Fibrin-Targeted PET Probes for the Detection of Thrombi

Abstract

There is an ongoing effort to develop better methods for noninvasive detection and characterization of thrombus. Here we describe the synthesis and evaluation of three new fibrin-targeted PET probes (FBP1, FBP2, FBP3). Three fibrin-specific peptides were conjugated as DOTA-monoamides at the C- and N- termini, and chelated with 64CuCl2. Probes were prepared with a specific activity ranging from 10 – 130 µCi/nmol. Both the peptides and the probes exhibited nanomolar dissociation constants (Kd) for the soluble fibrin fragment DD(E), although the Cu-DOTA derivatization resulted in a 2–3 fold loss in affinity relative to the parent peptide. Biodistribution and imaging studies were performed in a rat model of carotid artery thrombosis. For FBP1 and FBP2 at 120 min post injection, the vessel containing thrombus showed the highest concentration of radioactivity after the excretory organs, i.e. the liver and kidneys. This was confirmed ex vivo by autoradiography which showed > 4-fold activity in the thrombus containing artery compared to the contralateral artery. FBP3 showed much lower thrombus uptake and the difference was traced to greater metabolism of this probe. Hybrid MR-PET imaging with FBP1 or FBP2 confirmed that these probes were effective for detection of arterial thrombus in this rat model. Thrombus was visible on PET images as a region of high activity that corresponded to a region of arterial occlusion identified by simultaneous MR angiography. FBP1 and FBP2 represent promising new probes for the molecular imaging of thrombus.

In Vivo Molecular Imaging of Thrombosis and Thrombolysis Using a Fibrin-Binding Positron Emission Tomographic Probe

“…16 The cyclic peptide was previously shown to be effective for fibrin targeting. 14, 16 FBP7 was evaluated for in vitro fibrin affinity in the presence and absence of heparin and also for its plasma stability. Additional information regarding reagents and methods are reported in Supplemental Material.…”

“…Under isoflurane anesthesia, the right common carotid artery was exposed and crush injury was induced by clamping the vessel with a hemostat for 5 min, as previously described. 16 Injury was performed 1–2 cm proximal to the carotid bifurcation, using the same hemostat and by the same investigator to minimize variability.…”

“…as well as tissues obtained after euthanasia were measured. 16 Blood samples and urine were analyzed by radio-HPLC to evaluate the metabolic stability of FBP7. 16 We also measured the fraction of activity in each plasma sample that was capable of binding to fibrin immobilized on a well plate, and compared this to pure probe spiked into plasma as a means of assaying for functional probe.…”

“…16 Blood samples and urine were analyzed by radio-HPLC to evaluate the metabolic stability of FBP7. 16 We also measured the fraction of activity in each plasma sample that was capable of binding to fibrin immobilized on a well plate, and compared this to pure probe spiked into plasma as a means of assaying for functional probe. 16 The right and left common carotid arteries from crush experiments and the brain sections (2 mm-thick, coronal or horizontal) from stroke experiments were analyzed by autoradiography.…”

Multimodality Molecular Imaging of Cardiovascular Disease Based on Nanoprobes

“…Three fibrin-specific target peptides were labeled with 64 Cu after the conjugation with 1, 4,7, 10-tetraaza-cyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA). For MR angiography imaging, the results showed that there was an occlusion in the right carotid artery at the site of crush injury, and this vessel occlusion corresponds to the high radioactivity uptake displayed on PET images [32] (Fig. 3).…”