Future Platforms For In Vitro-Based Toxicity Testing

Mary McBride

Agilent Technologies

1 November 12, 2013

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Toxicity testing approaches

Traditional toxicology employs high-dose testing in animals with extrapolation to human-relevant doses

• time-consuming and expensive ($3B/year)

• requires exorbitant use of animals

•not amenable to high throughput

•questionable relevance to humans

Integrated systems toxicology is the integration of traditional toxicology approaches with high-content, high-throughput “omics” technologies.

• employ cells, cell lines, or tissues (preferably of human origin)

• rapid (high throughput) and inexpensive compared to animal tests

• enable mode-of-action elucidation

•need validation to be widely adopted

A comparison of traditional and integrated systems toxicity testing approaches

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Toxicity testing in the 21st century NRC report provided a vision and a strategy for shift to in vitro testing

NRC Report, 2007

Report Called for a Transformative Change to Toxicity Testing, with 4 Major Components:

Chemical Characterization: Physical and chemical properties, use, exposure routes, metabolites

Toxicity Pathways: Employ high-throughput cell-based assays (of human origin) with integrated ‘omics measurements to evaluate perturbations to relevant toxicity pathways (systems biology approach)

Targeted Testing: Conduct limited and directed testing using whole animals only until in vitro methods reliably predict outcomes

Dose-Response: Couple assay data with computational systems biology to address dose-response and in vitro-to-in vivo extrapolations

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Systems biology to map and model pathways Using known toxicity pathways to rapidly go from assays to risk assessments

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Data from dose-

response and

time-course

studies are

combined with

existing

pathway

knowledge and

bioinformatics

to produce a

CSB model,

which is, in turn,

used for IVIVE

and to establish

exposure limits.

Computational Systems Biology Pathway Model

In-vitro to in-vivo extrapolation

Compare to in-vivo

outcomes from MOA

studies

Validated Pathway Assay

(Stem) cell biology, high-throughput pathway assays

Multi-omics pathway analysis

Time course, dose response

Other data streams: Ca transients,

cell imaging, etc

Computational cell biology, control

theory, systems dynamics

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Cell-based assays for predictive toxicology In-vitro platforms for drug and chemical safety, toxicity testing and disease modeling

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Cardiomyocytes Chondrocytes Hepatocytes

• Proliferate extensively • Differentiate into any cell type • Recapitulate embryonic processes, providing

insights into development “windows” • GE enabling “personalized medicine” • Ethical/legal considerations (adult, induced) • Effects unknown; Long term studies needed

Primary Human Cells

• Limited availability • Variable quality • Phenotypic instability • Donor variability • Limited characterization

Human Stem Cells

Transformed Cell Lines

• May not recapitulate cell/tissue biology

• May lack key functional characteristics

• Inadequately represents human diversity

Animal Models

• Represent human biology?

• Resource intensive • Animal welfare issues

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3D culture simulates in-vivo cell environment Innovations in 3D culture are enabling more biologically-relevant results

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2D 3D

•Rigid inert substrates • Cells partially

interact •Not representative of

in vivo environment

• Porous, flexible (ECM gels) • Extensive cell-cell

communication and signaling •Better representation of in vivo environment with

micro-engineered controls

2D Cell Culture 3D Cell Culture

DARPA/FDA/NIH “Human-on-a-Chip”

Integrating “organ on a chip” microdevices to produce physiologically and pathologically accurate models of human organ systems

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Automated high-throughput screening Tox21C has demonstrated rapid screening of in-vitro assays

November 12, 2013

• 11,000 compounds assayed over 4 logs concentration

• Each compound tested against battery of >700 biochemical and cell-based assays

• Phase II assay panel to include more pathway-based assays (e.g., nuclear receptor, oxidative stress)

• Miniaturized assay volumes 2-6 uL in 1536-well plate

• Informatics pipeline for data processing, curve fitting & classification, extraction of SAR

