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1.
Clin Pharmacol Ther ; 107(1): 102-111, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31709525

RESUMO

This white paper presents principles for validating proarrhythmia risk prediction models for regulatory use as discussed at the In Silico Breakout Session of a Cardiac Safety Research Consortium/Health and Environmental Sciences Institute/US Food and Drug Administration-sponsored Think Tank Meeting on May 22, 2018. The meeting was convened to evaluate the progress in the development of a new cardiac safety paradigm, the Comprehensive in Vitro Proarrhythmia Assay (CiPA). The opinions regarding these principles reflect the collective views of those who participated in the discussion of this topic both at and after the breakout session. Although primarily discussed in the context of in silico models, these principles describe the interface between experimental input and model-based interpretation and are intended to be general enough to be applied to other types of nonclinical models for proarrhythmia assessment. This document was developed with the intention of providing a foundation for more consistency and harmonization in developing and validating different models for proarrhythmia risk prediction using the example of the CiPA paradigm.

2.
Circ J ; 84(1): 69-75, 2019 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-31801927

RESUMO

BACKGROUND: The impact of cold ambient temperature on out-of-hospital cardiac arrest (OHCA) in aged individuals caused by cardiovascular events in indoor environments has not been investigated sufficiently.Methods and Results:We conducted a case-crossover study. The relationship between OHCA caused by cardiovascular events and exposure to minimum temperature <0℃ was analyzed. Conditional logistic regression analysis was performed to estimate the odds ratios for the relationship between exposure to minimum temperature <0℃ and the risk of OHCA. Between January 1, 2011, and December 31, 2015, a total of 1,452 cases of OHCA were documented, and patients were screened for enrollment. A total of 458 individuals were enrolled in this analysis, and were divided into 2 groups of 110 (elderly group: 65-74 years old) and 348 (aged group: ≥75 years old). The aged individuals had a significant increased risk of OHCA after exposure to minimum temperature <0℃ (odds ratio [OR]: 1.528, 95% confidence interval [CI] 1.009-2.315, P=0.045). Cold ambient temperature was an especially significant increased risk for OHCA occurrence for males (OR: 1.997, 95% CI 1.036-3.773, P=0.039) and during winter (OR: 2.391, 95% CI 1.312-4.360, P=0.004) in the aged group. CONCLUSIONS: Cold ambient temperature significantly affected aged individuals (≥75 years old) experiencing an OHCA caused by cardiovascular events in indoor environments.

3.
J Pharmacol Toxicol Methods ; 98: 106593, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31158459

RESUMO

This commentary highlights and expands upon the thoughts conveyed in the lecture by Dr. Alan S. Bass, recipient of the 2017 Distinguished Service Award from the Safety Pharmacology Society, given on 27 September 2017 in Berlin, Germany. The lecture discussed the societal, scientific, technological, regulatory and economic events that dramatically impacted the pharmaceutical industry and ultimately led to significant changes in the strategic operations and practices of safety pharmacology. It focused on the emerging challenges and opportunities, and considered the lessons learned from drug failures and the influences of world events, including the financial crisis that ultimately led to a collapse of the world economies from which we are now recovering. Events such as these, which continue to today, challenge the assumptions that form the foundation of our discipline and dramatically affect the way that safety pharmacology is practiced. These include the latest scientific and technological developments contributing to the design and advancement of safe medicines. More broadly, they reflect the philosophical mission of safety pharmacology and the roles and responsibilities served by safety pharmacologists. As the discipline of Safety Pharmacology continues to evolve, develop and mature, the reader is invited to reflect on past experiences as a framework towards a vision of the future of the field.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Indústria Farmacêutica/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Animais , Humanos , Sociedades
6.
J Pharmacol Sci ; 138(4): 233-239, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30415824

