Uricosuric Agents Affect Plasma and Kidney Concentration of Adefovir via Inhibition of Oat1 and Mrp2 in Rats.

Uricosuric agents lower serum uric acid levels by increasing urinary excretion via inhibition of urate transporter 1 (URAT1), urate reabsorption transporter in the renal proximal tubules. Probenecid and benzbromarone have been used as uricosurics, but these drugs inhibit organic anion transporters (OATs) in addition to URAT1. In this study, we investigated whether uricosuric agents interacted with adefovir, known as a substrate for OAT1, using Sprague-Dawley (SD) rats. Furthermore, involvement of other transporters, multi-drug resistance protein 2 (MRP2) in this interaction was examined using Mrp2-deficient rats. Probenecid and lesinurad increased plasma adefovir concentrations and decreased kidney-to-plasma partition coefficient (Kp) in these rats, presumably by inhibiting Oat1. Although benzbromarone had no effect on plasma adefovir concentration, it increased the Kp to 141% in SD rats. Since this effect was abolished in Mrp2-deficient rats, together with the MRP2 inhibition study, it is suggested that benzbromarone inhibits Mrp2-mediated adefovir excretion from the kidney. In contrast, dotinurad, a novel uricosuric agent that selectively inhibits URAT1, had no effect on the plasma and kidney concentrations of adefovir. Therefore, due to the lack of interaction with adefovir, dotinurad is expected to have low drug-drug interaction risk mediated by OAT1, and also by MRP2.

A Novel Role of Uricosuric Agent Benzbromarone in BK Channel Activation and Reduction of Airway Smooth Muscle Contraction.

The uricosuric drug benzbromarone, widely used for treatment of gout, hyperpolarizes the membrane potential of airway smooth muscle cells, but how it works remains unknown. Here we show a novel role of benzbromarone in activation of large conductance calcium-activated K+ channels. Benzbromarone results in dose-dependent activation of macroscopic big potassium (BK) currents about 1.7- to 14.5-fold with an EC50 of 111 µM and shifts the voltage-dependent channel activation to a more hyperpolarizing direction about 10 to 54 mV in whole-cell patch clamp recordings. In single-channel recordings, benzbromarone decreases single BKα channel closed dwell time and increases the channel open probability. Coexpressing ß1 subunit also enhances BK activation by benzbromarone with an EC50 of 67 µM and a leftward shift of conductance-voltage (G-V) curve about 44 to 138 mV. Site-directed mutagenesis reveals that a motif of three amino acids 329RKK331 in the cytoplasmic linker between S6 and C-terminal regulator of potassium conductance (RCK) gating ring mediates the pharmacological activation of BK channels by benzbromarone. Further ex vivo assay shows that benzbromarone causes reduction of tracheal strip contraction. Taken together, our findings demonstrate that uricosuric benzbromarone activates BK channels through molecular mechanism of action involving the channel RKK motif of S6-RCK linker. Pharmacological activation of BK channel by benzbromarone causes reduction of tracheal strip contraction, holding a repurposing potential for asthma and pulmonary arterial hypertension or BK channelopathies. SIGNIFICANCE STATEMENT: We describe a novel role of uricosuric agent benzbromarone in big potassium (BK) channel activation and relaxation of airway smooth muscle contraction. In this study, we find that benzbromarone is an activator of the large-conductance Ca2+- and voltage-activated K+ channel (BK channel), which serves numerous cellular functions, including control of smooth muscle contraction. Pharmacological activation of BK channel by the FDA-approved drug benzbromarone may hold repurposing potential for treatment of asthma and pulmonary arterial hypertension or BK channelopathies.

An evaluation method for uric acid uptake inhibition using primary human proximal tubule epithelial cells treated with insulin.

The effects of uricosuric agents have been evaluated in vitro with indices of uric acid uptake into human urate transporter 1 (URAT1)-overexpressed oocytes or cells. In the present study, we evaluated a method using primary human renal proximal tubule epithelial cells (RPTECs). Pretreatment of RPTECs with insulin significantly increased the uptake of uric acid into these cells. The uric acid uptake was inhibited in a concentration-dependent manner by the URAT1 inhibitors benzbromarone and dotinurad. Therefore, effects of uricosuric agents can be evaluated by the novel method, which is closer to the physiological system compared with previous methods.

