RESUMO
The objective of the current study was to identify potential drug-drug interactions (DDIs) with the drug candidate fb-PMT, a novel anticancer thyrointegrin αvß3 antagonist. This was accomplished by using several in vitro assays to study interactions of fb-PMT with both cytochrome P450 (CYP) enzymes and drug transporters, two common mechanisms leading to adverse drug effects. In vitro experiments showed that fb-PMT exhibited weak reversible inhibition of CYP2C19 and CYP3A4. In addition, fb-PMT did not show time-dependent inhibition with any of the seven CYP isoforms tested, including 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4. Human liver microsomal incubations demonstrated that fb-PMT is stable. Potential transporter-mediated DDIs with fb-PMT were assessed with two ATP binding cassette (ABC) family transporters (P-glycoprotein and breast cancer resistance protein) using Caco2 cells and seven solute carrier family (SLC) transporters (organic cation transporter OCT2, organic anion transporters OAT1 and OAT3, organic anion transporter peptides OATP1B1 and OATP1B3, and the multidrug and toxic extrusion proteins MATE1 and MATE2-K using transfected HEK293 cells). Fb-PMT was not a substrate for any of the nine transporters tested in this study, nor did it inhibit the activity of seven of the transporters tested. However, fb-PMT inhibited the uptake of rosuvastatin by both OATP1B1 and OATP1B3 with half-maximal inhibitory concentrations greater than 3 and less than 10 µM. In summary, data suggest that the systemic administration of fb-PMT is unlikely to lead to DDIs through CYP enzymes or ABC and SLC transporters in humans.
Assuntos
Transportadores de Ânions Orgânicos Sódio-Independentes , Transportadores de Ânions Orgânicos , Humanos , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Células CACO-2 , Células HEK293 , Proteínas de Neoplasias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Interações Medicamentosas , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Ânions Orgânicos/metabolismoRESUMO
The use of hepatocytes to predict human hepatic metabolic clearance is the gold standard approach. However whilst enzymes are well characterised, knowledge gaps remain for transporters. Furthermore, methods to study specific transporter involvement are often complicated by overlapping substrate specificity. Selective substrates and inhibitors would aid investigations into clinically relevant pharmacokinetic effects. However, to date no consensus has been reached.This work defines selective hepatic uptake transporter substrates and inhibitors for the six main human hepatocyte transporters (OATP1B1, OATP1B3, OATP2B1, NTCP, OAT2 & OCT1), and demonstrates their use to rapidly characterise batches of human hepatocytes for uptake transporter activity. Hepatic uptake was determined across a range of substrate concentrations, allowing the definition of kinetic parameters and hence active and passive components. Systematic investigations identified a specific substrate and inhibitor for each transporter, with no overlap between the specificity of substrate and inhibitor for any given transporter.Early characterisation of compound interactions with uptake transporters will aid in early risk assessment and chemistry design. Hence, this work further highlights the feasibility of a refined methodology for rapid compound characterisation for the application of static and dynamic models, for early clinical risk assessment and guidance for the clinical development plan.
Assuntos
Descoberta de Drogas , Hepatócitos , Transportadores de Ânions Orgânicos , Humanos , Transporte Biológico , Descoberta de Drogas/métodos , Células HEK293 , Hepatócitos/metabolismo , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismoRESUMO
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
BACKGROUND: Letermovir is approved for prophylaxis of cytomegalovirus infection and disease in cytomegalovirus-seropositive hematopoietic stem-cell transplant (HSCT) recipients. OBJECTIVE: HSCT recipients are required to take many drugs concomitantly. The pharmacokinetics, absorption, distribution, metabolism, and excretion of letermovir and its potential to inhibit metabolizing enzymes and transporters in vitro were investigated to inform on the potential for drug-drug interactions (DDIs). METHODS: A combination of in vitro and in vivo studies described the absorption, distribution, metabolism, and routes of elimination of letermovir, as well as the enzymes and transporters involved in these processes. The effect of letermovir to inhibit and induce metabolizing enzymes and transporters was evaluated in vitro and its victim and perpetrator DDI potentials were predicted by applying the regulatory guidance for DDI assessment. RESULTS: Letermovir was a substrate of CYP3A4/5 and UGT1A1/3 in vitro. Letermovir showed concentration- dependent uptake into organic anionic transporting polypeptide (OATP)1B1/3-transfected cells and was a substrate of P-glycoprotein (P-gp). In a human ADME study, letermovir was primarily recovered as unchanged drug and minor amounts of a direct glucuronide in feces. Based on the metabolic pathway profiling of letermovir, there were few oxidative metabolites in human matrix. Letermovir inhibited CYP2B6, CYP2C8, CYP3A, and UGT1A1 in vitro, and induced CYP3A4 and CYP2B6 in hepatocytes. Letermovir also inhibited OATP1B1/3, OATP2B1, OAT3, OCT2, BCRP, BSEP, and P-gp. CONCLUSION: The body of work presented in this manuscript informed on the potential for DDIs when letermovir is administered both intravenously and orally in HSCT recipients.
