Experimental approaches in the targeting of photodynamic therapeutics

RÉSUMÉ : Chez l'homme, le manque de sélectivité des agents thérapeutiques représente souvent une limitation pour le traitement des maladies. Le ciblage de ces agents pour un tissu défini pourrait augmenter leur sélectivité et ainsi diminuer les effets secondaires en comparaison d'agents qui s'accumuleraient dans tout le corps. Cela pourrait aussi améliorer l'efficacité des traitements en permettant d'avoir une concentration localisée plus importante. Le ciblage d'agents thérapeutiques est un champ de recherche très actif. Les stratégies sont généralement basées sur les différences entre cellules normales et malades. Ces différences peuvent porter soit sur l'expression des molécules à leurs surfaces comme des récepteurs ou des transporteurs, soit sur les activités enzymatiques exprimées. Le traitement thérapeutique choisi ici est la thérapie photodynamique et est déjà utilisé pour le traitement de certains cancers. Cette thérapie repose sur l'utilisation de molécules qui réagissent à la lumière, les photosensibilisants. Elles absorbent l'énergie lumineuse et réagissent avec l'oxygène pour former des radicaux toxiques pour les cellules. Les photosensibilisants utilisés ici sont de deux natures : (i) soit ils sont tétrapyroliques (comme les porphyrines et chlorines), c'est à dire qu'ils sont directement activables par la lumière ; (ii) soit ce sont des prodrogues de photosensibilisants comme l'acide 5aminolévulinique (ALA) qui est transformé dans la cellule en protoporphyrine IX photosensibilisante. Dans le but d'augmenter la sélectivité des photosensibilisants, nous avons utilisé deux stratégies différentes : (i) le photosensibilisant est modifié par le greffage d'un agent de ciblage ; (ii) le photosensibilisant est incorporé dans des structures moléculaires de quelques centaines de nanomètres. Les sucres et l'acide folique sont des agents de ciblage largement établis et ont été utilisés ici car leurs récepteurs sont surexprimés à la surface de nombreuses cellules malades. Ainsi, des dérivés sucres ou acide folique de l'ALA ont été synthétisés et évalués in vitro sur de nombreuses lignées cellulaires cancéreuses. La stratégie utilisant l'acide folique est apparue incompatible avec l'utilisation de l'ALA puisque aucune photosensibilité n'a été induite par le composé. La stratégie utilisant les sucres a, par ailleurs, provoquée de bonnes photosensibilités mais pas d'augmentation de sélectivité. En parallèle, la combinaison entre les propriétés anticancéreuses des complexes métalliques au ruthénium avec les propriétés photosensibilisantes des porphyrines, a été évaluée. En effet, les thérapies combinées ont émergé il y a une dizaine d'années et représentent aujourd'hui de bonnes alternatives aux monothérapies classiques. Des ruthenium(I1)-arènes complexés avec la tetrapyridylporphyrine ont ainsi présenté de bonnes cytotoxicités et de bonnes phototoxicités pour des cellules de mélanomes. Des porphyrines ont aussi été compléxées avec des noyaux de diruthénium et ce type de dérivé a présenté de bonnes phototoxicités et une bonne sélectivité pour les cellules cancéreuses de l'appareil reproducteur féminin. L'incorporation de photosensibilisants tétrapyroliques a finalement été effectuée en utilisant des nanoparticules (NP) biocompatibles composées de chitosan et de hyaluronate. L'effet de ces NP a été évalué pour le traitement de la polyarthrite rhumatoïde (PR). Les NP ont d'abord été testées in vitro avec des macrophages de souris et les résultats ont mis en évidence de bonnes sélectivités et photosensibilités pour ces cellules. In vivo chez un modèle marin de la PR, l'utilisation de ces NP a révélé un plus grand temps de résidence des NP dans le genou de la souris en comparaison du temps obtenu avec le photosensibilisant seul. Le traitement par PDT a aussi démontré une bonne efficacité par ailleurs égale à celle obtenue avec les corticoïdes utilisés en clinique. Pour finir, les NP ont aussi démontré une bonne efficacité sur les myelomonocytes phagocytaires humains et sur les cellules contenues dans le liquide synovial de patients présentant une PR. Tous ces