Identifying candidate drivers of alcohol dependence-induced excessive drinking by assembly and interrogation of brain-specific regulatory networks

Background: A systems biology approach based on the assembly and interrogation of gene regulatory networks, or interactomes, was used to study neuroadaptation processes associated with the transition to alcohol dependence at the molecular level. Results: Using a rat model of dependent and non-dependent alcohol self-administration, we reverse engineered a global transcriptional regulatory network during protracted abstinence, a period when relapse rates are highest. We then interrogated the network to identify master regulator genes that mechanistically regulate brain region-specific signatures associated with dependent and non-dependent alcohol self-administration. Among these, the gene coding for the glucocorticoid receptor was independently identified as a master regulator in multiple brain regions, including the medial prefrontal cortex, nucleus accumbens, central nucleus of the amygdala, and ventral tegmental area, consistent with the view that brain reward and stress systems are dysregulated during protracted abstinence. Administration of the glucocorticoid antagonist mifepristone in either the nucleus accumbens or ventral tegmental area selectively decreased dependent, excessive, alcohol self-administration in rats but had no effect on non-dependent, moderate, alcohol self-administration. Conclusions: Our study suggests that assembly and analysis of regulatory networks is an effective strategy for the identification of key regulators of long-term neuroplastic changes within specific brain regions that play a functional role in alcohol dependence. More specifically, our results support a key role for regulatory networks downstream of the glucocorticoid receptor in excessive alcohol drinking during protracted alcohol abstinence

A New Strategy for Smoking Cessation: Characterization of a Bacterial Enzyme for the Degradation of Nicotine

Smoking is the leading cause of preventable diseases; thus, effective smoking cessation aids are crucial for reducing the prevalence of cigarette smoking and smoking-related illnesses. In our current campaign we offer a nicotine-degrading enzyme from <i>Pseudomonas putida</i>, NicA2, a flavin-containing protein. To explore its potential, a kinetic evaluation of the enzyme was conducted, which included determination of <i>K</i>_m, <i>k</i>_cat, buffer/serum half-life, and thermostability. Additionally, the catabolism profile of NicA2 was elucidated to assess the potential toxicity of the nicotine-derived products. In characterizing the enzyme, a favorable biochemical profile of the enzyme was discovered, making NicA2 a prospective therapeutic candidate. This approach provides a new avenue for the field of nicotine addiction therapy

Endocannabinoid Modulation of Scratching Response in an Acute Allergenic Model: A New Prospective Neural Therapeutic Target for Pruritus

Pruritus (itch) is a common cause of discomfort by dermatological disorders. Several peripherally and centrally mediated pathologies that induce pruritus do not generally respond to typical allergenic and anti-inflammatory treatments. In accordance, we employed an acute allergenic murine model to determine whether the endogenous cannabinoid system could be targeted to treat pruritus. Subcutaneous administration of the mast cell degranulator compound 48/80 evoked an intense, concentration-dependent scratching response. Systemic Δ9-tetrahydrocannabinol reduced the scratching response, although this effect was accompanied with hypomotility. Complementary genetic and pharmacological approaches to target fatty acid amide hydrolase (FAAH), the primary enzyme responsible for the degradation of the endocannabinoid anandamide, were evaluated in the compound 48/80 model. FAAH(-/-) mice and mice treated with the respective irreversible and reversible FAAH inhibitors, URB597 (cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester) and OL-135 [1-oxo-1-[5-(2-pyridyl)-2-yl]-7-phenylheptane], displayed comparable reductions in scratching to mice treated with common nonsedative allergenic treatments (loratadine and dexamethasone) but without affecting locomotor behavior. The antiscratching phenotype of FAAH-compromised mice was completely blocked by either genetic deletion or pharmacological antagonism of the CB1 receptor. Neural-specific conditional FAAH knockout (FAAH-NS) mice, which have FAAH exclusively restricted to neural tissues, showed a similar magnitude of scratching as wild-type mice. It is important that URB597 reduced compound 48/80-induced scratching in FAAH-NS mice, but it did not produce any further reduction in FAAH(-/-) mice. These findings indicate that neuronal FAAH suppression reduces the scratching response through activation of CB1 receptors. More generally, these are the first preclinical data suggesting that FAAH represents a novel target to treat pruritus without eliciting overt side effects

