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.

Interaction of Benzbromarone with Subdomains IIIA and IB/IIA on Human Serum Albumin as the Primary and Secondary Binding Regions.

Benzbromarone has been used for the treatment of gout for more than 30 years. Although it shows a high level of binding to plasma proteins (>99%), our knowledge of this binding is not sufficiently extensive to permit us to understand its pharmacokinetics and pharmacodynamics. To address this issue in more detail, we characterized the binding of benzbromarone to human serum albumin (HSA), the most abundant protein in plasma. Equilibrium dialysis and circular dichroism findings indicated that benzbromarone binds strongly to one primary as well as to multiple secondary sites on HSA and that the bromine atoms of benzbromarone play important roles in this high affinity binding. An X-ray crystallographic study revealed that benzbromarone molecules bind to hydrophobic pockets within subdomains IB, IIA, and IIIA. Inhibition experiments using site specific ligands (subdomain IB; fusidic acid, IIA; warfarin, IIIA; diazepam) indicated that the primary and secondary binding sites that benzbromarone binds to are within subdomains IIIA and IB/IIA, respectively. Lastly, a study of the effect of fatty acids on the benzbromarone-HSA interaction suggested that benzbromarone, when displaced from subdomain IIIA by sodium oleate, could transfer to subdomains IB or IIA. Thus, these data will permit more relevant assessments of the displacement interactions of benzbromarone especially in cases of co-administered drugs or endogenous compounds that also bind to subdomain IIIA. In addition, the findings presented herein will also be useful for designing drug combination therapy in which pharmacokinetic and pharmacodynamic performance need to be controlled.

Developing, optimizing, and assessing a green electrophoretic method for determination of benzbromarone and allopurinol with its active metabolite in biological and pharmaceutical matrices.

A combination of allopurinol and benzbromarone is a common gout treatment protocol. A suboptimal response to allopurinol in patients is very common due to its pharmacokinetics variability. Moreover, the safe doses of benzbromarone is very crucial in patients with hepatic diseases. This raised the inquisitiveness to develop and optimize a capillary zone electrophoresis method for the determination of allopurinol and benzbromarone in their coformulation and in the presence of oxypurinol, the active metabolite of allopurinol, in biological and pharmaceutical matrices. The method greenness profile was assessed using green metric tools the "National Environmental Method Index," the "Analytical Eco-Scale," and the "Green Analytical Procedure Index" by which the method proved to be ecofriendly. The method was successfully applied for the analysis of the pharmaceutical preparation and urine samples spiked with both drugs and the active metabolite. The linearity range was 25.0-250.0 µg/mL for benzbromarone, 50.0-350.0 µg/mL for allopurinol, and 100.0-500.0 µg/mL for oxypurinol. The recoveries were 99.60 ± 0.67, 99.89 ± 0.98, and 98.71 ± 1.18% for benzbromarone, allopurinol, and oxypurinol, respectively. The analysis results indicate potential usefulness of capillary zone electrophoresis as a competitive and greener method of analysis in biological and quality control labs.

Repurposing Benzbromarone for Familial Amyloid Polyneuropathy: A New Transthyretin Tetramer Stabilizer.

Transthyretin (TTR) is a homotetrameric protein involved in human amyloidosis, including familial amyloid polyneuropathy (FAP). Discovering small-molecule stabilizers of the TTR tetramer is a therapeutic strategy for these diseases. Tafamidis, the only approved drug for FAP treatment, is not effective for all patients. Herein, we discovered that benzbromarone (BBM), a uricosuric drug, is an effective TTR stabilizer and inhibitor against TTR amyloid fibril formation. BBM rendered TTR more resistant to urea denaturation, similarly to iododiflunisal (IDIF), a very potent TTR stabilizer. BBM competes with thyroxine for binding in the TTR central channel, with an IC50 similar to IDIF and tafamidis. Results obtained by isothermal titration calorimetry (ITC) demonstrated that BBM binds TTR with an affinity similar to IDIF, tolcapone and tafamidis, confirming BBM as a potent binder of TTR. The crystal structure of the BBM-TTR complex shows two molecules binding deeply in the thyroxine binding channel, forming strong intermonomer hydrogen bonds and increasing the stability of the TTR tetramer. Finally, kinetic analysis of the ability of BBM to inhibit TTR fibrillogenesis at acidic pH and comparison with other stabilizers revealed that benzbromarone is a potent inhibitor of TTR amyloidogenesis, adding a new interesting scaffold for drug design of TTR stabilizers.

