RT Journal Article
SR Electronic
T1 Characterization of HKI-272 Covalent Binding to Human Serum Albumin
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 1083
OP 1093
DO 10.1124/dmd.110.032292
VO 38
IS 7
A1 Jianyao Wang
A1 Xiao Xian Li-Chan
A1 Jim Atherton
A1 Lin Deng
A1 Robert Espina
A1 Linning Yu
A1 Peter Horwatt
A1 Steven Ross
A1 Susan Lockhead
A1 Syed Ahmad
A1 Appavu Chandrasekaran
A1 Aram Oganesian
A1 JoAnn Scatina
A1 Abdul Mutlib
A1 Rasmy Talaat
YR 2010
UL http://dmd.aspetjournals.org/content/38/7/1083.abstract
AB The study was initiated as an observation of incomplete extraction recovery of N-(4-(3-chloro-4-(2-pyridinylmethoxy)anilino)-3-cyano-7-ethoxy-6-quinolyl)-4-(dimethylamino)-2-butenamide (HKI-272) from human plasma. The objective of this study was to 1) identify the binding site(s) of HKI-272 to human plasma protein(s); 2) characterize the nature of the binding; and 3) evaluate the potential reversibility of the covalent binding. After incubation of [14C]HKI-272 with human plasma, the mixture was directly injected on liquid chromatography/mass spectrometry (LC/MS), and an intact molecular mass of HKI-272 human serum albumin (HSA) adduct was determined to be 66,999 Da, which is 556 Da (molecular mass of HKI-272) larger than the measured molecular mass of HSA (66,443 Da). For peptide mapping, the incubation mixture was separated with SDS-polyacrylamide gel electrophoresis followed by tryptic digestion combined with LC/tandem MS. A radioactive peptide fragment, LDELRDEGKASSAK [amino acid (AA) residue 182–195 of albumin], was confirmed to covalently bind to HKI-272. In addition, after HCl hydrolysis, a radioactive HKI-272-lysine adduct was identified by LC/MS. After combining the results of tryptic digestion and HCl hydrolysis, the AA residue of Lys190 of HSA was confirmed to covalently bind to HKI-272. A standard HKI-272-lysine was synthesized and characterized by NMR. The data showed that the adduct was formed via Michael addition with the ε-amine of lysine attacking to the β-carbon of the amide moiety of HKI-272. Furthermore, reversibility of the covalent binding of HKI-272 to HSA was shown when a gradual release of HKI-272 was observed from protein pellet of HKI-272-treated human plasma after resuspension in phosphate buffer, pH 7.4, at 37°C for 18 h. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics