Next-Generation Safety Assessment Tools for Advancing In Vivo to In Vitro Translation

Next-Generation Safety
Assessment Tool for Advancing
In Vivo to In Vitro Translation
Commercial Lead in Immunology
ImmuONE
Louis Scott, PhD
Victoria Hutter, PhD
Chief Scientific Officer
ImmuONE

© 2 0 2 4 IMMUONE
INNOVATIVE SOLUTIONS FOR
INHALATION SAFETY
In vitro specialists for immune responses
ImmuONE webinar
Wednesday 28th February 2024
Prof Victoria Hutter
Chief Scientific Officer, ImmuONE

© 2 0 2 4 IMMUONE
• Overview of in vitro inhalation toxicity assessment
• The models
• The exposure
• The assessment
• The interpretation
• Past, present and future – next generation tools and assessments
• How in vitro respiratory models complement and replace in vivo models
title
Overview

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title
In vitro inhalation toxicity assessment
The
model
The
exposure
The assessment
methodology
The
interpretation

• Specialized functions
• Upper & lower respiratory tract
• Conducting & pulmonary airways
• Inhalation toxicity typically has airway
epithelial focus
The respiratory tract
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The cellular composition of the airway epithelia differs
significantly
(a) In the upper airways, ciliated cells are
interspersed with secretory cells
(b) At lower levels they are interspersed with
club cells
(c) 2 cell types in the alveolar regions-squamous
cell Types I and II
The respiratory tract
Biological barriers: cells
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• Lung lining fluid
- Total lung volume 15-70 mL
- Ions but low protein content
• Conducting airways: mucus
- Mucopolysaccharide
- Influence on diffusion and absorption
• Respiratory airways: alveolar lining fluid
- Contains phospholipid surfactant and protein
- Monolayer coverage
- Also pools of excess lining fluid
Biological barriers: lining fluid
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Increasing complexity
Epithelial
2D cell lines
Epithelial cell lines
cultured in 3D at the
air-liquid interface
(ALI)
Primary human
cells 3D ALI
Commercial primary
human tissues 3D ALI
Spheroids and
organoids
Lung on a chip
Co-culture cell
line/primary models
cultured at the ALI
Models
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• Homo- or heterogenous populations
• Continuous cell lines has acquired the ability to proliferate indefinitely, either through genetic or
artificial modifications
• Lung specific cell models
• Bronchial epithelial – Calu-3, 16HBE14o-, BEAS-2B,
NuLi,
• Alveolar epithelial – A549, H441, hAeLVi
• Fibroblast – MRC5, CCD19-Lu
• Endothelial
• Alveolar macrophages – THP1, U937
Cell lines
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• Epithelial cells cultures at the air-liquid interface (ALI) generate a model
more morphologically representative of the airway epithelium than
cells cultured under submerged conditions
• Columnar morphology
• Increased cilia-like structures
• Increased glycoprotein secretions
• Reduced TEER/ZO1 staining
Submerged or LLI ALI Calu-3 cells at the ALI
Air-liquid interface culture
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• Primary cells are isolated directly from humans using
enzymatic or mechanical methods
• Biologically relevant
• Physiologically similar
• Short life
• Patient variability
• Commercial airways tissues
• Combine multiple donors
NHBE primary cells at ALI
Primary cell culture models
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Variety of co-culture models in literature
• epithelial, endothelial, fibroblast, immune
(alveolar macrophage)
Cells selected depending on response/mechanism
of interest
• irritation/viability – epithelial
• inflammation – macrophage (epithelial
limited)
• sensitisation – epithelial, endothelial,
macrophage, dendritic cells
ImmuLUNG
Co-culture of human alveolar macrophage-
like cells with alveolar epithelial cells
✓ 24 Transwell® format
✓ Immortalised cells from lung
origin
✓ Air-liquid interface culture
✓ Up to 14 days in culture (in house)
Co-culture models
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3D structures composed of multiple cells
Organoids
• complex clusters of organ-specific cells
• made from stem cells or progenitor cells
• self-assemble when given a scaffolding
• disease modelling, drug discovery, regenerative medicine,
