Knudsen - Lush Prize Conference 2014

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1 Is One R the new Three Rs? Lisbeth E. Knudsen Professor, PhD, University of Copenhagen, Department of Public Health [email protected] Line Mathiesen Thit Aarøe Mørck Pernille Winton Hansen Maria Helena Guerra Andersen Sara Højriis FACULTY OF HEALTH AND MEDICINE SCIENCES UNIVERSITY OF COPENHAGEN Lisbeth E. Knudsen LUSH Nov 2014

Transcript of Knudsen - Lush Prize Conference 2014

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Is One R the new Three Rs?

Lisbeth E. KnudsenProfessor, PhD, University of Copenhagen, Department of Public Health

[email protected] Mathiesen

Thit Aarøe MørckPernille Winton Hansen

Maria Helena Guerra Andersen Sara Højriis

 

FACULTY OF HEALTH AND MEDICINE SCIENCESUNIVERSITY OF COPENHAGEN

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picture© ChemSec

REACH in a nutshell

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Major impact of in silico and in vitro tests

billions of Euro, millions of animals, decades of testing

EU Chemical legislation: REACH

(Registration, Evaluation, Authorisation of Chemicals)

Investment into safety 30,000 chemicals > 1 t per year to be assessed

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Categories of medicines concerned by animal testing for Quality Control purposes

Vaccines for human use and for veterinary useBlood productsBiological and biotechnological productsAntibioticsRadiopharmaceuticals

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The TGN1412 (CD28-superMAB) disaster• Monoclonal CD28 agonistic antibody (T cell co-activator) developed as a

drug for leukemia and rheumatoid arthritis• First-in-man study in London 2006 led to catastrophic systemic multi-

organ failure in six young male volunteers• Animal studies (macaques, mice and rats) showed no adverse effects at

500x the dose used in the human phase I study

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News.bbc.co.uk

Modified from: Alegre et al. (2001), Nat rev immunol

Antigen-presenting cell

CD28

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The TGN1412 (CD28-superMAB) disaster

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• Animal >< humans?• Immunologically naïve animals (raised in sterile conditions)

with few memory T cells• Species differences (no CD28 receptors on memory cells)

• In healthy young men: massive memory cell activation cytokine storm

• Could have been detected by in vitro assays

Stebbings et al. (2007), J immunol. Modified from Eastwood et al. (2010), Br J Pharmacol

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Directive 2010/63/EU on the protection of animals used for scientific purposes requires, as a legal obligation, to apply the Three Rs in all aspects of the care and use of animals. Furthermore, a number of roles under the Directive have a specific responsibility in ensuring information on the Three Rs are made available for those who need it. Equally, several categories of personnel as well as Animal Welfare Bodies or National Committees, should take the opportunities to capture and disseminate new Three Rs innovations for the benefit of the wider user community, science and animals.

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PROBLEMs in getting engagement in replacement

TRADITIONS favouring animal studiesEstablished procedures (SOPS) in the Laboratory, previous publications, age of professorPREJUDICES/SCEPTICISM towards introducing new methods Will not work, no comparative measures, not scientificLACK OF RELEVANCE which can be stated asIn our area we have always used animal models

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PROBLEMs in getting engagement in replacement

LACK OF EXAMPLESThe 3R area is still developing and need more substantiationValidation is ongoingHuman relevanceConcerted research programs in H2020

LACK OF TOOLSDatabases under development/diverseNo full overview

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European Consensus Platform for Alternatives

•International Not-For-Profit Organisation in Belgium (Europe)•Members : National Consensus Platforms on Alternatives with 4 parties :

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WHAT ?

Government

Animal Welfare

Academia

Industry

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The purposes of ecopa are to facilitate the exchange of scientific information, expertise and experience between NCPs, industry, science animal welfare and EU and government institutions to enhance the further development and implementation of refinement, reduction and replacement (3R-concept) in animal experimentation in Europe and worldwide.

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Previously ECOPA participated in EU-research projects with the role of dissemination and public awareness. The new board of ECOPA wanted to stress the importance of local activities within the 3Rs – major emphasis on replacement which was the original remit of ECOPA.

However with the new directive also covering research much emphasis has been devoted to 3Rs and ECOPA is half heatedly taking the refinement and reduction on board as the Replacement R (1R) is our preference. 

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The Danish 3R-Center

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Background

Political decision to establish 3R-Center in Denmark, June ‘13

Unique, powerful and important collaboration - government

- pharmaceutical industry

- animal welfare organizations

Board elected- initial board meeting November ‘13

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Laboratory animals in Denmark

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Board

- Christine Nellemann

- Jan Lund Ottesen

- Axel Kornerup Hansen

- Lisbeth E. Knudsen

- Erwin L. Roggen

- Peter Bollen

- Adrian Smith

- Peter Bie (observer) Secretariat

- Tom Bengtsen

- Anne Sofie Grove

- Rasmus Normann Nielsen

- Mia Vorslund-Kiaer

Organization

Board

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CollaboratorsNationally- Animal welfare organisations- Animal welfare bodies- Pharmaceutical industry- Universities- Danish Ministry of the Environment /PARERE- The Danish Health and Medicines Authority / EMA

Internationally- 3R Research Foundation (Switzerland)- NC3R (U.K.)- ZEBET (Germany)- EURL-ECVAM (EU)-CCAC (Canada), -CAAT (USA)

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Mission

- Initiate activities that may lead to the immediate implementation of the 3Rs

- Provide a forum for collaboration, discussion, exchange and dissemination of information on the 3Rs

- Initiate research projects and recommend funds allocation of resources within the area

