Development of cyclodextrin-hydrogel polymeric systems in scCO2 for drug delivery

Thesis for the Degree of Master of Science in Bioorganic Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Development of electrospun Ion jelly fibers® for drug delivery

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Process engineering of liver cells for drug testing applications

Dissertação para obtenção do Grau de Doutor em Bioengenharia

The inhibitory activity of synthetic compounds and ions against transporters of multi-drug resistant bacteria

RESUMO: Sessenta e três derivados de hidantoína foram utilizados para avaliar possíveis efeitos de modulação na actividade das bombas de efluxo (BE) na Salmonella NCTC 13349 utilizando um método fluorimétrico semi-automático. Nenhum dos compostos apresentaram actividade anti-bacteriana até concentrações de 240 mg/L. Entre todos os compostos, SZ-7 demonstrou possuir propriedades de modulação de effluxo na presença de glucose. Para testar esta actividade, estirpes de Salmonella resistentes à ciprofloxacina, induzidas a elevados níveis de resistência com sobre-expressão de BE, foram expostas ao SZ-7. Este derivado afectou a susceptibilidade das estirpes à ciprofloxacina. Uma vez que os 63 compostos estudados apresentaram pouco efeito inibitório /cumulativo, apesar de serem conhecidos pelos seus efeitos moduladores de BE-dependentes de iões em eucariotas, foi questionado o papel dos iões na regulação de BE bacterianas, que poderão influenciar a eficácia de novos compostos. Para este estudo, utilizamos a Escherichia coli AG100 como modelo, devido ao extenso conhecimento no que respeita a estrutura e actividade das BE. Devido à importância de iões de cálcio (Ca2+) nos canais de transporte membranar e na actividade de ATPases, a sua actividade na modulação do efluxo foi investigada. De resultados anteriormente obtidos concluiu-se que a pH 5 o efluxo é independente de energia metabólica; contudo, a pH 8 é absolutamente dependente, sendo que o Ca2+ é indispensável para manter a actividade das ATPases bacterianas. A acumulação/effluxo de EtBr pela E. coli AG100 foi determinada na presença/ausência de Ca2+, clorpromazina (inibidor de ligação de Ca2+ a proteínas), e ácido etilenodiamino tetra-acético (quelante de Ca2+). Acumulação/effluxo aumentou a pH 8, contudo o Ca2+ reverte estes efeitos evidenciando a sua importância no funcionamento das BE bacterianas. Em resumo este trabalho colocou em evidência que muitos aspectos bioquímicos e bioenergéticos devem ser tomados em consideração no estudo da resistência bacteriana mediada por BE.------- ABSTRACT: Sixty-three hydantoin derivatives were evaluated for their modulating effects on efflux pump (EP) activity of Salmonella NCTC 13349 utilizing a semi-automatic fluorometric method. None of the compounds presented antibacterial activities at concentrations as high as 240 mg/L. Among all compounds, SZ-7 showed possible efflux modulating activity in the presence of glucose, indicative of a potential EP inhibitor. To verify its potential effects, ciprofloxacin-resistant Salmonella strains, induced to high level resistance with over-expressing EPs, were exposed to SZ-7. This derivative affected the susceptibility of the ciprofloxacin-resistant strains. Since the 63 compounds studied had very low inhibitory/accumulative effects, even though their known for being efficient in modulating ion-driven eukaryotic EPs, we questioned whether ions had a leading role in regulating bacterial EPs, influencing the effectiveness of new compounds. For this study we used Escherichia coli AG100 as a model, due to the extensive knowledge on its EPs structure and activity. Owing the importance of calcium ions (Ca2+) for membrane transport channels and activity of ATPases, the role of Ca2+ was investigated. From previous results we concluded that at pH 5 efflux is independent of metabolic energy; however, at pH 8 it is entirely dependent of metabolic energy and the Ca2+ ions are essential to maintain the activity of bacterial ATPases. Accumulation and efflux of ethidium bromide (EtBr) by E. coli AG100 was determined in the presence and absence of Ca2+, chlorpromazine (inhibitor of Ca2+-binding to proteins), and ethylenediaminetetraacetic acid (Ca2+ chelator). Accumulation of EtBr increased at pH 8; however Ca2+ reversed these effects providing information as to the importance of this ion in the regulation of bacterial EP systems. Overall this work puts in evidence that many biochemical and bioenergetic aspects related to the strains physiology need to be taken into consideration in bacterial drug resistance mediated by EPs.