Assuntos
Acetatos , Biotransformação , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/imunologia , Vias de Eliminação de Fármacos/fisiologia , Interações Medicamentosas , Quinazolinas , Distribuição Tecidual/fisiologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Acetatos/metabolismo , Acetatos/farmacocinética , Adulto , Animais , Antivirais/metabolismo , Antivirais/farmacocinética , Citocromo P-450 CYP3A/metabolismo , Glucuronosiltransferase/metabolismo , Voluntários Saudáveis , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Masculino , Conduta do Tratamento Medicamentoso/normas , Proteínas de Neoplasias/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Quinazolinas/metabolismo , Quinazolinas/farmacocinética , RatosRESUMO
Objectives: To determine whether the pharmacokinetic parameters of Gd-EOB-DTPA can identify the difference in liver function in a rat hepatectomy model. Methods: A total of 56 eight-week-old male Sprague-Dawley rats were divided into the following groups: control group without hepatectomy (n = 16), 70% hepatectomy group (n = 14), and 90% hepatectomy group (n = 26). On postoperative day 2, Gd-EOB-DTPA (0.1 mmol/kg) was injected intravenously and serial blood samples were obtained. Pharmacokinetic analysis was performed using a noncompartmental method. Statistical analysis was performed using one-way analysis of variance and post hoc pairwise group comparisons. Results: After excluding 6 rats that died unexpectedly, blood samples were obtained from 16, 14, and 20 rats in the control group, 70% hepatectomy group, and 90% hepatectomy group. There was a significant increase in area under the concentration-time curve from time zero to the time of the last measurable concentration between the 70% and 90% hepatectomy group (P < 0.001). The volume of distribution at steady state was significantly decreased between the control and 70% hepatectomy group (P < 0.001). The clearance was significantly different in all pairwise group comparisons (P < 0.001). Conclusions: The vascular clearance of Gd-EOB-DTPA can identify the difference in liver function in a rat hepatectomy model.
Assuntos
Gadolínio DTPA/farmacocinética , Hepatectomia/métodos , Testes de Função Hepática/métodos , Fígado/fisiologia , Taxa de Depuração Metabólica , Animais , Meios de Contraste/farmacocinética , Fígado/cirurgia , Masculino , Transportadores de Ânions Orgânicos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
1. Accurate predictions of clinical transporter-mediated drug-drug interactions (DDI) from in vitro data can be challenging when compounds have poor solubility and/or high nonspecific binding. Additionally, current DDI predictions for compounds with high plasma-protein binding assume that the unbound fraction in plasma is 0.01, if the experimental value is less than 0.01 or cannot be determined. This approach may result in an overestimation of DDI risk. To overcome these challenges, it may be beneficial to conduct inhibition studies under physiologically relevant conditions. 2. Here, IC50 values, determined in the presence of 4% bovine serum albumin approximating human plasma albumin concentrations, were successfully used to predict DDI for uptake transporters, OATP1B1/1B3, OCT1/2, OAT1/3 and MATE1/2K. 3. The IC50 values of reference inhibitors with 4% bovine serum albumin, considered total IC50, were comparable to the predicted values based on nominal IC50 values determined under protein-free conditions and unbound fraction in plasma. Calculation of R-total and Cmax/IC50,total values using total plasma exposure and total IC50 values explained the clinical DDI or absence of it for these inhibitors. 4. These results suggest that IC50 determinations in the presence of 4% albumin can be used, in the context of clinical total exposure, to predict DDI involving uptake transporters.
