Dissertations / Theses on the topic 'Doxorubicin'

La doxorubicine induit un stress oxydant, des dommages à l’ADN conduisant aussi bien à la mort des cellules cancéreuses que des cardiomyocytes. De nos jours, plusieurs hypothèses non reliées à la mort cellulaire et impliquant d’autres mécanismes ou l’altération des signalisations cardiaques telles que la signalisation β-adrénergique ont émergé. Cette thèse a donc pour objectif l’étude du rôle d’Epac, facteur d’échange directement activé par l’AMP cyclique, lui-même produit après stimulation β-adrénergique, dans la cardiotoxicité induite par la doxorubicine. En effet, la doxorubicine induit une cardiomyopathie dilatée 15 semaines après traitement associée à une altération de l’homéostasie calcique. Ces altérations sont corrélées à la modulation temps et dose-dépendantes de la signalisation d’Epac. Cette même altération globale de la signalisation d’Epac a également été observée in vitro après 24h de traitement à la dox. De plus, l’inhibition spécifique d’Epac 1 a permis la prévention des dommages à l’ADN et de façon subséquente de la mort des cardiomyocytes. L’invalidation du gène d’Epac1 chez la souris a également permis la prévention in vivo des altérations de l’homéostasie calcique ainsi que de la fonction cardiaque induite par la dox. Enfin, l’inhibition d’Epac n’interfère pas avec l’efficacité antitumorale de la doxorubicine sur différentes lignées cancéreuses. En conclusion, nous avons identifié Epac comme nouvelle cible thérapeutique de la cardiotoxicité induite par la dox permettant sa prévention sans réduire l’efficacité du traitement anticancéreux
The mechanisms underlying doxorubicin (Dox)-induced cardiotoxicity involve reactive oxygen species generation, DNA intercalation and topoisomerase II (TopII) inhibition which trigger DNA damage, oxidative stress, alteration of calcium homeostasis and lead to cardiomyocyte death. Now, evidences have emerged that Dox may promote cardiotoxicity by alternative mechanisms or by signaling pathways modulation including β-adrenergic signaling unrelated directly to cell death. This study provides in vitro and in vivo evidence of the guanine exchange factor directly activated by Epac role, a guanine exchange factor directly activated by cyclic AMP produced after β-AR stimulation, in cardiotoxicity induced by doxorubicin. Indeed, Dox leads to the development of a dilated cardiomyopathy (DCM) 15 weeks post treatment in mice associated with calcium homeostasis abnormalities. These alterations were associated with time- and dose-dependent alterations of Epac signaling. The same alterations of Epac signaling were observed in vitro after 24h of dox treatment. Furthermore, we first showed that the specific pharmacologic or genetic inhibition of Epac1 but not Epac2 prevents the deleterious effects of Dox in vitro. These cardioprotection were confirmed in vivo in transgenic Knock-out Epac1 mice. Epac 1 inhibition did not interfere with the attempted Dox antitumor efficiency on tumor cell lines. Altogether, these findings identify the cAMP-binding protein, Epac, as a potential therapeutic target of dox-induced cardiotoxicity

Cancer is a group of diseases caused by uncontrolled cellular proliferation and dissemination. After heart disease, cancer is the second most common cause of death in the United States. Main treatment approaches for cancer are surgery, radiotherapy, chemotherapy, and immunotherapy approaches. However, cancer cells have ability to develop resistance to conventional chemotherapy thus lowering the efficacy of those chemotherapeutic agents including doxorubicin (DOX). DOX has been used for the treatment of various cancers. It is usually administered via continuous intravenous infusion. Nevertheless, the use of soluble DOX is often limited by its low therapeutic index. It has been reported that DOX-induced cardiotoxicity is a life-threatening adverse effect and DOX is also a potent vesicant that can cause tissue necrosis following injections. Therefore, this dissertation investigated alternative delivery approaches for DOX including systemic and local delivery systems for enhancing antitumor efficacy while reducing the side effects of free DOX. The first part of this research aimed at developing a formulation capable of actively targeting DOX to tumors. Advances in nanotechnology have provided new ways to delivering DOX into the body and to tumor sites. Among all active targeting ligands developed to date, cRGD peptide (cyclic arginylglycylaspartic acid) occupies a unique position owing to its inherent safety, biocompatibility, and targeting ability. Thus, cRGD was used here to decorate the surface of DOX-loaded PLGA-PEG nanoparticles (NPs) using two independent crosslink reactions, EDC-NHS and thiol-maleimide reactions. The results showed that the different modification reactions yielded NPs of similar size (110-140 nm diameter). All formulations exhibited provided similar burst release phases (of DOX) over the first 12 h followed by sustained release for up to 200 h. For in vivo antitumor activity, C57BL/6J mice carrying melanoma tumors were administered with cRGD-modified DOX-loaded PLGA-PEG NPs (equivalent to 8 μg DOX) by intravenous injection once every other day for up to four doses. Tumor volumes and survival were recorded. The toxicity of this therapy was examined using serum biomarkers including bilirubin, alanine aminotransferase (ALT), and aspartate transaminase (AST). Histopathology of organs (heart, lung, spleen, liver and kidney) was evaluated using hematoxylin and eosin staining (H&E) after euthanizing the treated mice. The results indicated that the cRGD-modified DOX-loaded PLGA-PEG NPs using PLGA-PEG-maleimide polymers (cRGD-DOX-M) demonstrated higher antitumor activity as compared to other groups (p < 0.05). Finally, administration of cRGD-modified DOX-loaded PLGA-PEG NPs had no significant effect on total bilirubin, serum ALT, serum AST levels or animal weight (P > 0.05). There were no signs of tissue damage in any of the tested organs as evaluated by H&E staining. The second part of this dissertation proposed to evaluate the therapeutic effect of combining chemotherapy and immunotherapy in a murine melanoma model. In this study, DOX-loaded PLGA-PEG NPs and anti-programmed death 1 (anti-PD-1) antibodies were chosen as the model of chemotherapy and immunotherapy, respectively. Anti-PD-1 antibodies have shown a great deal of promise in the treatment of melanoma in the clinic. In this study, DOX-loaded PLGA-PEG NPs were administered IV at a dose of 8 µg of DOX/dose per mouse once every other day (total of four injections). Mice in combination treatment groups were also administered with 200 µg of anti-PD-1 solution via intraperitoneal (IP) injection every 3 days for five doses. The combination therapy demonstrated higher antitumor efficacy in vivo as compared to control, soluble DOX, monotherapy of DOX-loaded PLGA-PEG NPs or anti-PD1 solution (p<0.05). Moreover, in vivo safety studies were investigated, and the results suggested that the combination therapy was safe. Lastly, DOX-loaded PLGA-PEG millirods were successfully fabricated by a hot-melt extrusion technique and characterized for in vitro release. It was demonstrated that DOX released from the millirods could be controlled by coating with polylactide (PLA). The locally implanted uncoated DOX- loaded PLGA millirods provided significantly greater antitumor activity against melanoma tumors in mice compared to naïve group and PLA-coated DOX-loaded PLGA millirods. Antitumor activity of the millirods was related to the release profile of DOX from the millirods. PLA-coated DOX-loaded millirods exhibited slower release of DOX compared to uncoated DOX-loaded millirods which probably explains the shorter survival time of mice treated with this formulation. Moreover, skin samples from tumor-free mice were also analyzed. The results demonstrated that uncoated and PLA-coated DOX-loaded millirods could be administered peritumorally without causing local skin necrosis. In conclusion, the novel systemic delivery system and local delivery system of DOX presented here have the potential to be used as alternative approaches for cancer therapy.

Insensitivity to chemotherapy is an ongoing issue in cancer treatment, one that appears to be highly dependent on patient-specific variations. It has been shown clinically that while a subset of patients will successfully respond to a particular chemotherapeutic regimen, there exists another subset of patients who when exposed to the same course of therapy will remain resistant to treatment or exhibit signs of relapse after treatment has been administered. This discrepancy raises interesting questions regarding the role that patient-specific variations play in controlling the efficacy of chemotherapy treatment regimens. Doxorubicin (Dox) is a common chemotherapeutic agent used in the treatment of a variety of solid tumors and leukemias and resistance to Dox treatment is a major issue in cancer chemotherapy, oftentimes leading to patient relapse. To gain a deeper understanding of the processes that influence Dox resistance, we must first understand the mechanisms that underlie and contribute to Dox's toxicity. To this end, the metabolic reactions that activate Dox have been implicated as major determinants of Dox cytoxicity and as possible factors that control Dox resistance in cancer cells. There are several lines of evidence that redox-dependent metabolism plays a large role in Dox toxicity. The Dox bioactivation network is comprised of a system of reduction/oxidation (redox) reactions that lead to the formation of toxic Dox metabolites and reactive oxygen species (ROS). Moreover, multi-drug resistant acute lymphoblastic leukemia cells derived from relapsed patients have elevated levels of the antioxidant glutathione and show insensitivity to Dox treatment. The redox dependence of Dox bioactivation, the understanding that Dox treatment generates ROS, and the evidence that Dox resistant cells exhibit increased antioxidant capacity, suggest the possibility that redox pathways modulate the efficacy of Dox treatment in cancer cells. The overall objectives of the proposed dissertation, therefore, were to investigate how the redox properties of the Dox bioactivation network influence Dox toxicity in acute lymphoblastic leukemia cells, and to provide evidence that cell-specific variations in the intracellular levels of these redox components influences the degree to which Dox treatment will induce cancer cell death. The significant findings of this study are that the redox reactions involved in Dox metabolism are dual-natured, containing a toxicity-generating module characterized by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent Dox reductive conversion, as well as an ROS signal-generating module characterized by NADPH- and oxygen-dependent Dox redox cycling. The balance between the coupled redox reactions that comprise the toxicity- and ROS signal-generating modules of Dox bioactivation determines the sensitivity-phenotype of leukemia cells and phenotypic changes in the Dox-sensitivity of leukemia cells can be induced by the successful modulation of the Dox bioactivation network through the pharmacological inhibition of NADPH in a concentration- and cell type-dependent manner. This study highlights the importance of the intracellular redox network in controlling chemotherapy-induced ROS. The unequal distribution in antioxidant burden across the various intracellular antioxidant enzymes suggests a significant role for NADPH supply, as controlled by the enzyme glucose-6-phosphate dehydrogenase (G6PD), to the intracellular ROS buffering capacity of cells during instances of oxidative stress. Changes in G6PD activity were shown to promote protein-S-glutathionylation during oxidative stress conditions, thereby implicating G6PD in the modulation of redox-sensitive signal transduction pathways. The intracellular glutathione redox balance, a measure of the intracellular redox environment, can effectively regulate Dox-induced NF-κB signal transduction in leukemia cells. The systematic modulation of intracellular glutathione redox balance in leukemia cells by N-acetylcysteine (NAC) revealed an important role for protein S-glutathionylation mechanisms in the control of NF-κB signal transduction induced by Dox treatment. These findings identify the glutathione redox network as a potential therapeutic target for the systematic modulation of Dox sensitivity in cancer cells and elucidate the complex role that antioxidants such as NAC can play in modulating the effectiveness of Dox chemotherapy treatment regimens. Lastly, this study highlights the need for and the capacity of computational models to accurately describe the complex redox-reactions that contribute to Dox metabolism in leukemia cells. This study is groundbreaking in its use of computational modeling to analyze reversible electron transfer events between proteins using mass-action kinetics. The models developed in this study can accurately explain cytosolic doxorubicin bioactivation, intracellular hydrogen peroxide clearance, and kinase-specific S-glutathionylation, thereby showing that the use of comprehensive and/or relatively simple computational models can provide semi-quantitative predictions about the behavior of redox systems in mammalian cells as they relate to Dox-induced toxicity and Dox-induced cell signaling.

Less than half of all breast cancer patients respond to the anthracycline agents Epirubicin and Doxorubicin, the most effective agents available. Of those that do respond initially many eventually fail to do so. In this thesis some of the possible reasons for this failure are explored and ways of improving responses are also discussed. Doxorubicin uptake to breast cancers is examined in patients undergoing mastectomy who are given a dose pre-operatively. A range of uptakes is seen, but all tumours contained measurable levels of Doxorubicin. Intra-tumoural distribution is assessed using the auto fluorescent properties of Doxorubicin. There was variability of distribution but no gross penetration barriers were observed. To assess the range of 'inherent' variation in the sensitivity of breast cancers individual tumour cells and cells from distant normal breast were cultured in vitro from fresh specimens. Successful cultures were achieved in thirty four out of seventy three tumour samples (46%). Sixteen out of fifty for normal breast samples grew in vitro (29.6%). Chemosensitivity to Doxorubicin was measured using a short term clonogenic assay. There was a wide range in tumour sensitivities (1 x 10^-7M to 6 x 10^-9M) and also a marked interpatient variation in cells from normal breast (4 x 10^-8M - 7 x 10^-9M). In vitro sensitivities are correlated with the expression of the MDR-1 gene and gst π gene and long term clinical follow up was carried out to assess eventual responses to chemotherapy drugs and survival. The ability of a range of resistance modifying agents to overcome resistance to Doxorubicin was measured using the cell line MCF-Adr^Res , and Quinidine was found to be the most effective resistance modifier.

