RESUMEN
Intestinal permeability and neutrophil activity are closely linked to inflammatory bowel disease (IBD) pathophysiology. Here we discuss two techniques for assessing permeability and neutrophil activity in mouse IBD models using near infrared (NIR) detection. To address the limitation of visible light readouts-namely high background-IRDye 800CW was used to enable rapid, non-terminal measurements of intestinal permeability. The increased sensitivity of NIR readouts for colon permeability is shown using dextran sulfate sodium (DSS) and anti-CD40 murine colitis models in response to interleukin-22 immunoglobulin Fc (IL22Fc) fusion protein and anti-p40 monoclonal antibody treatments, respectively. In addition to enhanced permeability, elevated levels of neutrophil elastase (NE) have been reported in inflamed colonic mucosal tissue. Activatable NIR fluorescent probes have been extensively used for disease activity evaluation in oncologic animal models, and we demonstrate their translatability using a NE-activatable reagent to evaluate inflammation in DSS mice. Confocal laser endomicroscopy (CLE) and tissue imaging allow visualization of spatial NE activity throughout diseased colon as well as changes in disease severity from IL22Fc treatment. Our findings with the 800CW dye and the NE probe highlight the ease of their implementation in preclinical IBD research.
Asunto(s)
Enfermedades Inflamatorias del Intestino/diagnóstico , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Imagen Óptica/métodos , Animales , Transporte Biológico , Biomarcadores , Modelos Animales de Enfermedad , Inmunohistoquímica , Enfermedades Inflamatorias del Intestino/etiología , Elastasa de Leucocito/metabolismo , Ratones , Microscopía Confocal , Permeabilidad , Espectroscopía Infrarroja CortaRESUMEN
We describe the design, synthesis, and structure-activity relationships (SARs) of a series of 2-aminobenzothiazole inhibitors of Rho kinases (ROCKs) 1 and 2, which were optimized to low nanomolar potencies by use of protein kinaseâ A (PKA) as a structure surrogate to guide compound design. A subset of these molecules also showed robust activity in a cell-based myosin phosphatase assay and in a mechanical hyperalgesia in vivo pain model.
Asunto(s)
Benzotiazoles/farmacología , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Benzotiazoles/síntesis química , Benzotiazoles/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Quinasas Asociadas a rho/metabolismoRESUMEN
A novel 4-aminocyclopentapyrrolidine series of N-type Ca(2+) channel blockers have been discovered. Enantioselective synthesis of the 4-aminocyclopentapyrrolidines was enabled using N-tert-butyl sulfinamide chemistry. SAR studies demonstrate selectivity over L-type Ca(2+) channels. N-type Ca(2+) channel blockade was confirmed using electrophysiological recording techniques. Compound 25 is an N-type Ca(2+) channel blocker that produces antinociception in inflammatory and nociceptive pain models without exhibiting cardiovascular or motor liabilities.
Asunto(s)
Acetamidas/síntesis química , Analgésicos/síntesis química , Bloqueadores de los Canales de Calcio/síntesis química , Canales de Calcio Tipo N/química , Pirrolidinas/química , Pirrolidinas/síntesis química , Acetamidas/farmacología , Acetamidas/uso terapéutico , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Conducta Animal/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/uso terapéutico , Canales de Calcio Tipo N/metabolismo , Modelos Animales de Enfermedad , Masculino , Dolor/tratamiento farmacológico , Pirrolidinas/farmacología , Pirrolidinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Blockade of voltage-gated Ca²âº channels on sensory nerves attenuates neurotransmitter release and membrane hyperexcitability associated with chronic pain states. Identification of small molecule Ca²âº channel blockers that produce significant antinociception in the absence of deleterious hemodynamic effects has been challenging. In this report, two novel structurally related compounds, A-686085 and A-1048400, were identified that potently block N-type (IC50=0.8 µM and 1.4 µM, respectively) and T-type (IC50=4.6 µM and 1.2 µM, respectively) Ca²âº channels in FLIPR based Ca²âº flux assays. A-686085 also potently blocked L-type Ca²âº channels (EC50=0.6 µM), however, A-1048400 was much less active in blocking this channel (EC50=28 µM). Both compounds dose-dependently reversed tactile allodynia in a model of capsaicin-induced secondary hypersensitivity with similar potencies (EC50=300-365 ng/ml). However, A-686085 produced dose-related decreases in mean arterial pressure at antinociceptive plasma concentrations in the rat, while A-1048400 did not significantly alter hemodynamic function at supra-efficacious plasma concentrations. Electrophysiological studies demonstrated that A-1048400 blocks native N- and T-type Ca²âº currents in rat dorsal root ganglion neurons (IC50=3.0 µM and 1.6 µM, respectively) in a voltage-dependent fashion. In other experimental pain models, A-1048400 dose-dependently attenuated nociceptive, neuropathic and inflammatory pain at doses that did not alter psychomotor or hemodynamic function. The identification of A-1048400 provides further evidence that voltage-dependent inhibition of neuronal Ca²âº channels coupled with pharmacological selectivity vs. L-type Ca²âº channels can provide robust antinociception in the absence of deleterious effects on hemodynamic or psychomotor function.
