Construction of nanoassemblies from degradable components is desired for packaging and controlled release of active therapeutics, and eventual biodegradability in vivo. In this study, shell crosslinked micelles composed of biodegradable poly(lactide) (PLA) core were prepared by the self-assembly of an amphiphilic diblock copolymer synthesized by a combination of ring opening polymerization (ROP) and reversible addition-fragmentation chain transfer (RAFT) polymerization. Enzymatic degradation of the PLA cores of the nanoparticles was achieved upon the addition of proteinase K (PK). Kinetic analyses and comparison of the properties of the nanomaterials as a function of degradation extent will be discussed. Building upon our findings from selective-excavation of the PLA core, enzyme- and redox-responsive nanoparticles were constructed for the encapsulation and stimuli-responsive release of an antitumor drug. This potent chemotherapeutic, otherwise poorly soluble in water was dispersed into aqueous solution by the supramolecular co-assembly with an amphiphilic block copolymer, and the release from within the core of these nanoparticles were gated by crosslinking the hydrophilic shell region with a reduction-responsive crosslinker. Enzyme- and reduction-triggered release behavior of the antitumor drug was demonstrated along with their remarkably high in vitro efficacy. As cationic nanoparticles are a promising class of transfection agents for nucleic acid delivery, in the next part of the study, synthetic methodologies were developed for the conversion of the negatively-charged shell of the enzymatically-degradable shell crosslinked micelles to positively-charged cationic nanoparticles for the complexation of nucleic acids. These degradable cationic nanoparticles were found to efficiently deliver and transfect plasmid DNA in vitro. The hydrolysis of the PLA core and crosslinkers of the nanocarriers may provide a mechanism for their programmed disassembly within endosomes, which would in-turn promote endosomal disruption by osmotic swelling, and release of active therapeutics from the polymeric assemblies. In the last part, a comparative degradation study was performed between the anionic and cationic micellar assemblies in the presence of two model enzymes, and electrostatic interaction-mediated preferential hydrolysis was demonstrated between the oppositely-charged enzyme-micelle pairs. These findings may be of potential significance toward the design of charge-mediated enzyme-responsive nanomaterials that are capable of undergoing environmentally-triggered therapeutic release, disassembly or morphological alterations under selective enzyme conditions.

Paclitaxel (Taxol®) is now widely used against breast, ovarian and non-small-cell lung cancer. Anticancer agents generally have narrow therapeutic indices, often with myelosuppression (mainly neutropenia) as dose-limiting side effect. A further complicating factor is that paclitaxel when given as Taxol® has a nonlinear pharmacokinetic (PK) behaviour in plasma. Identifying risk groups more sensitive to chemotherapy due to either a PK or pharmacodynamic (PD) interindividual variability is of importance. The aim of the thesis was to develop predictive mechanism-based PK and PD models applicable for paclitaxel. PK and PK/PD models were developed for patient data from studies with relatively frequent sampling or sparse sampling schedules. Population analyses were performed using the software NONMEM. A pharmacokinetic model describing unbound, total plasma and blood concentrations of paclitaxel from known binding mechanisms was developed and validated. The nonlinear PK in plasma could to a large extent be explained by the micelle forming vehicle Cremophor EL (CrEL) and the unbound drug showed linear PK. Besides a binding component directly proportional to concentrations of CrEL, the model included both linear and nonlinear binding components in plasma and blood. Further, relations between the PK parameters and different demographic factors, including polymorphisms in the cytochrome P450s involved in paclitaxel metabolism, were investigated. A semi-physiological PD model for chemotherapy-induced myelosuppression was developed and applied to different anticancer drugs. The model included a self-renewal for proliferating cells, transit compartments describing the delay in observed myelosuppression and a feedback parameter reflecting the effect on the bone marrow from growth factors that can result in an overshoot in white blood cells. The system-related parameters estimated showed consistency across drugs and the difference in the drug-related parameter reflected the relative bone marrow toxicity of the drugs. Relations between demographic factors and the PD parameters were identified. The developed mechanism-based models promote a better understanding of paclitaxel PK and PD and may be used as tools in dosing individualisation and in development of dosing strategies for new administration forms and new drugs in the same area.

