Melanocyte-Stimulating Hormone Resulting in Enhanced Radioactivity Localization and Retention in Melanoma

[CANCER RESEARCH 64, 1411–1418, February 15, 2004] Radioiodination of Rhenium Cyclized ␣-Melanocyte-Stimulating Hormone Resulting in Enhanced Radioactivity Localization and Retention in Melanoma

Zhen Cheng,1 Jianqing Chen,2 Thomas P. Quinn,2 and Silvia S. Jurisson1 Departments of 1Chemistry and 2Biochemistry, University of Missouri-Columbia, Columbia, Missouri

ABSTRACT ing tumors (1–6). The clearance of radiohalogenated MAbs and peptides from normal tissues is generally faster than that for radiom- ␣ ␣ Radiohalogenated -melanocyte-stimulating hormone ( -MSH) ana- etal labeled MAbs, and a variety of radiohalogens are available for logs were proposed for melanoma imaging and potential radiotherapy potential imaging and therapeutic applications, giving radiohaloge- because ␣-MSH receptors are overexpressed on both mouse and human melanoma cell lines. However, biodistribution studies in tumor-bearing nated proteins an important role in radiopharmaceutical development mice with radiohalogenated ␣-MSH peptides showed very rapid tumor for cancer diagnosis and treatment (7, 8). However, rapid degradation radioactivity wash out due to lysosomal degradation of the radiohaloge- in vivo is often observed for internalized radiohalogenated biomol- nated complex after internalization, which decreased the therapeutic ef- ecules such as 131I-labeled peptides and MAbs radiolabeled with ficacy significantly (R. Stein et al., Cancer Res., 55: 3132–3139, 1995; P. K. conventional chloramine-T or Iodogen methods. Dehalogenation and Garg et al., Bioconjugate Chem., 6: 493–501, 1995.). The melanoma- proteolysis generally decrease the residence time of radiohalogenated 3,4,10 7 ␣ targeting metallopeptide ReO[Cys ,D-Phe ] -MSH3–13 (ReCCMSH) proteins or peptides in the target tumor. was shown to possess high tumor uptake and retention properties (J. Chen Several halogen labeling approaches have been developed to de- et al., Cancer Res., 60: 5649–5658, 2000). Therefore, three peptides, crease dehalogenation and increase tumor cell retention. Conjugation Ac-Lys-ReCCMSH(Arg11), Ac-D-Lys-ReCCMSH(Arg11), and [Nle4,D- Phe7]␣-MSH (NDP) (for comparison), labeled with N-succinimidyl labeling agents such as N-succinimidyl 3-iodobenzoate (SIB) or N- 4-[125I]iodobenzoate (125I-PIB), were prepared and evaluated in vitro and succinimidyl 4-iodobenzoate (PIB) have been reported to be inert to in vivo to develop radiohalogenated ␣-MSH peptide analogs with high dehalogenation; thus, MAbs labeled with N-succinimidyl 3-iodoben- tumor uptake, retention, and favorable biodistribution characteristics. In zoate showed higher accumulation in a human tumor model and vitro cell binding and internalization data showed that approximately 90% enhanced therapeutic efficacy (9, 10). Additional methods to increase of radioiodinated peptides were internalized at2hincultured B16/F1 radiohalogen retention in the tumor cell have involved the use of melanoma cells. Cellular retention studies showed that the receptor-bound positively charged molecules and nonmetabolizable adducts. For ex- ␣ radioiodinated linear -MSH analog NDP was released from the cells into ample, N-succinimidyl 5-iodo-3-pyridinecarboxylate-labeled Mabs the medium very quickly, whereas significant amounts of cell-associated were shown to resist cellular washout due to the trapping of positively radioactivity remained in the cells for Ac-Lys(125I-3- or 4-iodobenzoate 11 125 11 charged catabolites [pyridinium cations (11, 12)]. The use of N- (IBA))-ReCCMSH(Arg ) and Ac-D-Lys( I-IBA)-ReCCMSH(Arg ). The in vitro data clearly demonstrate that rhenium cyclization signifi- succinimidyl 5-iodo-3-pyridinecarboxylate for radioiodination (12), cantly enhanced peptide trapping in the cells, as did D-amino acid incor- the polycationic peptide [D-KRYRR (13)], and nonmetabolizable poration. The combination of these two effects resulted in a 2.9-fold carbohydrate-tyramine adducts (14–17) has enhanced cellular reten- increase in the retention of radioactivity for Ac-D-Lys(125I-IBA)-ReCCM- tion of radioactivity; however, low radiolabeling yields and MAb SH(Arg11) relative to 125I-IBA-NDP at 4 h. In vivo studies also showed that aggregation have been problems associated with the carbohydrate- 125 11 Ac-D-Lys( I-IBA)-ReCCMSH(Arg ) exhibited extremely high radioac- tyramine adducts (14, 18–20). 125 tivity accumulation and prolonged retention in the tumor. Ac-D-Lys( I- The presence of ␣-melanocyte-stimulating hormone (␣-MSH) re- IBA)-ReCCMSH(Arg11) and Ac-Lys(125I-IBA)-ReCCMSH(Arg11) exhib- ceptors on multiple murine and human melanoma cell lines has led to ited much higher tumor uptake at 24 h after injection compared with 125I-IBA-NDP [7.18% injected dose/gram (ID/g), 4.92% ID/g, and 0.26% the development of radiolabeled peptides for potential melanoma ␣ ID/g, respectively]. Ac-D-Lys(125I-IBA)-ReCCMSH(Arg11) also showed detection and treatment. -MSH receptors display nanomolar to sub- very fast whole body clearance and low nonspecific radioactivity accumu- nanomolar affinities for ␣-MSH peptides and are rapidly internalized lation in normal tissues compared with 125I-IBA-NDP and Ac-Lys(125I- on ligand binding (21, 22). High receptor affinity and specificity make IBA)-ReCCMSH(Arg11). A tumor:blood ratio of 34.3 was observed for ␣-MSH peptide analogs attractive vehicles for delivering radionu- Ac-D-Lys(125I-IBA)-ReCCMSH(Arg11) at 24 h postinjection, whereas val- clides to melanoma cells (23–25). Efforts to develop radiohalogenated 125 11 ues of 4.3 and 2.0 were observed for Ac-Lys( I-IBA)-ReCCMSH(Arg ) ␣-MSH analogs for melanoma targeting have been disappointing for 125 and I-IBA-NDP, respectively. The biodistribution data clearly demon- the most part. Radiolabeling of the gold standard ␣-MSH analog, strate that both rhenium cyclization and D-Lys incorporation enhanced 4 7 125 2 [Nle ,D-Phe ]␣-MSH (NDP), directly with I at the Tyr residue the tumor localization and retention of the radiolabel. Therefore Ac-D- resulted in very slow clearance (low tumor:blood ratios) and in vivo Lys-ReCCMSH(Arg11) is an excellent candidate for additional therapeutic studies. deiodination, making it unsuitable for melanoma radioimaging or radiotherapy (26). NDP labeled with N-succinimidyl-3-iodobenzoate 125 11 125 INTRODUCTION ( I-SIB) at Lys residues to give I-3- or 4-iodobenzoate (IBA)- NDP (conjugate labeling approach) resulted in increased affinity