résultats suggèrent que les deux stratégies de ciblage peuvent être efficaces pour les agents thérapeutiques. Afm d'obtenir de bons résultats, il est toutefois nécessaire de réaliser une analyse minutieuse de la cible et du mode d'action de l'agent thérapeutique. Concernant les perspectives, la combinaison des deux stratégies c'est à dire incorporer des agents thérapeutiques dans des nanostructures porteuses d'agents de ciblage, représente probablement une solution très prometteuse. SUMMARY : In humans, the lack of selectivity of drugs and their high effective concentrations often represent limitations for the treatment of diseases. Targeting the therapeutical agents to a defined tissue could enhance their selectivity and then diminish their side effects when compared to drugs that accumulate in the entire body and could also improve treatment efûciency by allowing a localized high concentration of the agents. Targeting therapeutics to defined cells in human pathologies is a main challenge and a very active field of research. Strategies are generally based on the different behaviors and patterns of expression of diseased cells compared to normal cells such as receptors, proteases or trans-membrane carriers. The therapeutic treatment chosen here is the photodynamic therapy and is already used in the treatment of many cancers. This therapy relies on the administration of a photosensitizer (PS) which will under light, react with oxygen and induce formation of reactive oxygen species which are toxic for cells. The PSs used here are either tetrapyrolic (i. e. porphyries and chlorins) or prodrugs of PS (5-aminolevulinic acid precursor of the endogenous protoporphyrin Imo. In order to improve PS internalization and selectivity, we have used two different strategies: the modification of the PSs with diseased cell-targeting agents as well as their encapsulation into nanostructures. Sugars and folic acid are well established as targeting entities for diseased cells and were used here since their transporters are overexpressed on the surface of many cancer cells. Therefore sugar- and folic acid-derivatives of 5-aminolevulinic acid (ALA) were synthesized and evaluated in vitro in several cancer cell lines. The folic acid strategy appeared to be incompatible with ALA since no photosensitivity was induced while the strategy with sugars induced good photosensitivites but no increase of selectivity. Alternatively, the feasibility of combining the antineoplastic properties of ruthenium complexes with the porphyrin's photosensitizing properties, was evaluated since combined therapies have emerged as good alternatives to classical treatments. Tetrapyridylporphyrins complexed to ruthenium (I17 arenes presented good cytotoxicities and good phototoxicities toward melanoma cells. Porphyries were also complexed to diruthenium cores and this type of compound presented good phototoxicities and good selectivity for female reproductive cancer cells. The encapsulation of tetrapyrolic PSs was finally investigated using biocompatible nanogels composed of chitosan and hyaluronate. The behavior of these nanoparticles was evaluated for the treatment of rheumatoid arthritis (RA). They were first tested in vitro in mouse macrophages and results revealed good selectivities and phototoxicities toward these cells. In vivo in mice model of RA, the use of such nanoparticles instead of free PS showed longer time of residence in mice knees. Photodynamic protocols also demonstrated good efficiency of the treatment comparable to the corticoid injection used in the clinic. Finally our system was also efficient in human cells using phagocytic myelomonocytes or using cells of synovial fluids taken from patients with RA. Altogether, these results revealed that both strategies of modification or encapsulation of drugs can be successful in the targeting of diseased cells. However, a careful analysis of the target and of the mode of action of the drug, are needed in order to obtain good results. Looking ahead to the future, the combination of the two strategies (i.e. drugs loaded into nanostructures bearing the targeting agents) would represent probably the best solution.

