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1.
ACS Chem Neurosci ; 12(21): 4020-4036, 2021 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-34676751

RESUMO

Synthetic cannabinoid receptor agonists (SCRAs) remain a prolific class of new psychoactive substances (NPS) and continue to expand rapidly. Despite the recent identification of hydroxybenzotriazole (HOBt) containing SCRAs in synthetic cannabis samples, there is currently no information regarding the pharmacological profile of these NPS with respect to human CB1 and CB2 receptors. In the current study, a series consisting of seven HOBt indole-, indazole-, and 7-azaindole-carboxylates bearing a range of N-alkyl substituents were synthesized and pharmacologically evaluated. Competitive binding assays at CB1 and CB2 demonstrated that all analogues except a 2-methyl-substituted derivative had low affinity for CB1 (Ki = 3.80-43.7 µM) and CB2 (Ki = 2.75-18.2 µM). A fluorometric functional assay revealed that 2-methylindole- and indole-derived HOBt carboxylates were potent and efficacious agonists of CB1 (EC50 = 12.0 and 63.7 nM; Emax = 118 and 120%) and CB2 (EC50 = 10.9 and 321 nM; Emax = 91 and 126%). All other analogues incorporating indazole and 7-azaindole cores and bearing a range of N1-substituents showed relatively low potency for CB1 and CB2. Additionally, a reporter assay monitoring ß-arrestin 2 (ßarr2) recruitment to the receptor revealed that the 2-methylindole example was the most potent and efficacious at CB1 (EC50 = 131 nM; Emax = 724%) and the most potent at CB2 (EC50 = 38.2 nM; Emax = 51%). As with the membrane potential assay, the indazole and other indole HOBt carboxylates were considerably less potent at both receptors, and analogues comprising a 7-azaindole core showed little activity. Taken together, these data suggest that NNL-3 demonstrates little CB1 receptor activity and is unlikely to be psychoactive in humans. NNL-3 is likely an unintended SCRA manufacturing byproduct. However, the synthesis of NNL-3 analogues proved simple and general, and some of these showed potent cannabimetic profiles in vitro, indicating that HOBt esters of this type may represent an emerging class of SCRA NPS.


Assuntos
Agonistas de Receptores de Canabinoides , Ésteres , Agonistas de Receptores de Canabinoides/farmacologia , Humanos , Indazóis/farmacologia , Receptor CB1 de Canabinoide , Receptor CB2 de Canabinoide , Receptores de Canabinoides , Transdução de Sinais
2.
Int J Mol Sci ; 22(17)2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34502379

RESUMO

The Endocannabinoid System (ECS) is primarily responsible for maintaining homeostasis, a balance in internal environment (temperature, mood, and immune system) and energy input and output in living, biological systems. In addition to regulating physiological processes, the ECS directly influences anxiety, feeding behaviour/appetite, emotional behaviour, depression, nervous functions, neurogenesis, neuroprotection, reward, cognition, learning, memory, pain sensation, fertility, pregnancy, and pre-and post-natal development. The ECS is also involved in several pathophysiological diseases such as cancer, cardiovascular diseases, and neurodegenerative diseases. In recent years, genetic and pharmacological manipulation of the ECS has gained significant interest in medicine, research, and drug discovery and development. The distribution of the components of the ECS system throughout the body, and the physiological/pathophysiological role of the ECS-signalling pathways in many diseases, all offer promising opportunities for the development of novel cannabinergic, cannabimimetic, and cannabinoid-based therapeutic drugs that genetically or pharmacologically modulate the ECS via inhibition of metabolic pathways and/or agonism or antagonism of the receptors of the ECS. This modulation results in the differential expression/activity of the components of the ECS that may be beneficial in the treatment of a number of diseases. This manuscript in-depth review will investigate the potential of the ECS in the treatment of various diseases, and to put forth the suggestion that many of these secondary metabolites of Cannabis sativa L. (hereafter referred to as "C. sativa L." or "medical cannabis"), may also have potential as lead compounds in the development of cannabinoid-based pharmaceuticals for a variety of diseases.


Assuntos
Canabinoides/farmacologia , Endocanabinoides/metabolismo , Endocanabinoides/fisiologia , Ansiedade/tratamento farmacológico , Agonistas de Receptores de Canabinoides/farmacologia , Cannabis/metabolismo , Doenças Cardiovasculares/tratamento farmacológico , Depressão/tratamento farmacológico , Comportamento Alimentar/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Neurogênese/efeitos dos fármacos , Dor/tratamento farmacológico , Receptores de Canabinoides/metabolismo
3.
J Med Chem ; 64(13): 9354-9364, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34161090

RESUMO

Cannabidiol (CBD), the second most abundant of the active compounds found in the Cannabis sativa plant, is of increasing interest because it is approved for human use and is neither euphorizing nor addictive. Here, we design and synthesize novel compounds taking into account that CBD is both a partial agonist, when it binds to the orthosteric site, and a negative allosteric modulator, when it binds to the allosteric site of the cannabinoid CB2 receptor. Molecular dynamic simulations and site-directed mutagenesis studies have identified the allosteric site near the receptor entrance. This knowledge has permitted to perform structure-guided design of negative and positive allosteric modulators of the CB2 receptor with potential therapeutic utility.


