Pharmacokinetics/Pharmacodynamics of Y‐700, A Novel Xanthine Oxidase Inhibitor,in Rats and Man

The pharmacokinetics and pharmacodynamics of a novel xanthine oxidase (XO) inhibitor, Y‐700, were evaluated in rats and healthy male volunteers. In a rat model of hyperuricemia, oral Y‐700 (0.3–10 mg/kg) showed a more potent and a longer‐lasting hypouricemic action than allopurinol. A single oral dosing of Y‐700 (5, 20 or 80 mg) to volunteers caused a dose‐dependent reduction of serum uric acid levels indicating close relationship to plasma concentrations of the compound. In addition, Y‐700 was hardly excreted in urine but mainly excreted in feces in rats and volunteers. These results suggested that Y‐700 is a new effective inhibitor of XO in rats and humans with high oral bioavailability being predominantly eliminated via the liver unlikely to allopurinol.     

Novel insights into interactions between mitochondria and xanthine oxidase in acute cardiac volume overload

Xanthine oxidoreductase (XOR) is increased in the left ventricle (LV) of humans with volume overload (VO), and mitochondrial inhibition of the respiratory chain occurs in animal models of VO. Because mitochondria are both a source and a target of reactive oxygen and nitrogen species, we hypothesized that activation of XOR and mitochondrial dysfunction are interdependent. To test this we used the aortocaval fistula (ACF) rat model of VO and a simulation of the stretch response in isolated adult cardiomyocytes with and without the inhibitor of XOR, allopurinol, or the mitochondrially targeted antioxidant MitoQ. Xanthine oxidase (XO) activity was increased in cardiomyocytes from ACF vs sham rats (24h) without an increase in XO protein. A twofold increase in LV end-diastolic pressure/wall stress and a decrease in LV systolic elastance with ACF were improved when allopurinol treatment (100mg/kg) was started at ACF induction. Subsarcolemmal State 3 mitochondrial respiration was significantly decreased in ACF and normalized by allopurinol. Cardiomyocytes subjected to 3h cyclical stretch resulted in an increase in XO activity and mitochondrial swelling, which was prevented by allopurinol or MitoQ pretreatment. These studies establish an early interplay between cardiomyocyte XO activation and bioenergetic dysfunction that may provide a new target that prevents progression to heart failure in VO.     

Xanthine oxidase and mitochondria contribute to vascular superoxide anion generation in DOCA-salt hypertensive rats

Vascular superoxide anion (O(2)(*-)) levels are increased in DOCA-salt hypertensive rats. We hypothesized that the endothelin (ET)-1-induced generation of ROS in the aorta and resistance arteries of DOCA-salt rats originates partly from xanthine oxidase (XO) and mitochondria. Accordingly, we blocked XO and the mitochondrial oxidative phosphorylation chain to investigate their contribution to ROS production in mesenteric resistance arteries and the aorta from DOCA-salt rats. Systolic blood pressure rose in DOCA-salt rats and was reduced after 3 wk by apocynin [NAD(P)H oxidase inhibitor and/or radical scavenger], allopurinol (XO inhibitor), bosentan (ET(A/B) receptor antagonist), BMS-182874 (BMS; ET(A) receptor antagonist), and hydralazine. Plasma uric acid levels in DOCA-salt rats were similar to control unilaterally nephrectomized (UniNx) rats, reduced with allopurinol and bosentan, and increased with BMS. Levels of thiobarbituric acid-reacting substances were increased in DOCA-salt rats versus UniNx rats, and BMS, bosentan, and hydralazine prevented their increase. Dihydroethidium staining showed reduced O(2)(*-) production in mesenteric arteries and the aorta from BMS- and bosentan-treated DOCA-salt rats compared with untreated DOCA-salt rats. Increased O(2)(*-) derived from XO was reduced or prevented by all treatments in mesenteric arteries, whereas bosentan and BMS had no effect on aortas from DOCA-salt rats. O(2)(*-) generation decreased with in situ treatment by tenoyltrifluoroacetone and CCCP, inhibitors of mitochondrial electron transport complexes II and IV, respectively, whereas rotenone (mitochondrial complex I inhibitor) had no effect. Our findings demonstrate the involvement of ET(A) receptor-modulated O(2)(*-) derived from XO and from mitochondrial oxidative enzymes in arteries from DOCA-salt rats.     

