Dissertations / Theses on the topic 'P-glycoprotein inhibitors'

Thesis (Ph. D.)--School of Pharmacy. University of Missouri--Kansas City, 2007.
"A dissertation in pharmaceutical science and pharmacology." Advisor: Ashim K. Mitra. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed July 16, 2008. Includes bibliographical references (leaves 231-248). Online version of the print edition.

The objective of this dissertation is to study the mechanism by which HIV protease inhibitors enter into the central nervous system (CNS) and breast milk of rats, and what effects MDR modulators have on the distribution and metabolism of HIV protease inhibitors. The transporter P-glycoprotein (P-gp) has been shown to limit the distribution of HIV protease inhibitors into the CNS of rodents. This thesis examined the effects of GF120918, an MDR modulator, on the CNS distribution of amprenavir, an HIV protease inhibitor, in rats. GF120918 significantly increased the unbound CNS concentrations of amprenavir without altering the unbound blood concentrations of amprenavir. The results of these studies show that GF120918 can inhibit P-gp at the blood brain barrier (BBB) to increase the unbound CNS concentration of amprenavir and potentially other HIV protease inhibitors. Many first generation MDR modulators inhibited both P-gp transport and CYP3A metabolism. Therefore, a principal goal of this thesis was to determine if GF120918 could selectively inhibit P-gp transport without inhibiting CYP3A metabolism. Using in vitro (human) and in vivo (rat) studies, GF120918 selectively inhibited P-gp at the BBB without inhibiting CYP3A metabolism. The transporter MRP1 has been shown to both transport HIV protease inhibitors and expressed in the CNS. Studies contained in the thesis have shown that mrp1 is not localized to the BBB of rats, therefore, mrp1 is unlikely to play a significant role in the distribution of HIV protease inhibitors into the CNS of rats. The distribution of nelfinavir, an HIV protease inhibitor, into rat breast milk was studied in the thesis as a first approach in understanding the extent to which HIV protease inhibitors can accumulate into milk. The concentration of nelfinavir in rat milk was approximately half that of plasma. P-gp protein expression was detected in lactating rat mammary tissue. However, GF120918 showed no effect on the distribution of nelfinavir into rat milk suggesting that P-gp does not play a significant role in the distribution of HIV protease inhibitors into milk.

Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2004.
"A dissertation in pharmaceutical science and chemistry." Advisor: Ashim K. Mitra. Typescript. Vita. Description based on contents viewed Feb. 27, 2006; title from "catalog record" of the print edition. Includes bibliographical references (leaves 175-199). Online version of the print edition.

Die Therapie der HIV Infektion besteht aus Kombination mehrerer antiretroviraler Substanzen und birgt ein erhöhtes Risiko an Arzneimittelwechselwirkungen. Das bekannte Problem der Virusresistenz kann zudem durch Enzyminduktion begünstigt werden. Das Ziel der vorliegenden Arbeit lag in Untersuchungen zu Einflüssen der Virustatika auf die Expression von Cytochrom P450 Enzymen: 1A1, 1B1, 3A4 sowie der P-Glykoproteins (MDR1) an immortalisierten Zellsystemen. Die Protease Inhibitoren Indinavir, Nelfinavir, Ritonavir und Saquinavir induzierten die Regulation der mRNA Expression über den Aryl-Kohlenwasserstoff-Rezeptor (AhR) und den Pregnan-X-Rezeptor (PXR) dosisabhängig und signifikant. Die Nukleosidischen Reverse Transkriptase Inhibitoren Zalcitabin, Zidovudin und Lamivudin sowie der Nicht-Nukleosidische Inhibitor Nevirapin zeigten induktive Eigenschaften nur für die AhR Zielgene CYP1A1 und CYP1B1. Amprenavir und Efavirenz aktivierten die PXR-Regulation. Die möglichen Auswirkungen der Induktion der untersuchten Gene wurden ausführlich diskutiert. Die molekularen Grundlagen der interindividuell variierenden Aktivität von CYP3A wurden in einer Probandenstudie untersucht. Es wurden die mRNA Expression in den Leukozyten, die Aktivität des Enzyms und einige bekannte Polymorphismen unter Einwirkung von Rifampicin untersucht und diskutiert.
The therapy of HIV infection requires a combination of several antiretroviral substances accompanying risk factors for drug-drug interactions. Moreover, virus resistance can be promoted by enzyme induction caused by antiretroviral drugs. The aim of the study was to investigate the influences of antiretroviral substances on the expression of cytochrome P450 enzymes: 1A1, 1B1, 3A4 and p-glycoprotein (MDR1) using immortalized cell systems. The protease inhibitors indinavir, nelfinavir, ritonavir and saquinavir induced significantly the regulation of mRNA expression through the aryl hydrocarbon receptor (AhR) and the pregnane-x-receptor (PXR) in a concentration-dependent manner. The nucleoside reverse transcriptase inhibitors zalcitabine, zidovudine and lamivudine and the non-nucleoside reverse transcriptase inhibitor nevirapine showed inductive properties only for the AhR target genes CYP1A1 and CYP1B1.Amprenavir and efavirenz activated the PXR target genes. Potentially effects of the described induction are discussed. In a second part of the work, the molecular mechanisms of the individual varying activity of the CYP3A enzyme were investigated applying an in vivo study. CYP3A4 mRNA expression and rifampicin mediated induction in leucocytes were correlated with systemic enzyme activity under induction and known polymorphisms.

