Allysine-Targeted Molecular MRI Enables Early Prediction of Chemotherapy Response in Pancreatic Cancer.

Neoadjuvant therapy is routinely used in pancreatic ductal adenocarcinoma (PDAC), but not all tumors respond to this treatment. Current clinical imaging techniques are not able to precisely evaluate and predict the response to neoadjuvant therapies over several weeks. A strong fibrotic reaction is a hallmark of a positive response, and during fibrogenesis, allysine residues are formed on collagen proteins by the action of lysyl oxidases. Here, we report the application of an allysine-targeted molecular MRI probe, MnL3, to provide an early, noninvasive assessment of treatment response in PDAC. Allysine increased 2- to 3-fold after one dose of neoadjuvant therapy with FOLFIRINOX in sensitive human PDAC xenografts in mice. Molecular MRI with MnL3 could specifically detect and quantify fibrogenesis in PDAC xenografts. Comparing the MnL3 signal before and 3 days after one dose of FOLFIRINOX predicted subsequent treatment response. The MnL3 tumor signal increased by 70% from day 0 to day 3 in mice that responded to subsequent doses of FOLFIRINOX, whereas no signal increase was observed in FOLFIRINOX-resistant tumors. This study indicates the promise of allysine-targeted molecular MRI as a noninvasive tool to predict chemotherapy outcomes. Significance: Allysine-targeted molecular MRI can quantify fibrogenesis in pancreatic tumors and predict response to chemotherapy, which could guide rapid clinical management decisions by differentiating responders from nonresponders after treatment initiation.

Noninvasive Quantification of Radiation-Induced Lung Injury Using a Targeted Molecular Imaging Probe.

PURPOSE: Radiation-induced lung injury (RILI) is a progressive inflammatory process seen after irradiation for lung cancer. The disease can be insidious, often characterized by acute pneumonitis followed by chronic fibrosis with significant associated morbidity. No therapies are approved for RILI, and accurate disease quantification is a major barrier to improved management. Here, we sought to noninvasively quantify RILI using a molecular imaging probe that specifically targets type 1 collagen in mouse models and patients with confirmed RILI. METHODS AND MATERIALS: Using a murine model of lung radiation, mice were imaged with EP-3533, a type 1 collagen probe, to characterize the development of RILI and to assess disease mitigation after losartan treatment. The human analog probe 68Ga-CBP8, targeting type 1 collagen, was tested on excised human lung tissue containing RILI and was quantified via autoradiography. 68Ga-CBP8 positron emission tomography was used to assess RILI in vivo in 6 human subjects. RESULTS: Murine models demonstrated that probe signal correlated with progressive RILI severity over 6 months. The probe was sensitive to mitigation of RILI by losartan. Excised human lung tissue with RILI had increased binding versus unirradiated control tissue, and 68Ga-CBP8 uptake correlated with collagen proportional area. Human imaging revealed significant 68Ga-CBP8 uptake in areas of RILI and minimal background uptake. CONCLUSIONS: These findings support the ability of a molecular imaging probe targeted at type 1 collagen to detect RILI in preclinical models and human disease, suggesting a role for targeted molecular imaging of collagen in the assessment of RILI.

Detection of Pulmonary Fibrosis with a Collagen-Mimetic Peptide.

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology that is characterized by excessive deposition and abnormal remodeling of collagen. IPF has a mean survival time of only 2-5 years from diagnosis, creating a need to detect IPF at an earlier stage when treatments might be more effective. We sought to develop a minimally invasive probe that could detect molecular changes in IPF-associated collagen. Here, we describe the design, synthesis, and performance of [68Ga]Ga·DOTA-CMP, which comprises a positron-emitting radioisotope linked to a collagen-mimetic peptide (CMP). This peptide mimics the natural structure of collagen and detects irregular collagen matrices by annealing to damaged collagen triple helices. We assessed the ability of the peptide to detect aberrant lung collagen selectively in a bleomycin-induced mouse model of pulmonary fibrosis using positron emission tomography (PET). [68Ga]Ga·DOTA-CMP PET demonstrated higher and selective uptake in a fibrotic mouse lung compared to controls, minimal background signal in adjacent organs, and rapid clearance via the renal system. These studies suggest that [68Ga]Ga·DOTA-CMP identifies fibrotic lungs and could be useful in the early diagnosis of IPF.

