In vitro characterization of [(18)F]-florbetaben, an Aβ imaging radiotracer

PurposeAmyloid-β (Aβ) plaques are a major pathological hallmark of Alzheimer's disease (AD). The noninvasive detection of Aβ plaques may increase the accuracy of clinical diagnosis as well as monitor therapeutic interventions. While [¹¹C]-PiB is the most widely used Aβ positron emission tomography (PET) radiotracer, due to the short half-life of ¹¹C (20 min), its application is limited to centers with an on-site cyclotron and ¹¹C radiochemistry expertise. Therefore, novel [¹⁸F] (half-life 110 min)-labeled Aβ PET tracers have been developed. We have demonstrated that [¹⁸F]-florbetaben-PET can differentiate individuals diagnosed with AD from healthy elderly, Parkinson's disease and frontotemporal lobe dementia (FTLD-tau) patients. While [¹⁸F]-florbetaben-PET retention matched the reported postmortem distribution of Aβ plaques, the nature of [¹⁸F]-florbetaben binding to other pathological lesions comprising misfolded proteins needs further assessment. The objective of this study was to determine whether Florbetaben selectively binds to Aβ plaques in postmortem tissue specimens containing mixed pathological hallmarks (i.e., tau and α-synuclein aggregates).MethodHuman AD, FTLD-tau and dementia with Lewy bodies (DLB) brain sections were analyzed by [¹⁸F]-florbetaben autoradiography and [³H]-florbetaben high-resolution emulsion autoradiography and [¹⁹F]-florbetaben fluorescence microscopy.ResultsBoth autoradiographical analyses demonstrated that Florbetaben exclusively bound Aβ plaques in AD brain sections at low nanomolar concentrations. Furthermore, at concentrations thousand-folds higher than those during a PET scan, [¹⁹F]-florbetaben did not bind to α-synuclein or tau aggregates in DLB and FTLD-tau brain sections, respectively. Detection of [¹⁹F]-florbetaben staining by fluorescence microscopy in several AD brain regions demonstrated that Florbetaben identified Aβ plaques in all brain regions examined.ConclusionThis study provides further evidence that [¹⁸F]-florbetaben-PET is a highly selective radiotracer to assess Aβ plaque deposition in the brain.     

Comparison of the binding characteristics of [18F]THK-523 and other amyloid imaging tracers to Alzheimer��s disease pathology

Purpose

Extensive deposition of senile plaques and neurofibrillary tangles in the brain is a pathological hallmark of Alzheimer??s disease (AD). Although several PET imaging agents have been developed for in vivo detection of senile plaques, no PET probe is currently available for selective detection of neurofibrillary tangles in the living human brain. Recently, [¹⁸F]THK-523 was developed as a potential in vivo imaging probe for tau pathology. The purpose of this study was to compare the binding properties of [¹⁸F]THK-523 and other amyloid imaging agents, including PiB, BF-227 and FDDNP, to synthetic protein fibrils and human brain tissue.

Methods

In vitro radioligand binding assays were conducted using synthetic amyloid ??₄₂ and K18??K280-tau fibrils. Nonspecific binding was determined by the addition of unlabelled compounds at a concentration of 2???M. To examine radioligand binding to neuropathological lesions, in vitro autoradiography was conducted using sections of AD brain.

Results

[¹⁸F]THK-523 showed higher affinity for tau fibrils than for A?? fibrils, whereas the other probes showed a higher affinity for A?? fibrils. The autoradiographic analysis indicated that [¹⁸F]THK-523 accumulated in the regions containing a high density of tau protein deposits. Conversely, PiB and BF-227 accumulated in the regions containing a high density of A?? plaques.

Conclusion

These findings suggest that the unique binding profile of [¹⁸F]THK-523 can be used to identify tau deposits in AD brain.     

Biodistribution studies of two 18F-labeled pyridinylphenyl amides as subtype selective radioligands for the dopamine D3 receptor

IntroductionDopamine D3 receptors are implicated in various neuropsychiatric diseases, drug abuse and alcoholism, but specific agents for D3 molecular imaging are lacking. We evaluated two in vitro selective fluorine-18-labeled radioligand candidates ([¹⁸F]5 and [¹⁸F]6) for positron emission tomography (PET) imaging of D3 receptor availability in the brain.MethodsBiodistribution was evaluated in Sprague–Dawley rats using ex vivo autoradiography and small-animal PET. Protein binding studies were conducted in human plasma and cerebrospinal fluid.Results[¹⁸F]5 showed rapid blood–brain barrier penetration and fast washout after intravenous injection, whereas the rat brain penetration of [¹⁸F]6 was lower. The total distribution volume (V_T) of [¹⁸F]5 was 20–26 mL g^− 1 throughout brain. Co-injection with the D3 antagonist BP897 resulted in globally increased cerebral washout of [¹⁸F]5 and [¹⁸F]6, but SUV analysis and parametric mapping of binding potential (BP_ND) relative to the cerebellum did not reveal specific binding of either ligand in D3-rich brain regions, i.e. the ventral striatum. However, there was substantial displaceable binding of [¹⁸F]5, and to a lesser extent [¹⁸F]6, in the pituitary.ConclusionThese radioligands reveal dopamine D3 receptors in the pituitary, but are not suitable for PET imaging of in brain, possibly due to low specific signal relative to the globally high V_T.     

