RESUMEN
OBJECTIVES: To evaluate the early tumor vasculature disrupting effects of the AVE8062 molecule and the feasibility of dynamic contrast-enhanced ultrasonography (DCE-US) in the quantitative assessment of these effects. MATERIAL AND METHODS: AVE8062 was administered at a single dose (41 mg/kg) to 40 melanoma-bearing nude mice, which were all imaged before and after drug administration (5 + 15 minutes, 1, 6, and 24 hours). Using an ultrasound scanner (Aplio, Toshiba), intratumor vessels were counted in power Doppler mode and tumor microvasculature was assessed in a specific harmonic mode associated with a perfusion and quantification software for contrast-uptake quantification (Sonovue, Bracco). The peak intensity (PI), time-to-PI (T PI), and full-width at half maximum (FWHM) were extracted from the time-intensity curves expressed as linear raw data. Histologic analysis evaluated microvessel density (MVD) and necrosis at each time point studied. Statistical significance was estimated (paired sum rank and Mann-Whitney tests) to evaluate drug activity and to compare its efficacy at the different time points. RESULTS: In power Doppler mode, intratumoral vessels depletion started 15 minutes postinjection (32%, P = 0.004) and the decrease was maximal at 6 hours (51%, P = 0.002). PI decreased by 3.5- and 45.7-fold at 1 and 6 hours, respectively, compared with preinjection values (P = 0.016 and P = 0.008). The decrease at 6 hours was significantly different from the variation at 1 hour (P = 0.0012) and at 24 hours (P = 0.0008). T PI and FWHM showed a significant increase exclusively at 6 hours (P = 0.0034, P = 0.0039). Histology revealed significantly decreased MVD and increased necrosis at 24 hours (P < 0.01). CONCLUSION: DCE-US allowed quantitative in vivo evaluation of the functional effects of AVE8062, which was found most effective on tumoral microvasculature 6 hours after its administration. A clinical phase-1 study of AVE8062 is ongoing using the same ultrasonography methodology before and 6 and 24 hours postadministration.
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Inhibidores de la Angiogénesis , Antineoplásicos , Medios de Contraste , Melanoma/diagnóstico por imagen , Microburbujas , Piel/irrigación sanguínea , Animales , Femenino , Melanoma/irrigación sanguínea , Melanoma/patología , Ratones , Necrosis , Factores de Tiempo , UltrasonografíaRESUMEN
The development of suitable Cystic Fibrosis (CF) models for preclinical bench tests of therapeutic candidates is challenging. Indeed, the validation of molecules to rescue the p.Phe508del-CFTR channel (encoded by the Cystic Fibrosis Transmembrane conductance Regulator gene carrying the p.Phe508del mutation) requires taking into account their overall effects on the epithelium. Suberoylanilide Hydroxamic Acid (SAHA), a histone deacetylase inhibitor (HDACi), was previously shown to be a CFTR corrector via proteostasis modulation in CFTR-deficient immortalized cells. Here, we tested SAHA effects on goblet cell metaplasia using an ex vivo model based on the air-liquid interface (ALI) culture of differentiated airway epithelial cells obtained by nasal scraping from CF patients and healthy controls. Ex vivo epithelium grew successfully in ALI cultures with significant rise in the expression of CFTR and of markers of airway epithelial differentiation compared to monolayer cell culture. SAHA decreased CFTR transcript and protein levels in CF and non-CF epithelia. Whereas SAHA induced lysine hyperacetylation, it did not change histone modifications at the CFTR promoter. SAHA reduced MUC5AC and MUC5B expression and inhibited goblet epithelial cell differentiation. Similar effects were obtained in CF and non-CF epithelia. All the effects were fully reversible within five days from SAHA withdrawal. We conclude that, ex vivo, SAHA modulate the structure of airway epithelia without specific effect on CFTR gene and protein suggesting that HDACi cannot be useful for CF treatment.
