RESUMO
High-throughput mass spectrometry (MS) has witnessed rapid advancements and has found extensive applications across various disciplines. It enables the fast and accurate analysis of large sample sets, delivering a 10-fold or greater enhancement in analytical throughput when compared to conventional LC-MS methods. However, the signal duration in these high-throughput MS technologies is typically confined to a narrow range, presenting challenges for workflows demanding prolonged signal durations. In this study, we introduce a method that enables precise modulation of the signal duration on an acoustic ejection mass spectrometry (AEMS) system while ensuring high signal reproducibility. This flexibility allows for simultaneous and precise analysis of a significantly greater number of MS/MS transitions in high-throughput MS environments. Additionally, it offers a unique approach for parameter optimization and method development with minimal sample volume requirements. This advancement enhances the efficiency of MS-based analyses across diverse applications and facilitates broader utilization of MS technologies in high-throughput settings, including data-dependent acquisition (DDA) and data-independent acquisition (DIA).
RESUMO
The gut microbiota possesses diverse metabolic activities, but its contribution toward heterogeneous toxicological responses is poorly understood. In this study, we investigated the role of the liver-gut microbiota axis in underpinning the hepatotoxicity of tacrine. We employed an integrated strategy combining pharmacokinetics, toxicology, metabonomics, genomics, and metagenomics to elucidate and validate the mechanism of tacrine-induced hepatotoxicity in Lister hooded rats. Pharmacokinetic studies in rats demonstrated 3.3-fold higher systemic exposure to tacrine in strong responders that experienced transaminitis, revealing enhanced enterohepatic recycling of deglucuronidated tacrine in this subgroup, not attributable to variation in hepatic disposition gene expression. Metabonomic studies implicated variations in gut microbial activities that mapped onto tacrine-induced transaminitis. Metagenomics delineated greater deglucuronidation capabilities in strong responders, based on differential gut microbial composition (e.g., Lactobacillus, Bacteroides, and Enterobacteriaceae) and approximately 9% higher ß-glucuronidase gene abundance compared with nonresponders. In the validation study, coadministration with oral ß-glucuronidase derived from Escherichia coli and pretreatment with vancomycin and imipenem significantly modulated the susceptibility to tacrine-induced transaminitis in vivo. CONCLUSION: This study establishes pertinent gut microbial influences in modifying the hepatotoxicity of tacrine, providing insights for personalized medicine initiatives. (Hepatology 2018;67:282-295).
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Tacrina/toxicidade , Animais , Biópsia por Agulha , Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Testes de Função Hepática , Masculino , Distribuição Aleatória , Ratos , Ratos Endogâmicos , Valores de Referência , Índice de Gravidade de Doença , Tacrina/farmacocinética , Tacrina/farmacologiaRESUMO
The drug discovery process increasingly relies on high-throughput sample analysis to accelerate the identification of viable drug candidates. Recently, chromatographic-free high-throughput mass spectrometry (HT-MS) technologies have emerged, significantly increasing sample readout speed and enabling the analysis of large sample sets. These HT-MS platforms continuously acquire data from various samples into a single data file, presenting challenges in applying distinctive data acquisition methods to specific samples. This study introduces a novel approach that integrates real-time sample loading status to activate sample-specific MS/MS data acquisition methods on the high-throughput acoustic ejection mass spectrometry platform. Effective method switching and high signal reproducibility were demonstrated across different data acquisition window durations in multiple reaction monitoring (MRM), high-resolution MRM (MRMHR), and information-dependent acquisition modes. This advancement provides a user-friendly and robust solution to the method-setting challenges of HT-MS, expanding the implementation of HT-MS platforms in drug discovery and other high-throughput analytical applications.
