Le protocole TICE (Taxol, Ifosfamide, Carboplatine et Etoposide) représente le traitement standard du cancer germinal réfractaire en première ligne ou en rechute de mauvais pronostic. Une étude de phase II a été réalisée consistant en une adaptation de posologie du carboplatine (utilisé à haute dose) basée sur un suivi thérapeutique pharmacologique (TDM) de ses concentrations ultrafiltrables (UF) alors que les pratiques habituelles se limitent à des doses calculées à partir d'une valeur cible de l'aire sous la courbe (AUC) des concentrations UF et la clairance UF prédite du patient. Les analyses pharmacocinétiques effectuées dans le cadre de cette thèse ont permis de démontrer la faisabilité du TDM ainsi que sa performance dans la maîtrise de l'AUC du carboplatine utilisé dans le protocole TICE. Cependant, la réalisation de cette pratique est limitée, dans certains hôpitaux, par les contraintes matérielles et humaines liées à l'obtention des concentrations UF par l'ultrafiltration des prélèvements plasmatiques. Une méthodologie a donc été développée et validée pour permettre la réalisation du TDM à partir des concentrations plasmatiques totales. Par ailleurs, l'étude pharmacocinétique réalisée pour l'étoposide, médicament associé au carboplatine dans la phase d'intensification de ce protocole, indique que le calcul actuel de la dose en fonction de la surface corporelle s'accompagne d'une variabilité interindividuelle limitée de l'exposition et qu'il n'y a pas lieu de pratiquer un TDM pour ce médicament. Enfin, l'implication de différents facteurs génétiques correspondant, d'une part, à la toxicité auditive du carboplatine et, d'autre part, à la pharmacologie de l'étoposide a été aussi évaluée. En conclusion, ces travaux permettront d'améliorer la prise en charge des patients traités par ce protocole à haute dose de carboplatine et étoposide et, au-delà de cette indication thérapeutique, notre connaissance de ces deux médicaments cytotoxiques importants. / The TI-CE protocol ((Taxol, Ifosfamide, Carboplatin and Etoposide) is the standard treatment of germ cell tumor refractory to first-line chemotherapy or relapsed germ cell tumor having unfavorable prognostic features. A phase II study was conducted and consisted in adapting the dose of (high dose) carboplatin using therapeutic drug monitoring (TDM) of unbound concentrations instead of the usual method of dose individualization based on a target area under the curve (AUC) of unbound concentrations and predicted unbound clearance. Pharmacokinetic analyses carried out in the context of this thesis have demonstrated the feasibility of conducting the TDM as well as its performance in terms of controlling the variability of AUC of carboplatin in the TI-CE protocol. However, the use of this practice is limited, in some hospitals, by material and human constraints related to the ultrafiltration of plasma samples to obtain unbound concentrations. A method was developed and validated to enable the use of total plasma concentrations for the TDM instead of unbound concentrations. Furthermore, the pharmacokinetic study of etoposide, used in combination with carboplatin during the dose intensification phase of the protocol, showed that the usual dose calculation method based on body surface area is associated with a low interindividual variability of exposure and that TDM is, therefore, not necessary for this drug. Finally, the role of different genetic factors in the ototoxicity of carboplatin and in the pharmacology of etoposide was also assessed. In conclusion, these analyses help to improve the level of care of patients treated with this protocol of high dose carboplatin and etoposide as well as our current knowledge of these two important cytotoxic drugs.

