Endocannabinoid brain mechanisms and addiction

内源性大麻素脑机制和成瘾

• 批准号: 10267531
• 负责人: Eliot Gardner
• 金额: $ 72.02万
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10267531
• 关键词: AM 251; AM630; Absence of pain sensation; Abstinence; Addictive Behavior; Address; Agonist; Animal Model; Animals; Antibodies; Attenuated; Behavior; Beta-caryophyllene; Binding; Biochemical; Biological Assay; Brain; C-terminal; CB1 receptor antagonist; CB2 knockout; CNR1 gene; CNR2 gene; Cannabinoids; Cannabis; Catalepsy; Chemicals; Clinical Trials; Cocaine; Cues; Detection; Diet; Disputes; Dose; Electrical Stimulation of the Brain; Endocannabinoids; Enzymes; Epitopes; Equilibrium; Exposure to; Fc Receptor; GPR55 receptor; Genes; Genetic; Genotype; Goals; Heroin; High Pressure Liquid Chromatography; Human; Hyperalgesia; Immune; Intravenous; Intraventricular; Knockout Mice; Laboratory Animals; Laboratory Rat; Locomotion; Mediating; Messenger RNA; Methamphetamine; Microdialysis; Microinjections; Midbrain structure; Modeling; Mus; N-terminal; Neurotransmitters; Nicotine; Nicotine Dependence; Nicotine Withdrawal; Opiate Addiction; Opioid; Peptides; Pharmaceutical Preparations; Pharmacology; Play; Polymerase Chain Reaction; Property; Proteins; Psychological reinforcement; RNA; Rattus; Receptor Activation; Relapse; Reporting; Research; Research Project Grants; Reverse Transcription; Rewards; Rodent; Rodent Model; Role; SR 141716A; Sampling; Self Administration; Signal Transduction; Specificity; Spleen; Stress; System; Techniques; Terpenes; Testing; Tetrahydrocannabinol; Therapeutic; Western Blotting; Wild Type Mouse; Withdrawal; Work; addiction; analog; anxiety-like behavior; base; cannabinoid drug; conditioned place preference; craving; density; depressive symptoms; dopaminergic neuron; drug of abuse; immunoreactivity; in vivo; lateral ventricle; mRNA Expression; methamphetamine effect; mutant; natural hypothermia; nicotine seeking behavior; obesity treatment; overexpression; polyclonal antibody; pre-clinical; preclinical study; psychostimulant; receptor; response; rimonabant; side effect; smoking cessation; therapy development

During the present reporting period, significant progress was made on this research project. Cannabinoid CB1 receptors (CB1Rs) have been shown to be a promising target in medication development for the treatment of addiction. However, clinical trials with SR141716A (rimonabant, a selective CB1R antagonist/inverse agonist) for the treatment of obesity and smoking cessation failed due to unwanted side effects. Recent preclinical studies suggest that the neutral CB1R antagonist AM4113 may retain the therapeutic anti-addictive effects of SR141716A in nicotine self-administration models with fewer unwanted side effects. However, little is known about whether AM4113 is also effective for other drugs of abuse, such as opioids and psychostimulants, and whether it produces depressive side effects similar to SR141716A in experimental animals. We therefore studied whether systemic administration of AM4113 (3 and 10 mg/kg) alters heroin, cocaine, or methamphetamine effects. We found that AM4113 dose-dependently inhibited i.v. self-administration of heroin but not cocaine or methamphetamine, whereas SR141716A (3 and 10 mg/kg) dose-dependently inhibited the self-administration of heroin and methamphetamine but not cocaine. In the electrical brain-stimulation reward (BSR) paradigm, SR141716A (3 and 10 mg/kg) dose-dependently increased the BSR stimulation threshold (i.e., decreased brain reward), but AM4113 had no effect on BSR at the same doses, suggesting that SR141716A may produce aversive effects while AM4113 may not. Together, these findings show that neutral CB1R antagonists such as AM4113 deserve further research as a new class of CB1R-based medications for the treatment of opioid addiction without SR141716A-like aversive effects. Growing evidence indicates the presence of functional cannabinoid CB2 receptors (CB2Rs) in the brain. However, this finding is disputed because of the specificity of CB2R antibody signals. We used two strains of currently available partial CB2-knockout (CB2-KO) mice as controls, four anti-rat or anti-mouse CB2R antibodies, and mRNA quantification to further address this issue. Western blot assays using the four antibodies detected a CB2R-like band at 40 kD in both the brain and spleen. Notably, more bands were detected in the brain than in the spleen, and specific immune peptides blocked band detection. Immunohistochemical assays also detected CB2-like immunostaining in mouse midbrain dopamine neurons. CB2R deletion in CB2-KO mice may reduce or leave CB2R-like immunoreactivity unaltered depending on antibody epitope. Antibodies