For many years, the nasal route of administration as part of a therapeutic treatment has been used. This route of administration is easy to implement, especially due to its non-invasiveness the ease of administration that it affords for the patient. In addition, it is suitable for chronic treatment as well as for an emergency situation when the patient is unconscious. For instance, the administration of benzodiazepines, such as midazolam, may be done to stop convulsions in a patient.Traditionally, intranasal administration was mainly borrowed to target a local effect (e.g. treatment of a cold with a decongestant agent). Subsequently, its application for systemic delivery (e.g. treatment of migraine with triptans) was more and more frequently considered. However, the administration of a drug in the nasal cavities for systemic delivery still remains limited. Indeed, even if the intravenous route has several major limitations such as its invasiveness or the pain generated during administration, it remains more widely used than the intranasal route. This can be explained, on the one hand, by the knowledge that was relatively limited regarding the nasal delivery but also because of the unavailability of nasal devices allowing precise control of the nasal administration (i.e. accurate dose delivery, strong deposition in the nasal cavity, etc).Subsequently, the intranasal route has led to a third therapeutic targeting, namely, the “nose-to-brain pathway”. In that case, the nasal cavity was considered as an opportunity to access the central nervous system (CNS). Indeed, the nose-to-brain delivery allows reaching the brain while bypassing the blood-brain barrier which is known to be a major obstacle to the diffusion of drugs in the CNS. Moreover, the passage through the nasal cavity would allow the administration of sensitive molecules (e.g. biopharmaceuticals) while avoiding excessive enzymatic degradation.Therefore, the nose-to-brain pathway appears to be an attractive route for the delivery of unstable molecules, requiring an access to the brain to reach their site of action. In this context, the therapeutic target that has been selected was "cachexia". It is a complex metabolic syndrome associated with underlying illness and characterized by loss of muscle with or without loss of fat mass. It usually results in particular from undernutrition and a generalized inflammatory state in the patient. In order to treat this syndrome and to restore the appetite in these patients, the goal was to use ghrelin (GHRL) as a model drug. GHRL is a peptide hormone that exhibits, among other effects, an orexigenic action. This biopharmaceutical needs to reach its receptors, located in the hypothalamus, to exert its therapeutic effect.In this study, the goal was to develop a formulation that was able to protect GHRL during its nasal administration, while increasing its residence time to promote its diffusion through the nasal olfactory epithelium.In the first part of the project, GHRL was mainly characterized in terms of stability (e.g. temperature and pH), but also in terms of surface charge. These results allowed selecting the most suitable strategy of formulation as well as the optimal storage conditions. After these preformulation evaluations, it was decided to work on the development of a liquid formulation. The first formulation was based on micelles composed of lipids with polyethylene glycol "DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino (polyethylene glycol) -2000] (ammonium salt)" as hydrophilic group. This type of pegylated lipids have already shown, in many scientific studies, interesting properties in the context of intranasal administration, especially in terms of mucopenetration. With a slight adaptation of the protocol found in the literature, it was possible to obtain micelles of an adequate size (~15 nm). The micelles produced also showed good ability to encapsulate GHRL with an encapsulation rate of 98%, but micelles of DSPE-PEG failed to increase the GHRL diffusion through epithelial layer. This step is essential in order to obtain high GHRL levels in the brain. The formulation containing DSPE-PEG micelles has thus been abandoned.Still in the goal of combining lipid excipients with hydrophilic polymer, another formulation strategy based on liposomes coated with chitosan has been considered. Since GHRL has a positive charge at physiological pH, anionic liposomes have been developed to get a high loading. Three types of liposomes have been produced: anionic, neutral and cationic. The objective was to evaluate the influence of the liposomes charge on GHRL encapsulation. By working with anionic liposomes, the loading could be 46% higher than that obtained from the cationic liposomes. In order to evaluate a potential relation between the amount of GHRL that was encapsulated in the liposomes and the amount of GHRL that could potentially be degraded in the presence of enzyme, the three types of liposomes were exposed to trypsin. Following enzyme exposure, anionic liposomes showed enzymatic protection 4 times higher than cationic liposomes. These anionic liposomes have also shown high GHRL protection in the presence of another enzyme with another mechanism of digestion, namely, carboxylesterase-1. Subsequently, isothermal titration calorimetry tests