Development of a Physiologically Based Pharmacokinetic and Pharmacodynamic Model to Quantitate Biomarkers of

Exposure to Organophosphorus Insecticides

Charles Timchalk, Ahmed Kousba and Torka S. PoetBattelle, Pacific Northwest Division, Richland WA

QQ PON1 Polymorphism

1.E-08

1.E-07

1.E-06

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1.E-01

Brai

n O

xon

(AUC

)

5 mg/kg

0.5 mg/kg

0.05 mg/kg

0.005 mg/kg

Lowest PON1 Activity

#7 Variability

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Time (hrs)

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trol

Dermal 5 mg/kgOral 0.5 mg/kg

#5 Rat & Human Validation Studies #6 Saliva Biomonitoring

Blood TCP

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0 3 6 9 12Time (hrs)

umol

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Saliva TCP

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Plasma ChE Inhibition

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TCP Kinetics

ChE InhibitionSaliva ChE Inhibition

0255075

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Free Esterase Oxon-Esterase Aged Complex

Synthesis of New Esterase

“Free”CPF-Oxon +

Degradation of Esterase Released TCP Metabolite

mole hr-1

Regeneration

hr -1

B-Esterase (B-EST) Inhibition(shaded compartments)

CPF-Oxon

Km1, Vmax1

Compartment Model Ke B-EST (AChE, BuChE, CaE)

A-EST* Km3,4, Vmax3,4

BloodV

enou

s B

lood

Arte

rial B

lood

Fat

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Rapid Perfused

Diaphragm

Brain

Liver

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Oxon

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ous

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odSlowly Perfused

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rial B

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Qc

Qfo Cvfo

Qso Cvso

Qro Cvro

Qdo Cvdo

Qbo Cvbo

Qlo Cvlo

Intestine

KsI

KaI

TCP

SkinQsk Cvsk

GavageExposure

DermalExposure

Kp

SA

* Liver and blood only.

Dietary Exposure

Kzero

hr -1

InhibitionuM hr-1 -1

hr -1

Fa Fa

##4 PBPK/PD Model4 PBPK/PD Model

#8 Age-Dependent Response

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PND17 Neonate Rat~4x

Brain AChE Inhibition, Oral 15 mg/kgBrain AChE Inhibition, Oral 15 mg/kg

Background:This project entails development and validation of a physiologically based

pharmacokinetic and pharmacodynamic (PBPK/PD) model (see #1) for the organophosphorus insecticide chlorpyrifos (CPF) to quantitate dosimetry and acetylcholinesterase (AChE) inhibition in young rats and children.

Chlorpyrifos is metabolized to an active oxon metabolite which is a potent inhibitor of AChE. Toxicity is due to the inhibition of AChE resulting in a broad range of neurotoxic effects (see #2).

It is hypothesized that an age-dependent decrement in chlorpyrifos metabolism correlates with increased sensitivity of young animals and potentially children. A balance between the contribution of various parameters like metabolism can increase or decrease the potential toxicity (see #3).

PBPK/PD models (see #4) allow for the integration of all the key parameters associated with toxicity and can be used to quantitate both the dosimetry and biological response (AChE inhibition), over a range of doses, exposure conditions (single vs. repeated), and exposure routes (oral, dermal or inhalation). This tool can be used by risk assessors to make a more biologically based assessment for risk associated with exposure to organophosphorus insecticides.

Dose BioavailabilityMetabolic ActivationMetabolic Detoxification A-Esterase CaE

Increased Toxicity Decreased Toxicity

DoseBioavailabilityMetabolic ActivationMetabolic Detoxification A-Esterase CaE

##33 OP Pharmacokinetic BalanceOP Pharmacokinetic Balance

Exposure Tissue Dose Oxon

Tissue Interaction

Tissue Interaction

AChEInhibition Neurotoxicity

Pharmacokinetics

Pharmacodynamics

##11

N

Cl Cl

ClOP

SC2H5O

C2H5O

N

Cl

Cl

Cl

HO

ChlorpyrifosS

P-450

N

Cl Cl

ClOP

OC2H5O

C2H5O Oxon

OHP

SC2H5O

C2H5ODiethylthiophosphate

3,5,6-trichloro-2-pyridinol (TCP)

OHP

OC2H5O

C2H5ODiethylphosphate

N

Cl

Cl

Cl

Conjugates- O

Sulfate or glucuronides of TCP

P-450

ToxicityAChE Inhibition

##2 Metabolism2 Metabolism

Results:

The PBPK/PD model has been validated using dosimetry and dynamic response (cholinesterase (ChE) inhibition) data obtained from animal and human studies. Figure #5 illustrates the plasma ChE response (data points) and PBPK/PD model fit (lines) for human volunteers exposed to chlorpyrifos both dermally and orally. The model does an excellent job of fitting a range of experimental data.

To evaluate the potential utility of saliva for biomonitoring, studies were undertaken to measure the amount of metabolite (TCP) present in saliva and the degree of salivary ChE inhibition following a oral exposure to chlorpyrifos (see Figure #6). These results suggest that saliva may be useful for biomonitoring for organophosphorus insecticides.

To assess the impact of variability associated with detoxification by human metabolism in adults a Monte Carlo analysis was conducted over a broad ranges of doses (see Figure #7). A metabolic polymorphism has the greatest impact on dosimetry (brain oxon AUC) at doses that overwhelm other detoxification pathways.

The PBPK/PD model was modified to allometrically scale (based on body weight) the age-dependent development of metabolizing enzymes and ChE enzyme activity, and simulations were compared against experimental data. These simulations (see Figures #8 and #9) are consistent with differences in the acute toxicity response between neonatal and adult rats (4-fold difference in sensitivity). However, the model also suggest that metabolism in neonates my be adequate at environmentally relevant exposure concentrations.

Groups Brain CPF-Oxon AUC (moles L-1 hr-1)

  0.5 mg/kg

1.0 mg/kg

5 mg/kg

10 mg/kg

50 mg/kg

Adult Rat 0.02 0.03 0.13 0.31 1.64

Neonatal Rat (PND4) 0.02 (1.0)

0.06 (2.0)

0.36 (2.8)

0.77 (2.5)

6.51** (4.0)

Values in parenthesis are the ratio of brain CPF-oxon AUC for neonatal: adult rats.**Lethal dose level.

##9 Dose Comparison Adult vs. Neonatal Rat9 Dose Comparison Adult vs. Neonatal Rat

Conclusions & Impact:This EPA-STAR project has resulted in the development and validation of an integrated PBPK/PD

model for organophosphorus insecticides that can be used to quantitate age-dependent dosimetry and dynamic response following exposure to chlorpyrifos. This model can be used to address risk assessment issues specifically dealing with children’s susceptibility and cumulative risk.

The model framework can be readily extended to other important organophosphorus and carbamate insecticides.

The quantitation of key metabolites and ChE activity in saliva following in vivo exposure represents an important opportunity for development of non-invasive biomonitoring technology for the rapid detection of organophosphorus insecticide exposure. This approach can be readily adapted to other important pesticides and potentially used as a tool for the rapid assessment of exposure to chemical warfare nerve agents.