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Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Masculino , Mucina 5AC/genética , Mucina 5B/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Vorinostat , Adulto JovenRESUMEN
We investigated the molecular events involved in the long-lasting reduction of adipose mass by the selective CB1 antagonist, SR141716. Its effects were assessed at the transcriptional level both in white (WAT) and brown (BAT) adipose tissues in a diet-induced obesity model in mice. Our data clearly indicated that SR141716 reversed the phenotype of obese adipocytes at both macroscopic and genomic levels. First, oral treatment with SR141716 at 10 mg/kg/d for 40 days induced a robust reduction of obesity, as shown by the 50% decrease in adipose mass together with a major restoration of white adipocyte morphology similar to lean animals. Second, we found that the major alterations in gene expression levels induced by obesity in WAT and BAT were mostly reversed in SR141716-treated obese mice. Importantly, the transcriptional patterns of treated obese mice were similar to those obtained in the CB1 receptor knockout mice fed a high-fat regimen and which are resistant to obesity, supporting a CB1 receptor-mediated process. Functional analysis of these modulations indicated that the reduction of adipose mass by the molecule resulted from an enhanced lipolysis through the induction of enzymes of the beta-oxidation and TCA cycle, increased energy expenditure, mainly through futile cycling (calcium and substrate), and a tight regulation of glucose homeostasis. These changes accompanied a significant cellular remodeling and contributed to a reduction of the obesity-related inflammatory status. In addition to a transient reduction of food consumption, increases of both fatty acid oxidation and energy expenditure induced by the molecule summate leading to a sustained weight loss. Altogether, these data strongly indicate that the endocannabinoid system has a major role in the regulation of energy metabolism.
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Metabolismo Energético/efectos de los fármacos , Lipólisis/efectos de los fármacos , Obesidad/tratamiento farmacológico , Piperidinas/farmacología , Pirazoles/farmacología , Receptor Cannabinoide CB1/antagonistas & inhibidores , Adipocitos/efectos de los fármacos , Adiponectina/genética , Animales , Citoesqueleto/efectos de los fármacos , Grasas de la Dieta/administración & dosificación , Ingestión de Energía , Perfilación de la Expresión Génica , Glucosa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Fenotipo , Piperidinas/uso terapéutico , Pirazoles/uso terapéutico , Rimonabant , Termogénesis/efectos de los fármacosRESUMEN
SR31747A is a sigma ligand with potent antiproliferative activity against tumor cells and for which three binding proteins have been identified to date: (a) SRBP-1 (also called sigma 1); (b) HIS; and (c) sigma 2. In this study, we characterized an additional SR31747A binding site, i.e., SRBP-2 (SR31747A-binding protein 2). Using an in silico screening approach, we identified this novel sequence, which exhibits 41% homology with HSI. The 1142-bp cDNA was found to encode a 206 amino acid protein not related to SRBP-1. Northern blot analysis of SRBP-2 mRNA expression revealed a single 1.1-kb transcript that was widely expressed in organs; the liver was particularly enriched, and the brain showed the lowest abundance. A murine homologue that exhibited a similar expression pattern was also characterized. Subcellular localization analysis using specific polyclonal antibodies revealed that SRBP-2 had the same nuclear membrane and endoplasmic reticulum localization as other members of the SR31747A-binding protein family. Considering SRBP-2-binding properties, pharmacological analysis clearly highlighted that SRBP-2 was distinct from sigma 2. Scatchard plot analysis revealed K(d) values of 10 and 3 nM for SR31747A and Tamoxifen, respectively. In contrast with HSI, the protein also did not exhibit detectable isomerase activity. When analyzing SRBP-2 expression in human breast cancer biopsies, we obtained evidence that SRBP-2 expression, together with SRBP-1 and HSI, may be of interest as a prognostic marker. These findings demonstrated that SRBP-2 represents an additional molecular target for SR31747A, which could help to understand the immunosuppressive and antiproliferative effects of the molecule.
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Proteínas Portadoras/análisis , Receptores Opioides , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Proteínas Portadoras/genética , Proteínas Portadoras/farmacología , Femenino , Humanos , Hibridación in Situ , Hibridación Fluorescente in Situ , Ratones , Datos de Secuencia Molecular , Pronóstico , ARN Mensajero/análisis , Conejos , Receptores sigma , Esteroide Isomerasas/metabolismo , Células Tumorales Cultivadas , Receptor Sigma-1RESUMEN
Exhaustive analysis of the location of the peripheral benzodiazepine receptor (PBR) both at the subcellular and the tissue level is warranted to gain a better understanding of its biological roles. To date, many studies have been performed in animal models, such as rat, mouse, and pig, that yielded important information. However, only a few reports were dedicated to the analysis of PBR expression in humans. To enlarge on previous studies, we investigated PBR expression in different human organs using the monoclonal antibody 8D7 that specifically recognized the human PBR. First, we performed electron microscopic analysis that for the first time unambiguously demonstrated the localization of the PBR on the outer mitochondrial membrane. Second, focusing our analysis on human tissues for which information on PBR expression is sparse (lung, stomach, small intestine, colon, thyroid, adrenal gland, pancreas, breast, prostate, ovary), we found that PBR exhibits selective localization. This characterization of PBR localization in human tissues should provide important insights for the understanding of PBR functions.
