Production of radioiodinated prosthetic group for indirect protein labeling

Monoclonal antibodies and their fragments and, more recently, radiolabeled peptides have been extensively studied in order to develop radiopharmaceuticals for diagnostic and therapy in Nuclear Medicine. The radioiodination of proteins can be done by a direct method, with radioiodine being incorporated in to a tyrosine residue of the protein by electrophilic substitution. The main problem in the use of radioiodinated proteins, is that they are often dehalogenated in vivo by the action of specific enzymes, probably because of the structural similarity between iodophenyl groups and thyroid hormones. Several protein radioiodination methods have been developed in order to minimize this in vivo dehalogenation using prosthetic groups for indirect labeling. In this case, the radioiodine is first incorporated in to the prosthetic group that is subsequently attached to a terminal amino group or to a ε-amino group of lysine residue. The aim of this work is to obtain a radioiodinated prosthetic group for indirect labeling of proteins. The prosthetic group selected was the N-succinimidyl-4-radioiodine benzoate (SIB), obtained by the iodination of the p-bromobenzoic acid followed by the reaction with TSTU (0-(N-succinimidyl)-N,N,N',N'-tetramethyl uronium tetrafluoroborate) The results of these studies showed that the p-radio iodobenzoic acid was obtained with a radiochemical purity greater than 92% and a labeling yield of about 65%. Some reaction parameters were studied like temperature, time and Cu Cl mass (cataliser). The SIB was quantitatively obtained from p-radio iodobenzoic acid, using basic medium and after removing the water from the reaction using an nitrogen stream. The kinetic of this reaction is very fast with complete consumption of the p-radioiodebenzoic acid after 5 minutes. The coupling of the SIB prosthetic group to the protein was studied using Human Immunoglobulin (IgG) as a protein model. In a comparative way, the same protein was used on direct labeling studies using the Iodogen method. The yield observed by indirect method was low when compared to the direct method, with the major part of the activity remaining in the reaction vial, what suggests that the tridimensional structure of the antibody may difficult the SIB interaction with the protein amino groups. Swiss mice (normal animals for control and animals with infection focus developed on the right foot by terebentine injection) were injected with radioiodinated IgG obtained by direct and indirect method. The comparison of the biological distribution results showed a fast blood clearance, better organ/background relations (infection focus), and low uptake in thyroid and stomach (P<0,01) for the protein labeled by the indirect method, what suggests a greater in vivo stability. The method developed makes it possible to label peptides with 131I or 123I in the future, even those peptides without tyrosine residues, and use them used in diagnostic and therapy with in vivo stability. (author)