Isoforms enzyme induction

A large variety of substances have been shown to be inducers, probably numbering several 100 and including many drugs. Table 5.21 shows some inducers of different CYPs. Some of these inducers will be specific form one CYP isoform, but others will induce a number of different isoforms, complicating the effect on metabolism. Also, although enzyme induction occurs most commonly in the liver, enzymes can be induced in other tissues also, sometimes to a greater extent. 

Besides enzyme activity, enzyme induction assays can also utilize mRNA and enzyme protein level as endpoints. Gene expression studies now can be performed using branch-chained DNA and microarray techniques. Protein level quantification in general is performed using isoform-specific antibodies and Western blotting. Enzyme activity represents the most relevant endpoint for drug-drug interaction evalua-... 

Table 4 Drug metabolizing enzyme (P450 isoforms (CYP), UDP-glucuronosyl transferase (UGT) and phenol sulfotransferase (PST)) substrates that can be used for the in situ measurement of activity in the human hepatocyte enzyme induction assay.

Immortalized hepatocytes A major drawback of the use of primary hepatocytes is that hepatocytes can not be expanded in culture. To overcome this problem, researchers have embarked on the immortalization of primary hepatocyte cultures (e.g. Bayad et al. 1991 Li et al. 2005). Currently, immortalized human hepatocyte cell lines are available commercially and may represent convenient screening experimental systems for enzyme induction studies. Unfortunately, at the time of this writing, there are no peer-reviewed publications on the application of human immortalized hepatocytes in induction studies. It is important to ensure that the induced isoforms in the cell lines are the mature P450 isoforms rather than the embryonic forms. For instance, the use of HepG2 cells may not be appropriate as the embryonic P450 isoforms CYP1A1... 

Interactions. Enzyme induction of CYP 3A, e.g. by rifampicin, reduces the plasma concentration of itraconazole. Additionally, its affinity for several P450 isoforms, notably CYP 3A4, causes it to inhibit the oxidation of a number of drugs, including phenytoin, warfarin, cyclosporine, tacrolimus, midazolam, triazolam, cisapride and terfenidine (see above), increasing their intensity and/or duration of effect. 

The isoform-specific substrates described earlier for CYP inhibition studies are generally used for enzyme induction studies. 

Enzyme induction. Some of the enzymes responsible for biotransformation may be induced by exposure to chemicals and other factors (such as diabetes). Induction requires repeated exposure. If only one isoform of the enzyme (e.g. of cytochrome P-450) is induced the route of metabolism/proportion of metabolites may be changed as well as the rate. The amount of enzyme is increased (and the overall... 

Some commercially available phenotyping kits use an array of coumarin analogs designed to be relatively isozyme-specific substrates (probes) that are metabolized to products with easily measurable spectral characteristics. Other commercially available kits use microsomes from baculovirus-infected cells that overexpress individual human GYP isoforms and fluorescent substrates (Vivid substrates) that can be incorporated into 1536 well formats. These simple systems do not readily lend themselves to the in vitro study of enzyme induction, however. The prediction of xenobiotic alteration of the expression of GYP activity in vivo from in vitro experiments will be discussed more completely in the chapter on drug—drug interactions. 

Inductive interactions can be of a concern with UGTs. Similar to some of the P450 enzymes, the nuclear hormone receptors CAR and PXR regulate induction of UGT1A1, one of the intestinal UGT isoforms and may contribute to... 

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