cancer research and tumour pathophysiology
Spheroids
• simple clusters of broad-ranging cells
• stick together without the need for a scaffold
• can’t self-assemble or regenerate
• understanding tumour microenvironments
Spheroids and organoids
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• Simulate human physiology within the system
• Mechanical forces - breathing
• Physiological flow - microfluidics
• Closer human physiological representation of the cells/tissues
Organ-on-a-chip models
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What is physiological relevance
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• Contain all lung cell types present in
tissue (at the time of slicing)
• Can be maintained for 3-4 weeks
• Acute and chronic
Precision cut lung slices
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Right cell types for
the desired
response/function
Which model?
Increasing
complexity not
always needed
- establish clear
benefit of adding
more cell types
Optimal for
exposure/test item
(media, system)
Optimal for the
right assessment
techniques
Compatible with
the assessment
endpoints
Skill of the end user
and time to
optimise
Model selection
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• Human lung physiology (vs rat)
• Particle characteristics
• Deposition
• Interaction with lining fluid
• Cellular exposure
Lung physiology
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Regional deposition
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• Deposition modelling
• Broad range that covers toxic to non-toxic
• mg/area
• Lung area 100-130m2
• In vitro area (24 well plate – 0.33cm2)
Dosing in vitro
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• Solution
• Suspension
• Aerosol
In vitro exposure
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• Exposure is reasonably certain
• If aqueous solubility is acceptable
• Non-specific binding
• Potential for cellular metabolism
• Potential interaction with cell culture
medium
• Less physiologically relevant
• Flooding the airways with liquid
Dosing in solution
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• Liquid aerosols
• 15-300 µL nebulised volume
• 3-6 min exposure time
Dosing using aerosols
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Dry powder
• Powder placed in loading system
• Expansion chamber sealed
• Powder aerosolised under high pressure
breaks up agglomerates)
• Powder settles on samples (gravitational
sedimentation)
Dosing using aerosols
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Continuous flow exposure
• Gases, complex mixtures, particles
• 6-24h
• Grams of material
Continuous dosing
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Does it kill cells?
Does it affect cell/tissue
function/change cell behaviour?
No
Can they recover?
Yes
Yes
No
Yes
( )
No
Mechanistic
assessment
• how does it kill cells?
• how is cell/tissue
function affected?
• what is the recovery
time?
• Adverse Outcome
Pathways (AOP)
In vitro assessment
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• Viability
- PI, LDH
- mitochondrial activity
- ATP
• Barrier
- TEER
- paracellular marker
• Histology
Untreated
Standard assessments: epithelial
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• Cilia beating frequency • Mucociliary clearance
Other epithelial assessments
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• Inflammation
• Cytokines
• Cell surface markers
• Markers for cell activation
• Growth factors
• Prostaglandins
• Acute phase proteins (CRP)
• Oxidative stress (ROS, RNS)
• Individual cell
characterisation and
population characterisation
Other stress/inflammatory assessments
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• What is the mechanism of death?
• What is the mechanism of inflammation?
• sensitisation, irritant
• Is there the potential for longer term
toxicology/airway remodelling?
• fibrosis
• Not all cells in a model/tissue will be have
the same
• average population characteristics
• individual cell characteristics
Genomics (DNA)
Transcriptomics (RNA)
Proteomics (Protein)
Mechanistic driven assessment
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Multi-endpoint analysis for mechanism-driven risk
assessment
Exposure: Solution vs
suspension vs aerosol
Multiple endpoint assessment

