* DuPont Haskell Laboratory for Health and Biomedical Sciences, Newark, Delaware 19714; and
The K.S. Crump Group, Inc., ICF Consulting, Research Triangle Park, North Carolina 27709
Received February 15, 2002; accepted May 20, 2002
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ABSTRACT |
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Key Words: PBPK model; endocrine system; estradiol; risk assessment; endocrine-active compound (EAC).
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INTRODUCTION |
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Estradiol is carried in the plasma in two forms, bound to plasma binding proteins, and to a lesser extent, as "free" estradiol. In the human, binding to the sex hormone binding globulin (SHBG) and albumin restrict the free fraction of estradiol in plasma. Albumin and -fetoprotein (AFP) are the corresponding rat proteins. The free form is presumed to be the pharmacologically active fraction, which is capable of passing through the lipid bilayer and equilibrating with tissues. However, under specific conditions, protein bound E2 appears to be available for tissue uptake (Mendel, 1992
; Pardridge and Mietus, 1979a
). In vivo, free plasma concentrations of estradiol are regulated by feedback control (hypothalamic-pituitary-gonadal axis) and binding to albumin, SHBG or AFP (Andersen et al., 1997
).
Interest has recently grown in the use of E2 as a prototypical estrogenic endocrine-active compound (EAC). EACs are a structurally diverse group of chemicals that have impacts on the endocrine system, some of which act specifically by binding to the estrogen receptor (Andersen and Barton, 1999). Comparatively, the biology of these ligands is poorly characterized, and given their number, efforts to evaluate their comparative binding or transactivating potency have centered on comparisons made to the endogenous ligand E2 (Danzo, 1997
; Jobling et al., 1995
; Klotz et al., 1996
; Milligan et al., 1998
; Shelby et al., 1996
; Soto et al., 1995
; Stahl et al., 1998
). Rankings must be based on more than external dose since pharmacologically active plasma and tissue concentrations and potency of these compounds are determined by the interaction of a variety of processes that are compound specific: binding affinity to the ER and plasma binding proteins, ligand receptor competition, metabolic and total clearance rates, and tissue:blood partition coefficients among others. Pharmacodynamic processes (regulation of serum estrogenicity, enzyme induction, and regulation of tissue receptor levels) will also influence the nature and magnitude of toxicity following exposure. In addition, the relevant dose metrics (plasma, tissue, total, or free concentrations) must be determined.
To better appreciate the physiologic responses produced by E2 in target tissues and their dose-response relationships, it is necessary to understand E2 pharmacokinetics and the factors controlling its disposition within the quantitative framework of a physiologically based pharmacokinetic model. Reliable PBPK models will be useful for conducting dose-response analysis, establishment of appropriate dose metrics for ER agonists, and understanding impacts of exposure route, dose, and timing of target tissue levels. While several pharmacokinetic analyses have been conducted for E2 in rat and human plasma using compartmental (Eisenfeld, 1967) and model-independent (Kuhnz et al., 1993
) approaches, a PBPK model for estradiol has not been developed. Construction of a PBPK model for E2 is the initial and necessary step in the development of a biologically motivated quantitative tool for evaluating the dose-response characteristics of EACs.
There are three primary objectives for the work presented here: (1) Construct a PBPK model to describe the uptake, distribution, and clearance of E2 in the rat and human following oral and iv administration; (2) Evaluate the impact of plasma protein binding on tissue uptake in the liver and uterus and free plasma concentrations of E2; and (3) Identify physiological and biochemical parameters that have the most significant impact on E2 pharmacokinetics, plasma, hepatic, and uterine E2 concentrations, and use this information to establish a research agenda to collect the necessary data to develop a more complete PBPK model for E2. In addition, we sought to describe the framework for, and importance of using, a quantitative model that integrates the processes that influence EAC pharmacokinetics and tissue response (receptor binding affinity, receptor distribution, restriction of free EAC concentrations via plasma binding proteins, tissue kinetics, and clearance) for evaluating the biological activity of EACs.
