Covalent binding mechanism of Furmonertinib and Osimertinib with human serum albumin .

As third-generation tyrosine kinase inhibitors, furmonertinib and osimertinib exhibit better efficacy than first and second generation TKIs in patients with advanced non-small-cell lung cancer. However, radioactive pharmacokinetics studies showed that parent-related components remain in human plasma for at least 21 days after oral administration. Similar pharmacokinetic profiles were found in pyrotinib and neratinib, which have been identified to covalently bind with human serum albumin at Lys-190, leading to low extraction recovery in protein precipitation. However, the binding mechanism of Furmonertinib and Osimertinib in human plasma has not been confirmed. Comprehensive techniques were used to investigate the mechanism of this binding, including UHPLC coupled with HRMS and online/offline radioactivity profiling. SDS-PAGE and further autoradiography were also used to detect drug-protein adducts. We found that most furmonertinib exists in the human plasma following ex vivo incubation in the form of protein-drug adducts. Only lysine-furmonertinb adducts were found in pronase digests. A standard reference of lysine-furmonertinib was synthesized and confirmed by NMR. Through peptide mapping analysis, we confirmed that furmonertinib almost exclusively binds with HSA in plasma following ex vivo incubation, via Michael addition at Lys-195 and Lys-199, instead of Lys-190. Two peptides found to bond with furmonertinib were ASSAKQR and LKCASLQK. Osimertinib was also found to bond with Lys-195 and Lys-199 of HSA via peptide mapping analysis. Significance Statement Here we report, that furmonertinib and osimertinib can covalently bind with human serum albumin at the site of Lys-195 and Lys-199 instead of Lys -190, leading to the long duration of drug-protein adducts in the human body.