Protein adducts from the anti-HIV drug abacavir – possible biomarkers of drug toxicity

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Novel approaches for culturing hepatocytes for drug testing applications

Dissertation presented to obtain the Ph.D degree in Biochemisry, Biotechnology

Development of chitosan-based microparticles for pulmonary drug delivery

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Nevirapine in an animal model of pre-diabetes: study of drug pharmacokinetic and its effects on fasting glycemia and insulin resistance

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Programas de cuidados integrados para pessoas com esquizofrenia ou perturbação esquizoafetiva : estudo sobre a exequibilidade, implementação e resultados de um programa de cuidados integrados para pessoas com esquizofrenia ou perturbação esquizoafectiva em Portugal

RESUMO: De acordo com o estado da arte, existem intervenções psicofarmacológicas, psicológicas e psicossocias, com evidência científica dos seus resultados, no tratamento de pessoas com esquizofrenia e perturbação esquizoafectiva. No entanto, muitos destes doentes, não procuram ajuda dos serviços de saúde mental, não recebem os referidos cuidados ou não são detectados nem seguidos por estes. Esta realidade levou ao desenvolvimento de programas integrados, intervenções e estudos mais específicos, nomeadamente para tentar ultrapassar os obstáculos na acessibilidade aos cuidados de saúde e na continuidade de seguimento destes doentes. No conjunto das dificuldades apuradas, as questões da exequibilidade (feasibility) e da implementação, têm tido particular relevo na literatura científica recente, bem como a melhor forma de vencer as respectivas barreiras e adaptar essas intervenções às varias realidades, culturas e recursos. Objectivos: Objectivos gerais:1) Avaliar a exequibilidade e a implementação inicial de um programa de cuidados integrados, para pessoas com esquizofrenia ou perturbação esquizoafectiva, no contexto clínico das equipas de saúde mental comunitárias de um departamento de psiquiatria do Serviço Nacional de Saúde, em Portugal, com os recursos materiais e humanos existentes; 2) Avaliar o impacto deste programa, nestes doentes e na respectiva prestação de cuidados de saúde mental. Metodologia. Elaborámos um programa de cuidados integrados (Programa Integrar) com base no modelo clínico de case management, com seguimento mantido e integrado. Cada doente passou a ter um terapeuta de referência, um plano individual de cuidados e manteve o seguimento com o seu psiquiatra assistente. Foram seleccionadas intervenções, nomeadamente, psicoeducativas, familiares, estratégias para lidar com os sintomas e a doença, prevenção de recaídas e intervenções para melhorar o funcionamento social e ocupacional. A estas intervenções foi sempre associado o tratamento psicofarmacológico. O estudo delineado incluiu dois componentes: avaliação da exequibilidade e implementação inicial do programa de cuidados integrados (componente A) e avaliação do impacto deste programa (componente B), através de um estudo de intervenção, prospectivo, naturalista, não aleatorizado e não ontrolado. A amostra do estudo resultou das sucessivas referenciações, para o Programa Integrar, de pessoas com os diagnósticos de esquizofrenia ou perturbação esquizoafectiva, seguidas nas cinco equipas de saúde mental comunitárias do Departamento de Psiquiatria do Centro Hospitalar de Lisboa Ocidental, com uma área assistencial correspondente a uma população de, aproximadamente, 400 000 pessoas. Definimos etapas, estratégias, parâmetros e indicadores para o estudo da exequibilidade do programa. Efectuámos a monitorização e a avaliação de tarefas, procedimentos e intervenções recomendadas aos terapeutas de referência. Realizámos duas avaliações, uma no início do programa e outra após um ano de intervenção. Foram avaliadas as seguintes dimensões (com indicação do acrónimo do instrumento de avaliação utilizado entre parêntesis): psicopatologia (BPRS), depressão (MADRS), necessidades (CAN), incapacidade (DAS), actividade social e ocupacional (SOFAS), atitude em relação à medicação (DAI), insight (SAI), qualidade de vida (WHOQOL-S) e satisfação (POCS). Resultados: Dos 146 doentes que foram incluídos no estudo, 97 (66%) eram do sexo masculino e 49 (34%) do sexo feminino, com uma idade média de 36 anos. Destes oentes,116 (79,4%)tinham o diagnóstico (ICD10) de esquizofrenia e 30 (20,6%) de perturbação esquizoafectiva. Os restantes dados sociodemográficos eram típicos de populações afins em serviços de saúde mental