RESUMEN
Cannabidiol (CBD) is a non-psychoactive component of Cannabis which has recently received regulatory consideration for the treatment of intractable forms of epilepsy such as the Dravet and the Lennox-Gastaut syndromes. The mechanisms of the antiepileptic effects of CBD are unclear, but several pre-clinical studies suggest the involvement of ion channels. Therefore, we have evaluated the effects of CBD on seven major cardiac currents shaping the human ventricular action potential and on Purkinje fibers isolated from rabbit hearts to assess the in vitro cardiac safety profile of CBD. We found that CBD inhibits with comparable micromolar potencies the peak and late components of the NaV1.5 sodium current, the CaV1.2 mediated L-type calcium current, as well as all the repolarizing potassium currents examined except Kir2.1. The most sensitive channels were KV7.1 and the least sensitive were KV11.1 (hERG), which underly the slow (IKs) and rapid (IKr) components, respectively, of the cardiac delayed-rectifier current. In the Purkinje fibers, CBD decreased the action potential (AP) duration more potently at half-maximal than at near complete repolarization, and slightly decreased the AP amplitude and its maximal upstroke velocity. CBD had no significant effects on the membrane resting potential except at the highest concentration tested under fast pacing rate. These data show that CBD impacts cardiac electrophysiology and suggest that caution should be exercised when prescribing CBD to carriers of cardiac channelopathies or in conjunction with other drugs known to affect heart rhythm or contractility.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Cannabidiol/farmacología , Ventrículos Cardíacos/efectos de los fármacos , Corazón/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Animales , Cannabidiol/toxicidad , Canalopatías/complicaciones , Humanos , Técnicas In Vitro , Canal de Potasio KCNQ1/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.5/efectos de los fármacos , Técnicas de Placa-Clamp , Ramos Subendocárdicos/efectos de los fármacos , ConejosRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Automated patch clamp (APC) instruments enable efficient evaluation of electrophysiologic effects of drugs on human cardiac currents in heterologous expression systems. Differences in experimental protocols, instruments, and dissimilar site procedures affect the variability of IC50 values characterizing drug block potency. This impacts the utility of APC platforms for assessing a drug's cardiac safety margin. We determined variability of APC data from multiple sites that measured blocking potency of 12 blinded drugs (with different levels of proarrhythmic risk) against four human cardiac currents (hERG [IKr], hCav1.2 [L-Type ICa], peak hNav1.5, [Peak INa], late hNav1.5 [Late INa]) with recommended protocols (to minimize variance) using five APC platforms across 17 sites. IC50 variability (25/75 percentiles) differed for drugs and currents (e.g., 10.4-fold for dofetilide block of hERG current and 4-fold for mexiletine block of hNav1.5 current). Within-platform variance predominated for 4 of 12 hERG blocking drugs and 4 of 6 hNav1.5 blocking drugs. hERG and hNav1.5 block. Bland-Altman plots depicted varying agreement across APC platforms. A follow-up survey suggested multiple sources of experimental variability that could be further minimized by stricter adherence to standard protocols. Adoption of best practices would ensure less variable APC datasets and improved safety margins and proarrhythmic risk assessments.
