Anthony P. Belanger

In Vivo Detection of Age- and Disease-Related Increases in Neuroinflammation by 18F-GE180 TSPO MicroPET Imaging in Wild-Type and Alzheimer's Transgenic Mice

Alzheimers disease (AD) is the most common cause of dementia. Neuroinflammation appears to play an important role in AD pathogenesis. Ligands of the 18 kDa translocator protein (TSPO), a marker for activated microglia, have been used as positron emission tomography (PET) tracers to reflect neuroinflammation in humans and mouse models. Here, we used the novel TSPO-targeted PET tracer 18F-GE180 (flutriciclamide) to investigate differences in neuroinflammation between young and old WT and APP/PS1dE9 transgenic (Tg) mice. In vivo PET scans revealed an overt age-dependent elevation in whole-brain uptake of 18F-GE180 in both WT and Tg mice, and a significant increase in whole-brain uptake of 18F-GE180 (peak-uptake and retention) in old Tg mice compared with young Tg mice and all WT mice. Similarly, the 18F-GE180 binding potential in hippocampus was highest to lowest in old Tg > old WT > young Tg > young WT mice using MRI coregistration. Ex vivo PET and autoradiography analysis further confirmed our in vivo PET results: enhanced uptake and specific binding (SUV75%) of 18F-GE180 in hippocampus and cortex was highest in old Tg mice followed by old WT, young Tg, and finally young WT mice. 18F-GE180 specificity was confirmed by an in vivo cold tracer competition study. We also examined 18F-GE180 metabolites in 4-month-old WT mice and found that, although total radioactivity declined over 2 h, of the remaining radioactivity, ∼90% was due to parent 18F-GE180. In conclusion, 18F-GE180 PET scans may be useful for longitudinal monitoring of neuroinflammation during AD progression and treatment. SIGNIFICANCE STATEMENT Microglial activation, a player in Alzheimers disease (AD) pathogenesis, is thought to reflect neuroinflammation. Using in vivo microPET imaging with a novel TSPO radioligand, 18F-GE180, we detected significantly enhanced neuroinflammation during normal aging in WT mice and in response to AD-associated pathology in APP/PS1dE9 Tg mice, an AD mouse model. Increased uptake and specific binding of 18F-GE180 in whole brain and hippocampus were confirmed by ex vivo PET and autoradiography. The binding specificity and stability of 18F-GE180 was further confirmed by a cold tracer competition study and a metabolite study, respectively. Therefore, 18F-GE180 PET imaging may be useful for longitudinal monitoring of neuroinflammation during AD progression and treatment and may also be useful for other neurodegenerative diseases.

Imaging Macrophage and Hematopoietic Progenitor Proliferation in Atherosclerosis

RATIONALE Local plaque macrophage proliferation and monocyte production in hematopoietic organs promote progression of atherosclerosis. Therefore, noninvasive imaging of proliferation could serve as a biomarker and monitor therapeutic intervention. OBJECTIVE To explore (18)F-FLT positron emission tomography-computed tomography imaging of cell proliferation in atherosclerosis. METHODS AND RESULTS (18)F-FLT positron emission tomography-computed tomography was performed in mice, rabbits, and humans with atherosclerosis. In apolipoprotein E knock out mice, increased (18)F-FLT signal was observed in atherosclerotic lesions, spleen, and bone marrow (standardized uptake values wild-type versus apolipoprotein E knock out mice, 0.05 ± 0.01 versus 0.17 ± 0.01, P<0.05 in aorta; 0.13 ± 0.01 versus 0.28 ± 0.02, P<0.05 in bone marrow; 0.06 ± 0.01 versus 0.22 ± 0.01, P<0.05 in spleen), corroborated by ex vivo scintillation counting and autoradiography. Flow cytometry confirmed significantly higher proliferation of macrophages in aortic lesions and hematopoietic stem and progenitor cells in the spleen and bone marrow in these mice. In addition, (18)F-FLT plaque signal correlated with the duration of high cholesterol diet (r(2)=0.33, P<0.05). Aortic (18)F-FLT uptake was reduced when cell proliferation was suppressed with fluorouracil in apolipoprotein E knock out mice (P<0.05). In rabbits, inflamed atherosclerotic vasculature with the highest (18)F-fluorodeoxyglucose uptake enriched (18)F-FLT. In patients with atherosclerosis, (18)F-FLT signal significantly increased in the inflamed carotid artery and in the aorta. CONCLUSIONS (18)F-FLT positron emission tomography imaging may serve as an imaging biomarker for cell proliferation in plaque and hematopoietic activity in individuals with atherosclerosis.