Assuntos
Interações Medicamentosas , Concentração Inibidora 50 , Proteínas de Membrana Transportadoras/metabolismo , Humanos , Transportadores de Ânions Orgânicos/metabolismoRESUMO
[18F]Fluciclovine (trans-1-amino-3-[18F]fluorocyclobutanecarboxylic acid; anti-[18F]FACBC), a positron emission tomography tracer used for the diagnosis of recurrent prostate cancer, is transported via amino acid transporters (AATs) with high affinity (Km: 97-230 µM). However, the mechanism underlying urinary excretion is unknown. In this study, we investigated the involvement of AATs and drug transporters in renal [18F]fluciclovine reuptake. [14C]Fluciclovine (trans-1-amino-3-fluoro[1-14C]cyclobutanecarboxylic acid) was used because of its long half-life. The involvement of AATs in [14C]fluciclovine transport was measured by apical-to-basal transport using an LLC-PK1 monolayer as model for renal proximal tubules. The contribution of drug transporters herein was assessed using vesicles/cells expressing the drug transporters P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance-associated protein 4 (MRP4), organic anion transporter 1 (OAT1), organic anion transporter 3 (OAT3) , organic cation transporter 2 (OCT2), organic anion transporting polypeptide 1B1 (OATP1B1), and organic anion transporting polypeptide 1B3 (OATP1B3). The apical-to-basal transport of [14C]fluciclovine was attenuated by l-threonine, the substrate for system alanine-serine-cysteine (ASC) AATs. [14C]Fluciclovine uptake by drug transporter-expressing vesicles/cells was not significantly different from that of control vesicles/cells. Fluciclovine inhibited P-gp, MRP4, OAT1, OCT2, and OATP1B1 (IC50 > 2.95 mM). Therefore, system ASC AATs may be partly involved in the renal reuptake of [18F]fluciclovine. Further, given that [18F]fluciclovine is recognized as an inhibitor with millimolar affinity for the tested drug transporters, slow urinary excretion of [18F]fluciclovine may be mediated by system ASC AATs, but not by drug transporters.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Ácidos Carboxílicos/metabolismo , Ciclobutanos/metabolismo , Rim/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico , Linhagem Celular , Células HEK293 , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismoRESUMO
1. Cabotegravir (CAB; GSK1265744) is a potent HIV integrase inhibitor in clinical development as an oral lead-in tablet and long-acting injectable for the treatment and prevention of HIV infection. 2. This work investigated if CAB was a substrate for efflux transporters, the potential for CAB to interact with drug-metabolizing enzymes and transporters to cause clinical drug interactions, and the effect of CAB on the pharmacokinetics of midazolam, a CYP3A4 probe substrate, in humans. 3. CAB is a substrate for Pgp and BCRP; however, its high intrinsic membrane permeability limits the impact of these transporters on its intestinal absorption. 4. At clinically relevant concentrations, CAB did not inhibit or induce any of the CYP or UGT enzymes evaluated in vitro and had no effect on the clinical pharmacokinetics of midazolam. 5. CAB is an inhibitor of OAT1 (IC50 0.81 µM) and OAT3 (IC50 0.41 µM) but did not or only weakly inhibited Pgp, BCRP, MRP2, MRP4, MATE1, MATE2-K, OATP1B1, OATP1B3, OCT1, OCT2 or BSEP. 6. Based on regulatory guidelines and quantitative extrapolations, CAB has a low propensity to cause clinically significant drug interactions, except for coadministration with OAT1 or OAT3 substrates.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Inibidores de Integrase de HIV/química , Midazolam/administração & dosagem , Piridonas/administração & dosagem , Transportadores de Cassetes de Ligação de ATP/metabolismo , Administração Oral , Adolescente , Adulto , Idoso , Animais , Fármacos Anti-HIV/farmacocinética , Área Sob a Curva , Citocromo P-450 CYP3A/química , Cães , Relação Dose-Resposta a Droga , Interações Medicamentosas , Feminino , Infecções por HIV/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Células Madin Darby de Rim Canino , Masculino , Midazolam/farmacocinética , Pessoa de Meia-Idade , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Piridonas/farmacocinética , Adulto JovemRESUMO
Rifabutin, used to treat HIV-infected tuberculosis, shows highly variable drug exposure, complicating dosing. Effects of SLCO1B1 polymorphisms on rifabutin pharmacokinetics were investigated in 35 African HIV-infected tuberculosis patients after multiple doses. Nonlinear mixed-effects modeling found that influential covariates for the pharmacokinetics were weight, sex, and a 30% increased bioavailability among heterozygous carriers of SLCO1B1 rs1104581 (previously associated with low rifampin concentrations). Larger studies are needed to understand the complex interactions of host genetics in HIV-infected tuberculosis patients. (This study has been registered at ClinicalTrials.gov under registration no. NCT00640887.).