with epitopes at the receptor-deleted region detected a significant reduction in CB2R band density and immunostaining in N-terminal-deleted Deltagen and C-terminal-deleted Zimmer strain CB2-KO mice. Other antibodies with epitopes at the predicted receptor-undeleted regions detected similar band densities and immunostaining in wild-type and CB2-KO mice. Quantitative RT-PCR assays detected CB2 mRNA expression using probes that targeted upstream or downstream gene sequences but not the probe that targeted the gene-deleted sequence in Deltagen or Zimmer CB2-KO mice. These findings suggest that none of the tested four polyclonal antibodies are highly mouse CB2R-specific. Non-specific binding may be related to the expression of mutant or truncated CB2R-like proteins in partial CB2-KO mice and the use of anti-rat CB2 antibodies because the epitopes are different between rat and mouse CB2Rs. Further, we re-examined the roles of both CB1 and CB2 receptors in the effects of cannabis. We used electrical brain-stimulation reward (BSR) to evaluate the effects of various cannabinoid drugs on brain reward in laboratory rats and the roles of CB1 and CB2 receptors activation in brain reward function(s). Two mixed CB1/CB2 receptor agonists, delta-9-tetrahydrocannabinol (THC) and WIN55,212-2, produced biphasic effects - mild enhancement of BSR at low doses but inhibition at higher doses. The CB1R antagonist AM251 attenuated the low dose-enhanced BSR, while the CB2R receptor AM630 attenuated high dose-inhibited BSR. Rats were also treated with selective CB1R and CB2R agonists, which produced BSR enhancement and inhibition, respectively. We conclude that the subjective effects of cannabis depend on the balance of these opposing effects, and explain why cannabis can be either rewarding or aversive in humans, as expression of CB1Rs and CB2Rs may differ in the brains of different subjects. Further, we studied whether combining CB1R antagonism with CB2R agonism produces additive anti-addiction effects. Delta-8-tetrahydrocannabivarin (delta-8-THCV) is a delta-9-THCV analogue with combined CB1 antagonist/CB2 agonist properties. We tested delta-8-THCV in seven different rodent models relevant to nicotine dependence - nicotine self-administration, cue-triggered nicotine-seeking behavior following forced abstinence, nicotine-triggered reinstatement of nicotine-seeking behavior, acquisition of nicotine-induced conditioned place preference, anxiety-like behavior induced by nicotine withdrawal, somatic withdrawal signs induced by nicotine withdrawal, and hyperalgesia induced by nicotine withdrawal. Delta-8-THCV significantly attenuated intravenous nicotine self-administration and both cue-induced and nicotine-induced relapse to nicotine-seeking behavior in rats. Delta-8-THCV also significantly attenuated nicotine-induced conditioned place preference and nicotine withdrawal in mice. We conclude that delta-8-THCV may have therapeutic potential for the treatment of nicotine dependence. We also suggest that tetrahydrocannabivarins be tested for anti-addiction efficacy in a broader range of preclinical animal models, against other addictive drugs, and eventually in humans. We also studied cannabinoid-induced tetrad effects - analgesia, hypothermia, catalepsy, and suppressed locomotion, which were previously believed to be mediated by the activation of cannabinoid CB1Rs. Given recent findings of CB2 and GPR55 receptors in the brain, we examined whether these receptors are also involved in cannabinoid action. We found that administration of delta-9-THC, WIN55212-2, or XLR11 produced dose-dependent tetrad effects in wild-type (WT) mice. Genetic deletion or pharmacological blockade of CB1Rs abolished tetrad effects produced by all three cannabinoids. Unexpectedly, genetic deletion of CB2Rs abolished analgesia and catalepsy produced by delta-9-THC or WIN55212-2, but not by XLR11. Microinjections of delta-9-THC into the lateral ventricles also produced tetrad effects in WT, but not in CB1-KO mice. CB2-KO mice displayed a reduction in intraventricular delta-9-THC-induced analgesia and catalepsy. In contrast, genetic deletion of GPR55 receptors caused enhanced responses to delta-9-THC or WIN55212-2. Antagonism of CB1Rs, CB2Rs, or GPR55Rs produced alterations similar to those observed in each genotype mouse line. These findings suggest that in addition to CB1Rs, both CB2Rs and GPR55Rs are also involved in pharmacological effects produced by cannabinoids. CB1Rs/CB2Rs, in contrast to GPR55Rs, appear to play opposite roles in cannabinoid actions. Finally, we found that beta-Caryophyllene, a dietary terpenoid, inhibits nicotine taking and nicotine seeking behaviors in rodents.

