Diarylidene-N-Methyl-4-Piperidones and Spirobibenzopyrans as Antioxidant and Anti-Inflammatory Agents.

Curcumin has antioxidant properties resulting from its radical scavenging ability and inhibition of inflammation-associated factors. However, its lack of solubility, instability, and poor bioavailability are impediments to its therapeutic use. As potential alternatives, we synthesized and performed chemical analysis of thirty diarylidene-N-methyl-4-piperidone (DANMP), diheteroarylidene-N-methyl-4-piperidone (DHANMP), and spirobibenzopyran (SBP) derivatives, one of which was also characterized by single crystal X-ray diffraction. All compounds were evaluated for antioxidant activity via 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and for drug-like properties in silico. A subset of five compounds was investigated in terms of aqueous solubilities, which were significantly improved compared to that of curcumin. In vitro assessments of cellular and anti-inflammatory effects were conducted via real time polymerase chain reaction (RT-PCR) and Griess assays to evaluate the presence of inflammatory/activated (M1) markers and production of nitric oxide (NO) species, which are associated with inflammation. The five compounds reduced levels of markers and NO to extents similar to or better than curcumin in inflamed cells, and showed no adverse effects on cell viability. We show that these compounds possess anti-inflammatory properties and may be used as curcumin-substitutes with improved characteristics.

Macrophage-Encapsulated Bioorthogonal Nanozymes for Targeting Cancer Cells.

Macrophages migrate to tumor sites by following chemoattractant gradients secreted by tumor cells, providing a truly active targeting strategy for cancer therapy. However, macrophage-based delivery faces challenges of cargo loading, control of release, and effects of the payload on the macrophage vehicle. We present a strategy that employs bioorthogonal "nanozymes" featuring transition metal catalysts (TMCs) to provide intracellular "factories" for the conversion of prodyes and prodrugs into imaging agents and chemotherapeutics. These nanozymes solubilize and stabilize the TMCs by embedding them into self-assembled monolayer coating gold nanoparticles. Nanozymes delivered into macrophages were intracellularly localized and retained activity even after prolonged (72 h) incubation. Significantly, nanozyme-loaded macrophages maintained their inherent migratory ability toward tumor cell chemoattractants, efficiently killing cancer cells in cocultures. This work establishes the potential of nanozyme-loaded macrophages for tumor site activation of prodrugs, providing readily tunable dosages and delivery rates while minimizing off-target toxicity of chemotherapeutics.

Targeted Optical Imaging Agents in Cancer: Focus on Clinical Applications.

Molecular imaging is an emerging strategy for in vivo visualization of cancer over time based on biological mechanisms of disease activity. Optical imaging methods offer a number of advantages for real-time cancer detection, particularly in the epithelium of hollow organs and ducts, by using a broad spectral range of light that spans from visible to near-infrared. Targeted ligands are being developed for improved molecular specificity. These platforms include small molecule, peptide, affibody, activatable probes, lectin, and antibody. Fluorescence labeling is used to provide high image contrast. This emerging methodology is clinically useful for early cancer detection by identifying and localizing suspicious lesions that may not otherwise be seen and serves as a guide for tissue biopsy and surgical resection. Visualizing molecular expression patterns may also be useful to determine the best choice of therapy and to monitor efficacy. A number of these imaging agents are overcoming key challenges for clinical translation and are being validated in vivo for a wide range of human cancers.

Surface-Modified Macrophages Facilitate Tracking of Breast Cancer-Immune Interactions.

The immune system has been found to play key roles in cancer development and progression. Macrophages are typically considered to be pro-inflammatory cells but can also facilitate pro-oncogenic activities via associations with tumors and metastases. The study of macrophages and their interactions within the context of cancer microenvironments is stymied by the lack of a system to track them. We present a cell-based strategy for studying cancer-immune cell interactions by chemically modifying the surfaces of macrophages with fluorophores. Two widely used methods are employed, affecting cell surface proteins and glycans via NHS-ester and Staudinger ligation reactions, respectively. We show that these modifications do not interfere with macrophage responses to chemoattractants and that interactions with cancer cells can be readily monitored. This work describes the development of macrophage-based imaging agents for tumor detection and assessment of interactions between immune cells and cancers.

