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Examples Illustrating Potential Applications of IVIVE in Chemical Assessment
Miyoung Yoon, PhD
Director, Biokinetics and Biosimulations
ScitoVation, LLC
Conflict of Interest Statement
I disclose that I have no conflicts of interest.
The presented research and case studies were supported by
– American Chemistry Council Long-Range
Research Initiative - QIVIVE/Paraben MoS case
study.
– Council for Advancing Pyrethroids Human Risk
Assessment (CAPHRA) – Early life PBPK case
study with pyrethroids.
Paradigm Shift in Toxicity Testing and Safety Assessment
The 2007 NAS Report on Toxicity Testing in the 21st
Century
Establishing safe human exposures based on cellular
assays
– Modern biology tools to predict mode-of-action in vitro
– Computational approaches to assist translation of the
results in the context of human safety
Multiple initiatives in North America and the EU to move
away from animal testing to mode-of-action based in vitro
assays
Traditional Safety Assessment
Human Safe Exposure Estimates
(Chemical concentration in environment)
Animal Exposures
(Administered dose in toxicity studies)
PK Modeling (Chemical concentration in
the body)
Estimation of Human Safe Exposure based on In Vitro Toxicity Assay Results
In vivo Human
Safe Exposure Estimate
In Vitro
Toxicity Assays
?
QIVIVE - Translation of In Vitro Assay Results in the Context of Human Safety
Potential Target
Tissue
PB/PK Model
In vivo Human
Safe Exposure Estimate
In Vitro
Toxicity Assays
In Vitro
Kinetic Assays QSAR
QSPR
Information
on assays
conditions
Prediction of
chemical kinetics Nature of Toxicity
• Metabolite-ID
• Absorption
• Distribution
• Metabolism
• Excretion
Reverse Dosimetry
(modified from Yoon et al. 2012)
CSBP Dose-
response
modeling
QIVIVE Opportunities in Chemical Safety Assessment Determined by
In vitro toxicity assays
• HT screening vs. Fit-For-Purpose
• Screening for hazards vs. dose
response
• Parent chemical only vs. inclusion of
metabolites
In vitro kinetic assays
• Chemical properties (domains of
applicability)
• Parent disappearance vs. metabolites
formation
• Types of in vitro systems (short vs. long
term culture, system complexity)
Purpose of IVIVE
• Screening/ prioritization (e.g., MoE
analysis)
• Predicting human safe exposure
• Parent vs. metabolite effects
• Interpretation of human data (e.g.,
reverse dosimetry)
• Sensitive population
Level of PB/PK model complexity
• Steady-state exposure calculation (e.g.,
HT-dosimetry) vs. time course
• Extrapolation of in vitro dose-response
to in vivo
• Ability to describe exposure-dose-effect
continuum
Meeting the Needs from Both Sectors
The common goal is to increase efficiency AND
human relevancy in chemical safety assessment
• HT- screening for prioritization of
compounds for further testing
• Safety assessment and
regulatory decisions
• Fit-for-purpose assays guiding
rapid decisions/early selection of
chemicals for product
development
• Meeting regulatory requirement
Regulatory Agencies Industry
PBPK
(Parent &
metabolites)
Domains of Applicability – An Example with In Vitro Metabolism Assays
Primary hepatocytes
Parent CLint
Primary hepatocytes
Parent Clint
+ Metabolite formation rates
Long term hepatocyte
culture
Parent Clint
+ Metabolite formation rates
+ In vitro biokinetics
Organotypic culture in
bioreactor
Parent Clint
+ Metabolite formation rates
+ In vitro biokinetics
+ Toxicity/efficacy
Chemical spaces covered
• Rapid clearance
• Low to moderate lipophilic
• Parent only
IVIVE applicability
• Rapid clearance
• Low to moderate lipophilic
• Parent + some metabolites
• Rapid + slow clearance
• Improved accuracy for lipophilic
