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1.
World J Gastroenterol ; 25(32): 4580-4597, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31528088

RESUMO

Chronic delta hepatitis is the most severe form of viral hepatitis affecting nearly 65 million people worldwide. Individuals with this devastating illness are at higher risk for developing cirrhosis and hepatocellular carcinoma. Delta virus is a defective RNA virus that requires hepatitis B surface antigen for propagation in humans. Infection can occur in the form of a co-infection with hepatitis B, which can be self-limiting, vs superinfection in a patient with established hepatitis B infection, which often leads to chronicity in majority of cases. Current noninvasive tools to assess for advanced liver disease have limited utility in delta hepatitis. Guidelines recommend treatment with pegylated interferon, but this is limited to patients with compensated disease and is efficacious in about 30% of those treated. Due to limited treatment options, novel agents are being investigated and include entry, assembly and export inhibitors of viral particles in addition to stimulators of the host immune response. Future clinical trials should take into consideration the interaction of hepatitis B and hepatitis D as suppression of one virus can lead to the activation of the other. Also, surrogate markers of treatment efficacy have been proposed.


Assuntos
Antivirais/uso terapêutico , Coinfecção/tratamento farmacológico , Hepatite B Crônica/tratamento farmacológico , Hepatite D Crônica/tratamento farmacológico , Superinfecção/tratamento farmacológico , Terapias em Estudo/métodos , Antivirais/farmacologia , Coinfecção/epidemiologia , Coinfecção/virologia , Quimioterapia Combinada/métodos , Carga Global da Doença , Antígenos de Superfície da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/epidemiologia , Hepatite B Crônica/virologia , Hepatite D Crônica/epidemiologia , Hepatite D Crônica/virologia , Vírus Delta da Hepatite/imunologia , Vírus Delta da Hepatite/patogenicidade , Humanos , Interferon-alfa/farmacologia , Interferon-alfa/uso terapêutico , Lipopeptídeos/farmacologia , Lipopeptídeos/uso terapêutico , Transportadores de Ânions Orgânicos Dependentes de Sódio/antagonistas & inibidores , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Literatura de Revisão como Assunto , Superinfecção/epidemiologia , Superinfecção/virologia , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Resultado do Tratamento , Montagem de Vírus/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos
2.
Curr Med Sci ; 39(3): 403-409, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31209810

RESUMO

Accumulation of lactate in tumor has been linked to poor prognosis of oral squamous cell carcinoma (OSCC), but the underlying mechanism remained largely uncertain. Previous studies have suggested that presence of cancer stem cells (CSCs) closely correlated with cellular malignancy of OSCC. Here, using 3D organoid culture model, we investigated whether lactate promoted CSCs phenotype in primary OSCC cells. We generated organoids using fresh OSCC specimens and verified that organoids recapitulated histopathology and cellular heterogeneity of parental tumor. Organoids were then transfected with a Wnt reporter to visualize Wnt activity. The sphere forming assay demonstrated that high Wnt activity functionally designated CSCs population in OSCC cells. Further investigations indicated that lactate treatment promoted Wnt activity and increased the expression of CSCs (i.e. CD133+ cells) in organoids. Moreover, silencing monocarboxylate transporter 1 (MCT1), the prominent path for lactate uptake in human tumor with siRNA significantly impaired organoid forming capacity of OSCC cells. Together, our study demonstrated that lactate can promote CSCs phenotype of OSCC, and MCT1 may be a therapeutic target against OSCC growth.


Assuntos
Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Ácido Láctico/farmacologia , Transportadores de Ácidos Monocarboxílicos/genética , Neoplasias Bucais/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Simportadores/genética , Via de Sinalização Wnt/efeitos dos fármacos , Antígeno AC133/genética , Antígeno AC133/metabolismo , Idoso , Proteína Axina/genética , Proteína Axina/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Masculino , Pessoa de Meia-Idade , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Via de Sinalização Wnt/genética
3.
Biomed Res Int ; 2019: 8941046, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31240228

RESUMO

Chloride (Cl-) homeostasis is an essential process involved in neuronal signalling and cell survival. Inadequate regulation of intracellular Cl- interferes with synaptic signalling and is implicated in several neurological diseases. The main inhibitory neurotransmitter of the central nervous system is γ-aminobutyric acid (GABA). GABA hyperpolarises the membrane potential by activating Cl- permeable GABAA receptor channels (GABAAR). This process is reliant on Cl- extruder K+-Cl- cotransporter 2 (KCC2), which generates the neuron's inward, hyperpolarising Cl- gradient. KCC2 is encoded by the fifth member of the solute carrier 12 family (SLC12A5) and has remained a poorly understood component in the development and severity of many neurological diseases for many years. Recent advancements in next-generation sequencing and specific gene targeting, however, have indicated that loss of KCC2 activity is involved in a number of diseases including epilepsy and schizophrenia. It has also been implicated in neuropathic pain following spinal cord injury. Any variant of SLC12A5 that negatively regulates the transporter's expression may, therefore, be implicated in neurological disease. A recent whole exome study has discovered several causative mutations in patients with epilepsy. Here, we discuss the implications of KCC2 in neurological disease and consider the evolving evidence for KCC2's potential as a therapeutic target.


