RESUMEN
Dotinurad was developed as a uricosuric agent, inhibiting urate (UA) reabsorption through the UA transporter URAT1 in the kidneys. Due to its high selectivity for URAT1 among renal UA transporters, we investigated the mechanism underlying this selectivity by identifying dotinurad binding sites specific to URAT1. Dotinurad was docked to URAT1 using AutoDock4, utilizing the AlphaFold2-predicted structure. The inhibitory effects of dotinurad on wild-type and mutated URAT1 at the predicted binding sites were assessed through URAT1-mediated [14C]UA uptake in Xenopus oocytes. Nine amino acid residues in URAT1 were identified as dotinurad-binding sites. Sequence alignment with UA-transporting organic anion transporters (OATs) revealed that H142 and R487 were unique to URAT1 among renal UA-transporting OATs. For H142, IC50 values of dotinurad increased to 62, 55, and 76 nM for mutated URAT1 (H142A, H142E, and H142R, respectively) compared with 19 nM for the wild type, indicating that H142 contributes to URAT1-selective interaction with dotinurad. H142 was predicted to interact with the phenyl-hydroxyl group of dotinurad. The IC50 of the hydroxyl group methylated dotinurad (F13141) was 165 µM, 8420-fold higher than dotinurad, suggesting the interaction of H142 and the phenyl-hydroxyl group by forming a hydrogen bond. Regarding R487, URAT1-R487A exhibited a loss of activity. Interestingly, the URAT1-H142A/R487A double mutant restored UA transport activity, with the IC50 value of dotinurad for the mutant (388 nM) significantly higher than that for H142A (73.5 nM). These results demonstrate that H142 and R487 of URAT1 determine its selectivity for dotinurad, a uniqueness observed only in URAT1 among UA-transporting OATs. SIGNIFICANCE STATEMENT: Dotinurad selectively inhibits the urate reabsorption transporter URAT1 in renal urate-transporting organic ion transporters (OATs). This study demonstrates that dotinurad interacts with H142 and R487 of URAT1, located in the extracellular domain and unique among OATs when aligning amino acid sequences. Mutations in these residues reduce affinity of dotinurad for URAT1, confirming their role in conferring selective inhibition. Additionally, the interaction between dotinurad and URAT1 involving H142 is found to mediate hydrogen bonding.
Asunto(s)
Transportadores de Anión Orgánico , Ácido Úrico , Uricosúricos , Animales , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico/genética , Ácido Úrico/metabolismo , Ácido Úrico/farmacología , Sitios de Unión , Humanos , Uricosúricos/farmacología , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Xenopus laevis , Riñón/metabolismo , Riñón/efectos de los fármacos , Oocitos/metabolismo , Oocitos/efectos de los fármacos , Benzotiazoles/farmacología , Simulación del Acoplamiento MolecularRESUMEN
Controlling RAS mutant cancer progression remains a significant challenge in developing anticancer drugs. Whereas Ras G12C-covalent binders have received clinical approval, the emergence of further mutations, along with the activation of Ras-related proteins and signals, has led to resistance to Ras binders. To discover novel compounds to overcome this bottleneck, we focused on the concurrent and sustained blocking of two major signaling pathways downstream of Ras. To this end, we synthesized 25 drug-drug conjugates (DDCs) by combining the MEK inhibitor trametinib with Akt inhibitors using seven types of linkers with structural diversity. The DDCs were evaluated for their cell permeability/accumulation and ability to inhibit proliferation in RAS-mutant cell lines. A representative DDC was further evaluated for its effects on signaling proteins, induction of apoptosis-related proteins, and the stability of hepatic metabolic enzymes. These in vitro studies identified a series of DDCs, especially those containing a furan-based linker, with promising properties as agents for treating RAS-mutant cancers. Additionally, in vivo experiments in mice using the two selected DDCs revealed prolonged half-lives and anticancer efficacies comparable to those of trametinib. The PK profiles of trametinib and the Akt inhibitor were unified through the DDC formation. The DDCs developed in this study have potential as drug candidates for the broad inhibition of RAS-mutant cancers.
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Antineoplásicos , Neoplasias , Animales , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Transducción de Señal , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Mutación , Inhibidores de la Angiogénesis/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Línea Celular TumoralRESUMEN
We recently reported that the gastrointestinal (GI) fluid volume is influenced by the solution osmolality, and proposed that this effect may play a role in beverage-drug interactions. Here, we investigated whether osmolality-dependent fluid secretion can explain the difference in the magnitudes of fruit juice-drug interactions depending on the type of fruit juice (grapefruit juice (GFJ), orange juice (OJ), and apple juice (AJ)). The osmolality of GFJ, OJ, and AJ used in this study was found to be 552, 686, and 749 mOsm/kg, respectively. Measurements of intestinal fluid movement following beverage administration by the in situ closed-loop technique revealed the following rank order for fluid volume in rat ileum: AJ > OJ > GFJ > purified water, suggesting that water movement is dependent on the osmolality of these beverages. Such changes in GI fluid volume are expected to alter the luminal drug concentration, potentially contributing to the magnitude of beverage-drug interactions. Indeed, in vivo pharmacokinetic study in rats revealed that the plasma concentration of atenolol, a low-permeability drug, was the highest after oral administration in purified water, followed by GFJ and OJ, and was the lowest after administration in AJ. In contrast, antipyrine, a high-permeability drug, showed no significant difference in plasma concentration after administration in purified water and fruit juices, suggesting that the absorption of high-permeability drugs is less affected by solution osmolality. Our findings indicate that differences in the magnitude of beverage-drug interactions can be at least partly explained by differences in the osmolality of the beverages ingested.
