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Recent years have seen an increased effort in the development of peptide based radiopharmaceuticals for nuclear medicine applications in imaging and therapy. This field is of particular interest in the development of new cancer imaging and treatment strategies. Major developments in the molecular biology of cancer have brought forth the discovery of a number of receptor systems and other cell surface molecules that can be utilized as molecular targets for peptide based agents. Although such a strategy is very appealing and shows great potential for application in humans, there are as yet very few radiopharmaceuticals developed based on this scheme that have made it into clinical practice. Many different factors contribute to the generation of a successful radiopharmaceutical. Among these, a thorough and efficient preclinical evaluation process is necessary to single out those lead compounds that show more promise and are most likely to be winners in clinical applications. To maximize the efficacy of pre-clinical testing one must utilize currently available technology to the best extent possible. Expertise in different areas of drug development is indispensable for this type of research. This work will analyze currently available methods utilized to evaluate radiopharmaceuticals being developed, from compound design to evaluation in pre-clinical animal models.

Regulatory peptides and their analogs are being extensively investigated as radiopharmaceuticals for cancer imaging. In particular, cholecystokinin (CCK) receptors of the subtype B (CCK-BR) have been shown to be overexpressed in certain neuroendocrine tumors including medullary thyroid cancer. Our recent work has focused on new methods to radiolabel the CCK8 peptide with ¹¹¹In or ^99mTc for the purpose of developing radiopharmaceuticals for in vivo CCK-B receptor imaging. Labeling of CCK8 with ¹¹¹In was achieved at the N-terminus of the peptide by adding, in solid phase, a glutamate coupled diethylenetriaminepentaacetic acid (DTPA) moiety through a glycine linker, yielding DTPA–Glu–G–CCK8. For labeling with ^99mTc, the CCK8 peptide was modified at its N-terminus by introducing, in the following order—cysteine, glycine, and a diphenylphosphinopropionyl moiety—giving a 10-residue peptide derivative, Phos–GC–CCK8. A cell culture model was developed for the purpose of evaluating the binding properties of these two ligands. The human epidermoid carcinoma cell line, A431, was transfected with a plasmid containing the full coding sequence of the human CCK-BR under a strong viral promoter, obtaining a number of receptors in the range of 2–5 × 10⁶ per cell. Control cells were transfected with vector alone. An animal tumor model utilizing these two cell lines was developed to evaluate the specificity of interaction with the CCK-BR and biodistribution properties of the compounds. CCK-BR positive and control cells were subcutaneously injected in opposite flanks of CD1 female nude mice in order to obtain xenografts differing only in their ability to express CCK-B receptors. High performance liquid chromatography (HPLC) and other chromatographic methods were utilized to assess stability of the radiolabeled compounds after injection. Both ¹¹¹In–DTPA–Glu–G–CCK8 and ^99mTc–Phos–GC–CCK8 showed similar binding affinities for cultured CCK-BR expressing cells, with dissociation constants in the range of 20–40 nM. With the two xenograft approach, we were able to demonstrate specific interaction with the receptor of both CCK analogs in our animal model. The data obtained shows rapid specific localization of both compounds on the CCK-BR overexpressing xenografts. Both tracers show rapid plasma clearance of unbound peptide. Clearance of ¹¹¹In–DTPA–Glu–G–CCK8 appears to be preferentially through the kidneys, whereas ^99mTc–Phos–GC–CCK8 clearance occurs both through kidneys and the hepatobiliary system. Both our labeling approaches appear adequate for clinical use of peptide based radiopharmaceuticals, although ^99mTc–Phos–GC–CCK8 shows elevated accumulation in the gastrointestinal tract, which causes high background activity. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 66: 370–380, 2002

We have studied the expression of cell polarity in hybrids between two rat thyroid epithelial cells: FRT and FRTL-5. FRT cells are polarized but do not express tissue-specific properties, FRTL-5 are unpolarized and express many thyroid-specific genes. A and express many thyroid-specific genes. A pool of 170 hybrid clones and five independent clones were characterized. The chromosome complement was that expected from 1:1 fusion of the parental cells. No chromosome loss was observed for several generations. All hybrids were polarized as judged from: (1) morphology, (2) transepithelial resistance, (3) preferential secretion of several proteins either through the apical (e.g. thyroglobulin) or through the basolateral pole, and (4) basolateral trapping of iodide. On the other hand, the expression of thyroid-specific markers: thyroglobulin synthesis and secretion, trapping of iodide, thyrotropin-dependent growth and expression of specific membrane antigens, were greatly reduced or inhibited in the pool and in the isolated clones. We also found that reduction of thyroglobulin synthesis was correlated with the loss of activity of the trans-acting factor TgTF1. We conclude that cell polarity, a property of FRT cells, is dominant in the hybrids whereas thyroid differentiation is recessive.

We compared the surface envelope glycoprotein distribution and the budding polarity of four RNA viruses in Fischer rat thyroid (FRT) cells and in CaCo-2 cells derived from a human colon carcinoma. Whereas both FRT and CaCo-2 cells sort similarly influenza hemagglutinin and vesicular stomatitis virus (VSV) G protein, respectively, to apical and basolateral membrane domains, they differ in their handling of two togaviruses, Sindbis and Semliki Forest virus (SFV). By conventional EM Sindbis virus and SFV were shown to bud apically in FRT cells and basolaterally in CaCo-2 cells. Consistent with this finding, the distribution of the p62/E2 envelope glycoprotein of SFV, assayed by immunoelectronmicroscopy and by domain-selective surface biotinylation was predominantly apical on FRT cells and basolateral on CaCo-2 cells. We conclude that a given virus and its envelope glycoprotein can be delivered to opposite membrane domains in epithelial cells derived from different tissues. The tissue specificity in the polarity of virus budding and viral envelope glycoprotein distribution indicate that the sorting machinery varies considerably between different epithelial cell types.