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3-Nitropropionic acid (3-NP), a potent irreversible inhibitor of mitochondrial complex II enzyme, leads to mitochondrial dysfunction and oxidative stress in Huntington's disease (HD) rat model. In this study, biochemical assays were used to demonstrate the presence of oxidative stress and mitochondrial dysfunction in 3-NP early stage HD rat models. Gas chromatography time-of-flight mass spectrometry (GC/TOFMS) was applied to analyze metabolites in brain and plasma of 3-NP-treated and vehicle-dosed rats. The orthogonal partial least-squares discriminant analysis (OPLS-DA) model generated using brain metabolic profiles robustly differentiated the 3-NP early stage HD rat model from the control. Metabonomic characterization of the 3-NP HD rat model facilitated the detection of biomarkers that define the physiopathological phenotype of early stage HD and elucidated the treatment effect of galantamine. Brain marker metabolites that were identified based on the OPLS-DA model were associated with altered glutathione metabolism, oxidative stress, and impaired energy metabolism. The treatment effect of galantamine in early stage HD could not be concluded mechanistically using the brain metabotype. Our study confirmed that GC/TOFMS is a strategic and complementary platform for the metabonomic characterization of 3-NP induced neurotoxicity in the early stage HD rat model.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Doença de Huntington/induzido quimicamente , Doença de Huntington/metabolismo , Metaboloma , Metabolômica/métodos , Nitrocompostos/toxicidade , Propionatos/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiologia , Modelos Animais de Doenças , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Galantamina/farmacologia , Doença de Huntington/patologia , Doença de Huntington/fisiopatologia , Mitocôndrias/metabolismo , Nootrópicos/farmacologia , Estresse Oxidativo , Ratos , Ratos Sprague-DawleyRESUMO
Physiological alterations that may change pharmacological response accompany aging. Pharmacokinetic/pharmacodynamic properties of cholinesterase inhibitors (ChEIs) used in the treatment of Alzheimer's disease, donepezil, tacrine, and galantamine, were investigated in an aged Lister hooded rat model. Intravenous and oral 6-h blood sampling profiles in old (30 months old) and young (7 months old) rats revealed pharmacokinetic changes similar to those in humans with an approximately 40% increase in C(max) of galantamine and prolonged t(1/2) (1.4-fold) and mean residence time (1.5-fold) of donepezil. Tacrine disposition was maintained with age, and area under the concentration-time curve and clearance in old rats were similar to those in young rats for all drugs tested as was bioavailability. Old rats showed a trend of increased pharmacodynamic sensitivity (<20%) to ChEIs in cholinesterase activity assays, which was attributed to pharmacokinetic effects because a trend of higher blood and brain concentrations was seen in the old rats although brain/blood ratios remained unaffected. Enhanced cholinergic-mediated behaviors such as tremor, hypothermia, salivation, and lacrimation were also observed in the old rats, which could not be accounted for by a similar magnitude of change in pharmacokinetics. A decrease in expression of muscarinic acetylcholine receptor subtype 2 detected in old rat brains was postulated to play a role. Greater age effects in both pharmacokinetics and pharmacodynamics of donepezil and tacrine were seen in previous studies with Fischer 344 rats, indicating a potential risk in overreliance on this rat strain for aging studies.
Assuntos
Envelhecimento , Inibidores da Colinesterase/farmacocinética , Galantamina/farmacocinética , Indanos/farmacocinética , Piperidinas/farmacocinética , Tacrina/farmacocinética , Animais , Disponibilidade Biológica , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Inibidores da Colinesterase/efeitos adversos , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Colinesterases/sangue , Colinesterases/metabolismo , Donepezila , Relação Dose-Resposta a Droga , Galantamina/efeitos adversos , Galantamina/metabolismo , Galantamina/farmacologia , Meia-Vida , Hipotermia/induzido quimicamente , Indanos/efeitos adversos , Indanos/metabolismo , Indanos/farmacologia , Masculino , Taxa de Depuração Metabólica , Piperidinas/efeitos adversos , Piperidinas/metabolismo , Piperidinas/farmacologia , Distribuição Aleatória , Ratos , Ratos Endogâmicos , Receptor Muscarínico M2/metabolismo , Salivação/efeitos dos fármacos , Tacrina/efeitos adversos , Tacrina/metabolismo , Tacrina/farmacologia , Lágrimas/efeitos dos fármacos , Lágrimas/metabolismo , Tremor/induzido quimicamenteRESUMO
In this study, the correlation between the metabolic profiles of rats undergoing cognition enhancement drug therapy and their associated cognitive behavioral outcomes were investigated. Male Lister Hooded rats were administered either with donepezil, galantamine, or vehicle and subjected to Atlantis watermaze training and novel object recognition tests. An UPLC/MS/MS method was developed to profile 21 neurologically related metabolites in the rat brains. Pharmacologically induced behavioral changes were compared subsequently with the metabolic fluctuations of neurologically related metabolites from multiple neurotransmitter pathways using multivariate and univariate statistical analyses. Significant improvements in cognitive behavioral outcomes were demonstrated in the rats administered with donepezil and galantamine using both AWM training (P < 0.05) and NOR (P < 0.05) tests as compared to those dosed with the vehicle. This corroborated with the significant elevation of eight prominent biomarkers after the cognitive enhancement therapy. An orthogonal partial least-squares discriminant analysis model generated using only the 8 metabolites identified as discriminating the drug-dosed rats from the vehicle-dosed rats gave a Q(2) = 0.566, receiver operator characteristic (ROC) AUC = 1.000, using 7-fold cross validation. Our study suggests that metabolic profiling of rat brain is a potential complementary strategy to the cognitive behavioral tasks for characterizing neurobiological responses to cognition enhancement drug testing.
