The Influence of Maturation on Rat and Human Physiological

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Chapter 5

The Influence of Maturation on Rat and Human Physiological Processes Involving Protein and Lipoprotein Binding, Gastrointestinal Absorption, and Blood Brain Permeability and Transport of Pyrethroids J. V. Bruckner,*,1 T. G. Osmitiz,2 S. Anand,3 D. Minnema,4 W. Schmitt,5 N. Assaf,6 and J. Zastre1 1College

of Pharmacy, University of Georgia, Athens, Georgia 30602 Strategies, Charlottesville, Virginia 22902 3Dupont Haskell Laboratories, Newark, New Jersey 19714 4Syngenta Crop Protection, Greensboro, North Carolina 27419 5Bayer Crop Sciences, 40789 Monheim, Germany 6Valent Biosciences, Libertyville, Illinois 60048 *E-mail: [email protected] 2Science

The widespread use of pyrethroids as insecticides has resulted in exposure of much of the U.S. populace, including pregnant women and children. Greater susceptibility of preweanling rats to high doses of pyrethroids has led to concern that infants and children may be more sensitive than adults to neurotoxic effects at contemporary exposure levels. Research has shown that preweanling rats’ low metabolic detoxification capacity is a major contributor to elevated blood and brain levels of the neurotoxic parent compounds. The Council for the Advancement of Pyrethroid Human Risk Assessment (CAPHRA) is initiating a series of research projects to learn more about factors that may contribute to age-dependent sensitivity to pyrethroids, and for their incorporation into physiological models capable of accurately predicting target organ (brain) dosimetry and toxicity in different age-groups for different exposure scenarios. In our own laboratory, CAPHRA is sponsoring investigations of age- and

© 2012 American Chemical Society In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

species-dependent: pyrethroid transportation in blood (plasma protein and lipoprotein binding); tissue:blood distribution; and blood-brain barrier (BBB) gastrointestinal (GI) barrier efficiency, including the potential role of GI and BBB efflux transporters. Experiments are underway with Caco-2 cells to characterize GI membrane flux and to learn whether pyrethroids are substrates for P-glycoprotein or other transporters.


Introduction Concern for the last 3 decades led to formation of a committee by the National Research Council (NRC) to assess exposures and risks posed to infants and children by dietary pesticides (1). Recommendations in the committee’s report (2) had some far-reaching consequences, including passage of the Pediatric Research Equity Act of 2003 and the Food Quality Protection Act (FQPA) of 1996. The FQPA dictated that an additional safety factor of 10X be used in risk assessments when pediatric data for a pesticide are unavailable. This is invariably the case. The children’s safety factor of 10X is considered to take into account potential age-related pharmacokinetic (PK) and pharmacodynamics (PD) differences of 3.16X each (3). The NRC (2) advocated development of physiologically-based (PB) PK and PD models for use in predicting the PK and PD of chemicals that cannot be studied directly in children. Pyrethroids, synthetic derivatives of naturally-occurring pyrethrins, have largely replaced organochlorine and organophosphate insecticides in the U.S. and Europe (4). The primary mode of neurotoxicity of pyrethroids is interaction of the parent compounds with voltage-sensitive sodium channels. Pyrethroids are widely used in agriculture and households for insect control. Thus, exposure of the general population is quite common, as is oral and dermal exposure of potentially sensitive subpopulations [e.g., pregnant women, children and infants (5, 6)]. Anatomical, physiological and biochemical changes that occur as a child grows can alter the absorption, distribution, metabolism and elimination (ADME) of chemicals, thereby altering target organ (e.g., brain) dosimetry and adverse effects. Postnatal neurological development is also a complex process involving cell division, differentiation, and migration during structuring of the central nervous system (CNS). Unfortunately, there are few animal PK or PD data on pyrethroids in immature animals, and a data vacuum for infants and children.

Age-Dependent Acute Neutotoxicity in Rats: A Primary Cause Preweanling rats have been shown to be more sensitive than adults to neurotoxic effects of high doses of several pyrethroids. The susceptibility of rat pups to cypermethrin and permethrin was reported to be inversely related to age (7). Sheets et al. (8) also observed that preweanling pups were much more susceptible to deltamethrin (DLM) lethality than adults: oral LD50 values for 11-, 21- and 72-day-old rats were 5, 11 and 81 mg/kg, respectively. At the time of death, comparable brain DLM levels were present in weanling and adult rats that 56 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


received 12 and 80 mg DLM/kg orally, respectively. Sheets et al. (8) proposed this disparity in brain dosimetry was due to the immature animals’ limited metabolic detoxification capacity. Data supporting this hypothesis were not forthcoming until 12 years later. Anand et al. (9) demonstrated in vitro that intrinsic clearance of DLM by liver cytochrome P450s and liver and plasma carboxylesterases rose progressively in male rats between 10 and 40 days of age. Correspondingly, blood and brain DLM concentrations (Cmax and AUC) of the neurotoxic parent compound diminished substantially with age (9, 10). Brain dosimetry was more closely correlated with the magnitude of salivation and tremors than blood levels. Gastrointestinal (GI) absorption in different age-groups appeared to be rapid but incomplete. Bioavailability was just 15 – 18% (11). Fat and skin accumulated large amounts of the highly lipophilic chemical and served as slow-release depots. Surprisingly, brain DLM concentrations were only ~ 20% of blood concentrations (10, 11). The two did not parallel one another closely over time. Much remains to be learned about physiological and biochemical processes and barriers that govern pyrethroid PK and contribute to age-dependent differences.

