HNSCC accounts for 7 percent of all new cancer occurrences. Despite currently available treatments, there continues to be a high mortality and recurrence rate in HNSCC. Well over 50 percent of all cancer patients receive chemotherapy as a standard treatment. However, only 5 percent of these cases have been shown to help with treatment of the disease. Formerly, two options were available for drug testing: in vivo animal models, and in vitro two-dimensional models. While in vivo models remain the most representative, their use is burdened by high costs, time constraints, and ethical concerns. 2D models are simple to use and cost effective, although they have been shown to produce inaccurate data regarding chemotherapeutic drug resistance due to their 2D arrangement and altered gene expression. Researchers for the past decade have been working to create 3D models that more accurately represent in vivo systems in order to evaluate chemotherapeutic efficacy and improve clinical outcomes. In line with this agenda, novel 3D head and neck cancer models were created out of electrospun synthetic polymers seeded with either HN6 or HN12 cancer cells. The models were then treated with chemotherapeutic drugs (either paclitaxel or cisplatin), and, after 72 hours, subjected to a live-dead assay in order to determine the cytotoxic effects of the drugs. 2D cultures of HN6 and HN12 were also and subject to a WST-1 assay after 72 hours. The results of the treated-scaffold assays were then compared to the results of the 2D culture assays, and, as predicted, the cancer cells in a 3D culture system proved to be more resistant to chemotherapeutic drugs. The underlying assumption for this study being that a 3D culture system based on precisely defined structural parameters would provide a practical environment to screen therapeutics for anti-cancer efficacy. To prove this, 3D scaffolds of three different fiber sizes were developed by electrospinning different concentrations of Poly(L-lactic acid) (“PLLA”) (55mg/ml, 115mg/ml, and 180mg/ml) onto a mandrel that was perforated to allow for increased porosity. The resultant small, medium, and large scaffolds were then subjected to concentrated hydrochloric acid (HCl) pretreatment in order to make them less hydrophobic. Different fiber diameters represented different ECM environments for both HN6 and HN12. It was proven that both cell types thrived best in small fibers (55mg/ml-115mg/ml) than in large fibers. It was also reaffirmed through live-dead anlaysis of cells seeded on 3D scaffolds and treated with IC90 values of cisplatin that the head and neck cancer cells were more resistant which is more representative to the 3D environment of cancer cells in vivo.

Resistance to chemotherapeutics is a widespread phenomenon in cancer cells that may counteract the successful therapy of many patients. In resistant cells, higher level of ABC transporters, among others, often can be detected. This high level of ABC transporters represents a suspected mechanism of acquired cancer resistance. We studied the molecular mechanism of resistance to taxanes in cancer cells using SK-BR-3 and MCF-7 breast cancer cell lines. We analyzed the effect of paclitaxel on apoptosis induction in the originally sensitive cells of these lines as compared to their counterpart resistant cells, developed by gradual adaptation to paclitaxel. In resistant cells of the SK-BR-3 and MCF-7 lines, we did not detected ongoing induction of apoptosis but we did detect significantly increased expression of ABCB1 transporter after paclitaxel application. By silencing the expression of the transport via employment of small interfering RNA (siRNA), we tested the role of the ABCB1 transporter in cells resistant to paclitaxel. We found that resistant cells with silenced expression of the ABCB1 transporter had a statistically significant increase of sensitivity to paclitaxel as compared to control resistant cells with high expression of this transporter. Along with increased sensitivity, we demonstrated...

