Search

Preclinical and clinical work suggests that mifepristone (glucocorticoid receptor antagonist), may be a viable treatment for alcohol use disorder (AUD). The aim of this work was to translate our preclinical mifepristone study using yohimbine (α2 receptor antagonist) stress-induced reinstatement of alcohol-seeking to a clinical setting. This was a Phase 1/2, outpatient, cross-over, randomized, double-blind, placebo-controlled trial with non-treatment-seeking individuals with AUD (N=32). We investigated the safety, alcohol craving and consumption after oral administration of mifepristone (600mg daily for a week) in a human laboratory study comprised of administration of yohimbine in a cue-reactivity procedure and alcohol self-administration. Outcomes were assessed using Generalized Estimating Equations and mediation and moderation analyses assessed mechanisms of action and precision medicine targets. We did not observe serious adverse events related to the study drugs or study procedure and mild to moderate non-serious adverse events were reported by both study conditions. Also, there was no statistically-significant difference between the mifepristone and placebo in the hemodynamic response, alcohol subjective effects and pharmacokinetics parameters. Mifepristone significantly reduced alcohol craving and increased cortisol levels. Mifepristone-induced cortisol increase was not a mediator of alcohol craving. Moderation analysis with family history density of AUD (FHDA) and mifepristone, suggested that reduced craving was present in individuals withlow, but nothighFHDA. Mifepristone, compared to placebo, did not reduce alcohol consumption in the laboratory or in a naturalistic setting. This study successfully translated a preclinical paradigm to a human laboratory study confirming safety, tolerability and efficacy of mifepristone in an alcohol paradigm. Mediation analysis showed that the effect of mifepristone on craving was not related to mifepristone-induced increases in cortisol and moderation of FHDA suggested the importance of evaluating AUD endophenotypes for pharmacotherapies.Clinical trial registrationClinicaltrials.gov;NCT02243709IND/FDA121984, mifepristone and yohimbine (Holder: Haass-Koffler)

Background and aims Andrographis paniculata is an annual herbaceous plant which belongs to the Acanthaceae family. Extracts from this plant have shown hepatoprotective, anti-inflammatory and antidiabetic properties, at least in part, through activation of the nuclear receptor Peroxisome Proliferator-Activated Receptor-gamma (PPAR γ). Recent evidence has demonstrated that activation of PPARγ reduces alcohol drinking and seeking in Marchigian Sardinian (msP) alcohol-preferring rats. Methods The present study evaluated whether A. paniculata reduces alcohol drinking and relapse in msP rats by activating PPARγ. Results Oral administration of an A. paniculata dried extract (0, 15, 150 mg/kg) lowered voluntary alcohol consumption in a dose-dependent manner and achieved ~65% reduction at the dose of 450 mg/kg. Water and food consumption were not affected by the treatment. Administration of Andrographolide (5 and 10 mg/kg), the main active component of A. paniculata, also reduced alcohol drinking. This effect was suppressed by the selective PPARγ antagonist GW9662. Subsequently, we showed that oral administration of A. paniculata (0, 150, 450 mg/kg) prevented yohimbine- but not cues-induced reinstatement of alcohol seeking. Conclusions Results point to A. paniculata-mediated PPARγactivation as a possible therapeutic strategy to treat alcohol use disorder.

The ongoing rise in opioid use disorder (OUD) has made it imperative to better model the individual variation within the human population that contributes to OUD vulnerability. Using animal models that capture such variation can be a useful tool. Individual variation in novelty-induced locomotion is predictive of substance use disorder (SUD) propensity. In this model, rats are characterized as high-responders (HR) or low-responders (LR) using a median split based on distance travelled during a locomotor test, and HR rats are generally found to exhibit a more SUD vulnerable behavioral phenotype.The HR/LR model has commonly been used to assess behaviors in male rats using psychostimulants, with limited knowledge of the predictive efficacy of this model in females or the use of an opioid as the reward. In the current study, we assessed several behaviors across the different phases of drug addiction (heroin taking, refraining, and seeking) in over 500 male and female heterogeneous stock rats run at two geographically separate locations. Rats were characterized as HRs or LRs within each sex for analysis.Overall, females exhibit a more OUD vulnerable phenotype relative to males. Additionally, the HR/LR model was predictive of OUD-like behaviors in male, but not female rats. Furthermore, phenotypes did not differ in anxiety-related behaviors, reacquisition of heroin-taking, or punished heroin-taking behavior in either sex.These results emphasize the importance of assessing females in models of individual variation in SUD and highlight limitations in using the HR/LR model to assess OUD propensity.

Alcohol use disorder (AUD) is a significant public health concern, contributing to a myriad of social, psychological, and physiological issues. Despite substantial efforts within the alcohol research field, promising preclinical findings have failed to translate to clinical use, highlighting the necessity to develop safe and effective pharmacological probes with the ability to be used in preclinical and clinical research. Yohimbine, an α2 adrenergic receptor antagonist, is a well-validated pharmacological tool that has been widely employed in alcohol studies to evaluate noradrenergic activation. This scoping systematic review examines published literature in rodent and human studies involving the use of yohimbine relevant to alcohol research. We conducted a systematic literature review of MEDLINE, Embase, Web of Science Core Collection, CINAHL, PsycInfo, and Cochrane Central Register of Controlled Trials to identify: (1) Experimental Characteristics and Methodology, (2) Sex Differences, (3) Neurochemical Systems and Brain Regions, and (4) Discussion of Applications for Medication Development. Sixty-seven (62 preclinical and 5 clinical) studies were identified meeting the stated criteria, comprising extensive evidence supporting the use of yohimbine as a safe, titratable pharmacological agent for translational alcohol research. Support for the use of yohimbine as a fully translational tool, however, is hindered by limited available findings from human laboratory studies, as well as a dearth of studies examining sex differences in yohimbine's mechanistic actions. Additional consideration should be given to further translational modeling, ideally allowing for parallel preclinical and clinical assessment of yohimbine, methodological assessment of neurochemical systems and brain regions.

