Made available in DSpace on 2014-06-12T16:31:09Z (GMT). No. of bitstreams: 2 arquivo6049_1.pdf: 9661385 bytes, checksum: b6f80e4ca0d817f46550e8c8614f26ea (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2004 / A introdução de novos agentes anti-retrovirais no mercado tem sido mais freqüente, fato este que evidencia a constante necessidade de obter agentes terapêuticos mais eficazes e com menores efeitos colaterais. Devido ao grande número de casos de portadores do HIV no mundo e à capacidade do vírus em sofrer mutação, nos meados da década de 90, uma nova classe de agentes anti-retrovirais foi desenvolvida, os inibidores da protease, dentre eles destacando-se o Sulfato de Indinavir. Este, tem como produto de referência no mercado o CRIXIVAN®, fabricado pela indústria farmacêutica Merck Sharp & Dohme® e disponibilizado para atender ao Programa de SIDA do Ministério da Saúde a um custo relativamente alto. Este estudo foi realizado em parceria com o Laboratório de Tecnologia dos Medicamentos do Departamento de Ciências Farmacêuticas da UFPE e o Laboratório Farmacêutico do Estado de Pernambuco (LAFEPE). Sendo assim, fez-se necessário o desenvolvimento farmacotécnico-industrial do Sulfato de Indinavir 400 mg cápsulas, o qual contemplou os estudos de pré-formulação qualitativa de excipientes, os testes de bancada e a transposição para escala industrial, buscando uma forma farmacêutica estável, produzida a baixo custo e com qualidade atendendo às Boas Práticas de Fabricação e, conseqüentemente permitindo um maior número de portadores do HIV acesso ao medicamento. Outro objetivo do estudo foi o desenvolvimento e a validação da metodologia analítica para quantificação da matéria-prima e produto acabado, por Cromatografia Líquida de Alta Eficiência (CLAE), onde foram definidos os parâmetros analíticos para o método e as especificações para os produtos em análise. Dando continuidade, foi iniciado, para as cápsulas de Sulfato de Indinavir, o estudo de estabilidade, tomando-se como base a Resolução RE n° 560, publicada em 02 de abril de 2002 pela Agência Nacional de Vigilância Sanitária (ANVISA), que preconiza a utilização de parâmetros para realização da estabilidade acelerada e de longa duração. Também foi efetuado um estudo comparativo do produto desenvolvido Sulfato de Indinavir LAFEPE® e o de referência CRIXIVAN®. Os estudos realizados com as cápsulas desenvolvidas demonstraram que eles são equivalentes entre si, sendo esta a etapa que antecede o estudo de bioequivalência para permitir a disponibilização do Sulfato de Indinavir LAFEPE como Medicamento Genérico para o mercado nacional

Metabolomics approaches have recently been used to understand the complex molecular interactions of biological systems. One popular area in which these methods are being developed is to understand the biochemical changes during abiotic and biotic stresses; for example, how a cell may respond to a drug. Since metabolites are the end products of gene expression, these can be used to indicate the result of the activities and interaction of the cell or organism with its environment. The investigation of the level and compositional changes of metabolites against metabolic stresses such as chemotherapeutic treatment (drug exposure) are required to understand more fully abiotic perturbation to biological systems. The aim of this project was to understand the metabolic effect that the anti-viral drugs indinavir and lopinavir (currently used by HIV patients) have on HPV-related cervical cancer cell lines by measuring changes in metabolism using a wide range of analytical techniques; including Fourier transform infrared (FT-IR) and Raman spectroscopies, and gas and liquid chromatography-mass spectrometry (GC and LC-MS). The analyses and interpretation of the large volumes of complex multidimensional data generated by metabolomics approaches were performed with a combination of multivariate data analysis techniques such as principal components analysis (PCA) and canonical variates analysis (CVA), as well as univariate approaches such as N-Way analysis of variance (ANOVA). By combining biochemical imaging, metabolite fingerprinting and footprinting, and metabolite profiling, with multi- and uni-variate analyses, the actions and effects of the anti-viral drugs were investigated. FT-IR spectroscopy was initially used to generate global biochemical finger- and foot-prints, and Raman spectroscopy was employed to investigate intracellular distribution of metabolites, and other cellular species, as well as the localisation of drug molecules within cells. FT-IR spectroscopy ascertained that the intra- and extra-cellular metabolomes were being directly influenced in a fashion that correlated with increasing anti-viral dosing; these effects were phenotypic rather than measurements of the drug level. Raman imaging spectroscopy indicated that the indinavir but not lopinavir was being compartmentalised within the cell nucleus, but only in HPV early protein 6 (E6) expressing cells. This observation was further confirmed by fractionation of cell samples into nuclear and cytoplasmic fractions and assessing the indinavir concentrations via LC-MS. Finally, LC-MS and GC-MS metabolite profiling were employed to investigate changes in the intracellular metabolome in response to the anti-viral compounds across a range of physiologically relevant concentrations and in the presence and absence of the E6 oncoprotein. General effects of both anti-viral compounds included the regulation of metabolites such as glutathione, octenedionoic and octadecenoic acids, which may be involved in stress related responses, reduced levels of sugars and sugar-phosphates indicating a potential arrest of glycolysis, and reduced levels of malic acid indicating potential decreased flux into the TCA cycle; all indicating that central metabolism was being reduced. Finally, LC-MS based quantification indicated that in the presence of E6, lopinavir was actively removed from the cell, whereas the indinavir intracellular concentration increased concomitantly with the level of dosing. These investigations have revealed that metabolomics approaches are an apt tool for the study of anti-viral effects within cell cultures, but improvements need to be made with respect to the major limitation of metabolite identification.

