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