The PBPK model developed here provides a flexible tool for integrating existing biological knowledge of the processes governing E2 pharmacokinetics, evaluating their significance and impact, as well as providing predictions of in vivo binding potency and tissue dosimetry. The final model will be central to ongoing efforts to characterize the toxicity and dose-response relationships for the prototypical EAC, E2, and by extension, other EACs.
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MATERIALS AND METHODS |
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Parameterization of PB-PK model.
Models were written in ACSL simulation language (MGA Software, Concord, MA) using ACSL Graphic ModellerTM. Parameter estimations and sensitivity analyses were conducted with ACSL OptimizeTM and ACSL MathTM. Simulations of rat and human data were conducted with the same model, and differed only in species- and sex-specific physiologic parameters.
The various model parameters were either obtained directly from the available literature or inferred from experimental data. Where feasible, inferred parameters were validated using independent data sets. Validation of these parameter values is presented in the Results section.
Physiologic parameters.
The rat and human organ blood flows and volumes used for the PBPK model are listed in Table 1 (ILSI, 1994
). Uterine blood flow and organ size are reported for both intact and ovariectomized rats (Bruce, 1976
; Kerr et al., 1992
; Zhang et al., 1995
). Although treatment of ovariectomized rats with E2 increases uterine blood flow greater than 100% in 2 h (Zhang et al., 1995
), this parameters was assumed to be time-invariant since the iv bolus data sets have few data points past 2 h. Likewise, uterine volume was held constant since there is only approximately a 25% increase in uterine weight within 3 h of treatment with E2 (Kerr et al., 1992
; Zhang et al., 1995
). Uterine weight for nonpregnant women is a function of age and parity. The value used here is for women approximately 30 years of age who bore 13 children during their lifetime (Langlois, 1970
). Uterine weight for nulligravidis women > 60 years old was used to approximate uterine volume in postmenopausal or ovariectomized women (Langlois, 1970
). Cardiac output is reported in terms of plasma as opposed to whole blood since this is more consistent with the available data sets and simplified the treatment of plasma protein binding.
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Tissue and plasma binding.
Model descriptions of ERs and ER binding were simplified to be consistent with the level of detail of the experimental data and the availability of parameter values. The following assumptions were made: (1) ER concentrations in tissues are time and treatment invariant; (2) ERs exist as a single type and are not separated into and ß subtypes (Verheugen et al., 1984
); (3) Type II estrogen binding sites (Clark and Mani, 1994
) do not affect E2 disposition; and (4) both cytosolic and nuclear ER receptor are equally available in tissues.
Total estrogen receptor content (Bmax) and binding affinities (Kd) were obtained for most organs in rats, however, information for humans is somewhat limited (Table 3). When necessary, values for human tissues were extrapolated from rat values based on organ weight. ER content in some tissues is dependent on sexual maturity and hormonal status, therefore several values are reported for comparison purposes. As evident from Table 3
, there was a significant amount of variability in published tissue receptor content, which is probably due to differences in strain, age, and hormonal status as well as methods used for tissue preparation and analysis. ER content in adult male and female rat liver ranged between 0.020.04 nmol/liver. Uterine and anterior pituitary ER content ranged between 0.0020.012 nmol/uterus and 0.070.2 fmol/pituitary in adult rats, respectively. An ER dissociation constant of 0.25 nM was used in the model and is consistent with the range of values (0.10.5 nM) reported in the literature (Table 3
). Values for Kd (
12 nM) reported by Notides (1970) and Clark and Peck (1979) are somewhat higher than the more commonly accepted range (0.10.5 nM) and were therefore considered outliers.
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Metabolic clearance of estradiol.
The metabolism of E2 in rats and humans is similar in that hepatic microsomal enzymes transform E2 into estrone as well as other hydroxylated metabolites (Martucci and Fishman, 1993). Excretion of these products occurs through the urine or bile as sulfate or glucuronide conjugates and very little systemic conversion (
5%) back to E2 occurs (Lobo and Cassidenti, 1992
). Clearance of estradiol in the model was attributed solely to metabolism and assumed to take place in the liver and central plasma compartment. In addition, it was assumed that there is no endogenous production of E2, either through de novo synthesis or regeneration from conjugates, estrone metabolites, or enterohepatic recirculation.
