Qingshou Chen

Synthesis and Preclinical Evaluation of Folate-NOTA-Al18F for PET Imaging of Folate-Receptor-Positive Tumors

UNLABELLED Folate-receptor-targeted PET radiotracers can potentially serve as versatile imaging agents for the diagnosis, staging, and prediction of response to therapy of patients with folate-receptor (FR)-expressing cancers. Because current FR-targeted PET reagents can be compromised by complex labeling procedures, low specific activities, poor radiochemical yields, or unwanted accumulation in FR negative tissues, we have undertaken to design an improved folate-PET agent that might be more amenable for clinical development. For this purpose, we have synthesized a folate-NOTA-Al(18)F radiotracer and examined its properties both in vitro and in vivo. METHODS Radiochemical synthesis of folate-NOTA-Al(18)F was achieved by incubating (18)F(-) with AlCl3 for 2 min followed by heating in the presence of folate-NOTA for 15 min at 100 °C. Binding of folate-NOTA-Al(18)F to FR was quantitated in homogenates of KB and Cal51 tumor xenografts in the presence and absence of excess folic acid as a competitor. In vivo imaging was performed on nu/nu mice bearing either FR+ve (KB cell) or FR-ve (A549 cell) tumor xenografts, and specific accumulation of the radiotracer in tumor and other tissues was assessed by high-resolution micro-PET and ex vivo biodistribution in the presence and absence of excess folic acid. Image quality of folate-NOTA-Al(18)F was compared with that of (99m)Tc-EC20, a clinically established folate-targeted SPECT imaging agent. RESULTS Total radiochemical synthesis and purification of folate-NOTA-Al(18)F was completed within 37 min, yielding a specific activity of 68.82 ± 18.5 GBq/μmol, radiochemical yield of 18.6 ± 4.5%, and radiochemical purity of 98.3 ± 2.9%. Analysis of FR binding revealed a Kd of ∼1.0 nM, and micro-PET imaging together with ex vivo biodistribution analyses demonstrated high FR-mediated uptake in an FR+ tumor and the kidneys. CONCLUSIONS Folate-NOTA-Al(18)F constitutes an easily prepared FR-targeted PET imaging agent with improved radiopharmaceutical properties and high specificity for folate receptor expressing tumors. Given its improved properties over (99m)Tc-EC20 (i.e., higher resolution, shorter image acquisition time, etc.), we conclude that folate-NOTA-Al(18)F constitutes a viable alternative to (99m)Tc-EC20 for use in identification, diagnosis, and staging of patients with FR-expressing cancers.

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

BackgroundFolate receptor β (FRβ) is involved in facilitating cellular uptake of folates and anti-folates (such as methotrexate (MTX)). In rheumatoid arthritis, FRβ 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.MethodsArthritic 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.ResultsPET 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.ConclusionThis 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.

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-β). 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.

Aluminum fluoride-18 labeled folate enables in vivo detection of atherosclerotic plaque inflammation by positron emission tomography

Inflammation plays an important role in the development of atherosclerosis and its complications. Because the folate receptor β (FR-β) is selectively expressed on macrophages, an FR targeted imaging agent could be useful for assessment of atherosclerotic inflammation. We investigated aluminum fluoride-18-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid conjugated folate (18F-FOL) for the detection of atherosclerotic plaque inflammation. We studied atherosclerotic plaques in mice, rabbits, and human tissue samples using 18F-FOL positron emission tomography/computed tomography (PET/CT). Compound 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG) was used as a comparison. Firstly, we found that the in vitro binding of 18F-FOL co-localized with FR-β-positive macrophages in carotid endarterectomy samples from patients with recent ischemic symptoms. We then demonstrated specific accumulation of intravenously administered 18F-FOL in atherosclerotic plaques in mice and rabbits using PET/CT. We noticed that the 18F-FOL uptake correlated with the density of macrophages in plaques and provided a target-to-background ratio as high as 18F-FDG, but with considerably lower myocardial uptake. Thus, 18F-FOL PET/CT targeting of FR-β-positive macrophages presents a promising new tool for the in vivo imaging of atherosclerotic inflammation.

SAT0650 [18F]FLUORO-PEG-FOLATE pet: a novel imaging technique to visualize rheumatoid arthritis

Background Imaging arthritis activity in rheumatoid arthritis (RA) patients using PET macrophage tracers holds promise for both early diagnostics and monitoring response to therapy (1,2). Previously, (R)-[11C]PK11195 has been used, but this macrophage tracer is limited due to high background uptake, especially in bone and bone marrow. Recently, a novel macrophage tracer, [18F]fluoro-PEG-folate, was developed, which showed excellent targeting of the folate receptor beta on activated macrophages in synovial tissue in a preclinical arthritic rat model (3). Objectives To assess the value of [18F]fluoro-PEG-folate PET-CT for imaging inflamed joints in patients with clinically active RA. Methods Nine RA patients with at least two clinically inflamed hand joints were included. PET-CT scans of the hands were acquired after intravenous administration of either 185 MBq of [18F]fluoro-PEG-folate (n=6) or 425 MBq of (R)-[11C]PK11195 (n=3). Volumes of Interest (VOI) were drawn over joints with visually marked uptake and Standardized Uptake Values (SUVs) were calculated. Background VOIs were drawn on metacarpal bone in order to calculate Target-to-Background (T/B) ratios. Results No side effects were observed, establishing the safety of [18F]fluoro-PEG-folate for use in humans. [18F]fluoro-PEG-folate clearly showed uptake in arthritic joints, as shown in Figure 1. In patients scanned with [18F]fluoro-PEG-folate, 25 positive joints were seen, with a minimum of two joints per patient. Clinical arthritis was confirmed in 10 of these 25 joints, and was absent in 15 positive joints, suggesting the presence of subclinical inflammation. Whilst both [18F]fluoro-PEG-folate and (R)-[11C]PK11195 accumulated in arthritic joints, [18F]fluoro-PEG-folate showed a significantly lower background uptake than (R)-[11C]PK11195 (SUV of 0.18 vs 0.75; p<0.001) respectively. T/B-ratios were significantly higher for [18F]fluoro-PEG-folate (3.60vs 1.72, p=0.009). Conclusions This first in patient study clearly demonstrates the potential of [18F]fluoro-PEG-folate PET-CT as macrophage tracer to image both clinically and sub-clinically affected joints in RA patients. [18F]fluoro-PEG-folate showed better characteristics for arthritis imaging than the established tracer (R)-[11C]PK11195 because of its lower background signal. References Gent YY, et al. J Rheumatology. 2014; 41: 2145–52. Gent YY, et al. Arthritis Rheum. 2012; 64: 62–6. Gent YY, et al. Arthritis Res Ther. 2013; 15: R37. Disclosure of Interest None declared