Les petites molécules bioactives sont des acteurs clés dans la recherche biomédicale et les études de chimie biologiques, pour leur application potentielle comme sondes moléculaires pour enquêter les systèmes biologiques, et la possibilité de développer de nouveaux traitements puissants et sélectifs. Dans ce travail, nous avons exploré l'utilisation de plusieurs classes de petites molécules capables d'interférer avec des cibles biologiquement significatives, comme les acides nucléiques et les lysosomes, et déclencher des réponses biologiques. A partir de l'étoposide, l'un des agents anti-néoplasiques le plus employé en clinique, nous avons développé une nouvelle classe de petites molécules actives capables de cibler différemment les deux isoformes humaines de la topoisomérase II, TOP2A et TOP2B, qui jouent des rôles différents dans l'inhibition de la croissance des cellules cancéreuses, et le développement de tumeurs malignes secondaires. Dans un autre projet, nous avons utilisé une stratégie d'étiquetage « click in situ » pour enquêter sur la localisation subcellulaire d'un nouvel inhibiteur de la réaction d'échange nucléotidique des protéines Arf, un outil potentiel pour l'étude du trafic cellulaire et la signalisation liés a ces protéines. Enfin, nous avons étudié l'origine de l'efficacité d'un nouveau agent de ciblage du compartiment lysosomal: l'artesumycin, un hybride moléculaire des petites molécules bioactives la marmycine A et l'artésunate, qui possède une activité antiproliferative accrue en comparaison avec les deux produits naturels utilisés indépendamment. Nos résultats fournissent une précieuse contribution dans ces domaines de recherche. / Bioactive small molecules are key players in biomedical research and chemical biology studies given their potential application as molecular probes to investigate biological system, together with the possibility to develop new potent and selective therapeutics. In this work, we explored the use of several classes of small molecules able to interfere with therapeutically relevant targets, from nucleic acids to lysosomes, and evaluate ensuing biological responses. Starting from etoposide, one of the most clinically employed anti-neoplastic agents, we developed a new class of active small molecules able to differentially target the two human topoisomerase II isoforms, TOP2A and TOP2B, which have been shown to play different roles in inhibiting cancer cell growth and initiating secondary malignancies. In a separate project, we aimed to use an in situ click labelling strategies to investigate the subcellular localization of a new inhibitor of the nucleotide exchange reaction of Arf proteins, a potential tool for the study of Arf-related cellular trafficking and signalling. Finally, we studied the origins of the synergy displayed by the new lysosome targeting agent artesumycin, a molecular hybrid of the bioactive small molecules marmycin A and artesunate, which has been shown to possess enhanced antiproliferative activity in comparison to the two natural product used independently. Our results provide valuable contributions for future advances in these research areas.

Fármacos antiproliferativos vêm sendo utilizados em procedimentos de angioplastia, onde são administrados localmente por meio de \"stents\" farmacológicos. Embora seja possível que um tratamento sistêmico apresente maior eficiência, os efeitos colaterais dos fármacos antiproliferativos disponíveis constituem uma grande limitação. Estudos recentes em nosso laboratório demonstraram que uma nanoemulsão lipídica rica em colesterol concentra-se em regiões onde a proliferação celular é maior, possivelmente devido a uma maior demanda por colesterol para a síntese de membranas celulares. Assim, consideramos a possibilidade de utilização desta nanoemulsão como veículo de fármacos antiproliferativos para o tratamento da aterosclerose. No presente trabalho, dezesseis coelhos brancos, machos, da raça New Zealand, pesando aproximadamente 3 kg, foram submetidos a uma dieta contendo 1% de colesterol durante quatro semanas e divididos em dois grupos: um grupo recebendo uma injeção endovenosa contendo apenas solução salina e outro grupo tratado com uma associação de etoposídeo e metotrexato, veiculados na nanoemulsão lipídica. A análise da morfometria macroscópica mostra que o tratamento reduz as áreas de lesão aterosclerótica em aproximadamente 84% (razão da área de lesão sobre área total 0,57±0,2 nos animais controle versus 0,089±0,05 nos animais tratados, p<0,05). Houve redução de aproximadamente 3 vezes na razão da íntima-média dos animais tratados em relação aos animais controle. Adicionalmente, observamos