Assuntos
Canais de Cloreto/metabolismo , Neurônios/metabolismo , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Ácido gama-Aminobutírico/farmacologia , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Cloretos/metabolismo , Epilepsia/metabolismo , Marcação de Genes , Homeostase , Humanos , Potenciais da Membrana , Neuralgia/metabolismo , Neurotransmissores/farmacologia , Fosforilação , Esquizofrenia/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Traumatismos da Medula Espinal , Simportadores/genética
4.
PLoS One ; 14(6): e0218459, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31233523

RESUMO

Interruption of bile acid recirculation through inhibition of the apical sodium-dependent bile acid transporter (ASBT) is a promising strategy to alleviate hepatic cholesterol accumulation in non-alcoholic steatohepatitis (NASH), and improve the metabolic aspects of the disease. Potential disease-attenuating effects of the ASBT inhibitor volixibat (5, 15, and 30 mg/kg) were investigated in high-fat diet (HFD)-fed Ldlr-/-.Leiden mice over 24 weeks. Plasma and fecal bile acid levels, plasma insulin, lipids, and liver enzymes were monitored. Final analyses included liver histology, intrahepatic lipids, mesenteric white adipose tissue mass, and liver gene profiling. Consistent with its mechanism of action, volixibat significantly increased the total amount of bile acid in feces. At the highest dose, volixibat significantly attenuated the HFD-induced increase in hepatocyte hypertrophy, hepatic triglyceride and cholesteryl ester levels, and mesenteric white adipose tissue deposition. Non-alcoholic fatty liver disease activity score (NAS) was significantly lower in volixibat-treated mice than in the HFD controls. Gene profiling showed that volixibat reversed the inhibitory effect of the HFD on metabolic master regulators, including peroxisome proliferator-activated receptor-γ coactivator-1ß, insulin receptor, and sterol regulatory element-binding transcription factor 2. Volixibat may have beneficial effects on physiological and metabolic aspects of NASH pathophysiology.


Assuntos
Benzotiepinas/farmacologia , Metabolismo Energético/efeitos dos fármacos , Glicosídeos/farmacologia , Reguladores do Metabolismo de Lipídeos/farmacologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/antagonistas & inibidores , Simportadores/antagonistas & inibidores , Tecido Adiposo Bege/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Biomarcadores , Modelos Animais de Doenças , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Fatores de Risco
5.
J Pharmacol Exp Ther ; 370(1): 84-91, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31010842

RESUMO

The illicit use of γ-hydroxybutyric acid (GHB), and its prodrug, γ-butyrolactone (GBL), results in severe adverse effects including sedation, coma, respiratory depression, and death. Current treatment of GHB/GBL overdose is limited to supportive care. Recent reports indicate that GHB-related deaths are on the rise; a specific treatment may reduce lethality associated with GHB/GBL. Pretreatment with inhibitors of monocarboxylate transporter 1 (MCT1), a transporter that mediates many of the processes involved in the absorption, distribution (including brain uptake), and elimination of GHB/GBL, has been shown to prevent GHB-induced respiratory depression by increasing the renal clearance of GHB. To identify whether MCT1 inhibition is an effective treatment of GHB overdose, the impact of two MCT1 inhibitors, (S)-5-(4-hydroxy-4-methylisoxazolidine-2-carbonyl)-1-isopropyl-3-methyl-6-((3-methyl-5-(trifluoromethyl)-1H-pyrazol-4-yl)methyl)thieno[2,3-day]pyrimidine-2,4(1H,3H)-dione (AZD3965) and 6-[(3,5-dimethyl-1H-pyrazol-4-yl)methyl]-5-[[(4S)-4-hydroxy-2-isoxazolidinyl]carbonyl]-3-methyl-1-(2-methylpropyl)thieno[2,3-day]pyrimidine2,4(1H,3H)-dione (AR-C155858), on the toxicokinetics and toxicodynamics of GHB/GBL was assessed when the administration of the inhibitor was delayed 60 and 120 minutes (post-treatment) after administration of GHB/GBL. AR-C155858 and AZD3965 reduced the toxicodynamic effects of GHB when GHB was administered intravenously, orally, or orally as the prodrug GBL. The impact of these inhibitors on GHB toxicokinetics was dependent on the route of GHB administration and the delay between GHB/GBL administration and administration of the MCT1 inhibitor. The reduction in GHB plasma exposure did not explain the observed effect of MCT1 inhibition on GHB-induced respiratory depression. The efficacy of MCT1 inhibition on GHB toxicodynamics is likely driven by the pronounced reduction in GHB brain concentrations. Overall, this study indicates that inhibition of MCT1 is an effective treatment of GHB/GBL overdose.


