RESUMEN
Selenocompounds (SeCs) are promising therapeutic agents for a wide range of diseases including cancer. The treatment results are heterogeneous and dependent on both the chemical species and the concentration of SeCs. Moreover, the mechanisms of action are poorly revealed, which most probably is due to the detection methods where the quantification is based on the total selenium as an element. To understand the mechanisms underlying the heterogeneous cytotoxicity of SeCs and to determine their pharmacokinetics, we investigated selenium speciation of six SeCs representing different categories using liquid chromatography-mass spectrometry (LC-MS) and X-ray absorption spectroscopy (XAS) and the cytotoxicity using leukemic cells. SeCs cytotoxicity was correlated with albumin binding degree as revealed by LC-MS and XAS. Further analysis corroborated the covalent binding between selenol intermediates of SeCs and albumin thiols. On basis of the Se-S model, pharmacokinetic properties of four SeCs were for the first time profiled. In summary, we have shown that cytotoxic SeCs could spontaneously transform into selenol intermediates that immediately react with albumin thiols through Se-S bond. The heterogeneous albumin binding degree may predict the variability in cytotoxicity. The present knowledge will also guide further kinetic and mechanistic investigations in both experimental and clinical settings.
Asunto(s)
Albúminas/química , Selenio/metabolismo , Albúmina Sérica Humana/química , Albúmina Sérica Humana/metabolismo , Animales , Cistina/análogos & derivados , Cistina/farmacocinética , Cistina/farmacología , Humanos , Masculino , Espectrometría de Masas/métodos , Ratones , Ratones Endogámicos C57BL , Compuestos de Organoselenio/farmacocinética , Compuestos de Organoselenio/farmacología , Unión Proteica/efectos de los fármacos , Selenometionina/farmacocinética , Selenometionina/farmacología , Espectroscopía de Absorción de Rayos X/métodosRESUMEN
N-acetylcysteine amide (NACA) is the amide derivative of N-acetylcysteine (NAC) that is rapidly converted to NAC after systemic administration. It has emerged as a promising thiol antioxidant for multiple indications; however, the pharmacokinetic property is yet unclear due to lack of an accurate quantification method. The present investigation aimed to develop an analytical method for simultaneous quantification of NACA and NAC in plasma. A new reagent (2-(methylsulfonyl)-5-phenyl-1,3,4-oxadiazole, MPOZ) was introduced for thiol stabilization during sample processing and storage. Further, we utilized tris (2-carboxyethyl) phosphine (TCEP) to reduce the oxidized forms of NACA and NAC. After derivatization, NACA-MPOZ and NAC-MPOZ were quantified using liquid chromatography-mass spectrometry (LC-MS). The new method was validated and found to have high specificity, linearity, accuracy, precision, and recovery for the quantification of NACA and NAC in plasma. Furthermore, the formed derivatives of NACA and NAC were stable for 48 h under different conditions. The method was utilized in pharmacokinetic study which showed that the bioavailability of NACA is significantly higher than NAC (67% and 15%, respectively). The pharmacokinetic of NACA obeyed a two-compartment open model. The glutathione (GSH)-replenishing capacity was found to be three to four-fold higher after the administration of NACA compared to that observed after the administration of NAC. In conclusion, the present method is simple, robust and reproducible, and can be utilized in both experimental and clinical studies. NACA might be considered as a prodrug for NAC. Furthermore, this is the first report describing the pharmacokinetics and bioavailability of NACA in mouse.
Asunto(s)
Acetilcisteína/análogos & derivados , Profármacos/farmacocinética , Acetilcisteína/sangre , Acetilcisteína/farmacocinética , Animales , Disponibilidad Biológica , Femenino , Glutatión/metabolismo , Humanos , Ratones Endogámicos BALB C , Compuestos de Sulfhidrilo/químicaRESUMEN
Aroylated phenylenediamines (APDs) are novel inducers of innate immunity enhancing cathelicidin gene expression in human bronchial epithelial cell lines. Here we present two newly developed APDs and aimed at defining the response and signaling pathways for these compounds with reference to innate immunity and antimicrobial peptide (AMP) expression. Induction was initially defined with respect to dose and time and compared with the APD Entinostat (MS-275). The induction applies to several innate immunity effectors, indicating that APDs trigger a broad spectrum of antimicrobial responses. The bactericidal effect was shown in an infection model against Pseudomonas aeruginosa by estimating bacteria entering cells. Treatment with a selected APD counteracted Pseudomonas mediated disruption of epithelial integrity. This double action by inducing AMPs and enhancing epithelial integrity for one APD compound is unique and taken as a positive indication for host directed therapy (HDT). The APD effects are mediated through Signal transducer and activator of transcription 3 (STAT3) activation. Utilization of induced innate immunity to fight infections can reduce antibiotic usage, might be effective against multidrug resistant bacteria and is in line with improved stewardship in healthcare.