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Citrus paradisi , Citrus sinensis , Malus , Ratas , Animales , Jugos de Frutas y Vegetales , Interacciones Alimento-Droga , Bebidas/análisis , Concentración Osmolar , Agua , FrutasRESUMEN
The aim of this study was firstly to investigate the effect of membrane permeability on the intestinal availability (Fg ) of 10 cytochrome P450 3A4 substrates with differing permeability (Papp ) and metabolic activity (CLint ) using Madin-Darby canine kidney II (MDCKII) cells expressing human CYP3A4 (MDCKII/CYP3A4 cells), and secondly to confirm the essential factors by simulations. A membrane permeation assay using MDCKII/CYP3A4 cells showed a significant correlation between human intestinal extraction ratio (ER) (Eg (=1 - Fg )) and in vitro cellular ER (r = 0.834). This relationship afforded better predictability of Eg values than the relationship between Eg and CLint,HIM values obtained from human intestinal microsomes (r = 0.598). An even stronger correlation was observed between 1 - Fa ·Fg and ER (r = 0.874). Simulation with a cellular kinetic model indicated that ER is sensitive to changes of PSpassive and CLint values, but not to the intracellular unbound fraction (fu,cell ) or P-gp-mediated efflux (PSP - gp ). It may be concluded that, based on the concentration-time profile of drugs in epithelial cells, transmembrane permeability influences Fg (or ER) and drug exposure time to metabolizing enzymes for P450 substrate.
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Citocromo P-450 CYP3A , Absorción Intestinal , Humanos , Animales , Perros , Citocromo P-450 CYP3A/metabolismo , Intestinos , Permeabilidad de la Membrana Celular , PermeabilidadRESUMEN
Urate transporter 1 (URAT1) is a transporter responsible for uric acid (UA) reabsorption by renal proximal tubules and a pharmacological target of uricosuric agents. Probenecid and benzbromarone have been used as uricosuric agents, while dotinurad was recently approved in Japan. Notably, the in vitro IC 50 of dotinurad on URAT1 is not strong enough to explain its in vivo uricosuric effect estimated based on clinical unbound plasma concentrations, suggesting the presence of mechanisms other than competition with UA uptake at the extracellular domain of URAT1 (cis-inhibition). In this study, trans-inhibition was hypothesized as the mechanism underlying URAT1 inhibition by dotinurad, wherein intracellularly accumulated dotinurad inactivates URAT1. In URAT1-expressing Madin-Darby Canine Kidney-II cells and Xenopus oocytes, pre-incubation with dotinurad potentiated the inhibitory effect more than co-incubation alone, but this effect was not observed with benzbromarone or probenecid. Under co-incubation, dotinurad inhibited UA uptake in a competitive manner (cis-inhibition). When we pre-injected dotinurad directly into oocytes and immediately measured [14C]UA uptake without coincubation (only trans-inhibition), dotinurad noncompetitively inhibited UA uptake. URAT1 is an exchange transporter for UA and monocarboxylates such as nicotinic acid (NA). Pre-injected dotinurad and extracellular UA attenuated and facilitated efflux of [3H]NA, respectively, whereas pre-injection of benzbromarone or probenecid did not affect it, suggesting that dotinurad exhibits trans-inhibition by attenuating URAT1-mediated efflux of monocarboxylates, which is a driving force for UA uptake by URAT1. Accordingly, dotinurad ameliorates URAT1-mediated UA reabsorption by both cis- and trans-inhibition, explaining its clinically stronger uricosuric effect than that estimated by the in vitro IC50 value. SIGNIFICANCE STATEMENT: The uricosuric agent dotinurad inhibits uric acid reabsorptive transporter (URAT) 1 with a clinical potency stronger than that estimated from IC 50 obtained by in vitro URAT1 inhibition. This in vivo-in vitro discrepancy was explained by the trans-inhibition effect of dotinurad on URAT1. Trans-inhibition was due to the attenuation of monocarboxylates efflux via URAT1, which is a driving force for URAT1-mediated exchange transport of uric acid. Overall, this is the first study to experimentally demonstrate trans-inhibition mechanism of URAT1.
