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The fac-[M(CO)3(PyA)(P)] and cis-trans-[M(CO)2(PyA)(P)2] neutral complexes (M is Re or 99mTc), based on the mixed ligand strategy with pyrazine-2-carboxylic acid (PyAH) as the bidentate N,O and triphenylphosphine as the monodentate P ligand, are presented. Through the employment of the anhydride of pyrazine-2,3-dicarboxylic acid (PyDA), the PyAH scaffold was conveniently derivatized with the model bioactive amine 1-(2-methoxyphenyl)piperazine, the active part of the 5-HT1A antagonist WAY100635. Reaction of either PyAH or the pharmacophore-bearing PyAH ligand (L1H) with fac-[M(CO)3]+ core in water yielded the intermediate fac-[M(CO)3(PyA)(H2O)] complexes. The labile aqua ligand was easily replaced by PPh3 to yield the fac-[Re(CO)3(PyA)(PPh3)] complexes, while in toluene under reflux, the cis-trans-[Re(CO)2(PyA)(PPh3)2] complexes were obtained. The latter complexes were alternatively obtained from mer-[Re(CO)3(PPh3)2Cl] by refluxing with the PyA ligand in toluene. The analogous 99mTc complexes were synthesized quantitatively, showing excellent stability in competition studies. The methodology described herein represents a practical procedure for the effective integration of the fac-[M(CO)3]+ core with amine-bearing biologically active compounds for diagnosis/therapy.

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A new prosthetic group is reported for 18F-labelling of peptides and proteins based on the chemoselective ligation of potassium acyltrifluoroborates (KATs) and hydroxylamines without any detectable 18F/19F isotope exchange at the acyltrifluoroborate moiety. The new building block is appended via a common amide bond at room temperature with no need for protecting groups which enables an effective orthogonal 18F-radiolabelling.

A new prosthetic group is reported for 18F-labelling of peptides and proteins based on the chemoselective ligation of potassium acyltrifluoroborates (KATs) and hydroxylamines without any detectable 18F/19F isotope exchange at the acyltrifluoroborate moiety. The new building block is appended via a common amide bond at room temperature with no need for protecting groups which enables an effective orthogonal 18F-radiolabelling.

The costimulatory molecule CD80 is an early marker for immune activation. It is upregulated on activated antigen-presenting cells. We aimed at developing a tracer for imaging CD80 by positron emission tomography (PET). Novel CD80 ligands were synthesized and tested by SPR for affinity to human CD80 (hCD80) and displacement of endogenous ligands. Several compounds bound with one-digit nanomolar affinity to hCD80 and displaced CTLA-4 and CD28 at nanomolar concentrations. A structure-affinity relationship study revealed relevant moieties for strong affinity to hCD80 and positions for further modifications. Lead compound MT107 (7f) was radiolabeled with carbon-11. In vitro, [11C]MT107 showed specific binding to hCD80-positive tissue and high plasma protein binding. In vivo, [11C]MT107 accumulated in liver, gall bladder, and intestines but only scarcely in hCD80-positive xenografts. The unfavorable in vivo performance may result from high plasma protein binding and extensive biliary excretion.

A shear stress-induced atherosclerosis mouse model was characterized for its expression of inflammation markers with focus on CD80. With this model, we evaluated two positron emission tomography (PET) radiotracers targeting CD80 as well as 2-deoxy-2-[18F]fluoro-d-mannose ([18F]FDM) in comparison with 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG). A flow constrictive cuff implanted around the common carotid artery in apolipoprotein E knockout mice resulted in plaque formation. CD80 expression levels and plaque histopathology were evaluated. Serial PET/X-ray computed tomography scans were performed to follow inflammation. Plaque formation with increased levels of CD80 was observed. Histologically, plaques presented macrophage-rich and large necrotic areas covered by a thin fibrous cap. Of the CD80-specific tracers, one displayed an increased uptake in plaques by PET. Both [18F]FDG and [18F]FDM accumulated in atherosclerotic plaques. This mouse model presented, similar to humans, an increased expression of CD80 which renders it suitable for non-invasively targeting CD80-positive immune cells and evaluating CD80-specific radiotracers.

