RESUMO
This study aims to evaluate the effect of combined use of the racemic flavanone Naringenin (NRG) and the protein sericin as TNF-α blockers. Sericin (SMs) and (R/S) NRG-loaded Sericin (SNRGMs) microparticles were prepared by spray-drying, characterized in terms of morphology and particle size distribution, and encapsulation efficiency was determined. Concerning morphology and particle size distribution of microparticles, results indicated that they were not affected by the presence of NRG. The encapsulation efficiency was almost quantitative (93%), thus proving that sericin can be advantageously loaded with (R/S) NRG. Biological evaluation of (R/S) NRG, SMs and SNRGMs was then performed in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (hPBMC). SNRGMs resulted cytotoxic at the higher dose used (200 µg/mL) and the effect was greater than (R/S) NRG alone. Moreover, even if sericin alone was not effective in suppressing LPS-induced serum TNF-α levels, SNRGMs loaded with 9.3% of (R/S) NRG were significantly more potent than (R/S) NRG alone. In summary, this study provides the proof of concept that sericin-based microspheres loaded with TNF-α-blockers could contribute to the down regulation of the cytokine and represents the starting point for the development of new topical formulations for the treatment of middle-stage psoriasis.
Assuntos
Flavanonas/uso terapêutico , Microesferas , Psoríase/tratamento farmacológico , Sericinas/química , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Células Cultivadas , Química Farmacêutica , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Flavanonas/química , Flavanonas/farmacologia , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/toxicidade , Tamanho da Partícula , Sericinas/farmacologia , Sericinas/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Previous work has established the existence of dystrophin-nitric oxide (NO) signaling to histone deacetylases (HDACs) that is deregulated in dystrophic muscles. As such, pharmacological interventions that target HDACs (that is, HDAC inhibitors) are of potential therapeutic interest for the treatment of muscular dystrophies. In this study, we explored the effectiveness of long-term treatment with different doses of the HDAC inhibitor givinostat in mdx mice--the mouse model of Duchenne muscular dystrophy (DMD). This study identified an efficacy for recovering functional and histological parameters within a window between 5 and 10 mg/kg/d of givinostat, with evident reduction of the beneficial effects with 1 mg/kg/d dosage. The long-term (3.5 months) exposure of 1.5-month-old mdx mice to optimal concentrations of givinostat promoted the formation of muscles with increased cross-sectional area and reduced fibrotic scars and fatty infiltration, leading to an overall improvement of endurance performance in treadmill tests and increased membrane stability. Interestingly, a reduced inflammatory infiltrate was observed in muscles of mdx mice exposed to 5 and 10 mg/kg/d of givinostat. A parallel pharmacokinetic/pharmacodynamic analysis confirmed the relationship between the effective doses of givinostat and the drug distribution in muscles and blood of treated mice. These findings provide the preclinical basis for an immediate translation of givinostat into clinical studies with DMD patients.
Assuntos
Carbamatos/uso terapêutico , Inibidores de Histona Desacetilases/uso terapêutico , Distrofia Muscular de Duchenne/tratamento farmacológico , Animais , Carbamatos/farmacologia , Células Cultivadas , Teste de Esforço , Fibrose/tratamento farmacológico , Fibrose/patologia , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/fisiopatologia , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , CorridaRESUMO
(RS)-Naringenin is a flavanone well-known for its beneficial health-related properties, such as its anti-inflammatory activity. The preparative enantioselective chromatographic resolution of commercial (RS)-naringenin was performed on a Chiralpak AD-H column (500×50 mm i.d., dp 20 µm) using MeOH as eluent. The developed method is in accordance with the principles of green chemistry, since the environmental impact was lowered by recycling of the eluent, and allowed the production of gram amounts of each enantiomer with high purity (chemical purity >99%, enantiomeric excess (ee) >94%). Racemic and enantiomeric naringenin were subjected to an exhaustive in vitro investigation of anti-inflammatory activity, aimed at evaluating the relevance of chirality. The assay with cultured human peripheral blood mononuclear cells (hPBMC) activated by phytohemagglutinin A revealed that (R)-naringenin was more effective in inhibiting T-cell proliferation than the (S)-enantiomer and the racemate. Moreover, (R)-naringenin significantly reduced proinflammatory cytokine levels such as those of TNF-α and, with less potency, IL-6. These results evidenced the anti-inflammatory potential of naringenin and the higher capacity of (R)-naringenin to inhibit both in vitro hPBMC proliferation and cytokine secretion at non toxic doses. Thus, (R)-naringenin is a promising candidate for in vivo investigation.