RESUMEN
The proximal tubule plays an important role in the kidney and is a major site of drug interaction and toxicity. Analysis of kidney toxicity via in vitro assays is challenging, because only a few assays that reflect functions of drug transporters in renal proximal tubular epithelial cells (RPTECs) are available. In this study, we aimed to develop a simple and reproducible method for culturing RPTECs by monitoring organic anion transporter 1 (OAT1) as a selection marker. Culturing RPTECs in spherical cellular aggregates increased OAT1 protein expression, which was low in the conventional two-dimensional (2D) culture, to a level similar to that in human renal cortices. By proteome analysis, it was revealed that the expression of representative two proximal tubule markers was maintained and 3D spheroid culture improved the protein expression of approximately 7% of the 139 transporter proteins detected, and the expression of 2.3% of the 4,800 proteins detected increased by approximately fivefold that in human renal cortices. Furthermore, the expression levels of approximately 4,800 proteins in three-dimensional (3D) RPTEC spheroids (for 12 days) were maintained for over 20 days. Cisplatin and adefovir exhibited transporter-dependent ATP decreases in 3D RPTEC spheroids. These results indicate that the 3D RPTEC spheroids developed by monitoring OAT1 gene expression are a simple and reproducible in vitro experimental system with improved gene and protein expressions compared with 2D RPTECs and were more similar to that in human kidney cortices. Therefore, it can potentially be used for evaluating human renal proximal tubular toxicity and drug disposition. SIGNIFICANCE STATEMENT: This study developed a simple and reproducible spheroidal culture method with acceptable throughput using commercially available RPTECs by monitoring OAT1 gene expression. RPTECs cultured using this new method showed improved mRNA/protein expression profiles to those in 2D RPTECs and were more similar to those of human kidney cortices. This study provides a potential in vitro proximal tubule system for pharmacokinetic and toxicological evaluations during drug development.
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Riñón , Proteína 1 de Transporte de Anión Orgánico , Humanos , Riñón/metabolismo , Proteína 1 de Transporte de Anión Orgánico/genética , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Túbulos Renales Proximales/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Expresión Génica , Células Epiteliales/metabolismoRESUMEN
OBJECTIVES: Stroke patients frequently exhibit loss of independence of urination, and their lower urinary tract symptoms change with the phase of stroke. However, it is unclear whether switching prescribed drugs for lower urinary tract symptoms during hospitalization from acute care wards to convalescence rehabilitation wards affects patients' independence of urination at discharge. It is also unclear whether the impact of switching varies by stroke type. This retrospective cohort study aimed to examine these issues. MATERIALS AND METHODS: We analyzed 990 patients registered in the Kaga Regional Cooperation Clinical Pathway for Stroke database during 2015-2019. Prescriptions for lower urinary tract symptoms from pre-onset to convalescence rehabilitation were surveyed. Logistic regression analysis was performed to examine the association between switching drugs and independence of urination based on bladder management and voiding location at discharge. Stroke types were also examined in subgroup analyses. RESULTS: About 21 % of patients had their lower urinary tract symptoms prescriptions switched during hospitalization. Switching was positively associated with independence of bladder management (odds ratio 1.65, 95 % confidence interval 1.07 to 2.49) and voiding location (odds ratio 2.72, 95 % confidence interval 1.72 to 4.37). Similar associations were observed in different stroke types. CONCLUSIONS: Approximately 20 % of patients had their lower urinary tract symptoms medications switched upon transfer from acute to convalescence rehabilitation wards. Switching was significantly associated with improved urinary independence at discharge. Consistent results were observed across different stroke types, suggesting that switching medications contributes to urinary independence after stroke, regardless of the etiology or severity of stroke.
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Síntomas del Sistema Urinario Inferior , Rehabilitación de Accidente Cerebrovascular , Accidente Cerebrovascular , Humanos , Micción , Convalecencia , Estudios Retrospectivos , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/complicaciones , Síntomas del Sistema Urinario Inferior/diagnóstico , Síntomas del Sistema Urinario Inferior/tratamiento farmacológico , Síntomas del Sistema Urinario Inferior/etiologíaRESUMEN
AIM: Identification of blood-brain barrier (BBB) uptake transporters is a major challenge in the research and development of central nervous system (CNS) drugs. However, conventional methods that consider known drug uptake characteristics have failed at identifying the responsible transporter molecule. The present study aimed at identifying aripiprazole uptake transporters in BBB model hCMEC/D3 cells using a knockdown screening study targeting various transporters, including uncharacterized ones. METHODS: We evaluated the effect of 214 types of siRNA targeting transporters on the uptake of aripiprazole, an atypical antipsychotic drug, in hCMEC/D3 cells. Aripiprazole uptake was determined using Xenopus oocytes expressing the candidate genes extracted from the siRNA screening assay. RESULTS: The estimated unbound brain to plasma concentration ratio (Kp,uu,brain) of aripiprazole was estimated as 0.67 in wild-type mice and 1.94 in abcb1a/1b/abcg2 knockout mice, suggesting the involvement of both uptake and efflux transporters in BBB permeation. According to siRNA knockdown screening studies, organic cation/carnitine transporter 2 (OCTN2) and long-chain fatty acid transporter 1 (FATP1) were identified as candidate genes. The uptake of aripiprazole by hCMEC/D3 cells was decreased by OCTN2 inhibitors, but not by FATP1 inhibitors. A partially increased uptake of aripiprazole was observed in OCTN2-expressing Xenopus oocytes. Finally, to evaluate transporter-mediated BBB permeation of drugs, the reported and estimated Kp,uu,brain values were summarized. CONCLUSIONS: A knockdown screening study in combination with Kp,uu,brain values showed that aripiprazole was a potential substrate of OCTN2. The technique described in this study can be applied to identifying novel BBB transporters for CNS drugs.
