Comparison of the In Vivo Distribution of Four Different Annexin A5 Adducts in Rhesus Monkeys

Abstract

Annexin A5 has been used for the detection of apoptotic cells, due to its ability to bind to phosphatidylserine (PS). Four different labeled Annexin A5 adducts were evaluated in rhesus monkey, with radiolabeling achieved via 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Of these adducts differing conjugation methods were employed which resulted in nonspecific radiolabeling (AxA5-I), or site-specific radiolabeling (AxA5-II). A nonbinding variant of Annexin A5 was also evaluated (AxA5-IINBV), conjugation here was site specific. The fourth adduct examined had both specific and nonspecific conjugation techniques employed (AxA5-IImDOTA). Blood clearance for each adduct was comparable, while appreciable uptake was observed in kidney, liver, and spleen. Significant differences in uptake of AxA5-I and AxA5-II were observed, as well as between AxA5-II and AxA5-IINBV. No difference between AxA5-II and AxA5-IImDOTA was observed, suggesting that conjugating DOTA nonspecifically did not affect the in vivo biodistribution of Annexin A5.

Radiopharmacological evaluation of 18F-labeled phosphatidylserine-binding peptides for molecular imaging of apoptosis

“…Wild-type annexin V was randomly functionalized with macrocyclic chelator NOTA through bioconjugation of p-NCS-Bn-NOTA via thiourea formation to amine groups present in the protein (22 lysine residues and one N-terminal amine group) as described by McQuade et al before [14]. Binding of 64 Cu-NOTA-annexin V to PS was challenged with different concentrations of peptides LIKKPF, PGDLSR, FBz-LIKKPF, FBz-PGDLSR, FBAM-CLIKKPF, FBAM-CPGDLSR and wild-type annexin V as internal reference.…”

“…Apoptosis was induced by the injection of 38 mg/kg etoposide and 100 mg/kg cyclophosphamide on two consecutive days into EL4 tumor-bearing C57Bl6 mice following the literature procedure [14,15]. EL4 tumor apoptosis was assessed histologically using TUNEL assay analysis.…”

“…For this purpose we have developed a novel competitive radiometric PS binding assay using 64 Cu-labeled NOTA-annexin V as radiotracer to assess binding properties of various small peptides to immobilized PS. 64 Culabeled annexin V is a suitable radiotracer for the radiometric PS binding assay due to the favorable long half-life of 64 Cu (t 1/2 = 12.7 h) and the straightforward synthesis of 64 Cu-NOTA-annexin V according to a report recently published by McQuade et al [14]. Radiopharmacological evaluation of radiolabeled peptides included determination of their metabolic stability, cellular uptake studies in camptothecin-induced apoptotic human T lymphocyte Jurkat cells, biodistribution, and dynamic PET imaging experiments using a chemotherapy-induced apoptotic murine EL4 tumor model.…”

Tissue Distribution Studies of Protein Therapeutics Using Molecular Probes: Molecular Imaging

“…The distribution of excreted or catabolized labels can confound the imaging of certain tissues, especially in and around the hepatobiliary system and gut, the kidneys, and urinary bladder. When imaging abdominal sites, for example, this can be a limitation and has been a major driver in the selection among imaging agents based on annexin V (22,23).…”