Kinetic and mechanistic investigation toward the characterization of tucatinib inhibition of CYP3A4.

A highly selective tyrosine kinase inhibitor of the human epidermal growth factor receptor 2, tucatinib, has shown remarkable efficacy and safety for treatment of adult patients with advanced, unresectable, or metastatic human epidermal growth factor receptor 2 positive breast cancer, including those with brain metastases. The purpose of this study was to evaluate in vitro the potential to inhibit cytochrome P450 (P450) isoforms for tucatinib. Preliminary in vitro P450 inhibition survey revealed that tucatinib was a weak inhibitor of CYP1A2, CYP2B6, CYP2C19, and CYP2D6 with IC values >20 μM. Follow-up kinetic studies generated K values of tucatinib for CYP2C8, CYP2C9, and CYP3A of 0.17, 4.57, and 0.80 μM, respectively; moreover, the inhibition mechanism was determined as competitive inhibition from all 3 P450 isoforms. Of the 7 P450 isoforms in human liver microsomes, tucatinib showed time-dependent inhibition of only CYP3A4. Detailed mechanistic kinetic experiments suggest that tucatinib inactivated CYP3A-mediated midazolam 1'-hydroxylation with a mean k value of 0.011 min and a mean K value of 0.54 μM. Detailed experiments to describe the mechanism of the observed tucatinib inactivation of CYP3A4 revealed that loss of enzyme activity is not the result of heme destruction (ie, adduct formation or bleaching), as evidenced by the presence of a CO binding spectra and absence of any loss of intact heme. Investigations were conducted to explore whether tucatinib could act as a quasi-irreversible inhibitor and form a metabolite-intermediate (MI) complex with CYP3A4. The results suggest that the inhibition by tucatinib was not reversed upon the addition of ferricyanide and no MI complex spectrum was observed. Mass spectrometric interrogation surrounding the potential basis for tucatinib inactivation of CYP3A4 via apoprotein adduct revealed that tucatinib forms covalent adduct with Cys239 of CYP3A4 only in the presence of NADPH. In summary, the results from the kinetic evaluation of tucatinib inhibition of CYP3A4 suggest that tucatinib is a modest mechanism-based inactivator of CYP3A4. Mechanistic studies conducted to describe the root cause of the inactivation revealed that the mechanism of tucatinib inactivation of CYP3A4 is not explained by modification of the heme or formation of an MI complex. However, through targeted proteomics the basis for the observed CYP3A4 inactivation by tucatinib was identified as being the formation of a NADPH-dependent covalent adduct between tucatinib and Cys239. SIGNIFICANCE STATEMENT: Time-dependent inhibition of CYP3A4 by novel chemical entities has major implications in drug development. This study reports the mechanistic nature of irreversible mechanism-based inactivation of CYP3A4 by tucatinib and proposes a potential bioactivation pathway to account for the results observed.