Synthesis and Evaluation of a Monomethyl Auristatin E─Integrin αβ 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 αβ are emerging as powerful tools to overcome these challenges. The development of an integrin αβ-selective PDC was achieved by combining the therapeutic efficacy of the cytotoxic drug monomethyl auristatin E with the selectivity of the αβ-binding peptide (αβ-BP) and with the ability of positron emission tomography (PET) imaging by copper-64.

Preclinical Evaluation of Ga- and Lu-Labeled Integrin αβ-Targeting Radiotheranostic Peptides.

The integrin αβ, 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 αβ-targeting peptide, DOTA-5G () radiolabeled with Ga, for PET/CT imaging and Lu 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 ().

A Comparison of Evans Blue and 4-(-Iodophenyl)butyryl Albumin Binding Moieties on an Integrin αβ 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 αvβ6 binding peptide (αvβ6-BP); a peptide that is currently being used for positron emission tomography (PET) imaging in patients with metastatic cancer. The ABM-modified αvβ6-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-αvβ6-BP ([64Cu]1) and [64Cu]Cu DOTA-IP-αvβ6-BP ([64Cu]2).

Evaluation of Copper-64-Labeled αβ-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 [F]AlF NOTA-K(ABM)-αβ-BP, where αβ-BP is a peptide with high affinity for the cell surface receptor integrin αβ that is overexpressed in several cancers, and the ABM is an iodophenyl-based moiety. [F]AlF NOTA-K(ABM)-αβ-BP demonstrated prolonged blood circulation compared to the non-ABM parent peptide, resulting in high, αβ-targeted uptake with continuously improving detection of αβ(+) tumors using PET/CT.

αβ-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 αβ 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.

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

Purpose: The αβ-BP peptide selectively targets the integrin αβ, a cell surface receptor recognized as a prognostic indicator for several challenging malignancies. Given that the 4-[F]fluorobenzoyl (FBA)-labeled peptide is a promising PET imaging agent, radiolabeling via aluminum [F]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-αβ-BP (1) and NOTA-K(ABM)-αβ-BP (2) were synthesized on solid phase, radiolabeled with aluminum [F]fluoride, and evaluated in vitro (integrin ELISA, albumin binding, cell studies) and in vivo in mouse models bearing paired DX3puroβ6 [αβ(+)]/DX3puro [αβ(-)], and for [F]AlF 2, BxPC-3 [αβ(+)] cell xenografts (PET imaging, biodistribution).

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

Purpose: The study was undertaken to develop and evaluate the potential of an integrin αβ-binding peptide (αβ-BP) for noninvasive imaging of a diverse range of malignancies with PET.

Experimental Design: The peptide αβ-BP was prepared on solid phase and radiolabeled with 4-[F]fluorobenzoic acid. testing included ELISA, serum stability, and cell binding studies using paired αβ-expressing and αβ-null cell lines.

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.

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([F]FBA). Herein is reported the first on-resin cyclization and F-radiolabeling of a cyclic peptide (cRGDyK) in an overall peptide synthesis yield of 88% (cRGDyK(NH)) 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 αvβ₆-Targeted ¹⁸F Peptide on Pharmacokinetics and Tumor Uptake.

Unlabelled: 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-(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.

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.

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.

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.

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).

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).

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.

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.

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.

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.

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.

Use of a peptide derived from foot-and-mouth disease virus for the noninvasive imaging of human cancer: generation and evaluation of 4-[18F]fluorobenzoyl A20FMDV2 for in vivo imaging of integrin alphavbeta6 expression with positron emission tomography.

Expression of the epithelial-specific integrin alphavbeta6 is low or undetectable in most adult tissues but may be increased during wound healing and inflammation and is up-regulated dramatically by many different carcinomas, making alphavbeta6 a promising target for the in vivo detection of cancer using noninvasive imaging. In addition, alphavbeta6 is recognized as promoting invasion and correlates with aggressive behavior of human cancers and thus agents that recognize alphavbeta6 specifically in vivo will be an essential tool for the future management of alphavbeta6-positive cancers. Recently, we identified the peptide NAVPNLRGDLQVLAQKVART (A20FMDV2), derived from foot-and-mouth disease virus, as a potent inhibitor of alphavbeta6.

Solid-phase synthesis of 2-[18F]fluoropropionyl peptides.

The 2-[(18)F]fluoropropionic (2-[(18)F]FPA) acid is used as a prosthetic group for radiolabeling proteins and peptides for targeted imaging using positron emission tomography (PET). Radiolabeling of compounds with more than one acylable functional group can lead to complex mixtures of products; however, peptides can be labeled regioselectively on the solid phase. We investigated the use of a solid-phase approach for the preparation of 2-[(18)F]fluoropropionyl peptides.

Dibenzotetraaza crown ethers: a new family of crown ethers based on o-phenylenediamine.

Dibenzotetraaza (DBTA) crown ethers possess two o-phenylenediamine moieties. They are homologues of dibenzo crown ether phase-transfer catalysts and were prepared from the condensation of benzimidazoles with oligo(ethyleneglycol) dichlorides and oligo(ethyleneglycol) ditosylates. Compounds with ring sizes ranging from 18-crown-6 to 42-crown-14 were prepared.