Expression of the epithelial-specific integrin A v B 6 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 A v B 6 a promising target for the in vivo detection of cancer using noninvasive imaging. In addition, A v B 6 is recognized as promoting invasion and correlates with aggressive behavior of human cancers and thus agents that recognize A v B 6 specifically in vivo will be an essential tool for the future management of A v B 6 -positive cancers. Recently, we identified the peptide NAVPNLRGDLQV-LAQKVART (A20FMDV2), derived from foot-and-mouth disease virus, as a potent inhibitor of A v B 6 . Using flow cytometry and ELISA, we show that this peptide is highly selective, inhibiting A v B 6 -ligand binding with a IC 50 of 3 nmol/L, an activity 1,000-fold more selective for A v B 6 than for other RGDdirected integrins (A v B 3 , A v B 5 , and A 5 B 1 ). A20FMDV2 was radiolabeled on solid-phase using 4-[18 F]fluorobenzoic acid, injected into mice bearing both A v B 6 -negative and A v B 6 -positive (DX3puro/DX3puroB6 cell lines) xenografts and imaged using a small animal positron emission tomography (PET) scanner. Rapid uptake (<30 min) and selective retention (>5 h) of radioactivity in the A v B 6 -positive versus the A v B 6 -negative tumor, together with fast renal elimination of nonspecifically bound activity, resulted in specific imaging of the A v B 6 -positive neoplasm. These data suggest that PET imaging of A v B 6 -positive tumors is feasible and will provide an important new tool for early detection and improved management of many types of cancers. [Cancer Res 2007;67(16):7833-40]

The cell surface receptor A v B 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 A v B 6 as a prognostic biomarker linked to poor survival. We have recently shown the feasibility of imaging A v B 6 in vivo by positron emission tomography (PET) using the peptide [18 F]FBA-A20FMDV2. Here, we describe improved A v B 6 imaging agents and test their efficacy in a mouse model with endogenous A v B 6 expression. The modified compounds maintained high affinity for A v B 6 and >1,000-fold selectivity over related integrins (by ELISA) and showed significantly improved A v B 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 A v B 6 expression) or the BxPC-3 human pancreatic carcinoma cell line (endogenous A v B 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 A v B 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.

Purpose: The study was undertaken to develop and evaluate the potential of an integrin a v b 6-binding peptide (a v b 6-BP) for noninvasive imaging of a diverse range of malignancies with PET. Experimental Design: The peptide a v b 6-BP was prepared on solid phase and radiolabeled with 4-[ 18 F]fluorobenzoic acid. In vitro testing included ELISA, serum stability, and cell binding studies using paired a v b 6-expressing and a v b 6-null cell lines. In vivo evaluation (PET/CT, biodistribution, and autoradiography) was performed in a mouse model bearing the same paired a v b 6-expressing and a v b 6-null cell xenografts. A first-inhuman PET/CT imaging study was performed in patients with metastatic lung, colon, breast, or pancreatic cancer. Results: [ 18 F]a v b 6-BP displayed excellent affinity and selectivity for the integrin a v b 6 in vitro [IC 50 (a v b 6) ¼ 1.2 nmol/L vs IC 50 (a v b 3) >10 mmol/L] in addition to rapid target-specific cell binding and internalization (72.5% AE 0.9% binding and 52.5% AE 1.8%, respectively). Favorable tumor affinity and selectivity were retained in the mouse model and excretion of unbound [ 18 F]a v b 6-BP was rapid, primarily via the kidneys. In patients, [ 18 F]a v b 6-BP was well tolerated without noticeable adverse side effects. PET images showed significant uptake of [ 18 F]a v b 6-BP in both the primary lesion and metastases, including metastasis to brain, bone, liver, and lung. Conclusions: The clinical impact of [ 18 F]a v b 6-BP PET imaging demonstrated in this first-inhuman study is immediate for a broad spectrum of malignancies.

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 3 different radiolabeling approaches and report the effects on PET imaging and pharmacokinetics. The prosthetic groups did have an influence; metabolites with significantly different polarities were observed.Non-invasive PET a imaging has become a widely used tool for the detection of many diseases. 1 Among the available positron emitting nuclides [ 18 F]fluorine is particularly widely used because it can be produced on demand in medical cyclotrons and combines favorable decaycharacteristics (T 1/2 = 110 min, mode of decay: 97% β + , maximum β + energy = 0.64 MeV) with relative chemical versatility. 2 As new disease-specific imaging targets (e.g. cell surface receptors) are being identified, there is an increased demand for targeted radiotracers. 3 Peptides are receiving much attention for in vivo cancer detection because excellent, tissue-specific uptake can be achieved. Relying on well-established synthetic chemistries, peptides are readily produced and modified. 3 Strategies include automated syntheses with incorporation of unnatural amino acids, peptidomimetics, and cyclization, among others, to develop compounds with desirable pharmacokinetic properties. To make peptides amendable for PET imaging the [ 18 F]fluorine-radiolabel is introduced using small molecules (prosthetic groups). Examples of [ 18 F]-labeled peptides for PET imaging include octreotide, 4 vasoactive intestinal peptide, 5 integrin specific peptides, 6-8 N ε -(γ-glutamyl)lysine, 9 neurotensin analogs, 10 human Cpeptide, 11 and insulin. 12The prosthetic group approach involves at least two synthetic steps: Incorporation of [ 18 F] fluorine into the prosthetic group, and attachment to the peptide. Generally, the prosthetic group itself should not negatively affect receptor binding, and the synthetic approach should be applicable to many different peptide substrates with minimal synthetic modifications. For this, Recently, our group successfully used the copper-catalyzed Huisgen 1,3-dipolar cycloaddition ("click" chemistry) 20, 21 to conjugate ω-[ 18 F]fluoroalkynes to peptides functionalized with 3-azidopropionic acid (Table 1 , Scheme 1). 22 The formation of 1,4-disubstituted 1,2,3-triazoles proceeded smoothly under mild conditions and the radiolabeled peptides were obtained in a short period of time. Subsequently, this approach has been applied for radiolabeling of different substrates with [ 18 F]fluorine. 10, 23-25 As a result of ongoing improvements in 1,3-dipolar cycloaddition-chemistry and because of its versatility and short reaction times "click" radiochemistry promises to become a widely used tool for preparation of radiotracers.Here we...

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, sitespecific 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.high-throughput screening ͉ in vivo imaging ͉ microPET ͉ radiolabeled peptides ͉ positron emission tomography C ombinatorial chemistry (1, 2) and phage display (3) techniques have become essential tools for the production of large compound libraries, which can be rapidly produced for the discovery of new drugs. Designing and implementing high-throughput screening (HTS) approaches to identify lead compounds that show affinity for a biological target from these large libraries, wherein millions of new compounds may exist, is often accomplished through in vitro screening. In general, the 2 approaches by which this can be accomplished are either solution-phase screening techniques (4-11), generally used by drug discovery programs, or solid-phase screening, which has garnered interest for the evaluation of libraries prepared using combinatorial chemistry.Though there are many reports outlining high-throughput approaches for in vitro screening, few exist where the sole purpose is to develop high-throughput in vivo methodologies for the identification of new molecular imaging agents. While the feasibility of using magnetic resonance imaging (MRI) for high-throughput applications has been explored with both the development of scanner technology and the scanning of multiple animals in parallel (12)(13)(14)(15)(16)(17)(18)(19)(20), positron emission tomography (PET) has received very little attention for high-throughput applications. This is likely the result of the unique set of problems that arise when considering using the latter imaging modality. The half-lives of the radioactive isotopes commonly used for PET ar...