INTRODUCTION:Newly developed antineoplastic drugs have resulted in improvements in morbidity and mortality from many forms of cancers. However, some of these new chemotherapeutic agents have potentially lethal side effects, which are now being exposed with their widespread use. Gemcitabine is a nucleoside analog, which is a commonly used agent for various solid organ malignancies. Phase 1 and 2 trials with gemcitabine did not show significant risk for cardiotoxicity / however, with its widespread clinical use over the last decade, a few cases of cardiotoxicity related to gemcitabine use have been reported. Cardiomyopathy after the use of gemcitabine monotherapy is extremely rare / and only one such case has been reported in detail previously.CASE PRESENTATION:We report a case of a 56-year-old African American man with pancreatic cancer who presented with signs and symptoms of congestive heart failure after being treated with gemcitabine for two cycles (six doses). A two-dimensional echocardiography showed left ventricular ejection fraction of 15 to 20 percent with global hypokinesia. With the absence of significant risk factors for coronary artery disease and a strong temporal relationship with the initiation of chemotherapy, it was concluded that our patient's cardiomyopathy was related to the use of gemcitabine. Gemcitabine was discontinued and our patient responded well to standard heart failure therapy. Two months later, a repeat echocardiogram showed significant improvements in left ventricular systolic function.CONCLUSIONS:Gemcitabine should be considered as a potential cause of cardiomyopathy in patients receiving chemotherapy with this drug. We need further studies to look into potential mechanisms and treatments of gemcitabine-induced cardiac dysfunction.

Imexon is an iminopyrrolidone aziridine which previously has shown activity against a variety of human cancer types, including multiple myeloma and pancreatic adenocarcinoma. Recently, mechanistic studies in the MIA PaCa-2 human pancreatic cancer cell line have demonstrated binding to sulfhydryls, build-up of reactive oxygen species (ROS), perturbations in mitochondrial membrane potential (MMP), and activation of caspases 3, 8 and 9. Because imexon binds sulfhydryls and generates ROS, it was hypothesized that imexon would have considerable activity against pancreatic cancer by promoting oxidative stress in cells which are already oxidatively challenged and in combination with gemcitabine by interacting with key sulfhydryl-dependent enzymes involved with gemcitabine metabolism. In vitro anti-tumor activity of imexon and gemcitabine was evaluated in PANC-1, MIA PaCa-2, MutJ, and BxPC-3 human pancreatic cancer cell lines. Interactions between imexon and gemcitabine were assessed with simultaneous drug exposure at a fixed (imexon: gemcitabine) ratio using median effect analysis. The PANC-1, MutJ, and BxPC-3 cells demonstrated synergy with combination treatment. Severe combined immune deficient (SCID) mice bearing PANC-1 cells treated with imexon and gemcitabine demonstrated tumor growth inhibition and regression. Imexon inhibited ribonucleotide reductase (RNR) at drug concentrations ≥100 μMol. This is similar to the selective RNR inhibitor hydroxyurea, suggesting that imexon may enhance gemcitabine-mediated inhibition of RNR as a mechanism of synergy. An S phase accumulation of PANC-1 cells occurred at ≥300 μMol imexon at 24 hr. This was associated with a ≥2-fold increase of radiolabeled gemcitabine incorporation into PANC-1 DNA at ≥100 μMol imexon. Therefore the mechanisms of synergy between imexon and gemcitabine appear to include: (1) cell cycle arrest in S-phase, and (2) inhibition of RNR. Both actions would increase the uptake of the active metabolite, gemcitabine-triphosphate, (GEM-TP), into DNA. Arresting cells in S-phase would increase the time of cellular incorporation of deoxynucleotides, including GEM-TP, into DNA. Similarly, RNR inhibition reduces the availability of normal deoxynucleotides which compete with GEM-TP for incorporation. Overall, these data demonstrate that imexon is uniquely synergistic with gemcitabine in vitro and in vivo and support the rationale for combining the agents in clinical trials for the treatment of pancreatic cancer.

