Route-Specific Differences in Distribution Characteristics of Octamethylcyclotetrasiloxane in Rats: Analysis Using PBPK Models

Ramesh Sarangapani*,1, Justin Teeguarden{dagger}, Melvin E. Andersen{ddagger}, Richard H. Reitz§ and Kathleen P. Plotzke,2

* ICF Consulting, P.O. Box 14348, Research Triangle Park, North Carolina 27709; {dagger} Environ Corporation, 933 Mesa Lane, Collegeville, Pennsylvania 19426; {ddagger} Department of Environmental Health, CETT/Foothills Campus, Colorado State University, Ft. Collins, Colorado 80523; § RHR Toxicology Consulting Services, Midland, Michigan 48640; and Toxicology, Health and Environmental Sciences, Dow Corning Corporation, Mail CO3101, Midland, Michigan 48686

Received July 25, 2002; accepted October 18, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Octamethylcyclotetrasiloxane (D4) is used in selected consumer products and has a potential for human exposure from multiple routes. Here we develop a physiologically based pharmacokinetic (PBPK) model to describe the tissue dosimetry, plasma concentration, and clearance in the rat following inhalation and dermal, oral, and iv exposure. An initial multiroute PBPK model, based on a previously published inhalation PBPK model for D4, provided excellent fits to the observed concentration time course of D4 metabolites in urine and D4 exhalation rate following dermal exposures. However, the pharmacokinetics of D4, following oral and iv exposure, were sensitive to the mode of entry into the blood compartment. A refined model, describing delivery of D4 from the GI tract to the nonexchangeable/deep blood compartment, provided the best fits to observed plasma D4, exhaled D4, and D4 metabolites excreted in the urine following oral exposure. Pharmacokinetics following iv administration was best described by delivery of D4 directly into the deep blood compartment, possibly reflecting a kinetically identifiable characteristic of the administration of D4 as an emulsion for the intravenous route of exposure. This model-based analysis indicates that the pharmacokinetics of D4 delivered by the inhalation or dermal routes is similar, and is different from the iv or oral delivery routes.

Key Words: inhalation, oral; dermal; iv; multiroute; pharmacokinetics; PBPK model; siloxane.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Octamethylcyclotetrasiloxane (D4) is primarily used as an intermediate in the manufacturing of high molecular weight silicone polymers. A secondary use of D4 is as a vehicle or ingredient in consumer and precision cleaning products. Exposure to D4 in humans is possible via the inhalation, dermal, and oral routes. Following subchronic inhalation exposures to high concentrations of D4 in rats, reproductive effects, as well as induction of hepatic CYP2B1/2 and associated hypertrophy, have been observed (McKim et al., 2000Go, 1999Go; Stump et al., 2000Go). As part of ongoing research to understand the pharmacokinetics of D4, a physiologically based pharmacokinetic (PBPK) model for inhaled D4 was constructed in the rat, and this model was validated against measured D4 concentrations from multiple tissues following single and repeated exposure to a wide range of D4 exposure concentrations (Andersen et al., 2001Go). This analysis revealed unusual pharmacokinetics for D4 resulting from its unusual combination of a low blood:air partition coefficient and a high fat:blood partition coefficient. Inhaled D4 is sequestered in various lipid pools that act as depots limiting the redistribution of D4 within the body, and resulting in a significant fraction of absorbed dose persisting in a bound form postexposure. Free D4, on the other hand, is rapidly cleared from the blood through pulmonary gas exchange. Free D4 is believed to be the biologically active form responsible for hepatic effects observed in rats (Sarangapani et al., 2002Go).

A multiroute PBPK model for D4, once extended to humans, would be used to support estimation and extrapolation of tissue dosimetry, specifically, the amount of biologically active ("free") D4 in humans exposed via multiple routes across dose, dose route, and species. A successful multiroute PBPK model would identify and address route-specific differences in disposition, metabolism, and bioavailability of D4, and could be used for exposure assessment or to reconstruct human exposure from measurements of tissue concentrations in humans. Since chemical toxicity is a consequence of the delivery of active forms of toxic compounds to target tissues, where specific chemical interactions initiate organ-level responses, PBPK models that permit estimation of pertinent tissue-dose metrics are a useful tool to implement mode-of-action driven risk assessments.

In this paper, an extension to the existing inhalation model (Andersen et al., 2001Go) is developed to quantitatively characterize the retention, distribution, and elimination of D4 and its hydrolysis and oxidation products following dermal, oral, and intravenous (iv) exposures in the rat. Concentrations of D4 in plasma, exhaled breath, and urinary metabolite excretion data, measured following dermal, oral, or iv dosing, formed the basis for the model extension and validation. A major focus of this work was to identify and accommodate route-specific pharmacokinetic behaviors, and as such, the results of previously published simulations of inhalation exposures are not repeated here (Andersen et al., 2001Go) This PBPK model is intended to provide the basis for estimating tissue dosimetry of D4 and for supporting extrapolations of tissue dose across dose, dose route, and species for D4, and possibly other volatile cyclic methyl siloxanes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Experimental data from inhalation exposures.
Single-exposure and multiple-exposure studies have been performed in which groups of 10 male and 10 female F344 rats were exposed by inhalation to 7, 70, or 700 ppm of D4 (Plotzke et al., 2000Go). In single-exposure studies, these animals were exposed to 14C-D4 vapor for 6 h in a cylindrical flow-past, nose-only inhalation chamber following a conditioning period of 4 days. In multiple-exposure studies, after receiving the same conditioning, animals received a 6-h exposure of unlabeled D4 for 14 consecutive days, followed by a 6-h exposure of 14C-D4 vapor on the 15th day. Immediately after the last exposure, rats were placed in glass metabolism cages for collection of urine and feces and expired air. Excreta were collected at 6, 12, and 24 h and subsequently at each 24-h interval, up to 168 h postexposure. Expired volatiles were collected at 1, 2, 4, 6, 9, 12, and 24 h and subsequently at each 24-h interval, up to 168 h postexposure. In addition to these two end points, blood, perirenal fat, lung tissue, and liver tissue samples were processed for radioactivity measurements and chemical analysis of parent D4 at similar time points (Varaprath et al., 1998Go, 2000Go). These data sets (Plotzke et al., 2000Go) formed the basis of development of an inhalation PBPK model for D4 (Andersen et al., 2001Go).

