Profiling alveolar macrophage responses to inhaled compounds using in vitro high content image analysis.

One of the main hurdles in the development of new inhaled medicines is the frequent observation of foamy macrophage (FM) responses in non-clinical studies in experimental animals, which raises safety concerns and hinders progress into clinical trials. We have investigated the potential of a novel multi-parameter high content image analysis (HCIA) assay as an in vitro safety screening tool to predict drug induced FM. Rat (NR8383) and human U937-derived alveolar macrophages were exposed in vitro to a panel of model compounds with different biological activity, including inhaled bronchodilators, inhaled corticosteroids (ICS), phospholipidosis inducers and proapoptotic agents. An HCIA was utilized to produce drug-induced cell response profiles based on individual cell health, morphology and lipid content parameters. The profiles of both rat and human macrophage cell lines differentiated between cell responses to marketed inhaled drugs and compounds known to induce phospholipidosis and apoptosis. Hierarchical clustering of the aggregated data allowed identification of distinct cell profiles in response to exposure to phospholipidosis and apoptosis inducers. Additionally, in NR8383 cell responses formed two distinct clusters, associated with increased vacuolation with or without lipid accumulation. U937 cells presented a similar trend but appeared less sensitive to drug exposure and presented a narrower range of responses. These results indicate that our multi-parameter HCIA assay is suitable to generate characteristic drug-induced macrophage response profiles, thus enabling differentiation of foamy macrophage phenotypes associated with phospholipidosis and apoptosis. This approach shows great potential as pre-clinical in vitro screening tool for safety assessment of candidate inhaled medicines.

Predicting Skin Permeability by Means of Computational Approaches: Reliability and Caveats in Pharmaceutical Studies.

The skin is the main barrier between the internal body environment and the external one. The characteristics of this barrier and its properties are able to modify and affect drug delivery and chemical toxicity parameters. Therefore, it is not surprising that permeability of many different compounds has been measured through several in vitro and in vivo techniques. Moreover, many different in silico approaches have been used to identify the correlation between the structure of the permeants and their permeability, to reproduce the skin behavior, and to predict the ability of specific chemicals to permeate this barrier. A significant number of issues, like interlaboratory variability, experimental conditions, data set building rationales, and skin site of origin and hydration, still prevent us from obtaining a definitive predictive skin permeability model. This review wants to show the main advances and the principal approaches in computational methods used to predict this property, to enlighten the main issues that have arisen, and to address the challenges to develop in future research.

Morphometric Characterization of Rat and Human Alveolar Macrophage Cell Models and their Response to Amiodarone using High Content Image Analysis.

PURPOSE: Progress to the clinic may be delayed or prevented when vacuolated or "foamy" alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures. METHODS: Human (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation. RESULTS: Cell health, morphology and lipid content were comparable (p < 0.05) for both cell lines and the primary macrophages in terms of vacuole number, size and lipid content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed. CONCLUSIONS: A high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.

In Vitro Multiparameter Assay Development Strategy toward Differentiating Macrophage Responses to Inhaled Medicines.

Although foamy macrophages (FMΦ) are commonly observed during nonclinical development of medicines for inhalation, there are no accepted criteria to differentiate adaptive from adverse FMΦ responses in drug safety studies. The purpose of this study was to develop a multiparameter in vitro assay strategy to differentiate and characterize different mechanisms of drug-induced FMΦ. Amiodarone, staurosporine, and poly(vinyl acetate) nanoparticles were used to induce distinct FMΦ phenotypes in J774A.1 cells, which were then compared with negative controls. Treated macrophages were evaluated for morphometry, lipid accumulation, gene expression, apoptosis, cell activation, and phagocytosis. Analysis of vacuolization (number/area vacuoles per cell) and phospholipid content revealed inducer-dependent distinctive patterns, which were confirmed by electron microscopy. In contrast to the other inducers, amiodarone increased vacuole size rather than number and resulted in phospholipid accumulation. No pronounced dysregulation of transcriptional activity or apoptosis was observed in response to sublethal concentrations of all inducers. Functionally, FMΦ induction did not affect macrophage activation by lipopolysaccharide, but it reduced phagocytic capacity, with different patterns of induction, severity, and resolution observed with the different inducers. An in vitro multiparameter assay strategy is reported that successfully differentiates and characterizes mechanisms leading to FMΦ induction by different types of agents.

Arsenic trioxide downregulates cancer procoagulant activity in MCF-7 and WM-115 cell lines in vitro.

