Objective: The aim of this study was to investigate inherent in vitro permeability, metabolism, and cytotoxicity of idebenone - an active used to protect skin as an anti-aging agent -and compare it to idebenone linoleate. Methods: Idebenone and idebenone linoleate were investigated in pig ear skin and melanoma (B16: F10 mouse) cells. Diffusion experiments were conducted at 37 °C (bath temperature) using Franz diffusion cells. Authentic metabolite samples were synthetically prepared. Samples were analyzed using liquid chromatography-mass spectrometry/mass spectrometry. Cell viability was determined via the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Results: Idebenone was shown to permeate across viable porcine ear tissue; there was no evidence that idebenone linoleate permeated across porcine ear tissue after 4 h. Idebenone was metabolized to idebenone acid in both pig ear and mouse melanocytes; only minor idebenone linoleate metabolism was observed. Idebenone displayed delayed in vitro toxicity (via MTT assay) in melanocytes, while idebenone linoleate displayed no such in vitro toxicity. Conclusions: The in vitro metabolism and cytotoxicity results suggest that metabolic activation of idebenone is the likely culprit that activates the skin irritation mechanism via idebenone in vivo usage. An idebenone ester (e.g. idebenone linoleate) appears to provide a superior in vitro safety profile over idebenone.

Dysfunction within complex I (CI) of the mitochondrial electron transport system has been implicated in a number of disease states ranging from cardiovascular diseases to neuro-ophthalmic indications. Herein, we provide three novel approaches to model and study the impacts of injury on the function of CI. Cardiovascular ischemia/reperfusion (I/R) injury has long been recognized as a leading contributor to CI dysfunction. Aside from the physical injury that occurs in the tissue during the ischemic period, the production of high levels of reactive oxygen species (ROS) upon reperfusion, led by reverse electron transport (RET) from CI, causes significant damage to the cell. With over 700,000 people in the US set to experience an ischemic cardiac event annually, the need for a pharmacological intervention is paramount. Unfortunately, current pharmacological approaches to treat I/R related injury are limited and the ones that have shown efficacy have often done so with mixed results. Among the current approaches to treat I/R injury antioxidants have shown some promise to help preserve mitochondrial function and assuage tissue death. The studies described herein have provided new, more physiologically matched, methods for assessing the impact of potential therapeutic interventions in I/R injury. With these methods we evaluated the efficacy of the coenzyme-Q derivative idebenone, a proposed antioxidant. Surprisingly, in both chemically induced models of I/R and I/R in the intact heart, we see no antioxidant-based mechanism for rescue. The mechanistic insight we gained from these models of I/R injury directed us to further examine CI dysfunction in greater detail. Through the use of two cutting edge genetic engineering approaches, CRISPR/Cas9 and Artificial Site-specific RNA Endonucleases (ASRE), we have been able to directly edit the mitochondria to accurately model CI dysfunction in disease. The use of these genetic engineering technologies have provided first in class methods for modeling three unique mitochondrial diseases. The culmination of these projects has provided tremendous insight into the role of CI in disease and have taken a significant step towards elucidating potential therapeutic avenues for targeting decrements in mitochondrial function. / Doctor of Philosophy / Within the mitochondria, “the powerhouse of the cell,” exists a series of five enzyme complexes that produce 90% of the energy for our cells need to function. The largest of these enzymes, complex I (CI), plays an important role in ensuring proper mitochondrial function. Injury to CI contributes to a number of diseases, but surprisingly few options exist to treat complex I. One of the most prevalent forms of CI dysfunction can be seen in ischemia/ reperfusion injury, a form of which is most commonly recognized as a heart attack. Surprisingly, the American Heart Association reports that in the next year over 700,000 people in the US will suffer from an ischemic event. With such a profound impact on the population, the need for new therapeutic developments is extremely high. Some current therapeutic approaches have been shown to be effective at treating cardiac dysfunction, but few address the dysfunction that occurs in the mitochondria. Here we test both a method for modeling these ischemia/reperfusion-based injuries and a potential therapeutic for treating these injuries within the context of CI dysfunction. We further evaluate CI dysfunction by using both established genetic engineering approaches as well as a completely new method to model CI disease. Through the use of two cutting edge genetic engineering approaches, we have been able to directly edit components of the mitochondria to accurately model CI dysfunction in disease. The use of these genetic engineering technologies have provided a first-in-class method for modeling three unique mitochondrial diseases. The culmination of these projects has provided tremendous insight into the role of CI in disease and have taken a significant step towards elucidating potential therapeutic avenues for targeting decrements in mitochondrial function.

