RESUMEN
AIMS: Long-term or recreational use of ketamine affects the urinary system and can result in ketamine-induced cystitis (KIC). Rodent models of KIC are important to study KIC pathophysiology and are paramount to the future development of therapies for this painful condition. This review aims to provide a summary of rodent models of KIC, focusing on disease induction, experimental methods, and pathological features of the model. METHOD: A literature search was performed using the National Center for Biotechnology Information (NCBI) Pubmed database up to March 2021. 20 articles met the inclusion criteria and were finally selected. RESULTS: There are considerable variations in the rodent models used for studying KIC in terms of the strain of the animal being used; dose, duration, and route of ketamine administration to induce KIC, and assessment of pathological features. CONCLUSION: KIC remains difficult to fully recapitulate in humans. Improved characterization of KIC models and the experimental parameters and meticulous discussion on translational limitations are required to improve the translational value of research using rodent models of KIC.
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Cistitis , Ketamina , Animales , Cistitis/inducido químicamente , Ketamina/toxicidad , RoedoresRESUMEN
The cannabinoid type 1 (CB1 ) receptor and the dopamine type 2 (D2 ) receptor are co-localized on medium spiny neuron terminals in the globus pallidus where they modulate neural circuits involved in voluntary movement. Physical interactions between the two receptors have been shown to alter receptor signaling in cell culture. The objectives of the current study were to identify the presence of CB1 /D2 heteromers in the globus pallidus of C57BL/6J male mice, define how CB1 /D2 heteromer levels are altered following treatment with cannabinoids and/or antipsychotics, and determine if fluctuating levels of CB1 /D2 heteromers have functional consequences. Using in situ proximity ligation assays, we observed CB1 /D2 heteromers in the globus pallidus of C57BL/6J mice. The abundance of the heteromers increased following treatment with the nonselective cannabinoid receptor agonist, CP55,940. In contrast, treatment with the typical antipsychotic haloperidol reduced the number of CB1 /D2 heteromers, whereas the atypical antipsychotic olanzapine treatment had no effect. Co-treatment with CP55,940 and haloperidol had similar effects to haloperidol alone, whereas co-treatment with CP55,940 and olanzapine had similar effects to CP55,940. The observed changes were found to have functional consequences as the differential effects of haloperidol and olanzapine also applied to γ-aminobutyric acid release in STHdhQ7/Q7 cells and motor function in C57BL/6J male mice. This work highlights the clinical relevance of co-exposure to cannabinoids and different antipsychotics over acute and prolonged time periods.
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Antipsicóticos/administración & dosificación , Agonistas de Receptores de Cannabinoides/administración & dosificación , Cannabinoides/administración & dosificación , Receptor Cannabinoide CB1/metabolismo , Receptores de Dopamina D2/metabolismo , Animales , Línea Celular Transformada , Quimioterapia Combinada , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor Cannabinoide CB1/agonistasRESUMEN
The endothelin receptor A (ETA) and endothelin receptor B (ETB) are G protein-coupled receptors that are co-expressed in vascular smooth muscle cells. Endothelin-1 (ET-1) activates endothelin receptors to cause microvascular vasoconstriction. Previous studies have shown that heteromerization between ETA and ETB prolongs Ca2+ transients, leading to prolongation of Gαq-dependent signaling and sustained vasoconstriction. We hypothesized that these effects are in part mediated by the resistance of ETA/ETB heteromers to ß-arrestin recruitment and subsequent desensitization. Using bioluminescence resonance energy transfer 2 (BRET2), we found that ETB has a relatively equal affinity to form either homomers or heteromers with ETA when co-expressed in the human embryonic kidney 293 (HEK293) cells. When co-expressed, activation of ETA and ETB by ET-1 caused a heteromer-specific reduction and delay in ß-arrestin-2 recruitment with a corresponding reduction and delay in ET-1-induced ETA/ETB co-internalization. Furthermore, the co-expression of ETA and ETB inhibited ET-1-induced ß-arrestin-1-dependent extracellular signal-regulated kinase (ERK) phosphorylation while prolonging ET-1-induced Gαq-dependent ERK phosphorylation. ETA/ETB heteromerization mediates the long-lasting vasoconstrictor response to ET-1 by the prolongation of Gαq-dependent signaling and inhibition of ß-arrestin function.
