* Battelle Pacific Northwest Division, Chemical Dosimetry, PO Box 999, Richland, Washington 99352;
Dow AgroSciences, 9330 Zionsville, Indianapolis, Indiana 46268; and
The Dow Chemical Co., Midland, Michigan 48674
Received August 16, 2001; accepted November 28, 2001
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ABSTRACT |
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Key Words: physiologically based pharmacokinetics; organophosphate insecticide; chlorpyrifos; esterase inhibition.
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INTRODUCTION |
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Detoxification reactions are believed to share a common phosphooxythiran intermediate and represent critical biotransformation steps required for toxicity (Neal, 1980). Differences in the ratio of activation to detoxification are believed to be associated with sensitivity to OPs (Ma and Chambers, 1994
). Hepatic and extrahepatic A-esterase (A-EST, EC 3.1.1.1.2) can effectively metabolize CPF-oxon to TCP (nontoxic metabolite) and diethylphosphate without inactivating the enzyme (Sultatos and Murphy, 1983
). B-Esterases (B-EST) such as carboxylesterase (CaE, EC 3.1.1.1), and butyrylcholinesterase (BuChE, EC 3.1.1.8) can likewise detoxify CPF-oxon; however, these B-ESTs become irreversibly bound (1:1 ratio) to the CPF-oxon and thereby become inactivated (Chanda et al., 1977; Clement, 1984
). Studies in both humans and rodents indicate that TCP represents the primary metabolite of CPF, although glucuronide and sulfate conjugates of TCP have likewise been observed (Bakke et al., 1976
; Nolan et al., 1984
).
Based on the extensive understanding of the biochemical interactions between OPs and AChE, the relationship between OP activation and detoxification is of critical importance in determining the toxicological response to OP insecticides (Ma and Chamber, 1984). In this regard, Gearhart et al. (1990) suggested that physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models capable of predicting the relationship between OP exposure and AChE inhibition are useful for evaluating the risk associated with a given OP exposure. Although a limited number of PBPK/PD models for OP insecticides and nerve agents have been published in the literature (Abbas and Hayton, 1997; Gearhart et al., 1990
; Maxwell et al, 1988
; Sultatos, 1990
), there are currently no models for the phosphorothionate insecticide CPF.
The primary objective of the research reported herein was to develop and validate a PBPK/PD model for CPF in rats and humans. This model quantitatively integrates target tissue dosimetry and dynamic response describing the pharmacokinetics of CPF, CPF-oxon, and TCP, as well as the associated cholinesterase (ChE) inhibition kinetics in blood components (plasma and red blood cell (RBC)) and selected tissues, including the brain. To develop and validate the model, pharmacokinetic/pharmacodynamic studies were conducted in both rats and humans to quantitate dosimetry and dynamic response over a range of CPF doses. In addition, the model was further evaluated against available dosimetry and dynamic response data in the published literature.
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MATERIALS AND METHODS |
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Animals.
Male Fischer 344 (F344) rats (Charles River Laboratories Inc., Raleigh, NC) were utilized for the pharmacokinetic/pharmacodynamic studies (time points, 10 min12 h postdosing) and were 1011 weeks of age at the time of dosing. Female F344 rats were purchased from the same supplier and were also utilized to assess the pharmacodynamic response (time point, 24 h postdosing) and were 7 weeks of age at the start of the study. Animals were housed one per cage (wire mesh or plastic), and maintained on a 12-h photocycle, at controlled temperature (2224°C), and relative humidity (4452%) and allowed access to municipal tap water and Purina certified rodent chow ad libitum, except where otherwise noted.
Model structure.