Multiplexed Targeting of Barrett's Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft in Vivo and Human Specimens ex Vivo.

Esophageal adenocarcinoma (EAC) is a molecularly heterogeneous disease that is rising rapidly in incidence and has poor prognosis. We developed a heterobivalent peptide to target detection of early Barrett's neoplasia by combining monomer heptapeptides specific for either EGFR or ErbB2 in a heterodimer configuration. The structure of a triethylene glycol linker was optimized to maximize binding interactions to the surface receptors on cells. The Cy5.5-labeled heterodimer QRH*-KSP*-E3-Cy5.5 demonstrated specific binding to each target and showed 3-fold greater fluorescence intensity and 2-fold higher affinity compared with those of either monomer alone. Peak uptake in xenograft tumors was observed at 2 h postinjection with systemic clearance by ∼24 h in vivo. Furthermore, ligand binding was evaluated on human esophageal specimens ex vivo, and 88% sensitivity and 87% specificity were found for the detection of either high-grade dysplasia (HGD) or EAC. This peptide heterodimer shows promise for targeted detection of early Barrett's neoplasia in clinical study.

Harnessing Biology to Deliver Therapeutic and Imaging Entities via Cell-Based Methods.

The accumulation of therapeutic and imaging agents at sites of interest is critical to their efficacy. Similarly, off-target effects (especially toxicity) are a major liability for these entities. For this reason, the use of delivery vehicles to improve the distribution characteristics of bio-active agents has become ubiquitous in the field. However, the majority of traditionally employed, cargo-bearing platforms rely on passive accumulation. Even in cases where "targeting" functionalities are used, the agents must first reach the site in order for the ligand-receptor interaction to occur. The next stage of vehicle development is the use of "recruited" entities, which respond to biological signals produced in the tissues to be targeted, resulting in improved specificities. Recently, many advances have been made in the utilization of cells as delivery agents. They are biocompatible, exhibit excellent circulation lifetimes and tissue penetration capabilities, and respond to chemotactic signals. In this Minireview, we will explore various cell types, modifications, and applications where cell-based delivery agents are used.

In vivo near-infrared imaging of ErbB2 expressing breast tumors with dual-axes confocal endomicroscopy using a targeted peptide.

ErbB2 expression in early breast cancer can predict tumor aggressiveness and clinical outcomes in large patient populations. Accurate assessment with physical biopsy and conventional pathology can be limited by tumor heterogeneity. We aim to demonstrate real-time optical sectioning using a near-infrared labeled ErbB2 peptide that generates tumor-specific contrast in human xenograft breast tumors in vivo. We used IRDye800CW as the fluorophore, validated performance characteristics for specific peptide binding to cells in vitro, and investigated peak peptide uptake in tumors using photoacoustic tomography. We performed real-time optical imaging using a handheld dual-axes confocal fluorescence endomicroscope that collects light off-axis to reduce tissue scattering for greater imaging depths. Optical sections in either the vertical or horizontal plane were collected with sub-cellular resolution. Also, we found significantly greater peptide binding to pre-clinical xenograft breast cancer in vivo and to human specimens of invasive ductal carcinoma that express ErbB2 ex vivo. We used a scrambled peptide for control. Peptide biodistribution showed high tumor uptake by comparison with other organs to support safety. This novel integrated imaging strategy is promising for visualizing ErbB2 expression in breast tumors and serve as an adjunct during surgery to improve diagnostic accuracy, identify tumor margins, and stage early cancers.

Detection of Sessile Serrated Adenomas in the Proximal Colon Using Wide-Field Fluorescence Endoscopy.

