RESUMEN
The incidence of systemic lupus erythematosus (SLE) is about nine times higher in women than in men, and the underlying mechanisms that contribute to this gender bias are not fully understood. Previously, using lupus-prone (SWR × NZB)F1 (SNF1) mice, we have shown that the intestinal immune system could play a role in the initiation and progression of disease in SLE, and depletion of gut microbiota produces more pronounced disease protection in females than in males. Here, we show that the gut permeability features of lupus-prone female SNF1 mice at juvenile ages directly correlate with the expression levels of pro-inflammatory factors, faecal IgA abundance and nAg reactivity and the eventual systemic autoantibody levels and proteinuria onset. Furthermore, we observed that the disease protection achieved in female SNF1 mice upon depletion of gut microbiota correlates with the diminished gut inflammatory protein levels, intestinal permeability and circulating microbial DNA levels. However, faecal microbiota transplant from juvenile male and females did not result in modulation of gut inflammatory features or permeability. Overall, these observations suggest that the early onset of intestinal inflammation, systemic autoantibody production and clinical stage disease in lupus-prone females is linked to higher gut permeability in them starting at as early as juvenile age. While the higher gut permeability in juvenile lupus-prone females is dependent on the presence of gut microbes, it appears to be independent of the composition of gut microbiota.
Asunto(s)
Autoinmunidad , Lupus Eritematoso Sistémico , Femenino , Humanos , Masculino , Ratones , Animales , Funcion de la Barrera Intestinal , Sexismo , Ratones Endogámicos NZB , Autoanticuerpos , Modelos Animales de EnfermedadRESUMEN
PURPOSE: A same-day PET imaging agent capable of measuring PD-L1 status in tumors is an important tool for optimizing PD-1 and PD-L1 treatments. Herein we describe the discovery and evaluation of a novel, fluorine-18 labeled macrocyclic peptide-based PET ligand for imaging PD-L1. METHODS: [18F]BMS-986229 was synthesized via copper mediated click-chemistry to yield a PD-L1 PET ligand with picomolar affinity and was tested as an in-vivo tool for assessing PD-L1 expression. RESULTS: Autoradiography showed an 8:1 binding ratio in L2987 (PD-L1 (+)) vs. HT-29 (PD-L1 (-)) tumor tissues, with >90% specific binding. Specific radioligand binding (>90%) was observed in human non-small-cell lung cancer (NSCLC) and cynomolgus monkey spleen tissues. Images of PD-L1 (+) tissues in primates were characterized by high signal-to-noise, with low background signal in non-expressing tissues. PET imaging enabled clear visualization of PD-L1 expression in a murine model in vivo, with 5-fold higher uptake in L2987 (PD-L1 (+)) than in control HT-29 (PD-L1 (-)) tumors. Moreover, this imaging agent was used to measure target engagement of PD-L1 inhibitors (peptide or mAb), in PD-L1 (+) tumors as high as 97%. CONCLUSION: A novel 18F-labeled macrocyclic peptide radioligand was developed for PET imaging of PD-L1 expressing tissues that demonstrated several advantages within a nonhuman primate model when compared directly to adnectin- or mAb-based ligands. Clinical studies are currently evaluating [18F]BMS-986229 to measure PD-L1 expression in tumors.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Dominio de Fibronectina del Tipo III , Radioisótopos de Flúor , Neoplasias Pulmonares , Proteínas Recombinantes , Humanos , Ratones , Animales , Antígeno B7-H1/metabolismo , Ligandos , Macaca fascicularis/metabolismo , Tomografía de Emisión de Positrones/métodos , Péptidos/químicaRESUMEN
It is estimated that more than 170 million people are infected with hepatitis C virus (HCV) worldwide. Clinical trials have demonstrated that, for the first time in human history, the potential exists to eradicate a chronic viral disease using combination therapies that contain only direct-acting antiviral agents. HCV non-structural protein 5A (NS5A) is a multifunctional protein required for several stages of the virus replication cycle. NS5A replication complex inhibitors, exemplified by daclatasvir (DCV; also known as BMS-790052 and Daklinza), belong to the most potent class of direct-acting anti-HCV agents described so far, with in vitro activity in the picomolar (pM) to low nanomolar (nM) range. The potency observed in vitro has translated into clinical efficacy, with HCV RNA declining by ~3-4 log10 in infected patients after administration of single oral doses of DCV. Understanding the exceptional potency of DCV was a key objective of this study. Here we show that although DCV and an NS5A inhibitor analogue (Syn-395) are inactive against certain NS5A resistance variants, combinations of the pair enhance DCV potency by >1,000-fold, restoring activity to the pM range. This synergistic effect was validated in vivo using an HCV-infected chimaeric mouse model. The cooperative interaction of a pair of compounds suggests that NS5A protein molecules communicate with each other: one inhibitor binds to resistant NS5A, causing a conformational change that is transmitted to adjacent NS5As, resensitizing resistant NS5A so that the second inhibitor can act to restore inhibition. This unprecedented synergistic anti-HCV activity also enhances the resistance barrier of DCV, providing additional options for HCV combination therapy and new insight into the role of NS5A in the HCV replication cycle.
