In vitro macrophage assays for selection of drug candidates for preclinical development. Philippa Allen GSK

In vitro macrophage assays for selection of drug candidates for preclinical development. Philippa Allen GSK

Role of macrophages in the lung First line of host defence to inhaled matter. Regulate immune responses and inflammation. Phagocytose and destroy invading microorganisms. Uptake of environmental particulate material. Clearance. Inflammatory response E.g. induction of TNF. Toxic response. Particle overload and loss of function. Macrophage uptake of titanium dioxide

Alveolar macrophages Unique alveolar macrophage phenotype in the lung microenvironment. Alveolar macrophages show remarkable plasticity. Responses can be diverse. Key lung findings in vivo include inflammatory infiltration and macrophage foamyness.

What data do we need to demonstrate whether a macrophage response to inhaled drugs is adverse or non-adverse? A photomicrograph of a bronchoalveolar-lavage specimen shows an alveolar macrophage with a segmented nucleus (Giemsa stain, 400). François Paraf, M.D. Centre Hospitalier Universitaire Dupuytren, 87042 Limoges, France

Reduce Attrition of Inhaled Drugs We are withdrawing molecules in development due to unforeseen inflammatory responses of a variable nature. macrophages appear to play a major role. Macrophage and inflammatory responses are having an impact

Aims Define the likely biological responses in vitro to drug substance selected for the inhaled route. Increase scientific knowledge around lung macrophage responses. Introduce early assessments of potential hazard prior to candidate selection. Enhance toxicology studies to maximise output. Seek to develop inhaled drugs with a greater chance of success.

Objectives To implement a set of in vitro assays to assess the macrophage response to drug. Evaluate compounds pre candidate selection. Recommendations on compounds with improved probability of success. Advise on in vivo studies End points Observations. Combine in vitro and in vivo data to identify key decision making parameters.

In vitro advantages Control throughput, screen and rank compounds. Expensive time consuming in vivo studies. Many cellular models available. Cell lines. Nasal, tracheal, bronchial, alveolar epithelium, alveolar macrophages. Primary cells. Tracheal, bronchial, alveolar epithelium. Co-culture. Epithelium + macrophages + dendritic cells. 3D culture. Polarised, air-liquid interface, secreting mucous. Lung on a chip...

Lung on a chip (Wyss Institute, Harvard) 3D culture mounted on a flexible porous membrane. Air-liquid interface. Mechanical ventilation. Mimic the steady state lung & reproduce stress and strain. Transparent device allows real time fluorescence microscopy measurements. From 3D cell culture to organs-onchips. Dongeun Huh, Geraldine A. Hamilton and Donald E. Ingber. Trends in Cell Biology. December 2011, Vol. 21, No. 12.

In vitro macrophage screen Assays to assess Cell health Cell function Cell activation Cell morphology

Cell health Viability test Trypan blue Neutral red Plate based toxicity assays. LDH release Cell titer glo (ATP) MTT Flow cytometry live/dead discrimination. Nuclear dye of membrane compromised cells DAPI, DRAQ7, PI, 7-AAD etc

Macrophage Function Phagocytosis. Functional capacity in terms of phagocytic ability may be compromised following drug treatment. Flow cytometry to quantitate phagocytosis of apoptotic neutrophils by macrophages.

LDH % of signal window phagocytosis % of basal Phagocytic ability reflects viability Cell viability in culture Phagocytosis 100 140 80 60 GSK5 GSK6 120 100 40 80 60 20 0 40 20 GSK5 GSK6-20 0.1 1 10 100 1000 log [ug/ml] 0 1 10 100 1000 log[ug/ml] GSK5 was terminated in development due to the generation of foamy alveolar macrophages in rodents and lung inflammatory infiltrates in dogs. GSK6 successfully progressed into an inhaled medicine.

Oxidative Stress Reactive oxygen species (ROS) production. Glutathione (GSH:GSSG). Lipid peroxidation. Heme oxygenase-1 (HO-1) expression. Tearful relations: oxidative stress, inflammation and eye diseases.tais Hitomi Wakamatsu; Murat Dogru; Kazuo Tsubota. Arq. Bras. Oftalmol. vol.71 no.6 São Paulo Nov./Dec. 2008

Macrophage Activation Cytokine & chemokine induction post compound treatment. ELISA, MSD etc It is a challenge to determine which cytokines/chemokines are important and how to interpret the response.? Which cytokines & chemokines are chosen?? What does the cytokine/chemokine profile mean?? Does the profile relate in vivo?? Is the profile driving pathology?

Macrophage Activation Phenotypic changes of macrophages. Activation marker expression; MHCII, CD40 etc M1/M2 polarisation Qiao Yang, Yu Shi, Jiliang He, Zhi Chen. The evolving story of macrophages in acute liver failure. Immunology Letters, Volume 147, Issues 1 2, September October 2012, Pages 1 9 Is macrophage phenotype important and would this aid the understanding of adverse/non adverse responses to inhaled drugs?

Macrophage Morphology Brightfield / phase contrast microscopy. Initial indication of cell health and particle uptake. Foamy cells.

Basic microscopy Foamy cells Toxicity

Macrophage Morphology Brightfield / phase contrast microscopy. Initial indication of cell health and particle uptake. Foamy cells. Confocal microscopy. Particle uptake. Physical changes.

Confocal microscopy More detail of foamyness. UV fluorescence associated with cells indicating compound presence.

Macrophage Morphology Brightfield / phase contrast microscopy. Initial indication of cell health and particle uptake. Foamy cells. Confocal microscopy. Particle uptake. Physical changes. Cellular granularity by flow cytometry.

% SSC above control Cellular granularity 800 Granularity 600 400 200 0 1 10 100 1000 Log [ug/ml] Compare granularity as % of control (untreated cells) for compounds.

Alveolar macrophage in vitro risk assessment strategy Chemistry candidates In vitro Macrophage assessment Toxicity. Inflammatory. Physical changes. Compound specific. Nature of target. In vivo Candidate selection More informative selection of compound to take forward.

Acknowledgements Inhaled Sciences Dave Hassall Graham Somers Nicola Bevan Safety Assessment Anita Naidoo Paul McGill Supporting colleagues