A NOVEL APPROACH FOR THE IDENTIFICATION OF CANDIDATE DRIVER LESIONS IN BREAST CANCER BASED ON THE COMPARISON OF THE MUTATIONAL PROFILES OF A PRIMARY TUMOUR AND ITS MATCHED MAMMOSPHERES AND XENOGRAFT.

The clinical management of breast cancer patients is complicated by the high genetic heterogeneity of this disease, which makes the standardization of treatments, the prediction of prognosis and therapy response, and the development of personalized therapies difficult. Nevertheless, the advent of high-throughput genomics screenings based on microarray or next-generation sequencing (NGS) technologies has greatly enhanced our understanding of the genomic landscapes underlying breast cancer development and progression. Such discoveries are now allowing clinicians to tailor therapies based on the molecular subtype of the tumour (luminal, basal and HER2). NGS studies have also started to provide insights into the range of molecular profiles of tumour cells from the same tumour, and have shown that in some breast cancers a high level of intratumoral genetic heterogeneity exists. The findings from these studies support a scenario in which breast tumours can be either: i) monogenomic, comprised of a single clonal cell population; ii) or polygenomic, composed of several related clonal subpopulations. The co-existence of different cancer driver genetic lesions in polygenomic tumours might contribute to treatment failure in some cases, as relapse could be driven by the expansion of a subpopulation of cells intrinsically resistant to the therapy. Importantly, cancer genetic heterogeneity has been recapitulated in experimental settings using cancer stem cells (CSCs) xenografted in mouse models. We hypothesized that the mutational events that drive the onset and progression of breast tumours lie within the CSC compartment. To explore this possibility, we analysed and compared the mutational profiles of a primary breast tumour and its matched mammospheres (source of CSC-derived population), patient-derived xenograft (PDX) and PDX-derived mammospheres using Whole Exome Sequencing (WES). We setup a NGS approach to look for rare mutations in the primary tumour that may be present in the CSC compartment using low amounts of DNA input. We optimised an experimental protocol in which the genomic DNA (gDNA) of each sample was subjected to Whole Genome Amplification (WGA) prior to performing WES. This enabled us to obtain a sufficient amount of DNA ( 65 3 \u3bcg) to perform WES. We also introduced a filtering step in our analysis, based on the Xenome software, for PDX-derived samples to eliminate possible contamination from murine DNA. Our study allowed us to characterize the genetic profiles of CSCs and to identify cancer-relevant mutations that could drive breast cancer onset and progression. We identified 15 candidate driver mutations in 11 genes that were enriched, in terms of mutation frequency, within primary tumour-derived mammospheres and the PDX. Together with these mutations, we identified 4 mutations in 4 genes, not enriched, but shared among all analysed samples, which likely represent \u201cfounder\u201d mutations. Based on our results, we will now endeavour to determine the clinical relevance of the candidate driver mutations identified in our study by determining their prevalence in independent patient cohorts. Having optimised the protocol for NGS of matched primary tumour, PDX and mammosphere populations, we will also extend our mutational analysis to additional breast tumours for the identification of more driver mutations and for the deconvolution of intratumoral genetic heterogeneity of breast cancer. Understanding the driving mutational forces of breast tumours and relative mechanisms involved is paramount for the development of more effective therapeutic strategies

INJECTION MOLDING/MICROMOLDING APPLICATIONS TO DRUG DELIVERY

In the present work the application potential of injection molding (IM) and micromolding (\ub5IM) for the manufacturing of drug products was investigated. These techniques are largely employed in the plastics industry to process thermoplastic polymers into objects with different size, shape and possibly many details, and they could offer several advantages in the pharmaceutical area, mainly related to versatility, patentability, scalability and production costs (continuous manufacturing). Processes and equipment generally employed as well as current pharmaceutical applications already proposed in the literature were preliminarily reviewed. Drug delivery systems (DDSs) in the form of gastro-resistant containers based on HPMCAS were afterwards designed and manufactured by \ub5IM. Notably, such DDSs represent a step forward in the field as they may provide a ready-to-use alternative to enteric-coated dosage forms. Moreover, the feasibility by hot-processing techniques (hot melt extrusion and IM) of prolonged-release hydrophilic matrices and immediate release tablets was demonstrated, which could help promoting the use of continuous manufacturing in the pharmaceutical production areas

Transferencia de un fondo de comercio como aporte para la constitución de sociedad comercial

