Inhibition of mucus secretion by niclosamide and benzbromarone in airways and intestine.

The Ca2+ activated Cl- channel TMEM16A (anoctamin 1; ANO1) is expressed in secretory epithelial cells of airways and intestine. Previous studies provided evidence for a role of ANO1 in mucus secretion. In the present study we investigated the effects of the two ANO1-inhibitors niclosamide (Niclo) and benzbromarone (Benz) in vitro and in vivo in mouse models for cystic fibrosis (CF) and asthma. In human CF airway epithelial cells (CFBE), Ca2+ increase and activation of ANO1 by adenosine triphosphate (ATP) or ionomycin was strongly inhibited by 200 nM Niclo and 1 µM Benz. In asthmatic mice airway mucus secretion was inhibited by intratracheal instillation of Niclo or Benz. In homozygous F508del-cftr mice, intestinal mucus secretion and infiltration by CD45-positive cells was inhibited by intraperitoneal injection of Niclo (13 mg/kg/day for 7 days). In homozygous F508del-cftr rats intestinal mucus secretion was inhibited by oral application of Benz (5 mg/kg/day for 60 days). Taken together, well tolerated therapeutic concentrations of niclosamide and benzbromarone corresponding to plasma levels of treated patients, inhibit ANO1 and intracellular Ca2+ signals and may therefore be useful in inhibiting mucus hypersecretion and mucus obstruction in airways and intestine of patients suffering from asthma and CF, respectively.

Benzbromarone Induces Targeted Degradation of HSP47 Protein and Improves Hypertrophic Scar Formation.

Fibrotic diseases are characterized by the abnormal accumulation of collagen in the extracellular matrix, leading to the functional impairment of various organs. In the skin, excessive collagen deposition manifests as hypertrophic scars and keloids, placing a substantial burden on patients and the healthcare system worldwide. HSP47 is essential for proper collagen assembly and contributes to fibrosis. However, identifying clinically applicable HSP47 inhibitors has been a major pharmaceutical challenge. In this study, we identified benzbromarone (BBR) as an HSP47 inhibitor for hypertrophic scarring treatment. BBR inhibited collagen production and secretion in fibroblasts from patients with keloid by binding to HSP47 and inhibiting the interaction between HSP47 and collagen. Interestingly, BBR not only inhibits HSP47 but also acts as a molecular glue degrader that promotes its proteasome-dependent degradation. Through these molecular mechanisms, BBR effectively reduced hypertrophic scarring in mini pigs and rats with burns and/or excisional skin damage. Thus, these findings suggest that BBR can be used to clinically treat hypertrophic scars and, more generally, fibrotic diseases.

Urate-lowering drugs for chronic kidney disease with asymptomatic hyperuricemia and hypertension: a randomized trial.

INTRODUCTION: Xanthine oxidase (XO) inhibitors may slow down chronic kidney disease (CKD) progression. The comparative effectiveness of the different urate-lowering drugs is unknown. The aim of this study was to determine whether urate-lowering therapy with an XO inhibitor (febuxostat) and that with a uricosuric drug (benzbromarone) are comparable in slowing renal function decline in patients with CKD complicated with hypertension and hyperuricemia. METHODS: This study was an open-label randomized parallel-group clinical trial of 95 patients with stage G3 CKD in Japan. The patients had hypertension and hyperuricemia without a history of gout. They were randomized to receive febuxostat ( n  = 47; febuxostat group) or benzbromarone ( n  = 48; benzbromarone group) and titrated to reduce their serum urate level to <6.0 mg/dl. The primary end-point was change in estimated glomerular filtration rate (eGFR) from baseline to 52 weeks. The secondary end-points included changes in uric acid level, blood pressure, urinary albumin-to-creatinine ratio, and XO activity. RESULTS: Of the 95 patients, 88 (92.6%) completed the trial. There were no significant differences in change in eGFR (in ml/min/1.73 m 2 ) between the febuxostat [-0.23, 95% confidence interval (CI), -2.00 to 1.55] and benzbromarone (-2.18, 95% CI, -3.84 to -0.52) groups (difference, 1.95; 95% CI, -0.48 to 4.38; P  = 0.115) nor in the secondary end-points, except for XO activity. Febuxostat significantly reduced XO activity ( P  = 0.010). There were no significant differences in primary and secondary outcomes between the groups. A decrease in eGFR was significantly less in the febuxostat group than that of the benzbromarone group in the CKDG3a, but not in CKDG3b, in the subgroup analysis. There were no adverse effects specific to either drug. CONCLUSIONS: No significant differences were found in the effects of febuxostat and benzbromarone in renal function decline in stage G3 CKD complicated with hyperuricemia and hypertension.

