In Vitro and in Vivo Comparison of Human Escherichia coli Heat-Stable Peptide Analogues Incorporating the ¹¹¹In-DOTA Group and Distinct Linker Moieties

Abstract

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.

Pre-clinical evaluation of [111In]-benzyl-DOTA-ZHER2:342, a potential agent for imaging of HER2 expression in malignant tumors

“…The same inertness necessitates the use of elevated temperatures for chelation, and the literature often reports on labeling at 80-100˚C using long reaction times (28,35,36). In our case, >95% labeling efficiency was obtained within 5 min at a lower temperature, 60˚C.…”

Influence of Chelate Conjugation on a Newly Identified Tumor-Targeting Peptide

“…The MCF-7 cells lines showed faster binding kinetics in vitro as compared with the FRO82-2; in vivo the MCF-7 tumors showed more activity in the tumor than the FRO82-2, which could be the result of the faster peptide binding of the MCF-7 tumor cells. The fast renal clearance is caused by the hydrophilic chelator DOTA (15). Consequently, derivatives with a prolonged circulation time to provide the time necessary for successful interaction with the target cells in vivo must be investigated as a next step (16).…”

In vitro and in vivo Evaluation of 111In-labeled E. coli Heat-Stable Enterotoxin Analogs for Specific Targeting of Human Breast Cancers

“…IC 50 analysis of both peptides in competition with 125 I-F 19 -ST h (1-19) using both ER + and ER ) human breast cancer cell lines demonstrates that these peptides have affinities for human breast cancer cells similar to those for human colon cancer lines (Figure 2, Table 1) [20,23]. Scatchard analyses of binding of 125 I-F 19 -ST h (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) to human breast cancer cells indicates that cell surface receptor concentrations range between 40,000 and 110,000 sites/cell for the T-47D and MDA-MB-231 lines, respectively (Figure 3).…”

“…Other reagents were purchased from Aldrich Chemical Company, Bachem, Nova Biochem, Quiagen, Invitrogen, and Applied Biosystems. 111 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) were synthesized according to a previously published procedure [20]. Human breast cancer MDA-MB-231 and T-47D cells as well as human colon cancer T-84 cells were obtained from American Type Culture Collection (ATCC) and maintained and grown for use in these studies in the University of Missouri Cell and Immunology Core facilities.…”

“…ST h and related peptides are bacterial mimics of guanylin peptides, and are the highest affinity ligands known for the GC-C receptor [19]. The high expression of GC-C on primary and metastatic colorectal adenocarcinomas has catalyzed the development of radiolabeled ST h analogs for in vivo imaging and therapy of colorectal cancer [20][21][22][23][24]. Here we report that ST h analogs bind with high affinity to a cell surface ST h binding protein (STBP) expressed in both estrogen receptor positive (ER + ) and estrogen receptor negative (ER ) ) human breast cancer cell lines.…”