Assuntos
Antituberculosos/farmacocinética , Transportadores de Ânions Orgânicos/genética , Polimorfismo de Nucleotídeo Único , Rifabutina/farmacocinética , Tuberculose Pulmonar/tratamento farmacológico , Adulto , Antituberculosos/administração & dosagem , Antituberculosos/sangue , Área Sob a Curva , Contagem de Linfócito CD4 , Coinfecção , Etambutol/administração & dosagem , Feminino , Expressão Gênica , Infecções por HIV/patologia , Infecções por HIV/virologia , Humanos , Isoniazida/administração & dosagem , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Método de Monte Carlo , Transportadores de Ânions Orgânicos/metabolismo , Pirazinamida/administração & dosagem , Rifabutina/administração & dosagem , Rifabutina/sangue , Rifampina/administração & dosagem , Fatores Sexuais , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologiaRESUMO
Probe drug cocktails are used clinically to assess the potential for drug-drug interactions (DDIs), and in particular, DDIs resulting from coadministration of substrates and inhibitors of cytochrome P450 enzymes. However, a probe drug cocktail has not been identified to assess DDIs involving inhibition of drug transporters. We propose a cocktail consisting of the following substrates to explore the potential for DDIs caused by inhibition of key transporters: digoxin (P-glycoprotein, P-gp), rosuvastatin (breast cancer resistance protein, BCRP; organic anion transporting polypeptides, OATP), metformin (organic cation transporter, OCT; multidrug and toxin extrusion transporters, MATE), and furosemide (organic anion transporter, OAT). Furosemide was evaluated in vitro, and is a substrate of OAT1 and OAT3, with Km values of 38.9 and 21.5 µM, respectively. Furosemide was also identified as a substrate of BCRP, OATP1B1, and OATP1B3. Furosemide inhibited BCRP (50% inhibition of drug transport: 170 µM), but did not inhibit OATP1B1, OATP1B3, OCT2, MATE1, and MATE2-K at concentrations below 300 µM, and P-gp at concentrations below 2000 µM. Conservative approaches for the estimation of the likelihood of in vivo DDIs indicate a remote chance of in vivo transporter inhibition by these probe drugs when administered at low single oral doses. This four component probe drug cocktail is therefore proposed for clinical evaluation.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Digoxina/metabolismo , Furosemida/metabolismo , Metformina/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Rosuvastatina Cálcica/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/fisiologia , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Interações Medicamentosas/fisiologia , Células HEK293 , Humanos , Proteínas de Neoplasias/metabolismoRESUMO
Drug transporter proteins recruit to pharmacological barrier tissues and profoundly affect the ADME properties of a large number of drugs. In vitro assays optimized for drug transporters have grown into routine tools in the determination of molecular level interactions as well as prediction of barrier penetration and system level pharmacokinetics. Regulatory position mandates increasing interest in the application of these assays during drug development.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Bioensaio/métodos , Descoberta de Drogas/métodos , Transportadores de Ânions Orgânicos/metabolismo , Preparações Farmacêuticas , Farmacocinética , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Animais , Sítios de Ligação , Transporte Biológico , Membrana Celular/metabolismo , Interações Medicamentosas , Humanos , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Preparações Farmacêuticas/metabolismo , Especificidade por SubstratoRESUMO
Aggregate or spheroid culture has emerged as a more biologically relevant method for screening pharmaceutical compounds and understanding exact mechanism of action. Here in, the aggregate approach applied to the freshwater toxins, microcystins, further unearths exact mechanism(s) of toxicity and provides a markedly improved in vitro predictor of toxicity. Microcystins result in acute intoxication by binding covalently to protein phosphatase 1/2A, resulting in hepatocellular necrosis, hemorrhaging and death. Hepatocellular uptake by organic anion transporting polypeptides (OATPs), in addition to other intracellular sequelae, is considered essential for toxicity. In aggregate HepG2, expression of OAT1B1 and OATP1B3 significantly increased relative to monolayer culture. Uptake of two fluorescently labeled substrates significantly increased in aggregates compared with monolayer, confirmed by inhibition of uptake with known competitive substrates. Increased reaction oxygen species (ROS) production occurred following a three-hour exposure of microcystin LR at concentrations from 100 nM to 100 µM, with reversal by ROS scavengers, in contrast with no response in monolayers. These results suggest monolayer culture inadequately predict intracellular effects of microcystins and support evidence that aggregate culture more closely approximates in vivo form and function. The approach results in more reliable prediction of microcystin toxicity in vitro.
