Abstract
99mTc-HYNIC-annexin A5 can be considered as a benchmark in the field of apoptosis imaging. However, 99mTc-HYNIC-annexin A5 has characteristics of high uptake and long retention in non-target tissues such as kidney and liver. To minimize this problem, we developed a novel 99mTc-labeled annexin A5 using a bis(hydroxamamide) derivative [C3(BHam)2] as a bifunctional chelating agent, and evaluated its usefulness as an imaging agent for detecting apoptosis. The amino group of C3(BHam)2 was converted to a maleimide group, and was coupled to thiol groups of annexin A5 pretreated with 2-iminothiolane. 99mTc labeling was performed by a ligand exchange reaction with 99mTc-glucoheptonate. Biodistribution experiments for both 99mTc-C3(BHam)2-annexin A5 and 99mTc-HYNIC-annexin A5 were performed in normal mice. In addition, in tumor-bearing mice, the relationship between the therapeutic effects of chemotherapy (5-FU) and the tumor accumulation of 99mTc-C3(BHam)2-annexin A5 just after the first treatment of 5-FU was evaluated. 99mTc-C3(BHam)2-annexin A5 was prepared with a radiochemical purity of over 95%. In biodistribution experiments, 99mTc-C3(BHam)2-annexin A5 had a much lower kidney accumulation of radioactivity than 99mTc-HYNIC-annexin A5. In the organs for metabolism, such as liver and kidney, radioactivity after the injection of 99mTc-HYNIC-annexin A5 was residual for a long time. On the other hand, radioactivity after the injection of 99mTc-C3(BHam)2-annexin A5 gradually decreased. In therapeutic experiments, tumor growth in the mice treated with 5-FU was significantly inhibited. Accumulation of 99mTc-C3(BHam)2-annexin A5 in tumors significantly increased after 5-FU treatment. The accumulation of radioactivity in tumor correlated positively with the counts of TUNEL-positive cells. These findings suggest that 99mTc-C3(BHam)2-annexin A5 may contribute to the efficient detection of apoptotic tumor response after chemotherapy.
Highlights
Annexin A5 is a 36-kDa human protein with a high affinity for phosphatidyl serine (PS)
Tricine has been used most widely for protein labeling, since it provides 99mTc-HYNIC-labeled polypeptides with high radiochemical yields and high specific activities in a short reaction time [4,5]. 99mTc-HYNIC-annexin A5 can be considered as a benchmark in the field of apoptosis imaging since this tracer is the most extensively investigated and best characterized apoptosisdetecting radioligand to date [6]
The number of C3(BHam)2 ligands introduced per molecule of annexin A5 was estimated by measuring the number of thiol groups in the annexin A5 before and after the conjugation reaction with compound 3 using 4,49-dithiodipyridine [10]
Summary
Annexin A5 is a 36-kDa human protein with a high affinity for phosphatidyl serine (PS). PS is normally retained on the intracellular face of the cell membrane. For that reason, radiolabeled annexin A5 can be used to detect cell death in vivo [1,2]. Since most polypeptides do not possess binding sites to form 99mTc chelates of high in vivo stability, appropriate chelating molecules are incorporated into polypeptide molecules to prepare 99mTclabeled peptides for in vivo applications. Hydrazinonicotinamide (HYNIC) is one of the most attractive bifunctional chelating agents for the labeling of peptides and proteins with 99mTc. It was reported that HYNIC acts as a monodentate or bidentate ligand to form a mixed ligand 99mTc complex in the presence of appropriate coligands [3]. Several coligands have been reported such as glucoheptonate, tricine, ethylene diamine diacetic acid (EDDA), and ternary ligand systems containing tricine and water-soluble phosphines or tricine and imine-N-containing heterocycles
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