Made available in DSpace on 2014-08-13T14:50:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-04-23Bitstream added on 2014-08-13T17:59:47Z : No. of bitstreams: 1 000773667_20170423.pdf: 241128 bytes, checksum: 2eb6497bc7ab87727dcefc224bee3822 (MD5) Bitstreams deleted on 2017-04-24T11:16:39Z: 000773667_20170423.pdf,. Added 1 bitstream(s) on 2017-04-24T11:17:26Z : No. of bitstreams: 1 000773667.pdf: 2224116 bytes, checksum: e342a365c65436e77801c3e655df11ec (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O darunavir, antirretroviral inibidor de protease, compõe a terapia considerada altamente eficaz no tratamento da síndrome da imunodeficiência humana adquirida, e é um dos fármacos disponibilizados gratuitamente pelo Sistema Único de Saúde (SUS). Os produtos genéricos têm contribuído para a manutenção e abrangência desta política de distribuição gratuita de medicamentos, ao reduzir os custos desses produtos. Neste propósito, este estudo busca fornecer informações sobre o comportamento físicoquímico e estabilidade do fármaco e da formulação, diante de diferentes condições climáticas, bem como, sobre sua solubilidade e liberação a partir da formulação. Além disso, pretende apresentar ferramentas in vitro e in silico que contribuam, no futuro próximo, para a produção de novos produtos contendo darunavir que apresentem alta qualidade, especialmente, genéricos do medicamento referência. Para tanto, a solubilidade do fármaco foi investigada em diferentes meios. A baixa solubilidade aquosa do fármaco foi verificada, e os meios adequados aos testes de dissolução foram escolhidos. O perfil de dissolução in vitro do darunavir e de absorção in vivo a partir papel dos comprimidos de Prezista® contendo 300 mg de fármaco foram investigados. Obtevese um perfil de dissolução discriminativo para o produto e um método de quantificação por espectrofotometria derivada foi desenvolvido e validade. Uma correlação entre os dados de fração dissolvida in vitro e fração absorvida in vivo do darunavir foi alcançada (r2= 0,95). Este é um método preditivo para avaliar o desempenho in vivo do darunavir a partir de comprimidos de liberação imediata, com base em dados in vitro, e é importante para minimizar o uso de animais e seres humanos em experimentos durante o desenvolvimento novos produtos farmacêuticos contendo darunavir. Além disso, uma ferramenta in silico foi desenvolvida e validada para a previsão da ... / Darunavir, a protease inhibitor, constitute the highly active antiretroviral therapy against the acquired immunodeficiency syndrome. It is one of agents given free by the Brazilian Health System (SUS). Generic products have contributed to the maintenance and the policy of free distribution of medicines by the decreasing of costs of these products. In this way, this study seeks to provide information on the physical and chemical behavior and stability of the drug and formulation, facing different weather conditions, as well as provide information on their solubility and release from the formulation. It also aims to provide in vitro and in silico tools to contribute in the near future, for the production of new products containing darunavir, especially generic medicines. Therefore, the solubility of the drug was investigated in different media. The low aqueous solubility of the drug was observed, and suitable media for dissolution testing were chosen. The in vitro dissolution profile of darunavir and the in vivo absorption from Prezista® (tablets containing 300 mg of drug) were investigated. It was obtained a discriminating dissolution profile of the product and a method of quantification by spectrophotometry derivative was developed and validated. A correlation between the data of in vitro dissolved fraction and in vivo absorbed fraction of darunavir was achieved (r2 = 0.95). This is a predictive method to evaluate the in vivo performance of darunavir from immediate-release tablets, based on in vitro data, and it is important to minimize the use of animals and humans in experiments during the development of new medicines containing darunavir. In addition, an in silico tool was developed and validated for predicting the oral bioavailability of darunavir formulations, from their dissolution profile. For this, physiologically based pharmacokinetic modeling was applied through Simcyp® software. These, in vitro and in silico tools, assist in ... / FAPESP: 11/05836-4

