Associação de capsaicina com anestésicos locais para aumento da analgesia

Orientadores: Eneida de Paula, Laura de Oliveira NascimentoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Os anestésicos locais (AL) são imprescindíveis na prática médica, porém eles não permitem bloqueio seletivo das fibras nociceptivas e falham em anestesiar tecidos inflamados. A capsaicina (CAP) é um composto pouco hidrossolúvel utilizado como analgésico. Há relatos na literatura de que a injeção de AL seguida de CAP leva a diminuição do bloqueio motor, em animais. Os AL e CAP são também utilizados individualmente, em formulações tópicas, para tratamento da dor crônica. Esta tese relata pela primeira vez o efeito analgésico de uma associação medicamentosa entre AL e CAP em tecido inflamado e, também, descreve o desenvolvimento de uma formulação tópica dos mesmos. Na parte 1 da tese, a CAP foi complexada com HP-ß-ciclodextrina, aumentando sua solubilidade aquosa em 20 vezes. Esse complexo foi liofilizado com ciclo otimizado e caracterizado por Calorimetria Diferencial de varredura, Difração de Raios-X e microscopia eletrônica de varredura. Essas técnicas forneceram evidências da complexação entre a ciclodextrina e CAP, enquanto a Ressonância Magnética Nuclear (RMN) comprovou a formação de complexo de inclusão. O liofilizado (ressuspenso em solução contendo o AL mepivacaina) foi administrado em camundongos, resultando em diminuição do bloqueio motor do nervo ciático e, em outro teste, aumento significativo da anestesia em tecido inflamado. A parte 2 da tese relata o desenvolvimento de uma nova formulação contendo exclusivamente CAP e o AL lidocaína (LDC). A mistura equimolar dos fármacos produziu um sistema líquido à temperatura ambiente, com padrão amorfo relatado por Difração de Raios-X. A espectroscopia de infra-vermelho indicou interação intermolecular entre os compostos, confirmada por medidas de RMN. Esta técnica indicou formação de ponte de hidrogênio intermolecular, entre os grupos carbonila (CAP) e amida (LDC), o que explica a redução da temperatura de fusão dos componentes, para abaixo de 25 °C, caracterizando uma mistura eutética profunda. A mistura [LDC]1[CAP]1 revelou-se altamente estável e aumentou a lipofilicidade dos fármacos, característica que pode ser útil para maior permeação dos mesmos na pele. Ambas partes desta tese geraram resultados promissores para futura aplicação clínica da associação medicamentosa entre AL e CAP. Na Parte 1, foi obtida uma formulação parenteral que possibilitou atingir efeito desejável de anestesia, apesar da condição de inflamação. Na Parte 2, o estudo de pré-formulação revelou uma nova alternativa de medicação tópica para o tratamento da dor neuropáticaAbstract: Local anesthetics (LA) are crucial in medical practice to numb parts of the body. However, LA do not allow selective blocking of nociceptive fibers and fail to anesthetize inflamed tissues. Capsaicin (CAP) is a poorly hydrosoluble compound used as an analgesic. There are reports in the literature that the injection of LA followed by CAP leads to a decrease in motor blockade in animals. LA and CAP are also used individually in topical formulations for the treatment of chronic pain. This thesis reports for the first time the analgesic effect of a drug combination (LA and CAP) on inflamed tissue and also describes the development of a topical formulation of them. In part 1 CAP was complexed with HP-ß-cyclodextrin, which increases its water solubility by 20 times. The complex was lyophilized under an optimized cycle and characterized by Differential Scanning Calorimetry, X-ray Diffraction and Scanning Electron Microscopy. These techniques provided evidence on the complexation between cyclodextrin and CAP, while Nuclear Magnetic Resonance (NMR) proved the inclusion complex formation. The lyophilized complex (resuspended in a solution with mepivacaine) was injected in mice, resulting in: i) decreased motor blockade of the sciatic nerve; ii) significant increase of anesthesia in a mechanical test, in inflamed tissue. In part 2 of this thesis, a new formulation containing exclusively the lidocaine (LA) and capsaicin was reported. The equimolar mixture of these drugs produced a liquid system at room temperature, with an amorphous pattern reported by X-ray diffraction. Infrared spectroscopy indicated intermolecular interaction between the compounds, as confirmed by NMR. This technique indicated a strong intermolecular hydrogen bond between the carbonyl (CAP) and amide (LDC) groups, which explains the reduction of the melting temperature of the components to below 25 ° C, characterizing a deep eutectic mixture. The [LDC]1[CAP]1 mixture proved to be highly stable and it increased the lipophilicity of the drugs, which may lead to improve their permeation into the skin. Both parts of this thesis have generated promises for future clinical application of the combination of drugs (LA and CAP). In Part 1, a parenteral formulation was developed that achieved desirable anesthesia effect, despite the inflammatory condition. In Part 2, the pre-formulation study provided a new topical formulation suitable for the treatment of neuropathic painDoutoradoFármacos, Medicamentos e Insumos para SaúdeDoutora em Ciências2015/11804-9 ; 2017/13004-5; 2014/14457-5FAPES

