RESUMEN
Coordinated cell function requires a variety of subcellular organelles to exchange proteins and lipids across physical contacts that are also referred to as membrane contact sites. Such organelle-to-organelle contacts also evoke interest because they can appear in response to metabolic changes, immune activation, and possibly other stimuli. The microscopic size and complex, crowded geometry of these contacts, however, makes them difficult to visualize, manipulate, and understand inside cells. To address this shortcoming, we deposited endoplasmic reticulum (ER)-enriched microsomes purified from rat liver or from cultured cells on a coverslip in the form of a proteinaceous planar membrane. We visualized real-time lipid and protein exchange across contacts that form between this ER-mimicking membrane and lipid droplets (LDs) purified from the liver of rat. The high-throughput imaging possible in this geometry reveals that in vitro LD-ER contacts increase dramatically when the metabolic state is changed by feeding the animal and also when the immune system is activated. Contact formation in both cases requires Rab18 GTPase and phosphatidic acid, thus revealing common molecular targets operative in two very different biological pathways. An optical trap is used to demonstrate physical tethering of individual LDs to the ER-mimicking membrane and to estimate the strength of this tether. These methodologies can potentially be adapted to understand and target abnormal contact formation between different cellular organelles in the context of neurological and metabolic disorders or pathogen infection.
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Retículo Endoplásmico , Gotas Lipídicas , Animales , Células Cultivadas , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Gotas Lipídicas/inmunología , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos , Microsomas Hepáticos/química , Membranas Mitocondriales/metabolismo , Ácidos Fosfatidicos/metabolismo , Ratas , Proteínas de Unión al GTP rab/metabolismoRESUMEN
The identification of novel 4-hydroxy-2-quinolone-3-carboxamide antibacterials with improved properties is of great value for the control of antibiotic resistance. In this study, a series of N-heteroaryl-substituted 4-hydroxy-2-quinolone-3-carboxamides were developed using the bioisosteric replacement strategy. As a result of our research, we discovered the two most potent GyrB inhibitors (WBX7 and WBX18), with IC50 values of 0.816 µM and 0.137 µM, respectively. Additional antibacterial activity screening indicated that WBX18 possesses the best antibacterial activity against MRSA, VISA, and VRE strains, with MIC values rangingbetween0.5and 2 µg/mL, which was 2 to over 32 times more potent than that of vancomycin. In vitro safety and metabolic stability, as well as in vivo pharmacokinetics assessments revealed that WBX18 is non-toxic to HUVEC and HepG2, metabolically stable in plasma and liver microsomes (mouse), and displays favorable in vivo pharmacokinetic properties. Finally, docking studies combined with molecular dynamic simulation showed that WBX18 could stably fit in the active site cavity of GyrB.
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Antibacterianos , Girasa de ADN , Pruebas de Sensibilidad Microbiana , Inhibidores de Topoisomerasa II , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Humanos , Girasa de ADN/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/síntesis química , Relación Estructura-Actividad , Animales , Estructura Molecular , Relación Dosis-Respuesta a Droga , Ratones , Células Hep G2 , Simulación del Acoplamiento Molecular , Microsomas Hepáticos/metabolismo , Microsomas Hepáticos/químicaRESUMEN
Zotizalkib (ZTK, TPX-0131) is a fourth-generation highly effective inhibitor of wild-type anaplastic lymphoma kinase (ALK) and ALK-resistant mutations that can penetrate the central nervous system. It exhibited greater potency compared to all five officially approved ALK inhibitors. The aim of this study was to develop a rapid, accurate, eco-friendly, and highly sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for measuring the concentration of ZTK in human liver microsomes (HLMs). The validation aspects of the current UHPLC-MS/MS methodology in the HLMs were conducted in accordance with the bioanalytical method validation standards specified by the US Food and Drug Administration. ZTK and encorafenib were separated using an Agilent C8 column (Eclipse Plus) and an isocratic mobile phase. The calibration curve for the developed ZTK exhibited a linear relationship within the concentration range of 1-3000 ng/mL. The results from the Analytical Green-ness Metric Approach program (0.76) suggested that the created method demonstrated a significant degree of environmental sustainability. The in vitro half-life (t1/2) and intrinsic clearance (Clint) of ZTK were determined to be 15.79 min and 51.35 mL/min/kg, respectively that suggests the ZTK exhibits characteristics similar to those of a medication with a high extraction ratio. These approaches are crucial for the progress of novel pharmaceutical development, especially in improving metabolic stability.
