Synthesis and Evaluation of a Monomethyl Auristatin E─Integrin αvß6 Binding Peptide-Drug Conjugate for Tumor Targeted Drug Delivery.

Many anticancer drugs exhibit high systemic off-target toxicities causing severe side effects. Peptide-drug conjugates (PDCs) that target tumor-specific receptors such as integrin αvß6 are emerging as powerful tools to overcome these challenges. The development of an integrin αvß6-selective PDC was achieved by combining the therapeutic efficacy of the cytotoxic drug monomethyl auristatin E with the selectivity of the αvß6-binding peptide (αvß6-BP) and with the ability of positron emission tomography (PET) imaging by copper-64. The [64Cu]PDC-1 was produced efficiently and in high purity. The PDC exhibited high human serum stability, integrin αvß6-selective internalization, cell binding, and cytotoxicity. Integrin αvß6-selective tumor accumulation of the [64Cu]PDC-1 was visualized with PET-imaging and corroborated by biodistribution, and [64Cu]PDC-1 showed promising in vivo pharmacokinetics. The [natCu]PDC-1 treatment resulted in prolonged survival of mice bearing αvß6 (+) tumors (median survival: 77 days, vs αvß6 (-) tumor group 49 days, and all other control groups 37 days).

Preclinical Evaluation of 68Ga- and 177Lu-Labeled Integrin αvß6-Targeting Radiotheranostic Peptides.

The integrin αvß6, an epithelium-specific cell surface receptor, is overexpressed on numerous malignancies, including the highly lethal pancreatic ductal adenocarcinomas. Here, we developed and tested a novel αvß6-targeting peptide, DOTA-5G (1) radiolabeled with 68Ga, for PET/CT imaging and 177Lu for treatment. With the goal to develop a radiotheranostic, further modifications were made for increased circulation time, renal recycling, and tumor uptake, yielding DOTA-albumin-binding moiety-5G (2). Methods: Peptides 1 and 2 were synthesized on solid phase, and their affinity for αvß6 was assessed by enzyme-linked immunosorbent assay. The peptides were radiolabeled with 68Ga and 177Lu. In vitro cell binding, internalization, and efflux of 68Ga-1 and 177Lu-2 were evaluated in αvß6-positive BxPC-3 human pancreatic cancer cells. PET/CT imaging of 68Ga-1 and 68Ga-2 was performed on female nu/nu mice bearing subcutaneous BxPC-3 tumors. Biodistribution was performed for 68Ga-1 (1 and 2 h after injection), 68Ga-2 (2 and 4 h after injection), and 177Lu-1 and 177Lu-2 (1, 24, 48, and 72 h after injection). The 177Lu-2 biodistribution data were extrapolated for human dosimetry data estimates using OLINDA/EXM 1.1. Therapeutic efficacy of 177Lu-2 was evaluated in mice bearing BxPC-3 tumors. Results: Peptides 1 and 2 demonstrated high affinity (<55 nM) for αvß6 by enzyme-linked immunosorbent assay. 68Ga-1, 68Ga-2, 177Lu-1, and 177Lu-2 were synthesized in high radiochemical purity. Rapid in vitro binding and internalization of 68Ga-1 and 177Lu-2 were observed in BxPC-3 cells. PET/CT imaging and biodistribution studies demonstrated uptake in BxPC-3 tumors. Introduction of the albumin-binding moiety in 177Lu-2 resulted in a 5-fold increase in tumor uptake and retention over time. Based on the extended dosimetry data, the dose-limiting organ for 177Lu-2 is the kidney. Treatment with 177Lu-2 prolonged median survival by 1.5- to 2-fold versus controls. Conclusion: 68Ga-1 and 177Lu-2 demonstrated high affinity for the integrin αvß6 both in vitro and in vivo, were rapidly internalized into BxPC-3 cells, and were stable in mouse and human serum. Both radiotracers showed favorable pharmacokinetics in preclinical studies, with predominantly renal excretion and good tumor-to-normal-tissue ratios. Favorable human dosimetry data suggest the potential of 177Lu-2 as a treatment for pancreatic ductal adenocarcinoma.

A Comparison of Evans Blue and 4-(p-Iodophenyl)butyryl Albumin Binding Moieties on an Integrin αvß6 Binding Peptide.

