RESUMEN
Metabolic abnormalities play a pivotal role in various pathological conditions, necessitating the quantification of specific metabolites for diagnosis. While mass spectrometry remains the primary method for metabolite measurement, its limited throughput underscores the need for biosensors capable of rapid detection. Previously, we reported that pillar[6]arene with 12 carboxylate groups (P6AC) forms host-guest complexes with 1-methylnicotinamide (1-MNA), which is produced in vivo by nicotinamide N-methyltransferase (NNMT). P6AC acts as a biosensor by measuring the fluorescence quenching caused by photoinduced electron transfer upon 1-MNA binding. However, the low sensitivity of P6AC makes it impractical for detecting 1-MNA in unpurified biological samples. In this study, we found that P6A with 12 sulfonate groups (P6AS) is a specific and potent supramolecular host for 1-MNA interactions even in biological samples. The 1-MNA binding affinity of P6AS in water was found to be (5.68 ± 1.02) × 106 M-1, which is approximately 700-fold higher than that of P6AC. Moreover, the 1-MNA detection limit of P6AS was determined to be 2.84 × 10-7 M, which is substantially lower than that of P6AC. Direct addition of P6AS to culture medium was sufficient to quantify 1-MNA produced by cancer cells. Furthermore, this sensor was able to specifically detect 1-MNA even in unpurified human urine. P6AS therefore enables rapid and high-throughput quantification of 1-MNA, and further improvement of our strategy will contribute to the establishment of high-throughput screening of NNMT inhibitors, diagnosis of liver diseases, and imaging of human cancer cells in vivo.
RESUMEN
Potential inhibition of the breast cancer resistance protein (BCRP), a drug efflux transporter, is a key issue during drug development, and the use of its physiologic substrates as biomarkers can be advantageous to assess inhibition. In this study, we aimed to identify BCRP substrates by an untargeted metabolomic approach. Mice were orally administered lapatinib to inhibit BCRP in vivo, and plasma samples were assessed by liquid chromatography/time of flight/mass spectrometry with all-ion fragmentation acquisition and quantified by liquid chromatography with tandem mass spectrometry. A differential metabolomic analysis was also performed for plasma from Bcrp -/- and wild-type mice. Plasma peaks of food-derived isoflavone metabolites, daidzein sulfate (DS), and genistein sulfate (GS) increased after lapatinib administration and in Bcrp -/- mice. Administration of lapatinib and another BCRP inhibitor febuxostat increased the area under the plasma concentration-time curve (AUC) of DS, GS, and equol sulfate (ES) by 3.6- and 1.8-, 5.6- and 4.1-, and 1.6- and 4.8-fold, respectively. BCRP inhibitors also increased the AUC and maximum plasma concentration of DS and ES after coadministration with each parent compound. After adding parent compounds to the apical side of induced pluripotent stem cell-derived small intestinal epithelial-like cells, DS, GS, and ES in the basal compartment significantly increased in the presence of lapatinib and febuxostat, suggesting the inhibition of intestinal BCRP. ATP-dependent uptake of DS and ES in BCRP-expressing membrane vesicles was reduced by both inhibitors, indicating inhibition of BCRP-mediated DS and ES transport. Thus, we propose the first evidence of surrogate markers for BCRP inhibition. SIGNIFICANCE STATEMENT: This study performed untargeted metabolomics to identify substrates of BCRP/ABCG2 to assess changes in its transport activity in vivo by BCRP/ABCG2 inhibitors. Food-derived isoflavone sulfates were identified as useful markers for evaluating changes in BCRP-mediated transport in the small intestine by its inhibitors.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/efectos de los fármacos , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Isoflavonas/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Animales , Biomarcadores , Neoplasias de la Mama/metabolismo , Cromatografía Líquida de Alta Presión , Resistencia a Antineoplásicos/genética , Genisteína/farmacología , Humanos , Células Madre Pluripotentes Inducidas , Isoflavonas/química , Lapatinib/farmacología , Ratones , Ratones Noqueados , Sulfatos/farmacología , Espectrometría de Masas en TándemRESUMEN
Quantitative assessment of drug-drug interactions (DDIs) via organic anion transporting polypeptide (OATP) 1B1 is one of the key issues in drug development. Although OATP1B1 inhibition exhibits unique characteristics, including preincubation dependence for some inhibitors, a limited approach has been attempted based on the static model that considers such preincubation dependence in the prediction of DDIs via OATP1B1. The present study aimed to establish the prediction of DDIs via OATP1B1 using preincubation-dependent inhibitors based on the static model and incorporating both inactivation and recovery of OATP1B1 activity. Cyclosporine A was selected as a preincubation-dependent inhibitor, as well as five substrates that include probes and pharmaceuticals. The inhibition ratio (R value) calculated on the basis of a conventional static model, considering inhibition of OATP1B1 and contribution ratio of OATP1B1 to the overall hepatic uptake, was much lower than the reported AUC ratio, even when IC50 values were estimated after preincubation conditions. Conversely, the R value that was estimated by considering inactivation and recovery parameters was closer to the AUC ratio. The R value that was calculated assuming the complete contribution of OATP1B1 was much higher than the AUC ratio, avoiding false-negative prediction. The R value estimated by considering inactivation and recovery for another combination of a preincubation-dependent inhibitor, asunaprevir, and substrate drug, rosuvastatin, was also closer to the AUC ratio. Thus, R values calculated based on such OATP1B1 kinetics would be potential alternative indexes for the quantitative prediction of OATP1B1-mediated DDIs using preincubation-dependent inhibitors, although this prediction is affected by estimation of the contribution ratio of substrates. SIGNIFICANCE STATEMENT: Static model-based quantitative prediction of organic anion transporting polypeptide 1B1-mediated drug-drug interactions induced by preincubation-dependent inhibitors was newly proposed to avoid false-negative prediction.