Selectivity of febuxostat, a novel non-purine inhibitor of xanthine oxidase/xanthine dehydrogenase

The purine analogue, allopurinol, has been in clinical use for more than 30 years as an inhibitor of xanthine oxidase (XO) in the treatment of hyperuricemia and gout. As consequences of structural similarities to purine compounds, however, allopurinol, its major active product, oxypurinol, and their respective metabolites inhibit other enzymes involved in purine and pyrimidine metabolism. Febuxostat (TEI-6720, TMX-67) is a potent, non-purine inhibitor of XO, currently under clinical evaluation for the treatment of hyperuricemia and gout. In this study, we investigated the effects of febuxostat on several enzymes in purine and pyrimidine metabolism and characterized the mechanism of febuxostat inhibition of XO activity. Febuxostat displayed potent mixed-type inhibition of the activity of purified bovine milk XO, with Ki and Ki' values of 0.6 and 3.1 nM respectively, indicating inhibition of both the oxidized and reduced forms of XO. In contrast, at concentrations up to 100 muM, febuxostat had no significant effects on the activities of the following enzymes of purine and pyrimidine metabolism: guanine deaminase, hypoxanthine-guanine phosphoribosyltransferase, purine nucleoside phosphorylase, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. These results demonstrate that febuxostat is a potent non-purine, selective inhibitor of XO, and could be useful for the treatment of hyperuricemia and gout.     

Renierol from marine sponge Haliclona.SP.: a natural inhibitor of xanthine oxidase with hypouricemic effects

The purpose of this study was to evaluate the inhibitory effect of renierol, extracted from marine sponge Halicdona.SP., on xanthine oxidase (XO) and its hypouricemic effect in vivo. Renierol and a positive control, allopurinol, were tested for their effects on XO activity by measuring the formation of uric acid and superoxide radical from xanthine. Renierol inhibited XO in a concentration-dependent and competitive manner. IC(50) value was 1.85 microg.ml(-1) through the measuring of uric acid and was 1.36 microg.ml(- 1) through the measuring of superoxide radical. Renierol was found to have an in vivo hypouricemic activity against potassium oxonate-induced hyperuricaemia in mice. After oral administration of renierol at doses of 10, 20 and 30 mg.kg(- 1), there was a significant decrease in the serum urate level (4.08 +/- 0.09 mg.dl(- 1), P < 0.01), (3.47 +/- 0.11 mg.dl(- 1), P < 0.01) and (3.12 +/- 0.08 mg.dl(- 1), P < 0.01), when compared to the hyperuricaemic control (6.74 +/- 0.23 mg.dl(- 1)). Renierol was a potent XO inhibitor with hypouricemic activity in mice.     

Synthesis and structure-activity relationships of 1-phenylpyrazoles as xanthine oxidase inhibitors

A series of 1-phenylpyrazoles was evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds prepared, 1-(3-cyano-4-neopentyloxyphenyl)pyrazole-4-carboxylic acid (Y-700) had the most potent enzyme inhibition and displayed longer-lasting hypouricemic action than did allopurinol in a rat model of hyperuricemia induced by the uricase inhibitor potassium oxonate.     

Xanthine oxidase-derived reactive oxygen metabolites contribute to liver necrosis: protection by 4-hydroxypyrazolo[3,4-d]pyrimidine

Xanthine oxidase (XO) generates reactive oxygen metabolites (ROM) as a by-product while catalyzing their reaction. The present study implicates these ROM in the pathogenesis of liver necrosis produced in rats by the intraperitoneal administration of thioacetamide (TAA; 400 mg/kg b.wt.). After 16 h of TAA administration, the activity of rat liver XO increased significantly compared to that of the control group. At the same time, the level of serum marker enzymes of liver necrosis (aminotransferases and alkaline phosphatase) and tissue malondialdehyde content also increased in TAA treated rats. Tissue malondialdehyde concentration is an indicator of lipid peroxidation and acts as a useful marker of oxidative damage. Pretreatment of rats with XO inhibitor (4-hydroxypyrazolo[3,4-d]pyrimidine; allopurinol (AP)) followed by TAA could lower the hepatotoxin-mediated rise in malondialdehyde level as well as the level of marker enzymes associated with liver necrosis. The survival rate also increased in rats given AP followed by the lethal dose of TAA. In either case, the effect of AP was dose-dependent. Results presented in the paper indicate that increased production of XO-derived ROM contributes to liver necrosis, which can be protected by AP.     

Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta

Evidence indicates that both the Rho/Rho kinase signaling pathway and reactive oxygen species (ROS) such as superoxide and H(2)O(2) are involved in the pathogenesis of hypertension. This study aimed to determine whether ROS-induced vascular contraction is mediated through activation of Rho/Rho kinase. Rat aortic rings (endothelium denuded) were isolated and placed in organ chambers for measurement of isometric force development. ROS were generated by a xanthine (X)-xanthine oxidase (XO) mixture. The antioxidants tempol (3 mM) and catalase (1,200 U/ml) or the XO inhibitor allopurinol (400 microM) significantly reduced X/XO-induced contraction. A Rho kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl-N-4-pyridil)cyclohexanecarboxamide dihydrochloride (Y-27632), decreased the contraction in a concentration-dependent manner; however, the Ca(2+)-independent protein kinase C inhibitor rottlerin did not have an effect on X/XO-induced contraction. Phosphorylation of the myosin light chain phosphatase target subunit (MYPT1) was increased by ROS, and preincubation with Y-27632 blocked this increased phosphorylation. Western blotting for cytosolic and membrane-bound fractions of Rho showed that Rho was increased in the membrane fraction by ROS, suggesting activation of Rho. These observations demonstrate that ROS-induced Ca(2+) sensitization is through activation of Rho and a subsequent increase in Rho kinase activity but not Ca(2+)-independent PKC.     

Allopurinol attenuates L-NAME induced cardiomyopathy comparable to blockade of angiotensin receptor

It is widely recognized that L-NAME exposed rats develop myocardial fibrosis and hypertrophy. The aim of this study was to evaluate the contribution of xanthine oxidase (XO) to these phenomena using allopurinol, isolated or associated with olmesartan. Thirty adult male Wistar rats were divided into 5 groups (n=6) and studied for 5 weeks: L group (L-NAME, 40mg/kg/day); L+A group (L-NAME and allopurinol, 40 mg/kg/day); L+O group (L-NAME and olmesartan, 15mg/kg/day); L+A+O group (L-NAME, allopurinol, and olmesartan); and control group. L-NAME caused arterial hypertension and cardiomyocyte hypertrophy. Hypertension was prevented by olmesartan, but not by allopurinol. There was an increase of left ventricular mass index in the L-NAME group that was prevented by allopurinol, olmesartan and by the combination of both. The increase in mean cardiomyocyte transversal area caused by L-NAME was prevented by the allopurinol and olmesartan combination, or by olmesartan used as monotherapy, but not by allopurinol alone. There was a reduction in the myocardial vascularization index caused by L-NAME which was abolished by allopurinol or by olmesartan, but not by the association. L-NAME caused a reduction in the total number of cardiomyocyte nuclei. This was prevented by olmesartan alone or associated with allopurinol, but not by allopurinol alone. We conclude that XO has an important contribution to adverse cardiac remodeling in L-NAME exposed animals. Moreover, allopurinol acts without interfering with L-NAME induced hypertension. The protective action of this drug is comparable to the results obtained with olmesartan. Antioxidative mechanisms are proposed to account for the pressure independent effects of allopurinol.     

Implication of xanthine oxidase in muscle oxidative stress in COPD patients

Objective: The aim of this study was to determine the implication of xanthine oxidase (XO) in the exercise-induced muscle oxidative stress and muscle dysfunction of these patients.

Methods: A randomized, crossover and double-blind study was conducted in nine severe COPD patients, who performed a localized quadriceps endurance test after oral treatment with allopurinol, a XO inhibitor or placebo. Redox status was studied in arterial and venous femoral blood before and after the endurance test.

Results: In placebo condition, muscle exercise resulted in a significant increase in AOPP and isoprostanes, with a significant increase in the venoarterial difference (v-a) in isoprostanes after exercise as compared with before (p<0.05). In contrast, allopurinol treatment prevented the elevation in AOPP levels and v-a isoprostanes after exercise. However, no significant improvement in quadriceps muscle endurance was observed, but allopurinol treatment seemed to preserve muscle strength properties.

Conclusion: This study demonstrates that XO is implicated in the exercise-induced muscle oxidative stress of COPD patients. Allopurinol administration seemed to improve only some muscle properties. Therefore other sources of muscle oxidative stress should be implicated in muscle dysfunction observed in these patients.     

