Comparative studies of midazolam metabolism in chickens, turkeys, ring-necked pheasant and bobwhite quail: In vitro, in vivo and physiologically-based pharmacokinetic modeling
TABLE OF CONTENTS TITLE PAGE i LIST OF FIGURES vi LIST OF TABLES vii ABSTRACT 1 CHAPTER 1. Introduction to Comparative Studies of Midazolam Metabolism in Chickens, Turkeys, Ring-necked Pheasants and Bobwhite Quail 3 Background 4 Midazolam as a Model Compound for Hepatic Oxidative Metabolism 6 Evidence for CYP3A Existence and Activity in Poultry Species 9 Specific Objectives 12 References 13 CHAPTER 2. Hepatic Microsomal Pharmacokinetic and Inhibition Studies with Prototypical CYP3A Substrates and Inhibitors thereof. 17 Abstract 18 Introduction 19 Methods 22 Results 27 Discussion 33 References 40 iv
CHAPTER 3. Plasma Pharmacokinetics of Midazolam in Chickens, Turkeys, Pheasants and Bobwhite Quail 44 Abstract 45 Introduction 46 Methods 48 Results 54 Discussion 61 Conclusion 65 References 66 CHAPTER 4. Physiologically-based Pharmacokinetic Modeling of Midazolam in Gamebirds and Poultry 73 Abstract 74 Introduction 75 Methods 76 Results 90 Discussion 105 Conclusion 107 Appendix 109 References 113 SUMMARY AND CONCLUDING REMARKS 116 v
LIST OF FIGURES 1.1 Midazolam metabolism 8 2.1a. Initial rate of 1-hydroxymidazolam formation in hepatic microsomes incubated with 1.25-50uM midazolam 29 2.1b. Initial rate of 4-hydroxymidazolam formation in hepatic microsomes incubated with 1.25-50uM midazolam 30 3.1. Compartmental pharmacokinetic fits to plasma midazolam concentration following IV administration 55 3.2. Compartmental pharmacokinetic fits to plasma 1-hydroxymidazolam concentration following IV administration 55 3.3 Compartmental pharmacokinetic fits to plasma 4-hydroxymidazolam concentration following IV administration 56 3.4. Plasma midazolam levels in vivo vs. Km values determined in vitro 64 4.1. PBPK model for midazolam residues in chickens 83 4.2. Final model fits to chicken tissue residue data showing predicted lines and observed data points 91 4.3. Standardized residual plots for line fits of each chicken tissue 92 4.4. Predicted versus observed mean plots for each chicken tissue 93 4.5. Final model fits to turkey tissue residue data showing predicted lines and observed data points 95 4.6. Final model fits to pheasant tissue residue data showing predicted lines and observed data points 96 vi
4.7. Final model fits to quail tissue residue data showing predicted lines and observed data points 97 4.8. Standardized residual plots for line fits of each turkey tissue 98 4.9. Standardized residual plots for line fits of each pheasant tissue 99 4.10. Standardized residual plots for line fits of each quail tissue 100 4.11. Predicted versus observed mean plots for each turkey tissue 101 4.12. Predicted versus observed mean plots for each pheasant tissue 102 4.13. Predicted versus observed mean plots for each quail tissue 103 vii
LIST OF TABLES 2.1. Michaelis-Menton parameters calculated from hepatic microsomal metabolism of midazolam using Winnonlin models 28 2.2. Calculated IC50 values for ketoconazole inhibition of midazolam metabolism 32 3.1. Compartmental analysis of plasma drug concentration-time curve following IV administration 57 3.2. Noncompartmental analysis of plasma drug concentration-time curve following IV administration 58 4.1 Tissue midazolam limit of detection and limit of quantification 81 4.2 Chicken hematocrit, cardiac output and tissue blood flows from the literature. 84 4.3. Physiologic data from 10 birds of each species used to build the original PBPK model for chicken and validate the model for turkey, pheasant and quail.... 85 4.4. Physiologic data of birds from the drug residue study used to optimize (chicken) and validate (others) the PBPK model 88 4.5. Model fitting optimized parameters 89 4.6. Model validation results comparing the model predicted tissue concentrations to observed mean concentrations at each slaughter time point 104 vm
1 ABSTRACT Comparatively little is known about the ability of commercially raised poultry and gamebirds to metabolize therapeutic drugs. The goal of this research was to use a combination of in vitro, in vivo and physiologically-based pharmacokinetic (PBPK) studies to characterize metabolism of a cytochrome P450 3 A substrate in four closely related poultry and gamebird species. Cytochrome P450 3A (CYP3A) enzymes, found primarily in the liver but also in the kidneys and other organs of mammals, are one of the major oxidative metabolic pathways. Studies using hepatic microsomes from chickens, turkeys, pheasant and bobwhite quail were conducted with midazolam as a substrate of CYP3A metabolism. Inhibition of midazolam metabolism was also measured in vitro using ketoconazole as an inhibitor. All four avian species produced 1-hydroxymidazolam as the major metabolite and 4-hydroxymidazolam as a minor metabolite. Ketoconazole inhibited the 4- hydroxymidazolam more than the 1-hydroxymidazolam and pheasant and quail appeared most sensitive to inhibition. To better assess underlying metabolic processes in our four avian species, whole animal pharmacokinetic and tissue residue studies were conducted after midazolam intravenous administration. Pharmacokinetic profiles were similar with regard to area under the drug concentration-time curve. Tissue residue studies also resulted in similar profiles. There was some variation in the later time points for all tissues, with some birds clearing the drug faster than others. Due to the sparse nature of the pharmacokinetic data
