Synthesis and evaluation of 2-amino-5-(4-[18F]fluorophenyl)pent-4-ynoic acid ([18F]FPhPA): A novel 18F-labeled amino acid for oncologic PET imaging

Introduction¹⁸ F-labeled amino acids are important PET radiotracers for molecular imaging of cancer. This study describes synthesis and radiopharmacological evaluation of 2-amino-5-(4-[¹⁸ F]fluorophenyl)pent-4-ynoic acid ([¹⁸ F]FPhPA) as a novel amino acid radiotracer for oncologic imaging.Methods¹⁸ F]FPhPA was prepared using Pd-mediated Sonogashira cross-coupling reaction between 4-[¹⁸ F]fluoroiodobenzene ([¹⁸ F]FIB) and propargylglycine. The radiopharmacological profile of [¹⁸ F]FPhPA was evaluated in comparison with O-(2-[¹⁸ F]fluoroethyl)-L-tyrosine ([¹⁸ F]FET) using the murine breast cancer cell line EMT6 involving cellular uptake studies, radiotracer uptake competitive inhibition experiments and small animal PET imaging.Results¹⁸ F]FPhPA was prepared in 42 ± 10% decay-corrected radiochemical yield with high radiochemical purity >95% after semi-preparative HPLC purification. Cellular uptake of L-[¹⁸ F]FPhPA reached a maximum of 58 ± 14 % radioactivity/mg protein at 90 min. Lower uptake was observed for racemic and D-[¹⁸ F]FPhPA.Radiotracer uptake inhibition studies by synthetic and naturally occurring amino acids suggested that Na⁺-dependent system ASC, especially ASCT2, and Na⁺-independent system L are important amino acid transporters for [¹⁸ F]FPhPA uptake into EMT6 cells. Small animal PET studies demonstrated similar high tumor uptake of [¹⁸ F]FPhPA in EMT6 tumor-bearing mice compared to [¹⁸ F]FET reaching a maximum standardized uptake value (SUV) of 1.35 after 60 min p.i.. Muscle uptake of [¹⁸ F]FPhPA was higher (SUV_30min = 0.65) compared to [¹⁸ F]FET (SUV_30min = 0.40), whereas [¹⁸ F]FPhPA showed a more rapid uptake and clearance from the brain compared to [¹⁸ F]FET.ConclusionL-[¹⁸ F]FPhPA is the first ¹⁸ F-labeled amino acid prepared through Pd-mediated cross-coupling reaction.Advances in Knowledge and Implications for patient CareL-[¹⁸ F]FPhPA displayed promising properties as a novel amino acid radiotracer for molecular imaging of system ASC and system L amino acid transporters in cancer.     

In vitro and in vivo evaluation of the effects of aluminum [18F]fluoride radiolabeling on an integrin αvβ6-specific peptide

IntroductionIncorporation of fluorine-18 (¹⁸F) into radiotracers by capturing ionic [¹⁸F]-species can greatly accelerate and simplify radiolabeling for this important positron emission tomography (PET) radioisotope. Among the different strategies, the incorporation of aluminum [¹⁸F]fluoride (Al[¹⁸F]^2 +) into NOTA chelators has recently emerged as a robust approach to peptide radiolabeling. This study presents Al[¹⁸F]^2 +-radiolabeling of an α_vβ₆ integrin-targeted peptide (NOTA-PEG₂₈-A20FMDV2) and its in vitro and in vivo evaluation.MethodsAluminum [¹⁸F]fluoride was prepared at r.t. from [¹⁸F]fluoride (40 MBq–11 GBq) and introduced into NOTA-PEG₂₈-A20FMDV2 (1) in sodium acetate (pH 4.1; 100°C, 15 min). The radiotracer Al[¹⁸F] NOTA-PEG₂₈-A20FMDV2 (2) was purified by HPLC, formulated in PBS and evaluated in vitro (stability; binding and internalization in α_vβ₆(+) and α_vβ₆(−) cells) and in vivo (paired α_vβ₆(+) and α_vβ₆(−) xenograft mice: PET/CT, biodistribution, tumor autoradiography and metabolites).ResultsThe radiotracer 2 was prepared in 90 ± 6 min (incl. formulation; n = 3) in 19.3 ± 5.4% decay corrected radiochemical yield (radiochemical purity: > 99%; specific activity: 158 ± 36 GBq/μmol) and was stable in PBS and serum (2 h). During in vitro cell binding studies, 2 showed high, α_vβ₆-targeted binding (α_vβ₆(+): 42.4 ± 1.2% of total radioactivity, ratio (+)/(−) = 8.4/1) and internalization (α_vβ₆(+): 28.3 ± 0.5% of total radioactivity, (+)/(−) = 11.7/1). In vivo, 2 maintained α_vβ₆-targeted binding (biodistribution; 1 h: α_vβ₆(+): 1.74 ± 0.38% ID/g, (+)/(−) = 2.72/1; 4 h: α_vβ₆(+): 1.21 ± 0.56% ID/g, (+)/(−) = 4.0/1; 11% intact 2 in tumor at 1 h), with highest uptake around the tumor edge (autoradiography). Most of the radioactivity cleared rapidly in the urine within one hour, but a significant fraction remained trapped in the kidneys (4 h: 229 ± 44% ID/g).ConclusionThe Al[¹⁸F]/NOTA-based radiolabeling was rapid and efficient, and the radiotracer 2 showed good α_vβ₆-selectivity in vitro and in vivo. However, in contrast to A20FMDV2 labeled with covalently bound [¹⁸F]-prosthetic groups (e.g., [¹⁸F]fluorobenzoic acid), 2 demonstrated significant trapping in kidneys, similar to radiometal-labeled chelator-analogs of 2.     

