In this thesis, improved techniques for biomolecule analysis using capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and hyphenations between those have been presented.A pre-concentration method which is possible to apply in both techniques, has also been investigated. In this work the off-line MS mode has been used either in the form of fractionation (Paper I) or by incorporating the MALDI target in the CE separation system (Paper II).In Paper I, a protocol for CE-MALDI analysis of cyanogen bromide digested bacteriorhodopsin (BR) peptides as model integral membrane protein peptides were established. Also, an improved protocol for partially automated manufacturing of a concentration MALDI-target plate is presented. The design of the targets was suitable for the fractions from the CE. A novel technique for the integration of CE to MALDI-MS using a closed-open-closed system is presented in Paper II, where the open part is a micro canal functioning as a MALDI target window. A protein separation was obtained and detected with MALDI-MS analysis in the micro canal. A method has been developed for detection of monosaccharides originating from hydrolysis of a single wood fiber performed in a micro channel, with an incorporated electromigration pre-concentration step preceding CE analysis in Paper III. The pre-concentration showed to be highly complex due to the fact that several parameters are included that affecting each other. In Paper IV a protocol using enzymatic digestion, MALDI-TOF-MS and CE with laser induced fluorescence (LIF) detection for the investigation of the degree of substitution of fluorescein isothiocyanate (FITC) to bovine serum albumin (BSA), as a contact allergen model system for protein-hapten binding in the skin, is presented. The intention of a further CE-MALDI hyphenation has been considered during the work. In Paper V 2,6-dihydroxyacetophenone (DHAP) was investigated, showing promising MALDI-MS matrix properties for hydrophobic proteins and peptides. 2,5-dihydroxybenzoic acid (DHB) was undoubtedly the better matrix for the hydrophilic proteins, but its performance for the larger and hydrophobic peptides was not optimal. Consequently, DHAP can be used as a compliment matrix for improved analysis of hydrophobic analytes. / QC 20101214

[pt] As doenças trasmitidas pelo sangue, assim como a necessidade de bancos de sangue para um pronto auxílio em casos de acidentes tem estimulado esforços para desenvolver substitutos do sangue. A albumina serica humana (HSA do ingles Human Serum Albumin) é a proteína mais abundante no plasma sanguíneo. A molécula heme é a transportadora de oxigênio no sangue. Portanto, um estudo detalhado da interação HSA/heme seria útil em pesquisas que visam tornar o complexo HSA-heme em um substituto do sangue. Nesta tese, foram usadas técnicas de dinâmica molecular e ferramentas estatísticas para estudar o sistema HSA-heme em solvente explícito. Tanto o ligante quanto a proteína foram também estudados separadamente em meio aquoso. Dentre outros resultados, nosso estudo revelou a organização da água circundante, os efeitos da ligação do heme na HSA, os mecanismos moleculares da ligação do heme, os movimentos coletivos da proteína livre e ligada, assim como também os aminoácidos que atuam como dobradiças moleculares na mudança conformacional que sofre a proteína ao ligar o heme. / [en] Diseases transmitted through the blood, as well as the need for blood banks to help in case of accidents, stimulated efforts to develop blood substitutes. The human serum albumin (HSA) is the most abundant protein in blood plasma. The heme molecule is the carrier of oxygen in the blood. Therefore, a detailed study of the interaction HSA/heme could give useful insights in the research aimed to convert the HSA-heme complex into a blood substitute. In this thesis, molecular dynamics techniques and statistical tools were applied to study the HSA-heme system in explicit solvent. Both ligand and protein were also studied separately in aqueous medium. Among other results, our study reveals the organization of the surrounding water, the effects of the heme upon its binding to HSA, the molecular mechanisms for heme binding, the collective motions of the protein with and without the heme, as well as the amino acids that act as molecular hinges in the conformational change between the free and bound forms of the protein.

