Nicholas J. Rotile

Pycup—A Bifunctional, Cage-like Ligand for 64Cu Radiolabeling

In developing targeted probes for positron emission tomography (PET) based on (64)Cu, stable complexation of the radiometal is key, and a flexible handle for bioconjugation is highly advantageous. Here, we present the synthesis and characterization of the chelator pycup and four derivatives. Pycup is a cross-bridged cyclam derivative with a pyridyl donor atom integrated into the cross-bridge resulting in a pentadentate ligand. The pycup platform provides kinetic inertness toward (64)Cu dechelation and offers versatile bioconjugation chemistry. We varied the number and type of additional donor atoms by alkylation of the remaining two secondary amines, providing three model ligands, pycup2A, pycup1A1Bn, and pycup2Bn, in 3-4 synthetic steps from cyclam. All model copper complexes displayed very slow decomplexation in 5 M HCl and 90 °C (t1/2: 1.5 h for pycup1A1Bn, 2.7 h for pycup2A, 20.3 h for pycup2Bn). The single crystal crystal X-ray structure of the [Cu(pycup2Bn)](2+) complex showed that the copper was coordinated in a trigonal, bipyramidal manner. The corresponding radiochemical complexes were at least 94% stable in rat plasma after 24 h. Biodistribution studies conducted in Balb/c mice at 2 h postinjection of (64)Cu labeled pycup2A revealed low residual activity in kidney, liver, and blood pool with predominantly renal clearance observed. Pycup2A was readily conjugated to a fibrin-targeted peptide and labeled with (64)Cu for successful PET imaging of arterial thrombosis in a rat model, demonstrating the utility of our new chelator in vivo.

In vivo molecular imaging of thrombosis and thrombolysis using a fibrin-binding positron emission tomographic probe.

Background—Fibrin is a major component of arterial and venous thrombi and represents an ideal candidate for molecular imaging of thrombosis. Here, we describe imaging properties and target uptake of a new fibrin-specific positron emission tomographic probe for thrombus detection and therapy monitoring in 2 rat thrombosis models. Methods and Results—The fibrin-binding probe FBP7 was synthesized by conjugation of a known short cyclic peptide to a cross-bridged chelator (CB-TE2A), followed by labeling with copper-64. Adult male Wistar rats (n=26) underwent either carotid crush injury (mural thrombosis model) or embolic stroke (occlusive thrombosis model) followed by recombinant tissue-type plasminogen activator treatment (10 mg/kg, IV). FBP7 detected thrombus location in both animal models with a high positron emission tomographic target-to-background ratio that increased over time (>5-fold at 30–90 minutes, >15-fold at 240–285 minutes). In the carotid crush injury animals, biodistribution analysis confirmed high probe uptake in the thrombotic artery (≈0.5%ID/g; >5-fold greater than blood and other tissues of the head and thorax). Similar results were obtained from ex vivo autoradiography of the ipsilateral versus contralateral carotid arteries. In embolic stroke animals, positron emission tomographic–computed tomographic imaging localized the clot in the internal carotid/middle cerebral artery segment of all rats. Time-dependent reduction of activity at the level of the thrombus was detected in recombinant tissue-type plasminogen activator–treated rats but not in vehicle-injected animals. Brain autoradiography confirmed clot dissolution in recombinant tissue-type plasminogen activator–treated animals, but enduring high thrombus activity in control rats. Conclusions—We demonstrated that FBP7 is suitable for molecular imaging of thrombosis and thrombolysis in vivo and represents a promising candidate for bench-to-bedside translation.

Multisite Thrombus Imaging and Fibrin Content Estimation With a Single Whole-Body PET Scan in Rats

