Physiologically Based Pharmacokinetics – Lecture

II

Melvin AndersenMelvin AndersenCIIT Centers for Health ResearchCIIT Centers for Health Research

October 27, 2006October 27, 2006University of North CarolinaUniversity of North Carolina

I. PBPK Models for the Metabolism of Methylene Chloride and Application in Risk Assessment - EasyEasy

II. Thinking about Pharmacokinetics while thinking more deeply about terms such as exposure and mixture? - HarderHarder

TODAY’S TOPICSTODAY’S TOPICS

I. Metabolism of Inhaled Dihalomethanes In Vivo:

Two Pathways – How can we measure them?

H

H Cl

Cl

StainlessSteel

BellowsPump

~ 2.0L/min

CO2

Scrubber

ParticulateFilter

O2 Monitor

PressureGauge

InjectionPort

Ice Filled Pan forH2O Condensation

Desiccator JarChamber

~ 100 mL/min

INTEGRATOR

Gas Chromatograph

5 mL GasSampling Loop

Gas Uptake and Metabolic Parameters

Vial equilibration and Partitioning

Experiments to Get Some of the Experiments to Get Some of the Needed Parameters for a PBPK Needed Parameters for a PBPK ModelModel

Tissue Partition Coefficients

Chamber Volume

Alveolar Space

Lung Blood

Fat Tissue Group

Muscle TissueGroup

Richly PerfusedTissue Group

LiverMetabolizingTissue Group( )

MetabolitesVmax

Km

Cart

Ql

Cart

Qr

Cart

Qm

Cart

Qt

Cart

Qc

Calv (Cart/Pb)

QalvQalv

Cinh

Qc

Cven

Cvt

Cvm

Cvr

Cvl

kf

Adding a Different Exposure ScenarioAdding a Different Exposure Scenario

Gas Uptake Methods Estimate Metabolic Parameters: – Vmax and Km- kf

Ch

am

ber

loss

wit

h C

H2B

rCl

Saturable process

Linear process

Motivation for Studying Bromide

– We studied bromide concentrations in plasma after 4-hr exposures of rats to dibromomethane at various concentrations.

– From data, determined production rates of bromide ion during exposure and thereby estimated biochemical constants (Vmax and Km and kfc) for metabolism of CH2Br2 or CH2BCl in rats.

– Examine the effect of inducers and inhibitors on bromide production curves to see if we can discover the biochemical pathways of dihalomethane metabolism.

Formation and Distribution of Bromide from Oxidation of Dibromomethane

A complete distributional model for CH2Br2 with hepatic metabolism via Cytochrome P450 2E1 oxidation and GST.

kf

Plasma inorganic bromide concentrations on ambient concentrations of CH2BrCl following 4-hr exposures using naive, 2,3-EP, and pyrazole-pretreated rats. The smooth curves were generated by the PBPK.

• 2,3-EP reduces liver GSH

• Pyrazole blocks microsomal oxidation

HbCO concentrations in naïve, 2,3-EP-, and pyrazole-pretreated animals following 4-hr exposures to CH2BrCl.

What about carbon monoxide?

METHYLENE CHLORIDE - 1987

• Causes cancer in mouse lung and mouse liver by inhalation, but not in mice exposed via drinking water.

• Metabolized by two pathways, each producing a reactive metabolite. Oxidation to formyl chloride and GST-conjugation to chloromethylglutathione

• Either pathway could produce a mutagenic metabolite. Which is it?

H

H Cl

Cl

rate

exposure level - PPM

G SH

CYP 2E 1

METHYLENE CHLO RIDE METABOLISM:

C H2C l2 C l-C -H

O C Y P 2E1

GST

G S -CH Cl

ReactionPathw ays

Reaction Kinetics

1000

METHYLENE CHLORIDE - 1987 H

H Cl

Cl

The two pathways contribute differentially at high and low exposure concentrations in rodents, as noted in studies with bromide release.

METHYLENE CHLORIDE - 1987

Responses related to intensity of tissue exposure to short-lived, spontaneously reactive intermediates.

Dose metric for PBPK modeling was estimated by

(amount metabolite formed )/tissue volume/time

Evaluate relationship between daily tissue exposure and cancer in the two-year mouse bioassay.