Assuntos
Produtos Biológicos/farmacologia , Canabidiol/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Desenho de Fármacos , Receptor CB2 de Canabinoide/agonistas , Sítio Alostérico/efeitos dos fármacos , Produtos Biológicos/síntese química , Produtos Biológicos/química , Canabidiol/síntese química , Canabidiol/química , Agonistas de Receptores de Canabinoides/síntese química , Agonistas de Receptores de Canabinoides/química , Cannabis/química , Relação Dose-Resposta a Droga , Humanos , Simulação de Dinâmica Molecular , Estrutura Molecular , Relação Estrutura-Atividade
4.
Sci Rep ; 11(1): 10611, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34012003

RESUMO

The first synthetic cannabinoid receptor agonists (SCRAs) were designed as tool compounds to study the endocannabinoid system's two predominant cannabinoid receptors, CB1R and CB2R. Unfortunately, novel SCRAs now represent the most rapidly proliferating novel psychoactive substances (NPS) of abuse globally. Unlike ∆9-tetrahydrocannabinol, the CB1R and CB2R partial agonist and the intoxicating constituent of Cannabis, many SCRAs characterized to date are full agonists of CB1R. Gaining additional insight into the pharmacological activity of these SCRAs is critical to assess and regulate NPSs as they enter the marketplace. The purpose of this study was to assess select SCRAs recently identified by Canadian police, border service agency, private companies and the illicit market as potential CB1R and CB2R agonists. To this end, fifteen SCRAs were screened for in vitro activity and in silico interactions at CB1R and CB2R. Several SCRAs were identified as being highly biased for cAMP inhibition or ßarrestin2 recruitment and receptor subtype selectivity between CB1R and CB2R. The indazole ring and halogen-substituted butyl or pentyl moieties were identified as two structural features that may direct ßarrestin2 bias. Two highly-biased SCRAs-JWH-018 2'-napthyl-N-(3-methylbutyl) isomer (biased toward cAMP inhibition) and 4-fluoro MDMB-BINACA (biased toward ßarrestin2 recruitment) displayed unique and differential in vivo activity in mice. These data provide initial insight into the correlations between structure, signalling bias, and in vivo activity of the SCRAs.


Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Animais , Temperatura Corporal , Células CHO , Agonistas de Receptores de Canabinoides/química , Catalepsia , Colforsina/farmacologia , Cricetulus , AMP Cíclico/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Modelos Moleculares , Nociceptividade/efeitos dos fármacos , beta-Arrestina 2/metabolismo
5.
Arch Pharm Res ; 44(4): 402-413, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33811300

RESUMO

Over the last decade, new psychoactive substances (NPS) have continuously been the focus of the international society since their emergence on the illicit drug market. NPS can be classified into six groups including; synthetic cannabinoid receptor agonists (SCRAs), stimulants, opioids, dissociatives, sedatives/hypnotics, and classic hallucinogens with psychoactive effects. These are sold as "herbal incense," "bath salts," "legal highs," and "research chemicals". They can be synthesized easily with slight changes in the chemical moieties of known psychoactive substances. NPS are sold worldwide via on- and off-line markets without proper scientific evaluation regarding their safety or harmfulness. Abuse of NPS poses a serious public health issue, and systematic studies on their adverse effects are lacking. Therefore, it would be meaningful to collect currently available data in order to understand NPS and to establish viable solutions to cope with the various health issues related to them. In this article, we reviewed the general pharmacological characteristics, recent findings, and adverse effects of representative NPS; SCRAs. SCRAs are known as the most commonly abused NPS. Most SCRAs, cannabinoid receptor 1 and cannabinoid receptor 2 agonists, are often associated with severe toxicities, including cardiotoxicity, immunotoxicity, and even death, unlike natural cannabinoid Δ9-Tetrahydrocannabinol.


Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Psicotrópicos/farmacologia , Receptores de Canabinoides/metabolismo , Agonistas de Receptores de Canabinoides/efeitos adversos , Agonistas de Receptores de Canabinoides/síntese química , Humanos , Estrutura Molecular , Psicotrópicos/efeitos adversos , Psicotrópicos/síntese química
6.
Cells ; 10(3)2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33809893

RESUMO

AIMS: The endocannabinoid system is a complex cell-signaling network through which endogenous cannabinoid ligands regulate cell function by interaction with CB1 and CB2 cannabinoid receptors, and with the novel cannabinoid receptor GPR55. CB1, CB2, and GPR55 are expressed by islet ß-cells where they modulate insulin secretion. We have previously shown that administration of the putative CB2 antagonist/inverse agonist JTE 907 to human islets did not affect the insulinotropic actions of CB2 agonists and it unexpectedly stimulated insulin secretion on its own. In this study, we evaluated whether the lack of antagonism could be related to the ability of JTE 907 to act as a GPR55 agonist. MATERIALS AND METHODS: We used islets isolated from human donors and from Gpr55+/+ and Gpr55-/- mice and quantified the effects of incubation with 10 µM JTE 907 on dynamic insulin secretion, apoptosis, and ß-cell proliferation by radioimmunoassay, luminescence caspase 3/7 activity, and immunofluorescence, respectively. We also measured islet IP1 and cAMP accumulation using fluorescence assays, and monitored [Ca2+]i elevations by Fura-2 single cell microfluorometry. RESULTS: JTE 907 significantly stimulated insulin secretion from islets isolated from human donors and islets from Gpr55+/+ and Gpr55-/- mice. These stimulatory effects were accompanied by significant elevations of IP1 and [Ca2+]i, but there were no changes in cAMP generation. JTE 907 also significantly reduced cytokine-induced apoptosis in human and mouse islets and promoted human ß-cell proliferation. CONCLUSION: Our observations show for the first time that JTE 907 acts as a Gq-coupled agonist in islets to stimulate insulin secretion and maintain ß-cell mass in a GPR55-independent fashion.


Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Dioxóis/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Quinolonas/farmacologia , Receptores de Canabinoides/efeitos dos fármacos , Adulto , Animais , Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Fosfatos de Inositol/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Ligantes , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Receptores de Canabinoides/genética , Receptores de Canabinoides/metabolismo , Técnicas de Cultura de Tecidos
7.
Eur J Med Chem ; 220: 113354, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33915369

RESUMO

We report the development and extensive structure-activity relationship evaluation of a series of modified coumarins as cannabinoid receptor ligands. In radioligand, and [35S]GTPγS binding assays the CB receptor binding affinities and efficacies of the new ligands were determined. Furthermore, we used a ligand-based docking approach to validate the empirical observed results. In conclusion, several crucial structural requirements were identified. The most potent coumarins like 3-butyl-7-(1-butylcyclopentyl)-5-hydroxy-2H-chromen-2-one (36b, Ki CB2 13.7 nM, EC50 18 nM), 7-(1-butylcyclohexyl)-5-hydroxy-3-propyl-2H-chromen-2-one (39b, Ki CB2 6.5 nM, EC50 4.51 nM) showed a CB2 selective agonistic profile with low nanomolar affinities.


Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Cumarínicos/farmacologia , Receptores de Canabinoides/metabolismo , Animais , Células CHO , Agonistas de Receptores de Canabinoides/síntese química , Agonistas de Receptores de Canabinoides/química , Células Cultivadas , Cumarínicos/síntese química , Cumarínicos/química , Cricetulus , Relação Dose-Resposta a Droga , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade
8.
Mol Cell Biochem ; 476(9): 3285-3301, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33886060

RESUMO

Colorectal cancer (CRC) is between the top three occurring cancers worldwide. The anticancer effects of Cannabinoid receptor 2 (CB2) agonist (GW833972A) in the presence and absence of its inverse agonist (SR144528) on Human colorectal adenocarcinoma cells (HT-29) was investigated. Following cell viability assays on HT-29 and HFF cells, the molecular mechanism(s) of cytotoxicity and apoptotic pathways of cell death were analyzed. The anticancer effects of CB2 agonist were measured with tumor cell migration and colony-forming assays. Real-time PCR and Western blotting techniques were used to examine any alterations in the expression of apoptotic genes. A concentration and time-dependent cytotoxicity of CB2 agonist with IC50 value of 24.92 ± 6.99 µM was obtained. The rate of lipid peroxidation was elevated, while the TNF-α concentration was declined, significantly (p < 0.05). CB2 agonist (50 µM) reduced the colony-forming capability by 83% and tumor cell migration by 50%. Apoptotic effects of CB2 agonist were revealed with the increase of apoptotic cells in Acridine orange/Ethidium bromide staining, clear DNA fragmentation, pro-apoptotic genes and proteins upregulation (Caspase-3 and p53), and significant downregulation of anti-apoptotic Bcl-2. All assessments demonstrated that CB2 agonist-induced effects were reversed by CB2 inverse agonist. These data suggest that CB2 agonists at micro-molar concentrations might be considered in the CRC treatment, and their effectiveness attributes to the apoptosis induction via upregulation of caspase-3 and p53 and downregulation of Bcl-2.


