Addressing the metabolic activation potential of new leads in drug discovery: a case study using ion trap mass spectrometry and tritium labeling techniques

Metabolic activation of drug candidates to electrophilic reactive metabolites that can covalently modify cellular macromolecules may result in acute and/or idiosyncratic immune system-mediated toxicities in humans. This presents a significant potential liability for the future development of these compounds as safe therapeutic agents. We present here an example of an approach where sites of metabolic activation within a new drug candidate series were rapidly identified using online liquid chromatography/multi-stage mass spectrometry on an ion trap mass spectrometer. This was accomplished by trapping the reactive intermediates formed upon incubation of compounds with rat and human liver microsomes as their corresponding glutathione conjugates and mass spectral characterization of these thiol adducts. Based on the structures of the GSH adducts identified, potential sites and mechanisms of bioactivation within the chemical structure were proposed. These metabolism studies were interfaced with iterative structural modifications of the chemical series in order to block these bioactivation sites within the molecule. This strategy led to a significant reduction in the propensity of the compounds to undergo metabolic activation as evidenced by reductions in the irreversible binding of radioactivity to liver microsomal material upon incubation of tritium-labeled compounds with this in vitro system. With the efficiency and throughput achievable with such an approach, it appears feasible to identify and address the metabolic activation potential of new drug leads during routine metabolite identification studies in an early drug discovery setting.     

Intracellular fate of hydrocarbons

In previous work, purification procedures and zymogram analysis conducted with supernatants of crude extracts from aerobic mycelium of the YR-1 strain of Mucor circinelloides isolated from petroleum-contaminated soils indicated the existence of only one soluble alcohol oxidase (sAO) activity. In the present work enzymatic activity of alcohol oxidase (AO) was also detected in the mixed membrane fraction (MMF) of a high-speed centrifugation procedure after drastic ballistic cellular homogenization to break the mycelium from strain YR-1. When mycelial cells were gently broken by freezing the mycelium with liquid nitrogen, smashing in a mortar, and submitting the samples to an isopycnic sucrose gradients (10–60% sucrose), AO activity was detected in particular and discrete fractions of the gradient, showing specific density values quite different from the density of peroxisomes. The results suggest that there could be a different intracellular pattern of distribution of the microsomal fraction in aerobically grown mycelium depending on the carbon source used in the culture media, including alcohols and hydrocarbons, but not in glucose. In working with particulate fractions, we found two AO activities: a new membrane alcohol oxidase (mAO) activity and the sAO. Both activities appear to be located in the inner of the cells in specific compartments different from the peroxisomes, so mAO could be in the membrane of these compartments and sAO in the lumen of the vesicles. We also assayed other enzymatic activities involved in hydrocarbon biodegradation to establish its intracellular location and other enzymatic activities such as peroxidase to use them as intracellular markers of different organelles. In the case of monooxygenase, the first enzymatic step in the hydrocarbon biodegradation pathway, its location was in the same fractions where AOs were located, suggesting the existance of a specific organelle that contains the enzymatic activities involved in hydrocarbon biodegradation.     

Carotenoid incorporation into microsomes: yields, stability and membrane dynamics

The carotenoids beta-carotene (BC), lycopene (LYC), lutein (LUT), zeaxanthin (ZEA), canthaxanthin (CTX) and astaxanthin (ASTA) have been incorporated into pig liver microsomes. Effective incorporation concentrations in the range of about 1-6 nmol/mg microsomal protein were obtained. A stability test at room temperature revealed that after 3 h BC and LYC had decayed totally whereas, gradually, CTX (46%), LUT (21%), ASTA (17%) and ZEA (5%) decayed. Biophysical parameters of the microsomal membrane were changed hardly by the incorporation of carotenoids. A small rigidification may occur. Membrane anisotropy seems to offer only a small tolerance for incorporation of carotenoids and seems to limit the achievable incorporation concentrations of the carotenoids into microsomes. Microsomes instead of liposomes should be preferred as a membrane model to study mutual effects of carotenoids and membrane dynamics.     

