BSA. Nonspecific binding (NSB) was defined in the presence of 1 µM unlabeled NPY. After incubating the cells for 90 min at room temperature, the incubation was terminated by centrifugation at 2000 × g and 4 °C for 5 min. The pellets were washed twice with 400 µL PBS (137 mM NaCl, 8 mM Na2HPO4/KH2PO4, pH 7.4), centrifuged, and resuspended in 100 µL PBS. The cell suspension was mixed with 3 mL scintillation cocktail and radioactivity was determined by using a beta-counter. IC50 values of the binding curves were calculated by nonlinear regression on a sigmoidal dose-response based model by using GraphPad Software PRISM 3.0. Each experiment was performed 2-3 times in triplicate and Ki ( SEM values were calculated, using the Cheng-Prusoff equation (17). The following KD values of the radioligand 3H-NPY at the respective receptors were used: 0.18 nM at the Y1 receptor, 0.45 nM at the Y2 receptor, and 1.8 nM at the Y5 receptor. Confocal Laser Scanning Microscopy (CLSM). For confocal laser scanning microscopy, the cells were grown on Falcon Cell Culture Inserts with Cyclopore membranes or a LaboratoryTak Chamber Slides for the observation of unfixed cells. In order to perform measurements, the Leica Confocal System TCS SP2 was used at excitation/emission wavelengths of 488/514 nm argon laser, respectively. Metabolic Stability in Human Blood Plasma. The proteolytic degradation of the CF-labeled peptide was determined in Vitro in human blood plasma obtained from healthy donors. 0.1 mL of plasma was incubated at 37 °C with 10 µM CFlabeled peptide for 0, 3, 6, 12, and 24 h. Then, samples were precipitated with 40 µL acetonitrile/ethanol (50:50, v/v) by incubation in ice for 30 min followed by centrifugation at 8000 rpm at 4 °C for 15 min. The supernatant was filtered through HPLC certified 220 µm filters, analyzed by HPLC on a Vydac RP18-column (4.6 × 250 mm; 5 µm/ 300 Å, Merck Hitachi, Darmstadt, Germany), and monitored by fluorescence detection. A linear gradient with 0.1% TFA in water (A) and 0.08% in acetonitrile (B) from 25% to 50% B in A over 60 min at a flow rate of 600 µL min-1 was used. The fluorescence labeled metabolites collected from each HPLC chromatogram were identified by MALDI-ToF as possible cleavage sites from the parent peptide.

Figure 3. (A) Cellular uptake of CF-[Ahx5-24]NPY in SMS-KAN cells. Cells were incubated with the CF-labeled peptide at a final concentration of 1 µm at 37 °C for 120 min. The CLSM view shows a slice at 3.08 µm from Z-scans (from -4.50 to 2.04 µm with an increment interval of 0.38 µm). (B) For the determination of nonspecific adsorption of the ligand, BHK cells which do not express NPY receptors were used as negative control. They were treated in parallel and observed in CLSM under the same parameters.

Inoculation of MCF-7 Tumor Cells in Immunodeficient Mice. In general, for biodistribution studies nude mice (female); app. 18 g NMRI nu/nu were used. First of all, three days before tumor cell inoculation, subcutaneous implantation of estrogen pellet in the left shoulder was affected. Mice were anesthesized with Isofluran Curamed. MCF-7 cells were resuspended in Dulbecco′s PBS with Ca2+/Mg2+ and Matrigel Matrix with growth factor. Subcutaneous injection of 100 µL of the cell suspension containing similarly 2 × 107 cells occurred in the right shoulder of the mice 16 days before biodistribution. Biodistribution of Lys(111In-DOTA)4-[Phe7, Pro34]NPY in MCF-7 Tumor-Bearing Mice. The biodistribution of Lys(111In-DOTA)4-[Phe7, Pro34]NPY was studied in nude mice bearing MCF-7 tumors. Adjacent to the induction of light anesthesia with Isofluran Curamed, the animals were injected with the radiolabeled peptide via a lateral tail vein at a radioactive dose of 88 kBq 111In. Following injection, animals were individually housed in metabolism cages with food and water to allow separate collection of voided urine and feces. Animals were killed after 0.5 h, 1.0 h, 2.0 h, 4.0 h, and 24 h by decapitation under anesthesia with Isofluran Curamed. Each carcass was dissected and the organs and tissues assayed for radioactivity (18).

