Production of laminar extrudates containing particles of a model drug processed by supercritical fluids

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2015

Photochemical control of rheology

The main objective of the research work developed in the framework of this PhD thesis was the preparation and development of novel photorheological fluids. This was pursued following two distinct strategies. The first one focused on the synthesis of tripodal compounds functionalized with photodimerizable moieties of cinnamic acid, coumarin and anthracene. Two sets of compounds were prepared, varying the central unit as well as spacers resulting in molecules with different solubilities and molecular weight. All compounds were characterized towards their photochemical properties and all exhibited photoreactivity upon irradiation with ultra-violet light. In particular, both coumarin derivatives exhibited the greatest photopolymerization reactivity, resulting in the formation of dendrimeric nanoparticles or in the increase of viscosity of organic solutions. The second strategy was focused on the careful design of photosensitive ionic liquids, based on the results of several quantitative structure-property relationship studies. Thus, photosensitive ionic liquids were synthesized bearing cinnamic acid or coumarin moieties in the organic cation. Upon irradiation, all compounds exhibited reactivity, which resulted in changes in their physical properties, such as melting point or viscosity. In addition, novel coumarin chromophores with different photophysical and photochemical properties were developed. It is expected that these compounds may find application in the preparation of new photosensitive ionic liquids.

Therapeutics bioconjugates: evaluation of iminoboronates as payload delivery system for cancer

The advent of bioconjugation impacted deeply the world of sciences and technology. New biomolecules were found, biological processes were understood, and novel methodologies were formed due to the fast expansion of this area. The possibility of creating new effective therapies for diseases like cancer is one of big applications of this now big area of study. Off target toxicity was always the problem of potent small molecules with high activity towards specific tumour targets. However, chemotherapy is now selective due to powerful linkers that connect targeting molecules with affinity to interesting biological receptors and cytotoxic drugs. This linkers must have very specific properties, such as high stability in plasma, no toxicity, no interference with ligand affinity nor drug potency, and at the same time, be able to lyse once inside the target molecule to release the therapeutic warhead. Bipolar environments between tumour intracellular and extracellular medias are usually exploited by this linkers in order to complete this goal. The work done in this thesis explores a new model for that same task, specific cancer drug delivery. Iminoboronates were studied due to its remarkable selective stability towards a wide pH range and endogenous molecules. A fluorescence probe was design to validate this model by creating an Off/On system and determine the payload release location in situ. A process was optimized to synthetize the probe 8-(1-aminoethyl)-7-hydroxy-coumarin (1) through a reductive amination reaction in a microwave reactor with 61 % yield. A method to conjugate this probe to ABBA was also optimized, obtaining the iminoboronate in good yields in mild conditions. The iminoboronate model was studied regarding its stability in several simulated biological environments and each half-life time was determined, showing the conjugate is stable most of the cases except in tumour intracellular systems. The construction of folate-ABBA-coumarin bioconjugate have been made to complete this evaluation. The ability to be uptaken by a cancer cell through endocytosis process and the conjugation delivery of coumarin fluorescence payload are two features to hope for in this construct.

Synthèse de 4-désoxy hexopyrannoses, C-disaccharides et C-glycosides biologiquement actifs

