Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 37 (6), 1242-50

Characterization of Dasatinib and Its Structural Analogs as CYP3A4 Mechanism-Based Inactivators and the Proposed Bioactivation Pathways

Affiliations

Characterization of Dasatinib and Its Structural Analogs as CYP3A4 Mechanism-Based Inactivators and the Proposed Bioactivation Pathways

Xiaohai Li et al. Drug Metab Dispos.

Erratum in

  • Drug Metab Dispos. 2009 Oct;37(10):2118. Vojkovsky, Tomas [added]

Abstract

Dasatinib was approved in 2006 for the treatment of imatinib-resistant chronic myelogenous leukemia and functions primarily through the inhibition of BCR-ABL and Src kinase. Dasatinib is extensively metabolized in humans by CYP3A4. In this study, we report that the bioactivation of dasatinib by CYP3A4 proceeds through a reactive intermediate that leads to CYP3A4 inactivation with K(I) = 6.3 microM and k(inact) = 0.034 min(-1). The major mechanism of inactivation proceeds through hydroxylation at the para-position of the 2-chloro-6-methylphenyl ring followed by further oxidation, forming a reactive quinone-imine, similar to the reactive intermediates formed by acetaminophen and diclofenac. Formation of a reactive imine-methide was also detected but appears to be a minor pathway. When glutathione was added to human liver microsomal incubations, dasatinib-glutathione adducts were detected. Numerous dasatinib analogs were synthesized in an effort to understand what modifications would block the formation of reactive intermediates during dasatinib metabolism. It is interesting to note that blocking the site of hydroxylation with a methyl group was not effective because a reactive imine-methide was formed, nor was blocking the site with fluorine because the fluorine was removed through an oxidative defluorination mechanism and the reactive quinone-imine was still formed. Numerous analogs are presented that did effectively block the formation of glutathione adducts and prevent the inactivation of CYP3A4.

Figures

Fig. 1
Fig. 1
Time- and concentration-dependent inactivation of CYP3A4 by dasatinib. Six concentrations of dasatinib (0, 2.5, 5, 10, 20, and 40 μM) were incubated with human liver microsomes. Aliquots were removed and assayed for remaining CYP3A4 activity at various time points. Each point represents the mean from three separate replicates that do not differ by more than 10%. The remaining activity versus incubation time was plotted, and the slopes for the individual dasatinib concentrations were fit to a Kitz-Wilson plot (inset). The calculated KI and kinact for CYP3A4 inactivation were 6.3 μM and 0.034 min−1, respectively.
Fig. 2
Fig. 2
Chromatographic separation of dasatinib-glutathione adducts generated in incubations with HLM and GSH. The incubation had 40 μM dasatinib, 1 mM NADPH, 5 mM GSH, and 1 mg/ml human liver microsomes (A); 1 μM ketoconazole, was added to incubation B. D-2OH-GSH (m/z 825.2 → 347.2) corresponds to a dihydroxylated dasatinib-glutathione adduct. D-OH-GSH (m/z 809.2 → 347.2) corresponds to a hydroxylated dasatinib-glutathione adduct. D-GSH (m/z 793.2 → 347.2) corresponds to a dasatinib-glutathione adduct. The internal standard (IS) is 100 nM carbamazepine (m/z 237.0 → 194.0).
Fig. 3
Fig. 3
Fragmentation patterns of dasatinib and hydroxylated-glutathione adducts. Dasatinib-glutathione adducts were generated in an incubation mixture containing 40 μM dasatinib, 1 mM NADPH, 5 mM GSH, and 1 mg/ml human liver microsomes. The enhanced product ion scan is shown for dasatinib (A), D-OH-GSH (B), and D-2OH-GSH (C). The structure depicted represents one possible regioisomer. Rel. Int., relative intensity; amu, atomic mass units.
Fig. 4
Fig. 4
Fragmentation patterns of dasatinib-glutathione adducts (A) and (B). Dasatinib-glutathione adducts were generated in an incubation mixture containing 40 μM dasatinib, 1 mM NADPH, 5 mM GSH, and 1 mg/ml human liver microsomes. The enhanced product ion scan is shown for D-GSH (A) and D-GSH (B). The structures depicted are proposed adducts. Rel. Int., relative intensity; amu, atomic mass units.
Fig. 5
Fig. 5
Proposed scheme for CYP3A4-mediated metabolic activation of dasatinib. Dasatinib hydroxylation leads to the formation of a chemically reactive para-quinoneimine intermediate (major pathway) or hydrogen abstraction of the methyl leads to an ortho-imine-methide intermediate (minor pathway). These reactive intermediates react with glutathione to form corresponding GSH adducts or may react with CYP3A4 and lead to enzyme inactivation.*, proposed sites of glutathione adduction.

Similar articles

See all similar articles

Cited by 19 PubMed Central articles

See all "Cited by" articles

MeSH terms

LinkOut - more resources

Feedback