RESUMEN
Organophosphate flame retardants (OPFRs) are environmental pollutants of increasing interest, widely distributed in the environment and exerting possible deleterious effects towards the human health. The present study investigates in vitro their possible interactions with human drug transporters, which are targets for environmental chemicals and actors of their toxicokinetics. Some OPFRs, i.e., tris(2-butoxyethyl) phosphate (TBOEP), tris(1,3-dichloroisopropyl) phosphate (TDCPP), tri-o-cresyl phosphate (TOCP) and triphenyl phosphate (TPHP), were found to inhibit activities of some transporters, such as organic anion transporter 3 (OAT3), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter 2 (OCT2) or breast cancer resistance protein (BCRP). These effects were concentration-dependent, with IC50 values ranging from 6.1 µM (for TDCPP-mediated inhibition of OCT2) to 51.4 µM (for TOCP-mediated inhibition of BCRP). OPFRs also blocked the transporter-dependent membrane passage of endogenous substrates, notably that of hormones. OAT3 however failed to transport TBOEP and TPHP. OPFRs additionally repressed mRNA expressions of some transporters in cultured human hepatic HepaRG cells, especially those of OAT2 and OCT1 in response to TOCP, with IC50 values of 2.3 µM and 2.5 µM, respectively. These data therefore add OPFRs to the expanding list of pollutants interacting with drug transporters, even if OPFR concentrations required to impact transporters, in the 2-50 µM range, are rather higher than those observed in humans environmentally or dietarily exposed to these chemicals.
Asunto(s)
Contaminantes Ambientales , Retardadores de Llama , Tritolilfosfatos , Humanos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Retardadores de Llama/toxicidad , Proteínas de Neoplasias , Proteínas de Transporte de Membrana/genética , Contaminantes Ambientales/toxicidadRESUMEN
HepaRG cells are highly-differentiated human hepatoma cells, which are increasingly recognized as a convenient cellular model for in vitro evaluation of hepatic metabolism, transport, and/or toxicity of drugs. The present study was designed to evaluate whether HepaRG cells can also be useful for studying drug-mediated inhibition of canalicular and/or sinusoidal hepatic efflux of bile acids, which constitutes a major mechanism of drug-induced liver toxicity. For this purpose, HepaRG cells, initially loaded with the bile acid taurocholate (TC), were reincubated in TC-free transport assay medium, in the presence or absence of calcium or drugs, before analysis of TC retention. This method allowed us to objectivize and quantitatively measure biliary and sinusoidal efflux of TC from HepaRG cells, through distinguishing cellular and canalicular compartments. In particular, time-course analysis of the TC-free reincubation period of HepaRG cells, that is, the efflux period, indicated that a 20 min-efflux period allowed reaching biliary and sinusoidal excretion indexes for TC around 80% and 60%, respectively. Addition of the prototypical cholestatic drugs bosentan, cyclosporin A, glibenclamide, or troglitazone during the TC-free efflux phase period was demonstrated to markedly inhibit canalicular and sinusoidal secretion of TC, whereas, by contrast, incubation with the noncholestatic compounds salicylic acid or flumazenil was without effect. Such data therefore support the use of human HepaRG cells for in vitro predicting drug-induced liver toxicity (DILI) due to the inhibition of hepatic bile acid secretion, using a biphasic TC loading/efflux assay.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Ácido Taurocólico/farmacología , Ácido Taurocólico/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Ácidos y Sales Biliares/farmacología , Ácidos y Sales Biliares/metabolismoRESUMEN
Interactions of the Janus kinase (JAK) inhibitor ruxolitinib with solute carriers (SLCs) remain incompletely characterised. The present study was therefore designed to investigate this issue.The interactions of ruxolitinib with SLCs were analysed using transporter-overexpressing human embryonic kidney HEK293 cells. Substrate accumulation was detected by spectrofluorimetry, liquid chromatography coupled to tandem mass spectrometry or scintillation counting.Ruxolitinib was found to potently inhibit the activities of organic anion transporter 3 (OAT3), organic cation transporter 2 (OCT2), multidrug and toxin extrusion 1 (MATE1) and MATE2-K (half maximal inhibitory concentration (IC50) < 10 µM). It blocked OAT1, OAT4, OATP1B1, OATP1B3, OATP2B1 and OCT3, but in a weaker manner (IC50 > 10 µM), whereas OCT1 was not impacted. No time-dependent inhibition was highlighted. When applying the US Food and Drug Administration (FDA) criteria for transporters-related drug-drug interaction risk, OCT2 and MATE2-K, unlike MATE1 and OAT3, were predicted to be in vivo inhibited by ruxolitinib. Cellular uptake studies additionally indicated that ruxolitinib is a substrate for MATE1 and MATE2-K, but not for OAT3 and OCT2.Ruxolitinib in vitro blocked activities of most of SLC transporters. Only OCT2 and MATE-2K may be however clinically inhibited by the JAK inhibitor, with the caution for OCT2 that in vitro inhibition data were generated with an FDA-non recommended fluorescent substrate. Ruxolitinib MATEs-mediated transport may additionally deserve attention for its possible pharmacological consequences in MATE-positive cells.
