Design, synthesis, and biological evaluation of 3-phenyl substituted pyridine derivatives as potential dual inhibitors of XOR and URAT1.

Xanthine oxidoreductase (XOR) and uric acid transporter 1 (URAT1) are two most widely studied targets involved in production and reabsorption of uric acid, respectively. Marketed drugs almost target XOR or URAT1, but sometimes, single agents might not achieve aim of lowering uric acid to ideal value in clinic. Thus, therapeutic strategies of combining XOR inhibitors with uricosuric drugs were proposed and implemented. Based on our initial work of virtual screening, A and B were potential hits for dual-targeted inhibitors on XOR/URAT1. By docking A/B with XOR/URAT1 respectively, compounds I1-7 were designed to get different degree of inhibition effect on XOR and URAT1, and I7 showed the best inhibitory effect on XOR (IC50 = 0.037 ± 0.001 µM) and URAT1 (IC50 = 546.70 ± 32.60 µM). Further docking research on I7 with XOR/URAT1 led to the design of compounds II with the significantly improved inhibitory activity on XOR and URAT1, such as II11 and II15. Especially, for II15, the IC50 of XOR is 0.006 ± 0.000 µM, superior to that of febuxostat (IC50 = 0.008 ± 0.000 µM), IC50 of URAT1 is 12.90 ± 2.30 µM, superior to that of benzbromarone (IC50 = 27.04 ± 2.55 µM). In acute hyperuricemia mouse model, II15 showed significant uric acid lowering effect. The results suggest that II15 had good inhibitory effect on XOR/URAT1, with the possibility for further investigation in in-vivo models of hyperuricemia.

Xanthine Oxidoreductase Inhibitors Suppress the Onset of Exercise-Induced AKI in High HPRT Activity Urat1-Uox Double Knockout Mice.

BACKGROUND: Hereditary renal hypouricemia type 1 (RHUC1) is caused by URAT1/SLC22A12 dysfunction, resulting in urolithiasis and exercise-induced AKI (EIAKI). However, because there is no useful experimental RHUC1 animal model, the precise pathophysiologic mechanisms underlying EIAKI have yet to be elucidated. We established a high HPRT activity Urat1-Uox double knockout (DKO) mouse as a novel RHUC1 animal model for investigating the cause of EIAKI and the potential therapeutic effect of xanthine oxidoreductase inhibitors (XOIs). METHODS: The novel Urat1-Uox DKO mice were used in a forced swimming test as loading exercise to explore the onset mechanism of EIAKI and evaluate related purine metabolism and renal injury parameters. RESULTS: Urat1-Uox DKO mice had uricosuric effects and elevated levels of plasma creatinine and BUN as renal injury markers, and decreased creatinine clearance observed in a forced swimming test. In addition, Urat1-Uox DKO mice had increased NLRP3 inflammasome activity and downregulated levels of Na+-K+-ATPase protein in the kidney, as Western blot analysis showed. Finally, we demonstrated that topiroxostat and allopurinol, XOIs, improved renal injury and functional parameters of EIAKI. CONCLUSIONS: Urat1-Uox DKO mice are a useful experimental animal model for human RHUC1. The pathogenic mechanism of EIAKI was found to be due to increased levels of IL-1ß via NLRP3 inflammasome signaling and Na+-K+-ATPase dysfunction associated with excessive urinary urate excretion. In addition, XOIs appear to be a promising therapeutic agent for the treatment of EIAKI.

Influence of xanthine oxidoreductase inhibitor, topiroxostat, on body weight of diabetic obese mice.

Plasma xanthine oxidoreductase (XOR) activity is high in metabolic disorders such as diabetic mellitus, obesity, or overweight. Thus, this study investigated whether the XOR inhibitor, topiroxostat, affected body weight. Male db/db mice were fed standard diets with or without topiroxostat for 4 weeks. Body weight and food intake were constantly monitored, along with monitoring plasma biochemical markers, including insulin and XOR activity. Additionally, hepatic hypoxanthine and XOR activity were also documented. Single regression analysis was performed to determine the mechanism. Topiroxostat treatment suppressed weight gain relative to the vehicle without any impact on food intake. However, the weight of fat pads and hepatic and muscle triglyceride content did not change. Topiroxostat decreased the plasma uric acid and increased hepatic hypoxanthine in response to the inhibition of XOR activity. Plasma ketone body and free fatty acid were also increased. Moreover, fat weight was weakly associated with plasma XOR activity in the diabetic state and was negatively associated with ketone body by topiroxostat. These results suggested that topiroxostat amplified the burning of lipids and the salvage pathway, resulting in predisposing the body toward catabolism. The inhibition of plasma XOR activity may contribute to weight loss.

