Formation Of GSH Adducts Research Articles

On the Role of ROS and Glutathione in the Mode of Action Underlying Nrf2 Activation by the Hydroxyanthraquinone Purpurin.

Purpurin is a major anthraquinone present in the roots of Rubia cordifolia (madder). Purpurin is known to activate Nrf2 (Nuclear transcription factor erythroid 2-related factor 2) EpRE (electrophile responsive element) mediated gene expression as a potential beneficial effect. This study aimed to elucidate the balance between the electrophilicity or pro-oxidant activity of purpurin underlying the Nrf2 induction. For this, Nrf2 activation with modified intracellular glutathione (GSH) levels was measured in an Nrf2 CALUX reporter gene assay. In addition, both cell-free and intracellular ROS formation of purpurin with modified (intracellular) GSH levels at different pH were quantified using the DCF-DA assay. GSH adduct formation was evaluated by UPLC and LC-TOF-MS analysis. GSH and GSSG levels following purpurin incubations were quantified by LC-MS/MS. We show that Nrf2 induction by purpurin was significantly increased in cells with buthionine sulfoximine depleted GSH levels, while Nrf2 induction was decreased upon incubation of the cells with N-acetylcysteine being a precursor of GSH. In cell-free incubations, ROS formation increased with increasing pH pointing at a role for the deprotonated form of purpurin. Upon incubations of purpurin with GSH at physiological pH, GSH adduct formation appeared negligible (<1.5% of the added purpurin). The addition of GSH resulted in conversion of GSH to GSSG and significantly reduced the ROS formation. Together these results demonstrate that Nrf2 induction by purpurin originates from intracellular ROS formation and not from its electrophilicity, which becomes especially relevant when intracellular GSH levels can no longer scavenge the ROS. The present study demonstrated that the efficiency of intracellular Nrf2 activation by purpurin and related anthraquinones will depend on (i) their pKa and level of deprotonation at the intracellular pH, (ii) the oxidation potential of their deprotonated form and (iii) the intracellular GSH levels. Thus, the Nrf2 induction by purpurin depends on its pro-oxidant activity and not on its electrophilicity.

Diminishing GSH-Adduct Formation of Tricyclic Diazepine-based Mutant IDH1 Inhibitors.

Mutant isocitrate dehydrogenase 1 (IDH1) has been identified as an attractive oncology target for which >70% of grade II and III gliomas and ∼10% of acute myeloid leukemia (AML) harbor somatic IDH1 mutations. These mutations confer a neomorphic gain of function, leading to the production of the oncometabolite (R)-2-hydroxyglutarate (2-HG). We identified and developed a potent, selective, and orally bioavailable brain-penetrant tricyclic diazepine scaffold that inhibits mutant IDH1. During the course of in vitro metabolism studies, GSH-adduct metabolites were observed. The hypothesis for GSH-adduct formation was driven by the electron-rich nature of the tricyclic core. Herein, we describe our efforts to reduce the electron-rich nature of the core. Ultimately, a strategy focused on core modifications to block metabolic hot spots coupled with substitution pattern changes (C8 N → C linked) led to the identification of new tricyclic analogues with minimal GSH-adduct formation across species while maintaining an overall balanced profile.

Multiwell photocatalytic microreactor device integrating drug biotransformation modeling and sample preparation on a MALDI target

Metabolic conversion of drugs to chemically reactive products, known as bioactivation, is considered to underlie the adverse drug reactions such as idiosyncratic hepatotoxicity. As bioactivation of most drugs involves phase I oxidation, UV-induced TiO2 photocatalytic oxidation (UV/TiO2-PCO) can be used as a fast, simple and inexpensive non-enzymatic method of oxidative metabolism simulation allowing for rapid preliminary screening of candidate drugs for reactive metabolite formation. We developed and evaluated a novel prototype 96-well on-target UV/TiO2-photocatalytic microreactor setup (PCμR96) that integrates drug metabolism simulation and sample preparation directly on a MALDI target. PCμR96 allows to sequentially perform UV/TiO2-PCO of xenobiotics, GSH or protein adduct formation, protein digestion and sample concentration, followed by surface- or matrix-assisted laser desorption ionization (SALDI/MALDI) mass spectrometry (MS). Amodiaquine (AQ), a well-studied antimalarial drug that is known to undergo oxidative biotransformation, was selected as a model compound. Oxidation products of AQ and their protein adducts were obtained using PCμR96 and identified by SALDI and MALDI MS/MS analysis. The achieved AQ oxidation products were in good agreement with the known AQ metabolites produced in biological systems. Two of the products were proved to be reactive towards cysteyl thiols of human globin.

