Complex Metabolism of the Novel Neurosteroid, Ganaxolone, in Humans: A Unique Challenge for Metabolites in Safety Testing Assessment.

The human pharmacokinetics, metabolism, and excretion of [14C]-ganaxolone (GNX) were characterized in healthy male subjects (n = 8) following a single 300-mg (150 µCi) oral dose. GNX exhibited a short half-life of 4 hours in plasma, whereas total radioactivity had a half-life of 413 hours indicating extensive metabolism to long-lived metabolites. Identification of the major GNX circulating metabolites required extensive isolation and purification for liquid chromatography-tandem mass spectrometry analysis, together with in vitro studies, NMR spectroscopy, and synthetic chemistry support. This revealed that the major routes of GNX metabolism involved hydroxylation at the 16α-hydroxy position, stereoselective reduction of the 20-ketone to afford the corresponding 20α-hydroxysterol, and sulfation of the 3α-hydroxy group. This latter reaction yielded an unstable tertiary sulfate, which eliminated the elements of H2SO4 to introduce a double bond in the A ring. A combination of these pathways, together with oxidation of the 3ß-methyl substituent to a carboxylic acid and sulfation at the 20α position, led to the major circulating metabolites in plasma, termed M2 and M17. These studies, which led to the complete or partial identification of no less than 59 metabolites of GNX, demonstrated the high complexity of the metabolic fate of this drug in humans and demonstrated that the major circulating products in plasma can result from multiple sequential processes that may not be easily replicated in animals or with animal or human in vitro systems. SIGNIFICANCE STATEMENT: Studies on the metabolism of [14C]-ganaxolone in humans revealed a complex array of products that circulated in plasma, the two major components of which were formed via an unexpected multi-step pathway. Complete structural characterization of these (disproportionate) human metabolites required extensive in vitro studies, along with contemporary mass spectrometry, NMR spectroscopy, and synthetic chemistry efforts, which served to underscore the limitations of traditional animal studies in predicting major circulating metabolites in man.

The evolution of the British Journal of Anaesthesia: the first 100 years.

At this centenary of the British Journal of Anaesthesia (BJA) in 2023, six of its 12 editors/editors-in-chief detail developments over the decades that have led to the BJA becoming a high-impact international scientific journal. As a charity, the BJA supports academic research and training in anaesthesia, critical care, and pain medicine including funding of research grants and postgraduate education. Building on this foundation, the BJA continues to innovate as it aims to become fully electronic, expand into open access publishing, and increase the diversity of its editorial board.

Selection on vocal output affects laryngeal morphology in rats.

Although laryngeal morphology often reflects adaptations for vocalization, the structural consequences of selection for particular aspects of vocal behavior remain poorly understood. In this study, we investigated the effects of increased ultrasonic calling in pups on the adult larynx morphology in selectively bred rat lines. Laryngeal morphology was assessed using multiple techniques: mineralized cartilage volumes were compared in 3D-models derived from microCT scans, internal structure was compared using clearing and staining procedures combined with microscopy, cellular structure was compared using histology and microscopy, and element composition was assessed with scanning energy dispersive X-ray spectroscopy. Our results show that adult rats from lines bred to produce ultrasonic calls at higher rates as pups have shorter vocal folds and a more mineralized thyroid cartilage compared to rats bred to produce ultrasonic calls at lower rates. The change in vocal fold length appears to account for differences in low-frequency calls in these two rat lines. We suggest that the observed increases in mineralization of the thyroid cartilage in the high-ultrasound lineage provide increased reinforcement of the laryngeal structure during ultrasonic call production. Our findings therefore demonstrate an effect of selection for vocal behavior on laryngeal morphology, with acoustic consequences.

Tunable Enzymatic Synthesis of the Immunomodulator Lipid IVA To Enable Structure-Activity Analysis.