Experimental data from intravenous administration.
D4 pharmacokinetic behavior following a single iv dose has been evaluated in male and female rats (Kirkpatrick, 1995Go). Ten male and ten female Sprague-Dawley (CD) rats (195–225 g, Harlan Olac Ltd, Bicester, Oxon, U.K.) per dose group were acclimated for 5 days and administered 7 mg/kg D4 intravenously. D4 was administered as an emulsion, 1:1:7 ethanol, Emulphor EL620 (Rhone Poulenc, NJ) and saline (0.9% w/v Baxter Healthcare, Ltd, Thetford, Norfolk, U.K.). Blood samples were taken at 11 time points between 0 and 48 h following dosing. An additional group of 5 female and 5 male rats were administered 7 mg D4/kg and moved to glass metabolism cages for separate collection of urine, feces, and expired air, to demonstrate a good recovery of radioactivity (98 and 93% of administered dose for males and females, respectively). Radioactivity in blood, urine, and expired air (as 14CO2 from combusted charcoal trap contents) were quantified by liquid scintillation counting in terms of D4 equivalents.

Experimental data from oral exposures.
Three groups of rats (female Fischer 344, 149–177 g, Charles River Laboratories, Raleigh, NC) received oral doses of 300 mg D4/kg (25 mCi 14C-D4), either in a neat form or in simethicone (Dow Corning® 7–9254 LVA) or in corn oil (Mazola) (Plotzke, 1998Go). One group of 50 rats per formulation (5 animals per time point, 10 time points) was used for determination of plasma D4. Following dosing, the blood group animals were placed in stainless steel metabolism cages. Five animals per time point were anesthetized and blood was collected at 15 and 60 min, 6, 12, 24, 48, 72, 96, 120, and 144 h. D4 content in blood samples was determined by GC/MS (Varaprath et al., 1998Go, 2000Go).

One group of 5 rats was used for mass balance and determination of D4 and metabolites in excreta (urine, feces) and expired air. The excreta group also served as the 168-h time point for blood collection. Excreta groups were acclimated in all glass metabolism cages for at least 2 days prior to administration of test material. Immediately following the administration of 14C-D4 by oral gavage, the animals in the excreta group were housed individually in glass metabolism cages suitable for the separate collection of excreta (urine, feces) and of expired air (14CO2 and other volatiles). Urine, feces, and expired CO2 were collected at 6, 12, 24, 48, 72, 96, 120, 144, and 168 h. Expired volatiles were collected at 1, 2, 4, 6, 24, 48, 72, 96, 120, 144, and 168 h postexposure. Urine and feces were collected over dry ice, expired 14CO2 was trapped in a single 4-N potassium hydroxide (KOH) trap, and expired volatiles were collected via charcoal tubes. Total radioactivity (14C-D4/metabolites) in excreta, expired CO2 and volatiles (as toluene extracts of the charcoal traps or directly as aliquots from the KOH trap), and blood was determined by liquid scintillation counting. Mass balance calculations demonstrated good recovery of radioactivity following administration of D4 in corn oil (93.2%), simethicone (92.74%), and as neat D4 (82.9%).

Experimental data from dermal exposures.
Female Fischer 344/CrlBR rats (Charles River Breeding Laboratories) were exposed by the dermal route to 25, 12, or 5 mg of neat 14C-D4 applied over a skin surface area of approximately 2.5 cm2 for a 24-h duration (Jovanovic et al., 2000Go). In this dermal exposure experiment, the skin was semioccluded with a charcoal basket to capture volatilized D4. The charcoal basket was removed after 24 h and the dosed skin area was washed. Each exposure group contained 2 control and 24 treated animals. Blood kinetics data were collected via jugular cannula in 8 rats, and excreta (urine, feces) and exhaled volatiles and 14CO2 were collected (as described previously) in 16 rats placed in glass metabolism cages after the start of dosing. Urine, feces, and expired CO2 were collected at 6, 12, 24, 48, 72, 96, 120, 144, and 168 h. Expired volatiles were collected at 1, 2, 4, 6, 24, 48, 72, 96, 120, 144, and 168 h postexposure. Plasma D4 concentration was assessed at 0.5, 1, 2, 4, 6, 10, 24, and 168 h postexposure. The plasma D4 concentrations were very low in the high-dose group and were below detection limit in the two lowest dose groups. Radioactivity was determined by liquid scintillation counting as described previously. Total parent D4 in exhaled air was determined by GC/MS (Varaprath et al., 1998Go, 2000Go) following toluene extraction of the charcoal traps. Mass balance calculations demonstrated good recovery of radioactivity following dermal administration of D4. At 168 h postexposure, total dose recovery at all three dose levels was higher than 91%.