Hypouricemic agents reduce indoxyl sulfate excretion by inhibiting the renal transporters OAT1/3 and ABCG2.

Indoxyl sulfate (IS) accumulates in the body in chronic kidney disease (CKD). In the renal proximal tubules, IS excretion is mediated by OAT1/3 and ABCG2. These transporters are inhibited by some hypouricemic agents; OATs by probenecid and benzbromarone, ABCG2 by febuxostat and benzbromarone. Thus, we evaluated whether hypouricemic agents including dotinurad, a novel selective urate reabsorption inhibitor with minimal effect on OATs or ABCG2, affect IS clearance in rats. Intact and adenine-induced acute renal failure rats were orally administered hypouricemic agents, and both endogenous IS and exogenously administered stable isotope-labeled d4-IS in the plasma and kidney were measured. Our results demonstrated that OATs inhibitors, such as probenecid, suppress IS uptake into the kidney, leading to increased plasma IS concentration, whereas ABCG2 inhibitors, such as febuxostat, cause renal IS accumulation remarkably by suppressing its excretion in intact rats. The effects of these agents were reduced in adenine-induced acute renal failure rats, presumably due to substantial decrease in renal OAT1/3 and ABCG2 expression. Dotinurad did not significantly affected the clearance of IS under both conditions. Therefore, we suggest that hypouricemic agents that do not affect OATs and ABCG2 are effective therapeutic options for the treatment of hyperuricemia complicated by CKD.

Effect of Serum Urate Lowering With Allopurinol on Blood Pressure in Young Adults: A Randomized, Controlled, Crossover Trial.

OBJECTIVE: To determine whether serum urate reduction with allopurinol lowers blood pressure (BP) in young adults and the mechanisms mediating this hypothesized effect. METHODS: We conducted a single-center, randomized, double-blind, crossover clinical trial. Adults ages 18-40 years with baseline systolic BP ≥120 and <160 mm Hg or diastolic BP ≥80 and <100 mm Hg, and serum urate ≥5.0 mg/dl for men or ≥4.0 mg/dl for women were enrolled. Main exclusion criteria included chronic kidney disease, gout, or past use of urate-lowering therapies. Participants received oral allopurinol (300 mg daily) or placebo for 1 month followed by a 2-4 week washout and then were crossed over. Study outcome measures were change in systolic BP from baseline, endothelial function estimated as flow-mediated dilation (FMD), and high-sensitivity C-reactive protein (hsCRP) levels. Adverse events were assessed. RESULTS: Ninety-nine participants were randomized, and 82 completed all visits. The mean ± SD age was 28.0 ± 7.0 years, 62.6% were men, and 40.4% were African American. In the primary intent-to-treat analysis, systolic BP did not change during the allopurinol treatment phase (mean ± SEM -1.39 ± 1.16 mm Hg) or placebo treatment phase (-1.06 ± 1.08 mm Hg). FMD increased during allopurinol treatment periods compared to placebo treatment periods (mean ± SEM 2.5 ± 0.55% versus -0.1 ± 0.42%; P < 0.001). There were no changes in hsCRP level and no serious adverse events. CONCLUSION: Our findings indicate that urate-lowering therapy with allopurinol does not lower systolic BP or hsCRP level in young adults when compared with placebo, despite improvements in FMD. These findings do not support urate lowering as a treatment for hypertension in young adults.

Anoctamin 1 antagonism potentiates conventional tocolytic-mediated relaxation of pregnant human uterine smooth muscle.