The Lipid A family of glycolipids, found in the outer membranes of all Gram-negative bacteria, exhibits considerable structural diversity in both lipid and glycan moieties. The lack of facile methods to prepare analogues of these natural products represents a major roadblock in understanding the relationship between their structure and immunomodulatory activities. Here we present a modular, cell-free multienzymatic platform to access these structure-activity relationships. By individually purifying 19 Escherichia coli proteins and reconstituting them in vitro in the presence of acetyl-CoA, UDP- N-acetylglucosamine, NADPH, and ATP, we have developed a system capable of synthesizing Lipid IVA, the first bioactive intermediate in the Lipid A pathway. Our reconstituted multienzyme system revealed considerable promiscuity for orthologs with distinct substrate specificity, as illustrated by swapping enzymes from distantly related cyanobacterial and Pseudomonas species. Analysis of the agonistic and antagonistic activities of the resulting products against the THP-1 human monocytic cell line revealed hitherto unrecognized trends, while opening the door to harnessing the potent biological activities of these complex glycolipid natural products.

In vitro and in vivo metabolite identification of a novel benzimidazole compound ZLN005 by liquid chromatography/tandem mass spectrometry.

RATIONALE: A novel benzimidazole compound ZLN005 was previously identified as a transcriptional activator of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in certain metabolic tissues. Upregulation of PGC-1α by ZLN005 has been shown to have a beneficial effect in a diabetic mouse model and in a coronary artery disease model in vitro. ZLN005 could also have therapeutic potential in neurodegenerative diseases involving down-regulation of PGC-1α. Given the phenotypic efficacy of ZLN005 in several animal models of human disease, its metabolic profile was investigated to guide the development of novel therapeutics using ZLN005 as the lead compound. METHODS: ZLN005 was incubated with both rat and human liver microsomes and S9 fractions to identify in vitro metabolites. Urine from rats dosed with ZLN005 was used to identify in vivo metabolites. Extracted metabolites were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using a hybrid linear ion trap triple quadrupole mass spectrometer in full scan, enhanced product ion scan, neutral loss scan and precursor scan modes. Metabolites in plasma and brain of ZLN005-treated rats were also profiled using multiple reaction monitoring. RESULTS: Identified in vitro transformations of ZLN005 include mono- and dihydroxylation, further oxidation to carboxylic acids, and mono-O-glucuronide and sulfate conjugation to hydroxy ZLN005 as well as glutathione conjugation. Identified in vivo metabolites are mainly glucuronide and sulfate conjugates of dihydroxyl, carboxyl, and hydroxy acid of the parent compound. The parent compound as well as several major phase I metabolites were found in rat plasma and brain. CONCLUSIONS: Using both in vitro and in vivo methods, we elucidated the metabolic pathway of ZLN005. Phase I metabolites with hydroxylation and carboxylation, as well as phase II metabolites with glucuronide, sulfate and glutathione conjugation, were identified.

Small-Volume Injections: Evaluation of Volume Administration Deviation From Intended Injection Volumes.

BACKGROUND: In the perioperative period, anesthesiologists and postanesthesia care unit (PACU) nurses routinely prepare and administer small-volume IV injections, yet the accuracy of delivered medication volumes in this setting has not been described. In this ex vivo study, we sought to characterize the degree to which small-volume injections (≤0.5 mL) deviated from the intended injection volumes among a group of pediatric anesthesiologists and pediatric postanesthesia care unit (PACU) nurses. We hypothesized that as the intended injection volumes decreased, the deviation from those intended injection volumes would increase. METHODS: Ten attending pediatric anesthesiologists and 10 pediatric PACU nurses each performed a series of 10 injections into a simulated patient IV setup. Practitioners used separate 1-mL tuberculin syringes with removable 18-gauge needles (Becton-Dickinson & Company, Franklin Lakes, NJ) to aspirate 5 different volumes (0.025, 0.05, 0.1, 0.25, and 0.5 mL) of 0.25 mM Lucifer Yellow (LY) fluorescent dye constituted in saline (Sigma Aldrich, St. Louis, MO) from a rubber-stoppered vial. Each participant then injected the specified volume of LY fluorescent dye via a 3-way stopcock into IV tubing with free-flowing 0.9% sodium chloride (10 mL/min). The injected volume of LY fluorescent dye and 0.9% sodium chloride then drained into a collection vial for laboratory analysis. Microplate fluorescence wavelength detection (Infinite M1000; Tecan, Mannedorf, Switzerland) was used to measure the fluorescence of the collected fluid. Administered injection volumes were calculated based on the fluorescence of the collected fluid using a calibration curve of known LY volumes and associated fluorescence.To determine whether deviation of the administered volumes from the intended injection volumes increased at lower injection volumes, we compared the proportional injection volume error (loge [administered volume/intended volume]) for each of the 5 injection volumes using a linear regression model. Analysis of variance was used to determine whether the absolute log proportional error differed by the intended injection volume. Interindividual and intraindividual deviation from the intended injection volume was also characterized. RESULTS: As the intended injection volumes decreased, the absolute log proportional injection volume error increased (analysis of variance, P < .0018). The exploratory analysis revealed no significant difference in the standard deviations of the log proportional errors for injection volumes between physicians and pediatric PACU nurses; however, the difference in absolute bias was significantly higher for nurses with a 2-sided significance of P = .03. CONCLUSIONS: Clinically significant dose variation occurs when injecting volumes ≤0.5 mL. Administering small volumes of medications may result in unintended medication administration errors.