BACKGROUND & AIMS: Many cancers in the proximal colon develop via from sessile serrated adenomas (SSAs), which have flat, subtle features that are difficult to detect with conventional white-light colonoscopy. Many SSA cells have the V600E mutation in BRAF. We investigated whether this feature could be used with imaging methods to detect SSAs in patients. METHODS: We used phage display to identify a peptide that binds specifically to SSAs, using subtractive hybridization with HT29 colorectal cancer cells containing the V600E mutation in BRAF and Hs738.St/Int cells as a control. Binding of fluorescently labeled peptide to colorectal cancer cells was evaluated with confocal fluorescence microscopy. Rats received intra-colonic 0.0086 mg/kg, 0.026 mg/kg, or 0.86 mg/kg peptide or vehicle and morbidity, mortality, and injury were monitored twice daily to assess toxicity. In the clinical safety study, fluorescently labeled peptide was topically administered, using a spray catheter, to the proximal colon of 25 subjects undergoing routine outpatient colonoscopies (3 subjects were given 2.25 µmol/L and 22 patients were given 76.4 µmol/L). We performed blood cell count, chemistry, liver function, and urine analyses approximately 24 hours after peptide administration. In the clinical imaging study, 38 subjects undergoing routine outpatient colonoscopies, at high risk for colorectal cancer, or with a suspected unresected proximal colonic polyp, were first evaluated by white-light endoscopy to identify suspicious regions. The fluorescently labeled peptide (76.4 µmol/L) was administered topically to proximal colon, unbound peptide was washed away, and white-light, reflectance, and fluorescence videos were recorded digitally. Fluorescence intensities of SSAs were compared with those of normal colonic mucosa. Endoscopists resected identified lesions, which were analyzed histologically by gastrointestinal pathologists (reference standard). We also analyzed the ability of the peptide to identify SSAs vs adenomas, hyperplastic polyps, and normal colonic mucosa in specimens obtained from the tissue bank at the University of Michigan. RESULTS: We identified the peptide sequence KCCFPAQ and measured an apparent dissociation constant of Kd = 72 nM and an apparent association time constant of K = 0.174 min-1 (5.76 minutes). During fluorescence imaging of patients during endoscopy, regions of SSA had 2.43-fold higher mean fluorescence intensity than that for normal colonic mucosa. Fluorescence labeling distinguished SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity. The peptide had no observed toxic effects in animals or patients. In the analysis of ex vivo specimens, peptide bound to SSAs had significantly higher mean fluorescence intensity than to hyperplastic polyps. CONCLUSIONS: We have identified a fluorescently labeled peptide that has no observed toxic effects in animals or humans and can be used for wide-field imaging of lesions in the proximal colon. It distinguishes SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity. This targeted imaging method might be used in early detection of premalignant serrated lesions during routine colonoscopies. ClinicalTrials.gov ID: NCT02156557.

Overexpressed Claudin-1 Can Be Visualized Endoscopically in Colonic Adenomas In Vivo.

BACKGROUND & AIMS: Conventional white-light colonoscopy aims to reduce the incidence and mortality of colorectal cancer (CRC). CRC has been found to arise from missed polypoid and flat precancerous lesions. We aimed to establish proof-of-concept for real-time endoscopic imaging of colonic adenomas using a near-infrared peptide that is specific for claudin-1. METHODS: We used gene expression profiles to identify claudin-1 as a promising early CRC target, and performed phage display against the extracellular loop of claudin-1 (amino acids 53-80) to identify the peptide RTSPSSR. With a Cy5.5 label, we characterized binding parameters and showed specific binding to human CRC cells. We collected in vivo near-infrared fluorescence images endoscopically in the CPC;Apc mouse, which develops colonic adenomas spontaneously. With immunofluorescence, we validated specific peptide binding to adenomas from the proximal human colon. RESULTS: We found a 2.5-fold increase in gene expression for claudin-1 in human colonic adenomas compared with normal. We showed specific binding of RTSPSSR to claudin-1 in knockdown and competition studies, and measured an affinity of 42 nmol/L and a time constant of 1.2 minutes to SW620 cells. In the mouse, we found a significantly higher target-to-background ratio for both polypoid and flat adenomas compared with normal by in vivo images. On immunofluorescence, we found significantly greater intensity for human adenomas (mean ± SD, 25.5 ± 14.0) vs normal (mean ± SD, 9.1 ± 6.0) and hyperplastic polyps (mean ± SD, 3.1 ± 3.7; P = 10-5 and 8 × 10-12, respectively), and for sessile serrated adenomas (mean ± SD, 20.1 ± 13.3) vs normal and hyperplastic polyps (P = .02 and 3 × 10-7, respectively). CONCLUSIONS: Claudin-1 is overexpressed in premalignant colonic lesions, and can be detected endoscopically in vivo with a near-infrared, labeled peptide.