RESUMEN
Heterologous expression and lesioning studies were conducted to identify possible subunit assembly partners in nicotinic acetylcholine receptors (nAChR) containing alpha6 subunits (alpha6(*) nAChR). SH-EP1 human epithelial cells were transfected with the requisite subunits to achieve stable expression of human alpha6beta2, alpha6beta4, alpha6beta2beta3, alpha6beta4beta3, or alpha6beta4beta3alpha5 nAChR. Cells expressing subunits needed to form alpha6beta4beta3alpha5 nAChR exhibited saturable [(3)H]epibatidine binding (K(d) = 95.9 +/- 8.3 pM and B(max) = 84.5 +/- 1.6 fmol/mg of protein). The rank order of binding competition potency (K(i)) for prototypical nicotinic compounds was alpha-conotoxin MII (6 nM) > nicotine (156 nM) approximately methyllycaconitine (200 nM) > alpha-bungarotoxin (>10 microM), similar to that for nAChR in dopamine neurons displaying a distinctive pharmacology. 6-Hydroxydopamine lesioning studies indicated that beta3 and alpha5 subunits are likely partners of the alpha6 subunits in nAChR expressed in dopaminergic cell bodies. Similar to findings in rodents, quantitative real-time reverse transcription-polymerase chain reactions of human brain indicated that alpha6 subunit mRNA expression was 13-fold higher in the substantia nigra than in the cortex or the rest of the brain. Thus, heterologous expression studies suggest that the human alpha5 subunit makes a critical contribution to alpha6beta4beta3alpha5 nAChR assembly into a ligand-binding form with native alpha6(*)-nAChR-like pharmacology and of potential physiological and pathophysiological relevance.
Asunto(s)
Receptores Nicotínicos/biosíntesis , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Línea Celular , Membrana Celular/metabolismo , ADN Complementario/biosíntesis , ADN Complementario/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Humanos , Masculino , ARN/biosíntesis , ARN/aislamiento & purificación , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Relación Estructura-Actividad , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismoRESUMEN
Mutations of the parkin gene are the most frequent cause of early onset autosomal recessive parkinsonism (EO-AR). Here we show that inactivation of the parkin gene in mice results in motor and cognitive deficits, inhibition of amphetamine-induced dopamine release and inhibition of glutamate neurotransmission. The levels of dopamine are increased in the limbic brain areas of parkin mutant mice and there is a shift towards increased metabolism of dopamine by MAO. Although there was no evidence for a reduction of nigrostriatal dopamine neurons in the parkin mutant mice, the level of dopamine transporter protein was reduced in these animals, suggesting a decreased density of dopamine terminals, or adaptative changes in the nigrostriatal dopamine system. GSH levels were increased in the striatum and fetal mesencephalic neurons from parkin mutant mice, suggesting that a compensatory mechanism may protect dopamine neurons from neuronal death. These parkin mutant mice provide a valuable tool to better understand the preclinical deficits observed in patients with PD and to characterize the mechanisms leading to the degeneration of dopamine neurons that could provide new strategies for neuroprotection.
Asunto(s)
Conducta Animal/efectos de los fármacos , Dopamina/metabolismo , Silenciador del Gen , Inhibidores de la Captación de Neurotransmisores/metabolismo , Ubiquitina-Proteína Ligasas/genética , Alelos , Animales , Secuencia de Bases , Temperatura Corporal/genética , Peso Corporal/genética , Catecolaminas/antagonistas & inhibidores , Células Cultivadas , Dopamina/farmacocinética , Inhibidores Enzimáticos/farmacología , Exones , Femenino , Homocigoto , Intrones , Masculino , Ratones , Ratones Transgénicos , Monoaminooxidasa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Eliminación de Secuencia , Ubiquitina-Proteína Ligasas/metabolismo , alfa-Metiltirosina/farmacologíaRESUMEN