Una de las competencias de un contador, según la ley 20.488 de ejercicio profesional, art. 13, inciso 9, es intervenir en las operaciones de transferencia de fondos de comercio sin perjuicio de las facultades reservadas a otros profesionales en la ley 11.867. Cualquier transferencia de un fondo de comercio y en especial cuando se trata de su aporte para constituir una sociedad, es una operación compleja dado que requiere dar cumplimiento a un conjunto de normas legales y además implica la realización de trámites ante la Dirección de Personas Jurídicas de Mendoza (DPJ), la Administración Federal de Ingresos Públicos (AFIP), la Dirección de Registros Públicos y Archivo Judicial, la Administración Tributaria Mendoza - Dirección General de Rentas de Mendoza (ATM - DGR), el Boletín Oficial de Mendoza (BO), la Subsecretaría de Trabajo y Seguridad Social de Mendoza (STySS) y la Municipalidad que corresponda al departamento donde se encuentra el fondo de comercio. Los objetivos principales de este trabajo son: 1. Brindar un resumen sobre los principales conceptos teóricos relativos a una transferencia de fondo de comercio: concepto, naturaleza jurídica y elementos de un fondo de comercio; intereses protegidos por la ley 11.867. 2. Estudiar el aporte societario de un fondo de comercio, teniendo en cuenta, la normativa societaria, comercial, impositiva, previsional y laboral. 3. Desarrollar en forma cronológica, las distintas etapas a cumplir para llevar a cabo el aporte societario de un fondo de comercio dando cumplimiento a todas las disposiciones legales y reglamentarias.Fil: Melocchi, Carlos Matías. Universidad Nacional de Cuyo. Facultad de Ciencias Económicas

3D printed multi-compartment capsular devices for two-pulse oral drug delivery

In the drug delivery area, versatile therapeutic systems intended to yield customized combinations of drugs, drug doses and release kinetics have drawn increasing attention, especially because of the advantages that personalized pharmaceutical treatments would offer. In this respect, a previously proposed capsular device able to control the release performance based on its design and composition, which could extemporaneously be filled, was improved to include multiple separate compartments so that differing active ingredients or formulations may be conveyed. The compartments, which may differ in thickness and composition, resulted from assembly of two hollow halves through a joint also acting as a partition. The systems were manufactured by fused deposition modeling (FDM) 3D printing, which holds special potential for product personalization, and injection molding (IM) that would enable production on a larger scale. Through combination of compartments having wall thickness of 600 or 1200\u3bcm, composed of promptly soluble, swellable/erodible or enteric soluble polymers, devices showing two-pulse release patterns, consistent with the nature of the starting materials, were obtained. Systems fabricated using the two techniques exhibited comparable performance, thus proving the prototyping ability of FDM versus IM

Gastroresistant capsular device prepared by injection molding

In the present work, the possibility of manufacturing by injection molding (IM) a gastro-resistant capsular device based on hydroxypropyl methyl cellulose acetate succinate (HPMCAS) was investigated. By performing as an enteric soluble container, such a device may provide a basis for the development of advantageous alternatives to coated dosage forms. Preliminarily, the processability of the selected thermoplastic polymer was evaluated, and the need for a plasticizer (polyethylene glycol 1500) in order to counterbalance the glassy nature of the molded items was assessed. However, some critical issues related to the physical/mechanical stability (shrinkage and warpage) and opening time of the device after the pH change were highlighted. Accordingly, an in-depth formulation study was carried out taking into account differing release modifiers potentially useful for enhancing the dissolution/disintegration rate of the capsular device at intestinal pH values. Capsule prototypes with thickness of 600 and 900 \u3bcm containing Kollicoat\uae IR and/or Explotab\uae CLV could be manufactured, and a promising performance was achieved with appropriate gastric resistance in pH 1.2 medium and break-up in pH 6.8 within 1 h. These results would support the design of a dedicated mold for the development of a scalable manufacturing process

Non-uniform drug distribution matrix system (NUDDMat) for zero-order release of drugs with different solubility

