RESUMO
Organic anion transporting polypeptides (OATPs), OATP1B1 and OATP2B1 are membrane proteins mediating the cellular uptake of chemically diverse organic compounds. OATP1B1 is exclusively expressed in hepatocytes and plays a key role in hepatic detoxification. The ubiquitously expressed OATP2B1 promotes the intestinal absorption of orally administered drugs. Flavonoids are widely found in foods and beverages, and many of them can inhibit OATP function, resulting in food-drug interactions. In our previous work, we have shown that not only luteolin (LUT) and quercetin (Q), but also some of their metabolites can inhibit OATP1B1 and OATP2B1 activity. However, data about the potential direct transport of these flavonoids by OATPs have been incomplete. Hence, in the current study, we developed a simple, fluorescence-based method for the measurement of intracellular flavonoid levels. The method applies a cell-permeable small molecule (2-aminoethyl diphenylborinate, 2-APB), that, upon forming a complex with flavonoids, results in their fluorescence enhancement. This way the direct uptake of LUT and Q, and also their metabolites' could be investigated both by confocal microscopy and in a fluorescence plate reader in living cells. With this approach we identified quercetin-3'-O-sulfate, luteolin-3'-O-glucuronide, luteolin-7-O-glucuronide and luteolin-3'-O-sulfate as substrates of both OATP1B1 and OATP2B1. Our results highlight that OATP1B1 and OATP2B1 can be key participants in the transmembrane movement of LUT and Q conjugates with otherwise low cell permeability. In addition, the novel method developed in this study can be a good completion to existing fluorescence-based assays to investigate OATP function.
RESUMO
Zearalenone (ZEN) is a mycoestrogen produced by Fusarium fungi. ZEN is a frequent contaminant in cereal-based products, representing significant health threat. The major reduced metabolites of ZEN are α-zearalenol (α-ZEL) and ß-zearalenol (ß-ZEL). Since the toxicokinetic interactions of ZEN/ZELs with cytochrome P450 enzymes (CYPs) and organic anion transporting polypeptides (OATPs) have been barely characterized, we examined these interactions applying in vitro models. ZEN and ZELs were relatively strong inhibitors of CYP3A4 and moderate inhibitors of CYP1A2 and CYP2C9. Both CYP1A2 and CYP3A4 decreased ZEN and ß-ZEL concentrations in depletion assays, while only CYP1A2 reduced α-ZEL levels. OATPs tested were strongly or moderately inhibited by ZEN and ZELs; however, these mycotoxins did not show higher cytotoxicity in OATP-overexpressing cells. Our results help the deeper understanding of the toxicokinetic/pharmacokinetic interactions of ZEN, α-ZEL, and ß-ZEL.
Assuntos
Micotoxinas , Transportadores de Ânions Orgânicos , Zearalenona , Zeranol/análogos & derivados , Zearalenona/toxicidade , Citocromo P-450 CYP1A2 , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450 , PeptídeosRESUMO
Organic anion transporting polypeptides OATP1A2, OATP1B1, OATP1B3 and OATP2B1 are Na+ - and ATP-independent exchangers of large, organic compounds, encompassing structurally diverse xenobiotics, including various drugs. These OATPs influence intestinal absorption (OATP2B1), hepatic clearance (OATP1B1/3) and blood to brain penetration (OATP1A2, OATP2B1) of their drug substrates. Consequently, OATP-mediated drug or food interactions may lead to altered pharmacokinetics and toxicity. During drug development, investigation of hepatic OATP1B1 and OATP1B3 is recommended by international regulatory agencies. Most frequently, OATP-drug interactions are investigated in an indirect assay, i.e., by examining uptake inhibition of a radioactive or fluorescent probe. However, indirect assays do not distinguish between transported substrates and non-transported OATP inhibitors. To fill this hiatus, a novel assay, termed competitive counterflow (CCF) has been developed and has since been applied for several OATPs to differentiate between substrates and non-transported inhibitors. However, previous OATP CCF assays, with the exception of that for OATP1B1, used radioactive probes. In the current study, we demonstrate that sulforhodamine 101 or pyranine can be used as fluorescent probes in a CCF assay to identify transported substrates of OATP1A2, or OATPs 1B1, 1B3 and 2B1, respectively. With the help of the newly developed fluorescence-based CCF method, we identify the FDA-approved anti-protozoal drug, pentamidine as a unique substrate of OATP1A2. Furthermore, we confirm the selective, OATP1A2-mediated uptake of pentamidine in a cytotoxicity assay. Based on our results, OATP1A2 may be an important determinant of pentamidine transport through the blood-brain barrier.