The main focus of this project was to optimize a protocol for small molecule ligand co-purification from an in-vivo tissue source. For this purpose, I employed a transgenic zebrafish line called the pLT-gypsy, which expresses a fusion protein containing a tagged-NR LBD (Tiefenbach et al., 2010). The particular line I used to optimize the ligand identification protocol is the pLT-PPARγ zebrafish line, which expresses the tagged-PPARγ receptor's LBD (also called PPARγ-fusion protein). By using rosiglitazone (a known PPARγ ligand) as a positive control, I managed to optimize a protocol to purify the PPARγ-fusion protein and identify the co-purified ligand by mass spectrometry. This protocol can be used to identify the physiological/endogenous ligand for the PPARγ receptor as well as other orphan NRs. Compared to previous methods of ligand identification, this method allows for the identification of the ligand from the tissues where it is functional.

The main focus of this project was to optimize a protocol for small molecule ligand co-purification from an in-vivo tissue source. For this purpose, I employed a transgenic zebrafish line called the pLT-gypsy, which expresses a fusion protein containing a tagged-NR LBD (Tiefenbach et al., 2010). The particular line I used to optimize the ligand identification protocol is the pLT-PPARγ zebrafish line, which expresses the tagged-PPARγ receptor's LBD (also called PPARγ-fusion protein). By using rosiglitazone (a known PPARγ ligand) as a positive control, I managed to optimize a protocol to purify the PPARγ-fusion protein and identify the co-purified ligand by mass spectrometry. This protocol can be used to identify the physiological/endogenous ligand for the PPARγ receptor as well as other orphan NRs. Compared to previous methods of ligand identification, this method allows for the identification of the ligand from the tissues where it is functional.

Nanopartículas lipídicas têm sido desenvolvidas para aplicação tópica de fármacos e ativos cosméticos. Neste trabalho, foi proposta a primeira aplicação de um lipídeo natural não-refinado biodegradável e biocompatível - manteiga de cupuaçu (Theobroma grandiflorum) - para a preparação de nanopartículas lipídicas, as quais foram denominadas teosferas. As teosferas foram preparadas por emulsificação-evaporação do solvente (EES) e por homogeneização à alta pressão (HAP), apresentando tamanho nanométrico e distribuição granulométrica estreita quando preparadas por ambos os métodos. O trabalho teve continuidade com a preparação de teosferas pelo método de EES utilizando manteiga de cupuaçu ou sua mistura com óleo de castanha do Brasil (Bertholletia excelsa) - também derivado da biodiversidade Amazônica - visando a incorporação de antioxidantes. Idebenona (IDB) foi selecionada por sua conhecida ação antioxidante e pela sua utilização em formulações cosméticas antienvelhecimento. IDB foi incorporada nas teosferas com eficiência de encapsulação superior a 99%, sendo que os estudos de liberação in vitro mostraram que a liberação de IDB a partir das teosferas foi mais lenta em comparação à IDB livre. Estes experimentos foram capazes ainda de demonstrar as características elásticas das teosferas. Além disso, foi evidenciada in vitro a atividade antioxidante superior das teosferas contendo IDB em relação ao ativo livre. Visando possibilitar a aplicação tópica de teosferas contendo IDB, em um trabalho subseqüente, suspensões de teosferas preparadas por HAP foram incorporadas em géis hidrofílicos. As formulações apresentaram características pseudoplásticas e demonstraram efeito oclusivo in vitro, o qual foi dependente da composição dos colóides. Finalmente, os estudos de permeação in vitro utilizando pele humana demonstraram que teosferas e nanocápsulas de núcleo lipídico, utilizadas neste estudo de modo comparativo, modificaram a permeção da IDB, permitindo a acumulação do ativo nas camadas superficiais da pele. / Lipid nanoparticles have been developed for administration of active substances to the skin, both for pharmaceutical and cosmetic uses. In the present work, we proposed the