As AM404 does not activate cannabinoid receptors , the effects of this drug were suggested to result from the elevation of endogenous anandamide levels. However, recent findings suggest that AM404 also directly activates the vanilloid VR1 receptor , complicating the identification of its mechanism of action on ethanol self-administration. However, the effect of AM404 was not reversed or enhanced by pre-treatment with the competitive vanilloid VR1 receptor antagonist capsazepine, indicating that the inhibitory action of AM404 is not mediated through VR1 stimulation and may be derived from other targets in the endocannabinoid system. Following this rationale we studied the involvement of the cannabinoid CB1 receptor, the natural target of anandamide. In order to confirm its participation we first studied whether the cannabinoid receptor antagonist SR141716A reversed the actions of AM404. This pharmacological test was complicated by the inhibitory actions of SR141716A on ethanol self-administration that precluded the observation of a reversal of the actions of AM404. A second strategy was to compare the actions of AM404 with those of selective cannabinoid CB1 receptor agonists belonging to three of the four main classes of cannabinoid agonists: eicosanoids , aminoalkylindoles and classical cannabinoids. The effects of these compounds in ethanol self-administration are not similar to those of AM404. ACEA and WIN 55,212-2 reduced ethanol self-administration, although the component of motor inhibition of WIN 55,212-2 might be responsible for this effect. However, the classical cannabinoid receptor agonist HU-210 did not affect ethanol self-administration. We replicated this finding in a separate study in Marchigian Sardinian alcohol-preferring rats. These results indicate that the contribution of the CB1 receptors to AM404 cannot be supported. The similar profile of actions observed after systemic administration of either cannabinoid CB1 receptor agonists or antagonist seems to be challenging. It has been reported that both cannabinoid CB1 receptor agonists,sub irrigation cannabis such as tetrahydrocannabinol, CP55 940 and WIN 55,212-2, and cannabinoid receptor antagonist ⁄ inverse agonists, such as SR141716A, suppress operant behavior.
These reports stress the pleiotropic spectrum of actions found after the interference with endocannabinoid signaling. The complex roles of the endocannabinoid system on the regulation of GABA and glutamate synapses throughout the brain circuits processing the appetitive ⁄ motivational properties of ethanol might explain these findings. As an example, we have recently described that intracerebral injections of SR141716A only affect ethanol self administration in rats when the CB1 antagonist is infused in the prefrontal cortex but not in the hippocampus or dorsal striatum. Moreover, in this study, local blockade of fatty acid amidohydrolase, the main enzyme that degrades anandamide, enhances ethanol self-administration when injected into the prefrontal cortex. However, we cannot exclude additional targets such as noncloned cannabinoid-like receptors on which anandamide and WIN 55,212-2 may act. Thus, the present study stressed the need to clarify the growing complexity of endocannabinoid pharmacology, especially in the fifield of motivated behaviors. Although the present results exclude VR1, CB1 and CB2 receptors as the targets of the effects of AM404, we cannot exclude the contribution of endocannabinoids elevated by AM404 to the present actions, especially because the endocannabinoid system has been recently implicated in the neuroadaptations that occur during acute alcohol exposure, alcohol dependence and abstinence. Several studies have documented that endocannabinoid transmission is acutely inhibited by ethanol and becomes hyperactive during chronic ethanol administration, as revealed by the increase in the levels of endocannabinoids and the down-regulation of CB1 receptors. Thus, it is tempting to imagine that those compounds that increase endocannabinoid transmission, such as AM404, might be useful in reducing operant responses for ethanol. With the precautions derived from the non-CB1 profile of the effects of AM404, we propose that the increased levels of endogenous cannabinoids occurring during chronic ethanol administration contribute to facilitate the action of AM404; the neuroadaptations in the central nervous system associated with chronic ethanol intake lead to an increase in anandamide levels and this event could enhance the action of AM404 acting through the increased endogenous anadamide. However, we also demonstrate that the acute administration of AM404 was not able to suppress the relapse response for ethanol, i.e. the reinstatement of ethanol responding induced by the presentation of contextual cues associated with ethanol after a period of extinction. The differential response to AM404 in self-administration and relapse conditions may have a neuropharmacological basis in the recently described changes in endocannabinoid levels after chronic ethanol exposure.
A possible explanation for these differences may reside in the probable alterations induced by chronically consumed ethanol in the functionality of the receptor systems mediating the central effects of ethanol that sustain ethanol-drinking behavior in rats. These neuroadaptation processes might result in a decreased potency and efficacy of the ligands. The increased levels of anandamide observed during ethanol consumption may return to basal levels or even disappear and thereby AM404 could not be acting in such a situation.This hypothesis is supported by the results obtained recently by Gonzalez et al. who showed that the levels of endocannabinoids underwent significant changes in reward-related areas during relapse, showing the lowest values in this phase. The levels of both anandamide and 2-arachidonoyl-glycerol were significantly reduced when rats were allowed to relapse to alcohol consumption. Thus, the induction of compensatory mechanisms in the status of the endocannabinoid system may be determinant in the actions of AM404. The sensitivity to AM404 modulation of reward processes may be also dependent on the rate of motivation for the drug. The differential responses found after administration of AM404 in food and saccharin reinforcement with respect to ethanol might be explained by the different rewarding properties between natural rewards and drugs of abuse such as ethanol, the latter being more potent. In other words, AM404 may be efficacious only in those situations in which the motivation for the drug is stronger, indicating that this effect is linked to the strength of the hedonic properties of the reward rather than to a motivational state. In conclusion, our results showing that AM404 administration reduced ethanol self-administration open a new line of research for the development of therapies to reduce ethanol intake in alcoholic non abstinent patients.Alcohol use disorder is a chronic and relapsing condition for which current pharmacological treatments are only modestly effective. The development of efficacious medications for AUD remains a high research priority with recent emphasis on identifying novel molecular targets for AUD treatment and to efficiently screen new compounds aimed at those targets. To that end,vertical grow modulation of neuroimmune function represents a promising novel target for AUD. Chronic alcohol consumption produces a sustained inflammatory state, such that individuals with AUD have increased neuroinflammation throughout the brain, and alcohol-induced neuroinflammation is thought to contribute to chronic alcohol seeking behavior and to the behavioral and neurotoxic effects of alcohol. In rodents, lipopolysaccharide-induced neuroinflammation produces prolonged increases in alcohol consumption, and knocking out neuroimmune signaling genes attenuates alcohol preference and self-administration. Therefore, a medication that reduces proinflammatory signaling may produce anti-alcohol and neuroprotective effects that may be beneficial for the treatment of AUD. Ibudilast has been advanced as a novel addiction pharmacotherapy that targets neurotrophin signaling and neuroimmune function.
