actc conference may 2016 crack it: in vitro approaches · actc conference may 2016 crack it: in...
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ACTC Conference May 2016
Crack It: In vitro approaches
Dr Victoria Hutter and Dr Ewelina HoffmanUniversity of Hertfordshire
Safety challenges in developing new inhaled medicines• Safety is the 2nd most common
reason for halting the
development of new inhaled
drugs (approximately 30% of
cases)
• May be due to the design
considerations for lung-targeted
medicines
– High molecular weight
– Increased lipophilicity
– Poorly soluble compounds
Safety challenges in developing new inhaled medicines
Determining inhaled toxicity
• Use histopathological examination of
haemotoxylin and eosin stained tissue
sections as a primary endpoint
• Types of responses:
– Nasal and laryngeal irritation
– Lung irritation (epithelial
degeneration, ulceration, necrosis)
– Changes in alveolar macrophages
(increase in number, change in
morphology)
http://www.albany.edu/mcnaylab/sd.html
Determining inhaled toxicity
Macrophage responses
Slide subtitle in Arial
Forbes B et al., Adv Drug Deliv Rev. 2014, 71:15-33.
Types of macrophage response
• Simple minor
accumulations to more
solid aggregates co-
localised with hyperplastic
alveolar type 2 cells
• Localisation around
bronchial-alveolar
junctions
• Reversible reaction
• No reaction in regional
lymph nodes
• Development of more
severe inflammation
(emphysema or fibrosis)
• Inflammation penetrates
into the alveolar lumina
• Pronounced reaction in
lymph nodes
• Irreversible
• Associated with
infiltrating neutrophils or
lymphocytes
• Macrophages can
appear with a more
granular or pigmented
cytoplasm
• Can cause short term
damage to epithelial
cells of alveolar ducts
THE
GOOD
THE
BAD
THE
UGLY
Lewis DJ et al., J Appl Toxicol. 2014, 34(4):319-31
http://postercabaret.com/the-good-the-bad-and-the-ugly-movie-poster-by-billy-perkins-sold-out.html
Types of foamy macrophage response
Foamy Macrophage (FM) in vitro
• 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
Crack It Challenge: Inhalation Translation
• NC3R funded project supported by
industrial sponsors GSK, Envigo and
Pfizer
• 2 phase approach
– Phase 1: six months proof of
principle studies
– Phase 2: three years collaborative
research to answer problem
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
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
• Control compounds (amiodarone, LPS, ac-LDL)
• Inhaled drugs (β2 agonists, corticosteroids,
antimuscarinics)
• Particulates (TiO2, ZnO)
• Solution vs suspension vs aerosolisation
• Physical chemical properties (size, charge, shape)
• Screened for:
– Cell health
– Cell function
– Cell activation
– Cell morphology
Phase 1 Approach (UH)
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
WP1 – In vitro FM Toolkit
WP2 - Advanced understanding of FM
responses in vivo
WP3 - Non-invasive in vivo
methodology
WP4 – Translation, commercialisation
& engagement to
Project Manager: B. Forbes (KCL)
WP1 Leads
V. Hutter (UH) · H. Collins (KCL)
WP2 Leads
J. Bunch (NPL) · I. Mudway (KCL)
WP3 Leads
L. Dailey (KCL) · D. Murnane (UH)
WP4 Leads
C. Page (KCL) · R. Booth (NPL)
Phase 2 Consortium Inhalation Translation Platform
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
• Deon Hildebrand
• Doug Ball y
• 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