AIT conference October 2015 Crack It: In vitro approaches · AIT conference October 2015 Crack It:...
Transcript of AIT conference October 2015 Crack It: In vitro approaches · AIT conference October 2015 Crack It:...
AIT conference October 2015
Crack It: In vitro approaches
Dr Victoria HutterUniversity of Hertfordshire
Foamy Macrophage (FM)
• Term used to describe lung macrophages that have taken on a granular or vacuolated
cytoplasmic appearance when viewed by light microscopy
• Do FM constitute an adaptive or adverse response?
• Can we predict and/or monitor FM development?
Forbes et al., - Advanced Drug Delivery Reviews 71 (2014) 15-33
In vitro approaches
• Longitudinal
• Non-invasive
• Determine mechanistic behaviours
• Screen a large number of compounds and
concentrations simultaneously
• Predictions are only as good as the cell
culture models
Cell models available
• Cell lines
• Primary cells
• Co-culture
• 3D culture
• Lung on a chip
Huh D et al., Trends in cell biol. 2011; 21,12
Phase I study approach (UH)
Human and rat in vitro cell culture models
Added stimulus (inhaled drugs and known inducers
of FAM)
Range of established in vitro tests
Explored adaptive responses
Plausibility of non invasive testing/markers
Stimuli
• Control compounds
– amiodarone, LPS, ac-LDL
• Inhaled drugs
– β2 agonists (salbutamol, salmeterol)
– Corticosteriods (budesonide,
beclometasone fluticasone)
– Antimuscarinics (ipratropium)
• Particulates
– Titanium dioxide, zinc oxide
Dosing
• Presentation of the drug/particulate to a cell
can have a large impact on cell response
– Solutions/suspensions
– Aerosolised particles
• Characterisation of particulates
– Size
– Charge
– Shape
• Dose
– Concentration
– Surface area
Cell assessment
• Viability
• Activation
• Functionality
• Morphology
In vitro assessment
Viability
• To determine suitable
concentration ranges
• No significant differences
between human and rat
responses for any of the
compounds tested
• Significant differences
observed for salbutamol and
salmeterol responses
Human (U937) Rat (NR8383)
In vitro assessment
Phospholipidosis
Phospholipidosis Marker
Amiodarone concentration
(mg/ml)0.01 0.1 1 10 100 0.01 0.1 1 10 100 0.01 0.1 1 10 100
U937 (human) - - + + ++ - - - + + - - + ++ +
NR8383 (rat) - + ++ ++ ++ - - + ++ + - + ++ ++ +
Salbutamol concentration
(mg/ml)0.01 0.1 1 10 100 0.01 0.1 1 10 100 0.01 0.1 1 10 100
U937 (human) - - - - - - - - - - - - - - -
NR8383 (rat) - - - - + - - - - - - - - + +
Salmeterol concentration
(mg/ml)0.01 0.1 1 10 100 0.01 0.1 1 10 100 0.01 0.1 1 10 100
U937 (human) - - + + ++ - - - + + - - + + +
NR8383 (rat) - - + ++ ++ - - ++ ++ + - - + ++ ++
Immunological staining
(PIP2/LAMP2)
Flow cytometry
(Nile Red staining)
Cytology
(oil red o staining)
Results are shown as relative to untreated cells. – indicates no change (80-120% of
control); + indicates minor increase (120-200% of control); ++ indicates moderate
increase (200-400% of control).
In vitro assessment
Adaptive responses
• Calcium signalling increased
after addition of stimuli
• Further investigation to cell
signalling pathway (MAPK, Erk
etc)
• Cytokine release
– G-CSF, IL-1β, IL-6, IL-8, and IL-10 (human and rat)
– IL-13, IL-17α and IL-18 (rat only)
In vitro assessment
Vacuolation
Non-invasive strategies
Volatile organic compound detection and analysis
• Cells produce a variety of volatile compounds in response to stimuli
• Can be collected from the headspace of cell culture vessels or the exhaled breath of
animals/humans in a non-invasive manner
• Several volatile compounds were identified from the culture headspace
• Similarities and differences between rat and human cultures
• Increased in concentration over time (4h vs 24h)
CRACK IT Phase 1 (KCL)
• Model and measure FM responses
in vitro
• Establish a panel of inducers which
yield mechanistic data reflective of
different pathways of FM
development
• Generate a well-defined dose-
response and temporal data
At the end of phase 1 (KHN collaboration formed):
Development of a foamy macrophage in vitro toolkit
• Phase II – KHN consortium
– King’s College London
– University of Hertfordshire
– National Physical Laboratories
• Develop a foamy macrophage in vitro
toolkit
Non-invasive,
longitudinal monitoring
Influx of mononuclear
cells in thelung
Define FM phenotype
Measure inflammatory
response
Define rodent response to
inhaled pharmaceuticals
Understand the FM
response timeframe
Understand drug-
induced FM biology
CRACK IT Phase 2
NC3R CrackIT
Challenge
Academic Collaboration
Technology Providers
Sponsors
In vitro work package:
Phase 2 overview
Stage 1:
Compound selection
Plus and
additional 12
blinded
compounds
from the
sponsor
companies
Stage 2: Morphometric screening
In cell analyser 6000
• High throughput in vitro screen
• Rapid, automated method
• Images and numerical analysis
• Commercializable technology
Kumar et al., – AIT 2014
Hoffman et al., – Molecular Pharmaceutics 12 (2015) 2675-2687
Morphometric screening:
Viability determination
• Nuclei and cytoplasm area
• Cell health markers
Morphometric screening:
Vacuole characterisation and phagocytosis
• Cell area
• Vacuole formation in cytoplasm
• Phospholipids
• Neutral lipids
• Phagocytosis of beads
Hoffman et al., – Molecular Pharmaceutics 12 (2015) 2675-2687
• Full morphometric screening of rat (NR8383) and human (U937)
alveolar macrophage cell lines
• Detailed morphometric analysis to determine main parameters
predictive of an adverse response
• Further probe and characterise biological mechanism behind
response
• Link with WP 2 and 3 and IVIVC for sponsor compounds
Future plans:
Vacuole characterisation and phagocytosis
Acknowledgments
• Aateka Patel
• Abhinav Kumar
• Anna Morgan
• Anthony Holmes
• Ben Forbes
• Clive Page
• Chris Walton
• David Hassall
• David Jones
• Darragh Murnane
• Doug Ball
• Helen Collins
• Ian Mudway
• Jan Klapwijk
• Jo Ann Rhodes
• Josephine Bunch
• Jo Taylor
• Lea Ann Dailey
• Martin Bootman
• Richard Booth
• Rory Steven
• Simon Moore
• Val Millar
• Victoria Hutter
Funding body: NC3R NC/C013203/1