Preclinical evaluation of EpCAM-binding designed ankyrin repeat proteins (DARPins) as targeting moieties for bimodal near-infrared fluorescence and photoacoustic imaging of cancer.

PURPOSE: Fluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed ankyrin repeat proteins (DARPins) possess optimal pharmacokinetic and other properties for in vivo imaging. This study aims to evaluate the preclinical potential of epithelial cell adhesion molecule (EpCAM)-binding DARPins as targeting moieties for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of cancer. METHODS: EpCAM-binding DARPins Ac2, Ec4.1, and non-binding control DARPin Off7 were conjugated to IRDye 800CW and their binding efficacy was evaluated on EpCAM-positive HT-29 and EpCAM-negative COLO-320 human colon cancer cell lines. Thereafter, NIRF and PA imaging of all three conjugates were performed in HT-29_luc2 tumor-bearing mice. At 24 h post-injection, tumors and organs were resected and tracer biodistributions were analyzed. RESULTS: Ac2-800CW and Ec4.1-800CW specifically bound to HT-29 cells, but not to COLO-320 cells. Next, 6 nmol and 24 h were established as the optimal in vivo dose and imaging time point for both DARPin tracers. At 24 h post-injection, mean tumor-to-background ratios of 2.60 ± 0.3 and 3.1 ± 0.3 were observed for Ac2-800CW and Ec4.1-800CW, respectively, allowing clear tumor delineation using the clinical Artemis NIRF imager. Biodistribution analyses in non-neoplastic tissue solely showed high fluorescence signal in the liver and kidney, which reflects the clearance of the DARPin tracers. CONCLUSION: Our encouraging results show that EpCAM-binding DARPins are a promising class of targeting moieties for pan-carcinoma targeting, providing clear tumor delineation at 24 h post-injection. The work described provides the preclinical foundation for DARPin-based bimodal NIRF/PA imaging of cancer.

BF2-Azadipyrromethene Fluorophores for Intraoperative Vital Structure Identification.

A series of mono- and bis-polyethylene glycol (PEG)-substituted BF2-azadipyrromethene fluorophores have been synthesized with emissions in the near-infrared region (700-800 nm) for the purpose of fluorescence guided intraoperative imaging; chiefly ureter imaging. The Bis-PEGylation of fluorophores resulted in higher aqueous fluorescence quantum yields, with PEG chain lengths of 2.9 to 4.6 kDa being optimal. Fluorescence ureter identification was possible in a rodent model with the preference for renal excretion notable through comparative fluorescence intensities from the ureters, kidneys and liver. Ureteral identification was also successfully performed in a larger animal porcine model under abdominal surgical conditions. Three tested doses of 0.5, 0.25 and 0.1 mg/kg all successfully identified fluorescent ureters within 20 min of administration which was sustained up to 120 min. 3-D emission heat map imaging allowed the spatial and temporal changes in intensity due to the distinctive peristaltic waves of urine being transferred from the kidneys to the bladder to be identified. As the emission of these fluorophores could be spectrally distinguished from the clinically-used perfusion dye indocyanine green, it is envisaged that their combined use could be a step towards intraoperative colour coding of different tissues.

Cysteine Cathepsins in Breast Cancer: Promising Targets for Fluorescence-Guided Surgery.

The majority of breast cancer patients is treated with breast-conserving surgery (BCS) combined with adjuvant radiation therapy. Up to 40% of patients has a tumor-positive resection margin after BCS, which necessitates re-resection or additional boost radiation. Cathepsin-targeted near-infrared fluorescence imaging during BCS could be used to detect residual cancer in the surgical cavity and guide additional resection, thereby preventing tumor-positive resection margins and associated mutilating treatments. The cysteine cathepsins are a family of proteases that play a major role in normal cellular physiology and neoplastic transformation. In breast cancer, the increased enzymatic activity and aberrant localization of many of the cysteine cathepsins drive tumor progression, proliferation, invasion, and metastasis. The upregulation of cysteine cathepsins in breast cancer cells indicates their potential as a target for intraoperative fluorescence imaging. This review provides a summary of the current knowledge on the role and expression of the most important cysteine cathepsins in breast cancer to better understand their potential as a target for fluorescence-guided surgery (FGS). In addition, it gives an overview of the cathepsin-targeted fluorescent probes that have been investigated preclinically and in breast cancer patients. The current review underscores that cysteine cathepsins are highly suitable molecular targets for FGS because of favorable expression and activity patterns in virtually all breast cancer subtypes. This is confirmed by cathepsin-targeted fluorescent probes that have been shown to facilitate in vivo breast cancer visualization and tumor resection in mouse models and breast cancer patients. These findings indicate that cathepsin-targeted FGS has potential to improve treatment outcomes in breast cancer patients.

