RESUMEN
Src tyrosine kinase (TK), a redox-sensitive protein overexpressed in dystrophin-deficient muscles, can contribute to damaging signaling by phosphorylation and degradation of ß-dystroglycan (ß-DG). We performed a proof-of-concept preclinical study to validate this hypothesis and the benefit-safety ratio of a pharmacological inhibition of Src-TK in Duchenne muscular dystrophy (DMD). Src-TK inhibitors PP2 and dasatinib were administered for 5 weeks to treadmill-exercised mdx mice. The outcome was evaluated in vivo and ex vivo on functional, histological and biochemical disease-related parameters. Considering the importance to maintain a proper myogenic program, the potential cytotoxic effects of both compounds, as well as their cytoprotection against oxidative stress-induced damage, was also assessed in C2C12 cells. In line with the hypothesis, both compounds restored the level of ß-DG and reduced its phosphorylated form without changing basal expression of genes of interest, corroborating a mechanism at post-translational level. The histological profile of gastrocnemius muscle was slightly improved as well as the level of plasma biomarkers. However, amelioration of in vivo and ex vivo functional parameters was modest, with PP2 being more effective than dasatinib. Both compounds reached appreciable levels in skeletal muscle and liver, supporting proper animal exposure. Dasatinib exerted a greater concentration-dependent cytotoxic effect on C2C12 cells than the more selective PP2, while being less protective against H2O2 cytotoxicity, even though at concentrations higher than those experienced during in vivo treatments. Our results support the interest of Src-TK as drug target in dystrophinopathies, although further studies are necessary to assess the therapeutic potential of inhibitors in DMD.
Asunto(s)
Dasatinib , Distrofia Muscular Animal/tratamiento farmacológico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Inhibidores de Proteínas Quinasas , Pirimidinas , Familia-src Quinasas/antagonistas & inhibidores , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Dasatinib/farmacocinética , Dasatinib/farmacología , Dasatinib/uso terapéutico , Distroglicanos/genética , Distroglicanos/metabolismo , Hígado/metabolismo , Masculino , Ratones Endogámicos mdx , Fatiga Muscular/efectos de los fármacos , Fuerza Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Músculo Esquelético/fisiología , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Distrofia Muscular Animal/fisiopatología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/fisiopatología , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Reproducibilidad de los Resultados , TorqueRESUMEN
Ligand gated ion channels are involved in many pathophysiological processes and represent a relevant, although challenging, target for drug discovery. We propose an innovative electro-optical approach to their analysis able to derive membrane conductance values from the local membrane potential changes imposed by test current pulses and measured by fast voltage-sensitive fluorescent dyes. We exploited the potential of this proprietary method by developing a drug testing system called "ionChannel Optical High-content Microscope" (ionChannelΩ). This automated platform was validated by testing the responses of reference drugs on cells expressing different ligand-gated ion channels. Furthermore, a double-blind comparison with FLIPR and automated patch-clamp was performed on molecules designed to act as antagonists of the P2RX7 receptor. ionChannelΩ proved highly reliable in all tests, resulting faster and more cost-effective than electrophysiological techniques. Overall, ionChannelΩ is amenable to the study of ligand gated ion channels that are receiving less attention due to limitations in current assays.
Asunto(s)
Descubrimiento de Drogas/métodos , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos Activados por Ligandos/metabolismo , Microscopía/métodos , Imagen Óptica/métodos , Automatización de Laboratorios , Permeabilidad de la Membrana Celular/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Potenciales de la Membrana/efectos de los fármacos , Microscopía Fluorescente/métodos , Reproducibilidad de los ResultadosRESUMEN
Single-agent therapy with molecularly targeted agents has shown limited success in tumor growth control, mainly because escape or resistance mechanisms are activated once a signalling molecule is inhibited. Rational combinations of target-specific agents could counteract this response providing a useful strategy in cancer treatment. In this regard, the EGFR and mTOR inhibitors have been used together to generate a synergistic effect and maximize the efficacy of each individual agent. Overall, the in vivo and in vitro evidences support the utilization of combinations targeting EGFR and mTOR, for malignancies characterized by deregulated EGFR/PI3K/Akt/ mTOR signalling cascade; whereas the clinical experience points out that the assessment of the therapeutic value of such combination awaits further investigations.