Asunto(s)
Antivirales/farmacología , Compuestos de Bifenilo/farmacología , Farmacorresistencia Viral/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Imidazoles/farmacología , Proteínas no Estructurales Virales/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Carbamatos , Línea Celular , Sinergismo Farmacológico , Quimioterapia Combinada , Hepacivirus/metabolismo , Hepatitis C/virología , Hepatocitos/trasplante , Humanos , Ratones , Modelos Moleculares , Conformación Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Estructura Cuaternaria de Proteína/efectos de los fármacos , Pirrolidinas , Reproducibilidad de los Resultados , Valina/análogos & derivados , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Replicación Viral/efectos de los fármacosRESUMEN
The risk of developing systemic lupus erythematosus (SLE) is about 9 times higher in women as compared to men. Our recent report, which used (SWRxNZB) F1 (SNF1) mouse model of spontaneous lupus, showed a potential link between immune response initiated in the gut mucosa at juvenile age (sex hormone independent) and SLE susceptibility. Here, using this mouse model, we show that gut microbiota contributes differently to pro-inflammatory immune response in the intestine and autoimmune progression in lupus-prone males and females. We found that gut microbiota composition in male and female littermates are significantly different only at adult ages. However, depletion of gut microbes causes suppression of autoimmune progression only in females. In agreement, microbiota depletion suppressed the pro-inflammatory cytokine response of gut mucosa in juvenile and adult females. Nevertheless, microbiota from females and males showed, upon cross-transfer, contrasting abilities to modulate disease progression. Furthermore, orchidectomy (castration) not only caused changes in the composition of gut microbiota, but also a modest acceleration of autoimmune progression. Overall, our work shows that microbiota-dependent pro-inflammatory immune response in the gut mucosa of females initiated at juvenile ages and androgen-dependent protection of males contribute to gender differences in the intestinal immune phenotype and systemic autoimmune progression.
Asunto(s)
Autoinmunidad , Microbioma Gastrointestinal/inmunología , Lupus Eritematoso Sistémico/etiología , Lupus Eritematoso Sistémico/patología , Fenotipo , Factores de Edad , Animales , Biomarcadores , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Mediadores de Inflamación/metabolismo , Masculino , Metagenoma , Metagenómica/métodos , Ratones , Factores Sexuales , TranscriptomaRESUMEN
BACKGROUND: ß-Glucans (BGs), a group of complex dietary polysaccharides (CDPs), are available as dietary supplements. However, the effects of orally administered highly purified BGs on gut inflammation are largely unknown. OBJECTIVES: The aim of this study was to investigate the impact of orally administering highly purified, yeast-derived BG (YBG; ß-1,3/1,6-d-glucan) on susceptibility to colitis. METHODS: Eight-week-old C57BL/6 (B6) mice were used in a series of experiments. Experiment (Expt) 1: male and female mice were treated every day, for 40 d, with saline (control) or 250 µg YBG, followed by 2.5% (wt:vol) dextran sulfate sodium (DSS) in drinking water during days 30-35; and colitis severity and intestinal immune phenotype were determined. Expt 2: female B6 mice were treated with saline or YBG for 30 d and intestinal immune phenotype, gut microbiota composition, and fecal SCFA concentrations were determined. Expt 3: female B6 mice were treated as in Expt 2, given drinking water with or without antibiotics [Abx; ampicillin (1 g/L), vancomycin (0.5 g/L), neomycin (1 g/L), and metronidazole (1 g/L)] during days 16-30, and gut immune phenotype and fecal SCFA concentrations were determined. Expt 4: female B6 Foxp3-green fluorescent protein (-GFP) reporter mice were treated as in Expt 3, and intestinal T-regulatory cell (Treg) frequencies and immune phenotypes were determined. Expt 5: female mice were treated as in Expt 1, given drinking water with or without antibiotics during days 16-40, and colitis severity and intestinal cytokine production were determined. RESULTS: Compared with controls, the YBG group in Expt 1 exhibited