RESUMEN
BACKGROUND: Angiogenesis is a common finding in chronic inflammatory diseases; however, its role in multiple sclerosis (MS) is unclear. Central nervous system lesions from both MS and experimental autoimmune encephalomyelitis (EAE), the animal model of MS, contain T cells, macrophages and activated glia, which can produce pro-angiogenic factors. Previous EAE studies have demonstrated an increase in blood vessels, but differences between the different phases of disease have not been reported. Therefore we examined angiogenic promoting factors in MS and EAE lesions to determine if there were changes in blood vessel density at different stages of EAE. METHODS: In this series of experiments we used a combination of vascular casting, VEGF ELISA and immunohistochemistry to examine angiogenesis in experimental autoimmune encephalomyelitis (EAE). Using immunohistochemistry we also examined chronic active MS lesions for angiogenic factors. RESULTS: Vascular casting and histological examination of the spinal cord and brain of rats with EAE demonstrated that the density of patent blood vessels increased in the lumbar spinal cord during the relapse phase of the disease (p < 0.05). We found an increased expression of VEGF by inflammatory cells and a decrease in the recently described angiogenesis inhibitor meteorin. Examination of chronic active human MS tissues demonstrated glial expression of VEGF and glial and blood vessel expression of the pro-angiogenic receptor VEGFR2. There was a decreased expression of VEGFR1 in the lesions compared to normal white matter. CONCLUSIONS: These findings reveal that angiogenesis is intimately involved in the progression of EAE and may have a role in MS.
Asunto(s)
Inductores de la Angiogénesis/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Esclerosis Múltiple/patología , Neovascularización Patológica , Adulto , Anciano , Animales , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Cobayas , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/metabolismo , Ratas , Ratas Endogámicas Lew , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Pyrrolo[2,3-f]isoquinoline based amino acids, tetracyclic lactams and cyclic ketone analogues are described as novel MK2 inhibitors with IC(50) as low as 5nM and good selectivity profiles against a number of related kinases including ERK, p38alpha and JNKs. TNFalpha release was suppressed from human peripheral blood mononuclear cells (hPBMCs), and a representative compound inhibited LPS induced TNFalpha release in mice illustrating the potential of this series to provide orally active MK2 inhibitors.
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos/química , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Administración Oral , Aminoácidos/síntesis química , Aminoácidos/química , Aminoácidos/farmacología , Animales , Sitios de Unión , Cristalografía por Rayos X , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isoquinolinas/química , Cetonas/síntesis química , Cetonas/química , Cetonas/farmacología , Lactamas/síntesis química , Lactamas/química , Lactamas/farmacología , Ratones , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Pirroles/química , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Fingolimod (FTY720) represents the first in a new class of immune-modulators whose target is sphingosine-1-phosphate (S1P) receptors. It was first identified by researchers at Kyoto University and Yoshitomi Pharmaceutical as a chemical derivative of the ascomycete metabolite ISP-1 (myriocin). Unlike its natural product parent, FTY720 does not interfere with sphingolipid biosynthesis. Instead, its best characterized mechanism of action upon in vivo phosphorylation, leading to the active principle FTY720-P, is the rapid and reversible inhibition of lymphocyte egress from peripheral lymph nodes. As a consequence of S1P1 receptor internalization, tissue-damaging T-cells can not recirculate and infiltrate sites of inflammation such as the central nervous system (CNS). Furthermore, FTY720-P modulation of S1P receptor signaling also enhances endothelial barrier function. Due to its mode of action, FTY720 effectively prevents transplant rejection and is active in various autoimmune disease models. The most striking efficacy is in the multiple sclerosis (MS) model of experimental autoimmune encephalomyelitis, which has now been confirmed in the clinic. FTY720 demonstrated promising results in Phase II trials and recently entered Phase III in patients with relapsing MS. Emerging evidence suggests that its efficacy in the CNS extends beyond immunomodulation to encompass other aspects of MS pathophysiology, including an influence on the blood-brain-barrier and glial repair mechanisms that could ultimately contribute to restoration of nerve function. FTY720 may represent a potent new therapeutic modality in MS, combined with the benefit of oral administration.