Asunto(s)
Antibacterianos/farmacología , Bronquios/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fenilendiaminas/farmacología , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/metabolismo , Benzamidas/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Inmunidad Innata/efectos de los fármacos , Interleucina-8/genética , Interleucina-8/metabolismo , Infecciones por Pseudomonas/microbiología , Piridinas/farmacología , Factor de Transcripción STAT3/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , CatelicidinasRESUMEN
p-Xyleneselenocyanate (p-XSC) is one of the most investigated selenium compounds in cancer-prevention and -therapy. Despite the potent anticancer property, there is still no proper method to perform the quantitative analysis of p-XSC in plasma. In this investigation, we aimed at developing a method based on liquid chromatography-mass spectrometry (LC-MS) for the measurement of p-XSC in plasma. Direct deproteinization was first used to extract parent p-XSC from plasma, but failed to achieve high recovery rate (<2%) due to formation of selenium-sulfur bond between p-XSC and plasma protein. To overcome this problem, we modified the extraction method to three steps: (1) break the selenium-sulfur bond by tris(2-carboxyethyl)phosphine; (2) stabilize the newly formed intermediate selenol by N-ethylmaleimide; (3) deproteinization. This three-step method efficiently recovered bound p-XSC by more than 75%. In in vivo study, p-XSC was injected intravenously into mice and plasma was collected for LC-MS analysis. Consistently, p-XSC was undetectable in its parent form, whereas the bound form was readily quantified, employing the modified extraction method. In summary, we describe a novel, robust, and sensitive method for quantification of p-XSC in plasma. The present method will enable pharmacokinetic and pharmacodynamic studies of p-XSC in both clinical and preclinical settings.
RESUMEN
Bacterial resistance against classical antibiotics is a growing problem and the development of new antibiotics is limited. Thus, novel alternatives to antibiotics are warranted. Antimicrobial peptides (AMPs) are effector molecules of innate immunity that can be induced by several compounds, including vitamin D and phenyl-butyrate (PBA). Utilizing a luciferase based assay, we recently discovered that the histone deacetylase inhibitor Entinostat is a potent inducer of the CAMP gene encoding the human cathelicidin LL-37. Here we investigate a mechanism for the induction and also find that Entinostat up-regulates human ß-defensin 1. Analysis of the CAMP promoter sequence revealed binding sites for the transcription factors STAT3 and HIF-1α. By using short hairpin RNA and selective inhibitors, we found that both transcription factors are involved in Entinostat-induced expression of LL-37. However, only HIF-1α was found to be recruited to the CAMP promoter, suggesting that Entinostat activates STAT3, which promotes transcription of CAMP by increasing the expression of HIF-1α. Finally, we provide in vivo relevance to our findings by showing that Entinostat-elicited LL-37 expression was impaired in macrophages from a patient with a STAT3-mutation. Combined, our findings support a role for STAT3 and HIF-1α in the regulation of LL-37 expression.
Asunto(s)
Benzamidas/farmacología , Catelicidinas/genética , Inhibidores de Histona Desacetilasas/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Síndrome de Job/genética , Piridinas/farmacología , Factor de Transcripción STAT3/genética , Péptidos Catiónicos Antimicrobianos , Catelicidinas/agonistas , Catelicidinas/metabolismo , Genes Reporteros , Células HEK293 , Células HT29 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/agonistas , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Síndrome de Job/inmunología , Síndrome de Job/patología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Luciferasas/genética , Luciferasas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/patología , Cultivo Primario de Células , Regiones Promotoras Genéticas , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factor de Transcripción STAT3/agonistas , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Activación TranscripcionalRESUMEN
Innate immunity, the front line of our defence against pathogens, relies, to a great extent, on the production of antimicrobial peptides (AMPs). These peptides exhibit antimicrobial activity and immunomodulatory properties. In humans, AMPs include the defensins (α- and ß-families) and the cathelicidin, LL-37. Bacterial resistance against antibiotics is a growing concern, and novel antimicrobial strategies are needed urgently. Hence, the concept of strengthening immune defences against infectious microbes by inducing AMP expression may represent novel or complementary pharmaceutical interventions in the treatment or prevention of infections. We have developed and validated a robust cell-based reporter assay for LL-37 expression, which serves as a marker for a healthy epithelial barrier. This reporter assay can be a powerful tool for high-throughput screenings. We first employed our assay to screen a panel of histone deacetylase inhibitors and derivatives, and then the Prestwick Chemical Library of Food and Drug Administration-approved compounds. After hit confirmation and independent validation in the parental cell line we identified five novel inducers of LL-37. This reporter assay will help to identify novel drug candidates for the treatment and prevention of infections. Importantly, the pattern of hits obtained may suggest cellular pathways and key mediators involved in the regulation of AMP expression.