Das Ziel der vorliegenden Arbeit war die Evaluierung der Möglichkeit der Bildung von Einschlußverbindungen eines neuen Vitamin D3 Analogon mit nativem [beta]-Cyclodextrin, Dimethyl-[beta]-Cyclodextrin, [beta]-Cyclodextrin-Polymer, Maltosyl-[beta]-Cyclodextrin, Hydroxypropyl-[beta]-Cyclodextrin und Carboxyethyl-[beta]-Cyclodextrin. Die resultierenden Addukte wurden im Hinblick auf Zusammensetzung und Eigenschaften charakterisiert. Weiterhin wurden in vitro und in vivo Untersuchungen durchgeführt, um den Einfluß von [beta]-Cyclodextrinen, vor allem im Hinblick auf Retardierungseffekte, nach Einarbeitung in Salben für die Indikation Psoriasis zu interpretieren. Die Herstellung der festen Kavitate erfolgte in Abhängigkeit vom Cyclodextrin-Typ mittels Knetmethode oder Kopräzipitation. Die Assoziatbildung mit [beta]-Cyclodextrinen in Lösung führte zu einer deutlichen Verbesserung der Löslichkeit des Vitamin D3 Analogon. Zur Ermittlung von Löslichkeitsisothermen wurden von den Systemen Phasenlöslichkeitsdiagramme aufgenommen sowie Komplexstabilitätskonstanten berechnet. Zur Charakterisierung der festen dienten DSC-Untersuchungen. Die Anwesenheit von [beta]-Cyclodextrinen führte zu einer Reduktion der Freisetzungsrate der aktiven Verbindung in vitro, besonders im Falle von nativem [beta]-Cyclodextrin. Auch ein deutlich reduzierter Effekt im Hinblick auf die Epidermishyperplasie von Nacktmäusen ist in vivo demonstrierbar. Die Ergebnisse werden im Rahmen der Fragestellung eines Retardierungseffektes sowie einer besseren Hautverträglichkeit, systemischen Nebenwirkungen und lokaler Effektivität des neuen Vitamin D3 Analogon diskutiert. / The aim of the present work was to study the possibility of forming cavitates of a new Vitamin D3 analogue in native [beta]-cyclodextrin, dimethyl-[beta]-cyclodextrin, [beta]-cyclodextrin-polymer, maltosyl-[beta]-cyclodextrin, hydroxypropyl-[beta]-cyclodextrin and carboxyethyl-[beta]-cyclodextrin and to characterize the resulting adducts as regards their composition and properties. Furthermore, in vitro and in vivo studies will be conducted to examine the influence of the [beta]-cyclodextrins in view to retardation effects after incorporation in ointments chosen for the treatment of psoriasis. The solid inclusions are produced with the kneading method or by co-precipitation, depending on the type of cyclodextrin. Phase-solubility diagrams will be drawn and complex stability constants calculated. The formation of associates with the cyclodextrins leads to a substantial improvement of the solubility of the drug. DSC studies are used to characterize the solid associates. The presence of [beta]-cyclodextrins leads to a reduction of the release rate of the active substance in vitro, especially in the case of native [beta]-cyclodextrin. A much reduced effect with regard to epidermal hyperplasia of nude mice is also demonstrable in vivo. The findings are discussed within the framework of the question of retardation and better tolerance as regards skin irritation, systemic side effects and local efficacy of the Vitamin D3 analogue.