A diagram of the PBPK/PD model structure developed for CFP and CPF-oxon is illustrated in Figure 1 and is based on the model for diisopropylflurophosphate (DFP) in rats (Gearhart et al., 1990
). The current PBPK/PD model was developed to describe the time course of absorption, distribution, metabolism, and excretion of CPF, CPF-oxon, and TCP, and the inhibition of target esterases by CPF-oxon in the rat and human. The model assumes that the pharmacokinetic and pharmacodynamic response in rats and humans is independent of gender, which is consistent with the observed response in human males and females reported in this study. The absorption of CPF following oral administration in corn oil vehicle required the use of a two-compartment uptake model to simulate absorption, according to Staats et al. (1991). This two-compartment model incorporated 1st-order rate equations to describe systemic uptake and transfer between compartments. In addition, absorption of CPF from the diet was incorporated into the model to allow for the simulation of chronic dietary administration. In the chronic dietary studies with CPF, the doses (mg/kg/day) determined were based upon the measured rate of food consumption per day. To model the data, the absorbed dose was expressed as a constant zero-order rate (mg/h) over a 12-h interval. This assumption was reasonable since, in rats, the majority of the daily food intake occurs during the lights-off cycle (
12 h) (Jochemsen et al., 1993
). Equations to describe the dermal uptake of CPF into the skin compartment of humans were adapted from Poet et al. (2000). In this model, physiological and metabolic parameters (i.e., tissue volume, blood flow, and metabolic capacity) were scaled as a function of body weight, according to the methods of Ramsey and Andersen (1984). The CYP450-mediated activation and detoxification of CPF to CPF-oxon and TCP, respectively, was limited to the liver compartment. The model was linked to the CPF-oxon model that contained equations to describe the A-EST hydrolysis of CPF-oxon to TCP in both the liver and blood compartments. The CYP450 activation/detoxification and A-EST detoxification of CPF-oxon were all described as Michaelis-Menten processes. Interactions of the oxon with B-EST (AChE, BuChE, and CaE) were modeled as 2nd-order processes occurring in the liver, blood (plasma and RBC), brain, and diaphragm. The B-EST enzyme levels (µmol) in blood, brain, liver, and diaphragm were calculated based on the enzyme turnover rates and enzyme activities reported by Maxwell et. al. (1987), and these were based on a balance between basal degradation and enzyme resynthesis. Following exposure to CPF-oxon, the amount of available B-EST was determined by finding a balance between the bimolecular rate of inhibition and the rate of B-EST regeneration (reactivation and resynthesis). In this model, TCP was formed by the direct CYP450 metabolic conversion of CPF and through A-EST-mediated hydrolysis of CPF-oxon and B-EST binding of CPF-oxon, respectively. The blood kinetics and urinary elimination of TCP were described with a simple one-compartment model utilizing a 1st-order rate of urinary elimination.
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Biochemical constants.
The metabolism of CPF to TCP and CPF-oxon is mediated by CYP450 and is described using Michaelis-Menten kinetics. A range of Km and Vmax parameters has been determined in vitro, utilizing tissue obtained from both mice and rats (Ma and Chambers, 1994, 1995
; Mortensen et al., 1996
; Pond et al., 1995
; Sultatos and Murphy, 1983
; Sultatos et al., 1984
). The selection of a reasonable set of model parameters was determined by evaluating the overall goodness of fit of the model against the experimental data over the range of reported rate constants for enzyme affinity and activities. Sultatos et al. (1984) previously reported that CPF was extensively bound (
97%) to plasma proteins over a broad range of concentrations. It was also assumed that CPF-oxon would exhibit an even higher plasma protein binding based upon oxon reactivity, although the extent of CPF-oxon plasma protein binding has not yet been experimentally determined. In the current model structure, the total amount of absorbed CPF is directly added to the liver compartment. However, once in the systemic circulation, only nonbound (i.e., free) parent chemical or metabolite was capable of entering the tissue compartments.
B-esterase (B-EST) inhibition.
The model structure for the inhibition of B-EST (AChE, BuChE, and CaE) by CPF-oxon in plasma, RBC, diaphragm, and liver were likewise based on the model structure developed by Gearhart et al. (1990). However, when available, CPF-specific model parameters for bimolecular inhibition (Ki), reactivation (Kr), and aging (Ka) were incorporated into the model (Amitai et al., 1998; Carr and Chambers, 1996
). Reactivation and aging rates were not available for CPF-oxon inhibition of BuChE or CaE so these parameters were set at the rates for CPF-oxon interaction with AChE. The amount of enzyme (µmol), zero-order synthesis rate (µmol/h), and 1st-order degradation rate (h-1) of AChE, BuChE, and CaE for each of the tissue compartments was calculated as previously described (Gearhart et al. 1990
; Maxwell et al. 1987
). The CaE enzyme degradation rate (Kd) and Ki constants were estimated for each tissue by fitting the model to data obtained by Chanda et al. (1997). The Ki parameters for AChE and BuChE blood and tissue activity were estimated by fitting the experimental data from Figure 2
. The esterase recovery rates in the various tissue compartments were obtained from a previous study (Gearhart et al., 1990
) or by fitting the oral dose plasma BuChE data from Nolan et al. (1984).