La surexpression de protéines d’efflux, et tout particulièrement la P-glycoprotéine, est impliquée dans la multidrug résistance. Dans cette thèse, nous démontrons que le vandetanib, inhibiteur de tyrosine kinase, est à la fois substrat et inhibiteur de la P-glycoprotéine et qu’il est capable de réverser in vitro la résistance à la doxorubicine liée à la surexpression de la P-glycoprotéine.Nous nous sommes également intéressés à l’étude du passage transplacentaire du vandetanib et nous montrons que ce médicament traverse la barrière placentaire
Overexpression of ABC transporters, especially P-glycoprotein, is involved in multidrug resistance. In this study, we demonstrate that vandetanib, a tyrosine kinase inhibitor, is both substrate and inhibitor of P-glycoprotein and is able to reverse in vitro resistance to doxorubicin, linked to overexpression of P-glycoprotein.We also studied the placental transfer of vandetanib and we show that this drug crosses the placenta

Blood-brain barrier (BBB) is a unique membranous barrier, which segregates brain from the circulating blood. It works as a physical and metabolic barrier between the central nervous system (CNS) and periphery. In mammals, endothelial cells were shown to be of BBB and are characterized by the tight junctions along with efflux system which are responsible for the restriction of movement of molecules within the cells. Efflux system consists of multidrug resistance proteins such as P-glycoprotein (P-gp). P-gp removes substances out back from the brain to the blood before they reach to the brain. So the barrier is impermeable to many compounds such as amino acids, ions, small peptides and proteins, making it the most challenging factor for the development of new drugs for targeting CNS. Curcumin is a bioactive compound that has a number of health promoting benefits such as anti-inflammatory, anticancer, anti-oxidant agent; as well as a role in neurodegenerative diseases, but low oral bioavailability is the major limiting factor. Low water solubility and rapid metabolism are the two important factors responsible for poor bioavailability of curcumin. Galaxolide is a musk compound and previously known for the bioaccumulation of toxic components in the aquatic animals by interference with the activity of multidrug/multixenobiotic resistance efflux transporters (MDR/MXR). The bioavailability of curcumin can be enhanced when administered with galaxolide. This study was carried out to investigate the effect of galaxolide on the permeation of curcumin through the epithelial cell monolayers. MDCKII-MDR1 cell monolayer is used an in vitro blood-brain barrier model while Caco-2 monolayer is used as an in vitro intestinal model, which also expresses the P-glycoprotein. The curcumin and galaxolide were separately solubilised in the DMSO and used in combination to perform permeation study, to determine the effect of galaxolide on curcumin permeation through epithelial cell monolayers. The galaxolide shows an efflux protein inhibition activity and this activity was used to enhance permeation of curcumin through the Caco-2 monolayer. In summary, galaxolide is a novel permeation enhancer molecule, which can be used for the improvement of drug delivery of other bioactive compounds in future.

P-glycoprotein (p-gp), a member of the superfamily ATP-binding cassette (ABC) is known to be present in the absorptive enterocytes of the gastro-intestinal tract and many other tissues in the body where it acts mainly as a defence mechanism against exogenous assault. Defects in p-gp is speculated to result in the development of diseases as mutations in genes are causes of numerous diseases in the metabolic mosaic that underlies health. Due to the importance of p-gp, particularly in the intestines, mutation of the gene encoding this protein may lead to the presence of unusual compounds, xenobiotics in the body and the urine. It is thought that defective p-gp in the intestine might also lead to the absorption of some metabolites of bacterial origin and residue of digestion which normally would have been refluxed back into the gut by the p-gp. To investigate if defective p-gp may be involved in the manifestation of unusual compounds and organic acids in the urine, inhibition of intestinal p-gp was proposed. Grapefruit juice (GJ), a natural beverage commonly taken by the majority of the populace has been reported to inhibit p-gp activity in the intestine (Spahn-Langguth & Langguth, 2001). Grapefruit juice was administered to healthy subjects in this study and the sugars and organic acids content of the urine sample after administration was analysed and compared with the controls (urine samples taken from the same set of subjects before grapefruit juice administration). These were determined by thin layer chromatography and gas chromatography-mass spectrophotometry respectively. The thin layer chromatography revealed that there was no difference between the concentrations of sugars in the control and samples after the administration of grapefruit juice. This might indicate that the inhibition of p-gp or mutation of the gene encoding p-gp does not result in the presence of sugars in the urine. The analysis of organic acids by gas chromatography-mass spectrophotometry method showed a remarkable difference between the organic acids present in the controls and urine samples after the administration of grapefruit juice as well as their concentrations. The organic acids solely from microbial origin were statistically analysed and the results gave statistically significant increase in these organic acids in the adults. There was no statistically significant increase in the children. In conclusion, this study confirmed that grapefruit juice inhibits p-gp in the intestine and this resulted in the presence of unusual organic acids from microbial origin in the urine of the adults. The presence of some of these organic acids have been indicated in some metabolic disorders and are also known to give rise to toxic effects on brain, liver, muscle and other tissues. There is the need to do more study on p-gp expression in children so that its functional roles and effect of the mutation of the gene encoding this protein can be known.
Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.

BACKGROUND:Active HIV infection within the central nervous system (CNS) is confined primarily to microglia. The glial cell compartment acts as a viral reservoir behind the blood-brain barrier. It provides an additional roadblock to effective pharmacological treatment via expression of multiple drug efflux transporters, including P-glycoprotein. HIV/AIDS patients frequently suffer bacterial and viral co-infections, leading to deregulation of glial cell function and release of pro-inflammatory mediators including cytokines, chemokines, and nitric oxide.METHODS:To better define the role of inflammation in decreased HIV drug accumulation into CNS targets, accumulation of the antiretroviral saquinavir was examined in purified cultures of rodent microglia exposed to the prototypical inflammatory mediator lipopolysaccharide (LPS).RESULTS:3H]-Saquinavir accumulation by microglia was rapid, and was increased up to two-fold in the presence of the specific P-glycoprotein inhibitor, PSC833. After six or 24 hours of exposure to 10 ng/ml LPS, saquinavir accumulation was decreased by up to 45%. LPS did not directly inhibit saquinavir transport, and did not affect P-glycoprotein protein expression. LPS exposure did not alter RNA and/or protein expression of other transporters including multidrug resistance-associated protein 1 and several solute carrier uptake transporters.CONCLUSIONS:The decrease in saquinavir accumulation in microglia following treatment with LPS is likely multi-factorial, since drug accumulation was attenuated by inhibitors of NF-kappabeta and the MEK1/2 pathway in the microglia cell line HAPI, and in primary microglia cultures from toll-like receptor 4 deficient mice. These data provide new pharmacological insights into why microglia act as a difficult-to-treat viral sanctuary site.

The resistance to antiepileptic drugs (AEDs) remains a major unsolved therapeutic problem, which affects 30-40% of patients with epilepsy. The overexpression of multidrug efflux transporters, as the P-glycoprotein (P-gp), at the level of the blood-brain barrier of epileptic patients has been suggested as a key mechanism underlying the refractory epilepsy. Bearing this in mind, efforts have been made to search for therapeutically useful P-gp inhibitors. In an attempt to find potent and safer P-gp inhibitor drugs, a particular emphasis has been given to flavonoid compounds. Actually, apart from their potential value as P-gp inhibitors, these phytochemical compounds have been recognised as having a panoply of important pharmacological properties like anti-inflammatory, antioxidant, antitumoral, antimicrobial, antiviral, hormonal and even anticonvulsant effects. Taking this into account, the purpose of the present thesis was to conduct a comprehensive in vitro and in vivo evaluation of the potential of flavonoids as P-gp inhibitors, but also to explore a strategy of flavonoid/AED combined therapy as a possible approach to overcome the P-gp–mediated pharmacoresistance in epilepsy. This project involved the development and validation of appropriate and reliable bioanalytical techniques to support the accomplishment of the intended studies. Thus, high-performance liquid chromatography methods coupled with diode array detection (HPLC-DAD) were properly validated for the quantification of the target AEDs and some of their main metabolites in cell culture samples and in rat plasma and brain matrices. An additional HPLC-DAD technique was also developed to quantify several AEDs and metabolites in human plasma, which has shown to be a useful tool for the therapeutic drug monitoring in the clinical practice. According to the results of a set of in vitro assays, five out of eleven flavonoids tested, namely baicalein, (-)-epigallocatechin gallate [(-)-EPG], kaempferol, quercetin and silymarin demonstrated to have an interesting potential in inhibiting the P-gp activity. These promising flavonoids also promoted a significant increase in the intracellular accumulation of the AEDs carbamazepine (CBZ), oxcarbazepine (OXC) and phenytoin (PHT) and their active metabolites carbamazepine-10,11-epoxide (CBZ-E) and licarbazepine (LIC) in the Madin-Darby canine kidney cell line transfected with the human multidrug resistance-1 gene which encodes the human P-gp (MDCK-MRD1), showing up as important drug candidates to overcome the AED-resistance. Actually, excluding LTG, all the AEDs tested (CBZ, OXC and PHT) as well as their active metabolites (CBZ-E and LIC) were found to be P-gp substrates in the MDCK-MDR1 cells. Additionally, CBZ, CBZ-E, LIC, LTG, OXC and PHT promoted a statistically significant decrease of the intracellular concentration of rhodamine 123 (a classic P-gp probe substrate), suggesting an inducer effect on the functional activity of P-gp. An assessment of the potential synergic effects of baicalein, (-)-EPG, kaempferol, quercetin and silymarin on the P-gp inhibition was also performed, firstly in in vitro conditions and then in in vivo experiments. Indeed, when compared to their individual activity, some dual flavonoid combinations exhibited an increased potential in inhibiting the P-gp in the in vitro assays. Moreover, the