• Generates pharma actives rather than statistical “hits”

• Dramatically increases reliability

• Dramatically reduces false positives and false negatives

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Next-generation in-vitro assay development Need in-vitro assays to map pathway circuitry, understand perturbations and MOAs

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Various high throughput tests

Pr + L Pi - L

Reporter assay

Evaluate Results

Determine consistency of

observed MOAs and expected

targets

e.g., most likely reproductive

toxicity through E2 activation

Move on to

specific in vitro pathway assays for E2-pathway

Safety

Assessment E2-Pathway

AhR-Pathway

DNA-damage Pathway

Oxidative Stress Pathway

Mitochondrial Damage Pathways

“Validated” Toxicity Pathway Assays

CSBP Models in Vitro PK

EC10

Slide courtesy Mel Andersen, Hamner Institute , 2013

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Multi-omics pathway analysis of dose-response Illuminating biological understanding through a systems biology approach

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Production

Regulation

Genomics Proteomics Transcriptomics Metabolomics

Genes mRNA Protein Metabolite

Prevailing paradigm for biological information flow does not fully describe the system

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Multi-omics analysis in GeneSpring Pathway-centric approach to multi-omics research powered by GeneSpring Analytics

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Multi-omics pathway analysis of dose response

Agilent provides the most comprehensive technology portfolio of toxicity testing solutions

Treat Cells

Collect Data

Single-omic Analyses

Multi-omic Analyses

Human Biological Relevance

Sample Prep, Automation, Separation, Detection, Data Analysis, Data Integration

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A private sector partnership for toxicity testing Partnership is accelerating implementation of in vitro tox testing

–One representative from each partner –Development and oversight of research strategy and partner

interactions –Administrative budgeting, organization, etc.

Governance Board

EPA- Regulatory/policy, risk assessment, computational models and database hosting. NIH, FDA may be better positioned to join in 2014.

Provide instrumentation and expertise for genomic, metabolomic, proteomic, and transcriptomic studies, bioinformatics, data analysis and visualization tools

Technology

Direct and coordinate laboratory R&D. Develop assays and protocols. Analyze data. Work with public and private

partner in developing software and bioinformatics tools.

Academic Thought Leaders

Industry

Leverage $3M investments already made for Hamner estrogen-signaling case study approach; technology sector investments to extend/accelerate R&D

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Case studies using prototype pathways Using known toxicity pathways to rapidly go from assays to risk assessments

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1. Select well-studied prototype compounds targeting specific pathways Estrogen receptor mediated proliferation p53-DNA-damage and mutation PPAR-a and lipid metabolism Nrf2-Keap1 oxidative stress AhR liver induction and altered cell growth mitochondrial stress and toxicity

2. Design cell-based toxicity pathway assays to understand key portions of

the network that control dose-response behaviors

3. Refine new quantitative risk assessment tools, i.e., computational pathway models and in vitro to in vivo extrapolation

4. Integrate results into proposed health safety/risk assessments.

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Mapping and modeling toxicity pathways Expanding the map of nuclear reception activation for PPAR-a

Slide Courtesy Mel Andersen, Hamner Institute for Chemical Safety Sciences

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Case studies approach: Estrogen signaling Using known toxicity pathways to rapidly go from assays to risk assessments

Why the Estrogen Cell Signaling Pathway? • Leverage on-going E2-based pathway efforts (NIH grant, EPA, NIEHS) • High visibility (endocrine disruptors are top priority, EPA EDSP) • Already several validated in vitro assays, validated standards and reference compounds • LOTS of animal data

Use human breast and uterine cells, RUA

Map pathway (time/dose courses)

• Transcription factors

• Genomics

• Phosphoproteomics

• Calcium transients

• Metabolomics

Develop CSBP to establish exposure limits

Compare to in vivo uterotrophic responses and perform IVIVE

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Questions??

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Thank You!!

November 12, 2013