THE XOR INHIBITORY EFFECT OF ALLOPURINOL AND OXYPURINOL: WHERE AND HOW DO THESE COMPOUNDS WORK?
M. Sekine, K. Okamoto, K. Nagata, T. Nishino
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Science, The University of Tokyo, Japan
Introduction: Xanthine oxidoreductase (XOR) catalyzes the final two step reaction in purine metabolism, the oxidation of hypoxanthine to xanthine and xanthine to uric acid. Inhibitors of XOR are used as anti-gout drugs and divided into purine and non-purine analogs. Allopurinol, an analog of hypoxanthine, has been used for a long time and proven to be an effective drug, although it has not frequently but severe side effects such as Steven Jonson syndrome, most frequently in Asian people. Allopurinol is almost converted to oxypurinol in vivo by XOR or partially aldehyde oxidase (AO). Mechanistically, allopurinol itself serves as a substrate of XOR to reduce Mo (VI) of Moco possessed by the enzyme protein. The electrons are transferred to FAD via two iron-sulfur centers, and finally to NAD+ or oxygen. Oxypurinol inhibits by forming covalent binding with transiently formed Mo (IV) in this reaction. Therefore, allopurinol is a time-dependent suicide inhibitor. In contrast to allopurinol, xanthine is required as an enzyme reducing agent for oxypurinol itself to inhibit XOR. This work compares the XOR inhibitory effects of allopurinol and oxypurinol.
Methods: We measured activity by spectrophotometer and high-performance liquid chromatography (HPLC) using XOR purified from bovine milk.
Results: We present detailed data that the XOR inhibitory effect of oxypurinol was much less effective than that of allopurinol. Moreover, in the two-step reaction, the reaction of hypoxanthine to xanthine was less inhibited than the step of xanthine to uric acid formation. The half-life of the formed XOR-oxypurinol complex was approximately 1 hour at 37°C. Furthermore, we found that oxypurinol inhibited PNPase allosterically very strongly above a certain concentration.
Conclusion: The present data suggest that the inhibitory effect of oxypurinol itself in blood is not sufficient. We believe that the major contribution to lowering uric acid in blood after administration of allopurinol is mainly due to allopurinol itself according to suicide-manner inhibition mainly in the organs where XOR exists in large amounts, such as the liver, intestine, and others. The contribution of oxipurinol in blood should be minor in other organs where XOR is expressed in small amounts. Since a low concentration of allopurinol provides a sufficient inhibitory effect, it can be preferable to administer lower amounts in several divided doses per day as recommended before by G.B. Elion (Wellcome Co.). Recently performed or performing direct comparative clinical studies using the same protocol (once a day administration) between two kinds of inhibitor, allopurinol, and febuxostat, of those mechanisms and CPK are largely different, are questionable.