Gastroesophageal cancer is the sixth leading cause of cancer-related death worldwide. The 2 most common histologies are squamous cell carcinoma and adenocarcinoma, which has seen an increase in incidence correlating with an increase in obesity in developed countries. Gastroesophageal adenocarcinoma has a preponderance to metastasize early, making it a highly lethal cancer with a low 5-year survival rate of similar to 15-25%. Therefore, for the majority of patients, treatment focuses on palliation and prolongation of survival. Combination chemotherapy regimens, mostly platinum-based, have only modestly prolonged survival in patients with stage IV disease. Recently, it was discovered that the activation of the HER2 receptor plays an important role in a minority of adenocarcinomas of the distal esophagus and stomach. This introduced the treatment option of trastuzumab (Herceptin), a monoclonal antibody directed at the HER2 receptor, which has demonstrated improvement in overall and progression-free survival as noted in the ToGA trial. Currently, the role of Herceptin therapy beyond first-line therapy and outside of combination regimens is not well established. In this case report we review 2 cases of patients with gastroesophageal cancer, with HER2 overexpression, who achieved a robust response to trastuzumab in combination with chemotherapy and were able to maintain a durable response with maintenance trastuzumab monotherapy. (c) 2017 The Author(s) Published by S. Karger AG, Basel.

Tesis para optar al grado de Magíster en Marketing / Mae Tan Chui [Parte I] / Autor no autoriza el acceso a texto completo de su documento. / En Chile el cáncer de mama es la primera causa de muerte por tumores malignos en las mujeres, presentando una tasa de mortalidad observada de 14,5 por 100 mil mujeres en 2009 . La tasa de Años de Vida Potenciales Perdidos (AVPP) por cáncer de mama en la mujer es de 100 por 100 mil, ocupando el segundo lugar después del cáncer cervicouterino. Entre el 20 y el 25% de las mujeres enfermas de cáncer de mama desarrollará el tipo más agresivo, el cáncer de mama HER2-positivo, que se manifiesta por una sobreexpresión del gen HER2. Herceptin IV (trastuzumab intravenoso) es un anticuerpo monoclonal humanizado anti-HER2, importado y comercializado por Roche Chile, representación local de la farmacéutica suiza Roche, líder mundial en biotecnología. Este medicamento produce tasas de respuesta del 15 al 40%, cuando se utiliza en pacientes con cáncer de mama que sobreexpresan el HER2. A pesar de ser un medicamento que puede cambiar la vida de las mujeres, ha tenido fuertes barreras de entrada en el sistema público chileno. Sin embargo, en el sistema privado, es utilizado de forma integral desde su incorporación en la Guía Clínica de Cáncer de Mama en 2011 (recomendaciones del Gobierno chileno acerca del tratamiento de enfermedades). Los científicos de Roche han desarrollado Herceptin SC (trastuzumab subcutáneo), medicamento cuya formulación innovadora significará una mejora en la calidad de vida de las pacientes y un ahorro de costos para las instituciones que utilicen esta versión del medicamento. Mientras la administración de la formulación IV (la estándar y actual) tarda alrededor de 30 a 90 minutos, en esta nueva versión las pacientes demorarán sólo aproximadamente cinco minutos en su aplicación. Es un proceso menos invasivo que permite que los pacientes pasen menos tiempo en el hospital, lo que resulta importante en el contexto del cáncer de mama temprano, donde usualmente se aplica el medicamento durante 1 año. Herceptin SC se administra en forma de inyección subcutánea a una dosis fija de 600 mg donde no se requiere una dosis de carga ni ajustar la dosificación al peso del paciente.

