Abstract
6-mercaptopurine (6MP), an immunosuppressive and cytotoxic metabolite of azathioprine, is used therapeutically in the treatment of many inflammatory and immunological diseases. 6MP is known to undergo metabolism through oxidation by xanthine oxidase, S-methylation via thiopurine methyltransferase, and can be metabolized to form 6-thioguanine, which is believed to be the active metabolite. At sites of inflammation, reactive oxygen species (ROS) are generated, including H2O2 that inactivates superoxide dismutase. In the presence of HCO3-, peroxymonocarbonate (HCO4–) can potentially be produced, which enhances the oxidation potential of H2O2. H2O2 can also produce CO3●– through SOD peroxidase activity. Thiols have been shown to be oxidized by HCO4– or CO3●– but no studies have investigated the effect on 6MP.
HCO4– and CO3●– will oxidize 6-mercaptopurine at the sulfur atom, and SOD peroxidase activity will act as a catalyst.
UV-Vis spectrometry was used at room temperature to monitor the spectral changes (200 – 500 nm) using 20 µM 6MP and other purine analogs including its parent drug, azathioprine, and related metabolites including 6-methyl-MP, 6-thioguanine, 6-thioxanthine, and 6-thiouric acid. Reactions were run in 0.1 M KPO4 buffer pH 7.4 containing 250 µM DTPA, and were initiated by the addition of 25 mM HCO3– and 1 mM H2O2. The most prominent changes were observed with 6MP and 6-thioguanine; less changes were observed with 6-thioxanthine and 6-thiouric acid. Oxidation of 6MP by H2O2 was observed in the absence of HCO3– but was significantly enhanced using 12.5 and 25 mM HCO3–. The reactions were analyzed by high resolution LC/MS and were compared with reactions that contained SOD to catalyze SOD peroxidase activity. 6MP was detected at its expected m/z (151.0086, C5H4N4S) and the major product peak found corresponded to an m/z of 182.9981 (C5H4N4O2S), representing a sulfoxide product. SOD inclusion in these reactions produced the same products but with only 30 min incubation time vs. 24 h without SOD. The reaction of CO3●– with 6-MP was confirmed by EPR and spectrometric measurements. SOD activity was significantly enhanced by 6-MP but not azathioprine and the CO3●– radicals were scavenged by only 6-MP. 6MP was oxidized by H2O2/HCO3– and treated with HEK293 and HepG2 cells. 6MP-sulfoxide was significantly less cytotoxicity to both cell lines compared to unadulterated 6MP.
Exposure of 6MP to H2O2/HCO3– with and without SOD significantly enhanced its oxidation, and led to the formation of a 6MP-sulfoxide, which loses its cytotoxic activity and consequently ameliorates SOD activity.