12 research outputs found
Role of Human Organic Cation Transporter 1 (hOCT1) Polymorphisms in Lamivudine (3TC) Uptake and Drug-Drug Interactions
Lamivudine (3TC), a drug used in the treatment of HIV infection, needs to cross the plasma membrane to exert its therapeutic action. Human Organic cation transporter 1 (hOCT1), encoded by the SLC22A1 gene, is the transporter responsible for its uptake into target cells. As SLC22A1 is a highly polymorphic gene, the aim of this study was to determine how SNPs in the OCT1-encoding gene affected 3TC internalization and its interaction with other co-administered drugs. HEK293 cells stably transfected with either the wild type form or the polymorphic variants of hOCT1 were used to perform kinetic and drug-drug interaction studies. Protein co-immunoprecipitation was used to assess the impact of selected polymorphic cysteines on the oligomerization of the transporter. Results showed that 3TC transport efficiency was reduced in all polymorphic variants tested (R61C, C88R, S189L, M420del, and G465R). This was not caused by lack of oligomerization in case of variants located at the transporter extracellular loop (R61C and C88R). Drug-drug interaction measurements showed that co-administered drugs [abacavir (ABC), zidovudine (AZT), emtricitabine (FTC), tenofovir diproxil fumarate (TDF), efavirenz (EFV) and raltegravir (RAL)], differently inhibited 3TC uptake depending upon the polymorphic variant analyzed. These data highlight the need for accurate analysis of drug transporter polymorphic variants of clinical relevance, because polymorphisms can impact on substrate (3TC) translocation but even more importantly they can differentially affect drug-drug interactions at the transporter level
Role of human Organic Cation Transporter 1 (hOCT1) polymorphisms in lamivudine (3TC) uptake and drug-drug interactions.
Lamivudine (3TC), a drug used in the treatment of HIV infection, needs to cross the plasma membrane to exert its therapeutic action. Human Organic cation transporter 1 (hOCT1), encoded by the SLC22A1 gene, is the transporter responsible for its uptake into target cells. As SLC22A1 is a highly polymorphic gene, the aim of this study was to determine how SNPs in the OCT1-encoding gene affected 3TC internalization and its interaction with other co-administered drugs. HEK293 cells stably transfected with either the wild type form or the polymorphic variants of hOCT1 were used to perform kinetic and drug-drug interaction studies. Protein co-immunoprecipitation was used to assess the impact of selected polymorphic cysteines on the oligomerization of the transporter. Results showed that 3TC transport efficiency was reduced in all polymorphic variants tested (R61C, C88R, S189L, M420del, and G465R). This was not caused by lack of oligomerization in case of variants located at the transporter extracellular loop (R61C and C88R). Drug-drug interaction measurements showed that co-administered drugs [abacavir (ABC), zidovudine (AZT), emtricitabine (FTC), tenofovir diproxil fumarate (TDF), efavirenz (EFV) and raltegravir (RAL)], differently inhibited 3TC uptake depending upon the polymorphic variant analyzed. These data highlight the need for accurate analysis of drug transporter polymorphic variants of clinical relevance, because polymorphisms can impact on substrate (3TC) translocation but even more importantly they can differentially affect drug-drug interactions at the transporter level
P-glycoprotein (ABCB1) activity decreases raltegravir disposition in primary CD4+P-gp high cells and correlates with HIV-1 viral load
