16-1-2015

Organ-specific toxicity in
vitro: methodologies

Bas J. Blaauboer
Doerenkamp-Zbinden Chair
Institute for Risk Assessment Sciences (IRAS)
Utrecht University, the Netherlands

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Objectives of Predict-IV
Evaluation of drug effects in organ specific rat and
human in vitro models
Focus on liver, kidney and CNS as target organs.
Models already described in the literature
In vivo effect of model compounds well
characterized

Safety assessment based on toxico-dynamic effects

and cellular exposure using M&S (modelling &
simulation)

9 | Presentation Title | Presenter Name | Date | Subject | Business Use Only

Objectives of Predict-IV
Experiments to be performed:
Chronic-like long-term treatment
Circumventing un-specific cytotoxicity
Aiming for in vivo-relevant in vitro concentrations.
Exposure measurements (kinetic profiles)
Identification of new mechanisms by genomics and
validation of pathways by siRNA. Focusing on
functional effects
Exploring potential biomarker by proteomics and
metabonomics

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Cellular systems investigated

Liver:
Primary rat hepatocytes (collagen-
collagen sandwich and collagen-matrigel
sandwich)
Primary human hepatocytes (collagen-
matrigel sandwich)
HepRG (Human cell line)
Fibroblasts
11 | Presentation Title | Presenter Name | Date | Subject | Business Use Only

Cellular systems investigated

Kidney
RPTEC/hTERT1-cell line
Human primary cells RPTEC
Static and perfusion cultures
Norm-oxic and hypoxic conditions
CNS
Rat cortical primary culture
Re-aggregating brain cell cultures (embryonic
cells: neurons, astrocytes, oligodendrocytes,
microglica, stem cells)
BBB

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Selected compound/models
Liver :
Ciclosporin A Primary rat hepatocytes
(PRH)
Chlorpromazine Primary human
Amiodarone hepatocytes(PHH)
Ibuprofen
HepaRG
Kidney:
Ciclosporin A Renal cells (RPTEC/TERT1)
Adefovir dipivoxil Tissue: renal cortex
Cis-platin Cell Type: proximal tubule
epithelium
CNS:
Ciclosporin A
3D aggregating rat brain cells
Chlorpromazine
Diazepan 2D mouse brain cells
Amiodarone

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Technologies used
Determination of cellular exposure
(LCMS, NMR)
M&S: Prediction of in vivo doses
Transcriptomics (Illumina)
Proteomics (LCMS)
Metabonomics (LCMS)
High Content Imaging (HCI)
MEA (Electrical Multichannel Recordings)
Biochemical endpoints

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HCI Endpoints
Staining for specific pathologies
Hyperbilirubinemia (Inhibition of Mrp2-mediated
transport) [Carboxy-DCF (DA)]

Steatosis (Accumulation of neutral lipids)

[BODIPY 493/503 (4,4-difluoro-4-bora-3a,4a-
diaza-s-indacene)]

Phospholipidosis (Accumulation of
phospholipids) [LipidTOX Red]

Fluorescent Spots detected by

Cellomics ArrayScan Algorithms

Measurement of Spot Average Area

and Spot Average Intensity

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Treatment schedules Examples

CNS
Chronic-like treatment for all
cellular systems
Multiple treatment: Once daily or
every second day by cell culture
media changes
3 selected concentrations (L,M,H)
- Selected from IC10 from acute 24-48
hour incubations Liver
- Aim: Highest concentration should not
(or only slightly) become cytotoxic after
14 days.
Sampling during 14 days for various
endpoints
- Cell lysates and cell culture supernatant

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Predict-IVProfiling the toxicity of new drugs: a non

animal-based approach integrating toxicodynamics and
biokinetics

WP3:
Non animal-based models for in vitro kinetics and human
kinetic prediction

The ultimate goal is to contribute to the derivation of

NOEC values (relatively to drug safety) in model systems
based on human cells representative of in vivo target
organs, from which it would be possible to extrapolate the
corresponding in vivo dose.

Where we started from?

KaLy-Cell

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In vitro studies can be used to study the MoA

Need of translating information from the cell level,

to organs and subsequently to organisms and to
distinguish between adaption vs. adversity,
likely identifying actual in vitro marker of
adversity (Blaauboer et al, 2012).
Lack of information on actual exposure of cell: in
this respect in vitro biokinetics data providing the
actual level of cell exposure producing an in vitro
observed effect can improve the in vitro-in vivo
extrapolation.

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AMI in vitro biokinetics

3-compartment model

MDEA

Quantity of AMI/MDEA
measured in HepaRG (circles):
culture medium (black),
cytosol (red)
plastic vial walls (blue).
Top row: high doses
Bottom row: low doses

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Murk AJ, Rijntjes E, Blaauboer BJ, Clewell R, Crofton KM,

Dingemans MML, Furlow JD, Kavlock RJ, Kohrle J, Opitz R,
Visser TJ, Traas T, Xia M, Gutleb A (2013)
Mechanism-based testing strategy
using in vitro approaches for
identification of thyroid hormone
disrupting chemicals
Toxicology in Vitro 27, 13201346

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