suppressive effects on features of colitis, such as loss of body weight (by 47%; P < 0.001), shortening of colon (by 24%; P = 0.016), and histopathology severity score (by 45%; P = 0.01). The YBG group of Expt 2 showed a shift in the abundance of gut microbiota towards Bacteroides (by 16%; P = 0.049) and Verrucomicrobia (mean ± SD: control = 7.8 ± 0.44 vs. YBG = 21.0 ± 9.6%) and a reduction in Firmicutes (by 66%; P < 0.001). The YBG group also showed significantly higher concentrations of fecal SCFAs such as acetic (by 37%; P = 0.016), propionic (by 47%; P = 0.026), and butyric (by 57%; P = 0.013) acids. Compared with controls, the YBG group of Expt 2 showed higher frequencies of Tregs (by 32%; P = 0.043) in the gut mucosa. Depletion of gut microbiota in the YBG group of mice caused diminished fecal SCFA concentrations (Expt 3) and intestinal Treg frequencies (Expt 4). Compared with the YBG group, the YBG-(Abx) group of Expt 5 showed aggravated colitis features including loss of body weight (by >100%; P < 0.01) and colonic inflammation score (by 42%; P = 0.04). CONCLUSIONS: Studies using B6 mice show that dietary BGs are beneficial for promoting intestinal health when the gut microbiota is intact. However, these CDPs may produce adverse effects if gut microbiota is compromised.
Asunto(s)
Colitis/prevención & control , Microbioma Gastrointestinal/efectos de los fármacos , Polisacáridos/administración & dosificación , Saccharomyces cerevisiae/química , beta-Glucanos/administración & dosificación , Animales , Colitis/inducido químicamente , Colitis/inmunología , Sulfato de Dextran/farmacología , Ácidos Grasos Volátiles/análisis , Heces/química , Heces/microbiología , Femenino , Factores de Transcripción Forkhead/genética , Técnicas de Sustitución del Gen , Proteínas Fluorescentes Verdes/genética , Inmunidad/efectos de los fármacos , Intestinos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
In an effort to improve physical properties by introducing polar functionality into the bicyclic pyrimidine gamma-secretase modulator (GSM) clinical candidate BMS-932481, we prepared several oxidative products of BMS-932481. Among the analogs that were prepared, the C-5 alcohol 3 was identified as the predominant metabolite of BMS-932481 found in rat and human liver microsomes. Alcohol 3 was determined to be chemically unstable, leading to the hypothesis that 3 may lead to the production of reactive species both in vitro and in vivo.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Compuestos de Anilina/farmacología , Pirimidinas/farmacología , Compuestos de Anilina/química , Compuestos de Anilina/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Humanos , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Pirimidinas/química , Pirimidinas/metabolismo , Ratas , Relación Estructura-ActividadRESUMEN
The dietary supplement and prebiotic values of ß-glucan-rich products have been widely recognized and dietary approaches for modulating autoimmunity have been increasingly explored, we assess the impact of oral administration of high-purity yeast ß-glucan (YBG) on gut immune function, microbiota and type 1 diabetes (T1D) using mouse models. Oral administration of this non-digestible complex polysaccharide caused a dectin-1-dependent immune response involving increased expression of interleukin-10 (IL-10), retinaldehyde dehydrogenase (Raldh) and pro-inflammatory cytokines in the gut mucosa. YBG-exposed intestinal dendritic cells induced/expanded primarily Foxp3+ , IL-10+ and IL-17+ T cells, ex vivo. Importantly, prolonged oral administration of low-dose YBG at pre-diabetic stage suppressed insulitis and significantly delayed the appearance of T1D in non-obese diabetic (NOD) mice. Further, prolonged treatment with YBG showed increased Foxp3+ T-cell frequencies, and a significant change in the gut microbiota, particularly an increase in the abundance of Bacteroidetes and a decrease in the Firmicute members. Oral administration of YBG, together with Raldh-substrate and ß-cell antigen, resulted in better protection of NOD mice from T1D. These observations suggest that YBG not only has a prebiotic property, but also an oral tolerogenic-adjuvant-like effect, and these features could be exploited for modulating autoimmunity in T1D.
