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1.
PLoS One ; 15(2): e0228848, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32050000

RESUMO

We investigated the relation of 99mTc-MIBI uptake to mitochondrial membrane potential (MMP) in cancer cell lines and patient-derived tumor cells (PDCs). In T47D and HT29 cells with low MDR1 expression, FCCP dose-dependently reduced MMP and 99mTc-MIBI accumulation in similar patterns with nearly perfect linear relationships. T47D and HT29 cells with high MDR1 expression had low 99mTc-MIBI accumulation that was minimally affected by FCCP dose. In these cells, verapamil markedly increased 99mTc-MIBI accumulation to magnitudes that were excessive compared to MMP increase. Decreased plasma membrane potential by verapamil and its recovery by FCCP suggested that enhanced 99mTc-MIBI transport through modified plasma membranes contributed to the excess accumulation. Evaluation of three different colon cancer PDCs with low to modest MDR1 expression verified that FCCP significantly suppressed MMP and similarly reduced 99mTc-MIBI accumulation. Verapamil partially recovered both MMP and 99mTc-MIBI accumulation that was lowered by FCCP. Importantly, a high linear correlation was found (r = 0.865) between 99mTc-MIBI accumulation and MMP in these cells. These findings indicate that low baseline 99mTc-MIBI uptake that is markedly increased by verapamil represents cancer cells with high levels of MDR1 expression. However, in cancer cells with low or modest levels of MDR1 expression that do not markedly increase 99mTc-MIBI uptake by verapamil, the magnitude of uptake is largely dependent on cellular MMP.


Assuntos
Transporte Biológico/fisiologia , Potencial da Membrana Mitocondrial/fisiologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Tecnécio Tc 99m Sestamibi/metabolismo , Verapamil/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Células HT29 , Humanos , Compostos Radiofarmacêuticos/metabolismo , Células Tumorais Cultivadas
2.
Pharm Biol ; 58(1): 152-156, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31990625

RESUMO

Context: Hydroxycamptothecin (HCPT) has antitumor activity in various cancers, but its poor bioavailability and efflux limit its clinical application. Verapamil has been demonstrated to improve the bioavailability of many drugs. However, the effect of verapamil on the pharmacokinetics of HCPT was not clear.Objective: The effect of verapamil on the pharmacokinetics of HCPT was investigated to clarify the drug-drug interaction between HCPT and verapamil.Materials and methods: The pharmacokinetic profiles of oral administration of HCPT (50 mg/kg) in two group of Sprague-Dawley rats (six rats each), with pre-treatment of verapamil (10 mg/kg/day) for 7 days were investigated, with the group without verapamil pre-treatment as control. Additionally, the metabolic stability and transport of HCPT in the presence or absence of verapamil were also investigated with the employment of the rat liver microsomes and Caco-2 cell transwell model.Results: Verapamil significantly increased the peak plasma concentration (from 91.97 ± 11.30 to 125.30 ± 13.50 ng/mL), and decrease the oral clearance (from 63.85 ± 10.79 to 32.95 ± 6.17 L/h/kg). The intrinsic clearance rate was also significantly decreased (from 39.49 ± 0.42 to 28.64 ± 0.30 µL/min/mg protein) by the preincubation of verapamil. The results of Caco-2 cell transwell experiments showed the efflux of HCPT was inhibited by verapamil, as the efflux ratio decreased from 1.82 to 1.21.Discussion and conclusions: The system exposure of HCPT was increased by verapamil. Verapamil may exert this effect through inhibiting the activity of CYP3A4 or P-gp, which are related to the metabolism and transport of HCPT.


Assuntos
Antineoplásicos Fitogênicos/farmacocinética , Bloqueadores dos Canais de Cálcio/farmacologia , Camptotecina/análogos & derivados , Verapamil/farmacologia , Administração Oral , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Disponibilidade Biológica , Transporte Biológico , Células CACO-2 , Bloqueadores dos Canais de Cálcio/administração & dosagem , Camptotecina/administração & dosagem , Camptotecina/farmacocinética , Interações Medicamentosas , Humanos , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Ratos , Ratos Sprague-Dawley , Verapamil/administração & dosagem
3.
Molecules ; 25(2)2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31936160

