General Kinetics and Reverse Dosimetry

AXLR8-3 Meeting June 11 2012

Harvey Clewell & Mel Andersen

The Hamner Institutes for Health Sciences

-5-4-3-2-10123

0 50 100 150

Ln C

onc

(uM

)

Time (min)

Hepatic Clearance

Plasma Protein Binding

Estimated Renal Clearance

Reverse Dosimetry

Exposure Tissue Dose

ACC-LRI program had interests in tools to interpret human biomarkers. Hamner hired Harvey Clewell in 2005 to lead a PBPK based approach, led to a short course at Hamner and research publications, including a review paper.

http://www.thehamner.org/pbpk-course-2006

Relationship of Human Biomonitoring Data to Animal Toxicity Data

Chemical concentrations in human blood from biomonitoring studies

Human exposures (Chemical concentrations in

environment)

Chemical concentrations in animal blood in

toxicity studies

Animal exposures (Administered doses in

toxicity studies)

Pharmacokinetic modeling

Pharmacokinetic Modeling

Traditional risk assessment

Margin of safety

Forward dosim

etry

Reverse dosim

etry

Using Reverse Dosimetry to Interpret Human Biomonitoring Data

• Issue: – Detection of chemicals in human blood (“chemical trespass”) – Uncertain relationship to doses in animal toxicity studies

• Goal: – Reconstruct exposures – Compare to regulatory guidelines

(MCL, RfD, etc) • Tools:

– Pharmacokinetic (PBPK) models – Monte Carlo analysis of exposure variability and sampling

uncertainty • Products:

– Margins of safety – Objective interpretation of biomonitoring data

Example of Reverse Dosimetry for an Individual (Clewell et al, 1999)

• A PBPK model estimated an Iraqi woman’s exposure to methyl mercury (MeHg) through consumption of contaminated bread during pregnancy. – The exposure was estimated based on the concentrations of MeHg in

her hair

• Using the estimated ingestion rate, the model simulated:

– MeHg concentrations measured in the mother’s blood after she was admitted to the hospital

– MeHg concentration in the blood of the infant at birth

• The model then estimated fetal exposures, e.g.:

– The peak fetal blood concentration during gestation

– The average fetal brain concentration during the third trimester

0

50

100

150

200

250

300

350

400

450

0 200 400 600 800

Days

MeHg

in Ha

ir (ppm

)

0

1

2

3

4

5

6

MeHg

in Blo

od (pp

m)

Maternal hairMaternal bloodInfant blood

MaternalExposure

Pregnancy

42 µg/kg/day108 days

Example of Reverse Dosimetry with a PBPK Model Iraqi woman exposed during pregnancy

to grain contaminated with methylmercury

(Clewell et al. 1999)

Reverse Dosimetry with QSAR-Estimated Partition Coefficients and Metabolism Parameters

Exposure Parameters

Partition Coefficients

Kinetic Parameters

Physiological Parameters KTSD

CI

Fat TissuePF CF VF

Rapidly Perfused TissuesPR CR VR

Slowly Perfused TissuesPS CS VS

Skin

Gut TissuePG CG VG

QCLung CAQP CX

QF

QR

QS

QSkn

Liver TissuePL CL VL

StomachDuodenum PDOSE

Surface

P

KTD

KASKAD

VMAX, KMKF

CVGCVL

CVSkn

CVS

CVR

CVF

CVQC

QG

QL

KTSD

CI

Fat TissuePF CF VF

Rapidly Perfused TissuesPR CR VR

Slowly Perfused TissuesPS CS VS

Skin

Gut TissuePG CG VG

QCLung CAQP CX

QF

QR

QS

QSkn

Liver TissuePL CL VL

StomachDuodenum PDOSE

Surface

P

KTD

KASKAD

VMAX, KMKF

CVGCVL

CVSkn

CVS

CVR

CVF

CVQC

QG

QL

Time of the day

Blood Levels

QSAR

Bounding

(Liao et al. 2007)

Predicted Population Distribution of Trichloroethylene Blood Concentrations

LOG10(TCE concentration in blood [mg/L])

-7.0 -6.0 -5.0 -4.0 -3.0

Freq

uenc

y

0

100

200

300

400

500

600

QSPR-based Monte CarloPublished model-based Monte Carlo

Variability

Variability + Uncertainty

Reverse Dosimetry Will Be Necessary to Relate Nominal/Actual Concentrations in In Vitro Assays to the