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Instituto Nacional de Controle de Qualidade em Saúde / Passados quase 16 anos após o surgimento dos medicamentos genéricos no Brasil em 1999, sua aceitação por parte dos prescritores e da população ainda não chegou a níveis alcançados por países que adotaram a política há mais tempo. A principal desconfiança da população é em relação à qualidade dos produtos devido ao baixo custo e, associações que a palavra “genérico” tem conotação em determinadas frases como sendo algo de pouca qualidade ou incompleto, que não atingiu o esperado, carregando assim uma forma pejorativa de se referir a um produto. Apesar da qualidade destes produtos ser garantida pela resolução RDC n°17/2010 que regulamenta as boas práticas para fabricação de medicamentos, o controle de qualidade pós-mercado ainda deixa a desejar. A análise laboratorial do produto, salvo por motivos de renovação de registro, só é atualmente feita quando há denúncias devido a ocorrência de muitos casos de efeitos adversos ou ineficácia a cerca de um medicamento. Nestes casos, os testes realizados são relativos ao teor, características organolépticas, microbiológicas e potência. Porém, nenhuma dessas verificações gera resultados capazes de comprovar o real comportamento dos fármacos no organismo humano. Com isso, faz-se necessária a realização de estudos que comprovem a segurança e eficácia dos medicamentos genéricos disponíveis no mercado brasileiro para que haja uma maior adesão a este programa de governo. O objetivo deste trabalho foi de desenvolver um modelo animal para testar a biodisponibilidade/ bioequivalência dos medicamentos, uma vez que esses testes são feitos em humanos e possuem alto custo de realização além das implicações éticas. Os resultados obtidos confirmam que o fato dos antibióticos não apresentarem alterações na potência não pode ser associada a eficácia do tratamento. O teste de potência não reprovou nenhuma das formulações testadas, enquanto que a avaliação da bioequivalência in vivo foi negativa para um dos medicamentos. Nossos resultados comprovam a necessidade de revisão dos modelos de controle de qualidade utilizados atualmente. E, disponibiliza uma nova e importante opção para utilização no controle de qualidade de medicamentos e, que também pode ser utilizada no desenvolvimento de novos fármacos e medicamentos. / After almost 16 years since the beginning of the Brazilian generic medicines politics in 1999, the acceptance from physicians and the general population is unlike that in other countries that have adopted them for a longer time. The main distrust of consumers regarding the quality of the product lies on its lower cost and the association the word “generic” has with some expressions that infer something of poor quality or inefficient, which gives the product pejorative character. Even though the quality of the products is guaranteed by Resolution RDC No. 17/2010, which regulates good practice for the manufacturing of drugs, post-market quality control is still poor. The verification of the product, except for registration and renewal purposes, is currently only performed when there are reports or many cases of adverse events regarding a drug. In these cases, the tests made are content, organoleptic characteristics, microbiological and potency related. However, none of these tests yields results that prove the bioavailability of the drug. Thus, it is necessary to carry out studies to prove the safety and efficacy of generic drugs available in Brazilian market to promote a greater adherence to the program. The goal of this study was to develop an animal model to test the bioavailability / bioequivalence of drugs, once these tests are made in humans and have high costs to execution and ethical questions. The results confirm the fact that antibiotics that don’t present changes in potency can’t be associated with efficacy of treatment. The in vitro test didn’t reprove any of the tested compositions, whereas the in vivo bioequivalence assessment was negative for one the drugs. Our results show the need to review quality control models currently being used. And gives another and important option for the quality control of medicines and also can be used in new drugs development and medicines.