RESUMO

: Multidrug resistance (MDR) is a complicated ever-changing problem in cancer treatment, and P-glycoprotein (P-gp), a drug efflux pump, is regarded as the major cause. In the way of developing P-gp inhibitors, natural products such as phenolic acids have gotten a lot of attention recently. The aim of the present study was to investigate the modulating effects and mechanisms of caffeic acid on human P-gp, as well as the attenuating ability on cancer MDR. Calcein-AM, rhodamine123, and doxorubicin were used to analyze the interaction between caffeic acid and P-gp, and the ATPase activity of P-gp was evaluated as well. Resistance reversing effects were revealed by SRB and cell cycle assay. The results indicated that caffeic acid uncompetitively inhibited rhodamine123 efflux and competitively inhibited doxorubicin efflux. In terms of P-gp ATPase activity, caffeic acid exhibited stimulation in both basal and verapamil-stimulated activity. The combination of chemo drugs and caffeic acid resulted in decreased IC50 in ABCB1/Flp-InTM-293 and KB/VIN, indicating that the resistance was reversed. Results of molecular docking suggested that caffeic acid bound to P-gp through GLU74 and TRY117 residues. The present study demonstrated that caffeic acid is a promising candidate for P-gp inhibition and cancer MDR attenuation.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Ácidos Cafeicos/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Ácidos Cafeicos/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Quimioterapia Combinada , Fluoresceínas/farmacologia , Humanos , Simulação de Acoplamento Molecular , Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rodaminas/farmacologia , Verapamil/farmacologia
4.
Int J Cardiovasc Imaging ; 36(4): 703-712, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31950298

RESUMO

Cardiovascular magnetic resonance feature tracking (CMR-FT) is a novel technique for non-invasive assessment of myocardial motion and deformation. Although CMR-FT is standardized in humans, literature on comparative analysis from animal models is scarce. In this study, we measured the reproducibility of global strain under various inotropic states and the sample size needed to test its relative changes in pigs. Ten anesthetized healthy Landrace pigs were investigated. After baseline (BL), two further steps were performed: (I) dobutamine-induced hyper-contractility (Dob) and (II) verapamil-induced hypocontractility (Ver). Global longitudinal (GLS), circumferential (GCS) and radial strain (GRS) were assessed. This study shows a good to excellent inter- and intra-observer reproducibility of CMR-FT in pigs under various inotropic states. The highest inter-observer reproducibility was observed for GLS at both BL (ICC 0.88) and Ver (ICC 0.79). According to the sample size calculation for GLS, a small number of animals could be used for future trials.


Assuntos
Ventrículos do Coração/diagnóstico por imagem , Imagem Cinética por Ressonância Magnética , Contração Miocárdica , Função Ventricular Esquerda , Agonistas de Receptores Adrenérgicos beta 1/farmacologia , Anestesia Geral , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Dobutamina/farmacologia , Feminino , Ventrículos do Coração/efeitos dos fármacos , Modelos Animais , Contração Miocárdica/efeitos dos fármacos , Variações Dependentes do Observador , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Tamanho da Amostra , Sus scrofa , Função Ventricular Esquerda/efeitos dos fármacos , Verapamil/farmacologia
5.
Chem Biol Interact ; 315: 108892, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31704064

RESUMO

Oleanolic acid (OA) is a triterpenoid, widely found in plants and possesses antitumor activity in many cancer lines. However, cancer cells develop multidrug resistance (mdr) hindering the effect of anticancer drugs. P-glycoprotein (P-gp) is a major cause of mdr. Therefore, the cytotoxic effect of OA was evaluated on human breast cancer MDA-MB-231 and human liver cancer HepG2 with absence and presence of P-gp, respectively. OA reduced MDA-MB-231 viability in a dose dependent manner, whereas no remarkable effect was observed on HepG2 in the same range of concentrations (1-60 µM). Moreover, cytotoxicity studies were conducted in the presence of verapamil (20 mg/L), a P-gp inhibitor. OA exhibited the same effect on MDA-MB-231 in the absence and presence of verapamil. However, the cytotoxicity was greatly enhanced for HepG2 cells in the presence of verapamil (cell viability dropped from 63.7% to 25% after 72 h at 60 µM). The results were then confirmed in vivo on zebrafish embryos. Increased mortality and malformations were observed in verapamil pretreated group between 5 and 15 µM of OA compared to control; also, all embryos died at 20 µΜ OA and above. These results demonstrate that inhibiting P-gp enhances the chemotherapeutic activity of OA.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Ácido Oleanólico/farmacologia , Peixe-Zebra/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Feminino , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Verapamil/farmacologia
6.
Life Sci ; 244: 117153, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31830479

