ABSTRACT
Various aspects of radiopeptide receptor-mediated cell internalisation and externalization assays were assessed, including the integrity of externalized peptides and the effect of varying the pH and incubation time of the acid wash step (to remove surface receptor-bound ligand) on efficacy and cell viability. The observed intact proportion of externalized peptide was 5-10%, and acid wash buffers with pH 2.8 or below were found to be detrimental to cell viability and integrity, particularly following prolonged incubation times.
Subject(s)
Gastrins/pharmacokinetics , Indium Radioisotopes/pharmacokinetics , Organometallic Compounds/pharmacokinetics , Pancreatic Neoplasms/radiotherapy , Radiopharmaceuticals/pharmacokinetics , Animals , Cell Line, Tumor , Cell Survival/drug effects , Endocytosis/physiology , Exocytosis/physiology , Gastrins/chemistry , Gastrins/pharmacology , Indium Radioisotopes/chemistry , Indium Radioisotopes/pharmacology , Organometallic Compounds/chemical synthesis , Organometallic Compounds/pharmacology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Radiopharmaceuticals/chemical synthesis , Radiopharmaceuticals/pharmacology , RatsABSTRACT
Radiolabeled neuropeptides are widely explored for targeting tumours for either imaging or radiotherapeutic purposes. After binding to their receptors, these peptides are rapidly internalized into lysosomes, where they are degraded by proteolytic enzymes, such as cathepsins. The aim of this study was to investigate the effect of the inclusion of specific cleavage sites for cathepsin B into the peptide sequence. The cleavage site, GFLG, together with a series of dipeptides for pharmacokinetic modification of radiometabolites, were, therefore, inserted into a peptide that binds to the gastrin/CCK2 receptor. The receptor binding of the peptides was explored in AR42J cells, rates of internalization, and externalization of the radionuclide were measured and the nature of the radiometabolites explored. The effects of the modifications on biodistribution in tumor-bearing mice was explored by high-resolution single-photon emission computed tomography imaging. Differences in rates of externalization from tumor cells in vitro and in the rates of washout from tumor and kidney in vivo were observed. These results indicate that insertion of an enzymatic cleavage site, such as that for cathepsin B, into a neuropeptide appears to have an influence on the intracellular processing, which results in a change in the rate of egress of radioactivity from target and nontarget tissues.