Asunto(s)
Ascomicetos/química , Productos Biológicos/uso terapéutico , Inmunosupresores/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Glicoles de Propileno/uso terapéutico , Receptores de Lisoesfingolípidos/efectos de los fármacos , Esfingosina/análogos & derivados , Administración Oral , Animales , Productos Biológicos/administración & dosificación , Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Modelos Animales de Enfermedad , Clorhidrato de Fingolimod , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/aislamiento & purificación , Inmunosupresores/farmacología , Estructura Molecular , Glicoles de Propileno/administración & dosificación , Glicoles de Propileno/aislamiento & purificación , Glicoles de Propileno/farmacología , Esfingosina/administración & dosificación , Esfingosina/aislamiento & purificación , Esfingosina/farmacología , Esfingosina/uso terapéutico , Resultado del TratamientoRESUMEN
We describe here orally active and brain-penetrant cathepsin S selective inhibitors, which are virtually devoid of hERG K(+) channel affinity, yet exhibit nanomolar potency against cathepsin S and over 100-fold selectivity to cathepsin L. The new non-peptidic inhibitors are based on a 2-cyanopyrimidine scaffold bearing a spiro[3.5]non-6-yl-methyl amine at the 4-position. The brain-penetrating cathepsin S inhibitors demonstrate potential clinical utility for the treatment of multiple sclerosis and neuropathic pain.
Asunto(s)
Catepsinas/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/metabolismo , Pirimidinas/síntesis química , Pirimidinas/farmacología , Administración Oral , Animales , Encéfalo/efectos de los fármacos , Catepsina L , Técnicas Químicas Combinatorias , Cisteína Endopeptidasas , Humanos , Masculino , Estructura Molecular , Esclerosis Múltiple/tratamiento farmacológico , Dolor/tratamiento farmacológico , Pirimidinas/sangre , Pirimidinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
T cells and macrophages directed against myelin proteins orchestrate the inflammation process in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). So far, assessment of macrophages infiltration or structural alterations has been achieved by in vivo imaging. In this work, we show the infiltration of Cy5.5-labeled T lymphocytes into the brains of EAE rats by reflectance near-infrared fluorescence imaging. T lymphocytes were labeled with Cy5.5-Tat and administered intravenously to naïve or EAE animals. The highest fluorescence signal was observed for EAE animals, which received myelin-activated T cells during the acute phase of the disease. The temporal profile of fluorescence in this group paralleled the pattern of neurological impairment during the acute phase, the remittance and first relapses of EAE. No disease specific fluorescence pattern was observed for EAE animals, which received naïve T cells. However, uptake of Cy5.5-Tat by scavenger cells (e.g. macrophages) following death of labeled T cells in vivo prevents prolonged longitudinal studies. Our work demonstrates that Cy5.5-Tat labeling of T cells is suitable for in vivo fluorescence imaging of inflammation initiation in the EAE model. This approach may particularly be useful for evaluation of novel anti-inflammatory therapies.