Assuntos
4-Butirolactona/toxicidade , Overdose de Drogas/tratamento farmacológico , Hidroxibutiratos/toxicidade , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Pró-Fármacos/farmacologia , Pirimidinonas/farmacologia , Simportadores/antagonistas & inibidores , Tiofenos/farmacologia , Uracila/análogos & derivados , 4-Butirolactona/administração & dosagem , Administração Intravenosa , Administração Oral , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Overdose de Drogas/sangue , Overdose de Drogas/metabolismo , Hidroxibutiratos/administração & dosagem , Hidroxibutiratos/sangue , Hidroxibutiratos/farmacocinética , Masculino , Pirimidinonas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Tiofenos/uso terapêutico , Uracila/farmacologia , Uracila/uso terapêutico
6.
Artigo em Inglês | MEDLINE | ID: mdl-30917615

RESUMO

The sodium iodide symporter (NIS) is the plasma membrane glycoprotein that mediates active iodide transport in the thyroid and other tissues, such as the salivary, gastric mucosa, rectal mucosa, bronchial mucosa, placenta and mammary glands. In the thyroid, NIS mediates the uptake and accumulation of iodine and its activity is crucial for the development of the central nervous system and disease prevention. Since the discovery of NIS in 1996, research has further shown that NIS functionality and iodine transport is dependent on the activity of the sodium potassium activated adenosine 5'-triphosphatase pump (Na+, K+-ATPase). In this article, I review the molecular mechanisms by which F inhibits NIS expression and functionality which in turn contributes to impaired iodide absorption, diminished iodide-concentrating ability and iodine deficiency disorders. I discuss how NIS expression and activity is inhibited by thyroglobulin (Tg), tumour necrosis factor alpha (TNF-α), transforming growth factor beta 1 (TGF-ß1), interleukin 6 (IL-6) and Interleukin 1 beta (IL-1ß), interferon-γ (IFN-γ), insulin like growth factor 1 (IGF-1) and phosphoinositide 3-kinase (PI3K) and how fluoride upregulates expression and activity of these biomarkers. I further describe the crucial role of prolactin and megalin in regulation of NIS expression and iodine homeostasis and the effect of fluoride in down regulating prolactin and megalin expression. Among many other issues, I discuss the potential conflict between public health policies such as water fluoridation and its contribution to iodine deficiency, neurodevelopmental and pathological disorders. Further studies are warranted to examine these associations.


Assuntos
Fluoretos/toxicidade , Iodo/metabolismo , Simportadores/antagonistas & inibidores , Animais , Humanos , Saúde Pública
7.
Environ Int ; 126: 377-386, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30826616

RESUMO

In support of the Endocrine Disruptor Screening Program (EDSP), the U.S.EPA's Office of Research and Development (ORD) is developing high-throughput screening (HTS) approaches to identify chemicals that alter target sites in the thyroid hormone (TH) pathway. The sodium iodide symporter (NIS) is a transmembrane glycoprotein that mediates iodide uptake into the thyroid as the initial step of TH biosynthesis. Previously, we screened 293 ToxCast chemicals (ph1v2) using a HEK293T cell line expressing human NIS in parallel radioactive iodide uptake (RAIU) and cell viability assays to identify potential environmental NIS inhibitors. Here, we expanded NIS inhibitor screening for a set of 768 ToxCast Phase II (ph2) chemicals, and applied a novel computational toxicology approach based on the ToxPrint chemotype to identify chemical substructures associated with NIS inhibition. Following single-concentration screening (at 1 × 10-4 M with a 20% inhibition cutoff), 235 samples (228 chemicals) were further tested in multiple-concentration (1 × 10-9 - 1 × 10-4 M) format in both RAIU and cell viability assays. The 167 chemicals that exhibited significant RAIU inhibition were then prioritized using combined RAIU and cell viability responses that were normalized relative to the known NIS inhibitor sodium perchlorate. Some of the highest ranked chemicals, such as PFOS, tributyltin chloride, and triclocarban, have been previously reported to be thyroid disruptors. In addition, several novel chemicals were identified as potent NIS inhibitors. The present results were combined with the previous ph1v2 screening results to produce two sets of binary hit-calls for 1028 unique chemicals, consisting of 273 positives exhibiting significant RAIU inhibition, and 63 positives following application of a cell viability filter. A ToxPrint chemotype-enrichment analysis identified >20 distinct chemical substructural features, represented in >60% of the active chemicals, as significantly enriched in each NIS inhibition hit-call space. A shared set of 9 chemotypes enriched in both hit-call sets indicates stable chemotype signals (insensitive to cytotoxicity filters) that can help guide structure-activity relationship (SAR) investigations and inform future research.


Assuntos
Disruptores Endócrinos/toxicidade , Ensaios de Triagem em Larga Escala , Simportadores/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Humanos
8.
Cell Oncol (Dordr) ; 42(3): 303-318, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30790227