Assuntos
Anti-Inflamatórios/química , Flavanonas/química , Leucócitos Mononucleares/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citrus/química , Citocinas/metabolismo , Flavanonas/farmacologia , Química Verde , Humanos , Leucócitos Mononucleares/imunologia , Solanum lycopersicum/química , Malus/química , Estrutura Molecular , EstereoisomerismoRESUMO
Phytochemical investigation on the Amygdalus lycioides Spach branchelets resulted in the isolation of four chiral flavanones: (2R,3R)-Taxifolin, (2R,3R)-aromadendrin, (S)-5,7,3',5'-tetrahydroxyflavanone and (S)-naringenin. The flavanones were isolated by semi-preparative HPLC, their structures elucidated based on spectroscopic data and their absolute configuration assigned. As a part of our ethnobotanical-directed search for novel TNFα inhibitors, the bioassay-guided fractionation of the n-hexane-acetone (n-Hex-Ac, 1:1 v/v) Amygdalus lycioides Spach branchelets extract was performed. In this way, (S)-naringenin was identified as the constituent responsible for the TNFα blocking effect, being effective in vitro and in vivo after oral administration. This is the first investigation on bioactive secondary metabolites of Amygdalus lycioides Spach branchelets.
Assuntos
Flavonóis/química , Prunus/química , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Cromatografia Líquida de Alta Pressão , Flavonóis/isolamento & purificação , Flavonóis/farmacologia , Camundongos , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Ultravioleta , EstereoisomerismoRESUMO
Inhibition of histone deacetylases (HDAC) has been shown to modulate gene expression and cytokine production after stimulation with several stimuli. In the present study, the antiinflammatory effect of a potent HDACi, ITF2357, was explored in different experimental models of arthritis. In addition, the bone protective effect of ITF2357 was investigated in vitro. Treatment of acute arthritis (Streptococcus pyogenes cell wall [SCW] arthritis) with ITF2357 showed that joint swelling and cell influx into the joint cavity were reduced. Furthermore, the chondrocyte metabolic function was improved by treatment of ITF2357. The production of proinflammatory cytokines by synovial tissue was reduced after ITF2357 treatment. To examine the effect of HDAC inhibition on joint destruction, ITF2357 was applied to both rat adjuvant arthritis and mouse collagen type II arthritis. ITF2357 treatment both ameliorates the severity scores in arthritis models and prevents bone destruction. In an in vitro bone destruction assay, ITF2357 was highly effective at a dose of 100 nmol/L. In conclusion, inhibition of HDAC prevents joint inflammation and cartilage and bone destruction in experimental arthritis.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Experimental/fisiopatologia , Doenças Ósseas/prevenção & controle , Doenças das Cartilagens/prevenção & controle , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Articulações/imunologia , Articulações/patologia , Animais , Artrite Experimental/induzido quimicamente , Reabsorção Óssea/induzido quimicamente , Reabsorção Óssea/tratamento farmacológico , Colágeno/toxicidade , Interleucina-1beta/farmacologia , Articulações/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
ITF2357 (givinostat) is a histone deacetylase inhibitor with antiinflammatory properties at low nanomolar concentrations. We report here a phase I safety and pharmacokinetics trial in healthy males administered 50, 100, 200, 400 or 600 mg orally. After 50 mg, mean maximal plasma concentrations reached 104 nmol/L 2 h after dosing, with a half-life of 6.9 h. After 100 mg, maximal concentration reached 199 nmol/L at 2.1 h with a half-life of 6.0 h. Repeat doses for 7 consecutive days of 50, 100 or 200 mg resulted in nearly the same kinetics. There were no serious adverse effects (AEs) and no organ toxicities. However, there was a dose-dependent but transient fall in platelets. After 7 daily doses of 50 or 100 mg, the mean decrease in platelets of 17 and 25% was not statistically significant and returned to baseline within 14 d. Blood removed from the subjects after oral dosing was cultured ex vivo with endotoxin, and the release of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-1Ra, interferon (IFN)-γ and IL-10 was determined. Maximal reduction in IL-1ß, TNFα, IL-6 and IFNγ was observed 4 h after dosing but returned to baseline at 12 h. There was no significant reduction in IL-1Ra or IL-10. With daily dosing, the fall in cytokine production in blood cultures observed on day 7 was nearly the same as that of the first day. We conclude that dosing of 50 or 100 mg ITF2357 is safe in healthy humans and transiently but repeatedly reduces the production of proinflammatory cytokines without affecting production of antiinflammatory cytokines.
