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UnlabelledRadiotracers based on the peptide A20FMDV2 selectively target the cell surface receptor integrin αvβ6. This integrin has been identified as a prognostic indicator correlating with the severity of disease for several challenging malignancies. In previous studies of A20FMDV2 peptides labeled with 4-(18)F-fluorobenzoic acid ((18)F-FBA), we have shown that the introduction of poly(ethylene glycol) (PEG) improves pharmacokinetics, including increased uptake in αvβ6-expressing tumors. The present study evaluated the effect of site-specific C-terminal or dual (N- and C-terminal) PEGylation, yielding (18)F-FBA-A20FMDV2-PEG28 (4) and (18)F-FBA-PEG28-A20FMDV2-PEG28 (5), on αvβ6-targeted tumor uptake and pharmacokinetics. The results are compared with (18)F-FBA -labeled A20FMDV2 radiotracers (1- 3) bearing either no PEG or different PEG units at the N terminus.MethodsThe radiotracers were prepared and radiolabeled on solid phase. Using 3 cell lines, DX3puroβ6 (αvβ6+), DX3puro (αvβ6-), and BxPC-3 (αvβ6+), we evaluated the radiotracers in vitro (serum stability; cell binding and internalization) and in vivo in mouse models bearing paired DX3puroβ6-DX3puro and, for 5, BxPC-3 xenografts.ResultsThe size and location of the PEG units significantly affected αvβ6 targeting and pharmacokinetics. Although the C-terminally PEGylated 4 showed some improvements over the un-PEGylated (18)F-FBA-A20FMDV2 (1), it was the bi-terminally PEGylated 5 that displayed the more favorable combination of high αvβ6 affinity, selectivity, and pharmacokinetic profile. In vitro, 5 bound to αvβ6-expressing DX3puroβ6 and BxPC-3 cells with 60.5% ± 3.3% and 48.8% ± 8.3%, respectively, with a significant fraction of internalization (37.2% ± 4.0% and 37.6% ± 4.1% of total radioactivity, respectively). By comparison, in the DX3puro control 5: showed only 3.0% ± 0.5% binding and 0.9% ± 0.2% internalization. In vivo, 5: maintained high, αvβ6-directed binding in the paired DX3puroβ6-DX3puro model (1 h: DX3puroβ6, 2.3 ± 0.2 percentage injected dose per gram [%ID/g]; DX3puroβ6/DX3puro ratio, 6.5:1; 4 h: 10.7:1). In the pancreatic BxPC-3 model, uptake was 4.7 ± 0.9 %ID/g (1 h) despite small tumor sizes (20-80 mg).ConclusionThe bi-PEGylated radiotracer 5 showed a greatly improved pharmacokinetic profile, beyond what was predicted from individual N- or C-terminal PEGylation. It appears that the 2 PEG units acted synergistically to result in an improved metabolic profile including high αvβ6+ tumor uptake and retention.

Introduction The gonadotropin releasing hormone receptor (GnRH-R) has a well-described neuroendocrine function in the anterior pituitary. However, little is known about its function in the central nervous system (CNS), where it is most abundantly expressed in hippocampus and amygdala. Since peptide ligands based upon the endogenous decapetide GnRH do not pass the blood–brain-barrier, we are seeking a high-affinity small molecule GnRH-R ligand suitable for brain imaging by positron emission tomography. We have previously reported the radiosynthesis and in vitro evaluation of two novel [ 18 F]fluorinated GnRH-R ligands belonging to the furamide class of antagonists, with molecular weight less than 500 Da. We now extend this work using palladium coupling for the synthesis of four novel radioligands, with putatively reduced polar surface area and hydrophilicity relative to the two previously described compounds, and report the uptake of these 18 F-labeled compounds in brain of living rats. Methods We synthesized reference standards of the small molecule GnRH-R antagonists as well as mesylate precursors for 18 F-labeling. The antagonists were tested for binding affinity for both human and rat GnRH-R. Serum and blood stability in vitro and in vivo were studied. Biodistribution and PET imaging studies were performed in male rats in order to assess brain penetration in vivo . Results A palladium coupling methodology served for the synthesis of four novel fluorinated furamide GnRH receptor antagonists with reduced heteroatomic count. Radioligand binding assays in vitro revealed subnanomolar affinity of the new fluorinated compounds for both human and rat GnRH-R. The 18 F-GnRH antagonists were synthesized from the corresponding mesylate precursors in 5–15% overall radiochemical yield. The radiolabeled compounds demonstrated good in vivo stability. PET imaging with the 18 F-radiotracers in naive rats showed good permeability into brain and rapid washout, but absence of discernible specific binding in vivo . Conclusions The novel small molecule 18 F-fluorinated GnRH-R antagonist compounds show high receptor affinity in vitro , and may prove useful for quantitative autoradiographic studies in vitro . The compounds were permeable to the blood–brain barrier, but nonetheless failed to reveal significant specific binding in brain of living rats. Nonetheless, our approach may serve as a foundation for designing PET ligands suitable to image the GnRH-R distribution in brain. [ABSTRACT FROM AUTHOR]

Catalyst-free click reactions are effective chemical tools for synthesis of radiometal-based radiopharmaceuticals offering advantages towards preparation of non-toxic agents with high specific activity. In the present study the radiotracer [Cu]DOTA-ADIBON-Ala-PEG-A20FMDV2, [Cu] 3, was synthesized for positron emission tomography imaging of integrin αβ expressing tumors via a strain-promoted click reaction using both a 'pre-click' and 'post-click' approaches. The radiotracer, prepared in >99 % radiochemical yield, was evaluated in vitro (64.6 ± 2.8 % binding to αβ-positive cells vs. <5 % to αβ-negative cells) and in vivo (αβ-positive tumor uptake: 1.52 ± 0.16 % ID/g, 24 h p.i.). While the high initial renal uptake (76.2 ± 10.7 % ID/g at 1 h p.i.) was comparable to a previously reported radiotracer, [Cu]DOTA-PEG-A20FMDV2, [Cu] 3 showed notably improved renal clearance (11.3 ± 2.5 % ID/g at 24 h p.i.). Thus, the introduction of a chelator-strained alkyne system resulted in improved pharmacokinetics for the present radiotracer, highlighting the attractive prospects of strain-promoted click-based preparations in the construction of radiometalated bioconjugates for targeted molecular imaging and therapy. [ABSTRACT FROM AUTHOR]

Introduction: Incorporation 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 αvβ6 integrin-targeted peptide (NOTA-PEG28-A20FMDV2) and its in vitro and in vivo evaluation. Methods: Aluminum [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 αvβ6(+) and αvβ6(−) cells) and in vivo (paired αvβ6(+) and αvβ6(−) xenograft mice: PET/CT, biodistribution, tumor autoradiography and metabolites). Results: The 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, αvβ6-targeted binding (αvβ6(+): 42.4±1.2% of total radioactivity, ratio (+)/(−) = 8.4/1) and internalization (αvβ6(+): 28.3±0.5% of total radioactivity, (+)/(−) = 11.7/1). In vivo, 2 maintained αvβ6-targeted binding (biodistribution; 1 h: αvβ6(+): 1.74±0.38% ID/g, (+)/(−) = 2.72/1; 4 h: αvβ6(+): 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). Conclusion: The Al[18F]/NOTA-based radiolabeling was rapid and efficient, and the radiotracer 2 showed good αvβ6-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. [ABSTRACT FROM AUTHOR]