RESUMEN
The aim of this study was to evaluate the anti-edematogenic activity of X. americana L. (HEXA) hydroethanolic extract in ear edema models (acute and chronic) induced by croton oil and by different phlogistic agents (arachidonic acid, capsaicin, phenol and histamine), identifying the possible anti-edematogenic mechanism. HEXA demonstrated a significant anti-edematogenic effect at concentrations of 100-500⯵g/ear in ear edema induced by croton oil with higher inhibition of edema of 39.37. However, the concentrations of 100 and 200⯵g/ear were taken as a standard, demonstrating the effect in the chronic model induced by croton oil with inhibition of 61.62% and 48.74%. In the AA-induced ear edema model, HEXA showed inhibition of: 24.45% and 32.31%; capsaicin inhibition of 72.72% and 47.57%; phenol inhibition of 34% and 20.1%; and histamine inhibition of 31.8% and 21.62%. Then, the results were showed that HEXA demonstrated an anti-edematogenic effect in acute and chronic inflammation models, demonstrating a probable mechanism of action by the inhibition or modulation of key mediators of the inflammatory process. The chemical profile and presence of flavonoids guaranteeing a profile of activity similar to natural drugs that act or modulate the production of mediators of inflammations.
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Cromatografía Líquida de Alta Presión/métodos , Dermatitis/tratamiento farmacológico , Edema/tratamiento farmacológico , Olacaceae/química , Extractos Vegetales/uso terapéutico , Animales , Ácido Araquidónico/efectos adversos , Ácido Araquidónico/antagonistas & inhibidores , Capsaicina/efectos adversos , Capsaicina/antagonistas & inhibidores , Aceite de Crotón/toxicidad , Relación Dosis-Respuesta a Droga , Edema/inducido químicamente , Femenino , Histamina/efectos adversos , Antagonistas de los Receptores Histamínicos/uso terapéutico , Ratones , Fenol/efectos adversos , Fenol/antagonistas & inhibidoresRESUMEN
G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8-37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8-37-cholestanol, but not unconjugated CGRP8-37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8-37-cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund's adjuvant more effectively than unconjugated CGRP8-37 Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.
Asunto(s)
Proteína Similar al Receptor de Calcitonina/genética , Endocitosis/efectos de los fármacos , Endosomas/metabolismo , Nocicepción/fisiología , Dolor/fisiopatología , Transmisión Sináptica/efectos de los fármacos , Antagonistas Adrenérgicos/farmacología , Animales , Péptido Relacionado con Gen de Calcitonina/farmacología , Proteína Similar al Receptor de Calcitonina/antagonistas & inhibidores , Proteína Similar al Receptor de Calcitonina/metabolismo , Capsaicina/antagonistas & inhibidores , Capsaicina/farmacología , Colestanoles/farmacología , Clatrina/antagonistas & inhibidores , Clatrina/genética , Clatrina/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Endosomas/efectos de los fármacos , Formaldehído/antagonistas & inhibidores , Formaldehído/farmacología , Adyuvante de Freund/antagonistas & inhibidores , Adyuvante de Freund/farmacología , Regulación de la Expresión Génica , Inyecciones Espinales , Masculino , Ratones , Microtomía , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Nocicepción/efectos de los fármacos , Dolor/inducido químicamente , Dolor/genética , Dolor/prevención & control , Fragmentos de Péptidos/farmacología , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Ratas , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Beige adipocytes emerge postnatally within the white adipose tissue in response to certain environmental cues, such as chronic cold exposure. Because of its highly recruitable nature and relevance to adult humans, beige adipocytes have gained much attention as an attractive cellular target for antiobesity therapy. However, molecular circuits that preferentially promote beige adipocyte biogenesis remain poorly understood. We report that a combination of mild cold exposure at 17°C and capsinoids, a nonpungent analog of capsaicin, synergistically and preferentially promotes beige adipocyte biogenesis and ameliorates diet-induced obesity. Gain- and loss-of-function studies show that the combination of capsinoids and cold exposure synergistically promotes beige adipocyte development through the ß2-adrenoceptor signaling pathway. This synergistic effect on beige adipocyte biogenesis occurs through an increased half-life of PRDM16, a dominant transcriptional regulator of brown/beige adipocyte development. We document a previously unappreciated molecular circuit that controls beige adipocyte biogenesis and suggest a plausible approach to increase whole-body energy expenditure by combining dietary components and environmental cues.
