RESUMEN
Malignant gliomas are a type of primary brain tumour that originates in glial cells. Among them, glioblastoma multiforme (GBM) is the most common and the most aggressive brain tumour in adults, classified as grade IV by the World Health Organization. The standard care for GBM, known as the Stupp protocol includes surgical resection followed by oral chemotherapy with temozolomide (TMZ). This treatment option provides a median survival prognosis of only 16-18 months to patients mainly due to tumour recurrence. Therefore, enhanced treatment options are urgently needed for this disease. Here we show the development, characterization, and in vitro and in vivo evaluation of a new composite material for local therapy of GBM post-surgery. We developed responsive nanoparticles that were loaded with paclitaxel (PTX), and that showed penetration in 3D spheroids and cell internalization. These nanoparticles were found to be cytotoxic in 2D (U-87 cells) and 3D (U-87 spheroids) models of GBM. The incorporation of these nanoparticles into a hydrogel facilitates their sustained release in time. Moreover, the formulation of this hydrogel containing PTX-loaded responsive nanoparticles and free TMZ was able to delay tumour recurrence in vivo after resection surgery. Therefore, our formulation represents a promising approach to develop combined local therapies against GBM using injectable hydrogels containing nanoparticles.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Nanocompuestos , Nanopartículas , Adulto , Humanos , Glioblastoma/patología , Preparaciones de Acción Retardada/uso terapéutico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/patología , Temozolomida/uso terapéutico , Paclitaxel , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Hidrogeles/uso terapéutico , Línea Celular TumoralRESUMEN
The treatment for glioblastoma multiforme (GBM) has not changed for more than 20 years while the prognosis for the patients is still poor and most of them survive less than 1 year after diagnosis. The standard of care for GBM is comprised of surgical resection followed by radiotherapy and oral chemotherapy with temozolomide. The placement of carmustine wafers in the brain after tumour removal is added in cases of recurrent glioma. Significant research is underway to improve the GBM therapy outcome and patient quality of life. Biomaterials are in the front line of the research focus for new treatment options. Specially, biocompatible polymers have been proposed in hydrogel-based formulations aiming at injectable and localized therapies. These formulations can comprise many different pharmacological agents such as chemotherapeutic drugs, nanoparticles, cells, nucleic acids, and diagnostic agents. In this manuscript, we review the most recent formulations developed and tested both in vitro and in vivo using different types of hydrogels. Firstly, we describe three common types of thermo-responsive polymers addressing the advantages and drawbacks of their formulations. Then, we focus on formulations specifically developed for GBM treatment.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Neoplasias Encefálicas/tratamiento farmacológico , Carmustina , Glioblastoma/tratamiento farmacológico , Humanos , Recurrencia Local de Neoplasia , Polímeros , Calidad de VidaRESUMEN
In the process of synthesis of a new drug, as important as the drug itself is the formulation used, because the same compound can present a very different efficacy depending on how it is administered. In this work, we demonstrate how the antitumor capacity of a new octahedral organoruthenium complex, [Ru(ppy-CHO)(phen)2][PF6] is affected by its encapsulation in different types of mesoporous silica nanoparticles. The interactions between the Ru complex and the silica matrix and how these interactions are affected at two different pHs (7.4 and 5.4, mimicking physiological and endolysosomal acidic conditions, respectively) have been studied. The encapsulation has also been shown to affect the induction of apoptosis and necrosis and progression of the cell cycle compared to the free drug. The encapsulation of the Ru complex in nanoparticles functionalized with amino groups produced very high anticancer activity in cancer cells in vitro, especially against U87 glioblastoma cells, favoring cellular internalization and significantly increasing the anticancer capacity of the initial non-encapsulated Ru complex.