Synthesis and Preliminary Evaluation of 18-18F-Fluoro-4-Thia-Oleate as a PET Probe of Fatty Acid Oxidation

Fatty acid oxidation (FAO) is a major energy-providing process with important implications in cardiovascular, oncologic, neurologic, and metabolic diseases. A novel 4-thia oleate analog, 18-18F-fluoro-4-thia-oleate (18F-FTO), was evaluated in relationship to the previously developed palmitate analog 16-18F-fluoro-4-thia-palmitate (18F-FTP) as an FAO probe. Methods: 18F-FTO was synthesized from a corresponding bromoester. Biodistribution and metabolite analysis studies were performed in rats. Preliminary small-animal PET studies were performed with 18F-FTO and 18F-FTP in rats. Results: A practical synthesis of 18F-FTO was developed, providing a radiotracer of high radiochemical purity (>99%). In fasted rats, myocardial uptake of 18F-FTO (0.70 ± 0.30% dose kg [body mass]/g [tissue mass]) was similar to that of 18F-FTP at 30 min after injection. At 2 h, myocardial uptake of 18F-FTO was maintained, whereas 18F-FTP uptake in the heart was 82% reduced. Similar to 18F-FTP, 18F-FTO uptake by the heart was approximately 80% reduced at 30 min by pretreatment of rats with the CPT-I inhibitor etomoxir. Folch-type extraction analyses showed 70–90% protein-bound fractions in the heart, liver, and skeletal muscle, consistent with efficient trafficking of 18F-FTO to the mitochondrion with subsequent metabolism to protein-bound species. Preliminary small-animal PET studies showed rapid blood clearance and avid extraction of 18F-FTO and of 18F-FTP into the heart and liver. Images of 18F-FTO accumulation in the rat myocardium were clearly superior to those of 18F-FTP. Conclusion: 18F-FTO is shown to be a promising metabolically trapped FAO probe that warrants further evaluation.

18F-Florbetapir Binds Specifically to Myocardial Light Chain and Transthyretin Amyloid Deposits: Autoradiography Study.

Background—18F-florbetapir is a promising imaging biomarker for cardiac light chain amyloidosis (AL) and transthyretin amyloidosis (ATTR). Our aim, using human autopsy myocardial specimens, was to test the hypothesis that 18F-florbetapir binds specifically to myocardial AL and ATTR amyloid deposits. Methods and Results—We studied myocardial sections from 30 subjects with autopsy-documented AL (n=10), ATTR (n=10), and nonamyloid controls (n=10) using 18F-florbetapir and cold florbetapir compound and digital autoradiography. Total and nonspecific binding of 18F-florbetapir was determined using the maximum signal intensity values. Specific binding of 18F-florbetapir was calculated by subtracting nonspecific from total binding measurements (in decays per minute/mm2, DPM mm2) and was compared with cardiac structure and function on echocardiography and the histological extent of amyloid deposits. Diffuse or focally increased 18F-florbetapir uptake was noted in all AL and ATTR samples and in none of the control samples. Compared with control samples, mean 18F-florbetapir–specific uptake was significantly higher in the amyloid samples (0.94±0.43 versus 2.00±0.58 DPM/mm2; P<0.001), and in the AL compared with the ATTR samples (2.48±0.40 versus 1.52±0.22 DPM/mm2; P<0.001). The samples from subjects with atypical echocardiographic features of amyloidosis showed quantitatively more intense 18F-florbetapir–specific uptake compared with control samples (1.50±0.17 versus 0.94±0.43 DPM/mm2; P=0.004), despite smaller amyloid extent than in subjects with typical echocardiograms. Conclusions—18F-florbetapir specifically binds to myocardial AL and ATTR deposits in humans and offers the potential to screen for the 2 most common types of myocardial amyloid.