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Aclimatación , Adipocitos Beige/fisiología , Adipogénesis , Fármacos Antiobesidad/uso terapéutico , Capsaicina/análogos & derivados , Suplementos Dietéticos , Obesidad/prevención & control , Adipocitos Beige/citología , Adipocitos Beige/efectos de los fármacos , Adipocitos Beige/patología , Adipogénesis/efectos de los fármacos , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Agonistas de Receptores Adrenérgicos beta 2/uso terapéutico , Antagonistas de Receptores Adrenérgicos beta 2/farmacología , Antagonistas de Receptores Adrenérgicos beta 2/toxicidad , Animales , Fármacos Antiobesidad/agonistas , Fármacos Antiobesidad/antagonistas & inhibidores , Capsaicina/agonistas , Capsaicina/antagonistas & inhibidores , Capsaicina/química , Capsaicina/uso terapéutico , Células Cultivadas , Frío , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Metabolismo Energético/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hidrogenación , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Obesidad/inducido químicamente , Obesidad/metabolismo , Obesidad/patología , Consumo de Oxígeno/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Distribución Aleatoria , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Evodiae fructus (EF), a fruit of Evodia rutaecarpa Bentham, has long been used as an analgesic drug in traditional Chinese and Japanese medicine. However, the underlying molecular mechanism of its pharmacological action is unclear. Here, using calcium imaging, whole-cell patch-clamp recording, and behavioral analysis, we investigated the pharmacological action of EF and its principal compound, evodiamine, on the transient receptor potential (TRP) V1 channels. Dorsal root ganglion (DRG) neurons and TRPV1- or TRPA1-transfected human embryonic kidney-derived (HEK) 293 cells were used for calcium imaging or whole-cell patch-clamp recording. Twenty male adult Sprague-Dawley rats were used for the capsaicin-induced thermal hyperalgesia behavioral analyses. We found that evodiamine induced significant increases in intracellular calcium and robust inward currents in a subpopulation of isolated rat DRG neurons, most of which were also sensitive to capsaicin. The effect of evodiamine was completely blocked by capsazepine, a competitive antagonist of TRPV1. Evodiamine induced significant inward currents in TRPV1-, but not TRPA1-transfected HEK293 cells. Pretreatment with evodiamine reduced capsaicin-induced currents significantly. Furthermore, the in vivo pre-treatment of evodiamine suppressed thermal hyperalgesia induced by intraplantar injection of capsaicin in rats. These results identify that the analgesic effect of EF and evodiamine may be due to the activation and subsequent desensitization of TRPV1 in sensory neurons.
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Alcaloides/farmacología , Analgésicos/farmacología , Capsaicina/análogos & derivados , Hiperalgesia/metabolismo , Extractos Vegetales/farmacología , Quinazolinas/farmacología , Quinolinas/farmacología , Canales Catiónicos TRPV/agonistas , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Capsaicina/antagonistas & inhibidores , Capsaicina/farmacología , Relación Dosis-Respuesta a Droga , Evodia/química , Ganglios Espinales/fisiología , Células HEK293 , Calor , Humanos , Hiperalgesia/inducido químicamente , Masculino , Medicina Tradicional China , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
BACKGROUND: Currently available over-the-counter cough remedies historically have been criticized for lack of scientific evidence supporting their efficacy. Although the first-generation antihistamine diphenhydramine is classified as an antitussive by the United States Food and Drug Administration, to the authors' knowledge it has never been shown to inhibit cough reflex sensitivity in subjects with pathological cough. OBJECTIVE: To evaluate the effect of diphenhydramine on cough reflex sensitivity. SETTING: Montefiore Medical Center, an academic medical center in New York City. METHODS: Twenty two subjects with acute viral upper respiratory tract infection (common cold) underwent cough reflex sensitivity measurement employing capsaicin challenge on 3 separate days, 2 h after ingesting single doses of study drug (to coincide with peak blood concentrations), administered in randomized, double-blind manner: a multicomponent syrup containing diphenhydramine (25 mg), phenylephrine (10 mg), in a natural cocoa formulation; dextromethorphan (30 mg) syrup; and, placebo syrup. The standard endpoint of cough challenge was used: concentration of capsaicin inducing ≥5 coughs (C5). MAIN OUTCOME MEASURE: Effect on cough reflex sensitivity (C5). RESULTS: A significant difference (p = 0.0024) was established among groups, with pairwise analysis revealing a significant increase in mean log C5 (0.4 ± 0.55 (SD); p < 0.01) for the diphenhydramine-containing medication versus placebo, but not for dextromethorphan versus placebo. CONCLUSIONS: Our results provide the initial evidence of the ability of diphenhydramine to inhibit cough reflex sensitivity in subjects with acute pathological cough. Timing of cough reflex sensitivity measurement may not have allowed demonstration of maximal antitussive effect of dextromethorphan.
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Antitusígenos/farmacología , Tos/complicaciones , Tos/tratamiento farmacológico , Difenhidramina/farmacología , Difenhidramina/uso terapéutico , Reflejo/efectos de los fármacos , Infecciones del Sistema Respiratorio/complicaciones , Adulto , Antitusígenos/uso terapéutico , Capsaicina/efectos adversos , Capsaicina/antagonistas & inhibidores , Tos/inducido químicamente , Tos/prevención & control , Dextrometorfano/farmacología , Dextrometorfano/uso terapéutico , Método Doble Ciego , Femenino , Humanos , Masculino , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Adulto JovenRESUMEN
BACKGROUND AND PURPOSE: Nasal sensory nerves play an important role in symptoms associated with rhinitis triggered by environmental stimuli. Here, we propose that TRPV1 is pivotal in nasal sensory nerve activation and assess the potential of SB-705498 as an intranasal therapy for rhinitis. EXPERIMENTAL APPROACH: The inhibitory effect of SB-705498 on capsaicin-induced currents in guinea pig trigeminal ganglion cells innervating nasal mucosa was investigated using patch clamp electrophysiology. A guinea pig model of rhinitis was developed using intranasal challenge of capsaicin and hypertonic saline to elicit nasal secretory parasympathetic reflex responses, quantified using MRI. The inhibitory effect of SB-705498, duration of action and potency comparing oral versus intranasal route of administration were examined. KEY RESULTS: SB-705498 concentration-dependently inhibited capsaicin-induced currents in isolated trigeminal ganglion cells (pIC50 7.2). In vivo, capsaicin ipsilateral nasal challenge (0.03-1 mM) elicited concentration-dependent increases in contralateral intranasal fluid secretion. Ten per cent hypertonic saline initiated a similar response. Atropine inhibited responses to either challenge. SB-705498 inhibited capsaicin-induced responses by â¼50% at 10 mg·kg⻹ (oral), non-micronized 10 mg·mL⻹ or 1 mg·mL⻹ micronized SB-705498 (intranasal) suspension. Ten milligram per millilitre intranasal SB-705498, dosed 24 h prior to capsaicin challenge produced a 52% reduction in secretory response. SB-705498 (10 mg·mL⻹, intranasal) inhibited 10% hypertonic saline responses by 70%. CONCLUSIONS AND IMPLICATIONS: The paper reports the development of a guinea pig model of rhinitis. SB-705498 inhibits capsaicin-induced trigeminal currents and capsaicin-induced contralateral nasal secretions via oral and intranasal routes; efficacy was optimized using particle-reduced SB-705498. We propose that TRPV1 is pivotal in initiating symptoms of rhinitis.
