RESUMEN
OBJECTIVE: To explore the in vivo effects of PD-0200347, an alpha(2)delta ligand of voltage gated Ca(2+) channels, on cell signalling in osteoarthritic (OA) chondrocytes from an experimental dog model, and examine the effect of PD-0200347 on the major signalling pathways involved in OA cartilage degradation. METHODS: OA was surgically induced in dogs by sectioning the anterior cruciate ligament. OA dogs were divided into three groups and treated orally with (a) placebo; (b) 15 mg/kg/day PD-0200347, or (c) 90 mg/kg/day PD-0200347. The animals were killed 12 weeks after surgery. Cartilage specimens from femoral condyles and tibial plateaus were processed for immunohistochemistry. Specific antibodies against the phosphorylated form of PKCalpha, Ras, c-Raf, the MAP kinases Erk1/2, p38, JNK, and the transcription factors, CREB and Elk-1, were used. RESULTS: Levels of all the tested signalling mediators were increased in the placebo treated (OA) group compared with the normal group. PD-0200347 treatment significantly reduced the levels of the active forms of PKCalpha, c-Raf, Erk1/2, and Elk-1; however, the levels of the active forms of Ras, p38, JNK, and CREB were not affected by the PD-0200347 treatment. CONCLUSION: The action of PD-0200347 on OA chondrocytes is probably mediated through the inhibition of Erk1/2 activation via a Ras independent mechanism. This effect is associated with reduction of the activation of transcription factors such as Elk-1, which leads to the inhibition of the induction of the major catabolic factors involved in the degradation process of OA cartilage.
Asunto(s)
Condrocitos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/efectos de los fármacos , Osteoartritis/patología , Proteína Quinasa C-alfa/fisiología , Ácido gamma-Aminobutírico/farmacología , Animales , Proteína de Unión a CREB/metabolismo , Canales de Calcio/efectos de los fármacos , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Perros , Relación Dosis-Respuesta a Droga , Ligandos , MAP Quinasa Quinasa 4/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Osteoartritis/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Elk-1 con Dominio ets/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas ras/metabolismoRESUMEN
OBJECTIVE: To describe an in vivo model in the rat in which change in weight distribution is used as a measure of disease progression and efficacy of acetaminophen and two nonsteroidal anti-inflammatory drugs (NSAIDs) in a model of monosodium iodoacetate (MIA)-induced osteoarthritis (OA). METHODS: Intra-articular injections of MIA and saline were administered to male Wistar rats (175-200 g) into the right and left knee joints, respectively. Changes in hind paw weight distribution between the right (osteoarthritic) and left (contralateral control) limbs were utilized as an index of joint discomfort. Acetaminophen and two archetypal, orally administered NSAIDs, naproxen and rofecoxib, were examined for their ability to decrease MIA-induced change in weight distribution. RESULTS: A concentration-dependent increase in change in hind paw weight distribution was noted after intra-articular injection of MIA. Both naproxen and rofecoxib demonstrated the capacity to significantly (P<0.05) decrease hind paw weight distribution in a dose-dependent fashion, indicating that the change in weight distribution associated with MIA injection is susceptible to pharmacological intervention. CONCLUSION: The determination of differences in hind paw weight distribution in the rat MIA model of OA is a technically straightforward, reproducible method that is predictive of the effects of anti-inflammatory and analgesic agents. This system may be useful for the discovery of novel pharmacologic agents in human OA.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Artritis Experimental/fisiopatología , Osteoartritis/fisiopatología , Soporte de Peso , Acetaminofén/uso terapéutico , Analgésicos no Narcóticos/uso terapéutico , Animales , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/patología , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Miembro Posterior/fisiopatología , Yodoacetatos , Masculino , Osteoartritis/inducido químicamente , Osteoartritis/tratamiento farmacológico , Osteoartritis/patología , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
In this paper we describe a method for validating therapeutic gene targets in arthritic disease. Ribozymes are catalytic oligonucleotides capable of highly sequence-specific cleavage of RNA. We designed ribozymes that cleave the mRNA encoding stromelysin, a matrix metalloproteinase implicated in cartilage catabolism. Ribozymes were initially screened in cultured fibroblasts to identify sites in the mRNA that were accessible for binding and cleavage. Accessible sites for ribozyme binding were found in various regions of the mRNA, including the 5' untranslated region, the coding region, and the 3' untranslated region. Several ribozymes that mediated sequence-specific and dose-dependent inhibition of stromelysin expression were characterized. Site selection in cell culture was predictive of in vivo bioactivity. An assay for measuring cartilage catabolism in rabbit articular cartilage explants was developed. Ribozymes inhibited IL-1-stimulated stromelysin mRNA expression in articular cartilage explants, yet failed to inhibit proteoglycan degradation. This indicated that up-regulation of stromelysin was not essential for IL-1-induced cartilage catabolism. Broad applications of this approach in therapeutic target validation are discussed.
