Nadine Bauer

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

INTRODUCTION Click chemistry, particularly the Huisgen 1,3-dipolar cycloaddition of an alkyne with an azide, has quickly become popular for site-specific radiolabeling. Recently, strain-promoted click chemistries have been developed, eliminating the need for potentially toxic copper catalysts. This study presents radiolabeling of an α(v)β(6) integrin targeting peptide (A20FMDV2) via strain-promoted click using a fluorine-18 prosthetic group, and in vitro and in vivo evaluation. METHODS The radiotracer was prepared from and N(3)-PEG(7)-A20FMDV2 (ethanol; 10 min; 35-45 °C). HPLC-purified and formulated radiotracer 1 was evaluated in vitro by cell binding (DX3puroβ6, α(v)β(6)-positive; and DX3puro, α(v)β(6)-negative control) and serum stability, and in vivo using PET/CT imaging and biodistribution studies in mice. RESULTS The radiotracer 1 was readily prepared and purified (from 2: 40±4 min including HPLC, 11.9±3.2% decay corrected isolated radiochemical yield, >99% radiochemical purity, n=4) and displayed good stability (1 h: >99%, saline; 94.6%, serum). Strong α(v)β(6)-targeted binding was observed in vitro (DX3puroβ6 cells, 15 min: 43.2% binding, >6:1 for DX3puroβ6:DX3puro). In the mouse model DX3puroβ6-tumor binding was low (1 h: 0.47±0.28% ID/g, 4h: 0.14±0.09% ID/g) and clearing from the bloodstream was via the renal and hepatobiliary routes (urine: 167±84% ID/g at 1 h, 10.3±4.8% ID/g at 4 h; gall bladder: 95±33% ID/g at 1 h, 63±11% ID/g at 4 h). CONCLUSION Copper-free, strain-promoted click chemistry is an attractive, straightforward approach to radiolabeling. Although the [(18)F]FBA-C(6)-ADBIO-based prosthetic group did not interfere with α(v)β(6)-targeted binding in vitro, it did influence the pharmacokinetics, possibly due to its size and lipophilic nature.

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

INTRODUCTION Incorporation of fluorine-18 ((18)F) into radiotracers by capturing ionic [(18)F]-species can greatly accelerate and simplify radiolabeling for this important positron emission tomography (PET) radioisotope. Among the different strategies, the incorporation of aluminum [(18)F]fluoride (Al[(18)F](2+)) into NOTA chelators has recently emerged as a robust approach to peptide radiolabeling. This study presents Al[(18)F](2+)-radiolabeling of an α(v)β₆ integrin-targeted peptide (NOTA-PEG₂₈-A20FMDV2) and its in vitro and in vivo evaluation. METHODS Aluminum [(18)F]fluoride was prepared at r.t. from [(18)F]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[(18)F] NOTA-PEG₂₈-A20FMDV2 (2) was purified by HPLC, formulated in PBS and evaluated in vitro (stability; binding and internalization in α(v)β₆(+) and α(v)β₆(-) cells) and in vivo (paired α(v)β₆(+) and α(v)β₆(-) 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, α(v)β₆-targeted binding (α(v)β₆(+): 42.4 ± 1.2% of total radioactivity, ratio (+)/(-)=8.4/1) and internalization (α(v)β₆(+): 28.3 ± 0.5% of total radioactivity, (+)/(-)=11.7/1). In vivo, 2 maintained α(v)β₆-targeted binding (biodistribution; 1 h: α(v)β₆(+): 1.74 ± 0.38% ID/g, (+)/(-)=2.72/1; 4 h: α(v)β₆(+): 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[(18)F]/NOTA-based radiolabeling was rapid and efficient, and the radiotracer 2 showed good α(v)β₆-selectivity in vitro and in vivo. However, in contrast to A20FMDV2 labeled with covalently bound [(18)F]-prosthetic groups (e.g., [(18)F]fluorobenzoic acid), 2 demonstrated significant trapping in kidneys, similar to radiometal-labeled chelator-analogs of 2.