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Microsomas Hepáticos , Espectrometría de Masas en Tándem , Humanos , Microsomas Hepáticos/metabolismo , Microsomas Hepáticos/química , Cromatografía Líquida de Alta Presión , Estructura MolecularRESUMEN
Palbociclib (Ibrance; Pfizer) was approved for the management of metastatic breast cancer characterized by hormone receptor-positive/human epidermal growth factor receptor 2 negative status. The objective of this study was to create a fast, precise, environmentally friendly, and highly sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry approach for quantifying palbociclib (PAB) in human liver microsomes with the application for assessing metabolic stability. The validation features were performed in agreement with the bioanalytical method validation standards outlined by the US Food and Drug Administration. The StarDrop software (WhichP450 and DEREK modules) was used in screening the metabolic lability and structural alerts of PAB. The separation of PAB and encorafenib (as an internal standard) was achieved on a C8 column, employing an isocratic mobile phase. The inter-day and intra-day accuracy and precision ranged from -6.00% to 4.64% and from -2.33% to 3.13%, respectively. The constructed calibration curve displayed a linearity in the range of 1-3000 ng/mL. The sensitivity of the established approach was proven by the lower limit of quantification of 0.73 ng/mL. The Analytical GREEness calculator results revealed the high level of greenness of the developed method. The PAB's metabolic stability (t1/2 of 18.5 min and a moderate clearance (Clint) of 44.8 mL/min/kg) suggests a high extraction ratio medication that matched the WhichP450 software results.
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Microsomas Hepáticos , Piperazinas , Piridinas , Espectrometría de Masas en Tándem , Humanos , Piperazinas/metabolismo , Piperazinas/análisis , Piperazinas/química , Microsomas Hepáticos/metabolismo , Microsomas Hepáticos/química , Piridinas/metabolismo , Piridinas/química , Piridinas/análisis , Cromatografía Líquida de Alta Presión , Simulación por Computador , Antineoplásicos/análisis , Antineoplásicos/metabolismo , Antineoplásicos/químicaRESUMEN
Preparation of drug metabolites at the milligram scale is essential for determining the structure and toxicity of drug metabolites. However, their preparation using recombinant proteins and human liver microsomes (HLM) is often difficult because of technical and ethical issues. Reproducing human drug metabolism in food-derived microorganisms may be useful for overcoming these challenges. In this study, we identified an unknown metabolite of the anaesthetic drug lidocaine, which is metabolised by HLM. By screening for lidocaine metabolic activity in five types of foods (blue cheese, shiitake mushroom, natto, yoghurt, and dry yeast), we found that bacteria isolated from natto reproduced the lidocaine metabolic reaction that occurs in HLM. A fraction containing the unknown lidocaine metabolite was prepared through mass cultivation of a Bacillus subtilis standard strain, ethyl acetate extraction, open column chromatography, and HPLC purification. We identified the unknown metabolite as 3-(2,6-dimethylphenyl)-1-ethyl-2-methyl-4-imidazolidinone using NMR. Our results showed that food-derived microorganisms can produce large amounts of human drug metabolites via large-scale cultivation. Additionally, food microorganisms that can reproduce drug metabolism in humans can be used to examine drug metabolites at a low cost and without ethical issues.
