Drug Metabolism in Drug Design and Development Basic Concepts and Practice

metabolic turnover between individual enzymes, and the identification of

metabolites formed by individual enzymes. Additionally, for P450 enzymes, an

advantage is that the amounts of enzyme on a nanomolar basis within each

incubation can be controlled, which in addition to the relative amounts in the

liver, allow for quantitative predictions to clearance in humans by each P450.

This forms the basis of the ‘‘relative abundancein vitrotoin vivoextrapolation

approach’’ (Rodrigues, 1999). As stated above (Section 15.4.1), similar data for

UGT enzymes is lacking.

Perhaps the most important contribution to error in use of expressed

cytochrome P450s is in the interpretation and extrapolation of generated data.

The increased metabolic rate per milligram protein or per nanomole P450

relative to human liver microsomes can be overinterpreted in some cases

where experimental conditions are not appropriately controlled. For example,

there is potential for false positive results in some cases, where the rates of

metabolic turnover for one enzyme may overestimate the contribution

predicted from a single enzyme, based on subsequent data from other

experimental systems such as human liver microsomes with selective chemical

inhibitors.

15.6.2.3 Subcellular Fractions P450s are located in the endoplasmic

reticulum of the cell, thus the subcellular fraction utilized for P450 reaction

phenotyping is microsomes. Selective P450 inhibitors allow semiquantitation of

the relative contributions of each enzyme to thein vitrometabolism of a test

substance (Bjornsson et al., 2003). Table 15.1 shows a list of preferred

inhibitors of the major human cytochrome P450s––(Tucker et al., 2001). Other

enzymes expressed in human liver microsomes include flavin monooxygenase,

UGTs, glutathione-S-transferases, esterases, microsomal epoxide hydrolase,

and via impurity, measurable levels of monoamine oxidases.

TABLE 15.1 Preferred and accepted inhibitors for P450s (adapted from Tucker
et al., 2001).

P450 Preferred Acceptable

CYP1A2 Furafylline (1–10mM) a-Napthoflavone (can also activate

and inhibit CYP3A4)

CYP2A6 8-methoxypsoralen (20mM) Coumarin,

Sertraline (also inhibits CYP2D6)

CYP2C8 Montelukast (0.2mM)

CYP2C9 Sulfaphenazole (2.5–25mM) Ticlopidine (also inhibits CYP2D6)

Nootkatone (also inhibits CYP2A6)

CYP2C19

CYP2D6 Quinidine (2.5–25mM)

CYP3A4 Ketoconazole (1–2.5mM)

Troleandomycin Cyclosporine

490 REACTION PHENOTYPING