Uric acid-lowering therapy with benzbromarone in hypertension with asymptomatic hyperuricemia: a randomized study focusing left ventricular diastolic function.

BACKGROUND: Both hypertension and hyperuricemia are closely associated with the morbidity and mortality of heart failure with preserved ejection fraction (HFpEF). However, there is limited evidence on the effect of uric acid-lowering therapy on left ventricular (LV) diastolic function in this population. In this randomized study, we prescribed benzbromarone, a uric acid-lowering drug, to those with hypertension and asymptomatic hyperuricemia to investigate its clinical benefits by evaluating LV diastolic function, incidence of HFpEF and hospitalization for heart failure and cardiovascular death. METHODS: 230 participants were randomly assigned into two groups: uric acid-lowering group (benzbromarone) and control groups (without uric acid-lowering drug). The primary endpoint was LV diastolic function evaluated by echocardiography. The secondary endpoint of composite endpoints is the combination of new-onset HFpEF, hospitalization for heart failure and cardiovascular death. RESULTS: After a median of 23.5 months' follow-up (16-30 months), the primary endpoint reflected by E/e' in benzbromarone group reached a significant improvement when compared to control group (p <.001). Composite endpoints occurred in 11 patients of the control group while only 3 patients occurred in the benzbromarone group (p = .027). We also presented the favorable trend of freedom from the composite endpoints or new-onset HFpEF using Kaplan-Meier curve by log-rank test in benzbromarone group (p = .037 and p = .054). CONCLUSIONS: Our study demonstrated the efficiency of benzbromarone in hypertensive patients with concomitant asymptomatic hyperuricemia, including the benefits on ameliorating LV diastolic dysfunction as well as improving composite endpoints.

The TMEM16A blockers benzbromarone and MONNA cause intracellular Ca2+-release in mouse bronchial smooth muscle cells.

We investigated effects of TMEM16A blockers benzbromarone, MONNA, CaCCinhA01 and Ani9 on isometric contractions in mouse bronchial rings and on intracellular calcium in isolated bronchial myocytes. Separate concentrations of carbachol (0.1-10 µM) were applied for 10 min periods to bronchial rings, producing concentration-dependent contractions that were well maintained throughout each application period. Benzbromarone (1 µM) markedly reduced the contractions with a more pronounced effect on their sustained component (at 10 min) compared to their initial component (at 2 min). Iberiotoxin (0.3 µM) enhanced the contractions, but they were still blocked by benzbromarone. MONNA (3 µM) and CaCCinhA01 (10 µM) had similar effects to benzbromarone, but were less potent. In contrast, Ani9 (10 µM) had no effect on carbachol-induced contractions. Confocal imaging revealed that benzbromarone (0.3 µM), MONNA (1 µM) and CaCCinhA01 (10 µM) increased intracellular calcium in isolated myocytes loaded with Fluo-4AM. In contrast, Ani9 (10 µM) had no effect on intracellular calcium. Benzbromarone and MONNA also increased calcium in calcium-free extracellular solution, but failed to do so when intracellular stores were discharged with caffeine (10 mM). Caffeine was unable to cause further discharge of the store when applied in the presence of benzbromarone. Ryanodine (100 µM) blocked the ability of benzbromarone (0.3 µM) to increase calcium, while tetracaine (100 µM) reversibly reduced the rise in calcium induced by benzbromarone. We conclude that benzbromarone and MONNA caused intracellular calcium release, probably by opening ryanodine receptors. Their ability to block carbachol contractions was likely due to this off-target effect.

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.

Short-Term Hyperuricemia Leads to Structural Retinal Changes That Can be Reversed by Serum Uric Acid Lowering Agents in Mice.