RESUMO

PURPOSE: Increased glycolytic activity with accumulation of extracellular lactate is regarded as a hallmark of cancer. In lymphomas, FDG-PET has undeniable diagnostic and prognostic value, corroborating that these tumours are avid for glucose. However, the role of glycolytic metabolism-related molecules in lymphoma is not well known. Here, we aimed to evaluate the clinical and prognostic significance of a panel of glycolytic metabolism-related molecules in primary non-Hodgkin lymphomas (NHL) and to test in vitro the putative therapeutic impact of lactate transport inhibition. METHODS: We assessed, by immunohistochemistry, the expression of the metabolism-related molecules MCT1, MCT2, MCT4, CD147, GLUT1, LDHA and CAIX in both tumour and stroma compartments of tissue sections obtained from 104 NHL patients. In addition, the lymphoma-derived cell lines OZ and DOHH-2 were used to evaluate the effect of AZD3965 on their viability and on apoptosis induction, as well as on extracellular lactate accumulation. RESULTS: We found that expression of MCT1 in the NHL tumour compartment was significantly associated with a poor clinicopathological profile. We also found that MCT4 and CAIX were present in the stromal compartment and correlated with an aggressive phenotype, while MCT1 was absent in this compartment. In addition, we found that AZD3965-mediated disruption of MCT1 activity led to inhibited NHL cell viability and extracellular lactate accumulation, while increasing apoptotic cell death. CONCLUSIONS: Our results indicate that elevated glycolytic activity is associated with NHL aggressiveness, pointing at metabolic cooperation, mediated by MCT1 and MCT4, between tumour cells and their surrounding stroma. MCT1 may serve as a target to treat NHL (diffuse large B cell lymphoma) patients with high MCT1/low MCT4 expressing tumours. Further (pre-)clinical studies are required to allow the design of novel therapeutic strategies aimed at e.g. reprogramming the tumour microenvironment.


Assuntos
Biomarcadores Tumorais/metabolismo , Glicólise , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma não Hodgkin/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Estimativa de Kaplan-Meier , Linfoma Difuso de Grandes Células B/patologia , Linfoma não Hodgkin/patologia , Masculino , Pessoa de Meia-Idade , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Pirimidinonas/farmacologia , Simportadores/antagonistas & inibidores , Tiofenos/farmacologia , Adulto Jovem
9.
Am J Physiol Cell Physiol ; 316(2): C274-C284, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30649919

RESUMO

During aging, and development of atherosclerosis and cardiovascular disease (CVD), aortic vascular smooth muscle cells (VSMCs) transition from healthy contractile to diseased synthetic phenotypes. K-Cl cotransport (KCC) maintains cell volume and ion homeostasis in growth and differentiation, and hence is important for VSMC proliferation and migration. Therefore, KCC activity may play a role in the contractile-to-synthetic VSMC phenotypic transition. Early, medium, and late synthetic passage VSMCs were tested for specific cytoskeletal protein expression. KCC-mediated ouabain- and bumetanide-insensitive Rb+ (a K+ congener) influx was determined as Cl--dependent Rb+ influx at different external Rb+ and Cl- ion concentrations, [Rb+]o and [Cl-]o. Expressions of the cytoskeletal proteins α-actin, vimentin, and desmin fell from early through late synthetic VSMCs. KCC kinetic parameters, such as maximum velocity ( Vm), and apparent Cl- and Rb+ affinities ( Km), were calculated with Lineweaver-Burk, Hanes-Woolf, and Hill approximations. Vm values of both Rb+- and Cl--dependent influxes were of equal magnitude, commensurate with a KCC stoichiometry of unity, and rose threefold from early to late synthetic VSMCs. Hill coefficients for Rb+ and Cl- correlated with cell passage number, suggesting increased KCC ligand cooperativity. However, Km values for [Cl-]o were strikingly bimodal with 60-80 mM in early, ~20-30 mM in medium, and 60 mM in late passage cells. In contrast, Km values for [Rb+]o remained steady at ~17 mM. Since total KCC isoform expression was similar with cell passage, structure/function changes of the KCC signalosome may accompany the transition of aortic VSMCs from a healthy to a diseased phenotype.


Assuntos
Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Simportadores/metabolismo , Animais , Cardiotônicos/farmacocinética , Células Cultivadas , Relação Dose-Resposta a Droga , Cinética , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacocinética , Simportadores/agonistas , Simportadores/antagonistas & inibidores
10.
AAPS J ; 21(2): 13, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30617815

RESUMO

AR-C155858 and AZD3965, pyrrole pyrimidine derivatives, represent potent monocarboxylate transporter 1 (MCT1) inhibitors, with potential immunomodulatory and chemotherapeutic properties. Currently, there is limited information on the inhibitory properties of this new class of MCT1 inhibitors. The purpose of this study was to characterize the concentration- and time-dependent inhibition of L-lactate transport and the membrane permeability properties of AR-C155858 and AZD3965 in the murine 4T1 breast tumor cells that express MCT1. Our results demonstrated time-dependent inhibition of L-lactate uptake by AR-C155858 and AZD3965 with maximal inhibition occurring after a 5-min pre-incubation period and prolonged inhibition. Following removal of AR-C155858 or AZD3965 from the incubation buffer, inhibition of L-lactate uptake was only fully reversed after 3 and 12 h, respectively, indicating that these inhibitors are slowly reversible. The uptake of AR-C155858 was concentration-dependent in 4T1 cells, whereas the uptake of AZD3965 exhibited no concentration dependence over the range of concentrations examined. The uptake kinetics of AR-C155858 was best fitted to a Michaelis-Menten equation with a diffusional clearance component, P (Km = 0.399 ± 0.067 µM, Vmax = 4.79 ± 0.58 pmol/mg/min, and P = 0.330 ± 0.088 µL/mg/min). AR-C155858 uptake, but not AZD3965 uptake, was significantly inhibited by alpha-cyano-4-hydroxycinnamic acid, a known nonspecific inhibitor of MCTs 1, 2, and 4. AR-C155858 demonstrated a trend toward higher uptake at lower pH, a characteristic of proton-dependent MCT1. These findings provide evidence that AR-C155858 and AZD3965 exert slowly reversible inhibition of MCT1-mediated L-lactate uptake in 4T1 cells, with AR-C155858 representing a potential substrate of MCT1.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Pirimidinonas/farmacologia , Simportadores/antagonistas & inibidores , Tiofenos/farmacologia , Uracila/análogos & derivados , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ácidos Cumáricos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores/metabolismo , Uracila/farmacologia
11.
Acta Pharmacol Sin ; 40(7): 895-907, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30573812

