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Background: Anthracyclines, in particular Doxorubicin (DOX), are highly effective chemotherapeutic agents in the breast cancer setting, which are limited by their cardiotoxic side effects. Recently, the introduction of Trastuzumab (TRZ), a novel monoclonal antibody against the HER2 receptor, in the breast cancer setting compounds the issue of DOX mediated cardiac dysfunction. Amongst the potential mechanisms for the deleterious effects of this drug-induced cardiomyopathy, the relationship between nitric oxide synthase 3 (NOS3) and oxidative stress has gained recent attention. Objective: To determine the role of NOS3 in a clinically relevant female murine model of DOX+TRZ induced heart failure. Methods: A total of 120 C57Bl/6 female mice [60 wild type (WT) and 60 NOS3 knockout (NOS3-/-)] were treated with either 0.9% saline, DOX (20 mg/kg), TRZ (10 mg/kg), or DOX+TRZ. Serial echocardiography was performed daily for a total of 10 days, after which the mice were euthanized for histological and biochemical analyses. Results: As compared to WT, NOS3-/- mice demonstrated increased cardiotoxicity following treatment with DOX. This effect was potentiated with DOX+TRZ combination therapy. In WT female mice receiving DOX+TRZ, left ventricular ejection fraction (LVEF) decreased from 75±3% at baseline to 46±2% at day 10 (p<0.05). In the NOS3-/- group, LVEF decreased from 72±3% at baseline to 35±2% at day 10 (p<0.05). LVEF was significantly lower in NOS3-/- mice than WT at day 10 in those receiving DOX+TRZ (p<0.05). As compared to WT, NOS3-/- mice also demonstrated increased mortality following treatment with DOX+TRZ, corroborating the echocardiographic findings. Histological analysis using light and electron microscopy demonstrated increased loss of cell integrity including myofibrillar degradation, cytoplasmic vacuolization, and enlargement of the smooth endoplasmic reticulum in both the WT and NOS3-/- mice treated with DOX+TRZ. There was no significant difference, however, in the degree of cardiac remodeling between the WT and NOS3-/- groups. There was an increasing trend in the degree of cardiac apoptosis in both WT and NOS3-/- mice treated with DOX+TRZ therapy. Conclusion: Congenital absence of NOS3 potentiates the cardiotoxic effects of DOX+TRZ in an acute female murine model of chemotherapy-induced cardiomyopathy.

Background: Anthracyclines, in particular Doxorubicin (DOX), are highly effective chemotherapeutic agents in the breast cancer setting, which are limited by their cardiotoxic side effects. Recently, the introduction of Trastuzumab (TRZ), a novel monoclonal antibody against the HER2 receptor, in the breast cancer setting compounds the issue of DOX mediated cardiac dysfunction. Amongst the potential mechanisms for the deleterious effects of this drug-induced cardiomyopathy, the relationship between nitric oxide synthase 3 (NOS3) and oxidative stress has gained recent attention. Objective: To determine the role of NOS3 in a clinically relevant female murine model of DOX+TRZ induced heart failure. Methods: A total of 120 C57Bl/6 female mice [60 wild type (WT) and 60 NOS3 knockout (NOS3-/-)] were treated with either 0.9% saline, DOX (20 mg/kg), TRZ (10 mg/kg), or DOX+TRZ. Serial echocardiography was performed daily for a total of 10 days, after which the mice were euthanized for histological and biochemical analyses. Results: As compared to WT, NOS3-/- mice demonstrated increased cardiotoxicity following treatment with DOX. This effect was potentiated with DOX+TRZ combination therapy. In WT female mice receiving DOX+TRZ, left ventricular ejection fraction (LVEF) decreased from 75±3% at baseline to 46±2% at day 10 (p<0.05). In the NOS3-/- group, LVEF decreased from 72±3% at baseline to 35±2% at day 10 (p<0.05). LVEF was significantly lower in NOS3-/- mice than WT at day 10 in those receiving DOX+TRZ (p<0.05). As compared to WT, NOS3-/- mice also demonstrated increased mortality following treatment with DOX+TRZ, corroborating the echocardiographic findings. Histological analysis using light and electron microscopy demonstrated increased loss of cell integrity including myofibrillar degradation, cytoplasmic vacuolization, and enlargement of the smooth endoplasmic reticulum in both the WT and NOS3-/- mice treated with DOX+TRZ. There was no significant difference, however, in the degree of cardiac remodeling between the WT and NOS3-/- groups. There was an increasing trend in the degree of cardiac apoptosis in both WT and NOS3-/- mice treated with DOX+TRZ therapy. Conclusion: Congenital absence of NOS3 potentiates the cardiotoxic effects of DOX+TRZ in an acute female murine model of chemotherapy-induced cardiomyopathy.