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Pharmacokinetic/Pharmacodynamic Studies
To develop and validate the CPF PBPK/PD model, pharmacokinetic/pharmacodynamic studies were conducted in both rats and humans.
Rat.
The time course of CPF and CPF-oxon in the blood and the activity of ChE in the plasma, and AChE in brains of male (F344) rats were determined following oral administration of 100, 50, 10, 5, 1, and 0.5 mg CPF/kg of body weight. An additional group of female rats were administered the same doses of CPF and plasma; RBC, and selected tissue ChE activity was determined at 24 h postdosing. The dose solution was prepared in a corn oil vehicle and administered by gavage, and all animals were fasted for approximately 16 h prior to administration of CPF. Four animals per time point were humanely anesthetized, sacrificed, and tissues were collected at 10 and 20 min, and 1, 3, 6, 12, and 24 h postdosing for CPF, CPF-oxon, or ChE analysis. The blood was collected, via cardiac puncture, into heparinized syringes containing an acidic salt solution (2.5 N acetic acid/saturated sodium chloride), which effectively halted the enzymatic hydrolysis of CPF-oxon. These specimens were then analyzed for the presence of CPF and CPF-oxon by NCI-GC-MS as described by Brzak et al. (1998). The limit of quantitation (LOQ) for both CPF and CPF-oxon was determined to be 3 nmol/l of blood. An additional blood sample was collected at each time point, and centrifuged to separate the plasma and RBC (24 h postdosing only) for analysis of plasma ChE and RBC AChE activity. In addition, brains were removed, rapidly frozen by submersion in liquid nitrogen, and both the plasma and brains were then stored at 80°C until assayed for esterase activity. Brain and RBC AChE, and plasma ChE activity assays were performed with Boehringer Mannheim (Indianapolis, IN) reagents on a Hitachi 914 clinical chemistry analyzer utilizing acetylthiocholine (AcTh) as an enzyme substrate, as previously described (Ellman et al., 1961).
Human volunteer pharmacokinetic study.
A double-blind, placebo-controlled clinical pharmacokinetic study was conducted at MDS Harris Laboratory (Lincoln, NE), in accordance with all applicable U.S. guidelines as specified in Title 21 of the Code of Federal Regulations (parts 50, 56 and 321) and the International Guidelines for Human Testing as promulgated in the Declaration of Helsinki (1964; amended 1996). The overall objective of this study was to provide needed data to better define the pharmacokinetics of CPF in humans. In this study, groups of 6 male and 6 female volunteers received a single oral dose of 0, 0.5, 1, or 2 mg CPF/kg of body weight in capsule form. The volunteers were confined to the testing facility for the first 24-h posttreatment period, and their health status was closely monitored during this time, providing a mechanism to confirm the presence or absence of cholinergic or other treatment-related effects. After the initial 24-h interval, the volunteers were allowed to go home but continued to return to the testing facility for the collection of additional blood specimens. Blood was collected at each interval by venipuncture and the targeted collection intervals were 2, 4, 8, 12, 24, 36, 48, 72, 96, 120, 144, and 168 h following treatment. In addition to blood, urine specimens were collected prior to treatment and through 168 h postdosing. Blood and urine specimens were analyzed for the presence of CPF, CPF-oxon, and TCP by GC-NCI-MS (Brzak et al., 1998). The LOQ for CPF and CPF-oxon in blood was 3 nmol/l, whereas the TCP LOQ was determined to be 50 nmol/l. In urine specimens, the LOQ for CPF and CPF-oxon was 3 or 3.5 nmol/l and 1 nmol/l for TCP. In addition, blood specimens were centrifuged to separate plasma from RBC and the inhibition of RBC AChE was determined as previously described (Ellman et al., 1961
). The area-under-the-concentration curve (AUC0
) for blood and urinary TCP excretion were determined using the trapezoidal rule (Renwick, 1994
) and the extent of absorption was calculated based on the amount of TCP recovered in the urine, as described by Nolan et al. (1984).