Noninvasive Quantification of Radiation-Induced Lung Injury using a Targeted Molecular Imaging Probe.

Rationale: Radiation-induced lung injury (RILI) is a progressive inflammatory process commonly seen following irradiation for lung cancer. The disease can be insidious, often characterized by acute pneumonitis followed by chronic fibrosis with significant associated morbidity. No therapies are approved for RILI, and accurate disease quantification is a major barrier to improved management. Objective: To noninvasively quantify RILI, utilizing a molecular imaging probe that specifically targets type 1 collagen in mouse models and patients with confirmed RILI. Methods: Using a murine model of lung radiation, mice were imaged with EP-3533, a type 1 collagen probe to characterize the development of RILI and to assess disease mitigation following losartan treatment. The human analog probe targeted against type 1 collagen, 68Ga-CBP8, was tested on excised human lung tissue containing RILI and quantified via autoradiography. Finally, 68Ga-CBP8 PET was used to assess RILI in vivo in six human subjects. Results: Murine models demonstrated that probe signal correlated with progressive RILI severity over six-months. The probe was sensitive to mitigation of RILI by losartan. Excised human lung tissue with RILI had increased binding vs unirradiated control tissue and 68Ga-CBP8 uptake correlated with collagen proportional area. Human imaging revealed significant 68Ga-CBP8 uptake in areas of RILI and minimal background uptake. Conclusions: These findings support the ability of a molecular imaging probe targeted at type 1 collagen to detect RILI in preclinical models and human disease, suggesting a role for targeted molecular imaging of collagen in the assessment of RILI.Clinical trial registered with www.clinicaltrials.gov (NCT04485286, NCT03535545).

Fast detection of liver fibrosis with collagen-binding single-nanometer iron oxide nanoparticles via T1-weighted MRI.

SNIO-CBP, a single-nanometer iron oxide (SNIO) nanoparticle functionalized with a type I collagen-binding peptide (CBP), was developed as a T1-weighted MRI contrast agent with only endogenous elements for fast and noninvasive detection of liver fibrosis. SNIO-CBP exhibits 6.7-fold higher relaxivity compared to a molecular gadolinium-based collagen-binding contrast agent CM-101 on a per CBP basis at 4.7 T. Unlike most iron oxide nanoparticles, SNIO-CBP exhibits fast elimination from the bloodstream with a 5.7 min half-life, high renal clearance, and low, transient liver enhancement in healthy mice. We show that a dose of SNIO-CBP that is 2.5-fold lower than that for CM-101 has comparable imaging efficacy in rapid (within 15 min following intravenous injection) detection of hepatotoxin-induced liver fibrosis using T1-weighted MRI in a carbon tetrachloride-induced mouse liver injury model. We further demonstrate the applicability of SNIO-CBP in detecting liver fibrosis in choline-deficient L-amino acid-defined high-fat diet mouse model of nonalcoholic steatohepatitis. These results provide a platform with potential for the development of high relaxivity, gadolinium-free molecular MRI probes for characterizing chronic liver disease.

HER3 PET Imaging Predicts Response to Pan Receptor Tyrosine Kinase Inhibition Therapy in Gastric Cancer.

