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.     

Synthesis and biological evaluation of N-(2-[18F]Fluoropropionyl)-L-methionine for tumor imaging

IntroductionN-position radiolabeled amino acids, such as N-(2-[¹⁸F]fluoropropionyl)-L-methionine ([¹⁸F]FPMET) as a derivative of L-methionine (MET), can potentially serve as a PET tracer for tumor imaging. In the current study, radiosynthesis and biological evaluation of [¹⁸F]FPMET as a new PET tumor agent are performed.Methods[¹⁸F]FPMET was synthesized by reacting 4-nitrophenyl 2-[¹⁸F]fluoropropionate ([¹⁸F]NFP) with MET. In vitro competitive inhibition and protein incorporation experiments were performed with Hepa1-6 hepatoma cell lines. The biodistribution of [¹⁸F]FPMET was determined in S180 fibrosarcoma-bearing mice. PET/CT studies of [¹⁸F]FPMET were conducted in S180 fibrosarcoma-bearing mice, A549 lung adenocarcinoma-bearing nude mice, and PC-3 prostate cancer-bearing nude mice.Results[¹⁸F]FPMET was synthesized in 72% ± 4% uncorrected radiochemical yield (n = 10) from [¹⁸F]NFP. In vitro experiments showed that [¹⁸F]FPMET was primarily transported through Na⁺-dependent system A, system ASC, and system B^0,+, and was not incorporated into protein. Biodistribution and PET/CT imaging studies indicated that [¹⁸F]FPMET could delineate S180 fibrosarcoma, A549 lung adenocarcinoma, and PC-3 prostate cancer.ConclusionAn efficient synthesis of N-position [¹⁸F]labeled amino acids with a classic [¹⁸F]NFP prosthetic group is developed. The results support that [¹⁸F]FPMET seems to be a potential tracer for tumor imaging with PET.     

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.     

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.     

Radiosynthesis and biological evaluation of 5-(3-[18F]Fluoropropyloxy)-L-tryptophan for tumor PET imaging

Introduction[¹⁸F]FDG PET has difficulty distinguishing tumor from inflammation in the clinic because of the same high uptake in nonmalignant and inflammatory tissue. In contrast, amino acid tracers do not accumulate in inflamed tissues and thus provide an excellent opportunity for their use in clinical cancer imaging. In this study, we developed a new amino acid tracer 5-(3-[¹⁸F]Fluoropropyloxy)-L-tryptophan ([¹⁸F]-L-FPTP) by two-step reactions and performed its biologic evaluation.Methods[¹⁸F]-L-FPTP was prepared by [¹⁸F]fluoropropylation of 5-hydroxy-L-tryptophan disodium salt and purification on C18 cartridges. The biodistribution of [¹⁸F]-L-FPTP was determined in normal mice and the incorporation of [¹⁸F]-L-FPTP into tissue proteins was investigated. In vitro competitive inhibition experiments were performed with Hepa1-6 hepatoma cell lines. [¹⁸F]-L-FPTP PET imaging was performed on tumor-bearing and inflammation mice and compared with [¹⁸F]-L-FEHTP PET.ResultsThe overall uncorrected radiochemical yield of [¹⁸F]-L-FPTP was 21.1 ± 4.4% with a synthesis time of 60 min, the radiochemical purity was more than 99%. Biodistribution studies demonstrate high uptake of [¹⁸F]-L-FPTP in liver, kidney, pancreas, and blood at the early phase, and fast clearance in most tissues over the whole observed time. The uptake studies in Hepa1-6 cells suggest that [¹⁸F]-L-FPTP is transported by the amino acid transport system B^0,+, LAT2 and ASC. [¹⁸F]-L-FPTP displays good stability and is not incorporated into proteins in vitro. PET imaging shows that [¹⁸F]-L-FPTP can be a better potential PET tracer for differentiating tumor from inflammation than [¹⁸F]FDG and 5-(3-[¹⁸F]fluoroethyloxy)-L-tryptophan ([¹⁸F]-L-FEHTP), with high [¹⁸F]-L-FPTP uptake ratio (2.53) of tumor to inflammation at 60 min postinjection.ConclusionsUsing [¹⁸F]fluoropropyl derivatives as intermediates, the new tracer [¹⁸F]-L-FPTP was achieved with good yield and radiochemical purity, and the biological evaluation results of [¹⁸F]-L-FPTP showed that it was a hopeful tracer for PET tumor imaging.     

