This thesis describes the design and synthesis of molecules with potential roles in biomedicine, with an emphasis on molecular recognition in complex biological environments. The first chapter describes the synthesis and evaluation of compounds for use in nuclide therapy. Carboranes are frequently used in the development of drugs for Boron Neutron Capture Therapy. New routes for monohydroxylation at the B and C atoms of p-carborane have been developed. The Suzuki-Miyaura reaction has been applied to the cross-coupling of bis(neopentyl glycolato)diboron or bis(pinacolato)diboron and 2-I-p-carborane. The synthesized derivatives are important intermediates in the synthesis of a number of potentially biologically active carborane-containing molecules. The DNA intercalator doxorubicin has been functionalized to enable 125I labelling. The aim of combining the DNA intercalator with 125I was to achieve high delivery of cytotoxic radiation to the nucleus. The DNA-binding ability and cellular uptake of the synthesized compounds have been evaluated. One of the compounds bound strongly to DNA and had similar cellular uptake as daunorubicin, which makes the compound very interesting for further biological evaluation. The second chapter describes the use of polypeptide conjugates to broaden our knowledge of molecular recognition. The polypeptides consist of 42 amino acids each and are designed to fold into helix-loop-helix motifs that dimerize due to their amphiphilic character. The polypeptides are combined with a variety of small organic molecules. The incorporation of small aromatic molecules to influence the structure and dynamics of a polypeptide has been investigated. By attaching a dansyl group to the side chain of a lysine residue, the dynamics of the protein’s hydrophobic core where affected to such a degree that a native-like fold was formed. The polypeptide conjugates have also been used to study the binding and recognition of native proteins. High-affinity binders for chitinases and acetylcholine esterase have been developed and evaluated.

This thesis describes the synthesis and radiohalogenation of compounds of potential use for tumor targeting. The first section describes the synthesis and radioiodination of DNA intercalating compounds. The compounds are derivatives of 9-aminoacridine, and the anthracyclins daunorubicin and doxorubicin. The precursor compounds were labeled with 125I (T1/2 = 60 days), which is an Auger emitting nuclide. 125I decaying in the close vicinity of DNA is known to have a much higher cell killing effect than 125I decaying in the cytoplasm and some of the labeled compounds prepared in this thesis are currently being tested for use in targeted radionuclide therapy for cancer. The second section describes the radiobromination of closo-carboranes by subjecting the corresponding iodinated compounds to palladium-catalyzed halogen exchange using [76Br]bromide. The 76Br isotope (T1/2 = 16.2 h) is a positron emitting nuclide that is suitable for PET studies. Via the halogen exchange reaction good to excellent radiochemical yields of radiobrominated closo-carboranes were obtained. The results of the present study may prove to be applicable to pharmacokinetic studies of carboranes and their derivatives. The third and final section describes the indirect radiobromination of the trastuzumab anti-HER2 monoclonal antibody and of the anti-HER2 Affibody by means of an “one-pot” procedure using N-succinimidyl-5-(tributylstannyl)-3-pyridinecarboxylate (SPC) and ((4-hydroxyphenyl)ethyl))maleimide (HPEM), respectively. It was found that SPC and HPEM can be efficiently radiobrominated and thereafter coupled to the antibody and Affibody, respectively. The labeled proteins retained their capacity to bind specifically to HER2 expressing SKOV-3 cells in vitro. Application of this method to 76Br might enable the use of PET in the detection of HER2 expression in breast, ovarian, and urinary bladder carcinomas.