Imaging of amyloid deposition in human brain using positron emission tomography and [18F]FACT: comparison with [11C]PIB

Purpose

The characteristic neuropathological changes in Alzheimer’s disease (AD) are deposition of amyloid senile plaques and neurofibrillary tangles. The ¹⁸F-labeled amyloid tracer, [¹⁸F]2-[(2-{(E)-2-[2-(dimethylamino)-1,3-thiazol-5-yl]vinyl}-1,3-benzoxazol-6-yl)oxy]-3-fluoropropan-1-ol (FACT), one of the benzoxazole derivatives, was recently developed. In the present study, deposition of amyloid senile plaques was measured by positron emission tomography (PET) with both [¹¹C]Pittsburgh compound B (PIB) and [¹⁸F]FACT in the same subjects, and the regional uptakes of both radiotracers were directly compared.

Methods

Two PET scans, one of each with [¹¹C]PIB and [¹⁸F]FACT, were performed sequentially on six normal control subjects, two mild cognitive impairment (MCI) patients, and six AD patients. The standardized uptake value ratio of brain regions to the cerebellum was calculated with partial volume correction using magnetic resonance (MR) images to remove the effects of white matter accumulation.

Results

No significant differences in the cerebral cortical uptake were observed between normal control subjects and AD patients in [¹⁸F]FACT studies without partial volume correction, while significant differences were observed in [¹¹C]PIB. After partial volume correction, the cerebral cortical uptake was significantly larger in AD patients than in normal control subjects for [¹⁸F]FACT studies as well as [¹¹C]PIB. Relatively lower uptakes of [¹¹C]PIB in distribution were observed in the medial side of the temporal cortex and in the occipital cortex as compared with [¹⁸F]FACT. Relatively higher uptake of [¹¹C]PIB in distribution was observed in the frontal and parietal cortices.

Conclusion

Since [¹⁸F]FACT might bind more preferentially to dense-cored amyloid deposition, regional differences in cerebral cortical uptake between [¹¹C]PIB and [¹⁸F]FACT might be due to differences in regional distribution between diffuse and dense-cored amyloid plaque shown in the autoradiographic and histochemical assays of postmortem AD brain sections.     

Validation of an 18F-labeled biphenylalkyne as a positron emission tomography imaging agent for β-amyloid plaques

AimRecently, the feasibility of detecting amyloid plaques in the living brain by positron emission tomography (PET) imaging has been successfully demonstrated. As such, imaging β-amyloid (Aβ) plaques in the brain may further advance the differential diagnosis of the disease and allow clinicians to measure the effectiveness of therapeutic drugs aimed at lowering plaques in the brain. We report herein the preclinical validation of a potential ¹⁸F-labeled biphenylalkyne, AV-138, as a preliminary step toward developing the imaging agent for patients suspected of having Alzheimer's disease.MethodsIn vitro binding was carried out in the homogenates prepared from postmortem AD brains with [¹²⁵I]IMPY as the radioligand. [¹⁸F]AV-138 was successfully prepared using a tosylate precursor and Sumitomo modules for radiosynthesis. Similarly, specific binding of [¹⁸F]AV-138 (0.02–0.05 nM) to homogenates, prepared from gray and white matters of pooled AD patients and control subjects, was performed. Specific binding to Aβ plaques was measured by autoradiography in AD brain sections (n=11), and the same brain sections were fluorescently stained with thioflavin-S (TF-S). Images of both radiolabeling and fluorescent staining of plaques obtained by a phosphor imager were used for correlation image analysis.ResultsAs expected, AV-138 displayed a high binding affinity (K_i=2.4±0.7 nM) in AD gray matter homogenates (due to its high level of Aβ plaque accumulation). Specific binding can be clearly measured in the AD gray matter homogenates, but not in the AD white matters. Control brain homogenates, due to a lack of Aβ plaques, also showed no specific binding. Furthermore, in vitro autoradiography of postmortem AD brain sections showed that the high binding signal of [¹⁸F]AV-138 was specifically due to Aβ plaques. Fluorescent staining of plaques with TF-S correlated well with the radiolabeling of [¹⁸F]AV-138 in AD brain sections (r>0.90).ConclusionTaken together, these preliminary results strongly suggest that [¹⁸F]AV-138 is potentially useful for imaging Aβ plaques in the living human brain.     