Mestrado em Biomedicina Molecular
A Doxorubicina (DOX) é um agente antineoplásico de grande eficácia utilizado no tratamento de vários tipos de tumores. No entanto, a sua utilização clínica é limitada devido à sua toxicidade em vários órgãos, com destaque para o coração. Outros órgãos afectados por este fármaco incluem fígado, cérebro, rins e testículos. Algumas estratégias farmacológicas e não farmacológicas têm sido desenvolvidas de forma a contrariar os seus efeitos secundários tóxicos, incluindo suplementação com antioxidantes e, mais recentemente, exercício físico. Assim, o objectivo do presente estudo é avaliar o efeito da actividade física na funcionalidade testicular, bem como no stress oxidativo e apoptose, sugeridos para a acção tóxica da DOX. Trinta e seis ratos macho Sprag-Dawley foram divididos em 6 grupos: salino sedentário (SAL+SED), sedentáros tratados com doses sub-crónicas de DOX – injecções de 2mg.Kg-1 durante sete semanas (DOX+SED), salinos treinados na passadeira durante 12 semanas (SAL+TM), treinados tratados com DOX (DOX+TM), salinos realizando exercício voluntário em roda livre (SAL+FW) e tratados realizando exercício voluntário em roda livre (DOX+FW). Vinte e quatro horas depois da última sessão de exercício, os animais foram sacrificados, os espermatozóides foram obtidos e tratados para estudos de contagem e de motilidade. Os testículos foram recolhidos para posterior análise de marcadores de stress oxidativo (actividade da aconitase, concentrações de substâncias reactivas de ácido tiobarbiturico, malondialdeido (MDA) e de grupos sulfidril (-SH) e sinalização apoptotica (actividades das caspases 3,8 e 9). O tratamento com DOX induziu uma diminuição significativa na contagem e motilidade dos espermatozóides, independentemente da actividade física. Apesar de existir uma tendência para um aumento de MDA e diminuição de –SH com o tratamento com DOX, não foi detectado qualquer efeito significativo nos marcadores de stress oxidativo e apoptose. Não foi observado qualquer efeito do exercício nestes parâmetros. Concluindo, o exercício físico não influenciou o impacto que a DOX teve na funcionalidade testicular. Surpreendentemente, nem a DOX nem o exercício modularam o ambiente redox e a sinalização apoptótica nos testículos, considerando os marcadores analisados.
The anthracycline Doxorubicin (DOX) is a widely used antineoplastic agent against several tumors with high efficacy. However, the clinical use of this drug is limited by its dose-related toxicity in several organs with particular emphasis on the heart. Other organs affected by DOX include liver, brain, kidney and testes. Several pharmacological and non-pharmacological strategies have been designed to antagonize the toxic side effects of DOX, including antioxidant supplementation and, recently, physical exercise. Therefore, the aim of the present study is to analyze the effect of physical exercise in testes function as well as oxidative damage and apoptosis, suggested mechanisms by which DOX exerts its toxic effects. Thirty-six Sprag Dawley male rats were randomly divided into 6 groups as follows: Saline Sedentary (SAL+SED), Sedentary sub-chronically treated with DOX – 2mg.Kg-1 injections for 7 weeks (DOX+SED), Saline endurance treadmill trained for 12 weeks (SAL+TM), trained receiving DOX (DOX+TM), saline voluntary exercised in a free-wheel (SAL+FW) and voluntary exercised receiving DOX (DOX+FW). Twenty-four hours after the last exercise bout, animals were sacrificed; sperm was obtained and treated for counting and motility studies. Testes were harvested for tissues analysis of markers of oxidative stress and damage (aconitase activity, thiobarbituric acid reactive substances, as MDA, and sulfhydryl –SH groups content) and apoptotic signaling (caspases 3,8 and 9 activities). DOX treatment induced significant decrease in sperm count and motility, irrespective of exercise training status. Despite a tendency for MDA increase and –SH decrease with DOX treatment, no significant effect was detected in either markers of oxidative damage or apoptosis. No exercise effect was observed as well. In summary, chronic physical exercise did not influence DOX-induced testes dysfunction. Surprisingly, neither DOX nor exercise modulated testes redox environment and apoptotic signaling, at least seen by the measured markers.

La doxorubicine (DOX) a été utilisée dans le traitement de divers cancers, mais son administration est limitée par une toxicité dose-dépendante. Ses effets cytotoxiques sur les cellules ont montré des cardiotoxicités, d'hépatotoxicité, d'insufisance rénale. L'utilisation des antioxydants ont été explorés pour leurs propriétés de prévention du cancer et et de la toxicité induite par DOX. Le resvératrol (RSV) est un constituant polyphénolique de plusieurs aliments principalement de raisin et de vin. Récemment, son potentiel anticancéreux a été largement exploré, révélant son effet anti-prolifératif sur différentes lignées de cellules cancéreuses, in vitro et in vivo. Le RSV est également connu pour avoir des effets modulateurs sur l'apoptose, la migration et la croissance des cellules via diverses voies de signalisation. Bien que le RSV possède une grande valeur médicinale, ses applications en tant que médicament thérapeutique sont limitées. Des problèmes tels qu'une biodisponibilité orale faible et une solubilité aqueuse médiocre font du RSV un candidat peu fiable à des fins thérapeutiques. De plus, la digestion gastro-intestinale rapide du VRS constitue également un obstacle majeur à sa traduction clinique. Par conséquent, pour surmonter ces inconvénients, nous avons fabriqué des nanoparticules de PLGA/RSV, PLGA/DOX et PLGA/RSV/DOX. Les nanoparticules de RSV ont montré des résultats prometteurs dans son absorption par le système épithélial ainsi que la livraison améliorée au site cible et de réduire l'hépatotoxicité induite par la doxorubicine
Doxorubicin (DOX) has been used in the treatment of variety of cancers but its administration is limited by a dose-dependent toxicity. Its cytotoxic effects on malignant cells, have shown an increase in the risk of cardiotoxicity, hepatoxicity, renal insufisance. Antioxydants have been explored for both their cancer preventive properties and chemodulatory of DOX toxicity. Resveratrol (RSV) is a polyphenolic constituent of several dietary mainly of grapes and wine origin recently its anti-cancer potential has been extensively explored, revealing its anti-proliferative effect on different cancer cell lines, both in vitro and in vivo. RSV is also known to have modulatory effects on cell apoptosis, migration and growth via various signaling pathways. Though, RSV possesses great medicinal value, its applications as a therapeutic drug are limited. Problems like low oral bioavailability and poor aqueous solubility make RSV an unreliable candidate for therapeutic purposes. Additionally, the rapid gastrointestinal digestion of RSV is also a major barrier for its clinical translation. Hence, to overcome these disadvantages RSV-based nanodelivery systems have been considered in recent times.we used nanodelivery systems of RSV, DOX and DOX/RSV have shown promising results in its uptake by the epithelial system as well as enhanced delivery to the target site and reduce the hepatotoxicity induced by doxorubicin

In response to DNA damage, cells decrease global rates of protein synthesis to conserve energy and selectively translate mRNAs of proteins involved in the DNA damage response. Doxorubicin is a widely used chemotherapeutic that induces double strand DNA breaks. It might be expected that doxorubicin-induced DNA damage would rapidly inhibit global protein synthesis through the phosphorylation of eIF2α, as has been observed in response to UVB-induced DNA damage. However, in MCF10A cells, a delay of 9 hours was observed between DNA damage recognition and protein synthesis inhibition. Furthermore, eIF2α phosphorylation was not observed until 12 hours, and global protein synthesis inhibition was subsequently shown to be independent of eIF2α phosphorylation status. An alternative regulator of translation initiation is the mTORC1 target protein 4E-BP1. Doxorubicin-induced mTORC1 inhibition preceded eIF2α phosphorylation and correlated with the inhibition of global protein synthesis, suggesting that the DDR signalled through mTOR to regulate protein synthesis. Experiments using p53-/- MCF10A cells suggested that doxorubicin-induced mTORC1 inhibition was mediated by p53 activity, and p53-/- cells were shown to be more sensitive to doxorubicin-induced cell death. Interestingly, doxorubicin-and catalytic-inhibition of mTORC1 activity mediated the phosphorylation of eIF2α in a signalling mechanism that may be dependent on PP6, DNA-PKcs and GCN2 or PERK. Importantly, eIF2α phosphorylation was absent in response to doxorubicin in p53-/- cells, whereas catalytic inhibition of mTORC1 activity enhanced eIF2α phosphorylation. These data suggested a mechanism where p53-mediated mTORC1 inhibition signalled to enhance the phosphorylation of eIF2α.

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2008.
Includes bibliographical references (leaves 26-28).
Treatment of U20S cells with the chemotherapeutic drug Doxorubicin results in either apoptosis or cellular senescence. The pathway the cell takes is dependent upon the dosage of Doxorubicin administered to the cells. When a 10 [mu]M dose is administered Topoisomerase II is inhibited resulting in double stranded DNA breaks because the DNA is unable to relegate during synthesis. This is shown by lower levels of synthesis after analysis with Bro mo-2-deoxyuridine (BrdU) and Propidium Iodide (PI) staining. The cells are unable to recover from the severity of this damage and become apoptotic. When a 2 [mu]M dose is applied to the cells, a G2 arrest occurs. This is shown by lower levels of Cyclin B in the G2 phase during flow cytometry analysis and staining with PI. Apoptosis levels are monitored using cleaved Caspase 3 and cleaved PARP. The percentage of 10 [mu]M cells undergoing apoptosis increased steadily over 48 hours, while the 2 [mu]M and untreated cells maintained constant low levels of apoptosis. Both cellular senescence and apoptosis put a halt to cell proliferation.
by Katherine D. Weston.
S.B.

The anionic derivative of asymmetric triblock copolymer containing biocompatible chemically complementary polyacrylamide and poly(ethylene oxide) (PAAm-b-PEO-b-PAAm) was obtained. The micellization of the initial (TBC) and modified (TBC-COOH) copolymer samples in aqueous solution were investigated. Practically the same values of the critical micellization concentration and the Gibbs free micellization energy for both TBCs were found. The anticancer effects of the doxorubicin (DOX)-loaded micelles of the initial and modified triblock copolymers were studied on the tumor cells of human T-leukemia of Jurkat line, transformed leukemia of L929 line and mouse lymphatic leukemia of L1210 line. The fact of high efficacy of DOX/TBC and DOX/TBC-COOH compositions in compare with pure DOX was determined and discussed. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/34970

Sorafenib is a potent multi-kinase inhibitor and is the only FDA-approved first-line agent for the treatment of advanced hepatocellular carcinoma (HCC). Liver cancer stem cells (LCSCs) represent a small subset of cells within the HCC tumour mass with an important role in the tumour initiation, progression, recurrence and metastasis. Importantly, LCSCs are also the key cell types that are responsible for the acquisition of sorafenib resistance in HCC. Hence, LCSCs are an ideal target for overcoming sorafenib resistance and HCC therapy. Aptamers (also known as ‘chemical antibodies’) are a group of single-stranded nucleic acid (DNA or RNA) oligonucleotides. As aptamers have distinctive features, particularly favourable tumourpenetrating capacities, low toxicity and immunogenicity, they have been frequently used to deliver therapeutic payloads to cancer cells, and have achieved encouraging anti-tumour effects in various cancers. In this study, we observed that two sorafenib-resistant HCC cell lines derived from Huh7 and Hep3B cells were highly enriched with LCSCs. We developed aptamers specific to LCSC surface marker epithelial cell adhesion molecule (EpCAM) and CD133. These aptamers were then conjugated with Doxorubicin (Dox) to generate the therapeutic complexes EpCAM-apt- Dox (EP-apt-Dox) and CD133-apt-Dox (CD-apt-Dox). We have demonstrated that these therapeutic complexes can be specifically delivered to the EpCAM+ or CD133+ cells in vitro and the xenograft tumours derived from the sorafenib-resistant HCC cells in nude mice. In vitro studies have shown that combination of either EP-apt-Dox or CD-apt-Dox with sorafenib could not only significantly inhibit the proliferation and colony-forming ability but also induce high rates of apoptosis in sorafenib-resistant HCC cells. In animal studies, EP-apt-Dox or CDapt- Dox could sensitize the resistant xenograft tumours to sorafenib treatment. Importantly, the combination of EP-apt-Dox or CD-apt-Dox with sorafenib could exert synergistic anti-tumour effect as demonstrated by significant reduction in the rate of tumour growth and metastasis, and a significant improvement in animal survival rate. These therapeutic effects are likely due to the successful interruption of the self-renewal capacity and ultimately the depletion of the LCSCs. Moreover, treatment of EP-apt-Dox or CD-apt-Dox exhibited much lower toxicity in normal organs (such as heart and liver) than free Dox or sorafenib alone. This study may provide novel evidence that LCSCs are a major subset of cells that are responsible for sorafenib resistance, and that aptamer-based targeting of LCSCs possesses a great potential for overcoming sorafenib resistance and improving treatment outcomes in patients with advanced HCC.

Despite considerable progress in conventional cancer therapies, major challenges persist in the treatment of patients with advanced stage malignancies. Cancer immunotherapy (harnessing the immune system against tumors) has demonstrated limited success to date, partially due to the immunosuppressive environment generated by tumors. The mechanisms of cancer-induced immune suppression are multiple and include the promotion of immunosuppressive cells such as regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). MDSC expand in tumor-bearing hosts and play a central role in cancer immune evasion by inhibiting adaptive and innate immunity. Different approaches have been explored to negatively impact MDSC, each associated with specific pitfalls. In this study, we demonstrated that the anthracycline doxorubicin selectively eliminates MDSC in the spleen, blood and tumor beds. Furthermore, five days after doxorubicin treatment residual MDSC exhibited impaired suppressive function, which correlated with reduced reactive oxygen species (ROS) production, and down-regulation of arginase-1 and indoleamine 2,3-dioxygenase (IDO) expression. Of therapeutic relevance, the frequency of effector lymphocytes (CD4⁺ and CD8⁺ T cells) or natural killer cells (NK) to suppressive MDSC ratios was significantly increased following doxorubicin treatment of tumor-bearing mice. Importantly, the proportion of natural killer (NK) and cytotoxic T cells (CTL) expressing perforin and granzyme B and of CTL producing IFNγ was augmented following doxorubicin administration. The mechanism of doxorubicin-mediated elimination of MDSC was partly mediated by the increase of ROS production in MDSC at earlier time points after doxorubicin treatment. Consistently, MDSC isolated from gp91-/- mice were less sensitive to doxorubicin in vitro, and doxorubicin effects on MDSC in gp91-/- tumor-bearing mice were reduced. Of clinical significance, this drug efficiently combined with Th1 or Th17 lymphocytes to suppress tumor development and metastatic disease, resulting in better overall survival. MDSC isolated from patients with different types of cancer were also sensitive to doxorubicin-mediated cytotoxicity in vitro. Our results therefore indicate that doxorubicin may be used not only as a direct cytotoxic drug against tumor cells, but also as a potent immunomodulatory agent that selectively impairs MDSC-induced immunosuppression, thereby fostering the efficacy of T cell-based immunotherapy.

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Inadequate drug delivery to tumours is now recognised as a key factor that limits the efficacy of anticancer drugs. Extravasation and penetration of therapeutic agents through avascular tissue are critically important processes if sufficient drug is to be delivered to be therapeutic. The purpose of this study is to develop an in silico model that will simulate the transport of the clinically used cytotoxic drug doxorubicin across multicell layers (MCLs) in vitro. Three cell lines were employed: DLD1 (human colon carcinoma), MCF7 (human breast carcinoma) and NCI/ADR-Res (doxorubicin resistant and P-glycoprotein [Pgp] overexpressing ovarian cell line). Cells were cultured on transwell culture inserts to various thicknesses and doxorubicin at various concentrations (100 or 50 microM) was added to the top chamber. The concentration of drug appearing in the bottom chamber was determined as a function of time by HPLC-MS/MS. The rate of drug penetration was inversely proportional to the thickness of the MCL. The rate and extent of doxorubicin penetration was no different in the presence of NCI/ADR-Res cells expressing Pgp compared to MCF7 cells. A mathematical model based upon the premise that the transport of doxorubicin across cell membrane bilayers occurs by a passive "flip-flop" mechanism of the drug between two membrane leaflets was constructed. The mathematical model treats the transwell apparatus as a series of compartments and the MCL is treated as a series of cell layers, separated by small intercellular spaces. This model demonstrates good agreement between predicted and actual drug penetration in vitro and may be applied to the prediction of drug transport in vivo, potentially becoming a useful tool in the study of optimal chemotherapy regimes.