Purpose: Metabolic disorders have been implicated in ocular diseases, such as age-related macular degeneration (AMD) and diabetic retinopathy (DR). Recently, hyperuricemia (HUA) has been proposed as another risk factor for AMD, although no cause-and-effect experimental data have been published. In this study, we investigated whether HUA would initiate AMD or related retinal damages in hyperuricemic mice. Methods: HUA was induced in male ICR mice by dietary supplements of uric acid and oxonic acid potassium salt, with or without treatments by allopurinol or benzbromarone for various durations. Serum uric acid and angiotensin II concentrations were measured by enzyme-linked immunosorbent assay (ELISA) at regular intervals. The retinal damages were assessed by hematoxylin and eosin staining, immunostaining, and TUNEL assay. The cause-and-effect of HUA was compared among the study groups. Results: The results showed that the total thickness of photoreceptor inner and outer segments, as well as the thickness of the photoreceptor outer segment alone, were reduced under HUA. Furthermore, HUA elevated serum angiotensin II, which indicated activation of the renin-angiotensin system (RAS), leading to higher matrix metalloproteinase-2 (MMP-2) expression, and glial activation in the ganglion cell layer. HUA also led to the reduction of retinal pigment epithelium gap junction protein connexin-43 and apoptosis. Uric acid lowering agents, allopurinol or benzbromarone, were effective in ameliorating the impairments. Conclusions: HUA may pose as a causative factor of retinal injuries. The reduction of serum uric acid may reduce the detrimental effects caused by HUA.

Discovery of novel benzbromarone analogs with improved pharmacokinetics and benign toxicity profiles as antihyperuricemic agents.

Benzbromarone (BM) is a potent URAT1 inhibitor approved for the treatment of gout. However, the low URAT1-selectivity and hepatotoxcity limit its clinical use. To solve these problems, we rationally designed and synthesized a series of BM derivatives by chemotype hybridization and bioisosteric replacement. Most compounds exhibited potent inhibitory activities against URAT1 with IC50 values ranging from 5.83 µM to 0.80 µM. Among them, JNS4 exhibited the highest URAT1 inhibitory activity with an IC50 of 0.80 µM, comparable to that of BM (IC50 = 0.53 µM). Molecular dynamic simulations showed that JNS4 formed π-cation interaction with R477, the same as BM. Different from BM, JNS4 bound to W357 and H245 via π-π interactions and formed a hydrogen bond with S35, which might contribute to the high URAT1 binding affinity of JNS4. JNS4 hardly inhibited GLUT9 (IC50 > 20 µM), another urate reabsorption transporter. In addition, JNS4 showed little inhibitory effects against OAT1 and ABCG2 with IC50 of 4.04 µM and 10.16 µM, respectively. Importantly, JNS4 displayed higher in vivo urate-lowering effects at doses of 1-4 mg/kg in a mouse model of hyperuricemia, as compared to BM and lesinurad. Furthermore, JNS4 possessed favorable pharmacokinetic properties with an oral bioavailability of 55.28%, significantly higher than that of BM (36.11%). Moreover, JNS4 demonstrated benign toxicity profiles (no cytotoxicities against HepG2 and HK2 cells; no hepatic and renal toxicities observed in vivo). Collectively, these results suggest that JNS4 represents a novel, safe and selective URAT1 inhibitor with excellent druggabilities and is worthy of further investigation as an anti-hyperuricemic agent.

Beneficial herb-drug interaction of Gnaphalium affine extract on benzbromarone: A pharmacokinetic and pharmacodynamic study in rats.

