O presente trabalho é composto por 4 capítulos distintos. No primeiro intitulado \"Ciclodextrinas: tecnologia para melhoria da solubilidade de fármacos pouco solúveis\" aborda-se uma revisão das ciclodextrinas e de seus derivados, como também a formação dos complexos de inclusão. No segundo capítulo, \"Obtenção e caracterização de complexos ternários de saquinavir, β-ciclodextrina e polivinilpirrolidona\", foram avaliadas condições que poderiam influenciar na obtenção dos complexos, tais como: tempo de agitação na formação dos complexos, proporção equimolar de fármaco e ciclodextrina, solvente para solubilização do fármaco e porcentagem de polímero hidrossolúvel, concluindo-se que as melhores condições para obtenção dos complexos são 48 horas de agitação, razão equimolar 1:2 e 1:4, água para solubilização do fármaco e acréscimo de1% de polímero ao sistema. O terceiro capítulo, \"Influência do método de secagem na obtenção de complexos ternários de saquinavir\", teve o objetivo de obter e caracterizar complexos ternários de saquinavir, β-ciclodextrina e polivinilpirrolidona pelos métodos de secagem em estufa e liofilização, bem como, comparar a influência dos métodos de obtenção na solubilidade do fármaco, chegando-se ao aumento de 44 vezes da solubilidade do fármaco quando na forma de complexo SAQ:βCD:PVP de proporção equimolar 1:2 obtido por liofilização. O quarto, \"Perfil de dissolução de cápsulas e comprimidos contendo complexos ternários de saquinavir, ciclodextrina e polivinilpirrolidona\", teve o objetivo de comparar o perfil de dissolução de cápsulas de gelatina dura e comprimidos contendo sistemas ternários SAQ:βCD:PVP, obtidos pelos métodos de secagem em estufa e liofilização e o produto referência (Fortovase®). Os resultados indicam que os perfis de dissolução das formas farmacêuticas contendo complexos liofilizados apresentaram melhores resultados de eficiência de dissolução e que as cápsulas liberam mais prontamente o ativo que os comprimidos. / The present study has 4 captions. The first is called \"Cyclodextrins: Technology to improve the solubility of the little soluble drugs\" show a review of the cyclodextrins and its derivates, as well the formations of inclusion complexes. At the second caption, \"Preparation and characterization of the ternary complexes of the saquinavir, &#946-cyclodextrin and polyvinylpyrrolidone\", were evaluated some conditions that could influence in an obtention of the complexes like stirring time at the formation of the complexes, equimolar ratio of the drug and cyclodextrin, solvent for the solubility of the drug and percentage of the hydrosoluble polymer concluding that the best conditions for the formation of the complexes are: 48 hours of the stirring, equimolar ratio 1:2 and 1:4, water to solubilize the drug and addition of 1% of the polymer in the system. The third caption, \"Influence of the drying method at the formation of the ternary complexes of the saquinavir\", had the aim to prepare and characterize ternary complexes of the saquinavir, &#946-cyclodextrin and polyvinylpyrrolidone by the method of drying in equipment and lyophilization, as well to compare the influence of the methods of the obtention at the solubility of the drug, reaching the increasing of 44 times the solubility of the drug when at the form of the ternary inclusion complex (SAQ:βCD:PVP) at the equimolar ratio 1:2 obtained by lyophilization. The fourth and the last caption, \"Dissolution rate of the capsules and tablets of ternary complex of the saquinavir, cyclodextrin and polyvinylpyrrolidone, had the aim to compare the dissolution profile of capsules of the hard gelatine and tablets of ternary complexes SAQ:βCD:PVP obtained by the method of drying in equipment and lyophilization and the reference product (Fortovase®). The results indicate that the dissolution profiles of the pharmaceutical forms of lyophilized complexes show better results of the efficience of the dissolution and that the capsules liberate more efficient the active than the tablets.