Formulation of a Thermosensitive Imaging Hydrogel for Topical Application and Rapid Visualization of Tumor Margins in the Surgical Cavity.

BACKGROUND: Tumor-positive surgical margins during primary breast cancer (BCa) surgery are associated with a two-fold increase in the risk of local recurrence when compared with tumor-negative margins. Pathological microscopic evaluation of the samples only assesses about 1/10 of 1% of the entire volume of the removed BCa specimens, leading to margin under-sampling and potential local recurrence in patients with pathologically clean margins, i.e., false negative margins. In the case of tumor-positive margins, patients need to undergo re-excision and/or radiation therapy, resulting in increases in complications, morbidity, and healthcare costs. Development of a simple real-time imaging technique to identify residual BCa in the surgical cavity rapidly and precisely could significantly improve the quality of care. METHODS: A small-molecule, fluorescently quenched protease-substrate probe, AKRO-QC-ICG, was tested as part of a thermosensitive imaging gel formulated for topical application and imaging of the BCa surgical cavity. RESULTS: More than forty formulations of gel mixtures were investigated to enable easy fluid application and subsequent solidification once applied, preventing dripping and pooling in the surgical cavity. The final formulation was tested using human BCa orthotopic implants in nude and NSG patient-derived xenografts (PDX) mice. This formulation of Pluronic F-127/DMSO/AKRO-QC-ICG imaging gel was found to be a good solvent for the probe, with a desirable thermo-reversible solid-gel transition and mechanical strength for distribution of AKRO-QC-ICG on the surfaces of tissue. It demonstrated excellent ability to detect BCa tissue after 10 min exposure, with a high signal-to-noise ratio both in mouse xenografts and freshly excised human lumpectomy tissue. The in vivo efficacy of the AKRO-QC-ICG imaging gel to detect BCa revealed the levels of sensitivity/specificity = 0.92/1 in 12 nude mice, which was corroborated with the sensitivity/specificity = 0.94/1 in 10 PDX mice. CONCLUSIONS: Utilization of Pluronic F-127/DMSO/AKRO-QC-ICG imaging gel for topical application to detect BCa in the surgical cavity during surgery has the potential to reduce re-excisions, with consequent savings in healthcare costs and enhancement in patient quality of life.

First-in-Human Assessment of cRGD-ZW800-1, a Zwitterionic, Integrin-Targeted, Near-Infrared Fluorescent Peptide in Colon Carcinoma.

PURPOSE: Incomplete oncologic resections and damage to vital structures during colorectal cancer surgery increases morbidity and mortality. Moreover, neoadjuvant chemoradiotherapy has become the standard treatment modality for locally advanced rectal cancer, where subsequent downstaging can make identification of the primary tumor more challenging during surgery. Near-infrared (NIR) fluorescence imaging can aid surgeons by providing real-time visualization of tumors and vital structures during surgery. EXPERIMENTAL DESIGN: We present the first-in-human clinical experience of a novel NIR fluorescent peptide, cRGD-ZW800-1, for the detection of colon cancer. cRGD-ZW800-1 was engineered to have an overall zwitterionic chemical structure and neutral charge to lower nonspecific uptake and thus background fluorescent signal. We performed a phase I study in 11 healthy volunteer as well as a phase II feasibility study in 12 patients undergoing an elective colon resection, assessing 0.005, 0.015, and 0.05 mg/kg cRGD-ZW800-1 for the intraoperative visualization of colon cancer. RESULTS: cRGD-ZW800-1 appears safe, and exhibited rapid elimination into urine after a single low intravenous dose. Minimal invasive intraoperative visualization of colon cancer through full-thickness bowel wall was possible after an intravenous bolus injection of 0.05 mg/kg at least 2 hours prior to surgery. Longer intervals between injection and imaging improved the tumor-to-background ratio. CONCLUSIONS: cRGD-ZW800-1 enabled fluorescence imaging of colon cancer in both open and minimal invasive surgeries. Further development of cRGD-ZW800-1 for widespread use in cancer surgery may be warranted given the ubiquitous overexpression of various integrins on different types of tumors and their vasculature.