Radiohalogen-labeled biomolecules such as monoclonal antibodies (lower KD;10versus 140 pM) and significantly lower thyroid and (MAbs) and peptides have attracted intensive interest for the devel- stomach uptake than 125I-Tyr2-NDP in normal mice (7). Likewise, opment of diagnostic and therapeutic radiopharmaceuticals for target- succinimidyl-4-fluorobenzoate (18F-SFB) labeled NDP exhibited rapid clearance in normal mice; however, no in vivo data are reported Received 1/29/03; revised 11/24/03; accepted 12/11/03. in tumor-bearing mice (27). Grant support: Department of Energy Grant ER60661. A novel family of ␣-MSH analogs has been developed that incor- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with porates the transition metals rhenium or technetium directly into the 18 U.S.C. Section 1734 solely to indicate this fact. peptide’s structure to generate the cyclic ␣-MSH analog Requests for reprints: Silvia S. Jurisson, Department of Chemistry, 125 Chemistry 3,4,10 7 ␣ 99m 188 Building, University of Missouri, Columbia, Missouri 65211. Phone: (573) 882-2107; ReO[Cys ,D-Phe ] -MSH3–13 [ReCCMSH (23, 24)]. Tc/ Re 3,4,10 7 ␣ Fax: (573) 882-2754; E-mail: [email protected]. cyclized [Cys ,D-Phe ]- -MSH3–13 (Tc/Re-CCMSH) showed 1411

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2004 American Association for Cancer Research. RADIOIODINATED RHENIUM CYCLIZED ␣-MSH ANALOGS high tumor uptake, prolonged tumor retention, and high stability in Radioiodination of ␣-MSH Analogs. The peptides NDP, Ac-Lys-ReCC- both B16/F1 murine and TXM-13 human melanoma-bearing mouse MSH(Arg11), and Ac-D-Lys-ReCCMSH(Arg11)[5␮g/␮l in dimethyl formam- models (23, 24). Substitution of Arg11 for Lys11 in ReCCMSH re- ide] and 3 ␮lofN-diisopropylethylamine were added to 5 ␮lof[125I]PIB sulted in the analog, ReCCMSH(Arg11), which showed greater tumor (approximately 100 ␮Ci). The reaction mixtures were incubated for 20 min at uptake and lower kidney accumulation (28, 29). Therefore, ReCCM- room temperature. After incubation, the radioiodinated peptides were purified by RP-HPLC, lyophilized, and stored at –20°C until further use. The stability SH(Arg11) was used as a structural motif for radiohalogenation. of the radiolabeled complexes was determined in 0.01 M PBS (pH 7.4)/0.1% In this report, radioiodinated ReCCMSH(Arg11) ␣-MSH analogs BSA. were designed and synthesized to demonstrate the potential for incor- Cells and Culture Conditions. B16/F1 murine melanoma cells were cul- porating radiohalogens with improved tumor uptake and retention as tured in RPMI 1640 containing NaHCO3 (2 g/liter), which was supplemented 18 123 131 agents for melanoma detection ( F and I) and therapy ( I and with 10% heat-inactivated FCS, 2 mML-glutamine, and 48 mg of gentamicin. 211At). A commercially available reagent, 125I-PIB, was selected for The cells were expanded in 75-cm2 tissue culture flasks and kept in a humid- 11 radioiodination of ReCCMSH(Arg ) because of its strong resistance ified atmosphere of 5% CO2 at 37°C, with the medium changed every other ϩ to dehalogenation on incorporation into biomolecules (2). A Lys or day. A confluent monolayer was detached with 0.02% EDTA in Ca2 - and ϩ 11 2 D-Lys residue was added at position 2 in ReCCMSH(Arg ) for Mg -free 0.01 M PBS (pH 7.4) and dissociated into a single cell suspension incorporation of the radiohalogen at the Lys ⑀-amino group. The for further cell culture. In Vitro Cell Assays. Receptor binding affinity, internalization, and cellu- D-amino acid residue (D-Lys) should further improve the in vivo lar retention assays for the 125I-labeled peptides were performed using B16/F1 stability of the peptide. The syntheses, radioiodination chemistry, in murine melanoma cells lines. Cells were prepared by seeding at a density of vitro cell binding studies, and in vivo biodistribution studies in tumor- 0.2 million cells/well in 24-well tissue culture plates and allowed to attach bearing mice are reported. overnight. After washing once with the binding media (Modified Eagle’s Medium with 25 mM HEPES, 0.2% BSA, and 0.3 mM 1,10-phenanthroline), MATERIALS AND METHODS the cells were incubated at 25°C for 2 h with 100,000–500,000 dpm of the radiolabeled complex in 0.5 ml of binding media. The nonspecific binding was Chemicals. All 9-fluorenylmethyloxycarbonyl amino acids and resins were determined by coincubation with nonradiolabeled NDP at a final concentration purchased from Nova-Biochem Co. and Advanced Chemtech (Louisville, KY). of 10 ␮M. The cells were rinsed twice with 0.01 M PBS (pH 7.4)/0.2% BSA All other chemicals were obtained from Sigma-Aldrich Chemical Co. (St. and lysed in 0.5 ml of 1 M NaOH for 5 min, and the radioactivity was Louis, MO). [125I]PIB was purchased from DuPont New England Nuclear measured. The cell binding capacity was calculated as the percentage of LifeScience (Boston, MA; 2200 Ci/mmol). B16/F1 murine melanoma cells radioactivity bound to the cells divided by the total radioactivity added. All cell were obtained from American Type Culture Collection. An Applied Biosys- binding experiments were carried out in triplicate. tems (Foster City, CA) Synergy432A desktop solid-phase peptide synthesizer The apparent equilibrium dissociation constants (KD) of the peptides were and an ISCO Inc. (Lincoln, NE) high-performance liquid chromatography determined using