Study On The Metabolism-and Transport-mediated Hepatotoxicity Mechanism And Drug-drug Interaction Of Nimesulide

Liver-mediated drug metabolism and transport play an important role in the druginduced liver injury and drug-drug interactions.The metabolic activation of drugs can generate reactive metabolites,which may cause oxidative stress by depleting GSH,or covalently bind to cel ular proteins,thereby resulting in the toxicity.Inhibition of the hepatobiliary transporters responsible for bile acid uptake and efflux by the parent drug or its metabolites may cause the accumulation of bile acids in the liver,sequentially leading to cholestasis.Moreover,drugs can also alter the in vivo disposition of coadministrated drugs by influencing the bioactivities of metabolizing enzymes and transporters,which may further affect the efficacy and toxicity of coadministrated drugs.Nimesulide?NIM?is a nonsteroidal anti-inflammatory drug,and clinical treatment with NIM has been associated with severe hepatotoxicity.This thesis describes the mechanistic studies on metabolism-and transport-mediated hepatotoxicity of NIM,and explores the metabolizing enzymes-and transporters-mediated drug-drug interactions of NIM and commonly used drugs.The findings provide a theoretical basis for the rational use and safety evaluation of NIM.1.Metabolic activation of NIM and its role in NIM-induced hepatocellular toxicityNIM is a nonsteroidal anti-inflammatory drug that selectively inhibits cyclooxygenase-2.Severe hepatotoxicity has been observed in clinical practice.Studies have demonstrated that nitro-reduced NIM?M2?was capable of generating reactive metabolites under the catalysis of metabolizing enzymes,thus the metabolic activation of M2 was presumed to be responsible for the hepatotoxicity of NIM.However,the hepatocyte experiment showed that no toxicity was observed in primary rat hepatocytes after incubation with M2 for 24 h,whereas concentration-dependent toxicity was observed in the case of NIM,suggesting that metabolic activation of M2 was weakly correlated with the hepatotoxicity of NIM.This study aimed to investigate the potential metabolic activation pathways of NIM,and further evaluate their association with the hepatocellular toxicity of NIM.Two types of GSH conjugates were detected in the incubation of NIM with NADPHand GSH-supplemented human or rat liver microsomes.One was derived from the attachment of GSH to hydroxyl des-nitro NIM?NIM-OH-GSH?,and the other one was derived from the attachment of GSH to nitro-reduced NIM?NIM-NH₂-GSH?.The yield of NIM-OH-GSH in the NIM incubation was much higher than that in the NIM-OH incubation,and the presence of microsomal epoxide hydrolase greatly reduced the formation of NIM-OH-GSH in the NIM incubation,indicating that NIMOH-GSH was formed mainly from NIM via epoxidation,rather than from NIM-OH.CYP1A2 was the main enzyme catalyzing the generation of NIM-OH-GSH from NIM.NIM-NH₂-GSH was also detected in the nonenzymatic incubation containing NIM,NADPH and GSH,indicating that NIM-NH₂-GSH could be generated from the reductive activation of NIM.Both NADPH and GSH could act as reducing agents.Besides,the yield of NIM-NH₂-GSH in the cytosol incubations was much higher than that in the nonenzymatic incubation,and the formation was significantly suppressed by estradiol,a selective aldehyde oxidase?AO?inhibitor,suggesting the involvement of AO in the reductive activation of NIM.The structure of NIM-NH₂-GSH was confirmed using UPLC/Triple TOF MS and 1H NMR.A plausible mechanism for the reductive activation was proposed: in the presence of reducing agents,NIM probably underwent reduction from nitro to nitroso,then to hydroxylamine,and,final y,to amino;the nitroso intermediate was unstable and readily reacted with GSH,leading to the formation of a semimercaptal intermediate and a sulphenamide intermediate;the latter might be rearranged to form NIM-NH₂-GSH.Concenration-dependent toxicity was observed after the incubation of NIM in primary human and rat hepatocytes for 24 h,with IC50 values of 213 and 40 ?M,respectively.Inhibition of the oxidative and reductive activation of NIM by the nonspecific CYP inhibitor 1-aminobenzotriazole and selective AO inhibitor estradiol did not protect the cel s from NIM-mediated toxicity.Moreover,pretreating cel s with L-buthionine-sulfoximine?BSO,a GSH depletor?did not enhance the cytotoxicity of NIM.These results suggested that oxidative and reductive activation of NIM did not cause the hepatotoxicity and that the parent drug concentration was associated with the cytotoxicity.2.Transporters-mediated mechanism for NIM-induced cholestasisIn addition to the hepatocellular toxicity,cholestasis is also an important pattern of