The understanding of self-assembly processes is important for fabrication of well-defined structures with new functionalities for applications in the area of biomedical sciences, material sciences and electronics. In this thesis, two types of self-assembly processes are described: (1) self-assembly of fullerene derivatives in water and (2) self-assembly on surfaces using layer-by-layer (LbL) approach. The various interactions and parameters involved in the self-assembly are detailed in the introductory chapter 1. The various internal parameters like molecular geometry, intramolecular and intermolecular forces that guides the self-assembly process of amphiphiles in water are discussed. The experimental procedures used in the present thesis for the fabrication of nanostructures via self-assembly approach are also described. In the later part of the chapter, the LbL technique for fabrication of thin films and microcapsules is reviewed where various interactions involved in the growth of LbL assembly are discussed. The effect of ionic strength and pH on the growth and property of LbL assemblies is elaborated. A brief discussion of the materials used in the thesis ‒ fullerene, bovine serum albumin (BSA) and nanocrystalline cellulose (NCC) is also provided The self-assembly behaviour of amphiphilic fullerene derivatives are described in chapter 2. Fullerene is anisotropically substituted with five polar hydroxyl groups using organo-copper reagent. The derivative can interact in water via the van der Waals and hydrophobic interactions of the fullerene moiety as well as the intermolecular hydrogen bonding among the hydroxyl groups and also with water. The penta-hydroxy fullerene derivative self-assembles in water as vesicular structures. The size of these vesicles can be varied by modifying the kinetics of self-assembly which was done by changing the rate of addition of non-solvent (water) to the solution of the fullerene derivative. In the second derivative, the hydroxyl groups are substituted with less polar methoxy groups. The penta-methoxy fullerene derivative cannot participate in inter-molecular hydrogen bonding formation unlike the penta-hydroxy derivative but there is possibility of hydrogen bond formation with water where oxygens on methoxy group can act as hydrogen bond acceptor. The penta-methoxy fullerene does not show any vesicle formation in water. The computational simulation studies were carried out on the two fullerene derivatives to understand the self-assembly behaviour of these two derivatives. Furthermore, the vesicle structures formed by the penta-hydroxy fullerene derivative are used for entrapment of hydrophobic polymer, poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) and also hydrophilic dye, Rhodamine B. In both the cases, fluorescence quenching is observed due to electron transfer reaction with fullerene and hence these fullerene vesicles can be used to study the effect of confinement on electron transfer reactions and other chemical dynamics. The layer-by-layer self-assembly approach for the fabrication of biopolymeric thin films and microcapsules is discussed in the chapters 3 to 6. The biocompatible nanoparticles and nanofibers were used as the components of the assembly. In chapter 3, we have described fabrication of thin film of bovine serum albumin (BSA) nanoparticles via LbL approach using biopolymer chitosan as the complementary polymer. The driving force for the assembly growth of the assembly was the electrostatic and complementary hydrogen bond formation between the two components. The idea of incorporating nanoparticles in the thin film was that the nanoparticles can act as reservoirs for functional materials. The films were loaded with anticancer drug doxorubicin and show pH dependent release of the drug. The various interactions involved in the LbL assembly of BSA nanoparticles and polymers were investigated towards understanding the growth mechanism of the assembly in chapter 4. The understanding of the interactions involved in the assembly formation is important in order to modify the conditions of the assembly for enhancing the growth. It is inferred from the study reported in this chapter that not only the interaction of nanoparticles with polymers but also the inter-particle interactions are important factors in determining the growth of LbL assembly of nanoparticles/polymers. The growth of the assembly is enhanced on minimizing the inter-particle repulsions, which was achieved in case of BSA nanoparticles by modifying the pH of the assembly. We also utilized the LbL self-assembly approach for the delivery of lipophilic drugs. The lipophilic drugs are difficult to administer in the body due to their poor water solubility and hence show poor pharmacokinetic profile. The methods for incorporating hydrophobic drugs in LbL assembled thin films and microcapsules are described in chapters 5 and 6. In chapter 5, hydrophobic molecules binding property of albumin has been exploited for solubilisation of a water-insoluble molecule, pyrene (model drug) and hydrophobic drug, curcumin, by preparation of non-covalent conjugates with BSA. The interaction with BSA provided negative zeta potential to the previously uncharged molecules and hence they can be incorporated in the LbL assembled thin films and microcapsules using electrostatic as well as hydrogen bonding interaction with biopolymer, chitosan. The fabrication of protein encapsulated stable microcapsules with hydrophobic molecules incorporated in the shell of the microcapsules has also been demonstrated. The microcapsules were further capable of loading hydrophilic molecules like Rhodamine B. Thus, this approach can be employed for fabrication of multi-agent carrier for hydrophobic and hydrophilic drugs as well as therapeutic macromolecules. In chapter 6, we have incorporated nanocrystalline cellulose (NCC) LbL assembled thin films and microcapsules. The assembly formed was porous in nature due to the nano-fibrous morphology of NCC. The nanoassemblies can act as potential drug delivery carrier, which has been demonstrated by loading anticancer drug doxorubicin, and a lipophilic drug, curcumin. Doxorubicin hydrochloride, the salt form of the drug, doxorubicin, has good water solubility and hence can be postloaded in the assembly by diffusion from its aqueous solution. In the case of curcumin, which has limited solubility in water, a stable aqueous dispersion of the drug was prepared via noncovalent interaction with NCC prior to incorporation in the LbL assembly. The interaction of various other lipophilic drugs with NCC was analysed computationally.