Provide an approach to extrapolate to lower doses, dose routes, and between mouse and humans.

H

H Cl

Cl

PBPK Model Structure for Methylene Chloride

Attributes:

- Tissue Volumes - Blood Flows - Partition Coefficients - Metabolic Constants - Breathing Rate - Water Intake - Tissue metabolism

H

H Cl

Cl

Lung GasExchange

Lung TissueQC·CV

QP·CI QP·CX

QC·CA

GSTMFO

Richly PerfusedTissues

QR·CAQR·CVR

Slowly PerfusedTissues

QS·CAQS·CVS

FatQF·CAQF·CVF

ModeratelyPerfused Tissues

QM·CAQM·CVM

DrinkGI Tract

QG·CA

Liver

GSTMFO

QL·CA(QL+QG)·CVLQG·CVG

Com parison of Average Daily Values of Dose M easures and Tum or Incidence in Fem ale M ice after M ethylene Chloride Exposure

Control Inhalation Inhalation Drinking W ater (4000 ppm) (2000 ppm) (250 mg/kg/day)

L iver: C YP450* 0.0 3701 3575 5197 GST 0.0 1810 851 15 Parent 0.0 771 362 6.4 Tum ors (% ) 6 83 33 6

Lung: C YP450 0.0 1583 1531 1227 GST 0.0 256 123 1.0 Parent 0.0 794 381 3.1 Tum ors (% ) 6 85 63 6

* C YP 45 0 and G ST d oses: m g m e tabo lized /lite r t issue /day; P a ren t exposu re : m g /lite r-h r

Tissue Doses for CYP2E21 and GST Pathways of Metabolism H

H Cl

Cl

H

H Cl

Cl

LIVER DOSE LUNG DOSE

Interspecies Dose Comparison for Metabolites from the Glutathione Transferase Pathway

• The solid curve is calculated from the PBPK model for the mouse; the dashed curve is calculated for the human. The straight line is extrapolated based on a linear relationship, as was previously assumed. The difference between the upper and lower lines is about 70 to 80 fold.

Using PBPK Models - 1987Using PBPK Models - 1987

Identify toxic effects in animals and/or people

Evaluate available data on mode(s) of action, metabolism, chemistry of compound, metabolites and related chemicals

Describe potential mode(s) of action

Propose relation between response and tissue dose

Develop a PBPK model to calculate tissue dose(s)

Estimate tissue dose during toxic exposures with model

Estimate risk in humans assuming similar tissue response for equivalent target tissue dose

Rats exposed to 500 ppm CO for 2 hr.

% H

bC

ODevelop PBPK parameters for CO portion of model in absence of DHM exposures.

What about carbon monoxide? Can we develop a PBPK model as well? Sure…

Human volunteers were exposed to 50, 100, 200, and 500 ppm CO (Stewart et al., 1975).

Develop parameters for CO portion of PBPK model for humans.

Human exposure to 50 and 350 ppm dichloromethane.

Link DHM metabolism to CO production

Human exposure to 50 and 350 ppm dichloromethane: time course of blood carboxyhemoglobin.

Link DHM metabolism to CO production

What can we evaluate with a model of HbCO production from a Dihalomethane?

Metabolism to CO for methylene chloride first noted in a human volunteer study on carbon monoxide.

Volunteer doing paint stripping at home with solvents had high blood HBCO in the morning.

How could this happen?

Blood carboxyhemoglobin levels after half-hour exposures to 5159 ppm dichloromethane or 5000 ppm bromochloromethane (BCM). Triangles are for BCM; circles are for DCM.

Experimental design in rat study !!!What’s going on here? Why do the compounds have different time courses?