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Multimerización de Proteína , Receptor de Endotelina A/química , Receptor de Endotelina B/química , beta-Arrestinas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HEK293 , Humanos , Fosforilación , Estructura Cuaternaria de Proteína , Transducción de SeñalRESUMEN
Cannabinoid receptor 1 (CB1) activation has been reported to reduce transient receptor potential cation channel subfamily V member 1 (TRPV1)-induced inflammatory responses and is anti-nociceptive and anti-inflammatory in corneal injury. We examined whether allosteric ligands, can modulate CB1 signaling to reduce pain and inflammation in corneal hyperalgesia. Corneal hyperalgesia was generated by chemical cauterization of cornea in wildtype and CB2 knockout (CB2-/-) mice. The novel racemic CB1 allosteric ligand GAT211 and its enantiomers GAT228 and GAT229 were examined alone or in combination with the orthosteric CB1 agonist Δ8-tetrahydrocannabinol (Δ8-THC). Pain responses were assessed following capsaicin (1 µM) stimulation of injured corneas at 6 h post-cauterization. Corneal neutrophil infiltration was also analyzed. GAT228, but not GAT229 or GAT211, reduced pain scores in response to capsaicin stimulation. Combination treatments of 0.5% GAT229 or 1% GAT211 with subthreshold Δ8-THC (0.4%) significantly reduced pain scores following capsaicin stimulation. The anti-nociceptive effects of both GAT229 and GAT228 were blocked with CB1 antagonist AM251, but remained unaffected in CB2-/- mice. Two percent GAT228, or the combination of 0.2% Δ8-THC with 0.5% GAT229 also significantly reduced corneal inflammation. CB1 allosteric ligands could offer a novel approach for treating corneal pain and inflammation.
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Lesiones de la Cornea/tratamiento farmacológico , Dronabinol/análogos & derivados , Hiperalgesia/tratamiento farmacológico , Indoles/administración & dosificación , Inflamación/tratamiento farmacológico , Receptor Cannabinoide CB1/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Cauterización , Lesiones de la Cornea/complicaciones , Lesiones de la Cornea/etiología , Modelos Animales de Enfermedad , Dronabinol/administración & dosificación , Dronabinol/farmacología , Sinergismo Farmacológico , Técnicas de Inactivación de Genes , Hiperalgesia/metabolismo , Indoles/farmacología , Inflamación/etiología , Inflamación/metabolismo , Ligandos , Ratones , Receptor Cannabinoide CB1/química , Receptor Cannabinoide CB2/genética , Transducción de SeñalRESUMEN
BACKGROUND: Exogenous endophthalmitis is a potential complication of intraocular surgery and frequently results in visual impairment. Current treatment involves administration of intravitreal (IVT) antibiotics with or without vitrectomy surgery. Evidence for the use of adjunctive anti-inflammatory agents is conflicting. We set out to determine if bevacizumab, a humanized monoclonal IgG1 antibody targeted against vascular endothelial growth factor (VEGF), has anti-inflammatory properties in experimental models of Gram-positive and Gram-negative inflammation. METHODS: BALB/c mice were subjected to lipopolysaccharide- (LPS) or peptidoglycan- (PGN) induced ocular inflammation and treated with IVT bevacizumab. Iris microvasculature was imaged 6 hours following irritant/treatment using intravital microscopy (IVM) before the mice were euthanized and the eyes were enucleated immediately post-mortem. Following enucleation, levels of VEGF and 23 cytokines and chemokines (IL-1α, IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, TNF, KC, G-CSF, GM-CSF, Eotaxin, INF-γ, MCP-1, MIP-1α, MIP-1ß, RANTES) were quantified using a multiplex assay. RESULTS: Levels of VEGF were significantly increased during the inflammatory response, triggered by either PGN or LPS. Both the adherence of leukocytes to the iris vascular endothelium and the levels of pro-inflammatory cytokines and chemokines were significantly increased following administration of either irritant. Treatment with bevacizumab decreased levels of leukocyte adherence in LPS-treated eyes, however, not in PGN-treated eyes. Conversely, bevacizumab treatment decreased levels of cytokines and chemokines (TNF, IL-6, MCP-1, MIP-1α, MIP-1ß, RANTES, KC) in PGN-treated eyes, however, not in LPS-treated eyes. CONCLUSIONS: Within a 6-hour window bevacizumab had anti-inflammatory actions that were distinct in both Gram-positive (PIU) and Gram-negative (EIU) models, respectively. Given our findings, this would suggest that bevacizumab may have utility as an adjunctive therapy to IVT antibiotics and vitrectomy in the management of exogenous endophthalmitis.