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Comportamento Animal , Química Encefálica , Cognição , Metaboloma/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Química Encefálica/fisiologia , Cognição/efeitos dos fármacos , Donepezila , Galantamina/farmacologia , Indanos/farmacologia , Masculino , Piperidinas/farmacologia , Ratos , Ratos Endogâmicos , Espectrometria de Massas em TandemRESUMO
AIMS: The NADPH oxidase (NOX) family of enzymes catalyzes the formation of reactive oxygen species (ROS). NOX enzymes not only have a key role in a variety of physiological processes but also contribute to oxidative stress in certain disease states. To date, while numerous small molecule inhibitors have been reported (in particular for NOX2), none have demonstrated inhibitory activity in vivo. As such, there is a need for the identification of improved NOX inhibitors to enable further evaluation of the biological functions of NOX enzymes in vivo as well as the therapeutic potential of NOX inhibition. In this study, both the in vitro and in vivo pharmacological profiles of GSK2795039, a novel NOX2 inhibitor, were characterized in comparison with other published NOX inhibitors. RESULTS: GSK2795039 inhibited both the formation of ROS and the utilization of the enzyme substrates, NADPH and oxygen, in a variety of semirecombinant cell-free and cell-based NOX2 assays. It inhibited NOX2 in an NADPH competitive manner and was selective over other NOX isoforms, xanthine oxidase, and endothelial nitric oxide synthase enzymes. Following systemic administration in mice, GSK2795039 abolished the production of ROS by activated NOX2 enzyme in a paw inflammation model. Furthermore, GSK2795039 showed activity in a murine model of acute pancreatitis, reducing the levels of serum amylase triggered by systemic injection of cerulein. INNOVATION AND CONCLUSIONS: GSK2795039 is a novel NOX2 inhibitor that is the first small molecule to demonstrate inhibition of the NOX2 enzyme in vivo.
Assuntos
Aminopiridinas/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Sulfonamidas/farmacologia , Aminopiridinas/química , Animais , Células Cultivadas , Inibidores Enzimáticos/uso terapêutico , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos Endogâmicos C57BL , NADPH Oxidase 2 , NADPH Oxidases/antagonistas & inibidores , Pancreatite/tratamento farmacológico , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Sulfonamidas/químicaRESUMO
The 5-HT(6) receptor subtype is predominantly expressed in the central nervous system, and preclinical evidence suggests that it plays a critical role in the regulation of molecular pathways underlying cognitive function. Patients with schizophrenia show cognitive impairment as a fundamental symptom, and it is proposed that the procognitive properties of some antipsychotics such as olanzapine and clozapine would be, in part, due to the central blockade of 5-HT(6) receptors. In this study, we characterized the brain 5-HT(6) receptor occupancy of olanzapine, clozapine and chlorpromazine in relation to their pharmacokinetic profiles using in vivo [(3)H]GSK215083 binding assay in rat brain. Oral administration of olanzapine (3mg/kg), clozapine (30mg/kg) and chlorpromazine (30mg/kg) produced significant 5-HT(6) receptor occupancy in the brain, inhibiting radioligand binding by 88, 97 and 81%, respectively. The blood concentrations required to achieve significant occupancy were clinically achievable (9.6, 26.9 and 98.6nM for olanzapine, clozapine and chlorpromazine, respectively). This data provides preclinical evidence to support the hypothesis that brain 5-HT(6) antagonism contributes to the procognitive properties of antipsychotic drugs such as olanzapine and clozapine.
Assuntos
Antipsicóticos/farmacologia , Encéfalo/metabolismo , Receptores de Serotonina/metabolismo , Animais , Antipsicóticos/farmacocinética , Benzodiazepinas/farmacocinética , Benzodiazepinas/farmacologia , Encéfalo/efeitos dos fármacos , Clorpromazina/farmacocinética , Clorpromazina/farmacologia , Clozapina/farmacocinética , Clozapina/farmacologia , Relação Dose-Resposta a Droga , Masculino , Olanzapina , Piperazinas/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores de Serotonina/efeitos dos fármacos , Sulfonamidas/farmacologiaRESUMO
Oral administration of drugs to laboratory rodents typically is achieved by using the gavage technique. Although highly effective, this method occasionally can cause esophageal injury as well as restraint-associated distress, particularly with repeated use. The aim of this study was to assess an alternative oral dosing method that could reduce the distress and morbidity associated with standard gavage techniques. The palatability and pharmacokinetic profile of 2 medicines approved for the treatment of Alzheimer disease, donepezil and galantamine, were investigated in male Lister hooded rats by using a syringe-feeding method and compared with results from traditional gavage administration. In addition, the stimulant nicotine was tested by using the syringe-feeding method in a separate series of experiments. Animals reliably learned to drink voluntarily from the syringe, and latency to drink decreased rapidly. The addition of donepezil, galantamine, or nicotine to sucrose had no apparent effect on the palatability of the solution, although nicotine produced aversive effects that inhibited subsequent voluntary intake. Oral bioavailability was improved by using syringe feeding with donepezil but not galantamine. Both drugs improved cognitive performance in the novel object recognition test, with similar behavioral profiles between the 2 methods of administration. Our results suggest that the syringe-feeding technique is an effective alternative oral dosing method in rats.