assessment
Barrier properties
Epithelial-immune
interactions
Morphology
Surfactant
production
Viability

assessment
Cell size
Vacuolation profile
Cell shape
Cytotoxicity/cell
membrane integrity
Metabolic activity
Cell number
Barrier properties
Epithelial-immune
interactions
Morphology
Surfactant
production
Viability
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assessment
Cell size
Vacuolation profile
Cell shape
Polarisation (CD
markers)
Phagocytosis
Cytokines
Cell lipid profiles
Genetic analysis
Cytotoxicity/cell
membrane integrity
Metabolic activity
Cell number
Barrier properties
Epithelial-immune
interactions
Morphology
Surfactant
production
Viability
Proteomic analysis
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assessment
Cell size
Vacuolation profile
Cell shape
Polarisation (CD
markers)
Phagocytosis
Cytokines
Cell lipid profiles
Genetic analysis
Cytotoxicity/cell
membrane integrity
Metabolic activity
Cell number
Barrier properties
Epithelial-immune
interactions
Morphology
Surfactant
production
Viability
Proteomic analysis
Cell migration Cell velocity
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High content analysis (cell painting)
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4 h
24 h
48 h 48 h
4 h
24 h
48 h 48 h
4 h
24 h
48 h 48 h
Control Known toxicant Test item A
Phenotype fingerprinting for safety profiling
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air
control
known
toxicant
Phenotype can indicate toxicity mechanism
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MITOCHO NDRIA L A CTIVITY ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − − − − − − − − − − − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑
CELL A REA ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑
NUMBER OF VA CUO LES ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑
A REA O F VACUO LA TIO N ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑
Control
PBS
Compound A (low)
Compound A (high)
Compound B (low)
Compound B (high)
Compound C (low)
Compound C (high)
Compound D (low)
Compound D (high)
Control
PBS
Compound A (low)
Compound A (high)
Compound B (low)
Compound B (high)
MITOCHO NDRIA L A CTIVITY ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − − − − − − − − − − − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑
Control
PBS
Compound A (low)
Compound A (high)
Compound B (low)
Compound B (high)
Compound C (low)
Compound C (high)
Compound D (low)
Compound D (high)
Nuclei
Membrane
Permeability
Mitochondrial
Activity Cytoplasm Merged
Control
Compound C
(low)
Compound C
(high)
04
Test item (low conc)
Test item (high conc)
Phenotype indicative of toxicity mechanism
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• What is the purpose of the in vitro test
• Differentiate response
• Benchmark response
• Correlate response
• In vitro-in vivo correlation
• Appropriate endpoints for direct correlation (morphology, blood markers)
• Appropriate compound set with sufficient in vivo characterisation
• Mechanism-driven risk assessment
• Adverse Outcome Pathways (AOP)
• Integrated Approaches to Testing and Assessment (IATA)
• IATA NAMS for refining assessment of chlorothalonil
Interpretation and context
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Human in vitro
Rat in vivo
(lung pathology)
Rat ex vivo
(BAL)
Rat in vitro
Rat in vivo
Interspecies comparisons
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• Wide range of in vitro models, dosing systems and assessments available
• Defining the question and purpose of the in vitro assessment is key
• Does it kill cells?
• What is the long-term effect?
• What is the mechanism of toxicity/cell interaction?
• Examples of how in vitro respiratory models complement and potentially replace in vivo
models
Summary
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Profiling alveolar
macrophage responses to
inhaled compounds
Dr. Louis Scott
Commercial Lead in Immunology

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• Concerns over safety and initiation of immune
responses in the distal airways in preclinical
studies is one of the reasons over a third of
candidate inhaled compounds fail.
• Highly vacuolated or "foamy" alveolar
macrophages (FM) observed in pre-clinical rat
studies poses safety concerns, despite not
knowing the correlation to hindering progress of
promising candidates into humans
title
Background: “foamy” macrophages
in vivo
in vitro
Forbes et al., 2014

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• HCIA combines alveolar
macrophage morphology/ lipid
endpoints as an in vitro tool for
inhaled safety assessment.
• Morphometric endpoints that
directly correlate with in vivo
observation.
title
Purpose and methodology
in vivo in vitro

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title
HCIA screens
HCA screens overview
A - Cell Health and Morphology Assay
• Nuclear staining (Hoechst 33342)
• Mitochondrial membrane potential (MitoTracker Red)
• Membrane permeability (Image-It Dead Green)
• Cytoplasm staining (Cell Mask)
B - Lipid content and Phagocytosis Assay
• Nuclear staining (Hoechst 33342)
• Phospholipidosis detection (Lipidtox Red)
• Neutral lipids detection (Neutral lipid green)
• Phagocytosis activity (Crimson beads)
Lipid content staining
10 µM Imipramine, NR8383
Cell health staining,
Air-treated, BAL

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title
Median morphology parameters of untreated and amiodarone-
treated rat and human macrophage cell models.
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Number of vacuoles (human)
Number
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Number of vaculoes (rat)
Number
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Numbe
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Cell area (human)
Cell
area
[µm
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]
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Numb
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Cell area (rat)
Cell
area
[µm
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]
Vacuolation Cell area
Hoffman et al., 2017

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title
Median lipid parameters of untreated and amiodarone-treated
rat and human macrophage cell models.
Neutral lipids Phospholipids
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Relative
fluo
Phos
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Neutral Lipids (human)
Relative
fluorescence
intensity
Neutral
lipid
Green
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Relative
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Neutral Lipids (rat)
Relative
fluorescence
intensity
Neutral
lipid
Green
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Phospholipids (human)
Relative
fluorescence
intensity
Phospholipid
Red
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Phospholipids (rat)
Relative
fluorescence
intensity
Phospholipid
Red