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Inhibidores de la Angiogénesis/uso terapéutico , Bevacizumab/uso terapéutico , Infecciones Bacterianas del Ojo/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Uveítis/tratamiento farmacológico , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Infecciones Bacterianas del Ojo/etiología , Infecciones Bacterianas del Ojo/metabolismo , Infecciones por Bacterias Gramnegativas/etiología , Infecciones por Bacterias Gramnegativas/metabolismo , Infecciones por Bacterias Grampositivas/etiología , Infecciones por Bacterias Grampositivas/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/microbiología , Inyecciones Intravítreas , Lipopolisacáridos , Masculino , Ratones , Ratones Endogámicos BALB C , Peptidoglicano , Factores de Tiempo , Uveítis/metabolismo , Uveítis/microbiología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Central nervous system (CNS) injury, such as stroke or trauma, is known to increase susceptibility to various infections that adversely affect patient outcomes (CNS injury-induced immunodepression-CIDS). The endocannabinoid system (ECS) has been shown to have immunoregulatory properties. Therefore, the ECS might represent a druggable target to overcome CIDS. Evidence suggests that cannabinoid type 2 receptor (CB2R) activation can be protective during the early pro-inflammatory phase after CNS injury, as it limits neuro-inflammation and, therefore, attenuates CIDS severity. In the later phase post CNS injury, CB2R inhibition is suggested as a promising pharmacologic strategy to restore immune function in order to prevent infection.
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Enfermedades del Sistema Nervioso Central/metabolismo , Endocannabinoides/metabolismo , Traumatismos del Sistema Nervioso/metabolismo , Inmunidad Adaptativa , Animales , Enfermedades del Sistema Nervioso Central/diagnóstico , Enfermedades del Sistema Nervioso Central/etiología , Humanos , Inmunidad Innata , Neuroinmunomodulación , Transducción de Señal , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/metabolismo , Factores de Tiempo , Traumatismos del Sistema Nervioso/diagnóstico , Traumatismos del Sistema Nervioso/etiologíaRESUMEN
(1) Background: The cannabinoid 2 receptor (CB2R) is a promising anti-inflammatory drug target and development of selective CB2R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB2R ligands: CB2R agonists, RO6871304, and RO6871085, as well as a CB2R inverse agonist, RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB2R interactions, binding, cell signaling (ß-arrestin and cAMP) and early absorption, distribution, metabolism, excretion, and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity, in comparison to the reported CB2R ligand, HU910, using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB2R-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB2R-/- mice, and in vivo in WT mice with EIU using adoptive transfer of WT and CB2R-/- neutrophils, respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB2R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB2R and selectivity for CB2R > CB1R, with both ligands acting as full agonists in cAMP and ß-arrestin assays (EC50s in low nM range). When tested in EIU, topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist, RO6851228. The CB2R agonist, RO6871304, decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB2R-/-, and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB2R and have good immunomodulating actions in the eye. RO6871304 and RO6871085, as well as HU910, decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB2R agonists support selective targeting of CB2R for treating ocular inflammatory diseases.
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Antiinflamatorios/administración & dosificación , Agonistas de Receptores de Cannabinoides/administración & dosificación , Endotoxinas/efectos adversos , Receptor Cannabinoide CB2/antagonistas & inhibidores , Uveítis/tratamiento farmacológico , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Agonistas de Receptores de Cannabinoides/química , Agonistas de Receptores de Cannabinoides/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Masculino , Ratones , Ratones Noqueados , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Receptor Cannabinoide CB2/química , Receptor Cannabinoide CB2/genética , Transducción de Señal , Uveítis/inducido químicamente , Uveítis/inmunologíaRESUMEN
Activation of dopamine receptor 2 long (D2L) switches the signaling of type 1 cannabinoid receptor (CB1) from Gαi to Gαs, a process thought to be mediated through CB1-D2L heteromerization. Given the clinical importance of D2 antagonists, the goal of this study was to determine if D2 antagonists could modulate CB1 signaling. Interactions between CB1 and D2L, Gαi, Gαs, and ß-arrestin1 were studied using bioluminescence resonance energy transfer 2 (BRET(2)) in STHdh(Q7/Q7) cells. CB1-dependent extracellular regulated kinase (ERK)1/2, CREB phosphorylation, and CB1 internalization following cotreatment of CB1 agonist and D2 antagonist were quantified. Preassembled CB1-Gαi complexes were detected by BRET(2) Arachidonyl-2'-chloroethylamide (ACEA), a selective CB1 agonist, caused a rapid and transient increase in BRET efficiency (BRETEff) between Gαi-Rluc and CB1-green fluorescent protein 2 (GFP(2)), and a Gαi-dependent increase in ERK phosphorylation. Physical interactions between CB1 and D2L were observed using BRET(2) Cotreatment of STHdh(Q7/Q7) cells with ACEA and haloperidol, a D2 antagonist, inhibited BRETEff signals between Gαi-Rluc and CB1-GFP(2) and reduced the EMax and pEC50 of ACEA-mediated Gαi-dependent ERK phosphorylation. ACEA and haloperidol cotreatments produced a delayed and sustained increase in BRETEff between Gαs-Rluc and CB1-GFP(2) and increased the EMax and pEC50 of ACEA-induced Gαs-dependent cAMP response element-binding protein phosphorylation. In cells expressing CB1 and D2L treated with ACEA, binding of haloperidol to D2 receptors switched CB1 coupling from Gαi to Gαs In addition, haloperidol treatment reduced ACEA-induced ß-arrestin1 recruitment to CB1 and CB1 internalization. D2 antagonists allosterically modulate cannabinoid-induced CB1 coupling, signaling, and ß-arrestin1 recruitment through binding to CB1-D2L heteromers. These findings indicate that D2 antagonism, like D2 agonists, change agonist-mediated CB1 coupling and signaling.
