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Rewarding and reinforcing properties of alcohol are mediated by activation of the mesolimbic dopaminergic system1. This neurosystem is hypofunctional in the addicted brain, even beyond somatic and psychological signs of withdrawal. Boosting strategy on the dopaminergic tone could represent a valid approach to alcohol addiction treatment2. The effects of a new dopamine conjugate3 (2-amino-N-[2-(3,4-dihydroxy-phenyl)-ethyl]-3-phenyl-propionamide, DA-Phen) on operant behaviour and on withdrawal behaviour, following alcohol deprivation, were evaluated. The concentration of acetaldehyde (ACD), ethanol's first metabolite, as an indirect measure of the possible DA-Phen modulation in alcohol consumption, was also assessed. Male Wistar rats were tested in an operant paradigm, made by different phases: Habituation (Ethanol 5%), Training (Ethanol 20%), Extinction and Deprivation/Relapse (3 cycles). Rats were treated with DA-Phen (0.03 mmol/kg i.p) during abstinence, or during relapse. Behavioural reactivity and anxiety-like behaviour during withdrawal were also evaluated. At the end of described paradigm, animals were sacrificed, and DA-Phen distribution in organ homogenates was detected and quantified. Da-Phen promoted a reduction in alcohol intake by 50% from the second day of relapse (p

Rewarding and reinforcing properties of alcohol have been shown to be mediated by activation of the mesolimbic dopaminergic system. Experimental evidences suggest that mesolimbic dopamine system is hypofunctional in addicted brain; further, reduced dopaminergic activity outlasts somatic signs of withdrawal and may drive craving and relapse. Boosting strategy on dopaminergic neurons could represent a valid therapy. Effects of pharmacological manipulation of brain Dopaminergic receptors by a new dopamine conjugate, Phenylalanine-β(3,4dihydroxyphenyl)-etilamide (DA-Phen), on operant behaviour and on both acute and prolonged withdrawal symptoms during ethanol abstinence have been evaluated. Male Wistar rats were tested in an operant conditioning task: they were subjected to Habituation (Ethanol 5%), Training (Ethanol 20%), and Extinction sessions. Then, three cycles of Deprivation/Relapse were performed. Rats were administered with DA-Phen (0.03mmol/kg i.p) during abstinence, or during relapse, 2h prior the operant task session. Locomotor activity and anxiety-like behaviour during withdrawal were also evaluated. Da-Phen, administered during abstinence phase, or during relapse, was able to reduce alcoholic intake pattern by 50% from the second day of operant conditioning task (p

Acetaldehyde (ACD), ethanol first metabolite, is rewarding in rodents and humans; it induces “place preference”, is self-administer directly in the VTA, orally in an operant/conflict paradigm and increases DA neurons’ firing. This research aims at investigating DA2-receptor role in the reinstatement of acetaldehyde operant-drinking behaviour, following induction, maintenance and abstinence in the rat. Male Wistar rats are trained to orally self-administer ACD solution (3.2% v/v) or water, in an operant chamber under a FR1. Afterwards animals undergo cyclic periods of deprivation and relapse to ACD. The effect ofD2-receptor activation by quinpirole (0.03mg/kg,i.p.) on operant ACD self-administration is tested during relapse sessions. Rats show a peak-and-drop drinking pattern that reaches regular and higher values in the last training days. Quinpirole administration produces lever press reduction in ACD group when compared to basal intake(p

In recent years, with the increased use of antibiotics and immunosuppres-sive agents, there was an increased incidence of oral mycosis and Candida albicans is the most common etiologic agent in oropharyngeal candidiasis, especially in patients with HIV. The aim of this work is to develop a new dosage form containing miconazole (MN) to be topically applied on oral mucosa, allowing for a massive penetration of the drug in the tissue. The vehicle for the drug delivery was lipid microparticles incorporated in a hydrophilic gel. The lipospheres were obtained by hot melt encapsulation method using as matrix ingredients a mixtures of esters of fatty acids with higher fatty alcohols, having low melting point and a composition similar to that of some lipid membrane components The microparticles were characterized for particle size (by laser light scattering), entrapment efficiency (by UV-Vis analysis) morphology (optical microscopy) and physical state (DSC) The hydrophilic gel contained also lemon essence to enhance the palatability of product and increase the compliance of patient. The oral tissue penetration of MN released from the lipospheres alone or incorporated in gel was evaluated ex-vivo using franz diffusion cell and buccal porcine mucosa. The results were compared with behaviour of commercial Daktarin® oral gel. The lipid microparticles increase significantly the accumulative uptake of MN in buccal mucosa over the marketed gel and showed a considerably enhanced mucosa targeting effect, even at very low doses. These results indicate that the studied lipospheres/gel formulation may be a promising carrier for topical delivery of MN in the oral cavity.

At present, neuroscience literature dealing with Acetaldehyde (ACD), ethanol first metabolite, as a drug of abuse, reported conditioned taste aversion paradigm and conditioned place preference, following ICV or i.p. administration. No reports exist on ACD oral self-administration probably because of its high volatility. For this reason, this pilot study was aimed at the evaluation of ACD concentration in aqueous solutions, and consequently to the rat drinking behaviour when acetaldehyde was presented in a free-access paradigm together with water and a sweet solution. Preliminarly we investigated the loss of ACD content in aqueous standard solutions. For the investigation of ACD concentration, all standard samples (ACD concentration ranging from 2 to 90 mM) were analysed in triplicate at regular time intervals (t = 0 h,12 h, 24 h and 48h.) using UV-Vis method at ? = 276 nm. Despite of ACD great volatility, no appreciable loss of ACD has been found in all solutions tested. When adult male Wistar rats were subjected to a three bottle choice paradigm with ACD 9mM, saccharin 0,125% (w/v) plus methyl-paraben 0,1% (w/v), and tap water, the amount of fluid intake was monitored every 24 h, and bottles were refilled every day with fresh solutions. Our results demonstrate that rats do self-administer ACD, even in the presence of a sweet solution and that their preference toward the ACD gradually increases day by day. In conclusion our pilot study shows for the first time that ACD concentration is maintained in an aqueous solution within 24 hours and that rats spontaneously self-administer ACD in a free-access paradigm even in the presence of a saccharin solution, providing further evidence of the addictive properties of ACD.