competitive binding experiments with radioiodinated pep- Ϫ Ϫ (HPLC) system were used for peptide synthesis and purification. tides over a 10 14 to 10 6 (M) concentration range of NDP, using the method Peptide Synthesis and Rhenium Cyclization. NDP, Ac-Lys-CCM- described previously (30). B16/F1 cells were prepared as described above in SH(Arg11), and Ac-D-Lys-CCMSH(Arg11) were synthesized by conventional 24-well tissue culture plates and incubated at 25°C for 3 h with approximately solid-phase peptide synthesis methods using 9-fluorenylmethyloxycarbonyl/O- 100,000–500,000 dpm of 125I-labeled ␣-MSH analogs in 0.5 ml of binding benzotriazolyl-tetramethyluronium hexafluorophosphate chemistry. Peptide media containing NDP at different concentrations. The radioactivity in the identities were confirmed by electrospray ionization mass spectrometry (ESI- cells and in the media was collected separately and measured. The data were 11 11 MS). Ac-Lys-ReCCMSH(Arg ) and Ac-D-Lys-ReCCMSH(Arg ) were pre- processed with the RADLIG software program, and the KD values of the pared by cyclization with rhenium coordination using a transchelation reaction radioiodinated complexes were calculated using the Origin 6.1 software pro- with rhenium-glucoheptonate, as described previously (24), and they were gram and the Cheng-Prusoff equation. lyophilized and identified by ESI-MS after purification. Ac-SYS(Nle)EHdFR- Internalization and cellular retention of the three 125I-labeled peptides were 11 WGKPV-NH2 (NDP), Ac-KCCEHdFRWCRPV-NH2 [Ac-Lys-CCMSH(Arg )], examined in B16/F1 cells prepared as described above in 24-well tissue plates. 11 125 Ac-K-ReO[CCEHdFRWC]RPV-NH2 [Ac-Lys-ReCCMSH(Arg )], Ac-DKCCE- Approximately 50,000 dpm of I-labeled peptide in 0.5 ml of binding media 11 HdFRWCRPV-NH2 [Ac-D-Lys-CCMSH(Arg )], and Ac-DK-ReO[CCEHdFR- were added to each well containing cells and incubated at 25°C for 10 min to 11 WC]RPV-NH2 [Ac-D-Lys-ReCCMSH(Arg )] were synthesized and character- 4 h. Internalization of the radiolabeled peptide was determined by washing the ized, with the ESI-MS results for these peptides in agreement with the calculated cells with acidic buffer [40 mM sodium acetate (pH 4.5) containing 0.9% NaCl values. and 0.2% BSA] to remove the membrane-bound radiocomplex and then Preparation of the Nonradioactive, Iodinated ␣-MSH Analogs. The measuring the remaining internalized radioactivity. The cellular retention 125 peptides NDP, Ac-Lys-ReCCMSH(Arg11), and Ac-D-Lys-ReCCMSH(Arg11) properties of the membrane-bound and internalized I-labeled peptides were were dissolved in dimethyl formamide to a final concentration of 5 ␮g/␮l determined by incubating B16/F1 cells in binding media at 37°C with the solution. 4-Iodobenzoic acid (13.1 ␮l; 7.2 ␮g/␮l), diiospropylethylamine (0.25 radiolabeled complexes for 2 h. The binding media were then removed, the ␮l), and O-benzotriazolyl-tetramethyluronium hexafluorophosphate (13.4 ␮l; cells were washed twice with cold 0.01 M PBS (pH 7.4)/0.2% BSA, 0.5 ml of 10.8 ␮g/␮l) were mixed together in a small tube and allowed to sit at room culture media was added, and radioactivity release into the media was moni- temperature for 10 min. The peptide (29 ␮l; 5 ␮g/␮l) was then added, and the tored at various time points over a 4-h incubation period. resulting solution was incubated at room temperature for 20 min. The resulting Tissue Distribution of Radioiodinated Peptides. All animal studies were product, the iodinated peptide, was isolated by reversed phase HPLC (RP- carried out in compliance with federal and local institutional rules for the HPLC) using a gradient system of 0.1% trifluoroacetic acid in acetonitrile conduct of animal experimentation. C57 BL/6 female mice (7–8 weeks old) (solvent B) and 0.1% trifluoroacetic acid in water (solvent A). The gradient were inoculated s.c. in the right flank with 1 ϫ 106 cultured B16/F1 murine system was optimized for each peptide analog, ranging from an initial solvent melanoma cells. Nine to ten days after inoculation, when the tumors had grown composition of 18% to 28% solvent B (depending on the peptide) going to to a weight of ϳ500 mg, the mice received injection with 2 ␮Ci of 125I-labeled 90% solvent B by 23 min and returning to the initial solvent composition by peptide through the tail vein; the mice were housed separately, and their urine 32 min. Under the optimized solvent system for each peptide, retention times and feces were collected. Because the mice were not housed in metabolic ranged from 13 to 23 min. After lyophilization, the iodinated peptides were cages, the urine in the bladder of the mouse plus all urine in the cage was 11 identified by ESI-MS. Ac-SYS(Nle)EHdFRWGK(IBA)PV-NH2 [NDP(Lys - pooled for each time point. Precautions were taken to carefully separate the IBA)], Ac-K(IBA)-ReO[CCEHdFRWC]RPV-NH2 [Ac-Lys(IBA)-ReCCM- urine and feces, and absorbent paper was placed in each cage to soak up the 11 SH(Arg )], and Ac-DK(IBA)-ReO[CCEHdFRWC]RPV-NH2 [Ac-D-Ly- urine excreted by the mouse. The mice were sacrificed at different time points s(IBA)-ReCCMSH(Arg11)] were synthesized and characterized, with the from 30 min to 72 h postinjection (p.i.). Tumors and normal tissues of interest ESI-MS results consistent with the calculated formulations. were removed and weighed, and their radioactivity was measured using a 1412

