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The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo, optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency (K i 0.13-1.81 nM, depending on species) and a peripherally restricted action due to P-glycoprotein-mediated efflux from the brain. 3H and 14C labeled RNB-61 showed apparent K d values of <4 nM toward human CB2R in both cell and tissue experiments. The 6,800-fold selectivity over CB1 receptors and negligible off-targets in vitro, combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands.
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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
The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo, optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency (K i 0.13-1.81 nM, depending on species) and a peripherally restricted action due to P-glycoprotein mediated efflux from the brain. 3H and 14C labelled RNB-61 showed apparent K d values < 4 nM towards human CB2R in both cell and tissue experiments. The >6000-fold selectivity over CB1 receptors and negligible off-targets in vitro, combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands.
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Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs.
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The G-protein-coupled cannabinoid receptor type 2 (CB2R) is a key element of the endocannabinoid (EC) system. EC/CB2R signaling has significant therapeutic potential in major pathologies affecting humans such as allergies, neurodegenerative disorders, inflammation or ocular diseases. CB2R agonism exerts anti-inflammatory and tissue protective effects in preclinical animal models of cardiovascular, gastrointestinal, liver, kidney, lung and neurodegenerative disorders. Existing ligands can be subdivided into endocannabinoids, cannabinoid-like and synthetic CB2R ligands that possess various degrees of potency on and selectivity against the cannabinoid receptor type 1. This review is an account of granted CB2R ligand patents from 2010 up to the present, which were surveyed using Derwent Innovation®.
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Antiinflamatorios , Endocannabinoides , Animales , Humanos , Ligandos , Patentes como Asunto , Receptores de Cannabinoides , Transducción de SeñalRESUMEN
Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across applications, species, and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease.
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Enfermedad de Alzheimer/metabolismo , Colorantes Fluorescentes/química , Microglía/metabolismo , Receptor Cannabinoide CB2/análisis , Animales , Células CHO , Cricetulus , Modelos Animales de Enfermedad , Citometría de Flujo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Ligandos , Ratones , Simulación del Acoplamiento Molecular , Sondas Moleculares/química , Imagen Óptica , Sensibilidad y Especificidad , Transducción de SeñalRESUMEN
The cannabinoid type 2 (CB2) receptor has emerged as a valuable target for therapy and imaging of immune-mediated pathologies. With the aim to find a suitable radiofluorinated analogue of the previously reported CB2 positron emission tomography (PET) radioligand [11C]RSR-056, 38 fluorinated derivatives were synthesized and tested by in vitro binding assays. With a Ki (hCB2) of 6 nM and a selectivity factor of nearly 700 over cannabinoid type 1 receptors, target compound 3 exhibited optimal in vitro properties and was selected for evaluation as a PET radioligand. [18F]3 was obtained in an average radiochemical yield of 11 ± 4% and molar activities between 33 and 114 GBq/µmol. Specific binding of [18F]3 to CB2 was demonstrated by in vitro autoradiography and in vivo PET experiments using the CB2 ligand GW-405â¯833. Metabolite analysis revealed only intact [18F]3 in the rat brain. [18F]3 detected CB2 upregulation in human amyotrophic lateral sclerosis spinal cord tissue and may thus become a candidate for diagnostic use in humans.
