Folate Receptor ß-Targeted PET Imaging of Macrophages in Autoimmune Myocarditis.

Currently available imaging techniques have limited specificity for the detection of active myocardial inflammation. Aluminum 18F-labeled 1,4,7-triazacyclononane-N,N',N″-triacetic acid conjugated folate (18F-FOL) is a PET tracer targeting folate receptor ß (FR-ß), which is expressed on activated macrophages at sites of inflammation. We evaluated 18F-FOL PET for the detection of myocardial inflammation in rats with autoimmune myocarditis and studied the expression of FR-ß in human cardiac sarcoidosis specimens. Methods: Myocarditis was induced by immunizing rats (n = 18) with porcine cardiac myosin in complete Freund adjuvant. Control rats (n = 6) were injected with Freund adjuvant alone. 18F-FOL was intravenously injected, followed by imaging with a small-animal PET/CT scanner and autoradiography. Contrast-enhanced high-resolution CT or 18F-FDG PET images were used for coregistration. Rat tissue sections and myocardial autopsy samples from 6 patients with cardiac sarcoidosis were studied for macrophages and FR-ß. Results: The myocardium of 10 of 18 immunized rats showed focal macrophage-rich inflammatory lesions, with FR-ß expression occurring mainly in M1-polarized macrophages. PET images showed focal myocardial 18F-FOL uptake colocalizing with inflammatory lesions (SUVmean, 2.1 ± 1.1), whereas uptake in the remote myocardium of immunized rats and controls was low (SUVmean, 0.4 ± 0.2 and 0.4 ± 0.1, respectively; P < 0.01). Ex vivo autoradiography of tissue sections confirmed uptake of 18F-FOL in myocardial inflammatory lesions. Uptake of 18F-FOL in inflamed myocardium was efficiently blocked by a nonlabeled FR-ß ligand folate glucosamine in vivo. The myocardium of patients with cardiac sarcoidosis showed many FR-ß-positive macrophages in inflammatory lesions. Conclusion: In a rat model of autoimmune myocarditis, 18F-FOL shows specific uptake in inflamed myocardium containing macrophages expressing FR-ß, which were also present in human cardiac sarcoid lesions. Imaging of FR-ß expression is a potential approach for the detection of active myocardial inflammation.

Folate receptor-targeted positron emission tomography of experimental autoimmune encephalomyelitis in rats.

BACKGROUND: Folate receptor-ß (FR-ß) is a cell surface receptor that is significantly upregulated on activated macrophages during inflammation and provides a potential target for folate-based therapeutic and diagnostic agents. FR-ß expression in central nervous system inflammation remains relatively unexplored. Therefore, we used focally induced acute and chronic phases of experimental autoimmune encephalomyelitis (EAE) to study patterns of FR-ß expression and evaluated its potential as an in vivo imaging target. METHODS: Focal EAE was induced in rats using heat-killed Bacillus Calmette-Guérin followed by activation with complete Freund's adjuvant supplemented with Mycobacterium tuberculosis. The rats were assessed with magnetic resonance imaging and positron emission tomography/computed tomography (PET/CT) at acute (14 days) and chronic (90 days) phases of inflammation. The animals were finally sacrificed for ex vivo autoradiography of their brains. PET studies were performed using FR-ß-targeting aluminum [18F]fluoride-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid conjugated folate ([18F]AlF-NOTA-folate, 18F-FOL) and 18 kDa translocator protein (TSPO)-targeting N-acetyl-N-(2-[11C]methoxybenzyl)-2-phenoxy-5-pyridinamine (11C-PBR28). Post-mortem immunohistochemistry was performed using anti-FR-ß, anti-cluster of differentiation 68 (anti-CD68), anti-inducible nitric oxide synthase (anti-iNOS), and anti-mannose receptor C-type 1 (anti-MRC-1) antibodies. The specificity of 18F-FOL binding was verified using in vitro brain sections with folate glucosamine used as a blocking agent. RESULTS: Immunohistochemical evaluation of focal EAE lesions demonstrated anti-FR-ß positive cells at the lesion border in both acute and chronic phases of inflammation. We found that anti-FR-ß correlated with anti-CD68 and anti-MRC-1 immunohistochemistry; for MRC-1, the correlation was most prominent in the chronic phase of inflammation. Both 18F-FOL and 11C-PBR28 radiotracers bound to the EAE lesions. Autoradiography studies verified that this binding took place in areas of anti-FR-ß positivity. A blocking assay using folate glucosamine further verified the tracer's specificity. In the chronic phase of EAE, the lesion-to-background ratio of 18F-FOL was significantly higher than that of 11C-PBR28 (P = 0.016). CONCLUSION: Our EAE results imply that FR-ß may be a useful target for in vivo imaging of multiple sclerosis-related immunopathology. FR-ß-targeted PET imaging with 18F-FOL may facilitate the monitoring of lesion development and complement the information obtained from TSPO imaging by bringing more specificity to the PET imaging armamentarium for neuroinflammation.