nacionais. Do total de doentes (146) que iniciaram o estudo, 26 (18%) abandonaram o seguimento neste programa. Para o componente A da investigação (estudo de exequibilidade) salientamos: exerceram funções a totalidade (15) dos terapeutas de referência que receberam formação, 76 % efectuaram o número mínimo recomendado de sessões / ano por doente (≥18), 44,9 fizeram o número mínimo de sessões familiares pretendido (≥ 3). Nas intervenções mais específicas foram atingidos os objectivos em mais de 75% dos doentes, à excepção das intervenções domiciliárias (19,4%), prevenção do abuso de substâncias (45,4%) e do risco de suicídio (34,3%). O plano individual de cuidados foi realizado em 98 % dos doentes e em 38,9 % dos casos ocorreu a participação da família. Neste plano, a média de objectivos definidos foi de 5 e a média de objectivos atingidos correspondeu a 3 (p= 0,001). Na primeira avaliação, estavam a frequentar estruturas de reabilitação psicossocial 42 doentes (28,8%) e,12 meses após, esse número passou para 80 (74,1%).Também aumentou o número de doentes com actividade profissional a tempo completo, de 8 (7,4%) para 18 (16,7%). No componente B do estudo (avaliação do impacto do programa), em termos de psicopatologia, e para as pontuações médias globais do BPRS, ocorreu uma diminuição entre a primeira e a segunda avaliação (p=0,001), tal como nas subescalas: sintomas positivos (p=0,003), sintomas negativos (p=0,002), sintomas de mania (p=0,002) e sintomas de depressão/ansiedade (p=0,001). Na avaliação da depressão (p= 0,001) e da incapacidade (p=0,003), as diferenças foram significativas e favoráveis. O mesmo não sucedeu na atitude em relação à medicação (p=0,690) nem na escala de avaliação do insight (p=0,079). Em relação ao funcionamento social e ocupacional, qualidade de vida e satisfação dos doentes, ocorreu uma melhoria significativa da primeira para a segunda avaliação As necessidades sem resposta mais frequentes, na primeira avaliação, corresponderam aos itens: actividades diárias, contactos sociais, relações íntimas, relacionamento sexual, benefícios sociais, sintomas psicóticos, sofrimento psicológico, informação sobre a doença / tratamento e gestão/problemas de dinheiro. Para todos estes últimos nove itens, verificou-se uma diferença estatisticamente significativa, entre a primeira e a segunda avaliação, com diminuição destas necessidades, excepto nas relações íntimas, relacionamento sexual e nos problemas de dinheiro. Na distribuição dos três estados de necessidades, para todos os itens, diminuíram as necessidades sem resposta e as necessidades com resposta parcial e aumentaram as situações em que deixaram de se verificar necessidades relevantes. Dos resultados obtidos para outros indicadores clínicos e de utilização dos cuidados, será importante referir que na comparação do ano anterior com o ano em que decorreu o programa, o número de doentes da amostra internados diminuiu 64,1%, bem como a média do número de internamentos (p=0,001). Em relação à duração dos internamentos, no ano anterior ao programa, os 39 doentes internados, tiveram um total de dias de internamento de 1522, sendo que, no ano do programa, para os 14 doentes internados, o total foi de 523 dias. Em termos absolutos, ocorreu uma redução de 999 dias (menos 65,6% dias). Também se verificou uma diminuição de 45,6 % de recaídas (p=0,001).Discussão e conclusões A exequibilidade do programa de cuidados integrados permitiu a aplicação do modelo clínico de case management, com seguimento mantido e integrado, através do qual cada doente passou a ter um terapeuta de referência assim como, em 98% casos, um plano individual de cuidados. As famílias continuaram a ser o principal suporte para os doentes, mas surgiram dificuldades quando se pretendeu uma participação mais activa destas no tratamento.A diminuição do número e da duração dos internamentos constituíram importantes resultados com implicações não só em termos clínicos mas também económicos. Os valores obtidos, para as diferentes variáveis, também sugerem o impacto favorável do Programa Integrar a nível da psicopatologia, das necessidades, da incapacidade, do funcionamento social e ocupacional, da qualidade de vida e da satisfação dos doentes. O mesmo não sucedeu para o insight e para a mudança de atitudes dos doentes em relação à medicação, resultados que devem ser igualmente considerados em futuros reajustamentos deste programa ou no desenvolvimento de novos programas. Como principais conclusões podemos referir que: 1) Foi possível a exequibilidade de um programa de cuidados integrados inovador e a implementação