Les glucides constituent la classe de molécules organiques la plus abondante et ceux-ci jouent des rôles cruciaux dans divers processus biologiques. De part leur importance médicinale, la préparation des désoxy-sucres, des C-glycosides et des C-disaccharides est devenue un sujet de pointe en synthèse organique. De façon générale, cette thèse décrit une nouvelle synthèse de novo des 4-désoxy hexopyrannoses en plus de la préparation de C-glycosides biologiquement actifs. De plus, une attention particulière a été portée à la préparation de novo de 4-désoxy-C-disaccharides. Dans un premier temps, le catalyseur de Cr(III) de Jacobsen et un complexe binaphtol/titane ont été utilisés pour réaliser des hétéro-Diels-Alder énantiosélectives. Les dihydropyrannes ainsi générés ont été transformés en 4-désoxy hexopyrannoses présents dans la nature. De cette façon, un dérivé de l’acide ézoaminuroïque, un précurseur de la désosamine et de la néosidomycine, a été préparé suivant cette approche de novo. De plus, à titre comparatif, la néosidomycine a également été fabriquée selon une approche chiron, à partir du méthyl alpha-D-mannopyrannoside. Finalement, une évaluation biologique préliminaire de la néosidomycine a été effectuée sur une la concanavaline-A (Chapitre 2). Dans un deuxième temps, une allylation stéréosélective sur un aldéhyde lié via des liens C-C à une unité mannoside a permis de générer un alcool homoallylique. Cette dernière fonctionnalité a été transformée en 4-désoxy hexopyrannose de configuration D ou L. De cette façon, la préparation de pseudo 4-désoxy-C-disaccharides, de 4-désoxy-C-disaccharides et de pseudo 4-désoxy aza-C-disaccharides a facilement été réalisée. Les rapports diastéréoisomériques de la réaction d’allylation ont été déterminés en plus de la configuration absolue des nouveaux centres stéréogéniques formés. La transformation des alcools homoallyliques en pyrannes poly hydroxylés ou en lactames poly hydroxylés a été réalisée, en plus de la déprotection de certains membres de cette famille pour une évaluation biologique préliminaire sur la concanavaline-A (Chapitre 3). Finalement, la synthèse de C-glycosides biologiquement actifs a été réalisée selon deux volets: i) préparation de 3-C-mannopyrannosyl coumarines et ii) synthèse de C-galactosides, inhibiteurs de la lectine PA-IL. Pour ce faire, le couplage de Heck a été utilisé à partir d’un ester alpha,bêta-insaturé, attaché à une unité glycosidique via des liens C-C, pour générer un dérivé glycosyl cinnamate de méthyle. Cependant, lorsque le 2-iodophénol est utilisé comme partenaire de Heck, la coumarine correspondante a été isolée. Les dérivés C-galactopyrannosyl cinnamates de méthyle représentent de bons inhibiteurs monovalents de la PA-IL avec un Kd aussi bas que 37 micro M (Chapitre 4).

Light amplification in dye doped polymer films

We report unusual spectral narrowing and laser emission from polymer thin films doped with Coumarin 540 dye. The laser emission from the polymer films is found to be highly dependent upon the excitation length of the medium. Even a short length of 1.75 mm of the dye doped film gave rise to laser emission with FWHM of 0.3 nm for a pump intensity of 825 kW cm−2. The partial reflections from the broad lateral surfaces of the free standing films provided the optical feedback for the laser emission. Occurrence of well-resolved equally spaced resonant modes confirmed the effect of a Fabry–Perot-like optical cavity between the film surfaces

Light amplification in dye doped polymer films

We report unusual spectral narrowing and laser emission from polymer thin films doped with Coumarin 540 dye. The laser emission from the polymer films is found to be highly dependent upon the excitation length of the medium. Even a short length of 1.75 mm of the dye doped film gave rise to laser emission with FWHM of 0.3 nm for a pump intensity of 825 kW cm−2. The partial reflections from the broad lateral surfaces of the free standing films provided the optical feedback for the laser emission. Occurrence of well-resolved equally spaced resonant modes confirmed the effect of a Fabry–Perot-like optical cavity between the film surfaces.

A comparative study of energy transfer in dye mixtures in monomer and polymer matrices under pulsed laser excitation

This study was conducted to identify the concentration dependence of the operating wavelengths and the relative intensities in which a dye mixture doped polymer optical fibre can operate. A comparative study of the radiative and Forster type energy transfer processes in Coumarin 540:Rhodamine 6G, Coumarin 540:Rhodamine B and Rhodamine 6G:Rhodamine B in methyl methacrylate (MMA) and poly(methyl methacrylate) (PMMA) was done by fabricating a series of dye mixture doped polymer rods which have two emission peaks with varying relative intensities. These rods can be used as preforms for the fabrication of polymer optical fibre amplifiers operating in the multi-wavelength regime. The 445 nm line from an Nd:YAG pumped optical parametric oscillator (OPO) was used as the excitation source for the first two dye pairs and a frequency doubled Nd:YAG laser emitting at 532 nm was used to excite the Rh 6G:Rh B pair. The fluorescence lifetimes of the donor molecule in pure form as well as in the mixtures were experimentally measured in both monomer and polymer matrices by time-correlated single photon counting technique. The energy transfer rate constants and transfer efficiencies were calculated and their dependence on the acceptor concentration was analysed. It was found that radiative energy transfer mechanisms are more efficient in all the three dye pairs in liquid and solid matrices.

