Development and Validation of the Bioanalytical UHPLC–MS/MS Method for the Quantification of Branebrutinib and Metformin: Application for Preclinical Pharmacokinetic Assessments

Branebrutinib (BRNB) is a selective, oral Bruton's Tyrosine Kinase (BTK) inhibitor under clinical development. Metformin (MTF) exhibits antidiabetic properties and is recognized for enhancing chemotherapy efficacy by inhibiting cell proliferation and facilitating apoptosis via critical genes like TP53 and caspases. Consequently, research into diabetic patients co-morbid with B-cell malignancies has gained significance. This study compared in vivo pharmacokinetics of MTF and BRNB to elucidate their metabolic and transporter level interactions. A sensitive, reliable ultra-high performance liquid chromatography mass spectrometric (UHPLC-MS/MS) method was validated for efficiently quantifying analytes on Poroshell 120 EC-C18 column using ammonium bicarbonate and methanol. Mass transitions revealed m/z 130.0 → 60.0 (MTF), m/z 371.1 → 150.0 (BRNB), and m/z 260.1 → 56.1 (propranolol), and protein precipitation provided effective extraction. The method was validated as per USFDA, and it was found specific, selective, and sensitive at 10 ng/mL concentration and noticed linear, accurate, precise, and free from carry over and matrix effects. In vivo oral MTF (25 mg/kg) and BRNB (5 mg/kg) co-administration significantly increased MTF's Cmax (3.4-fold), AUC0-t (3.32-fold), and half-life (1.65-fold). Similarly, systemic exposure of BRNB followed an increased trend for Cmax (1.59-fold) and AUC0-t (2.1-fold), while its elimination rate remained unaltered. Integrated PK/PD studies support further concurrent MTF-BRNB therapies.