RESUMO

AIMS: Increased activity of calpain-1 and matrix metalloproteinase (MMP)-2 was observed in different models of arterial hypertension and contribute to thicken the left ventricle (LV) walls and to hypertrophy cardiac myocytes. MMP-2 activity may be regulated by calpain-1 via bioactive molecules activation such as transforming growth factor (TGF)-ß in cardiovascular diseases. This study analyzed whether calpain-1 causes cardiac hypertrophy and dysfunction by modulating the expression and activity of MMP-2 in renovascular hypertension. MAIN METHODS: Male Wistar rats were submitted to two kidneys, one clip (2K1C) model of hypertension or sham surgery and were treated with verapamil (VRP, 8 mg/kg/bid) by gavage from the second to tenth week post-surgery. Systolic blood pressure (SBP) was weekly assessed by tail-cuff plethysmography and morphological and functional parameters of LV were analyzed by echocardiography. MMP-2 activity was analyzed by in situ and gelatin zymography, while calpain-1 activity by caseinolytic assay. MMP-2, calpain-1, TGF-ß and MMP-14/TIMP-2 levels were identified in the LV by western blots. Fluorescence assays were performed to evaluate oxidative stress, MMP-2 and calpain-1 levels. KEY FINDINGS: SBP increased in 2K1C rats and was unaltered by VRP. However, VRP notably ameliorated hypertension-induced increase in LV thickness. VRP decreased hypertension-induced enhances in calpain-1 and MMP-2 activities, oxidative stress and mature TGF-ß levels. Treatment with VRP also decreased the accentuated MMP-14/TIMP-2 levels in 2K1C. SIGNIFICANCE: Treatment with VRP decreases calpain-1 and MMP-2 activities and also reduces TGF-ß and MMP-14/TIMP-2 levels in the LV of hypertensive rats, thus contributing to ameliorate cardiac hypertrophy.


Assuntos
Calpaína/metabolismo , Cardiomegalia/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Hipertensão/complicações , Metaloproteinase 2 da Matriz/metabolismo , Remodelação Ventricular/efeitos dos fármacos , Verapamil/farmacologia , Animais , Calpaína/genética , Cardiomegalia/etiologia , Masculino , Metaloproteinase 2 da Matriz/genética , Ratos , Ratos Wistar , Vasodilatadores/farmacologia
7.
Colloids Surf B Biointerfaces ; 186: 110682, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31846891

RESUMO

Over the past few years, cardiac tissue engineering has undergone tremendous progress. Various in vitro methods have been developed to improve the accuracy in the result of drug-induced cardiac toxicity screening. Herein, we propose a novel SU-8 cantilever integrated with an electromechanical-stimulator to enhance the maturation of cultured cardiac cells. The simultaneous electromechanical stimulation significantly enhances the contraction force of the cardiomyocytes, thereby increasing cantilever displacement. Fluorescence microscopy analysis was performed to confirm the improved maturation of the cardiomyocytes. After the initial experiments, the contractile behaviors of the cultured cardiomyocytes were investigated by measuring the mechanical deformation of the SU-8 cantilever. Finally, the proposed electromechanical-stimulator-integrated SU-8 cantilever was used to evaluate the adverse effects of different cardiac vascular drugs, i.e., verapamil, lidocaine, and isoproterenol, on the cultured cardiomyocytes. The physiology of the cardiac-drug-treated cardiomyocytes was examined with and without electrical stimulation of the cardiomyocytes. The experimental results indicate that the proposed cantilever platform can be used as a predictive assay system for preliminary cardiac drug toxicity screening applications.


Assuntos
Técnicas Biossensoriais , Compostos de Epóxi/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Polímeros/farmacologia , Animais , Técnicas Biossensoriais/instrumentação , Fármacos Cardiovasculares/química , Fármacos Cardiovasculares/farmacologia , Compostos de Epóxi/química , Isoproterenol/química , Isoproterenol/farmacologia , Lidocaína/química , Lidocaína/farmacologia , Fenômenos Mecânicos , Tamanho da Partícula , Polímeros/química , Propriedades de Superfície , Verapamil/química , Verapamil/farmacologia
8.
Anticancer Res ; 39(12): 6457-6462, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31810909