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Interacciones Farmacológicas , Eliminación Hepatobiliar/fisiología , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Modelos Biológicos , Área Bajo la Curva , Ciclosporina/farmacología , Evaluación Preclínica de Medicamentos/métodos , Células HEK293 , Eliminación Hepatobiliar/efectos de los fármacos , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacocinética , Concentración 50 Inhibidora , Isoquinolinas/farmacología , Hígado/metabolismo , Transportador 1 de Anión Orgánico Específico del Hígado/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Rosuvastatina Cálcica/farmacocinética , Sulfonamidas/farmacologíaRESUMEN
Understanding of the underlying mechanism of epilepsy is desired since some patients fail to control their seizures. The carnitine/organic cation transporter OCTN1/SLC22A4 is expressed in brain neurons and transports food-derived antioxidant ergothioneine (ERGO), L-carnitine, and spermine, all of which may be associated with epilepsy. This study aimed to clarify the possible association of this transporter with epileptic seizures. In both pentylenetetrazole (PTZ)-induced acute seizure and kindling models, ocnt1 gene knockout mice (octn1-/-) showed lower seizure scores compared with wild-type mice. Up-regulation of the epilepsy-related genes, c-fos and Arc, and the neurotrophic factor BDNF following PTZ administration was observed in the hippocampus of wild-type, but not octn1-/- mice. To find the OCTN1 substrate associated with the seizure, untargeted metabolomics analysis using liquid chromatography-quadrupole time-of-flight mass spectrometry was conducted on extracts from the hippocampus, frontal cortex, and plasma of both strains, leading to the identification of a plant alkaloid homostachydrine as a compound present in a lower concentration in octn1-/- mice. OCTN1-mediated uptake of deuterium-labeled homostachydrine was confirmed in OCTN1-transfected HEK293 cells, suggesting that this compound is a substrate of OCTN1. Homostachydrine administration increased PTZ-induced acute seizure scores and the expression of Arc in the hippocampus and that of Arc, Egr1, and BDNF in the frontal cortex. Conversely, administration of the OCTN1 substrate/inhibitor ERGO inhibited PTZ-induced kindling and reduced the plasma homostachydrine concentration. Thus, these results suggest that OCTN1 is at least partially associated with PTZ-induced seizures, which is potentially deteriorated by treatment with homostachydrine, a newly identified food-derived OCTN1 substrate.