BACKGROUND: Currently available tocolytic agents are not effective treatment for preterm labor beyond 48 h. A major reason is the development of maternal side effects which preclude the maintenance of an effective steady-state drug concentration. One strategy that can mitigate these side effects is utilizing synergistic drug combinations to reduce the drug concentrations necessary to elicit a clinical effect. We have previously shown that three anoctamin 1 (ANO1) antagonists mediate potent relaxation of precontracted human uterine smooth muscle (USM). In this study, we aimed to determine whether a combination of sub-relaxatory doses of tocolytic drugs in current clinical use [the L-type voltage-gated calcium channel (VGCC) blocker, nifedipine (NIF); and the ß2-adrenergic (ß2AR) agonist, terbutaline (TRB)] will potentiate USM relaxation with two ANO1 antagonists [benzbromarone (BB) and MONNA (MN)]. OBJECTIVE: This study sought to examine the synergistic potency and mechanistic basis of two ANO1 antagonists with currently available tocolytic drugs. Functional endpoints assessed included relaxation of pre-contracting pregnant human USM tissue, inhibition of intracellular calcium release, and reduction of spontaneous transient inward current (STIC) recordings in human uterine smooth muscle cells. METHODS: Human myometrial strips and primary human USM cells were used in organ bath and calcium flux experiments with different combinations of sub-threshold doses of ANO1 antagonists and terbutaline or nifedipine to determine if ANO1 antagonists potentiate tocolytic drugs. RESULTS: The combination of sub-threshold doses of two ANO1 antagonists and current tocolytic drugs demonstrate a significant degree of synergy to relax human pregnant USM compared to the effects achieved when these drugs are administered individually. CONCLUSION: A combination of sub-threshold doses of VGCC blocker and ß2AR agonist with ANO1 antagonists potentiates relaxation of oxytocin-induced contractility and calcium flux in human USM ex vivo. Our findings may serve as a foundation for novel tocolytic drug combinations.

Mechanism-Based Inactivation of Cytochrome P450 3A4 by Benzbromarone.

Benzbromarone (BBR), a potent uricosuric agent for the management of gout, is known to cause fatal fulminant hepatitis. Although the mechanism of BBR-induced idiosyncratic hepatotoxicity remains unelucidated, cytochrome P450 enzyme-mediated bioactivation of BBR to electrophilic reactive metabolites is commonly regarded as a key molecular initiating event. However, apart from causing aberrant toxicities, reactive metabolites may result in mechanism-based inactivation (MBI) of cytochrome P450. Here, we investigated and confirmed that BBR inactivated CYP3A4 in a time-, concentration-, and NADPH-dependent manner with K I, k inact, and partition ratio of 11.61 µM, 0.10 minutes-1, and 110, respectively. Coincubation with ketoconazole, a competitive inhibitor of CYP3A4, attenuated the MBI of CYP3A4 by BBR, whereas the presence of glutathione and catalase did not confer such protection. The lack of substantial recovery of enzyme activity postdialysis and after oxidation with potassium ferricyanide, combined with the absence of a Soret peak in spectral difference scans, implied that MBI of CYP3A4 by BBR did not occur through the formation of quasi-irreversible metabolite-intermediate complexes. Analysis of the reduced CO-difference spectrum revealed an ∼44% reduction in ferrous-CO binding and hinted that inactivation is mediated via irreversible covalent adduction to both the prosthetic heme moiety and the apoprotein. Finally, our in silico covalent docking analysis further suggested the modulation of substrate binding to CYP3A4 via the covalent adduction of epoxide-derived reactive intermediates of BBR to two key cysteine residues (Cys239 and Cys58) vicinal to the entrance of the orthosteric binding site. SIGNIFICANCE STATEMENT: Although the bioactivation of benzbromarone (BBR) to reactive metabolites has been well characterized, its potential to cause mechanism-based inactivation (MBI) of cytochrome P450 has not been fully investigated. This study reports the MBI of CYP3A4 by BBR via irreversible covalent adduction and develops a unique covalent docking methodology to predict the structural molecular determinants underpinning the inactivation for the first time. These findings lay the groundwork for future investigation of clinically relevant drug-drug interactions implicating BBR and mechanisms of BBR-induced idiosyncratic hepatotoxicity.

Increased Susceptibility of Atrial Fibrillation Induced by Hyperuricemia in Rats: Mechanisms and Implications.