Safety and activity of RRx-001 in patients with advanced cancer: a first-in-human, open-label, dose-escalation phase 1 study.

BACKGROUND: Epigenetic alterations have been strongly associated with tumour formation and resistance to chemotherapeutic drugs, and epigenetic modifications are an attractive target in cancer research. RRx-001 is activated by hypoxia and induces the generation of reactive oxygen and nitrogen species that can epigenetically modulate DNA methylation, histone deacetylation, and lysine demethylation. The aim of this phase 1 study was to assess the safety, tolerability, and pharmacokinetics of RRx-001. METHODS: In this open-label, dose-escalation, phase 1 study, we recruited adult patients (aged >18 years) with histologically or cytologically confirmed diagnosis of advanced, malignant, incurable solid tumours from University of California at San Diego, CA, USA, and Sarah Cannon Research Institute, Nashville, TN, USA. Key eligibility criteria included evaluable disease, Eastern Cooperative Group performance status of 2 or less, an estimated life expectancy of at least 12 weeks, adequate laboratory parameters, discontinuation of all previous antineoplastic therapies at least 6 weeks before intervention, and no residual side-effects from previous therapies. Patients were assigned to receive intravenous infusions of RRx-001 at increasing doses (10 mg/m(2), 16·7 mg/m(2), 24·6 mg/m(2), 33 mg/m(2), 55 mg/m(2), and 83 mg/m(2)) either once or twice-weekly for at least 4 weeks, with at least three patients per dose cohort and allowing a 2-week observation period before dose escalation. Samples for safety and pharmacokinetics analysis, including standard chemistry and haematological panels, were taken on each treatment day. The primary objective was to assess safety, tolerability, and dose-limiting toxic effects of RRx-001, to determine single-dose pharmacokinetics, and to identify a recommended dose for phase 2 trials. All analyses were done per protocol. Accrual is complete and follow-up is still on-going. This trial is registered with ClinicalTrials.gov, number NCT01359982. FINDINGS: Between Oct 10, 2011, and March 18, 2013, we enrolled 25 patients and treated six patients in the 10 mg/m(2) cohort, three patients in the 16·7 mg/m(2) cohort, three patients in the 24·6 mg/m(2) cohort, four patients in the 33 mg/m(2) cohort, three patients in the 55 mg/m(2), and six patients in the 83 mg/m(2) cohort. Pain at the injection site, mostly grade 1 and grade 2, was the most common adverse event related to treatment, experienced by 21 (84%) patients. Other common drug-related adverse events included arm swelling or oedema (eight [32%] patients), and vein hardening (seven [28%] patients). No dose-limiting toxicities were observed. Time constraints related to management of infusion pain from RRx-001 resulted in a maximally feasible dose of 83 mg/m(2). Of the 21 evaluable patients, one (5%) patient had a partial response, 14 (67%) patients had stable disease, and six (29%) patients had progressive disease; all responses were across a variety of tumour types. Four patients who had received RRx-001 were subsequently rechallenged with a treatment that they had become refractory to; all four responded to the rechallenge. INTERPRETATION: RRx-001 is a well-tolerated novel compound without clinically significant toxic effects at the tested doses. Preliminary evidence of activity is promising and, on the basis of all findings, a dose of 16·7 mg/m(2) was recommended as the targeted dose for phase 2 trials. FUNDING: EpicentRx (formerly RadioRx).