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title
Macrophage Responses to Amiodarone: ‘elevated’
responses
• No significant difference (p > 0.05) was observed
between untreated cells and those exposed to 10 μM
amiodarone (known inducer of phospholipidosis) when
comparing median values, despite observable
differences in histogram profiles.
• Average population statistical analysis was not a suitably
sensitive descriptor to depict the morphology and lipid
changes observed in cells exposed to amiodarone which
impacted less than 20% of the total cell population.
• Therefore, an alternative analytical approach which
included only the cell population with elevated
characteristics in comparison with the untreated cells
was considered.
Untreated Amiodarone

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title
Macrophage Responses to Amiodarone: ‘elevated’
responses
HCA screens overview
Parameter Inclusion criteria
Elevated mitochondrial
activity
% cells > mean mitochondrial activity + 2nd SD of the untreated cell
population
Elevated membrane
permeability
% cells > mean membrane permeability + 2nd SD of the untreated cell
population
Elevated cellular area % cells > mean cell area + 2nd SD of the untreated cell population
Elevated vacuole number
per cell
% cells > mean vacuole number per cell + 2nd SD of the untreated cell
population
Elevated vacuole area per
cell
% cells > mean vacuole area per cell + 2nd SD of the untreated cell
population
Elevated phospholipid
content
% cells > mean phospholipid content per cell + 2nd SD of the untreated cell
population
Elevated neutral lipid
content
% cells > mean neutral lipid content per cell + 2nd SD of the untreated cell
population
**Summary of diagnostic criteria employed in foamy macrophage investigation.
E Median value untreated
F Median value amiodarone
G Population of cells elevated number of vacuoles per cell

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title
Cell health, morphology and lipid multi-parameter profiles after
amiodarone exposure to rat macrophages
24h
48h • In contrast to median value
analysis, multi-parameter
profile analysis of the
elevated population
characteristics revealed an
impact on elevated
phospholipid content and
cell health markers as
expected for the
phospholipidosis inducer,
amiodarone.

title
In vivo to in vitro translation
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• Rat alveolar macrophages were more
sensitive to amiodarone exposure than
human cells.
• All alveolar lung macrophage cell
models require analysis of cell sub-
populations rather than average
population data.
• HCIA provides a method for profiling
human and rat alveolar macrophage
responses in vitro.

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• 12 compounds, categorized as FM- and
non-FM-inducers.
• In vitro tests deemed all compounds
suitable for animal studies.
• In vivo studies revealed that some
compounds led to macrophage
accumulation and FM responses.
• Compounds were discontinued from
further development due to potential
human safety issues.
title
Profiling of alveolar macrophage responses to
inhaled modalities

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title
CrackIT: profiling of alveolar macrophage
responses to inhaled modalities
Non-FM-inducers FM-inducers
salbutamol
hemisulphate
β2-agonist amiodarone
hydrochloride
cationic amphiphilic drug
formoterol
fumarate
β2-agonist imipramine
hydrochloride
dihydrate,
salmeterol
xinafoate
β2-agonist chlorpromazine
hydrochloride
ipratropium
bromide
muscarinic receptor
antagonist
dibucaine
hydrochloride
beclomethasone
dipropionate
steroidal antiflammatory drug staurosporine Pro-apoptotic
fluticasone
propionate
steroidal antiflammatory drug etoposide Pro-apoptotic
budesonide steroidal antiflammatory drug campthotecin Pro-apoptotic

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title
Multiparameter analysis of rat alveolar
macrophages
B C
Rat macrophages treated in vitro with Amiodarone in various concentrations for 24 hours
(A) 0µM
(B) 10µM
(C) 50µM
InCell Analyser images, magnification 40x
A

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title
Multiparameter analysis of rat alveolar
macrophages
0
50
100
150
24h
0
50
100
150
48h
%
Cell
Population
Untreated
Salbutamol
Formoterol
Salmeterol
Ipratropium
Beclomethasone
Fluticasone
Budesonide
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l
A
r
e
a
M
i
t
o
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
24h
Parameter
24h
C
e
l
l
C
o
u
n
t
C
e
l
l
H
e
a
l
t
h
N
u
c
A
r
e
a
N
u
c
/
C
e
l
l
A
r
e
a
M
i
t
o
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
0
50
100
150
48h
Parameter
%
Cell
Population
Untreated
Salbutamol
Formoterol
Salmeterol
Ipratropium
Beclomethasone
Fluticasone
Budesonide
0
50
100
150
48h
%
Cell
Population
Untreated
Amiodarone
Imipramine
Dibucaine
Chlorpromazine
Staurosporine
Etoposide
Campthotecin
C
e
l
l
C
o
u
n
t
C
e
l
l
H
e
a
l
t
h
N
u
c
A
r
e
a
N
u
c
/
C
e
l
l
A
r
e
a
M
i
t
o
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
0
50
100
150
24h
Parameter
%
Cell
Population
0
50
100
150
24h
%
Cell
Population
C
e
l
l
C
o
u
n
t
C
e
l
l
H
e
a
l
t
h
N
u
c
A
r
e
a
N
u
c
/
C
e
l
l
A
r
e
a
M
i
t
o
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
0
50
100
150
48h
Parameter
%
Cell
Population
Untreated
Salbutamol
Formoterol
Salmeterol
Ipratropium
Beclomethasone
Fluticasone
Budesonide
t
h
a
a
t
t
a
c
c
c
s
s
0
50
100
150
48h
%
Cell
Population
Untreated
Amiodarone
Imipramine
Dibucaine
Chlorpromazine
Staurosporine
Etoposide
Campthotecin