Evaluation of metabolism,plasma protein binding and other biological parameters after administration of (−)-[18 F]Flubatine in humans

Introduction(−)-[¹⁸ F]Flubatine is a PET tracer with high affinity and selectivity for the nicotinic acetylcholine α₄β₂ receptor subtype. A clinical trial assessing the availability of this subtype of nAChRs was performed. From a total participant number of 21 Alzheimer’s disease (AD) patients and 20 healthy controls (HCs), the following parameters were determined: plasma protein binding, metabolism and activity distribution between plasma and whole blood.MethodsPlasma protein binding and fraction of unchanged parent compound were assessed by ultracentrifugation and HPLC, respectively. The distribution of radioactivity (parent compound + metabolites) between plasma and whole blood was determined ex vivo at different time-points after injection by gamma counting after separation of whole blood by centrifugation into the cellular and non-cellular components. In additional experiments in vitro, tracer distribution between these blood components was assessed for up to 90 min.ResultsA fraction of 15% ± 2% of (−)-[¹⁸ F]Flubatine was found to be bound to plasma proteins. Metabolic degradation of (−)-[¹⁸ F]Flubatine was very low, resulting in almost 90% unchanged parent compound at 90 min p.i. with no significant difference between AD and HC. The radioactivity distribution between plasma and whole blood changed in vivo only slightly over time from 0.82 ± 0.03 at 3 min p.i. to 0.87 ± 0.03 at 270 min p.i. indicating the contribution of only a small amount of metabolites. In vitro studies revealed that (−)-[¹⁸ F]Flubatine was instantaneously distributed between cellular and non-cellular blood parts.Discussion(−)-[¹⁸ F]Flubatine exhibits very favourable characteristics for a PET radiotracer such as slow metabolic degradation and moderate plasma protein binding. Equilibrium of radioactivity distribution between plasma and whole blood is reached instantaneously and remains almost constant over time allowing both convenient sample handling and facilitated fractional blood volume contribution assessment.     

[18F]Fallypride: Metabolism studies and quantification of the radiotracer and its radiometabolites in plasma using a simple and rapid solid-phase extraction method

Introduction[¹⁸F]Fallypride, a fluorinated and substituted benzamide with high affinity for D₂/D₃ receptors, is a useful PET radioligand for the study of striatal/extrastriatal areas. Since [¹⁸F]fallypride is extensively metabolized in vivo and since PET examinations are long lasting in humans, the rapid measurement of the unchanged radiotracer in plasma is essential for the quantification of images. The present study aims: i) to evaluate if the radiometabolites of [¹⁸F]fallypride cross the blood–brain barrier in rodents, ii) to identify these radiometabolites in baboon plasma and iii) to develop a rapid solid phase extraction method (SPE) suitable for human applications to quantify both [¹⁸F]fallypride and its radiometabolites in plasma.MethodsThe metabolites P450-dependant in rat and human liver microsomes were characterized by LC–MS–MS and compared to those detected in vivo. Sequential solvent elution on Oasis®-MCX-SPE cartridges was used to quantify [¹⁸F]fallypride and its radiometabolites.ResultIn rat microsomal incubations, five metabolites generated upon N/O-dealkylation or hydroxylation at the pyrrolidine and/or at the benzamide moiety were identified. No radiometabolite was detected in the rat brain. N-dealkylated and hydroxylated derivatives were detected in human microsomal incubations as well as in baboon plasma. The use of SPE (total recovery 100.2% ± 2.8%, extraction yield 95.5% ± 0.3%) allowed a complete separation of [¹⁸F]fallypride from its radiometabolites in plasma and evaluate [¹⁸F]fallypride at 150 min pi to be 22% ± 5% of plasma radioactivity.ConclusionsThe major in vivo radiometabolites of [¹⁸F]fallypride were produced by N-dealkylation and hydroxylation. Allowing the rapid analysis of multiple plasma samples, SPE is a method of choice for the determination of [¹⁸F]fallypride until late images required for quantitative PET imaging in humans.     

Single-step radiosynthesis and in vivo evaluation of a novel fluorine-18 labeled hippurate for use as a PET renal agent

ObjectiveThe objective of this study was to investigate a new fluorine-18 labeled hippurate, m-cyano-p-[¹⁸ F]fluorohippurate ([¹⁸ F]CNPFH), as a potential radiopharmaceutical for evaluating renal function by PET.Methods[¹⁸ F]CNPFH was synthesized by a direct one-step nucleophilic aromatic substitution using an ¹⁸ F-for-[N(CH₃)₃]⁺-reaction. In vivo stability was determined by HPLC analysis of urine collected from a healthy rat at 30 min p.i. of [¹⁸ F]CNPFH. The plasma protein binding (PPB) and erythrocyte uptake of [¹⁸ F]CNPFH were determined using blood collected from healthy rats at 5 min p.i. Biodistribution studies were conducted in healthy rats at 10 min and 1 h p.i. of [¹⁸ F]CNPFH. Dynamic PET/CT imaging data were acquired in normal rats. For comparison, the same rats underwent an identical imaging study using the previously reported p-[¹⁸ F]fluorohippurate ([¹⁸ F]PFH) renal agent.Results[¹⁸ F]CNPFH demonstrated high in vivo stability with no metabolic degradation. The in vivo PPB and erythrocyte uptake of [¹⁸ F]CNPFH were found to be comparable to those of [¹⁸ F]PFH. Biodistribution and dynamic PET/CT imaging studies revealed a rapid clearance of [¹⁸ F]CNPFH primarily through the renal–urinary pathway. However, unlike [¹⁸ F]PFH, a minor (about 12%) fraction was eliminated via the hepatobiliary route. The PET-derived [¹⁸ F]CNPFH renograms revealed an average time-to-peak (T_max) of 3.2 ± 0.4 min which was similar to [¹⁸ F]PFH, but the average time-to-half-maximal activity (11.4 ± 2.8 min) was found to be higher than that of [¹⁸ F]PFH (7.1 ± 1.3 min).ConclusionsOur in vivo results indicate that [¹⁸ F]CNPFH has renogram characteristics similar to those of [¹⁸ F]PFH, however, the unexpected hepatobiliary elimination is adding undesirable background signal in the PET images.     