Chemotherapy is one of the common treatments in cancer therapy. The effectiveness of chemotherapy is limited by several factors one of which is the emergence of multidrug resistance (MDR). MDR is caused by the activity of diverse ATP binding cassette (ABC) transporters that pump drugs out of the cells. There are several drugs which have been used in treatment of cancer. One of them is doxorubicin that intercalates and inhibits DNA replication. However, doxorubicin has been found to cause development of MDR in tumors. It has been reported that there is a correlation between multidrug resistance and invasiveness of cancer cells. Vimentin is a type III intermediate filament protein that is expressed frequently in epithelial carcinomas correlating with invasiveness and also poor prognosis of cancer. There are several studies that have shown the connection between expression level of vimentin and invasiveness. In this study, MCF-7 cell line (MCF-7/S), which is a model cell line for human mammary carcinoma, and doxorubicin resistant MCF-7 cell line (MCF-7/Dox) were used. The resistant cell line was previously obtained by stepwise selection in our laboratory. The main purpose of this study was to investigate changes of metastatic behaviour in MCF-7/Dox cell line, after transient silencing of vimentin gene by siRNA. In conclusion, down-regulation of vimentin gene expression in MCF-7/Dox cell lines was expected to change the characteristics in migration and invasiveness shown by migration and invasion assays.

No description available.

Η θεραπεία του καρκίνου αποτελεί στις μέρες μας μία από τις μεγαλύτερες προκλήσεις της επιστημονικής κοινότητας παγκοσμίως. Τα συμβατικά φάρμακα που κυκλοφορούν εδώ και χρόνια προσβάλλουν τόσο τα καρκινικά όσο και τα υγιή κύτταρα. Το αποτέλεσμα είναι η πρόκληση σοβαρών παρενεργειών, οι οποίες δεν επιτρέπουν τη χορήγηση θεραπευτικών δόσεων. Η ανάπτυξη της νανοτεχνολογίας έχει βοηθήσει κατά πολύ το επιστημονικό πεδίο όσον αφορά τη διάγνωση και τη θεραπεία του καρκίνου. Τα νανοσωματίδια παρέχουν ένα νέο τρόπο χορήγησης των αντικαρκινικών φαρμάκων, λειτουργώντας ως φορείς που εξαγγειώνονται στην περιοχή του όγκου, επιτρέποντας με αυτόν τον τρόπο άμεση πρόσβαση στα καρκινικά κύτταρα. Τα συγκεκριμένα σωματίδια μπορούν να τροποποιηθούν κατάλληλα, ώστε να αποκτήσουν τη δυνατότητα να δεσμευθούν στις μεμβράνες των καρκινικών κυττάρων, στο μικροπεριβάλλον τους ή σε κυτταροπλασματικούς ή πυρηνικούς υποδοχείς. Κατά συνέπεια, μεταφέρονται στοχευμένα υψηλές συγκεντρώσεις του φαρμάκου στα καρκινικά κύτταρα και μειώνεται η τοξικότητα στους φυσιολογικούς ιστούς. Η ανάπτυξη τροποποιημένων νανοφορέων για τη χορήγηση των αντικαρκινικών φαρμάκων μπορεί να περιορίσει τα προβλήματα που συνδέονται με τα συμβατικά συστήματα χορήγησης, όπως έλλειψη εκλεκτικότητας και τοξικότητα. Οι νανοφορείς που έχουν μελετηθεί μέχρι σήμερα, εμφανίζουν σημαντικά προβλήματα, όπως δομική αστάθεια, δομική ετερογένεια και ελλειπή έλεγχο μεγέθους και σχήματος. Υπάρχει, συνεπώς, μια αυξανόμενη ανάγκη για ανάπτυξη προηγμένων συστημάτων μεταφοράς αντικαρκινικών φαρμάκων, στα οποία το σχήμα, το μέγεθος και η ικανότητα φόρτωσης να μπορούν να ρυθμιστούν κατάλληλα, προκειμένου να μεγιστοποιηθεί η αποτελεσματικότητα και να ελαχιστοποιηθεί η τοξικότητα των φαρμάκων. Τα φουλλερένια (C60) ανήκουν στην κατηγορία των νανοφορέων που μελετούνται εκτενώς για τη χορήγηση αντικαρκινικών παραγόντων. Η δυνατότητα των φουλλερενίων για την εκπλήρωση των παραπάνω προδιαγραφών έγκειται στη μεγάλη δυνατότητα τροποποίησης της βασικής τους δομής και την παρασκευή πληθώρας παραγώγων με επιθυμητές ιδιότητες. Το μεγαλύτερο μειονέκτημα αυτών των μορίων είναι η ελάχιστη διαλυτότητά τους στο νερό. Το πρόβλημα αυτό μπορεί να αντιμετωπιστεί με την πρόσδεση σε αυτά υδρόφιλων πολυμερών, όπως είναι η πολυαιθυλενογλυκόλη (PEG). Στην παρούσα εργασία, παρουσιάζονται τα αποτελέσματα της σύνθεσης ενός συζεύγματος