Orientador: Marlus Chorilli / Banca: Maria Vitória Lopes Badra Bentley / Banca: Renata Fonseca Vianna Lopez / Banca: Carlota de Oliveira Rangel Yagui / Banca: Raquel Mantuanelli Scarel Caminaga / Resumo: O câncer bucal é uma doença altamente incidente na população mundial, podendo provocar invalidez, deformidades faciais e morte. O tratamento de escolha é a quimioterapia e radioterapia, que causam vários efeitos adversos como xerostomia, mucosite, perda de cabelo, náusea, dores e vômitos. A administração de moléculas bioativas, como o peptídeo antitumoral CTT1, demonstrou ser um tratamento inovador e eficaz para o câncer bucal. No entanto, peptídeos apresentam baixa biodisponibilidade oral, o que diminui o desempenho clínico do tratamento. Portanto, a sua administração pela via bucal incorporado em sistemas nanoestruturados mucoadesivos precursores de cristais líquidos (SPCL) constituídos por álcool cetílico etoxilado 20 e propoxilado 5, ácido oleico e fase aquosa contendo água (FA), dispersão polimérica de quitosana (FQ), dispersão polimérica de polietilenoimina (FP) ou dispersão de quitosana associada a polietilenoimina (FPQ) surge como uma valiosa estratégia, visto que a mucosa bucal apresenta alta permeabilidade e alta irrigação sanguínea, o que pode aumentar a absorção do CTT1. Ademais, o SPCL possibilita a incorporação concomitante do CTT1 com o fotossensibilizante azul de metileno (AM), permitindo a associação da terapia fotodinâmica (TFD) à terapia peptídica antitumoral, o que pode potencializar o tratamento do câncer bucal. O objetivo do trabalho foi avaliar o potencial de SPCL mucoadesivos para administração bucal do peptídeo CTT1 associados à TFD no tratamento do c... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Oral cancer is a highly incident disease in the world population, and can cause disability, facial deformities and death. The treatment of choice is chemotherapy and radiotherapy, which cause various adverse effects like xerostomia, mucositis, hair loss, nausea, aches and vomiting. The administration of bioactive molecules, such as the antitumor peptide CTT1, has proven to be an innovative and effective treatment for oral cancer. However, peptides have low oral bioavailability, which decreases the clinical performance of the treatment. Therefore, its administration by the oral route incorporated in liquid crystal precursor (SPCL) mucoadhesive nanostructured systems consisting of ethoxylated ethoxylated 20 and propoxylated cetyl alcohol 5, oleic acid and aqueous phase containing water (FA), polymer dispersion of chitosan (CF), dispersion (FP) or polyethyleneimine-associated chitosan dispersion (FPQ) arises as a valuable strategy, since the buccal mucosa has high permeability and high blood supply, which may increase the uptake of CTT1. In addition, SPCL allows the simultaneous incorporation of CTT1 with the methylene blue photosensitizer (MA), allowing the association of photodynamic therapy (PDT) with antitumor peptide therapy, which may potentiate oral cancer treatment. The objective of this study was to evaluate the potential of mucoadhesive SPCL for buccal administration of the CTT1 peptide, as well as to evaluate the use of PDT using AM as a photosensitizer in oral cancer therapy. SPCL were characterized by polarized light microscopy (MLP), low-angle X-ray scattering (SAXS), in vitro mucoadhesion rheology and texture profile tests, which showed that all formulations acted as SPCL because (FPQ-P) and AM (FPQ-AM) were added to the liquid crystals after the addition of artificial saliva, and FPQ formed a more mucoadhesive and structured liquid crystal... (Complete abstract click electronic access below) / Doutor

A amiodarona é um dos fármacos mais usados para o tratamento de arritmias cardíacas, tanto ventriculares como supraventriculares. Apesar de sua eficácia, o uso da amiodarona está associado a vários efeitos adversos, incluindo a toxicidade pulmonar. O mecanismo pelo qual a amiodarona causa lesão nas células pulmonares humanas não é inteiramente conhecido, mas estudos em cultura de células hepáticas humanas e pulmonares de ratos têm sugerido que a disfunção mitocondrial e o estresse oxidativo têm um papel importante na citotoxicidade da amiodarona. Os compostos fenólicos, incluindo catequina e epicatequina são amplamente distribuídos na natureza e conhecidos por sua capacidade de reduzir o estresse oxidativo. Além disso, alguns compostos fenólicos são capazes de modular a atividade mitocondrial. Em vista disso, o objetivo deste trabalho foi avaliar a capacidade dos compostos fenólicos catequina e epicatequina em a disfunção mitocondrial e os danos oxidativos causados pela amiodarona em células de fibroblasto de pulmão humano (MRC-5). Para atingir os objetivos as células MRC-5 foram tratadas com diferentes concentrações de catequina e epicatequina e após foram expostas a amiodarona 100 μM. A disfunção mitocondrial foi determinada através da atividade do complexo I da cadeia de transporte de elétrons e a biossíntese de ATP usando kits específicos. A viabilidade