Asunto(s)
Antineoplásicos/farmacología , Receptores ErbB/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos/administración & dosificación , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Humanos , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
AIMS: Glucocorticoids are the sole drugs clinically used in Duchenne muscular dystrophy, in spite of the relevant side effects. Combination of glucocorticoids with synergistic drugs may be one strategy to lower doses and control side effects, meanwhile providing wider control of the complex pathology. This study is a preclinical evaluation of the effect of a combined treatment of α-methyl-prednisolone (PDN) with taurine, a safe aminoacid with positive effects on some pathology-related events. METHODS: PDN (1 mg/kg/day i.p.) and taurine (1 g/kg/day orally) were administered either alone or in combination, for 4-8 weeks to male dystrophic mdx mice chronically exercised on a treadmill. Effects were assessed in vivo and ex vivo with a variety of methodological approaches. RESULTS: In vivo, each treatment significantly increased fore limb strength, a marked synergistic effect being observed with the combination PDN + taurine. Ex vivo, PDN + taurine completely restored the mechanical threshold, an electrophysiological index of calcium homeostasis, of extensor digitorum longus myofibres and the benefit was greater than for PDN alone. In parallel, the overactivity of voltage-independent cation channels in dystrophic myofibres was reduced. No effects were observed on plasma levels of creatine kinase, while lactate dehydrogenase was decreased by taurine and, to a minor extent, by PDN + taurine. A similar histology profile was observed in PDN and PDN + taurine-treated muscles. PDN + taurine significantly increased taurine level in fast-twitch muscle and brain, by high-pressure liquid chromatography analysis. CONCLUSIONS: The combination PDN + taurine has additive actions on in vivo and ex vivo functional end points, with less evident advantages on histopathology and biochemical markers of the disease.
Asunto(s)
Glucocorticoides/administración & dosificación , Metilprednisolona/administración & dosificación , Músculo Esquelético/efectos de los fármacos , Distrofia Muscular Animal/tratamiento farmacológico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Taurina/administración & dosificación , Animales , Cromatografía Líquida de Alta Presión , Creatina Quinasa/sangre , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Quimioterapia Combinada , L-Lactato Deshidrogenasa/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Fuerza Muscular/efectos de los fármacos , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Técnicas de Placa-ClampRESUMEN
BACKGROUND AND PURPOSE: Statins and fibrates can produce mild to life-threatening skeletal muscle damage. Resting chloride channel conductance (gCl), carried by the ClC-1 channel, is reduced in muscles of rats chronically treated with fluvastatin, atorvastatin or fenofibrate, along with increased resting cytosolic calcium in statin-treated rats. A high gCl, controlled by the Ca(2+)-dependent protein kinase C (PKC), maintains sarcolemma electrical stability and its reduction alters muscle function. Here, we investigated how statins and fenofibrate impaired gCl. EXPERIMENTAL APPROACH: In rats treated with fluvastatin, atorvastatin or fenofibrate, we examined the involvement of PKC in gCl reduction by the two intracellular microelectrodes technique and ClC-1 mRNA level by quantitative real time-polymerase chain reaction. Direct drug effects were tested by patch clamp analysis on human ClC-1 channels expressed in human embryonic kidney (HEK) 293 cells. KEY RESULTS: Chelerythrine, a PKC inhibitor, applied in vitro on muscle dissected from atorvastatin-treated rats fully restored gCl, suggesting the involvement of this enzyme in statin action. Chelerythrine partially restored gCl in muscles from fluvastatin-treated rats but not in those from fenofibrate-treated rats, implying additional mechanisms for gCl impairment. Accordingly, a decrease of ClC-1 channel mRNA was found in both fluvastatin- and fenofibrate-treated rat muscles. Fenofibric acid, the in vivo metabolite of fenofibrate, but not fluvastatin, rapidly reduced chloride currents in HEK 293 cells. CONCLUSIONS AND IMPLICATIONS: Our data suggest multiple mechanisms underlie the effect of statins and fenofibrate on ClC-1 channel conductance. While statins promote Ca(2+)-mediated PKC activation, fenofibrate directly inhibits ClC-1 channels and both fluvastatin and fenofibrate impair expression of mRNA for ClC-1.