Serum albumin binding moieties (ABMs) such as the Evans blue (EB) dye fragment and the 4-(p-iodophenyl)butyryl (IP) have been used to improve the pharmacokinetic profile of many radiopharmaceuticals. The goal of this work was to directly compare these two ABMs when conjugated to an integrin αvß6 binding peptide (αvß6-BP); a peptide that is currently being used for positron emission tomography (PET) imaging in patients with metastatic cancer. The ABM-modified αvß6-BP peptides were synthesized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA) chelator for radiolabeling with copper-64 to yield [64Cu]Cu DOTA-EB-αvß6-BP ([64Cu]1) and [64Cu]Cu DOTA-IP-αvß6-BP ([64Cu]2). Both peptides were evaluated in vitro for serum albumin binding, serum stability, and cell binding and internalization in the paired engineered melanoma cells DX3puroß6 (αvß6 +) and DX3puro (αvß6 −), and pancreatic BxPC-3 (αvß6 +) cells and in vivo in a BxPC-3 xenograft mouse model. Serum albumin binding for [64Cu]1 and [64Cu]2 was 53−63% and 42−44%, respectively, with good human serum stability (24 h: [64Cu]1 76%, [64Cu]2 90%). Selective αvß6 cell binding was observed for both [64Cu]1 and [64Cu]2 (αvß6 (+) cells: 30.3−55.8% and 48.5−60.2%, respectively, vs. αvß6 (−) cells <3.1% for both). In vivo BxPC-3 tumor uptake for both peptides at 4 h was 5.29 ± 0.59 and 7.60 ± 0.43% ID/g ([64Cu]1 and [64Cu]2, respectively), and remained at 3.32 ± 0.46 and 4.91 ± 1.19% ID/g, respectively, at 72 h, representing a >3-fold improvement over the non-ABM parent peptide and thereby providing improved PET images. Comparing [64Cu]1 and [64Cu]2, the IP-ABM-αvß6-BP [64Cu]2 displayed higher serum stability, higher tumor accumulation, and lower kidney and liver accumulation, resulting in better tumor-to-organ ratios for high contrast visualization of the αvß6 (+) tumor by PET imaging.

Evaluation of Copper-64-Labeled αvß6-Targeting Peptides: Addition of an Albumin Binding Moiety to Improve Pharmacokinetics.

The incorporation of non-covalent albumin binding moieties (ABMs) into radiotracers results in increased circulation time, leading to a higher uptake in the target tissues such as the tumor, and, in some cases, reduced kidney retention. We previously developed [18F]AlF NOTA-K(ABM)-αvß6-BP, where αvß6-BP is a peptide with high affinity for the cell surface receptor integrin αvß6 that is overexpressed in several cancers, and the ABM is an iodophenyl-based moiety. [18F]AlF NOTA-K(ABM)-αvß6-BP demonstrated prolonged blood circulation compared to the non-ABM parent peptide, resulting in high, αvß6-targeted uptake with continuously improving detection of αvß6(+) tumors using PET/CT. To further extend the imaging window beyond that of fluorine-18 (t1/2 = 110 min) and to investigate the pharmacokinetics at later time points, we radiolabeled the αvß6-BP with copper-64 (t1/2 = 12.7 h). Two peptides were synthesized without (1) and with (2) the ABM and radiolabeled with copper-64 to yield [64Cu]1 and [64Cu]2, respectively. The affinity of [natCu]1 and [natCu]2 for the integrin αvß6 was assessed by enzyme-linked immunosorbent assay. [64Cu]1 and [64Cu]2 were evaluated in vitro (cell binding and internalization) using DX3puroß6 (αvß6(+)), DX3puro (αvß6(-)), and pancreatic BxPC-3 (αvß6(+)) cells, in an albumin binding assay, and for stability in both mouse and human serum. In vivo (PET/CT imaging) and biodistribution studies were done in mouse models bearing either the paired DX3puroß6/DX3puro or BxPC-3 xenograft tumors. [64Cu]1 and [64Cu]2 were synthesized in ≥97% radiochemical purity. In vitro, [natCu]1 and [natCu]2 maintained low nanomolar affinity for integrin αvß6 (IC50 = 28 ± 3 and 19 ± 5 nM, respectively); [64Cu]1 and [64Cu]2 showed comparable binding to αvß6(+) cells (DX3puroß6: ≥70%, ≥42% internalized; BxPC-3: ≥19%, ≥12% internalized) and ≤3% to the αvß6(-) DX3puro cells. Both radiotracers were ≥98% stable in human serum at 24 h, and [64Cu]2 showed a 6-fold higher binding to human serum protein than [64Cu]1. In vivo, selective uptake in the αvß6(+) tumors was observed with tumor visualization up to 72 h for [64Cu]2. A 3-5-fold higher αvß6(+) tumor uptake of [64Cu]2 vs [64Cu]1 was observed throughout, at least 2.7-fold improved BxPC-3-to-kidney and BxPC-3-to-blood ratios, and 2-fold improved BxPC-3-to-stomach ratios were noted for [64Cu]2 at 48 h. Incorporation of an iodophenyl-based ABM into the αvß6-BP ([64Cu]2) prolonged circulation time and resulted in improved pharmacokinetics, including increased uptake in αvß6(+) tumors that enabled visualization of αvß6(+) tumors up to 72 h by PET/CT imaging.

