RESUMO
Dabrafenib is a BRAF inhibitor used in combination treatment of malignant melanoma and non-small cell lung carcinoma. In this study, we aimed to characterize its interactions with cytochrome P450 (CYP) isoenzymes and ATP-binding cassette (ABC) efflux transporters that have critical impact on the pharmacokinetics of drugs and play a role in drug resistance development. Using accumulation assays, we showed that dabrafenib inhibited ABCG2 and, less potently, ABCB1 transporter. We also confirmed dabrafenib as a CYP2C8, CYP2C9, CYP3A4, and CYP3A5 inhibitor. Importantly, inhibition of ABCG2 and CYP3A4 by dabrafenib led to the potentiation of cytotoxic effects of mitoxantrone and docetaxel toward respective resistant cell lines in drug combination studies. On the contrary, the synergistic effect was not consistently observed in ABCB1-expressing models. We further demonstrated that mRNA levels of ABCB1, ABCG2, ABCC1, and CYP3A4 were increased after 24 h and 48 h exposure to dabrafenib. Overall, our data confirm dabrafenib as a drug frequently and potently interacting with ABC transporters and CYP isoenzymes. This feature should be addressed with caution when administering dabrafenib to patients with polypharmacy but also could be utilized advantageously when designing new dabrafenib-containing drug combinations to improve the therapeutic outcome in drug-resistant cancer.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/farmacologia , Inibidores das Enzimas do Citocromo P-450/farmacologia , Daunorrubicina/farmacologia , Imidazóis/farmacocinética , Mitoxantrona/farmacologia , Oximas/farmacocinética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Inibidores das Enzimas do Citocromo P-450/administração & dosagem , Daunorrubicina/administração & dosagem , Cães , Quimioterapia Combinada , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/administração & dosagem , Mitoxantrona/administração & dosagem , Oximas/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Methyl mercury (MeHg) is an organic highly toxic compound that is transported efficiently via the human placenta. Our previous data suggest that MeHg is taken up into placental cells by amino acid transporters while mercury export from placental cells mainly involves ATP binding cassette (ABC) transporters. We hypothesized that the ABC transporter multidrug resistance-associated protein (MRP)1 (ABCC1) plays an essential role in mercury export from the human placenta. Transwell transport studies with MRP1-overexpressing Madin-Darby Canine Kidney (MDCK)II cells confirmed the function of MRP1 in polarized mercury efflux. Consistent with this, siRNA-mediated MRP1 gene knockdown in the human placental cell line HTR-8/SVneo resulted in intracellular mercury accumulation, which was associated with reduced cell viability, accompanied by increased cytotoxicity, apoptosis, and oxidative stress as determined via the glutathione (GSH) status. In addition, the many sources claiming different localization of MRP1 in the placenta required a re-evaluation of its localization in placental tissue sections by immunofluorescence microscopy using an MRP1-specific antibody that was validated in-house. Taken together, our results show that (1) MRP1 preferentially mediates apical-to-basolateral mercury transport in epithelial cells, (2) MRP1 regulates the GSH status of placental cells, (3) MRP1 function has a decisive influence on the viability of placental cells exposed to low MeHg concentrations, and (4) the in situ localization of MRP1 corresponds to mercury transport from maternal circulation to the placenta and fetus. We conclude that MRP1 protects placental cells from MeHg-induced oxidative stress by exporting the toxic metal and by maintaining the placental cells' GSH status in equilibrium.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Glutationa/metabolismo , Compostos de Metilmercúrio/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Estresse Oxidativo , Placenta/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cães , Células Endoteliais , Feminino , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Células Madin Darby de Rim Canino , Compostos de Metilmercúrio/efeitos adversos , GravidezRESUMO
Maraviroc is a chemokine receptor 5 (CCR5) inhibitor used in the treatment of human immunodeficiency virus (HIV) that also shows therapeutic potential for several autoimmune, cancer, and inflammatory diseases that can afflict pregnant women. However, only limited information exists on the mechanisms underlying the transplacental transfer of the drug. We aimed to expand the current knowledge base on how maraviroc interacts with several placental ATP-binding cassette (ABC) efflux transporters that have a recognized role in the protection of a developing fetus: P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), and multidrug resistance protein 2 (ABCC2). We found that maraviroc does not inhibit any of the three studied ABC transporters and that its permeability is not affected by ABCG2 or ABCC2. However, our in vitro results revealed that maraviroc shows affinity for human ABCB1 and the endogenous canine P-glycoprotein (Abcb1) expressed in Madin-Darby canine kidney II (MDCKII) cells. Perfusion of rat term placenta showed accelerated transport of maraviroc in the fetal-to-maternal direction, which suggests that ABCB1/Abcb1 facilitates in situ maraviroc transport. This transplacental transport was saturable and significantly diminished after the addition of the ABCB1/Abcb1 inhibitors elacridar, zosuquidar, and