Les glioblastomes sont des tumeurs cérébrales qui sont extrêmement agressives, et qui, en dépit de l'arsenal thérapeutique (chirurgie, radiothérapie ou chimiothérapie), ne laissent pas plus de 16 mois d'espérance de vie aux patients. Dans le cadre de cette thèse, nous proposons d'utiliser certaines toxines en tant que vecteurs pour l'administration de médicaments anticancéreux, et notamment pour le traitement du gliome. Les travaux présentés ici se concentrent sur l’utilisation de variants de la maurocalcine (MCa) et des analogues de la chlorotoxine (CTX). La MCa est une toxine issue du venin du scorpion Scorpio maurus palmatus, qui est capable de pénétrer dans les cellules facilement et rapidement. Il a été prouvé que la MCa peut entrer dans la cellule avec une cargaison. C’est en exploitant cette capacité présente chez deux de ces variants que nous avons synthétisé avec succès deux nouveaux composés à base de cette toxine avec de la doxorubicine et un dérivé du platine. Les études de toxicité et de caractérisation de ces composés qui ont été réalisé on permit de mettre en évidence l’intérêt et le potentiel de la MCa. La seconde partie de ces travaux de thèse portée sur la CTX et des peptides semblables, également extrait de venin de scorpion. Ils ont la particularité de fixer / interagir uniquement avec les cellules cancéreuses d'origine gliale. Après une rapide caractérisation de ces analogues de la CTX, l’un d’eux la Lqh-8/6 a été utilisé avec succès pour l'administration ciblée de doxorubicine. L’ensemble des travaux menés durant cette thèse constitue une base de départ solide pour une amélioration des systèmes de vectorisation, surtout en cancérologie de molécules actives. De plus ces résultats mettent aussi en avant l’avantage de l’utilisation d’un système de couplage « universel » basé sur la chimie click
Glioblastoma are cerebral tumors that are extremely aggressive, and that, in spite of a battery of therapeutic interventions (surgery, radiotherapy or chemotherapy), leave no more than 16 months life expectancy to the patients. As part of this thesis, we propose to use some selected toxins as vectors for the delivery of anticancer drugs, and namely for the treatment of glioma. The works presented here concentrate on the use of variants of maurocalcine (MCa) and the analogues of chlorotoxine (CTX). MCa is a toxin from of the scorpion Maurus palmatus that has cell penetrating propriety. It has been proved that MCa can enter the cell with cargoes. While exploiting this present capacity to two of these variants we synthesized successfully two new compounds with this toxin with the doxorubicine and a by-product of the platinum. Toxicity studies and characterization of these compounds that have been made were permitted to highlight the interest and potential of the MCa. The second part of the thesis work focused on the CTX and similar peptides, also extracted from scorpion venom. They have the particularity to fix/ interact only with cancer cell from neuroectodermal origin. After a fast characterization of these analogues of CTX, one of them (Lqh-8/6) was successfully used for the targeted administration of doxorubicin. All work conducted during this thesis constitutes a solid starting point for an improvement of the systems of vectorization of active molecules, especially in cancer research Moreover, these results also emphasize the advantage of the use of a system of "universal" coupling based on the click chemistry

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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
IntroduÃÃo: A Doxorubicina (DOX), antineoplÃsico antracÃclico, à largamente utilizada no tratamento dos mais diversos tipos de tumores sÃlidos e neoplasias hematolÃgicas. A Cardiotoxicidade provocada pelo uso de antibiÃticos antracÃclicos tem sido observada hà algumas dÃcadas como fator agravante e limitante do uso terapÃutico da DOX. Apesar do conhecimento de inÃmeros fatores que podem mediar a induÃÃo da cardiotoxicidade pela DOX, os mecanismos fisiopatolÃgicos continuam nÃo esclarecidos. Objetivo: Avaliar o efeito da amifostina e glutationa na cardiotoxicidade aguda induzida por doxorubicina e estudar a morfologia do tecido cardÃaco de camundongos tratados com doxorubicina por microscopia de forÃa atÃmica. Material e MÃtodos: Camundongos C57BL/6 fÃmeas (n=8) foram tratados com doxorubicina (25mg/Kg i.p.) ou salina (0,2mL i.p.) e sacrificados 96 horas apÃs tratamento. Outro grupo experimental foi tratado com amifostina (AMF 25, 50 e 100 mg/Kg s.c.), glutationa (GLT 125, 250 e 500mg/Kg s.c.) ou salina 30 mim antes da injeÃÃo de doxorubicina, no caso da glutationa a administraÃÃo foi diÃria atà o dia do sacrifÃcio. Os parÃmetros analisados foram: eletrocardiograma, Ãndices cardÃaco e esplÃnico, dosagem de grupos sulfidrilas nÃo protÃicos, dosagem de CK e CK-MB, parÃmetros histolÃgicos, dosagem por ELISA de TNF-, IL-1 expressÃo por imunohistoquÃmica de TNF-, IL-1, iNOS, apoptose e anÃlise por microscopia de forÃa atÃmica. Resultados: O tratamento com AMF nas doses de 50 e 100mg/Kg e GLT 250 e 500 mg/Kg foi capaz de aumentar a percentagem de sobrevivÃncia dos animais que foram submetidos a cardiotoxicidade aguda induzida por DOX (25 mg/Kg) quando comparados com o grupo injetado somente com DOX. A AMF e GLT tambÃm foram capazes de prevenir, em comparaÃÃo ao grupo DOX (p<0,05), as alteraÃÃes nos valores eletrocardiogrÃficos, (aumento do QRS e QTc e diminuiÃÃo da amplitude de R), as alteraÃÃes nos Ãndices cardÃacos e esplÃnicos, a elevaÃÃo dos nÃveis sÃricos das enzimas CK e CK-MB, a reduÃÃo dos nÃveis de grupos sulfidrilas nÃo protÃicos no tecido cardÃaco e as alteraÃÃes histolÃgicas (degeneraÃÃo hidrÃpica e vacuolizaÃÃo, focos de hialinizaÃÃo de fibras cardÃacas, picnose e necrose) induzidas pela DOX (25mg/Kg). A DOX induziu aumento da marcaÃÃo imunohistoquÃmica para cÃlulas apoptÃticas e expressÃo de iNOS e diminuiu a expressÃo de TNF-. A AMF foi capaz de prevenir estas alteraÃÃes, sendo esta prevenÃÃo apenas discreta para a expressÃo de TNF-. A microscopia de forÃa atÃmica revelou alteraÃÃes morfolÃgicas nÃo vistas pela microscopia Ãptica e mostrou ser uma ferramenta valiosa na avaliaÃÃo de efeitos de drogas. ConclusÃo: Nossos resultados sugerem o efeito citoprotetor da amifostina pelo aumento da atividade da glutationa peroxidase no tecido cardÃaco e que esta se mostra tÃo eficiente quanto a droga de referencia dexrazoxane. A utilizaÃÃo da microscopia atÃmica introduz uma ferramenta de anÃlise comparativa em escala nanomÃtrica, tornando possÃvel observar a destruiÃÃo membranar cardÃaco condizente com dano oxidativo.
IntroduÃÃo: A Doxorubicina (DOX), antineoplÃsico antracÃclico, à largamente utilizada no tratamento dos mais diversos tipos de tumores sÃlidos e neoplasias hematolÃgicas. A Cardiotoxicidade provocada pelo uso de antibiÃticos antracÃclicos tem sido observada hà algumas dÃcadas como fator agravante e limitante do uso terapÃutico da DOX. Apesar do conhecimento de inÃmeros fatores que podem mediar a induÃÃo da cardiotoxicidade pela DOX, os mecanismos fisiopatolÃgicos continuam nÃo esclarecidos. Objetivo: Avaliar o efeito da amifostina e glutationa na cardiotoxicidade aguda induzida por doxorubicina e estudar a morfologia do tecido cardÃaco de camundongos tratados com doxorubicina por microscopia de forÃa atÃmica. Material e MÃtodos: Camundongos C57BL/6 fÃmeas (n=8) foram tratados com doxorubicina (25mg/Kg i.p.) ou salina (0,2mL i.p.) e sacrificados 96 horas apÃs tratamento. Outro grupo experimental foi tratado com amifostina (AMF 25, 50 e 100 mg/Kg s.c.), glutationa (GLT 125, 250 e 500mg/Kg s.c.) ou salina 30 mim antes da injeÃÃo de doxorubicina, no caso da glutationa a administraÃÃo foi diÃria atà o dia do sacrifÃcio. Os parÃmetros analisados foram: eletrocardiograma, Ãndices cardÃaco e esplÃnico, dosagem de grupos sulfidrilas nÃo protÃicos, dosagem de CK e CK-MB, parÃmetros histolÃgicos, dosagem por ELISA de TNF-, IL-1 expressÃo por imunohistoquÃmica de TNF-, IL-1, iNOS, apoptose e anÃlise por microscopia de forÃa atÃmica. Resultados: O tratamento com AMF nas doses de 50 e 100mg/Kg e GLT 250 e 500 mg/Kg foi capaz de aumentar a percentagem de sobrevivÃncia dos animais que foram submetidos a cardiotoxicidade aguda induzida por DOX (25 mg/Kg) quando comparados com o grupo injetado somente com DOX. A AMF e GLT tambÃm foram capazes de prevenir, em comparaÃÃo ao grupo DOX (p<0,05), as alteraÃÃes nos valores eletrocardiogrÃficos, (aumento do QRS e QTc e diminuiÃÃo da amplitude de R), as alteraÃÃes nos Ãndices cardÃacos e esplÃnicos, a elevaÃÃo dos nÃveis sÃricos das enzimas CK e CK-MB, a reduÃÃo dos nÃveis de grupos sulfidrilas nÃo protÃicos no tecido cardÃaco e as alteraÃÃes histolÃgicas (degeneraÃÃo hidrÃpica e vacuolizaÃÃo, focos de hialinizaÃÃo de fibras cardÃacas, picnose e necrose) induzidas pela DOX (25mg/Kg). A DOX induziu aumento da marcaÃÃo imunohistoquÃmica para cÃlulas apoptÃticas e expressÃo de iNOS e diminuiu a expressÃo de TNF-. A AMF foi capaz de prevenir estas alteraÃÃes, sendo esta prevenÃÃo apenas discreta para a expressÃo de TNF-. A microscopia de forÃa atÃmica revelou alteraÃÃes morfolÃgicas nÃo vistas pela microscopia Ãptica e mostrou ser uma ferramenta valiosa na avaliaÃÃo de efeitos de drogas. ConclusÃo: Nossos resultados sugerem o efeito citoprotetor da amifostina pelo aumento da atividade da glutationa peroxidase no tecido cardÃaco e que esta se mostra tÃo eficiente quanto a droga de referencia dexrazoxane. A utilizaÃÃo da microscopia atÃmica introduz uma ferramenta de anÃlise comparativa em escala nanomÃtrica, tornando possÃvel observar a destruiÃÃo membranar cardÃaco condizente com dano oxidativo.
Introduction: Doxorubicin (DOX) is an antineoplasic anthracyclic agent used on the treatment of several solid tumors and hematological cancers. DOX-induced cardiotoxicity has been studied for decades as a limiting factor on the anticancer therapy with this drug. Despite the current knowledge concerning the mechanisms of DOX-induced cardotoxicity, its pathophysiology is still not clear. Purpose: To evaluate of the amifostine and glutathione citoprotective effect on the DOX-induced acute cardiotoxicity and to study the morphology of cardiac tissue through the use of atomic force microscopy as a tool. Materials and Methods: C57BL/6 female mice were treated with doxorubicin (25mg/Kg i.p.) or saline (0,2mL i.p.) and sacrificed 96 hours after treatment. In another experimental setting, mice were given amifostine (AMF 25, 50 e 100 mg/Kg s.c.), glutathione (GLT 125, 250 e 500mg/Kg s.c.) or vehicle, 30 mim before the administration of DOX, except glutathione that was injected daily. Analitical parameters included: electrocardiograms, cardiac and spleen indices, non protein suphidrils groups levels, CK and CK-MB cardiac enzymes levels, histological analysis, cardiac levels of (TNF-, IL-1, iNOS) determined by ELISA, immunohistochemistry for TNF-, IL-1, iNOS, apoptosis and atomic force microscopy tissue analysis. Results: AMF (100mg/Kg) and GLT (250 e 500 mg/Kg) treatments were able of improve the survival rate of animals in spite of the injection of DOX (25 mg/Kg) in comparison to DOX-treated only group (p<0.05). A AMF e GLT were also able to prevent electrocardiographic changes (rising of QRS e QTc and reduced R amplitude), changes in the cardiac and spleen indices, the augmentation of blood levels of CK e CK-MB, reduction of non proteic suphidrils groups levels, and histological changes induced by DOX (25mg/Kg). DOX induced the augmentation of the immunostaining for apoptotic cells and iNOS what was prevented by the administration of amifostine. The atomic force microscopy reveals morphological changes on the tissue organizational structure which is not possible to be observed through optical microscopy. Conclusion: Our results suggest that the amifostine citoprotective effect on DOX-induced acute cardiotoxicity is due the rising of glutathione peroxidase activity in the cardiac tissue. The citoprotective effect of amifostine is as efficient as the reference drug dexrazoxane. The use of atomic force microscopy as a new pharmacological tool for comparative analysis in nanometric scale allow us to observe DOX-induced membrane destruction what is suggestive of oxidative stress process.
Introduction: Doxorubicin (DOX) is an antineoplasic anthracyclic agent used on the treatment of several solid tumors and hematological cancers. DOX-induced cardiotoxicity has been studied for decades as a limiting factor on the anticancer therapy with this drug. Despite the current knowledge concerning the mechanisms of DOX-induced cardotoxicity, its pathophysiology is still not clear. Purpose: To evaluate of the amifostine and glutathione citoprotective effect on the DOX-induced acute cardiotoxicity and to study the morphology of cardiac tissue through the use of atomic force microscopy as a tool. Materials and Methods: C57BL/6 female mice were treated with doxorubicin (25mg/Kg i.p.) or saline (0,2mL i.p.) and sacrificed 96 hours after treatment. In another experimental setting, mice were given amifostine (AMF 25, 50 e 100 mg/Kg s.c.), glutathione (GLT 125, 250 e 500mg/Kg s.c.) or vehicle, 30 mim before the administration of DOX, except glutathione that was injected daily. Analitical parameters included: electrocardiograms, cardiac and spleen indices, non protein suphidrils groups levels, CK and CK-MB cardiac enzymes levels, histological analysis, cardiac levels of (TNF-, IL-1, iNOS) determined by ELISA, immunohistochemistry for TNF-, IL-1, iNOS, apoptosis and atomic force microscopy tissue analysis. Results: AMF (100mg/Kg) and GLT (250 e 500 mg/Kg) treatments were able of improve the survival rate of animals in spite of the injection of DOX (25 mg/Kg) in comparison to DOX-treated only group (p<0.05). A AMF e GLT were also able to prevent electrocardiographic changes (rising of QRS e QTc and reduced R amplitude), changes in the cardiac and spleen indices, the augmentation of blood levels of CK e CK-MB, reduction of non proteic suphidrils groups levels, and histological changes induced by DOX (25mg/Kg). DOX induced the augmentation of the immunostaining for apoptotic cells and iNOS what was prevented by the administration of amifostine. The atomic force microscopy reveals morphological changes on the tissue organizational structure which is not possible to be observed through optical microscopy. Conclusion: Our results suggest that the amifostine citoprotective effect on DOX-induced acute cardiotoxicity is due the rising of glutathione peroxidase activity in the cardiac tissue. The citoprotective effect of amifostine is as efficient as the reference drug dexrazoxane. The use of atomic force microscopy as a new pharmacological tool for comparative analysis in nanometric scale allow us to observe DOX-induced membrane destruction what is suggestive of oxidative stress process.