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Epilepsia/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Piperidinas/farmacología , Convulsiones/metabolismo , Simportadores/metabolismo , Xenobióticos/farmacología , Animales , Antioxidantes/farmacología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteínas del Citoesqueleto/metabolismo , Epilepsia/inducido químicamente , Ergotioneína/farmacología , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Pentilenotetrazol , Piperidinas/metabolismo , Piperidinas/orina , Convulsiones/inducido químicamente , Simportadores/genética , Xenobióticos/metabolismo , Xenobióticos/orinaRESUMEN
PURPOSE: P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are xenobiotic transporters which pump out variety types of compounds, but information on their interaction with endogenous substrates in the skin is limited. The purpose of the present study was to clarify possible association of these transporters in dermal accumulation of inflammatory mediators. METHODS: Dermatitis model was constructed by repeated topical application of oxazolone in wild-type, and P-gp and BCRP gene triple knockout (Mdr1a/1b/Bcrp-/-) mice to observe difference in phenotype. Target metabolome analysis of 583 metabolites was performed using skin and plasma. RESULTS: Dermatitis and scratching behavior in dermatitis model of Mdr1a/1b/Bcrp-/- mice were more severe than wild-type mice, suggesting protective roles of these transporters. This hypothesis was supported by the metabolome analysis which revealed that concentration of histamine and other dermatitis-associated metabolites like urate and serotonin in the dermatitis skin, but not normal skin, of Mdr1a/1b/Bcrp-/- mice was higher than that of wild-type mice. Gene expression of P-gp and BCRP was reduced in oxazolone-treated skin and the skin of patients with atopic dermatitis or psoriasis. CONCLUSIONS: These results suggest possible association of these efflux transporters with dermal inflammatory mediators, and such association could be observed in the dermatitis skin.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Dermatitis/metabolismo , Histamina/metabolismo , Metaboloma/efectos de los fármacos , Proteínas de Neoplasias/genética , Piel/metabolismo , Animales , Eliminación de Gen , Humanos , Masculino , Ratones , Ratones NoqueadosRESUMEN
The aim of the present study is to construct and characterize a novel three-dimensional culture system for mouse neurons using the functional polymer, FP001. Stereoscopically extended neurites were found in primary mouse cortical neurons cultured in the FP001-containing medium. Neurons cultured with FP001 were distributed throughout the medium of the observation range whereas neurons cultured without FP001 were distributed only on the bottom of the dish. These results demonstrated that neurons can be three-dimensionally cultured using the FP001-containing medium. The mRNA expression of the glutamatergic neuronal marker vesicular glutamate transporter 1 in neurons cultured in the FP001-containing medium were higher than that in neurons cultured in the FP001-free medium. Expression of the matured neuronal marker, microtubule-associated protein 2 (MAP2) a,b, and the synapse formation marker, Synapsin I, in neurons cultured with FP001 was also higher than that in neurons cultured without FP001. The expression pattern of MAP2a,b in neurons cultured with FP001, but not that in neurons cultured without FP001, was similar to that in the embryonic cerebral cortex. Exposure to glutamate significantly increased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity in neurons cultured with FP001 compared to that in neurons cultured without FP001. These results suggested that glutamatergic neurotransmission in neurons three-dimensionally cultured in the FP001-containing medium may be upregulated compared to neurons two-dimensionally cultured in the FP001-free medium. Thus, neurons with the properties close to those in the embryonic brain could be obtained by three-dimensionally culturing neurons using FP001, compared to two-dimensional culture with a conventional adhesion method.
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Técnicas de Cultivo de Célula/métodos , Corteza Cerebral/citología , Medios de Cultivo/química , Neuronas/citología , Polisacáridos Bacterianos/química , Animales , Corteza Cerebral/embriología , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Proteínas Asociadas a Microtúbulos/metabolismo , Neuritas/metabolismo , Neuronas/metabolismo , Sinapsinas/metabolismoRESUMEN
Pazopanib is an orally active tyrosine kinase inhibitor that exhibits hepatotoxicity in some patients. Despite the clinical importance of its hepatic distribution, the transporter(s) responsible for hepatic uptake of pazopanib in humans remain undetermined. To characterize its hepatic uptake mechanism, we screened the effects of several transporter inhibitors, including tetrapentylammonium (TPeA) for organic cation transporters (OCTs) and cyclosporin A (CsA) for organic anion-transporting polypeptides (OATPs), on both plasma disappearance and hepatic distribution of pazopanib in mice after its i.v. administration. Among the inhibitors, TPeA largely reduced hepatic distribution and plasma clearance of pazopanib, whereas CsA showed only partial reduction. Pazopanib uptake by isolated mouse hepatocytes was similarly reduced by these inhibitors, suggesting that OCTs play a major role in the overall hepatic uptake of pazopanib in mice. In human embryonic kidney cell line HEK293 cells stably transfected with human OCT1, pazopanib uptake was significantly higher than that in vector-transfected cells. Moreover, pazopanib uptake by OCT1 became saturated and was inhibited by TPeA, but not by CsA, confirming that pazopanib is also a substrate of human OCT1. Importantly, OCT1-mediated uptake of a typical OCT1 substrate metformin was inhibited by pazopanib with an IC50 value of 0.253 µM, indicating that pazopanib has the potential for clinically relevant inhibition of human OCT1. Finally, pazopanib was taken up by cryopreserved human pooled hepatocytes in a time-dependent manner, and this uptake was largely reduced by TPeA but only partially reduced by CsA. Thus, the present findings suggest that OCT1 is responsible for hepatocellular uptake of pazopanib.