Kinetic analyses of trans-1-amino-3-[18F]fluorocyclobutanecarboxylic acid transport in Xenopus laevis oocytes expressing human ASCT2 and SNAT2

IntroductionTrans-1-amino-3-[¹⁸F]fluorocyclobutanecarboxylic acid (anti-[¹⁸F]FACBC) is a promising amino acid positron emission tomography (PET) radiotracer for visualizing prostate cancer. We previously showed that anti-FACBC is transported by amino acid transporters, especially by alanine-serine-cysteine transporter 2 (ASCT2), which is associated with tumor growth. We studied this affinity to assess the mechanism of anti-FACBC transport in prostate cancer cells.MethodsKinetic assays for trans-1-amino-3-fluoro-[1-¹⁴C]cyclobutanecarboxylic acid ([¹⁴C]FACBC) were performed in Xenopus laevis oocytes over-expressing either ASCT2 or sodium-coupled neutral amino acid transporter 2 (SNAT2), both of which are highly expressed in prostate cancer cells. We also examined the kinetics of [¹⁴C]FACBC uptake using mammalian cell lines over-expressing system L amino acid transporter 1 or 2 (LAT1 or LAT2).ResultsASCT2 and SNAT2 transported [¹⁴C]FACBC with Michaelis–Menten kinetics K_m values of 92.0 ± 32.3 μM and 222.0 ± 29.3 μM, respectively. LAT1 and LAT2 transported [¹⁴C]FACBC with Michaelis–Menten K_m values of 230.4 ± 184.5 μM and 738.5 ± 87.6 μM, respectively.ConclusionsBoth ASCT2 and SNAT2 recognize anti-FACBC as a substrate. Anti-FACBC has higher affinity for ASCT2 than for SNAT2, LAT1, or LAT2. The ASCT2-preferential transport of anti-[¹⁸F]FACBC in cancer cells could be used for more effective prostate cancer imaging.     

2-[18F]Fluoroethanol and 3-[18F]fluoropropanol: facile preparation,biodistribution in mice,and their application as nucleophiles in the synthesis of [18F]fluoroalkyl aryl ester and ether PET tracers

Introduction2-[¹⁸F]Fluoroethoxy and 3-[¹⁸F]fluoropropoxy groups are common moieties in the structures of radiotracers used with positron emission tomography. The objectives of this study were (1) to develop an efficient one-step method for the preparation of 2-[¹⁸F]fluoroethanol (2-[¹⁸F]FEtOH) and 3-[¹⁸F]fluoropropanol (3-[¹⁸F]FPrOH); (2) to demonstrate the feasibility of using 2-[¹⁸F]FEtOH as a nucleophile for the synthesis of 2-[¹⁸F]fluoroethyl aryl esters and ethers; and (3) to determine the biodistribution profiles of 2-[¹⁸F]FEtOH and 3-[¹⁸F]FPrOH in mice.Methods2-[¹⁸F]FEtOH and 3-[¹⁸F]FPrOH were prepared by reacting n-Bu₄N[¹⁸F]F with ethylene carbonate and 1,3-dioxan-2-one, respectively, in diethylene glycol at 165 °C and purified by distillation. 2-[¹⁸F]fluoroethyl 4-fluorobenzoate and 1-(2-[¹⁸F]fluoroethoxy)-4-nitrobenzene were prepared by coupling 2-[¹⁸F]FEtOH with 4-fluorobenzoyl chloride and 1-fluoro-4-nitrobenzene, respectively. Biodistribution and PET/CT imaging studies of 2-[¹⁸F]FEtOH and 3-[¹⁸F]FPrOH were performed in normal female Balb/C mice.ResultsThe preparation of 2-[¹⁸F]FEtOH and 3-[¹⁸F]FPrOH took 60 min, and their decay-corrected yields were 88.6 ± 2.0% (n = 9) and 65.6 ± 10.2% (n = 5), respectively. The decay-corrected yields for the preparation of 2-[¹⁸F]fluoroethyl 4-fluorobenzoate and 1-(2-[¹⁸F]fluoroethoxy)-4-nitrobenzene were 36.1 ± 5.4% (n = 3) and 27.7 ± 10.7% (n = 3), respectively. Imaging/biodistribution studies in mice using 2-[¹⁸F]FEtOH showed high initial radioactivity accumulation in all major organs followed by very slow clearance. On the contrary, by using 3-[¹⁸F]FPrOH, radioactivity accumulated in all major organs was cleared rapidly, but massive in vivo defluorination (31.3 ± 9.57%ID/g in bone at 1 h post-injection) was observed.ConclusionsUsing 2-[¹⁸F]FEtOH/3-[¹⁸F]FPrOH as a nucleophile is a competitive new strategy for the synthesis of 2-[¹⁸F]fluoroethyl/3-[¹⁸F]fluoropropyl aryl esters and ethers. Our biodistribution data emphasize the importance of in vivo stability of PET tracers containing a 2-[¹⁸F]fluoroethyl or 3-[¹⁸F]fluoropropyl group due to high background and high bone uptake resulting from 2-[¹⁸F]FEtOH and 3-[¹⁸F]FPrOH, respectively. This is especially important for their aryl ester derivatives which are prone to in vivo hydrolysis.     