Tobacco use disorder is a worldwide health problem for which available medications show limited efficacy. Nicotine is the psychoactive component of tobacco responsible for its addictive liability. Similar to other addictive drugs, nicotine enhances mesolimbic dopamine transmission. Inhibition of the fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the endocannabinoid anandamide (AEA), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), reduces nicotine-enhanced dopamine transmission and acquisition of nicotine self-administration in rats. Down-regulation of dopamine transmission by antagonists or partial agonists of the dopamine D3 receptor (DRD3) also reduced nicotine self-administration and conditioned place preference. Based on these premises, we evaluated the effect of ARN15381, a multitarget compound showing FAAH inhibition and DRD3 partial agonist activity in the low nanomolar range, on nicotine self-administration in rats. Pretreatment with ARN15381 dose dependently decreased self-administration of a nicotine dose at the top of the nicotine dose/response (D/R) curve, while it did not affect self-administration of a nicotine dose laying on the descending limb of the D/R curve. Conversely, pretreatment with the selective FAAH inhibitor URB597 and the DRD3 partial agonist CJB090 failed to modify nicotine self-administration independent of the nicotine dose self-administered. Our data indicates that the concomitant FAAH inhibition and DRD3 partial agonism produced by ARN15381 is key to the observed reduction of nicotine self-administration, demonstrating that a multitarget approach may hold clinical importance for the treatment of tobacco use disorder.

Alcohol Use Disorder (AUD) is a chronic disease that develops over the years. The complexity of the neurobiological processes contributing to the emergence of AUD and the neuroadaptive changes occurring during disease progression make it difficult to improve treatments. On the other hand, this complexity offers researchers the possibility to explore new targets. Over years of intense research several molecules were tested in AUD; in most cases, despite promising preclinical data, the clinical efficacy appeared insufficient to justify futher development. A prototypical example is that of corticotropin releasing factor type 1 receptor (CRF1R) antagonists that showed significant effectiveness in animal models of AUD but were largely ineffective in humans. The present article attempts to analyze the most recent venues in the development of new medications in AUD with a focus on the most promising drug targets under current exploration. Moreover, we delineate the importance of using a more integrated translational framework approach to correlate preclinical findings and early clinical data to enhance the probability to validate biological targets of interest.

The neuropeptide S (NPS) is the endogenous ligand of the NPS receptor (NPSR). The NPSR is widely expressed in brain regions that process emotional and affective behavior. NPS possesses a unique physio-pharmacological profile, being anxiolytic and promoting arousal at the same time. Intracerebroventricular NPS decreased alcohol consumption in alcohol-preferring rats with no effect in non-preferring control animals. This outcome is most probably linked to the anxiolytic properties of NPS, since alcohol preference is often associated with high levels of basal anxiety and intense stress-reactivity. In addition, NPSR mRNA was overexpressed during ethanol withdrawal and the anxiolytic-like effects of NPS were increased in rodents with a history of alcohol dependence. In line with these preclinical findings, a polymorphism of the NPSR gene was associated with anxiety traits contributing to alcohol use disorders in humans. NPS also potentiated the reinstatement of cocaine and ethanol seeking induced by drug-paired environmental stimuli and the blockade of NPSR reduced reinstatement of cocaine-seeking. Altogether, the work conducted so far indicates the NPS/NPSR system as a potential target to develop new treatments for alcohol and cocaine abuse. An NPSR agonist would be indicated to help individuals to quit alcohol consumption and to alleviate withdrawal syndrome, while NPSR antagonists would be indicated to prevent relapse to alcohol- and cocaine-seeking behavior.

Increasing sensitivity due to alcohol intake has been explored using molecular and cellular mechanisms of sensitization and adaptive biobehavioral changes as well as through negative experiences of altered function during withdrawal. However, within both a preclinical and human laboratory setting, little has been elucidated toward understanding the neural substrates of decreased sensitivity to alcohol effects, that is, alcohol tolerance. More paradigms assessing alcohol tolerance are needed. Tolerance can be assessed through both self-reported response (subjective) and observed (objective) measurements. Therefore, sensitivity to alcohol is an exploitable variable that can be utilized to disentangle the diverse alcohol use disorder (AUD) phenotypical profile. This literature review focuses on preclinical models and human laboratory studies to evaluate alcohol tolerance and its modulating factors. Increased understanding of alcohol tolerance has the potential to reduce gaps between preclinical models and human laboratory studies to better evaluate the development of alcohol-related biobehavioral responses. Furthermore, alcohol tolerance can be used as an AUD phenotypic variable in randomized clinical trials designed for developing AUD therapies. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Alcoholism is often associated with other forms of drug abuse, suggesting that innate predisposing factors may confer vulnerability to addiction to diverse substances. However, the neurobiological bases of these factors remain unknown. Here, we have used a combination of imaging, neurochemistry and behavioral techniques to investigate responses to the psychostimulant amphetamine in Marchigian Sardinian (msP) alcohol-preferring rats, a model of vulnerability to alcoholism. Specifically, we employed pharmacological magnetic resonance imaging to investigate the neural circuits engaged by amphetamine challenge, and to relate functional reactivity to neurochemical and behavioral responses. Moreover, we studied self-administration of cocaine in the msP rats. We found stronger functional responses in the extended amygdala, alongside with increased release of dopamine in the nucleus accumbens shell and augmented vertical locomotor activity compared with controls. Wistar and msP rats did not differ in operant cocaine self-administration under short access (2 hours) conditions, but msP rats exhibited a higher propensity to escalate drug intake following long access (6 hours). Our findings suggest that neurobiological and genetic mechanisms that convey vulnerability to excessive alcohol drinking also facilitate the transition from psychostimulants use to abuse.

