Assessment of Tofacitinib and Ruxolitinib and their Anti Inflammatory Effects on Myeloperoxidase

Abstract

Abstract

Myeloperoxidase (Mpo) is a heme-containing enzyme present in inflammation. Mpo catalyzes the production of hypochlorous acid (HCLO) by reacting with hydrogen peroxide (H2O2) and chloride (Cl-). Mpo together with H2O2 and a halide represents a potent oxidizing system that is involved in a variety of functions that include the killing of bacteria, and the lysis of mammalian cells and inflammatory mediators. To determine this inhibition of mpo we tested inhibitors that we know inhibit. To start ,we tested this inhibition by H2O2 dependence. Serial dilutions of H2O2 were made using 30% stock of H2O2 to determine the concentration of H2O2 necessary to produce the greatest fluorescence signal using 530nm excitation and 590 nm emission wavelength using a SpectraMax plate reader. This allowed us to see the greatest fluorescence value for that particular dilution. Fluorogenic peroxidase substrate 10-acetyl-3, 7-dihydrophenozazine (ADHP), MPO, was mixed in sodium acetate buffer at pH 5.6 in the absence and presence of H2O2 to serve as the negative and positive controls, respectively. Three compounds were tested for mpo inhibition, namely benzoic acid hydrazide (bah), tofacitinib, and ruxolitinib. The same conditions were used for each inhibitor. First we tested each inhibitor at a concentration of 10mM. Once we received data we did an one-way ANOVA test. After completing an ANOVA test comparing the positive control to each inhibitor it was determined that both bah and tofacitinib were significantly different but ruxolitinib was not. The concentrations of the inhibitors were 10mM, 5mM, 0.5mM and 1 mM respectively. The experiment was done again under the same conditions. After completing an ANOVA test and comparing each inhibitor with the positive control again it was determined that all inhibitors were significantly different at 1mM. This information does in fact tell us that all three inhibitors do inhibit mpo. After testing these inhibitors at different concentrations we determined the solubility of them is a factor, potentially a limiting factor in the inhibition of mpo. Further tests will be done to see what effect does the solubility have on mpo inhibition, if there is an effect.