celular foi avaliada através do ensaio de 3-[4,5- dimetiltiazol 2-il]-2,5 difenil brometo de tetrazolina. A atividade das enzimas superóxido dismutase e catalase foram determinadas espectrofotometricamente. Os danos oxidativos a lipídeos e proteínas foram verificados através dos ensaios de substâncias reativas ao acido tiobarbitúrico e a proteínas carboniladas, respectivamente, e os níveis de óxido nítrico foram avaliados usando o método de Griess. Os resultados mostraram que a amiodarona inibiu a atividade do complexo I da cadeia de transporte de elétrons em 53% e a biossíntese de ATP em 9,5% e tanto a catequina como a epicatequina foram capazes de evitar estes efeitos em todas as concentrações (5, 10, 20 μM) testadas. Verificou-se que a amiodarona reduziu a atividade das enzimas superóxido dismutase e catalase (indicando produção de superóxido e peróxido de hidrogênio) e aumentou os danos oxidativos a lipídeos e proteínas. Os compostos fenólicos catequina e epicatequina foram capazes de minimizar as alterações no metabolismo redox induzidos pela amiodarona e aumentar a viabilidade nas células MRC-5. Catequina e epicatequina reduziram a depleção de óxido nítrico causada pela amiodarona. Este trabalho mostrou, pela primeira vez, que o mecanismo de toxicidade da amiodarona em células MRC-5 está associado à disfunção mitocondrial, principal causa de geração de dano oxidativo celular e que estes efeitos tóxicos são em parte reduzidos pela catequina e epicatequina. Embora outros estudos sejam necessários, estes dados abrem novas perspectivas para estudos visando o desenvolvimento de medicamentos que minimizem os efeitos tóxicos da amiodarona. / Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2015-12-09T15:32:21Z No. of bitstreams: 1 Dissertacao Luciana Fernandes Silva Santos.pdf: 1790981 bytes, checksum: 4f3e8dbb7bc255b525503174b7e1a3d2 (MD5) / Made available in DSpace on 2015-12-09T15:32:21Z (GMT). No. of bitstreams: 1 Dissertacao Luciana Fernandes Silva Santos.pdf: 1790981 bytes, checksum: 4f3e8dbb7bc255b525503174b7e1a3d2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES. / Amiodarone is among the most widely used drugs for the treatment of ventricular and supraventricular cardiac arrhythmias. However, the use of amiodarone is associated with several side effects including pulmonary toxicity. The mechanism of amiodarone toxicity is not well known, but studies in human liver cells and rats lung cells have been suggested that mitochondrial dysfunction and oxidative stress play important role in the amiodarone cytotoxicity. Phenolic compounds, including catechin and epicatechin are widespread in nature and known for their ability to reduce oxidative stress. In addition, some phenolic compounds are able to modulate mitochondrial activity. Therefore, the objective of this study was to evaluate the ability of phenolic compounds catechin and epicatechin to minimize the mitochondrial dysfunction and oxidative damage induced by amiodarone in human lung fibroblast cells (MRC-5). To achieve the objectives, MRC-5 cells were treated with different concentrations of catechin and epicatechin and then amiodarone 100 μM. Mitochondrial dysfunction was determined by the activity of complex I of the electron transport chain and ATP biosynthesis using specific kits. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The activity of the enzymes superoxide dismutase and catalase were determined spectrophotometrically. The oxidative damage to lipids and proteins have been verified through the test substances reactive to the thiobarbituric acid and carbonyl protein, respectively, and nitric oxide levels were evaluated using the Griess method. The results showed that amiodarone inhibit 53% of the activity of complex I of the electron transport chain and 9.5% of ATP biosynthesis and both catechin and epicatechin were able to avoid these effects in all concentrations (5 10, 20 mM) tested. It was found that amiodarone reduced the superoxide dismutase and catalase activities (indicating the production of radicals superoxide and hydrogen peroxide) and increased oxidative damage to lipids and proteins. Phenolic compounds catechin and epicatechin were able to minimize alterations in the redox metabolism and increase in viability of MRC-5 cells. Furthermore, catechin and epicatechin reduced nitric oxide depletion caused by amiodarone. This study showed, for the first time, that toxicity of amiodarone in human lung cultured cells is associated, at least, in part, with mitochondrial dysfunction which was avoided by catechin and epicatechin. Although further studies are needed, these data open new perspectives for studies aiming the development of drugs that minimize the toxic effects of amiodarone.

