Abstract

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.