Microwave-assisted radiosynthesis of [18F]fluorinated fatty acid analogs

UNLABELLED Microwave reactors remain largely underutilized in the field of positron emission tomography (PET) chemistry. This is particularly unfortunate since microwave synthesis elegantly addresses two of the most critical issues of PET radiochemistry with short-lived radionuclides: reaction rate and side-product formation. In this study, we investigate the efficiency of synthesis of terminally [(18)F]fluorinated fatty acid analogs using a commercial microwave reactor in comparison with conventional heating (CH). METHODS The labeling precursors were methyl esters of terminally substituted alkyl bromides and iodides. Duration and temperatures of the [(18)F]fluorination reaction were varied. Chemical and radiochemical purities, and radiochemical yields were investigated for conventional (CH) and microwave-assisted (MW) radiosyntheses. RESULTS The results demonstrate that microwave heating enhanced [(18)F]fluoride incorporation to >95% (up to 55% improvement), while reducing reaction times to 2 min (∼ 10-fold reduction) or temperatures to 55-60 °C (20 °C reduction). Overall decay-corrected radiochemical yields of purified [(18)F]fluoro fatty acids were higher (MW = 49.0 ± 4.5%, CH = 23.6 ± 3.5%, P < .05) with microwave heating and side-products were notably fewer. CONCLUSION For routine synthesis of [(18)F]fluoro fatty acid analogs, microwave heating is faster, milder, cleaner, less variable and higher yielding than CH and therefore the preferred reaction method.

Structure dependence of long-chain [18F]fluorothia fatty acids as myocardial fatty acid oxidation probes.

In vivo imaging of regional fatty acid oxidation (FAO) rates would have considerable potential for evaluation of mammalian diseases. We have synthesized and evaluated 18F-labeled thia fatty acid analogues as metabolically trapped FAO probes to understand the effect of chain length, degree of unsaturation, and placement of the thia substituent on myocardial uptake and retention. 18-[18F]Fluoro-4-thia-(9Z)-octadec-9-enoic acid (3) showed excellent heart/background radioactivity concentration ratios along with highest retention in heart and liver. Pretreatment of rats with the CPT-1 inhibitor, POCA, caused >80% reduction in myocardial uptake of 16-[18F]fluoro-4-thiahexadecanoic acid (2) and 3, indicating high specificity for FAO. In contrast, 18-[18F]fluoro-4-thiaoctadecanoic acid (4) showed dramatically reduced myocardial uptake and blunted response to POCA. 18-[18F]Fluoro-6-thiaoctadecanoic acid (5) showed moderate myocardial uptake and no sensitivity of myocardial uptake to POCA. The results demonstrate relationships between structures of 18F-labeled thia fatty acid and uptake and their utility as FAO probes in various tissues.

Use of pressure-hold test for sterilizing filter membrane integrity in radiopharmaceutical manufacturing

The bubble point test is the de facto standard for postproduction filter membrane integrity test in the radiopharmaceutical community. However, the bubble point test depends on a subjective visual assessment of bubbling rate that can be obscured by significant diffusive gas flows below the manufacturers prescribed bubble point. To provide a more objective means to assess filter membrane integrity, this study evaluates the pressure-hold test as an alternative to the bubble point test. In our application of the pressure-hold test, the nonsterile side of the sterilizing filter is pressurized to 85% of the predetermined bubble point with nitrogen, the filter system is closed off from the pressurizing gas and the pressure is monitored over a prescribed time interval. The drop in pressure, which has a known relationship with diffusive gas flow, is used as a quantitative measure of membrane integrity. Characterization of the gas flow vs. pressure relationship of each filter/solution combination provides an objective and quantitative means for defining a critical value of pressure drop over which the membrane is indicated to be nonintegral. The method is applied to sterilizing filter integrity testing associated with the commonly produced radiopharmaceuticals, [(18)F]FDG and [(11)C]PIB. The method is shown to be robust, practical and amenable to automation in radiopharmaceutical manufacturing environments (e.g., hot cells).

New chemical and radiochemical routes to [18F]Rho6G-DEG-F, a delocalized lipophilic cation for myocardial perfusion imaging with PET

New chemical and radiochemical syntheses are described for the preparation of [18F]Rho6G-DEG-F, an 18F-labeled analogue of the fluoresecent dye rhodamine 6G, which has shown promise as myocardidal perfusion imaging agent. Tosylated precursors of [18F]Rho6G-DEG-F amenable to 18F-labeling were obtained either through a two-step synthesis from rhodamine 6G lactone (33% yield), or in one step from rhodamine 575 (64% yield), then purified by preparative C18 chromatography. Manual synthesis of [18F]Rho6G-DEG-F was achieved in a single radiochemical step from either the tosylate salt or the tosylate/formate double salt in DMSO under standard nucleophillic aliphatic 18F-fluorination conditions (K[18F]F/K2CO3/Kryptofix 2.2.2.). Incorporation of the [18F]F- was found to be satisfactory (≥34% by TLC), despite the protic character of the precursor molecules. [18F]Rho6G-DEG-F was manually synthesized in final decay-corrected radiochemical yields of 11-26% (tosylate salt) and 9-21% (tosylate/formate double salt). The protocol was transferred to an automated synthesis unit, where the product was obtained in 3-9% radiochemical yield (n=3) decay corrected to start-of-synthesis, >99% radiochemical purity, and a molar activity of 122-267 GBq/μmol (3.3-7.2 Ci/μmol).

