Discovery and validation of novel targets for pharmacotherapy of alcoholism

酒精中毒药物治疗新靶点的发现和验证

• 批准号: 8559275
• 负责人: Markus Heilig
• 金额: $ 320.35万
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 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/8559275
• 关键词: Affinity; Agonist; Alcohol consumption; Alcohol dependence; Alcoholism; Alcohols; Alleles; Amygdaloid structure; Animal Model; Animals; Anxiety; Automobile Driving; Behavior; Behavioral; Binding; Bioinformatics; Biological; Biological Assay; Brain; Brain region; Brain-Derived Neurotrophic Factor; Breeding; Calcium; Chemicals; Chronic; Clinical; Clinical Trials; Collaborations; Corticotropin-Releasing Hormone; Corticotropin-Releasing Hormone Receptors; Cues; Cyclic AMP; DNA; DNA Methylation; DNA Methylation Inhibition; Data; Desire for food; Development; Dopamine; Dose; Emotional; Engineering; Epigenetic Process; Exposure to; Extinction (Psychology); Follow-Up Studies; Functional disorder; Gene Expression; Gene Expression Profiling; Genes; Genetic; Genetic Polymorphism; Genomics; Goals; Heavy Drinking; Heterogeneity; Hormone Antagonists; Human; Hypermethylation; In Vitro; Infusion procedures; Injection of therapeutic agent; Intake; Intervention; Knockout Mice; Lead; Literature; Measures; Mediating; Medicine; Methylation; MicroRNAs; Microinjections; Modeling; Modification; Molecular; Motivation; Motor Activity; Mus; Naltrexone; Narcotic Antagonists; Neuropeptides; Nucleus Accumbens; Opioid; Opioid Receptor; Peripheral; Pharmaceutical Preparations; Phenotype; Phosphorylation; Play; Population; Positron-Emission Tomography; Preparation; Property; Proteins; Rattus; Receptor Gene; Recording of previous events; Regulation; Relapse; Rewards; Rice; Role; Running; Self Administration; Self Stimulation; Series; Signal Transduction; Stimulus; Stress; Substance P; Substance P Receptor; Synaptic Vesicles; TACR1 gene; Target Populations; Therapeutic; Time; Time Study; Transcript; Transgenic Organisms; United States National Institutes of Health; Up-Regulation; Validation; Variant; Ventral Striatum; Wistar Rats; Work; alcohol exposure; alcohol relapse; alcohol response; alcohol reward; alcohol seeking behavior; alcoholism pharmacotherapy; alcoholism therapy; biological adaptation to stress; drug candidate; follow-up; in vivo; inhibitor/antagonist; interest; mu opioid receptors; novel; pre-clinical; preference; prevent; programs; receptor; research study; response; therapeutic target

MP currently utilizes two types of animal models: animals that escalate their alcohol intake following a prolonged history of intermittent brain alcohol exposure (post-dependent), or animals that have interesting alcohol-related phenotypes as a function of genetic factors, such as rats selectively bred for high alcohol preference, or mice or rats that have been genetically engineered. These different models may tap into different biological mechanisms, and are therefore complementary. Voluntary alcohol consumption and its escalation over time are studied in several models. Relapse-like behavior is studied in animals using reinstatement models, in which lever-pressing for alcohol is first established, and then extinguished by removing the alcohol. Presentation of drug-associated cues or exposure to stress will lead to resumption of lever-pressing on the previously alcohol-delivering lever (reinstatement). Reinstatement induced by cue or stress is differentially sensitive to pharmacological manipulations, and evaluating candidate drugs for their ability to suppress reinstatement induced by the two different types of stimuli guides optimal selection of target populations in initial clinical trials. Information may also be gained on how treatments might be combined to obtain additive effects. Mu-opioid receptor gene (OPRM1) The opioid receptor antagonist naltrexone (NTX) is an established treatment for alcoholism, however there is considerable heterogeneity of responses in clinical populations. Recent evidence suggests that a non-synonymous SNP, A118G, found in the human OPRM1 gene encoding the mu-opioid receptor may modulate alcohol reward and therapeutic response to NTX. To establish the mechanistic role of human OPRM1 A118G variation, and to isolate the effects of this polymorphism, we created two lines of humanized mice carrying the respective human allele (118AA and 118GG). We observed a markedly enhanced dopamine (DA) release in the nucleus accumbens of 118GG mice in response to alcohol. This finding was consistent with a positron emission tomography study conducted in parallel in humans, which found a more vigorous DA response to alcohol in the ventral striatum in 118G carriers (Ramchandani et al., 2011). Further work with the humanized mouse lines revealed elevated alcohol consumption in 118GG mice, and experiments are underway to investigate whether 118GG mice will be preferentially or selectively sensitive to NTX (Thorsell et al., in preparation). In collaboration with a lab at UNC, we are also investigating the consequences of this receptor variation for brain reward as measured by intracranial self-stimulation. Substance P and its NK1 