αvß6-Targeted Molecular PET/CT Imaging of the Lungs After SARS-CoV-2 Infection.

The true impact and long-term damage to organs such as the lungs after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain to be determined. Noninvasive molecularly targeted imaging may play a critical role in aiding visualization and understanding of the systemic damage. We have identified αvß6 as a molecular target; an epithelium-specific cell surface receptor that is low or undetectable in healthy adult epithelium but upregulated in select injured tissues, including fibrotic lung. Herein we report the first human PET/CT images using the integrin αvß6-binding peptide (18F-αvß6-BP) in a patient 2 mo after the acute phase of infection. Minimal uptake of 18F-αvß6-BP was noted in normal lung parenchyma, with uptake being elevated in areas corresponding to opacities on CT. This case suggests that 18F-αvß6-BP PET/CT is a promising noninvasive approach to identify the presence and potentially monitor the persistence and progression of lung damage.

The Effects of an Albumin Binding Moiety on the Targeting and Pharmacokinetics of an Integrin αvß6-Selective Peptide Labeled with Aluminum [18F]Fluoride.

PURPOSE: The αvß6-BP peptide selectively targets the integrin αvß6, a cell surface receptor recognized as a prognostic indicator for several challenging malignancies. Given that the 4-[18F]fluorobenzoyl (FBA)-labeled peptide is a promising PET imaging agent, radiolabeling via aluminum [18F]fluoride chelation and introduction of an albumin binding moiety (ABM) have the potential to considerably simplify radiochemistry and improve the pharmacokinetics by increasing biological half-life. PROCEDURES: The peptides NOTA-αvß6-BP (1) and NOTA-K(ABM)-αvß6-BP (2) were synthesized on solid phase, radiolabeled with aluminum [18F]fluoride, and evaluated in vitro (integrin ELISA, albumin binding, cell studies) and in vivo in mouse models bearing paired DX3puroß6 [αvß6(+)]/DX3puro [αvß6(-)], and for [18F]AlF 2, BxPC-3 [αvß6(+)] cell xenografts (PET imaging, biodistribution). RESULTS: The peptides were radiolabeled in 23.0 ± 5.7 % and 22.1 ± 4.4 % decay-corrected radiochemical yield, respectively, for [18F]AlF 1 and [18F]AlF 2. Both demonstrated excellent affinity and selectivity for integrin αvß6 by ELISA (IC50(αvß6) = 3-7 nM vs IC50(αvß3) > 10 µM) and in cell binding studies (51.0 ± 0.7 % and 47.2 ± 0.7 % of total radioactivity bound to DX3puroß6 cells at 1 h, respectively, vs. ≤ 1.2 % to DX3puro for both compounds). The radiotracer [18F]AlF 1 bound to human serum at 16.3 ± 1.9 %, compared to 67.5 ± 1.0 % for the ABM-containing [18F]AlF 2. In vivo studies confirmed the effect of the ABM on blood circulation (≤ 0.1 % ID/g remaining in blood for [18F]AlF 1 as soon as 1 h p.i. vs. > 2 % ID/g for [18F]AlF 2 at 6 h p.i.) and higher αvß6(+) tumor uptake (4 h: DX3puroß6; [18F]AlF 1: 3.0 ± 0.7 % ID/g, [18F]AlF 2: 7.2 ± 0.7 % ID/g; BxPC-3; [18F]AlF 2: 10.2 ± 0.1 % ID/g). CONCLUSION: Both compounds were prepared using standard chemistries; affinity and selectivity for integrin αvß6 in vitro remained unaffected by the albumin binding moiety. In vivo, the albumin binding moiety resulted in prolonged circulation and higher αvß6-targeted uptake.

