Recommended conditions and inhibitors useful in identifying the role of drug-metabolizing enzymes

In Vitro SystemInhibitorRecommended ConditionsTests
HLM+/− NADPHP450, FMO versus other oxidases
HLM, hepatocytes+/− 1-Aminobenzotriazolea1 mM ∼30-min pretreatment at 37°C1-ABT is a broad specific mechanism-based inactivator of most cytochrome P450s
HLM∼2-min pretreatment at 45°CInactivates FMO
HLM, hepatocytes+/− Chlorgyline0.5–1 μM ∼10-min pretreatment at 37°CPotent and selective for MAO-A versus MAO-B; will also inhibit P450s: 2D6 > 1A2 > 2C19 > 3A
+/− Deprenyl0.5–1 μM ∼10-min pretreatment at 37°CPotent and selective for MAO-B versus MAO-A; little to no effect on cytochrome P450s
S9, hepatocytes, cytosol+/− 4-Methylpyrazole0.1–1.0 μMAlcohol dehydrogenase
S9, hepatocytes+/− Hydralazine25–50 μMAldehyde oxidase
Cytosol+/− Raloxifene100 nMAldehyde oxidase
HLM+/− BNPP, bis(4-nitrophenyl) phosphate<1 μMCarboxylesterase 1 and 2
HIM+/− Loperamide∼1 μMCarboxylesterase 2
S9, HLM, cytosol+/− Ascorbate100 μMPeroxidases
Cytosol+/− Allopurinol100 μMXanthine 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).