Fatty acid amide hydrolase (FAAH) is an enzyme that prominently degrades the major endocannabinoid N-arachidonoylethanolamine (anandamide). Inhibition of this enzyme leads to increased anandamide levels in brain regions that modulate stress and anxiety. Recently, we found that genetically selected Marchigian Sardinian alcohol-preferring (msP) rats display hyperactive FAAH in amygdalar regions that was associated with increased stress sensitivity and a hyper-anxious phenotype. Our previous work has also demonstrated that msPs display an innate preference for and excessive consumption of alcohol, potentially reflecting a form of self-medication to gain relief from hyper-anxious states. Here, we expand on our previous work by microinjecting the selective FAAH inhibitor URB597 (vehicle, 0.03, 0.1 and 1.0 μg per rat) into the central amygdala (CeA) and basolateral amygdala in msP versus non-selected Wistar rats to evaluate the effects of localized FAAH inhibition on operant alcohol self-administration and restraint-induced anxiety using the elevated plus maze. Intra-CeA URB597 significantly reduced alcohol self-administration in msP but not in Wistar rats. Intra-basolateral amygdala URB597 also attenuated alcohol drinking in msPs, although the effect was less pronounced relative to CeA treatment. In contrast, control experiments administering URB597 into the ventral tegmental area produced no genotypic differences in drinking. We also found that URB597 treatment in the CeA significantly reduced the anxiogenic effects of restraint stress in msPs, although no effects were detected in Wistars. Dysregulation of FAAH regulated systems in the major output region of the amygdala may drive the propensity for co-morbid expression of anxiety and excessive alcohol use.

Cocaine addiction is a multi-dimensional behavioral disorder characterized by a loss of control over cocaine taking despite of detrimental consequences. Structural MRI studies have revealed association between cocaine consumption and gray matter volume (GMV) in cocaine-addicted patients. However, the behavioral correlates of GMV in cocaine addiction are poorly understood. Here, we used a DSM-IV-based rat model of cocaine addiction with high face validity for structural imaging. According to three behavioral sub-dimensions of addiction, rats were separated into two groups showing either addict-like or non-addict-like behavior. These behavioral sub-dimensions were (1) the inability to refrain from drug-seeking and taking, (2) high motivation for the drug, and (3) maintained drug use despite negative consequences. In these rats, we performed structural MRI with voxel-based morphometry and analyzed the interaction of GMV with behavioral sub-dimensions in cocaine-addicted rats. Our major findings are that GMV differentially correlate with the inability to refrain from drug-seeking and taking in addict-like and non-addict-like rats within the somatosensory cortices and the amygdala. High motivation for the drug differentially correlates with GMV in addict-like and non-addict-like rats within the medial prefrontal cortex, and maintained drug use despite negative consequences differentially correlates with GMV in these two groups of rats within the periaqueductal gray. Our results demonstrate that the behavioral differences characterizing addict-like and non-addict-like rats in each behavioral sub-dimension of addiction are reflected by divergent covariance with GMV. We conclude that structural imaging provides specific neuroanatomical correlates of behavioral sub-dimensions of addiction.

High rates of relapse after prolonged abstinence are often triggered by exposure to drug-associated cues that induce drug craving. Incubation of drug craving is a phenomenon that consists of time-dependent increases in cue-induced drug craving during withdrawal. Plasticity mechanisms in the nucleus accumbens (NAc) underlie drug-seeking responses and involve changes in excitatory synaptic transmission's efficacy. In particular, the prefrontal cortex (PFC) glutamatergic input to the NAc core has been well characterized regarding cocaine-evoked plasticity following non-contingent versus contingent exposure to cocaine or alternatively after protracted abstinence. Still, the synaptic strength during the course of withdrawal compared to drug-naïve condition is unknown, since electrophysiological characterizations are mainly performed in brain slices or focus on distinct time points during cocaine-evoked plasticity in vivo. Here we used an incubation paradigm, in which rats had extended accessed to cocaine self-administration, and underwent cue-induced reinstatement at withdrawal day 1 and 30. Longitudinal in vivo field potential recordings in awake rats showed that chronic contingent exposure to cocaine strengthened the prelimbic PFC to NAc core pathway when compared to pre-cocaine condition. This strengthening was associated with decreased paired-pulse ratios (PPR), indicative of presynaptic enhancement of glutamate release, which persisted throughout withdrawal. Moreover, both field potential increase and PPR reduction after chronic cocaine exposure correlated with the number of cocaine infusions received during training. The present results together with previous findings of withdrawal-dependent postsynaptic enhancement of the PFC-NAc core pathway, suggest an additional presynaptic strengthening that is initiated during self-administration and maintained throughout abstinence in drug-seeking rats. These cocaine-driven neuroadaptations may provide a neural substrate for maladaptive processing of cues that can ultimately trigger craving and relapse.

Nociceptin/orphanin FQ (N/OFQ) is a 17 amino acid peptide that was deorphanized in 1995 and has been widely studied since. The role of the N/OFQ system in drug abuse has attracted researchers’ attention since its initial discovery. The first two scientific papers describing the effect of intracranial injection of N/OFQ appeared 20 years ago and reported efficacy of the peptide in attenuating alcohol intake, whereas heroin self-administration was insensitive. Since then more than 100 scientific articles investigating the role of the N/OFQ and N/OFQ receptor (NOP) system in drug abuse have been published. The present article provides an historical overview of the advances in the field with focus on three major elements. First, the most robust data supportive of the efficacy of NOP agonists in treating drug abuse come from studies in the field of alcohol research, followed by psychostimulant and opioid research. In contrast, activation of NOP appears to facilitate nicotine consumption. Second, emerging data challenge the assumption that activation of NOP is the most appropriate strategy to attenuate consumption of substances of abuse. This assumption is based first on the observation that animals carrying an overexpression of NOP system components are more prone to consume substances of abuse, whereas NOP knockout rats are less motivated to self-administer heroin, alcohol, and cocaine. Third, administration of NOP antagonists also reduces alcohol consumption. In addition, NOP blockade reduces nicotine self-administration. Hypothetical mechanisms explaining this apparent paradox are discussed. Finally, we focus on the possibility that co-activation of NOP and mu opioid (MOP) receptors is an alternative strategy, readily testable in the clinic, to reduce the consumption of psychostimulants, opiates, and, possibly, alcohol.

