Low Dose Metronomic (LDM) chemotherapy, combined with VEGF pathway inhibitors, is a highly effective strategy to coordinately inhibit angiogenesis and tumor growth. We have tested the efficacies of daily oral LDM topotecan alone and in combination with pazopanib, in three pediatric extracranial solid tumors mouse models. We also investigated the effect of prolonged combination therapy with the combination on tumor behavior in a neuroblastoma mouse xenograft model. In-vitro dose-response study of topotecan and pazopanib was conducted on several cell lines. In-vivo antitumor efficacies of drugs, as single agents and combination, were tested in immunodeficient mice models. For studying the mechanisms of resistance to our therapy, a time-response study (28, 56 and 80 days) was conducted in SK-N-BE(2) xenografts model, treated in same way as earlier. In vitro, topotecan caused a dose-dependent decrease in viabilities of all cell lines, while pazopanib did not. In vivo, the combination of topotecan and pazopanib demonstrated significant anti-tumor activity compared to the respective single agents in all models. Reductions in the levels of viable Circulating Endothelial Progenitors and/or Circulating Endothelial Cells and tumor microvessel density were correlated with tumor response and therefore confirmed the antiangiogenic activity of the regimens. However, the combination also caused significantly higher myelotoxicity than single agents. Pharmacokinetic study did not reveal any interaction between the two co-administered drugs. In the time-response study, we found that only combination treated animals survived till 80 days. However, tumors in these animals started growing gradually after 50 days. Unlike single agents, all three durations of combination treatment significantly lowered tumor microvessel densities, compared to the control. However, tumors treated with the combination for 56 and 80 days had higher pericyte coverage. The combination increased the hypoxia, angiogenic expression and proliferative index and caused metabolic reprogramming of tumor cells. We conclude that the combination of LDM topotecan and pazopanib has superior efficacy than either single agents, which is attributed to superior antiangiogenic activity. However, prolonged treatment with the combination can have additive myelotoxicity and may encounter adaptive resistance associated with metabolic reprogramming and increased proliferation of the tumor cells.

Low Dose Metronomic (LDM) chemotherapy, combined with VEGF pathway inhibitors, is a highly effective strategy to coordinately inhibit angiogenesis and tumor growth. We have tested the efficacies of daily oral LDM topotecan alone and in combination with pazopanib, in three pediatric extracranial solid tumors mouse models. We also investigated the effect of prolonged combination therapy with the combination on tumor behavior in a neuroblastoma mouse xenograft model. In-vitro dose-response study of topotecan and pazopanib was conducted on several cell lines. In-vivo antitumor efficacies of drugs, as single agents and combination, were tested in immunodeficient mice models. For studying the mechanisms of resistance to our therapy, a time-response study (28, 56 and 80 days) was conducted in SK-N-BE(2) xenografts model, treated in same way as earlier. In vitro, topotecan caused a dose-dependent decrease in viabilities of all cell lines, while pazopanib did not. In vivo, the combination of topotecan and pazopanib demonstrated significant anti-tumor activity compared to the respective single agents in all models. Reductions in the levels of viable Circulating Endothelial Progenitors and/or Circulating Endothelial Cells and tumor microvessel density were correlated with tumor response and therefore confirmed the antiangiogenic activity of the regimens. However, the combination also caused significantly higher myelotoxicity than single agents. Pharmacokinetic study did not reveal any interaction between the two co-administered drugs. In the time-response study, we found that only combination treated animals survived till 80 days. However, tumors in these animals started growing gradually after 50 days. Unlike single agents, all three durations of combination treatment significantly lowered tumor microvessel densities, compared to the control. However, tumors treated with the combination for 56 and 80 days had higher pericyte coverage. The combination increased the hypoxia, angiogenic expression and proliferative index and caused metabolic reprogramming of tumor cells. We conclude that the combination of LDM topotecan and pazopanib has superior efficacy than either single agents, which is attributed to superior antiangiogenic activity. However, prolonged treatment with the combination can have additive myelotoxicity and may encounter adaptive resistance associated with metabolic reprogramming and increased proliferation of the tumor cells.

