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

Either inadequate or excessive apoptosis (programmed cell death) is associa ted with many diseases. A method to image apoptosis in vivo, rather than re quiring histologic evaluation of tissue, could assist with therapewutic dec ision making in these disorders. Programmed cell death is associated with a well-choreographed series of events resulting in the cessation of normal c ell function, and the ultimate disappearance of the cell. One component of apoptosis is signaling adjacent cells that this cell is committing suicide by externalizing phosphatidylserine to the outer leaflet of the cell membra ne. Annexin V, a 32-kDa endogenous human protein, has a high affinity for m embrane-bound phosphatidylserine, We have coupled annexin V with the bifunc tional hydrazinonicotinamide reagent (HYNIC) to prepare technetium-99m HYNI C-annexin V and demonstrated localization of radioactivity in tissues under going 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 T c-99m-HYNIC- annexin V. Biodistribution studies were performed in groups of rats at 10-180 min after intravenous injection of Tc-99m-HYNIC-annexin V. In order to estimate the degree of apoptosis required for localization of T c-99m-annexin V in vivo, mice were treated with dexamethasone at doses rang ing from 1 to 20 mg/kg, 5 h prior to Tc-99m-HYNIC-annexin V administration, to induce rhymic apoptosis. Thymus was excised 1 h after radiolabeled HYNI C-annexin V injection; thymocytes were isolated, incubated with Hoechst 333 42 followed by propidium iodide, and analyzed on a fluorescence-activated c ell sorter, Each sorted cell population was counted in a scintillation coun ter. To test Tc-99m-HYNIC-annexin V as a tracer for external radionuclide i maging of apoptotic cell death, radionuclide imaging of Fas-defective mice (lpr-lpr mice) and wild-type mice treated with the antibody to Fas (anti-Fa s) was carried out 1 h post injection. Rat biodistribution studies demonstr ated a blood clearance half-time of less than 10 min for Tc-99m-HYNIC-annex in V, The kidneys had the highest concentration of radioactivity at all tim e points. Studies in the mouse thymus demonstrated a 40-fold increase in 99 mTc-HYNIC-annexin V concentration in apoptotic thymocytes compared with the viable cell population, A correlation of r=0.78 was found between radioact ivity and flow cytometric and histologic evidence of apoptosis. Imaging stu dies in the lpr/lpr and wild-type mice showed a substantial increase of act ivity 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 sis were obtained in wild-type mice 30 min after injection of 99mTc-HYNIC-annexin V, The imaging results were consistent with histologic analysis in these animals. In conlusion, th ese studies confirm the value of Tc-99m-HYNIC-annexin V uptake as a marker for the detection and quantification of apoptotic cells in vivo.