RESULTS Synthesis and Labeling of NPY Analogues. For our studies, we used the three NPY analogues [Phe7, Pro34]NPY,

1434 Bioconjugate Chem., Vol. 19, No. 7, 2008

[Ahx5-24]NPY, and [Ahx8-20]NPY as well as NPY. It has been shown that [Phe7, Pro34]NPY significantly binds to Y1 receptors with subnanomolar affinity (19). For [Ahx5-24]NPY and [Ahx8-20]NPY, selectively binding to Y2 receptors has been observed (13, 20). The peptides were produced with either a DOTA or a CF group (Figure 1). The labels were introduced manually to the resin-bound peptides either at the N-terminus or at the selectively deprotected Nε side chain of Lys4 while they were still bound to the resin. The most potent DOTAconjugated peptide was chelated with Ga(NO3)3 and InCl3. The optimum conditions at which we could obtain the Ga-DOTANPY analogue with a maximum yield (42%) and the In-DOTANPY analogue with a maximum yield (96%) included the incubation of DOTA-conjugated peptide in 0.4 M sodium acetate buffer and 0.4 M ammonium acetate buffer with 0.01 M aqueous solution of Ga(NO3)3 or InCl3 for 30 min at 37 °C. All peptides were purified and analyzed by RP-HPLC and the identity was confirmed by MALDI-ToF. A typical analytical data of the compound 2f are shown in Figure 2. In total, 23 peptides were produced and their sequences and analytical data are listed in Table 1. Numbering was performed according to the scheme described in Figure 1. Receptor Binding Affinity. The receptor affinity was tested at cell lines that selectively express single NPY receptor subtypes by the displacement of 1 nM [3H]propionyl-NPY. The experimental data showed an IC50 value of 2.0 ( 0.1 nM for NPY at Y1 receptor expressing SK-N-MC cells that was transferred to a Ki value of 0.3 ( 0.02 nM, by using the ChengPrusoff equation (17). Evaluation of the binding data obtained with the modified or labeled analogues is summarized in Table 1. As shown in Table 1, the unlabeled NPY showed maximum binding at all NPY receptor expressing cells. To test the effect of the chelator on receptor binding properties, compounds 2-4 were labeled with DOTA. The labeling of compound 2 as well as of NPY at the Nε side chain of Lys4 with DOTA showed better binding affinities at Y1/Y2 receptor expressing cells compared to the labeling at the N-terminus (compound 2a with pKi values 8.6 ( 0.2 vs 2b with 9.5 ( 0.2). A reversed effect was observed with compounds 3a and 3b at Y2 receptors with pKi values 8.8 ( 0.1 vs 8.5 ( 0.1, respectively. Although some loss in affinity was found for compound 2e (pKi ) 8.8 ( 0.1), nanomolar affinity was still found for the Ga-DOTA-labeled peptide. The same affinity could be observed for the In-DOTA-labeled peptide, compound 2f (pKi ) 8.8 ( 0.1). Furthermore, the DOTA conjugated compound 2a showed constant selectivity at Y1 receptor expressing SK-N-MC and MCF-7 cells compared to Y2 and Y5 receptor expressing SMSKAN and HEC-1B-Y5 cells (compound 2a with pKi values of 8.6 ( 0.2 at SK-N-MC vs 6.5 ( 0.1 and 7.1 ( 0.3 at SMSKAN and HEC-1B-Y5 cells). Same results could be observed for the compound 2b with DOTA at Nε side chain of Lys4 (compound 2b with pKi values of 9.5 ( 0.2 at SK-N-MC vs 7.2 ( 0.1 and 7.5 ( 0.1 at SMS-KAN and HEC-1B-Y5 cells). To determine whether the fluorescently labeled NPY analogues had any effect on receptor affinity, CF labeled peptides were tested as well. Interestingly, the binding was found to significantly dependent on the position of the labeling, as the NPY receptor selective N-terminally labeled peptides as well as NPY were found to be mostly more biologically active at the corresponding NPY Y1 or Y2 receptor. The effect was pronounced at the Y1 receptors especially in MCF-7 cells (compounds 1c and 1d with pKi values of 9.3 ( 0.2 vs 7.2 ( 0.1, respectively). Similar results were also obtained with Y2 receptor selective analogues at SMS-KAN cells (compounds 3c and 4c with pKi values of 8.2 ( 0.1 and 8.8 ( 0.1 vs pKi