The Role of Oxidative Stress in Hyperuricemia and Xanthine Oxidoreductase (XOR) Inhibitors.

Uric acid is the end product of purine metabolism in humans. Hyperuricemia is a metabolic disease caused by the increased formation or reduced excretion of serum uric acid (SUA). Alterations in SUA homeostasis have been linked to a number of diseases, and hyperuricemia is the major etiologic factor of gout and has been correlated with metabolic syndrome, cardiovascular disease, diabetes, hypertension, and renal disease. Oxidative stress is usually defined as an imbalance between free radicals and antioxidants in our body and is considered to be one of the main causes of cell damage and the development of disease. Studies have demonstrated that hyperuricemia is closely related to the generation of reactive oxygen species (ROS). In the human body, xanthine oxidoreductase (XOR) catalyzes the oxidative hydroxylation of hypoxanthine to xanthine to uric acid, with the accompanying production of ROS. Therefore, XOR is considered a drug target for the treatment of hyperuricemia and gout. In this review, we discuss the mechanisms of uric acid transport and the development of hyperuricemia, emphasizing the role of oxidative stress in the occurrence and development of hyperuricemia. We also summarize recent advances and new discoveries in XOR inhibitors.

Xanthine Oxidoreductase-Mediated Superoxide Production Is Not Involved in the Age-Related Pathologies in Sod1-Deficient Mice.

Reactive oxygen species (ROS) metabolism is regulated by the oxygen-mediated enzyme reaction and antioxidant mechanism within cells under physiological conditions. Xanthine oxidoreductase (XOR) exhibits two inter-convertible forms (xanthine oxidase (XO) and xanthine dehydrogenase (XDH)), depending on the substrates. XO uses oxygen as a substrate and generates superoxide (O2•-) in the catalytic pathway of hypoxanthine. We previously showed that superoxide dismutase 1 (SOD1) loss induced various aging-like pathologies via oxidative damage due to the accumulation of O2•- in mice. However, the pathological contribution of XO-derived O2•- production to aging-like tissue damage induced by SOD1 loss remains unclear. To investigate the pathological significance of O2•- derived from XOR in Sod1-/- mice, we generated Sod1-null and XO-type- or XDH-type-knock-in (KI) double-mutant mice. Neither XO-type- nor XDH-type KI mutants altered aging-like phenotypes, such as anemia, fatty liver, muscle atrophy, and bone loss, in Sod1-/- mice. Furthermore, allopurinol, an XO inhibitor, or apocynin, a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor, failed to improve aging-like tissue degeneration and ROS accumulation in Sod1-/- mice. These results showed that XOR-mediated O2•- production is relatively uninvolved in the age-related pathologies in Sod1-/- mice.

Renal Reabsorptive Transport of Uric Acid Precursor Xanthine by URAT1 and GLUT9.

Xanthine and hypoxanthine are intermediate metabolites of uric acid and a source of reactive oxidative species (ROS) by xanthine oxidoreductase (XOR), suggesting that facilitating their elimination is beneficial. Since they are reabsorbed in renal proximal tubules, we investigated their reabsorption mechanism by focusing on the renal uric acid transporters URAT1 and GLUT9, and examined the effect of clinically used URAT1 inhibitor on their renal clearance when their plasma concentration is increased by XOR inhibitor. Uptake study for [3H]xanthine and [3H]hypoxanthine was performed using URAT1- and GLUT9-expressing Xenopus oocytes. Transcellular transport study for [3H]xanthine was carried out using Madin-Darby canine kidney (MDCK)II cells co-expressing URAT1 and GLUT9. In in vivo pharmacokinetic study, renal clearance of xanthine was estimated based on plasma concentration and urinary recovery. Uptake by URAT1- and GLUT9-expressing oocytes demonstrated that xanthine is a substrate of URAT1 and GLUT9, while hypoxanthine is not. Transcellular transport of xanthine in MDCKII cells co-expressing URAT1 and GLUT9 was significantly higher than those in mock cells and cells expressing URAT1 or GLUT9 alone. Furthermore, dotinurad, a URAT1 inhibitor, increased renal clearance of xanthine in rats treated with topiroxostat to inhibit XOR. It was suggested that xanthine is reabsorbed in the same manner as uric acid through URAT1 and GLUT9, while hypoxanthine is not. Accordingly, it is expected that treatment with XOR and URAT1 inhibitors will effectively decrease purine pools in the body and prevent cell injury due to ROS generated during XOR-mediated reactions.

