RESUMO
Probenecid is used to treat gout and hyperuricemia as well as increase plasma levels of antiviral drugs and antibiotics. In vivo, probenecid mainly inhibits the renal SLC22 organic anion transporters OAT1 (SLC22A6), OAT3 (SLC22A8), and URAT1 (SLC22A12). To understand the endogenous role of these transporters in humans, we administered probenecid to 20 healthy participants and metabolically profiled the plasma and urine before and after dosage. Hundreds of metabolites were significantly altered, indicating numerous drug-metabolite interactions. We focused on potential OAT1 substrates by identifying 97 metabolites that were significantly elevated in the plasma and decreased in the urine, indicating OAT-mediated clearance. These included signaling molecules, antioxidants, and gut microbiome products. In contrast, urate was the only metabolite significantly decreased in the plasma and elevated in the urine, consistent with an effect on renal reuptake by URAT1. Additional support comes from metabolomics analyses of our Oat1 and Oat3 knockout mice, where over 50% of the metabolites that were likely OAT substrates in humans were elevated in the serum of the mice. Fifteen of these compounds were elevated in both knockout mice, whereas six were exclusive to the Oat1 knockout and 4 to the Oat3 knockout. These may be endogenous biomarkers of OAT function. We also propose a probenecid stress test to evaluate kidney proximal tubule organic anion transport function in kidney disease. Consistent with the Remote Sensing and Signaling Theory, the profound changes in metabolite levels following probenecid treatment support the view that SLC22 transporters are hubs in the regulation of systemic human metabolism.
Assuntos
Transportadores de Ânions Orgânicos , Proteínas de Transporte de Cátions Orgânicos , Animais , Ânions/metabolismo , Ânions/farmacologia , Humanos , Rim/metabolismo , Camundongos , Camundongos Knockout , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Probenecid/farmacologiaRESUMO
Persistent pain is sustained by maladaptive changes in gene transcription resulting in altered function of the relevant circuits; therapies are still unsatisfactory. The epigenetic mechanisms and affected genes linking nociceptive activity to transcriptional changes and pathological sensitivity are unclear. Here, we found that, among several histone deacetylases (HDACs), synaptic activity specifically affects HDAC4 in murine spinal cord dorsal horn neurons. Noxious stimuli that induce long-lasting inflammatory hypersensitivity cause nuclear export and inactivation of HDAC4. The development of inflammation-associated mechanical hypersensitivity, but neither acute nor basal sensitivity, is impaired by the expression of a constitutively nuclear localized HDAC4 mutant. Next generation RNA-sequencing revealed an HDAC4-regulated gene program comprising mediators of sensitization including the organic anion transporter OAT1, known for its renal transport function. Using pharmacological and molecular tools to modulate OAT1 activity or expression, we causally link OAT1 to persistent inflammatory hypersensitivity in mice. Thus, HDAC4 is a key epigenetic regulator that translates nociceptive activity into sensitization by regulating OAT1, which is a potential target for pain-relieving therapies.
Assuntos
Dor Crônica/patologia , Histona Desacetilases/metabolismo , Neuralgia/patologia , Dor Nociceptiva/patologia , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Corno Dorsal da Medula Espinal/metabolismo , Animais , Células Cultivadas , Dependovirus/genética , Feminino , Hipersensibilidade/patologia , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/genética , Corno Dorsal da Medula Espinal/citologiaRESUMO
As an expansion investigation of drug-drug interaction (DDI) from previous clinical trials, additional plasma endogenous metabolites were quantitated in the same subjects to further identify the potential biomarkers of organic anion transporter (OAT) 1/3 inhibition. In the single dose, open label, three-phase with fixed order of treatments study, 14 healthy human volunteers orally received 1000 mg probenecid alone, or 40 mg furosemide alone, or 40 mg furosemide at 1 hour after receiving 1000 mg probenecid on days 1, 8, and 15, respectively. Endogenous metabolites including kynurenic acid, xanthurenic acid, indo-3-acetic acid, pantothenic acid, p-cresol sulfate, and bile acids in the plasma were measured by liquid chromatography-tandem mass spectrometry. The Cmax of kynurenic acids was significantly increased about 3.3- and 3.7-fold over the baseline values at predose followed by the treatment of probenecid alone or in combination with furosemide respectively. In comparison with the furosemide-alone group, the Cmax and area under the plasma concentration-time curve (AUC) up to 12 hours of kynurenic acid were significantly increased about 2.4- and 2.5-fold by probenecid alone, and 2.7- and 2.9-fold by probenecid plus furosemide, respectively. The increases in Cmax and AUC of plasma kynurenic acid by probenecid are comparable to the increases of furosemide Cmax and AUC reported previously. Additionally, the plasma concentrations of xanthurenic acid, indo-3-acetic acid, pantothenic acid, and p-cresol sulfate, but not bile acids, were also significantly elevated by probenecid treatments. The magnitude of effect size analysis for known potential endogenous biomarkers demonstrated that kynurenic acid in the plasma offers promise as a superior addition for early DDI assessment involving OAT1/3 inhibition. SIGNIFICANCE STATEMENT: This article reports that probenecid, an organic anion transporter (OAT) 1 and OAT3 inhibitor, significantly increased the plasma concentrations of kynurenic acid and several uremic acids in human subjects. Of those, the increases of plasma kynurenic acid exposure are comparable to the increases of furosemide by OAT1/3 inhibition. Effect size analysis for known potential endogenous biomarkers revealed that plasma kynurenic acid is a superior addition for early drug-drug interaction assessment involving OAT1/3 inhibition.