Asunto(s)
Inhibidores de las Cinasas Janus , Preparaciones Farmacéuticas , Interacciones Farmacológicas , Células HEK293 , Humanos , Nitrilos , Proteínas de Transporte de Catión Orgánico , Pirazoles , PirimidinasRESUMEN
The search of substrates for solute carriers (SLCs) constitutes a major issue, owing notably to the role played by some SLCs, such as the renal electrogenic organic cation transporter (OCT) 2 (SLC22A2), in pharmacokinetics, drug-drug interactions and drug toxicity. For this purpose, substrates have been proposed to be identified by their cis-inhibition and trans-stimulation properties towards transporter activity. To get insights on the sensitivity of this approach for identifying SLC substrates, 15 various exogenous and endogenous OCT2 substrates were analysed in the present study, using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (DiASP) as a fluorescent OCT2 tracer substrate. All OCT2 substrates cis-inhibited DiASP uptake in OCT2-overexpressing HEK293 cells, with IC50 values ranging from 0.24 µM (for ipratropium) to 2.39 mM (for dopamine). By contrast, only 4/15 substrates, i.e., acetylcholine, agmatine, choline and metformin, trans-stimulated DiASP uptake, with a full suppression of the trans-stimulating effect of metformin by the reference OCT2 inhibitor amitriptyline. An analysis of molecular descriptors next indicated that trans-stimulating OCT2 substrates exhibit lower molecular weight, volume, polarizability and lipophilicity than non-trans-stimulating counterparts. Overall, these data indicated a rather low sensitivity (26.7%) of the trans-stimulation assay for identifying OCT2 substrates, and caution with respect to the use of such assay may therefore be considered.
Asunto(s)
Transportador 2 de Cátion Orgánico/metabolismo , Células HEK293 , Humanos , Estimulación QuímicaRESUMEN
The interactions of six neonicotinoid pesticides and one neonicotinoid metabolite with drug transporters have been characterized in vitro. Acetamiprid, clothianidin, imidacloprid, nitenpyram, thiacloprid and its metabolite thiacloprid amide, and thiamethoxam, each used at 100 µM, did not impair activity of the efflux pumps P-glycoprotein, multidrug resistance-associated proteins, and breast cancer resistance protein. They also did not inhibit that of the uptake transporters OATP1B1, OATP1B3, OAT4, and MATE1, whereas that of OATP2B1, OAT1, and MATE2-K was affected by only one of the seven neonicotinoids. Activity of OCT1 was moderately stimulated (up to 1.5-fold) by several neonicotinoids. By contrast, that of OAT3 and OCT2 was inhibited by most (OAT3), if not all (OCT2), neonicotinoids, with IC50 values in the 20 to 60 µM range for thiacloprid, likely not relevant to environmental exposure. Thiacloprid was moreover not transported by OAT3 and OCT2. Overall, these data suggest that neonicotinoid pesticides rather poorly interact with drug transporter activities.
