spielmann - lush prize conference 2014

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Der Landestierschutzbeaufragte

Dr. med. Horst Spielmann

Professor for Regulatory Toxikology

FU Berlin

& State Animal Welfare Officer - Berlin

„The way forward in implementing the 3Rs into research,

regulatory practice and academic education"

14 November 2014 London UK

Horst Spielmann LUSH Prize 2014

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State Animal Welfare Officer - Berlin

Berlin - this is a honorary office, which has been establsiehd only

in a few states (Länder) of the Federal Republic of Germany

Prof. Horst Spielmann

was appointed „state animal welfare officer – Berlin“

on 19 December 2012

Tasks/mission of the „State Animal Welfare Officer – Berlin“

Providing expert advice on all aspects of animal welfare to the „minister of

justice and consumer protection“ in particular on

- legal changes of the animal welfare policy of the State of Berlin and also

of the Federal government

- official contact point for citizens on all apects of animal welfare

providing advisory opinion to local adminsitrations of the 12 districts of

Berlin

initiation and promotion of proposals to improve animal welfare in Berlin

contact point for citizens, anmal welfare organizations, members of

parliament and the media

internet platform for animal wlelfare via http://www.berlin.de/lb/tierschutz/ Horst Spielmann LUSH Prize 2014

http://www.berlin.de/lb/tierschutz/

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Website of the State Animal Welfare Officer - Berlin


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● End of cosmetics testing in Europe 2013

● The US NAS concept “TT21C”- AOP approach: example sensitization

● Disease models: human in vitro models rather than animal models

● “robotic EST” & human Pluripotent Stem cell Test “hPST“ at ROCHE

● “Human-on-a-chip” technology

● Advancing regulatory science through innovation and worldwide networking

for alternative testing

● BB3R first “Graduate School for the 3Rs” at FU Berlin & U Potsdam, Germany

Topics


Seite 5 Horst Spielmann LUSH Prize 2014

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National

Academy of

Sciences

2007

“….. a not-so-distant future where all routine toxicity testing will be conducted in human cells or cell lines in vitro by evaluating perturbations of cellular responses in a suite of toxicity pathway assays…..”

Andersen and Krewski (2009). Toxicity Testing in the 21st Century: Bringing the Vision to Life. Tox. Sci., 107, 324-330.


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Biologic

Inputs

Normal

Biologic

Function

Morbidity

and

Mortality

Cell

Injury

Adaptive Stress

Responses

Early Cellular

Changes

Exposure

Tissue Dose

Biologic Interaction

Perturbation

Low Dose Higher Dose

Higher yet

Perturbation of Toxicity Pathways


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?

Epidermis Epidermis

Lymph

Node

Induction Elicitation

1. Skin

Penetration

2. Electrophilic

substance:

directly or via

auto-oxidation

or metabolism

3-4. Haptenation:

covalent

modification of

epidermal proteins

5-6. Activation

of epidermal

keratinocytes &

Dendritic cells

7. Presentation of

haptenated protein by

Dendritic cell resulting

in activation &

proliferation of

specific T cells

8-10. Allergic Contact

Dermatitis: Epidermal

inflammation

following re-exposure

to substance due to T

cell-mediated cell

death

Key Event 1 Key Event 2 + 3 Key Event 4 Adverse Outcome

Chemical

Structure &

Properties

Organism

Response

Organ

Response

Cellular

Response

Molecular

Initiating

Event

Adverse Outcome Pathway (AOP) for Skin Sensitization


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Strategy for Testing Skin Sensitisation Potential Without Animals

Combining Different Methods Addressing the Adverse Outcome Pathway

Susanne Kolle and colleagues 2012 BASF


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Increase of experimental animal numbers in Germany (& EU) reasons??

1. Transgenic animals - this increase is a world-wide problem

- a major reason is the increaed use of human diesease models in transgenic mice

2. Disease models - Cardiac and circularory disorders

- Immunologic dieseases

- Multiple Sclerosis

- Alzheimer‘s disease

- Infectious diseases

- Psychiatric dieseases including addiction

- Cancer & metastasis

REALITY: There are only a few established in vtro models for human dieseases

ENCOURAGING EXAMPLE: Infections (tuberculosis, candida, pneumonie)

Why ?? Defense mechnism against infections are based on „antiinfcktive

proteins“ via „toll-like“ receptors, which are different in „men and mice“

RECOMMENTATION FOR FUTURE RESEARCH

molecular pharmakology & toxicology using human cells & tissues EXAMPLE SKIN


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Are mouse models of oral Candida infection valid ?

C. albicans is usually not found in the oral microbial flora of the mouse infection/colonisation almost impossible

In addition significant difference between the human oral mucosa and the horny mulit-layer oral epithelium of the mouse

Buccal

epithelia

Buccal

epithelia

hard

palate Oral mucosa

Weindl et al.