Preclinical Development and First-in-Human Imaging of the Integrin αvß6 with [18F]αvß6-Binding Peptide in Metastatic Carcinoma.

PURPOSE: The study was undertaken to develop and evaluate the potential of an integrin αvß6-binding peptide (αvß6-BP) for noninvasive imaging of a diverse range of malignancies with PET. EXPERIMENTAL DESIGN: The peptide αvß6-BP was prepared on solid phase and radiolabeled with 4-[18F]fluorobenzoic acid. In vitro testing included ELISA, serum stability, and cell binding studies using paired αvß6-expressing and αvß6-null cell lines. In vivo evaluation (PET/CT, biodistribution, and autoradiography) was performed in a mouse model bearing the same paired αvß6-expressing and αvß6-null cell xenografts. A first-in-human PET/CT imaging study was performed in patients with metastatic lung, colon, breast, or pancreatic cancer. RESULTS: [18F]αvß6-BP displayed excellent affinity and selectivity for the integrin αvß6 in vitro [IC50(αvß6) = 1.2 nmol/L vs IC50(αvß3) >10 µmol/L] in addition to rapid target-specific cell binding and internalization (72.5% ± 0.9% binding and 52.5% ± 1.8%, respectively). Favorable tumor affinity and selectivity were retained in the mouse model and excretion of unbound [18F]αvß6-BP was rapid, primarily via the kidneys. In patients, [18F]αvß6-BP was well tolerated without noticeable adverse side effects. PET images showed significant uptake of [18F]αvß6-BP in both the primary lesion and metastases, including metastasis to brain, bone, liver, and lung. CONCLUSIONS: The clinical impact of [18F]αvß6-BP PET imaging demonstrated in this first-in-human study is immediate for a broad spectrum of malignancies.

In Vivo Tracking of Copper-64 Radiolabeled Nanoparticles in Lactuca sativa.

Engineered nanoparticles (NPs) are increasingly used in commercial products including automotive lubricants, clothing, deodorants, sunscreens, and cosmetics and can potentially accumulate in our food supply. Given their size it is difficult to detect and visualize the presence of NPs in environmental samples, including crop plants. New analytical tools are needed to fill the void for detection and visualization of NPs in complex biological and environmental matrices. We aimed to determine whether radiolabeled NPs could be used as a noninvasive, highly sensitive analytical tool to quantitatively track and visualize NP transport and accumulation in vivo in lettuce (Lactuca sativa) and to investigate the effect of NP size on transport and distribution over time using a combination of autoradiography, positron emission tomography (PET)/computed tomography (CT), scanning electron microscopy (SEM), and transition electron microscopy (TEM). Azide functionalized NPs were radiolabeled via a "click" reaction with copper-64 (64Cu)-1,4,7-triazacyclononane triacetic acid (NOTA) azadibenzocyclooctyne (ADIBO) conjugate ([64Cu]-ADIBO-NOTA) via copper-free Huisgen-1,3-dipolar cycloaddition reaction. This yielded radiolabeled [64Cu]-NPs of uniform shape and size with a high radiochemical purity (>99%), specific activity of  2.2 mCi/mg of NP, and high stability (i.e., no detectable dissolution) over 24 h across a pH range of 5-9. Both PET/CT and autoradiography showed that [64Cu]-NPs entered the lettuce seedling roots and were rapidly transported to the cotyledons with the majority of the accumulation inside the roots. Uptake and transport of intact NPs was size-dependent, and in combination with the accumulation within the roots suggests a filtering effect of the plant cell walls at various points along the water transport pathway.

Solid-phase synthesis and fluorine-18 radiolabeling of cycloRGDyK.

Solid-phase peptide synthesis, head-to-tail cyclization, and subsequent radiolabeling provided a reproducible, simple, rapid synthetic method to generate the cyclic peptide radiotracer cRGDyK([18F]FBA). Herein is reported the first on-resin cyclization and 18F-radiolabeling of a cyclic peptide (cRGDyK) in an overall peptide synthesis yield of 88% (cRGDyK(NH2)) and subsequent radiolabeling yield of 14 ± 2% (decay corrected, n = 4). This approach is generally applicable to the development of an automated process for the synthesis of cyclic radiolabeled peptides for positron emission tomography (PET).