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Cuerpo Estriado/citología , Antagonistas de los Receptores de Dopamina D2/farmacología , Neuronas/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptores de Dopamina D2/metabolismo , Animales , Ácidos Araquidónicos/farmacología , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Endocitosis/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Haloperidol/farmacología , Humanos , Cinética , Masculino , Ratones Endogámicos C57BL , Modelos Biológicos , Neuronas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Isoformas de Proteínas/metabolismo , Multimerización de Proteína/efectos de los fármacos , beta-Arrestinas/metabolismoRESUMEN
Huntington disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder with limited treatment options. Prior to motor symptom onset or neuronal cell loss in HD, levels of the type 1 cannabinoid receptor (CB1) decrease in the basal ganglia. Decreasing CB1 levels are strongly correlated with chorea and cognitive deficit. CB1 agonists are functionally selective (biased) for divergent signaling pathways. In this study, six cannabinoids were tested for signaling bias in in vitro models of medium spiny projection neurons expressing wild-type (STHdh(Q7/Q7)) or mutant huntingtin protein (STHdh(Q111/Q111)). Signaling bias was assessed using the Black and Leff operational model. Relative activity [ΔlogR (τ/KA)] and system bias (ΔΔlogR) were calculated relative to the reference compound WIN55,212-2 for Gαi/o, Gαs, Gαq, Gßγ, and ß-arrestin1 signaling following treatment with 2-arachidonoylglycerol (2-AG), anandamide (AEA), CP55,940, Δ(9)-tetrahydrocannabinol (THC), cannabidiol (CBD), and THC+CBD (1:1), and compared between wild-type and HD cells. The Emax of Gαi/o-dependent extracellular signal-regulated kinase (ERK) signaling was 50% lower in HD cells compared with wild-type cells. 2-AG and AEA displayed Gαi/o/Gßγ bias and normalized CB1 protein levels and improved cell viability, whereas CP55,940 and THC displayed ß-arrestin1 bias and reduced CB1 protein levels and cell viability in HD cells. CBD was not a CB1 agonist but inhibited THC-dependent signaling (THC+CBD). Therefore, enhancing Gαi/o-biased endocannabinoid signaling may be therapeutically beneficial in HD. In contrast, cannabinoids that are ß-arrestin-biased--such as THC found at high levels in modern varieties of marijuana--may be detrimental to CB1 signaling, particularly in HD where CB1 levels are already reduced.
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Supervivencia Celular/fisiología , Enfermedad de Huntington/metabolismo , Neuronas/metabolismo , Receptor Cannabinoide CB1/metabolismo , Animales , Cannabidiol/metabolismo , Cannabidiol/farmacología , Cannabinoides/metabolismo , Cannabinoides/farmacología , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Receptor Cannabinoide CB1/agonistas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Glaucoma is an irreversible blinding eye disease which produces progressive retinal ganglion cell (RGC) loss. Intraocular pressure (IOP) is currently the only modifiable risk factor, and lowering IOP results in reduced risk of progression of the disorder. The endocannabinoid system (ECS) has attracted considerable attention as a potential target for the treatment of glaucoma, largely due to the observed IOP lowering effects seen after administration of exogenous cannabinoids. However, recent evidence has suggested that modulation of the ECS may also be neuroprotective. This paper will review the use of cannabinoids in glaucoma, presenting pertinent information regarding the pathophysiology of glaucoma and how alterations in cannabinoid signalling may contribute to glaucoma pathology. Additionally, the mechanisms and potential for the use of cannabinoids and other novel agents that target the endocannabinoid system in the treatment of glaucoma will be discussed.
