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1.
Mol Ther Nucleic Acids ; 35(3): 102246, 2024 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-39027419

RESUMEN

Huntington's disease (HD) is an autosomal dominant disease caused by the expansion of cytosine-adenine-guanine (CAG) repeats in one copy of the HTT gene (mutant HTT, mHTT). The unaffected HTT gene encodes wild-type HTT (wtHTT) protein, which supports processes important for the health and function of the central nervous system. Selective lowering of mHTT for the treatment of HD may provide a benefit over nonselective HTT-lowering approaches, as it aims to preserve the beneficial activities of wtHTT. Targeting a heterozygous single-nucleotide polymorphism (SNP) where the targeted variant is on the mHTT gene is one strategy for achieving allele-selective activity. Herein, we investigated whether stereopure phosphorothioate (PS)- and phosphoryl guanidine (PN)-containing oligonucleotides can direct allele-selective mHTT lowering by targeting rs362273 (SNP3). We demonstrate that our SNP3-targeting molecules are potent, durable, and selective for mHTT in vitro and in vivo in mouse models. Through comparisons with a surrogate for the nonselective investigational compound tominersen, we also demonstrate that allele-selective molecules display equivalent potency toward mHTT with improved durability while sparing wtHTT. Our preclinical findings support the advancement of WVE-003, an investigational allele-selective compound currently in clinical testing (NCT05032196) for the treatment of patients with HD.

2.
Nucleic Acid Ther ; 33(5): 287-305, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37590469

RESUMEN

This white paper summarizes the recommendations of the absorption, distribution, metabolism, and excretion (ADME) Subcommittee of the Oligonucleotide Safety Working Group for the characterization of absorption, distribution, metabolism, and excretion of oligonucleotide (ON) therapeutics in nonclinical studies. In general, the recommended approach is similar to that for small molecule drugs. However, some differences in timing and/or scope may be warranted due to the greater consistency of results across ON classes as compared with the diversity among small molecule classes. For some types of studies, a platform-based approach may be appropriate; once sufficient data are available for the platform, presentation of these data should be sufficient to support development of additional ONs of the same platform. These recommendations can serve as a starting point for nonclinical study design and foundation for discussions with regulatory agencies.


Asunto(s)
Oligonucleótidos , Oligonucleótidos/uso terapéutico , Oligonucleótidos/farmacocinética
3.
World J Clin Cases ; 9(3): 758-763, 2021 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-33553417

RESUMEN

BACKGROUND: The in-stent restenosis (ISR) rates are reportedly inconsistent despite the increased use of second-generation drug eluting stent (DES). Although bioresorbable vascular scaffold (BVS) have substantial advantages with respect to vascular restoration, the rate of scaffold thrombosis is higher with BVS than with DES. Optimal treatment strategies have not been established for DES-ISR to date. CASE SUMMARY: We report on a case of a 60-year-old man patient with acute coronary syndrome. He had a history of ST-segment elevation myocardial infarction associated with very late scaffold thrombosis and treated with a DES. Coronary angiography revealed significant stenosis, suggesting DES-ISR on the previous BVS. Optical coherence tomography (OCT) identified a plaque rupture and a disrupted scaffold strut in the neointimal proliferation of DES. To treat the DES-ISR on the previous BVS, we opted for a drug-coated balloon (DCB) after a balloon angioplasty using a semi-compliant and non-compliant balloon. The patient did not experience adverse cardiovascular events on using a DCB following the use of intensive dual antiplatelet therapy and statin for 24 mo. CONCLUSION: This case highlights the importance of OCT as an imaging modality for characterizing the mechanism of target lesion failure. The use of a DCB following the administration of optimal pharmacologic therapy may be an optimal strategy for the treatment and prevention of recurrent BVS thrombosis and DES-ISR.

4.
Nat Commun ; 12(1): 847, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558503

RESUMEN

A large G4C2-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Neuronal degeneration associated with this expansion arises from a loss of C9orf72 protein, the accumulation of RNA foci, the expression of dipeptide repeat (DPR) proteins, or all these factors. We report the discovery of a new targeting sequence that is common to all C9orf72 transcripts but enables preferential knockdown of repeat-containing transcripts in multiple cellular models and C9BAC transgenic mice. We optimize stereopure oligonucleotides that act through this site, and we demonstrate that their preferential activity depends on both backbone stereochemistry and asymmetric wing design. In mice, stereopure oligonucleotides produce durable depletion of pathogenic signatures without disrupting protein expression. These oligonucleotides selectively protect motor neurons harboring C9orf72-expansion mutation from glutamate-induced toxicity. We hypothesize that targeting C9orf72 with stereopure oligonucleotides may be a viable therapeutic approach for the treatment of C9orf72-associated neurodegenerative disorders.


