RESUMO
Although the sodium channel blocker, mexiletine, is the first choice drug in myotonia, some myotonic patients remain unsatisfied due to contraindications, lack of tolerability, or incomplete response. More therapeutic options are thus needed for myotonic patients, which require clinical trials based on solid preclinical data. In previous structure-activity relationship studies, we identified two newly-synthesized derivatives of tocainide, To040 and To042, with greatly enhanced potency and use-dependent behavior in inhibiting sodium currents in frog skeletal muscle fibers. The current study was performed to verify their potential as antimyotonic agents. Patch-clamp experiments show that both compounds, especially To042, are greatly more potent and use-dependent blockers of human skeletal muscle hNav1.4 channels compared to tocainide and mexiletine. Reduced effects on F1586C hNav1.4 mutant suggest that the compounds bind to the local anesthetic receptor, but that the increased hindrance and lipophilia of the N-substituent may further strengthen drug-receptor interaction and use-dependence. Compared to mexiletine, To042 was 120 times more potent to block hNav1.4 channels in a myotonia-like cellular condition and 100 times more potent to improve muscle stiffness in vivo in a previously-validated rat model of myotonia. To explore toxicological profile, To042 was tested on hERG potassium currents, motor coordination using rotarod, and C2C12 cell line for cytotoxicity. All these experiments suggest a satisfactory therapeutic index for To042. This study shows that, owing to a huge use-dependent block of sodium channels, To042 is a promising candidate drug for myotonia and possibly other membrane excitability disorders, warranting further preclinical and human studies.
Assuntos
Miotonia/prevenção & controle , Canal de Sódio Disparado por Voltagem NAV1.4/fisiologia , Tocainide/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Relação Dose-Resposta a Droga , Canais de Potássio Éter-A-Go-Go/fisiologia , Humanos , Masculino , Mexiletina/farmacologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Miotonia/fisiopatologia , Ratos , Ratos Wistar , Reflexo de Endireitamento/efeitos dos fármacos , Teste de Desempenho do Rota-Rod , Tocainide/efeitos adversos , Tocainide/análogos & derivados , Tocainide/uso terapêutico , Bloqueadores do Canal de Sódio Disparado por Voltagem/efeitos adversos , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêuticoRESUMO
Mexiletine (Mex) has been recently appointed as an orphan-drug in myotonic-syndromes, being a potent use-dependent blocker of skeletal-muscle sodium channels (NaV1.4). Available evidences about a potential anti-oxidant effect of Mex and its tetramethyl-pyrroline-derivatives in vivo, suggest the possibility to further enlarge the therapeutic potential of Mex-like compounds in myopathies in which alteration of excitation-contraction coupling is paralleled by oxidative stress. In line with this and based on our previous structure-activity-relationship studies, we synthesized new compounds with a tetramethyl-pyrroline-ring on the amino-group of both Mex (VM11) and of its potent use-dependent isopropyl-derivative (CI16). The compounds were tested for their ability to block native NaV1.4 and to exert cyto-protective effects against oxidative-stress injury in myoblasts. Voltage-clamp-recordings on adult myofibers were performed to assess the tonic and use-dependent block of peak sodium-currents (INa) by VM11 and CI16, as well as Mex, VM11 and CI16 were 3 and 6-fold more potent than Mex in producing a tonic-block of peak sodium-currents (INa), respectively. Interestingly, CI16 showed a 40-fold increase of potency with respect to Mex during high-frequency stimulation (10-Hz), resulting the strongest use-dependent Mex-like compound so far. The derivatives also behaved as inactivated channel blockers, however the voltage dependent block was modest. The experimental data fitted with the molecular-modeling simulation based on previously proposed interaction of main pharmacophores with NaV1.4 binding-site. CI16 and VM11 were then compared to Mex and its isopropyl derivative (Me5) for the ability to protect C2C12-cells from H2O2-cytotoxicity in the concentration range effective on Nav1.4. Mex and Me5 showed a moderate cyto-protective effect in the presence of H2O2, Importantly, CI16 and VM11 showed a remarkable cyto-protection at concentrations effective for use-dependent block of NaV1.4. This effect was comparable to that of selected anti-oxidant drugs proved to exert protective effect in preclinical models of progressive myopathies such as muscular dystrophies. Then, the tetramethyl-pyrroline compounds have increased therapeutic profile as sodium channel blockers and an interesting cyto-protective activity. The overall profile enlarges therapeutic potential from channelopathies to myopathies in which alteration of excitation-contraction coupling is paralleled by oxidative-stress, i.e., muscular dystrophies.