Asunto(s)
Insecticidas/farmacología , Neonicotinoides/farmacología , Receptores de Superficie Celular/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/metabolismo , Línea Celular Tumoral , Interacciones Farmacológicas , Humanos , Insecticidas/farmacocinética , Neonicotinoides/metabolismo , Neonicotinoides/farmacocinética , Tiazinas/metabolismoRESUMEN
The inflammatory cytokine interleukin (IL)-6, which basically activates the Janus kinase (JAK)/ signal transducer and activator of transcription (STAT) signaling pathway, is well known to repress expression of hepatic cytochromes P-450 (P450s) and transporters. Therapeutic proteins, like monoclonal antibodies targeting IL-6 or its receptor, have consequently been demonstrated to restore full hepatic detoxification capacity, which results in inflammatory disease-related drug-drug interactions (idDDIs). In the present study, we investigated whether ruxolitinib, a small drug acting as a JAK1/2 inhibitor and currently used in the treatment of myeloproliferative neoplasms, may also counteract the repressing effects of IL-6 toward hepatic detoxifying systems. Ruxolitinib was found to fully inhibit IL-6-mediated repression of P450 (CYP1A2, CYP2B6, and CYP3A4) and transporter (NTCP, OATP1B1, and OCT1) mRNA levels in primary human hepatocytes and differentiated hepatoma HepaRG cells. Such effects were dose-dependent, with ruxolitinib EC50 values around 1.0-1.2 µM and thus close to ruxolitinib plasma levels that can be reached in patients. Moreover, they were associated with concomitant restoration of P450 and drug transporter activities in IL-6-exposed HepaRG cells. By contrast, ruxolitinib failed to suppress the repression of drug-detoxifying protein mRNA levels caused by IL-1ß The JAK inhibitor and anti-rheumatoid arthritis compound tofacitinib was additionally found to reverse IL-6-mediated suppression of P450 and transporter mRNA expressions. Taken together, our results demonstrated that small drugs acting as JAK inhibitors, like ruxolitinib, counteract IL-6-mediated repression of drug-metabolizing enzymes and drug transporters in cultured human hepatocytes. These JAK inhibitors may consequently be hypothesized to restore hepatic detoxification capacity for patients suffering from inflammatory diseases, which may in turn cause idDDIs.
Asunto(s)
Hepatocitos/efectos de los fármacos , Inactivación Metabólica/efectos de los fármacos , Interleucina-6/metabolismo , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 2/antagonistas & inhibidores , Pirazoles/farmacología , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones Farmacológicas/fisiología , Hepatocitos/metabolismo , Humanos , Nitrilos , Pirimidinas , Transducción de Señal/efectos de los fármacosRESUMEN
Drug transporters are now recognized as major actors in pharmacokinetics, involved notably in drug-drug interactions and drug adverse effects. Factors that govern their activity, localization and expression are therefore important to consider. In the present review, the implications of protein kinases C (PKCs) in transporter regulations are summarized and discussed. Both solute carrier (SLC) and ATP-binding cassette (ABC) drug transporters can be regulated by PKCs-related signaling pathways. PKCs thus target activity, membrane localization and/or expression level of major influx and efflux drug transporters, in various normal and pathological types of cells and tissues, often in a PKC isoform-specific manner. PKCs are notably implicated in membrane insertion of bile acid transporters in liver and, in this way, are thought to contribute to cholestatic or choleretic effects of endogenous compounds or drugs. The exact clinical relevance of PKCs-related regulation of drug transporters in terms of drug resistance, pharmacokinetics, drug-drug interactions and drug toxicity remains however to be precisely determined. This issue is likely important to consider in the context of the development of new drugs targeting PKCs-mediated signaling pathways, for treating notably cancers, diabetes or psychiatric disorders.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , Proteínas Transportadoras de Solutos/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transporte Biológico , Regulación de la Expresión Génica , Humanos , Isoenzimas/metabolismo , Preparaciones Farmacéuticas/metabolismo , Fosforilación , Proteínas Transportadoras de Solutos/genéticaRESUMEN
The heterodimeric L-type amino acid transporter (LAT) 1/CD98hc is overexpressed in lung cancers with a poor prognosis factor. Factors that contribute to LAT1/CD98hc overexpression in lung cells remain however to be determined, but the implication of atmospheric pollution can be suspected. The present study was therefore designed to analyze the effects of diesel exhaust particle (DEP) extract (DEPe) on LAT1/CD98hc expression in bronchial epithelial BEAS-2B cells. Exposure to DEPe up-regulated LAT1 and CD98hc mRNA levels in a concentration-dependent manner, with DEPe EC50 values (around 0.2 µg/mL) relevant to environmental situations. DEPe concomitantly induced LAT1/CD98hc protein expression and LAT1-mediated leucine accumulation in BEAS-2B cells. Inhibition of the aryl hydrocarbon receptor (AhR) pathway through the use of a chemical AhR antagonist or the siRNA-mediated silencing of AhR expression was next found to prevent DEPe-mediated induction of LAT1/CD98hc, indicating that this regulation depends on AhR, known to be activated by major chemical DEP components like polycyclic aromatic hydrocarbons. DEPe exposure was finally shown to induce mRNA expression and activity of matrix metalloproteinase (MMP)-2 in BEAS-2B cells, in a CD98hc/focal adhesion kinase (FAK)/extracellular regulated kinase (ERK) manner, thus suggesting that DEPe-mediated induction of CD98hc triggers activation of the integrin/FAK/ERK signaling pathway known to be involved in MMP-2 regulation. Taken together, these data demonstrate that exposure to DEPe induces functional overexpression of the amino acid transporter LAT1/CD98hc in lung cells. Such a regulation may participate to pulmonary carcinogenic effects of DEPs, owing to the well-documented contribution of LAT1 and CD98hc to cancer development.
