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Development of liposome-based drug delivery system to improve drug-like properties and anticancer activity of tyrosine kinase inhibitors with pyrazolo [3,4-d] pyrimidine structure.
Full text linkCancer is a multifactorial disease whose causes are still unknown. Frequently, mutations in several genes play an important role in cellular growth processes and, based on their specific roles, they are usually distinguished in proto-oncogenes and oncosuppressors. One of the most studied oncogenes is c-Src, encoding for a non-receptor tyrosine kinase protein that plays a multitude of roles in cell signalling. The activation of c-Src is involved in the control of many functions, including cell adhesion, growth, movement, and differentiation by a different set of cell surface receptors. c-Src is found to be over- expressed and mutationally activated in a wide variety of human cancers. Moreover, c- Src may have an influence on the development of the metastatic phenotype.
Significant progresses in the understanding of cancer biology have prompted extensive research within novel classes of anticancer drugs. A number of tyrosine kinase inhibitors targeting the c-Src tyrosine kinase (as well as related tyrosine kinases) have been developed for therapeutic use.
4-aminopyrazolo [3,4-d] pyrimidines compounds were firstly used as c-Src family kinases (SFKs) inhibitors in 1996 but later demonstrated to inhibit all SFKs members with IC50 values in the low nanomolar range. These compounds have been extensively used for studying the biological pathways of SFKs and, even though they are a class of promising anti-tumor compounds because of their good activity against several cancer cell lines, these molecules showed a poor aqueous solubility, limiting them as clinical drug candidates. In the continuing effort to find new anticancer agents, our group conducted large studies on a series of new c-Src inhibitors with a pyrazolo [3,4-d] pyrimidine scaffold.
The aim of this thesis was to improve the solubility profile and the pharmacokinetic properties of selected compounds, chosen on the base of their inhibitory activity on c- Src. To overcome the poor water solubility of these molecules, pyrazolo [3,4-d] pyrimidines compounds were encapsulated in liposomes formulations. Liposomes were prepared and characterized for size, ζ potential distribution and polydispersity index (PDI) by dynamic light scattering in order to obtain a homogeneous population of small unilamellar vesicles (SUV); subsequently, fluorescent liposomes were prepared to assess the ability of liposomes to interact with neuroblastoma (SH-SY5Y) and glioblastoma (U87) cells by confocal microscopy; finally, encapsulation efficiency and activity by cellular assays were studied to determine cytotoxicity, showing that
liposomes loaded with pyrazolo [3,4-d] pyrimidine compounds determine a cytotoxic effect in SH-SY5Y (Neuroblastoma) or U87 (Glioblastoma) cell line equal to or higher than the free compound solubilised in DMSO. Moreover, biodistribution of liposomes loaded with pyrazolo [3,4-d] pyrimidine was evaluated in male Sprague Dawley rats after 24h of treatment. Altogether, the obtained data strongly indicate that the encapsulation of pyrazolo [3,4-d] pyrimidine compounds in liposomes represent an effective method to improve the biodistribution of pyrazolo [3,4-d] pyrimidines and attribute a therapeutic efficacy to this novel formulation.
During my Ph.D. period, I also focused on the study of active targeting of liposomes against tumor areas that overexpress plasmin. The plasminogen/plasmin system plays a key role in tumor development and, in particular, in progression and metastasis. The aim of my project was to functionalize the surface of stealth liposomes encapsulating pyrazolo [3,4-d] pyrimidines with a peptide able to bind plasmin. Since plasmin is overexpressed in tumor areas, the bond between plasmin and the peptide in liposomes surface would lead to a destabilization of the liposomes and consequently to a greater drug release in the tumor site. The obtained liposomes were characterized after purification evaluating the size, the ζ potential distribution, the polydispersity index (PDI) and the encapsulation efficiency (EE). Furthermore, several tests to quantify the peptide bound to liposomes were performed, unfortunately with poor results. Moreover, peptide-bearing fluorescent liposomes were also prepared and tested on hepatocarcinoma cell line HepG2 to investigate any change in the cellular uptake. In addition, cytotoxicity assays on HepG2 cells were performed to test the efficacy of liposomes conjugated with the peptide with respect to non-conjugated liposomes, both encapsulating the selected compounds. Liposomes functionalized with the peptide resulted more stable and with better physicochemical properties than non-functionalized liposomes. Furthermore, confocal microscopy of fluorescent liposomes highlighted the ability of liposomes with the peptide to penetrate in hepatocarcinoma cells after 1h of treatment with respect to non-functionalized liposomes, not able to be internalized after the same time. Finally, cytotoxicity data showed that liposomes with the peptide have lower IC50 values than non-functionalized liposomes.