Thesis (Ph.D. )--University of Tennessee Health Science Center, 2007. / Title from title page screen (viewed on June 20, 2008 ). Research advisor: Clinton Stewart, Pharm.D. Document formatted into pages (xvi, 129 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 112-129).

Thesis (Ph.D.)--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on Sept. 17, 2008). Research advisor: Clinton Stewart, Pharm.D. Document formatted into pages (xiii, 105 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 80-102).

Syntheses of biodegradable PLLA homopolymers and PLLA-mPEG diblock copolymers for the formation of thermo-sensitive gels were performed. The sol-gel transition temperature of the matrix was adjusted by altering the length of each PEG and LA component. PLLA-mPEG biocompatible copolymers, having appropriate length of each block component, showed sols at around 42-45 oC, suitable for the injection, then a gel with subsequent rapid cooling to body temperature. Topotecan and camptothecin were selected as anti-cancer drugs. Both drugs can easily hydrolyze at physiological conditions (pH=7.4). This causes the loss of its activity, and it turns into inactive toxic carboxylate form from active lactone state. To keep those anti cancer drugs in the lactone form, they were efficiently loaded into PLLA-mPEG gels in different loading ratios. Their stability in gel was fully examined with HPLC and fluorescence spectroscopy. It was found that both drugs were highly stable and in active form in the prepared gels (&gt / 95 %). Then, both release profile of drugs at different loading ratios showed prolonged release over weeks. Mechanistic studies on the stabilization of CPT anti cancer drug with PLLA-mPEG gels were carried out using ATR-FTIR, confocal and optic microscopes. The cytotoxic efficacy of TPT in the PLLA-mPEG platform (PLLA-mPEG-TPT) was evaluated on LLC-1 and 4T1 cancer cell lines by MTT assay. In vivo, the administration of PLLA-mPEG-TPT to the mice bearing breast tumours established with 4T1 cells resulted in a significant reduction in tumour size and better survival percentages. Additionally, stabilization of CPT and TPT with gels may find another application on solid tumors in brain via local injection. A novel conducting polymer was successfully synthesized via electropolymerization of 1-(1H-pyrrol-1-yl)-2,5-di(thiophen-2-yl)-1H-pyrrole. The electrochemical and electro-optical properties of the corresponding polymer, which was the first example of polymer containing 1,1&rsquo / -bipyrrole units, were elaborated using electroanalytical and spectroscopic techniques. Cyclic voltammograms and electrooptical studies showed that the polymer has a stable and well-defined reversible redox process as well as electrochromic behavior. The processable polymer film also possessed a yellowish orange light emitter property.

Thesis (M. Sc.)--Carleton University, 2008. / Includes bibliographical references (p. 96-98). Also available in electronic format on the Internet.

Advancements in nanoparticle drug delivery of anticancer agents require mathematical models capable of predicting in vivo formulation performance from in vitro characterization studies. Such models must identify and incorporate the physicochemical properties of the therapeutic agent and nanoparticle driving in vivo drug release. This work identifies these factors for two nanoparticle formulations of anticancer agents using an approach which develops mechanistic mathematical models in conjunction with experimental studies. A non-sink ultrafiltration method was developed to monitor liposomal release kinetics of the anticancer agent topotecan. Mathematical modeling allowed simultaneous determination of drug permeability and interfacial binding to the bilayer from release data. This method also quantified the effects of topotecan dimerization and surface potential on total amount of drug released from these liposomal formulations. The pH-sensitive release of topotecan