Pode-se afirmar que no século XX originou-se uma indústria farmacêutica multinacional com extraordinária capacidade de pesquisa e desenvolvimento para produzir novos fármacos. Porém, contradizendo este potencial inovador, o número de fármacos introduzidos no mercado vem declinando desde 1960 e as oportunidades abertas com os avanços da biologia molecular, da genômica, da bioinformática e da química ainda não trouxeram os resultados esperados. No Brasil, a pesquisa científica tem obtido resultados de grande relevância, mas encontra muitas dificuldades para levar novos produtos ao mercado. O objetivo principal deste trabalho é discutir estratégias de fomento para a pesquisa e desenvolvimento de fármacos no país, procurando conciliar os requisitos técnicos e econômicos deste processo com as competências preexistentes. O referencial metodológico adotado enfatiza o papel determinante das relações econômicas na pesquisa e desenvolvimento de fármacos e procura encontrar caminhos compatíveis com a realidade nacional. Para tanto, discute as características técnicas e econômicas desta atividade, bem como a estratégia das empresas inovadoras e algumas experiências brasileiras nesta área do conhecimento. Fundamentado na análise de 766 novos fármacos introduzidos no mercado mundial entre 1984-2003, nos pilares econômicos do processo de inovação e no contexto político-institucional da pesquisa e desenvolvimento de fármacos, propõe alternativas para aprimorar o desenvolvimento científico e tecnológico do setor farmacêutico brasileiro. Muitas atividades relacionadas com a pesquisa e desenvolvimento de fármacos são realizadas no país, mas encontram-se dispersas nas principais universidades, centros e institutos de pesquisa. O mapeamento destas competências representa o ponto de partida para a criação de uma rede de inovação no setor farmacêutico. Um dos gargalos identificados neste trabalho é a fragilidade do suporte institucional para negociações de alta tecnologia, do qual fazem parte as patentes, os acordos de cooperação e a transferência de tecnologia. Para viabilizar, no Brasil, a incorporação de ferramentas de alta tecnologia empregadas no desenvolvimento de fármacos foi proposta a criação do Laboratório Nacional de Pesquisa e Desenvolvimento de Fármacos. Além de garantir sofisticação técnica, este Laboratório Nacional atuaria como instituição aberta, multidisciplinar, assumindo o papel de um centro de articulação das iniciativas voltadas para o desenvolvimento de fármacos, podendo gerar recursos para financiar a pesquisa e contribuir para o desenvolvimento científico e tecnológico. Acordos de cooperação com empresas inovadoras e organismos internacionais fazem parte das estratégias para captação de recursos. Linhas de pesquisa alinhadas com as necessidades do Sistema Único de Saúde e com outras políticas do setor público também devem nortear a pesquisa e desenvolvimento de fármacos no país. A exploração de novos alvos moleculares, articulada com projetos genômicos, inovações incrementais, doenças negligenciadas e produtos naturais são apontados como áreas estratégicas. A afirmação de que o Brasil reúne as condições necessárias para participar do processo de desenvolvimento de fármacos - hipótese primária deste trabalho - encontra sustentação nos argumentos apresentados e revela que as condições para a inovação tecnológica nunca foram tão favoráveis quanto agora. Estimular o debate acerca de estratégias que possam fomentar o desenvolvimento científico e tecnológico da pesquisa e desenvolvimento de fármacos no país representa a contribuição almejada por este trabalho. / It can be asserted that in the twentieth century a multinational pharmaceutical industry with extraordinary research and development capacity to produce new drugs arose. However, contradicting this innovative potential, the number of new chemical entities introduced in the market is declining since 1960 and the opportunities open with the progresses of molecular biology, genomics, bioinformatics and chemistry haven\'t brought the expected results yet. In Brazil, the scientific research has been obtaining results of great relevance, but a lot of difficulty is found to introduce new products into the market. The main purpose of this work is to discuss fomentation strategies for the drug research and development in the country, trying to reconcile technical and economic requirements of this process with the pre-existent competences. The adopted methodological referential emphasizes the decisive role of economic relationships in drug research and development and tries to find out compatible ways with the national reality. For that, it discusses the technical and economic characteristics of this activity, as well as the strategy of innovative companies and some Brazilian experiences in this knowledge area. Based on the analysis of 766 new chemical entities introduced in world market among 1984 to 2003, in economic pillars of the innovation process and in political-institutional context of drug