Improved Parkinsons disease motor score in a single-arm open-label trial of febuxostat and inosine.

BACKGROUND: Cellular energetics play an important role in Parkinsons disease etiology, but no treatments directly address this deficiency. Our past research showed that treatment with febuxostat and inosine increased blood hypoxanthine and ATP in healthy adults, and a preliminary trial in 3 Parkinson's disease patients suggested some symptomatic improvements with no adverse effects. METHODS: To examine the efficacy on symptoms and safety in a larger group of Parkinsons disease patients, we conducted a single-arm, open-label trial at 5 Japanese neurology clinics and enrolled thirty patients (nmales = 11; nfemales = 19); 26 patients completed the study (nmales = 10; nfemales = 16). Each patient was administered febuxostat 20 mg and inosine 500 mg twice-per-day (after breakfast and dinner) for 8 weeks. The primary endpoint was the difference of MDS-UPDRS Part III score immediately before and after 57 days of treatment. RESULTS: Serum hypoxanthine concentrations were raised significantly after treatment (Pre = 11.4 µM; Post = 38.1 µM; P < .0001). MDS-UPDRS Part III score was significantly lower after treatment (Pre = 28.1 ±â€Š9.3; Post = 24.7 ±â€Š10.8; mean ±â€ŠSD; P = .0146). Sixteen adverse events occurred in 13/29 (44.8%) patients, including 1 serious adverse event (fracture of the second lumbar vertebra) that was considered not related to the treatment. CONCLUSIONS: The results of this study suggest that co-administration of febuxostat and inosine is relatively safe and effective for improving symptoms of Parkinsons disease patients. Further controlled trials need to be performed to confirm the symptomatic improvement and to examine the disease-modifying effect in long-term trials.

Long-Term Safety and Effectiveness of the Xanthine Oxidoreductase Inhibitor, Topiroxostat in Japanese Hyperuricemic Patients with or Without Gout: A 54-week Open-label, Multicenter, Post-marketing Observational Study.

BACKGROUND AND OBJECTIVES: Topiroxostat, a selective xanthine oxidoreductase inhibitor, is used for the management of hyperuricemic patients with or without gout in Japan. Accumulating evidence has demonstrated the efficacy of topiroxostat for the treatment of hyperuricemia with or without gout. However, the safety and efficacy of topiroxostat in the clinical setting remain unclear, and there is little large-scale clinical evidence. We conducted a post-marketing observational study over 54 weeks. PATIENTS AND METHODS: Patients were centrally enrolled, and case report forms of 4491 patients were collected between April 2014 and March 2019 from 825 medical sites. RESULTS: Overall, 4329 patients were assessed for safety and 4253 patients for effectiveness. The overall incidence of adverse drug reactions was 6.95%, and the incidence rates of adverse drug reactions of gouty arthritis, hepatic dysfunction, and skin disorders, which are of special interest in this study, were 0.79%, 1.73%, and 0.95%, respectively. No case of serious gouty arthritis was observed. Serum urate levels decreased stably over time and showed a significant reduction rate at 54 weeks (21.19% ± 22.07%) and on the final visit (19.91% ± 23.35%) compared to the baseline. The rates for subjects who achieved serum uric acid levels ≤ 6.0 mg/dL at 18 and 54 weeks after administration were 43.80% and 48.28%, respectively. CONCLUSIONS: This study suggests that there is no particular concern about adverse drug reactions or the efficacy of topiroxostat for hyperuricemic patients with or without gout in a post-marketing setting in Japan.

Synthesis and bioevaluation of 1-phenylimidazole-4-carboxylic acid derivatives as novel xanthine oxidoreductase inhibitors.

