Immunohistochemical Study of the Localization of Ibrutinib-Protein Conjugates in the Rat Gastrointestinal Tract

Ibrutinib is an oral irreversible Bruton's tyrosine kinase (BTK) inhibitor that blocks BTK activity by forming covalent bonds with the thiol group of cysteine in the ATP-binding pocket via Michael addition. However, it also reacts with a variety of off-target nonspecific proteins. In this study, we attempted to generate a specific antibody against ibrutinib and develop an immunohistochemical method to detect the ibrutinib-protein conjugates. Ibrutinib has the same amino group as the nucleobase adenine. Paraformaldehyde fixation could not fix it to the tissue via this amino group. However, ibrutinib covalently binds to proteins such as BTKs to exert its action and is therefore immobilized in tissue as ibrutinib-protein conjugates. Thus, immunohistochemistry for ibrutinib detects the location of the ibrutinib-protein conjugates, that is, the sites of covalently bound to the tissue via Michael addition. Using this immunohistochemical method, we visualized the ibrutinib-protein conjugates in the rat gastrointestinal tract (gastric body, duodenum, jejunum, ileum, and colon). This study is the first to elucidate the location of the ibrutinib-protein conjugates in the rat gastrointestinal tract and helps to clarify the mechanism of ibrutinib-induced toxicity.