Asunto(s)
Proteína C9orf72/genética , Expansión de las Repeticiones de ADN/genética , Mutación/genética , Oligonucleótidos/química , Oligonucleótidos/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Proteína C9orf72/química , Exones/genética , Glutamatos/toxicidad , Intrones/genética , Ratones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estereoisomerismo
5.
Bioanalysis ; 10(22): 1781-1801, 2018 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-30488725

RESUMEN

The 2018 12th Workshop on Recent Issues in Bioanalysis (12th WRIB) took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LC-MS, hybrid ligand binding assay (LBA)/LC-MS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for LC-MS for small molecules, peptides, oligonucleotides and small molecule biomarkers. Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) and Part 3 (large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays) are published in volume 10 of Bioanalysis, issues 23 and 24 (2018), respectively.


Asunto(s)
Biomarcadores/análisis , Oligonucleótidos/análisis , Péptidos/análisis , Animales , Cromatografía Liquida , Humanos , Espectrometría de Masas , Philadelphia
6.
Coron Artery Dis ; 29(7): 589-596, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-29965838

RESUMEN

BACKGROUND: This study aimed to evaluate whether the coronary artery calcium score (CACS) measured with computed tomography coronary angiography (CTCA) predicts periprocedural myocardial infarction (PMI) in patients undergoing an elective percutaneous coronary intervention (PCI). PATIENTS AND METHODS: A total of 197 patients with stable angina underwent elective PCI after CTCA. We evaluated CACS using CTCA and assessed the clinical risk factors for PMI. PMI was defined as an elevation of troponin I levels exceeding five times the upper limit of normal within 24 h after PCI. Patients were followed up for major adverse cardiovascular events for a median of 4.6 years. RESULTS: The prevalence of PMI was 18.7% (37 patients) and patients with PMI showed a trend toward a higher CACS (721±779 vs. 498±842, P=0.142). The prevalence of PMI showed a positive correlation with the CACS distribution [8.0%, first interquartile range (IQR); 14.3%, second IQR; 22.4%, third IQR; 30.6%, fourth IQR; P=0.002]. The CACS cut-off value for PMI was greater than 113 (area under the curve: 0.670; 95% confidence interval: 0.600-0.736; P<0.001). Patients with CACS greater than 113 before PCI showed a higher prevalence of PMI (26.2 vs. 5.6%, odds ratio: 5.994; P<0.001). Multivariate analysis showed that CACS greater than 113 was the main predictor for PMI (odds ratio: 3.61, 95% confidence interval: 1.145-11.363; P=0.028). In this study, the cumulative incidence of major adverse cardiovascular event was higher in patients with PMI (54.1 vs. 10.6%; P<0.001). CONCLUSION: This study suggests that high CACS measured with CTCA influences the occurrence of PMI, which is associated with worse cardiovascular outcomes.


Asunto(s)
Angiografía por Tomografía Computarizada , Angiografía Coronaria/métodos , Enfermedad de la Arteria Coronaria/terapia , Infarto del Miocardio/epidemiología , Intervención Coronaria Percutánea/efectos adversos , Calcificación Vascular/terapia , Anciano , Biomarcadores/sangre , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Enfermedad de la Arteria Coronaria/epidemiología , Femenino , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Infarto del Miocardio/sangre , Infarto del Miocardio/diagnóstico , Valor Predictivo de las Pruebas , Prevalencia , República de Corea/epidemiología , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Índice de Severidad de la Enfermedad , Factores de Tiempo , Resultado del Tratamiento , Troponina I/sangre , Regulación hacia Arriba , Calcificación Vascular/diagnóstico por imagen , Calcificación Vascular/epidemiología
7.
Clin Cancer Res ; 22(22): 5527-5538, 2016 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-27780853

RESUMEN

PURPOSE: Non-small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK+) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib. EXPERIMENTAL DESIGN: A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib-ALK co-structure was determined. RESULTS: Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK+ cell lines, brigatinib inhibited native ALK (IC50, 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK+ tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses. CONCLUSIONS: Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK+, crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527-38. ©2016 AACR.


Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Compuestos Organofosforados/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Crizotinib , Células Hep G2 , Humanos , Neoplasias Pulmonares/metabolismo , Mutación/efectos de los fármacos , Pirazoles/farmacología , Piridinas/farmacología , Sulfonas/farmacología , Células U937
8.
J Med Chem ; 59(10): 4948-64, 2016 05 26.
Artículo en Inglés | MEDLINE | ID: mdl-27144831

RESUMEN

In the treatment of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase positive (ALK+) non-small-cell lung cancer (NSCLC), secondary mutations within the ALK kinase domain have emerged as a major resistance mechanism to both first- and second-generation ALK inhibitors. This report describes the design and synthesis of a series of 2,4-diarylaminopyrimidine-based potent and selective ALK inhibitors culminating in identification of the investigational clinical candidate brigatinib. A unique structural feature of brigatinib is a phosphine oxide, an overlooked but novel hydrogen-bond acceptor that drives potency and selectivity in addition to favorable ADME properties. Brigatinib displayed low nanomolar IC50s against native ALK and all tested clinically relevant ALK mutants in both enzyme-based biochemical and cell-based viability assays and demonstrated efficacy in multiple ALK+ xenografts in mice, including Karpas-299 (anaplastic large-cell lymphomas [ALCL]) and H3122 (NSCLC). Brigatinib represents the most clinically advanced phosphine oxide-containing drug candidate to date and is currently being evaluated in a global phase 2 registration trial.


Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Neoplasias Pulmonares/tratamiento farmacológico , Compuestos Organofosforados/farmacología , Fosfinas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Administración Oral , Quinasa de Linfoma Anaplásico , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Ratones , Ratones SCID , Conformación Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Compuestos Organofosforados/administración & dosificación , Compuestos Organofosforados/química , Fosfinas/farmacología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Pirimidinas/administración & dosificación , Pirimidinas/química , Ratas , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Relación Estructura-Actividad
9.
Nat Genet ; 45(1): 18-24, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23222956

RESUMEN

Atypical chronic myeloid leukemia (aCML) shares clinical and laboratory features with CML, but it lacks the BCR-ABL1 fusion. We performed exome sequencing of eight aCMLs and identified somatic alterations of SETBP1 (encoding a p.Gly870Ser alteration) in two cases. Targeted resequencing of 70 aCMLs, 574 diverse hematological malignancies and 344 cancer cell lines identified SETBP1 mutations in 24 cases, including 17 of 70 aCMLs (24.3%; 95% confidence interval (CI) = 16-35%). Most mutations (92%) were located between codons 858 and 871 and were identical to changes seen in individuals with Schinzel-Giedion syndrome. Individuals with mutations had higher white blood cell counts (P = 0.008) and worse prognosis (P = 0.01). The p.Gly870Ser alteration abrogated a site for ubiquitination, and cells exogenously expressing this mutant exhibited higher amounts of SETBP1 and SET protein, lower PP2A activity and higher proliferation rates relative to those expressing the wild-type protein. In summary, mutated SETBP1 represents a newly discovered oncogene present in aCML and closely related diseases.


Asunto(s)
Proteínas Portadoras/genética , Leucemia Mieloide Crónica Atípica BCR-ABL Negativa/genética , Mutación , Proteínas Nucleares/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN , Exoma , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Humanos , Leucemia Mieloide Crónica Atípica BCR-ABL Negativa/mortalidad , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Pronóstico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Fosfatasa 2/metabolismo , Análisis de Secuencia de ADN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
11.
J Exp Med ; 207(2): 339-44, 2010 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-20142433

RESUMEN

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2), are present in most gliomas and secondary glioblastomas, but are rare in other neoplasms. IDH1/2 mutations are heterozygous, and affect a single arginine residue. Recently, IDH1 mutations were identified in 8% of acute myelogenous leukemia (AML) patients. A glioma study revealed that IDH1 mutations cause a gain-of-function, resulting in the production and accumulation of 2-hydroxyglutarate (2-HG). Genotyping of 145 AML biopsies identified 11 IDH1 R132 mutant samples. Liquid chromatography-mass spectrometry metabolite screening revealed increased 2-HG levels in IDH1 R132 mutant cells and sera, and uncovered two IDH2 R172K mutations. IDH1/2 mutations were associated with normal karyotypes. Recombinant IDH1 R132C and IDH2 R172K proteins catalyze the novel nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of alpha-ketoglutarate (alpha-KG) to 2-HG. The IDH1 R132C mutation commonly found in AML reduces the affinity for isocitrate, and increases the affinity for NADPH and alpha-KG. This prevents the oxidative decarboxylation of isocitrate to alpha-KG, and facilitates the conversion of alpha-KG to 2-HG. IDH1/2 mutations confer an enzymatic gain of function that dramatically increases 2-HG in AML. This provides an explanation for the heterozygous acquisition of these mutations during tumorigenesis. 2-HG is a tractable metabolic biomarker of mutant IDH1/2 enzyme activity.


