Tacrolimus (FK 506) biotransformation in primary rat hepatocytes depends on extracellular matrix geometry.
Established in vitro models for studies of hepatic drug biotransformation include the use of primary hepatocytes. In normal liver the space of Disse provides the possibility of bilateral attachment to extracellular matrix for each hepatocyte. This configuration is disrupted by the cell isolation procedure of normal liver tissue, which delivers suspensions of round shaped cells. In standard culture configurations this unphysiologic cell shape terminates in a morphological dedifferentiation and inability to biotransform drugs. This study analyses the relevance of extracellular matrix geometry in hepatocyte monolayer configurations for expression and activity of cytochrome P450 3A. This enzyme is involved in the biotransformation of a large number of pharmaceuticals including the immunosuppressants tacrolimus and sirolimus. Morphological analysis of primary rat hepatocytes cultured with and without overlay of collagen type I was performed by transmission and scanning electron microscopy. Expression and activity of cytochrome P450 3A was studied by Western blot and the use of two model drugs specific for this enzyme. To this purpose the immunosuppressive drugs tacrolimus and sirolimus were used. Metabolites were analyzed by HPLC and HPLC/MS. Two sided attachment to extracellular matrix induces profound changes of the hepatocellular morphology in vitro resulting in the reconstitution of a polyhedric cell shape. This phenomenon is paralleled by an enhanced expression of cytochrome P450 3A and corresponding metabolic activity. As shown for tacrolimus biotransformation, the model may be useful to study complex metabolic patterns. In addition this model may facilitate studies of the kinetics of hepatocellular drug biotransformation in a setting with prolonged stability.
Bader
A
A
Institut für Allgemeine Pharmakologie, Medizinische Hochschule Hannover, Germany.
Knop
E
E
Böker
K H
KH
Crome
O
O
Frühauf
N
N
Gonschior
A K
AK
Christians
U
U
Esselmann
H
H
Pichlmayr
R
R
Sewing
K F
KF
D000418:Albumins / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001711:Biotransformation; D048429:Cell Size / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000648:ultrastructure; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005109:Extracellular Matrix / Q000648:ultrastructure*; D007166:Immunosuppressive Agents / Q000493:pharmacokinetics*; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D011090:Polyenes / Q000493:pharmacokinetics*; D051381:Rats; D017208:Rats, Wistar; D020123:Sirolimus; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000493:pharmacokinetics*
D000418:Albumins; D007166:Immunosuppressive Agents; D011090:Polyenes; D003094:Collagen; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D020123:Sirolimus; D016559:Tacrolimus