18F-Labeled phosphopeptide-cell-penetrating peptide dimers with enhanced cell uptake properties in human cancer cells

IntroductionPhosphopeptides represent interesting compounds to study and elucidate cellular protein phosphorylation/dephosphorylation processes underlying various signal transduction pathways. However, studies of phosphopeptide action in cells are severely constrained by the negatively charged phosphate moiety of the phosphopeptide resulting in poor transport through the cell membrane. The following study describes the synthesis and radiopharmacological evaluation of two ¹⁸F-labeled phosphopeptide-cell-penetrating peptide dimers. The polo-like kinase-1-binding hexaphosphopeptide H-Met-Gln-Ser-pThr-Pro-Leu-OH was coupled to cell-penetrating peptides (CPPs), either sC18, a cathelicidin-derived peptide, or the human calcitonin derivative hCT(18-32)-k7.MethodsRadiolabeling was accomplished with the prosthetic group N-succinimidyl 4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) using both, conventional and microfluidic-based bioconjugation of [¹⁸F]SFB to N-terminal end of phosphopeptide part of the peptide dimers. Cellular uptake studies in human cancer cell lines HT-29 and FaDu cells at 4 °C and 37 °C and small animal PET in BALB/c mice were utilized for radiopharmacological characterization.ResultsIsolated radiochemical yields ranged from 2% to 4% for conventional bioconjugation with [¹⁸F]SFB. Significantly improved isolated radiochemical yields of up to 26% were achieved using microfluidic technology. Cellular uptake studies of radiolabeled phosphopeptide and phosphopeptide-CPP dimers indicate enhanced internalization of 50% ID/mg protein after 2 h for both phosphopeptide dimers compared to the phosphopeptide alone (< 1% ID/mg protein). In vivo biodistribution of ¹⁸F-labeled peptide dimers was determined with small animal PET revealing a superior biodistribution pattern of sC18-containing peptide dimer MQSpTPL-sC18 [¹⁸F]4.Conclusion^[18F]SFB labeling of the phosphopeptide-CPP dimers using a microfluidic system leads to an improved chemoselectivity towards the N-terminal NH₂ group compared to the conventional labeling approach. Cell-penetrating peptide sC18 can be considered as an ideal molecular shuttle for intracellular delivery of the Plk1-PBD-binding hexaphosphopeptide as demonstrated by its favourable radiopharmacological profile.     

Evaluating Radioactive Analogs of Doxorubicin to Quantify ChemoFilter Binding and Whole-Body Positron Emission Tomography/Magnetic Resonance Imaging for Drug Biodistribution

PurposeTo evaluate radiolabeled doxorubicin (Dox) analogs as tracers of baseline Dox biodistribution in vivo during hepatic intra-arterial chemotherapy and to assess the efficacy of ChemoFilter devices to bind Dox in vitro.Materials and MethodsIn an in vitro static experiment, [fluorine-18]N-succinimidyl 4-fluorobenzoate ([¹⁸F]SFB) and [fluorine-18]fluorobenzoyl-doxorubicin ([¹⁸F]FB-Dox) were added to a beaker containing a filter material (Dowex cation exchange resin, single-stranded DNA (ssDNA) resin, or sulfonated polymer coated mesh). In an in vitro flow model, [¹⁸F]FB-Dox was added into a Dox solution in phosphate-buffered saline, and the solution flowed via a syringe column containing the filter materials. In an in vitro flow experiment, using micro-positron emission tomography (PET), images were taken as [¹⁸F]SFB and [¹⁸F]FB-Dox moved through a phantom. For in vivo biodistribution testing, a catheter was placed into the common hepatic artery of a swine, and [¹⁸F]FB-Dox was infused over 30 seconds. A 10-minute dynamic image and three 20-minute static images were acquired using 3T PET/MR imaging.ResultsIn the in vitro static experiment, [¹⁸F]FB-Dox demonstrated 76.7%, 88.0%, and 52.4% binding to the Dowex resin, ssDNA resin, and coated mesh, respectively. In the in vitro flow model, the first-pass binding of [¹⁸F]FB-Dox to the Dowex resin, ssDNA resin, and coated mesh was 76.7%, 74.2%, and 76.2%, respectively, and the total bound fraction was 80.9%, 84.6%, and 79.9%, respectively. In the in vitro flow experiment using micro-PET, the phantom demonstrated a greater amount of [¹⁸F]FB-Dox bound to both filter cartridges than of the control [¹⁸F]SFB. In in vivo biodistribution testing, the first 10 minutes depicted [¹⁸F]FB-Dox moving through the right upper quadrant of the abdomen. A region-of-interest analysis showed that the relative amount increased by 2.97 times in the gallbladder and 1.08 times in the kidney. The amount decreased by 0.74 times in the brain and 0.57 times in the heart.Conclusions[¹⁸F]FB-Dox can be used to assess Dox binding to ChemoFilters as well as in vivo biodistribution. This sets the stage for the evaluation of ChemoFilter effectiveness in reducing systemic toxicity from intra-arterial chemotherapy.     