Abstract A06: Biophysical and biochemical characterization of KRAS G12C inhibition through the SMARTTM platform

Abstract RAS proteins are small GTPases involved in cell proliferation, survival, and differentiation, and are mutationally activated in about a third of all human cancers. These mutations drive cancer by impairing GTPase activity so that the RAS protein is found predominantly in its GTP-bound “on” conformation. Most KRAS isoform mutations are located at codon 12, a glycine in the P-loop of the GTPase active site. KRAS mutations in which the glycine is mutated to a cysteine (G12C) are particularly common in lung cancer. Despite its prevalence as an important oncogene and decades of research, the RAS protein remains an unexploited cancer target. We have developed a novel platform, SMART (Small Molecule Assisted Receptor Targeting), to disrupt the protein-protein interactions of so-called “undruggable” targets. Our compounds bind the immunophilin protein Cyclophilin A (CypA), and subsequently form a ternary complex with the target protein, KRAS. An electrophilic moiety on the ligand selectively forms a covalent bond with the cysteine of GTP-KRAS G12C, thus strengthening the stability of the ternary complex, partially occupying the effector face, and thus occluding the binding of downstream effector proteins, such as RAF. In a separate presentation, we describe a wide breadth of data to support the advancement of a small molecule specifically targeting the activated form of KRAS, with a focus on the cellular pharmacology. In this presentation, we highlight biophysical and biochemical findings that support our novel strategy for targeting GTP-KRAS G12C. SPR reveals the CypA-compound binary KD. A novel SPR protocol method was developed to examine CypA-compound-KRAS ternary complex formation, exploiting the A-B-A injection system of the Biacore 8K. LC-MS is used to characterize the crosslinking of our compounds to KRAS G12C in the presence of CypA, and full kinact/KI analysis is conducted with select compounds; in combination with SPR studies, the noncovalent KD of the ternary complex can be determined. Intrinsic warhead reactivity is measured through GSH adduct formation. TR-FRET studies probe the ability of ternary complexes to inhibit the binding of RAF to KRAS G12C. ITC and BLI are used to confirm ternary complex formation for selected compounds. In addition to guiding SAR, these studies characterize the degree of presenter (CypA) dependence, specificity of compound binding to the G12C mutant and to the GTP nucleotide-bound form of KRAS, and the ability to predict cellular efficacy. Our kinetic crosslinking experiments reveal single-digit micromolar KIs and a good kinact/KI ratio for our compounds relative to published literature on compounds targeting GDP-KRAS G12C. When these values are compared to SPR findings, the noncovalent KD contributes largely to KI, and thus noncovalent interactions play a bigger role in our compound’s interaction with RAS as compared to known GDP-KRAS G12C agents. Citation Format: Minyun Zhou, Alexander Yuzhakov, Cindy C Benod, Linlong Xue, Alec D Silver, Ganesh Iyer, Sharon A. Townson, Meizhong Jin, Nicholas R. Perl, Anna Kohlmann, Alan S. Mann, Gizem Akcay, Earl W. May. Biophysical and biochemical characterization of KRAS G12C inhibition through the SMARTTM platform [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A06.