Altres ajuts: SAF2014-25560-RTo evaluate the role of P-glycoprotein (P-gp) and multidrug-resistant-protein 1 (MRP1) on raltegravir intracellular drug disposition in CD4+ T cells, investigate the effect of HIV-1 infection on P-gp expression and correlate HIV-1 viraemia with P-gp activity in primary CD4+ T cell subsets. The cellular accumulation ratio of [ 3 H]raltegravir was quantified in CD4+ T cell lines overexpressing either P-gp (CEM-P-gp) or MRP1 (CEM-MRP1) and in primary CD3+CD4+ T cells with high (P-gp high) and low P-gp activity (P-gp low); inhibition of efflux transporters was confirmed by the intracellular retention of calcein-AM. The correlation of P-gp activity with HIV-1 viraemia was assessed in naive and memory T cell subsets from 21 HIV-1-infected treatment-naive subjects. [ 3 H]Raltegravir cellular accumulation ratio decreased in CEM-P-gp cells (P < 0.0001). XR9051 (a P-gp inhibitor) and HIV-1 PIs reversed this phenomenon. Primary CD4+P-gp high cells accumulated less raltegravir (38.4% ± 9.6%) than P-gp low cells, whereas XR9051 also reversed this effect. In vitro HIV-1 infection of PBMCs and stimulation of CD4+ T cells increased P-gp mRNA and P-gp activity, respectively, while primary CD4+P-gp high T cells sustained a higher HIV-1 replication than P-gp low cells. A significant correlation between HIV-1 viraemia and P-gp activity was found in different CD4+ T cell subsets, particularly memory CD4+ T cells (r = 0.792, P < 0.0001). Raltegravir is a substrate of P-gp in CD4+ T cells. Primary CD4+P-gp high T cells eliminate intracellular raltegravir more readily than P-gp low cells and HIV-1 viraemia correlates with P-gp overall activity. Specific CD4+P-gp high T cell subsets could facilitate the persistence of viral replication in vivo and ultimately promote the appearance of drug resistance
CD69 expression potentially predicts response to bendamustine and its modulation by ibrutinib or idelalisib enhances cytotoxic effect in chronic lymphocytic leukemia
Clinical responses to bendamustine in chronic lymphocytic leukemia (CLL) are highly heterogeneous and no specific markers to predict sensitivity to this drug have been reported. In order to identify biomarkers of response, we analyzed the in vitro activity of bendamustine and the gene expression profile in primary CLL cells. We observed that mRNA expression of CD69 (CD69) and ITGAM (CD11b) constitute the most powerful predictor of response to bendamustine. When we interrogated the predictive value of the corresponding cell surface proteins, the expression of the activation marker CD69 was the most reliable predictor of sensitivity to bendamustine. Importantly, a multivariate analysis revealed that the predictive value of CD69 expression was independent from other clinico-biological CLL features. We also showed that when CLL cells were co-cultured with distinct subtypes of stromal cells, an upregulation of CD69 was accompanied by a reduced sensitivity to bendamustine. In agreement with this, tumor cells derived from lymphoid tumor niches harbored higher CD69 expression and were less sensitive to bendamustine than their peripheral blood counterparts. Furthermore, pretreatment of CD69 high CLL cases with the B-cell receptor (BCR) pathway inhibitors ibrutinib and idelalisib decreased CD69 levels and enhanced bendamustine cytotoxic effect. Collectively, our findings indicate that CD69 could be a predictor of bendamustine response in CLL patients and the combination of clinically-tested BCR signaling inhibitors with bendamustine may represent a promising strategy for bendamustine low responsive CLL cases
Impact of membrane transporters polymorphisms on nucleoside-derived drug bioavailability and action = Impacte dels polimorfismes en transportadors de membrana en la biodisponibilitat i acció de fàrmacs anàlegs de nucleòsids
Nucleoside-derived drugs are used for the treatment of haematological malignancies, viral infections and autoimmune or inflammatory diseases. These drugs require membrane transporters to be internalised and be active. Transporters implicated in nucleoside and nucleoside derivatives internalisation are concentrative nucleoside transporters (CNTs) encoded by SLC28 and equilibrative nucleoside transporters (ENTs) encoded by SLC29. Other transporters such as organic cation transporters (OCTs) encoded by SLC22 have been implicated in the transport of some antiviral nucleoside derivatives, too. Nucleoside transporters as well as hOCT1 have been detected in immune cells, as well as in polarised epithelia where they are asymmetrically distributed mediating a net flux of the natural nucleoside or the derivative.