PBPK model structure.
A PBPK model developed to study the disposition of inhaled D4 has been applied successfully to estimate a diverse set of tissue level dose metrics in rats, following a variety of exposure scenarios to D4 (Andersen et al., 2001Go). The initial multiroute model structure (Fig. 1Go), applied to analyze the disposition of D4 in rats following dermal, oral, and iv exposure, is simply an extension of a D4 inhalation model (Andersen et al., 2001Go) to other exposure routes. This initial model consists of multiple compartments representing fat, liver, lung, blood, GI tract, rapidly perfused tissues, skin, and other slowly perfused tissues (Fig. 1Go). These individual tissues are described as homogenous, well-mixed compartments and are connected by the systemic circulation. Following inhalation exposure to D4, the arterial blood that flows out of the lung compartment, at a rate equal to the cardiac output, supplies D4 to these tissue compartments. Parent D4 is stored in fat and other tissue compartments, depending on their physiological parameters; i.e., tissue volumes, regional blood perfusion rates, and tissue:blood partition coefficients. The venous blood exiting each compartment is equilibrated with the mixed-mean tissue concentration of D4 in the respective compartments. The venous effluents of the various compartments combine to yield a flow-averaged venous concentration of D4. The mixed venous blood returns to the lung at a flow rate equal to the cardiac output. Metabolism of D4 occurs in the liver by a single metabolic pathway following saturable (Michaelis-Menten) kinetics.



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FIG. 1. The initial multiroute PBPK model structure. This is an extension of the D4 inhalation PBPK model (Andersen et al., 2001Go) to include dermal, oral, and iv dosing. Topically applied D4 is practically transferred into the skin compartment and the remaining is captured in the charcoal basket following volatilization; orally administered D4 is practically transferred into the shallow liver compartment by a first-order uptake term and the remaining removed from the gut into the feces compartment; and D4 administered as an iv bolus is introduced into the venous blood as free D4 that is available for exhalation in the gas exchange region.

 
To simulate experimentally observed metabolite clearance, a simplified description of total metabolite kinetics was appended to the initial model. Metabolism of D4 results in the formation of a linear trimer and a monomeric siloxane unit. The initial model was not designed to simulate the concentration of individual metabolites of D4 in the plasma, rather it describes the clearance of the two metabolites produced from the oxidation/hydrolysis ring breaking, using first-order elimination rate constants for each of the two metabolites from the plasma to the urine. The concentration of metabolites in the plasma was calculated by dividing the total amount of metabolite in the body by a constant volume of distribution.

Since D4 is highly lipophilic, a sizable fraction of inhaled D4 is sequestered in various fat stores in the body. To account for the unusual physiochemical properties of this chemical, the initial model had deep compartments to represent the tissue-lipid pool in the lung, liver, and blood. The shallow tissue compartment containing free D4 is in equilibrium with the blood perfusing the tissues. The net transport of D4 between the deep and the shallow tissue compartments in the liver and lung was estimated as the product of a diffusional transfer coefficient and the concentration gradient of free D4 between the two compartments. The various fat depots in the body such as the perirenal, epididymal, and omental fat, as well, as the adipose component of tissues, are grouped and represented using two fat compartments in the initial model: one small fat compartment (~0.5% of body weight) that represents a sequestered fat depot in the body, such as the perirenal fat, and another diffuse fat compartment (~3.5% body weight) that constitutes the remaining mass of adipose tissue in the body. D4 in the deep blood compartment was formulated as a balance between a first-order transfer from the deep liver compartment and slow clearance into the fat compartment.

Following standard approaches to model exposure routes, the iv exposure was introduced in the initial model structure as a bolus dose into the venous blood compartment. The oral dose was introduced as a bolus dose to the GI tract compartment. D4 was cleared from the GI tract compartment by a first-order uptake term into the shallow liver tissue and by another first-order clearance term into the feces. Dermal exposure was modeled as a bolus dose to a (virtual) topical compartment placed on top of the dosed skin compartment. D4 was transported from the topical compartment, either by volatilization (into a charcoal basket compartment) or permeation into the dosed skin area, both represented by first-order clearance terms. The blood perfusing the dosed skin compartment was assumed to be in equilibrium with tissue D4 and contributed directly to the mixed venous circulation (Fig. 1Go).

The governing mass balance equations that describe the rate of change of parent D4 in the various compartments, and the total excreted metabolite in the urine appearing in the initial multiroute model, are similar to those presented elsewhere (Andersen et al., 2001Go) and are not repeated here. The resulting series of differential equations were solved by numerical integration using the Gear algorithm for stiff systems in ACSL® (Advanced Continuous-Simulation Language, AEgis Technologies, Huntsville, AL).

Model parameterization.
Physiological parameters such as tissue volumes and blood perfusion rates for the various organs were obtained from the literature (Brown et al., 1997Go). All other parameters used in the inhalation PBPK model, such as partition coefficients and hepatic metabolism parameters and diffusional transfer rates between deep and shallow pools, were retained unchanged in the multiroute model. Simulations of inhalation exposure pharamacokinetics, which are not repeated here, are not different in this version of the model from those previously published (Andersen et al., 2001Go). The remaining route-specific model parameters such as first-order absorption rate into the liver from the gut, first-order elimination rate from the gut into the feces, rate of volatilization from the topical compartment, and the penetration rate into the skin for the topical dose had to be estimated from the experimental data. A small subset of the available PK data was used for model calibration and the remaining was used in validating the model. The estimated model parameters are provided in Table 1Go.


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TABLE 1 Estimated Values of Parameters Used in the Initial and Refined Multiroute PBPK Rat Model for D4
 
Formal optimization of the additional parameters in the revised model was not conducted with commercially available software (e.g., ACSLOPT, Aegis Technologies). While optimization may increase the reproducibility of parameterizations when applied successfully, it is not without limitations when applied to complicated models. The values of the fitted parameters here reflect extensive exploration of the parameter space and represent best estimates of their values. Formal optimization would not be expected to change the value of the final parameters significantly.

PK data following dermal exposure were available at three different dose levels. D4 concentration time course data in charcoal basket (data not shown) and D4 in exhaled air from the high dose group (25 mg) were used to parameterize the model for the dermal exposure, and the remaining two lower dose groups were used to validate the model for this exposure route. The rate of volatilization was estimated by fitting the model to the D4 concentration time course in the charcoal basket, and the rate of penetration into the skin was estimated by fitting the model to the D4 concentration time course in exhaled air.

