Ratios of biliary glutathione disulfide (GSSG) to glutathione (GSH): a potential index to screen drug-induced hepatic oxidative stress in rats and mice.

Hepatotoxicity of drug candidates is one of the major concerns in drug screening in early drug discovery. Detection of hepatic oxidative stress can be an early indicator of hepatotoxicity and benefits drug selection. The glutathione (GSH) and glutathione disulfide (GSSG) pair, as one of the major intracellular redox regulating couples, plays an important role in protecting cells from oxidative stress that is caused by imbalance between prooxidants and antioxidants. The quantitative determination of the GSSG/GSH ratios and the concentrations of GSH and GSSG have been used to indicate oxidative stress in cells and tissues. In this study, we tested the possibility of using the biliary GSSG/GSH ratios as a biomarker to reflect hepatic oxidative stress and drug toxicity. Four compounds that are known to alter GSH and GSSG levels were tested in this study. Diquat (diquat dibromide monohydrate) and acetaminophen were administered to rats. Paraquat and tert-butyl hydroperoxide were administered to mice to induce changes of biliary GSH and GSSG. The biliary GSH and GSSG were quantified using calibration curves prepared with artificial bile to account for any bile matrix effect in the LC-MS analysis and to avoid the interference of endogenous GSH and GSSG. With four examples (in rats and mice) of drug-induced changes in the kinetics of the biliary GSSG/GSH ratios, this study showed the potential for developing an exposure response index based on biliary GSSG/GSH ratios for predicting hepatic oxidative stress.

Localization and quantification of drugs in animal tissues by use of desorption electrospray ionization mass spectrometry imaging.

Mass spectrometric imaging (MSI) has emerged as a powerful technique to obtain spatial arrangement of individual molecular ions in animal tissues. Ambient desorption electrospray ionization (DESI) technique is uniquely suited for such imaging experiments, as it can be performed on animal tissues in their native environment without prior treatments. Although MSI has become a rapid growing technique for localization of proteins, lipids, drugs, and endogenous compounds in different tissues, quantification of imaged targets has not been explored extensively. Here we present a novel MSI approach for localization and quantification of drugs in animal thin tissue sections. DESI-MSI using an Orbitrap mass analyzer in full scan mode was performed on 6 µm coronal brain sections from rats that were administered 2.5 mg/kg clozapine. Clozapine was localized and quantified in individual brain sections 45 min postdose. External calibration curves were prepared by micropipetting standards with internal standard (IS) on top of the tissues, and average response factors were calculated for the scans in which both clozapine and IS were detected. All response factors were normalized to area units. Quantifications from DESI-MSI revealed 0.2-1.2 ng of clozapine in individual brain sections, results that were further confirmed by extraction and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis.

Gender effects on rat metabolism of AMG 900, an orally available small molecule aurora kinase inhibitor.

AMG 900 is an orally available small molecule that is highly potent and selective as a pan-aurora kinase inhibitor. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. The metabolism of AMG 900 was investigated in both male and female rats. We conducted studies in bile-duct catheterized (BDC) rats where bile, urine and plasma were analyzed to obtain metabolism profiles for each gender. These studies identified gender differences in the metabolism profiles in bile. Bile contained the majority of the drug related material and contained little unchanged AMG 900 which indicated that metabolism was the prominent process in drug elimination. Although bile contained the same metabolites for both genders, the amount of specific metabolites differed. Male rats metabolized AMG 900 primarily through hydroxylation with subsequent sulfate conjugation on the pyrimidinyl-pyridine side-chain whereas female rats favored a different oxidation site on the thiophene ring's methyl group, which is then metabolized to a carboxylic acid with subsequent conjugation to an acyl glucuronide. CYP phenotyping identified the prominent isoforms as being gender specific or biased in the oxidative metabolism of AMG 900. The metabolism in male rats favored both CYP2C11 and CYP2A2 whereas females favored the CYP2C12. The prominent sulfate conjugate identified in the male rat bile could also be due to male biased metabolism since it has been reported that sulfate conjugation is more prevalent in male rats. All the prominent rat metabolism routes for AMG 900 either have male or female bias. These differences in the rat AMG 900 metabolism profiles in bile can be explained by gender specific P450CYP isoforms.

Identification of a potent, state-dependent inhibitor of Nav1.7 with oral efficacy in the formalin model of persistent pain.