Asunto(s)
Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Pulmón/efectos de los fármacos , Receptores de Hidrocarburo de Aril/metabolismo , Regulación hacia Arriba , Emisiones de Vehículos/toxicidad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Cadena Pesada de la Proteína-1 Reguladora de Fusión/genética , Humanos , Transportador de Aminoácidos Neutros Grandes 1/genética , Pulmón/citología , Pulmón/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Hidrocarburos Policíclicos Aromáticos/toxicidad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Hidrocarburo de Aril/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Organochlorine (OC) pesticides constitute a major class of persistent and toxic organic pollutants, known to modulate drug-detoxifying enzymes. In the present study, OCs were demonstrated to also alter the activity and expression of human hepatic drug transporters. Activity of the sinusoidal influx transporter OCT1 (organic cation transporter 1) was thus inhibited by endosulfan, chlordane, heptachlor, lindane, and dieldrine, but not by dichlorodiphenyltrichloroethane isomers, whereas those of the canalicular efflux pumps MRP2 (multidrug resistance-associated protein 2) and BCRP (breast cancer resistance protein) were blocked by endosulfan, chlordane, heptachlor, and chlordecone; this latter OC additionally inhibited the multidrug resistance gene 1 (MDR1)/P-glycoprotein (P-gp) activity. OCs, except endosulfan, were next found to induce MDR1/P-gp and MRP2 mRNA expressions in hepatoma HepaRG cells; some of them also upregulated BCRP. By contrast, expression of sinusoidal transporters was not impaired (organic anion-transporting polypeptide (OATP) 1B1 and OATP2B1) or was downregulated (sodium taurocholate co-transporting polypeptide (NTCP) and OCT1). Such regulations of drug transporter activity and expression, depending on the respective nature of OCs and transporters, may contribute to the toxicity of OC pesticides.
Asunto(s)
Hidrocarburos Clorados/farmacología , Proteínas de Transporte de Membrana/metabolismo , Plaguicidas/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Línea Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inactivación Metabólica , Hígado/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , Transportador 1 de Anión Orgánico Específico del Hígado , Células MCF-7/efectos de los fármacos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Transportador 1 de Catión Orgánico/metabolismo , Simportadores/genética , Simportadores/metabolismoRESUMEN
Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these environmental chemicals, the interactions of 15 SDHIs with activities of main human drug transporters implicated in pharmacokinetics were investigated in vitro. 5/15 SDHIs, i.e., benzovindiflupyr, bixafen, fluxapyroxad, pydiflumetofen and sedaxane, were found to strongly reduce activity of the renal organic anion transporter (OAT) 3, in a concentration-dependent manner (with IC50 values in the 1.0-3.9 µM range), without however being substrates for OAT3. Moreover, these 5/15 SDHIs decreased the membrane transport of estrone-3 sulfate, an endogenous substrate for OAT3, and sedaxane was predicted to inhibit in vivo OAT3 activity in response to exposure to the acceptable daily intake (ADI) dose. In addition, pydiflumetofen strongly inhibited the renal organic cation transporter (OCT) 2 (IC50 = 2.0 µM) and benzovindiflupyr the efflux pump breast cancer resistance protein (BCRP) (IC50 = 3.9 µM). Other human transporters, including organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 as well as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K were moderately or weakly inhibited by SDHIs, whereas P-glycoprotein, multidrug resistance-associated protein (MRP), OCT1 and OAT1 activities were not or only marginally impacted. Then, some human drug transporters, especially OAT3, constitute molecular targets for SDHIs. This could have toxic consequences, notably with respect to levels of endogenous compounds and metabolites substrates for the considered transporters or to potential SDHI-drug interactions. This could therefore contribute to putative health risk of these fungicides.