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Modelos Animales de Enfermedad , Mucosa Nasal/efectos de los fármacos , Sistema Nervioso Parasimpático/efectos de los fármacos , Parasimpatolíticos/uso terapéutico , Pirrolidinas/uso terapéutico , Rinitis Alérgica Perenne/tratamiento farmacológico , Canales Catiónicos TRPV/antagonistas & inhibidores , Urea/análogos & derivados , Administración Intranasal , Administración Oral , Animales , Antialérgicos/administración & dosificación , Antialérgicos/química , Antialérgicos/farmacología , Antialérgicos/uso terapéutico , Capsaicina/administración & dosificación , Capsaicina/antagonistas & inhibidores , Capsaicina/toxicidad , Células Cultivadas , Relación Dosis-Respuesta a Droga , Composición de Medicamentos , Femenino , Cobayas , Masculino , Mucosa Nasal/inervación , Mucosa Nasal/metabolismo , Sistema Nervioso Parasimpático/metabolismo , Sistema Nervioso Parasimpático/patología , Parasimpatolíticos/administración & dosificación , Parasimpatolíticos/química , Parasimpatolíticos/farmacología , Tamaño de la Partícula , Pirrolidinas/administración & dosificación , Pirrolidinas/química , Pirrolidinas/farmacología , Rinitis Alérgica , Rinitis Alérgica Perenne/metabolismo , Rinitis Alérgica Perenne/patología , Vías Secretoras/efectos de los fármacos , Fármacos del Sistema Sensorial/administración & dosificación , Fármacos del Sistema Sensorial/antagonistas & inhibidores , Fármacos del Sistema Sensorial/toxicidad , Canales Catiónicos TRPV/metabolismo , Ganglio del Trigémino/efectos de los fármacos , Ganglio del Trigémino/metabolismo , Ganglio del Trigémino/patología , Urea/administración & dosificación , Urea/química , Urea/farmacología , Urea/uso terapéuticoRESUMEN
During a migraine attack capsaicin-sensitive trigeminal sensory nerves release calcitonin gene-related peptide (CGRP), producing cranial vasodilatation and central nociception; hence, trigeminal inhibition may prevent this vasodilatation and abort migraine headache. This study investigated the role of spinal α2-adrenoceptors and their subtypes (i.e. α(2A), α(2B) and/or α(2C)-adrenoceptors) in the inhibition of the canine external carotid vasodilator responses to capsaicin. Anaesthetized vagosympathectomized dogs were prepared to measure arterial blood pressure, heart rate and external carotid conductance. The thyroid artery was cannulated for one-min intracarotid infusions of capsaicin, α-CGRP and acetylcholine. A cannula was inserted intrathecally for spinal (C1-C3) administration of 2-amino-6-ethyl-4,5,7,8-tetrahydro-6H-oxazolo-[5,4-d]-azepin-dihydrochloride (B-HT 933; a selective α2-adrenoceptor agonist) and/or the α2-adrenoceptor antagonists rauwolscine (α(2A/2B/2C)), 2-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole maleate (BRL44408; α(2A)), imiloxan (α(2B)) or acridin-9-yl-[4-(4-methylpiperazin-1-yl)-phenyl]amine (JP-1302; α(2C)). Infusions of capsaicin, α-CGRP and acetylcholine dose-dependently increased the external carotid conductance. Intrathecal B-HT 933 (1000 and 3100 µg) inhibited the vasodilator responses to capsaicin, but not those to α-CGRP or acetylcholine. This inhibition, abolished by rauwolscine (310 µg), was: (i) unaffected by 3,100 µg imiloxan; (ii) partially blocked by 310 µg of BRL44408 or 100 µg of JP-1302; and (iii) abolished by 1,000 µg of BRL44408 or 310 µg of JP-1302. Thus, intrathecal B-HT 933 inhibited the external carotid vasodilator responses to capsaicin. This response, mediated by spinal α2-adrenoceptors unrelated to the α(2B)-adrenoceptor subtype, resembles the pharmacological profile of α(2C)-adrenoceptors and, to a lesser extent, α(2A)-adrenoceptors.