Clinical human genetic studies have recently identified the tetrodotoxin (TTX) sensitive neuronal voltage gated sodium channel Nav1.7 (SCN9A) as a critical mediator of pain sensitization. Herein, we report structure-activity relationships for a novel series of 2,4-diaminotriazines that inhibit hNav1.7. Optimization efforts culminated in compound 52, which demonstrated pharmacokinetic properties appropriate for in vivo testing in rats. The binding site of compound 52 on Nav1.7 was determined to be distinct from that of local anesthetics. Compound 52 inhibited tetrodotoxin-sensitive sodium channels recorded from rat sensory neurons and exhibited modest selectivity against the hERG potassium channel and against cloned and native tetrodotoxin-resistant sodium channels. Upon oral administration to rats, compound 52 produced dose- and exposure-dependent efficacy in the formalin model of pain.

Identification of quinone imine containing glutathione conjugates of diclofenac in rat bile.

High-resolution accurate MS with an LTQ-Orbitrap was used to identify quinone imine metabolites derived from the 5-hydroxy (5-OH) and 4 prime-hydroxy (4'-OH) glutathione conjugates of diclofenac in rat bile. The initial quinone imine metabolites formed by oxidation of diclofenac have been postulated to be reactive intermediates potentially involved in diclofenac-mediated hepatotoxicity; while these metabolites could be formed using in vitro systems, they have never been detected in vivo. This report describes the identification of secondary quinone imine metabolites derived from 5-OH and 4'-OH diclofenac glutathione conjugates in rat bile. To verify the proposed structures, the diclofenac quinone imine GSH conjugate standards were prepared synthetically and enzymatically. The novel metabolite peaks displayed the identical retention times, accurate mass MS/MS spectra, and the fragmentation patterns as the corresponding authentic standards. The formation of these secondary quinone metabolites occurs only under conditions where bile salt homeostasis was experimentally altered. Standard practice in biliary excretion experiments using bile duct-cannulated rats includes infusion of taurocholic acid and/or other bile acids to replace those lost due to continuous collection of bile; for this experiment, the rats received no replacement bile acid infusion. High-resolution accurate mass spectrometry data and comparison with chemically and enzymatically prepared quinone imines of diclofenac glutathione conjugates support the identification of these metabolites. A mechanism for the formation of these reactive quinone imine containing glutathione conjugates of diclofenac is proposed.

Desorption electrospray ionization tissue imaging using heated nebulizing gas and high-resolution accurate mass spectra.

A new method for tissue imaging using desorption electrospray ionization (DESI) mass spectrometry is described. The technique utilizes a DESI source with a heated nebulizing gas and high-resolution accurate mass data acquired with an LTQ-Orbitrap mass spectrometer. The two-dimensional (2D) automated DESI ion source creates images using the ions that are collected under high-resolution conditions. The use of high-resolution mass detection significantly improves the image quality due to exclusion of interfering ions. The use of a heated nebulizing gas increases the signal intensity observed at lower gas pressure. The technique developed is highly compatible with soft tissue imaging due to the minimal surface destruction.

Discovery and optimization of substituted piperidines as potent, selective, CNS-penetrant alpha4beta2 nicotinic acetylcholine receptor potentiators.

The discovery of a series of small molecule alpha4beta2 nAChR potentiators is reported. The structure-activity relationship leads to potent compounds selective against nAChRs including alpha3beta2 and alpha3beta4 and optimized for CNS penetrance. Compounds increased currents through recombinant alpha4beta2 nAChRs, yet did not compete for binding with the orthosteric ligand cytisine. High potency and efficacy on the rat channel combined with good PK properties will allow testing of the alpha4beta2 potentiator mechanism in animal models of disease.

Synthesis and activity of substituted carbamates as potentiators of the alpha4beta2 nicotinic acetylcholine receptor.

The synthesis and structure-activity relationship of a series of carbamate potentiators of alpha4beta2 nAChR is reported herein. These compounds were highly selective for alpha4beta2 over other nAChR subtypes. In addition, compounds increased the response of alpha4beta2 nAChRs to acetylcholine, as measured with patch-clamp electrophysiology.

A novel bioactivation pathway for 2-[2-(2,6-dichlorophenyl)aminophenyl]ethanoic acid (diclofenac) initiated by cytochrome P450-mediated oxidative decarboxylation.