ritonavir. Our results indicate that neither ABCG2 nor ABCC2 influence maraviroc pharmacokinetic but that ABCB1/Abcb1 may be partly responsible for the decreased transplacental permeability of maraviroc to the fetus. The strong affinity of maraviroc to Abcb1 found in our animal models necessitates studies in human tissue so that maraviroc pharmacokinetics in pregnant women can be fully understood. SIGNIFICANCE STATEMENT: Antiretroviral drug maraviroc shows low toxicity and is thus a good candidate for prevention of mother-to-child transmission of human immunodeficiency virus when failure of recommended therapy occurs. Using in vitro cell-based experiments and in situ dually perfused rat term placenta, we examined maraviroc interaction with the placental ABC drug transporters ABCB1, ABCG2, and ABCC2. We demonstrate for the first time that placental ABCB1 significantly reduces mother-to-fetus transport of maraviroc, which suggests that ABCB1 may be responsible for the low cord-blood/maternal-blood ratio observed in humans.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Antagonistas dos Receptores CCR5/farmacocinética , Maraviroc/farmacocinética , Troca Materno-Fetal , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Animais , Antagonistas dos Receptores CCR5/uso terapêutico , Cães , Feminino , Feto/metabolismo , Infecções por HIV/tratamento farmacológico , Humanos , Células Madin Darby de Rim Canino , Maraviroc/uso terapêutico , Modelos Animais , Proteína 2 Associada à Farmacorresistência Múltipla , Permeabilidade , Placenta/metabolismo , Circulação Placentária , Gravidez , Complicações Infecciosas na Gravidez/tratamento farmacológico , RatosRESUMO
Alectinib is a tyrosine kinase inhibitor currently used as a first-line treatment of anaplastic lymphoma kinase-positive metastatic nonsmall cell lung cancer (NSCLC). In the present work, we investigated possible interactions of this novel drug with ATP-binding cassette (ABC) drug efflux transporters and cytochrome P450 (P450) biotransformation enzymes that play significant roles in the phenomenon of multidrug resistance (MDR) of cancer cells as well as in pharmacokinetic drug-drug interactions. Using accumulation studies in Madin-Darby canine kidney subtype 2 (MDCKII) cells alectinib was identified as an inhibitor of ABCB1 and ABCG2 but not of ABCC1. In subsequent drug combination studies, we demonstrated the ability for alectinib to effectively overcome MDR in ABCB1- and ABCG2-overexpressing MDCKII and A431 cells. To describe the pharmacokinetic interaction profile of alectinib in a complete fashion, its possible inhibitory properties toward clinically relevant P450 enzymes (i.e., CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, or CYP3A5) were evaluated using human P450-expressing insect microsomes, revealing alectinib as a poor interactor. Advantageously for its use in pharmacotherapy, alectinib further exhibited negligible potential to cause any changes in expression of ABCB1, ABCG2, ABCC1, CYP1A2, CYP3A4, and CYP2B6 in intestine, liver, and NSCLC models. Our in vitro observations might serve as a valuable foundation for future in vivo studies that could support the rationale for our conclusions and possibly enable providing more efficient and safer therapy to many oncological patients.
Assuntos
Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Carbazóis/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Biotransformação , Carbazóis/farmacocinética , Cães , Humanos , Células Madin Darby de Rim Canino , Piperidinas/farmacocinética , Inibidores de Proteínas Quinases/farmacocinéticaRESUMO
In spite of remarkable reduction in the number of children born with HIV due to antiretroviral therapy, concerns remain on the short- and long-term effects of antiretroviral drugs at the feto-placental unit. Cardio- and skeletal myopathies have been reported in children exposed to antiretroviral drugs prenatally. These conditions have also been described in perturbed placental transfer of l-carnitine, an essential co-factor in fatty acid oxidation. Due to limited fetal and placental synthesis, carnitine supply is maintained through the placental carnitine uptake from maternal blood by the organic cation/carnitine transporters OCTN1 and OCTN2 (SLC22A4 and SLC22A5, respectively). The aim of our study was to investigate potential inhibition of placental carnitine uptake by a broad range of antiretroviral drugs comprising nucleoside/nucleotide reverse transcriptase inhibitors (lamivudine, zidovudine, abacavir, tenofovir disoproxil fumarate), non-nucleoside reverse transcriptase inhibitors (rilpivirine, efavirenz, etravirine), protease inhibitors (ritonavir, lopinavir, atazanavir, saquinavir, tipranavir), integrase inhibitors (raltegravir, dolutegravir, elvitegravir) and viral entry inhibitor, maraviroc. Studies in choriocarcinoma BeWo cells and human placenta-derived models confirmed predominant expression and function of OCTN2 above OCTN1 in l-carnitine transport. Subsequent screenings in BeWo cells and isolated MVM vesicles revealed seven antiretroviral drugs as inhibitors of the Na+-dependent l-carnitine uptake, corresponding to OCTN2. Ritonavir, saquinavir and elvitegravir showed the highest inhibitory potential which was further confirmed for ritonavir and saquinavir in placental fresh villous fragments. Our data indicate possible impairment in placental and fetal supply of l-carnitine with ritonavir and saquinavir, while suggesting retained placental carnitine transport with the other antiretroviral drugs.