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Cannabinoides/uso terapéutico , Endocannabinoides/metabolismo , Glaucoma/tratamiento farmacológico , Glaucoma/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Animales , Agonistas de Receptores de Cannabinoides/metabolismo , Agonistas de Receptores de Cannabinoides/uso terapéutico , Cannabinoides/metabolismo , Humanos , Presión Intraocular/efectos de los fármacos , Fármacos Neuroprotectores/metabolismo , Receptores de Cannabinoides/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Modulation of type 1 cannabinoid receptor (CB1) activity has been touted as a potential means of treating addiction, anxiety, depression, and neurodegeneration. Different agonists of CB1 are known to evoke varied responses in vivo. Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor that can signal through multiple pathways. To understand cannabinoid-specific functional selectivity, different groups have examined the effect of individual cannabinoids on various signaling pathways in heterologous expression systems. In the current study, we compared the functional selectivity of six cannabinoids, including two endocannabinoids (2-arachidonyl glycerol (2-AG) and anandamide (AEA)), two synthetic cannabinoids (WIN55,212-2 and CP55,940), and two phytocannabinoids (cannabidiol (CBD) and Δ(9)-tetrahydrocannabinol (THC)) on arrestin2-, Gα(i/o)-, Gßγ-, Gα(s)-, and Gα(q)-mediated intracellular signaling in the mouse STHdh(Q7/Q7) cell culture model of striatal medium spiny projection neurons that endogenously express CB1. In this system, 2-AG, THC, and CP55,940 were more potent mediators of arrestin2 recruitment than other cannabinoids tested. 2-AG, AEA, and WIN55,212-2, enhanced Gα(i/o) and Gßγ signaling, with 2-AG and AEA treatment leading to increased total CB1 levels. 2-AG, AEA, THC, and WIN55,212-2 also activated Gα(q)-dependent pathways. CP55,940 and CBD both signaled through Gα(s). CP55,940, but not CBD, activated downstream Gα(s) pathways via CB1 targets. THC and CP55,940 promoted CB1 internalization and decreased CB1 protein levels over an 18-h period. These data demonstrate that individual cannabinoids display functional selectivity at CB1 leading to activation of distinct signaling pathways. To effectively match cannabinoids with therapeutic goals, these compounds must be screened for their signaling bias.
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Agonistas de Receptores de Cannabinoides/farmacología , Cannabinoides/farmacología , Neuronas/metabolismo , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/metabolismo , Animales , Ácidos Araquidónicos/farmacología , Arrestina/genética , Arrestina/metabolismo , Benzoxazinas/farmacología , Western Blotting , Células Cultivadas , Cuerpo Estriado/citología , Cuerpo Estriado/metabolismo , Ciclohexanoles/farmacología , Espinas Dendríticas/metabolismo , Dronabinol/farmacología , Endocannabinoides/farmacología , Transferencia Resonante de Energía de Fluorescencia , Proteínas de Unión al GTP/metabolismo , Glicéridos/farmacología , Ligandos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Modelos Biológicos , Morfolinas/farmacología , Naftalenos/farmacología , Neuronas/citología , Alcamidas Poliinsaturadas/farmacología , Receptor Cannabinoide CB1/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Microglia are resident immune cells of the central nervous system (CNS) and propagate inflammation following damage to the CNS, including the retina. Proliferative vitreoretinopathy (PVR) is a condition that can emerge following retinal detachment and is characterized by severe inflammation and microglial proliferation. The type 2 cannabinoid receptor (CB2) is an emerging pharmacological target to suppress microglial-mediated inflammation when the eyes or brain are damaged. CB2-knockout mice have exacerbated inflammation and retinal pathology during experimental PVR. We aimed to assess the anti-inflammatory effects of CB2 stimulation in the context of retinal damage and also explore the mechanistic roles of CB2 in microglia function. To target CB2, we used a highly selective agonist, HU-308, as well as its enantiomer, HU-433, which is a putative selective agonist. First, ß-arrestin2 and Gαi recruitment was measured to compare activation of human CB2 in an in vitro heterologous expression system. Both agonists were then utilized in a mouse model of PVR, and the effects on retinal damage, inflammation, and cell death were assessed. Finally, we used an in vitro model of microglia to determine the effects of HU-308 and HU-433 on phagocytosis, cytokine release, migration, and intracellular signaling. We observed that HU-308 more strongly recruited both ß-arrestin2 and Gαi compared to HU-433. Stimulation of CB2 with either drug effectively blunted LPS- and IFNγ-mediated signaling as well as NO and TNF release from microglia. Furthermore, both drugs reduced IL-6 accumulation, total caspase-3 cleavage, and retinal pathology following the induction of PVR. Ultimately, this work supports that CB2 is a valuable target for drugs to suppress inflammation and cell death associated with infection or sterile retinopathy, although the magnitude of effector recruitment may not be predictive of anti-inflammatory capacity.
