Comparative studies of drug-metabolizing enzymes in dog, monkey, and human small intestines, and in Caco-2 cells.
Drug-metabolizing enzymes were studied in subcellular fractions of dog, monkey, and human small intestines, and in the human adenocarcinoma cell line Caco-2, a commonly used in vitro absorption model. Immunoblot analysis indicated the presence of enzymes related to cytochrome P450 (CYP) 1A1/CYP1A2, CYP2D6, CYP3A, and carboxylesterases (ESs) in human and monkey intestines, and of CYP3A and ES in dog intestines. Catalytically, human and monkey intestines exhibited significant and comparable testosterone 6 beta-hydroxylase, (+)-bufuralol 1'-hydroxylase, and ES activities. In contrast, dog intestine possessed moderate testosterone 6 beta-hydroxylase, much lower ES, and undetectable bufuralol hydroxylase activities. In addition, low tolbutamide methylhydroxylase activity was observed in human and monkey intestines, but not in dog intestines. Of the phase I enzymes investigated, only ES was detected immunologically and functionally in Caco-2 cells. With respect to phase II enzymes, human and monkey intestines contained relatively high intestinal glucuronyltransferase, N-acetyltransferase (NAT), sulfotransferase, and glutathione S-transferase activities. Except for NAT, all phase II enzymes studied were detectable in dog intestines. In Caco-2 cells, acetaminophen sulfation activity was below the limit of detection, whereas all other conjugating activities were evident. Studies of enzyme kinetics and inhibition by known inhibitors of testosterone 6 beta-hydroxylase activity, the major intestinal mono-oxygenase in all species, revealed some similarities between the responsible enzymes. Comparative studies with human liver microsomes suggested the possible involvement of CYP3A enzymes in the intestinal catalysis of testosterone 6 beta-hydroxylation similar to those observed with human hepatic CYP3A. Further studies on ESs, however, revealed multiplicity and species and/or tissue differences in the microsomal and cytosolic enzymes. Based on kinetic studies, monkey intestines and Caco-2 cells possessed NAT activities, with properties similar to those in human intestine and liver. Overall, the results demonstrated that both the preparations of small intestines and Caco-2 cells exhibited significant drug-metabolizing enzyme activities, although several differences were noted between the intestinal enzymes in the animals or in the Caco-2 cells and those found in humans.
Prueksaritanont
T
T
Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA.
Gorham
L M
LM
Hochman
J H
JH
Tran
L O
LO
Vyas
K P
KP
D000328:Adult; D000368:Aged; D000818:Animals; D018938:Caco-2 Cells / Q000187:drug effects / Q000201:enzymology; D019388:Cytochrome P-450 CYP1A2 / Q000032:analysis*; D019389:Cytochrome P-450 CYP2D6 / Q000032:analysis*; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D004285:Dogs; D006801:Humans; D066298:In Vitro Techniques; D007421:Intestine, Small / Q000201:enzymology*; D008253:Macaca mulatta; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D008875:Middle Aged; D013045:Species Specificity; D013250:Steroid Hydroxylases / Q000032:analysis*
D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D019388:Cytochrome P-450 CYP1A2; D019389:Cytochrome P-450 CYP2D6; C022657:steroid hormone 6-beta-hydroxylase