Imaging Tumour ATB0,+ Transport Activity by PET with the Cationic Amino Acid O -2((2-[18F]fluoroethyl)methyl-amino)ethyltyrosine

Purpose: The concentrative amino acid transporter ATB0,+ (SLC6A14) is under evaluation as a target for anticancer therapy. An ATB0,+-selective positron emission tomography (PET) probe could advance preclinical drug development. We characterised the cationic tyrosine analogue O-2((2-[18F]fluoroethyl)methyl-amino)ethyltyrosine ([18F]FEMAET) as a PET probe for ATB0,+ activity. Procedures: Cell uptake was studied in vitro. ATB0,+ expression was quantified by real-time PCR. [18F]FEMAET accumulation in xenografts was investigated by small animal PET with mice. Results: [18F]FEMAET accumulated in PC-3 and NCI-H69 cancer cells in vitro. As expected for ATB0,+ transport, uptake was inhibited by LAT/ATB0,+ inhibitors and dibasic amino acids, and [18F]FEMAET efflux was only moderately stimulated by extracellular amino acids. ATB0,+ was expressed in PC-3 and NCI-H69 but not MDA-MB-231 xenografts. PET revealed accumulation in PC-3 and NCI-H69 xenografts and significant reduction by ATB0,+ inhibition. Uptake was negligible in MDA-MB-231 xenografts. Conclusion: ATB0,+ activity can be imaged in vivo by PET with [18F]FEMAET

Synthesis and preliminary biological evaluation of O -2((2-[18F]fluoroethyl)methylamino)ethyltyrosine ([18F]FEMAET) as a potential cationic amino acid PET tracer for tumor imaging

Amino acid transport is an attractive target for oncologic imaging. Despite a high demand of cancer cells for cationic amino acids, their potential as PET probes remains unexplored. Arginine, in particular, is involved in a number of biosynthetic pathways that significantly influence carcinogenesis and tumor biology. Cationic amino acids are transported by several cationic transport systems including, ATB0,+ (SLC6A14), which is upregulated in certain human cancers including cervical, colorectal and estrogen receptor-positive breast cancer. In this work, we report the synthesis and preliminary biological evaluation of a new cationic analog of the clinically used PET tumor imaging agent O-(2-[18F]fluroethyl)-l-tyrosine ([18F]FET), namely O-2((2-[18F]fluoroethyl)methylamino)ethyltyrosine ([18F]FEMAET). Reference compound and precursor were prepared by multi-step approaches. Radiosynthesis was achieved by no-carrier-added nucleophilic [18F]fluorination in 16-20% decay-corrected yields with radiochemical purity >99%. The new tracer showed good stability in vitro and in vivo. Cell uptake assays demonstrated that FEMAET and [18F]FEMAET accumulate in prostate cancer (PC-3) and small cell lung cancer cells (NCI-H69), with an energy-dependent mechanism. Small animal PET imaging with NCI-H69 xenograft-bearing mice revealed good tumor visualization comparable to [18F]FET and low brain uptake, indicating negligible transport across the blood-brain barrier. In conclusion, the non-natural cationic amino acid PET probe [18F]FEMAET accumulates in cancer cells in vitro and in vivo with possible involvement of ATB0,+

Whole-body and adipose tissue-specific mechanisms underlying the metabolic effects of fibroblast growth factor 21 in the Siberian hamster.

