<p>U ovoj disertaciji su ispitivani efekti žučnih kiselina na transportne procese kod<br />kojih se ispoljava efekat građenja molekulskih agregata (micele, me&scaron;ovite micele,<br />kompleks sa vodoničnim vezama itd). Ispitan je uticaj temperature na kritičnu micelarnu<br />koncnentraciju holne, deoksiholne i henodeoksiholnekiseline i njihovih keto derivata,<br />određena je entropija formiranja micele, koja je važan parametar ne samo u<br />samoasocijaciji žučnih kiselina već i u njihovoj interakciji sa hidrofobnim molekulima.<br />Određen je kompleks sa vodoničnim vezama izmeđ u lidokaina i žučnih kiselina,<br />regresiona jednačina koja povezuje strukturne parametre žučnih kiselina i ravnotežnu<br />konstantu formiranja tog kompleksa. Zatim je ispitivano delovanje žučnih kiselina u<br />hloroformu na kinetiku prelaza lidokaina i verapamila iz vodene faze u hloroform (model<br />za predtretman sa žučnim kiselinama) U ovom radu je određena i solubilizacija lecitina i<br />holesterola sa žučnim kiselinama.</p> / <p>In this work,&nbsp; effects of bile acids which form molecular&nbsp; aggregates (micelles,<br />mixed micelles, hydrogen complex etc.) on transportation processes were investigated.<br />Influence of temperature on critical micellar concentration of cholic, deoxyholic and<br />henodeoxycholic acids and its keto derivatives was&nbsp; examined. Also, micelle formation<br />entropy was determined. This is very important parameter for self-association of bile<br />acids and their interactions with hydrophobic molecules.<br />Hydrogen complex of lidocain and bile acids was investigated and regression equation<br />which connects structural parameters of bile acids&nbsp; and equilibrium constant of forming<br />this complex was established. After that, effects of bile acids on transfer kinetics of<br />lidocaine and verapamil from aqueous phase to chlorophorm was investigated. Also,<br />micellar solubilization of lecithin and cholesterolby bile acids was determined.</p>

Submitted by Priscila dos Santos Oliveira (priscilaspitty@hotmail.com) on 2018-08-29T22:43:22Z No. of bitstreams: 1 Merged_Priscila Oliveira.pdf: 1205779 bytes, checksum: 2ed8db0dfdefdc0a97d7a31fad8acaa9 (MD5) / Approved for entry into archive by Silvana Alvarez null (silvana@ict.unesp.br) on 2018-09-03T14:47:25Z (GMT) No. of bitstreams: 1 Oliveira_mp_me_sjc.pdf: 1205779 bytes, checksum: 2ed8db0dfdefdc0a97d7a31fad8acaa9 (MD5) / Made available in DSpace on 2018-09-03T14:47:25Z (GMT). No. of bitstreams: 1 Oliveira_mp_me_sjc.pdf: 1205779 bytes, checksum: 2ed8db0dfdefdc0a97d7a31fad8acaa9 (MD5) Previous issue date: 2018-08-06 / Resumo: A epidemiologia das infecções causadas por Candida sofreu mudanças nos últimos anos. Embora Candida albicans ainda seja o principal patógeno causador de candidíase, relatos recentes reportam o aumento da incidência de infecções causadas por espécies de Candida não-albicans. A toxicidade dos fármacos antifúngicos utilizados juntamente com o desenvolvimento da resistência tem se tornado um sério problema clínico. Com isso, atualmente existe a urgência na descoberta de novos compostos com atividades antifúngicas. Objetivos: avaliar a atividade antifúngica do fármaco Cloridrato de Verapamil, determinar a Concentração Inibitória Mínima (CIM) e a Concentração Fungicida Mínima (CFM), frente à Candida albicans ATCC 18804, Candida krusei ATCC 6258, Candida parapsilosis ATCC 90018 e Candida glabrata ATCC 9030. Além de avaliar a citotoxicidade do fármaco em queratinócitos humanos (HaCaT). Metodologia: a determinação da CIM foi realizada pela técnica de microdiluição de acordo com The European Committee on Antimicrobial Susceptibility Testing (EUCAST). A CFM foi determinada por plaqueamento em ágar Sabouraud de alíquotas provenientes das diluições do ensaio de microdiluição. A avaliação da citotoxicidade foi realizada pela técnica de redução da resazurina, sendo possível avaliar a atividade