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Antagonistas de Receptores Adrenérgicos alfa 2/uso terapéutico , Arteria Carótida Externa/efectos de los fármacos , Modelos Animales de Enfermedad , Trastornos Migrañosos/tratamiento farmacológico , Receptores Adrenérgicos alfa 2/metabolismo , Médula Espinal/efectos de los fármacos , Vasoconstrictores/uso terapéutico , Acetilcolina/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 2/administración & dosificación , Antagonistas de Receptores Adrenérgicos alfa 2/química , Analgésicos no Narcóticos/administración & dosificación , Analgésicos no Narcóticos/antagonistas & inhibidores , Analgésicos no Narcóticos/uso terapéutico , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Capsaicina/antagonistas & inhibidores , Capsaicina/toxicidad , Arteria Carótida Externa/fisiología , Vértebras Cervicales , Perros , Hemodinámica/efectos de los fármacos , Infusión Espinal , Masculino , Trastornos Migrañosos/inducido químicamente , Trastornos Migrañosos/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Receptores Adrenérgicos alfa 2/química , Flujo Sanguíneo Regional/efectos de los fármacos , Médula Espinal/metabolismo , Vasoconstrictores/administración & dosificación , Vasoconstrictores/antagonistas & inhibidores , Vasodilatadores/antagonistas & inhibidores , Vasodilatadores/toxicidadRESUMEN
OBJECTIVES: Ginsenoside Rg1 (GRg1), one of the major active constituents of Panax notoginseng, has shown anti-inflammatory and antinocioceptic activity, but its role in keratinocytes needs further study. We have examined the inhibitory effect of GRg1 on transient receptor potential vanilloid-1 (TRPV1) activation in keratinocyte HaCaT cells and explored its involved mechanism. METHODS: HEK 293T cells over-expressing exogenous TRPV1 were constructed and named HEK 293T-TRPV1 cells. The effects of GRg1 on production of interleukin-8 (IL-8) and prostaglandin E(2) (PGE(2) ), calcium influx, the expression of cyclooxygenase-2 (COX-2) and nuclear factor-κB (NF-κB) transcriptional activity in HEK 293T-TRPV1 and HaCaT cells were examined by ELISA, Fluo 3-AM fluorescence probe, Western blot and Dual-Luciferase Reporter Assay, respectively. KEY FINDINGS: The results showed that GRg1 blocked intracellular calcium by both capsaicin and proton activation in a TRPV1-dependent manner. Furthermore, GRg1 inhibited the expression of COX-2 and NF-κB transcriptional activity induced by capsaicin in keratinocytes. The inhibitory effect of GRg1 was similar to capsazepine, an antagonist of TRPV1. More importantly, GRg1 dose-dependently inhibited capsaicin-induced PGE(2) and IL-8 secretion in HaCaT cells and HEK 293T-TRPV1 cells. CONCLUSIONS: These data showed that GRg1 could inhibit TRPV1 mediated responses in HaCaT cells, indicating that GRg1 acted as a TRPV1 antagonist.
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Capsaicina/antagonistas & inhibidores , Fármacos del Sistema Nervioso Central/farmacología , Dinoprostona/metabolismo , Ginsenósidos/farmacología , Interleucina-8/metabolismo , Queratinocitos/efectos de los fármacos , Canales Catiónicos TRPV/metabolismo , Calcio/metabolismo , Capsaicina/farmacología , Células Cultivadas , Ciclooxigenasa 2/metabolismo , Medicamentos Herbarios Chinos/farmacología , Ensayo de Inmunoadsorción Enzimática , Células HEK293/efectos de los fármacos , Células HEK293/metabolismo , Humanos , Queratinocitos/metabolismo , FN-kappa B/metabolismoRESUMEN
Transient receptor potential vanilloid-1 (TRPV1) antagonists are widely viewed as next-generation pain therapeutics. However, these compounds cause hyperthermia, a serious side effect. TRPV1 antagonists differentially block three modes of TRPV1 activation: by heat, protons, and chemical ligands (e.g., capsaicin). We asked what combination of potencies in these three modes of TRPV1 activation corresponds to the lowest potency of a TRPV1 antagonist to cause hyperthermia. We studied hyperthermic responses of rats, mice, and guinea pigs to eight TRPV1 antagonists with different pharmacological profiles and used mathematical modeling to find a relative contribution of the blockade of each activation mode to the development of hyperthermia. We found that the hyperthermic effect has the highest sensitivity to the extent of TRPV1 blockade in the proton mode (0.43 to 0.65) with no to moderate sensitivity in the capsaicin mode (-0.01 to 0.34) and no sensitivity in the heat mode (0.00 to 0.01). We conclude that hyperthermia-free TRPV1 antagonists do not block TRPV1 activation by protons, even if they are potent blockers of the heat mode, and that decreasing the potency to block the capsaicin mode may further decrease the potency to cause hyperthermia.
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Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Fiebre/inducido químicamente , Fiebre/metabolismo , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/metabolismo , Animales , Capsaicina/antagonistas & inhibidores , Sistema Nervioso Central/fisiopatología , Simulación por Computador , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Fiebre/fisiopatología , Cobayas , Calor/efectos adversos , Masculino , Ratones , Ratones Noqueados , Neurofarmacología/métodos , Terapia de Protones , Ratas , Ratas Wistar , Fármacos del Sistema Sensorial/farmacologíaRESUMEN
Botulinum neurotoxin A (BoNT/A), the most toxic, naturally occurring protein, cleaves synapse-associated protein of 25 kDa and inhibits acetylcholine release from motor nerve endings (MNEs). This leads to paralysis of skeletal muscles. Our study demonstrates that capsaicin protects mouse neuromuscular junctions from the neuroparalytic effects of BoNT/A. Bilateral injection of BoNT/A near the innervation of the Extensor digitorum longus (EDL) muscle of adult Swiss-Webster mice inhibited the toe spread reflex (TSR). However, when capsaicin was coinjected bilaterally, or injected 4 or 8 h before injecting BoNT/A, the TSR remained normal. In animals that were pretreated with capsazepine, capsaicin failed to protect against the neuroparalytic effects of BoNT/A. In vivo analyses demonstrated that capsaicin protected muscle functions and electromygraphic activity from the incapacitating effects of BoNT/A. The twitch response to nerve stimulation was greater for EDL preparations isolated from mice injected with capsaicin before BoNT/A. Capsaicin pretreatment also prevented the inhibitory effects of BoNT/A on end-plate currents. Furthermore, pretreatment of Neuro 2a cells with capsaicin significantly preserved labeling of synaptic vesicles by FM 1-43. This protective effect of capsaicin was observed only in the presence of extracellular Ca(2+) and was inhibited by capsazepine. Immunohistochemistry demonstrated that MNEs express transient receptor potential protein of the vanilloid subfamily, TRPV1, the capsaicin receptor. Capsaicin pretreatment, in vitro, reduced nerve stimulation or KCl-induced uptake of BoNT/A into motor nerve endings and cholinergic Neuro 2a cells. These data demonstrate that capsaicin interacts with TRPV1 receptors on MNEs to reduce BoNT/A uptake via a Ca(2+)-dependent mechanism.
