RESUMEN
BACKGROUND: Postoperative pancreatic fistula is a common and serious complication following pancreaticoduodenectomy. Duct-to-mucosa pancreaticojejunostomy has been used in many centers to reconstruct pancreatic digestive continuity following pancreatoduodenectomy, however, its efficacy and safety are uncertain. OBJECTIVES: To assess the benefits and harms of duct-to-mucosa pancreaticojejunostomy versus other types of pancreaticojejunostomy for the reconstruction of pancreatic digestive continuity in participants undergoing pancreaticoduodenectomy, and to compare the effects of different duct-to-mucosa pancreaticojejunostomy techniques. SEARCH METHODS: We searched the Cochrane Library (2021, Issue 1), MEDLINE (1966 to 9 January 2021), Embase (1988 to 9 January 2021), and Science Citation Index Expanded (1982 to 9 January 2021). SELECTION CRITERIA: We included all randomized controlled trials (RCTs) that compared duct-to-mucosa pancreaticojejunostomy with other types of pancreaticojejunostomy (e.g. invagination pancreaticojejunostomy, binding pancreaticojejunostomy) in participants undergoing pancreaticoduodenectomy. We also included RCTs that compared different types of duct-to-mucosa pancreaticojejunostomy in participants undergoing pancreaticoduodenectomy. DATA COLLECTION AND ANALYSIS: Two review authors independently identified the studies for inclusion, collected the data, and assessed the risk of bias. We performed the meta-analyses using Review Manager 5. We calculated the risk ratio (RR) for dichotomous outcomes and the mean difference (MD) for continuous outcomes with 95% confidence intervals (CIs). For all analyses, we used the random-effects model. We used the Cochrane RoB 1 tool to assess the risk of bias. We used GRADE to assess the certainty of the evidence for all outcomes. MAIN RESULTS: We included 11 RCTs involving a total of 1696 participants in the review. One RCT was a dual-center study; the other 10 RCTs were single-center studies conducted in: China (4 studies); Japan (2 studies); USA (1 study); Egypt (1 study); Germany (1 study); India (1 study); and Italy (1 study). The mean age of participants ranged from 54 to 68 years. All RCTs were at high risk of bias. Duct-to-mucosa versus any other type of pancreaticojejunostomy We included 10 RCTs involving 1472 participants comparing duct-to-mucosa pancreaticojejunostomy with invagination pancreaticojejunostomy: 732 participants were randomized to the duct-to-mucosa group, and 740 participants were randomized to the invagination group after pancreaticoduodenectomy. Comparing the two techniques, the evidence is very uncertain for the rate of postoperative pancreatic fistula (grade B or C; RR 1.45, 95% CI 0.64 to 3.26; 7 studies, 1122 participants; very low-certainty evidence), postoperative mortality (RR 0.77, 95% CI 0.39 to 1.49; 10 studies, 1472 participants; very low-certainty evidence), rate of surgical reintervention (RR 1.12, 95% CI 0.65 to 1.95; 10 studies, 1472 participants; very low-certainty evidence), rate of postoperative bleeding (RR 0.85, 95% CI 0.51 to 1.42; 9 studies, 1275 participants; very low-certainty evidence), overall rate of surgical complications (RR 1.12, 95% CI 0.92 to 1.36; 5 studies, 750 participants; very low-certainty evidence), and length of hospital stay (MD -0.41 days, 95% CI -1.87 to 1.04; 4 studies, 658 participants; very low-certainty evidence). The studies did not report adverse events or quality of life outcomes. One type of duct-to-mucosa pancreaticojejunostomy versus a different type of duct-to-mucosa pancreaticojejunostomy We included one RCT involving 224 participants comparing duct-to-mucosa pancreaticojejunostomy using the modified Blumgart technique with duct-to-mucosa pancreaticojejunostomy using the traditional interrupted technique: 112 participants were randomized to the modified Blumgart group, and 112 participants were randomized to the traditional interrupted group after pancreaticoduodenectomy. Comparing the two techniques, the evidence is very uncertain for the rate of postoperative pancreatic fistula (grade B or C; RR 1.51, 95% CI 0.61 to 3.75; 1 study, 210 participants; very low-certainty evidence), postoperative mortality (there were no deaths in either group; 1 study, 210 participants; very low-certainty evidence), rate of surgical reintervention (RR 1.93, 95% CI 0.18 to 20.91; 1 study, 210 participants; very low-certainty evidence), rate of postoperative bleeding (RR 2.89, 95% CI 0.12 to 70.11; 1 study, 210 participants; very low-certainty evidence), overall rate of surgical complications (RR 1.10, 95% CI 0.80 to 1.51; 1 study, 210 participants; very low-certainty evidence), and length of hospital stay (15 days versus 15 days; 1 study, 210 participants; very low-certainty evidence). The study did not report adverse events or quality of life outcomes. AUTHORS' CONCLUSIONS: The evidence is very uncertain about the effects of duct-to-mucosa pancreaticojejunostomy compared to invagination pancreaticojejunostomy on any of the outcomes, including rate of postoperative pancreatic fistula (grade B or C), postoperative mortality, rate of surgical reintervention, rate of postoperative bleeding, overall rate of surgical complications, and length of hospital stay. The evidence is also very uncertain whether duct-to-mucosa pancreaticojejunostomy using the modified Blumgart technique is superior, equivalent or inferior to duct-to-mucosa pancreaticojejunostomy using the traditional interrupted technique. None of the studies reported adverse events or quality of life outcomes.