Assuntos
Antirretrovirais/toxicidade , Carnitina/metabolismo , Placenta/efeitos dos fármacos , Membro 5 da Família 22 de Carreadores de Soluto/antagonistas & inibidores , Transporte Biológico , Linhagem Celular Tumoral , Feminino , Humanos , Exposição Materna/efeitos adversos , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Placenta/metabolismo , Gravidez , Medição de Risco , Membro 5 da Família 22 de Carreadores de Soluto/metabolismo , SimportadoresRESUMO
Brivanib, a promising tyrosine kinase inhibitor, is currently undergoing advanced stages of clinical evaluation for solid tumor therapy. In this work, we investigated possible interactions of this novel drug candidate with ABC drug efflux transporters and cytochrome P450 (CYP450) drug-metabolizing enzymes that participate in cancer multidrug resistance (MDR) and pharmacokinetic drug-drug interactions (DDIs). First, in accumulation experiments with various model substrates, we identified brivanib as an inhibitor of the ABCB1, ABCG2, and ABCC1 transporters. However, in subsequent combination studies employing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide proliferation assays in both Madin-Darby canine kidney II (MDCKII) and A431 cellular models, only ABCG2 inhibition was revealed to be able to synergistically potentiate mitoxantrone effects. Advantageous to its possible use as MDR antagonist, brivanib's chemosensitizing properties were not impaired by activity of any of the MDR-associated ABC transporters, as observed in comparative viability assay in the MDCKII cell sublines. In incubation experiments with eight recombinant CYP450s, we found that brivanib potently inhibited CYP2C subfamily members and the CYP2B6 isoform. Finally, in induction studies, we demonstrated that brivanib upregulated ABCB1 and CYP1A2 messenger RNA levels in systemic cell models, although this interaction was not significantly manifested at a functional level. In conclusion, brivanib exhibits potential to cause clinically relevant pharmacokinetic DDIs and act as a modulator of ABCG2-mediated MDR. Our findings might be used as an important background for subsequent in vivo investigations and pave the way for the safe and effective use of brivanib in oncological patients.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Alanina/análogos & derivados , Biotransformação/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas/fisiologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Triazinas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Alanina/farmacologia , Animais , Linhagem Celular , Inibidores das Enzimas do Citocromo P-450/farmacologia , Cães , Humanos , Células Madin Darby de Rim CaninoRESUMO
Abacavir is a preferred antiretroviral drug for preventing mother-to-child human immunodeficiency virus transmission; however, mechanisms of its placental transfer have not been satisfactorily described to date. Because abacavir is a nucleoside-derived drug, we hypothesized that the nucleoside transporters, equilibrative nucleoside transporters (ENTs, SLC29A) and/or Na+-dependent concentrative nucleoside transporters (CNTs, SLC28A), may play a role in its passage across the placenta. To test this hypothesis, we performed uptake experiments using the choriocarcinoma-derived BeWo cell line, human fresh villous fragments, and microvillous plasma membrane (MVM) vesicles. Using endogenous substrates of nucleoside transporters, [3H]-adenosine (ENTs, CNT2, and CNT3) and [3H]-thymidine (ENTs, CNT1, and CNT3), we showed significant activity of ENT1 and CNT2 in BeWo cells, whereas experiments in the villous fragments and MVM vesicles, representing a model of the apical membrane of a syncytiotrophoblast, revealed only ENT1 activity. When testing [3H]-abacavir uptakes, we showed that of the nucleoside transporters, ENT1 plays the dominant role in abacavir uptake into placental tissues, whereas contribution of Na+-dependent transport, most likely mediated by CNTs, was observed only in BeWo cells. Subsequent experiments with dually perfused rat term placentas showed that Ent1 contributes significantly to overall [3H]-abacavir placental transport. Finally, we quantified the expression of SLC29A in first- and third-trimester placentas, revealing that SLC29A1 is the dominant isoform. Neither SLC29A1 nor SLC29A2 expression changed over the course of placental development, but there was considerable interindividual variability in their expression. Therefore, drug-drug interactions and the effect of interindividual variability in placental ENT1 expression on abacavir disposition into fetal circulation should be further investigated to guarantee safe and effective abacavir-based combination therapies in pregnancy.