Assuntos
Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/farmacocinética , Citocinas/sangue , Ácidos Hidroxâmicos/efeitos adversos , Ácidos Hidroxâmicos/farmacocinética , Adulto , Anti-Inflamatórios/administração & dosagem , Feminino , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Interferon gama/sangue , Proteína Antagonista do Receptor de Interleucina 1/sangue , Interleucina-10/sangue , Interleucina-1beta/sangue , Interleucina-6/sangue , Masculino , Fator de Necrose Tumoral alfa/sangue , Adulto JovemRESUMO
BACKGROUND: In the search of genetic determinants of Duchenne muscular dystrophy (DMD) severity, LTBP4, a member of the latent TGF-ß binding protein family, emerged as an important predictor of functional outcome trajectories in mice and humans. Nonsynonymous single-nucleotide polymorphisms in LTBP4 gene associate with prolonged ambulation in DMD patients, whereas an in-frame insertion polymorphism in the mouse LTBP4 locus modulates disease severity in mice by altering proteolytic stability of the Ltbp4 protein and release of transforming growth factor-ß (TGF-ß). Givinostat, a pan-histone deacetylase inhibitor currently in phase III clinical trials for DMD treatment, significantly reduces fibrosis in muscle tissue and promotes the increase of the cross-sectional area (CSA) of muscles in mdx mice. In this study, we investigated the activity of Givinostat in mdx and in D2.B10 mice, two mouse models expressing different Ltbp4 variants and developing mild or more severe disease as a function of Ltbp4 polymorphism. METHODS: Givinostat and steroids were administrated for 15 weeks in both DMD murine models and their efficacy was evaluated by grip strength and run to exhaustion functional tests. Histological examinations of skeletal muscles were also performed to assess the percentage of fibrotic area and CSA increase. RESULTS: Givinostat treatment increased maximal normalized strength to levels that were comparable to those of healthy mice in both DMD models. The effect of Givinostat in both grip strength and exhaustion tests was dose-dependent in both strains, and in D2.B10 mice, Givinostat outperformed steroids at its highest dose. The in vivo treatment with Givinostat was effective in improving muscle morphology in both mdx and D2.B10 mice by reducing fibrosis. CONCLUSION: Our study provides evidence that Givinostat has a significant effect in ameliorating both muscle function and histological parameters in mdx and D2.B10 murine models suggesting a potential benefit also for patients with a poor prognosis LTBP4 genotype.
Assuntos
Distrofia Muscular de Duchenne , Animais , Carbamatos , Modelos Animais de Doenças , Haplótipos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Proteínas de Ligação a TGF-beta Latente/genética , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genéticaRESUMO
Histone deacetylase (HDAC) inhibitors are small molecules inducing cell-cycle arrest, differentiation, and apoptosis, currently undergoing clinical trials as anticancer drugs. In addition, emerging evidence suggests HDAC inhibitors may have anti-inflammatory and immunomodulatory properties as well, although the molecular mechanisms remain poorly defined. Given the central role of dendritic cells (DC) in the induction and maintenance of the inflammatory and immune response, we investigated the effects of HDAC inhibitors on the maturation and activation of human monocyte-derived DC in the presence of LPS and IFN-gamma. Our results show that the production of T(H)1- and T(H)17-inducing cytokines, namely IL-12 and IL-23, was inhibited by trichostatin A (72% and 52%, respectively) and suberoylanilide hydroxamic acid (86% and 83%). Strikingly, HDAC inhibitors were effective if added simultaneously as well as after the proinflammatory challenge, and their effect was not associated to a reduction of expression or function of LPS/IFN-gamma receptors. These findings were confirmed in two different murine models. In addition, HDAC inhibitors selectively blocked the production of T(H)1-attracting chemokines CXCL9, CXCL10, and CXCL11. The reduction of T(H)1- and T(H)17-inducing cytokines as well as T(H)1-attracting chemokines may represent relevant mechanisms through which HDAC inhibitors at nonproapoptotic doses exert their immunomodulatory properties.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Interleucina-12/metabolismo , Interleucina-23/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Técnicas In Vitro , Interferon gama/metabolismo , Interleucina-12/antagonistas & inibidores , Interleucina-23/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interferon/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vorinostat , Receptor de Interferon gamaRESUMO
In the original publication, the 6th author's first name and the last name was inverted and incorrectly published. The correct author name is Letizia Ferella.
