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.Peer reviewedFinal Published versio

A quantitative structure-permeability relationship model for split-thickness skin absorption, reasoning for the choice of the database.

The skin is the largest organ in the human body, protecting the body from xenobiotic invasion (1). Local and systemic drugs may also be administered through the skin, therefore the need to measure the permeability of the skin to chemicals has long been apparent. The use of in vivo or in vitro techniques is time-consuming, since it is not only necessary to conduct a permeation study, but also to optimize experimental conditions and build analytical methods for each chemical. Moreover, it is not possible to assess the permeability of compounds not yet synthesised. An alternative option can be the development of Quantitative Structure-Permeability Relationships (QSPRs). These in silico models aim to form a relationship between the absorption of chemicals through the skin and their physico-chemical and/or structural properties (2). Knowing that permeability can be affected by different experimental conditions, the aim of this study is to build a QSPR based on uniform and consistent experimental conditions, but with a significant database size. Two different databases were compared: the first one was obtained only from Zhang et al (3), the second one was created from multiple literature sources, fulfilling the following conditions: - Data (log Kp values) were obtained by an in vitro diffusion system; - The membrane was human stratum corneum and viable epidermis; - The donor solvent was an aqueous solution; - No permeation enhancement technologies were used; - No association with other chemicals were considered. The geometrical structures of all chemicals were optimized with MM2 forcefield. Molecular descriptors and fingerprints were generated where possible. For each database, a wide range of Multi Linear Regression models were built using QSARins (4, 5) through a stepwise forward regression process. The models have been validated according to Golbraikh and Tropsha (6) criteria and the best ones have been selected according to the Multi-Criteria Decision Making (7). The model calculated from the data obtained from a single source shows better correlation, robustness, and predictivity, revealing a grade of uncertainty coming from an inter laboratory variability of the different sources used to build the database. REFERENCES 1. Baba H, Takahara J-i, Mamitsuka H. In Silico Predictions of Human Skin Permeability using Nonlinear Quantitative Structure–Property Relationship Models. Pharmaceutical Research. 2015;32(7):2360-71. 2. Moss GP, Cronin MTD. Quantitative structure–permeability relationships for percutaneous absorption: re-analysis of steroid data. International Journal of Pharmaceutics. 2002;238(1):105-9. 3. Zhang K, Chen M, Scriba GKE, Abraham MH, Fahr A, Liu X. Human Skin Permeation of Neutral Species and Ionic Species: Extended Linear Free Energy Relationship Analyses. Journal of Pharmaceutical Sciences. 2012;101(6):2034-44. 4. Gramatica P, Chirico N, Papa E, Cassani S, Kovarich S. QSARINS: A new software for the development, analysis, and validation of QSAR MLR models. Journal of Computational Chemistry. 2013;34(24):2121-32. 5. Gramatica P, Cassani S, Chirico N. QSARINS-chem: Insubria datasets and new QSAR/QSPR models for environmental pollutants in QSARINS. Journal of Computational Chemistry. 2014;35(13):1036-44. 6. Golbraikh A, Tropsha A. Beware of q2! Journal of Molecular Graphics and Modelling. 2002;20(4):269-76. 7. Keller HR, Massart DL, Brans JP. Multicriteria decision making: A case study. Chemometrics and Intelligent Laboratory Systems. 1991;11(2):175-89.Peer reviewedFinal Published versio

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

© 2015 Termedia Sp. z o. o. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.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.Peer reviewedFinal Published versio

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

© 2019 American Chemical Society.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.Peer reviewedFinal Accepted Versio

High content analysis of in vitro alveolar macrophage responses can provide mechanistic insight for inhaled product safety assessment

© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license. https://creativecommons.org/licenses/by/4.0/Assessing the safety of inhaled substances in the alveolar region of the lung requires an understanding of how the respired material interacts with both physical and immunological barriers. Human alveolar-like macrophages in vitro provide a platform to assess the immunological response in the airways and may better inform the understanding of a response to an inhaled challenge being adaptive or adverse. The aim of this study was to determine if a morphometric phenotyping approach could discriminate between different inhaled nicotine products and indicate the potential mechanism of toxicity of a substance. Cigarette smoke (CS) and e-liquids extracted into cell culture medium were applied to human alveolar-like macrophages in mono-culture (ImmuONE™) and co-culture (ImmuLUNG™) to test the hypothesis. Phenotype profiling of cell responses was highly reproducible and clearly distinguished the different responses to CS and e-liquids. Whilst the phenotypes of untreated macrophages were similar regardless of culture condition, macrophages cultured in the presence of epithelial cells were more sensitive to CS-induced changes related to cell size and vacuolation processes. This technique demonstrated phenotypical observations typical for CS exposure and indicative of the established mechanisms of toxicity. The technique provides a rapid screening approach to determine detailed immunological responses in the airways which can be linked to potentially adverse pathways and support inhalation safety assessment.Peer reviewe

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.Peer reviewedFinal Published versio

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 di_erential 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.Peer reviewedFinal Published versio

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

© 2023 The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/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.Peer reviewe

Effect of 6 weeks treatment with nebivolol on blood pressure reduction and echocardiographic indices in patients with essential hypertension