Methamphetamine Vaccines: Improvement through Hapten Design

Methamphetamine (MA) addiction is a serious public health problem, and current methods to abate addiction and relapse are currently ineffective for mitigating this growing global epidemic. Development of a vaccine targeting MA would provide a complementary strategy to existing behavioral therapies, but this has proven challenging. Herein, we describe optimization of both hapten design and formulation, identifying a vaccine that elicited a robust anti-MA immune response in mice, decreasing methamphetamine-induced locomotor activity

Long-Term Antagonism of κ Opioid Receptors Prevents Escalation of and Increased Motivation for Heroin Intake

The abuse of opioid drugs, both illicit and prescription, is a persistent problem in the United States, accounting for &gt;1.2 million users who require treatment each year. Current treatments rely on suppressing immediate withdrawal symptoms and replacing illicit drug use with long-acting opiate drugs. However, the mechanisms that lead to preventing opiate dependence are still poorly understood. We hypothesized that κ opioid receptor (KOR) activation during chronic opioid intake contributes to negative affective states associated with withdrawal and the motivation to take increasing amounts of heroin. Using a 12 h long-access model of heroin self-administration, rats showed escalation of heroin intake over several weeks. This was prevented by a single high dose (30 mg/kg) of the long-acting KOR antagonist norbinaltorphimine (nor-BNI), paralleled by reduced motivation to respond for heroin on a progressive-ratio schedule of reinforcement, a measure of compulsive-like responding. Systemic nor-BNI also significantly decreased heroin withdrawal-associated anxiety-like behavior. Immunohistochemical analysis showed prodynorphin content increased in the nucleus accumbens core in all heroin-exposed rats, but selectively increased in the nucleus accumbens shell in long-access rats. Local infusion of nor-BNI (4 μg/side) into accumbens core altered the initial intake of heroin but not the rate of escalation, while local injection into accumbens shell selectively suppressed increases in heroin intake over time without altering initial intake. These data suggest that dynorphin activity in the nucleus accumbens mediates the increasing motivation for heroin taking and compulsive-like responding for heroin, suggesting that KOR antagonists may be promising targets for the treatment of opioid addiction

Injection Route and TLR9 Agonist Addition Significantly Impact Heroin Vaccine Efficacy

Active immunization is an effective means of blocking the pharmacodynamic effects of drugs and holds promise as a treatment for heroin addiction. Previously, we demonstrated the efficacy of our first-generation vaccine in blocking heroin self-administration in rats, however, many vaccine components can be modified to further improve performance. Herein we examine the effects of varying heroin vaccine injection route and adjuvant formulation. Mice immunized via subcutaneous (sc) injection exhibited inferior anti-heroin titers compared to intraperitoneal (ip) and sc/ip coadministration injection routes. Addition of TLR9 agonist cytosine-guanine oligodeoxynucleotide 1826 (CpG ODN 1826) to the original alum adjuvant elicited superior antibody titers and opioid affinities compared to alum alone. To thoroughly assess vaccine efficacy, full dose–response curves were generated for heroin-induced analgesia in both hot plate and tail immersion tests. Mice treated with CpG ODN 1826 exhibited greatly shifted dose–response curves (10–13-fold vs unvaccinated controls) while non-CpG ODN vaccine groups did not exhibit the same robust effect (2–7-fold shift for ip and combo, 2–3-fold shift for sc). Our results suggest that CpG ODN 1826 is a highly potent adjuvant, and injection routes should be considered for development of small molecule–protein conjugate vaccines. Lastly, this study has established a new standard for assessing drugs of abuse vaccines, wherein a full dose–response curve should be performed in an appropriate behavioral task