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / nÃo hà / Paclitaxel (PCX) foi o 1 antineoplÃsico efetivo no tratamento de cÃnceres refratÃrios a quimioterapia convencional. Clinicamente, induz artralgias e mialgias de carÃter incapacitante que comprometem a qualidade de vida e limitam o tempo de tratamento antitumoral, acometendo atà 57% dos doentes. Apesar destas repercussÃes clÃnicas nada foi descrito atà o momento, visando elucidar o envolvimento de citocinas prÃ-inflamatÃrias, na gÃnese da atividade hipernociceptiva do PCX, jà que a droga compartilha com o LPS uma via de sinalizaÃÃo desencadeada por receptores Toll (TLR-4 e TLR-2) para geraÃÃo de genes que codificam TNF-&#945;. Dados da literatura sugerem que ocorra um âcross-talkâ entre esses dois membros da famÃlia Toll e, que agonistas diferentes de TLR-2 e TLR-4 sÃo capazes de induzir a ativaÃÃo de NF-&#945;B, AP1 e MAP kinase e a geraÃÃo de TNF-&#945;, citocina chave na cascata de liberaÃÃo de mediadores inflamatÃrios finais que atuam diretamente no nociceptor. Dados do laboratÃrio registraram que o zymosan (ZY) intrarticular em joelhos de ratos produz uma periartrite caracterÃstica da hipernocicepÃÃo no teste de incapacitaÃÃo articular (IA), e que PCX (8mg/kg) amplificou essa resposta quando se injetou  da dose do ZY. Tal amplificaÃÃo foi inibida com o prÃ-tratamento com inibidores de citocina e de prostanÃides. Objetivo. Investigar a participaÃÃo do TLR-4 e TNF-&#945; na gÃnese do efeito potencializador do PCX na artralgia experimental induzida por ZY. Material e MÃtodos. Ratos foram prÃ-tratados Sc com talidomida (TLD), pentoxifilina, dexametasona, indometacina ou celecoxib e estimulados com subdose de ZY (250&#956;g/animal; i-art). ApÃs a 1 medida do tempo de suspensÃo de pata (TSP) no teste de IA, os animais receberam PCX (8mg/kg; ip). Numa segunda etapa os ratos receberam durante trÃs dias consecutivos o prÃ-tratamento com atorvastatina (3, 10, 30mg/kg/dia; VO). Os seguintes parÃmetros foram avaliados: modulaÃÃo da hipernocicepÃÃo no teste de incapacitaÃÃo articular, dosagem de citocinas em lavado de joelho de ratos (TNF-&#945;, IL-1&#945;, Il-6, KC e CINC) e imunohistoquÃmica para TNF-&#945;, IL-1&#945; e TLR-4 no tecido sinovial. Resultados. Ficou demonstrado que PCX (8mg/kg) potencializa a artralgia experimental induzida por ZY em ratos avaliada pelo aumento significativo do TSP (p<0,001) na 4Âh de artrite em relaÃÃo ao controle no teste de IA. Tal efeito foi inibido de maneira significativa pelo prÃ-tratamento com TLD (45mg/kg) e essa inibiÃÃo foi associada à reduÃÃo dos nÃveis de TNF-&#945; produzido pelas cÃlulas do tecido sinovial no lavado articular e da marcaÃÃo imunohistoquÃmica para TNF-&#61537;. Da mesma forma a inibiÃÃo dessa resposta amplificadora do PCX foi ratificado pelo prÃ-tratamento com atorvastatina nas trÃs doses utilizadas no modelo, tambÃm sendo associado à diminuiÃÃo significativa dos nÃveis de TNF-&#61537; no lavado articular e visÃvel reduÃÃo na marcaÃÃo imunohistoquÃmica para TNF-&#61537;, IL-1&#61538; e TLR-4, nas trÃs doses utilizadas. ConclusÃes. PCX potencializa a hipernocicepÃÃo induzida por ZY por um mecanismo indireto sobre cÃlulas residentes da membrana sinovial que liberam TNF-&#61537; provavelmente pela ativaÃÃo da NF-&#61547;B via TLR-4/MD2, pois esse efeito potencializador foi inibido pela atorvastatina, um provÃvel antagonista de TLR-4. O TNF-&#61537; liberado age iniciando a cascata de mediadores envolvidos com a dor inflamatÃria, o que justifica em parte as artralgias dos pacientes em tratamento com PCX. / Paclitaxel (PCX) was the first effective antineoplastic medicine in the treatment of tumors that do not respond to conventional chemotherapy. Clinically, it induces incapacitating arthralgias and myalgias that interfere with the patient quality of life and limit the duration of the treatment. This is observed in up to 57% of the patients using the drug. Despite these clinical manifestations, nothing has been published that could explain the involvement of pro-inflammatory cytokines in the triggering of the hypernociceptive effect of PCX, even