dual combinations of (-)-EPG/silymarin and kaempferol/baicalein demonstrated a great potential in enhancing the intracellular accumulation of CBZ, OXC and PHT and their metabolites CBZ-E and LIC in the MDCK-MDR1 cells and such effects were comparable to those promoted by verapamil (the standard P-gp inhibitor). The effect of the combination of (-)-EPG/silymarin was also tested in transport assays of LIC (P-gp substrate) through MDCK-MDR1 cells mounted in Ussing chambers; as expected, this combination of flavonoids increased the apparent permeability coefficient of LIC. Overall, these in vitro findings were further supported by in vivo results. In fact, after the pretreatment of male Wistar rats with silymarin an increasing in the plasma concentrations of the studied AEDs (CBZ, OXC and PHT) was observed. Nevertheless, it should be highlighted that the main effects induced by silymarin were found on the OXC pharmacokinetics, for which was found a statistically significant increase in the peak plasma concentration (50%) and in the extent of systemic exposure (41%), having a direct impact on the drug concentrations reached in the brain. On the other hand, the use of dual combinations of (-)-EPG/silymarin on the inhibition of the activity of P-gp was also evaluated in vivo in Wistar rats, being noticeable the synergic potential of (-)-EPG/silymarin combinations in enhancing the degree of systemic exposure to OXC and LIC (a pharmacologically active metabolite of OXC), and it occurred in a comparable extent to that observed for verapamil (positive control). Indeed, the pretreatment of male Wistar rats with dual silymarin/(-)-EPG combinations originated peak plasma concentrations of OXC similar to those achieved in the presence of verapamil. Moreover, the effects promoted by silymarin/(-)-EPG combinations on the magnitude of systemic drug exposure were also reflected in the corresponding drug levels attained in the brain (biophase). Hence, according to our findings, it seems that the flavonoid/AED combined therapy can be thought as a promising approach that should continue to be exploited in order to overcome the P-gp–mediated pharmacoresistance. The availability of this in vitro and in vivo information also adds support to the efflux transporter hypothesis in explaining the pharmacoresistant epilepsy. Considering all its intrinsic potential and indisputable properties, the flavonoid-type compounds may emerge as an alternative to the available P-gp inhibitors for a prospective management of patients with drug-refractory epilepsy.
A epilepsia é uma das doenças neurológicas crónicas mais comuns e tem um grande impacto negativo na qualidade de vida dos doentes, sendo de extrema importância os esforços continuados para desenvolver novos fármacos antiepiléticos e novas abordagens terapêuticas. Contudo, apesar dos avanços significativos alcançados nos últimos anos, os fenómenos de farmacorresistência associados aos fármacos antiepiléticos são provavelmente um dos principais problemas no que diz respeito à terapêutica da epilepsia, afetando 30-40% dos doentes. De facto, esta situação tem proporcionado um ímpeto crescente para o desenvolvimento de abordagens terapêuticas inovadoras e mais eficazes. A sobreexpressão de transportadores de efluxo de fármacos, como a glicoproteína-P (P-gp), ao nível da barreira hematoencefálica, tem sido sugerida como um dos principais mecanismos subjacentes à epilepsia refratária. Desta forma, vários esforços têm sido realizados no sentido de descobrir e desenvolver inibidores da P-gp que sejam úteis de um ponto de vista terapêutico. No entanto, as múltiplas desvantagens apresentadas pelas primeiras gerações de inibidores da P-gp demonstraram a necessidade de encontrar agentes mais potentes e mais seguros, tendo sido dado um ênfase especial nos últimos anos a compostos flavonoides. Além do seu interesse potencial como inibidores da P-gp, uma panóplia de outras propriedades farmacológicas importantes têm sido atribuídas a estes compostos fitoquímicos, entre as quais, por exemplo, efeitos anti-inflamatórios, antioxidantes, antitumorais, antimicrobianos, antivirais, hormonais e, mesmo, anticonvulsivantes. Tendo em consideração todos estes aspetos, a presente tese teve como principal objetivo realizar uma avaliação abrangente, in vitro e in vivo, do papel potencial dos flavonoides como inibidores da P-gp, e também explorar uma estratégia de terapia combinada de flavonoides/fármacos antiepiléticos como possível abordagem para superar a farmacorresistência verificada na epilepsia. Este projeto envolveu o desenvolvimento e validação de técnicas bioanalíticas apropriadas e fiáveis para apoiar a execução dos estudos pretendidos. Neste contexto, métodos de cromatografia líquida de alta eficiência acoplados à deteção por fotodíodos (HPLC-DAD) foram devidamente desenvolvidos e validados para a quantificação dos fármacos antiepiléticos alvo de interesse e de alguns dos seus principais metabolitos em amostras de cultura celular e em matrizes de plasma e cérebro de rato. Uma técnica adicional de HPLC-DAD foi também desenvolvida para a quantificação de vários fármacos antiepiléticos e metabolitos em plasma humano, constituindo uma ferramenta útil