πεγκυλιωμένου μορίου φουλλερενίου (C60) με το αντικαρκινικό φάρμακο δοξορουμπικίνη, καθώς και η in vitro αξιολόγηση του τελικού προϊόντος. Στόχος είναι η αύξηση της αποτελεσματικότητας και η μείωση της τοξικότητας του φαρμάκου, μέσω στοχευμένης μεταφοράς στον καρκινικό όγκο. Ένα πεγκυλιωμένο σύζευγμα φουλλερενίου (C60) με δοξορουμπικίνη (FULL-PEG-DOX) συντέθηκε επιτυχώς σε έξι στάδια με συνολική απόδοση 25,7% και χαρακτηρίστηκε με 1H-NMR, IR και UV. Η δραστικότητα του τελικού προϊόντος, καθώς επίσης και δύο ενδιάμεσων προϊόντων (FULL-PEG και FULL-DOX) αλλά και του υδροχλωρικού άλατος της δοξορουμπικίνης ενάντια στον καρκίνο ελέγχθηκε in vitro σε καρκινικές σειρές κυττάρων MCF-7. Το τελικό προϊόν εμφάνισε ικανοποιητική αντικαρκινική δράση, κυρίως μέσω αναστολής του πολλαπλασιασμού των καρκινικών κυττάρων. Τα αποτελέσματα της μελέτης μπορούν να θεωρηθούν ιδιαίτερα ενθαρρυντικά και δικαιολογούν τη συνέχιση της ερευνητικής προσπάθειας για στοχευμένη χορήγηση δοξορουμπικίνης σε καρκινικά κύτταρα μέσω της πρόσδεσής της σε τροποποιημένα μόρια φουλλερενίου. / Cancer’s treatment is nowadays one of the most important challenges that the scientific community worldwide has to face. Conventional anticancer drugs, which are in the market many years, affect both cancer and normal cells. Hence, they cause serious side effects, which make the administration of therapeutic drug doses impossible. The development of nanotechnology has changed the scientific landscape in terms of cancer diagnosis and treatment. Nanoparticulate drug carriers provide a new mode of cancer drug delivery. They can extravasate at tumor site, allowing direct drug access to cancer cells. These particles allow exquisite modification for binding to cancer cell membranes, the microenvironment or to cytoplasmic or nuclear receptor sites. This results to the delivery of high drug concentrations to the targeted cancer cells with reduced toxicity to normal (healthy) tissue. The application of engineered nanocarriers for the delivery of anticancer drugs may alleviate the problems associated with conventional anticancer drug delivery systems, such as lack of selectivity and toxicity. Drug nanocarriers explored to date suffer from inherent limitations, including instability, structural heterogeneity and poor control over size and shape. There is an increasing need for advanced delivery agents for the anticancer drugs where shape, size, functionalization and loading capacity can be precisely tuned in order to maximize efficacy and minimize drug toxicity. Among the nanocarriers which are currently studied as drug delivery systems for anticancer agents are fullerenes (C60). The potential of fullerenes to address the above specifications lies in the immense scope for chemical derivatization to the basic structure. Except for their high toxicity to normal cells, the main drawback of these carbon molecules is their poor solubility to water. This problem can be overcome by attaching on fullerene particles hydrophilic polymers, such as polyethylene glycol (PEG). In the present work, the results of synthesis and in vitro biological evaluation of a pegylated fullerene conjugate with the potent anticancer drug Doxorubicin are represented. The main goal is to increase efficacy and reduce toxicity of doxorubicin, through targeted delivery to tumors, by conjugating the drug to pegylated fullerenes. A pegylated fullerene-doxorubicin conjugate has been successfully synthesized in 6 steps with a 25.7 % overall yield and was characterized by 1H-NMR, IR and UV. The final product, two intermediate products and doxorubicin hydrochloride were evaluated in vitro against MCF-7 cancer cells lines. The final product exhibited satisfactory anticancer activity mainly by inhibiting proliferation. The results of this study justify further investigation of the potential of pegylated fullerenes as targeted nanocarriers of doxorubicin.