Asunto(s)
Succinato Deshidrogenasa , Humanos , Succinato Deshidrogenasa/antagonistas & inhibidores , Succinato Deshidrogenasa/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/antagonistas & inhibidores , Transporte Biológico/efectos de los fármacos , Fungicidas Industriales/toxicidad , Fungicidas Industriales/farmacología , Inhibidores Enzimáticos/farmacología , Estrona/análogos & derivados , Estrona/metabolismo , Células HEK293 , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico/antagonistas & inhibidoresRESUMEN
The identification of substrates for solute carriers (SLCs) handling drugs is an important challenge, owing to the major implication of these plasma membrane transporters in pharmacokinetics and drug-drug interactions. In this context, the competitive counterflow (CCF) assay has been proposed as a practical and less expensive approach than the reference functional uptake assays for discriminating SLC substrates and non-substrates. The present article was designed to summarize and discuss key-findings about the CCF assay, including its principle, applications, challenges and limits, and perspectives. The CCF assay is based on the decrease of the steady-state accumulation of a tracer substrate in SLC-positive cells, caused by candidate substrates. Reviewed data highlight the fact that the CCF assay has been used to identify substrates and non-substrates for organic cation transporters (OCTs), organic anion transporters (OATs), and organic anion transporting polypeptides (OATPs). The performance values of the CCF assay, calculated from available CCF study data compared with reference functional uptake assay data, are, however, rather mitigated, indicating that the predictability of the CCF method for assessing SLC-mediated transportability of drugs is currently not optimal. Further studies, notably aimed at standardizing the CCF assay and developing CCF-based high-throughput approaches, are therefore required in order to fully precise the interest and relevance of the CCF assay for identifying substrates and non-substrates of SLCs.
RESUMEN
Human membrane drug transporters are recognized as major actors of pharmacokinetics; they also handle endogenous compounds, including hormones and metabolites. Chemical additives present in plastics interact with human drug transporters, which may have consequences for the toxicokinetics and toxicity of these widely-distributed environmental and/or dietary pollutants, to which humans are highly exposed. The present review summarizes key findings about this topic. In vitro assays have demonstrated that various plastic additives, including bisphenols, phthalates, brominated flame retardants, poly-alkyl phenols and per- and poly-fluoroalkyl substances, can inhibit the activities of solute carrier uptake transporters and/or ATP-binding cassette efflux pumps. Some are substrates for transporters or can regulate their expression. The relatively low human concentration of plastic additives from environmental or dietary exposure is a key parameter to consider to appreciate the in vivo relevance of plasticizer-transporter interactions and their consequences for human toxicokinetics and toxicity of plastic additives, although even low concentrations of pollutants (in the nM range) may have clinical effects. Existing data about interactions of plastic additives with drug transporters remain somewhat sparse and incomplete. A more systematic characterization of plasticizer-transporter relationships is needed. The potential effects of chemical additive mixtures towards transporter activities and the identification of transporter substrates among plasticizers, as well as their interactions with transporters of emerging relevance deserve particular attention. A better understanding of the human toxicokinetics of plastic additives may help to fully integrate the possible contribution of transporters to the absorption, distribution, metabolism and excretion of plastics-related chemicals, as well as to their deleterious effects towards human health.