BACKGROUND: Gnaphalium affine D. Don extract (GAD) enhanced efficacy and reduced toxicity of benzbromarone (BBR) in combination use. However, little is known about effects of GAD on the pharmacokinetics (PKs) and metabolic enzymes of BBR. PURPOSE: To investigate the pharmacokinetic (PK) and pharmacodynamic (PD) mechanism of the herb-drug interactions (HDIs) between GAD and BBR. STUDY DESIGN AND METHODS: Intragastric single BBR (4.5 or 50 mg/kg), single BBR (4.5 or 50 mg/kg) + single GAD (450 mg/kg, 2 h after BBR-administration), or single BBR (4.5 or 50 mg/kg) + multiple GAD (450 mg/kg/day, once daily for 7 days) were administered to both sexes for BBR PK studies in normal rats. Intragastric multiple BBR (4.5 mg/kg/day), or multiple BBR (4.5 mg/kg/day) + multiple GAD (450 mg/kg/day, 2 h after BBR-administration) were administered for BBR PK and PD studies in male rats with hyperuricemic nephropathy (HN). The cumulative anti-hyperuricemic effects of BBR and BBR+GAD were determined by plasma uric acid (UA) concentration-time curve and area under curve (AUCUA). An in vivo cocktail approach was employed to determine the effects of GAD on cytochrome P450 (CYP) 2C11(9) and 1A2 - mediated drug metabolism. RESULTS: In normal rats, the repeated dose administration of GAD induced a significant increase of BBR AUC and prolonged the mean residence time (MRT) (p < 0.05). systemic exposure to BBR and metabolically derived hydroxybenzbromarones was significantly greater in female compared with male rats (p < 0.05). In HN rats, post-administration of GAD resulted in significantly higher bioavailability and enterohepatic recycling (ER) of BBR relative to the BBR alone administrated group from the prolongation of terminal elimination half-life (T1/2) and MRT of BBR (p < 0.05). Significantly higher urate-lowering effect of BBR+GAD compared with BBR alone was generally observed at post-dosing most time points with a maximal effect of 84.3% (acute treatment), 71.4% (7-day subchronic treatment) and 82.5% (14-day subchronic treatment) reduction in UA levels. Additionally, GAD showed a significant inhibitory effect on CYP2C11(9)-mediated tolbutamide (probe substrate) metabolism with ≥ 1.25 but < 2-fold increase in AUCtolbutamide. CONCLUSIONS: PD synergism demonstrated with the BBR+GAD combination could be explained by the PK interaction observed partially from CYP2C11(9)-mediation and enterohepatic recycling.

TMEM16A blockers T16Ainh-A01 and benzbromarone do not modulate the regulation of sweating and cutaneous vasodilatation in humans in vivo.

NEW FINDINGS: What is the central question of this study? Do transmembrane member 16A (TMEM16A) blockers modulate the activation of heat loss responses of sweating and cutaneous vasodilatation? What are the main finding and its importance? Relative to the vehicle control site, TMEM16A blockers T16Ainh-A01 and benzbromarone had no effect on sweat rate or cutaneous vascular conductance during whole-body heating inducing a 1.1 ± 0.1°C increase in core temperature above baseline resting levels. These results suggest that TMEM16A blockers T16Ainh-A01 and benzbromarone do not modulate the regulation of sweating and cutaneous vasodilatation during whole-body heat stress. ABSTRACT: Animal and in vitro studies suggest that transmembrane member 16A (TMEM16A), a Ca2+ -activated Cl- channel, contributes to regulating eccrine sweating. However, direct evidence supporting this possibility in humans is lacking. We assessed the hypothesis that TMEM16A blockers attenuate sweating during whole-body heating in humans. Additionally, we assessed the associated changes in the heat loss response of cutaneous vasodilatation to determine if a functional role of TMEM16A may exist. Twelve young (24 ± 2 years) adults (six females) underwent whole-body heating using a water-perfused suit to raise core temperature 1.1 ± 0.1°C above baseline. Sweat rate and cutaneous vascular conductance (normalized to maximal conductance via administration of sodium nitroprusside) were evaluated continuously at four forearm skin sites treated continuously by intradermal microdialysis with (1) lactated Ringer's solution (control), (2) 5% dimethyl sulfoxide (DMSO) serving as a vehicle control, or (3) TMEM16A blockers 1 mM T16Ainh-A01 or 2 mM benzbromarone dissolved in 5% DMSO solution. All drugs were administered continuously via intradermal microdialysis. Whole-body heating increased core temperature progressively and this was paralleled by an increase in sweat rate and cutaneous vascular conductance at all skin sites. However, sweat rate (all P > 0.318) and cutaneous vascular conductance (all P ≥ 0.073) did not differ between the vehicle control site relative to the TMEM16A blocker-treated sites. Collectively, our findings indicate that TMEM16A blockers T16Ainh-A01 and benzbromarone do not modulate the regulation of sweating and cutaneous vasodilatation during whole-body heating in young adults in vivo.