• Parent + metabolites
• Rapid + slow clearance
• Improved accuracy for lipophilic
• Parent + metabolites
• Delayed metabolites
HT-IVIVE
(parent)
PBPK
(parent)
PBPK - PD
(Parent &
metabolites)
In vitro assay choices
Screening
Estimation
of Safe
Dose
Application: Prioritizing Compounds using In Vitro Effect Data and Exposure Estimation
Simple Implementation of IVIVE for HTS: Defining Dosimetry in High Throughput Toxicity Screens
Steady-state IVIVE - steady state plasma concentrations of parent chemicals were
calculated using in vitro hepatic clearance and plasma protein binding data
Triclosan
Pyrithiobac-sodium
AC
50
Concentr
ation (
µM
)
Emam
ecti
n B
enzo
ate
Bup
rofe
zin
Pyra
clos
trob
in
Etox
azol
e
Para
thio
n
Isox
aben
Pryr
ithi
obac
-sod
ium
Ben
tazo
ne
2,4-
D
Prop
etam
phos
Atr
azin
e
Bro
mac
il
Feno
xyca
rb
Rot
enon
e
Forc
hlor
fenu
ron
Met
hyl P
arat
hion
Cypr
odin
il
Isox
aflu
tole
Ace
tam
ipri
d
Tric
losa
n
Zoxa
mid
e
MG
K
Diu
ron
Ben
sulid
e
Oxy
tetr
acyc
line
DH
Dic
roto
phos
Thia
zopy
r
Tria
dim
efon
Met
ribu
zin
Fena
mip
hos
Clot
hian
idin
Bis
phen
ol-A
Ala
chlo
r
Ace
toch
lor
Dia
zoxo
n
Pyri
thio
bac-s
od
ium
Triclosan
Pyrithiobac-sodium
mg/k
g/d
ay
Emam
ecti
n B
enzo
ate
Bup
rofe
zin
Pyra
clos
trob
in
Etox
azol
e
Para
thio
n
Isox
aben
Pryr
ithi
obac
-sod
ium
Ben
tazo
ne
2,4-
D
Prop
etam
phos
Atr
azin
e
Bro
mac
il
Feno
xyca
rb
Rot
enon
e
Forc
hlor
fenu
ron
Met
hyl P
arat
hion
Cypr
odin
il
Isox
aflu
tole
Ace
tam
ipri
d
Tric
losa
n
Zoxa
mid
e
MG
K
Diu
ron
Ben
sulid
e
Oxy
tetr
acyc
line
DH
Dic
roto
phos
Thia
zopy
r
Tria
dim
efon
Met
ribu
zin
Fena
mip
hos
Clot
hian
idin
Bis
phen
ol-A
Ala
chlo
r
Ace
toch
lor
Dia
zoxo
n
Pyri
thio
bac-s
od
ium
Greater than 4 orders of magnitude
difference in potencies in vivo – this
simple Implementation of IVIVE for
HTS demonstrates that kinetics is
crucial
By themselves, in vitro assay effect
concentrations are quantitatively
meaningless for risk assessment
(Rotroff et al. 2010)
Application: In Vitro Based Safety Assessment
In Vitro-based Safety Assessment Tiered Testing Strategy
Hum
an data as evidence of chemical effects
Hum
an d
ata
for
valid
atio
n of
in
vitr
o
Bridging the Source to Outcome Continuum for Risk-based Decisions
• Population Lifecourse Exposure
to Health Effects Modeling
Platform (PLETHEM)
• Simulating the exposure of
human populations to
environmental chemicals
• PB/PK modeling - a critical
integration/bridging tool to make
risk-based decisions for
chemical safety under the new
toxicity testing paradigm
“PLETHEM’
Collaboration with US EPA NERL and NCCT
Parabens QIVIVE Study: Margin of Safety (MoS) Assessment
Human health concerns
– Widely used as preservatives (usually as a mixture) in a variety of
consumer products as well as food and pharmaceuticals
– Potential for endocrine affects (weak estrogenic activity with ER
receptor binding affinity ~ 100,000 fold < estrogen)
– Cumulative exposure to mixtures
Objectives
– Development of a PBPK model based on in vitro/in silico data (IVIVE)
– Estimation of population exposure (Css) using PBPK-reverse
dosimetry
– MoS analysis using in vitro PoD and the estimated in vivo Css
– Identify data gaps to improve IVIVE
Parabens QIVIVE - Key Steps
Human PBPK model:
– Physiological parameters for standard human
– QSAR estimation of partition coefficients – Peyret et al. (2010)
– Estimate metabolism and clearance rates from in vitro studies
Reverse dosimetry:
– Free (non-conjugated) paraben plasma concentration equivalent to 95
percentile urine conc. (NHANES)
Point of Departure in vitro:
– In vitro EC10 based on human cell line estrogenic potential assay
Safety Assessment: MOS =
In vitro EC10
In vivo plasma conc.