from unilamellar vesicles was subsequently evaluated with this method. A mechanistic model identified three permeable species in which the zwitterionic lactone form of topotecan was the most permeable. Ring-closing kinetics of topotecan from its carboxylate to lactone form were found to be rate-limiting for topotecan drug release in the neutral pH region. Models were also developed to non-invasively analyze release kinetics of actively-loaded liposomal formulations of topotecan in vivo. The fluorescence excitation spectra of released topotecan were used to observe release kinetics in aqueous solution and human plasma. Simulations of the intravesicular pH in the various release media indicated accelerated release in plasma was a consequence of increased intravesicular pH due to ammonia levels in the plasma instead of alterations in bilayer integrity. Further studies were performed to understand the roles of dimerization, ion-pairing, and precipitation on loading and release kinetics obtained from actively-loaded topotecan. Extension of this type of modeling for other types of nanoparticles was illustrated with doxorubicin-conjugated polymeric micelles. Mathematical modeling of experimental studies monitoring doxorubicin release identified conjugation stability during storage, hydrazone hydrolysis kinetics, and unconjugated doxorubicin partitioning affected micellar doxorubicin release. This work identifies several of the key parameters governing drug release from these liposomal and micellar nanoparticles and lays the framework for future development of in vivo release models for these formulations.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-02-14T16:45:23Z No. of bitstreams: 2 Dissertação - João Hélio Venâncio Gomes - 2015.pdf: 2182325 bytes, checksum: 05a2adca44bd1d62895d3e1db48e449c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-02-15T09:31:12Z (GMT) No. of bitstreams: 2 Dissertação - João Hélio Venâncio Gomes - 2015.pdf: 2182325 bytes, checksum: 05a2adca44bd1d62895d3e1db48e449c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-02-15T09:31:12Z (GMT). No. of bitstreams: 2 Dissertação - João Hélio Venâncio Gomes - 2015.pdf: 2182325 bytes, checksum: 05a2adca44bd1d62895d3e1db48e449c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-09-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Topotecan (TPT) is a potent cytotoxic agent used in the treatment of various tumors, and studies have reported its effectiveness in the treatment of melanoma. Local treatment of melanoma with TPT appears to be a viable alternative since conventional treatments result in scarring, pain, inflammation and possible recurrence. However, the permeation of hydrophilic drugs such as TPT, is quite difficult. The encapsulation of the drug into nanostructured lipid carriers (NLC) may facilitate TPT permeation to deeper skin layers. Therefore, the final formulation shall provide appropriate viscosity for easy application and remain in the desired location. Thus, the objective was to incorporate the CLN-TPT in hydrogels hydroxyethyl cellulose (NLC-TPT-HEC) and chitosan (NLC-TPT-QUIT) and evaluate the skin permeation of the merged formulations or not in gels. NLC were incorporated into the hydrogels and were characterized as mean diameter, polydispersity index (PdI), zeta potential, drug recovery (REC%) and encapsulation efficiency (EE%). The release profiles and in vitro permeation studies were carried out in Franz-type diffusion cells using synthetic membrane and porcine ear skin, respectively. To quantify the TPT, high-performance liquid chromatography (HPLC) was used and a method for its extraction and quantitation in different skin layers was developed. The NLC-TPT-HEC and NLC-TPT-QUIT obtained respectively mean diameters of 117.8 nm and 183.2 nm; PdI of 0.32 and 0.33 and zeta potential -12,0mV and 75,0mV. Approximately 60% of TPT was recovered at the end of the preparation of formulations and EE% remained higher than 85% after