Hypouricemic effect of 2,4-dihydroxybenzoic acid methyl ester in hyperuricemic mice through inhibiting XOD and down-regulating URAT1.

In this paper, we reported the hypouricemic effect of 2,4-dihydroxybenzoic acid methyl ester (DAE), a component of Ganoderma applanatum, in hyperuricemic mice through inhibiting XOD and down-regulating URAT1. Computationally, DAE showed a high similarity to allopurinol and depicted a high affinity in docking to XOD. In vitro, DAE exhibited an inhibitory effect against XOD. Importantly, DAE demonstrated a remarkable hypouricemic effect, decreasing serum uric acids (SUAs) of hyperuricemic mice (407 ± 31 µmol/L) to 195 ± 23, 145 ± 33 and 134 ± 16 µmol/L (P < 0.01) at the doses of 20, 40, and 80 mg/kg with a dose-dependent manner and showing efficacies at 54-68 %, which were close to the efficacies of allopurinol (61 %) and benzbromarone (57 %). DAE depicted higher and negatively dose-independent urinary uric acids in comparison with that of the hyperuricemic control, implying DAE exerted an uricosuric effect and also a reduction effect on uric acid production. Unlike toxic allopurinol and benzbromarone, no general toxicity on body weights and no negative influence on liver, kidney, spleen and thymus were observed for DAE. Mechanistically, DAE inhibited XOD activities in vivo. Moreover, DAE up-regulated OAT1 and down-regulated GLUT9, URAT1 and CNT2. Overall, DAE may present a hypouricemic effect through inhibiting XOD and up-regulating OAT1 and down-regulating GLUT9, URAT1 and CNT2. This work provided novel insights into the hypouricemic effect of DAE and G. applanatum.

Repurposing benzbromarone as antifolate to develop novel antifungal therapy for Candida albicans.

Fungal infections in humans are responsible for mild to severe infections resulting in systemic effects that cause a large amount of mortality. Invasive fungal infections are having similar symptomatic effects to those of COVID-19. The COVID-19 patients are immunocompromised in nature and have a high probability of developing severe fungal infections, resulting in the development of further complications. The existing antifungal therapy has associated problems related to the development of drug resistance, being sub-potent in nature, and the presence of undesirable toxic effects. The fungal dihydrofolate reductase is an essential enzyme involved in the absorption of dietary folic acid and its conversion into tetrahydrofolate, which is a coenzyme required for the biosynthesis of the fungal nucleotides. Thus, in the current study, an attempt has been made to identify potential folate inhibitors of Candida albicans by a computational drug repurposing approach. Based upon the molecular docking simulation-based virtual screening followed by the molecular dynamic simulation of the macromolecular complex, benzbromarone has been identified as a potential anti-folate agent for the development of a novel therapy for the treatment of candidiasis.

SGLT2 Inhibition and Uric Acid Excretion in Patients with Type 2 Diabetes and Normal Kidney Function.