Asunto(s)
Artritis/enzimología , Artritis/terapia , Marcación de Gen , ARN Catalítico/uso terapéutico , Animales , Artritis/genética , Artritis/metabolismo , Cartílago Articular/enzimología , Cartílago Articular/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Fibroblastos/enzimología , Marcación de Gen/métodos , Humanos , Hidrólisis , Inyecciones Intraarticulares , Masculino , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/fisiología , Inhibidores de la Metaloproteinasa de la Matriz , Técnicas de Cultivo de Órganos , ARN Catalítico/administración & dosificación , ARN Catalítico/metabolismo , Conejos , Reproducibilidad de los Resultados , Especificidad por Sustrato , Membrana Sinovial/enzimología , Membrana Sinovial/metabolismoRESUMEN
The phosphodiesterase-IV (PDE-IV) inhibitor, rolipram, is antiinflammatory in a number of animal models and inhibits the release of a variety of cytokines, including TNFalpha. Arthritis induced in rats by systemic reactivation with streptococcal cell walls (SCW) following intraarticular sensitization is a TNFalpha-dependent, delayed-type hypersensitivity (DTH) reaction. Rolipram administered during the reactivation phase dose-dependently inhibited hind paw edema through day 24, the day of peak swelling. PMN and T-cell recruitment to the arthritic joint were also attenuated in rolipram-treated rats. Histologic examination of ankle sections from rolipram-treated animals showed a marked attenuation of synovial inflammation. Mechanistic studies to determine the role of glucocorticoids in mediating rolipram action showed that the inhibitory effect of rolipram on swelling was not reversed by RU 486, a glucocorticoid antagonist. In contrast, RU 486 reversed the inhibitory effects of rolipram on TNFalpha secretion. To further evaluate the role of cAMP in the model, the beta-adrenergic receptor (betaAR) agonist isoproterenol was tested, and found to inhibit swelling but not the release of TNFalpha. These results are consistent with the view that the inhibitory effects of rolipram may be partially mediated by cAMP-dependent, but TNFalpha-independent, mechanisms. The betaAR antagonists propranolol and nadolol had no appreciable affect on the antiinflammatory effect of rolipram. However, rolipram reversed the lethal effects of the antagonists observed when either was administered alone. Apparently, beta-adrenergic mechanisms moderate the response to challenge, and rolipram treatment, presumably as a result of its effects on cAMP levels, reverses the toxic effect of the antagonists.
Asunto(s)
Artritis Infecciosa/tratamiento farmacológico , Inhibidores de Fosfodiesterasa/uso terapéutico , Rolipram/uso terapéutico , Infecciones Estreptocócicas/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Agonistas Adrenérgicos beta/uso terapéutico , Antagonistas Adrenérgicos beta/uso terapéutico , Animales , Antiinflamatorios/uso terapéutico , AMP Cíclico/metabolismo , Femenino , Miembro Posterior , Antagonistas de Hormonas/uso terapéutico , Isoproterenol/uso terapéutico , Mifepristona/uso terapéutico , Neutrófilos/efectos de los fármacos , Ratas , Ratas Endogámicas Lew , Linfocitos T/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Selective cyclooxygenase-2 (COX-2) inhibitors have been shown to be potent antiinflammatory agents with fewer side effects than currently marketed nonsteroidal antiinflammatory drugs (NSAIDs). Initial mass screening and subsequent structure-activity relationship (SAR) studies have identified 4b (PD138387) as the most potent and selective COX-2 inhibitor within the thiazolone and oxazolone series of di-tert-butylphenols. Compound 4b has an IC50 of 1.7 microM against recombinant human COX-2 and inhibited COX-2 activity in the J774A.1 cell line with an IC50 of 0.17 microM. It was inactive against purified ovine COX-1 at 100 microM and did not inhibit COX-1 activity in platelets at 20 microM. Compound 4b was also orally active in vivo with an ED40 of 16 mg/kg in the carrageenan footpad edema (CFE) assay and caused no gastrointestinal (GI) damage in rats at the dose of 100 mg/kg but inhibited gastric prostaglandin E2 (PGE2) production in rats' gastric mucosa by 33% following a dose of 100 mg/kg. The SAR studies of this chemical series revealed that the potency and selectivity are very sensitive to minor structural changes. A simple isosteric replacement led to the reversal of selectivity.