Remote loading of dibucaine into liposomes using transmembranar ionic gradient

Orientador: Eneida de PaulaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Os anestésicos locais (AL) são utilizados para se bloquear a sensação de dor em procedimentos clínicos e odontológicos. Porém, os AL são moléculas pequenas que facilmente se redistribuem no sitio de ação, limitando a duração da anestesia. A dibucaína (DBC) é um anestésico local da família das amino-amidas e tem uso predominante como anestésico tópico. Este trabalho teve por finalidade desenvolver um novo sistema de liberação sustentada para o anestésico local dibucaína baseado em lipossomas de fosfatidilcolina de soja hidrogenada e colesterol com gradiente iônico transmembranar, para uso parenteral. Primeiramente, avaliamos uma metodologia analítica para quantificação de DBC por cromatografia líquida de alta eficiência. Entao, foram então desenvolvidas formulações de lipossomas unilamelares (LUV) e multivesiculares (LMVV) com gradiente iônico (citrato e sulfato), para encapsulação da DBC a 0,012%. As formulações foram caracterizadas quanto à eficiência de encapsulação (%EE) potencial zeta, diâmetro e polidispersão. O potencial zeta de todas as formulações foi sempre negativo (ca. -30 mV). Formulações de LUV contendo sulfato (LUVDBC5.5+sulfin/7.4ex) apresentaram %EE (62,6% ± 4,3) significativamente maior que as demais formulações. As LUV apresentaram tamanho médio de 500 nm, sendo mais estáveis e menos polidispersas que as LMVV. As formulações LUV foram selecionadas para a continuidade do estudo, sendo caracterizadas morfologicamente por microscopia eletrônica de transmissão e testadas quanto a sua estabilidade química, liberação in vitro, viabilidade celular e atividade antinociceptiva em camundongos. As micrografias das LUV confirmaram a formação de vesículas esféricas e unilamelares. Nenhuma formulação apresentou níveis de peroxidação lipídica significativa (> 0,5% de lipídios totais) durante 150 dias de armazenamento a 4oC. A formulação LUVDBC5.5+sulfin/7.4ex apresentou o melhor perfil de liberação sustentada in vitro e aumento significativo no tempo de bloqueio sensorial (27 h) in vivo, em comparação com solução de DBC (11 h) de igual concentração. Nos testes de viabilidade celular não houve mudança significativa no perfil da citotoxicidade induzida por DBC, encapsulada ou não. Portanto, nesse trabalho reportamos o preparo e caracterização de uma formulação lipossomal de liberação sustentada para DBC, com alta eficiência de encapsulação e capaz de promover um significativo aumento (ca. 2,5 vezes) no tempo de analgesia in vivoAbstract: Local anesthetics (LA) are used in medical and dental procedures to decrease pain sensation. However, LA are small molecules that easily diffuse in the site of action, restricting the duration of anesthesia. Dibucaine (DBC) is an amino-amide local anesthetic, which major use as a topical agent. This study aimed the development of a new drug delivery system for dibucaine based on liposomes with transmembrane ion-gradient, for parenteral drug administration. First, an analytical methodology for DBC quantification through high performance liquid chromatography was determined. Then, large unilamellar (LUV) and multivesicular (MLVV) liposome formulations were prepared with internal ionic-gradients for the encapsulation of DBC (0.012 %). The formulations were characterized regarding their encapsulation efficiency (%EE), zeta potential, average size and polydispersity. The zeta potential of all formulations was always negative (ca. -30 mV). Sulfate-containing LUV formulations (LUVDBC5.5 sulfin/7.4out) showed 62.6% ± 4.3 EE, which was significantly higher than all other formulations. LUV presented an average size of 500 nm and proved to be less polidisperse and more stable than LMVV, being selected for further morphological (by transmission electron microscopy), storage chemical stability, in vitro release, cell viability and in vivo analgesic tests. Micrographs proved that LUVs were spherical and unilamellar shape. No formulation showed lipid peroxidation level higher than 0.5 % of total lipids during 150 days of storage at 4oC. LUVDBC5.5 sulfin/7.4out presented increased in vitro sustained release profile and significant increase in sensory block duration (27 h) in vivo, than a equivalent DBC solution (11 h). No changes in the cytotoxicity effect of the anesthetic were observed for DBC free or encapsulated. Therefore, this study reports the successful preparation and characterization of a liposomal formulation for the sustained release of DBC with high encapsulation efficiency and that is able to significantly prolong (ca. 2.5 times) the duration of analgesia in vivoMestradoFármacos, Medicamentos e Insumos para SaúdeMestra em Ciência