The goal of this project was to develop a non-toxic amphiphilic diblock copolymer nanoparticulate drug delivery system that will solubilize paclitaxel (PTX) and retain the drug in plasma. Methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) (MePEG-b-PCL) diblock copolymers loaded with PTX were characterized and their physicochemical properties were correlated with their performance as nanoparticulate drug delivery systems. A series of MePEG-b-PCL was synthesized with PCL blocks ranging from 2-104 repeat units and MePEG blocks of 17, 44 or 114 repeat units. All copolymers were water soluble and formed micelles except MePEG₁₁₄-b-PCL₁₀₄, which was water insoluble and formed nanospheres. Investigation of the effects of block length on the physicochemical properties of the nanoparticles was used to select appropriate copolymers for development as PTX nanoparticles. The critical micelle concentration, pyrene partition coefficient and diameter of nanoparticles were found to be dependent on the PCL block length. Copolymers based on a MePEG molecular weight of 750 g/mol were found to have temperature dependent phase behavior. Relationships between the concentration of micellized drug and the compatibility between the drug and core-forming block, as determined by the Flory-Huggins interaction parameter, and PCL block length were developed. Increases in the compatibility between PCL and the drug, as well as longer PCL block lengths resulted in increased drug solubilization. The physicochemical properties and drug delivery performance characteristics of MePEG₁₁₄-b-PCL₁₉ micelles and MePEG₁₁₄-b-PCL₁₀₄ nanospheres were compared. Nanospheres were larger, had a more viscous core, solubilized more PTX and released it slower, compared to micelles. No difference was seen in the hemocompatibility of the nanoparticles as assessed by plasma coagulation time and erythrocyte hemolysis. Micellar PTX had an in vitro plasma distribution similar to free drug. The majority of micellar PTX associated with the lipoprotein deficient plasma fraction (LPDP). In contrast, nanospheres were capable of retaining more of the encapsulated drug with significantly less PTX partitioning into the LPDP fraction. In conclusion, although both micelles and nanospheres were capable of solubilizing PTX and were hemocompatible, PTX nanospheres may offer the advantage of prolonged blood circulation, based on the in vitro plasma distribution data, which showed that nanospheres retained PTX more effectively.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Medical Sciences, Laboratory Medicine and Pathology. Title from pdf file main screen (viewed on October 31, 2009). Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Apr 14

Ο σκοπός της παρούσας μελέτης ήταν να εξετάσει τον ρόλο της ταξόλης (paclitaxel) και της καρβοπλατίνης σε σχέση με τις παραμέτρους του ανοσοποιητικού συστήματος σε ασθενείς πάσχοντες από μη μικροκυτταρικό καρκίνο του πνεύμονα και από καρκίνο των ωοθηκών· πριν, κατά τη διάρκεια και μετά από χημειοθεραπεία και η επίδραση που είχε ο ανωτέρω συνδυασμός φαρμάκων στην συνολική επιβίωση των ασθενών. Υλικό και Μέθοδος: Εξετάσθηκαν 24 ασθενείς με μη-μικροκυτταρικό καρκίνο του πνεύμονα και 20 με καρκίνο των ωοθηκών (όλοι μεταστατικοί), όπου χωρίστηκαν σε δύο ομάδες με κριτήριο την επιβίωση και που εν συνεχεία τους χορηγήθηκε συνδυασμός καρβοπλατίνης και ταξόλης για έξι θεραπευτικούς κύκλους. Ομάδα