High levels of serum uric acid is closely associated with atrial fibrillation (AF); nonetheless, the detailed mechanisms remain unknown. Therefore, this work examined the intricate mechanisms of AF triggered by hyperuricemia and the impact of the uricosuric agent benzbromarone on atrial remodeling in hyperuricemic rats. After adjusting baseline serum uric acid levels, a total of 28 healthy male adult Sprague Dawley rats were randomly divided into 4 groups, namely, control (CTR), hyperuricemia (oxonic acid potassium salt, OXO) and benzbromarone (+ BBR), and OXO withdrawal groups. Primary rat cardiomyocytes were cultured with uric acid for 24 h to investigate the direct influence of uric acid on cardiomyocytes. Results revealed that AF vulnerability and AF duration were dramatically greater in hyperuricemic rats (OXO group), while the atrial effective refractory periods (AERPs) were significantly shorter. Meanwhile, BBR treatment and withdrawal of 2% OXO administration remarkably reduced AF inducibility and shortened AF duration. Moreover, abnormal morphology of atrial myocytes, atrial fibrosis, apoptosis, and substantial sympathetic nerve sprouting were observed in hyperuricemic rats. Apoptosis and fibrosis of atria were partly mediated by caspase-3, BAX, TGF-ß1, and α-smooth muscle actin. Uric acid significantly induced primary rat cardiomyocyte apoptosis and fibrosis in vitro. Also, we found that sympathetic nerve sprouting was markedly upregulated in the atria of hyperuricemia rats, and was restored by BRB or absence of OXO administration. In summary, our study confirmed that AF induced by hyperuricemic rats occurred primarily via induction of atrial remodeling, thereby providing a novel potential treatment approach for hyperuricemia-related AF.

Novel Human Urate Transporter 1 Inhibitors as Hypouricemic Drug Candidates with Favorable Druggability.

Lesinurad, a human urate transporter 1 (URAT1) inhibitor approved as a medication for the treatment of hyperuricemia associated with gout in 2015, can cause liver and renal toxicity. Here, we modified all three structural components of lesinurad by applying scaffold hopping, bioisosterism, and substituent-decorating strategies. In a mouse model of acute hyperuricemia, 21 of the synthesized compounds showed increased serum uric acid (SUA)-reducing activity; SUA was about 4-fold lower in animals treated with 44, 54, and 83 compared with lesinurad or benzbromarone. In the URAT1 inhibition assay, 44 was over 8-fold more potent than lesinurad (IC50: 1.57 µM vs 13.21 µM). Notably, 83 also displayed potent inhibitory activity (IC50 = 31.73 µM) against GLUT9. Furthermore, we also preliminarily explored the effect of chirality on the potency of the promising derivatives 44 and 54. Compounds 44, 54, and 83 showed favorable drug-like pharmacokinetics and appear to be promising candidates for the treatment of hyperuricemia and gout.

Total glucosides of herbaceous peony (Paeonia lactiflora Pall.) flower attenuate adenine- and ethambutol-induced hyperuricaemia in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Herbaceous peony (Paeonia lactiflora Pall.) flower has been used widely in dietotherapy in China and other countries. It has good ethnopharmacological value in the treatment of various metabolic diseases. However, the molecular mechanisms by which it lowers serum uric acid are unknown. The development of pharmaceutical resources is very important. Here, we sought to elucidate the mode of action of herbaceous peony in terms of reducing uric acid levels. AIM OF THE STUDY: In the present research, the effects of the total glucosides of herbaceous peony flower were investigated in a rat hyperuricaemia model. Another aim of the study was to clarify the mechanism by which herbaceous peony flower (TGPF) lowers serum uric acid levels. MATERIALS AND METHODS: A hyperuricaemic rat model was induced via intragastric administration of 100 mg/kg adenine and 250 mg/kg ethambutol hydrochloride (EH) for 23 d. Then TongFengShu 600 mg/kg, allopurinol 42 mg/kg, or TGPF (50 mg/kg, 100 mg/kg, or 200 mg/kg) was administered 1 h after the adenine and EH treatments. RESULTS: TGPF improved weight loss and decreased serum UA, XOD, MCP-1, TNF-α, Cr, and BUN in the rats with hyperuricaemic nephropathy. TGPF downregulated renal URAT1 and GLUT9, upregulated renal OAT1, and ameliorated histopathological changes in the thymus, spleen, and kidney. CONCLUSION: TGPF is promising as a therapeutic agent against hyperuricaemia. It regulates the uric acid transporters and diminished serum uric acid levels, and alleviates renal pathology associated with hyperuricaemia.