RESUMO
Statin-induced skeletal muscle damage in rats is associated to the reduction of the resting sarcolemmal chloride conductance (gCl) and ClC-1 chloride channel expression. These drugs also affect the ClC-1 regulation by increasing protein kinase C (PKC) activity, which phosphorylate and close the channel. Also the intracellular resting calcium (restCa) level is increased. Similar alterations are observed in skeletal muscles of aged rats, suggesting a higher risk of statin myotoxicity. To verify this hypothesis, we performed a 4-5-weeks atorvastatin treatment of 24-months-old rats to evaluate the ClC-1 channel function by the two-intracellular microelectrodes technique as well as transcript and protein expression of different genes sensitive to statins by quantitative real-time-PCR and western blot analysis. The restCa was measured using FURA-2 imaging, and histological analysis of muscle sections was performed. The results show a marked reduction of resting gCl, in agreement with the reduced ClC-1 mRNA and protein expression in atorvastatin-treated aged rats, with respect to treated adult animals. The observed changes in myocyte-enhancer factor-2 (MEF2) expression may be involved in ClC-1 expression changes. The activity of PKC was also increased and further modulate the gCl in treated aged rats. In parallel, a marked reduction of the expression of glycolytic and mitochondrial enzymes demonstrates an impairment of muscle metabolism. No worsening of restCa or histological features was found in statin-treated aged animals. These findings suggest that a strong reduction of gCl and alteration of muscle metabolism coupled to muscle atrophy may contribute to the increased risk of statin-induced myopathy in the elderly.
Assuntos
Envelhecimento/fisiologia , Atorvastatina/efeitos adversos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Atrofia Muscular/induzido quimicamente , Envelhecimento/metabolismo , Animais , Atorvastatina/sangue , Atorvastatina/farmacocinética , Cálcio/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Creatina Quinase/sangue , Inibidores de Hidroximetilglutaril-CoA Redutases/sangue , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Fatores de Transcrição MEF2 , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologia , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Ratos WistarRESUMO
OBJECTIVE: We performed a clinical, functional, and pharmacologic characterization of the novel p.P1158L Nav1.4 mutation identified in a young girl presenting a severe myotonic phenotype. METHODS: Wild-type hNav1.4 channel and P1158L mutant were expressed in tsA201 cells for functional and pharmacologic studies using patch-clamp. RESULTS: The patient shows pronounced myotonia, slowness of movements, and generalized muscle hypertrophy. Because of general discomfort with mexiletine, she was given flecainide with satisfactory response. In vitro, mutant channels show a slower current decay and a rightward shift of the voltage dependence of fast inactivation. The voltage dependence of activation and slow inactivation were not altered. Mutant channels were less sensitive to mexiletine, whereas sensitivity to flecainide was not altered. The reduced inhibition of mutant channels by mexiletine was also observed using clinically relevant drug concentrations in a myotonic-like condition. CONCLUSIONS: Clinical phenotype and functional alterations of P1158L support the diagnosis of myotonia permanens. Impairment of fast inactivation is consistent with the possible role of the channel domain III S4-S5 loop in the inactivation gate docking site. The reduced sensitivity of P1158L to mexiletine may have contributed to the unsatisfactory response of the patient. The success of flecainide therapy underscores the usefulness of in vitro functional studies to help in the choice of the best drug for each individual.
Assuntos
Mutação , Miotonia Congênita/tratamento farmacológico , Miotonia Congênita/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêutico , Linhagem Celular , Criança , Diagnóstico Diferencial , Feminino , Flecainida/farmacologia , Flecainida/uso terapêutico , Humanos , Mexiletina/efeitos adversos , Mexiletina/farmacologia , Mexiletina/uso terapêutico , Miotonia Congênita/diagnóstico , Miotonia Congênita/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Testes Farmacogenômicos/métodos , Medicina de Precisão/métodos , Pesquisa Translacional Biomédica , Bloqueadores do Canal de Sódio Disparado por Voltagem/efeitos adversos , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologiaRESUMO
Muscle atrophy is a widespread ill condition occurring in many diseases, which can reduce quality of life and increase morbidity and mortality. We developed a new method using non-invasive ultrasonography to measure soleus and gastrocnemius lateralis muscle atrophy in the hindlimb-unloaded rat, a well-accepted model of muscle disuse. Soleus and gastrocnemius volumes were calculated using the conventional truncated-cone method and a newly-designed sinusoidal method. For Soleus muscle, the ultrasonographic volume determined in vivo with either method was linearly correlated to the volume determined ex-vivo from excised muscles as muscle weight-to-density ratio. For both soleus and gastrocnemius muscles, a strong linear correlation was obtained between the ultrasonographic volume and the muscle fiber cross-sectional area determined ex-vivo on muscle cryosections. Thus ultrasonography allowed the longitudinal in vivo evaluation of muscle atrophy progression during hindlimb unloading. This study validates ultrasonography as a powerful method for the evaluation of rodent muscle atrophy in vivo, which would prove useful in disease models and therapeutic trials.
