Development of a competitive enzyme-linked immunosorbent assay for therapeutic drug monitoring of afatinib

Abstract

Afatinib is an oral tyrosine kinase inhibitor (TKI) approved for treating advanced non-small cell lung cancer. It is necessary to develop a simple quantification method for TKIs in order to facilitate therapeutic drug monitoring (TDM) in clinical settings. This study sought to develop a simple and sensitive competitive enzyme-linked immunosorbent assay (ELISA) to quantify afatinib in plasma for routine pharmacokinetic applications. An anti-afatinib antibody was obtained using (S)-N-4-(3-chloro-4-fluorophenyl)-7-(tetrahydrofuran-3-yloxy)-quinazoline-4,6-diamine (CTQD), which has the same substructure as afatinib, as a hapten. Enzyme labeling of afatinib with horseradish peroxidase was similarly performed using CTQD. A simple competitive ELISA for afatinib was developed based on the principle of direct competition between afatinib and the enzyme marker for the anti-afatinib antibody, which had been immobilized on the plastic surface of a microtiter plate. Plasma afatinib concentrations below the limit of quantification of 30 pg/mL were reproducibly measurable. Also, the values of plasma afatinib levels measured from 20 patients were comparable with those measured by high-performance liquid chromatography, and there was a strong correlation between the values determined by both methods (Y = 0.976X – 0.207, r = 0.975). As indicated by its specificity and sensitivity, this newly developed ELISA for afatinib is an important tool for TDM and studies of the pharmacokinetics of afatinib.

Localization and Accumulation Studies of Dacomitinib in Rat Intestines and Skin by Immunohistochemistry

“…We previously generated a specific antibody against afatinib that recognizes a structure other than its 4-(dimethyl-amino)-2-butene moiety [7]. The structure of afatinib is very similar to that of dacomitinib (Fig.…”

“…Afatinib and dacomitinib have very similar chemical structures. The anti-afatinib serum that we previously developed is likely to cross-react with dacomitinib based on its specificity profile [7]. Therefore, in this present study, we tried to develop an IHC system to detect dacomitinib using the anti-afatinib serum to reveal the localization of orally administered dacomitinib in the intestines and skin, thereby elucidating the mechanism dacomitinib-induced diarrhea and skin toxicity.…”

“…E-mail: sait1102@life.sojo-u.ac.jp ErbB family receptors in vivo [2,8]. Therefore, we developed a specific antibody against afatinib and an immunohistochemistry (IHC) system that can detect EGFR-bound afatinib in tissues [7,13]. Using this IHC system, we revealed the localization of administered afatinib in the intestines and skin for the first time [13].…”

“…An ELISA for afatinib was performed according to our previous methods [7]. The underlying principle of ELISA is enzyme-labeled and unlabeled drugs competing for a purified immobilized antibody, followed by measuring marker enzyme activity of the resulting immunocomplex bound to the solid phase.…”

A UPLC–MS/MS method for simultaneous determination of nine antiepileptic drugs in human plasma and its application in TDM

“…On the one hand, the new method should be able to monitor as many drugs as possible simultaneously to minimize healthcare costs (LLopis et al, 2020). On the other hand, it should ensure TDM results are obtained quickly by optimizing sample processing and analyzing methods to guide doctors to adjust treatment regimens for patients (Sogawa, Saita, & Yamamoto, 2019).…”