Purpose: Bruton's tyrosine kinase (BTK) plays a crucial role in the development of B-cells. Selective BTK inhibitor blocks primary antigen-driven antibody response by interfering with cell signaling cascades and preventing key phosphorylation in B cells to prevent B cell dysfunction. Although covalent binding inhibitor raises concerns over potential toxicity caused by reaction with off-target proteins etc., they may offer long duration of action. Both marketed BTK drugs Ibrutinib and Acalabrutinib are covalent inhibitors. Glutathione (GSH) conjugate formation in in-vitro assay is a recognized indicator for potential in-vivo toxicity risk and mitigation. A simple reaction assay with GSH of 10 covalent BTK inhibitors to assess the chemical reactivity. Also verified by GSH conjugation in human liver microsomes.
Methods: Ten BTK covalent inhibitors reported in the literature were either obtained commercially or custom made. The inhibitors (10 µM) were incubated individually in potassium phosphate buffer (100 mM pH 7.4) in the presence of GSH (5 mM) at 37°C in a UPLC autosampler. Samples were injected automatically and repeatedly every half hour over 4 hours for LC-UV-MS analysis on a Waters Acquity UPLC system coupled with Sciex TripleTOF 5600. The half-life of the pseudo 1st order reaction with GSH was determined by monitoring the LC-UV peak area of parent drug at 254 nm. In this study, the inhibitors (10 µM) were also incubated in human liver microsomes (HLM) fortified with GSH (5mM) and NADPH (2mM), at 37°C for one hour. After protein crash, supernatant was analyzed as described above. Percentage of GSH conjugate formation was determined using LC-UV peak areas.
Results: In chemical reactivity assessment, Evobrutinib, Tirabrutinib and Acalabrutinib were relatively stable and had the longest half-life with 9.7, 8.8 and 4.1 hours respectively. PRN1008, Ibrutinib, Zanubrutinib and Branebrutinib were moderately stable with half-life 2.7, 2.2, 1.5 and 1.3 hours respectively. Spebrutinib, Olmutinib and Poseltinib were unstable with shortest half-life of 0.9, 0.6 and 0.5 hours respectively. These results correlated generally well with GSH trapping data in HLM (Figure I), except for an outlier PRN1008 (red dot). Furthermore, the measured chemical reactivity half-life is in an approximate correlation with biochemical potency BTK IC50 of these 10 inhibitors.
Conclusion: This present method has three advantages versus traditional UV detection methods (Barf et al., 2017) in GSH chemical reactivity assay. Firstly, thermostat autosampler was used as the chemical reaction incubator and subsequently sample injections from the same vial, reducing labor-consuming sample preparation and potential manual operation induced variation. Secondly, lower drug concentration (10 uM) incubation in this study avoids adding organic solvent to dissolve usually low soluble BTK inhibitors at a high concentration, thus maintaining buffer pH 7.4 in aqueous solution. Thirdly, MS and MS/MS data acquisition in-line with UV detection facilitates identification of GSH conjugates and other degradation products. For example, in addition to the major direct GSH conjugate with Olmutinib in buffer, two other minor products were formed including a demethylation product and a hydrolysis product with GSH conjugation. It appears that the potency of these 10 covalent binding inhibitors is partially driven by the electrophilicity of the “warhead”, whereas, the implication of the chemical reactivity ranking among other factors to the off-target toxicity potential is still yet to be studied.
Barf T, Covey T, Izumi R, Kar BVD, Gulrajani M, Lith BV, Hoek MV, Zwart ED, Mittag D, Demont D, Verkaik S, Krantz F, Pearson PG, Ulrich R, and Kaptein A (2017) Acalabrutinib (ACP-196): A Covalent Bruton Tyrosine Kinase Inhibitor with a Differentiated Selectivity and In Vivo Potency Profiles. J Pharmacol Exp Ther 363:240–252.