Identification of DNA and glutathione adducts in male Sprague–Dawley rats exposed to 1-bromopropane

ABSTRACTOccupational exposure of workers to 1-bromopropane (1-BP) has raised concerns in industry for many years. Despite the known toxicity of this chemical, molecular events attributed to exposure to 1-BP have not been extensively studied. The aim of the present study was to examine the effects of 1-BP exposure on adduct formation with DNA and glutathione (GSH) in male Sprague–Dawley rats in an attempt to determine the early stages of toxicity. Following 6 h after either single or daily exposure to 1-BP for 3 days, N7-propyl guanine and S-propyl GSH were quantified in several organs by using liquid chromatography-mass spectrometry (LC-MS/MS). The results showed that N7-propyl guanine was maximally formed in liver followed by spleen, testes, and lung in both dose- and time-dependent manners. However, DNA adduct was not detected in cardiac tissue. In the case of S-propyl GSH, this compound was formed in the following order in various organs: liver > testes > spleen > kidney > lung > heart. In a subsequent in vitro study, formation of N7-propyl guanine initiated by 1-BP in calf thymus DNA was not markedly affected by addition of liver homogenates, which indicated that this chemical may be acting as a direct alkylating agent. In contrast, an in vitro study with free GSH demonstrated that 1-BP reduced GSH and elevated production of S-propyl GSH, and that the production of this adduct was significantly higher in the presence of active liver homogenates. Data indicated that formation of GSH adducts initiated by 1-BP might be associated with an enzyme-driven process. Although further characterization is necessary, it would appear that N7-propyl guanine and S-propyl GSH might serve as useful markers in cases of exposure assessment of 1-BP.

Impact of glutathione modulation on the toxicity of the Fusarium mycotoxins deoxynivalenol (DON), NX-3 and butenolide in human liver cells

DON, NX-3 and butenolide (BUT) are secondary metabolites formed by Fusarium graminearum. Evidence for formation of DON-glutathione adducts exists in plants, and also in human liver (HepG2) cells mass spectrometric evidence for GSH-adduct formation was reported. NX-3 is a DON derivative lacking structural features for Thiol-Michael addition, while BUT has the structural requirements (conjugated double bond and keto group). In the present study, we addressed whether these structural differences affect levels of intracellular reactive oxygen species in HepG2 cells, and if intracellular GSH levels influence toxic effects induced by DON, NX-3 and BUT. Pre-treatment with an inhibitor of GSH bio-synthesis, L-buthionine-[S,R]-sulfoximine, aggravated substantially BUT-induced cytotoxicity (≥50 μM, 24 h), but only marginally affected the cytotoxicity of DON and NX-3 indicating that GSH-mediated detoxification is of minor importance in HepG2 cells. We further investigated whether BUT, a compound inducing alone low oral toxicity, might affect the toxicity of DON. Under different experimental designs with respect to pre- and/or co-incubations, BUT was found to contribute to the combinatorial cytotoxicity, exceeding the toxic effect of DON alone. The observed combinatorial effects underline the potential contribution of secondary metabolites like BUT, considered to be alone of low toxicological relevance, to the toxicity of DON or structurally related trichothecenes, arguing for further studies on the toxicological relevance of naturally occurring mixtures.

Evaluation of Strategies for the Assessment of Drug-Drug Interactions Involving Cytochrome P450 Enzymes.

Drug-drug interactions (DDIs) can occur when one drug alters the metabolism of another drug. Drug metabolism mediated by cytochrome P450 enzymes (CYPs) is responsible for the majority of metabolism of known drugs and inhibition of CYP enzymes is a well-known cause of DDIs. In the current study, the use of various human liver microsomes (HLM)-based methods to determine occurrence of CYP-mediated metabolism-dependent inhibition (MDI) and possible follow-up studies were evaluated. Human CYP inhibition was studied using the following methodologies: direct inhibition and (non-diluted) IC50-shift assays, a ferricyanide-based reversibility assay, a spectrophotometric metabolic intermediate complex (MIC) assay, and recording of reduced carbon monoxide (CO)-difference spectra. HLM incubations in the presence and absence of NADPH and glutathione (GSH) were performed to study the possible formation of CYP-dependent GSH adducts. HLM incubations with the radiolabeled inhibitors mifepristone and paroxetine were performed to study CYP-mediated covalent binding. Dihydralazine and furafylline displayed irreversible MDI of CYP1A2. Paroxetine displayed both quasi-irreversible and irreversible MDI of CYP2D6, formation of CYP-dependent GSH adducts was observed, while CYP-mediated covalent binding occurred which was decreased in the presence of GSH. Mifepristone displayed irreversible MDI of CYP3A4, formation of CYP-dependent GSH adducts was observed, while CYP-mediated covalent binding occurred which was decreased in the presence of GSH. Troleandomycin and verapamil displayed quasi-irreversible MDI of CYP3A4; MIC formation was observed, while no formation of CYP-dependent GSH adducts occurred. This study gives a representative overview of current methodologies that can be used to study CYP inhibition. The here presented strategy can be applied as a tool during risk evaluation of CYP-mediated DDIs.

