2015 nas interindividual variability workshop | september 30, 2015 integrating in vitro and in...
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2015 NAS Interindividual Variability Workshop | September 30, 2015
Integrating In Vitro and In Silico Methods to Evaluate Variability
Barbara A. WetmoreThe Hamner Institutes for Health SciencesResearch Triangle Park, NC USA [email protected]
2015 NAS Interindividual Variability Workshop | September 30, 2015
Broad-Based Movement in Toxicology Towards In Vitro Testing and Hazard Prediction
2015 NAS Interindividual Variability Workshop | September 30, 2015
HT Toxicity Testing DataDifficulty Translating Nominal Testing Concentrations
into In Vivo Doses
Knudsen et al. Toxicology 282:1-15, 2011
2015 NAS Interindividual Variability Workshop | September 30, 2015
Prioritization and Hazard Prediction Based on Nominal Concentrations Can Misrepresent Potential Health Risks
Protein Binding
Metabolic Clearance
Bioavailability
van de Waterbeemd and Gifford, Nat Rev Drug Disc 2:192, 2003
Reif et al. Environ Hlth Perspect 118:1714, 2010
2015 NAS Interindividual Variability Workshop | September 30, 2015
Incorporating Dosimetry with High-Throughput Screening Data
Human Hepatocytes
(10 donor pool)
HumanPlasma
(6 donor pool)
-5
-4
-3
-2
-1
0
1
2
3
0 50 100 150
Ln C
onc
(uM
)Time (min)
Nifedipine
1 uM initial
10 uM initial
Hepatic Clearance
Plasma Protein Binding
Population-Based In Vitro-to-In Vivo Extrapolation
Plasma Concentration at Steady State for 10,000
Healthy Individuals of Both Sexes from 20 to 50 Yrs Old
Setting the Stage…
2015 NAS Interindividual Variability Workshop | September 30, 2015
Using Reverse Dosimetry to Estimate Population-Based Oral Equivalent Doses
Upper 95th Percentile Css Among 10,000 Healthy
Individuals of Both Sexes from 20 to 50 Yrs Old
Plasma Concentration
ToxCast AC50 Value
Assay X (e.g., ACE inhibition)
Oral Exposure
Oral Dose Required to Achieve Steady State
Plasma Concentrations Equivalent to AC50
Oral Equivalent (mg/kg/day)
ToxCast AC50 (uM)
1 mg/kg/day
Upper 95th Percentile Css (uM)
=
1 mg/kg/dayOral Exposure
Metabolic and binding parameters
Setting the Stage…
2015 NAS Interindividual Variability Workshop | September 30, 2015
Incorporating Dosimetry and Exposure with HTS Data
2015 NAS Interindividual Variability Workshop | September 30, 2015
Primary Hepatocytes
-5
-4
-3
-2
-1
0
1
2
3
0 50 100 150
Ln C
onc
(uM
)
Time (min)
Nifedipine
1 uM initial
10 uM initial
Hepatic ClearanceCl in vitro Plasma Css
General Population
≠
Plasma Css
General Population
Dosimetry and Exposure Strategy Limited to General Population
2015 NAS Interindividual Variability Workshop | September 30, 2015
The Impact of Population Variability on Risk Assessment
Clearance results for full database (27 substrates).
from Ginsberg et al., 2002, Toxicol. Sci., 66, 185-200.
Rat
io to
you
ng a
dults
Age (years)
from Ginsberg et al., 2005, Environ. Health Persp., 113, 1243-49.
Clearance differences span acrossmultiple newborn and juvenile life stages…
… as well as geriatric life stages.
2015 NAS Interindividual Variability Workshop | September 30, 2015
Percent contribution of individual CYPs to total hepatic clearance of xanthines.
Ginsberg et al., 2004, J. Toxicol. Env. Health Pt. A, 67:297-329.
The Impact of Population Variability on Risk Assessment
Cresteil, 1998, Food Addit. Contam., 15:45-51.
Ontogeny of Human Hepatic CYP Isozymes Frequency for CYP2D6 alleles classified as functional, non-functional and reduced functioning for various subpopulations.
Bradford, 2002, Pharmacogenomics. 3(2):229-43.