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Encéfalo/metabolismo , Radioisótopos de Flúor/metabolismo , Neuroimagen/métodos , Tomografía de Emisión de Positrones/métodos , Piridinas/química , Radiofármacos/metabolismo , Receptor Cannabinoide CB2/metabolismo , Animales , Encéfalo/diagnóstico por imagen , AMP Cíclico/metabolismo , Radioisótopos de Flúor/química , Hepatocitos/metabolismo , Humanos , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Conformación Proteica , Radioquímica , Radiofármacos/química , Ratas , Ratas Wistar , Receptor Cannabinoide CB2/química , Relación Estructura-ActividadRESUMEN
(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
Age-related macular degeneration (AMD) is a progressive disease of the retinal pigment epithelium (RPE) and the retina leading to loss of central vision. Polymorphisms in genes involved in lipid metabolism, including the ATP-binding cassette transporter A1 (ABCA1), have been associated with AMD risk. However, the significance of retinal lipid handling for AMD pathogenesis remains elusive. Here, we study the contribution of lipid efflux in the RPE by generating a mouse model lacking ABCA1 and its partner ABCG1 specifically in this layer. Mutant mice show lipid accumulation in the RPE, reduced RPE and retinal function, retinal inflammation and RPE/photoreceptor degeneration. Data from human cell lines indicate that the ABCA1 AMD risk-conferring allele decreases ABCA1 expression, identifying the potential molecular cause that underlies the genetic risk for AMD. Our results highlight the essential homeostatic role for lipid efflux in the RPE and suggest a pathogenic contribution of reduced ABCA1 function to AMD.
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Transportador 1 de Casete de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Metabolismo de los Lípidos , Degeneración Retiniana/fisiopatología , Epitelio Pigmentado de la Retina/fisiopatología , Transportador 1 de Casete de Unión a ATP/deficiencia , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/deficiencia , Animales , Línea Celular , Modelos Animales de Enfermedad , Humanos , Inflamación/patología , Ratones , Células Fotorreceptoras/patologíaRESUMEN
Astrocytes are involved in non-cell-autonomous pathogenic cascades in amyotrophic lateral sclerosis (ALS); however, their role is still debated. We show that astrocytic NF-κB activation drives microglial proliferation and leukocyte infiltration in the SOD1 (G93A) ALS model. This response prolongs the presymptomatic phase, delaying muscle denervation and decreasing disease burden, but turns detrimental in the symptomatic phase, accelerating disease progression. The transition corresponds to a shift in the microglial phenotype showing two effects that can be dissociated by temporally controlling NF-κB activation. While NF-κB activation in astrocytes induced a Wnt-dependent microglial proliferation in the presymptomatic phase with neuroprotective effects on motoneurons, in later stage, astrocyte NF-κB-dependent microglial activation caused an accelerated disease progression. Notably, suppression of the early microglial response by CB2R agonists had acute detrimental effects. These data identify astrocytes as important regulators of microglia expansion and immune response. Therefore, stage-dependent microglia modulation may be an effective therapeutic strategy in ALS.
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Esclerosis Amiotrófica Lateral/inmunología , Astrocitos/inmunología , FN-kappa B/inmunología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/terapia , Animales , Astrocitos/patología , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Microglía/inmunología , Microglía/patología , Neuronas Motoras/inmunología , Neuronas Motoras/patología , FN-kappa B/genética , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/genética , Receptor Cannabinoide CB2/inmunología , Superóxido Dismutasa/genética , Superóxido Dismutasa/inmunología , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/inmunologíaRESUMEN
Genetic studies have linked age-related macular degeneration (AMD) to genes involved in high-density lipoprotein (HDL) metabolism, including ATP-binding cassette transporter A1 (ABCA1). The retinal pigment epithelium (RPE) handles large amounts of lipids, among others cholesterol, partially derived from internalized photoreceptor outer segments (OS) and lipids physiologically accumulate in the aging eye. To analyze the potential function of ABCA1 in the eye, we measured cholesterol efflux, the first step of HDL generation, in RPE cells. We show the expression of selected genes related to HDL metabolism in mouse and human eyecups as well as in ARPE-19 and human primary RPE cells. Immunofluorescence staining revealed localization of ABCA1 on both sides of polarized RPE cells. This was functionally confirmed by directional efflux to apolipoprotein AI (ApoA-I) of 3H-labeled cholesterol given to the cells via serum or via OS. ABCA1 expression and activity was modulated using a liver-X-receptor (LXR) agonist and an ABCA1 neutralizing antibody, demonstrating that the efflux was ABCA1-dependent. We concluded that the ABCA1-mediated lipid efflux pathway, and hence HDL biosynthesis, is functional in RPE cells towards both the basal (choroidal) and apical (subretinal) space. Impaired activity of the pathway might cause age-related perturbations of lipid homeostasis in the outer retina and thus may contribute to disease development and/or progression.