Assuntos
Biomarcadores Farmacológicos , Interações Medicamentosas/fisiologia , Furosemida/farmacologia , Ácido Cinurênico , Proteína 1 Transportadora de Ânions Orgânicos , Transportadores de Ânions Orgânicos Sódio-Independentes , Probenecid/farmacocinética , Adjuvantes Farmacêuticos/farmacocinética , Biomarcadores Farmacológicos/análise , Biomarcadores Farmacológicos/sangue , Cromatografia Líquida/métodos , Furosemida/farmacocinética , Voluntários Saudáveis , Humanos , Ácido Cinurênico/análise , Ácido Cinurênico/sangue , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Indoxyl sulfate (IS) accumulates in the body in chronic kidney disease (CKD). In the renal proximal tubules, IS excretion is mediated by OAT1/3 and ABCG2. These transporters are inhibited by some hypouricemic agents; OATs by probenecid and benzbromarone, ABCG2 by febuxostat and benzbromarone. Thus, we evaluated whether hypouricemic agents including dotinurad, a novel selective urate reabsorption inhibitor with minimal effect on OATs or ABCG2, affect IS clearance in rats. Intact and adenine-induced acute renal failure rats were orally administered hypouricemic agents, and both endogenous IS and exogenously administered stable isotope-labeled d4-IS in the plasma and kidney were measured. Our results demonstrated that OATs inhibitors, such as probenecid, suppress IS uptake into the kidney, leading to increased plasma IS concentration, whereas ABCG2 inhibitors, such as febuxostat, cause renal IS accumulation remarkably by suppressing its excretion in intact rats. The effects of these agents were reduced in adenine-induced acute renal failure rats, presumably due to substantial decrease in renal OAT1/3 and ABCG2 expression. Dotinurad did not significantly affected the clearance of IS under both conditions. Therefore, we suggest that hypouricemic agents that do not affect OATs and ABCG2 are effective therapeutic options for the treatment of hyperuricemia complicated by CKD.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Injúria Renal Aguda , Indicã/sangue , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Uricosúricos/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Injúria Renal Aguda/sangue , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/tratamento farmacológico , Animais , Masculino , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Ratos , Ratos WistarRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Aristolochic acid nephropathy (AAN) is a kidney disease caused by the administration of plants containing aristolochic acids (AAs). Aristolochic acid I (AAI) is the main toxic component in AAs. Organic anion transporters (OATs) 1 and 3 mediate the renal uptake of AAI, which is related to AAN. In our previous study, we found that anthraquinones derived from the herbal medicine Rheum palmatum L. (RP) inhibited both OAT1 and OAT3, with rhein exhibiting the greatest potency among the components. AIM OF THE STUDY: This study aimed to investigate the effects of rhein and RP extract on the pharmacokinetics and tissue distribution of AAI and its demethylated metabolite (8-hydroxy-aristolochic acid I [AAIa]) in rats. MATERIALS AND METHODS: Rhein and RP extract were used as OAT inhibitors, and AAI was used as the toxic substrate. The pharmacokinetics and tissue distribution of AAI and AAIa in rats following the intravenous injection of AAI (10 mg/kg) in the presence and absence of rhein (100 mg/kg) or RP extract (5 g crude drug/kg) were investigated. RESULTS: Co-administration with rhein increased AUC0-∞ of AAI and AAIa by 39 and 44%, respectively. However, the renal level of AAI was decreased to 50, 42, and 58% of those in rats treated with AAI alone at 5, 10, and 20 min after treatment, respectively, and the renal level of AAIa was decreased to 58, 57, and 61% of the level in rats treated with AAI alone, respectively, at these time points. In the RP extract co-administration group, AAI and AAIa plasma exposure was not significantly increased, but renal accumulation of AAI was decreased to 63, 58, and 68% of that in rats treated with AAI alone at 5, 10, and 20 min after treatment, respectively. In addition, renal accumulation of AAIa was decreased to 74, 70, and 70% of that in rats treated with AAI alone at 5, 10, and 20 min after treatment, respectively. CONCLUSIONS: This study indicated that co-administration with rhein significantly increased the plasma exposure of AAI and AAIa while decreased their renal accumulation in rats. RP extract reduced the renal accumulation of AAI and AAIa, but have no significant effect on their plasma exposure levels in rats.