Asunto(s)
Fístula Pancreática , Pancreaticoduodenectomía , Anciano , Humanos , Persona de Mediana Edad , Membrana Mucosa , Pancreatectomía/efectos adversos , Fístula Pancreática/etiología , Fístula Pancreática/prevención & control , Pancreaticoduodenectomía/efectos adversos , Pancreaticoduodenectomía/métodos , Pancreatoyeyunostomía/efectos adversos , Complicaciones Posoperatorias/etiología , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
The discovery of a series of substituted diarylether compounds as retinoic acid related orphan receptor γt (RORγt) agonists is described. Compound 1 was identified from deck mining as a RORγt agonist. Hit-to-lead optimization led to the identification of lead compound 5, which possesses improved potency (10x). Extensive SAR exploration led to the identification of a potent and selective compound 22, that demonstrated an improved pharmacokinetic profile and a dose-dependent pharmacodynamic response. However, when dosed in a MC38 syngeneic tumor model, no evidence of efficacy was observed. ©2020 Elsevier Science Ltd. All rights reserved.
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Éteres/farmacología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Tretinoina/farmacología , Animales , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Éteres/síntesis química , Éteres/química , Humanos , Ratones , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Células Th17 , Tretinoina/síntesis química , Tretinoina/químicaRESUMEN
BACKGROUND: This is the second update of a Cochrane Review first published in 2015 and last updated in 2018. Appendectomy, the surgical removal of the appendix, is performed primarily for acute appendicitis. Patients who undergo appendectomy for complicated appendicitis, defined as gangrenous or perforated appendicitis, are more likely to suffer postoperative complications. The routine use of abdominal drainage to reduce postoperative complications after appendectomy for complicated appendicitis is controversial. OBJECTIVES: To assess the safety and efficacy of abdominal drainage to prevent intraperitoneal abscess after appendectomy (irrespective of open or laparoscopic) for complicated appendicitis; to compare the effects of different types of surgical drains; and to evaluate the optimal time for drain removal. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL), Ovid MEDLINE, Ovid Embase, Web of Science, the World Health Organization International Trials Registry Platform, ClinicalTrials.gov, Chinese Biomedical Literature Database, and three trials registers on 24 February 2020, together with reference checking, citation searching, and contact with study authors to identify additional studies. SELECTION CRITERIA: We included all randomised controlled trials (RCTs) that compared abdominal drainage versus no drainage in people undergoing emergency open or laparoscopic appendectomy for complicated appendicitis. We also included RCTs that compared different types of drains and different schedules for drain removal in people undergoing appendectomy for complicated appendicitis. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane. Two review authors independently identified the trials for inclusion, collected the data, and assessed the risk of bias. We used the GRADE approach to assess evidence certainty. We included intraperitoneal abscess as the primary outcome. Secondary outcomes were wound infection, morbidity, mortality, hospital stay, hospital costs, pain, and quality of life. MAIN RESULTS: Use of drain versus no drain We included six RCTs (521 participants) comparing abdominal drainage and no drainage in participants undergoing emergency open appendectomy for complicated appendicitis. The studies were conducted in North America, Asia, and Africa. The majority of participants had perforated appendicitis with local or general peritonitis. All participants received antibiotic regimens after open appendectomy. None of the trials was assessed as at low risk of bias. The evidence is very uncertain regarding the effects of abdominal drainage versus no drainage on intraperitoneal abscess at 30 days (risk ratio (RR) 1.23, 95% confidence interval (CI) 0.47 to 3.21; 5 RCTs; 453 participants; very low-certainty evidence) or wound infection at 30 days (RR 2.01, 95% CI 0.88 to 4.56; 5 RCTs; 478 participants; very low-certainty evidence). There were seven deaths in the drainage group (N = 183) compared to one in the no-drainage group (N = 180), equating to an increase in the risk of 30-day mortality from 0.6% to 2.7% (Peto odds ratio 4.88, 95% CI 1.18 to 20.09; 4 RCTs; 363 participants; low-certainty evidence). Abdominal drainage may increase 30-day overall complication rate (morbidity; RR 6.67, 95% CI 2.13 to 20.87; 1 RCT; 90 participants; low-certainty evidence) and hospital stay by 2.17 days (95% CI 1.76 to 2.58; 3 RCTs; 298 participants; low-certainty evidence) compared to no drainage. The outcomes hospital costs, pain, and quality of life were not reported in any of the included studies. There were no RCTs comparing the use of drain versus no drain in participants undergoing emergency laparoscopic appendectomy for complicated appendicitis. Open drain versus closed drain There were no RCTs comparing open drain versus closed drain for complicated appendicitis. Early versus late drain removal There were no RCTs comparing early versus late drain removal for complicated appendicitis. AUTHORS' CONCLUSIONS: The certainty of the currently available evidence is low to very low. The effect of abdominal drainage on the prevention of intraperitoneal abscess or wound infection after open appendectomy is uncertain for patients with complicated appendicitis. The increased rates for overall complication rate and hospital stay for the drainage group compared to the no-drainage group are based on low-certainty evidence. Consequently, there is no evidence for any clinical improvement with the use of abdominal drainage in patients undergoing open appendectomy for complicated appendicitis. The increased risk of mortality with drainage comes from eight deaths observed in just under 400 recruited participants. Larger studies are needed to more reliably determine the effects of drainage on morbidity and mortality outcomes.
