RESUMEN
OBJECTIVE: The natural molecule α-lipoic acid has been shown to be partially cytoprotective through antioxidant and antiapoptotic mechanisms. To obtain an initial assessment of the safety and potential efficacy of a synthetic derivative, CMX-2043, in preventing ischemic complications of percutaneous coronary intervention (PCI) we conducted the Subjects Undergoing PCI and Perioperative Reperfusion Treatment (SUPPORT-1) trial, the first patient experience with this agent. METHODS AND RESULTS: SUPPORT-1 was a phase 2a, 6-center, international, placebo-controlled, randomized, double-blind trial. A total of 142 patients were randomized to receive a single intravenous bolus dose of drug or placebo administered 15-60 minutes before PCI. Cardiac biomarker assessments included serial measurements of creatine kinase myocardial band (CK-MB) at 6, 12, 18, and 24 hours after PCI and a single measurement of troponin T (TnT) at 24 hours. Peak concentrations of CK-MB and TnT were significantly reduced in the 2.4 mg/kg group compared with placebo (P = 0.05 and 0.03, respectively). No subject administered 2.4 mg/kg of CMX-2043 had an increase of CK-MB to ≥3X upper limit of normal versus 16% for placebo (P = 0.02); 16% of the 2.4-mg/kg dose group developed an elevation of TnT to ≥3X upper limit of normal versus 39% in the placebo group (P = 0.05). No drug-related serious adverse events were observed in any group. CONCLUSION: These data suggest that CMX-2043 may reduce PCI periprocedural myonecrosis and support further clinical evaluation of this novel agent for its potential cytoprotective effects.
Asunto(s)
Angioplastia Coronaria con Balón , Fármacos Cardiovasculares/uso terapéutico , Enfermedad de la Arteria Coronaria/terapia , Dipéptidos/uso terapéutico , Miocitos Cardíacos/efectos de los fármacos , Ácido Tióctico/análogos & derivados , Anciano , Angioplastia Coronaria con Balón/efectos adversos , Biomarcadores/sangre , Fármacos Cardiovasculares/efectos adversos , Fármacos Cardiovasculares/farmacocinética , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Forma MB de la Creatina-Quinasa/sangre , Dipéptidos/efectos adversos , Dipéptidos/farmacocinética , Método Doble Ciego , Femenino , Humanos , India , Masculino , Persona de Mediana Edad , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Necrosis , Estudios Prospectivos , Ácido Tióctico/efectos adversos , Ácido Tióctico/farmacocinética , Ácido Tióctico/uso terapéutico , Factores de Tiempo , Resultado del Tratamiento , Troponina T/sangre , Estados UnidosRESUMEN
α-Lipoic acid has been shown to provide cytoprotection in some tissues through antioxidant and antiapoptotic mechanisms. We have enhanced these properties by synthetic modification, resulting in a new chemical entity, CMX-2043, with proven efficacy in an animal model of cardiac ischemia-reperfusion injury. The present studies compare cytoprotective cellular pathways of R-α-lipoic acid and CMX-2043. Biochemical and cellular assays were used to compare antioxidant potency, tyrosine kinase activation, and protein kinase B (Akt) phosphorylation. CMX-2043 was more effective than lipoic acid in antioxidant effect, activation of insulin receptor kinase, soluble tyrosine kinase, and Akt phosphorylation. Activation of insulin-like growth factor 1 receptor was similar for both. CMX-2043 stimulation of Akt phosphorylation was abolished by the phosphatidylinositide 3-kinase inhibitor LY294002. Consistent with Akt activation, CMX-2043 reduced carbachol-induced calcium overload. The S-stereoisomer of CMX-2043 was less active in the biochemical assays than the R-isomer. These results are consistent with cytoprotection through activation of Akt and antioxidant action. CMX-2043 may thus provide a pharmacological approach to cytoprotection consistent with established anti-apoptotic mechanisms.