Studies on ASE, Lasing and NLO characteristics in Certain Laser Dyes

Observing the wide possibilities of fluorescent dyes, an exhaustive investigation is done in laser dyes mainly focusing on Coumarin 540 which has a very strong emission in the green region. The photophysics of the dye is studied in detail in a good number of solvent environments. The results of the amplified spontaneous emission and lasing behaviour in both dye solution and different polymer solid state matrices and the ptotostability of the these matrices are investigated using the photoacoustic technique and the same are also included in this thesis. The energy transfer behaviour in dye mixtures which could be utilized for laser studies and bio-analysis are also presented. The nonlinear characterization of Coumarin540 forms the last part of the experimental investigations presented in the thesis.

Studies in molecular mechanics using spectroscopic data exact force field and bond in homogeneity

This thesis deals with some studies in molecular mechanic using spectroscopic data. It includes an improvement in the parameter technique for the evaluation of exact force fields, the introduction of a new and simple algebraic method for the force field calculation and a study of asymmetric variation of bonding forces along a bond.

Coumarins and phenolic fingerprints of oak and Brazilian woods extracted by sugarcane spirit

A total of 25 sugarcane spirit extracts of six different Brazilian woods and oak, commonly used by cooperage industries for aging cachaca, were analyzed for the presence of 14 phenolic compounds (ellagic acid, gallic acid, vanillin, syringaldehyde, synapaldehyde, coniferaldehyde, vanillic acid, syringic acid, quercetin, trans-resveratrol, catechin, epicatechin, eugenol, and myricetin) and two coumarins (scopoletin and coumarin) by HPLC-DAD-fluorescence and HPLC-ESI-MS(n). Furthermore, an HPLC-DAD chromatographic fingerprint was build-up using chemometric analysis based on the chromatographic elution profiles of the extracts monitored at 280 nm. Major components identified and quantified in Brazilian wood extracts were coumarin, ellagic acid, and catechin, whereas oak extracts shown a major contribution of catechin, vanillic acid, and syringaldehyde. The main difference observed among oak and Brazilian woods remains in the concentration of coumarin, catechin, syringaldehyde, and coniferaldehyde. The chemometric analysis of the quantitative profile of the 14 phenolic compounds and two coumarins in the wood extracts provides a differentiation between the Brazilian wood and oak extracts. The chromatographic fingerprint treated by multivariate analysis revealed significant differences among Brazilian woods themselves and oak, clearly defining six groups of wood extracts: (i) oak extracts, (ii) jatoba extracts, (iii) cabreuva-parda extracts, (iv) amendoim extracts, (v) canela-sassafras extracts and (vi) pequi extracts.

Comparison of the analytical performance of electrophoresis microchannels fabricated in PDMS, glass, and polyester-toner

This paper compares the analytical performance of microchannels fabricated in PDMS, glass, and polyester-toner for electrophoretic separations. Glass and PDMS chips were fabricated using well-established photolithographic and replica-molding procedures, respectively. PDMS channels were sealed against three different types of materials: native PDMS, plasma-oxidized PDMS, and glass. Polyester-toner chips were micromachined by a direct-printing process using an office laser printer. All microchannels were fabricated with similar dimensions according to the limitations of the direct-printing process (width/depth 150 mu m/12 mu m). LIF was employed for detection to rule out any losses in separation efficiency due to the detector configuration. Two fluorescent dyes, coumarin and fluorescein, were used as model analytes. Devices were evaluated for the following parameters related to electrophoretic separations: EOF, heat dissipation, injection reproducibility, separation efficiency, and adsorption to channel wall.

