Multimodal Molecular Imaging Reveals High Target Uptake and Specificity of ¹¹¹In- and ⁶⁸Ga-Labeled Fibrin-Binding Probes for Thrombus Detection in Rats

Abstract

We recently showed the high target specificity and favorable imaging properties of 64Cu and Al18F positron emission tomography (PET) probes for non-invasive imaging of thrombosis. Here, our aim was to evaluate new derivatives labeled with either with 68Ga, 111In, or 99mTc as thrombus imaging agents for PET and single-photon emission computed tomography (SPECT). In this study, the feasibility and potential of these probes for thrombus imaging was assessed in detail in two animal models of arterial thrombosis. The specificity of the probes was further evaluated using a triple-isotope approach with multimodal SPECT/PET/CT imaging. Methods Radiotracers were synthesized using a known fibrin-binding peptide conjugated to NODAGA, DOTA-MA, or a diethylenetriamine ligand (DETA-PA), followed by labeling with 68Ga (FBP14, 68Ga-NODAGA), 111In (FBP15, 111In-DOTA-MA) or 99mTc (FBP16, 99mTc(CO)3-DETA-PA), respectively. PET or SPECT imaging, biodistribution, pharmacokinetics and metabolic stability were evaluated in rat models of mural and occlusive carotid artery thrombosis. In vivo target specificity was evaluated by comparing the distribution of the SPECT and PET probes with preformed 125I-labeled thrombi and with a non-binding control probe using SPECT/PET/CT imaging. Results All three radiotracers showed similar affinity to soluble fibrin fragment DD(E) (Ki = 0.53–0.83 μM). After the kidneys, the highest uptake of 68Ga-FBP14 and 111In-FBP15 was in the thrombus (1.0 ± 0.2% ID/g) with low off-target accumulation. Both radiotracers underwent fast systemic elimination (t1/2 = 8-15 min) through the kidneys, which led to highly conspicuous thrombi on PET and SPECT images. 99mTc-FBP16 displayed low target uptake and distribution consistent with aggregation and/or degradation. Triple isotope imaging experiments showed that both 68Ga-FBP14 and 111In-FBP15, but not the nonbinding derivative 64Cu-D-Cys-FBP8, detected the location of the 125I-labeled thrombus, confirming high target specificity. Conclusion 68Ga-FBP14 and 111In-FBP15 have high fibrin affinity and thrombus specificity, and represent useful PET and SPECT probes for thrombus detection.

Peptide-based fibrin-targeting probes for thrombus imaging

“…87 We also used the NODAGA derivative to construct 68 Ga-FBP14. 89 The DOTA-monoamide conjugate was radiolabelled with In-111 to give 111 In-FBP15. 89 The choice of the chelators was based on literature describing the superiority of NODAGA and DOTA for stable coordination of 68 Ga and 111 In in vivo , respectively.…”

“…89 The DOTA-monoamide conjugate was radiolabelled with In-111 to give 111 In-FBP15. 89 The choice of the chelators was based on literature describing the superiority of NODAGA and DOTA for stable coordination of 68 Ga and 111 In in vivo , respectively. 90 For 99m Tc we synthesised a new precursor using a bifunctional diethylenetriamine ligand (DETA-PA) conjugated to the same fibrin-binding peptide.…”

“…This precursor allowed radiolabelling with the tricarbonyl precursor fac -[ 99m Tc(CO) 3 (H 2 O) 3 ] + to give 99m Tc-FBP16. 89 All the probes were evaluated for their affinity for fibrin, imaging profile, metabolic stability, biodistribution and ex vivo autoradiography (Figure 14). Using PET-CT or SPECT-CT imaging, we could readily observe the thrombus with Al 18 F-FBP11, 68 Ga-FBP14, and 111 In-FBP15, with high thrombus-to-background ratios (Figure 14a).…”

“…This was further confirmed with blood samples collected over the course of the study and their analysis for fibrin binding (Figure 15c). 89 …”

“…The SPECT imaging showed co-localisation between 125 I and 111 In but PET imaging with 64 Cu-D-Cys-FBP8 did not reveal any significant uptake at the target site. 89 Using a gold nanoparticle CT contrast agent we performed CT angiography to confirm the location of the SPECT signals in the carotid artery. In a complementary study, rats bearing a 125 I-labeled thrombus were injected with 111 In-FBP15 and then with 68 Ga-FBP14.…”