P54 Expression of four porcine CYPs in E. coli and development of fluorogenic CYP inhibition assays.

Michael W. Voice , Cypex Ltd., Dundee, United Kingdom
Gillian M. Macintyre , Cypex Ltd., Dundee, United Kingdom
Michael P. Pritchard , Cypex Ltd., Dundee, United Kingdom
While the pathways of xenobiotic metabolism in humans are well characterized, they are less so in animal models used during drug development and in animals of commercial importance. With this in mind, we have, for the first time, expressed four porcine (Sus scrofa) CYPs, CYP1A2, CYP2C49, CYP3A39 and CYP2E1 in E. coli along with porcine NADPH P450 reductase. The activity of these enzymes was investigated using substrates routinely used in human CYP in vitro work. The Km values observed for the substrates tested were as follows, CYP1A2 / 7-ER, 0.03 µM (human 0.3 µM, dog 0.24 µM), CYP2C49 / tolbutamide, 500 µM (human CYP2C9 150 µM, 2C19 200 µM, dog CYP2C41 approx 1700 µM, CYP2C21 not determined), CYP3A39 / testosterone 50 µM, (human CYP3A4 20 µM, CYP3A5 60 µM, dog CYP3A12, CYP3A26 both 50 µM) and CYP2E1 (plus human cytochrome b5) 120 µM, (human with cytochrome b5 45 µM). CYP2C49 did not metabolise diclofenac to the 4’-hydroxy metabolite although an alternative metabolite peak was seen on HPLC analysis of the incubation. The Vmax for testosterone-6b-hydroxylase with CYP3A39 was very low compared to human CYP3A4 and 5 (~30 fold lower). The activity was raised by increasing the relative amount of co-expressed reductase and adding cytochrome b5 to the assay, resulting in a Vmax that was only 3 - 4 fold lower than the equivalent human CYP3A preparations. Subsequently a simple fluorescence based CYP inhibition assay for the four porcine CYPs was developed, based on Cypex’s inhibition screen for human and canine CYPs, which allows comparisons to be made between the three species. Substrates tested included diethoxyfluorescein (DEF), 7-ethoxyresorufin (7-ER), 7-benzyloxyquinoline (7-BQ), 7-methoxy-4-aminomethylcoumarin (MAMC), 7-methoxy-4-trifluoromethylcoumarin (MFC) and 3-cyano-7-ethoxycoumarin (CEC). The final pairings used were CYP1A2 / 7-ER, CYP2C49 / DEF, CYP3A39 / DEF and CYP2E1 / CEC. For each CYP a panel of ten known inhibitors of human CYPs was tested and, for each compound, the IC50 was compared to that obtained with human and canine CYPs (except for CYP2E1 for which we haven’t generated IC50 data for the human and canine ortholog). Differences were seen between the species, for example porcine CYP1A2 was not inhibited by paroxetine while the dog and human orthologs were (IC50 14 µM and 30 µM respectively). So far this work only covers one CYP isoform from each of four CYP families so it is entirely possible that other isoforms behave more like their human orthologs however, the isoforms chosen for expression are the ones that appear to be the most abundant members of their family in porcine liver1. The data highlight the possibility for species differences in metabolism to throw up unexpected results and the use of tools such as the recombinant enzymes described above may help when planning studies to mitigate complications that may arise from such differences.

1 Achour B., Barber J., Rostami_Hodjegan, A Cytochrome P450 Pig Liver Pie: Determination of Individual Cytochrome P450 Isoform Contents in Microsomes from Two Pig Livers Using Liquid Chromatography in Conjunction withMass Spectrometry (2011) Drug Met. Disp. 39(11) p2130-2134