Using chimeric mice with humanized livers to predict human drug metabolism and a drug-drug interaction.

Interspecies differences in drug metabolism have made it difficult to use preclinical animal testing data to predict the drug metabolites or potential drug-drug interactions (DDIs) that will occur in humans. Although chimeric mice with humanized livers can produce known human metabolites for test substrates, we do not know whether chimeric mice can be used to prospectively predict human drug metabolism or a possible DDI. Therefore, we investigated whether they could provide a more predictive assessment for clemizole, a drug in clinical development for the treatment of hepatitis C virus (HCV) infection. Our results demonstrate, for the first time, that analyses performed in chimeric mice can correctly identify the predominant human drug metabolite before human testing. The differences in the rodent and human pathways for clemizole metabolism were of importance, because the predominant human metabolite was found to have synergistic anti-HCV activity. Moreover, studies in chimeric mice also correctly predicted that a DDI would occur in humans when clemizole was coadministered with a CYP3A4 inhibitor. These results demonstrate that using chimeric mice can improve the quality of preclinical drug assessment.

Metabolomic-derived novel cyst fluid biomarkers for pancreatic cysts: glucose and kynurenine.

BACKGROUND: Better pancreatic cyst fluid biomarkers are needed. OBJECTIVE: To determine whether metabolomic profiling of pancreatic cyst fluid would yield clinically useful cyst fluid biomarkers. DESIGN: Retrospective study. SETTING: Tertiary-care referral center. PATIENTS: Two independent cohorts of patients (n = 26 and n = 19) with histologically defined pancreatic cysts. INTERVENTION: Exploratory analysis for differentially expressed metabolites between (1) nonmucinous and mucinous cysts and (2) malignant and premalignant cysts was performed in the first cohort. With the second cohort, a validation analysis of promising identified metabolites was performed. MAIN OUTCOME MEASUREMENTS: Identification of differentially expressed metabolites between clinically relevant cyst categories and their diagnostic performance (receiver operating characteristic [ROC] curve). RESULTS: Two metabolites had diagnostic significance-glucose and kynurenine. Metabolomic abundances for both were significantly lower in mucinous cysts compared with nonmucinous cysts in both cohorts (glucose first cohort P = .002, validation P = .006; and kynurenine first cohort P = .002, validation P = .002). The ROC curve for glucose was 0.92 (95% confidence interval [CI], 0.81-1.00) and 0.88 (95% CI, 0.72-1.00) in the first and validation cohorts, respectively. The ROC for kynurenine was 0.94 (95% CI, 0.81-1.00) and 0.92 (95% CI, 0.76-1.00) in the first and validation cohorts, respectively. Neither could differentiate premalignant from malignant cysts. Glucose and kynurenine levels were significantly elevated for serous cystadenomas in both cohorts. LIMITATIONS: Small sample sizes. CONCLUSION: Metabolomic profiling identified glucose and kynurenine to have potential clinical utility for differentiating mucinous from nonmucinous pancreatic cysts. These markers also may diagnose serous cystadenomas.

Liquid chromatography/mass spectrometry methods for measuring dipeptide abundance in non-small-cell lung cancer.