Membrane transporters can present polymorphic variants which can alter its ability to interact with the substrate. Of all studied transporters, hOCT1 is the most polymorphic, being its polymorphic variants implicated in pharmacokinetics and pharmacodynamics of some drugs used in clinics such as metformine among others.
In this thesis we show the importance polymorphic variants of hOCT1 have not only in lamivudine uptake but also in drug-drug interactions with other drugs co-administrated with lamivudine. Infected PBMCs showed an up-regulation of hOCT1 expression which would made it a better lamivudine target. We also discard the possibility that polymorphisms located at the extracellular loop might affect oligomerization.
hOCT1 has been also identified as a bendamustine transporter and hOCT1 polymorphic variants modulated bendamustine sensitivity. Bendamustine sensitivity in CLL cells ex vivo can partially be explained by the hOCT1 polymorphic variants they are carrying.
In case of DNMT inhibitors, nucleoside transporters are responsible for zebularine internalisation but not decitabine and mediate a apical-basolateral flux in polarised epithelia.
hOCT1 can mediate zebularine efflux which is impaired by the presence of polymorphic variants.
In this case polymorphic variants would confer sensitivity to zebularine treatment. In order to better understand the transporter-substrates interaction a hCNT3 model has been generated based on the recent crystallised V.cholerae nucleoside transporter. This model has been preliminary proved experimentally showing, so far, to be a good model.
In summary, this thesis highlights the importance drug transporters and their polymorphic variants play in drug bioavailability and actionLos fármacos derivados de nucleósidos son ampliamente utilizados en el tratamiento de enfermedades hematológicas, infecciones virales y enfermedades autoinmunes o inflamatorias. Estos fármacos necesitan de transportadores para ser internalizados en la célula y así, ser activos. Los transportadores implicados en la internalización de los nucleósidos y sus derivados son: los transportadores de nucleósidos concentrativos (CNTs) codificados por SLC28 y, los transportadores de nucleósidos equilibrativos (ENTs) codificados por SLC29. Otros transportadores, como los transportadores de cationes orgánicos (OCTs) codificados por SLC22, están implicados también en el transporte de algunos análogos usados en el tratamiento del SIDA. Los transportadores de nucleósdios y hOCT1 han sido detectados en células de sistema inmune, así como también, en epitelio polarizado donde se encuentran distribuidos asimétricamente mediando un flujo neto de nucleósidos y de sus derivados. Los transportadores de membrana pueden presentar polimorfismos alterando su habilidad de interacción con el sustrato. De todos los transportadores estudiados, hOCT1 es el más polimórfico, estando relacionadas sus variantes con la farmacocinética y farmacodinámica de algunos fármacos, como la metformina.
En esta tesis mostramos la importancia que tienen estas variantes polimórficas, no solamente en la internalización de lamivudina, sino también en las interacciones de ésta con otros fármacos co-administrados. La infección de PBMCs con VIH mostró un aumento en la expresión de hOCT1, convirtiéndolos en mejor diana. Se descartó también la posibilidad de que los polimorfismos localizados en el loop extracelular, pudieran afectar la oligomerización del transportador.
hOCT1 se ha identificado también como transportador de bendamustina, observándose a su vez, que sus variantes polimórficas modificaban la sensibilidad al fármaco. La sensibilidad de células de CLL ex vivo puede ser debida, en parte, a las variantes polimórficas que presentan.
En el caso de los inhibidores de la ADN metiltransferasa (DNMT), observamos que los transportadores de nucleósidos son los responsables de la captación de zebularina pero no de decitabina, mediando un flujo de esta apical-basolateral en un modelo de epitelio polarizado. hOCT1 es capaz de mediar la extrusión de zebularina, en cambio, no lo son las variantes polimórficas estudiadas. Estos resultados estarían indicando que los polimorfismos serían responsables de la sensibilidad al tratamiento con zebularina.
Para poder entender mejor la interacción transportador-sustrato, se generó un modelo preliminar de hCNT3 basado en el cristal de CNT de V. cholerae.