que o tratamento reduz a migração de macrófagos na camada íntima do arco aórtico destes animais. Houve amplificação do efeito terapêutico em função da associação destes fármacos e redução na toxicidade. Portanto, a associação destes fármacos antiproliferativos veiculados em um sistema nanoparticulado pode ser promissora para o tratamento da aterosclerose. Estudos em humanos deverão confirmar o potencial desta proposta na terapêutica cardiovascular. / Drug eluting stents have been used in angioplastic procedures to deliver antiproliferative drugs directly to arterial tissue. Presumably, a systemic treatment could be more effective than local administration, but the side-effects of available antiproliferative drugs limit this approach. In recent studies, we have shown that a cholesterol-rich nanoemulsion concentrates in sites of high cell proliferation, possibly due to a high demand of cholesterol to membrane cell synthesis. Thus, we sought to test the emerging hypothesis that our nanoemulsion could be used as a vehicle to antiproliferative drugs for systemic treatment of atherosclerosis. In the present study, sixteen New Zealand White rabbits weighing from 3.2 to 3.8 kg were submitted to a diet containing 1% cholesterol during 4 weeks and then they were split in two groups: a group treated with an intravenous injection of saline solution and a group treated with an association of etoposide and methotrexate delivered in our lipid nanoemulsion. We have shown by macroscopic morphometry that the treatment reduced the lesion areas by roughly 84% (lesion area/total area ratio 0,57±0,2 in control animals, versus 0,089±0,05 in treated animals, p<0,05). The intima-media ratio was reduced by three fold in treated animals. Furthermore, we have observed a reduction in macrophage migration to the arterial intima. The association of etoposide and methotrexate resulted in a synergistic effect without increasing the toxicity. In conclusion, the association of antiproliferative drugs delivered in a nanoemulsion is a promising approach for atherosclerosis treatment. Further studies in humans are needed to confirm the potential of this treatment in cardiovascular therapeutics.

No description available.

Failure of chemotherapy in cancer patients because of development of drug resistance is a major problem. Alterations of DNA repair mechanisms and drug targets are among the important resistance mechanisms which are developed against topoisomerase II inhibitors etoposide and doxorubicin. Modifications in the expression levels of mismatch repair (MMR) genes due to resistance to topoisomerase II inhibitors are involved in breast cancer. In this study, etoposide resistant sublines were developed from MCF7 breast cancer cell line (MCF7/S) and the expression levels of TOP2A and two important MMR genes MSH2 and MLH1 were examined by real time qPCR. Previously developed doxorubicin resistant cells were also studied for comparison. Etoposide resistant sublines MCF7/1000E, MCF7/1250E and MCF7/2000E were approximately 2, 3 and 4 fold resistant relative to parental MCF7/S cells, respectively. MLH1, MSH2 and TOP2A expressions decreased in both etoposide and doxorubicin resistant sublines relative to MCF7/S cells. Expression levels of TOP2A in resistant sublines differ between 10-95 percent of the expression levels in the parental cells. In the sublines MCF7/200E, MCF7/500E, MCF7/750E and MCF7/1000E a decrease in TOP2A gene expression was determined. In sublines MCF7/1250E and MCF7/2000E fluctuations in the expression levels were observed. Among the doxorubicin resistant sublines (MCF7/600D and MCF7/1000D), in MCF7/1000D which is more resistant to doxorubicin, TOP2A expression level was higher. Expression levels of MSH2 decreased regularly as the resistance increased. However, in MCF7/1250E significant increase relative to MCF7/1000E was observed. In MCF7/2000E, expression levels of MSH2 again significantly decreased to 41 percent of the levels in parental cell line. Expression levels of MLH1 decreased significantly (18-58 percent) in etoposide resistant sublines relative to MCF7/S cells. In doxorubicin resistant sublines, a decrease in MLH1 gene expression was observed in MCF7/1000D. It can be concluded from the results that decrease in the expression levels of TOP2A, MSH2 and MLH1 genes may contribute to resistance together. Above a certain resistance level, sublines may develop new strategies for acquiring higher resistance. Whenever a strategy becomes limited, new strategies emerge. New approaches developed to overcome resistance in cancer chemotherapy should consider the molecular basis of resistance in different stages of the disease.