Assuntos
Transportadores de Ânions Orgânicos , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Pentamidina , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Fluorescência , Transporte Biológico , PeptídeosRESUMO
Aza-BODIPY dyes have recently come to attention owing to their excellent chemical and photophysical properties. In particular, their absorption and emission maxima can efficiently be shifted to the red or even to the NIR spectral region. On this basis, aza-BODIPY derivatives are widely investigated as fluorescent probes or phototherapeutic agents. Here we report the synthesis of a set of novel aza-BODIPY derivatives as potential photosensitizers for use in photodynamic therapy. Triazolyl derivatives were obtained via Cu(I)-catalyzed azide-alkyne cycloaddition as the key step. In vitro photodynamic activities of the newly synthesized compounds were evaluated on the A431 human epidermoid carcinoma cell line. Structural differences influenced the light-induced toxicity of the test compounds markedly. Compared to the initial tetraphenyl aza-BODIPY derivative, the compound bearing two hydrophilic triethylene glycol side chains showed substantial, more than 250-fold, photodynamic activity with no dark toxicity. Our newly synthesized aza-BODIPY derivative, acting in the nanomolar range, might serve as a promising candidate for the design of more active and selective photosensitizers.
Assuntos
Fotoquimioterapia , Fármacos Fotossensibilizantes , Humanos , Compostos de Boro/química , Linhagem CelularRESUMO
Orally administered small molecules may have important therapeutic potential in treating COVID-19 disease. The recently developed antiviral agents, Molnupiravir and Nirmatrelvir, have been reported to be efficient treatments, with only moderate side effects, especially when applied in the early phases of this disease. However, drug-drug and drug-transporter interactions have already been noted by the drug development companies and in the application notes. In the present work, we have studied some of the key human transporters interacting with these agents. The nucleoside analog Molnupiravir (EIDD-2801) and its main metabolite (EIDD-1931) were found to inhibit CNT1,2 in addition to the ENT1,2 nucleoside transporters; however, it did not significantly influence the relevant OATP transporters or the ABCC4 nucleoside efflux transporter. The active component of Paxlovid (PF-07321332, Nirmatrelvir) inhibited the function of several OATPs and of ABCB1 but did not affect ABCG2. However, significant inhibition was observed only at high concentrations of Nirmatrelvir and probably did not occur in vivo. Paxlovid, as used in the clinic, is a combination of Nirmatrelvir (viral protease inhibitor) and Ritonavir (a "booster" inhibitor of Nirmatrelvir metabolism). Ritonavir is known to inhibit several drug transporters; therefore, we have examined these compounds together, in relevant concentrations and ratios. No additional inhibitory effect of Nirmatrelvir was observed compared to the strong transporter inhibition caused by Ritonavir. Our current in vitro results should help to estimate the potential drug-drug interactions of these newly developed agents during COVID-19 treatment.