Folate-PEG-NOTA-Al18F: A New Folate Based Radiotracer for PET Imaging of Folate Receptor-Positive Tumors.

The folate receptor (FR) has been established as a promising target for imaging and therapy of cancer (FR-α), inflammation, and autoimmune diseases (FR-ß). Several folate based PET radiotracers have been reported in the literature, but an 18F-labeled folate-PET imaging agent with optimal properties for clinical translation is still lacking. In the present study, we report the design and preclinical evaluation of folate-PEG12-NOTA-Al18F (1), a new folate-PET agent with improved potential for clinical applications. Radiochemical synthesis of 1 was achieved via a one-pot labeling process by heating folate-PEG12-NOTA in the presence of in situ prepared Al18F for 15 min at 105 °C, followed by HPLC purification. Specific binding of 1 to FR was evaluated on homogenates of KB (FR-positive) and A549 (FR-deficient) tumor xenografts in the presence and absence of excess folate. In vivo tumor imaging with folate-PEG12-NOTA-Al18F was compared to imaging with 99mTc-EC20 using nu/nu mice bearing either KB or A549 tumor xenografts. Specific accumulation of 1 in tumor and other tissues was assessed by high-resolution micro-PET and ex vivo biodistribution in the presence and absence of excess folate. Radiosynthesis of 1 was accomplished within ∼35 min, affording pure radiotracer 1 in 8.4 ± 1.3% (decay corrected) radiochemical yield with ∼100% radiochemical purity after HPLC purification and a specific activity of 35.8 ± 15.3 GBq/mmol. Further in vitro and in vivo examination of 1 demonstrated highly specific FR-mediated uptake in FR+ tumor, with Kd of ∼0.4 nM (KB), and reduced accumulation in liver. Given its facile preparation and improved properties, the new radiotracer, folate-PEG12-NOTA-Al18F (1), constitutes a promising tool for identification and classification of patients with FR overexpressing cancers.

In-vivo monitoring of anti-folate therapy in arthritic rats using [18F]fluoro-PEG-folate and positron emission tomography.

BACKGROUND: Folate receptor ß (FRß) is involved in facilitating cellular uptake of folates and anti-folates (such as methotrexate (MTX)). In rheumatoid arthritis, FRß is expressed on synovial macrophages and recently has been explored as a biomarker for imaging in arthritic rats using the folate-based positron emission tomography (PET) tracer [18F]fluoro-PEG-folate. The purpose of this study was to examine whether this folate tracer can also be used to monitor therapeutic efficacy of MTX in arthritic rats. METHODS: Arthritic rats received either no treatment or MTX therapy (1 mg/kg, either 2× or 4×). Healthy rats did not receive any arthritic induction or therapy. [18F]fluoro-PEG-folate PET-CT scans (60 min) were performed before and after MTX therapy. Following PET, the ex-vivo tissue distribution of radioactivity was determined in excised knees and multiple tissues. Synovial macrophage infiltration in knee sections was quantified by immunohistochemistry using ED1 and ED2 antibodies. RESULTS: PET scans clearly visualized increased uptake of [18F]fluoro-PEG-folate in arthritic knees compared with contralateral knees. Significantly lower standard uptake values (1.5-fold, p < 0.01) were observed in arthritic knees of both MTX-treated groups after therapy, approximating the levels seen in healthy rats. Consistently, ex-vivo tissue distribution demonstrated a 2-4-fold lower tracer uptake in the arthritic knee of 2× and 4× MTX-treated rats, respectively, compared with control rats. These results were corroborated with significantly reduced (2-4-fold, p < 0.01) ED1-positive and ED2-positive synovial macrophages in arthritic knees of the MTX-treated rats compared with those of the control rats. CONCLUSION: This study in arthritic rats underscores the potential and usefulness of [18F]fluoro-PEG-folate PET as a therapeutic monitoring tool of MTX therapy and potentially other anti-folate treatment of arthritis.