Asunto(s)
Bacteroidetes/fisiología , Diabetes Mellitus Tipo 1/inmunología , Carbohidratos de la Dieta/uso terapéutico , Microbioma Gastrointestinal/inmunología , Linfocitos T Reguladores/inmunología , Familia de Aldehído Deshidrogenasa 1 , Animales , Diabetes Mellitus Tipo 1/dietoterapia , Diabetes Mellitus Tipo 1/microbiología , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/metabolismo , Humanos , Tolerancia Inmunológica , Inmunidad , Inmunomodulación , Interleucina-10/metabolismo , Isoenzimas/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Prebióticos , Retinal-Deshidrogenasa/metabolismoRESUMEN
Though the current therapies are effective at clearing an early stage prostate cancer, they often fail to treat late-stage metastatic disease. We aimed to investigate the molecular mechanisms underlying the anticancer effects of a natural triterpenoid, ganoderic acid DM (GA-DM), on two human prostate cancer cell lines: the androgen-independent prostate carcinoma (PC-3), and androgen-sensitive prostate adenocarcinoma (LNCaP). Cell viability assay showed that GA-DM was relatively more toxic to LNCaP cells than to PC-3 cells (IC50 s ranged 45-55 µM for PC-3, and 20-25 µM for LNCaP), which may have occurred due to differential expression of p53. Hoechst DNA staining confirmed detectable nuclear fragmentation in both cell lines irrespective of the p53 status. GA-DM treatment decreased Bcl-2 proteins while it upregulated apoptotic Bax and autophagic Beclin-1, Atg5, and LC-3 molecules, and caused an induction of both early and late events of apoptotic cell death. Biochemical analyses of GA-DM-treated prostate cancer cells demonstrated that caspase-3 cleavage was notable in GA-DM-treated PC-3 cells. Interestingly, GA-DM treatment altered cell cycle progression in the S phase with a significant growth arrest in the G2 checkpoint and enhanced CD4 + T cell recognition of prostate tumor cells. Mechanistic study of GA-DM-treated prostate cancer cells further demonstrated that calpain activation and endoplasmic reticulum stress contributed to cell death. These findings suggest that GA-DM is a candidate for future drug design for prostate cancer as it activates multiple pathways of cell death and immune recognition.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/inmunología , Triterpenos/farmacología , Calpaína/metabolismo , Caspasa 3/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Humanos , Masculino , Células PC-3 , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
1. Terbinafine (TBF), a common antifungal agent, has been associated with rare incidences of hepatotoxicity. It is hypothesized that bioactivation of TBF to reactive intermediates and subsequent binding to critical cellular proteins may contribute to this toxicity. In the present study, we have characterized the bioactivation pathways of TBF extensively in human, mouse, monkey, dog and rat liver microsomes and hepatocytes. 2. A total of twenty glutathione conjugates of TBF were identified in hepatocytes; thirteen of these conjugates were also detected in liver microsomes. To the best of our knowledge, only two of these conjugates have been reported previously. The conjugates were categorized into three groups based on their mechanism of formation: (a) alkene/alkyne oxidation followed by glutathione conjugation, with or without N-demethylation, (b) arene oxidation followed by glutathione conjugation, with or without N-demethylation, and (c) N-dealkylation followed by glutathione conjugation of the allylic aldehyde, alcohol and acid intermediates. 3. Differences were observed across species in the contributions of these pathways toward overall metabolic turnover. We conclude that, in addition to the glutathione conjugates known to form by Michael addition to the allylic aldehyde, there are other pathways involving the formation of arene oxides and alkene/alkyne epoxides that may be relevant to the discussion of TBF-mediated idiosyncratic drug reactions.
Asunto(s)
Glutatión/metabolismo , Hepatocitos/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , Terbinafina/farmacocinética , Animales , Antifúngicos/metabolismo , Antifúngicos/farmacocinética , Perros , Haplorrinos , Hepatocitos/metabolismo , Humanos , Masculino , Ratones , Microsomas Hepáticos/metabolismo , Ratas , Espectrometría de Masas en Tándem , Terbinafina/metabolismoRESUMEN
1. Due to its unique C-C and C-H bonding properties, conformational preferences and relative hydrophilicity, the cyclopropyl ring has been used as a synthetic building block in drug discovery to modulate potency and drug-like properties. During an effort to discover inhibitors of the hepatitis C virus non-structural protein 5B with improved potency and genotype-coverage profiles, the use of a pyrimidinylcyclopropylbenzamide moiety linked to a C6-substituted benzofuran or azabenzofuran core scaffold was explored in an effort to balance antiviral potency and metabolic stability. 2. In vitro metabolism studies of two compounds from this C6-substituted series revealed an NADPH-dependent bioactivation pathway leading to the formation of multiple glutathione (GSH) conjugates. Analysis of these conjugates by LC-MS and NMR demonstrated that the cyclopropyl group was the site of bioactivation. Based on the putative structures and molecular weights of the cyclopropyl-GSH conjugates, a multi-step mechanism was proposed to explain the formation of these metabolites by P450. This mechanism involves hydrogen atom abstraction to form a cyclopropyl radical, followed by a ring opening rearrangement and reaction with GSH. 3. These findings provided important information to the medicinal chemistry team which responded by replacing the cyclopropyl ring with a gem-dimethyl group. Subsequent compounds bearing this feature were shown to avert the bioactivation pathways in question.