Asunto(s)
Contaminantes Ambientales , Plásticos , Humanos , Plásticos/toxicidad , Plásticos/metabolismo , Toxicocinética , Plastificantes/toxicidad , Proteínas de Transporte de Membrana , Contaminantes Ambientales/toxicidad , Interacciones FarmacológicasRESUMEN
Chimeric mice with humanized liver are thought to represent a sustainable source of isolated human hepatocytes for in vitro studying detoxification of drugs in humans. Because drug transporters are now recognized as key-actors of the hepatic detoxifying process, the present study was designed to characterize mRNA expression and activity of main hepatic drug transporters in cryopreserved human hepatocytes isolated from chimeric TK-NOG mice and termed HepaSH cells. Such cells after thawing were shown to exhibit a profile of hepatic solute carrier (SLC) and ATP-binding cassette (ABC) drug transporter mRNA levels well correlated to those found in cryopreserved primary human hepatocytes or human livers. HepaSH cells used either as suspensions or as 24 h-cultures additionally displayed notable activities of uptake SLCs, including organic anion transporting polypeptides (OATPs), organic anion transporter 2 (OAT2) or sodium-taurocholate co-transporting polypeptide (NTCP). SLC transporter mRNA expression, as well as SLC activities, nevertheless fell in HepaSH cells cultured for 120 h, which may reflect a partial dedifferentiation of these cells with time in culture in the conventional monolayer culture conditions used in the study. These data therefore support the use of cryopreserved HepaSH cells as either suspensions or short-term cultures for drug transport studies.
Asunto(s)
Hígado , Transportadores de Anión Orgánico , Humanos , Ratones , Animales , Suspensiones , Hígado/metabolismo , Hepatocitos/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , ARN Mensajero/metabolismoRESUMEN
Paracetamol/acetaminophen (N-acetyl-p-aminophenol, APAP) is a top selling analgesic used in more than 600 prescription and non-prescription pharmaceuticals. To study efficiently some of the potential undesirable effects associated with increasing APAP consumption (e.g., developmental disorders, drug-induced liver injury), there is a need to improve current APAP biomonitoring methods that are limited by APAP short half-life. Here, we demonstrate using high-resolution mass spectrometry (HRMS) in several human studies that APAP thiomethyl metabolite conjugates (S-methyl-3-thioacetaminophen sulfate and S-methyl-3-thioacetaminophen sulphoxide sulfate) are stable biomarkers with delayed excretion rates compared to conventional APAP metabolites, that could provide a more reliable history of APAP ingestion in epidemiological studies. We also show that these biomarkers could serve as relevant clinical markers to diagnose APAP acute intoxication in overdosed patients, when free APAP have nearly disappeared from blood. Using in vitro liver models (HepaRG cells and primary human hepatocytes), we then confirm that these thiomethyl metabolites are directly linked to the toxic N-acetyl-p-benzoquinone imine (NAPQI) elimination, and produced via an overlooked pathway called the thiomethyl shunt pathway. Further studies will be needed to determine whether the production of the reactive hepatotoxic NAPQI metabolites is currently underestimated in human. Nevertheless, these biomarkers could already serve to improve APAP human biomonitoring, and investigate, for instance, inter-individual variability in NAPQI production to study underlying causes involved in APAP-induced hepatotoxicity. Overall, our findings demonstrate the potential of exposomics-based HRMS approach to advance towards a better precision for human biomonitoring.
Asunto(s)
Acetaminofén , Monitoreo Biológico , Humanos , Acetaminofén/toxicidad , Acetaminofén/química , Acetaminofén/metabolismo , Espectrometría de Masas , Hígado , Biomarcadores/metabolismo , Sulfatos/metabolismoRESUMEN
Chimeric mice with humanized livers constitute an attractive emergent experimental model for investigating human metabolism and disposition of drugs. The present review was designed to summarize key findings about the use of this model for studying human hepatic drug transporters, which are now recognized as important players in pharmacokinetics and consequently have to be considered from a regulatory perspective during pharmaceutical drug development. The reviewed data indicate that chimeric mice with humanized livers have been successfully used for analysing the implications of human hepatic drug transporters for drug hepatobiliary elimination, drug-drug interactions and drug-induced cholestasis. Such transporter studies have been performed in vivo with chimeric mice and/or in vitro with human hepatocytes isolated from humanized liver and used either in suspension or in culture. The residual presence of mouse hepatocytes and the potential morphological/histological alterations of the humanized liver, as well as its immunodeficient mouse environment, have, however, to be considered when using chimeric mice with humanized livers for transporter studies. Finally, if the proof of concept of applying chimeric mice with humanized livers to hepatic drug transport is established, more experimental data on this topic, including from standardization approaches, are likely required to completely and accurately demonstrate the robustness, convenience and added value of this chimeric mouse model for drug transporter studies.