Evaluation of 18F-labeled BODIPY dye as potential PET agents for myocardial perfusion imaging

IntroductionDespite the great potential of positron emission tomography/computed tomography (PET/CT) in cardiovascular disease imaging, one of the major limitations is the availability of PET probes with desirable half-lives and reasonable cost. In this report, we hypothesized that lipophilic cationic BODIPY dye could be selectively accumulated in cardiac muscle, possibly for the development of novel PET myocardial perfusion imaging (MPI) probes.MethodsA ¹⁸F-labeled BODIPY dye ([¹⁸F]1) was synthesized efficiently through a fluoride exchange reaction catalyzed by the Lewis acid tin chloride (SnCl₄). The compound was first evaluated by a cellular uptake assay in vitro, followed by biodistribution and microPET imaging studies in vivo.Results[¹⁸F]1 was obtained in more than 90% labeling yield, with > 98% radiochemical purity. The HEK-293 cellular uptake assay showed that the preferential uptake of [¹⁸F]1 could be related to the cell membrane potential. The biodistribution data demonstrated high levels of [¹⁸F]1 accumulation in the heart. In the biodistribution study in mice, the radioactivity uptake in the heart, blood, liver and lung was 3.01 ± 0.44, 0.39 ± 0.09, 0.69 ± 0.07, 1.71 ± 0.27%ID/g, respectively, at 3 h post-injection (p.i.). The heart-to-lung and heart-to-liver ratios are 1.76 ± 0.14 and 4.37 ± 0.51 at 3 h p.i., respectively. Volume-of-interest analysis of the microPET images correlated well with the biodistribution studies in mice. The heart was clearly visualized in normal rats, with 0.72 ± 0.18, 0.69 ± 0.18, 0.67 ± 0.20 and 0.59 ± 0.17%ID/g uptake at 0.5, 1, 2 and 4 h p.i., respectively.Conclusions¹⁸F-labeled BODIPY dye showed good heart uptake and heart-to-blood and heart-to-lung contrast. A ¹⁸F-labeled BODIPY dyes may represent a new category of cationic PET agents for myocardial perfusion imaging.     

Synthesis and radiopharmacological evaluation of a high-affinity and metabolically stabilized 18F-labeled bombesin analogue for molecular imaging of gastrin-releasing peptide receptor-expressing prostate cancer

IntroductionBombesin (BBN) and BBN analogues have attracted much attention as high-affinity ligands for selective targeting of the gastrin-releasing peptide (GRP) receptor. GRP receptors are overexpressed in a variety of human cancers including prostate cancer. Radiolabeled BBN derivatives are promising diagnostic probes for molecular imaging of GRP receptor-expressing prostate cancer. This study describes the synthesis and radiopharmacological evaluation of various metabolically stabilized fluorobenzoylated bombesin analogues (BBN-1, BBN-2, BBN-3).MethodsThree fluorobenzoylated BBN analogues containing an aminovaleric (BBN-1, BBN-2), or an aminooctanoic acid linker (BBN-3) were tested in a competitive binding assay against ¹²⁵I-[Tyr⁴]-BBN for their binding potency to the GRP receptor. Intracellular calcium release in human prostate cancer cells (PC3) was measured to determine agonistic or antagonistic profiles of fluorobenzoylated BBN derivatives. Bombesin derivative BBN-2 displayed the highest inhibitory potency toward GRP receptor (IC₅₀ = 8.7 ± 2.2 nM) and was subsequently selected for radiolabeling with fluorine-18 (¹⁸F) through acylation with N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). The radiopharmacological profile of ¹⁸F-labeled bombesin [¹⁸F]BBN-2 was evaluated in PC3 tumor-bearing NMRI nude mice involving metabolic stability studies, biodistribution experiments and dynamic small-animal PET studies.ResultsAll fluorobenzoylated BBN derivatives displayed high inhibitory potency toward the GRP receptor (IC₅₀ = 8.7–16.7 nM), and all compounds exhibited antagonistic profiles as determined in an intracellular calcium release assay. The ¹⁸F-labeled BBN analogue [¹⁸F]BBN-2 was obtained in 30% decay-corrected radiochemical yield with high radiochemical purity > 95% after semi-preparative HPLC purification. [¹⁸F]BBN-2 showed high metabolic stability in vivo with 65% of the radiolabeled peptide remaining intact after 60 min p.i. in mouse plasma. Biodistribution experiments and dynamic small-animal PET studies demonstrated high tumor uptake of [¹⁸F]BBN-2 in PC3 xenografts (2.75 ± 1.82 %ID/g after 5 min and 2.45 ± 1.25 %ID/g after 60 min p.i.). Specificity of radiotracer uptake in PC3 tumors was confirmed by blocking experiments.ConclusionThe present study demonstrates that ¹⁸F-labeled BBN analogue [¹⁸F]BBN-2 is a suitable PET radiotracer with favorable metabolic stability in vivo for molecular imaging of GRP receptor-positive prostate cancer.     