RESUMO

BACKGROUND/AIM: Nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the NAD+ biosynthetic pathway, is a drug target of potent anticancer candidates, including FK866 and other reported NAMPT inhibitors. However, it is known that NAMPT point-mutations render resistance to specific NAMPT inhibitors in several cancer cells. We investigated the resistance mechanisms of NAMPT inhibitor FK866 in human colorectal cancer (CRC) cells. MATERIALS AND METHODS: We used CRC human cell line HCT116 to determine the expression profiles of FK866-sensitive parental HCT116 cells and FK866-resistant HCT116 (HCT116RFK866) cells by DNA microarray analysis. The levels of multidrug resistance protein 1 (MDR1) were assessed via western blot. In addition, we analyzed the sensitivity of FK866 in parental HCT116 cells and HCT116RFK866 cells by co-treatment with MDR1 inhibitor verapamil. RESULTS: Our results revealed an association between ATP-binding cassette (ABC) transporter gene ABCB1 and resistance to NAMPT inhibitor FK866 in both HCT116RFK866 cells and parental HCT116 cells. The expression of ABCB1, which encodes MDR1, was lower in HCT116RFK866 cells than in parental HCT116 cells. Furthermore, the protein level of MDR1/ATP-binding cassette sub-family B member 1 (ABCB1) was 0.5-fold lower in HCT116RFK866 cells than in parental HCT116 cells. Additionally, HCT116RFK866 cells showed improved sensitivity to FK866 when co-treated with verapamil, an ABCB1 inhibitor. Interestingly, the efficacy of FK866 in parental HCT116 cells was the same for the treatment with FK866 alone or in combination with verapamil. CONCLUSION: The change in expression of ABCB1 plays a key role in CRC drug resistance to NAMPT inhibitor FK866. This suggests that the MDR1/ABCB1 mechanism may regulate the resistance of anticancer NAMPT inhibitor FK866.


Assuntos
Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Acrilamidas/farmacologia , Neoplasias Colorretais/metabolismo , Citocinas/antagonistas & inibidores , Regulação para Baixo , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Piperidinas/farmacologia , Verapamil/farmacologia
9.
Int J Med Sci ; 16(12): 1621-1630, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31839750

RESUMO

The goal of this study was to examine the effect of lipid emulsion on the vasodilation induced in isolated endothelium-denuded rat aortae by a toxic dose of amlodipine. We examined the effects of lipid emulsion and verapamil on amlodipine-induced vasodilation. We also examined the effects of a mixture of lipid emulsion and amlodipine, as well as the centrifuged aqueous extract (CAE) obtained by ultracentrifuging such a mixture and then removing the upper lipid layer, on amlodipine-induced vasodilation. The effect of lipid emulsion on the amlodipine concentration was examined. Lipid emulsion attenuated amlodipine-induced vasodilation in isolated aortae. Both CAE and lipid emulsion containing amlodipine inhibited amlodipine-induced vasodilation. However, there was no significant difference in amlodipine-induced vasodilation between aortae treated with CAE and those treated with lipid emulsion containing amlodipine. Verapamil inhibited amlodipine-induced vasodilation. Lipid emulsion decreased the concentration of amlodipine. Lipid emulsion attenuated the vasodilation induced by a toxic amlodipine dose in NaF-precontracted aortae. The data show that lipid emulsion inhibited the vasodilation induced by a toxic amlodipine dose in isolated rat aortae by reducing the concentration of amlodipine. Amlodipine-induced vasodilation seems to be mediated mainly by blockade of L-type calcium channels and partially by inhibition of the Rho-kinase pathway.


Assuntos
Anlodipino/farmacologia , Aorta/efeitos dos fármacos , Lipídeos/farmacologia , Vasodilatação/efeitos dos fármacos , Anlodipino/toxicidade , Animais , Aorta/fisiopatologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/genética , Emulsões/farmacologia , Humanos , Lipídeos/antagonistas & inibidores , Lipídeos/química , Masculino , Técnicas de Cultura de Órgãos , Ratos , Transdução de Sinais/efeitos dos fármacos , Vasodilatação/genética , Vasodilatação/fisiologia , Verapamil/farmacologia , Quinases Associadas a rho/genética
10.
Pharm Biol ; 57(1): 787-791, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31747844

RESUMO

Context: Oridonin has been traditionally used in Chinese treatment of various cancers, but its poor bioavailability limits its therapeutic uses. Verapamil can enhance the absorption of some drugs with poor oral bioavailability. Whether verapamil can enhance the bioavailability of oridonin is still unclear.Objective: This study investigated the effect of verapamil on the pharmacokinetics of oridonin in rats and clarified its main mechanism.Materials and methods: The pharmacokinetic profiles of oral administration of oridonin (20 mg/kg) in Sprague-Dawley rats with two groups of six animals each, with or without pre-treatment of verapamil (10 mg/kg/day for 7 days) were investigated. The effects of verapamil on the transport and metabolic stability of oridonin were also investigated using Caco-2 cell transwell model and rat liver microsomes.Results: The results showed that verapamil could significantly increase the peak plasma concentration (from 146.9 ± 10.17 to 193.97 ± 10.53 ng/mL), and decrease the oral clearance (from 14.69 ± 4.42 to 8.09 ± 3.03 L/h/kg) of oridonin. The Caco-2 cell transwell experiments indicated that verapamil could decrease the efflux ratio of oridonin from 1.67 to 1.15, and the intrinsic clearance rate of oridonin was decreased by the pre-treatment with verapamil (40.06 ± 2.5 vs. 36.09 ± 3.7 µL/min/mg protein).Discussion and conclusions: These results indicated that verapamil could significantly change the pharmacokinetic profile of oridonin in rats, and it might exert these effects through increasing the absorption of oridonin by inhibiting the activity of P-gp, or through inhibiting the metabolism of oridonin in rat liver. In addition, the potential drug-drug interaction should be given special attention when verapamil is used with oridonin. Also, the dose of oridonin should be carefully selected in the clinic.