para apoiar a monitorização terapêutica destes fármacos na prática clínica. De acordo com os resultados obtidos num conjunto de ensaios in vitro, cinco dos onze flavonoides testados, nomeadamente, a baicaleína, a (-)-epigalocatequina galato [(-)-EPG], o kaempferol, a quercetina e a silimarina demonstraram ter um potencial interessante na inibição da atividade da P-gp. Estes flavonoides promoveram também um aumento significativo da acumulação intracelular dos fármacos antiepiléticos carbamazepina (CBZ), oxcarbazepina (OXC) e fenitoína (PHT), e dos seus metabolitos ativos carbamazepina-10,11-epóxido (CBZ-E) e licarbazepina (LIC), na linha celular Madin-Darby canine kidney, transfetada com o gene de resistência a múltiplos fármacos 1 (MDR1) que codifica para a P-gp humana (MDCK-MRD1), evidenciando-se como candidatos promissores para reverter a resistência associada aos fármacos antiepiléticos. Além disso, com exceção da lamotrigina (LTG), todos os fármacos antiepiléticos testados (CBZ, PHT e OXC), assim como os seus respetivos metabolitos ativos (CBZ-E e LIC), demonstraram ser substratos da P-gp neste modelo celular. Adicionalmente, a CBZ, CBZ-E, LIC, LTG, OXC e PHT promoveram uma diminuição significativa nas concentrações intracelulares de rodamina 123 (um substrato “sonda” clássico da P-gp), o que sugere um efeito indutor, mediado pelos próprios fármacos e metabolitos, da atividade funcional da P-gp. Reconhecendo-se as várias propriedades biológicas atribuídas aos flavonoides e tendo-se em consideração que misturas complexas de flavonoides são consumidas diariamente na nossa dieta, não se poderá ignorar o seu potencial farmacológico sinérgico. Neste sentido, foi concretizada uma avaliação dos potenciais efeitos sinérgicos da baicaleína, (-)-EPG, kaempferol, quercetina e silimarina relativamente à inibição da P-gp, primeiro in vitro e depois in vivo. Na verdade, comparativamente aos resultados individuais obtidos, algumas combinações duais de flavonoides apresentaram um potencial acrescido para inibição da P-gp in vitro. Além disso, algumas dessas combinações, nomeadamente (-)-EPG/silimarina e kaempferol/baicaleína, proporcionaram um aumento substancial da acumulação intracelular de CBZ, OXC e PHT, bem como dos seus metabolitos CBZ-E e LIC, na linha celular MDCK-MDR1, sendo estes efeitos comparáveis aos obtidos com o verapamilo, inibidor de referência da P-gp. Assim, enquanto os flavonoides baicaleína, (-)-EPG, kaempferol, quercetina e silimarina testados individualmente aumentaram em 1,2 a 31 vezes a acumulação intracelular dos fármacos antiepiléticos e metabolitos ativos considerados nas células MDCK-MDR1, as combinações duais desses flavonoides permitiram alcançar aumentos de cerca de 1,5 a 76 vezes. Adicionalmente, o efeito da combinação da (-)-EPG com a silimarina foi também avaliado em ensaios de transporte com a LIC (substrato da P-gp) através de uma monocamada de células MDCK-MDR1 montadas em câmaras de Ussing; como esperado, esta combinação aumentou o coeficiente de permeabilidade aparente da LIC. Em geral, os resultados dos estudos in vitro foram suportados pelos resultados dos estudos in vivo. De facto, após pré-tratamento de ratos Wistar machos com silimarina observou-se um aumento nas concentrações plasmáticas dos fármacos antiepiléticos alvo (CBZ, OXC e PHT). No entanto, é de ressaltar que os principais efeitos induzidos pela silimarina foram notados na farmacocinética da OXC, para a qual foi observado um aumento estatisticamente significativo na sua concentração plasmática máxima (50%) e na sua extensão de exposição sistémica (41%), tendo tais efeitos um impacto direto nas concentrações farmacológicas alcançadas no cérebro. Por outro lado, o uso de combinações duais de (-)-EPG/silimarina na inibição da atividade da P-gp foi também avaliado in vivo em ratos Wistar, sendo evidente o potencial sinérgico destesdois flavonoides no aumento do grau de exposição sistémica à OXC e LIC (metabolito farmacologicamente ativo da OXC), o que ocorreu numa extensão comparável à observada para o verapamilo (controlo positivo). De facto, o pré-tratamento de ratos Wistar com combinações de silimarina/(-)-EPG originou concentrações plasmáticas máximas de OXC semelhantes àquelas obtidas na presença do verapamilo. Além disso, os efeitos promovidos pelas combinações de silimarina/(-)-EPG no que diz respeito à magnitude da exposição sistémica ao fármaco foram também refletidos nos níveis de OXC e LIC alcançados no cérebro (biofase). Assim, de acordo com os nossos resultados, a terapia combinada de flavonoides/fármacos antiepiléticos pode ser considerada como uma abordagem promissora que deve continuar a ser explorada no sentido de superar a farmacorresistência mediada pela P-gp. A disponibilidade desta informação resultante de ensaios in vitro e in vivo contribui também para apoiar a hipótese do envolvimento de transportadores de efluxo, em particular da P-gp, na condição de epilepsia farmacorresistente. Considerando todo o seu potencial intrínseco e propriedades biológicas indiscutíveis, os compostos de tipo flavonoide podem então emergir como uma alternativa aos inibidores da P-gp disponíveis para um tratamento prospetivo dos doentes com epilepsia refratária.