Asunto(s)
Analgésicos/administración & dosificación , Bloqueadores de los Canales de Calcio/administración & dosificación , Hemodinámica/fisiología , Neuronas/fisiología , Dimensión del Dolor , Piperidonas/administración & dosificación , Piperidonas/química , Administración Oral , Animales , Relación Dosis-Respuesta a Droga , Células HEK293 , Hemodinámica/efectos de los fármacos , Humanos , Masculino , Neuronas/efectos de los fármacos , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Ratas , Ratas Sprague-DawleyRESUMEN
Positive modulation of the neuronal nicotinic acetylcholine receptor (nAChR) α4ß2 subtype by selective positive allosteric modulator NS-9283 has shown to potentiate the nAChR agonist ABT-594-induced anti-allodynic activity in preclinical neuropathic pain. To determine whether this benefit can be extended beyond neuropathic pain, the present study examined the analgesic activity and adverse effect profile of co-administered NS-9283 and ABT-594 in a variety of preclinical models in rats. The effect of the combined therapy on drug-induced brain activities was also determined using pharmacological magnetic resonance imaging. In carrageenan-induced thermal hyperalgesia, co-administration of NS-9283 (3.5 µmol/kg, i.p.) induced a 6-fold leftward shift of the dose-response of ABT-594 (ED(50)=26 vs. 160 nmol/kg, i.p.). In the paw skin incision model of post-operative pain, co-administration of NS-9283 similarly induced a 6-fold leftward shift of ABT-594 (ED(50)=26 vs. 153 nmol/kg). In monoiodo-acetate induced knee joint pain, co-administration of NS-9283 enhanced the potency of ABT-594 by 5-fold (ED(50)=1.0 vs. 4.6 nmol/kg). In pharmacological MRI, co-administration of NS-9283 was shown to lead to a leftward shift of ABT-594 dose-response for cortical activation. ABT-594 induced CNS-related adverse effects were not exacerbated in presence of an efficacious dose of NS-9283 (3.5 µmol/kg). Acute challenge of NS-9283 produced no cross sensitization in nicotine-conditioned animals. These results demonstrate that selective positive allosteric modulation at the α4ß2 nAChR potentiates nAChR agonist-induced analgesic activity across neuropathic and nociceptive preclinical pain models without potentiating ABT-594-mediated adverse effects, suggesting that selective positive modulation of α4ß2 nAChR by PAM may represent a novel analgesic approach.