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ResearchGoals

- Work to decrease the use of and the collective strain on laboratory animals- Initiate and support research, development and validation within 3R- Develop and promote alternatives to animal experimentation- Initiate/promote research alliances and opportunities for further education

Initiatives

- Distribute research funding- Grant applications in 2014: 18 (4 projects supported)- Publish information regarding and results from the supported projects

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Supported projects 2014

i. Pathological and immunological consequences of basic experimental animal procedures in mice

ii. Reducing the group size in studies of dermatitis by standardization of the gut microbiota

iii. Refinement of animal models of pain: Establishment of strategies to alleviate avoidable pain in rat models for pain and inflammation

iv. Establishment of an In vitro model for diabetic atherosclerosis

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International collaboration

Goals-Keep focus on international alliances-Collaborate with other 3R Centers internationally

Initiatives-Participate in international meetings, councils, seminars and networks concerning 3R and Best Practice-Organise meetings with other 3R Centers worldwide

- NC3R, ZEBET, 3R Research Foundation, EURL-ECVAM-Host an international conference -Maintain good international relations-Participate in” World Congress on Alternatives and Animal Use in the Life Sciences”

- Oral and poster presentations-Connect with national parties involved in international 3R collaborations

- PARERE national contact person- The Danish Health and Medicines Authority contact involved in the 3R work in EMA

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My Research

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Adverse effects from fetal exposures

Premature birth

Low birth weight

Fetal programming

cardiovascular diseasesReproductive damage

Neurodev Diabetes

etc....

DrugsAlcoholDiabetesNutritionAir pollutionMedicine

Prenatal exposure

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Biomarkers in environmental/food healthBiomarkers in environmental/food health researchresearch

Environmental/foodconcentration(external dose)

dose-responserelationship

toxic effects

internaldose

biologicallysignificantdose

alterationsin structure/function

earlybiologicaleffects

biomarkers of individual susceptibility (genetic or acquired)

biomarkers of exposure biomarkers of risk

Biomarkers of exposure (internal dose or biologically significant dose)

Biomarkers of risk (early biological effects or alterations in structure and function of cellular macromolecules)

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Biomonitering

• Blood

• Umbilical cord blood

• Urine

• Hair

• Placenta tissue

• Breast milk

• House dust

• Indoor air

• Questionnaires

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For at ændre ”Enhedens navn” og ”Sted og dato”:

Klik i menulinjen, vælg ”Indsæt” > ”Sidehoved / Sidefod”.Indføj ”Sted og dato” i feltet for dato og ”Enhedens navn” i Sidefod

Human exposure

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Chemical exposure

Effects of exposure:Traditional methods- Animal studies- Cell toxicology assays

Biomarker of exposure-Internal dose

Biomarker of effect- Early response

Human biomonitoring:Measurement for total exposure

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Human biomonitoring

• EU project DEMOCOPHES (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale)

• 145 mother-child pairs in Denmark• Two areas (urban and rural)• Sample collection (urine, hair and blood)• Questionnaire (mothers)

Thit Aarøe Mørck

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64 biomarkers of exposure and effects

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Phthalates

 Parabens PCBs

PFASsPBDEs

Paracetamol

Phenols

Mercury

Cotinine

Cadmium

Dioxin-like activity

Micronuclei frequency

URINE BLOOD

HAIR

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Ex vivo placenta transportLine Mathiesen

95% N2 + 5% CO2

95% O2 + 5% CO2

Maternal

Fetal

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BeWo cells

Human choriocarcinoma cell line

Monolayer with tight junctions (b30)

Trophoblastic cell functions

Transport pathways

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From mother to child

BDE-28

0 100 200 300 4000

100

200

300r = 0.86p <0.001

Maternelt plasma(pg/g lw)

talt

pla

sma

(pg

/g lw

)

Plasma FM-ratio

Ex vivo FM-ratio

BDE-28 0.45 -

BDE-47 0.30 0.47

BDE-99 - 0.25

BB-153 0.18 -

BDE-153 0.15 -

Frederiksen et al., Int. J. Hyg. Environ. Health, (in press); Frederiksen & Vorkamp et al., Environ. Health.

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I have come to the conclusion the 3 Rs together are today not compatible with different scientific disciplines, not normally working together  forced to collaborate. This is my experience from the discussions in the new  Danish 3R centre – strongly associated with the Animal Experiments Inspectorate (abbreviated AEI).I do of course give the 3R work a try in the 3R Centre and emphasize the task of educating and encouraging young researchers in seeking information about animal free research and applying this. For animal free to become the 1R.

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Acknowledgements

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NewGenerisJos Kleinjans et alMischeline Kirch-Volders et alKirsi Vähänkangas et alPäivi Myllynen et alHarrie Besselink et alMargareta Törnquist et alAndrew Collins et alManolis Kogevinas et alhttp://www.newgeneris.org/

Democophes/CophesAll European countriesMarike Kollosa-GehringGreet SchoetersArgelia CastanoReinhard and Anke Joashttp://www.eu-hbm.info/

NanoTESTMaria Dusinska et alMargaret Saunders et al

University of CopenhagenÅse Marie HansenZorana Jovanovic AndersenSteffen Loft

University Hospital CopenhagenMorten Hedegård, Morten Dziengiel, Leif Kofoed, Lisa Maroun, Lise Grupe

Southern Denmark UniversityJesper Bo Nielsen, Philippe Grandjean, Tina Kold Jensen

Aarhus University/NERIEva Bonefeld Jørgensen, Herman Autrup, Katrin Vorkamp

DTU-Food: Christine NellemannAnne Marie Vinggaard

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