Asunto(s)
Canales de Cloruro/efectos de los fármacos , Cloruros/metabolismo , Ácidos Grasos Monoinsaturados/farmacología , Fenofibrato/farmacología , Ácidos Heptanoicos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Hipolipemiantes/farmacología , Indoles/farmacología , Músculo Esquelético/efectos de los fármacos , Pirroles/farmacología , Potenciales de Acción , Animales , Atorvastatina , Benzofenantridinas/farmacología , Calcio/metabolismo , Línea Celular , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Electromiografía , Activación Enzimática , Ácidos Grasos Monoinsaturados/toxicidad , Fenofibrato/toxicidad , Fluvastatina , Ácidos Heptanoicos/toxicidad , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/toxicidad , Hipolipemiantes/toxicidad , Indoles/toxicidad , Masculino , Músculo Esquelético/metabolismo , Técnicas de Placa-Clamp , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/toxicidad , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , TransfecciónRESUMEN
The role of tumour necrosis factor (TNF)-alpha or cyclo-oxygenase-2 (COX-2) eicosanoids in dystrophinopathies has been evaluated by chronically treating (4-8 weeks) adult dystrophic mdx mice with the anti-TNF-alpha etanercept (0.5 mg/kg) or the COX-2 inhibitor meloxicam (0.2 mg/kg). Throughout the treatment period the mdx mice underwent a protocol of exercise on treadmill in order to worsen the pathology progression; gastrocnemious muscles from exercised mdx mice showed an intense staining for TNF-alpha by immunohistochemistry. In vivo, etanercept, but not meloxicam, contrasted the exercise-induced forelimb force drop. Electrophysiological recordings ex vivo, showed that etanercept counteracted the decrease in chloride channel function (gCl), a functional index of myofibre damage, in both diaphragm and extensor digitorum longus (EDL) muscle, meloxicam being effective only in EDL muscle. None of the drugs ameliorated calcium homeostasis detected by electrophysiology and/or spectrofluorimetry. Etanercept, more than meloxicam, effectively reduced plasma creatine kinase (CK). Etanercept-treated muscles showed a reduction of connective tissue area and of pro-fibrotic cytokine TGF-beta1 vs. untreated ones; however, the histological profile was weakly ameliorated. In order to better evaluate the impact of etanercept treatment on histology, a 4-week treatment was performed on 2-week-old mdx mice, so to match the first spontaneous degeneration cycle. The histology profile of gastrocnemious was significantly improved with a reduction of degenerating area; however, CK levels were only slightly lower. The present results support a key role of TNF-alpha, but not of COX-2 products, in different phases of dystrophic progression. Anti-TNF-alpha drugs may be useful in combined therapies for Duchenne patients.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Eicosanoides/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Creatina Quinasa/sangre , Creatina Quinasa/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Electrofisiología , Etanercept , Inmunoglobulina G/farmacología , Inmunohistoquímica , Inmunosupresores/farmacología , Masculino , Meloxicam , Ratones , Ratones Endogámicos mdx , Microelectrodos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Condicionamiento Físico Animal , Receptores del Factor de Necrosis Tumoral , Tiazinas/farmacología , Tiazoles/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: The 2-propyl-1,4 benzoxazine (AM10) shows a peculiar behaviour in skeletal muscle, inhibiting or opening the ATP-sensitive K(+) (KATP) channel in the absence and presence of ATP, respectively. We focused on tissue selectivity and mechanism of action of AM10 by testing its effects on pancreatic KATP channels by means of both in vitro and in vivo investigations. EXPERIMENTAL APPROACH: In vitro, patch-clamp recordings were performed in native pancreatic beta cells and in tsA201 cells expressing the Kir6.2 Delta C36 channel. In vivo, an intraperitoneal glucose tolerance test was performed in normal mice. KEY RESULTS: In contrast with what observed in the skeletal muscle, AM10, in whole cell perforated mode, did not augment KATP current (I(KATP)) of native beta cells but it inhibited it in a concentration-dependent manner (IC(50): 11.5 nM; maximal block: 60%). Accordingly, in current clamp recordings, a concentration-dependent membrane depolarization was observed. On excised patches, AM10 reduced the open-time probability of KATP channels without altering their single channel conductance; the same effect was observed in the presence of trypsin in the bath solution. Moreover, AM10 inhibited, in an ATP-independent manner, the K(+) current resulting from expressed Kir6.2 Delta C36 (maximal block: 60% at 100 microM; IC(50): 12.7 nM) corroborating an interaction with Kir. In vivo, AM10 attenuated the glycemia increase following a glucose bolus in a dose-dependent manner, without, at the dose tested, inducing fasting hypoglycaemia. CONCLUSION AND IMPLICATIONS: Altogether, these results help to gain insight into a new class of tissue specific KATP channel modulators.
Asunto(s)
Adenosina Trifosfato/farmacología , Aminopiridinas/farmacología , Benzoxazinas/farmacología , Glucemia/efectos de los fármacos , Hipoglucemiantes/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Rectificación Interna/efectos de los fármacos , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Prueba de Tolerancia a la Glucosa , Humanos , Técnicas In Vitro , Células Secretoras de Insulina/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Ratones , Mutación , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Valores de Referencia , Factores de Tiempo , TransfecciónRESUMEN
Proteins with a short half-life are potential sites of pancreatic ss cell dysfunction under pathophysiological conditions. In this study, mouse islets were used to establish which step in the regulation of insulin secretion is most sensitive to inhibition of protein synthesis by 10 microM cycloheximide (CHX). Although islet protein synthesis was inhibited approximately 95% after 1 h, the inhibition of insulin secretion was delayed and progressive. After long (18-20 h) CHX-treatment, the strong (80%) inhibition of glucose-, tolbutamide-, and K(+)-induced insulin secretion was not due to lower insulin stores, to any marked impairment of glucose metabolism or to altered function of K(+)-ATP channels (total K(+)-ATP currents were however decreased). It was partly caused by a decreased Ca(2+) influx (whole-cell Ca(2+) current) resulting in a smaller rise in cytosolic Ca(2+) ([Ca(2+)](i)). The situation was very different after short (2-5 h) CHX-treatment. Insulin secretion was 50-60% inhibited although islet glucose metabolism was unaffected and stimulus-induced [Ca(2+)](i) rise was not (2 h) or only marginally (5 h) decreased. The efficiency of Ca(2+) on secretion was thus impaired. The inhibition of insulin secretion by 15 h of CHX treatment was more slowly reversible (>4 h) than that of protein synthesis. This reversibility of secretion was largely attributable to recovery of a normal Ca(2+) efficiency. In conclusion, inhibition of protein synthesis in islets inhibits insulin secretion in two stages: a rapid decrease in the efficiency of Ca(2+) on exocytosis, followed by a decrease in the Ca(2+) signal mediated by a slower loss of functional Ca(2+) channels. Glucose metabolism and the regulation of K(+)-ATP channels are more resistant. Proteins with a short half-life appear to be important to ensure optimal Ca(2+) effects on exocytosis, and are the potential Achille's heel of stimulus-secretion coupling.