Assuntos
Apoptose , Agonistas de Receptores de Canabinoides/farmacologia , Canabinoides/farmacologia , Neoplasias Colorretais/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Receptor CB2 de Canabinoide/agonistas , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Humanos , Células Tumorais Cultivadas
9.
Neurosci Lett ; 753: 135883, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33838258

RESUMO

We have previously reported that the repeated exposure to cannabinoids upregulates and enhances the activity of serotonin 2A (5-HT2A) and dopamine 2 (D2) receptors and facilitates the formation of D2-5-HT2A receptor heterodimers in the rat prefrontal cortex and two neuronal cell lines. Because the repeated exposure to cannabinoids has been associated with adverse neuropsychiatric disorders, this study investigated the mechanisms that underly the cannabinoid-mediated regulation of D2 receptor expression in a neuronal cell model, CLU213 cells. We initially tested the effects of repeated exposure (72 h) to a non-selective cannabinoid agonist (1 nM CP55940), a selective CB1 receptor agonist (15 nM ACEA), or a selective CB2 receptor drug (1 nM GP1a) on the expression of postsynaptic D2 (D2L) receptors in CLU213 cells. Repeated CP55940, GP1a, or ACEA treatments significantly increased D2L receptor protein levels (99 % ± 7%, 30 % ± 7%, and 39 % ± 5% increases compared with control levels, respectively). Repeated exposure to both GP1a and ACEA increased D2L receptor protein levels by 73 % ± 8%. Interestingly, CP55940 and GP1a, but not ACEA, upregulated D2 mRNA. Using cells that were stably transfected with short-hairpin RNA (shRNA) lentiviral particles targeting CB2 receptors, G protein-coupled receptor kinase 5 (GRK5), and ß-arrestin 2, we found that CB2 receptors regulated D2 expression through a mechanism that is dependent on GRK5, ß-arrestin 2, and extracellular signal-related kinase (ERK)1/2. We also found that repeated exposure to either ACEA or GP1a selectively stimulated the protein and mRNA expression of GRK proteins. ACEA significantly upregulated GRK2 proteins, whereas GP1a upregulated GRK5 protein expression. Our results identified mechanisms associated with the upregulation of D2 receptors in neuronal cells after the repeated exposure to cannabinoids. These data can shed light on the mechanisms that can be targeted to prevent potential adverse effects, while simultaneously determining the therapeutic benefits of cannabinoids.


Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Neurônios/efeitos dos fármacos , Receptor CB2 de Canabinoide/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Linhagem Celular , Dopamina/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Neurônios/metabolismo , Ratos , Receptor CB2 de Canabinoide/agonistas , Regulação para Cima/efeitos dos fármacos , beta-Arrestina 2/metabolismo
10.
Life Sci ; 276: 119407, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33794254

RESUMO

AIMS: The aim of the study was to investigate the interaction between cannabinoid CB1/CB2 and lysophosphatidic acid (LPA) receptors in controlling neuronal signaling and fate. METHODS: HT22 hippocampal cells were treated with different cannabinoid and LPA receptor agonists and antagonists. Western blot and immunofluorescence microscopy were used to study intracellular signaling and the expression of apoptotic markers. Cell viability was determined by a luminescence assay. KEY FINDINGS: Cannabinoid agonists induced activation of both ERK1/2 and p38 MAP kinases. The effects of the CB1/CB2 receptor agonist HU210 were antagonized by the CB1 antagonist rimonabant, whereas the responses to the CB2 agonist JWH133 were blocked by the CB2 antagonist SR144528. HU210 reduced the apoptotic cell death induced by the pro-inflammatory cytokine TNF-α, whereas JWH133 enhanced the cytokine cytotoxicity. Blockade of ERK1/2 and p38 MAPK activation abrogated the HU210 pro-survival and the JWH133 pro-apoptotic effects, respectively. HU210 and the endocannabinoid anandamide, but not JWH133, potentiated ERK1/2 stimulation by LPA and the tricyclic antidepressant amitriptyline acting through the LPA1 receptor. HU210 enhanced amitriptyline-stimulated CREB phosphorylation and protection against TNF-α-induced apoptosis, whereas JWH133 had no effect. ERK1/2 stimulation by either HU210 or amitriptyline was dependent on fibroblast growth factor receptor (FGF-R) kinase activity and the combination of the two stimulants induced FGF-R phosphorylation. Moreover, the CB1 receptor was found to co-immunoprecipitate with the LPA1 receptor. CONCLUSIONS: In HT22 hippocampal cells CB1 and CB2 receptors differentially regulate TNF-α-induced apoptosis and CB1 receptors positively interact with amitriptyline-stimulated LPA1 in promoting FGF-R-mediated ERK1/2 signaling and neuroprotection.