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.

With increasing environmental awareness and natural resource limitations, researchers must begin to incorporate sustainability into their process and product designs. One target for green engineering is in reaction and separation design. This is typically done in a wasteful and often toxic manner with organic solvents and lack of recycle. The following thesis discusses alternatives to these costly separations by means of ionic liquids, benign extraction, separation with carbon dioxide, and near critical water. Ionic liquids are combined with carbon dioxide to induce melting point depressions of up to 124 degrees Celsius. Using this system as a reaction medium will offer control over the reaction phases while utilizing green solvents. Benign extractions are performed on both ferulic acid and on proteins from biomass by replacing alkaline solvents and costly protein separation techniques with simple liquid-liquid extraction. This means simpler systems and less waste than from previous methods. This thesis also discusses an opportunity for more efficient separation and recycle of a pharmaceutical catalyst, Mn-Salen. Using carbon dioxide with the organic aqueous tunable solvent system, the reaction can be run homogeneously and the product and catalyst separated heterogeneously, thus creating an extremely efficient process. Lastly, near critical water is used as an extraction and reaction medium by extracting ferulic acid from Brewers Spent Grain and then catalyzing its transformation to 4-vinylguaiacol. In this manner a simple, benign process is used to turn waste into valuable chemicals. Although somewhat different, each of the studied processes strives to eliminate waste and toxicity of many commonly used reaction and separation techniques, thus creating safe and sustainable processes.

Magister Scientiae - MSc / Indinavir is a potent and well tolerated protease inhibitor drug used as a component of the highly active antiretroviral therapy (HAART) of HIV/AIDS, which results in pharmacokinetics that may be favourable or adverse. These drugs work by maintaining a plasma concentration that is sufficient to inhibit viral replication and thereby suppressing a patient’s viral load. A number of antiretroviral drugs, including indinavir, undergo metabolism that is catalysed by cytochrome P450-3A4 enzyme found in the human liver microsomes. The rate of drug metabolism influences a patient’s response to treatment as well as drug interactions that may lead to life-threatening toxic conditions, such as haemolytic anaemia, kidney failure and liver problems. Therapeutic drug monitoring (TDM) during HIV/AIDS treatment has been suggested to have a potential to reduce drug toxicity and optimise individual therapy. A fast and reliable detection technique, such as biosensing, is therefore necessary for the determination of a patient’s metabolic profile for indinavir and for appropriate dosing of the drugs. In this study biosensors developed for the determination of ARV drugs comprised of cysteamine self-assembled on a gold electrode, on which was attached 3-mercaptopropionic acid-capped palladium telluride (3-MPA-PdTe) or thioglycolic acid-capped palladium telluride (TGA-PdTe) quantum dots that are cross-linked to cytochrome P450-3A4 (CYP3A4) in the presence of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide. The quantum dots were synthesized in the presence of capping agents (3-MPA or TGA) to improve their stability, solubility and biocompatibility. The capping of PdTe quantum dots with TGA or 3-MPA was confirmed by FTIR, where the SH group absorption band disappeared from the spectra of 3-MPA-PdTe and TGA-PdTe. The particle size of the quantum dots (< 5 nm) was estimated from high resolution transmission electron microscopy (HRTEM) measurements. Optical properties of the materials were confirmed by UV-Vis spectrophotometry which produced absorption iii bands at ~320 nm that corresponded to energy band gap values of 3 eV (3.87 eV) for TGAPdTe (3-MPA-PdTe) quantum dots. The electrocatalytic properties of the quantum dots biosensor systems were studied by cyclic voltammetry (CV) for which the characteristic reduction peak at 0.75 V was used to detect the response of the biosensor to indinavir. Results for indinavir biosensor constructed with 3-MPA-SnSe quantum dots are also reported in this thesis. The three biosensors systems were very sensitive towards indinavir; and gave low limits of detection (LOD) values of 3.22, 4.3 and 6.2 ng/mL for 3-MPA-SnSe, 3-MPA-PdTe and TGA-PdTe quantum dots biosensors, respectively. The LOD values are within the ‘maximum plasma concentration’ (Cmax) value of indinavir (5 - 15 ng/mL) normally observed 8 h after drug intake.