RESUMO
PURPOSE: Aberrant expression and activity of histone deacetylases (HDACs) sustain glioblastoma (GBM) onset and progression, and, therefore, HDAC inhibitors (HDACi) represent a promising class of anti-tumor agents. Here, we analyzed the effects of ITF2357 (givinostat), a pan-HDACi, in GBM models for its anti-neoplastic potential. METHODS: A set of GBM- and patient-derived GBM stem-cell lines was used and the ITF2357 effects on GBM oncophenotype were investigated in in vitro and in vivo xenograft models. RESULTS: ITF2357 inhibited HDAC activity and affected GBM cellular fate in a dose-dependent manner by inducing G1/S growth arrest (1-2.5 µM) or caspase-mediated cell death (≥ 2.5 µM). Chronic treatment with low doses (≤ 1 µM) induced autophagy-mediated cell death, neuronal-like phenotype, and the expression of differentiation markers, such as glial fibrillar actin protein (GFAP) and neuron-specific class III beta-tubulin (Tuj-1); this reduces neurosphere formation from patient-derived GBM stem cells. Autophagy inhibition counteracted the ITF2357-induced expression of differentiation markers in p53-expressing GBM cells. Finally, in in vivo experiments, ITF2357 efficiently passed the blood-brain barrier, so rapidly reaching high concentration in the brain tissues, and significantly affected U87MG and U251MG growth in orthotopic xenotransplanted mice. CONCLUSIONS: The present findings provide evidence of the key role played by HDACs in sustaining transformed and stem phenotype of GBM and strongly suggest that ITF2357 may have a clinical potential for the HDACi-based therapeutic strategies against GBM.
Assuntos
Carbamatos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Histona Desacetilases/química , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Apoptose , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Histone deacetylase 6 (HDAC6) is a peculiar HDAC isoform whose expression and functional alterations have been correlated with a variety of pathologies such as autoimmune disorders, neurodegenerative diseases, and cancer. It is primarily a cytoplasmic protein, and its deacetylase activity is focused mainly on nonhistone substrates such as tubulin, heat shock protein (HSP)90, Foxp3, and cortactin, to name a few. Selective inhibition of HDAC6 does not show cytotoxic effects in healthy cells, normally associated with the inhibition of Class I HDAC isoforms. Here, we describe the design and synthesis of a new class of potent and selective HDAC6 inhibitors that bear a pentaheterocyclic central core. These compounds show a remarkably low toxicity both in vitro and in vivo and are able to increase the function of regulatory T cells (Tregs) at well-tolerated concentrations, suggesting a potential clinical use for the treatment of degenerative, autoimmune diseases and for organ transplantation.
Assuntos
Desacetilase 6 de Histona/antagonistas & inibidores , Ácidos Hidroxâmicos/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/metabolismo , Histonas/metabolismo , Camundongos , Isoformas de Proteínas , Baço/citologia , Linfócitos T Reguladores , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Adjuvant arthritis (AA) can be induced in Lewis rats by immunization with Mycobacterium tuberculosis (Mt) in oil. We have investigated the modulation of AA by mycobacterial 10-kDa heat shock protein (hsp10), administered according to several protocols known to induce immune tolerance and immune deviation. Subcutaneous immunization with hsp10 in aqueous solution did not induce a cellular immune response, evaluated as delayed-type hypersensitivity (DTH) reaction, although anti-hsp10 antibodies, mainly of the IgG2a isotype, were detected in serum of treated animals. When rats were pretreated with hsp10 in aqueous solution before AA induction, no effects were seen on arthritis-induced joint swelling, although osteolysis and lymphocyte infiltration were slightly decreased. When other routes of administration were attempted, the strongest suppression was seen in the group of animals which received four intranasal (i.n.) administrations of protein and a subsequent challenge of hsp10 in incomplete Freund's adjuvant (IFA). We also found that the extent of disease suppression among the different groups of animals correlated with serum anti-hsp10 antibody levels. These antibodies mostly belonged to the IgG2a subtype, suggesting that immune deviation may play a role in the mechanism of disease suppression by hsp10.