Asunto(s)
Antivirales , Benzamidas , Sistema Enzimático del Citocromo P-450/metabolismo , Hepacivirus , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/farmacocinética , Antivirales/farmacología , Benzamidas/farmacocinética , Benzamidas/farmacología , Humanos , RatasRESUMEN
An asymmetric synthesis of the major metabolite of the calcitonin gene-related peptide recepotor antagonist BMS-846372 is presented. The variously substituted cyclohepta[b]pyridine ring system represents an underexplored ring system and showed some unexpected chemistry. Reactivities of epoxide and ketone functional groups on the cycloheptane ring were extensively controlled by a remote bulky TIPS group. The rate difference of the hydrogenolysis between two diastereomeric epoxide intermediates shed some light on the mechanism of epoxide hydrogenolysis, and further, deuterium labeling studies revealed more mechanistic details on this well-known chemical transformation for the first time.
Asunto(s)
Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina , Compuestos Epoxi/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Hidrólisis , Análisis Espectral/métodosRESUMEN
During a medicinal chemistry campaign to identify inhibitors of the hepatitis C virus nonstructural protein 5B (RNA-dependent RNA polymerase), a bicyclo[1.1.1]pentane was introduced into the chemical scaffold to improve metabolic stability. The inhibitors bearing this feature, 5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)-4-fluorophenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide (1) and 5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide (2), exhibited low turnover in incubations with liver S9 or hepatocytes (rat, human), with hydroxylation of the bicyclic moiety being the only metabolic pathway observed. In subsequent disposition studies using bile-duct-cannulated rats, the metabolite profiles of bile samples revealed, in addition to multiple products of bicyclopentane-oxidation, unexpected metabolites characterized by molecular masses that were 181 Da greater than those of 1 or 2. Further LC/MSn and NMR analysis of the isolated metabolite of 1 demonstrated the presence of a phosphocholine (POPC) moiety bound to the methine carbon of the bicyclic moiety through an ester bond. The POPC conjugate of the NS5B inhibitors was assumed to result from two sequential reactions: hydroxylation of the bicyclic methine to a tertiary alcohol and addition of POPC by CDP-choline: 1,2-diacylglycerol cholinephosphotransferase, an enzyme responsible for the final step in the biosynthesis of phosphatidylcholine. However, this pathway could not be recapitulated using CDP-choline-supplemented liver S9 or hepatocytes due to inadequate formation of the hydroxylation product in vitro. The observation of this unexpected pathway prompted concerns about the possibility that 1 and 2 might interfere with routine phospholipid synthesis. These results demonstrate the participation in xenobiotic metabolism of a process whose function is ordinarily limited to the synthesis of endogenous compounds.
RESUMEN
Daclatasvir is a first-in-class, potent, and selective inhibitor of the hepatitis C virus nonstructural protein 5A replication complex. In support of nonclinical studies during discovery and exploratory development, liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance were used in connection with synthetic and radiosynthetic approaches to investigate the biotransformation of daclatasvir in vitro and in cynomolgus monkeys, dogs, mice, and rats. The results of these studies indicated that disposition of daclatasvir was accomplished mainly by the release of unchanged daclatasvir into bile and feces and, secondarily, by oxidative metabolism. Cytochrome P450s were the main enzymes involved in the metabolism of daclatasvir. Oxidative pathways included δ-oxidation of the pyrrolidine moiety, resulting in ring opening to an aminoaldehyde intermediate followed by an intramolecular reaction between the aldehyde and the proximal imidazole nitrogen atom. Despite robust formation of the resulting metabolite in multiple systems, rates of covalent binding to protein associated with metabolism of daclatasvir were modest (55.2-67.8 pmol/mg/h) in nicotinamide adenine dinucleotide phosphate (reduced form)-supplemented liver microsomes (human, monkey, rat), suggesting that intramolecular rearrangement was favored over intermolecular binding in the formation of this metabolite. This biotransformation profile supported the continued development of daclatasvir, which is now marketed for the treatment of chronic hepatitis C virus infection.
