Orientadores: André Deeke Sasse, Carmen Silvia Passos Lima / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T01:24:11Z (GMT). No. of bitstreams: 1 Lima_JoaoPaulodaSilveiraNogueira_D.pdf: 5411736 bytes, checksum: d1c0f5a06bb4361a4b7416cd43d94af5 (MD5) Previous issue date: 2012 / Resumo: Introdução: a superioridade de regimes baseados em camptotecinas sobre os regimes baseados em etoposidio para câncer de pulmão de células pequenas, doença extensa, (CPCP-DE) e assunto amplamente debatido com achados contraditórios nos estudos randomizados que compararam estas combinações. Variações farmacogenomicas entre etnias surgiram como hipótese para justificar estas diferenças. Com objetivo de elucidar esta duvida e mensurar a extensão de um eventual beneficio, realizamos uma revisão sistemática e metalize. Métodos: Revisão sistemática da literatura com metanalise. Foram elegíveis estudos randomizados controlados que comparassem o uso de análogo de platina associado ou a camptotecina (irinotecano ou topotecana) ou ao etoposidio como tratamento de primeira linha para CPCP-DE. Buscamos estes estudos nas bases PubMed, EMBASE, CENTRAL, LILACS e nos sítios eletrônicos dos congressos da ASCO, ESMO, ECCO e IASLC. As avaliações de sobrevida global (SG) e sobrevida livre de progressão mediana (SLP) foram apresentadas como hazard ratio (HR) enquanto a taxa de resposta tumoral (RT), toxicidades graves e sobrevida global em um e dois anos foram expressas através de odds ratio (OR). Os intervalos de confiança de 95% foram calculados para cada desfecho. A metalize utilizou método de efeitos randomicos, sendo a heterogeneidade entre estudos expressa através do índice de heterogeneidade I². Avaliação de subgrupos conforme origem geográfica dos estudos foi realizada e o teste de interação foi utilizado para identificar eventuais diferenças. Irinotecano-platina (IP) e topotecana-platina (TP) foram avaliados separadamente. Resultados: 615 referencias foram avaliadas, sendo selecionados nove estudos (3527 pacientes). Dois estudos compararam TP versus EP enquanto sete compararam IP versus EP. A metanalise demonstrou que IP e capaz de aumentar a sobrevida global (1965 pacientes, HR = 0,87; 95% CI 0,80-0,95; P=0,002; I²=0%) e sobrevida global em um ano (HR = 0,74; IC95% 0,60-0,91; P = 0,004; I² = 14%), sem contudo, aumentar claramente a sobrevida livre de progressão, ou resposta tumoral. IP levou a maior incidência de toxicidades gastrointestinais e menos hematológicas. A avaliação de subgrupos conforme origem geográfica não identificou diferença na extensão de beneficio em sobrevida global e em um ano (interação ocidente versus oriente P = 0,34 e P = 0,08 respectivamente) com IP. Na analise de sobrevida global em dois anos, IP e superior a EP em pacientes europeus e asiáticos, sem contudo trazer ganho para pacientes da América do Norte (OR 1,05; IC95% 0,65-1,69; P = 0,85; interação América do Norte versus Europa, P = 0,009; interação América do Norte versus Ásia, P = 0,02). IP aumenta a sobrevida livre de progressão para pacientes orientais (HR = 0,61; IC95% 0,45 - 0,84; P = 0,01) sem ter impacto nos pacientes ocidentais (HR = 0,96; IC95% 0,82 - 1,13; P = 0,65%) com diferença estatisticamente significativa entre estes dois grupos (interação ocidente versus oriente, P = 0,03). O teste de interação não identificou impacto em taxa de resposta tumoral ao esquema IP conforme a origem geográfica (interação ocidente versus oriente P = 0,07). Nas diversas toxicidades avaliadas, não houve diferença entre pacientes ocidentais e orientais. A metanalise de TP versus EP não foi possível devido a heterogeneidade em todos os desfechos avaliados. Conclusão: O presente estudo confirma que IP aumenta sobrevida global tanto para pacientes ocidentais quanto orientais. A sobrevida livre de progressão sob o regime IP e a sobrevida global em dois anos parecem diferir conforme a origem geográfica, enquanto a taxa de resposta e tolerância não. IP pode ser considerado um novo padrão terapêutico para CPCP-DE em todo o globo, com diferenças regionais que merecem avaliação mais profunda / Abstract: Background: Superiority of camptothecin regimens over etoposide - both combined with platinum analogs - in extensive disease small cell lung cancer (ED-SCLC) has been extensively debated, with contradictory results in randomized trials worldwide. Ethnic and pharmacogenomical issues were hypothesized as major causes for these divergent findings. A systematic review was sought to elucidate this confounding scenario. Methods: Randomized controlled trials comparing first-line camptothecin (either irinotecan or topotecan)-platinum doublets (CP) versus etoposide-platinum doublets (EP) in ED-SCLC patients were searched in PubMed, EMBASE, and CENTRAL databases, ESMO, ASCO, and IASLC meeting proceedings. Meta-analyses were performed using random-effects model. Overall survival (OS) and Progression Free survival (PFS) were pooled as hazard ratio (HR), whereas response rate and toxicity were computed as odds ratio (OR). Pertinent 95% confidence intervals (CI) were calculated for all outcomes. Heterogeneity was measured by I². Subgroup analyses were undertaken comparing the geographical area of study and interaction tests were used to evaluate any existing differences found among subgroups. Irinotecanplatinum (IP) and topotecan-platinum (TP) trials were evaluated separately. Results: Nine studies (3527 patients) were included. IP improved median OS (1965 patients; HR = 0.87; 95% CI 0.80- 0.95; P=0.002; I²=0%) and one year OS (HR = 0.74; IC95% 0.60-0.91; P = 0.004; I² = 14%). PFS meta-analysis of IP versus EP was not feasible due to impending heterogeneity (I²=80%), as response rate meta-analysis (I²=56%; absolute response rates: IP = 52%, EP = 50%). There was more severe gastrointestinal toxicity with IP than EP, but less hematological toxicity. The median, and the one year OS analyses according to geographical area demonstrated that eastern and western patients experienced similar benefit from IP (interaction test Western trials versus Eastern trial, P=0.34 and P=0.08 respectively); however the two year OS analysis according to continental origin stated that European and Asian patients derived benefit from IP, whereas North Americans did not (interaction North America versus Europe, P = 0.009; North America versus Asia, P = 0.02). IP improved progression free survival in the Eastern trial (HR = 0.61; CI95% 0.45 - 0.84; P = 0.01), while it had no impact in Western trials (HR = 0.90; CI95% 0.78 - 1.03; P = 0.13; I² = 24%) with difference according to geographical origin (interaction test Western versus Eastern trials, P = 0.03). There was no identifiable difference in response rate to IP due to trial origin (interaction test, P = 0.07). In all toxicity sought, there was no suggestion of different toxicity according to trial origin. The metaanalysis of TP was not reliable due to impending heterogeneity in all outcomes. Conclusion: The present meta-analysis demonstrated that IP improved median and one year OS for both Western and Eastern patients. The activity of IP in terms of PFS and two year OS may be subject of geographical origin of patients ,while IP tolerance seemed to be stable. The present findings corroborate the role of IP as a new standard of care for SCLC-ED worldwide, with regional differences that merit further appraisal / Doutorado / Clinica Medica / Doutor em Clínica Médica