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Absceso/prevención & control , Apendicectomía/efectos adversos , Apendicitis/cirugía , Drenaje/métodos , Peritonitis/prevención & control , Complicaciones Posoperatorias/prevención & control , HumanosRESUMEN
Aortic valve sclerosis is a highly prevalent, poorly characterized asymptomatic manifestation of calcific aortic valve disease and may represent a therapeutic target for disease mitigation. Human aortic valve cusps and blood were obtained from 333 patients undergoing cardiac surgery (n = 236 for severe aortic stenosis, n = 35 for asymptomatic aortic valve sclerosis, n = 62 for no valvular disease), and a multiplex assay was used to evaluate protein expression across the spectrum of calcific aortic valve disease. A subset of six valvular tissue samples (n = 3 for asymptomatic aortic valve sclerosis, n = 3 for severe aortic stenosis) was used to create RNA sequencing profiles, which were subsequently organized into clinically relevant gene modules. RNA sequencing identified 182 protein-encoding, differentially expressed genes in aortic valve sclerosis vs. aortic stenosis; 85% and 89% of expressed genes overlapped in aortic stenosis and aortic valve sclerosis, respectively, which decreased to 55% and 84% when we targeted highly expressed genes. Bioinformatic analyses identified six differentially expressed genes encoding key extracellular matrix regulators: TBHS2, SPARC, COL1A2, COL1A1, SPP1, and CTGF. Differential expression of key circulating biomarkers of extracellular matrix reorganization was observed in control vs. aortic valve sclerosis (osteopontin), control vs. aortic stenosis (osteoprotegerin), and aortic valve sclerosis vs. aortic stenosis groups (MMP-2), which corresponded to valvular mRNA expression. We demonstrate distinct mRNA and protein expression underlying aortic valve sclerosis and aortic stenosis. We anticipate that extracellular matrix regulators can serve as circulating biomarkers of early calcific aortic valve disease and as novel targets for early disease mitigation, pending prospective clinical investigations.
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Estenosis de la Válvula Aórtica/sangre , Estenosis de la Válvula Aórtica/genética , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Calcinosis/sangre , Calcinosis/genética , Ácidos Nucleicos Libres de Células/metabolismo , Osteopontina/metabolismo , Osteoprotegerina/metabolismo , Transcriptoma , Anciano , Válvula Aórtica/cirugía , Estenosis de la Válvula Aórtica/cirugía , Secuencia de Bases , Biomarcadores/metabolismo , Calcinosis/cirugía , Estudios de Casos y Controles , Ácidos Nucleicos Libres de Células/genética , Matriz Extracelular/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteopontina/genética , Osteoprotegerina/genética , ARN Mensajero/genética , RNA-SeqRESUMEN
An ability to characterize the cellular composition and spatial organization of the tumor microenvironment (TME) using multiplexed IHC has been limited by the techniques available. Here we show the applicability of multiplexed ion beam imaging (MIBI) for cell phenotype identification and analysis of spatial relationships across numerous tumor types. Formalin-fixed paraffin-embedded (FFPE) samples from tumor biopsies were simultaneously stained with a panel of 15 antibodies, each labeled with a specific metal isotope. Multi-step processing produced images of the TME that were further segmented into single cells. Frequencies of different cell subsets and the distributions of nearest neighbor distances between them were calculated using this data. A total of 50 tumor specimens from 15 tumor types were characterized for their immune profile and spatial organization. Most samples showed infiltrating cytotoxic T cells and macrophages present amongst tumor cells. Spatial analysis of the TME in two ovarian serous carcinoma images highlighted differences in the degree of mixing between tumor and immune cells across samples. Identification of admixed PD-L1+ macrophages and PD-1+ T cells in an urothelial carcinoma sample allowed for the detailed observations of immune cell subset spatial arrangement. These results illustrate the high-parameter capability of MIBI at a sensitivity and resolution uniquely suited to understanding the complex tumor immune landscape including the spatial relationships of immune and tumor cells and expression of immunoregulatory proteins.