Asunto(s)
Carbocianinas , Encefalitis/patología , Encefalomielitis Autoinmune Experimental/patología , Técnicas Inmunológicas , Linfocitos T/inmunología , Animales , Encefalitis/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente/métodos , Productos del Gen tat , VIH/genética , Microscopía Confocal , Ratas , Ratas Endogámicas Lew , Espectroscopía Infrarroja Corta , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
FTY720 (fingolimod) is an oral sphingosine 1-phosphate (S1P) receptor modulator under development for the treatment of multiple sclerosis (MS). To elucidate its effects in the central nervous system (CNS), we compared functional parameters of nerve conductance in the DA rat model of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) after preventive and therapeutic treatment. We demonstrate that prophylactic therapy protected against the emergence of EAE symptoms, neuropathology, and disturbances to visual and somatosensory evoked potentials (VEP, SEP). Moreover, therapeutic treatment from day 25 to 45 markedly reversed paralysis in established EAE and normalized the electrophysiological responses, correlating with decreased demyelination in the brain and spinal cord. The effectiveness of FTY720 in this model is likely due to several contributing factors. Evidence thus far supports its role in the reduction of inflammation and preservation of blood-brain-barrier integrity. FTY720 may also act via S1P receptors in glial cells to promote endogenous repair mechanisms that complement its immunomodulatory action.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Inmunosupresores/uso terapéutico , Glicoles de Propileno/uso terapéutico , Esfingosina/análogos & derivados , Análisis de Varianza , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/inmunología , Modelos Animales de Enfermedad , Estimulación Eléctrica , Encefalomielitis Autoinmune Experimental/inducido químicamente , Potenciales Evocados Somatosensoriales/efectos de los fármacos , Potenciales Evocados Visuales/efectos de los fármacos , Femenino , Clorhidrato de Fingolimod , Estudios Longitudinales , Proteínas de la Mielina , Glicoproteína Asociada a Mielina , Glicoproteína Mielina-Oligodendrócito , Conducción Nerviosa/efectos de los fármacos , Ratas , Tiempo de Reacción/efectos de los fármacos , Esfingosina/uso terapéutico , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Factores de TiempoRESUMEN
FTY720 [2-amino-2-[2-(4-octylphenyl) ethyl]propane-1,3-diol hydrochloride] is an oral sphingosine-1-phosphate receptor modulator under development for the treatment of multiple sclerosis (MS). The drug is phosphorylated in vivo by sphingosine kinase 2 to its bioactive form, FTY720-P. Although treatment with FTY720 is accompanied by a reduction of the peripheral lymphocyte count, its efficacy in MS and experimental autoimmune encephalomyelitis (EAE) may be due to additional, direct effects in the central nervous system (CNS). We now show that FTY720 localizes to the CNS white matter, preferentially along myelin sheaths. Brain trough levels of FTY720 and FTY720-P in rat EAE are of the same magnitude and dose dependently increase; they are in the range of 40 to 540 ng/g in the brain tissue at efficacious doses and exceed blood concentrations severalfold. In a rat model of chronic EAE, prolonged treatment with 0.03 mg/kg was efficacious, but limiting the dosing period failed to prevent EAE despite a significant decrease in blood lymphocytes. FTY720 effectiveness is likely due to a culmination of mechanisms involving reduction of autoreactive T cells, neuroprotective influence of FTY720-P in the CNS, and inhibition of inflammatory mediators in the brain.
Asunto(s)
Encéfalo/metabolismo , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Inmunosupresores/farmacocinética , Esclerosis Múltiple/tratamiento farmacológico , Glicoles de Propileno/farmacocinética , Esfingosina/análogos & derivados , Administración Oral , Animales , Autorradiografía , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Clorhidrato de Fingolimod , Recuento de Linfocitos , Fosforilación , Glicoles de Propileno/administración & dosificación , Glicoles de Propileno/uso terapéutico , Ratas , Ratas Endogámicas Lew , Esfingosina/administración & dosificación , Esfingosina/farmacocinética , Esfingosina/uso terapéuticoRESUMEN
In vivo tracking of macrophage migration is feasible by labeling cells with ultra-small particles of iron oxide (USPIO). It is demonstrated that it is possible to monitor distinct patterns of macrophage migration during the early states of inflammation in a rodent model of chronic relapsing experimental autoimmune encephalomyelitis (EAE). As previous MRI studies showed that EAE inflammation processes are clearly linked to macrophage infiltration in the brain, a longitudinal protocol for macrophage visualization was designed, where USPIOs were injected repeatedly during the acute phase of the disease, the remitting phase and the first relapse. In addition to USPIO-enhanced MRI, blood-brain barrier (BBB) damage, magnetization transfer ratios (MTRs) and neurological impairment were assessed as classical markers for central nervous system (CNS) inflammation and tissue damage. During the acute phase, animals showed severe paralysis of the hind paws, intense accumulation of macrophages in brain tissue and some diffuse patterns of BBB disruption. While USPIO-accumulation completely disappeared after the acute phase, residual damage of the BBB remained detectable in some lesions during the remitting phase. During the first relapse, the accumulation of USPIO-loaded cells was less pronounced but still detectable. The time course of MTR, which is used as a marker for myelin loss, was linked to the infiltration of macrophages during the acute phase.