Human microglia play a pivotal role in neurological diseases, but few targeted therapies that directly modulate microglial state or function exist due to an incomplete understanding of microglial heterogeneity. This thesis aims to advance our understanding of microglial heterogeneity by using single-cell RNA sequencing to profile live human microglia from autopsies or surgical resections across diverse neurological diseases and using computational tools to infer chemical and genetic regulators of specific microglial substates. Chapter 1 provides an overview of microglial ontogeny, function, and known heterogeneity, especially in disease contexts. It also describes the steadily increasing disease burden seen in neurological disease as well as the lack of efficacious treatments and future directions for microglia-targeted therapies. Chapter 2 focuses on microglial heterogeneity in an understudied disease, ALS, describing population structure shifts seen in ALS across cortex and spinal cord. Chapter 3 instead focuses on exploring underlying cross-disease microglial population structure, identifying subsets with metabolic and functional properties, as well as subsets enriched in susceptibility genes for neurodegenerative disease. We then demonstrate applications of this type of data by using our resource to annotate other datasets. Chapter 4 leverages this data in another way, by identifying and validating candidates for chemically and genetically inducing subtype-specific states in vitro. Notably, we show that Camptothecin downregulates the transcriptional signature of disease-enriched subsets and upregulates a signature previously shown to be depleted in Alzheimer’s. Finally, I review our findings and discuss future directions for the field.

No description available.

No description available.

We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 µm. The diffusive limit of competition for media occurred with a pitch of ≥1250 µm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

No description available.