18F-Florbetapir Binds Specifically to Myocardial Light Chain and Transthyretin Amyloid DepositsCLINICAL PERSPECTIVE

Background—18F-florbetapir is a promising imaging biomarker for cardiac light chain amyloidosis (AL) and transthyretin amyloidosis (ATTR). Our aim, using human autopsy myocardial specimens, was to test the hypothesis that 18F-florbetapir binds specifically to myocardial AL and ATTR amyloid deposits. Methods and Results—We studied myocardial sections from 30 subjects with autopsy-documented AL (n=10), ATTR (n=10), and nonamyloid controls (n=10) using 18F-florbetapir and cold florbetapir compound and digital autoradiography. Total and nonspecific binding of 18F-florbetapir was determined using the maximum signal intensity values. Specific binding of 18F-florbetapir was calculated by subtracting nonspecific from total binding measurements (in decays per minute/mm2, DPM mm2) and was compared with cardiac structure and function on echocardiography and the histological extent of amyloid deposits. Diffuse or focally increased 18F-florbetapir uptake was noted in all AL and ATTR samples and in none of the control samples. Compared with control samples, mean 18F-florbetapir–specific uptake was significantly higher in the amyloid samples (0.94±0.43 versus 2.00±0.58 DPM/mm2; P<0.001), and in the AL compared with the ATTR samples (2.48±0.40 versus 1.52±0.22 DPM/mm2; P<0.001). The samples from subjects with atypical echocardiographic features of amyloidosis showed quantitatively more intense 18F-florbetapir–specific uptake compared with control samples (1.50±0.17 versus 0.94±0.43 DPM/mm2; P=0.004), despite smaller amyloid extent than in subjects with typical echocardiograms. Conclusions—18F-florbetapir specifically binds to myocardial AL and ATTR deposits in humans and offers the potential to screen for the 2 most common types of myocardial amyloid.

18F-Florbetapir Binds Specifically to Myocardial Light Chain and Transthyretin Amyloid DepositsCLINICAL PERSPECTIVE: Autoradiography Study

Background—18F-florbetapir is a promising imaging biomarker for cardiac light chain amyloidosis (AL) and transthyretin amyloidosis (ATTR). Our aim, using human autopsy myocardial specimens, was to test the hypothesis that 18F-florbetapir binds specifically to myocardial AL and ATTR amyloid deposits. Methods and Results—We studied myocardial sections from 30 subjects with autopsy-documented AL (n=10), ATTR (n=10), and nonamyloid controls (n=10) using 18F-florbetapir and cold florbetapir compound and digital autoradiography. Total and nonspecific binding of 18F-florbetapir was determined using the maximum signal intensity values. Specific binding of 18F-florbetapir was calculated by subtracting nonspecific from total binding measurements (in decays per minute/mm2, DPM mm2) and was compared with cardiac structure and function on echocardiography and the histological extent of amyloid deposits. Diffuse or focally increased 18F-florbetapir uptake was noted in all AL and ATTR samples and in none of the control samples. Compared with control samples, mean 18F-florbetapir–specific uptake was significantly higher in the amyloid samples (0.94±0.43 versus 2.00±0.58 DPM/mm2; P<0.001), and in the AL compared with the ATTR samples (2.48±0.40 versus 1.52±0.22 DPM/mm2; P<0.001). The samples from subjects with atypical echocardiographic features of amyloidosis showed quantitatively more intense 18F-florbetapir–specific uptake compared with control samples (1.50±0.17 versus 0.94±0.43 DPM/mm2; P=0.004), despite smaller amyloid extent than in subjects with typical echocardiograms. Conclusions—18F-florbetapir specifically binds to myocardial AL and ATTR deposits in humans and offers the potential to screen for the 2 most common types of myocardial amyloid.