As part of a continuing study, we designed and synthesized four series of 1-phenylimidazole-4-carboxylic acid derivatives as xanthine oxidoreductase (XOR) inhibitors, evaluated their in vitro inhibitory potencies against XOR and hypouricemic effects in mice, and determined their structure-activity relationships (SARs). Most of the compounds exhibited in vitro XOR inhibition at the nanomolar level. In comparison to febuxostat (half-maximal inhibitory concentration [IC50] value of 7.0 nM), compounds Ie and IVa exhibited the most promising XOR inhibitory effects with IC50 values of 8.0 and 7.2 nM, respectively. In the potassium oxonate/hypoxanthine-induced acute and long-term hyperuricemia mouse models, compounds Ie and IVa displayed significant hypouricemic potencies (P < 0.05), that were slightly weaker than and similar to febuxostat, respectively. More interestingly, both compounds showed a capacity to improve kidney damage by decreasing creatinine and urea nitrogen levels compared to the long-term hyperuricemia mouse group (P < 0.05), while febuxostat showed no significant effect.

Anti-hyperuricemia activity and toxicity prediction of a novel xanthine oxidoreductase inhibitor.

A potent xanthine oxidoreductase inhibitor (LS087) was recently proved to exhibit a similar hypouricemic potency to febuxostat. A hyperuricemia model induced by potassium oxonate and hypoxanthine was proposed in specific pathogen-free male Kunming mice, and the serum urea nitrogen, creatinine and uric acid levels were measured after oral administration of LS087. Furthermore, renal histopathology was conducted by staining with hematoxylin and eosin, periodic acid-Schiff and Masson's trichrome stains, respectively. The results showed that the levels of serum urea nitrogen and uric acid significantly decreased compared with the model group, but the level of creatinine showed no significant changes. The pathological abnormalities in kidney tubules were improved after LS087 administration. Ten metabolites (M1-M10) of LS087 were identified after a single oral dosing of 10 mg/kg in rats. M6 was the primary LS087 metabolite in vivo with a pathway of methylation. The toxicity and potential risks of LS087 and its metabolites were predicted using the ProTox-II software. LS087 and the major metabolites (M2, M3, M5, M6, M7 and M8) were predicted to have no potential hepatotoxicity, but some metabolites with a total rate of <1% (M1, M4, M9, and M10) showed potential hepatotoxicity. M1 and M8 showed potential carcinogenicity. The LS087 biotransformation pathway in rat was well characterized.

Febuxostat, a Xanthine Oxidoreductase Inhibitor, Decreases NLRP3-dependent Inflammation in Macrophages by Activating the Purine Salvage Pathway and Restoring Cellular Bioenergetics.

The nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome mediates caspase-1 activation and IL-1ß processing and is implicated in autoinflammatory as well as other chronic inflammatory diseases. Recent studies have demonstrated that xanthine oxidoreductase (XOR) inhibition attenuated IL-1ß secretion in activated macrophages, but the detailed mechanism of inhibition remains unclear. In this study, we report that febuxostat, an inhibitor of XOR, suppressed NLRP3 inflammasome-mediated IL-1ß secretion and cell death by two mechanisms: in a mitochondrial ROS (mitoROS)-dependent and mitoROS-independent manner. MitoROS-independent effects of febuxostat were mediated by an increase of intracellular ATP and improved mitochondrial energetics via the activation of purine salvage pathway. Our findings suggest that cellular bioenergetics are important in regulating NLRP3 activation, and XOR inhibition may be clinically relevant in NLRP3-related inflammatory diseases.

The effects of topiroxostat on vascular function in patients with hyperuricemia.

Xanthine oxidoreductase (XOR) inhibitors, such as allopurinol and febuxostat, inhibit the catalysis of serum uric acid (SUA) synthesis. In doing so, they are thought to improve vascular endothelial function in patients with hyperuricemia and cardiovascular risk by reducing increases in SUA and reactive oxygen species levels. We performed a retrospective cohort study to evaluate the effects of topiroxostat, a novel XOR inhibitor, on vascular function measured by flow-mediated dilation (FMD) on ultrasonography. In total, 23 patients with hyperuricemia were enrolled. After approximately 8 weeks, topiroxostat was associated with a significant increase in the peak percentage change in diameter (∆FMD) from 4.53% ± 2.09% to 5.54% ± 3.08% (P = .045). It also significantly reduced the SUA levels from 7.31 ± 1.43 to 5.44 ± 1.11 mg/dL (P < .001). Although further studies are needed to validate these results, it appears that topiroxostat improves vascular endothelial function in patients with hyperuricemia.

