OAT3 Participates in Drug-Drug Interaction between Bentysrepinine and Entecavir through Interactions with M8-A Metabolite of Bentysrepinine-In Rats and Humans In Vitro.

Bentysrepinine (Y101) is a novel phenylalanine dipeptide for the treatment of hepatitis B virus. Renal excretion played an important role in the elimination of Y101 and its metabolites, M8 and M9, in healthy Chinese subjects, although the molecular mechanisms of renal excretion and potential drug-drug interactions (DDIs) remain unclear. The present study aimed to determine the organic anion transporters (OATs) involved in the renal disposition of Y101 and to predict the potential DDI between Y101 and entecavir, the first-line agent against HBV and a substrate of OAT1/3. Pharmacokinetic studies and uptake assays using rat kidney slices, as well as hOAT1/3-HEK293 cells, were performed to evaluate potential DDI. The co-administration of probenecid (an inhibitor of OATs) significantly increased the plasma concentrations and area under the plasma concentration-time curves of M8 and M9 but not Y101, while reduced renal clearance and the cumulative urinary excretion of M8 were observed in rats. The time course of Y101 and M8 uptake via rat kidney slices was temperature-dependent. Moreover, the uptake of M8 was inhibited significantly by probenecid and benzylpenicillin, but not by p-aminohippurate or tetraethyl ammonium. M8 was found to be a substrate of hOAT3, but Y101 is not a substrate of either hOAT1 or hOAT3. Additionally, the entecavir inhibited the uptake of M8 in the hOAT3-transfected cells and rat kidney slices in vitro. Interestingly, no significant changes were observed in the pharmacokinetic parameters of Y101, M8 or entecavir, regardless of intravenous or oral co-administration of Y101 and entecavir in rats. In conclusion, M8 is a substrate of OAT3 in rats and humans. Furthermore, M8 also mediates the DDI between Y101 and entecavir in vitro, mediated by OAT3. We speculate that it would be safe to use Y101 with entecavir in clinical practice. Our results provide useful information with which to predict the DDIs between Y101 and other drugs that act as substrates of OAT3.

Asparagine and Glutamine Residues Participate in Protein Covalent Binding by Epoxide Metabolite of 8-Epidiosbulbin E Acetate In Vitro and In Vivo.

Dioscorea Bulbifera L. (DBL), an effective traditional Chinese medicine, has been restricted because of multiple reports that it can cause severe hepatotoxicity. 8-Epidiosbulbin E acetate (EEA), one of the main components of DBL, can induce severe liver injury. It has been reported that EEA can be metabolized by CYP3A to the corresponding cis-enedial intermediate which alkylates the lysine residues of proteins to form pyrroline derivatives. The present study unexpectedly found that the reactive intermediate reacted with the amide groups of asparagine (Asn) and glutamine (Gln) residues of hepatic proteins of mice treated with EEA. The amide-derived protein modification increased with the increase in the dose administered. Like the adduction of the primary amine of lysine residues, the electrophilic metabolite reacted with the amide groups of Asn and Gln residues to offer the corresponding pyrrolines. The structures of the pyrrolines were confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy.

Evidence for adduction of biologic amines with reactive metabolite of 8-epidiosbulbin E acetate in vitro and in vivo.

Dioscorea bulbifera L. (DBL) is one of traditional Chinese medicines and has been used for the treatment of goiter, tumor and carbuncles. However, clinic application of the herbal medicine has been limited, due to reported severe hepatotoxicity. 8-Epidiosbulbin E acetate (EEA), one of the major components of DBL, can cause severe liver damage. The furan ring of EEA is metabolized by CYP3A4 to a cis-enedial reactive intermediate prone to react amino and/or thiol groups of amino acid residues. In this study, we investigated the interaction of the reactive intermediate with biologic amines. EEA-derived biologic amine adducts, including spermidine, spermine, putrescine, ornithine, lysine and glutamine were detected in cultured mouse primary hepatocytes treated with EEA. Only spermidine adduct was observed in bile of mice given EEA. The detection of the adducts was established by labeling with bromobenzyl mercaptan and LC-MS/MS analysis. Exposure of EEA resulted in concentration dependent cytotoxicity in hepatocytes. Pretreatment with spermidine attenuated the susceptibility of cells to the cytotoxicity of EEA, because of the compensation of the depleted spermidine.

Glutathione conjugation and protein modification resulting from metabolic activation of venlafaxine in vitro and in vivo.