Adrenergic pathway activation enhances brown adipose tissue metabolism: A [18 F]FDG PET/CT study in mice

ObjectivePharmacologic approaches to study brown adipocyte activation in vivo with a potential of being translational to humans are desired. The aim of this study was to examine pre- and postsynaptic targeting of adrenergic system for enhancing brown adipose tissue (BAT) metabolism quantifiable by [¹⁸ F]fluoro-2-deoxyglucose ([¹⁸ F]FDG) positron emission tomography (PET)/computed tomography (CT) in mice.MethodsA β₃-adrenoreceptor selective agonist (CL 316243), an adenylyl cyclase enzyme activator (forskolin) and a potent blocker of presynaptic norepinephrine transporter (atomoxetine), were injected through the tail vein of Swiss Webster mice 30 minutes before intravenous (iv) administration of [¹⁸ F]FDG. The mice were placed on the PET/CT bed for 30 min PET acquisition followed by 10 min CT acquisition for attenuation correction and anatomical delineation of PET images.ResultsActivated interscapular (IBAT), cervical, periaortic and intercostal BAT were observed in 3-dimentional analysis of [¹⁸ F]FDG PET images. CL 316243 increased the total [¹⁸ F]FDG standard uptake value (SUV) of IBAT 5-fold greater compared to that in placebo-treated mice. It also increased the [¹⁸ F]FDG SUV of white adipose tissue (2.4-fold), and muscle (2.7-fold), as compared to the control. There was no significant difference in heart, brain, spleen and liver uptakes between groups. Forskolin increased [¹⁸ F]FDG SUV of IBAT 1.9-fold greater than that in placebo-treated mice. It also increased the [¹⁸ F]FDG SUV of white adipose tissue (2.2-fold) and heart (5.4-fold) compared to control. There was no significant difference in muscle, brain, spleen, and liver uptakes between groups. Atomoxetine increased [¹⁸ F]FDG SUV of IBAT 1.7-fold greater than that in placebo-treated mice. There were no significant differences in all other organs compared to placebo-treated mice except liver (1.6 fold increase). A positive correlation between SUV levels of IBAT and CT Hounsfield unit (HU) (R² = 0.55, p < 0.001) and between CT HU levels of IBAT and liver (R² = 0.69, p < 0.006) was observed.ConclusionsThe three pharmacologic approaches reported here enhanced BAT metabolism by targeting different sites in adrenergic system as measured by [¹⁸ F]FDG PET/CT.     

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.     

Comparison of trans-1-amino-3-[18 F]fluorocyclobutanecarboxylic acid (anti-[18 F]FACBC) accumulation in lymph node prostate cancer metastasis and lymphadenitis in rats

IntroductionTrans-1-amino-3-[¹⁸ F]fluorocyclobutanecarboxylic acid (anti-[¹⁸ F]FACBC) is a positron emission tomography (PET) tracer used to visualize prostate cancer (PCa). In this study, we investigated the differences in anti-[¹⁸ F]FACBC accumulation between metastatic and inflamed lymph node (LN) lesions.MethodsA PCa LN metastasis (PLM) model was developed by inoculating a rat PCa cell line, MAT-Ly-Lu-B2, into popliteal LNs of Copenhagen rats. Acute lymphadenitis (AL) was induced by injecting concanavalin A (Con A) into the hind footpad, and chronic lymphadenitis (CL) was induced by daily injection of Con A into the tissues surrounding the popliteal LNs for 2 weeks. Main lesions of all animal models were established in lumbar and/or inguinal LNs. Biodistribution and dynamic PET imaging data were acquired after tracer injection. T2-weighted magnetic resonance (MR) images were registered with PET images.ResultsIn the biodistribution study, the uptake ratios of PLM-to-lymphadenitis in lesional lumbar and inguinal LNs were 0.97 − 1.57 and 1.47 − 2.08 at 15 and 60 min post-anti-[¹⁸ F]FACBC injection respectively. In PET imaging, the lesional lumbar LNs of CL and PLM, but not of AL, were visualized on anti-[¹⁸ F]FACBC-PET/MR fusion images without disturbance from radioactivity from urine, and the rank order of anti-[¹⁸ F]FACBC accumulation at 50 − 60 post-injection in lesional lumbar LNs was PLM > CL > AL.ConclusionsAnti-[¹⁸ F]FACBC accumulation in LNs with PLM was higher than that in inflamed LNs.Advances in knowledgeThe study showed that although low but significant levels of anti-[¹⁸ F]FACBC uptake by chronic inflamed lesions might cause false-positives in anti-[¹⁸ F]FACBC-PET in some PCa patients, uptake of the tracer at acutely inflamed sites was minimal.Implications for patient careThe findings of this study suggest the potential of Anti-[¹⁸ F]FACBC for distinguishing between tumors and acute inflammation in clinical practice.     