Research towards the non-invasive imaging of atherosclerotic plaques is of high clinical priority as early recognition of vulnerable plaques may reduce the incidence of cardiovascular events. The fibroblast activation protein alpha (FAP) was recently proposed as inflammation-induced protease involved in the process of plaque vulnerability. In this study, FAP mRNA and protein levels were investigated by quantitative polymerase chain reaction and immunohistochemistry, respectively, in human endarterectomized carotid plaques. A published boronic-acid based FAP inhibitor, MIP-1232, was synthetized and radiolabeled with iodine-125. The potential of this radiotracer to image plaques was evaluated by in vitro autoradiography with human carotid plaques. Specificity was assessed with a xenograft with high and one with low FAP level, grown in mice. Target expression analyses revealed a moderately higher protein level in atherosclerotic plaques than normal arteries correlating with plaque vulnerability. No difference in expression was determined on mRNA level. The radiotracer was successfully produced and accumulated strongly in the FAP-positive SK-Mel-187 melanoma xenograft in vitro while accumulation was negligible in an NCI-H69 xenograft with low FAP levels. Binding of the tracer to endarterectomized tissue was similar in plaques and normal arteries, hampering its use for atherosclerosis imaging., Molecules, 20 (2), ISSN:1420-3049

Antibody-drug conjugates (ADCs) have demonstrated clinical benefits that have led to the recent FDA approval of KADCYLA and ADCETRIS. Most ADCs that are currently in clinical use or development, including ADCETRIS, are produced by chemical conjugation of a toxin via either lysine or cysteine residues, inevitably leading to heterogeneous products with variable drug-to-antibody ratios (DARs). Here, we describe the in vitro and in vivo characterization of four novel ADCs that are based on the anti-CD30 antibody cAC10, which has the same polypeptide backbone as ADCETRIS, and compare the results with the latter. Bacterial transglutaminase (BTG) was exploited to site-specifically conjugate derivatives of monomethyl auristatin E (all comprising a cleavable linker) to the glutamine at positions 295 and 297 of cAC10, thereby yielding homogeneous ADCs with a DAR of 4. In vitro cell toxicity experiments using two different CD30-positive cell lines (Karpas 299 and Raji-CD30(+)) revealed comparable EC50 values for ADCETRIS (1.8 ± 0.4 and 3.6 ± 0.6 ng/mL, respectively) and the four cAC10-based ADCs (2.0 ± 0.4 to 4.9 ± 1.0 ng/mL). Quantitative time-dependent in vivo biodistribution studies (3-96 h p.i.) in normal and xenografted (Karpas 299 cells) SCID mice were performed with a selected (125)I-radioiodinated cAC10 ADC and compared with that of (125)I-ADCETRIS. The chemo-enzymatically conjugated, radioiodinated ADC showed higher tumor uptake (17.84 ± 2.2% ID/g 24 h p.i.) than (125)I-ADCETRIS (10.5 ± 1.8% ID/g 24 h p.i.). Moreover, (125)I-ADCETRIS exhibited higher nontargeted liver and spleen uptake. In line with these results, the maximum tolerated dose of the BTG-coupled ADC (60 mg/kg) was significantly higher than that of ADCETRIS (18 mg/kg) in rats. These results suggest that homogeneous ADCs display improved pharmacokinetics and better therapeutic indexes compared to those of chemically modified ADCs with variable DARs.

Aiming at a convenient way toward the synthesis of terminal (fluoroalkyl)silanes, the effect of (diethylamino)sulfur trifluoride (DAST) on some terminal hydroxyalkylsilanes was investigated. Reaction of DAST with a substituted (hydroxyethyl)diphenylsilane led to the formation of the desired (2-fluoroethyl)diphenylsilane in considerable amounts although the competing elimination route was still the favored mechanism. This new reaction was studied under various parameters with the best conditions yielding the substitution product in a ratio of 1:4 over the undesired elimination product. In a different approach trying to optimize the outcome of the reaction, the presence of two bulky 2,4,6-trimethoxyphenyl (TMOP) groups on the silicon atom favored the elimination route.