The mechanism of Arhalofenate in alleviating hyperuricemia-Activating PPARγ thereby reducing caspase-1 activity.

Hyperuricemia (HUA) is an important risk factor for renal diseases and contributes to gout. Arhalofenate (Arha) has been proved to have uricosuric activity as an inhibitor of URAT1, organic anion transporter 4 (OAT4) and OAT10. However, the effects of Arha on HUA remain unknown. The objective of this study was to investigate whether Arha could alleviate HUA and uncovered the underlying mechanism in vitro. HK-2 cells were exposed to uric acid (UA) to simulate HUA in vitro. Then cells were treated with Arha, caspase-1 inhibitor Belnacasan (Beln), caspase-11 inhibitor Wedelolactone (Wede) and PPARγ inhibitor Mifobate, respectively. The alteration of cell proliferation, inflammation, pyroptosis and expression of related proteins were detected. Results showed that UA exposure inhibited cell viability and increased IL-1ß and IL-18 generation in a concentration dependent manner. Meanwhile, UA activated the cleavage of gasdermin D (GSDMD), enhanced the protein expression of URAT1, OAT4, TLR4, caspase-1, and caspase-11 and reduced PPARγ expression. While the presence of Arha or Beln enhanced cell viability and inhibited cleavage of GSDMD. Wede slightly increased cell viability but failed to prevent GSDMD cleavage. The expression of related proteins except caspase-11was also recovered by Arha. Beln and Wede partially rescued related proteins level except PPARγ compared with model group. Besides, the co-treatment of Mifobate blunted the effects of Arha on cell viability and expression of GSDMD, TLR4, and caspase-1. In conclusion, Arha inhibited UA transport as well as preventing inflammation and pyroptosis via activating PPARγ thereby blocking caspase-1 activation of HUA in vitro.

Pharmacokinetic/pharmacodynamic modeling and simulation of dotinurad, a novel uricosuric agent, in healthy volunteers.

This study aimed to investigate the pharmacokinetic and pharmacodynamic (PK/PD) profiles of dotinurad, a novel uricosuric agent, and to construct a PK/PD model to predict serum urate (SUA) levels after dotinurad administration in healthy men. PK/PD model was constructed using single-dose study data considering the physiological features of urate handling. Model validation was performed by comparing the predicted SUA levels with the SUA levels in a multiple-dose study. Dotinurad was absorbed rapidly, and its exposure increased proportionally in the tested dose ranges (0.5-20 mg) after a single-dose administration. The PK model after oral administration was described using a one-compartment model with first-order absorption. Effects on SUA and renal urate excretion of dotinurad increased with dose escalation but were apparently saturable at a dose >5 mg. The simple maximal effect (Emax) model was selected as the PD model of dotinurad on renal urate reabsorption, resulting in an estimated Emax of 0.51. The plasma concentration at the half-maximal effect of dotinurad was 196 ng/mL. Other PD parameters were calculated from the change in SUA level or urinary excretion of urate before and after dotinurad administration. The predicted SUA levels, using the PK/PD model, were well-fitted with the observed values. The constructed PK/PD model of dotinurad appropriately described the profiles of dotinurad plasma concentrations and SUA level in multiple administration study.

URC102, a potent and selective inhibitor of hURAT1, reduced serum uric acid in healthy volunteers.

OBJECTIVE: URC102, a novel and potent inhibitor of human uric acid transporter 1 (hURAT1), is currently under clinical development to treat patients with gout. We performed a randomized, double-blind, placebo-controlled, phase I study to evaluate the safety, tolerability, pharmacodynamic, and pharmacokinetic profiles of URC102 after single and multiple oral administration in healthy male subjects. METHODS: Thirty-one Koreans and 23 Caucasians received a single dose of URC102 at 1-30 mg and 1-10 mg, respectively, while 44 Koreans received URC102 once-daily for 7 days at 1-20 mg. We evaluated safety and tolerability throughout the study, and serially determined serum uric acid, the fractional excretion of uric acid and URC102 concentrations. RESULTS: URC102 was well tolerated over the dose range of 1-10 mg after single and multiple administration. URC102 rapidly reduced serum uric acid, which was maintained over the entire treatment period. Furthermore, URC102 increased the area-under-the-effect curve over 168 h for fractional excretion of uric acid in a dose-dependent manner. The maximum plasma concentration and the area under the plasma concentration-time curve of URC102 increased dose-proportionally. The pharmacokinetic and pharmacodynamics characteristics of URC102 were similar in Koreans and Caucasians. CONCLUSION: URC102 was safe and effectively lowered serum uric acid, which should be tested and confirmed in patients with hyperuricaemia and/or gout through further studies. TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT01953497 and NCT02524678.