PURPOSE: New generation of receptor tyrosine kinase inhibitors (RTKIs) have shown to improve survival in many solid tumors. However, an imaging biomarker is needed for patient selection and prediction of treatment response. This study evaluates the use of quantitative changes of HER3 on 68 Ga-NOTA-HER3P1 PET/MRI for prediction of early response to pan-RTKIs in gastric cancer (GCa). PROCEDURES: GCa cell lines were evaluated for expression of RTKs, and downstream signaling pathways (AKT and MAPK). Cell viability was assessed following 24-72 h of treatment with 0.01-1 µmol/L of afatinib, a pan-RTKI. HER3-expressing afatinib-sensitive (NCI-N87) and resistant cells (SNU16) were selected for evaluation of changes in RTKs expression and downstream pathways, with 24-72 h of 0.1 µmol/L afatinib treatment. 68 Ga-NOTA-HER3P1 PET/MRI was performed in subcutaneous NCI-N87 and SNU16 xenografts (nu:nu, n = 12/group) at baseline and 4 days after afatinib treatment (10 mg/kg, PO, daily). Temporal changes in PET measures were correlated to HER3 expression in tumors, tumor growth rate, and treatment response. RESULTS: With afatinib therapy, NCI-N87 cells showed increased total HER3 expression, and reduction of other RTKs and downstream nodes within 72 h, while SNU16 cells showed no significant change in total HER3 and downstream nodes. 68 Ga-HER3P1 PET/MRI showed increased uptake in NCI-N87 and no significant change in SNU16 tumors (day 4 vs. baseline SUVmean: 3.8 ± 0.7 vs. 1.6 ± 0.6, p < 0.05 in NCI-N87, and 1.5 ± 0.7 vs. 1.7 ± 0.7, p > 0.05 in SNU16). These findings were in concordance with HER3 expression in histopathological analyses and tumor growth over 3 weeks of treatment (mean tumor volume in treated vs. control: 11 ± 17 mm3 vs. 293 ± 79 mm3, p < 0.001 in NCI-N87, and 238 ± 91 mm3 vs. 282 ± 35 mm3, p > 0.05 in SNU16). CONCLUSIONS: Quantitative changes in HER3 PET could be used to predict response to pan-RTKI within few days after initiation of treatment and can help with personalizing GCa management.

Molecular MRI quantification of extracellular aldehyde pairs for early detection of liver fibrogenesis and response to treatment.

Liver fibrosis plays a critical role in the evolution of most chronic liver diseases and is characterized by a buildup of extracellular matrix, which can progress to cirrhosis, hepatocellular carcinoma, liver failure, or death. Now, there are no noninvasive methods available to accurately assess disease activity (fibrogenesis) to sensitively detect early onset of fibrosis or to detect early response to treatment. Here, we hypothesized that extracellular allysine aldehyde (LysAld) pairs formed by collagen oxidation during active fibrosis could be a target for assessing fibrogenesis with a molecular probe. We showed that molecular magnetic resonance imaging (MRI) using an extracellular probe targeting these LysAld pairs acts as a noninvasive biomarker of fibrogenesis and demonstrated its high sensitivity and specificity in detecting fibrogenesis in toxin- and dietary-induced mouse models, a cholestasis rat model of liver fibrogenesis, and in human fibrotic liver tissues. Quantitative molecular MRI was highly correlated with fibrogenesis markers and enabled noninvasive detection of early onset fibrosis and response to antifibrotic treatment, showing high potential for clinical translation.

Hyperpolarized [1-13C]Pyruvate Magnetic Resonance Spectroscopic Imaging for Evaluation of Early Response to Tyrosine Kinase Inhibition Therapy in Gastric Cancer.