Redirecting anti-viral CTL against cancer cells by surface targeting of monomeric MHC class I-viral peptide conjugated to antibody fragments.

To combine the advantage of both the tumor targeting capacity of high affinity monoclonal antibodies (mAbs) and the potent killing properties of cytotoxic T lymphocytes (CTL), we investigated the activity of conjugates made by coupling single Fab' fragments, from mAbs specific for tumor cell surface antigens, to monomeric HLA-A2 complexes containing the immunodominant influenza-matrix peptide 58-66. In solution, the monovalent 95 kDa Fab-HLA-A2/Flu conjugates did not activate influenza-specific CTL. However, when targeted to tumor cells expressing the relevant tumor-associated antigen, the conjugates induced CTL activation and efficient tumor cell lysis, as a result of MHC/peptide surface oligomerization. The highly specific and sensitive in vitro cytotoxicity results presented suggest that injection of Fab-MHC/peptide conjugates could represent a new form of immunotherapy, bridging antibody and T lymphocyte attack on cancer cells.

Noninvasive imaging of 5-HT3 receptor trafficking in live cells: from biosynthesis to endocytosis.

Sequential stages in the life cycle of the ionotropic 5-HT(3) receptor (5-HT(3)R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT(3)R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT(3)R-specific antagonist. After novel protein synthesis was chemically blocked, the 5-HT(3) R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quantitatively to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT(3) receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 mum/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT(3) receptors was observed within minutes after exposure to an extracellular agonist. Our orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking.

Effectiveness of interventions targeting frequent users of emergency departments: a systematic review

L'article publié de le cadre de cette thèse est intitulé "Effectiveness of interventions targeting frequent users of emergency departments: A systematic review." Il a été publié par les "Annals of Emergency Medicine (AEM)" en juillet 2011. Le titre en français pourrait être: "Efficacité des interventions ciblant les utilisateurs fréquents des services d'urgence: Une revue systématique." Le titre du journal américain pourrait être: "Annales de Médecine d'Urgence". Il s'agit du journal du "Collège Américain des Médecins d'Urgence", en anglais "American College of Emergency Physicians (ACEP)". L'article a été soumis à l'AEM en raison de l'intérêt que ses rédacteurs en chef portent pour le sujet des utilisateurs fréquents des services d'urgence, démontré depuis plus de dix ans par la publication de nombreux articles dans ce domaine. Le facteur d'impact de l'AEM est de surcroît le plus important des journaux d'urgence, assurant ainsi une large diffusion des articles publiés. Lors de sa publication, l'article a été accompagné d'un éditorial signé par le Docteur Maria C. Raven, médecin au Centre Hospitalier de Bellevue à New York, Etats-Unis.¦Contexte et enjeux¦La Direction Générale du Centre Hospitalier Vaudois (CHUV) finance, dans le cadre du plan stratégique 2009-2013, un axe "populations vulnérables". Cet axe est porté en grande partie par des projets développés au sein de la Policinlique Médicale Universitaire et l'Unité des Populations Vulnérables qui prend en charge, enseigne la prise en charge et s'interroge sur la prise en charge des personnes les plus vulnérables. C'est dans ce contexte que nous avons été amenés à réfléchir à l'existence éventuelle de marqueurs de vulnérabilité; l'utilisation fréquente des services d'urgence par certains individus constitue à n'en pas douter l'un de ces marqueurs. Il existe une importante littérature décrivant en détail ces utilisateurs fréquents des services d'urgence, raison pour laquelle nous avons décidé de faire un pas supplémentaire et de nous interroger sur l'efficacité des interventions (quelle qu'elles soient) ciblant cette population particulière. Nous avons ainsi entrepris une revue systématique de la littérature scientifique médicale et sociale pour approfondir cette question, et c'est précisément le résultat de cette recherche qui constitue ce travail de thèse.¦Conclusions et perspectives¦Les utilisateurs fréquents des services d'urgence sont des individus particulièrement vulnérables, et ce aussi bien aux Etats-Unis, qu'en Europe ou en Australie: ils présentent par exemple une mortalité supérieure aux autres utilisateurs des urgences; ils sont également plus à risque de présenter une consommation abusive d'alcool ou de drogues, une maladie mentale, ou une maladie chronique. Ils sont plus souvent sans abri, sans assurance et d'un statut socio-économique bas.¦De nombreuses interventions on été développées pour prendre en charge les utilisateurs fréquents des urgences. Le but de ces interventions est d'une part de réduire la consommation des services d'urgence et d'autre part d'améliorer la santé au sens large de ces patients vulnérables. C'est en ces termes (réduction de la fréquence d'utilisation des services d'urgence et amélioration de la santé) que l'efficacité d'une intervention est mesurée.¦Parmi l'ensemble des interventions étudiées, l'une semble particulièrement efficace pour réduire le nombre de visites aux urgences et améliorer un certain nombre de marqueurs sociaux (accès à un logement ou à une assurance-maladie). Cette intervention est appelée "case management" (ou "gestion de cas", difficile tentative de traduction de ce concept en français), et consiste en une approche multidisciplinaire (médecins, infirmiers, assistants sociaux) fournissant un service individualisé, dans le cadre de l'hôpital et souvent également dans la communauté. L'approche consiste à évaluer les besoins précis du patient, à l'accompagner dans son parcours de soin, à l'orienter si nécessaire et à mettre en place autour de lui un réseau communiquant de manière adaptée.¦Le "case management" ayant montré son efficacité dans la prise en charge des utilisateurs fréquents des services d'urgence, y-compris en termes de coûts, notre conclusion va dans le sens d'encourager les hôpitaux à évaluer l'importance de ce phénomène dans leur propre pratique et à mettre en place des équipes de prise en charge de ces patients, dans le double but de soutenir des patients particulièrement vulnérables et de réduire la consommation des services d'urgence. Suite à la réflexion suscitée par ce travail de thèse, une telle équipe a été mise en place en 2010, dans un cadre de recherche-action, au niveau du CHUV. Ce projet est dirigé par le Dr Patrick Bodenmann, responsable de l'Unité Populations Vulnérables de la Policlinique Médicale Universitaire de Lausanne. Le Dr Bodenmann est également le directeur de cette thèse et le dernier auteur de la revue systématique.