Asunto(s)
Barrera Hematoencefálica , Proteínas de Transporte de Membrana , Animales , Aripiprazol/farmacología , Transporte Biológico , Encéfalo , Ratones , ARN Interferente Pequeño/genéticaRESUMEN
The type of method adopted for the evaluation of drug-induced kidney injury (DIKI) plays an important role during the drug discovery process. In the present study, the usefulness of cultured rat kidney tissue slices maintained on gas-permeable poly(dimethylsiloxane) (PDMS) plates for DIKI was assessed by monitoring the ATP content as a marker of cell viability. The amount of ATP in the kidney slices cultured on the PDMS plates was higher than that in the slices cultured on gas-impermeable polystyrene plates. The protein expression of organic cation transporter-2 (Oct2) was maintained for 3 d. Cisplatin showed a time- and concentration-dependent reduction in ATP in the slices with a half-effective concentration value of 24 µM, which was alleviated by cimetidine, an Oct2 inhibitor, suggesting that cisplatin-induced kidney injury in the cultured slices was regulated by the basolateral uptake transporter Oct2. Furthermore, the intensity of platinum anticancer drug-induced nephrotoxicity in the cultured slices was consistent with that of the in vivo study. In conclusion, the primary culture of rat kidney tissue slices on gas-permeable plates is expected to aid in the prediction of the extent of nephrotoxicity of drugs, even when transporters are responsible for the accumulation of drugs in kidney tissues.
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Antineoplásicos , Platino (Metal) , Animales , Antineoplásicos/metabolismo , Antineoplásicos/toxicidad , Cisplatino/efectos adversos , Riñón , Proteínas de Transporte de Catión Orgánico/metabolismo , Platino (Metal)/metabolismo , RatasRESUMEN
Cluster of differentiation (CD) 38, a major enzyme for nicotinamide adenine dinucleotide (NAD+) degradation, plays a key role in inflammation. Meanwhile, intracellular NAD+ decline is also associated with inflammatory responses. However, whether CD38 activation is involved in gouty inflammation has not been elucidated. The present study aimed to clarify the role of CD38 in monosodium urate crystals (MSU)-triggered inflammatory responses. The results showed that MSU crystals increased the protein expression of CD38 in time- and concentration-dependent manner in THP-1 macrophages and mouse bone marrow-derived macrophages (BMDMs). Moreover, intracellular NAD+ levels were reduced by MSU crystals along with the increased IL-1ß release. However, CD38 inhibition by 78c elevated intracellular NAD+ levels and suppressed IL-1ß release in MSU crystals-treated THP-1 macrophages and BMDMs. Interestingly, CD38 inhibition without significant elevation of intracellular NAD+ also decreased IL-1ß release driven by MSU crystals in THP-1 macrophages. In conclusion, the present study revealed that MSU crystals could activate CD38 with the ensuing intracellular NAD+ decline to promote inflammatory responses in THP-1 macrophages and BMDMs, while CD38 inhibition could suppress MSU crystals-triggered inflammatory responses, indicating that CD38 is a potential therapeutic target for gout.