Assuntos
Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/fisiopatologia , Atrofia Muscular/diagnóstico por imagem , Atrofia Muscular/fisiopatologia , Ultrassonografia , Animais , Masculino , Ratos , Ratos WistarRESUMO
Muscle disuse produces severe atrophy and a slow-to-fast phenotype transition in the postural Soleus (Sol) muscle of rodents. Antioxidants, amino-acids and growth factors were ineffective to ameliorate muscle atrophy. Here we evaluate the effects of nandrolone (ND), an anabolic steroid, on mouse skeletal muscle atrophy induced by hindlimb unloading (HU). Mice were pre-treated for 2-weeks before HU and during the 2-weeks of HU. Muscle weight and total protein content were reduced in HU mice and a restoration of these parameters was found in ND-treated HU mice. The analysis of gene expression by real-time PCR demonstrates an increase of MuRF-1 during HU but minor involvement of other catabolic pathways. However, ND did not affect MuRF-1 expression. The evaluation of anabolic pathways showed no change in mTOR and eIF2-kinase mRNA expression, but the protein expression of the eukaryotic initiation factor eIF2 was reduced during HU and restored by ND. Moreover we found an involvement of regenerative pathways, since the increase of MyoD observed after HU suggests the promotion of myogenic stem cell differentiation in response to atrophy. At the same time, Notch-1 expression was down-regulated. Interestingly, the ND treatment prevented changes in MyoD and Notch-1 expression. On the contrary, there was no evidence for an effect of ND on the change of muscle phenotype induced by HU, since no effect of treatment was observed on the resting gCl, restCa and contractile properties in Sol muscle. Accordingly, PGC1α and myosin heavy chain expression, indexes of the phenotype transition, were not restored in ND-treated HU mice. We hypothesize that ND is unable to directly affect the phenotype transition when the specialized motor unit firing pattern of stimulation is lacking. Nevertheless, through stimulation of protein synthesis, ND preserves protein content and muscle weight, which may result advantageous to the affected skeletal muscle for functional recovery.
Assuntos
Atrofia Muscular/tratamento farmacológico , Nandrolona/farmacologia , Anabolizantes/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Cálcio/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Membro Posterior/efeitos dos fármacos , Membro Posterior/fisiopatologia , Contração Isométrica/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Atrofia Muscular/genética , Atrofia Muscular/fisiopatologia , Biossíntese de Proteínas/efeitos dos fármacosRESUMO
Myotonia congenita (MC) is an inherited muscle disease characterized by impaired muscle relaxation after contraction, resulting in muscle stiffness. Both recessive (Becker's disease) or dominant (Thomsen's disease) MC are caused by mutations in the CLCN1 gene encoding the voltage-dependent chloride ClC-1 channel, which is quite exclusively expressed in skeletal muscle. More than 200 CLCN1 mutations have been associated with MC. We provide herein a detailed clinical, molecular, and functional evaluation of four patients with recessive MC belonging to three different families. Four CLCN1 variants were identified, three of which have never been characterized. The c.244A>G (p.T82A) and c.1357C>T (p.R453W) variants were each associated in compound heterozygosity with c.568GG>TC (p.G190S), for which pathogenicity is already known. The new c.809G>T (p.G270V) variant was found in the homozygous state. Patch-clamp studies of ClC-1 mutants expressed in tsA201 cells confirmed the pathogenicity of p.G270V, which greatly shifts the voltage dependence of channel activation toward positive potentials. Conversely, the mechanisms by which p.T82A and p.R453W cause the disease remained elusive, as the mutated channels behave similarly to WT. The results also suggest that p.G190S does not exert dominant-negative effects on other mutated ClC-1 subunits. Moreover, we performed a RT-PCR quantification of selected ion channels transcripts in muscle biopsies of two patients. The results suggest gene expression alteration of sodium and potassium channel subunits in myotonic muscles; if confirmed, such analysis may pave the way toward a better understanding of disease phenotype and a possible identification of new therapeutic options.