Anti-cancer drugs gain access to solid tumors via the blood, and must penetrate tissue to reach all viable cancer cells. This thesis aims to compare the distribution of anticancer drugs in normal tissues and tumours, to examine whether drug distribution is modifiable and quantifiable in solid tumours, and to determine whether extracellular drug distribution can be improved by modifying intracellular drug distribution. The time-dependent spatial distribution of three anticancer drugs, doxorubicin, mitoxantrone and topotecan, were studied in normal tissues and tumours. Ten minutes after drug administration, there was fairly uniform distribution in the heart, kidney and liver whereas drug distribution within tumours was limited to perivascular regions. Doxorubicin distribution in P-glycoprotein (PgP) over-expressing tumours was compared to that in wild-type tumours and changes in distribution were evaluated with the use of PgP inhibitors. There was better doxorubicin distribution in PgP-over-expressing tumours compared to wild-type tumours, and pretreatment of PgP-over-expressing tumours with PgP inhibitors decreased doxorubicin distribution. These data suggest that reduced uptake of drug into cells may enhance extracellular drug distribution, and the dual effects of PgP inhibitors (increased drug uptake in proximal cells, but poorer drug distribution) may explain, in part, why these agents have not provided clinical benefit. The effect of the proton pump inhibitor pantoprozole on intracellular and extracellular drug distribution was determined. Pantoprazole increased endosomal pH in cells, leading to less sequestration of doxorubicin within them, and increased the toxicity of doxorubicin for cultured cells. In wild-type MCF7 tumours, pretreatment with pantoprazole enhanced doxorubicin distribution and tumour growth delay without apparent increase in toxicity. These studies have led to initiation of a phase I clinical trial of pantoprazole and doxorubicin for patients with solid tumours. The work completed in this thesis demonstrates that drug distribution can be modified and that these changes can be quantified, and may correlate with improved anti-tumour effects. Improving drug distribution through the use of proton pump inhibitors may be an effective strategy to improve chemotherapeutic efficacy.

Anti-cancer drugs gain access to solid tumors via the blood, and must penetrate tissue to reach all viable cancer cells. This thesis aims to compare the distribution of anticancer drugs in normal tissues and tumours, to examine whether drug distribution is modifiable and quantifiable in solid tumours, and to determine whether extracellular drug distribution can be improved by modifying intracellular drug distribution. The time-dependent spatial distribution of three anticancer drugs, doxorubicin, mitoxantrone and topotecan, were studied in normal tissues and tumours. Ten minutes after drug administration, there was fairly uniform distribution in the heart, kidney and liver whereas drug distribution within tumours was limited to perivascular regions. Doxorubicin distribution in P-glycoprotein (PgP) over-expressing tumours was compared to that in wild-type tumours and changes in distribution were evaluated with the use of PgP inhibitors. There was better doxorubicin distribution in PgP-over-expressing tumours compared to wild-type tumours, and pretreatment of PgP-over-expressing tumours with PgP inhibitors decreased doxorubicin distribution. These data suggest that reduced uptake of drug into cells may enhance extracellular drug distribution, and the dual effects of PgP inhibitors (increased drug uptake in proximal cells, but poorer drug distribution) may explain, in part, why these agents have not provided clinical benefit. The effect of the proton pump inhibitor pantoprozole on intracellular and extracellular drug distribution was determined. Pantoprazole increased endosomal pH in cells, leading to less sequestration of doxorubicin within them, and increased the toxicity of doxorubicin for cultured cells. In wild-type MCF7 tumours, pretreatment with pantoprazole enhanced doxorubicin distribution and tumour growth delay without apparent increase in toxicity. These studies have led to initiation of a phase I clinical trial of pantoprazole and doxorubicin for patients with solid tumours. The work completed in this thesis demonstrates that drug distribution can be modified and that these changes can be quantified, and may correlate with improved anti-tumour effects. Improving drug distribution through the use of proton pump inhibitors may be an effective strategy to improve chemotherapeutic efficacy.