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Inhibidores de la Ciclooxigenasa/síntesis química , Isoenzimas/metabolismo , Oxazoles/síntesis química , Fenoles/síntesis química , Prostaglandina-Endoperóxido Sintasas/metabolismo , Tiazoles/síntesis química , Administración Oral , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/toxicidad , Carragenina/toxicidad , Línea Celular , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores de la Ciclooxigenasa/toxicidad , Dinoprostona/antagonistas & inhibidores , Edema/inducido químicamente , Edema/tratamiento farmacológico , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Humanos , Hiperalgesia/tratamiento farmacológico , Masculino , Proteínas de la Membrana , Ratones , Oxazoles/química , Oxazoles/farmacología , Oxazoles/toxicidad , Fenoles/química , Fenoles/farmacología , Fenoles/toxicidad , Ratas , Ratas Sprague-Dawley , Úlcera Gástrica/inducido químicamente , Úlcera Gástrica/patología , Relación Estructura-Actividad , Tiazoles/química , Tiazoles/farmacología , Tiazoles/toxicidadRESUMEN
Two isoforms of the cyclooxygenase (COX) enzyme have been identified: COX-1, which is expressed constitutively, and COX-2, which is induced in inflammation. Recently, it has been shown that selective COX-2 inhibitors have antiinflammatory activity and lack the GI side effects typically associated with NSAIDs. Initial mass screening and subsequent SAR studies have identified 6b (PD164387) as a potent, selective, and orally active COX-2 inhibitor. It had IC50 values of 0.14 and 100 microM against recombinant human COX-2 and purified ovine COX-1, respectively. It inhibited COX-2 activity in the J774A.1 cell line with an IC50 of 0.18 microM and inhibited COX-1 activity in platelets with an IC50 of 3.1 microM. The choline salt of compound 6b was also orally active in vivo with an ED40 of 7. 1 mg/kg in the carrageenan footpad edema (CFE) assay. In vivo studies in rats at a dose of 100 mg/kg showed that this compound inhibited gastric prostaglandin E2 (PGE2) production in gastric mucosa by 77% but caused minimal GI damage. SAR studies of this chemical series revealed that the potency and selectivity are very sensitive to minor structural changes.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Inhibidores de la Ciclooxigenasa/síntesis química , Isoenzimas/metabolismo , Fenoles/síntesis química , Prostaglandina-Endoperóxido Sintasas/metabolismo , Tiadiazoles/síntesis química , Administración Oral , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/toxicidad , Plaquetas/efectos de los fármacos , Plaquetas/enzimología , Carragenina/toxicidad , Línea Celular , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores de la Ciclooxigenasa/toxicidad , Dinoprostona/antagonistas & inhibidores , Edema/inducido químicamente , Edema/tratamiento farmacológico , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Humanos , Hiperalgesia/tratamiento farmacológico , Técnicas In Vitro , Masculino , Proteínas de la Membrana , Ratones , Fenoles/química , Fenoles/farmacología , Fenoles/toxicidad , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/antagonistas & inhibidores , Ovinos , Úlcera Gástrica/inducido químicamente , Úlcera Gástrica/patología , Relación Estructura-Actividad , Tiadiazoles/química , Tiadiazoles/farmacología , Tiadiazoles/toxicidadRESUMEN
Nonsteroidal anti-inflammatory drugs often cause development of significant GI lesions. Selective inhibitors of prostaglandin G/H synthase/cyclooxygenase-2 (PGHS-2) enzyme and some dual inhibitors of PGHS/5-lipoxygenase (5-LO) enzymes have been reported to be potent anti-inflammatory compounds that carry a much lower risk of having GI irritating effects. We have evaluated the anti-inflammatory effect and the GI safety profile of three new anti-inflammatory compounds: the selective PGHS-2 inhibitors NS-398 and PD 138387 and the PGHS/5-LO dual inhibitor PD 137968. All the compounds tested showed an anti-inflammatory activity in the carragenan footpad edema test in rats. None of these compounds caused either gastric damage 4 h after p.o. administration of 100 mg/kg in rats or inhibition of PGE2 synthesis in the stomach. However, when administered p.o. at an effective anti-inflammatory dose to rats with pre-existing acetic acid-induced gastric ulcer, NS-398 caused a statistically significant delay of ulcer healing. No impairment of the ulcer healing was observed with the other compounds evaluated. Derivatives of 2,6-di-tert-butylphenol, whose members may act as PGHS-1/PGHS-2 inhibitors, selective PGHS-2 inhibitors or PGHS/5-LO dual inhibitors, are novel anti-inflammatory compounds that are devoid of GI irritating effects and do not affect the rate of pre-existing gastric ulcer healing.