Asunto(s)
Hepatocitos , Hígado , Animales , Quimera/metabolismo , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Tasa de Depuración Metabólica , RatonesRESUMEN
Endotoxemia-induced inflammation may impact the activity of hepatocyte transporters, which control the hepatobiliary elimination of drugs and bile acids. 99mTc-mebrofenin is a non-metabolized substrate of transporters expressed at the different poles of hepatocytes. 99mTc-mebrofenin imaging was performed in rats after the injection of lipopolysaccharide (LPS). Changes in transporter expression were assessed using quantitative polymerase chain reaction of resected liver samples. Moreover, the particular impact of pharmacokinetic drug-drug interactions in the context of endotoxemia was investigated using rifampicin (40 mg/kg), a potent inhibitor of hepatocyte transporters. LPS increased 99mTc-mebrofenin exposure in the liver (1.7 ± 0.4-fold). Kinetic modeling revealed that endotoxemia did not impact the blood-to-liver uptake of 99mTc-mebrofenin, which is mediated by organic anion-transporting polypeptide (Oatp) transporters. However, liver-to-bile and liver-to-blood efflux rates were dramatically decreased, leading to liver accumulation. The transcriptomic profile of hepatocyte transporters consistently showed a downregulation of multidrug resistance-associated proteins 2 and 3 (Mrp2 and Mrp3), which mediate the canalicular and sinusoidal efflux of 99mTc-mebrofenin in hepatocytes, respectively. Rifampicin effectively blocked both the Oatp-mediated influx and the Mrp2/3-related efflux of 99mTc-mebrofenin. The additive impact of endotoxemia and rifampicin led to a 3.0 ± 1.3-fold increase in blood exposure compared with healthy non-treated animals. 99mTc-mebrofenin imaging is useful to investigate disease-associated change in hepatocyte transporter function.
RESUMEN
Because of a narrow therapeutic index and a wide inter- and intra-patient variability, therapeutic drug monitoring of the immunosuppressant drug tacrolimus (TAC) based on whole-blood concentrations (Cblood) is mandatory in solid organ transplant recipients. Using peripheral blood mononuclear cells concentrations (CPBMC) could improve patient outcomes. The poor correlation between Cblood and CPBMC makes hypothesize that drug transporters are implicated in the intracellular accumulation of TAC. The aim of this work was therefore to clinically study: i) the role of genetic variants and ii) the effect of mRNA and protein expression of 4 drug transporters on the TAC CPBMC/blood ratio. In addition, functional in vitro experiments were performed to mechanistically validate the clinical observations. Genetic variants of ABCB1/P-gp and SLC28A3/CNT3 did not influence TAC CPBMC in liver transplant recipients (LTR). ABCC2/MRP2 at the mRNA level; ABCB1/P-gp, SLC28A3/CNT3 and SLC29A1/ENT1 at the protein level; correlated with the CPBMC/blood in kidney and LTR. In vitro results suing transporter-expressing cells confirmed that TAC is substrate of P-gp but not MRP2, whereas experiments remained inconclusive for CNT3 and ENT1. In conclusion, the genetic-transcription-protein-functional approach presented in this work provides new insights in the understanding of TAC transport at the T lymphocyte plasma membrane.
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Trasplante de Hígado , Tacrolimus , Humanos , Leucocitos Mononucleares/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Citocromo P-450 CYP3A/metabolismo , Linfocitos T , Inmunosupresores , RiñónRESUMEN
Introduction: Janus kinase inhibitors (JAKinibs) constitute an emerging and promising pharmacological class of anti-inflammatory or anti-cancer drugs, used notably for the treatment of rheumatoid arthritis and some myeloproliferative neoplasms.Areas covered: This review provides an overview of the interactions between marketed JAKinibs and major uptake and efflux drug transporters. Consequences regarding pharmacokinetics, drug-drug interactions and toxicity are summarized.Expert opinion: JAKinibs interact in vitro with transporters in various ways, as inhibitors or as substrates of transporters or as regulators of transporter expression. This may theoretically result in drug-drug interactions (DDIs), with JAKinibs acting as perpetrators or as victims, or in toxicity, via impairment of thiamine transport. Clinical significance in terms of DDIs for JAKinib-transporter interactions remains however poorly documented. In this context, the in vivo unbound concentration of JAKinibs is likely a key parameter to consider for evaluating the clinical relevance of JAKinibs-mediated transporter inhibition. Additionally, the interplay with drug metabolism as well as possible interactions with transporters of emerging importance and time-dependent inhibition have to be taken into account. The role drug transporters may play in controlling cellular JAKinib concentrations and efficacy in target cells is also an issue of interest.