The Effect of Bi-Terminal PEGylation of an Integrin αvß6-Targeted ¹8F Peptide on Pharmacokinetics and Tumor Uptake.

UNLABELLED: Radiotracers based on the peptide A20FMDV2 selectively target the cell surface receptor integrin αvß6. 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 αvß6-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 αvß6-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. METHODS: The radiotracers were prepared and radiolabeled on solid phase. Using 3 cell lines, DX3puroß6 (αvß6+), DX3puro (αvß6-), and BxPC-3 (αvß6+), we evaluated the radiotracers in vitro (serum stability; cell binding and internalization) and in vivo in mouse models bearing paired DX3puroß6-DX3puro and, for 5, BxPC-3 xenografts. RESULTS: The size and location of the PEG units significantly affected αvß6 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 αvß6 affinity, selectivity, and pharmacokinetic profile. In vitro, 5 bound to αvß6-expressing DX3puroß6 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, αvß6-directed binding in the paired DX3puroß6-DX3puro model (1 h: DX3puroß6, 2.3 ± 0.2 percentage injected dose per gram [%ID/g]; DX3puroß6/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 αvß6+ tumor uptake and retention.

Cerenkov luminescence imaging of αv ß6 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 ß6 and their future use in tumor targeted radiotherapy. Cell binding and internalization in αv ß6 -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 ß6 -negative control cells. Biodistribution studies showed maximum αv ß6 -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 ß6 -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.

In vitro and in vivo evaluation of the effects of aluminum [¹8F]fluoride radiolabeling on an integrin αvß6-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)ß6 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)ß6(+) and α(v)ß6(-) cells) and in vivo (paired α(v)ß6(+) and α(v)ß6(-) 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)ß6-targeted binding (α(v)ß6(+): 42.4 ± 1.2% of total radioactivity, ratio (+)/(-)=8.4/1) and internalization (α(v)ß6(+): 28.3 ± 0.5% of total radioactivity, (+)/(-)=11.7/1). In vivo, 2 maintained α(v)ß6-targeted binding (biodistribution; 1 h: α(v)ß6(+): 1.74 ± 0.38% ID/g, (+)/(-)=2.72/1; 4 h: α(v)ß6(+): 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)ß6-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.

Evaluation of an integrin αvß6-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)ß(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 (DX3puroß6, α(v)ß(6)-positive; and DX3puro, α(v)ß(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)ß(6)-targeted binding was observed in vitro (DX3puroß6 cells, 15 min: 43.2% binding, >6:1 for DX3puroß6:DX3puro). In the mouse model DX3puroß6-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)ß(6)-targeted binding in vitro, it did influence the pharmacokinetics, possibly due to its size and lipophilic nature.

Optimization of the solid-phase synthesis of [18F] radiolabeled peptides for positron emission tomography.

Establishing improved methods for the radiolabeling of peptides with fluorine-18 via solid-phase peptide synthesis (SPPS) is desirable for the efficient synthesis of peptide-based molecular imaging agents. This work focuses on the development of a standardized platform to facilitate the reliable and efficient synthesis of high-purity fluorine-18 radiolabeled peptides for in vivo imaging with positron emission tomography (PET). Seven commercially available resins were selected for solid-phase radiolabeling of the model peptide VQAAIDYING with 4-[(18)F]fluorobenzoic acid ([(18)F]FBA). A wide range of radiochemical yields (18.8 ± 1.5% to 41.2 ± 5.3%) was obtained using standard conditions (coupling: 3 eq amino acid, 3 eq HATU, 6 eq DIPEA, 1.5 h, r.t.; cleavage: 94% TFA, 3 h, r.t.). After modification of coupling reagents and employing heated reactions to 37°C, radiochemical yields were improved by as much as 35.3% over standard conditions. When the optimized conditions were applied to the synthesis of [(18)F]FBA-PEG(28)-A20FMDV2, which targets the α(v)ß(6) integrin in vivo, radiochemical yields improved by as much as 73.4% over those obtained using standard coupling and cleavage conditions. This platform can be utilized to improve the synthesis of peptide-based molecular probes for molecular imaging with PET.