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RESULTS |
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The time course of plasma ChE and brain AChE inhibition were also determined in rats through 24 h of postdosing following single oral gavage administration of CPF at doses ranging from 0.5 to 100 mg/kg, and the experimental results and model simulations are illustrated in Figures 2 and 4. In these experiments, CPF produced a dose-dependent reduction in both plasma ChE and brain AChE activity. As might be expected at any given dose, plasma esterase was inhibited to a greater extent than brain. The maximum inhibition of plasma ChE was observed at 3 to 6 h after the 0.5 to 10 mg/kg doses, and 3 to 12 h after the 50 and 100 mg/kg doses; doses of 50 mg/kg or greater resulted in a maximum inhibition of plasma ChE (
90%). Inhibition of brain AChE occurred at dose levels of 10 mg/kg and higher. Maximum inhibition of brain AChE occurred from 6 to 12 h postdosing at doses of 10 and 50 mg/kg, and at 3 to 12 h after the 100-mg/kg dose.
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The extent of RBC AChE inhibition was likewise determined at 24 h postdosing in rats orally administered CPF at single doses ranging from 0.5 to 100 mg/kg. In addition, single dose RBC AChE inhibition data (4 h postdosing) at doses ranging from 0.15 to 15 mg/kg was obtained from Zheng et al. (2000). The resulting inhibition and model-simulated prediction of the enzyme inhibition at 4 and 24 h postdosing are presented in Table 3. The inhibition of the RBC AChE activity was dose-dependent; however the RBC's were less sensitive to inhibition than plasma ChE, and a no-effect level was seen at doses of 1 mg/kg or less. The apparent Ki for the RBC's was less than for plasma AChE (
100 vs. 243 µM/h, respectively), and the reactivation rate (Kr) was set at 0.04/h to accommodate a faster reactivation, through 24 h postdosing, that could not be accounted for by RBC resynthesis alone. With these parameters, the model dose response was consistent with the experimental data, although the model slightly underpredicted the peak 4-h inhibition (pred/obs ratios 0.942.07), while slightly overpredicting the 24 h postdosing data (pred/obs ratios 0.340.94). Overall, the model predictions were generally within a factor of 2 of the experimental data.
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Nolan et al. (1984) reported that the blood CPF concentrations following either oral or dermal exposure were extremely low (<0.085 nmol/ml) and exhibited no consistent temporal pattern; however the levels of the major metabolite TCP were readily quantifiable in both blood and urine following either dermal or oral administration. Therefore, the PBPK/PD model was used to characterize the TCP pharmacokinetics. Based on the amount of TCP excreted in the urine, it was determined that 70% ± 11 and 1.28% ± 0.83 of the oral and dermal dose of CPF was absorbed in these volunteers, respectively (Nolan et al., 1984). The time course of TCP in the blood and urine of these human volunteers, and the resulting model simulations following either a single oral 0.5-mg/kg or a dermal 5 mg/kg dose of CPF are presented in Figure 6
. Following the oral or dermal dose, peak plasma TCP concentrations were 4.69 and 0.44 µmol/l and were attained at both 5 and 24 h postdosing. In addition, peak TCP urine excretion was achieved by 24 h postdosing for both the oral and dermal treatments, and the time course of urine excretion was very comparable for both dosing routes. Overall, the model provided a reasonable prediction of the route-dependent blood TCP kinetics following CPF exposure in humans.
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Although only limited CPF blood time course data was available from the controlled human exposure (see Fig. 8) the model was capable of accurately describing the CPF blood kinetics. To further validate the capability of the model to accurately describe the pharmacokinetics of CPF, the time course of CPF in serum from an individual who ingested a concentrated formulation of CPF (Drevenkar et al., 1993
) was modeled and the results are presented in Figure 11
. The subject was a 25-year-old male reported to have drunk 3060 ml of a commercially available insecticide that contained CPF as the active ingredient. The subject was admitted to the hospital 2 to 5 h after ingestion of the pesticide formulation, and CPF was quantitated in the serum for up to 15 days after poisoning. The samples were likewise analyzed for CPF-oxon; however the oxon was not detectable in any of the samples (Drevenkar et al., 1993
). The PBPK/PD model was capable of accurately describing the overall pharmacokinetics of CPF in the blood following a single acute exposure. Based on the observed CPF blood time course, the model estimated that an acutely toxic dose of
180 mg/kg was needed to achieve the highest concentrations of CPF (110 µmol/l) that were detected in the serum. This predicted dosage is well within the range of reported acute LD50 doses (85504 mg/kg) in animals, and is a reasonably good approximation of a dosage that is capable of producing the reported acute toxicological responses in humans (Gaines 1969
; McCollister et al., 1974
).