research and development, alternatives are proposed to straighten out the scientific and technological development of Brazilian pharmaceutical sector. Many activities related to the drug research and development are accomplished in the country, but they are scattered in the main universities, research centers and institutes. The charting of these competences represents the start up to create an innovation net in the pharmaceutical section. One of the bottlenecks identified in this work is the fragility of institutional support for high technology negotiations, of which patents, cooperation agreements and technology transference make part. To make it possible, in Brazil, incorporation of high technology tools used in drug development, creation of National Laboratory of Drug Research and Development was proposed. Besides guaranteeing technical sophistication, this National Laboratory would act as an open institution, multidisciplinary, shouldering the role of an articulation center of initiatives aiming drug development, being able to generate resources to finance research and to contribute to scientific and technological development. Cooperation agreements with innovative companies and international organisms are part of strategies to raise funds research fields aligned with the needs of the Brazilian Unique Health System (SUS) and with other policies of Public Sector also must direct the drug research and development in the country. New molecular targets evaluation articulated in genomic process, incremental innovations, neglected diseases and natural products are pointed out as strategic areas. The statement that Brazil has conditions of participating in the process of drug development - primary hypothesis of this work - finds back-up in reported arguments and reveals that conditions for technological innovation have never been as favorable as now. To stimulate the debate concerning strategies that can foment scientific and technological development of drug research and development in the country represents the contribution aimed for this work.

Contemporary cases of crowdsourcing (CS) and open source development (OS) related to drug development have been selected and studied. Contemporary examples of CS/OS from within and outside of the pharmaceutical industries have been presented to give a background and suggest possible benefits and problems. The main criteria for selection have been that the case must seek to advance drug development and must use crowdsourcing or open source as a mechanism. The cases found in our search show a large diversity in terms of application, usage, and possible implications for the pharmaceutical industry. We found that crowdsourcing within a scientific problem context produced good results, but that open source initiatives were either poorly financed and not successful or focused on neglected diseases made possible through strong backing by non-profit organizations. An analysis of which the pharmaceutical companies where that showed activity on the platforms identified R&D-intensive and biotech companies as the most active. Contract research organizations (CROs) and generics manufacturers (GMs) showed almost complete absence. We argue that GMs are not likely to be interested in this kind of R&D, but CROs are an untapped resource. Finally we propose a hypothetical model that takes into account all the findings from our study and the literature. This model is based on a limited type of open source with a limited number of partners making use of the untapped CRO resource through crowdsourcing. / Ett antal pågående fall av samverkans-modellen crowdsourcing (CS) samt fenomenet av ”öppen-innovation”, open-innovation (OI) har studerats i kontexten läkemedelsutveckling. I denna fall-studien presenteras ett antal fall där CS och OI används både inom och utom läkemedelsindustrin. Detta har gjorts för att skapa en bättre förståelse för fenomenen samt att tydliggöra problem och att belysa fördela med öppen-innovation och olika typer av samverkans-modeller. Huvudkriteriet för utvalen av fall har varit ett avancerat stadium i läkemedelsutvecklingen samt att något av de två verktygen OI eller CS används aktivt i utvecklingsarbetet. Fall-studien klargör en tydlig differens mellan olika applikations-typer och hur man i verkligheten implementerar dessa verktyg i forskningsarbete. Vår fall-studie indikerar goda resultat då man använder samverkans-modellen CS inom forskningsarbete, men att ”öppen-innovation” OI inte gav samma goda resultat då immateriella mekanismer bromsar öppenheten i läkemedelsindustrin. Inom läkemedels-utvecklingen för Negleted Diseases fann vi däremot en större aktivitet vilken grundar sig på icke-vinst-drivande aktörers intressen. Genom en analys av aktörer inom läkemedelsindustrin har vi kunna framtyda att forsknings-och utveckligns-företag samt Biotech som de som mest ser och utnyttjar potentiallen i CS och OI. Contract research organizations (CROs) och Generics Manufacturers (GMs) är nästan uteslutande frånvarande i resultaten från vår analys. Våra resultat visar att GM-företagen inte har mycket intresse av varken CS eller OI, men att för CRO-företagen finns det vinning i att utnyttja dessa resurser. Slutligen förespråkar vi en fortsatt forskning i ämnet baserat på fall-studien för att kunna ta fram en hypotetisk-CS modell, vilken skulle baseras på ett begränsat antal av samarbetspartners vars potentiella vinning skulle visas genom utnyttjandet av samverkans-modellen CS. / Johan Evaldsson +733-706822