Zwanziger et al.

values of 8.0 ( 0.1 and 7.7 ( 0.1 of compounds 3d and 4d). All of these binding data at various receptors are summarized in Table 1. These fluorescent labeled compounds were further used in confocal microscopy for visualization of the receptorligand interaction in internalization assays to investigate uptake of compounds into the cells. For all analogues, receptor subtype selective uptake could be demonstrated. Figure 3 shows the cellular uptake of compound 3c into SMS-KAN cells by using confocal laser scanning microscopy (CLSM). Metabolic Stability in Human Blood Plasma. The metabolic stability and the main cleavage product in human blood plasma of [Lys(CF)4, Phe7, Pro34]NPY, compound 2d, was determined. The intact peptide found after 3 h incubation was about 95%, after 6 h as 88%, after 12 h as 74%, and after 24 h about 71%. Only one main cleavage product could be detected for the NPY analogue after 24 h, [Lys(CF)4, Phe7, Pro34]NPY(1-35) with about 25-30%. Figure 4 shows the analytical data of metabolic degradation in human blood plasma after 24 h and the time course of degradation of [Lys(CF)4, Phe7, Pro34]NPY. Radiolabeling. [Phe7,Pro34]NPY has been shown to be the compound with the most significant Y1-receptor preference with subnanomolar affinity (19). Accordingly, we used this NPY analogue for radiolabeling and biodistribution studies in MCF-7 tumor-bearing mice for detection of breast cancers. 111In-DOTA radiolabeling of [Lys(DOTA)4, Phe7, Pro34]NPY is also dependent on different labeling conditions, like temperature, pH, and reaction time. Optimized conditions were found for the incubation of DOTA-NPY analogue in 2.5 M saline solution and 111InCl3 (41 MBq) in 0.04 M HCl for 15 min at 75 °C with a radiochemical yield of 80%. In contrast, a longer incubation time of 20 min and a temperature of 70 °C led to a radiochemical yield of only 6% (Figure 5A). The radiolabeled peptide was analyzed by radio-HPLC. A typical radio-HPLC chromatogram of the compound 2g is shown in Figure 5B. Biodistribution. The evaluation of [Lys(111In-DOTA)4, Phe7, Pro34]NPY for in ViVo receptor targeting was performed by using biodistribution in MCF-7 tumor-bearing mice. Therefore, the nude mice were successfully inoculated with the Y1 receptor expressing MCF-7 cell line in the right shoulder. The animals were injected with the 111In-labeled peptide via a lateral tail vein (inject dose of 87.67 kBq). The radiolabeled peptide was not removed from the unlabeled peptide. Separated organs, tissues, and the blood were weighed and assayed for radioactivity using a gamma-counter for detection of 111In after 0.5 h, 1.0 h, 2.0 h, 4.0 h, and 24 h respectively. Microsoft Excel analysis program was applied to calculate %ID/g (percent of injected dose per gram organ, tissue, or blood). The biodistribution study data of the compound 2g demonstrated extensive accumulation in the kidney and a low tumor uptake. After intravenous injection of the 111In-labeled peptide, maximum tumor uptake of 1.7%ID/g was reached after 30 min. A significant accumulation of 86.8%ID/g after 4 h was reached in the kidney. Furthermore, an increase in the uptake was found in the bone with a maximum of 1.62%ID/g after 24 h. A relatively lower concentration was detected in spleen (1.10%ID/g after 24 h). In the adrenals and blood, 111In-labeled peptide concentration decreased over 24 h. The complete results of the biodistribution study are summarized in Table 2.