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Gemcitabine (dFdC) is an effective chemotherapeutic nucleoside analogue for treatment of cancers and solid tumors. Gemcitabine’s chemotherapeutic effect is limited by its rapid intracellular deamination by cytidine deaminase into the inactive uracil derivative. Herein, I designed and synthesized two sets of gemcitabine analogues: i) a 4-N-alkyl gemcitabine analogue containing a β-keto sulfonate moiety, and ii) clickable analogues possessing silicon-fluoride acceptor building blocks. Both of these sets of analogues undergo efficient fluorination, including fluorination protocols compatible with 18F labeling. The synthesis of the 4-N-alkyl gemcitabine analogue bearing β-keto sulfonate moiety began with reaction of 4-N-tosylgemcitabine with 1-amino-10-undecene, followed by a series of oxidation and sulfonation steps which yielded the β-keto sulfonate analogues. The coupling of gemcitabine with carboxylic acids using peptide coupling conditions afforded 4-N-alkanoyl analogues with a terminal alkyne or azido moiety. Click reaction of these 4-N-alkanoyl analogues with dialkylsilyl building blocks gave 4-N-alkanoylsilanegemcitabine analogue. Reaction of 4-N-tosylgemcitabine with vii functionalized azidoalkyl amines provided 4-N-alkylgemcitabine with a terminal azido group. Coupling of the latter with dialkylsilyl building block provided 4-N-alkylsilanegemcitabine. Fluorination of 4-N-alkyl gemcitabine analogues with β-keto sulfonate moieties and of the trisubstituted silane derivatives with KF and 18-Crown-6 (CH3CN/75°C/0.5-1h), gave the corresponding fluorinated 4-N-alkyl and alkanoyl gemcitabine analogues under conditions that are compatible with protocols for positron emission tomography (PET) 18F labeling. The [18F] 4-N-alkyl and alkanoyl silane gemcitabine analogues were successfully synthesized on microscale and macroscale radiochemical protocols. The biodistribution of [18F] 4-N-alkyl gemcitabine analogue was analyzed via PET imaging. The cytotoxicity activity of the silane gemcitabine analogues were studied in cancer L1210 and HEK293 cell lines and their cellular uptake were investigated using HPLC analysis and fluorescence microscopy. Reduction of ribono-1,4-lactones and gulono-1,4-lactone as well as ribono-1,5-lactone and glucono-1,5-lactones with LTBH (1.2 equiv.) in CH2Cl2 at 0 °C for 30 min provided the corresponding pentose or hexose hemiacetals in chemoselective fashion and in high yields. Commonly used in carbohydrate chemistry protecting groups such as trityl, benzyl, silyl, acetals and to some extent acyls are compatible with this reduction.

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Les nanoparticules de Gemcitabine-Squalène (Gem-Sq), synthétisées suivant le concept de « squalénisation », ont montré des activités anticancéreuses très supérieures à celles obtenues en présence de Gem libre. Néanmoins, leur PEGylation, c’est-à-dire leur décoration par du poly(éthylène glycol)-squalène (PEG-Sq) pour augmenter leur temps de demi-vie plasmatique, s’est avérée infructueuse du fait d’une déstructuration colloïdale. Par ailleurs, aucune stratégie de fonctionnalisation pour effectuer un ciblage actif de cellules cancéreuses, n’est à ce jour disponible. Au cours de cette thèse, nous avons donc cherché à résoudre ces problèmes. Après une étude bibliographique portant sur la conception de nanoparticules de prodrogues lipidiques, dans