Radiosynthesis and biological evaluation of alpha-[F-18]fluoromethyl phenylalanine for brain tumor imaging

ObjectivesRadiolabeled amino acids have proven utility for imaging brain tumors in humans, particularly those that target system L amino acid transport. We have prepared the novel phenylalanine analogue, α-[¹⁸F]fluoromethyl phenylalanine (FMePhe, 9), as part of an effort to develop new system L tracers that can be prepared in high radiochemical yield through nucleophilic [¹⁸F]fluorination. The tumor imaging properties of both enantiomers of this new tracer were evaluated through cell uptake, biodistribution and microPET studies in the mouse DBT model of high grade glioma.MethodsThe non-radioactive form of 9 and the cyclic sulfamidate labeling precursor were prepared from commercially available racemic α-benzylserine. Racemic [¹⁸F]9 was prepared from the labeling precursor in two steps using standard[¹⁸F]fluoride nucleophilic reaction conditions followed by acidic deprotection. The individual enantiomers [¹⁸F]9a and [¹⁸F]9b were isolated using preparative chiral HPLC. In vitro uptake inhibition assays were performed with each enantiomer using DBT cells. Biodistribution and microPET/CT studies were performed with each enantiomer in male BALB/c mice at approximately 2 weeks after implantation of DBT tumor cells.ResultsRadiolabeling of the cyclic sulfamidate precursor 5 provides racemic [¹⁸F]9 in high radiochemical yield (60%–70%, n = 4) and high radiochemical purity (> 96%, n = 4). In vitro uptake assays demonstrate that both [¹⁸F]9a and [¹⁸F]9b undergo tumor cell uptake through system L transport. The biodistribution studies using the single enantiomers [¹⁸F]9a and [¹⁸F]9b demonstrated good tumor uptake with lower uptake in most normal tissues, and [¹⁸F]9a had higher tumor uptake than [¹⁸F]9b. MicroPET imaging demonstrated good tumor visualization within 10 min of injection, rapid uptake of radioactivity, and tumor to brain ratios of approximately 6:1 at 60 min postinjection.ConclusionsThe novel PET tracer, [¹⁸F]FMePhe, is readily synthesized in good yield from a cyclic sulfamidate precursor. Biodistribution and microPET studies in the DBT model demonstrate good tumor to tissue ratios and tumor visualization, with enantiomer [¹⁸F]9a having higher tumor uptake. However, the brain availability of both enantiomers was lower than expected for system L substrates, suggesting the [¹⁸F]fluorine group in the β-position affects uptake of these compounds by system L transporters.     

Preliminary evaluation of 1′-[18F]fluoroethyl-β-D-lactose ([18F]FEL) for detection of pancreatic cancer in nude mouse orthotopic xenografts

