In Vitro Assessment of Methylene Blue Hydrate as a Multiple CYP450 Inhibitor and a Mechanism-Based Inhibitor

Methylene blue hydrate (MBH) is used to treat methemoglobinemia and urinary tract infections. MBH is also used as a dye or staining agent to make certain body fluids and tissues. Recently, MBH is developed as an investigational drug for Alzheimer’s disease ^{1), 2), 3)}. Despite many uses in a range of different fields, there are no known drug interactions of MBH. Drug-drug interactions (DDIs) remain an important issue in the drug discovery and development. Especially, cytochrome P450 (CYP450) inhibition and mechanism-based inhibition (MBI) might cause clinically significant DDIs ^{4), 5)}. Consequently, to assess the risk of DDIs of MBH, we studied the potential for CYP450 inhibition and MBI of MBH on multiple CYP450 enzymes using human liver microsomes. For CYP450 IC₅₀ determinations, multiple concentrations of MBH were prepared and pre-incubated with human liver microsomes and post-incubated with substrate and NADPH at 37.5°C to initiate the reaction. The reactions were terminated by combining with ice-cold acetonitrile and the supernatant from each sample after centrifugation was analyzed by LC/MS/MS. MBH at a concentration of 10 µM showed more than 87% inhibition for six CYP450 enzymes, CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4 (using two substrates). IC₅₀ value for 1A2 was 0.63 µM, 2C8 was 2.66 µM, 2C9 was 1.46 µM, 2C19 was 0.33 µM, 2D6 was 1.67 µM, 3A4 using nifedipine as a substrate was 2.71 µM, and 3A4 using testosterone as a substrate was 1.01 µM, respectively. As a result, MBH showed moderate to potent inhibition (IC₅₀ < 10 µM) in tested six CYP450 isoforms. To evaluate whether MBH is also possible to show MBI, we further performed MBI test using human liver microsomes and major CYP450 enzymes with industry recommended probe substrates ^{4), 5), 6)}. MBH at a concentration of 1 µM showed similar MBI on CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4 (using two substrates) by comparison with reference compounds known as selective mechanism-based inhibitors. These data indicate that MBH has the potential as a multiple CYP450 inhibitor and mechanism-based inhibitor of CYP1A2, 2C8, 2C9, 2C19, 2D6 and 3A4.

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