The rate of hepatic E2 metabolic clearance (RMET, nmol/h) was defined as the product of intrinsic clearance (Clint) and the free E2 concentration in the liver tissue (CLfree). This first approximation of clearance, represented as a first order process, is reasonable assuming that endogenous estradiol concentrations under the experimental conditions are subsaturating. Hepatic and extrahepatic clearance of E2 is predominately described as metabolic clearance rates (equivalent to intrinsic clearance; l/h). This composite term reflects the contribution of all metabolic processes (oxidation, sulfation, etc.) involved in the clearance of E2, and is well characterized for rats and humans in the literature (Ball et al., 1983; Hembree et al., 1969
). Use of intrinsic clearance rates limits uncertainties associated with constructing a composite metabolic rate based on Vmax/Km data for the various (Badawi et al., 2001
) E2 metabolizing enzymes. Fitted intrinsic clearance values of 3.0 and 1.0 l/h were used for male and female rats, respectively, while values of 500 and 150 l/h were used for men and women, respectively (Table 2
). Under steady state conditions, the corresponding hepatic E2 extraction for rats is 0.71 (female)0.84 (male), and for humans is 0.75 (female)0.85 (male) based on liver blood flow and the intrinsic clearance rates.
Total metabolic clearance rates (MCRs) in rats and humans typically exceed hepatic blood flow indicating E2 is subject to extrahepatic metabolism (blood flow in rats and humans are 1.1 and 62.5 l/h, respectively). In humans, it has been estimated that this may account for as much as 1525% of total E2 metabolism (Longcope et al., 1968
), which is equivalent to clearance rates of
620 l/h. In view of this, an additional term for E2 metabolism was included in the central plasma compartment where the rate of extrahepatic metabolism was defined as the product of the extrahepatic clearance rate (ClEH) and the total circulating E2 concentration. Extrahepatic clearance rates of 0.1 l/h and 510 l/h were assigned for rats and humans, respectively (Table 2
), corresponding to extrahepatic E2 extractions of 0.083 for the rat and 0.0740.14 for the human.
Modeling approach.
The general procedure for evaluating the PBPK model was first to calibrate the model against 1 data set and then validate against secondary (independent) data sets when available.
Three general categories of simulations were run for both rats and humans based on different routes of administration: (1) iv infusion, (2) iv bolus, and (3) po or intraduodenal. Details of these simulations and the test data sets used for calibration and validation are described below and in Table 4.
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RESULTS |
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The predicted distributions of E2 between free, albumin- and SHBG-bound states are shown in Table 5. In the absence of binding proteins, 100% of E2 is was free. In plasma containing albumin and low adult levels of
FP, such as rat plasma (BMAXA = 4.1 x 105 nM, KDA = 2.3 x 104 nM), 94.7% of E2 is bound to albumin and 5.3% is free. This distribution is consistent with in vitro dialysis experiments that show only 6.0% of E2 is dialyzable in Ringer's solution containing 4% albumin (Verheugen et al., 1984
). In human plasma, which in addition to albumin (BMAXA = 5 x 105 nM) contains the high affinity E2 binding protein SHBG (BMAXG = 2040 nM), free E2 decreases. Modeled free fractions of E2 in male serum (2.5%) was identical to the value measured by ultrafiltration dialysis (Nagel et al., 1998
). Similarly, free fractions in nonpregnant female (1.9%) and pregnant female plasma (0.4%) generally agreed well with those measured experimentally (
2 and 0.5% for nonpregnant and pregnant women, respectively; Dunn, 1983
).
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Plasma and Tissue E2 Concentrations in Rats
Intravenous administration studies.