Venlafaxine (VLF), an antidepressant agent, is widely used to combat major depressive disorders, particularly for the treatment of selective serotonin reuptake inhibitor-resistant depression. VLF has been shown to cause liver injury. The present study aimed to investigate the metabolic activation of VLF and explore the mechanisms of hepatotoxicity induced by VLF.One glutathione (GSH) conjugate and one cysteine conjugate were both detected in mouse and human liver microsomal incubations containing VLF and GSH or cysteine. The two conjugates were also detected in cultured mouse primary hepatocytes and bile of rats after exposure to VLF. The in vitro and in vivo studies demonstrated that VLF was metabolized to a quinone methide intermediate reactive to GSH and cysteine residues of hepatic protein. The observed protein covalent binding revealed dose-dependency. The metabolic activation of VLF was P450-dependent, and CYP3A4 was found as the predominant enzyme involved in the bioactivation process.These findings facilitate better understanding of the metabolic activation-hepatotoxicity relationship of VLF and provide chemists with information about new potential structural alerts during drug design process.

Metabolic activation of aegeline mediated by CYP2C19.

Aegeline (AGL) is a natural alkaloidal amide mainly isolated from the leaves and fruits of tropical plant Aegle marmelos, with multiple pharmacological activities.As one component of several dietary supplements, AGL caused a series of acute and chronic liver injuries. Nevertheless, the mechanisms of AGL-induced hepatotoxicity remain unclear. This study was conducted to identify reactive metabolite(s), to determine related metabolic pathways, and define the possible association of the bioactivation with AGL cytotoxicity.A demethylation metabolite (M1) and a GSH conjugate (M2) were detected in rat liver microsomal incubations containing AGL and GSH. The two metabolites were both found in bile of rats and rat primary hepatocytes after AGL administration.Recombinant P450 enzyme incubations showed that CYP2C19 was the principal enzyme catalysing this metabolic activation.Ticlopidine, a selective inhibitor of CYP2C19, decreased the formation of M1 and M2 in hepatocytes and attenuated the susceptibility of hepatocytes to the cytotoxicity of AGL. The results suggest that AGL was metabolized to a p-quinone methide intermediate which could in part participate in AGL-induced cytotoxicity.

Metabolic Activation of Pirfenidone Mediated by Cytochrome P450s and Sulfotransferases.

Pirfenidone is approved for the treatment of idiopathic pulmonary fibrosis. Idiosyncratic drug reactions, due to clinical application of pirfenidone, have been documented, even along with death cases resulting from acute liver failure. The present study aimed at the investigation of metabolic activation of pirfenidone possibly participating in the reported adverse reactions. Pirfenidone-derived glutathione/N-acetylcysteine (GSH/NAC) conjugates were detected in microsomal/primary hepatocyte incubations after exposure to pirfenidone. The GSH/NAC conjugates were also observed in bile and urine of rats given pirfenidone, respectively. The observation of the conjugates suggests the formation of a quinone methide intermediate derived from pirfenidone. The intermediate was possibly generated through two pathways. First, pirfenidone was directly metabolized to the quinone methide intermediate via dehydrogenation; second, pirfenidone was oxidized to 5-hydroxymethyl pirfenidone, followed by sulfation to a benzyl alcohol-sulfate derivative. The findings facilitate the understanding of the mechanisms of pirfenidone-induced idiosyncratic toxicity and assist medicinal chemists to minimize toxicities in the development of new pharmaceutical agents.

Evidence for Polyamine, Biogenic Amine, and Amino Acid Adduction Resulting from Metabolic Activation of Diosbulbin B.

Dioscorea bulbifera L. (DBL), a traditional Chinese medicine, is a well-known herb with hepatotoxicity, and the biochemical mechanisms of the toxic action remain unknown. Diosbulbin B (DSB), a major component of DBL, can induce severer liver injury which requires cytochrome P450-catalyzed oxidation of the furan ring. It is reported that a cis-enedial reactive intermediate resulting from metabolic activation of DSB can react with thiols and amines to form pyrrole or pyrroline derivatives. In this study, we investigated the interaction of the reactive intermediate with polyamines, biogenic amines, and amino acids involved in the polyamine metabolic pathway, including putrescine, spermidine, spermine, histamine, arginine, ornithine, lysine, glutamine, and asparagine. Seven DSB-derived amine adducts were detected in microsomal incubations supplemented with DSB and individual amines. Six adducts were observed in cultured rat primary hepatocytes after exposure to DSB. DSB was found to induce apoptosis and cell death in time- and concentration-dependent manners. Apparently, the observed apoptosis was associated with the detected amine adduction. The findings facilitate the understanding of the mechanisms of toxic action of DSB.