IBUD inhibits phosphodiesterases 4 and 10 and macrophage migration inhibitory factor . As PDE4 and MMIF are critically involved in proinflammatory signaling, and PDE10 negatively regulates neurotrophin expression, the inhibition of these molecules by IBUD has been theorized to reduce neuroinflammation and promote neurotrophin expression . In support, IBUD enhances neurotrophin expression, reduces pro-inflammatory cytokine release, and attenuates neuronal death. In rodents, IBUD has been demonstrated to reduce ethanol intake by approximately 50% both under conditions of maintenance and relapse testing. These recent preclinical findings for IBUD support prior studies indicating pharmacological inhibition of PDE4 and PDE10 decreases alcohol intake. To advance medications development for AUD, our laboratory has recently completed a randomized, double-blind, placebo-controlled crossover laboratory study of IBUD in non-treatment seeking individuals with AUD. This study tested the safety, tolerability, and initial human laboratory efficacy of IBUD on measures of subjective response to alcohol, as well as cue- and stress-induced changes in craving and mood. Twenty-four individuals completed two separate 7-day intensive outpatient protocols which included daily visits for medication administration and testing. Upon reaching a stable target dose of IBUD , participants completed a stress-exposure session, an alcohol cue-exposure session, and an IV alcohol administration session. Results indicated that IBUD was well tolerated and associated with mood improvements during stress- and alcohol-cue exposures, and with reduction in tonic levels of alcohol craving. Exploratory analyses revealed that among individuals with higher depressive symptomatology, IBUD attenuated the stimulant and positive mood-altering effects of alcohol. Given the promising pre-clinical and initial human laboratory findings, we will conduct a large-scale randomized clinical trial of IBUD in treatment-seeking participants with AUD. Additionally,we will collect psychosocial stress- and alcohol-cuerelated neuroimaging data as part of the trial. As an exploratory aim, we will also collect proinflammatory biomarkers from participants over the course of the trial.At the initial in-person screening visit and prior to conducting any research-related procedures, a trained member of the study team will conduct the three-part consent process which details the procedures that take place during the screening process. First, participants will be asked to read and provide verbal consent for the breathalyzer. If the breathalyzer test is 0.000 g/dl, study staff will read and discuss the written informed consent outlining procedures for the initial screening visit with the participant. Once the participant has asked questions and has a clear understanding of the procedures, the participant will sign the consent form. If the participant is found eligible to continue to the medical screening visit, a second written consent form outlining the study purpose, procedures, potential risks, and benefits will be reviewed and signed in the presence of the study physician in a private, confidential setting. Only physician investigators who are continuously involved in the research and qualified to answer questions regarding the nature of the subject’s participation and the alternatives to participation will obtain the second informed consent.At the first initial in-person screening visit, participants will read and sign a consent form that outlines the procedures for collecting biological specimens such as a urine sample for a toxicology screen and pregnancy test at each study visit. At the medical screening visit, participants will sign the experimental consent form that includes the electrocardiogram and blood sample for a comprehensive metabolic panel and complete blood cell count in order to evaluate overall health and determine eligibility. In addition, participants will be asked to consent to the collection of biological specimens during the study such as a blood sample for neuroinflammation assays at every study visit, salivary cortisol samples, and brain imaging at the week 4 visit. Participant data and biological specimens used to evaluate eligibility and study compliance will be disposed of after testing. Data and specimens germane to the research study such as de-identified neuroinflammation assays and salivary cortisol samples will be owned by the University of California or by a third party designated by the University. Participants will be asked to indicate if they permit part of the sample to be shared with other researchers and/or used in future studies. However, the samples for this study will be used for the specified analyses and will not be stored for secondary analysis.The trial is placebo-controlled, due to there being no universal standard-of-care medication for AUD treatment. Since the standard treatment for AUD is therapist-delivered behavioral support, all subjects will be provided the computer-based, NIAAA-developed Take Control program. This decision is supported by a recent study comparing computer-delivered Take Control to Therapist-Delivered Platforms , which found comparable drinking outcomes and higher medication adherence in the Take Control trials, suggesting that Take Control is a comparable and cost-efficient alternative to TDP in AUD clinical trials.The study physicians will be available to participants for the entire duration of the study. The study physicians will call every participant at the end of the first week on the study medication to discuss and manage any adverse events. Study staff will also notify the study physicians of any adverse events recorded during the follow-up visits. Side effects will be collected through an open-ended question asking participants to report any adverse events they may be experiencing. If study medication adjustments are required due to safety concerns, it is up to the study physicians’ discretion to make a dose adjustment or terminate medication.