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Biomarcadores de Tumor/metabolismo , Diagnóstico por Imagen/métodos , Neoplasias/diagnóstico por imagen , Microambiente Tumoral , Antígeno B7-H1/metabolismo , Diagnóstico Diferencial , Humanos , Macrófagos/metabolismo , Neoplasias/clasificación , Receptor de Muerte Celular Programada 1/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Linfocitos T Citotóxicos/metabolismoRESUMEN
INTRODUCTION: Tenascin-C is a marker of interstitial fibrosis. We assessed whether plasma Tenascin-C differed between heart failure with preserved ejection fraction (HFpEF) and asymptomatic controls and related to clinical outcomes. MATERIALS AND METHODS: Prospective, observational study of 172 age- and sex-matched subjects (HFpEF n = 130; controls n = 42, age 73 ± 9, males 50%) who underwent phenotyping with 20 plasma biomarkers, echocardiography, cardiac MRI and 6-minute-walk-testing. The primary endpoint was the composite of all-cause death/HF hospitalisation. RESULTS: Tenascin-C was higher in HFpEF compared to controls (13.7 [10.8-17.3] vs (11.1 [8.9-12.9] ng/ml, p < 0.0001). Tenascin-C correlated positively with markers of clinical severity (NYHA, E/E', BNP) and plasma biomarkers reflecting interstitial fibrosis (ST-2, Galectin-3, GDF-15, TIMP-1, TIMP-4, MMP-2, MMP-3, MMP-7, MMP-8), cardiomyocyte stress (BNP, NTpro-ANP), inflammation (MPO, hs-CRP, TNFR-1, IL6) and renal dysfunction (urea, cystatin-C, NGAL); p < 0.05 for all. During follow-up (median 1428 days), there were 61 composite events (21 deaths, 40 HF hospitalizations). In multivariable Cox regression analysis, Tenascin-C (adjusted hazard ratio [HR] 1.755, 95% confidence interval [CI] 1.305-2.360; p < 0.0001) and indexed extracellular volume (HR 1.465, CI 1.019-2.106; p = 0.039) were independently associated with adverse outcomes. CONCLUSIONS: In HFpEF, plasma Tenascin-C is higher compared to age- and sex-matched controls and a strong predictor of adverse outcomes. Trial registration: ClinicalTrials.gov: NCT03050593.
Asunto(s)
Biomarcadores/sangre , Insuficiencia Cardíaca/sangre , Pronóstico , Tenascina/sangre , Adulto , Anciano , Femenino , Galectina 3/sangre , Factor 15 de Diferenciación de Crecimiento/sangre , Insuficiencia Cardíaca/patología , Humanos , Masculino , Persona de Mediana Edad , Volumen Sistólico/genética , Inhibidor Tisular de Metaloproteinasa-1/sangreRESUMEN
Substituted benzyloxy aryl compound 2 was identified as an RORγt agonist. Structure based drug design efforts resulted in a potent and selective tricyclic compound 19 which, when administered orally in an MC38 mouse tumor model, demonstrated a desired pharmacokinetic profile as well as a dose-dependent pharmacodynamic response. However, no perceptible efficacy was observed in this tumor model at the doses investigated.
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Compuestos de Bencilo/farmacología , Compuestos Heterocíclicos/farmacología , Receptores de Ácido Retinoico/agonistas , Animales , Compuestos de Bencilo/química , Relación Dosis-Respuesta a Droga , Femenino , Compuestos Heterocíclicos/química , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Relación Estructura-Actividad , Receptor de Ácido Retinoico gammaRESUMEN
Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a marked improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling in vitro assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells. This manuscript reports the assay development and medicinal chemistry leading to the development of NCT-502 and -503 reported in Pacold et al. (2016).
Asunto(s)
Inhibidores Enzimáticos/farmacología , Fosfoglicerato-Deshidrogenasa/antagonistas & inhibidores , Piperazinas/farmacología , Tiourea/análogos & derivados , Tiourea/farmacología , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Estructura Molecular , Fosfoglicerato-Deshidrogenasa/genética , Fosfoglicerato-Deshidrogenasa/metabolismo , Piperazinas/síntesis química , Piperazinas/química , Relación Estructura-Actividad , Tiourea/síntesis química , Tiourea/químicaRESUMEN
Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC50 of 1.1 µm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.
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Puntos de Control del Ciclo Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Ciclina D1/metabolismo , Endopeptidasas/metabolismo , Linfoma de Células del Manto/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Inhibidores de Proteasas/farmacología , Proteolisis/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Ciclina D1/genética , Daño del ADN , Reparación del ADN , Endopeptidasas/genética , Humanos , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/metabolismo , Proteínas de Neoplasias/genética , Inhibidores de Proteasas/química , Ubiquitina TiolesterasaRESUMEN
Hutchinson-Gilford Progeria Syndrome (HGPS) is an early onset lethal premature aging disorder caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A. The presence of progerin causes extensive morphological, epigenetic and DNA damage related nuclear defects that ultimately disrupt tissue and organismal functions. Hypothesis-driven approaches focused on HGPS affected pathways have been used in attempts to identify druggable targets with anti-progeroid effects. Here, we report an unbiased discovery approach to HGPS by implementation of a high-throughput, high-content imaging based screening method that enables systematic identification of small molecules that prevent the formation of multiple progerin-induced aging defects. Screening a library of 2816 FDA approved drugs, we identified retinoids as a novel class of compounds that reverses aging defects in HGPS patient skin fibroblasts. These findings establish a novel approach to anti-progeroid drug discovery.
