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, 38 (7), 1238-45

Cytochrome P450-mediated Bioactivation of the Epidermal Growth Factor Receptor Inhibitor Erlotinib to a Reactive Electrophile

Affiliations

Cytochrome P450-mediated Bioactivation of the Epidermal Growth Factor Receptor Inhibitor Erlotinib to a Reactive Electrophile

Xiaohai Li et al. Drug Metab Dispos.

Abstract

The epidermal growth factor receptor tyrosine kinase inhibitor erlotinib (ERL) is approved for treatment of non-small-cell lung cancer. Numerous reports of ERL-associated toxicities are consistent with immune-mediated toxicity, including drug-induced hepatitis, interstitial lung disease, Stevens-Johnson syndrome, and toxic epidermal necrolysis. Although the mechanism of toxicity has not been established, we present evidence that reactive intermediates are formed during the metabolism of ERL, which can covalently conjugate to the cysteine group of the peptide-mimetic GSH. Seven ERL-GSH conjugates were identified in incubations with hepatic microsomes. Cytochrome P450 (P450)-dependent adducts are proposed to be formed via reactive epoxide and electrophilic quinone-imine intermediates. In incubations of human liver microsomes, intestinal microsomes, pulmonary microsomes, and recombinant P450s, CYP3A4 was the primary enzyme responsible for the bioactivation of ERL; however, CYP1A1, CYP1A2, CYP3A5, and CYP2D6 were capable of catalyzing the bioactivation as well. During the metabolism of ERL, CYP3A4 and CYP3A5 are irreversibly inactivated by ERL in a time- and concentration-dependent manner. Inactivation was not dependent on oxidation of the ERL alkyne group to form a reactive oxirene or ketene, as shown by synthesizing analogs where the alkyne was replaced with a cyano group. CYP1A1, CYP1A2, and CYP2D6 were not inactivated despite catalyzing the formation of ERL-GSH adducts.

Figures

Fig. 1
Fig. 1
Time- and concentration-dependent inactivation of CYP3A4 by ERL. Incubations containing 0.5 mg/ml HLM and 1 mM NADPH in 100 mM phosphate buffer, pH 7.4, were incubated with the following ERL concentrations: 0, 5, 10, 20, and 40 μM. A, at the indicated time points, the remaining CYP3A4 activity was measured by a midazolam hydroxylation assay. Each point represents the mean of triplicate incubation. The observed inactivation rate constants, Kobs, were calculated from the slopes of the regression lines in A. B, the hyperbolic plot of Kobs versus ERL concentration was used to calculate kinetic constants. Potential preservation of CYP3A4 activity by the addition of 5 mM GSH or 1000 units of superoxide dismutase and catalase to incubations containing 0.5 mg/ml HLM, 1 mM NADPH, and 20 μM ERL was evaluated (C).
Fig. 2
Fig. 2
Formation of ERL-GSH adducts. A, ERL-GSH adducts were detected in 1-h incubations containing 2 mg/ml HLM, 1 mM NADPH, 40 μM ERL, and 5 mM GSH in 100 mM phosphate buffer, pH 7.4. Parallel incubations to those in A were prepared without GSH; after 1 h, the solution was directly applied to a solid-phase extraction column. The column was washed with 20 bed volumes of water and eluted with acetonitrile. The acetonitrile was evaporated, and ERL and its metabolites were reconstituted in phosphate buffer with 5 mM GSH and incubated at 37°C for 4 h (B).
Fig. 3
Fig. 3
Tandem mass spectrometry spectrum of the MH+ ion m/z 715.3 of ERL-G5. The origins of the characterized ions are as indicated.
Fig. 4
Fig. 4
H NMR spectrum of ERL-G6.
Fig. 5
Fig. 5
Effect of NADPH and superoxide dismutase plus catalase on the formation rate of ERL-G6 in recombinant P450 incubations. All the incubations had 100 pmol/ml P450, 5 mM GSH, and 20 μM ERL in 100 mM phosphate buffer, pH 7.4. Filled bars contained 1 mM NADPH; open bars did not have NADPH; and hashed bars had 1 mM NADPH plus 1000 units of superoxide dismutase and 1000 units of catalase.
Fig. 6
Fig. 6
ERL-G5 formation by recombinant P450 enzymes. ERL-G5 generation was compared in incubations containing 100 pmol/ml P450, 1 mM NADPH, 5 mM GSH, and 40 μM ERL (A). Relative ERL-G5 concentration was determined by comparison of the peak area of ERL-G5 to an internal standard. The enzyme activities were expressed as the percentage of CYP3A4 activity and are an average of two measurements. Correlation analysis of ERL-G5 formation to formation of the para-hydroxyaniline metabolite in HLM and recombinant P450 is shown in plot B.
Fig. 7
Fig. 7
CYP3A and CYP1A inhibition on ERL-G5 formation. A, ERL-G5 production was measured in incubations where the CYP3A inhibitor ketoconazole (1 μM) was added to human pulmonary (smoker), intestinal, and hepatic microsomes. B, inhibition of CYP3A (1 μM ketoconazole) and CYP1A (20 μM α-naphthoflavone) was tested in incubations containing pulmonary microsomes from smokers (S) and nonsmokers (NS). All the incubations contained 2 mg/ml microsomal protein, 40 μM ERL, 5 mM GSH, and 1 mM NADPH in 100 mM phosphate buffer, pH 7.4. The values were an average of two replicates.
Fig. 8
Fig. 8
Influence of human microsomal epoxide hydrolase on the formation of ERL-G5. The addition of 1 mg/ml human microsomal epoxide hydrolase was evaluated in incubations of HLM and recombinant CYP3A4 and CYP1A1. Incubations containing 2 mg/ml HLM or 100 pmol/ml recombinant P450, 1 mM NADPH, 5 mM GSH, and 40 μM ERL in 100 mM phosphate buffer, pH 7.4. The values were an average of two replicates.
Scheme 1
Scheme 1
Proposed mechanism of ERL-GSH adduct formation.

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