Applications of New Multi-Organ-Chip Tools for Toxicity Assessment

Reyk Horland, PhDTissUse GmbHOudenarder Str. 1613347 Berlin, Germanyreyk.horland@tissuse.com

Conflict of Interest Statement

Dr. Horland is employed by an entity that manufactures and/or distributes a material that is the subject of this session

Overview/Objectives

• Overview of Microphysiological Systems (MPS)

• Introduction to Multi-Organ-Chips (MOC)

• The MOC-based Skin-Liver Co-culture Mode

• Cosmetics Europe Case Study

Types of Microphysiological Systems (MPS)

Marx et al. ALTEX 2016

Components of MPS

Organs-on-Chips Can Recreate Complex Biological Functions of Tissues

The Multi-Organ-Chip (MOC)

COMSOL Multiphysics® 5.2.

Standard cell culture inserts(96-/12-/24-well format)

Features:

Size of a standard microscope slide On-chip micro-pump enabling

pulsatile flow Suitable for iPSC-derived cells,

primary cells, 3D tissues and cell lines

Compatible with life tissue imaging Plug-in option for insert-based

barrier models

Components of a Qualified MPS-Based Assay

The HUMIMIC AutoLab• Automated chip

operation (24 chips per robot)

• Integrated cold storage for different liquids

• Automatic media exchange, liquid sampling, microscopy, etc.

• Robot facility with customized number ofrobots available

MPS-Based Assays in Industrial AdoptionDMPK/Safety Disease Modelling/Efficacy

Chip-Based Human Skin-Liver Co-cultureEvaluate effects of different application routes

Chip-Based Skin-Liver Co-culture Model

Retinoic Acid Metabolite Analyses

repeated systemic exposure Repeated application

peak

area

M10

repeated topical exposure

M10

Cosmetics Europe Partnership

Aim of the Project

HUMIMIC Technology

skin- and liver-specific

metabolism ?

organ-specific toxicity ?

hepatic enzyme

activities ?

Clearance of chemicals ?

Inter-action of organ systems

?

route of exposure ?

• Evaluation of HUMIMIC Technology to contribute tosafety assessment for subacute, repeated dose systemic toxicity

• Multi-Organ-Chip model to investigate the interaction of skin- and liver-specific metabolism of cosmetics chemicals after single and repeated dermal and systemic exposure

Selected Organ Models

Experimental Design

Project Phase I – POC Chemical Selection

Phytochemical – St. John’s wort

Activates CYP3A4 and CYP2C9 via PXR

Cosmetics/Dermatics

Antidepressants - NT reuptake inhibitor

Pesticide (occupational)

Ointment against scabies

Permethrin Hyperforin

Skin Model Histology over 5 days of Repeated Exposure

Project Phase I – POC Chemical Selection

Permethrin Hyperforin

Focus: Metabolites

Systemic vs. Topical Application: Permethrin Metabolite Kinetics

• Metabolite kinetics of single topical application were different from a single systemic application

• Repeat topical application resulted in similar metabolic profile to repeated systemic application–only M2 and M16 were present at lower concentrations

Project Phase I – POC Chemical Selection

Permethrin Hyperforin

Focus: XME induction

Hyperforin: Cytotoxicity

• Hyperforin exerted strong cytoxicity towards liver organoids in pre-experiments but acceptabletoxicity in MOC experiments at tested concentration

Hyperforin: XME Gene Modulation

• Liver organoids show XME gene modulation by hyperforin and respond differently to topical vs. systemic application at early time points

∆∆ct ∆∆

ct fo

ldch

ange

toSC

∆∆ct

Predefined Success Criteria for Project Phase 1

Deliverable

Maintenance of skin and liver organoid structure and functionality in MOC

Transferability of MOC method to other labs

High intra- and inter-laboratory reproducibility

Demonstration of route effects on metabolism of POC chemicals

Verification of application frequency effects on metabolism of POC chemicals

Demonstrate that application route and frequency affects XME levels in liver organoids

Project Phase 2 – Chemical Selection

Criteria Genistein4-amino-2-

hydroxytoluene (AHT)