though it is known that the drug shares with LPS a signaling pathway started by Toll-like receptors (TLR-2 and TLR-4) that activates genes coding for TNF-&#945;. The literature suggests that there is a crosstalk between these two members of the Toll family and that different agonists of TLR-2 and TLR-4 are able to induce the activation of NF-kB, AP1 and MAP kinase in the generation of TNF-&#945;, a key cytokines in the cascade liberating the final inflammatory mediators that act directly on the nociceptor. Data obtained in laboratory show that the injection of zymozan into rat knee-joints produces a periarthritis characteristic of the hypernociception seen in the knee joint incapacitation test and that PCX (8mg/kg) amplified the response when  of the zymozan (ZY) doses was injected. The amplification was inhibited when animals were pre-treated with inhibitors of cytokines and prostanoids. Objective: To study the role of TNF-&#945; and TLR-4 on the initiation of the potentiating effect of PCX on the experimental arthralgia induced by ZY. Material and Methods: Rats were pre-treated Sc with thalidomide, pentoxifiline, dexametazone, indometacin and celecoxib and then stimulated with an intra-articular subdoses of ZY (250&#956;g/animal). After the first measurement of the paw elevation time in the knee joint incapacitation test, the animals were treated with PCX (8mg/kg ip). On a second trial, rats were treated for three consecutive days with atorvastatin (3, 10, 30mg/kg/day; VO). The following parameters were evaluated: modulation of the effect on the knee joint incapacitation test (JIT), amount of cytokines in the ratâs knee lavage (TNF-&#945;, IL-1 &#946;, IL-6, KC and CINC) and immunohistochemistry for TNF-&#945;, IL-1&#946; and TLR-4 on synovial tissue. Results: It was shown that PCX (8mg/kg) potentiates the experimental arthralgia induced by ZY in the rats as evaluated by the significant increase in paw elevation time (p<0.001) at the 4th h of arthritis in relations to controls. Such effect was significantly inhibited by pre-treatment with thalidomide (45mg/kg) and the inhibition was associated with a decrease in the amount of TNF-&#945; produced by synovial tissue cells and detected in the joint lavage and in the immunohistochemistry for TNF-&#945;. Likewise the inhibition of the amplifying response to PCX was seen with pre-treatment with atorvastatin at the three doses used in the experiment, which was also associated with a lower TNF-&#945; in the joint lavage and perceptible decrease in the immunohistochemistry for TNF-&#945;, IL-1&#946; and TLR-4. Conclusions: PCX potentiates the hypernociception induced by ZY through an indirect effect on synovial membrane resident cells that release TNF-&#945; probably through activation of the NF-kB pathway by TLR-4/MD2, since the potentiating effect was inhibited by atorvastatin, a TLR-4 antagonist. Released TNF-&#945; act starting the cascade of mediators involved in the inflammatory pain and this partially explains the arthralgia in patients treated with PCX.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-09-03T21:30:23Z No. of bitstreams: 2 DISSET Correção FINAL 03-07-14 FOLHA PDF.pdf: 1467526 bytes, checksum: 196b53fbd9a59de359b14f8fd82c2ee5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-09-03T21:30:23Z (GMT). No. of bitstreams: 2 DISSET Correção FINAL 03-07-14 FOLHA PDF.pdf: 1467526 bytes, checksum: 196b53fbd9a59de359b14f8fd82c2ee5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-01-29 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Paclitaxel (PTX) is a natural product extracted from the bark of the Pacific Yew and has numerous antitumor actions, including skin cancers. The topical treatment of skin and pre-cancerous lesions cancer is desired, since the systemic treatment has many side effects However, PTX to be incorporated into formulations suitable for it to penetrate the stratum corneum and skin tumors reached. Lipid nanoparticles have potential to increase drug retention in the stratum corneum, thus providing controlled release and great percutaneous absorption. Within this context, the aim