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Hígado/metabolismo , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Pirimidinas/farmacocinética , Sulfonamidas/farmacocinética , Administración Intravenosa , Animales , Ciclosporina/farmacología , Células HEK293 , Hepatocitos/metabolismo , Humanos , Indazoles , Concentración 50 Inhibidora , Hígado/citología , Masculino , Metformina/farmacocinética , Ratones , Ratones Endogámicos ICR , Factor 1 de Transcripción de Unión a Octámeros/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirimidinas/administración & dosificación , Compuestos de Amonio Cuaternario/farmacología , Sulfonamidas/administración & dosificación , Distribución Tisular , TransfecciónRESUMEN
Brain immune cells, i.e., microglia, play an important role in the maintenance of brain homeostasis, whereas chronic overactivation of microglia is involved in the development of various neurodegenerative disorders. Therefore, the regulation of microglial activation may contribute to their treatment. The aim of the present study was to clarify the functional expression of carnitine/organic cation transporter OCTN1/SLC22A4, which recognizes the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a substrate in vivo, in microglia and its role in regulation of microglial activation. Primary cultured microglia derived from wild-type mice (WT-microglia) and mouse microglial cell line BV2 exhibited time-dependent uptake of [3H]- or d9-labeled ERGO. The uptake was markedly decreased in cultured microglia from octn1 gene knockout mice (octn1 -/--microglia) and BV2 cells transfected with small interfering RNA targeting the mouse octn1 gene (siOCTN1). These results demonstrate that OCTN1 is functionally expressed in murine microglial cells. Exposure of WT-microglia to ERGO led to a significant decrease in cellular hypertrophy by LPS-stimulation with concomitant attenuation of intracellular reactive oxygen species (ROS), suggesting that OCTN1-mediated ERGO uptake may suppress cellular hypertrophy via the inhibition of ROS production with microglial activation. The expression of mRNA for interleukin-1ß (IL-1ß) after LPS-treatment was significantly increased in octn1 -/--microglia and siOCTN1-treated BV2 cells compared to the control cells. Meanwhile, treatment of ERGO minimally affected the induction of IL-1ß mRNA by LPS-stimulation in cultured microglia and BV2 cells. Thus, OCTN1 negatively regulated the induction of inflammatory cytokine IL-1ß, at least in part, via the transport of unidentified substrates other than ERGO in microglial cells.
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Transporte Biológico/efectos de los fármacos , Proteínas Portadoras/metabolismo , Ergotioneína/farmacología , Proteínas de la Membrana/metabolismo , Microglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Carnitina , Proteínas Portadoras/genética , Células Cultivadas , Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Proteínas de la Membrana/genética , Ratones , Microglía/metabolismo , Neuronas/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , SimportadoresRESUMEN
PURPOSE: Solute carrier SLC22A4 encodes the carnitine/organic cation transporter OCTN1 and is associated with inflammatory bowel disease, although little is known about how this gene is linked to pathogenesis. The aim of the present study was to identify endogenous substrates that are associated with gastrointestinal inflammation. METHODS: HEK293/OCTN1 and mock cells were incubated with colon extracts isolated from dextran sodium sulfate-induced colitis mice; the subsequent cell lysates were mixed with the amino group selective reagent 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), to selectively label OCTN1 substrates. Precursor ion scanning against the fragment ion of APDS was then used to identify candidate OCTN1 substrates. RESULTS: Over 10,000 peaks were detected by precursor ion scanning; m/z 342 had a higher signal in HEK293/OCTN1 compared to mock cells. This peak was detected as a divalent ion that contained four APDS-derived fragments and was identified as spermine. Spermine concentration in peripheral blood mononuclear cells from octn1 gene knockout mice (octn1-/-) was significantly lower than in wild-type mice. Lipopolysaccharide-induced gene expression of inflammatory cytokines in peritoneal macrophages from octn1-/- mice was lower than in wild-type mice. CONCLUSIONS: The combination metabolomics approach can provide a novel tool to identify endogenous substrates of OCTN1.
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Proteínas de Transporte de Catión Orgánico/metabolismo , Animales , Colitis/inducido químicamente , Colitis/metabolismo , Colon/química , Colon/metabolismo , Citocinas/metabolismo , Células HEK293 , Humanos , Enfermedades Inflamatorias del Intestino/metabolismo , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Metabolómica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Transporte de Catión Orgánico/genética , Espermina/metabolismo , Simportadores , Extractos de Tejidos/farmacologíaRESUMEN
PURPOSE: Monoamine oxidases (MAOs) are non-CYP enzymes that contribute to systemic elimination of therapeutic agents, and localized on mitochondrial membranes. The aim of the present study was to validate quantitative estimation of metabolic clearance of MAO substrate drugs using human liver microsomes (HLMs). METHODS: Three MAO substrate drugs, sumatriptan, rizatriptan and phenylephrine, as well as four CYP substrates were selected, and their disappearance during incubation with HLMs or mitochondria (HLMt) was measured. Metabolic clearance (CL) was then calculated from the disappearance curve. RESULTS: CL obtained in HLMs for sumatriptan and a typical MAO substrate serotonin was correlated with that obtained in HLMt among ten human individual livers. Hepatic intrinsic clearance (CLint,vitro) estimated from CL in HLMs was 14-20 and 2-5 times lower than in vivo hepatic intrinsic clearance (CLint,vivo) obtained from literature for MAO and CYP substrates, respectively. Utilization of HLMs for quantitatively assessing metabolic clearance of MAO substrates was further validated by proteomics approach which has revealed that numerous proteins localized on inner and outer membranes of mitochondria were detected in both HLMs and HLMt. CONCLUSION: CLint,vitro values of MAO substrate drugs can be quantitatively estimated with HLMs and could be used for semi-quantitative prediction of CLint,vivo values.