Assuntos
Antraquinonas/farmacologia , Ácidos Aristolóquicos/farmacocinética , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Extratos Vegetais/farmacologia , Rheum , Animais , Antraquinonas/isolamento & purificação , Ácidos Aristolóquicos/administração & dosagem , Ácidos Aristolóquicos/sangue , Ácidos Aristolóquicos/toxicidade , Biotransformação , Desmetilação , Injeções Intravenosas , Rim/metabolismo , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Nefropatias/prevenção & controle , Masculino , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Extratos Vegetais/isolamento & purificação , Ratos Sprague-Dawley , Rheum/química , Distribuição TecidualRESUMO
BACKGROUND AND OBJECTIVES: Citrus flavonoids are not only components of daily nutrition, they are also promoted as dietary supplements and are important ingredients in traditional medicines. Interactions of flavonoids with synthetic drugs represent an often neglected issue. We therefore investigated in vitro whether the polymethoxyflavones nobiletin, sinensetin, and tangeretin and the flavonoid rutinosides didymin, hesperidin, and narirutin can inhibit human organic anion transporting polypeptides (OATP) 1B1, 1B3, and 2B1, which are important transporters mediating drug-drug and food-drug interactions. METHODS: Inhibition was investigated by quantifying the decreased uptake of the fluorescent OATP1B1 and OATP1B3 substrate 8-fluorescein-cAMP in HEK293 cells overexpressing OATP1B1 or OATP1B3 and of the fluorescent OATP2B1 substrate 4',5'-dibromofluorescein in HEK293 cells overexpressing OATP2B1. RESULTS: We demonstrate that all flavonoids investigated inhibit OATP2B1 in the lower micromolar range (IC50 between 1.6 and 14.2 µM), but only the polymethoxyflavones also inhibit OATP1B1 and 1B3 (IC50 between 2.1 and 21 µM). CONCLUSIONS: All flavonoids investigated might contribute to the intestinal OATP2B1-based interactions with drugs observed with citrus juices or fruits. In contrast, the concentration of the polymethoxyflavones after consumption of citrus juices or fruits is most likely too low to reach relevant systemic concentrations and thus to inhibit hepatic OATP1B1 and OATP1B3, but there might be a risk when they are consumed as medicines or as dietary supplements.
Assuntos
Citrus/química , Flavonas/farmacologia , Interações Alimento-Droga , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Bebidas , Células HEK293 , Humanos , Fígado/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Relação Estrutura-AtividadeRESUMO
In light of the recent pandemic, favipiravir (Avigan®), a purine nucleic acid analog and antiviral agent approved for use in influenza in Japan, is being studied for the treatment of coronavirus disease 2019 (COVID-19). Increase in blood uric acid level is a frequent side effect of favipiravir. Here, we discussed the mechanism of blood uric acid elevation during favipiravir treatment. Favipiravir is metabolized to an inactive metabolite M1 by aldehyde oxidase and xanthine oxidase, and excreted into urine. In the kidney, uric acid handling is regulated by the balance of reabsorption and tubular secretion in the proximal tubules. Favipiravir and M1 act as moderate inhibitors of organic anion transporter 1 and 3 (OAT1 and OAT3), which are involved in uric acid excretion in the kidney. In addition, M1 enhances uric acid reuptake via urate transporter 1 (URAT1) in the renal proximal tubules. Thus, favipiravir is thought to decrease uric acid excretion into urine, resulting in elevation of uric acid levels in blood. Elevated uric acid levels were returned to normal after discontinuation of favipiravir, and favipiravir is not used for long periods of time for the treatment of viral infection. Thus, the effect on blood uric acid levels was subclinical in most studies. Nevertheless, the adverse effect of favipiravir might be clinically important in patients with a history of gout, hyperuricemia, kidney function impairment (in which blood concentration of M1 increases), and where there is concomitant use of other drugs affecting blood uric acid elevation.