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Isoenzimas/efectos de los fármacos , Inhibidores de la Lipooxigenasa , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Úlcera Gástrica/fisiopatología , Ácido Acético , Animales , Antiinflamatorios no Esteroideos/toxicidad , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Dinoprostona/biosíntesis , Mucosa Gástrica/efectos de los fármacos , Indometacina/farmacología , Masculino , Proteínas de la Membrana , Nitrobencenos/farmacología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Sulfonamidas/farmacologíaRESUMEN
Intra-articular injection of streptococcal cell wall Ag followed by i.v. challenge ("reactivation") results in a destructive lymphocyte-dependent monoarticular arthritis. To further define the role of immune mechanisms in the model, Abs to Th1 and Th2-related cytokines were evaluated. Treatment of rats with antibodies to IL-4 reduced swelling, while treatment with anti-IL-10 or anti-IFN-gamma either had no effect or slightly enhanced the inflammatory response. These results suggest that Th-2 immune mechanisms may be, at least in part, operative in the model. To more precisely define the role of IL-4, the effects of anti-IL-4 on monocyte chemoattractant protein-1 (MCP-1) expression were evaluated. Initial studies demonstrated that mRNA (as determined by in situ hybridization) and protein (as determined by immunofluorescence) for MCP-1 were detectable in inflamed synovial tissue in a time-dependent manner. Anti-IL-4 treatment significantly reduced the expression of mRNA for MCP-1 24 and 72 h after reactivation. In addition, anti-MCP-1 inhibited swelling and reduced influx of (111)In-labeled T cells. These data suggest that the reactivation model of streptococcal cell wall Ag-induced arthritis is Th-2 dependent, and that an inter-relationship exists between IL-4 and the expression of MCP-1.
Asunto(s)
Artritis/inmunología , Quimiocina CCL2/fisiología , Interferón gamma/fisiología , Interleucina-10/fisiología , Interleucina-4/fisiología , Peptidoglicano/administración & dosificación , Streptococcus/inmunología , Animales , Anticuerpos Bloqueadores/administración & dosificación , Artritis/etiología , Artritis/patología , Movimiento Celular/inmunología , Quimiocina CCL2/análisis , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Femenino , Inmunohistoquímica , Hibridación in Situ , Inyecciones Intraarticulares , Inyecciones Intravenosas , Interferón gamma/inmunología , Interleucina-10/inmunología , Interleucina-4/inmunología , Ratas , Ratas Endogámicas Lew , Bazo/citología , Bazo/inmunología , Linfocitos T/patologíaRESUMEN
Intraarticular injection of streptococcal cell wall (SCW) antigen followed by intravenous challenge results in a T cell-mediated monoarticular arthritis ill female Lewis rats. Initial studies showed that this reactivation response to intravenous SCW antigen is dependent on the presence of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) and that the early phase of swelling is neutrophil-dependent. Neutrophil depletion or passive immunization with antibodies to P-selectin or macrophage inflammatory protein-2 reduced the intensity of ankle edema and the influx of neutrophils. After the first few days, however, the arthritic response is mediated primarily by mononuclear cells. Joint tissues showed up-regulation of mRNA for monocyte chemotactic protein-1 (MCP-1), which could be inhibited in part by anti-IL-4; treatment of rats with antibodies to IL-4 or MCP-1 significantly suppressed development of ankle edema and histopathological evidence of inflammation. Antibodies to interferon-gamma or IL-10 had no effect. Treatment with anti-MCP-1 also suppressed influx of (111)In-labeled T cells into the ankle joint. These data suggest that the late, mononuclear-dependent phase of SCW-induced arthritis in female Lewis rats requires cytokines that up-regulate MCP-1, which in turn may facilitate recruitment and extravasation of mononuclear cells into the joint.