PURPOSE: To evaluate the use of hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopic imaging (HP-13C MRSI) for quantitative measurement of early changes in glycolytic metabolism and its ability to predict response to pan-tyrosine kinase inhibitor (Pan-TKI) therapy in gastric cancer (GCa). PROCEDURES: Pan-TKI afatinib-sensitive NCI-N87 and resistant SNU16 human GCa cells were assessed for GLUT1, hexokinase-II (HKII), lactate dehydrogenase (LDHA), phosphorylated AKT (pAKT), and phosphorylated MAPK (pMAPK) at 0-72 h of treatment with 0.1 µM afatinib. Subcutaneous NCI-N87 tumor-bearing nude mice underwent [18F]FDG PET/MRI and HP-13C MRSI at baseline and 4 days after treatment with afatinib 10 mg/kg/day or vehicle (n = 10/group). Changes in PET and HP-13C MRSI metabolic parameters were compared between the two groups. Imaging findings were correlated with tumor growth and histopathology over 3 weeks of treatment. RESULTS: In vitro analysis showed a continuous decrease in LDHA, pAKT, and pMAPK in NCI-N87 compared to SNU16 cells within 72 h of treatment with afatinib, without a significant change in GLUT1 and HKII in either cell type. [18F]FDG PET of NCI-N87 tumors showed no significant change in PET measures at baseline and day 4 of treatment in either treatment group (SUVmean day 4/day 0: 2.7 ± 0.42/2.34 ± 0.38, p = 0.57 in the treated group vs. 1.73 ± 0.66/2.24 ± 0.43, p = 0.4 in the control group). HP-13C MRSI demonstrated significantly decreased lactate-to-pyruvate ratio (L/P) in treated tumors (L/P day 4/day 0: 0.83 ± 0.30/1.10 ± 0.20, p = 0.012 vs. 0.94 ± 0.20/0.98 ± 0.30, p = 0.75, in the treated vs. control group, respectively). Response to afatinib was confirmed with decreased tumor size over 3 weeks (11.10 ± 16.50 vs. 293.00 ± 79.30 mm3, p < 0.001, treated group vs. control group, respectively) and histopathologic evaluation. CONCLUSIONS: HP-13C MRSI is a more representative biomarker of early metabolic changes in response to pan-TKI in GCa than [18F]FDG PET and could be used for early prediction of response to targeted therapies.

Imaging High-Risk Atherothrombosis Using a Novel Fibrin-Binding Positron Emission Tomography Probe.

BACKGROUND AND PURPOSE: High-risk atherosclerosis is an underlying cause of cardiovascular events, yet identifying the specific patient population at immediate risk is still challenging. Here, we used a rabbit model of atherosclerotic plaque rupture and human carotid endarterectomy specimens to describe the potential of molecular fibrin imaging as a tool to identify thrombotic plaques. METHODS: Atherosclerotic plaques in rabbits were induced using a high-cholesterol diet and aortic balloon injury (N=13). Pharmacological triggering was used in a group of rabbits (n=9) to induce plaque disruption. Animals were grouped into thrombotic and nonthrombotic plaque groups based on gross pathology (gold standard). All animals were injected with a novel fibrin-specific probe 68Ga-CM246 followed by positron emission tomography (PET)/magnetic resonance imaging 90 minutes later. 68Ga-CM246 was quantified on the PET images using tissue-to-background (back muscle) ratios and standardized uptake value. RESULTS: Both tissue-to-background (back muscle) ratios and standardized uptake value were significantly higher in the thrombotic versus nonthrombotic group (P<0.05). Ex vivo PET and autoradiography of the abdominal aorta correlated positively with in vivo PET measurements. Plaque disruption identified by 68Ga-CM246 PET agreed with gross pathology assessment (85%). In ex vivo surgical specimens obtained from patients undergoing elective carotid endarterectomy (N=12), 68Ga-CM246 showed significantly higher binding to carotid plaques compared to a D-cysteine nonbinding control probe. CONCLUSIONS: We demonstrated that molecular fibrin PET imaging using 68Ga-CM246 could be a useful tool to diagnose experimental and clinical atherothrombosis. Based on our initial results using human carotid plaque specimens, in vivo molecular imaging studies are warranted to test 68Ga-CM246 PET as a tool to stratify risk in atherosclerotic patients.

Quantitative, noninvasive MRI characterization of disease progression in a mouse model of non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is an increasing cause of chronic liver disease characterized by steatosis, inflammation, and fibrosis which can lead to cirrhosis, hepatocellular carcinoma, and mortality. Quantitative, noninvasive methods for characterizing the pathophysiology of NASH at both the preclinical and clinical level are sorely needed. We report here a multiparametric magnetic resonance imaging (MRI) protocol with the fibrogenesis probe Gd-Hyd to characterize fibrotic disease activity and steatosis in a common mouse model of NASH. Mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) to induce NASH with advanced fibrosis. Mice fed normal chow and CDAHFD underwent MRI after 2, 6, 10 and 14 weeks to measure liver T1, T2*, fat fraction, and dynamic T1-weighted Gd-Hyd enhanced imaging of the liver. Steatosis, inflammation, and fibrosis were then quantified by histology. NASH and fibrosis developed quickly in CDAHFD fed mice with strong correlation between morphometric steatosis quantification and liver fat estimated by MRI (r = 0.90). Sirius red histology and collagen quantification confirmed increasing fibrosis over time (r = 0.82). Though baseline T1 and T2* measurements did not correlate with fibrosis, Gd-Hyd signal enhancement provided a measure of the extent of active fibrotic disease progression and correlated strongly with lysyl oxidase expression. Gd-Hyd MRI accurately detects fibrogenesis in a mouse model of NASH with advanced fibrosis and can be combined with other MR measures, like fat imaging, to more accurately assess disease burden.