Development of [18F]afatinib as new TKI-PET tracer for EGFR positive tumors

IntroductionAfatinib is an irreversible ErbB family blocker that was approved for the treatment of EGFR mutated non-small cell lung cancer in 2013. Positron emission tomography (PET) with fluorine-18 labeled afatinib provides a means to obtain improved understanding of afatinib tumor disposition in vivo. PET imaging with [¹⁸F]afatinib may also provide a method to select treatment responsive patients. The aim of this study was to label afatinib with fluorine-18 and evaluate its potential as TKI-PET tracer in tumor bearing mice.MethodsA radiochemically novel coupling, using peptide coupling reagent BOP, was explored and optimized to synthesize [¹⁸F]afatinib, followed by a metabolite analysis and biodistribution studies in two clinically relevant lung cancer cell lines, xenografted in nude mice.ResultsA reliable [¹⁸F]afatinib radiosynthesis was developed and the tracer could be produced in yields of 17.0 ± 2.5% calculated from [¹⁸F]F⁻ and >98% purity. The identity of the product was confirmed by co-injection on HPLC with non-labeled afatinib. Metabolite analysis revealed a moderate rate of metabolism, with >80% intact tracer in plasma at 45 min p.i. Biodistribution studies revealed rapid tumor accumulation and good retention for a period of at least 2 hours, while background tissues showed rapid clearance of the tracer.ConclusionWe have developed a method to synthesize [¹⁸F]afatinib and related fluorine-18 labeled 4-anilinoquinazolines. [¹⁸F]Afatinib showed good stability in vivo, justifying further evaluation as a TKI-PET tracer.     

Peripheral metabolism of [F]FDDNP and cerebral uptake of its labelled metabolites

[¹⁸F]FDDNP is a positron emission tomography (PET) tracer for determining amyloid plaques and neurofibrillary tangles in the brain in vivo. In order to quantify binding of this tracer properly, a metabolite-corrected plasma input function is required. The purpose of the present study was to develop a sensitive method for measuring [¹⁸F]FDDNP and its radiolabelled metabolites in plasma. The second aim was to assess whether these radiolabelled metabolites enter the brain.In humans, there was extensive metabolism of [¹⁸F]FDDNP. After 10 min, more than 80% of plasma radioactivity was identified as polar ¹⁸F-labelled fragments, probably formed from N-dealkylation of [¹⁸F]FDDNP. These labelled metabolites were reproduced in vitro using human hepatocytes. PET studies in rats showed that these polar metabolites can penetrate the blood–brain barrier and result in uniform brain uptake.     

Whole-body biodistribution and brain PET imaging with [18F]AV-45, a novel amyloid imaging agent — a pilot study

PurposeThe compound (E)-4-(2-(6-(2-(2-(2-¹⁸F-fluoroethoxy)ethoxy)ethoxy) pyridin-3-yl)vinyl)-N-methylbenzenamine ([¹⁸F]AV-45) is a novel radiopharmaceutical capable of selectively binding to β-amyloid (Aβ) plaques. This pilot study reports the safety, biodistribution, and radiation dosimetry of [¹⁸F]AV-45 in human subjects.MethodsIn vitro autoradiography and fluorescent staining of postmortem brain tissue from patients with Alzheimer's disease (AD) and cognitively healthy subjects were performed to assess the specificity of the tracer. Biodistribution was assessed in three healthy elderly subjects (mean age: 60.0±5.2 years) who underwent 3-h whole-body positron emission tomography (PET)/computed tomographic (CT) scans after a bolus injection of 381.9±13.9 MBq of [¹⁸F]AV-45. Another six subjects (three AD patients and three healthy controls, mean age: 67.7±13.6 years) underwent brain PET studies. Source organs were delineated on PET/CT. All subjects underwent magnetic resonance imaging (MRI) for obtaining structural information.ResultsIn vitro autoradiography revealed exquisitely high specific binding of [¹⁸F]AV-45 to postmortem AD brain sections, but not to the control sections. There were no serious adverse events throughout the study period. The peak uptake of the tracer in the brain was 5.12±0.41% of the injected dose. The highest absorbed organ dose was to the gallbladder wall (184.7±78.6 μGy/MBq, 4.8 h voiding interval). The effective dose equivalent and effective dose values for [¹⁸F]AV-45 were 33.8±3.4 μSv/MBq and 19.3±1.3 μSv/MBq, respectively.Conclusion[¹⁸F]AV-45 binds specifically to Aβ in vitro, and is a safe PET tracer for studying Aβ distribution in human brain. The dosimetry is suitable for clinical and research application.     