The use of liposomes exhibiting a transmembrane pH gradient (inside acidic) to accumulate doxorubicin into the interior aqueous compartment has been shown to achieve drug trapping efficiencies in excess of 98% in a manner which is independent of lipid composition. Doxorubicin entrapment appears to be most efficient at 60°C with 100% drug accumulation occurring after 5 minutes. An increase in internal buffering capacity and trap volume of the vesicles significantly enhances doxorubicin sequestration. Initial drug to lipid ratios as high as 2:1 (wt:wt) have been used, although trapping efficiencies fall below 95% at drug to lipid ratios in excess of 1:2 (wt:wt). As vesicle size is decreased the initial drug to lipid ratio must be reduced to 1:10 to maintain high trapping efficiencies. In addition to effecting efficient doxorubicin entrapment, the transmembrane pH gradient also reduces the rate of doxorubicin leakage. For example, in liposomes exhibiting a pH gradient greater than 2 units, release is less than 5% of the encapsulated doxorubicin over 24 hours at 37°C whereas release rates are significantly higher in the absence of a pH gradient. Finally, a procedure for a rapid colorimetric test for determining the amount of unencapsulated doxorubicin is described. The test is based on a spectral shift of doxorubicin peak absorption from 480nm to approximately 600nm upon addition of alkali to the liposomal doxorubicin. The resulting color change of untrapped drug from orange to purple can be quantitated spectrophotometrically or visually.
Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate

Daunorubicin and its congener, doxorubicin, are two of the most important anti-tumor antibiotics used in cancer chemotherapy. Both agents exhibit a broad range of activity against solid tumors and hematologic malignancies, yet their clinical potential is limited by severe dosedependent cumulative cardiotoxicity. So far, over 2000 analogs have been synthesized comprising numerous chemical modifications, substitutions and conjugations to the tetracyclic ring, the side chain or the amino-sugar. However, only few of those have reached the stage of clinical development and approval. Furthermore no analog offers a definite advantage over daunorubicin and doxorubicin in terms of activity or toxicity. The search for a better anthracycline may consequently require a radically different approach. These drugs are synthesized by a type II polyketide synthase consisting of a number of fully dissociable enzymes. Although the structural, regulatory and resistance genes of the daunorubicinldoxorubicin biosynthetic clusters have been identified, their functions characterized and a general scheme for the biosynthetic pathway has been proposed, little is known about the structural organization of these enzymes within the PKS and how this affects catalysis. Furthermore, the exact mechanism of starter unit selection remains elusive. Knowledge of the above would help us modify the biosynthetic pathway in order to engineer new anthracycline natural products with improved activity and pharmacological properties. To accomplish these goals, all dps proteins were expressed and purified to homogeneity. Two enzymes, the ketosynthase subunits (DpsAlB) and the malonyl transferase (DpsD) were expressed for the first time. Functional interactions between the dps components were then identified in vitro by ITC and 1FT and, based on the interactions data, a model for the architectural organization of the PKS was proposed. The roles of DpsC and DpsD as the starter unit specifying enzymes were also examined in detail. Our analysis showed that, contrary to previous reports, DpsD seems to be the starter unit specifying enzyme. Finally, crystals of DpsC and DpsE were obtained and diffraction data collected that could aid in structure elucidation for these components.

A tumor targeted dendrimer based drug delivery system was designed and synthesized to carry chemotherapy drug doxorubicin. Polyamidoamine (PAMAM) dendrimer G4.5 was chosen as the underlying carrier. Anionic G4.5 is a good option for drug delivery as it consists of 128 surface groups, is less cytotoxic and favorably biodistributed. The delivery system was synthesized using a layer-by layer arrangement of three functional entities: chemotherapy drug doxorubicin, monoclonal antibody Cetuximab against EGF receptor, and polyethylene glycol (PEG). Doxorubicin was attached via an acid-sensitive hydrazon linkage to the dendrimer. Macromolecules are taken in by cells through endocytosis. pH inside the early endosomes to lysosomes ranges from pH 6 to 4.5. These acidic conditions are favorable for release of drug bound to the dendrimer vehicle through acid-sensitive linkage. 35% of all solid tumors of brain express exceptionally high EGF receptors whereas normal brain tumors express less EGFR. This makes the EGFR a potent targeting moiety for targeted drug delivery. Cetuximab will serve as a targeting ligand to help the delivery system target tumor cells. PEG was incorporated as a linker between Cetuximab and dendrimer to avoid reticuloendothelial system (RES) uptake of the system, increase biocompatibility, increase drug half-life and other shortcomings associated with nanomaterials. Nuclear magnetic resonance spectroscopy (NMR), fluorescence anisotropy, and western blotting were used to confirm the conjugation of PEG, doxorubicin and cetuximab to the dendrimer. The synthesized delivery system was characterized using ultraviolet-visible spectroscopy (UV-Vis) to approximate the number of doxorubicin attached. Dynamic light scattering (DLS) and zeta potential were used to analyze the change in size and surface properties of dendrimer during the synthesis. Doxorubicin release studies were conducted at different pHs. Maximum doxorubicin was released at pH 4.5 indicating the successful acid-sensitive linkage between the drug and dendrimer. Cytotoxicity studies indicated that the addition of PEG increased the biocompatibility as compared to free doxorubicin whereas; combination of doxorubicin and cetuximab exerted a significant toxic effect over a period of 72 hours. The cellular uptake of the delivery system was higher than that of free doxorubicin. Free DOX localized mainly in the nucleus whereas, CTX-G4.5-PEG-DOX conjugate localized within both cytoplasm and nucleus after 6 hour incubation. The synthesized delivery system represents a potential targeted drug delivery system.

Prior studies have demonstrated the effect of diazoxide in protecting against apoptosis via mitochondrial KATP channel opening in vitro. The current investigations are designed to determine if sildenafil, a phosphodiesterase-5 inhibitor and known mitochondrial KATP channel opener, would protect against chronic doxorubicin cardiomyopathy both in vivo and in vitro.Male ICR mice were randomized to 1 of 4 treatments: saline, sildenafil (0.7 mg/kg IP), doxorubicin (5 mg/kg IP), and sildenafil (0.7 mg/kg IP)+doxorubicin. Apoptosis was determined using the terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling and in situ oligo ligation methods. Desmin distribution was determined via immunofluorescence. Bcl-2 was analyzed by Western blot. Left ventricular function was measured in Langendorff mode. Electrocardiographical analysis measured changes indicative of doxorubicin cardiotoxicity (ST-prolongation). In vitro studies using adult ventricular cardiomyocytes were exposed to doxorubicin (1 μM), sildenafil (1 μM) with or without NG-nitro-L-arginine methyl ester (L-NAME; 100 μM), or 5-hydroxydecanoate (5-HD; 100 μM) 1 hour before doxorubicin and incubated for 18 hours. Doxorubicin-treated mice demonstrated increased apoptosis and desmin disruption, which was attenuated in the sildenafil+doxorubicin group. Bcl-2 decreased in the doxorubicin group but was maintained at basal levels in the sildenafil+doxorubicin group. Left ventricular developed pressure and rate pressure product were significantly depressed in the doxorubicin group but attenuated in the sildenafil+doxorubicin group. ST-interval significantly increased in the doxorubicin group over 8 weeks. In the sildenafil+doxorubicin group, ST-interval remained unchanged from baseline. Doxorubicin significantly increased apoptosis, caspase-3 activation, and disruption of mitochondrial membrane potential in vitro,. In contrast, sildenafil significantly protected against doxorubicin cardiotoxicity; however, protection was abolished by both L-NAME and 5-HD. Cell viability studies using spectrophotometer and flow cytometric techniques demonstrated that sildenafil did not affect the antitumor efficacy of doxorubicin in PC-3 cells in vitro. In fact, flow cytometry data indicate that sildenafil, when combined with doxorubicin, was synergistic in the antineoplastic action of doxorubicin. Prophylactic treatment with sildenafil prevented apoptosis and left ventricular dysfunction in a chronic model of doxorubicin-induced cardiomyopathy. Moreover, these studies provide relevant clinical data on the safety and efficacy of sildenafil, leading the way for clinical trials in humans receiving doxorubicin chemotherapy.

Multidrug resistance is a phenomenon associated with the emergence of simultaneous cross-resistance to the cytotoxic actions of a wide variety of structurally and functionally unrelated antineoplastic agents. One of the agents to which cross-resistance is commonly observed is doxorubicin, a commonly used antineoplastic. Studies were undertaken to determine the mechanism of doxorubicin resistance in multidrug resistant 8226 human myeloma cells. When sensitive and resistant cells were exposed to the same extracellular concentration of doxorubicin there was a decrease in the quantity of DNA lesions in the resistant subline which corresponded to a decrease in doxorubicin accumulation. When the extracellular concentration of drug was adjusted to yield equivalent intracellular levels these differences were removed. Studies utilizing an isolated nuclei system revealed no differences in the formation of DNA lesions between the sensitive and resistant cells when exposed to the same concentration of drug. Studies were undertaken to determine if the resistant subline had an increased capacity to detoxify doxorubicin via glutathione-based enzyme systems. The activities of glutathione-s-transferase and glutathione peroxidase were not found to be elevated in the resistant subline. There was a significant elevation in the nonprotein sulfhydryl content of the resistant cells as compared to the drug-sensitive line. This elevation was unstable in the absence of doxorubicin, displaying a steady decline until reaching baseline levels found in the sensitive cells. The decrease in NPSH content in the resistant line was not accompanied by an alteration in doxorubicin resistance. Thus, it appears that glutathione-based enzymatic detoxification is not causally related to doxorubicin resistance in 8226 human myeloma cells. Verapamil, an agent shown by previous studies to modulate doxorubicin resistance, led to an increase in the formation of doxorubicin-induced DNA lesions in the resistant cells secondary to an increase in intracellular drug accumulation. It had no effect on doxorubicin-induced DNA lesions or drug accumulation in the sensitive cells. Verapamil thus appears to be reversing doxorubicin resistance by increasing drug accumulation and thereby enhancing DNA damage. Under these circumstances there was a good correlation between doxorubicin accumulation, DNA damage, and cytotoxicity in the 8226 cells. The conclusion is drawn that drug accumulation accounts for the majority of doxorubicin resistance in the 8226 human myeloma cell line.

The current studies were designed to explore the protective effects of pifithrin-α and melatonin against doxorubicin-induced cardiotoxicity. Doxorubicin was injected at a dose of 22.5 mg/kg (i.p.) in mice to induce cardiotoxic effects. Meanwhile, doxorubicin caused a significant increase of cardiac cell apoptosis following injection (14.2 ± 1.1% for doxorubicin-5 d vs. 1.8 ± 0.12% for control, P < 0.01). Ribonuclease protection assays and Western blot analyses revealed that doxorubicin upregulated the p53-dependent genes Bax, BclxL, and MDM2 at least 2-fold. p53 was phosphorylated at Ser 15 in mouse hearts 1 h following doxorubicin injection, and p38 and ERK1/2 MAPKs mediated the phosphorylation of p53. In addition, caspases-3 and -9 were activated 24 h after doxorubicin injection. A p53 inhibitor, pifithrin-α, inhibited doxorubicin-induced apoptosis when administered at a dose of 2.2 mg/kg. Pifithrin-α abolished p53 transactivation activity, but did not influence doxorubicin-induced phosphorylation at Ser 15. By effectively inhibiting the expression of p53-dependent genes, pifithrin-α blocked doxorubicin-induced activation of caspases-3 and -9, thereby preventing cardiac apoptosis. In addition, pifithrin-α attenuated doxorubicin-induced structural and functional damages, without diminishing its anti-tumor efficacy on p53-null PC-3 cancer cells. The protective effects of melatonin and its metabolite 6-hydroxymelatonin on doxorubicin-induced cardiac dysfunction were evaluated in an isolated perfused mouse hearts and in vivo doxorubicin-treated mice. While perfusion of mouse hearts with 5 μM doxorubicin for 60 min resulted in a 50% suppression of HRxLVDP and a 50% reduction of coronary flow, pre-exposure of hearts to 1 μM melatonin or 6-hydroxymelatonin eased the cardiac dysfunction. In addition, administration of melatonin or 6-hydroxymelatonin (2 mg/kg/d) significantly attenuated doxorubicin-induced cardiac dysfunction, myocardial lesions, and cardiac cell apoptosis. Melatonin and 6-hydroxymelatonin significantly improved the survival rate of doxorubicin-treated mice. Another melatonin analog, 8-methoxy-2-propionamidotetralin, did not show any convincing protection on either animal survival or on in vitro cardiac function, presumably due to its lack of free radical-scavenging activity. Finally, neither melatonin nor 6-hydroxymelatonin compromised the anti-tumor activity of doxorubicin in cultured PC-3 cells. These studies suggest that pifithrin-α and melatonin have significant therapeutic potential for patients suffering doxorubicin-induced cardiotoxicity.