OBJECTIVE: Fibroblast growth factor 21 (FGF21) has been shown to rapidly lower body weight in the Siberian hamster, a preclinical model of adiposity. This induced negative energy balance mediated by FGF21 is associated with both lowered caloric intake and increased energy expenditure. Previous research demonstrated that adipose tissue (AT) is one of the primary sites of FGF21 action and may be responsible for its ability to increase the whole-body metabolic rate. The present study sought to determine the relative importance of white (subcutaneous AT [sWAT] and visceral AT [vWAT]), and brown (interscapular brown AT [iBAT]) in governing FGF21-mediated metabolic improvements using the tissue-specific uptake of glucose and lipids as a proxy for metabolic activity. METHODS: We used positron emission tomography-computed tomography (PET-CT) imaging in combination with both glucose (18F-fluorodeoxyglucose) and lipid (18F-4-thiapalmitate) tracers to assess the effect of FGF21 on the tissue-specific uptake of these metabolites and compared responses to a control group pair-fed to match the food intake of the FGF21-treated group. In vivo imaging was combined with ex vivo tissue-specific functional, biochemical, and molecular analyses of the nutrient uptake and signaling pathways. RESULTS: Consistent with previous findings, FGF21 reduced body weight via reduced caloric intake and increased energy expenditure in the Siberian hamster. PET-CT studies demonstrated that FGF21 increased the uptake of glucose in BAT and WAT independently of reduced food intake and body weight as demonstrated by imaging of the pair-fed group. Furthermore, FGF21 increased glucose uptake in the primary adipocytes, confirming that these in vivo effects may be due to a direct action of FGF21 at the level of the adipocytes. Mechanistically, the effects of FGF21 are associated with activation of the ERK signaling pathway and upregulation of GLUT4 protein content in all fat depots. In response to treatment with FGF21, we observed an increase in the markers of lipolysis and lipogenesis in both the subcutaneous and visceral WAT depots. In contrast, FGF21 was only able to directly increase the uptake of lipid into BAT. CONCLUSIONS: These data identify brown and white fat depots as primary peripheral sites of action of FGF21 in promoting glucose uptake and also indicate that FGF21 selectively stimulates lipid uptake in brown fat, which may fuel thermogenesis

In vivo imaging of the 1A receptors of serotonin (5-HT1A) in the brain with compounds of Tc-99m