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Receptores de GABA-A/metabolismo , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Sistema Endocrino/metabolismo , Humanos , Inmunohistoquímica , Microscopía Electrónica , Especificidad de Órganos , Receptores de GABA-A/inmunología , Fracciones Subcelulares/metabolismoRESUMEN
In this study, we documented the effects of different peripheral benzodiazepine receptor (PBR) ligands: PK 11195, Ro5-4864 and the newly described SSR 180575 on the development of pulmonary inflammation in vivo. To this aim, we used MRL/lpr mice that develop pathological signs similar to the human lupus erythematosus (LE) signs. We found that a chronic treatment (at 3 mg/kg per i.p. for 30 days) with PBR ligands had a significant beneficial therapeutic action and decreased the inflammatory pulmonary responses and alveolitis onset. When analyzing PBR expression in inflamed tissues, we observed that in addition to the infiltrated leukocytes, PBR was expressed in the bronchial epithelium, and especially we evidenced for the first time that PBR in expressed in Clara cells. Interestingly, we observed that PBR expression in those cells was reduced when MRL/lpr mice developed the pathology and restored upon PBR ligand treatment. These original findings support a role of PBR in pulmonary inflammatory process and suggest new therapeutic applications in auto immune disorders for specific potent PBR ligands.
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Antagonistas de Receptores de GABA-A , Neumonía/tratamiento farmacológico , Receptores de GABA-A/metabolismo , Acetamidas/química , Acetamidas/metabolismo , Acetamidas/uso terapéutico , Animales , Benzodiazepinonas/química , Benzodiazepinonas/metabolismo , Benzodiazepinonas/uso terapéutico , Indoles/química , Indoles/metabolismo , Indoles/uso terapéutico , Inyecciones Intraperitoneales , Isoquinolinas/química , Isoquinolinas/metabolismo , Isoquinolinas/uso terapéutico , Ligandos , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos MRL lpr , Estructura Molecular , Neumonía/patología , Receptores de GABA-A/análisisRESUMEN
Mrl/Lpr mice develop inflammatory pathologies similar to human lupus erythematosus (LE). In that model, we showed a protective effect of different peripheral benzodiazepine receptor (PBR) ligands: PK 11195, Ro5-4864 and the newly described SSR180575 on the development of the cutaneous lesions. Specifically, we evidenced that a chronic treatment at 3 mg/kg per i.p. for 30 days prevented acanthosis, hyperkeratosis and generation of dermal infiltrates as compared with control untreated mice. In addition, using a specific polyclonal anti mouse PBR antibody, we characterized PBR expression in the skin lesions, and we observed that PBR expression in the epidermal component was increased when Mrl/Lpr mice developed the pathology and diminished upon PBR ligand treatment. PBR expression modulation together with the protective effects of its ligands further reinforce the role that PBR may play in the regulation of inflammation processes. Provided the exact mechanism of action that accounts for PBR action in that process is elucidated, these data support new therapeutic applications for specific potent PBR ligands.
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Lupus Eritematoso Cutáneo/patología , Receptores de GABA-A/fisiología , Piel/patología , Animales , Anticuerpos Bloqueadores/farmacología , Modelos Animales de Enfermedad , Agonistas de Receptores de GABA-A , Isoquinolinas , Ligandos , Lupus Eritematoso Cutáneo/inmunología , Ratones , Ratones Endogámicos MRL lprRESUMEN
Sigma (sigma) receptors were initially proposed as a subtype of opiate receptors, and bind several psychoactive compounds. They are classified into sigma 1 (sigma1) and sigma 2 (sigma2) subtypes. The characterization of these subunits, and the discovery of new specific sigma receptor ligands, demonstrated that sigma receptors belong to a specific entity distinct from opiate receptors. Radioligand-binding data have recently demonstrated that the sigma1 subtype is related to a sterol isomerase, which is involved in the cholesterol biosynthesis pathway, and also to another protein of unknown function, SRBP2 (SR-31747 binding-protein 2), which shares a high homology with this enzyme. This complex group of proteins also binds molecules devoid of central effect, which demonstrate potent anti-inflammatory properties, and so are potentially useful in pathologies where pro-inflammatory cytokines are involved, particularly rheumatoid arthritis, Crohn's disease or psoriasis. In addition, the two sigma receptor subtypes and their two related proteins are also expressed on tumor cells, where they could be of prognostic relevance, and their ligands could potentially be used in the detection and targeting of tumors.