O câncer de mama constitui o segundo tipo de câncer mais frequente no mundo e o mais comum entre as mulheres, representando uma das principais causas de morte. O tamoxifeno é um fármaco antiestrogênico utilizado para o tratamento deste tipo de câncer desde 1971 e ainda é o mais utilizado nos casos de tumores mamários que expressam receptores de estrógeno. Apesar de apresentar resultados significativamente positivos, seu efeito antiestrogênico não se restringe apenas ao sítio tumoral causando, com isso, efeitos colaterais graves que podem deixar sequelas. A proposta deste trabalho foi desenvolver sistemas de liberação nanoparticulados à base de PLA e PLA-PEG para veiculação do tamoxifeno, como uma estratégia para o potencial aumento da segurança e da eficácia deste fármaco através de um possível direcionamento passivo ao sítio de ação, devido à permeabilidade vascular aumentada destas regiões tumorais. As nanopartículas foram preparadas pela técnica de nanoprecipitação e apresentaram diâmetro médio inferior a 200 nm para a maioria das formulações. Foram avaliados três estabilizantes, o poloxamer 407, o poloxamer 188 e o polissorbato 80, este último proporcionou maior eficiência de encapsulação, 86,7% e 100%, nas nanopartículas de PLA e PLA-PEG, respectivamente. Quanto à composição das nanopartículas de PLA-PEG, o polímero utilizado inicialmente (PLA(1000)-PEG(750)) apresentou distribuição de tamanho heterogênea, perfil multimodal e alto índice de polidispersividade. Assim, este polímero foi substituído pelo PLA(5000)-PEG(1000), que apresentou distribuição de tamanho uniforme, perfil monomodal e baixo índice de polidispersividade. A caracterização por microscopia eletrônica de varredura comprovou a homogeneidade no tamanho de partícula, mostrando seu formato esférico. As análises de espectrofotometria no infravermelho e calorimetria diferencial exploratória sugeriram que não ocorreu nenhum tipo de interação ou reação entre o fármaco e os demais componentes das formulações. Dois métodos analíticos para a determinação do tamoxifeno foram validados com sucesso por CLAE e espectroscopia UV-vis. O perfil de liberação in vitro do tamoxifeno a partir das nanopartículas de PLA apresentou característica sustentada e alcançou 50% em 180 h, tendo sido totalmente liberado após 288 h. Já as nanopartículas de PLA(5000)-PEG(1000) liberaram apenas 16,9% do fármaco após 216 h. A liberação do fármaco a partir das nanopartículas foi muito mais lenta comparada ao tamoxifeno não encapsulado, evidenciando a vantagem da incorporação do fármaco em nanopartículas compostas por PLA e PLA-PEG. No estudo do perfil de concentração plasmática em ratas Wistar, não foi possível detectar o fármaco e seu principal metabólito pelo método por CLAE desenvolvido, sugerindo que os sistemas nanoparticulados tenham extravasado rapidamente para os órgãos. / Breast cancer is the second most frequent type of cancer in the world and it is the most common among women, representing a major cause of death. Tamoxifen is an antiestrogen drug used in the treatment of this type of cancer since 1971 and it is the most employed drug in the treatment of breast cancer subtypes that expresses estrogen receptors. Despite presenting significantly positive results, its antiestrogen effect is not restricted to the tumour site, causing, as consequence, severe side effects. The purpose of this work was to develop nanostructured drug delivery systems based on PLA and PLA-PEG loaded with tamoxifen, as a strategy to potentially increase the safety and efficacy of this drug through a possible passive accumulation the site of action, due to the enhanced vascular permeability of tumour sites. Nanoparticles were prepared by the nanoprecipitation technique and presented average diameter smaller than 200 nm for the majority of the formulations. Three stabilizing adjuvants were analysed, poloxamer 407, poloxamer 188 and polysorbate 80 and the last one yielded the highest encapsulation efficiency, 86.7% and 100%, for the PLA and PLA-PEG nanoparticles, respectively. Regarding the PLA-PEG nanoparticles composition, the first polymer employed was (PLA(1000)-PEG(750)), which presented heterogeneous particle size distribution, multimodal profile and high polydispersity index. So, it was replaced by PLA(5000)-PEG(1000), which exhibited uniform particle size distribution, monomodal profile and low polydispersity index. The characterization by scanning electron microscopy confirmed the homogeneity of particles size, evidencing their spherical shape. Infrared spectrophotometry and differential scanning calorimetry analysis suggested that any interaction or reaction had occurred between the drug and the other components of the formulations. Two analytical methods for tamoxifen quantification were successfully validated by HPLC and UV-vis spectroscopy. In vitro tamoxifen release profile from PLA nanoparticles presented sustained release and reached 50% in 180 h, being completely released after 288 h, whereas PLA(5000)-PEG(1000) nanoparticles released only 16.9% of tamoxifen after 216 h. Drug release from nanoparticles was much slower compared to the non-encapsulated tamoxifen, showing the advantage of nanoparticles composed of PLA and PLA-PEG. In the plasmatic concentration profile study carried out in Wistar rats, it was not possible to detect tamoxifen or its main metabolite by the HPLC method, suggesting that nanoparticles quickly extravased to organs.