RESUMEN
Introduction: Preclinical studies suggest that cannabinoid receptor type 2 (CB2R) activation has a therapeutic effect in animal models on chronic inflammation and vascular permeability, which are key pathological features of diabetic retinopathy (DR). A novel CB2R agonist, triazolopyrimidine RG7774, was generated through lead optimization of a high-throughput screening hit. The aim of this study was to characterize the pharmacology, absorption, distribution, metabolism, elimination, and toxicity (ADMET) profile of RG7774, and to explore its potential for managing the key pathological features associated with retinal disease in rodents. Methods: The in vitro pharmacology of RG7774 was investigated for CB2R binding and receptor activation using recombinant human and mouse CB2R expression in Chinese hamster ovary cells, and endogenous CB2R expression in human Jurkat cells, and rat and mouse spleen cells. The ADMET profile was evaluated and the effects of RG7774 on retinal permeability, leukocyte adhesion, and choroidal neovascularization (CNV) were investigated in rodent models of retinal disease. Pharmacokinetic (PK) parameters and the exposure-response relationship were characterized in healthy animals and in animals with laser-induced CNV. Results: RG7774 was found to be a potent (EC50: 2.8 nM and Ki: 51.3 nM), selective, and full CB2R agonist with no signs of cannabinoid receptor type 1 (CB1R) binding or activation. The ligand showed a favorable ADMET profile and exhibited systemic and ocular exposure after oral delivery. Functional potency in vitro translated from recombinant to endogenous expression systems. In vivo, orally administered RG7774 reduced retinal permeability and leukocyte adhesion in rodents with lipopolysaccharide (LPS)-induced uveitis and streptozotocin (STZ)-induced DR, and reduced lesion areas in rats with laser-induced CNV with an ED50 of 0.32 mg/kg. Anatomically, RG7774 reduced the migration of retinal microglia to retinal lesions. Discussion: RG7774 is a novel, highly selective, and orally bioavailable CB2R agonist, with an acceptable systemic and ocular PK profile, and beneficial effects on retinal vascular permeability, leukocyte adhesion, and ocular inflammation in rodent animal models. Results support the development of RG7774 as a potential treatment for retinal diseases with similar pathophysiologies as addressed by the animal models.
RESUMEN
BACKGROUND: Changes in leukocyte-endothelial and microvascular perfusion are hallmark events in inflammation. Thus, protection of the intestinal microcirculation represents a pivotal therapeutic target in systemic inflammation and sepsis. The endocannabinoid system (ECS) modulates a number of critical homeostatic functions and has been associated with anti-inflammatory responses. Our study aimed to examine intestinal leukocyte adhesion and capillary perfusion following selective inhibition of the endocannabinoid degradation enzyme, fatty acid amide hydrolase (FAAH), in experimental sepsis (endotoxemia). METHODS: Five groups of rats were used: controls, endotoxemia [lipopolysaccharide (LPS)], FAAH inhibitor URB597 (0.3 mg/kg)+LPS, URB597 (0.6 mg/kg)+LPS, and URB597 (0.6 mg/kg)+cannabinoid 2 receptor (CB2R) antagonist (AM630)+LPS. After 2 h, intravital microscopy was performed to quantify intestinal leukocyte recruitment and functional capillary density (FCD), as well as macrohemodynamic monitoring and histological examinations. RESULTS: LPS induced a significant increase in leukocyte adhesion in collecting and postcapillary submucosal venules and a decrease in intestinal FCD. URB597 pretreatment prevented the LPS-induced increase in leukocyte adhesion in intestinal venules and a decrease in intestinal FCD. The administration of the CB2R inhibitor, AM630, with URB597 reversed the protective effects of URB597 on the LPS-induced increase in leukocyte adhesion in intestinal venules, but not URB597's effect on the intestinal FCD. CONCLUSIONS: FAAH inhibition prevents the LPS-induced increase in leukocyte adhesion and improves the capillary perfusion of the gut. This might be mediated in part by CB2R activation. Our study encourages further investigation into the therapeutic potential of drugs targeting the ECS in sepsis.