Equivalent In Vivo Human Exposure

In Vitro Toxicity Assays

EC50 or Data on Concentration-

Response Estimate of Human

Equivalent Dose

QIVIVE*

* Quantitative In Vitro to In Vivo Extrapolation

QIVIVE Approach

Potential Target Tissue

Biokinetic Model

In Vivo Human Toxicty Estimate

In vitro Dynamics

In Vitro Kinetics

QSAR QSPR

Metabolite ID

Metabolite ID

Metabolite ID

Nature of Toxicity

Hepatic clearance Intestinal uptake / metabolism

Renal clearance Partitioning

QIVIVE

Defining Dosimetry and Exposure in High Throughput Toxicity Screens - 1st Generation Approach

Provided by ToxCast

Data Generated In Vitro

398 In Vitro ToxCast Assays

ToxCast AC50 Values

Estimated Target Tissue Bioactivity

Concentration

Metabolic Stability Plasma Protein Binding

Toxicokinetic Parameters

Upper Level of Human

Exposure

Predicted Assay Oral Equivalent

Doses

Data Obtained from Registration Documents

In Vitro-to-In Vivo Extrapolation

Chemicals with Potential to Perturb Cellular Pathways at Relevant

Human Exposure Levels Computational Modeling

Rusty Thomas

Distribution of AC50 Values for the In Vitro ToxCast Assays (Rotroff et al. 2010)

Triclosan

Pyrithiobac-sodium

AC

50C

once

ntra

tion

(µM

)

Emam

ectin

Ben

zoat

eBu

prof

ezin

Pyra

clost

robi

nEt

oxaz

ole

Para

thio

nIso

xabe

nPr

yrith

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c-so

dium

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azon

e2,

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Prop

etam

phos

Atra

zine

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acil

Feno

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rbRo

teno

neFo

rchl

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nM

ethy

l Par

athi

onCy

prod

inil

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etam

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osan

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mid

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uron

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ulid

e Ox

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cline

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Dicr

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zopy

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idin

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heno

l-AAl

achl

orAc

etoc

hlor

Diaz

oxon

Pyrit

hiob

ac-s

odiu

m

Reverse Dosimetry Exposure Modeling for Interpreting In Vitro Assay Results

-5-4-3-2-10123

0 50 100 150

Ln C

onc

(uM

)

Time (min)

Hepatocellular Clearance

Plasma Protein Binding

Estimated Renal

Clearance

Prediction of in vivo clearance

Reverse Dosimetry

Equivalent Exposure

Concentration from in vitro

assay

In Vitro Assays for Characterizing Steady-State Pharmacokinetics

Human Hepatocytes

(10 donor pool)

Human Plasma

(6 donor pool)

Add Chemical (1 and 10 uM)

Add Chemical (1 and 10 uM)

Remove Aliquots at 15, 30, 60,

120 min

Analytical Chemistry

Analytical Chemistry

-5-4-3-2-10123

0 50 100 150

Ln C

onc

(uM

)

Time (min)

Hepatic Clearance

Plasma Protein Binding

Equilibrium Dialysis

At Low Concentrations the Kinetics are Linear

[Conc]SS = DR * BW

ClExtrinsic

ClHepatic ClRenal

FU, BFPortal, MHepatic FU, GFR

*Assume 100% GI absorption.

Dis

trib

utio

n of

Ora

l Equ

ival

ent

Valu

es a

nd

Esti

mat

ed E

xpos

ures

(m

g/kg

/day

)

Distribution of Oral Equivalent Values for the ToxCast Assays and Comparison with Exposures

Triclosan

Pyrithiobac-sodiumEm

amec

tin B

enzo

ate

Bupr

ofez

inPy

raclo

stro

bin

Etox

azol

ePa

rath

ion

Isoxa

ben

Pryr

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sodi

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one

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razin

e Br

omac

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noxy

carb

Rote

none

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hlor

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ron

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hyl P

arat

hion

Cypr

odin

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xaflu

tole

Acet

amip

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anZo

xam

ide

MGK

Diur

onBe

nsul

ide

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etra

cycli

ne D

HDi

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dim

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ochl

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on

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ac-s

odiu

m

(Rotroff et al., Toxicol. Sci., 2010)