Sole reliance on pharmacokinetic data for a “generic” population could lead to a significant underestimation of risk
to a susceptible lifestage or subpopulation
2015 NAS Interindividual Variability Workshop | September 30, 2015
CYP1A2
CYP2D6
CYP2C8
CYP2E1
UGT1A4
CYP3A5
CYP3A4
CYP2C19
UGT1A1
CYP2C9
CYP2B6
UGT2B7
Population-based In Vitro-In Vivo Extrapolation
Primary Hepatocytes
-5
-4
-3
-2
-1
0
1
2
3
0 50 100 150
Ln C
onc
(uM
)
Time (min)
Nifedipine
1 uM initial
10 uM initial
Hepatic ClearanceCl in vitro Plasma Css
General Population
CYP1A2
CYP…
CYP3A4
UGT…
ClrCYP1A2
ClrCYP…
ClrCYP3A4
ClrUGT…
Plasma Css for:
Neonates
Northern Europeans
Asians
Children
And so on…
Intrinsic Clearance Rates
rCYP1A2
rCYP3A4
rCYP…
rUGT …
2015 NAS Interindividual Variability Workshop | September 30, 2015
Integrating High-Throughput Pharmacokinetics with the ToxCast In Vitro Assays
Reverse Dosimetry
Oral Exposure
Plasma Concentration
ToxCast AC50 Value
Oral Dose Required for Specific Subpopulations to
Achieve Steady State Plasma Concentrations
Equivalent to In Vitro Bioactivity
~600 In Vitro ToxCast Assays
Ora
l Equ
ival
ent
Dos
e (m
g/kg
/day
)
What are humans exposed to?
?
?
?
Chemical
In VitroBioactivity
Recombinant Enzyme
MetabolismHuman Plasma Protein Binding
Population-Based IVIVE Model
Steady State Plasma Concentrations for Different
Lifestages or Subpopulations
Least Sensitive Assay
MostSensitive Assay
Wetmore et al., 2014, Toxicol Sci, 142(1):210-214
2015 NAS Interindividual Variability Workshop | September 30, 2015
0 20 40 60 80-4
-3
-2
-1
0
1 CYP3A4 CYP3A5 CYP2C19CYP2C8CYP2C9CYP1A2CYP2C18
Time (min)
Ln
Co
nce
ntr
atio
n (
uM
)
0 20 40 60 80-0.4
-0.2
0.0
0.2
0.4
0.6
UGT1A4
Time (min)
Ln
Co
nce
ntr
atio
n ( M
)
0 20 40 60 80-10
-8
-6
-4
-2
0
2CYP2E1CYP2C9CYP1A2CYP2C19CYP2B6
Time (min)
Ln
Co
nce
ntr
atio
n ( M
)
No UGT metabolism detected
Carbaryl Difenoconazole
Recombinant Isozyme Clearance Rates
2015 NAS Interindividual Variability Workshop | September 30, 2015
Incorporating Recombinant Phase I and II Enzyme Data into IVIVE Modeling
Clint = intrinsic clearanceISEF = Clint, HLM
Clint, rCYP x HLM CYP abundanceHLM = human liver microsomes
rCYP = recombinant CYP isoform(pmol P450 / mg protn)
(dimensionless)
(uL / min / mg protn)
(uL / min*pmol P450)
12.25
3.8
5.4
16.5
0.2
3.41.9
140.3
33
4.3 1A22A62B62C82C92C182C192D62E12J23A4
Hepatic CYP Isozyme Abundance in Healthy Adults
(% of Total)
Scaling rCYP Data to HLM using intersystem extrapolation factors
2015 NAS Interindividual Variability Workshop | September 30, 2015
Combining Isozyme Clearance and Abundance Data to Determine Fraction Metabolized
Isozyme No. Chemicals% fm > 5%
% fm Range Chemicals with% fm > 5%
CYP1A2 3 0.4 - 91.4 Bensulide, Carbaryl, Fludioxonil
CYP2C9 6 2.1-63.1 Azoxystrobin, Bensulide, Carbaryl, Difenoconazole, Haloperidol,
Tebupirimfos
CYP3A4 7 1.0-80.2 Acetochlor, Azoxystrobin, Bensulide, Difenoconazole, Haloperidol, Lovastatin
Tebupirimfos
CYP3A5 2 1.4-6.4 Lovastatin, Tebupirimfos
UGT1A1 2 2.6-19.3 Haloperidol, Tebupirimfos
UGT1A4 3 0.1-12.1 Difenoconazole, Haloperidol, Lovastatin
2015 NAS Interindividual Variability Workshop | September 30, 2015
Carbaryl
0.01
0.1
1
Cs
s a
t1
M (
M)
(5th
-95t
h %
ile)
Fludioxonil
0.1
1
10
Cs
s a
t1
M (
M)
(5th
-95t
h %
ile)
Comparison of Css Values Derived Across Multiple Lifestages and Subpopulations
Upper 95th percentile Css
Lifestage or Subpopulation(Age (yr) or Ethnic)
Lifestage or Subpopulation(Age (yr) or Ethnic)
Health
y
Chines
e
Japa
nese
North
Europ
ean
Diabet
ic
Renal
Insu
fficien
cy6-
20 2-6
0.5-
20-
0.5
Health
y
Chines
e
Japa
nese
North
Europ
ean
Diabet
ic
Renal
Insu
fficien
cy6-
20 2-6
0.5-
20-
0.5
HKAF =11.4 HKAF =11.5
2015 NAS Interindividual Variability Workshop | September 30, 2015
Difenoconazole
0.01
0.1
1
Cs
s a
t1
M (
M)
(5th
-95t
h %
ile)
Acetochlor
0.001
0.01
0.1
1
Cs
s a
t1
M (
M)
(5th
-95t
h %
ile)
Comparison of Css Values Derived Across Multiple Lifestages and Subpopulations
Lifestage or Subpopulation(Age (yr) or Ethnic)
Lifestage or Subpopulation(Age (yr) or Ethnic)
Health
y
Chines
e
Japa
nese
North
Europ
ean
Diabet
ic
Renal
Insu
fficien
cy6-
20 2-6
0.5-
20-
0.5
Health
y
Chines
e
Japa
nese
North
Europ
ean
Diabet
ic
Renal
Insu
fficien
cy6-
20 2-6
0.5-
20-
0.5
HKAF =3.5
HKAF =6.7
2015 NAS Interindividual Variability Workshop | September 30, 2015
Wetmore et al., 2014, Toxicol Sci. 142(1):210-214.