Asunto(s)
Artritis Experimental/inmunología , Quimiocina CCL2/biosíntesis , Factores Quimiotácticos/inmunología , Citocinas/inmunología , Monocinas/inmunología , Neutrófilos/inmunología , Selectina-P/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos/farmacología , Artritis Experimental/patología , Pared Celular/inmunología , Quimiocina CXCL2 , Edema , Femenino , Inmunización Pasiva , Interferón gamma/inmunología , Interleucina-10/inmunología , Interleucina-4/inmunología , Articulaciones/inmunología , Articulaciones/patología , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas Lew , Streptococcus/inmunología , Transcripción GenéticaRESUMEN
Immune arthritis in rat ankle joints was induced by intra-articular injection of streptococcal cell was extract (SCW), followed 21 days later by i.v. injection of SCW. This results in a monoarticular arthritis characterized by an influx of neutrophils and mononuclear cells, a 35-fold increase in urinary excretion of 8-hydroxy-deoxyguanosine (8-OH-dGUA; an index of free radical production), ankle edema, and joint damage/destruction. Neutrophil depletion substantially reduced the intensity of ankle edema. Ab-induced blockade of P-selectin or ICAM-1 also reduced the intensity of ankle edema and the influx of neutrophils. Blockade of TNF-alpha or IL-1 resulted in nearly complete and persistent reduction in ankle edema and profound reductions in the accumulation of neutrophils and mononuclear cells in affected joints. Finally, blocking of macrophage-inflammatory protein-2 reduced ankle edema and neutrophil accumulation during the first 2 days after i.v. challenge with SCW. These data indicate that SCW-induced arthritis is neutrophil dependent and that the recruitment of neutrophils and subsequent joint edema requires ICAM-1, P-selectin, and macrophage-inflammatory protein-2, as well as TNF-alpha and IL-1.
Asunto(s)
Artritis/inmunología , Factores Quimiotácticos/fisiología , Molécula 1 de Adhesión Intercelular/fisiología , Monocinas/fisiología , Neutrófilos/fisiología , Selectina-P/fisiología , Peptidoglicano/inmunología , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Artritis/etiología , Quimiocina CXCL2 , Desoxiguanosina/análogos & derivados , Desoxiguanosina/biosíntesis , Desoxiguanosina/orina , Modelos Animales de Enfermedad , Edema/patología , Femenino , Inyecciones Intravenosas , Interleucina-1/fisiología , Neutropenia/inmunología , Peptidoglicano/administración & dosificación , Ratas , Ratas Endogámicas Lew , Factores de Tiempo , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Airway instillation of bacterial lipopolysaccharide (LPS) into rat lungs induces neutrophil accumulation, which is known to be intercellular adhesion molecule-1 (ICAM-1)-dependent. In the present study, ICAM-1 messenger RNA (mRNA) of whole lung was found to increase by 20-fold in this inflammatory model. This increase was reduced by 81% after treatment of animals with anti-tumor necrosis factor-alpha (TNF-alpha) antibody and by 37% after treatment with anti-interleukin-1 (IL-1) antibody. The same interventions reduced whole-lung ICAM-1 protein by 85% and 25%, respectively. The studies were extended to assess the locale in lung of ICAM-I upregulation. Lung vascular ICAM-1 content, which was assessed by vascular fixation of [125I]anti-ICAM-1, rose 4-fold after airway instillation of LPS. This rise was also TNF-alpha-dependent. Under the same experimental conditions, fixation of [125I]anti-ICAM-1 to airway surfaces increased 11-fold in a TNF-alpha-dependent manner. In situ hybridization and immunohistochemical analyses of lung tissue revealed ICAM-1 upregulation in the bronchiolar epithelium and in peribronchiolar smooth muscle. Soluble ICAM-1 could also be detected in bronchoalveolar lavage fluids (BALFs) of animals after intratracheal instillation of LPS. Retrieved alveolar macrophages showed a small, significant, and transient increase in surface expression of ICAM-1. These data indicate, at the very least, a dual compartmentalized (vascular and airway) upregulation of ICAM-1 after airway instillation of LPS. This upregulation requires TNF-alpha and IL-1. The functional significance of upregulated airway ICAM-1 remains to be determined.