Comparing In Vitro Bioactive Doses with Exposure

Fent

in H

ydro

xide

Clo

prop

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noxy

fen

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ine

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0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

10000

100000O

ral E

quiv

alen

t Dos

e or

Est

imat

ed E

xpos

ure

(mg/

kg/d

ay)

Lact

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0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

10000

100000

Ora

l Equ

ival

ent D

ose

or E

stim

ated

Exp

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e(m

g/kg

/day

)

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1

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100

1000

10000

100000

Ora

l Equ

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or E

stim

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Exp

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e(m

g/kg

/day

)

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0.00001

0.0001

0.001

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0.1

1

10

100

1000

10000

100000

Ora

l Equ

ival

ent D

ose

or E

stim

ated

Exp

osur

e(m

g/kg

/day

)

Wetmore et al., Tox Sci., 2011

A total of 9.9% of ToxCast Phase I chemicals have in vitro bioactivity at oral equivalent doses that overlap with the most highly exposed subpopulation.

Comparison of in vitro-to-in vivo extrapolation results with estimates based on in vivo PK

Chemical

PK- or PBPK-Derived

Css (µM)

IVIVE

Cssa,b (uM)

IVIVE

Caco-2c Css

a,b uM)

IVIVE, Fub=0.99

Css (uM)

IVIVE Fub=0.99, Caco-2c

Css (uM)

2,4-dichlorophenoxyacetic acid 9.05-90.05 39.25 40.34 39.25 40.34

Bisphenol-A < 0.13d 0.35 0.40 0.06 0.07

Cacodylic acid 1.80 3.06 tbve 3.06 tbve

Carbaryl 0.03 0.04 0.04 0.03 0.03

Fenitrothion 0.03 17.91 --f 0.10 --f

Lindane 0.46 13.21 tbve 0.07 tbve Oxytetracycline dihydrate 0.36 2.00 0.44 2.00 0.44 Parathion 0.17 24.63 --f 0.14 --f Perfluorooctanoic acid

20,120 g 55.34 g 58.19 g 0.4 g 0.4 g

Picloram 0.27 57.63 32.01 0.37 0.19

Thiabendazole 0.45 13.76 15.20 13.76 15.20

Triclosan 2-10 1.56 1.59 0.01 0.01

Conclusions

• In vitro assays for hepatocyte clearance, plasma protein binding, and Caco-2 transport can reduce uncertainty regarding the chronic in vivo doses that are equivalent to in vitro toxicity assay concentrations from over 4 orders of magnitude to less than 2

• More complicated approaches are necessary for toxicity mediated by metabolites, for acute exposures, and for compounds with more varied physicochemical properties

Liver blood

Liver tissue

http://www.thehamner.org/pbpk-course-2010

Tools for QIVIVE explained:

Key research needed to support QIVIVE

In Vitro Liver Bioreactor for Metabolite Identification & Kinetics

Ed LeCluyse

Other Bioreactor Applications

• Assessing liver toxicity on repeat dosing • Establishing factors affecting clearance for

chemicals with broad ranges of physical chemical properties

• Modeling the platform to identify and modify liver micorenvironments

• Create an in vitro ADME platform for assessing kinetics and tissue responses

• Support human on a chip approaches

Microfluidic Human on a Chip

(Sung et al. 2010)

Considerations for scale-up to a human on a chip – based on physiological sizes of organs and flow characteristics

Building a Human ADME mimic for Human on a chip that may have broader applications

Research Area

Characterization of free concentration in cell-based assays - binding - metabolism - active transport

In vitro models - concurrent intestinal absorption/metabolism - dermal absorption - blood/brain barrier - hepatocyte clearance - pathway/metabolite ID/kinetics (organotypic) - renal clearance

Data collection to support QSPR modeling - metabolite identification - protein binding in cell-based assays - tissue partitions (some classes of compounds) - restricted vs unrestricted hepatic clearance - metabolism rates - gut absorption/metabolism (non-druglike cmpds) - transporter substrates/renal clearance

QIVIVE case studies - classes of physicochemical properties - different metabolism pathways - parent vs stable metabolite vs reactive metabolite - portal of entry vs liver vs remote toxicity

Development of generic PBPK modeling platforms - user friendly, open access - database for physiological parameters - inhalation, dermal, and oral exposure - multiple parallel metabolic pathways

Kinetic research needs to support in vitro based risk assessment

Thank you for your attention Questions