Croubels, Siska

De Bock, Lies

Deforce, Dieter

Devreese, Mathias

Gasthuys, Elke

Millecam, Joske

Van Bocxlaer, Jan

English

Ghent University Academic Bibliography

A NEW APPROACH IN PEDIATRIC DRUG DESIGN: THE MATURATION OF HEPATIC CYTOCHROME P450 ENZYMES IN CONVENTIONAL PIGS USING ENZYME ACTIVITY AND PROTEOMICS1DEPARTMENT OF PHARMACOLOGY, TOXICOLOGY AND BIOCHEMISTRY, FACULTY OF VETERINARY MEDICINE, GHENT UNIVERSITY, MERELBEKE, BELGIUM ; 2DEPARTMENT OF PHARMACEUTICS, FACULTY OF PHARMACEUTICAL SCIENCES, GHENT UNIVERSITY, GHENT, BELGIUM ; 3DEPARTMENT OF BIOANALYSIS, FACULTY OF PHARMACEUTICAL SCIENCES, GHENT UNIVERSITY, GHENT, BELGIUMJoske Millecam1, Elke Gasthuys1, Mathias Devreese1, Dieter Deforce2, Lies De Bock3, Jan Van Bocxlaer3, Siska Croubels1ConclusionBoth the absolute amount of protein and the activity per gram liver shows anincrease with age, suggesting growth and maturation of the CYP450isoenzymes. This maturation of the CYP450 isoenzymes is also observed inhuman, suggesting the piglet might be a suitable animal model for preclinicalPK/PD research.Results01002003004005006000 5 10 15 20 25 301-Hydroxy-midazolam formation (pmol/min/g liver)age (weeks)BIOTRANSFORMATION RATE OF MIDAZOLAMmedian median male median female male female010020030040050060070080090010000 5 10 15 20 25 304-Hydroxy-tolbutamide formation (pmol/min/g liver)age (weeks)BIOTRANSFORMATION RATE OF TOLBUTAMIDEmedian median male median female male female0123456780 5 10 15 20 25 306-Hydroxy-chlorzoxazone formation(nmol/min/g liver)age (weeks)BIOTRANSFOMATION RATE OF CHLORZOXAZONEmedian median male median female male femaleObjectives and methodsDevelopment of appropriate animal models taking growth and maturation into account is pivotal for pediatric preclinical pharmacokinetic and pharmacodynamic (PK/PD) research. Literaturereports have demonstrated a high homology between human and porcine CYP450 enzymes in adults, suggesting the pig as a suited animal model for PK/PD and safety studies (1, 2).However data regarding the ontogeny of porcine hepatic CYP enzymes are lacking. The aim of this research was to gain more insight in the development of the CYP450 enzymes by in vitroenzyme activity experiments and through proteomics.The in vitro CYP450 enzyme activity of the following probe substrates was measured in microsomes: midazolam (CYP3A), tolbutamide (CYP2C) and chlorzoxazone (CYP2E). Themicrosomes were prepared of each time 16 pigs (8 ♂ and 8 ♀, Hybrid sow x Piétrain boar) aging 2 days, 4 weeks, 8 weeks and 6-7 months. The corresponding metabolites were quantifiedusing a validated UHPLC-MS/MS method (3). Furthermore, the microsomal protein per gram liver (MPPGL) was determined (4). In addition to these in vitro activity experiments, the CYPisoenzymes in the same microsomes were determined by high definition data directed analysis (HD-DDA) mass spectrometry. The data analysis was performed using Progenesis QI.A total of 20 CYP isoenzymes were identified of which 12 had 2 or more unique peptides. A pig liver pie out of the average amount of protein for the 6 and 7 months old pigs wascalculated. This was considered to be 100% to recalculate the proportion of the younger age categories as shown above. At puberty significant sex differences (P < 0,05) wereobserved.AcknowledgementsThis study was supported by the “Agency for Innovation by Science and Technology in Flanders (IWT)” (IWT 141427).1. Gasthuys E et al. The Potential Use of Piglets as Human Pediatric Surrogate for Preclinical Pharmacokinetic and Pharmacodynamic Drug Testing. Curr Pharm Des. 2016;22(26):4069-85.2. Gasthuys E et al. Repetitive urine and blood sampling in neonatal and weaned piglets for pharmacokinetic and pharmacodynamic modelling in drug discovery: a pilot study. Lab Anim. 2017:23677217692372.3. De Bock L et al. Development and validation of a fast and sensitive UPLC-MS/MS method for the quantification of six probe metabolites for the in vitro determination of cytochrome P450 activity. Talanta. 2012;89:209-16.4. Guengerich FP et al. Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase. Nature protocols. 2009;4(9):1245-51.ContactJoske.Millecam@UGent.beMathias.Devreese@UGent.beLaboratory of Pharmacology and Toxicology, GLP compliantGhent UniversitySafepedrug.euThe microsomal activity of the three substrates, expressed as pmol metabolite formed per minute and per milligram microsomal protein, increased with age. Significant sexdifferences were observed at 8 weeks of age for the three substrates and at 6 months of age for chlorzoxazone (P < 0,05 ; data not shown). The activity per gram liver, ascalculated with the MPPGL and shown above, also showed a maturation profile. However the sex differences are no longer statistically significant. The increase in microsomalactivity is reflected in an increase in CYP450 proteins in the microsomes.male 2 days male 4 weeks male 8 weeksmale 6 monthsCYP2D6CYP4A21CYP3A46CYP3ACYP1A2CYP2B22CYP2A19CYP2C35CYP2C33v4CYP3A22CYP2C49CYP51A1CYP2E1/1CYP2E1/2CYP2C36CYP20A1CYP2C34female 2 days female 4 weeks female 8 weeks female 7 months

A new approach in pediatric drug design : the development of a pediatric pig model, part II : the maturation of hepatic cytochrome P450 enzymes using enzyme activity and proteomics

https://biblio.ugent.be/publication/8527438/file/8527439

A new approach in pediatric drug design : the development of a pediatric pig model, part II : the maturation of hepatic cytochrome P450 enzymes using enzyme activity and proteomics

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