Molecular mechanism of an adverse drug–drug interaction of allopurinol and furosemide in gout treatment

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Gout patients receiving a combination of allopurinol and furosemide require higher allopurinol doses to achieve the target serum urate (SU) of <6mg/dl (Stamp et al., 2012) [1]. Our study aimed to identify the molecular basis for this observation. We used a fluorimetric assay to determine the impact of furosemide and oxypurinol (the active metabolite of allopurinol) on xanthine oxidase (XO) activity. Immunoblot analysis quantified expression of XO and AMP-kinase (AMPK) in drug-treated human liver (HepG2) and primary kidney (HRCE) cells. In silico analysis identified miR-448 as a potential XO-regulator, whose expression level in HepG2 cells was examined by qPCR.Fluorimetric experiments revealed no direct interactions between XO and furosemide, nor did the combination of oxypurinol/furosemide alter the XO inhibition profile of oxypurinol. In HepG2 cells, we found a significant decrease in XO protein expression following oxypurinol treatment, which was abolished after co-incubation with furosemide. Probenecid alone or in combination with furosemide reduced XO protein expression significantly. qPCR analysis of miR-448 in HepG2 cells mirrored the drug-dependent changes in XO protein expression. In addition, oxypurinol and the combination of oxypurinol/furosemide significantly down-regulated AMPK protein expression in HRCE cells.In conclusion, we show for the first time that besides the established effects of allopurinol on the purine synthetic pathway the efficiency of allopurinol treatment of gout patients is based on two further complementary mechanisms, the direct inhibition of XO activity by the allopurinol metabolite oxypurinol and a down-regulation of XO protein expression. The latter is compromised by addition of furosemide and might explain why patients receiving furosemide therapy require higher allopurinol doses. miR-448 was identified as a potential drug-dependent XO regulator. Finally, down-regulation of AMPK protein expression in HRCE cells by administration of oxypurinol/furosemide reveals a possible new mechanism of renal drug-induced hyperuricemia.