Assuntos
Fármacos Anti-HIV/metabolismo , Didesoxinucleosídeos/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Proteínas de Transporte de Nucleosídeos/metabolismo , Placenta/metabolismo , Adenosina/metabolismo , Animais , Transporte Biológico/fisiologia , Linhagem Celular Tumoral , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , Feminino , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Nucleosídeos/metabolismo , Gravidez , Ratos , Ratos WistarRESUMO
Rilpivirine (TMC278) is a highly potent nonnucleoside reverse transcriptase inhibitor (NNRTI) representing an effective component of combination antiretroviral therapy (cART) in the treatment of HIV-positive patients. Many antiretroviral drugs commonly used in cART are substrates of ATP-binding cassette (ABC) and/or solute carrier (SLC) drug transporters and, therefore, are prone to pharmacokinetic drug-drug interactions (DDIs). The aim of our study was to evaluate rilpivirine interactions with abacavir and lamivudine on selected ABC and SLC transporters in vitro and assess its importance for pharmacokinetics in vivo Using accumulation assays in MDCK cells overexpressing selected ABC or SLC drug transporters, we revealed rilpivirine as a potent inhibitor of MDR1 and BCRP, but not MRP2, OCT1, OCT2, or MATE1. Subsequent transport experiments across monolayers of MDCKII-MDR1, MDCKII-BCRP, and Caco-2 cells demonstrated that rilpivirine inhibits MDR1- and BCRP-mediated efflux of abacavir and increases its transmembrane transport. In vivo experiments in male Wistar rats confirmed inhibition of MDR1/BCRP in the small intestine, leading to a significant increase in oral bioavailability of abacavir. In conclusion, rilpivirine inhibits MDR1 and BCRP transporters and may affect pharmacokinetic behavior of concomitantly administered substrates of these transporters, such as abacavir.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Didesoxinucleosídeos/metabolismo , Interações Medicamentosas/fisiologia , Absorção Intestinal/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Rilpivirina/metabolismo , Animais , Transporte Biológico/fisiologia , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Didesoxinucleosídeos/farmacologia , Cães , Humanos , Lamivudina/metabolismo , Lamivudina/farmacologia , Células Madin Darby de Rim Canino , Masculino , Ratos , Ratos Wistar , Inibidores da Transcriptase Reversa/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Rilpivirina/farmacologiaRESUMO
1. Emtricitabine is a nucleoside reverse transcriptase inhibitor used in combination antiretroviral therapy of HIV (cART). Although active transport mechanisms are believed to mediate tubular secretion of the drug into urine, the responsible transporter and its potential to cause pharmacokinetic drug--drug interactions (DDI) has not been identified so far. The aim of this study was to investigate whether drug transporters P-gp (ABCB1), BCRP (ABCG2), MRP2 (ABCC2), OCT1 (SLC22A1), OCT2 (SLC22A2) or MATE1 (SLC47A1) can mediate active transcellular transfer of emtricitabine. 2. We employed transport assays in polarized monolayers of MDCK cells stably expressing P-gp, BCRP, MRP2, OCT1, OCT2 and/or MATE1. Among the transporters studied only MATE1 accelerated basal-to-apical transport of emtricitabine over a wide range of concentrations (6 nM to 1 mM). The transport was enhanced by an oppositely directed pH gradient and significantly reduced (p < 0.001) at low temperature in MDCK-MATE1, MDCK-OCT1/MATE1 and MDCK-OCT2/MATE1 cells. Co-administration of cimetidine or ritonavir decreased MATE1-mediated transport of emtricitabine by up to 42 and 39%, respectively (p < 0.01) and augmented intracellular accumulation of emtricitabine (p < 0.05). 3. We demonstrate emtricitabine as a substrate of MATE1 and suggest that MATE1 might cause DDI between emtricitabine and other co-administrated drugs including antiretrovirals.