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Senescencia Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Lamina Tipo A/antagonistas & inhibidores , Imagen Molecular/métodos , Retinoides/farmacología , Línea Celular Transformada , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Senescencia Celular/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Epigénesis Genética , Fibroblastos/metabolismo , Fibroblastos/patología , Histonas/genética , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Cultivo Primario de Células , Progeria/genética , Progeria/metabolismo , Progeria/patología , Bibliotecas de Moléculas Pequeñas/farmacología , Transfección , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Two mutant forms (R132H and R132C) of isocitrate dehydrogenase 1 (IDH1) have been associated with a number of cancers including glioblastoma and acute myeloid leukemia. These mutations confer a neomorphic activity of 2-hydroxyglutarate (2-HG) production, and 2-HG has previously been implicated as an oncometabolite. Inhibitors of mutant IDH1 can potentially be used to treat these diseases. In this study, we investigated the mechanism of action of a newly discovered inhibitor, ML309, using biochemical, cellular, and biophysical approaches. Substrate binding and product inhibition studies helped to further elucidate the IDH1 R132H catalytic cycle. This rapidly equilibrating inhibitor is active in both biochemical and cellular assays. The (+) isomer is active (IC50 = 68 nm), whereas the (-) isomer is over 400-fold less active (IC50 = 29 µm) for IDH1 R132H inhibition. IDH1 R132C was similarly inhibited by (+)-ML309. WT IDH1 was largely unaffected by (+)-ML309 (IC50 >36 µm). Kinetic analyses combined with microscale thermophoresis and surface plasmon resonance indicate that this reversible inhibitor binds to IDH1 R132H competitively with respect to α-ketoglutarate and uncompetitively with respect to NADPH. A reaction scheme for IDH1 R132H inhibition by ML309 is proposed in which ML309 binds to IDH1 R132H after formation of the IDH1 R132H NADPH complex. ML309 was also able to inhibit 2-HG production in a glioblastoma cell line (IC50 = 250 nm) and had minimal cytotoxicity. In the presence of racemic ML309, 2-HG levels drop rapidly. This drop was sustained until 48 h, at which point the compound was washed out and 2-HG levels recovered.
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Acetamidas/farmacología , Bencimidazoles/farmacología , Fenómenos Biofísicos , Inhibidores Enzimáticos/farmacología , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Isocitrato Deshidrogenasa/genética , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/genética , Mutación , Acetamidas/metabolismo , Acetamidas/farmacocinética , Animales , Bencimidazoles/metabolismo , Bencimidazoles/farmacocinética , Línea Celular Tumoral , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacocinética , Humanos , Isocitrato Deshidrogenasa/metabolismo , Ratones , Proteínas Mutantes/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacocinética , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Dystonias are a group of chronic movement-disabling disorders for which highly effective oral medications or disease-modifying therapies are lacking. The most effective treatments require invasive procedures such as deep brain stimulation. In this study, we used a high-throughput assay based on a monogenic form of dystonia, DYT1 (DYT-TOR1A), to screen a library of compounds approved for use in humans, the NCATS Pharmaceutical Collection (NPC; 2816 compounds), and identify drugs able to correct mislocalization of the disease-causing protein variant, ∆E302/3 hTorsinA. The HIV protease inhibitor, ritonavir, was among 18 compounds found to normalize hTorsinA mislocalization. Using a DYT1 knock-in mouse model to test efficacy on brain pathologies, we found that ritonavir restored multiple brain abnormalities across development. Ritonavir acutely corrected striatal cholinergic interneuron physiology in the mature brain and yielded sustained correction of diffusion tensor magnetic resonance imaging signals when delivered during a discrete early developmental window. Mechanistically, we found that, across the family of HIV protease inhibitors, efficacy correlated with integrated stress response activation. These preclinical results identify ritonavir as a drug candidate for dystonia with disease-modifying potential.
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Distonía , Inhibidores de la Proteasa del VIH , Animales , Encéfalo/diagnóstico por imagen , Distonía/tratamiento farmacológico , Ratones , Chaperonas Moleculares , Fenotipo , RitonavirRESUMEN
Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are oncogenic for a number of malignancies, primarily low-grade gliomas and acute myeloid leukemia. We report a medicinal chemistry campaign around a 7,7-dimethyl-7,8-dihydro-2H-1λ2-quinoline-2,5(6H)-dione screening hit against the R132H and R132C mutant forms of isocitrate dehydrogenase (IDH1). Systematic SAR efforts produced a series of potent pyrid-2-one mIDH1 inhibitors, including the atropisomer (+)-119 (NCATS-SM5637, NSC 791985). In an engineered mIDH1-U87-xenograft mouse model, after a single oral dose of 30 mg/kg, 16 h post dose, between 16 and 48 h, (+)-119 showed higher tumoral concentrations that corresponded to lower 2-HG concentrations, when compared with the approved drug AG-120 (ivosidenib).
