Metabolic Activation,hepatotoxicity And Structural Optimization Of Benzbromarone

Gout is a form of inflammatory arthritis caused by elevation of blood urate levels(a medical condition known as hyperuricemia),which crystallize and deposit into the joints and surrounding tissues.With the improvement of people’s living standard and the change of life style,the prevalence of gout has increased substantially and the incidence is trending younger.Gout has become a common disease that threatens human health.Benzbromarone(BBR),as an anti-gout agent,shows the strongest uricosuric effect at present.However,BBR has been withdrawn from most of the European markets due to several reported cases linking the drug to acute liver damage,including some instances of fatalities.Still,BBR is widely used in many Asian countries such as China.It is urgent to clarify the mechanism of BBR-induced idiosyncratic hepatotoxicity to ensure its safety use in clinic.It is widely accepted that cytochrome P450-mediated metabolic activation is most frequently responsible for drug-induced idiosyncratic hepatotoxicity.The resulting electrophilic reactive metabolites can react with nucleophilic sites in protein to form stable protein covalent adduction,possibly triggering liver toxicities.Studies on P450-mediated metabolic activation of BBR and the covalent modification of proteins by its reactive metabolites facilitate the illumination of the molecular mechanism of hepatotoxicity.The correlation between the structure,bioactivation,and BBR-induced hepatotoxicity can be defined by manipulating the structure of BBR and specifically blocking its metabolic activation pathway.At the same time,it provides theoretical foundations for the design and development of new anti-gout agents with high efficiency and low toxicity.The studies performed are summarized as below.1.Studies on cytochrome P450-mediated epoxidation of BBRBBR is a benzofuran derivative.It has been reported that many compounds containing benzene ring undergo epoxidized metabolic pathway via cytochrome P450,and the resulting epoxide is suggested to cause drug-induced liver injury.The objective of the present study was to identify the epoxide metabolite of BBR through in vitro incubation with liver microsomes,chemical synthesis,and in vivo metabolic study.An epoxide metabolite was trapped by N-acetylcysteine(NAC)or glutathione(GSH)in mouse liver microsomal incubation systems after exposure to BBR.One NAC conjugate(BBR-NAC-1)and GSH conjugate(BBR-GSH-1)were detected by LC-MS/MS in MRM mode.One NAC conjugate(BBR-NAC-2)derived from the reported o-quinone-1 metabolite of BBR was also detected.The structure of BBR-NAC-lwas identified to be BBR with NAC attached to C6 position by chemical synthesis following 1H-NMR,13C-NMR and HMBC analysis.One GSH conjugate and one NAC conjugate with the same chromatographic and mass spectrometric identities as BBR-NAC-1 and BBR-GSH-1 were also detected in bile and urine of BBR-treated mice,indicating that BBR was metabolized to epoxide intermediate in vivo.Microsomal incubations with human recombinant P450 enzymes and specific inhibitors of P450 enzymes indicate that P450 3A was the prime enzyme responsible for the epoxidation of BBR.2.Investigation of ipso-substitution of BBR in miceIt is reported that BBR also undergoes ipso-substitution metabolism via P450 in vitro with the formation of catechol and hydroquinone by oxidative debromination and decarbonylation,respectively.Their oxidized form,o-quinone and p-quinone,are known electrophilic reagents that may be involved in the hepatotoxicity process of BBR.However,there is no further evidence for this proposal in vivo.This study aimed to investigate the ipso-substitution pathway of BBR in mice.Solid phase extraction was used to enrich and purify urine and bile samples,and one NAC conjugate(BBR-NAC-3)and GSH conjugate(BBR-GSH-2)were detected in urine and bile of BBR-treated mice by LC-MS/MS,respectively.The NAC and GSH conjugates showed the same chromatographic and mass spectrometric identities as that for the products generated in microsomal incubations.The MS/MS spectra indicated that BBR-NAC-3 or BBR-GSH-2 was derived from o-quinone-2 intermediate through ipso-substitution metabolism of BBR.In addition,BBR-NAC-3 was chemically synthesized for metabolite identification.3.Determine BBR-derived hepatic protein covalent binding in mice and define its correlation with BBR-induced hepatotoxicityModification of key proteins by the electrophilic intermediates of drugs through metabolic activation is suggested to be one of the important cytotoxic mechanisms.The objectives of this study were to identify the interaction of the reactive metabolites(epoxide and o-quinone-2)of BBR with cysteine residues of proteins and to define the correlation between the protein adduction and hepatotoxicity induced by BBR.Due to the large molecular weight and complex structure of protein adductions,pronase E and chymotrypsin were used to completely hydrolyze protein adductions into small molecular cysteine conjugates which can be analyzed by