Fluorescent nanoprobes as a biomarker for increased vascular permeability: implications in diagnosis and treatment of cancer and inflammation.

This article describes the use of a fluorescent nanoprobe as a functional biomarker for the identification of increased vascular permeability in cancer/arthritis disease models. Synthesis of the fluorescent nanoprobe was achieved by passive loading of a fluorophore inside the nanoparticle using thin film hydration method. The outer layer of the nanoprobe was decorated with poly(ethylene glycol) arms to increase the bioavailability of the fluorophore. Stability studies of the nanoprobe showed that the particles were stable up to 70 days. The uptake and internalization of the fluorescent nanoprobe inside target cells was confirmed by fluorescence microscopy studies. Co-localization of the probe with the target tissue in vivo was unambiguously identified using intravital microscopy. Results from in vivo imaging studies showed that the particles had a long half-life in the circulation and passively targeted tumor or arthritic tissue. The increased and specific uptake of the fluorescent nanoprobe in tumor/arthritic tissue is attributed to an enhanced permeation and retention (EPR) effect. Use of an optical method to validate anti-inflammatory drugs in an arthritis disease model is demonstrated in this study. In general, this methodology could be used for detection of leaky vasculature in different pathological states.

In vivo assessments of mucus dynamics in the rat lung using a Gd-Cy5.5-bilabeled contrast agent for magnetic resonance and optical imaging.

Dysfunctions in mucociliary clearance are associated with the accelerated loss of lung function in several respiratory diseases. Approaches enabling the in vivo visualization of mucus dynamics in rodents at high resolution and sensitivity would be beneficial for experimental lung research. We describe the synthesis and characterization of two bilabeled amino dextran-based probes binding specifically to mucin. Labeling of secreted mucus and of mucin in goblet cells in the lungs of lipopolysaccharide-challenged rats has been demonstrated in vivo with near-infrared fluorescence and MRI and confirmed by histology. The effects of uridine triphosphate were then studied in lipopolysaccharide-challenged rats by simultaneously administering the imaging probe and the compound. The data suggest that uridine triphosphate increased the mucociliary clearance, but at the same time induced a release of mucin from goblet cells, thus not contributing to the overall reduction of mucus in the lung. The approach outlined here enables one to derive information on mucus clearance, as well as secretion. Such a global view on mucus dynamics may prove invaluable when testing new pharmacological agents aimed at improving mucociliary clearance.

Investigating pharmacology in vivo using magnetic resonance and optical imaging.

The better and earlier a disease can be diagnosed and characterized, the greater the chance of being able to intervene in this process with a chemical entity. This is the rationale for the use of in vivo imaging techniques in the drug discovery and development process. In this article we address the value of two imaging modalities in this area, i.e. magnetic resonance imaging (MRI) and optical imaging. The multiparametric nature of MRI enables anatomical, functional, metabolic and, to a certain extent, also cellular and target-related information to be obtained noninvasively at high spatial resolution. This favours characterization of a disease state and the corresponding drug intervention. The noninvasiveness of MRI strengthens the link between preclinical and clinical pharmaceutical research. The high sensitivity of optical techniques enables molecular information to be obtained in vivo. Within pharmacological research, the main applications of optical techniques relate to the early drug discovery process and acquisition of target-related information. However, potential clinical applications of optical imaging are also emerging. The complementary character of both imaging modalities renders them useful in various portions of the drug discovery process, from early target selection and validation to clinical studies.

In vivo monitoring the fate of Cy5.5-Tat labeled T lymphocytes by quantitative near-infrared fluorescence imaging during acute brain inflammation in a rat model of experimental autoimmune encephalomyelitis.

T cells and macrophages directed against myelin proteins orchestrate the inflammation process in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). So far, assessment of macrophages infiltration or structural alterations has been achieved by in vivo imaging. In this work, we show the infiltration of Cy5.5-labeled T lymphocytes into the brains of EAE rats by reflectance near-infrared fluorescence imaging. T lymphocytes were labeled with Cy5.5-Tat and administered intravenously to naïve or EAE animals. The highest fluorescence signal was observed for EAE animals, which received myelin-activated T cells during the acute phase of the disease. The temporal profile of fluorescence in this group paralleled the pattern of neurological impairment during the acute phase, the remittance and first relapses of EAE. No disease specific fluorescence pattern was observed for EAE animals, which received naïve T cells. However, uptake of Cy5.5-Tat by scavenger cells (e.g. macrophages) following death of labeled T cells in vivo prevents prolonged longitudinal studies. Our work demonstrates that Cy5.5-Tat labeling of T cells is suitable for in vivo fluorescence imaging of inflammation initiation in the EAE model. This approach may particularly be useful for evaluation of novel anti-inflammatory therapies.

In vivo mouse imaging and spectroscopy in drug discovery.

Imaging modalities such as micro-computed tomography (micro-CT), micro-positron emission tomography (micro-PET), high-resolution MRI, optical imaging, and high-resolution ultrasound have become invaluable tools in preclinical pharmaceutical research. They can be used to non-invasively investigate, in vivo, rodent biology and metabolism, disease models, and pharmacokinetics and pharmacodynamics of drugs. The advantages and limitations of each approach usually determine its application, and therefore a small-rodent imaging laboratory in a pharmaceutical environment should ideally provide access to several techniques. In this paper we aim to illustrate how these techniques may be used to obtain meaningful information for the phenotyping of transgenic mice and for the analysis of compounds in murine models of disease.

In vivo detection of amyloid-beta deposits by near-infrared imaging using an oxazine-derivative probe.

