Background/Purpose: The xanthine oxidase (XO) inhibitor allopurinol is the most commonly used urate lowering therapy in gout. Allopurinol is metabolized to oxypurinol, which is responsible for most of the XO inhibition. Co-morbidities, such as hypertension, are common in gout patients and result in concomitant therapy with diuretics such as furosemide, which is known to inhibit renal urate clearance. It has been reported that patients receiving furosemide and allopurinol have higher serum urate (SU) and oxypurinol concentrations compared to patients’ not receiving furosemide. The aim of this study was to identify the molecular basis for this observation.

Methods: The effect of oxypurinol and furosemide (alone and in combination) on XO activity was determined using a cell-free in vitro assay. HepG2 cells were incubated for 24 hours with oxypurinol (250µM), furosemide (1mM), probenecid (250µM) or a combination of oxypurinol/furosemide or probenecid/furosemide. XO protein expression was determined by Western blot analysis. In silico analysis revealed that miR-448 could act as a potential regulator of XO expression by binding to the 3’UTR of the XO gene. miR-448 expression was examined in the presence of oxypurinol, furosemide, probenecid or a combination  by qPCR.

Results: Oxypurinol inhibited XO activity, an effect that was maintained in the presence of furosemide within the examined physiologically-relevant activity range of 1-7 mU/mL. There was a significant reduction in XO gene expression in HepG2 cells after incubation with oxypurinol. This reduction in XO gene expression was blocked by the addition of furosemide (Fig 1). Probenecid also led to a reduction in XO gene expression, but this effect was not reversed by the addition of furosemide.  miR-448 showed a mirrored expression pattern  to XO gene expression in the presence of oxypurinol, furosemide, probenecid or a combination . However, these results did not reach statistical significance.

Conclusion: Oxypurinol may have effects on urate production by down-regulation of XO gene expression as well as by inhibiting XO function. Furosemide in combination with oxypurinol appears to have effects on XO expression, which may contribute to the observed increase in SU in addition to its effects on renal urate clearance. miR-448 may be a drug dependent regulator of XO expression. These effects on XO gene expression may contribute to the relative inefficiency of allopurinol in urate lowering in patients receiving furosemide. The lack of effect of probenecid, in combination with furosemide, on XO expression suggests it may be a better therapeutic option in patients receiving furosemide. Further clinical studies in patients with gout receiving different urate lowering therapies with and without furosemide are required.

Figure 1: XO expression in HepG2 cells treated with oxypurinol (250µM), furosemide (1mM), probenecid (250µM) or a combination compared to non-treated cells (Ctrl). *p<0.05)