Development of egg Pc/cholesterol/α-tocopherol liposomes with ionic gradients to deliver ropivacaine

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULORopivacaine (RVC) is an aminoamide local anesthetic widely used in surgical procedures. Studies with RVC encapsulated in liposomes and complexed in cyclodextrins have shown good results, but in order to use RVC for lengthy procedures and during the postoperative period, a still more prolonged anesthetic effect is required. This study therefore aimed to provide extended RVC release and increased upload using modified liposomes. Three types of vesicles were studied: (i) large multilamellar vesicle (LMV), (ii) large multivesicular vesicle (LMVV) and (iii) large unilamellar vesicle (LUV), prepared with egg phosphatidylcholine/cholesterol/α-tocopherol (4:3:0.07 mol%) at pH 7.4. Ionic gradient liposomes (inside: pH 5.5, pH 5.5 + (NH4)2SO4 and pH 7.4 + (NH4)2SO4) were prepared and showed improved RVC loading, compared to conventional liposomes (inside: pH 7.4). An high-performance liquid chromatography analytical method was validated for RVC quantification. The liposomes were characterized in terms of their size, zeta potential, polydispersion, morphology, RVC encapsulation efficiency (EE(%)) and in vitro RVC release. LMVV liposomes provided better performance than LMV or LUV. The best formulations were prepared using pH 5.5 (LMVV 5.5in) or pH 7.4 with 250 mM (NH4)2SO4 in the inner aqueous core (LMVV 7.4in + ammonium sulfate), enabling encapsulation of as much as 2% RVC, with high uptake (EE(%) ∼70%) and sustained release (∼25 h). The encapsulation of RVC in ionic gradient liposomes significantly extended the duration of release of the anesthetic, showing that this strategy could be a viable means of promoting longer-term anesthesia during surgical procedures and during the postoperative period.Ropivacaine (RVC) is an aminoamide local anesthetic widely used in surgical procedures. Studies with RVC encapsulated in liposomes and complexed in cyclodextrins have shown good results, but in order to use RVC for lengthy procedures and during the postoperative period, a still more prolonged anesthetic effect is required. This study therefore aimed to provide extended RVC release and increased upload using modified liposomes. Three types of vesicles were studied: (i) large multilamellar vesicle (LMV), (ii) large multivesicular vesicle (LMVV) and (iii) large unilamellar vesicle (LUV), prepared with egg phosphatidylcholine/cholesterol/α-tocopherol (4:3:0.07 mol%) at pH 7.4. Ionic gradient liposomes (inside: pH 5.5, pH 5.5 + (NH4)2SO4 and pH 7.4 + (NH4)2SO4) were prepared and showed improved RVC loading, compared to conventional liposomes (inside: pH 7.4). An high-performance liquid chromatography analytical method was validated for RVC quantification. The liposomes were characterized in terms of their size, zeta potential, polydispersion, morphology, RVC encapsulation efficiency (EE(%)) and in vitro RVC release. LMVV liposomes provided better performance than LMV or LUV. The best formulations were prepared using pH 5.5 (LMVV 5.5in) or pH 7.4 with 250 mM (NH4)2SO4 in the inner aqueous core (LMVV 7.4in + ammonium sulfate), enabling encapsulation of as much as 2% RVC, with high uptake (EE(%) ∼70%) and sustained release (∼25 h). The encapsulation of RVC in ionic gradient liposomes significantly extended the duration of release of the anesthetic, showing that this strategy could be a viable means of promoting longer-term anesthesia during surgical procedures and during the postoperative period261110FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2011/21735-