RESUMO

Cardiac safety assessment is challenging because a better understanding of torsadogenic mechanisms beyond hERG blockade and QT interval prolongation is necessary for patient safety. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a new human cell-based platform to assess cardiac safety in non-clinical testing during drug development. The multi-electrode array (MEA) platform is a promising electrophysiological technology to assess QT interval prolongation and proarrhythmic potential of drug candidates using hiPSC-CMs. The Japan iPS Cardiac Safety Assessment (JiCSA) has established an MEA protocol to evaluate the applicability of hiPSC-CMs for assessing the torsadogenic potential of compounds and completed a large-scale validation study using 60 compounds. During our study, an international multi-site study of hiPSC-CMs was performed by the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative using 28 compounds. We have comparatively analyzed our JiCSA datasets with those of CiPA using the CiPA logistical and ordinal linear regression model. Regardless of the protocol differences, the evaluation results of the 28 compounds were very similar and highly predictable for torsadogenic risks. Thus, an MEA-based approach using hiPSC-CMs would be a standard testing method to evaluate proarrhythmic potentials. This review paper would provide new insights into the hiPSC-CMs/MEA method required for its regulatory use.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/efeitos dos fármacos , Torsades de Pointes/induzido quimicamente , Bioensaio , Humanos , Miócitos Cardíacos/fisiologia , Medição de Risco
7.
Sensors (Basel) ; 18(11)2018 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-30428530

RESUMO

This paper proposes a new approach to visualizing spatial variation of plant status in a tomato greenhouse based on farm work information operated by laborers. Farm work information consists of a farm laborer's position and action. A farm laborer's position is estimated based on radio wave strength measured by using a smartphone carried by the farm laborer and Bluetooth beacons placed in the greenhouse. A farm laborer's action is recognized based on motion data measured by using smartwatches worn on both wrists of the farm laborer. As experiment, harvesting information operated by one farm laborer in a part of a tomato greenhouse is obtained, and the spatial distribution of yields in the experimental field, called a harvesting map, is visualized. The mean absolute error of the number of harvested tomatoes in each small section of the experimental field is 0.35. An interview with the farm manager shows that the harvesting map is useful for intuitively grasping the states of the greenhouse.

8.
Br J Pharmacol ; 175(17): 3435-3452, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29745425

RESUMO

BACKGROUND AND PURPOSE: To date, proposed in silico models for preclinical cardiac safety testing are limited in their predictability and usability. We previously reported a multi-scale heart simulation that accurately predicts arrhythmogenic risk for benchmark drugs. EXPERIMENTAL APPROACH: We created a comprehensive hazard map of drug-induced arrhythmia based on the electrocardiogram (ECG) waveforms simulated under wide range of drug effects using the multi-scale heart simulator described here, implemented with cell models of human cardiac electrophysiology. KEY RESULTS: A total of 9075 electrocardiograms constitute the five-dimensional hazard map, with coordinates representing the extent of the block of each of the five ionic currents (rapid delayed rectifier potassium current (IKr ), fast (INa ) and late (INa,L ) components of the sodium current, L-type calcium current (ICa,L ) and slow delayed rectifier current (IKs )), involved in arrhythmogenesis. Results of the evaluation of arrhythmogenic risk based on this hazard map agreed well with the risk assessments reported in the literature. ECG databases also suggested that the interval between the J-point and the T-wave peak is a superior index of arrhythmogenicity when compared to the QT interval due to its ability to characterize the multi-channel effects compared with QT interval. CONCLUSION AND IMPLICATIONS: Because concentration-dependent effects on electrocardiograms of any drug can be traced on this map based on in vitro current assay data, its arrhythmogenic risk can be evaluated without performing costly and potentially risky human electrophysiological assays. Hence, the map serves as a novel tool for use in pharmaceutical research and development.