Asunto(s)
Analgésicos/uso terapéutico , Azetidinas/uso terapéutico , Agonistas Nicotínicos/uso terapéutico , Oxadiazoles/uso terapéutico , Dolor/tratamiento farmacológico , Piridinas/uso terapéutico , Receptores Nicotínicos/metabolismo , Regulación Alostérica , Analgésicos/administración & dosificación , Analgésicos/efectos adversos , Animales , Azetidinas/administración & dosificación , Azetidinas/efectos adversos , Conducta Animal/efectos de los fármacos , Temperatura Corporal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Quimioterapia Combinada , Imagen por Resonancia Magnética , Masculino , Agonistas Nicotínicos/administración & dosificación , Agonistas Nicotínicos/efectos adversos , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Oxadiazoles/administración & dosificación , Oxadiazoles/efectos adversos , Dolor/metabolismo , Piridinas/administración & dosificación , Piridinas/efectos adversos , Ratas , Ratas Sprague-DawleyRESUMEN
Despite the increasing interest in TRPA1 channel as a pain target, its role in cold sensation and body temperature regulation is not clear; the efficacy and particularly side effects resulting from channel blockade remain poorly understood. Here we use a potent, selective, and bioavailable antagonist to address these issues. A-967079 potently blocks human (IC(50): 51 nmol/L, electrophysiology, 67 nmol/L, Ca(2+) assay) and rat TRPA1 (IC(50): 101 nmol/L, electrophysiology, 289 nmol/L, Ca(2+) assay). It is >1000-fold selective over other TRP channels, and is >150-fold selective over 75 other ion channels, enzymes, and G-protein-coupled receptors. Oral dosing of A-967079 produces robust drug exposure in rodents, and exhibits analgesic efficacy in allyl isothiocyanate-induced nocifensive response and osteoarthritic pain in rats (ED(50): 23.2 mg/kg, p.o.). A-967079 attenuates cold allodynia produced by nerve injury but does not alter noxious cold sensation in naive animals, suggesting distinct roles of TRPA1 in physiological and pathological states. Unlike TRPV1 antagonists, A-967079 does not alter body temperature. It also does not produce locomotor or cardiovascular side effects. Collectively, these data provide novel insights into TRPA1 function and suggest that the selective TRPA1 blockade may present a viable strategy for alleviating pain without untoward side effects.
Asunto(s)
Regulación de la Temperatura Corporal/efectos de los fármacos , Canales de Calcio/metabolismo , Frío/efectos adversos , Hiperalgesia/tratamiento farmacológico , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Dolor/fisiopatología , Sensación/fisiología , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Temperatura Corporal/efectos de los fármacos , Temperatura Corporal/fisiología , Regulación de la Temperatura Corporal/genética , Regulación de la Temperatura Corporal/fisiología , Péptido Relacionado con Gen de Calcitonina/metabolismo , Calcio/metabolismo , Canales de Calcio/genética , Células Cultivadas , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Ganglios Espinales/patología , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Humanos , Hiperalgesia/fisiopatología , Concentración 50 Inhibidora , Isotiocianatos/farmacología , Imagen por Resonancia Magnética/métodos , Masculino , Ratones , Proteínas del Tejido Nervioso/genética , Neuronas/efectos de los fármacos , Oximas/farmacología , Oximas/uso terapéutico , Dolor/tratamiento farmacológico , Dolor/genética , Dolor/metabolismo , Dimensión del Dolor/métodos , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Sensación/efectos de los fármacos , Umbral Sensorial/efectos de los fármacos , Canal Catiónico TRPA1 , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Canales de Potencial de Receptor Transitorio/genética , TritioRESUMEN
Na(v)1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons. It has been implicated in the pathophysiology of inflammatory and neuropathic pain, and we envisioned that selective blockade of Na(v)1.8 would be analgesic, while reducing adverse events typically associated with non-selective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 6-aryl-2-pyrazinecarboxamides, which are potent blockers of the human Na(v)1.8 channel and also block TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons. Selected derivatives display selectivity versus human Na(v)1.2. We further demonstrate that an example from this series is orally bioavailable and produces antinociceptive activity in vivo in a rodent model of neuropathic pain following oral administration.
Asunto(s)
Neuralgia/tratamiento farmacológico , Pirazinas/química , Bloqueadores de los Canales de Sodio/química , Canales de Sodio/química , Administración Oral , Animales , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Ganglios Espinales/citología , Humanos , Microsomas/metabolismo , Canal de Sodio Activado por Voltaje NAV1.8 , Neuronas/metabolismo , Pirazinas/farmacocinética , Pirazinas/uso terapéutico , Ratas , Bloqueadores de los Canales de Sodio/farmacocinética , Bloqueadores de los Canales de Sodio/uso terapéutico , Canales de Sodio/metabolismo , Relación Estructura-ActividadRESUMEN
A series of aryl-substituted nicotinamide derivatives with selective inhibitory activity against the Na(v)1.8 sodium channel is reported. Replacement of the furan nucleus and homologation of the anilide linker in subtype-selective blocker A-803467 (1) provided potent, selective derivatives with improved aqueous solubility and oral bioavailability. Representative compounds from this series displayed efficacy in rat models of inflammatory and neuropathic pain.