RESUMO

The manipulation of bile acid (BA) homeostasis by blocking the ileal apical Na+-dependent bile salt transporter (ASBT/SLC10A2) may have therapeutic effects in nonalcoholic fatty liver disease. We developed a novel ASBT inhibitor, an N-(3,4-o-dichlorophenyl)-2-(3-trifluoromethoxy) benzamide derivative referred to as IMB17-15, and investigated its therapeutic effects and the molecular mechanisms underlying the effects. Syrian golden hamsters were challenged with high-fat diet (HFD) to induce NAFLD and were subsequently administered 400 mg/kg IMB17-15 by gavage daily for 21 days. Serum, liver, and fecal samples were collected for further analysis. Plasma concentration-time profiles of IMB17-15 were also constructed. The human hepatocyte cell line HL-7702 was treated with Oleic acid (OA) with or without IMB17-15. Western blotting and real-time PCR were used to study the molecular mechanisms of IMB17-15. We found that IMB17-15 inhibited ASBT and subsequently suppressed ileal farnesoid X receptor (FXR) and FXR-activated fibroblast growth factor15/19 (FGF15/19) expression, which reduced the hepatic phosphorylated extracellular regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) levels and upregulated the cholesterol 7α-hydroxylase (CYP7A1) activity. Additionally, IMB17-15 stimulated adenosine monophosphate (AMP)-activated protein kinase (AMPKα) phosphorylation and enhanced peroxisome proliferator activated receptor α (PPARα) expression and thus promoted triglyceride (TG) oxidation and high-density lipoprotein cholesterol (HDL-c) metabolism through an ASBT-independent mechanism. In conclusion, a novel ASBT inhibitor known as IMB17-15 protected hamsters against HFD-induced NFALD by manipulating BA and lipid homeostasis. IMB17-15 also reduced lipid deposition in human hepatic cell lines, indicating that it may be useful as a therapy for NAFLD patients.


Assuntos
Benzamidas/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Transportadores de Ânions Orgânicos Dependentes de Sódio/antagonistas & inibidores , Sulfonamidas/uso terapêutico , Simportadores/antagonistas & inibidores , Animais , Benzamidas/farmacocinética , Benzamidas/toxicidade , Linhagem Celular , Citocinas/metabolismo , Dieta Hiperlipídica , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fígado/patologia , Masculino , Mesocricetus , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Sulfonamidas/farmacocinética , Sulfonamidas/toxicidade
12.
Nat Commun ; 9(1): 5245, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30532032

RESUMO

Sodium-dependent glucose transporters (SGLTs) exploit sodium gradients to transport sugars across the plasma membrane. Due to their role in renal sugar reabsorption, SGLTs are targets for the treatment of type 2 diabetes. Current therapeutics are phlorizin derivatives that contain a sugar moiety bound to an aromatic aglycon tail. Here, we develop structural models of human SGLT1/2 in complex with inhibitors by combining computational and functional studies. Inhibitors bind with the sugar moiety in the sugar pocket and the aglycon tail in the extracellular vestibule. The binding poses corroborate mutagenesis studies and suggest a partial closure of the outer gate upon binding. The models also reveal a putative Na+ binding site in hSGLT1 whose disruption reduces the transport stoichiometry to the value observed in hSGLT2 and increases inhibition by aglycon tails. Our work demonstrates that subtype selectivity arises from Na+-regulated outer gate closure and a variable region in extracellular loop EL5.


Assuntos
Glucose/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/metabolismo , Sódio/metabolismo , Simportadores/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Feminino , Humanos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Florizina/metabolismo , Florizina/farmacologia , Ligação Proteica , Transportador 1 de Glucose-Sódio/genética , Transportador 1 de Glucose-Sódio/metabolismo , Transportador 2 de Glucose-Sódio/genética , Transportador 2 de Glucose-Sódio/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Simportadores/antagonistas & inibidores , Simportadores/genética , Xenopus laevis
13.
Cell Rep ; 25(11): 3047-3058.e4, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30540938