Assuntos
Microcistinas/toxicidade , Testes de Toxicidade/métodos , Técnicas de Cultura de Células , Sobrevivência Celular , Células Hep G2 , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado , Microcistinas/análise , Microtecnologia , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Espécies Reativas de Oxigênio , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de SolutoRESUMO
The absorption, distribution, metabolism, and excretion (ADME) of drugs in vivo are to a large extent dependent on different transport and metabolism routes. Elucidation of this complex transport-metabolism interplay is a major challenge in drug development and at present no in vitro models suitable for this purpose are at hand. The aim of this study was to develop flexible, well-controlled, easy-to-use, integrated cell models, where drug transport and drug metabolism processes could be studied simultaneously. HEK293 cells stably transfected with the organic anion transporting polypeptide 1B1 (OATP1B1) were subjected to either transient transfection or adenoviral infection to introduce the genes expressing cytochrome P450 3A4 (CYP3A4), NADPH cytochrome P450 oxidoreductase (POR), cytochrome b5 (CYB5A), and multidrug resistance protein 1 (MDR1), in different combinations. Thereafter, the time and concentration-dependent transport and metabolism of two well-characterized statins, atorvastatin (acid and lactone forms) and simvastatin (acid form), were determined in the different models. The results show that CYP3A4-dependent metabolism of the more hydrophilic atorvastatin acid was dependent on OATP1B1 uptake and influenced by MDR1 efflux. In contrast, the metabolism of the more lipophilic atorvastatin lactone was not affected by active transport, whereas the metabolism of simvastatin acid was less influenced by active transport than atorvastatin acid. Our results, together with the models being applicative for any combination of drug transporters and CYP metabolizing enzymes of choice, provide proof-of-concept for the potential of the new integrated cell models presented as valuable screening tools in drug discovery and development.
Assuntos
Transporte Biológico/fisiologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adenoviridae , Linhagem Celular , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Citocromos b5/genética , Citocromos b5/metabolismo , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Sinvastatina/análogos & derivados , Sinvastatina/metabolismoRESUMO
Drug-induced liver injury (DILI) is a clinically important adverse drug reaction, which prevents the development of many otherwise safe and effective new drugs. Currently, there is a lack of sensitive and specific biomarkers that can be used to predict, assess and manage this toxicity. The aim of this work was to evaluate gadoxetate-enhanced MRI as a potential novel biomarker of hepatobiliary transporter inhibition in the rat. Initially, the volume fraction of extracellular space in the liver was determined using gadopentetate to enable an estimation of the gadoxetate concentration in hepatocytes. Using this information, a compartmental model was developed to characterise the pharmacokinetics of hepatic uptake and biliary excretion of gadoxetate. Subsequently, we explored the impact of an investigational hepatobiliary transporter inhibitor on the parameters of the model in vivo in rats. The investigational hepatobiliary transporter inhibitor reduced both the rate of uptake of gadoxetate into the hepatocyte, k1 , and the Michaelis-Menten constant, Vmax , characterising its excretion into bile, whereas KM values for biliary efflux were increased. These effects were dose dependent and correlated with effects on plasma chemistry markers of liver dysfunction, in particular bilirubin and bile acids. These results indicate that gadoxetate-enhanced MRI provides a novel functional biomarker of inhibition of transporter-mediated hepatic uptake and clearance in the rat. Since gadoxetate is used clinically, the technology has the potential to provide a translatable biomarker of drug-induced perturbation of hepatic transporters that may also be useful in humans to explore deleterious functional alterations caused by transporter inhibition.