For the oral gavage dose, absorption and transfer rate constants were estimated for each carrier fluid: corn oil, simethicone, and neat. For each of the carrier fluids, the elimination rate from the gut was estimated by matching model-derived feces D4 concentrations to experimental measured values. Similarly, the absorption rate from the gut into the liver was estimated by visually fitting the model predictions for D4 excretion rate in urine to measured data. The uptake rate constant was highest with corn oil as the carrier and lowest for simethicone, with neat falling in the middle. This behavior is consistent with the observation that uptake of lipophilic compounds by the GI tract and transport via the lymphatics are influenced by dietary fat concentrations (Roth et al., 1993bGo). D4 concentrations in plasma and data on exhaled D4 were used to validate the model following oral exposure.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Initial model fit to PK data from inhalation and dermal exposures.
The multiroute PBPK D4 model provides excellent fits to the observed blood and exhaled air concentrations of parent D4 or total blood radioactivity (D4 and metabolites), and acceptable fits to urinary excretion rates of D4 metabolites following single inhalation exposures to 700 ppm D4 in male and female F344 rats (Fig. 2Go). The initial model provides good fits to all the tissue concentration data (Andersen et al., 2001Go), and captures the biphasic nature of the tissue clearance characterized by rapid elimination of free D4 from tissue in the initial times after the inhalation exposure and the more gradual elimination at later time points following cessation of inhalation exposure.



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FIG. 2. Plots comparing initial model simulations to experimentally observed D4 concentrations in plasma, exhalation rate of D4, and urinary excretion rate of D4 metabolites in male and female F344 rats following a single 6-h exposure to 700 ppm D4. Error bar represents standard error of the mean.

 
The physiological parameters, rate constants for movement of D4 into and out of deep and shallow tissue compartments, and the metabolic constants used to fit the inhalation PBPK model were retained for simulations of the other exposure routes (i.e., dermal, oral, and iv). Without changes to model parameters, the multiroute model provided good fits to the excretion rate time course of D4 metabolites in urine across the modeled dose range (2.5–10 mg/cm2 skin) following dermal exposure (Fig. 3Go). The multiroute model predictions were compared to the observed D4 elimination rate in exhaled air and urine in female F344 rats during (0–24 h) and postexposure (24–160 h) at the medium (12 mg) and low (5 mg) dose group and the total D4 plasma concentration in terms of D4 equivalents at the high (25 mg) dose group (Fig. 3Go). The model provided reasonable overall fits to the rate of excretion of D4 metabolites in urine and very good fits of exhalation rate of D4 and plasma concentration. As explained in Materials and Methods, the topical D4 dose was applied in a semioccluded manner, with a charcoal basket on top of the dosed skin area to capture volatilized D4. Model simulations indicate that the high vapor pressure of D4 (i.e., high volatility) results in almost 98.5% of the applied dose being volatilized and captured in the charcoal basket. The model predicts that only 1.5% of the topical dose is absorbed into the skin (Fig. 4Go). Of this relatively small absorbed fraction, nearly 90% is eliminated rapidly in the exhaled air during exposure, and the remaining amount is cleared by urinary excretion. Only 0.3% of the absorbed dose is retained in the body at 24 h postexposure (Fig. 4Go).



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FIG. 3. Plots comparing initial model simulations to experimentally observed exhalation rate of D4, total D4 plasma concentration, and urinary excretion rate of D4 metabolites in female F344 rats during and following a 24-h topical dose of 25, 12, or 5 mg 14C-D4/cm2 applied over a 2.5 cm2 skin surface area. Error bar represents standard error of the mean.

 


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FIG. 4. Model simulations of percent D4 volatilized or absorbed into the skin compartment and relative fractional elimination of the absorbed dose by exhalation and urinary excretion following topical doses of 25, 12, or 5 mg 14C-D4/cm2 applied over a 2.5 cm2 skin surface area.

 
Initial model fit to PK data from oral and iv exposures.
In contrast to the predictions of PK data following inhalation or dermal exposures with the initial model, the initial multiroute model provides a poor fit to PK data from oral and iv exposures. The initial model underestimated D4 exhalation rate and plasma concentrations by several orders of magnitude in the postexposure period following oral exposure (Fig. 5Go), whereas the model provided good fits to excretion rate in urine and feces following oral exposure (Fig. 6Go). The latter agreement is not unexpected since the absorption and elimination parameters in the gut were optimized against the observed urine and feces excretion rates. Similar discrepancies were apparent when the initial model was used to predict the D4 exhalation rate and plasma time course of all three vehicles for the oral dose administrations, i.e., in corn oil, in simethicone, or neat. The degree of uptake varied for the three vehicles. On an average, about 57% of the administered dose is absorbed in the gut following corn oil gavage, and the remainder is excreted in feces. The absorbed fraction is approximately 35 and 14% following oral gavage, using neat or simethicone as carriers, respectively.



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FIG. 5. Plots comparing initial (thin line) and refined (thick line) model simulations to experimentally observed D4 concentrations in plasma and exhalation rate of D4 in female F344 rats following a single oral doses of 300 mg D4/kg either in corn oil, simethicone, or a neat form. Error bar represents standard error of the mean.

 


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FIG. 6. Plots comparing initial (thin line) and refined (thick line) model simulations to experimentally observed urinary and fecal excretion rate of D4 in female F344 rats following single oral doses of 300 mg D4/kg either in corn oil, simethicone, or in neat. Error bars represent standard error of the mean.

 
Modeling delivery of administered D4 directly to the blood compartment (as free D4) from the iv exposure resulted in very poor fits to the total D4 (parent plus metabolite) concentration in plasma, exhaled D4 in air, and the excretion rate of D4 metabolites in the urine (Fig. 7Go). The model predicted that concentration of total plasma radioactivity equivalents would drop sharply immediately after exposure, due to rapid exhalation and tissue uptake dictated by the low blood:air partition coefficient and the high fat:blood partition coefficient. The former leads to rapid exhalation clearance of D4 postexposure, making insufficient quantities of D4 available for hepatic metabolism and redistribution in the body after exposure. With both iv and oral dosing, the discrepancies between the initial model predictions and blood concentrations indicate that a large amount of D4 in plasma is unavailable for exhalation or equilibration with tissues.