Assuntos
Arritmias Cardíacas/fisiopatologia , Ventrículos do Coração/fisiopatologia , Canais Iônicos/antagonistas & inibidores , Modelos Biológicos , Adulto , Arritmias Cardíacas/induzido quimicamente , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Eletrocardiografia , Análise de Elementos Finitos , Humanos
9.
Bioorg Med Chem ; 26(9): 2508-2513, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29673716

RESUMO

The physicochemical properties of 4-substituted carbamazepine derivatives were investigated. It was elucidated that the 4-substitution is not effective in reducing the rotations (E/Z) about the N-C1' axes around the outer carbamoyl moiety. However, the atropisomers were isolated with high stereochemical stability, meaning that the 4-substitution reduced the butterfly motion of the tricyclic ring system efficiently. The Cl/CH3-substituted carbamazepine derivatives showed greater inhibitory effects on hNav1.2 channel currents compared with carbamazepine, although no difference in the activity between enantiomers was observed.


Assuntos
Carbamazepina/análogos & derivados , Carbamazepina/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Animais , Células CHO , Carbamazepina/síntese química , Carbamazepina/química , Cricetulus , Humanos , Conformação Molecular , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Estereoisomerismo , Temperatura Ambiente , Termodinâmica
10.
J Clin Pharmacol ; 57(1): 96-104, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27338807

RESUMO

Lemborexant is a novel dual orexin receptor antagonist being developed to treat insomnia. Its potential to cause QT prolongation was evaluated using plasma concentration-response (CR) modeling applied to data from 2 multiple ascending-dose (MAD) studies. In the primary MAD study, placebo or lemborexant (2.5 to 75 mg) was administered for 14 consecutive nights. In another MAD study designed to "bridge" pharmacokinetic and safety data between Japanese and non-Japanese subjects (J-MAD), placebo or lemborexant (2.5, 10, or 25 mg) was administered for 14 consecutive nights. QT intervals were estimated using a high-precision measurement technique and evaluated using a linear mixed-effects CR model, for each study separately and for the pooled data set. When each study was analyzed separately, the slopes of the CR relationship were shallow and not statistically significant. In the pooled analysis, the slope of the CR relationship was -0.00002 milliseconds per ng/mL (90%CI, -0.01019 to 0.01014 milliseconds). The highest observed Cmax was 400 ng/mL, representing a margin 8-fold above exposures expected for the highest planned clinical dose. The model-predicted QTc effect at 400 ng/mL was 1.1 milliseconds (90%CI, -3.49 to 5.78 milliseconds). In neither the J-MAD study nor the pooled analysis was an effect of race identified. CR modeling of data from early-phase clinical studies, including plasma levels far exceeding those anticipated clinically, indicated that a QT effect >10 milliseconds could be excluded. Regulatory agreement with this methodology demonstrates the effectiveness of a CR modeling approach as an alternative to thorough QT studies.


Assuntos
Descoberta de Drogas/métodos , Eletrocardiografia/efeitos dos fármacos , Síndrome do QT Longo/induzido quimicamente , Modelos Biológicos , Antagonistas dos Receptores de Orexina/administração & dosagem , Antagonistas dos Receptores de Orexina/efeitos adversos , Adulto , Relação Dose-Resposta a Droga , Método Duplo-Cego , Eletrocardiografia/métodos , Feminino , Humanos , Síndrome do QT Longo/diagnóstico , Síndrome do QT Longo/fisiopatologia , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Adulto Jovem
11.
J Pharmacol Toxicol Methods ; 84: 111-127, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27956204

