Practical Tips

1. Studies are usually performed at 1 mM, but once the enzyme(s) involved has been identified, performing the incubations at multiple concentrations is useful for determining the role of enzymes with different Km and Vmax.

2. In vitro and in vivo metabolite identification are necessary parts of reaction phenotyping to ensure that the right enzymes are being examined.

3. Typically performed monitoring drug disappearance from the incubation. When CLint is low, it is important that the metabolites are monitored.

The rate of metabolism is how fast a compound is metabolized.

The extent of metabolism is the contribution of metabolism by a certain pathway to the overall CL.

So a compound can have a slow rate of metabolism, yet the extent of metabolism can be high.

5.6.1.1 P450-Based Reaction Phenotyping

Even though reaction phenotyping can be conducted for various enzymes, these studies are most often used for P450 enzymes. For other enzyme systems, the recombinant enzymes and inhibitors that can be used are listed in Chap. 2.

5.6.1.2 Practical Tips

1. 1 -Aminobenzotriazole (ABT; 1 mM) is preincubated for 15-30 min with liver microsomes (0.5-2 mg/mL) or hepatocytes (0.5-1 x 106 cells/mL) to inactivate P450 activity. The metabolites formed are not P450 dependent.

2. When using recombinant human P450 isoforms, equal concentrations of each isoform are usually used. Based on the quantities of P450 isoforms in an average liver, the contribution of the isoforms can be recalculated (see Table 2.5).

3. Both chemical and antibody selective inhibitors can be used with liver microsomes. Typically, chemical inhibitors are used, perhaps because of their low cost.

4. Correlation analysis studies in multiple liver microsomes with characterized P450 isoform activities are not often conducted.

This is for several reasons, including complications that can occur when multiple enzymes are involved.

In general, any metabolic pathway that contributes to fm < 50% of the CLint of a drug does not contribute to drug interactions with AUC fold change <2 (see Table 5.8).

For polymorphic enzymes that are a major component of the CLint, a clinical study comparing poor and extensive meta-bolizers is required to determine variability in the extent of drug interactions with an inhibitor in different populations.

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