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.

First in man study of [18F]fluoro-PEG-folate PET: a novel macrophage imaging technique to visualize rheumatoid arthritis.

Non-invasive imaging of arthritis activity in rheumatoid arthritis (RA) patients using macrophage PET holds promise for early diagnosis and therapeutic response monitoring. Previously obtained results with macrophage tracer (R)-[11C]PK11195 were encouraging, but the imaging signal could be further improved by reduction of background uptake. Recently, the novel macrophage tracer [18F]fluoro-PEG-folate was developed. This tracer showed excellent targeting of the folate receptor ß on activated macrophages in synovial tissue in a preclinical arthritic rat model. We performed three substudies to investigate the biodistribution, potential for imaging arthritis and kinetic properties of [18F]fluoro-PEG-folate in RA patients. Firstly, biodistribution demonstrated fast clearance of [18F]fluoro-PEG-folate from heart and blood vessels and no dose limiting uptake in organs. Secondly, [18F]fluoro-PEG-folate showed uptake in arthritic joints with significantly lower background and hence significantly higher target-to-background ratios as compared to reference macrophage tracer (R)-[11C]PK11195. Lastly, dynamic scanning demonstrated fast tracer uptake in affected joints, reaching a plateau after 1 minute, co-existing with a rapid blood clearance. In conclusion, this first in man study demonstrates the potential of [18F]fluoro-PEG-folate to image arthritis activity in RA with favourable imaging characteristics of rapid clearance and low background uptake, that allow for detection of inflammatory activity in the whole body.

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.

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 ß (FR-ß) 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-ß-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-ß-positive macrophages presents a promising new tool for the in vivo imaging of atherosclerotic inflammation.

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.

Selective Inhibition of STAT3 Phosphorylation Using a Nuclear-Targeted Kinase Inhibitor.

The discovery of compounds that selectively modulate signaling and effector proteins downstream of EGFR could have important implications for understanding specific roles for pathway activation. A complicating factor for receptor tyrosine kinases is their capacity to be translocated to the nucleus upon ligand engagement. Once localized in subcellular compartments like the nucleus, the roles for EGFR take on additional features, many of which are still being revealed. Additionally, nuclear localization of EGFR has been implicated in downstream events that have significance for therapy resistance and disease progression. The challenges to addressing the differential roles for EGFR in the nucleus motivated experimental approaches that can selectively modulate its subcellular function. By adding modifications to the established EGFR kinase inhibitor gefitinib, an approach to small molecule conjugates with a unique nuclear-targeting peptoid sequence was tested in both human and murine breast tumor cell models for their capacity to inhibit EGF-stimulated activation of ERK1/2 and STAT3. While gefitinib alone inhibits both of these downstream effectors, data acquired here indicate that compartmentalization of the gefitinib conjugates allows for pathway specific inhibition of STAT3 while not affecting ERK1/2 signaling. The inhibitor conjugates offered a more direct route to evaluate the role of EGF-stimulated epithelial-to-mesenchymal transition in these breast cancer cell models. These conjugates revealed that STAT3 activation is not involved in EGF-induced EMT, and instead utilization of the cytoplasmic MAP kinase signaling pathway is critical to this process. This is the first example of a conjugate kinase inhibitor capable of partitioning to the nucleus and offers a new approach to enhancing kinase inhibitor specificity.

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.

Synthesis and Preclinical Evaluation of Folate-NOTA-Al(18)F 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.

Associations between thyroid autoantibody status and abnormal pregnancy outcomes in euthyroid women.