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2004 American Association for Cancer Research. RADIOIODINATED RHENIUM CYCLIZED ␣-MSH ANALOGS gamma counter. The radioactivity uptake in the tumor and normal tissues was expressed as a percentage of the injected radioactive dose per gram of tissue (% ID/g) or as a percentage of the injected dose (% ID). Statistical Method. Statistical analysis was performed using the Student’s t test for unpaired data. A 95% confidence level was chosen to determine the significance between compounds, with P Ͻ 0.05 being significantly different. HPLC Analysis of Catabolites in Urine. Urine samples were obtained during the animal experiments and pooled together for each time point. They were diluted with 500 ␮l of 0.9% NaCl and filtered through Centricon-10 filters to isolate the low molecular weight (Ͻ10,000) catabolites. The filtrate and filter were counted on a gamma counter. The filtrates were analyzed by RP-HPLC under the identical conditions used for analyzing the original radiolabeled compound. Fractions were collected in 30-s intervals and counted on a gamma counter.

RESULTS Peptide Synthesis, Iodination, and Radioiodination. Three ␣-MSH analogs [the rhenium cyclized CCMSH analogs Ac-Lys- 11 11 ReCCMSH(Arg ) and Ac-D-Lys-ReCCMSH(Arg ) (Fig. 1) and NDP] were radioiodinated with 125I-PIB for biological investigation. All peptides were synthesized using conventional solid-phase peptide synthesis methods, followed by a rhenium cyclization reaction. The nonradioactive iodinated peptides were prepared with 4-iodobenzoic acid to allow for characterization of the radioiodinated analogs of these peptides. All of the peptides were purified by RP-HPLC and characterized by ESI-MS. The radioiodinated products, which have retention times similar to the nonradioactive iodinated peptides under the same HPLC gradients, were used for biological evaluation. The effects of rhenium cyclization on the biological properties of the radiolabeled peptide were compared using the radioiodinated linear peptide NDP and the two rhenium cyclized peptides. The difference between radioiodinated D-Lys and L-Lys tumor uptake and retention was assessed. Fig. 2. Cell binding and internalization over time for 125I-IBA-NDP (A), Ac-Lys(125I- Because the amount of receptor present on the tumor cells is 11 125 11 IBA)-ReCCMSH(Arg )(B), and Ac-D-Lys( I-IBA)-ReCCMSH(Arg )(C) with limited, the radiopharmaceutical (agonist) should be of high specific B16/F1 cells at 37°C. Bound radioactivity (ࡗ) and internalized activity (f) are expressed activity. The conjugation of 125I-PIB with the peptides in dimethyl as a percentage of total activity. formamide and N-diisopropylethylamine was usually higher than 80%. The radioiodinated NDP, Ac-Lys-ReCCMSH(Arg11), and Ac- 11 labeled counterparts by HPLC, with the difference in retention times D-Lys-ReCCMSH(Arg ) were easily separated from their nonradio- of radiolabeled and unlabeled peptide being greater than 10 min, making the specific activity of the three radioiodinated peptides nearly the same as the specific activity of the starting radioiodination agent, 125I-PIB (2200 Ci/mmol). The radiochemical stability of the 125I-PIB-labeled analogs was evaluated in PBS (pH 7.4). Over a 24-h period of incubation at 25°C in PBS, only the radiolabeled peptide peak was observed by RP- HPLC. The lyophilized radiolabeled complex could be stored at –20°C for 2 weeks without any observed degradation. Cell Binding and Internalization. Fig. 2 shows the cell binding and internalization of 125I-IBA-NDP, Ac-Lys(125I-IBA)-ReCCMSH(Arg11), 125 11 and Ac-D-Lys( I-IBA)-ReCCMSH(Arg ) in B16/F1 cells at 37°C over a 4-h incubation period. The cell binding observed for 125I-IBA-NDP at 1 h of incubation was 12%, and this decreased to 3.5% at 4 h. However, the receptor binding for Ac-Lys(125I-IBA)-ReCCMSH(Arg11) and 125 11 Ac-D-Lys( I-IBA)-ReCCMSH(Arg ) steadily increased over the 4-h incubation time to 4.2% and 5.1%, respectively. The three radiolabeled analogs showed rapid internalization with approximately 80% internalization of the receptor-bound radiolabeled complexes within 5 min of incubation. The internalized versus total binding percentages did not change significantly with the incubation time. Cellular Retention. Cellular retention of the radiolabeled analogs as a function of time is shown in Fig. 3. After a 2-h incubation with B16/F1 cells, the cells were returned to the cell culture medium and 11 Fig. 1. The structure of Ac-D-Lys(*I-IBA)-ReCCMSH(Arg ). incubated at 37°C for a 4-h period. The receptor-bound radioiodinated 1413

125 125 11 125 11 Table 1 Biodistribution comparison of I-IBA-NDP (NDP), Ac-Lys( I-IBA)-ReCCMSH(Arg ) (Lys), and Ac-D-Lys( I-IBA)-ReCCMSH(Arg )(D-Lys) in B16/F1 murine melanoma-bearing C57 BL/6 mice at 30 min, 2 h, 4 h, and 24 h after injection 30 min 2 h

NDP Lys D-Lys NDP Lys D-Lys Tissues (% ID/g)a Tumor 5.34 Ϯ 1.35b 9.30 Ϯ 1.62 9.00 Ϯ 2.21 1.29 Ϯ 0.97c 14.94 Ϯ 2.34 17.69 Ϯ 4.13 Blood 2.90 Ϯ 0.17c 13.03 Ϯ 1.71c 7.26 Ϯ 0.43 0.62 Ϯ 0.18c 7.72 Ϯ 0.49 6.25 Ϯ 1.37 Heart 1.23 Ϯ 0.30c 4.54 Ϯ 0.48c 3.30 Ϯ 0.42 0.40 Ϯ 0.30c 3.10 Ϯ 0.49 2.43 Ϯ 0.48 Lung 2.96 Ϯ 0.47c 9.81 Ϯ 4.37 12.05 Ϯ 2.54 1.12 Ϯ 0.40c 8.84 Ϯ 0.46 8.19 Ϯ 1.46 Spleen 1.45 Ϯ 0.37c 12.66 Ϯ 5.30 7.13 Ϯ 1.31 0.08 Ϯ 0.15c 4.83 Ϯ 0.29c 3.33 Ϯ 0.36 Liver 6.72 Ϯ 0.49c 17.57 Ϯ 3.10 21.74 Ϯ 2.80 3.03 Ϯ 2.41c 10.53 Ϯ 0.46c 13.97 Ϯ 0.71 Kidney 26.12 Ϯ 2.34c 12.19 Ϯ 2.05 13.20 Ϯ 1.03 10.36 Ϯ 1.99 9.23 Ϯ 0.57c 11.74 Ϯ 0.94 Muscle 0.61 Ϯ 0.23c 1.45 Ϯ 0.46 1.53 Ϯ 0.24 0.33 Ϯ 0.24 0.75 Ϯ 0.27 0.83 Ϯ 0.34 %ID Stomach 1.61 Ϯ 1.11 0.74 Ϯ 0.12 0.77 Ϯ 0.15 1.01 Ϯ 0.59 0.70 Ϯ 0.31 1.12 Ϯ 0.30 Intestines 20.74 Ϯ 2.75c 4.60 Ϯ 0.40 3.92 Ϯ 0.40 22.41 Ϯ 2.18c 5.84 Ϯ 0.49 6.10 Ϯ 0.81 Urined 38.47 Ϯ 4.93c 16.08 Ϯ 2.60 15.62 Ϯ 2.07 66.13 Ϯ 1.79c 40.75 Ϯ 1.72b 37.59 Ϯ 1.22 Thyroid 0.17 Ϯ 0.07 0.70 Ϯ 0.27 0.65 Ϯ 0.45 0.02 Ϯ 0.03 0.38 Ϯ 0.23 0.31 Ϯ 0.21 Uptake ratio of tumor:normal tissue Tumor:blood 1.8 0.7 1.2 1.9 1.8 2.8 Tumor:muscle 9.3 7.2 6.0 4.5 21.9 25.1