There are currently two types of intra-arterial drug-delivery system (DDS) in clinical use in the palliative treatment of primary liver cancer. The chemotherapeutic drug doxorubicin (DOX) can be formulated into a drug-in-lipiodol emulsion (LIPDOX) or a microparticulate drug-eluting bead system (DEBDOX). To facilitate development of future DDSs, we need to understand the release and local distribution of drug from these DDSs into the complex, in vivo, pathological environment. The overall aim of this project was to assess and improve understanding of the in vivo release of DOX from LIPDOX and DEBDOX and its local disposition in the liver. These processes were investigated in detail in a multisampling-site, healthy pig model and in human patients with liver cancer. The mechanisms involved in DOX disposition were studied by examining potential interactions between DOX and lipiodol and/or cyclosporine A (CsA) in pigs.   In this project, the main elimination pathway for DOX and its primary metabolite doxorubicinol (DOXol) was via bile; their extensive canalicular carrier-mediated transport (e.g. ATP-binding cassette transporters ABCB1, ABCC1, ABCC2 and ABCG2) was inhibited by CsA. CsA had no effect on the carbonyl and aldo-keto reductases responsible for the metabolism of DOX into DOXol. LIPDOX released DOX more rapidly and to a greater extent into the circulation than DEBDOX, which had only released 15% of the dose in patients after 24 hrs. The systemic exposure to DOX was lower for DEBDOX than for LIPDOX. Greater fractions of DOXol were formed in blood and bile with LIPDOX than with DEBDOX. This may have been because DOX was more widely distributed into regions with increased metabolic capacity or because of increased intracellular uptake when DOX was delivered in LIPDOX. The excipient lipiodol in the LIPDOX formulation did not interact with transporters, enzymes or membranes that would explain the increased cellular uptake of DOX. In conclusion, the release of DOX from DEBDOX is more controlled in vivo than that from LIPDOX, indicating that DEBDOX is a more robust pharmaceutical product. The formulations for future optimized DDSs should therefore be more similar to DEBDOX than to LIPDOX.

O Sarcoma de Ewing (ES) é um raro tumor de ossos e tecidos moles com uma característica translocação cromossomal, a fusão EWS/FLI-1, que atua sobre diversos processos oncogênicos. O desenvolvimento da resistência à quimioterapia é comum no tumor e continua como uma das principais causas na falha do tratamento. O objetivo desse estudo foi avaliar a expressão de genes após a indução de resistência em linhagens celulares de ES. Foi selecionado um conjunto de genes (CCAR1, TUBA1A, POLDIP2, SMARCA4 e SMARCB1) a partir da mineração da literatura em resistência tumoral para duas drogas utilizadas na terapia de ES, doxorrubicina e vincristina. Descrevemos a expressão de cada gene selecionado antes e após as linhagens SK-ES-1 serem submetidas a um protocolo de indução de resistência para ambos os fármacos, que obteve êxito ao induzir as células à resistência. A expressão relativa dos níveis de mRNA foi avaliada e foi encontrada em maior expressão para os genes SMARCA4, SMARCB1 e POLDIP2, e em menor expressão para os genes TUBA1A e CCAR1, quando comparadas às linhagens de controle não-resistentes de cada quimioterápico. Os resultados sugerem o envolvimento de mecanismos de reparo de dano ao DNA, remodelamento de cromatina via SWI/SNF, atividade de microtúbulos e atividade spliceossomal nos processos de resistência quimioterápica em ES. / Ewing Sarcoma (ES) is a rare bone and soft tissue tumor with a characteristic chromosomal translocation, the fusion protein EWS/FLI-1, that drives several oncogenic processes. The development of resistance to chemotherapy is common and remains as the main cause of treatment failure. The goal of this study was to evaluate the expression of selected genes in ES cell lines after induction of resistance. A set of genes (CCAR1, TUBA1A, POLDIP2, SMARCA4 and SMARCB1) was data mined from tumoral resistance literature for two drugs used in ES therapy, doxorubicin and vincristine. We describe the expression of each selected gene before and after SK-ES-1 cell lines were exposed to a drug resistance inducing protocol for doxorubicin and vincristine. Cell lines were successfully induced to be resistant to doxorubicin and vincristine. The relative mRNA expression levels were upregulated for genes SMARCA4, SMARCB1 and POLDIP2 and downregulated for genes TUBA1A and CCAR1, when comparing resistant and non-resistant ES cell lines for each drug. The results suggest involvement of repair pathways, SWI/SNF chromatin remodeling, microtubule and spliceosomal activity processes in drug resistance mechanisms in ES.

