RESUMEN
BMS-299897 is a gamma-secretase inhibitor that was effective in reducing amyloid beta-peptide (A beta) in transgenic mice and guinea pigs. Therefore, pharmacokinetic and drug metabolism studies were conducted in animals to support its clinical development. The compound appeared to have low to intermediate total body clearance and was orally bioavailable (24-100%). The oral absorption of BMS-299897 from solid dosage forms appeared to be dissolution rate-limited. BMS-299897 was distributed into extravascular space (V(ss) >or= 1.3 l kg(-1)), including brain (brain-to-plasma ratio = 0.13-0.50). BMS-299897 appeared to be a P-glycoprotein (P-gp) substrate as the brain-to-plasma ratio was two-fold higher in the mdr1a knockout mouse as compared with the wild-type. Apparent autoinduction by BMS-299897 was observed in murine and rat efficacy and toxicity studies. In vitro, BMS-299897 was a weaker inducer of cytochrome P450 3A4 (CYP3A4) and a weaker transactivator of human pregnane X receptor (hPXR) as compared with rifampicin. Induction of human UGT1A and UGT2B was evaluated in primary human hepatocytes, but the results were inconclusive. A low potential for autoinduction in humans was predicted at a clinical dose of 250 mg and the prediction was consistent with the findings from a clinical multiple-dose study with BMS-299897 in probable Alzheimer's patients.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Butiratos/farmacocinética , Citocromo P-450 CYP3A/biosíntesis , Sistema Enzimático del Citocromo P-450/biosíntesis , Hepatocitos/enzimología , Hidrocarburos Halogenados/farmacocinética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adulto , Animales , Antibióticos Antituberculosos/farmacocinética , Antibióticos Antituberculosos/farmacología , Disponibilidad Biológica , Encéfalo/enzimología , Butiratos/farmacología , Células Cultivadas , Perros , Inducción Enzimática/efectos de los fármacos , Femenino , Cobayas , Hepatocitos/citología , Humanos , Hidrocarburos Halogenados/farmacología , Masculino , Ratones , Ratones Noqueados , Receptor X de Pregnano , Ratas , Ratas Sprague-Dawley , Receptores de Esteroides/metabolismo , Rifampin/farmacocinética , Rifampin/farmacología , Especificidad de la EspecieRESUMEN
gamma-Secretase inhibitors are one promising approach to the development of a therapeutic for Alzheimer's disease (AD). gamma-Secretase inhibitors reduce brain beta-amyloid peptide (Abeta), which is believed to be a major contributor in the etiology of AD. Transgenic mice overexpressing the human beta-amyloid precursor protein (APP) are valuable models to examine the dynamics of Abeta changes with gamma-secretase inhibitors in plaque-free and plaque-bearing animals. BMS-299897 2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoic acid, a gamma-secretase inhibitor, showed dose- and time dependent reductions of Abeta in brain, cerebrospinal fluid (CSF), and plasma in young transgenic mice, with a significant correlation between brain and CSF Abeta levels. Because CSF and brain interstitial fluid are distinct compartments in composition and location, this correlation could not be assumed. In contrast, aged transgenic mice with large accumulations of Abeta in plaques showed reductions in CSF Abeta in the absence of measurable changes in plaque Abeta in the brain after up to 2 weeks of treatment. Hence, CSF Abeta levels were a valuable measure of gamma-secretase activity in the central nervous system in either the presence or absence of plaques. Transgenic mice were also used to examine potential side effects due to Notch inhibition. BMS-299897 was 15-fold more effective at preventing the cleavage of APP than of Notch in vitro. No changes in the maturation of CD8(+) thymocytes or of intestinal goblet cells were observed in mice treated with BMS-299897, showing that it is possible for gamma-secretase inhibitors to reduce brain Abeta without causing Notch-mediated toxicity.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Química Encefálica/efectos de los fármacos , Química Encefálica/genética , Endopeptidasas/fisiología , Inhibidores de Proteasas/farmacología , Envejecimiento/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/líquido cefalorraquídeo , Animales , Ácido Aspártico Endopeptidasas , Western Blotting , Separación Celular , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunoprecipitación , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Placa Amiloide/patología , Receptores Notch , Linfocitos T/metabolismoRESUMEN