NIM-induced hepatotoxicity.At present,the mechanim of NIM-induced cholestasis remains unclear.Inhibition of bile acid transporters by the parent drug or its metabolites has been implicated in drug-induced cholestasis.In the present study,the cholestatic effect of NIM in rats was elucidated,and the effects of NIM and its main metabolites on bile acid transporters were studied using sandwich-cultured human and rat hepatocytes and isolated suspended rat hepatocytes.Oral administration of 100 mg/kg/day NIM to Wistar rats for 5 days increased the levels of plasma total bile acids?TBA?,alkaline phosphatase?ALP?,alanine aminotransferase?ALT?,a nd aspartate aminotransferase?AST?to 1.49-,1.31-,1.60-,and 1.29-fold of control,respectively.Besides,the levels of cholic acid?CA?,deoxycholic acid?DCA?,chenodeoxycholic acid?CDCA?,and ursodeoxycholic acid?UDCA?in rat plasma significantly increased;the levels of CA,CDCA,and glycochenodeoxycholic acid?GCDCA?in rat liver also markedly increased.The concentrations of NIM and its main metabolites in rat plasma and liver at 24 h after the last dose were quantified by using the liquid chromatography-tandem mass spectrometry?LC-MS/MS?method.In rat plasma,the concentrations of NIM,4'-hydroxynimesulide?M1?,nitro-reduced NIM?M2?and acetylated metabolite of nitroreduced NIM?M4?were 45.4,21.0,0.037 and 0.180 ?M,respectively;in rat liver,the concentrations of NIM,M1,M2 and M4 were 17.3,23.6,1.8 and 13.4 ?M,respectively.In sandwich-cultured rat hepatocytes,NIM and M1 reduced the biliary excretion index?BEI?and in vitro biliary clearance?Clbiliary?of d8-TCA in a concentrationdependent manner,and a greater extent of reduction was found in Clbiliary than in BEI,suggesting that NIM and M1 inhibited the BSEP-mediated excretion of d8-TCA into the canalicular lumen,and they exerted a greater inhibitory effect on the uptake than on the efflux of d8-TCA.We further evaluated the inhibitory effects of NIM and M1 on NTCP-and OATP-mediated d8-TCA uptake by rat hepatocytes.NIM and M1 inhibited the uptake transporters of d8-TCA for NTCP at IC50 values of 21.3 and 25.0 ?M,respectively,and for OATPs at IC50 values of 45.6 and 39.4 ?M,respectively.Moreover,NIM and M1 decreased the BEI of CDF in a concentration-dependent manner,indicating that NIM and M1 inhibited MRP2-mediated CDF excretion.By contrast,M2 and M4 did not inhibit or only marginally inhibited these transporters.Similar results were obtained in sandwich-cultured human hepatocytes,suggesting that NIM and M1 could also inhibit human bile acid transporters.The current results demonstrated that both NIM and M1 were capable of inhibiting the uptake transporters NTCP and OATPs,and the efflux transporters BSEP and MRP2,which may negatively impact bile acid homeostasis.The inhibition of bile acid transporters by NIM and M1 was possibly one of the biologic mechanisms of NIMinduced cholestasis.3.Transporters-mediated drug-drug interaction of NIM and acetaminophenIt was reported that the coadministration of combination of NIM and acetaminophen?PA?would cause a synergistic antinociception in mice.PA may cause severe hepatotoxicity when taken in overdose,and the hepatotoxicity has been attributed to the CYP450-mediated metabolic activation.A previous study reported that troglitazone enhanced the metabolic activation of PA by inducing hepatic CYP3A4 in rats,thus potentiating the hepatotoxicity.In addition,acetaminophen glucuronide?PA-Glu?is a substrate for both hepatic canalicular efflux transporter MRP2 and basolateral efflux transporter MRP3.The hepatobiliary disposition of PA and PA-Glu may be affected by changes in the acticity of these transporters.This study aimed to investigate whether NIM would affect the hepatobiliary disposition of PA by modulating the activites of metabolizing enzymes and transporters,thereby influencing the hepatotoxicity and efficiency of PA.Oral administration of 100 mg/kg/day NIM to Wistar rats for 5 days decreased the t1/2,MRT and AUC of PA by 69%,65% and 53%,respectively,but did not change the tmax and Cmax of PA;the pretreatment also decreased the t1/2 and MRT of PA-Glu by 72% and 54%,respectively,increased the Cmax and AUC of PA-Glu by 2.59-and 0.85-fold,respectively,but did not alter the tmax of PA-Glu.After NIM pretreatment,the accumulated excretion of PA in 0-24 h urine of rats significantly decreased,but the accumulated excretion of PA-Glu did not change.Besides,the biliary excretion of both PA and PA-Glu significantly decreased.In contrast,the single dose of 100 mg/kg/day NIM to Wistar rats almost did not influence any pharmacokinetic parameter of PA and PA-Glu in rats.There was no significant difference in the glucuronide formation of 4-MU and PA in rat liver microsomes prepared