Tracer level radiochemistry to clinical dose preparation of 177Lu-labeled cyclic RGD peptide dimer

AimIntegrin α_vβ₃ plays a significant role in angiogenesis during tumor growth and metastasis, and is a receptor for the extracellular matrix proteins with the exposed arginine(R)-glycine(G)-aspartic acid(D) tripeptide sequence. The over-expression of integrin α_vβ₃ during tumor growth and metastasis presents an interesting molecular target for both early detection and treatment of rapidly growing solid tumors. Considering the advantages of ¹⁷⁷Lu for targeted radiotherapy and enhanced tumor targeting capability of cyclic RGD peptide dimer, an attempt has been made to optimize the protocol for the preparation of clinical dose of ¹⁷⁷Lu labeled DOTA-E[c(RGDfK)]₂ (E = Glutamic acid, f = phenyl alanine, K = lysine) as a potential agent for targeted tumor therapy.Methods¹⁷⁷Lu was produced by thermal neutron bombardment on enriched Lu₂O₃ (82% in ¹⁷⁶Lu) target at a flux of 1 × 10¹⁴ n/cm².s for 21 d. Therapeutic dose of ¹⁷⁷Lu-DOTA-E[c(RGDfK)]₂ (7.4 GBq) was prepared by adding the aqueous solution of the ligand and ¹⁷⁷LuCl₃ to 0.1 M NH₄OAC buffer containing gentisic acid and incubating the reaction mixture at 90 °C for 30 min. The yield and radiochemical purity of the complex was determined by HPLC technique. Parameters, such as, ligand-to-metal ratio, pH of the reaction mixture, incubation time and temperature were varied using tracer quantity of ¹⁷⁷Lu (37 MBq) in order to arrive at the optimized protocol for the preparation of clinical dose. Biological behavior of the radiotracer prepared was studied in C57/BL6 mice bearing melanoma tumors.Results¹⁷⁷Lu was produced with a specific activity of 950 ± 50 GBq/mg (25.7 ± 1.4 Ci/mg) and radionuclidic purity of 99.98%. A careful optimization of several parameters showed that ¹⁷⁷Lu-DOTA-E[c(RGDfK)]₂ could be prepared with adequately high radiochemical purity using a ligand-to-metal ratio ~ 2. Based on these studies therapeutic dose of the agent with 7.4 GBq of ¹⁷⁷Lu was formulated in ~ 63 GBq/μM specific activity with high yield (98.2 ± 0.7%), radiochemical purity and in vitro stability. Biodistribution studies carried out in C57/BL6 mice bearing melanoma tumors revealed specific accumulation of the radiolabeled conjugate in tumor (3.80 ± 0.55% ID/g at 30 min p.i.) with high tumor to blood and tumor to muscle ratios. However, the uptake of the radiotracer in the tumor was found to be reduced to 1.51 ± 0.32 %ID/g at 72 h p.i.ConclusionsThe present work successfully demonstrates the formulation of an optimized protocol for the preparation of ¹⁷⁷Lu labeled DOTA-E[c(RGDfK)]₂ for PRRT applications using ¹⁷⁷Lu produced by direct neutron activation in a medium flux research reactor.     

Synthesis and 18F-labeling of the analogues of Omecamtiv Mecarbil as a potential cardiac myosin imaging agent with PET

IntroductionCardiac myosin is a potential molecular target for heart failure imaging since its changes can be used to assess the function of heart. In this study, two analogues of Omecamtiv Mecarbil, which is the first selective activator of cardiac myosin, were synthesized and radio-labeled with ¹⁸F. Then the radio-compounds were evaluated as potential cardiac myosin imaging agent.MethodsThe labeling precursor and the nonradioactive compounds were synthesized and characterized by IR, ¹H NMR, ¹³C NMR and MS analysis. By substituting bromo of precursors with ¹⁸F, the radiolabeled compounds [¹⁸F]8 and [¹⁸F]10 were prepared and further evaluated for their in vitro physicochemical properties, stabilities, protein binding assay and ex vivo biodistribution.ResultsStarting with [¹⁸F]F^- Kryptofix 2.2.2./K₂CO₃ solution, the total reaction time for [¹⁸F]8 and [¹⁸F]10 was about 40 min respectively, with final high-performance liquid chromatography purification included. Typical decay-corrected radiochemical yield stayed at 12.47% ± 3.30% (n = 8), the radiochemical purity, 98% or more. Their specific activity was estimated as 50 GBq/μmol. Both [¹⁸F]8 and [¹⁸F]10 could be stable after incubation in water at room temperature and in serum or binding buffer at 37 °C for 3 h. Biodistribution in normal mice showed that both [¹⁸F]8 and [¹⁸F]10 have good heart uptake at 2 min post-injection time. Compound [¹⁸F]10 has better heart retention and higher heart to background ratios than those of [¹⁸F]8. In vitro protein binding assay demonstrates that [¹⁸F]10 may have high affinity with myosin from bovine heart.Conclusion[¹⁸F]8 and [¹⁸F]10 were synthesized with good radiochemical yield and high radiochemical purity (> 98%). One of the compounds ([¹⁸F]10) has higher bovine heart myosin binding affinity and better heart/liver ratio. It will be further evaluated as a potent cardiac myosin imaging agent in normal and systolic heart failure model with positron emission tomography in the future.     