Assuntos
Apoptose , Agonistas de Receptores de Canabinoides/farmacologia , Hipocampo/patologia , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Inibidores da Captação Adrenérgica/farmacologia , Amitriptilina/farmacologia , Animais , Sobrevivência Celular , Células Cultivadas , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Receptor CB1 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/agonistas , Receptores de Ácidos Lisofosfatídicos/genética , Transdução de Sinais
11.
Nat Neurosci ; 24(5): 658-666, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33737752

RESUMO

Cannabinoids reduce tremor associated with motor disorders induced by injuries and neurodegenerative disease. Here we show that this effect is mediated by cannabinoid receptors on astrocytes in the ventral horn of the spinal cord, where alternating limb movements are initiated. We first demonstrate that tremor is reduced in a mouse model of essential tremor after intrathecal injection of the cannabinoid analog WIN55,212-2. We investigate the underlying mechanism using electrophysiological recordings in spinal cord slices and show that endocannabinoids released from depolarized interneurons activate astrocytic cannabinoid receptors, causing an increase in intracellular Ca2+, subsequent release of purines and inhibition of excitatory neurotransmission. Finally, we show that the anti-tremor action of WIN55,212-2 in the spinal cords of mice is suppressed after knocking out CB1 receptors in astrocytes. Our data suggest that cannabinoids reduce tremor via their action on spinal astrocytes.


Assuntos
Astrócitos/metabolismo , Tremor Essencial/metabolismo , Interneurônios/metabolismo , Receptores de Canabinoides/metabolismo , Medula Espinal/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Benzoxazinas/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Modelos Animais de Doenças , Interneurônios/efeitos dos fármacos , Camundongos , Morfolinas/farmacologia , Naftalenos/farmacologia , Medula Espinal/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia
12.
J Bone Joint Surg Am ; 103(11): 984-991, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-33759484

RESUMO

BACKGROUND: After spinal surgery and other orthopaedic procedures, most patients receive opioids for pain, leading to potential complications such as pseudarthrosis and opioid abuse associated with long-term use. As an alternative, the endocannabinoid system has been shown to have antinociceptive activity, while contributing to bone homeostasis via the CB1 and CB2 cannabinoid receptors. This study evaluates the impact of the cannabinoid receptor agonist WIN55,212-2 (WIN55) on osteogenic differentiation in vitro as well as bone regeneration and spinal fusion in a preclinical rat model. METHODS: Primary rat bone marrow stromal cells were cultured in standard or osteogenic media and exposed to vehicle alone or WIN55. Runx2 and Alkaline phosphatase (Alpl) were quantified via qPCR (quantitative real-time polymerase chain reaction), followed by assessment of ALP activity and matrix mineralization. For in vivo evaluation, 45 female Sprague Dawley rats (n = 15 per group) underwent L4-L5 posterolateral spinal fusion with bilateral placement of collagen scaffolds preloaded with low-dose rhBMP-2 (recombinant human bone morphogenetic protein-2; 0.5 µg/implant). Postoperatively, rats received the vehicle alone or 0.5 or 2.5 mg/kg WIN55 via daily intraperitoneal injections for 5 days. Bone regeneration and spinal fusion were assessed using radiography, manual palpation-based fusion scoring, microcomputed tomography imaging, and histology. RESULTS: mRNA expression levels of Runx2 and Alp were similar among cells treated with vehicle alone and WIN55. Likewise, exposure to WIN55 did not inhibit ALP activity or bone matrix mineralization. In this animal model, no significant differences were found among groups with regard to mean fusion score, fusion rate, or new bone volume. CONCLUSIONS: WIN55 showed no adverse impact on osteogenic differentiation, bone regeneration, and spinal fusion. This supports that cannabinoid receptor agonists should be further investigated as a potential alternative approach for postoperative analgesia following spinal fusion and other orthopaedic procedures requiring bone-healing. CLINICAL RELEVANCE: The identification of alternative treatments for postoperative pain following orthopaedic surgical procedures is crucial in combating the ongoing opioid abuse crisis. The endocannabinoid system may represent a viable alternative target for addressing orthopaedic postoperative pain.


Assuntos
Benzoxazinas/farmacologia , Regeneração Óssea/efeitos dos fármacos , Agonistas de Receptores de Canabinoides/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Morfolinas/farmacologia , Naftalenos/farmacologia , Osteogênese/efeitos dos fármacos , Fusão Vertebral , Animais , Proteína Morfogenética Óssea 2/administração & dosagem , Feminino , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/cirurgia , Período Pós-Operatório , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Tecidos Suporte , Tomografia Computadorizada por Raios X , Fator de Crescimento Transformador beta/administração & dosagem
13.
Pharmacol Biochem Behav ; 205: 173182, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33774007