Antioxidant enzymes activity in subcellular fraction of freshwater prawnsM. malcolmsonii andM. lamarrei lamarrei

Subcellular distribution of Superoxide dismutase (SOD), catalase (CAT), selenium (SC) dependent glutathione peroxidase, and Se-independent glutathione peroxidase (GSH-Px) activities were detected in different tissues (hepatopancreas, muscle, and gill) of freshwater prawnsMacrobrachium malcolmsonii andMacrobrahium lamarrei lamarrei. CAT and SOD were found almost equally between the mitochondrial and cytosolic fraction. Both Se-dependent and Se-independent GSH-Px activities were mainly found in cytosolic fraction.     

Metabolism studies on prim‐O‐glucosylcimifugin and cimifugin in human liver microsomes by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

Prim‐O‐glucosylcimifugin (PGCN) and cimifugin (CN) are major constituents of Radix Saposhnikoviae that have antipyretic, analgesic and anti‐inflammatory pharmacological activities. However, there were few reports with respect to the metabolism of PGCN and CN in vitro. In this paper, we describe a strategy using ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) for fast analysis of the metabolic profile of PGCN and CN in human liver microsomes. In total, five phase I metabolites of PGCN, seven phase I metabolites and two phase II metabolites of CN were identified in the incubation of human liver microsomes. The results revealed that the main phase I metabolic pathways of PGCN were hydroxylation and hydrolysis reactions. The phase I metabolic pathways of CN were found to be hydroxylation, demethylation and dehydrogenation. Meanwhile, the results indicated that O‐glucuronidation was the major metabolic pathway of CN in phase II metabolism. The specific UDP‐glucuronosyltransferase (UGT) enzymes responsible for CN glucuronidation metabolites were identified using recombinant UGT enzymes. The results indicated that UGT1A1, UGT1A9, UGT2B4 and UGT2B7 might play major roles in the glucuronidation of CN. Overall, this study may be useful for the investigation of metabolic mechanism of PGCN and CN, and it can provide reference and evidence for further pharmacodynamic experiments. Copyright © 2016 John Wiley & Sons, Ltd.     

Metabolism of human insulin after subcutaneous administration: A possible means to uncover insulin misuse

The misuse of insulin for performance enhancement in sport or as toxic agent has frequently been reported in the past. In contrast to synthetic insulin analogues, the administration of recombinant human insulin is hardly recognized by mass spectrometry. The present study was designed to uncover the misuse of recombinant human insulin for doping control purposes as well as for forensic applications. It is hypothesized that an altered metabolite profile of circulating insulin prevails after subcutaneous administration due to exposure of insulin to epidermal proteases.     

Metabolic profiles of ribociclib in rat and human liver microsomes using liquid chromatography combined with electrospray ionization high-resolution mass spectrometry

Ribociclib is a highly specific CDK4/6 inhibitor. Determination of the metabolism of ribociclib is required during the drug development stage. In this study, metabolic profiles of ribociclib were investigated using rat and human liver microsomes. Metabolites were structurally identified by liquid chromatography electrospray ionization high-resolution mass spectrometry operated in positive-ion mode. The metabolites were characterized by accurate masses, MS² spectra and retention times. With rat and human liver microsomes, a total of 10 metabolites were detected and further identified. No human-specific metabolites were detected. The metabolic pathways of ribociclib were oxygenation, demethylation and dealkylation. Most importantly, two glutathione (GSH) adducts were identified in human liver microsomes fortified with GSH. The formation of the GSH adducts was hypothesized to be through the oxidation of electron-rich 1,4-benzenediamine to a 1,4-diiminoquinone intermediate, which is highly reactive and can be trapped by GSH to form stable metabolites. The current study provides an overview of the metabolic profiles of ribociclib in vitro, which will be of great help in understanding the efficacy and toxicity of this drug.     

Characterization and in vitro phase I microsomal metabolism of designer benzodiazepines — an update comprising adinazolam,cloniprazepam, fonazepam, 3‐hydroxyphenazepam,metizolam and nitrazolam

