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-F-19-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-R-1,R-4,F-19-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-F-19-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 In-111-DOTA-R-1,R-4,F-19-STh(1-19) and In-111-DOTA-11AUN-F-19-STh(1-19) analogues both had longer residence times in the blood than did the In-111-DOTA-F-19-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.