Α. Ασθενείς με καλή επιβίωση (>12 μήνες για μη-μικροκυτταρικό καρκίνο του πνεύμονα, >30 μήνες για καρκίνο των ωοθηκών) Ομάδα Β. Ασθενείς με «φτωχή» επιβίωση (<12 μήνες για μη-μικροκυτταρικό καρκίνο του πνεύμονα, <18 μήνες για καρκίνο των ωοθηκών) Την ίδια χρονική περίοδο εξετάσθηκαν οι λεμφοκυτταρικοί υποπληθυσμοί (CD3, CD4, CD8, CD56, CD34) καθώς και οι κυτταροκίνες ιντερλευκίνη-3 (IL-3) και ιντερφερόνη-γ (IFN-γ), σε σχέση με την ποιότητα ζωής και το προσδόκιμο επιβίωσης κατά την διάρκεια της χημειοθεραπευτικής αγωγής. Η στατιστική ανάλυση των αποτελεσμάτων έγινε με την μέθοδο ANOVA. Αποτελέσματα: Από την εξαγωγή των αποτελεσμάτων παρατηρήθηκε μία στατιστικώς σημαντική διαφορά ανάμεσα στις τιμές των λεμφοκυτταρικών 207 υποπληθυσμών CD4 και CD4/CD8 μετά από χημειοθεραπεία μεταξύ των δύο ομάδων ασθενών Α και Β (p=0,001 και p=0,006). Αυτό σημαίνει ότι η περαιτέρω αύξηση του αριθμού των βοηθητικών Τ-λεμφοκυττάρων (T-helper) μετά από χημειοθεραπεία συμβάλλει θετικά στο προσδόκιμο επιβίωσης. Επιπροσθέτως, στατιστικώς ενδιαφέρων σε σημείο που να μπορούμε να μιλήσουμε και για προγνωστικό παράγοντα, ήταν η διαφορά ανάμεσα στις τιμές της ιντερφερόνης-γ μεταξύ των ομάδων Α και Β πριν και μετά τη χημειοθεραπεία (p=0,039 και p=0,027, αντιστοίχως). Οι ασθενείς με υψηλές τιμές ιντερλευκίνης-3 παρουσίαζαν επίσης χαμηλή τοξικότητα. Συμπεράσματα: Στην παρούσα μελέτη η προσπάθεια μας επικεντρώθηκε στο να καταδείξουμε την επίδραση που ασκείται, από την χρήση του συνδυασμού καρβοπλατίνης-ταξόλης, στους λεμφοκυτταρικούς υποπληθυσμούς και στις κυτταροκίνες καθώς και την επιρροή που ασκούν και τα δύο αυτά στοιχεία του ανοσοποιητικού συστήματος στο προσδόκιμο επιβίωσης και στην εν γένει ποιότητα ζωής. / The aim of the present study was to exam the role of Paclitaxel (Taxane) and Carboplatin in the parameters of the immune system in patients with non-smallcell lung cancer and in patients with ovarian cancer before, during and after chemotherapy treatment, and the effect of this combination in the overall survival of the patients. Methods: 24 patients with non-small-cell lung cancer (NSCLC) and 20 patients with ovarian cancer (all in stage IIIb-IV) were treated with a combination of paclitaxel and carboplatin for six treatment cicles and they were separated into two groups in terms of survival. GROUP (A). Long survival (>12 months for NSCLC, >30 months for ovarian Ca) GROUP (B). Long survival (<12 months for NSCLC, <18 months for ovarian Ca) At the same time we combined the relevant parameters (CD3, CD4, CD8, CD56, CD34, IL-3, IFN-γ) with the quality of life during treatment with chemotherapy. The results were analyzed using ANOVA system. Results: We observed a significant statistical difference between the values of CD4 and CD4/CD8 after chemotherapy between group A and group B (p=0,001 and p=0,006). This means that the further increase of T-helper cells after chemotherapy has a better prognosis concerning survival. In addiction, statistically interesting, which we may call a prognostic factor, was the difference in values of IFN-γ between individuals of groups and B before and after chemotherapy (p=0,039 and p=0,027, respectively). Patients with high IL-3 had little chance of toxicity. Conclusions: In the current study we tried to demonstrate the effects from the use of the combination of carboplatin-paclitaxel in the whole population of Tcells/ cytokines and the reaction of them in the quality of life.