receptor Substance P mediates stress responses, and blockade of the receptor for substance P, NK1R, produces anti-stress effects. We have shown that genetic deletion of NK1R in mice leads to markedly decreased alcohol preference, a lack of conditioned place preference for alcohol, and an absence of escalated intake over time. Pharmacological blockade of NK1 receptors using the NK1 antagonist L-703,606 suppresses alcohol consumption similar to genetic deletion of the receptor (Thorsell et al., 2010). We recently collaborated with Dr. Kenner Rice to resynthesize a literature compound, L-822429, with high affinity for the rat NK1 receptor. L-822429 dose-dependently suppressed stress-induced reinstatement of alcohol seeking in Wistar rats, but had no effect on cue-induced reinstatement, suggesting the possibility of additive effects of an NK1R antagonist and opioid antagonists such naltrexone (Schank et al., 2011). In follow-up work using genetically selected alcohol preferring P-rats, L-822429 dose-dependently suppressed self-administration rates in P-rats but was ineffective in non-dependent Wistar rats. NK1R receptor was upregulated in several brain regions in P rats, with highest expression in the central amygdala (CeA); microinjections of the antagonist into this region mimicked systemic effects(Schank et al., submitted). Together, these finding further support NK1R antagonism as a candidate target for the treatment of alcoholism. Corticotropin-Releasing Hormone (CRH) and its CRH1 receptor Elevated self-administration of alcohol and an increased behavioral sensitivity to stress in the post-dependent state is in large part mediated by an up-regulation of the CRH1 subtype of CRH receptors in the amygdala (Sommer et al., 2008). We had previously identified a series of CRH antagonists with suitable properties for clinical development (Gehlert et al., 2007; Thorsell et al., unpublished data). Two of the candidates, pexacerfont and GSK561679, were selected to enter experimental medicine studies in our clinical program under CRADAs with Bristol Myers Squibb and GlaxoSmithKline respectively. Those studies are currently underway. Neuropeptide S (NPS) NPS suppresses anxiety and appetite in experimental animals, and initial work in collaboration with the Ciccocioppo lab indicated that NPS signaling may play a role in relapse to alcohol seeking (Canella et al., 2009). We developed a screenable assay for the NPS receptor, and in collaboration with the NIH Chemical Genomics Center (NCGC) identified a lead molecule, NCG001865684, that was determined to be brain penetrant upon peripheral administration in rats. In vitro, NCGC00185684 shows biased antagonist properties, and preferentially blocks ERK-phosphorylation over intracellular cAMP- or calcium-responses to NPS. In vivo, pretreatment with NCG001865684 was found to block alcohol-induced ERK-phosphorylation in the rat CeA, a region involved in regulation of alcohol intake. NCGC00185684 also decreases operant alcohol self-administration, and lowers alcohol reward as measured using progressive ratio responding. These effects are behaviorally specific because they do not influence locomotor activity or reinstatement responding following extinction. Taken together, these data provide an initial validation of the NPSR as a therapeutic target in alcoholism (Thorsell et al., submitted). Gene expression profiling and epigenetic modifications A series of projects are being carried out looking at changes in gene expression and epigenetic modifications (microRNA and methylation) in the post-dependent rat model to identify potential pharmacological targets. Bioinformatic analysis of the initial runs has identified several interesting, differentially expressed transcripts as well as several cases of miR-mediated changes in gene expression(Tapocik et al., 2012). A follow up study determined the functional role of one microRNA, miR-206, in alcohol dependence. Inhibition of miR-206 in the mPFC decreased alcohol consumption and preference in post-dependent rats but not control rats. In addition, miR-206 was shown to bind and inhibit the expression of BDNF (Tapocik et al., in preparation). In collaboration with Dr. Paul Kenny, we have determined that miR-206 knockout mice consume and prefer alcohol significantly less than WT mice at pharmacologically active doses of alcohol (Tapocik et al., in preparation). We also found that DNA methylation in the mPFC is regulated by a history of alcohol dependence, and contributes to the persistent escalation in alcohol consumption observed in post-dependent rats. ICV infusion of a DNA methylation inhibitor normalized both mPFC DNA methylation and alcohol consumption in post-dependent rats. Conversely, systemic injections of a methyl donor induced DNA hypermethylation in the mPFC of non-dependent rats, and escalated alcohol consumption. The expression of genes encoding synaptic vesicle proteins is altered following a history of alcohol dependence. This was prevented by DNA methylation inhibition, suggesting that DNA methylation regulates behavioral adaptations to chronic alcohol exposure (Barbier et al., submitted).