first use of a none-refined natural biodegradable and biocompatible lipid – Cupuaçu seed butter (Theobroma grandiflorum) – for the preparation of lipid nanoparticles, which were called theospheres. Theospheres were prepared by emulsification-solvent evaporation (ESE) and by high pressure homogenization technique (HPH), presenting size in nanometrical range and narrow particle size distribution for both methods. Taking these results into account, the next step of this work was the preparation of theospheres by ESE method using Cupuaçu seed butter with or without Brazil nut (Bertholletia excelsa) seed oil - another ingredient derived from an Amazonian fruit - intending the encapsulation of an antioxidant. Idebenone (IDB) has been selected due to its known antioxidant action and because it has been used in antiaging cosmetic formulations. IDB was incorporated in the theospheres presenting encapsulation efficiency higher than 99%. The in vitro release evaluation demonstrated that the release of IDB from theospheres was lower than that of free drug. Besides, the in vitro release study highlighted the elastic characteristics of theospheres. Additionally, IDB-loaded theospheres showed higher antioxidant activity compared to free IDB. Viewing the cutaneous administration, theosphere suspensions prepared by HPH technique were incorporated into hydrogels. The rheograms of the semi-solid formulations exhibited a non-Newtonian behavior presenting pseudoplastic characteristics. In vitro occlusion study highlighted the dependence of the occlusive effect on the lipidic composition of the theospheres. Finally, in vitro human skin permeation studies showed that theospheres and lipid-core nanocapsules, used in this study in a comparative way, changed the permeation of IDB, increasing the accumulative amount of IDB in the upper skin layer.

Nanopartículas lipídicas têm sido desenvolvidas para aplicação tópica de fármacos e ativos cosméticos. Neste trabalho, foi proposta a primeira aplicação de um lipídeo natural não-refinado biodegradável e biocompatível - manteiga de cupuaçu (Theobroma grandiflorum) - para a preparação de nanopartículas lipídicas, as quais foram denominadas teosferas. As teosferas foram preparadas por emulsificação-evaporação do solvente (EES) e por homogeneização à alta pressão (HAP), apresentando tamanho nanométrico e distribuição granulométrica estreita quando preparadas por ambos os métodos. O trabalho teve continuidade com a preparação de teosferas pelo método de EES utilizando manteiga de cupuaçu ou sua mistura com óleo de castanha do Brasil (Bertholletia excelsa) - também derivado da biodiversidade Amazônica - visando a incorporação de antioxidantes. Idebenona (IDB) foi selecionada por sua conhecida ação antioxidante e pela sua utilização em formulações cosméticas antienvelhecimento. IDB foi incorporada nas teosferas com eficiência de encapsulação superior a 99%, sendo que os estudos de liberação in vitro mostraram que a liberação de IDB a partir das teosferas foi mais lenta em comparação à IDB livre. Estes experimentos foram capazes ainda de demonstrar as características elásticas das teosferas. Além disso, foi evidenciada in vitro a atividade antioxidante superior das teosferas contendo IDB em relação ao ativo livre. Visando possibilitar a aplicação tópica de teosferas contendo IDB, em um trabalho subseqüente, suspensões de teosferas preparadas por HAP foram incorporadas em géis hidrofílicos. As formulações apresentaram características pseudoplásticas e demonstraram efeito oclusivo in vitro, o qual foi dependente da composição dos colóides. Finalmente, os estudos de permeação in vitro utilizando pele humana demonstraram que teosferas e nanocápsulas de núcleo lipídico, utilizadas neste estudo de modo comparativo, modificaram a permeção da IDB, permitindo a acumulação do ativo nas camadas superficiais da pele. / Lipid nanoparticles have been developed for administration of active substances to the skin, both for pharmaceutical and cosmetic uses. In the present work, we proposed the first use of a none-refined natural biodegradable and biocompatible lipid – Cupuaçu seed butter (Theobroma grandiflorum) – for the preparation of lipid nanoparticles, which were called theospheres. Theospheres were prepared by emulsification-solvent evaporation (ESE) and by high pressure homogenization technique (HPH), presenting size in nanometrical range and narrow particle size distribution for both methods. Taking these results into account, the next step of this work was the preparation of theospheres by ESE method using Cupuaçu seed butter with or without Brazil nut (Bertholletia excelsa) seed oil - another ingredient derived from an Amazonian fruit - intending the encapsulation of an antioxidant. Idebenone (IDB) has been selected due to its known antioxidant action and because it has been used in antiaging cosmetic formulations. IDB was incorporated in the theospheres presenting encapsulation efficiency higher than 99%. The in vitro release evaluation demonstrated that the release of IDB from theospheres was lower than that of free drug. Besides, the in vitro release study highlighted the elastic characteristics of theospheres. Additionally, IDB-loaded theospheres showed higher antioxidant activity compared to free IDB. Viewing the cutaneous administration, theosphere suspensions prepared by HPH technique were incorporated into hydrogels. The rheograms of the semi-solid formulations exhibited a non-Newtonian behavior presenting pseudoplastic characteristics. In vitro occlusion study highlighted the dependence of the occlusive effect on the lipidic composition of the theospheres. Finally, in vitro human skin permeation studies showed that theospheres and lipid-core nanocapsules, used in this study in a comparative way, changed the permeation of IDB, increasing the accumulative amount of IDB in the upper skin layer.

Idebenone is an antioxidant, a synthetic derivative of coenzyme Q10, with several applications, such as neuroprotection. However, this drug has low solubility in water, besides is irritant substance and has chemical instability. Hence, idebenone-loaded nano-organized systems have been developed, such as polymeric nanocapsules (NC). Vegetable oils containing antioxidants such as coconut oil and palm oil can be interesting for the composition of these particles. In this way, the aim of this work was to develop NC suspensions containing different oils as core (palm, coconut or medium chain triglycerides) for delivery of neuroprotective agent idebenone in order to compare the behavior of these systems concerning physico-chemical stability, photostability, controlled release, and conversion to redispersible solid dosage forms (lyophilized products). The nanocapsules were prepared by interfacial deposition of preformed polymer. As the results, it was possible to prepare poly(Ɛ-caprolactone) NC suspensions and palm oil (PO/PCL) or Eudragit® RS100 and coconut oil (OC/EUD) containing idebenone (1.0 mg/mL) with appropriate physico-chemical characteristics. Parameters such as the proportion of aqueous phase/organic phase, type of both surfactant (low HLB) and polymer influenced the optimization of these systems. For comparison, corresponding formulations were prepared with medium chain triglycerides (TCM/PCL or TCM/EUD), using the same conditions. The suspensions had an average diameter between 166 and 216nm, low polydispersity index (0.085 to 0.142), positive or negative zeta potential, depending on the characteristics of the polymer, and high encapsulation efficiency. The maintenance of average particle diameter and low polydispersity index have be verified during stability study (room temperature and exposed or not to light) for 75 days. However, the idebenone content significantly decreased in this period, with influence on the type of polymer. Thereafter, photostability study has shown that the suspensions NC OP/PCL (UVC/UVA) and TCM/PCL (UVA) were able to significantly reduce the degradation of idebenone (content: 53,7-76,1%) compared to an aqueous micellar (content: 31,2-63,1%) dispersion containing the drug. In addition, these systems were able to promoting drug controlled release (t1/2 26 h), without burst effect, showing