Copper-Free Click for PET: Rapid 1,3-Dipolar Cycloadditions with a Fluorine-18 Cyclooctyne.

The strain-promoted click 1,3-dipolar cycloaddition reactions involving azides and cyclooctynes for the synthesis of triazoles offer the advantage of being able to be performed in biological settings via copper-free chemistries. While strained reagents conjugated to optical dyes and radiometal conjugates have been reported, cyclooctyne reagents labeled with fluorine-18 ((18)F) and radiochemically evaluated in a copper-free click reaction have yet to be explored. This report describes the conversion of a bifunctional azadibenzocyclooctyne (ADIBO) amine to the (18)F-labeled cyclooctyne 4, the subsequent fast copper-free 1,3-dipolar cycloaddition reaction with alkyl azides at 37 °C (>70% radiochemical conversion in 30 min), and biological evaluations (serum stability of >95% at 2 h). These findings demonstrate the excellent reactivity of the (18)F-labeled cyclooctyne 4 with readily available azides that will allow future work focusing on rapid copper-free in vitro and in vivo click chemistries for PET imaging using (18)F-labeled cyclooctyne derivatives of ADIBO.

High-throughput in vivo screening of targeted molecular imaging agents.

The rapid development and translation of targeted molecular imaging agents from bench to bedside is currently a slow process, with a clear bottleneck between the discovery of new compounds and the development of an appropriate molecular imaging agent. The ability to identify promising new molecular imaging agents, as well as failures, much earlier in the development process using high-throughput screening techniques could save significant time and money. This work combines the advantages of combinatorial chemistry, site-specific solid-phase radiolabeling, and in vivo imaging for the rapid screening of molecular imaging agents. A one-bead-one-compound library was prepared and evaluated in vitro, leading to the identification of 42 promising lead peptides. Over 11 consecutive days, these peptides, along with a control peptide, were successfully radiolabeled with 4-[(18)F]fluorobenzoic acid and evaluated in vivo using microPET. Four peptides were radiolabeled per day, followed by simultaneous injection of each individual peptide into 2 animals. As a result, 4 promising new molecular imaging agents were identified that otherwise would not have been selected based solely on in vitro data. This study is the first example of the practical application of a high-throughput screening approach using microPET imaging of [(18)F]-labeled peptides for the rapid in vivo identification of potential new molecular imaging agents.

Targeted in vivo imaging of integrin alphavbeta6 with an improved radiotracer and its relevance in a pancreatic tumor model.

The cell surface receptor alpha(v)beta(6) is epithelial specific, and its expression is tightly regulated; it is low or undetectable in adult tissues but has been shown to be increased in many different cancers, including pancreatic, cervical, lung, and colon cancers. Studies have described alpha(v)beta(6) as a prognostic biomarker linked to poor survival. We have recently shown the feasibility of imaging alpha(v)beta(6) in vivo by positron emission tomography (PET) using the peptide [(18)F]FBA-A20FMDV2. Here, we describe improved alpha(v)beta(6) imaging agents and test their efficacy in a mouse model with endogenous alpha(v)beta(6) expression. The modified compounds maintained high affinity for alpha(v)beta(6) and >1,000-fold selectivity over related integrins (by ELISA) and showed significantly improved alpha(v)beta(6)-dependent binding in cell-based assays (>60% binding versus <10% for [(18)F]FBA-A20FMDV2). In vivo studies using either a melanoma cell line (transduced alpha(v)beta(6) expression) or the BxPC-3 human pancreatic carcinoma cell line (endogenous alpha(v)beta(6) expression) revealed that the modified compounds showed significantly improved tumor retention. This, along with good clearance of nonspecifically bound activity, particularly for the new radiotracer [(18)F]FBA-PEG(28)-A20FMDV2, resulted in improved PET imaging. Tumor/pancreas and tumor/blood biodistribution ratios of >23:1 and >47:1, respectively, were achieved at 4 hours. Significantly, [(18)F]FBA-PEG(28)-A20FMDV2 was superior to 2-[(18)F]fluoro-2-deoxy-d-glucose ([(18)F]FDG) in imaging the BxPC-3 tumors. Pancreatic ductal adenocarcinoma is highly metastatic and current preoperative evaluation of resectability using noninvasive imaging has limited success, with most patients having metastases at time of surgery. The fact that these tumors express alpha(v)beta(6) suggests that this probe has significant potential for the in vivo detection of this malignancy, thus having important implications for patient care and therapy.