La résistance des cellules cancéreuses à la chimiothérapie est une cause majeure de l’échec thérapeutique. Des études suggèrent qu’une adaptation du métabolisme énergétique pourrait jouer un rôle dans cette résistance. Ce travail de thèse montre que la résistance des cellules cancéreuses mammaires MCF-7dox à la doxorubicine est associée à une diminution de l’activité du complexe I de la chaîne respiratoire mitochondriale et à un métabolisme des cardiolipines (CL) particulier (diminution de la quantité de CL et augmentation de la quantité de MLCL, la forme immature des CL). Nos résultats montrent aussi que les mitochondries des cellules MCF-7dox expriment deux pompes d’efflux ATP-dépendantes (BCRP et MRP1) qui participent à limiter la quantité de doxorubicine accumulée dans ces mitochondries. De plus, l’activité de ces deux transporteurs dépend partiellement de l’ATP mitochondrial dont l’efficacité de synthèse est améliorée dans les cellules MCF-7dox. D’autre part, nous montrons que l’effet sensibilisant du DHA à la doxorubicine dans les cellules MCF-7dox implique un stress oxydant mitochondrial et s’accompagne d’une diminution de l’efficacité de la synthèse d’ATP. / Resistance of cancer cells to chemotherapy is a major cause of treatment failure. Studies have suggested that an adaptation of energy metabolism may play a role in the development of this resistance. The present work shows that resistance of the breast cancer cell line MCF-7dox to doxorubicin is associated with decreased activity of the mitochondrial respiratory chain complex I and particularly altered cardiolipin (CL) metabolism, (decreased CL levels and increased MLCL levels, the immature form of the CL). Our results also show that mitochondria from MCF-7dox cells express two ATP-dependent efflux pumps (BCRP and MRP1) limiting the accumulation of doxorubicin in these mitochondria. In addition, the activity of these two transporters is partially dependent on mitochondrial ATP synthesis which efficiency is improved in MCF-7dox cells. Moreover, we show that the sensitizing effect of DHA to doxorubicin in MCF-7dox cells is regulated by mitochondrial oxidative stress and is accompanied by a decrease in ATP synthesis efficiency.