Glutathione Conjugation and Protein Adduction Derived from Oxidative Debromination of Benzbromarone in Mice.

Benzbromarone (BBR), a uricosuric agent, has been known to induce hepatotoxicity, and its toxicity has a close relation to cytochrome P450-mediated metabolic activation. An oxidative debromination metabolite of BBR has been reported in microsomal incubations. The present study attempted to define the oxidative debromination pathway of BBR in vivo. One urinary mercapturic acid (M1) and one glutathione (GSH) conjugate (M2) derived from the oxidative debromination metabolite were detected in BBR-treated mice after solid phase extraction. M1 and M2 shared the same chromatographic behavior and mass spectral identities as those detected in N-acetylcysteine/GSH- and BBR-fortified microsomal incubations. The structure of M1 was characterized by chemical synthesis, along with mass spectrometry analysis. In addition, hepatic protein modification that occurs at cysteine residues (M'3) was observed in mice given BBR. The observed protein adduction reached its peak 4 hours after administration and occurred in a dose-dependent manner. A GSH conjugate derived from oxidative debromination of BBR was detected in livers of mice treated with BBR, and the formation of the GSH conjugate apparently took place earlier than the protein adduction. In summary, our in vivo work provided strong evidence for the proposed oxidative debromination pathway of BBR, which facilitates the understanding of the mechanisms of BBR-induced hepatotoxicity. SIGNIFICANCE STATEMENT: This study investigated the oxidative debromination pathway of benzbromarone (BBR) in vivo. One urinary mercapturic acid (M1) and one glutathione (GSH) conjugate (M2) derived from the oxidative debromination metabolite were detected in BBR-treated mice. M1 and M2 were also observed in microsomal incubations. The structure of M1 was characterized by chemical synthesis followed by mass spectrometry analyses. More importantly, protein adduction derived from oxidative debromination of BBR (M'3) was observed in mice given BBR, and occurred in dose- and time-dependent manners. The success in detection of GSH conjugate, urinary N-acetylcysteine conjugate, and hepatic protein adduction in mice given BBR provided solid evidence for in vivo oxidative debromination of BBR. The studies allowed a better understanding of the metabolic activation of BBR.

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.

Metabolic Epoxidation Is a Critical Step for the Development of Benzbromarone-Induced Hepatotoxicity.

Benzbromarone (BBR) is effective in the treatment of gout; however, clinical findings have shown it can also cause fatal hepatic failure. Our early studies demonstrated that CYP3A catalyzed the biotransformation of BBR to epoxide intermediate(s) that reacted with sulfur nucleophiles of protein to form protein covalent binding both in vitro and in vivo. The present study attempted to define the correlation between metabolic epoxidation and hepatotoxicity of BBR by manipulating the structure of BBR. We rationally designed and synthesized three halogenated BBR derivatives, fluorinated BBR (6-F-BBR), chlorinated BBR (6-Cl-BBR), and brominated BBR (6-Br-BBR), to decrease the potential for cytochrome P450-mediated metabolic activation. Both in vitro and in vivo uricosuric activity assays showed that 6-F-BBR achieved favorable uricosuric effect, while 6-Cl-BBR and 6-Br-BBR showed weak uricosuric efficacy. Additionally, 6-F-BBR elicited much lower hepatotoxicity in mice. Fluorination of BBR offered advantage to metabolic stability in liver microsomes, almost completely blocked the formation of epoxide metabolite(s) and protein covalent binding, and attenuated hepatic and plasma glutathione depletion. Moreover, the structural manipulation did not alter the efficacy of BBR. This work provided solid evidence that the formation of the epoxide(s) is a key step in the development of BBR-induced hepatotoxicity.

