Elke Gasthuys

The Potential Use of Piglets as Human Pediatric Surrogate for Preclinical Pharmacokinetic and Pharmacodynamic Drug Testing

Pediatric drug research is still substandard, not reaching the same quality level as adult drug research. Despite the efforts made by the Food and Drug Administration and European Medicines Agency to reduce off-label use in children, the lack of clinical studies involving the pediatric population still stands. Pharmacokinetic and pharmacodynamics studies (PK/PD) taking growth and maturation into account are necessary to rationalize dosing strategies in children. Currently, traditional animal models such as rats, mice, dogs and primates are used to conduct pharmacokinetic and pharmacodynamic studies, however age-related trials are rather uncommon. Moreover, these species have several shortcomings as animal models, such as a different physiology and anatomy of the gastrointestinal tract in dogs or the ethical aspects for the use of primates. In contrast, piglets might be potential biomedical pediatric animal models because of the good resemblance with humans, anatomically, physiologically and biochemically. This review summarizes the comparative anatomy and physiology and postnatal development of piglets and infants, focusing on six major topics, namely growth, cardiovascular system, gastrointestinal tract, liver, kidney and integument. Furthermore, the application of piglets as animal model in pediatric PK/PD research is discussed.

Postnatal Maturation of the Glomerular Filtration Rate in Conventional Growing Piglets As Potential Juvenile Animal Model for Preclinical Pharmaceutical Research

Adequate animal models are required to study the preclinical pharmacokinetics (PK), pharmacodynamics (PD) and safety of drugs in the pediatric subpopulation. Over the years, pigs were presented as a potential animal model, since they display a high degree of anatomical and physiological similarities with humans. To assess the suitability of piglets as a preclinical animal model for children, the ontogeny and maturation processes of several organ systems have to be unraveled and compared between both species. The kidneys play a pivotal role in the PK and PD of various drugs, therefore, the glomerular filtration rate (GFR) measured as clearance of endogenous creatinine (Jaffe and enzymatic assay) and exo-iohexol was determined in conventional piglets aging 8 days (n = 16), 4 weeks (n = 8) and 7 weeks (n = 16). The GFR data were normalized to bodyweight (BW), body surface area (BSA) and kidney weight (KW). Normalization to BSA and KW showed an increase in GFR from 46.57 to 100.92 mL/min/m2 and 0.49 to 1.51 mL/min/g KW from 8 days to 7 weeks of age, respectively. Normalization to BW showed a less pronounced increase from 3.55 to 4.31 mL/min/kg. The postnatal development of the GFR was comparable with humans, rendering the piglet a convenient juvenile animal model for studying the PK, PD and safety of drugs in the pediatric subpopulation. Moreover, to facilitate the assessment of the GFR in growing piglets in subsequent studies, a formula was elaborated to estimate the GFR based on plasma creatinine and BW, namely eGFR =1.879 × BW1.092/Pcr0.600.

The ontogeny of cytochrome P450 enzyme activity and protein abundance in conventional pigs in support of preclinical pediatric drug research

Since the implementation of several legislations to improve pediatric drug research, more pediatric clinical trials are being performed. In order to optimize these pediatric trials, adequate preclinical data are necessary, which are usually obtained by juvenile animal models. The growing piglet has been increasingly suggested as a potential animal model due to a high degree of anatomical and physiological similarities with humans. However, physiological data in pigs on the ontogeny of major organs involved in absorption, distribution, metabolism, and excretion of drugs are largely lacking. The aim of this study was to unravel the ontogeny of porcine hepatic drug metabolizing cytochrome P450 enzyme (CYP450) activities as well as protein abundances. Liver microsomes from 16 conventional pigs (8 males and 8 females) per age group: 2 days, 4 weeks, 8 weeks, and 6–7 months were prepared. Activity measurements were performed with substrates of major human CYP450 enzymes: midazolam (CYP3A), tolbutamide (CYP2C), and chlorzoxazone (CYP2E). Next, the hepatic scaling factor, microsomal protein per gram liver (MPPGL), was determined to correct for enzyme losses during the fractionation process. Finally, protein abundance was determined using proteomics and correlated with enzyme activity. No significant sex differences within each age category were observed in enzyme activity or MPPGL. The biotransformation rate of all three substrates increased with age, comparable with human maturation of CYP450 enzymes. The MPPGL decreased from birth till 8 weeks of age followed by an increase till 6–7 months of age. Significant sex differences in protein abundance were observed for CYP1A2, CYP2A19, CYP3A22, CYP4V2, CYP2C36, CYP2E_1, and CYP2E_2. Midazolam and tolbutamide are considered good substrates to evaluate porcine CYP3A/2C enzymes, respectively. However, chlorzoxazone is not advised to evaluate porcine CYP2E enzyme activity. The increase in biotransformation rate with age can be attributed to an increase in absolute amount of CYP450 proteins. Finally, developmental changes were observed regarding the involvement of specific CYP450 enzymes in the biotransformation of the different substrates.

Population Pharmacokinetic Modeling of a Desmopressin Oral Lyophilisate in Growing Piglets as a Model for the Pediatric Population

Desmopressin is used to treat primary nocturnal enuresis in children. Over the years, various formulations of desmopressin were commercialized of which the sublingual melt tablet is preferred in the pediatric population, despite the lack of full PK studies in this population. A full PK study was performed in growing conventional piglets to evaluate if this juvenile animal model can provide supplementary information to complement the information gap in the pediatric population. A desmopressin sublingual melt tablet (120 μg) was administered to 32 male piglets aged 8 days, 4 weeks, 7 weeks, and 6 months (each group n = 8). Population PK (pop-PK) analysis was performed to derive the PK parameters, the between- and within-subject variabilities and the effects of covariates. Desmopressin demonstrated two-compartmental PK, with a dual, sequential absorption process, and linear elimination. Body weight was the only significant covariate on clearance and on apparent volume of distribution of the central compartment. In human pediatric trials, no double peak in the absorption phase was observed in the plasma concentration-time curves, possibly due to the sparse sampling strategy applied in those pediatric studies. Therefore, it is recommended to perform additional studies, based on the sampling protocol applied in the current study.