Assuntos
COVID-19 , Ritonavir , Humanos , Ritonavir/farmacologia , SARS-CoV-2 , Nucleosídeos , Tratamento Farmacológico da COVID-19 , Proteínas de Membrana Transportadoras , Antivirais/farmacologiaRESUMO
Organic anion transporting polypeptides (OATPs) were found to readily deliver membrane impermeable, tetrazine bearing fluorescent probes into cells. This feature was explored in OATP3A1 conditioned bio-orthogonal labeling schemes of various intracellular proteins in live cells. Confocal microscopy and super-resolution microscopy (STED) studies have shown that highly specific and efficient staining of the selected intracellular proteins can be achieved with the otherwise non-permeable probes when OATP3A1 is present in the cell membrane of cells. Such a transport protein linked bio-orthogonal labeling scheme is believed to be useful in OATP3A1 activity-controlled protein expression studies in the future.
Assuntos
Transportadores de Ânions Orgânicos , Transportadores de Ânions Orgânicos/metabolismo , Proteínas/metabolismo , Corantes FluorescentesRESUMO
In humans, approximately 70% of drugs are eliminated through the liver. This process is governed by the concerted action of membrane transporters and metabolic enzymes. Transporters mediating hepatocellular uptake of drugs belong to the SLC (Solute carrier) superfamily of transporters. Drug efflux either toward the portal vein or into the bile is mainly mediated by active transporters of the ABC (ATP Binding Cassette) family. Alteration in the function and/or expression of liver transporters due to mutations, disease conditions, or co-administration of drugs or food components can result in altered pharmacokinetics. On the other hand, drugs or food components interacting with liver transporters may also interfere with liver function (e.g., bile acid homeostasis) and may even cause liver toxicity. Accordingly, certain transporters of the liver should be investigated already at an early stage of drug development. Most frequently radioactive probes are applied in these drug-transporter interaction tests. However, fluorescent probes are cost-effective and sensitive alternatives to radioligands, and are gaining wider application in drug-transporter interaction tests. In our review, we summarize our current understanding about hepatocyte ABC and SLC transporters affected by drug interactions. We provide an update of the available fluorescent and fluorogenic/activable probes applicable in in vitro or in vivo testing of these ABC and SLC transporters, including near-infrared transporter probes especially suitable for in vivo imaging. Furthermore, our review gives a comprehensive overview of the available fluorescence-based methods, not directly relying on the transport of the probe, suitable for the investigation of hepatic ABC or SLC-type drug transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Fígado , Humanos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Fluorescência , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Interações Medicamentosas , Preparações Farmacêuticas/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Luteolin and naringenin are flavonoids found in various foods/beverages and present in certain dietary supplements. After a high intake of these flavonoids, their sulfate and glucuronide conjugates reach micromolar concentrations in the bloodstream. Some pharmacokinetic interactions of luteolin and naringenin have been investigated in previous studies; however, only limited data are available in regard to their metabolites. In this study, we aimed to investigate the interactions of the sulfate and glucuronic acid conjugates of luteolin and naringenin with human serum albumin, cytochrome P450 (CYP2C9, 2C19, and 3A4) enzymes, and organic anion transporting polypeptide (OATP1B1 and OATP2B1) transporters. Our main findings are as follows: (1) Sulfate conjugates formed more stable complexes with albumin than the parent flavonoids. (2) Luteolin and naringenin conjugates showed no or only weak inhibitory action on the CYP enzymes examined. (3) Certain conjugates of luteolin and naringenin are potent inhibitors of OATP1B1 and/or OATP2B1 enzymes. (4) Conjugated metabolites of luteolin and naringenin may play an important role in the pharmacokinetic interactions of these flavonoids.