New Targeting Social Need Action Plan 2000 (PDF 2000 KB)

This report sets out New Targeting Social Need actions for 2000.

Tackling Equality and Targeting Social Need - Improving the Infrastucture (PDF 101 KB)

This paper considers two recent initiatives in Northern Ireland relating to targeting social need and inequalities. While the paper deals with each separately they should be seen as complementary. Therefore, many of the points made in relation to one are also pertinent to the other. åÊ

Tackling Equality and Targeting Social Need (PDF 80 KB)

Statistics Users Council Annual Conference This paper considers two recent initiatives in NI relating to targeting social need, and inequalities. While the paper deals with each separately they should be seen as complementary. Therefore, many of the points made in relation to one are also pertinent to the other. åÊ

Zymogen activation and subcellular activity of subtilisin kexin isozyme 1/site 1 protease.

The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B'/B followed by the herein newly identified C'/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B'/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1-P8) and P1' are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates.

Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes

Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania) amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME) as substrate, phenylmethylsulphone fluoride (PMSF) and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK) as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate), but also in a crude plasma membrane fraction (2.0-fold). Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP), with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

Vascular-endothelial-growth-factor (VEGF) targeting therapies for endocrine refractory or resistant metastatic breast cancer.

BACKGROUND: Vascular-endothelial-growth-factor (VEGF) is a key mediator of angiogenesis. VEGF-targeting therapies have shown significant benefits and been successfully integrated in routine clinical practice for other types of cancer, such as metastatic colorectal cancer. By contrast, individual trial results in metastatic breast cancer (MBC) are highly variable and their value is controversial. OBJECTIVES: To evaluate the benefits (in progression-free survival (PFS) and overall survival (OS)) and harms (toxicity) of VEGF-targeting therapies in patients with hormone-refractory or hormone-receptor negative metastatic breast cancer. SEARCH METHODS: Searches of CENTRAL, MEDLINE, EMBASE, the Cochrane Breast Cancer Group's Specialised Register, registers of ongoing trials and proceedings of conferences were conducted in January and September 2011, starting in 2000. Reference lists were scanned and members of the Cochrane Breast Cancer Group, experts and manufacturers of relevant drug were contacted to obtain further information. No language restrictions were applied. SELECTION CRITERIA: Randomised controlled trials (RCTs) to evaluate treatment benefit and non-randomised studies in the routine oncology practice setting to evaluate treatment harms. DATA COLLECTION AND ANALYSIS: We performed data collection and analysis according to the published protocol. Individual patient data was sought but not provided. Therefore, the meta-analysis had to be based on published data. Summary statistics for the primary endpoint (PFS) were hazard ratios (HRs). MAIN RESULTS: We identified seven RCTs, one register, and five ongoing trials from a total of 347 references. The published trials for VEGF-targeting drugs in MBC were limited to bevacizumab. Four trials, including a total of 2886 patients, were available for the comparison of first-line chemotherapy, with versus without bevacizumab. PFS (HR 0.67; 95% confidence interval (CI) 0.61 to 0.73) and response rate were significantly better for patients treated with bevacizumab, with moderate heterogeneity regarding the magnitude of the effect on PFS. For second-line chemotherapy, a smaller, but still significant benefit