We investigated whether thyroid autoantibody status influences pregnancy outcomes in euthyroid women, by comparing abnormal pregnancy outcome rates between those who tested positive for thyroid autoantibodies (Ab+) and those who tested autoantibody-negative (Ab-). Euthyroid pregnant women (n=7,641) underwent tests for serum thyroid peroxidase antibody (TPOAb) and thyroglobulin antibody (TgAb). The subjects were divided into 4 groups according to thyroid antibody status: TPOAb-/TgAb- (92.9%); TPOAb+/TgAb- (3.2%); TPOAb-/TgAb+ (2.0%); and TPOAb+/TgAb+ (1.9%). The incidence rates of the following abnormal pregnancy outcomes were compared among the 4 groups and analyzed by Fisher's exact test: gestational diabetes, gestational hypertension, placenta previa, placental abruption, premature rupture of fetal membrane (PROM), intrauterine growth restriction, fetal distress, fetal anomalies, stillbirth, preterm birth, and low birth weight. Among the 4 groups, there were no significant differences in age, gestational age, or in the incidence rates of abnormal pregnancy outcomes, except for PROM and low birth weight. The highest incidence rates for PROM and low birth weight were in the TPOAb-/TgAb+ and TPOAb+/TgAb+ subjects, respectively. TgAb positivity and TPOAb positivity were associated with PROM and low birth weight, respectively. Underlying factors that govern the association between thyroid autoantibodies and PROM and low birth weight require further investigation.

Effects of subclinical hypothyroidism on maternal and perinatal outcomes during pregnancy: a single-center cohort study of a Chinese population.

OBJECTIVE: Adverse maternal outcomes and perinatal complications are closely associated with overt maternal hypothyroidism, but whether these complications occur in women with subclinical hypothyroidism (SCH) during pregnancy remains controversial. The aim of this study was to evaluate the effects of SCH on maternal and perinatal outcomes during pregnancy. METHODS: A prospective study of data from 8012 pregnant women (371 women with SCH, 7641 euthyroid women) was performed. Maternal serum samples were collected in different trimesters to examine thyroid hormone concentrations. SCH was defined as a thyroid stimulating hormone concentration exceeding the trimester-specific reference value with a normal free thyroxine concentration. The occurrence of maternal outcomes, including gestational hypertension (GH), gestational diabetes mellitus, placenta previa, placental abruption, prelabor rupture of membranes (PROM), and premature delivery; and perinatal outcomes, including intrauterine growth restriction (IUGR), fetal distress, low birth weight (LBW; live birth weight ≤ 2500 g), stillbirth, and malformation, was recorded. Logistic regression with adjustment for confounding demographic and medical factors was used to determine the risks of adverse outcomes in patients with SCH. RESULTS: Compared with euthyroid status, SCH was associated with higher rates of GH (1.819% vs. 3.504%, P = 0.020; χ2 = 7.345; odds ratio (OR), 2.243; 95% confidence interval (CI), 1.251-4.024), PROM (4.973% vs. 8.625%, P = 0.002; χ2 = 72.102; adjusted OR, 6.014; 95% CI, 3.975-9.099), IUGR (1.008% vs. 2.965%, <0.001; χ2 = 13.272; adjusted OR, 3.336; 95% CI, 1.745-6.377), and LBW (1.885% vs. 4.582%, P<0.001; χ2 = 13.558; adjusted OR, 2.919; 95% CI, 1.650-5.163). CONCLUSIONS: The results of this study indicate that pregnant women with SCH had increased risks of GH and PROM, and their fetuses and infants had increased risks of IUGR and LBW. Thus, routine maternal thyroid function testing is necessary to improve maternal and perinatal outcomes.

Screening strategies for thyroid disorders in the first and second trimester of pregnancy in China.

BACKGROUND: Thyroid dysfunction during pregnancy is associated with multiple adverse outcomes, but whether all women should be screened for thyroid disorders during pregnancy remains controversial. OBJECTIVE: To evaluate the effectiveness of the targeted high risk case-finding approach for identifying women with thyroid dysfunction during the first and second trimesters of pregnancy. METHODS: Levels of thyroid stimulating hormone (TSH), free thyroxine (FT4), and thyroid peroxidase antibodies (TPOAb) were measured in 3882 Chinese women during the first and second trimester of pregnancy. All tested women were divided into the high risk or non-high risk groups, based on their history, findings from physical examination, or other clinical features suggestive of a thyroid disorder. Diagnosis of thyroid disorders was made according to the standard trimester-specific reference intervals. The prevalence of thyroid disorders in each group was determined, and the feasibility of a screening approach focusing exclusively on high risk women was evaluated to estimate the ability of finding women with thyroid dysfunction. RESULTS: The prevalence of overt hypothyroidism or hyperthyroidism in the high risk group was higher than in the non-high risk group during the first trimester (0.8% vs 0, χ2 = 7.10, p = 0.008; 1.6% vs 0.2%, χ2 = 7.02, p = 0.008, respectively). The prevalence of hypothyroxinemia or TPOAb positivity was significantly higher in the high risk group than in the non-high risk group during the second trimester (1.3% vs 0.5%, χ2 = 4.49, p = 0.034; 11.6% vs 8.4%, χ2 = 6.396, p = 0.011, respectively). The total prevalence of hypothyroidism or hyperthyroidism and the prevalence of subclinical hypothyroidism or hyperthyroidism were not statistically different between the high risk and non-high risk groups, for either the first or second trimester. CONCLUSION: The high risk screening strategy failed to detect the majority of pregnant women with thyroid disorders. Therefore, we recommend universal screening of sTSH, FT4, and TPOAb during the first trimester and second trimester of pregnancy.