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Interacciones Farmacológicas , Inhibidores de las Cinasas Janus/farmacología , Proteínas de Transporte de Membrana/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Humanos , Preparaciones Farmacéuticas/metabolismo , Farmacocinética , Tiamina/metabolismo , Factores de TiempoRESUMEN
Interleukin (IL)-22 is a cytokine up-regulated in inflammatory situations and known to exert various hepatic effects. The potential impact of IL-22 towards liver drug detoxifying proteins remains nevertheless unknown, but may be important to determine owing to the well-established alterations of liver detoxification occuring during inflammation. The present study was therefore designed to analyze the effects of IL-22 towards drug metabolizing enzyme and drug transporter expression and activity in cultured human hepatic cells. Exposure of differentiated hepatoma HepaRG cells or primary human hepatocytes to 10 ng/mL IL-22 was found to repress mRNA expression of cytochrome P-450 (CYP) 1A2, CYP3A4, CYP2B6 and CYP2C9 and of the sinusoidal sodium-taurocholate co-transporting polypeptide (NTCP); such IL-22 effects were concentration-dependent for CYP3A4 (IC50 = 1.7 ng/mL), CYP2B6 (IC50 = 0.9 ng/mL) and NTCP (IC50 = 1.8 ng/mL). Activity of CYP1A2 (phenacetin O-deethylation), CYP3A4 (midazolam hydroxylation) and CYP2B6 (bupropion hydroxylation), as well as that of NTCP (taurocholate uptake) were concomitantly decreased in IL-22-treated HepaRG cells; by contrast, activity of organic anion transporter polypeptides (OATPs) (estrone-3-sulfate uptake) and of organic cation transporter (OCT) 1 (tetra-ethylammonium uptake) remained unchanged. IL-22 was next found to activate the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) 3 pathway, whose inhibition by the JAK inhibitor ruxolitinib fully prevented the IL-22-mediated CYP3A4, CYP2B6 and NTCP repression in HepaRG cells. This JAK-dependent down-regulation of hepatic drug detoxifying proteins, notably of CYPs, by IL-22 may contribute to alteration of pharmacokinetics in patients suffering from acute and chronic inflammatory diseases and may be the source of drug-drug interactions.
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Hepatocitos/fisiología , Interleucinas/metabolismo , Pirazoles/farmacocinética , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Regulación de la Expresión Génica , Células Hep G2 , Humanos , Inactivación Metabólica , Interleucinas/genética , Quinasas Janus/metabolismo , Nitrilos , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Pirimidinas , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Simportadores/genética , Simportadores/metabolismo , Interleucina-22RESUMEN
Human membrane drug transporters are recognized as major actors of pharmacokinetics. Pesticides also interact with human drug transporters, which may have consequences for pesticide toxicokinetics and toxicity. The present review summarizes key findings about this topic. In vitro assays have demonstrated that some pesticides, belonging to various chemical classes, modulate drug transporter activity, regulate transporter expression and/or are substrates, thus bringing the proof of concept for pesticide-transporter relationships. The expected low human concentration of pesticides in response to environmental exposure constitutes a key-parameter to be kept in mind for judging the in vivo relevance of such pesticide-transporter interactions and their consequences for human health. Existing data about interactions of pesticides with drug transporters remain, however, rather sparse; more extensive and systematic characterization of pesticide-transporter relationships, through the use of high throughput in vitro assays and/or in silico methods, is, therefore, required. In addition, consideration of transporter polymorphisms, pesticide mixture effects and physiological and pathological factors governing drug transporter expression may help to better define the in vivo relevance of pesticide-transporter interactions in terms of toxicokinetics and toxicity for humans. © 2019 Society of Chemical Industry.