Biochemical Pharmacology, Vol. 45. No. 10, pp. 2003-2010, 1993. Printed in Great Britain. -295@3 $6.00 + 0.00 @ 1993. Pergamon Press Ltd SUPEROXIDE IS AN ANTAGONIST OF ANTI- INFLAMMATORY DRUGS THAT INHIBIT HYPOCHLOROUS ACID PRODUCTION BY MYELOPEROXIDASE zyxwvutsrqponmlkjihgfedcbaZYXWVU ANTHONY J. KE~LE,* CRAIG A. GEDYE and CHRISTINE C. WINTERBOURN Free Radical Research Unit, Department of Pathology, Christchurch School of Medicine, Christchurch, New Zealand (Received 23 December 1992; accepted 18 February 1993) Abstract-Myeloperoxidase, the most abundant enzyme in neutrophils, catalyses the conversion of hydrogen peroxide and chloride to hypochlorous acid. This potent oxidant has the potential to cause considerable tissue damage in many inflammatory diseases. We have investigated the ability of dapsone, diclofenac, primaquine, sulfapyridine and benzocaine to inhibit hypochlorous acid production by stimulated human neutrophils. The drugs were also tested against purified myeloperoxidase using xanthine oxidase to generate hydrogen peroxide and superoxide. The inhibitory effects of the drugs on hypochlorous acid production, either by cells stimulated with phorbol my&ate acetate or by myeloperoxidase and xanthine oxidase, were significantly less than those determined with mye- loperoxidase and reagent hydrogen peroxide. Comparable potency was observed only when superoxide dismutase was present to remove superoxide. We also observed that with the xanthine oxidase system, inhibition of hypochlorous acid production by dapsone decreased markedly as the concentration of myeloperoxidase increased. Dapsone was a poor inhibitor of hypochlorous acid production by neutrophils stimulated with opsonized zymosan, regardless of the presence of superoxide dismutase. With this phagocytic stimulus, catalase inhibited hypochlorous acid formation by only 60%, which indicates that a substantial amount of the hypochlorous acid detected originated from within phagosomes. Thus, it is apparent that dapsone is unable to affect intraphagosomal conversion of hydrogen peroxide to hypo- chlorous acid. All the drugs inhibit myeloperoxidase reversibly by trapping it as its inactive redox intermediate, compound II. We propose that superoxide limits the potency of the drugs by reducing compound II back to the active enzyme. Furthermore, under conditions where the activity of mye- loperoxidase exceeds that of the hydrogen peroxide-generating system, which is most likely to occur in phagosomes, partial inhibition of myeloperoxidase need not affect hypochlorous acid production. We conclude that drugs that inhibit myeloperoxidase by converting it to compound II are unlikely to be effective against hypochlorous acid-mediating tissue damage. With the realization that neutrophils contribute to tissue damage in many inflammatory pathologies [ 11, considerable effort is being directed at blocking their destructive potential. Much of this effort is focused on stopping the production of reactive oxidants. When stimulated, neutrophils discharge superoxide, which is converted to secondary oxidants including hydrogen peroxide and hypochlorous acid [2]. Hypochlorous acid is the most reactive oxidant produced by neutrophils in appreciable amounts [3]. It is highly damaging to proteins [l, 41 and reacts with unsaturated fatty acids to form chlorohydrins that are likely to destabilize cell membranes [5]. Thus, hypochlorous acid could be responsible for much of the inflammatory tissue damage inflicted by neutrophils. Hypochlorous acid is produced from hydrogen peroxide and chloride by myeloperoxidase [6]. This is the most abundant enzyme of neutrophils, constituting 2-5% of their dry weight [3]. It plays a central role in host defence, but it is not absolutely essential for microbial killing since myeloperoxidase- deficient individuals are generally in good health [7]. * Corresponding author. Tel. (64) 3 364 0565; FAX (64) 3 364 0534. In direct contrast, people with chronic granulomatous disease have serious infections due to the inability of their neutrophils to generate superoxide [S]. Therefore, targeting myeloperoxidase has the potential to eliminate much of the inflammatory damage inflicted by reactive oxidants without compromising host defence. In the last few years a wide range of anti- inflammatory drugs have been shown to inhibit the formation of hypochlorous acid [S14]. However, they may be less effective at inhibiting hypochlorous acid production by neutrophils as compared to purified myeloperoxidase. We found that dapsone and sulfapyridine inhibit myeloperoxidase by greater than 90% at a concentration of 5@I [15], yet at 1OOpM these drugs are unable to completely inhibit halogenation or hypochlorous acid-dependent cytotoxicity by neutrophils [X-18]. Recently we showed that several anti-inflammatory drugs are potent inhibitors of myeloperoxidase [15]. The drugs act by trapping myeloperoxidase at compound II, which is an inactive redox intermediate of the enzyme [2]. We have also shown that superoxide reduces compound II back to the active enzyme and boosts production of hypochlorous acid [19,20]. In this paper we investigate whether the interaction of 2003