O câncer de mama é a segunda principal causa de morte entre as mulheres nos países em desenvolvimento, devido ao seu alto grau de malignidade. O tratamento baseia-se, principalmente, em terapias hormonais, uma vez que as células deste tipo de tumor expressam, em sua maioria, um elevado número de receptores hormonais, responsáveis pela regulação do crescimento do mesmo. O tamoxifeno é um fármaco da classe dos moduladores seletivos de receptores de estrógeno, que atua através do antagonismo à ativação de tais receptores por este hormônio, reduzindo, assim, a taxa de crescimento celular do tecido tumoral. Embora o tratamento com tamoxifeno seja altamente efetivo, este se relaciona a severos efeitos colaterais dosedependentes. O objetivo central deste trabalho foi desenvolver sistemas micelares de longo tempo de circulação contendo tamoxifeno, preparados à base do fosfolipídeo DSPE-PEG(n), associado ou não ao derivado de vitamina E TPGS, para administração intravenosa, capazes de permitir um acúmulo maior do fármaco no sítio tumoral devido a suas dimensões nanométricas, permitindo, desta forma, a redução da dose e a consequente redução dos efeitos colaterais. A determinação da eficiência de encapsulação e a quantificação do tamoxifeno no estudo de liberação in vitro a partir dos sistemas obtidos foram realizadas por CLAE, utilizando métodos previamente validados. Os melhores resultados foram alcançados com as formulações à base de DSPE-PEG(2000) e TPGS, preparadas pelo método de evaporação do solvente, as quais apresentaram diâmetro médio inferior a 20 nm, baixo índice de polidispersividade e eficiência de encapsulação entre 70 e 95%. A análise por microscopia eletrônica de transmissão evidenciou o formato esférico e comprovou a homogeneidade do tamanho das partículas. Os sistemas foram caracterizados, ainda, por espectrofotometria no infravermelho para avaliação de possíveis interações entre os componentes das formulações. O perfil de liberação in vitro demonstrou que após 168 h, no máximo cerca de 30% do fármaco foi liberado, verificando-se que o aumento na quantidade de TPGS na formulação reduziu a porcentagem de tamoxifeno liberado. A baixa taxa de liberação in vitro sugere que a maior parte do fármaco mantenha-se no interior da estrutura micelar durante o período de permanência no sangue, favorecendo a chegada da nanoestrutura íntegra ao sítio tumoral. No estudo do perfil de concentração plasmática em ratas Wistar, não foi possível detectar o fármaco e seu principal metabólito pelo método por CLAE desenvolvido, sugerindo que os sistemas micelares tenham extravasado rapidamente para os órgãos. / Breast cancer is the second main cause of death among women in development countries due to their high malignance grade. The treatment is mainly based on hormonal therapies, once the cells of the majority of mammary tumors express a high number of hormone receptors, responsible for the tumor growth. Tamoxifen is a selective estrogen receptor modulator drug, acting through the antagonism of the activation of the estrogen receptor, reducing thus the tumor growing rate. Despite the treatment with tamoxifen is highly effective, it is related to severe dose-dependent side effects. The central objective of this work was the development of long time circulation micelles containing tamoxifen, prepared with the phospholipid DSPEPEG(n) and TPGS, a vitamin E derivative, by the method of solvent evaporation, for intravenous administration, able to allow a higher accumulation of the drug at the tumoral site due to their nanometric dimensions, leading to a reduction in the dose and consequently in the side effects. The determination of the encapsulation efficiency and the quantification of tamoxifen in the in vitro release profile study from the micellar systems were carried out by HPLC, using methods previously validated. The best results were achieved with the formulations based on DSPE-PEG(2000) and TPGS, which showed mean particle diameter less than 20 nm, low polydispersity index and encapsulation efficiency ranging from 70 to 95%. The transmition electronic microscopy pointed the spherical shape and proved the homogeneity of particle size. The systems were also characterized by infrared spectrophotometry to identify eventual interactions among the components of the formulations. The in vitro release profile study showed that after 168 h, a maximum of about 30% of tamoxifen was released, evidencing that the increase of the TPGS amount in the formulation reduced the amount of tamoxifen released. The low rate of in vitro release drug suggests that the major part of the drug will remain encapsulated during the period of blood permanence, favoring the arrival of the intact nanostructure at the tumoral site. During the evaluation of the plasmatic concentration profile, conducted with Wistar rats, it was not possible to detect neither the tamoxifen nor its main metabolite, suggesting that the intact micelles may have quickly accumulated in the organs.