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Inhibidores Enzimáticos/química , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Piridonas/química , Animales , Encéfalo/metabolismo , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Femenino , Glicina/análogos & derivados , Glicina/uso terapéutico , Semivida , Humanos , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Ratones , Ratones Desnudos , Microsomas Hepáticos/metabolismo , Mutagénesis Sitio-Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Piridinas/uso terapéutico , Piridonas/metabolismo , Piridonas/uso terapéutico , Ratas , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AIMS: Enhanced risk stratification of patients with aortic stenosis (AS) is necessary to identify patients at high risk for adverse outcomes, and may allow for better management of patient subgroups at high risk of myocardial damage. The objective of this study was to identify plasma biomarkers and multimarker profiles associated with adverse outcomes in AS. METHODS AND RESULTS: We studied 708 patients with calcific AS and measured 49 biomarkers using a Luminex platform. We studied the correlation between biomarkers and the risk of (i) death and (ii) death or heart failure-related hospital admission (DHFA). We also utilized machine-learning methods (a tree-based pipeline optimizer platform) to develop multimarker models associated with the risk of death and DHFA. In this cohort with a median follow-up of 2.8 years, multiple biomarkers were significantly predictive of death in analyses adjusted for clinical confounders, including tumour necrosis factor (TNF)-α [hazard ratio (HR) 1.28, P < 0.0001], TNF receptor 1 (TNFRSF1A; HR 1.38, P < 0.0001), fibroblast growth factor (FGF)-23 (HR 1.22, P < 0.0001), N-terminal pro B-type natriuretic peptide (NT-proBNP) (HR 1.58, P < 0.0001), matrix metalloproteinase-7 (HR 1.24, P = 0.0002), syndecan-1 (HR 1.27, P = 0.0002), suppression of tumorigenicity-2 (ST2) (IL1RL1; HR 1.22, P = 0.0002), interleukin (IL)-8 (CXCL8; HR 1.22, P = 0.0005), pentraxin (PTX)-3 (HR 1.17, P = 0.001), neutrophil gelatinase-associated lipocalin (LCN2; HR 1.18, P < 0.0001), osteoprotegerin (OPG) (TNFRSF11B; HR 1.26, P = 0.0002), and endostatin (COL18A1; HR 1.28, P = 0.0012). Several biomarkers were also significantly predictive of DHFA in adjusted analyses including FGF-23 (HR 1.36, P < 0.0001), TNF-α (HR 1.26, P < 0.0001), TNFR1 (HR 1.34, P < 0.0001), angiopoietin-2 (HR 1.26, P < 0.0001), syndecan-1 (HR 1.23, P = 0.0006), ST2 (HR 1.27, P < 0.0001), IL-8 (HR 1.18, P = 0.0009), PTX-3 (HR 1.18, P = 0.0002), OPG (HR 1.20, P = 0.0013), and NT-proBNP (HR 1.63, P < 0.0001). Machine-learning multimarker models were strongly associated with adverse outcomes (mean 1-year probability of death of 0%, 2%, and 60%; mean 1-year probability of DHFA of 0%, 4%, 97%; P < 0.0001). In these models, IL-6 (a biomarker of inflammation) and FGF-23 (a biomarker of calcification) emerged as the biomarkers of highest importance. CONCLUSIONS: Plasma biomarkers are strongly associated with the risk of adverse outcomes in patients with AS. Biomarkers of inflammation and calcification were most strongly related to prognosis.
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Estenosis de la Válvula Aórtica , Calcinosis , Insuficiencia Cardíaca , Biomarcadores , Humanos , Péptido Natriurético Encefálico , Fragmentos de Péptidos , PronósticoRESUMEN
Muscle and fat cells translocate GLUT4 (glucose transporter 4) to the plasma membrane when stimulated by insulin. Usually, this event is measured in differentiated adipocytes, myotubes, or cell lines overexpressing tagged GLUT4 by immunostaining. However, measurement of the translocation in differentiated adipocytes or myotubes or GLUT4 overexpressing cell lines is difficult because of high assay variability caused by either the differentiation protocol or low assay sensitivity. We recently reported the identification of a novel splice variant of AS160 (substrate of 160kDa), namely AS160_v2, and showed that its coexpression with GLUT4 in L6 myoblasts increased the insulin-stimulated glucose uptake rate due to an increased amount of GLUT4 on the cell surface. L6 cells, which coexpress myc-tagged GLUT4 and AS160_v2, can be efficiently used to generate an assay useful for identifying compounds that affect cellular responses to insulin. We compared the EC(50) values for radioactive glucose uptake and GLUT4 translocation of different insulins and several small molecules to validate the assay. The use of L6 cells overexpressing AS160_v2 can be considered as a novel tool for the characterization of molecules modulating insulin signaling and GLUT4 translocation, and an image-based assay increases our confidence in the mode of action of the compounds identified.
