Physiologically Based Pharmacokinetics – Lecture II Melvin Andersen CIIT Centers for Health Research October 27, 2006 University of North Carolina

Easy I.PBPK Models for the Metabolism of Methylene Chloride and Application in Risk Assessment - Easy Harder II.Thinking about Pharmacokinetics while thinking more deeply about terms such as exposure and mixture? - Harder TODAY’S TOPICS

I.Metabolism of Inhaled Dihalomethanes In Vivo: Two Pathways – How can we measure them?

Stainless Steel Bellows Pump ~ 2.0 L/min CO 2 Scrubber Particulate Filter O 2 Monitor Pressure Gauge Injection Port Ice Filled Pan for H 2 O Condensation Desiccator Jar Chamber ~ 100 mL/min INTEGRATOR Gas Chromatograph 5 mL Gas Sampling Loop Gas Uptake and Metabolic Parameters Vial equilibration and Partitioning Experiments to Get Some of the Needed Parameters for a PBPK Model

Tissue Partition Coefficients

Chamber Volume Alveolar Space Lung Blood Fat Tissue Group Muscle Tissue Group Richly Perfused Tissue Group Liver Metabolizing Tissue Group () Metabolites V max KmKm C art QlQl QrQr QmQm QtQt QcQc C alv (C art /Pb) Q alv C inh QcQc C ven C vt C vm C vr C vl kf Adding a Different Exposure Scenario

Gas Uptake Methods Estimate Metabolic Parameters: – Vmax and Km - kf Chamber loss with CH 2 BrCl 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 (V max and K m and kfc) for metabolism of CH 2 Br 2 or CH 2 BCl 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 CH 2 BrCl 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 CH 2 BrCl. What about carbon monoxide?

METHYLENE CHLORIDE 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?

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

METHYLENE CHLORIDE 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.

PBPK Model Structure for Methylene Chloride Attributes: - Tissue Volumes - Blood Flows - Partition Coefficients - Metabolic Constants - Breathing Rate - Water Intake - Tissue metabolism Lung Gas Exchange Lung Tissue QC · CV QP · CIQP · CX QC · CA GSTMFO Richly Perfused Tissues QR · CAQR · CVR Slowly Perfused Tissues QS · CAQS · CVS Fat QF · CAQF · CVF Moderately Perfused Tissues QM · CAQM · CVM Drink GI Tract QG · CA Liver GSTMFO QL · CA(QL+QG) · CVL QG · CVG

Tissue Doses for CYP2E21 and GST Pathways of Metabolism

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 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. % HbCO Develop 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

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? & 98

Atrazine Ethyl Isopropyl DACT RAMatra = (Vmaxatra*Catra) / (Catra + Katra*(1+Ciso/Kiso + Cethyl/Kethyl + Cdact/Kdact)) Catra/Katra RAMiso = (Vmaxiso*Ciso) / (Ciso + Kiso*(1+ Cethyl/Kethyl + Catra/Katra + Cdact/Kdact)) Catra/Katra RAMethyl=(Vmaxethyl*Cethyl)/ (Cethyl + KEthyl*(1+ Catra/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) CYP 2E1

Complex dose and time dependencies Hexane Exposure - 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 X X X Tissue Concentration Time You can be wrong! Collect Needed Data Metabolic Constants Tissue Solubility Tissue Volumes Blood and Air Flows Experimental System Model Equations Define Realistic Model Liver Fat Body Lung Air