Rational design of polymer-lipid nanoparticles for docetaxel delivery

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCONSEJO NACIONAL DE INVESTIGACIONES CIENTIFICAS Y TECNICAS (ARGENTINA)In this work, a stable nanocarrier for the anti-cancer drug docetaxel was rational designed. The nanocarrier was developed based on the solid lipid nanoparticle preparation process aiming to minimize the total amount of excipients used in the final formul1755664FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCONSEJO NACIONAL DE INVESTIGACIONES CIENTIFICAS Y TECNICAS (ARGENTINA)FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCONSEJO NACIONAL DE INVESTIGACIONES CIENTIFICAS Y TECNICAS (ARGENTINA)14/25372-014/14457-50131-201

Rational design of polymer-lipid nanoparticles for docetaxel delivery

In this work, a stable nanocarrier for the anti-cancer drug docetaxel was rational designed. The nanocarrier was developed based on the solid lipid nanoparticle preparation process aiming to minimize the total amount of excipients used in the final formulations. A particular interest was put on the effects of the polymers in the final composition. In this direction, two poloxoamers -Pluronic F127 and F68- were selected. Some poloxamers are well known to be inhibitors of the P-glycoprotein efflux pump. Additionally, their poly-ethylene-oxide blocks can help them to escape the immune system, making the poloxamers appealing to be present in a nanoparticle designed for the treatment of cancer. Within this context, a factorial experiment design was used to achieve the most suitable formulations, and also to identify the effects of each component on the final (optimized) systems. Two final formulations were chosen with sizes 90%) and its release dynamics from formulations were measured, showing that the polymer-lipid nanoparticle is suitable as a drug delivery system for the treatment of cancer.Fil: Albano, Juan Manuel Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Ribeiro, Lígia Nunes de Morais. Universidade Estadual de Campinas; BrasilFil: Couto, Verônica Muniz. Universidade Estadual de Campinas; BrasilFil: Barbosa Messias, Mariana. Universidade Estadual de Campinas; BrasilFil: Rodrigues da Silva, Gustavo Henrique. Universidade Estadual de Campinas; BrasilFil: Breitkreitz, Márcia Cristina. Universidade Estadual de Campinas; BrasilFil: de Paula, Eneida. Universidade Estadual de Campinas; BrasilFil: Pickholz, Mónica Andrea. Universidade Estadual de Campinas; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentin

Brazilian Flora 2020: Leveraging the power of a collaborative scientific network

International audienceThe shortage of reliable primary taxonomic data limits the description of biological taxa and the understanding of biodiversity patterns and processes, complicating biogeographical, ecological, and evolutionary studies. This deficit creates a significant taxonomic impediment to biodiversity research and conservation planning. The taxonomic impediment and the biodiversity crisis are widely recognized, highlighting the urgent need for reliable taxonomic data. Over the past decade, numerous countries worldwide have devoted considerable effort to Target 1 of the Global Strategy for Plant Conservation (GSPC), which called for the preparation of a working list of all known plant species by 2010 and an online world Flora by 2020. Brazil is a megadiverse country, home to more of the world's known plant species than any other country. Despite that, Flora Brasiliensis, concluded in 1906, was the last comprehensive treatment of the Brazilian flora. The lack of accurate estimates of the number of species of algae, fungi, and plants occurring in Brazil contributes to the prevailing taxonomic impediment and delays progress towards the GSPC targets. Over the past 12 years, a legion of taxonomists motivated to meet Target 1 of the GSPC, worked together to gather and integrate knowledge on the algal, plant, and fungal diversity of Brazil. Overall, a team of about 980 taxonomists joined efforts in a highly collaborative project that used cybertaxonomy to prepare an updated Flora of Brazil, showing the power of scientific collaboration to reach ambitious goals. This paper presents an overview of the Brazilian Flora 2020 and provides taxonomic and spatial updates on the algae, fungi, and plants found in one of the world's most biodiverse countries. We further identify collection gaps and summarize future goals that extend beyond 2020. Our results show that Brazil is home to 46,975 native species of algae, fungi, and plants, of which 19,669 are endemic to the country. The data compiled to date suggests that the Atlantic Rainforest might be the most diverse Brazilian domain for all plant groups except gymnosperms, which are most diverse in the Amazon. However, scientific knowledge of Brazilian diversity is still unequally distributed, with the Atlantic Rainforest and the Cerrado being the most intensively sampled and studied biomes in the country. In times of “scientific reductionism”, with botanical and mycological sciences suffering pervasive depreciation in recent decades, the first online Flora of Brazil 2020 significantly enhanced the quality and quantity of taxonomic data available for algae, fungi, and plants from Brazil. This project also made all the information freely available online, providing a firm foundation for future research and for the management, conservation, and sustainable use of the Brazilian funga and flora