2 collected, a bootstrapping technique was employed to estimate pharmacokinetic parameters and an estimate of their variability within the study population. To obtain a more mechanistic understanding of hepatic metabolism in our avian species, a PBPK model was developed for the metabolism of midazolam in each species. This model was optimized for the chicken and then applied to the other species, changing only the body weights and organ volumes as appropriate. The PBPK model as designed for the chicken was surprisingly accurate at predicting midazolam tissue drug concentrations in the other species.
CHAPTER 1 3 Introduction to Comparative Studies of Midazolam Metabolism in Chickens, Turkeys, Ring-necked Pheasants and Bobwhite Quail
4 Background Despite the economic importance of poultry, the increasing number of pet birds being treated in a veterinary setting and the increased number of studies done on environmental contaminants and their effects on wild birds, little is known about avian drug metabolism compared to mammals. Detailed studies to assess efficacy and/or residues in edible tissues in commercially raised minor avian species (species other than chicken or turkey) are rarely carried out due to lack of resources. Currently, there are only two therapeutic drugs approved for use in pheasant and three for use in quail, compared to twenty-six and twenty-eight compounds for broiler chickens and turkeys, respectively (FARAD). Game birds raised for food are routinely treated with therapeutic drugs for which pharmacokinetic data are only available in chickens. With no other information available, dosage regimens and withdrawal times for extra-label drug use in minor species are usually based on the assumption that the minor species' clearances are similar to those in major species. In a clinical setting, pet birds are often treated with drugs approved for mammalian use that may not have been tested in the avian species being treated. There is a serious need for a better understanding of avian metabolic processes to accurately predict drug efficacy and any resulting tissue drug residues. An approach that has proven useful in elucidating the underlying mechanisms of mammalian metabolic processes and predicting toxic responses is the use of physiologically-based pharmacokinetic (PBPK) models. In contrast to classic pharmacokinetic (PK) methods, PBPK models are biologically motivated and consider physiologic, physicochemical and biochemical processes. Developing a PBPK model
5 forces the organization of current knowledge of a compound and/or species and allows for hypothesis testing of proposed mechanisms of an organism's clearance of a drug/toxicant (Conolly and Andersen, 1991). It is especially useful in making predictions between species or routes of exposure (Krishnan and Andersen, 2001). Thus, PBPK models have found extensive use in risk assessment but are only recently finding use to predict drug residues in food animals (Craigmill, 2003; Buur et al., 2006). The predictive power of a PBPK model is dependent upon accurate estimations/assumptions of the various metabolic processes involved. These include those processes that affect delivery of the compound to the organs involved, such as blood flow to the tissues and partition coefficients of a drug for various tissues, as well as the rate constants of the enzymes responsible for the metabolism of the compound. Each tissue and metabolic process is represented by mass balance differential equations, which are then solved simultaneously to approximate chemical uptake, transfer between and within tissues, and metabolism/excretion within an animal. Thus, it is crucial to include both what is known about the compound and the species being modeled and to characterize the physiological, physicochemical and biochemical parameters affecting these processes. For example, Clewell et al. reported that although there are a number of PBPK models for perchlorethylene, only the model that included the kinetics of the principal metabolite trichloroacetic acid accurately predicts urinary excretion of low doses of perchloroethylene in humans (Clewell et al, 2005). However, it is important to balance an accurate description of the biologic and physical processes affecting a compound's metabolism with keeping the model as simple as possible. This ensures that the most important processes are considered and the model remains flexible.