Referat: Background: Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease of humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. However, the available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to the probable similarities in cell metabolism among tumor and trypanosoma cells, some of the current registered drugs against HAT were derived from cancer chemotherapeutic research. Here too, for the first time, we have demonstrated that the simple ester, ethyl pyruvate, comprises such properties. On the other hand initial studies have confirmed the efficacy of protease inhibitors in treatment of Trypanosoma cruzi, Plasmodium falciparum and Leishmania major. However, studies on efficacy and specific proteases inhibition using HIV-1 protease inhibitors on T. brucei cells remain untouched. Methodology/Principal findings: The current study covers efficacy and corresponding target evaluation of ethyl pyruvate and HIV-1 protease inhibitors (ritonavir and saquinavir) on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, zymography, phase contrast microscopic video imaging and ex vivo drug toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki=3.0±0.29 mM). The potential of this compound as an anti-trypanosomal drug is also strengthened by its fast acting property, killing cells within three hours post exposure. This was demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, this drug produced minimal side effects in human erythrocytes and is known to easily cross the blood-brain-barrier (BBB) which makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug resistance tests indicate irreversible killing of cells and a low chance of drug resistance development under applied experimental conditions. In addition to ethyl pyruvate our experimental study on HIV-1 protease inhibitors showed that both ritonavir (RTV) (IC50=12.23 µM) and saquinavir (SQV) (IC50=11.49 µM) effectively inhibited T. brucei cells proliferation. The major proteases identified in these cells were the cysteine- (~29kDa Mr) and metallo- (~66kDa Mr) proteases. Their proteolytic activity was, however, not hampered by either of these two protease inhibitors. Conclusion/Significance: Our results present ethyl pyruvate as a safe and fast acting drug. Hence, because of its predefined property to easily cross the BBB, it can probably be a new candidate agent to treat the heamolymphatic as well as neurological stages of sleeping sickness. Similarly, HIV-1 protease inhibitors, SQV and RTV, exhibited their antitrypanosomal potential but require further anlysis to identify their specific targets.:Bibliographic description ii Acronyms iii 1. Introduction 1 1.1. Disease background 1 1.2. Epidemiological distribution and disease transmission dynamics 1 1.3. Biology and life cycle of the trypanosomatidea 3 1.4. Public health significance 4 1.5. Clinical stages and disease progression 5 1.6. Current challenges of disease control 6 1.7. Current drugs and their clinical applications 9 1.8. Targets for drug discovery 12 1.8.1. Energy metabolism 12 1.8.2. Proteolysis 17 1.9. Ethyl pyruvate 18 1.10. HIV-1 Protease Inhibitors 21 2. Aim of the study 22 3. Materials and Methods 24 4. Results 31 5. Discussion 45 6. Conclusion 53 7. Supporting information 54 8. Summary 56 9. References 62 Erklärung über die eigenständige Abfassung der Arbeit 77 Curriculum vitae 78 Publications and Presentations 81 Acknowledgement 83

HIV-1 protease (PR) inhibitors (PIs) are important anti-HIV drugs for the treatment of AIDS and have shown great success in reducing mortality and prolonging the life of HIV-infected individuals. However, the rapid development of drug resistance is one of the major factors causing the reduced effectiveness of PIs. Consequently, various drug resistant mutants of HIV-1 PR have been extensively studied to gain insight into the mechanisms of drug resistance. In this study, the crystal structures, dimer stabilities, and kinetics data have been analyzed for wild type PR and over 10 resistant mutants including PRL24I, PRI32V, PRM46L, PRG48V, PRI50V, PRF53L, PRI54V, PRI54M, PRG73S and PRL90M. These mutations lie in varied structural regions of PR: adjacent to the active site, in the inhibitor binding site, the flap or at protein surface. The enzymatic activity and inhibition were altered in mutant PR to various degrees. Crystal structures of the mutants complexed with a substrate analog inhibitor or drugs indinavir, saquinavir and darunavir were determined at resolutions of 0.84 – 1.50 Å. Each mutant revealed distinct structural changes, which are usually located at the mutated residue, the flap and inhibitor binding sites. Moreover, darunavir was shown to bind to PR at a new site on the flap surface in PRI32V and PRM46L. The existence of this additional inhibitor binding site may explain the high effectiveness of darunavir on drug resistant mutants. Moreover, the unliganded structure PRF53L had a wider separation at the tips of the flaps than in unliganded wild type PR. The absence of flap interactions in PRF53L suggests a novel mechanism for drug resistance. Therefore, this study enhanced our understanding of the role of individual residues in the development of drug resistance and the structural basis of drug resistance mechanisms. Atomic resolution crystal structures are valuable for the design of more potent protease inhibitors to overcome the drug resistance problem.

HIV-1 protease is the most effective target for drugs to treat AIDS, however, the long-term therapeutic efficiency is restricted by the rapid development of drug resistant variants. To better understand the molecular basis of drug resistance, crystallographic and kinetic studies were applied to wild-type HIV-1 protease (PR) and drug-resistant mutants, PRV82A, and PRI84V, in complex with substrate analogues, the current drug saquinavir and the new inhibitor UIC-94017 (TMC-114). UIC-94017 was also studied with mutants PRD30N and PRI50V. The drug-resistant mutations V82A, I84V, D30N and I50V participate in substrate binding. Eighteen crystal structures were refined at resolutions of 0.97-1.60A. The high accuracy of the atomic resolution crystal structures helps understand the reaction mechanism of HIV-1 PR. Different binding modes are observed for different types of inhibitors. The substrate analogs have more extended interactions with PR subsites up to S5-S5', while the clinical inhibitors maximize the contacts within S2-S2'. Hydrophobic interactions are the major force for saquinavir binding since it was designed with enhanced hydrophobic groups based on substrate side-chains. In contrast, the new clinical inhibitor UIC-94017 was designed to mimic the hydrogen bonds between substrates and PR. UIC-94017 forms polar interactions with the PR main-chain atoms of Asp29/30, which have been proposed to be critical for its potency against resistant HIV. The mutants showed different structural and kinetic effects, depending on the inhibitor and location of the mutations. The observed structural changes were consistent with the relative inhibition data. Both PRI84V and PRI50V lost favorable hydrophobic interactions with inhibitor compared with PR. Similarly, in PRD30N the UIC-94017 had a water-mediated interaction with the side-chain of Asn30 rather than the direct interaction observed in PR. However, PRV82A compensated for the mutation by shifts of the backbone of Ala82. Furthermore, the complexes of PRV82A showed smaller shifts relative to PR, but more movement of the peptide analog, compared to complexes with clinical inhibitors. The structures suggest that substrate analogs have more flexibility than the drugs to accommodate the structural changes caused by mutation, which may explain how HIV can develop drug resistance while retaining the ability of PR to hydrolyze natural substrates.