DISCUSSION The prerequisite for radiolabeled receptor-binding peptides as diagnostic or therapeutic agents include therapeutic efficacy and favorable pharmacokinetics, such as rapid and high tumor uptake, extended tumor retention, high organ ratio of tumor to normal organ, and rapid whole-body clearance (21). In a metalloradiopharmaceutical, the targeting biomolecule is only one part of the whole molecule and the development of an efficient BFC and new

Chemically Modified Analogues of NPY for Tumor Targeting

Bioconjugate Chem., Vol. 19, No. 7, 2008 1435

Figure 4. (A) HPLC chromatogram from 20% to 50% ACN in 60 min and MALDI spectra of fluorescence labeled metabolites of [Lys(CF)4, Phe7, Pro34]NPY, compound 2d after 24 h incubation at 37 °C in human blood plasma. (B) Time course of degradation after incubating compound 2d with blood plasma at 37 °C.

radiolabeling technologies is equally important. For the receptorbased therapeutic radiopharmaceuticals, it is very important to remember that the receptor population is often limited. The use of a large amount of unlabeled bifunctional chelator-biomolecule (BFC-BM) conjugate may block the binding of the radiolabeled BFC-BM conjugate at the receptor sites. Therefore, the BFC must have very high labeling efficiency (fast and high-yield labeling) and form metal complexes with high specific activity. The choice of BFC is largely dependent upon the nature and oxidation state of the metal ion and requires a good understanding of the coordination chemistry of any given radiometal. Another point of fundamental concern in the design of radiometallated peptide constructs is to ensure the in ViVo stability of the radiometal incorporated by the ligand framework. In ViVo stability can be controlled by virtue of the high thermodynamic stability and the kinetic inertness of a specific metal chelation framework (22–24). Because the DOTA macrocyclic ligand system is well-known to form kinetically inert and thermodynamically stable chelates with indium, yttrium, and lanthanides, it has received wide acceptance for in ViVo applications with several trivalent radiometals, not only for scintigraphy but also for peptide receptor radionuclide therapy (PRRT) (25–27). Peptide ligands can be derivatized with DOTA without loss of receptor specificity and binding affinity. Recently, [DOTA0, Tyr3]octreotate, labeled with the β- and γ-particle-emitting radionuclide 177Lu, was reported to be very successful in terms of tumor regression and animal survival in a rat model (28).

Bifunctional chelating agents (BFCAs) can be incorporated into peptides by using either the prelabeling or postlabeling approach. In the postlabeling approach, a BFCA is first attached to the peptide and then the radionuclide is coupled to the free chelating group of the BFCA (29). This approach can be combined with the advantages of solid-phase peptide synthesis (SPPS). Applying a molar excess of BFCA, complete coupling to the resin-bound peptide can be achieved, and unreacted BFCA is easily washed away. Subsequent cleavage from the resin yields the highly pure BFCA-peptide conjugate. Accordingly, NPY receptor-selective derivatives on Rink amide resin were synthesized, selectively deprotected Nε side chain of Lys4 by hydrazine in DMF and coupled the BFCA either at the Nε side chain of Lys4 or at the N-terminus, by using the postlabeling approach. Furthermore, by incorporation of Phe7 and Pro34 into NPY sequence, Y1 receptor subtype selectivity (19) and by replacing the sequence (5-24)NPY or (8-20)NPY with the spacer 6-aminohexanoic acid (Ahx), Y2 receptor subtype selectivity were achieved (30). This can be confirmed by the binding data of compounds 2 and 3 determined with SK-N-MC and SMS-KAN cells with (pKi values 10.3 ( 0.2 vs 7.6 ( 0.1) and (pKi values 6.8 ( 0.1 vs 9.2 ( 0.3), respectively. The compounds conjugated with DOTA at the Nε side chain of Lys4 showed higher affinity compared to the N-terminally conjugated compounds with respect to native NPY and the NPY Y1 receptor selective analogue. These effects could be due to

1436 Bioconjugate Chem., Vol. 19, No. 7, 2008

Zwanziger et al.