6 A PBPK model for the disposition of midazolam in surgical patients has been reported, based on scaling from rat data and incorporating human physiology, and applied with partial success to individual drug clearance studies in humans (Bjorkman et al, 2001). These authors also applied their model successfully to predict midazolam disposition in infants and children (Bjorkman, 2004). However, the model they developed is fairly complicated and relies on a number of assumptions and detailed individual physiological measurements that are not available for avian species. Yet, for reasons discussed below, the drug midazolam was chosen for the comparative metabolic studies in avians and subsequent PBPK model development presented here. Midazolam as a Model Compound for Hepatic Oxidative Metabolism To make the model as simple as possible and applicable to a wide range of compounds, a model compound was chosen with straightforward metabolism. Midazolam is a benzodiazepine used clinically in human and veterinary medicine (including avian medicine) to induce sedation. It is the focus of the current study for two reasons. First, in mammals midazolam is cleared almost exclusively by hepatic metabolism, (oxidation by cytochrome P450 enzymes followed by glucuronidation) which makes it a good candidate for studying the metabolic processes of the liver in avians. Second, midazolam is a prototypical cytochrome P450 3A (CYP3A) marker substrate in mammals (Kronbach et al, 1989; Kobayashi et al, 2002), which may allow us some insight into this enzyme system in avians. A majority of therapeutic drugs, including macrolide antibiotics, as well as other compounds such as imidazole fungicides, are CYP3A substrates. This has implications not only for poultry but for pet
7 birds in a clinical setting and wild bird populations as well. In addition, both in vitro and in vivo kinetic data are available for midazolam in rats and humans, and tissue partition coefficients are available for rats. Midazolam (MDZ) undergoes oxidative metabolism via CYP 3A in the liver to the 1-hydroxy, 4-hydroxy or 1,4-dihydroxy metabolites (minor), which are then conjugated with glucuronic acid in the liver and excreted in bile or in the urine (Fig. 1). In pharmacokinetic studies with radioactively labeled drug, urinary excretion of conjugated drug accounted for approximately 90%, 30-40% and 10% of total radioactivity in humans, dogs and rats, respectively (Heizmann and Ziegler, 1981; Woo etal, 1981). Midazolam has been reported to be specific, though not selective, for the CYP3A isoform in humans (Kronbach et al, 1989). However, there is a small contribution from CYP2C11 to midazolam metabolism in rats (4-OH MDZ more so than 1-OH MDZ) and mice (Perloff et al, 2000; Kobayashi et al, 2002). While this has not been confirmed in avian species it is a logical place to start. The plasma half-life of the parent compound given intravenously range from 0.67, 0.9 to 1.6-3 hours in rat, dog and human, respectively (Vree et al, 1981; Mandema et al, 1992; Ma and Lau, 1996; Schwagmeier et al, 1998). The alpha-hydroxymethyl metabolite is pharmacologically active although its lower level of activity (5%) and short half-life (11 minutes) are not thought to contribute much to midazolam's overall pharmacologic effect (Pieri, 1983). The rat microsomal Vmax and apparent Km values for the formation of the 4-hydroxy metabolite (major) are 5.9 nmol min"1 mg protein"1 and 24.5uM and for the alpha-hydroxy metabolite (minor) are 2.0 nmol min"1 mg protein"1 and 32.3uM (Ghosal et al, 1996).
Midazolam 1 -hydroxymidazolam 4-hydroxymidazolam glucuronide glucuronide Figure 1. Midazolam metabolism.