monoexponential profile (t1/2< 3.0 h for free drug). The lyophilization of suspensions, employing trehalose as soluble carbohydrate, resulted in suitable and redispersible products (content of 96-100%, less than 3.6% moisture; 0.8-1.2 index), presenting several spherical structures, including in colloidal range, featuring the presence of NC in these dried products. In the stability study (room temperature/protection from light and moisture), it was observed that the lyophilized products were able to delay or decrease the degradation of idebenone compared to suspensions of origin, regardless of the both type of polymer (PCL/EUD) and oil (OP/OC/TCM). In conclusion, the developed systems are promising for the controlled release of neuroprotective drug idebenone. / A idebenona é um antioxidante, derivado sintético da coenzima Q10, com várias aplicações, como em neuroproteção. No entanto, este fármaco possui baixa solubilidade em água, potencial irritativo e instabilidade química, fato que tem despertado o interesse em sua associação a sistemas nano-organizados. Dentre estes, destacam-se as nanocápsulas poliméricas (NC). Óleos vegetais, contendo compostos antioxidantes, como óleo de coco e de palma, podem ser interessantes para a composição destas partículas. Neste sentido, o objetivo deste trabalho foi desenvolver suspensões de NC contendo diferentes óleos como núcleo (palma, coco ou triglicerídeos de cadeia média), almejando à veiculação do neuroprotetor idebenona, de forma a comparar o comportamento dos sistemas quanto à estabilidade físico-química, fotoestabilidade, controle de liberação, além da conversão em formas farmacêuticas sólidas redispersíveis (liofilizados). As NC foram preparadas pelo método de deposição interfacial de polímero pré-formado. Conforme os resultados, foi possível preparar suspensões de NC de poli(ε-caprolactona) e óleo de palma (OP/PCL) ou de Eudragit® RS100 e óleo de coco (OC/EUD), contendo idebenona (1,0 mg/mL), com características físico-químicas adequadas, sendo que fatores como proporção das fases aquosa/orgânica, tipo de tensoativo de baixo EHL e de polímero influenciaram a otimização dos sistemas. Para fins comparativos, formulações correspondentes foram preparadas com triglicerídeos de cadeia média (TCM/PCL ou TCM/EUD), empregando as mesmas condições. As suspensões apresentaram diâmetros médios entre 166 e 216 nm, baixos índices de polidispersão (0,085-0,142), potencial zeta positivo ou negativo, dependendo das características do polímero e elevada eficiência de encapsulamento. Quando estas suspensões foram submetidas a estudo de estabilidade, à temperatura ambiente e expostas ou não à luz, durante 75 dias, verificou-se manutenção dos diâmetros médios de partículas e baixos índices de polidispersão. Entretanto, o teor de idebenona decaiu significativamente neste período, com influência significativa do tipo de polímero. Após, estudo de fotoestabilidade demonstrou que as suspensões de NC OP/PCL (UVC/UVA) e TCM/PCL (UVA) foram capazes de reduzir significativamente a degradação da idebenona (teor remanescente: 53,7-76,1%) em comparação a uma dispersão micelar aquosa contendo o fármaco (teor remanescente: 31,2-63,1%), além de promoverem liberação controlada da mesma (t1/2 26 h), sem efeito burst, com perfil ajustado ao modelo monoexponencial (t1/2<3,0 h para fármaco livre). A liofilização das suspensões, empregando trealose, um carboidrato solúvel, resultou em produtos adequados (teor entre 96-100 %; umidade inferior a 3,6 %), redispersíveis (índice de ressuspensão entre 0,8-1,2) e com a presença de inúmeras estruturas esféricas, inclusive na faixa coloidal, caracterizando a presença das NC nestes produtos secos. No estudo de estabilidade (temperatura ambiente/proteção da luz e da umidade), observou-se que os produtos liofilizados foram capazes de retardar ou diminuir significativamente a degradação da idebenona em comparação às suspensões de origem, independentemente do tipo de polímero (PCL/EUD) e de óleo (OP/OC/TCM). Sendo assim, os sistemas desenvolvidos são promissores para a liberação controlada do neuroprotetor idebenona.