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Lidocaína , Microsomas Hepáticos , Humanos , Microsomas Hepáticos/metabolismo , Microsomas Hepáticos/química , Lidocaína/metabolismo , Lidocaína/química , Lidocaína/análisis , Bacillus subtilis/metabolismo , Estructura Molecular , Cromatografía Líquida de Alta PresiónRESUMEN
Multiple Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase inhibitors such as Palmostatin M (Palm M) block N-Ras depalmitoylation, but lack specificity and target several enzymes displaying depalmitoylase activity. Here, we describe ABD957, a potent and selective covalent inhibitor of the ABHD17 family of depalmitoylases, and show that this compound impairs N-Ras depalmitoylation in human acute myeloid leukemia (AML) cells. ABD957 produced partial effects on N-Ras palmitoylation compared with Palm M, but was much more selective across the proteome, reflecting a plasma membrane-delineated action on dynamically palmitoylated proteins. Finally, ABD957 impaired N-Ras signaling and the growth of NRAS-mutant AML cells in a manner that synergizes with MAP kinase kinase (MEK) inhibition. Our findings uncover a surprisingly restricted role for ABHD17 enzymes as regulators of the N-Ras palmitoylation cycle and suggest that ABHD17 inhibitors may have value as targeted therapies for NRAS-mutant cancers.
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Membrana Celular/metabolismo , Hidrolasas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Proteínas ras/metabolismo , Proliferación Celular , Células Cultivadas , Humanos , Leucemia Mieloide Aguda/patología , Leucemia Promielocítica Aguda/patología , Lipoilación , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura MolecularRESUMEN
BACKGROUND: The continuous introduction of new synthetic cannabinoid (SC) subtypes and analogues remains a major problem worldwide. Recently, a new "OXIZID" generation of SCs surfaced in seized materials across various countries. Hence, there is an impetus to identify urinary biomarkers of the OXIZIDs to detect their abuse. METHODS: We adapted our previously reported two-pronged approach to investigate the metabolite profiles and disposition kinetics of 4 OXIZID analogues, namely, BZO-HEXOXIZID (MDA-19), BZO-POXIZID (5C-MDA-19), 5F-BZO-POXIZID (5F-MDA-19), and BZO-CHMOXIZID (CHM-MDA-19). First, bottom-up in vitro incubation experiments comprising metabolite identification, metabolic stability, and reaction phenotyping were performed using human liver microsomes and recombinant human cytochrome P450 enzymes. Second, top-down analysis of authentic urine samples from drug abusers was performed to corroborate the in vitro findings and establish a panel of urinary biomarkers. RESULTS: A total of 42 to 51 metabolites were detected for each OXIZID, and their major metabolic pathways included N-alkyl and phenyl hydroxylation, oxidative defluorination (for 5F-BZO-POXIZID), oxidation to ketone and carboxylate, amide hydrolysis, and N-dealkylation. The OXIZIDs were metabolically unstable, mainly metabolized by cytochromes P3A4, P3A5, and P2C9, and demonstrated mechanism-based inactivation of cytochrome P3A4. Integrating with the results of 4 authentic urine samples, the parent drug and both N-alkyl and phenyl mono-hydroxylated metabolites of each OXIZID were determined as suitable urinary biomarkers. CONCLUSIONS: Drug enforcement agencies worldwide may apply these biomarkers in routine monitoring procedures to identify abusers and counter the escalation of OXIZID abuse.
Asunto(s)
Cannabinoides , Humanos , Cannabinoides/análisis , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Hidroxilación , Oxidación-Reducción , Biomarcadores/metabolismoRESUMEN
RATIONALE: Tirabrutinib is an orally administered Bruton's tyrosine kinase (BTK) inhibitor developed for the treatment of autoimmune disorders and haematological malignancies. The goals of this study were to identify the metabolites of tirabrutinib and to propose the metabolic pathways. METHODS: Tirabrutinib was individually incubated with rat, dog and human liver microsomes at 37°C for 1 h. To trap the potential reactive metabolites, glutathione (GSH) was incorporated into the incubation samples. The incubation samples were analysed using ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry (UHPLC-HRMS). The metabolites were identified and characterized by exact masses, product ions and retention times. RESULTS: A total of 18 metabolites, including four GSH conjugates, were identified and characterized in terms of elemental compositions and product ions. The metabolic pathways of tirabrutinib included amide hydrolysis, O-dealkylation, mono-oxygenation, di-oxygenation and GSH conjugation. Among these metabolites, M10 was the most abundant metabolite. Compared with dog, rat has the closer metabolic profiles to humans, and thus it would be more suitable for toxicity study. CONCLUSIONS: This study provides valuable data regarding the in vitro metabolism of tirabrutinib, which may be helpful for further safety assessment of this drug.
