Direct and indirect radioiodination of protein: comparative study of chemotactic peptide labeling

The development of simple methods for protein radioiodination have stimulated the use of radioiodinated peptides in vivo. There are two basic methods for labeling proteins with radioiodine: direct labeling, reaction of an electrophilic radioiodine with functional activated groups on protein, like the phenol ring in the tyrosine residue, and the conjugation of a previously radioiodinated molecule to the protein, referred as indirect method. The great problem related to the direct radioiodination of proteins is the in vivo dehalogenation. This problem can be minimized if a non-phenolic prosthetic group is used in the indirect radioiodination of the peptide. The ATE prosthetic group, N-succinimidyl 3-(tri-n-butylstannyl) benzoate, when radioiodinated by electrophilic iododestannilation produces N-succinimidyl 3-[123l/131l] iodine benzoate (SIB) that is subsequently conjugated to the protein by the acylation of the lysine group. There are many radiopharmaceuticals employed in scintigraphic images of infection and inflammation used with some limitations. These limitations stimulated the improvement of a new class of radiopharmaceuticals, the receptor-specific related labeled peptides, as the mediators of the inflammatory response, that presents high affinity by receptors expressed in the inflammation process, and fast clearance from blood and non-target tissues. One of these molecules is the synthetic chemotactic peptide fNleLFNIeYK that presents potent chemotaxis for leukocytes, with high affinity by the receptors presented in polymorphonuclear leukocytes and mononuclear phagocytes. The objective of this work included the synthesis of ATE prosthetic group and comparative radioiodination of the chemotactic peptide fNleLFNIeYK by direct and indirect methods, with radiochemical purity determination and evaluation of in vivo and in vitro stability of the compounds. This work presented an original contribution in the comparative biological distribution studies of the chemotactic peptide labeled by direct and indirect methods. The ATE was obtained with satisfactory yield (90,7%). The conditions for ATE radioiodination were p H 3 - 4, 50 μmol of t-butylhydroperoxide and 30 minutes of reaction. The SIB was purified to remove the unreacted ATE and some radiochemical impurities presented in the reaction mixture. The peptide radioiodinated by direct method was obtained in a short reaction time (10 minutes), with high radiochemical purity (> 96%) and in vitro stability (48 hours under refrigeration). The biodistribution studies developed in normal Swiss mice and in mice with inflammatory focus developed by the administration of turpentine in the right thigh showed the ability of the compound to concentrate in the inflammatory focus as evidenced by the higher uptake in the inflamed thigh when compared to the normal thigh (p< 0.05, Student t) in all studied time. However, the uptake in thyroid increased in time due to the in vivo dehalogenation of the compound. The peptide radioiodinated by indirect method was also obtained with high radiochemical purity but only after high performance liquid chromatography purification (> 99%) and was stable in vitro (24 hours under refrigeration). Despite the indirect labeling procedure was laborious, time consuming and the product was obtained with low radiochemical yield (26,3%), the labeled peptide showed specificity by inflammatory focus and in vivo stability confirmed by the lower thyroid uptake when compared with the peptide labeled by direct method. The great in vivo stability of the peptide labeled by indirect method justifies the study of new and alternative purification procedure that reduces process time and increases the final yield. (author)