Asunto(s)
Molécula 1 de Adhesión Intercelular/genética , Lipopolisacáridos/farmacología , Pulmón/irrigación sanguínea , Pulmón/química , Animales , Anticuerpos/farmacología , Western Blotting , Expresión Génica/efectos de los fármacos , Hibridación in Situ , Molécula 1 de Adhesión Intercelular/análisis , Molécula 1 de Adhesión Intercelular/inmunología , Interleucina-1/inmunología , Pulmón/citología , Macrófagos Alveolares/química , Masculino , Músculo Liso Vascular/química , Músculo Liso Vascular/efectos de los fármacos , Neutrófilos/inmunología , ARN Mensajero/análisis , Ratas , Ratas Endogámicas , Factores de Tiempo , Factor de Necrosis Tumoral alfa/inmunología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
OBJECTIVE: To evaluate the effects of the phospholipase A2 (PLA2) inhibitor manoalide on cartilage degradation, stromelysin expression, and inflammatory cell accumulation in rabbits treated intraarticularly with recombinant human interleukin-1 alpha (rHuIL-1 alpha). METHODS: Rabbits were given an intraarticular injection of rHuIL-1 alpha. At various time points over a 24-hour period, the rabbits were euthanized and the articular space was lavaged with sterile PBS. The proteoglycan content of the lavage fluid was measured using a dimethylmethylene blue assay. PLA2 activity and differential cell counts were also measured. The femur was removed and cartilage proteoglycan content determined. In some experiments, levels of synovial stromelysin messenger RNA (mRNA) were assessed. Manoalide or vehicle was administered 30 minutes before the rHuIL-1 alpha injection. RESULTS: The rHuIL-1 alpha-induced arthritic response is characterized by significant accumulation of inflammatory cells, loss of proteoglycan from the condylar cartilage, and induction of mRNA for stromelysin. PLA2 activity was also elevated in synovial fluids from rHuIL-1 alpha-injected joints. Pretreatment with manoalide (0.3 mg/joint) significantly inhibited PLA2 activity in the synovial fluid, prevented the loss of proteoglycan from the condylar cartilage, and reduced proteoglycan levels in lavage fluids. However, manoalide either had no effect on, or stimulated, cell accumulation. To assess the relationship between the induction of PLA2 and stromelysin, levels of stromelysin mRNA were measured in synovial tissue from manoalide- and vehicle-treated joints. Stromelysin message levels were significantly suppressed in a dose-dependent manner. CONCLUSION: These studies demonstrate that manoalide is a potent inhibitor of inflammation and cartilage catabolism, and suggest that PLA2 is involved in the pathophysiology of rHuIL-1 alpha-induced arthritis in rabbits.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Cartílago/efectos de los fármacos , Metaloendopeptidasas/genética , Inhibidores de Fosfodiesterasa/farmacología , Fosfolipasas A/antagonistas & inhibidores , Líquido Sinovial/citología , Terpenos/farmacología , Animales , Artritis/inducido químicamente , Cartílago/metabolismo , Recuento de Células/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Humanos , Inyecciones Intraarticulares , Interleucina-1 , Masculino , Metaloproteinasa 3 de la Matriz , Proteínas de Neoplasias/genética , Fosfolipasas A/metabolismo , Fosfolipasas A2 , ARN Mensajero/metabolismo , Conejos , Proteínas Recombinantes , Líquido Sinovial/enzimologíaRESUMEN
The surface levels of ICAM-1 and E-selectin on activated endothelial cells can be reduced by 3-alkoxybenzo[b]thiophene-2-carboxamides. This property is shared by several N-alkylthiopyridine substituted imides. Combining structural elements of these two diverse series lead to a new class of small molecule inhibitors of adhesion molecule expression.
Asunto(s)
Antiinflamatorios/farmacología , Selectina E/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Piridinas/farmacología , Tiofenos/farmacología , Antiinflamatorios/síntesis química , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Selectina E/biosíntesis , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Piridinas/síntesis química , Relación Estructura-Actividad , Tiofenos/síntesis químicaRESUMEN
Streptococcus pyogenes infections in humans may be associated with severe clinical manifestations, including adult respiratory distress syndrome and a toxic shock-like syndrome. These observations have led to the investigation of products of group A streptococci that may contribute to increased virulence. Streptococcal pyrogenic exotoxin B is a highly conserved precursor of an extracellular cysteine protease that is secreted by S. pyogenes. We investigated the ability of this streptococcal cysteine protease (SCP) to act synergistically with either streptococcal cell wall antigen (SCW) or streptolysin-O (SLO) to augment lung injury in rats. Intratracheal administration of either SCW or SLO alone caused lung injury, as measured by pulmonary vascular leak. Bronchoalveolar lavage (BAL) fluid analysis showed that SCW induced neutrophil accumulation and appearance of interleukin-1beta and tumor necrosis factor alpha. In contrast, SLO induced neither neutrophil influx nor significant cytokine elevations in BAL fluids. Intratracheal administration of SCP with either SCW or SLO resulted in synergistic augmentation of lung vascular permeability and accumulation of BAL neutrophils. The synergy was reduced when SCP was either heat inactivated or coinstilled with a peptide inhibitor of the protease. SCP in the presence of SCW resulted in a significant increase in BAL fluid tumor necrosis factor alpha content but not in immunoreactive interleukin-1beta. Moreover, the copresence of SAP with SAW resulted in increased BAL fluid nitrite-nitrate levels, indicative of nitric oxide production. These data demonstrate that SCP acts synergistically with other S. pyogenes products (SCW or SLO) to increase tissue injury and provide additional evidence that SCP may function as an important virulence factor in group A streptococcal infections.