Assuntos
Emtricitabina/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Inibidores da Transcriptase Reversa/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Cães , Células Madin Darby de Rim CaninoRESUMO
Lamivudine is one of the antiretroviral drugs of choice for the prevention of mother-to-child transmission (MTCT) in HIV-positive women. In this study, we investigated the relevance of drug efflux transporters P-glycoprotein (P-gp) (MDR1 [ABCB1]), BCRP (ABCG2), MRP2 (ABCC2), and MATE1 (SLC47A1) for the transmembrane transport and transplacental transfer of lamivudine. We employed in vitro accumulation and transport experiments on MDCK cells overexpressing drug efflux transporters, in situ-perfused rat term placenta, and vesicular uptake in microvillous plasma membrane (MVM) vesicles isolated from human term placenta. MATE1 significantly accelerated lamivudine transport in MATE1-expressing MDCK cells, whereas no transporter-driven efflux of lamivudine was observed in MDCK-MDR1, MDCK-MRP2, and MDCK-BCRP monolayers. MATE1-mediated efflux of lamivudine appeared to be a low-affinity process (apparent Km of 4.21 mM and Vmax of 5.18 nmol/mg protein/min in MDCK-MATE1 cells). Consistent with in vitro transport studies, the transplacental clearance of lamivudine was not affected by P-gp, BCRP, or MRP2. However, lamivudine transfer across dually perfused rat placenta and the uptake of lamivudine into human placental MVM vesicles revealed pH dependency, indicating possible involvement of MATE1 in the fetal-to-maternal efflux of the drug. To conclude, placental transport of lamivudine does not seem to be affected by P-gp, MRP2, or BCRP, but a pH-dependent mechanism mediates transport of lamivudine in the fetal-to-maternal direction. We suggest that MATE1 might be, at least partly, responsible for this transport.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Lamivudina/metabolismo , Placenta/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Cães , Feminino , Humanos , Células Madin Darby de Rim Canino , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Gravidez , Ratos , Ratos WistarRESUMO
1. Purine cyclin-dependent kinase inhibitors have recently been recognised as promising candidates for the treatment of various cancers. While pharmacodynamic properties of these compounds are relatively well understood, their pharmacokinetics including possible interactions with placental transport systems have not been characterised to date. 2. In this study, we investigated transplacental passage of olomoucine II and purvalanol A in rat focusing on possible role of p-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and/or multidrug resistance-associated proteins (ABCCs). Employing the in situ method of dually perfused rat term placenta, we demonstrate transplacental passage of both olomoucine II and purvalanol A against the concentration gradient in foetus-to-mother direction. Using several ATP-binding cassette (ABC) drug transporter inhibitors, we confirm the participation of ABCB1, ABCG2 and ABCCs transporters in the placental passage of olomoucine II, but not purvalanol A. 3. Transplacental passage of olomoucine II and purvalanol A from mother to foetus is significantly reduced by active transporters, restricting thereby foetal exposure and providing protection against harmful effects of these xenobiotics. Importantly, we demonstrate that in spite of their considerable structural similarity, the two molecules utilise distinct placental transport systems. These facts should be kept in mind when introducing these prospective anticancer candidates and/or their analogues into the clinical area.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Placenta/metabolismo , Purinas/farmacocinética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Trifosfato de Adenosina/química , Animais , Transporte Biológico Ativo , Cromatografia Líquida de Alta Pressão , Feminino , Exposição Materna , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Placenta/efeitos dos fármacos , Gravidez , Prenhez , Purinas/administração & dosagem , Ratos , Ratos Wistar , Roscovitina , Trofoblastos/efeitos dos fármacos , Xenobióticos/químicaRESUMO
Zidovudine (AZT) is one of the most frequently used antiretroviral drugs in prevention of perinatal transmission of HIV. However, safety concerns on AZT use in pregnancy still persist as severe side effects are associated with AZT exposure in children. In our study we aimed to contribute to current knowledge on AZT transplacental transport and to evaluate potential involvement of the main human drug efflux ATP-binding cassette (ABC) transporters, p-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and multidrug resistance-associated proteins 2 and 5 (ABCC2 and ABCC5) in the disposition of AZT between mother and fetus. In order to elucidate this issue we investigated the effect of selected ABC transporters on AZT transepithelial transport across MDCKII cell monolayers. In addition we used the in situ method of dually perfused rat term placenta to further study the role of ABC transporters in AZT transplacental transport. In vitro studies revealed significant effect of ABCB1 and ABCG2 on AZT transport which was subsequently confirmed also on organ level. Lamivudine, an antiretroviral agent commonly co-administered with AZT, did not affect ABC transporter-mediated AZT transfer.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Fármacos Anti-HIV/farmacocinética , Placenta/metabolismo , Zidovudina/farmacocinética , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Acridinas/farmacologia , Animais , Cães , Interações Medicamentosas , Feminino , Técnicas In Vitro , Indometacina/farmacologia , Lamivudina/farmacologia , Células Madin Darby de Rim Canino , Proteína 2 Associada à Farmacorresistência Múltipla , Gravidez , Ratos Wistar , Tetra-Hidroisoquinolinas/farmacologiaRESUMO