Assuntos
Artrite Experimental/imunologia , Artrite Experimental/prevenção & controle , Chaperonina 10/imunologia , Mycobacterium tuberculosis/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Artrite Experimental/diagnóstico por imagem , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/imunologia , Densidade Óssea , Chaperonina 10/administração & dosagem , Feminino , Hipersensibilidade Tardia/imunologia , Tolerância Imunológica/imunologia , Imunização Passiva , Radiografia , Ratos , Ratos Endogâmicos Lew , Fatores de TempoRESUMO
We investigated whether clinically achievable concentrations of the histone deacetylase (HDAC) inhibitors givinostat and hydroxyurea induce synergistic cytotoxicity in Jak2(V617F) cells in vitro and through which possible mechanism. Givinostat and hydroxyurea at low doses potentiated the pro-apoptotic effects of each other in the Jak2(V617F) HEL and UKE1 cell lines. Givinostat induced 6.8%-20.8% and hydroxyurea (HU) 20.4%-42.4% cell death alone and 35.8%-75.3% in combination. The effect was statistically significant using the median effect Chou-Talalay method, resulting in a combination index less than 1, indicating synergy. Givinostat alone induced cell cycle arrest of the cell lines in G0/G1 and hydroxyurea in S phase, whereas both drugs together led to a G1 block. At the molecular level, hydroxyurea counteracted the induction of p21CDKN1A by Givinostat and potentiated caspase 3 activation, explaining at least in part the increased apoptosis observed in presence of both compounds. We also verified the effect of the same drugs in colony assays of freshly isolated Jak2(V617F) polycythemia vera cells. In this case, low doses of the compounds were additive to each other. These results suggest that combined treatment with givinostat and hydroxyurea is a potential strategy for the management of Jak2(V617F) myeloproliferative neoplasms.
Assuntos
Apoptose/efeitos dos fármacos , Carbamatos/farmacologia , Hidroxiureia/farmacologia , Janus Quinase 2/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Substituição de Aminoácidos , Antineoplásicos/farmacologia , Western Blotting , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Sinergismo Farmacológico , Humanos , Janus Quinase 2/genética , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Mutação , Transtornos Mieloproliferativos/sangue , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Policitemia Vera/sangue , Policitemia Vera/metabolismo , Policitemia Vera/patologiaRESUMO
A number of Lys-Pro-containing short peptides have been described as possessing a variety of biological activities in vitro. Because of limited metabolic stability, however, their efficacy in vivo is uncertain. To exploit the pharmacological potential of Lys-Pro-containing short peptides, we synthesized a series of chemically modified forms of these peptides. One of them, ITF1697 (Gly-(Nalpha-Et)Lys-Pro-Arg) was stable in vivo and particularly efficacious in experimental models of disseminated endotoxemia and of cardiovascular disorders. Using intravital fluorescence microscopy, we studied the peptide cellular and molecular basis of protection in the Syrian hamster cheek pouch microcirculation subjected to ischemia/reperfusion (I/R) and in pressure elevation-induced proinflammatory responses in isolated Sprague-Dawley rat lungs. Continuous intravenous infusion of ITF1697 at 0.1 to 100 mug/kg/min nearly completely protected the cheek pouch microcirculation from I/R injury as measured by decreased vascular permeability and increased capillary perfusion. Adhesion of leukocytes and platelets to blood vessels was strongly inhibited by the peptide. ITF1697 exerted its activity at the early stages of endothelial activation and inhibited P-selectin and von Willebrand factor secretion. Further mechanistic studies in the rat lung preparation revealed that the peptide inhibited the intracellular Ca(2+)-dependent fusion of Weibel-Palade bodies with the plasma membrane. The ability of ITF1697 to inhibit the early functions of activated endothelial cells, such as the exocytosis of Weibel-Palade bodies, represents a novel and promising pharmacological tool in model of pathologies of a variety of microvascular disorders.