Assuntos
Diterpenos de Caurano/metabolismo , Diterpenos de Caurano/farmacocinética , Verapamil/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Células CACO-2 , Interações Medicamentosas , Humanos , Taxa de Depuração Metabólica/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Verapamil/sangue
11.
Bull Exp Biol Med ; 168(2): 187-192, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31776956

RESUMO

The whole-cell patch-clamp technique was used to examine the effect of gadolinium Gd3+ (a non-specific blocker of mechanically gated current IMGCh, a component of late current IL) on ionic currents in insolated rat ventricular cardiomyocytes alone and in combination with the blockers of L-type calcium currents (ICaL) nifedipine (10 µM) or verapamil (1 µM). In K+in/K+out or Cs+in/Cs+out media, blockade of ICaL produced no effect on IL at negative potentials, but inhibited IL at positive ones. In K+in/K+out medium, Gd3+ (5 µM) decreased the net persistent current (Inp) at -45 mV from 198.6±6.4 to 96.7±9.5 pA over 15 min. Gd3+ alone or in combination with ICaL blockers shifted the reversal potential of IL to more negative values. At negative potentials, Gd3+ decreased IK1 and inward current including IMGCh. At positive potentials, Gd3+ alone or in combination with ICaL blockers decreased IL. When applied for 15 min in Cs+in/Cs+out medium at -45 mV, Gd3+ produced no effect on net current and inward and outward components of IL. Thus, Gd3+ can be viewed as a specific blocker of IMGCh only in Cs+ medium.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Gadolínio/farmacologia , Transporte de Íons/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Canais de Cálcio/metabolismo , Césio/metabolismo , Ventrículos do Coração/citologia , Masculino , Nifedipino/farmacologia , Técnicas de Patch-Clamp , Canais de Potássio/metabolismo , Ratos , Verapamil/farmacologia
12.
Acta Pharm ; 69(4): 607-619, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31639087

RESUMO

Flavonoids are natural polyphenolic compounds present in a wide spectrum of plants that have a beneficial effect on human health. In the context of cardiovascular diseases related to plaque and thrombus formation, flavonoids exhibit an anti-aggregatory effect. Previously, it has been reported that all tested flavonoids exhibit an antiaggregatory effect on platelet aggregation when measured by impedance aggregometry on whole blood, in the test of aggregation induced by adenosine diphosphate (ADP). As not all flavonoids have the same targets within signaling pathways, an assumption of a common non-specific mechanism related to lipophilicity is to be considered. To test this hypothesis, reverse-phase thin layer chromatography was used to assess the lipophilicity of flavonoids; impedance aggregometry was used for testing of platelet aggregation and flow cytometry to monitor the influence of flavonoids on platelet activation. Lipophilicity analysis showed a highly negative correlation of logP and MINaAC for groups of flavones and flavanones. As determined by flow cytometry, the exposition of receptors necessary for the promotion of platelet activation and primary clot formation was diminished, i.e., lowered expression of the activated form of integrin αIIbß3 was observed in the presence of flavanone. Platelet membrane stabilization by flavonoids as a mechanism of antiaggregatory effect has been supported by impedance aggregometry experiments when specific inhibitors of platelet aggregation signaling pathways (U73122, indomethacin, verapamil) were used in the presence of a weak (ADP) and a strong (TRAP-6) agonist of aggregation. While individual flavonoids can have specific targets within aggregation signaling pathways, all flavonoids share a common non-specific mechanism of platelet aggregation inhibition related to their lipophilicity and membrane stabilization that, to some extent, contributes to their antiaggregatory effect.