Asunto(s)
Barrera Hematoencefálica/patología , Encéfalo/patología , Encefalomielitis Autoinmune Experimental/patología , Hierro , Macrófagos/patología , Imagen por Resonancia Magnética/métodos , Fibras Nerviosas Mielínicas/patología , Óxidos , Enfermedad Aguda , Animales , Movimiento Celular , Medios de Contraste , Dextranos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Encefalitis/patología , Femenino , Óxido Ferrosoférrico , Aumento de la Imagen/métodos , Nanopartículas de Magnetita , Ratas , Ratas Endogámicas Lew , Remisión EspontáneaRESUMEN
A test library with three novel p38alpha inhibitory scaffolds and a narrow set of substituents was prepared. Appropriate combination of substituent and scaffold generated potent p38alpha inhibitors, for example, pyrazolo[3,4-b]pyridine 9, pyrazolo[3,4-d]pyrimidine 18a and pyrazolo[3,4-b]pyrazine 23b with potent in vivo activity upon oral administration in animal models of rheumatoid arthritis.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Pirazinas/administración & dosificación , Pirazoles/administración & dosificación , Piridinas/administración & dosificación , Pirimidinas/administración & dosificación , Administración Oral , Animales , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Ratones , Estructura Molecular , Pirazinas/síntesis química , Pirazinas/química , Pirazoles/síntesis química , Pirazoles/química , Piridinas/síntesis química , Piridinas/química , Pirimidinas/síntesis química , Pirimidinas/química , Ratas , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cinnamides as novel CCR1 antagonist chemotypes are described with high affinity to human and rodent receptors. A1B1 and A4B7 showed oral activity in the mouse collagen induced arthritis.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Receptores de Quimiocina/antagonistas & inhibidores , Administración Oral , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Humanos , Ratones , Ratas , Receptores CCR1 , Relación Estructura-ActividadRESUMEN
PURPOSE: To examine the efficacy of FTY720 as a new agent to reduce inflammatory activity in an animal model of multiple sclerosis (MS) by in vivo macrophage tracking. MATERIAL AND METHODS: FTY720 was used for treatment of rats in a model of chronic relapsing experimental autoimmune encephalomyelitis (EAE) at an oral dose of 0.3 mg/kg/day. Magnetic resonance imaging (MRI) based on in vivo tracking of macrophages labeled with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, immunohistological staining (IHC), and neurological readouts was used to study the burden of disease in treated and untreated animals. RESULTS: While untreated animals showed severe paralysis of the hind paws, intense accumulation of macrophages in brain tissue, and areas of blood-brain barrier (BBB) disruption, FTY720-treated animals displayed no signs of inflammatory activity or neurological impairment. These observations were made for both acute phase and first relapse. CONCLUSION: Tracking of macrophages by MRI provides direct evidence of the immunomodulatory efficacy of FTY720 in the EAE model and correlates well with neurological symptoms and histology.
