RESUMEN
RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) accompanying increased production of inflammatory factors and adaptation of the mitochondrial metabolism to a hyperproliferative state. However, all the drugs in clinical use target pulmonary vascular dilatation, which may not be effective for patients with advanced PAH. OBJECTIVE: We aimed to discover a novel drug for PAH that inhibits PASMC proliferation. METHODS AND RESULTS: We screened 5562 compounds from original library using high-throughput screening system to discover compounds which inhibit proliferation of PASMCs from patients with PAH (PAH-PASMCs). We found that celastramycin, a benzoyl pyrrole-type compound originally found in a bacteria extract, inhibited the proliferation of PAH-PASMCs in a dose-dependent manner with relatively small effects on PASMCs from healthy donors. Then, we made 25 analogs of celastramycin and selected the lead compound, which significantly inhibited cell proliferation of PAH-PASMCs and reduced cytosolic reactive oxygen species levels. Mechanistic analysis demonstrated that celastramycin reduced the protein levels of HIF-1α (hypoxia-inducible factor 1α), which impairs aerobic metabolism, and κB (nuclear factor-κB), which induces proinflammatory signals, in PAH-PASMCs, leading to reduced secretion of inflammatory cytokine. Importantly, celastramycin treatment reduced reactive oxygen species levels in PAH-PASMCs with increased protein levels of Nrf2 (nuclear factor erythroid 2-related factor 2), a master regulator of cellular response against oxidative stress. Furthermore, celastramycin treatment improved mitochondrial energy metabolism with recovered mitochondrial network formation in PAH-PASMCs. Moreover, these celastramycin-mediated effects were regulated by ZFC3H1 (zinc finger C3H1 domain-containing protein), a binding partner of celastramycin. Finally, celastramycin treatment ameliorated pulmonary hypertension in 3 experimental animal models, accompanied by reduced inflammatory changes in the lungs. CONCLUSIONS: These results indicate that celastramycin ameliorates pulmonary hypertension, reducing excessive proliferation of PAH-PASMCs with less inflammation and reactive oxygen species levels, and recovered mitochondrial energy metabolism. Thus, celastramycin is a novel drug for PAH that targets antiproliferative effects on PAH-PASMCs.
Asunto(s)
Miocitos del Músculo Liso/efectos de los fármacos , Naftoquinonas/farmacología , Hipertensión Arterial Pulmonar/tratamiento farmacológico , Pirroles/farmacología , Resorcinoles/farmacología , Animales , Células Cultivadas , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Metabolismo Energético/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Hipoxia/complicaciones , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Indoles/toxicidad , Masculino , Metaboloma/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Monocrotalina/toxicidad , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/biosíntesis , Naftoquinonas/uso terapéutico , Estrés Oxidativo , Hipertensión Arterial Pulmonar/inducido químicamente , Arteria Pulmonar/citología , Pirroles/uso terapéutico , Pirroles/toxicidad , Ratas , Especies Reactivas de Oxígeno/metabolismo , Resorcinoles/uso terapéutico , Factores de Transcripción/fisiologíaRESUMEN
RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling with aberrant pulmonary artery smooth muscle cells (PASMCs) proliferation, endothelial dysfunction, and extracellular matrix remodeling. OBJECTIVE: Right ventricular (RV) failure is an important prognostic factor in PAH. Thus, we need to elucidate a novel therapeutic target in both PAH and RV failure. METHODS AND RESULTS: We performed microarray analysis in PASMCs from patients with PAH (PAH-PASMCs) and controls. We found a ADAMTS8 (disintegrin and metalloproteinase with thrombospondin motifs 8), a secreted protein specifically expressed in the lung and the heart, was upregulated in PAH-PASMCs and the lung in hypoxia-induced pulmonary hypertension (PH) in mice. To elucidate the role of ADAMTS8 in PH, we used vascular smooth muscle cell-specific ADAMTS8-knockout mice (ADAMTSΔSM22). Hypoxia-induced PH was attenuated in ADAMTSΔSM22 mice compared with controls. ADAMTS8 overexpression increased PASMC proliferation with downregulation of AMPK (AMP-activated protein kinase). In contrast, deletion of ADAMTS8 reduced PASMC proliferation with AMPK upregulation. Moreover, deletion of ADAMTS8 reduced mitochondrial fragmentation under hypoxia in vivo and in vitro. Indeed, PASMCs harvested from ADAMTSΔSM22 mice demonstrated that phosphorylated DRP-1 (dynamin-related protein 1) at Ser637 was significantly upregulated with higher expression of profusion genes (Mfn1 and Mfn2) and improved mitochondrial function. Moreover, recombinant ADAMTS8 induced endothelial dysfunction and matrix metalloproteinase activation in an autocrine/paracrine manner. Next, to elucidate the role of ADAMTS8 in RV function, we developed a cardiomyocyte-specific ADAMTS8 knockout mice (ADAMTS8ΔαMHC). ADAMTS8ΔαMHC mice showed ameliorated RV failure in response to chronic hypoxia. In addition, ADAMTS8ΔαMHC mice showed enhanced angiogenesis and reduced RV ischemia and fibrosis. Finally, high-throughput screening revealed that mebendazole, which is used for treatment of parasite infections, reduced ADAMTS8 expression and cell proliferation in PAH-PASMCs and ameliorated PH and RV failure in PH rodent models. CONCLUSIONS: These results indicate that ADAMTS8 is a novel therapeutic target in PAH.