Assuntos
Amidas/efeitos adversos , Antivirais/efeitos adversos , Infecções por Coronavirus/tratamento farmacológico , Hiperuricemia/induzido quimicamente , Pneumonia Viral/tratamento farmacológico , Pirazinas/efeitos adversos , Ácido Úrico/sangue , Aldeído Oxidase/metabolismo , Amidas/farmacocinética , Amidas/urina , Antivirais/farmacocinética , Biotransformação , COVID-19 , Interações Medicamentosas , Humanos , Hiperuricemia/fisiopatologia , Rim/metabolismo , Nefropatias/metabolismo , Estrutura Molecular , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Pandemias , Pirazinas/farmacocinética , Pirazinas/urina , Xantina Oxidase/metabolismoRESUMO
The ß-lactam penicillin antibiotic cloxacillin (CLX) presents wide inter-individual pharmacokinetics variability. To better understand its molecular basis, the precise identification of the detoxifying actors involved in CLX disposition and elimination would be useful, notably with respect to renal secretion known to play a notable role in CLX elimination. The present study was consequently designed to analyze the interactions of CLX with the solute carrier transporters organic anion transporter (OAT) 1 and OAT3, implicated in tubular secretion through mediating drug entry at the basolateral pole of renal proximal cells. CLX was first shown to block OAT1 and OAT3 activity in cultured OAT-overexpressing HEK293 cells. Half maximal inhibitory concentration (IC50 ) value for OAT3 (13 µm) was however much lower than that for OAT1 (560 µm); clinical inhibition of OAT activity and drug-drug interactions may consequently be predicted for OAT3, but not OAT1. OAT3, unlike OAT1, was next shown to mediate CLX uptake in OAT-overexpressing HEK293 cells. Kinetic parameters for this OAT3-mediated transport of CLX (Km = 10.7 µm) were consistent with a possible in vivo saturation of this process for high CLX plasma concentrations. OAT3 is consequently likely to play a pivotal role in renal CLX secretion and consequently in total renal CLX elimination, owing to the low plasma unbound fraction of the antibiotic. OAT3 genetic polymorphisms as well as co-administered drugs inhibiting in vivo OAT3 activity may therefore be considered as potential sources of CLX pharmacokinetics variability.
Assuntos
Antibacterianos/farmacologia , Cloxacilina/farmacologia , Rim/efeitos dos fármacos , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Antibacterianos/farmacocinética , Cloxacilina/farmacocinética , Relação Dose-Resposta a Droga , Interações Medicamentosas , Células HEK293 , Humanos , Rim/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Eliminação RenalRESUMO
Rheumatoid arthritis patients can be prescribed a combination of immunosuppressive drug leflunomide (LEF) and the antiviral drug acyclovir to reduce the high risk of infection. Acyclovir is a substrate of organic anion transporter (OAT) 1/3 and multidrug resistance-associated protein (MRP) 2. Considering the extraordinarily long half-life of LEF's active metabolite teriflunomide (TER) and the kidney injury risk of acyclovir, it is necessary to elucidate the potential impact of LEF on the disposition of acyclovir. Here we used a specific MRP inhibitor MK571 and probenecid (OAT1/3 and MRP2 inhibitor) to assess the effects of MRP2 and OAT1/3 on the pharmacokinetics and tissue distribution of acyclovir in rats. We showed that LEF and probenecid, but not MK571 significantly increased the plasma concentration of acyclovir. However, kidney and liver exposures of acyclovir were increased when coadministered with LEF, probenecid or MK571. The kidney/plasma ratio of acyclovir was increased to approximately 2-fold by LEF or probenecid, whereas it was increased to as much as 14.5-fold by MK571. Consistently, these drugs markedly decreased the urinary excretion of acyclovir. TER (0.5-100 µmol/L) dose-dependently increased the accumulation of acyclovir in MRP2-MDCK cells with an IC50 value of 4.91 µmol/L. TER (5 µmol/L) significantly inhibited the uptake of acyclovir in hOAT1/3-HEK293 cells. These results suggest that LEF/TER increased the kidney accumulation of acyclovir by inhibiting the efflux transporter MRP2, which increased its kidney/plasma ratio and renal injury risk. However, the inhibitory effects of LEF/TER on OAT1/3 reduced the tubular cells' uptake of acyclovir and increased the plasma concentration.