Assuntos
Citocromo P-450 CYP3A , Transportadores de Ânions Orgânicos , Humanos , Citocromo P-450 CYP3A/metabolismo , Glucuronídeos , Luteolina/farmacologia , Albumina Sérica Humana/metabolismo , Sulfatos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Flavonoides/farmacologia , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP2C19/metabolismoRESUMO
Organic anion transporting polypeptide 3A1 (OATP3A1, encoded by the SLCO3A1 gene) is a prostaglandin, oligopeptide, and steroid/thyroid hormone transporter with wide tissue distribution, expressed, e.g., in the human brain and testis. Although the physiological importance of OATP3A1 has not yet been clarified, based on its expression pattern, substrate recognition, and evolutionary conservation, OATP3A1 is a potential pharmacological target. Previously, two isoforms of OATP3A1, termed as V1 and V2, have been characterized. Here, we describe the cloning and functional characterization of a third isoform, OATP3A1_V3. The mRNA of isoform V3 is formed by alternative splicing and results in an OATP3A1 protein with an altered C-terminus compared to isoforms V1 and V2. Based on quantitative PCR, we demonstrate the widespread expression of SLCO3A1_V3 mRNA in human organs, with the highest expression in the brain and testis. By generation of an isoform V3-specific antibody and immunostaining, we show that the encoded protein is expressed in the human choroid plexus, neurons, and both germ and Sertoli cells of the testis. Moreover, we demonstrate that in contrast to isoform V1, OATP3A1_V3 localizes to the apical membrane of polarized MDCKII cells. Using HEK-293 cells engineered to overexpress OATP3A1_V3, we verify the protein's functionality and identify dehydroepiandrosterone sulfate as a novel OATP3A1 substrate. Based on their distinct expression patterns but overlapping functions, OATP3A1 isoforms may contribute to transcellular (neuro)steroid transport in the central nervous system.
RESUMO
Organic anion-transporting polypeptide 2B1 (OATP2B1) is a multispecific transporter mediating the cellular uptake of steroids and numerous drugs. OATP2B1 is abundantly expressed in the intestine and is also present in various tumors. Increased steroid hormone uptake by OATP2B1 has been suggested to promote progression of hormone dependent tumors. 13α-estrones are effective inhibitors of endogenous estrogen formation and are potential candidates to inhibit proliferation of hormone dependent cancers. Recently, we have identified a variety of 13α/ß-estrone-based inhibitors of OATP2B1. However, the nature of this interaction, whether these inhibitors are potential transported substrates of OATP2B1 and hence may be enriched in OATP2B1-overexpressing cells, has not yet been investigated. In the current study we explored the antiproliferative effect of the most effective OATP2B1 inhibitor 13α/ß-estrones in control and OATP2B1-overexpressing A431 carcinoma cells. We found an increased antiproliferative effect of 3-O-benzyl 13α/ß-estrones in both mock transfected and OATP2B1-overexpressing cells. However, C-2 halogenated 13α-estrones had a selective OATP2B1-mediated cell growth inhibitory effect. In order to demonstrate that increased sensitization can be attributed to OATP2B1-mediated cellular uptake, tritium labeled 2-bromo-13α-estrone was synthesized for direct transport measurements. These experiments revealed increased accumulation of [3H]2-bromo-13α-estrone due to OATP2B1 function. Our results indicate that C-2 halogenated 13α-estrones are good candidates in the design of anti-cancer drugs targeting OATP2B1.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Estrona/farmacologia , Moduladores de Transporte de Membrana/farmacologia , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Antineoplásicos/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Estrona/análogos & derivados , Estrona/metabolismo , Humanos , Moduladores de Transporte de Membrana/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
Transporters in the human liver play a major role in the clearance of endo- and xenobiotics. Apical (canalicular) transporters extrude compounds to the bile, while basolateral hepatocyte transporters promote the uptake of, or expel, various compounds from/into the venous blood stream. In the present work we have examined the in vitro interactions of some key repurposed drugs advocated to treat COVID-19 (lopinavir, ritonavir, ivermectin, remdesivir and favipiravir), with the key drug transporters of hepatocytes. These transporters included ABCB11/BSEP, ABCC2/MRP2, and SLC47A1/MATE1 in the canalicular membrane, as well as ABCC3/MRP3, ABCC4/MRP4, SLC22A1/OCT1, SLCO1B1/OATP1B1, SLCO1B3/OATP1B3, and SLC10A1/NTCP, residing in the basolateral membrane. Lopinavir and ritonavir in low micromolar concentrations inhibited BSEP and MATE1 exporters, as well as OATP1B1/1B3 uptake transporters. Ritonavir had a similar inhibitory pattern, also inhibiting OCT1. Remdesivir strongly inhibited MRP4, OATP1B1/1B3, MATE1 and OCT1. Favipiravir had no significant effect on any of these transporters. Since both general drug metabolism and drug-induced liver toxicity are strongly dependent on the functioning of these transporters, the various interactions reported here may have important clinical relevance in the drug treatment of this viral disease and the existing co-morbidities.
