[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.