Neuropeptide S (NPS) displays unique pharmacological properties and induces both anxiolytic and pro-stress/arousal activities. Previous studies performed using Wistar rats demonstrated that NPS facilitated alcohol and cocaine seeking but did not affect alcohol or cocaine consumption.Here, we investigated the effects of NPS in Marchigian Sardinian alcohol-preferring (msP) rats, a rat strain characterized by excessive alcohol consumption comorbid with heightened anxiety and depressive-like phenotypes. Specifically, we evaluated the effect of NPS on operant alcohol self-administration by msP rats compared to Wistar rats. The effect of NPS on cue-induced reinstatement of alcohol seeking in msP rats was also evaluated. Finally, using the open field test (OFT) and the elevated plus maze (EPM), we evaluated the effects of NPS on locomotor activity and anxiety.NPS reduced alcohol self-administration but did not affect cue-induced reinstatement in the msP rat. In addition, NPS induced reinstatement of extinguished alcohol seeking in Wistar rats without affecting alcohol intake. In the EPM task, NPS, in accordance with its anxiolytic activity, increased the time spent in the open arm of the arena by msP rats, although this effect was not observed in Wistar rats.These data suggest that the effect of NPS is strongly influenced by the genetic background of the animal. In Wistar rats, NPS acts as a pro-arousal agent to promote the reinstatement of alcohol seeking. However, when alcohol drinking is motivated by or associated with a state of pathological anxiety, NPS attenuates alcohol consumption and seeking due to its anxiolytic activity.

Cocaine addiction is a widespread psychiatric condition still waiting for approved efficacious medications. Previous studies suggested that simultaneous activation of nociceptin opioid (NOP) and mu opioid (MOP) receptors could be a successful strategy to treat cocaine addiction, but the paucity of molecules co-activating both receptors with comparable potency has hampered this line of research. Cebranopadol is a non-selective opioid agonist that at nanomolar concentration activates both NOP and MOP receptors and that recently reached phase-III clinical trials for cancer pain treatment. Here, we tested the effect of cebranopadol on cocaine self-administration (SA) in the rat. We found that under a fixed-ratio-5 schedule of reinforcement, cebranopadol (25 and 50 µg/kg) decreased cocaine but not saccharin SA, indicating a specific inhibition of psychostimulant consumption. In addition, cebranopadol (50 µg/kg) decreased the motivation for cocaine as detected by reduction of the break point measured in a progressive-ratio paradigm. Next, we found that cebranopadol retains its effect on cocaine consumption throughout a 7-day chronic treatment, suggesting a lack of tolerance development toward its effect. Finally, we found that only simultaneous blockade of NOP and MOP receptors by concomitant administration of the NOP antagonist SB-612111 (30 mg/kg) and naltrexone (2.5 mg/kg) reversed cebranopadol-induced decrease of cocaine SA, demonstrating that cebranopadol activates both NOP and classical opioid receptors to exert its effect. Our data, together with the fairly advanced clinical development of cebranopadol and its good tolerability profile in humans, indicate that cebranopadol is an appealing candidate for cocaine addiction treatment.

The DSM5-based dimensional diagnostic approach defines substance use disorders on a continuum from recreational drug use to habitual and ultimately addicted behavior. Biomarkers that are indicative of recreational drug use and addicted behavior are lacking. We performed a translational [18F]-fluorodeoxyglucose-positron emission tomography (FDG-PET) study in the multi-dimensional 0/3crit model of cocaine addiction. Addict-like (3crit) and non-addict-like (0crit) rats, which shared identical life conditions and levels of cocaine self-administration, were acquired for FDG-PET under baseline conditions and following cocaine and yohimbine challenges. Compared to cocaine-naive control rats, 0crit animals showed higher glucose uptake in the caudate putamen (CPu) and medial prefrontal cortex (mPFC) respect to naive controls. 3crit animals did not show this adaptive higher glucose utilization, but had lower uptake in several cortical areas. Both cocaine and yohimbine challenges affected glucose uptake in control rats in several brain sites, but not in 0crit and 3crit rats, indicating that impaired glucose mobilization in response to these challenges is not specifically associated with addictive behavior. Compared to 0crit, 3crit rats showed higher reinstatement responses, which were negatively associated with glucose uptake in the ventral tegmental area. Data indicate that cocaine non-addict- and addict-like phenotypes are associated with several potential biomarkers. Specifically, we propose that increased glucose uptake in the CPu and mPFC is a function of controlled drug use, whereas a loss of striatal and prefrontal metabolic activity and reduced uptake in cortical areas are indicative of addictive behavior.

Background Pioglitazone is a selective peroxisome proliferator-activated receptor γ (PPARγ) agonist used for the treatment of insulin resistance and type 2 diabetes. Previous studies conducted in our laboratory showed that activation of PPARγ by pioglitazone reduces alcohol drinking, stress-induced relapse, and alcohol withdrawal syndrome in rats. Pioglitazone was not able to prevent relapse elicited by alcohol cues. Conversely, the nonselective opioid antagonist naltrexone has been shown to reduce alcohol drinking and cue- but not stress-induced relapse in rodents. Methods Based on these findings, this study was sought to determine the efficacy of pioglitazone and naltrexone combination on alcohol intake and relapse behavior. Genetically selected alcohol-preferring Marchigian Sardinian (msP) rats were used for the study. Results Pioglitazone (10 and 30 mg/kg) and naltrexone (0.25 and 1.0 mg/kg) each individually reduced alcohol drinking in msP rats. The combination of the 2 drugs resulted in a more potent alcohol drinking reduction than single agents. Confirming previous studies, pioglitazone (10 and 30 mg/kg) significantly reduced relapse induced by the pharmacological stressor yohimbine (1.25 mg/kg) but not by cues predictive of alcohol availability. Conversely, naltrexone reduced reinstatement of drug seeking elicited by alcohol cues but not by yohimbine. Conclusions The drug combination was effective in reducing both relapse behaviors. These findings open new vistas in the use pioglitazone in combination with naltrexone for the treatment of alcoholism.