the peptides labeled with CF at the N-terminus showed mostly higher affinity compared to the CF-labeling at the Nε side chain of Lys4. This also suggests that the type of modifier is involved in the Y-receptor-ligand interaction. A metabolic stability study in human blood plasma of [Lys(CF)4, Phe7, Pro34]NPY was performed. The peptide was found to be enzymatically more stable than native NPY (halflife of 24.66 ( 1.38 h) in Vitro (31). In addition, only one main cleavage product [Lys(CF)4, Phe7, Pro34]NPY(1-35) could be detected after 24 h incubation (31). Furthermore, first results of the in ViVo behavior of the Y1 receptor selective neuropeptide Y (NPY) analogue [Phe7, Pro34]NPY could be obtained. Our data show that [Lys(DOTA)4, Phe7, Pro34]NPY was successfully labeled with 111 In at 75 °C for 15 min with a radiochemical yield of 80%. In contrast, by using a temperature of 70 °C and an incubation time of 20 min only a radiochemical yield of 6% could be observed. This result suggests that higher reaction temperatures could lead to more flexibility of the cage-like structure of DOTA followed by a better complexation of 111In. However, higher temperatures could lead to degradation of the peptide. Different retention times in radio-HPLC (20.9 min vs 18.9 min) could be explained by distinct concentrations of the unlabeled peptide. In the past, strong predominance of the Y1 receptor subtype was shown in breast carcinomas (9). Thus, the nude mice for the biodistribution study were inoculated with the Y1 receptor expressing MCF-7 breast cancer cell line. Our results demonstrate a high accumulation of the 111In-DOTA-labeled [Phe7, Pro34]NPY in the kidney with 86.8% ID/g after 4 h. This is a common phenomenon for radiometal-chelated peptides, especially for peptides with DOTA as BFC (32). On the other hand, our biodistribution results show a low tumor uptake of the peptide with 1.7% ID/g after 30 min. These data indicate that the interaction between the 111Inlabeled peptide and the breast cancer tumor, which expresses the Y1 receptor subtype, was disturbed by the faster accumulation in the kidney. Another reason for the low tumor uptake could be the lack of separation of the unlabeled peptide, which might have resulted in binding competition of the unlabeled and 111In-labeled peptide in ViVo. Moreover, it has been shown that the native ligand NPY exhibits rapid proteolysis in humans with a half-life of only 4 min (33). The differences between the metabolic stability in the body

Figure 5. (A) Radio-HPLC chromatogram from 25% to 50% ACN in 30 min of [Lys(111In-DOTA)4, Phe7, Pro34]NPY, compound 2g, after 20 min incubation at 70 °C. The radio-HPLC chromatogram shows a radiochemical yield of only 6%. (B) Radio-HPLC chromatogram from 25% to 50% ACN in 30 min of [Lys(111In-DOTA)4, Phe7, Pro34]NPY, compound 2g, after 15 min incubation at 75 °C. The radio-HPLC chromatogram shows a radiochemical yield of 80%.

less steric hindrance encountered by more spatial availability of the N-terminus produced by labeling of the chelator at the Nε side chain of Lys4. Our results show that N-terminal flexibility is a crucial factor for the interaction of the bioconjugate with the receptor. Otherwise, the N-terminally DOTAconjugated NPY Y2 receptor selective analogue showed higher affinity than that conjugated at the Nε side chain of Lys4. These data can be explained by the reduced influence of the truncated N-terminus of [Ahx5-24]NPY in receptor binding compared to NPY or [Phe7, Pro34]NPY. Carboxyfluorescein possesses two carboxyl groups as possible reaction centers, the intrinsic carboxyl group of fluorescein in position 2 and the additional carboxyl group in position 4(5). Since the fluorescence-properties of fluorescein depend on the presence of the free carboxyl-group in position 2, this group has to remain unmodified during reaction. Our data show that Table 2. Biodistribution of [Lys(111In-DOTA)4, Phe7, Pro34]NPY time

0.5 h

1.0 h

2.0 h

4.0 h

24 h

weight (g)

29.79

31.68

30.73

27.69

27.36

1.52 ( 0.27 3.34 ( 0.56 60.54 ( 8.80 3.04 ( 0.35 1.52 ( 0.20 1.62 ( 0.02 0.11 ( 0.01 0.50 ( 0.04 1.70 ( 0.29 0.75 ( 0.07 1.66 ( 0.51 1.93 ( 0.36 3.77 ( 0.38 3.74 ( 0.98 1.21 ( 0.15 1.58 ( 0.13 1.62 ( 0.28 0.89 ( 0.18 1.13 ( 0.17 1.69 ( 0.22

