Discovery and validation of novel targets for pharmacotherapy of alcoholism

MP目前使用了两种类型的动物模型：一种是在长时间间歇性脑酒精暴露（后依赖）后酒精摄入量增加的动物，另一种是由于遗传因素而具有有趣的酒精相关表型的动物，如选择性繁殖的高酒精偏好大鼠，或者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。

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).