Objective—Thrombosis is a leading cause of morbidity and mortality worldwide. Current diagnostic strategies rely on imaging modalities that are specific for distinct vascular territories, but a thrombus-specific whole-body imaging approach is still missing. Moreover, imaging techniques to assess thrombus composition are underdeveloped, although therapeutic strategies may benefit from such technology. Therefore, our goal was to test whether positron emission tomography (PET) with the fibrin-binding probe 64Cu-FBP8 allows multisite thrombus detection and fibrin content estimation. Approach and Results—Thrombosis was induced in Sprague-Dawley rats (n=32) by ferric chloride application on both carotid artery and femoral vein. 64Cu-FBP8-PET/CT imaging was performed 1, 3, or 7 days after thrombosis to detect thrombus location and to evaluate age-dependent changes in target uptake. Ex vivo biodistribution, autoradiography, and histopathology were performed to validate imaging results. Arterial and venous thrombi were localized on fused PET/CT images with high accuracy (97.6%; 95% confidence interval, 92–100). A single whole-body PET/MR imaging session was sufficient to reveal the location of both arterial and venous thrombi after 64Cu-FBP8 administration. PET imaging showed that probe uptake was greater in younger clots than in older ones for both arterial and venous thrombosis (P<0.0001). Quantitative histopathology revealed an age-dependent reduction of thrombus fibrin content (P<0.001), consistent with PET results. Biodistribution and autoradiography further confirmed the imaging findings. Conclusions—We demonstrated that 64Cu-FBP8-PET is a feasible approach for whole-body thrombus detection and that molecular imaging of fibrin can provide, noninvasively, insight into clot composition.

Effect of Chelate Type and Radioisotope on the Imaging Efficacy of 4 Fibrin-Specific PET Probes

Thrombus formation plays a major role in cardiovascular diseases, but noninvasive thrombus imaging is still challenging. Fibrin is a major component of both arterial and venous thrombi and represents an ideal candidate for imaging of thrombosis. Recently, we showed that 64Cu-DOTA–labeled PET probes based on fibrin-specific peptides are suitable for thrombus imaging in vivo; however, the metabolic stability of these probes was limited. Here, we describe 4 new probes using either 64Cu or aluminum fluoride (Al18F) chelated to 2 NOTA derivatives. Methods: Probes were synthesized using a known fibrin-specific peptide conjugated to either NODAGA (FBP8, FBP10) or NOTA-monoamide (FBP9, FBP11) as chelators, followed by labeling with 64Cu (FBP8 and FBP9) or Al18F (FBP10 and FBP11). PET imaging efficacy, pharmacokinetics, biodistribution, and metabolic stability were assessed in a rat model of arterial thrombosis. Results: All probes had similar nanomolar affinity (435–760 nM) for the soluble fibrin fragment DD(E). PET imaging allowed clear visualization of thrombus by all probes, with a 5-fold or higher thrombus-to-background ratio. Compared with the previous DOTA derivative, the new 64Cu probes FBP8 and FBP9 showed substantially improved metabolic stability (>85% intact in blood at 4 h after injection), resulting in high uptake at the target site (0.5–0.8 percentage injected dose per gram) that persisted over 5 h, producing increasingly greater target-to-background ratios. The thrombus uptake was 5- to 20-fold higher than the uptake in the contralateral artery, blood, muscle, lungs, bone, spleen, large intestine, and heart at 2 h after injection and 10- to 40-fold higher at 5 h. The Al18F derivatives FBP10 and FBP11 were less stable, in particular the NODAGA conjugate (FBP10, <30% intact in blood at 4 h after injection), which showed high bone uptake and low thrombus-to-background ratios that decreased over time. The high thrombus-to-contralateral ratios for all probes were confirmed by ex vivo biodistribution and autoradiography. The uptake in the liver (<0.5 percentage injected dose per gram), kidneys, and blood were similar for all tracers, and they all showed predominant renal clearance. Conclusion: FBP8, FBP9, and FBP11 showed excellent metabolic stability and high thrombus-to-background ratios and represent promising candidates for imaging of thrombosis in vivo.

Combined magnetic resonance elastography and collagen molecular magnetic resonance imaging accurately stage liver fibrosis in a rat model