Asunto(s)
Biotransformación/fisiología , Imidazoles/metabolismo , Pirrolidinas/metabolismo , Animales , Bilis/metabolismo , Carbamatos , Cromatografía Líquida de Alta Presión/métodos , Sistema Enzimático del Citocromo P-450/metabolismo , Perros , Haplorrinos , Hepatocitos/metabolismo , Humanos , Macaca fascicularis , Espectroscopía de Resonancia Magnética/métodos , Masculino , Espectrometría de Masas/métodos , Ratones , Ratones Endogámicos BALB C , Microsomas Hepáticos/metabolismo , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Valina/análogos & derivadosRESUMEN
ß-Glucans are naturally occurring polysaccharides in cereal grains, mushrooms, algae, or microbes, including bacteria, fungi, and yeast. Immune cells recognize these ß-glucans through a cell surface pathogen recognition receptor called Dectin-1. Studies using ß-glucans and other Dectin-1 binding components have demonstrated the potential of these agents in activating the immune cells for cancer treatment and controlling infections. In this study, we show that the ß-glucan from Saccharomyces cerevisiae induces the expression of immune regulatory cytokines (IL-10, TGF-ß1, and IL-2) and a tolerogenic enzyme (IDO) in bone marrow-derived dendritic cells as well as spleen cells. These properties can be exploited to modulate autoimmunity in the NOD mouse model of type 1 diabetes (T1D). Treatment of prediabetic NOD mice with low-dose ß-glucan resulted in a profound delay in hyperglycemia, and this protection was associated with increase in the frequencies of Foxp3(+), LAP(+), and GARP(+) T cells. Upon Ag presentation, ß-glucan-exposed dendritic cells induced a significant increase in Foxp3(+) and LAP(+) T cells in in vitro cultures. Furthermore, systemic coadministration of ß-glucan plus pancreatic ß cell Ag resulted in an enhanced protection of NOD mice from T1D as compared with treatment with ß-glucan alone. These observations demonstrate that the innate immune response induced by low-dose ß-glucan is regulatory in nature and can be exploited to modulate T cell response to ß cell Ag for inducing an effective protection from T1D.
Asunto(s)
Diabetes Mellitus Tipo 1/prevención & control , Polisacáridos Fúngicos/farmacología , Inmunidad Innata/efectos de los fármacos , Lectinas Tipo C/inmunología , Saccharomyces cerevisiae/química , beta-Glucanos/farmacología , Animales , Citocinas/genética , Citocinas/inmunología , Células Dendríticas/inmunología , Células Dendríticas/patología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Relación Dosis-Respuesta Inmunológica , Polisacáridos Fúngicos/química , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/patología , Lectinas Tipo C/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Linfocitos T/inmunología , Linfocitos T/patología , beta-Glucanos/químicaRESUMEN
A recent medicinal chemistry campaign to identify positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGluR5) led to the discovery of potent compounds featuring an oxazolidinone structural core flanked by biaryl acetylene and haloaryl moieties. However, biotransformation studies of some of these mGluR5 PAMs demonstrated the formation of glutathione (GSH) conjugates. The conjugates in question were formed independently of NADPH as the main products in liver microsomes and liver cytosol (rat and human) and exhibited masses that were 307 u greater than their respective substrates, indicating the involvement of a reductive step in the formation of these metabolites. To further characterize the relevant metabolic sequences, GSH conjugates of (4R,5R)-5-(3-fluorophenyl)-4-(5-(pyrazin-2-ylethynyl)pyridin-3-yl)oxazolidin-2-one and (4R,5R)-5-(4-fluorophenyl)-4-(6-((3-fluoropyridin-2-yl)ethynyl)pyridin-2-yl)oxazolidin-2-one were biosynthesized and isolated. Subsequent analysis by NMR showed that GSH had reacted with the acetylene carbon atoms of these mGluR5 PAMs, suggesting a conjugate addition mechanism and implicating cytosolic and microsomal GSH S-transferases (GSTs) in catalysis. Interestingly, five closely related mGluR5 PAMs were not similarly prone to the formation of GSH conjugates in vitro. These compounds also featured acetylenes, but were flanked by either phenyl or cyclohexyl rings, which indicated that the formation of GSH conjugates was influenced by proximal functional groups that modulated the electron density of the triple bond and/or differences in enzyme-substrate specificity. These results informed an ongoing drug-discovery effort to identify mGluR5 PAMs with drug-like properties and a low risk of reactivity with endogenous thiols.