Wstęp Badania, w których porównano leki &#946;-adrenolityczne nowych generacji z &#946;-adrenolitykami starszych generacji pod względem wpływu na wysokość ciśnienia i wybrane parametry hemodynamiczne u chorych z nadciśnieniem tętniczym pierwotnym, nie dostarczają jednoznacznych wyników. Głównym celem pracy była ocena skuteczności hipotensyjnej &#946;-adrenolityku nowej generacji &#8212; nebiwololu &#8212; w porównaniu z wpływem wywieranym przez atenolol. Przeanalizowano również wpływ leczenia nebiwololem na wybrane parametry echokardiograficzne. Oceniano wpływ terapii na tolerancję wysiłku u chorych z nadciśnieniem tętniczym pierwotnym. Materiał i metody Badaniami objęto 76 mężczyzn z nadciśnieniem tętniczym pierwotnym łagodnym i umiarkowanym (wg ESH/ESC z 2003 roku: odpowiednio stopień 1 i 2) w wieku od 22 do 61 lat (średnio: 43,6 &#177; 8,2 roku). W warunkach wyjściowych oraz po 6 tygodniach leczenia nebiwololem (5 mg raz dziennie) oraz atenololem (50 mg raz dziennie) odpowiednio u 39 i 37 chorych wykonywano następujące badania: całodobową automatyczną rejestrację ciśnienia tętniczego (ABPM), badanie echokardiograficzne (ECHO), EKG całodobowe metodą Holtera (EKG-24 h) oraz elektrokardiograficzny test wysiłkowy. Wyniki W grupie leczonej nebiwololem efekt hipotensyjny w zakresie SBP i DBP uzyskano odpowiednio u 87% i 69%. Średnie wartości ciśnienia tętniczego z całej doby obniżały się odpowiednio o 13 &#177; 8/8 &#177; 8 mm Hg, średnie wartości ciśnienia z dnia o 14 &#177; 9/10 &#177; 7 mm Hg, średnie wartości z okresu nocy o 9 &#177; 8/6 &#177; 8 mm Hg (p < 0,01 dla wszystkich różnic). Nie stwierdzono istotnych statystycznie różnic między grupami. W grupie przyjmującej nebiwolol prędkość fali E w warunkach wyjściowych wynosiła 0,75 &#177; 0,15 m/s i po leczeniu wzrosła do 0,81 &#177; 0,2 m/s (p = 0,05), prędkość fali A w warunkach wyjściowych wynosiła 0,66 &#177; 0,08 m/s, a w momencie zakończenia leczenia 0,64 &#177; 0,1 m/s (NS). Stwierdzono wzrost wartości współczynnika E/A z 1,14 &#177; 0,25 do 1,3 &#177; 0,23 (p < 0,001). Podczas testu wysiłkowego &#8212; w momencie zakończenia leczenia w porównaniu z wartościami wyjściowymi &#8212; wartości maksymalnego SBP i DBP zmniejszyły się o 18 &#177; 26 mm Hg (p < 0,001) i o 3 &#177; 14 mm Hg (NS) pod wpływem leczenia nebiwololem. Wnioski Uzyskane wyniki wskazują na dużą skuteczność hipotensyjną nebiwololu u wysokiego odsetka chorych z nadciśnieniem tętniczym pierwotnym. W przeprowadzonej analizie stwierdzono korzystny kierunek zmian w ocenie wybranych parametrów echokardiograficznych. Nie wykazano niekorzystnego wpływu nebiwololu na zdolność do wykonywania wysiłku fizycznego &#8212; zaobserwowano, że omawiany lek wywiera umiarkowanie zaznaczony wpływ na poprawę tolerancji wysiłku u chorych z nadciśnieniem tętniczym pierwotnym.Background The results of clinical studies comparing antihypertensive efficacy of newer and older beta blockers and their effect on echocardiographic indices are inconclusive. The aim of the study was to evaluate the effect of nebivolol on blood pressure reduction and on selected echocardiographic indices in patients with essential hypertension. The effect of treatment on exercise tolerance was also investigated. Material and methods The study included 76 male patients with mild to moderate (ESH/ESC: grade 1 and 2) essential hypertension (mean age: 43.6 &#177; 8.2 years). In all subjects at baseline and after 6 weeks of nebivolol treatment (5 mg once daily) or atenolol (50 mg once daily) (39 and 37 pts respectively) ABPM, Echo, ECG Holter and exercise test were performed. Results In the nebivolol group SBP and DBP reduction was noticed in 87% and 69% of patients with essential hypertension. Based on ABPM mean SBP and DBP during the whole day, daytime and nighttime decreased: 13 &#177; 8/8 &#177; 8 mm Hg, 14 &#177; 9/10 &#177; 7 mm Hg and 9 &#177; 8/6 &#177; 8 mm Hg respectively (p < 0.01 for all differences). In the nebivolol group E wave was increased between two examinations from 0.75 &#177; 0.15 m/s to 0.81 &#177; 0.2 m/s (p = 0.05), there were no significant changes in A wave 0.66 &#177; 0.08 m/s vs. 0.64 &#177; 0.1 m/s (NS). Ratio E/A was increased during the time of observation from 1.14 &#177; 0.25 to 1.3 &#177; 0.23 (p < 0.001). During the exercise test in the nebivolol group maximal SBP and DBP decreased by 18 &#177; 26 mm Hg (p < 0.001) and 3 &#177; 14 mm Hg (NS) respectively after 6 weeks of treatment. Conclusions Our results indicate high antihypertensive efficacy of nebivolol in patients with essential hypertension. We also observed an improvement of selected echocardiographic indices. Nebivolol was not impairing the exercise tolerance in patients with essential hypertension

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

© The Author(s) 2017. This article is an open access publication. Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.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.Peer reviewedFinal Published versio