9 Reports of microsomal metabolism to the 1-hydroxy metabolite vary widely for humans (190-4380 pmoles/mg protein*min) and are somewhat more consistent for rats, dogs and monkeys (2000, 1110 and 2481 pmoles/mg protein*min, respectively) (Sharer et al, 1995; Ghosal et al, 1996). The reported Km values for the formation of 1- hydroxymidazolam in humans are 2.5-8.6 uM compared to 32.3 uM for rats, which reflects the fact that the 1-hydroxy metabolite is the major metabolite in humans (Kronbach et al, 1989; Sharer et al, 1995; Ghosal et al, 1996; Perloff et al, 2000) Investigating the metabolism of this compound in chickens, turkeys, pheasant and bobwhite quail, for the purpose of building and validating a predictive model, has yielded comparative data on several levels of organization. Evidence for CYP3A Existence and Activity in Poultry Species The majority of work concerning avian metabolism has been done to understand the hepatic biotransformation of environmental contaminants, not therapeutic compounds (Giorgi et al, 2000), (Kennedy et al, 1996), (Riviere et al, 1985), (Ronis et al, 1994). Many recent studies on avian phase I metabolic enzymes involve work with chicken embryos (Sinclair and Sinclair, 1993), (Ratz et al, 1997). This is because the chicken is an inexpensive model to study both the aryl hydrocarbon (Ah) receptor and the human disease porphyria. In addition, much of the research in avian toxicology focuses on the CYP1A family because of its critical role in the metabolism of environmental toxicants and the implications to wild bird populations (Ronis and Walker, 1989). To date, no game bird isozymes have been identified and only one turkey (1A5) and five specific chicken P450 isozymes (1A4,1A5,2F1, 2F2 and 3A37) have been identified and
10 sequenced (Rifkind et al. 1994, Hobbs et al. 1986, Hansen and May 1989, Gupta et al. 1990, Ourlin et al. 2000,Yip and Coulombe, 2006). In humans approximately 50% of therapeutic drugs are metabolized by the CYP3A family (Hardman, 2001) but this has not yet been adequately studied in avians. Macrolide antibiotics and imidazole fungicides are also CYP3A substrates, which has implications not only for poultry but for pet birds in a clinical setting and wild bird populations as well. The presence of CYP3A has been confirmed in the chicken and there is supportive evidence for its existence in other poultry species. An isoform of CYP3A, designated CYP 3A37, has been cloned and functionally expressed in the chicken (Ourlin et al, 2000). This isoform shares 57-62% amino acid sequence identity with mammalian CYPs in the 3A family and appears to be regulated in a similar manner with induction of the gene and subsequent increases in mRNA leading to increased enzyme activity (Ourlin et al, 2000). Chicken CYP3A37 is induced by the classic mammalian CYP3A inducers metyrapone, dexamethasone and pregnenolone-16a- carbonitrile but not by rifampicin (Ourlin et al, 2000). Recently, induction of CYP3A37 by phenobarbital was demonstrated in one-week old chicks of different strains (Goriya et al, 2005). Other studies have demonstrated cytochrome P450 3A-like activities in chickens, turkeys, pheasants and bobwhite quail using compounds known to be metabolized by CYP3A in mammalian species. Chicken hepatic microsomes have shown activity toward erythromycin and triacetyloleandomycin (Nebbia et al, 2001) and turkey hepatic microsomes have been reported to show nifedipine oxidase activity comparable to humans (Klein et al, 2000). Ring-necked pheasants show hepatic microsomal activity
11 toward erythromycin but this activity is not induced by dexamethasone (in contrast to chickens) or triacetyloleandomycin (a potent CYP3A inducer in rodents) (Lorr et al, 1989; Giorgi et al, 2000). Ronis et al. noted that while clotrimazole is a potent inducer of CYP3A in rats, as judged from erythromycin N-demethylase activity and induction of a protein recognized by rabbit anti-rat CYP3A2, it has no effect in bobwhite quail (Ronis et al, 1994). Thus, there appears to be a CYP3A-like enzyme present in all four of these closely related species but it may be differentially regulated. Other supportive evidence for the existence of CYP3A enzymes in avian species is their detection using antibodies raised against CYP3A enzymes from mammalian species. Proteins have been detected in chickens that cross-react to antibodies raised in sheep against rabbit CYP3A4 and raised in rabbit against rat CYP3A2 (Lorr et al, 1989; Ronis et al., 199'4; Coulet et al, 1996). It is difficult to determine how similar poultry species are with regard to CYP3A capabilities as none of the studies reported to date directly compared all four species using the same substrate. The current study compared the in vitro hepatic microsomal metabolic capabilities of avian food species with regard to a compound known to be a marker substrate for CYP3A in mammals, midazolam (MDZ). Midazolam is a benzodiazepine that undergoes oxidative metabolism via cytochrome P450's in the liver to the 1-hydroxy, 4-hydroxy or 1,4-dihydroxy metabolites (minor), which are then conjugated with glucuronic acid and excreted. Another reason for choosing midazolam is that it is used to sedate wild quail for short-term handling (Day and Roge, 1996) and to anesthetize or stop seizures in pet birds in clinical medicine settings (Forbes, 1998;
12 Machin and Caulkett, 1998). Thus, information gained from this study may be useful to a wide range of researchers/clinicians. Specific Objectives Therapeutic drugs approved for use in major food species (chicken and turkey) are often used in an extralabel manner to treat diseases in minor species (pheasant and bobwhite quail). Detailed studies to assess efficacy and/or residues in edible tissues in minor species are rarely carried out due to lack of resources. With no other information available, dosage regimens and withdrawal times in minor species are usually based on the assumption that the minor species' clearances are similar to those in major species. The goal of this research was to develop a PBPK model that accurately predicts the in vivo pharmacokinetics of midazolam in chickens as a model for hepatic drug clearance and to extend this model to other similar species. In the process, the research would enhance our knowledge of avian hepatic metabolism, both in vitro and in vivo, and provide comparative information with rodents and with other galliform species (turkeys, pheasant, bobwhite quail). A successful model will provide a good starting point for predicting pharmacokinetic profiles of similarly metabolized drugs in the chicken, and perhaps, in other avian species. The goals of this research were therefore to: 1: Establish in vitro Michaelis-Menten rate constants for metabolism of midazolam for use in the PBPK model and for comparative purposes. 2: Develop a PBPK model for the pharmacokinetics of midazolam in chickens. 3: Validation of the PBPK model using data obtained from in vivo pharmacokinetic studies of midazolam in chickens, turkeys, pheasant and bobwhite quail.