Bromofatty aldehyde derived from bromine exposure and myeloperoxidase and eosinophil peroxidase modify GSH and protein

α-Chlorofatty aldehydes (α-ClFALDs) and α-bromofatty aldehydes (α-BrFALDs) are produced in activated neutrophils and eosinophils. This study investigated the ability of α-BrFALD and α-ClFALD to react with the thiols of GSH and protein cysteinyl residues. Initial studies showed that 2-bromohexadecanal (2-BrHDA) and 2-chlorohexadecanal (2-ClHDA) react with GSH producing the same fatty aldehyde-GSH adduct (FALD-GSH). In both synthetic and cellular reactions, FALD-GSH production was more robust with 2-BrHDA compared with 2-ClHDA as precursor. NaBr-supplemented phorbol myristate acetate (PMA)-activated neutrophils formed more α-BrFALD and FALD-GSH compared with non-NaBr-supplemented neutrophils. Primary human eosinophils, which preferentially produce hypobromous acid and α-BrFALD, accumulated FALD-GSH following PMA stimulation. Mice exposed to Br2 gas had increased levels of both α-BrFALD and FALD-GSH in the lungs, as well as elevated systemic plasma levels of FALD-GSH in comparison to mice exposed to air. Similar relative reactivity of α-ClFALD and α-BrFALD with protein thiols was shown using click analogs of these aldehydes. Collectively, these data demonstrate that GSH and protein adduct formation are much greater as a result of nucleophilic attack of cysteinyl residues on α-BrFALD compared with α-ClFALD, which was observed in both primary leukocytes and in mice exposed to bromine gas.

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.

Cytochrome P450 3A4 and CYP3A5-Catalyzed Bioactivation of Lapatinib.

Metabolic activation of the dual-tyrosine kinase inhibitor lapatinib by cytochromes CYP3A4 and CYP3A5 has been implicated in lapatinib-induced idiosyncratic hepatotoxicity; however, the relative enzyme contributions have not been established. The objective of this study was to examine the roles of CYP3A4 and CYP3A5 in lapatinib bioactivation leading to a reactive, potentially toxic quinoneimine. Reaction phenotyping experiments were performed using individual human recombinant P450 enzymes and P450-selective chemical inhibitors. Lapatinib metabolites and quinoneimine-glutathione (GSH) adducts were analyzed using liquid chromatography-tandem mass spectrometry. A screen of cDNA-expressed P450s confirmed that CYP3A4 and CYP3A5 are the primary enzymes responsible for quinoneimine-GSH adduct formation using lapatinib or O-dealkylated lapatinib as the substrate. The mean kinetic parameters (Km and kcat) of lapatinib O-dealkylation revealed that CYP3A4 was 5.2-fold more efficient than CYP3A5 at lapatinib O-dealkylation (CYP3A4 kcat/Km = 6.8 μM(-1) min(-1) versus CYP3A5 kcat/Km = 1.3 μM(-1) min(-1)). Kinetic analysis of GSH adduct formation indicated that CYP3A4 was also 4-fold more efficient at quinoneimine-GSH adduct formation as measured by kcat (maximum relative GSH adduct levels)/Km (CYP3A4 = 0.0082 vs. CYP3A5 = 0.0021). In human liver microsomal (HLM) incubations, CYP3A4-selective inhibitors SR-9186 and CYP3cide reduced formation of GSH adducts by 78% and 72%, respectively, compared with >90% inhibition by the pan-CYP3A inhibitor ketoconazole. The 16%-22% difference between CYP3A- and CYP3A4-selective inhibition indicates the involvement of remaining CYP3A5 activity in generating reactive metabolites from lapatinib in pooled HLMs. Collectively, these findings support the conclusion that both CYP3A4 and CYP3A5 are quantitatively important contributors to lapatinib bioactivation.