THE AIM OF THE STUDY: To analyze human breast cancer cell line MCF-7 and human malignant melanoma cell line WM-115 in order to characterize the cellular expression of CP and to evaluate whether ATO may affect this activity, as well as the viability of the cells. MATERIAL AND METHODS: The inhibitory effect of arsenic trioxide on the proliferation of MCF-7 and WM-115 cells were measured with MTT test. The activity of cancer procoagulant after ATO exposure was determined by a specific three-stage chromogenic assay. RESULTS: ATO decreased the CP activity in a dose- and time-dependent manner in MCF-7 cells with no effect on cell proliferation at the same time. However, it affected the CP activity of WM-115 cells in a different way. Reduction in CP activity was followed by an increase after 48 h incubation. The cells viability results showed dose-and time-correlated response within high arsenic concentrations. CONCLUSIONS: Arsenic trioxide downregulates the CP expression in human breast cancer and melanoma cells.

[Arsenic trioxide: impact on the growth and differentiation of cancer cells and possible use in cancer therapy]. / Trójtlenek arsenu: wplyw na procesy wzrostu i róznicowania komórek nowotworowych oraz mozliwe zastosowanie w terapii choroby nowotworowej.

Arsenic trioxide (As2O3) has recently been identified as an effective drug in different types of cancer therapy. It is a useful pharmacological agent in acute promyelocytic leukemia (APL) treatment, especially the form that is resistant to conventional chemotherapy with all-trans retinoic acid (ATRA). What is more, laboratory data suggest that As2O3 is also active when it comes to several solid tumor cell lines. However, the mechanism of action is not fully understood. As2O3 in high doses triggers apoptosis, while in lower concentrations it induces partial differentiation. The As2O3 mechanism of action involves effects on mitochondrial transmembrane potential which lead to apoptosis. It also acts on the activity of JNK kinase, glutathione, caspases, NF-ĸB nuclear factor or pro- and antiapoptotic proteins. This publication presents the current knowledge about the influence of arsenic trioxide in cancer cells.

Longitudinal characterization of TK6 cells sequentially adapted to animal product-free, chemically defined culture medium: considerations for genotoxicity studies.

Introduction: In vitro approaches are an essential tool in screening for toxicity of new chemicals, products and therapeutics. To increase the reproducibility and human relevance of these in vitro assessments, it is advocated to remove animal-derived products such as foetal bovine serum (FBS) from the cell culture system. Currently, FBS is routinely used as a supplement in cell culture medium, but batch-to-batch variability may introduce inconsistency in inter- and intra-lab assessments. Several chemically defined serum replacements (CDSR) have been developed to provide an alternative to FBS, but not every cell line adapts easily and successfully to CDSR-supplemented medium, and the long-term effect on cell characteristics remains uncertain. Aim: The aim of this study was to adapt the TK6 cell line to animal-product free CDSR-supplemented medium and evaluate the long-term effects on cell health, growth, morphology, phenotype, and function. This included a provisional assessment to determine the suitability of the transitioned cell line for standardised genotoxicity testing using the "in vitro mammalian cell micronucleus test" (OECD TG 487). Materials and methods: Gradual adaptation and direct adaptation methodologies were compared by assessing the cell proliferation, size and viability every passage until the cells were fully adapted to animal-free CDSR. The metabolic activity and membrane integrity was assessed every 4-8 passages by PrestoBlue and CytoTox-ONE™ Homogeneous Membrane Integrity Assay respectively. A detailed morphology study by high content imaging was performed and the expression of cell surface markers (CD19 and CD20) was conducted via flow cytometry to assess the potential for phenotypic drift during longer term culture of TK6 in animal-free conditions. Finally, functionality of cells in the OECD TG 487 assay was evaluated. Results: The baseline characteristics of TK6 cells cultured in FBS-supplemented medium were established and variability among passages was used to set up acceptance criteria for CDSR adapted cells. TK6 were adapted to CDSR supplemented medium either via direct or gradual transition reducing from 10% v/v FBS to 0% v/v FBS. The cell growth rate was compromised in the direct adaptation and therefore the gradual adaptation was preferred to investigate the long-term effects of animal-free CDSR on TK6 cells. The new animal cells showed comparable (p > 0.05) viability and cell size as the parent FBS-supplemented cells, with the exception of growth rate. The new animal free cells showed a lag phase double the length of the original cells. Cell morphology (cellular and nuclear area, sphericity) and phenotype (CD19 and CD20 surface markers) were in line (p > 0.05) with the original cells. The new cells cultured in CDSR-supplemented medium performed satisfactory in a pilot OECD TG 487 assay with compounds not requiring metabolic activation. Conclusion: TK6 cells were successfully transitioned to FBS- and animal product-free medium. The new cell cultures were viable and mimicked the characteristics of FBS-cultured cells. The gradual transition methodology utilised in this study can also be applied to other cell lines of interest. Maintaining cells in CDSR-supplemented medium eliminates variability from FBS, which in turn is likely to increase the reproducibility of in vitro experiments. Furthermore, removal of animal derived products from cell culture techniques is likely to increase the human relevance of in vitro methodologies.