Hepatic fibrosis is associated with an overproduction of matrix proteins and a pathological increase of liver stiffness. Noninvasive magnetic resonance (MR) quantification of matrix can be assessed with a collagen‐binding molecular MR probe and stiffness by MR elastography, complementary techniques. This study used both imaging techniques to more accurately stage hepatic fibrosis in a rat model. Thirty rats with varying levels of diethylnitrosamine‐induced liver fibrosis were imaged before and 45 minutes after injection of collagen‐specific probe EP‐3533. MR elastography was performed in the same imaging session. Changes in liver relaxation rate post–EP‐3533 and liver stiffness were compared to the collagen proportional area determined by histology and to Ishak scoring using receiver operating characteristic analysis. Collagen imaging was most sensitive to early fibrosis, while elastography was more sensitive to advanced fibrosis. This complementary feature enabled the formulation of a composite model using multivariate analysis of variance. This model incorporated the discriminating advantages of both MR techniques, resulting in more accurate staging throughout fibrotic progression. Conclusion: Collagen molecular MR imaging is complementary to MR elastography, and combining the two techniques in a single exam leads to increased diagnostic accuracy for all stages of fibrosis. (Hepatology 2017;65:1015‐1025)

Radiation Dosimetry of the Fibrin-Binding Probe 64Cu-FBP8 and Its Feasibility for PET Imaging of Deep Vein Thrombosis and Pulmonary Embolism in Rats

The diagnosis of deep venous thromboembolic disease is still challenging despite the progress of current thrombus imaging modalities and new diagnostic algorithms. We recently reported the high target uptake and thrombus imaging efficacy of the novel fibrin-specific PET probe 64Cu-FBP8. Here, we tested the feasibility of 64Cu-FBP8 PET to detect source thrombi and culprit emboli after deep vein thrombosis and pulmonary embolism (DVT-PE). To support clinical translation of 64Cu-FBP8, we performed a human dosimetry estimation using time-dependent biodistribution in rats. Methods: Sprague–Dawley rats (n = 7) underwent ferric chloride application on the femoral vein to trigger thrombosis. Pulmonary embolism was induced 30 min or 2 d after DVT by intrajugular injection of a preformed blood clot labeled with 125I-fibrinogen. PET imaging was performed to detect the clots, and SPECT was used to confirm in vivo the location of the pulmonary emboli. Ex vivo γ counting and histopathology were used to validate the imaging findings. Detailed biodistribution was performed in healthy rats (n = 30) at different time points after 64Cu-FBP8 administration to estimate human radiation dosimetry. Longitudinal whole-body PET/MR imaging (n = 2) was performed after 64Cu-FBP8 administration to further assess radioactivity clearance. Results: 64Cu-FBP8 PET imaging detected the location of lung emboli and venous thrombi after DVT-PE, revealing significant differences in uptake between target and background tissues (P < 0.001). In vivo SPECT imaging and ex vivo γ counting confirmed the location of the lung emboli. PET quantification of the venous thrombi revealed that probe uptake was greater in younger clots than in older ones, a result confirmed by ex vivo analyses (P < 0.001). Histopathology revealed an age-dependent reduction of thrombus fibrin content (P = 0.006), further supporting the imaging findings. Biodistribution and whole-body PET/MR imaging showed a rapid, primarily renal, body clearance of 64Cu-FBP8. The effective dose was 0.021 mSv/MBq for males and 0.027 mSv/MBq for females, supporting the feasibility of using 64Cu-FBP8 in human trials. Conclusion: We showed that 64Cu-FBP8 PET is a feasible approach to image DVT-PE and that radiogenic adverse health effects should not limit the clinical translation of 64Cu-FBP8.

Uncoupling of the profibrotic and hemostatic effects of thrombin in lung fibrosis

Fibrotic lung disease, most notably idiopathic pulmonary fibrosis (IPF), is thought to result from aberrant wound-healing responses to repetitive lung injury. Increased vascular permeability is a cardinal response to tissue injury, but whether it is mechanistically linked to lung fibrosis is unknown. We previously described a model in which exaggeration of vascular leak after lung injury shifts the outcome of wound-healing responses from normal repair to pathological fibrosis. Here we report that the fibrosis produced in this model is highly dependent on thrombin activity and its downstream signaling pathways. Direct thrombin inhibition with dabigatran significantly inhibited protease-activated receptor-1 (PAR1) activation, integrin αvβ6 induction, TGF-β activation, and the development of pulmonary fibrosis in this vascular leak-dependent model. We used a potentially novel imaging method - ultashort echo time (UTE) lung magnetic resonance imaging (MRI) with the gadolinium-based, fibrin-specific probe EP-2104R - to directly visualize fibrin accumulation in injured mouse lungs, and to correlate the antifibrotic effects of dabigatran with attenuation of fibrin deposition. We found that inhibition of the profibrotic effects of thrombin can be uncoupled from inhibition of hemostasis, as therapeutic anticoagulation with warfarin failed to downregulate the PAR1/αvβ6/TGF-β axis or significantly protect against fibrosis. These findings have direct and important clinical implications, given recent findings that warfarin treatment is not beneficial in IPF, and the clinical availability of direct thrombin inhibitors that our data suggest could benefit these patients.