Comparison of Css Values Derived Across Multiple Lifestages and Subpopulations
2015 NAS Interindividual Variability Workshop | September 30, 2015
Agreement between In Vivo and IVIVE-derived Css Values using Recombinant
CYP-based Clearance Rates
ChemicalIn vivo PKCss (mM)
IVIVECss (mM)
Carbaryl 0.030 0.046
Haloperidol 0.090-0.126 0.029
Lovastatin 0.004-0.009 0.001
2015 NAS Interindividual Variability Workshop | September 30, 2015
Estimated Chemical-Specific Toxicokinetic Adjustment Factors
Chemical Median Css for Healthy Population
95th Percentile
Css for Most Sensitive
Most Sensitive
Estimated HKAF
% Contribution of Isozyme
Differences to Average HKAF
Acetochlor 0.026 0.15 Neonatal 6.7 86
Azoxystrobin 0.099 0.66 Neonatal 6.7 86
Bensulide 0.241 0.97 Neonatal 4.0 79
Carbaryl 0.043 0.49 Neonatal 11.4 87
Difenoconazole 0.201 0.49Renal
Insufficiency 3.5 99
Fludioxonil 0.38 4.37 Neonatal 11.5 87
Haloperidol 0.029 0.14 Neonatal 4.9 83
Lovastatin 0.001 0.009 Neonatal 6.5 90
Tebupirimfos 0.107 0.38Renal
Insufficiency 3.5 15
2015 NAS Interindividual Variability Workshop | September 30, 2015
Acetochlor
<1 1-2
3-5
6-12
Femal
es
20-5
0
50-8
00.0001
0.001
0.01
0.1
1
10
100
1000
10000
100000
Ora
l Eq
uiv
alen
t D
ose
or
Est
imat
ed E
xpo
sure
(m
g/k
g/d
ay) Difenoconazole
<1 1-2
3-5
6-12
Femal
es
20-5
0
50-8
00.0001
0.001
0.01
0.1
1
10
100
1000
10000
100000
Ora
l Eq
uiv
alen
t D
ose
or
Est
imat
ed E
xpo
sure
(m
g/k
g/d
ay)
♦●▼ ■▲■▲ ♦ ӿ
Matching Oral Equivalent Doses and Exposure Estimates for Different Populations
●ӿ
▼♠ ♠
Subpopulation or Lifestage(Age (yr) or Ethnic)
Subpopulation or Lifestage(Age (yr) or Ethnic)
2015 NAS Interindividual Variability Workshop | September 30, 2015
Matching Oral Equivalent Doses and Exposure Estimates for Different Populations
2015 NAS Interindividual Variability Workshop | September 30, 2015
Incorporating Population TD Variability with PK Variability
Abdo et al. 2015, Environ Int Sep 17; 85:147-155.
2015 NAS Interindividual Variability Workshop | September 30, 2015
Key Points
• First comprehensive attempt to combine physiologic and PK differences to quantitate variability anticipated between life stage-, ethnic- and disease-based populations.
• First effort to evaluate PK variability in a manner that could 1) identify sensitive populations and 2) replace use of default safety factors in risk assessment.
• Subpopulation- or lifestage- based pharmacodynamic differences will also contribute to the variable susceptibilities that may be observed following chemical exposure.
• Tools are emerging that allow the incorporation of pharmacokinetic with pharmacodynamic variability.
2015 NAS Interindividual Variability Workshop | September 30, 2015
Acknowledgements
Hamner - Institute for Chemical Safety Sciences
Rusty Thomas (EPA-NCCT)John Wambaugh (EPA-NCCT)Lisa M. Almond (Simcyp)Masoud Jamei (Simcyp)
External Collaborators
American Chemistry Council –Long Range Initiative
Funding
Brittany AllenMel AndersenHarvey Clewell Alina EfremenkoEric HealyTimothy ParkerReetu SinghMark SochaskiLonglong Yang