Introduction: The effect of renal impairment (RI) on systemic exposure is assessed in phase I with RI studies and/or in phase III with population pharmacokinetic analysis. Regulatory review has indicated that the estimated effect of RI from the two methods may differ. Aim: To map the estimated effect of RI on systemic exposure based on phase I and III data, to investigate if the estimated effect based on the two data sources differ and to investigate causes to this potential discrepancy. Methods: Marketing authorisation applications (MAA) were scrutinised with focus on impact of RI on systemic exposure estimated based on phase I and III data. In addition, a simulation-estimation study was performed to explore causes to discrepancies. Phase I and III data were simulated and analysed with non-compartmental analysis (NCA) and population analysis. The phase III data were simulated under several alternative conditions thought to be potential sources for discrepancies,  such as uncertainty in creatinine clearance (CLCR) measurements and varying number of subjects. Results: Six examples were found in MAAs in which a discrepancy was observed, where phase III tended to estimate a lower effect of RI compared with phase I. In the simulation-estimation study, the NCA of phase I data over-predicted the effect of RI on systemic exposure, while the population analysis of phase III data estimated the effect of RI without bias. Uncertainty in CLCR measurement in the phase III data resulted in under-prediction of the effect of RI on systemic exposure. Conclusions: A discrepancy in the estimated effect of RI on systemic exposure between phase I and III was observed in existing MAAs. The NCA of phase I RI study and uncertain CLCR measurements were identified as possible reasons to the discrepancy.

One of the primary contributing factors to the escalating costs of health care is the high cost of innovator pharmaceutical products. As a consequence, health authorities in various countries and in particular in the developing world have identified generic prescribing and generic substitution as possible strategies to contain the escalating costs of health care provision. There is therefore a need for formulation scientists in developing countries to invest more time in the research and development of generic formulations. Clobetasol 17-propionate (CP) generic cream formulations containing 0.05% w/w of the drug were manufactured and characterized using in vitro testing. Formulation development studies were preceded by the development and validation of an RP-HPLC with UV detection for the quantitation and characterization of CP in innovator and generic cream formulations during formulation development and assessment studies. Furthermore the in vitro release ates of CP release from innovator and generic cream formulations were monitored using a validated in vitro release test method developed in these studies. The formulation of CP cream products was accomplished using a variety of commercially available mixed primary emulsifiers, such as Estol® 1474, Ritapro® 200, Emulcire® 61 WL and Gelot® 64. Successful formulations were selected based on their ability to remain physically stable immediately after manufacture and for 24 hours after storage at room temperature (22°C). Estol® 1474 was found to produce an unstable cream and was therefore not investigated further. The other three emulgents produced stable creams, but only the in vitro release profile of CP from a cream manufactured to contain Gelot® 64 was found to be statistically similar to that of the innovator formulation. Therefore the cream containing Gelot® 64 was selected as the most appropriate prototype generic cream formulation and was characterized in vitro in terms of CP content, viscosity, pH and in vitro release rate. Data generated from these studies were compared to those of the innovator product, Dermovate® cream, using statistical methods. The CP content, pH and in vitro release rate data of the CP formulation were similar to those of the innovator product, however the intrinsic viscosity of Dermovate® cream was almost three (3) times greater than the intrinsic viscosity of the test formulation developed using Gelot® 64. The CP cream formulation developed in these studies was stored for 4 weeks at 40 ± 2°C and 25 ± 5% RH in an incubator and the formulation was found to be stable. A formulation has been developed and assessed and found to be suitable for use as a topical semi-solid dosage form for CP.