RESUMO

Highly glycolytic cancer cells prevent intracellular acidification by excreting the glycolytic end-products lactate and H+ via the monocarboxylate transporters 1 (MCT1) and 4 (MCT4). We report that syrosingopine, an anti-hypertensive drug, is a dual MCT1 and MCT4 inhibitor (with 60-fold higher potency on MCT4) that prevents lactate and H+ efflux. Syrosingopine elicits synthetic lethality with metformin, an inhibitor of mitochondrial NADH dehydrogenase. NAD+, required for the ATP-generating steps of glycolysis, is regenerated from NADH by mitochondrial NADH dehydrogenase or lactate dehydrogenase. Syrosingopine treatment leads to high intracellular lactate levels and thereby end-product inhibition of lactate dehydrogenase. The loss of NAD+ regeneration capacity due to combined metformin and syrosingopine treatment results in glycolytic blockade, leading to ATP depletion and cell death. Accordingly, ATP levels can be partly restored by exogenously provided NAD+, the NAD precursor nicotinamide mononucleotide (NMN), or vitamin K2. Thus, pharmacological inhibition of MCT1 and MCT4 combined with metformin treatment is a potential cancer therapy.


Assuntos
Ácido Láctico/metabolismo , Metformina/farmacologia , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Proteínas Musculares/antagonistas & inibidores , NAD/metabolismo , Neoplasias/metabolismo , Simportadores/antagonistas & inibidores , Mutações Sintéticas Letais , Ácidos/metabolismo , Animais , Linhagem Celular Tumoral , Metabolismo Energético/efeitos dos fármacos , Humanos , Espaço Intracelular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Reserpina/análogos & derivados , Reserpina/farmacologia , Simportadores/metabolismo
14.
AAPS J ; 21(1): 3, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30397860

RESUMO

Monocarboxylate transporter 1 (MCT1), also known as a L-lactate transporter, is a potential therapeutic target in cancer. The objectives of this study were to evaluate efficacy and assess concentration-effect relationships of AR-C155858 (a selective and potent MCT1 inhibitor) in murine 4T1 breast cancer cells and in the 4T1 tumor xenograft model. Western blotting of 4T1 cells demonstrated triple negative breast cancer (TNBC) characteristics and overexpression of MCT1 and CD147 (a MCT1 accessory protein), but absence of MCT4 expression. AR-C155858 inhibited the cellular L-lactate uptake and cellular proliferation at low nanomolar potencies (IC50 values of 25.0 ± 4.2 and 20.2 ± 0.2 nM, respectively). In the xenograft 4T1 mouse model of immunocompetent animals, AR-C155858 (10 mg/kg i.p. once daily) had no effect on tumor volume and weight. Treatment with AR-C155858 resulted in slightly increased tumor lactate concentrations; however, the changes were not statistically significant. AR-C155858 was well tolerated, as demonstrated by the unchanged body weight and blood lactate concentrations. Average blood and tumor AR-C155858 concentrations (110 ± 22 and 574 ± 245 nM, respectively), 24 h after the last dose, were well above the IC50 values. These data indicate that AR-C155858 penetrated 4T1 xenograft tumors and was present at high concentrations but was ineffective in decreasing tumor growth. Evaluations of AR-C155858 in other preclinical models of breast cancer are needed to further assess its efficacy.


Assuntos
Neoplasias Mamárias Experimentais/tratamento farmacológico , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Simportadores/antagonistas & inibidores , Tiofenos/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Uracila/análogos & derivados , Animais , Basigina/metabolismo , Linhagem Celular Tumoral/transplante , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Simportadores/metabolismo , Tiofenos/farmacologia , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/patologia , Uracila/farmacologia , Uracila/uso terapêutico
15.
Cell Physiol Biochem ; 50(3): 924-935, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30355947

RESUMO

BACKGROUND/AIMS: Increasing evidence shows that reprogramming of energy metabolism is a hallmark of cancer. Considering the emergence of microRNAs as crucial modulators of cancer, this study aimed to better understand the molecular mechanisms of miR-124 in regulating glycolysis in human pancreatic cancer. METHODS: RT-PCR was used to investigate the expression of monocarboxylate transporters (MCTs) in pancreatic ductal adenocarcinoma (PDAC) patient samples and the PANC-1 cell line. A public database and immunochemistry were used for comprehensive analysis of MCT1 expression. The targeting of MCT1 by miR-124 was predicted by software and validated for the MCT1 3'-UTR by dual-luciferase reporter analysis. Cell proliferation, apoptosis, migration, xenografting, and the intracellular pH and L-lactate levels were assessed. Hypoxia-inducible factor-α (HIF-1α) and lactate dehydrogenase A (LDH-A) expression levels were determined by RT-PCR and western blotting. RESULTS: MCT1 expression was higher in PDAC tissue than in normal tissue. Inhibition of MCT1 affected lactate metabolism, resulting in a higher intracellular pH and less proliferation of PANC-1 cells. MCT1 was the target gene of miR-124. In in vitro experiments, miR-124 inhibited the glycolytic activity of PANC-1 cells by targeting MCT1, further decreasing the tumor phenotype by increasing the intracellular pH through LDH-A and HIF-1α. In in vivo experiments, overexpression of miR-124 and silencing of MCT1 significantly inhibited tumor growth. CONCLUSION: miR-124 inhibits the progression of PANC-1 by targeting MCT1 in the lactate metabolic pathway. Our findings provide novel evidence for further functional studies of miR-124, which might be useful for future therapeutic approaches to PDAC.