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Toxinas Botulínicas Tipo A/antagonistas & inhibidores , Toxinas Botulínicas Tipo A/toxicidad , Capsaicina/uso terapéutico , Fármacos Neuromusculares/antagonistas & inhibidores , Fármacos Neuromusculares/toxicidad , Unión Neuromuscular/efectos de los fármacos , Fármacos Neuroprotectores , Acetilcolina/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Capsaicina/análogos & derivados , Capsaicina/antagonistas & inhibidores , Capsaicina/farmacología , Línea Celular , Electrofisiología , Inmunohistoquímica , Técnicas In Vitro , Ratones , Microscopía Confocal , Neuronas Motoras/efectos de los fármacos , Fuerza Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/inervación , Terminaciones Nerviosas/efectos de los fármacos , Sinapsis/efectos de los fármacos , Canales Catiónicos TRPV/metabolismoRESUMEN
Transient receptor potential vanilloid 1 (TRPV1) receptor antagonists have gained much attention for their potential to treat inflammatory and neuropathic pain. However, systemic administration of TRPV1 antagonists induces a period of hyperthermia, a potential liability for small molecule development. Here we characterize the effects of the TRPV1 antagonist A-425619 on body temperature (T(b)) in the rat when administered: (1) alone at different times of the circadian cycle, (2) as repeated hourly or daily treatment, (3) as pre-treatment to prevent capsaicin-induced hypothermia, (4) to capsaicin-desensitized animals, and (5) prior to a heat challenge. Changes in T(b) were compared with compound exposure data, locomotor activity, and time course of efficacy in inflammatory pain models. Without affecting locomotor activity, oral administration of A-425619 induced a transient period of hyperthermia that was followed by a period of hypothermia, a profile unique among reported TRPV1 antagonists. Repeated hourly administration of A-425619 produced an increase in T(b) similar to a single administration. A-425619 had no effect on T(b) when administered to capsaicin-desensitized rats. The duration of A-425619-induced hyperthermia, but not hypothermia, was dependent on the time of the circadian cycle when administered. Pre-treatment with A-425619 attenuated capsaicin-induced hypothermia and did not potentiate T(b) or alter thermoregulatory behavioral responses during a heat challenge. These results indicate that A-425619-induced hyperthermia is transient, circadian-dependent, not related to exposure levels, locomotor activity, or time course of analgesic action, and does not affect the ability to thermoregulate during a heat challenge.
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Regulación de la Temperatura Corporal/efectos de los fármacos , Temperatura Corporal/efectos de los fármacos , Ritmo Circadiano/efectos de los fármacos , Fiebre/inducido químicamente , Isoquinolinas/farmacología , Canales Catiónicos TRPV/antagonistas & inhibidores , Urea/análogos & derivados , Administración Oral , Animales , Temperatura Corporal/fisiología , Regulación de la Temperatura Corporal/fisiología , Capsaicina/antagonistas & inhibidores , Ritmo Circadiano/fisiología , Esquema de Medicación , Fiebre/metabolismo , Fiebre/fisiopatología , Hipotálamo/efectos de los fármacos , Hipotálamo/fisiología , Hipotermia/inducido químicamente , Hipotermia/metabolismo , Hipotermia/fisiopatología , Masculino , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Ratas , Ratas Sprague-Dawley , Canales Catiónicos TRPV/metabolismo , Factores de Tiempo , Urea/farmacologíaRESUMEN
Although the cannabinoid agonists R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazin-6-yl)-(1-naphthalenyl) methanone mesylate [WIN 55,212-2 (WIN)] and (R,S)-3-(2-iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole (AM1241) exert peripheral antihyperalgesia in inflammatory pain models, the mechanism for cannabinoid-induced inhibition of nociceptive sensory neurons has not been fully studied. Because TRPV1 and TRPA1 channels play important roles in controlling hyperalgesia in inflammatory pain models, we investigated their modulation by WIN and AM1241. The applications of WIN (>5 microM) and AM1241 (>30 microM) inhibit responses of sensory neurons to capsaicin and mustard oil. To determine potential mechanisms for the inhibition, we evaluated cannabinoid effects on nociceptors. WIN and AM1241 excite sensory neurons in a concentration-dependent manner via a nonselective Ca2+-permeable channel. The expression of TRP channels in CHO cells demonstrates that both WIN and AM1241 activate TRPA1 and, by doing so, attenuate capsaicin and mustard oil responses. Using TRPA1-specific small interfering RNA or TRPA1-deficient mice, we show that the TRPA1 channel is a sole target through which WIN and mustard oil activate sensory neurons. In contrast, AM1241 activation of sensory neurons is mediated by TRPA1 and an unknown channel. The knockdown of TRPA1 activity in neurons completely eliminates the desensitizing effects of WIN and AM1241 on capsaicin-activated currents. Furthermore, the WIN- or AM1241-induced inhibition of capsaicin-evoked nocifensive behavior via peripheral actions is reversed in TRPA1 null-mutant mice. Together, this study demonstrates that certain cannabinoids exert their peripheral antinocifensive actions via activation of the TRPA1 channel on sensory neurons.