在本报告期间，该研究项目取得了重大进展。大麻素CB1受体（CB1R）已被证明是用于治疗成瘾的药物开发的有希望的靶标。然而，SR141716A（Rimonabant，选择性CB1R拮抗剂/逆激动剂）的临床试验治疗肥胖和戒烟，由于不需要的副作用而失败了。最近的临床前研究表明，中性的CB1R拮抗剂AM4113可能会保留SR141716A在尼古丁自我管理模型中的治疗性抗吸毒作用，其副作用较少。但是，对于AM4113是否对其他滥用药物（例如阿片类药物和心理刺激物）以及它是否产生类似于实验动物SR141716A的抑郁副作用，对AM4113是否也有效。因此，我们研究了AM4113（3和10 mg/kg）的全身给药是否会改变海洛因，可卡因或甲基苯丙胺的作用。我们发现AM4113剂量依赖性地抑制了静脉注射。海洛因而非可卡因或甲基苯丙胺的自我给药，而SR141716A（3和10 mg/kg）剂量依赖性地抑制了海洛因和甲基苯丙胺的自我给药，但不能抑制可卡因。在电脑刺激奖励（BSR）范式中，SR141716A（3和10 mg/kg）剂量依赖性地增加了BSR刺激阈值（即降低了大脑奖励），但AM4113对BSR没有对BSR的影响，这表明SR1411716A可能会产生AM4113113113113113113113113111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111VIOL AM4113效应。总之，这些发现表明，中性的CB1R拮抗剂（例如AM4113）值得进一步研究，这是一种新的基于CB1R的药物，用于治疗阿片类药物成瘾，而无需SR141716A类似于类似厌恶的厌恶。越来越多的证据表明大脑中存在功能性大麻素CB2受体（CB2R）。但是，由于CB2R抗体信号的特异性，这一发现是有争议的。我们使用了两种当前可用的部分CB2敲除（CB2-KO）小鼠的菌株作为对照，四种抗鼠或抗小鼠CB2R抗体以及mRNA量化，以进一步解决此问题。使用四种抗体的蛋白质印迹测定在大脑和脾脏中检测到40 kd的CB2R样带。值得注意的是，在大脑中检测到的条带比脾脏更多，并且特定的免疫肽阻断了带状的检测。免疫组织化学测定还检测到小鼠中脑多巴胺神经元中的CB2样免疫染色。 CB2-KO小鼠中的CB2R缺失可能会根据抗体表位减少或留下类似CB2R的免疫反应性。在受体排列区域的表位抗体检测到N末端删除的三角形和C末端删除的Zimmer菌株CB2-KO小鼠的CB2R带密度显着降低和免疫染色。在预测的受体非骨骼区域处的其他具有表位的抗体检测到野生型和CB2-KO小鼠中相似的带密度和免疫染色。定量RT-PCR分析使用针对上游或下游基因序列的探针检测到CB2 mRNA表达，而不是针对Deltagen或Zimmer CB2-KO小鼠中基因删除序列的探针。这些发现表明，测试过的四种多克隆抗体都不是高度小鼠CB2R特异性的。非特异性结合可能与部分CB2-KO小鼠中突变体或截短的CB2R样蛋白的表达以及使用抗RAT CB2抗体的使用有关，因为大鼠和小鼠CB2R之间的表位不同。此外，我们重新检查了CB1和CB2受体在大麻作用中的作用。我们使用电脑刺激奖励（BSR）来评估各种大麻素药物对实验室大鼠脑奖励的影响，以及CB1和CB2受体在脑奖励功能中激活CB1和CB2受体的作用（S）。两种混合的CB1/CB2受体激动剂，即Delta-9-四氢大麻醇（THC）和Win55,212-2，产生了双相作用 - 低剂量以低剂量但抑制更高剂量的BSR轻度增强。 