Assuntos
Canais de Cloreto/genética , Miotonia Congênita/genética , Mutação Puntual , Adulto , Linhagem Celular , Canais de Cloreto/química , Canais de Cloreto/fisiologia , Cloretos/metabolismo , Consanguinidade , Sequência Conservada , Éxons/genética , Feminino , Genes Recessivos , Estudos de Associação Genética , Humanos , Ativação do Canal Iônico , Masculino , Músculo Esquelético/patologia , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Adulto JovemRESUMO
Metaglidasen is a fibrate-like drug reported as a selective modulator of peroxisome proliferator-activated receptorâ γ (PPARγ), able to lower plasma glucose levels in the absence of the side effects typically observed with thiazolidinedione antidiabetic agents in current use. Herein we report an improved synthesis of metaglidasen's metabolically active form halofenic acid (R)-2 and that of its enantiomer (S)-2. The activity of the two stereoisomers was carefully examined on PPARα and PPARγ subtypes. As expected, both showed partial agonist activity toward PPARγ; the investigation of PPARα activity, however, led to unexpected results. In particular, (S)-2 was found to act as a partial agonist, whereas (R)-2 behaved as an antagonist. X-ray crystallographic studies with PPARγ were carried out to gain more insight on the molecular-level interactions and to propose a binding mode. Given the adverse effects provoked by fibrate drugs on skeletal muscle function, we also investigated the capacity of (R)-2 and (S)-2 to block conductance of the skeletal muscle membrane chloride channel. The results showed a more beneficial profile for (R)-2, the activity of which on skeletal muscle function, however, should not be overlooked in the ongoing clinical trials studying its long-term effects.
Assuntos
Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Músculo Esquelético/efeitos dos fármacos , PPAR alfa/metabolismo , PPAR gama/metabolismo , Animais , Cristalografia por Raios X , Células Hep G2 , Humanos , Ligantes , Masculino , Simulação de Acoplamento Molecular , Músculo Esquelético/metabolismo , PPAR alfa/agonistas , PPAR alfa/antagonistas & inibidores , PPAR gama/agonistas , PPAR gama/antagonistas & inibidores , Ratos WistarRESUMO
Hypertension is often associated with skeletal muscle pathological conditions related to function and metabolism. The mechanisms underlying the development of these pathological conditions remain undefined. Because calcium homeostasis is a biomarker of muscle function, we assessed whether it is altered in hypertensive muscles. We measured resting intracellular calcium and store-operated calcium entry (SOCE) in fast- and slow-twitch muscle fibers from normotensive Wistar-Kyoto rats and spontaneously hypertensive rats (SHRs) by cytofluorimetric technique and determined the expression of SOCE gene machinery by real-time PCR. Hypertension caused a phenotype-dependent dysregulation of calcium homeostasis; the resting intracellular calcium of extensor digitorum longus and soleus muscles of SHRs were differently altered with respect to the related muscle of normotensive animals. In addition, soleus muscles of SHR showed reduced activity of the sarcoplasmic reticulum and decreased sarcolemmal calcium permeability at rest and after SOCE activation. Accordingly, we found an alteration of the expression levels of some SOCE components, such as stromal interaction molecule 1, calcium release-activated calcium modulator 1, and transient receptor potential canonical 1. The hypertension-induced alterations of calcium homeostasis in the soleus muscle of SHRs occurred with changes of some functional outcomes as excitability and resting chloride conductance. We provide suitable targets for therapeutic interventions aimed at counterbalancing muscle performance decline in hypertension, and propose the reported calcium-dependent parameters as indexes to predict how the antihypertensive drugs could influence muscle function.
Assuntos
Cálcio/metabolismo , Hipertensão/fisiopatologia , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/metabolismo , Animais , Cafeína/metabolismo , Cálcio/análise , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Homeostase , Humanos , Masculino , Contração Muscular/fisiologia , Fibras Musculares de Contração Lenta/fisiologia , Músculo Esquelético/fisiologia , Fenótipo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Although the sodium channel blocker mexiletine is considered the first-line drug in myotonia, some patients experiment adverse effects, while others do not gain any benefit. Other antimyotonic drugs are thus needed to offer mexiletine alternatives. In the present study, we used a previously-validated rat model of myotonia congenita to compare six marketed sodium channel blockers to mexiletine. Myotonia was induced in the rat by injection of anthracen-9-carboxylic acid, a muscle chloride channel blocker. The drugs were given orally and myotonia was evaluated by measuring the time of righting reflex. The drugs were also tested on sodium currents recorded in a cell line transfected with the human skeletal muscle sodium channel hNav1.4 using patch-clamp technique. In vivo, carbamazepine and propafenone showed antimyotonic activity at doses similar to mexiletine (ED50 close to 5mg/kg); flecainide and orphenadrine showed greater potency (ED50 near 1mg/kg); lubeluzole and riluzole were the more potent (ED50 near 0.1mg/kg). The antimyotonic activity of drugs in vivo was linearly correlated with their potency in blocking hNav1.4 channels in vitro. Deviation was observed for propafenone and carbamazepine, likely due to pharmacokinetics and multiple targets. The comparison of the antimyotonic dose calculated in rats with the current clinical dose in humans strongly suggests that all the tested drugs may be used safely for the treatment of human myotonia. Considering the limits of mexiletine tolerability and the occurrence of non-responders, this study proposes an arsenal of alternative drugs, which may prove useful to increase the quality of life of individuals suffering from non-dystrophic myotonia. Further clinical trials are warranted to confirm these results.