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ADP-Ribosil Ciclasa 1/genética , Interleucina-1beta/genética , Macrófagos/efectos de los fármacos , Glicoproteínas de Membrana/genética , Ácido Úrico/farmacología , ADP-Ribosil Ciclasa 1/agonistas , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Cristalización , Femenino , Regulación de la Expresión Génica , Gota/etiología , Gota/genética , Gota/metabolismo , Gota/patología , Humanos , Hiperuricemia/etiología , Hiperuricemia/genética , Hiperuricemia/metabolismo , Hiperuricemia/patología , Inflamación , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , NAD/metabolismo , Cultivo Primario de Células , Transducción de Señal , Células THP-1RESUMEN
Plant-derived nanoparticles exert cytoprotective effects on intestinal cells by delivering their cargo to intestinal tissues. We previously reported that apple-derived nanoparticles (APNPs) downregulate the mRNA of the human intestinal transporter organic anion-transporting peptide 2B1 (OATP2B1)/SLCO2B1 and that the 3'-untranslated region (3'UTR) is required for the response to APNPs. Here, we investigated the involvement of microRNAs (miRNAs) in APNPs in suppressing OATP2B1 expression to demonstrate that APNP macromolecules directly interact with intestinal tissues. Using in silico analysis, seven apple miRNAs were predicted as candidate miRNAs that interact with the SLCO2B1-3'UTR. The APNP-mediated decrease in luciferase activity of pGL3/SLCO2B1-3'UTR was abrogated by inhibitors of mdm-miR-160a-e, -7121a-c, or -7121d-h. Each miRNA mimic reduced the endogenous expression of SLCO2B1 mRNA in Caco-2 cells. The luciferase activity of the truncated pGL3/SLCO2B1-3'UTR, which contains approximately 200 bp around each miRNA recognition element (MRE), was decreased by the miR-7121d-h mimic but decreased little by the other mimics. APNP also reduced the luciferase activity of truncated pGL3/SLCO2B1-3'UTR containing an MRE for miR-7121d-h. Thus, we demonstrated that mdm-miR-7121d-h contributes to the APNP-mediated downregulation of intestinal OATP2B1. Accordingly, plant macromolecules, such as miRNAs, may directly interact with intestinal tissues via nanoparticles. SIGNIFICANCE STATEMENT: This study demonstrates that mdm-miR7121d-h contained in apple-derived nanoparticles downregulated the mRNA expression of SLCO2B1 by interacting with SLCO2B1-3'-untranslated region directly and that SLCO2B1 mRNA might also be decreased by mdm-miR160a-e and -7121a-c indirectly. This finding that the specific apple-derived microRNAs influence human intestinal transporters provides a novel concept that macromolecules in foods directly interact with and affect the intestinal function of the host.
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Genes de Plantas/fisiología , Intestinos , Malus , Transportadores de Anión Orgánico/metabolismo , Regiones no Traducidas 3' , Células CACO-2 , Citoprotección , Regulación de la Expresión Génica de las Plantas , Humanos , Intestinos/metabolismo , Intestinos/patología , Malus/química , Malus/metabolismo , MicroARNs , Nanopartículas/metabolismo , Fitoquímicos/metabolismoRESUMEN
Literature reports that Poria cocos reduces blood lipid levels; however, the underlying mechanism remains unclear. Blood lipid levels are closely related to the enterohepatic circulation of bile acids, where uptake transporters playing a significant role. P. cocos extract is commonly used in traditional prescriptions and food supplements in China. We investigated the effects of P. cocos and its five triterpene acids on bile acid uptake transporters, including intestinal apical sodium-dependent bile acid transporter (ASBT) and hepatic sodium/taurocholate cotransporting polypeptide (NTCP). Triterpene acids were fingerprinted by high-performance liquid chromatography-TripleTOF and quantified by ultraperformance liquid chromatography/tandem mass spectrometry. The inhibitory effect of P. cocos and its five major representative triterpene acids on ASBT and NTCP was investigated by in vitro assays using Xenopus oocytes expressing ASBT and NTCP. P. cocos extract exhibited significant inhibitory effects with half-maximum inhibition constants of 5.89 µg/ml and 14.6 µg/ml for NTCP and ASBT, respectively. Among five triterpene acids, poricoic acid A, poricoic acid B, and polyporenic acid C significantly inhibited NTCP function. Poricoic acid A, poricoic acid B, and dehydrotumulosic acid significantly inhibited ASBT function. The representative triterpene acid, poricoic acid A, was identified as a competitive inhibitor of NTCP with an inhibitory constant of 63.4 ± 18.7 µM. In conclusion, our results indicate that both P. cocos extract and its major triterpenes are competitive inhibitors of ASBT and NTCP. Accordingly, it was suggested that competitive inhibition of these bile acid transporters is one of the underlying mechanisms for the hypolipidemic effect of P. cocos. SIGNIFICANCE STATEMENT: Poria cocos, a commonly used Chinese herbal medicine and food supplement, demonstrates significantly inhibitory effects on the function of apical sodium-dependent bile acid transporter and sodium/taurocholate cotransporting polypeptide. P. cocos has potential to reduce the blood lipid through inhibition of these uptake transporters in enterohepatic circulation of bile acid.