Several studies describing the plasma, and in some cases uterine and liver kinetics in rats following iv bolus or infusion of E2 were available for model calibration and validation. De Hertogh et al. (1970; 1971a,b; 1973) infused intact female rats with E2 at a rate of 6, 65, or 180 ng/h for 27 h and measured E2 in plasma, uterus, and liver. An initial loading dose of 0.8 and 8.0 was administered to the 6 and 65 ng/h dose groups, respectively, prior to initiating the infusion. Similar data sets for ovariectomized female rats infused for 48 h (104 and 290 ng/h) were also evaluated (Ball et al., 1983). Data for male rats was limited to rats given an E2 loading dose (
27 ng) followed by a 27 ng/h infusion (Farrell et al., 1988
). Eisenfeld (1967) reported E2 concentrations in tissues (plasma, uterus, and pituitary) after iv bolus doses of 2.5 and 25 µg/kg to ovariectomized rats. Data for intact female rats (1.4 µg/kg) were obtained from Larner and Hochberg (1985). E2 plasma concentrations in male rats were available following iv doses of 5, 10, and 20 µg (Bawaarshi-Hassar et al., 1989
). This same study provided data sets for intraduodenal administration of E2 (5, 10, and 20 µg) and are the only data found for assessing gastrointestinal absorption of E2 in rats (Bawaarshi-Hassar et al., 1989
).
Model predictions of steady-state E2 concentrations in plasma of male and female rats (intact and ovariectomized) following iv infusion of E2 were in good agreement with all experimental data sets. The best correspondence to the data for intact (Fig. 3) and ovariectomized (Ball et al., 1983
; results not shown) female rats was seen using an intrinsic clearance (Clint) of 1.0 l/h and an extrahepatic clearance rate (EHC) of 0.1 l/h. Infusion data for male rats (Fig. 4
) were best fit with an intrinsic clearance of 3.0 l/h and extrahepatic clearance rate of 0.1 l/h (Farrell et al., 1988
). Simulations of plasma E2 concentrations (Fig. 5
) in male rats after iv administration of 5, 10, and 20 µg corresponded well with the experimental (Bawaarshi-Hassar et al., 1989
) data. A higher partition coefficient (PS = 2) and male-specific hepatic binding protein (Bmax = 65 nmol/liver, Kd = 35 nM) provided the best model fits for all 3 doses.
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The model successfully fit data sets for E2 plasma pharmacokinetics following iv bolus dosing in both intact (Larner and Hochberg, 1985) and ovariectomized rats (Eisenfeld, 1967
). The initial distribution and elimination phases fit well for both data sets (Figs. 6 and 7
); however, additional binding in the liver compartment (20 nmol/liver) was needed to extend the predicted terminal elimination phase in ovariectomized rats.
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Intraduodenal administration.
One study was available for assessing gastrointestinal absorption of E2 in rats. Bawaarshi-Nassar reported plasma E2 concentrations following intraduodenal administration of 5, 10, and 20 µg of E2 (Bawaarshi-Hassar et al., 1989). The absorption rate and intrinsic clearance were adjusted manually to arrive at single values of each (KO = 1.7 h-1, CLINT = 25.0 l/h) that achieved the best fit for the 3 dose groups. Simulations of E2 uptake from the intestinal tract after intraduodenal administration of 5, 10, and 20 µg corresponded well with the data for the high and low dose, but was overpredicted at the middle dose (Fig. 8
). Fractional absorption (FO) was set to 1.0 since E2 absorption from the gut appears complete when given at low doses by the po route (Kuhnz et al., 1993
).
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Model predictions of steady-state E2 plasma concentrations following iv infusions of E2 (Fig. 9) were in good agreement with data for both human female (Hembree et al., 1969
) and male subjects (Longcope and Tait, 1971
; results not shown). The Bmax for both albumin and SHBG were set to zero in the liver plasma compartment, and separate male and female intrinsic clearance rates were estimated by fitting the plasma E2 time course. Greater hepatic and extrahepatic clearance rates were required in simulations for men compared to women (Table 2
). Using the same model parameters as for the iv infusions the simulated plasma concentration-time curve in premenopausal women following iv bolus administration of 0.3 mg E2 was in good agreement with the observed data (Kuhnz et al., 1993
; Fig. 10
).