1.36 ( 0.36 3.23 ( 0.61 63.01 ( 9.40 2.28 ( 0.15 0.97 ( 0.10 1.09 ( 0.13 0.09 ( 0.04 0.38 ( 0.02 1.08 ( 0.40 0.52 ( 0.09 1.29 ( 0.33 1.29 ( 0.06 2.93 ( 0.36 3.61 ( 2.46 0.86 ( 0.05 1.18 ( 0.21 1.51 ( 0.58 0.71 ( 0.04 0.91 ( 0.11 1.43 ( 0.05

1.08 ( 0.16 3.41 ( 0.23 77.11 ( 3.30 1.48 ( 0.12 0.86 ( 0.08 0.95 ( 0.07 0.07 ( 0.01 0.55 ( 0.12 0.74 ( 0.07 0.33 ( 0.09 1.13 ( 0.23 0.87 ( 0.14 2.41 ( 0.33 5.05 ( 2.23 0.64 ( 0.06 1.38 ( 0.41 1.31 ( 0.15 0.74 ( 0.05 0.78 ( 0.04 1.46 ( 0.40

1.37 ( 0.19 4.20 ( 0.30 86.83 ( 11.60 1.48 ( 0.13 0.89 ( 0.21 1.02 ( 0.21 0.08 ( 0.01 0.48 ( 0.14 0.73 ( 0.11 0.42 ( 0.10 1.25 ( 0.31 1.07 ( 0.09 2.23 ( 0.52 3.04 ( 1.36 0.64 ( 0.12 1.14 ( 0.22 1.41 ( 0.34 0.84 ( 0.13 0.86 ( 0.13 1.52 ( 0.24

1.10 ( 0.24 3.25 ( 0.99 67.60 ( 13.30 0.48 ( 0.14 1.62 ( 0.11 0.36 ( 0.04 0.03 ( 0.003 0.68 ( 0.12 0.52 ( 0.05 0.24 ( 0.09 1.21 ( 0.39 0.99 ( 0.10 0.32 ( 0.12 1.15 ( 0.56 0.49 ( 0.09 0.75 ( 0.15 0.89 ( 0.16 0.65 ( 0.18 0.63 ( 0.13 1.03 ( 0.10

spleen liver kidney lung bone heart brain fat thyroid muscle tumor skin blood tail stomach ovary uterus intestine pancreas adrenals

%ID/g

Chemically Modified Analogues of NPY for Tumor Targeting

and the blood can be confirmed by our results of the fast degradation of the 111In-DOTA-labeled [Phe7, Pro34]NPY in mice compared to the half-life of [Lys(CF)4, Phe7, Pro34]NPY in human blood plasma. The development of metabolically more stable NPY analogues is a crucial step for radiopharmaceutical application.However, Y1 receptor selective NPY analogues show good capability for future applications in tumor targeting and therapy, confirmed by some tumor uptake of [Lys(111In-DOTA)4, Phe7, Pro34]NPY. Accordingly, we clearly could demonstrate a novel application of modified BFCA-conjugated NPY Y1/Y2 receptor selective analogues. Competitive binding assays showed surprisingly high affinity to the desired receptors. Based on structure-activity relationships of NPY, we found potential candidates for metal complexation. By using SPPS, stable, well-defined BFCA DOTA conjugated analogues for Ga or In and 111In complexation were synthesized. These analogues are stable and selectively maintained the binding with the desired receptor. Our data show that, in spite of replacement/ substitution of amino acids, introduction of spacer, and incorporation of an organometallic moiety, the binding potency of the ligand at the receptor was only slightly influenced. Moreover, the in ViVo data show the possibility of using NPY Y1 receptor selective analogues for future applications in tumor therapy and diagnosis, because some, although still low, tumor uptake in MCF-7 tumor-bearing mice was observed. Our in Vitro and in ViVo data demonstrate that modified NPY analogues are expandable candidates for further developments in the field of tumor targeting.

ACKNOWLEDGMENT The authors thank Regina Reppich for excellent mass spectrometry and Christina Dammann for technical assistance in cell culture. This work was financially supported by DFG (FOR630, Be1264/ 9-1), EFRE (Grant # 3370701481201), and by Bayer Schering Pharma.