In conclusion, data obtained showed that it was obtained a good functionalized liposomal formulation able to be recognized by hepatocarcinoma cells secreting
plasmin. Further in vivo tests will be performed to study the ability of these liposomes to release the compound in tumor areas over-expressing plasmin
Molecular insights to the bioactive form of BV02, a reference inhibitor of 14-3-3σ protein-protein interactions
No full textBV02 is a reference inhibitor of 14-3-3 protein-protein interactions, which is currently used as chemical biology tool to understand the role of 14-3-3 proteins in pathological contexts. Due to chemical instability in certain conditions, its bioactive form has remained unclear. Here, we use NMR spectroscopy to prove for the first time the direct interaction between the molecule and 14-3-3σ, and to depict its bioactive form, namely the phthalimide derivative 9. Our work provides molecular insights to the bioactive form of the 14-3-3 PPI inhibitor and facilitates further development as candidate therapeutic agent
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Improvement of pyrazolo[3,4-d]pyrimidines pharmacokinetic properties: Nanosystem approaches for drug delivery
Full text linkPyrazolo[3,4-d]pyrimidines are a class of compounds with a good activity against several cancer cell lines. Despite the promising anticancer activity, these molecules showed a poor aqueous solubility. This issue could threat the future development of pyrazolo[3,4-d]pyrimidines as clinical drug candidates. With the aim of improving their solubility profile and consequently their pharmacokinetic properties, we have chosen four compounds (1-4) on the base of their anti-neuroblastoma activity and we have developed albumin nanoparticles and liposomes for the selected candidates. Albumin nanoparticles and liposomes were prepared and characterized regarding size and ? -potential distribution, polidispersity index, entrapment efficiency and activity against SH-SY5Y human neuroblastoma cell line. The most promising nanosystem, namely LP-2, was chosen to perform further studies: confocal microscopy, stability and drug release in physiological conditions, and biodistribution. Altogether, the obtained data strongly indicate that the encapsulation of pyrazolo[3,4-d]pyrimidines in liposomes represent an effective method to overcome the poor water solubility
Plasmin-Binding Tripeptide-Decorated Liposomes Loading Pyrazolo[3,4- d]pyrimidines for Targeting Hepatocellular Carcinoma
No full textHepatocellular carcinoma (HCC) is one of the most fatal cancer types worldwide. HCC cells were proved to overexpress c-Src and Sgk1, a tyrosine and a serine-threonine kinase, respectively, whose role is crucial for the development and progression of the tumor. Pyrazolo[3,4-d]pyrimidine derivatives are a class of tyrosine kinase inhibitors that have shown good activity against HepG2. HCC cells were also proved to overexpress plasmin, which is localized on the cell surface bound to its receptors. In this study, a tripeptide with sequence d-Ala-Phe-Lys, which binds a specific reactive site of plasmin, was synthesized and characterized. This tripeptide was used to decorate liposomes encapsulating three selected pyrazolo[3,4-d]pyrimidines. Liposomes bearing tripeptide have been characterized, not showing remarkable differences with respect to the corresponding tripeptide-free liposomes. In vitro HepG2 cell uptake profiles and cytotoxicities showed that the presence of the tripeptide on the liposomal membrane surface improves the cell-penetrating ability of liposomes and increases the activity of two of the three tested compounds
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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