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