Asunto(s)
Encéfalo/patología , Medios de Contraste , Encefalomielitis Autoinmune Experimental/diagnóstico , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Hierro , Macrófagos/patología , Imagen por Resonancia Magnética , Óxidos , Glicoles de Propileno/uso terapéutico , Enfermedad Aguda , Animales , Barrera Hematoencefálica , Encéfalo/metabolismo , Dextranos , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Óxido Ferrosoférrico , Clorhidrato de Fingolimod , Compuestos Heterocíclicos , Inmunohistoquímica , Nanopartículas de Magnetita , Compuestos Organometálicos , Ratas , Ratas Endogámicas Lew , Receptores Acoplados a Proteínas G/agonistas , Receptores Lisofosfolípidos , Recurrencia , EsfingosinaRESUMEN
A library of trisubstituted oxazoles, thiazoles, imidazoles (1,2,4- and 2,4,5-substituted) and imidazo[1,2-b]pyridines was prepared and evaluated in vitro as p38alpha inhibitors and in vivo in several models of rheumatoid arthritis. Four structures--32, 37, 45 and 59--were identified as potent inhibitors of p38alpha with high efficacy in the LPS induced TNFalpha release model in the mouse, the adjuvant induced arthritis and the collagen induced arthritis in the rat with ED50s between 1.0 and 9.5 mg/kg p.o.
Asunto(s)
Antirreumáticos/síntesis química , Piridinas/síntesis química , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Antirreumáticos/farmacología , Antirreumáticos/uso terapéutico , Artritis Experimental/inducido químicamente , Artritis Experimental/tratamiento farmacológico , Colágeno , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Imidazoles/química , Lipopolisacáridos/farmacología , Ratones , Oxazoles/química , Piridinas/farmacología , Piridinas/uso terapéutico , Ratas , Relación Estructura-Actividad , Tiazoles/química , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Benzoylpyridines and benzophenones were synthesized and evaluated in vitro as p38alpha inhibitors and in vivo in several models of rheumatoid arthritis. Oral activity was found to depend upon substitution: 1,1-dimethylpropynylamine substituted benzophenone 10b (IC50: 14 nM) and pyridinoyl substituted benzimidazole 17b (IC50: 21 nM) showed highest efficacy and selectivity with ED50s of 9.5 and 8.6 mg/kg p.o. in CIA.
Asunto(s)
Benzofenonas/síntesis química , Inhibidores Enzimáticos/síntesis química , Boca/metabolismo , Piridinas/síntesis química , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/tratamiento farmacológico , Benzofenonas/farmacología , Benzofenonas/uso terapéutico , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Concentración 50 Inhibidora , Piridinas/farmacología , Piridinas/uso terapéutico , Ratas , Relación Estructura-ActividadRESUMEN
2,6-Diamino-3,5-difluoropyridinyl substituted pyridinylimidazoles, -pyrroles, -oxazoles, -thiazoles and -triazoles have been identified as novel p38alpha inhibitors. Pyridinylimidazole 11 potently inhibited LPS-induced TNFalpha in mice, showed good efficacy in the established rat adjuvant (ED(50): 10 mg/kg po b.i.d.) and collagen induced arthritis (ED(50): 5 mg/kg po b.i.d.) with disease modifying properties based on histological analysis of the joints.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Piridonas/química , Piridonas/farmacología , Relación Estructura-Actividad , Animales , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/patología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/uso terapéutico , Humanos , Lipopolisacáridos/farmacología , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estructura Molecular , Piridonas/uso terapéutico , Ratas , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Immunosuppressant drugs such as cyclosporin have allowed widespread organ transplantation, but their utility remains limited by toxicities, and they are ineffective in chronic management of autoimmune diseases such as multiple sclerosis. In contrast, the immune modulating drug FTY720 is efficacious in a variety of transplant and autoimmune models without inducing a generalized immunosuppressed state and is effective in human kidney transplantation. FTY720 elicits a lymphopenia resulting from a reversible redistribution of lymphocytes from circulation to secondary lymphoid tissues by unknown mechanisms. Using FTY720 and several analogs, we show now that FTY720 is phosphorylated by sphingosine kinase; the phosphorylated compound is a potent agonist at four sphingosine 1-phosphate receptors and represents the therapeutic principle in a rodent model of multiple sclerosis. Our results suggest that FTY720, after phosphorylation, acts through sphingosine 1-phosphate signaling pathways to modulate chemotactic responses and lymphocyte trafficking.