linear ␣-MSH analog NDP showed radioactivity release from the cells The specific/nonspecific binding ratios for 125I-IBA-NDP, Ac- 125 11 125 into the medium very quickly, with only 59% remaining bound to the Lys( I-IBA)-ReCCMSH(Arg ), and Ac-D-Lys( I-IBA)-ReCCM- receptors at 10 min and decreasing to 11% at 4 h. Although 69% and SH(Arg11) were 14.8, 7.9, and 5.0, respectively. The apparent equi- 34% of the cell-associated radioactivity for Ac-Lys(125I-IBA)-ReCC- librium dissociation constants were determined using nonradioactive MSH(Arg11) remained in the cells at 10 min and 4 h, respectively, NDP as a competitor and calculated using Origin 6.1 software. The 125 11 D 125 125 11 Ac- -Lys( I-IBA)-ReCCMSH(Arg ) exhibited the slowest recep- KD values for I-IBA-NDP, Ac-Lys( I-IBA)-ReCCMSH(Arg ), 125 11 tor-bound radioactivity wash out rate of the analogs, 82% and 43% of and Ac-D-Lys( I-IBA)-ReCCMSH(Arg ) were 1.02 Ϯ 0.48 ϫ Ϫ11 Ϫ11 Ϫ11 the radioactivity associated with the cells at 10 min and 4 h, respec- 10 , 1.41 Ϯ 0.14 ϫ 10 , and 2.08 Ϯ 0.04 ϫ 10 M, respectively. These in vitro data clearly demonstrate that rhenium cycliza- 125 tively. The KD value for I-IBA-NDP determined is similar to the Ϫ11 tion significantly enhances peptide trapping and retention in the cells, reported value of 1.0 Ϯ 0.5 ϫ 10 M (7). as does D-amino acid incorporation. The combination of these two Murine Melanoma C57 Mouse Model. The in vivo biodistribu- 11 effects resulted in a 2.9-fold increase in the retention of radioactivity tion of radioiodinated NDP, Ac-Lys-ReCCMSH(Arg ), and Ac-D- 125 11 125 for Ac-D-Lys( I-IBA)-ReCCMSH(Arg ) relative to I-IBA-NDP Lys-ReCCMSH(Arg11) was examined in a B16/F1 melanoma tumor- at4h. bearing mouse model. Biodistribution data are presented in Table 1. Receptor Binding. In vitro receptor binding of radioiodinated The tumor uptake values for 125I-IBA-NDP were significantly lower ␣-MSH analogs was performed using the murine B16/F1 cell line. 125 11 than that of Ac-D-Lys( I-IBA)-ReCCMSH(Arg ) for all time points investigated (0.05 Ͻ P Ͻ 0.01 at 0.5 h and P Ͻ 0.01 at 2, 4, and 24 h). 125 The highest tumor uptake observed for Ac-D-Lys( I-IBA)-ReCCM- SH(Arg11) was 17.69 Ϯ 4.13% ID/g at 2 h p.i., compared with 5.34 Ϯ 1.35% ID/g for radioiodinated NDP at 0.5 h p.i. (Table 1). The tumor uptake observed for the L-Lys analog was lower than that observed for the D-Lys analog at most time points, although the difference was not statistically significant (P Ͼ 0.05). The radioiodinated NDP quickly washed out of the tumor, with only 0.50 Ϯ 0.20% ID/g remaining at 4 h. The radioiodinated rhenium cyclized peptides showed significantly higher tumor retention, with 4.92 Ϯ 0.48 and 7.18 Ϯ 2.14% ID/g remaining for Ac-Lys(125I-IBA)- 11 125 11 ReCCMSH(Arg ) and Ac-D-Lys( I-IBA)-ReCCMSH(Arg ), respectively, at 24 h p.i. These values are approximately 19 and 28 times greater than the tumor retention observed for 125I-IBA-NDP. The thyroid (Ͻ1% ID) and stomach (Ͻ1.61% ID) accumulations at all time points were very low (Table 1), suggesting that these 125I- PIB-labeled peptides are stable to deiodination. 125I-IBA-NDP showed lower uptake in the blood and other blood- rich organs (e.g., heart, lung, spleen, and liver) than did Ac-D- Lys(125I-IBA)-ReCCMSH(Arg11) at the earlier time points [0.5, 2, and4h(P Ͻ 0.01)], although there were no significant differences between these two compounds in most of the normal tissues at 24 h. 125 125 Fig. 3. The percentage retention of receptor-bound I-IBA-NDP (ࡗ), Ac-Lys( I- 125 11 11 125 11 The Ac-Lys( I-IBA)-ReCCMSH(Arg ) showed significantly IBA)-ReCCMSH(Arg )(f), and Ac-D-Lys( I-IBA)-ReCCMSH(Arg )(Œ) radioac- 125 11 tivity in B16/F1 cells over time at 37°C. higher blood uptake than Ac-D-Lys( I-IBA)-ReCCMSH(Arg )at 1414

Table 1 Continued 4h 24 h

NDP Lys D-Lys NDP Lys D-Lys

0.50 Ϯ 0.20c 14.94 Ϯ 2.34 15.10 Ϯ 1.38 0.26 Ϯ 0.12c 4.92 Ϯ 0.48 7.18 Ϯ 2.14 0.59 Ϯ 0.40c 8.12 Ϯ 1.23c 2.43 Ϯ 0.24 0.16 Ϯ 0.10 1.20 Ϯ 0.40c 0.17 Ϯ 0.14 0.06 Ϯ 0.10c 2.25 Ϯ 0.24c 1.21 Ϯ 0.18 0.11 Ϯ 0.13 0.51 Ϯ 0.38 0.19 Ϯ 0.14 0.41 Ϯ 0.11c 5.36 Ϯ 0.78b 3.75 Ϯ 0.65 0.09 Ϯ 0.09 0.43 Ϯ 0.33 0.17 Ϯ 0.19 0.29 Ϯ 0.34c 4.76 Ϯ 1.21b 1.71 Ϯ 0.36 0.54 Ϯ 0.38 1.22 Ϯ 0.72 0.66 Ϯ 0.40 0.39 Ϯ 0.13c 6.92 Ϯ 0.84 6.45 Ϯ 0.58 0.05 Ϯ 0.02c 0.62 Ϯ 0.08 0.55 Ϯ 0.11 3.61 Ϯ 0.41c 7.59 Ϯ 1.43 8.57 Ϯ 0.87 0.23 Ϯ 0.17c 0.77 Ϯ 0.22b 1.18 Ϯ 0.19 0.20 Ϯ 0.16 0.58 Ϯ 0.09b 0.34 Ϯ 0.16 0.11 Ϯ 0.14 0.13 Ϯ 0.11 0.07 Ϯ 0.11

0.20 Ϯ 0.08 0.46 Ϯ 0.24 0.51 Ϯ 0.25 0.02 Ϯ 0.02b 0.08 Ϯ 0.07 0.16 Ϯ 0.08 4.36 Ϯ 0.76c 9.44 Ϯ 0.65 10.20 Ϯ 0.70 0.37 Ϯ 0.40b 0.78 Ϯ 0.30 1.35 Ϯ 0.59 88.03 Ϯ 3.86c 50.19 Ϯ 2.28 56.01 Ϯ 4.74 96.05 Ϯ 2.14c 80.51 Ϯ 1.21b 76.34 Ϯ 2.38 0.12 Ϯ 0.02 0.40 Ϯ 0.22 0.26 Ϯ 0.09 0.02 Ϯ 0.03 0.06 Ϯ 0.05 0.02 Ϯ 0.03

