Technetium-99m HYNIC-annexin V: a potential radiopharmaceutical for the in-vivo detection of apoptosis

Annexin V, a 32-kDa endogenous human protein, has a high affinity for membrane-bound phosphatidylserine. We have coupled annexin V with the bifunctional hydrazinonicotinamide reagent (HYNIC) to prepare technetium-99m HYNIC-annexin V and demonstrated localization of radioactivity in tissues undergoing apoptosis in vivo. In this report we describe the results of a series of experiments in mice and rats to characterize the biologic behavior of 99mTc-HYNIC- annexin V. Biodistribution studies were performed in groups of rats at 10-180 min after intravenous injection of 99mTc-HYNIC-annexin V. In order to estimate the degree of apoptosis required for localization of 99mTc-annexin V in vivo, mice were treated with dexamethasone at doses ranging from 1 to 20 mg/kg, 5 h prior to 99mTc-HYNIC-annexin V administration, to induce thymic apoptosis. Thymus was excised 1 h after radiolabeled HYNIC-annexin V injection; thymocytes were isolated, incubated with Hoechst 33342 followed by propidium iodide, and analyzed on a fluorescence-activated cell sorter. Each sorted cell population was counted in a scintillation counter. To test 99mTc-HYNIC-annexin V as a tracer for external radionuclide imaging of apoptotic cell death, radionuclide imaging of Fas-defective mice (lpr/lpr mice) and wild-type mice treated with the antibody to Fas (anti-Fas) was carried out 1 h post injection. Rat biodistribution studies demonstrated a blood clearance half-time of less than 10 min for 99mTc-HYNIC-annexin V. The kidneys had the highest concentration of radioactivity at all time points. Studies in the mouse thymus demonstrated a 40-fold increase in 99mTc-HYNIC-annexin V concentration in apoptotic thymocytes compared with the viable cell population. A correlation of r=0.78 was found between radioactivity and flow cytometric and histologic evidence of apoptosis. Imaging studies in the lpr/lpr and wild-type mice showed a substantial increase of activity in the liver of wild-type mice treated with anti-Fas, while there was no significant change, irrespective of anti-Fas administration, in lpr/lpr mice. Excellent images of hepatic apoptosis were obtained in wild-type mice 30 min after injection of 99mTc-HYNIC-annexin V. The imaging results were consistent with histological analysis in these animals. (orig.)