Gallium-68-dotamaleimide-Cys165-annexin A5

The gallium-68 (Ga)-dotamaleimide-Cys165-annexin A5 (AnxA5) conjugate, abbreviated as Ga-Cys165-AnxA5, is a radionuclide agent developed by Bauwens et al. for phosphatidylserine (PS)-targeted positron emission tomography (PET) of tumor cell apoptosis after chemotherapy and radiotherapy (1). Ga has a half-life of 68 min and emits positrons with β = 1.9 MeV. Evaluation of tumor response to therapy is traditionally based on volumetric and morphological changes before and after treatment (2); these changes, however, occur rather late after effective therapy. Recent studies have shown that successful treatment induces apoptosis of tumor cells at the very early stage, and externalization of PS is one of the earliest events in apoptosis (3, 4). PS is a membrane phospholipid normally restricted to the inner leaflet of the lipid bilayer. Within a few hours of the apoptotic stimulus, however, PS externalizes to the outer leaflet of the cell membrane and is readily accessible to exogenous molecules. These findings indicate that the early response of tumors to therapy can be evaluated by detection of apoptosis imaging of externalized PS (2, 3, 5). AnxA5 is a cellular protein of 36 kDa and specifically binds to membrane-bound PS with nanomolar affinity in the presence of calcium (Ca) (3, 6, 7). AnxA5 has no immunogenicity or toxicity when used . These features of AnxA5 make it a valuable agent for apoptosis detection. AnxA5 was first radiolabeled with I and later with other radionuclides, including Tc, F, In, and Ga (1, 5, 6). Both preclinical and clinical studies have shown that AnxA5-based radionuclides are promising tracers for the rapid dection of apoptosis. However, there are several issues that remain to be resolved (1, 2). First is the suboptimal pharmacokinetics of radiolabeled AnxA5 agents. A high nonspecific accumulation is often observed in the liver and kidneys. Second is the decreased binding affinity caused by radiolabeling and other conjugations. Optimal binding of AnxA5 to PS also requires a micromolar concentration of Ca. Third is the inability of AnxA5 agents to differentiate apoptosis from necrosis because PS is also externalized during necrosis. To maintain the binding affinity of AnxA5, Tait et al. applied a site-specific labeling technique in the radiolabeling of AnxA5, and they have shown that specific labeling in the non-binding region of AnxA5 improves the detection of apoptosis compared to random labeling of amino residues (8). Using a similar method, Bauwens et al. synthesized two radionuclide agents, Ga-Cys2-AnxA5 and Ga-Cys165-AnxA5 (1). Cys2-AnxA5 and Cys165-AnxA5 are two variants of AnxA5, and they contain a single available Cys residue at the 2-position and at the 165-position, respectively. Both Cys residues are located at the concave side of AnxA5, opposite the convex side that harbors the Ca/PS binding sites. Covalent coupling of large structures such as liposomes and iron nanoparticles thiol-chemistry to the Cys2-AnxA5 or Cys165-AnxA5 exhibits no side effect on the PS binding activity (8-11). Bauwens et al. showed that both Ga-Cys2-AnxA5 and Ga-Cys165-AnxA5 exhibit a clear binding to apoptotic cells and are promising tracers for the rapid evaluation of cancer therapy (1). No significant differences between the two analogs were observed in the data obtained from and analyses. This chapter summarizes the data obtained with Ga-Cys165-AnxA5, and another chapter in MICAD summarizes the data obtained with Ga-Cys2-AnxA5.