LITERATURE CITED (1) Heppeler, A., Froidevaux, S., Eberle, A. N., and Maecke, H. R. (2000) Receptor targeting for tumor localisation and therapy with radiopeptides. Curr. Med. Chem. 7, 971–94. (2) Tatemoto, K., Carlquist, M., and Mutt, V. (1982) Neuropeptide Y-a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 296, 659–60. (3) Grundemar, L. B., S.R. (1997) Neuropeptide Y and drug deVelopments, p 396, Academic Press, San Diego. (4) Michel, M. C., Beck-Sickinger, A., Cox, H., Doods, H. N., Herzog, H., Larhammar, D., Quirion, R., Schwartz, T., and Westfall, T. (1998) XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol. ReV. 50, 143–50. (5) Beck-Sickinger, A. (1997) The importance of various parts of the NPY molecule for receptor recognition. Neuropept. Drug DeV. 107–126. (6) Korner, M., Waser, B., and Reubi, J. C. (2005) Neuropeptide Y receptors in renal cell carcinomas and nephroblastomas. Int. J. Cancer 115, 734–41. (7) Korner, M., Waser, B., and Reubi, J. C. (2004) Neuropeptide Y receptor expression in human primary ovarian neoplasms. Lab. InVest. 84, 71–80. (8) Reubi, J. C., Korner, M., Waser, B., Mazzucchelli, L., and Guillou, L. (2004) High expression of peptide receptors as a novel target in gastrointestinal stromal tumours. Eur. J. Nucl. Med. Mol. Imaging 31, 803–10. (9) Reubi, J. C., Gugger, M., Waser, B., and Schaer, J. C. (2001) Y(1)-mediated effect of neuropeptide Y in cancer: breast carcinomas as targets. Cancer Res. 61, 4636–41.

Bioconjugate Chem., Vol. 19, No. 7, 2008 1437 (10) Breeman, W. A., Bakker, W. H., De Jong, M., Hofland, L. J., Kwekkeboom, D. J., Kooij, P. P., Visser, T. J., and Krenning, E. P. (1996) Studies on radiolabeled somatostatin analogues in rats and in patients. Q. J. Nucl. Med. 40, 209–20. (11) Moser, C., Bernhardt, G., Michel, J., Schwarz, H., and Buschauer, A. (2000) Cloning and functional expression of the hNPY Y5 receptor in human endometrial cancer (HEC-1B) cells. Can. J. Physiol. Pharmacol. 78, 134–42. (12) Rist, B., Ingenhoven, N., Scapozza, L., Schnorrenberg, G., Gaida, W., Wieland, H. A., and Beck-Sickinger, A. G. (1997) The bioactive conformation of neuropeptide Y analogues at the human Y2-receptor. Eur. J. Biochem. 247, 1019–28. (13) Rist, B., Wieland, H. A., Willim, K. D., and Beck-Sickinger, A. G. (1995) A rational approach for the development of reducedsize analogues of neuropeptide Y with high affinity to the Y1 receptor. J. Pept. Sci. 1, 341–8. (14) Bloomberg, G. B., A, D., Gargaro, A. R., and Tanner, M. J. A. (1993) Synthesis of a branched cyclic peptide using a strategy employing Fmoc chemistry and two additional orthogonal protecting groups. Tetrahedron Lett. 34, 4709–4712. (15) Weber, P. J., Bader, J. E., Folkers, G., and Beck-Sickinger, A. G. (1998) A fast and inexpensive method for N-terminal fluorescein-labeling of peptides. Bioorg. Med. Chem. Lett. 8, 597– 600. (16) Gali, H., Sieckman, G. L., Hoffman, T. J., Owen, N. K., Chin, D. T., Forte, L. R., and Volkert, W. A. (2001) In vivo evaluation of an 111In-labeled ST-peptide analog for specific-targeting of human colon cancers. Nucl. Med. Biol. 28, 903–9. (17) Cheng, H. C. (2001) The power issue: determination of KB or Ki from IC50. A closer look at the Cheng-Prusoff equation, the Schild plot and related power equations. J. Pharmacol. Toxicol. Methods 46, 61–71. (18) Chen, J., Giblin, M. F., Wang, N., Jurisson, S. S., and Quinn, T. P. (1999) In vivo evaluation of 99mTc/188Re-labeled linear R-melanocyte stimulating hormone analogs for specific melanoma targeting. Nucl. Med. Biol. 26, 687–93. (19) Soll, R. M., Dinger, M. C., Lundell, I., Larhammer, D., and Beck-Sickinger, A. G. (2001) Novel analogues of neuropeptide Y with a preference for the Y1-receptor. Eur. J. Biochem. 268, 2828–37. (20) Rist, B., Zerbe, O., Ingenhoven, N., Scapozza, L., Peers, C., Vaughan, P. F., McDonald, R. L., Wieland, H. A., and BeckSickinger, A. G. (1996) Modified, cyclic dodecapeptide analog of neuropeptide Y is the smallest full agonist at the human Y2 receptor. FEBS Lett. 394, 169–73. (21) Miao, Y., Owen, N. K., Fisher, D. R., Hoffman, T. J., and Quinn, T. P. (2005) Therapeutic efficacy of a 188Re-labeled R-melanocyte-stimulating hormone peptide analog in murine and human melanoma-bearing mouse models. J. Nucl. Med. 46, 121–9. (22) Otte, A., Herrmann, R., Heppeler, A., Behe, M., Jermann, E., Powell, P., Maecke, H. R., and Muller, J. (1999) Yttrium-90 DOTATOC: first clinical results. Eur. J. Nucl. Med. 26, 1439– 47. (23) Heppeler, A. F., S., Maecke, H. R., Jermann, E., Behe, M., Powell, P., and Hennig, M. (1999) Radiometal-labelled macrocyclic chelator-derivatised somatostatin analogue with superb tumour-targeting properties and potential for receptormediated internal radiotherapy. Chem. Eur. J. 5, 1974–1981. (24) Behr, T. M., Jenner, N., Behe, M., Angerstein, C., Gratz, S., Raue, F., and Becker, W. (1999) Radiolabeled peptides for targeting cholecystokinin-B/gastrin receptor-expressing tumors. J. Nucl. Med. 40, 1029–44. (25) Kwekkeboom, D. J., Mueller-Brand, J., Paganelli, G., Anthony, L. B., Pauwels, S., Kvols, L. K., O’Dorisio, T, M., Valkema, R., Bodei, L., Chinol, M., Maecke, H. R., and Krenning, E. P. (2005) Overview of results of peptide receptor radionuclide therapy with 3 radiolabeled somatostatin analogs. J. Nucl. Med. 46, 62S–6S. (26) Ingenhoven, N., and Beck-Sickinger, A. G. (1997) Fluorescent labelled analogues of neuropeptide Y for the characterization of