the incorporation of the particles in the gels. The NLCTPT-HEC and NLC-TPT-QUIT demonstrated a significantly lower drug release (p <0.05) than the drug incorporated in the hydrogel and in NLC aqueous dispersion, demonstrating a potentiation in controlling the release of TPT. The NLC-TPT formulations CLN-TPT-HEC and CLN-TPT-QUIT increased respectively 1.93, 2.37 and 2.06 times the permeation of the drug into the deeper layers of the skin, compared to unloaded drug in same formulations. The NLCTPT-HEC / QUIT showed a lower permeation of the drug into the deeper skin layers when compared with the CLN-TPT dispersed in water. The controlled release resulted in a lower amount of drug available for permeation. Thus, the formulations allow control of permeation through the control of the drug release, which can meet different needs. The gel QUIT, for example, decreased the amount of drug retained in the EC and increases the amount of TPT permeated to the deeper layers. The developed formulations have potential use for topical treatment of melanoma. / O topotecano (TPT) é um potente agente citotóxico utilizado no tratamento de diversos tumores, e estudos têm relatado a sua eficácia no tratamento de melanoma. O tratamento local de melanoma com TPT parece ser uma alternativa viável, visto que os tratamentos convencionais resultam em cicatrizes desagradáveis, dor, inflamação e possíveis recidivas. Entretanto, a permeação de fármacos hidrofílicos, como o TPT, é bastante difícil. A encapsulação deste fármaco em carreadores lipídicos nanoestruturados (CLN) poderá facilitar a permeação do TPT para as camadas mais profundas da pele. Para tanto, a formulação final deve apresentar viscosidade adequada para facilitar a aplicação e manter-se no local desejado. Desta forma, o objetivo do trabalho foi incorporar os CLN-TPT em hidrogéis de hidroxietilcelulose (CLN-TPT-HEC) e quitosana (CLN-TPT-QUIT) e avaliar a permeação cutânea do TPT a partir das diferentes formulações. Os CLN incorporados nos hidrogéis foram caracterizados quanto ao diâmetro médio, índice de polidispersividade (PdI), potencial zeta, recuperação (REC%) e eficiência de encapsulação (EE%). Os perfis de liberação e permeação in vitro foram determinados utilizando células de difusão tipo Franz, utilizando membrana sintética e pele de orelha suína, respectivamente. Para a quantificação do TPT utilizou-se metodologia desenvolvida e validada por cromatografia líquida de alta eficiência (CLAE), e um método para a sua extração das camadas da pele foi desenvolvido. Os CLN-TPT-HEC e CLNTPT-QUIT apresentaram, respectivamente, diâmetros médios de 117,8 nm e 183,2 nm; PdI de 0,32 e 0,33 e potencial zeta -12,0mV e 75,9mV. Aproximadamente 60% do TPT foi recuperado ao final do preparo das formulações e a EE% manteve-se maior que 85% após a incorporação das partículas nos géis. Os CLN-TPT-HEC e CLN-TPT-QUIT demonstraram uma liberação significativamente menor (p<0,05) do que do fármaco incorporado nos hidrogéis e nos CLN em dispersão aquosa, demonstrando uma potencialização no controle da liberação do TPT, quando os CLN estão dispersos nos hidrogéis. As formulações CLN-TPT, CLN-TPT-HEC e CLN-TPT-QUIT aumentaram respectivamente 1,93, 2,37 e 2,06 vezes a permeação do fármaco para as camadas mais profundas da pele, em relação ao fármaco não encapsulado (nas mesmas formulações). Os CLN-TPT-HEC/QUT demonstraram menor permeação do fármaco para as camadas mais profundas da pele quando comparado com o CLN-TPT disperso em água. O controle da liberação resultou em uma quantidade menor de fármaco disponível para permeação. Desta forma, as formulações permitiram o controle da permeação através do controle da liberação do fármaco, podendo atender a diferentes necessidades. O gel de QUIT, por exemplo, diminuiu a quantidade de fármaco retido no EC e aumentou a quantidade de TPT permeado para as camadas mais profundas. As formulações desenvolvidas têm potencial de utilização para tratamento tópico do melanoma.