RATIONALE: Metabolomic profiling is a promising methodology of identifying candidate biomarkers for disease detection and monitoring. Although lung cancer is among the leading causes of cancer-related mortality worldwide, the lung tumor metabolome has not been fully characterized. METHODS: We utilized a targeted metabolomic approach to analyze discrete groups of related metabolites. We adopted a dansyl [5-(dimethylamino)-1-naphthalene sulfonamide] derivatization with liquid chromatography/mass spectrometry (LC/MS) to analyze changes of metabolites from paired tumor and normal lung tissues. Identification of dansylated dipeptides was confirmed with synthetic standards. A systematic analysis of retention times was required to reliably identify isobaric dipeptides. We validated our findings in a separate sample cohort. RESULTS: We produced a database of the LC retention times and MS/MS spectra of 361 dansyl dipeptides. Interpretation of the spectra is presented. Using this standard data, we identified a total of 279 dipeptides in lung tumor tissue. The abundance of 90 dipeptides was selectively increased in lung tumor tissue compared to normal tissue. In a second set of validation tissues, 12 dipeptides were selectively increased. CONCLUSIONS: A systematic evaluation of certain metabolite classes in lung tumors may identify promising disease-specific metabolites. Our database of all possible dipeptides will facilitate ongoing translational applications of metabolomic profiling as it relates to lung cancer.

A three-parameter model for classifying anurans into four genera based on advertisement calls.

The vocalizations of anurans are innate in structure and may therefore contain indicators of phylogenetic history. Thus, advertisement calls of species which are more closely related phylogenetically are predicted to be more similar than those of distant species. This hypothesis was evaluated by comparing several widely used machine-learning algorithms. Recordings of advertisement calls from 142 species belonging to four genera were analyzed. A logistic regression model, using mean values for dominant frequency, coefficient of variation of root-mean square energy, and spectral flux, correctly classified advertisement calls with regard to genus with an accuracy above 70%. Similar accuracy rates were obtained using these parameters with a support vector machine model, a K-nearest neighbor algorithm, and a multivariate Gaussian distribution classifier, whereas a Gaussian mixture model performed slightly worse. In contrast, models based on mel-frequency cepstral coefficients did not fare as well. Comparable accuracy levels were obtained on out-of-sample recordings from 52 of the 142 original species. The results suggest that a combination of low-level acoustic attributes is sufficient to discriminate efficiently between the vocalizations of these four genera, thus supporting the initial premise and validating the use of high-throughput algorithms on animal vocalizations to evaluate phylogenetic hypotheses.

Identification of drug targets by chemogenomic and metabolomic profiling in yeast.

OBJECTIVE: To advance our understanding of disease biology, the characterization of the molecular target for clinically proven or new drugs is very important. Because of its simplicity and the availability of strains with individual deletions in all of its genes, chemogenomic profiling in yeast has been used to identify drug targets. As measurement of drug-induced changes in cellular metabolites can yield considerable information about the effects of a drug, we investigated whether combining chemogenomic and metabolomic profiling in yeast could improve the characterization of drug targets. BASIC METHODS: We used chemogenomic and metabolomic profiling in yeast to characterize the target for five drugs acting on two biologically important pathways. A novel computational method that uses a curated metabolic network was also developed, and it was used to identify the genes that are likely to be responsible for the metabolomic differences found. RESULTS AND CONCLUSION: The combination of metabolomic and chemogenomic profiling, along with data analyses carried out using a novel computational method, could robustly identify the enzymes targeted by five drugs. Moreover, this novel computational method has the potential to identify genes that are causative of metabolomic differences or drug targets.

Preclinical evaluation of the metabolism and disposition of RRx-001, a novel investigative anticancer agent.

