Caco enzyme activity

Drugs may also undergo hydrolysis by intestinal esterases (hydrolases), more specifically carboxylesterases (EC 3.1.1.1) in the intestinal lumen and at the brush border membrane [58, 59]. It has been shown that intestinal hydrolase activity in humans was closer to that of the rat than the dog or Caco-2 cells [60]. In these studies, six propranolol ester prodrugs and p-nitrophenylacetate were used as substrates, and the hydrolase activity found was ranked in the order human > rat Caco-2 cells > dog for intestinal microsomes. The rank order in hydrolase activity for the intestinal cytosolic fraction was rat > Caco-2 cells = human > dog. The hydrolase activity towards p-nitrophenylacetate and tenofovir disoproxil has also been reported in various intestinal segments from rats, pigs and humans. The enzyme activity in intestinal homogenates was found to be both site-specific (duodenum > jejunum > ileum > colon) and species-dependent (rat > man > Pig)-... 

Intestinal Caco-2 cells provide useful models for assessing the effect of xeno-biotics on enzyme activity. Thus, Faist et al.291 have studied the effect of melanoidins both on a Phase-I enzyme, NADPH cytochrome c reductase, and a Phase-II enzyme, glutathione S-transferase. Where the results achieved a significant level, activity was reduced, thus implying that detoxification would be less efficient in the presence of the melanoidins tested. However, some other results pointed the other way (see Chapter 6). 

When applying any of these models it is crucial to understand the main transport mechanisms as well as the metabolic route and characterization of the activity of the transporter/enzyme involved. It is well recognized that the activities of carrier-mediated processes in Caco-2 cells are considerably lower than in vivo [20, 42, 48] therefore, it is crucial to extrapolate in vitro cell culture data to the in vivo situation with great care [18, 20, 42, 48], This is especially important when carrier-mediated processes are involved, as evidenced by a recent report which showed significant differences in gene expression levels for transporters, channels and metabolizing enzymes in Caco-2 cells than in human duodenum [48], If an animal model is used, then potential species differences must also be considered [18, 20, 45],... 

Cell monolayers grown on permeable culture inserts form confluent mono-layers with barrier properties and can be used for drug absorption experiments. The most well-known cell line for the in vitro determination of intestinal drug permeability is the human colon adenocarcinoma Caco-2 [20, 21], The utility of the Caco-2 cell line is due to its spontaneous differentiation to enterocytes under conventional cell culture conditions upon reaching confluency on a porous membrane to resemble the intestinal epithelium. This cell model displays small intestinal carriers, brush borders, villous cell model, tight junctions, and high resistance [22], Caco-2 cells express active transport systems, brush border enzymes, and phase I and II enzymes [22-24], Permeability models... 

In summary, CACO-2 cells express many enzymes characteristic for intestinal cells involved in drag metabolism. The major drug metabolising enzyme, CYP 3A, is active only in selected clones pointing to polyclonal origin of CACO-2 and the necessity to characterize CACO-2 cells extensively for growth and experimental conditions used for given experiments. 

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