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DISCUSSION |
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In developing the CPF model it became obvious that, although a considerable amount of hazard evaluation research has been performed on CPF, only a limited number of pharmacokinetic studies were available for model development. Therefore, both rat and human pharmacokinetic/pharmacodynamic studies were conducted to provide the needed experimental data to develop and validate the model. Utilizing recently developed sensitive (i.e., low ppb range) analytical methods (Brzak et al., 1998) it was feasible to quantitate CPF, CPF-oxon, and TCP following in vivo exposures to CPF and to directly link dosimetry with the observed ChE and AChE inhibition kinetics in both rats and humans.
The PBPK/PD model provides important insights into the route- and dose formulation-dependent absorption of CPF in rats and humans. Due to its poor aqueous solubility, CPF dose solutions are generally prepared in an oil-based matrix (e.g., corn oil) and administered to rodents by oral gavage. Previous studies have shown that the dosing vehicle can modify the amount and rate of absorption, and corn oil in particular has been shown to result in lower peak and increased time to maximum blood concentrations (Cmax) (Withey et al., 1983). Hence, to describe the uptake of CPF from the GI tract of rats, we needed a two-compartment GI tract model similar to the one used to describe the uptake of chlorinated solvents (Staats et al., 1991
). This model modification resulted in a more accurate simulation of CPF uptake than was feasible with the single-compartment model, although it did not enable the model to adequately fit the peak blood (3 h postdosing) CPF concentration. In addition to a slower overall rate of uptake, due to corn oil, the amount of CPF orally absorbed in rats was estimated to be
80%. This estimate was based on pharmacokinetic studies where rats were administered 14C-labeled CPF and the 14C activity was determined in tissues and excreta (Dow Chemical, unpublished report). Differences in the extent of CPF absorption were also clearly evident when comparing the human CPF pharmacokinetics conducted in this study with the results from human kinetic studies conducted under different dosing conditions. Nolan et al. (1984) deposited a known amount of CPF onto a lactose tablet, which was subsequently swallowed with water. This dose preparation resulted in
70% of the orally administered dose being recovered as TCP. In contrast, the CPF dose in the current human kinetic study was prepared by adding CPF and lactose as dry powders into a dissolvable capsule that was likewise swallowed with water, however only 2035% of this oral dose was recovered as TCP. Based on these experimental findings, the extent of CPF absorption appears to be dependent upon its physical form when administered. The question of bioavailability is of particular importance when interpreting a ChE response based only upon the administered dose, without taking into full consideration how much of that dose was actually absorbed. Therefore, interpretation of pharmacodynamic response data would certainly be strengthened with a concurrent assessment of bioavailability within the same study. This analysis could readily be accomplished by quantitating blood or urinary TCP concentrations. However, for the purposes of the rat PBPK/PD model development, the oral absorption of CPF, using a corn oil dosing matrix, was set at 80% for all model simulations. This assumption was made while fully recognizing that it represents an oversimplification, since the extent of absorption can be drastically different depending on the conditions of the dose solution (i.e., formulation), dosing method, and variations in species, gender, or strain (Kararli, 1995
).
In reality the majority of both occupational and environmental exposures to OPs are primarily associated with the dermal route, which has been reported to account for more than 90% of the systemically absorbed dose in humans (Aprea et al., 1994). Therefore, an understanding of percutaneous absorption of OPs is critical for quantitatively determining a systemic dose. The extent of in vivo dermal absorption of CPF in humans has previously been reported as low (13%; Griffin et al., 1999
; Nolan et al., 1984
). This is consistent with the low in vivo dermal absorption potential reported in humans exposed to diazinon, isofenphos, and malathion, which ranged from 2.53.9% of the applied dose (Wester et al., 1983
; 1992
; 1993
). In the current PBPK/PD model, the dermal uptake of CPF in humans was well described by estimating a CPF permeability coefficient (Kp) of 4.81 x 10-5/cm/h utilizing previously published dermal absorption data in humans (Nolan et al., 1984
). The model presented herein quantitatively predicts the concentration of CPF, CPF-oxon, and TCP in blood, tissues, and excreta in both rats, and humans following exposure, either orally or through direct skin contact, with CPF. These results are consistent with TCP being the major metabolite formed that attained a blood concentration of 100-fold higher than the parent compound (see Fig. 8
). Likewise, the pharmacokinetic studies conducted in rats suggest that levels of CPF in the blood exceed the CPF-oxon concentrations. These results clearly indicate that even when employing sensitive analytical techniques, quantifiable concentrations of both CPF and CPF-oxon in blood are only obtainable when relatively high doses of CPF (>5 mg/kg) were administered, and even then, it was only detectable for a limited time after dosing. This inability to adequately quantitate both CPF and CPF-oxon blood concentrations over a range of dose levels has made it particularly challenging to accurately model dosimetry, particularly at low doses (<10 mg/kg).