Plasmodium falciparum 1–deoxy–D–xylulose–5 phosphatereductoisomerase (PfDXR) plays a role in isoprenoid biosynthesis in the malaria parasite and is absent in the human host, making this parasite enzyme an attractive target for antimalarial drug design. To characterize PfDXR, it is necessary to produce large quantities of the enzyme in a soluble and functional form. However, the over–production of malarial proteins in prokaryotic host systems often results in the formation of truncated proteins or insoluble protein aggregates. A heterologous expression system was developed for the production of active PfDXR using codon harmonization and tight control of expression in the presence of lac repressor. Yields of up to 2 mg/l of enzyme were reported using the optimised expression system, which is 8 to 10– fold greater than previously reported yields. The kinetic parameters Km, Vmax and kcat were determined for PfDXR; values reported in this study were consistent with those reported in the literature for other DXR enzymes. A three–dimensional model of the malarial drug target protein PfDXR was generated, and validated using structure–checking programs and protein docking studies. Structural and functional features unique to PfDXR were identified using the model and comparative sequence analyses with apicomplexan and non–apicomplexan DXR proteins. Residues Val44 and Asn45, essential for NADPH binding; and catalytic hatch residues Lys224 and Lys226, which are unique to the species of Plasmodium, were mutated to resemble those of E. coli DXR. Interestingly,these mutations resulted in significant reductions in substrate affinity, when compared to the unmutated PfDXR. Mutant enzymes PfDXR(VN43,44AG) and PfDXR(KK224,226NS) also demonstrated a decreased ability to turnover substrate by 4–fold and 2–fold respectively. This study indicates a difference in the role of the catalytic hatch of PfDXR with regards to the way in which it captures substrates. The study also highlights subtle differences in cofactor binding to PfDXR, compared with the well characterized EcDXR enzyme. The validated PfDXR model was also used to develop a novel efficient in silico screening method for potential tool compounds for use in the rational design of novel DXR inhibitors. Following in silico screening of 46 potential DXR inhibitors, a two–tiered in vitro screening approach was undertaken. DXR inhibition was assessed for the 46 novel compounds using an NADPH– ependant DXP enzyme inhibition assay and antimalarial potential was assessed using P.falciparum–infected erythrocyte growth assays. Select compounds were tested in human cells in order to determine whether they were toxic to the host. From the parallel in silico and in vitro drug screening, it was evident that only a single compound demonstrated reasonable potential binding to DXR (determined using in silico docking), inhibited DXR in vitro and inhibited P. falciparum growth, without being toxic to human cells. Its potential as a lead compound in antimalarial drug development is therefore feasible. Two outcomes were evident from this work. Firstly, analogues of known antimalarial natural products can be screened against malaria, which may then lead towards the rational design of novel compounds that are effective against a specific antimalarial drug target enzyme, such as PfDXR. Secondly, the rational design of novel compounds against a specific antimalarial drug target enzyme can be untaken by adopting a coupled in silico and in vitro approach to drug discovery.