Novel fluorine-18 PET radiotracers based on flumazenil for GABAA imaging in the brain

IntroductionTwo 7-fluoroimidazobenzodiazepines (AH114726 and GEH120348), analogs of flumazenil, were labeled with fluorine-18 and evaluated as alternative radioligands for in vivo imaging of the GABA_A/benzodiazepine receptor by comparing them to [¹¹C]flumazenil in rhesus monkey.MethodsRadiotracers were prepared from the corresponding nitro-precursors in an automated synthesis module, and primate imaging studies were conducted on a Concorde MicroPET P4 scanner. The brain was imaged for 60 (12 × 5 min frames) or 90 min (18 × 5 min frames), and data was reconstructed using the 3D MAP algorithm. Specificity of [¹⁸F]AH114726 and [¹⁸F]GEH120348 was confirmed by displacement studies using unlabeled flumazenil.Results[¹⁸F]GEH120348 and [¹⁸F]AH114726 were obtained in 13–24% yields (end of synthesis) with high chemical (> 95%) and radiochemical (> 99%) purities, and high specific activities (2061 ± 985 Ci/mmol). The in vivo pharmacokinetics of [¹⁸F]AH114726 and [¹⁸F]GEH120348 were determined in a non-human primate and directly compared with [¹¹C]flumazenil. Both fluorine-18 radioligands showed time-dependent regional brain distributions that correlated with the distribution of [¹¹C]flumazenil and the known concentrations of GABA_A/benzodiazepine receptors in the monkey brain. [¹⁸F]AH114726 exhibited maximal brain uptake and tissue time-radioactivity curves that were most similar to [¹¹C]flumazenil. In contrast, [¹⁸F]GEH120348 showed higher initial brain uptake but very different pharmacokinetics with continued accumulation of radioactivity into the cortical regions of high GABA/benzodiazepine receptor concentrations and very little clearance from the regions of low receptor densities. Rapid washout of both radiotracers occurred upon treatment with unlabeled flumazenil.ConclusionThe ease of the radiochemical synthesis, together with in vivo brain pharmacokinetics most similar to [¹¹C]flumazenil, support that [¹⁸F]AH114726 is a suitable option for imaging the GABA_A receptor.     

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.     

Evaluation of an integrin αvβ6-specific peptide labeled with [18F]fluorine by copper-free,strain-promoted click chemistry

IntroductionClick chemistry, particularly the Huisgen 1,3-dipolar cycloaddition of an alkyne with an azide, has quickly become popular for site-specific radiolabeling. Recently, strain-promoted click chemistries have been developed, eliminating the need for potentially toxic copper catalysts. This study presents radiolabeling of an α_vβ₆ integrin targeting peptide (A20FMDV2) via strain-promoted click using a fluorine-18 prosthetic group, and in vitro and in vivo evaluation.MethodsThe radiotracer [¹⁸F]FBA-C₆-ADIBON₃-PEG₇-A20FMDV2 (1) was prepared from [¹⁸F]FBA-C₆-ADIBO (2) and N₃-PEG₇-A20FMDV2 (ethanol; 10 min; 35–45 °C). HPLC-purified and formulated radiotracer 1 was evaluated in vitro by cell binding (DX3puroβ6, α_vβ₆-positive; and DX3puro, α_vβ₆-negative control) and serum stability, and in vivo using PET/CT imaging and biodistribution studies in mice.ResultsThe radiotracer 1 was readily prepared and purified (from 2: 40 ± 4 min including HPLC, 11.9 ± 3.2% decay corrected isolated radiochemical yield, > 99% radiochemical purity, n = 4) and displayed good stability (1 h: > 99%, saline; 94.6%, serum). Strong α_vβ₆-targeted binding was observed in vitro (DX3puroβ6 cells, 15 min: 43.2% binding, > 6:1 for DX3puroβ6:DX3puro). In the mouse model DX3puroβ6-tumor binding was low (1 h: 0.47 ± 0.28% ID/g, 4 h: 0.14 ± 0.09% ID/g) and clearing from the bloodstream was via the renal and hepatobiliary routes (urine: 167 ± 84% ID/g at 1 h, 10.3 ± 4.8% ID/g at 4 h; gall bladder: 95 ± 33% ID/g at 1 h, 63 ± 11% ID/g at 4 h).ConclusionCopper-free, strain-promoted click chemistry is an attractive, straightforward approach to radiolabeling. Although the [¹⁸F]FBA-C₆-ADBIO-based prosthetic group did not interfere with α_vβ₆-targeted binding in vitro, it did influence the pharmacokinetics, possibly due to its size and lipophilic nature.     

Effect of co-ligands on chemical and biological properties of 99mTc(III) complexes [99mTc(L)(CDO)(CDOH)2BMe] (L = Cl,F, SCN and N3; CDOH2 = cyclohexanedione dioxime)