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Cannabinoides/farmacología , Capsaicina/antagonistas & inhibidores , Capsaicina/toxicidad , Planta de la Mostaza/toxicidad , Neuronas Aferentes/efectos de los fármacos , Aceites de Plantas/toxicidad , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Células CHO , Cannabinoides/uso terapéutico , Células Cultivadas , Cricetinae , Cricetulus , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Planta de la Mostaza/metabolismo , Neuronas Aferentes/metabolismo , Dolor/inducido químicamente , Dolor/tratamiento farmacológico , Dolor/metabolismo , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Aceites de Plantas/metabolismo , Ratas , Ratas Sprague-Dawley , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/genética , Canales de Potencial de Receptor Transitorio/fisiologíaRESUMEN
Vanilloid receptor 1 (VR1) is a noxious receptor and a novel target for pain therapy. Cochinchinenin B (6-hydroxy-7-methoxy-3-(4'-hydroxybenzyl) chromone; CB) is one of the small-molecular components from the flavonoids of Dragon's Blood, a well-known herbal medicine to treat various types of pain. Using whole-cell patch clamp technique, we found that capsaicin (CAP)-activated currents (ICAP) was inhibited by CB with an IC50 of 0.92 mM in acutely isolated rat dorsal root ganglion (DRG) neurons. The inhibition was reversible and not competitive. We also found that the inhibition was neither voltage- nor agonist-dependent. The bind site was on the extracellular part of the channel since intracellular application of CB did not alter the inhibition effect on ICAP. In addition, CB inhibited CAP-evoked depolarization under current-clamp condition. Our findings indicate that CB may be a candidate in developing new analgesic drugs targeting the VR1 receptor.
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Chalcona/análogos & derivados , Ganglios Espinales/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Nociceptores/efectos de los fármacos , Dolor/tratamiento farmacológico , Canales Catiónicos TRPV/antagonistas & inhibidores , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , Capsaicina/antagonistas & inhibidores , Capsaicina/farmacología , Células Cultivadas , Chalcona/farmacología , Chalcona/uso terapéutico , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Femenino , Ganglios Espinales/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Estructura Molecular , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Neuronas Aferentes/metabolismo , Nociceptores/metabolismo , Dolor/metabolismo , Dolor/fisiopatología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Fármacos del Sistema Sensorial/antagonistas & inhibidores , Canales Catiónicos TRPV/metabolismoRESUMEN
A series of trisubstituted pyrimidines were synthesized to improve aqueous solubility of our first TRPV1 clinical candidate (1; AMG 517), while maintaining potent TRPV1 inhibitory activity. Structure-activity and structure-solubility studies led to the identification of compound 26. The aqueous solubility of 26 (>or=200microg/mL, 0.01 HCl; 6.7microg/mL, phosphate buffered saline (PBS); 150microg/mL, fasted-state simulated intestinal fluid (SIF)) was significantly improved over 1. In addition, compound 26 was found to be orally bioavailable (rat F(oral)=24%) and had potent TRPV1 antagonist activity (capsaicin IC(50)=1.5nM) comparable to that of 1.
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Pirimidinas/síntesis química , Pirimidinas/farmacología , Canales Catiónicos TRPV/antagonistas & inhibidores , Animales , Benzotiazoles/química , Benzotiazoles/farmacología , Células CHO , Capsaicina/antagonistas & inhibidores , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos , Concentración de Iones de Hidrógeno , Piperazinas/síntesis química , Piperazinas/farmacología , Pirimidinas/química , Ratas , Ratas Sprague-Dawley , Solubilidad/efectos de los fármacos , Relación Estructura-Actividad , Canales Catiónicos TRPV/biosíntesis , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/fisiologíaRESUMEN
UNLABELLED: Vanilloid receptor subunit 1 (TRPV1) is an integrator of physical and chemical stimuli in the peripheral nervous system. This receptor plays a key role in the pathophysiology of inflammatory pain. Thus, the identification of receptor antagonists with analgesic and anti-inflammatory activity in vivo is an important goal of current neuropharmacology. Here, we report that [L-arginyl]-[N-[2,4-dichlorophenethyl]glycyl]-N-(2,4-dichlorophenethyl) glycinamide (H-Arg-15-15C) is a channel blocker that abrogates capsaicin and pH-evoked TRPV1 channel activity with submicromolar activity. Compound H-Arg-15-15C preferentially inhibits TRPV1, showing marginal block of other neuronal receptors. Compound H-Arg-15-15C acts as a noncompetitive capsaicin antagonist with modest voltage-dependent blockade activity. The compound inhibited capsaicin-evoked nerve activity in afferent fibers without affecting mechanically activated activity. Notably, administration of compound H-Arg-15-15C prevented the irritant activity of a local administration of capsaicin and formalin and reversed the thermal hyperalgesia evoked by injection of complete Freund's adjuvant. Furthermore, it attenuated carrageenan-induced paw inflammation. Compound H-Arg-15-15C specifically decreased inflammatory conditions without affecting normal nociception. Taken together, these findings demonstrate that compound H-Arg-15-15C is a channel blocker of TRPV1 with analgesic and anti-inflammatory activity in vivo at clinically useful doses and substantiate the tenet that TRPV1 plays an important role in the etiology of chronic inflammatory pain. PERSPECTIVE: This study reports the design of a potent TRPV1 noncompetitive antagonist that exhibits anti-inflammatory and analgesic activity in preclinical models of acute and chronic pain. This compound is a lead for analgesic drug development.