Ano1 mediates pressure-sensitive contraction frequency changes in mouse lymphatic collecting vessels.

Lymphatic collecting vessels exhibit spontaneous contractions with a pressure-dependent contraction frequency. The initiation of contraction has been proposed to be mediated by the activity of a Ca2+-activated Cl- channel (CaCC). Here, we show that the canonical CaCC Anoctamin 1 (Ano1, TMEM16a) plays an important role in lymphatic smooth muscle pacemaking. We find that isolated murine lymphatic muscle cells express Ano1, and demonstrate functional CaCC currents that can be inhibited by the Ano1 inhibitor benzbromarone. These currents are absent in lymphatic muscle cells from Cre transgenic mouse lines targeted for Ano1 genetic deletion in smooth muscle. We additionally show that loss of functional Ano1 in murine inguinal-axillary lymphatic vessels, whether through genetic manipulation or pharmacological inhibition, results in an impairment of the pressure-frequency relationship that is attributable to a hyperpolarized resting membrane potential and a significantly depressed diastolic depolarization rate preceding each action potential. These changes are accompanied by alterations in action potential shape and duration, and a reduced duration but increased amplitude of the action potential-induced global "Ca2+ flashes" that precede lymphatic contractions. These findings suggest that an excitatory Cl- current provided by Ano1 is critical for mediating the pressure-sensitive contractile response and is a major component of the murine lymphatic action potential.

Safety and tolerability of available urate-lowering drugs: a critical review.

INTRODUCTION: Urate-lowering therapy (ULT) is the cornerstone of gout management, which is a widespread chronic illness characterized by hyperuricemia, arthropathy, tophus development, and urolithiasis. Since asymptomatic increased serum urate levels are associated with a higher risk of cardiovascular, renal and metabolic disorders, a larger use of ULTs in the general population is expected in the near future. AREAS COVERED: This review will focus on the safety and tolerability profile of the available urate-lowering drugs: xanthine oxidase inhibitors (XOIs), uricosuric agents and injectable uricases. EXPERT OPINION: Older drugs for ULT like allopurinol are well studied and extensively described from typical adverse effects (mild skin rash) to unusual fatal reactions, while febuxostat seems to be overall well tolerated. More evidence is required to define the safety profile of topiroxostat, arhalofenate, tranilast, and sulfinpyrazone. Furthermore, there are some unanswered questions about the pharmacological interactions of probenecid and the hepatotoxicity of benzbromarone. Despite a limited use in clinical practice, combination therapy with lesinurad or verinurad and XOI is not frequently accompanied by side effects. Rasburicase and pegloticase are usually well tolerated with some specific exceptions. Before prescribing UL drugs, physicians should take into account their safety profile tailoring the treatment on the patient characteristics.

Characterization of Stereoselective Metabolism, Inhibitory Effect on Uric Acid Uptake Transporters, and Pharmacokinetics of Lesinurad Atropisomers.

Lesinurad [Zurampic; 2-(5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-ylthio)], a selective inhibitor of uric acid reabsorption transporters approved for the treatment of gout, is a racemate of two atropisomers. The objective of this investigation was to evaluate the stereoselectivity of metabolism, the inhibitory potency on kidney uric acid reabsorption transporters (URAT1 and OAT4), and the clinical pharmacokinetics of the lesinurad atropisomers. Incubations with human liver microsomes (HLM), recombinant CYP2C9, and recombinant CYP3A4 were carried out to characterize the stereoselective formation of three metabolites: M3 (hydroxylation), M4 (a dihydrodiol metabolite), and M6 (S-dealkylation). The formation of M3 in HLM with atropisomer 1 was approximately twice as much as that with atropisomer 2, whereas formation of M4 with atropisomer 1 was 8- to 12-fold greater than that with atropisomer 2. There were no significant differences in the plasma protein binding among lesinurad and the atropisomers. Following oral administration of 400 mg lesinurad once daily for 14 days to healthy human volunteers, the systemic exposure (C max at steady state and area under the concentration-time curve from time zero to the time of dosing interval) of atropisomer 1 was approximately 30% lower than that of atropisomer 2, whereas renal clearance was similar. In vitro cell-based assays using HEK293 stable cells expressing URAT1 and OAT4 demonstrated that atropisomer 2 was approximately 4-fold more potent against URAT1 than atropisomer 1 and equally active against OAT4. In conclusion, lesinurad atropisomers showed stereoselectivity in clinical pharmacokinetics, metabolism, and inhibitory potency against URAT1.

