RESUMEN
Immunosuppressive drugs are widely used for the treatment of immune-mediated diseases and inflammation, but the toxicity and side effects of the available immunosuppressors make the search of new agents of great relevance. Here, we evaluated the immunomodulatory activity of an N-acylhydrazone derivative, (E)-N'-(3,4-dimethoxybenzylidene)-4-methoxybenzohydrazide (LASSBio-1386), a phosphodiesterase-4 (PDE-4) inhibitor. LASSBio-1386 inhibited lymphocyte activation in a concentration-dependent fashion, decreasing lymphoproliferation and IFN-γ and IL-2 production stimulated by anti-CD3/CD28 mAbs or concanavalin A (Con A) and inducing cell-cycle arrest in the G0/G1 phase. These effects were not blocked by RU486, a glucocorticoid receptor (GR) antagonist, indicating an effect independent of glucocorticoid receptor activation. Combination index-isobologram analysis indicates a synergistic effect between LASSBio-1386 and dexamethasone in lymphoproliferation inhibition. LASSBio-1386 presented immunomodulatory action in macrophage cultures, as observed by a significant and concentration-dependent decrease in NO and TNF-α production, an effect achieved by reducing IĸB expression and NF-κB activation. In the mouse model of endotoxic shock, LASSBio-1386 at 50 and 100â¯mg/kg protected 50 and 85% of mice against LPS-induced lethality, respectively. In agreement to its in vitro action, treatment with 100â¯mg/kg of LASSBio-1386 reduced TNF-α and IL-1ß serum levels, while increased IL-6 and IL-10. Finally, LASSBio-1386 reduced the paw edema in a BSA-induced delayed-type hypersensitivity model. These findings demonstrate the immunomodulatory and immunosuppressant effects of LASSBio-1386 and indicate this molecule is a promising pharmacologic agent for immune-mediated diseases.
Asunto(s)
Hidrazonas/farmacología , Hipersensibilidad Tardía/tratamiento farmacológico , Inmunosupresores/farmacología , Lipopolisacáridos/toxicidad , Inhibidores de Fosfodiesterasa 4/farmacología , Choque/tratamiento farmacológico , Animales , Benzamidas , Citocinas/genética , Citocinas/metabolismo , Dexametasona/farmacología , Antagonistas de Hormonas/farmacología , Hidrazonas/química , Macrófagos , Masculino , Ratones , Ratones Endogámicos BALB C , Mifepristona/farmacología , Estructura Molecular , FN-kappa B/genética , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Células RAW 264.7RESUMEN
Betulinic acid (BA) is a naturally occurring triterpenoid with several biological properties already described, including immunomodulatory activity. Here we investigated the immunomodulatory activity of eight semi-synthetic amide derivatives of betulinic acid. Screening of derivatives BA1-BA8 led to the identification of compounds with superior immunomodulatory activity than BA on activated macrophages and lymphocytes. BA5, the most potent derivative, inhibited nitric oxide and TNFα production in a concentration-dependent manner, and decreased NF-κB activation in Raw 264.7 cells. Additionally, BA5 inhibited the proliferation of activated lymphocytes and the secretion of IL-2, IL-4 IL-6, IL-10, IL-17A and IFNÉ£, in a concentration-dependent manner. Flow cytometry analysis in lymphocyte cultures showed that treatment with BA5 induces cell cycle arrest in pre-G1 phase followed by cell death by apoptosis. Moreover, BA5 also inhibited the activity of calcineurin, an enzyme that plays a critical role in the progression of cell cycle and T lymphocyte activation. BA5 has a synergistic inhibitory effect with dexamethasone on lymphoproliferation, showing a promising profile for drug combination. Finally, we observed immunosuppressive effects of BA5 in vivo in mouse models of lethal endotoxemia and delayed type hypersensitivity. Our results reinforce the potential use of betulinic acid and its derivatives in the search for potent immunomodulatory drugs.