Biomonitoring of OPs and their metabolites in blood and urine has been used to provide a quantitative assessment of dosimetry in human poison victims following acute high-dose exposure (Drevenkar et al., 1993; Vasilic et al., 1992
), and for the assessment of secondary exposures (Loewenherz et al., 1997
; Richter et al., 1992
). A major advantage in utilizing the analysis of intact pesticide or specific metabolites in body fluids versus ChE depressions is that it enables identification of specific chemical agent(s) that may be associated with the observed esterase inhibition (Ellenhorn and Barceloux, 1988
; Lotti et al., 1986
), and as previously mentioned, can be used to better determine bioavailabilty. The utilization of PBPK/PD models such as the one developed for CPF enables the quantitative integration of dosimetry with biological response. The model can predict the extent of esterase inhibition (see Fig. 7
) based on the observed concentrations of either the parent compound (see Figs. 8 and 11
) or metabolites (see Figs. 6, 8, 9
). Hence this PBPK/PD model provides an integrated assessment of CPF dosimetry and biological response in both rats and humans for a number of exposure scenarios.
The PBPK/PD model integrates CPF dosimetry with a pharmacodynamic model capable of predicting stoichiometric B-EST inhibition, and is consistent with other pharmacodynamic models such as the one developed for soman in the rat (Maxwell et al., 1988). The extent and rate of B-EST inhibition and recovery is dependent upon the amount of available enzyme, the Ki, and the amount of time the B-EST is exposed to the available oxon (Vale, 1998
). As previously noted, the amount of available B-EST binding sites (µmol) in general followed the order CaE >> BuChE
AChE (Maxwell et al., 1987
), whereas the Ki rates followed the order: BuChE >> AChE > CaE. Initial model simulations utilized relatively higher Ki values determined with purified BuChE and AChE enzyme (Amitai et al., 1998
), which severely over predicted the extent of ChE and AChE inhibition (data not shown). In this regard, Mortensen et al. (1998) noted differences in in vitro sensitivity (i.e., IC50s) of AChE when comparing the inhibition of purified enzyme against crude tissue homogenates. Hence it is not unreasonable to expect that the Ki values determined with purified enzymes would not adequately describe the inhibition response obtained with crude enzyme preparations. Therefore, an apparent Ki was determined for CPF-oxon inhibition of B-EST enzymes that was qualitatively consistent with the Ki values measured with purified enzymes (Amitai et al., 1998
). The model simulation suggests that BuChE is the most sensitive to CPF-oxon inhibition due to a higher apparent Ki (2.0 x 103 µM/h) and relatively low amounts (
1600 µmol total in rat) of available BuChE in the tissues. In contrast, the model predicts that CaE is the least sensitive to CPF-oxon, and appears to be due to the combination of an estimated low apparent Ki (20 µM/h), and high amounts of available enzyme (2 x 106 µmol total in rat). Finally, the sensitivity of AChE enzyme to CPF-oxon inhibition resulted from a moderate (between BuChE and CaE) apparent Ki (243 µM/h), and relatively low amounts (
1000 µmol total in rat) of available AChE enzyme. Although these apparent Ki parameter estimates provide a reasonable description of the B-EST inhibition kinetics, it is clear that the model would be further strengthened by experimentally determining these Ki parameters under physiological conditions that more accurately reflect in vivo activity. In addition, uncertainty associated with model estimates of CPF-oxon concentration raises the possibility that the model overestimates of CPF-oxon concentration, particularly at high doses, may have contributed to excessive esterase inhibition predictions. Additional quantitative data on CPF-oxon blood and/or tissue concentrations and Ki parameter estimates may help resolve this question.