SGLT2 inhibition and renal urate excretion: role of luminal glucose, GLUT9, and URAT1.

Inhibitors of the Na+-glucose cotransporter SGLT2 enhance urinary glucose and urate excretion and lower plasma urate levels. The mechanisms remain unclear, but a role for enhanced glucose in the tubular fluid, which may interact with tubular urate transporters, such as the glucose transporter GLUT9 or the urate transporter URAT1, has been proposed. Studies were performed in nondiabetic mice treated with the SGLT2 inhibitor canagliflozin and in gene-targeted mice lacking the urate transporter Glut9 in the tubule or in mice with whole body knockout of Sglt2, Sglt1, or Urat1. Renal urate handling was assessed by analysis of urate in spontaneous plasma and urine samples and normalization to creatinine concentrations or by renal clearance studies with assessment of glomerular filtration rate by FITC-sinistrin. The experiments confirmed the contribution of URAT1 and GLUT9 to renal urate reabsorption, showing a greater contribution of the latter and additive effects. Genetic and pharmacological inhibition of SGLT2 enhanced fractional renal urate excretion (FE-urate), indicating that a direct effect of the SGLT2 inhibitor on urate transporters is not absolutely necessary. Consistent with a proposed role of increased luminal glucose delivery, the absence of Sglt1, which by itself had no effect on FE-urate, enhanced the glycosuric and uricosuric effects of the SGLT2 inhibitor. The SGLT2 inhibitor enhanced renal mRNA expression of Glut9 in wild-type mice, but tubular GLUT9 seemed dispensable for the increase in FE-urate in response to canagliflozin. First evidence is presented that URAT1 is required for the acute uricosuric effect of the SGLT2 inhibitor in mice.

Overexpression of Uric Acid Transporter SLC2A9 Inhibits Proliferation of Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated mortality worldwide. Although the mechanisms of HCC progression are not well understood, recent studies demonstrated the potential contribution of uric acid transporter SLC2A9 to tumor suppression. However, the roles and underlying mechanisms are still unknown. We aimed to study the roles and mechanisms of SLC2A9 in HCC. The present study showed that SLC2A9 expression was decreased in human HCC tissues and cell lines. In addition, overexpression of SLC2A9 inhibited HCC cell proliferation. SCL2A9 induced HCC cell apoptosis by inhibiting the expression of caspase 3. Our study also revealed that upregulation of SLC2A9 reduced intracellular reactive oxygen species (ROS) accumulation. Furthermore, SLC2A9 increased the mRNA and protein expression of tumor suppressor p53 in HCC cells. Probenecid inhibits SLC2A9-mediated uric acid transport, which promotes cell proliferation, inhibits cell apoptosis, induces intracellular ROS, and decreases the expression of p53 in HCC cells. Therefore, the present study demonstrated that SLC2A9 may be a novel tumor suppressor gene and a potential therapeutic target in HCC.

Synthesis of novel benzbromarone derivatives designed to avoid metabolic activation.

We synthesized six novel BBR derivatives that were designed to avoid metabolic activation via ipso-substitution and evaluated for their degree of toxicity and hURAT1 inhibition. It was found that all of the derivatives demonstrate lower cytotoxicity in mouse hepatocytes and lower levels of metabolic activation than BBR, while maintaining their inhibitory activity toward the uric acid transporter. We propose that these derivatives could serve as effective uricosuric agents that have much better safety profiles than BBR.