The phenotypic and functional characteristics of activated T cells and recruited unactivated T cells at an inflammatory site were examined using a V beta 4+ myelin basic protein-specific T cell clone in a passively transferred model of experimental allergic encephalomyelitis. A high percentage of the T cells isolated from the central nervous system (CNS) were V beta 4+. This population exhibited the characteristics of activated T cells based on the proportion of cells in the blast state, their ability to proliferate in response to IL-2 or CNS Ag, and their expression of activation/memory cell markers. Activated V beta 4+ T cells were also observed in the periphery. Large numbers of V beta 4- T cells, which are entirely host-recruited, were also found in the CNS, where they demonstrated the properties of memory cells. There were differences in adhesion molecule expression between CNS V beta 4+ T cells and peripheral V beta 4+ T cells, although both populations were in activated state. V beta 4+ T cells at the site of Ag expression (the spinal cord) demonstrated higher levels of LFA-1 and CD44, but lower levels of VLA-4 and intercellular adhesion molecule-1, than did V beta 4+ T cells in the spleen. In contrast, the levels of all of these adhesion molecules on recruited V beta 4- T cells were higher in the CNS than in the periphery. This experimental model allows the detailed characterization of different T cell populations isolated from the same inflammatory site.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Memoria Inmunológica/inmunología , Activación de Linfocitos , Médula Espinal/patología , Linfocitos T/inmunología , Animales , Células Cultivadas , Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/patología , Receptores de Hialuranos/biosíntesis , Inmunización Pasiva , Integrina alfa4beta1 , Integrinas/biosíntesis , Molécula 1 de Adhesión Intercelular/biosíntesis , Interleucina-2/inmunología , Activación de Linfocitos/inmunología , Antígeno-1 Asociado a Función de Linfocito/biosíntesis , Ratones , Ratones Endogámicos , Proteína Básica de Mielina/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Interleucina-2/biosíntesis , Receptores Mensajeros de Linfocitos/biosíntesis , Médula Espinal/inmunología , Bazo/citologíaRESUMEN
Anti-tumor necrosis factor (TNF) antibodies inhibit passively transferred experimental allergic encephalomyelitis (EAE) in SJL mice. The possibility that this occurs through interference in TNF's upregulation of endothelial cell adhesion molecules was investigated. Expression of both vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on spinal cord vessels increased during EAE. The upregulation of VCAM-1 was markedly reduced or prevented by anti-TNF treatment. Leukocytic infiltration was 15-fold lower in anti-TNF-treated than diseased animals. Spinal cord endothelial expression of VCAM-1, though not ICAM-1 or fibronectin, positively correlated with the extent of T cell, B cell or monocyte infiltration in each animal.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Moléculas de Adhesión Celular/análisis , Encefalomielitis Autoinmune Experimental/terapia , Factor de Necrosis Tumoral alfa/fisiología , Animales , Células Cultivadas , Encefalomielitis Autoinmune Experimental/patología , Femenino , Fibronectinas/análisis , Humanos , Leucocitos/patología , Ratones , Receptores de Antígeno muy Tardío/análisis , Médula Espinal/química , Molécula 1 de Adhesión Celular VascularRESUMEN
Protein carboxylmethyltransferase (PCM) is an enzyme whose function in eucaryotic cells remains controversial. Early studies suggested that protein carboxylmethylation subserved a regulatory, post-translational role in such diverse processes as secretion, neuronal receptor function, chemotaxis, and cellular differentiation. Later work strongly supported a totally unrelated role for this enzyme, i.e., the repair of spontaneously altered aspartate residues in cellular proteins. More recent evidence, however, suggests that a distinct, membrane-associated PCM catalyzes the methylation of alpha-carboxyl groups of C-terminal cysteines on discrete proteins. In view of these recent investigations, the data supporting a regulatory role for PCM are critically discussed and re-evaluated. There now appears to be compelling evidence that PCM(s) subserves both repair and regulatory functions in eucaryotic cells, catalyzing post-translational modifications of proteins involved in cell division, hormonal secretion, calmodulin-associated events and the interaction of guanyl nucleotide-linked proteins with the cell membrane.