inicial desse programa, para doentes com esquizofrenia ou perturbação esquizoafectiva, com os recursos humanos e materiais existentes, no contexto clínico das equipas de saúde mental comunitárias, de um departamento de psiquiatria e saúde mental, em Portugal; 2) Na avaliação do impacto do programa, os resultados obtidos indiciam potencialidades de aplicação, deste programa de cuidados integrados, com vista à melhoria clínica e psicossocial destes doentes. Devem ser realizados estudos de replicação, ou complementares à presente investigação, no entanto, os dados obtidos são encorajadores para o desenvolvimento de programas similares, a nível nacional e internacional, que possam beneficiar um grupo mais alargado de doentes.------------ABSTRACT: Although there are psychological and psychosocial interventions well supported by scientific evidence, which show benefit when combined with psychopharmacological treatments, we know that a significant number of people with schizophrenia or schizoaffective disorders, do not seek help from mental health services, do not receive the care mentioned and are not detected or followed-up by them. This reality led to the development of integrated programs, interventions and more specific studies, to try to overcome the obstacles in the accessibility to the health services and on the follow-up of these patients. Amongst the barriers identified, feasibility and implementation of those programs have been of special relevance in recent scientific literature, as well as the best way to overcome such difficulties and adapt the interventions to the various realities, cultures and resources. Objectives: General objectives were defined: 1) Assessment of the feasibility and initial implementation of an integrated care program, for people with schizophrenia or schizoaffective disorder, in the clinical setting of community mental health teams, in a psychiatric department from the national health service in Portugal; 2) Impact evaluation of the integrated care program, for these patients and their mental health care delivery. Methods: We drew up an integrated care program (Program Integrar) based on the clinical case management model, with continuous and integrated follow-up. Each patient got one case manager, an individual care plan and kept the same psychiatrist. Were selected the appropriated interventions, namely: psycho-educative, family-based interventions, strategies for dealing with the symptoms and the disorder, relapse prevention and interventions to improve social and occupational functioning. These interventions were always associated with psychopharmacological treatment. The investigation was outline with two parts: assessment of the feasibility and initial implementation of the Program Integrar (part A of the study) and impact evaluation of the program (part B of the study). We designed a naturalistic, prospective, intervention study, non-randomized and without control group. Our chosen sample was made with successive referrals of patients with the diagnosis of schizophrenia or schizoaffective disorder, followedup in one of the five community mental health teams of the Psychiatric Department of Centro Hospitalar Lisboa Ocidental, with a catchment area for a population of about 400 000 people. Different stages, strategies, criteria and indicators for studying the feasibility of the program and its implementation were set and the tasks, procedures and recommended interventions of the case managers were monitored and evaluated. We did two assessments with an interval of one year and we evaluated the following dimensions (the acronym of the assessment instrument used in brackets): psychopathology (BPRS), depression (MADRS), needs (CAN), disability (DAS), social and occupational functioning (SOFAS), attitude toward medication (DAI), insight (SAI), quality of life (WHOQOL-S) and satisfaction (POCS). Results: Of the 146 patients who started the study, 97 (66%) were male and 49 (34%) females with a mean age of 36 years. Of these, 116 (79,4%) were diagnosed (ICD10) with schizophrenia and 30 (20,6%) with schizoaffective disorder. The other socio-demographic data were typical of populations within Portuguese mental health services. Of all patients (146), who started the program, 26 (18%) of patients left the program (program dropout rate). Of the regarding part A of the study, which focused on feasibility, the following is of note: all professionals who had been trained for this purpose (15) acted as case manager, 76% did the recommended minimum number of sessions / year per patient (≥18) and 44,9% did