Le domaine des nanosciences suscite un intérêt croissant au sein de la communauté scientifique cherchant à créer et à développer des objets sur mesure adaptés à de nombreuses applications. Depuis une vingtaine d'années, le développement de nano-objets pour leur utilisation en nanomédecine (diagnostic, thérapie, vectorisation, encapsulation et relargage contrôlé de principes actifs etc...) est en plein essor. En effet, l'avantage de travailler à l'échelle nanométrique est de pouvoir combiner au sein d'un même objet diverses propriétés et fonctionnalités complémentaires faisant de ces nano-matériaux de véritables plateformes pouvant être modulées à souhait. Ainsi, pour la conception de ces nouveaux objets, il est crucial de pouvoir contrôler finement la taille, la structure, la morphologie ainsi que la composition chimique tout en explorant l'influence de la réduction de la taille sur les propriétés physico-chimiques de ces nano-objets. En regard de ces problématiques, ce travail de thèse est dédié à la synthèse contrôlée de nanoparticules de réseaux de coordination paramagnétiques, pour leur utilisation en tant qu'agents de contraste T1 pour l'IRM. D'autre part, la synthèse de nanoparticules de type cœur-coquille permettant de faire croître de manière contrôlée des objets en solution, par l'ajout d'une coquille constituée d'un réseau de même nature ou de nature différente que les particules servant de germes, sera exploitée. Cette synthèse ouvrira la voie à la conception d'objets multimodaux, détectables par différentes techniques d'imagerie médicale, ainsi qu'à l'élaboration de nanoparticules à visée thérapeutique. Pour la mise au point de ces nanoparticules paramagnétiques, les réseaux de coordination à ponts cyanures ont été choisis et plus particulièrement les analogues du bleu de Prusse. Cette famille de matériaux présente de nombreux avantages pour la synthèse d'agents de contraste T1 pour l'IRM, de systèmes multimodaux ou encore de nano-objets alliant diagnostic et thérapie. En effet, la grande proportion d'atomes de surface, la structure tridimensionnelle rigide, la microporosité ainsi que la grande versatilité chimique de ces réseaux de coordination font de ces nanoparticules d'analogue du bleu de Prusse d'excellents candidats pour leur utilisation dans le domaine de l'imagerie médicale ainsi que de la thérapie. Au cours de ce travail de thèse, la synthèse de ces nanoparticules d'analogue du bleu de Prusse sera finement contrôlée afin d'explorer et d'optimiser les différents paramètres pouvant influencer les propriétés de ces nano-objets. Ainsi, différentes questions seront abordées au cours de cette étude afin de comprendre l'influence de la taille, de la composition chimique, de la microporosité, de l'enrobage ou encore de la localisation des ions paramagnétiques sur les propriétés des nanoparticules synthétisées, dans le but d'exalter les performances de ces nano-objets innovants. / In the last twenty years, working at the nanoscale in order to create and develop new nano-objets adapted to a large variety of applications is an important challenge among the scientific community. Particularly, tailoring nanoparticles for nanomedicine (diagnosis, therapy, drug delivery etc…) has become an attractive area for many scientific groups. Indeed, working at the nanoscale offers the possibility to combine several properties and functionalities within a single objet. To understand the effect of size reduction on the physico-chemical properties of these nano-objects, it is crucial to control precisely lots of parameters as the size, the shape, the structure and also the chemical composition of nanoparticles. This thesis is focused on the synthesis and characterization of new cyano-bridged coordination network nanoparticles as T1 contrast agents for magnetic resonance imaging. Moreover, the synthesis of core-shell nanoparticles allowing the controlled growth of nanoparticles in aqueous solution, by adding a shell composed by a different or an identical network to that of the core, will be exploited. This strategy of synthesis will lead to the design of multimodal nanoparticles as well as theragnostic nano-objets. For the elaboration of these paramagnetic nanoparticles, we chose Prussian blue analogue coordination networks that present many advantages for the synthesis of T1 contrast agents due to the high proportion of surface atoms, to the tridimensional rigid structure, to the microporosity and also to the large chemical versatility. In this work, the study and the optimization of the different physico-chemical parameters that could influence the properties and the performances of these nanoparticles as T1 contrast agents for MRI will be presented. In this way, the influence of size reduction, chemical composition, kind of coating and location of paramagnetic ions on relaxivity properties of these innovative nano-materials will be discuss.