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The model did not perform as well for orally administered E2 as it did for intravascular doses. Simulations of E2 plasma kinetics in women who received 18 mg of micronized E2 are shown in Figure 11. Initially, a Clint of 150 l/h, equivalent to the value identified in the infusion studies was used, and the po absorption and movement from the GI tract compartment to the feces was manually adjusted to obtain the best fit. The rate constant for po uptake varied from 0.005/h to 0.013/h and the rate constant for fecal elimination varied from 0.0/h to 0.04/h. Peak concentrations were approximated, but the Tmax was off by several hours for all 6 data sets. Although the model gives reasonable predictions of the peak plasma concentrations, it fails to accurately predict the time at which maximum plasma concentrations are achieved and the general shape of the curve.
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DISCUSSION |
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The extensive binding of E2 by both albumin and SHBG, allows only a small percentage (25%) of E2 to circulate unbound (Dunn, 1983; Pardridge, 1986
). The early work of Brodie et al.(1960) introduced the idea that only unbound drug in plasma is available to interact with membrane receptors or diffuse into tissues to elicit pharmacologic effects. Single pass extraction experiments in rats designed to study E2 uptake into target organs indicate binding to either albumin or SHBG can differentially affect tissue availability of E2 (Pardridge and Mietus, 1979a
,b
; Verheugen et al., 1984
). In most cases E2 extraction is significantly greater than would be expected for uptake of unbound E2 alone. Simulations of hepatic extraction predicting limited uptake of E2 by the liver in the presence of albumin (0.24) and even less extraction with both albumin and SHBG (0.03) are inconsistent with in vivo experiments, which report a single pass extraction of
0.85 (Verheugen et al., 1984
). Pardridge et al. has demonstrated that in vitro protein binding constants for E2 are not predictive of in vivo uptake in the liver, brain, or uterus (Pardridge, 1981
, 1986
; Pardridge and Landaw, 1985
). Instead, an apparent Kd (Kd(app)) for individual organs can be used to reflect better the bioavailable E2 fraction in the plasma passing through each organ, where Kd(app) is related to the in vitro Kd by the E2 flux rate through the capillary membrane and capillary transit time (Pardridge and Landaw, 1985
). Where the capillary transit time is large relative to the E2 disassociation constant and the plasma tissue concentrations gradient is also large (for highly cleared tissues like the liver), protein binding restricts tissue uptake less than would be predicted based on free concentrations (Mendel, 1992
). In the PBPK model presented here, a Kd(app) was used only for the uterus and slowly perfused tissue compartments. The Kd(app) in the liver plasma compartment was set high enough to effectively disable protein binding; E2 was free for hepatic uptake and clearance. It appears that capillary transit time is sufficiently long relative to dissociation rate of E2 from the plasma binding proteins that protein binding does not significantly restrict hepatic extraction, but rather acts as a reservoir for E2. This suggests that hepatic uptake of E2 is dependent on the total E2 concentration in the blood and not simply free E2, a concept consistent with the "free hormone transport hypothesis" proposed by Mendel (1992).
Because tissue E2 data are only available for adult rats, where only albumin binding is significant, the potential for SHBG to restrict E2 uptake in most human tissues cannot be evaluated. Model simulations of E2 kinetics in humans were only predictive when hepatic plasma protein binding was eliminated, suggesting that at least E2 uptake by the liver is not significantly restricted by SHBG. This has been confirmed experimentally by Pardridge and Mietus (1979a) who found >82% of E2 in human plasma is extracted on a single pass through the rat liver. The intrinsic clearance rates used in the model led to similar, extensive, extraction of E2 by the liver. Extraction will increase with transit time, which may be different in humans.