le but d’établir un constat récent de l’état de l’art dans ce domaine, nous avons proposé une voie de synthèse pour obtenir des nanoparticules multifonctionnelles (i.e., thérapeutique, fluorescentes et ciblées) à base de Gem-Sq, et ce par co-auto-assemblage des composés conjugués de Rhodamine-Sq, Gem-Sq et Biotin-Sq. Ces nanoparticules ont montré une internalisation plus importante dans les cellules cancéreuses et une meilleure efficacité thérapeutique que les nanoparticules de Gem-Sq non-fonctionnalisées. Dans un deuxième temps, nous avons apporté une solution au problème de la PEGylation des nanoparticules de Sq via la synthèse et l’utilisation de composés conjugués de type Gem-poly(méthacrylate de squalène). Ces prodrogues macromoléculaires ont été synthétisées par polymérisation radicalaire contrôlée et plus précisément par la technique RAFT. Les nanoparticules obtenues par auto-assemblage en solution aqueuse sont stables et présentent des activités anticancéreuses importantes sur différentes lignées cellulaires. Leur PEGylation par ajout de Sq-PEG durant la formulation s’est avérée possible et n’a pas conduit à une déstabilisation colloïdale. Enfin, j’ai participé à l’élaboration d’une nouvelle famille de nanoparticules de prodrogues macromoléculaires qui a consisté à faire croitre de courtes chaines de polyisoprène (PI) à partir de la Gem, donnant ainsi des conjugués de type Gem-PI, capables de s’auto-assembler sous la forme de nanoparticules avec une activité anticancéreuse in vitro et in vivo. / Gemcitabine-Squalene (Gem-Sq) nanoparticles have been synthesized from the “squalenoylation” approach and have shown superior anticancer activities compared to those obtained with free Gem. However, their PEGylation, that is their coating with poly(ethylene glycol)-squalene (PEG-Sq) in order to increase their circulation time, has been unsuccessful, leading to colloidal disassembly. In addition, to the best of our knowledge, there is not functionalization strategy yet available to perform active targeting against cancer. During this PhD thesis, we have been looking for solutions to tackle these two important problems. After a littérature survey about the design of lipidic prodrug nanoparticles, in order to establish a pretty accurate picture of the domain, we have reported a synthetic approach towards multifunctional Sq-based nanoparticles (i.e., therapeutic, fluorescent and targeted), through the co-self-assembly of the different Sq-based materials; that is Rhodamine-Sq, Gem-Sq and Biotin-Sq. These nanoparticles have demonstrated a greater internalization into cancer cells and a greater therapeutic effect than non-functionalized Gem-Sq nanoparticles. In the next step, we have provided a solution to the PEGylation issue by synthetizing Gem-poly(squalenoyl methacrylate) macromolecular prodrugs. These materials have been prepared by controlled/living radical polymerization and especially the RAFT technique. The resulting nanoparticles exhibited significant anticancer activities against various cancer cells and can be successfully PEGylated by the addition of Sq-PEG during their formulation. Eventually, I have participated to the design of a new family of macromolecular prodrugs obtained from the growing of short polyisoprene (PI) chains from Gem, leading to Gem-PI nanoparticles after self-assembly of Gem-PI. The nanoparticles led to significant anticancer activity both in vitro and in vivo.