Assuntos
Mexiletina/uso terapêutico , Músculo Esquelético/efeitos dos fármacos , Miotonia Congênita/tratamento farmacológico , Bloqueadores dos Canais de Sódio/uso terapêutico , Animais , Carbamazepina/farmacologia , Carbamazepina/uso terapêutico , Modelos Animais de Doenças , Flecainida/farmacologia , Flecainida/uso terapêutico , Células HEK293 , Humanos , Mexiletina/farmacologia , Orfenadrina/farmacologia , Orfenadrina/uso terapêutico , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Propafenona/farmacologia , Propafenona/uso terapêutico , Ratos , Ratos Wistar , Riluzol/farmacologia , Riluzol/uso terapêutico , Bloqueadores dos Canais de Sódio/farmacologia , Tiazóis/farmacologia , Tiazóis/uso terapêuticoRESUMO
On the basis of a 3D-QSAR study, a new generation of tocainide analogues were designed and synthesized as voltage-gated skeletal muscle sodium channel blockers. Data obtained by screening new compounds by means of Hille-Campbell Vaseline gap voltage-clamp recordings showed that the elongation of the alkyl chain and the introduction of lipophilic and sterically hindered groups on the amino function enhance both potency and use-dependent block. The results provide additional indications about the structural requirement of pharmacophores for further increasing potency and state-dependent block and allowed us to identify a new tocainide analogue (6f) with a favorable pharmacodynamic profile to be proposed as a valid candidate for studies aimed at evaluating its usefulness in the treatment of myotonias.
Assuntos
Antiarrítmicos/farmacologia , Músculo Esquelético/efeitos dos fármacos , Tocainide/análogos & derivados , Tocainide/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/síntese química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Antiarrítmicos/síntese química , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Humanos , Modelos Moleculares , Técnicas de Patch-Clamp , Ligação Proteica , Relação Quantitativa Estrutura-Atividade , Ratos , Albumina Sérica/metabolismo , Canais de Sódio/efeitos dos fármacos , Relação Estrutura-Atividade , Tocainide/síntese químicaRESUMO
Myotonia congenita (MC) is caused by loss-of-function mutations of the muscle ClC-1 chloride channel. Clinical manifestations include the variable association of myotonia and transitory weakness. We recently described a cohort of recessive MC patients showing, at a low rate repetitive nerves stimulation protocol, different values of compound muscle action potential (CMAP) transitory depression, which is considered the neurophysiologic counterpart of transitory weakness. From among this cohort, we studied the chloride currents generated by G190S (associated with pronounced transitory depression), F167L (little or no transitory depression), and A531V (variable transitory depression) hClC-1 mutants in transfected HEK293 cells using patch-clamp. While F167L had no effect on chloride currents, G190S dramatically shifts the voltage dependence of channel activation and A531V reduces channel expression. Such variability in molecular mechanisms observed in the hClC-1 mutants may help to explain the different clinical and neurophysiologic manifestations of each ClCN1 mutation. In addition we examined five different mutations found in compound heterozygosis with F167L, including the novel P558S, and we identified additional molecular defects. Finally, the G190S mutation appeared to impair acetazolamide effects on chloride currents in vitro.