Multimodal molecular imaging reveals high target uptake and specificity of 111In and 68Ga labeled fibrin-binding probes for thrombus detection in rats

We recently showed the high target specificity and favorable imaging properties of 64Cu and Al18F PET probes for noninvasive 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 SPECT. In this study, the feasibility and potential of these probes for thrombus imaging was assessed in detail in 2 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 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid monoamide (DOTA-MA), or a diethylenetriamine ligand (DETA-propanoic acid [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 nonbinding control probe using SPECT/PET/CT imaging. Results: All 3 radiotracers showed affinity similar to soluble fibrin fragment DD(E) (inhibition constant = 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 percentage injected dose per gram), with low off-target accumulation. Both radiotracers underwent fast systemic elimination (half-life, 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 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.

Type I collagen–targeted PET probe for pulmonary fibrosis detection and staging in preclinical models

Positron emission tomography with a probe targeting type I collagen enables detection, staging, and treatment response monitoring in lung fibrosis. Focusing on fibrosis Although fibrosis is known to play a role in the progression of multiple diseases, affecting heart, lung, liver, and skin, among other organs, it remains difficult to visualize and diagnose noninvasively. To address this, Désogère and colleagues developed an imaging probe for positron emission tomography that detects type I collagen, an extracellular matrix protein present in fibrotic tissues. The probe detected fibrotic lung tissue in two mouse models of bleomycin-induced pulmonary fibrosis and in samples of human lungs from patients with idiopathic pulmonary fibrosis, where higher probe uptake correlated with regions of increasing fibrosis. Pulmonary fibrosis is scarring of the lungs that can arise from radiation injury, drug toxicity, environmental or genetic causes, and for unknown reasons [idiopathic pulmonary fibrosis (IPF)]. Overexpression of collagen is a hallmark of organ fibrosis. We describe a peptide-based positron emission tomography (PET) probe (68Ga-CBP8) that targets collagen type I. We evaluated 68Ga-CBP8 in vivo in the bleomycin-induced mouse model of pulmonary fibrosis. 68Ga-CBP8 showed high specificity for pulmonary fibrosis and high target/background ratios in diseased animals. The lung PET signal and lung 68Ga-CBP8 uptake (quantified ex vivo) correlated linearly (r2 = 0.80) with the amount of lung collagen in mice with fibrosis. We further demonstrated that the 68Ga-CBP8 probe could be used to monitor response to treatment in a second mouse model of pulmonary fibrosis associated with vascular leak. Ex vivo analysis of lung tissue from patients with IPF supported the animal findings. These studies indicate that 68Ga-CBP8 is a promising candidate for noninvasive imaging of human pulmonary fibrosis.

Molecular imaging of oxidized collagen quantifies pulmonary and hepatic fibrogenesis

Fibrosis results from the dysregulation of tissue repair mechanisms affecting major organ systems, leading to chronic extracellular matrix buildup, and progressive, often fatal, organ failure. Current diagnosis relies on invasive biopsies. Noninvasive methods today cannot distinguish actively progressive fibrogenesis from stable scar, and thus are insensitive for monitoring disease activity or therapeutic responses. Collagen oxidation is a universal signature of active fibrogenesis that precedes collagen crosslinking. Biochemically targeting oxidized lysine residues formed by the action of lysyl oxidase on collagen with a small-molecule gadolinium chelate enables targeted molecular magnetic resonance imaging. This noninvasive direct biochemical elucidation of the fibrotic microenvironment specifically and robustly detected and staged pulmonary and hepatic fibrosis progression, and monitored therapeutic response in animal models. Furthermore, this paradigm is translatable and generally applicable to diverse fibroproliferative disorders.