Insulin resistance is an early marker of Type II diabetes. Excessive lipid accumulation in muscle and liver leads to insulin resistance, and lowering tissue lipids causes an enhancement of insulin action. The enzyme AMP-activated protein kinase (AMPK) is activated when cellular energy levels are compromised, such as during exercise; this enhances fuel oxidation and inhibits energy consuming processes. The hypothesis in this thesis was that activating AMPK in a lipid-induced insulin resistant state leads to tissue lipid reduction and improved insulin sensitivity. Insulin resistant high-fat fed (HF-) rats were administered 5-aminoimidazole-4-carboxamide-1-??-D-ribofuranoside (AICAR), a specific AMPK activator. During an euglycaemic hyperinsulinaemic clamp performed 24h later, HF-rats showed increased whole body, muscle and liver insulin action, independent of changes in PKB-phosphorylation. The liver had reduced triglycerides, malonyl-CoA and increased IkB-a content. A lowering of muscle malonyl-CoA was consistent with conditions favouring increased lipid utilisation. Normal, chow-fed rats also showed improved insulin action post-AICAR. Further studies showed that basal glucose uptake was not increased 24h after AICAR, suggesting that AMPK activation had caused an increase in insulin sensitivity. Diacylglycerols and triglycerides, but not ceramides, were reduced in the liver of AICAR treated HF-rats, suggesting lipid reduction as a likely mediator of enhanced liver insulin action. These lipid species were not reduced in muscle. AICAR administration to HF-rats lowered plasma glucose and fatty acids (FA) acutely, probably due to increased muscle glucose uptake and FA oxidation. Glycogen was reduced in liver and increased in muscle, suggesting glucose mobilisation from liver to muscle. Adrenergic blockade excluded the sympathetic nervous system in the acute AICAR effects. AMPK was activated in white muscle and liver of HF-rats immediately after AICAR, the same tissues that exhibited later improved insulin sensitivity. Tracer technologies used to investigate glucose and lipid fluxes showed that AMPK activation in white muscle simultaneously increased both glucose and FA uptake and their metabolism, with glucose also being stored as glycogen. The liver showed lower lipid synthesis, consistent with reduced liver lipid accumulation observed 24h post-AICAR. In conclusion, these results suggest that activation of AMPK leads to selective tissue lipid reduction and improved insulin action, and is a potential target for the treatment of insulin resistance and type II diabetes.

Insulin resistance is an early marker of Type II diabetes. Excessive lipid accumulation in muscle and liver leads to insulin resistance, and lowering tissue lipids causes an enhancement of insulin action. The enzyme AMP-activated protein kinase (AMPK) is activated when cellular energy levels are compromised, such as during exercise; this enhances fuel oxidation and inhibits energy consuming processes. The hypothesis in this thesis was that activating AMPK in a lipid-induced insulin resistant state leads to tissue lipid reduction and improved insulin sensitivity. Insulin resistant high-fat fed (HF-) rats were administered 5-aminoimidazole-4-carboxamide-1-??-D-ribofuranoside (AICAR), a specific AMPK activator. During an euglycaemic hyperinsulinaemic clamp performed 24h later, HF-rats showed increased whole body, muscle and liver insulin action, independent of changes in PKB-phosphorylation. The liver had reduced triglycerides, malonyl-CoA and increased IkB-a content. A lowering of muscle malonyl-CoA was consistent with conditions favouring increased lipid utilisation. Normal, chow-fed rats also showed improved insulin action post-AICAR. Further studies showed that basal glucose uptake was not increased 24h after AICAR, suggesting that AMPK activation had caused an increase in insulin sensitivity. Diacylglycerols and triglycerides, but not ceramides, were reduced in the liver of AICAR treated HF-rats, suggesting lipid reduction as a likely mediator of enhanced liver insulin action. These lipid species were not reduced in muscle. AICAR administration to HF-rats lowered plasma glucose and fatty acids (FA) acutely, probably due to increased muscle glucose uptake and FA oxidation. Glycogen was reduced in liver and increased in muscle, suggesting glucose mobilisation from liver to muscle. Adrenergic blockade excluded the sympathetic nervous system in the acute AICAR effects. AMPK was activated in white muscle and liver of HF-rats immediately after AICAR, the same tissues that exhibited later improved insulin sensitivity. Tracer technologies used to investigate glucose and lipid fluxes showed that AMPK activation in white muscle simultaneously increased both glucose and FA uptake and their metabolism, with glucose also being stored as glycogen. The liver showed lower lipid synthesis, consistent with reduced liver lipid accumulation observed 24h post-AICAR. In conclusion, these results suggest that activation of AMPK leads to selective tissue lipid reduction and improved insulin action, and is a potential target for the treatment of insulin resistance and type II diabetes.