Drug abuse represents a considerable burden of disease and has enormous economic impacts on societies. Over the years, few medications have been developed for clinical use. Their utilization is endowed with several limitations, including partial efficacy or significant side effects. On the other hand, the successful advancement of these compounds provides an important proof-of-concept for the feasibility of drug development programs in addiction. In recent years, a wealth of information has been generated on the psychological mechanisms, genetic or epigenetic predisposing factors, and neurobiological adaptations induced by drug consumption that interact with each other to contribute to disease progression. It is now clear that addiction develops through phases, from initial recreational use to excessive consumption and compulsive drug seeking, with a shift from positive to negative reinforcement driving motivated behaviors. A greater understanding of these mechanisms has opened new vistas in drug development programs. Researchers’ attention has been shifted from investigation of classical targets associated with reward to biological substrates responsible for negative reinforcement, impulse loss of control and maladaptive mechanisms resulting from protracted drug use. From this research, several new biological targets for the development of innovative therapies have started to emerge. The present article offers an overview of targets currently under scrutiny for the development of new medications for addiction. This work is not exhaustive but rather it provides a few examples of how this research has advanced in recent years by virtue of studies carried out in our laboratory.

Drug abuse represents a considerable burden of disease and has enormous economic impacts on societies. Over the years, few medications have been developed for clinical use. Their utilization is endowed with several limitations, including partial efficacy or significant side effects. On the other hand, the successful advancement of these compounds provides an important proof of concept for the feasibility of drug development programs in addiction. In recent years, a wealth of information has been generated on the psychological mechanisms, genetic or epigenetic predisposing factors, and neurobiological adaptations induced by drug consumption that interact with each other to contribute to disease progression. It is now clear that addiction develops through phases, from initial recreational use to excessive consumption and compulsive drug seeking, with a shift from positive to negative reinforcement driving motivated behaviors. A greater understanding of these mechanisms has opened new vistas in drug development programs. Researchers' attention has been shifted from investigation of classical targets associated with reward to biological substrates responsible for negative reinforcement, impulse loss of control, and maladaptive mechanisms resulting from protracted drug use. From this research, several new biological targets for the development of innovative therapies have started to emerge. This chapter offers an overview of targets currently under scrutiny for the development of new medications for addiction. This work is not exhaustive but rather it provides a few examples of how this research has advanced in recent years by virtue of studies carried out in our laboratory.

Neuropeptide S (NPS) is the endogenous ligand for the previously orphan G-protein-coupled-receptor, now termed NPS receptor (NPSR). NPS has both anxiolytic and pro-arousal properties and decreases food intake. In this work we use a rat model of palatable food intake to test in vivo different analogs of human NPS developed in our laboratories and characterized in previous in vitro experiments as partial agonists ([Ala 3 ]NPS and [Aib 5 ]NPS), or antagonists ([ d -Cys( t Bu) 5 ]NPS and [ t Bu- d -Gly 5 ]NPS). Our results confirmed that intracerebroventricular (ICV) injection of NPS (1 nmol) decreases standard chow intake in food restricted rats as well as in freely feeding animals fed with standard or palatable food diets. [Aib 5 ]NPS (30 and 60 nmol), like NPS, reduced palatable food intake, thus confirming in vivo its ability to activate NPSR. [Ala 3 ]NPS (60 nmol) did not affect palatable food intake per se but blocked the anorectic effect of NPS, thus suggesting its ability to function as an antagonist in this model. Finally, [ d -Cys( t Bu) 5 ]NPS (20–60 nmol) and [ t Bu- d -Gly 5 ]NPS (10–30 nmol), described in previous in vitro studies as pure NPSR antagonists, did not affect palatable food intake when given alone, but fully blocked the anorectic effect of NPS. These results provide an important characterization of the pharmacological properties of these NPS analogs in vivo . Of particular relevance are the data showing that [ d -Cys( t Bu) 5 ]NPS and [ t Bu- d -Gly 5 ]NPS behave as pure antagonists at NPSR regulating food intake, indicating that these molecules are suitable tools for further investigation of the physiopharmacology of the NPS/NPSR system.