BACKGROUND:Active HIV infection within the central nervous system (CNS) is confined primarily to microglia. The glial cell compartment acts as a viral reservoir behind the blood-brain barrier. It provides an additional roadblock to effective pharmacological treatment via expression of multiple drug efflux transporters, including P-glycoprotein. HIV/AIDS patients frequently suffer bacterial and viral co-infections, leading to deregulation of glial cell function and release of pro-inflammatory mediators including cytokines, chemokines, and nitric oxide.METHODS:To better define the role of inflammation in decreased HIV drug accumulation into CNS targets, accumulation of the antiretroviral saquinavir was examined in purified cultures of rodent microglia exposed to the prototypical inflammatory mediator lipopolysaccharide (LPS).RESULTS:3H]-Saquinavir accumulation by microglia was rapid, and was increased up to two-fold in the presence of the specific P-glycoprotein inhibitor, PSC833. After six or 24 hours of exposure to 10 ng/ml LPS, saquinavir accumulation was decreased by up to 45%. LPS did not directly inhibit saquinavir transport, and did not affect P-glycoprotein protein expression. LPS exposure did not alter RNA and/or protein expression of other transporters including multidrug resistance-associated protein 1 and several solute carrier uptake transporters.CONCLUSIONS:The decrease in saquinavir accumulation in microglia following treatment with LPS is likely multi-factorial, since drug accumulation was attenuated by inhibitors of NF-kappabeta and the MEK1/2 pathway in the microglia cell line HAPI, and in primary microglia cultures from toll-like receptor 4 deficient mice. These data provide new pharmacological insights into why microglia act as a difficult-to-treat viral sanctuary site.

α-Tocopherol has a well known antioxidant action but is also considered likely to exert significant non-antioxidant effects in cell membranes. Due to its lipophilic nature α-tocopherol inserts into biological membranes where it influences the organisation of the component lipids and may therefore influence biophysical parameters including the membrane dipole potential. The dipole potential has been demonstrated to modulate the function of several membrane associated proteins and perturbation of this physical parameter by α-tocopherol may prove to be a significant non-antioxidant mechanism underlying several of its cellular effects. This study investigates the influence of α-tocopherol, and the non-antioxidant structural analogue α-tocopherol succinate, on the membrane dipole potential employing fluorescence spectroscopy techniques with the dipole potential sensitive probe Di-8-ANEPPS. Similar techniques are utilised with the surface potential sensitive probe FPE to investigate the interaction of the charged α-tocopherol succinate molecule with membranes. α-Tocopherol and α-tocopherol succinate are shown to decrease the dipole potential of egg-phosphatidylcholine vesicles and Jurkat T-lymphocyte cell membranes. This effect is placed in the context of the significant influence of membrane cholesterol oxidation on the dipole potential. 7-ketocholesterol, an oxidised form of cholesterol, significantly influences several cellular processes and is thought to mediate these effects, in part, through its physical effects on the cell membrane. These include altering the composition, and therefore biophysical properties, of rafts; structures which are considered to support the function of a host of membrane proteins. This study attempts to correlate the effect of 7-ketocholesterol on the dipole potential of microdomains with the influence of the oxysterol on the function of two microdomains associated receptors: P-glycoprotein and the insulin receptor, assessed by determining the extent of ligand binding using flow fluorocytometry. α-Tocopherol has been suggested to inhibit the raft-mediated effects of 7-ketocholesterol and the influence of this molecule on the effect of 7-ketocholesterol on the dipole potential are investigated as a potential mechanism for this inhibition. It is hypothesized that α-tocopherols may protect against the deleterious effects of cholesterol oxidation in cell membranes by excluding 7-ketocholesterol from specific microdomains, of which rafts are a subset, acting to preserve their dipole potential and maintain the function of the proteins they support. However, where significant cholesterol oxidation has previously occured the concurrent changes in the microdomain landscape of the membrane is suggested to prevent α-tocopherol succinate from eliciting this protective effect.