En resumen, esta tesis muestra la importancia que tienen los transportadores y sus variantes polimórficas, en la biodisponibilidad y acción de los fármacos
Impact of membrane transporters polymorphisms on nucleoside-derived drug bioavailability and action = Impacte dels polimorfismes en transportadors de membrana en la biodisponibilitat i acció de fàrmacs anàlegs de nucleòsids
[spa] Los fármacos derivados de nucleósidos son ampliamente utilizados en el tratamiento de enfermedades hematológicas, infecciones virales y enfermedades autoinmunes o inflamatorias. Estos fármacos necesitan de transportadores para ser internalizados en la célula y así, ser activos. Los transportadores implicados en la internalización de los nucleósidos y sus derivados son: los transportadores de nucleósidos concentrativos (CNTs) codificados por SLC28 y, los transportadores de nucleósidos equilibrativos (ENTs) codificados por SLC29. Otros transportadores, como los transportadores de cationes orgánicos (OCTs) codificados por SLC22, están implicados también en el transporte de algunos análogos usados en el tratamiento del SIDA. Los transportadores de nucleósdios y hOCT1 han sido detectados en células de sistema inmune, así como también, en epitelio polarizado donde se encuentran distribuidos asimétricamente mediando un flujo neto de nucleósidos y de sus derivados. Los transportadores de membrana pueden presentar polimorfismos alterando su habilidad de interacción con el sustrato. De todos los transportadores estudiados, hOCT1 es el más polimórfico, estando relacionadas sus variantes con la farmacocinética y farmacodinámica de algunos fármacos, como la metformina. En esta tesis mostramos la importancia que tienen estas variantes polimórficas, no solamente en la internalización de lamivudina, sino también en las interacciones de ésta con otros fármacos co-administrados. La infección de PBMCs con VIH mostró un aumento en la expresión de hOCT1, convirtiéndolos en mejor diana. Se descartó también la posibilidad de que los polimorfismos localizados en el loop extracelular, pudieran afectar la oligomerización del transportador. hOCT1 se ha identificado también como transportador de bendamustina, observándose a su vez, que sus variantes polimórficas modificaban la sensibilidad al fármaco. La sensibilidad de células de CLL ex vivo puede ser debida, en parte, a las variantes polimórficas que presentan. En el caso de los inhibidores de la ADN metiltransferasa (DNMT), observamos que los transportadores de nucleósidos son los responsables de la captación de zebularina pero no de decitabina, mediando un flujo de esta apical-basolateral en un modelo de epitelio polarizado. hOCT1 es capaz de mediar la extrusión de zebularina, en cambio, no lo son las variantes polimórficas estudiadas. Estos resultados estarían indicando que los polimorfismos serían responsables de la sensibilidad al tratamiento con zebularina. Para poder entender mejor la interacción transportador-sustrato, se generó un modelo preliminar de hCNT3 basado en el cristal de CNT de V. cholerae. En resumen, esta tesis muestra la importancia que tienen los transportadores y sus variantes polimórficas, en la biodisponibilidad y acción de los fármacos.[eng] Nucleoside-derived drugs are used for the treatment of haematological malignancies, viral infections and autoimmune or inflammatory diseases. These drugs require membrane transporters to be internalised and be active. Transporters implicated in nucleoside and nucleoside derivatives internalisation are concentrative nucleoside transporters (CNTs) encoded by SLC28 and equilibrative nucleoside transporters (ENTs) encoded by SLC29. Other transporters such as organic cation transporters (OCTs) encoded by SLC22 have been implicated in the transport of some antiviral nucleoside derivatives, too. Nucleoside transporters as well as hOCT1 have been detected in immune cells, as well as in polarised epithelia where they are asymmetrically distributed mediating a net flux of the natural nucleoside or the derivative. Membrane transporters can present polymorphic variants which can alter its ability to interact with the substrate. Of all studied transporters, hOCT1 is the most polymorphic, being its polymorphic variants implicated in pharmacokinetics and pharmacodynamics