Asunto(s)
Citoprotección/efectos de los fármacos , Dipéptidos/farmacología , Activadores de Enzimas/farmacología , Ácido Tióctico/análogos & derivados , Ácido Tióctico/farmacología , Células A549 , Animales , Células CHO , Calcio/metabolismo , Cardiotónicos/farmacología , Cricetinae , Cricetulus , Citoprotección/fisiología , Relación Dosis-Respuesta a Droga , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
α-Lipoic acid (LA) has been shown to offer protection against ischemia-reperfusion injury (IRI) in multiple organ systems. N-[(R)-1,2-dithiolane-3-pentanoyl]-L-glutamyl-L-alanine (CMX-2043), a novel analogue of LA, was studied as part of a preclinical development program intended to identify safe and efficacious drug candidates for prevention or reduction in myocardial IRI. This study was designed to evaluate the efficacy of CMX-2043 in an animal model of myocardial IRI and to establish effective dosing conditions. CMX-2043 or placebo was administered at different doses, routes, and times in male Sprague-Dawley rats subjected to 30-minute left coronary artery ligation. Fluorescent microsphere injection defined the area at risk (AR). Animals were euthanized 24 hours after reperfusion, and the hearts were excised, sectioned, and stained with triphenyltetrazolium. Cytoprotective effectiveness was determined by comparing the unstained myocardial infarction zone (MI) to the ischemic AR. The reduction in the MI-AR ratio was used as the primary measure of drug efficacy relative to placebo injections. Treatment with CMX-2043 reduced myocardial IRI as measured by the MI-AR ratio and the incidence of arrhythmia. The compound was effective when administered by injection, both before and during the ischemic injury and at reperfusion. The most efficacious dose was that administered 15 minutes prior to the ischemic event and resulted in a 36% (P < .001) reduction in MI-AR ratio compared to vehicle control.
Asunto(s)
Fármacos Cardiovasculares/administración & dosificación , Dipéptidos/administración & dosificación , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/patología , Ácido Tióctico/análogos & derivados , Administración Oral , Animales , Arritmias Cardíacas/etiología , Arritmias Cardíacas/prevención & control , Fármacos Cardiovasculares/farmacocinética , Citoprotección , Dipéptidos/farmacocinética , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Inyecciones Intravenosas , Masculino , Infarto del Miocardio/etiología , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/etiología , Daño por Reperfusión Miocárdica/patología , Ratas Sprague-Dawley , Ácido Tióctico/administración & dosificación , Ácido Tióctico/farmacocinéticaRESUMEN
Although much has been learned about basic mechanisms of cell invasion, the genes whose expression is required for this process by malignant cell lines have remained obscure. We assessed invasion through Matrigel using EGF as a chemoattractant and gene expression profiles using oligonucleotide microarrays for 22 non-small cell lung cancer cell lines. The expression of 22 genes were significantly correlated (p < 0.001) with the measured invasion index. Cluster analysis demonstrated that gene expression profiles classify the cell lines into low and high invasive subgroups. Considering invasiveness as a dichotomous variable, Bayesian analysis was used to identify genes that have the highest probability of being differentially expressed between the high and low invasion groups. This analysis identified 16 genes whose expression was associated with invasiveness. "Leave one out" cross validation was 91% accurate. Nine genes were identified in both correlation and Bayesian analyses. Seven of the nine genes were negatively associated with invasion and four of those genes are plasma membrane proteins. The two genes with the highest inverse association with invasion, TACSTD1 and CLDN3, are involved with cell adhesion and cell-cell interactions, respectively. Interestingly, the gene with the highest positive association with invasion, SERPINE1 (PAI-1), is a protease inhibitor. These and the other genes identified by both analyses represent targets for further study to assess their importance in non-small cell lung cancer invasion and metastasis.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Invasividad Neoplásica , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/secundario , Biomarcadores de Tumor , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Adhesión Celular , Colágeno/metabolismo , Combinación de Medicamentos , Humanos , Laminina/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteoglicanos/metabolismo , Células Tumorales CultivadasRESUMEN
CMX-2043 is an α-lipoic acid analogue targeted to reduction of cellular injury and organ damage due to ischaemia-reperfusion injury (IRI). It has been shown to be effective in a rat model of cardiac IRI. The studies here reported evaluate its safety and pharmacokinetic profile in preparation for human clinical studies in procedures associated with IRI. Safety and tolerability were tested in standard pre-clinical in vitro and animal models and in a Phase 1 human clinical trial. CMX-2043 did not bind to a wide range of receptors and specific targets at approximately 4 µg/mL (10 µM). It was not mutagenic by Ames assay, did not produce chromosome aberrations in Chinese hamster ovary (CHO) cells, and was negative for clastogenic potential. Toxicological studies in rats including both single and 14-day repeat intravenous doses and in dogs (single intravenous dose) with a 2-week recovery period were conducted. The NOAEL in rats and dogs was 30 and >10 mg/kg, respectively. No serious adverse events were reported in a placebo-controlled, sequential dose escalation Phase 1 clinical trial. The low toxicity in the pre-clinical studies and the absence of adverse events in the Phase 1 trial have supported investigation of CMX-2043 in a human efficacy trial.