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Colesterol/metabolismo , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismo , Animales , Western Blotting , Células Cultivadas , Células Epiteliales/metabolismo , Humanos , Ratones , Reacción en Cadena de la Polimerasa , PorcinosRESUMEN
The cannabinoid CB2 receptor (CB2R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB2R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB2R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB2R agonists to study the role of CB2R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research.
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Agonistas de Receptores de Cannabinoides/farmacología , Neuronas/efectos de los fármacos , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Transducción de Señal , Animales , Compuestos Bicíclicos con Puentes/farmacología , Células CHO , Cannabinoides/farmacología , Línea Celular Tumoral , Cricetulus , Expresión Génica , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Cinética , Ligandos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Neuronas/citología , Neuronas/metabolismo , Unión Proteica , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB2/genéticaRESUMEN
The cannabinoid receptor 2 (CB2) system is described to modulate various pathological conditions, including inflammation and fibrosis. A series of new heterocyclic small-molecule CB2 receptor agonists were identified from a high-throughput screen. Lead optimization gave access to novel, highly potent, and selective (over CB1) triazolopyrimidine derivatives. A preliminary structure-activity relationship was established, and physicochemical properties in this compound class were significantly improved toward better solubility, lipophilicity, and microsomal stability. An optimized triazolopyrimidine derivative, (3S)-1-[5-tert-butyl-3-[(1-cyclopropyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol (39), was tested in a kidney ischemia-reperfusion model, in which it showed efficacy at a dose of 10 mg kg(-1) (p.o.). A significant depletion of the three measured kidney markers indicated a protective role of CB2 receptor activation toward inflammatory kidney damage. Compound 39 was also protective in a model of renal fibrosis. Oral treatment with 39 at 3 mg kg(-1) per day significantly decreased the amount of fibrosis by â¼ 40% which was induced by unilateral ureter obstruction.
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Antiinflamatorios no Esteroideos/farmacología , Agonistas de Receptores de Cannabinoides/farmacología , Inflamación/tratamiento farmacológico , Enfermedades Renales/tratamiento farmacológico , Pirimidinas/farmacología , Receptor Cannabinoide CB2/agonistas , Triazoles/farmacología , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Agonistas de Receptores de Cannabinoides/síntesis química , Agonistas de Receptores de Cannabinoides/química , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/químicaRESUMEN
2-Methoxyestradiol (2-ME), a metabolite of estradiol with little affinity for estrogen receptors, inhibits proliferation of vascular smooth muscle cells; however, the molecular mechanisms underlying this effect are incompletely understood. Our previous work shows that 2-ME inhibits initiation (blocks phosphorylation of ERK and Akt) and progression (reduces cyclin expression and increases expression of cyclin inhibitors) of the mitogenic pathway and interferes with mitosis (disrupts tubulin organization). Because the RhoA/ROCK1 pathway (RhoA â ROCK1 â myosin phosphatase targeting subunit â myosin light chain) is involved in cytokinesis, herein we tested the concept that 2-ME also blocks the RhoA/ROCK1 pathway. Because of the potential importance of 2-ME for preventing/treating vascular diseases, experiments were conducted in female human aortic vascular smooth muscle cells. Microarray transcriptional profiling suggested an effect of 2-ME on the RhoA/ROCK1 pathway. Indeed, 2-ME blocked mitogen-induced GTP-bound RhoABC expression and membrane-bound RhoA, suggesting interference with the activation of RhoA. 2-ME also reduced ROCK1 expression, suggesting reduced production of the primary downstream signaling kinase of the RhoA pathway. Moreover, 2-ME inhibited RhoA/ROCK1 pathway downstream signaling, including phosphorylated myosin phosphatase targeting subunit and myosin light chain; the ROCK1 inhibitor H-1152 mimicked these effects of 2-ME; both 2-ME and H-1152 blocked cytokinesis. 2-ME also reduced the expression of tissue factor, yet another downstream signaling component of the RhoA/ROCK1 pathway. We conclude that 2-ME inhibits the pathway RhoA â ROCK1 â myosin phosphatase targeting subunit â myosin light chain, and this likely contributes to the reduced cytokinesis in 2-ME treated HASMCs.