Tissue Partition Coefficients

N N

NH

CH2CH3

N

CH(CH3)2

H

Cl

N

N N

NH

CH2CH3

N

CH(CH3)2

H

SG

N

N N

NH

CH2CH3

N

H

H

Cl

N

N N

NH

H

N

CH(CH3)2

H

Cl

N

N N

NH

CH2CH3

N

H

H

SG

N

N N

NH

H

N

H

H

Cl

N

N N

NH

H

N

H

H

SG

N

N N

NH

H

N

CH(CH3)2

H

SG

N

GLUTATHIONE CONJUGATES

CONJUGATE / DECHLORINATED METABOLITE ELIMINATION

Krbc CHLORO - TRIAZINES

CYP450 CYP450

CYP450CYP450

GSH

GSH

GSH

I. II.

b) c)

d)

a)Incubations conducted at 4 different atrazine concentrations for 90 min.

All 4 chlorinated triazines were followed in the incubation media.

What do you expect from a study of this kind?

McMullin, T.S. (2005). Integrating tissue dosimetry and mode of action to evaluate atrazine dose response. PhD Thesis, Colorado State University. In press at Toxicology in vitro

Iso Ethyl

DACT

II. Atrazine Metabolism & Inhibition in vitro

Results with atrazine metabolism in rat hepatocytes look quite odd...

What’s going on here? Any Ideas?

1.7

266

44 & 98

Atrazine

Ethyl Isopropyl

DACT

RAMatra = (Vmaxatra*Catra) / (Catra + Katra*(1+Ciso/Kiso + Cethyl/Kethyl + Cdact/Kdact))

RAMatra = (Vmaxatra*Catra) / (Catra + Katra*(1+Ciso/Kiso + Cethyl/Kethyl + Cdact/Kdact))

RAMiso = (Vmaxiso*Ciso) / (Ciso + Kiso*(1+ Cethyl/Kethyl + Catra/KatraCatra/Katra + Cdact/Kdact))

RAMethyl=(Vmaxethyl*Cethyl)/ (Cethyl + KEthyl*(1+Catra/KatraCatra/Katra + Ciso/Kiso + Cdact/Kdact))

Accounting for inhibition of metabolic pathways by multiple substrates

DACT dose-response at 90 minutes comparing model simulations (A) without and (B) with competitive metabolic inhibition terms. Lines represent model simulations.

The non-linear behavior of DACT formation required a model that included competitive inhibition, where high [ATRA] inhibit further metabolism of Iso or Ethyl.

Could it be competitive inhibition???

Hexane (Hx) Exposures & Mixtures

Hx induces changes in mean nerve conduction velocity.

It is more potent at 1000 ppm than at 3000 ppm!

What gives rise to this behavior?

Hexane exposures produce 2,5-HD – the actual neurotoxicant.

Baker and Rikert (1979)

Blood concentrations of 2,5-HD are complexly related to inhaled Hx and have very unintuitive relationships over time.

After cessation of Hx exposure in the 2 higher concentration groups, 2,5-HD actually increases over time.

Where have you seen this behavior?

Hexane PBPK Modeling

Interactions arise from two primary sources:

– competition for a common enzyme required for sequential steps in Hx oxidation

- differential properties of Hx (low blood:air partitioning) versus m-n-BK (much higher blood:air partitioning)

Clewell & Andersen (1984)

OH

O OH

OH

O

O

O

OH

Clearance/Filtration

CYP 2E1

CYP 2E1

Complex dose and time dependencies

HexaneExposure

- Clewell & Andersen (1984)

At higher Hx exposures, 2,5-HD increases after exposure cessation.

Inhibitory interactions present during exposure are released as Hx is rapidly exhaled.

Looks a lot like the atrazine story from the in vitro studies…

Do you believe me? Do you want to buy a bridge?

How could we test if this idea worked with other situations?

Designer Mixture – A lipophilic compound (DBM) metabolized to CO and a poorly soluble anesthetic, isoflurane (ISO), in the air.

What happens to CO?

Develop a PBPK model with inhibition between ISO and DBM. Can you explain why you see the bump?

What do we mean by exposure?

Physiologically Based Pharmacokinetic (PBPK) Modeling

Refine Model Structure

Make Predictions

X

X

X

X

X

XX X

Tis

sue C

on

centr

ati

on

Time

You can be wrong!

Collect NeededData

Metabolic Constants

Tissue Solubility

Tissue Volumes

Blood and Air Flows

Experimental System

Model Equations

Define Realistic Model

Liver

Fat

Body

Lung

Air