Assuntos
Sistema Biliar/metabolismo , Biomarcadores/metabolismo , Meios de Contraste , Gadolínio DTPA , Fígado/metabolismo , Imageamento por Ressonância Magnética , Proteínas de Membrana Transportadoras/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Espaço Extracelular/metabolismo , Gadolínio DTPA/farmacocinética , Células HEK293 , Hepatócitos/metabolismo , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Ratos , Ratos WistarRESUMO
The inhibitory effect of boceprevir (BOC), an inhibitor of hepatitis C virus nonstructural protein 3 protease was evaluated in vitro against a panel of drug-metabolizing enzymes and transporters. BOC, a known substrate for cytochrome P450 (P450) CYP3A and aldo-ketoreductases, was a reversible time-dependent inhibitor (k(inact) = 0.12 minute(-1), K(I) = 6.1 µM) of CYP3A4/5 but not an inhibitor of other major P450s, nor of UDP-glucuronosyltransferases 1A1 and 2B7. BOC showed weak to no inhibition of breast cancer resistance protein (BCRP), P-glycoprotein (Pgp), or multidrug resistance protein 2. It was a moderate inhibitor of organic anion transporting polypeptide (OATP) 1B1 and 1B3, with an IC(50) of 18 and 4.9 µM, respectively. In human hepatocytes, BOC inhibited CYP3A-mediated metabolism of midazolam, OATP1B-mediated hepatic uptake of pitavastatin, and both the uptake and metabolism of atorvastatin. The inhibitory potency of BOC was lower than known inhibitors of CYP3A (ketoconazole), OATP1B (rifampin), or both (telaprevir). BOC was a substrate for Pgp and BCRP but not for OATP1B1, OATP1B3, OATP2B1, organic cation transporter, or sodium/taurocholate cotransporting peptide. Overall, our data suggest that BOC has the potential to cause pharmacokinetic interactions via inhibition of CYP3A and CYP3A/OATP1B interplay, with the interaction magnitude lower than those observed with known potent inhibitors. Conversely, pharmacokinetic interactions of BOC, either as a perpetrator or victim, via other major P450s and transporters tested are less likely to be of clinical significance. The results from clinical drug-drug interaction studies conducted thus far are generally supportive of these conclusions.
Assuntos
Antivirais/metabolismo , Inibidores Enzimáticos/metabolismo , Enzimas/metabolismo , Fígado/enzimologia , Moduladores de Transporte de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Prolina/análogos & derivados , Animais , Antivirais/toxicidade , Biotransformação , Células CHO , Cricetinae , Cricetulus , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A , Cães , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/toxicidade , Enzimas/genética , Feminino , Glucuronosiltransferase/metabolismo , Humanos , Cinética , Células LLC-PK1 , Fígado/efeitos dos fármacos , Transportador 1 de Ânion Orgânico Específico do Fígado , Células Madin Darby de Rim Canino , Masculino , Moduladores de Transporte de Membrana/toxicidade , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Microssomos Hepáticos/enzimologia , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Oxirredutases/metabolismo , Prolina/metabolismo , Prolina/toxicidade , Proteínas Recombinantes/metabolismo , Suínos , TransfecçãoRESUMO
PURPOSE: To apply physiologically-based pharmacokinetic (PBPK) modeling to investigate the consequences of reduction in activity of hepatic and intestinal uptake and efflux transporters by cyclosporine and its metabolite AM1. METHODS: Inhibitory potencies of cyclosporine and AM1 against OATP1B1, OATP1B3 and OATP2B1 were investigated in HEK293 cells +/- pre-incubation. Cyclosporine PBPK model implemented in Matlab was used to assess interaction potential (+/- metabolite) against different processes (uptake, efflux and metabolism) in liver and intestine and to predict quantitatively drug-drug interaction with repaglinide. RESULTS: Cyclosporine and AM1 were potent inhibitors of OATP1B1 and OATP1B3, IC(50) ranging from 0.019-0.093 µM following pre-incubation. Cyclosporine PBPK model predicted the highest interaction potential against liver uptake transporters, with a maximal reduction of >70% in OATP1B1 activity; the effect on hepatic efflux and metabolism was minimal. In contrast, 80-97% of intestinal P-gp and CYP3A4 activity was reduced due to the 50-fold higher cyclosporine enterocytic concentrations relative to unbound hepatic inlet. The inclusion of AM1 resulted in a minor increase in the predicted maximal reduction of OATP1B1/1B3 activity. Good predictability of cyclosporine-repaglinide DDI and the impact of dose staggering are illustrated. CONCLUSIONS: This study highlights the application of PBPK modeling for quantitative prediction of transporter-mediated DDIs with concomitant consideration of P450 inhibition.