Diclofenac (2-[2-(2,6-dichlorophenyl)aminophenyl]ethanoic acid), a nonsteroidal antiinflammatory drug, undergoes bioactivation by cytochrome P450 oxidation to chemically reactive metabolites that are capable of reacting with endogenous nucleophiles such as glutathione (GSH) and proteins and that may play a role in the idiosyncratic hepatotoxicity associated with the drug. Here, we investigated the ability of diclofenac to be metabolized to 2-(2,6-dichloro-phenylamino)benzyl-S-thioether glutathione (DPAB-SG) in incubations with rat liver microsomes (RLMs) and human liver microsomes (HLMs) fortified with NADPH and GSH. Thus, after incubation of diclofenac (50 microM) with liver microsomes (1 mg protein/ml), the presence of DPAB-SG was detected in both RLM and HLM incubation extracts by liquid chromatography-tandem mass spectrometry techniques. The formation of DPAB-SG was NADPH-, concentration-, and time-dependent. Coincubation of diclofenac (10 microM) with ketoconazole (1 microM), an inhibitor of cytochrome P450 (P450) 3A4, with HLMs led to a 75% decrease in DPAB-SG formation. However, in contrast, coincubation with the P450 2C9 inhibitor sulfaphenazole (10 microM) or the P450 2D6 inhibitor quinidine (40 microM) led to a 1.9- and 1.6-fold increase in DPAB-SG production, respectively. From these data, we propose that P450 3A4 mediates the oxidative decarboxylation of diclofenac, resulting in the formation of a transient benzylic carbon-centered free radical intermediate that partitions between elimination (o-imine methide production) and recombination (alcohol formation) pathways. The benzyl alcohol intermediate, which was not analyzed for in the present studies, if formed could undergo dehydration to provide a reactive o-imine methide species. The o-imine methide intermediate then is proposed to react covalently with GSH, forming DPAB-SG.

Identification of a novel glutathione conjugate of diclofenac by LTQ-Orbitrap.

High resolution accurate MS with an LTQ-Orbitrap identified two novel metabolites of diclofenac in rat bile and rat and human hepatocyte incubations: a benzyl-S-glutathione conjugate and 2-(2,6-dichlorophenylamino) benzoic acid. A mechanism for the bioactivation of diclofenac involving decarboxylation is proposed.

DPP IV inhibitor blocks mescaline-induced scratching and amphetamine-induced hyperactivity in mice.

Dipeptidyl peptidase IV (DPP IV) is a ubiquitous membrane-bound enzyme that cleaves the two N-terminal amino acids from peptides with a proline or alanine residue in the second position from the amino end. Potential substrates for DPP IV include several neuropeptides, suggesting a role for DPP IV in neurological processes. We have developed a potent DPP IV inhibitor (IC50 = 30 nM), 1-(2-amino-3-methyl-butyryl)-azetidine-2-carbonitrile (AMAC), which has shown efficacy in two established models of psychosis: mescaline-induced scratching and amphetamine-induced hyperactivity. In the mescaline-induced scratching model, AMAC treatment before mescaline administration reduced the number of scratching paroxysms by 68% (P < 0.01). The compound showed a dose-dependent effect, inhibiting significantly at 6, 20 and 60 mg/kg (37%, 39% and 68%, respectively). In the amphetamine-induced hyperactivity model, 50 and 60 mg/kg AMAC, given before injection of amphetamine, significantly reduced hyper-locomotion by 65% and 76%, respectively. Additionally, AMAC showed no significant activity in binding assays for 20 receptors thought to be involved in the pathology of schizophrenia, including dopamine, serotonin and glutamate. A structurally similar analog, 1-(2-dimethylamino-3-methyl-butyryl)-azetidine-2-carbonitrile (DAMAC), that does not inhibit DPP IV, was inactive in both models. Taken together, these data suggest that the antipsychotic effects of AMAC are the result of DPP IV inhibition.

Identification of a novel mitochondrial protein ("mitoNEET") cross-linked specifically by a thiazolidinedione photoprobe.