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FIG. 7. Plots comparing initial (thin line) and refined (thick line) model simulations to experimentally observed exhalation rate of D4 and urinary excretion rate of D4 metabolites in male and female SD rats following a single iv dose of 7 mg/kg D4 administered as an emulsion. Error bar represents the standard error of the mean.

 
PBPK model refinement.
The initial multiroute model structure assumes that intravenously injected D4 and orally administered D4 are available for gas exchange in the lung and metabolism in the liver in the same manner as free D4 that is inhaled or introduced dermally. Hence, predicted kinetic behaviors from the initial PBPK model were in poor correspondence with the exhaled air, excreted urine, and plasma D4 concentration following oral and iv exposure. Following intravenous administration, D4 is likely to remain in the form of a microemulsion that does not dissipate rapidly into the free pool of D4 in blood. Similarly, orally administered D4 may be transported in association with chylomicrons and other blood lipoproteins and thus may not be completely available for tissue interactions as free D4. To account for these route-specific physiological properties, the initial model was changed to account for persistent retention of "nonexchangeable" D4 in blood. Here, nonexchangeable refers to D4 in a deep compartment that is not directly exchangeable with blood, but is exchangeable with the shallow blood compartment.

The oral route was revised to be consistent with slow uptake (conceptualized as lymphatic system transport) and delivery of a nonexchangeable form of D4 into the blood. D4 was first delivered as a bolus input into the GI tract compartment, with a first-order uptake into a deep blood compartment, after a short time delay in the refined multiroute model (Fig. 8Go). D4 bound in this deep storage pool was assumed to diffuse reversibly to the exchangeable blood pool. Consistent with the liver and lung tissue, the net transport of D4 between the deep and the shallow blood compartments was proportional to a diffusional transfer coefficient and the concentration gradient of free D4 between the two compartments. D4 bound to the blood lipoproteins may be selectively sequestered in the fat depots and in the liver and modeled as first-order clearance from the deep blood compartment. Consistent with the earlier inhalation model, transfer of hepatic D4 into the blood lipoproteins was modeled as a first-order uptake term into deep blood. The iv exposure route was revised and D4 was introduced as a bolus dose into the deep blood, rather than exchangeable blood compartment (Fig. 8Go). The governing mass balance equations for the various compartments in the refined multiroute model are derived in the Appendix and model parameters are presented in Table 1Go.



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FIG. 8. Refinements on the initial multiroute PBPK that include (1) two-way transport between the blood lipoprotein pool and venous blood compartment as also the blood lipoprotein pool and shallow liver compartment; (2) orally administered D4 transferred into the lymph flow by a first-order uptake term from the gut and subsequently moved into the blood lipoprotein pool after a short delay; and (3) D4 administered as an iv bolus transferred into the blood lipoprotein pool that is not freely available for exhalation in the gas exchange region.

 
Refined model fit to PK data from oral and iv exposures.
The dose route-specific changes improved simulations of D4 pharmacokinetics following oral and iv exposure (Figs. 5–7GoGoGo). Delivery of D4 administered as an iv bolus directly into the deep blood compartment, or delayed delivery from the GI tract into the deep blood from the oral gavage dose, reduced the fraction of free D4 in the blood available for exhalation, thus improving fits to both plasma D4 concentration and D4 exhalation rate (Figs. 5–7GoGoGo). The increased retention of D4 in the blood also improved fits to the urinary excretion rate of D4 metabolites. The refined model fits of the inhalation and the dermal PK data sets were essentially indistinguishable from those produced by the initial model (data not shown), indicating that the refinements to the blood lipoprotein pool do not impact plasma concentration following inhalation or dermal exposure. The presence of a nonexchangeable compartment in the blood is quantitatively less important for inhalation/dermal exposure since D4 is not administered directly into these compartments, as is the case with oral/iv exposure.

A comparison of relative fractional clearance of D4 by exhalation and excretion following inhalation, dermal, oral, or iv exposures shows that greater than 90% of absorbed dose is cleared by gas exchange in the lung for inhalation/dermal routes and the remaining (less than 10%) is excreted in urine. Only a very small fraction of the absorbed dose (less than 0.3%) is retained in the body, postexposure from inhalation/dermal routes (Fig. 9Go). However, for the oral/iv routes, the relative significance of exhalation and excretion is approximately equal (~49%) and a much larger fraction (~2%) is retained in the fat depots postexposure (Fig. 9Go).



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FIG. 9. Plot comparing model-derived relative fractional clearance of D4 by exhalation (expired volatiles) and excretion (urinary and fecal elimination), and fraction retained in the body following inhalation, dermal, oral, and iv exposure. These estimates are based on cumulative clearance at 168 h postexposure following a single 6-h exposure to 700 ppm D4, a 24-h exposure to a 25-mg 14C-D4/cm2 topical dose applied over a 2.5 cm2 skin surface area, a single oral dose of 300 mg D4/kg in a neat form, and a single iv dose of 7 mg/kg D4 administered as an emulsion.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Initial work modeling the pharmacokinetic behavior of inhaled D4 in the rat showed that this compound had very unusual distributional characteristics. Although D4 has low blood:air and high fat:blood partition coefficients, it persisted in blood after cessation of the inhalation exposure at much higher than expected levels. In addition, the blood:air concentration ratio increased with time after exposure. This persistence and variable partitioning appeared to be due to the presence of D4 in blood in a form unavailable for gas exchange in the lung, presumably bound in a lipid compartment in the blood. The initial model has been extended to allow parameterization of the model following oral, dermal or iv administration of D4, and identification of route-specific differences in disposition and clearance (metabolic and pulmonary). Pharmacokinetic modeling of D4 following oral, iv, or dermal administration further reinforces our understanding of the unique pharmacokinetic properties of this compound, including storage in "deep" tissue compartments, and unexpected route-specific pharmacokinetic behaviors.