Assuntos
Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Antivirais/farmacologia , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Fígado/efeitos dos fármacos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alanina/farmacologia , Alanina/uso terapêutico , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Comorbidade , Reposicionamento de Medicamentos , Humanos , Fígado/metabolismo , Fígado/patologia , Transportador 1 de Ânion Orgânico Específico do Fígado/antagonistas & inibidores , Lopinavir/química , Lopinavir/metabolismo , Lopinavir/farmacologia , Lopinavir/uso terapêutico , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Ritonavir/química , Ritonavir/metabolismo , Ritonavir/farmacologia , Ritonavir/uso terapêutico , SARS-CoV-2/isolamento & purificação , Especificidade por Substrato , Tratamento Farmacológico da COVID-19RESUMO
Organic anion-transporting polypeptides, OATP1B1, OATP1B3, and OATP2B1 are multispecific membrane proteins mediating the hepatocellular uptake of structurally diverse endo- and exogenous compounds, including various kinds of drugs. Co-administration of OATP1B/2B1 substrates may lead to altered pharmacokinetics or even toxicity. Therefore, the study of the interaction with these OATPs is essential in drug development and is recommended by international regulatory agencies, the FDA, EMA, and PMDA. In general, radiolabeled indicators are used to measure drug interactions of OATPs, and, lately, fluorescent probes are also gaining wider application in OATP tests. However, all of the currently available methods (either radioactive or fluorescence-based) comprise multiple steps, including the removal of the indicator in the end of the experiment. Hence, they are not ideally suited for high-throughput screening. In the current study, in order to find an indicator allowing real-time assessment of hepatic OATP function, we searched for an activatable fluorogenic OATP substrate. Here, we show that 8-acetoxypyrene-1,3,6-trisulfonate (Ace), a fluorogenic derivative of the hepatic OATP substrate pyranine (8-hydroxypyrene-1,3,6-trisulfonate) enters the cells via OATP1B1/3 or OATP2B1 function. In living cells, Ace is then converted into highly fluorescent pyranine, allowing "no-wash" measurement of OATP function and drug interactions. Furthermore, we demonstrate that Ace can be used in an indirect assay termed as competitive counterflow suitable to distinguish between transported substrates and inhibitors of OATP1B1. The fluorescence-based methods described here are unique and open the way toward high-throughput screening of interactions between new molecular entities and OATPs.
Assuntos
Corantes Fluorescentes/análise , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo , Animais , Sulfonatos de Arila/análise , Sulfonatos de Arila/química , Sulfonatos de Arila/metabolismo , Linhagem Celular , Sobrevivência Celular , Corantes Fluorescentes/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Fígado/metabolismoRESUMO
During the COVID-19 pandemic, several repurposed drugs have been proposed to alleviate the major health effects of the disease. These drugs are often applied with analgesics or non-steroid anti-inflammatory compounds, and co-morbid patients may also be treated with anticancer, cholesterol-lowering, or antidiabetic agents. Since drug ADME-tox properties may be significantly affected by multispecific transporters, in this study, we examined the interactions of the repurposed drugs with the key human multidrug transporters present in the major tissue barriers and strongly affecting the pharmacokinetics. Our in vitro studies, using a variety of model systems, explored the interactions of the antimalarial agents chloroquine and hydroxychloroquine; the antihelmintic ivermectin; and the proposed antiviral compounds ritonavir, lopinavir, favipiravir, and remdesivir with the ABCB1/Pgp, ABCG2/BCRP, and ABCC1/MRP1 exporters, as well as the organic anion-transporting polypeptide (OATP)2B1 and OATP1A2 uptake transporters. The results presented here show numerous pharmacologically relevant transporter interactions and may provide a warning on the potential toxicities of these repurposed drugs, especially in drug combinations at the clinic.