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Ácidos y Sales Biliares/antagonistas & inhibidores , Ácidos y Sales Biliares/metabolismo , Productos Biológicos/aislamiento & purificación , Triterpenos/aislamiento & purificación , Wolfiporia , Animales , Productos Biológicos/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Espectrometría de Masas en Tándem/métodos , Triterpenos/farmacología , Xenopus laevisRESUMEN
ATP-binding cassette sub-family G member 2 (ABCG2) is a homodimeric ATP-binding cassette (ABC) transporter that not only has a key role in helping cancer cells to evade the cytotoxic effects of chemotherapy, but also in protecting organisms from multiple xeno- and endobiotics. Structural studies indicate that substrate and inhibitor (ligands) binding to ABCG2 can be differentiated quantitatively by the number of amino acid contacts, with inhibitors displaying more contacts. Although binding is the obligate initial step in the transport cycle, there is no empirical evidence for one amino acid being primarily responsible for ligand binding. By mutagenesis and biochemical studies, we demonstrated that the phylogenetically conserved amino acid residue, F439, was critical for both transport and the binding of multiple substrates and inhibitors. Structural modeling implied that the π-π interactions from each F439 monomer mediated the binding of a surprisingly diverse array of structurally unrelated substrates and inhibitors and that this symmetrical π-π interaction "clamps" the ligand into the binding pocket. Key molecular features of diverse ABCG2 ligands using the π-π clamp along with structural studies created a pharmacophore model. These novel findings have important therapeutic implications because key properties of ligands interacting with ABCG2 have been disovered. Furthermore, mechanistic insights have been revealed by demonstrating that for ABCG2 a single amino acid is essential for engaging and initiating transport of multiple drugs and xenobiotics.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/química , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Células HEK293 , Humanos , Lapatinib/análogos & derivados , Lapatinib/farmacología , Ratones , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
PURPOSE: This study aims to understand the effect of salvage enzyme activity on the saturable kinetics of facilitated cellular uptake of purine nucleobase by developing a cellular kinetic model incorporating equilibrative nucleobase transporter 1 (ENBT1) and adenine phosphoribosyltransferase (APRT), with adenine as a model nucleobase. METHODS: A cellular kinetic model incorporating the functions of ENBT1 and APRT was developed using Napp software and employed for model-based analysis of the cellular disposition of adenine. RESULTS: Simulation analysis using the developed cellular kinetic model could account for the experimentally observed time-dependent changes in the Km(app) value of adenine for ENBT1-mediated uptake. At a long experimental time, the model shows that uptake of adenine is rate-limited by APRT, enabling determination of the Km value for APRT. At early time, the rate-limiting step for adenine uptake is ENBT1-mediated transport, enabling determination of the Km value for ENBT1. Further simulations showed that the effect of experimental time on the Km(app) value for ENBT1-mediated uptake is dependent on the APRT expression level. CONCLUSION: Our findings indicate that both enzyme expression levels and experimental time should be considered when using cellular uptake studies to determine the Km values of purine nucleobases for facilitated transporters.
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Transporte Biológico/fisiología , Proteínas de Transporte de Nucleósido Equilibrativas/metabolismo , Purinas/metabolismo , Adenina/metabolismo , Animales , Línea Celular , Perros , Cinética , Células de Riñón Canino Madin DarbyRESUMEN
PURPOSE: Food-derived nanoparticles exert cytoprotective effects on intestinal cells by delivering their cargo, which includes macromolecules such as microRNAs and proteins, as well as low-molecular weight compounds. We previously reported that apple-derived nanoparticles (APNPs) downregulate the expression of human intestinal transporter OATP2B1/SLCO2B1 mRNA. To verify the involvement of the cargo of APNPs in affecting the expression of transporters, we characterized the uptake mechanism of APNPs in intestinal cells. METHODS: The uptake of fluorescent PKH26-labeled APNPs (PKH-APNPs) into Caco-2, LS180, and HT-29MTX cells was evaluated by confocal microscopy and flow cytometry. RESULTS: The uptake of PKH-APNPs was prevented in the presence of clathrin-dependent endocytosis inhibitors, chlorpromazine and Pitstop2. Furthermore, PKH-APNPs were incorporated by the HT29-MTX cells, despite the disturbance of the mucus layer. Additionally, the decrease in SLCO2B1 mRNA by APNPs was reversed by Pitstop 2 in Caco-2 cells, indicating that APNPs decrease SLCO2B1 by being incorporated via clathrin-dependent endocytosis. CONCLUSIONS: We demonstrated that clathrin-dependent endocytosis was mainly involved in the uptake of APNPs by intestinal cells, and that the cargo in the APNPs downregulate the mRNA expression of SLCO2B1. Therefore, APNPs could be a useful tool to deliver large molecules such as microRNAs to intestinal cells.
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Intestinos/patología , Malus/química , Nanopartículas/química , Nanopartículas/metabolismo , Transporte Biológico , Células CACO-2 , Clatrina/metabolismo , Endocitosis , Colorantes Fluorescentes/química , Regulación de la Expresión Génica/efectos de los fármacos , Células HT29 , Humanos , Intestinos/citología , Imagen Óptica , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal , Distribución TisularRESUMEN
In vitro transport studies across cells grown on culture inserts are widely used for evaluating pharmacokinetic characteristics such as intestinal membrane permeability. However, measurements of the apparent permeability coefficient of highly lipophilic compounds are often limited by transport across the membrane filters, not by transport across the cultured cells. To overcome this concern, we have investigated the utility of a high-porosity membrane honeycomb film (HCF) for transcellular transport studies. Using the HCF inserts, the apparent permeability coefficient (Papp) of the drugs tested in LLC-PK1 and Caco-2 cells tended to increase with an increase in lipophilicity, reaching a maximum Papp value at Log D higher than 2. In contrast, using the commercially available Track-Etched membrane (TEM) inserts, a maximum value was observed at Log D higher than 1. The basolateral to apical transport permeability Papp(BLâAP) of rhodamine 123 across LLC-PK1 cells that express P-glycoprotein (P-gp) cultured on HCF inserts and TEM inserts was 2.33 and 2.39 times higher than the reverse directional Papp(APâBL) permeability, respectively. The efflux ratio (Papp(B-A)/Papp(A-B)) of rhodamine 123 in LLC-PK1 expressing P-gp cells using HCF inserts was comparable to that obtained using TEM inserts, whereas the transported amount in both directions was significantly higher when using the HCF inserts. Accordingly, due to the higher permeability and high porosity of HCF membranes, it is expected that transcellular transport of high lipophilic as well as hydrophilic compounds and substrate recognition of transporters can be evaluated more accurately by using HCF inserts.
