Interaction of γ-glutamyltranspeptidase with ibuprofen-S-acyl-glutathione in vitro and in vivo in human.

Ibuprofen is metabolized to chemically reactive acyl glucuronide and S-acyl-CoA metabolites that are proposed to transacylate glutathione (GSH) forming ibuprofen-S-acyl-GSH (I-SG) in vivo. Herein, we report the detection of novel metabolites of ibuprofen, namely ibuprofen-N-acyl-cysteinylglycine (I-N-CG), ibuprofen-N-acyl-cysteine (I-N-C), and the mercapturic acid conjugate, ibuprofen-S-acyl-N-acetylcysteine (I-S-NAC), in urine from an ibuprofen-dosed volunteer. Thus, analysis of ibuprofen-dosed (Advil, 800 mg, Pfizer, Madison, NJ) human urine extracts by sensitive liquid chromatography tandem mass spectrometric detection resulted in the identification of I-N-CG, I-N-C, and I-S-NAC derivatives as minor metabolites (6.0, 1.7, and 0.2 µg excreted 10-hours postadministration, respectively). I-N-CG is proposed to be formed from the degradation of I-SG by γ-glutamyltranspeptidase (γ-GT)-mediated cleavage of the γ-glutamyl group, leading to an unstable ibuprofen-S-acyl-cysteinylglycine (I-S-CG) intermediate that undergoes spontaneous S to N intramolecular rearrangement. Then, dipeptidase-mediated cleavage of glycine from I-N-CG leads to the formation of I-N-C. Treatment of racemic I-SG (100 µM) in vitro with commercially available bovine kidney γ-GT (0.1 units/ml) in buffer at pH 7.4 and 37°C resulted in its complete degradation, yielding (R)- and (S)-I-N-CG after 15 minutes of incubation. In vitro enzyme kinetic studies with bovine kidney γ-GT incubated separately with (R)- and (S)-I-SG isomers revealed no enantioselective degradation. Results from these studies provided evidence that ibuprofen is metabolized in human to reactive transacylating-type intermediates that react with GSH, forming I-SG thioester that, following degradation by γ-GT and dipeptidase enzymes and following S to N intramolecular rearrangement, leads to the urinary excretion of the I-N-CG and I-N-C amide-linked conjugates, respectively.

Generation of statin drug metabolites through electrochemical and enzymatic oxidations.

The generation of key drug metabolites for the purpose of their complete structural characterization, toxicity testing, as well as to serve as standards for quantitative studies, is a critical step in the pharmaceutical discovery and development cycle. Here, we utilized electrochemistry/mass spectrometry for the detection and subsequent generation of six phase I metabolites of simvastatin and lovastatin. Both simvastatin and lovastatin are widely used for the treatment of hypercholesterolemia. There are known drug-drug interaction issues of statin therapy, and it has been suggested that the oxidative metabolites may contribute to the cholesterol-lowering effect of both statins. Of the known phase I metabolites of simvastatin and lovastatin, none are commercially available, and chemical means for the synthesis of a very few of them have been previously reported. Here, we report that electrochemical oxidation of less than 1 mg each of simvastatin and lovastatin led to the generation of three oxidative metabolites of each parent to allow complete nuclear magnetic resonance characterization of all six metabolites. The yields obtained by the electrochemical approach were also compared with incubation of parent drug with commercially available bacterial mutant CYP102A1 enzymes, and it was found that the electrochemical approach gave higher yields than the enzymatic oxidations for the generation of most of the observed oxidative metabolites in this study.

Stereoselective flunoxaprofen-S-acyl-glutathione thioester formation mediated by acyl-CoA formation in rat hepatocytes.

