Background and Aims:
Vascular adhesion protein-1 (VAP-1) is an endothelial glycoprotein present on the endothelial surface of tissues affected by infection/inflammation that recruit lymphocytes from blood. VAP-P1 is a VAP-1 selective peptide (sequence GGGGKGGGG) which, according to previous studies, conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and labelled with Ga-68, could be used as imaging agent capable to differentiate between normal bone healing and bone infection.
The aim of this paper is to study the potential of 1,4,7-triazacyclononane-triacetic acid (NOTA)-conjugated and Cu-64 labelled VAP-P1 peptide for possible clinical applications.
Synthesized peptide VAP-P1 was conjugated with NOTA-SCN, purified using semi-prep HPLC and lyophilized. The conjugate was then labelled with Cu-64. In vitro plasma protein binding and stability in human serum in time were determined using transfected Chinese Hamster Ovary cells (CHO) that express human VAP-1 and HT-29 (negative control). Each experiment was performed in octaplicate, and the data were reported as percentage of total added activity.
The peptide was conjugated with NOTA chelator and labelled with 68Ga and 64Cu with radiochemical purity ≥ 95 %. Radiolabelled analogue [Cu-64]NOTA-VAP-P1 presented here was rapidly internalized into CHO cells versus negative control HT-29. The internalization of the surface bound peptide was time-dependent. It sharply increased during the first 30 min reaching internalization levels of 80–90 %.
In vitro studies showed that NOTA-VAP-P1 labelled with Cu-64 may provide a faster and a simpler method of diagnostics of inflammation/infection compared to the method of labelled leucocytes. The concept deserves to be tested in vivo.
Key words: Cu-64, NOTA-VAP-P1, VAP-P1, VAP-1, labelling, conjugation, PET, CHO cells, plasma protein binding, human serum stability