Boldine, the major alkaloid from the Chilean Boldo tree, is used in traditional medicine to support bile production, but evidence to support this function is controversial. We analyzed the choleretic potential of boldine, including its molecular background. The acute- and long-term effects of boldine were evaluated in rats either during intravenous infusion or after 28-day oral treatment. Infusion of boldine instantly increased the bile flow 1.4-fold in healthy rats as well as in animals with Mrp2 deficiency or ethinylestradiol induced cholestasis. This effect was not associated with a corresponding increase in bile acid or glutathione biliary excretion, indicating that the effect is not related to stimulation of either bile acid dependent or independent mechanisms of bile formation and points to the osmotic activity of boldine itself. We subsequently analyzed bile production under conditions of changing biliary excretion of boldine after bolus intravenous administration and found strong correlations between both parameters. HPLC analysis showed that bile concentrations of boldine above 10 µM were required for induction of choleresis. Importantly, long-term pretreatment, when the bile collection study was performed 24-h after the last administration of boldine, also accelerated bile formation despite undetectable levels of the compound in bile. The effect paralleled upregulation of the Bsep transporter and increased biliary clearance of its substrates, bile acids. We consequently confirmed the ability of boldine to stimulate the Bsep transcriptional regulator, FXR receptor. In conclusion, our study clarified the mechanisms and circumstances surrounding the choleretic activity of boldine.
Assuntos
Aporfinas/farmacologia , Bile/metabolismo , Colagogos e Coleréticos/farmacologia , Fígado/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/agonistas , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Administração Oral , Animais , Aporfinas/administração & dosagem , Aporfinas/metabolismo , Colagogos e Coleréticos/administração & dosagem , Colagogos e Coleréticos/metabolismo , Cães , Etinilestradiol/farmacologia , Feminino , Glutationa/metabolismo , Células Hep G2 , Eliminação Hepatobiliar , Humanos , Infusões Intravenosas , Cinética , Fígado/metabolismo , Células Madin Darby de Rim Canino , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Osmose , Ratos Endogâmicos Lew , Ratos Transgênicos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transfecção , Regulação para CimaRESUMO
The tetratricopeptide repeat (TPR) structural motif is known to occur in a wide variety of proteins present in prokaryotic and eukaryotic organisms. The TPR motif represents an elegant module for the assembly of various multiprotein complexes, and thus, TPR-containing proteins often play roles in vital cell processes. As the TPR profile is well defined, the complete TPR protein repertoire of a bacterium with a known genomic sequence can be predicted. This provides a tremendous opportunity for investigators to identify new TPR-containing proteins and study them in detail. In the past decade, TPR-containing proteins of bacterial pathogens have been reported to be directly related to virulence-associated functions. In this minireview, we summarize the current knowledge of the TPR-containing proteins involved in virulence mechanisms of bacterial pathogens while highlighting the importance of TPR motifs for the proper functioning of class II chaperones of a type III secretion system in the pathogenesis of Yersinia, Pseudomonas, and Shigella.
Assuntos
Motivos de Aminoácidos/genética , Bactérias/patogenicidade , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , VirulênciaRESUMO
In our previous study, we described synchronized activity of organic cation transporter 3 (OCT3/SLC22A3) and multidrug and toxin extrusion 1 (MATE1/SLC47A1) transporter in the passage of organic cations across the rat placenta and the role of these transporters in fetal defense; in this study, we hypothesized that changes in placental levels of OCT3 and MATE1 throughout gestation might affect the fetal protection and detoxification. Using quantitative RT-PCR, Western blot analysis, and immunohistochemistry, we were able to detect Oct3/OCT3 and Mate1/MATE1 expression in the rat placenta as early as on Gestation Day (gd) 12 with increasing tendency toward the end of pregnancy. Comparing first versus third trimester human placenta, we observed stable expression of OCT1 and decreasing expression of OCT2 and OCT3 isoforms. Contrary to the current literature, we were able to detect also MATE1/MATE2 isoforms in the human placenta, however, with considerable inter- and intraindividual variability. Using infusion of 1-methyl-4-phenylpyridinium (MPP(+)), a substrate of OCT and MATE transporters, into pregnant dams, we investigated the protective function of the placenta against organic cations at different gds. The highest amount of MPP(+) reached the fetus on gd 12 while from gd 15 onward, maternal-to-fetal transport of MPP(+) decreased significantly. We conclude that increased expression of placental OCT3 and MATE1 along with general maturation of the placental tissues results in significantly lower transport of MPP(+) from mother to fetus. In contrast, decreasing expression of OCT3 and MATE1 in human placenta indicates these transporters may play a role in fetal protection preferentially at earlier stages of gestation.
