alternatives to animal testing in the 21 century · 21st century technologies. developmental...
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Acceptableseverity of experiments
Different views“ethical standards”
vs.possiblebenefit
vs.availablealternatives
• Better science• Less animals• Human relevance• Faster and cheaper results
• Information• Grants• Concepts• New tools, quality control• EU branch, policy program
Scientific American 2005
Research Drug development Clinical trials
92% fail:- 20% tox not
predicted- 40% no efficacy
Average cost$1,4 billion
1 in 100 patientsin hospitals diesfrom adversedrug reactions
$4-11 billionForbes 2012
95% fail(Arrowsmith2012)
47 drugswithdrawnsince 1990
Clinical Studies- Mostly randomized,
double-blind, and placebo-controlled
- Usually multi-centric- Ethical review- Good Clinical Practice- Skilled professionals- Urge to publish / register- Evidence-based Medicine
Basic Research- Mostly unblinded, single
laboratory- Learning on the job- No quality assurance- Limited replicates- Pressure to publish
• Humans are not 70 kg-rats…• Young animals, artificial diseases,
unrealistic treatments, lack of covariables (comorbidity, other treatments)
• Few evaluations, e.g. stroke, sepsis, multiple sclerosis, show disappointing results
• Lack of reproducibility by industry of academic preclinical studies (11-25%)
Limitations of (animal-based) drug development
The third scientific culture: Toxicology
• Internationally harmonized protocols
• Good Laboratory Practice
• Outdated methods• Precaution• Mechanistic thinking
as “mustard after the meal”
Genotoxic: sugarGenotoxic: salt
Protected against TCDD in eggs
Same calculationfor alcohol:One glass per345 years
Protected against minute amounts of pesticides
23 of 31 tested coffee ingredients carcinogenic
Natural pesticides 10,000x more, 35 of 63 carcinogenic
Enjoy!!!
R22 harmful if swallowed (LD50 = 150-200mg/kg in rats)R 36 irritant to eyesR 37 respiratory irritantR 38 irritant to skinNot carcinogenic, but co-carcinogen (promotor)Unclear mutagenicityEmbryonic malformations incat, dog, rat, mice, rabbit, monkey
Unlikely to be brought to the market today
If this gives you headache, take an aspirin!
43 – 60% interspecies correlation3% well tested chemicals8% somewhat tested chemicals
100,000+ chemicals in consumer products
Let’s not beat a dead horse* talking once again about the shortcomings of toxicology
*Completely inappropriate coming from CAAT
3R Success storiesOECD acceptance of
validated methods:• 1999-2001
- Refine: Painless testfor skin sensitisation
- Reduce: Animal numbers for acute tox.from 45 to 8
• 2004 Replace:- phototoxicity- skin corrosion
Traditional 3Rs methods will not be the solution to the problem
• Little perspective for complex endpoints• 2/3 fail validation• hardly solved the cosmetics 7th amendment challenge for 2009, no way for 2013
Limitations of in vitro models Mycoplasma Dedifferentiation favored by
growth conditions and cell selection Cells are bored to death Lack of oxygenLack of metabolism and defense Unknown fate of test
compounds in culture Tumor origin of many cellsCell identity
http://www.hpacultures.org.uk/services/celllineidentityverification/misidentifiedcelllines.jsp
Hello, HeLa….
Since 1967 cell line contamina-tions known:10-20% HeLa18-36% total
wrong
1968: 18 / 18 HeLa1974: 9 / 20 HeLa1976: 30% of 246 wrong (14% wrong species)1977: 41 of 279 wrong1981: about 100 contaminations in cells from
103 sources 1984: 35% of 257 wrong1999: 15% of 189 wrong2003: 82 of 550 wrong2007: 18 of 100 wrong
HeLa genome (Landry et al., 2013)“HeLa cells contain one extra version of most chromosomes, with up to five copies of some. Many genes were duplicated even more extensively, with four, five or six copies sometimes present, instead of the usual two. Furthermore, large segments of chromosome 11 and several other chromosomes were reshuffled like a deck of cards, drastically altering the arrangement of the genes.”Ewen Callawa in Nature
Tox-20c
Tox-21c
Evidence-based Tox.
Omics, high-content, HTSBio-informatics& -engineering
Pathwaysof Tox (PoT)HumanToxome
IntegratedTestingStrategiesITS
Organo-typicculturesHuman-on-Chip
The basic concepts
Validation effort (1 + 1)2
Tests: 1 + 1 = 2
Predictive relevance: 1 + 1 >> 2Applicability domain 1 + 1 < 1
• Interim decision points• Probabilistic / Bayesian approaches• Modeling and Machine Learning
Jaworska, J., and S. Hoffmann. 2010.Integrated Testing Strategy (ITS) -Opportunities to better use existing data and guide future testing in toxicology. ALTEX 27: 231–242.
The future of ITS
Bayesian network ITSfor skin sensitization
Stem cells &Organo-typicculture & High-content
Human-on-chipMicrophysiologicalsystems
21st century technologies
Developmentalneurotoxicityanimal test $1.4 million, 1400 animals per substance
- Autism doubled last decade
- Least tested health effect (<150 substances of 100k in products, 70m synthesized)May 12-14, 2014
Philadelphia
A 3D model of human brain development for studying gene/environment
interactions
School of Public HealthCAAT
Kennedy Krieger InstituteSchool of Medicine
Neural Differentiation of iPSCs in 3D
Nanog/Oct4/DAPIiPSCs
NPCs
Astrocytes
GFAP/NEF/DAPI
MAP2/s100b/DAPI
NeuronsNestin/MAP2/DAPI
MAP2/GABA/DAPI
MAP2/TH/DAPI
MAP2/VGLUT/DAPI
NPCs
GABA
Dopamin
Glutamin
Data processing Data acquisition
Generic workflow
Toxicant exposure
-omics
Bio-informatics: information extraction POT identification and
validation
Why metabolomics & miRNomics?