Metabolic clearance of E2 was restricted to the liver and plasma compartments of the model. In the liver, the rate of elimination was defined as the product of intrinsic clearance (Clint) and the free E2 concentration in liver tissue (Clfree). The total plasma metabolic clearance rate (MCRP), 0.45 l/h for the female rat, falls well within the reported range of values (MCRP = 0.360.97 l/h (Ball et al., 1983; De Hertogh et al., 1970
; Kono et al., 1981
; Larner and Hochberg, 1985
; Tapper and Brown-Grant, 1975
). The same analysis of Clint for men and women yield MCRPs consistent with values reported for humans (Hembree et al., 1969
; Longcope et al., 1968
; Longcope and Tait, 1971
). Hepatic extraction was between 0.70 and 0.85 for rats and humans, consistent with the observed first pass extraction rate of 0.85 in rats (Verheugen et al., 1984
). When expressed as a percentage of the total MCR, extrahepatic clearance represent
22% of total clearance, and is consistent with the range of 1525% reported by Longcope et al.(1968). The model required higher Clint values for male rats and humans as compared to females. This is consistent with the MCR differences reported between sexes and may be due to inherently greater activity of some enzymes involved in E2 metabolism. For example, 17
-2- and 4-hydroxylase activities are
5-fold higher in microsomes from male Sprague-Dawley rats as compared to females (Dannan et al., 1986
).
Hepatic blood flow appears to be one of the most important sources of experimental variability in determining the overall metabolic clearance of E2 (Longcope and Tait, 1971). This is confirmed by this PBPK model by estimated hepatic clearance rates and a sensitivity analysis that indicated near perfusion limited hepatic clearance.
As confirmed by the sensitivity analysis, the E2 concentration achieved in plasma under steady state infusion conditions is dependent on only the infusion and clearance rates (Gibaldi and Perrier, 1982), and is independent of other parameters such as partition coefficients, receptor binding, blood flows, diffusional clearance rates, and organ volumes. However, these parameters do have an impact on the time required to reach steady state and the shape of the concentration-time curve, as well as steady-state concentrations in tissues. Altering Kd not only changes the shape of the plasma concentration-time curve, but also impacts the steady-state concentration of E2 in the uterus. This is an important consideration in light of the recent finding of two distinct ER subtypes (ER
and ERß) possessing different dissociation constants and which have differential tissue distribution (Brandenberger et al., 1997
; Kuiper et al., 1997
). Uncertainty around ER content and binding affinities will greatly impact model predictions of uterine tissue dose and ultimately estimates of receptor occupation and tissue response characteristics.
The uterine ER content used in the model provided reasonable fits to all of the data sets, however, the uptake of E2 by the uterus appeared dependent on hormonal status. Uptake behavior of E2 in the ovariectomized rat, but not intact rats, was best characterized as diffusion-limited. E2 stimulation of uterine hyperemia, edema, albumin (Peterson and Spaziani, 1971), and trypan blue uptake (Cecil et al., 1966
) in ovariectomized rats are all indications of enhanced uterine vascular permeability (Cullinan-Bove and Koos, 1993
). The diffusion-limited behavior needed to describe E2 uptake in the ovariectomized rat may reflect a reduced state of vascular permeability compared to the intact state where the uterus is continuously exposed to endogenous E2. Because estrogen receptor (ER) binding is included in the uterus compartment of the model, estimates of receptor occupancy as a tissue dose metric are easily obtained. Assuming the E2 production rate in the rat ovary is
8 ng/h, model simulations predict approximately 2% occupancy of ER in the uterus, which is identical to the estimate made by Eisenfeld (1967). Sensitivity of uterine E2 concentrations to both ER Bmax and Kd indicates uncertainty around these parameters would introduce significant error into tissue dosimetry estimates such as receptor occupancy. For this reason, ER values that are relevant to species, strain, age, and hormonal status should be used in the model when possible.
E2 distribution in the rat and human were clearly dependent on model parameters such as tissue partitioning and binding and diffusional clearance rates. Improved fits to the experimental iv data were achieved by increasing receptor binding in the slowly perfused compartment or increasing binding to a nonreceptor binding site in the liver. For male rats, this may represent the male-specific hepatic binding protein described by Rogerson and Eagon (1986), especially since the reported (Kd = 3143 nM and Bmax = 20 nmol/liver) and model estimated binding constants (Kd = 35 nM and Bmax = 65 nmol/liver) are similar. The need for additional binding in the lumped tissue compartment and in the female liver simply imply there is additional binding (specific or nonspecific) that is not accounted for at the model's current level of detail.