Effects of Greek legume plant extracts on xanthine oxidase,catalase and superoxide dismutase activities

Legumes are considered to have beneficial health implications, which have been attributed to their phytochemical content. Polyphenols are considered the most important phytochemical compounds extensively studied for their antioxidant properties. The aim of the present study was to examine the effects of potent antioxidant legume plant extracts on xanthine oxidase (XO), catalase (CAT) and superoxide dismutase (SOD) activities. XO exerts a dual role, as it is the major contributor of free radicals during exercise while it generates uric acid, the most potent antioxidant molecule in plasma. CAT and SOD are two of the main enzymes of the antioxidant defence of tissues. We demonstrate that the majority of the extracts inhibited XO activity, but they had no effect on CAT inhibition and SOD induction when used at low concentrations. These results imply that the tested extracts may be considered as possible source of novel XO inhibitors. However, we have shown that allopurinol administration, a known XO inhibitor, before exercise reduces performance and induces oxidative stress in rats. Considering the fact that the extracts examined had an inhibitory effect on XO activity, possibly posing a restriction in their characterization as antioxidants, phytochemical antioxidant administration before exercise should probably be reconsidered.     

Renierol from marine sponge Haliclona.SP.: A natural inhibitor of xanthine oxidase with hypouricemic effects

The purpose of this study was to evaluate the inhibitory effect of renierol, extracted from marine sponge Halicdona.SP., on xanthine oxidase (XO) and its hypouricemic effect in vivo. Renierol and a positive control, allopurinol, were tested for their effects on XO activity by measuring the formation of uric acid and superoxide radical from xanthine. Renierol inhibited XO in a concentration-dependent and competitive manner. IC₅₀ value was 1.85 μg·ml^{? 1} through the measuring of uric acid and was 1.36 μg.ml^{? 1} through the measuring of superoxide radical. Renierol was found to have an in vivo hypouricemic activity against potassium oxonate-induced hyperuricaemia in mice. After oral administration of renierol at doses of 10, 20 and 30 mg.kg^{? 1}, there was a significant decrease in the serum urate level (4.08 ± 0.09 mg.dl^{? 1}, P < 0.01), (3.47 ± 0.11 mg.dl^{? 1}, P < 0.01) and (3.12 ± 0.08 mg.dl^{? 1}, P < 0.01), when compared to the hyperuricaemic control (6.74 ± 0.23 mg.dl^{? 1}). Renierol was a potent XO inhibitor with hypouricemic activity in mice.     

Generation of oxidative stress contributes to the development of pulmonary hypertension induced by hypoxia.

Chronic hypoxia causes pulmonary hypertension and right ventricular hypertrophy associated with pulmonary vascular remodeling. Because hypoxia might promote generation of oxidative stress in vivo, we hypothesized that oxidative stress may play a role in the hypoxia-induced cardiopulmonary changes and examined the effect of treatment with the antioxidant N-acetylcysteine (NAC) in rats. NAC reduced hypoxia-induced cardiopulmonary alterations at 3 wk of hypoxia. Lung phosphatidylcholine hydroperoxide (PCOOH) increased at days 1 and 7 of the hypoxic exposure, and NAC attenuated the increase in lung PCOOH. Lung xanthine oxidase (XO) activity was elevated from day 1 through day 21, especially during the initial 3 days of the hypoxic exposure. The XO inhibitor allopurinol significantly inhibited the hypoxia-induced increase in lung PCOOH and pulmonary hypertension, and allopurinol treatment only for the initial 3 days also reduced the hypoxia-induced right ventricular hypertrophy and pulmonary vascular thickening. These results suggest that oxidative stress produced by activated XO in the induction phase of hypoxic exposure contributes to the development of chronic hypoxic pulmonary hypertension.     