in terms of PFS could be demonstrated for patients treated with bevacizumab (HR 0.85; 95% CI 0.73 to 0.98), as well as a benefit in tumour response. However, OS did not differ significantly, neither in first- (HR 0.93; 95% CI 0.84 to 1.04), nor second-line therapy (HR 0.98; 95% CI 0.83 to 1.16). Quality of life (QoL) was evaluated in four trials but results were published for only two of these with no relevant impact. Subgroup analysis stated a significant greater benefit for patients with previous (taxane) chemotherapy and patients with hormone-receptor negative status. Regarding toxicity, data from RCTs and registry data were consistent and in line with the known toxicity profile of bevacizumab. While significantly higher rates of adverse events (AEs) grade III/IV (odds ratio (OR) 1.77; 95% CI 1.44 to 2.18) and serious adverse events (SAEs) (OR 1.41; 95% CI 1.13 to 1.75) were observed in patients treated with bevacizumab, rates of treatment-related deaths were lower in patients treated with bevacizumab (OR 0.60; 95% CI 0.36 to 0.99). AUTHORS' CONCLUSIONS: The overall patient benefit from adding bevacizumab to first- and second-line chemotherapy in metastatic breast cancer can at best be considered as modest. It is dependent on the type of chemotherapy used and limited to a prolongation of PFS and response rates in both first- and second-line therapy, both surrogate parameters. In contrast, bevacizumab has no significant impact on the patient-related secondary outcomes of OS or QoL, which indicate a direct patient benefit. For this reason, the clinical value of bevacizumab for metastatic breast cancer remains controversial.

Targeting burn prevention in the paediatric population : a prospective study of children's burns in the Lausanne area.

QUESTION UNDER STUDY: Domestic accidents are an important problem in paediatric medicine. This study was designed to gain a better understanding of burn mechanisms and target prevention. METHODS: Children treated for burn lesions in the Department of Paediatric Surgery between August 2004 and August 2005 were included in this prospective study. The burn mechanisms, the children's ages and the circumstances in which children were burned as well as their home environment variables were analyzed. RESULTS: The current study included eighty-nine patients, aged between 2 months and 15 years. Seventy-eight percent were less than 5 years old. More than half were boys. Hot liquid scalding was the most frequent mechanism. There does not seem to be an increased risk in the immigrant population or in low economic status families. In most cases, an adult person was present at time of injury. CONCLUSIONS: If we were to describe the highest "at risk" candidate for a burn in our region, it would be a boy aged 15 months to 5 years who is burned by a cup of hot liquid on his hand, at home, around mealtime, in the presence of one or both parents. Reduced attention in the safe domestic setting is probably responsible.

Cilengitide: an integrin-targeting arginine-glycine-aspartic acid peptide with promising activity for glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM), a highly invasive and vascular cancer, responds poorly to conventional cytotoxic therapy. Integrins, widely expressed in GBM and tumor vasculature, mediate cell survival, migration and angiogenesis. Cilengitide is a potent alphavbeta3 and alphavbeta5 integrin inhibitor. OBJECTIVE: To summarize the preclinical and clinical experience with cilengitide for GBM. METHODS: Preclinical studies and clinical trials evaluating cilengitide for GBM were reviewed. RESULTS/CONCLUSIONS: Cilengitide is active and synergizes with external beam radiotherapy in preclinical GBM models. In clinical trials for recurrent GBM, single-agent cilengitide has antitumor benefits and minimal toxicity. Among newly diagnosed GBM patients, single-arm studies incorporating cilengitide into standard external beam radiotherapy/temozolomide have shown encouraging activity with no increased toxicity and have led to a planned randomized Phase III trial.