RESUMO

Cannabinoid (CB) receptor agonists show robust antinociceptive effects in various pain models. However, most of the clinically potent CB1 receptor-active drugs derived from cannabis are considered concerning due to psychotomimetic side effects. Selective CB receptor ligands that do not induce CNS side effects are of clinical interest. The venoms of marine snail Conus are a natural source of various potent analgesic peptides, some of which are already FDA approved. In this study we evaluated the ability of several Conus venom extracts to interact with CB1 receptor. HEK293 cells expressing CB1 receptors were treated with venom extracts and CB1 receptor internalization was analyzed by immunofluorescence. Results showed C. textile (C. Tex) and C. miles (C. Mil) samples as the most potent. These were serially subfractionated by HPLC for subsequent analysis by internalization assays and for analgesic potency evaluated in the formalin test and after peripheral nerve injury. Intrathecal injection of C. Tex and C. Mil subfractions reduced flinching/licking behavior during the second phase of formalin test and attenuated thermal and mechanical allodynia in nerve injury model. Treatment with proteolytic enzymes reduced CB1 internalization of subfractions, indicating the peptidergic nature of CB1 active component. Further HPLC purification revealed two potent antinociceptive subfractions within C. Tex with CB1 and possible CB2 activity, with mild to no side effects in the CB tetrad assessment. CB conopeptides can be isolated from these active Conus venom-derived samples and further developed as novel analgesic agents for the treatment of chronic pain using cell based or gene therapy approaches.


Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Dor Crônica/tratamento farmacológico , Venenos de Moluscos/farmacologia , Analgésicos/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Agonistas de Receptores de Canabinoides/administração & dosagem , Canabinoides/farmacologia , Dor Crônica/metabolismo , Caramujo Conus/química , Terapia Genética/métodos , Células HEK293 , Humanos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Injeções Espinhais , Venenos de Moluscos/administração & dosagem , Medição da Dor/efeitos dos fármacos , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/metabolismo , Ratos , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo
14.
Am J Physiol Renal Physiol ; 320(5): F859-F865, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33749323

RESUMO

Bladder afferents play a pivotal role in bladder function such as urine storage and micturition as well as conscious sensations such as urgency and pain. Endocannabinoids are ligands of cannabinoid 1 and 2 (CB1 and CB2) receptors but can influence the activity of a variety of G protein-coupled receptors as well as ligand-gated and voltage-gated channels. It is still not known which classes of bladder afferents are influenced by CB1 and CB2 receptor agonists. This study aimed to determine the role of CB2 receptors in two major classes of afferents in the guinea pig bladder: mucosal and muscular-mucosal. The mechanosensitivity of these two classes was determined by an ex vivo extracellular electrophysiological recording technique. A stable analog of endocannabinoid anandamide, methanandamide (mAEA), potentiated the mechanosensitivity of mucosal bladder afferents in response to stroking. In the presence of a transient receptor potential vanilloid 1 antagonist (capsazepine), the effect of mAEA switched from excitatory to inhibitory. A selective CB2 receptor agonist, 4-quinolone-3-carboxyamide (4Q3C), significantly inhibited the mechanosensitivity of mucosal bladder afferents to stroking. In the presence of a CB2 receptor antagonist, the inhibitory effect of 4Q3C was lost. mAEA and 4Q3C did not affect responses to stretch and/or mucosal stroking of muscular-mucosal afferents. Our findings revealed that agonists of CB2 receptors selectively inhibited the mechanosensitivity of capsaicin-sensitive mucosal bladder afferents but not muscular-mucosal afferents. This may have important implications for understanding of the role of endocannabinoids in modulating bladder function and sensation in health and diseases.NEW & NOTEWORTHY This article describes, for the first time, to our knowledge, the direct inhibitory effect of cannabinoid 2 receptor agonists on guinea pig mucosal bladder afferents. The cannabinoid 2 receptor is involved in pain and inflammation, suggesting that this may be a viable target for treatment of bladder disorders such as cystitis.


Assuntos
Ácidos Araquidônicos/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Mecanotransdução Celular/efeitos dos fármacos , Membrana Mucosa/inervação , Músculo Liso/inervação , Neurônios Aferentes/efeitos dos fármacos , Receptor CB2 de Canabinoide/agonistas , Bexiga Urinária/inervação , Animais , Canfanos/farmacologia , Antagonistas de Receptores de Canabinoides/farmacologia , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Endocanabinoides/metabolismo , Feminino , Cobaias , Ligantes , Neurônios Aferentes/metabolismo , Pirazóis/farmacologia , Receptor CB2 de Canabinoide/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismo
15.
Neuron ; 109(9): 1513-1526.e11, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33770505

RESUMO

Recent advances in neuroscience have positioned brain circuits as key units in controlling behavior, implying that their positive or negative modulation necessarily leads to specific behavioral outcomes. However, emerging evidence suggests that the activation or inhibition of specific brain circuits can actually produce multimodal behavioral outcomes. This study shows that activation of a receptor at different subcellular locations in the same neuronal circuit can determine distinct behaviors. Pharmacological activation of type 1 cannabinoid (CB1) receptors in the striatonigral circuit elicits both antinociception and catalepsy in mice. The decrease in nociception depends on the activation of plasma membrane-residing CB1 receptors (pmCB1), leading to the inhibition of cytosolic PKA activity and substance P release. By contrast, mitochondrial-associated CB1 receptors (mtCB1) located at the same terminals mediate cannabinoid-induced catalepsy through the decrease in intra-mitochondrial PKA-dependent cellular respiration and synaptic transmission. Thus, subcellular-specific CB1 receptor signaling within striatonigral circuits determines multimodal control of behavior.