A glicação é uma reação não enzimática que ocorre entre proteínas e açúcares redutores e, é responsável pela formação de adultos e de ligações cruzadas entre proteínas, como por exemplo: a pentosidina, produto final de glicação avançada que se acumula em vários tecidos ao longo do tempo. A glicação é deletéria para o organismo e está associada a modificações estruturais em proteínas e alterações de suas funções específicas, tais como: atividade enzimática, capacidade de ligação e tempo de vida de proteínas, além de ser responsável pela produção de espécies reativas de oxigênio (EROS). O mecanismo de formação da pentosidina envolve reações oxidativas e, uma das estratégias para minimizá-Ia é o aumento da atividade antioxidante nos tecidos. A pariparoba (Pothomorphe umbellata (L.) Miq) demonstrou atividade antioxidante in vitro e in vivo quando aplicada sobre a pele. Essa atividade foi atribuída ao 4-nerolidilcatecol (4-NC), que se mostrou 10 vezes mais potente que o α-tocoferol. Os extratos de pariparoba também inibiram a lipoperoxidação espontânea da pele em camundongos sem pelo. Neste trabalho empregou-se o modelo de glicação de albumina de soro bovino (BSA) frente à D-ribose, com avaliação da fluorescência produzida pela pentosidina formada na reação. Avaliou-se igualmente a atividade do 4-NC em diferentes concentrações sobre a reação de glicação da BSA em presença de D-ribose após 24 horas, empregando-se a aminoguanidina como controle positivo. Nas condições experimentais o 4-NC não foi capaz de inibir a reação de glicação, ao contrário da aminoguanidina. Foi também utilizado modelo para avaliação da propriedade contrátil de fibroblastos em matriz tridimensional de gel de colágeno, glicado e não glicado com D-ribose. O 4-NC na concentração de 100 µM permitiu a manutenção da propriedade contrátil de fibroblastos em gel colágeno glicado. Estudos de glicação em maiores períodos de tempo devem ser realizados visando a confirmar a possível atividade anti-glicação deste composto. / Glycation is a non enzymatic reaction which occurs between proteins and reductor sugars, responsible for the formation of adducts and crosslinkers between proteins, such as, pentosidine, an advanced glycation end-product (AGE) which accumulates in many tissues during aging. AGEs accumulation is deleterious to the body and is associated with structural modifications in proteins and imbalance in their specific functions, such as: enzymatic activity, binding capacity, protein turnover and also responsible for the production of reactive oxygen species (ROS). The mechanism of pentosidine formation involves oxidative reactions. One of the strategies to reduce pentosidine formation is by increasing antioxidant activity in tissues. Pariparoba (Pothomorphe umbellata (L.) Miq. has showed antioxidant activity in vitro and in vivo when applied on the skin. This activity was attributed to 4-nerolydilcatechol (4-NC), which is 10 times more potent than α-tocopherol. Extracts of Pariparoba also inhibited the spontaneous lipid peroxidation in the skin of hairless mice. In this work, the bovine serum albumin (BSA) model for glycation with D-ribose, evaluated by pentosidine fluorescence spectroscopy was employed. The activity of 4¬NC was evaluated in different concentrations in this model after 24 hours. Aminoguanidine was used as positive control. In this experimental condition, 4-NC was not capable to inhibit the BSA glycation. We also evaluated the contractile properties of fibroblasts on tridimensional matriz of collagen gel glycated or not with D-ribose. 4-NC (100 µM) was able to keep the contractile capacity of fibroblasts in glycated collagen. Studies of glycation in longer periods of time should be made in order to further evaluate the possible anti-glycation activity of this compound.

Produtos de glicação avançada, carbamilação e estresse oxidativo contribuem como fatores de risco não tradicionais para a aterosclerose na doença renal crônica (DRC), em parte, por prejudicarem o metabolismo lipídico e por representarem um mecanismo de injúria memorizado ao longo do desenvolvimento da doença renal. A albumina sérica, isolada de animais com DRC, reduz a remoção de colesterol mediado por apoA-I e subfrações de HDL, prejudicando o fluxo de colesterol de macrófagos arteriais ao fígado por meio do transporte reverso de colesterol. Objetivo: Avaliou-se a influência do tratamento com N-acetilcisteína (NAC) em ratos com DRC sobre a concentração plasmática de produtos de oxidação e glicação avançada e o reflexo sobre os efeitos da albumina sérica sobre o efluxo de colesterol e o estresse de retículo endoplasmático em macrófagos. Métodos: Ratos Wistar com 2 meses de idade, pesando aproximadamente 200-250g foram submetidos à nefrectomia 5/6 e mantidos por 60 dias (grupo DRC) com ou sem tratamento com N-acetilcisteína na água (600mg/L), após o 7° dia de indução da DRC (grupo DRC + NAC). Animais controles foram falso-operados (grupo C) e um subgrupo submetido ao tratamento com NAC (C + NAC). No início e no final do estudo foram determinadas as concentrações plasmáticas de glicose, colesterol (CT), triglicérides (TG), ureia, creatinina e na urina, excreção urinária de proteína de 24 h. AGE totais, pentosidina, TBARS (marcador de peroxidação lipídica) e pressão arterial sistólica (PAS) foram determinados no final do estudo. A albumina sérica foi isolada por cromatografia rápida para separação de proteínas e purificada por extração alcoólica. Macrófagos J774 foram incubados por 18 h com as albuminas dos diferentes grupos experimentais para determinação do conteúdo dos receptores de HDL (ABCA-1 e ABCG-1) e de marcadores de estresse de retículo