THE CORTICOTROPIN-RELEASING FACTOR (CRF) peptide system has been long proposed to be involved in drug addiction and alcoholism (Dave and Eskay, 1986; Hawley et al., 1994; Inder et al., 1995; Koob, 1999; Rivier et al., 1984; Wand and Dobs, 1991; Wilkins and Gorelick, 1986). In mammals this system is composed of 4 endogenous ligands, namely, CRF, Urocortin (Ucn)1, Ucn 2, and Ucn 3 (Bale and Vale, 2004). These 4 ligands act on CRF1 receptors, CRF2 receptors, and/or the CRF-binding protein with different affinities (Bale and Vale, 2004). Discovered first of the 4 ligands, CRF has attracted the most attention in the addiction field. However, evidence is accumulating that Ucn 1 also may be important for addiction-related behaviors, specifically, for alcohol self-administration (Ryabinin and Weitemier, 2006). In the brain, Ucn 1 is primarily synthesized in the pericolumotor Ucn-containing population of neurons (pIIIu) and, to a lesser extent, in the lateral superior olive (May et al., 2008; Vaughan et al., 1995). The pIIIu was originally thought to be the Edinger-Westphal nucleus, a brain region primarily involved in ocular functions (Bittencourt et al., 1999; Kozicz et al., 1998; Vaughan et al., 1995). Later, it was realized that this brain region has been misidentified in rodents and humans, and it was subsequently renamed: at first, as the non-preganglionic Edinger-Westphal nucleus (Ryabinin et al., 2005; Weitemier and Ryabinin, 2005), and subsequently, as the pIIIu (May et al., 2008). The pIIIu exerts its actions via projections to the lateral septum, dorsal raphe, spinal cord, and other brain regions (Bachtell et al., 2003; Bittencourt et al., 1999; Weitemier et al., 2005). Substantial evidence implicating Ucn 1 in alcohol self-administration has been obtained in mouse experimental models. First, various alcohol drinking paradigms in mice have demonstrated preferential induction of Fos immunoreactivity in pIIIu (Bachtell et al., 1999; Ryabinin et al., 2001, 2003; Sharpe et al., 2005). Second, higher levels of Ucn 1 immunoreactivity in pIIIu have been found in ethanol-naive, C57BL/6J mice, an inbred strain with spontaneously high alcohol preference, than in alcohol-avoiding DBA/2J mice, which show low ethanol preference (Bachtell et al., 2002b; Weitemier et al., 2005). In addition, differences in Ucn1 immunoreactivity were observed between several mouse lines selectively-bred for differences in alcohol intake, ethanol-induced place preference, ethanol-induced hypothermia, ethanol-induced loss of righting reflex or ethanol withdrawal-induced convulsions (Bachtell et al., 2002b, 2003; Kiianmaa et al., 2003; Ryabinin and Tsivkovskaia, 2004; Turek et al., 2008). Third, electrolytic lesions of pIII blocked alcohol preference in C57BL/6J mice (Bachtell et al., 2004). Fourth, injections of picomolar doses of Ucn 1, but not CRF, into the mouse lateral septum selectively attenuated alcohol self-administration (Ryabinin et al., 2008). Evidence implicating Ucn 1 in alcohol self-administration in other species also exists, but is less substantial and some-what conflicting. Operant alcohol self-administration in alcohol-preferring AA rats (Weitemier et al., 2001) and beer drinking in Sprague–Dawley rats (Topple et al., 1998) each selectively induced c-Fos immunoreactivity in pIIIu. Additionally, higher levels of Ucn 1 immunoreactivity in pIIIu have been observed in the HAD1 and HARF alcohol-preferring rats (each selectively bred for high alcohol intake), compared to their respective alcohol-avoiding LAD1 and LARF lines (selectively bred for low alcohol intake) (Turek et al., 2005). In contrast, high alcohol-preferring inbred P rats have shown lower Ucn 1 immunoreactivity in pIIIu than low alcohol-preferring inbred NP rats, and high alcohol preferring HAD2 and AA lines have not shown significant differences in Ucn 1 immunoreactivity from low alcohol-preferring LAD2 and ANA lines (Turek et al., 2005). Still, further meta-analysis has indicated that the number of Ucn 1-immunoreactive fibers in the lateral septum, a target of pIIIu neurons (Bittencourt et al., 1999; Weitemier et al., 2005), is significantly higher in the 5 lines of alcohol-preferring rats relative to their corresponding alcohol-avoiding lines (Turek et al., 2005). Thus, while overall the majority of the data in rats support previous data in mice, the relationship between Ucn 1 expression and alcohol preference in genetically-selected rats would benefit from further investigation. Some of the inconsistencies in the reported results might stem from limitations in the design of previous studies. Of importance, previous studies on Ucn 1 expression were only done bi-directionally, comparing alcohol-preferring versus avoiding rodent lines, which could complicate interpretation of the resulting data. Comparison of alcohol-preferring rats versus outbred rats unselected for alcohol preference could be more informative for confirming involvement of Ucn 1 in alcohol “preference.” Additionally, previous studies in selectively bred rats did not analyze females. It was shown previously that Ucn 1 gene expression can be regulated by estrogen (Derks et al., 2007; Haeger et al., 2006), and therefore Ucn 1’s contribution to alcohol intake might be affected by sex. Previous work showed that the pIII area also has a significant subpopulation of dopaminergic neurons (Bachtell et al., 2002a; Gaszner and Kozicz, 2003). Midbrain dopamine (DA) systems are known to contribute to mechanisms of addiction (Kiianmaa et al., 2003; Nestler et al., 1993; Wise, 1998), and previous research suggested differential dopaminergic innervation in structures related to reward behaviors in rats selectively bred for alcohol preference (Casu et al., 2002a). Thus, this study tested the hypothesis that selective breeding for alcohol preference also affected this subpopulation of dopaminergic neurons in pIII. Tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of monoamines, is a precursor of both noradrenaline and dopamine, and was used as a marker of noradrenergic and dopaminergic activity (Casu et al., 2002a). To address the above issues, we used 2 sublines derived from the Sardinian preferring (sP) rats, with their respective nonselectively-bred Wistar pairs in our analyses. The sP rat lines represent unique animal models for human alcohol consumption. The breeding program of sP rats started in 1981 at the University of Cagliari, Italy, with a heterogeneous population of outbred Wistar rats. After 40 generations of mating the sP rats displayed average daily alcohol consumption ranging 6 to 7 g/kg and met the majority of several fundamental requirements for a successful animal model of alcoholism (Colombo et al., 2006). Starting from the 13th generation of sP rats, a subset of these animals were used for separate selective breeding for alcohol preference at the University of Camerino, Italy. After 20 further generations of selective breeding, this subline was re-named the Marchigian Sardinian preferring rats (msP). Similar to sP rats, msP rats display innate preference for alcohol characterized by: binge-like drinking behavior, consumption of pharmacologically significant daily doses of 7 to 8 g/kg of EtOH (Ciccocioppo et al., 2006), low aversion to alcohol (Ciccocioppo et al., 1999; Polidori et al., 1998), and spontaneous drinking of large amounts of alcohol from the very first homecage presentation (Ciccocioppo et al., 2006). At the 32nd generation of breeding of sP rats, a subset of sP rats were provided by Dr. G.L. Gessa to The Scripps Research Institute and maintained there without further selective breeding. These rats have been designated as Scr:sP. The Scr:sP rats continue to show alcohol preference (consuming voluntarily about 5 to 6 g/kg of EtOH daily) and display high levels of anxiety-like behavior (Sabino et al., 2006, 2007). A genetic divergence has emerged between the msP and Scr:sP substrains including specific single nucleotide polymorphisms in the CRF1 gene present in msP but not Scr:sP or corresponding Wistar control lines (Hansson et al., 2006). Therefore, while behavioral profiles of both msP and Scr:sP rats are similar in mimicking several aspects of the human alcoholic population, the underlying genes involved in regulation of alcohol intake partly differ between the 2 sublines, resulting in 2 rat models of alcoholism complementary to each other. Using these 2 selectively bred preferring rat lines (Scr:sP and msP) versus nonselectively bred Wistar rats, we tested the hypothesis that pIII is involved in the predisposition to high alcohol consumption in rats. To test this hypothesis, we analyzed Ucn 1 and TH immunoreactivity in pIII of male and female alcohol-preferring animals (Scr:sP and msP rats) compared to control Wistar rats.