Small Molecules for Multi-Wavelength Near-Infrared Fluorescent Mapping of Regional and Sentinel Lymph Nodes in Colorectal Cancer Staging.

Assessing lymph node (LN) status during tumor resection is fundamental for the staging of colorectal cancer. Current guidelines require a minimum of 12 LNs to be harvested during resection and ultra-staging regional lymph nodes by sentinel lymph node (SLN) assessment is being extensively investigated. The current study presents novel near-infrared (NIR) fluorescent dyes for simultaneous pan lymph node (PanLN; regional) and SLN mapping. PanLN-Forte was intravenously injected in mice and assessed for accumulation in regional LNs. SLN800 was injected intradermally in mice, after which the collection and retention of fluorescence in SLNs were measured using indocyanine green (ICG) and its precursor, SLN700, as references. LNs in the cervical, inguinal, jejunal, iliac, and thoracic basins could clearly be distinguished after a low dose intravenous injection of PanLN-Forte. Background fluorescence was significantly lower compared to the parent compound ZW800-3A (p < 0.001). SLN700 and SLN800 specifically targeted SLNs with fluorescence being retained over 40-fold longer than the current clinically used agent ICG. Using SLN700 and SLN800, absolute fluorescence in SLN was at least 10 times higher than ICG in second-tier nodes, even at 1 hour post-injection. Histologically, the fluorescent signal localized in the LN medulla (PanLN-Forte) or sinus entry (SLN700/SLN800). PanLN-Forte and SLN800 appear to be optimal for real-time NIR fluorescence imaging of regional and SLNs, respectively.

Multimodal image-guided surgery of HER2-positive breast cancer using [111In]In-DTPA-trastuzumab-IRDye800CW in an orthotopic breast tumor model.

BACKGROUND: Combining modalities using dual-labeled antibodies may allow preoperative and intraoperative tumor localization and could be used in image-guided surgery to improve complete tumor resection. Trastuzumab is a monoclonal antibody against the human epidermal growth factor-2 (HER2) receptor and dual-labeled trastuzumab with both a fluorophore (IRDye800CW) and a radioactive label (111In) can be used for multimodal imaging of HER2-positive breast cancer. The aim of this study was to demonstrate the feasibility of HER2-targeted multimodal imaging using [111In]In-DTPA-trastuzumab-IRDye800CW in an orthotopic breast cancer model. METHODS: Trastuzumab was conjugated with p-isothiocyanatobenzyl (ITC)-diethylenetriaminepentaacetic acid (DTPA) and IRDye800CW-NHS ester and subsequently labeled with 111In. In a dose escalation study, the biodistribution of 10, 30, and 100 µg [111In]In-DTPA-trastuzumab-IRDye800CW was determined 48 h after injection in BALB/c nude mice with orthotopic high HER2-expressing tumors. Also, a biodistribution study was performed in a low HER2-expressing breast cancer model. In addition, multimodal image-guided surgery was performed in each group. Autoradiography, fluorescence microscopy, and immunohistochemically stained slices of the tumors were compared for co-localization of tumor tissue, HER2 expression, fluorescence, and radiosignal. RESULTS: Based on the biodistribution data, a 30 µg dose of dual-labeled trastuzumab (tumor-to-blood ratio 13 ± 2) was chosen for all subsequent studies. [111In]In-DTPA-trastuzumab-IRDye800CW specifically accumulated in orthotopic HER2-positive BT474 tumors (101 ± 7 %IA/g), whereas uptake in orthotopic low HER2-expressing MCF7 tumor was significantly lower (1.2 ± 0.2 %IA/g, p = 0.007). BT474 tumors could clearly be visualized with both micro-SPECT/CT, fluorescence imaging and subsequently, image-guided resection was performed. Immunohistochemical analyses of BT474 tumors demonstrated correspondence in fluorescence, radiosignal, and high HER2 expression. CONCLUSIONS: Dual-labeled trastuzumab showed specific accumulation in orthotopic HER2-positive BT474 breast tumors with micro-SPECT/CT and fluorescence imaging and enabled image-guided tumor resection. In the clinical setting, [111In]In-DTPA-trastuzumab-IRDye800CW could be valuable for preoperative detection of (metastatic) tumors by SPECT/CT imaging, and intraoperative localization by using a gamma probe and fluorescence image-guided surgery to improve radical resection of tumor tissue in patients with HER2-positive tumors.