A dissertation submitted to the Faculty of Science, University of Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. March 2015 / Lung cancer is currently the most deadly form of cancer due to the fact that metastasis occurs in the lymph nodes making it difficult to remove by surgical means. Chemotherapy has been the most successful method of treatment, although it has been harmful to human health as a consequence of non-specific cytotoxicity. There has been, therefore, a growing interest in cancer research to develop alternative cancer treatments, which are less toxic. Currently plant-derived drugs are perceived to be more effective as they display both cytotoxic activity and are less harmful to overall human health. Thus the aim of the study was to determine the cytotoxic effects of the plant Lobostemon fruticosus on A549 cells. The IC50 of the methanol and butanol extracts of L. fruticosus were obtained at 40 μg/ml and 50 μg/ml, respectively. DNA fragmentation was observed after 48 hour exposure to treatments, indicating that the plant extracts induced apoptosis. Cell cycle analysis indicated that the plant extracts inhibited cell cycle progression at the sub-G0 phase, which indicated that the cells had undergone apoptosis. RT-PCR showed that the expression of p53 was down-regulated; however, p21 and Bax were up-regulated in all treatments. LC-MS identified that the compounds from the plant extracts are known apoptotic inducers. The results lead to the conclusion that the extracts of L. fruticosus, induce cell death in A549 cells. The plant extracts induced a p53-independent apoptotic mechanism, which was mediated by Bax and p21. Key words: Lobostemon fruticosus, camptothecin, taxol, Non-small cell lung cancer (NSCLC)

The survival for high-grade glioma patients is poor and the treatment may cause severe side effects. A common obstacle in the treatment is chemoresistance. To improve the quality of life and prolong survival for these patients prognostic biomarkers and new approaches for chemotherapy are needed. To this end, a strategy to evade chemoresistance was evaluated by combining chemotherapeutic drugs with agents inhibiting resistance mechanisms identified by a bioinformatic analysis (paper I). The prognostic value of 13 different proteins was analyzed in this thesis (papers II-IV). Two of them, p38 mitogen-activated protein kinase (MAPK) and protein tyrosine phosphatase non-receptor type 6 (PTPN6, also known as SHP1) were analyzed for their potential as targets in combination chemotherapy (in paper III and IV, respectively).   We found that: PTPN6 expression and methylation status may be important for survival of anaplastic glioma patients, p38 MAPK phosphorylation may be a potential negative prognostic biomarker for high-grade glioma patients and FGF2 expression may be a potential negative prognostic biomarker for proneural glioma patients. PTPN6 may be a useful target for combination chemotherapy with cisplatin, melphalan or bortezomib in high-grade gliomas. The following drug combinations; camptothecin combined with an EGFR or RAC1 inhibitor, imatinib combined with a Notch or RAC1 inhibitor, temozolomide combined with an EGFR or FAK inhibitor and vandetanib combined with a p38 MAPK inhibitor may be useful combination chemotherapy for high-grade gliomas.

Block copolymer nanoparticles (NPs) have gained great attention among researcher for various medical application mainly due to their extraordinary optical, chemical, and biological properties. The current thesis presents design of multifunctional polymeric NPs for imaging and drug delivery system (DDS) with an in-vitro study of their participation in drug release and cell viability. The NPs were synthesized using reversible addition chain fragmentation transfer (RAFT)-mediated emulsion polymerization via polymerization induce self-assembly (PISA) approach. The environment-friendly emulsion polymerization process of n-buytl acrylate (n-BA) in water is highly efficient. The process produced uniform NPs which would have control over the particle size and molecular weight of the compound. Herein we report a novel simultaneous encapsulation of camptothecin (CPT) and Nile red (NR) into poly(ethylene glycol) methyl ether methacrylate-co-N-hydroxyethyl acrylamide-b-poly n-buytlacrylate (PEGA-co-HEAA)-b-P(n-BA) during the particles formation with a small particle size of 66 nm, high conversion ~80% and encapsulation efficiency of ~50%. The In vitro drug release of the CPT from the NPs exhibited an initial burst (70-80%) within 6h. cell viability was evaluated for the NPs against RAW 264.7 cell line, which indicated the designed NPs are biocompatible and not toxic.