Oral absorption kinetics of E2 in rats and humans presented some difficulty. Only intraduodenal administration data were available for the male rat, while the dosage form for humans was micronized E2 (Estrace®). Although the model consistently provided reasonable estimates for the maximum plasma E2 concentrations reached, the shape of the curves did not correspond to the data. Likely, this is due to the complex nature of E2 dissolution and gut transit rates of micronized E2 that are not addressed in the model. It was necessary to increase hepatic extraction from 0.75 to 0.91 in the human and from 0.84 to 0.98 in the rate in order to fit the rat and human data; otherwise, plasma concentrations were over predicted. To accomplish these increases, intrinsic clearance (Clint) was increased from 3.0 l/h to 25.0 l/h and from 150 l/h to 500 l/h in humans and rats, respectively. Oral bioavailability of E2 is only 25% in both rats and humans (Bawaarshi-Hassar et al., 1989; Kuhnz et al., 1993
) and is the result of first-pass metabolism by the intestinal mucosa and liver (Longcope et al., 1985
; Meli et al., 1968
). Using a higher intrinsic clearance value for the oral route of administration may account for additional metabolism in the gut wall. Alternative approachesslower uptake and lower first pass metabolismwere explored and improved simulations of oral route pharmacokinetics. These more speculative analyses only suggest that additional experimental work is called for to improve our understanding of oral uptake of E2, and were therefore not presented here.
The PBPK model developed here provides a flexible tool for integrating existing biological knowledge of the processes governing E2 pharmacokinetics, evaluating their significance and impact, as well as providing predictions of in vivo binding potency and tissue dosimetry. Overall, the simulations from our PBPK model are consistent with E2 pharmacokinetics in rats and in humans for a variety of dosing regimens. The model will be central to ongoing efforts to characterize the toxicity and dose-response relationships for the prototypical EAC, E2, and by extension, to other EACs.
Quantitative Framework for Evaluating Responses to E2 and Other EAC Compounds
The distinction between adverse and normal responses to endocrine-active compounds is a function of the timing, magnitude, and duration of target tissue exposure (PK), as well as the affinity for the receptor (e.g., ER) and the potency of the compound. Efforts to quantify the potential for EACs to cause toxicity initially focused on in vitro measures of receptor affinity. Without adequate treatment of the processes controlling pharmacokineticsabsorption, tissue distribution, metabolic and other clearances and restriction of free concentrations by plasma protein bindingthe results of these experiments can be misleading.
The E2 model presented here is a biologically motivated quantitative framework that simultaneously treats both pharmacokinetic processes and receptor binding, and is readily adaptable to other EACs. Integration of this model with response models for specific tissues will ultimately create more biologically realistic models for studying the pharmacodynamics of E2 as well as other EACs.
The model is best used as an integrated part of an experimental program, used to evaluate experimental data and guide the development and evolution of and effective experimental agenda. The initial step, population of the model with in vitro derived parameter estimates of clearance, tissue partitioning, plasma protein binding, and receptor binding, facilitates evaluation of processes controlling tissue dosimetry, allowing informed decisions regarding the priority of additional experimental work. After necessary revisions to initial parameter estimates based on additional experimental work have been made and satisfactory representations of pharmacokinetics are achieved, the model can be linked to pharmacodynamic models of common response assays such as the uterotrophic response to complete the construction of a biologically motivated dose-response model. Models such as this are the best available tool for integrating quantitative knowledge regarding the processes which ultimately determine the potency of endocrine-active compounds. As such they should be seen as a useful, flexible tool for evaluating and interpreting experimental data, making informed decisions regarding research priorities directed at evaluation of the overall potency of these compounds and well as making well supported predictions.
Uncertainties, Data Gaps, and Recommendations
The nature of constructing PBPK models requires a simplification and reduction of the complexities found in biological systems, and thus by default introduces a degree of model uncertainty. In addition, the test data sets and model parameters, which were obtained from numerous sources, are potential sources of parameter uncertainty derived from measurement and sampling errors. The following discussion points address some of these uncertainties and recommend potential research to fill data gaps.