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Transportador de Glucosa de Tipo 4/metabolismo , Mioblastos/metabolismo , Transporte de Proteínas , Animales , Proteínas Activadoras de GTPasa/biosíntesis , Proteínas Activadoras de GTPasa/metabolismo , Glucosa/metabolismo , Insulina/fisiología , Microscopía Confocal , Ratas , Reproducibilidad de los ResultadosRESUMEN
Little data are available regarding the determinants and prognostic significance of serum albumin in Heart Failure with Preserved Ejection Fraction (HFpEF). We sought to examine the phenotypic correlates of albumin and its independent prognostic implications in HFpEF. We analyzed data from 3,254 subjects enrolled the TOPCAT trial. We stratified subjects according to tertiles of albumin and examined differences in various phenotypic traits between these strata, including 8 protein biomarkers selected ad hoc and measured from frozen samples available in a subset of participants (nâ¯=â¯372). We also assessed the relationship between albumin and the trial primary endpoint. Lower albumin was associated with older age, black race, and greater prevalence of NYHA class III-IV, peripheral arterial disease, atrial fibrillation and diabetes mellitus. Lower albumin was also associated with increased levels of several inflammatory biomarkers, markers of liver fibrosis, albuminuria, and greater arterial stiffness, diastolic dysfunction and pulmonary hypertension. Albumin was a strong predictor of the primary trial endpoint, even after adjustment for the MAGGIC risk score (hazard ratio [HR] 0.72, confidence interval [CI] 0.67 to 0.78; p <0.0001) and prespecified traditional risk factors (HR 0.78, CI 0.71 to 0.85; p <0.0001). Lower albumin was strongly associated with a worse prognosis even well within normal ranges (>3.5 g/dL), with a sharp increase in risk between 4.6 and 3.6 g/dL. In conclusion, albumin is an integrated marker of various adverse processes in HFpEF, including inflammation, subclinical liver disease, arterial stiffness, and renal disease. Albumin is a powerful risk predictor independent of traditional risk prediction models, even within normal ranges.
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Biomarcadores/metabolismo , Insuficiencia Cardíaca/metabolismo , Albúmina Sérica/metabolismo , Anciano , Método Doble Ciego , Femenino , Insuficiencia Cardíaca/tratamiento farmacológico , Humanos , Masculino , Persona de Mediana Edad , Antagonistas de Receptores de Mineralocorticoides/uso terapéutico , Pronóstico , Factores de Riesgo , Espironolactona/uso terapéutico , Volumen SistólicoRESUMEN
Quantitatively determining in vivo achievable drug concentrations in targeted organs of animal models and subsequent target engagement confirmation is a challenge to drug discovery and translation due to lack of bioassay technologies that can discriminate drug binding with different mechanisms. We have developed a multiplexed and high-throughput method to quantify drug distribution in tissues by integrating high content screening (HCS) with U-Net based deep learning (DL) image analysis models. This technology combination allowed direct visualization and quantification of biologics drug binding in targeted tissues with cellular resolution, thus enabling biologists to objectively determine drug binding kinetics.
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Cadherinas/inmunología , Carbocianinas , Aprendizaje Profundo , Colorantes Fluorescentes , Ensayos Analíticos de Alto Rendimiento/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Inmunoconjugados/metabolismo , Animales , Cadherinas/metabolismo , Colon/metabolismo , Descubrimiento de Drogas/métodos , Intestino Delgado/metabolismo , Ratones , Distribución TisularRESUMEN
OBJECTIVES: This study sought to assess if clinical phenogroups differ in comprehensive biomarker profiles, cardiac and arterial structure/function, and responses to spironolactone therapy. BACKGROUND: Previous studies identified distinct subgroups (phenogroups) of patients with heart failure with preserved ejection fraction (HFpEF). METHODS: Among TOPCAT (Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist Trial) participants, we performed latent-class analysis to identify HFpEF phenogroups based on standard clinical features and assessed differences in multiple biomarkers measured from frozen plasma; cardiac and arterial structure/function measured with echocardiography and arterial tonometry; prognosis; and response to spironolactone. RESULTS: Three HFpEF phenogroups were identified. Phenogroup 1 (n = 1,214) exhibited younger age, higher prevalence of smoking, preserved functional class, and the least evidence of left ventricular (LV) hypertrophy and arterial stiffness. Phenogroup 2 (n = 1,329) was older, with normotrophic concentric LV remodeling, atrial fibrillation, left atrial enlargement, large-artery stiffening, and biomarkers of innate immunity and vascular calcification. Phenogroup 3 (n = 899) demonstrated more functional impairment, obesity, diabetes, chronic kidney disease, concentric LV hypertrophy, high renin, and biomarkers of tumor necrosis factor-alpha-mediated inflammation, liver fibrosis, and tissue remodeling. Compared with phenogroup 1, phenogroup 3 exhibited the highest risk of the primary endpoint of cardiovascular death, heart failure hospitalization, or aborted cardiac arrest (hazard ratio [HR]: 3.44; 95% confidence interval [CI]: 2.79 to 4.24); phenogroups 2 and 3 demonstrated similar all-cause mortality (phenotype 2 HR: 2.36; 95% CI: 1.89 to 2.95; phenotype 3 HR: 2.26, 95% CI: 1.77 to 2.87). Spironolactone randomized therapy was associated with a more pronounced reduction in the risk of the primary endpoint in phenogroup 3 (HR: 0.75; 95% CI: 0.59 to 0.95; p for interaction = 0.016). Results were similar after excluding participants from Eastern Europe. CONCLUSIONS: We identified important differences in circulating biomarkers, cardiac/arterial characteristics, prognosis, and response to spironolactone across clinical HFpEF phenogroups. These findings suggest distinct underlying mechanisms across clinically identifiable phenogroups of HFpEF that may benefit from different targeted interventions.