Assuntos
Exocitose/efeitos dos fármacos , Oligopeptídeos/farmacologia , Traumatismo por Reperfusão/metabolismo , Corpos de Weibel-Palade/efeitos dos fármacos , Animais , Cricetinae , Masculino , Pressão , Ratos , Ratos Sprague-Dawley , Corpos de Weibel-Palade/metabolismoRESUMO
Pharmacological manipulation of gene expression is considered a promising avenue to reduce postischemic brain damage. Histone deacetylases (HDACs) play a central role in epigenetic regulation of transcription, and inhibitors of HDACs are emerging as neuroprotective agents. In this study, we investigated the effect of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on histone acetylation in control and ischemic mouse brain. We report that brain histone H3 acetylation was constitutively present at specific lysine residues in neurons and astrocytes. It is noteworthy that in the ischemic brain tissue subjected to 6 h of middle cerebral artery occlusion, histone H3 acetylation levels drastically decreased, without evidence for a concomitant change of histone acetyl-transferase or deacetylase activities. Treatment with SAHA (50 mg/kg i.p.) increased histone H3 acetylation within the normal brain (of approximately 8-fold after 6 h) and prevented histone deacetylation in the ischemic brain. These effects were accompanied by increased expression of the neuroprotective proteins Hsp70 and Bcl-2 in both control and ischemic brain tissue 24 h after the insult. It is noteworthy that at the same time point, mice injected with SAHA at 25 and 50 mg/kg had smaller infarct volumes compared with vehicle-receiving animals (28.5% and 29.8% reduction, p < 0.05 versus vehicle, Student's t test). At higher doses, SAHA was less efficient in increasing Bcl-2 and Hsp70 expression and did not afford significant ischemic neuroprotection (13.9% infarct reduction). Data demonstrate that pharmacological inhibition of HDACs promotes expression of neuroprotective proteins within the ischemic brain and underscores the therapeutic potential of molecules inhibiting HDACs for stroke therapy.
Assuntos
Isquemia Encefálica/prevenção & controle , Encéfalo/irrigação sanguínea , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Animais , Western Blotting , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , VorinostatRESUMO
Inhibitors of histone deacetylases (HDAC) are being studied for their antiproliferative effects in preclinical cancer trials. Recent studies suggest an anti-inflammatory role for this class of compounds. Because inflammatory bowel disease is associated with an increased risk of malignancies, agents with antiproliferative and anti-inflammatory properties would be of therapeutic interest. HDAC inhibitors from various classes were selected and evaluated for their in vitro capacity to suppress cytokine production and to induce apoptosis and histone acetylation. Valproic acid (VPA) and suberyolanilide hydroxamic acid (SAHA) were chosen for further studies in dextran sulfate sodium- and trinitrobenzene sulfonic acid-induced colitis in mice. In vitro, inhibition of HDAC resulted in a dose-dependent suppression of cytokine synthesis and apoptosis induction requiring higher concentrations of HDAC inhibitors for apoptosis induction compared with cytokine inhibition. Oral administration of either VPA or SAHA reduced disease severity in dextran sulfate sodium-induced colitis. The macroscopic and histologic reduction of disease severity was associated with a marked suppression of colonic proinflammatory cytokines. In parallel to the beneficial effect observed, a dose-dependent increase in histone 3 acetylation at the site of inflammation was shown under VPA treatment. Furthermore, SAHA as well as VPA treatment resulted in amelioration of trinitrobenzene sulfonic acid-induced colitis, which was associated with an increase of apoptosis of lamina propria lymphocytes. Inhibitors of HDAC reveal strong protective effects in different models of experimental colitis by inducing apoptosis and suppressing proinflammatory cytokines, thereby representing a promising class of compounds for clinical studies in human inflammatory bowel disease.