The association of ethanol's reinforcing effects with specific environmental stimuli is thought to be a critical factor for relapse risk in alcoholism. This study examined in rats the effects of a newly deorphanized neuropeptide receptor and its cognate ligand, Neuropeptide S (NPS), on ethanol consumption and reinstatement of ethanol-seeking by environmental cues previously associated with ethanol availability. In the self-administration experiments, the stable response rates observed for ethanol reinforcement were not modified by intracerebroventricular (ICV) injection of NPS (1.0 and 2.0 nmol per rat). In the reinstatement experiments, ethanol-associated cues induced robust rates of ethanol seeking, which were highly resistant to extinction over repeated sessions of reinstatement testing. ICV NPS treatment (1.0, 2.0 and 4.0 nmol per rat) resulted in a significant increase of ethanol seeking elicited by ethanol-associated cues. In contrast, NPS did not affect the reinstatement of responding to water-paired stimuli. Site-specific NPS injection (0.1 and 0.5 nmol per rat) into the lateral hypothalamus also reinstated extinguished responding to ethanol. This effect was selectively blocked by pre-treatment with the hypocretin-1/orexin-A antagonist SB-334867 (10 mg/kg, i.p.). At the dose tested, SB-334867 did not modify alcohol reinstatement per se. These results provide the first demonstration that activation of NPS receptors in the LH intensifies relapse to ethanol-seeking elicited by environmental conditioning factors. This effect is selective, and is mediated by activation of LH hypocretin neurones. Based on the present findings, we also predict that antagonism at NPS receptors could represent a novel pharmacological approach to alcohol relapse treatment.

Rationale: A major clinical concern with the use of cannabinoid receptor 1 (CB1) direct agonists is that these compounds increase alcohol drinking and drug abuse-related behaviours. As an alternative approach, CB1-receptor-mediated activity can be facilitated by increasing anandamide levels with the use of hydrolase fatty acid amide hydrolase (FAAH) inhibitors. Objective: Using the selective FAAH inhibitor URB597, we investigated whether activation of the endogenous cannabinoid tone increases alcohol abuse liability, as what happens with the CB1 receptor direct agonists. Materials and methods: URB597 was tested on alcohol self-administration in Wistar rats and on homecage alcohol drinking in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats. In Wistar rats, URB597 effects on alcohol-induced anxiety and on stress-, yohimbine- and cue-induced reinstatement of alcohol seeking were also evaluated. For comparison, the effect of the CB1 receptor antagonist rimonabant on ethanol self-administration was also tested. Results: Under our experimental condition, intraperitoneal (IP) administration of URB597 (0.0, 0.3 and 1.0 mg/kg) neither increased voluntary homecage alcohol drinking in msP rats nor facilitated fixed ratio 1 and progressive ratio alcohol self-administration in nonselected Wistars. In the reinstatement tests, the compound did not have effects on cue-, footshock stress- and yohimbine-induced relapse. Conversely, URB597 completely abolished the anxiogenic response measured during withdrawal after an acute IP administration of alcohol (3.0 g/kg). Rimonabant (0.0, 0.3, 1.0 and 3.0 mg/kg) significantly reduced ethanol self-administration. Conclusions: Results demonstrate that activation of the endocannabinoid anandamide system by selective inhibition of FAAH does not increase alcohol abuse risks but does reduce anxiety associated to alcohol withdrawal. We thus can speculate that medication based on the use of endocannabinoid system modulators such as URB597 may offer important advantages compared to treatment with direct CB1 receptor activators. © 2008 Springer-Verlag.

It has been proposed that gonadotropin-releasing hormone (GnRH) plays an autocrine/paracrine regulatory role in mammalian and fish ovaries. The marine teleost gilthead seabream is an interesting model since, during the life span of the fish, gonadal tissues develop first as testes, which then regress allowing the development of ovarian follicles. Recent studies carried out in ovaries of the gilthead seabream have demonstrated that various GnRH transcripts as well as GnRH splicing variants are expressed. The mRNA level of several GnRH forms in the female and male areas of the switching gonad, and their possible role in this process, were further investigated. The results here reported show that sGnRH, cGnRH-II, and sbGnRH transcripts are locally expressed during gilthead seabream gonadal differentiation; the expression of the three GnRH forms was found to differ among the morphologically defined areas of the switching gonad, as demonstrated by applying reverse transcription-polymerase chain reaction (RT-PCR), together with in situ hybridization, and semiquantitative PCR analyses. Moreover, the hypothesis that GnRH forms may regulate testicular regression via an apoptotic mechanism was investigated by analyzing the different areas of switching gonads for caspase-3 activity as a measure of apoptosis. Our results showed a marked increase of caspase-3 activity in the area corresponding to the regressing testes in which a significant decrease of testosterone production was also found. The present findings demonstrate that the changes in the endogenous GnRH transcripts could be related with the gonadal differentiation in gilthead seabream, and that exogenous GnRH plays a role by stimulating apoptosis in the degenerating testis.