Adiponectin and peroxisome proliferator-activated receptor-γ gene polymorphisms and gene-gene interactions with type 2 diabetes.

AIMS: To investigate whether gene polymorphisms of both adiponectin and peroxisome proliferator-activated receptor gamma (PPARγ) influence type 2 diabetes mellitus (T2DM) respectively in the Han people of the Wenzhou region of China and whether the interaction of gene polymorphism between adiponectin and PPARγ influences T2DM in the same subjects. MAIN METHODS: This study included 198 patients with T2DM and 255 healthy individuals. Polymerase chain reaction-restriction fragment length polymorphism analyses were used to detect single nucleotide polymorphisms (SNPs). Logistic regression and multifactor dimensionality reduction (MDR) methods were used to analyze gene-gene interactions. KEY FINDINGS: The frequency distribution of adiponectin SNP11377 was not different (p=0.792), but the frequency of CC, CG and GG genotypes showed the difference between two groups (T2DM: 57.1%, 33.3%, and 9.6%; control: 53.7%, 41.6%, and 4.7%, respectively; p=0.047). Adiponectin SNP45, SNP276 and PPAR γ SNPp12a were equally distributed between the two groups (p=0.586, 0.119, 0.437, respectively), and there were no significant differences in genotype frequencies between the two groups (p=0.751, 0.144, 0.479, respectively). Linkage disequilibrium existed between SNP11377 and SNP45 (p<0.001) and SNP45 and SNP276 (p<0.001). Haplotype analyses showed no significant differences between the T2DM and control groups. According to the logistic regression and MDR gene-gene interaction analyses, SNP11377GG and SNP276GT interactions increased the risk of T2DM (odds ratio=6.984, p=0.012). SIGNIFICANCE: Adiponectin SNP11377 and SNP276 gene-gene interactions are associated with the increased risk of T2DM in this population.

Total synthesis of iejimalide B.

Iejimalide B, a structurally unique 24-membered polyene macrolide having a previously underutilized mode of anticancer activity, was synthesized according to a strategy employing Julia-Kocienski olefinations, a palladium-catalyzed Heck reaction, a palladium-catalyzed Marshall propargylation, a Keck-type esterification, and a palladium-catalyzed macrolide-forming, intramolecular Stille coupling of a highly complex cyclization substrate. The overall synthesis is efficient (19.5% overall yield for 15 linear steps) and allows for more practical scaled-up synthesis than previously reported strategies that differed in the order of assembly of key subunits and in the method of macrocyclization. The present synthesis paves the way for efficient preparation of analogues for drug development efforts.

Total synthesis of absinthin.

(+)-Absinthin, a structurally unique triterpene, has been efficiently constructed in nine reaction steps and in 18.6% overall yield from O-acetylisophotosantonic lactone. The synthesis features Mitsunobu arylselenylation, oxidative elimination of allylic arylselenides, biomimetic dimerization via regio- and stereospecific Diels-Alder reaction, and a four-step stereochemical inversion of a highly sterically congested tertiary alcohol. This approach has not only tackled the formidable synthetic challenges in assembling structurally complex (+)-absinthin but also paved an efficient synthetic route to a series of medicinally attractive absinthin analogues.