Rifabutin boosts rifampicin accumulation in THP-1-derived M2 macrophages by inhibiting P-glycoprotein efflux activity

M2 macrophages show higher efflux activity of the P-glycoprotein (P-gp) drug transporter and lower rifampicin uptake than M1 macrophages. Accordingly, in M2 macrophages rifampicin accumulation should be restored by potent P-gp inhibitors such as rifabutin. THP-1 cells were differentiated (200 nM phorbol-12-myristate 13-acetate for 72 h) and polarized (20 ng/mL interleukin 4 and interleukin 13 for 48 h) to M2 macrophages. P-gp inhibition by rifabutin was assessed through flow cytometry using rhodamine 123 (substrate) and compared to zosuquidar (positive P-gp inhibitor control). Ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS/MS) was used to evaluate the cellular rifampicin accumulation after 2 h of exposure to 0.05, 0.1, or 0.5 µM rifampicin, with or without co-treatment of cells with rifabutin (0.01 µM, 0.1 µM, 1 µM, 10 µM). Rifabutin (IC₅₀ 0.8 µM) but not rifampicin inhibited P-gp efflux activity in M2 cells. When exposed to 0.05 µM rifampicin only, M2 cells accumulated 5.9 ± 1.1 ng rifampicin/ng protein, but took up significantly more rifampicin when co-treated with rifabutin (e.g. 10 µM rifabutin: 13.7 ± 3.5 ng rifampicin/ng protein). The same concentration-dependent boosting effect by rifabutin was detectable for the 0.1 µM and 0.5 µM rifampicin exposure levels. Together, rifabutin concentration-dependently inhibits P-gp and enhances rifampicin accumulation in M2 macrophages during concomitant drug exposure.