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 IC 50 values for the F 19 -ST h (2-19) and F 9 -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 111 In-DOTA-F 19 -ST h (2-19) analog, and significantly reduced tumor localization (0.27 + 0.08 %ID/g) for the 111 In-DOTA-F 9 -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.