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Imidazoles/química , Imidazoles/metabolismo , Microsomas Hepáticos/metabolismo , Pirimidinas/química , Pirimidinas/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Perros , Humanos , Espectrometría de Masas , Metaboloma , Microsomas Hepáticos/química , RatasRESUMEN
Structure-based design was utilized to optimize 6,6-diaryl substituted dihydropyrone and hydroxylactam to obtain inhibitors of lactate dehydrogenase (LDH) with low nanomolar biochemical and single-digit micromolar cellular potencies. Surprisingly the replacement of a phenyl with a pyridyl moiety in the chemical structure revealed a new binding mode for the inhibitors with subtle conformational change of the LDHA active site. This led to the identification of a potent, cell-active hydroxylactam inhibitor exhibiting an in vivo pharmacokinetic profile suitable for mouse tumor xenograft study.
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Inhibidores Enzimáticos/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Lactamas/farmacología , Animales , Línea Celular , Perros , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , L-Lactato Deshidrogenasa/metabolismo , Lactamas/química , Ratones , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Hepatitis B virus (HBV) core protein, the building block of the HBV capsid, plays multiple roles in viral replication, and is an attractive target for development of antiviral agents with a new mechanism of action. In addition to the heteroaryldihydropyrimidines (HAPs), sulfamoylbenzamides (SBAs), dibenzothiazepine derivatives (DBTs), and sulfamoylpyrrolamides (SPAs) that inhibit HBV replication by modulation of viral capsid assembly and are currently under clinical trials for the treatment of chronic hepatitis B (CHB), other chemical structures with activity to modulate HBV capsid assembly have also been explored. Here we describe our continued optimization of a benzamide originating from our high throughput screening. A new bicyclic carboxamide lead featuring an electron deficient non-planar core structure was discovered. Evaluations of its ADMET (absorption, distribution, metabolism, excretion and toxicity) and pharmacokinetic (PK) profiles demonstrate improved metabolic stability and good bioavailability.
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Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Quinolinas/farmacología , Animales , Antivirales/síntesis química , Antivirales/química , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Quinolinas/síntesis química , Quinolinas/química , Relación Estructura-Actividad , Proteínas del Núcleo Viral , Replicación Viral/efectos de los fármacosRESUMEN
Phosphatidyl inositol (4,5)-bisphosphate (PI(4,5)P2) plays several key roles in human biology and the lipid kinase that produces PI(4,5)P2, PIP5K, has been hypothesized to provide a potential therapeutic target of interest in the treatment of cancers. To better understand and explore the role of PIP5K in human cancers there remains an urgent need for potent and specific PIP5K inhibitor molecules. Following a high throughput screen of the AstraZeneca collection, a novel, moderately potent and selective inhibitor of PIP5K, 1, was discovered. Detailed exploration of the SAR for this novel scaffold resulted in the considerable optimization of both potency for PIP5K, and selectivity over the closely related kinase PI3Kα, as well as identifying several opportunities for the continued optimization of drug-like properties. As a result, several high quality in vitro tool compounds were identified (8, 20 and 25) that demonstrate the desired biochemical and cellular profiles required to aid better understanding of this complex area of biology.
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Amidas/farmacología , Inhibidores Enzimáticos/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Amidas/química , Amidas/metabolismo , Animales , Células CACO-2 , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ratas , Relación Estructura-ActividadRESUMEN
Esters are one of the major functional groups present in the structures of prodrugs and bioactive compounds. Their presence is often associated with hydrolytic lability. In this paper, we describe a comparative chemical and biological stability of homologous esters and isosteres in base media as well as in rat plasma and rat liver microsomes. Our results provided evidence for the hydrolytic structure lability relationship and demonstrated that the hydrolytic stability in plasma and liver microsome might depend on carboxylesterase activity. Molecular modelling studies were performed in order to understand the experimental data. Taken together, the data could be useful to design bioactive compounds or prodrugs based on the correct choice of the ester subunit, addressing compounds with higher or lower metabolic lability.