Inhibition of Xanthine Oxidase by Allopurinol Prevents Skeletal Muscle Atrophy: Role of p38 MAPKinase and E3 Ubiquitin Ligases

Alterations in muscle play an important role in common diseases and conditions. Reactive oxygen species (ROS) are generated during hindlimb unloading due, at least in part, to the activation of xanthine oxidase (XO). The major aim of this study was to determine the mechanism by which XO activation causes unloading-induced muscle atrophy in rats, and its possible prevention by allopurinol, a well-known inhibitor of this enzyme. For this purpose we studied one of the main redox sensitive signalling cascades involved in skeletal muscle atrophy i.e. p38 MAPKinase, and the expression of two well known muscle specific E3 ubiquitin ligases involved in proteolysis, the Muscle atrophy F-Box (MAFbx; also known as atrogin-1) and Muscle RING (Really Interesting New Gene) Finger-1 (MuRF-1). We found that hindlimb unloading induced a significant increase in XO activity and in the protein expression of the antioxidant enzymes CuZnSOD and Catalase in skeletal muscle. The most relevant new fact reported in this paper is that inhibition of XO with allopurinol, a drug widely used in clinical practice, prevents soleus muscle atrophy by ∼20% after hindlimb unloading. This was associated with the inhibition of the p38 MAPK-MAFbx pathway. Our data suggest that XO was involved in the loss of muscle mass via the activation of the p38MAPK-MAFbx pathway in unloaded muscle atrophy. Thus, allopurinol may have clinical benefits to combat skeletal muscle atrophy in bedridden, astronauts, sarcopenic, and cachexic patients.     

6-(N-benzoylamino)purine as a novel and potent inhibitor of xanthine oxidase: inhibition mechanism and molecular modeling studies

The inhibition of xanthine oxidase (XO) activity by the purine analogue 6-(N-benzoylamino)purine was evaluated and compared with the standard inhibitor, allopurinol and the parent compound adenine. 6-(N-benzoylamino)purine is a highly potent inhibitor of XO (IC50 = 0.45 microM) and comparable to allopurinol (IC50 = 0.80 microM). Furthermore, 6-(N-benzoylamino)purine neither produced any enzymatic superoxide nor reduced XO by an electron transfer reaction unlike allopurinol. 6-(N-benzoylamino)purine (Ki = 0.0475 microM) is about 10000-fold more potent as a XO inhibitor compared to the only known purine analogue 8-bromoxanthine (Ki = 400 microM). 6-(N-Benzoylamino)purine is a competitive inhibitor of XO and the inhibition was not completely reversed even at 100 microM xanthine concentration. The calculated interaction energy [Ecomplex - (Eligand + Eprotein)] of -30.5, -22.6, and -17.2 kcal/mol, respectively, of 6-(N-benzoylamino)purine, 8-bromoxanthine and the parent compound adenine provided the rationale for the better enzyme inhibitory activity of 6-(N-benzoylamino)purine. To understand the role of the benzamido group in the inhibition process, molecular docking studies were carried out and it was revealed that the hydrogen bonding interactions involving N-7 of the purine ring and the N-H of Arg880, N-H of the purine ring and OH of Thr1010, as well as non-bonded interactions of the benzamido group of 6-(N-benzoylamino)purine with amino acid residues Gly799, Glu802, Phe914, Ala1078, Ala1079 and Glu1261 in the active site of XO play an important role in the stabilization of the E-I complex.     

Chronic xanthine oxidase inhibition following myocardial infarction in rabbits: effects of early versus delayed treatment

Xanthine oxidase (XO) expression is increased in the failing heart, and animal studies in rodents and dogs showed that XO inhibition with allopurinol can improve left ventricular (LV) function and myocardial oxygen efficiency in the failing heart. The purpose of this study was to determine whether chronic XO inhibition by allopurinol or febuxostat, an investigational, potent non-purine, selective inhibitor of XO, could prevent or treat the progression of congestive heart failure (CHF) induced by coronary artery ligation in rabbits, a species that exhibits low intrinsic XO activity similar to humans. One day after coronary ligation, rabbits were assigned to one of four groups (n = 7-8/group): control group (vehicle for 49 days), early treatment (prevention) group (febuxostat for 49 days), and two delayed-treatment groups (vehicle for 21 days followed by either febuxostat or allopurinol for 28 days). An echocardiogram of the LV was obtained on Days 0 (prior to surgery), 21, and 49. Control rabbits developed CHF by Day 21 (significant reduction in LV shortening fraction and ejection fraction, thinning of the LV posterior wall, and increases in LV internal dimensions and end-diastolic volume). Early preventive treatment with febuxostat significantly lessened the reduction of LV function when compared to vehicle on both Days 21 and 49. These cardiac functional improvements were accompanied by moderately less severe changes in LV dimensional parameters relative to vehicle controls. In contrast, when treatments with XO inhibitors were started after the establishment of CHF, no significant relative improvements in cardiac functional or dimensional parameters were observed. These results suggest that chronic preventive treatment with an XO inhibitor initiated shortly after myocardial infarction can delay or prevent the onset of CHF, and that XO inhibition initiated after establishment of the disease does not offer cardiac protection. In contrast to previous rodent studies which do suggest a cardiovascular (CV) benefit of delayed XO inhibition, the results of this rabbit study are in keeping with those of recently completed studies in severe CHF patients treated with oxypurinol, the active metabolite of allopurinol, in which no clinical benefit was observed. This may be due to the fact that rodents have relatively high levels of XO activity, while the levels in rabbits and humans are intrinsically low, suggesting that the rabbit may be the preferred model for investigating the role of XO in CV diseases.     