Tesis para optar al grado de Magíster en Marketing / Delpiano, Carla [Parte III] / Autor no autoriza el acceso a texto completo de su documento. / Apoyándonos en la historia de é xito que ha tenido Herceptin IV, se desarrollan las tácticas promocionales para la entrada del nuevo producto al mercado, antes de que la competencia obtenga los permisos necesarios para anunciar su llegada. Basándonos en los resultados entregados por el análisis situacional y la investigación de mercado, se obtienen tres pilares fundamentales para las tácticas del período analizado, las que se convertirán en el hilo conductor de promoción: 1. Desmitificar percepciones acerca de medicamentos subcutáneos 2. Gen erar evidencia de los beneficios para asegurar continuidad de uso 3. Fidelizar a los stakeholders con la historia de Herceptin Siguiendo con lo que se ha estado haciendo durante estos años con la versión intravenosa del medicamento , se propone acompaña r con actividades especiales a cada uno de nuestros stakeholders, incluyendo por primera vez con fuerza a las pacientes, quienes habían sido dejadas en un segundo plano, mientras la compañía se enfocaba en los oncólogos y en los pagadores. Durante un año, y a través de información científica respaldada por los buenos resultados que se han obtenido en otros lugares del mundo, se pretende demostrar los beneficios que se logran con la utilización de este producto, el que mejorar de gran forma la calidad de vida d e las pacientes y que trae ahorros en los costos de administración del medicamento en las instituciones de salud

Tesis para optar al grado de Magíster en Marketing / Gutiérrez, César [Parte II] / Autor no autoriza el acceso a texto completo de su documento / A continuación se presentan los posibles escenarios para el desarrollo de la nueva presentación subcutánea de Herceptin, la cual tiene como base el mantener su estrategia de enfoque por diferenciación, caracterizando las mejoras continuas del producto y no su precio. Posteriormente se exponen las fortalezas y debilidades derivadas de la información del entorno, donde se identifica que Herceptin subcutáneo es la forma más rápida de aplicar un medicamento para cáncer de mama y como debilidad la falta de experiencia de los stakeholders con medicamentos subcutáneos. Los objetivos de ventas están enfocados principalmente en el sector público, esperándose un crecimiento de 122%, esto, gracias a la apertura de protocolos de tratamiento con Herceptin que determinan las bases para el tratamiento de cáncer de mama tipo HER2+. Se determinaron los mercados objetivos, divididos en macro y microsegmentos, en los cuales se buscarán alcanzar los objetivos de marketing (tasa conversión de Herceptin IV a SC en un 24%) por medio de estrategias enfocadas en neutralizar los nuevos competidores que entran al mercado, pero en presentación intravenosa (IV). Por último, se determina la estrategia de posicionamiento gracias a los estudios de análisis multidimensional y análisis factorial, los cuales dan las bases para descifrar el posicionamiento de las principales terapias (en el cáncer de mama) en la mente de los médicos, así como los atributos que ellos valoran de éstos, en los cuales, se debería dar más importancia a la hora de determinar las tácticas en terreno para transmitir el mensaje promocional.

There is a lack of stability data for in-use parenteral drugs. Manufacturers state a shelf-life of 24 hours for infusions based on microbiological contamination. The lack of data is of particular significance with protein-based drugs where action is determined by their complex structure. A range of techniques are required to assess stability, including biological assessment to support other data. There has been an increase in published data but often the few studies that address in-use stability are incomplete as they do not employ biological assessment to assess potency. Trastuzumab is an antibody-based drug used to treat cancers where the Epidermal Growth Factor Receptor 2 (HER2) is over expressed or over abundant on the cell surface. Trastuzumab infusions have been assigned by the manufacturer to be stable for 24 hours at temperatures not exceeding 30 oC. If stability is shown beyond this point it would enable extended storage and administration. To this end, methods were selected and developed with biological assessment central to the approach to assess clinically relevant infusion concentrations (0.5 mg/mL and 6.0 mg/mL) and a sub-clinical infusion concentration (0.1 mg/mL). This may enhance instability and provide opportunity to study degradation. A Cell Counting Kit CCK8 (Sigma Aldrich) was ultimately adopted as a basis for a colorimetric assay to assess cell viability. Attenuated Total Reflectance Infra-Red Spectroscopy and Size Exclusion Chromatography methods were developed to evaluate secondary structure and aggregation respectively. These methods were applied to a shelf-life study (43 days) as a collaboration with Quality Control North West (NHS) and Clatterbridge Centre for Oncology NHS Foundation Trust, Clatterbridge Hospital. There was no evidence of degradation and no loss efficacy for clinically relevant infusions (0.5 mg/mL and 6.0 mg/mL) over 43 days, whilst the sub-clinical infusions (0.1 mg/mL) developed particles after 7 days of storage between 2 oC and 8 oC. Furthermore, evidence of stability at day 119 gave increased confidence for the data from earlier time points. This work assisted in the shelf-life being recommended to be extended to 28 days for Trastuzumab stored in polyolefin IV bags at concentrations between 0.5 mg/mL and 6.0 mg/mL with 0.9% saline between 2 oC and 8 oC. However, infusions with concentrations below 0.5 mg/mL were not recommended for storage.

