Biochemical pharmacology

Allosteric activation of midazolam CYP3A5 hydroxylase activity by icotinib - Enhancement by ketoconazole.

PMID 27666601


Icotinib (ICO), a novel small molecule and a tyrosine kinase inhibitor, was developed and approved recently in China for non-small cell lung cancer. During screening for CYP inhibition potential in human liver microsomes (HLM), heterotropic activation toward CYP3A5 was revealed. Activation by icotinib was observed with CYP3A-mediated midazolam hydroxylase activity in HLM (∼40% over the baseline) or recombinant human CYP3A5 (rhCYP3A5) (∼70% over the baseline), but not in the other major CYPs including rhCYP3A4. When co-incubated with selective CYP3A4 inhibitor CYP3cide or monoclonal human CYP3A4 inhibitory antibody in HLM, the activation was extended to ∼60%, suggesting CYP3A5 might be the isozyme involved. Further, the relative activation was enhanced to ∼270% in rhCYP3A5 in the presence of ketoconazole. The activation was substrate and pathway dependent and observed only in the formation of 1'-OH-midazolam, and not 4-OH-midazolam, 6β-OH-testosterone, or oxidized nifedipine. The activation requires the presence of cytochrome b5 and it is only observed in the liver microsomes of dogs, monkeys, and humans, but not in rats and mice. Kinetic analyses of 1'-OH-midazolam formation showed that ICO increased the Vmax values in HLM and rhCYP3A5 with no significant changes in Km values. By adding CYP3cide with ICO to the incubation, the Vmax values increased 2-fold over the CYP3cide control. Addition of ketoconazole with ICO alone or ICO plus CYP3cide resulted in an increase in Vmax values and decrease in Km values compared to their controls. This phenomenon may be attributed to a new mechanism of CYP3A5 heterotropic activation, which warrants further investigation.

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