Radiosynthesis and in vivo evaluation of fluorinated huprine derivates as PET radiotracers of acetylcholinesterase

IntroductionDeveloping positron emission tomography (PET) radiotracers for non-invasive study of the cholinergic system is crucial to the understanding of neurodegenerative diseases. Although several acetylcholinesterase (AChE) PET tracers radiolabeled with carbon-11 exist, no fluorinated radiotracer is currently used in clinical imaging studies. The purpose of the present study is to describe the first fluorinated PET radiotracer for this brain enzyme.MethodsThree structural analogs of huprine, a specific AChE inhibitor presenting high affinity towards AChE in vitro, were synthesized and labeled with fluorine-18 via a mesylate/fluoro-nucleophilic aliphatic substitution: ([¹⁸ F]-FHUa, [¹⁸ F]-FHUb and [¹⁸ F]-FHUc). Initial biological evaluation included in vitro autoradiography in rat with competition with an AChE inhibitor at different concentrations, and microPET-scan on anesthetized rats. In vivo PET studies in anesthetized cat focused on [¹⁸ F]-FHUa.Results and ConclusionsAlthough radiosynthesis of these huprine analogs was straightforward, they showed poor brain penetration potential, partially reversed after pharmacological inhibition of P-glycoprotein. These results indicated that current huprine analogs are not suitable for PET mapping of brain AChE receptors, but require physicochemical modulation in order to increase brain penetration.     

Rapid and one-step radiofluorination of bioactive peptides: Potential PET radiopharmaceuticals

In an attempt to develop a new and rapid method for labeling peptides with ¹⁸F, we have synthesized MUC1-[¹⁸F]SFB and BBN-[¹⁸F]SFB peptide conjugates using a convenient and one-step simple reactions. Radiochemical yields for MUC1-[¹⁸F]SFB and BBN-[¹⁸F]SFB peptide conjugates were greater than 70% in less than 30 min synthesis time, thus amenable for automation for the radiofluorination of peptides. in vitro tests on T47D breast cancer cells showed that the significant amounts of the radioconjugates were associated with cell fractions and held sufficient affinities and specificities toward T47D cell line. These radioconjugates may be useful as molecular probes for detecting and staging of breast cancer and monitoring tumor response to treatment.     

Efficient synthesis of a fluorine-18 labeled biotin derivative

IntroductionThe natural occurring vitamin biotin, also known as vitamin H or vitamin B₇, plays a major role in various metabolic reactions. Caused by its high binding affinity to the protein avidin with a dissociation constant of about 10^- 15 M the biotin-avidin system was extensively examined for multiple applications. We have synthesized a fluorine-18 labeled biotin derivative [¹⁸F]4 for a potential application in positron emission tomography (PET).MethodsMesylate precursor 3 was obtained by an efficient two-step reaction via a copper catalyzed azide-alkyne cycloaddition (CuAAC) from easily accessible starting materials. [¹⁸F]4 was successfully synthesized by a nucleophilic radiofluorination of precursor 3. A biodistribution study by means of small-animal PET imaging in wt-mice was performed and serum stability was examined.ResultsCompound [¹⁸F]4 was obtained from precursor compound 3 with an average specific activity of 16 GBq/μmol within 45 min and a radiochemical yield of 45 ± 5% (decay corrected). [¹⁸F]4 demonstrated only negligible decomposition in human serum. A qualitative binding study revealed the high affinity of the synthesized biotin derivative to avidin. Blocking experiments with native biotin showed that binding was site-specific. Biodistribution studies showed that [¹⁸F]4 was cleared quickly and efficiently from the body by hepatobiliary and renal elimination.ConclusionAn efficient synthesis for [¹⁸F]4 was established. In vivo characteristics were determined and demonstrated the pharmacokinetic behaviour of [¹⁸F]4.     