Asunto(s)
Proteínas ADAMTS/deficiencia , Insuficiencia Cardíaca/metabolismo , Hipertensión Arterial Pulmonar/metabolismo , Disfunción Ventricular Derecha/metabolismo , Proteínas ADAMTS/antagonistas & inhibidores , Proteínas ADAMTS/genética , Adulto , Animales , Células Cultivadas , Sistemas de Liberación de Medicamentos/tendencias , Femenino , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/patología , Humanos , Masculino , Mebendazol/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Hipertensión Arterial Pulmonar/tratamiento farmacológico , Hipertensión Arterial Pulmonar/patología , Distribución Aleatoria , Disfunción Ventricular Derecha/tratamiento farmacológico , Disfunción Ventricular Derecha/patologíaRESUMEN
Objectives: The main goal of this research was to develop better tablet formulations by utilizing solid dispersions (SDs) and coprocessing excipients composite to achieve a better release rate of poor water-soluble gliclazide. Methods: The solvent evaporation method made SDs of gliclazide with different carriers carplex 67, carplex 80, and carplex FPS 500 (weight ratio, 1:1). The drug release patterns of the SDs were all evaluated and optimized. The SDs were illustrated by using scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray powder diffraction (PXRD), and Fourier transform infrared spectroscopy (FTIR). Tablet batches FGC-1 to 8 were made using gliclazide-carplex 67 solid dispersions (GC67-SDs) and the co-processed composite of excipients, namely starch-MCC-povidone (SMP) and lactose-MCC-povidone-sodium starch glycolate (LMPS), prepared with coprocessing technology. We evaluated these batches by conducting physicochemical tests and comparing them to the existing commercial brand. Results: In a water medium, the release of gliclazide from SDs peaked within the first 30 min, showing a roughly 5â¼6-fold increase compared to plain gliclazide. This quick dissolution rate may be due to the amorphization of the drug, which improved the specific surface area, and increased wettability caused by the hydrophilic properties of carplex particles. This has been confirmed through SEM, DSC, FTIR, and PXRD analysis. All FGC formulations had satisfactory pre-compression factor results, while the post-compression parameters indicated good mechanical strength and homogeneity across the blend. All produced tablets met the weight variation, friability, and disintegration time limit set by the compendia. Through in vitro drug release testing, it was discovered that all FGC tablet batches had consistent and nearly identical release results compared to SDs of gliclazide. However, the FGC-5 to 8 batches containing LMPS composites were determined to be the most effective formulations. In the first 30 min in a water medium, the percentage of drug generated from the FGC-8 tablets involving GC67-SDs and co-processed composite LMPS-4 is approximately 3.5 times higher than the average release of currently marketed products (MPs). After storing the selected FGC tablet batches for three months at 40 °C and 75 % RH, there were no noticeable alterations in the amount of drug and drug release profiles across the batches. Conclusion: Based on these findings, it appears that using the carplex silica-based SDs approach, along with gliclazide and co-processing excipients composite, could result in significant benefits compared to the current commercial brands. This approach could be effectively utilized to create solid dosage forms for drugs that have low solubility in water.