In vivo photoacoustic tomography of EGFR overexpressed in hepatocellular carcinoma mouse xenograft.

EGFR is a promising cell surface target for in vivo imaging that is highly overexpressed in hepatocellular carcinoma (HCC), a common cancer worldwide. Peptides penetrate easily into tumors for deep imaging, and clear rapidly from the circulation to minimize background. We aim to demonstrate use of an EGFR specific peptide to detect HCC xenograft tumors in mice with photoacoustic imaging. Nude mice implanted with human HCC cells that overexpress EGFR were injected intravenously with Cy5.5-labeled EGFR and scrambled control peptides respectively. Photoacoustic images collected from 0 to 24 h. Photoacoustic signal peaked in tumors at 3 h post-injection. Images from 0 to 1.8 cm beneath the skin revealed increased target-to-background (T/B) ratio from tumors. The T/B ratio was significantly greater for the EGFR versus control peptide. Clearance of signal was observed by ∼24 h. EGFR overexpression was validated with immunofluorescence and immunohistochemistry. A peptide specific for EGFR delivered systemically can detect HCC xenograft tumors in vivo with photoacoustic imaging.

Gastrointestinal imaging in 2015: Emerging trends in endoscopic imaging.

Several key papers published in 2015 highlight important emerging trends in endoscopic imaging that promise to improve patient diagnosis and guidance of therapy. These studies reflect the future role for 'smart' contrast agents and fluorescence endoscopes to provide a molecular basis for disease detection, identify precancerous lesions and determine optimal choice of therapy.

Multimodal endoscope can quantify wide-field fluorescence detection of Barrett's neoplasia.

BACKGROUND AND STUDY AIMS: To demonstrate the clinical use of a multimodal endoscope with a targeted fluorescently labeled peptide for quantitative detection of Barrett's neoplasia. PATIENTS AND METHODS: We studied 50 patients with Barrett's esophagus using a prototype multimodal endoscope with a fluorescently labeled peptide. Co-registered fluorescence and reflectance images were converted to ratios to correct for differences in distance and geometry over the image field of view. The ratio images were segmented using a unique threshold that maximized the variance between high and low intensities to localize regions of high grade dysplasia (HGD) and esophageal adenocarcinoma (EAC). RESULTS: Early neoplasia (HGD and EAC) was identified with 94 % specificity and 96 % positive predictive value at a threshold of 1.49. The mean results for HGD and EAC were significantly greater than those for squamous/Barrett's esophagus and low grade dysplasia by one-way analysis of variance (ANOVA). The receiver operator characteristic curve for detection of early neoplasia had an area under the curve of 0.884. No adverse events associated with the endoscope or peptide were found. CONCLUSION: A multimodal endoscope can quantify fluorescence images from targeted peptides to localize early Barrett's neoplasia. (ClinicalTrials.gov number NCT01630798.).

Design and Synthesis of Near-Infrared Peptide for in Vivo Molecular Imaging of HER2.

We report the development, characterization, and validation of a peptide specific for the extracellular domain of HER2. This probe chemistry was developed for molecular imaging by using a structural model to select an optimal combination of amino acids that maximize the likelihood for unique hydrophobic and hydrophilic interactions with HER2 domain 3. The sequence KSPNPRF was identified and conjugated with either FITC or Cy5.5 via a GGGSK linker using Fmoc-mediated solid-phase synthesis to demonstrate flexibility for this chemical structure to be labeled with different fluorophores. A scrambled sequence was developed for control by altering the conformationally rigid spacer and moving both hydrophobic and hydrophilic amino acids on the C-terminus. We validated peptide specificity for HER2 in knockdown and competition experiments using human colorectal cancer cells in vitro, and measured a binding affinity of kd = 21 nM and time constant of k = 0.14 min(-1) (7.14 min). We used this peptide with either topical or intravenous administration in a preclinical model of colorectal cancer to demonstrate specific uptake in spontaneous adenomas and to show feasibility for real time in vivo imaging with near-infrared fluorescence. We used this peptide in immunofluorescence studies of human proximal colon specimens to evaluate specificity for sessile serrated and sporadic adenomas. Improved visualization can be used endoscopically to guide tissue biopsy and detect premalignant lesions that would otherwise be missed. Our peptide design for specificity to HER2 is promising for clinical translation in molecular imaging methods for early cancer detection.