endoplasmático (chaperonas Grp 78, Grp94 e proteína dissulfeto isomerase, PDI) por imunolbot e efluxo de colesterol, mediado por apo A-I e HDL2. Para isto, as células foram previamente enriquecidas com LDL-acetilada e 14C-colesterol. Macrófagos foram também incubados isoladamente com concentrações crescentes de NAC para avaliação do conteúdo dos receptores de HDL. Resultados: Ao final do estudo, o peso corporal foi 10% menor no grupo DRC em comparação ao C (p=0,006). Esta alteração foi prevenida pelo tratamento com NAC. A PAS (mmHg) foi maior no grupo DRC (130 ± 3) em comparação ao grupo DRC+NAC (109±3; p=0,0004). Ureia, creatinina, CT, TG (mg/dL), proteinúria (mg/24 h), AGE total, pentosidina (unidades arbitrárias de fluorescência) e TBARS (nmol/mL) foram maiores nos grupos DRC em comparação ao grupo C (122 ± 8 vs. 41 ± 0,9 ; 0,9 ± 0,07 vs. 0,4 ± 0,03; 151 ± 6 vs. 76 ± 2,7; 83 ± 4 vs. 51,5 ± 3; 46 ± 2,5 vs. 14 ± 0,9; 32620 ± 673 vs. 21750 ± 960; 16700 ± 1370 vs. 5314 ± 129; 6,6 ± 0,5 vs. 2 ± 0,2, respectivamente) (p < 0,0001) e nos grupos DRC+NAC em comparação ao grupo C+NAC (91,4 ± 5 vs. 40 ± 0,9 ; 0,6 ± 0,02 vs. 0,3 ± 0,02; 126 ± 7,5 vs. 76 ± 2,6; 73 ± 6 vs. 68 ± 4; 51 ± 3,5 vs. 18,4 ± 1,5; 24720 ± 1114 vs. 20040 ± 700; 10080 ± 748 vs. 5050 ± 267; 4,5 ± 0,5 vs. 1,8 ± 0,2, respectivamente) (p < 0,0001). No grupo DRC + NAC, PAS, CT, ureia, creatinina, AGE total, pentosidina e TBARS foram, respectivamente, 17% (p=0,0004), 17% (p=0,02), 25% (p=0,02), 33% (p=0,06), 24% (p < 0,0001), 40% (p=0,0008), 28% (p=0,009) menores do que no grupo DRC. A glicemia foi maior nos grupos C + NAC (107+-4,6) e DRC + NAC (107+-2,6) em comparação ao C (96+-1,8) e DRC (98+-1,6), respectivamente. Macrófagos tratados com albumina-DRC apresentaram maior conteúdo de PDI (5 vezes; p=0,02 e 7 vezes p=0,02) e Grp94 (66 %; p =0,02 e 20 %; p=0,02) quando comparados aos tratados com albumina-C ou albumina-DRC + NAC, respectivamente. O conteúdo do receptor ABCA-1 foi menor 87% e 70% (p < 0,01) nos macrófagos tratados com albumina-C+NAC e albumina-DRC, respectivamente em comparação com albumina-C. O conteúdo de ABCG-1 foi, respectivamente, 4 e 7,5 vezes maior nos macrófagos tratados com albumina-C+NAC e albumina-DRC+NAC em comparação as respectivas situações sem tratamento. O efluxo de colesterol mediado por apo A-I foi 59 % e 70 % (p < 0,0001) menor nos macrófagos tratados com albumina-C+NAC e albumina-DRC, respectivamente em comparação a albumina-C. O efluxo de colesterol mediado pela HDL2 foi 52 % maior nos macrófagos tratados com albumina-C+NAC em comparação as células tratadas com albumina-C. Não houve diferença no conteúdo do receptor ABCA-1 nos macrófagos tratados com concentrações crescentes NAC por 8 h. No entanto, após 18 h, o ABCA-1 diminuiu 50 %, 69 % e 72 % nos macrófagos tratados respectivamente com 10 mM, 20 mM e 30 mM de NAC isoladamente em comparação aos macrófagos controles. O conteúdo de ABCG-1 nos macrófagos tratados com NAC, em 8 h e 18 h não sofreu alteração. Conclusão: A N-acetilcisteína reduz produtos de oxidação e glicação avançada no plasma de animais com DRC e previne o estresse de RE em macrófagos, induzido pela albumina isolada destes animais. Apesar de diminuir o conteúdo de ABCA-1 e o efluxo de colesterol mediado por apo A-I, a NAC aumenta o conteúdo de ABCG-1. Desta forma, a NAC pode contribuir para atenuar os efeitos deletérios da albumina modificada na DRC sobre o acúmulo lipídico em macrófagos, contribuindo para a prevenção da aterosclerose / Advanced glycation, carbamylation and oxidative stress c contribute to atherosclerosis in chronic kidney disease (CKD) as nontraditional risk factors. They impair lipid metabolism and promote a long last injury during the development of CKD. Serum albumin isolated from CKD-animals reduces cholesterol efflux mediated by apoa A-I and HDl subfractions, impairing the cholesterol flux from arterial wall macrophage to the the liver by the reverse cholesterol transport (RCT).Objective: In the present study it was analyzed the influence of N-acetylcysteine treatment in CKD-rats in plasma concentration of lipid peroxides and advanced glycation end products and the effect of serum albumin in macrophage cholesterol efflux and endoplasmic reticulum stress development. Methods: Two months male Wistar weighting 200-250g were submitted to a 5/6 nephrectomized maintained for 60 days (CKD group) treated or not with N-acetylcysteine in water (600 mg/L), after the seventh day of CKD induction (CKD+NAC group). Sham animals were false-operated (SHAM group) and a subgroup was treated with NAC (SHAM+NAC group). In the basal and final periods it was determined plasma concentration of glucose, total cholesterol (TC), triglycerides (TG), urea, creatinine and 24h-urinary protein excretion (UPE). Total AGE, pentosidine, thiobarbituric reactive substances (TBARS) levels and systolic blood pressure (SBP) were measured at the final period only. Serum albumin was isolated by fast protein liquid chromatography and purified by alcoholic extraction. J774 macrophage were incubated for 18 h with albumin isolated from the experimental groups in order to determine the content of HDL receptors and markers of endoplasmic reticulum stress (Grp78, Grp94 and protein dissulfide isomerase, PDI) by immunioblot and cholesterol efflux mediated by apo A-I and HDL2. For this, cells were previously overloaded with acetylated LDL and 14C-cholesterol. Macrophage were also incubated with different concentrations of NAC alone in order to measure HDL-receptors and cholesterole efflux. Results: In the end of the protocol, body weight was 10% lower in CKD group in comparison to SHAM group (p=0.006). This change was