Recommended conditions and inhibitors useful in identifying the role of drug-metabolizing enzymes
| In Vitro System | Inhibitor | Recommended Conditions | Tests |
|---|---|---|---|
| HLM | +/− NADPH | P450, FMO versus other oxidases | |
| HLM, hepatocytes | +/− 1-Aminobenzotriazolea | 1 mM ∼30-min pretreatment at 37°C | 1-ABT is a broad specific mechanism-based inactivator of most cytochrome P450s |
| HLM | ∼2-min pretreatment at 45°C | Inactivates FMO | |
| HLM, hepatocytes | +/− Chlorgyline | 0.5–1 μM ∼10-min pretreatment at 37°C | Potent and selective for MAO-A versus MAO-B; will also inhibit P450s: 2D6 > 1A2 > 2C19 > 3A |
| +/− Deprenyl | 0.5–1 μM ∼10-min pretreatment at 37°C | Potent and selective for MAO-B versus MAO-A; little to no effect on cytochrome P450s | |
| S9, hepatocytes, cytosol | +/− 4-Methylpyrazole | 0.1–1.0 μM | Alcohol dehydrogenase |
| S9, hepatocytes | +/− Hydralazine | 25–50 μM | Aldehyde oxidase |
| Cytosol | +/− Raloxifene | 100 nM | Aldehyde oxidase |
| HLM | +/− BNPP, bis(4-nitrophenyl) phosphate | <1 μM | Carboxylesterase 1 and 2 |
| HIM | +/− Loperamide | ∼1 μM | Carboxylesterase 2 |
| S9, HLM, cytosol | +/− Ascorbate | 100 μM | Peroxidases |
| Cytosol | +/− Allopurinol | 100 μM | Xanthine oxidase |
FMO, flavin monooxygenase; HIM, human intestinal microsomes; MAO, monoamine oxidase.
↵a 1-Aminobenzotriazole (1-ABT) is generally considered to be a nonselective mechanism-based inactivator of human cytochrome P450 enzymes. Whereas the activities of P450s 2A6 and 3A4 are essentially eliminated upon 30-minute pretreatment with 1-ABT, the other human P450s are less affected, with at least 20% activity remaining after pretreatment, with the exception of CYP2C9, with roughly 60% activity remaining after pretreatment. This demonstrates that one should be cautious when using 1-ABT as a nonselective P450 inhibitor in vitro and not automatically assume any remaining metabolic activity being non-P450–mediated after 1-ABT pretreatment (Linder et al., 2009).