5-Hydroxytryptamine (5-HT) is involved in the pathogenesis and treatment of a number of CNS disorders, Today, several types and subtypes of 5-HT receptors have been identified and the in vivo imaging of these receptors is a promising research area for the investigation of the distribution, function and role of the 5-HT receptors in the CNS pathophysiology. 5-HT1A in particular, is a receptor subtype directly involved in major CNS disorders like depression schizophrenia and anxiety and despite the great variety of 5-HT1A radioligands for PET imaging there is still a scope for the development of Tc-labelled 5-HT1A ligands for SPECT imaging mainly because technetium’s optimal nuclear properties and readily availability. To this direction, the introduction of the versatile precursor [M(CO)3]+, M= Tc/Re for the labeling of biomolecules gave a new thrust for radiopharmaceutical development. In this study, we report the design, synthesis, characterization and pharmacological evaluation of novel rhenium/technetium at the oxidation state +I. These complexes are neutral, lipophilic and carry the 2-methoxyphenyl piperazine moiety, a fragment of the true 5-HT1A antagonist WAY 100635 in order to possess all the theoretical requirements to bind in vivo with the 5-HT1A serotonin receptors in the brain. For the design of the Re(I) complexes, the tricarbonyl core M(CO)3, M= Tc/Re was chosen as the metal center stabilized by ligands that are either monoamide derivatives of picollinamino N,N diacetic acid (PADA) or cysteine S-thioethers derivatives. For both type of ligands it has been established that they bind very efficiently with the [M(CO)3]+ organometallic core via their [NNO] or [SNO] donor atom set respectively. For the synthesis of the PADA ligands, a convenient, one pot synthetic method was found for their preparation in high yields that includes the formation of the PADA anhydride and its subsequent reaction with the appropriate amine for the formation of the amide. Due to its sensitivity, the PADA anhydride was not isolated and the final products were synthesized by reacting equimolar quantities of the corresponding amine in the PADA anhydride solution. The novel reaction and the coordination chemistry of the complexes was studied using benzylamine, pyrrolidine and aniline as model compound for primary and secondary aliphatic amines and aromatic amines respectively, leading to ligand L1H -L3H and the corresponding complexes 1-3 whose structure was elucidated with NMR analysis and X-ray crystallography. This synthetic method simplifies to some extent the incorporation of a biological active molecule which contains an amine group to the NNO donor atom system of PADA ligand providing thus an easy way for labelling a pharmacophore moiety with technetium or rhenium. Subsequently the pharmacophore structure of 2-methoxyphenylpiperazine was incorporated in the PADA molecule yielding ligands L4-L5H. The [SNO] ligands L6H-L8H were prepared under under Schauten - Bauman conditions [2] by reacting the appropriate S-amino acid with the bromide 4. All ligands were characterized with NMR and IR spectroscopy. ...................................................................................................................Η σύγχρονη έρευνα στον τομέα των ραδιοφαρμάκων του Tc/Re στοχεύει στην επισήμανση βιολογικά δραστικών ενώσεων με τα στοιχεία αυτά με σκοπό την ανάπτυξη εξειδικευμένων ραδιοφαρμάκων προς ένα συγκεκριμένο βιολογικό στόχο. Οι υποδοχείς 5-ΗΤ1Α στο ΚΝΣ παρουσιάζουν ιδιαίτερο ενδιαφέρον γιατί μεταβολές στη συγκέντρωση και τη λειτουργικότητά τους φαίνεται να εμπλέκονται σε νευρολογικές και ψυχολογικές διαταραχές όπως κατάθλιψη, άγχος, σχιζοφρένεια, νόσο του Alzheimer και βουλιμία. Επομένως η απεικόνιση των υποδοχέων αυτών in vivo θα μπορούσε να δώσει πληροφορίες για τον τρόπο που αυτοί μεταβάλλονται σε παθολογικές καταστάσεις και να αποτελέσει μέσο για την έγκαιρη διάγνωσή τους με την ευρέως χρησιμοποιούμενη τεχνολογία SPECT. Κύριος στόχος της παρούσας εργασίας ήταν ο σχεδιασμός, η σύνθεση, ο, χαρακτηρισμός και η φαρμακολογική αξιολόγηση νέων ενώσεων του ρηνίου και τεχνητίου με κατάλληλες ιδιότητες για την in vivo απεικόνιση των υποδοχέων 5-ΗΤ1Α στον εγκέφαλο. Προς αυτόν το σκοπό χρησιμοποιήθηκε ο τρικαρβόνυλο πυρήνας των μετάλλων [Μ(CO)3+, M = Tc/Re] λόγω των ιδανικών φυσικοχημικών του χαρακτηριστικών και στα σύμπλοκα ενσωματώθηκε με κατάλληλο τρόπο η φαρμακοφόρος δομή της 1-(2-μεθοξυφαινυλο)-πιπεραζίνης που αποτελεί τμήμα του «πραγματικού» ανταγωνιστή των υποδοχέων 5-ΗΤ1Α της σεροτονίνης, WAY 100635, η οποία αποτελεί την απαραίτητη δομική μονάδα για την εμφάνιση συγγένειας για τους συγκεκριμένους υποδοχείς. Για τον σχεδιασμό των συμπλόκων επιλέχτηκε το μεταλλικό κέντρο να σταθεροποιείται από συναρμοτές που να είναι είτε μονοάμιδο παράγωγα του πυριδινυλο-2-μεθυλαμινο-Ν-Ν-διοξικού οξέος (PADA, σύστημα από άτομα δότες [ΝΝΟ]) είτε θειαιθέρες θειούχων αμινοξέων (σύστημα από άτομα δότες [SNO]) για τα οποία δύο συστήματα έχει βρεθεί ότι συναρμόζονται πολύ καλά με το συγκεκριμένο πυρήνα δίνοντας ουδέτερα και σταθερά σύμπλοκα. Για τη παρασκευή των συναρμοτών - παράγωγα του PADA ευρέθη εύχρηστο συνθετικό σχήμα μέσω του ανυδρίτη του PADA με επίδραση σε αυτόν ισομοριακής ποσότητας της αντίστοιχης αμίνης. Δοκιμάστηκαν καταρχήν οι απλές αμίνες βενζυλαμίνη, πυρρολιδίνη και ανιλίνη ως πρότυπες ενώσεις για αλειφατικές και αρωματικές αμίνες αντίστοιχα οπότε και ελήφθηκαν οι ζητούμενοι συναρμοτές. Η μέθοδος αυτή αποτελεί ουσιαστικά τρόπο ενσωμάτωσης οποιασδήποτε βιολογικά δραστικής αμίνης στο μόριο του PADA και άρα επισήμανσή της με Tc-99m. Οι συναρμοτές πρότυπα χρησιμοποιήθηκαν για την σύνθεση των αντίστοιχων συμπλόκων με ρήνιο προκειμένου να διερευνηθεί η χημεία συναρμογής οπότε και διαπιστώθηκε ότι τα σύμποκα που σχηματίζονται είναι όντως του τύπου [Re(CO)3(NNO)] όπως συμβαίνει και με το πρότυπο μόριο του PADA. ..........................................................................................................................................

