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

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

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

Experiments to Get Some of the Needed Parameters for a PBPK Model Gas Chromatograph Experiments to Get Some of the Needed Parameters for a PBPK Model Gas Chromatograph Stainless Steel Bellows Pump 5 m. L Gas Sampling Loop Particulate Filter O 2 Monitor CO 2 Scrubber Pressure Gauge INTEGRATOR Injection Port ~ 2. 0 L/min ~ 100 m. L/min Ice Filled Pan for H 2 O Condensation Desiccator Jar Chamber Gas Uptake and Metabolic Parameters Vial equilibration and Partitioning

Tissue Partition Coefficients Tissue Partition Coefficients

Adding a Different Exposure Scenario Chamber Volume Qalv Cinh Qc Cven Cvt Qalv Alveolar Adding a Different Exposure Scenario Chamber Volume Qalv Cinh Qc Cven Cvt Qalv Alveolar Space Calv (Cart/Pb) Qc Lung Blood Cart Fat Tissue Group Qt Cart Qm Cvm Muscle Tissue Group Qr Cvr Richly Perfused Tissue Group Cart Liver Ql Cvl ( Metabolizing Tissue Group kf Vmax Km ) Cart Metabolites

Chamber loss with CH 2 Br. Cl Gas Uptake Methods Estimate Metabolic Parameters: – Chamber loss with CH 2 Br. Cl Gas Uptake Methods Estimate Metabolic Parameters: – Vmax and Km - kf Linear process Saturable process

Motivation for Studying Bromide – We studied bromide concentrations in plasma after 4 -hr 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 Km 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 kf A complete distributional model Formation and Distribution of Bromide from Oxidation of Dibromomethane kf A complete distributional model for CH 2 Br 2 with hepatic metabolism via Cytochrome P 450 2 E 1 oxidation and GST.

 • 2, 3 -EP reduces liver GSH • Pyrazole blocks microsomal oxidation Plasma • 2, 3 -EP reduces liver GSH • Pyrazole blocks microsomal oxidation Plasma inorganic bromide concentrations on ambient concentrations of CH 2 Br. Cl following 4 -hr exposures using naive, 2, 3 -EP, and pyrazolepretreated rats. The smooth curves were generated by the PBPK.

What about carbon monoxide? Hb. CO concentrations in naïve, 2, 3 -EP-, and pyrazole-pretreated What about carbon monoxide? Hb. CO concentrations in naïve, 2, 3 -EP-, and pyrazole-pretreated animals following 4 -hr exposures to CH 2 Br. Cl.

METHYLENE CHLORIDE - 1987 • Causes cancer in mouse lung and mouse liver by 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?

METHYLENE CHLORIDE - 1987 The two pathways contribute differentially at high and low exposure METHYLENE CHLORIDE - 1987 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 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.

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

Tissue Doses for CYP 2 E 21 and GST Pathways of Metabolism Tissue Doses for CYP 2 E 21 and GST Pathways of Metabolism

Interspecies Dose Comparison for Metabolites from the Glutathione Transferase Pathway LIVER DOSE LUNG DOSE Interspecies Dose Comparison for Metabolites from the Glutathione Transferase Pathway LIVER DOSE LUNG DOSE • 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 - 1987 Identify toxic effects in animals and/or people Evaluate available Using 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

% Hb. CO What about carbon monoxide? Can we develop a PBPK model as % Hb. CO What about carbon monoxide? Can we develop a PBPK model as well? Sure… Rats exposed to 500 ppm CO for 2 hr. Develop PBPK parameters for CO portion of model in absence of DHM exposures.

Develop parameters for CO portion of PBPK model for humans. Human volunteers were exposed Develop parameters for CO portion of PBPK model for humans. Human volunteers were exposed to 50, 100, 200, and 500 ppm CO (Stewart et al. , 1975).

Link DHM metabolism to CO production Human exposure to 50 and 350 ppm dichloromethane. Link DHM metabolism to CO production Human exposure to 50 and 350 ppm dichloromethane.

Link DHM metabolism to CO production 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.

What can we evaluate with a model of Hb. CO production from a Dihalomethane? What can we evaluate with a model of Hb. CO 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?

Experimental design in rat study !!! What’s going on here? Why do the compounds Experimental design in rat study !!! What’s going on here? Why do the compounds have different time courses? Blood carboxyhemoglobin levels after half-hour exposures to 5159 ppm dichloromethane or 5000 ppm bromochloromethane (BCM). Triangles are for BCM; circles are for DCM.

Tissue Partition Coefficients Tissue Partition Coefficients

II. Atrazine Metabolism & Inhibition in vitro Incubations conducted at 4 different atrazine concentrations II. Atrazine Metabolism & Inhibition in vitro Incubations conducted at 4 different atrazine concentrations for 90 min. All 4 chlorinated triazines were followed in the incubation media. Ethyl Iso What do you expect from a study of this kind? DACT Mc. Mullin, T. S. (2005). Integrating tissue dosimetry and mode of action to evaluate atrazine dose response. Ph. D Thesis, Colorado State University. In press at Toxicology in vitro

Results with atrazine metabolism in rat hepatocytes look quite odd. . . What’s going Results with atrazine metabolism in rat hepatocytes look quite odd. . . What’s going on here? Any Ideas? 44 & 98 266 1. 7

Accounting for inhibition of metabolic pathways by multiple substrates RAMatra = (Vmaxatra*Catra) / (Catra Accounting for inhibition of metabolic pathways by multiple substrates RAMatra = (Vmaxatra*Catra) / (Catra + Katra*(1+Ciso/Kiso + Cethyl/Kethyl + Cdact/Kdact)) Atrazine Ethyl Isopropyl DACT RAMethyl=(Vmaxethyl*Cethyl)/ (Cethyl + KEthyl*(1+Catra/Katra + Ciso/Kiso + Cdact/Kdact)) RAMiso = (Vmaxiso*Ciso) / (Ciso + Kiso*(1+ Cethyl/Kethyl + Catra/Katra + Cdact/Kdact))

Could it be competitive inhibition? ? ? DACT dose-response at 90 minutes comparing model Could it be competitive inhibition? ? ? 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.

Hexane (Hx) Exposures & Mixtures Hx induces changes in mean nerve conduction velocity. It 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. Blood concentrations of 2, Hexane exposures produce 2, 5 -HD – the actual neurotoxicant. 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. Baker and Rikert (1979) Where have you seen this behavior?

Hexane PBPK Modeling CYP 2 E 1 Interactions arise from two primary sources: CYP Hexane PBPK Modeling CYP 2 E 1 Interactions arise from two primary sources: CYP 2 E 1 – 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)

Complex dose and time dependencies At higher Hx exposures, 2, 5 -HD increases after Complex dose and time dependencies At higher Hx exposures, 2, 5 -HD increases after exposure cessation. Hexane Exposure 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? - Clewell & Andersen (1984) How could we test if this idea worked with other situations?

Designer Mixture – A lipophilic compound (DBM) metabolized to CO and a poorly soluble 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 Lung Body Fat Metabolic Constants Tissue Solubility Tissue Volumes Physiologically Based Pharmacokinetic (PBPK) Modeling Lung Body Fat Metabolic Constants Tissue Solubility Tissue Volumes Blood and Air Flows Experimental System Liver Model Equations Define Realistic Model Collect Needed Data Refine Model Structure You can be wrong! Tissue Concentration Air X X X X Time Make Predictions