Nanopartículas lipídicas têm sido desenvolvidas para aplicação tópica de fármacos e ativos cosméticos. Neste trabalho, foi proposta a primeira aplicação de um lipídeo natural não-refinado biodegradável e biocompatível - manteiga de cupuaçu (Theobroma grandiflorum) - para a preparação de nanopartículas lipídicas, as quais foram denominadas teosferas. As teosferas foram preparadas por emulsificação-evaporação do solvente (EES) e por homogeneização à alta pressão (HAP), apresentando tamanho nanométrico e distribuição granulométrica estreita quando preparadas por ambos os métodos. O trabalho teve continuidade com a preparação de teosferas pelo método de EES utilizando manteiga de cupuaçu ou sua mistura com óleo de castanha do Brasil (Bertholletia excelsa) - também derivado da biodiversidade Amazônica - visando a incorporação de antioxidantes. Idebenona (IDB) foi selecionada por sua conhecida ação antioxidante e pela sua utilização em formulações cosméticas antienvelhecimento. IDB foi incorporada nas teosferas com eficiência de encapsulação superior a 99%, sendo que os estudos de liberação in vitro mostraram que a liberação de IDB a partir das teosferas foi mais lenta em comparação à IDB livre. Estes experimentos foram capazes ainda de demonstrar as características elásticas das teosferas. Além disso, foi evidenciada in vitro a atividade antioxidante superior das teosferas contendo IDB em relação ao ativo livre. Visando possibilitar a aplicação tópica de teosferas contendo IDB, em um trabalho subseqüente, suspensões de teosferas preparadas por HAP foram incorporadas em géis hidrofílicos. As formulações apresentaram características pseudoplásticas e demonstraram efeito oclusivo in vitro, o qual foi dependente da composição dos colóides. Finalmente, os estudos de permeação in vitro utilizando pele humana demonstraram que teosferas e nanocápsulas de núcleo lipídico, utilizadas neste estudo de modo comparativo, modificaram a permeção da IDB, permitindo a acumulação do ativo nas camadas superficiais da pele. / Lipid nanoparticles have been developed for administration of active substances to the skin, both for pharmaceutical and cosmetic uses. In the present work, we proposed the first use of a none-refined natural biodegradable and biocompatible lipid – Cupuaçu seed butter (Theobroma grandiflorum) – for the preparation of lipid nanoparticles, which were called theospheres. Theospheres were prepared by emulsification-solvent evaporation (ESE) and by high pressure homogenization technique (HPH), presenting size in nanometrical range and narrow particle size distribution for both methods. Taking these results into account, the next step of this work was the preparation of theospheres by ESE method using Cupuaçu seed butter with or without Brazil nut (Bertholletia excelsa) seed oil - another ingredient derived from an Amazonian fruit - intending the encapsulation of an antioxidant. Idebenone (IDB) has been selected due to its known antioxidant action and because it has been used in antiaging cosmetic formulations. IDB was incorporated in the theospheres presenting encapsulation efficiency higher than 99%. The in vitro release evaluation demonstrated that the release of IDB from theospheres was lower than that of free drug. Besides, the in vitro release study highlighted the elastic characteristics of theospheres. Additionally, IDB-loaded theospheres showed higher antioxidant activity compared to free IDB. Viewing the cutaneous administration, theosphere suspensions prepared by HPH technique were incorporated into hydrogels. The rheograms of the semi-solid formulations exhibited a non-Newtonian behavior presenting pseudoplastic characteristics. In vitro occlusion study highlighted the dependence of the occlusive effect on the lipidic composition of the theospheres. Finally, in vitro human skin permeation studies showed that theospheres and lipid-core nanocapsules, used in this study in a comparative way, changed the permeation of IDB, increasing the accumulative amount of IDB in the upper skin layer.