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Carboxilesterasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Ésteres/farmacología , Profármacos/farmacología , Animales , Carboxilesterasa/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Ésteres/sangre , Ésteres/química , Hidrólisis , Masculino , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Estructura Molecular , Profármacos/química , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
T-LAK-cell-originated protein kinase (TOPK), a novel member of the mitogen-activated protein kinase family, is considered an effective therapeutic target for skin inflammation. In this study, a series (A - D) of paeonol derivatives was designed and synthesised using a fragment growing approach, and their anti-inflammatory activities against lipopolysaccharide (LPS)-induced nitric oxide production in RAW264.7 cells were tested. Among them, compound B12 yielded the best results (IC50 = 2.14 µM) with low toxicity (IC50 > 50 µM). Preliminary mechanistic studies indicated that this compound could inhibit the TOPK-p38/JNK signalling pathway and phosphorylate downstream related proteins. A murine psoriasis-like skin inflammation model was used to determine its therapeutic effect.
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Acetofenonas/farmacología , Antiinflamatorios no Esteroideos/farmacología , Descubrimiento de Drogas , Inflamación/tratamiento farmacológico , Piel/efectos de los fármacos , Acetofenonas/síntesis química , Acetofenonas/química , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inflamación/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Estructura Molecular , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Transducción de Señal/efectos de los fármacos , Piel/metabolismo , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: (S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-butyl-1H-indazole-3carboxamide (ADB-BUTINACA) is an emerging synthetic cannabinoid that was first identified in Europe in 2019 and entered Singapore's drug scene in January 2020. Due to the unavailable toxicological and metabolic data, there is a need to establish urinary metabolite biomarkers for detection of ADB-BUTINACA consumption and elucidate its biotransformation pathways for rationalizing its toxicological implications. METHODS: We characterized the metabolites of ADB-BUTINACA in human liver microsomes using liquid chromatography Orbitrap mass spectrometry analysis. Enzyme-specific inhibitors and recombinant enzymes were adopted for the reaction phenotyping of ADB-BUTINACA. We further used recombinant enzymes to generate a pool of key metabolites in situ and determined their metabolic stability. By coupling in vitro metabolism and authentic urine analyses, a panel of urinary metabolite biomarkers of ADB-BUTINACA was curated. RESULTS: Fifteen metabolites of ADB-BUTINACA were identified with key biotransformations being hydroxylation, N-debutylation, dihydrodiol formation, and oxidative deamination. Reaction phenotyping established that ADB-BUTINACA was rapidly eliminated via CYP2C19-, CYP3A4-, and CYP3A5-mediated metabolism. Three major monohydroxylated metabolites (M6, M12, and M14) were generated in situ, which demonstrated greater metabolic stability compared to ADB-BUTINACA. Coupling metabolite profiling with urinary analysis, we identified four urinary biomarker metabolites of ADB-BUTINACA: 3 hydroxylated metabolites (M6, M11, and M14) and 1 oxidative deaminated metabolite (M15). CONCLUSIONS: Our data support a panel of four urinary metabolite biomarkers for diagnosing the consumption of ADB-BUTINACA.
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Cannabinoides , Trastornos Relacionados con Sustancias , Biomarcadores/metabolismo , Cannabinoides/análisis , Cromatografía Liquida/métodos , Humanos , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Psicotrópicos/metabolismoRESUMEN
In early drug development, drug-drug interaction risk is routinely assessed using human liver microsomes (HLMs). Nonspecific binding of drugs to HLMs can affect the determination of accurate enzyme parameters (Km, Ki, KI). Previously, we described a novel in vitro model consisting of HLMs bound to magnetizable beads [HLM-magnetizable-beads system (HLM-beads)]. The HLM-beads enable rapid separation of HLMs from incubation media by applying a magnetic field. Here, HLM-beads were further characterized and evaluated as a tool to assess HLM nonspecific binding of small molecules. The free fractions (fu,mic) of 13 compounds (chosen based on their pKa values) were determined using HLM-beads under three HLM concentrations (0.025, 0.50, and 1.0 mg/ml) and compared with those determined by equilibrium dialysis. Most fu,mic values obtained using HLM-beads were within 0.5- to 2-fold of the values determined using equilibrium dialysis. The highest fold difference were observed for high binders itraconazole and BIRT2584 (1.9- to 2.9-fold), as the pronounced adsorption of these compounds to the equilibrium dialysis apparatus interfered with their fu,mic determination. Correlation and linear regression analysis of the fu,mic values generated using HLM-beads and equilibrium dialysis was conducted. Overall, a good correlation of fu,mic values obtained by the two methods were observed, as the r and R2 values from correlational analysis and linear regression analysis were >0.9 and >0.89, respectively. These studies demonstrate that HLM-beads can produce comparable fu,mic values as determined by equilibrium dialysis while reducing the time required for this type of study from hours to only 10 minutes and compound apparatus adsorption. SIGNIFICANCE STATEMENT: This work introduces a new method of rapidly assessing nonspecific microsomal binding using human liver microsomes bound to magnetizable beads.