RESUMO
Mutations in the ABCC6 gene result in calcification diseases such as pseudoxanthoma elasticum or Generalized Arterial Calcification of Infancy. Generation of antibodies recognizing an extracellular (EC) epitope of ABCC6 has been hampered by the short EC segments of the protein. To overcome this limitation, we immunized bovine FcRn transgenic mice exhibiting an augmented humoral immune response with Human Embryonic Kidney 293 cells cells expressing human ABCC6 (hABCC6). We obtained a monoclonal antibody recognizing an EC epitope of hABCC6 that we named mEChC6. Limited proteolysis revealed that the epitope is within a loop in the N-terminal half of ABCC6 and probably spans amino acids 338-347. mEChC6 recognizes hABCC6 in the liver of hABCC6 transgenic mice, verifying both specificity and EC binding to intact hepatocytes.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Epitopos/imunologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Animais , Epitopos/genética , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genéticaRESUMO
Organic anion-transporting polypeptide 3A1 (OATP3A1) is a membrane transporter mediating the cellular uptake of various hormones such as estrone-3-sulfate, prostaglandins E1 and E2 and thyroxine. OATP3A1 is widely expressed in the human body and its presence in tissue-blood barriers, neurons and muscle cells marks it as a potential pharmacological target. Herein we demonstrate that an otherwise membrane impermeant, zwitterionic fluorescent coumarin probe, bearing a sulfonate function is a potent substrate of human OATP3A1, thus readily transported into HEK-293-OATP3A1 cells allowing functional investigation and the screen of drug interactions of the OATP3A1 transporter. At the same time, dyes lacking either the sulfonate motif or the coumarin scaffold showed a dramatic decrease in affinity or even a complete loss of transport. Furthermore, we observed a distinct inhibition/activation pattern in the OATP3A1-mediated uptake of closely related fluorescent coumarin derivatives differing only in the presence of the sulfonate moiety. Additionally, we detected a synergistic effect between one of the probes tested and the endogenous OATP substrate estrone-3-sulfate. These data, together with docking results indicate the presence of at least two cooperative substrate binding sites in OATP3A1. Besides providing the first sensitive probe for testing OATP3A1 substrate/inhibitor interactions, our results also help to understand substrate recognition and transport mechanism of the poorly characterized OATP3A1. Moreover, coumarins are good candidates for OATP3A1-targeted drug delivery and as pharmacological modulators of OATP3A1.
Assuntos
Cumarínicos/metabolismo , Cumarínicos/farmacologia , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacologia , Transportadores de Ânions Orgânicos/metabolismo , Cumarínicos/química , Corantes Fluorescentes/química , Células HEK293 , Humanos , Transportadores de Ânions Orgânicos/química , Estrutura Secundária de Proteína , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologiaRESUMO
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.