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Analgésicos/farmacología , Antiinflamatorios/farmacología , Arginina/análogos & derivados , Glicina/análogos & derivados , Canales Iónicos/antagonistas & inhibidores , Nociceptores/efectos de los fármacos , Dolor/tratamiento farmacológico , Canales Catiónicos TRPV/antagonistas & inhibidores , Analgésicos/síntesis química , Analgésicos/química , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/química , Arginina/síntesis química , Arginina/química , Arginina/farmacología , Células COS , Capsaicina/antagonistas & inhibidores , Chlorocebus aethiops , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Femenino , Glicina/síntesis química , Glicina/química , Glicina/farmacología , Inflamación/tratamiento farmacológico , Inflamación/fisiopatología , Canales Iónicos/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Nociceptores/metabolismo , Nociceptores/fisiopatología , Oocitos , Dolor/fisiopatología , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiología , Canales Catiónicos TRPV/metabolismo , XenopusRESUMEN
Vanilloid receptor type 1 (TRPV1) is a ligand-gated nonselective cation channel that is considered to be an important integrator of various pain stimuli such as endogenous lipids, capsaicin, heat, and low pH. In addition to expression in primary afferents, TRPV1 is also expressed in the CNS. To test the hypothesis that the CNS plays a differential role in the effect of TRPV1 antagonists in various types of pain, the analgesic effects of two TRPV1 antagonists with similar in vitro potency but different CNS penetration were compared in vivo. Oral administration of either A-784168 (1-[3-(trifluoromethyl)pyridin-2-yl]-N-[4-(trifluoromethylsulfonyl)phenyl]-1,2,3,6-tetrahydropyridine-4-carboxamide) (good CNS penetration) or A-795614 (N-1H-indazol-4-yl-N'-[(1R)-5-piperidin-1-yl-2,3-dihydro-1H-inden-1-yl]urea) (poor CNS penetration) blocked capsaicin-induced acute pain with the same potency. In complete Freund's adjuvant (CFA)-induced chronic inflammatory pain, oral administration of either compound blocked thermal hyperalgesia with similar potency. Furthermore, intraplantar or intrathecal administration of A-784168 blocked CFA-induced thermal hyperalgesia, suggesting that both peripheral and CNS TRPV1 receptors may play a role in inflammatory thermal hyperalgesia. The effects of the two TRPV1 antagonists were further assessed in models presumably mediated by central sensitization, including CFA- and capsaicin-induced mechanical allodynia and osteoarthritic pain. In these models, the potency of the two compounds was similar after intrathecal administration. However, when administered orally, A-784168, with good CNS penetration, was much more potent than A-795614. Together, these results demonstrate that TRPV1 receptors in the CNS play an important role in pain mediated by central sensitization. In addition, these results demonstrate that significant CNS penetration is necessary for a TRPV1 antagonist to produce broad-spectrum analgesia.
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Analgésicos/farmacocinética , Sistema Nervioso Central/efectos de los fármacos , Nociceptores/efectos de los fármacos , Dolor/tratamiento farmacológico , Canales Catiónicos TRPV/antagonistas & inhibidores , Administración Oral , Analgésicos/metabolismo , Animales , Artralgia/tratamiento farmacológico , Artralgia/metabolismo , Artralgia/fisiopatología , Calcio/metabolismo , Capsaicina/antagonistas & inhibidores , Línea Celular , Células Cultivadas , Sistema Nervioso Central/metabolismo , Modelos Animales de Enfermedad , Humanos , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Indazoles/farmacología , Mediadores de Inflamación/antagonistas & inhibidores , Inyecciones Espinales , Masculino , Nociceptores/metabolismo , Dolor/metabolismo , Dolor/fisiopatología , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Sulfonas/farmacología , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Resultado del Tratamiento , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
BACKGROUND: We previously reported that endogenous prostaglandin I(2), generated by a mild irritant, sensitised calcitonin gene related peptide (CGRP) containing sensory nerves and facilitated the release of CGRP and gastric mucosal protection against ethanol. Administration of capsaicin also inhibited ethanol induced gastric mucosal injury through immediate release of CGRP from primary sensory neurones, which is termed the neural emergency system. In the present study, we tested whether endogenous prostaglandin I(2) also modulates the cytoprotective action of capsaicin using prostaglandin I receptor knockout mice (IP(-/-)). METHODS: The stomachs of IP(-/-) or their wild-type counterparts (IP(+/+)), anaesthetised with urethane (1.225 g/kg), were doubly cannulated from the oesophageal and duodenal sides, and the gastric mucosa was perfused (1 ml/min) with physiological saline. Perfusate was changed to 50% ethanol alone, or 50% ethanol containing capsaicin (16 approximately 1600 micro M). The injured area was estimated at the end of each perfusion experiment. In some animals, CGRP-(8-37), a CGRP antagonist (0.3 mg/kg), or indomethacin (1 mg/kg) was intravenously injected before perfusion of 50% ethanol containing capsaicin. RESULTS: Capsaicin inhibited the injured area in a dose dependent manner. Fifty per cent ethanol containing capsaicin (480 micro M) immediately increased intragastric levels of CGRP although 50% ethanol alone did not. The protective action of capsaicin (480 micro M) against ethanol was completely abolished by intravenous injection of CGRP-(8-37). Indomethacin also inhibited the protective action of capsaicin, and this was accompanied by reduced levels of intragastric CGRP. Intragastric levels of prostaglandin E(2) were not increased by capsaicin treatment but those of 6-keto-prostaglandin F(1alpha), a metabolite of prostaglandin I(2), were markedly increased. No protective action of capsaicin was observed in IP(-/-) which lacked the ability to increase intragastric CGRP levels in response to ethanol containing capsaicin. The CGRP content of the stomach from untreated IP(-/-) did not differ from those in IP(+/+). Capsaicin (160 micro M) together with intragastric perfusion of beraprost sodium (PGI(2) analogue, 2.5 micro g/ml) showed enhanced protection against ethanol induced injury. This enhanced protection was completely blocked by intravenous injection of CGRP-(8-37). CONCLUSIONS: The present results suggest that endogenous prostaglandin I(2) enhances the protective action of the capsaicin mediated neural emergency system against ethanol induced gastric mucosal injury through enhancement of CGRP release.