Immunochemical Detection of Protein Modification Derived from Metabolic Activation of 8-Epidiosbulbin E Acetate.

Furanoid 8-epidiosbulbin E acetate (EEA) is one of the most abundant diterpenoid lactones in herbal medicine Dioscorea bulbifera L. (DB). Our early work proved that EEA could be metabolized to EEA-derived cis-enedial (EDE), a reactive intermediate, which is required for the hepatotoxicity observed in experimental animals exposed to EEA. Also, we found that EDE could modify hepatic protein by reaction with thiol groups and/or primary amines of protein. The present study was inclined to develop polyclonal antibodies to detect protein modified by EDE. An immunogen was prepared by reaction of EDE with keyhole limpet hemocyanin (KLH), and polyclonal antibodies were raised in rabbits immunized with the immunogen. Antisera collected from the immunized rabbits demonstrated high titers evaluated by enzyme-linked immunosorbent assays (ELISAs). Immunoblot analysis showed that the polyclonal antibodies recognized EDE-modified bovine serum albumin (BSA) in a hapten load-dependent manner but did not cross-react with native BSA. Competitive inhibition experiments elicited high selectivity of the antibodies toward EDE-modified BSA. The antibodies allowed us to detect and enrich EDE-modified protein in liver homogenates obtained from EEA-treated mice. The developed immunoprecipitation technique, along with mass spectrometry, enabled us to succeed in identifying multiple hepatic proteins of animals given EEA. We have successfully developed polyclonal antibodies with the ability to recognize EDE-derived protein adducts, which is a unique tool for us to define the mechanisms of toxic action of EEA.

Nitidine Chloride Is a Mechanism-Based Inactivator of CYP2D6.

Nitidine chloride (NC) is a benzophenanthridine alkaloid isolated from the roots of Zanthoxylum nitidum (Roxb.) DC, a widely used traditional herbal medicine. Several reports have revealed NC's multiple pharmacologic properties. The inhibitory effects of NC on human cytochrome P450 enzymes were investigated in the present study. We found that NC caused time- and concentration-dependent inhibition of CYP2D6, and more than 50% of CYP2D6 activity was suppressed after a 15-minute incubation with NC at 100 µM in the primary incubation mixtures, with KI of 4.36 µM, kinact of 0.052 minute-1, and a partition ratio of approximately 290. Moreover, the loss of CYP2D6 activity required the presence of NADPH. Superoxide dismutase/catalase and glutathione showed minor protection against the NC-induced enzyme inhibition. Quinidine as a competitive inhibitor of CYP2D6 slowed down the inactivation by NC. Trapping experiments using N-acetylcysteine demonstrated that quinone and/or carbene intermediate(s) were/was generated in human liver microsomal incubations with NC. In addition, potassium ferricyanide prevented the enzyme from the inactivation mediated by NC, which provided evidence that inhibition of CYP2D6 resulted from heme destruction by the formation of a carbene-iron complex. CYP1A2 was found to be the major enzyme involved in the generation of NC quinone metabolites. In conclusion, NC is a mechanism-based inactivator of CYP2D6. The generation of a carbene intermediate might be mainly responsible for the enzyme inactivation.

Development of Polyclonal Antibodies for Detection of Diosbulbin B-Derived cis-Enedial Protein Adducts.

Diosbulbin B (DSB), a major component of herbal medicine Dioscorea bulbifera L. (DB), can be metabolized to an electrophilic intermediate, DSB-derived cis-enedial (DDE). DDE was suggested to contribute to the hepatotoxicity observed in experimental animals and humans after their exposure to DSB. Our previous work found that DDE reacted with primary amino and/or sulfhydryl groups of hepatic protein. The objective of the study was to develop polyclonal antibodies that can recognize DDE-derived protein adducts. Immunogens synthesized from DDE and keyhole limpet hemocyanin were employed to raise polyclonal antibodies in rabbits. An enzyme-linked immunosorbent assay (ELISA) demonstrated high titers of antisera obtained from immunized rabbits. Immunoblot analysis showed that DDE-modified bovine serum albumin (BSA) was recognized by the obtained polyclonal antibodies in a concentration-dependent manner and without cross-reaction to native BSA. Competitive ELISA and competitive immunoblot analyses defined the specificity of the antibodies to recognize BSA modified by DDE. Immunoblot analysis also detected a multitude of chemiluminescent bands with a variety of molecular weights in liver homogenates that were harvested from mice treated with DSB. In summary, we have successfully raised polyclonal antibodies to detect protein adducts derived from DDE.