the minimum number of family sessions desired (≥ 3). For the more specific interventions the parameters set out were met for more than 75% of patients, with the exception of domiciliar interventions (19.4%), prevention of substance abuse (45.4%) and suicide risk prevention(34.3%). The individual care plan was done for 98% of patients and in 38,9% of cases this involved family participation. For this plan the mean objectives defined were 5 and in average was achieved 3 (p=0,001). On the first assessment, 42 patients (28.8%) were attending psychosocial rehabilitation structures and 12 months later that number rose up to 80 (74,1%). Regarding their employment status, in the first assessment 8 (7,4%) were in full time employment and in the second evaluation the number rise to 18 (16,7%). For part B of the study (impact program evaluation), in terms of psychopathology, global mean scores for the BPRS, decreased (p=0,001), as did the four sub scales: positive symptoms (p=0,003); negative symptoms (p=0,002); manic symptoms (p=0,002) and symptoms of depression/anxiety (p=0,001). Both in the evaluation of depression (p=0,001), as in the assessment of disability (p=0,003), the differences were significant. However, this was not the case with attitudes towards medication (p=0,690) and with insight evaluation (p=0,079). In relation to social and occupational functioning, quality of life and patient satisfaction there was a statistically significant improvement from the first to the second assessment. The most commonly unmet needs in the first assessment were daily activities, social contacts, intimate relationships, sexual relations, social benefits, psychotic symptoms,psychological distress, information about the disorder / treatment and money problems money management. Of these, in the second assessment, all of those nine unmet needs showed significant improvement, excepted intimate relationships, sexual relations and Money problems / money management. In the distribution of the three states of needs for all items, it happened a decreased in unmet needs and partially met needs and increased in the situations where relevant needs were no longer found. For other clinical indicators it is important to note, when we compared the year prior to this program and the year after, there were fewer hospitalizations (reduction of 64,1% of admissions) and in the mean number of admissions (p=0,001). Regarding the length of hospitalization in the year prior to the program, the 39 patients admitted had a total of 1522 hospital days, and in the year of the program for the 14 hospitalized patients, the total was 523 days. In absolute terms, there was a reduction of 999 days (65,6%). There was also a 45,6% reduction of relapses (p = 0,001). Discussion and Conclusions: The feasibility of the integrated care program allowed the application of the clinical case management model, with continuous follow-up. Each patient got a case manager and in 98% of the cases they also got an individual plan of care. Families continued to be the main support for patients but, difficulties occurred when it was claimed a more active participation. The decrease in the number and duration of admissions were important findings with implications not only in clinical terms but also in economic field. The achieved results for the different variables can also indicate the favorable impact of this program, at the level of psychopathology, needs, disability, social and occupationa functioning, quality of life and patient satisfaction. The same did not happen for the evaluation of insight and in the changes of attitudes towards medication. These data should also be considered for future readjustments of this program and for the developing of new programs.Finally, the two-overview conclusions are: 1) It was possible the feasibility of an integrated care program and initial implementation of this innovative program, for patients with schizophrenia or schizoaffective disorder, with the human and material resources available in the clinical context of the community mental health teams, in a psychiatry and mental health department of the national health service in Portugal; 2) In assessing the impact of the program, the results suggest potential application of this integrated care program, to improve clinical state and psychosocial variables for these patients. There should be done studies to replicate these results, however the results obtained are promising for the development of similar programs at nationally and internationally level, that could benefit a wider group of patients.