T-cell acute lymphoblastic leukemia (T-ALL) is a lymphoid disorder that results from an over proliferation of immature lymphocytes in the blood and bone marrow. It has been determined that 60% of patients stricken with T-ALL aberrantly express TAL-1 and have been shown to respond poorly to chemotherapy. This research sought to determine if TAL-1 influences the expression of the Bcl-2 family members Bcl-2 (anti-apoptotic), Bad and Bax (pro-apoptotic). TAL-1 and Bcl-2 levels were elevated while Bad and Bax levels were lower in etoposide-treated Jurkat cells as compared to TRAIL-treated and dual-treated Jurkat cells in which TAL-1 and Bcl-2 levels were lower while Bad and Bax levels were elevated. These results suggest TAL-1 up-regulates Bcl-2 and suppress Bad and Bax expression in response to etoposide treatment, thus inducing an anti-apoptotic response in the cell. These results also suggest that TRAIL and the dual treatment of etoposide and TRAIL down-regulates TAL-1 and Bcl-2 expression while up-regulating Bad and Bax, thus inducing a pro-apoptotic response in the cell. / Department of Biology

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Fármacos antiproliferativos vêm sendo utilizados em procedimentos de angioplastia, onde são administrados localmente por meio de \"stents\" farmacológicos. Embora seja possível que um tratamento sistêmico apresente maior eficiência, os efeitos colaterais dos fármacos antiproliferativos disponíveis constituem uma grande limitação. Estudos recentes em nosso laboratório demonstraram que uma nanoemulsão lipídica rica em colesterol concentra-se em regiões onde a proliferação celular é maior, possivelmente devido a uma maior demanda por colesterol para a síntese de membranas celulares. Assim, consideramos a possibilidade de utilização desta nanoemulsão como veículo de fármacos antiproliferativos para o tratamento da aterosclerose. No presente trabalho, dezesseis coelhos brancos, machos, da raça New Zealand, pesando aproximadamente 3 kg, foram submetidos a uma dieta contendo 1% de colesterol durante quatro semanas e divididos em dois grupos: um grupo recebendo uma injeção endovenosa contendo apenas solução salina e outro grupo tratado com uma associação de etoposídeo e metotrexato, veiculados na nanoemulsão lipídica. A análise da morfometria macroscópica mostra que o tratamento reduz as áreas de lesão aterosclerótica em aproximadamente 84% (razão da área de lesão sobre área total 0,57±0,2 nos animais controle versus 0,089±0,05 nos animais tratados, p<0,05). Houve redução de aproximadamente 3 vezes na razão da íntima-média dos animais tratados em relação aos animais controle. Adicionalmente, observamos que o tratamento reduz a migração de macrófagos na camada íntima do arco aórtico destes animais. Houve amplificação do efeito terapêutico em função da associação destes fármacos e redução na toxicidade. Portanto, a associação destes fármacos antiproliferativos veiculados em um sistema nanoparticulado pode ser promissora para o tratamento da aterosclerose. Estudos em humanos deverão confirmar o potencial desta proposta na terapêutica cardiovascular. / Drug eluting stents have been used in angioplastic procedures to deliver antiproliferative drugs directly to arterial tissue. Presumably, a systemic treatment could be more effective than local administration, but the side-effects of available antiproliferative drugs limit this approach. In recent studies, we have shown that a cholesterol-rich nanoemulsion concentrates in sites of high cell proliferation, possibly due to a high demand of cholesterol to membrane cell synthesis. Thus, we sought to test the emerging hypothesis that our nanoemulsion could be used as a vehicle to antiproliferative drugs for systemic treatment of atherosclerosis. In the present study, sixteen New Zealand White rabbits weighing from 3.2 to 3.8 kg were submitted to a diet containing 1% cholesterol during 4 weeks and then they were split in two groups: a group treated with an intravenous injection of saline solution and a group treated with an association of etoposide and methotrexate delivered in our lipid nanoemulsion. We have shown by macroscopic morphometry that the treatment reduced the lesion areas by roughly 84% (lesion area/total area ratio 0,57±0,2 in control animals, versus 0,089±0,05 in treated animals, p<0,05). The intima-media ratio was reduced by three fold in treated animals. Furthermore, we have observed a reduction in macrophage migration to the arterial intima. The association of etoposide and methotrexate resulted in a synergistic effect without increasing the toxicity. In conclusion, the association of antiproliferative drugs delivered in a nanoemulsion is a promising approach for atherosclerosis treatment. Further studies in humans are needed to confirm the potential of this treatment in cardiovascular therapeutics.

