51
Thomas Hartung & CAAT team Alternatives to Animal Testing in the 21 st Century

Alternatives to Animal Testing in the 21 Century · 21st century technologies. Developmental neurotoxicity animal test $1.4 million, 1400 ... • 108 genes involved in estrogen receptor

  • Upload
    lythu

  • View
    215

  • Download
    0

Embed Size (px)

Citation preview

Thomas Hartung & CAAT team

Alternatives to Animal Testing in the 21st Century

Acceptableseverity of experiments

Different views“ethical standards”

vs.possiblebenefit

vs.availablealternatives

Often forgotten: there was

good reason to introduce

animal experiments,

e.g. Draizeeye test

• Better science• Less animals• Human relevance• Faster and cheaper results

• Information• Grants• Concepts• New tools, quality control• EU branch, policy program

Scientific American 2005

Not to forget:We can also do a lot for reduction and refinement

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

The temple of biomedicalscience

Toxicology

Basic research

Clinical studies

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• Outdated methods• Precaution• Mechanistic thinking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!

There is some good reason for regulating new products….

More and more innovative products with even more new chemicals….

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

Not physiological situations in vitro…

… but designed artifacts

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

Our vision

NIH:Human Toxome

DTRA:INGOTS

NCATS:3D iPSC(human)

FDA:DNTox-21c(rat)

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)

ALTEX 201330, 209-225

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

The difficulty lies, not in the new ideas,

but in escaping from the old ones.

John Maynard Keynes

(1883 - 1946)