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Introdução: O câncer de ovário é o mais prevalente e letal câncer ginecológico. A quimioterapia é um componente importante do tratamento sistêmico clássico com uma combinação de um agente platinado e um taxano, usualmente. Invariavelmente, câncer de ovário avançado torna-se resistente à quimioterapia. Objetivos: Com base em dados recentes que demonstram um possível papel das neurotrofinas na regulação de quimiosensibilidade, decidimos estudar o impacto do fator neurotrófico derivado de cérebro (BDNF) sobre a atividade antitumoral de diferentes classes de agentes antineoplásicos. Métodos: Para avaliar um possível efeito sinérgico entre BDNF e diferentes combinações de tratamento para câncer de ovário, as células foram expostas a cisplatina, etoposideo, doxorrubicina e paclitaxel concomitantemente com BDNF durante 48 horas. Administração sequencial de BDNF e quimioterapia foi realizada para avaliar o potencial de BDNF em modificar a resposta ao tratamento quimioterápico dependendo de qual agente é aplicado em primeiro lugar. Resultados: Houve uma redução da viabilidade de células OVCAR-3 quando expostas a cisplatina, doxorubicina e etoposideo concomitantemente com BDNF em 61,18% (SE±1.12, p=0.002), 38,96% (SE±1.08, p=0.001) e 49,63% (SE±1.17, p<0.001), respectivamente. BDNF também reduziu significativamente o efeito do paclitaxel e doxorrubicina quando usado antes da quimioterapia com uma redução de efeito de 53,46% (SE±3.48, p=0.001) e 48,25% (SE±1.25, p=0.018), respectivamente. Além disso, o BDNF utilizado sequencialmente à doxorrubicina foi capaz de reverter a quimiotoxicidade deste agente em 37,77% (SE±1.25, p=0.018). Conclusão: Utilizando a linhagem celular de câncer de ovário (OVCAR-3), BDNF exibiu um efeito sinérgico quando administrado concomitantemente com os agentes citotóxicos doxorrubicina, etoposideo e cisplatina. Observamos também um efeito protetor de BDNF quando aplicado 24 horas antes de doxorrubicina e paclitaxel. Notavelmente, quando BDNF foi administrado após a exposição a agentes antineoplásicos, uma reversão da citotoxicidade foi observada apenas para a doxorrubicina e não para os outros agentes. / Background: Ovarian cancer is the most prevalent and lethal of gynecological malignancies. Chemotherapy is an important component of the systemic treatment with a combination of a platinum complex and a taxane one of the classic treatments. Invariably, advanced ovarian cancer becomes resistant to chemotherapy. Objective: Based on recent data demonstrating a possible role of neurotrophins regulating chemosensitivity, we decided to study the impact of brain-derived neurotrophic factor (BDNF) on the antitumor activity of different classes of antineoplastic agents. Methods: Primarily, to evaluate a possible synergistic effect of BDNF and different ovarian cancer treatments combination, cells were exposed to cisplatin, etoposide, doxorubicin and paclitaxel concomitantly with BDNF for 48 hours. Sequential administration of BDNF and any of the agents was carried out to evaluate if BDNF has the potential of enhancing or protecting cells from the effects of treatment depending of each agent is applied first. Results: There were a reduction in viability of OVCAR-3 cells exposed to cisplatin, doxorubicin and etoposide when used concomitantly with BDNF in 61.18% (SE 1.12, p=0.002), 38.96% (SE 1.08, p=0.001) and 49.63% (SE 1.17, p<0.001) respectively. We also found that BDNF reduced significantly the effect of paclitaxel and doxorubicin when used before chemotherapy with a reduction of effect of 53.46% (SE±3.48, p=0.001) and 48.25% (SE±1.25, p=0.018), respectively. Furthermore, BDNF used sequentially to doxorubicin was able to reverse the chemotoxicity of this agent in 37.77% (SE 1.25, p=0.018). Conclusion: In conclusion, using the human ovarian carcinoma cell line OVCAR-3, BDNF exhibited a synergistic effect when administered concomitantly to the cytotoxic agents doxorubicin, etoposide and cisplatin. We have also observed a protective effect of BDNF when applied 24 hours before doxorubicin and paclitaxel. Notably, when BDNF was administered after the exposure to the antineoplastic agents, a reversal of cytotoxicity was observed only for doxorubicin and not for the other agents.