Assuntos
Canais de Cloreto/genética , Mutação , Miotonia Congênita/genética , Fenótipo , Potenciais de Ação/fisiologia , Canais de Cloreto/metabolismo , Células HEK293 , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Miotonia Congênita/metabolismo , Miotonia Congênita/fisiopatologiaRESUMO
Emerging evidences suggest that Ca(2+)activated-K(+)-(BK) channel is involved in the regulation of cell viability. The changes of the cell viability observed under hyperkalemia (15 mEq/L) or hypokalemia (0.55 mEq/L) conditions were investigated in HEK293 cells expressing the hslo subunit (hslo-HEK293) in the presence or absence of BK channel modulators. The BK channel openers(10(-11)-10(-3)M) were: acetazolamide(ACTZ), Dichlorphenamide(DCP), methazolamide(MTZ), bendroflumethiazide(BFT), ethoxzolamide(ETX), hydrochlorthiazide(HCT), quercetin(QUERC), resveratrol(RESV) and NS1619; and the BK channel blockers(2 x 10(-7)M-5 x 10(-3)M) were: tetraethylammonium(TEA), iberiotoxin(IbTx) and charybdotoxin(ChTX). Experiments on cell viability and channel currents were performed using cell counting kit-8 and patch-clamp techniques, respectively. Hslo whole-cell current was potentiated by BK channel openers with different potency and efficacy in hslo-HEK293. The efficacy ranking of the openers at -60 mV(Vm) was BFT> ACTZ >DCP ≥RESV≥ ETX> NS1619> MTZ≥ QUERC; HCT was not effective. Cell viability after 24 h of incubation under hyperkalemia was enhanced by 82+6% and 33+7% in hslo-HEK293 cells and HEK293 cells, respectively. IbTx, ChTX and TEA enhanced cell viability in hslo-HEK293. BK openers prevented the enhancement of the cell viability induced by hyperkalemia or IbTx in hslo-HEK293 showing an efficacy which was comparable with that observed as BK openers. BK channel modulators failed to affect cell currents and viability under hyperkalemia conditions in the absence of hslo subunit. In contrast, under hypokalemia cell viability was reduced by -22+4% and -23+6% in hslo-HEK293 and HEK293 cells, respectively; the BK channel modulators failed to affect this parameter in these cells. In conclusion, BK channel regulates cell viability under hyperkalemia but not hypokalemia conditions. BFT and ACTZ were the most potent drugs either in activating the BK current and in preventing the cell proliferation induced by hyperkalemia. These findings may have relevance in disorders associated with abnormal K(+) ion homeostasis including periodic paralysis and myotonia.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/metabolismo , Potássio/metabolismo , Bendroflumetiazida/farmacologia , Linhagem Celular , Charibdotoxina/farmacologia , Diclorofenamida/farmacologia , Etoxzolamida/farmacologia , Humanos , Metazolamida/farmacologia , Peptídeos/farmacologia , Canais de Potássio Cálcio-Ativados/agonistas , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Tetraetilamônio/farmacologiaRESUMO
The sodium channel blocker mexiletine is considered the first-line drug in myotonic syndromes, a group of muscle disorders characterized by membrane over-excitability. We previously showed that the ß-adrenoceptor modulators, clenbuterol and propranolol, block voltage-gated sodium channels in a manner reminiscent to mexiletine, whereas salbutamol and nadolol do not. We now developed a pharmacological rat model of myotonia congenita to perform in vivo preclinical test of antimyotonic drugs. Myotonia was induced by i.p. injection of 30 mg/kg of anthracene-9-carboxylic acid (9-AC), a muscle chloride channel blocker, and evaluated by measuring the time of righting reflex (TRR). The TRR was prolonged from <0.5 s in control conditions to a maximum of â¼4 s, thirty minutes after 9-AC injection, then gradually recovered in a few hours. Oral administration of mexiletine twenty minutes after 9-AC injection significantly hampered the TRR prolongation, with an half-maximum efficient dose (ED(50)) of 12 mg/kg. Both propranolol and clenbuterol produced a dose-dependent antimyotonic effect similar to mexiletine, with ED(50) values close to 20 mg/kg. Antimyotonic effects of 40 mg/kg mexiletine and propranolol lasted for 2 h. We also demonstrated, using patch-clamp methods, that both propranolol enantiomers exerted a similar block of skeletal muscle hNav1.4 channels expressed in HEK293 cells. The two enantiomers (15 mg/kg) also showed a similar antimyotonic activity in vivo in the myotonic rat. Among the drugs tested, the R(+)-enantiomer of propranolol may merit further investigation in humans, because it exerts antimyotonic effect in the rat model, while lacking of significant activity on the ß-adrenergic pathway. This study provides a new and useful in vivo preclinical model of myotonia congenita in order to individuate the most promising antimyotonic drugs to be tested in humans.