It is well established that alterations in the functionality of dopaminergic transmission are associated with neurodegenerative diseases such as Parkinson’s Disease. The purpose of this study was to investigate the activity of a new dopamine amino-acid conjugate: L-phenylalanine-β-(3,4-dihydroxyphenyl) etilamide(DA-Phe) in the central nervous system, by assessing its influence on different behavioural parameters in the rat. Preliminarily, we tested the in vitro stability in plasmatic environment following the disappearance of DA-Phen from human plasma and the concurrent appearance of dopamine. Using rat brain homogenate, we also evaluated the level of DA-Phen cleavage by cerebral enzymes and the correspondent production of dopamine in situ. Our in vitro data suggested that DA-Phen fulfills the pro-drug criteria and could be considered as a very helpful molecule able to cross the blood brain barrier and to explicate a CNS action. On the basis of these results, we assessed some central pharmacological effects of DA-Phen on male Wistar rats, by the evaluation of some behavioural outcomes following i.p. administration of the prodrug. Firstly, we assessed the effects of DA-Phen on behavioural reactivity in the Open Field (O.F.). The Elevated Plus Maze (EPM) test was also used in order to reveal anxiogenic- or anxiolytic-like properties of the administered compound. Both these tests highlight the conflict between the innate fear that rodents have for the central area (O.F) or open arms (EPM) of a novel maze versus their desire to explore new environments. The forced swim test (FST)is the most widely used experimental model for assessing antidepressant activity of drugs, acting on noradrenergic and dopaminergic transmission. The test is based on the observation that, when placed into a cylinder filled with water from which they cannot escape, after a time of active swimming or climbing, rodents develop immobility, as a measure of despair behaviour. Therefore immobility time is considered as a measure of depressive-like beaviour. Antidepressant drugs can reduce the amount of immobility, or delay its onset, and/or increase active escape behaviours displayed during the FST. For evaluating also the effects of DA-Phen on spatial learning and memory, the Morris water maze (MWM) was performed. In this test the rat is placed in a pool where he must use and remember visual cues located in the room in order to find a platform hidden underneath the surface of the water. The ability of the animal to retrieve and retain information acquired, or flexibility to purge and re-learn new strategies, can be determined using specific parameters (Escape Latency and Percentage of time spent in a specific quadrant) in a Reversal and a Probe trial. Briefly, our results suggest that DA-Phen is able to affect behavioural reactivity and emotionality in the Open Field (p

Stress-related neuropeptides are involved in setting up alcohol addiction. Ethanol is able to acutely induce CRH and ACTH release, while cronically a dampered response of the hypothalamus -pituitary- adrenal (HPA) axis has been observed. Also neuropeptide Y (NPY) has been shown to modulate ethanol consumption, and its central expression seems inversely correlated to ethanol intake. Recent in vivo and in vitro evidence have highlighted the key role of acetaldehyde (ACD), ethanol first metabolite, as a mediator of the central effects of ethanol, even as modulator of the neuropeptidergic transmission in the rat brain. The aim of this study was to investigate NPY immunoreactivity following a 4-day binge ACD treatment in hippocampus and nucleus accumbens, two areas particulary involved in addictive-related learning and reward processes. Wistar rats have been intragastrically administered with ACD (450mg/kg) five times per day, for four days, and have been sacrificed at early (16h) and late (72h) withdrawal. Our results show: i) an increased NPY expression at 16h and 72h after the last administration in hippocampus (p< 0.001; p< 0.001) and nucleus accumbens (p< 0.05; p< 0.001) compared to controls. ii) an increase in the number of NPY-positive neurons in the hippocampus (p< 0.001) and nucleus accumbens (p< 0.001) in late withdrawal with respect to early withdrawal. These findings are consistent with the hypothesis that NPY levels increase in a time- and region-specific fashion to reset homeostasis in the brain during withdrawal; and that ACD is able to affect neuropeptidergic transmission as well as ethanol, playing thus a key role in the neuroadaptative changes that characterize ethanol addiction.

Drug delivery to the CNS is subject to the permeability limitations imposed by the BBB that regulates movements of actives in and out of the brain. During the drug discovery phase a key aspect could be the selection of the compounds properties crucial for brain penetration. Novel CNS-targeted neurotherapeutics should possess the optimal characteristics that allow passive diffusion through the BBB via the transcellular route, or have the structural features necessary to serve as a substrate for one of the endogenous transport systems of the BBB. An attractive and rewarding chemistry-based strategy, employed to increase the CNS transport of poorly penetrating therapeutic agents, is the transient chemical modification. This approach could improve physicochemical, biopharmaceutical, pharmacokinetic and drug delivery properties of active agents that overcome barriers to a drug’s usefulness. With the aim of mask functional groups and modify the physicochemical properties relevant to bioavailability, in this work CNS-actives were covalently linked to different aminoacidic moieties. The chemical structure of the derivatives and Log D were determined. Chemical stability was evaluated in simulated biological fluids. Enzymatic stability was assessed ex vivo. The effects of derivatives were observed in vitro and their availability was measured in rat brain tissue. Finally, cellular toxicity was established. Our data support that aminoacids could be considered as good masking agents for CNS-targeted drugs.