mitocondrial das células HaCaT. Resultados: o fármaco Cloridrato de Verapamil demonstrou atividade antifúngica contra as quatro espécies patogênicas de Candida, com o valor de CIM de 1250 µM; valor de CFM maior que 1250 µM, sendo assim este fármaco foi considerado fungistático. Além disso, o Cloridrato de Verapamil não apresentou citotoxicidade nas concentrações avaliadas no estudo, pois, a redução de células viáveis nas concentrações mais elevadas não ultrapassa 30%. Com relação à redução de biomassa em biofilme de Candida, após tratamento com Cloridrato de Verapamil, houve redução (de 10 a 20%) para as quatro espécies de Candida em estudo, quando utilizadas concentrações de fármaco correspondentes a CIM; quando utilizadas concentrações de fármaco correspondentes a cinco vezes o valor de CIM, houve um aumento significativo na redução de biomassa (de 25% a 60%) em biofilme formado pelas quatro espécies de Candida. Conclusão: o fármaco Cloridrato de Verapamil apresentou atividade antifúngica para Candida albicans e Candida não-albicans, sendo considerado um fármaco fungistático, além de não apresentar citotoxicidade em queratinócitos humanos, e demonstrar atividade na redução de biomassa em biofilme formado pelas quatro espécies de Candida. Demais estudos são necessários para verificar a ação desse fármaco em diferentes mecanismos de virulência frente a células de Candida spp. / Abstract: The epidemiology of Candida infections has changed in recent years. Although Candida albicans is still the main pathogen causing candidiasis, recent reports have reported an increase in the incidence of infections caused by nonalbicans Candida species. The toxicity of the antifungal drugs used together with the development of resistance has become a serious clinical problem. With this, there is now an urgency in the discovery of new compounds with antifungal activities. Objectives: To evaluate the antifungal activity of the drug Verapamil Hydrochloride, to determine the Minimum Inhibitory Concentration (MIC) and the Minimum Fungicidal Concentration (CFM) against Candida albicans ATCC 18804, Candida krusei ATCC 6258, Candida parapsilosis ATCC 90018 and Candida glabrata ATCC 9030 In addition to evaluating the cytotoxicity of the drug in human keratinocytes (HaCaT). Methodology: MIC determination was performed by the microdilution technique according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST). The CFM was determined by plating on Sabouraud agar from aliquots from the dilutions of the microdilution assay. The evaluation of cytotoxicity was performed using the resazurin reduction technique, and it was possible to evaluate the mitochondrial activity of HaCaT cells. Results: The drug Verapamil Hydrochloride demonstrated antifungal activity against the four pathogenic species of Candida, with MIC value of 1250 μM; CFM value greater than 1250 μM, so this drug was considered fungistatic. In addition, Verapamil Hydrochloride did not present cytotoxicity in the units evaluated in the study, because the reduction of viable cells in the most cells is not exceeded by 30%. Regarding the biomass reduction in Candida biofilm, after treatment with Verapamil Hydrochloride, there was a reduction (from 10 to 20%) for the four Candida species in study, when the use of drug corresponding to MIC; when the use of drug corresponding to 5 times of MIC, there was a significant increase in the biomass reduction (from 25% to 60%) in the biofilm molded by the four Candida species. Conclusion: Thus, the drug Verapamil Hydrochloride led to the antifungal activity of Candida albicans and non-albicans Candida species, being considered a fungicidal drug, besides not presenting cytotoxicity in human serotonizers, and were submitted to biomass reduction in biofilm molded by four Candida species. More studies are needed to verify the action of the drug on different mechanisms of virulence against Candida spp cells.

