RESUMEN
Tumor necrosis factor-alpha (TNF-alpha), a cytokine secreted by activated monocytes/macrophages and T lymphocytes, has been implicated in several disease states, including rheumatoid arthritis, inflammatory bowel disease, septic shock, and osteoporosis. Monocyte/macrophage production of TNF-alpha is dependent on the mitogen-activated protein kinase p38. RWJ 67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol -2-yl]-3-butyn-1-ol) inhibited the release of TNF-alpha by lipopolysaccharide (a monocyte stimulus)-treated human peripheral blood mononuclear cells with an IC(50) of 3 nM, as well as the release of TNF-alpha from peripheral blood mononuclear cells treated with the superantigen staphylococcal enterotoxin B (a T cell stimulus), with an IC(50) value of 13 nM. This compound was approximately 10-fold more potent than the literature standard p38 kinase inhibitor SB 203580 in all p38 dependent in vitro systems tested. RWJ 67657 inhibited the enzymatic activity of recombinant p38alpha and beta, but not gamma or delta, in vitro and had no significant activity against a variety of other enzymes. In contrast, SB 203580 significantly inhibited the tyrosine kinases p56 lck and c-src (IC(50) = 5 microM). RWJ 67657 did not inhibit T cell production of interleukin-2 or interferon-gamma and did not inhibit T cell proliferation in response to mitogens. RWJ 67657 inhibited TNF-alpha production in lipopolysaccharide-injected mice (87% inhibition at 50 mg/kg) and in rats (91% inhibition at 25 mg/kg) after oral administration. Based on these favorable biological properties, RWJ 67657 may have use as a treatment for inflammatory diseases.
Asunto(s)
Imidazoles/farmacología , Macrófagos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Monocitos/metabolismo , Proteínas Quinasas/metabolismo , Piridinas/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Antígenos/inmunología , División Celular/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Enterotoxinas/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratas , Ratas Endogámicas Lew , Staphylococcus/fisiología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoAsunto(s)
Aminopiridinas/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Proteínas Quinasas Activadas por Mitógenos , Pirroles/farmacología , Animales , Artritis Experimental/patología , Humanos , Técnicas In Vitro , Interleucina-1/antagonistas & inhibidores , Lipopolisacáridos , Masculino , Ratones , Ratones Endogámicos BALB C , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Ratas , Ratas Endogámicas Lew , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Protein tyrosine kinases play key roles in cellular physiology. Specific inhibitors of these enzymes are important laboratory tools and may prove to be novel therapeutic agents. In this report we describe a new class of tyrosine kinase inhibitor, synthetic oligodeoxynucleotides (ODNs). An ODN is described which specifically inhibits p210bcr-abl tyrosine kinase autophosphorylation in vitro with a Ki of 0.5 microM. Inhibition is non-competitive with respect to ATP. The effects upon inhibitory activity of ODN structure modifications are described. The inhibition described is not mediated by classical antisense mechanisms and represents an example of the recently recognized aptameric properties of ODNs.
Asunto(s)
Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Oligodesoxirribonucleótidos/farmacología , Células 3T3 , Animales , Secuencia de Bases , Línea Celular , ADN , Proteínas de Fusión bcr-abl/metabolismo , Ratones , Datos de Secuencia Molecular , FosforilaciónRESUMEN
Obtaining high transfection efficiencies and achieving appropriate intracellular concentrations and localization are two of the most important barriers to the implementation of gene targeted therapy. The efficiency of endogenous uptake of oligodeoxynucleotides (ODNs) varies from cell type to cell type and may be a limiting factor of antisense efficacy. The use of electroporation to obtain high intracellular concentrations of a synthetic ODN in essentially 100% of viable cells is described. It is also shown that the transfected ODNs initially localize to the nucleus and remain there for at least 48 hours. The cellular trafficking of electroporated ODNs is shown to be an energy dependent process. Targeting of the c-myc proto-oncogene of U937 cells by electroporation of phosphorothioate-modified ODNs results in rapid and specific suppression of this gene at ODN concentrations much lower than would otherwise be required. This technique appears to be applicable to a variety of cell types and may represent a powerful new investigate tool as well as a promising approach to the ex vivo treatment of hematologic disorders.
Asunto(s)
Electricidad , Genes myc , Oligonucleótidos Antisentido/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Secuencia de Bases , Western Blotting , División Celular , Línea Celular , Supervivencia Celular , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Ratones , Datos de Secuencia Molecular , Fosfatos , Proteínas Proto-Oncogénicas c-myc/biosíntesis , TransfecciónRESUMEN
We report experiments in the rat demonstrating the feasibility of intraventricular administration of oligodeoxynucleotides (ODNs) as a regional treatment approach to disorders within the central nervous system (CNS). Although we find little intrinsic nuclease activity in cerebrospinal fluid (CSF), phosphodiester ODNs are rapidly degraded by brain-associated alpha-exonuclease activity. Phosphorothioate ODNs, however, appear resistant to degradation in the CNS and, after intraventricular administration, we find they are cleared in a manner consistent with CSF bulk flow. Continuous infusion of ODN at 1.5 nmol/hr by miniosmotic pump can maintain micromolar concentrations of intact phosphorothioate ODN in CSF for at least 1 week without obvious neurologic or systemic toxicity. After infusion, extensive brain penetration and marked cellular uptake, especially by astrocytic cells, is demonstrated.
Asunto(s)
Oligodesoxirribonucleótidos/administración & dosificación , Animales , Secuencia de Bases , Femenino , Inyecciones Intraventriculares , Microscopía Fluorescente , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/farmacocinética , Ratas , Ratas Sprague-Dawley , Tionucleótidos/química , Distribución TisularAsunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Genes myc/genética , Oligonucleótidos Antisentido/farmacología , Proto-Oncogenes , Animales , Diferenciación Celular/efectos de los fármacos , ADN sin Sentido/farmacología , Ratones , Neoplasias del Sistema Nervioso , ARN sin Sentido/farmacología , Células Madre/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Using a panel of anti-CD3-TCR monoclonal antibodies (OKT3 A-E), it appears possible to separate the ability to cause surface antigen modulation from inhibition of MLR or induction of mitosis. OKT3D, an antibody that recognizes the CD3 antigen at a site that can be differentiated from the epitopes recognized by other members of this panel by competition binding, does not cause antigen modulation when incubated with human T cells for up to 3 days. Despite this, OKT3D is mitogenic and is capable of blocking MLR. Two different isotypes were produced from the OKT3D clone, IgG1 and IgG2b. The IgG2b isotype of OKT3D blocked MLR even in individuals unable to respond mitogenically to this antibody. Use of members of this panel may now permit dissection of the types of signals delivered by the CD3-TCR complex inducing mitosis, receptor modulation, and other T-cell responses.