Assuntos
MicroRNAs/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores/metabolismo , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Lactatos/metabolismo , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Simportadores/antagonistas & inibidores , Simportadores/genética
16.
Drug Metab Pharmacokinet ; 33(6): 270-274, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30341000

RESUMO

Medication therapy is the first line of treatment in the management of epilepsy. Fetal exposure to valproic acid (VPA), an antiepileptic drug, poses an elevated risk of teratogenicity in early pregnancy. Some studies have reported that monocarboxylate transporters (MCTs) may be involved in the placental transport of VPA. However, it has not been determined which MCTs contribute to VPA transport into the placenta. Therefore, the aim of this study was to determine how MCTs contribute to VPA transport into the placenta using the human placenta choriocarcinoma cell line JEG-3. VPA uptake was investigated using JEG-3 cells and radiolabeled VPA. MCT expression in JEG-3 cells was detected using RT-PCR and western blotting. Knockdown of MCTs was carried out using siRNAs. VPA uptake into JEG-3 cells was pH- and concentration-dependent, and described by using the Michaelis-Menten equation (Km = 0.95 ± 0.17 mM; Vmax = 19.3 ± 1.21 nmol/mg protein/15 s). MCT1 and MCT4 expression was found in JEG-3 cells, and typical MCT inhibitors significantly inhibited VPA uptake into JEG-3 cells. However, knockdown of MCT1 and MCT4 did not alter VPA uptake. In conclusion, VPA transport is mediated by a proton-dependent transporter in JEG-3 cells, but not by MCT1 and MCT4.


Assuntos
Transportadores de Ácidos Monocarboxílicos , Proteínas Musculares , Prótons , Simportadores , Ácido Valproico/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , RNA Interferente Pequeno/farmacologia , Simportadores/antagonistas & inibidores , Simportadores/genética , Simportadores/metabolismo , Ácido Valproico/antagonistas & inibidores
17.
Mol Cancer Ther ; 17(11): 2285-2296, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30115664

RESUMO

The lactate transporter SLC16A1/monocarboxylate transporter 1 (MCT1) plays a central role in tumor cell energy homeostasis. In a cell-based screen, we identified a novel class of MCT1 inhibitors, including BAY-8002, which potently suppress bidirectional lactate transport. We investigated the antiproliferative activity of BAY-8002 in a panel of 246 cancer cell lines and show that hematopoietic tumor cells, in particular diffuse large B-cell lymphoma cell lines, and subsets of solid tumor models are particularly sensitive to MCT1 inhibition. Associated markers of sensitivity were, among others, lack of MCT4 expression, low pleckstrin homology like domain family A member 2, and high pellino E3 ubiquitin protein ligase 1 expression. The antitumor effect of MCT1 inhibition was less pronounced on tumor xenografts, with tumor stasis being the maximal response. BAY-8002 significantly increased intratumor lactate levels and transiently modulated pyruvate levels. In order to address potential acquired resistance mechanisms to MCT1 inhibition, we generated MCT1 inhibitor-resistant cell lines and show that resistance can occur by upregulation of MCT4 even in the presence of sufficient oxygen, as well as by shifting energy generation toward oxidative phosphorylation. These findings provide insight into novel aspects of tumor response to MCT1 modulation and offer further rationale for patient selection in the clinical development of MCT1 inhibitors. Mol Cancer Ther; 17(11); 2285-96. ©2018 AACR.


Assuntos
Aminobenzoatos/farmacologia , Biomarcadores Tumorais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Sulfonas/farmacologia , Simportadores/antagonistas & inibidores , Aminobenzoatos/química , Animais , Transporte Biológico/efeitos dos fármacos , Radioisótopos de Carbono , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fluorescência , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Camundongos SCID , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Pirimidinonas/farmacologia , Ácido Pirúvico/metabolismo , Sulfonas/química , Simportadores/metabolismo , Tiofenos/farmacologia , Resultado do Tratamento , Xenopus laevis
18.
Cell Physiol Biochem ; 49(2): 479-488, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30157477

