Development and biological evaluation of radiolabeled tracer agents for in vivo visualization of necrosis

With increasing knowledge on cell death, it has become clear that both apoptosis and necrosis play an important role in a variety of pathologies ranging from cancer to stroke. Radiolabeled tracer agents for SPECT or PET imaging that can differentiate between apoptotic and necrotic cell death would allow in vivo localization and identification of the type of cell death at every time point in these pathologies. Molecular imaging of cell death using specific radiolabeled tracer agents would thereby provide valuable information that could prove useful in both pre-clinical research and clinical diagnosis, prognosis and therapy management in a variety of diseases. Although several necrosis-avid tracer agents for application in nuclear medicine have been studied, none of these tracer agents has optimal imaging characteristics in terms of specificity, sensitivity or kinetics. Therefore we have developed and evaluated a number of novel necrosis-avid tracer agents, radiolabeled with the generator produced SPECT radioisotope 99mTc or positron emitter 68Ga, based on our experience with non-porphyr in necrosis-avid contrast agents for application in MRI. In a first approach bis-DTPA-PA, which had previously been shown to display selective uptake in necrotic tissue in different animal models of necrosis labeled with two paramagnetic Gd3+ atoms or 99mTc(CO)3, was labeled with the generator produced positron emitter 68Ga. The tracer was evaluated using different in vitro, in vivo and ex vivo techniques in normal mice and indifferent animal models of necrosis and apoptosis. Although the tracer showed a high and selective uptake in necrotic tissue, allowing in vivo visualization of necrosis using PET, the limited in vivo stability of the tracer resulted in an unfavourable biodistribution profile. The second lead compound, bis-DTPA-BI, was labeled with both 68Ga and 99mTc(CO)3 as technetium-99m has almost ideal physical properties for imaging in addition to its low cost and continuous availability. While both the 68Ga and99mTc(CO)3 complex of bis-DTPA-BI displayed a selective uptake in necrotic tissue in different animal models of necrosis, biodistribution studies revealed a more favourable biodistribution profile for both complexes of bis- DTPA-BI when compared to the radiolabeled bis-DTPA-PA complexes. As the results from the in vivo stability studies clearly demonstrated a limited in vivo stability of the Ga-DTPA complex, leading to a less favourable biodistribution profile of the tracer agent, analogs of pamoic acid and benzylidene-bis-indole were synthesized in which the acyclic chelator DTPA was replaced by the macrocyclic chelator DOTA which can form stable complexes with 68Ga. Biological evaluation of the resulting 68Ga-bis-DOTA-PA and 68Ga-bis-DOTA-BI in different animal models of necrosis and apoptosis confirmed that replacement of the DTPA chelator by the macrocyclic chelator DOTA had not compromised the necrosis avidity of the tracer agents while in vivo stability and biodistribution profile of the two tracers had clearly improved. In conclusion, several different necrosis-avid tracer agents based upon the pamoic acid and benzylidene-bis-indole structure were developed and labeled with generator produced radioisotopes for SPECT or PET imaging. Although all radiolabeled complexes displayed a selective uptake in necrotic tissue, 68Ga-bis-DOTA- BI shows the highest potential as a radiolabeled tracer agent for in vivo visualization of necrosis using positron emission tomography because of the fast kinetics and favourable biodistribution profile of the tracer.nrpages: 186status: publishe