Asunto(s)
Antineoplásicos/farmacología , Complejos de Coordinación/farmacología , Portadores de Fármacos/química , Nanopartículas/química , Dióxido de Silicio/química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Complejos de Coordinación/química , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Rutenio/químicaRESUMEN
The human amylin is a pancreatic peptide hormone found in hyperhormonemic state along with insulin in subclinical diabetes. Amylin has been associated with the pathology of type 2 diabetes, particularly due to its ability to assembly into toxic oligomers and amyloid specimens. On the other hand, some variants such as murine amylin has been described as non-amyloidogenic, either in vitro or in vivo. Recent data have demonstrated the amyloid propensity of murine amylin and the therapeutic analogue pramlintide, suggesting a universality for amylin amyloidosis. Here, we report the amyloidogenesis of murine amylin, which showed lower responsivity to the fluorescent probe thioflavin T compared to human amylin, but presented highly organized fibrilar amyloid material. The aggregation of murine amylin also resulted in the formation of cytotoxic specimens, as evaluated in vitro in INS-1 cells. The aggregation product from murine amylin was responsive to a specific antibody raised against amyloid oligomers, the A11 oligomer antibody. Pancreatic islets of wild-type Swiss male mice have also shown responsivity for the anti-oligomer, indicating the natural abundance of such specimen in rodents. These data provide for the first time evidences for the toxic nature of oligomeric assemblies of murine amylin and its existence in wild-type, non-transgenic mice.
Asunto(s)
Amiloide/inmunología , Anticuerpos/farmacología , Células Secretoras de Insulina/inmunología , Polipéptido Amiloide de los Islotes Pancreáticos/inmunología , Polipéptido Amiloide de los Islotes Pancreáticos/toxicidad , Agregación Patológica de Proteínas/inmunología , Animales , Anticuerpos/inmunología , Humanos , Células Secretoras de Insulina/patología , Masculino , Ratones , Agregación Patológica de Proteínas/patologíaRESUMEN
The secretory granule of the pancreatic ß-cells is a zinc-rich environment copopulated with the hormones amylin and insulin. The human amylin is shown to interact with zinc ions with major contribution from the single histidine residue, which is absent in amylin from other species such as cat, rhesus and rodents. We report here the interaction of murine amylin with zinc ions in vitro. The self-assembly of murine amylin is tightly regulated by zinc and pH. Ion mobility mass spectrometry revealed zinc interaction with monomers and oligomers. Nuclear magnetic resonance confirms the binding of zinc to murine amylin. The aggregation process of murine amylin into amyloid fibrils is accelerated by zinc. Collectively these data suggest a general role of zinc in the modulation of amylin variants oligomerization and amyloid fibril formation.
Asunto(s)
Polipéptido Amiloide de los Islotes Pancreáticos/química , Zinc/farmacología , Amiloide/biosíntesis , Amiloide/efectos de los fármacos , Animales , Concentración de Iones de Hidrógeno , Polipéptido Amiloide de los Islotes Pancreáticos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Agregado de Proteínas/efectos de los fármacosRESUMEN
Amylin is a pancreatic peptide hormone co-secreted along with insulin by the ß-cells. It is found in amyloid deposits in both type 2 diabetic individuals and elder non-diabetic. The triple proline amylinomimetic compound (25,28,29-Pro-human amylin) named pramlintide was designed aiming to solve the solubility and amyloid characteristics of human amylin. We have found by using ion mobility spectrometry-based mass spectrometry that pramlintide is able to assembly into multimers. Pramlintide formed amyloid fibrils in vitro in a pH-dependent kinetic process within a few hours, as followed by thioflavin T, quantification of soluble peptide and further characterized by transmission electron microscopy, atomic force microscopy and X-ray diffraction. These data indicate that pramlintide can form amyloid fibers.