Assuntos
Ciclosporina/farmacologia , Inibidores do Citocromo P-450 CYP3A , Inibidores Enzimáticos/farmacologia , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Ciclosporina/metabolismo , Ciclosporina/farmacocinética , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Células HEK293 , Humanos , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado , Modelos Biológicos , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de SolutoRESUMO
Mesna and its dimer, dimesna, are coadministered for mitigation of ifosfamide- and cisplatin-induced toxicities, respectively. Dimesna is selectively reduced to mesna in the kidney, producing its protective effects. In vitro screens of uptake and efflux transporters revealed saturable uptake by renal organic anion transporters OAT1, OAT3, and OAT4. Efflux transporters breast cancer resistance protein; multidrug and toxin extrusion 1 (MATE1); multidrug resistance proteins MRP1, MRP2, MRP4, and MRP5; and P-glycoprotein (Pgp) significantly reduced dimesna accumulation. Further investigation demonstrated that renal apical efflux transporters MATE1, MRP2, and Pgp were also capable of mesna efflux. Administration of OAT inhibitor probenecid to healthy subjects significantly increased combined mesna and dimesna plasma exposure (91% ± 34%) while decreasing the renal clearance due to net secretion (67.0% ± 12.7%) and steady-state volume of distribution (45.2% ± 13.4%). Thus, the kidney represents a significant sink of total mesna, whereas function of renal drug transporters facilitates clearance in excess of glomerular filtration rate and likely the presence of active mesna in the urine. Loss of renal transporter function due to genetic variability or drug-drug interactions may decrease the efficacy of chemoprotectants, increasing the risk of ifosfamide- and cisplatin-induced toxicities.
Assuntos
Rim/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mesna/farmacocinética , Substâncias Protetoras/farmacocinética , Adulto , Feminino , Taxa de Filtração Glomerular , Células HeLa , Humanos , Masculino , Mesna/análogos & derivados , Pessoa de Meia-Idade , Transportadores de Ânions Orgânicos/metabolismo , Probenecid/farmacologia , Distribuição Tecidual , Adulto JovemRESUMO
OBJECTIVE: The pharmaceutical industry currently offers six different statins in Spain and there is one more soon to be available. Choosing the most appropriate drug and dose is determined by the therapeutic target (reduction in LDL-C levels). Statin doses that decrease LDL-C at the same percentage are considered equivalent. Evaluating the pharmacokinetic characteristics of each statin can be useful when setting selection criteria, helping to determine which statin may be more appropriate for a patient based on their individual characteristics and on the other co-administered drugs. METHODS: We reviewed the pharmacokinetics properties of each statin and its possible involvement in drug interactions. RESULTS: CYP3A4 was responsible for the metabolism of lovastatin, simvastatin and atorvastatin; fluvastatin depends on CYP2C9; P-glycoprotein is responsible for decreased atorvastatin, pravastatin, simvastatin and lovastatin concentrations. The OATPA1B1 transporter involved in all statins' access to the hepatocyte, except for fluvastatin, is essential for rosuvastatin and pravastatin. These circumstances cause those drugs inhibiting or inducing isoenzymes or transporters' activity not to have the same effect on the different statins. CONCLUSION: The pharmacokinetics is important when choosing the best statin and could be a limitation in the use of interchange therapeutic programmes when other drugs are present.