Assuntos
Plaquetas/efeitos dos fármacos , Flavonoides/fisiologia , Agregação Plaquetária/efeitos dos fármacos , Estrenos/farmacologia , Humanos , Indometacina/farmacologia , Pirrolidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Verapamil/farmacologia
13.
Eur J Med Chem ; 183: 111726, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31585275

RESUMO

Multidrug resistance (MDR) is a main cause of chemotherapy failure and patient death. This situation usually involves a glycoprotein (P-gp) mediated drug efflux, resulting in a low cellular drug concentration and insensitivity. Here we report the design, synthesis and evaluation of novel (+/-)-securinine bivalents as P-gp inhibitors in vitro and in vivo. MTT assays reflected that bivalent mimetics of securinine particularly the virosecurinine bivalent mimetic 8C showed promissing MDR reversal potential in both P-gp highly expressed cell line HepG2/DOX and MCF-7/ADM. At a 10 µM concentration, 8C can entirely reverse the resistance of HepG2/DOX to doxorubicin (DOX), and is more effective than the positive control verapamil (VRP). Fluorescence, flow cytometry, and DOX efflux assays demonstrated that 8C can facilitate the accumulation and diminish the efflux of intracellular DOX. Molecular docking analysis and western blot assays indicated that 8C accomplished this by competitively inhibiting the activity of P-gp rather than by affecting its expression. Compound 8C was also observed to reverse drug resistance effectively in xenograft models when combined with DOX. This study lays a foundation for the discovery of (+/-)-securinine ramifications as P-gp inhibitors and provides a promising lead compound 8C as a P-gp mediated MDR reversal agent.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Alcaloides , Antineoplásicos , Azepinas , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Lactonas , Piperidinas , Alcaloides/química , Alcaloides/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Azepinas/química , Azepinas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Compostos Heterocíclicos de Anel em Ponte/química , Compostos Heterocíclicos de Anel em Ponte/farmacologia , Humanos , Lactonas/química , Lactonas/farmacologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Piperidinas/química , Piperidinas/farmacologia , Verapamil/farmacologia
14.
Cell Physiol Biochem ; 53(2): 337-354, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31373783

RESUMO

BACKGROUND/AIMS: The availability of truly maturated cardiomyocytic subtypes is a major prerequisite for cardiovascular cell replacement therapies. Pluripotent stem cells provide a suitable source for the development of new strategies to overcome enormous hurdles such as yield, purity and safety of in vitro generated cells. METHODS: To address these issues, we have refined existing forward programming protocols by combining forced exogenous overexpression of the early cardiovascular transcription factor Nkx2.5 with a αMHC-promoter-based antibiotic selection step. Additionally, we applied small molecules such as ascorbic acid to enhance cardiomyogenic differentiation efficiency. Subsequently, we evaluated the cell fate of the resulting cardiomyocytes on the mRNA as well as protein levels. The latter was performed using high-resolution confocal microscopy. Furthermore, we examined the response of the cells` beating activities to pharmacological substance administration. RESULTS: Our results reveal an apparent influence of Nkx2.5 on the cell fate of ESC-derived cardiomyocytes. Resulting single cells exhibit characteristics of early ventricular cardiomyocytes, such as sarcomeric marker expression, spontaneous beating frequency, and distinct L-type calcium channel occurrence. CONCLUSION: Therefore, we demonstrate cardiovascular subtype forward programming of ESCs using a combination of transcription factors along with small molecule administration. However, our findings also underline current assumptions, that a terminal maturation of PSC derived cardiomyocytes in vitro is still an unsolved problem which urgently needs to be addressed in the field.


Assuntos
Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Proteína Homeobox Nkx-2.5/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Ácido Ascórbico/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Proteína Homeobox Nkx-2.5/antagonistas & inibidores , Proteína Homeobox Nkx-2.5/genética , Camundongos , Microscopia Confocal , Miócitos Cardíacos/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Verapamil/farmacologia
15.
Mol Pharmacol ; 96(4): 485-492, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31391290

RESUMO

Diltiazem is a widely prescribed Ca2+ antagonist drug for cardiac arrhythmia, hypertension, and angina pectoris. Using the ancestral CaV channel construct CaVAb as a molecular model for X-ray crystallographic analysis, we show here that diltiazem targets the central cavity of the voltage-gated Ca2+ channel underneath its selectivity filter and physically blocks ion conduction. The diltiazem-binding site overlaps with the receptor site for phenylalkylamine Ca2+ antagonist drugs such as verapamil. The dihydropyridine Ca2+ channel blocker amlodipine binds at a distinct site and allosterically modulates the binding sites for diltiazem and Ca2+ Our studies resolve two distinct binding poses for diltiazem in the absence and presence of amlodipine. The binding pose in the presence of amlodipine may mimic a high-affinity binding configuration induced by voltage-dependent inactivation, which is favored by dihydropyridine binding. In this binding pose, the tertiary amino group of diltiazem projects upward into the inner end of the ion selectivity filter, interacts with ion coordination Site 3 formed by the backbone carbonyls of T175, and alters binding of Ca2+ to ion coordination Sites 1 and 2. Altogether, our results define the receptor site for diltiazem and elucidate the mechanisms for pore block and allosteric modulation by other Ca2+ channel-blocking drugs at the atomic level. SIGNIFICANCE STATEMENT: Calcium antagonist drugs that block voltage-gated calcium channels in heart and vascular smooth muscle are widely used in the treatment of cardiovascular diseases. Our results reveal the chemical details of diltiazem binding in a blocking position in the pore of a model calcium channel and show that binding of another calcium antagonist drug alters binding of diltiazem and calcium. This structural information defines the mechanism of drug action at the atomic level and provides a molecular template for future drug discovery.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Diltiazem/farmacologia , Regulação Alostérica , Anlodipino/química , Anlodipino/farmacologia , Animais , Sítios de Ligação , Bloqueadores dos Canais de Cálcio/química , Cristalografia por Raios X , Diltiazem/química , Humanos , Modelos Moleculares , Conformação Proteica , Verapamil/farmacologia
16.
PLoS One ; 14(7): e0204712, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31283756

