L’arthrose affecte une dizaine de millions de français. La présence de microcristaux calciques notamment ceux de pyrophosphates de calcium (CPP) dihydratés (Ca2P2O72H2O ; mono- et/ou triclinique : respectivement m- et/ou t-CPPD) au sein de l’articulation est l’un des facteurs aggravant identifié de cette maladie. Cependant, les conditions et mécanismes de formation de ces cristaux ainsi que l’ordre d’apparition de ces deux phases de CPPD restent méconnus ou n’ont pas encore été entièrement décrits et les traitements existants sont seulement symptomatiques. Dans ce mémoire nous nous sommes intéressés à l’étude de l’effet d’additifs ioniques sur la formation et les cinétiques de croissance cristalline de cristaux de CPP qui constitue une étape clé pour la mise au point à moyen ou long terme d’une stratégie thérapeutique visant à empêcher la formation, transformer ou dissoudre ces cristaux. La synthèse in vitro par précipitation des phases pures de CPP hydratés d’intérêt biologique (m-CPPD, t-CPPD et CPP monoclinique tétrahydraté ) a été réalisée afin d’identifier les paramètres opératoires conduisant à leur apparition et d’analyser l’effet d’additifs ioniques (Mg2+, Cu2+, Zn2+, Fe3+ ou S2O32-) lors de leur précipitation en conditions maîtrisées. La méthode de synthèse établie par Gras et al. a été transposée en réacteur agité. Il s’agit d’une précipitation avec ajout simultané de deux solutions mères respectivement de nitrate de calcium et de pyrophosphate de potassium dans une solution tampon d’acétate d’ammonium. Les composés obtenus ont été caractérisés par diffraction des rayons X, résonance magnétique nucléaire du solide, spectroscopies FTIR et RAMAN, microscopie électronique à balayage (MEB) et spectrométrie à plasma à couplage inductif. Grâce à une analyse semi-quantitative par diffraction des RX, nous avons montré que la présence d’additifs ioniques (Mg2+, Zn2+, Fe3+ ou Cu2+) lors de la précipitation change significativement les proportions des phases de CPP du produit final. Ainsi nous montrons que les ions Mg2+, Cu2+ ou Zn2+ orientent la formation de cristaux de m-CPPD lors de la précipitation en conditions maîtrisées de t-CPPD, et selon les conditions l’ion Fe3+ peut totalement inhiber la cristallisation de t-CPPD. Des analyses fines par RMN du solide 31P ont permis de déterminer les modes d’action de ces additifs. Les ions Cu2+ et Mg2+ ont notamment agi par substitution au Ca2+ dans la maille de CPP. Nous avons aussi mis en œuvre la méthode de croissance cristalline à composition constante pour étudier la germination et la croissance cristalline à température, pH, sursaturation et force ionique constants de t-CPPD sur une semence de t-CPPD en présence ou non d’additif ionique. La semence est introduite dans une solution sursaturée métastable de CPP à pH 6,2 incluant l’additif ionique et placée dans un réacteur agité et thermostaté à 37°C. La sursaturation du milieu est calculée à l’aide du logiciel Phreeqc et les temps de germination et vitesses initiales de croissance cristalline ont été déterminés. Les cristaux ont été analysés par spectroscopie FTIR et MEB. Nous avons mis en évidence l’influence d’additifs ioniques (Mg2+, Zn2+ ou Cu2+) sur les paramètres cinétiques de germination et croissance cristalline de t-CPPD. Ainsi les ions Cu2+ et Mg2+ ont respectivement inhibé partiellement et totalement la croissance cristalline de t-CPPD. Les résultats obtenus dans cette étude pourraient aider à expliquer la formation in vivo de cristaux de m-CPPD au sein des articulations de patients arthritiques alors que cette phase pure est difficile à obtenir in vitro par précipitation sans ions autres que Ca2+ et P2O74-. Ces résultats cinétiques pourront donner une indication sur l’évolution du ratio des deux phases (m-CPPD et tCPPD) formées in vivo dans les articulations arthrosiques et des pistes de traitement pour éviter la formation de ces cristaux inflammatoires ou orienter la formation de phases non inflammatoires. / Osteoarthritis (OA) affects about ten million people in France. The presence of microcrystals including those of calcium pyrophosphate (CPP) dihydrates (Ca2P2O7•2H2O; mono- and/or triclinic: m-CPPD and/or t-CPPD respectively) in the joints has been identified as an aggravating factor of OA. However the conditions of formation of both CPPD phases and their order of appearance remain unknown or have not been totally described and the existing treatments are only symptomatic. In the present work we were interested in the effect of ionic additives on the formation and the kinetics of the crystallization of CPP which is a key step in the medium to long term development of a therapeutic strategy to prevent the formation of these crystals, their transformation or their dissolution. At first the in vitro synthesis by precipitation of hydrated CPP pure phases of biological interest (mCPPD, t-CPPD and monoclinic CPP tetrahydrated ) have been realized to identify the operating parameters (concentration, pH, temperature) leading to their formation and to analyze the effect of ionic additives (Mg2+, Cu2+, Zn2+, Fe3+ or S2O32-) on their precipitation under controlled conditions. To achieve this objective, the synthesis method developed by Gras et al. was transposed in stirred reactor. It consists in a precipitation by simultaneous addition of two reagent solutions of calcium nitrate and potassium pyrophosphate in an ammonium acetate buffer solution. Powders synthesized were analyzed by X-ray diffraction, solid state nuclear magnetic resonance, FTIR and RAMAN spectroscopies, scanning electron microscopy and inductively coupled plasma atomic emission spectroscopy. Thanks to a semiquantitative analysis by X-ray diffraction, we showed that the presence of ionic additives (Mg2+, Zn2+, Fe3+ or Cu2+) during the precipitation significantly modified the proportions of CPP phases constituting the final product. We showed that Mg2+, Cu2+ or Zn2+ promote the formation of m-CPPD crystals during the precipitation under controlled conditions for t-CPPD phase synthesis and depending on the conditions Fe3+ ion can completely inhibit t-CPPD phase crystallization. 