Assuntos
Encéfalo/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Transdução de Sinais/fisiologia , Transmissão Sináptica/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Agonistas de Receptores de Canabinoides/farmacologia , Antagonistas de Receptores de Canabinoides/farmacologia , Catalepsia/induzido quimicamente , Membrana Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Nociceptividade/efeitos dos fármacos , Nociceptividade/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos
16.
J Med Chem ; 64(7): 3870-3884, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33761251

RESUMO

We report the development of novel cannabinergic probes that can stabilize the cannabinoid receptors (CBRs) through tight binding interactions. Ligand design involves the introduction of select groups at a judiciously chosen position within the classical hexahydrocannabinol template (monofunctionalized probes). Such groups include the electrophilic isothiocyanato, the photoactivatable azido, and the polar cyano moieties. These groups can also be combined to produce bifunctionalized probes potentially capable of interacting at two distinct sites within the CBR-binding domains. These novel compounds display remarkably high binding affinities for CBRs and are exceptionally potent agonists. A key ligand (27a, AM11245) exhibits exceptionally high potency in both in vitro and in vivo assays and was designated as "megagonist," a property attributed to its tight binding profile. By acting both centrally and peripherally, 27a distinguishes itself from our previously reported "megagonist" AM841, whose functions are restricted to the periphery.


Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Canabinoides/farmacologia , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Analgésicos/síntese química , Analgésicos/metabolismo , Analgésicos/farmacologia , Animais , Regulação da Temperatura Corporal/efeitos dos fármacos , Células CHO , Agonistas de Receptores de Canabinoides/síntese química , Agonistas de Receptores de Canabinoides/metabolismo , Canabinoides/síntese química , Canabinoides/metabolismo , Cricetulus , Humanos , Ligantes , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Simulação de Acoplamento Molecular , Ratos
17.
Adv Exp Med Biol ; 1297: 97-109, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33537939

RESUMO

The cannabinoids are a family of chemical compounds that can be either synthesized or naturally derived. These compounds have been shown to modulate a wide variety of biological processes. In this chapter, the studies detailing the effects of cannabinoids on sleep in laboratory animals are reviewed. Both exogenous and endogenous cannabinoids generally appear to decrease wakefulness and alter rapid eye movement (REM) and non-REM sleep in animal models. In addition, cannabinoids potentiate the effects of sedative-hypnotic drugs. However, the individual contributions of each cannabinoid on sleep processes is more nuanced and may depend on the site of action in the central nervous system. Many studies investigating the mechanism of cannabinoid effects on sleep suggest that the effects of cannabinoids on sleep are mediated via cannabinoid receptors; however, some evidence suggests that some sleep effects may be elicited via non-cannabinoid receptor-dependent mechanisms. More research is necessary to fully elucidate the role of each compound in modulating sleep processes.


Assuntos
Agonistas de Receptores de Canabinoides , Canabinoides , Animais , Animais de Laboratório , Agonistas de Receptores de Canabinoides/farmacologia , Canabinoides/farmacologia , Receptores de Canabinoides , Sono
18.
Pharmacol Biochem Behav ; 203: 173119, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33508249

RESUMO

The endocannabinoid system, which spans the central and peripheral nervous systems and regulates many biologic processes, is an important target for probe discovery and medications development. Whereas the earliest endocannabinoid receptor probes were derivatives of the non-selective phytocannabinoids isolated from Cannabis species, modern drug discovery techniques have expanded the definitions of what constitutes a CB1R or CB2R cannabinoid receptor ligand. This review highlights recent advances in synthetic cannabinoid receptor chemistry and pharmacology. We provide examples of new CB1R- and CB2R-selective probes, and discuss rational approaches to the design of peripherally-restricted agents. We also describe structural classes of positive- and negative allosteric modulators (PAMs and NAMs) of CB1R and CB2R. Finally, we introduce new opportunities for cannabinoid receptor probe development that have emerged in recent years, including biased agonists that may lead to medications lacking adverse effects.


Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Antagonistas de Receptores de Canabinoides/farmacologia , Canabinoides/farmacologia , Extratos Vegetais/farmacologia , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , Receptores Artificiais/agonistas , Receptores Artificiais/antagonistas & inibidores , Regulação Alostérica , Sítio Alostérico , Animais , Cannabis/química , Descoberta de Drogas/métodos , Endocanabinoides/metabolismo , Humanos , Ligantes , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo
19.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33468648

RESUMO

Recurrent excitatory neural networks are unstable. In the hippocampus, excitatory mossy cells (MCs) receive strong excitatory inputs from dentate granule cells (GCs) and project back onto the proximal dendrites of GCs. By targeting the ipsi- and contralateral dentate gyrus (DG) along the dorsoventral axis of the hippocampus, MCs form an extensive recurrent excitatory circuit (GC-MC-GC) whose dysregulation can promote epilepsy. We recently reported that a physiologically relevant pattern of MC activity induces a robust form of presynaptic long-term potentiation (LTP) of MC-GC transmission which enhances GC output. Left unchecked, this LTP may interfere with DG-dependent learning, like pattern separation-which relies on sparse GC firing-and may even facilitate epileptic activity. Intriguingly, MC axons display uniquely high expression levels of type-1 cannabinoid receptors (CB1Rs), but their role at MC-GC synapses is poorly understood. Using rodent hippocampal slices, we report that constitutively active CB1Rs, presumably via ßγ subunits, selectively inhibited MC inputs onto GCs but not MC inputs onto inhibitory interneurons or CB1R-sensitive inhibitory inputs onto GCs. Tonic CB1R activity also inhibited LTP and GC output. Furthermore, brief endocannabinoid release from GCs dampened MC-GC LTP in two mechanistically distinct ways: during induction via ßγ signaling and before induction via αi/o signaling in a form of presynaptic metaplasticity. Lastly, a single in vivo exposure to exogenous cannabinoids was sufficient to induce this presynaptic metaplasticity. By dampening excitatory transmission and plasticity, tonic and phasic CB1R activity at MC axon terminals may preserve the sparse nature of the DG and protect against runaway excitation.


Assuntos
Giro Denteado/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Receptor CB1 de Canabinoide/genética , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Animais Recém-Nascidos , Agonistas de Receptores de Canabinoides/farmacologia , Antagonistas de Receptores de Canabinoides/farmacologia , Canabinoides/farmacologia , Giro Denteado/citologia , Giro Denteado/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Expressão Gênica , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Interneurônios/citologia , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Camundongos , Piperidinas/farmacologia , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Transmissão Sináptica
20.
Biomed Pharmacother ; 136: 111283, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33482616

RESUMO

BACKGROUND AND PURPOSE: The endocannabinoid system became a promising target for osteoarthritis (OA) treatment. Functional selectivity of cannabinoids may increase their beneficial properties while reducing side effects. The aim of the present study was to evaluate the analgesic potential of two functionally biased CB2 agonists in different treatment regimens to propose the best pharmacological approach for OA management. EXPERIMENTAL APPROACH: Two functionally selective CB2 agonists were administered i.p. - JWH133 (cAMP biased) and GW833972A (ß-arrestin biased), in a chemically induced model of OA in rats. The drugs were tested in acute and chronic treatment regimens. Analgesic effects were assessed by pressure application measurement and kinetic weight bearing. X-ray microtomography was used for the morphometric analysis of the femur's subchondral bone tissue. Underlying biochemical changes were analysed via RT-qPCR. KEY RESULTS: Dose-response studies established the effective dose for both JWH133 and GW833972A. In chronic treatment paradigms, JWH133 was able to elicit analgesia throughout the course of the experiment, whereas GW833972A lost its efficacy after 2 days of treatment. Later studies revealed improvement in subchondral bone architecture and decrement of matrix metalloproteinases and proinflammatory factors expression following JWH133 chronic treatment. CONCLUSION AND IMPLICATIONS: Data presents analgesic and disease-modifying potential of CB2 agonists in OA treatment. Moreover, the study revealed more pronounced tolerance development for analgesic effects of the ß-arrestin biased CB2 agonist GW833972A. These results provide a better understanding of the molecular underpinnings of the anti-nociceptive potential of CB2 agonists and may improve drug development processes for any cannabinoid-based chronic pain therapy.


Assuntos
Analgésicos/farmacologia , Artralgia/prevenção & controle , Agonistas de Receptores de Canabinoides/farmacologia , Canabinoides/farmacologia , Articulações/efeitos dos fármacos , Osteoartrite/prevenção & controle , Limiar da Dor/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Receptor CB2 de Canabinoide/agonistas , Animais , Artralgia/etiologia , Artralgia/metabolismo , Artralgia/fisiopatologia , Modelos Animais de Doenças , Tolerância a Medicamentos , Ácido Iodoacético , Articulações/metabolismo , Articulações/fisiopatologia , Masculino , Osteoartrite/induzido quimicamente , Osteoartrite/metabolismo , Osteoartrite/fisiopatologia , Ratos Wistar , Receptor CB2 de Canabinoide/metabolismo , Transdução de Sinais , Fatores de Tempo
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