of some drugs used in clinics such as metformine among others. In this thesis we show the importance polymorphic variants of hOCT1 have not only in lamivudine uptake but also in drug-drug interactions with other drugs co-administrated with lamivudine. Infected PBMCs showed an up-regulation of hOCT1 expression which would made it a better lamivudine target. We also discard the possibility that polymorphisms located at the extracellular loop might affect oligomerization. hOCT1 has been also identified as a bendamustine transporter and hOCT1 polymorphic variants modulated bendamustine sensitivity. Bendamustine sensitivity in CLL cells ex vivo can partially be explained by the hOCT1 polymorphic variants they are carrying. In case of DNMT inhibitors, nucleoside transporters are responsible for zebularine internalisation but not decitabine and mediate a apical-basolateral flux in polarised epithelia. hOCT1 can mediate zebularine efflux which is impaired by the presence of polymorphic variants. In this case polymorphic variants would confer sensitivity to zebularine treatment. In order to better understand the transporter-substrates interaction a hCNT3 model has been generated based on the recent crystallised V.cholerae nucleoside transporter. This model has been preliminary proved experimentally showing, so far, to be a good model. In summary, this thesis highlights the importance drug transporters and their polymorphic variants play in drug bioavailability and actio
Role of human Organic Cation Transporter 1 (hOCT1) polymorphisms in lamivudine (3TC) uptake and drug-drug interactions.
Lamivudine (3TC), a drug used in the treatment of HIV infection, needs to cross the plasma membrane to exert its therapeutic action. Human Organic cation transporter 1 (hOCT1), encoded by the SLC22A1 gene, is the transporter responsible for its uptake into target cells. As SLC22A1 is a highly polymorphic gene, the aim of this study was to determine how SNPs in the OCT1-encoding gene affected 3TC internalization and its interaction with other co-administered drugs. HEK293 cells stably transfected with either the wild type form or the polymorphic variants of hOCT1 were used to perform kinetic and drug-drug interaction studies. Protein co-immunoprecipitation was used to assess the impact of selected polymorphic cysteines on the oligomerization of the transporter. Results showed that 3TC transport efficiency was reduced in all polymorphic variants tested (R61C, C88R, S189L, M420del, and G465R). This was not caused by lack of oligomerization in case of variants located at the transporter extracellular loop (R61C and C88R). Drug-drug interaction measurements showed that co-administered drugs [abacavir (ABC), zidovudine (AZT), emtricitabine (FTC), tenofovir diproxil fumarate (TDF), efavirenz (EFV) and raltegravir (RAL)], differently inhibited 3TC uptake depending upon the polymorphic variant analyzed. These data highlight the need for accurate analysis of drug transporter polymorphic variants of clinical relevance, because polymorphisms can impact on substrate (3TC) translocation but even more importantly they can differentially affect drug-drug interactions at the transporter level
P-glycoprotein ( ABCB1
Altres ajuts: SAF2014-25560-RTo evaluate the role of P-glycoprotein (P-gp) and multidrug-resistant-protein 1 (MRP1) on raltegravir intracellular drug disposition in CD4+ T cells, investigate the effect of HIV-1 infection on P-gp expression and correlate HIV-1 viraemia with P-gp activity in primary CD4+ T cell subsets. The cellular accumulation ratio of [ 3 H]raltegravir was quantified in CD4+ T cell lines overexpressing either P-gp (CEM-P-gp) or MRP1 (CEM-MRP1) and in primary CD3+CD4+ T cells with high (P-gp high) and low P-gp activity (P-gp low); inhibition of efflux transporters was confirmed by the intracellular retention of calcein-AM. The correlation of P-gp activity with HIV-1 viraemia was assessed in naive and memory T cell subsets from 21 HIV-1-infected treatment-naive subjects. [ 3 H]Raltegravir cellular accumulation ratio decreased in CEM-P-gp cells (P < 0.0001). XR9051 (a P-gp inhibitor) and HIV-1 PIs reversed this phenomenon. Primary CD4+P-gp high cells accumulated less raltegravir (38.4% ± 9.6%) than P-gp low cells, whereas XR9051 