Notre travail s’est focalisé sur l’utilisation du trastuzumab‐immunoPET afin d’étudier et deguider une approche nouvelle visant à surmonter la résistance au médicament trastuzumab,causée par la surexpression de MUC4 dans le cancer du sein.Pour ce faire, nous avons préparé et utilisé du 89Zr‐trastuzumab dans le but de suivresa capacité de liaison au récepteur HER2 ainsi que son accumulation dans des cellulescancéreuses mammaires. Ensuite, nous avons formulé l’hypothèse que des agentsmucolytiques, tels que la N‐Acétylcystéine (NAC), en démêlant les réseaux formés par lesmucines, permettent l’amélioration de la captation du radiotraceur in vitro et in vivo. Eneffet, l’addition du NAC a occasionné une accumulation significative de 89Zr‐trastuzumab,sans altération ni changement de l’affinité de liaison au récepteur. Ceci semble égalementproduire une meilleure sensibilité des imageries PET dans le modèle animal choisi.Dans une seconde étape, nous avons évalué, dans un modèle murin de cancer du seinrésistant au trastuzumab et surexprimant la MUC4, si cette captation accrue se traduit parun bénéfice thérapeutique en utilisant le NAC combiné au trastuzumab. Nous avons obtenuun effet inhibiteur qui réduit de moitié la croissance tumorale, comparable à celui observépour la tumeur mammaire sensible au trastuzumab (implantée dans le même animal).En conclusion, notre étude démontre l’efficacité de l’utilisation de traceurs PETsurtout à visée théranostique, comme c’est le cas du 89Zr‐trastuzumab, pour étudier etévaluer la résistance aux médicaments ciblés apparentés au radiotraceur lui‐même. Ellepropose l’utilisation du NAC pour améliorer l’accessibilité du récepteur pour le radiotraceurainsi que pour le médicament « froid » ouvrant, de ce fait, une perspective vers uneutilisation clinique chez un sous‐type de patientes atteintes d’un cancer du sein. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Breast cancers with overexpression or amplification of the HER2 tyrosine kinase receptor are more aggressive, resistant to chemotherapy, and associated with a worse prognosis. Currently, these breast cancers are treated with the monoclonal antibody trastuzumab (Herceptin®). Unfortunately, not all patients respond to trastuzumab drug therapy; some patients show de novo resistance, while others acquire resistance during treatment. This thesis describes our RNAi studies to identify novel regulators of the HER2 signaling pathway in breast cancer. Three kinome-wide siRNA screens were performed on five HER2 amplified and seven HER2 non-amplified breast cancer cell lines, two normal breast cell lines, as well as two HER2-positive breast cancer cell lines with acquired trastuzumab resistance and their isogenic trastuzumab-sensitive controls. To understand the main kinase drivers of HER2 signaling, we performed a comprehensive screen that selected against growth inhibitors of the non-HER2 amplified breast cancer cell lines. This screen identified the loss of the HER2/HER3 heterodimer as the most prominent selective inhibitor of HER2-amplified breast cancers. In a trastuzumab sensitization screen on five trastuzumab-treated breast cancer cell lines, we identified several siRNA against the PI3K pathway as well as various other signaling pathways that inhibited proliferation. Finally, in a screen for acquired trastuzumab resistance, PKCη and its downstream targets were identified. Loss of PKCη resulted in a decrease in G1/S transition and upregulation of the cyclin dependent kinase inhibitor p27. Initial data suggest that PKCη promotes p27 ubiquitination and degradation. Taken together, these studies provide novel insight into the complex signaling of HER2-positive breast cancers and the mechanisms of resistance to trastuzumab therapy. This work describes how various kinases can modulate cell proliferation, and points to possible novel drug targets for the treatment of HER2-positive breast cancers.