Novel Series of Dihydropyridinone P2X7 Receptor Antagonists.

Identification of singleton P2X7 inhibitor 1 from HTS gave a pharmacophore that eventually turned into potential clinical candidates 17 and 19. During development, a number of issues were successfully addressed, such as metabolic stability, plasma stability, GSH adduct formation, and aniline mutagenicity. Thus, careful modification of the molecule, such as conversion of the 1,4-dihydropyridinone to the 1,2-dihydropyridinone system, proper substitution at C-5″, and in some cases addition of fluorine atoms to the aniline ring allowed for the identification of a novel class of potent P2X7 inhibitors suitable for evaluating the role of P2X7 in inflammatory, immune, neurologic, or musculoskeletal disorders.

Metabolomics reveals the formation of aldehydes and iminium in gefitinib metabolism

Gefitinib (GEF), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is widely used for the treatment of cancers, particularly non-small cell lung cancer. However, its clinical use is limited by multiple adverse effects associated with GEF, such as liver and lung injuries, severe nausea, and diarrhea. Although, the exact mechanism of GEF adverse effects are still unknown, xenobiotic-induced bioactivation is thought to play a significant role in GEF induced toxicity. Using a metabolomic approach, we investigated the metabolic pathways of GEF in human and mouse liver microsomes. Thirty four GEF metabolites and adducts were identified and half of them are novel. The potential reactive metabolites, two aldehydes and one iminium, were identified for the first time. The previously reported GSH adducts and primary amines were observed as well. The aldehyde and iminium pathways were further confirmed by using methoxylamine and potassium cyanide as trapping reagents. Using recombinant CYP450 isoforms, CYP3A4 inhibitor, and S9 from Cyp3a-null mice, we confirmed CYP3A is the major enzyme contributing to the formation of aldehydes, GSH adducts, and primary amines in liver. Multiple enzymes contribute to the formation of iminium. This study provided us more knowledge of GEF bioactivation and enzymes involved in metabolic pathways, which can be utilized for understanding the mechanism of adverse effects associated with GEF and predicting possible drug-drug interactions. Further studies are suggested to determine the roles of these bioactivation pathways in GEF toxicity.

Irreversible binding of an anticancer compound (BI-94) to plasma proteins

1. We investigated the mechanisms responsible for the in vivo instability of a benzofurazan compound BI-94 (NSC228148) with potent anti-cancer activity.2. BI-94 was stable in MeOH, water, and in various buffers at pHs 2.5–5, regardless of the buffer composition. In contrast, BI-94 was unstable in NaOH and at pHs 7–9, regardless of the buffer composition. BI-94 disappeared immediately after spiking into mice, rat, monkey, and human plasma. BI-94 stability in plasma can be only partially restored by acidifying it, which indicated other mechanisms in addition to pH for BI-94 instability in plasma.3. BI-94 formed adducts with the trapping agents, glutathione (GSH) and N-acetylcysteine (NAC), in vivo and in vitro via nucleophilic aromatic substitution reaction. The kinetics of adduct formation showed that neutral or physiological pHs enhanced and accelerated GSH and NAC adduct formation with BI-94, whereas acidic pHs prevented it. Therefore, physiological pHs not only altered BI-94 chemical stability but also enhanced adduct formation with endogenous nucleophiles. In addition, adduct formation with human serum albumin-peptide 3 (HSA-T3) at the Cys34 position was demonstrated.4. In conclusion, BI-94 was unstable at physiological conditions due to chemical instability and irreversible binding to plasma proteins.