Preparation and evaluation of ethyl [18F]fluoroacetate as a proradiotracer of [18F]fluoroacetate for the measurement of glial metabolism by PET

IntroductionChanges in glial metabolism in brain ischemia, Alzheimer's disease, depression, schizophrenia, epilepsy and manganese neurotoxicity have been reported in recent studies. Therefore, it is very important to measure glial metabolism in vivo for the elucidation and diagnosis of these diseases. Radiolabeled acetate is a good candidate for this purpose, but acetate has little uptake in the brain due to its low lipophilicity. We have designed a new proradiotracer, ethyl [¹⁸F]fluoroacetate ([¹⁸F]EFA), which is [¹⁸F]fluoroacetate ([¹⁸F]FA) esterified with ethanol, to increase the lipophilicity of fluoroacetate (FA), allowing the measurement of glial metabolism.MethodsThe synthesis of [¹⁸F]EFA was achieved using ethyl O-mesyl-glycolate as precursor. The blood–brain barrier permeability of ethyl [1-¹⁴C]fluoroacetate ([¹⁴C]EFA) was estimated by a brain uptake index (BUI) method. Hydrolysis of [¹⁴C]EFA in the brain was calculated by the fraction of radioactivity in lipophilic and water fractions of homogenized brain. Using the plasma of five animal species, the stability of [¹⁴C]EFA was measured. Biodistribution studies of [¹⁸F]EFA in ddY mice were carried out and compared with [¹⁸F]FA. Positron emission tomography (PET) scanning using common marmosets was performed for 90 min postadministration. At 60 min postinjection of [¹⁸F]EFA, metabolite studies were performed. Organs were dissected from the marmosets, and extracted metabolites were analyzed with a thin-layer chromatography method.ResultsThe synthesis of [¹⁸F]EFA was accomplished in a short time (29 min) and with a reproducible radiochemical yield of 28.6±3.6% (decay corrected) and a high radiochemical purity of more than 95%. In the brain permeability study, the BUI of [¹⁴C]EFA was 3.8 times higher than that of sodium [1-¹⁴C]fluoroacetate. [¹⁴C]EFA was hydrolyzed rapidly in rat brains. In stability studies using the plasma of five animal species, [¹⁴C]EFA was stable only in primate plasma. Biodistribution studies in mice showed that the uptake of [¹⁸F]EFA in selected organs was higher than that of [¹⁸F]FA. From nonprimate PET studies, [¹⁸F]EFA was initially taken into the brain after injection. Metabolites related to the tricarboxylic acid (TCA) cycle were detected in common marmoset brain.Conclusion[¹⁸F]EFA rapidly enters the brain and is then converted into TCA cycle metabolites in the brains of common marmosets. [¹⁸F]EFA shows promise as a proradiotracer for the measurement of glial metabolism.     

18F-labeled styrylpyridines as PET agents for amyloid plaque imaging

Positron emission tomography (PET) imaging of beta-amyloid (Abeta) plaques in the brain is a potentially valuable tool for studying the pathophysiology of Alzheimer's disease (AD). It may also be applicable for measuring the effectiveness of therapeutic drugs aimed at lowering Abeta plaques in the brain. We have successfully reported a series of 18F-labeled fluoropegylated stilbenes for PET imaging studies. Encouraging results clearly demonstrated the usefulness of 18F-labeled stilbenes as potential Abeta plaque-imaging agents. In the present study, we applied a similar approach to a styrylpyridine backbone structure. Among all derivatives examined, (E)-2-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)-5-(4-dimethylaminostyryl)-pyridine (2) displayed high binding affinity in postmortem AD brain homogenates (Ki=2.5+/-0.4 nM, with [125I]IMPY as radioligand). No-carrier-added [18F]2 was successfully prepared by [18F]fluoride displacement of the corresponding tosylate precursor with a high labeling yield (30-40%) and a high radiochemical purity (>99%). Specific activity at the end of synthesis was determined to be 1500-2000 Ci/mmol. The tracer [18F]2 showed adequate lipophilicity (log P=3.22). In vivo biodistribution of [18F]2 in normal mice exhibited excellent initial brain penetration and rapid washout (7.77% and 1.03% dose/g in the brain at 2 and 30 min after intravenous injection, respectively)--properties that are highly desirable for Abeta-plaque-specific brain imaging agents. Autoradiography of AD brain sections and homogenate binding with postmortem AD brain tissues confirmed the high binding signal of [18F]2 due to the presence of Abeta plaques. These preliminary results suggest that novel PET tracers may be potentially useful for the imaging of Abeta plaques in the living human brain.     