preserved by treatment with NAC. SBP (mmHg) was higher in CKD group (130±3) in comparison to CKD+NAC (109±3; p=0.0004). Urea, creatinine, TC, TG (mg/dL), UPE (mg/24 h), total AGE, pentosidine (arbitrary units of fluorescence) and TBARS (nmol/mL) were higher in CKD group in comparison to SHAM (122±8 vs. 41 ± 0.9; 0.9 ± 0.07 vs. 0.4 ± 0.03; 151 ± 6 vs. 76±2.7; 83 ± 4 vs. 51.5 ± 3; 46 ± 2.5 vs. 14 ± 0.9; 32620 ± 673 vs. 21750 ± 960; 16700 ± 1370 vs. 5314 ± 129; 6.6 ± 0.5 vs. 2 ± 0.2, respectively) (p < 0.0001) and in CKD+NAC in comparison to C+NAC (91.4±5 vs. 40±0.9 ; 0.6±0.02 vs. 0.3 ± 0.02; 126±7.5 vs. 76 ± 2.6; 73±6 vs. 68±4; 51 ± 3.5 vs. 18.4±1.5; 24720 ± 1114 vs. 20040±700; 10080±748 vs. 5050 ± 267; 4.5±0.5 vs. 1.8±0.2, respectively) (p < 0.0001). In CKD+NAC group, SBP, TC, urea, creatinine, total AGE, pentosidine and TBARS were, respectively, 17 % (p=0.0004), 17 % (p=0.02), 25 % (p=0.02), 33 % (p=0.06), 24 % (p<0.0001), 40 % (p=0.0008), 28 % (p=0.009) lower than CKD group. Glycemia was higher in SHAM+NAC (107+-4.6) and CKD+NAC (107+-2.6) in comparison to SHAM (96+-1.8) and CKD group (98+-1.6), respectively. Macrophages treat with CKD-albumin presented higher content of PDI (5 times; p=0.02 e 7 times p=0.02) and Grp94 (66 %; p=0.02 e 20 %; p=0.02) when compared to SHAM-albumin and CKD+NAC-albumin- treated cells, respectively. ABCA-1 protein content was 87 % and 70 % (p < 0.01) lower in macrophages treated with SHAM+NAC-albumin and CKD-albumin, respectively compared with SHAM-albumin. ABCG-1 protein level was respectively 4 and 7.5 times higher in macrophages treated with SHAM+NAC-albumin and CKD+NAC-albumin in comparison to their respective controls without treatment. The cholesterol efflux mediated by apo A-I was 59 % and 70 % (p < 0.0001) lower in macrophages treated with SHAM+NAC-albumin and CKD-albumin, respectively, when compared to SHAM-albumin. The HDL2-mediated cholesterol efflux was 52 % higher in macrophages treated with SHAM+NAC-albumin compared to macrophages treated with SHAM-albumin. No difference was observed in the ABCA-1 protein level in macrophages treated with crescent concentrations of NAC alone for 8 h. Nonetheless, after 18 h, ABCA-1 was 50 %, 69 % and 72 % reduced in macrophages treated, respectively, with 10 mM, 20 mM and 30 mM NAC in comparison to control cells. ABCG-1 content in macrophages treated with NAC for 8 h and 18 h was not changed. Conclusion: NAC reduces plasma lipid peroxidation and AGE in CKD animals and prevents the endoplasmic reticulum stress induced by CKD-albumin in macrophages. Despite diminishing ABCA-1 and apo A-I-mediated cholesterol efflux, NAC increases ABCG-1. Then, NAC may contribute to attenuate the deleterious effects of the in vivo modified albumin on lipid accumulation in macrophages helping to prevent atherosclerosis in CKD

La prédiction des paramètres pharmacocinétiques/toxicocinétiques (PK/TK), tels que la clairance intrinsèque (CLint) et la clairance hépatique (CLh) des médicaments, demeure un défi majeur en modélisation quantitative. Selon « l’hypothèse du médicament libre », seul le médicament libre peut traverser la membrane plasmique et la CLh de ce médicament est calculée en fonction de sa fraction libre (fup). Néanmoins, la captation hépatique facilitée par l’albumine (ALB) représente clairement une violation de « l’hypothèse du médicament libre ». Cette captation hépatique se base sur la possibilité que le complexe ALB-médicament puisse assurer un apport supplémentaire en médicament aux hépatocytes. Ainsi, cela pourrait expliquer en grande partie les sous-prédictions observées de CLh. Par ailleurs, certains médicaments peuvent se lier fortement à plusieurs protéines plasmatiques telles que l’ALB et l’alpha-1-glycoprotéine acide (AGP). Ainsi, la forte liaison d’un même médicament à l’ALB, à l’AGP, ou aux deux, pourrait avoir des répercussions bien distinctes sur la prédiction de ces paramètres PK/TK. Cependant, aucune étude n’a été faite pour simuler la différence entre leurs effets. L’objectif principal de cette thèse est donc d’évaluer (avec plus d’exactitude et de précision), pour une série de médicaments, en condition in vivo (ou in situ), ces répercussions en présence des deux protéines plasmatiques, conjointement ou séparément. En outre, il est indispensable de vérifier si une approche générique en modélisation peut être appliquée. Cette thèse est répartie en trois objectifs spécifiques. Le premier est de proposer un arbre décisionnel pour faciliter la sélection des approches prédictives appropriées de CLhin vivo pour des médicaments ayant des caractéristiques différentes. Le second est d’évaluer les répercussions de fortes liaisons aux deux protéines plasmatiques ALB et AGP sur la CLh de deux xénobiotiques choisis (perampanel (PER) et fluoxétine (FLU)) ; ces médicaments ont de fortes affinités pour les deux protéines et un métabolisme exclusif (ou prédominant) dans le foie. Et, le dernier est de développer et valider un nouveau modèle prédictif de CLh pour les xénobiotiques ayant le potentiel de se lier fortement dans le plasma, à l’ALB ainsi qu’à l’AGP. Dans un premier temps, des données in vitro rapportées chez l’humain ont été colligées pour 19 médicaments (substrats des transporteurs OAT2 et OATP1B1), et ont été ensuite utilisées dans six modèles d’extrapolation in vitro-in vivo (IVIVE) pour prédire lesdits paramètres. Après une comparaison statistique, les résultats ont montré que l’approche 2 (c’est-à-dire « fup-adjusted model ») qui se base sur la captation hépatique facilitée par l’ALB, avait la meilleure performance prédictive. Cependant, l’approche 5 (c’est-à-dire « Extended Clearance Model ») qui se base sur le