Objetivo: O objetivo do presente estudo foi descrever a relação entre as concentrações plasmáticas totais e livres tumorais do etoposídeo (ETO) e a inibição do crescimento do tumor observada em ratos Wistar portadores de tumor Walker- 256 (W256) utilizando a modelagem farmacocinética/farmacodinâmica (PK/PD). Métodos: Os procedimentos com animais foram aprovados no CEUA/UFRGS sob o número 22302. Os experimentos de farmacocinética foram realizados para determinar concentrações plasmáticas e livres em duas regiões do tumor sólido W256 através de microdiálise. Após a administração do ETO nas doses de 10 ou 20 mg/kg i.v. bolus em ratos Wistar portadores de tumor W256, amostras de sangue e microdialisado de tecido do centro e periferia do tumor foram coletadas simultaneamente, até 7 h pós-dose, para determinar o fator de penetração no tumor. Um método analítico por CLAE-UV foi desenvolvido e validado para quantificação do etoposídeo nas amostras de plasma e dialisado. Os experimentos de farmacodinâmica foram conduzidos em ratos portadores de tumor W256 que receberam ETO 5 e 10 mg/kg i.v. bolus uma vez ao dia por 8 e 4 dias, respectivamente. O volume dos tumores foram monitorados diariamente durante 30 dias. Análise não-compartimental dos dados de PK foi realizada no WinNonlin®. A modelagem dos dados PK e PK/PD foi realizada no Monolix®, utilizando abordagem populacional. Os dados PK/PD foram analisados usando o modelo Simeoni TGI modificado através da introdução de uma função Emax para descrever a relação nãolinear entre a concentração plasmática e tumoral e o efeito. Resultados e Discussão: O método por CLAE-UV foi desenvolvido e validado para quantificar as amostras de ETO em plasma e tecido. A penetração do ETO no tumor foi maior na periferia (61 ± 15 % e 61 ± 29 %) do que no centro do tumor (34 ± 6 % e 28 ± 11 %) após administração das doses 10 e 20 mg/kg, respectivamente (ANOVA, α = 0.05). Um modelo de 4 compartimentos compreendendo uma distribuição saturável (cinética de Michaelis-Menten) nos compartimentos tumorais a partir do compartimento central modelou simultaneamente os perfis de concentração-tempo do ETO em plasma e em ambas regiões do tumor. O modelo populacional PK/PD Simeoni TGI–Emax foi capaz de descrever o efeito antitumoral dependente do regime de administração do ETO utilizando concentrações totais plasmáticas ou livres no tumor, resultando em um maior k2max (potência máxima) para as concentrações livres (25,8 mL.μg-1.dia-1 - intratumoral vs. 12,6 mL.μg-1.dia-1 - plasma total). Conclusões: Os resultados mostram que a utilização das concentrações livres do fármaco no tumor para a modelagem PK/PD pode fornecer um melhor entendimento da relação farmacocinética e farmacodinâmica e melhoram a capacidade de previsão do modelo, considerando que a eficácia dos fármacos antineoplásicos no tratamento de tumores sólidos é dependente da capacidade do fármaco em se distribuir no tecido tumoral. / Objective: The aim of this study was to describe the relationship between total plasma and free interstitial tumor etoposide (ETO) concentrations and the drug tumor growth inhibition observed in a Walker-256 (W256) tumor-bearing Wistar rat model using the pharmacokinetic/pharmacodynamic (PK/PD) modeling. Methods: The experiments with animals were approved by CEUA/UFRGS (protocol number 22302). Pharmacokinetic experiments were conducted to determine total plasma and free intratumoral concentrations in two regions of W256 solid tumor by microdialysis. After