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Citocinesis/fisiología , Estradiol/análogos & derivados , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , 2-Metoxiestradiol , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Células Cultivadas , Citocinesis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Estradiol/administración & dosificación , Femenino , Humanos , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
The goal of this study was to determine whether and how adenosine affects the proliferation of human coronary artery smooth muscle cells (HCASMCs). In HCASMCs, 2-chloroadenosine (stable adenosine analogue), but not N(6)-cyclopentyladenosine, CGS21680, or N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide, inhibited HCASMC proliferation (A2B receptor profile). 2-Chloroadenosine increased cAMP, reduced phosphorylation (activation) of ERK and Akt (protein kinases known to increase cyclin D expression and activity, respectively), and reduced levels of cyclin D1 (cyclin that promotes cell-cycle progression in G1). Moreover, 2-chloroadenosine inhibited expression of S-phase kinase-associated protein-2 (Skp2; promotes proteolysis of p27(Kip1)) and upregulated levels of p27(Kip1) (cell-cycle regulator that impairs cyclin D function). 2-Chloroadenosine also inhibited signaling downstream of cyclin D, including hyperphosphorylation of retinoblastoma protein and expression of cyclin A (S phase cyclin). Knockdown of A2B receptors prevented the effects of 2-chloroadenosine on ERK1/2, Akt, Skp2, p27(Kip1), cyclin D1, cyclin A, and proliferation. Likewise, inhibition of adenylyl cyclase and protein kinase A abrogated 2-chloroadenosine's inhibitory effects on Skp2 and stimulatory effects on p27(Kip1) and rescued HCASMCs from 2-chloroadenosine-mediated inhibition. Knockdown of p27(Kip1) also reversed the inhibitory effects of 2-chloroadenosine on HCASMC proliferation. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 µmol/L for 7 days) downregulated vascular expression of Skp2, upregulated vascular expression of p27(Kip1), and reduced neointima hyperplasia by 71% (P<0.05; neointimal thickness: control, 37 424±18 371 pixels; treated, 10 352±2824 pixels). In conclusion, the adenosine/A2B receptor/cAMP/protein kinase A axis inhibits HCASMC proliferation by blocking multiple signaling pathways (ERK1/2, Akt, and Skp2) that converge at cyclin D, a key G1 cyclin that controls cell-cycle progression.
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Adenosina/farmacología , Proliferación Celular/efectos de los fármacos , Ciclina D/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , 2-Cloroadenosina/farmacología , Animales , Western Blotting , Proliferación Celular/genética , Células Cultivadas , Vasos Coronarios/citología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclina D/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Masculino , Miocitos del Músculo Liso/metabolismo , Interferencia de ARN , Ratas Endogámicas WKY , Receptor de Adenosina A1/genética , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2B/genética , Receptor de Adenosina A2B/metabolismo , Proteínas Quinasas Asociadas a Fase-S/genética , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Transducción de Señal/genéticaRESUMEN
As part of our efforts to develop CB2 PET imaging agents, we investigated 2,5,6-substituted pyridines as a novel class of potential CB2 PET ligands. A total of 21 novel compounds were designed, synthesized, and evaluated for their potency and binding properties toward human and rodent CB1 and CB2. The most promising ligand 6a was radiolabeled with carbon-11 to yield 16 ([(11)C]RSR-056). Specific binding of 16 to CB2-positive spleen tissue of rats and mice was demonstrated by in vitro autogadiography and verified in vivo in PET and biodistribution experiments. Furthermore, 16 was evaluated in a lipopolysaccharid (LPS) induced murine model of neuroinflammation. Brain radioactivity was strikingly higher in the LPS-treated mice than the control mice. Compound 16 is a promising radiotracer for imaging CB2 in rodents. It might serve as a tool for the investigation of CB2 receptor expression levels in healthy tissues and different neuroinflammatory disorders in humans.