A decrease in the drug release rate over time typically affects the performance of hydrophilic matrices for oral prolonged release. To address such an issue, a Non-Uniform Drug Distribution Matrix (NUDDMat) based on hypromellose was proposed and demonstrated to yield zero-order release. The system consisted of 5 overlaid layers, applied by powder layering, having drug concentration decreasing from the inside towards the outside of the matrix according to a descending staircase function. In the present study, manufacturing and performance of the described delivery platform were evaluated using drug tracers having different water solubility. Lansoprazole, acetaminophen and losartan potassium were selected as slightly (SST), moderately (MST) and highly (HST) soluble tracers. By halving the thickness of the external layer, which contained no drug, linear release of HST and MST was obtained. The release behavior of the NUDDMat system loaded with a drug having pH-independent solubility was shown to be consistent in pH 1.2, 4.5 and 6.8 media. Based on these results, feasibility of the NUDDMat platform by powder layering was demonstrated using drugs having different physico-technological characteristics. Moreover, its ability to generate zero-order release was proved in the case of drugs with water solubility in a relatively wide range

Evaluation of Hot-Melt Extrusion and Injection Molding for Continuous Manufacturing of Immediate-Release Tablets

The exploitation of hot-melt extrusion and injection molding for the manufacturing of immediate-release (IR) tablets was preliminarily investigated in view of their special suitability for continuous manufacturing, which represents a current goal of pharmaceutical production because of its possible advantages in terms of improved sustainability. Tablet-forming agents were initially screened based on processability by single-screw extruder and micromolding machine as well as disintegration/dissolution behavior of extruded/molded prototypes. Various polymers, such as low-viscosity hydroxypropylcellulose, polyvinyl alcohol, polyvinyl alcohol-polyethylene glycol graft copolymer, various sodium starch glycolate grades (e.g., Explotab\uae CLV) that could be processed with no need for technological aids, except for a plasticizer, were identified. Furthermore, the feasibility of both extruded and molded IR tablets from low-viscosity hydroxypropylcellulose or Explotab\uae CLV was assessed. Explotab\uae CLV, in particular, showed thermoplastic properties and a very good aptitude as a tablet-forming agent, starting from which disintegrating tablets were successfully obtained by either techniques. Prototypes containing a poorly soluble model drug (furosemide), based on both a simple formulation (Explotab\uae CLV and water/glycerol as plasticizers) and formulations including dissolution/disintegration adjuvants (soluble and effervescent excipients) were shown to fulfill the USP 37 dissolution requirements for furosemide tablets

In vitro and human pharmacoscintigraphic evaluation of an oral 5-ASA delivery system for colonic release

5-aminosalicylic acid (5-ASA) is the most widely used drug for the treatment of ulcerative colitis. The benefits of targeted delivery of 5-ASA to the large intestine are well known, resulting in reduced systemic absorption and increased local concentrations at the disease site. In the present study, a 5-ASA colon delivery system based on the time-dependent strategy, exploiting the relatively consistent small intestinal transit time (SITT), was manufactured and evaluated in vitro as well as in vivo. The system was obtained by successive spray-coating of an immediate-release tablet core with low-viscosity HPMC and Eudragit (R) L. The enteric film was effective in preventing release during the acidic stage of the in vitro test, while the HPMC coating brought about reproducible lag phases prior to release in phosphate buffer medium. A gamma-scintigraphy investigation pointed out that, following administration to fasted and fed volunteers, disintegration of the units never occurred prior to colon arrival. In all cases, a lag time preceded the appearance of the drug and its N-acetyl metabolite in the blood-stream, which was found to correlate with the time of disintegration in a linear mode. The plasma levels of the drug and metabolite as well as their cumulative urinary recovery were relatively low with respect to those reported when 5-ASA is delivered to the small bowel

Novel hydrophilic matrix system with non-uniform drug distribution for zero-order release kinetics

A decrease in the release rate over time is typically encountered when dealing with hydrophilic matrix systems for oral prolonged release due to progressive increase of the distance the drug molecules have to cover to diffuse outwards and reduction of the area of the glassy matrix at the swelling front. In order to solve this issue, a novel formulation approach based on non-uniform distribution of the active ingredient throughout the swellable polymer matrix was proposed and evaluated. Various physical mixtures of polymer (high-viscosity hypromellose) and drug tracer (acetaminophen), having decreasing concentrations of the latter, were applied by powder-layering onto inert core seeds. The resulting gradient matrices showed to possess satisfactory physico-technological characteristics, with spherical shape and consistent thickness of the layers sequentially applied. The non-uniform matrix composition pursued was confirmed by Raman mapping analysis. As compared with a system having uniform distribution of the drug tracer, the multi-layer formulations were proved to enhance linearity of release. The simple design concept, advantageous technique, which involves no solvents nor high-impact drying operations, and the polymeric material of established use make the delivery platform hereby proposed a valuable strategy to improve the performance of hydrophilic matrix systems