99mTc-labeled duramycin as a novel phosphatidylethanolamine-binding molecular probe

With only 19 amino acids, duramycin is the smallest known polypeptide that has a defined 3-dimensional binding structure. Duramycin binds phosphatidylethanolamine (PtdE) at a 1:1 ratio with high affinity and exclusive specificity. As an abundant binding target, PtdE is a major phospholipid and accounts for about 20% of the phospholipid content in mammalian cellular membranes. PtdE is externalized to the surface of apoptotic cells and also becomes accessible in necrotic cells because of compromised plasma membrane integrity. Given the unique physicochemical properties of duramycin and the availability of PtdE in acute cell death, the goal of this study was to develop and evaluate Tc-99m-duramycin as a novel molecular probe for imaging PtdE. Methods: Duramycin is covalently modified with succinimidyl 6-hydrazinonicotinate acetone hydrazone (HYNIC) and labeled with Tc-99m using a coordination chemistry involving tricine-phosphine coligands. The retention of PtdE-binding activities was confirmed using competition assays with PtdE-containing liposomes. The blood clearance, pharmacokinetics, and biodistribution of Tc-99m-duramycin were measured in rats. Finally, Tc-99m-duramycin binding to acute cell death in vivo was demonstrated using a rat model of acute myocardial infarction induced by ischemia and reperfusion and confirmed using autoradiography and histology. Results: HYNIC-derivatized duramycin with 1:1 stoicheometry was synthesized and confirmed by mass spectrometry. The radiolabeling efficiency was 80%-85%, radiochemical purity was 78%-89%, and specific activity wets 54 GBq. The radiotracer was purified with high-performance liquid chromatography radiodetection before use. The specific uptake of Tc-99m-duramycin in apoptotic cells, compared with that in viable control cells, was enhanced by more than 30-fold. This binding was competitively diminished in the presence of PtdE-containing liposomes but not by liposomes consisting of other phospholipid species. Intravenously injected Tc-99m-duramycin has favorable pharmacokinetic and biodistribution profiles: it quickly clears from the circulation via the renal system, with a blood half-life of less than 4 min in rats. The hepatic and gastrointestinal uptake were very low. Tc-99m-duramycin is completely unmetabolized in vivo, and the intact agent is recovered from the urine. Combined with a fast clearance and low hepatic background, the avid binding of Tc-99m-duramycin to the infarcted myocardium quickly becomes conspicuous shortly after injection. The uptake of radioactivity in infarcted tissues was confirmed by autoradiography and histology. Conclusion: Tc-99m-duramycin is a stable, low-molecular-weight PtdE-binding radiopharmaceutical, with favorable in vivo imaging profiles. It is a strong candidate as a molecular probe for PtdE imaging and warrants further development and characterization.