RESUMO

BACKGROUND: We present a new family of ECG biomarkers for assessing drug effects on ventricular repolarization. We show that drugs blocking inward (depolarizing) ion currents cause a relative increase of the T vector velocity (TVV) and accelerate repolarization, while drugs blocking outward ion currents cause a relative decrease of the TVV and delay repolarization. The results suggest a link between the TVV and the instantaneous change of the cellular action potentials that may contribute to bridge the gap between the surface ECG and myocardial cellular processes. METHODS: We measure TVV as the time required to reach X% of the total Trajectory length of the T vector loop, denoted as TrX. Applied to data from two FDA funded studies (22+22 subjects, 5232+4208 ECGs) which target ECG effects of various ion-channel blocking drugs, the TrX effect profiles indicate increasingly delayed electrical activity over the entire repolarization process for drugs solely reducing outward potassium current (dofetilide, moxifloxacin). For drugs eliciting block of the inward sodium or calcium currents (mexiletine, lidocaine), the TrX effect profiles were consistent with accelerated electrical activity in the initial repolarization phase. For multichannel blocking drugs (ranolazine) or drug combinations blocking multiple ion currents (dofetilide + mexiletine, dofetilide + lidocaine), the overall TrX effect profiles indicate a superposition of the individual TrX effect profiles. RESULTS: The parameter Tr40c differentiates pure potassium channel blocking drugs from multichannel blocking drugs with an area under the ROC curve (AUC) of 0.90, CI = [0.88 to 0.92]. This is significantly better than the performance of J-Tpeakc (0.81, CI = [0.78 to 0.84]) identified as the best parameter in the second FDA study. Combining the ten parameters Tr10c to Tr100c in a logistic regression model further improved the AUC to 0.94, CI = [0.92 to 0.96]. CONCLUSIONS: TVV analysis substantially improves assessment of drug effects on cardiac repolarization, providing a plausible and improved mechanistic link between drug effects on ionic currents and overall ventricular repolarization reflected in the body surface ECG. TVV contributes to an enhanced appraisal of the proarrhythmic risk of drugs beyond QTc prolongation and J-Tpeakc.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Coração/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Adulto , Bases de Dados Factuais , Método Duplo-Cego , Eletrocardiografia/métodos , Feminino , Humanos , Lidocaína/farmacologia , Masculino , Mexiletina/farmacologia , Fenetilaminas/farmacologia , Ranolazina/farmacologia , Sulfonamidas/farmacologia , Verapamil/farmacologia , Adulto Jovem
17.
J Toxicol Sci ; 44(7): 441-457, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31270301

RESUMO

The objective of this study is to assess the response of telemetered common marmosets to multiple cardiac ion channel inhibitors and to clarify the usefulness of this animal model in evaluating the effects of drug candidates on electrocardiogram (ECG). Six multiple cardiac ion channel inhibitors (sotalol, astemizole, flecainide, quinidine, verapamil and terfenadine) were orally administered to telemetered common marmosets and changes in QTc, PR interval and QRS duration were evaluated. Drugs plasma levels were determined to compare the sensitivity in common marmosets to that in humans. QTc prolongation was observed in the marmosets dosed with sotalol, astemizole, flecainide, quinidine, verapamil and terfenadine. PR prolongation was noted after flecainide and verapamil administration, and QRS widening occurred following treatment with flecainide and quinidine. Drugs plasma levels associated with ECG changes in marmosets were similar to those in humans, except for verapamil-induced QTc prolongation. Verapamil-induced change is suggested due to body temperature decrease. These results indicate that telemetered common marmoset is a useful animal for evaluation of the ECG effects of multiple cardiac ion channel inhibitors and the influence of body temperature change should be considered in the assessment.