Assuntos
Aciclovir/farmacocinética , Rim/metabolismo , Leflunomida/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Aciclovir/administração & dosagem , Aciclovir/metabolismo , Administração Intravenosa , Animais , Células Cultivadas , Crotonatos/administração & dosagem , Crotonatos/metabolismo , Crotonatos/farmacologia , Cães , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Hidroxibutiratos , Leflunomida/administração & dosagem , Leflunomida/metabolismo , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/metabolismo , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Nitrilas , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Probenecid/administração & dosagem , Probenecid/metabolismo , Probenecid/farmacologia , Propionatos/administração & dosagem , Propionatos/metabolismo , Propionatos/farmacologia , Quinolinas/administração & dosagem , Quinolinas/metabolismo , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Toluidinas/administração & dosagem , Toluidinas/metabolismo , Toluidinas/farmacologiaRESUMO
Multiple doses of tenofovir disoproxil fumarate (TDF) together with emtricitabine is effective for HIV preexposure prophylaxis (PrEP). TDF is converted to tenofovir (TFV) in circulation, which is subsequently cleared via tubular secretion by organic ion transporters (OATs; OAT1 and OAT3). Using in vitro kinetic parameters for TFV and the OAT1 and OAT3 inhibitor probenecid, a bottom-up physiologically-based pharmacokinetic model was successfully developed for the first time that accurately describes the probenecid-TFV interaction. This model predicted an increase in TFV plasma exposure by 60%, which was within 15% of the observed clinical pharmacokinetic data, and a threefold decrease in renal cells exposure following coadministration of a 600 mg TDF dose with 2 g probenecid. When compared with multiple-dose regimens, a single-dose probenecid-boosted TDF regimen may be effective for HIV PrEP and improve adherence and safety by minimizing TFV-induced nephrotoxicity by reducing TFV accumulation in renal cells.
Assuntos
Fármacos Anti-HIV/farmacocinética , Modelos Biológicos , Probenecid/farmacologia , Tenofovir/farmacocinética , Fármacos Anti-HIV/administração & dosagem , Interações Medicamentosas , Combinação Emtricitabina e Fumarato de Tenofovir Desoproxila/administração & dosagem , Combinação Emtricitabina e Fumarato de Tenofovir Desoproxila/farmacocinética , Infecções por HIV/prevenção & controle , Humanos , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Profilaxia Pré-Exposição/métodos , Probenecid/administração & dosagemRESUMO
Stellera chamaejasme L. is a traditional Chinese medicine with a long history to treat stubborn skin ulcer, and it also has antiviral and antitumor effects. Neochamaejasmine B (NCB), Neochamaejasmine A (NCA) and Chamaechromone (CMC) are the major components in dried roots of Stellera chamaejasme L.. Our studies suggested that NCB, NCA and CMC are inhibitors of Organic anion transporter 1 (OAT1). OAT1 is encoded by solute carrier family 22 member 6 gene (SLC22A6) in humans and plays a critical role in the organic anion drug uptake and excretion in the kidney. Lamivudine is the typical substrate of OAT1 and is frequently used in combination with other antiviral drugs in clinical antiviral treatments. The aim of this study is to investigate the interaction and its mechanism between these bi-flavone components in Stellera chamaejasme L. and lamivudine via OAT1 both in vitro and in vivo. In vitro, the uptake studies in Madin-Darby canine kidney (MDCK) cells overexpressing OAT1 suggested that NCB inhibited the uptake of 6-CFL and lamivudine.Similar results were obtained for NCA and CMC. NCB was a noncompetitive and competitive inhibitor interaction with OAT1. IC50 values of NCB, NCA and CMC for inhibiting OAT1-mediated lamivudine transport were 2.46, 8.35 and 0.61 µmol·L-1, respectively. In vivo, the pharmacokinetic results of lamivudine in rats showed that the mean area under the plasma concentration-time curve (AUC0-∞) and maximal plasma concentration (Cmax) of lamivudine after co-administration is increased 2.94-fold and 1.87-fold, respectively, compared to lamivudine administration alone. The results of interactions between lamivudine and these bi-flavone components in Stellera chamaejasme L. extracts via OAT1 in vivo are consistent with studies in vitro. The inhibition of OAT1-mediated uptake of lamivudine by NCB, NCA and CMC is the possible mechanism for Stellera chamaejasme L. extracts improving the oral bioavailability of lamivudine in rats.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Flavonoides/farmacologia , Lamivudina/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Thymelaeaceae/química , Animais , Biflavonoides/farmacologia , Disponibilidade Biológica , Transporte Biológico/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/química , Feminino , Flavonas/farmacologia , Flavonoides/química , Humanos , Concentração Inibidora 50 , Lamivudina/farmacocinética , Células Madin Darby de Rim Canino , Masculino , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Ratos Sprague-DawleyRESUMO