Identification of Epoxide-Derived Metabolite(s) of Benzbromarone.

Benzbromarone (BBR) is a benzofuran derivative that has been quite useful for the treatment of gout; however, it was withdrawn from European markets in 2003 because of reported serious incidents of drug-induced liver injury. BBR-induced hepatotoxicity has been suggested to be associated with the formation of a quinone intermediate. The present study reported epoxide-derived intermediate(s) of BBR. An N-acetylcysteine (NAC) conjugate derived from epoxide metabolite(s) was detected in both microsomal incubations of BBR and urine samples of mice treated with BBR. The NAC conjugate was identified as 6-NAC BBR. Ketoconazole suppressed the bioactivation of BBR to the epoxide intermediate(s), and the CYP3A subfamily was the primary enzyme responsible for the formation of the epoxide(s). The present study provided new information on metabolic activation of BBR.

Cysteine-Based Protein Adduction by Epoxide-Derived Metabolite(s) of Benzbromarone.

Benzbromarone (BBR) is a therapeutically useful uricosuric agent but can also cause acute liver injury. The hepatotoxicity of BBR is suggested to be associated with its metabolic activation. Our recent metabolic study demonstrated that BBR was metabolized to epoxide intermediate(s) by cytochrome P450 3A, and the intermediate(s) was reactive to N-acetylcysteine. The objectives of the present study were to determine the chemical identity of the interaction of protein with the epoxide intermediate(s) of BBR and to define the association of the protein modification with hepatotoxicity induced by BBR. Microsomal incubation study showed that the reactive intermediate(s) covalently modified microsomal protein at cysteine residues. Such adduction was also observed in hepatic protein obtained from liver of mice given BBR. The protein covalent binding occurred in time- and dose-dependent manners. Pretreatment with ketoconazole attenuated BBR-induced protein modification and hepatotoxicity, while pretreatment with dexamethasone or buthionine sulfoximine potentiated the protein adduction and hepatotoxicity induced by BBR. A good correlation was observed between BBR-induced hepatotoxicity and the epoxide-derived hepatic protein modification in mice. The present study provided in-depth mechanistic insight into BBR-induced hepatotoxicity.

Development of a cell viability assay to assess drug metabolite structure-toxicity relationships.

Many adverse drug reactions are caused by the cytochrome P450 (CYP)-dependent activation of drugs into reactive metabolites. In order to reduce attrition due to metabolism-induced toxicity and to improve the safety of drug candidates, we developed a simple cell viability assay by combining a bioactivation system (human CYP3A4, CYP2D6 and CYP2C9) with Hep3B cells. We screened a series of drugs to explore structural motifs that may be responsible for CYP450-dependent activation caused by reactive metabolite formation, which highlighted specific liabilities regarding certain phenols and anilines.

Metabolic activation of hepatotoxic drug (benzbromarone) induced mitochondrial membrane permeability transition.

The risk of drug-induced liver injury (DILI) is of great concern to the pharmaceutical industry. It is well-known that metabolic activation of drugs to form toxic metabolites (TMs) is strongly associated with DILI onset. Drug-induced mitochondrial dysfunction is also strongly associated with increased risk of DILI. However, it is difficult to determine the target of TMs associated with exacerbation of DILI because of difficulties in identifying and purifying TMs. In this study, we propose a sequential in vitro assay system to assess TM formation and their ability to induce mitochondrial permeability transition (MPT) in a one-pot process. In this assay system, freshly-isolated rat liver mitochondria were incubated with reaction solutions of 44 test drugs preincubated with liver microsomes in the presence or absence of NADPH; then, NADPH-dependent MPT pore opening was assessed as mitochondrial swelling. In this assay system, several hepatotoxic drugs, including benzbromarone (BBR), significantly induced MPT in a NADPH-dependent manner. We investigated the rationality of using BBR as a model drug, since it showed the most prominent MPT in our assay system. Both the production of a candidate toxic metabolite of BBR (1',6-(OH)2 BBR) and NADPH-dependent MPT were inhibited by several cytochrome P450 (CYP) inhibitors (clotrimazole and SKF-525A, 100µM). In summary, this assay system can be used to evaluate comprehensive metabolite-dependent MPT without identification or purification of metabolites.