Background The application of nanotechnology in the medical field is called nanomedicine. This novel sector have got a lot of interest from many investigators nowadays due to the important development that happened in the last decades, in particular in cancer treatment. Cancer nanomedicine has been applied in different domains such as drug delivery, nanopharmaceuticals and nanodevices. The application of nanotechnology to pharmaceutical science allowed to build up system based on at least two stage vectors (drug/nanomaterial). New formulations based on that platform show often an improvement in drug pharmacokinetics (PK), bioavailability and biodistribution owing enhanced permeability and retention (EPR) effect to passively target tumor both decreasing side effect of free drug. Among natural nanovectors, exosomes (exo) were first described in 1981 as extracellular nanovesicles with a size range of 50-200 nm. Exosomes are produced by cells embedded of cellular information and represent a formidable natural cargo for long distance communication. The name "fedexosome" denotes the general idea that exosomes could deliver cargo that conveniently manipulated could be of help for patients therapy. Aims To develop exosomes loaded with doxorubicin (DOX) To test the cytotoxic effect of exoDOX (exosomal doxorubincin) in vitro using cell lines models compared to the parental drug (DOX) To test the antitumor activity and the toxic side effects of exoDOX compared to the parental DOX in in vivo experimental models To test tissue biodistribution and pharmacokinetics (PK) of exoDOX compared to DOX in vivo experimental models To define the maximum tolerated dose (MTD) of exoDOX and DOX in in vivo experimental models Materials and Methods Purified exosomes from cell lines were loaded with DOX and characterized by nanoparticle tracking analysis (NTA), Scanning/Transmission electron microscopy (SEM/TEM) and western blot. The anti-tumoral effects of exoDOX were tested in vitro (MDA-MB-231 breast, HCT-116, LoVo and DLD1 colon and STOSE ovarian cancer cell lines) and in vivo using nude and FVB/N mice as breast and ovarian cancer models. The antitumor effect was assessed by measuring the tumor volumes. The toxic effects were evaluated by following the body weight and through histopathological analyses of mice organs. The biodistribution and PK of exoDOX and DOX were assessed by mass spectrometry (LC-MS). Results • In vitro studies showed no increased cytotoxic effect (cell viability) of exoDOX compared to DOX in all the investigated cell lines . • Similar results were observed in the in vivo models indicating no significant differences in the tumor volume after treating mice with the same concentrations of exoDOX and DOX. • In vivo toxicity analysis showed a significant reduction of cardio-toxic side effects by using exoDOX compared to free DOX. Mass spectrometry studies showed that the accumulation of exoDOX in the heart was reduced by about 40% compared to free DOX when using the same concentration of active drug. ExoDOX avoids heart toxicity by partially limiting the crossing of DOX through the myocardial endothelial cells. For this reason, mice can be treated with higher concentration of exoDOX thus increasing the efficacy of DOX as demonstrated in breast and ovarian mouse tumors. - Conclusions Differently from previously published papers that focused on the efficacy of the doxorubicin encapsulated in exosomes, in this thesis for the first time, we demonstrated that unmodified exosomes loaded with DOX are less toxic than free DOX by altering the biodistribution of the drug, these results were published in Nanomedicine (Lond) Journal in 2015. ExoDOX is safer and more effective than free DOX using breast cancer model and importantly was confirmed using the first spontaneous transformed syngeneic model of high-grade serous ovarian cancer which open the road for providing a new therapeutic opportunity, which was published recently in Nanomedicine (Lond) Jounral in 2016.
Introduzione L'applicazione delle nanotecnologie in medicina è chiamata nanomedicina. Questo settore è motivo di interesse da parte di molti ricercatori dovuto agli importanti avanzamenti avvenuti negli ultimi decenni, in particolare nel trattamento del cancro. In oncologia la nanomedicina è stata applicata in diversi settori quali la costruzione di nuovi sistemi di veicolazione del farmaco e nano dispositivi per la diagnosi. L'applicazione delle nanotecnologie alle scienze farmaceutiche ha permesso di costruire sistemi basati su almeno due vettori (farmaco/nanomateriali). Le nuove formulazioni spesso mostrano un miglioramento del profilo di farmacocinetica (PK), la biodisponibilità e biodistribuzione dimostrando una migliorata permeabilità e ritenzione passiva nel tumore (EPR effect) diminuendo gli effetti collaterali del farmaco libero. Tra i nanovettori naturali, gli esosomi (exo) sono stati descritti nel 1981 come nanovescicole extracellulari con una gamma di dimensioni da 50-200 nm. Gli esosomi sono prodotti dall invaginazione dalla conseguente gemmazione della membrana cellulare consentendo il caricamento di acidi nucleici e di componenti citoplasmatiche rappresentando formidabile mezzo naturale per la comunicazione a lunga distanza. Il nome "fedexosome" denota l'idea generale che exosomes potevano consegnare contenuti che adeguatamente ingegnerizzato potrebbe essere di aiuto per la terapia di pazienti. Scopo • Sviluppare esosomi caricati con doxorubicina (DOX) • Verificare l'effetto citotossico dell'exoDOX exosomal doxorubincin in vitro rispetto al farmaco libero (DOX), utilizzando modelli cellulari. • Testare l'attività antitumorale e la tossicità dell'exoDOX rispetto alla DOX libera in modelli sperimentali in vivo. • Valutare la biodistribuzione nei tessuti e la farmacocinetica (PK) dell'exoDOX rispetto al DOX in modelli sperimentali in vivo. • Definire la dose massima tollerata (MTD) dell' exoDOX e DOX in modelli sperimentali in vivo. Materiali e metodi Gli esosomi purificati da linee cellulari sono stati caricati con DOX e caratterizzati tramite nanoparticle tracking analysis (NTA), microscopia a scansione/ trasmissione elettronica (SEM/TEM) e western blot. Gli effetti anti-tumorali dell'exoDOX sono stati valutati in vitro in linee cellulari di tumore alla mammella (MDA-MB-231), colon (HCT-116, LoVo e DLD1) e ovaio (STOSE) e in vivo utilizzando topi nudi e FVB/N come modelli di tumore della mammella e dell'ovaio. L'effetto antitumorale è stato valutato misurando il volume del tumore. Gli effetti tossici sono stati determinati monitorando il peso corporeo e attraverso analisi istopatologiche degli organi dei topi. La biodistribuzione e la PK dell'exoDOX e DOX sono state definite mediante spettrometria di massa (LC-MS). Risultati • Studi in vitro hanno dimostrato un aumento dell'effetto citotossico (vitalità cellulare) dell'exoDOX rispetto alla DOX in tutte le linee cellulari esaminate. • Risultati simili sono stati ottenuti nei modelli in vivo e indicano differenze significative nel volume del tumore dopo aver trattato i topi con le stesse concentrazioni dell'exoDOX e DOX. • Le analisi della tossicità in vivo hanno mostrato una riduzione significativa degli effetti collaterali cardio-tossici dell'exoDOX rispetto alla DOX libera. Dalle analisi di spettrometria di massa è emerso un minore accumulo dell'exoDOX a livello del cuore di circa il 40% rispetto alla DOX libera a parità di concentrazione di farmaco attivo. • La minore tossicità cardiaca della exoDOX è dovuta ad un ridotto passaggio della DOX attraverso le cellule endoteliali del miocardio. Pertanto, come dimostrato in tumori alla mammella e all'ovaio, l'efficacia terapeutica della DOX può essere ottimizzata trattando i topi con una maggiore concentrazione dell' exoDOX. Conclusioni A differenza di quanto riportato in letteratura circa l'efficacia della doxorubicina incapsulata negli esosomi, dal nostro studio, pubblicato nel 2015 nella rivista Nanomedicine, emerge che: - la tossicità della DOX veicolata dagli esosomi è minore rispetto al farmaco libero; -la biodistribuzione della DOX veicolata dagli esosomi è diversa da quella del farmaco libero. In conclusione, lo studio effettuato dimostra che l'exoDOX è più biocompatibile ed efficace della DOX libera in modelli di tumore alla mammella. Inoltre, come da noi pubblicato di recente sulla rivista Nanomedicine, tale risultato è stato confermato con studi su un modello singenico di carcinoma ovarico sieroso, aprendo così la strada ad un nuovo approccio terapeutico per il cancro all'ovaio.

La doxorubicine (DXR) est l'un des médicaments les plus efficaces en chimiothérapie, mais sonapplication clinique est limitée par ses effets cardiotoxiques. Malgré des décennies de recherche, sesmécanismes pathogéniques ne sont toujours pas entièrement compris. Il s'ensuit qu'aucun traitementsatisfaisant, curatif ou préventif, n'existe. Dans cette étude, nous recherchons les mécanismes designalisation cellulaire impliqués. Deux modèles expérimentaux de toxicités, aigue d'une part (coeurisolé et perfusé de rat avec la DXR), et chronique d'autre part (rat traité à la DXR), ont permis deréaliser une étude ciblée (sur les voies de signalisation énergétiques) et deux études systémiques(phosphoprotéomique et transcriptomique). Les résultats combinés de ces travaux ont montré que laDXR modifiait le niveau de phosphorylation (activation) ou l'expression génique de protéinesimpliquées dans trois domaines fonctionnels distincts : métabolisme énergétique, réponses au stress,et structure/fonction du sarcomère. (i) Métabolisme énergétique : nous avons confirmé la surprenanteinhibition de l'AMPK, probablement provoquée par un contrôle négatif exercé par des partenaires designalisation (Akt et ERK), plutôt que par une modification des kinases activatrices en amont. Nousavons également montré l'augmentation du niveau de phosphorylation de la PDH, ce qui, en inhibantl'enzyme, ralentit le cycle de Krebs. Cependant, nous avons également observé un phénomènecompensatoire de surexpression de gènes codant pour des enzymes de la glycolyse et du cycle deKrebs ; (ii) Réponses au stress : dans nos modèles, la DXR génère des stress énergétique,génotoxique et oxydatif. Cependant, seuls quelques mécanismes compensatoires sont activés (lesvoies de signalisation de DNA-PK-Akt-GSK3, diverses chaperonnes). Les autres semblent êtreinhibées suggérant que l'amoindricement des réponses au stress serait un des mécanismes de lacardiotoxicité de la DXR; (iii) Structure/fonction du sarcomère: L'augmentation de la phosphorylationde la desmine ainsi que la réduction du nombre de transcrits codant pour des protéines essentiellesau développement cardiaque normal pourraient être la cause de la désorganisation du réseaumyofibrillaire. En conclusion, ces résultats révèlent potentiellement de nouveaux mécanismes de lacardiotoxicité induite par la DXR et permettent d'envisager de nouvelles cibles moléculaires pour ledéveloppement de stratégies protectrices.

Aims: Previous studies have shown that tumour associated macrophages (TAMs) limit the efficacy of chemotherapy agents like paclitaxel in mouse tumours. Furthermore, perivascular (PV) MRC1+ TAMs stimulate tumour regrowth after their exposure to cyclophosphamide. The aim of this thesis was to investigate the presence and origin of these PV cells in orthotopic mammary (TS1) tumours after doxorubicin (DOX) treatment. Attempts were also made to characterise their interaction with the tumour vasculature in such tumours. Methods and Results: When TS1 tumours had become established in the mammary fat pads of FVB/N mice, their hosts were treated with a single injection of DOX or PBS. 48 hours later, mice were culled and their tumours removed for analysis. Immunofluorescent staining of tumours sections revealed the presence of increased numbers of MRC1+ TAMs in the well-vascularised (normoxic) stromal areas of TS1 tumours, compared to their less well-vascularised, tumour cell islands. Moreover, the number of these cells making direct contact with the tumour vasculature increased after DOX. These cells were mature Gr-1- cells, rather than newly recruited monocytes or immature TAMs. They were not seen to associate with vessels of a particular size. DOX had no effect on the luminal area, patency or pericyte coverage of tumour blood vessels but increased the expression of VegfA mRNA by CD31+ endothelial cells. Moreover, both endothelial cells, and other, as yet undefined cells, upregulated mRNA for Angiopoietin-2, Cx3cl1, Osteopontin and Plgf in response to DOX. Conclusions: DOX increases the number of MRC1+ TAMs associated with blood vessels in TS1 tumours, possibly in response to various genes upregulated by tumour endothelial cells (and other cells in the tumour microenvironment). The impact of these on the recruitment, retention and/or activation of TAMs in the PV niche merits further investigation.