MP 目前使用两种类型的动物模型：在长期间歇性脑酒精暴露（后依赖性）后逐渐增加酒精摄入量的动物，或作为遗传因素的函数而具有有趣的酒精相关表型的动物，例如选择性繁殖的高酒精偏好大鼠，或经过基因工程改造的小鼠或大鼠。这些不同的模型可能利用不同的生物机制，因此是互补的。 通过多个模型研究了自愿饮酒及其随时间的推移而增加的情况。使用恢复模型在动物中研究类似复发的行为，其中首先建立对酒精的杠杆按压，然后通过去除酒精来消除。出现与药物相关的提示或暴露于压力将导致恢复对先前的酒精输送杆的按压（恢复）。由提示或压力引起的恢复对药理学操作的敏感度不同，评估候选药物抑制两种不同类型刺激引起的恢复的能力可指导初始临床试验中目标人群的最佳选择。还可以获得有关如何组合治疗以获得附加效果的信息。 Mu-阿片受体基因 (OPRM1) 阿片受体拮抗剂纳曲酮 (NTX) 是一种既定的酒精中毒治疗方法，但临床人群的反应存在相当大的异质性。最近的证据表明，在编码 mu-阿片受体的人类 OPRM1 基因中发现的非同义 SNP A118G 可能调节酒精奖赏和对 NTX 的治疗反应。为了确定人类 OPRM1 A118G 变异的机制作用，并分离这种多态性的影响，我们创建了两个携带各自人类等位基因（118AA 和 118GG）的人源化小鼠品系。我们观察到 118GG 小鼠的伏核中对酒精的反应显着增强了多巴胺 (DA) 的释放。这一发现与在人类中平行进行的正电子发射断层扫描研究一致，该研究发现 118G 携带者的腹侧纹状体对酒精有更强烈的 DA 反应（Ramchandani 等，2011）。对人源化小鼠系的进一步研究显示 118GG 小鼠的酒精消耗量增加，并且正在进行实验以研究 118GG 小鼠是否会优先或选择性地对 NTX 敏感（Thorsell 等人，正在准备中）。我们还与北卡罗来纳大学的一个实验室合作，研究这种受体变异对通过颅内自我刺激测量的大脑奖赏的影响。 P物质及其NK1受体 P 物质介导应激反应，阻断 P 物质受体 NK1R，可产生抗应激作用。 我们已经证明，NK1R 基因缺失的小鼠会导致酒精偏好显着降低，缺乏对酒精的条件性位置偏好，并且不会随着时间的推移而增加摄入量。使用 NK1 拮抗剂 L-703,606 对 NK1 受体进行药理学阻断，可抑制饮酒，类似于受体的基因删除（Thorsell 等，2010）。我们最近与 Kenner Rice 博士合作重新合成了一种文献化合物 L-822429，它对大鼠 NK1 受体具有高亲和力。 L-822429 剂量依赖性地抑制 Wistar 大鼠应激诱导的酒精寻求恢复，但对提示诱导的恢复没有影响，这表明 NK1R 拮抗剂和阿片类拮抗剂（如纳曲酮）可能存在累加效应（Schank 等，2011）。在使用基因选择的酒精偏好 P 大鼠的后续工作中，L-822429 剂量依赖性地抑制 P 大鼠的自我给药率，但对非依赖性 Wistar 大鼠无效。 P 大鼠多个脑区的 NK1R 受体表达上调，其中杏仁核中央 (CeA) 表达最高；将拮抗剂显微注射到该区域模拟了全身效应（Schank 等人提交）。