Comparison of trapping profiles between d-peptides and glutathione in the identification of reactive metabolites

Qualitative trapping profile of reactive metabolites arising from six structurally different compounds was tested with three different d-peptide isomers (Peptide 1, gly–tyr–pro–cys–pro–his-pro; Peptide 2, gly–tyr–pro–ala–pro–his–pro; Peptide 3, gly–tyr–arg–pro–cys–pro–his–lys–pro) and glutathione (GSH) using mouse and human liver microsomes as the biocatalyst. The test compounds were classified either as clinically “safe” (amlodipine, caffeine, ibuprofen), or clinically as “risky” (clozapine, nimesulide, ticlopidine; i.e., associated with severe clinical toxicity outcomes). Our working hypothesis was as follows: could the use of short different amino acid sequence containing d-peptides in adduct detection confer any add-on value to that obtained with GSH? All “risky” agents’ resulted in the formation of several GSH adducts in the incubation mixture and with at least one peptide adduct with both microsomal preparations. Amlodipine did not form any adducts with any of the trapping agents. No GSH and peptide 2 and 3 adducts were found with caffeine, but with peptide 1 one adduct with human liver microsomes was detected. Ibuprofen produced one Peptide 1-adduct with human and mouse liver microsomes but not with GSH. In conclusion, GSH still remains the gold trapping standard for reactive metabolites. However, targeted d-peptides could provide additional information about protein binding potential of electrophilic agents, but their clinical significance needs to be clarified using a wider spectrum of chemicals together with other safety estimates.

Post-acquisition analysis of untargeted accurate mass quadrupole time-of-flight MS(E) data for multiple collision-induced neutral losses and fragment ions of glutathione conjugates.

Analytical methods to assess glutathione (GSH) conjugate formation based on mass spectrometry usually take advantage of the specific fragmentation behavior of the glutathione moiety. However, most methods used for GSH adduct screening monitor only one specific neutral loss or one fragment ion, even though the peptide moiety of GSH adducts shows a number of other specific neutral fragments and fragment ions which can be used for identification. Nine reference drugs well known to form GSH adducts were incubated with human liver microsomes. Mass spectrometric analysis was performed with a quadrupole time-of-flight mass spectrometer in untargeted accurate mass MS(E) mode. The data analysis and evaluation was achieved in an automated approach with software to extract and identify GSH conjugates based on the presence of multiple collision-induced neutral losses and fragment ions specific for glutathione conjugates in the high-energy MS spectra. In total 42 GSH adducts were identified. Eight (18%) adducts did not show the neutral loss of 129 but were identified based on the appearance of other GSH-specific neutral losses or fragment ions. In high-energy MS(E) spectra the GSH-specific fragment ions of m/z 308 and 179 as well as the neutral loss of 275 Da were complementary to the commonly used neutral loss of 129 Da. Further, one abundant (yet unpublished) GSH conjugate of troglitazone formed in human liver microsomes was found. A software-aided approach was developed to reliably retrieve GSH adduct formation data out of untargeted complex full scan QTOFMS(E) data in a fast and efficient way. The present approach to detect and analyze multiple collision-induced neutral losses and fragment ions of glutathione conjugates in untargeted MS(E) data might be applicable to higher throughput to assess reactive metabolite formation in drug discovery.