PBPK Modeling of Pyrethroids to Date The primary attribute of a validated PBPK model in risk assessment is its ability to accurately predict the time-course of bioactive moieties in target tissues for different exposure scenarios. Mirfazaelian et al. (12) published the first PBPK model for a pyrethroid in 2006. It reasonably simulated the time-course of DLM in the blood, brain, fat and other tissues of adult rats. The model was subsequently adapted to immature rats and used to forecast DLM concentrations over time following oral dosing of 10-, 21-, 40- and 90-day-old animals (13). Age-dependent changes in oxidative and hydrolytic clearance [measured in vitro by Anand et al. (9)] and age-specific organ weights [obtained with a generalized Michaelis-Menten model (14)] were used in the immature rat PBPK model. Description of the PK of DLM in the brain as diffusion- rather than flow-limited resulted in better agreement of simulated and empirical data. Godin et al. (15) subsequently modified the adult rat model for DLM to describe all compartments as diffusion-limited and scaled it to adult humans.

PBPK Modeling of Pyrethroids in Children: Planning There is considerably more to be learned about maturational changes in physiological processes that impact the ADME of pyrethroids. It is important to identify processes in addition to metabolism that significantly influence the PK of pyrethroids. It is then necessary to determine which of these factors are age-dependent and to characterize their ontogeny in rats, as an animal model. Accurate rate constants and other values for the key model input parameters for different age-groups need to be obtained. Thereby, it will be possible to construct PBPK models which more closely reflect different stages of maturation. The improved models should provide more reliable predictions of pyrethroid PK at different stages of development. It is essential, of course, to carefully consider the 57 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


relevance of such developmental changes in rats to humans. Whenever possible, age-specific in vivo or in vitro human parameters should be utilized in modeling. This research strategy was discussed at a meeting of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP) (16). It was decided that PBPK modeling was an appropriate experimental approach to address the important question of “Whether there are differences in the sensitivity of juveniles/children and adults to pyrethroids”? Discussants and presenters at that meeting included representatives of the Pyrethroid Technical Working Group. This consortium of pyrethroid distributors and manufacturers, now known as the Council for the Advancement of Pyrethroid Human Risk Assessment (CAPHRA), endorsed this experimental approach and is now sponsoring a multicenter research effort to answer the question of whether there are age-dependent differences in sensitivity to pyrethroids at contemporary exposure levels.

Overview of Our Ongoing Research In the current CAPHRA-sponsored research project in our laboratories, we are investigating several age-dependent ADME processes to learn whether they significantly impact the PK of selected pyrethroids and should be incorporated into future PBPK models. These processes and parameters include: (a) transportation in the bloodstream (i.e., plasma protein binding and lipoprotein incorporation); (b) blood:tissue deposition/partition coefficients; (c) blood-brain barrier (BBB) and GI barrier efficency; and (d) potential role of BBB and GI membrane transporters in pyrethroid efflux.

Role of BBB and GI Transportation in Pyrethroid Efflux The immature BBB in very young rodents and humans contributes to relatively high brain levels of a variety of heavy metals, drugs and other compounds (17, 18). The fully differentiated (mature) BBB consists of highly specialized capillary endothelial cells with complex tight junctions, pericytes embedded in a basement membrane, perivascular macrophages and overlapping astrocytic endfeet (19). Kim et al. (11) found that levels of DLM in the brain of orally-dosed adult rats were < 20% of blood levels. It might be anticipated that the brain, with its relatively high lipid content, would retain relatively large amounts of lipophilic pyrethroids. If the BBB limits the passage of pyrethroids into the CNS, an immature BBB in young rats or humans may contribute to the elevated brain levels we have seen in young rats. Plasma:brain ratios were significantly higher in 10-day-old rats than in 21-, 40- and 90-day old rats for the initial 1 – 2 hours following oral dosing with DLM (10, 11). In vivo infusion experiments will be conducted to learn whether the BBB inhibits passage of pyrethroids, and if so whether its ability is age-dependent. P-glycoprotein (P-gp) is an important component of the BBB (20). P-gp extrudes a variety of structurally-unrelated lipophilic compounds, though it has not been established whether pyrethroids are substrates for P-gp or other transporters. One research group (21) reported that cypermethrin significantly 58 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