The experimental work presented in this thesis was carried out at the Health Sciences Research Centre, Faculty of Health Sciences, University of Beira Interior (CICS-UBI) and at the Center for Neuroscience and Cell Biology (CNC), University of Coimbra

Little is known about potential influences of kinase pathway modulation on expression and activity of P-glycoprotein (P-gp). A protein kinase inhibitor (PKI) library was screened, to determine its effects on activity and expression of P-gp, in various cell lines. Cell lines were incubated with PKI for 24 h. Subsequent P-gp substrate accumulation studies were performed. Changes in P-gp activity and/or expression ≥ 25% compared to control were considered hits. Kinase pathways identified as P-gp activity hits were examined for their ability to modulate permeability. PKI families GSK-3, Craf1 and VEGFR2 and Tie-2, significantly modulated P-gp activity in the MDCK cell line. PKI families GSK-3, Iκκ and Jnk2/3 significantly modulated P-gp activity in the Caco-2 cell line. Few P-gp activity hits significantly modulated P-gp expression. PKIs modulate P-gp activity more than P-gp expression in a cell line dependent manner, excluding GSK-3 PKI family, which appears to be cell line independent.

碩士
長庚大學
生物醫學研究所
102
The overexpression of the serine/threonine specific polo-like kinase 1 (Plk1) has been detected in various types of cancer, and thus has fast become an attractive therapeutic target for cancer therapy. Plk1 inhibitors BI 2536, volasertib and GSK461364, were designed to selectively inhibit cancer cell proliferation by promoting G2/M cell cycle arrest at nanomolar concentrations. Unfortunately, alike most chemotherapeutic agents, the development of acquired resistance to Plk1 inhibitors is prone to present a significant therapeutic challenge. One of the most common mechanisms for acquired resistance in cancer chemotherapy is associated with the overexpression of ATP-binding cassette (ABC) transporters ABCB1, ABCC1 and ABCG2. Here, we discovered that in human cancer cells, the overexpression of ABCB1 and/or ABCG2 can lead to acquired resistance to three selective Plk1 inhibitors, BI 2536, volasertib and GSK461364. Moreover, these Plk1 inhibitors stimulate the ATPase activity of ABCB1 and ABCG2, as well as competitively inhibit the drug substrate transport mediated by ABCB1 and ABCG2. More significantly, the reduced chemosensitivity and Plk1 inhibitors-mediated G2/M cell cycle arrest in cancer cells overexpressing either ABCB1 or ABCG2 can be significantly restored in the presence of selective inhibitor of ABCB1 and ABCG2. Taken together, our findings indicate that in order to circumvent ABCB1 or ABCG2-mediated acquired resistance to Plk1 inhibitors, a combined regimen of Plk1 inhibitors and modulators or clinically active drugs that potently inhibit the function of ABC drug transporters, should be considered as a potential treatment strategy in the clinic.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Radek Machan Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Determination of permeability and active transport of selected butyrylcholinesterase inhibitors in vitro European Medicine Agency (EMA) and Food and Drug Administration agency (FDA) emphasise drug membrane permeability and drug-drug interactions on ABC transporters expressed in physiological barriers should be investigated for compounds in preclinical studies or for those already clinically used but evidence free. In this work we aimed to assess the capability of several experimental butyrylcholinesterase inhibitors that had been designed to treat dementia to permeate blood-brain barrier and to elucidate role of ATP-binding (ABC) cassette transporters in this transport. For this purpose, we employed in vitro bidirectional transport study across monolayers formed by polarized and highly differentiated Caco-2 cells. The permeability values gained from measurements were similar to values of several commonly used drugs for treatment of CNS disorders (e.g. antidepressants, antiepileptics). In addition, the compounds showed values of efflux ratio (basolateral- to-apical/apical-to-basolateral) approximately one...

Tese de mestrado, Química Farmacêutica e Terapêutica, Universidade de Lisboa, Faculdade de Farmácia, 2013
The present dissertation focuses on the relevance of macrocyclic diterpenes as leads for natural product-based anticancer drug development. In particular, the applicability of this type of compounds as P-glycoprotein (P-gp) inhibitors is thoroughly explored, given the well-known role of P-gp overexpression in the emergence of many multidrug resistant cancers, a major public health problem worldwide. Herein, a small library of macrocyclic lathyrane diterpenes was developed through molecular derivatization, using epoxyboetirane A as starting material. This compound had been previously isolated from Euphorbia boetica in large amounts, thus allowing several chemical transformations in the lathyrane scaffold, including hydrolysis, reduction and acylation reactions. Some of these transformations furthermore involved a base-catalyzed epoxide migration commonly referred to as the Payne rearrangement, as well as a Michael addition and an α-ketol rearrangement. Overall, twenty three new epoxyboetirane A derivatives were prepared, in addition to three formerly reported compounds, having all been fully characterized by onedimensional (1H-NMR, 13C-NMR, DEPT) and bidimensional (HMQC, HMBC and NOESY) nuclear magnetic resonance experiments, as well as by mass spectrometry. The antiproliferative and cytotoxic effects of the synthesized compounds were assessed on multidrug resistant mouse lymphoma and human colon adenocarcinoma cells. Some of them were furthermore tested on mouse embryonic fibroblasts so as to investigate their selective cytotoxic potential. Their ability to modulate P-gp was also evaluated on both cancer cell lines, by the rhodamine-123 accumulation assay, aiming to explore the effect of the implemented chemical transformations on P-gp-binding affinity. The collective analysis of the biological results and physicochemical properties of all twenty six molecules successfully enabled the establishment of structure-activity relationships, which was the ultimate goal of this project. To conclude, some of the most active derivatives were tested together with doxorubicin in a combination chemotherapy experimental model. All in all, most of them demonstrated to be extremely promising as multidrug resistance modifiers, attesting their usefulness in future rational anticancer drug development.