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.

Identification of CYP isozymes involved in benzbromarone metabolism in human liver microsomes.

Benzbromarone (BBR) is metabolized to 1'-hydroxy BBR and 6-hydroxy BBR in the liver. 6-Hydroxy BBR is further metabolized to 5,6-dihydroxy BBR. The aim of this study was to identify the CYP isozymes involved in the metabolism of BBR to 1'-hydroxy BBR and 6-hydroxy BBR and in the metabolism of 6-hydroxy BBR to 5,6-dihydroxy BBR in human liver microsomes. Among 11 recombinant P450 isozymes examined, CYP3A4 showed the highest formation rate of 1'-hydroxy BBR. The formation rate of 1'-hydroxy BBR significantly correlated with testosterone 6ß-hydroxylation activity in a panel of 12 human liver microsomes. The formation of 1'-hydroxy BBR was completely inhibited by ketoconazole in pooled human liver microsomes. On the other hand, the highest formation rate of 6-hydroxy BBR was found in recombinant CYP2C9. The highest correlation was observed between the formation rate of 6-hydroxy BBR and diclofenac 4'-hydroxylation activity in 12 human liver microsomes. The formation of 6-hydroxy BBR was inhibited by tienilic acid in pooled human liver microsomes. The formation of 5,6-dihydroxy BBR from 6-hydroxy BBR was catalysed by recombinant CYP2C9 and CYP1A2. The formation rate of 5,6-dihydroxy BBR was significantly correlated with diclofenac 4'-hydroxylation activity and phenacetin O-deethylation activity in 12 human liver microsomes. The formation of 5,6-dihydroxy BBR was inhibited with either tienilic acid or α-naphthoflavone in human liver microsomes. These results suggest that (i) the formation of 1'-hydroxy BBR and 6-hydroxy BBR is mainly catalysed by CYP3A4 and CYP2C9, respectively, and (ii) the formation of 5,6-dihydroxy BBR is catalysed by CYP2C9 and CYP1A2 in human liver microsomes.

Formation of reactive metabolites of benzbromarone in humanized-liver mice.

Benzbromarone, a uricosuric drug, has the potential to cause serious hepatotoxicity. Several studies have shown the formation of reactive metabolites of benzbromarone and their association with hepatotoxicity in mice. However, it is unknown whether those reactive metabolites are generated in humans in vivo. In the present study, we firstly investigated the pharmacokinetic profiles of benzbromarone in chimeric TK-NOG mice transplanted with human hepatocytes (humanized-liver mice) and then investigated whether reactive metabolites could be generated. The area under the plasma concentration-time curve ratio of benzbromarone and its major metabolites (benzbromarone: 1'-hydroxy benzbromarone: 6-hydroxy benzbromarone) in humanized-liver mice was 1: 1.2: 0.7, which was similar to that reported in humans. In addition, glutathione conjugates and their further metabolites derived from the epoxidation of the benzofuran ring and 1',6-dihydroxylation of benzbromarone were detected in the livers, urine and plasma. Furthermore, their peak intensities in mass spectrometry showed markedly higher levels compared with those of TK-NOG mice. These results suggested that the metabolic profiles of benzbromarone in humanized-liver mice were similar to those in humans and that the reactive metabolites detected in humanized-liver mice could be generated and are associated with the benzbromarone-induced hepatotoxicity in humans.