Flunoxaprofen (FLX) is a chiral nonsteroidal anti-inflammatory drug that was withdrawn from clinical use because of concerns of potential hepatotoxicity. FLX undergoes highly stereoselective chiral inversion mediated through the FLX-S-acyl-CoA thioester (FLX-CoA) in favor of the (R)-(-)-isomer. Acyl-CoA thioester derivatives of acidic drugs are chemically reactive species that are known to transacylate protein nucleophiles and glutathione (GSH). In this study, we investigated the relationship between the stereoselective metabolism of (R)-(-)- and (S)-(+)-FLX to FLX-CoA and the subsequent transacylation of GSH forming FLX-S-acyl-glutathione (FLX-SG) in incubations with rat hepatocytes in suspension. Thus, when hepatocytes (2 million cells/ml) were treated with (R)-(-)- or (S)-(+)-FLX (100 microM), both FLX-CoA and FLX-SG were detected by sensitive liquid chromatography-tandem mass spectrometry techniques. However, these derivatives were observed primarily from (R)-(-)-FLX incubation extracts, for which the formation rates of FLX-CoA and FLX-SG were rapid, reaching maximum concentrations of 42 and 2.8 nM, respectively, after 6 min of incubation. Incubations with (S)-(+)-FLX over 60 min displayed 8.1 and 2.7% as much FLX-CoA and FLX-SG area under the concentration versus time curves, respectively, compared with corresponding incubations with (R)-(-)-FLX. Coincubation of lauric acid (1000 microM) with (R)-(-)-FLX (10 microM) led to the complete inhibition of FLX-CoA formation and a 98% inhibition of FLX-SG formation. Reaction of authentic (R,S)-FLX-CoA (2 microM) with GSH (10 mM) in buffer (pH 7.4, 37 degrees C) showed the quantitative formation of FLX-SG after 3 h of incubation. Together, these results demonstrate the stereoselective transacylation of GSH in hepatocyte incubations containing (R)-(-)-FLX, which is consistent with bioactivation by stereoselective (R)-FLX-CoA formation.

Qualitative assessment of extractables from single-use components and the impact of reference standard selection.

The advent of single-use bioprocess systems used for the delivery, storage or manufacture of biopharmaceuticals has introduced a new potential source for extractables and leachables (E&L) as these systems are comprised of polymeric materials. Several industry working groups, the FDA and USP have issued guidance and draft guidance on E&L analyses for a variety of applications. These documents typically indicate that mass spectrometry should be applied for discovery of E&L's but provide little guidance as to the exact analytical methodology which should be used. We investigated the extractable profiles of a model single-use bioprocessing system consisting of a single-use bioprocess bag, connector tubing, and a hydrophilic disk filter including filter housing. Extractions were performed in water, ethanol, ethanol/water (50:50) and saline solutions. Extracts were analyzed using a stepwise analytical methodology including a variety of screening and mass spectrometry methods We then used this model system to demonstrate the use of recursive feature finding to automatically detect unique extractables followed by statistical filtering to focus on differentially present extractables which were above the analytical evaluation threshold (AET). We further show the significant affects of standard selection on the number of compounds determined to be above AET when reducing liquid chromatography-mass spectrometry (LC/MS) data. A relative response factor database consisting of 14 structurally diverse commercially available polymer additives was used to arrive at an LC/MS identification threshold. The results of this study demonstrate that significant care should be taken when selecting standards for LC/MS analysis to avoid under reporting of extractables and leachables.

Simultaneous and stereospecific analysis of warfarin oxidative metabolism using 2D LC/Q-TOF.

BACKGROUND: Warfarin is a widely used racemic anticoagulant with narrow therapeutic range and wide interindividual response to treatment. This is due to the extensive and differential clearance of R- and S-warfarin with the involvement of several polymorphic CYP450 enzymes resulting in the formation of several stereoisomeric oxidative metabolites. RESULTS: A stereospecific 2DLC/Q-TOF method was developed for the simultaneous identification and quantitation of hydroxylated warfarin metabolites from a single sample analysis. Using this method metabolites from rat microsomal and plated hepatocyte incubations with R-, S- and (R/S)-warfarin were estimated. CONCLUSION: Multiheart cutting with high resolution MS and MS/MS analysis is suggested as a viable approach for achiral-chiral separation of metabolites of warfarin and other chiral or prochiral drugs.

Application of diffusion-edited NMR spectroscopy for the structural characterization of drug metabolites in mixtures.

Diffusion-edited NMR spectroscopy is used to enable the structural characterization of low level metabolites in the presence of endogenous compounds, and organic solvents. We compared data from standard one-dimensional (1D) (1)H, 1D NOESY-presaturation, and 1D diffusion-edited experiments run on 20 microg and 100 microg samples of ethacrynic acid glutathione thioether (EASG) and a previously unreported metabolite of mefenamic acid, mefenamic acid glutathione thioester (MSG). The 1D NOESY-presaturation technique gave spectra with the best signal-to-noise (S/N) ratio, approximately three times that observed with the standard (1)H experiment, with respect to the metabolite signals. However, it was not selective for solvent signals as overlapping metabolite signals were also suppressed by this technique. In some cases, these signals were key to determining the site of glutathione attachment on the parent molecule. 1D NOESY-presaturation spectra also produced baseline distortions and inconsistent integration values. By comparison, 1D diffusion-edited experiments were found to selectively and simultaneously remove multiple solvent signals, resolve overlapping metabolite signals, and provide more uniform integration for metabolite signals overlapping with or proximal to solvent peaks, without producing baseline distortions. However, the diffusion-edited experiments caused significant signal attenuation of the metabolite signals when compared with a standard (1)H spectrum. Partially purified metabolites isolated from biological matrices were also characterized by using two-dimensional diffusion-ordered spectroscopy (DOSY). DOSY spectra acquired on a sample of EASG purified from rat bile proved useful in 'separating' the signals of EASG, from those of a co-eluting bile acid and parent drug ethacrynic acid (EA) in the diffusion-dimension in regions where there was no spectral overlap. In the low-field regions of high overlap, the DOSY experiment did not effectively separate the signals from the individual components. Diffusion based experiments provide a way to determine the total number of components that are present in a metabolite sample as well as an ability to identify them based on the chemical shift information, without the need for laborious chromatography on small samples.