Polymer Architecture Effects on Poly(N,N-Diethyl Acrylamide)-b-Poly(Ethylene Glycol)-b-Poly(N,N-Diethyl Acrylamide) Thermoreversible Gels and Their Evaluation as a Healthcare Material.

Thermoreversible gels which transition between liquid-like and solid-like states when warmed have enabled significant novel healthcare technologies. Poly(N,N-diethyl acrylamide) (PDEA) is a thermoresponsive polymer which can be used as a trigger to form thermoreversible gels, however its use in these materials is limited and crucial design principles are unknown. Herein ABA copolymers with the structure PDEA-b-poly(ethylene glycol) (PEG)-b-PDEA are synthesized to give four block copolymers with varied molecular weight of PDEA and PEG blocks. Rheometry on solutions of the block copolymers reveals that high molecular weight PEG blocks are required to form thermoreversible gels with predominantly solid-like behavior. Furthermore, small-angle X-ray scattering elucidates clear differences in the nanostructure of the copolymer library which can be linked to distinct rheological behaviors. A thermoreversible gel formulation based on PDEA (20 kDa)-b-PEG (10 kDa)-b-PDEA (20 kDa) is designed by optimizing the polymer concentration and ionic strength. It is found that the gel is mucoadhesive, stable, and non-toxic, as well as giving controlled release of a hydrophobic drug. Overall, this study provides insight into the effect of polymer architecture on the nanostructure and rheology of PDEA-b-PEG-b-PDEA and presents the development of a highly functional thermoreversible gel with high promise for healthcare applications.

High Content Image Analysis as a Tool to Morphologically Distinguish Macrophage Activation and Determine Its Importance for Foamy Alveolar Macrophage Responses.

Introduction: Lung diseases are an increasing global health burden affecting millions of people worldwide. Only a few new inhaled medicines have reached the market in the last 30 years, in part due to foamy alveolar macrophage (FAM) responses observed in pre-clinical rat studies. The induction mechanism and signaling pathways involved in the development of highly vacuolated 'foamy' phenotype is not known. Furthermore, it has not been determined if these observations are adaptive or adverse responses. Aim: To determine if high content image analysis techniques can distinguish between alveolar macrophage activation (LPS/IFN-γ activated and IL-4 activated macrophages) and if this could be applied to understanding the generation of 'foamy' macrophage phenotypes. Methods: NR8383 rat alveolar macrophages were stimulated with a mix of cytokines (LPS/IFN-γ or IL-4) for 24 h. The cells were further exposed to FAM inducing-compounds amiodarone and staurosporine. Following 24 h incubation, phagocytosis and lipid accumulation were measured using flow cytometry and high content image analysis techniques. The alveolar macrophages responses after exposure to cytokines were assessed by evaluation: (i) cell surface and biochemical markers such as: nitric oxide production, arginase-1 activity and MRC-1 receptor expression (ii) cellular morphology (iii) cellular functionality (phagocytic activity and lipids accumulation). Results: Macrophages activated with LPS/IFN-γ showed distinct morphological (increased vacuolation) features and functionality (increased lipidosis, decreased phagocytic activity). Foamy macrophage phenotypes induced by amiodarone also displayed characteristics of proinflammatory macrophages (significantly increased nitric oxide production, increased vacuolation and lipidosis and decreased phagocytosis). In contrast, staurosporine treatment resulted in increased NO production, as well as arginase-1 activity. Conclusion: High content image analysis was able to determine distinct differences in morphology between non-activated and LPS/IFN-γ activated macrophages, characterized by increased vacuolation and lipidosis. When exposed to compounds that induce a FAM phenotype, healthy non-activated macrophages displayed proinflammatory (amiodarone) or pro-apoptotic (staurosporine) characteristics but these responses were independent of a change in activation status. This technique could be applied in early drug discovery safety assessment to identify immune responses earlier and increase the understanding of alveolar macrophage responses to new molecules challenge in development of new inhalation therapies, which in turn will enhance decision-making in an early safety assessment of novel drug candidates.

[All-trans retinoic acid (ATRA) in prevention and cancer therapy]. / Kwas calkowicie trans-retinowy (ATRA) w prewencji i terapii nowotworów.

Retinoids are useful pharmacological agents in therapy and prevention of cancer. All-trans retinoic acid (ATRA) is applied in chemoprevention and differentiation therapy of some cancers with particularly impressive results in the management of acute promyelocytic leukemia (APL). ATRA plays a major role in regulating growth and differentiation of a wide variety of normal and malignant cell types. ATRA mediates these effects by regulating gene transcription. Nuclear retinoic acid receptors (RARs) are considered to be the mediators of most of the effects of ATRA on gene expression. We present a current state of knowledge on the effects of ATRA on cell growth and differentiation as well as describe RARs and their role in the cellular mechanism of ATRA action. A particular attention was paid to the effects of ATRA on proliferation and differentiation of cancer cells.