Insights into the epidemiology of drug-resistant Streptococcus pneumoniae and closely-related streptococci in the era of conjugate vaccines

In Portugal, the introduction of the seven-valent pneumococcal conjugate vaccine (PCV7) has led to significant changes in the population structure of Streptococcus pneumoniae. However, the levels of antimicrobial resistance have not decreased and have been a matter of concern. (...)

The patent expiry as a major inflection point in a branded Rx drug's life and strategies to counter generic competition - exemplified at Pfizer's blockbuster drug Lipitor in the USA

A large number of expensive, but highly profitable branded prescription drugs will go off-patent in the USA between 2011 and 2015. Their revenues are crucial to fund the immense costs associated with the development of an innovative drug. The rising cost pressure on pharmaceutical stakeholders has increased the demand for more affordable medications, as provided by the branded drug's generic counterpart. Yet, research based incumbents are moving beyond the traditional late lifecycle strategies and deploy more aggressive tactics in order to protect their brands, as seen with Pfizer's Lipitor!. It is doubtful, whether these efforts will help the blockbuster business model to resist current market conditions.

Novel antibiotic-loaded orthopaedic bone cements: insights on drug release profiles and biocompatibility

Acrylic bone cement (BC) is widely used as an anchor of artificial joints. Bacterial infection due to biofilm formation and inflammation are common and difficult to treat problems associated with commercial available BC formulations. Research on novel BC compositions is urgently needed. The main objective of this thesis was to develop a new biocompatible antibiotic-loaded BC with improved release profile. To achieve that aim several additives were incorporated, as an antibiotic (levofloxacin) to combat bacterial growth, an anti-inflammatory drug (diclofenac) to decrease the inflammatory process and two well-known and broadly used biopolymers, alginate and chitosan in order to increase matrix porosity, and in this way to intensify the amount of released drug. Novel BC formulations were tested in order to find the most suitable one that had potential to proceed to clinical application. Numerous tests were conducted as: a) evaluation of drug release profiles in different biomimetic media, b) mechanical and surface studies, c) microbiological activity testing against Staphylococcus aureus and d) in vitro biocompatibility assays (fibroblasts and osteoblasts). In general, the addition of biopolymers increased drug release, didn’t compromised BC mechanical properties and increased BC hydrophilicity. Microbiological testing revealed that Lev[BC]Chi was the only matrix that reduced significantly biofilm formation. On the contrary, alginate and diclofenac loading into BC seemed to increase biofilm growth. Biocompatibility studies showed some decrease in cell viability, in particularly on osteoblasts, mainly due to the high amounts of released drugs. In conclusion, the present work has shown that the matrix with more potential to proceed in further investigations was Lev[BC]Chi. Other conditions (namely additives and drugs concentrations) should be evaluated with the other tested BC matrices before being discharged.

Contribution to drug discovery and development for tauopathies using yeast as a model

This work aimed to contribute to drug discovery and development (DDD) for tauopathies, while expanding our knowledge on this group of neurodegenerative disorders, including Alzheimer’s disease (AD). Using yeast, a recognized model for neurodegeneration studies, useful models were produced for the study of tau interaction with beta-amyloid (Aβ), both AD hallmark proteins. The characterization of these models suggests that these proteins co-localize and that Aβ1-42, which is toxic to yeast, is involved in tau40 phosphorylation (Ser396/404) via the GSK-3β yeast orthologue, whereas tau seems to facilitate Aβ1-42 oligomerization. The mapping of tau’s interactome in yeast, achieved with a tau toxicity enhancer screen using the yeast deletion collection, provided a novel framework, composed of 31 genes, to identify new mechanisms associated with tau pathology, as well as to identify new drug targets or biomarkers. This genomic screen also allowed to select the yeast strain mir1Δ-tau40 for development of a new GPSD2TM drug discovery screening system. A library of unique 138 marine bacteria extracts, obtained from the Mid-Atlantic Ridge hydrothermal vents, was screened with mir1Δ-tau40. Three extracts were identified as suppressors of tau toxicity and constitute good starting points for DDD programs. mir1Δ strain was sensitive to tau toxicity, relating tau pathology with mitochondrial function. SLC25A3, the human homologue of MIR1, codes for the mitochondrial phosphate carrier protein (PiC). Resorting to iRNA, SLC25A3 expression was silenced in human neuroglioma cells, as a first step towards the engineering of a neural model for replicating the results obtained in yeast. This model is essential to understand the mechanisms of tau toxicity at the mitochondrial level and to validate PiC as a relevant drug target. The set of DDD tools here presented will foster the development of innovative and efficacious therapies, urgently needed to cope with tau-related disorders of high human and social-economic impact.