Comparative evaluation of three 64Cu-labeled E. coli heat-stable enterotoxin analogues for PET imaging of colorectal cancer.

Analogues of the E. coli heat-stable enterotoxin (STh) are currently under study as both imaging and therapeutic agents for colorectal cancer. Studies have shown that 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 analogue linked to N-terminal 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 and 3.2 nM. In vivo, tumor localization was similar for all three compounds, with 1.2-1.3%ID/g at 1 h pi and 0.58-0.83%ID/g at 4 h pi. The principal difference between the three compounds related to uptake in nontarget tissues, principally kidney and liver. At 1 h 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 analogues results in higher tumor/nontarget tissue ratios at 1 h 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.

Radiometallated peptides targeting guanylate cyclase C and the urokinase-type plasminogen activator receptor.

Research is currently underway worldwide into the development of receptor-specific radiopharmaceuticals for the imaging and treatment of cancer. The successful clinical development of radiolabeled somatostatin analogs for imaging and treatment of cancers overexpressing somatostatin receptors has catalyzed further preclinical investigation of other radiolabeled peptides for molecular imaging and peptide-receptor radiotherapy, including such well-studied peptide vectors as cholecystokinin, neurotensin, bombesin and RGD peptides. Within this larger context, this article will focus on the current status of two more recent additions to the list of molecular imaging targets - guanylate cyclase C, a specific marker for colorectal cancer, and the urokinase plasminogen activator receptor, a cell-surface receptor overexpressed in diverse cancer types.

Synthesis and characterization of an (111)In-labeled peptide for the in vivo localization of human cancers expressing the urokinase-type plasminogen activator receptor (uPAR).

This study describes the synthesis and preliminary biologic evaluation of an (111)In-labeled 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-betaAla)(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) values 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 h postinjection 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)-labeled peptide from the blood pool, with 0.12 +/- 0.06% ID/g remaining in blood at 4 h pi. Elimination was seen primarily via the renal/urinary route, with 83.9 +/- 2.2% ID in the urine at the same time point. 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.

In vitro and in vivo evaluation of 177Lu- and 90Y-labeled E. coli heat-stable enterotoxin for specific targeting of uroguanylin receptors on human colon cancers.

The human E. coli heat-stable enterotoxin (ST(h), amino acid sequence N1SSNYCCELCCNPACTGCY19) binds specifically to the guanylate cyclase C (GC-C) receptor, which is present in high density on the apical surface of normal intestinal epithelial cells as well as on the surface of human colon cancer cells. Analogs of ST(h) are currently being used as vectors targeting human colon cancers. Previous studies in our laboratory have focused on development of 111Indium-labeled ST(h) analogs for in vivo imaging applications. Here, we extend the scope of this work to include targeting of the therapeutic radionuclides 90Y and 177Lu. The peptide DOTA-F19-ST(h)(1-19) was synthesized using conventional Fmoc-based solid-phase techniques and refolded in dilute aqueous solution. The peptide was purified by RP-HPLC and characterized by MALDI-TOF MS and in vitro receptor binding assay. The DOTA-conjugate was metallated with nonradioactive Lu(III)Cl3 and Y(III)Cl3, and IC50 values of 2.6+/-0.1 and 4.2+/-0.9 nM were determined for the Lu- and Y-labeled peptides, respectively. 177Lu(III)Cl3 and 90Y(III)Cl3 labeling yielded tracer preparations that were inseparable by C18 RP-HPLC, indicating that putative differences between Lu-, Y- and In coordination spheres are not observed in the context of labeled ST(h) peptides. In vivo biodistribution studies of the 177Lu-labeled peptide in severe combined immunodeficient (SCID) mice bearing T-84 human cancer tumor xenografts showed rapid clearance from the bloodstream, with >90 %ID in the urine at 1 h pi. Localization of the tracer within tumor xenografts was 1.86+/-0.91 %ID/g at 1 h pi, a value higher than for all other tissues with the exception of kidney (2.74+/-0.24 %ID/g). At 24 h pi, >98 %ID was excreted into the urine, and 0.35+/-0.23 %ID/g remained in tumor, again higher than in all other tissues except kidney (0.91+/-0.46 %ID/g). Biodistribution results at 24 h pi for the 90Y-labeled peptide mirrored those for the 177Lu analog, in agreement with the identical behavior of the labeled analogs by C18 RP-HPLC. These results demonstrate the ability of 177Lu- and 90Y-labeled ST(h) molecules to specifically target GC-C receptors expressed on T-84 human colon cancer cells.

Selective targeting of E. coli heat-stable enterotoxin analogs to human colon cancer cells.

BACKGROUND: Radiolabeled analogs of the E. coli heat-stable enterotoxin (ST(h)) are currently under study as imaging and therapeutic agents for colorectal cancer. The aim of these studies is to compare in vitro and in vivo characteristics of two novel ST(h) analogs with appended DOTA chelating moieties. MATERIALS AND METHODS: ST(h) analogs were synthesized with pendant N-terminal DOTA moieties and radiolabeled with indium-111. In vitro cell binding was studied using cultured T-84 human colorectal cancer cells, and in vivo biodistribution studies were carried out using T-84 human colorectal tumor xenografts in SCID mice. RESULTS: Competitive radioligand binding assays employing T-84 human colon cancer cells demonstrated similar IC50 values for the F19-ST(h)(2-19) and F9-ST(h)(6-19) analogs. Addition of DOTA to the N-terminus of these peptides elicited distinctly different effects on binding affinities in vitro, effects that were largely unchanged by metallation with nonradioactive (nat)In. In vivo pharmacokinetic studies in SCID mice bearing T-84 human colon cancer-derived tumor xenografts demonstrated tumor uptake of 0.74 +/- 0.1% ID/g at 4 h post-injection (p.i.) for the 111In-DOTA-F19-ST(h)(2-19) analog, and significantly reduced tumor localization (0.27 + 0.08 % ID/g) for the 111In-DOTA-F9-ST(h)(6-19) analog. CONCLUSION: These results demonstrate that placement of a DOTA moiety immediately adjacent to Cys 6 in ST(h) significantly inhibits receptor binding in vitro and in vivo, highlighting the need for intervening spacer residues between the pharmacophore and the DOTA chelating moiety in effective ST(h)-based radiopharmaceutical constructs.