Route-specific D4 disposition.
The processes that govern delivery of D4 to the blood compartment are similar between the inhalation and dermal routes of exposure. Air phase delivery of D4 to the blood from the pulmonary region and dermal delivery of D4 following topical exposure occur by partitioning and passive diffusion controlled by the free D4 concentration gradient. The form of D4 delivered to the blood is not expected to differ for these two routes. D4 is delivered to the blood as free D4, in what can be described as a molecular infusion-like process. Thus, the tissue distribution, metabolism, and clearance of D4 would be similar for these routes of exposure, and the parameterization for distribution and clearance determined by fitting multiple inhalation data sets (Andersen et al., 2001Go) provides accurate simulations of pharmacokinetics following dermal exposure, given a standard model of dermal absorption of D4.

D4 delivered by oral or iv routes appears to enter the blood compartment in a form different from that for the inhalation and dermal routes, which are diffusion-controlled processes. D4 most likely enters the blood as lipid-like particles when administered as an emulsion by the iv route. For the oral route, significant amounts of D4 may be delivered via the lymphatics within the lipid core of chylomicrons and other lipoproteins (Roth et al., 1993bGo). Refining the oral route submodel to approximate some pharmacokinetic characteristics of delivery of D4 through the lymphatics to the deep blood compartment led to improved fits to the observed plasma D4, exhaled D4, and urinary metabolite elimination rate. Similarly, modifying the iv submodel to deliver D4, not as free D4 into the blood but directly into the blood lipoprotein pool (deep blood), restricted clearance of D4 from the blood and significantly improved simulations of plasma and exhaled D4 as well as metabolic clearance (urinary metabolites). The route-specific differences in pharmacokinetics and the biologically motivated refinements necessary to represent them imply that the disposition and clearance of D4 is strongly controlled by its distribution in the blood lipoproteins (deep blood). Inhalation and dermal are the major routes of exposure for D4 in humans. Given the route-specific nature of D4 pharmacokinetics, PK data collected using iv or oral routes of exposure are not going to be useful in understanding the bioavailabilty or tissue kinetics of D4 for the inhalation or dermal routes. A lack of understanding of these route-specific differences in D4 pharmacokinetics and the consequent misrepresentation of these processes in a PBPK model (Luu and Hutter, 2001Go) can result in misleading and erroneous conclusions (Andersen et al., 2002). Adequately characterizing these route-specific differences and incorporating them into PBPK models will be critical for conducting model-based interspecies and dose-route extrapolation for D4.

Deep compartments.
Preferential storage in lipid-rich tissues such as fat and, if hepatic metabolism is limited, the liver, is a common characteristic of lipophilic chemicals. These tissues act as depots with slow time constants for equilibration with blood, and lead to identifiable pharmacokinetics. D4 is a highly lipophilic compound with an uncharacteristically low blood:air partition coefficient. These attributes lead to blood and tissue pharmacokinetics that are unusually sensitive to fat and other tissue storage (Andersen et al., 2001Go). Successful modeling of D4 concentrations or its metabolites in the lung, liver, fat, and blood tissue, as well as exhaled D4 concentrations, required the inclusion of standard and "slow" tissue and blood depots. The latter were referred to as "deep" compartments, reflecting slower kinetics of equilibrium with blood. The physiological correlates of these deep compartments are not known with certainty. Slow and fast depots of endogenous lipophilic compounds such as cholesterol have been described for most tissues (Oh et al., 1976Go). It is possible that these slow depots are common to all tissues, but tissue size, and in the case of D4, the tissues for which concentration data are available, limit the sensitivity of the model to the presence of these compartments.

Liver and adipose tissue exchange cholesterol, here a proxy for lipophilic chemicals, into two kinetically independent pools. Elimination kinetics identifies a slow and fast depot in both tissues (Oh et al., 1976Go), consistent with a membrane-free pool and an intracellular pool. The origin of the intracellular pool may differ from tissue to tissue, representing the lipid environment of the endoplasmic reticulum and/or the membranes of the various organelles or some cell type-specific lipid pool. Given the two pools in the liver, it appears unnecessary to invoke the possibility of an inducible binding protein, as was identified for TCDD (Leung et al., 1990Go; Poland et al., 1989Go; van Birgelen and van den Berg, 2000Go). However, specific physiological and biochemical structures exist that may also represent specific deep compartments for the lung and blood compartments.

The physical chemical properties of hydrophobic compounds such as hormones, fat-soluble vitamins, and xenobiotics like D4 severely limit their solubility in blood. Transport of D4 through the blood seems to be controlled by the movement of fat-soluble carriers that provide a hydrophobic microenvironment (Roth et al., 1993aGo). Serum proteins such as albumin can bind lipophilic compounds, but the affinity and capacity of these proteins for lipophilic compounds is greatly exceeded by the plasma lipoprotein pool (Koller-Lucae et al., 1997Go; Vost and Maclean 1984Go). Ocatadecane, hexadecane, DDT, and BP are carried in the blood primarily in the lipoprotein pool (Vost and Maclean, 1984Go). While it appears that the solutes carried within the core triglyceride pool are available for metabolism and uptake by tissues, hydrocarbons administered intravenously in chylomicrons, for instance, have been shown to be "transferable" to the liver (Vost and Maclean, 1984Go). It is possible that a fraction of this pool is less available and represents the deep blood compartment required to describe all routes of administration. This distribution to lipoproteins would be consistent with blood half-lives, which differ significantly between the various components of the lipoprotein pool. High-density lipoproteins (HDL) have a half-life on the order of several days, while very low density lipoproteins (LDL) have a half-life of several hours (Wasan and Morton, 1996Go). Lipophilic compounds such as D4 may also be distributed to, and carried in, erythrocyte membranes (Koller-Lucae et al., 1997Go), which may also be described with the addition of a deep blood compartment description. The model structure, which describes transport of D4 from the lipoprotein pool to the liver and sequestration in a blood lipid pool is consistent with these observations. In contrast, we assume that "free" D4 in the blood is freely available to exchange with tissues and air, behavior that is consistent with the available pharmacokinetic data. The hypotheses regarding the availability of lipoprotein-bound D4 may be tested and verified by determining the distribution of free and lipoprotein-bound D4 in venous blood following exposure by these various routes.