A maioria dos agentes terapêuticos, frequentemente prescritos são formulados e comercializados sob a forma racêmica, embora para alguns deles, já tenha sido demonstrado que os efeitos farmacológicos e ou tóxicos estejam relacionados apenas a um dos enantiômeros. Além disso, é conhecido o fato de que os enantiômeros podem apresentar perfis farmacocinéticos e farmacodinâmicos diferentes. O estudo avaliou a influência da exposição inalatória ao vapor de gasolina e ao etanol combustível na farmacocinética enantiosseletiva dos fármacos verapamil, ibuprofeno e fluoxetina. Ratos machos Wistar foram divididos em 09 grupos: controle, gasolina, etanol combustível. A exposição aos solventes foi realizada em câmara de exposição do tipo apenas pelo nariz, durante 6 horas/dia, cinco dias por semana, durante 6 semanas. A análise das AUCs foram calculadas diretamente no intervalo de zero a infinito com base na Quadratura de Gauss- Laguerre. As concentrações correspondentes aos tempos foram estimadas por interpolação polinomial. A comparação dos valores de AUC e Cl/f obtidos para cada fármaco e para cada Grupo exposto e seu respectivo Controle, foi realizada através da construção de Intervalos de Confiança, ao nível de 95%. A farmacocinética do verapamil, do ibuprofeno e da fluoxetina é enantiosseletiva. Os dados mostram que a exposição inalatória de ratos ao etanol combustível na concentração de 2 LEOSTEL mostrou indução do CYP2C através da redução do AUC e do aumento do clearance aparente do enantiômero (+)-(S)-ibuprofeno, inibição do CYP2D indicada pelo aumento da AUC e redução do clearance aparente do enantiômero (-)-(R)- fluoxetina e indução do CYP3A evidenciada por redução dos valores de AUC e aumento dos valores de clearance aparente de ambos os enantiômeros do verapamil. A exposição inalatória de ratos à gasolina na concentração de 2-LEOTWA também mostrou indução do CYP2C denotada pela redução do AUC e do aumento do clearance aparente de ambos os enantiômeros do ibuprofeno, inibição do CYP2D indicada pelo aumento dos valores de AUC e redução dos valores de clearance aparente de ambos enantiômeros da fluoxetina e, em não alteração do CYP3A evidenciada pela obtenção de valores de AUC e clearance aparente do verapamil similares aos do grupo controle. / Most therapeutic agents frequently used are formulated and sold under the racemic form, although for some of them, it has been demonstrated that the pharmacological or toxic and are associated only with one of the enantiomers. The study evaluated the influence of inhalation exposure to vapor of gasoline and ethanol in the enantioselective pharmacokinetics of the drug verapamil, ibuprofen and fluoxetine. Male Wistar rats were divided into 09 groups: control, gasoline, ethanol. The exposure was carried out in solvent exposure chamber by nose only exposure system for 6 hours / day, five days per week for six weeks. The analysis of the AUC were calculated directly in the range of zero to infinity on the basis of Quadrature Gauss-Laguerre. The concentrations corresponding to the times were estimated by polynomial interpolation. The comparison of AUC and Cl/f obtained for each drug and for each exposed group and its respective control, was accomplished through the construction of confidence intervals, at 95%. In conclusion, the pharmacokinetics of verapamil, ibuprofen and fluoxetine is enantioselective. The data show that inhalation exposure of rats to ethanol at a concentration of 2-LEO STEL showed induction CYP2C by reducing of the AUC and increase the apparent clearance of the enantiomer (+)-(S)-ibuprofen, inhibition of CYP2D indicated AUC increase and the reduction in the apparent clearance of the enantiomer (-)-(R)-fluoxetine and CYP3A induction as evidenced by reduction in AUC and increase and the values of apparent clearance of both enantiomers of verapamil. Inhalation exposure of rats to gasoline in a concentration of 2-LEO-TWA also showed induction CYP2C denoted by the reduction of AUC and increase and the apparent clearance of both enantiomers of ibuprofen, inhibition of CYP2D indicated by the increase in AUC and reduction values of apparent clearance of both enantiomers of fluoxetine and does not change the CYP3A evidenced by obtaining AUC and apparent clearance of verapamil similar to the control group.