The effect of dotinurad [(3,5-dichloro-4-hydroxyphenyl)(1,1-dioxo-1,2-dihydro-3H-1λ 6-1,3-benzothiazol-3-yl)methanone] was compared with that of commercially available uricosuric agents-namely, benzbromarone, lesinurad, and probenecid. Its effect on urate secretion transporters was evaluated using probe substrates for respective transporters. Dotinurad, benzbromarone, lesinurad, and probenecid inhibited urate transporter 1 (URAT1) with IC50 values of 0.0372, 0.190, 30.0, and 165 µM, respectively. Dotinurad weakly inhibited ATP-binding cassette subfamily G member 2 (ABCG2), organic anion transporter 1 (OAT1), and OAT3, with IC50 values of 4.16, 4.08, and 1.32 µM, respectively, indicating higher selectivity for URAT1. The hypouricemic effects of dotinurad and benzbromarone were evaluated in Cebus monkeys. Dotinurad, at doses of 1-30 mg/kg, concomitantly decreased plasma urate levels and increased fractional excretion of urate (FEUA) in a dose-dependent manner. On the contrary, benzbromarone, at a dose of 30 mg/kg, showed a modest effect on plasma urate levels. The inhibitory effect of dotinurad on urate secretion transporters was evaluated in Sprague-Dawley rats, with sulfasalazine and adefovir as probe substrates of ABCG2 and OAT1, respectively. Drugs, including febuxostat as a reference ABCG2 inhibitor, were administered orally before sulfasalazine or adefovir administration. Dotinurad had no effect on urate secretion transporters in vivo, whereas benzbromarone, lesinurad, probenecid, and febuxostat increased the plasma concentrations of probe substrates. These results suggested dotinurad is characterized as a selective urate reabsorption inhibitor (SURI), which is defined as a potent URAT1 inhibitor with minimal effect on urate secretion transporters, including ABCG2 and OAT1/3, because of its high efficacy in decreasing plasma urate levels compared with that of other uricosuric agents. SIGNIFICANCE STATEMENT: Our study on the inhibitory effects on urate transport showed that dotinurad had higher selectivity for urate transporter 1 (URAT1) versus ATP-binding cassette subfamily G member 2 (ABCG2) and organic anion transporter (OAT) 1/3 compared to other uricosuric agents. In Cebus monkeys, dotinurad decreased plasma urate levels and increased fractional excretion of urate in a dose-dependent manner. To determine the inhibitory effect of dotinurad on urate secretion transporters, we studied the movement of substrates of ABCG2 and OAT1 in rats. Dotinurad had no effect on these transporters, whereas the other uricosuric agents increased the plasma concentrations of the substrates. These results suggested dotinurad as a potent and selective urate reabsorption inhibitor is characterized by increased efficacy with decreasing plasma urate levels.
Assuntos
Benzotiazóis/farmacologia , Uricosúricos/farmacocinética , 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 , Animais , Benzotiazóis/efeitos adversos , Benzotiazóis/farmacocinética , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Haplorrinos , Humanos , Masculino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Ácido Úrico/sangue , Ácido Úrico/urina , Uricosúricos/efeitos adversosRESUMO
Organic anion transporters (OATs) are membrane proteins within the Solute carrier family 22 (SLC22). They play important roles in cellular uptake of various organic compounds, and due to their expression in barrier tissues of major excretory and non-excretory organs are considered as crucial elements in absorption and distribution of a wide range of endobiotic and xenobiotic compounds. Based on our previous work and initial insights on SLC22 members in zebrafish (Danio rerio), in this study we aimed at in vitro characterization of Oat1 and Oat3 transporters and understanding of their interaction with potential physiological substrates. We first performed synteny analysis to describe in more detail orthological relationship of zebrafish oat1 and oat3 genes. We then developed stable cell lines overexpressing Oat1 and Oat3, and identified Lucifer yellow as Oat1 model fluorescent substrate (Kmâ¯=â¯11.4⯵M) and 6-carboxyfluorescein as Oat3 model substrate (Kmâ¯=â¯5.8⯵M). Initial identification performed using the developed assays revealed Kreb's cycle intermediates, bilirubin, bile salts and steroid hormones as the most potent of Oat1 and Oat3 interactors, with IC50 values in micromolar range. Finally, we showed that bilirubin, deoxycholic acid, α-ketoglutarate, pregnenolone, estrone-3-sulfate and corticosterone are in vitro substrates of zebrafish Oat1, and bilirubin and deoxycholic acid are Oat3 substrates. In conclusion, using the approach described, structural and functional similarities of both transporters to human and mammalian orthologs are revealed, their broad ligand selectivity confirmed, potent interactors among endobiotic compounds identified, and first indications of their potential physiological role(s) in zebrafish obtained.
Assuntos
Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Ligação Proteica , Transporte Proteico , Proteínas de Peixe-Zebra/antagonistas & inibidoresRESUMO
A bioactivity guided program exploring the interaction of phytochemicals in the entire plant Primula macrocalyx with the organic anion transporters (OAT1 and OAT3) and microorganisms led to the elucidation of ten known flavones (1-4, 6-10, 12) and two previously undescribed flavones (5, 11). The structures of the compounds were determined by extensive analysis of spectroscopic data, as well as by comparison with data from previous reports. Two known flavones (9, 12) are reported for the first time from the family Primulaceae. All compounds were evaluated for inhibition of OAT1 and OAT3. Six flavones (2, 3, 6-8, 12) showed potent inhibitory activity on OAT1, while seven flavones (2, 3, 6-9, 12) showed marked inhibitory activity on OAT3, with IC50 ≤ 10.0 µM. Antimicrobial activities of crude fractions against sixteen microorganisms were tested to give a target yeast strain Candida rugosa for further evaluation of MICs on the isolates. Three flavones (7, 8, 12) showed marked antifungal activity with MIC < 2.0 µM. To our knowledge, this study is the first to evaluate these flavones as inhibitors of the OAT1 and OAT3, and as antifungal agents.
Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Flavonas/farmacologia , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Primula/química , Antifúngicos/química , Flavonas/química , Células HEK293 , HumanosRESUMO
Andrographolide, a major bioactive compound isolated from Andrographis paniculata (Burm. F.) Nees, was evaluated for its effects on the hOAT1 membrane transporter. Substrate determination and inhibition of hOAT1-mediated uptake transport assay was carried out using recombinant CHO-hOAT1 cells. The results showed that the uptake ratio of andrographolide was less than 2.0 at all concentrations tested, indicating that andrographolide is not a hOAT1 substrate. Andrographolide has no significant effects on the p-aminohippuric acid uptake and on the mRNA and protein expression of hOAT1. In conclusion, andrographolide may not pose a drug-herb interaction risk related to hOAT1.
Assuntos
Diterpenos/farmacologia , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Animais , Células CHO , Proliferação de Células/efeitos dos fármacos , Cricetulus , Diterpenos/farmacocinética , Interações Ervas-Drogas , Humanos , Simulação de Acoplamento Molecular , Proteína 1 Transportadora de Ânions Orgânicos/análise , Proteína 1 Transportadora de Ânions Orgânicos/química , Proteína 1 Transportadora de Ânions Orgânicos/genética , Probenecid/química , Probenecid/farmacologia , Ácido p-Aminoipúrico/farmacocinéticaRESUMO
A continuous phytochemical study on the seeds of Senna obtusifolia (Syn.: Cassia obtusifolia) led to the isolation of a new anthraquinone analogue, obtusifolin-2-O-ß-D-(6'-O-α,ß-unsaturated butyryl)-glucopyranoside (1) and a new eurotinone analogue, epi-9-dehydroxyeurotinone-ß-D-glucopyranoside (2). Their structures were established mainly by NMR and MS experiments as well as the necessary chemical evidences. Their inhibitory effects on two organic anion transporters (OAT1 and OAT3) were investigated and the results showed that 1 exhibited a strongly specific inhibitory effect on OAT1 at 100 µM.
Assuntos
Antraquinonas/isolamento & purificação , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Sementes/química , Senna/química , Antraquinonas/química , Antraquinonas/farmacologia , Humanos , Estrutura Molecular , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidoresRESUMO
PURPOSE: Nitisinone inhibits the cytochrome P450 (CYP) subfamilies CYP2C9, CYP2D6, and CYP2E1 and the organic anion transporter (OAT) isoforms OAT1 and OAT3 in vitro. Since the effect of nitisinone on these enzymes and transporters in humans is still unknown, the purpose of this study was to evaluate the effect of nitisinone on these CYP subfamilies and OAT isoforms. METHODS: This was an open-label, nonrandomized, two-arm, phase 1 study (EudraCT: 2016-004297-17) in healthy volunteers. The substrates (tolbutamide, metoprolol, and chlorzoxazone for the respective CYPs and furosemide for the OATs) were administered as single doses, before and after 15 days of once daily dosing of 80 mg nitisinone, to determine the AUC∞ ratios ([substrate+nitisinone]/[substrate]). Nitisinone pharmacokinetics, safety, and tolerability were also assessed, and blood and urine were collected to determine substrate and nitisinone concentrations by LC-MS/MS. RESULTS: Thirty-six subjects were enrolled with 18 subjects included in each arm. The least square mean ratio (90% confidence interval) for AUC∞ was 2.31 (2.11-2.53) for tolbutamide, 0.95 (0.88-1.03) for metoprolol, 0.73 (0.67-0.80) for chlorzoxazone, and 1.72 (1.63-1.81) for furosemide. Clinically relevant nitisinone steady-state concentrations were reached after 12 days: mean Cav,ss of 94.08 µM. All treatments were well tolerated, and no safety concerns were identified. CONCLUSIONS: Nitisinone did not affect CYP2D6 activity, was a weak inducer of CYP2E1, and was a weak inhibitor of OAT1 and OAT3. Nitisinone was a moderate inhibitor of CYP2C9, and treatment may therefore result in increased plasma concentrations of comedications metabolized primarily via this enzyme. CLINICAL TRIAL REGISTRY IDENTIFICATION: EudraCT 2016-004297-17.