Designer benzodiazepines represent an emerging class of new psychoactive substances. While other classes of new psychoactive substances such as cannabinoid receptor agonists and designer stimulants are mainly consumed for hedonistic reasons, designer benzodiazepines may also be consumed as ‘self‐medication’ by persons suffering from anxiety or other psychiatric disorders or as stand‐by ‘antidote’ by users of stimulant and hallucinogenic drugs. In the present study, five benzodiazepines (adinazolam, cloniprazepam, fonazepam, 3‐hydroxyphenazepam and nitrazolam) and one thienodiazepine (metizolam) offered as ‘research chemicals’ on the Internet were characterized and their main in vitro phase I metabolites tentatively identified after incubation with pooled human liver microsomes. For all compounds, the structural formula declared by the vendor was confirmed by nuclear magnetic resonance spectroscopy, gas chromatography–mass spectrometry (MS), liquid chromatography MS/MS and liquid chromatography quadrupole time‐of‐flight MS analysis. The detected in vitro phase I metabolites of adinazolam were N‐desmethyladinazolam and N‐didesmethyladinazolam. Metizolam showed a similar metabolism to other thienodiazepines comprising monohydroxylations and dihydroxylation. Cloniprazepam was metabolized to numerous metabolites with the main metabolic steps being N‐dealkylation, hydroxylation and reduction of the nitro function. It has to be noted that clonazepam is a metabolite of cloniprazepam, which may lead to difficulties when interpreting analytical findings. Nitrazolam and fonazepam both underwent monohydroxylation and reduction of the nitro function. In the case of 3‐OH‐phenazepam, no in vitro phase I metabolites were detected. Formation of licensed benzodiazepines (clonazepam after uptake of cloniprazepam) and the sale of metabolites of prescribed benzodiazepines (fonazepam, identical to norflunitrazepam, and 3‐hydroxyphenazepam) present the risk of incorrect interpretation of analytical findings. Copyright © 2016 John Wiley & Sons, Ltd.     

In vitro metabolism of corydaline in human liver microsomes and hepatocytes using liquid chromatography-ion trap mass spectrometry

Corydaline is a pharmacologically active isoquinoline alkaloid isolated from Corydalis tubers. It exhibits the antiacetylcholinesterase, antiallergic, antinociceptive, and gastric emptying activities. The purposes of this study were to establish in vitro metabolic pathways of corydaline in human liver microsomes and hepatocytes by identification of their metabolites using liquid chromatography-ion trap mass spectrometry. Human liver microsomal incubation of corydaline in the presence of an NADPH-generating system resulted in the formation of nine metabolites, namely, four O-desmethylcorydaline [M1 (yuanhunine), M2 (9-O-desmethylcorydaline), M3 (isocorybulbine), and M4 (corybulbine)], three di-O-desmethylcorydaline [M5 (9,10-di-O-desmethylcorydaline), M6 (2,10-di-O-desmethylcorydaline), and M7 (3,10-di-O-desmethylcorydaline)], M8 (hydroxyyuanhunine), and M9 (hydroxycorydaline). Incubation of corydaline in human hepatocytes produced four metabolites including M1, M5, M6, and M9. O-Demethylation and hydroxylation were the major metabolic pathways for the metabolism of corydaline in human liver microsomes and hepatocytes.     

Preparative Chromatographic Isolation of Caulophine from Radix caulophylli and its Metabolism In Vivo and In Vitro

Caulophine, as a novel alkaloid, was found in Radix caulophylli and has anti-myocardial ischemia activities. To conduct product development research on Radix caulophylli and caulophine, a preparative high-performance liquid chromatography method for the preparation of caulophine was investigated. Preparative HPLC was optimized to allow fraction I to be separated first and the caulophine was isolated following the second round of preparative HPLC. The purity of caulophine was >98%, which was assessed using analytical HPLC. Then, a highly sensitive and specific liquid chromatography-mass spectrometric method for the excretion and metabolism of caulophine in vivo was investigated. The metabolism to caulophine glucuronide conjugate was studied in rat liver microsomes or dog liver microsomes in vitro systems. Biosamples were pretreated by solid phase extraction (SPE) and analyzed by LC-MS with electrospray ionization (ESI) interface. Method validation results showed within-day and between-day precision was 1.14–6.21% and 5.45–9.78%, respectively, and average recoveries for all matrices were greater than 80%. The limits of detection for this method were determined to be up to 1 ng · mL^?1 of caulophine. Excretion of caulophine in rat results indicated that the total cumulative excretion of caulophine was high, with greater than 50% of the treatment dose being excreted. Two metabolites including glucuronide conjugate and N-oxide of caulophine were found in rat urine and feces by LC-MS. Moreover, the same caulophine glucuronide conjugate was observed in rat liver microsomes system.