CB1R拮抗剂AM251减弱了低剂量增强的BSR，而CB2R受体AM630减弱了高剂量抑制的BSR。还用选择性CB1R和CB2R激动剂处理大鼠，分别产生BSR增强和抑制作用。我们得出的结论是，大麻的主观效应取决于这些相反效应的​​平衡，并解释了为什么大麻在人类中可以有意义或厌恶性，因为CB1RS和CB2RS的表达在不同受试者的大脑中可能有所不同。此外，我们研究了将CB1R拮抗作用与CB2R激动剂相结合是否产生添加剂抗添加作用。 Delta-8-四氢大麻酶（Delta-8-THCV）是具有CB1拮抗剂/CB2激动剂特性的Delta-9-THCV类似物。 We tested delta-8-THCV in seven different rodent models relevant to nicotine dependence - nicotine self-administration, cue-triggered nicotine-seeking behavior following forced abstinence, nicotine-triggered reinstatement of nicotine-seeking behavior, acquisition of nicotine-induced conditioned place preference, anxiety-like behavior induced by nicotine withdrawal, somatic withdrawal signs induced by尼古丁的戒断和尼古丁戒断引起的痛觉过敏。 Delta-8-THCV显着减弱了静脉内尼古丁自我给药，以及提示诱导的和尼古丁引起的大鼠尼古丁寻求尼古丁行为的复发。 Delta-8-THCV还显着减弱了小鼠尼古丁诱导的条件偏好和尼古丁的戒断。我们得出的结论是，Delta-8-THCV可能具有治疗尼古丁依赖性的治疗潜力。我们还建议，在更广泛的临床前动物模型中，针对其他成瘾性药物，最终在人类中测试了四氢大麻素的抗添加功效。我们还研究了大麻素诱导的四局效应 - 镇痛，体温过低，培养和抑制运动，以前认为这是由大麻素CB1RS的激活介导的。考虑到大脑中CB2和GPR55受体的最新发现，我们检查了这些受体是否也参与大麻素作用。我们发现施用Delta-9-THC，WIN55212-2或XLR11在野生型（WT）小鼠中产生了剂量依赖性的Tetrad效应。所有三种大麻素产生的CB1RS的遗传缺失或药理学阻断了CB1RS的二甲虫效应。出乎意料的是，CB2RS的遗传缺失消除了由Delta-9-THC或Win555212-2产生的镇痛和细胞蛋白，但不是XLR11。 Delta-9-Thc对侧心室的显微注射也会在WT中产生tetrad效应，但在CB1-KO小鼠中却没有。 CB2-KO小鼠表现出脑室内delta-9-thc诱导的镇痛和细胞培养的降低。相反，GPR55受体的遗传缺失导致对Delta-9-THC或Win55212-2的反应增强。 CB1R，CB2RS或GPR55RS的拮抗作用类似于在每个基因型小鼠系中观察到的变化。这些发现表明，除了CB1RS外，CB2RS和GPR55RS都参与了大麻素产生的药理作用。与GPR55RS相比，CB1RS/CB2RS在大麻素动作中似乎起着相反的作用。最后，我们发现一种饮食中的萜类化合物β-蛋黄蛋白丁烯抑制啮齿动物中的尼古丁服用和寻求尼古丁的行为。