Ce travail, qui fait partie d’un projet européen, « NANOTHER », est focalisé sur la fonctionnalisation de nanoparticules polymériques et lipidiques fonctionnalisées par des anticorps Herceptine® pour cibler des cellules du cancer du sein HER2+. Deux stratégies de fonctionnalisation ont été étudiées : une a reposé sur l’utilisation de protéines de fusion, l’Anx5-ZZ, composée d’Annexine A5 et deux domaines Z homologues de la protéine A de Staphylococcus aureus qui peuvent se fixer des anticorps d’une manière orientée par leur fragment cristallisable ; l’autre a porté sur le couplage direct d’anticorps modifiés pour exposer des groupes sulfhydryles aux nanoparticules exposant des groupes maléimides.La première partie concerne le développement d’un agent de ciblage simplifié du complexe l’Anx5-ZZ-anticorps, à savoir l’Anx5-scFv (single-chain variable fragment). Puisque la cible n’avait pas été décidée au début de ce travail, deux scFvs ont été utilisé comme système modèle. L’expression de protéines de fusion a été essayée chez Escherichia Coli avec différentes constructions de protéines de fusion, différentes conditions d’expression et différentes souches bactériennes. Toutes les protéines sont soient agrégées soient non surexprimées.La deuxième partie consiste à fonctionnaliser les polymersomes par l’Herceptine® via l’Anx5-ZZ. D’abord, nous avons validé une méthode de modification de la surface de polymersome pour présenter des groupes maléimides. Ensuite, le couplage covalent de l’Anx5(SH)-ZZ aux polymersomes-maléimide a été réalisé et quantifié. Nous avons obtenu maximum 30 Anx5-ZZ par polymersome. Puis, la liaison d’affinité d’anticorps aux polymersomes-Anx5-ZZ a été caractérisée, réalisée et quantifiée. Pour 30 Anx5-ZZ par polymersome, nous avons 60 Herceptine® par polymersome. Cependant, l’efficacité de ciblage de ces systèmes est très faible.La troisième partie consiste à fonctionnaliser les liposomes par l’Herceptine® via couplage direct. Tout d’abord, la modification de l’Herceptine® pour présenter des groupes SH a été caractérisée et contrôlée. Ensuite, le couplage covalent d’Herceptine®-SH aux liposomes-maléimides a été réalisé et quantifié. L’étude de ciblage montre que les liposomes fonctionnalisés par une molécule d’Herceptine® sont capable de cibler les cellules HER2+. / This work, which is part of a European project "NANOTHER", focus on the functionalization of polymeric and lipidic nanoparticles by Herceptin® to target HER2+ cancer cells. Two functionalization strategies were studied: one was based on the use of a fusion protein, Anx5-ZZ, composed of Annexin A5 and two Z domains which are homologous with the protein A of Staphylococcus aureus that can bind antibodies by their crystallizable fragment in a oriented way; the other focused on the direct coupling of modified antibodies exposing sulfhydryl groups to nanoparticles exposing maleimid groups.The first part concerns the development of a targeting agent simplified from the Anx5-ZZ-antibody complex, namely Anx5-scFv (single-chain variable fragment). Since the target had not been decided at the beginning of this work, two scFvs were used as model system. The expression of fusion proteins was tested in Escherichia coli with different fusion protein constructions, different expression conditions and different bacterial strains. All proteins are either aggregated or non-overexpressed.The second part is to functionalize the polymersomes by Herceptin® via Anx5-ZZ. First, we validated a method for modifying the surface of polymersome to expose maleimid groups. Then, the covalent coupling of Anx5(SH)-ZZ to polymersomes-maleimid was performed and quantified. We obtained maximum 30 Anx5-ZZ per polymersome. Then, the affinity binding of antibodies to polymersomes-Anx5-ZZ was characterized, performed and quantified. For 30 Anx5-ZZ per polymersome, we have 60 Herceptin® per polymersome. However, the targeting efficiency of this system is very low.The third part consists in functionalizing the liposomes by Herceptin® via direct coupling. Firstly, the modification of Herceptin® to expose SH groups was characterized and controlled. Then, the covalent coupling of Herceptin®-SH to liposomes exposing maleimid groups was performed and quantified. The targeting study shows that liposomes functionalized with one Herceptin® are able to target HER2+ cells.