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Microsomas Hepáticos/metabolismo , Monoaminooxidasa/metabolismo , Fenilefrina/metabolismo , Sumatriptán/metabolismo , Triazoles/metabolismo , Triptaminas/metabolismo , Humanos , Cinética , Tasa de Depuración Metabólica , Fenilefrina/farmacología , Sumatriptán/farmacología , Triazoles/farmacología , Triptaminas/farmacologíaRESUMEN
Bile acids are synthesized from cholesterol in the liver and subjected to multiple metabolic biotransformations in hepatocytes, including oxidation by cytochromes P450 (CYPs) and conjugation with taurine, glycine, glucuronic acid, and sulfate. Mice and rats can hydroxylate chenodeoxycholic acid (CDCA) at the 6ß-position to form α-muricholic acid (MCA) and ursodeoxycholic acid (UDCA) to form ß-MCA. However, MCA is not formed in humans to any appreciable degree and the mechanism for this species difference is not known. Comparison of several Cyp-null mouse lines revealed that α-MCA and ß-MCA were not detected in the liver samples from Cyp2c-cluster null (Cyp2c-null) mice. Global bile acid analysis further revealed the absence of MCAs and their conjugated derivatives, and high concentrations of CDCA and UDCA in Cyp2c-null mouse cecum and feces. Analysis of recombinant CYPs revealed that α-MCA and ß-MCA were produced by oxidation of CDCA and UDCA by Cyp2c70, respectively. CYP2C9-humanized mice have similar bile acid metabolites as the Cyp2c-null mice, indicating that human CYP2C9 does not oxidize CDCA and UDCA, thus explaining the species differences in production of MCA. Because humans do not produce MCA, they lack tauro-ß-MCA, a farnesoid X receptor antagonist in mouse that modulates obesity, insulin resistance, and hepatosteatosis.
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Ácidos Cólicos/biosíntesis , Sistema Enzimático del Citocromo P-450/fisiología , Animales , Expresión Génica , Células Hep G2 , Humanos , Cinética , Hígado/enzimología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción , Especificidad de la EspecieRESUMEN
PURPOSE: Delayed plasma concentration profiles of the active irinotecan metabolite SN-38 were observed in cancer patients with severe renal failure (SRF), even though SN-38 is eliminated mainly via the liver. Here, we examined the plasma concentrations of unbound SN-38 in such patients. METHODS: Plasma unbound concentrations were examined by ultrafiltration. Physiologically-based pharmacokinetic (PBPK) models of irinotecan and SN-38 were established to quantitatively assess the principal mechanism for delayed SN-38 elimination. RESULTS: The area under the plasma unbound concentration-time curve (AUC(u)) of SN-38 in SRF patients was 4.38-fold higher than that in normal kidney patients. The unbound fraction of SN-38 was also 2.6-fold higher in such patients, partly because SN-38 protein binding was displaced by the uremic toxin 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF). This result was supported by correlation of the unbound fraction of SN-38 with the plasma CMPF concentration, which negatively correlated with renal function. PBPK modeling indicated substantially reduced influx of SN-38 into hepatocytes and approximately one-third irinotecan dose for SRF patients to produce an unbound concentration profile of SN-38 similar to normal kidney patients. CONCLUSION: The AUC(u) of SN-38 in SRF cancer patients is much greater than that of normal kidney patients primarily because of the reduced hepatic uptake of SN-38.