A novel approach in paediatric drug design : the conventional pig as juvenile animal model

Background To date, the paediatric subpopulation is often neglected during drug development. The main reasons are limited economic profit of drugs adapted to children, ethical concerns for performing paediatric clinical trials and lack of appropriate preclinical animal models and methodologies, taking maturation and metabolic de-velopment into account. Lack of clinical trials and conse-quently the lack of paediatric formulations frequently leads to off-label use of drugs in the paediatric subpopulation, which may lead to inappropriate dosage regimens and/or increased toxicity (Kimland et al, 2012). Since children are no small adults, extrapolation from adult clinical trials is not recommended. Therefore other strategies, such as suited animal models taking growth and maturation into account, should be investigated. Traditional animal mod-els including rodents, dogs and non-human primates, have already been explored, but seem to be insufficient due to either differences in physiology and ADME pro-cesses or ethical concerns. The aim of the present study was to determine whether the conventional pig could be a feasible juvenile animal model to study the pharma-cokinetic processes of drugs, since its striking anatomi-cal and physiological resemblances with humans. More specifically, the ontogeny of the glomerular filtration rate (GFR) and cytochrome P450 (CYP450) liver enzymes was assessed and compared to human maturation data. Methods An extensive literature search was performed based on the comparative anatomy and physiology of pigs and humans. The main focus of this meta-analysis was growth and ontogeny of the major organ systems involved in the pharmacokinetic processes of drugs, namely gastro-intestinal tract, liver and kidney. The GFR of conventional pigs was determined in four age categories using three different techniques, namely creatinine clear-ance in plasma and urine determined with Jaffe reaction and enzymatic method, and clearance of exo-iohexol. The ontogeny of the CYP450 enzymes was determined by in vitro activity experiments in liver microsomes of the same age categories next to the determination of the amount of CYP proteins by high definition data directed analysis (HD-DDA) mass spectrometry. Results Literature reports demonstrated that devel-opmental variability in ADME processes was most pronounced at birth and neonatal stage of life. The piglet might be a more appropriate juvenile animal model for PK studies when reaching infancy. An easy-to-apply creatinine equation was developed to estimate the GFR in growing piglets and to provide a useful tool in preclinical porcine studies. Furthermore, the maturation profile of GFR in pig-lets was comparable to humans. The in vitro metabolic capacity of the CYP enzymes increased with age which is probably due to maturation of the enzymes itself as well as to an increase in absolute amount of CYP proteins. Conclusion These data supports the use of the conven-tional pig as juvenile animal model, although additional studies are required to fully elucidate the suitability of the piglet preclinical animal model.

The ontogeny of hepatic CYP3A, CYP2C and CYP2E enzyme activity in conventional pigs

Introduction: The anatomy and physiology of pigs is closely related to human characteristics. Therefore, the use of pigs as an animal model to study the pharmacokinetic (PK) behavior of drugs in therapeutic subpopulations, including pediatrics, could be of interest. One of the key PK processes, biotransformation, is primarily mediated by the cytochrome P450 (CYP) enzyme system. Literature reports have demonstrated a high homology between human and porcine CYP3A, 2C and 2E in adults, namely at least 75, 62 and 79% amino acid sequence identity, respectively. However, data regarding the ontogeny of porcine CYP enzymes are lacking. Therefore, in order to assess whether piglets might serve as a model for pediatric PK studies, knowledge regarding the ontogeny of the CYP enzymes in pigs is mandatory. Materials and methods: Liver samples were collected immediately after euthanasia from 16 pigs (8 males and 8 females, Hybrid sow x Pietrain boar) of different ages (2 days, 4 and 8 weeks, 6 months-old). Samples were snap-frozen and stored at <-80°C until analysis. Microsomes were prepared by a differential centrifugation method. Midazolam, tolbutamide and chlorzoxazone probe drugs were used to determine the in vitro CYP3A, 2C and 2E catalytic activity, respectively. The corresponding metabolites, namely 1-hydroxy-midazolam, 4-hydroxy-tolbutamide and 6-hydroxy-chlorzoxazone, were quantified using a validated UHPLC-MS/MS method (1). Furthermore, the microsomal protein per gram of liver was determined as it is an important scaling factor in the extrapolation of the obtained in vitro enzyme activities to in vivo (2). Results and conclusions: The biotransformation of midazolam, tolbutamide and chlorzoxazone increased with age. The mean (±SD) CYP3A activity was 60.5 (±45.7) and 83.3 (±20.7) pmol/min/mg protein at the age of 2 days, 971.1 (±367.8) and 1072.7 (±371.7) pmol/min/mg protein at the age of 4 weeks and 723.4 (±146.3) and 1134.7 (±282.6) pmol/min/mg protein at 8 weeks of age for the barrows and sows, respectively. CYP2C activity at the same ages increased from 20.1 (±12.3) and 29.1 (±18.5) to 78.3 (±25.6) and 106.7 (±69.1) and 103.5 (±39.6) and 170.2 (±71.9) pmol/min/mg protein, while the activity of CYP2E was 539.3 (±251.0) and 643.3 (±220.3), 747.7 (±134.8) and 948.9 (±246.2) and 957.9 (±221.7) and 1549.8 (±345.0) pmol/min/mg protein, respectively for the barrows and sows. Significant sex differences (P<0.05) were only observed at 8 weeks of age. These data show similar trends with human CYP ontogeny.