transport facilité, en était une très pertinente à appliquer pour les substrats de transporteurs membranaires. Lesdits substrats seraient potentiellement moins affectés par l’ALB. Ainsi, un arbre décisionnel a été proposé pour choisir rapidement et judicieusement la meilleure approche IVIVE servant à prédire la CLhin vivo pour chaque xénobiotique en présence de l’ALB. Dans un deuxième temps, les médicaments PER et FLU ont été sélectionnés à partir d’une collecte de données (N= 1907 médicaments) en fonction de certains critères (avoir un métabolisme exclusif ou prédominant dans le foie, pas de transport facilité par les transporteurs membranaires, une haute affinité pour les deux protéines ALB et AGP, et un ratio de liaison à l’AGP sur celle à l’ALB proche de l’unité). Cette sélection a été réalisée pour faire des expériences sur des foies isolés et perfusés de rats (IPRL), en présence et en absence des protéines ALB et AGP (c’est-à-dire quatre scénarios IPRL). Les résultats IPRL ont démontré que PER est faiblement à moyennement métabolisé (extraction hépatique= 0,2-0,7), tandis que FLU est fortement métabolisé (extraction hépatique= 0,8-0,99). Le modèle Michaelis-Menten a été ajusté aux cinétiques métaboliques, et différents paramètres Vmax, Km et Km, u ont été obtenus de ce modèle. À de faibles concentrations libres pour les deux médicaments (c’est-à-dire à des concentrations thérapeutiques) et en présence des protéines plasmatiques, les valeurs de CLint non liée ont augmenté pour PER (avec l’ALB et le mélange des deux protéines (MIX)) et FLU (avec l’ALB, l’AGP et le MIX) par rapport à celles obtenues du scénario sans protéine (sauf pour PER avec AGP, lesdites valeurs ont diminué). Par ailleurs, les calculs des ratios CLint (SANS versus AVEC protéine) ont permis d’indiquer l’occurrence d’une facilitation de la captation hépatique de médicaments par l’ALB ou l’AGP. Ces ratios ont aussi permis de vérifier si la cinétique métabolique pour PER et FLU suivait soit « l’hypothèse du médicament libre » soit celle de « la captation hépatique facilitée par les protéines plasmatiques ». Dans un dernier temps, une nouvelle approche prédictive de CLh (approche WO-to-MIX) est développée en se basant sur une nouvelle notion de liaison fractionnelle et en intégrant dans le « fup-adjusted model » de nouveaux paramètres tels que la fraction liée à l’ALB (fB-ALB) et celle liée à l’AGP (fB-AGP) à partir du scénario MIX. Ce modèle est basé sur la captation facilitée par l’ALB. Contrairement à l’approche WO-to-MIX, le « well-stirred model » (ou modèle conventionnel) est basé sur l’hypothèse du médicament libre. Ensuite, les paramètres Vmax et Km obtenus in situ pour PER et FLU lors des expériences IPRL sans protéines, ont été utilisés en combinaison avec le paramètre intrant de la fraction libre ajustée (fup-adjusted) pour le « fup-adjusted model » ou avec la fraction libre (fup) pour le « well-stirred model ». Une comparaison des performances prédictives globales des deux modèles a été faite. Les performances prédictives du nouveau modèle étaient prometteuses, en particulier pour FLU qui montrait le plus haut degré de captation hépatique médiée par l’ALB, par rapport au modèle conventionnel. L’approche WO-to-MIX est une première validation d’un nouveau modèle d’extrapolation proposé pour les médicaments comme FLU qui se lient à l’ALB et à l’AGP. Néanmoins, le modèle conventionnel reste utile à utiliser pour les médicaments comme PER. L’exactitude de prédiction était inférieure pour ce dernier médicament probablement parce que la captation hépatique par l’ALB ne semble pas être maximale, et, par conséquent, l’utilisation de fup-adjusted a surestimé la CLhin vivo. Par conséquent, plus de travail est nécessaire en particulier pour PER. Cette thèse démontre qu’une seule approche générique pour prédire la CLh n’existe pas. Néanmoins, le choix d’une approche IVIVE ayant une performance prédictive satisfaisante est maintenant possible. Les résultats de cette thèse contribuent à : 1) mieux comprendre les répercussions sur les paramètres PK/TK de la forte liaison des médicaments à l’ALB et à l’AGP ; 2) choisir la meilleure approche prédictive de CLh sur la base de l’affinité du xénobiotique (médicament ou contaminant) pour chacune des protéines plasmatiques et des mécanismes impliqués dans le foie ; et 3) prédire la CLh avec précision et exactitude des xénobiotiques qui se lient aux deux protéines plasmatiques. Ces approches IVIVE pour la CLh pourront assurément être intégrées dans des modèles PK/TK à base physiologique pour les xénobiotiques afin d’améliorer la prédiction de leur pharmacocinétique et d’accélérer le processus de développement de médicaments. / The prediction of pharmacokinetic/toxicokinetic (PK/TK) parameters such as intrinsic clearance (CLint) and hepatic clearance (CLh) for highly bound drugs is a major challenge in quantitative modeling. According to the ‘free drug hypothesis’, only the free drug can pass through the plasma membrane and the CLh of this drug is calculated according to its free fraction (fup). Nevertheless, the hepatic uptake facilitated by albumin (ALB) is a violation of the ‘free drug hypothesis’. This facilitated hepatic uptake is based on the possibility that the ALB-drug complex may provide additional drug intake to the hepatocytes. Thus, this could largely explain the underpredictions of CLh. In addition, some drugs can bind extensively in plasma, and to several plasma proteins such as ALB and alpha-1-glycoprotein acid (AGP). Thus, the high binding of the same drug to either ALB or AGP, or to both, could have distinct impacts on the prediction of these PK/TK parameters. However, no