BACKGROUND AND OBJECTIVES: Sodium-glucose transporter 2 (SGLT2) inhibitor-induced uric acid lowering may contribute to kidney-protective effects of the drug class in people with type 2 diabetes. This study investigates mechanisms of plasma uric acid lowering by SGLT2 inhibitors in people with type 2 diabetes with a focus on urate transporter 1. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We conducted an analysis of two randomized clinical trials. First, in the Renoprotective Effects of Dapagliflozin in Type 2 Diabetes study, 44 people with type 2 diabetes were randomized to dapagliflozin or gliclazide for 12 weeks. Plasma uric acid, fractional uric acid excretion, and hemodynamic kidney function were measured in the fasted state and during clamped euglycemia or hyperglycemia. Second, in the Uric Acid Excretion study, ten people with type 2 diabetes received 1 week of empagliflozin, urate transporter 1 blocker benzbromarone, or their combination in a crossover design, and effects on plasma uric acid, fractional uric acid excretion, and 24-hour uric acid excretion were measured. RESULTS: In the Renoprotective Effects of Dapagliflozin in Type 2 Diabetes study, compared with the fasted state (5.3±1.1 mg/dl), acute hyperinsulinemia and hyperglycemia significantly reduced plasma uric acid by 0.2±0.3 and 0.4±0.3 mg/dl (both P<0.001) while increasing fractional uric acid excretion (by 3.2%±3.1% and 8.9%±4.5%, respectively; both P<0.001). Dapagliflozin reduced plasma uric acid by 0.8±0.8 during fasting, 1.0±1.0 in hyperinsulinemic-euglycemic state, and 0.8±0.7 mg/dl during hyperglycemic conditions (P<0.001), respectively, whereas fractional uric acid excretion in 24-hour urine increased by 3.0%±2.1% (P<0.001) and 2.6%±4.5% during hyperinsulinemic-euglycemic conditions (P=0.003). Fractional uric acid excretion strongly correlated to fractional glucose excretion (r=0.35; P=0.02). In the Uric Acid Excretion study, empagliflozin and benzbromarone both significantly reduced plasma uric acid and increased fractional uric acid excretion. Effects of combination therapy did not differ from benzbromarone monotherapy. CONCLUSIONS: In conclusion, SGLT2 inhibitors induce uric acid excretion, which is strongly linked to urinary glucose excretion and is attenuated during concomitant pharmacologic blockade of urate transporter 1. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Renoprotective Effects of Dapagliflozin in Type 2 Diabetes (RED), NCT02682563; SGLT2 Inhibition: Uric Acid Excretion Study (UREX), NCT05210517.

The Protective Effects of Benzbromarone Against Propofol-Induced Inflammation and Injury in Human Brain Microvascular Endothelial Cells (HBMVECs).

It has been widely reported that severe neurotoxicity can be induced by the application of propofol, which is closely related to the disruption of the blood-brain barrier (BBB) induced by inflammation and injury in the human brain microvascular endothelial cells (HBMVECs). Benzbromarone is a classic anti-gout agent that has been recently reported to exert anti-inflammatory and anti-oxidative stress effects. In the present study, we aim to investigate the protective property of Benzbromarone against propofol-induced injury on HBMVECs and the underlying mechanism. CCK8 assay was used to detect the cell viability of treated HBMVECs. Oxidative stress in HBMVECs was evaluated by measuring the levels of MDA and mitochondrial ROS. ELISA and qRT-PCR assay were used to determine the production of IL-1ß, IL-8, MCP-1, ICAM-1, and VCAM-1 by treated HBMVECs. Calcein-AM staining was utilized to evaluate the attachment of U937 monocytes to HBMVECs. The expression level of Egr-1 was determined by qRT-PCR and Western blot assay. Firstly, the decreased cell viability of HBMVECs induced by propofol was significantly elevated by treatment with Benzbromarone. The increased levels of MDA and mitochondrial ROS induced by propofol were dramatically suppressed by Benzbromarone. Secondly, the excessive production of inflammatory factors (IL-1ß, IL-8, and MCP-1) and adhesion molecules (ICAM-1 and VCAM-1) triggered by propofol was pronouncedly inhibited by Benzbromarone. Benzbromarone ameliorated propofol-induced attachment of U937 monocytes to HBMVECs. Lastly, Benzbromarone downregulated propofol-induced expression of the transcriptional factor Egr-1 in HBMVECs. Benzbromarone protected against propofol-induced inflammation and injury through suppressing Egr-1 in human brain vascular endothelial cells.

Differential Reversible and Irreversible Interactions between Benzbromarone and Human Cytochrome P450s 3A4 and 3A5.

