Primary porcine enterocyte and hepatocyte cultures to study drug oxidation reactions.
1. Primary porcine hepatocytes and enterocytes were isolated and cultured in Williams' E medium for up to 10 days to investigate potential organ differences in the metabolism of the immunosuppressive compound tacrolimus (FK 506) and of two investigational drugs (KC11346 and KC12291). Using LC-MS (FK506) and HPLC-FL (KC 11346/12291) a number of metabolites with identical mass and/or identical retention time could be detected. 2. In the case of tacrolimus hepatocytes and enterocytes produced the same spectrum of metabolites, e.g. bisdemethyl-tacrolimus, demethyl-tacrolimus, demethyl-hydroxy-tacrolimus and hydroxy-tacrolimus, albeit at varying intensities. 3. Treatment of enterocyte cultures with dexamethasone increased the overall metabolite formation very significantly (up to 36 fold). 4. The metabolism of tacrolimus was also studied with preparations of insect cells, that express specifically high levels of individual human cytochrome P450 (CYP) isoenzymes. All metabolites could be generated with microsomal preparations specifically expressing CYP3A4, but hydroxy-tacrolimus was exclusively produced by CYP3A5. 5. In the case of the investigational drugs KC 11346 and KC 12291 only three metabolites were formed by cultured enterocytes whereas hepatocytes produced 10 and 20 metabolites, respectively. 6. When assessed at the protein level CYP1A and CYP3A were expressed in cultures of porcine enterocytes for up to 10 days but porcine hepatocytes expressed additionally CYP2C9/10. 7. In conclusion, primary enterocytes and hepatocytes can be successfully cultured for several days while maintaining mono-oxygenase activity and may therefore be used as a tool for studying intestinal and hepatic metabolism.
Bader
A
A
Leibniz Research Laboratories for Biotechnology and Artificial Organs, Forschungszentrum der MHH, Podbielskistr. 380, D-30659, Hannover, Germany. Augustinus.Bader@GBF.de
Hansen
T
T
Kirchner
G
G
Allmeling
C
C
Haverich
A
A
Borlak
J T
JT
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases; D015415:Biomarkers; D001711:Biotransformation; D015153:Blotting, Western; D002478:Cells, Cultured; D003470:Culture Media; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D020895:Enterocytes / Q000187:drug effects*; D006801:Humans; D007166:Immunosuppressive Agents / Q000378:metabolism / Q000493:pharmacokinetics; D007527:Isoenzymes / Q000378:metabolism; D008099:Liver / Q000166:cytology* / Q000187:drug effects; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D013552:Swine; D016559:Tacrolimus / Q000378:metabolism / Q000493:pharmacokinetics
D000893:Anti-Inflammatory Agents; D015415:Biomarkers; D003470:Culture Media; D007166:Immunosuppressive Agents; D007527:Isoenzymes; D004364:Pharmaceutical Preparations; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D016559:Tacrolimus