Metal ions often play critical roles in protein structure and function. Engineered metal-binding sites in peptides and proteins have been widely used to enhance structural integrity, stabilize biologically active conformations, and confer novel enzymatic activities (1-3). Biochemical and structural analyses of transition-metal coordination by proteins and peptides have traditionally focused on zinc, copper, manganese, and iron because of their roles in important biological processes (4-7). Other transition metals not found in natural proteins have coordination, isotopic, and chemical properties that make them attractive for peptide and protein engineering. Rhenium (Re) and technetium (Tc) are group VIIB transition metals that share similar coordination geometries and form stable complexes with amine and amide nitrogens, carboxylate oxygens, and thiolate and thioether sulfurs, with a strong preference for thiolate sulfurs (8). Radioactive isotopes of Re and Tc have significant medical applications because of the nature of their associated radiation and physical half-life properties.The synthesis and characterization of radiolabeled antibodies, peptides, and steroid hormones as in vivo tumor-imaging and therapeutic agents is an active area of cancer research today. These molecules specifically target tumor cells by virtue of their high specificities for receptors and antigens present on the surfaces of these cells. In one commonly used approach, metallic radionuclides such as 186 Re, 188

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Receptor binding peptides labeled with medically important radionuclides such as technetium and rhenium are an important tool for the imaging and treatment of many forms of cancer. This paper describes a method of labeling peptides with rhenium using a natural amino acid chelating moiety. The structural characteristics of this chelate moiety, N-acetyl-cysteine-glycine-cysteine-glycine (NAc-CGCG) complexed with nonradioactive rhenium, have been investigated. The stability of this peptide-metal complex has been evaluated on the tracer level using radioactive rhenium-186. The rhenium-bound peptide has been appended to the N termini of receptor binding alpha-melanocyte stimulating hormone (alpha-MSH, NAc-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) fragments via solid phase peptide synthesis. Bioassays and receptor binding studies of the resulting complexes demonstrate that the fragments retained biological activity and exhibited receptor binding constants ranging from 0.3 to 1.1 nM. This method could provide a general means of labeling bioactive peptide fragments that would simplify product purification and characterization.

This study describes the synthesis and preliminary biologic evaluation of an 111 Inlabeled peptide antagonist of the urokinase-type plasminogen activator receptor (uPAR) as a potential probe for assessing metastatic potential of human breast cancer in vivo. The peptide (NAc-dD-CHA-F-dS-dR-Y-L-W-S-βAla) 2 -K-K(DOTA)-NH 2 was synthesized and conjugated with the DOTA chelating moiety via conventional Solid-Phase Peptide Synthesis (SPPS), purified by reversed-phase HPLC, and characterized by MALDI-TOF MS and receptor binding assay. In vitro receptor binding studies demonstrated an IC 50 of 240 ± 125 nM for the peptide, compared with IC 50 's of 0.44 ± 0.02 and 0.75 ± 0.01 nM for the amino terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA) and full-length uPA, respectively. In vivo biodistribution studies were carried out using SCID mice bearing MDA-MB-231 human breast cancer xenografts. Biodistribution data was collected at 1, 4, and 24 hr post-injection of 111 In-DOTA-peptide, and compared with data obtained using a scrambled control peptide, as well as with data obtained using wild-type ATF radiolabeled with I-125. Biodistribution studies showed rapid elimination of the 111 In-labeled peptide from the blood pool, with 0.12 ± 0.06% ID/g remaining in blood at 4 hr pi. Elimination was seen primarily via the renal/ urinary route, with 83.9 ± 2.2%ID in the urine at the same timepoint. Tumor uptake at this time was 0.53 ± 0.11%ID/g, resulting in tumor: blood and tumor: muscle ratios of 4.2 and 9.4, respectively. Uptake in tumor was significantly higher than that obtained using a scrambled control peptide that showed no specific binding to uPAR (p < 0.05). In vitro and ex vivo results both suggested that the magnitude of tumor-specific binding was reduced in this model by endogenous expression of uPA. The results indicate that radiolabeled peptide uPAR antagonists may find application in the imaging and therapy of uPAR-expressing breast cancers in vivo.