Asunto(s)
Células/enzimología , Células Eucariotas/enzimología , Proteína Metiltransferasas/fisiología , Animales , Diferenciación Celular , División CelularRESUMEN
Protein carboxylmethyltransferase has been proposed to play a role in the regulation of neuroblastoma differentiation (Kloog et al., 1983). When we investigated this hypothesis further, different results for methyl ester formation were obtained when measured in acid-precipitated proteins and in proteins separated by acidic polyacrylamide gel electrophoresis, following the incubation of intact neuroblastoma cells with [3H]methionine. These unexpected findings led to the development of a modified assay using S-[3H]-adenosylmethionine as the radiolabeled precursor for quantitating carboxyl methylation in intact cells. Data obtained from either acid-precipitated proteins or those separated on an electrophoresis gel following S-[3H]adenosylmethionine incubation directly correlated with data obtained from proteins separated by electrophoresis following [3H]methionine incubation. Using each of the three methods, an approximately twofold increase in the carboxyl methylation of cellular proteins was detected in neuroblastoma cells differentiated by reducing the serum concentration from 10 to 0.5%, but not in those cells differentiated with either 5 mM hexamethylene bisacetamide or 2% dimethyl sulfoxide. The finding that all detectable methyl acceptor proteins are increasingly methylated following 0.5% serum treatment and that this modification is substoichiometric suggests that protein carboxyl methylation is not an essential component of the differentiation process in neuroblastoma cells.
Asunto(s)
Neuroblastoma/metabolismo , Proteína Metiltransferasas/metabolismo , Proteína O-Metiltransferasa/metabolismo , S-Adenosilmetionina/metabolismo , Animales , Diferenciación Celular , Línea Celular , Electroforesis en Gel de Poliacrilamida , Ratones , Neuroblastoma/patología , Proteínas/aislamiento & purificación , Proteínas/metabolismo , Técnica de Dilución de Radioisótopos , S-Adenosilhomocisteína/metabolismo , TritioRESUMEN
The noncompetitive S-adenosylhomocysteine (AdoHcy) hydrolase antagonist adenosine dialdehyde (AD) has been shown to suppress the growth of cultured C-1300 murine neuroblastoma (MNB) cells. The enzymatic sites at which AD and other nucleoside analogues exert their cytotoxic effects have been postulated to include protein carboxylmethyltransferase (PCM), AdoHcy hydrolase, and ribonucleotide reductase. AD (10(-5) M) increased PCM activity 350% in suspensions prepared from disrupted cells after 72 h of drug exposure; in contrast, 3-deazaadenosine (10(-4) M) increased PCM activity 57%, whereas AdoHcy and sinefungin had no effect. When intact MNB cells were incubated with AD for varying time periods up to 72 h and then pulse labeled with the S-adenosylmethionine precursor L-[3H]-methionine, AD (10(-8) to 5 X 10(-6) M) produced a concentration-dependent inhibition of protein carboxylmethylation which persisted for up to 6 h. Following extended periods of AD treatment (48 to 72 h), AD (10(-6) to 10(-5) M) produced a 250% increment in protein carboxylmethylation, similar in magnitude to that observed in disrupted cell preparations. This increase in carboxylmethylation was observed at timepoints when AdoHcy hydrolase activity remained suppressed. The inhibitory effect of AD on AdoHcy hydrolase activity was maximal within 4 h and still apparent after 72 h of incubation. In contrast, AD treatment had no effect on the ribonucleotide reductase activity of MNB cells. These data suggest that the cytotoxic effect of AD on MNB cells results directly from its inhibition of AdoHcy hydrolase activity and indirectly through its suppression of methyltransferase enzyme systems. The potential linkage between the observed long-term elevations in PCM activity and AD-induced cytotoxicity remains to be defined.