总之，这些发现进一步支持 NK1R 拮抗作用作为治疗酒精中毒的候选靶点。 促肾上腺皮质激素释放激素 (CRH) 及其 CRH1 受体 在依赖后状态下，自我饮酒的增加和对压力行为敏感性的增加在很大程度上是由杏仁核中 CRH 受体的 CRH1 亚型上调介导的（Sommer 等，2008）。我们之前已经鉴定了一系列具有适合临床开发特性的 CRH 拮抗剂（Gehlert 等人，2007 年；Thorsell 等人，未发表数据）。其中两个候选药物 pexacerfont 和 GSK561679 被选入我们分别与百时美施贵宝和葛兰素史克合作的 CRADA 临床项目中的实验医学研究。这些研究目前正在进行中。 神经肽S (NPS) NPS 抑制实验动物的焦虑和食欲，与 Ciccocioppo 实验室合作的初步研究表明，NPS 信号可能在酗酒复发中发挥作用（Canella 等，2009）。我们开发了一种针对 NPS 受体的筛选检测方法，并与 NIH 化学基因组中心 (NCGC) 合作，鉴定了一种先导分子 NCG001865684，经测定，该分子在大鼠外周给药后具有脑渗透性。在体外，NCGC00185684 显示出偏向的拮抗剂特性，并且优先阻断 ERK 磷酸化，而不是细胞内 cAMP 或钙对 NPS 的反应。在体内，NCG001865684 预处理可阻断大鼠 CeA（参与调节酒精摄入的区域）中酒精诱导的 ERK 磷酸化。 NCGC00185684 还减少了操作性酒精自我管理，并降低了使用渐进比率响应测量的酒精奖励。这些影响在行为上是特定的，因为它们不影响运动活动或灭绝后的恢复反应。总而言之，这些数据初步验证了 NPSR 作为酒精中毒治疗靶点的作用（Thorsell 等人提交）。 基因表达谱和表观遗传修饰 一系列项目正在开展，研究依赖后大鼠模型中基因表达和表观遗传修饰（microRNA 和甲基化）的变化，以确定潜在的药理学靶点。 初始运行的生物信息学分析已经确定了几个有趣的差异表达转录本以及 miR 介导的基因表达变化的几个案例（Tapocik 等人，2012）。一项后续研究确定了一种 microRNA（miR-206）在酒精依赖中的功能作用。抑制 mPFC 中的 miR-206 会降低依赖性大鼠的饮酒量和偏好，但不会降低对照大鼠的饮酒量和偏好。此外，miR-206 被证明可以结合并抑制 BDNF 的表达（Tapocik 等人，正在准备中）。我们与 Paul Kenny 博士合作，确定在药理活性剂量的酒精下，miR-206 敲除小鼠的饮酒量和偏好程度显着低于 WT 小鼠（Tapocik 等人，准备中）。 我们还发现，mPFC 中的 DNA 甲基化受到酒精依赖史的调节，并导致酒精依赖后大鼠的酒精消耗量持续增加。 ICV 输注 DNA 甲基化抑制剂可使依赖后大鼠的 mPFC DNA 甲基化和饮酒量正常化。相反，全身注射甲基供体会诱导非依赖性大鼠 mPFC 中的 DNA 高甲基化，并增加饮酒量。 编码突触小泡蛋白的基因的表达在酒精依赖史后发生改变。这是通过 DNA 甲基化抑制来阻止的，这表明 DNA 甲基化调节对长期酒精暴露的行为适应（Barbier 等人提交）。