Optimization of automated radiosynthesis of [18F]AV-45: a new PET imaging agent for Alzheimer's disease

IntroductionAccumulation of β-amyloid (Aβ) aggregates in the brain is linked to the pathogenesis of Alzheimer's disease (AD). Imaging probes targeting these Aβ aggregates in the brain may provide a useful tool to facilitate the diagnosis of AD. Recently, [¹⁸F]AV-45 ([¹⁸F]5) demonstrated high binding to the Aβ aggregates in AD patients. To improve the availability of this agent for widespread clinical application, a rapid, fully automated, high-yield, cGMP-compliant radiosynthesis was necessary for production of this probe. We report herein an optimal [¹⁸F]fluorination, de-protection condition and fully automated radiosynthesis of [¹⁸F]AV-45 ([¹⁸F]5) on a radiosynthesis module (BNU F-A2).MethodsThe preparation of [¹⁸F]AV-45 ([¹⁸F]5) was evaluated under different conditions, specifically by employing different precursors (-OTs and -Br as the leaving group), reagents (K222/K₂CO₃ vs. tributylammonium bicarbonate) and deprotection in different acids. With optimized conditions from these experiments, the automated synthesis of [¹⁸F]AV-45 ([¹⁸F]5) was accomplished by using a computer-programmed, standard operating procedure, and was puri?ed on an on-line solid-phase cartridge (Oasis HLB).ResultsThe optimized reaction conditions were successfully implemented to an automated nucleophilic fluorination module. The radiochemical purity of [¹⁸F]AV-45 ([¹⁸F]5) was >95%, and the automated synthesis yield was 33.6±5.2% (no decay corrected, n=4), 50.1±7.9% (decay corrected) in 50 min at a quantity level of 10–100 mCi (370–3700 MBq). Autoradiography studies of [¹⁸F]AV-45 ([¹⁸F]5) using postmortem AD brain and Tg mouse brain sections in the presence of different concentration of “cold” AV-136 showed a relatively low inhibition of in vitro binding of [¹⁸F]AV-45 ([¹⁸F]5) to the Aβ plaques (IC50=1–4 μM, a concentration several order of magnitude higher than the expected pseudo carrier concentration in the brain).ConclusionsSolid-phase extraction purification and improved labeling conditions were successfully implemented into an automated synthesis module, which is more convenient, highly efficient and simpler in operation than using a semipreparative high-performance liquid chromatography method. This new, automated procedure for preparation of [¹⁸F]AV-45 ([¹⁸F]5) is suitable for routine clinical application.     

Radiation dosimetry of the translocator protein ligands [18F]PBR111 and [18F]PBR102

IntroductionThe translocator protein (TSPO) ligands [¹⁸F]PBR111 and [¹⁸F]PBR102 show promise for imaging neuroinflammation. Our aim was to estimate the radiation dose to humans from primate positron emission tomography (PET) studies using these ligands and compare the results with those obtained from studies in rodents.Methods[¹⁸F]PBR111 and [¹⁸F]PBR102 PET–computed tomography studies were carried out in baboons. The cumulated activity in the selected source organs was obtained from the volume of interest time–activity curves drawn on coronal PET slices and adjusted for organ mass relative to humans. Radiation dose estimates were calculated in OLINDA/EXM Version 1.1 from baboon studies and compared with those calculated from Sprague–Dawley rat tissue concentration studies, also adjusted for relative organ mass.ResultsIn baboons, both ligands cleared rapidly from brain, lung, kidney and spleen and more slowly from liver and heart. For [¹⁸F]PBR111, the renal excretion fraction was 6.5% and 17% for hepatobiliary excretion; for [¹⁸F]PBR102, the renal excretion was 3.0% and 15% for hepatobiliary excretion. The estimated effective dose in humans from baboon data was 0.021 mSv/MBq for each ligand, whilst from rat data, the estimates were 0.029 for [¹⁸F]PBR111 and 0.041 mSv/MBq for [¹⁸F]PBR102.ConclusionBiodistribution in a nonhuman primate model is better suited than the rat model for the calculation of dosimetry parameters when translating these ligands from preclinical to human clinical studies. Effective dose calculated from rat data was overestimated compared to nonhuman primate data. The effective dose coefficient for both these TSPO ligands determined from PET studies in baboons is similar to that for [¹⁸F]FDG.     