31P solid state NMR fine analyzes have allowed determining the mode of action of these ionic additives. Thus, Cu2+ or Mg2+ ions have substituted for Ca2+ in the CPP lattice. Additionally, we have implemented the method of constant composition crystal growth to study the nucleation and crystal growth at constant temperature, pH, supersaturation and ionic strength of tCPPD on seeds of t-CPPD crystals in the presence or not of ionic additive. The seed was introduced into a supersaturated metastable solution of calcium pyrophosphate at pH 6.2 including the ionic additive and placed in a stirred reactor maintained at 37°C. Supersaturation of the medium was calculated using Phreeqc software and nucleation time and the initial rate of crystal growth were determined. The resulting crystals were analyzed by FTIR spectroscopy and scanning electron microscopy. We were able to highlight the influence of ionic additives (Mg2+, Zn2+ or Cu2+) on the kinetic parameters of nucleation and crystal growth of t-CPPD. Thereby, Cu2+ and Mg2+ ions have respectively inhibited partially and totally the growth of the tCPPD crystals. These results may help to explain why m-CPPD phase is identified in vivo in the joint of arthritic patients while this pure phase is difficult to obtain by in vitro precipitation (very narrow temperature-pH domain in a medium free of any ionic additives). These results could also provide indications on the order of appearance and potential evolutions of m-CPPD and tCPPD phases formed in vivo in osteoarthritic joints and provide some clues to avoid the formation of CPPD crystals with inflammatory potential or orient the formation of noninflammatory CPP phases

Addition of a small amount of polar solvent (i.e. modifier) which contains an ionic component (i.e. additive) to a CO2 mobile phase has shown major improvement in the elution of ionic analytes via packed column supercritical fluid chromatography (SFC). Firstly, we focused on the elution of sodium arylsulfonate analytes by using various ionic additives, such as lithium acetate, ammonium acetate, tetramethylammonium acetate, tetrabutylammonium acetate, and ammonium chloride. The analytes were successfully eluted with all additives with good peak shape under isocratic/isobaric/isothermal conditions. Three stationary phases with different degrees of deactivation were considered. They were conventional Cyanopropyl, Deltabond Cyanopropyl, and non-chemically bonded silica. The effect of additive concentration and additive functionality on retention was also investigated. Secondly, solid state NMR of the silica packing material before and after being flushed with supercritical CO2 modified by methanol containing the ionic additives was performed to gain some insight into the retention mechanism(s). A fraction of silanol protons were undetected after being treated with the mobile phase which suggested replacement by the cationic component of the additive. CaChe calculations were carried out on several of the additives in an attempt to explain why different ionic additives produce different effects on chromatographic retention. Modification of the stationary phase and ion pairing with the analyte are two possible retention mechanisms being considered. As ion-pair formation was considered to be one of the retention mechanisms, the use of sodium sulfonates as mobile phase additives to elute secondary and quaternary ammonium salts was then studied. Propranolol HCl, benzyltrimethylammonium chloride, and cetylpyridium chloride were chosen as the probe analytes. Sodium ethansulfonate, sodium 1-heptanesulfonate, and sodium 1-decanesulfonate were studied as mobile phase additives. The analytes were successfully eluted from Deltabond Cyano phase within 5 minutes, but were retained strongly without additive or with ammonium acetate as the additive. An Ethylpyridine column showed dramatic advantages on the elution of these ammonium analytes. No additive was required to elute these ionic compounds. Protonation of some fraction of the pyridine functional groups and the deactivation of active silanol sites were believed to be the major mechanisms responsible for this behavior. Lastly, we successfully eluted large peptides (up to 40 mers) containing a variety of acidic and basic residues in SFC. We used trifluoroacetic acid as additive in a CO2/methanol mobile phase to suppress deprotonation of peptide carboxylic acid groups and to protonate peptide amino groups. The Ethylpyridine column was used for the majority of this work. The relatively simple mobile phase was compatible with mass spectrometric (MS) detection. To our knowledge, this is the first report of the elution of peptides of this size with a simple, MS-compatible mobile phase. Fast analysis speed, the possibility of coupling multiple columns to achieve desired resolution, a normal-phase retention mechanism, and less use of organic solvents are the advantages of SFC approach for peptide separation. / Ph. D.