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Medios de Contraste/química , Medios de Contraste/farmacocinética , Tomografía de Emisión de Positrones/métodos , Receptor Cannabinoide CB2/análisis , Animales , Azetidinas/química , Azetidinas/farmacocinética , Células CHO , Cricetulus , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Estabilidad de Medicamentos , Humanos , Masculino , Ratones Endogámicos , Ácidos Picolínicos/química , Ácidos Picolínicos/farmacocinética , Piridinas/química , Ratas Wistar , Receptor Cannabinoide CB1/análisis , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/genética , Receptor Cannabinoide CB2/metabolismo , Distribución TisularRESUMEN
Chronic renal disease (CRD) accelerates the development of atherosclerosis. The potent protease cathepsin S cleaves elastin and generates bioactive elastin peptides, thus promoting vascular inflammation and calcification. We hypothesized that selective cathepsin S inhibition attenuates atherogenesis in hypercholesterolemic mice with CRD. CRD was induced by 5/6 nephrectomy in high-fat high-cholesterol fed apolipoprotein E-deficient mice. CRD mice received a diet admixed with 6.6 or 60 mg/kg of the potent and selective cathepsin S inhibitor RO5444101 or a control diet. CRD mice had significantly higher plasma levels of osteopontin, osteocalcin, and osteoprotegerin (204%, 148%, and 55%, respectively; P < 0.05), which were inhibited by RO5444101 (60%, 40%, and 36%, respectively; P < 0.05). Near-infrared fluorescence molecular imaging revealed a significant reduction in cathepsin activity in treated mice. RO5444101 decreased osteogenic activity. Histologic assessment in atherosclerotic plaque demonstrated that RO5444101 reduced immunoreactive cathepsin S (P < 0.05), elastin degradation (P = 0.01), plaque size (P = 0.01), macrophage accumulation (P < 0.01), growth differentiation factor-15 (P = 0.0001), and calcification (alkaline phosphatase activity, P < 0.01; osteocalcin, P < 0.05). Furthermore, cathepsin S inhibitor or siRNA significantly decreased expression of growth differentiation factor-15 and monocyte chemotactic protein-1 in a murine macrophage cell line and human primary macrophages. Systemic inhibition of cathepsin S attenuates the progression of atherosclerotic lesions in 5/6 nephrectomized mice, serving as a potential treatment for atherosclerosis in patients with CRD.
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Apolipoproteínas E/deficiencia , Aterosclerosis/patología , Catepsinas/antagonistas & inhibidores , Fallo Renal Crónico/enzimología , Fallo Renal Crónico/patología , Animales , Arterias/enzimología , Arterias/patología , Aterosclerosis/complicaciones , Biomarcadores/sangre , Catepsinas/metabolismo , Quimiocina CCL2/metabolismo , Factor 15 de Diferenciación de Crecimiento/metabolismo , Humanos , Interferón gamma/farmacología , Fallo Renal Crónico/sangre , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Osteogénesis/efectos de los fármacos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Calcificación Vascular/complicaciones , Calcificación Vascular/patologíaRESUMEN
A series of highly potent & selective adamantane derived CB2 agonists was identified in a high-throughput screen. A SAR was established and physicochemical properties were significantly improved. This was accompanied by potency of the compounds on the Q63R variant and varying ß-arrestin data which will support the insight into their relevance for the in vivo situation.