were performed to better understand the interaction mechanisms between GHRL and anionic liposomes. This technique showed that hydrophobic interactions between both compounds were predominant. The coating of anionic liposomes by chitosans was performed and confirmed by an increase of the mean diameter (+48 nm) and charge (+6 mV) as well as by the modification of the morphology of the liposomes. This coating of liposomes with chitosans was supposed to confer additional properties to the formulation such as mucoadhesion and permeation enhancement. These both effects can be obtained thanks to the positive charge of chitosans which allows adhering to the mucins of the mucus, on the one hand, and thanks to the opening of the epithelial tight junctions that enhances drug permeation, on the other hand. The chitosan coating allowed increasing the fixation of the liposomes to mucins by about twenty percent compared to uncoated liposomes. In addition, the "absorption promoter" effect of chitosans was confirmed on cells culture. Then, the formulation was introduced into two distinct nasal devices intended for the administration of liquid nasal sprays, namely, the VP3 device from Aptar Pharma and the SP270 device from Nemera. The aerosols produced by each device allowed generating droplets characterized by a mean diameter higher than 10µm, leading to potential satisfactory impaction onto the olfactory region instead of diffusion throughout posterior region of the nasal cavities. In the second part of the work, a dry formulation was produced by spray-drying from the liquid dispersion of coated liposomes. The objective was to increase the stability of GHRL during storage as well as to enhance its remanence and diffusion through the olfactory epithelium. The optimized parameters allowed producing a powder characterized by a mean diameter higher than 10 μm with an acceptable yield. The powder produced exhibited a low residual moisture and showed good homogeneity in terms of GHRL content. Then, a comparative study was carried out between the powder and the liquid formulation to compare the GHRL stability over time during storage at different temperatures (4°C and 25°C) but also their ability to fix mucins. In both cases, the dry powder showed better results The powder was also re-dispersed in aqueous phase to evaluate the ability of the liposomes to be reconstituted without modifying their physicochemical properties (e.g. size distribution, charges, stability). It was demonstrated that the majority of the initial properties could be preserved after reconstitution (i.e. rate of encapsulation). Similarly to the liquid formulation, the powder was loaded into a specific device developed for the nasal administration of powders that allows targeting the olfactory region to optimize the nose-to-brain transfer. The device, "UDS - Unit Dose System " from Aptar Pharma, has shown excellent properties in terms of particle size distribution in the aerosol but also in terms of targeting the olfactory zone. The latest was studied by means of "nasal cast" that is a 3-printed model of artificial nasal cavities. After impaction in the different cavities of the cast, it was possible to quantify the amount of GHRL that was deposited in the olfactory zone. Using our optimized formulation in combination with the device developed by Aptar, it was shown that 52% of the powder was impacted onto the area corresponding to the olfactory region. Such data demonstrated the relative difficulty to target this section of the nasal cavities.Finally, the formulation loaded with fluorescent GHRL was intranasally administered in mice. It was demonstrated that GHRL could reach the brain after intranasal administration of the formulation and that the formulation was essential to allow this transfer to the brain.The administration of such biopharmaceutical by nose-to-brain with this formulation seems to be an interesting alternative to exploit. However, additional studies to quantify this transfer more precisely, to better define its kinetics and also to evaluate the efficacy of the treatment should be carried out. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Made available in DSpace on 2016-07-01T11:59:26Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 1.pdf: 7298045 bytes, checksum: 81f5fe7a39c309b83b7b15679d38d8b6 (MD5) Previous issue date: 2014 / Made available in DSpace on 2016-07-21T14:39:33Z (GMT). No. of bitstreams: 2 1.pdf: 7298045 bytes, checksum: 81f5fe7a39c309b83b7b15679d38d8b6 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Fármacos/Farmanguinhos. Rio de Janeiro, RJ, Brasil. / A Etionamida (ETA) é um fármaco usado no tratamento da tuberculose pulmonar e extrapulmonar, junto com outros tuberculostáticos, nos casos de falência de tratamentos, resistência ou intolerância a outros fármacos, ou abandono de tratamento. A ETA está descrita na literatura como praticamente insolúvel em água e possui rápida absorção por via oral, o que sugere ser um fármaco de classe II do sistema de classificação biofarmacêutica (SCB), ou seja, baixa solubilidade e alta permeabilidade. Para estes fármacos, é importante o conhecimento dos fatores físicos que influenciam na sua dissolução a partir do produto final, além de se investigar condições para ensaios de perfis de dissolução que possam