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Lesión Renal Aguda/complicaciones , Antineoplásicos Fitogénicos/sangre , Antineoplásicos Fitogénicos/uso terapéutico , Camptotecina/análogos & derivados , Neoplasias/complicaciones , Neoplasias/tratamiento farmacológico , Lesión Renal Aguda/sangre , Lesión Renal Aguda/fisiopatología , Camptotecina/sangre , Camptotecina/metabolismo , Camptotecina/uso terapéutico , Humanos , Irinotecán , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/fisiopatología , Modelos Biológicos , Neoplasias/sangre , Neoplasias/fisiopatología , Unión ProteicaRESUMEN
The aim of the present study is to discover multidrug resistance-associated protein (MRP) inhibitors with neuroblastoma-selective cytotoxicity by means of fluorescence assay with a membrane-permeable fluorescent dye, Fluo-8 AM, based on our observation that gene expression of Mrp3 in neuroblastoma Neuro2a cells was remarkably higher than that in primary cultured cortical neurons, as determined by real-time PCR. Neuro2a cells showed minimal fluorescence upon incubation with Fluo-8 AM. However, blocking of Mrp3 efflux function by small interfering RNA (siRNA) transfection or inhibition with probenecid resulted in significant dye accumulation, observed as an increase of fluorescence. Interestingly, Mrp3 siRNA or probenecid treatment also resulted in increased cytotoxicity, as evidenced by decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-reducing activity of Neuro2a, with a concomitant increase in release of lactate dehydrogenase. On the other hand, primary cultured neurons exhibited higher fluorescence intensity after incubation with Fluo-8 AM regardless of addition of probenecid. Also, probenecid only minimally affected MTT-reducing activity. Thus, probenecid showed selective cytotoxicity towards Neuro2a cells. Based on these findings, we screened a series of established therapeutic agents for ability to induce Fluo-8 accumulation in Neuro2a cells. Several uricosuric and nonsteroidal anti-inflammatory drugs were identified, and these drugs were confirmed to decrease MTT-reducing activity selectively in Neuro2a. There was a negative linear correlation between Fluo-8 accumulation and cytotoxicity of these agents. Although the compounds identified here are insufficiently potent for practical application, further screening to discover higher-affinity MRP3 inhibitors using larger chemical libraries may uncover drug candidates with potent neuroblastoma-selective cytotoxicity.
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Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Neuroblastoma/tratamiento farmacológico , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Antiinflamatorios no Esteroideos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Femenino , Ratones , Ratones Endogámicos ICR , Neuroblastoma/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Embarazo , Uricosúricos/farmacologíaRESUMEN
5-Aminosalicylic acid (5-ASA) is an orally administered therapeutic agent for inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease. We hypothesized that the absorption of 5-ASA is mediated by the polyspecific carnitine/organic cation transporter (OCTN1/SLC22A4), based on the similarity of chemical structure between 5-ASA and other OCTN1 substrates. Therefore, we examined the involvement of this transporter in the disposition of 5-ASA in vivo by using octn1 gene knockout (octn1(-/-)) mice. After oral administration of 5-ASA, the plasma concentrations of 5-ASA and its primary metabolite, N-acetyl-5-aminosalicylate (Ac-5-ASA), in octn1(-/-) mice were much lower than those in wild-type mice. The time required to reach maximum plasma concentration was also delayed in octn1(-/-) mice. On the other hand, the plasma concentration profiles of both 5-ASA and Ac-5-ASA after intravenous administration of 5-ASA (bolus or infusion) were similar in the two strains. Uptake of 5-ASA from the apical to the basal side of isolated small-intestinal tissues of octn1(-/-) mice, determined in an Ussing-type chamber, was lower than that in wild-type mice. Further, uptake of 5-ASA in HEK293 cells stably transfected with the OCTN1 gene, assessed as the sum of cell-associated 5-ASA and Ac-5-ASA, was higher than that in HEK293 cells transfected with the vector alone. Overall, these results indicate that OCTN1 is involved, at least in part, in the gastrointestinal absorption of 5-ASA.