The effects of a chronic treatment with the anti-glutamate and sodium channel modulating neuroprotective agent riluzole on the degeneration of dopamine-containing neurons were studied in the brain of weaver mutant mice. In these animals, as in Parkinson's disease, dopaminergic neurons of the nigro-striatal pathway undergo spontaneous and progressive cell death. Homozygous weaver mice were orally treated twice a day with either 8 mg/kg riluzole or placebo for 2 months. Quantification of tyrosine-hydroxylase and dopamine-transporter axonal immunostaining in the striatum revealed that riluzole significantly increased the density of striatal dopaminergic nerve terminals. These results suggest that riluzole protects dopaminergic processes in the weaver mice and/or promotes their neuroplasticity.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Glicoproteínas de Membrana , Proteínas del Tejido Nervioso , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson/tratamiento farmacológico , Canales de Potasio de Rectificación Interna , Riluzol/farmacología , Sustancia Negra/efectos de los fármacos , Animales , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Esquema de Medicación , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Homocigoto , Proteínas de Transporte de Membrana/biosíntesis , Ratones , Ratones Mutantes Neurológicos , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Canales de Potasio/genética , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/patología , Sustancia Negra/metabolismo , Sustancia Negra/patología , Tiempo , Tirosina 3-Monooxigenasa/biosíntesisRESUMEN
Excessive release of glutamate, a potent excitatory neurotransmitter, is thought to play an important role in a variety of acute and chronic neurological disorders, suggesting that excitatory amino acid antagonists may have broad therapeutic potential in neurology. Here, we describe the synthesis, pharmacological properties and neuroprotective activity of 9-carboxymethyl-imidazo-[1-2a]indeno[1-2e]pyrazin-4-one-2-carboxylic acid (RPR117824), an original selective AMPA antagonist. RPR117824 can be obtained through a six-step synthesis starting from (1-oxo-indan-4-yl) acetic acid, which has been validated on a gram-scale with an overall yield of 25%. Monosodium or disodium salts of the compound exhibit excellent solubility in saline (> or = 10 g/L), enabling intravenous administration. RPR117824 displays nanomolar affinity (IC(50)=18 nM) for AMPA receptors and competitive inhibition of electrophysiological responses mediated by AMPA receptors heterologously expressed in Xenopus oocytes (K(B)=5 nM) and native receptors in rat brain slices (IC(50)=0.36 microM). In in vivo testing, RPR117824 behaves as a powerful blocker of convulsions induced in mice or rats by supramaximal electroshock or chemoconvulsive agents such as pentylenetetrazole, bicuculline, isoniazide, strychnine, 4-aminopyridine and harmaline with half maximal effective doses ranging from 1.5 to 10 mg/kg following subcutaneous or intraperitoneal administration. In disease models in rats and gerbils, RPR117824 possesses significant neuroprotective activity in global and focal cerebral ischemia, and brain and spinal cord trauma.
Asunto(s)
Anticonvulsivantes/síntesis química , Imidazoles/farmacología , Fármacos Neuroprotectores/síntesis química , Pirazinas/farmacología , Receptores AMPA/antagonistas & inhibidores , Animales , Anticonvulsivantes/química , Anticonvulsivantes/farmacología , Lesiones Encefálicas/tratamiento farmacológico , Isquemia Encefálica/tratamiento farmacológico , Modelos Animales de Enfermedad , Gerbillinae , Imidazoles/síntesis química , Imidazoles/química , Masculino , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Oocitos , Unión Proteica , Pirazinas/síntesis química , Pirazinas/química , Ensayo de Unión Radioligante , Ratas , Daño por Reperfusión/tratamiento farmacológico , Convulsiones/tratamiento farmacológico , Compresión de la Médula Espinal/tratamiento farmacológico , Sinapsis/efectos de los fármacos , XenopusRESUMEN
Nitric oxide production in the cerebellum and induction of long-term potentiation (LTP) in the hippocampus have some characteristics in common: both phenomena are induced by activation of N-methyl-d-aspartate receptors and both are highly dependent on calcium-mediated processes. Here we provide evidence that endogenous nitric oxide production is necessary for synaptic plasticity in the CA1 hippocampus of the rat. LTP recorded in slices was blocked in a concentration-dependent manner by the nitric oxide synthase inhibitors l-NG-nitroarginine and l-NG-nitroarginine methyl ester, but l-NG-monomethylarginine was only marginally active. Bathing the slices with haemoglobin, a protein that scavenges nitric oxide, also resulted in a concentration-dependent blockade of LTP. Nitric oxide released locally from hydroxylamine produced a stable potentiation of synaptic transmission that was not additive with LTP induced by high-frequency stimulation. These results are fully consistent with the presumed retrograde messenger role of nitric oxide in LTP.