Thiazolidinediones address underlying causes of type 2 diabetes, although their mechanism of action is not clearly understood. The compounds are thought to function as direct activators of the nuclear receptor PPARgamma (peroxisome proliferator-activated receptor-gamma), although pioglitazone, the weaker agonist of the two thiazolidinediones now in clinical use, seems to have more useful effects on circulating lipids. We have used tritiated pioglitazone and a photoaffinity cross-linker to identify a novel binding site in mitochondria. A saturable binding site for [3H]pioglitazone was solubilized from the membranes with CHAPS and migrated as a large complex by size exclusion chromatography. The binding correlated with a <17-kDa protein (m17), marked by a photoaffinity cross-linker, in both subcellular location and selectivity of competition by analogs. The protein was isolated and identified by mass spectrometry analysis and NH2-terminal sequencing. Three synthetic peptides with potential antigenic properties were synthesized from the predicted nontransmembrane sequence to generate antibodies in rabbits. Western blots show that this protein, which we have termed "mitoNEET," is located in the mitochondrial fraction of rodent brain, liver, and skeletal muscle, showing the identical subcellular location and migration on SDS-PAGE as the protein cross-linked specifically by the thiazolidinedione photoprobe. The protein exists in low levels in preadipocytes, and expression increases exponentially in differentiated adipocytes. The synthetic protein bound to solid phase associated with a complex of solubilized mitochondrial proteins, including the trifunctional beta-oxidation protein. It is possible that thiazolidinedione modification of the function of the mitochondrial target may contribute to lipid lowering and/or antidiabetic actions.

Studies on the chemical reactivity of diclofenac acyl glucuronide with glutathione: identification of diclofenac-S-acyl-glutathione in rat bile.

Diclofenac, a nonsteroidal anti-inflammatory drug, is metabolized to a reactive acyl glucuronide that has been proposed to mediate toxic adverse drug reactions associated with its use. In the present study, we examined the ability of diclofenac acyl glucuronide (D-1-O-G) to transacylate glutathione (GSH) in vitro in buffer and in vivo in rats. Thus, in vitro reactions of D-1-O-G (100 microM) with GSH (10 mM) at pH 7.4 and 37 degrees C showed a linear time-dependent formation of diclofenac-S-acyl-glutathione (D-SG, 3 microM/h) through 60 min of incubation, reaching a maximum of 3.7 microM after 2 h of incubation. The major reaction that occurred was acyl migration of D-1-O-G (t1/2, 54 min) to less reactive isomers. The D-SG thioester product was shown to be unstable by degrading primarily to 1-(2,6-dichlorophenyl)indolin-2-one and by hydrolysis to diclofenac. After administration of diclofenac to rats (200 mg/kg), bile was collected and analyzed for D-SG by liquid chromatography-tandem mass spectrometry. Results indicated the presence of D-SG, which was confirmed by coelution with synthetic standard and by its tandem mass spectrum. When the reactivity of D-SG (100 microM) was compared with D-1-O-G (100 microM) in vitro in reactions with N-acetylcysteine (NAC, 10 mM), results showed the quantitative reaction of D-SG with NAC after 30 min of incubation, whereas only approximately 1% of D-1-O-G reacted to form diclofenac-S-acyl-NAC at the same time point. Results from these studies indicate that GSH reacts with D-1-O-G in vitro, and presumably in vivo, to form D-SG, and that the product D-SG thioester is chemically more reactive in transacylation-type reactions than the D-1-O-G metabolite.

Cross-linking in the living cell locates the site of action of oxazolidinone antibiotics.

Oxazolidinone antibiotics, an important new class of synthetic antibacterials, inhibit protein synthesis by interfering with ribosomal function. The exact site and mechanism of oxazolidinone action has not been elucidated. Although genetic data pointed to the ribosomal peptidyltransferase as the primary site of drug action, some biochemical studies conducted in vitro suggested interaction with different regions of the ribosome. These inconsistent observations obtained in vivo and in vitro have complicated the understanding of oxazolidinone action. To localize the site of oxazolidinone action in the living cell, we have cross-linked a photoactive drug analog to its target in intact, actively growing Staphylococcus aureus. The oxazolidinone cross-linked specifically to 23 S rRNA, tRNA, and two polypeptides. The site of cross-linking to 23 S rRNA was mapped to the universally conserved A-2602. Polypeptides cross-linked were the ribosomal protein L27, whose N terminus may reach the peptidyltransferase center, and LepA, a protein homologous to translation factors. Only ribosome-associated LepA, but not free protein, was cross-linked, indicating that LepA was cross-linked by the ribosome-bound antibiotic. The evidence suggests that a specific oxazolidinone binding site is formed in the translating ribosome in the immediate vicinity of the peptidyltransferase center.