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Técnicas de Cultivo de Célula/instrumentación , Evaluación Preclínica de Medicamentos/instrumentación , Rodamina 123/farmacocinética , Células CACO-2 , Evaluación Preclínica de Medicamentos/métodos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , PermeabilidadRESUMEN
We reported that bleomycin (BLM)-induced pulmonary fibrosis was exacerbated in the prostaglandin transporter gene (Slco2a1)-deficient mice (Slco2a1(-/-)). Because cigarette smoke (CS) contributes to creating a profibrotic milieu in the respiratory region, the present study aimed to investigate the impact of CS on SLCO2A1-associated pathogenesis in the lungs of BLM-instilled mice. Bronchoalveolar lavage (BAL) fluid cell analysis indicated more severe inflammation in Slco2a1(-/-) on day 5 after BLM intratracheal instillation, and Slco2a1 deletion increased mRNA expression of pro-inflammatory cytokines (Tnf-α and Il-1ß) and chemokine (Ccl5) in BAL cells. Male Slco2a1(-/-) exhibited significantly higher amounts of released Il-1ß in BAL fluid, compared with female Slco2a1(-/-), male or female Slco2a1(+/+) group. The amount of PGE2 collected in BAL fluid tended to increase in Slco2a1(-/-) compared with Slco2a1(+/+) group, whereas the PGE2 concentrations in lung tissues were comparable between both groups. Besides, PGE2 accumulated more in BAL fluid of male than that of female mice. Therefore, Slco2a1-deficient male mice were found to be more susceptible to BLM-treatment. Moreover, CS extracts (CSE) significantly reduced initial PGE2 uptake by rat type1 alveolar epithelial cell-like (AT1-L) cells and human SLCO2A1-transfected cells. Exposure of AT1-L cells to CSE resulted in decreased mRNA expression of Slco2a1, suggesting that CS modulates SLCO2A1 function. These results indicate that exacerbated lung inflammation is attributed to an increase in Il-1ß peptide and PGE2 accumulation in the alveolar space, which exhibits a male predominance. SLCO2A1 inhibition by CSE is considered to be a new rationale for the lung toxicity of CS.
Asunto(s)
Bleomicina/toxicidad , Pulmón/efectos de los fármacos , Transportadores de Anión Orgánico/genética , Fibrosis Pulmonar/inducido químicamente , Productos de Tabaco/toxicidad , Contaminación por Humo de Tabaco/efectos adversos , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/genética , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Femenino , Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Noqueados , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/metabolismo , Fumar/efectos adversosRESUMEN
The acetylcholinesterase inhibitor, acotiamide, improves gastric motility and is clinically used to treat functional dyspepsia. The present study aimed to identify the transporters involved in the distribution of acotiamide in stomach tissue. Acotiamide uptake by the gastric cancer-derived model cell line, Hs746 T, was Na+- and pH-independent. The initial uptake velocity of acotiamide was saturable with increasing concentrations of acotiamide and was inhibited by selective serotonin reuptake inhibitors, which are potent inhibitors of the plasma membrane monoamine transporter (PMAT). The uptake of acotiamide by PMAT gene-transfected HEK293 cells was saturable, with similar Km (197.9 µM) values to those of uptake by Hs 746T cells (106 µM). Moreover, immunoreactivity of PMAT was found in the gastric smooth muscle and vascular endothelial cells. These results suggest that PMAT contributes to the distribution of acotiamide in the stomach, where it exerts its pharmacological effects.