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Amidohidrolasas/antagonistas & inhibidores , Endocannabinoides/metabolismo , Endotoxemia/fisiopatología , Leucocitos/metabolismo , Amidohidrolasas/metabolismo , Animales , Benzamidas/farmacología , Antagonistas de Receptores de Cannabinoides/farmacología , Capilares/metabolismo , Carbamatos/farmacología , Adhesión Celular , Modelos Animales de Enfermedad , Indoles/farmacología , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Leucocitos/efectos de los fármacos , Lipopolisacáridos/toxicidad , Masculino , Ratas , Ratas Endogámicas Lew , Receptor Cannabinoide CB2/efectos de los fármacos , Receptor Cannabinoide CB2/metabolismoRESUMEN
Acute corneal pain is a common complaint that causes significant distress to patients and continues to challenge therapeutic avenues for pain management. Current topical treatment options have marked limitations in terms of both efficacy and safety, thus often prompting the adjunctive use of systemic analgesics, including opioids. In general, there have not been extensive advancements in pharmacologic options for the management of corneal pain over the past several decades. Despite this, multiple promising therapeutic avenues exist which hold the potential to transform the ocular pain landscape, including druggable targets within the endocannabinoid system. This review will summarize the current evidence base for topical nonsteroidal anti-inflammatory drugs, anticholinergic agents, and anesthetics before focusing on several potential avenues in the setting of acute corneal pain management, including autologous tear serum, topical opioids and endocannabinoid system modulators.
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Analgésicos , Endocannabinoides , Humanos , Endocannabinoides/uso terapéutico , Analgésicos/uso terapéutico , Dolor/tratamiento farmacológico , Antiinflamatorios no Esteroideos/uso terapéutico , Manejo del Dolor , Analgésicos OpioidesRESUMEN
Intraocular pressure (IOP) is the primary risk factor for glaucoma, a blinding eye disease. Cannabinoid agonists have long been known to decrease IOP, suggesting they may be useful in glaucoma treatment. However, the specific mechanism by which cannabinoids generate this ocular hypotensive effect remains unknown. The current evidence suggests the cannabinoids reduce IOP through actions at cannabinoid 1 (CB(1)) receptors within the eye, and adrenergic receptors (ARs) may also contribute to this action of cannabinoids. Considering this, the present study aimed to elucidate the mechanism behind the ocular hypotensive properties of cannabinoids through the use of mice genetically lacking either cannabinoid receptors or ßARs. Cannabinoid agonists, ßAR antagonists, and ßAR agonists decreased IOP in wild-type mice and CB(2)(-/-) mice. In contrast, none of these compounds were found to reduce IOP in ßAR(-/-) or CB(1)(-/-) mice. Desensitization of the ßARs and depletion of catecholamines in wild-type mice also eliminated the ability of the cannabinoid agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2) to reduce IOP, strongly implicating a role for both ßARs and catecholamines in the ocular hypotensive properties of cannabinoids. Finally, CB(1) receptors were shown to colocalize with tyrosine hydroxylase, a marker for adrenergic neurons. Taken together, these findings suggest that ßARs are required for the ocular hypotensive properties of cannabinoids, and cannabinoids reduce IOP by acting as indirect sympatholytics and inhibiting norepinephrine release within the eye.
