Substrate-dependent modulation of the catalytic activity of CYP3A by erlotinib

Abstract

Aim: To ascertain the effects of erlotinib on CYP3A, to investigate the amplitude and kinetics of erlotinib-mediated inhibition of seven major CYP isoforms in human liver microsomes (HLMs) for evaluating the magnitude of erlotinib in drug-drug interaction in vivo.

Methods: The activities of 7 major CYP isoforms (CYP1A2, CYP2A6, CYP3A, CYP2C9, CYP2D6, CYP2C8, and CYP2E1) were assessed in HLMs using HPLC or UFLC analysis. A two-step incubation method was used to examine the time-dependent inhibition of erlotinib on CYP3A.

Results: The activity of CYP2C8 was inhibited with an IC(50) value of 6.17±2.0 μmol/L. Erlotinib stimulated the midazolam 1'-hydroxy reaction, but inhibited the formation of 6β-hydroxytestosterone and oxidized nifedipine. Inhibition of CYP3A by erlotinib was substrate-dependent: the IC(50) values for inhibiting testosterone 6β-hydroxylation and nifedipine metabolism were 31.3±8.0 and 20.5±5.3 μmol/L, respectively. Erlotinib also exhibited the time-dependent inhibition on CYP3A, regardless of the probe substrate used: the value of K(I) and k(inact) were 6.3 μmol/L and 0.035 min(-1) for midazolam; 9.0 μmol/L and 0.045 min(-1) for testosterone; and 10.1 μmol/L and 0.058 min(-1) for nifedipine.

Conclusion: The inhibition of CYP3A by erlotinib was substrate-dependent, while its time-dependent inhibition on CYP3A was substrate-independent. The time-dependent inhibition of CYP3A may be a possible cause of drug-drug interaction, suggesting that attention should be paid to the evaluation of erlotinib's safety, especially in the context of combination therapy.

Figure 1

Reversible inhibition of CYP3A and CYP2C8 by erlotinib. A1: Inhibition by erlotinib of testosterone 6β-hydroxylation activity. A2: Dixon plot of the inhibitory effect of erlotinib on testosterone 6β-hydroxylation (TS) activity. A3: Lineweaver-Burk plot of the inhibitory effect of erlotinib on testosterone 6β-hydroxylation activity. B1: Inhibition by erlotinib of paclitaxel 6α-hydroxylation activity. B2: Dixon plot of the inhibitory effect of erlotinib on paclitaxel 6α-hydroxylation activity. B3: Lineweaver-Burk plot of the inhibitory effect of erlotinib on paclitaxel 6α-hydroxylation activity. C1: Inhibition by erlotinib of nifedipine oxidation activity. C2: Dixon plot of the inhibitory effect of erlotinib on nifedipine oxidation activity. C3: Lineweaver-Burk plot of the inhibitory effect of erlotinib on nifedipine oxidation activity.

Figure 2

Activation of midazolam 1′-hydroxylation by 1−20 μmol/L erlotinib.

Figure 3

Single point inactivation of CYP3A by erlotinib measured using midazolam, testosterone and nifedipine as probe substrates in HLM. The concentrations for erlotinib were 50 μmol/L, 75 μmol/L and 75 μmol/L when using midazolam, testosterone and nifedipine as probe substrates, respectively. The concentration of the positive control inhibitor TAO was 250 μmol/L. Each data point represents the mean±SD of duplicate incubations.

Figure 4

Time- and concentration-dependent inactivation of CYP3A by erlotinib. (A) At the indicated time points, the remaining CYP3A activity was measured by a midazolam 1′-hydroxylation (A1), testosterone 6β-hydroxylation (A2) or nifedipine oxidation (A3) assay. Each point represents the mean of triplicate incubations. The observed inactivation rate constants, k_obs, were calculated from the slopes of the regression lines in A. (B) The hyperbolic plot of k_obs versus erlotinib concentration was used to calculate kinetic constants.