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17 CHAPTER 2 Hepatic Microsomal Pharmacokinetic and Inhibition Studies with Prototypical CYP3A Substrates and Inhibitors thereof1 1 This manuscript has been published: Cortright, K.A. & Craigmill, A.L. (2006) Cytochrome P450- dependent metabolism of midazolam in hepatic microsomes from chickens, turkeys, pheasant and bobwhite quail. Journal of Veterinary Pharmacology and Therapeutics, 29, 468-476.
18 ABSTRACT In vitro putative cytochrome P450 3A mediated activity, and inhibition thereof, were measured in four avian species using midazolam as a substrate and ketoconazole as an inhibitor. All species produced 1-hydroxymidazolam (1-OH MDZ) to a much greater extent than 4-hydroxymidazolam (4-OH MDZ). Calculated Vmaxapparent values for formation of 1-OH MDZ were 117 ± 17, 239 ± 108, 437 + 168, and 201 ± 55 pmol/mg protein*min and Kmapparent values were 2.1 + 0.8, 2.4 + 1.6, 6.7 + 5.1 and 3.2 + 2.1 uM for chicken, turkey, pheasant and bobwhite quail, respectively. For the formation of 4- OH MDZ the Vmaxapparent values were 21 + 10, 94 + 46, 144 + 112, and 68 + 30 pmol/mg protein*min and Kmapparent values for 4-OH MDZ formation were 12.4 +10.1, 18.0 + 10.8, 38.6 + 34.7 and 29.1 + 10.1 uM for chicken, turkey, pheasant and bobwhite quail, respectively. In all four species, ketoconazole inhibited the production of both major metabolites of midazolam, with 4-OH MDZ formation more sensitive to inhibition than 1-OH MDZ. Pheasant and bobwhite quail appeared most sensitive to ketoconazole inhibition.
19 INTRODUCTION Comparatively little is known about the ability of commercially raised poultry and gamebirds to metabolize therapeutic drugs as compared to other species. In particular, their ability to metabolize substrates via oxidative pathways, such as those mediated by the family of enzymes known as the cytochrome P450 enzymes, is not as well understood. A need for basic research in this area stems from real-world situations in which poultry are routinely exposed to feed additives and therapeutic drugs, many of which are metabolized via these enzymes. Species differences in drug metabolism affect not only therapeutic efficacy but also drug residues of concern to consumers. The current study was undertaken to obtain an estimation of the putative cytochrome P450 3 A (CYP3 A) activities in these species using a marker drug. In humans approximately 50% of therapeutic drugs are metabolized by the CYP3A family (Hardman, 2001) and this may be true in avian species as well. The presence of CYP3A has been confirmed in the chicken and there is supportive evidence for its existence in other poultry species. An isoform of CYP3A, designated CYP 3A37, has been cloned and functionally expressed in the chicken (Ourlin et al, 2000). In addition, the chicken genome was recently sequenced, which should help identify more avian cytochrome P450 isoforms (Hillier et al, 2004). Previous studies have demonstrated cytochrome P450 3A-like activities in chickens, turkeys, pheasants and bobwhite quail using compounds known to be metabolized by CYP3A in mammalian species (Ronis et al, 1994; Giorgi et al, 2000; Klein et al, 2000; Nebbia et al, 2001). Another piece of supportive evidence for the existence of CYP3A enzymes in avian