Superoxide scavenging activity of pirfenidone–iron complex

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Abstract

Pirfenidone (PFD) is focused on a new anti-fibrotic drug, which can minimize lung fibrosis etc. We evaluated the superoxide (O2) scavenging activities of PFD and the PFD–iron complex by electron spin resonance (ESR) spectroscopy, luminol-dependent chemiluminescence assay, and cytochrome c reduction assay. Firstly, we confirmed that the PFD–iron complex was formed by mixing iron chloride with threefold molar PFD, and the complex was stable in distillated water and ethanol. Secondary, the PFD–iron complex reduced the amount of O2 produced by xanthine oxidase/hypoxanthine without inhibiting the enzyme activity. Thirdly, it also reduced the amount of O2 released from phorbor ester-stimulated human neutrophils. PFD alone showed few such effects. These results suggest the possibility that the O2 scavenging effect of the PFD–iron complex contributes to the anti-fibrotic action of PFD used for treating idiopathic pulmonary fibrosis.

Section snippets

Materials and methods

Chemicals. PFD was purchased from Tocris Bioscience (Bristol, UK); 5,5-dimethyl-1-pyrroline N-oxide (DMPO; purity, 99%), diethylene-triamine-pentaacetic acid (DTPA) and 1,10-phenanthroline from Dojindo Laboratories (Kumamoto, Japan); Cu, Zn-superoxide dismutase (SOD), hypoxanthine cytochrome c, and phorbol myristate acetate (PMA) from Sigma Chemical Co. (St. Louis, MO, USA); dimethyl sulfoxide, iron chloride hexahydrate (FeCl3), and ethanol from Nakalai Tesque Inc., (Kyoto, Japan); Ascorbic

Formation of PFD–iron complex

The spectra of PFD, the PFD–iron complex, and iron chloride at pH 6.8 are shown in Fig. 1. The UV/vis spectrum of the PFD–iron complex was distinct from that of PFD or iron chloride. To elucidate the binding abilities of PFD and iron, we used the competitive 1,10-phenanthroline method. As for the results, we confirmed that the chelating constant of the PFD–iron complex was 5.95. As this value was about half that of EDTA, this suggests that the PFD–iron complex might be formed in a biological

Discussion

In this study, we found the superoxide scavenging activity of the PFD–iron complex in enzymatic and cellular systems. These results suggest that the O2 scavenging effect of the PFD–iron complex contributes to the anti-fibrotic action of PFD used for treating IPF.

The present study demonstrated that a stable PFD–iron complex was produced by the 3:1 reaction between PFD and iron chloride in ethanol and water, and its chelating constant enabled easy formation of the complex in a biological system (

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    Mitani Yoshihiro and Keizo Sato contributed equally to this work.

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