CYP2C9-mediated metabolic activation of losartan detected by a highly sensitive cell-based screening assay.

Drug-induced hepatotoxicity is a major problem in drug development, and reactive metabolites generated by cytochrome P450s are suggested to be one of the causes. CYP2C9 is one of the major enzymes in hepatic drug metabolism. In the present study, we developed a highly sensitive cell-based screening system for CYP2C9-mediated metabolic activation using an adenovirus vector expressing CYP2C9 (AdCYP2C9). Human hepatocarcinoma HepG2 cells infected with our constructed AdCYP2C9 for 2 days at multiplicity of infection 10 showed significantly higher diclofenac 4'-hydroxylase activity than human hepatocytes. AdCYP2C9-infected cells were treated with several hepatotoxic drugs, resulting in a significant increase in cytotoxicity by treatment with losartan, benzbromarone, and tienilic acid. Metabolic activation of losartan by CYP2C9 has never been reported, although the metabolic activations of benzbromarone and tienilic acid have been reported. To identify the reactive metabolites of losartan, the semicarbazide adducts of losartan were investigated by liquid chromatography-tandem mass spectrometry. Two CYP2C9-specific semicarbazide adducts of losartan (S1 and S2) were detected. S2 adduct formation suggested that a reactive metabolite was produced from the aldehyde metabolite E3179, but a possible metabolite from S1 adduct formation was not produced via E3179. In conclusion, a highly sensitive cell-based assay to evaluate CYP2C9-mediated metabolic activation was established, and we found for the first time that CYP2C9 is involved in the metabolic activation of losartan. This cell-based assay system would be useful for evaluating drug-induced cytotoxicity caused by human CYP2C9.

The uricosuric benzbromarone disturbs the mitochondrial redox homeostasis and activates the NRF2 signaling pathway in HepG2 cells.

The uricosuric benzbromarone is a mitochondrial toxicant associated with severe liver injury in patients treated with this drug. Since dysfunctional mitochondria can increase mitochondrial superoxide (O2•-) production, we investigated the consequences of benzbromarone-induced mitochondrial oxidative stress on the hepatic antioxidative defense system. We exposed HepG2 cells (a human hepatocellular carcinoma cell line) to increasing concentrations of benzbromarone (1-100 µM) for different durations (2-24 h), and investigated markers of antioxidative defense and oxidative damage. At high concentrations (≥50 µM), benzbromarone caused accumulation of mitochondrial superoxide (O2•-) and cellular reactive oxygen species (ROS). At concentrations >50 µM, benzbromarone increased the mitochondrial and cellular GSSG/GSH ratio and increased the oxidized portion of the mitochondrial thioredoxin 2. Benzbromarone stabilized the transcription factor NRF2 and caused its translocation into the nucleus. Consequently, the expression of the NRF2-regulated antioxidative proteins superoxide dismutase 1 (SOD1) and 2 (SOD2), glutathione peroxidase 1 (GPX1) and 4 (GPX4), as well as thioredoxin 1 (TRX1) and 2 (TRX2) increased. Finally, upregulation of NRF2 by siRNA-mediated knock-down of KEAP1 partially protected HepG2 cells from benzbromarone-induced membrane damage and ATP depletion. In conclusion, benzbromarone increased mitochondrial O2•- accumulation and activates the NRF2 signaling pathway in HepG2 cells, thereby strengthening the cytosolic and mitochondrial antioxidative defense. Impaired antioxidative defense may represent a risk factor for benzbromarone-induced hepatotoxicity.

Assessment of unconventional antimicrobial compounds for the control of 'Candidatus Liberibacter asiaticus', the causative agent of citrus greening disease.