Paraben Metabolism
Paraben PBPK Model Structure
(Campbell et al., 2015)
* Biological scaling of
in vitro-derived
metabolic parameters
(IVIVE)
• Point of Departure
– Two screens of estrogenic activity were included
• ERLUX – reporter gene (luminescence)
• E-SCREEN – mammary epithelial tumor cell (cell
proliferation)
– EC10 (µg/L) from assay
– Cumulative toxicity was additive at the EC10 for the
17 compound mixture
– Relative Potency Factor = EC10 of a paraben
EC10 of butyl paraben
Margin of Safety
Margin of Safety Adult Female
Parabens QIVIVE Summary
• Paraben PBPK model successfully developed using in vitro metabolism
data and QSAR-predicted tissue partitioning
• IVIVE-PBPK used for reverse dosimetry to estimate plasma Css based
on NHANES urinary biomonitoring data
• MoS calculated based on in vitro EC10 and general population exposure
levels – lowest for female consistent with exposure assessment based
on product use
• Current limitations
• Data gaps for full metabolic pathways – e.g., conjugation
• Oral bioavailability partly informed from rat in vivo data – a new in
vitro tool (human CES-2 expressed Caco-2 cells) is in development
in-house
Improving In Vitro Tools to Increase Confidence in QIVIVE – Oral Bioavailability
• Butyl paraben is preferentially metabolized by CES-2
• Next step: incorporation of the gut description into the whole body PBPK model of parabens
Addressing Sensitive Populations in In Vitro-based Assessment: Pyrethroid case study
Age-specific internal dose
Life stage PBPK MODEL
Age-dependent physiological changes
Age-dependent biochemical changes
Age-appropriate exposure
(Exposure prediction tools)
Requires in vitro data-based
parameterization
(Yoon and Clewell, 2016)
Major Challenges in Developing Early Life PBPK models for Chemicals
Human in vivo data is necessarily limited (in contrast to pharmaceuticals), leading to a reliance on in vitro data. However:
– Pediatric tissues samples are very difficult to obtain and sample quality is generally uncertain
– Addressing human variability is challenging
IVIVE with expressed enzymes in conjunction with enzyme ontogeny of expression as an alternative approach
In Vitro Metabolism Studies for IVIVE Pyrethroids Case Study
Microsomes
Cytosol
Subcellular fractions
Age-specific liver donors
Human expressed
enzymes
Individual CYP or
CES enzymes
CESs
CYPs + CESs
Recombinant enzymes
Human Rat
Building a Generic Model For Pyrethroids For Risk Assessment
Human Age-dependent
PBPK Model
Human age-specific
parameters in vitro
Human Age-relevant
Exposure
(CARES or SHEDS)
Rat Age-dependent
PBPK Model
Rat age-specific
parameters in vitro
IVIVE
Step 1: Proof of concept
in the rat
IVIVE
Step 2: Human modeling
for case compounds
Generic PBPK model
for pyrethroids
Step 4: Exercising the generic
model for risk assessment in
different life stages
Step 3: Read across strategies for model
parameterization built upon the case studies
Targeted in vitro for
compound specific
metabolism parameters
Early Age PBPK Model for Deltamethrin
Human relevant
Exposure
Age-appropriate
metabolism
parameters from
in vitro data
IVIVE Rapidly
Liver
QBR
QF
QS
QR
QH
GI
tissue
QGI
Metabolism
(CES and
CYP)
Plasma
Oral dose
Fecal excretion
Lumen
Brain
Fat
Slowly
QGI
QL
QBR
QF
QS
QR
Lymphatic absorption Human
Growth
Physiology
Average adult Clint
for CES-1
IVIVE
(careful
consideration of in
