La mucoviscidose est une maladie génétique mortelle qui limite ou empêche la production de composés antimicrobiens tels que l’hypothiocyanite (OSCN-) et la lactoferrine. L’objectif de cette étude est de produire un médicament contenant ces deux composés antimicrobiens (10 mL). Cependant l’hypothiocyanite est instable et nécessite une production juste avant administration. Pour cela, une unité de production de médicament portable, destinée à une utilisation par le patient à domicile, est développée avec un appareil réutilisable comprenant le système de pilotage du procédé et une cassette jetable composée par le circuit fluidique et le module membranaire. Le développement du circuit fluidique associé à un module membranaire nécessaire à la purification de l’hypothiocyanite, présent dans le milieu réactionnel, en prenant en compte les contraintes pharmaceutiques, constitue le verrou scientifique et technologique de cette thèse. Au travers de deux géométries membranaires testées, l’étude des paramètres opératoires pour la réalisation de la réaction enzymatique (mécanique des fluides, ultrafiltration, réaction) a permis de mieux appréhender et d’optimiser la production d’hypothiocyanite mais aussi de mettre en évidence les paramètres clés de l’élimination de la glycérine, présente initialement dans les membranes. En parallèle des essais cliniques modifiant les contraintes imposées, l’unité de production et la cassette jetable développées ont permis d’obtenir des résultats proches de ces nouvelles contraintes. / Cystic fibrosis is a fatal genetic disease that limits or prevents antimicrobial compounds such as hypothiocyanite (OSCN-) or lactoferrin. The aim of this study is to produce a drug with those two antimicrobial compounds (10 mL). However hypothiocyanite is unstable and requires production just prior to administration. In order to do so, a portable drug production unit, to be used by the patient at home, is developed. It is made with a reusable device including a control process system and a disposable cassette composed by a fluidic circuit and a membrane module. The scientific and technological challenge of this work is the development of a fluidic circuit incorporating the membrane module for the purification of hypothiocyanite present in a reaction medium while taking into account pharmaceutical constraints. Through two membrane geometries tested, the study of operating parameters to achieve enzymatic reaction (fluid mechanics, ultrafiltration and reaction) allowed to better understand and to optimize the hypothiocyanite production. Furthermore it highlighted the important parameters of the removal of glycerin, initially contained in the membranes. Finally the production unit and the disposable cassette allowed to obtain results close to the specifications. Even though these specifications were redefined and more stringent for clinical trials.

Mammalian heme peroxidases are fascinating due to their unique peculiarity of oxidizing (pseudo)halides under physiologically relevant conditions. These proteins are able either to incorporate oxidized halides into substrates adjacent to the active site or to generate different oxidized (pseudo)halogenated species, which can take part in multiple (pseudo)halogenation and oxidation reactions with cell and tissue constituents. The present article reviews basic biochemical and redox mechanisms of (pseudo)halogenation activity as well as the physiological role of heme peroxidases. Thyroid peroxidase and peroxidasin are key enzymes for thyroid hormone synthesis and the formation of functional cross-links in collagen IV during basement membrane formation. Special attention is directed to the properties, enzymatic mechanisms, and resulting (pseudo)halogenated products of the immunologically relevant proteins such as myeloperoxidase, eosinophil peroxidase, and lactoperoxidase. The potential role of the (pseudo)halogenated products (hypochlorous acid, hypobromous acid, hypothiocyanite, and cyanate) of these three heme peroxidases is further discussed

Background: The heme enzyme lactoperoxidase is found in body secretions where it significantly contributes to the humoral immune response against pathogens. After activation the peroxidase oxidizes thiocyanate to hypothiocyanite which is known for its microbicidal properties. Yet several pathologies are accompanied by a disturbed hypothiocyanite production which results in a reduced immune defense. Methods: The results were obtained by measuring enzyme-kinetic parameters using UV–vis spectroscopy and a standardized enzyme-kinetic test system as well as by the determination of second order rate constants using stopped-flow spectroscopy. Results: In this study we systematically tested thirty aromatic substrates for their efficiency to promote the lactoperoxidase-mediated hypothiocyanite production by restoring the native ferric enzyme state. Thereby hydrophobic compounds with a 3,4-dihydroxyphenyl partial structure such ashydroxytyrosol and selected flavonoids emerged as highly efficient promotors of the (pseudo-)halogenating lactoperoxidase activity. Conclusions: This study discusses important structure-function relationships of efficient aromatic LPO substrates and may contribute to the development of new agents to promote lactoperoxidase activity in secretory fluids of patients. Significance: This study may contribute to a better understanding of the (patho-)physiological importance of the (pseudo-)halogenating lactoperoxidase activity. The presented results may in future lead to the development of new therapeutic strategies which, by reactivating lactoperoxidase-derived hypothiocyanite production, promote the immunological activity of this enzyme.

Background: The heme enzyme lactoperoxidase is found in body secretions where it significantly contributes to the humoral immune response against pathogens. After activation the peroxidase oxidizes thiocyanate to hypothiocyanite which is known for its microbicidal properties. Yet several pathologies are accompanied by a disturbed hypothiocyanite production which results in a reduced immune defense. Methods: The results were obtained by measuring enzyme-kinetic parameters using UV–vis spectroscopy and a standardized enzyme-kinetic test system as well as by the determination of second order rate constants using stopped-flow spectroscopy. Results: In this study we systematically tested thirty aromatic substrates for their efficiency to promote the lactoperoxidase-mediated hypothiocyanite production by restoring the native ferric enzyme state. Thereby hydrophobic compounds with a 3,4-dihydroxyphenyl partial structure such ashydroxytyrosol and selected flavonoids emerged as highly efficient promotors of the (pseudo-)halogenating lactoperoxidase activity. Conclusions: This study discusses important structure-function relationships of efficient aromatic LPO substrates and may contribute to the development of new agents to promote lactoperoxidase activity in secretory fluids of patients. Significance: This study may contribute to a better understanding of the (patho-)physiological importance of the (pseudo-)halogenating lactoperoxidase activity. The presented results may in future lead to the development of new therapeutic strategies which, by reactivating lactoperoxidase-derived hypothiocyanite production, promote the immunological activity of this enzyme.