Three human Escherichia coli heat-stable peptide (STh) analogues, each containing a DOTA chelating group, were synthesized by SPPS and oxidative refolding and compared in in vitro and in vivo systems. One analogue, DOTA-F19-STh(1-19), contains an N-terminal DOTA group attached via an amide bond linkage to an STh moiety which is essentially wild-type except for a Tyr to Phe alteration at position 19 of the molecule. A second analogue, DOTA-R1,4,F19-STh(1-19), differs from the first in that asparagine residues in positions 1 and 4 have been altered to arginine residues in order to examine the effect of positively charged groups in the linker domain. A third analogue, DOTA-11AUN-F19-STh(1-19), differs from the first in that it incorporates an 11-aminoundecanoic acid spacer group between the DOTA group and the first asparagine residue. In vitro competitive binding assays utilizing T-84 human colon cancer cells demonstrated that significant alterations to the N-terminal region of the STh molecule were well tolerated and did not significantly affect binding affinity of STh for the guanylyl cyclase C (GC-C) receptor. Internalization and efflux studies of the indium-labeled species demonstrated that inclusion of positive charge in the linker moiety inhibits internalization of the compound within tumor cells. The characteristics of the three analogues were compared in an in vivo model utilizing T-84 human colon cancer cell xenografts in SCID mice. Clearance of all analogues was rapid, primarily via renal excretion into the urine, with >89% ID excreted into the urine at 1 h pi for all analogues. The 111In-DOTA-R1,4,F19-STh(1-19) and 111In-DOTA-11AUN-F19-STh(1-19) analogues both had longer residence times in the blood than did the 111In-DOTA-F19-STh(1-19) analogue, probably accounting for increased %ID/g values for tumors and nontarget tissues at 1 h pi. At 4 h pi, significant differences between analogues were only seen with respect to metabolic routes of excretion, indicating that increased blood residence time did not result in increased tumor residualization. Reduction of hepatic uptake of these compounds, however, could have significance in the development of agents for the imaging of hepatic metastases. The ability to manipulate in vivo pharmacodynamics and tumor uptake of radiolabeled STh peptides through modification of linker moieties is under continuing investigation in order to produce optimal imaging and therapeutic radiopharmaceuticals.

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Analogs of the E. coli heat-stable enterotoxin (STh) are currently under study as both imaging and therapeutic agents for colorectal cancer. Studies have shown the guanylate cyclase C (GC-C) receptor is commonly expressed in colorectal cancers. It has also been shown that STh peptides inhibit the growth of tumor cells expressing GC-C. The ability to determine GC-C status of tumor tissue using in vivo molecular imaging techniques would provide a useful tool for the optimization of GC-C-targeted therapeutics. In this work we have compared receptor binding affinities, internalization/efflux rates, and in vivo biodistribution patterns of an STh analog linked to Nterminal DOTA, TETA, and NOTA chelating moieties and radiolabeled with Cu-64. The peptide F 19 -STh(2-19) was N-terminally labeled with three different chelating groups via NHS ester activation and characterized by RP-HPLC, ESI-MS, and GC-C receptor binding assays. The purified conjugates were radiolabeled with Cu-64 and used for in vitro internalization/efflux, in vivo biodistribution, and in vivo PET imaging studies. In vivo experiments were carried out using SCID mice bearing T84 human colorectal cancer tumor xenografts. Incorporation of DOTA-, TETA-, and NOTA-chelators at the N-terminus of the peptide F 19 -STh(2-19) resulted in IC 50 's between 1.2-3.2 nM. In vivo, tumor localization was similar for all three compounds, with 1.2-1.3 %ID/g at 1 hr pi and 0.58-0.83 %ID/g at 4 hr pi. The principal difference between the three compounds related to uptake in nontarget tissues, principally kidney and liver. At 1 hr pi, 64 Cu-NOTA-F 19 -STh(2-19) demonstrated significantly (p < 0.05) lower uptake in liver than 64 Cu-DOTA-F 19 -STh(2-19) (0.36 ± 0.13 vs. 1.21 ± 0.65 %ID/g), and significantly (p < 0.05) lower uptake in kidney than 64 Cu-TETA-F 19 -STh(2-19) (3.67 ± 1.60 vs. 11.36 ± 2.85 %ID/g). Use of the NOTA chelator for coordination of Cu-64 in the context of E. coli heat-stable enterotoxin analogs results in higher tumor:nontarget tissue ratios at 1 hr pi than either DOTA or TETA macrocycles. Heat-stable enterotoxin-based radiopharmaceuticals such as these provide a means of noninvasively determining GC-C receptor status in colorectal cancers by PET.