Introduction^99mTc-Teboroxime ([^99mTcCl(CDO)(CDOH)₂BMe]) is a member of the BATO (boronic acid adducts of technetium dioximes) class of ^99mTc(III) complexes. This study sought to explore the impact of co-ligands on solution stability, heart uptake and myocardial retention of [^99mTc(L)(CDO)(CDOH)₂BMe] (^99mTc-Teboroxime: L = Cl; ^99mTc-Teboroxime(F): L = F; ^99mTc-Teboroxime(SCN): L = SCN; and ^99mTc-Teboroxime(N₃): L = N₃).MethodsRadiotracers ^99mTc-Teboroxime(L) (L = F, SCN and N₃) were prepared by reacting ^99mTc-Teboroxime with NaF, NaSCN and NaN₃, respectively. Biodistribution and imaging studies were carried out in Sprague–Dawley rats. Image quantification was performed to compare their heart retention and liver clearance kinetics.ResultsComplexes ^99mTc-Teboroxime(L) (L = F, SCN and N₃) were prepared in high yield with high radiochemical purity. All new radiotracers were stable for > 6 h in the kit matrix. In its HPLC chromatogram, ^99mTc-Teboroxime showed one peak at ~ 15.5 min, which was shorter than that of ^99mTc-Teboroxime(F) (~ 16.4 min). There were two peaks for ^99mTc-Teboroxime(SCN) at 16.5 and 18.3 min. ^99mTc-Teboroxime(N₃) appeared as a single peak at 18.4 min. Their heart retention and liver clearance curves were best fitted to the bi-exponential decay function. The half-times of fast/slow components were 1.6 ± 0.4/60.7 ± 8.9 min for ^99mTc-Teboroxime, 0.8 ± 0.2/101.7 ± 20.7 min for ^99mTc-Teboroxime(F), 1.2 ± 0.3/84.8 ± 16.6 min for ^99mTc-Teboroxime(SCN), and 2.9 ± 0.9/51.6 ± 5.0 min for ^99mTc-Teboroxime(N₃). The 2-min heart uptake followed the order of ^99mTc-Teboroxime (3.00 ± 0.37%ID/g) > ^99mTc-Teboroxime(N₃) (2.66 ± 0.01 %ID/g) ≈ ^99mTc-Sestamibi (2.55 ± 0.46 %ID/g) > ^99mTcN-MPO (2.38 ± 0.15 %ID/g). ^99mTc-Teboroxime remains the best in first-pass extraction. The best image acquisition window is 0–5 min for ^99mTc-Teboroximine and 0–15 min for ^99mTc-Teboroximine(N₃).ConclusionCo-ligands had significant impact on the heart uptake and myocardial retention of complexes [^99mTc(L)(CDO)(CDOH)₂BMe] (L = Cl, F, SCN and N₃). Future studies should be directed towards minimizing the liver uptake and radioactivity accumulation in the blood vessels while maintaining their high heart uptake.     

PET quantification of pancreatic VMAT 2 binding using (+) and (−) enantiomers of [18 F]FP-DTBZ in baboons

ObjectivesThe exact cause(s) of apparent overestimation of β cell mass (BCM) with vesicular monoamine transporter type 2 (VMAT2) PET imaging in type 1 diabetes (T1D) is unknown. The objectives were to estimate in baboons non-displaceable binding of [¹⁸ F]fluoropropyl (FP)-(+)-dihydrotetrabenazine (DTBZ) with its inactive enantiomer, [¹⁸ F]FP-(?)-DTBZ, to validate the use of the reference tissue (renal cortex or spleen) in VMAT2 quantification; and also to compare specific pancreatic VMAT2 binding with that of the striatum in the same baboon brains because high specific binding signal for the pancreas would be desirable for its accurate quantification.MethodsBaboons (Papio ursinus) had multiple dynamic abdominal and brain PET scans each for 2 h with (+) and (?) enantiomers on separate occasions. Data were analyzed by compartmental models to estimate non-displaceable (V_ND) and specific (V_S) VMAT2 binding in respective organs.Results[¹⁸ F]FP-DTBZ PET showed excellent target tissue signal and specific VMAT2 binding in the pancreas (Vs = 41 ± 11 mL/cm³) at nearly 80% that of the striatum. Directly estimated non-displaceable binding in the pancreas (V_ND = 12 ± 1 mL/cm³) was similar to that of the renal cortex, spleen or cerebellum.Conclusion¹⁸ FFP-DTBZ PET shows excellent specific VMAT2 binding in both the pancreas and striatum in baboons. The renal cortex or spleen as the reference tissue in VMAT2 quantification appears supported. However further studies appear warranted to directly estimate pancreatic non-displaceable binding in humans including T1D patients and also to clarify the cause of the apparent overestimation of BCM in T1D.     

Monitoring tumor response with [18F]FMAU in a sarcoma-bearing mouse model after liposomal vinorelbine treatment

ObjectivePrevious studies have shown that the accumulation level of FMAU in tumor is proportional to its proliferation rate. This study demonstrated that 2′-deoxy-2′-[¹⁸F]fluoro-β-d-arabinofuranosyluracil ([¹⁸F]FMAU) is a promising PET probe for noninvasively monitoring the therapeutic efficacy of 6% PEGylated liposomal vinorelbine (lipo-VNB) in a subcutaneous murine NG4TL4 sarcoma mouse model.MethodsFemale syngenic FVB/N mice were inoculated with NG4TL4 cells in the right flank. After tumor size reached 150 ± 50 mm³ (day 0), lipo-VNB (5 mg/kg) was intravenously administered on days 0, 3 and 6. To monitor the therapeutic efficacy of lipo-VNB, [¹⁸F]FMAU PET was employed to evaluate the proliferation rate of tumor, and it was compared with that observed from [¹⁸F]FDG/[¹⁸F]fluoroacetate PET. The expression of proliferating cell nuclear antigen (PCNA) in tumor during treatment was determined by semiquantitative analysis of immunohistochemical staining.ResultsA significant inhibition (p < 0.001) in tumor growth was observed on day 3 after a single dose treatment. The tumor-to-muscle ratio (T/M) derived from [¹⁸F]FMAU-PET images of lipo-VNB-treated group declined from 2.33 ± 0.16 to 1.26 ± 0.03 after three doses of treatment, while that of the control remained steady. The retarded proliferation rate of lipo-VNB-treated sarcoma was confirmed by PCNA immunohistochemistry staining. However, both [¹⁸F]FDG and [¹⁸F]fluoroacetate microPET imaging did not show significant difference in T/M between the therapeutic and the control groups throughout the entire experimental period.ConclusionLipo-VNB can effectively impede the growth of NG4TL4 sarcoma. [¹⁸F]FMAU PET is an appropriate modality for early monitoring of the tumor response during the treatment course of lipo-VNB.     