Imaging tumour ATB0,+ transport activity by PET with the cationic amino acid O-2((2-[18F]fluoroethyl)methyl-amino)ethyltyrosine

PURPOSE The concentrative amino acid transporter ATB(0,+) (SLC6A14) is under evaluation as a target for anticancer therapy. An ATB(0,+)-selective positron emission tomography (PET) probe could advance preclinical drug development. We characterised the cationic tyrosine analogue O-2((2-[(18)F]fluoroethyl)methyl-amino)ethyltyrosine ([(18)F]FEMAET) as a PET probe for ATB(0,+) activity. PROCEDURES Cell uptake was studied in vitro. ATB(0,+) expression was quantified by real-time PCR. [(18)F]FEMAET accumulation in xenografts was investigated by small animal PET with mice. RESULTS [(18)F]FEMAET accumulated in PC-3 and NCI-H69 cancer cells in vitro. As expected for ATB(0,+) transport, uptake was inhibited by LAT/ATB(0,+) inhibitors and dibasic amino acids, and [(18)F]FEMAET efflux was only moderately stimulated by extracellular amino acids. ATB(0,+) was expressed in PC-3 and NCI-H69 but not MDA-MB-231 xenografts. PET revealed accumulation in PC-3 and NCI-H69 xenografts and significant reduction by ATB(0,+) inhibition. Uptake was negligible in MDA-MB-231 xenografts. CONCLUSION ATB(0,+) activity can be imaged in vivo by PET with [(18)F]FEMAET

Imaging Tumour ATB0,+ Transport Activity by PET with the Cationic Amino Acid O-2((2-[18F]fluoroethyl)methyl-amino)ethyltyrosine

Purpose The concentrative amino acid transporter ATB0,+ (SLC6A14) is under evaluation as a target for anticancer therapy. An ATB0,+-selective positron emission tomography (PET) probe could advance preclinical drug development. We characterised the cationic tyrosine analogue O-2((2-[18F]fluoroethyl)methyl-amino)ethyltyrosine ([18F]FEMAET) as a PET probe for ATB0,+ activity. Procedures Cell uptake was studied in vitro. ATB0,+ expression was quantified by real-time PCR. [18F]FEMAET accumulation in xenografts was investigated by small animal PET with mice. Results [18F]FEMAET accumulated in PC-3 and NCI-H69 cancer cells in vitro. As expected for ATB0,+ transport, uptake was inhibited by LAT/ATB0,+ inhibitors and dibasic amino acids, and [18F]FEMAET efflux was only moderately stimulated by extracellular amino acids. ATB0,+ was expressed in PC-3 and NCI-H69 but not MDA-MB-231 xenografts. PET revealed accumulation in PC-3 and NCI-H69 xenografts and significant reduction by ATB0,+ inhibition. Uptake was negligible in MDA-MB-231 xenografts. Conclusion ATB0,+ activity can be imaged in vivo by PET with [18F]FEMAET.ISSN:1860-2002ISSN:1536-163

Synthesis and biological evaluation of (18)F-labeled Fluoroethoxy tryptophan analogues as potential PET tumor imaging agents

As a continuation of our research efforts toward the development of tryptophan-based radiotracers for tumor imaging with positron emission tomography (PET), three new fluoroethoxy tryptophan analogues were synthesized and evaluated in vivo. These new tracers (namely, 4-(2-[(18)F]fluoroethoxy)-dl-tryptophan ([(18)F]4-FEHTP), 6-(2-[(18)F]fluoroethoxy)-dl-tryptophan ([(18)F]6-FEHTP), and 7-(2-[(18)F]fluoroethoxy)-dl-tryptophan ([(18)F]7-FEHTP) carry the fluoroethoxy side chain either at positions 4-, 6-, or 7- of the indole core. Reference compounds and precursors were synthesized by multistep approaches. Radiosynthesis was accomplished by no-carrier-added nucleophilic (18)F-fluorination following either an indirect approach (O-alkylation of the corresponding hydroxytryptophan with [(18)F]fluoroethyltosylate) or a direct approach (nucleophilic [(18)F] fluorination using a protected mesyl precursor). Radiochemical yields (decay corrected) for both methods were in the range of 10-18%. Small animal PET imaging with xenograft-bearing mice revealed the highest tumor/background ratio for [(18)F]6-FEHTP which, in a direct comparison, outperformed the other two tryptophan tracers and also the well-established tyrosine analogue O-(2-[(18)F]fluoroethyl)-l-tyrosine ([(18)F]l-FET). Investigation of the transport mechanism of [(18)F]6-FEHTP in small cell lung cancer cells (NCI-H69) revealed that it is most probably taken up exclusively via the large neutral amino acid transporter(s) (LAT)

Synthesis and Biological Evaluation of Quinoxaline Derivatives for PET Imaging of the NMDA Receptor