administration of ETO 10 or 20 mg/kg i.v. bolus to W256 tumorbearing Wistar rats, blood and tissue microdialysate samples from tumor center and periphery were simultaneously collected up to 7h to determine the tumor penetration factor. An analytical HPLC-UV method was developed and validated for quantification of ETO in plasma and microdialysate samples. The pharmacodynamic experiments were conducted in W256 tumor-bearing rats that received ETO 5 or 10 mg/kg i.v. bolus every day for 8 and 4 days, respectively. Tumor volumes were monitored daily for 30 days. Non-compartmental analysis of PK data was performed in WinNonlin®. The PK and PK/PD modeling by population approach were performed using Monolix®. PK/PD data were analyzed using a modification of Simeoni TGI model by introducing an Emax function to describe the nonlinear relationship between tumor and plasma concentrations and effect. Results and Discussion: The HLPCUV method was developed and validated to determine plasma and tissue samples of ETO. ETO tumor penetration was higher in the tumor periphery (61 ± 15 % and 61 ± 29 %) than center (34 ± 6 % and 28 ± 11 %) following 10 and 20 mg/kg doses, respectively (ANOVA, α = 0.05). A 4-compartment structural model comprising a saturable distribution (Michaelis-Menten kinetics) into the tumor compartments from the central compartment simultaneously described the ETO concentration–time profiles in plasma and both tumor regions. The PK/PD population Simeoni TGI–Emax model was capable of describing the schedule-dependent antitumor effects of ETO using total plasma or free tumor concentrations obtained in a W256-tumor bearing Wistar rat model, resulting in higher k2max (maximal potency) for free concentrations (25.8 mL.μg-1.day-1 - intratumoral vs. 12.6 mL.μg-1.day-1 total plasma). Conclusions: The results showed that the use of free intratumoral drug concentrations in the PK/PD modeling can provide a better understanding of the pharmacokinetics and pharmacodynamics relationship and improve the forecasting ability of the models considering that the efficacy of antineoplastic drugs in the treatment of solid tumors is dependent on the drug ability to distribute into the tumor.

Echinacea is an herbal supplement commonly used as an immune system stimulant to prevent infections, such as the common cold or flu. Echinacea has been documented as a cyctochrome P450 (CYP) 3A4 inhibitor in vitro, but no formal studies have been conducted in humans. Etoposide is a cytotoxic, topoisomerase II inhibitor, chemotherapeutic agent used in the treatment of lung cancer. Etoposide is primarily metabolized by CYP 3A4. We report the first possible drug–herbal interaction between Echinacea and etoposide. A 61-year-old gentleman newly diagnosed with nonsmall cell lung cancer began concurrent chemoradiation with cisplatin and etoposide. He was admitted to the hospital on day 8 of his first cycle and found to be thrombocytopenic. His platelet count eventually reached a nadir of 16 × 103/L, requiring platelet transfusion support. Upon admission, it was discovered he was taking Echinacea, which was discontinued. He received his next cycle of chemotherapy without taking Echinacea. His platelet count decreased to a nadir of 44 × 103/L, but he did not require platelet transfusions. Echinacea likely contributed to this patient's profound thrombocytopenia and should be avoided in patients receiving etoposide and possibly other chemotherapeutic drugs that are CYP 3A4 substrates.