Fibrotic Response to Neoadjuvant Therapy Predicts Survival in Pancreatic Cancer and Is Measurable with Collagen-Targeted Molecular MRI.

PURPOSE: To evaluate the prognostic value of posttreatment fibrosis in human PDAC patients, and to compare a type I collagen targeted MRI probe, CM-101, to the standard contrast agent, Gd-DOTA, for their abilities to identify FOLFIRINOX-induced fibrosis in a murine model of PDAC. EXPERIMENTAL DESIGN: Ninety-three chemoradiation-treated human PDAC samples were stained for fibrosis and outcomes evaluated. For imaging, C57BL/6 and FVB mice were orthotopically implanted with PDAC cells and FOLFIRINOX was administered. Mice were imaged with Gd-DOTA and CM-101. RESULTS: In humans, post-chemoradiation PDAC tumor fibrosis was associated with longer overall survival (OS) and disease-free survival (DFS) on multivariable analysis (OS P = 0.028, DFS P = 0.047). CPA increased the prognostic accuracy of a multivariable logistic regression model comprised of previously established PDAC risk factors [AUC CPA (-) = 0.76, AUC CPA (+) = 0.82]. In multiple murine orthotopic PDAC models, FOLFIRINOX therapy reduced tumor weight (P < 0.05) and increased tumor fibrosis by collagen staining (P < 0.05). CM-101 MR signal was significantly increased in fibrotic tumor regions. CM-101 signal retention was also increased in the more fibrotic FOLFIRINOX-treated tumors compared with untreated controls (P = 0.027), consistent with selective probe binding to collagen. No treatment-related differences were observed with Gd-DOTA imaging. CONCLUSIONS: In humans, post-chemoradiation tumor fibrosis is associated with OS and DFS. In mice, our MR findings indicate that translation of collagen molecular MRI with CM-101 to humans might provide a novel imaging technique to monitor fibrotic response to therapy to assist with prognostication and disease management.

Improving the reactivity of hydrazine-bearing MRI probes for in vivo imaging of lung fibrogenesis.

Pulmonary fibrosis (PF) is the pathologic accumulation of extracellular matrix components in lung tissue that result in scarring following chronic lung injury. PF is typically diagnosed by high resolution computed tomography (HRCT) and/or invasive biopsy. However, HRCT cannot distinguish old injury from active fibrogenesis. We previously demonstrated that allysine residues on oxidized collagen represent an abundant target during lung fibrogenesis, and that magnetic resonance imaging (MRI) with a small-molecule, gadolinium-containing probe, Gd-Hyd, could specifically detect and stage fibrogenesis in a mouse model. In this work, we present an improved probe, Gd-CHyd, featuring an N,N-dialkyl hydrazine which has an order of magnitude both greater reactivity and affinity for aldehydes. In a paired study in mice with bleomycin induced lung injury we show that the improved reactivity and affinity of Gd-CHyd results in significantly higher lung-to-liver contrast, e.g. 77% higher at 45 min post injection, and slower lung clearance than Gd-Hyd. Gd-CHyd enhanced MRI is >60-fold higher in bleomycin injured mouse lungs compared to uninjured mice. Collectively, our data indicate that enhancing hydrazine reactivity and affinity towards allysine is an effective strategy to significantly improve molecular MRI probes for lung fibrogenesis.

Advanced MRI of Liver Fibrosis and Treatment Response in a Rat Model of Nonalcoholic Steatohepatitis.