RRx-001 has shown promise as a novel cancer therapeutic agent. The disposition of RRx-001 was evaluated in vitro and after intravenous administration to rats. At both 24 and 168 h after a single intravenous administration of ¹4C-RRx-001 (10 mg/kg), the majority of radiolabel was in the blood. The recovery of label in excreta was quite low, but the major route of radiolabel excretion was via the kidney, with approximately 26% in the urine by the first 8 h and decreasing amounts in all subsequent collections to a total of 36.3% by 168 h. The partitioning of total radioactivity in red blood cells (RBCs) and plasma was determined after in vitro addition to human, rat, dog, and monkey whole blood at 1 and 20 µM. In rat, at 30 min, approximately 75% of the radioactivity is associated with RBCs and 25% with plasma. In human, at 30 min, approximately 25% of the radioactivity is associated with RBCs and 75% with plasma. Analysis by liquid chromatography/radiodetection/mass spectrometry showed that ¹4C-RRx-001 reacted rapidly with whole blood to give four major soluble metabolites: the GSH and Cys adducts of RRx-001 (M1 and M2) and the corresponding mononitro GSH and Cys adducts (M3 and M4). Human Hb was incubated with cold RRx-001 in buffer, and a standard proteomics protocol was used to separate and identify the tryptic peptides. Standard peptide collision-induced fragment ions supported the structure of the peptide GTFATLSELHCDK with the alkylation on the Cys-93 locus of the Hb ß chain.

Identification of metabolites in Withania sominfera fruits by liquid chromatography and high-resolution mass spectrometry.

RATIONALE: Withania somnifera is a rich source of biologically active secondary metabolites. Our earlier investigations on the fruits of this plant have yielded novel compounds, withanamides, with potent antioxidant activity and protective effect on ß-amyloid-induced cytotoxicity in vitro. However, several minor compounds present in the fruits have not been characterized previously which may contribute to the observed biological activities. METHODS: Liquid chromatography (LC) coupled with high-resolution mass spectrometry (HRMS) with data-dependent and targeted MS/MS experiments were conducted to elucidate the structure of observed metabolites. RESULTS: A total of 62 metabolites identified included 32 withanamides, 22 withanolides, 3 steroidal saponins, 2 lignanamides, feruloyl tyramine, methoxy feruloyl tyramine and a diglucoside of hydroxyl palmitic acid. The structures of these compounds were proposed based on accurate masses of the molecular and fragment ions. Several of these new compounds identified from the profile were derived from withanamides with variations in aliphatic and/or glycosyl moieties. In addition, six new withanolides, a new hydroxy fatty acid diglucoside and several known compounds in the extract were identified. CONCLUSIONS: The current study revealed the presence of several new and known compounds in Withania somnifera fruits. High-resolution MS and MS/MS experiments provide an extremely effective approach to derive the structures of plant secondary metabolites including isomeric compounds.

Discovery of G2019S-Selective Leucine Rich Repeat Protein Kinase 2 inhibitors with in vivo efficacy.

Mutations in the Leucine Rich Repeat Protein Kinase 2 gene (LRRK2) are the most common genetic causes of Parkinson's Disease (PD). The G2019S mutation is the most common inherited LRRK2 mutation, occurs in the kinase domain, and results in increased kinase activity. We report the discovery and development of compound 38, an indazole-based, G2019S-selective (>2000-fold vs. WT) LRRK2 inhibitor capable of entering rodent brain (Kp = 0.5) and selectively inhibiting G2019S-LRRK2. The compounds disclosed herein present a starting point for further development of brain penetrant G2019S selective inhibitors that hopefully reduce lung phenotype side-effects and pave the way to providing a precision medicine for people with PD who carry the G2019S mutation.

Exploration of piperidine-4-yl-aminopyrimidines as HIV-1 reverse transcriptase inhibitors. N-Phenyl derivatives with broad potency against resistant mutant viruses.

Further investigation of the recently reported piperidine-4-yl-aminopyrimidine class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) has been carried out. Thus, preparation of a series of N-phenyl piperidine analogs resulted in the identification of 3-carboxamides as a particularly active series. Analogs such as 28 and 40 are very potent versus wild-type HIV-1 and a broad range of NNRTI-resistant mutant viruses. Synthesis, structure-activity relationship (SAR), clearance data, and crystallographic evidence for the binding motif are discussed.

Metabolic activation of nevirapine in human liver microsomes: dehydrogenation and inactivation of cytochrome P450 3A4.