Assuntos
Cicloexanonas/farmacologia , Inibidores Enzimáticos/farmacologia , Nitrobenzoatos/farmacologia , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Adolescente , Adulto , Área Sob a Curva , Cicloexanonas/efeitos adversos , Cicloexanonas/farmacocinética , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/farmacocinética , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Nitrobenzoatos/efeitos adversos , Nitrobenzoatos/farmacocinética , Especificidade por Substrato , Adulto JovemRESUMO
Phytochemical investigation of Camphorosma lessingii has resulted in the isolation of four previously unreported isoflavones (1: -4: ) and eight known compounds (5: -12: ). Nine of these compounds (1: -6, 8: -10: ) are reported for the first time from members of the family Amaranthaceae. The structures of all isolated compounds were determined by spectroscopic methods, primarily one-dimensional and two-dimensional nuclear magnetic resonance and mass spectrometry. The absolute configuration of 6: was confirmed by circular dichroism. Inhibition of the organic anion transporters, OAT1 and OAT3, by the isolated compounds was evaluated. Among them, 7, 2'-dihydroxy- 6,8-dimethoxyisoflavone (1: ), 2'-hydroxy-6,7,8-trimethoxyisoflavone (2: ), 6,2'-dihydroxy-7,8-dimethoxyisoflavone (3: ), and 7-methoxyflavone (5: ) showed a significant inhibitory effect on 6-carboxyfluorescein uptake mediated by OAT1 and OAT3.
Assuntos
Chenopodiaceae/química , Isoflavonas/farmacologia , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Dicroísmo Circular , Células HEK293 , Humanos , Isoflavonas/química , Isoflavonas/isolamento & purificação , Espectroscopia de Ressonância Magnética , Espectrometria de MassasRESUMO
PURPOSE: To evaluate association of the dose-dependent effect of rifampicin, an OATP1B inhibitor, on the plasma concentration-time profiles among OATP1B substrates drugs and endogenous substrates. METHODS: Eight healthy volunteers received atorvastatin (1 mg), pitavastatin (0.2 mg), rosuvastatin (0.5 mg), and fluvastatin (2 mg) alone or with rifampicin (300 or 600 mg) in a crossover fashion. The plasma concentrations of these OATP1B probe drugs, total and direct bilirubin, glycochenodeoxycholate-3-sulfate (GCDCA-S), and coproporphyrin I, were determined. RESULTS: The most striking effect of 600 mg rifampicin was on atorvastatin (6.0-times increase) and GCDCA-S (10-times increase). The AUC0-24h of atorvastatin was reasonably correlated with that of pitavastatin (r2 = 0.73) and with the AUC0-4h of fluvastatin (r2 = 0.62) and sufficiently with the AUC0-24h of rosuvastatin (r2 = 0.32). The AUC0-24h of GCDCA-S was reasonably correlated with those of direct bilirubin (r2 = 0.74) and coproporphyrin I (r2 = 0.78), and sufficiently with that of total bilirubin (r2 = 0.30). The AUC0-24h of GCDCA-S, direct bilirubin, and coproporphyrin I were reasonably correlated with that of atorvastatin (r2 = 0.48-0.70) [corrected]. CONCLUSION: These results suggest that direct bilirubin, GCDCA-S, and coproporphyrin I are promising surrogate probes for the quantitative assessment of potential OATP1B-mediated DDI.
Assuntos
Antibióticos Antituberculose/sangue , Antibióticos Antituberculose/farmacologia , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Proteína 1 Transportadora de Ânions Orgânicos/sangue , Rifampina/sangue , Rifampina/farmacologia , Adulto , Estudos Cross-Over , Relação Dose-Resposta a Droga , Voluntários Saudáveis , Humanos , Masculino , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologia , Espectrometria de Massas em Tandem/métodosRESUMO
Cheminformatics datasets used in classification problems, especially those related to biological or physicochemical properties, are often imbalanced. This presents a major challenge in development of in silico prediction models, as the traditional machine learning algorithms are known to work best on balanced datasets. The class imbalance introduces a bias in the performance of these algorithms due to their preference towards the majority class. Here, we present a comparison of the performance of seven different meta-classifiers for their ability to handle imbalanced datasets, whereby Random Forest is used as base-classifier. Four different datasets that are directly (cholestasis) or indirectly (via inhibition of organic anion transporting polypeptide 1B1 and 1B3) related to liver toxicity were chosen for this purpose. The imbalance ratio in these datasets ranges between 4:1 and 20:1 for negative and positive classes, respectively. Three different sets of molecular descriptors for model development were used, and their performance was assessed in 10-fold cross-validation and on an independent validation set. Stratified bagging, MetaCost and CostSensitiveClassifier were found to be the best performing among all the methods. While MetaCost and CostSensitiveClassifier provided better sensitivity values, Stratified Bagging resulted in high balanced accuracies.