Chanda et al. (1997) evaluated the tissue-specific effects of CPF on both CaE and ChE, utilizing in vivo and in vitro comparisons. They reported that the maximum inhibition of plasma CaE, following an 80-mg CPF/kg dose, was 40% of control (Chanda et al., 1997
), whereas, in the current study, a comparable level of plasma ChE (50:50BuChE: AChE) inhibition was achieved at a dose level of
5 mg CPF/kg (see Fig, 2
). These results are consistent with the observed differences in apparent Ki's, and the amounts of available B-EST enzymes in the rat. Likewise, these results are consistent with the observation that B-EST inhibition is an integrated function of target-tissue dosimetry, esterase affinity for CPF-oxon, and the number of available esterase binding sites in each tissue compartment. In this regard, improvements in the predictive capability of the current model may be achieved by conducting studies that better characterize the time course of specific esterase activities (i.e., AChE, BuChE, and CaE) in both blood and tissue compartments over a broad range of CPF exposures.
It has been generally accepted that the biological bases of risk assessments are strengthened by the utilization of an internal dose surrogate versus the administered dose in cross-species extrapolations (Andersen, 1995). A determination of dose-rate effect may also be important, since under the Food Quality Protection Act (FQPA) there is concern about the risk associated with pesticide residue exposures where dermal contact and/or ingestion of low levels of pesticide residue on food constitute important exposure routes (U.S. EPA, 1998
). In this case, an evaluation of maximum blood concentration (Cmax) and/or blood AUC is of relevance since these dose surrogates may be particularly sensitive to dosing rate (Corley, et al., 1994
). To illustrate this point, a simulation of the blood CPF concentration and plasma ChE response in rats following either a single bolus dose of 10 mg CPF/kg or the same dose fractionated (3 x 3.3 mg/kg at 8-h intervals) over a 24-h period is presented in Figure 12
. Based on model simulations, the Cmax for blood CPF was
0.05 µmol/l, whereas the same dose when fractionated over a 24-h period attained a Cmax of
0.02 µmol/l. Although the blood concentration for the fractionated dose was slightly less than half of that obtained following the bolus administration, repetitive dosing did result in the blood levels of CPF being maintained at elevated levels until dosing ceased (
24 h). The potential impact of dividing the dose was particularly evident when comparing the model simulation of ChE inhibition (see Fig. 12 B
). Compared to bolus administration, dividing the dose reduced the peak inhibition by
10% (
27% of control). These simulations infer that the administration of CPF as a large bolus dose, results in a greater maximum plasma ChE inhibition than when the same dose is administered over a prolonged time period (i.e., divided). Based on these model simulations, future studies should consider experimentally determining the impact of dose-rate on both CPF dosimetry and dynamic response.
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Although the current PBPK/PD model describes the pharmacokinetics and pharmacodynamics of CPF over a broad dose range (0.5100 mg/kg), it is important to recognize that even the lowest doses evaluated in the current study are still significantly greater than typical aggregate (total dietary and residential) human exposures. In both adults and children, potential aggregate exposures to CPF are estimated to range from 0.46 to 1.7 µg/kg/day, which is 294- to 1000-fold below the lowest dose (0.5 mg/kg) used in this study (Gibson et al., 1999
; Hill et al., 1995
; Quackenboss et al., 1998
; Shurdut et al., 1998
; ). Based upon the observed dose response, where CPF and CPF-oxon were nondetectable and blood ChE activity was minimally depressed at doses of 0.5 mg/kg, it is hypothesized that a significant first-pass metabolism will be observed at environmentally relevant doses. In this regard, a number of recent studies have demonstrated that intestinal epithelial cells have CYP450 metabolic capacity and are capable of significantly altering oral bioavailability of drugs and chemicals in animals and humans (Hall et al., 1999
; Obach et al., 2001
; Paine et al., 1999
; Schmiedlin-Ren et al., 1993
; Watkins, 1992
; Zhang et al., 1999
). A number of the CYP450 isoforms residing within the intestines have been shown to metabolize a number of OP insecticides including parathion, diazinon, and CPF (Butler and Murray, 1997
; Fabrizi et al., 1999
; Sams et al., 2000
). In addition, P-glycoproteins (multidrug resistance proteins) are located in the apical borders of intestinal cells, and are known to be upregulated and bound by CPF-oxon (Lanning et al., 1996
). Thus, an unknown amount of first-pass CPF detoxification and activation should occur in the intestines, and CPF-oxon that is generated in enterocytes would be subject to removal by P-glycoproteins. Since the current model does not incorporate intestinal metabolism or P-glycoprotein removal of CPF-oxon, it is anticipated that the model overestimates low-dose oral bioavailability, thereby potentially overpredicting dosimetry and dynamic response. Therefore, to address this important question, research is ongoing to further refine the PBPK/PD model with the inclusion of a metabolically active intestinal compartment, and associated studies are being conducted to validate the model response at environmentally relevant exposures.
