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The Application of Adverse
Outcome Pathways (AOPs)
for Risk Assessment
Crina Heghes, Principal Global Alliance Manager
12th May 2020
Chris Barber, CEO
Overview
• Supporting tox decisions in an evolving landscape
• AOPs – a useful way to organise knowledge
• Utility of AOPs
• Lhasa’s AOP development
• Introducing Kaptis – a decision support system for risk assessment
• What Kaptis will solve
• Future direction
Evolving landscape for non-animal studies
time
Animal
studies
In vitro
studies
In silico
studies
Routine Targeted Confirmatory
Informs
selection
Run to
increase
confidence
now
Sufficient
for
decision
Informs
selection
Run to
increase
confidence
Sufficient
for
decision
+10 years
Acceptance of non-animal
Acceptance of in silico
+5 years
Routine* Targeted Confirmatory Not needed
+15 years
Limited Applicability Comprehensive
Comprehensive
Accepted
The SupportThe DecisionThe Problem
Given this
biological target..
Given this assay
result..
Given this (real or
potential) AO..
AO = adverse outcome
What AO should I
expect?
What assay should I
run?
What is known about this
biology/mechanism?
How certain is this
relationship?
What data supports this?
Where is the evidence?
A toxicity decision support system
Given this
modality..
AOP = adverse outcome pathway
AOP
Discovery
AOP
Organisation
PBPK
Modelling
Toxicity
Decision
Support
Key Event
Reasoning
Predictive
Models
A Toxicity (Safety) Decision Support System
AOP = adverse outcome pathway
AOP
Discovery
AOP
Organisation
PBPK
Modelling
Toxicity
Decision
Support
Key Event
Reasoning
Predictive
Models
A Toxicity (Safety) Decision Support System
AOPs – a useful way to organise knowledge
What is an AOP?
Molecular Cellular Organ
Individual
Why use AOPs?
• Effort to move toward an integrated approach to testing and assessment (IATA)
• It is likely that many different types of evidence will be needed to replace traditional
animal models
• Combining evidence from different sources into an overall conclusion can be a significant
challenge
• What is the assay actually measuring?
• How closely is this assay linked to the adverse outcome?
• How does this result relate to findings from other assays/models?
• Adverse outcome pathways (AOPs) provide a framework to contextualise these assays
In vitro (Endpoint, Cell Line,
metabolic competency)
In vivo(Endpoint, Species,
route of admin.)
In Chemico(Endpoint, Conditions)
?
In Silico
Integration into new guidance/regulatory paradigms
QSAR
Pharmacological/ toxicological potency prediction
QSAR
Pharmacological/ toxicological pathway prediction
Emerging Assays
IATA
ICH Q3A
ICH S1
OECD Skin Sensitisation Guidelines
QSAR
Toxicity prediction
Emerging Assays
IATA
ICH M7
QSAR
AOPs are a good framework
• Alternative methods are emerging
• We need to know how to use these
AOPs present a good framework to
arrange this knowledge
MIE KE AO
Utility of AOPs
Exploratory toxicology
How to utilise AOPs
MIE KEn AOMIE – molecular initiating event
KE – key event
AO – adverse outcome
How to utilise AOPs
Assay Models
MIE – molecular initiating event
KE – key event
AO – adverse outcomeMIE KEn AO
Receptor
bindingQSAR
binding
Animal
in vivo
Receptor
activityStructural
alerts
Alternative
method
QSAR
activity
How to utilise AOPs
Assay Models
Query compound
MIE KEn AO
Receptor
bindingQSAR
binding
Animal
in vivo
Receptor
activityStructural
alerts
Alternative
method
QSAR
activity
How to utilise AOPs
MIE KEn AO
Receptor
bindingQSAR
binding
Animal
in vivo
Receptor
activityStructural
alerts
Alternative
method
QSAR
activity
Assay Models
Query compound
How to utilise AOPs
• Models associated to events provide the entry points to AOPs and return the
predictions for AOs and KEs
MIE KEn AO
+ve
eqv.
+ve
Receptor
bindingQSAR
binding
Animal
in vivo
Receptor
activityStructural
alerts
Alternative
method
QSAR
activity
Assay Models
Query compound
How to utilise AOPs
• Models associated to events provide the entry points to AOPs and return the
predictions for AOs and KEs
• Association of assays will guide researchers to perform the appropriate
experiments
• Data associated with the pathways will allow experts to answer questions like
“what do compounds like mine do?”
MIE KEn AO
Assay Models
Query compound+ve
eqv.
+ve
Receptor
bindingQSAR
binding
Animal
in vivo
Receptor
activityStructural
alerts
Alternative
method
QSAR
activity
How to utilise AOPs
• Data associated with the pathways will allow experts to answer questions like “what
do compounds like mine do?”
MIE KEn AO
1 2 3
Assay
Query compound
Compound Similarity 1: Binding 2: Activity 3: In vivo
X 99 Active Agonist Teratogenic
Y 84 Active Mixed No data
Z 72 Inactive Inactive No data
Receptor
binding
Animal
in vivo
Receptor
activity
MIE KEn AO
How to utilise AOPs
• Evidence used to develop each key event relationship (KER) is provided
• Enabling expert review
Supporting Evidence:
1. Biological plausibility of relationship
2. Empirical evidence
3. Essentiality
Lhasa’s AOPs development
How Derek Nexus predicts toxicity
Query compound Adverse outcome
Molecular initiating event Key event
Rearranging Derek Nexus knowledge
Molecular
initiating
event
Adverse
outcomeKey
eventKey
event
Molecular Cellular Organ Individual
Benzophenone Pregnane X
receptor (PXR) binding
PXR activation
CYP3A upregulation
Cell proliferation
increase
CYP2B upregulation
Reactive oxygen species
increase
Carcinogenicity
Using Adverse Outcome Pathways
Molecular
Initiating Event
Adverse Outcome (animal/human)
Key Events (KE)
Assayin vitro, in vivo,
clinical, in silico
Adverse Outcome Pathway (AOP)
Mechanistic, knowledge-based
“is the assay relevant for this KE?”