Metabolomics analysis elucidates unique influences on purine / pyrimidine metabolism by xanthine oxidoreductase inhibitors in a rat model of renal ischemia-reperfusion injury.

BACKGROUND: Clinically applied as anti-gout drugs, xanthine oxidoreductase (XOR) inhibitors, especially the potent, selective, non-purine-analog XOR inhibitors febuxostat and topiroxostat, exert organ-protective effects. We tested the hypothesis that preservation of tissue concentrations of high-energy phosphates, such as ATP and ADP, contributes to organ-protective effects through CE-TOFMS metabolomics. METHODS: Rats were subjected to 30 min of renal ischemia-reperfusion (I/R) injury 60 min after oral administration of 10 mg/kg febuxostat, 10 mg/kg topiroxostat, 50 mg/kg allopurinol, or vehicle. RESULTS: In non-purine-analog XOR inhibitor-treated groups, renal concentrations of high-energy phosphates were greater before and after I/R injury, and renal adenine compounds were less depleted by I/R injury than in the vehicle and allopurinol groups. These findings were well in accordance with the proposed hypothesis that the recomposition of high-energy phosphates is promoted by non-purine-analog XOR inhibitors via the salvage pathway through blockade of hypoxanthine catabolism, whereas non-specific inhibitory effects of allopurinol on purine/pyrimidine enzymes impede this re-synthesis process. CONCLUSIONS: This metabolic approach shed light on the physiology of the organ-protective effects of XOR inhibitors.

Diminishing Inflammation by Reducing Oxidant Generation: Nitrated Fatty Acid-Mediated Inactivation of Xanthine Oxidoreductase.

Inhibition of xanthine oxidoreductase (XOR) has proven beneficial in a plethora of inflammatory disease processes due to a net reduction in pro-inflammatory oxidants and secondary nitrating species. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OA-NO2) are also noted to display a broad spectrum of anti-inflammatory effects via interaction with critical signaling pathways. An alternative process in which nitrated fatty acids may extend anti-inflammatory actions is via inactivation of XOR, a process that is more effective than allo/oxypurinol-mediated inhibition. Herein, we describe the molecular aspects of nitrated fatty acid-associated inactivation of XOR, identify specificity via structure function relationships and discuss XOR as a crucial component of the anti-inflammatory portfolio of nitrated fatty acids.

Hepatocyte-Specific Ablation or Whole-Body Inhibition of Xanthine Oxidoreductase in Mice Corrects Obesity-Induced Systemic Hyperuricemia Without Improving Metabolic Abnormalities.

Systemic hyperuricemia (HyUA) in obesity/type 2 diabetes facilitated by elevated activity of xanthine oxidoreductase (XOR), which is the sole source of uric acid (UA) in mammals, has been proposed to contribute to the pathogenesis of insulin resistance/dyslipidemia in obesity. Here, the effects of hepatocyte-specific ablation of Xdh, the gene encoding XOR (HXO), and whole-body pharmacologic inhibition of XOR (febuxostat) on obesity-induced insulin resistance/dyslipidemia were assessed. Deletion of hepatocyte Xdh substantially lowered liver and plasma UA concentration. When exposed to an obesogenic diet, HXO and control floxed (FLX) mice became equally obese, but systemic HyUA was absent in HXO mice. Despite this, obese HXO mice became as insulin resistant and dyslipidemic as obese FLX mice. Similarly, febuxostat dramatically lowered plasma and tissue UA and XOR activity in obese wild-type mice without altering obesity-associated insulin resistance/dyslipidemia. These data demonstrate that hepatocyte XOR activity is a critical determinant of systemic UA homeostasis, that deletion of hepatocyte Xdh is sufficient to prevent systemic HyUA of obesity, and that neither prevention nor correction of HyUA improves insulin resistance/dyslipidemia in obesity. Thus, systemic HyUA, although clearly a biomarker of the metabolic abnormalities of obesity, does not appear to be causative.

Long-term renal outcomes of APRT deficiency presenting in childhood.