The PBPK/PD model that was developed for CPF is capable of quantitating target tissue dosimetry and dynamic response in both rats and humans, and is a useful tool to quantitatively assess risk associated with CPF exposure as well as helping to design and focus future experimental research. The capability of the model to accurately predict dosimetry and response is limited by the adequacy of the model parameters and limitations of the experimental data. As with all models, developing and validating a PBPK/PD model is an iterative process and highlights the current limitations of our understanding of critical biological processes that help identify important data gaps. Additional research could provide a better characterization of the absorption kinetics and dosimetry for CPF and CPF-oxon under various dosing conditions. Of particular importance are experimental data to better understand the extent of CPF absorption at environmentally relevant exposures, and to evaluate the potential role of gut metabolism and CPF-oxon protein binding following low-dose exposures. Additional studies could better characterize the time course and dose response for RBC AChE inhibition, since this pharmacodynamic response is particularly relevant as a potential biomarker for exposure. Better parameter estimates are needed for the binding of CPF-oxon with B-EST and in particular the CaE tissue inhibition kinetic parameters for both rats and humans. In conclusion, this CPF PBPK/PD model quantitatively estimates target tissue dosimetry and AChE inhibition, and is an effective framework for future OP model development, and for establishing a biologically based risk assessment for CPF exposure under a variety of scenarios.
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APPENDIX |
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Oral absorption (gavage).
The absorption of CPF following oral administration in corn oil vehicle was modeled utilizing a two-compartment model. The rate of change and amount of CPF in the compartments is described as
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Oral absorption (dietary).
To model dietary administration of CPF, the dietary update was modeled as a zero-order process, and was based on the equations used to describe uptake from drinking water (Andersen et al., 1987 and Corley et al., 1994
) as
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Dermal absorption.
The rate of change in the amount of CPF absorbed through the skin (ASK, µmol) was described based on previous dermal uptake PBPK models (Corley et al., 1994; McDougal et al., 1986
) as
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Liver compartment.
The rate of change in the amount of CPF in the liver (AH, µmol) is described as the rate of free CPF entering the liver as absorbed dose from the GI tract, or through the systemic circulation minus the rate of free CPF leaving the liver unchanged, or by metabolism to either CPF-oxon or TCP as
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Protein binding of CPF and CPF-oxon.
The protein binding of CPF (FBc) and CPF-oxon (Fbo) were set at a constant fraction (%). Only free (i.e., nonbound fraction) CPF, or CPF-oxon were allowed to partition into and out of each tissue compartment and be available for metabolism (i.e., CYP450, B-EST, A-EST). The concentration of bound CPF or CPF-oxon in both arterial and venous blood was continuously calculated as a function of the free CPF or CPF-oxon in arterial blood or in venous blood draining each tissue compartment to maintain mass balance; the protein binding in plasma was calculated by the equations
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Liver compartment CPF-oxon model.
The liver compartment represents the linkage between the models for CPF and CPF-oxon (See Fig. 1). The rate of change in the amount of CPF-oxon in the liver (AHo, µmol) is defined as the rate of input from the metabolism of CPF to CPF-oxon (dAML1dt), plus the input from the hepatic artery, minus the rate of loss into the venous blood draining the liver (CVHof), where the blood concentrations available for partitioning of CPF-oxon into and out of the liver are not bound to protein (i.e., free). Additional loss of free CPF-oxon is associated with hepatic A-EST and B-EST metabolism. according to the equations
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Tissue concentrations of CPF or CPF-oxon.
The rate of change of CPF or CPF-oxon in tissues that neither form nor eliminate CPF-oxon is similar to that of Corley et al. (1994), as
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B-EST tissue inhibition by CPF-oxon.
The equations describing the tissue (i.e., blood, brain, diaphragm, and liver) inhibition of AChE, BuChE, and CaE by CPF-oxon are similar to those used to describe B-EST inhibition by DFP (Gearhart, et al., 1990). For example, the equations describing ChE inhibition in the liver compartments are
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One-compartment model TCP elimination.
The pharmacokinetics of TCP in blood and urine were modeled utilizing a simple one-compartment analysis to describe blood and urinary elimination, where
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ACKNOWLEDGMENTS |
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NOTES |
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Presented in part at the 37th annual meeting of the Society of Toxicology, March, 1998, Seattle, Washington.
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