of this study was to develop and characterize solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) containing the antineoplastic PTX and evaluate its permeation into the pig ear skin in vertical diffusion cells type "Franz". Quantification of PTX paclitaxel was performed by high performance liquid chromatography. The NLS and CLNs were obtained by the method of dilution of the microemulsion containingcetylpyridinium chloride, glyceryl behenate, triglycerides of caprylic / capric acid, polysorbate 80 and sorbitan trioleate 85. The particles were characterized by medium size, PdI, zeta potential, encapsulation efficiency, drug loading and recovery. Stability studies were carried out for a period of 30 days with storage at 4 °C (± 2 °C). Theskin permeation studies of the PTX nanoparticles were conducted in “Franz” type diffusion cells in pig ear skin. The NLS obtained showed average size of 314.1 ± 10.9 to 335.9 ± 0.9nm. The CLN obtained with more oil in the lipid matrix (CLN100)showed average size 270.6 ± 13.5 nm. The encapsulation efficiency of the systemsobtained was above 90% when 3.75% was added PTX formulations. The stability studies revealed a trend in increasing the size of the particles PdI along the storage period, but these differences are not statistically significant. The CLN100 increased about 3 times the amount of drug in the stratum corneum (SC) as compared to the administration of unencapsulated drug and also increased by 1.5 times the amount of PTX in the SC in relation to the topical application of other lipid particles. Thus, the lipid particles appear to be promising systems for topical application of PTX. / O paclitaxel (PTX) é um produto natural extraído da casca do Teixo do Pacífico e possui númeras ações antitumorais, inclusive em neoplasias cutâneas. O tratamento tópico do câncer de pele e de lesões pré-cancerígenas é almejado, visto que o tratamento sistêmico possui diversos efeitos colaterais. Entretanto, o PTX precisa ser incorporado em formulações adequadas para que o mesmo penetre no estrato córneo e atinja os tumores cutâneos. As nanopartículas lipídicas apresentampotencial para aumentar a retenção de fármacos no estrato córneo, proporcionandouma liberação controlada e um aumento na absorção percutânea. Dentro destecontexto, o objetivo deste trabalho foi desenvolver e caracterizar nanopartículaslipídicas sólidas (NLS) e carreadores lipídicos nanoestruturados (CLN) contendo o antineoplásico PTX e avaliar sua permeação cutânea em pele de orelha de suíno em células de difusão vertical tipo “Franz”. A quantificação do paclitaxel PTX foi realizada por cromatografia líquida de alta eficiência. As NLS e CLN foram obtidas pelo método de diluição da microemulsão contendo cloreto de cetilpiridínio, behenato de glicerila, triglicerídeos do ácido cáprico/ caprílico, polissorbato 80 e trioleato de sorbitano 85. As partículas foram caracterizadas quanto ao tamanho médio, PdI, potencial zeta, eficiência de encapsulação, carga de fármaco e recuperação. Os estudos de estabilidade foram realizados por um período de 30 dias com armazenamento a 4°C (± 2°C). Os estudos de permeação cutânea do PTX nas nanopartículas foram realizados em células de difusão tipo “Franz” em pele de orelha de suíno. As NLS obtidas apresentaram tamanho médio entre 314,1 ± 10,9 a 335,9 ± 0,9nm. Os CLN obtidos com mais óleo na matriz lipídica (CLN100), apresentaram tamanho médio 270,6 ± 13,5nm. A eficiência de encapsulação dos sistemas obtidos foi superior a 90% quando 3,75% de PTX foi adicionado as formulações. Nos estudos de estabilidade observou-se uma tendência no aumento do tamanho e PdI das partículas ao longo do período de armazenamento, porém estas diferenças não são estatisticamente significativas. Os CLN100 aumentaram aproximadamente 3 vezes a quantidade de fármaco no estrato córneo (EC) quando comparados com a administração do fármaco não encapsulado e, ainda ,aumentaram 1,5 vezes a quantidade de PTX no EC em relação à aplicação tópica das demais partículas lipídicas. Desta forma, as partículas lipídicas parecem ser sistemas promissores para aplicação tópica do PTX.