IntroductionEarly detection of pancreatic cancer could save many thousands of lives. Non-invasive diagnostic imaging, including PET with [¹⁸F]FDG, has inadequate resolution for detection of small (2–3 mm) pancreatic tumours. We demonstrated the efficacy of PET imaging with an ¹⁸F-labelled lactose derivative, [¹⁸F]FEDL, that targets HIP/PAP, a biomarker that is overexpressed in the peritumoural pancreas. We developed another analogue, 1-[¹⁸F]fluoroethyl lactose ([¹⁸F]FEL), which is simpler to synthesise, for the same application. We conducted a preliminary evaluation of the new probe and its efficacy in detecting orthotopic pancreatic carcinoma xenografts in mice.MethodsXenografts were developed in nude mice by injecting L3.6pl/GL⁺ pancreatic carcinoma cells into the pancreas of each mouse. Tumour growth was monitored by bioluminescence imaging (BLI); accuracy of BLI tumour size estimates was verified by MRI in two representative mice. When the tumour size reached approximately 2–3 mm, the animals were injected with [¹⁸F]FEL (3.7 MBq) and underwent static PET/CT scans. Blood samples were collected at 2, 5, 10, 20 and 60 min after [¹⁸F]FEL injection to track blood clearance. Following imaging, animals were sacrificed and their organs and tumours/pancreatic tissue were collected and counted on a gamma counter. Pancreas, including tumour, was frozen, sliced and used for autoradiography and immunohistochemical analysis of HIP/PAP expression.ResultsTumour growth was rapid, as observed by BLI and MRI. Blood clearance of [¹⁸F]FEL was bi-exponential, with half-lives of approximately 3.5 min and 40 min. Mean accumulation of [¹⁸F]FEL in the peritumoural pancreatic tissue was 1.29 ± 0.295 %ID/g, and that in the normal pancreas of control animals was 0.090 ± 0.101 %ID/g. [¹⁸F]FEL was cleared predominantly by the kidneys. Comparative analysis of autoradiographic images and immunostaining results demonstrated a correlation between [¹⁸F]FEL binding and HIP/PAP expression.Conclusion[¹⁸F]FEL may be useful for non-invasive imaging of early-stage pancreatic tumours by PET. The results warrant further studies.     

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.     

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.     

[18 F]-(fluoromethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)propan-2-ol ([18 F FPTC) a novel PET-ligand for cerebral beta-adrenoceptors

Cerebral β‐adrenergic receptors (β‐ARs) play important roles in normal brain and changes of β-AR expression are associated with several neuropsychiatric illnesses. Given the high density of β‐AR in several brain regions, quantification of β‐AR levels using PET is feasible. However, there is a lack of radiotracers with suitable biological properties and meeting safety requirements for use in humans. We developed a PET tracer for β‐AR by ¹⁸ F‐fluorination of 1-((9H-carbazol-4-yl)oxy)-3-4(4-((2-(2-(fluoromethoxy)-ethoxy)methyl)-1H-1,2,3-triazol-1-yl)propan-2-ol (¹⁸ F-FPTC).Methods[¹⁸ F] FPTC was synthesized by Cu(I)-catalyzed alkyne-azide cycloaddition. First, ¹⁸ F‐PEGylated alkyne was prepared by ¹⁸ F‐fluorination of the corresponding tosylate. Next ¹⁸ F‐PEGylated alkyne was reacted with an azidoalcohol derivative of 4‐hydroxycarbazol in the presence of the phosphoramidite Monophos as a ligand and Cu(I) as a catalyst. After purification with radio‐HPLC, the binding properties of [¹⁸ F FPTC were tested in β‐AR‐expressing C6‐glioma cells in vitro and in Wistar rats in vivo using microPET.ResultsThe radiochemical yield of ¹⁸ F‐PEGylated alkyne was 74%–89%. The click reaction to prepare [¹⁸ F]FPTC proceeded in 10 min with a conversion efficiency of 96%. The total synthesis time was 55 min from the end of bombardment. Specific activities were > 120 GBq/μmol. Propranolol strongly and dose-dependently inhibited the binding of both [¹²⁵I]-ICYP and [¹⁸ F]FPTC to C6 glioma cells, with IC₅₀ values in the 50–60 nM range. However, although both FPTC and propranolol inhibited cellular [¹²⁵I]ICYP binding, FPTC decreased [¹²⁵I]ICYP uptake by only 25%, whereas propranolol reduced it by 83%. [¹⁸ F]FPTC has the appropriate lipophilicity to penetrate the blood brain barrier (logP + 2.48). The brain uptake reached a maximum within 2 min after injection of 20–25 MBq [¹⁸ F]FPTC. SUV values ranged from 0.4 to 0.6 and were not reduced by propranolol. Cerebral distribution volume of the tracer (calculated from a Logan plot) was increased rather than decreased after propranolol treatment.Conclusion‘Click chemistry’ was successfully applied to the synthesis of [¹⁸ F]FPTC resulting in high radiochemical yields. [¹⁸ F]FPTC showed specific binding in vitro, but not in vivo. Based on the logP value and its ability to block [¹²⁵I]ICYP binding to C6 cells, FPTC may be a lead to suitable cerebral β-AR ligands.     