DNA
RNA
Proteins
Genotype
Metabolites
Genomics(25,000 genes)
Transcriptomics(100,000 mRNAs)
Proteomics(1,000,000 proteins)
Metabolomics(5,000‐7,000 metabolites)
Phenotype
Mammalian Species
miRNAs miRNomics(2,042 miRNAs)
An atmosphere of departure in toxicology
New technologies from biotech and (bio-)informatics revolution
Mapping of pathways of toxicity (PoT)
NAS vision report Tox-21c
“We propose a shift from primarily in vivo animal studies to in vitro assays, in vivo assays with lower organisms, and computational modeling for toxicity assessments” F. Collins, NIH, 2008
“With an advanced field ofregulatory science, new tools, including functional genomics, proteomics, metabolomics, high-throughput screening, andsystems biology, we can
replace current toxicology assays with tests that incorporate the mechanistic underpinnings of disease and of underlying toxic side effects.” M.A. Hamburg, FDA 2011
The concept of (finite number of) pathways of toxicity
Annotation to:- Hazard- Toxicant
(class)- Cell type- Species
Comprehensive list (Human Toxome) Negatives
Toxicant
Toxicant
Initiatives implementing Tox-21c
Organization Approach Purpose Outcome
US EPA & Tox21 (ToxCast Program)
High‐throughput testing
Chemical prioritization(initially)
“Biological signatures”
Hamner Institute Case studies “Just do it” Proof‐of‐principle
NIH project(CAAT‐US) Pathway mapping Pathway ID &
annotation Human Toxome
Mapping the Human Toxome by Systems Toxicology
Hewitt et al., 2005. Science, 307:1572-1573
Endocrine disruption • Use “omics” to map PoT for endocrine disruption
• Develop software tools
• Identify PoT
• Develop a process for PoT annotation, validation
• Establish public database on PoT.
www.humantoxome.com
What this project is not….
…an endocrine disruptor screening project
…a test development project
…an academic publish or perish project
…a five year project
Use for PoT identification:• Homeostasis under stress,
i.e. signatures of tox• Critical cell infrastructures
• Network knowledge• Reference models• Reference toxicants
Toxicity
Cell System
Transcriptomics
Metabolomics
Bioinformatics
Pre-Validated
Matured
IntegratedBiology /Clustering
Standardization
Bottle-neck
Metabolite-ID
Validation?
Similar studies, dissimilar genes
Meta-analysis of transcriptomics studies
All MCF-7 cells24h, 1nM estradiol, whole genome Affymetrix chips
Ochsner et al. Cancer Res 2009
BU_0.01nM-2_2013
BU_0.01nM-1_2013
BU_0.01nM-3_2013
BU_0.1nM-3_2013BU_0.1nM-2_2013
BU_0.1nM-1_2013
BU_1nM-3_2013BU_1nM-2_2013
BU_1nM-1_2013
JHU_0.01nM_2013
JHU_0.1nM_2013JHU_1nM_2013
BU_1nM-2_2012BU_1nM-1_2012
GU_E2 1nM_2012GU_E2+ICI_2012
Microarray data comparison among BU, JHU and GU samples – 24 h samples
• 108 genes involved in estrogen receptor signaling pathway (ESR1) were selected using Qiagen Super-array library
• good concordance between sites for this select group of genes
BU 2012
JHU 2013
GU 2012
Agilent Customized Software for Human Toxome Mapping
GeneSpring Workgroup Plus (released) – server-based collaborative bioinformatics environment in Amazon cloud accessible to all members of the consortium
Agilent BridgeDB (released) – enhanced mapping metabolites onto biological pathways
MassHunter ProFinder – new recursive batch extraction for metabolomics
Correlation Analysis in GeneSpring for transcriptomics & metabolomics
Methods Automation in MPP KEGG content in Pathway Architect Pathways-to-PCDL
Feature Finding
MassHunter Qual
Identify
ID Browser
Alignment & Statistics
MPP
Pathways
Pathway Architect
PoToMaC -The Pathways of Toxicty Mapping CenterTransformative Research Grant: Mapping the Human Toxomeby Systems Toxicology
7 companies, 3 stakeholders
European branch?
Frontloading of toxicology
“fail early, fail cheap”
Anticipate human or regulatory problems?
“test early, develop clean”
Green Toxicology
2006-7: Publication / 1st conferenceMar 2011: US EBTCOct 2011: Secretariat at CAAT
www.ebtox.comJan 2012: First conference hosted by EPAJun 2012: EU EBTCDiverse working groupsJul 2013: Symposium at IUTOX, Seoul,
KoreaSep 2013: Symposium at EuroTox,
Interlaken, SwitzerlandSystematic reviews increasingly embraced by IRIS and NTP
Global perspective
• Global economy needs global regulatory standards
• Opportunities for export and contract testing
• No transition until last important market changes
• Investment into 21st
century technologies for new economies