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Carnitina/metabolismo , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Absorción Intestinal , Intestino Delgado/metabolismo , Mesalamina/farmacocinética , Transportador 1 de Catión Orgánico/metabolismo , Animales , Disponibilidad Biológica , Transporte Biológico Activo , Células HEK293 , Humanos , Masculino , Proteínas de la Membrana , Mesalamina/sangre , Mesalamina/uso terapéutico , Ratones , Ratones Noqueados , Transportador 1 de Catión Orgánico/genética , Eliminación de Secuencia , TransfecciónRESUMEN
Eltrombopag (ELT), an orally available thrombopoietin receptor agonist, is a substrate of organic anion transporting polypeptide 1B1 (OATP1B1), and coadministration of ELT increases the plasma concentration of rosuvastatin in humans. Since the pharmacokinetic mechanism(s) of the interaction is unknown, the present study aimed to clarify the drug interaction potential of ELT at transporters. The OATP1B1-mediated uptake of ELT was inhibited by several therapeutic agents used to treat lifestyle diseases. Among them, rosuvastatin was a potent inhibitor with an IC(50) of 0.05 µM, which corresponds to one-seventh of the calculated maximum unbound rosuvastatin concentration at the inlet to the liver. Nevertheless, a simulation study using a physiologically based pharmacokinetic model predicted that the effect of rosuvastatin on the pharmacokinetic profile of ELT in vivo would be minimal. On the other hand, ELT potently inhibited uptake of rosuvastatin by OATP1B1 and human hepatocytes, with an IC(50) of 0.1 µM. However, the results of the simulation study indicated that inhibition of OATP1B1 by ELT can only partially explain the clinically observed interaction with rosuvastatin. ELT also inhibited transcellular transport of rosuvastatin in MDCKII cells stably expressing breast cancer resistance protein (BCRP), and was found to be a substrate of BCRP. The interaction of ELT with rosuvastatin can be almost quantitatively explained on the assumption that intestinal secretion of rosuvastatin is essentially completely inhibited by ELT. These results suggest that BCRP in small intestine may be the major target for interaction between ELT and rosuvastatin in humans.
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Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Benzoatos/farmacocinética , Plaquetas/efectos de los fármacos , Fluorobencenos/farmacocinética , Hidrazinas/farmacocinética , Intestino Delgado/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Transportadores de Anión Orgánico/antagonistas & inhibidores , Pirazoles/farmacocinética , Pirimidinas/farmacocinética , Sulfonamidas/farmacocinética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Adulto , Animales , Benzoatos/farmacología , Transporte Biológico , Plaquetas/citología , Simulación por Computador , Perros , Interacciones Farmacológicas , Fluorobencenos/sangre , Células HEK293 , Hepatocitos/metabolismo , Humanos , Hidrazinas/farmacología , Células LLC-PK1 , Transportador 1 de Anión Orgánico Específico del Hígado , Células de Riñón Canino Madin Darby , Masculino , Ratones , Modelos Biológicos , Proteínas de Neoplasias/genética , Transportadores de Anión Orgánico/genética , Pirazoles/farmacología , Pirimidinas/sangre , Receptores de Trombopoyetina/agonistas , Rosuvastatina Cálcica , Sulfonamidas/sangre , PorcinosRESUMEN
Oral administration of the food-derived antioxidant amino acid ergothioneine (ERGO) results in its efficient distribution in the brain and enhances cognitive function. However, effect of ERGO deficiency on cognitive impairment and the underlying mechanisms remain unknown. We revealed that cognitive function and hippocampal neurogenesis were lower in mice fed an ERGO-free diet than in those fed the control diet. Furthermore, ERGO supplementation to achieve the control diet ERGO levels reversed these effects and restored ERGO concentrations in the plasma and hippocampus. The ERGO-induced recovery of cognitive function and hippocampal neurogenesis was blocked by inhibiting the neurotrophic factor receptor tropomyosin receptor kinase B (TrkB), with a concomitant reduction in hippocampal phosphorylated TrkB, suggesting the involvement of TrkB in these events in mice. Phosphorylated TrkB was also detected in extracellular vesicles (EVs) derived from serum of volunteers who had been orally administered placebo or ERGO-containing tablets. Importantly, the ratio of serum EV-derived phosphorylated TrkB was significantly higher in the ERGO-treated group than in the placebo-treated group and was positively correlated with both serum ERGO concentrations and several cognitive domain scores from Cognitrax. Altogether, TrkB phosphorylation is involved in ERGO-induced cognitive enhancement in mice, and TrkB phosphorylation levels in serum EVs may quantitatively represent ERGO-induced cognitive enhancement in humans.
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Breast cancer resistance protein (BCRP) is expressed on hepatic bile canalicular membranes; however, its impact on substrate drug disposition is limited. This study proposes an in vivo knockdown approach using adeno-associated virus encoding short hairpin RNA (shRNA) targeting the bcrp gene (AAV-shBcrp) to clarify the substrate, the overall disposition of which is largely governed by hepatic Bcrp. The disposition of the tyrosine kinase inhibitor, regorafenib, was first examined in bcrp gene knockout (Bcrp-/-) and wild-type (WT) mice, as it was sequentially converted to active metabolites M - 2 and M - 5, which are BCRP substrates. After oral administration of regorafenib, plasma and liver concentrations of M - 5, but not regorafenib, were higher in Bcrp-/- than WT mice. To directly examine the role of hepatic Bcrp in M - 5 disposition, M - 5 was intravenously injected into mice three weeks after the intravenous injection of AAV-shBcrp, when mRNA of Bcrp in the liver (but not the small intestine) was downregulated. AAV-shBcrp-treated mice showed higher M - 5 concentration in plasma and liver, but lower biliary excretion than the control mice, indicating the fundamental role of hepatic Bcrp in M - 5 disposition. This is the first application of AAV-knockdown strategy to clarify the pharmacokinetic role of xenobiotic efflux transporters in the liver.
