In Vitro Assessment of the In Vivo Stability of Cu-64 Radiopharmaceuticals

Research Plans: The successful development of Cu-64 radiopharmaceuticals depends upon retention of the Cu-64 atom in the radiopharmaceutical. To date, the focus has been on the development of chelators that better retain Cu-64, but there has been no effort to develop an effective method by which improved retention may be measured. In the absence of a suitable analytical method, the stability of Cu-64 radiopharmaceuticals is estimated indirectly, with decreased liver uptake suggesting higher in vivo complex stability. But this approach is inadequate for radiopharmaceuticals, such as radiolabeled antibodies, that are expected to accumulate in the liver even when there is no free Cu-64 present. The absence of such a method has also hampered efforts to systematically evaluate the chemical factors that may give rise to improved retention. The objective of this project is to develop and validate such a method. Accomplishments: The two primary accomplishments of this project will be 1) the development and validation of a method to measure the stability of Cu-64 radiopharmaceuticals and 2) the determination of the chemical factors that define the in vivo stability of Cu 64 radiopharmaceuticals. Because Cu(II) is extremely labile, the in vivo stability of Cu-64 radiopharmaceuticals is not primarily determined by the amount of “free” Cu that is present at any given time or by the thermodynamic stability constants, but rather by the rate at which Cu is lost from the complex, the dissociation rate constant, kd. The dissociation rate constants of the Cu-64 complexes from a series of bifunctional chelators (BFCs) will be measured using Free Ion Selective Radiotracer Extraction (FISRE), a technique originally developed to measure bioavailable Cu in environmental samples. FISRE will also be applied to the determination of the kd’s of a series of reference Cu-64 complexes to determine the chemical factors that define the in vivo stability of Cu-64 radiopharmaceuticals. Potential Benefits: The FISRE method that will be used in this project, once validated, will provide researchers with a core technology by which the stability of Cu 64 radiopharmaceuticals can be accurately measured. In the short-term, we expect to produce extensive data regarding the stability of Cu-64 complexes of ligands of radiopharmaceutical interest, primarily those that are most commonly used as BFCs (e.g., DOTA, TETA). These data will provide a quantitative basis for deciding which ligands may be best suited for use as BFCs, data that is not currently available. In the intermediate term, we expect that these results will facilitate the development of new Cu-64 radiopharmaceuticals by providing a quantitative approach to assessing the stability of Cu-64 chelates. This innovative methodology will enable investigators to quantitatively compare the ability of different BFCs to retain Cu-64 in vivo. The benefits of this approach will be best seen in the development of Cu-64-labeled monoclonal antibodies where the accumulation of antibodies in the liver obviates liver uptake as an effective surrogate measure of Cu-64 lability. In the longer-term, we anticipate an improvement in the way in which various diseases (especially cancer) are detected, diagnosed, staged, and treated. This method will also enable researchers to distinguish differences in biodistribution that may arise from differences in charge, lipophilicity, etc. from those that may arise from loss of Cu-64 from the chelator. Last, this novel quantitative tool will allow investigators to evaluate the chemical factors that determine the in vivo stability of Cu-64 radiopharmaceuticals—laying the groundwork for the future development of more effective Cu-64 radiopharmaceuticals. Once the feasibility of this method is established, it can also be used to evaluate the stability of other metalloradiopharmaceuticals including those based on Ga-68, a radionuclide that is showing great promise in tumor imaging