The observed sequestration of D4 into the lungs of rats treated by the inhalation route exceeds that which can be described based on the lipid content of the lung (Andersen et al., 2001Go), which is relatively low compared to organs such as the liver (Anderson et al., 1993Go). A "deep lung" compartment, which has a large time constant for D4, was used to model the observed accumulation and loss of D4 from the lung compartment in the previously published PBPK model (Andersen et al., 2001Go). Excellent simulations were obtained when the deep compartment was approximately 2% of lung volume, which is consistent with the fraction of the lung represented by Clara cells (Jones, 1992; Martin, 1993; Mercer et al., 1994Go). The Clara cell is the site of accumulation of the methylsulfonyl derivatives of PCBs and of parent PCBs (Anderson et al., 1993Go; Lund et al., 1988Go; Stripp et al., 1996Go). The accumulation of the metabolites of PCBs has been causally linked to the presence of Clara cell secretory protein (CCSP). This structure is a transport protein for lipophilic substances in plasma and bronchioalveolar fluid (Burmeister et al., 2001Go), and is preferentially expressed in Clara cells, relative to Type II pulmonary epithelial cells (Lag et al., 2000Go). Endogenous ligands for CCSP include other lipophilic compounds such as retinoic acids and the steroid hormone progesterone (Lopez de Haro et al., 1988Go). The capacity of CCSP to bind lipophilic molecules makes it a candidate for binding other lipophilic molecules such as D4.

The number and size of Clara cells in the rat lung have been determined by quantitative morphometric analysis, and these studies report a total of 17.3 x 106 Clara cells in the rat lung (Mercer et al., 1994Go). Martin and coworkers (Martin et al., 1993Go) reported a Clara cell fraction (fraction of total lung cells) of 2.8% for F344 rats. A smaller fraction of 0.8% was reported in an earlier study in Sprague-Dawley rats (Jones et al., 1982Go). Based on terminal bronchiolar surface area, epithelial cell thickness, the fractional density of Clara cells, and whole lung volume (Mercer et al., 1994Go), we estimated that Clara cells occupy ~1% of the total lung volume in the rat. This volume fraction compares favorably to the volume of the deep compartment in the lung. The binding of D4 to CCSP would be consistent with the finding that a small volume compartment in the lung, of approximately the size of the Clara cell volume, retains D4.

The biological identity of all of the pharmacokinetically identifiable deep compartments described in the model are not yet known with certainty, but given the influence they have on the simulation of D4 kinetics, it is of interest to conduct experiments designed to test specific hypotheses regarding their identity. Characterizing the distribution of D4, specifically the amount of free D4, and the D4 associated with the lipid core of the various lipoproteins in portal and venous blood, as well as that found in the lymph system following oral exposure, would establish the extent to which uptake in the lymphatics occurs. Extending these analyses to other routes of administration would establish any dose-route dependencies of blood distribution, and the relative importance of blood lipoproteins for transport and storage in the blood for each route of administration. Such experiments might bring new insights into the processes that influence uptake, distribution, and clearance of other lipophilic compounds.

Summary
As part of ongoing research in understanding the pharmacokinetics of D4, a physiologically based pharmacokinetic (PBPK) model has been constructed to describe the tissue dosimetry, plasma concentration, and clearance in the rat following inhalation, dermal, oral, and iv exposure. The failure of a previous PBPK model for D4 to accurately describe its kinetics is credited with bringing to light route-specific differences in pharmacokinetics that otherwise may have been overlooked. This ability to evaluate expectations by simulation is strength of PBPK model-based approaches to analyzing the pharmacokinetics of novel compounds. The multiroute D4 model provides reasonably good fits to the dose route-specific pharmacokinetic behaviors of D4. The inhalation model that incorporates a simple description for dermal uptake provided excellent fits to the observed concentration time course of D4 metabolites in urine and D4 exhalation rates following dermal exposures. However, the pharmacokinetics of D4 following oral and iv exposure were sensitive to the mode of entry into the blood compartment. A refined model, describing delivery of D4 from the GI tract to the deep blood compartment as D4 within the triglyceride core in chylomicrons via the lymphatic system, and transport of D4 from the deep blood compartment to the two fat compartments provided the best fits to observed plasma D4, exhaled D4, and D4 metabolites excreted in the urine following oral exposure. Pharmacokinetics following iv administration was best described by delivery of D4 directly into the deep blood compartment, reflecting a kinetically identifiable characteristic of the administration of D4 as an emulsion for the intravenous route of exposure. This implies that the physical forms of D4 delivered by the inhalation and dermal routes are similar to each other and different from the form delivered by the iv or oral routes.


