Knudsen - Lush Prize Conference 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
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
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)
Fø
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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