study has yet explored how to simulate the difference between these impacts. The main objective of this thesis is therefore to evaluate (with accuracy and precision) for a series of drugs, in the in vivo (or in situ) condition, these impacts in the presence of the two plasma proteins, jointly or separately. Also, it is important to verify if a generic model can be applied. This thesis is divided into three specific objectives. The first is to propose a decision tree to facilitate the selection of appropriate predictive approaches of CLhin vivo for drugs with different characteristics. The second is to assess the impacts of extensive binding to the two plasma proteins ALB and AGP on the CLh of two selected xenobiotics (perampanel (PER) and fluoxetine (FLU)); these drugs have strong affinities to both proteins and an exclusive (or predominant) metabolism in the liver. And the last objective is to develop and validate a new predictive model of CLh for xenobiotics with the potential to bind extensively to ALB as well as to AGP. Firstly, in vitro data obtained in humans were collected for 19 drugs (i.e. substrates of OAT2 and OATP1B1 transporters) and were then used in six in vitro-to-in vivo (IVIVE) extrapolation models to predict these PK/TK parameters. After a statistical comparison, the results showed that the approach 2 (i.e. ‘fup-adjusted model’) that is based on the ALB-facilitated hepatic uptake, had the best predictive performance. However, the approach 5 (i.e. ‘Extended Clearance Model’) that is based on the membrane transporter-mediated uptake, was very relevant to apply for the substrates of membrane transporters. These substrates would potentially be less affected by ALB. Thus, a decision tree has been proposed to quickly and judiciously select the best IVIVE approach to predict CLhin vivo for each xenobiotic in the presence of ALB. Secondly, the PER and FLU drugs were selected from a data collection of 1907 drugs depending on certain criteria (exclusive or predominant metabolism in the liver, no transport facilitated by membrane transporters, high affinity for the two proteins ALB and AGP, and having a binding ratio between AGP and ALB close to the unity). This selection was made to conduct experiments using the isolated and perfused rat liver (IPRL) apparatus, in the presence, and in the absence of the ALB and AGP proteins (i.e. four IPRL scenarios). The IPRL results showed that PER is low to moderately metabolized (hepatic extraction= 0.2-0.7), while FLU is highly metabolized (hepatic extraction= 0.8-0.99). The Michaelis-Menten model was fitted to the obtained metabolic kinetics, and different parameters Vmax, Km and Km, u were obtained from the model. At low free concentrations for both drugs (i.e. therapeutic concentrations) and in the presence of plasma proteins, the values of unbound CLint increased for PER (with ALB and the mixture of the two proteins (MIX)) and FLU (with ALB, AGP and MIX); when compared to those obtained from the protein-free scenario (except for PER with AGP, the unbound CLint values decreased). In addition, the calculations of CLint ratios (WITHOUT versus WITH protein) indicated the occurrence of a hepatic uptake facilitated by ALB or AGP. These ratios also helped in verifying whether the metabolic kinetics for PER and FLU followed either ‘the free drug hypothesis’ or that of ‘plasma protein-facilitated hepatic uptake’. Finally, a new predictive approach of CLh (WO-to-MIX approach) was developed based on a new notion of fractional binding and incorporating new parameters such as the ALB bound fraction (fB-ALB) and the AGP bound fraction (fB-AGP) from the MIX scenario into the ‘fup-adjusted model’. This model is based on the ‘ALB-facilitated hepatic uptake’. Unlike the WO-to-MIX approach, the ‘well-stirred model’ is based on ‘the free drug hypothesis’. Then, the Vmax and Km parameters that were obtained in situ for PER and FLU from the protein-free IPRL experiments, were used in combination with the fup-adjusted input parameter for the ‘fup-adjusted model’ or with the free fraction (fup) for the ‘well-stirred model’. A comparison of the two models’ overall predictive performances was made. The predictive performances of the new model were promising for FLU, which showed the highest degree of ‘ALB-mediated hepatic uptake’, compared to the conventional model. This WO-to-MIX approach is a first validation of a novel extrapolation model suggested for drugs such as FLU that bind to both ALB and AGP. The well-stirred model remains however a useful tool to predict the clearance for drugs such as PER. The prediction accuracy was lower for the latter drug probably because the ALB-mediated hepatic uptake does not seem to be maximal, and, hence, the use of fup-adjusted has overestimated its CLhin vivo. Therefore, more work is needed particularly for PER. This thesis shows that a generic approach to predict the CLh in vivo does not exist. Nevertheless, the choice of an IVIVE approach with satisfactory predictive performances is now possible. The results of this thesis contribute to: 1) better understand the impacts on the PK/TK parameters of extensive drug binding to ALB and AGP; 2) choose the best predictive approach to CLh based on the affinity of xenobiotic (drug or contaminant) to each of the plasma proteins and the mechanisms involved in the liver; and 3) predict accurately and with precision the output CLh of xenobiotics that bind to the two plasma proteins. These IVIVE approaches for CLh can certainly be integrated into physiologically based PK/TK models for xenobiotics to improve the prediction of their pharmacokinetics and to accelerate the drug development process.