Ovarian cancer is one of the most common female cancer diseases in the world today and in Sweden more than 800 new cases are diagnosed every year. The standard treatment consists of chemotherapy with paclitaxel in combination with carboplatin after initial cytoreductive surgery. The response to treatment and the severity of adverse drug reactions after chemotherapy varies greatly among individuals, and one of the most important factors responsible for these differences is now recognized to be the genetic variability. One of the major obstacles to successful treatment is drug resistance. Several potential mechanisms have been suggested for the resistance to paclitaxel, such as mutations in the target protein β-tubulin, single nucleotide polymorphisms (SNPs) in the gene ABCB1, which encodes the transport protein P-glycoprotein. P-glycoprotein can mediate efflux of various drugs from cancer cells as well as from the circulation into the intestinal lumen, and overexpression and/or high activity leads to drug resistance and/or increased elimination. Another reason might be the high interindividual variability of paclitaxel plasma concentrations, which has been suggested to be influenced by variability in metabolic enzymes, such as CYP2C8 and CYP3A4, and transport proteins e.g. P-glycoprotein. In the studies constituting this thesis we have investigated the possibilities of predicting the pharmacokinetics of paclitaxel as well as the tumor response and adverse drug reactions after chemotherapy in the preparation of personalized chemotherapy. We studied the correlation between the response and the presence of mutations in the dominant β-tubulin gene and SNPs in ABCB1. DNA from 40 ovarian tumors was screened for sequence variations in the β-tubulin gene without finding any, showing that β-tubulin mutations are rare and unlikely to be a clinically relevant resistance mechanism for paclitaxel. The SNPs G2677T/A and C3435T in the ABCB1 gene were determined in 53 ovarian cancer tumors from patients with poor (progressive disease or relapse within one year) or good (disease-free survival of more than one year) response to paclitaxel-carboplatin chemotherapy. Patients homozygously mutated for G2677T/A had a higher probability of responding to chemotherapy. There was also a dose-dependent influence of the number of mutated alleles on the response to paclitaxel treatment. No correlation was found for the C3435T variant. By using a newly developed quantitative LC/MS method for the simultaneous determination of paclitaxel and its hydroxymetabolites in human plasma we assessed the individual elimination of paclitaxel in 33 ovarian cancer patients. The patients were genotyped for SNPs in the ABCB1, CYP2C8 and CYP3A4 genes and their in vivo CYP3A4 enzyme activity, tumor response and toxicity, especially the neurotoxicity, were determined. Patients heterozygous for G/A in position 2677 in ABCB1 had a significantly higher clearance of paclitaxel than patients with the wild type or homozygously mutated, but not compared to patients carrying the G/T alleles. A lower clearance of paclitaxel was also found for patients heterozygous for CYP2C8*3 when stratified according to the ABCB1 G2677T/A genotype. The CYP3A4 enzyme activity in vivo affected the relative influence of CYP2C8 and CYP3A4 on the metabolism, but not the total clearance of paclitaxel. The exposure to paclitaxel was correlated to the neurotoxicity, but not to the treatment response. In conclusion, our findings suggest that the SNP G2677T/A in the ABCB1 gene, but not β-tubulin mutations, might be a predictor for paclitaxel response and that the interindividual variability in paclitaxel pharmacokinetics might be predicted by ABCB1 and CYP2C8 genotypes and provide useful information for individualized chemotherapy. / Ovarialcancer (äggstockscancer) är en av de vanligaste cancerformerna hos kvinnor i Sverige idag. Behandlingen består vanligen av