Asunto(s)
Amiloide/síntesis química , Polipéptido Amiloide de los Islotes Pancreáticos/química , Benzotiazoles , Humanos , Concentración de Iones de Hidrógeno , Hipoglucemiantes/química , Técnicas In Vitro , Cinética , Espectrometría de Masas , Microscopía , Solubilidad , Tiazoles , Difracción de Rayos XRESUMEN
Amylin is a pancreatic hormone cosecreted with insulin that exerts unique roles in metabolism and glucose homeostasis. The therapeutic restoration of postprandial and basal amylin levels is highly desirable in diabetes mellitus. Protein conjugation with the biocompatible polymer polyethylene glycol (PEG) has been shown to extend the biological effects of biopharmaceuticals. We have designed a PEGylated human amylin by using the aminoreactive compound methoxylpolyethylene glycol succinimidyl carbonate (mPEGsc). The synthesis in organic solvent resulted in high yields of monoPEGylated human amylin, which showed large stability against aggregation, an 8 times increase in half-life in vivo compared to the non-conjugated amylin, and pharmacological activity as shown by modulation of cAMP production in MCF-7 cell line, decrease in glucagon and modulation of glycemia following subcutaneous administration in mice. Altogether these data reveal the potential use of PEGylated human amylin for the restoration of fasting amylin levels.
Asunto(s)
Polipéptido Amiloide de los Islotes Pancreáticos/química , Polietilenglicoles/química , Animales , Glucemia/efectos de los fármacos , AMP Cíclico/metabolismo , Glucagón/metabolismo , Homeostasis/efectos de los fármacos , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/farmacología , Cinética , Células MCF-7 , Masculino , Ratones , Polietilenglicoles/farmacología , Solventes/químicaRESUMEN
We modified amylin chemically by conjugating methoxyl polyethyleneglycol succinimidyl carbonate (mPEGSC) of varying molecular weights (1 kDa, 2 kDa and 5 kDa). The reaction occurred within a few minutes, resulting in at least four distinct PEGylated products. The reaction products were separated by reversed-phase chromatography and identified by mass-spectrometry. The monoPEGylated and diPEGylated amylin products were generated rapidly through conjugation to the two amino groups of the N-terminal lysine residue. Both PEGylated amylin products bound to the receptor activity-modifying protein 1 (RAMP1). Pharmacological evaluation by subcutaneous administration in mice of monoPEGylated and diPEGylated amylin obtained with mPEG-SC 5 kDa revealed that both compounds modulated glycemia for longer times than unmodified amylin. Collectively, these data demonstrate the potential of bioconjugation with mPEG for the design of amylin therapeutics with sustained action.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polietilenglicoles/síntesis química , Animales , Células COS , Chlorocebus aethiops , Polipéptido Amiloide de los Islotes Pancreáticos/síntesis química , Proteínas de la Membrana/química , Ratones , Polietilenglicoles/química , Unión Proteica , Proteína 1 Modificadora de la Actividad de Receptores/química , Proteína 1 Modificadora de la Actividad de Receptores/metabolismoRESUMEN
Amylin is a pancreatic hormone that plays important roles in overall metabolism and in glucose homeostasis. The therapeutic restoration of postprandial and basal amylin levels is highly desirable for patients with diabetes who need to avoid glucose excursions. Protein conjugation with polyethylene glycol (PEG) has long been known to be a convenient approach for extending the biological effects of biopharmaceuticals. We have investigated the reactivity of amylin with methoxy polyethylene glycol succinimidyl carbonate and methoxy polyethylene glycol succinimidyl propionate, which have an average molecular weight of 5 kDa. The reaction, which was conducted in both aqueous and organic (dimethyl sulfoxide) solvents, occurred within a few minutes and resulted in at least four detectable products with distinct kinetic phases. These results suggest a kinetic selectivity for PEGylation by succinimidyl derivatives; these derivatives exhibit enhanced reactivity with primary amine groups, as indicated by an evaluation of the remaining amino groups using fluorescamine. The analysis of tryptic fragments from mono- and diPEGylated amylin revealed that conjugation occurred within the 1-11 amino acid region, most likely at the two amine groups of Lys(1). The reaction products were efficiently separated by C-18 reversed phase chromatography. Binding assays confirmed the ability of mono- and diPEGylated amylin to interact with the amylin co-receptor receptor activity-modifying protein 2. Subcutaneous administration in mice revealed the effectiveness of monoPEG-amylin and diPEG-amylin in reducing glycemia; both compounds exhibited prolonged action compared to unmodified amylin. These features suggest the potential use of PEGylated amylin to restore basal amylin levels.