Assuntos
Fármacos Anti-HIV/metabolismo , Maraviroc/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Placenta/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Acridinas/farmacologia , Animais , Fármacos Anti-HIV/sangue , Fármacos Anti-HIV/farmacologia , Linhagem Celular Tumoral , Cães , Interações Medicamentosas , Feminino , Regulação da Expressão Gênica , Humanos , Células Madin Darby de Rim Canino , Maraviroc/sangue , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/genética , Perfusão , Placenta/efeitos dos fármacos , Circulação Placentária , Gravidez , Ritonavir/farmacologia , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/antagonistas & inibidores , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/genética , Tetra-Hidroisoquinolinas/farmacologiaRESUMO
Chrysin is an abundant flavonoid in nature, and it is also contained by several dietary supplements. Chrysin is highly biotransformed in the body, during which conjugated metabolites chrysin-7-sulfate and chrysin-7-glucuronide are formed. These conjugates appear at considerably higher concentrations in the circulation than the parent compound. Based on previous studies, chrysin can interact with biotransformation enzymes and transporters; however, the interactions of its metabolites have been barely examined. In this in vitro study, the effects of chrysin, chrysin-7-sulfate, and chrysin-7-glucuronide on cytochrome P450 enzymes (2C9, 2C19, 3A4, and 2D6) as well as on organic anion-transporting polypeptides (OATPs; 1A2, 1B1, 1B3, and 2B1) and ATP binding cassette [P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance protein (BCRP)] transporters were investigated. Our observations revealed that chrysin conjugates are strong inhibitors of certain biotransformation enzymes (e.g., CYP2C9) and transporters (e.g., OATP1B1, OATP1B3, OATP2B1, and BCRP) examined. Therefore, the simultaneous administration of chrysin-containing dietary supplements with medications needs to be carefully considered due to the possible development of pharmacokinetic interactions. SIGNIFICANCE STATEMENT: Chrysin-7-sulfate and chrysin-7-glucuronide are the major metabolites of flavonoid chrysin. In this study, we examined the effects of chrysin and its conjugates on cytochrome P450 enzymes and on organic anion-transporting polypeptides and ATP binding cassette transporters (P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2). Our results demonstrate that chrysin and/or its conjugates can significantly inhibit some of these proteins. Since chrysin is also contained by dietary supplements, high intake of chrysin may interrupt the transport and/or the biotransformation of drugs.
Assuntos
Inibidores das Enzimas do Citocromo P-450/farmacocinética , Suplementos Nutricionais , Flavonoides/farmacocinética , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Transportadores de Ânions Orgânicos/metabolismoRESUMO
Detoxification in hepatocytes is a strictly controlled process, in which the governed action of membrane transporters involved in the uptake and efflux of potentially dangerous molecules has a crucial role. Major transporters of hepatic clearance belong to the ABC (ATP Binding Cassette) and Solute Carrier (SLC) protein families. Organic anion-transporting polypeptide OATP1B1 (encoded by the SLCO1B1 gene) is exclusively expressed in the sinusoidal membrane of hepatocytes, where it mediates the cellular uptake of bile acids, bilirubin, and also that of various drugs. The removal of toxic molecules from hepatocytes to the bile is accomplished by several ABC transporters, including P-glycoprotein (ABCB1), MRP2 (ABCC2) and BCRP (ABCG2). Owing to their pharmacological relevance, monitoring drug interaction with OATP1B1/3 and ABC proteins is recommended. Our aim was to assess the interaction of recently identified fluorescent OATP substrates (various dyes used in cell viability assays, pyranine, Cascade Blue hydrazide (CB) and sulforhodamine 101 (SR101)) (Bakos et al., 2019; Patik et al., 2018) with MRP2 and ABCG2 in order to find fluorescent probes for the simultaneous characterization of both uptake and efflux processes. Transport by MRP2 and ABCG2 was investigated in inside-out membrane vesicles (IOVs) allowing a fast screen of the transport of membrane impermeable substrates by efflux transporters. Next, transcellular transport of shared OATP and ABC transporter substrate dyes was evaluated in MDCKII cells co-expressing OATP1B1 and MRP2 or ABCG2. Our results indicate that pyranine is a general substrate of OATP1B1, OATP1B3 and OATP2B1, and we find that the dye Live/Dead Violet and CB are good tools to investigate ABCG2 function in IOVs. Besides their suitability for MRP2 functional tests in the IOV setup, pyranine, CB and SR101 are the first dual probes that can be used to simultaneously measure OATP1B1 and MRP2 function in polarized cells by a fluorescent method.