Medication development for cocaine-addicted patients is difficult, and many promising preclinical candidates have failed in clinical trials. One reason for the difficulty in translating preclinical findings to the human condition is that drug testing is typically conducted in behavioral procedures in which animals do not show addiction-like traits. Recently, a DSM-IV-based animal model has been developed that allows studying the transition to an addiction-like behavior. Changes in synaptic plasticity are involved in the transition to cocaine addiction. In particular, it has been shown that metabotropic glutamate receptor 2/3 (mGluR2/3)-mediated long-term depression is suppressed in the prelimbic cortex in addict-like rats. We therefore hypothesized that cocaine-seeking in addict-like rats could be treated with an mGluR2/3 agonist. Indeed, addict-like rats that were treated systemically with the mGluR2/3 agonist LY379268 (0, 0.3, and 3 mg/kg) showed a pronounced reduction in cue-induced reinstatement of cocaine-seeking. In an attempt to dissect the role played by mGluR2 and mGluR3 in cue-induced reinstatement, we analyzed the mRNA expression patterns in several relevant brain areas but did not find any significant differences between cocaine addict-like and non-addict-like rats, suggesting that the behavioral differences observed are due to translational rather than transcriptional regulation. Another possibility to study the contributions of mGluR2 and mGluR3 in mediating addictive-like behavior is the use of knockout models. Because mGluR2 knockouts cannot be used in operant procedures due to motoric impairment, we only tested mGluR3 knockouts. These mice did not differ from controls in reinstatement, suggesting that mGluR2 receptors are critical in mediating addictive-like behavior.

Recent behavioral, pharmacological and molecular findings have linked the NPS system to drug dependence. Most of the evidence supports the possibility that increased NPS activity may contribute to shaping vulnerability to addiction, especially relapse. However, data suggesting that the anxiolytic-like properties of NPS may have protective effects on addiction have been also published. In addition, evidence from conditioned place preference experiments, though not unequivocal, suggests that NPS per se is devoid of motivational properties. Intriguingly, several effects of NPS on drugs of abuse appear to be mediated by downstream activation of brain corticotrophin releasing factor (CRF) and hypocretin-1/orexin-A (Hcrt-1/Ox-A) systems. The major objective of the present article is to review the existing work on NPS and addiction. Particular attention is devoted to the interpretation of findings revealing complex neuroanatomical and functional interactions between NPS, CRF, and the Hcrt-1/Ox-A systems. Original data aimed at shedding light on the role of NPS in reward processing are also shown. Finally, existing findings are discussed within the framework of addiction theories, and the potential of the NPS system as a treatment target for addiction is analyzed.

Neuropeptide S (NPS) is a recently discovered neurotransmitter that binds to its cognate G-protein coupled receptor, NPSR. Previous studies have shown that central administration of this peptide induces anxiolytic-like effects, promotes arousal and inhibits feeding in the same dose range. In the present study, we sought to investigate further the unique physiopharmacological profile of the NPS system by characterizing its effects on palatable food consumption in rats and comparing it with the effect of the classical anxiolytic benzodiazepine midazolam. The results demonstrated that midazolam (5.0 or 10.0 mg ⁄kg) increases palatable food consumption, while intracerebroventricular (ICV) administration of NPS markedly reduces it. The anorectic effect of NPS (0.1‐ 1.0 nmol per rat, ICV) was prevented by ICV pretreatment with the NPSR antagonist [d-Cys(tBU) 5 ]NPS (20.0‐60.0 nmol per rat). Pretreatment with the nonselective corticotrophin-releasing factor receptor (CRF) antagonist alpha-helical CRF 9‐41 (6.25 and 12.5 nmol per rat) completely reversed the hypophagic action of CRF (0.4 nmol per rat, ICV) but did not prevent the anorectic effect of ICV NPS (1.0 nmol per rat). Brain site-specific microinjection experiments revealed that NPS markedly inhibits palatable food intake if administered into the paraventricular nucleus of the hypothalamus (PVN). A similar but smaller and shorter lasting reduction of feeding was observed following intra-lateral hypothalamus administration, whereas no effect was observed following injection into the central amygdala. The present study demonstrates that NPS evokes a potent inhibition of palatable food consumption and that the PVN is an important site of action for its effect.

This article summarizes the proceedings of a symposium held at the conference on “Alcoholism and Stress: A Framework for Future Treatment Strategies” in Volterra, Italy, May 6–9, 2008. Chaired by Markus Heilig and Roberto Ciccocioppo, this symposium offered a forum for the presentation of recent data linking neuropetidergic neurotransmission to the regulation of different alcohol related behaviours in animals and in humans. Dr. Donald Gehlert described the development of a new corticotrophin releasing factor (CRH) receptor 1 antagonist and showed its efficacy in reducing alcohol consumption and stress-induced relapse in different animal models of alcohol abuse. Dr. Andrey Ryabinin reviewed recent findings in his laboratory indicating a role of the urocortin 1 (Ucn) receptor system in the regulation of alcohol intake. Dr. Annika Thorsell showed data supporting the significance of the neuropetide Y (NPY) receptor system in the modulation of behaviours associated with a history of ethanol intoxication. Dr. Roberto Ciccocioppo focused his presentation on the nociceptin/orphanin FQ (N/OFQ) receptors as treatment targets for alcoholism. Finally, Dr. Markus Heilig showed recent preclinical and clinical evidence suggesting that neurokinin 1 (NK1) antagonism may represent a promising new treatment for alcoholism. Collectively, these investigators highlighted the significance of neuropeptidergic neurotransmission in the regulation of neurobiological mechanisms of alcohol addiction. Data also revealed the importance of these systems as treatment targets for the development of new medication for alcoholism.

Individual Variations in the Mechanisms of Nicotine Seeking: A Key for Research on Nicotine Dependence