Asunto(s)
Cisteína Endopeptidasas/toxicidad , Enfermedades Pulmonares/etiología , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/patogenicidad , Animales , Proteínas Bacterianas , Pared Celular , Interleucina-1/análisis , Pulmón/patología , Masculino , Ratones , Neutrófilos/fisiología , Nitratos/metabolismo , Nitritos/metabolismo , Ratas , Estreptolisinas/toxicidad , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Catalytic RNA molecules, or ribozymes, have generated significant interest as potential therapeutic agents for controlling gene expression. Although ribozymes have been shown to work in vitro and in cellular assays, there are no reports that demonstrate the efficacy of synthetic, stabilized ribozymes delivered in vivo. We are currently utilizing the rabbit model of interleukin 1-induced arthritis to assess the localization, stability, and efficacy of exogenous antistromelysin hammerhead ribozymes. The matrix metalloproteinase stromelysin is believed to be a key mediator in arthritic diseases. It seems likely therefore that inhibiting stromelysin would be a valid therapeutic approach for arthritis. We found that following intraarticular administration ribozymes were taken up by cells in the synovial lining, were stable in the synovium, and reduced synovial interleukin 1 alpha-induced stromelysin mRNA. This effect was demonstrated with ribozymes containing various chemical modifications that impart nuclease resistance and that recognize several distinct sites on the message. Catalytically inactive ribozymes were ineffective, thus suggesting a cleavage-mediated mechanism of action. These results suggest that ribozymes may be useful in the treatment of arthritic diseases characterized by dysregulation of metalloproteinase expression.
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Artritis/inducido químicamente , Articulación de la Rodilla/efectos de los fármacos , Metaloendopeptidasas/biosíntesis , ARN Catalítico/farmacología , Membrana Sinovial/efectos de los fármacos , Animales , Artritis/fisiopatología , Secuencia de Bases , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica , Hibridación in Situ , Inyecciones Intraarticulares , Interleucina-1/farmacología , Masculino , Metaloproteinasa 3 de la Matriz , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , ARN Catalítico/metabolismo , ARN Mensajero/biosíntesis , Conejos , Ribonucleasas/metabolismoRESUMEN
This Phase I trial explores the use of high-dose 90Y conjugated to the antibreast cancer monoclonal antibody BrE-3 and autologous hematologic cell support in the treatment of women with stage four breast cancer. Nine women with heavily pretreated disease were enrolled. All of the patients had BrE-3-positive tumors by immunostaining and were treated with increasing doses of 90Y (15 mCi/m2, 3 patients), 20 mCi/M2 (six patients), and a fixed (50 mg) dose of BrE-3. 111In-labeled BrE-3 (5 mCi) was given simultaneously for scanning purposes. The only toxicity noted was hematological. Grade 4 platelet toxicity requiring transfusion support occurred in four patients. Grade 4 WBC toxicity was seen in two patients that resolved in 3-9 days. All hematological nadirs occurred approximately 25 days after treatment. Objective partial responses were noted in 4 of 8 (50%) patients with measurable tumors. Dose escalation is ongoing.