A presente dissertação pretende explorar a importância dos diterpenos macrocíclicos enquanto protótipos na descoberta de novos agentes anticancerígenos de origem natural. Em particular, a aplicabilidade deste tipo de compostos enquanto inibidores da glicoproteína-P (P-gp) é amplamente analisada, tendo em conta o já conhecido papel da sobreexpressão da P-gp no surgimento de vários tipos de cancro multirresistentes, um problema grave de saúde pública. Neste trabalho, foi criada uma pequena biblioteca de diterpenos macrocíclicos com o esqueleto do latirano por derivatização molecular, utilizando o epoxiboetirano A como material de partida. Este composto havia sido previamente isolado de Euphorbia boetica em quantidades suficientes para permitir uma série de transformações químicas no esqueleto original, incluíndo reacções de hidrólise, redução e acilação. Algumas envolveram ainda uma migração de funções epóxido usualmente citada como o rearranjo de Payne, uma adição de Michael e o rearranjo de uma α-hidroxicetona. No total, foram vinte e três os novos derivados preparados, para além de outros três compostos já descritos na literatura. Todos eles foram totalmente caracterizados através de experiências de ressonância magnética nuclear unidimensional (1H-RMN, 13C-RMN e DEPT) e bidimensional (HMQC, HMBC e NOESY), e por espectrometria de massa. Os efeitos antiproliferativo e citotóxico dos compostos sintetizados foram avaliados em células multirresistentes de linfoma de rato e de adenocarcinoma de cólon humano. Alguns foram ainda testados em fibroblastos de rato de forma a investigar o seu potencial citotóxico selectivo. Para além disso, a actividade moduladora da P-gp destes compostos foi avaliada em ambas as linhas celulares de cancro tendo em vista a exploração do efeito das transformações químicas implementadas na sua afinidade para a P-gp. A análise dos resultados culminou no estabelecimento de relações estruturaactividade, objectivo primordial deste projecto. Por fim, alguns dos derivados mais activos foram testados em conjunto com a doxorrubicina num modelo experimental de quimioterapia combinada. No geral, estes compostos mostraram ser extremamente promissores enquanto moduladores da multirresistência farmacológica, atestando a sua utilidade no desenvolvimento racional de novos agentes anticancerígenos.
The project was financed by Fundação para a Ciência e a Tecnologia (FCT) (PTDC/QUI-QUI/099815/2008, PTDC/QEQ-MED/0985/2012, Pest-OE/SAU/UI4013/2011)

碩士
國立陽明大學
傳統醫藥學研究所
89
ABSTRACT Sinomenine, which has structural similarities to morphine, is a herbal ingredient isolated from Sinomenium acutum, and used for amelioration of arthrities. This study uses microdialysis technique to investigate the pharmacokinetics of sinomenine in rat blood and bile, and its interaction in the presence of P-glycoprotein mediated transport modulator. In addition, in order to investigate the interaction of sinomenine and cytochrome P450 from aspect of pharmacokinetics, proadifen, a cytochrome P450 inhibitor, grapefruit juice, and bioflavonoids are subsequently administered. Microdialysis probes were inserted into the jugular vein/right atrium and bile duct of male Sprague-Dawley rats for simultaneous biological fluid sampling. Following sinomenine administration, the pharmacokinetics of protein-unbound sinomenine in rat blood appears in a dose-dependent manner at ranges of 3 to 30 mg kg-1. The disposition of sinomenine in rat bile shown to be a slow elimination phase, reaching a peak concentration at 20-40 min after sinomenine administration. A system consisting of a hepato-duodenal shunt microdialysis model was used to investigate the hepatobiliary excretion of sinomenine. The area under the concentration versus time curve (AUC) for unbound sinomenine in bile was significantly greater than that for sinomenine in blood at dosages of 3, 10 and 30 mg kg-1. The observations indicated active hepatobiliary excretion of sinomenine. However, at a dose of 20 mg kg-1 of cyclosporin A 10 min prior to sinomenine administration, the AUC for unbound sinomenine in bile was significantly reduced at the dosages of 10 and 30 mg kg-1. The blood-to-bile coefficient of distribution (k=AUCbile/AUCblood) of sinomenine following cyclosporin A treatment, the coefficient of distribution was reduced in the cyclosporin A treated group. The result reveals that hepatobiliary excretion of sinomenine is markedly affected by P-glycoprotein related modulator. With treatment of proadifen (10 mg kg-1), a cytochrome P450 inhibitor, 10 min prior to sinomenine (10 mg kg-1) administration, the AUC for unbound sinomenine in blood was increased. Similar result was also observed in the bile in which the AUC of unbound sinomenine in bile was increased as compared with sinomenine. These results indicate that the metabolism and hepatobiliary excretion of sinomenine may be blocked by the enzyme inhibitor of cytochrome P450 in the biotransformation in rat. In the pretreatment of grapefruit juice (10 ml kg-1) at 4 h prior to sinomenine (10 mg kg-1) administration, the AUC for unbound sinomenine in both blood and bile were significantly increased. These results suggest that the grapefruit juice may decelerate the disposition of sinomenine in rat. However, this circumstance was blocked on the concurrent treatment with cyclosporin A (20 mg