Aims: 1. Determination of AGEs (Advanced Glycation End products) in patients with various types of nephropathy. 2. Association AGEs with nutritional parameters and anemia. 3. Influence of renal parameters on sRAGE (soluble form of Receptor for Advanced Glycation End products) levels. 4. Technics and proceeding methods of the podocytes cultivation. 5. Determination of urine podocytes. Methods: We determined fluorescent AGEs by spectrofluorometry, sRAGE by Enzyme-Linked ImmunoSorbent Assay (ELISA). Podocytes were passaged and identified immunocytochemically. Podocytes in urine were specified by flow cytometry method. Results: 1. We did not find significant differences in AGEs serum levels among various types of nephropathy, even though the pathogenesis differs. 2. The albumin and prealbumin levels positively and haemoglobin levels negatively correlate with AGEs in patients with CKD grade 1-5, without necessity of dialysis. 3. Serum sRAGE levels are increased in patients with decreased renal function independently on the course of renal disease. 4. We implemented the methods and technics of podocyte cultivation. 5. Urine podocytes observation and confirmation that podocyturia relates to disease activity. Conclusion: We confirmed that AGEs serum levels depend more on renal function than the type of...

Aims: 1. Determination of AGEs (Advanced Glycation End products) in patients with various types of nephropathy. 2. Association AGEs with nutritional parameters and anemia. 3. Influence of renal parameters on sRAGE (soluble form of Receptor for Advanced Glycation End products) levels. 4. Technics and proceeding methods of the podocytes cultivation. 5. Determination of urine podocytes. Methods: We determined fluorescent AGEs by spectrofluorometry, sRAGE by Enzyme-Linked ImmunoSorbent Assay (ELISA). Podocytes were passaged and identified immunocytochemically. Podocytes in urine were specified by flow cytometry method. Results: 1. We did not find significant differences in AGEs serum levels among various types of nephropathy, even though the pathogenesis differs. 2. The albumin and prealbumin levels positively and haemoglobin levels negatively correlate with AGEs in patients with CKD grade 1-5, without necessity of dialysis. 3. Serum sRAGE levels are increased in patients with decreased renal function independently on the course of renal disease. 4. We implemented the methods and technics of podocyte cultivation. 5. Urine podocytes observation and confirmation that podocyturia relates to disease activity. Conclusion: We confirmed that AGEs serum levels depend more on renal function than the type of...

Recombinant human bone morphogenic proteins (rhBMPs) are extensively studied and employed clinically for treatment of various bone defects. Current clinical delivery vehicles suffer wasteful burst releases that mandate supra-physiological dosing driving concerns over safety and cost. It was therefore investigated whether a unique drug delivery vehicle sequestered within a composite scaffold could lower the burst release of rhBMP-2. PLGA-calcium phosphate tri-phasic composite scaffolds delivered model protein BSA with burst release of ~13% and sustained kinetics of 0.5-1.5% BSA/day up to 45 days. rhBMP-2 was delivered with zero burst release however at much lower levels, totaling 0.09% to 0.9 % release over 10 days, but had up to 6.3-fold greater bioactivity than fresh rhBMP-2 (p<0.05). In conclusion, the three-phase composite scaffold can deliver bioactive proteins with a reduced burst release and sustained secondary kinetics.

Recombinant human bone morphogenic proteins (rhBMPs) are extensively studied and employed clinically for treatment of various bone defects. Current clinical delivery vehicles suffer wasteful burst releases that mandate supra-physiological dosing driving concerns over safety and cost. It was therefore investigated whether a unique drug delivery vehicle sequestered within a composite scaffold could lower the burst release of rhBMP-2. PLGA-calcium phosphate tri-phasic composite scaffolds delivered model protein BSA with burst release of ~13% and sustained kinetics of 0.5-1.5% BSA/day up to 45 days. rhBMP-2 was delivered with zero burst release however at much lower levels, totaling 0.09% to 0.9 % release over 10 days, but had up to 6.3-fold greater bioactivity than fresh rhBMP-2 (p<0.05). In conclusion, the three-phase composite scaffold can deliver bioactive proteins with a reduced burst release and sustained secondary kinetics.