© 2 0 2 4 IMMUONE
title
Multiparameter analysis of human alveolar
macrophages
Human macrophages treated in vitro with amiodarone in various concentrations for 24 hours
(A) 0µM
(B) 1µM
(C) 10µM
(D) 50µM
InCell Analyser images, magnification 40x
A
B C
A B C D

© 2 0 2 4 IMMUONE
title
Multiparameter analysis of human alveolar
macrophages
C
e
l
l
C
o
u
n
t
C
e
l
l
H
e
a
l
t
h
N
u
c
A
r
e
a
N
u
c
/
C
e
l
l
A
r
e
a
M
i
t
o
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
0
50
100
150
24h
Parameter
%
Cell
Population
0
50
100
150
24h
%
Cell
Population
C
e
l
l
C
o
u
n
t
C
e
l
l
H
e
a
l
t
h
N
u
c
A
r
e
a
N
u
c
/
C
e
l
l
A
r
e
a
M
i
t
o
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
0
50
100
150
48h
Parameter
%
Cell
Population
Untreated
Salbutamol
Formoterol
Salmeterol
Ipratropium
Beclomethasone
Fluticasone
Budesonide
t
t
t
0
50
100
150
48h
%
Cell
Population
Untreated
Amiodarone
Imipramine
Dibucaine
Chlorpromazine
Staurosporine
Etoposide
Campthotecin
e
a
e
a
I
n
t
I
n
t
e
a
a
c
a
c
a
c
d
s
d
s
24h
n
t
l
t
h
e
a
e
a
I
n
t
I
n
t
e
a
a
c
a
c
a
c
d
s
d
s
0
50
100
150
48h
%
Cell
Population
Untreated
Salbutamol
Formoterol
Salmeterol
Ipratropium
Beclomethasone
Fluticasone
Budesonide
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
24h
Parameter
24h
C
e
l
l
C
o
u
n
t
C
e
l
l
H
e
a
l
t
h
N
u
c
A
r
e
a
N
u
c
/
C
e
l
l
A
r
e
a
M
i
t
o
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
0
50
100
150
48h
Parameter
%
Cell
Population
Untreated
Salbutamol
Formoterol
Salmeterol
Ipratropium
Beclomethasone
Fluticasone
Budesonide
t
h
a
a
t
t
a
c
c
c
s
s
0
50
100
150
48h
%
Cell
Population
Untreated
Amiodarone
Imipramine
Dibucaine
Chlorpromazine
Staurosporine
Etoposide
Campthotecin

Summary
© 2 0 2 4 IMMUONE
• All alveolar lung macrophage cell models responded to challenge with amiodarone, but detection of
these required analysis of cell sub-populations rather than average population data.
• Human alveolar macrophages profiles showed that cells were less sensitive to stimuli.
• HCIA-derived profiles differentiated between safe and unsafe responses in macrophages.
• Multi-parameter methodology has the potential to offer an early non-clinical screening tool to predict
the safety of candidate inhaled medicines.
• The comparison of in vitro cell responses from a variety of rat ex vivo and human and rat in vitro
models allows for a direct comparisons to be made.

IN VITRO INNOVATION SERVICES FOR
EMPOWERED DECISION MAKING
© 2 0 2 4 IMMUONE
ImmuONE provides innovative respiratory in vitro cell
culture products and contract research services for
inhalation safety testing. Our focus is supporting
companies and researchers to understand the
interaction and safety implications of inhaled products
with immune cells in the airways.

© 2 0 2 4 IMMUONE
Text box
Delivering in vitro solutions
In vitro services for inhalation
testing
In vitro alveolar macrophage
assays
Immune-competent in vitro
human airways models

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