Dissertação de Mestrado apresentada ao Programa de Pós-Graduação em Ciências da Saúde como requisito parcial para obtenção do título de Mestre em Ciências da Saúde. / O uso clínico do paclitaxel (PAC) como agente quimioterápico é limitado pelos seus efeitos adversos, especialmente a indução de dor. Embora seja bem conhecido que o tratamento prolongado com o paclitaxel poderia levar à dor neuropática, a administração única de PAC também pode provocar uma síndrome de dor aguda, o que se acredita ser causada pela sensibilização de nociceptores. O receptor de potencial transitório vaniloide tipo 1 (TRPV1) é expresso principalmente nos nociceptores, onde desempenha um papel chave na detecção de vários estímulos dolorosos nocivos. O TRPV1 foi implicado no desenvolvimento da dor neuropática induzida pelo PAC, mas o seu papel na síndrome da dor aguda relacionada com o PAC é desconhecido. Assim, o objetivo deste estudo foi observar a participação do canal TRPV1 em um modelo de síndrome de dor aguda induzida por PAC em ratos. Para isso foram utilizados ratos Wistar machos (200-300g). O modelo de síndrome de dor aguda utilizado foi a administração de PAC intraperitoneal (1 mg/kg, i.p., dose única). O limiar mecânico e a latência ao calor foram avaliadas na pata traseira direita dos animais, estes valores foram medidos utilizando filamentos de von Frey com intensidade crescente (6-100g) ou o teste de Hargreaves (através de um feixe de luz radiante de 60 W), respectivamente. Para avaliar a participação do receptor TRPV1 neste modelo foi utilizado o antagonista seletivo (SB-366791; 0,5 mg/kg; i.p.) ou provocada a desensibilização das fibras TRPV1 positivas com o uso de um agonista TRPV1 potente (resiniferatoxina, 200 μg/kg, s.c.; administrado 7 dias antes da injeção de PAC). A hiperalgesia química causada pela capsaicina foi observada pela administração intraplantar de capsaicina (0,01 nmol/pata/100 μL). Também foram realizados ensaios para avaliar o aumento da expressão do receptor TRPV1 após a adminstração de PAC como o western blot (amostras do gânglio da raiz dorsal - GRD ou nervo ciático) e a imunofluorescência (GRD). A sensibilização do receptor TRPV1 foi avaliada utilizando a técnica de influxo de cálcio provocado pela capsaicina (500 nM) em GRD em cultura e expostos ao PAC (50 μM, 24 horas antes). A administração de PAC causou o densenvolvimento de alodínia mecânica e hiperalgesia ao calor 24 horas após a administração. O antagonista do canal TRPV1 ou a dessensibilização das fibras sensoriais TRPV1-positivas com o uso de um agonista TRPV1 foram capazes de reduzir a alodinia mecânica e a hiperalgesia calor induzida por PAC. Também observamos o desenvolvimento de hiperalgesia química para o agonista TRPV1 capsaicina, aumento da imunorreatividade do receptor TRPV1 e do número de neurônios TRPV1 positivos de pequeno diâmetro em gânglios da raiz dorsal (GRD, L3-L5) 24 horas após a administração de PAC. Além disso, 24 horas de incubação com PAC em culturas de neurônios de GRD aumentou não só a percentagem de neurônios que responderam a capsaicina, mas também a amplitude de resposta causada pela capsaicina. Em conclusão, após a administração única de PAC o aumento da sensibilização e expressão do receptor TRPV1 poderia contribuir para o desenvolvimento de alodínia mecânica e hiperalgesia ao calor. Dessa forma, o bloqueio do receptor TRPV1 poderia ser explorado como um possível alvo para o tratamento da síndrome de dor aguda associada ao PAC.