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O câncer cervical representa a terceira causa de morte por câncer em mulheres e está associado a infecções persistentes pelo Vírus do Papiloma Humano (HPV) em mais de 99% dos casos. Evidências sólidas mostram que a expansão de Células Mielóides Supressoras (MDSC) e fatores solúveis como a interleucina IL-10 são importantes mediadores da evasão tumoral à resposta imunológica contra tumores, que limita a eficácia de imunoterapias. Dada a importância epidemiológica dos tumores induzidos por HPV e a necessidade eminente do desenvolvimento de imunoterapias ativas contra essas lesões, desenvolvemos durante os últimos 12 anos, uma estratégia vacinal terapêutica baseada em DNA. Esta vacina codifica a proteína E7 do HPV-16 fusionada à glicoproteína D (gD) do Vírus Herpes Simplex do tipo 1 (HSV-1), denominada pgDE7h. O presente trabalho propôs a investigação de estratégias que restringem a imunossupressão, baseadas em um plasmídeo que codifica o receptor solúvel da citocina IL-10 (pIL10R) e da quimioterapia com gencitabina, na potencialização da vacina pgDE7h para controlar o crescimento de tumores que expressam as proteínas E6 e E7 do HPV-16 em modelo murino (células TC-1). Foi possível observar um atraso no crescimento do tumoral a partir da combinação da vacina com o pIL-10R. Adicionalmente, a utilização da eletroporação como método de entrega dos plasmídeos aumentou a proteção terapêutica para 90% e 60% quando os animais foram imunizados 5 ou 14 dias após o desafio, respectivamente. A combinação dos plasmídeos foi ainda capaz de elevar o número de linfócitos T CD8+ E7-específicos ativados, encontrados sistemicamente e no sítio tumoral. No consócio da imunização com a pgDE7h à gencitabina, foi possível observar um efeito sinérgico dessa associação, que aumentou a proteção antitumoral de 20% para 100% de animais livres de tumor, concomitante ao controle da expansão de populações imunossupressoras no baço. Além disso, a quimioimunoterapia administrada até 14 dias após o desafio com as células tumorais mostrou-se protetora em modelo de recidivas, simuladas por meio de transplantes subsequentes de células tumorais. Na busca de um protocolo vacinal com maior viabilidade clínica, combinamos a coadministração dos plasmídeos pgDE7h e pIL- 10R com o tratamento com gencitabina. Esta combinação terapêutica mostrou-se mais eficiente, onde se observou um aumento robusto da ativação de linfócitos T CD8+ E7-espefíficos, com regressão completa de tumores pré-estabelecidos. Em suma, esses resultados evidenciam que a associação de abordagens terapêuticas pode superar barreiras imunológicas presentes no ambiente tumoral e aumentar as chances de sucesso clínico do tratamento proposto. / Cervical cancer represents the third cause of cancer death among women, which is associated with persistent Human Papilloma Virus (HPV) infection in more than 99% of cases. Solid evidences show that the expansion of Myeloid-derived Suppressor Cells (MDSC) and soluble factors, such as IL-10, are important mediators of immune evasion mechanisms expressed by tumor cells, that limits the efficacy of immunotherapeutic approaches. Given the epidemiological importance of HPVinduced tumors and the crucial importance of developing active immunotherapies against neoplasias, during the past 12 years, we have been developing a DNA-based therapeutic vaccine strategy encoding the HPV-16 E7 protein fused to the Herpes Simplex Virus type 1 (HSV-1) glycoprotein D (gD), named pgDE7h. This work aimed to investigate the potential role of immunossupression restriction using a plasmid encoding the soluble IL-10 receptor (pIL10R) and the chemotherapeutic drug gemcitabine to enhance in enhancing the pgDE7h vaccine potency in the control of tumors expressing HPV-16 E6 and E7 proteins in a murine model (TC-1 cells). It was possible to observe a tumor growth delay after combining the vaccine with pIL-10R. Additionally, the use of electroporation as a plasmid delivery method provided a therapeutic protection of 90% and 60% when the animals were immunized 5 or 14 days after challenge, respectively. The combination of plasmids was also capable to increase substantially the numbers of activatedE7-specific CD8+ T lymphocytes both systemically and at the tumor site. When pgDE7h immunization was combined to chemotherapy, we observed a synergistic effect, which increased the antitumor protection from 20% to 100% and promoted the control of immunosuppressive cell populations in the spleen. It was possible to maintain the high antitumour protective effects of the chemo-immunotherapy when the animals were immunized 14 days after tumor cell challenge and tumor relapses after subsequent challenges with tumor cells. To search a vaccine protocol with greater applicability in a clinical setting, we combined co-administration of pgDE7h and pIL-10R and gemcitabine treatment. Our data showed that the combined treatment induced a robust increase in the activation of CD8+ T lymphocytes and a complete regression of pre-established tumors. All together, these results show that the combination of therapeutic approaches can overcome immunological barriers present in the tumor environment and increase the chances of clinical success of the propose therapeutic treatment.

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