Orientadores: Cesar Costapinto Santana, Marco Aurélio Cremasco / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-23T03:10:20Z (GMT). No. of bitstreams: 1 Perna_RafaelFirmani_D.pdf: 3196327 bytes, checksum: 4240581b6c5c11e40e59fac8ddddd657 (MD5) Previous issue date: 2013 / Resumo: O fármaco racêmico verapamil tem sido utilizado no tratamento de doenças cardiovasculares relacionadas à hipertensão arterial, arritmias supraventriculares e angina pectoris. Seus enantiômeros apresentam biodisponibilidades distintas e exibem diferentes propriedades farmacológicas. O enantiômero S-(-)-verapamil apresenta maior potencial cardiovascular quando comparado ao seu antípoda óptico R-(+)-verapamil; este, por sua vez, exibe ação antitumoral, atuando como inibidor hepatocarcinogênico. Diante deste contexto, o presente trabalho teve como objetivo a determinação de parâmetros envolvidos na separação dos enantiômeros do fármaco em escala semi-preparativa visando obtê-los, opticamente puros, em unidade cromatográfica contínua do tipo leito móvel simulado. O desenvolvimento da separação ocorreu em colunas quirais recheadas com fase estacionária tris (3,5-dimetilfenilcarbamato) de amilose utilizando a mistura de n-hexano, isopropanol, etanol e dietilamina (90: 5: 5: 0,1 %, em volume) como fase móvel. Experimentos de pulsos com soluções diluídas dos enantiômeros do verapamil foram realizados para diferentes temperaturas (15 a 35 ºC) e vazões (1,0 a 2,5 mL min-1) visando determinar as constantes de equilíbrio de adsorção (constantes de Henry) e os parâmetros cromatográficos, termodinâmicos e de transferência de massa; ao passo que, experimentos com soluções diluídas dos traçadores (compostos inertes) foram realizados para se obter as porosidades do sistema. Os fatores de retenção apresentaram valores considerados ideais para separações enantioméricas (2,0 < k < 6,0) para vazões e temperaturas avaliadas. Com relação à seletividade e resolução cromatográficas, foram obtidos valores de 1,34 e 1,70, respectivamente, para temperatura de 25 ºC e vazão de 1,0 mL min-1, o que comprova a completa separação dos enantiômeros na coluna semi-preparativa. As constantes de Henry apresentaram-se relativamente elevadas, sendo que o enantiômero R-(+)-verapamil apresentou maior afinidade pela fase estacionária com maiores valores para a constante de equilíbrio de adsorção. O coeficiente de dispersão axial sofreu pouca variação entre os enantiômeros; entretanto, o coeficiente de transferência de massa global foi considerado relativamente rápido com valores superiores a 65,70 e 45,32 min-1 para os enantiômeros S-(-) e R-(+), respectivamente. Os parâmetros termodinâmicos confirmaram a maior afinidade do R-(+)-verapamil pela coluna quiral com maiores valores negativos de entalpia de adsorção. Constatou-se também que os fenômenos entálpicos regem a separação enantiomérica na fase estacionária avaliada. Experimentos com soluções concentradas da mistura racêmica foram realizados com a finalidade de se determinar as isotermas de adsorção competitivas e os perfis de eluição sob condições de sobrecarga da coluna cromatográfica. As isotermas de adsorção apresentaram comportamento não-linear para a faixa de concentração utilizada e foram satisfatoriamente ajustadas ao modelo de Langmuir Competitivo Modificado. O estudo de sobrecarga mostrou a possibilidade da separação do verapamil racêmico nas escalas semi-preparativa e preparativa. Finalmente, foram determinados os parâmetros operacionais do processo contínuo multicolunas para os modos VARICOL® e LMS e, obtidos os valores das variáveis de desempenho (pureza, produtividade, recuperação e consumo de solvente) das respectivas unidades. Os resultados obtidos mostraram-se promissores à separação dos enantiômeros do verapamil e, de fato, representaram o primeiro conjunto de dados experimentais em escala mundial decorrentes do uso da cromatografia contínua operada em leito móvel simulado / Abstract: The racemic drug verapamil has been used in the treatment of cardiovascular diseases related to hypertension, supraventricular arrhythmias, and angina pectoris. Its enantiomers have different bioavailability and pharmacological properties. The S-(-)-verapamil presents more cardiovascular potential when compared to its optical antipode R-(+)-verapamil. This, in turn, exhibits antitumor activity, acting as a hepatocarcinogenic inhibitor. Given this context, the present study aimed to determine the parameters involved in the separation of the enantiomers of the drug in semi-preparative scale in order to obtain them, optically pure, in continuous chromatographic unit type simulated moving bed. The development occurred in separation columns packed with chiral stationary phase amylose tris (3,5-dimethylphenylcarbamate) using a