RESUMO

INTRODUCTION: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are anticipated to be a useful tool for conducting proarrhythmia risk assessments of drug candidates. However, a torsadogenic risk prediction paradigm using hiPSC-CMs has not yet been fully established. METHODS: Extracellular field potentials (FPs) were recorded from hiPSC-CMs using the multi-electrode array (MEA) system. The effects on FPs were evaluated with 60 drugs, including 57 with various clinical torsadogenic risks. Actual drug concentrations in medium were measured using the equilibrium dialysis method with a Rapid Equilibrium Dialysis device. Relative torsade de pointes (TdP) scores were determined for each drug according to the degree of FP duration prolongation and early afterdepolarization occurrence. The margins were calculated from the free concentration in medium and free effective therapeutic plasma concentration. Each drug's results were plotted on a two-dimensional map of relative TdP risk scores versus margins. RESULTS: Each drug was categorised as high, intermediate, or low risk based on its location within predefined areas of the two-dimensional map. We categorised 19 drugs as high risk; 18 as intermediate risk; and 17 as low risk. We examined the concordance between our categorisation of high and low risk drugs against the torsadogenic risk categorisation in CredibleMeds®. Our system demonstrated high concordance, as reflected in a sensitivity of 81%, specificity of 87%, and accuracy of 83%. DISCUSSION: These results indicate that our torsadogenic risk assessment is reliable and has a potential to replace the hERG assay for torsadogenic risk prediction, however, this system needs to be improved for the accurate of prediction of clinical TdP risk. Here, we propose a novel drug induced torsadogenic risk categorising system using hiPSC-CMs and the MEA system.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Cardiotoxinas/toxicidade , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Torsades de Pointes/induzido quimicamente , Potenciais de Ação/fisiologia , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Medição de Risco , Torsades de Pointes/patologia , Torsades de Pointes/fisiopatologia
12.
PLoS One ; 11(12): e0167348, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27923051

RESUMO

The aims of this study were to (1) characterize basic electrophysiological elements of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that correspond to clinical properties such as QT-RR relationship, (2) determine the applicability of QT correction and analysis methods, and (3) determine if and how these in-vitro parameters could be used in risk assessment for adverse drug-induced effects such as Torsades de pointes (TdP). Field potential recordings were obtained from commercially available hiPSC-CMs using multi-electrode array (MEA) platform with and without ion channel antagonists in the recording solution. Under control conditions, MEA-measured interspike interval and field potential duration (FPD) ranged widely from 1049 to 1635 ms and from 334 to 527 ms, respectively and provided positive linear regression coefficients similar to native QT-RR plots obtained from human electrocardiogram (ECG) analyses in the ongoing cardiovascular-based Framingham Heart Study. Similar to minimizing the effect of heart rate on the QT interval, Fridericia's and Bazett's corrections reduced the influence of beat rate on hiPSC-CM FPD. In the presence of E-4031 and cisapride, inhibitors of the rapid delayed rectifier potassium current, hiPSC-CMs showed reverse use-dependent FPD prolongation. Categorical analysis, which is usually applied to clinical QT studies, was applicable to hiPSC-CMs for evaluating torsadogenic risks with FPD and/or corrected FPD. Together, this results of this study links hiPSC-CM electrophysiological endpoints to native ECG endpoints, demonstrates the appropriateness of clinical analytical practices as applied to hiPSC-CMs, and suggests that hiPSC-CMs are a reliable models for assessing the arrhythmogenic potential of drug candidates in human.


Assuntos
Cromanos/farmacologia , Cisaprida/farmacologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/citologia , Piperidinas/farmacologia , Piridinas/farmacologia , Sulfonamidas/farmacologia , Células Cultivadas , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Modelos Lineares , Modelos Cardiovasculares , Torsades de Pointes/induzido quimicamente , Torsades de Pointes/fisiopatologia
13.
Nature ; 538(7625): 344-349, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27602946

RESUMO

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.


Assuntos
Antimaláricos/síntese química , Antimaláricos/farmacologia , Azetidinas/uso terapêutico , Descoberta de Drogas , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Animais , Antimaláricos/administração & dosagem , Antimaláricos/uso terapêutico , Compostos Azabicíclicos/administração & dosagem , Compostos Azabicíclicos/síntese química , Compostos Azabicíclicos/farmacologia , Compostos Azabicíclicos/uso terapêutico , Azetidinas/administração & dosagem , Azetidinas/efeitos adversos , Azetidinas/farmacologia , Citosol/enzimologia , Modelos Animais de Doenças , Feminino , Fígado/efeitos dos fármacos , Fígado/parasitologia , Macaca mulatta/parasitologia , Malária Falciparum/prevenção & controle , Malária Falciparum/transmissão , Masculino , Camundongos , Fenilalanina-tRNA Ligase/antagonistas & inibidores , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Plasmodium falciparum/citologia , Plasmodium falciparum/enzimologia , Segurança
14.
Kyobu Geka ; 69(3): 191-5, 2016 Mar.
Artigo em Japonês | MEDLINE | ID: mdl-27075284