Investigating the Suitability of High Content Image Analysis as a Tool to Assess the Reversibility of Foamy Alveolar Macrophage Phenotypes In Vitro.

Many potential inhaled medicines fail during development due to the induction of a highly vacuolated or "foamy" alveolar macrophage phenotype response in pre-clinical studies. There is limited understanding if this response to an inhaled stimulus is adverse or adaptive, and additionally if it is a transient or irreversible process. The aim of this study was to evaluate whether high content image analysis could distinguish between different drug-induced foamy macrophage phenotypes and to determine the extent of the reversibility of the foamy phenotypes by assessing morphological changes over time. Alveolar-like macrophages derived from the human monocyte cell line U937 were exposed for 24 h to compounds known to induce a foamy macrophage phenotype (amiodarone, staurosporine) and control compounds that are not known to cause a foamy macrophage phenotype in vitro (fluticasone and salbutamol). Following drug stimulation, the cells were rested in drug-free media for the subsequent 24 or 48 h. Cell morphometric parameters (cellular and nuclear area, vacuoles numbers and size) and phospholipid content were determined using high content image analysis. The foamy macrophage recovery was dependent on the mechanism of action of the inducer compound. Amiodarone toxicity was associated with phospholipid accumulation and morphometric changes were reversed when the stimulus was removed from culture environment. Conversely cells were unable to recover from exposure to staurosporine which initiates the apoptosis pathway. This study shows that high content analysis can discriminate between different phenotypes of foamy macrophages and may contribute to better decision making in the process of new drug development.

Comparison of Oral, Intranasal and Aerosol Administration of Amiodarone in Rats as a Model of Pulmonary Phospholipidosis.

'Foamy' alveolar macrophages (FAM) observed in nonclinical toxicology studies during inhaled drug development may indicate drug-induced phospholipidosis, but can also derive from adaptive non-adverse mechanisms. Orally administered amiodarone is currently used as a model of pulmonary phospholipidosis and it was hypothesized that aerosol administration would produce phospholipidosis-induced FAM that could be characterized and used in comparative inhalation toxicology. Han-Wistar rats were given amiodarone via (1) intranasal administration (6.25 mg/kg) on two days, (2) aerosol administration (3 mg/kg) on two days, (3) aerosol administration (10 mg/kg) followed by three days of 30 mg/kg or (4) oral administration (100 mg/kg) for 7 days. Alveolar macrophages in bronchoalveolar lavage were evaluated by differential cell counting and high content fluorescence imaging. Histopathology and mass-spectrometry imaging (MSI) were performed on lung slices. The higher dose aerosolised amiodarone caused transient pulmonary inflammation (p < 0.05), but only oral amiodarone resulted in FAM (p < 0.001). MSI of the lungs of orally treated rats revealed a homogenous distribution of amiodarone and a putative phospholipidosis marker, di-22:6 bis-monoacylglycerol, throughout lung tissue whereas aerosol administration resulted in localization of both compounds around the airway lumen. Thus, unlike oral administration, aerosolised amiodarone failed to produce the expected FAM responses.

Quantitative assessment of nanoparticle surface hydrophobicity and its influence on pulmonary biocompatibility.

To date, the role of nanoparticle surface hydrophobicity has not been investigated quantitatively in relation to pulmonary biocompatibility. A panel of nanoparticles spanning three different biomaterial types, pegylated lipid nanocapsules, polyvinyl acetate (PVAc) and polystyrene nanoparticles, were characterized for size, surface charge, and stability in biofluids. Surface hydrophobicity of five nanoparticles (50-150nm) was quantified using hydrophobic interaction chromatography (HIC) and classified using a purpose-developed hydrophobicity scale: the HIC index, range from 0.00 (hydrophilic) to 1.00 (hydrophobic). This enabled the relationship between the nanomaterial HIC index value and acute lung inflammation after pulmonary administration to mice to be investigated. The nanomaterials with low HIC index values (between 0.50 and 0.64) elicited little or no inflammation at low (22cm(2)) or high (220cm(2)) nanoparticle surface area doses per animal, whereas equivalent surface area doses of the two nanoparticles with high HIC index values (0.88-0.96) induced neutrophil infiltration, elevation of pro-inflammatory cytokines and adverse histopathology findings. In summary, a HIC index is reported that provides a versatile, discriminatory, and widely available measure of nanoparticle surface hydrophobicity. The avoidance of high (HIC index>~0.8) surface hydrophobicity appears to be important for the design of safe nanomedicines for inhalation therapy.