Evaluation of [64Cu]Cu-DOTA and [64Cu]Cu-CB-TE2A chelates for targeted positron emission tomography with an alphavbeta6-specific peptide.

Significant upregulation of the integrin alpha(v)beta(6) has been described as a prognostic indicator in several cancers, making it an attractive target for tumor imaging. This study compares variants of a PEGylated alpha(v)beta(6)-targeting peptide, bearing either an [(18)F]fluorobenzoyl prosthetic group ([(18)F]FBA-PEG-A20FMDV2) or different [(64)Cu]copper chelators (DOTA-PEG-A20FMDV2, CB-TE2A-PEG-A20FMDV2). The compounds were evaluated in vitro by enzyme-linked immunosorbent assay (against the integrin alpha(v)beta(6) and the closely related integrin alpha(v)beta(3)) and by cell labeling (alpha(v)beta(6)-positive DX3purobeta6/alpha(v)beta(6)-negative DX3puro) and in vivo using micro-positron emission tomography in a mouse model bearing paired DX3purobeta6/Dx3puro xenografts. In vitro, all three compounds showed excellent alpha(v)beta(6)-specific binding (50% inhibitory concentration [IC(50)](alpha(v)beta(6)) = 3 to 6 nmol/L; IC(50)(alpha(v)beta(3)) > 10 micromol/L). In vivo, they displayed comparable, preferential uptake for the alpha(v)beta(6)-expressing xenograft over the alpha(v)beta(6)-negative control (> 4:1 ratio at 4 hours postinjection). Whereas [(64)Cu]Cu-DOTA-PEG-A20FMDV2 resulted in increased levels of radioactivity in the liver, [(64)Cu]Cu-CB-TE2A-PEG-A20FMDV2 did not. Significantly, both (64)Cu-labeled tracers showed unexpectedly high and persistent levels of radioactivity in the kidneys (> 40% injected dose/g at 4 and 12 hours postinjection). The findings underscore the potential influence of the prosthetic group on targeted in vivo imaging of clinically relevant markers such as alpha(v)beta(6). Despite identical targeting peptide moiety and largely equal in vitro behavior, both (64)Cu-labeled tracers displayed inferior pharmacokinetics, making them in their present form less suitable candidates than the (18)F-labeled tracer for in vivo imaging of alpha(v)beta(6).

Synthesis of 5- and 6-substituted 2-(4-dimethylaminophenyl)-1,3-benzoxazoles and their in vitro and in vivo evaluation as imaging agents for amyloid plaque.

A series of novel 5- and 6-substituted 2-(4-dimethylaminophenyl)-1,3-benzoxazoles was synthesized and their potential as imaging probes for Alzheimer's Disease (AD)-related amyloid plaque was evaluated in vitro and in vivo. In vitro binding affinities for Abeta1-40 peptide of several of these compounds were in the low-nanomolar range . The lowest K(i) of 9.3nM was found for N-(2-(4-(dimethylamino)phenyl)-1,3-benzoxazol-5-yl)-4-iodobenzamide (1e). Its (123)I-radiolabeled form ([(123)I]1e) was subsequently prepared by iododestannylation of the corresponding tributylstannyl precursor and evaluated in vivo in a baboon model using SPECT imaging. Contrary to our expectations, 1e did not cross the blood-brain barrier (BBB) to any significant extent.

In vivo positron emission tomography (PET) imaging with an alphavbeta6 specific peptide radiolabeled using 18F-"click" chemistry: evaluation and comparison with the corresponding 4-[18F]fluorobenzoyl- and 2-[18F]fluoropropionyl-peptides.

Numerous radiolabeled peptides have been utilized for in vivo imaging of a variety of cell surface receptors. For applications in PET using [(18)F]fluorine, peptides are radiolabeled via a prosthetic group approach. We previously developed solution-phase (18)F-"click" radiolabeling and solid-phase radiolabeling using 4-[(18)F]fluorobenzoic and 2-[(18)F]fluoropropionic acids. Here we compare the three different radiolabeling approaches and report the effects on PET imaging and pharmacokinetics. The prosthetic groups did have an effect; metabolites with significantly different polarities were observed.