discriminá-los, isoladamente ou em formulações, com o objetivo de se obter um produto final elegível para um estudo de bioequivalência frente ao medicamento de referência. Foi realizado estudo de solubilidade em HCl 0,1 N, tampão acetato pH 4,5 e tampão fosfato pH 6,8, com amostra de ETA obtida no mercado. Estes estudos demonstraram que a ETA possui alta solubilidade em HCl 0,1 N e baixa solubilidade em tampão acetato pH 4,5 e tampão fosfato pH 6,8, considerando-se o critério de solubilidade do SCB, sugerindo que estes últimos meios seriam mais discriminativos para estudos de perfil de dissolução. O tamanho de partícula da ETA foi reduzido utilizando-se os processos de moagem (ETA-MOÍDA) e micronização (ETA-MICRO). As amostras de ETA, ETA-MOÍDA e ETA-MICRO, foram avaliadas quanto suas características de dissolução (molhabilidade, dispersão e intrínseca), físicas (distribuição granulométrica, microscopia, fluidez, DSC, TGA, DRXP, FTIR e FTNIR) e físico-químicas (teor, pH, umidade e substâncias relacionadas), além de serem utilizadas em formulações de núcleos de comprimidos revestidos produzidos pelos processos de compressão direta e granulação úmida. Os processos de moagem e micronização impactaram em alterações na distribuição granulométrica, morfologia dos cristais, fluidez e dissolução da ETA, porém não resultaram em alterações físico-químicas e de arranjo cristalino interno (polimofismo). As formulações fabricadas por granulação úmida apresentaram melhores características de fluidez, quando comparados os valores do índice de compressibilidade, razão de Hausner e fluxo por orifícios. Entretanto, as formulações fabricadas por compressão direta não apresentaram variação de peso durante o processo de compressão que indicasse fluidez inapropriada, com exceção daquela que utilizou ETAMICRO. Foram avaliados os perfis de dissolução das formulações frente ao medicamento de referência Trecator®, sendo evidenciado que a condição descrita nos compêndios oficiais, HCl 0,1 N, não foi suficiente para discriminá-las, sendo os perfis de dissolução considerados semelhantes quando calculados os fatores diferença (F1) e semelhança (F2). Quando alterado o meio de dissolução para tampão acetato pH 4,5 e tampão fosfato pH 6,8, foi possível evidenciar diferença entre as formulações, tendo aquelas que utilizaram ETA-MOÍDA maior semelhança frente ao Trecator®. Portanto, pôde-se concluir que para obtenção de formulações futuramente elegíveis a um estudo de bioequivalência, é necessário tamanho de partícula específico para a ETA, sugerindo-se d90 ~ 113,26 µm e d50 ~ 32,32 µm, sendo obtidas duas formulações candidatas a continuidade do desenvolvimento, não finalizado neste trabalho, uma fabricada por compressão direta e outra fabricada por granulação úmida. / The Ethionamide (ETA) is a drug used to treatment of pulmonary and extrapulmonary tuberculosis, together with other antituberculosis drugs in cases of failure of treatment, resistance or intolerance to other drugs, and abandonment of treatment. ETA is described as practically insoluble in water and has rapid oral absorption, which suggests that it a drug class II in the biopharmaceutical classification system (BCS), low solubility and high permeability. For these drugs, it is important to know the physical factors that influence in dissolution from the final product, and investigate conditions for the dissolution profiles that could discriminate them, alone or in formulations, with the goal of obtaining one product eligible for the bioequivalence study, compared to the reference product. Solubility studies were performed in 0.1 N HCl, acetate buffer pH 4.5 and phosphate buffer pH 6.8, with a sample of ETA obtained on the market. These studies have shown that the ETA has a high solubility in 0.1 N HCl and low solubility in acetate buffer pH 4.5 and phosphate buffer pH 6.8, considering the criteria of solubility of BCS, suggesting that these last mediums will be more discriminative for studies of the dissolution profile. The particle size of the ETA was reduced using the process of milling (ETA-MILLED) and micronization (ETA-MICRO). Samples of ETA, ETA-MILLED, and ETA-MICRO were evaluated for their dissolution characteristics (wetting, dispersing dissolution and intrinsic dissolution), physical properties (particle size distribution, microscopy, flow, DSC, TGA, XRPD, FTIR and FTNIR) and physicochemical characteristics (assay, pH, moisture and related substances), besides being used in formulations of cores coated tablets produced by direct compression and wet granulation. The processes of milling and micronization resulted in changes in particle size distribution, crystal morphology, flowability and dissolution of ETA, but did not result in physicochemical and internal crystalline arrangement (polymorphism) changes. The formulations manufactured by wet granulation showed better flow characteristics, when comparing the values of the compressibility index, Hausner ratio and flow through holes. However the formulations manufactured by direct compression did not show weight variation during the compression process to indicate inappropriate flowability, except that they used ETA-MICRO. The dissolution profiles front reference product (Trecator®) were evaluated and shown that the condition described in official compendium, 0.1 N HCl, was not enough to discriminate against them, and the dissolution profiles were similar when calculated factors difference (F1) and similarity (F2), for all formulations. When the dissolution medium was changed (acetate buffer pH 4.5 or phosphate buffer pH 6.8) was possible to identify differences between formulations and those used ETAMILLED resulted in a greater similarity to the front Trecator®. Therefore, we concluded that to obtain future formulations eligible to a bioequivalence study would require specific particle size for the ETA (d90 ~ d50 ~ 113.26 µm and 32.32 µm), and were obtained two formulations candidates to continue the development, one manufactured by direct compression and other manufactured by wet granulation.