from the NIM-treated and the nontreated rats,indicating that five-day oral administration of NIM had no effect on the activity of hepatic UDPglucuronosyltransferase.Compared with the control group,the MRP2 mRNA level decreased by 51%,and the MRP3 mRNA leve increased by 7.42-fold in the liver of NIM-treated rats.The above-mentioned results indicated that the NIM pretreatment could decrease the enterohepatic circulation of PA-Glu by inhibiting MRP2 and inducing MRP3,resulting in the increased systematic exposure of PA-Glu and decreased systematic exposure of PA.Moreover,the NIM pretreatment greatly reduced the accumulated excretion of PAcys and PA-NAC in 0-24 h urine of rats by 76% and 75%,respectively,indicating that the metabolic activation of PA significantly decreased,thus resulting in the decreased hepatotoxicity.4.AO-mediated drug-drug interaction of NIM and methotrexateA recent study demonsrated that the combination of NIM and methotrexate?MTX?led to an increased risk of liver injury in adolescents.MTX was mainly metabolized by aldehyde oxidase?AO?to form 7-OH MTX,the latter was more toxic than MTX in rats.Thus,we presumed that NIM could induce AO,wich may result in the increased formation of the toxic metabolite 7-OH MTX.Oral administration of 100 mg/kg/day NIM to Wistar rats for five days increased the Cmax and AUC of MTX by 0.66-and 1.05-fold,respectively,and increased the Cmax and AUC of 7-OH MTX by 3.31-and 5.49-fold,respectively.Compared with the control group,the content of MTX in rat liver decreased by 34%,and the content of 7-OH MTX increased by 1.51-fold at 4 h after MTX administration.Moreover,the accumulated excretion of MTX and 7-OH MTX in 0-24 h urine increased by 0.80-and 4.19-fold,respectively,and the the accumulated excretion of 7-OH MTX in 0-24 h feces increased by 1.08-fold.There was no significant difference in the accumulated excretion of MTX in 0-24 h feces between the NIM-treated rats and the nontreated rats,which was because most of the parent drug was not absorbed and directly excreted into the feces.NIM increased the hepatic AO activity of rats in a dose-dependent manner.Compared with the control group,the formation of 7-OH MTX in rat liver cytosols prepared from rats at NIM doses of 20,50 and 100 mg/kg/day increased by 0.90-,2.16-and 2.66-fold,respectively.The AO-mediated metabolite formation from SGX523,JNJ-38877605 and NIM in rat liver cytosols prepared from rats at a 100 mg/kg/day dose also increased by 2.36-,2.84-and 1.69-fold,respectively.RT-PCR assay demonstrated that the mRNA levels of AOX1 and AOX3 in rat liver did not significantly change.Western Blot assay showed that the protein level of AOX1 increased in a dosedependent manner,and the protein level of AOX3 decreased in a dose-dependent manner.The hepatocyte experiments also demonstrated that the AO acticity in both primary rat and human hepatocytes could be induced by NIM.Compared with the control group,10,20 and 50 ?M NIM increased the yield of 7-OH MTX in rat hepatocytes by 0.50-,1.80-and 3.89-fold,respectively;10 ?M NIM also increased the yield of 7-OH MTX in human hepatocytes by 0.75-fold.The current study demonstrated that NIM could increase the formation of the toxic metabolite 7-OH MTX from MTX by inducing hepatic AO,which may increase the risk of MTX hepatotoxicity.5.ConclusionThis thesis demonstrated the metabolism-and transporters-mediated hepatotoxicity mechanism and drug-drug interactions of NIM.The metabolic activation of NIM did not cause the hepatocellular toxicity.Both NIM and its hydroxylated metabolite inhibited the bile acid transporters NTCP,OATPs,BSEP and MRP2,which was possibly one of the biologic mechanisms of NIM-induced cholestasis.Moreover,the inhibition of MRP2 and induction of MRP3 by NIM decreased the enterohepatic circulation of the acetaminophen glucuronide,thereby resulting in the decreased systematic exposure of acetaminophen,possibly influencing its efficiency.NIM could also induce AO,leading to increased formation of the toxic metabolite 7-hydroxy methotrexate from methotrexate,which may increase the risk of methotrexate hepatotoxicity.The current results suggested the significant contribution of liver-mediated drug metabolism and transport to the drug-induced liver injury and drug-drug interactions.During drug development,it is necessary to indentify the stable metabolites of drug and investigate its inhibitory and inducing effects on common metabolizing enzymes?such as CYP450?and transporters?such as P-gp and BCRP?.Besides,it is also important to identify reactive metabolites of drug and study its effects on specific metabolizing enzymes and transporters,such as AO and MRP2,in order to avoid the adverse events in clinical practice.