Alopurinol y su papel en el tratamiento de la sarcopenia

Xanthine oxidase (XO) is an enzyme that catalyzes the oxidation of hypoxanthine to xanthine and uric acid and plays an important role in purine catabolism. The purine analogue, allopurinol, is a well-known inhibitor of XO widely used in the clinical management of gout and conditions associated with hyperuricemia. More recent data indicate that allopurinol reduces oxidative stress and improves vascular function in several cardiometabolic diseases, prolongs exercise time in angina, and improves the efficiency of cardiac contractility in heart failure. XO also plays an important role in free radical generation during skeletal muscle contraction and thus, it has been related to the muscle damage associated to exhaustive exercise. Several research groups have shown the protective effect of allopurinol in the prevention of this type of damage.     

N-ethylmaleimide inhibits xanthine oxidase activity with no detectable change in xanthine dehydrogenase activity in rabbit liver

The effect of an alkylating agent, N-ethylmaleimide (NEM), on the activities of xanthine oxidase (XO) and xanthine dehydrogenase (XD) in the presence and absence of Cu2+ or trypsin in the cytosolic fraction from rabbit liver was examined. At concentrations ranging from 0.25 to 2.0 microM, allopurinol, which is generally considered to be a XO inhibitor, suppressed the XD activity (41.5-93.4% inhibition) in addition to the XO activity (28.6-88.4% inhibition) under basal conditions, without the addition of Cu2+ or trypsin. In contrast, NEM (100-400 microM) inhibited the XO activity (35.7-85.7% inhibition) without affecting the XD activity. Also, NEM inhibited the Cu2+- and trypsin-induced XO activities, but did not affect the XD activity at the same concentration range. These results demonstrate that NEM can be a selective inhibitor of XO activity in rabbit liver.     

A Reappraisal of Xanthine Dehydrogenase and Oxidase in Hypoxic Reperfusion Injury: the Role of NADH as an Electron Donor

Xanthine oxidase (XO) is conventionally known as a generator of reactive oxygen species (ROS) which contribute to hypoxic-reperfusion injury in tissues. However, this role for human XO is disputed due to its distinctive lack of activity towards xanthine, and the failure of allopurinol to suppress reperfusion injury. In this paper, we have employed native gel electrophore-sis together with activity staining to investigate the role human xanthine dehydrogenase (XD) and XO in hypoxic reperfusion injury. This approach has provided information which cannot be obtained by conventional spectrophotometric assays. We found that both XD and XO of human umbilical vein endothelial cells (HUVECs) and lymphoblastic leukaemic cells (CEMs) catalysed ROS generation by oxidising NADH, but not hypoxanthine. The conversion of XD to XO was observed in both HUVECs and CEMs in response to hypoxia, although the level of conversion varied. Purified human milk XD generated ROS more efficiently in the presence of NADH than in the presence of hypoxanthine. This NADH oxidising activity was blocked by the FAD site inhibitor, diphenyleneiodo-nium (DPI), but was not suppressible by the molybdenum site inhibitor, allopurinol. However, in the presence of both DPI and allopwinol the activities of XD/XO were completely blocked with either NADH or hypoxanthine as substrates. We conclude that both human XD and XO can oxidise NADH to generate ROS. Therefore, the conversion of XD to XO is not necessary for post-ischaemic ROS generation. The hypoxic-reperfusion injury hypothesis should be reappraised to take into account the important role played by XD and XO in oxidising NADH to yield ROS.     

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

Gout patients receiving a combination of allopurinol and furosemide require higher allopurinol doses to achieve the target serum urate (SU) of <6 mg/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.