As Alzheimer's disease pathogenesis is associated with the formation of insoluble aggregates of amyloid beta-peptide, approaches allowing the direct, noninvasive visualization of plaque growth in vivo would be beneficial for biomedical research. Here we describe the synthesis and characterization of the near-infrared fluorescence oxazine dye AOI987, which readily penetrates the intact blood-brain barrier and binds to amyloid plaques. Using near-infrared fluorescence imaging, we demonstrated specific interaction of AOI987 with amyloid plaques in APP23 transgenic mice in vivo, as confirmed by postmortem analysis of brain slices. Quantitative analysis revealed increasing fluorescence signal intensity with increasing plaque load of the animals, and significant binding of AOI987 was observed for APP23 transgenic mice aged 9 months and older. Thus, AOI987 is an attractive probe to noninvasively monitor disease progression in animal models of Alzheimer disease and to evaluate effects of potential Alzheimer disease drugs on the plaque load.

Near-infrared fluorescence imaging and histology confirm anomalous edematous signal distribution detected in the rat lung by MRI after allergen challenge.

PURPOSE: To address the issue concerning the predominant location, on the left anatomic side, of edematous signals detected by magnetic resonance imaging (MRI) in the lungs of actively sensitized rats following intratracheal (IT) allergen challenge. MATERIALS AND METHODS: Near-infrared fluorescence (NIRF) imaging was used to detect the lobular distribution in the lungs of normal rats of an IT instilled fluorescent dye, Cy5.5. Actively sensitized Brown Norway rats were examined by MRI 24 hours after IT administration of ovalbumin. The perivascular edema was quantified by histology in the different lobes of lungs removed from the same animals immediately after the MRI acquisitions. RESULTS: An uneven distribution of Cy5.5 was found, predominantly on the left lobe, paralleling the localized development of allergic pulmonary inflammation in the left lobe detected as edematous signal by MRI and confirmed by histology. The patterns of the distributions of the dye between and within the lobes were very similar to those of perivascular edema assessed histologically. CONCLUSION: The data indicate a relationship between the molecular deposition of the dye detected by NIRF in the lungs and the distribution of allergen eliciting the development of pulmonary inflammation in actively rats. The combination of MRI with NIRF imaging may provide important information in preclinical pharmacologic research in the area of airway diseases. While MRI is able to address the effects of compounds on the inflammatory response in models of airways diseases, NIRF imaging may provide important insights on drug distribution and interaction in the lung, being thus suited for molecular imaging studies.

Noninvasive assessment of gastric emptying by near-infrared fluorescence reflectance imaging in mice: pharmacological validation with tegaserod, cisapride, and clonidine.

Noninvasive near-infrared fluorescence reflectance imaging (FRI) is an in vivo technique to assess physiological and molecular processes in the intact organism. Here we describe a method to assess gastric emptying in mice. TentaGel beads with covalently bound cyanine dye (Cy5.5) conjugates as fluorescent probe were administered by oral gavage. The amount of intragastric beads/label was derived from the fluorescence signal intensity measured in a region of interest corresponding to the mouse stomach. The FRI signal intensity decreased as a function of time reflecting gastric emptying. In control mice, the gastric half-emptying time was in agreement with literature data. Pharmacological modulation of gastric motility allowed the evaluation of the sensitivity of the FRI-based method. Gastric emptying was either stimulated or inhibited by treatment with the 5-HT(4) receptor agonists tegaserod (Zelnorm) and cisapride or the alpha(2)-receptor agonist clonidine, respectively. Tegaserod and cisapride dose-dependently accelerated gastric emptying. In contrast, clonidine dose-dependently delayed gastric emptying. In conclusion, FRI using fluorescently labeled beads allows the reliable determination of gastric emptying as well as the assessment of pharmacological interventions. The technique thus offers the potential to characterize molecular targets and pathways involved in physiological regulation and pharmacological modulation of gastric emptying.

Conformational studies on 1,2-di- and 1,2,3-trisubstituted heterocycles. A spectroscopic and theoretical study of 3-acylaminopicolinic Acid derivatives and their N-oxides.

Theoretical studies involving minimization of model 3-propanoylaminopicolinic acids (10d-trans, 10d-cis), methyl ester (10a), and corresponding -N-oxide derivatives (10b, 10c-trans, 10c-cis) using AM1 gave conformations contrary to both sound chemical intuition and experimental data. RHF ab initio calculations using the 6-31G and 6-31G basis sets on the other hand corroborated spectroscopic data. 3-Amidopicolinic acid derivatives (7a-9a, 7b-9b, 7c-9c, 9d) were prepared and studied by NMR and IR spectroscopy. The results show that a strong intramolecular hydrogen bond between amide-H and the 2-carboxyl substituent results in a planar molecular conformation. This is particularly profound in the 3-acylaminopicolinic acid N-oxides (c-series). When the 2-substituent is a methyl ester on the other hand, repulsion between N-oxide and ester functions induces twisting of the carbomethoxy group out of the plane of the aromatic ring. The type of method used in molecular modeling can have profound impact on the final theoretical result in the case of the above-mentioned class of compounds. Our results indicate, that it is advisable to employ ab initio methods for modeling these types of compounds, and further, that the choice of basis set used for such calculations should depend on the type of information required. Thus, for most purposes pertaining to molecular conformation the 6-31G basis set provides sufficiently sound data in relatively short CPU time. For data related to electronic properties such as involvement of the N-oxide function or spectroscopic information such as IR frequencies or (1)H or (15)N NMR chemical shifts, the use of polarization functions as contained in the 6-31G basis set seems to be a must.