1438 Bioconjugate Chem., Vol. 19, No. 7, 2008 cells expressing NPY receptor subtypes. J. Recept. Signal Transduct. Res. 17, 407–18. (27) Gordon, E. A., Kohout, T. A., and Fishman, P. H. (1990) Characterization of functional neuropeptide Y receptors in a human neuroblastoma cell line. J. Neurochem. 55, 506–13. (28) Erion, J. L. B., J. E., Schmidt, M.A., Wilhelm, R. R., and Srinivasan, A. (1999) High radiotherapeutic efficacy of [Lu-177]DOTA-Y(3)-octreotate in a rat tumor model. J. Nucl. Med. 40, 223. (29) Liu, S., and Edwards, D. S. (1999) 99mTc-labeled small peptides as diagnostic radiopharmaceuticals. Chem. ReV. 99, 2235–68. (30) Cabrele, C., and Beck-Sickinger, A. G. (2000) Molecular characterization of the ligand-receptor interaction of the neuropeptide Y family. J. Pept. Sci. 6, 97–122.

Zwanziger et al. (31) Khan, I. U., Reppich, R., and Beck-Sickinger, A. G. (2007) Identification of neuropeptide Y cleavage products in human blood to improve metabolic stability. Biopolymers 88, 182–9. (32) Froidevaux, S., Calame-Christe, M., Tanner, H., and Eberle, A. N. (2005) Melanoma targeting with DOTA-R-melanocytestimulating hormone analogs: structural parameters affecting tumor uptake and kidney uptake. J. Nucl. Med. 46, 887–95. (33) Pernow, J., Lundberg, J. M., and Kaijser, L. (1987) Vasoconstrictor effects in vivo and plasma disappearance rate of neuropeptide Y in man. Life Sci. 40, 47–54.

BC7004297