RESUMO

The damage to the intervalvular fibrous trigone (IVFT) by infective endocarditis makes combined aortic and mitral valve replacement difficult. We performed Manouguian's double valve replacement for such a case and obtained a good result. A 81-year-old male underwent emergency operation due to active prosthetic valve endocarditis. He had a history of receiving combined aortic and mitral valve replacement because of active infective endocarditis at the age of 74 and redo aortic valve replacement 3 years after that. The infectious lesion extended from the mitral annulus to the IVFT and the aortic annulus, and it caused the prosthetic valve detachment from the aortic annulus. Manouguian's double valve replacement was required for radical resection and reconstruction of the IVFT. No recurrent infection or paravalvular leakage was observed during 49months follow up period. Manouguian's procedure is useful for complete resection of the infected IVFT and makes combined aortic and mitral valve replacement safer.


Assuntos
Valva Aórtica/cirurgia , Implante de Prótese de Valva Cardíaca/métodos , Próteses Valvulares Cardíacas/efeitos adversos , Valva Mitral/cirurgia , Infecções Relacionadas à Prótese/cirurgia , Idoso de 80 Anos ou mais , Humanos , Masculino , Reoperação
15.
Sci Adv ; 1(4): e1400142, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-26601174

RESUMO

To save time and cost for drug discovery, a paradigm shift in cardiotoxicity testing is required. We introduce a novel screening system for drug-induced arrhythmogenic risk that combines in vitro pharmacological assays and a multiscale heart simulator. For 12 drugs reported to have varying cardiotoxicity risks, dose-inhibition curves were determined for six ion channels using automated patch clamp systems. By manipulating the channel models implemented in a heart simulator consisting of more than 20 million myocyte models, we simulated a standard electrocardiogram (ECG) under various doses of drugs. When the drug concentrations were increased from therapeutic levels, each drug induced a concentration-dependent characteristic type of ventricular arrhythmia, whereas no arrhythmias were observed at any dose with drugs known to be safe. We have shown that our system combining in vitro and in silico technologies can predict drug-induced arrhythmogenic risk reliably and efficiently.

16.
PLoS One ; 8(9): e75040, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086433

RESUMO

Serotonin 1A (5-HT1A) receptors have been mechanistically implicated in micturition control, and there has been a need for an appropriate biomarker surrogating the potency of a provisional drug acting on this receptor system for developing a new therapeutic approach to overactive bladder (OAB). Here, we analyzed the occupancy of 5-HT1A receptors in living Sprague-Dawley rat brains by a novel candidate drug for OAB, E2110, using positron emission tomography (PET) imaging, and assessed the utility of a receptor occupancy (RO) assay to establish a pharmacodynamic index translatable between animals and humans. The plasma concentrations inducing 50% RO (EC50) estimated by both direct and effect compartment models were in good agreement. Dose-dependent therapeutic effects of E2110 on dysregulated micturition in different rat models of pollakiuria were also consistently explained by achievement of 5-HT1A RO by E2110 in a certain range (≥ 60%). Plasma drug concentrations inducing this RO range and EC50 would accordingly be objective indices in comparing pharmacokinetics-RO relationships between rats and humans. These findings support the utility of PET RO and plasma pharmacokinetic assays with the aid of adequate mathematical models in determining the in vivo characteristics of a drug acting on 5-HT1A receptors and thereby counteracting OAB.