1.0 1.9 6.3 2.0 4.3 34.3 3.4 26.6 59.9 3.8 242.1 1046.7 a Percentage of injected doselgram (% ID/g) or percentage of injected dose (%ID), n ϭ 4. b 0.05 Ͼ P Ͼ 0.01. c P Ͻ 0.01. d Urine, urine and feces at 24 h after injection. most time points (P Ͻ 0.01) and also had higher radioactivity accu- Analysis of Catabolites via RP-HPLC. To better understand the mulation in most other normal tissues, but with no significance. differences in the biological patterns of the iodinated rhenium cy- Although the three radiolabeled peptides were eliminated primarily clized peptides and the linear peptide NDP, catabolites from both the through the kidneys into the urine, the clearance for the radioiodinated in vitro cell culture studies and in vivo from urine were analyzed by linear peptide NDP was much more rapid than that for the radioiodi- RP-HPLC. nated rhenium cyclized peptides. The urine activity was The supernatants from in vitro cell culture assays at the 4 h time 88.03 Ϯ 3.86% ID for 125I-IBA-NDP at 4 h p.i., whereas it was only point were collected, separated according to molecular weight using Ϯ Ϯ 56.01 4.74% ID and 50.19 2.28% ID were observed for Ac-D- Centricon-10 filters (Mr 10,000 cutoff), and counted. The major Lys(125I-IBA)-ReCCMSH(Arg11) and Ac-Lys(125I-IBA)-ReCCM- difference observed between the catabolites of the radioiodinated SH(Arg11), respectively. The two ReCCMSH peptides exhibited linear peptide and the rhenium cyclized peptides generated from in higher gastrointestinal (GI) clearance, with feces radioactivity of vitro incubation with B16/F1 melanoma cells was that the majority of 12.18 Ϯ 1.57% ID/g and 16.71 Ϯ 3.30% ID/g at 24 h p.i., respec- the activity for 125I-IBA-NDP was in the low molecular weight 125 11 125 tively, for Ac-Lys( I-IBA)-ReCCMSH(Arg ) and Ac-D-Lys( I- (LMW) form (67% versus 24% for the Re cyclized peptides), whereas IBA)-ReCCMSH(Arg11) versus 3.18 Ϯ 2.27% ID/g for 125I-IBA- 76% of the radioactivity for both radioiodinated rhenium cyclized NDP. The reason for the increased GI excretion for Ac-Lys(125I- peptides was in the high molecular weight-associated form (versus 11 125 11 IBA)-ReCCMSH(Arg ) and Ac-D-Lys( I-IBA)-ReCCMSH(Arg ) 33% for 125I-IBA-NDP). is not clear; however, it is not due to the Re cyclization of the peptide. Urine samples collected from animal experiments were pooled, 99mTc/188Re-CCMSH exhibits predominantly urinary clearance, separated on Centricon-10 filters into high molecular weight and 111In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Ͼ10,000) and LMW (Ͻ10,000) catabolites, and then counted. The (DOTA)-ReCCMSH shows lower GI uptake and clearance than the results are presented in Table 2. The majority of the catabolites for 111 non-rhenium cyclized In-DOTA-CCMSH (31). these radioiodinated peptides are present in the LMW forms in the The radiolabeled rhenium cyclized peptides exhibited higher radio- urine, and there were no significant differences in the percentage of activity uptake in normal tissues (e.g., kidneys) than did radioiodi- LMW species for three complexes at various time points (Table 2). nated NDP; however, the rhenium-incorporated peptides showed high The LMW species were analyzed to determine the nature of the tumor uptake and retention, resulting in higher tumor:kidneys ratios catabolites excreted from mice receiving 125I-IBA-NDP, Ac-Lys(125I- 11 125 11 than observed for NDP at all time points investigated (Table 1). IBA)-ReCCMSH(Arg ), and Ac-D-Lys( I-IBA)-ReCCMSH(Arg ) Additionally, the D-Lys analog exhibited higher tumor uptake and (Fig. 4). The identities of the peaks were determined by comparison 125 faster blood clearance than the L-Lys analog. Ac-D-Lys( I-IBA)- with nonradioactive standards of the expected catabolites, 6-[N-(3- 11 ReCCMSH(Arg ) showed the highest tumor:blood and tumor:muscle iodobenzamido)]-2-aminocaproic acid (IBA-Lys), 2-[N-(3-iodobenz- ratios at most time points, with ratios of 34.3 and 1046.7 observed at amido)]acetic acid (IBA-Gly), and 3-iodobenzoic acid (IBA; Refs. 27 24 h p.i. (Table 1). and 32), analyzed under the same HPLC conditions. The hydrophi- licity order of these potential catabolites is iodide Ͼ IBA-Gly Ͼ IBA- Ͼ Table 2 Percentage of the low molecular weight catabolites present in the urine Lys IBA (27, 32), which is their order of expected elution under the collected from the mice receiving 125I-IBA-NDP (NDP), Ac-Lys(125I-IBA)- gradient system used in this study. 11 125 11 ReCCMSH(Arg ) (Lys), and Ac-D-Lys( I-IBA)-ReCCMSH(Arg )(D-Lys) at various 125 time points after injection The radiochromatogram of I-IBA-NDP indicated that all of the % ϭ (low molecular weight catabolites activity Ϭ total activity in the urine) ϫ 100. radiolabeled complex decomposed rapidly to small radioiodinated conjugates such as [125I]IBA-Lys, [125I]IBA-Gly, and some unknown Time (h) NDP (%) Lys (%) D-Lys (%) species. However, a small amount of Ac-Lys(125I-IBA)-ReCCM- 0.5 93.8 81.5 97.2 11 ϭ 2 92.1 82.2 81.4 SH(Arg ) peptide (approximate RT 18.5 min) remained in the 4 89.0 73.1 70.7 urine. The amount of small radioiodinated conjugates increased grad- 24 92.0 81.9 79.3 125 ually as a function of time. On the other hand, for Ac-D-Lys( I- 1415

dehalogenation in vivo. This combination resulted in high tumor uptake and retention for the radiohalogenated peptide. The in vitro receptor binding and in vivo experiments demonstrated that attachment of an iodobenzoate group at either Lys1 in rhenium cyclized CCMSH or Lys11 in NDP did not alter their bioactivity. Low