mixture of n-hexane, isopropanol, ethanol, and diethylamine (90: 5: 5: 0.1 %, by volume) as mobile phase. Pulse experiments with dilute solutions of the verapamil enantiomers were performed at different temperatures (15 to 35 ºC) and flow rates (1.0 to 2.5 mL min-1) to determine the equilibrium constants adsorption (Henry's constants) and chromatographic, thermodynamic, and mass transfer parameters; whereas experiments with dilute solutions of the tracers (inert compound) were performed to obtain the porosity of the system. The capacity factors had values considered ideal for enantiomeric separations (2.0 < k < 6.0) for the flow rate and temperature evaluated. Regarding the chromatographic selectivity and resolution values of 1.34 and 1.70 were obtained, respectively, for temperature of 25 ºC and flow rate of 1.0 mL min-1, which proves the complete separation of the enantiomers in the semi-preparative column. Henry's constants were relatively high, and the R-(+)-verapamil had a higher affinity for the stationary phase with the highest values for the adsorption equilibrium constant. Axial dispersion coefficient showed little variation between the enantiomers; however, the overall mass transfer coefficient was considered relatively fast to values of 65.70 and 45.32 min-1 to S-(-) and R-(+) enantiomers, respectively. Thermodynamics parameters confirm the major affinity of R-(+)-verapamil with high negative values of adsorption enthalpy. It was also found that the enthalpic phenomena govern the enantiomeric separation. Experiments with concentration solutions of the racemic mixture were conducted in order to determine the competitive adsorption isotherm, and the elution profiles under overload conditions of the chromatographic column. The competitive isotherms showed a nonlinear behavior to the range of concentration investigated and the Langmuir Competitive Modified model represents satisfactorily the adsorption data. The study of overload of the column carried through in high concentration showed the possibility of the separation of racemic verapamil using semi-preparative and preparative scale. Finally, the operating parameters of the multicolumn continuous process (VARICOL® and LMS units), and the values of the performance variables (purity, productivity, recovery, and solvent consumption) were determined. The results obtained were promising to the separation of the verapamil enantiomers and, in fact, represented the first set of experimental data worldwide from the use of the continuous chromatography operated in simulated moving bed / Doutorado / Desenvolvimento de Processos Biotecnologicos / Doutor em Engenharia Química

O excesso de tecido cicatricial (quelóides e cicatrizes hipertróficas) é um defeito do processo de cicatrização das feridas, caracterizado por um aumento na produção da matriz extracelular. Neste estudo, fibroblastos dérmicos humanos tratados com 50 <font face=\"symbol\">mM verapamil apresentaram discreta modificação na distribuição dos microfilamentos e alteraram sua morfologia de fusiformes para estrelados/arredondados. Estes efeitos poderiam estar associados a baixos níveis de cálcio citosólico. Esta hipótese foi confirmada através marcação de fibroblastos tratados com calcium green. Observamos também, que o verapamil inibiu a proliferação celular em 64,4%, aumentou a secreção de MMP1 e diminuiu o colágeno sintetizado pelos fibroblastos, sem aparentes efeitos citotóxicos. O metabolismo celular do cálcio está aparentemente relacionado a produção da matriz extracelular e portanto as patologias hipertróficas da cicatrização (quelóides e cicatrizes hipertróficas) podem responder ao tratamento com bloqueadores do canal de cálcio (verapamil). / Excessive scar tissue (keloids and hypertrophic scars) is a defective wound healing process characterized by overproduction of extracellular matrix. In the present study human dermal fibroblasts treated with 50 <font face=\"symbol\">mM verapamil changed their normal spindle-shaped morphology to stellate/rounded and showed discrete reorganization of microfilaments We hypothesized that these effects would be associated to lower levels of cytosolic Ca2+. Indeed, short time loading with calcium green confirmed that verapamil-treated fibroblasts exhibited lower intracellular calcium levels. We also observed that verapamil decrease cellular proliferation by 64.4%, increase the secretion of MMP1 and decrease synthesis of collagen in cultured fibroblasts. This alterations induced by verapamil are not associated with cytotoxic effects. The cellular calcium metabolism appears to regulate extracellular matrix production and so those hypertrophic disorders of wound healing (keloids and hypertrophic scars) may respond to therapy with calcium antagonist drugs (verapamil).