Asunto(s)
Adamantano/análogos & derivados , Agonistas de Receptores de Cannabinoides/química , Agonistas de Receptores de Cannabinoides/farmacología , Receptor Cannabinoide CB2/agonistas , Adamantano/farmacología , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
Apolipoprotein A-I (apoA-I) exerts many potentially anti-atherogenic properties and is therefore attractive for prevention and therapy of coronary heart disease. Since induction of apoA-I production by small molecules has turned out as difficult, application of exogenous apoA-I is pursued as an alternative therapeutic option. To counteract fast renal filtration of apoA-I, a trimeric high-molecular weight variant of apoA-I (TripA) was produced by recombinant technology. We compared TripA and apoA-I for important properties in reverse cholesterol transport. Reconstituted high-density lipoproteins (rHDL) containing TripA or apoA-I together with palmitoyl-2-oleyl-phosphatidylcholine (POPC) differed slightly by size. Compared to apoA-I, TripA activated lecithin:cholesterol acyltransferase (LCAT) with similar maximal velocity but concentration leading to half maximal velocity was slightly reduced (K(m)=2.1±0.3µg/mL vs. 0.59±0.06µg/mL). Both in the lipid-free form and as part of rHDL, TripA elicited cholesterol efflux from THP1-derived macrophages with similar kinetic parameters and response to liver-X-receptor activation as apoA-I. Lipid-free TripA is bound and transported by aortic endothelial cells through mechanisms which are competed by apoA-I and TripA and inhibited by knock-down of ATP-binding cassette transporter (ABC) A1. Pre-formed TripA/POPC particles were bound and transported by endothelial cells through mechanisms which are competed by excess native HDL as well as reconstituted HDL containing either apoA-I or TripA and which involve ABCG1 and scavenger receptor B1 (SR-BI). In conclusion, apoA-I and TripA show similar in vitro properties which are important for reverse cholesterol transport. These findings are important for further development of TripA as an anti-atherosclerotic drug.
Asunto(s)
Apolipoproteína A-I/metabolismo , Aterosclerosis/metabolismo , Fármacos Cardiovasculares/metabolismo , Enfermedad Coronaria/metabolismo , Células Endoteliales/metabolismo , Proteínas Recombinantes/metabolismo , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Apolipoproteína A-I/química , Apolipoproteína A-I/farmacología , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/patología , Fármacos Cardiovasculares/química , Fármacos Cardiovasculares/farmacología , Bovinos , Enfermedad Coronaria/tratamiento farmacológico , Enfermedad Coronaria/patología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Silenciador del Gen , Humanos , Cinética , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Fosfatidilcolinas/metabolismo , Unión Proteica , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologíaRESUMEN
AIMS: mast cells have been shown to accumulate in the adventitia of human atherosclerotic plaques and were recently demonstrated by us to contribute to plaque progression and instability. In this study, we investigated whether selective inhibition of mast cell chymases would affect the lesion development and stability. METHODS AND RESULTS: the protease inhibitor RO5066852 appeared to be a potent inhibitor of chymase activity in vitro and ex vivo. With this inhibitor, we provide three lines of evidence that chymase inhibition can prevent many pro-atherogenic activities. First, oral administration of RO5066852 reduced spontaneous atherosclerosis in the thoracic aorta of apoE(-/-) mice. Second, chymase inhibition prevented the accelerated plaque progression observed in apoE(-/-) mice that were exposed to repetitive episodes of systemic mast cell activation. Furthermore, RO5066852 enhanced lesional collagen content and reduced necrotic core size. Third, RO5066852 treatment almost completely normalized the increased frequency and size of intraplaque haemorrhages observed in apoE(-/-) mice after acute perivascular mast cell activation in advanced atherosclerosis. CONCLUSION: our data indicate that chymase inhibition can inhibit pro-atherogenic and plaque destabilizing effects which are associated with perivascular mast cell activation. Our study thus identifies pharmacological chymase inhibition as a potential therapeutic modality for atherosclerotic plaque stabilization.