Assuntos
Astemizol/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Callithrix , Eletrocardiografia/efeitos dos fármacos , Flecainida/farmacologia , Modelos Animais , Quinidina/farmacologia , Medição de Risco/métodos , Sotalol/farmacologia , Telemetria , Terfenadina/farmacologia , Verapamil/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Astemizol/sangue , Temperatura Corporal/fisiologia , Bloqueadores dos Canais de Cálcio/sangue , Flecainida/sangue , Masculino , Quinidina/sangue , Sotalol/sangue , Terfenadina/sangue , Verapamil/sangue , Bloqueadores do Canal de Sódio Disparado por Voltagem/sangue
19.
Pharm Biol ; 57(1): 407-411, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31230510

RESUMO

Context: Puerarin and triptolide are sometimes used together for the treatment of disease in Chinese clinics; however, the drug-drug interaction between puerarin and triptolide is still unknown. Objective: This study investigates the effects of puerarin on the pharmacokinetics of triptolide in rats and clarifies its main mechanism. Materials and methods: The pharmacokinetic profiles of oral administration of triptolide (1 mg/kg) in Sprague-Dawley rats with (test group, n = 6) or without pretreatment (control group, n = 6) with puerarin (100 mg/kg/day for seven days) were investigated. The effects of puerarin on the transport and metabolic stability of triptolide were also investigated using Caco-2 cell transwell model and rat liver microsomes. Results: The results showed that puerarin could significantly increase the peak plasma concentration (from 187.25 ± 15.36 to 219.67 ± 21.52 ng/mL), and decrease its oral clearance (from 4.92 ± 0.35 to 62.46 ± 3.75 ± 0.19 L/h/kg). The Caco-2 cell transwell experiments indicated that puerarin could decrease the efflux ratio of triptolide from 2.70 to 1.33, and the intrinsic clearance rate of triptolide was decreased by the pretreatment with puerarin (38.8 ± 4.7 vs. 32.9 ± 6.5 µL/min/mg protein). Discussion and conclusions: Puerarin could significantly change the pharmacokinetic profiles of triptolide in rats, and it might exert these effects through increasing the absorption of triptolide by inhibiting the activity of P-gp, or through inhibiting the metabolism of triptolide in rat liver. The results also showed that the dose of triptolide should be decreased when these drugs were co-administered.


Assuntos
Anti-Inflamatórios/farmacologia , Diterpenos/farmacocinética , Isoflavonas/farmacologia , Fenantrenos/farmacocinética , Administração Oral , Animais , Células CACO-2 , Diterpenos/química , Interações Medicamentosas , Compostos de Epóxi/química , Compostos de Epóxi/farmacocinética , Humanos , Isoflavonas/química , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Fenantrenos/química , Ratos , Ratos Sprague-Dawley , Verapamil/farmacologia
20.
Molecules ; 24(9)2019 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-31060204

RESUMO

Arterial pressure of each new breeding spontaneous Phase-1 hypertension (P1-HT) rat was recorded for 5 min by intravascular femoral artery catheter that served as a reference value prior to treatment. In the acute antihypertensive test, 0.36 g/kg Bwt of Plantago asiatica seed extract (PSE) was administered, via gavage feeding, to P1-HT rats, and the arterial pressures were continuously recorded for 1 h. The acute antihypertensive effects of PSE on P1-HT rats appeared within 15 min after PSE administration and lasted over 1 h with systolic pressure decreased 31.5 mmHg and diastolic pressure decreased 18.5 mmHg. The systolic pressure decreased 28 mmHg and diastolic pressure decreased 16 mmHg in P1-HT rats when simultaneously compared with verapamil hydrochloride (reference drug), whereas there were no significant differences in the pretreated reference values of acute PSE treatment and the untreated control. In the chronic test, P1-HT rats received 0.36 g/kg Bwt day of PSE or equal volume of water for 4 weeks via oral gavage, and the lower blood pressure tendencies of chronic PSE treatment were also found when compared with the controls. The antihypertensive values of PSE were also confirmed in spontaneously hypertensive rats (SHRs). Oral administration with PSE can effectively moderate blood pressure within an hour, while taking PSE daily can control the severity of hypertension, suggesting PSE is a potentially antihypertensive herb.


Assuntos
Hipertensão/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Plantago/química , Verapamil/administração & dosagem , Administração Oral , Animais , Pressão Sanguínea/efeitos dos fármacos , Modelos Animais de Doenças , Esquema de Medicação , Humanos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Ratos , Ratos Sprague-Dawley , Sementes/química , Verapamil/farmacologia
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