Mounting evidence has revealed that despite the high degree of sequence homology between cytochrome P450 3A isoforms (i.e., CYP3A4 and CYP3A5), they have the propensities to exhibit vastly different irreversible and reversible interactions with a single substrate. We have previously established that benzbromarone (BBR), a potent uricosuric agent used in the management of gout, irreversibly inhibits CYP3A4 via mechanism-based inactivation (MBI). However, it remains unelucidated if CYP3A5-its highly homologous counterpart-is susceptible to inactivation by BBR. Using three structurally distinct probe substrates, we consistently demonstrated that MBI was not elicited in CYP3A5 by BBR. Our in silico covalent docking models and molecular dynamics simulations suggested that disparities in the susceptibilities toward MBI could be attributed to the specific effects of BBR covalent adducts on the F-F' loop. Serendipitously, we also discovered that BBR reversibly activated CYP3A5-mediated rivaroxaban hydroxylation wherein apparent V max increased and K m decreased with increasing BBR concentration. Fitting data to the two-site model yielded interaction factors α and ß of 0.44 and 5.88, respectively, thereby confirming heterotropic activation of CYP3A5 by BBR. Furthermore, heteroactivation was suppressed by the CYP3A inhibitor ketoconazole in a concentration-dependent manner and decreased with increasing preincubation time, implying that activation was incited via binding of parent BBR molecule within the enzymatic active site. Finally, noncovalent docking revealed that CYP3A5 can more favorably accommodate both BBR and rivaroxaban in concert as compared with CYP3A4, which further substantiated our experimental observations. SIGNIFICANCE STATEMENT: Although it has been previously demonstrated that benzbromarone (BBR) inactivates CYP3A4, it remains uninterrogated whether it also elicits mechanism-based inactivation in CYP3A5, which shares ∼85% sequence similarity with CYP3A4. This study reported that BBR exhibited differential irreversible and reversible interactions with both CYP3A isoforms and further unraveled the molecular determinants underpinning their diverging interactions. These data offer important insight into differential kinetic behavior of CYP3A4 and CYP3A5, which potentially contributes to interindividual variabilities in drug disposition.

Functional and Pharmacological Comparison of Human and Mouse Na+/Taurocholate Cotransporting Polypeptide (NTCP).

The Na+/taurocholate cotransporting polypeptide (NTCP) is located in the basolateral membrane of hepatocytes, where it transports bile acids from the portal blood back into hepatocytes. Furthermore, NTCP has a role for the hepatic transport of some drugs. Extrapolation of drug transport data from rodents to humans is not always possible, because species differences in the expression level, localization, affinity, and substrate selectivity of relevant transport proteins must be considered. In the present study, a functional comparison of human NTCP (hNTCP) and mouse Ntcp (mNtcp) showed similar Km values of 67 ± 10 µM and 104 ± 9 µM for the probe substrate estrone-3-sulfate as well as of 258 ± 42 µM and 199 ± 13 µM for the drug rosuvastatin, respectively. IC50 values for the probe inhibitor cyclosporine A were 3.1 ± 0.3 µM for hNTCP and 1.6 ± 0.4 µM for mNtcp. In a drug and pesticide inhibitory screening on both transporters, 4 of the 15 tested drugs (cyclosporine A, benzbromarone, MK571, and fluvastatin) showed high inhibitory potency, but only slight inhibition was observed for the 13 tested pesticides. Among these compounds, only four drugs and three pesticides showed significant differences in their inhibition pattern on hNTCP and mNtcp. Most pronounced was the difference for benzbromarone with a fivefold higher IC50 for mNtcp (27 ± 10 µM) than for hNTCP (5.5 ± 0.6 µM).In conclusion, we found a strong correlation between the transport kinetics and inhibition pattern among hNTCP and mNtcp. However, specific compounds, such as benzbromarone, showed clear species differences. Such species differences have to be considered when pharmacokinetic data are transferred from rodent to humans.

Novel monocyclic amide-linked phenol derivatives without mitochondrial toxicity have potent uric acid-lowering activity.

Although benzbromarone (BBR) is a conventional, highly potent uricosuric drug, it is not a standard medicine because it causes rare but fatal fulminant hepatitis. We transformed the bis-aryl ketone structure of BBR to generate novel monocyclic amide-linked phenol derivatives that should possess uric acid excretion activity without adverse properties associated with BBR. The derivatives were synthesized and tested for uric acid uptake inhibition (UUI) in two assays using either urate transporter 1-expressing cells or primary human renal proximal tubule epithelial cells. We also evaluated their inhibitory activity against mitochondrial respiration as a critical mitochondrial toxicity parameter. Some derivatives with UUI activity had no mitochondrial toxicity, including compound 3f, which effectively lowered the plasma uric acid level in Cebus apella. Thus, 3f is a promising candidate for further development as a uricosuric agent.

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.