KD and high tumor uptake values for the radioiodinated peptides were observed. The biodistribution of NDP in B16/F1 murine melanoma- bearing C57 BL/6 mice labeled with 125I using chloramine T was reported in our previous work (26). From the biodistribution data, the conjugation labeling approach showed several advantages over direct Fig. 4. RP-HPLC radiochromatogram of urine samples obtained from tumor-bearing 125 125 11 labeling, including significantly lower stomach and thyroid uptake mice receiving I-IBA-NDP (A), Ac-Lys( I-IBA)-ReCCMSH(Arg )(B), and Ac-D- Lys(125I-IBA)-ReCCMSH(Arg11)(C) at various time points after injection. The arrows at than was observed using the chloramine-T method at all time points the baseline of each chromatogram designate the retention time of the original radioio- investigated, indicating a decrease in the in vivo deiodination of the Ϫ dinated peptide (22.2, 18.3, and 18.0 min, respectively, for A C). radiolabeled complex. The iodination site generated by the 125I-PIB labeling method is structurally dissimilar to iodotyrosine, a compound that is susceptible to multiple endogenous deiodinases. The stability to IBA)-ReCCMSH(Arg11), the majority of radioactivity remained in its deiodination of conjugation labeling agents such as PIB and SIB has original form for up to 24 h p.i. In addition, catabolite analysis of both been demonstrated repeatedly (7, 32). iodinated L-Lys and D-Lys containing rhenium cyclized peptides re- NDP labeled with 125I-PIB demonstrated faster clearance from vealed one large unknown peak eluting at a later time (RT ϭ 25 min). normal tissues and the body than peptides directly labeled with 125I. These results clearly demonstrate that the order of in vivo stability of 125 11 At 4 h p.i., 88.03% ID of the radioactivity was in the urine for these three peptides is Ac-D-Lys( I-IBA)-ReCCMSH(Arg ) Ͼ Ac- 125I-IBA-NDP, whereas only 42.67% ID was present in the urine for Lys(125I-IBA)-ReCCMSH(Arg11) Ͼ 125I-IBA-NDP. directly labeled 125I-NDP. 125I-NDP showed significantly higher radioactivity accumulation in the blood and blood-rich organs, such as DISCUSSION the heart, lung, spleen, and liver, and higher muscle uptake than 125 The presence of ␣-MSH receptors on melanoma cells and the fact I-IBA-NDP. In addition, our data are comparable with the biodis- 125 125 that the receptors undergo rapid internalization on ligand binding have tribution data for I-IBA-NDP and I-NDP reported in normal led scientists to explore the possibility of using peptide ligands as mice (7). 125 carriers to deliver radionuclides for detection and treatment of mela- The I-PIB labeling approach appears to increase tumor uptake 125 noma (21). Because radioiodinated MAbs or peptides usually exhibit and retention compared with direct labeling. I-IBA-NDP exhibited faster clearance rates from normal tissues compared with radiometal- a higher tumor:blood ratio at both 30 min and 4 h, demonstrating labeled ones, and a variety of radiohalogens are available for possible improved tumor uptake and retention for the conjugation labeling 11 imaging and therapeutic purposes, halogenated proteins continue to method. The reason for this may be that Lys is closer to the binding 2 125 play an important role in radiopharmaceutical development for cancer sequence His-D-Phe-Arg-Trp than Tyr . When proteolysis occurs, I diagnosis and treatment (7, 8). However, rapid release of radioactivity on Lys11 may remain associated with a peptide fragment retaining its from tumors after internalization of radiohalogenated peptides re- receptor binding capability, therefore increasing the tumor uptake. mains a major concern to be resolved before therapeutic trials (14, The ReCCMSH motif plays a major role in improving the tumor 33). uptake and retention of the radioiodinated complexes. Both 125I- In this study, a peptide consisting of three segments was designed labeled ReCCMSH analogs exhibited significantly higher tumor up- 125 11 125 and evaluated, namely, Ac-[ I-PIB]-D-Lys-ReCCMSH(Arg ). take and retention than the I-labeled linear peptide NDP, most ReCCMSH(Arg11) is the receptor targeting component that resists likely a result of high in vivo stability, as demonstrated by the urine proteolytic degradation, D-Lys serves as a residue for label conjuga- catabolite analysis of the three radiolabeled complexes. The use of tion and inhibits peptide degradation, and the PIB label reduces D-Lys coupling rather than L-Lys with ReCCMSH increased the in

Table 3 Comparison of in vivo distribution and uptake ratio of tumor:blood between 125I-IBA-NDP and 111In-DOTA-NDP in B16/F1 murine melanoma-bearing C57 BL/6 mice over time postinjection 30 min 4 h 24 h

125I 111Inb 125I 111Inb 125I 111Inb Tissues (% ID/g)a Tumor 5.34 Ϯ 1.35c 8.31 Ϯ 2.02 0.50 Ϯ 0.20d 7.45 Ϯ 2.38 0.26 Ϯ 0.12d 2.47 Ϯ 0.79 Blood 2.90 Ϯ 0.17d 0.59 Ϯ 0.06 0.59 Ϯ 0.40 0.11 Ϯ 0.03 0.16 Ϯ 0.10 0.02 Ϯ 0.02 Lung 2.96 Ϯ 0.47d 0.67 Ϯ 0.20 0.41 Ϯ 0.11c 0.35 Ϯ 0.14 0.09 Ϯ 0.09 0.25 Ϯ 0.18 Liver 6.72 Ϯ 0.49d 0.58 Ϯ 0.06 0.39 Ϯ 0.13 0.56 Ϯ 0.11 0.05 Ϯ 0.02d 0.38 Ϯ 0.11 Kidney 26.12 Ϯ 2.34d 8.96 Ϯ 2.24 3.61 Ϯ 0.41d 12.9 Ϯ 2.49 0.23 Ϯ 0.17d 10.2 Ϯ 2.72 %ID Intestines 20.74 Ϯ 2.75d 1.40 Ϯ 0.63 4.36 Ϯ 0.76c 1.61 Ϯ 0.47 0.37 Ϯ 0.40c 1.03 Ϯ 0.17 Urinee 38.47 Ϯ 4.93 35.97 Ϯ 4.37 88.03 Ϯ 3.86c 58.56 Ϯ 4.05 96.05 Ϯ 2.14d 75.05 Ϯ 5.25 Uptake ratio of tumor:normal tissues T/B 1.8 14.2 1.0 73.4 2.0 226.2 T/M 9.3 1.9 3.4 2.3 3.8 3.7 a % ID/g, percentage of injected dose/gram (n ϭ 4); % ID, percentage of injected dose; T/B, tumor:blood; T/M, tumor:muscle. b From Ref. 28. c 0.05 Ͼ P Ͼ 0.01. d P Ͻ 0.01. e Urine, urine and feces at 24 h after injection. 1416

125 11 111 Table 4 Comparison over time after injection of the in vivo distribution and uptake ratio of tumor:blood between Ac-D-Lys( I-IBA)-ReCCMSH(Arg ) and In-DOTA- ReCCMSH(Arg11) in B16/F1 murine melanoma-bearing C57BL/6 mice 30 min 4 h 24 h