    APPENDIX
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Here we develop the mass balance equations for the various tissue compartments in the refined PBPK model that consists of shallow and deep compartments for the lung, liver, and blood in addition to compartments to represent fat, richly perfused tissue, poorly perfused tissue, gut, and the skin area, dosed with D4. Inhaled D4 in alveolar air equilibrates rapidly with arterial blood in the shallow lung compartment. Mass conservation in the lung stipulates that the rate of change of amount of D4 (mg/h) in the shallow lung compartment is a balance between mass transferred from alveolar ventilation, arterial blood perfusion, and diffusional transport to the deep lung tissues. The governing equations for mass balance in the shallow and deep lung compartments are:

where Clu and Cdlu are D4 concentrations (mg/l) in the shallow and deep lung compartments, respectively; Vlu and Vdlu are the volume (in liters) of shallow and deep lung compartments, respectively; Cin is the inhaled D4 concentration (mg/l), Ca is D4 concentration (mg/l) in the arterial blood, Qp and Qc are the alveolar ventilation (l/h) and cardiac output (l/h), respectively; Plu:b, Pb:a, and Pdlu:b are the lung:blood, blood:air, and deep lung:blood partition coefficients, respectively; and Dlu is the diffusional clearance (l/h) from the lung to the deep lung compartment. In this formulation, free D4 in the arterial blood equilibrates very rapidly with D4 in the alveolar air and free D4 in the lung tissue. Hence, the ratio of D4 concentration in arterial blood to that in alveolar air and the shallow lung tissue is a constant specified by the blood:air and tissue:blood partition coefficients, respectively.

The rate of change of D4 in the liver is a balance between amounts gained from blood perfusion and transfer from the blood lipoprotein pool and the cumulative amount lost due to metabolism, transfer to deep liver tissue, and elimination of mass into the mobile lipid pool in blood. The rate of change of parent D4 in the shallow and deep liver compartments are:

where Vli and Vdli are the volume (in liters) of the shallow and deep liver compartments, respectively, Cli and Cdli are the mixed-mean D4 concentrations (mg/l) in the shallow and deep liver compartments, respectively, Pli:b and Pdli:b are the liver:blood and deep liver:blood partition coefficients, respectively, Vmax,t (mg/h) and Km (mg/l) are the Michaelis-Menten kinetic parameters for D4 metabolism in the liver; K1:db and Kdb:l are the first-order elimination rate (/h) of D4 from the liver to the deep blood and deep blood to liver, respectively, and Dli is the diffusional clearance (l/h) from the liver to the deep liver compartment. The refined model estimates urinary clearance as a product of first-order elimination rate constants and the amount of each of the two metabolites produced from the oxidation of D4 in the liver.

The rate of change in amount of D4 in the slowly and richly perfused tissues is equal to the difference in flux into the tissue due to arterial blood flow, and efflux due to free concentration in the venous blood. The governing equation is given by:

where Vt is the volume (in liters) of the richly or poorly perfused tissue compartment, Qt is the corresponding blood perfusion rate (l/h) to the tissue compartment, and Ct/Pt:b is the "free" D4 concentration in these tissue (mg/l).

Chemical mass balance equations in the fat compartments and skin compartment dosed with D4 are similar to those in the richly perfused and poorly perfused tissues, except for an additional transfer term. For the fat compartments, this is a first-order transfer rate from the blood lipoprotein to the fat. For the skin compartment, it is a first-order transfer rate from the topical dose into the skin. The governing equations in the fat and skin compartments are respectively:


where Vf and Vsk are the volume (in liters) of the fat and skin compartments, respectively, Qf and Qsk are the corresponding blood perfusion rates (l/h) to the fat and skin compartments, respectively, Cf/Pf:b is the "free" D4 concentration (mg/l) in fat, Kdb:f is the D4 transfer rate (/h) from deep blood to fat, Ksk is the first-order transfer rate (/h) into the skin compartment, and Mtop is the D4 topical dose (mg).

The mass balance in the venous blood compartment is determined by the flow averaged D4 concentration exiting all the tissue compartments and the transfer to the blood lipoprotein pool (deep blood compartment). The governing equation is given by:

where Vb is the blood volume (in liters), Cb is the venous blood concentration (mg/l), and Db is the diffusional clearance between the nonexchangeable and exchangeable blood compartments. Consistent with the formulation for D4 kinetics in liver and lung tissues, the net transport of D4 between the deep and the shallow blood compartments was assumed to be proportional to a diffusional transfer coefficient and the concentration gradient of free D4 between the two compartments.

D4 in the nonexchangeable/deep blood compartment is assumed to be in equilibrium with free D4 in the exchangeable blood compartment. D4 bound to the blood lipoproteins is assumed to be selectively sequestered in the fat depots and in the liver. These processes are modeled as first-order clearance terms from the deep blood compartment. Consistent with the earlier inhalation model (Andersen et al., 2001Go), transfer of hepatic D4 into the blood lipoproteins is modeled as a first-order uptake term into deep blood. Furthermore, D4 administered as an iv bolus or D4 absorbed from the GI tract following an oral gavage dose is transported to the deep blood compartment in the refined multiroute PBPK model (Fig. 8Go), instead of being transported to the shallow blood compartment or the liver, respectively, as is implemented in the initial model. The rate of change of D4 in the nonexchangeable/deep blood compartment is then given by:

where Vdb is the volume (in liters) of the deep blood compartment (assumed to be 2% of the blood volume), Cdb is D4 concentration (mg/l) in deep blood, Kg:db is the first-order transfer rate (/h) of D4 from the gut to the deep blood compartment, Mg:db is the amount in the gut, and M0 is the initial amount of D4 in the deep blood compartment set equal to the iv bolus dose.


    ACKNOWLEDGMENTS
 
This work was supported, in part, by the Silicones Environmental, Health, and Safety Council of North America. We thank Dow Corning Corporation for support for this work and also appreciate the thoughtful review of early drafts of this manuscript by Ms. Robinan Gentry, The K. S. Crump Group, Inc.


    NOTES
 
1 Present address: Novartis Pharmaceuticals, One Health Plaza, East Hanover, NJ 07936. Back

2 To whom correspondence should be addressed. Fax: (989) 496-5595. E-mail: kathy.plotzke{at}dowcorning.com. Back


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