Assuntos
Antiporters/metabolismo , Feto/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Placenta/metabolismo , Animais , Western Blotting , Feminino , Desenvolvimento Fetal , Humanos , Imuno-Histoquímica , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
Inhibition of cyclin-dependent kinases by specific small molecules, purine cyclin-dependent kinase inhibitors (CDKi), has become a promising strategy for cancer treatment. Although pharmacodynamic properties of these compounds have been studied extensively, their pharmacokinetic behavior has not been addressed in detail. In this study, we investigated possible inhibitory effect of five purine CDKi on breast cancer resistance protein (ABCG2) transport activity employing in vitro transport and accumulation methods in MCDKII cells transduced with human ABCG2. Hoechst 33342 and glyburide were used as model ABCG2 substrates for these experiments. In addition, in situ method of dually perfused rat term placenta was utilized to confirm our in vitro results at the organ level. Fumitremorgin C was used as a model inhibitor of ABCG2 for comparison purposes. We demonstrate significant inhibition of ABCG2 by four of the five CDKi tested. Regarding their ABCG2-inhibitory potencies, the investigated compounds can be ranked as follows: purvalanol A>olomoucine II≈fumitremorgin C>roscovitine≈bohemine, with slight differences among substrates, concentrations and methods used. Based on our findings, it is reasonable to expect a substantial impact of the studied CDKi on the pharmacokinetic and pharmacodynamic behavior of concomitantly administered ABCG2 substrates. Moreover, using combination index method of Chou-Talalay, we confirmed that the strongest inhibitors, purvalanol A and olomoucine II, can synergistically potentiate cytostatic effect of mitoxantrone, an ABCG2 substrate, in ABCG2 expressing cell lines.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Citostáticos/farmacologia , Mitoxantrona/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Purinas/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Benzimidazóis/metabolismo , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Glibureto/metabolismo , Humanos , Proteínas de Neoplasias/metabolismoRESUMO
Low curability of patients diagnosed with acute myeloid leukemia (AML) must be seen as a call for better understanding the disease's mechanisms and improving the treatment strategy. Therapeutic outcome of the crucial anthracycline-based induction therapy often can be compromised by a resistant phenotype associated with overexpression of ABCB1 transporters. Here, we evaluated clinical relevance of ABCB1 in a context of the FMS-like tyrosine kinase 3 (FLT3) inhibitor midostaurin in a set of 28 primary AML samples. ABCB1 gene expression was absolutely quantified, confirming its association with CD34 positivity, adverse cytogenetic risk, and unachieved complete remission (CR). Midostaurin, identified as an ABCB1 inhibitor, increased anthracycline accumulation in peripheral blood mononuclear cells (PBMC) of CD34+ AML patients and those not achieving CR. This effect was independent of FLT3 mutation, indicating even FLT3- AML patients might benefit from midostaurin therapy. In line with these data, midostaurin potentiated proapoptotic processes in ABCB1-overexpressing leukemic cells when combined with anthracyclines. Furthermore, we report a direct linkage of miR-9 to ABCB1 efflux activity in the PBMC and propose miR-9 as a useful prognostic marker in AML. Overall, we highlight the therapeutic value of midostaurin as more than just a FLT3 inhibitor, suggesting its maximal therapeutic outcomes might be very sensitive to proper timing and well-optimized dosage schemes based upon patient's characteristics, such as CD34 positivity and ABCB1 activity. Moreover, we suggest miR-9 as a predictive ABCB1-related biomarker that could be immensely helpful in identifying ABCB1-resistant AML phenotype to enable optimized therapeutic regimen and improved treatment outcome.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP , Leucemia Mieloide Aguda , MicroRNAs , Estaurosporina , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Antraciclinas/farmacologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação , Inibidores de Proteínas Quinases/farmacologia , Estaurosporina/análogos & derivados , Estaurosporina/farmacologiaRESUMO