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Péptido Relacionado con Gen de Calcitonina/metabolismo , Capsaicina/uso terapéutico , Epoprostenol/fisiología , Etanol/efectos adversos , Mucosa Gástrica/efectos de los fármacos , 6-Cetoprostaglandina F1 alfa/metabolismo , Animales , Péptido Relacionado con Gen de Calcitonina/análisis , Capsaicina/antagonistas & inhibidores , Inhibidores de la Ciclooxigenasa/administración & dosificación , Relación Dosis-Respuesta a Droga , Etanol/administración & dosificación , Mucosa Gástrica/metabolismo , Indometacina/administración & dosificación , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
We examined the effect of the subcutaneous infiltration of ketamine, lidocaine and saline before injury on capsaicin-induced pain and hyperalgesia. Twelve healthy volunteers participated in two separate, randomized, double-blind, placebo-controlled crossover experiments. In experiment 1, 100 micrograms capsaicin was injected intradermally in one volar forearm 10 min after the skin had been pretreated with lidocaine 20.0 mg in 2.0 ml or 0.9% saline 2.0 ml at the capsaicin injection site. In experiment 2, a similar capsaicin test was given 10 min after the skin had been pretreated with ketamine 5 mg in 2.0 ml or 0.9% saline 2.0 ml. To control for possible systemic effects, the capsaicin injection site was pretreated by injection of saline into the skin and the contralateral arm was treated with active drug, and vice versa. Outcome measures were spontaneous pain, pain evoked by punctate and brush stimuli, and areas of brush-evoked and punctate-evoked hyperalgesia. Lidocaine reduced all measures compared with placebo (P < 0.001), whereas ketamine failed to change any measures. Pain scores and areas of hyperalgesia were not affected when the contralateral site was infiltrated with ketamine or lidocaine. Lidocaine produced no side-effects, whereas ketamine produced paraesthesia, dizziness and sleepiness in six out of 24 (25%) cases. Blocking peripheral sodium channels with locally administered lidocaine reduces spontaneous pain and capsaicin-induced hyperalgesia but local block with the NMDA-type glutamate receptor antagonist ketamine has no effect on capsaicin-induced pain and hyperalgesia.
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Analgésicos/uso terapéutico , Anestésicos Locales/uso terapéutico , Hiperalgesia/prevención & control , Ketamina/uso terapéutico , Lidocaína/uso terapéutico , Análisis de Varianza , Capsaicina/antagonistas & inhibidores , Estudios Cruzados , Método Doble Ciego , Humanos , Hiperalgesia/inducido químicamente , Masculino , Dimensión del Dolor , TactoRESUMEN
Capsaicin is a natural product of Capsicum peppers, excitatory effects of which have been shown to be mediated by the recently cloned vanilloid receptor 1 (VR1). Since previous studies have shown that capsaicin inhibits protein synthesis, experiments were performed to investigate whether this effect is mediated by VR1 receptor on cultured monkey kidney cells (Vero cells). The capsaicin uptake was assessed in cellular homogenate and in medium by high-performance liquid chromatography (HPLC) separation and quantification on C18 reverse-phase column and fluorescence detection. Toxic effects were assessed by incorporation of [3H]L-leucine into cellular proteins in the presence of capsazepine, the VR1 vanilloid receptor antagonist and Ruthenium red or tyrosine or calcium. Capsazepine (1 to 256 microM) did not modify the uptake rate of capsaicin for incubation times up to 24 h and did not antagonize capsaicin-induced protein synthesis inhibition. It rather inhibited protein synthesis per se from 100 to 256 microM. Ruthenium red which blocks mitochondrial calcium uptake, inhibited protein synthesis and did not antagonise or increase synergistically the effects of capsaicin. Interestingly in a medium deprived of calcium and supplemented by calcium chloride (10-50 microM) the protein synthesis inhibition induced by capsaicin is antagonised somehow. There was no prevention of capsaicin diffusion into the cells. Tyrosine, which seems to be the best preventive agent of capsaicin inhibitory effects, prevents its metabolism but not its diffusion. Capsaicin might enter cells by diffusion and interfere with protein synthesis machinery by competition with tyrosine which in turn prevents the metabolism of capsaicin. The results of the present study suggest that cell responses to capsaicin may be transduced through at least two molecular pathways, one involving VR1, since the receptor antagonist capsazepine fails to prevent the inhibitory effect of capsaicin in Vero cells of renal origin.