Assuntos
Agonistas Adrenérgicos beta/uso terapêutico , Antagonistas Adrenérgicos beta/uso terapêutico , Modelos Animais de Doenças , Miotonia/tratamento farmacológico , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293 , Humanos , Miotonia/fisiopatologia , Distribuição Aleatória , Ratos , Ratos Wistar , Resultado do TratamentoRESUMO
The molecular composition and drug responses of calcium-activated K(+) (BK) channels of skeletal muscle are unknown. Patch-clamp experiments combined with transcript scanning of the Kcnma1 gene encoding the alpha subunit of the BK channel were performed in rat slow-twitch soleus (Sol) and fast-twitch flexor digitorum brevis (FDB) skeletal muscles. Five splicing products of the Kcnma1 gene were isolated from Sol and FDB: the e17, e22, +29 aa, Slo27 and Slo0 variants. RT-PCR analysis demonstrated that the expression of e22 and Slo0 were 80-90% higher in FDB than Sol, whereas the expression of Slo27 was 60% higher in Sol than FDB, and the +29 aa variant was equally expressed in both muscle types. No beta 1-4 subunits were detected. In Sol, a large BK current with low Ca(2+) sensitivity was recorded. The BK channel of Sol also showed a reduced response to BK channel openers, such as NS1619, acetazolamide and related drugs. In FDB, a reduced BK current with high Ca(2+) sensitivity and an enhanced drug response was recorded. The total BK RNA content, which was 200% higher in Sol than in FDB, correlated with the BK currents in both muscles. Drug responses primarily correlated with e22 and Slo0 expression levels in FDB and to Slo27 expression in Sol muscle. In conclusion, phenotype-dependent BK channel biophysical and pharmacological properties correlated with the expression levels of the variants in muscles. These findings may be relevant to conditions affecting postural muscles, such as prolonged bed-rest, and to diseases affecting fast-twitch muscles, such as periodic paralysis. Down-regulation or up-regulation of the variants associated with pathological conditions may affect channel composition and drug responses.
Assuntos
Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Moduladores de Transporte de Membrana/farmacologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Splicing de RNA/genética , Acetazolamida/farmacologia , Sequência de Aminoácidos , Animais , Benzimidazóis/farmacologia , Cálcio/metabolismo , Perfilação da Expressão Gênica , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Dados de Sequência Molecular , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/efeitos dos fármacos , Fibras Musculares de Contração Lenta/metabolismo , Proteínas Mutantes/metabolismo , Técnicas de Patch-Clamp , Fenótipo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Sítios de Splice de RNA/genética , Splicing de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The 2H-1,4-benzoxazine derivatives are novel drugs structurally similar to nucleotides; however, their actions on the pancreatic ß cell ATP-sensitive K+ (KATP) channel and on glucose disposal are unknown. Therefore, the effects of the linear/branched alkyl substituents and the aliphatic/aromatic rings at position 2 of the 2H-1,4-benzoxazine nucleus on the activity of these molecules against the pancreatic ß cell KATP channel and the Kir6.2ΔC36 subunit were investigated using a patch-clamp technique. The effects of these compounds on glucose disposal that followed glucose loading by intraperitoneal glucose tolerance test and on fasting glycemia were investigated in normal mice. The 2-n-hexyl analog blocked the KATP (IC50 = 10.1 × 10â»9 M) and Kir6.2ΔC36 (IC50 = 9.6 × 10â»9 M) channels, which induced depolarization. In contrast, the 2-phenyl analog was a potent opener (drug concentration needed to enhance the current by 50% = 0.04 × 10â»9 M), which induced hyperpolarization. The ranked order of the potency/efficacy of the analog openers was 2-phenyl > 2-benzyl > 2-cyclohexylmethyl. The 2-phenylethyl and 2-isopropyl analogs were not effective as blockers/openers. The 2-n-hexyl (2-10 mg/kg) and 2-phenyl analogs (2-30 mg/kg) reduced and enhanced the glucose areas under the curves, respectively, after glucose loading in mice. These compounds did not affect the fasting glycemia as is observed with glibenclamide. The linear alkyl chain and the aromatic ring at position 2 of the 1,4-benzoxazine nucleus are the determinants, which confer the KATP channel blocking action with glucose-lowering effects and the opening action with increased glucose levels, respectively. The opening/blocking actions of these compounds mimic those that were observed with ATP and ADP. The results support the use of these compounds as novel antidiabetic drugs.
