Potent mechanism-based inhibition of CYP3A4 by imatinib explains its liability to interact with CYP3A4 substrates

Abstract

Background and purpose: Imatinib, a cytochrome P450 2C8 (CYP2C8) and CYP3A4 substrate, markedly increases plasma concentrations of the CYP3A4/5 substrate simvastatin and reduces hepatic CYP3A4/5 activity in humans. Because competitive inhibition of CYP3A4/5 does not explain these in vivo interactions, we investigated the reversible and time-dependent inhibitory effects of imatinib and its main metabolite N-desmethylimatinib on CYP2C8 and CYP3A4/5 in vitro.

Experimental approach: Amodiaquine N-deethylation and midazolam 1'-hydroxylation were used as marker reactions for CYP2C8 and CYP3A4/5 activity. Direct, IC(50) -shift, and time-dependent inhibition were assessed with human liver microsomes.

Key results: Inhibition of CYP3A4 activity by imatinib was pre-incubation time-, concentration- and NADPH-dependent, and the time-dependent inactivation variables K(I) and k(inact) were 14.3 µM and 0.072 in(-1) respectively. In direct inhibition experiments, imatinib and N-desmethylimatinib inhibited amodiaquine N-deethylation with a K(i) of 8.4 and 12.8 µM, respectively, and midazolam 1'-hydroxylation with a K(i) of 23.3 and 18.1 µM respectively. The time-dependent inhibition effect of imatinib was predicted to cause up to 90% inhibition of hepatic CYP3A4 activity with clinically relevant imatinib concentrations, whereas the direct inhibition was predicted to be negligible in vivo.

Conclusions and implications: Imatinib is a potent mechanism-based inhibitor of CYP3A4 in vitro and this finding explains the imatinib-simvastatin interaction and suggests that imatinib could markedly increase plasma concentrations of other CYP3A4 substrates. Our results also suggest a possibility of autoinhibition of CYP3A4-mediated imatinib metabolism leading to a less significant role for CYP3A4 in imatinib biotransformation in vivo than previously proposed.

Figure 1

Molecular structures of imatinib, its main metabolite N-desmethylimatinib and two of its minor metabolites, piperidine N-oxide imatinib and pyridine N-oxide imatinib.

Figure 2

Effects of imatinib and N-desmethylimatinib on amodiaquine N-deethylation (CYP2C8 marker reaction) and midazolam 1′-hydroxylation (CYP3A4/5 marker reaction) with or without a 30 min pre-incubation in the presence of NADPH. Incubations were conducted in HLM incubations (0.1 mg·mL⁻¹ protein) with 2 µM substrate. Pre-incubation of imatinib or N-desmethylimatinib for 30 min without NADPH did not increase the inhibition of the marker reactions (data not shown). Data points are mean ± SD values of triplicate incubations.

Figure 3

Effects of piperidine N-oxide imatinib (10 µM) and pyridine N-oxide imatinib (10 µM) on N-deethylation of 2 µM amodiaquine (A) and 1′-hydroxylation of 2 µM midazolam (B) in HLM incubations (0.1 mg·mL⁻¹ protein), and the effect of 30 min pre-incubation with NADPH on inhibition of the 1′-hydroxylation of 2 µM midazolam by 10 µM imatinib (C) in recombinant CYP3A4 (3.3 pmol·mL⁻¹) and CYP3A5 (6.7 pmol·mL⁻¹) incubations. The inhibition by lower concentrations of piperidine N-oxide and pyridine N-oxide imatinib (0.1 and 1 µM) on the marker reactions was <15% and is not illustrated. Pre-incubation of imatinib without NADPH did not increase inhibition in CYP3A4 and CYP3A5 incubations. Data points represent mean ± SD values of duplicate incubations.

Figure 4

Double reciprocal (Lineweaver–Burk) plots for direct inhibition of amodiaquine N-deethylation and midazolam 1′-hydroxylation by different concentrations of imatinib (A and B, respectively), and by N-desmethylimatinib (C and D, respectively) in HLM incubations (0.1 mg·mL⁻¹ protein). The inhibition of CYP2C8 and CYP3A4/5 activity by imatinib were both best described by a mixed full inhibition mechanism, whereas that by N-desmethylimatinib on CYP2C8 activity was best described by a mixed full inhibition and that on CYP3A4/5 activity by a full competitive inhibition model. Data points are mean ± SD values of duplicate incubations.

Figure 5

Pre-incubation time- and concentration-dependent inhibition of midazolam 1′-hydroxylation by imatinib (0–128 µM) in HLM incubations (0.5 mg·mL⁻¹). Aliquots were removed from the pre-incubation mixtures at indicated time points and diluted 20-fold for measurement of residual CYP3A4 activity. The rate of inactivation of CYP3A4 activity by each inhibitor concentration (K_obs) was determined by linear regression analysis of the natural logarithm of the percentage of activity remaining versus pre-incubation time data (A). The K_i and k_inact were calculated by linear regression of the double-reciprocal plot of the K_obs versus inhibitor concentration [I] (B) and by non-linear regression analysis of the K_obs versus imatinib concentration data according to the equation described in Methods (C). Incubations were conducted in triplicate.

Figure 6

Prediction of the effect of imatinib on the pharmacokinetics of CYP3A4 substrates in vivo (AUC_po(I)/AUC_po(C)), assuming that intestinal bioavailability is unaffected (F_G(I)/F_G(C)= 1). The figures illustrate how the effect of imatinib on the AUC of the substrate depends on several factors: the concentration of imatinib at the enzyme site, the fraction of the substrate metabolized by CYP3A4 (f_m× f_m,CYP3A4 varying from 0.5 to 1.0) and on the CYP3A4 half-life. The simulation was carried out using the equation for mechanism-based inhibition for imatinib concentrations ranging from 0 to 0.7 µM. During treatment with 400 mg of imatinib mesilate daily, the average unbound C_trough and C_max concentrations in plasma approximate to 0.1 and 0.25 µM, respectively, whereas the unbound C_max of imatinib in the portal vein was estimated as 0.65 µM (indicated by the blue areas).