Synthesis and preclinical evaluation of [11C-carbonyl]PF-04457845 for neuroimaging of fatty acid amide hydrolase

IntroductionFatty acid amide hydrolase (FAAH) has a significant role in regulating endocannabinoid signaling in the central nervous system. As such, FAAH inhibitors are being actively sought for pain, addiction, and other indications. This has led to the recent pursuit of positron emission tomography (PET) radiotracers targeting FAAH. We report herein the preparation and preclinical evaluation of [¹¹C-carbonyl]PF-04457845, an isotopologue of the potent irreversible FAAH inhibitor.MethodsPF-04457845 was radiolabeled at the carbonyl position via automated [¹¹C]CO₂-fixation. Ex vivo brain biodistribution of [¹¹C-carbonyl]PF-04457845 was carried out in conscious rats. Specificity was determined by pre-administration of PF-04457845 or URB597 prior to [¹¹C-carbonyl]PF-04457845. In a separate experiment, rats injected with the title radiotracer had whole brains excised, homogenized and extracted to examine irreversible binding to brain parenchyma.ResultsThe title compound was prepared in 5 ± 1% (n = 4) isolated radiochemical yield based on starting [¹¹C]CO₂ (decay uncorrected) within 25 min from end-of-bombardment in > 98% radiochemical purity and a specific activity of 73.5 ± 8.2 GBq/μmol at end-of-synthesis. Uptake of [¹¹C-carbonyl]PF-04457845 into the rat brain was high (range of 1.2–4.4 SUV), heterogeneous, and in accordance with reported FAAH distribution. Saturable binding was demonstrated by a dose-dependent reduction in brain radioactivity uptake following pre-treatment with PF-04457845. Pre-treatment with the prototypical FAAH inhibitor, URB597, reduced the brain radiotracer uptake in all regions by 71–81%, demonstrating specificity for FAAH. The binding of [¹¹C-carbonyl]PF-04457845 to FAAH at 40 min post injection was irreversible as 98% of the radioactivity in the brain could not be extracted.Conclusions[¹¹C-carbonyl]PF-04457845 was rapidly synthesized via an automated radiosynthesis. Ex vivo biodistribution studies in conscious rodents demonstrate that [11C PF-04457845 is a promising candidate radiotracer for imaging FAAH in the brain with PET. These results coupled with the known pharmacology and toxicology of PF-04457845 should facilitate clinical translation of this radiotracer.     

Automation of the radiosynthesis and purification procedures for [18F]Fluspidine preparation,a new radiotracer for clinical investigations in PET imaging of σ1 receptors in brain

The radiosynthesis of [¹⁸F]Fluspidine, a potent σ₁ receptor imaging probe for pre-clinical/clinical studies, was implemented on a TRACERlab^TM FX F-N synthesizer. [¹⁸F]2 was synthesized in 15 min at 85 °C starting from its tosylate precursor. Purification via semi-preparative RP-HPLC was investigated using different columns and eluent compositions and was most successful on a polar RP phase with acetonitrile/water buffered with NH₄OAc. After solid phase extraction, [¹⁸F]Fluspidine was formulated and produced within 59±4 min with an overall radiochemical yield of 37±8%, a radiochemical purity of 99.3±0.5% and high specific activity (176.6±52.0 GBq/µmol).     

Uptake of O-(2-[18F]fluoroethyl)-L-tyrosine in reactive astrocytosis in the vicinity of cerebral gliomas

PET using O-(2-[¹⁸F]fluoroethyl)-L-tyrosine (¹⁸F-FET) allows improved imaging of tumor extent of cerebral gliomas in comparison to MRI. In experimental brain infarction and hematoma, an unspecific accumulation of ¹⁸F-FET has been detected in the area of reactive astrogliosis which is a common cellular reaction in the vicinity of cerebral gliomas. The aim of this study was to investigate possible ¹⁸F-FET uptake in the area of reactive gliosis in the vicinity of untreated and irradiated rat gliomas.MethodsF98-glioma cells were implanted into the caudate nucleus of 33 Fisher CDF rats. Sixteen animals remained untreated and in 17 animals the tumor was irradiated by Gamma Knife 5–8 days after implantation (2/50 Gy, 3/75 Gy, 6/100 Gy, 6/150 Gy). After 8–17 days of tumor growth the animals were sacrificed following injection of ¹⁸F-FET. Brains were removed, cut in coronal sections and autoradiograms of ¹⁸F-FET distribution were produced and compared with histology (toluidine blue) and reactive astrogliosis (GFAP staining). ¹⁸F-FET uptake in the tumors and in areas of reactive astrocytosis was evaluated by lesion to brain ratios (L/B).ResultsLarge F98-gliomas were present in all animals showing increased ¹⁸F-FET-uptake which was similar in irradiated and non-irradiated tumors (L/B: 3.9 ± 0.8 vs. 4.0 ± 1.3). A pronounced reactive astrogliosis was noted in the vicinity of all tumors that showed significantly lower ¹⁸F-FET-uptake than the tumors (L/B: 1.5 ± 0.4 vs. 3.9 ± 1.1). The area of ¹⁸F-FET-uptake in the tumor was congruent with histological tumor extent in 31/33 animals. In 2 rats irradiated with 150 Gy, however, high ¹⁸F-FET uptake was noted in the area of astrogliosis which led to an overestimation of the tumor size.ConclusionsReactive astrogliosis in the vicinity of gliomas generally leads to only a slight ¹⁸F-FET-enrichment that appears not to affect the correct definition of tumor extent for treatment planning.