Isolation, identification, and quantification of potential defensive compounds in the viceroy butterfly and its larval host-plant, Carolina willow.

The viceroy-monarch and viceroy-queen butterfly associations are classic examples of mimicry. These relationships were originally classified as Batesian, or parasitic, but were later reclassified as Müllerian, or mutalistic, based on predator bioassays. The Müllerian reclassification implies that viceroy is unpalatable because it too is chemically defended like the queen and the monarch. However, unlike the queen and the monarch, the viceroy defensive chemistry has remained uncharacterized. We demonstrate that the viceroy butterfly (Limenitis archippus, Nymphalidae) not only sequesters nonvolatile defensive compounds from its larval host-plant, the Carolina willow (Salix caroliniana, Salicaceae), but also secretes volatile defensive compounds when disturbed. We developed liquid chromatography-mass spectrometry-mass spectrometry methods to identify a set of phenolic glycosides shared between the adult viceroy butterfly and the Carolina willow, and solid phase microextraction and gas chromatography-mass spectrometry methods to identify volatile phenolic compounds released from stressed viceroy butterflies. In both approaches, all structures were characterized based on their mass spectral fragmentation patterns and confirmed with authentic standards. The phenolics we found are known to deter predator attack in other prey systems, including other willow-feeding insect species. Because these compounds have a generalized defensive function at the concentrations we described, our results are consistent with the Müllerian reclassification put forth by other researchers based on bioassay results. It seems that the viceroy butterfly possesses chemical defenses different from its monarch and queen butterfly counterparts (phenolic glycosides vs. cardiac glycosides, respectively), an unusual phenomenon in mimicry warranting future study.

(4R,4aR,6S,7S,7aS)-6-Hydroxy-7-hydroxymethyl-4-methylperhydrocyclopenta[c]pyran-1-one chloroform solvate from Valeriana laxiflora.

The structure of an iridolactone isolated from Valeriana laxiflora was established as (4R,4aR,6S,7S,7aS)-6-hydroxy-7-hydroxymethyl-4-methylperhydrocyclopenta[c]pyran-1-one chloroform solvate, C(10)H(16)O(4).CHCl(3). The two rings are cis-fused. The delta-lactone ring adopts a slightly twisted half-chair conformation with approximate planarity of the lactone group and the cyclopentane ring adopts an envelope conformation. The hydroxy group, the hydroxymethyl group and the methyl group all have beta orientations. The absolute configuration was determined using anomalous dispersion data enhanced by the adventitious inclusion of a chloroform solvent molecule. Hydrogen bonding, crystal packing and ring conformations are discussed in detail.

A novel antibacterial iridoid and triterpene from Caiophora coronata.

Bioassay-guided fractionation of the antibacterial CH(2)Cl(2)-MeOH extract obtained from the aerial parts of the Argentinean plant Caiophora coronata led to the isolation of a new triterpene, 1beta,3beta-dihydroxyurs-12-en-27-oic acid, 1, and a new iridoid, 1alpha-methoxy-6alpha,10-dihydroxyisoepiiridomyrmecin (caiophoraenin), 2, along with the known iridoid isoboonein 3. Their structures were established by spectroscopic techniques (1D and 2D NMR, HRFABMS, FTIR). The MIC values of isolated compounds were determined against methicillin-sensitive (MSSA) and -resistant (MRSA) strains of Staphylococcus aureus, Bacillus subtilis (BS), vancomycin-resistant Enterococcus faecium (VREF), Escherichia coli (EC), E. coli imp (ECimp), and Candida albicans (CA). Compound 1 was found active against BS, MSSA, MRSA, VREF, and ECimp with MIC values of 2, 4, 4, 4, and 16 microg/mL, respectively.