LC-MS/MS.Only one cysteine conjugate(BBR-Cys-1)was detected in the digestion mixtures of microsomal protein after incubation with BBR.The product generated in the enzymatic hydrolysis of 6-NAC-BBR previously synthesized in our laboratory showed the same retention time and mass spectrometric identities as BBR-Cys-1,suggesting that BBR-Cys-1 was derived from cysteine-based protein adduction by epoxide metabolite of BBR.BBR-Cys-1 was also detected in the digestion mixtures of liver homogenates obtained from mice given BBR,and occurred in time-and dose-dependent manners,indicating that the epoxide metabolite produced by BBR in mice could covalently bound to liver protein.No protein adduction derived from o-quinone-2 metabolite of BBR via ipso-substitution was observed both in vitro and in vivo studies.The correlation between BBR-induced hepatotoxicity and the epoxide-derived hepatic protein modification was investigated by induction of P450 3A or inhibition of P450 3A activity.After pretreatment with ketoconazole,an inhibitor of P450 3A,significant increase in AUC of the time course of plasma BBR concentrations in mice was observed.While pretreatment with ketoconazole exhibited a protective effect against BBR-induced liver injury,significantly reduced the formation of hepatic BBR-Cys-1,and attenuated the hepatic and plasma GSH depletion induced by BBR.Increased protein covalent binding(BBR-Cys-1)was observed in BBR exposed mice after pretreatment with dexamethasone,an inducer of P4503A,along with the increased susceptibility of mice to the hepatotoxicity of BBR.In addition,mice pretreatment with BSO,an inhibitor of glutathione synthetase,was carried out to further investigate the correlation between protein modification and BBR-induced hepatotoxicity.BSO pretreatment was found to significantly increase the production of BBR-Cys-1 and potentiate the hepatotoxicity of BBR in mice.Given together,cysteine residues of hepatic protein were modified by the epoxide metabolite of BBR catalyzed by P450 3A.The protein covalent binding showed a good correlation with BBR-induced hepatotoxicity.4.Metabolic activation-based design of uricosuric BBR derivatives with low potential of toxicity and favourable anti-gout activityThe important role of P450 3A-mediated epoxidation in BBR-induced hepatotoxicity is clarified.Structural optimization of BBR was carried out in this study by introducing halogen atoms to the metabolically labile site of epoxidation to reduce BBR elicited hepatotoxicity.Three BBR derivatives,namely 6-F-BBR,6-C1-BBR,and 6-Br-BBR were obtained by chemical total synthesis,and their structures were confirmed by 1H-NMR,13C-NMR,and high resolution mass spectrometry.The premise of attenuation is to ensure its pharmacological activity.In vitro pharmacological activity was evaluated by monitoring the amounts of[14C]-urate uptake in MDCK cells that stably expressed human uric acid transporter 1(hURAT1).6-F-BBR showed the strongest inhibitory effect on[14C]-urate uptake.In vivo pharmacological activity was investigated by measuring the effect of BBR and its derivatives on serum uric acid(SUA)in short-term hyperuricemic model rats.The SUA levels in hyperuricemic rats were achieved by BBR administration,followed by the group of 6-F-BBR.6-F-BBR was identified as the most effective compound via in vivo and in vitro pharmacological studies.It was selected for further toxicity and metabolism investigations through six aspects,including hepatotoxicity,metabolic stability,pharmacokinetic behaviors,metabolic activation(epoxidation),GSH depletion,and protein modification.(1)Mice were administrated BBR or 6-F-BBR by oral gavage at 50 mg/kg for 7 consecutive days.After pretreatment with BSO,the elevations of serum ALT and AST activities induced by BBR were significantly higher than those induced by 6-F-BBR.Vacuolar degeneration around the central vein was observed in BBR-treated mice,and there was no obvious lesion in the liver of mice given 6-F-BBR.(2)6-F-BBR demonstrated longer half-life and smaller in vitro intrinsic clearance compared with BBR in the metabolic stability studies of mouse,rat,and human liver microsomes.(3)Pharmacokinetic studies showed that 6-F-BBR achieved Cmax and AUC values over twice as high as those of BBR-treated rats,and displayed a much lower in vivo clearance than BBR.(4)Only minor epoxide-derived NAC conjugate(BBR-NAC-1)was detected in both mouse and human microsomal incubations with 6-F-BBR.(5)No epoxide-derived cysteine adduct(BBR-Cys-1)was observed in digestion mixtures of liver homogenates obtained from mice given 6-F-BBR.(6)6-F-BBR demonstrated less potential to deplete plasma and hepatic GSH than BBR at the same dose.In conclusion,a BBR derivative(6-F-BBR)with low toxicity and favourable pharmacological activity was designed and synthesized by introducing fluorine atom into the metabolically labile site of epoxidation.This study is a successful example of drug metabolism-guided rational drug modification,which lays a foundation for the design and development of anti-gout drugs with high efficiency and low toxicity.