Asunto(s)
Dipéptidos/uso terapéutico , Daño por Reperfusión/tratamiento farmacológico , Ácido Tióctico/análogos & derivados , Administración Intravenosa , Adolescente , Adulto , Animales , Células CHO , Cricetinae , Cricetulus , Dipéptidos/administración & dosificación , Perros , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Masculino , Pruebas de Mutagenicidad , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Ácido Tióctico/administración & dosificación , Ácido Tióctico/uso terapéutico , Adulto JovenRESUMEN
Crotoxin (CTX), the major component of the venom from the South American rattlesnake (Crotalus durissus terrificus) has diverse toxic effects, including pre-synaptic neurotoxicity. Among these, the effect of CTX in the heart is the least understood. In this study, we explored the effect(s) of CTX on the electrophysiological activity of neonatal rat cardiomyocytes (NRCM). By using patch, voltage-, and current-clamping techniques, we found that, in NRCM, CTX strongly potentiates L-type Ca2+ currents, an important contributor to the cardiac action potential (AP) and excitation-contraction (EC) coupling. External addition of CTX produced a rapid increase in L-type Ca2+ currents. Addition of verapamil (10 microM) an L-type Ca2+ channel blocker, completely blocked the CTX-induced increase in the currents. A detailed kinetic analysis of AP in NRCM revealed that CTX caused both an elongation of AP duration (APD) and an increase of its amplitude, while a decrease of firing frequency. In addition, increasing concentrations of CTX completely blocked the AP as well as the beating of NRCM. The data indicate that CTX is a potent modulator of L-type Ca2+ currents, which provides a possible mechanistic correlation for the cardiotoxicity of the toxin, and may offer potential clinical implications in the control of cardiac function.
Asunto(s)
Agonistas de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Crotoxina/farmacología , Miocitos Cardíacos/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Animales Recién Nacidos , Células Cultivadas , Electrofisiología , Frecuencia Cardíaca/efectos de los fármacos , Cinética , Miocitos Cardíacos/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Retículo Sarcoplasmático/efectos de los fármacosRESUMEN
Acquisition of motility is an important step in malignant progression of tumor cells and involves dynamic changes in actin filament architecture orchestrated by many actin binding proteins. A role for the actin-binding protein gelsolin has been demonstrated in fibroblast motility. In this report, we investigated the role of gelsolin in bronchial epithelial cell motility. The non-tumorigenic bronchial epithelial cell line, NL20 migrated towards EGF in a modified Boyden chamber cell motility assay. However, the tumorigenic NL20-TA cell line derived from the NL20 cells and lacking gelsolin, did not migrate towards EGF. Ectopic expression of gelsolin in NL20-TA cells restored the EGF response, while motility of NL20-TA derived cells towards serum, PDGF, and fibronectin was independent of gelsolin expression. PI3-kinase inhibition failed to block EGF-stimulated motility in gelsolin transfected NL20-TA cells. Furthermore, EGF stimulated a motility response in cells lacking gelsolin in the presence of fibronectin or fibrinogen that was blocked with PI3-kinase inhibition. Thus, EGF-stimulated motility in NL20 cells and its derivatives are gelsolin dependent and PI3-kinase independent, while fibronectin and fibrinogen enhances EGF-stimulated motility through a pathway independent of gelsolin and dependent upon PI3-kinase.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/efectos de los fármacos , Gelsolina/metabolismo , Pulmón/citología , Anticuerpos Monoclonales/metabolismo , Calcio/metabolismo , Línea Celular , Línea Celular Tumoral , Cromonas/farmacología , Medio de Cultivo Libre de Suero , Inhibidores Enzimáticos/farmacología , Células Epiteliales/metabolismo , Estrenos/farmacología , Fibrinógeno/farmacología , Fibronectinas/farmacología , Humanos , Modelos Biológicos , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Pirrolidinonas/farmacología , Suero/metabolismo , Transfección , Fosfolipasas de Tipo C/metabolismoRESUMEN
Tuberous sclerosis is a hamartoma syndrome due to mutations in TSC1 or TSC2 in which cardiac rhabdomyomas are seen in approximately 60% of patients. These lesions have an unusual natural history as they are usually most prominent immediately after birth and spontaneously resolve in most cases. To develop a mouse model of this lesion, we used a conditional, floxed allele of Tsc1 and a modified myosin light chain 2v allele in which cre recombinase expression occurs in ventricular myocytes. Mice with ventricular loss of Tsc1 had a median survival of 6 months and developed a dilated cardiomyopathy with the occurrence of scattered foci of enlarged ventricular myocytes. The enlarged cells were periodic acid-Schiff positive indicating the presence of excess glycogen and expressed elevated levels of phospho-S6, similar to findings in patient rhabdomyoma cells. The observations confirm that rhabdomyomas occur through a two hit mechanism of pathogenesis. However, the mice showed no evidence of fetal/neonatal demise, and there was no evidence of proliferation in the lesions. We propose that these differences are due to the timing of loss of Tsc1 in the ventricular myocytes and/or the truncated gestational period in the mouse compared with humans, during which progestational hormones may accentuate the growth of patient rhabdomyomas.