In Vitro Metabolism of 3,4-Methylenedioxymethamphetamine in Human Hepatocytes

Users of the illicit drug, 3,4-methylenedioxymethamphetamine (MDMA), show signs of neurotoxicity. However, the precise mechanism of neurotoxicity caused by use of MDMA has not yet been elucidated. Synthetic glutathione (GSH) conjugates of MDMA are transported into the brain by the GSH transporter and subsequently produce neurotoxicity. The objective of this research is to show direct evidence of the formation of GSH adducts of MDMA in human hepatocytes. High-performance liquid chromatography coupled with tandem mass spectrometry was utilized to examine in vitro incubations of MDMA with cryopreserved human hepatocytes. The use of hydrophilic liquid chromatography in combination with linear ion trap mass spectrometry permitted the identification of two possible GSH metabolites. Enhanced product ion scans of m/z = 499 and 487 amu of extracts from hepatocytes treated with 1.0 mM MDMA show a distinct fragmentation pattern (m/z 194.2, 163, 135, 105), suggesting the formation of MDMA-GSH conjugate, MDMA-SG and 3,4-dihydroxymethamphetamine-SG. The formation of an MDMA-GSH conjugate was further supported by the apparent lack of the same fragmentation pattern from hepatocyte samples without MDMA treatment. The results generated from this study yield valuable qualitative and quantitative information about the neurotoxic thioether metabolites formed from MDMA in humans.

Application of Interleukin-22 Mediates Protection in Experimental Acetaminophen-Induced Acute Liver Injury

Acetaminophen (APAP, paracetamol)-induced hepatotoxicity, although treatable by timely application of N-acetylcysteine, can be fatal. Because it is among the common causes of acute liver failure in intensive care units and in light of its gradually increasing incidence, the need for novel therapeutic strategies aimed at severe intoxication is apparent. Recently, it has been shown that IL-22, a STAT3-activating cytokine, has the capability to mediate liver protection. Herein, the protective potential of IL-22 in murine APAP-induced hepatotoxicity was assessed. Intravenous administration of prophylactic IL-22 significantly reduced serum alanine aminotransferase levels and histopathologic damage in APAP-induced liver injury, a process that coincided with increased hepatocyte proliferation invivo. Concomitant gene expression analysis revealed hepatic induction of genes prototypically up-regulated by the IL-22/STAT3 axis, among others suppressor of cytokine signaling-3, lipocalin-2, and α1-antichymotrypsin. Notably, in a translational setting of therapeutic treatment 2 hours after APAP, IL-22 supported protection in the context of suboptimal N-acetylcysteine dosing. IL-22 likewise connected to augmented hepatocyte proliferation in this experimental setting. As detected by analysis of inflammatory cytokine production, systemically applied IL-22 did not display acute immunomodulation/stimulation in otherwise untreated or endotoxemic mice. Those latter observations clearly confirm acute tolerability of systemically applied IL-22. Observations presented altogether suggest that therapeutic IL-22 administration is a conceivable tissue-protective regimen aimed at hard-to-treat patients with severe APAP-induced hepatotoxicity.

Bioactivation of 2-(Alkylthio)-1,3,4-Thiadiazoles and 2-(Alkylthio)-1,3-Benzothiazoles

Certain functional groups/structural motifs are known to generate chemically reactive metabolites that can covalently modify essential cellular macromolecules and, therefore, have the potential to disrupt biological function and elicit idiosyncratic adverse drug reactions. In this report, we describe the bioactivation of 5-substituted 2-(alkylthio)-1,3,4-thiadiazoles and 2-(alkylthio)-1,3-benzothiazoles, which can be added to the growing list of structural alerts. When 5-substituted 2-(methylthio)-1,3,4-thiadiazoles and 2-(methylthio)-1,3-benzothiazole were incubated with pooled human liver microsomes in the presence of NADPH and GSH, unusual GSH adducts were formed. Characterization of these GSH adducts by high-resolution mass spectrometry indicated the replacement of the methylthio- group by GSH, and NMR experiments ascertained the proposed structures. On the basis of the metabolic profile change in incubation samples with/without GSH, we proposed that the GSH adduct formation involved two steps: (1) enzymatic oxidation of the alkylthio- group to form sulfoxide and sulfone and (2) nucleophilic displacement of the formed sulfoxide and sulfone by GSH. The proposed mechanism was confirmed by the formation of the same GSH adduct from the incubation of synthetically prepared sulfoxide and sulfone compounds in buffer. We found the sulfur oxidation step was significantly inhibited (80-100%) by preincubation with 1-aminobenzotriazole but was much less affected by thermoinactivation (0-45%), suggesting that the sulfoxidation step is primarily catalyzed by cytochrome P450s and not by flavin monooxygenases. We also investigated the presence of this bioactivation pathway in more than a dozen compounds containing 2-(alkylthio)-1,3,4-thiadiazole and 2-(alkylthio)-1,3-benzothiazoles. The common GSH adduct formation pathway demonstrated by current studies raises a new structural alert and potential liability in drug safety when 2-alkylthio derivatives of 1,3-benzothiazoles and 1,3,4-thiadiazoles are incorporated in drug design.