Asunto(s)
Alquinos/farmacocinética , Regulación Alostérica/efectos de los fármacos , Glutatión/metabolismo , Hígado/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismo , Alquinos/química , Alquinos/farmacología , Animales , Citosol/metabolismo , Humanos , Hígado/citología , Fase II de la Desintoxicación Metabólica , Microsomas Hepáticos/metabolismo , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Ratas , Relación Estructura-ActividadRESUMEN
Certain aromatic nitriles are well-known inhibitors of cysteine proteases. The mode of action of these compounds involves the formation of a reversible or irreversible covalent bond between the nitrile and a thiol group in the active site of the enzyme. However, the reactivity of these aromatic nitrile-substituted heterocycles may lead inadvertently to nonspecific interactions with DNA, protein, glutathione, and other endogenous components, resulting in toxicity and complicating the use of these compounds as therapeutic agents. In the present study, the intrinsic reactivity and associated structure-property relationships of cathepsin K inhibitors featuring substituted pyridazines [6-phenylpyridazine-3-carbonitrile, 6-(4-fluorophenyl)pyridazine-3-carbonitrile, 6-(4-methoxyphenyl)pyridazine-3-carbonitrile, 6-p-tolylpyridazine-3-carbonitrile], pyrimidines [5-p-tolylpyrimidine-2-carbonitrile, 5-(4-fluorophenyl)pyrimidine-2-carbonitrile], and pyridines [5-p-tolylpicolinonitrile and 5-(4-fluorophenyl)picolinonitrile] were evaluated using a combination of computational and analytical approaches to establish correlations between electrophilicity and levels of metabolites that were formed in glutathione- and N-acetylcysteine-supplemented human liver microsomes. Metabolites that were characterized in this study featured substituted thiazolines that were formed following rearrangements of transient glutathione and N-acetylcysteine conjugates. Peptidases including γ-glutamyltranspeptidase were shown to catalyze the formation of these products, which were formed to lesser extents in the presence of the selective γ-glutamyltranspeptidase inhibitor acivicin and the nonspecific peptidase inhibitors phenylmethylsulfonyl fluoride and aprotinin. Of the chemical series mentioned above, the pyrimidine series was the most susceptible to metabolism to thiazoline-containing products, followed, in order, by the pyridazine and pyridine series. This trend was in keeping with the diminishing electrophilicity across these series, as demonstrated by in silico modeling. Hence, mechanistic insights gained from this study could be used to assist a medicinal chemistry campaign to design cysteine protease inhibitors that were less prone to the formation of covalent adducts.
Asunto(s)
Microsomas Hepáticos/metabolismo , Modelos Químicos , Nitrilos/metabolismo , Piridazinas/metabolismo , Piridinas/metabolismo , Pirimidinas/metabolismo , Tiazoles/metabolismo , Cromatografía Liquida , Humanos , Espectroscopía de Resonancia Magnética , Espectrofotometría Ultravioleta , Espectrometría de Masas en TándemRESUMEN
BMS-932481 was designed to modulate ɣ-secretase activity to produce shorter and less amyloidogenic peptides, potentially averting liabilities associated with complete enzymatic inhibition. Although it demonstrated the intended pharmacology in the clinic, BMS-932481 unexpectedly caused drug-induced liver injury (DILI) in a multiple ascending dose study characterized by dose- and exposure-dependence, delayed onset manifestation, and a high incidence of hepatocellular damage. Retrospective studies investigating the disposition and probable mechanisms of toxicity of BMS-932481 are presented here. These included a mass balance study in bile-duct-cannulated rats and a metabolite profiling study in human hepatocytes, which together demonstrated oxidative metabolism followed by biliary elimination as the primary means of disposition. Additionally, minimal protein covalent binding in hepatocytes and lack of bioactivation products excluded reactive metabolite formation as a probable toxicological mechanism. However, BMS-932481 and 3 major oxidative metabolites were found to inhibit the bile salt export pump (BSEP) and multidrug resistance protein 4 (MRP4) in vitro. Considering human plasma concentrations, the IC50 values against these efflux transporters were clinically meaningful, particularly in the high dose cohort. Active uptake into human hepatocytes in vitro suggested the potential for hepatic levels of BMS-932481 to be elevated further above plasma concentrations, enhancing DILI risk. Conversely, measures of mitochondrial functional decline in hepatocytes treated with BMS-932481 were minimal or modest, suggesting limited contributions to DILI. Collectively, these findings suggested that repeat administration of BMS-932481 likely resulted in high hepatic concentrations of BMS-932481 and its metabolites, which disrupted bile acid transport via BSEP and MRP4, elevating serum biomarkers of liver injury.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide , Enfermedad Hepática Inducida por Sustancias y Drogas , Humanos , Ratas , Animales , Estudios Retrospectivos , Hígado/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Hepatocitos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Ácidos y Sales Biliares/metabolismoRESUMEN