This research has focused on the development of novel substrate mimics as potential DXR inhibitors of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), an essential enzyme in the mevalonate-independent pathway for the biosynthesis of isoprenoids in Plasmodium falciparum. DXR mediates the isomerisation and reduction of 1-deoxy-D-xylulose-5-phosphate (DOXP) into 2C-methyl-D-erithrytol 4-phosphate (MEP) and has been validated as an attractive target for the development of novel anti-malarial chemotherapeutic agents. Reaction of various amines with specially prepared 4-phosphonated crotonic acid in the presence of the peptide coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), has afforded a series of amido-phosphonate esters in moderate to good yields (48% - 73%) which, using a RuCl₃/CeCl₃/NaIO₄ catalyst system, have been dihydroxylated to furnish the dihydroxy-amido phosphonate ester pro-drugs; subsequent hydrolysis under microwave irradiation has afforded the corresponding phosphonic acids. A second series of potential inhibitors viz., 3-substituted aniline-derived phosphonate esters, their corresponding phosphonic acids and mono-sodium salts, have also been successfully synthesised. In these compounds, the essential functional groups are separated by one, two, three or four methylene groups, Deprotonation of the 3-substituted aniline substrates, followed by reaction with the appropriate ω-chloroalkanoyl chloride produced the ω-chloroamide intermediates, which were subjected to the Michaelis-Arbuzov reaction to afford the diethyl phosphonate esters in moderate to good yields (48% - 74%). Microwave-assisted TMSBrmediated cleavage of the phosphonate esters furnished the phosphonic acids, neutralisation of which afforded the mono-sodium salts. Furan-derived phosphate esters and phosphonic acids have been prepared as conformationally-restricted DOXP analogues. Functionalization at C-5 of the trityl-protected furan was achieved using the Vilsmeier-Haack formylation and Friedel-Crafts acylation reactions and, following de-tritylation, phosphorylation and oximation, using hydroxylamine hydrochloride, the novel oxime derivatives have been isolated as a third series of potential DXR inhibitors in very good yields (87% - 96%). Finally, in order to exploit an additional binding pocket in the PƒDXR active site, a series of N-benzylated phosphoramidic derivatives were obtained in seven steps from the starting material, diethyl phosphoramidate. The known inhibitors, fosmidomycin and its acetyl derivative FR900098, were also successfully synthesised as standards for STD-NMR binding and inhibition assays. In all, over 200 compounds (136 novel) have been prepared and appropriately characterised using 1-and 2-D NMR and IR spectroscopic analysis and, where necessary, HRMS or combustion analysis. Saturation Transfer Difference (STD) protein-NMR experiments, undertaken using selected compounds, have revealed binding of most of the ligands examined to EcDXR. Computersimulated docking studies have also been used to explore the preferred ligand-binding conformations and interactions between the ligands and essential DXR active-site residues, while DXR-enzyme inhibition assays of selected synthesised ligands have revealed certain patterns of inhibitory activity.

Using a mechanism-based approach to drug discovery, efforts have been directed towards developing novel ATP mimics that can act as GS inhibitors. The purine-based systems, adenosine, adenine and allopurinol, were identified as possible scaffolds for potential ATP mimics, while various meta-disubstituted benzenoid compounds, 3-aminobenzonitrile, 3-aminophenol, resorcinol, 3-aminobenzyl alcohol, 3-hydroxybenzoic acid and 3-aminobenzoic acid have been explored as adenine analogues. These compounds were treated with different alkylating and acylating agents. Allylation of all the substrates was achieved using allyl bromide and N-9 alkylation of protected allopurinol was effected using a number of specially prepared Baylis-Hillman adducts. Acylation of the benzenoid precursors with chloroacetyl chloride, acetoxyacetyl chloride, acryloyl chloride and specially prepared 2,3,4,5,6-pentaacetylgluconoyl chloride afforded the corresponding mono- and /or diacylated products in varying yields (4-96%). Elaboration of the alkylated and acylated products has involved the reaction of hydroxy systems with diethyl chloro phosphate and chloro derivatives with triethyl phosphite in Arbuzov-type reactions to afford phosphorylated products. In all cases, products were fully characterized using 1- and 2-D NMR analysis and, where appropriate, high-resolution mass spectrometry. The application of Modgraph and ChemWindow NMR prediction programmes has been explored and the resulting data have been compared with experimental chemical shift assignments to confirm chemical structures and, in some cases, to establish the position of allylation or acylation. Experimental assignments were found to be generally comparable with the Modgraph data, but not always with the ChemWindow values. The docking of selected products in the 'active-site' of GS and their structural homology with ATP, both in their free and bound conformations have been studied using the ACCELERYS Cerius² platform. All the selected ATP mimics exhibit some form of interaction with the 'active-site' residues, and a number of them appear to be promising GS ligands.