tumörreducerande kirurgi följd av kemoterapi med paklitaxel och karboplatin. Målsättningen med detta avhandlingsarbete har varit att förbättra cytostatikabehandlingen (cellgiftsbehandlingen) med framförallt paklitaxel vid ovarialcancer genom att lägga grunden för individualisering av doser och förutsäga tumörsvaret vid behandlingen. Ett problem med dagens cancerbehandling är att många cancerceller så småningom blir resistenta mot olika cytostatika. För att angripa den mest resistenta cellen innan den induceras att öka uttrycket av, eller utveckla, fler resistensmekanismer vore det en fördel om vi före behandlingen kunde prediktera vilken dos av cytostatika som är bäst lämpad för individen samt om tumören kommer att reagera på behandlingen eller ej. En av de viktigaste faktorerna för skillnader i behandlingseffekt tros vara genetiska variationer mellan olika individer. I våra studier har vi använt genetiska metoder för att studera om vi kan prediktera tumörsvaret vid behandlingen genom att bestämma mutationer i genen för paklitaxels målprotein, β-tubulin, samt bestämma genetiska variationer i ABCB1-genen, kodande för transportproteinet P-glykoprotein. Tanken är att ett förändrat målprotein eller en förändrad förmåga hos cancercellerna eller kroppen att transportera ut paklitaxel skulle leda till en skillnad i påverkan på tumören. DNA från 40 ovarialtumörer analyserades utan att en enda sekvensvariation hittades i genen för β-tubulin, vilket tyder på att genetiska förändringar i genen för β-tubulin sannolikt inte är en klinisk relevant resistensmekanism. De normalt förekommande genetiska variationerna G2677T/A och C3435T i ABCB1-genen bestämdes i DNA från 53 ovarialtumörer där behandlingen endera givit en bra (tumörfri minst ett år) eller dålig (progression av tumören eller tumörfri mindre än ett år) anti-tumöreffekt. Patienter som var dubbelmuterade i position 2677 dvs hade endera T/T eller T/A (A/A hittades inte i materialet) i denna position hade en högre sannolikhet att få ett bra anti-tumörsvar vid behandlingen. Även antalet muterade baser påverkade utfallet, ju fler muterade baser i position 2677, desto högre sannolikhet att få ett bra svar på behandlingen. Andelen T eller A var också högre i den grupp av patienter som fått en lyckad behandling. För att kunna prediktera patientens individuella förmåga att bryta ner paklitaxel studerade vi inverkan av sekvensvariationer i generna för de nedbrytande enzymerna, CYP2C8 och CYP3A4, och transportproteinet P-glykoprotein (genen ABCB1) på eliminationen av läkemedlet i kroppen. Vi utvecklade en metod för att mäta paklitaxelkoncentrationerna i blodet och använde den för att studera hur snabbt 33 ovarialcancer patienter eliminerade cytostatikat från blodbanan. Hos dessa patienter bestämde vi förekomsten av kända genetiska variationer i generna ABCB1, CYP2C8 och CYP3A4 samt deras CYP3A4 enzymaktivitet i kroppen. Biverkningarna och tumörsvaret vid behandlingen utvärderades också. Eliminationen av paklitaxel hos dessa patienter var beroende av vilken bas som fanns i position 2677 i ABCB1-genen och förekomsten av den genetiska varianten CYP2C8*3. Enzymaktiviteten hos CYP3A4 kunde inte påvisas påverka eliminationen av paklitaxel utan snarare vilket enzym, CYP2C8 eller CYP3A4, som var relativt dominant i respektive patient. Exponeringen av paklitaxel korrelerade till den neurologiska påverkan som patienten orsakades av cytostatikat, men kunde inte korreleras till tumörsvaret vid slutet av cytostatikabehandlingen. Sammanfattningsvis ger patientens genetiska variationer i ABCB1, men inte β-tubulin, information om behandlingsutfallet. Genetiska variationer i CYP2C8 och ABCB1 påverkar patientens förmåga att eliminera paklitaxel och kan förhoppningsvis användas för att individualisera doserna. Vår förhoppning är att resultaten i denna avhandling skall kunna användas för att individualisera och ytterligare förbättra cytostatikabehandlingen vid ovarialcancer.