Assuntos
Piperidinas/farmacologia , Piperidinas/farmacocinética , Tomografia por Emissão de Pósitrons , Receptor 5-HT1A de Serotonina/metabolismo , Bexiga Urinária Hiperativa/diagnóstico por imagem , Bexiga Urinária Hiperativa/tratamento farmacológico , 8-Hidroxi-2-(di-n-propilamino)tetralina/administração & dosagem , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Administração Oral , Animais , Simulação por Computador , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Piperidinas/química , Piperidinas/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Reflexo/efeitos dos fármacos , Colículos Superiores/efeitos dos fármacos , Fatores de Tempo , Bexiga Urinária Hiperativa/fisiopatologia , Micção/efeitos dos fármacos
17.
Hum Exp Toxicol ; 32(10): 1028-37, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23424208

RESUMO

Channels responsible for slowly activating delayed-rectifier potassium current (I(Ks)) are composed of KCNQ1 and KCNE1 subunits, and these channels play a role in the repolarization of cardiac action potentials. Recently, we showed that the antihyperlipidemic drug probucol, which induces QT prolongation, decreases the I(Ks) after 24-h treatment. In the present study, we investigated the effects of three cholesterol-lowering agents (probucol, an enhancer of cholesterol efflux; simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor; and triparanol, a 3ß-hydroxysterol-▵24-reductase inhibitor) on cholesterol synthesis, the KCNQ1 current (I KCNQ1), and the I(Ks) to clarify the differences in the modes of action of these agents on the I(Ks). Probucol did not inhibit cholesterol synthesis and had no effect on I KCNQ1, while I(Ks) decreased after 24-h treatment. Simvastatin inhibited cholesterol synthesis and decreased I KCNQ1 and I(Ks). Additionally, the activation kinetics of I(Ks) became faster, compared with that of control I(Ks). Triparanol inhibited cholesterol synthesis but did not reduce I KCNQ1 and I(Ks). However, the activation kinetics of I(Ks) became faster. Our data indicated that the mechanism by which probucol inhibits I(Ks) was not mediated by the inhibition of cholesterol synthesis but depended on an interaction with the KCNQ1/KCNE1 complex. Meanwhile, the reduction in cholesterol induced by simvastatin and triparanol is one of the mechanisms that affects the kinetics of I(ks).


Assuntos
Anticolesterolemiantes/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Probucol/farmacologia , Sinvastatina/farmacologia , Triparanol/farmacologia , Animais , Células CHO , Colesterol/metabolismo , Cricetulus
18.
J Pharmacol Toxicol Methods ; 67(1): 16-24, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23138150

RESUMO

INTRODUCTION: IonWorks automated patch clamp systems are being widely used for ion channel drug discovery, but the perforated patch mode of these systems makes it difficult to obtain a steady intracellular Ca(2+) concentration ([Ca(2+)](i)). This difficulty prevents obtaining high-quality data regarding Ca(2+)-activated channels such as BK and SK channels. We examined the methods for stabilizing [Ca(2+)](i) in the IonWorks Quattro automated patch clamp system to evaluate BK channels. METHODS: Electrophysiological recordings were performed using the single-hole or population patch clamp mode of IonWorks Quattro. To increase [Ca(2+)](i), ionomycin was used. The variation in the BK current and the effect of BK channel modulators were examined in the presence and absence of an intracellular Ca(2+) chelator, BAPTA-AM (20µM). RESULTS: BK current activated by step pulses to +100mV in the presence of ionomycin exhibited large variation (ranging from 0.086 to 11nA). In individual cells, oscillation of the current amplitude was observed when five repetitive pulses were applied at 0.1Hz. Approximately 30% of cells exhibited current variation exceeding 20% when the variation was calculated using the first and third pulses. However, BAPTA-AM treatment before current measurement decreased the number of cells displaying large variation (>20%) to 5%. In the presence of BAPTA-AM, the BK channel modulators NS1619 and 12,14-dichlorodehydroabietic acid increased the BK current at concentrations of 10µM or more showing clear concentration dependency, whereas in its absence, the effect of both compounds was detected only at 30µM. DISCUSSION: The main finding of this study is that the [Ca(2+)](i) variation in the basal condition is very large and hinders the accurate evaluation of compounds in Ca(2+)-activated ion channels. The application of BAPTA-AM and ionomycin greatly improved the precision of BK channel screening, and this method should be applicable to other Ca(2+)-activated ion channels such as SK channels.