The model assumes E2 pharmacokinetics in plasma and tissues are independent of the pharmacologic activity of E2. In ovariectomized and immature animals, E2 alters uterine blood flow, vascular permeability, uterine size, and receptor levels. This list of physiologic responses implies E2 may affect its own kinetics. This could be evaluated by linking the current PBPK model to a pharmacodynamic response model for E2 effects on uterine physiology. Furthermore, an integrated model of this type will help elucidate the relationships between E2 pharmacokinetics and pharmacodynamics. Clearly, this model can be extended to compliment other work on endocrine-active compounds. Sensitivity analyses clearly indicated that ER content and binding affinity were important determinants of tissue E2 concentrations. Although ER levels have been reported for many tissues, uses of these data are confounded by nonstandardized assay methodology, inconsistent presentation of the results, and the use of surgically or hormonally manipulated test animals. All of these issues make use of published tissue receptor levels problematic. Ideally, ER tissue measurements and pharmacokinetic studies should be matched as closely as possible in terms of strain, age, and hormonal status.
Simulation results of E2 pharmacokinetics in intact and ovariectomized rats suggest tissue uptake and plasma pharmacokinetics are dependent on hormonal status. Not enough data are currently available to confirm this, pointing to a need for additional plasma and tissue kinetic studies designed to examine E2 kinetics in mature intact and ovariectomized rats.
ER binding in tissues was described using equilibrium binding constants. In order to describe the long terminal elimination phase seen following iv bolus dosing, additional binding in the liver and poorly perfused tissue compartment was required beyond the expected ER tissue content. An alternate approach worth exploring is to use a kinetic description for receptor binding in tissues that explicitly describe ligand on- and off-rates for the receptor and ligand-receptor interactions with DNA binding sites. This approach may better describe the tissue retention of E2 and not require additional binding sites in the model. A further advantage of this approach is that the measurements of E2-ER to DNA binding regions would provide a dosimeter closer to the final tissue response. It is not clear whether the data to validate such a model are available.
As mentioned above, tissue ER content was assumed to be time invariant. This is clearly an oversimplification of true receptor dynamics, which include the processes of synthesis, degradation, recycling, and replenishment (Clark et al., 1977; Katzenellenbogen, 1980
). These processes may become very important in explaining E2 tissue kinetics during multiple or prolonged exposures to E2 and ultimately influence tissue response. Exploration of this would clearly be of value for specific tissue response models such as those under investigation for the uterus at ICF Kaiser.
ER subtypes were not distinguished in the model. Differences in binding affinity for E2 and tissue distribution patterns may impact tissue dose estimates. A useful extension of the current model would be to parameterize the model so each target tissue contained appropriate ER subtype binding constants.
Estimates of intrinsic hepatic clearance could be refined by studies designed to determine the actual kinetic constants for the biotransformation of E2 to estrone. Estrone is formed by the further oxidation of estradiol at the C17 position and is the primary metabolite of estradiol seen upon iv and oral administration (Kuhnz et al., 1993). Furthermore, the kinetics of estrone may be worth including in future models since there is some speculation that estrone is converted to E2 in target tissues (Lobo and Cassidenti, 1992
).
The absence of time course data for target organs such as the brain, gonads, and storage compartments such as those comprising the richly and poorly perfused tissues (muscle, fat, kidney) also limit the current description of E2 pharmacokinetics. Regulatory control of serum estrogenicity is expected to provide some buffering against exposure to E2, but the range over which this compensation is effective and the timing and degree of control are not known. Adequate understanding of these processes may be particularly important for simulation of chronic exposures, where the compensation is more likely than for short infusions or bolus dose experiments.
Although the E2 response data is plentiful for rats, direct comparison between studies or with E2 pharmacokinetic studies are extremely difficult due to the diversity of experimental designs employed. Typically, an effort is made to maximize response to E2 by surgical or pharmacological manipulation of the test animals. Unfortunately, corresponding tissue and plasma E2 concentration data are not usually available for comparison. A standard set of pharmacokinetic studies for the most commonly used response testing paradigms would help address this problem.
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ACKNOWLEDGMENTS |
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REFERENCES |
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