Background: The molecular and cellular mechanisms corresponding to the compensatory and maladaptive hypertrophy and remodeling of the left ventricle with chronic doxorubicin (DOX) treatment are currently unclear. Non-invasive methods of determining these changes are still deficient. To investigate these changes, 8 groups of rats in 4 different studies including a control saline group of the same age, gender and strain were evaluated for cardiac morphology and function including: (1) DOX dose response using a cumulative dose of 7.5mg/kg, and 15mg/kg in 8-10 week old female Sprague-Dawley (SD) rats, (2) strain differences were investigated in response to a cumulative dose of 15mg/kg in 8-10 week old female Fisher (F344) rats compared to the SD rats treated with same dose, (3) the role of gender and aging were studied in response to DOX at a cumulative dose of 3mg/kg in male and female neonates, and (4) combined losartan and a cumulative dose of 15mg/kg of DOX in 8-10 week old female SD rats compared to controls of saline and 15mg/kg treated SD rats. Method: Onset of cardiac toxicity was assessed by echocardiography and the rat model of heart failure was developed when the fractional shortening declined ≤ 40%. The mean arterial pressure and single-photon-emission computer tomography scanning and Tc-99m-HYNIC-Annexin V were performed at week 10 to analyze blood pressure and quantify apoptosis, respectively. All rats were euthanized at week 10 except for the neonates and two of the 7.5mg/kg-treated SD rats that were left alive for study of long -term cardiac side effects. The heart and kidney tissues were harvested for protein isolation and histopathological studies. Blood samples were collected for hematological and lipid profile analysis in all the rats. Results: A dose- and time-dependent increase in LVmass coincided with a parallel increase in MAP, kidney damage, expression of myocardial erbB2, heat shock protein 90 Akt, mTOR, GSK-3β, TGF-β, pSMAD2, and cardiomyocyte apoptosis in SD rats treated with 7.5mg/kg and 15mg/kg of DOX at week 10. The 7.5 kg/kg treatment showed adaptive hypertrophy whereas the 15mg/kg treatment group showed maladaptive hypertrophy. However decompensation was apparent by week 14 in other rats treated with 7.5mg/kg. LVmass, FS, MAP, kidney damage, red blood cells and blood lipid levels were not significantly altered in the F344 rats compared to the 15 mg/kg-treated SD rats. Losartan supplementation reduced the left ventricular hypertrophy, improved myocardial contractility, and reduced TGF-β expression compared to the DOX-treated SD rats. The 3mg/kg of DOX in neonates induced cardiac toxicity and deaths in about 60% of males 50 weeks after treatment; the females instead developed mammary tumors. Conclusion: The results of this study suggest that age, gender, and strain differences are risks factors for doxorubicin-induced harmful reno-cardiovascular toxicity. The inhibition of TGF-β expression by losartan can be used in prevention of chronic doxorubicin-induced cardiac toxicity without interfering with its anti-tumor activities.

Thesis (M.S.)--Baylor University, 2008. / Includes bibliographical references (p. 60-67)

Thesis (M.S.)--Brigham Young University. Dept. of Chemical Engineering, 2007. / Includes bibliographical references (p. 79-82).