Prostate cancer is one of the leading causes of death in men aged 40-55. Genistein isoflavone (4', 5', 7-trihydroxyisoflavone) is a dietary phytochemical with demonstrated anti-tumor activities in a variety of cancers. Topotecan Hydrochloride (Hycamtin) is an FDA-approved chemotherapy drug, primarily used for secondary treatment of ovarian,cervical and small cell lung cancers. This study was to demonstrate the potential anticancer activities and synergy of topotecan-genistein combination in LNCaP prostate cancer cells. The potential efficacy and mechanism of topotecan/genistein-induced cell death was investigated... Results: The overall data indicated that i) both genistein and topotecan induce cellular death in LNCaP cells, ii) topotecan-genistein combination was significantly more efficacious in reducing LNCaP cell viabiligy compared to either genistein or topotecan alone, iii) in all cases, cell death was primarily through apoptosis, via the activation of the intrinsic pathway, iv) ROS levels were increased and VEGF expression was diminished significantly with the topotecan-genistein combination treatment, v) genetic analysis of topotecan-genistein treatment groups showed changes in genetic expression levels in pathway specific apoptotic genes.... Conclusion: Treatments involving topotecan-genistein combination may prove to be an attractive alternative phytotherapy of adjuvant therapy for prostate cancer. / by Vanessa P. Hèormann. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Le protozoaire Leishmania est à l’origine d’une maladie tropicale négligée qui peut s’avérer mortelle si elle n’est pas traitée adéquatement. Dans les dernières années, l’efficacité des médicaments utilisés dans le combat de la leishmaniose a grandement diminué en raison de la résistance du parasite à ceux-ci. Bien que le Topotecan (TPT), présentement utilisé comme antitumoral, ait démontré une activité antileishmaniale puissante (EC50 de l’ordre des nanomolaires), son efficacité pourrait être compromise par l’émergence de la résistance du parasite d’autant plus que des résistances tumorales cliniques au TPT ont déjà été rapportées. Dans cette étude, les mécanismes de résistance au TPT par Leishmania infantum ont été caractérisés d’un point de vue moléculaire. Le génome complet des parasites sélectionnés résistants au TPT (16 x EC50) (TPT700.1, TPT700.2, TPT700.3) a été séquencé. Le rôle dans la résistance des différents éléments génétiques identifiés a été confirmé à l’aide d’une complémentation par nucléofection épisomale dans le parasite sauvage et a été étudié avec des simulations computationnelles. Aucune amplification ni délétion n’a été identifiée et seulement une variation mineure du nombre de chromosomes a été observée. Cependant, un polymorphisme d’un seul nucléotide non synonyme a été identifié dans le gène de la grande sous-unité de la topoisomérase IB (TOP IB), la cible du TPT, chez chacun des parasites mutants résistants conférant des niveaux de résistance variables (TPT700.1 F187Y > TPT700.3 W232R > TPT700.2 G191A > sauvage). De plus, des modélisations in sillico ont permis d’illustrer la proximité de ces substitutions d’acides aminés au site catalytique de TOP IB ainsi qu’au site de liaison du TPT. En conclusion, ces résultats suggèrent qu’une mutation ponctuelle dans la grande sous-unité TOP IB est suffisante pour engendrer des hauts niveaux de résistance (environ 24 x EC50) chez TPT700.1F187Y, TPT700.2G191A et TPT700.3 W232R. TPT pourrait être considéré comme un modèle pharmacologique pour l’étude de la résistance chez Leishmania. / Leishmania, a protozoan parasite, causes a neglected tropical disease that is fatal if left untreated. In recent years, the effectiveness of the drugs used to tackle leishmaniasis has decreased dramatically due to the emergence of drug resistant parasites. Even though Topetecan (TPT), currently employed as an anti-tumoral drug, has shown strong anti-leishmanial activity (its EC50 being measured in nanomoles), its efficiency may be compromised by the resistance developped by the parasites, similarly to the resistance already recorded by tumoral cells to the drug. In this study, the mecanisms of resistance to TPT by Leishmania infantum were caracterised at the molecular level. The whole genome of parasites resistant to TPT (16 x EC50) (TPT700.1, TPT700.2, TPT700.3) was sequenced. The role of various genetic elements in the resistance mecanisms was confirmed via a complementation by episomal nucleofection in the wild type and was studied with the help of computational models. Neither amplications nor deletions were identified and only a minor variation in the chromosome number was observed. However, a non-synonymous single nucleotide polymorphism was identified in the gene coding the large subunit of topoisomerase IB, TPT’s target, within each of the resistant mutant parasites confering variable levels of resistance (TPT700.1 F187Y > TPT700.3 W232R > TPT700.2 G191A > wild type). Furthermore, in sillico models highlighted the proximity of these amino acid substitutions to the catalytic site of topoisomerase IB and also to the binding pockets of TPT. In conclusion, these results suggest that a point mutation in the large subunit of TOP IB is sufficient to confer high levels of resistance (about 24 x EC50) to TPT700.1F187Y, TPT700.2G191A and TPT700.3 W232R. Therefore, TPT can be considered a pharmacological tool to study resistance in Leishmania.