RESUMO

BACKGROUND/AIMS: The aim of this study was to investigate the transport properties and utilization of methionyl-methionine dipeptide (Met-Met) in ß-casein (ß-CN) synthesis in bovine mammary epithelial cells (BMECs). METHODS: The transport properties were studied for the effects of time, pH, concentration, temperature and inhibitors using Met-Met-FITC in BMECs. BMECs were treated with different concentrations of Met-Met (0, 20, 40, 80, 120 and 160 µg/ml). In several experiments, the cells were treated with Janus kinase 2 (JAK2) inhibitor (tyrphostin AG-490, 50 µM) and mammalian target of rapamycin (mTOR) inhibitor (rapamycin, 100 ng/ml). RESULTS: The uptake of Met-Met-FITC by BMECs was rapid during the first fifteen minutes and became saturated after 15 minutes. The transport of Met-Met-FITC in BMECs exhibited a Michaelis constant of 52.4 µM and maximum transport velocity of 14.8 pmol/min/mg protein. The uptake of Met-Met-FITC in BMECs was pH-dependent, peaked at pH 6.5 and was significantly inhibited by other peptides, including Met-Lys, Lys-Lys, Gly-Met, Gly-Leu and Met-Leu. Knocking down the peptide transporter 2 (PepT2) with small interference RNA markedly decreased Met-Met-FITC uptake. Met-Met concentration-dependently increased the PepT2 expression and ß-CN synthesis in BMECs with an optimal concentration of 80 µg/ml. At 80 µg/ml, Met-Met also enhanced the cell viability and cyclin D1 expression and promoted cell cycle transition from G1 phase to S phase. In addition, 80 µg/ml Met-Met increased the mRNA abundance of JAK2 and signal transducer and activator of transcription 5 (STAT5) and enhanced the phosphorylation of JAK2, STAT5, mTOR, p70 ribosomal S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1. The inhibition of JAK2 and mTOR significantly decreased Met-Met-induced increase in cell viability and ß-CN synthesis in BMECs. CONCLUSION: Our data elucidated the properties of peptide transporter and its effect on ß-CN synthesis in BMECs. Met-Met, taken up by PepT2, enhances cell proliferation and promotes ß-CN synthesis by activating JAK2-STAT5 and mTOR signaling pathways in BMECs.


Assuntos
Caseínas/metabolismo , Dipeptídeos/metabolismo , Animais , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ciclina D1/metabolismo , Dipeptídeos/química , Dipeptídeos/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Fluoresceína-5-Isotiocianato/química , Concentração de Íons de Hidrogênio , Janus Quinase 2/metabolismo , Glândulas Mamárias Animais/citologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Simportadores/antagonistas & inibidores , Simportadores/genética , Simportadores/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima/efeitos dos fármacos
19.
Adipocyte ; 7(4): 277-284, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30161013

RESUMO

Despite increased knowledge of nutrient intake regulation and energy homeostasis, treatment options for obesity remain limited. Food reward consists of two branches: gustatory and post-ingestive nutritive information. Drosophila lacking dSLC5A11 (sodium/glucose cotransporter 6-SGLT6) prefer L-glucose over D-glucose independently of their state of satiety. Human SGLT6 is an active transporter of myo-inositol and D-glucose. We investigated expression of SGLT6 in human tissue and found a significant expression in the small intestine and brain. The preference between a metabolizable and a non-metabolizable sugar was tested in 3 mouse models with a selective and potent SGLT6 inhibitor. No influence on sugar preference was seen with SGLT6 inhibition. These studies suggest that SGLT6 does not play a significant role in nutrient sensing in mammals.


Assuntos
Fármacos Antiobesidade/farmacologia , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/metabolismo , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Animais , Fármacos Antiobesidade/farmacocinética , Fármacos Antiobesidade/uso terapêutico , Células CACO-2 , Preferências Alimentares/efeitos dos fármacos , Glucose/metabolismo , Células HEK293 , Humanos , Inositol/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular
20.
Neurogastroenterol Motil ; 30(12): e13448, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30129138

RESUMO

BACKGROUND: Chronic constipation affects 14%-17% of the population. Elobixibat, a novel, ileal bile acid transporter (IBAT) inhibitor, has been approved as a new chronic constipation drug in Japan in January 2018. The present study aimed to examine the pharmacological effects of elobixibat on colonic motility in conscious dogs using a telemetry system. METHODS: Six male beagle dogs were surgically implanted with strain gauge force transducers for gastrointestinal (GI) motility recording. The motility index was calculated from GI motility at each recording site in conscious and nonrestraint dogs. The fasted dogs were orally administered elobixibat (3, 10, or 30 mg kg-1 ) or 30 mg kg-1 of sennoside as positive control or vehicle using a crossover design and washout period of more than 6 days. One hour after drug administration, the dogs were fed chow, and GI motility and defecation were observed for 10 hours; GI motility was quantified to calculate giant migrating contractions (GMCs). Fecal bile acids (BAs) were determined as well. KEY RESULTS: Elobixibat and sennoside significantly increased the number of defecations, fecal wet weight, and water content within 10 hours after administration. Elobixibat dose-dependently decreased the time to first bowel movement, increased the amount of total fecal BAs, and rapidly induced mild GMCs during defecation; however, higher strength of GMCs was observed with sennoside. CONCLUSIONS & INFERENCE: Elobixibat induces bowel movements faster than sennoside through a different mechanism. Elobixibat locally inhibits IBAT in the ileal lumen, leading to elevated fecal BAs in the colon and induced mild GMCs during defecation.


Assuntos
Defecação/efeitos dos fármacos , Dipeptídeos/farmacologia , Fármacos Gastrointestinais/farmacologia , Complexo Mioelétrico Migratório/efeitos dos fármacos , Tiazepinas/farmacologia , Animais , Estado de Consciência , Cães , Íleo/efeitos dos fármacos , Masculino , Transportadores de Ânions Orgânicos Dependentes de Sódio/antagonistas & inibidores , Simportadores/antagonistas & inibidores
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