Results available that indicate chemical could alter XME metabolism

Yes No

In vivo data available Yes Yes

Relevant topical exposure Yes Yes

Sufficient dermal penetration Yes Yes

Optional: Systemic in vivo application route

Yes: Consumer exposure and plasma levels known Yes

Stable & soluble Yes Yes

Not cost-prohibitive No: 250 g = €132 No: 25 g = €42

Chemical suited to liver organoid XME capacity

Yes – CYPs, UGT and SULTs presentYes – NAT, UGT and

SULTs present

Skin’s First Pass Effect is Crucial for AHT’s Safe Use

AHT: Dose and Application Route Dependent Metabolization

A H T - s k in

N-A

ce tyl-A

H T - s k in

A H T - l iv

e r

N-A

ce tyl-A

H T - l iv

e r

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

3 0 0 0

(A ) 2 .5 µ M A H T - s e p a r a t e c o m p a r t m e n t s

nM

in

co

mp

art

me

nt

A H T A H TN -A c -A H T N -A c -A H T

S k in

c o m p a r t m e n t

L iv e r

c o m p a r t m e n t

91 0

3 1

0 1 21 5

N u m b e r s = % to ta l N -a c e t y la te d

A H T - s k in

N-A

ce tyl-A

H T - s k in

A H T - l iv

e r

N-A

ce tyl-A

H T - l iv

e r

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

( B ) 1 0 0 µ M A H T - s e p a r a t e c o m p a r t m e n t s

nM

in

co

mp

art

me

nt

A H T A H TN -A c -A H T N -A c -A H T

S k in

c o m p a r t m e n t

L iv e r

c o m p a r t m e n t

85 3

5 1 02 3

N u m b e r s = % to ta l N -a c e t y la te d

A H T - 2 .5

uM

l-AH T -

2 .5 u

M

A H T - 1 0 0 u

M

A H T - 1 0 0 u

M

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

2 5 0 0 0

( C ) 2 .5 v s 1 0 0 µ M - T o t a l a m o u n t in c ir c u it

nM

in

co

mp

art

me

nt

A H T A H TN -A c -A H T N -A c -A H T

2 . 5 µ M 1 0 0 µ M

9 1 0 2 7 7 53

N u m b e r s = % to ta l N -a c e t y la te d

A H T 1 5 m in

A H T 3 0 m in

A H T 6 0 m in

N -A c - A H T 1 5 m in

N -A c - A H T 3 0 m in

N -A c - A H T 6 0 m in

A H T

N -ace ty

l-AH T

A H T -su lfa

te

A H T -glu

cu ron id

e

N -ace ty

l-AH T -s

u lfate

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

AU

C (

nM

.h)

S y s t e m ic

T o p ic a l2 7 5 %

6 4 %

1 0 4 %

4 6 %2 8 %

Genistein: Stability of Phase 2 XMEs Over Time

1 2 3 4 50

2×1 0 5

4×1 0 5

6×1 0 5

8×1 0 5

(A ) G e n is t e in 6 0 n M t o p ic a l

T im e (d a y s )

XME

rate

(met

abol

ite

PA/d

ay)

1 2 3 4 50

5 .0×1 0 5

1 .0×1 0 6

1 .5×1 0 6

2 .0×1 0 6

( B ) G e n is t e in 6 0 n M s y s t e m ic

T im e (d a y s )

XME

rate

(met

abol

ite

PA/d

ay)

1 2 3 4 50

5 .0×1 0 6

1 .0×1 0 7

1 .5×1 0 7

2 .0×1 0 7

2 .5×1 0 7

(C ) G e n is t e in 1µ M s y s t e m ic

T im e (d a y s )

XME

rate

(met

abol

ite

PA/d

ay)

G lu c u r o n id a t io n

S u lf a t io n

Project Phase 2: ResultsDeliverable Achieved

Excellent intra-laboratory reproducibility

Maintenance of skin and liver organoid structure and functionality in the Chip2

Excellent intra-laboratory reproducibility

Transfer of MOC method to 2nd Lab

Results reproducible in 2nd Lab

Demonstration of route effects on metabolism of 2 chemicals

Verification of application frequency effects on metabolism of 2 chemicals

Demonstration that the route and frequency of application results in different effects on the gene expression in EpiDerm and liver organoid models

Tiered Testing Framework for Hazard Identification

Thomas et al. (2019) The Next Generation Blueprint of Computational Toxicology at the U.S. Environmental Protection Agency

Acknowledgements

Uwe MarxIlka Maschmeyer Jochen KühnlThi Phuong Tao

Nicky HewittThamée RingsSilke Gerlach

Katrin Brandmair

for MS-analyses

Andreas Schepky

A Big Thank You to the Cosmetics Europe ADME Task Force for Scientific and Financial Support

Nicky HewittMartina Klaric (formerly)

Ian Sorrell

Corie EllisonCathy LesterCarine Jacques-Jamin

Camille GénièsHélène Duplan

Sébastien GrégoireAndreas SchepkyDaniela Lange

Eric FabianYurika Fujita