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Desarrollo de Medicamentos/métodos , Interacciones Farmacológicas , Campos Magnéticos , Tasa de Depuración Metabólica , Microsomas Hepáticos , Unión Proteica , Sitios de Unión , Inhibidores Enzimáticos/farmacocinética , Humanos , Microsomas Hepáticos/química , Microsomas Hepáticos/fisiología , Reproducibilidad de los Resultados , Medición de Riesgo/métodosRESUMEN
Capsaicin, primarily known as the pungent ingredient in hot peppers, is rapidly metabolized in the human body by enzymatic processes altering the pharmacological as well as toxicological properties. Herein, the oxidative transformation of capsaicin was investigated in vitro with electrochemistry as well as human liver microsomal incubations. The reaction mixtures were analyzed with liquid chromatography-mass spectrometry. Structure elucidation involved accurate mass measurements and multistage tandem mass spectrometry experiments. In total, 126 transformation products were detected. Electrochemistry provided evidence for 101 transformation products and the microsomal incubations for 46 species. 21 compounds were observed with both approaches. Identified oxidative pathways likely occurring during the phase I metabolism included dehydrogenation, O-demethylation, and hydroxylation reactions as well as combinations thereof. Furthermore, trapping of reactive intermediates either with glutathione or with electrochemically activated ribonucleosides provided evidence for the possible production of phase II metabolites and covalent adducts with a genetic material. Evidence for the occurrence of some capsaicin metabolites in humans was obtained by urine screening.
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Capsaicina/metabolismo , Técnicas Electroquímicas , Capsaicina/orina , Cromatografía Liquida , Voluntarios Sanos , Humanos , Espectrometría de Masas , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Oxidación-ReducciónRESUMEN
Binimetinib is a selective MEK1/2 inhibitor, which is indicative of melanoma. We aimed to investigate the inhibitory effect of binimetinib on cytochrome P450 using human liver microsomes. Binimetinib was demonstrated to display reversible and time-dependent inhibitory effects on human CYP1A2. Binimetinib can inhibit the activity of phenacetin deethylation with IC50 of 5.6 µM. A 30 min preincubation of binimetinib with NADPH-supplemented human liver microsomes raised a significant left IC50 shift (6.5-fold), from 5.69-0.88 µM. The inactivation parameters Kinact and KI were 0.063 min-1 and 15.47 µM, and the half-life of inactivation was 11 min. Glutathione (GSH) and catalase/superoxide exhibited minor or no protective effect on binimetinib-induced enzyme inactivation. Trapping experiment by GSH induced a detection of GSH adduct, of which the formation was believed to be through the oxidation of electron-rich 1,4-benzenediamine to reactive 1,4-diiminoquinone species. Cytochrome P450 3A4, 2C9, and 2D6 were involved in the bioactivation of binimetinib. In conclusion, binimetinib was proven to display reversible and time-dependent inhibitory effect on CYP1A2, which may have implications for the toxicity of binimetinib.