Made available in DSpace on 2015-03-26T13:47:16Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1204854 bytes, checksum: 3444b450baa838c9ffdcfffb1cfd08bd (MD5) Previous issue date: 2013-07-05 / Some drugs, such as doxorubicin, among other mechanisms act by triggering apoptosis, which not only affects the tumor but also normal cells in the body. Therefore, the study of the side effects of chemotherapy, seeking a less aggressive treatment for cancer patients, justifies this work. The objective was to investigate the hematological and gastrointestinal effects of doxorubicin and tamoxifen when used alone or in combination in male mice with or without the Ehrlich ascites tumor, with or without hormone therapy with estradiol cypionate. A total of 40 Balb / C male mice were divided into groups of 5 animals each, making 8 groups. At the end of 21 days, blood samples were collected for CBC (Complete Blood Count), aiming at evaluating parameters such as RBC count and hematocrit, hemoglobin, white blood cells count and platelets count. Samples of the stomach and intestines of the animals treated with hydrochloride doxorubicin, were also collected for histopathologic analysis, evaluating when present, the degree of damage to the epithelium and the degree of necrosis and apoptosis glands. The evaluation of the number of erythrocytes and the hematocrit value was significantly decreased (p <0.05) in groups I and VIII when compared to the other groups. Hemoglobin showed a significant decrease in group I in relation to groups II, III, IV and V. Counting the number of leukocytes showed a significant increase (p <0.05) when comparing groups I and VI with the other groups. The main histopathological changes evidenced in the stomach and small intestine were necrosis and apoptosis and loss of glands lining epithelium, and the group VII showed a slight decrease in the degree of lesions in group V. / Alguns medicamentos, como o cloridrato de doxorrubicina, atuam dentre outros mecanismos, desencadeando as vias da apoptose, que atingem não só o tumor, mas também as células normais do organismo. Por isso, o estudo dos efeitos colaterais dos quimioterápicos, buscando um tratamento menos agressivo para o paciente oncológico, justifica a realização deste trabalho. O objetivo foi verificar possíveis efeitos hematológicos e gastrointestinais das drogas doxorrubicina e tamoxifeno quando utilizadas em terapia ou em conjunto, em camundongos machos portadores ou não do tumor ascítico de Ehrlich, com e sem terapia hormonal com cipionato de estradiol. Foram utilizados 40 camundongos Balb/C machos, divididos em grupos de 5 animais cada, totalizando-se 8 grupos. Ao final de 21 dias, foi coletado sangue para a realização de hemograma, avaliando-se parâmetros como contagem de hemácias e hematócrito, dosagem de hemoglobina, contagem de leucócitos totais e contagem de plaquetas e amostras do estômago e intestino dos animais tratados com cloridrato de doxorrubicina, foram coletadas para análise histopatológica, avaliando-se quando presente, o grau das lesões no epitélio e o grau de necrose e apoptose das glândulas. A avaliação do número de hemácias e do valor do hematócrito apresentou diminuição significativa (p<0,05) nos grupos I e VIII quando comparado aos demais grupos. A dosagem de hemoglobina demonstrou diminuição significativa no grupo I em relação aos grupos II, III, IV e V. A contagem do numero de leucócitos demonstrou aumento significativo (p<0,05) quando se compararam os grupos I e VI com os demais grupos. As principais alterações histopatológicas evidenciadas no estomago e intestino delgado foram necrose e apoptose de glândulas e perda de epitélio de revestimento, sendo que o grupo VII demonstrou leve diminuição no grau das lesões em relação ao grupo V.

Food additives are used by the food industry in order to enhance the aroma, flavor and texture of foods. Of all the additives used colors are the most genotoxic. The azo dyes, derived from oil, used by industries, are the tartrazine and red #40. The SMART wing test was used to verify the possible genotoxic effects of colors in somatic cells of Drosophila melanogaster. For this evaluation were used larvae of 48 hours, descendants of the Standard Cross (ST) and High Bioactivation (HB) that who were treated with tartrazine in concentrations 1.5, 2.5 and 3.0 mg / mL and 5, 10 and 20 mg / mL for the red #40. The results showed significant increase of spots in the ST descendants for tartrazine and HB for the red #40. We concluded that the artificial colors tartrazine and red #40 are genotoxic in the doses used for the study. / Os aditivos alimentares são utilizados pela indústria alimentícia com o intuito de melhorar o aroma, o sabor e a textura dos alimentos. De todos os aditivos utilizados os corantes são os mais genotóxicos. Os corantes azóicos, derivados do petróleo, utilizados pelas indústrias, são o tartrazina e vermelho 40. O teste SMART de asa foi utilizado para verificar os possíveis efeitos genotóxicos destes corantes em células somáticas de Drosophila melanogaster. Para esta avaliação foram utilizadas larvas de 48 horas, descendentes do cruzamento padrão (ST) e alta bioativação (HB) que foram tratadas com tartrazina nas concentrações 1,5, 2,5 e 3,0 mg/mL e 5, 10 e 20 mg/mL para o vermelho 40. Os resultados mostraram aumento significativo de manchas nos descendentes ST para tartrazina e HB para o vermelho 40. Concluímos que os corantes artificiais tartrazina e vermelho 40 são genotóxicos nas doses utilizadas para o estudo. / Mestre em Genética e Bioquímica