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Insuficiencia Cardíaca/tratamiento farmacológico , Espironolactona/uso terapéutico , Volumen Sistólico/fisiología , Remodelación Ventricular/fisiología , Anciano , Ecocardiografía , Femenino , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Antagonistas de Receptores de Mineralocorticoides/uso terapéutico , Fenotipo , Pronóstico , Resultado del Tratamiento , Remodelación Ventricular/efectos de los fármacosRESUMEN
BACKGROUND: Better risk stratification strategies are needed to enhance clinical care and trial design in heart failure with preserved ejection fraction (HFpEF). OBJECTIVES: The purpose of this study was to assess the value of a targeted plasma multi-marker approach to enhance our phenotypic characterization and risk prediction in HFpEF. METHODS: In this study, the authors measured 49 plasma biomarkers from TOPCAT (Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist) trial participants (n = 379) using a Multiplex assay. The relationship between biomarkers and the risk of all-cause death or heart failure-related hospital admission (DHFA) was assessed. A tree-based pipeline optimizer platform was used to generate a multimarker predictive model for DHFA. We validated the model in an independent cohort of HFpEF patients enrolled in the PHFS (Penn Heart Failure Study) (n = 156). RESULTS: Two large, tightly related dominant biomarker clusters were found, which included biomarkers of fibrosis/tissue remodeling, inflammation, renal injury/dysfunction, and liver fibrosis. Other clusters were composed of neurohormonal regulators of mineral metabolism, intermediary metabolism, and biomarkers of myocardial injury. Multiple biomarkers predicted incident DHFA, including 2 biomarkers related to mineral metabolism/calcification (fibroblast growth factor-23 and OPG [osteoprotegerin]), 3 inflammatory biomarkers (tumor necrosis factor-alpha, sTNFRI [soluble tumor necrosis factor-receptor I], and interleukin-6), YKL-40 (related to liver injury and inflammation), 2 biomarkers related to intermediary metabolism and adipocyte biology (fatty acid binding protein-4 and growth differentiation factor-15), angiopoietin-2 (related to angiogenesis), matrix metalloproteinase-7 (related to extracellular matrix turnover), ST-2, and N-terminal pro-B-type natriuretic peptide. A machine-learning-derived model using a combination of biomarkers was strongly predictive of the risk of DHFA (standardized hazard ratio: 2.85; 95% confidence interval: 2.03 to 4.02; p < 0.0001) and markedly improved the risk prediction when added to the MAGGIC (Meta-Analysis Global Group in Chronic Heart Failure Risk Score) risk score. In an independent cohort (PHFS), the model strongly predicted the risk of DHFA (standardized hazard ratio: 2.74; 95% confidence interval: 1.93 to 3.90; p < 0.0001), which was also independent of the MAGGIC risk score. CONCLUSIONS: Various novel circulating biomarkers in key pathophysiological domains are predictive of outcomes in HFpEF, and a multimarker approach coupled with machine-learning represents a promising strategy for enhancing risk stratification in HFpEF.
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Biomarcadores/sangre , Insuficiencia Cardíaca/sangre , Aprendizaje Automático , Anciano , Femenino , Insuficiencia Cardíaca/mortalidad , Humanos , Masculino , Persona de Mediana Edad , Medición de Riesgo , Estados Unidos/epidemiologíaRESUMEN
Nonalcoholic steatohepatitis represents a significant and rapidly growing unmet medical need. The development of novel therapies has been hindered in part, by the limitations of existing preclinical models. There is a strong need for physiologically relevant in vivo and in vitro liver fibrosis models that are characterized by better translational predictability. In this study, we used the InSphero 3D InSightTM three-dimensional (3D) human liver microtissue (3D-hLMT) system prepared by co-culturing primary human hepatocytes with hepatic stellate cells, Kupffer cells and endothelial cells to develop a model of NASH with a severe fibrotic phenotype. In our model, palmitic acid (PA) induced a robust proinflammatory and profibrogenic phenotype in the 3D-hLMT. PA significantly increased several markers of the inflammatory and profibrotic process including gene expression of collagens, α-sma, tissue inhibitor of matrix metalloprotease 1 (timp1) and the stellate cell activation marker pdgfrß as well as secreted CXCL8 (IL8) levels. We also observed TGFß pathway activation, increase in active collagen synthesis and significant overall increase in tissue damage in the 3D-hLMTs. Immunohistochemistry analysis demonstrated the upregulation of collagen, cleaved caspase 3 as well as of the PDGFRß protein. We further validated the model using a phase 3 clinical compound, GS-4997, an apoptosis signal-regulating kinase 1 (ASK-1) inhibitor and showed that GS-4997 significantly decreased PA induced profibrotic and proinflammatory response in the 3D-hLMTs with decreases in apoptosis and stellate cell activation in the microtissues. Taken together we have established and validated an in vitro 3D-hLMT NASH model with severe fibrotic phenotype that can be a powerful tool to investigate experimental compounds for the treatment of NASH.