The Effect of Bi-Terminal PEGylation of an Integrin αvβ6–Targeted 18F Peptide on Pharmacokinetics and Tumor Uptake

Radiotracers 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 4-18F-fluorobenzoic acid (18F-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 18F-FBA-A20FMDV2-PEG28 (4) and 18F-FBA-PEG28-A20FMDV2-PEG28 (5), on αvβ6-targeted tumor uptake and pharmacokinetics. The results are compared with 18F-FBA–labeled A20FMDV2 radiotracers (1–3) bearing either no PEG or different PEG units at the N terminus. Methods: The 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. Results: The 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 18F-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). Conclusion: The 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.

Brain penetrant small molecule 18F-GnRH receptor (GnRH-R) antagonists: Synthesis and preliminary positron emission tomography imaging in rats

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.

Radiosynthesis of high affinity fluorine-18 labeled GnRH peptide analogues: in vitro studies and in vivo assessment of brain uptake in rats

Gonadotropin releasing hormone (GnRH) is recognized as an important neuromodulator affecting behavior and has been associated with the progression of Alzheimers disease. The peptide has been shown to have a bidirectional transport through the blood–brain-barrier (BBB), which may account for the cognitive effects of systemically administered GnRH. In this study, four novel 18F-GnRH peptide analogues were synthesized and their in vitro and in vivo characteristics studied in male rats. GnRH peptides were assembled by solid-phase peptide synthesis, either as the full length D-Lys6-GnRH (pyroGlu1-His2-Trp3-Ser4-Tyr5-D-Lys6-Leu7-Arg8-Pro9-Gly10-NH2) or as D-Lys6-desGly10-GnRH-NHEt. In all, four GnRH peptide analogues were synthesized and reacted with N-succinimidyl-4-fluorobenzoate (SFB) to yield the fluorinated versions. Binding affinities of the analogues were determined in a competitive binding assay for both human and rat GnRH receptors. Ki-values for the GnRH peptides were found to be subnanomolar, with D-Lys6(FBA)-desGly10-GnRH-NHEt (7) being most potent with a Ki-value of around 50 pM for GnRH receptor species. Radiolabeling was performed using N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) in 33.3 ± 12.8% isolated decay corrected (d.c.) yield within 1.5–2 h. Rat serum stability over 2 h revealed minor degradation (≤5%). For in vivo studies, 18F-peptides (4–30 MBq) were injected intravenously via the tail vein into rats and brain uptake was evaluated by means of dynamic PET (2 h) followed by biodistribution studies. PET showed limited or no uptake in brain for the 18F-peptides which predominantly cleared rapidly by renal excretion. Specific binding in the pituitary gland was confirmed for the 18F-peptide, 7, by blocking with the GnRH agonist buserelin.

Cerenkov luminescence imaging of αvβ6 integrin expressing tumors using 90Y‐labeled peptides

Cerenkov luminescence imaging (CLI) is an emerging preclinical molecular imaging modality that tracks the radiation emitted in the visible spectrum by fast moving charged decay products of radionuclides. The aim of this study was in vitro and in vivo evaluation of the two radiotracers, (90) Y-DOTA-PEG28 -A20FMDV2 ((90) Y-1) and (90) Y-DOTA-Ahx-A20FMDV2 ((90) Y-2) (>99% radiochemical purity, 3.7 GBq/µmol specific activity) for noninvasive assessment of tumors expressing the integrin αv β6 and their future use in tumor targeted radiotherapy. Cell binding and internalization in αv β6 -positive cells was (90) Y-1: 10.1 ± 0.8%, 50.3 ± 2.1%; (90) Y-2: 22.4 ± 1.7%, 44.7 ± 1.5% with <5% binding to αv β6 -negative control cells. Biodistribution studies showed maximum αv β6 -positive tumor uptake of the radiotracers at 1-h post injection (p.i.) ((90) Y-1: 0.64 ± 0.15% ID/g; (90) Y-2: 0.34 ± 0.11% ID/g) with high renal uptake (>25% ID/g at 24 h). Because of the lower tumor uptake and high radioactivity accumulation in kidneys (that could not be reduced by pre-administration of either lysine or furosemide), the luminescence signal from the αv β6 -positive tumor was not clearly detectable in CLI images. The studies suggest that CLI is useful for indicating major organ uptake for both radiotracers; however, it reaches its limitation when there is low signal-to-noise ratio.