Due to the biological complexity of the N-methyl-d-aspartate receptor (NMDAR), the development of a positron emission tomography radiotracer for the imaging of NMDAR has met with limited success. Recent studies have established the presence of GluN2A subunit of the NMDAR in the heart and its role in the regulation of intracellular calcium levels. In our efforts to develop an imaging agent for the GluN2A subunit, we designed three new compounds based on a quinoxaline scaffold. The synthesis of the analogues was based on a two-step Kabachnik–Fields reaction in sequence with Suzuki cross-coupling and acid hydrolysis. They exhibited comparable high binding affinity values below 5 nm. A two-step radiolabeling procedure was successfully developed for the synthesis of [18F]1. [18F]1 was obtained in a modest overall radiochemical yield of 5.5 ± 4.2%, a good specific radioactivity of 254 ± 158 GBq/μmol, and a radiochemical purity > 99%. While compounds 2 and 3 showed comparable binding affinity towards NMDAR, sluggish radiolabeling, prevented their further evaluation. For [18F]1, in vitro autoradiography on rat heart slices demonstrated heterogeneous but unspecific accumulation, whereas for the brain a high in vitro specificity towards NMDAR, could be demonstrated

Synthesis and biological evaluation of (18)F-labeled fluoropropyl tryptophan analogs as potential PET probes for tumor imaging

In the search for an efficient, fluorine-18 labeled amino acid based radiotracer for tumor imaging with positron emission tomography (PET), two new tryptophan analogs were synthesized and characterized in vitro and in vivo. Both are tryptophan alkyl-derivatives, namely 2-(3-[(18)F]fluoropropyl)-dl-tryptophan ([(18)F]2-FPTRP) and 5-(3-[(18)F]fluoro-propyl)-dl-tryptophan ([(18)F]5-FPTRP). Standard reference compounds and precursors were prepared by multi step approaches. Radiosynthesis was achieved by no-carrier-added nucleophilic [(18)F]fluorination in 29-34% decay corrected yields with radiochemical purity over 99%. In vitro cell uptake assays showed that both compounds are substrates for amino acid transport and enter small cell lung cancer cells (NCI-H69) most probably almost exclusively via large neutral amino acids transporter(s) (LAT). Small animal PET imaging with xenograft bearing mice revealed high tumor/background ratios for [(18)F]2-FPTRP comparable to the well established tyrosine analog O-(2-[(18)F]fluroethyl)-l-tyrosine ([(18)F]FET). Radiometabolite studies showed no evidence of involvement of a biotransformation step in tumor accumulation

Synthesis and preliminary biological evaluation of O-2((2-[(18)F]fluoroethyl)methylamino)ethyltyrosine ([(18)F]FEMAET) as a potential cationic amino acid PET tracer for tumor imaging

Amino acid transport is an attractive target for oncologic imaging. Despite a high demand of cancer cells for cationic amino acids, their potential as PET probes remains unexplored. Arginine, in particular, is involved in a number of biosynthetic pathways that significantly influence carcinogenesis and tumor biology. Cationic amino acids are transported by several cationic transport systems including, ATB(0,+) (SLC6A14), which is upregulated in certain human cancers including cervical, colorectal and estrogen receptor-positive breast cancer. In this work, we report the synthesis and preliminary biological evaluation of a new cationic analog of the clinically used PET tumor imaging agent O-(2-[(18)F]fluroethyl)-L-tyrosine ([(18)F]FET), namely O-2((2-[(18)F]fluoroethyl)methylamino)ethyltyrosine ([(18)F]FEMAET). Reference compound and precursor were prepared by multi-step approaches. Radiosynthesis was achieved by no-carrier-added nucleophilic [(18)F]fluorination in 16-20% decay-corrected yields with radiochemical purity >99%. The new tracer showed good stability in vitro and in vivo. Cell uptake assays demonstrated that FEMAET and [(18)F]FEMAET accumulate in prostate cancer (PC-3) and small cell lung cancer cells (NCI-H69), with an energy-dependent mechanism. Small animal PET imaging with NCI-H69 xenograft-bearing mice revealed good tumor visualization comparable to [(18)F]FET and low brain uptake, indicating negligible transport across the blood-brain barrier. In conclusion, the non-natural cationic amino acid PET probe [(18)F]FEMAET accumulates in cancer cells in vitro and in vivo with possible involvement of ATB(0,+)