A funcionalização de superfície nas nanocápsulas contendo doxorrubicina com o peptídeo RGD é uma estratégia promissora devido a ligação preferencial na integrina αvβ3 expressa em células tumorais. Este estudo objetivou o desenvolvimento, caracterização e estudos biológicos de nanocápsulas multiparedes com doxorrubicina e funcionalizadas com RGD. Para isso, na primeira etapa do trabalho foi realizada a síntese do peptídeo RGD. Os produtos obtidos foram caracterizados por análises de infravermelho e RMN de 1H. Na segunda etapa foram desenvolvidas formulações de nanocápsulas com doxorrubicina ou cloridrato de doxorrubicina, e, nanocápsulas multiparedes revestidas com quitosana, íons zinco, RGD ou fenilalanina. Essas suspensões foram caracterizadas através da determinação do pH, diâmetro de partícula por diferentes técnicas, potencial zeta, eficiência de encapsulação e eficiência de associação do RGD na superfície da nanopartícula. Na terceira etapa, foram realizados ensaios de viabilidade celular por MTT após 24 e 72h com as formulações desenvolvidas em células de câncer de mama (MCF7) e glioblastoma humano (U87MG). As formulações apresentaram diferentes valores de citotoxicidade e, utilizando o Gráfico de Pareto foi possível determinar os fatores que exercem maior influencia. Em células MCF7 foi a concentração de fármaco e tempo de tratamento e, nas células U87MG além desses fatores, a funcionalização mostrou-se determinante. Além disso, foi avaliada a captação das nanocápsulas funcionalizadas com RGD e fenilalanina após 24h nas células tumorais e células de queratinócitos humanos (HaCat), com diferentes níveis de expressão da integrina αvβ3. O estudo mostrou menores valores de captação nas células HaCat (sem expressão de integrina αvβ3) para as duas formulações testadas. Finalmente as nanocápsulas funcionalizadas com RGD apresentaram maior captação em células U87MG com maior expressão da integrina αvβ3. / The surface functionalization in nanocapsules containing doxorubicin with RGD peptide is a promising strategy due to preferential binding in the αvβ3 integrin expressed on tumor cells. This study aimed the development, characterization, and biological studies of multiwall nanocapsules containing doxorubicin and functionalized with RGD. For this reason, in the first stage of this study the synthesis of RGD peptide was performed and the products characterized by infrared analysis and 1H NMR. Besides, nanocapsules formulations were developed containing doxorubicin or doxorubicin hydrochloride, and multiwall nanocapsules coated with chitosan, zinc ions, RGD or phenylalanine. These suspensions were characterized by pH determination, particle diameter by different techniques, zeta potential, encapsulation efficiency, and association efficiency of RGD on the surface of the nanoparticle. Additionally, it was performed cell viability assays by MTT after 24 and 72 hours with formulations developed in breast cancer (MCF7) and human glioblastoma cells (U87MG). Formulations showed different cytotoxicity values. The Pareto chart was possible to determine factors that have more influence. In MCF7 cells was drug concentration and treatment time, and U87MG cells, besides these factors, the functionalization was decisive. Furthermore, it was performed the cellular uptake of nanocapsules functionalized with RGD or phenylalanine after 24 hours in tumor cells and human keratinocyte cells (HaCaT), with different levels of expression αvβ3 integrin. The study showed less uptake in HaCaT cells (without expression αvβ3 integrin) for the two formulations applied, and the nanocapsules functionalized with RGD showed more uptake in U87MG cells, with higher expression of integrin αvβ3.