Assuntos
Anticolesterolemiantes/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipercolesterolemia/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Anticolesterolemiantes/efeitos adversos , Anticolesterolemiantes/farmacocinética , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2C9 , Indústria Farmacêutica , Interações Medicamentosas , Prescrições de Medicamentos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado , Transportadores de Ânions Orgânicos/metabolismo , EspanhaRESUMO
AIM: To investigate the effect of quercetin on organic anion transporting polypeptide 1B1 (OATP1B1) activities in vitro and on the pharmacokinetics of pravastatin, a typical substrate for OATP1B1 in healthy Chinese-Han male subjects. METHODS: Using human embryonic kidney 293 (HEK293) cells stably expressing OATP1B1, we observed the effect of quercetin on OATP1B1-mediated uptake of estrone-3-sulphate (E3S) and pravastatin. The influence of quercetin on the pharmacokinetics of pravastatin was measured in 16 healthy Chinese-Han male volunteers receiving a single dose of pravastatin (40 mg orally) after co-administration of placebo or 500 mg quercetin capsules (once daily orally for 14 days). RESULTS: Quercetin competitively inhibited OATP1B1-mediated E3S uptake with a K(i) value of 17.9 ± 4.6 µm and also inhibited OATP1B1-mediated pravastatin uptake in a concentration dependent manner (IC(50) , 15.9 ± 1.4 µm). In healthy Chinese-Han male subjects, quercetin increased the pravastatin area under the plasma concentration - time curve (AUC(0,10 h) and the peak plasma drug concentration (C(max)) to 24% (95% CI 15, 32%, P < 0.001) and 31% (95% CI 20, 42%, P < 0.001), respectively. After administration of quercetin, the elimination half-life (t(1/2) ) of pravastatin was prolonged by 14% (95% CI 4, 24%, P = 0.027), with no change in the time to reach C(max) (t(max) ). Moreover, quercetin decreased the apparent clearance (CL/F) of pravastatin by 18% (95% CI 75, 89%, P < 0.001). CONCLUSIONS: These findings suggest that quercetin inhibits the OATP1B1-mediated transport of E3S and pravastatin in vitro and also has a modest inhibitory influence on the pharmacokinetics of pravastatin in healthy Chinese-Han male volunteers. The effects of quercetin on other OATP1B1 substrate drugs deserve further investigation.
Assuntos
Antioxidantes/farmacologia , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Quercetina/farmacologia , Área Sob a Curva , Povo Asiático , Células Cultivadas , Estudos Cross-Over , Método Duplo-Cego , Interações Medicamentosas , Estrogênios/metabolismo , Estrona/análogos & derivados , Estrona/metabolismo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Transportadores de Ânions Orgânicos/metabolismo , Pravastatina/farmacocinética , Adulto JovemRESUMO
Hepatic disposition plays a significant role in the pharmacokinetics and pharmacodynamics of a variety of drugs. Sinusoidal membrane transporters have been shown to participate in the hepatic disposition of many pharmaceuticals. Two sinusoidal membrane transporters with an established role in hepatic disposition are OATP1B1 and OATP1B3 (organic anion-transporting polypeptides 1B1 and 1B3, respectively). OATP1B1 and OATP1B3 have been implicated in the hepatic uptake of statin drugs, and polymorphisms linked to OATP1B1 have been associated with deleterious patient endpoints. As a result, OATP1B1 and OATP1B3 represent sites for potential drug-drug interactions. Numerous methods exist for identifying potential drug-drug interactions with transporters. However, relatively few offer the convenience and speed of fluorescence-based assays. Here a fluorescence-based assay was developed for measuring the OATP1B1- and OATP1B3-mediated transport of 8-fluorescein-cAMP (8-FcA). The OATP1B1- and OATP1B3-mediated transport of 8-FcA was time dependent and saturable (K(m)=2.9 and 1.8 microM, V(max)=0.20 and 0.33 pmol/min/cm(2), respectively). Molecules known to interact with OATPs, including cyclosporin A, rifampicin, and glibenclamide, each demonstrated concentration-dependent inhibition of 8-FcA transport by OATP1B1 and OATP1B3. The in vitro fluorescence-based assays described here using 8-FcA as the substrate are convenient and rapid and have utility in screening drug candidates for potential drug-drug interactions with OATP1B1 and OATP1B3.