C57BL/6 mice


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Candida infection – Reconstructed human Epidermis (RhE) Model Günther Weindl, Inst. Pharmazie, FU Berlin

basal

PMNs

Filter

Filter

c. albicans

apical

basal

0 – 6 hrs

or

0 -12 hrs

6 – 12 hrs

or

12 -24 hrs

Candidiasis model → infected RhE


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Local candidia infection (Weindl)

colonisation

surface and deeper infections

virulence

factors

host-

factors

• hydrolytic enzymes

(e.g. SAPs)

• dimorphism

• adhesion factors

• phenotypic switching

• skin, mucosa, pH

• antimikrobial peptides

• secretory IgA

• toll-like receptors

• (makrophages, T-cells,

IgG/M, complement etc.)

microbiology

immunology


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human polymorphonuclear leukocytes (PMN) are inducing

up-regulation of expression of TLR4 in human epithelial cells

this defence mechanism is absent in the mouse system

C. albicans + PMN

C. albicans

211,2 6,6

148,6 5,0

LDH (U/l)

24h

24h

n=6, *p < 0.05

nd = nicht detektiert Weindl et al. (2007) J Clin Invest Schaller et al. (2004) Microbiology


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Summary– candidiasis infection model (Weindl)

Chemotaxis von

PMN PMN

PMN

Freisetzung von

IL-1α, IL-1β, IL-6,

TNF, IL-8, …

Epithel

Hochregulation

von TLR4

Epithelium

no release of cytokines

C. albicans colonisation

Epithel

Protektion

C. albicans Infektion

Epithel

Freisetzung von

IL-8, GM-CSF,...


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12 October 2014


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Safety testing of cosmetic ingredients 2013 (source Cosmetics Europe)

< 3 %

35-50 %

< 10 %

< 1 %

50-80 %

< 3 %

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The validated embryonic stem cell test to predict embryotoxicity in vitro hanging

drop culture. Embryoid bodies for the differentiation of embryonic stem cells in

the embryonic stem cell test are generated by pipetting a single-cell suspension

onto the lid of a cell culture dish. The cells aggregate at the bottom of the drop

by gravitational force, thereby forming the embryoid body.

Nature Protocols Vol. 6, June 2011

Seiler A & Spielmann H

The validated embryonic stem cell test to predict embryotoxicity in vitro

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Automated EST Platform Set-up at Roche Implemented since Q1, 2012

Compound

liquid handling for preparing serial

dilutions and compound

plates

Warming

rack for buffer, media,

cells RoMa Arm moves plates in/out

of incubator

8-channel

pipettor uses

sterile, disposable tips

Centrifuge spins down EB into

well for adherence

and further

differentiation

Slide Claudia McGinnis

Position for

HDCP plate for EB

culture/differentiation

Staeubli Robotic

Arm turns HDCP lid

over for liquid handling

/media changes

Cytomax

Incubator for 10 d cytotox/

differentiation

incubations

Horst Spielmann

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Toxicological Sciences 137, 76–90, 2014

human Pluripotent Stem cell Test (hPST) SOX17 and DAPI TC50 values

Dose-dependent effect of

Thalidomide

on mesendoderm formation. Plot of SOX17 and DAPI TC50 values for

71 tested pharmaceutical compounds

94% accuracy

97% sensitivity

& 92% specificity


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Partnering “Human-on-a-chip” disease models

• Transplantation model

• Autoimmune disease model !?


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Microfabrication of human organs-on-chips

Donald E. Ingber et al. Wyss Institute/Harvard

| VOL.8 NO.11 | 2013 | nature protocols

Lung on a chip


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Microfabrication of human organs-on-chips

Donald E. Ingber et al. Wyss Institute/Harvard

| VOL.8 NO.11 | 2013 | nature protocols

Gut-on-a-chip


PRAGUE Czech Republic

www.wc9prague.org

9th World Congress on Alternatives and Animal Use

in Life Sciences

24 – 28 August 2014


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“Human-on-a-chip” – Advancing regulatory science …

Suzanne Fitzpatrick US-FDA Center for Food

Safety & Applied Nutrition Jufeng Wang National Center

for Safety

Evaluation for

Drugs

Hajime Kojima JaCVAM

Sonja Beken FAHMP,

Chair of the

Expert Group

on 3R’s at the

EMA

Sergey Tsyb Deputy Minister of

industry and trade

(responsible for Pharma

2020 program)

“Human-on-a-chip Experts”

Andries van de Meer, Wyss Institute, Boston

Uwe Marx, Technische Universität, Berlin Horst Spielmann LUSH Prize 2014

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Session I-3b: Regulatory science panel discussion .-

“Human-on-a-chip” Advancing regulatory science through innovation

and worldwide networking for alternative testing


Regulatory science panel discussion Prague 28 August 2014

Statement of Intend

Taking into account the large scale projects ongoing on a global

scale with regards to the human-on-a-chip technologies and the

potential interest for global regulatory authorities of different

sectors, it is considered important that a systematic mechanism

for exchange of information is being set up.

The latter should take advantage of the collaborative initiatives

already established for different sectors (e.g. EMA/FDA qualification

exercises, ICH, ...). Such a forum could also allow for cross

sectorial discussions on qualification criteria and performance

standards in order to foster possible qualification …. Horst Spielmann LUSH Prize 2014

Prof. Dr. Monika Schäfer-Korting

Institut für Pharmazie

Berlin-Brandenburg

Graduate School

& Research Platform for the 3Rs

COORDINATOR

Prof. Dr. Monika Schäfer-Korting

Vice-President of the Freie Universität Berlin

http://www.bb3r.de/en/index.html

http://www.bb3r.de/en/index.html

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11 Institutions

3 Junior-

Professors

12 PhD positions

Funding by

Fedral Ministry

for Research &

Technology

BMBF

Integrated Graduate School

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Der Landestierschutzbeaufragte

Dr. med. Horst Spielmann

Professor for Regulatory Toxikology

FU Berlin

& State Animal Welfare Officer - Berlin

Thank You for Your Attention

spielmann - lush prize conference 2014

Science

aspects of animal welfare

animal welfare policy

animal wlelfare

state of berlin

anmal welfare organizations

cell lines

routine toxicity testing

horst spielmannprofessor