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Antagonistas Adrenérgicos beta/farmacología , Benzoxazinas/farmacología , Agonistas de Receptores de Cannabinoides , Moduladores de Receptores de Cannabinoides/farmacología , Presión Intraocular/efectos de los fármacos , Morfolinas/farmacología , Naftalenos/farmacología , Receptores Adrenérgicos beta/metabolismo , Simpaticolíticos/farmacología , Neuronas Adrenérgicas/efectos de los fármacos , Neuronas Adrenérgicas/fisiología , Agonistas Adrenérgicos beta/farmacología , Animales , Catecolaminas/fisiología , Ritmo Circadiano , Evaluación Preclínica de Medicamentos , Ojo/efectos de los fármacos , Presión Intraocular/fisiología , Latanoprost , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Hipotensión Ocular/inducido químicamente , Prostaglandinas F Sintéticas/farmacología , Receptores de Cannabinoides/metabolismoRESUMEN
The human brain requires adequate cerebral blood flow to meet the high demand for nutrients and to clear waste products. With age, there is a chronic reduction in cerebral blood flow in small resistance arteries that can eventually limit proper brain function. The endothelin system is a key mediator in the regulation of cerebral blood flow, but the contributions of its constituent receptors in the endothelial and vascular smooth muscle layers of cerebral arteries have not been well defined in the context of aging. We isolated posterior cerebral arteries from young and aged Fischer 344 rats, as well as ETB receptor knock-out rats and mounted the vessels in plexiglass pressure myograph chambers to measure myogenic tone in response to increasing pressure and targeted pharmacological treatments. We used an ETA receptor antagonist (BQ-123), an ETB receptor antagonist (BQ-788), endothelin-1, an endothelin-1 synthesis inhibitor (phosphoramidon), and vessel denudation to dissect the roles of each receptor in aging vasculature. Aged rats exhibited a higher myogenic tone than young rats, and the tone was sensitive to the ETA antagonist, BQ-123, but insensitive to the ETB antagonist, BQ-788. By contrast, the tone in the vessels from young rats was raised by BQ-788 but unaffected by BQ-123. When the endothelial layer that is normally enriched with ETB1 receptors was removed from young vessels, myogenic tone increased. However, denudation of the endothelial layer did not influence vessels from aged animals. This indicated that endothelial ETB1 receptors were not functional in the vessels from aged rats. There was also an increase in ETA receptor expression with age, whereas ETB receptor expression remained constant between young and aged animals. These results demonstrate that in young vessels, ETB1 receptors maintain a lower myogenic tone, but in aged vessels, a loss of ETB receptor activity allows ETA receptors in vascular smooth muscle cells to raise myogenic tone. Our findings have potentially important clinical implications for treatments to improve cerebral perfusion in older adults with diseases characterized by reduced cerebral blood flow.
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Arterias Cerebrales , Receptor de Endotelina B , Vasoconstricción , Animales , Técnicas de Inactivación de Genes , Masculino , Ratas , Ratas Endogámicas F344RESUMEN
We apply the magic methyl effect to improve the potency/efficacy of GAT211, the prototypic 2-phenylindole-based cannabinoid type-1 receptor (CB1R) agonist-positive allosteric modulator (ago-PAM). Introducing a methyl group at the α-position of nitro group generated two diastereomers, the greater potency and efficacy of erythro, (±)-9 vs threo, (±)-10 constitutes the first demonstration of diastereoselective CB1R-allosteric modulator interaction. Of the (±)-9 enantiomers, (-)-(S,R)-13 evidenced improved potency over GAT211 as a CB1R ago-PAM, whereas (+)-(R,S)-14 was a CB1R allosteric agonist biased toward G protein- vs ß-arrestin1/2-dependent signaling. (-)-(S,R)-13 and (+)-(R,S)-14 were devoid of undesirable side effects (triad test), and (+)-(R,S)-14 reduced intraocular pressure with an unprecedentedly long duration of action in a murine glaucoma model. (-)-(S,R)-13 docked into both a CB1R extracellular PAM and intracellular allosteric-agonist site(s), whereas (+)-(R,S)-14 preferentially engaged only the latter. Exploiting G-protein biased CB1R-allosteric modulation can offer safer therapeutic candidates for glaucoma and, potentially, other diseases.
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Agonistas de Receptores de Cannabinoides/uso terapéutico , Glaucoma/tratamiento farmacológico , Indoles/uso terapéutico , Receptor Cannabinoide CB1/agonistas , Sitio Alostérico , Animales , Células CHO , Agonistas de Receptores de Cannabinoides/síntesis química , Agonistas de Receptores de Cannabinoides/metabolismo , Cricetulus , Células HEK293 , Hipocampo/citología , Humanos , Indoles/síntesis química , Indoles/metabolismo , Presión Intraocular/efectos de los fármacos , Ligandos , Masculino , Ratones Endogámicos C57BL , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Neuronas/efectos de los fármacos , Receptor Cannabinoide CB1/química , Receptor Cannabinoide CB1/metabolismo , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Seven-transmembrane G protein-coupled receptors (GPCRs) represent the single largest family of cell surface receptors. Signaling through these receptors is controlled by changes in the conformation of the receptor from inactive to active conformations, which in turn lead to the activation of multiple downstream signaling pathways. To facilitate greater diversity in signaling responses, many of these receptors are capable of adopting several distinct active conformations, in which each couples preferentially to its own set of downstream signaling partners. Because these unique signaling responses result from specific receptor active conformations, GPCR signaling may be directed toward these selective responses through either strength-of-signal effects resulting from partial agonism or through biased agonism and functional selectivity, resulting from the selective stabilization of one active conformation over the others. This review uses the CB(1) cannabinoid receptor as a specific example to highlight the contribution of two important aspects of GPCR function-orthosteric ligand binding and receptor heterodimerization-toward directed GPCR signaling.