There is a global rise in the use of herbal products in combination with allopathic medicines, while most patients do not inform their health care providers of the use of these natural products. Both pharmacodynamic and pharmacokinetic interactions between herbal products and conventional drugs must be avoided for the wellbeing of the patient. Increasing evidence from in vitro and in vivo studies indicate that changed drug pharmacokinetics by co-administered herbs may be attributed to modulation of efflux drug transporters such as P-glycoprotein (P-gp). Garlic (Allium sativum), lemon (Citrus limonum) and beetroot (Beta vulgaris) are widely used by human immunodeficiency virus (HIV) patients, especially following the pronouncement by a former President of South Africa and the Ministers of Health at that time who promoted the use of these botanicals in HIV patients. The aim of this study was to measure the bi-directional in vitro transport of indinavir, a protease inhibitor, in the presence of crude extracts and pure phytoconstituents of A. sativum (L-alliin and diallyl disulphide), C. limonum (hesperidin and eriocitrin) and B. vulgaris (betaine monohydrate and ß-carotene) across excised porcine intestinal tissue in Sweetana-Grass diffusion chambers. In the negative control group, the transport of indinavir alone (200 M) was determined with no modulator added. In the positive control group, the transport of indinavir was determined in the presence of verapamil (100 M), a known P-gp related efflux inhibitor. The control experiments were used to indicate that the effects of the test compounds were caused by their action and not by chance interferences or external factors. Samples collected at pre-determined time intervals were analysed by means of a validated high performance liquid chromatography (HPLC) method and the transport was expressed as the apparent permeability coefficient (Papp) and the transepithelial flux (J) from which the efflux ratio (ER) and the net flux (Jnet) values were calculated. Statistical analysis was used to compare the results of the test compounds with the control groups in order to indicate significant differences. The mean ER value for indinavir in the negative control group was 1.41 ± 0.170 and in the positive control group it was 0.56 ± 0.0426. Statistically significant (p < 0.05) inhibition of indinavir efflux as indicated by reduced ER values was obtained for L-alliin (ER = 0.280 ± 0.030), diallyl disulphide (ER = 0.505 ± 0.034) and ß-carotene (ER = 0.664 ± 0.075). Inhibition of indinavir efflux will lead to increased transport and therefore a potentially higher bioavailability. Statistically significant (p < 0.05) promotion of indinavir efflux as indicated by increased ER values was obtained for C. limonum crude extract (ER = 5.551 ± 0.575) and hesperidin (ER = 3.385 ± 0.477), which potentially may lead to lower bioavalability. B. vulgaris crude extract (p = 0.8452), betaine monohydrate (p = 0.9982), A. sativum crude extract (p = 0.7161) and eriocitrin (p = 0.4431) displayed no statistically significant effect compared to the negative control group on indinavir transport across excised porcine intestinal tissue. The results from this study demonstrate that L-alliin, diallyl disulphide and ß-carotene have an inhibitory effect on indinavir efflux, which may significantly increase indinavir plasma levels after oral administration. C. limonum crude extract and hesperidin promote indinavir efflux, which may significantly reduce indinavir plasma levels. These pharmacokinetic interactions between certain drugs and plant extracts may negatively affect the anti-retroviral treatment of HIV patients, but deliberate and controlled inclusion of L-alliin, diallyl disulphide and ß-carotene in dosage forms may possibly cause more effective delivery of protease inhibitors after oral administration resulting in less frequent dosing intervals. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