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Bencimidazoles/farmacología , Citocromo P-450 CYP1A2/metabolismo , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Bencimidazoles/química , Inhibidores Enzimáticos del Citocromo P-450/química , Relación Dosis-Respuesta a Droga , Humanos , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Factores de TiempoRESUMEN
Covalent drugs are newly developed and proved to be successful therapies in past decades. However, the pharmacokinetics (PK) and pharmacodynamic (PD) studies of covalent drugs now ignore the drug and metabolite-protein modification. The low abundance of modified proteins also prevents its investigation. Herein, a simple, selective, and sensitive liquid chromatography-mass spectrometry (LC-MS)/MS quantitative method was established based on the mechanism of a drug and its metabolite-protein adducts using osimertinib as an example. Five metabolites with covalent modification potential were identified. The drug and its metabolite-cysteine adducts released from modified proteins by a mixed hydrolysis method were developed to characterize the level of the modified proteins. This turned the quantitative objects from proteins or peptides to small molecules, which increased the sensitivity and throughput of the quantitative approach. Accumulation of protein adducts formed by osimertinib and its metabolites in target organs was observed in vivo and long-lasting modifications were noted. These results interpreted the long duration of the covalent drugs' effect from the perspective of both parent and the metabolites. In addition, the established method could also be applied in blood testing as noninvasive monitoring. This newly developed approach showed great feasibility for PK and PD studies of covalent drugs.
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Acrilamidas/análisis , Compuestos de Anilina/análisis , Quimotripsina/metabolismo , Cisteína/análisis , Hígado/efectos de los fármacos , Acrilamidas/metabolismo , Acrilamidas/farmacología , Compuestos de Anilina/metabolismo , Compuestos de Anilina/farmacología , Animales , Bovinos , Cromatografía Liquida , Cisteína/metabolismo , Cisteína/farmacología , Femenino , Humanos , Hidrólisis , Hígado/metabolismo , Masculino , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en TándemRESUMEN
RATIONALE: Daporinad is a novel and potent inhibitor of nicotinamide phosphoribosyl transferase with potential antineoplastic and antiangiogenic activities. We aimed to explore the metabolites of daporinad generated from liver microsomes and to propose metabolic pathways. METHODS: The metabolites were generated by individually incubating daporinad (10 µM) with liver microsomes at 37°C for 60 min. The metabolites were identified by ultra-high-performance liquid chromatography/quadrupole-orbitrap mass spectrometry (UPLC/Q-Orbitrap-MS) using electrospray ionization in positive ion mode. They were deduced by accurate MS and MS/MS data. RESULTS: In total, 16 metabolites were found and their identities were characterized. In rat, dog and human, they were minor; in monkey, M11 was the most abundant. Daporinad was metabolized mainly through N-dealkylation, amide hydrolysis, hydrogenation, oxygenation and dehydrogenation. There was no human-specific metabolite. CONCLUSIONS: The current study provided an overview of the metabolism of daporinad, which is helpful in predicting in vivo metabolites and in selecting animal species for toxicology studies.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Microsomas Hepáticos/química , Animales , Perros , Haplorrinos , Humanos , Ratas , Especificidad de la EspecieRESUMEN
RATIONALE: Methysticin is a naturally occurring ingredient isolated from Piper methysticum Forst. The metabolic profile of methysticin is unknown. The goal of this study was to elucidate the metabolism of methysticin using rat and human liver microsomes and hepatocytes. METHODS: The incubation samples were analyzed using ultra-high-performance liquid chromatography coupled with quadrupole/orbitrap high-resolution mass spectrometry (UHPLC-HRMS). The structures of the metabolites were characterized based on the elemental composition, exact mass, and product ions. RESULTS: A total of 10 metabolites were detected and identified. Among these metabolites, M4 (ring opening of 1,3-benzodioxole) was the predominant metabolite in rat and human liver microsomes. M4 and its glucuronide conjugate (M2) were the major metabolites in rat and human hepatocytes. The metabolic pathways of methysticin are summarized as follows: (a) oxidative ring opening of 1,3-benzodioxole forms the catechol derivative (M4), which subsequently undergoes glucuronidation (M1 and M2), methylation (M8), and sulfation (M7). (b) Demethylation to yield desmethyl methysticin (M6), followed by glucuronidation (M3 and M5). (c) Hydroxylation (M9 and M10). CONCLUSIONS: For the first time, this study provides new information on the in vitro metabolic profiles of methysticin, which facilitates an understanding of the disposition of this bioactive ingredient.