BACKGROUND: Adenine phosphoribosyltransferase (APRT) deficiency is a hereditary purine metabolism disorder that causes kidney stones and chronic kidney disease (CKD). The purpose of this study was to examine the course of APRT deficiency in patients who presented in childhood. METHODS: The disease course of 21 (35%) patients in the APRT Deficiency Registry of the Rare Kidney Stone Consortium, who presented with manifestations of APRT deficiency and/or were diagnosed with the disorder before the age of 18 years, was studied. The effect of pharmacotherapy on renal manifestations and outcomes was thoroughly assessed. RESULTS: Fourteen children were placed on allopurinol, 100 (25-200) mg/day, at the age of 2.6 (0.6-16.5) years. Six of these patients had experienced kidney stone events and three had developed acute kidney injury (AKI) prior to allopurinol treatment. During 18.9 (1.7-31.5) years of pharmacotherapy, stones occurred in two patients and AKI in three. Six adult patients started allopurinol treatment, 200 (100-300) mg/day, at age 29.8 (20.5-42.4) years. Five of these patients had experienced 28 stone episodes and AKI had occurred in two. Stone recurrence occurred in four patients and AKI in two during 11.2 (4.2-19.6) years of allopurinol therapy. Lack of adherence and insufficient dosing contributed to stone recurrence and AKI during pharmacotherapy. At latest follow-up, estimated glomerular filtration rate (eGFR) was 114 (70-163) and 62 (10-103) mL/min/1.73 m2 in those who initiated treatment as children and adults, respectively. All three patients with CKD stages 3-5 at the last follow-up were adults when pharmacotherapy was initiated. CONCLUSION: Timely diagnosis and treatment of APRT deficiency decreases renal complications and preserves kidney function.

Identification of new drug-like compounds from millets as Xanthine oxidoreductase inhibitors for treatment of Hyperuricemia: A molecular docking and simulation study.

Xanthine oxidoreductase plays an important role in formation of uric acid and its regulation during purine catabolism. Uncontrolled expression of this enzyme is responsible for overproduction and deposition of uric acid in blood that is potentially injurious because it can breakdown DNA and protein molecules, triggering many diseases. Human Xanthine oxidoreductase (HsXOR) is considered to be a pharmacological target for the treatment of hyperuricemia. Many of the HsXOR-inhibitor drugs such as Febuxostat and Allopurinol are known to have significant adverse effects. Therefore, there is an urgent need to develop new HsXOR-inhibitor drugs with less or no toxicity for the long-term treatment or prevention of hyperuricemia-related diseases. Many nutritious and medical functions have been reported in millets. Present work deals with identification of millet derived compounds in terms of their interaction with target, HsXOR through molecular docking and dynamic simulation studies. Of thirty two chosen compounds, Luteolin and Quercitin showed more binding affinity with HsXOR than reference drugs, Febuxostat and Allopurinol. Molecular dynamics simulations (20 ns long) revealed that Luteolin-protein complex was energetically more stable than Quercitin-protein complex. The millet derived compounds i.e. Luteolin and Quercitin showed binding energy -9.7 kcal/mol whereas the known drugs i.e. Febuxostat and Allopurinol showed binding energy -8.0 kcal/mol and -5.5 kcal/mol respectively. Based on the study, Luteolin possess high potential to be considered for trial as an inhibitor of HsXOR as it may regulate the pathway by inhibiting HsXOR. Further investigations are proposed to consider Luteolin for developing future drugs from millets and other natural sources.

Protective Effects of Topiroxostat on an Ischemia-Reperfusion Model of Rat Hearts.

BACKGROUND: Ischemia/reperfusion (I/R) injury triggers cardiac dysfunctions via creating reactive oxygen species (ROS). Because xanthine oxidase (XO) is one of the major enzymes that generate ROS, inhibition of XO is expected to suppress ROS-induced I/R injury. However, it remains unclear whether XO inhibition really yields cardioprotection during I/R. The protective effects of the XO inhibitors, topiroxostat and allopurinol, on cardiac I/R injury were evaluated.Methods and Results:Using isolated rat hearts, ventricular functions, occurrence of arrhythmias, XO activities and thiobarbituric acid reactive substances (TBARS) productions and myocardial levels of adenine nucleotides before and after I/R, and cardiomyocyte death markers during reperfusion, were evaluated. Topiroxostat prevented left ventricular dysfunctions and facilitated recovery from arrhythmias during I/R. Allopurinol and the antioxidant, N-acetylcysteine (NAC), exhibited similar effects at higher concentrations. Topiroxostat inhibited myocardial XO activities and TBARS productions after I/R. I/R decreased myocardial levels of ATP, ADP and AMP, but increased that of xanthine. While topiroxostat, allopurinol or NAC did not change myocardial levels of ATP, ADP or AMP after I/R, all of the agents decreased the level of xanthine. They also decreased releases of CPK and LDH during reperfusion. CONCLUSIONS: Topiroxostat showed protective effects against I/R injury with higher potency than allopurinol or NAC. It dramatically inhibited XO activity and TBARS production, suggesting suppression of ROS generation.

