Letters to the Editor: Fatal fulminant hepatic failure associated with benzbromarone

The objective of the study was to compare the relative effects of benzbromarone and allopurinol on the risk of developing chronic kidney disease in persons with asymptomatic hyperuricemia.

A retrospective cohort study was conducted to analyze a 2003–2015 national database including all claims data of 2 million beneficiaries in Taiwan. Asymptomatic hyperuricemia was defined as follows: persons using urate-lowering drugs who never developed gout flares. The benzbromarone group included persons ages 20–84 that had asymptomatic hyperuricemia and received benzbromarone alone. The allopurinol group included persons ages 20–84 that had asymptomatic hyperuricemia and received allopurinol alone. The maximum follow-up time was set as 5 years in this study. The main outcome was defined as follows: persons were newly diagnosed with chronic kidney disease. A Cox proportional hazards regression analysis was performed to test the association between variables and the risk of chronic kidney disease.

After propensity score matching, 9107 persons in the benzbromarone group and 4554 persons in the allopurinol group were eligible for the study. Approximately 71% of the study subjects were males. The mean age was 56 years old. The incidence rate of chronic kidney disease was lower in the benzbromarone group than in the allopurinol group (1.18 versus 1.99/per 100 person-years, incidence ratio = 0.60, and 95% confidence interval = 0.52–0.68).The Cox proportional hazards regression analysis disclosed that after adjusting for co-variables, there was a decreased risk of developing chronic kidney disease in the benzbromarone group as compared with the allopurinol group (hazard ratio = 0.59, 95% confidence interval = 0.52–0.67 and P<0.001).

The use of benzbromarone is associated with a lower hazard of developing chronic kidney disease as compared to allopurinol use among persons ages 20–84 with asymptomatic hyperuricemia. More studies are needed to confirm our findings.

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.

As a widely used anti-gout drug, benzbromarone has been found to induce hepatic toxicity in patients during clinical treatment. Previous studies have reported that benzbromarone is metabolized via cytochrome P450, thus causing mitochondrial toxicity in hepatocytes. In this study, we found that benzbromarone significantly aggravated hepatic steatosis in both obese db/db mice and high fat diet (HFD)-induced obese (DIO) mouse models. However, benzbromarone had less effect on the liver of lean mice. It was found that the expression of mRNAs encoding lipid metabolism and some liver-specific genes were obviously disturbed in benzbromarone-treated DIO mice compared to the control group. The inflammatory and oxidative stress factors were also activated in the liver of benzbromarone-treated DIO mice. In accordance with the in vivo results, an in vitro experiment using human hepatoma HepG2 cells also confirmed that benzbromarone promoted intracellular lipid accumulation under high free fatty acids (FFAs) conditions by regulating the expression of lipid metabolism genes. Importantly, prolonged treatment of benzbromarone significantly increased cell apoptosis in HepG2 cells in the presence of high FFAs. In addition, in benzbromarone-treated hyperuricemic patients, serum transaminase levels were positively correlated with patients' obesity level.

This study demonstrated that benzbromarone aggravated hepatic steatosis in obese individuals, which could subsequently contribute to hepatic cell injury, suggesting a novel toxicological mechanism in benzbromarone-induced hepatotoxicity.

Benzbromarone is a uricosuric drug metabolized predominantly by cytochrome P450 2C9 from in vitro findings. Human CYP2C9 exhibits extensive genetic polymorphism and numbers of clinic studies have demonstrated that CYP2C9 genetic polymorphism has a significant influence on the pharmacokinetics of benzbromarone. But in vitro study on the interaction between CYP2C9 allelic isoforms and benzbromarone was rare. Here, an LC–MS/MS method was established and validated to determine the concentration of benzbromarone in different CYP2C9 enzyme incubation systems for the drug-enzyme interaction study. By selecting appropriate internal standard and optimizing separation system, including mobile phase, sample solvent and gradient elution condition, this LC–MS/MS method was developed with fine linearity (r² ≥ 0.996), good reproducibility (RSD ≤ 6.6%), high stability (92.37–114.67%), efficient recovery (91.23–109.82%) and acceptable matrix effect (110.54–115.31%). Based on this method, the interaction between 3 CYP2C9 allelic isoforms and benzbromarone was researched by kinetics parameters (K_m, V_max, Cl_int). As a result, CYP2C9*1 displayed the highest metabolic activity towards benzbromarone, CYP2C9*2 showed a little lower catalytic activity than CYP2C9*1 (relative clearance/*1 = 85.86%), CYP2C9*3 showed the lowest catalytic activity (relative clearance/*1 = 21.57%). The result illustrated that various CYP2C9 allelic isoforms showed different enzymatic activities towards benzbromarone, which could offer effective consultation for personalized administration in clinic.

Benzbromarone (BBR) is a potent uricosuric drug that can cause serious liver injury. Our recent study suggested that 1′-hydroxy BBR, one of major metabolites of BBR, is metabolized to a cytotoxic metabolite that could be detoxified by glutathione (GSH). The aim of this study was to clarify whether GSH adducts are formed from 1′-hydroxy BBR in human liver microsomes (HLM). Incubation of 1′-hydroxy BBR with GSH in HLM did not result in the formation of GSH adducts, but 1′,6-dihydroxy BBR was formed. In addition, incubation of 1′,6-dihydroxy BBR with GSH in HLM resulted in the formation of three novel GSH adducts (M1, M2 and M3). The structures of M1 and M2 were estimated to be GSH adducts in which the 1-hydroxyethyl group at the C-2 position and the hydroxyl group at the C-1′ position of 1′,6-dihydroxy BBR were substituted by GSH, respectively. We also found that the 6-hydroxylation of 1′-hydroxy BBR is mainly catalyzed by CYP2C9 and that several CYPs and/or non-enzymatic reaction are involved in the formation of GSH adducts from 1′,6-dihydroxy BBR. The results indicate that 1′-hydroxy BBR is metabolized to reactive metabolites via 1′,6-dihydroxy BBR formation, suggesting that these reactive metabolites are responsible for BBR-induced liver injury.

Benzbromarone is an uricosuric structurally related to amiodarone and a known mitochondrial toxicant. The aim of the current study was to improve our understanding in the molecular mechanisms of benzbromarone-associated hepatic mitochondrial toxicity. In HepG2 cells and primary human hepatocytes, ATP levels started to decrease in the presence of 25–50 μM benzbromarone for 24–48 h, whereas cytotoxicity was observed only at 100 μM. In HepG2 cells, benzbromarone decreased the mitochondrial membrane potential starting at 50 μM following incubation for 24 h. Additionally, in HepG2 cells, 50 μM benzbromarone for 24 h induced mitochondrial uncoupling,and decreased mitochondrial ATP turnover and maximal respiration. This was accompanied by an increased lactate concentration in the cell culture supernatant, reflecting increased glycolysis as a compensatory mechanism to maintain cellular ATP. Investigation of the electron transport chain revealed a decreased activity of all relevant enzyme complexes. Furthermore, treatment with benzbromarone was associated with increased cellular ROS production, which could be located specifically to mitochondria. In HepG2 cells and in isolated mouse liver mitochondria, benzbromarone also reduced palmitic acid metabolism due to an inhibition of the long-chain acyl CoA synthetase. In HepG2 cells, benzbromarone disrupted the mitochondrial network, leading to mitochondrial fragmentation and a decreased mitochondrial volume per cell. Cell death occurred by both apoptosis and necrosis. The study demonstrates that benzbromarone not only affects the function of mitochondria in HepG2 cells and human hepatocytes, but is also associated with profound changes in mitochondrial structure which may be associated with apoptosis.