There is a global rise in the use of herbal products in combination with allopathic medicines, while most patients do not inform their health care providers of the use of these natural products. Both pharmacodynamic and pharmacokinetic interactions between herbal products and conventional drugs must be avoided for the wellbeing of the patient. Increasing evidence from in vitro and in vivo studies indicate that changed drug pharmacokinetics by co-administered herbs may be attributed to modulation of efflux drug transporters such as P-glycoprotein (P-gp). Garlic (Allium sativum), lemon (Citrus limonum) and beetroot (Beta vulgaris) are widely used by human immunodeficiency virus (HIV) patients, especially following the pronouncement by a former President of South Africa and the Ministers of Health at that time who promoted the use of these botanicals in HIV patients. The aim of this study was to measure the bi-directional in vitro transport of indinavir, a protease inhibitor, in the presence of crude extracts and pure phytoconstituents of A. sativum (L-alliin and diallyl disulphide), C. limonum (hesperidin and eriocitrin) and B. vulgaris (betaine monohydrate and ß-carotene) across excised porcine intestinal tissue in Sweetana-Grass diffusion chambers. In the negative control group, the transport of indinavir alone (200 M) was determined with no modulator added. In the positive control group, the transport of indinavir was determined in the presence of verapamil (100 M), a known P-gp related efflux inhibitor. The control experiments were used to indicate that the effects of the test compounds were caused by their action and not by chance interferences or external factors. Samples collected at pre-determined time intervals were analysed by means of a validated high performance liquid chromatography (HPLC) method and the transport was expressed as the apparent permeability coefficient (Papp) and the transepithelial flux (J) from which the efflux ratio (ER) and the net flux (Jnet) values were calculated. Statistical analysis was used to compare the results of the test compounds with the control groups in order to indicate significant differences. The mean ER value for indinavir in the negative control group was 1.41 ± 0.170 and in the positive control group it was 0.56 ± 0.0426. Statistically significant (p < 0.05) inhibition of indinavir efflux as indicated by reduced ER values was obtained for L-alliin (ER = 0.280 ± 0.030), diallyl disulphide (ER = 0.505 ± 0.034) and ß-carotene (ER = 0.664 ± 0.075). Inhibition of indinavir efflux will lead to increased transport and therefore a potentially higher bioavailability. Statistically significant (p < 0.05) promotion of indinavir efflux as indicated by increased ER values was obtained for C. limonum crude extract (ER = 5.551 ± 0.575) and hesperidin (ER = 3.385 ± 0.477), which potentially may lead to lower bioavalability. B. vulgaris crude extract (p = 0.8452), betaine monohydrate (p = 0.9982), A. sativum crude extract (p = 0.7161) and eriocitrin (p = 0.4431) displayed no statistically significant effect compared to the negative control group on indinavir transport across excised porcine intestinal tissue. The results from this study demonstrate that L-alliin, diallyl disulphide and ß-carotene have an inhibitory effect on indinavir efflux, which may significantly increase indinavir plasma levels after oral administration. C. limonum crude extract and hesperidin promote indinavir efflux, which may significantly reduce indinavir plasma levels. These pharmacokinetic interactions between certain drugs and plant extracts may negatively affect the anti-retroviral treatment of HIV patients, but deliberate and controlled inclusion of L-alliin, diallyl disulphide and ß-carotene in dosage forms may possibly cause more effective delivery of protease inhibitors after oral administration resulting in less frequent dosing intervals. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

Philosophiae Doctor - PhD / Breast cancer is regarded as the most common cancer in South Africa and its rate of occurrence is increasing. About one in every 31 South African women are at the risk of developing breast cancer and early diagnosis and treatment guarantee 90% survival rate. Tamoxifen is the drugs of choice for the treatment of all stages of breast cancer. The drug binds with estrogen receptor (ER) to minimize the transcription of estrogen dependent genes. However, nearly 50% of ER-positive breast cancer patients either become resistant or fail to respond to tamoxifen resulting in a serious clinical challenge in breast cancer management. The Grand Health Challenges of South Africa includes the development of cost effective diagnostic systems suitable for early detection of diseases and drug resistivity for timely invention and better patient management. / 2020-08-31