inhibited P-gp-mediated transport of tetramethylrosamine into proteoliposomes, but fenvalerate was less effective. Another group (22) found fenvalerate did not inhibit doxorubicin transport in vitro, but permethrin had a modest effect. Inhibition/modulation does not necessarily infer these pyrethroids are P-gp substrates. Developmental profiles of P-gp in the brain have been well characterized in rodents (23, 24) and primates (25). P-gp expression and CNS efflux function increased during maturation in each species. Thus, both functional and structural immaturity of the BBB may lead to increased deposition of pyrethroids in the brain of neonates and infants. The human brain microvessel endothelial cell line, hCMEC/D3, displays many BBB markers and properties of brain endothelium in vivo, such as tight junction formation. hCMEC/D3 cells also express a number of Solute Carrier and ATP Binding Cassette transporters. This cell line will be utilized to determine whether selected pyrethroids are substrates for specific carrier systems and to characterize their transport properties. The age-dependency of GI transporters and their potential role in oral absorption of pyrethroids is being examined in Caco-2 cells. This human adenocarcinoma cell line is the “gold standard” for in vitro evaluation of GI absorption and transport (26). Caco-2 cells undergo spontaneous enterocyte differentiation and polarization, once they reach confluency in culture (Figure 1). Completely differentiated monolayers of Caco-2 cells display microvilli and brush border hydrolases on their apical surface, as well as endogenous transport systems. Because of their structural and functional homology with human intestinal epithelium, they can be utilized to reliably assess both passive and carrier-mediated uptake of xenobiotics. The polarization of fully differentiated Caco-2 cell monolayers permits the measurement of transepithelial flux and the calculation of apparent permeability coefficients (Papp) (27). The cells are grown on collagen-coated polycarbonate (Transwell®) membrane inserts, which provide discrete apical and basolateral chambers (Figure 2). We have initiated studies of three pyrethroids: DLM, cis-, and trans-permethrin. Accurate measurements of Papp of such highly lipophilic compounds requires special consideration (28). The apical to basolateral (AP→BL) flux is measured by placing 14C-radiolabeled DLM, cisand trans-permethrin [in pH 7.4 Hanks Bufford Salt Solution (HBSS) containing 10 mM HEPES + 0.1 bovine serum albumin (BSA)] on the cells’ apical surface (Figure 2). At selected time-points, the Transwell® inserts are lifted out and placed into a new well containing fresh HBSS buffer. The cumulative amount of pyrethroid appearing in the BL chamber is determined, and flux is plotted as a function of time. Papp values are calculated and expressed in cm/sec. A similar approach is used to measure the basolateral to apical flux (BL→AP). 14C-DLM, cis- or trans-permethrin placed on the cells’ basolateral surface. Samples are then withdrawn at selected time-ponts from the apical chamber and analyzed for their 14C content, in order to determine PappBL→AP. Transepithelial electrical resistance and 3H-mannnitol flux are monitored to ensure cell monolayer integrity. It should be kept in mind that BL→AP flux is a function of pyrethroid diffusion and transport. AP→BL flux is the result of diffusion minus transport. An efflux ratio (PappBL→AP/PappAP→BL) of > 2 is indicative of a compound that is a substrate for membrane efflux transporters. 59 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 1. Caco-2 cells grown to confluency are shown at top. Cross sectional view at bottom of cells with prominent nuclei and microvilli.

Figure 2. Schematic depiction of the Caco-2 cell monolayer system for measuring bidirectional flux of pyrethroids. 60 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


We have completed some pilot studies of DLM, cis- and trans-permethrin accumulation by Caco-2 cell monolayers. It is apparent in Figure 3 that uptake of these compounds is linear between 0.5 and 5 minutes. Additional uptake during the next 15 to 30 minutes is quite modest (Figure 4). In Figure 5, uptake during a 3-minute period is plotted as a function of pyrethroid concentration. Cellular accumulation diminishes somewhat with increasing concentration, suggesting the possibility of a saturable transport process in this low range. It is not possible to work with higher concentrations to demonstrate saturation of uptake of these compounds, due to their very low water solubility. Pyrethroid uptake by the cells is reduced at the lower temperature (4°C). This could also be indicative of the presence of an active transport process, although the rate of passive diffusion decreases with decrease in temperature. Experiments have been undertaken to quantify transepithelial flux.

Figure 3. Mean uptake of 25 nM each of Deltamethrin (DLM) and Cis- and trans-permethrin (Cis ) and (Trans) by Caco-2 cells during 5 min. Each point represents the mean ± SE for 3-4 replicates.

Additional studies with inhibitors of P-gp and other transporters will be performed, as will be competitive inhibition experiments with known substrates, in order to establish whether/which active transporters may be involved in membrane flux of the pyrethroids. If active transport is demonstrated, the Michaelis-Menten parameters Vmax and Km will be determined for use as PBPK model input parameters. The data developed above will be considered for incorporation into PBPK models, as appropriate, for early life stage exposures of humans to pyrethroids. 61 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 4. Mean uptake of 25 nM each of Deltamethrin (DLM) and Cis- and trans-permethrin (Cis ) and (Trans) by Caco-2 cells during 30 min. Each point represents the mean ± SE for 3-4 replicates.

Figure 5. Concentration-and time-dependent uptake of deltamethrin, cis- and trans-permethrin by Caco-2 cells. Each point represents the mean ± SE for 3-4 replicates. 62 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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The Influence of Maturation on Rat and Human Physiological

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