Assuntos
Corantes Fluorescentes , Transportadores de Ânions Orgânicos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Interações Medicamentosas , Hepatócitos , Proteínas de Neoplasias , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de SolutoRESUMO
Human OATP2B1 encoded by the SLCO2B1 gene is a multispecific transporter mediating the cellular uptake of large, organic molecules, including hormones, prostaglandins and bile acids. OATP2B1 is ubiquitously expressed in the human body, with highest expression levels in pharmacologically relevant barriers, like enterocytes, hepatocytes and endothelial cells of the blood-brain-barrier. In addition to its endogenous substrates, OATP2B1 also recognizes clinically applied drugs, such as statins, antivirals, antihistamines and chemotherapeutic agents and influences their pharmacokinetics. On the other hand, OATP2B1 is also overexpressed in various tumors. Considering that elevated hormone uptake by OATP2B1 results in increased cell proliferation of hormone dependent tumors (e.g. breast or prostate), inhibition of OATP2B1 can be a good strategy to inhibit the growth of these tumors. 13-epiestrones represent a potential novel strategy in the treatment of hormone dependent cancers by the suppression of local estrogen production due to the inhibition of the key enzyme of estrone metabolism, 17ß-hydroxysteroid-dehydrogenase type 1 (HSD17ß1). Recently, we have demonstrated that various phosphonated 13-epiestrones are dual inhibitors also suppressing OATP2B1 function. In order to gain better insights into the molecular determinants of OATP2B1 13-epiestrone interaction we investigated the effect of C-2 and C-4 halogen or phenylalkynyl modified epiestrones on OATP2B1 transport function. Potent inhibitors (with EC50 values in the low micromolar range) as well as non-inhibitors of OATP2B1 function were identified. Based on the structure-activity relationship (SAR) of the various 13-epiestrone derivatives we could define structural elements important for OATP2B1 inhibition. Our results may help to understand the drug/inhibitor interaction profile of OATP2B1, and also may be a useful strategy to block steroid hormone entry into tumors.
Assuntos
Estrona/farmacologia , Transportadores de Ânions Orgânicos/metabolismo , Linhagem Celular Tumoral , Estrona/análogos & derivados , Estrona/química , Humanos , Transportadores de Ânions Orgânicos/química , Transportadores de Ânions Orgânicos/genética , Relação Estrutura-AtividadeRESUMO
Organic anion-transporting polypeptide 1A2 (OATP1A2), expressed in the human blood-brain barrier, promotes drug uptake from the blood and hence can be exploited for central nervous system-targeted drug delivery. The thyroid transporter OATP1C1, expressed in the choroid plexus and in astrocytes, is also a potential pharmacological target. Based on their established pharmacological relevance, screening the drug interaction profile of OATP1A2 and OATP1C1 is highly desirable. However, drug interaction screens require suitable model systems and functional assays. In the current study, uptake of a set of cell-impermeable fluorescent dyes was screened in HEK-293 and A431 cell lines overexpressing OATP1A2 and OATP1C1. Based on the uptake of fluorescent dye substrates, a functional assay was developed, which was used to characterize OATP inhibitors/substrates. We identify Live/Dead Green (LDG), Live-or-Dye 488, and sulforhodamines 101, G, and B as novel fluorescent substrates of OATP1A2 and OATP1C1. We show that LDG uptake is proportional to OATP1A2/1C1 expression, allowing the isolation of cells expressing high transporter levels. Additionally, dye uptake can be used to characterize the drug interaction pattern of OATP1A2 and OATP1C1. We demonstrate that third-generation P-glycoprotein inhibitors elacridar, tariquidar, and zosuquidar inhibit OATP1A2 function. Increased toxicity of elacridar in OATP1A2-expressing cells suggests that OATP1A2 may modulate the distribution of this compound. The fluorescence-based assays developed in the current study are a good alternative of radioligand-based tests and pave the way toward high-throughput screens for OATP1A2/1C1 drug interaction studies.