Objective: The cytotoxic property of anti-EGFR-1 monoclonal-antibody nimotuzumab modified with nuclear localization sequence and radiolabeled with 111In was evaluated in trastuzumab-resistant breast cancer cells. Methods: 111In-nimotuzumab-NLS was constructed and its immunoreactivity was determined. Cellular and nuclear uptake was evaluated by cell fractionation. Finally, the cytotoxicity of conjugates (111In-nimotuzumab/111In-nimotuzumab-NLS) was studied by clonogenic assays. Results: The immunoreactivity of 111In-nimotuzumab-NLS was conserved. 111In-nimotuzumab-NLS exhibited 2-fold higher nuclear translocation as compared to 111In-nimotuzumab in MDA-MB-468 cells. Nuclear importation of 111In-nimotuzumab-NLS in MDA-MB-468 cells was 4-fold and 6-fold higher than moderate and low EGFR expressing cell lines, respectively. Clonogenic survival (CS) for MDA-MB-468 cells showed 111In-nimotuzumab-NLS to be 10-folds and 60-folds more potent than 111In-nimotuzumab and nimotuzumab, respectively. Moderate killing for TrR1 and MDA-MB-231 was observed. 111In-hEGF showed significantly higher cytotoxicity and 2-fold higher γ-H2AX foci integrated density/nuclear-area as compared to 111In-nimotuzumab-NLS. Preserved selectivity of 111In-nimotuzumab-NLS makes it an excellent drug for treating cancers.

Objective: The cytotoxic property of anti-EGFR-1 monoclonal-antibody nimotuzumab modified with nuclear localization sequence and radiolabeled with 111In was evaluated in trastuzumab-resistant breast cancer cells. Methods: 111In-nimotuzumab-NLS was constructed and its immunoreactivity was determined. Cellular and nuclear uptake was evaluated by cell fractionation. Finally, the cytotoxicity of conjugates (111In-nimotuzumab/111In-nimotuzumab-NLS) was studied by clonogenic assays. Results: The immunoreactivity of 111In-nimotuzumab-NLS was conserved. 111In-nimotuzumab-NLS exhibited 2-fold higher nuclear translocation as compared to 111In-nimotuzumab in MDA-MB-468 cells. Nuclear importation of 111In-nimotuzumab-NLS in MDA-MB-468 cells was 4-fold and 6-fold higher than moderate and low EGFR expressing cell lines, respectively. Clonogenic survival (CS) for MDA-MB-468 cells showed 111In-nimotuzumab-NLS to be 10-folds and 60-folds more potent than 111In-nimotuzumab and nimotuzumab, respectively. Moderate killing for TrR1 and MDA-MB-231 was observed. 111In-hEGF showed significantly higher cytotoxicity and 2-fold higher γ-H2AX foci integrated density/nuclear-area as compared to 111In-nimotuzumab-NLS. Preserved selectivity of 111In-nimotuzumab-NLS makes it an excellent drug for treating cancers.