Prognostic value of 3′-Deoxy-3′-18F-Fluorothymidine ([18F] FLT PET) in patients with recurrent malignant gliomas

Introduction3′-deoxy-3′-18F-fluorothymidine ([¹⁸F] FLT) PET has been proven to be of value in diagnosis and assessment of glioma grading, in differentiating tumor recurrence from necrosis, in response assessment and in predicting overall survival (OS) in the primary high grade glioma. In this study, we evaluated the value of [¹⁸F] FLT PET-CT in predicting the OS of patients with recurrent malignant glioma.MethodsFifty-six patients with recurrent malignant glioma were enrolled in this prospective study. The PET-CT and contrast-enhanced MRI scans were performed in all patients. Tumor volume was determined from both PET image (proliferative volume, PV) and MRI image (Vol-MRI). Patients were followed up clinically until death. The likelihood of using PET-derived parameters of SUV_max, tumor-to-normal (T/N) ratio, and PV to predict the OS of patients were assessed in comparison with Vol-MRI and other clinical parameters.ResultThe follow up periods for all patients ranged from 1.5 to 35.6 months with median of 9.8 months. Univariate analysis showed that the following parameters were significantly correlated with OS: grade of primary tumor (p = 0.042), Karnofsky performance score (KPS) (p = 0.041), T/N ratio (p < 0.01), Vol-MRI (p = 0.041), and PV (p < 0.001). However, multivariate Cox regression showed that only the PV (p < 0.001) and T/N ratio (p = 0.001) were independent predictors. The thresholds to predict OS were 16.88 cm³ for PV and 10.94 for T/N ratio. Kaplan–Meier analyses using these thresholds showed a significant discrimination between short and long OS groups (p < 0.001).ConclusionThe PV and T/N ratio of tumor on [¹⁸F] FLT PET-CT are independent predictors of survival in patients with recurrent malignant glioma. The PV on [¹⁸F] FLT PET seems to be more predictive than tumor volume on T1-weighted MRI for OS.     

[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.     

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.     

Kinetic analysis of FDG in rat liver: Effect of dietary intervention on arterial and portal vein input

IntroductionDietary conditions may affect liver [¹⁸F]FDG kinetics due to arterial and portal vein (PV) input. The purpose of this study was to evaluate kinetic models of [¹⁸F]FDG metabolism under a wide range of dietary interventions taking into account variations in arterial (HA) and portal vein (PV) input.MethodsThe study consisted of three groups of rats maintained under different diet interventions: 12 h fasted, 24 h fasted and those fed with high fructose diet. [¹⁵O]H₂O PET imaging was used to characterize liver flow contribution from HA and PV to the liver's dual input function (DIF). [¹⁸F]FDG PET imaging was used to characterize liver metabolism. Differences in [¹⁸F]FDG kinetics in HA, PV and liver under different diet interventions were investigated. An arterial to PV Transfer Function (TF) was optimized in all three dietary states to noninvasively estimate PV activity. Finally, two compartment 3-parameter (2C3P), two compartment 4-parameter (2C4P), two compartment 5-parameter (2C5P), and three compartment 5-parameter (3C5P) models were evaluated and compared to describe the kinetics of [¹⁸F]FDG in the liver across diet interventions. Sensitivity of the compartmental models to ratios of HA to PV flow fractions was further investigated.ResultsDifferences were found in HA and PV [¹⁸F]FDG kinetics across 12 h fasted, 24 h fasted and high fructose fed diet interventions. A two exponential TF model was able to estimate portal activity in all the three diet interventions. Statistical analysis suggests that a 2C3P model configuration was adequate to describe the kinetics of [¹⁸F]FDG in the liver under wide ranging dietary interventions. The net influx of [¹⁸F]FDG was lowest in the 12 h fasted group, followed by 24 h fasted group, and high fructose diet.ConclusionsA TF was optimized to non-invasively estimate PV time activity curve in different dietary states. Several kinetic models were assessed and a 2C3P model was sufficient to describe [¹⁸F]FDG liver kinetics despite differences in HA and PV kinetics across wide ranging dietary interventions. The observations have broader implications for the quantification of liver metabolism in metabolic disorders and cancer, among others.     

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.     

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.     

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.