Schweinfurthins are intriguing natural product chemotherapeutics due to their potent yet selective activity and their unknown mechanism of growth inhibition in cancer. Much progress has been made in characterizing the intracellular effects of the schweinfurthins since they were first isolated from Macaranga schweinfurthii in 1986. Here, the L-type calcium channel and P- glycoprotein (Pgp) inhibitor verapamil has been found to enhance schweinfurthin- induced growth inhibition. Verapamil induces an increase in the intracellular concentration of a fluorescent schweinfurthin. However, the synergistic relationship between the schweinfurthins and verapamil is complex and not obvious in that verapamil fails to increase the intracellular concentration of a schweinfurthin analogue that is a known substrate of Pgp. Schweinfurthins are also found to induce alterations to cholesterol homeostasis by increasing the expression of the cholesterol efflux pump ABCA1 in an apparent liver X receptor- independent fashion. In addition, schweinfurthin treatment blunts epidermal growth factor downstream activation and phosphorylation of Akt. Lastly, a schweinfurthin-resistant cell line has been created and characterized for resistance to schweinfurthin-induced growth inhibition. The variety of intracellular effects characteristic of schweinfurthin treatment described here provide mechanistic framework for identifying the potential target and mechanism of growth inhibition for the schweinfurthins.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成24年3月26日 櫛山泰規氏の博士論文として提出された

Resistance to the avermectin/milbemycin class of anthelmintics in nematodes has become a serious problem worldwide due to their unrestricted usage. Resistance to these compounds is attributed to the over-expression of the transport protein, P-glycoprotein (P-gp). P-gp acts by pumping drug molecules out from the cell or organism, P-gp efflux activity can be blocked using multidrug resistance (MDR) modulators associated with chemotherapy to enhance their therapeutic effect. A series of experiments was undertaken to determine if the association of the anthelmintics, ivermectin (IVM) and moxidectin (MOX), and MDR modulators would increase the anthelmintics' efficacy against resistant parasites. Using an in vitro migration assay, IVM and MOX in the presence or absence of verapamil (VRP), CL347,099 and cyclosporin A (CyA) were used against IVM- and MOX-selected strains of H. contortus. The modulators alone had no effect on reducing the number of migrating larvae, IVM and MOX had a significant increase in efficacy of 52.7 and 58,3% respectively, when used in association with VRP, above that obtained with the anthelmintics alone. CL347,099 was also able to significantly increase the IVM and MOX efficacy by 24.2 and 38.9%, respectively. The effect of IVM and MOX in combination with VRP and CL347,099 was determined in jirds infected with selected strains of H. contortus. The combinations of VRP with either IVM or MOX significantly reduced worm counts of the selected strains compared with the untreated controls, whereas IVM or MOX alone did not. CL347,099 plus MOX combination was significantly more efficacious than moxidectin alone against the selected strains. To evaluate the effect of VRP on the pharmacokinetic behaviour of the anthelmintics IVM and MOX, the drug combination was given to sheep. The IVM plus VRP treatment resulted in an increase of the pharmacokinetic parameters of IVM. The peak concentration (83%) and area under the curve (54%) were significantly differen

Chemotherapy is used in the treatment of cancer. Unfortunately, drugs often fail due to multidrug resistance (MDR) caused by P-glycoprotein (P-gp1or ABCB1) and the multidrug resistance-associated protein (MRP1 or ABCC1). These proteins bind and transport drugs out of cancer cells, thereby conferring MDR. / The second chapter of this thesis addresses an unexplained phenomenon that accompanies P-gp1 expression, collaterally sensitive to verapamil. The collective results of this work demonstrated that treatment of cells that over-express P-gp1 with verapamil induces apoptosis. Furthermore, the findings show that the ATPase activity of P-gp1 was activated by verapamil. The degree of ATPase activation was proportional to the level of apoptosis and the increased demand for ATP resulted in the production of reactive oxygen species (ROS). Finally, the production of ROS led to cell death mediated by apoptosis in that experimental model system. / Chapters three and four are devoted to understanding the binding characteristics of MRP1 with two of its physiological substrates, glutathione (GSH) and leucotriene C4(LTC4). Photoreactive derivatives of these substrates were synthesised to address this objective, IAAGSH and IAALTC4. Photolabelling and transport studies showed that these derivatives have similar binding characteristics as the native compounds. In addition, photolabelling of MRP1 occurred with a high specificity with both compounds. IAAGSH and IAALTC 4 were also used to determine the locations of GSH and LTC4 binding sites. This was accomplished using MRP1-variants containing hemagglutinin (HA) epitopes at specific locations in the amino acid sequence. Through photoaffinity labelling, immunoprecipitation, and trypsin digestion, a map of binding sites for IAAGSH or IAALTC4 was obtained. Both LTC4 and GSH bound to transmembrane (TM) regions 10-11 and 16-17 which have been previously implicated in drug binding. Furthermore, novel binding sites for both substrates were discovered. IAALTC4 photolabelled a novel site within the first five TMs (TMD0) of MRP1, whereas IAAGSH labelled two cytoplasmic regions (L1 and L0). These may represent specific binding sites for LTC4 and GSH. / The work within this thesis explores some of the biochemical characteristics of Pgp1 and MRP1 that are not directly related to drug resistance and may lead to new strategies in cancer treatment.

No description available.