also reversed this effect. In vitro HIV-1 infection of PBMCs and stimulation of CD4+ T cells increased P-gp mRNA and P-gp activity, respectively, while primary CD4+P-gp high T cells sustained a higher HIV-1 replication than P-gp low cells. A significant correlation between HIV-1 viraemia and P-gp activity was found in different CD4+ T cell subsets, particularly memory CD4+ T cells (r = 0.792, P < 0.0001). Raltegravir is a substrate of P-gp in CD4+ T cells. Primary CD4+P-gp high T cells eliminate intracellular raltegravir more readily than P-gp low cells and HIV-1 viraemia correlates with P-gp overall activity. Specific CD4+P-gp high T cell subsets could facilitate the persistence of viral replication in vivo and ultimately promote the appearance of drug resistance
Role of Human Organic Cation Transporter 1 (hOCT1) Polymorphisms in Lamivudine (3TC) Uptake and Drug-Drug Interactions
Altres ajuts: This study was also supported by Deutsche Forschungsgemeinschaft Grant KO 872/6-1 to HK. CA was recipient of predoctoral fellowships FPI from Ministerio de Ciencia e Innovación.Lamivudine (3TC), a drug used in the treatment of HIV infection, needs to cross the plasma membrane to exert its therapeutic action. Human Organic cation transporter 1 (hOCT1), encoded by the SLC22A1 gene, is the transporter responsible for its uptake into target cells. As SLC22A1 is a highly polymorphic gene, the aim of this study was to determine how SNPs in the OCT1-encoding gene affected 3TC internalization and its interaction with other co-administered drugs. HEK293 cells stably transfected with either the wild type form or the polymorphic variants of hOCT1 were used to perform kinetic and drug-drug interaction studies. Protein co-immunoprecipitation was used to assess the impact of selected polymorphic cysteines on the oligomerization of the transporter. Results showed that 3TC transport efficiency was reduced in all polymorphic variants tested (R61C, C88R, S189L, M420del, and G465R). This was not caused by lack of oligomerization in case of variants located at the transporter extracellular loop (R61C and C88R). Drug-drug interaction measurements showed that co-administered drugs [abacavir (ABC), zidovudine (AZT), emtricitabine (FTC), tenofovir diproxil fumarate (TDF), efavirenz (EFV) and raltegravir (RAL)], differently inhibited 3TC uptake depending upon the polymorphic variant analyzed. These data highlight the need for accurate analysis of drug transporter polymorphic variants of clinical relevance, because polymorphisms can impact on substrate (3TC) translocation but even more importantly they can differentially affect drug-drug interactions at the transporter level
A service-learning experience in secondary education to promote STEM learning through collaboration between research and education centers
The needs of the digital revolution and the knowledge-based economy impose a transformation of traditional education to improve technical and scientific knowledge and include alternative abilities. This work presents a service-learning initiative with multiple goals: to improve scientific, technical, engineering, and mathematic (STEM) knowledge at school, to enhance students’ engagement, and to make people aware of the repercussions of poor indoor air quality. The initiative involves four actors: the administration which steers the initiative, students who are the receivers of the knowledge, teachers as facilitators of the activity and research centers as expert references. Within this service-learning initiative, a real-life project has been proposed, focusing on measuring carbon dioxide (CO2) levels in classrooms and correlating them with indoor airborne infection transmission. Reference experts have developed two systems provided to teachers and students for project implementation. The project involves an engineering step where students set up the systems, and a scientific step where students hypothesize, develop experiments, analyse data, and communicate results gaining experience with the scientific method. Through the combined efforts and appropriate allocation of responsibilities, this experience has yielded excellent results in STEM knowledge transmission and has proven effective in fostering student commitment to their learning process