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Dependovirus , Ratones , Animales , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Proteínas de Neoplasias/metabolismo , Hígado/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/metabolismo , Ratones NoqueadosRESUMEN
A fetal pituitary hormone, oxytocin which causes uterine contractions, increases throughout gestation, and its increase reaches 10-fold from week 32 afterward. Oxytocin is, on the other hand, degraded by placental leucine aminopeptidase (P-LAP) which exists in both terminal villi and maternal blood. Maternal blood P-LAP increases with advancing gestation under the control of non-genomic effects of progesterone, which is also produced from the placenta. Progesterone is converted to estrogen by CYP17A1 localized in the fetal adrenal gland and placenta at term. The higher oxytocin concentrations in the fetus than in the mother demonstrate not only fetal oxytocin production but also its degradation and/or inhibition of leakage from fetus to mother by P-LAP. Until labor onset, the pregnant uterus is quiescent possibly due to the balance between increasing fetal oxytocin and P-LAP under control of progesterone. A close correlation exists between the feto-placental and maternal units in the placental circulation, although the blood in the two circulations does not necessarily mix. Fetal maturation results in progesterone withdrawal via the CYP17A1 activation accompanied with fetal oxytocin increase. Contribution of fetal oxytocin to labor onset has been acknowledged through the recognition that the effect of fetal oxytocin in the maternal blood is strictly regulated by its degradation by P-LAP under the control of non-genomic effects of progesterone. In all senses, the fetus necessarily takes the initiative in labor onset.
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Hypothalamic neurons regulate body homeostasis by sensing and integrating changes in the levels of key hormones and primary nutrients (amino acids, glucose, and lipids). However, the molecular mechanisms that enable hypothalamic neurons to detect primary nutrients remain elusive. Here, we identified l-type amino acid transporter 1 (LAT1) in hypothalamic leptin receptor-expressing (LepR-expressing) neurons as being important for systemic energy and bone homeostasis. We observed LAT1-dependent amino acid uptake in the hypothalamus, which was compromised in a mouse model of obesity and diabetes. Mice lacking LAT1 (encoded by solute carrier transporter 7a5, Slc7a5) in LepR-expressing neurons exhibited obesity-related phenotypes and higher bone mass. Slc7a5 deficiency caused sympathetic dysfunction and leptin insensitivity in LepR-expressing neurons before obesity onset. Importantly, restoring Slc7a5 expression selectively in LepR-expressing ventromedial hypothalamus neurons rescued energy and bone homeostasis in mice deficient for Slc7a5 in LepR-expressing cells. Mechanistic target of rapamycin complex-1 (mTORC1) was found to be a crucial mediator of LAT1-dependent regulation of energy and bone homeostasis. These results suggest that the LAT1/mTORC1 axis in LepR-expressing neurons controls energy and bone homeostasis by fine-tuning sympathetic outflow, thus providing in vivo evidence of the implications of amino acid sensing by hypothalamic neurons in body homeostasis.
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Hipotálamo , Transportador de Aminoácidos Neutros Grandes 1 , Ratones , Animales , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Hipotálamo/metabolismo , Obesidad/metabolismo , Neuronas/metabolismo , Homeostasis/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismoRESUMEN
This study aimed to quantitatively clarify the critical factors responsible for the superior antitumor efficacy of a liposomal gemcitabine (2,2-difluorodeoxycytidine; dFdC) formulation, FF-10832, compared with dFdC. The underlying hypothesis is the different exposure of tumors to its active metabolite, dFdC triphosphate (dFdCTP), between the two formulations. Therefore, physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models for encapsulated and unencapsulated dFdC were constructed considering the tumor dFdCTP concentration as an index of antitumor activity. To estimate drug the parameters, the time profiles of encapsulated and unencapsulated dFdC in the blood and those of dFdC and dFdCTP in tumors were measured following the intravenous bolus administration of FF-10832 or dFdC. dFdC metabolism and transport in the liver S9 fraction and isolated hepatocytes, respectively, were experimentally determined. The tumor growth curve in a mouse xenograft model following the administration of FF10832 and dFdC was also used to construct the PD model. The sensitivity analysis of the PBPK/PD model revealed the critical factors affecting antitumor efficacy, which included the total and intratumor tissue uptake clearances for liposomal formulation and the cytidine deaminase and deoxycytidine deaminase activities in tumors. Thus, these parameters are potential biomarkers for predicting the efficacy of the liposomal formulation of dFdC.