Thesis (MSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Introduction The discovery of Doxorubicin in the 1960s has drastically improved the survival rates of cancer patients, however, its success is limited by dose-dependent cardiotoxicity. While much of the literature has focused on acute cardiotoxicity which is minor and generally reversible, chronic cardiotoxicity poses a serious threat to cancer survivors since it can lead to dilative cardiomyopathy, congestive heart failure and even death. The mechanisms that contribute to cardiotoxicity are still a matter of controversy, however, oxidative stress-induced myocardial damage and apoptosis are thought to be the major role players. Reperfusion injury, also characterized by oxidative stress and apoptosis, occurs as a result of restoring blood flow to an ischemic heart. Fortunately, pre- and post-conditioning are techniques employed to minimize this damage and are thought to do so by activating the reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways. The RISK pathway involves the pro-survival kinases, Erk1/2 and Akt, while the SAFE pathway, triggered by TNF-α, involves Jak2 and STAT3. Since both reperfusion injury and Doxorubicin-induced cardiotoxicity share similar characteristics, this study aimed to determine whether the RISK and SAFE pathways are activated in response to long-term Doxorubicin treatment. Furthermore, this study aimed to determine whether TNF-α is produced during treatment, since its role in Doxorubicin-induced cardiotoxicity is still relatively unknown. Methods H9c2 cardiomyocytes and differentiated C2C12 myotubes were treated daily with increasing concentrations of Doxorubicin for a total of 120 hours. Cell viability, apoptosis and necrosis were assessed using the MTT, Caspase-Glo® 3/7 and lactate dehydrogenase assays respectively. TNF-α production was measured using Quantikine® ELISA kits and various assays were used to assess oxidative stress, anti-oxidant capacity and anti-oxidant status. The protein expression of the RISK and SAFE pathways were analysed by western blotting using both phospho-specific and total antibodies. Results and Discussion Treatment with Doxorubicin caused a time- and dose-dependent decrease in cell viability in both cell lines and this was accompanied by an increase in apoptosis. In the H9c2 cardiomyocytes, treatment with 0.2 μM Doxorubicin yielded significant levels of TNF-α after 120 hours and we can speculate that these low levels partially protected the cells from the toxic effects of Doxorubicin by activating the SAFE pathway, since both Jak2 and STAT3 were phosphorylated at this concentration. Treatment with 1 μM Doxorubicin caused a larger and biphasic pattern of TNF-α release, which may have then contributed to the decrease in cell viability, since the SAFE pathway was not activated at this concentration. Akt was phosphorylated during the first 72 hours of treatment with the low dose of Doxorubicin, but chronic treatment prevented this phosphorylation. While Erk1/2 was not phosphorylated at all at the low dose of Doxorubicin, neither Akt nor Erk1/2 was phosphorylated at the high dose and their inhibition may contribute to the cardiotoxic effects of Doxorubicin. In the C2C12 myotubes, a significant amount of TNF-α was produced after 120 hours of treatment with the low dose of Doxorubicin. Treatment with the high dose of Doxorubicin induced significant TNF-α production at every time point. While STAT3 was phosphorylated at the serine residue after treatment with the low dose of Doxorubicin, treatment with the high dose induced phosphorylation at the tyrosine residue in a time-dependent manner. p-Jak2 expression was significantly down-regulated at both concentrations of Doxorubicin, suggesting that STAT3 proteins can by-pass activation by Jak2. The Erk1/2 leg of the RISK pathway was also not activated for the majority of the treatment period, however, p-Akt expression was increased at the low concentration of Doxorubicin relative to total Akt expression. Conclusion These observations indicate that treatment with Doxorubicin causes a severe, dose-dependent loss in viability which is likely to mediated by high concentrations of TNF-α (induced by high concentrations of Doxorubicin) and down-regulation of protective signaling pathways. TNF-α may confer partial protection at low concentrations by activating the SAFE pathway. However, activation of the SAFE pathway could not provide sufficient protection from Doxorubicin, most probably because the RISK pathway was not simultaneously activated. Our results also clearly highlight the differences between acute and chronic treatment since a single high dose of Doxorubicin produced vastly different responses to cumulative treatment with a low dose. Before one can extrapolate these results into the clinical setting, further research is required to provide a better understanding of the RISK and SAFE pathways and whether stimulation thereof will provide a protective effect. In addition, although our study has shown that TNF-α is produced in response to Doxorubicin treatment, its true role, whether beneficial or detrimental, remains to be determined.
AFRIKAANSE OPSOMMING: Inleiding Die ontdekking van Doksorubisien (DOKS) in die 1960’s het die oorlewingsyfer van kankerpasiënte drasties verhoog, maar DOKS-gebruik gaan egter ook gepaard met dosis-afhanklike kardiotoksisiteit. Terwyl die literatuur grootliks fokus op akute kardiotoksisiteit, wat minimaal en algemeen omkeerbaar is, hou kroniese kardiotoksisiteit ‘n ernistige bedreiging vir kankeroorlewendes in, aangesien dit kan lei tot dilatiewe kardiomiopatie, kongestiewe hartversaking, en selfs dood. Die spesfikieke meganismes wat bydrae tot kardiotoksisiteit is tans steeds onbekend, maar oksidatiewe stres-geinduseerde miokardiale skade en apoptose word beskou as hoof bydraende faktore. Reperfussie skade, ook gekaraktiseer deur die teenwoordigheid van oksidatiewe stres en apoptose, kom voor as gevolg van die herstel van bloedtoevoer na ‘n isgemiese hart. Om die skade te minimaliseer word voor- en nakondisionerings tegnieke geïmplimenteer wat die RSHK (Reperfussie Skade Herwinnings Kinase) en OAFV (Oorlewerings Aktiverings Faktor Versterkings)-weë aktiveer. Die RSHK weg maak gebruik van pro-oorlewings kinases Erk1/2 en Akt, terwyl die TNF-α geaktiveerde OAFV weg Jak2 en STAT3 betrek. Aangesien beide reperfussie skade en DOKS-geinduseerde kardiotoksisiteit soortgelyke eienskappe deel, is die doel van hierdie studie om vas te stel of die RSHK en OAFV-weë geaktiveer word in langtermyn DOKS behandeling. Boonop is nog ‘n doel van hierdie studie om vas te stel of TNF-α geproduseer word tydens behandeling, aangesien die rol daarvan in DOKS-geinduseerde kardiotoksisiteit steeds onbekend is. Metodes H9c2 kardiomiosiet en gedifferensieerde C2C12 miobuise was daagliks behandel met toenemende konsentrasies van Dox vir 120 ure. Die effekte van DOKS op sel lewensvatbaarheid, apoptose en nekrose is onderskeidelik ondersoek deur middel van die MTT, Caspase-Glo® 3/7 en LDH toetse. TNF-α produksie is bepaal deur van die Quantikine® toets gebruik te maak, en verskeie metodes is gebuik om die oksidatiewe stres, anti-oksidantkapasiteit en anti-oksidantstatus te bepaal. Die proteïenuitdrukking van die RSHK (Erk1/2 en Akt) en OAFV (Jak2 en STAT3) weë was ontleed deur middel van westerse afklattingstegniek deur van beide fosfospesifieke en totale teenliggaampies gebruik te maak. Resultate en Bespreking Behandeling met DOKS het ‘n tyd en dosis-afhanklike afname in sel lewensvatbaarheid in beide sellyne veroorsaak, wat gepaard gegaan het met ‘n toename in apoptose. In die H9c2 kardiomiosiete, het ‘n lae DOKS dosisbehandeling (0.2 μM) betekenisvolle vlakke van TNF-α na 120 uur opgelewer en ons kan spekuleer dat hierdie lae vlakke gedeeltelik die selle van die toksiese effekte van DOKS deur die aktivering van die OAFV weg beskerm het omrede beide Jak2 en STAT3 by hierdie konsentrasie gefosforileer is. Die hoë DOKS dosis (1 μM) het ‘n groter en bifasiese patroon van TNF-α vrystelling vertoon, wat kon bydra tot die DOKS-geinduseerde afname in sel lewensvatbaarheid. Akt is gedurende die eerste 72 uur van behandeling gefosforileer met die lae DOKS dosis, maar kroniese behandeling het hierdie fosforilering verhoed. Terwyl Erk1/2 glad nie gefosforileer is by die lae DOKS dosis nie, is nie Akt of Erk1/2 by die hoë dosis gefosforileer nie, en kan hierdie inhibering bydrae tot die kardiotoksiese effekte van DOKS. In die C2C12 miobuise, is ‘n betekenisvolle hoeveelheid TNF-α na 120 uur van behandeling geproduseer by die lae DOKS dosis. Behandeling met die hoë DOKS dosis het betekenisvolle TNF-α produksie geinduseer by elke tydspunt. Terwyl STAT3 gefosforileer is by die serienresidu na behandeling met die lae DOKS dosis, het behandeling met die hoë dosis fosforilering by die tirosienresidu op ’n tydsafhanklike wyse plaasgevind. p-Jak2 uitdrukking was betekenisvol verminder by beide DOKS konsentrasies, wat aanduidend is dat die STAT3 proteïene nie geaktiveer hoef te word deur Jak2 nie. Die Erk1/2 been van die RSHK weg is ook nie geaktiveer gedurende die oorhoofse behandelingstydperk nie, alhoewel, p-Akt wel uitgedruk is by die lae konsentrasie van DOKS relatief tot die totale Akt uitdrukking. Gevolgtrekkings Die resultate van hierdie studie toon dat DOKS-behandeling tot ‘n dosis-afhanklike verlies in sel lewensvatbaarheid lei. Hierdie effek word waarskynlik bemiddel deur die teenwoordigheid van hoë konsentrasies TNF-α, en ook die afregulering van die beskermende seinweë. TNF-α kan moontlik gedeeltelike beskerming bied by lae konsentrasies deur aktivering van die OAFV weg. Die aktivering van die OAFV weg kon egter nie voldoende beskerming teen DOKS bied nie; moontlik as gevolg van die afwesigheid van die gelyktydige RSHK weg aktivering. Ons resultate vertoon die verskille tussen die akute en kronies behandeling aangesien ‘n enkele hoë-dosis van DOKS, in vergelyking met ‘n kumulatiewe lae-dosis, grootliks verskillende resultate opgelewer het. Voordat hierdie resultate klinies verder ondersoek kan word is verdere navorsing nodig om TNF-α en die RSHK en OAFV-weë beter te verstaan, en om vas te stel of stimulering van hierdie seinoordragpaaie ‘n beskermende effek teweeg sal bring.

Doxorubicin is one of the most effective anti-cancer agents; however its use is associated with adverse cardiac effects, including cardiomyopathy and progressive heart failure. Mitochondrial function and integrity are crucial for cellular processes in general and play an important role during diseased development. These characteristics of the mitochondria make them the prime target for treatments for majority of diseases and in particular of the cardiovascular system. The mitochondria are also considered to play an integral role in the manifestation of the cardiotoxic effects of compounds such as doxorubicin. The current project is designed to investigate the cardiotoxic effects of doxorubicin at tissue, cellular and protein level. In addition, it is investigated whether the inhibition of the mitochondrial permeability transition pore (mPTP) with cyclosporin A (CsA) or the inhibition of mitochondrial fission with the mitochondrial division inhibitor (mdivi-1) protects against the detrimental effects of doxorubicin on cardiac function. We also investigated whether co-treatment of doxorubicin with either CsA or mdivi-1 has any negative interaction with the cytotoxicity of doxorubicin against cancer cells. Langendorff results indicated that doxorubicin caused a time dependent reduction in the haemodynamic function of the heart as well as causing an increase in the infarct size to risk ratio in both naïve conditions and in conditions of ischaemia and reperfusion. Detrimental effects of doxorubicin on cardiac function were abrogated by co-treatment of doxorubicin with CsA or mdivi-1 in naïve conditions and in conditions of ischaemia and reperfusion. Cell viability data of isolated cardiac myocytes revealed that doxorubicin caused a concentration dependant decrease in the viability of neonatal cardiac myocytes as well as causing a reduction in the time taken to depolarisation and hypercontracture under sustained oxidative stress, all of which were prevented when co-treated with either CsA or mdivi-1. Doxorubicin significantly elevated the levels of p-Akt, p-Erk, p-Drp1 and p-p53. Co-treatment with CsA prevented the increase in the levels of p-Akt and p-Erk caused by doxorubicin in both naïve and IR condition whereas mdivi-1 prevented the increase in the levels of p-Erk, p-Drp1 and p-p53 and caused further increase in the levels of p-Akt. Using sinusoidal muscle length change during contraction and relaxation, it is demonstrated that doxorubicin caused a decrease in the power output, peak force and force during shorting. Detrimental effects of doxorubicin on work-loop contraction were abrogated when doxorubicin was co-administered with CsA. To conclude, results demonstrated that doxorubicin caused cardiotoxicity at tissue, cellular and protein level in both naïve conditions and in conditions of ischaemia and reperfusion injury. In addition, it is shown that the inhibition of mitochondrial permeability transition pore with CsA or the inhibition of the mitochondrial fission with mdivi-1 protect against doxorubicin-induced toxicity without affecting its anti-cancer properties.

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A doxorrubicina, ou adriamicina, é uma droga utilizada como agente antineoplásico no tratamento de tumores sólidos e neoplasias hematológicas, principalmente. Apesar de seu amplo uso, apresenta como efeito colateral mais importante a cardiotoxicidade. A toxicidade crônica é bastante conhecida e estudada e cursa com miocardiopatia dilatada e quadro clínico clássico de insuficiência cardíaca. A toxicidade aguda, por cursar com quadro clínico pouco exuberante e manifestar-se através de alterações eletrocardiográficas, é muito pouco diagnosticada e seu mecanismo fisiopatológico não é totalmente conhecido. Os mecanismos envolvidos na toxicidade cardíaca são distintos dos mecanismos de ação da droga e são múltiplos: aumento do estresse oxidativo, aumento da apoptose e alteração na dinâmica intracelular do cálcio. Nossa hipótese é que ocorra lesão estrutural e funcional cardíaca, precocemente, após infusão da doxorrubicina. O objetivo do presente estudo foi avaliar a função ventricular esquerda agudamente após administração de doxorrubicina, a expressão gênica das proteínas reguladoras do trânsito de cálcio, a atividade de metaloproteinases 2 e 9 no miocárdio e alterações das citocinas inflamatórias no miocárdio de ratos tratados com a droga. Para isso, foram utilizados ratos Wistar machos adultos (n=35), que foram submetidos à infusão intraperitoneal de dose única de doxorrubicina de 20 mg/Kg ou volume equivalente de salina (grupo controle). Os animais foram eutanasiados 48 horas após injeção da droga. Todos os ratos foram submetidos ao ecocardiograma antes e 48 horas após a injeção da doxorrubicina. Além da avaliação da função cardíaca in vivo pelo ecocardiograma, a função ventricular esquerda foi avaliada in vitro através de estudo do coração isolado, segundo preparação...
Doxorubicin, or adriamycin, is a drug used as an antineoplastic agent in the treatment of solid tumors and hematologic malignancies. Despite of its use, there are several side effects, and the most important is cardiotoxicity. Chronic toxicity is well known and studied. It presented with dilated cardiomyopathy and clinical features of heart failure. The acute toxicity has mild clinical signs manifesting usually as electrocardiographic changes. Probably, this acute effect is underdiagnosed and the pathophysiological mechanism is not fully understood. The mechanisms involved in cardiac toxicity are multiple, and include increased oxidative stress, increased apoptosis and alteration in intracellular calcium dynamics. Our hypothesis is that structural and functional damage occurs in the heart early after infusion of doxorubicin. The purpose of this study was to evaluate left ventricular function acutely after doxorubicin administration. In addition, gene expression of calcium regulatory proteins, activity of metalloproteinases 2 and 9, and inflammatory cytokines in the myocardium of rats treated with this drug will also be evaluated. Thus, we used adult male Wistar rats (n = 35) who received a single-dose, by intraperitoneal infusion, of doxorubicin (20 mg / kg) or equivalent volume of saline (control group). After 48 hours of drug injection the rats were euthanized. All animals were submitted to echocardiography before drug infusion and immediately before euthanasia. Besides in vivo cardiac function evaluation by echocardiography. In vitro left ventricular function was assessed by isolated perfusion heart study, according to Langendorff preparation. We evaluated interstitial collagen and myocyte hypertrophy by light microscopy, and cardiac tissue metalloproteinases 2 and 9 activity was assessed by zymography. In addition... (Complete abstract click electronic access below)

Orientador: Rosângela Gonçalves Peccinini
Banca: Hérida Regina Nunes Salgado
Banca: Regina Helena Costa Queiroz
Resumo: 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 ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: 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)
Mestre