MEMS-based multiphoton endomicroscope for repetitive imaging of mouse colon.

We demonstrate a handheld multiphoton endomicroscope with 3.4 mm distal diameter that can repetitively image mouse colon in vivo. A 2D resonant MEMS mirror was developed to perform beam scanning in a Lissajous pattern. The instrument has an effective numerical aperture of 0.63, lateral and axial resolution of 2.03 and 9.02 µm, respectively, working distance of 60 µm, and image field-of-view of 300 × 300 µm(2). Hoechst was injected intravenously in mice to stain cell nuclei. We were able to collect histology-like images in vivo at 5 frames/sec, and distinguish between normal and pre-malignant colonic epithelium.

EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging.

OBJECTIVES: Colorectal cancer initially lies dormant as dysplasia, a premalignant state that provides an opportunity for early cancer detection. Dysplasia can be flat in morphology, focal in size, and patchy in distribution, and thus it appears "invisible" on conventional wide-field endoscopy. AIMS: We aim to develop and validate a peptide that is specific for epidermal growth factor receptor (EGFR), a cell surface target that is overexpressed in colonic adenomas and is readily accessible for imaging. METHODS: We expressed and purified the extracellular domain of EGFR for use with phage display to identify a peptide QRHKPRE that binds to domain 2 of this target. A near-infrared fluorescence endoscope was used to perform in vivo imaging to validate specific peptide binding to spontaneous colonic adenomas in a mouse model with topical administration. We also validated specific peptide binding to human colonic adenomas on immunohistochemistry and immunofluorescence. RESULTS: After labeling with Cy5.5, we validated specific peptide binding to EGFR on knockdown and competition studies. Peptide binding to cells occurred within 2.46 min and had an affinity of 50 nm. No downstream signaling was observed. We measured a target-to-background ratio of 4.0±1.7 and 2.7±0.7, for polyps and flat lesions, respectively. On immunofluorescence of human colonic specimens, greater intensity from peptide binding to dysplasia than normal was found with a 19.4-fold difference. CONCLUSIONS: We have selected and validated a peptide that can be used as a specific contrast agent to identify colonic adenomas that overexpress EGFR in vivo on fluorescence endoscopy.

Targeted therapy of colorectal neoplasia with rapamycin in peptide-labeled pegylated octadecyl lithocholate micelles.

Many powerful drugs have limited clinical utility because of poor water solubility and high systemic toxicity. Here, we formulated a targeted nanomedicine, rapamycin encapsulated in pegylated octadecyl lithocholate micelles labeled with a new ligand for colorectal neoplasia, LTTHYKL peptide. CPC;Apc mice that spontaneously develop colonic adenomas were treated with free rapamycin, plain rapamycin micelles, and peptide-labeled rapamycin micelles via intraperitoneal injection for 35days. Endoscopy was performed to monitor adenoma regression in vivo. We observed complete adenoma regression at the end of therapy. The mean regression rate for peptide-labeled rapamycin micelles was significantly greater than that for plain rapamycin micelles, P<0.01. On immunohistochemistry, we observed a significant reduction in phospho-S6 but not ß-catenin expression and reduced tumor cell proliferation, suggesting greater inhibition of downstream mTOR signaling. We observed significantly reduced renal toxicity for peptide-labeled rapamycin micelles compared to that of free drug, and no other toxicities were found on chemistries. Together, this unique targeted micelle represents a potential therapeutic for colorectal neoplasia with comparable therapeutic efficacy to rapamycin free drug and significantly less systemic toxicity.

Imaging: Dynamic imaging of gut function--allowing the blind to see.

Improved methods are needed to dynamically image gut behaviour to assess whether neuromuscular degenerative disease is present. So-called nanonaps (soluble nanoformulated naphthalocyanines) have been developed for oral administration to pass through the intestines and provide high contrast for visualizing bowel motion on photoacoustic imaging. Moreover, radiotracer labelling of these nanoparticles facilitates multimodal detection using PET.