Made available in DSpace on 2014-06-11T19:28:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-05-02Bitstream added on 2014-06-13T20:36:54Z : No. of bitstreams: 1 assumpcao_jucv_me_arafcf.pdf: 678214 bytes, checksum: 9acfe9078c5e33e1edcaa979ba032dca (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / Neste estudo investigou-se o perfil farmacocinético da DOX administrada na forma de microemulsão lipídica, em dose única i.v (6 mg/kg), a ratos Wistar (n=12; 250 g) e comparou-se ao perfil farmacocinético da doxorrubicina administrada na forma de cloridrato em solução aquosa. Ainda, avaliou-se a atividade da CKMb nos dois grupos de animais antes e após a administração das formulações com o objetivo de evidenciar a cardiotoxicidade do produto. Para a avaliação do perfil farmacocinético foram colhidas amostras seriadas de sangue (0 – 16 h) através de cânulas previamente implantadas na veia femoral, e para a avaliação da atividade de CKMb foram colhidas amostras de sangue nos tempos zero, 1 h e 12 h após a administração das formulações. Para a determinação da doxorrubicina em amostras de sangue e nas formulações, desenvolveu- se e validou-se um método analítico por HPLC com detecção por fluorescência (exc= 480 nm; em= 560 nm), empregando-se coluna Xterra (C18, 5 µm, 3,9 x 150 mm) e fase móvel composta por 25% de acetonitrila e 75% de água na presença de ácido fórmico (0,1%) e de solução de amônia 25% (0,1%), pH 3,0 , com fluxo de 0,8 mL/min, em modo gradiente. Para as determinações da atividade da CKMb utilizou-se o kit labtest. O método analítico desenvolvido demonstrou limites de confiança adequados para a sua aplicação na determinação da DOX em formulações e em amostras de plasma para a avaliação do seu perfil farmacocinético. Os parâmetros farmacocinéticos que apresentaram diferenças estatisticamente significativas (p<0,05, Mann-Whitney) entre a microemulsão e solução aquosa, apresentados como média (IC 95), foram respectivamente: Vd (L/kg) = 38,23 (24,94 – 51,50) vs 68,85 (55,69 – 82,00); tss (h) = 45,33 ( 39,45 – 58,20) vs 33,23 ( 27,7 – 38,75); β(h-1 )= 0,0014 (0,00072 – 0,00208) vs 0,00078... / The present study investigated the DOX pharmacokinetic profile when administered as a lipid microemulsion in a single dose (6 mg/kg, IV) to Wistar rats (n=12; 250g) and compared it to the pharmacokinetic profile of doxorubicin administered as hydrochloride in aqueous solution. With the purpose of demonstrating the cardiotoxicity of the product it also evaluated the CKMB activity in both animal groups before and after administration of the formulations. Serial blood samples were obtained (0 – 16h) through cannulas previously implanted into the femoral vein to evaluate the pharmacokinetic profile. To evaluate the CKMB activity, blood samples were collected after the formulation was administered at times zero, 1h and 12h. An analytical method by HPLC with fluorescence detection (exc= 480 nm; em= 560 nm) was developed and validated for the determination of doxorubicin in blood samples and in the formulations. The column used as stationary phase was Xterra  (C18, 5 µm, 3.9 x 150 mm) and the mobile phase was composed of 25% of acetonitrile and 75% of water in presence of formic acid (0.1%) and ammonia solution 25% (0.1%), pH 3.0 at a flow rate of 0.8 mL/min, gradient mode. The developed method demonstrated appropriate safety limits to its use in determining doxorubicin in formulations and in plasma samples, and therefore to evaluate the DOX pharmacokinetic profile. For the determination of CKMB activity it was used the Labtest. The pharmacokinetic parameters that showed statistically significant differences (p<0.05, Mann-Whitney) between the microemulsion and the aqueous solution, presented as medians (IC 95), were respectively: Vd (L/kg) = 38.23 (24.94 – 51.50) vs 68.85 (55.69 – 82.00); tss (h) = 45.33 (39.45 – 58.20) vs 33.23 ( 27.70 – 38.75); β(h-1 )= 0.0014 (0.00072 – 0.00208) vs 0.00078 ( 0.0004386 – 0.001076) and t1/2 (h) = 9.24 (5.31 – 13.17) vs 16.51 ... (Complete abstract click electronic access below)