Nevirapine, a non-nucleoside HIV-1 reverse transcriptase inhibitor, has been associated with incidences of skin rash and hepatotoxicity in patients. Although the mechanism of idiosyncratic hepatotoxicity remains unknown, it is proposed that metabolic activation of nevirapine and subsequent covalently binding of reactive metabolites to cellular proteins play a causative role. Studies were initiated to determine whether nevirapine undergoes cytochrome P450 (P450)-mediated bioactivation in human liver microsomes to electrophilic intermediates. Liquid chromatography-tandem mass spectrometry analysis of incubations containing nevirapine and NADPH-supplemented microsomes in the presence of glutathione (GSH) revealed the formation of a GSH conjugate derived from the addition of the sulfydryl nucleophile to nevirapine. No other GSH conjugates were detected, including conjugates of oxidized metabolites of nevirapine. These findings are consistent with a bioactivation sequence involving initial P450-catalyzed dehydrogenation of the aromatic nucleus with a 4-methyl group in nevirapine to an electrophilic quinone methide intermediate, which is subsequently attacked by glutathione yielding the sulfydryl conjugate. Formation of the nevirapine GSH conjugate was primarily catalyzed by heterologously expressed recombinant CYP3A4 and, to a lesser extent, CYP2D6, CYP2C19, and CYP2A6. In addition, the quinone methide reactive metabolite was a mechanism-based inactivator of CYP3A4, with inactivation parameters K(I) = 31 microM and k(inact) = 0.029 min(-1), respectively. It is proposed that formation of the quinone methide intermediate may represent a rate-limiting step in the initiation of nevirapine-mediated hepatotoxicity.

Screening and characterization of reactive metabolites using glutathione ethyl ester in combination with Q-trap mass spectrometry.

The present study describes a new analytical approach for the detection and characterization of chemically reactive metabolites using glutathione ethyl ester (GSH-EE) as the trapping agent in combination with hybrid triple quadrupole linear ion trap mass spectrometry. Polarity switching was applied between a negative precursor ion (PI) survey scan and the positive enhanced product ion (EPI) scan. The negative PI scan step was carried out monitoring the anion at m/z 300, corresponding to deprotonated gamma-glutamyl-dehydroalanyl-glycine ethyl ester originating from the GSH-EE moiety. Samples resulting from incubations in the presence of GSH-EE were cleaned and concentrated by solid-phase extraction, followed by the PI-EPI analysis. Unambiguous identification of GSH-EE-trapped reactive metabolites was greatly facilitated by the unique survey scan of the anion at m/z 300, which achieved less background interference, in particular, from endogenous glutathione adducts present in human liver microsomes. Further structural characterization was achieved by analyzing positive MS(2) spectra that featured rich fragments without mass cutoff and were acquired in the same liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The effectiveness and reliability of this approach was evaluated using a number of model compounds in human liver microsomal incubations, including acetaminophen, amodiaquine, carbamazepine, 4-ethylphenol, imipramine and ticlopidine. In addition, iminoquinone reactive metabolites of mianserin were trapped and characterized for the first time using this method. Compared to neutral loss (NL) scanning assays using GSH as the trapping agent, the results have demonstrated superior selectivity, sensitivity, and reliability of this current approach.

Using LC Retention Times in Organic Structure Determination: Drug Metabolite Identification.

BACKGROUND: There is a continued need for improvements in the efficiency of metabolite structure elucidation. OBJECTIVE: We propose to take LC Retention Time (RT) into consideration during the process of structure determination. METHODS: Herein, we develop a simple methodology that employs a Chromatographic Hydrophobicity Index (CHI) framework for standardizing LC conditions and introduce and utilize the concept of a predictable CHI change upon Phase 1 biotransformation (CHIbt). Through the analysis of literature examples, we offer a Quantitative Structure-Retention Relationship (QSRR) for several types of biotransformation (especially hydroxylation) using physicochemical properties (clogP, hydrogen bonding). RESULTS: The CHI system for retention indexing is shown to be practical and simple to implement. A database of CHIbt values has been created from re-incubation of 3 compounds and from analysis of an additional 17 datasets from the literature. Application of this database is illustrated. CONCLUSION: In our experience, this simple methodology allows complementing the discovery efforts that saves resources for in-depth characterization using NMR.