Biological applicability of the assay
“is the assay relevant for this
chemical?”
Chemical applicability of the assay
“is the KEs relevant for the AO?”
Predictivity of the assay for the AO
Lhasa AOP development
• Two key streams of AOP development
• Carcinogenicity – genotoxic and non-genotoxic
• Developmental and reproductive toxicity (DART)
• Literature based approach to AOP development
• Knowledge extracted from Derek Nexus and public literature
• Work supported by the Kaptis Consortium
Summary of carcinogenicity AOP progress
• Investigation of 310 alerts in Derek Nexus identified 37 MIEs
• 37 AOPs have been developed (many with multiple pathways)
Number of MIEs 37
Number of AOPs 37
Number of KEs 368
Number of KERs 511
Number of pathways 448*
Number of non-genotoxic AOPs 15
Number of genotoxic AOPs 22
* Pathways ending in cancer
Non-genotoxic
mechanisms
Genotoxic
mechanisms
Carcinogenicity
AOP network
Summary of DART AOP progress
AOP networks MIEs (excluding
isoforms)
Endpoints
RAR 5 Teratogenicity
Thyroid receptor 8 Embryo-foetal lethality,
Neurodevelopmental toxicity
24 more developed 40 Teratogenicity, Fertility,
Embryo-foetal lethality,
Neurodevelopmental toxicity
• Harvesting Derek Nexus has identified 56 MIEs/putative AOPs
• Several of these have been developed into AOPs/AOP networks
Steroidal
nuclear receptors
Folate pathway
DART MIEs grouped
by their biological role
Introducing Kaptis
What is Kaptis and our vision
A tool to support decision-making in toxicology
A framework for exploring toxicological hazard and risk assessment using a
combination of knowledge and assay data in the context of adverse outcome
pathways (AOPs).
MIE KE AO
Applying Kaptis to decision-making
within risk assessment
Applying Kaptis throughout the development cycle
Molecular
Initiating Event
Adverse Outcome (animal/human)
Key Events (KE)
Discovery
“what is the
consequence of hitting
this target?”
Early Development
“which assay best
predicts the (human)
outcome?”
Late Development
“what adverse outcome
might I see?”
The SupportThe DecisionThe Problem
Given this
biological target..
Given this assay
result..
Given this (real or
potential) AO..
AO = adverse outcome
What AO should I
expect?
What assay should I
run?
What is known about this
biology/mechanism?
How certain is this
relationship?
What data supports this?
Where is the evidence?
A toxicity decision support system
Given this
modality..
What is the consequence of hitting this target?
The DecisionThe Problem
Given this biological
target..
Given this AO..
What AO should I
expect?
What assay should I
run?Given this assay result..
Given this modality..
What is the consequence of hitting this target?
What is the consequence of hitting this target?
AOP Map
Supporting evidence
The DecisionThe Problem
Given this biological
target..
Given this AO..
What AO should I
expect?
What assay should I
run?Given this assay result..
Given this modality..
Which assay could I run?
Which assay could I run?
The DecisionThe Problem
Given this biological
target..
Given this AO..
What AO should I
expect?
What assay should I
run?Given this assay result..
Given this modality..
I have seen this AO… What could potentially cause it?
I have seen this AO… What could potentially cause it?
I have seen this AO… What could potentially cause it?
All pathways known to lead to cleft palate formation
Potentially a battery of binding and activity-based assays could be run to identify/rule out a mechanism
Cleft palate
Future direction – incorporating in silico models
We could associate predictive models (QSAR) to pathways to access the relevant knowledge that way:
GR
agonism
GR
binding
Cleft
palateKE KE
MIE model based on
binding data:
Endpoint = MIE
Prediction = Positive
Confidence = 0.8
Applicability = In domain
Derek Nexus alert
built on in vitro data
Derek Nexus alert
built on in vivo data
Future direction – interpreting results from a screening sequence
Screenshot from research version
= adverse outcome
= molecular initiating event
= key event
= model
= assay
AOPs are simplifications of complex biological networks
• It is not inevitable that triggering a MIE will result in the AO
• Measured activity in an assay associated with a KE does not ‘guarantee’ an AO will be
observed
Reasoning is key in using AOPs to make predictions of AOs
Future direction - reasoning between evidence on AOPs
Reasoning can consider
• Biological relevance of evidence – e.g. in vitro, in vivo, in silico
• Evidence applicability domain – e.g. is the assay result available appropriate to test
the pathway and KE being proposed
• Observational evidence
• Sensitivity and specificity of an assay (for predicting an AO)
• The performance of assays in an AOP for reference compounds
• Proximity to adverse outcome –maybe used in combination with observational
evidence
Future direction - reasoning between evidence on AOPs
Reasoning is key to avoid creating excessive numbers of false
positive predictions
Summary
• Decision-making in toxicity needs to combine
• Evidence from different domains (in silico, in vitro, in vivo)
• Biological knowledge of the underlying mechanisms
• Evidence from analogues (similar chemicals)
• Varied and complex data sources
• This is best addressed through pre-competitive collaboration using
• Knowledge from world-leading scientists
• Knowledge derived from proprietary data
• A systematic, validated approach to reasoning
• Kaptis is being developed to support expert decision-making
• Improve predictivity, reduce costs, increase confidence, reduce animal testing..
Questions?
Thank you!
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