Asunto(s)
Isocitrato Deshidrogenasa/genética , Leucemia Mieloide Aguda/enzimología , Mutación , Catálisis , Glutaratos/sangre , Humanos , Isocitrato Deshidrogenasa/metabolismo , Ácidos Cetoglutáricos/metabolismo , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/genética , NADP/metabolismo , Proteínas Recombinantes/metabolismo
12.
Nature ; 462(7274): 739-44, 2009 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-19935646

RESUMEN

Mutations in the enzyme cytosolic isocitrate dehydrogenase 1 (IDH1) are a common feature of a major subset of primary human brain cancers. These mutations occur at a single amino acid residue of the IDH1 active site, resulting in loss of the enzyme's ability to catalyse conversion of isocitrate to alpha-ketoglutarate. However, only a single copy of the gene is mutated in tumours, raising the possibility that the mutations do not result in a simple loss of function. Here we show that cancer-associated IDH1 mutations result in a new ability of the enzyme to catalyse the NADPH-dependent reduction of alpha-ketoglutarate to R(-)-2-hydroxyglutarate (2HG). Structural studies demonstrate that when arginine 132 is mutated to histidine, residues in the active site are shifted to produce structural changes consistent with reduced oxidative decarboxylation of isocitrate and acquisition of the ability to convert alpha-ketoglutarate to 2HG. Excess accumulation of 2HG has been shown to lead to an elevated risk of malignant brain tumours in patients with inborn errors of 2HG metabolism. Similarly, in human malignant gliomas harbouring IDH1 mutations, we find markedly elevated levels of 2HG. These data demonstrate that the IDH1 mutations result in production of the onco-metabolite 2HG, and indicate that the excess 2HG which accumulates in vivo contributes to the formation and malignant progression of gliomas.


Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Glutaratos/metabolismo , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Arginina/genética , Neoplasias Encefálicas/patología , Dominio Catalítico , Línea Celular , Cristalografía por Rayos X , Progresión de la Enfermedad , Pruebas de Enzimas , Glioma/genética , Glioma/metabolismo , Glioma/patología , Histidina/genética , Histidina/metabolismo , Humanos , Ácidos Cetoglutáricos/metabolismo , Modelos Moleculares , Mutación/genética , Conformación Proteica
13.
Anal Biochem ; 359(2): 151-60, 2006 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-17081492

RESUMEN

Detection and analysis of DNA adducts resulting from endogenous or exogenous exposures to carcinogens are essential not only for quantifying biologically effective doses but also for establishing relationships between exposure and cancer risk. We have developed and validated a procedure of high sensitivity and specificity based on fluorescence labeling of DNA adducts combined with high-performance liquid chromatography-laser-induced fluorescence detection. The fluorescent dye 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY FL) was used to label the deoxynucleoside adducts N-(2'-deoxyguanosine-8-yl)-4-aminobiphenyl and N-(2'-deoxyguanosine-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and the base adduct aflatoxin B(1)-formamidopyrimidine by acylation. The labeling reaction was carried out on adducts at 1pmol to 30nmol concentrations at 25 degrees C for 4h in dichloromethane with 200- to 5000-fold excess of BODIPY FL. BODIPY FL and its activating agents 1,3-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were used at a molar ratio of 1:2:2. Under these conditions, all of the above adducts were quantitatively converted to bis-labeled products, as confirmed by mass spectrometry. Sites of derivatization of adduct deoxynucleosides were established primarily by nuclear magnetic resonance and by collision-induced dissociation mass spectrometric analysis, which indicated that the bis-BODIPY groups were located predominantely on the 3'- and 5'-hydroxyl groups of the deoxyribose ring.


Asunto(s)
Aflatoxina B1/aislamiento & purificación , Compuestos de Boro/química , Aductos de ADN/aislamiento & purificación , Desoxiguanosina/análogos & derivados , Exposición a Riesgos Ambientales/análisis , Colorantes Fluorescentes/química , Aflatoxina B1/análogos & derivados , Aflatoxina B1/química , Compuestos de Aminobifenilo/química , Animales , Biomarcadores/análisis , Carcinógenos/química , Carcinógenos/aislamiento & purificación , Aductos de ADN/química , Imidazoles , Indicadores y Reactivos , Mutágenos/química , Mutágenos/aislamiento & purificación , Pirimidinas/aislamiento & purificación , Sensibilidad y Especificidad
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