Cytochrome P450 Pig Liver Pie: Determination of Individual Cytochrome P450 Isoform Contents in Microsomes from Two Pig Livers Using Liquid Chromatography in Conjunction with Mass Spectrometry

Abstract

ABSTRACT:The cytochrome P450 (P450) family of enzymes is a major player in the metabolism of therapeutic drugs available on the market, and the development of novel drugs has to take into account these enzymes in the fate of new drugs. Testing the pharmacokinetic behavior of new drugs in animals is a common part of the drug development process. Pigs are increasingly used for this purpose because of their similarity of enzymatic pattern to humans. In this study, adult Suffolk White pig liver microsomal samples were analyzed using mass-spectrometry-based techniques to identify and relatively quantify the porcine hepatic P450 enzymes. The total corrected microsomal protein content (milligrams of protein per gram of liver tissue) was estimated at 32.6 and 36.2 mg/g liver tissue in two samples, and the main identified liver P450 subfamilies were CYP1A, CYP2A, CYP2C, CYP2D, CYP2E, and CYP3A.Label-free quantification was performed using the exponentially modified protein abundance index, and the highest abundance enzymes were CYP2A19 at 34% and CYP2D25 at 26% of the total identified drug-metabolizing P450 enzymes. The highest abundance subfamilies were CYP2A (34%), CYP2C (16%), CYP2D (26%), and CYP3A (14%). Moreover, primary sequence alignment was used to identify human homologs of the identified porcine P450s. Porcine CYP1A2 and CYP2E1 were shown to be equivalent to human CYP1A2 and CYP2E1, respectively. Porcine CYP2A19 has the highest sequence homology to human CYP2A6 and CYP2A13, and pig CYP2C33v4 and CYP2C49 are the porcine equivalent of human CYP2C9 and CYP2C18, respectively. Both identified pig CYP3A enzymes (CYP3A29 and CYP39) were highly homologous to CYP3A4/5.

Simultaneous pharmacokinetics assessment of caffeine, warfarin, omeprazole, metoprolol, and midazolam intravenously or orally administered to Microminipigs

“…2) in vitro findings using liver microsomes from male Microminipigs. Further studies could be useful, such as a follow up to a preliminarily report of liver P450 2D content in pigs (Achour, et al, 2011). In conclusion, the results of this study suggest that the Microminipig is a suitable animal model for use in biological experiments for comparisons of pharmacokinetics of drugs in humans.…”

Choice of LC-MS Methods for the Absolute Quantification of Drug-Metabolizing Enzymes and Transporters in Human Tissue: a Comparative Cost Analysis

“…The cost values in this paper are based on the quantification of the abundance of cytochrome P450 (P450) and uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes and drug transporters in human tissue with QconCAT, AQUA, and label-free methods (7,(25)(26)(27)(28)(29)(30)(31)(32). The cost of comprehensive Fig.…”

Pigs in Toxicology

“…Porcine CYP2A19 represents 34% of hepatic CYP in the pig and has 90.1% DNA sequence homology to human CYP2A13 and 87.2% homology to human CYP2A6 (Achour, Barber, and Rostami-Hodjegan 2011). Porcine CYP2A19 is 99% homologous between Göttingen and conventional breed pigs.…”

“…In humans, there are high interindividual variances in CYP2D due to polymorphisms. CYP2D25 comprises 25.5% of hepatic CYP in the pig and is 77.6% homologous to human CYP2D6 (Achour, Barber, and Rostami-Hodjegan 2011). CYP2D has not been fully examined in the pig, and many of the substrates that are specific for human CYP2D are also metabolized by porcine CYP2B (Skaanild 2006).…”

“…Porcine CYP2C33v4 has only 62.6% homology to human CYP2C9 (Achour, Barber, and Rostami-Hodjegan 2011). Porcine CYP2C enzymes show some cross-reactivity toward many human test substrates, but not those specific for human CYP2C, making extrapolation between pigs and humans for CYP2C difficult (Skaanild 2006;Skaanild and Friis 2008;Thorn 2012).…”