Evaluation of ¹⁸F-annexin V as a PET imaging agent in an animal model of apoptosis

Annexin V is a 36-kDa protein that binds with high affinity to phosphatidylserine lipids in the cell membrane. Because one of the earliest measurable events in apoptosis is the eversion of phosphatidylserine from the inner membrane leaflet to the outer cell surface, annexin V has proven useful for detecting the earliest stages of apoptosis. Methods: Annexin V was radiolabeled with ¹⁸F using N-succinimidyl-4-¹⁸F- fluorobenzoic acid chemistry, to a specific activity of 555-925 kBq/μg of protein. ¹⁸F-Annexin V (14.8-51.8 MBq) was administered intravenously to rats after pretreatment with cycloheximide (5 mg/kg) to induce liver apoptosis, and the injected rats were imaged by PET over 2 h. After imaging, rats were dissected and individual organs were weighed and counted. Results: Pretreatment of rats with cycloheximide resulted in a 3- to 9-fold increase in uptake of ¹⁸F-annexin V in the liver of treated animals at 2 h, compared with controls. By morphologic analysis, treated livers showed a 3- to 6-fold higher level of apoptosis than controls, with higher levels also seen with longer exposure to cycloheximide. Terminal deoxynucleotide end-labeling (TUNEL) assays performed on liver slices showed that cycloheximide induced a 5- to 8-fold increase in the number of TUNEL-positive nuclei. These TUNEL results correlated with the uptake of ¹⁸F-annexin V in dissected liver tissue, with an r² value of 0.89. Biodistribution analysis of normal rats showed highest uptake of ¹⁸F-annexin V in the kidneys and urinary bladder, indicating rapid renal clearance of ¹⁸F-annexin V metabolites. Conclusion: The PET data, the organ-specific uptake data from dissection, and the morphologic and TUNEL measures of apoptosis together indicate that ¹⁸F-annexin V binds specifically to apoptotic tissues in this model of chemically induced apoptosis in rat liver. The short physical half-life of ¹⁸F-annexin V and the rapid clearance of its metabolites to the urinary system suggest that ¹⁸F-annexin V will be useful in early assessment of the clinical response to cancer therapy in individual patients.