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Neoplasias de la Mama/terapia , Trasplante de Células Madre Hematopoyéticas , Radioinmunoterapia , Radioisótopos de Itrio/uso terapéutico , Adulto , Anciano , Animales , Anticuerpos Monoclonales/uso terapéutico , Terapia Combinada , Femenino , Humanos , Ratones , Persona de Mediana Edad , Ácido Pentético/uso terapéuticoRESUMEN
Protein kinase C (PKC) regulates a variety of signal transduction events implicated in the pathogenesis of inflammation, including the biosynthesis of inflammatory cytokines and superoxide and the activation of phospholipase A2. Because of the significant role of PKC in these inflammatory processes, we evaluated a specific and potent inhibitor of C kinase for efficacy in several in vitro and in vivo murine models of inflammation. Unlike the relatively nonspecific kinase inhibitor staurosporine, the bisindolylmaleimide 3-[1-[-3-(dimethylaminopropyl]-1H-indol-3-yl]- 4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (Gö 6850) demonstrated increased selectivity for C kinase in purified enzyme assays (respective IC50 values (microM) for Gö 6850 and staurosporine: protein kinase C (0.032, 0.009); myosin light-chain kinase (0.6, 0.01); protein kinase G (4.6, 0.018); protein kinase A (33, 0.04); tyrosine kinase1 (94, 0.4); tyrosine kinase2 (> 100, > 1)). Topically applied Gö 6850 inhibited phorbol myristate acetate-induced edema, neutrophil influx and vascular permeability in murine epidermis in a dose- and time-dependent manner at levels comparable to indomethacin. In a murine model of delayed type hypersensitivity, Gö 6850 inhibited dinitrofluorobenzene-induced contact dermatitis with and ID50 value of 150 micrograms/ear. Cellular studies in mouse peritoneal macrophages demonstrated that Gö 6850 was a potent inhibitor of phorbol myristate acetate-induced prostaglandin E2 production. Superoxide production in phorbol myristate acetate-stimulated murine neutrophils was also inhibited by Gö 6850 (IC50 = 88 nM).(ABSTRACT TRUNCATED AT 250 WORDS)
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Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios/farmacología , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Maleimidas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Animales , Ácido Araquidónico/antagonistas & inhibidores , Dinoprostona/metabolismo , Femenino , Hipersensibilidad Tardía/tratamiento farmacológico , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Neutrófilos/efectos de los fármacos , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/antagonistas & inhibidoresRESUMEN
BACKGROUND & AIMS: CI-959 is an anti-inflammatory agent that inhibits neutrophil adhesion, respiratory burst, and mast cell histamine release in vitro. In view of the emerging role of neutrophils in gastric erosive damage, the goals of this study were to assess the gastric cytoprotective effects of CI-959 and identify the mechanism responsible for this action. METHODS: Cytoprotective effects in the rat nonsteroidal anti-inflammatory drug and ethanol erosion models were assessed using image analysis. The in vivo effects of CI-959 on gastric acid secretion, arachidonic acid metabolism, and intracellular sulfhydryl and leukocyte adhesion were also examined. RESULTS: CI-959 protected prophylactically against the erosive damage induced by aspirin, indomethacin, and ethanol with 50% effective doses (ED50s) of 0.05, 1.0, and 0.07 mg/kg administered orally, respectively. When administered after indomethacin or ethanol, CI-959 had no effect on the healing of erosive damage. CI-959 did not alter gastric acid secretion, arachidonic acid metabolism, or intracellular sulfhydryl levels. In vivo, CI-959 blocked leukocyte adhesion in intravital microscopy studies using indomethacin (ED50, < 5 mg/kg orally) or platelet-activating factor (50% inhibiting concentration, approximately 10 mumol/L) as the adhesion stimulus. CONCLUSIONS: The most likely mechanism responsible for the cytoprotective effects of CI-595 is its inhibitory effects on leukocyte trafficking and/or adhesion.
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Antiinflamatorios no Esteroideos/farmacología , Mucosa Gástrica/efectos de los fármacos , Antagonistas de los Receptores Histamínicos/farmacología , Leucocitos/efectos de los fármacos , Tetrazoles/farmacología , Tiofenos/farmacología , Animales , Ácido Araquidónico/metabolismo , Aspirina/efectos adversos , Adhesión Celular/efectos de los fármacos , Dinoprostona/metabolismo , Perros , Etanol/efectos adversos , Femenino , Ácido Gástrico/metabolismo , Indometacina/efectos adversos , Leucocitos/fisiología , Leucotrieno C4/metabolismo , Factor de Activación Plaquetaria/farmacología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Compuestos de Sulfhidrilo/metabolismoRESUMEN
A variety of benzylideneoxazoles, -thiazoles, and -imidazoles derived from 2,6-di-tert-butylphenol were prepared and evaluated as dual inhibitors of 5-lipoxygenase and cyclooxygenase in rat basophilic leukemia (RBL-1) cells. The target compounds exhibit varying degrees of selectivity toward the two enzymes. Several compounds are orally active in the rat carageenan footpad edema (CFE) and mycobacterium footpad edema (MFE) antiinflammatory models. Structure-activity relationships are discussed. From this work, (Z)-5-[[3,5-bis(1,1-dimethylethyl)-4- hydroxyphenyl]-methylene]-2-imino-4-thiazolidinone methanesulfonate salt (CI-1004) was identified as a potent dual inhibitor of 5-lipoxygenase (IC50 = 0.77 microM) and cyclooxygenase (IC50 = 0.39 microM), with oral activity (ID40 = 0.6 mg/kg) in the rat MFE model of inflammation.