Assuntos
Colite/tratamento farmacológico , Colite/metabolismo , Histonas/química , Histonas/metabolismo , Acetilação , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/farmacologia , Apoptose/efeitos dos fármacos , Colite/etiologia , Colite/patologia , Citocinas/biossíntese , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Feminino , Inibidores de Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Ácidos Hidroxâmicos/farmacologia , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Ácido Valproico/administração & dosagem , Ácido Valproico/farmacologia , VorinostatRESUMO
We studied inhibition of histone deacetylases (HDACs), which results in the unraveling of chromatin, facilitating increased gene expression. ITF2357, an orally active, synthetic inhibitor of HDACs, was evaluated as an anti-inflammatory agent. In lipopolysaccharide (LPS)-stimulated cultured human peripheral blood mononuclear cells (PBMCs), ITF2357 reduced by 50% the release of tumor necrosis factor-alpha (TNFalpha) at 10 to 22 nM, the release of intracellular interleukin (IL)-1alpha at 12 nM, the secretion of IL-1beta at 12.5 to 25 nM, and the production of interferon-gamma (IFNgamma) at 25 nM. There was no reduction in IL-8 in these same cultures. Using the combination of IL-12 plus IL-18, IFNgamma and IL-6 production was reduced by 50% at 12.5 to 25 nM, independent of decreased IL-1 or TNFalpha. There was no evidence of cell death in LPS-stimulated PBMCs at 100 nM ITF2357, using assays for DNA degradation, annexin V, and caspase-3/7. By Northern blotting of PBMCs, there was a 50% to 90% reduction in LPS-induced steady-state levels of TNFalpha and IFNgamma mRNA but no effect on IL-1beta or IL-8 levels. Real-time PCR confirmed the reduction in TNFalpha RNA by ITF2357. Oral administration of 1.0 to 10 mg/kg ITF2357 to mice reduced LPS-induced serum TNFalpha and IFNgamma by more than 50%. Anti-CD3-induced cytokines were not suppressed by ITF2357 in PBMCs either in vitro or in the circulation in mice. In concanavalin-A-induced hepatitis, 1 or 5 mg/kg of oral ITF2357 significantly reduced liver damage. Thus, low, nonapoptotic concentrations of the HDAC inhibitor ITF2357 reduce pro-inflammatory cytokine production in primary cells in vitro and exhibit anti-inflammatory effects in vivo.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Mediadores da Inflamação/farmacologia , Sequência de Aminoácidos , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Humanos , Inflamação/patologia , Inflamação/prevenção & controle , Mediadores da Inflamação/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Zea mays/enzimologiaRESUMO
Suberoylanilide hydroxamic acid (SAHA) is a hydroxamic acid-containing hybrid polar molecule; SAHA specifically binds to and inhibits the activity of histone deacetylase. Although SAHA, like other inhibitors of histone deacetylase, exhibits antitumor effects by increasing expression of genes regulating tumor survival, we found that SAHA reduces the production of proinflammatory cytokines in vivo and in vitro. A single oral administration of SAHA to mice dose-dependently reduced circulating TNF-alpha, IL-1-beta, IL-6, and IFN-gamma induced by lipopolysaccharide (LPS). Administration of SAHA also reduced hepatic cellular injury in mice following i.v. injection of Con A. SAHA inhibited nitric oxide release in mouse macrophages stimulated by the combination of TNF-alpha plus IFN-gamma. Human peripheral blood mononuclear cells stimulated with LPS in the presence of SAHA released less TNF-alpha, IL-1-beta, IL-12, and IFN-gamma (50% reduction at 100-200 nM). The production of IFN-gamma stimulated by IL-18 plus IL-12 was also inhibited by SAHA (85% at 200 nM). However, SAHA did not affect LPS-induced synthesis of the IL-1-beta precursor, the IL-1 receptor antagonist, or the chemokine IL-8. In addition, IFN-gamma induced by anti-CD3 was not suppressed by SAHA. Steady-state mRNA levels for LPS-induced TNF-alpha and IFN-gamma in peripheral blood mononuclear cells were markedly decreased, whereas IL-8 and IL-1-beta mRNA levels were unaffected. Because SAHA exhibits antiinflammatory properties in vivo and in vitro, inhibitors of histone deacetylase may stimulate the expression of genes that control the synthesis of cytokines and nitric oxide or hyperacetylate other targets.