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Benzamidas/metabolismo , Transporte Biológico/efectos de los fármacos , Proteínas de Transporte de Nucleósido Equilibrativas/metabolismo , Mucosa Gástrica/metabolismo , Estómago/efectos de los fármacos , Tiazoles/metabolismo , Acetilcolinesterasa/metabolismo , Línea Celular , Inhibidores de la Colinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacología , Dispepsia/tratamiento farmacológico , Dispepsia/metabolismo , Células Endoteliales/metabolismo , Células HEK293 , Humanos , Músculo Liso/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/metabolismoRESUMEN
Xanthine and hypoxanthine are intermediate metabolites of uric acid and a source of reactive oxidative species (ROS) by xanthine oxidoreductase (XOR), suggesting that facilitating their elimination is beneficial. Since they are reabsorbed in renal proximal tubules, we investigated their reabsorption mechanism by focusing on the renal uric acid transporters URAT1 and GLUT9, and examined the effect of clinically used URAT1 inhibitor on their renal clearance when their plasma concentration is increased by XOR inhibitor. Uptake study for [3H]xanthine and [3H]hypoxanthine was performed using URAT1- and GLUT9-expressing Xenopus oocytes. Transcellular transport study for [3H]xanthine was carried out using Madin-Darby canine kidney (MDCK)II cells co-expressing URAT1 and GLUT9. In in vivo pharmacokinetic study, renal clearance of xanthine was estimated based on plasma concentration and urinary recovery. Uptake by URAT1- and GLUT9-expressing oocytes demonstrated that xanthine is a substrate of URAT1 and GLUT9, while hypoxanthine is not. Transcellular transport of xanthine in MDCKII cells co-expressing URAT1 and GLUT9 was significantly higher than those in mock cells and cells expressing URAT1 or GLUT9 alone. Furthermore, dotinurad, a URAT1 inhibitor, increased renal clearance of xanthine in rats treated with topiroxostat to inhibit XOR. It was suggested that xanthine is reabsorbed in the same manner as uric acid through URAT1 and GLUT9, while hypoxanthine is not. Accordingly, it is expected that treatment with XOR and URAT1 inhibitors will effectively decrease purine pools in the body and prevent cell injury due to ROS generated during XOR-mediated reactions.
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Proteínas de Transporte de Anión/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Transportadores de Anión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Xantina/farmacocinética , Animales , Proteínas de Transporte de Anión/antagonistas & inhibidores , Benzotiazoles/administración & dosificación , Perros , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Células de Riñón Canino Madin Darby , Modelos Animales , Nitrilos/administración & dosificación , Oocitos , Transportadores de Anión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/genética , Piridinas/administración & dosificación , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación Renal/efectos de los fármacos , Ácido Úrico/metabolismo , Xantina/sangre , Xantina/metabolismo , Xantina/orina , Xantina Deshidrogenasa/antagonistas & inhibidores , Xantina Deshidrogenasa/metabolismo , Xenopus laevisRESUMEN
Prostaglandin E2 (PGE2) in the hypothalamus is a principal mediator of the febrile response. However, the role of organic anion transporting polypeptide 2A1 (OATP2A1/SLCO2A1), a prostaglandin transporter, in facilitating this response is unknown. Here, we investigated the effect of Slco2a1 deficiency on the body core temperature (Tc) and on the PGE2 concentration in hypothalamus interstitial fluid (Cisf) and CSF (Ccsf) of lipopolysaccharide (LPS; 100 µg/kg, i.p.)-treated mice of both sexes. Slco2a1-/- mice did not develop a febrile response. Ccsf was increased in Slco2a1+/+ and Slco2a1-/- mice, and Ccsf of Slco2a1-/- mice was well maintained at 5 h after LPS injection (1160 pg/ml) compared with Slco2a1+/+ mice (316 pg/ml). A microdialysis study revealed that Cisf peaked at 2 h after LPS injection in Slco2a1+/+ mice (841 pg/ml), whereas the increase in Cisf was negligible in Slco2a1-/- mice. The PGE2 plasma concentration in Slco2a1-/- mice (201 pg/ml) was significantly higher than that in Slco2a1+/+ mice (54 pg/ml) at 1 h after LPS injection, whereas the two groups showed similar PGE2 concentrations in the hypothalamus. Strong Oatp2a1 immunoreactivity was observed in F4/80-positive microglia and perivascular cells and in brain capillary endothelial cells. The changes in Tc and Cisf seen in LPS-injected Slco2a1+/+ mice were partially attenuated in monocyte-/macrophage-specific Slco2a1-/- (Slco2a1Fl/Fl/LysMCre/+) mice. Thus, OATP2A1 facilitates the LPS-induced febrile response by maintaining a high level of Cisf, possibly by regulating PGE2 secretion from F4/80-positive glial cells and/or facilitating PGE2 transport across the blood-brain barrier. These findings suggest that OATP2A1 is a useful therapeutic target for neuroinflammation.SIGNIFICANCE STATEMENT Fever is a physiological response caused by pyrogen-induced release of prostaglandin E2 (PGE2) in the hypothalamus, which plays a central role in regulating the set-point of body temperature. However, it is unclear whether the prostaglandin transporter OATP2A1/SLCO2A1 is involved in this response. We show here that LPS-induced fever is associated with increased PGE2 concentration in hypothalamus interstitial fluid (Cisf), but not in CSF (Ccsf), by means of a microdialysis study in global Slco2a1-knock-out mice and monocyte-/macrophage-specific Slco2a1-knock-out mice. The results suggest that OATP2A1 serves as a regulator of Cisf in F4/80-positive glial cells. OATP2A1 was detected immunohistochemically in brain capillary endothelial cells and, therefore, may also play a role in PGE2 transport across the blood-brain barrier.