Assuntos
Ionóforos de Cálcio/farmacologia , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ionomicina/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Técnicas de Patch-Clamp/normas , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/normas , Ácido Egtázico/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp/métodos
19.
Arterioscler Thromb Vasc Biol ; 32(6): 1400-9, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22556334

RESUMO

OBJECTIVE: Recently, we reported that angiopoietin-like protein 2 (Angptl2) functions in various chronic inflammatory diseases. In the present study, we asked whether Angptl2 and its associated chronic inflammation contribute to abdominal aortic aneurysm (AAA). METHODS AND RESULTS: Immunohistochemistry revealed that Angptl2 is abundantly expressed in infiltrating macrophages within the vessel wall of patients with AAA and in a CaCl(2)-induced AAA mouse model. When Angptl2-deficient mice were used in the mouse model, they showed decreased AAA development compared with wild-type mice, as evidenced by reduction in aneurysmal size, less severe destruction of vessel structure, and lower expression of proinflammatory cytokines and matrix metalloproteinase-9. However, no difference in the number of infiltrating macrophages within the aortic aneurysmal vessel wall was observed between genotypes. AAA development was also significantly suppressed in wild-type mice that underwent Angptl2-deficient bone marrow transplantation. Expression levels of proinflammatory cytokines and metalloproteinase-9 in Angptl2-deficient macrophages were significantly decreased, and those decreases were rescued by treatment of Angptl2 deficient macrophages with exogenous Angptl2. CONCLUSIONS: Macrophage-derived Angptl2 contributes to AAA development by inducing inflammation and degradation of extracellular matrix in the vessel wall, suggesting that targeting the Angptl2-induced inflammatory axis in macrophages could represent a new strategy for AAA therapy.


Assuntos
Angiopoietinas/metabolismo , Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/metabolismo , Macrófagos/metabolismo , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/deficiência , Angiopoietinas/genética , Animais , Aorta Abdominal/imunologia , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/imunologia , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/prevenção & controle , Transplante de Medula Óssea , Cloreto de Cálcio , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Genótipo , Humanos , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo
20.
J Cardiovasc Pharmacol ; 59(4): 377-86, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22189896

RESUMO

Indirect effects of drugs on ion channel expression levels on plasma membrane are focused as the cause of QT prolongation, and we explored the chronic effects of QT-prolonging drugs on the slow component of the delayed-rectifier potassium current (IKs). Chinese Hamster Ovary cells expressing IKs channels were constructed by transfecting KCNQ1/KCNE1 genes, and the IKs values were measured using IonWorks Quattro in the population patch-clamp mode. After 24 hours of treatment with IKs blockers (HMR1556, L-768673, or chromanol 293B) or human Ether-à-go-go related gene channel trafficking inhibitors (amiodarone,17-AAG, brefeldin A, pentamidine, thioridazine, or probucol), brefeldin A, pentamidine, and probucol decreased IKs. Probucol, which is a cholesterol-lowering drug and clinically reported to cause QT prolongation, potently inhibited the IKs in a concentration-dependent manner, with a half maximal inhibitory concentration of 149.1 nM. A reduction in the IKs by 1 µM of probucol was observed beginning 2 hours after treatment, and the current was reduced by about 80% at 24 hours. The activation and deactivation time constants of residual IKs currents became faster compared with that in the vehicle-treatment group. Acute application of probucol did not directly inhibit IKs channels at concentrations of up to 10 µM. Western blotting analysis indicated the reduction of multimeric complex of KCNQ1 proteins by probucol treatment but not monomeric form. These results suggest that chronic probucol treatment may contribute to QT prolongation in humans by decreasing the functional IKs channel complexes.


Assuntos
Anticolesterolemiantes/toxicidade , Síndrome do QT Longo/induzido quimicamente , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Probucol/toxicidade , Animais , Anticolesterolemiantes/administração & dosagem , Western Blotting , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Canais de Potássio Éter-A-Go-Go/genética , Humanos , Canal de Potássio KCNQ1/genética , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Probucol/administração & dosagem , Fatores de Tempo , Transfecção
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