Assuntos
Benzoxazinas/farmacologia , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Canais KATP/agonistas , Canais KATP/antagonistas & inibidores , Animais , Área Sob a Curva , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Relação Dose-Resposta a Droga , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Fenômenos Eletrofisiológicos/fisiologia , Jejum/sangue , Glucose/farmacologia , Teste de Tolerância a Glucose , Glibureto/farmacologia , Células HEK293 , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Canais KATP/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , Técnicas de Patch-Clamp , Canais de Potássio Corretores do Fluxo de Internalização/agonistas , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Azida Sódica/farmacologia , Tolbutamida/farmacologiaRESUMO
The first synthesis of m-hydroxymexiletine (MHM) has been accomplished. MHM displayed hNav1.5 sodium channel blocking activity, and tests indicate it to be â¼2-fold more potent than the parent mexiletine and to have more favorable toxicological properties than mexiletine. Thus, MHM and possible related prodrugs might be studied as agents for the treatment of arrhythmias, neuropathic pain, and myotonias in substitution of mexiletine (metabolite switch), which has turned out to be tainted with common toxicity.
Assuntos
Antiarrítmicos/síntese química , Mexiletina/análogos & derivados , Mexiletina/metabolismo , Canais de Sódio/fisiologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antiarrítmicos/metabolismo , Antiarrítmicos/toxicidade , Aorta/efeitos dos fármacos , Aorta/fisiologia , Ataxia/induzido quimicamente , Barreira Hematoencefálica/metabolismo , Células CACO-2 , Cobaias , Células HEK293 , Coração/efeitos dos fármacos , Coração/fisiologia , Frequência Cardíaca/efeitos dos fármacos , Humanos , Ativação do Canal Iônico , Mexiletina/síntese química , Mexiletina/toxicidade , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.5 , Permeabilidade , Estereoisomerismo , Vasodilatadores/síntese química , Vasodilatadores/toxicidadeRESUMO
[2-(2-Aminopropoxy)-1,3-phenylene]dimethanol 1 and 4-(2-aminopropoxy)-3-(hydroxymethyl)-5-methylphenol 2, two dihydroxylated analogs of mexiletine - a well known class IB anti-arrhythmic drug - were synthesized and used as pharmacological tools to investigate the blocking-activity requirements of human skeletal muscle, voltage-gated sodium channel. The very low blocking activity shown by newly synthesized compounds corroborates the hypothesis that the presence of a phenolic group in the para-position to the aromatic moiety and/or benzylic hydroxyl groups on the aromatic moiety of local anesthetic-like drugs impairs either the transport to or the interaction with the binding site in the pore of Na(+) channels.
Assuntos
Antiarrítmicos/farmacologia , Mexiletina/análogos & derivados , Mexiletina/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismo , Antiarrítmicos/síntese química , Antiarrítmicos/química , Sítios de Ligação , Linhagem Celular , Desenho de Fármacos , Humanos , Hidroxilação , Mexiletina/síntese química , Mexiletina/química , Músculo Esquelético/metabolismo , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Canais de Sódio/genética , Relação Estrutura-AtividadeRESUMO
New chiral mexiletine analogs were synthesized in their optically active forms and evaluated in vitro as use-dependent blockers of skeletal muscle sodium channels. Tests carried out on sodium currents of single muscle fibers of Rana esculenta demonstrated that all of them exerted a higher use-dependent block than mexiletine. The most potent analog, (S)-3-(2,6-dimethylphenoxy)-1-phenylpropan-1-amine (S)-(5), was six-fold more potent than (R)-Mex in producing a tonic block. As observed with mexiletine, the newly synthesized compounds exhibit modest enantioselective behavior, that is more evident in 3-(2,6-dimethylphenoxy)butan-1-amine (3).
Assuntos
Mexiletina/síntese química , Mexiletina/farmacologia , Músculos/efeitos dos fármacos , Miotonia/tratamento farmacológico , Bloqueadores dos Canais de Sódio/farmacologia , Animais , Mexiletina/análogos & derivadosRESUMO
The preparation of a new series of 2-aryloxy-3-phenyl-propanoic acids, resulting from the introduction of a linker into the diphenyl system of the previously reported PPARalpha/gamma dual agonist 1, allowed the identification of new ligands with improved potency on PPARalpha and unchanged activity on PPARgamma. For the most interesting stereoisomers S-2 and S-4, X-ray studies in PPARgamma and docking experiments in PPARalpha provided a molecular explanation for their different behavior as full and partial agonists of PPARalpha and PPARgamma, respectively. Due to the adverse effects provoked by hypolipidemic drugs on skeletal muscle function, we also investigated the blocking activity of S-2 and S-4 on skeletal muscle membrane chloride channel conductance and found that these ligands have a pharmacological profile more beneficial compared to fibrates currently used in therapy.