Lymph node metastasis is a critical prognostic factor for lung cancer. Effective therapy to control lymphatic metastasis may improve survival. The work described in this thesis focuses on the development of a microparticulate lymphatic targeting system, which can be applied as an adjuvant therapy in the control of lymphatic metastasis in lung cancer. The study shows that intrapleural administered colloidal particulates are predominantly taken up by regional lymphatic tissue in rat models including healthy rats, rats bearing orthotopic lung tumours and rats following pneumonectomy. The effect of particle size on lymphatic particle distribution was examined by intrapleural administration of 111In-aminopolystyrene beads. Approximately 2 µm is a suitable size for intrapleural lymphatic targeting. Biodegradable polylactide-co-glycolide (PLGA) microparticles containing the anticancer agent paclitaxel (PTX) were subsequently formulated in the desired size by spray drying. PLGA-PTX microspheres were incorporated into a biodegradable and biocompatible gelatin sponge matrix to form an implantable lymphatic targeted drug delivery system. The system was characterized in vitro and its lymphatic targeting ability was examined in vivo. Fluorescence labeled microspheres embedded within the sponge were selectively taken up by regional lymphatics as the sponge matrix disintegrated following intrapleural implantation. A pharmacokinetic study showed that the total PTX exposure in lymphatic tissue was dramatically higher than that achieved through intravenous administration. The peak plasma drug concentration, which governs systemic toxicity, was significantly reduced. The low but persistent detection of plasma PTX indicates that PTX was control released from the system after intrapleural implantation. In a therapeutic efficacy study performed in the H460 orthotopic lung cancer model, gelatin sponges containing PLGA-PTX microspheres were placed in the pleural cavity as an adjuvant treatment after surgical resection of the primary lung tumour. Trans-lymphatic chemotherapy resulted in a significantly lower incidence of lymphatic tumour recurrence (20%) compared to no treatment and placebo control animals (100%). PLGA-PTX microspheres were seen in regional lymphatic tissue over 4 weeks after the sponge placement. It is concluded that the trans-lymphatic targeting drug delivery system described in this thesis may improve the control of lymphatic metastasis in lung cancer.

Lymph node metastasis is a critical prognostic factor for lung cancer. Effective therapy to control lymphatic metastasis may improve survival. The work described in this thesis focuses on the development of a microparticulate lymphatic targeting system, which can be applied as an adjuvant therapy in the control of lymphatic metastasis in lung cancer. The study shows that intrapleural administered colloidal particulates are predominantly taken up by regional lymphatic tissue in rat models including healthy rats, rats bearing orthotopic lung tumours and rats following pneumonectomy. The effect of particle size on lymphatic particle distribution was examined by intrapleural administration of 111In-aminopolystyrene beads. Approximately 2 µm is a suitable size for intrapleural lymphatic targeting. Biodegradable polylactide-co-glycolide (PLGA) microparticles containing the anticancer agent paclitaxel (PTX) were subsequently formulated in the desired size by spray drying. PLGA-PTX microspheres were incorporated into a biodegradable and biocompatible gelatin sponge matrix to form an implantable lymphatic targeted drug delivery system. The system was characterized in vitro and its lymphatic targeting ability was examined in vivo. Fluorescence labeled microspheres embedded within the sponge were selectively taken up by regional lymphatics as the sponge matrix disintegrated following intrapleural implantation. A pharmacokinetic study showed that the total PTX exposure in lymphatic tissue was dramatically higher than that achieved through intravenous administration. The peak plasma drug concentration, which governs systemic toxicity, was significantly reduced. The low but persistent detection of plasma PTX indicates that PTX was control released from the system after intrapleural implantation. In a therapeutic efficacy study performed in the H460 orthotopic lung cancer model, gelatin sponges containing PLGA-PTX microspheres were placed in the pleural cavity as an adjuvant treatment after surgical resection of the primary lung tumour. Trans-lymphatic chemotherapy resulted in a significantly lower incidence of lymphatic tumour recurrence (20%) compared to no treatment and placebo control animals (100%). PLGA-PTX microspheres were seen in regional lymphatic tissue over 4 weeks after the sponge placement. It is concluded that the trans-lymphatic targeting drug delivery system described in this thesis may improve the control of lymphatic metastasis in lung cancer.