Asunto(s)
Niacinamida/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Administración Oral , Animales , Disponibilidad Biológica , Niacinamida/química , Niacinamida/farmacocinética , Ratas , Bloqueadores de los Canales de Sodio/administración & dosificación , Bloqueadores de los Canales de Sodio/química , Bloqueadores de los Canales de Sodio/farmacocinética , Relación Estructura-ActividadRESUMEN
UNLABELLED: Activation of Rho kinase (ROCK) has been shown to play a role in neuronal regeneration and development of posttraumatic neuropathic pain. The ROCK inhibitor Fasudil, used clinically for the treatment of vasospasm, was used to investigate the analgesic profile of a ROCK inhibitor. Fasudil was evaluated in different preclinical models of neuropathic, osteoarthritic (OA), and inflammatory pain as well as capsaicin-induced acute pain and secondary mechanical hypersensitivity. In addition, Fasudil was tested in in vivo electrophysiology to determine the mechanism by which Fasudil produces analgesia. Fasudil at the highest dose tested (30 mg/kg) significantly attenuated mechanical allodynia in spinal-nerve ligation (SNL; 77%), chronic constriction injury (CCI; 53%), capsaicin-induced secondary mechanical hypersensitivity (63%), sodium iodoacetate-induced OA pain (88%), and capsaicin-induced acute flinching behaviors (56%). However, Fasudil (at 30 mg/kg) failed to attenuate or had only modest effects on inflammatory thermal hyperalgesia following carrageenan injection and mechanical allodynia following Complete Freund's Adjuvant (CFA) injection. Fasudil produced ED(50) of 10.8 mg/kg in the SNL, and 5.7 mg/kg in the OA pain models. The ED(50) and 95% CI could not be obtained in the other models. Furthermore, administration of Fasudil (10 mg/kg, iv) significantly reduced both spontaneous and evoked firing of wide dynamic range (WDR) neurons in SNL, but not sham rats. Finally, Fasudil significantly decreased exploratory behaviors at 30 mg/kg. These results suggest that the acute administration of a ROCK inhibitor produces efficacy in both neuropathic and nociceptive pain states at doses devoid of locomotor side effects, with specific effects on WDR neurons. PERSPECTIVE: In this article, the potential analgesic effects of Fasudil in a range of preclinical pain models were assessed. Fasudil was shown to have efficacy in neuropathic and nociceptive pain models. These findings may help identify new therapeutic treatments for pain in the clinic.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Modelos Animales de Enfermedad , Dolor/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/síntesis química , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Masculino , Dolor/diagnóstico , Dolor/enzimología , Inhibidores de Proteínas Quinasas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Quinasas Asociadas a rho/fisiologíaRESUMEN
Most animal models of pain cannot separate the sensory and affective components of pain. One model that has been used to assess affective pain is the place escape avoidance paradigm (PEAP). The aim of the current study is two-fold. First, validate PEAP with Complete Freund's Adjuvant (CFA)-induced inflammation for the assessment of the affective component of pain using the reference analgesics celecoxib, diclofenac and duloxetine; fluoxetine and scopolamine were tested as negative controls. Secondly, determine if there is a difference in efficacy in PEAP in comparison to the effects of the same compounds on von Frey-evoked mechanical allodynia in CFA animals. All compounds were tested in mechanical allodynia, place escape/avoidance, and for potentially confounding side effects in locomotor activity. Results show that celecoxib, diclofenac, and duloxetine significantly increased the time spent on the side associated with stimulation of the injured paw, whereas fluoxetine and scopolamine had no effect. Higher doses of celecoxib, diclofenac, duloxetine, and fluoxetine were required to attenuate von Frey-evoked mechanical allodynia. In the side effect assays, only fluoxetine decreased locomotor activity at doses used in PEAP. These results show that in inflammatory pain induced by CFA injection, PEAP is more sensitive to the effects of pain relieving compounds than mechanical allodynia. Fluoxetine showed efficacy in the mechanical allodynia test, but not PEAP, whereas duloxetine showed efficacy in mechanical allodynia and PEAP. These studies show that methods other than reflex based measures of pain such as affective pain models could be more predictive of efficacy/potency in the clinic.