Preclinical Strategy to Reduce Clinical Hepatotoxicity Using in Vitro Bioactivation Data for &gt;200 Compounds

Drug-induced liver injury is the most common cause of market withdrawal of pharmaceuticals, and thus, there is considerable need for better prediction models for DILI early in drug discovery. We present a study involving 223 marketed drugs (51% associated with clinical hepatotoxicity; 49% non-hepatotoxic) to assess the concordance of in vitro bioactivation data with clinical hepatotoxicity and have used these data to develop a decision tree to help reduce late-stage candidate attrition. Data to assess P450 metabolism-dependent inhibition (MDI) for all common drug-metabolizing P450 enzymes were generated for 179 of these compounds, GSH adduct data generated for 190 compounds, covalent binding data obtained for 53 compounds, and clinical dose data obtained for all compounds. Individual data for all 223 compounds are presented here and interrogated to determine what level of an alert to consider termination of a compound. The analysis showed that 76% of drugs with a daily dose of <100 mg were non-hepatotoxic (p < 0.0001). Drugs with a daily dose of ≥100 mg or with GSH adduct formation, marked P450 MDI, or covalent binding ≥200 pmol eq/mg protein tended to be hepatotoxic (∼ 65% in each case). Combining dose with each bioactivation assay increased this association significantly (80-100%, p < 0.0001). These analyses were then used to develop the decision tree and the tree tested using 196 of the compounds with sufficient data (49% hepatotoxic; 51% non-hepatotoxic). The results of these outcome analyses demonstrated the utility of the tree in selectively terminating hepatotoxic compounds early; 45% of the hepatotoxic compounds evaluated using the tree were recommended for termination before candidate selection, whereas only 10% of the non-hepatotoxic compounds were recommended for termination. An independent set of 10 GSK compounds with known clinical hepatotoxicity status were also assessed using the tree, with similar results.

Novel Bioactivation Mechanism of Reactive Metabolite Formation from Phenyl Methyl-Isoxazoles

Recently, we described a series of phenyl methyl-isoxazole derivatives as novel, potent, and selective inhibitors of the voltage-gated sodium channel type 1.7 (Bioorg Med Chem Lett 21:3871-3876, 2011). The lead compound, 2-chloro-6-fluorobenzyl [3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]carbamate, showed unprecedented GSH and cysteine reactivity associated with NADPH-dependent metabolism in trapping studies using human liver microsomes. Additional trapping experiments with close analogs and mass spectra and NMR analyses suggested that the conjugates were attached directly to the 5'-methyl on the isoxazole moiety. We propose a mechanism of bioactivation via an initial oxidation of the 5'-methyl generating a stabilized enimine intermediate and a subsequent GSH attack on the 5'-methylene. Efforts to ameliorate reactive metabolite generation were undertaken to minimize the potential risk of toxicity. Formation of reactive metabolites could be significantly reduced or prevented by removing the 5'-methyl, by N-methylation of the carbamate; by replacing the nitrogen with a carbon or removing the nitrogen to obtain a carboxylate; or by inserting an isomeric 5'-methyl isoxazole. The effectiveness of these various chemical modifications in reducing GSH adduct formation is in line with the proposed mechanism. In conclusion, we have identified a novel mechanism of bioactivation of phenyl 5-methyl-isoxazol-4-yl-amines. The reactivity was attenuated by several modifications aimed to prevent the emergence of an enimine intermediate. Whether 5'-methyl isoxazoles should be considered a structural alert for potential formation of reactive metabolites is dependent on their context, i.e., 4'-nitrogen.