Characterisation of nickel(II) extraction by 2-hydroxy-5-nonylacetophenone oxime (LIX 84) in a micellar phase

The properties of the nickel(II)/2-hydroxy-5-nonylacetophenone oxime (HNAPO), an active ingredient in LIX 84, extraction system were characterised in a micellar system. The extinction coefficient, λ_max of HNAPO (316 nm) and the Ni²⁺ complex (387 nm) in a neutral micellar system, poly dispersed octa-ethyleneglycol mono-n-dodecyl ether (G₁₂A₈) were determined as 3100 and 3500 M⁻¹ cm⁻¹, respectively. HNAPO was found to have a neutral micellar phase and bulk aqueous phase pK_a of 11.5 and 12.5, respectively. The extraction equilibrium constant, K_ex, was determined to be 10^−8.0, and the deviation from theory observed at high pH can be accounted for by consideration of the competition for nickel(II) ions by hydroxide ions and HNAPO. A micellar phase of octa-ethyleneglycol mono-n-dodecyl ether (C₁₂E₈) was determined to be an appropriate model of the free oil/water interface from the solubilised location of HNAPO. Utilising the interfacial probe, 4-heptadecyl-7-hydroxy coumarin (HHC) allowed the determination of the electrostatic surface potential of mixed micelles of G₁₂A₈ and sodium dodecyl sulphate (SDS) or dodecyl trimethyl ammonium chloride (DTAC). The electrostatic surface potential was a linear function of the number of additional surfactant monomers within the G₁₂A₈ micelle, for the concentration range studied. For G₁₂A₈/DTAC mixed micelles, the surface potential was given by +1.1 mV per DTAC molecule per micelle, and for G₁₂A₈/SDS mixed micelles the relationship was −1.4 mV per SDS molecule per micelle.

Efeito da vitamina K da dieta na anticoagulação oral crônica : evidências prospectivas observacionais e randomizadas

Base Teórica. Embora vários relatos de casos tenham sugerido uma associação entre a ingesta de vitamina K e a instabilidade da anticoagulação oral, o impacto clínico de diferentes quantidades de vitamina K dietética na anticoagulação oral crônica nunca foi prospectivamente estabelecido. Métodos. Características clínicas e estimativas semi-quantitativas sobre a ingesta de vitamina K foram avaliadas, prospectivamente, em 230 visitas ambulatoriais de pacientes anticoagulados em um hospital público universitário (protocolo observacional). Treze pacientes ambulatoriais anticoagulados cronicamente e estáveis foram arrolados em um ensaio clínico randomizado cruzado de intervenção dietética de 4 dias com aumento e diminuição de 5 vezes da sua ingesta habitual de vitamina K (protocolo experimental randomizado). Resultados. Protocolo observacional: Na análise univariada, identificamos uma associação estatisticamente significativa, progressiva e inversa entre um escore global de ingesta de vitamina K e diferentes níveis de anticoagulação. Na análise multivariada, a ingesta de vitamina K dietética foi independentemente associada com níveis subterapêuticos e anticoagulação excessiva (ambos valores de p =0,04), após ajuste para escolaridade, renda, uso de novos fármacos, aderência à anticoagulação oral e intercorrências clínicas. Protocolo randomizado: Após a dieta restrita em vitamina K, observamos um aumento significativo nos valores do INR (de 2,6 ± 0,5 para 3,3 ± 1,0, p=0,03; do início para o dia 7) enquanto, após a dieta enriquecida, identificamos uma diminuição significativa e mais precoce no INR (de 3,2 ± 0,9 para 2,8 ± 0,7, p=0,005; do início para o dia 4). O efeito da ingesta de vitamina K sobre a variação do INR foi particularmente influenciada pela ingesta média habitual da vitamina. Conclusões. Nossos dados prospectivos reforçam o conceito de que a interação vitamina K e fármacos cumarínicos é de fato real e clinicamente relevante, devendo ser reconhecida como um fator importante e independente que interfere com a estabilidade da anticoagulação oral crônica.

Co-cultivation of plant cells as a technique for the elicitation of secondary metabolite production

Cells of Mikania glomerata, Cephaelis ipecacuanha and Maytenus aquifolia were co-cultured in a two-phase system using filter paper as a solid support. The species were co-cultured in all possible paired combinations. Interaction between Mikania and Maytenus cells resulted in increased biomass production of Maytenus cells, but the friedelin content was reduced. Co-cultivation of Cephaelis and Mikania cells enhanced coumarin content, but inhibited the growth of Mikania cells. However, yield of emetine as well as Cephaelis biomass accumulation were positively stimulated by the co-cultivation. Results indicate a possible occurrence of allelopathy in such a system.

Intestinal anti-inflammatory activity of esculetin and 4-methylesculetin in the trinitrobenzenesulphonic acid model of rat colitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)