In vivo evaluation of 18F-labeled TCO for pre-targeted PET imaging in the brain

IntroductionThe tetrazine-trans-cylooctene cycloaddition using radiolabeled tetrazine or radiolabeled trans-cyclooctene (TCO) has been reported to be a very fast, selective and bioorthogonal reaction that could be useful for in vivo radiolabeling of molecules. We wanted to evaluate the in vivo biodistribution profile and brain uptake of ¹⁸F-labeled TCO ([¹⁸F]TCO) to assess its potential for pre-targeted imaging in the brain.MethodsWe evaluated the in vivo behavior of [¹⁸F]TCO via an ex vivo biodistribution study complemented by in vivo μPET imaging at 5, 30, 60, 90, 120 and 240 min post tracer injection. An in vivo metabolite study was performed at 5 min, 30 min and 120 min post [¹⁸F]TCO injection by RP-HPLC analysis of plasma and brain extracts. Incubation with human liver microsomes was performed to further evaluate the metabolite profile of the tracer.ResultsμPET imaging and ex-vivo biodistribution revealed an high initial brain uptake of [¹⁸F]TCO (3.8%ID/g at 5 min pi) followed by a washout to 3.0%ID/g at 30 min pi. Subsequently the brain uptake increased again to 3.7%ID/g at 120 min pi followed by a slow washout until 240 min pi (2.9%ID/g). Autoradiography confirmed homogenous brain uptake. On the μPET images bone uptake became gradually visible after 120 min pi and was clearly visible at 240 min pi. The metabolite study revealed a fast metabolization of [¹⁸F]TCO in plasma and brain into three main polar radiometabolites.ConclusionsAlthough [¹⁸F]TCO has previously been described to be a useful tracer for radiolabeling of tetrazine modified targeting molecules, our study indicates that its utility for in vivo chemistry and pre-targeted imaging will be limited. Although [¹⁸F]TCO clearly enters the brain, it is quickly metabolized with a non-specific accumulation of radioactivity in the brain and bone.     

Neuroimaging of the vesicular acetylcholine transporter by a novel 4-[18F]fluoro-benzoyl derivative of 7-hydroxy-6-(4-phenyl-piperidin-1-yl)-octahydro-benzo[1,4]oxazines

Phenylpiperidinyl-octahydro-benzo[1,4]oxazines represent a new class of conformationally restrained vesamicol analogues. Derived from this morpholine-fused vesamicol structure, a new fluorine-18-labeled 4-fluorobenzoyl derivative ([¹⁸F]FBMV) was synthesized with an average specific activity of 75 GBq/μmol and a radiochemical purity of 99%. The radiolabeling method included an exchange reaction of a 4-nitro group of the precursor by fluorine-18, a reduction procedure to eliminate excess of the nitro compound, followed by a high-performance liquid chromatography purification. [¹⁸F]FBMV demonstrates (i) a moderate lipophilic character with a logD_pH7.0 1.8±0.10; (ii) a considerable binding affinity to the vesicular acetylcholine transporter (VAChT) (K_i=27.5 nM), as determined using PC12 cells transfected with a VAChT cDNA, and a low affinity to σ_1,2 receptors (K_i >3000 nM); (iii) a good uptake into the rat and pig brains; (iv) a typical accumulation in the VAChT-containing brain regions; and (v) an approximately 20% reduction in cortical tracer binding after a specific cholinergic lesion using 192IgG-saporin.[¹⁸F]FBMV exhibits another PET marker within the group of vesamicol derivatives that demonstrates potentials in imaging brain cholinergic deficits, while its usefulness in clinical practice must await further investigation.     

Synthesis, biodistribution and PET studies in rats of (18)F-Labeled bridgehead fluoromethyl analogues of WAY-100635

IntroductionIn vitro screening of fluoromethyl bridge-fused ring (BFR) analogues of WAY-100635 (5a, 5b and 5c) has shown a high binding affinity and a good selectivity for the 5-HT_1A receptor. As these compounds were designed to provide PET ligands with high metabolic stability, they are now radiolabeled with fluorine-18 and investigated in vivo.MethodsBFR precursors were synthesized and reacted with fluorine-18 in dry MeCN in the presence of 2,2,2-kryptofix and K₂CO₃. In rats, biodistribution and PET studies were performed using [¹⁸F]5a, [¹⁸F]5b and [¹⁸F]5c. The binding specificity was determined by administration of non-labeled WAY-100635 prior to the radiolabeled ligands.Results[¹⁸F]5 ligands were synthesized in overall radiochemical yields of 24%–45%, respectively with a radiochemical purity of > 98%. Relatively good hippocampus to cerebellum ratios of 5.55, 4.79 and 5.45, respectively were reached at 45 min pi. However, PET studies indicated defluorination of the radioligands by showing high accumulation of radioactivity in the bones in the order of [¹⁸F]5a ≈ [¹⁸F]5b > [¹⁸F]5c.ConclusionAlso in vivo, the radioligands bind preferentially to the 5-HT_1A receptor. Unfortunately, no metabolic stability with regard to defluorination was observed in rats.