125I 111Inb 125I 111Inb 125I 111Inb Tissues (% ID/g)a Tumor 9.00 Ϯ 2.21c 16.6 Ϯ 3.62 15.10 Ϯ 1.38 17.41 Ϯ 5.61 7.18 Ϯ 2.14 8.19 Ϯ 1.63 Blood 7.26 Ϯ 0.43c 1.70 Ϯ 0.14 2.43 Ϯ 0.24c 0.08 Ϯ 0.03 0.17 Ϯ 0.14 0.06 Ϯ 0.04 Lung 12.05 Ϯ 2.54c 1.66 Ϯ 0.40 3.75 Ϯ 0.65c 0.13 Ϯ 0.04 0.17 Ϯ 0.19 0.24 Ϯ 0.14 Liver 21.74 Ϯ 2.80c 0.66 Ϯ 0.05 6.45 Ϯ 0.58c 0.30 Ϯ 0.04 0.55 Ϯ 0.11d 0.32 Ϯ 0.04 Kidney 13.20 Ϯ 1.03c 9.31 Ϯ 0.98 8.57 Ϯ 0.87 7.37 Ϯ 1.13 1.18 Ϯ 0.19c 5.64 Ϯ 0.52 %ID Intestines 3.92 Ϯ 0.40c 0.96 Ϯ 0.15 10.20 Ϯ 0.70c 0.50 Ϯ 0.24 1.35 Ϯ 0.59 0.56 Ϯ 0.06 Urinee 15.62 Ϯ 2.07c 76.03 Ϯ 2.58 56.01 Ϯ 4.74c 89.81 Ϯ 4.22 76.34 Ϯ 2.38c 92.65 Ϯ 1.01 Uptake ratio of tumor:normal tissues T/Bf 1.2 9.7 6.3 254 34.3 95 T/M 6.0 47.4 59.9 193.2 1046.7 42.2 a % ID/g, percentage of injected dose/gram (n ϭ 4). b From Ref. 28. c P Ͻ 0.01. d 0.05 Ͼ P Ͼ 0.01. e Urine, urine and feces at 24 h after injection. f T/B, tumor:blood; T/M, tumor:muscle.

125 11 vivo stability of the iodinated complex, thus contributing to higher seen in Table 4. Both Ac-D-Lys( I-IBA)-ReCCMSH(Arg ) and 111 11 tumor retention for the D-Lys analog. In-DOTA-ReCCMSH(Arg ) showed good tumor uptake and re- The ReCCMSH motif also resulted in iodinated complexes with tention, although the iodinated analog was slower to reach its maximal significantly higher nonspecific cell binding than iodinated NDP. The uptake. The clearance rate of the 111In-labeled analog was faster than 125 125 high nonspecific binding properties of Ac-D-orL-Lys( I-IBA)- that for the I-labeled analog from most normal tissues, except the ReCCMSH(Arg11) might be responsible for their high blood activity, kidneys. The 111In-labeled peptide cleared primarily through the uri- as well as the high molecular weight catabolites observed in both cell nary system, whereas the 125I-labeled analog showed both urinary and culture supernatant and urine samples. These complexes appear to be GI clearance. The kidney clearance of the 125I-labeled analog was more lipophilic than 125I-IBA-NDP based on HPLC retention times, much better than that seen for the 111In-labeled analog, although its and this would result in greater hepatobiliary excretion, which is initial uptake was higher. Similar tumor uptake and significantly observed (Table 1). The more rapid clearance rate from blood and lower kidney uptake for the iodinated peptide compared with the 125 11 111 11 other normal tissues for Ac-D-Lys( I-IBA)-ReCCMSH(Arg ) com- In-labeled peptide suggest that Ac-D-Lys-ReCCMSH(Arg )la- 131 211 pared with the L-Lys analogs, however, is not understood at this point. beled with therapeutic halogens such as I and At might have The long-term goal of our research is to develop therapeutic radio- some advantages for melanoma therapy over DOTA-ReCCM- pharmaceuticals for melanoma treatment based on radiolabeled SH(Arg11) labeled with ␣- and ␤-emitting radiometals. 125 11 ␣-MSH analogs. Because ␣-MSH analogs labeled with either radio- In conclusion, Ac-D-Lys( I-IBA)-ReCCMSH(Arg ) exhibits halogens (i.e., 131I and 211At) or radiometals (90Y, 177Lu, and 212Bi) high tumor uptake and retention, comparable with 111In-DOTA-ReC- are potential candidates for melanoma targeting, it is important CMSH(Arg11). It is also rapidly cleared from normal tissues. The to compare the distributions of radiohalogenated (125I-labeled) and favorable biodistribution characteristics of this molecule make it a radiometalated (111In-labeled) ␣-MSH analogs. A comparison of superb candidate for conjugation with radionuclides used for treat- 125 111 125 11 the biodistributions of I-IBA-NDP and In-DOTA-NDP and ment of melanoma. Ac-D-Lys( I-IBA)-ReCCMSH(Arg ) was de- 125 11 111 of Ac-D-Lys( I-IBA)-ReCCMSH(Arg ) and In-DOTA-ReCC- veloped as an iodinated ␣-MSH analog, which exhibits high tumor MSH(Arg11) is listed in Tables 3 and 4, respectively. uptake and retention and rapid normal tissue clearance. To the best of Table 3 compares the biodistribution data for the two radiolabeled our knowledge, this is the first iodinated peptide developed with such linear NDP analogs, 125I-IBA-NDP and 111In-DOTA-NDP, and it is long tumor retention. Melanoma radiotherapy experiments will be clear that their biodistribution patterns are different. The tumor uptake performed based on this molecule. for the 111In-labeled complex was approximately 10 times that of the 125I-labeled peptide at 24 h p.i.; however, it cleared at a slower rate from normal tissues. The uptake for the dose-limiting kidneys was ACKNOWLEDGMENTS significantly higher at 24 h p.i. for 111In-DOTA-NDP than for 125I- We express our gratitude to Drs. Timothy J. Hoffman, Wynn Volkert, and IBA-NDP (10.2% ID/g versus 0.23% ID/g). These observations can Susan L. Deutscher for helpful discussions and assistance and Dr. Nellie K. be attributed to the different metabolic patterns of radioiodinated Owen and Donna Whitener for technical support. versus radiometal chelated peptides. The catabolites from the iodinated peptide (i.e., IBA, IBA-Lys, and IBA-Gly) are most likely expelled from the cells and excreted from the body quickly. However, REFERENCES the free radiometal and/or the radiometal chelate complex remained in 1. Wilbur, D. S. Radiohalogenation of proteins: an overview of radionuclides, labeling the cells after metabolism, thus resulting in an apparent higher radio- methods, and reagents for conjugate labeling. Bioconjugate Chem., 3: 433–470, activity accumulation for those organs (34–38). The clearance routes 1992. were also different, with 111In-DOTA-NDP clearing predominantly 2. Wilbur, D. S., Hadley, S. W., Hylarides, M. D., Abrams, P. G., Beaumier, P. L., Morgan, A. C., Reno, J., and Fritzberg, A. R. 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Zhen Cheng, Jianqing Chen, Thomas P. Quinn, et al.

Cancer Res 2004;64:1411-1418.

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