Synthesis, structure, and biological applications of α-fluorinated ß-amino acids and derivatives.

This review gives a broad overview of the state of play with respect to the synthesis, conformational properties, and biological activity of α-fluorinated ß-amino acids and derivatives. General methods are described for the preparation of monosubstituted α-fluoro-ß-amino acids (Scheme 1). Nucleophilic methods for the introduction of fluorine predominantly involve the reaction of DAST with alcohols derived from α-amino acids, whereas electrophilic sources of fluorine such as NFSI have been used in conjunction with Arndt-Eistert homologation, conjugate addition or organocatalyzed Mannich reactions. α,α-Difluoro-ß-amino acids have also been prepared using DAST; however, this area of synthesis is largely dominated by the use of difluorinated Reformatsky reagents to introduce the difluoro ester functionality (Scheme 9). α-Fluoro-ß-amino acids and derivatives analyzed by X-ray crystal and NMR solution techniques are found to adopt preferred conformations which are thought to result from stereoelectronic effects associated with F located close to amines, amides, and esters (Figs. 2-6). α-Fluoro amide and ß-fluoro ethylamide/amine effects can influence the secondary structure of α-fluoro-ß-amino acid-containing derivatives including peptides and peptidomimetics (Figs. 7-9). α-Fluoro-ß-amino acids are also components of a diverse range of bioactive anticancer (e.g., 5-fluorouracil), antifungal, and antiinsomnia agents as well as protease inhibitors where such fluorinated analogs have shown increased potency and spectrum of activity.

Diastereoselective synthesis of aliphatic α,α-difluoro-ß3-amino esters via a sonocatalyzed Reformatsky reaction.

(R)-2-Phenylglycine ethyl ester was found to be a cheap and effective auxiliary for the preparation of aliphatic α,α-difluoro-ß(3)-amino esters via a Reformatsky reaction performed under sonication conditions. The products were obtained in good to high yield and ≥96:4 dr, thus providing a new stereoselective route to this under-represented class of compounds. A facile one-pot removal of the phenylglycine moiety and concomitant Boc protection subsequently afforded the corresponding Boc-protected ß(3)-amino esters in excellent yield.

Enantioselective synthesis of α-fluoro-ß(3)-amino esters: synthesis of enantiopure, orthogonally protected α-fluoro-ß(3)-lysine.

The scope of a tandem conjugate addition-fluorination sequence performed on α,ß-unsaturated esters using the enantiopure lithium amide derived from (S)-N-benzyl-N-(α-methylbenzyl)amine, and the electrophilic fluorinating agent N-fluorobenzenesulfonimide has been investigated. Using this method, α-fluoro-ß(3)-amino esters can be obtained in up to quantitative yield and 80:20 to >99:1 dr. This simple methodology does not rely on the use of α-amino acids from the chiral pool and thus provides the potential for the preparation of enantiopure α-fluoro-ß(3)-amino acids with a wide variety of side chains. Its utility was demonstrated through the synthesis of orthogonally protected (2S,3S)-α-fluoro-ß(3)-lysine.