The medical treatment of pregnant women, as well as their fetuses, has become a common clinical practice in developed countries. Therefore, detailed knowledge of maternofetal pharmacokinetics, including the role of drug-efflux transporters in the fetoplacental unit, is crucial to optimize drug choice and dosage schemes and to avoid or exploit possible drug-drug interactions on placental transporters in order to assure appropriate drug levels in the mother and/or fetus. Breast cancer resistance protein (BCRP, ABCG2) is the most recent member of ATP-binding cassette drug-efflux transporters that has been associated with resistance in cancer chemotherapy. Importantly, ABCG2 has also been localized in various normal tissues, affecting the pharmacokinetics of several xenobiotics as well as a number of physiological substances. Extensive expression of ABCG2 in tissue barriers, such as the blood-brain barrier, intestine, testis, or placenta, suggests that ABCG2 plays an important role in the protection of sensitive tissues against toxins. In the placenta, ABCG2 has been experimentally evidenced to actively pump its substrates in the fetal-to-maternal direction and to play an important role in transplacental pharmacokinetics, fetal protection, and detoxication. Further, ABCG2 expression in embryonic and fetal membranes over the course of pregnancy helps ensure proper function of the fetoplacental unit. In this review, we summarize the current knowledge regarding expression and function of ABCG2 in the fetoplacental unit during the development of the fetus and overview the aspects of transplacental pharmacokinetics, ABCG2 regulation, and clinical significance of the transporter for pharmacotherapy in pregnancy.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Feto/fisiologia , Proteínas de Neoplasias/metabolismo , Gravidez/fisiologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Ensaios Clínicos como Assunto , Embrião de Mamíferos/fisiologia , Feminino , Humanos , Proteínas de Neoplasias/genética , Placenta/metabolismo , Polimorfismo Genético , Distribuição Tecidual , Xenobióticos/metabolismoRESUMO
Pharmacotherapy of acute myeloid leukemia (AML) remains challenging, and the disease has one of the lowest curability rates among hematological malignancies. The therapy outcomes are often compromised by the existence of a resistant AML phenotype associated with overexpression of ABCB1 and ABCG2 transporters. Because AML induction therapy frequently consists of anthracycline-like drugs, their efficiency may also be diminished by drug biotransformation via carbonyl reducing enzymes (CRE). In this study, we investigated the modulatory potential of the CDK4/6 inhibitors abemaciclib, palbociclib, and ribociclib on AML resistance using peripheral blood mononuclear cells (PBMC) isolated from patients with de novo diagnosed AML. We first confirmed inhibitory effect of the tested drugs on ABCB1 and ABCG2 in ABC transporter-expressing resistant HL-60 cells while also showing the ability to sensitize the cells to cytotoxic drugs even as no effect on AML-relevant CRE isoforms was observed. All tested CDK4/6 inhibitors elevated mitoxantrone accumulations in CD34+ PBMC and enhanced accumulation of mitoxantrone was found with abemaciclib and ribociclib in PBMC of FLT3-ITD- patients. Importantly, the accumulation rate in the presence of CDK4/6 inhibitors positively correlated with ABCB1 expression in CD34+ patients and led to enhanced apoptosis of PBMC in contrast to CD34- samples. In summary, combination therapy involving CDK4/6 inhibitors could favorably target multidrug resistance, especially when personalized based on CD34- and ABCB1-related markers.
RESUMO
Special attention is required when pharmacological treatment is indicated for a pregnant woman. P-glycoprotein (MDR1) is a well-known transporter localized in the maternal blood-facing apical membrane of placental syncytiotrophoblast and is considered to play an important role in protecting the developing fetus. Maraviroc, a MDR1 substrate that is registered for treatment of HIV infection, shows a low toxicity profile, suggesting favorable tolerability also if administered to pregnant women. Nevertheless, there is only poor understanding to date regarding the extent to which it permeates across the placental barrier and what are the transport mechanisms involved. Endeavoring to clarify the passage of maraviroc across placenta, we used in this study the method of closed-circuit perfusion of maraviroc across human placental cotyledon. The data obtained confirmed slight involvement of MDR1, but they also suggest possible interaction with other transport system(s) working in the opposite direction from that of MDR1. Complementary in vitro studies, including cellular experiments on choriocarcinoma BeWo cells as well as transporter-overexpressing MDCKII and A431 cell lines and accumulation in placental fresh villous fragments, revealed maraviroc transport by MRP1, OATP1A2, and OATP1B3 transporters. Based on mRNA expression data in the placental tissue, isolated trophoblasts, and fetal endothelial cells, especially MRP1 and OATP1A2 seem to play a crucial role in cooperatively driving maraviroc into placental tissue. By the example of maraviroc, we show here the important interplay of transporters in placental drug handling and its possibility to overcome the MDR1-mediated efflux.