Thesis (M.S.)--University of North Carolina at Wilmington, 2005. / Includes bibliographical references (leaves: 57-60)

SUMMARYThe diversity of cancer molecular origins associated with the genetic variability of patients has encouraged the development of chemotherapeutic treatments adapted not only to the target tumor, but also to a specific patient. This personalized strategy is based on cancer biomarkers allowing a better identification and characterization of each tumor where predictive biomarkers provide the distinction between various factors indicative of the response to the treatment. In this context, several studies highlighted the role of the solute carrier transporter family 22 (solute carriers 22 or SLC22) in the uptake of platinum anticancer drugs. This mechanism being not well understood, our work intends to establish the potential role of SLC22 member A18 (SLC22A18) as predictive biomarker in the aim to help to a better targeted chemotherapeutic strategy for each patient. We optimized a system overexpressing SLC22A18 stably or transiently in HeLa cancer cell line. SLC22A18 expression was confirmed by qRT-PCR, western blotting, microscopy and flow cytometry. The cell lines were treated with taxane, anthracyclin, vinca alkaloid and nitrosoureas anticancer drug families. We showed that doxorubicin, camptothecin, chloroquine, tetracycline and carmustin had no effect on the cell viability assays suggesting that they are not substrates of SLC22A18. Interestingly, the cell line was sensitized in the presence of antimitotic drug with a sensitivity factor of 2.7 in the presence of paclitaxel, 1.4 with docetaxel, 1.8 with vinblastin and 2.2 in the presence of vincristine. To confirm these results, we elaborated a SLC22A18 knockdown cell line in HS683 cells using siRNA technology. The downexpression of SLC22A18 was correlated to a tendency to resist to the accumulation of paclitaxel thereby confirming the previous results. Simultaneously, a knockout cell line was established using the transcription activator-like effectors nuclease (TALEN) technology in U373 cell line. Our studies constitute a robust base of knowledge for further investigation on SLC22A18 transporter as a predictive biomarker promoting antimitotic treatment in tumors where this transporter is detected. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Nanoparticles from biodegradable polymers are considered one of the most promising systems for biomedical application as drug delivery systems. Therefore, the synthesis and characterization of a new aliphatic biodegradable copolyester named PBS/PBDL (poly(butylene succinate-co- butylene dilinoleate)) intended to the application as drug delivery system is reported in the thesis. Surfactant-free biodegradable and narrowly distributed, nanosized spherical particles (RH < 60 nm) have been produced from the biodegradable material by applying a single-step nanoprecipitation protocol. The size of the generated polymer nanoparticles (PNPs) could be controlled by adjusting the polymer concentration, the choice of organic solvent, mixing different organic solvents or by changing temperature and ionic strength. By optimizing such parameters sub-100 nm uniform PNPs can be produced through this methodology including the advantage and ability to scale-up production. The nanoparticles structure was characterized in detail by employing a variety of scattering techniques and transmission electron microscopy (TEM). Combined static light scattering (SLS) and dynamic light scattering (DLS) measurements suggested that the nanoparticles comprise a porous core conferring them a non-compact characteristic. Their porosity...