Reduction of nitrosative stress by methane: Neuroprotection through xanthine oxidoreductase inhibition in a rat model of mesenteric ischemia-reperfusion.

Our aim was to characterize the main components of the nitrosative response with quantitative changes of the nitrergic myenteric neurons in adjacent intestinal segments after transient superior mesenteric artery occlusion. We also tested the hypothesis that exogenous methane may modulate the evolution of nitroxidation by influencing xanthine oxidoreductase (XOR) activity. The microcirculatory consequences of a 50 min ischemia or ischemia-reperfusion were investigated in anesthetized rats (n = 124) inhaling normoxic air with or without 2.2% methane. XOR activities, nitrogen monoxide (NO), nitrite/nitrate (NOx), and nitrotyrosine levels were measured, together with relative nitrergic neuron ratios from duodenum, ileum and colon samples. The effects of methane on XOR were also examined in vitro. The intramural flow stopped only in the ileum during ischemia. The highest baseline XOR activity was found in the duodenum, which increased further during ischemia. NO and nitrotyrosine levels rose, and the nNOS-immunopositive neuron ratio and NOx level both dropped. Reperfusion uniformly elevated XOR activity and nitrotyrosine formation, with the highest level attained in the duodenum, where the nitrergic neuron ratio remained depressed. These alterations were eliminated in methane-treated animals, XOR activity and nitrotyrosine formation decreased in all sites, and the duodenal nitrergic neuron ratio was re-established. The inhibitory effect of methane on XOR-linked nitrate reductase activity was also demonstrated in vitro. With segment-specific microcirculatory alterations, the risk for nitrosative stress is highest in transiently hypoxic tissues with high endogenous XOR activities. The XOR-inhibitory effect of methane can reduce nitroxidation and protects the nitrergic neuron population in such conditions.

Uric acid-lowering and renoprotective effects of topiroxostat, a selective xanthine oxidoreductase inhibitor, in patients with diabetic nephropathy and hyperuricemia: a randomized, double-blind, placebo-controlled, parallel-group study (UPWARD study).

BACKGROUND: Hyperuricemia is supposed to be an independent risk factor for kidney dysfunction in diabetic patients. We attempted to examine the uric acid-lowering effect and the renoprotective effect of topiroxostat, a selective xanthine oxidoreductase inhibitor, in patients with diabetic nephropathy and hyperuricemia in this pilot study. METHODS: The study design was randomized, double-blind, placebo-controlled, parallel-group study. A total of 65 patients with hyperuricemia and diabetic nephropathy with microalbuminuria were enrolled and assigned to either the topiroxostat group or the placebo group. Topiroxostat (stepwise dosing from 40 to 160 mg/day) or matching placebo was administered BID for 28 weeks. The primary endpoint was a change in the urinary albumin-to-creatinine ratio in the first-morning-void urine sample. Secondary endpoints were changes in the estimated glomerular filtration rate and the serum uric acid level. RESULTS: At 28 weeks, there was no significant difference in the percent change from baseline in the urinary albumin-to-creatinine ratio between the two groups (topiroxostat: 0 vs. placebo: 17%, p = 0.3206), but the changes in the estimated glomerular filtration rate (- 0.2 vs. - 4.0 mL/min/1.73 m2, p = 0.0303) and the serum uric acid level (- 2.94 vs. - 0.20 mg/dL, p < 0.0001) were significantly different between the topiroxostat and placebo groups. Gouty arthritis occurred in 1 patient in the placebo group and no patients in the topiroxostat group. CONCLUSION: These findings support that diabetic nephropathy combined with hyperuricemia may be associated with kidney dysfunctions. Topiroxostat provides strict control of the serum uric acid level preventing decline of eGFR in these patients.