kg-1) administration. In a group with simultaneous administeration with grapefruit juice, cyclosporin A and sinomenine, the bile-to-blood coefficient of distribution was significantly decreased from with or without cyclosporin A, respertively 4.74+0.58 and 0.56±0.06. These results indicate that grapefruit juice enlarge the concentration of sinomenine in both blood and bile of rat. However, a P-glycoprotein inhibitor, cyclosporin A, blocked this phenomenon. To determine if there is an effect of sinomenine concentration on the extent of the pharmacokinetic interaction between sinomenine and bioflavonoids, a dosage of 10 mg kg-1 of each flavonoids was administered 10 min prior to sinomenine (10 mg kg-1) administration. These flavonoids, quercetin, rutin, naringin, naringenin, hesperetin and hesperidin, are grapefruit juice ingredients which exert the modulatory function on both P-glycoprotein and cytochrome P450. The results indicate that all of those flavonoids increased the sinomenine AUC in blood and decreased the AUC in bile. The bile-to-blood coefficient of distribution of sinomenine was significantly decreased on the concomitant treatment of sinomenine and each of bioflavonoids. In conclusion, sinomenine goes through hepatobiliary elimination against the concentration gradient from blood to bile. Our results reveal that the hepatobiliary excretion of sinomenine is regulated by the P-glycoprotein, and sinomenine metabolism catalyzed by cytochrome P450. In addition, grapefruit juice and flavonoids prolong the sinomenine concentration in blood and reduce its excretion from hepatobiliary pathway.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Dominika Bezděková Supervisor: doc. PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: IN VITRO STUDY OF DRUG-DRUG INTERACTIONS OF HIV PROTEASE INHIBITOR DARUNAVIR ON EFFLUX ABC TRANSPORTERS Abstract: Darunavir is a drug used in the therapy of HIV belonging to the group of protease inhibitors. These protease inhibitors are used as a part of the combination antiretroviral therapy. For the increase of bioavailability, darunavir is always used in combination with ritonavir or cobicistat. As the CYP3A4 and ABCB1 (P-glycoprotein) transporter substrate, darunavir is a drug with a high potential to drug interactions. Considering the amount of adverse effects that can be caused by darunavir, it is necessary to know these drug interactions for the safety of therapy. Inhibition of the intestinal ABCB1 by the co-administrated drugs could also lead to the increased bioavailability of darunavir and to reduction of frequency of administration leading to a cheaper therapy. This thesis studies the drug-drug interactions of darunavir with in vitro methods using two cell lines - MDCKII and Caco-2 cells. The results from the transport of darunavir across the MDCKII cell monolayer indicates that darunavir is a ABCB1...

Glycosphingolipid (GSL) metabolism is a complex process involving proteins and enzymes at distinct locations within the cell. Mammalian GSLs are typically based on glucose or galactose, forming glucosylceramide (GlcCer) and galactosylceramide (GalCer). Most GSLs are derived from GlcCer, which is synthesized on the cytosolic leaflet of the Golgi, while all subsequent GSLs are synthesized on the lumenal side. We have utilized both pharamacological and genetic manipulation approaches to selectively regulate GSL metabolism and better understand its mechanistic details. We have developed analogues of GlcCer and GalCer by substituting the fatty acid moiety with an adamanatane frame. The resulting adamantylGSLs are more water-soluble than their natural counterparts. These analogues selectively interfere with GSL metabolism at particular points within the metabolic pathway. At 40 µM, adaGlcCer prevents synthesis of all GSLs downstream of GlcCer, while also elevating GlcCer levels, by inhibiting lactosylceramide (LacCer) synthase and glucocerebrosidase, respectively. AdaGalCer specifically reduces synthesis of globotriaosylceramide (Gb3) and downstream globo-series GSLs. AdaGalCer also increases Gaucher disease N370S glucocerebrosidase expression, lysosomal localization and activity. AdaGSLs, therefore, have potential as novel therapeutic agents in diseases characterized by GSL anomalies and as tools to study the effects of GSL modulation. Two predominant theories have been developed to explain how GlcCer accesses the Golgi lumen: one involving direct translocation from the cytosolic-to-lumenal leaflet of the Golgi by the ABC transporter P-glycoprotein (P-gp, ABCB1, MDR1), and the other involving retrograde transport of GlcCer by FAPP2 to the ER, followed by entry into the vesicular transport system for Golgi lumenal access. To examine the in vivo involvement of P-gp in GSL metabolism, we generated a knockout model by crossbreeding the Fabry disease mouse with the P-gp knockout mouse. HPLC analyses of tissue Gb3 levels revealed a tissue-specific reduction in MDR1/Fabry mice. TLC analyses, however, did not show such reduction. In addition, we performed a gene knockdown study using siRNA against P-gp and FAPP2. Results show these siRNA to have distinct effects on GSL levels that are cell-type specific. These results give rise to the prospect of unique therapeutic approaches by targeting P-gp or FAPP2 for synthesis inhibition of particular GSL pathways.