Made available in DSpace on 2016-03-08T13:58:53Z (GMT). No. of bitstreams: 2 13.pdf: 24054067 bytes, checksum: f2cedc51d13f66361d03cd66f3c46b36 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Fármacos/Farmanguinhos. Rio de Janeiro, RJ, Brasil. / A nimesulida é um fármaco anti-inflamatório não esteroidal (AINE), com seletividade preferencialmente para a COX-2. A nimesulida é um ácido fraco, praticamente insolúvel em água e está classificado como um fármaco de Classe II do Sistema de Classificação Biofarmacêutica. Além disso, apresenta polimorfismo, sendo descrito na literatura o polimorfo I e II, que demonstram algumas diferenças identificadas através da análise térmica, difração de raios X e RMN no estado sólido. Nesse trabalho, amostras do fármaco nimesulida obtidas de diferentes fabricantes, sendo duas amostras micronizadas e uma não micronizada, foram caracterizadas através de análise térmica, técnicas espectroscópicas, análise morfológica, ensaios de fluidez e avaliação biofarmacêutica. Com os resultados obtidos, verificou-se que as amostras são de um mesmo polimorfo, não apresentam boa fluidez e as variações dos tamanhos de partícula influenciam nos ensaios de avaliação biofarmacêutica. O delineamento das formulações e a escolha do processo produtivo foram conduzidos com base nos resultados obtidos nos ensaios de caracterização do fármaco. Os lotes galênicos foram avaliados através da realização de perfis de dissolução utilizando os parâmetros preconizados pela Farmacopeia Brasileira (FB 5, 2010) e comparados com o medicamento de referência Nisulid®. As formulações propostas apresentaram diferentes comportamentos de dissolução, podendo destacar a escolha do tensoativo, do aglutinante e as etapas de adição do tensoativo e desintegrante como fundamentais para a obtenção de resultados mais favoráveis. Foi selecionado o lote galênico que apresentou valores de cedência mais próximos do medicamento de referência e, em seguida, avaliou-se o comportamento em meios de dissolução contendo diferentes concentrações de tensoativo, com o objetivo de verificar se a concentração de 2% (v/v) de polissorbato 80, preconizado pela Farmacopeia Brasileira, estaria superestimando a biodisponibilidade do ativo em condições in vivo. Os resultados obtidos demonstraram que o percentual de tensoativo presente no meio de dissolução impacta diretamente na quantidade de fármaco dissolvida. A formulação selecionada demonstrou resultados promissores para prosseguir com a fabricação dos biolotes e realização do estudo de equivalência farmacêutica. / Nimesulide is a nonsteroidal antiinflammatory drug (NSAID), preferably with selectivity for COX-2. Nimesulide is a weak acid, almost insoluble in water and classified as a Class II drug Biopharmaceutical Classification System. Moreover, the literature has been described polymorph I and II, which show some differences identified by thermal analysis, diffraction X-ray and NMR in the solid state. In this study, drug nimesulide samples were obtained from different manufacturers, two micronized and one non-micronized samples were characterized by thermal analysis, spectroscopic techniques, morphological analysis, flowability and biopharmaceutical evaluation. The results showed that the samples belong to the same polymorph, do not have good flowability and particle size variations affect the biopharmaceutical evaluation tests. The design of the formulations and the choice of the production process were carried out based on the results obtained in the characterization of the drug tests. Pilot batches were evaluated by conducting dissolution profiles using the parameters established by the Brazilian Pharmacopoeia (FB 5, 2010) and compared with the reference Nisulid®. The proposed formulations show different dissolution behavior, and may highlight the choice of the surfactant, binder and adding steps of the surfactant and binding essential for obtaining most favorable results. The closest pilot batch dissolution values of the reference product was selected and then evaluated the behavior of dissolution media containing different concentrations of surfactant, in order to verify if the concentration of 2% (v/v) polysorbate 80, recommended by the Brazilian Pharmacopoeia, would be overestimating the bioavailability of the drug in vivo conditions. The results showed that the percentage of surfactant present in the dissolution medium directly impacts the amount of dissolved drug. The selected formulation demonstrated promising results to proceed with the manufacture of biobatches and the pharmaceutical equivalence study.