The goal of this thesis was to investigate magnetic iron oxide nanoparticles as potential carriers for doxorubicin from a purely chemical point of view. It involved spectroscopic studies of doxorubicin. This part of the research was extremely important because it provided us with an understanding of the stability and reactivity of doxorubicin. This information allowed us to establish limits for accurate quantitative determination of this drug using spectrofluorometry. Secondly I investigated the interactions of doxorubicin and related anthracyclines with ferric ions. The latter are known to form complexes with anthracyclines and are believed to be involved in the generation of adverse effects during anticancer therapy. This problem is significant from the viewpoint of anthracycline therapy, as iron ions are distributed throughout human body, but it is also of extreme importance from the viewpoint of designing iron oxide based drug carriers because every metal oxide exists in solution in equilibrium with its metal ions. The goal of my thesis was to understand the role of different anthracycline substituents in the process of iron complexation. These studies allowed us to understand the nature of iron binding as well as the means of preventing this process from occurring. Our final goal was to synthesize magnetic iron oxide nanoparticles and to prepare a stable colloid in water, which could function as a doxorubicin carrier. This goal was achieved by means of covering nanoparticles with a citrate coating. Citrates adsorbed to the nanoparticle surface not only stabilized the nanoparticles electrostatically, preventing them from aggregating in water, but also preventing anthracyclines from complexation with solution phase ferric ions. Additionally, this type of coating allowed us to create a high loading content of doxorubicin. In this work I had to overcome many difficulties related to doxorubicin, nanoparticles or both. One challenge was devising a method that would allow us to determine concentration of doxorubicin attached to nanoparticles quantitatively. This was not a trivial task as nanospheres are known to scatter light as the function of their size. Additionally I had to limit the photoreactivity of doxorubicin in solution. For this reason I developed a novel method of doxorubicin determination called the ternary system method. It allowed us to quantify the amount of doxorubicin present on the nanoparticle surface. This approach enables us to determine the concentration of a drug attached to the carrier, which is extremely important from a clinical standpoint . This method also allowed us to determine the isotherm of doxorubicin’s adsorption and the Gibbs free energy of binding. This work has resulted in publishing five original articles in internationally renowned journals: Journal of Physical Chemistry A (impact factor 2012: 2.771), Journal of Physical Chemistry B (2 articles, impact factor 2012: 3.607), Journal of Physical Chemistry C (impact factor 2012: 4.814) and RSC Advances (partial impact factor 2012: 2.562). One review article has been published in Bioelectrochemistry (impact factor 2012: 3.947). Furthermore this work resulted in two oral presentations at the international conferences: 43rd IUPAC World Chemistry Congress in San Juan, Puerto Rico, and 7th World Congress in Oxidation Catalysis Saint. Louis, USA and in three poster presentation at: 2nd Conference Innovation in Drug Delivery in Aix-en-Provence, France and 1st Warsaw-Cambridge Young Scientists Meeting in Warsaw, Poland (2 poster presentations).
Doksorubicyna jest wysoce skutecznym lekiem przeciwnowotworowym należącym do grupy antracyklin. Pomimo wysokiej skuteczności, doksorubicyna wykazuje szereg cech ograniczających jej szerokie stosowanie m.in. brak możliwości wchłaniania leku z przewodu pokarmowego czy powstawanie rozmaitych skutków ubocznych, w tym zagrażających życiu chorób serca. Badania kliniczne wskazały na wprost proporcjonalną zależność prawdopodobieństwa powstawania chorób serca od przyjętej dawki doksorubicyny. Jednym ze sposobów zwiększenia skuteczności terapii przeciwnowotworowej jest zastosowanie celowanego transportu leków, dzięki któremu lek (doksorubicyna) mógłby być kierowany bezpośrednio w miejsce chorobotwórcze zmniejszając przy tym narażenie innych organów na niepotrzebne skutki uboczne. Wśród różnych metod stosowanych w celowanym transporcie leków, zastosowanie magnetycznych nośników leków (takich jak magnetyczne nanocząstki tlenków żelaza) wydaje się być bardzo obiecujące. Magnetyczne nośniki są w stanie transportować większą ilość leku do tkanek poddanych działaniu zewnętrznego pola magnetycznego. Celem mojej pracy doktorskiej są studia nad magnetycznymi nanocząstkami tlenków żelaza jako nośnikami doksorubicyny. W mojej pracy skoncentrowałem się na zagadnieniach utrudniających stosowanie magnetycznych nanocząstek tlenków żelaza jako nośnika doksorubicyny, które mogą być zrozumiane z perspektywy fizykochemicznych właściwości badanych związków. Moja rozprawa doktorska została podzielona na dwie części. Pierwsza z nich – część literaturowa, przedstawia aktualny stan wiedzy dotyczący nanocząstek i doksorubicyny przedstawioną w oparciu o dostępną literaturę naukową. Druga część przedstawia rezultaty mojej pracy, opublikowane w czasopismach naukowych. Część ta została podzielona na trzy rozdziały: pierwszy- poświęcony właściwościom spektroskopowym i fotoreaktywnością doksorubicyny, drugi- dotyczący oddziaływań antracyklin z jonami żelaza (III), oraz trzeci- przedstawiający syntezę magnetycznych nanocząstek tlenków żelaza, oddziaływanie z doksorubicyną oraz metodę pozwalającą na ilościowe wyznaczenie stężenia doksorubicyny obecnej na powierzchni nanocząstek. Rozdział przedstawiający właściwości spektroskopowe i fotoreaktywność doksorubicyny jest istotny, ponieważ pokazuje ograniczenia w zastosowaniu metod spektroskopowych dla ilościowego oznaczenia antracyklin. Doksorubicyna pod wpływem światła ultrafioletowego ulega redukcji do odpowiadającej jej formy dihydrochinonu, która nie fluoryzuje w zakresie emisyjnym doksorubicyny. Związek ten ulega jednak ponownemu utlenieniu do doksorubicyny reagując z tlenem rozpuszczonym w roztworze. W wyniku tej reakcji następuje regeneracja cząsteczki doksorubicyny oraz utworzenie dodatkowej cząsteczki nadtlenku wodoru. Nadtlenek wodoru jest również znany jako substancja powodująca uszkodzenia tkanki mięśnia sercowego, dlatego istotne jest odpowiednie przechowywanie roztworów doksorubicyny w naczyniach pochłaniających promieniowanie z zakresu nadfioletu. W tym rozdziale przedstawiłem również mechanizm nieodwracalnej fotodegradacji doksorubicyny. Proces ten jest dwuetapowy. Końcowym produktem tego procesu jest kwas 3-metoksysalicylowy. Produkt pośredni nie jest wystarczająco stabilny, abym mógł go odpowiednio scharakteryzować. Dwuetapowość tego procesu jest jednak dobrze widoczna na podstawie analizy profilów kinetycznych reakcji fotodegradacji. Rozdział ten dostarcza istotnych informacji dotyczących kontroli zakresu i intensywności światła służącego do oznaczenia doksorubicyny. W kolejnym rozdziale, który poświęcony jest oddziaływaniom antracyklin z jonami żelaza(III) przedstawiłem wyniki opisujące wpływ poszczególnym podstawników na trwałość powstających kompleksów. W literaturze naukowej przedstawiono teorię o wpływie kompleksów antracyklina-żelazo(III) na występowaniem skutków ubocznych. Problem ten jest zatem ważny zarówno z punktu widzenia tradycyjnej terapii, ponieważ jony żelaza są rozpowszechnione w ludzkim organizmie, jak również z punktu widzenia zastosowania nanocząstek żelaza jako nośników doksorubicyny, ponieważ każdy tlenek metalu występuje w roztworze w równowadze z jonami metalu. W tym rozdziale scharakteryzowałem wpływ podstawnika metoksylowego, grupy hydroksylowej oraz podstawnika cukrowego – daunosaminy na zdolność tworzenia kompleksów z jonami żelaza(III). Prace te pozwoliły na pełniejsze zrozumienie mechanizmu tworzenia kompleksów antracyklin z jonami Fe3+ oraz pozwoliły na zaprojektowanie pokrycia dla magnetyczynych nanocząstek tlenków żelaza, które znacząco poważnie ogranicza zdolność tworzenia kompleksu doksorubicyna-żelazo(III). Ostateczny cel mojej pracy został przedstawiony w trzecim rozdziale. Opisałem w nim wykorzystaną metodę syntezy magnetycznych nanocząstek tlenków żelaza oraz ich pokrycie cytrynianami, które wykazują zdecydowanie wyższe powinnowactwo do jonów Fe3+, niż cząsteczki antracykliny. Otrzymane nanocząstki utworzyły stabilny koloid w środowisku wodnym. Cytryniany obecne na powierzchni nanocząstek elektrostatycznie ograniczały zdolność nanocząstek do samoagregacji oraz uniemożliwiały wytworzenie toksycznych kompleksów doksorubicyna-Fe3+. Zaproponowana przeze mnie metoda pokrycia zapewniła dużą efektywność w procesie adsorpcji doksorubicyny na powierzchni nanocząstek w porównaniu z metodami zaproponowanymi przez inne zespoły badawcze. W rozdziale tym istotnym elementem jest również omówienie nowej metody, którą wykorzystałem dla oznaczenia doksorubicyny zaadsorbowanej do powierzchni nanocząstek. Jest to metoda, która pozwala uniknąć kłopotów związanych z obecnością nanocząstek w badanej próbce. Nanocząstki, ze względu na swój rozmiar, efektywnie rozpraszają światło w zakresie wykorzystywanym w spektroskopii fluorescencyjnej i absorpcyjnej. W mojej metodzie pomiar polega na zastosowaniu układu trójfazowego zawierającego: nanocząstki, wodę oraz octan etylu. Octan etylu nie miesza się z wodą. Doksorubicyna wykazuje niewielką rozpuszczalność w octanie etylu, lecz jej kwantowa wydajność fluorescencji jest w tym rozpuszczalniku znacznie wyższa niż w roztworze wodnym, co jest gwarantem wysokiej czułości stosowanej metody. Nanocząstki pokryte cytrynianami nie przedostają się do fazy organicznej. Doksorubicyna obecna w układzie woda/octan etylu występuje w równowadze, dzięki czemu możliwy jest pomiar jej fluorescencji w fazie organicznej. Po wprowadzenie nanocząstek tlenków żelaza pokrytych cytrynianami do fazy wodnej znaczna część doksorubicyny ulega adsorpcji do powierzchni nanocząstek. Zmniejsza się tym samym stężenie doksorubicyny w fazie wodnej oraz w fazie organicznej, ponieważ równowaga musi być zachowana. Poprzez porównanie intensywności fluorescencji przed i po wprowadzeniu nanocząstek możliwe jest dokładne określenie ilości przyłączonej doksorubicyny do powierzchni magnetycznych nanocząstek. Dzieki temu, że pomiar odbywa się w fazie organicznej nie obserwujemy żadnych efektów optycznych związanych z obecnością nanocząstek w badanej próbce. Podstawą mojej rozprawy doktorskiej jest cykl pięciu oryginalnych artykułów naukowych mojego współautorstwa, opublikowanych w uznanych czasopismach o zasięgu międzynarodowym: Journal of Physical Chemistry A (impact factor 2012: 2.771), Journal of Physical Chemistry B (dwie publikacje, impact factor 2012: 3.607), Journal of Physical Chemistry C (impact factor 2012: 4.814) i RSC Advances (partial impact factor 2012: 2.562) oraz jedna praca przeglądowa opublikowana w Bioelectrochemistry (impact factor 2012: 3.947). Prezentowane badania zaowocowały również dwoma wystąpieniami ustnymi na 43rd IUPAC World Chemistry Congress w San Juan, Puerto Rico, oraz 7th World Congress in Oxidation Catalysis w Saint Louis, USA oraz trzema prezentacjami w formie plakatu: 2nd Conference Innovation in Drug Delivery in Aix-en-Provence, Francja oraz 1st Warsaw-Cambridge Young Scientists Meeting w Warszawie, Polska (dwa plakaty). Przedstawiona praca doktorska włącza się w nurt badań, których celem jest zrozumienie oddziaływań i reaktywności doksorubicyny oraz wnosi wkład do pełniejszego poznania nanocząstek tlenków żelaza jako potencjalnego nośnika leków.

碩士
臺北醫學大學
生物醫學材料研究所
93
Using the Liposomal-Doxorubicin has indeed lowered the cardiac toxicity of Doxorubicin. However, release from the liposomes after accumulated in the cancerous tissue was too slow and resulted in low concentrations of doxorubicin in the target cells, and thus limits the effect of the treatment. In this study, we used the red light to activate the Hematoporphyrin encapsulated in the lipid bilayer in the Liposomal-Doxorubicin-Hematoporphyrin system. After being activated, singlet oxygen as well as free radicals were formed and resulted in peroxidation of tht lipids, de-stablized the liposomes, and subsequently released the encapsulated Doxorubicin. Liposomes were prepared by the traditional film-hydration method. Cholesterol was added to stabilize Hematoporphyrin and Doxorubicin in the liposomes. Release results showed that by increasing the cholesterol concentration or decreasing the L-α-Phosphatidylcholine (Egg PC) in the formulation, drug release was slower accordingly. After irradiated by 635 nm red light, drug release from the liposomes was faster and the bilayer permeability of the liposomes was increased. Cell toxicity tests indicated that the toxicity was higher when the dosed cultured cells were exposed to higher light irradiation.

博士
國立臺灣大學
生化學研究所
87
Steric stabilization by polyethylene glycol (PEG) can reduce opsonization of the liposome by plasma proteins. It has a higher plasma area under the concentration-time curve (AUC), which is believed to correlate with better therapeutic efficacy. However, the presence of large molecules on the liposomal surface may reduce interactions of liposomes with cells and hinder the entry of liposomes into the tumor tissue. Using a stable liposomal system composed of distearoyl phosphatidylcholine (DSPC)/cholesterol, we examined the effect of PEG (mol. wt.: 2000) on the pharmacokinetics and on the efficacy of liposomal doxorubicin with C-26 syngeneic tumor model in the BALB/c mice. The plasma AUC of liposomal doxorubicin with 6 mol % PEG-modified distearoylphosphatidylethanolamine (PEG-DSPE) was about twice that of liposomal doxorubicin without PEG at various dosages, regardless of tumor bearing or not. Paradoxically, the tumor concentration of liposomal doxorubicin without PEG was higher. The 72-hour tumor AUC was 1.44 times that of liposomal doxorubicin with 6% PEG-DSPE. The tumor-accumulation efficiency (Te=AUCTumor/AUCPlasma) of liposomal doxorubicin without PEG was 0.87 and this was more than twice that of the liposomal doxorubicin with 6% PEG-DSPE (0.31). At the dose of 10 mg/kg, although both liposomal groups were better than free drug group in terms of clinic-relevant parameters, including toxicity, tumor shrinkage, and survival, there was no difference between two liposomal drug groups. In this stable liposome system, surface coating with PEG (PEGylation) offered no benefit for liposomal doxorubicin in the C-26 tumor model. To enhance the therapeutic index of liposomal doxorubicin, just increasing plasma AUC by PEGylation may not be satisfactory. Pegylated liposomes results in smaller distribution volume and longer circulation time in blood and thus may improve drug targeting. The characteristics and therapeutic efficacy of immunoliposomes with similar liposomal formulation have never been studied in lymphoma models. We have developed immunoliposomes conjugated with S5A8 monoclonal antibody (MAb), an anti-idiotype antibody to 38C13 murine B-cell lymphoma, and loaded them with doxorubicin using an ammonium sulfate gradient. Purified antibodies were covalently coupled to the termini of PEG on the surface of small unilamellar liposomes. Cell binding and internalization ability of these immunoliposomes was estimated by a fluorescence assay using a pH-sensitive fluorescent dye (HPTS). In vitro cytotoxicity of doxorubicin encapsulated in immunoliposomes was greater for idiotype-positive 38C13 cells than that for idiotype-negative variant (V1-1) of this cell line. In syngeneic C3H/HeN mice, doxorubicin encapsulated in immunoliposomes exhibited long circulation time and was more effective in prolonging survival of mice bearing 38C13 tumor than non-targeted liposomal doxorubicin or free doxorubicin plus empty immunoliposomes. Our results demonstrate the superiority of targeted therapy with these immunoliposomes, and may have potential in cancer treatment.