Cerenkov luminescence imaging of αv β6 integrin expressing tumors using (90) Y-labeled peptides.

Cerenkov luminescence imaging (CLI) is an emerging preclinical molecular imaging modality that tracks the radiation emitted in the visible spectrum by fast moving charged decay products of radionuclides. The aim of this study was in vitro and in vivo evaluation of the two radiotracers, (90) Y-DOTA-PEG28 -A20FMDV2 ((90) Y-1) and (90) Y-DOTA-Ahx-A20FMDV2 ((90) Y-2) (>99% radiochemical purity, 3.7 GBq/µmol specific activity) for noninvasive assessment of tumors expressing the integrin αv β6 and their future use in tumor targeted radiotherapy. Cell binding and internalization in αv β6 -positive cells was (90) Y-1: 10.1 ± 0.8%, 50.3 ± 2.1%; (90) Y-2: 22.4 ± 1.7%, 44.7 ± 1.5% with <5% binding to αv β6 -negative control cells. Biodistribution studies showed maximum αv β6 -positive tumor uptake of the radiotracers at 1-h post injection (p.i.) ((90) Y-1: 0.64 ± 0.15% ID/g; (90) Y-2: 0.34 ± 0.11% ID/g) with high renal uptake (>25% ID/g at 24 h). Because of the lower tumor uptake and high radioactivity accumulation in kidneys (that could not be reduced by pre-administration of either lysine or furosemide), the luminescence signal from the αv β6 -positive tumor was not clearly detectable in CLI images. The studies suggest that CLI is useful for indicating major organ uptake for both radiotracers; however, it reaches its limitation when there is low signal-to-noise ratio.

Cerenkov luminescence imaging ofαvβ6integrin expressing tumors using90Y-labeled peptides: Cerenkov imaging using90Y-labeled peptides

Cerenkov luminescence imaging (CLI) is an emerging preclinical molecular imaging modality that tracks the radiation emitted in the visible spectrum by fast moving charged decay products of radionuclides. The aim of this study was in vitro and in vivo evaluation of the two radiotracers, (90) Y-DOTA-PEG28 -A20FMDV2 ((90) Y-1) and (90) Y-DOTA-Ahx-A20FMDV2 ((90) Y-2) (>99% radiochemical purity, 3.7 GBq/µmol specific activity) for noninvasive assessment of tumors expressing the integrin αv β6 and their future use in tumor targeted radiotherapy. Cell binding and internalization in αv β6 -positive cells was (90) Y-1: 10.1 ± 0.8%, 50.3 ± 2.1%; (90) Y-2: 22.4 ± 1.7%, 44.7 ± 1.5% with <5% binding to αv β6 -negative control cells. Biodistribution studies showed maximum αv β6 -positive tumor uptake of the radiotracers at 1-h post injection (p.i.) ((90) Y-1: 0.64 ± 0.15% ID/g; (90) Y-2: 0.34 ± 0.11% ID/g) with high renal uptake (>25% ID/g at 24 h). Because of the lower tumor uptake and high radioactivity accumulation in kidneys (that could not be reduced by pre-administration of either lysine or furosemide), the luminescence signal from the αv β6 -positive tumor was not clearly detectable in CLI images. The studies suggest that CLI is useful for indicating major organ uptake for both radiotracers; however, it reaches its limitation when there is low signal-to-noise ratio.