Asunto(s)
Neoplasias Cardíacas/metabolismo , Células Musculares/metabolismo , Miocardio/metabolismo , Rabdomioma/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Alelos , Animales , Miosinas Cardíacas/genética , Proliferación Celular , Glucógeno/metabolismo , Neoplasias Cardíacas/genética , Neoplasias Cardíacas/patología , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Integrasas/genética , Ratones , Células Musculares/patología , Mutación , Miocardio/patología , Cadenas Ligeras de Miosina/genética , Fosforilación , Rabdomioma/genética , Rabdomioma/patología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Esclerosis Tuberosa/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
Tuberous sclerosis complex (TSC) is a familial tumor disorder for which there is no effective medical therapy. Disease-causing mutations in the TSC1 or TSC2 gene lead to increased mammalian target of rapamycin (mTOR) kinase activity in the conserved mTOR signaling pathway, which regulates nutrient uptake, cell growth, and protein translation. The normal function of TSC1 and TSC2 gene products is to form a complex that reduces mTOR kinase activity. Thus, mTOR kinase inhibition may be a useful targeted therapeutic approach. Elevated interferon-gamma (IFN-gamma) expression is associated with decreased severity of kidney tumors in TSC patients and mouse models; therefore, IFN-gamma also has therapeutic potential. We studied cohorts of Tsc2+/- mice and a novel mouse model of Tsc2-null tumors in order to evaluate the efficacy of targeted therapy for TSC. We found that treatment with either an mTOR kinase inhibitor (CCI-779, a rapamycin analog) or with IFN-gamma reduced the severity of TSC-related disease without significant toxicity. These results constitute definitive preclinical data that justify proceeding with clinical trials using these agents in selected patients with TSC and related disorders.
Asunto(s)
Modelos Animales de Enfermedad , Interferón gamma/uso terapéutico , Proteínas Represoras/fisiología , Sirolimus/análogos & derivados , Sirolimus/uso terapéutico , Esclerosis Tuberosa/tratamiento farmacológico , Proteínas Supresoras de Tumor/fisiología , Animales , Cistoadenoma/metabolismo , Cistoadenoma/patología , Cistoadenoma/prevención & control , Quimioterapia Combinada , Hemangioma/metabolismo , Hemangioma/patología , Hemangioma/prevención & control , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Neoplasias Renales/prevención & control , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas Experimentales/prevención & control , Ratones , Ratones Noqueados , Ratones Desnudos , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Represoras/genética , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR , Esclerosis Tuberosa/metabolismo , Esclerosis Tuberosa/patología , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
In this study, the presence of Na(+)-permeable cation channels was determined and characterized in LLC-PK1 cells, a renal tubular epithelial cell line with proximal tubule characteristics derived from pig kidney. Patch-clamp analysis under cell-attached conditions indicated the presence of spontaneously active Na(+)-permeable cation channels. The channels displayed nonrectifying single channel conductance of 11 pS, substates, and an approximately 3:1 Na(+)/K(+) permeability-selectivity ratio. The Na(+)-permeable cation channels were inhibited by pertussis toxin and reactivated by G protein agonists. Cation channel activity was observed in quiescent cell-attached patches after vasopressin stimulation. The addition of protein kinase A and ATP to excised patches also induced Na(+) channel activity. Spontaneous and vasopressin-induced Na(+) channel activity were inhibited by extracellular amiloride. To begin assessing potential molecular candidates for this cation channel, both reverse transcription-PCR and immunocytochemical analyses were conducted in LLC-PK1 cells. Expression of porcine orthologs of the alphaENaC and ApxL genes were found in LLC-PK1 cells. The expression of both gene products was confirmed by immunocytochemical analysis. Although alphaENaC labeling was mostly intracellular, ApxL labeled to both the apical membrane and cytoplasmic compartments of subconfluent LLC-PK1 cells. Vasopressin stimulation had no effect on alphaENaC immunolabeling but modified the cellular distribution of ApxL, consistent with an increased membrane-associated ApxL. The data indicate that proximal tubular LLC-PK1 renal epithelial cells express amiloride-sensitive, Na(+)-permeable cation channels, which are regulated by the cAMP pathway, and G proteins. This channel activity may implicate previously reported epithelial channel proteins, although this will require further experimentation. The evidence provides new clues as to potentially relevant Na(+) transport mechanisms in the mammalian proximal nephron.