Progressive destruction of pancreatic islet ß-cells by immune cells is a primary feature of type 1 diabetes (T1D) and therapies that can restore the functional ß-cell mass are needed to alleviate disease progression. Here, we report the use of mesenchymal stromal/stem cells (MSCs) for the production and delivery of Gastrin, a peptide hormone that is produced by intestinal cells and foetal islets and can increase ß-Cell mass, to promote protection from T1D. A single injection of syngeneic MSCs that were engineered to express Gastrin (Gastrin-MSCs) caused a significant delay in hyperglycaemia in non-obese diabetic (NOD) mice compared to engineered control-MSCs. Similar treatment of early-hyperglycaemic mice caused the restoration of euglycemia for a considerable duration, and these therapeutic effects were associated with the protection of, and/or higher frequencies of, insulin-producing islets and less severe insulitis. While the overall immune cell phenotype was not affected profoundly upon treatment using Gastrin-MSCs or upon in vitro culture, pancreatic lymph node cells from Gastrin-MSC treated mice, upon ex vivo challenge with self-antigen, showed a Th2 and Th17 bias, and diminished the diabetogenic property in NOD-Rag1 deficient mice suggesting a disease protective immune modulation under Gastrin-MSC treatment associated protection from hyperglycaemia. Overall, this study shows the potential of production and delivery of Gastrin in vivo, by MSCs, in protecting insulin-producing ß-cells and ameliorating the disease progression in T1D.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Gastrinas , Islotes Pancreáticos , Trasplante de Células Madre Mesenquimatosas , Animales , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Gastrinas/genética , Células Madre Mesenquimatosas , Ratones , Ratones Endogámicos NODRESUMEN
Recent mouse knockout studies identified adapter protein-2-associated kinase 1 (AAK1) as a viable target for treating neuropathic pain. BMS-986176/LX-9211 (4), as a highly selective, CNS-penetrable, and potent AAK1 inhibitor, has advanced into phase II human trials. On exploring the structure-activity relationship (SAR) around this biaryl alkyl ether chemotype, several additional compounds were found to be highly selective and potent AAK1 inhibitors with good druglike properties. Among these, compounds 43 and 58 showed very good efficacy in two neuropathic pain rat models and had excellent CNS penetration and spinal cord target engagement. Both compounds also exhibited favorable physicochemical and oral pharmacokinetic (PK) properties. Compound 58, a central pyridine isomer of BMS-986176/LX-9211 (4), was 4-fold more potent than 4 in vitro and showed lower plasma exposure needed to achieve similar efficacy compared to 4 in the CCI rat model. However, both 43 and 58 showed an inferior preclinical toxicity profile compared to 4.
Asunto(s)
Anestésicos Generales , Neuralgia , Animales , Éteres/uso terapéutico , Ratones , Neuralgia/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Ratas , Médula Espinal , Relación Estructura-ActividadRESUMEN
Recent mouse knockout studies identified adapter protein-2 associated kinase 1 (AAK1) as a viable target for treating neuropathic pain. Potent small-molecule inhibitors of AAK1 have been identified and show efficacy in various rodent pain models. (S)-1-((2',6-Bis(difluoromethyl)-[2,4'-bipyridin]-5-yl)oxy)-2,4-dimethylpentan-2-amine (BMS-986176/LX-9211) (34) was identified as a highly selective, CNS penetrant, potent AAK1 inhibitor from a novel class of bi(hetero)aryl ethers. BMS-986176/LX9211 (34) showed excellent efficacy in two rodent neuropathic pain models and excellent central nervous system (CNS) penetration and target engagement at the spinal cord with an average brain to plasma ratio of 20 in rat. The compound exhibited favorable physicochemical and pharmacokinetic properties, had an acceptable preclinical toxicity profile, and was chosen for clinical trials. BMS-986176/LX9211 (34) completed phase I trials with good human pharmacokinetics and minimum adverse events and is currently in phase II clinical trials for diabetic peripheral neuropathic pain (ClinicalTrials.gov identifier: NCT04455633) and postherpetic neuralgia (ClinicalTrials.gov identifier: NCT04662281).