SECTION ON MOLECULAR PATHOPHYSIOLOGY? ? Medications development: Target discovery and validation.? ? Our group utilizes rodent models, molecular studies and pharmacological approaches to identifying novel mechanisms and treatment targets, and validating them for human development. ? ? Two categories of models for target discovery? ? Pre-existing genetic susceptibility factors? ? Genetic selection for high alcohol preference is a well established approach to search for alleles that contribute to high alcohol consumption. Our group has over the years collaborated with two laboratories where such lines have been developed, that of Hyytia (Helsinki, Finland: Alko Alcohol preferring, or AA line ) and of Ciccocioppo (Camerino, Italy: marchigian-sardinian preferring, or msP line). Although selected for high alcohol preference, AA and msP lines are phenotypically very different, illustrating the multitude of factors that contribute to high voluntary alcohol intake. Behavioral characterization by our laboratory in collaboration with the Finnish and Italian groups has shown that the AA line has a high degree of behavioral disinhibition (impulsivity), while recent work (see below) has shown msP rats to be anxious / stress-sensitive.? ? Long-term neuroadaptations? ? In recent years there has been a realization that a shift in motivational and neural mechanisms that occurs as dependence evolves, and the development of animal models that allow a study of these neuroadaptive processes. Our group has been at the forefront of this development. We've had previously demonstrated that prolonged exposure of the rat brain to repeated cycles of intoxication and mild withdrawal results in a long-term up-regulation of voluntary alcohol intake, encoded by long-term changes in gene expression patterns (Rimondini et al. 2002). Work in this report period has establiblished that animals with a history of dependence also have abnormal stress sensitivity, manifested as long-term up-regulated fear suppressio of behavior; and respond to stress with increased voluntary alcohol consumption (Sommer et al, e-pub 2007; for review see Heilig and Koob 2007). ? ? Discovery and validation of novel targets? ? Hypothesis driven studies: focus on stress and negative affect? ? Stress is a major factor triggering relapse both in alcoholics and in animal models. Our recent findings point to several stress-related neuropeptide systems as a key category of targets, which are selectively sensitive in the post-dependent state. ? ? Corticotropin-Releasing Hormone (CRH) and its CRH1 receptor? ? We recently found that both the elevated self-administration of alcohol and the 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. e-pub 2007). This converges with our recent findings of a stress-sensitive / anxious behavioral phenotype in the msP rat, accompanied by an innate up-regulation of CRH1 receptors in the amygdala and several other brain regions in this line (Hansson et al. 2006). The up-regulated CRH1 gene expression in msP rats is driven by a genetic polymorphism in the promoter region of the receptor gene in this line. Using the selective CRH1 antagonist antalarmin we have shown that this accounts both for the elevated self-administration of alcohol and the anxious phenotype of msP rats. These findings validate the CRH1 receptor as a treatment target. Based on this, we developed a collaborative agreement (CRADA) with Eli Lilly and Co, and identified a CRH antagonist with suitable properties for clinical use which is now in clinical development (Gehlert et al. 2007). A series of back-up compounds has also been evaluated.? ? Neuropeptide Y (NPY)? ? NPY is a potent endogenous anti-stress compound and counteracts the behavioral stress effects of CRH. Recent studies have shown that potentiation of NPY signaling, by NPY-Y1 receptor agonists, or by blockade of presynaptic Y2 autoreceptors, suppresses post-dependent drinking, while leaving basal intake of alcohol in non-dependent animals unaffected (reviewed in Thorsell et al. 2006). Based on these observations, we have developed a collaborative agreement with Johnson and Johnson Pharmaceuticals, and are currently evaluating non-peptide compounds targeting the Y2 receptor in order to identify molecules that could be developed for clinical use.? ? Substance P and its NK1 receptor? ? The prototypical member of the neurokinin family, Substance P, and its preferred neurokinin-1 (NK1) receptor are expressed throughout the fear-processing pathways of the brain, including the amygdala, hippocampus, hypothalamus and periaqueductal grey. Central injection of substance P agonists produces a range of defensive behavioral and cardiovascular reactions in animals, including conditioned place aversion, avoidance of the open arms of the elevated plus maze, potentiation of the acoustic startle response, and cardiovascular changes resembling the defense response to threatening stimuli. Furthermore, studies suggest that blockade of NK1 receptors might provide a novel mechanism by which to alleviate anxiety and other stress disorders. However, the majority of available compounds have only low affinity for the rat and mouse NK1 receptor making pharmacological studies difficult in these species. In order to circumvent these issues we are beginning to examine the phenotype of genetically deficient NK1R-/- mice with regards to ethanol sensitivity and intake. Preliminary data indicate markedly decrease alcohol preference and increased alcohol sensitivity. These data parallel a clinical study in our clinical section, where an NK1 antagonist has been evaluated for its ability to suppress alcohol cravings.? ? Unbiased approaches to target discovery ? ? A typical strategy has been to subject the models mentioned above to analysis of differential gene expression in key brain areas that are thought to control ethanol self-administration and negative affect. Brain regions of particular interest are the amygdala complex, cingulate cortex and hippocampus. One strategy has employed Affymetrix DNA microarray analysis for a primary screen, followed by in situ and/or quantitative (TaqMan) PCR confirmation. More recently, high-throughput in situ hybridization techniques for panels of candidate transcripts have been employed. These approaches have established several differentially expressed genes/gene clusters:? ? Glutamatergic genes? ? GLAST, essential for removal of extracellular glutamate, is up-regulated in the post-dependent state. An attempt is right now ongoing to study this mechanism using null-mutants for the GLAST-gene. These might provide a useful model for the neuroadapted dependent state. Preliminary data indicate that GLAST null mutants do not display excessive alcohol intake initially, but escalate to much higher levels than wildtypes over time. The GLAST mutants also show a more stress-sensitive phenotype, as well as hyperlocomotion. This model can be used to evaluate whether compounds that reduce glutamatergic drive, such as agonists at presynaptic mGluR2/3 autoreceptors that inhibit release of glutamate, are capable of normalizing a hyperglutamatergic state, and through this suppress alcohol intake.? ? Beta arrestin 2 ? ? A DNA-microarray screen in the preferring AA line has identified an over-expression of beta arrestin 2 (BARR2). We subsequently found that this is due to the presence of a unique genetic variant (haplotype) of this gene in the AA rats. BARR2 is crucial for regulation of signalling through a large number of G-protein coupled receptors, and signalling studies are ongoing to clarify the underlying mechanisms.? ? Relevance? ? These studies identify targets for novel alcoholism treatments.
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