[pt] Os derivados da camptotecina (CPT), irinotecana (CPT-11) e topotecana (TPT) são usados para o tratamento do câncer, sendo a CPT um potencial contaminante em medicamentos anticâncer a base de CPT-11 ou TPT. Neste trabalho, a fosforimetria na temperatura ambiente em substrato sólido (SSRTP) foi proposta como técnica analítica para a determinação do princípio ativo do anticâncer injetável a base de CPT-11 e de traços do contaminante CPT em formulações farmacêuticas anticâncer. As características fosforescentes dos dois analitos foram estudadas de modo univariado em função de diversos parâmetros experimentais, como o tipo e a quantidade de sal de átomo pesado indutor de fosforescência, influência do valor do pH do tampão usado na solução do analito e quantidade de surfactante modificador de superfície da celulose. Uma vez definidos os fatores relevantes, o planejamento fatorial do tipo composto central foi realizado para a determinação de traços do contaminante (CPT) com o intuito de estudar os efeitos principais e as possíveis interações entre os fatores de resposta visando à escolha da melhor condição experimental. As melhores condições foram obtidas usando substratos de celulose contendo 332 microgramas de TlNO3 (indutor da fosforescência) em solução carreadora contendo tampão Britton-Robinson (pH 10,5). Para a CPT-11 a otimização foi realizada apenas pela abordagem univariada, com as seguintes condições escolhidas: 662 microgramas de Pb(NO3)2 em substratos de celulose contendo 577 microgramas de SDS. A CPT pôde ser determinada seletivamente em matrizes contendo 40 vezes mais TPT, em concentração, usando a determinação no ponto isodiferencial (367 nm) da derivada de segunda ordem do espectro de excitação. Para matrizes contendo CPT-11, esse desempenho foi mais limitado, pois a CPT só foi determinada seletivamente em misturas contendo até 5 vezes mais CPT-11. Para cada uma das condições selecionadas foram realizados estudos para obtenção dos parâmetros de desempenho. Em ambos os casos, a resposta analítica teve comportamento linear (homocedático) em função da massa de CPT ou de CPT-11 presentes no substrato de celulose. Os limites de detecção e de quantificação absolutos ficaram na ordem do ng. Um estudo detalhado da estimativa da incerteza de medição também foi realizado e a incerteza combinada associada à medição de fosforescência da CPT foi de até 16 por cento. O método foi aplicado na quantificação de CPT-11 em soluções injetáveis com 97,5 mais ou menos 5,5 por cento de recuperação e na determinação de CPT em medicamentos a base de TPT (101,5 mais ou menos 3,5 por cento de recuperação medindo em 367 nm do espectro de excitação após derivação de segunda ordem) e nas matrizes urina (102,5 mais ou menos 3,5 por cento de recuperação) e saliva (102,5 mais ou menos 4,5 por cento de recuperação), ambas fortificadas com CPT e usando-se detecção em 570 nm do espectro de emissão de varredura normal. Testes comparativos entre a SSRTP e a HPLC-DF foram realizados e os resultados foram satisfatórios para um nível de 95 por cento de confiança. Uma comparação entre diferentes substratos foi também realizada para avaliar requisitos práticos, variabilidade de sinal do analito e do branco, o que indicou vantagens do substrato de nylon sobre o de celulose. / [en] Irinotecan (CPT-11) and topotecan (TPT) are employed for cancer treatment and camptothecin (CPT) is a potential contaminant in anti-cancer drugs based on CPT-11 or TPT. In this work, solid substrate room-temperature phosphorimetry (SSRTP) was proposed as analytical technique for the quantification of CPT-11 in anti-cancer drugs and for the determination of traces of CPT in CPT-11 and TPT based anti-cancer pharmaceutical formulations. The phosphorescence characteristics of the analytes have been studied and experimental conditions (type and amount of the heavy atom salts used to induce phosphorescence, influence of the pH of the analyte carrier solution and the amount of surface modifier) were optimized in an univariate way. For the method aiming the determination of CPT, a further optimization using a central composite design was made in order to identify the main effects and possible interactions among factors. The best conditions had been achieved using cellulose substrate containing 332 micrograms of TlNO3 (phosphorescence inducer) and analyte carrier solution containing Britton-Robinson buffer (pH 10.5). For the CPT-11, best conditions were achieved in cellulose substrates containing 577 micrograms of SDS and 662 micrograms of Pb(NO3)2 The selective determination of CPT could be performed in samples containing a higher amount of TPT (40 times) if the signal measurement is made at the isodifferential wavelength (367 nm) of the second derivative excitation spectra. For samples containing CPT-11, selective determination of CPT could be made in samples containing CPT-11/CPT molar proportion no higher than 5. Parameters of merit have been obtained for both methods. Analytical responses presented linear behavior in the in working range from the limit of quantification up to at least 348.0 ng of CPT or 440.2 ng of CPT-11 (deposited in the center of the substrate). Absolute limits of detection and quantification were 26.8 and 42.3 ng for CPT and 79.6 and 99.9 ng for CPT-11. A detailed metrological study was performed for the measurement of CPT and the combined uncertainty associated to the phosphorescence measurement was 16 percent. The method was applied for the quantification of CPT-11 in injectable solutions with recovery of 97.5 more or less 5.5 percent. For CPT, recovery in TPT based pharmaceutical formulation, previously fortified with the analyte, was 101.5 more or less 3.5 percent (measurement made at 367 nm of the second derivative excitation spectra). In analyte fortified urine and saliva, recoveries were respectively 102.5 more or less 3.5 percent and 102.5 more or less 4.5 percent (using non-derived spectra and detention at 570 nm of the emission band). Comparative tests between the SSRTP and HPLC-DF have been made and the results agreed (at a 95 percent confidence level). A comparison using different substrates (nylon and cellulose) was also performed in order to evaluate practical aspects, analyte signal intensity and the variability of the analyte and blank signals. The result indicated advantages in using nylon substrates for the phosphorimetric determination of CPT.