Radiometallation of receptor-specific peptides for diagnosis and treatment of human cancer.

Radiolabeled, receptor-specific peptides are becoming increasingly popular as targeting vectors for the design and development of new diagnostic and therapeutic radiopharmaceuticals. The over-expression of functioning receptors on a variety of human cancers makes this method of drug development a viable tool for tumor targeting in vivo. This review describes some of the more recent efforts that are currently underway towards development of new receptor-specific radiopharmaceuticals. Diagnostic/therapeutic radionuclides, specific metal co-ordinating ligands/chelating systems, spacer technology, radiolabeling protocols, and specific peptides/peptide conjugates will be discussed in detail.

In vivo imaging of human colorectal cancer using radiolabeled analogs of the uroguanylin peptide hormone.

BACKGROUND: Uroguanylin is an endogenous peptide agonist that binds to the guanylate cyclase C receptor (GC-C). GC-C is overexpressed in human colorectal cancer (CRC), and exposure of GC-C-expressing cells to GC-C agonists results in cell cycle arrest and/or apoptosis, highlighting the therapeutic potential of such compounds. This study describes the first use of radiolabeled uroguanylin analogs for in vivo detection of CRC. MATERIALS AND METHODS: The peptides uroguanylin and E(3)-uroguanylin were N-terminally labeled with the DOTA chelating group via NHS ester activation and characterized by RP-HPLC, ESI-MS, and GC-C receptor binding assays. The purified conjugates were radiolabeled with In-111 and used for in vivo biodistribution and SPECT imaging studies. In vivo experiments were carried out using SCID mice bearing T84 human colorectal cancer tumor xenografts. RESULTS: Alteration of the position 3 aspartate residue to glutamate resulted in increased affinity for GC-C, with IC(50) values of 5.0+/-0.3 and 9.6+/-2.9 nM for E(3)-uroguanylin and DOTA-E(3)-uroguanylin, respectively. In vivo, (111)In-DOTA-E(3)-uroguanylin demonstrated tumor uptake of 1.17+/-0.23 and 0.61+/-0.07% ID/g at 1 and 4 h post injection, respectively. The specificity of tumor localization was demonstrated by coinjection of 3 mg/kg unlabeled E(3)-uroguanylin, which reduced tumor uptake by 69%. Uptake in kidney, however, was dramatically higher for the uroguanylin peptides than for previously characterized radiolabeled E. coli heat-stable enterotoxin (STh) analogs targeting GC-C, and was also inhibited by coinjection of unlabeled peptide in a fashion not previously observed. CONCLUSION: Use of uroguanylin-targeting vectors for in vivo imaging of colorectal cancers expressing GC-C resulted in tumor uptake that paralleled that of higher affinity heat-stable enterotoxin peptides, but also resulted in increased kidney uptake in vivo.

In vitro and in vivo evaluation of 111In-labeled E. coli heat-stable enterotoxin analogs for specific targeting of human breast cancers.

Research into the interaction between the E. coli heat-stable enterotoxin (STh) and the guanylin receptor guanylate cyclase C (GC-C) has generated >100 synthetic analogs of the peptide, several of which have been investigated as imaging or therapeutic agents for colorectal cancers. The evidence presented here suggests that in addition to STh binding to GC-C expressing cell lines derived from human colon, STh also specifically binds to an as yet unidentified receptor expressed in high densities on the surface of cell lines derived from human breast cancers. In vitro whole-cell crosslinking studies using 125I-labeled F19-STh(1-19) demonstrate that the putative STh binding protein migrates as an approximately 120-125 kDa species by SDS-PAGE, significantly smaller than the glycosylated GC-C molecule found in the T84 human colon cancer cell line. RT-PCR using total RNA isolated from breast and colon cancer cell lines indicates that GC-C transcripts are undetectable in human breast cancer cell lines and abundant in human colon cancer cell lines. In vitro competitive binding studies using STh analogs and the estrogen receptor positive (ER+) T-47D cell line demonstrated IC50 values between 2.6 and 8.5 nM. Similar studies on the estrogen receptor negative (ER-) cell line MDA-MB-231 showed IC50's between 5.6 and 9.9 nM. Saturation binding analysis revealed receptor expression to fall between 40,000 and 120,000 sites per cell in these cell lines, receptor abundances equal to or greater than the abundance of GC-C in colorectal cancer cell lines. STh binding to these cells, although of similar affinity to STh binding to GC-C, is distinguishable from it on the basis of its ligand specificity. The characteristics of STh analogs as radiopharmaceutical agents were tested in an in vivo model utilizing T-47D human breast cancer cell xenografts in SCID mice. Clearance of STh analogs was rapid, primarily via renal excretion into the urine, with >85% ID excreted into the urine at 1 h p.i. Tumor uptake at 1 h p.i. in T-47D tumor cell xenografts was 0.67+/-0.23% ID/g, and was significantly decreased (p<0.05) upon co-administration of 4 mg/kg unlabeled STh. These results suggest that STh may find application for the imaging and treatment of breast cancer.

In vitro and in vivo comparison of human Escherichia coli heat-stable peptide analogues incorporating the 111In-DOTA group and distinct linker moieties.

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.