Biochemical and Biophysical Research Communications
Molecular mechanism of an adverse drug–drug interaction of allopurinol and furosemide in gout treatment
Introduction
The xanthine oxidase (XO) inhibitor allopurinol, or rather its pharmacologically active metabolite oxypurinol [2], is the most common clinical treatment for abnormally high serum urate (SU) levels in gout patients [3], [4]. Frequently, patients with gout also suffer from co-morbidities such as cardiac or kidney problems that require additional drug treatment with diuretics, particularly furosemide. Belonging to the class of loop diuretics, furosemide primarily acts by inhibiting NKCC2, the luminal Na+/K+/2Cl- symporter in the thick ascending limb of the loop of Henle. Studies have shown that the combination of furosemide and oxypurinol decreases the urinary excretion of uric acid and oxypurinol [5], and initially it was speculated that this interaction might render the hypouricemic effect of allopurinol more potent. A more recent clinical report by Stamp and co-workers [1] showed that patients receiving allopurinol and furosemide indeed exhibited increased plasma oxypurinol levels as well as increased SU. Compared to patients receiving a similar allopurinol dose alone, however, they needed higher allopurinol doses to achieve the target SU of <6 mg/dl. This observation indicates that the hypouricemic effect of allopurinol is in fact attenuated by the addition of furosemide. Increased SU after co-administration of furosemide is generally thought to be due to the plasma volume reduction or secondary enhancement of sodium re-absorption that is coupled to urate. However, the molecular mechanisms underlying this drug interaction are not yet understood.
Oxypurinol works by competitively inhibiting the XO enzyme (Fig. 1A), but it is unknown whether furosemide exhibits a direct effect on XO as well or if it hampers the binding of oxypurinol to XO. The latter hypothesis could explain the observed adverse effect of the combination of furosemide and allopurinol. Even if direct interactions may be excluded, there are diverse regulatory pathways within the cell that could be subject to changes by the combination of furosemide and oxypurinol: (1) different expression of XO, (2) changes in levels of regulatory proteins or miRNAs, (3) changes in urate/drug transporter expression or function.
The latter has already partly been addressed. Renal secretion of diuretics such as furosemide or thiazides is crucial for their pharmacologic effect. However, this mechanisms of diuretic secretion has been recognised to cause high SU or hyperuricemia (SU > 7 mg/dl) [6], [7]. We and others have shown that organic anion transporters such as OAT1 and OAT3 [8], OAT4 ([8], [9], [10]), NPT4 [11] or MRP4 [12] are involved in the renal secretion of diuretics such as furosemide (Fig. 1B). Since all of these transporters are also uric acid transporters, the concomitance of diuretics and urate alters normal excretion patterns: Diuretics can either inhibit the luminal secretion of uric acid (in case of unidirectional transporters such as NPT4 or MRP4) or the excretion of the diuretic can simultaneously facilitate the uptake of uric acid from the luminal side (OAT1–4). Both mechanisms lead to hyperuricemia.
Recently, studies have shown that AMP-kinase (AMPK) is involved in renal uric acid transport in avian renal proximal tubules [13]. Under stress conditions AMPK is activated, resulting in a decrease of uric acid secretion via MRP4, which is the only renal uric acid secreting transporter in birds. This illustrates that AMPK can be pivotal in the regulation of transporters that are involved in the clearance of urate and furosemide. Assuming a similar mechanism exists in humans, furosemide treatment could alter urate transport via activation of AMPK. Whether AMPK is affected by furosemide in human proximal renal tubule cells is currently unknown.
The aim of this study was to elucidate these drug interactions on a molecular level. Firstly, we studied direct interactions between oxypurinol and furosemide (alone and in combination) on purified XO enzyme in a cell-free assay. We then explored the effects of these drugs on expression levels of XO in cultured human liver cells (HepG2) by immunoblot. Moreover, we identified miR-448 as a potential XO-regulator and analysed its expression levels in drug-treated HepG2 cells. We also analysed AMPK expression in drug-treated primary human renal cortical epithelial (HRCE) cells to look at kidney specific drug effects on possible regulation of uric acid transport.
Section snippets
Xanthine oxidase assay
Direct interactions between oxypurinol and/or furosemide and purified XO enzyme were tested using the fluorimetric Amplex Red Xanthine/Xanthine Oxidase Assay (Invitrogen) according to the manufacturer’s protocol. For an activity standard curve, XO concentrations between 0 and 4 mU/ml were used. From this curve a concentration well within the linear part of the curve (0.75 mU/ml) was chosen for further experiments. Oxypurinol concentrations in the range from 0 to 300 μM and furosemide
No direct interactions between furosemide and XO
We first examined the activity of a series of XO concentrations in the range of 0–4 mU/ml (Fig. 2A). From this curve we chose the concentration of 0.75 mU/ml for further experiments and tested the effect of various concentrations of oxypurinol or furosemide (Fig. 2B) on XO activity. As expected for a direct inhibitor, oxypurinol revealed a dose-dependent inhibition of XO activity. Contrary, furosemide alone (up to 1200 μM) had no effect on XO activity, nor did it alter oxypurinol-mediated XO
Discussion
From our experiments on the interactions of oxypurinol and furosemide on purified XO protein, we conclude that furosemide does not directly interact with XO and does not influence binding of oxypurinol to XO. These findings imply a more complex drug effect within the cellular environment.
We also demonstrate for the first time that the efficiency of oxypurinol in lowering SU could also be due to a suppression of XO protein expression in addition to its known direct inhibitory effect on XO
Conclusion
Our findings widen the understanding of the molecular mechanisms of these commonly used drugs and pinpoint adverse drug interactions. Thus, this study could pave the way to designing further studies on drug-induced molecular changes and interactions, eventually leading to the finding of more efficient treatment options for gout or other rheumatic diseases. Furthermore, this study highlights the potential usefulness of incorporating the patient’s miRNA status into the treatment strategy, getting
Conflict of interest
The authors report no competing interests.
Funding
This work was supported by an Otago Medical School Laurenson Research Grant (Otago Medical Research Foundation).
Acknowledgments
We would like to acknowledge Dr. Martin Fronius and Dr. Jeff Erickson for help with editing and proofreading of the manuscript.
References (25)
Oxypurinol as an inhibitor of xanthine oxidase-catalyzed production of superoxide radical
Biochem. Pharmacol.
(1988)- et al.
Effect of furosemide on renal excretion of oxypurinol and purine bases
Metabolism
(2001) - et al.
Human sodium phosphate transporter 4 (hNPT4/SLC17A3) as a common renal secretory pathway for drugs and urate
J. Biol. Chem.
(2010) - et al.
Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets
Cell
(2005) - et al.
Plasma urate level is directly regulated by a voltage-driven urate efflux transporter URATv1 (SLC2A9) in humans
J. Biol. Chem.
(2008) Repressed expression of the human xanthine oxidoreductase gene. E-box and TATA-like elements restrict ground state transcriptional activity
J. Biol. Chem.
(2000)- et al.
MicroRNA pharmacogenomics: post-transcriptional regulation of drug response
Trends Mol. Med.
(2011) - et al.
Furosemide increases plasma oxypurinol without lowering serum urate – a complex drug interaction: implications for clinical practice
Rheumatology (Oxford)
(2012) - et al.
The treatment of gout and disorders of uric acid metabolism with allopurinol
Can. Med. Assoc. J.
(1966) Clinical practice. Gout
N. Engl. J. Med.
(2011)