vitro biokinetics)
Enzyme
ontogeny
CES-1 rapidly
mature after birth
(Hines et al., 2016)
Ontogeny on liver weight
and blood flow (Clewell et
al., 2004; Wu et al., 2015)
The estimated average Clints for
each age
PBPK modeling
Clint_in vivo
20 year old 10 year old
5 year old
1 year old Metabolic rates
(Clint) in vitro
Building Age-specific Deltamethrin Model using IVIVE
In Vitro
Metabolism
Measurement
Simulated Target Tissue Exposure Across Ages
Single dose of deltamethrin 0.005 mg/kg was used for preliminary Monte Carlo simulation
1yr 5 yr 10 yr
Adult
Age
Bra
in C
max
Mode of Absorption Affecting In Vivo Free Concentration
Simulation results based on preliminary metabolism data for rats
Illustration of Lymphatic Absorption and Bypass of Hepatic First-Pass
Restricted Hepatic Clearance Maybe due to Sequestration in Lipoproteins
Pyrethroids Early Life QIVIVE Summary
• PBPK models to provide age-specific dosimetry information
• Dynamics of growth physiology were incorporated in the PBPK
models (life stage modeling)
• Biochemical parameters and their maturation profiles for life stage
modeling were provided using in vitro data (IVIVE)
• Further in vitro PK studies are being conducted to complete the
generic life stage PBPK modeling platform for pyrethroids
• The main goal is the use of the model predicted early age dosimetry
information in safety assessment for infants and children (CSAF)
Use of QIVIVE for Early Age Safety Assessment for Potential Neurotoxicity
Human Age-
dependent PBPK Model
Target tissue concentration
Early age population (Child-adult CSAF)
Age-appropriate Exposure
MoS based on neurotoxicity testing
results (in vitro, in vivo)
Safe exposure guidelines
Equivalent human
exposure
MoS
Different Approaches to Evaluate PBPK Models for Safety Assessment
Traditional PBPK
Parameterization based on the
in vivo (animal) PK data
Capability to perform
interspecies PK extrapolation
to predict humans
Evaluation largely determined
by the model performance
based on the in vitro animal or
human data
In vitro/in silico-based PBPK
Parameterization based on the in
vitro and in silico methods
Capability to coherently integrate
diverse inputs to predict human PK
Evaluation based on in vitro/in silico
tools
Relevance/quality
Validity of the IVIVE method
Evaluation using the available human
data
Acknowledgements
ScitoVation Gina Song Harvey Clewell Mel Andersen
Hamner Yuansheng Zhao Xuyeing Sun Huali Wu Cory Strope
Ramboll Jerry Campbell Jr.
Funding American Chemistry Council Long-Range Research Initiative (QIVIVE/Paraben) Council for Advancing Pyrethroids Human Risk Assessment (CAPHRA, early life pyrethroids)
U of GA Jim Bruckner lab
LFR/CXR Brian Lake team
Medical College of Wisconsin Gail McCarver/Ron Hines Lab
References
Campbell et al., 2015. Toxicology, 332:67-76 (PMID:25839974).
Clewell et al., 2004. Toxicological Science, 79:381–393 (PMID:
15056818).
Hines et al., 2016. Drug Metabolism and Disposition, 44:959-966 (PMID:
26825642).
Peyret et al., 2010. Toxicology and Applied Pharmacology, 249:197–207
(PMID: 20869379).
Rotroff et al., 2010. Toxicological Science, 117:348-358 (PMID:
20639261).
Wu et al., 2015. Environment International, 82:61–68 (PMID: 26043300).
Yoon and Clewell, 2016. Toxicological Research, 32:15-20 (PMID:
26977255).
Yoon et al., 2012. Critical Reviews in Toxicology, 42:633-652 (PMID:
22667820).