In this study, newly identified small molecules were examined for efficacy against 'Candidatus Liberibacter asiaticus' in commercial groves of sweet orange (Citrus sinensis) and white grapefruit (Citrus paradisi) trees. We used benzbromarone and/or tolfenamic acid delivered by trunk injection. We evaluated safety and efficacy parameters by performing RNAseq of the citrus host responses, 16S rRNA gene sequencing to characterize citrus-associated microbial communities during treatment, and qRT-PCR as an indirect determination of 'Ca. L. asiaticus' viability. Analyses of the C. sinensis transcriptome indicated that each treatment consistently induced genes associated with normal metabolism and growth, without compromising tree viability or negatively affecting the indigenous citrus-associated microbiota. It was found that treatment-associated reduction in 'Ca. L. asiaticus' was positively correlated with the proliferation of several core taxa related with citrus health. No symptoms of phytotoxicity were observed in any of the treated trees. Trials were also performed in commercial groves to examine the effect of each treatment on fruit productivity, juice quality and efficacy against 'Ca. L. asiaticus'. Increased fruit production (15%) was observed in C. paradisi following twelve months of treatment with benzbromarone and tolfenamic acid. These results were positively correlated with decreased 'Ca. L. asiaticus' transcriptional activity in root samples.

Human MRP2 exports MC-LR but not the glutathione conjugate.

Water contamination by cyanobacterial blooms is a worldwide health hazard to humans as well as livestock. Exposure to Microcystins (MCs), toxins produced by various cyanobacterial or blue green algae found in poorly treated drinking water or contaminated seafood such as fish or prawns are associated with hepatotoxicity, nephropathy and neurotoxicity and in extreme cases, death in humans. MC congeners, currently >240 known, differ dramatically in their uptake kinetics, i.e. their uptake via OATP1B1 and OATP1B3, in OATP overexpressing human HEK293 cells and primary human hepatocytes. It is thus likely that MC congeners will also differ with respect to the cellular efflux of the parent and conjugated congeners, e.g. via MRPs, MDRs, BCRP or BSEP. Consequently, the role and kinetics of different human efflux transporters - MRP, MDR, BCRP and BSEP in MC efflux was studied using insect membrane vesicles overexpressing the human transporters of interest. Of the efflux transporters investigated, MRP2 displayed MC transport. Michaelis-Menten kinetics displayed mild co-operativity and thus allosteric behavior of MRP2. MC transport by MRP2 was MC congener-specific, whereby MC-LF was transported more rapidly than MC-LR and -RR. Other human transporters (BCRP, BSEP, MRP1,3,5, MDR1) tested in this study did not exhibit interaction with MC. Although MRP2 showed specific MC transport, the MC-LR-GSH conjugate, was not transported suggesting the involvement of other transporters than MRP2 for the conjugate efflux.

Benzbromarone in the treatment of gout.

BACKGROUND: Benzbromarone is a uricosuric drug that has been used in the treatment of gout over the last 30 years. Due to its potent inhibition of the dominant apical (luminal) urate exchanger in the human proximal tubule URAT1, it reduces the urate reabsorption, diminishing serum urate levels and therefore preventing gout flares. Through several clinical trials, Benzbromarone has been proved effective and safe, inclusive in patients with chronic kidney disease and as combination therapy with allopurinol. Due to hepatotoxicity reports, it was withdrawn from the European market by the manufacturer, however many authors have questioned the product's withdrawal due to a lack of clinical evidence in order to support its hepatotoxicity. Benzbromarone is still available in several European countries, New Zealand, Brazil and several other countries. Despite the product's marketing over more than 20 years after the first hepatotoxicity reports, we have found only five reports in our literature search, and no prospective or retrospective study correlating hepatotoxicity with benzbromarone use. SHORT CONCLUSION: Benzbromarone is a safe and effective molecule for the treatment of gout. However, due to in vitro and in vivo data related to hepatotoxicity, it is prudent to prescribe it with some caution, especially for patients with an already known liver condition.

Identification of novel glutathione adducts of benzbromarone in human liver microsomes.

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.