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Adamantano/análogos & derivados , Antiinflamatorios/farmacología , Antineoplásicos/farmacología , Receptores sigma/efectos de los fármacos , Adamantano/farmacología , Animales , Ciclohexanos/farmacología , Humanos , Interleucina-1/antagonistas & inhibidores , Ligandos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
In this study, the effects of different peripheral benzodiazapine receptor ligands: PK 11195 [1-(2-chloro-phenyl)-N-methyl-N-(1-methylpropyl)-1-isoquinoline carboxamide], Ro5-4864 [7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one] and the newly described SSR 180575 (7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridozine[4,5-b] indole-1-acetamide) were analysed on the progression and severity of rheumatoid arthritis in vivo in the Mrl/lpr mice model, following chronic treatment (at 3 mg/kg, i.p. for 30 days). We found that peripheral benzodiazepine receptor ligands have significant beneficial therapeutic action on the development of spontaneous rheumatoid arthritis-like signs. Concomitantly, we mapped immunoreactive peripheral benzodiazepine receptor in inflamed tissues, and we observed that in addition to the infiltrated leukocytes, peripheral benzodiazepine receptor was expressed in synovial membranes, at the cartilage pannus junction and in chondrocytes. Interestingly, we observed that peripheral benzodiazepine receptor expression in chondrocytes was reduced when Mrl/lpr mice developed the pathology and restored upon peripheral benzodiazepine receptor ligand treatment. Altogether, our data provide further evidence of a role played by peripheral benzodiazepine receptor in the regulation of inflammation processes and support new therapeutic applications for specific potent peripheral benzodiazepine receptor ligands.
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Artritis Reumatoide/patología , Receptores de GABA-A/fisiología , Acetamidas/farmacología , Animales , Anticuerpos Bloqueadores/farmacología , Antirreumáticos/farmacología , Benzodiazepinonas/farmacología , Western Blotting , Línea Celular , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Condrocitos/patología , Antagonistas de Receptores de GABA-A , Miembro Posterior/patología , Indoles/farmacología , Isoquinolinas/farmacología , Articulaciones/patología , Ligandos , Masculino , Metotrexato/farmacología , Ratones , Ratones Endogámicos MRL lpr , Microscopía Confocal , Membrana Sinovial/patologíaRESUMEN
SSR125329A ([(Z)-3-(4-Adamantan-2-yl-3,5-dichloro-phenyl)-allyl]-cyclohexyl-ethyl-amine) is a new ligand exhibiting high affinity for sigma(1) and sigma(2) receptors and for the human Delta8-Delta7-sterol isomerase. Here we show that this molecule has potent immunoregulatory properties both in vitro and in vivo. SSR125329A inhibited staphylococcal enterotoxin B-induced mouse splenocyte proliferation in vitro, whereas in vivo it enhanced lipopolysaccharide-induced systemic release of interleukin-10 while simultaneously inhibiting tumor necrosis factor-alpha (TNF-alpha) synthesis. It also prevented graft-versus-host disease in B6D2F1 mice and protected Mrl/lpr mice against the development of its spontaneous rheumatoid-like syndrome. There is high interplay of pro- and anti-inflammatory cytokines in inflammatory processes, particularly in human rheumatoid arthritis. The results of this study provide substantial evidence that sigma receptor ligands may represent a new effective approach for rheumatoid arthritis treatment.
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Adamantano/farmacología , Antiinflamatorios/farmacología , Receptores sigma/metabolismo , Adamantano/análogos & derivados , Animales , Artritis Reumatoide/patología , Artritis Reumatoide/prevención & control , Unión Competitiva , División Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Enterotoxinas/farmacología , Femenino , Enfermedad Injerto contra Huésped/prevención & control , Interleucina-10/metabolismo , Ligandos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos , Subunidades de Proteína/metabolismo , Receptores sigma/efectos de los fármacos , Bazo/citología , Bazo/efectos de los fármacos , Esteroide Isomerasas/metabolismo , Síndrome , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Intrarenal distribution and function(s) of the peripheral benzodiazepine receptor (PBR) remain uncertain. The goals of this study were to (1) develop a specific anti-rat PBR antibody and (2) map intrarenal immunoreactive PBR (irPBR) in untreated rats and in rats that received chronic angiotensin II infusion (200 ng/kg per min, subcutaneously, 17 d). A polyclonal rabbit antibody was raised against the C-terminal end of rat PBR (aa 159 to 169). The antibody specifically recognized a single 18-kD protein in whole kidney extracts, and confocal microscopy showed exclusive mitochondrial localization of irPBR in cultured rat glial C6 cells. In control rats, irPBR was found along thick ascending limbs of Henle's loops, including the macula densa area, along distal tubules, and along collecting ducts. Vascular smooth-muscle cells were PBR-positive. General irPBR distribution was unaffected by angiotensin II treatment (systolic BP, 205 +/- 9 mmHg). However, irPBR appeared in parietal glomerular epithelial cells, atrophic proximal tubules, and infiltrating mononuclear cells. In conclusion, the results suggest previously unsuspected roles of PBR in the control of glomerular dynamics and in proximal tubular injury/repair processes.