Most chemical techniques used to produce antibody-drug conjugates (ADCs) result in a heterogeneous mixture of species with variable drug-to-antibody ratios (DAR) which will potentially display different pharmacokinetics, stability, and safety profiles. Here we investigated two strategies to obtain homogeneous ADCs based on site-specific modification of deglycosylated antibodies by microbial transglutaminase (MTGase), which forms isopeptidic bonds between Gln and Lys residues. We have previously shown that MTGase solely recognizes Gln295 within the heavy chain of IgGs as a substrate and can therefore be exploited to generate ADCs with an exact DAR of 2. The first strategy included the direct, one-step attachment of the antimitotic toxin monomethyl auristatin E (MMAE) to the antibody via different spacer entities with a primary amine functionality that is recognized as a substrate by MTGase. The second strategy was a chemo-enzymatic, two-step approach whereby a reactive spacer entity comprising a bio-orthogonal thiol or azide function was attached to the antibody by MTGase and subsequently reacted with a suitable MMAE-derivative. To this aim, we investigated two different chemical approaches, namely, thiol-maleimide and strain-promoted azide-alkyne cycloaddition (SPAAC). Direct enzymatic attachment of MMAE-spacer derivatives at an 80 molar excess of drug yielded heterogeneous ADCs with a DAR of between 1.0 to 1.6. In contrast to this, the chemo-enzymatic approach only required a 2.5 molar excess of toxin to yield homogeneous ADCs with a DAR of 2.0 in the case of SPAAC and 1.8 for the thiol-maleimide approach. As a proof-of-concept, trastuzumab (Herceptin) was armed with the MMAE via the chemo-enzymatic approach using SPAAC and tested in vitro. Trastuzumab-MMAE efficiently killed BT-474 and SK-BR-3 cells with an IC50 of 89.0 pM and 21.7 pM, respectively. Thus, the chemo-enzymatic approach using MTGase is an elegant strategy to form ADCs with a defined DAR of 2. Furthermore, the approach is directly applicable to a broad variety of antibodies as it does not require prior genetic modifications of the antibody sequence.

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.

Aiming at developing mechanism-based amino acid (18)F-PET tracers for tumor imaging, we synthesized two (18)F-labeled analogues of 5-hydroxy-l-[β-(11)C]tryptophan ([(11)C]5HTP) whose excellent in vivo performance in neuroendocrine tumors is mainly attributed to its decarboxylation by aromatic amino acid decarboxylase (AADC), an enzyme overexpressed in these malignancies. Reference compounds and precursors were synthesized following multistep synthetic approaches. Radiosynthesis of tracers was accomplished in good radiochemical yields (15-39%), high specific activities (45-95 GBq/μmol), and excellent radiochemical purities. In vitro cell uptake was sodium-independent and was inhibited ≥95% by 2-amino-2-norbornanecarboxylic acid (BCH) and ∼30% by arginine. PET imaging in mice revealed distinctly high tumor/background ratios for both tracers, outperforming the well-established O-(2-[(18)F]fluoroethyl)tyrosine ([(18)F]FET) tracer in a head-to-head comparison. Biological evaluation revealed that the in vivo performance is most probably independent of any interaction with AADC. Nevertheless, the excellent tumor visualization qualifies the new tracers as interesting probes for tumor imaging worthy for further investigation.

We report a novel prosthetic group based on a heterocyclic methylsulfone derivative for the rapid, stable, and chemoselective (18)F-labeling of thiol-containing (bio)molecules under mild aqueous reaction conditions. Compared to established maleimide approaches, the new methodology displays some clear advantages for imaging probe development.

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.

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).

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., Molecular Imaging and Biology, 16 (3), ISSN:1860-2002, ISSN:1536-1632

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. ...........................................................................................................................................

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.

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,+., Amino acids, 46 (8), ISSN:0939-4451, ISSN:1438-2199