碩士
長庚大學
生化與生醫工程研究所
98
The purposes of this study for the multifunctional nanoparticles - mediated cancer chemotherapy drug delivery, their multifunction includes the following steps. Firstly, the drug carrier can be controlled by superparamagnetism. Secondly, the chemotherapy drug (doxorubicin, DOX) was entrapped in the natural polymeric carrier to form nanoparticles with low toxicity. Then the surface modification of drug carrier was conjugated with the tumor-specific targeting ligand as the folic acid (FA) We developed the new purpose of the multifunctional chitosan (Cs) nanoparticles-mediated DOX release for cancer therapy. The magnetic nanoparticles (MNP) to form DOX-MNP complexes with DOX was used the negative charge molecule tripolyphospahte (TPP) as the linker and the surface of DOX-MNP complexes was conjugated with the Cs or the Cs-grafted FA to form the doxorubicin - magnetic chitosan nanoparticles (DOX-MCNP) or the doxorubicin - magnetic chitosan nanoparticles modified with folic acid (DOX-MCNPmFA). From the results of these DOX-MCNP and DOX-MCNPmFA, the complexes were not only nanoscale of particle size (around 200 nm) but also with high encapsulation efficiency (above 90%).The data also showed the DOX-MCNPmFA mediated the DOX release with lower dosage of the DOX after optimization of parameters such as the concentration of MNP. The MCNPmFA constitutes a useful approach for the future design of drug carriers.

Dissertação de mestrado em Bioengenharia
Cancer is the leading cause of death in the world. Cancer research is continuously growing aiming to achieve more efficient therapies and early diagnostics. Different challenges arise, concerning the development of efficient drug delivery systems compared to conventional therapies, such as chemotherapy. New formulations promoting a controlled drug distribution, potentiating selective and efficient pharmaceutical actions, have been developed. Nanogels, produced by self-assembly of chemical modified natural polymers, are suitable for this purpose since they are able to encapsulate the hydrophobic drugs, physically or chemically. The use of labile linkages, to stabilize drugs, allows a selective drug release, such as pH-sensitive hydrazone. Doxorubicin is a drug currently used in chemotherapy, however, a major drawback remains its toxicity to healthy tissues, when used in high dosages, and the development of multi-drug resistance during prolonged treatment. Doxorubicin can be conjugated with hyaluronic acid, a natural polymer abundant in the human body, via hydrazone or amide linkages. The main goals of this work consist in the development of hyaluronic acid-based nanogels for cancer therapy with doxorubicin, as well as the incorporation of 𝘺-Fe2O3 into the nanogels to develop a theranostic formulation. Chemical modifications were performed on the hyaluronic acid to obtain an amphiphilic polymer grafted with doxorubicin via hydrazone or amide linkage. Doxorubicin content, average size and polydispersity index were evaluated. The most promising nanogels were further characterized concerning release profile at different pH, cytotoxicity and physical incorporation of 𝘺-Fe2O3. Hyaluronic acid-doxorubicin conjugates, via hydrazone, were produced in PBS pH 7.4, containing 22 μg DOX/mg, an average size of 100 nm and a polydispersity index around 0.5. The conjugation, via amide, was performed in DMSO, leading to a doxorubicin content of 29 μg DOX/mg, an average size of 70 nm and a polydispersity index around 0.45. The release studies indicated a satisfactory release at pH 5.0 (lysosomal pH) although exhibiting some release at pH 7.4 (extracellular pH). 𝘺-Fe2O3 stabilization into nanogels, designed as nanomagnetogels, lead to 0.6-0.9 mM of stabilized 𝘺-Fe2O3. Concerning the cytotoxicity assay performed using A549 cell line, hyaluronic acid-doxorubicin conjugate via amide presented a fast action and promoted a decrease in cell viability. In summary, hyaluronic acid-doxorubicin nanogels were produced using a pH-sensitive linkage, hydrazone, and amide linkage. The nanogels exhibited interesting characteristics for drug delivery applications envisaging more effective therapies, even though further optimizations are required. 𝘺-Fe2O3 incorporation was accomplished allowing imaging detection for diagnostic purposes or therapy evaluation along with the controlled drug release.
O cancro é a principal causa de morte no mundo. A investigação na área do cancro está em evolução contínua, e tem como objetivo alcançar terapias mais eficientes e diagnósticos precoces. Diferentes desafios vão surgindo, relativamente ao desenvolvimento de sistemas de entrega de fármacos eficientes em comparação com as terapias convencionais, como a quimioterapia. Têm sido desenvolvidas novas formulações que promovam uma distribuição controlada do fármaco, potenciando uma ação farmacêutica seletiva e eficiente. Os nanogéis, produzidos por auto-organização de polímeros naturais quimicamente modificados, são adequados para este objetivo, uma vez que permitem encapsular fármacos hidrofóbicos, de forma física ou química. O uso de ligações lábeis, para estabilizar os fármacos, permite uma libertação seletiva, tais como as ligações hidrazona, sensíveis ao pH. A doxorrubicina é um fármaco usado atualmente em quimioterapia, contudo, o seu maior problema é a toxicidade em tecidos saudáveis, quando usada em doses elevadas, e o desenvolvimento de multirresistência durante tratamentos prolongados. A doxorrubicina pode ser conjugada ao ácido hialurónico, que é um polímero natural abundante no corpo humano, através de uma ligação hidrazona ou amida. Os principais objetivos deste projeto consistem no desenvolvimento de nanogéis de ácido hialurónico e doxorubicina para tratamento do cancro, assim como a incorporação de 𝘺-Fe2O3 nos nanogéis, para o desenvolvimento de formulações teranósticas. O ácido hialurónico foi modificado quimicamente para se obter um polímero anfifílico, o qual foi conjugado com a doxorrubicina por ligação hidrazona ou amida. A quantidade de doxorrubicina ligada, o tamanho médio e o índice de polidispersidade foram avaliados. Os nanogéis mais promissores foram ainda estudados em ensaios de libertação a diferentes pH, citotoxicidade e incorporação de 𝘺-Fe2O3. Os conjugados de ácido hialurónico e doxorrubicina por ligação hidrazona foram produzidos em tampão PBS pH 7,4, contendo 22 μg DOX/mg, tamanho médio de 100 nm e um índice de polidispersidade de cerca de 0,5. A conjugação por ligação amida foi efetuada em DMSO, o que conduziu a um conteúdo de doxorrubicina de 29 μg DOX/mg, tamanho médio de 70 nm e um índice de polidispersidade de cerca de 0,45. Os estudos de libertação indicam uma libertação satisfatória a pH 5,0 (pH dos lissossomas), contudo a pH 7,4 (pH extracelular) verificou-se também alguma libertação. A estabilização de 𝘺-Fe2O3 nos nanogéis, designados de nanomagnetogéis, levou à estabilização de 0,6 a 0,9 mM de 𝘺-Fe2O3. Os ensaios de citotoxicidade foram realizados com a linha celular A549, e o conjugado de ácido hialurónico por ligação amida apresentou uma atuação rápida e levou a um decréscimo na viabilidade celular. Em suma, foram produzidos nanogéis de ácido hialurónico e doxorrubicina usando uma ligação sensível ao pH, hidrazona, e ligação amida. Os nanogéis mostraram características interessantes para sistemas de entrega de fármacos, permitindo assim terapias mais eficientes, apesar de ainda serem necessárias algumas otimizações. A incorporação de 𝘺-Fe2O3 foi conseguida nos nanogéis, o que pode permitir a sua deteção por técnicas de imagem para diagnóstico ou para avaliação da terapêutica, ao mesmo tempo que se faz a libertação controlada do fármaco.

Trabalho de Projeto do Mestrado Integrado em Engenharia Biomédica apresentado à Faculdade de Ciências e Tecnologia
Medical breakthroughs nowadays depend almost entirely on scientific research which relies in elaborating numerous hypotheses and running them through continuous and exhausting processes and experiments. These processes usually generate large amounts of data which are normally processed and treated with standard statistical methods that do not live up to the demands imposed by the technological advances that demark our era. Doxorubicin (DOX) is an antitumor anthracycline antibiotic used for treating several types of cancer, such as breast cancer, Hodgkin’s disease and leukemia. Although mitochondrial disruption is an early and sensitive marker of DOX cardiotoxicity, how metabolic stress contributes to the development of cardiomyopathy still needs to be clarified. To address this problem, an experimental dataset was built at the MitoXT laboratory using a model of metabolic inhibition of perfused hearts from Saline (SAL) and DOX-treated Wistar rats to identify metabolic alterations caused by an acute DOX treatment. The hearts were removed and perfused with three diferent energy substrates such as glucose, Galactose plus Glutamine (GG) and Octanoate plus Malate (OM). Separately, glycolytic (Iodoacetate (IODO)) and oxidative phosphorylation (Rotenone (ROT) or Potassium Cyanide (KCN)) inhibitors were added to the distinctive metabolic perfusion buffers, aiming at detecting mitochondrial defects in the DOX-treated group. In this work we applied techniques and computational tools to organise and mine the generated data in order to ex- pose hidden patterns. Our conclusions suggest the exclusion of the protocol concerning the hearts perfusion with OM, confirming the original analysis. Additionally, we suggest that to spare time, means and animals, future experiments could only execute the glucose perfusion protocol. We also suggested that ANT and LDH transcripts expression, absolute weight diference, and Peroxisome proliferator-activated receptor-gamma coactivator (PGC-1alpha) ratio are the most relevant features to be considered for this problem. Finally, we established a classifier capable of an automatic distinction between DOX- and SAL- treated groups. Thus, we not only contributed to a better understanding of how metabolic stress contributes to the development of cardiomyopathy, by selecting which parameters show greater disparity between treatments, but we also confirmed that a detailed data analysis driven by Machine Learning allows a better exploration of these biological datasets enabling new discoveries and breakthroughs in this field.
Hoje em dia, progressos na área da medicina são quase sempre influenciados pela investigação científica, a qual se apoia na elaboração de hipóteses e processos experimentais para as validarem. Geralmente, estes processos geram grandes quantidades de dados, os quais são, posteriormente, processados e tratados com o auxílio de métodos estatísticos tradicionais, ficando, muitas vezes, aquém das expectativas impostas pelos avanços tecnológicos que marcam a nossa era. A Doxorubicina (DOX) é um fármaco antitumoral utilizado no tratamento de diversos tipos de cancro, como cancro da mama, doença de Hodgkin's e leucemia. E embora a disrupção mitocondrial seja um indicador sensível e precoce da cardiotoxicidade provoca pela DOX, subsiste ainda sobre debate a razão pela qual o stress metabólico contribui para o desenvolvimento de cardiomiopatia. Assim foi elaborado, no laboratório Mito-XT, um modelo de inibição metabólica em corações perfurados de ratos Wistar, tratados com solução salina ou com DOX, de modo a evidenciar as alteraçõesmetabólicas causadas por este fármaco. Os corações foram removidos e perfurados com três substratos cardíacos diferentes: glucose, galactose e glutamina, e octanoato e malato. Separadamente, foram adicionados, aos distintos tampões metabólicos da perfusão, os inibidores glicolíticos (iodoacetato), e de fosforilação oxidativa (rotenona e cianeto) com o objetivo de detectar defeitos metabólicos ocultos nos grupos tratados com o fármaco. Assim, neste estudo aplicaram-se diversas técnicas e ferramentas computacionais, incluindo algoritmos de aprendizagem automática, com o objetivo de expor padrões desconhecidos nos dados recolhidos, analisando e estruturando o dataset, de forma a estabelecer, também, um classificador capaz de distinguir automaticamente os grupos tratados com e sem DOX.As conclusões deste trabalho, verificaram a análise original dos dados, confirmando a exclusão do protocolo de perfusão correspondente ao substrato octanoato e malato, devido a não manifestar quaisquer conclusões relevantes. Adicionalmente, sugerimos que para poupar tempo, fundos e animais se deveria, apenas, implementar o protocolo relativo à perfusão com glucose. Verificámos, também, que os parâmetros mais importantes para o problema em questão são: a informação genética relativa aos transcriptos ANT e LDH, a informação proteica e a diferença dos pesos das amostras.Deste modo, este trabalho não só revelou informação importante referente à contribuição do stress metabólico para o desenvolvimento cardiomiopatia, visto que foram selecionadas as features que melhor identificam as amostras tratadas com e sem o fármaco, como também confirmou que uma análise detalhada, utilizando abordagens provenientes de ML, permitem uma melhor exploração de datasets biológicos, revelando novas informações que podem levar aos progressos inicialmente mencionados.

碩士
長庚大學
化工與材料工程學系
99
We prepared magnetic oleoylchitosan nanoparticles (MOCNP) containing an anticancer drug (doxorubicin; DOX) based on ionic gelation process of OCS and sodium tripolyphosphate (TPP) in order to develop an efficient drug delivery system for cancer treatment. MOCNP were 200~250 nm in size and with a spherical shape. The drug encapsulation efficiency was up to 90% with drug loading efficiency at 40%. In vitro release studies showed pH-dependent with rapid release at pH 5.5, whereas at pH 7.4 there was a sustained release after a burst release. After modification with folic acid (FA), EE and LE were not significantly different. The anti-tumor effects of these drug-containing particles were examined using human glioblastoma cells (U87) in vitro and evaluated via MTT assay. MOCNP showed no cytotoxicity, but nanoparticles containing the anticancer drug showed higher antitumor effect when compared to free DOX. The fluorescence imaging results indicated nanoparticles could be uptaken by U87 cells. These results suggested that nanoparticles may be a promising carrier for DOX delivery in cancer therapy. In addition, MOCNP may provide a therapeutic benefit by delivering drugs efficiently to magnetically targeted tumor tissues.

碩士
高雄醫學大學
藥學研究所
97
A field-amplified sample stacking and capillary electrophoresis with UV detection (CE-UV) is described for the determination of doxorubucin and doxorubicinol in human plasma. After sample pretreatment, field-amplified sample stacking method is applied for sensitive improvement. The sample was employed with an electrokinetic injection of 10 kV for 30 sec. CE separation of doxorubicin and doxorubicinol from human plasma was performed at 250C using a background electrolyte consisting of phosphate buffer (50 mM, pH 6.5) containing 16% ethylene glycol and methanol 60%. Using metformin as an internal standard (I.S.), the linear range of the method for the determination of doxorubicin and doxorubicinol was over 20.0-100.0 ng/mL. The LOD of the doxorubibin and doxorubicinol in human plasma was 5 ng/mL(S/N=3, 10 kV, 30 sec) and 10 ng/mL (S/N=3, 10 kV, 30 sec) respectively. For optimization of the procedure, the pH and strength of phosphate buffer, the volume of ethylene glycol and methanol would be discussed. Application of the method to the determination of doxorubicin and doxorubicinol in human plasma proved to be feasible.