Human Epidermal Growth Factor Receptor 2 (HER2) is a cell surface receptor tyrosine kinase and plays a role in the signal pathways leading to cell proliferation and differentiation. Overexpression of HER2 is found in various cancers including breast, ovarian, gastric, colon, and non-small-cell lung cancers, which makes it an attractive target for cancer therapy. Specific antibodies, peptides and small molecules are developed by scientists to bind with HER2 as therapeutical agents, dimerization inhibitors and biological makers. Among these molecules, antibodies showed excellent binding affinity and specificity toward HER2. However, uses of antibodies are limited by their high cost of production, long development time, limited ability to penetrate tumor tissue and immunogenicity. Many of these limitations are due to the high molecular weight of antibodies. Compared to antibodies, peptides and small molecule that selectively recognize HER2 have advantages in solubility, permeability and immunogenicity. So far, the design of all peptides and small molecules for binding with HER2 either utilize phage display technique or rely on computational screen of large library of millions of small molecules. These approaches all suffer from the drawbacks of tedious, labor intensive, and time consuming as well as uncertainty of outcome. In this study, it was hypothesized that a novel approach based on molecular interactions of HER2-Pertuzumab complex and Knob-Socket model can be developed to design antibody mimics for targeting HER2. All designed antibody mimics were simulated and docked with HER2 using Molecular Operating Environment (MOE) software to estimate binding energy and analyze the detail interaction map. A series of mimics were then synthesized and characterized. HER2 positive breast cancer cells MDA-MB-361 and ZR-75-1 were used in confocal microscopic and flow cytometric studies to evaluate the binding specificity of all antibody mimics to HER2 in vitro, while human embryonic kidney cell (HEK293) was used as control. After incubation with antibody mimics, high fluorescence intensities were observed on MDA-MB-361 and ZR-75-1 cells, while only background fluorescence were observed on HEK293 cells. Surface plasma resonance (SPR) studies showed that all antibody mimics bind to HER2 protein with KD value in range of 55.4 nM- 525.5 nM. Western blot technique was used to evaluate inhibition capability of antibody mimics on phosphorylation of HER2 downstream signaling Akt and MAPK pathways that were crucial for cell differentiation and survival. When treated with antibody mimics at 10µM for 24 h, more than 85% phosphorylation of Akt pathway was inhibited while phosphorylation of MAPK pathway was not affected. This finding proved that antibody mimics could bind to HER2 extracellular domain and selectively inhibit the dimerization between HER2 and HER3 to block phosphorylation of Akt pathway in a similar way as Pertuzumab. In addition, in vivo studies on tumor bearing nude mice were carried out to investigate the distribution and binding specificity of antibody mimics towards HER2 positive tumor after injecting through vein tail. Signal intensity ratio (SIR) of tumor to muscle revealed about 10-fold increase in tumor retention of HER2-PEP11 compared to the Cy7.5 carboxylic acid and Cy7.5-HER2-PEP22, which confirmed excellent in vivo binding specificity of antibody mimic HER2-PEP11 to HER2 positive tumor. In conclusion, this study demonstrated that a rational design of antibody mimics with both binding specificity and affinity towards HER2 based on the molecular interaction between Pertuzumab and HER2 and Knob-Socket model is feasible.