Background Liver biopsy is the reference standard to diagnose nonalcoholic steatohepatitis (NASH) but is invasive with potential complications. Purpose To evaluate molecular MRI with type 1 collagen-specific probe EP-3533 and allysine-targeted fibrogenesis probe Gd-Hyd, MR elastography, and native T1 to characterize fibrosis and to assess treatment response in a rat model of NASH. Materials and Methods MRI was performed prospectively (June-November 2018) in six groups of male Wistar rats (a) age- and (b) weight-matched animals received standard chow (n = 12 per group); (c) received choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) for 6 weeks or (d) 9 weeks (n = 8 per group); (e) were fed 6 weeks of CDAHFD and switched to standard chow for 3 weeks (n = 12); (f) were fed CDAHFD for 9 weeks with daily treatment of elafibranor beginning at week 6 (n = 14). Differences in imaging measurements and tissue analyses among groups were tested with one-way analysis of variance. The ability of each imaging measurement to stage fibrosis was quantified by using area under the receiver operating characteristic curve (AUC) with quantitative digital pathology (collagen proportionate area [CPA]) as reference standard. Optimal cutoff values for distinguishing advanced fibrosis were used to assess treatment response. Results AUC for distinguishing fibrotic (CPA >4.8%) from nonfibrotic (CPA ≤4.8%) livers was 0.95 (95% confidence interval [CI]: 0.91, 1.00) for EP-3533, followed by native T1, Gd-Hyd, and MR elastography with AUCs of 0.90 (95% CI: 0.83, 0.98), 0.84 (95% CI: 0.74, 0.95), and 0.65 (95% CI: 0.51, 0.79), respectively. AUCs for discriminating advanced fibrosis (CPA >10.3%) were 0.86 (95% CI: 0.76, 0.97), 0.96 (95% CI: 0.90, 1.01), 0.84 (95% CI: 0.70, 0.98), and 0.74 (95% CI: 0.63, 0.86) for EP-3533, Gd-Hyd, MR elastography, and native T1, respectively. Gd-Hyd MRI had the highest accuracy (24 of 26, 92%; 95% CI: 75%, 99%) in identifying responders and nonresponders in the treated groups compared with MR elastography (23 of 26, 88%; 95% CI: 70%, 98%), EP-3533 (20 of 26, 77%; 95% CI: 56%, 91%), and native T1 (14 of 26, 54%; 95% CI: 33%, 73%). Conclusion Collagen-targeted molecular MRI most accurately detected early onset of fibrosis, whereas the fibrogenesis probe Gd-Hyd proved most accurate for detecting treatment response. © RSNA, 2020 Online supplemental material is available for this article.

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

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.

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 (64)Cu-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. (64)Cu-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 (64)Cu-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 (64)Cu-FBP8-PET is a feasible approach for whole-body thrombus detection and that molecular imaging of fibrin can provide, noninvasively, insight into clot composition.

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

UNLABELLED: 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 (64)Cu-FBP8. Here, we tested the feasibility of (64)Cu-FBP8 PET to detect source thrombi and culprit emboli after deep vein thrombosis and pulmonary embolism (DVT-PE). To support clinical translation of (64)Cu-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 (125)I-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 (64)Cu-FBP8 administration to estimate human radiation dosimetry. Longitudinal whole-body PET/MR imaging (n = 2) was performed after (64)Cu-FBP8 administration to further assess radioactivity clearance. RESULTS: (64)Cu-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 (64)Cu-FBP8. The effective dose was 0.021 mSv/MBq for males and 0.027 mSv/MBq for females, supporting the feasibility of using (64)Cu-FBP8 in human trials. CONCLUSION: We showed that (64)Cu-FBP8 PET is a feasible approach to image DVT-PE and that radiogenic adverse health effects should not limit the clinical translation of (64)Cu-FBP8.

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

UNLABELLED: 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 (64)Cu-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 (64)Cu or aluminum fluoride (Al(18)F) 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 (64)Cu (FBP8 and FBP9) or Al(18)F (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 (64)Cu 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 Al(18)F 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.