Is 2-Hydroxypropyl-ß-cyclodextrin a Suitable Carrier for Central Administration of Δ9 -Tetrahydrocannabinol? Preclinical Evidence.

Preclinical Research Δ9 -Tetrahydrocannabinol (THC) is a hydrophobic compound that has a potent antinociceptive effect in animals after intrathecal (IT) or intracerebroventricular (ICV) administration. The lack of a suitable solvent precludes its IT administration in humans. 2-Hydroxypropyl-ß-cyclodextrin (HPßCD) increases the water solubility of hydrophobic drugs and is approved for IT administration in humans. To investigate whether HPßCD might be a suitable carrier for ICV administration of THC in rats, two formulations containing THC complexed with HPßCD (30 and 135 µg of THC per animal) and vehicle were administered to Wistar rats. The antinociceptive effect (using the tail flick test), locomotor activity, and body temperature were evaluated. ICV injection of 135 µg of THC/HPßCD complex increased tail flick latency, reduced locomotor activity, and had a dual effect on body temperature. The 30 µg THC/HPßCD formulation only produced a hyperthermic effect. All animals appeared healthy, with no difference between the groups. These results were similar to those obtained in other preclinical studies in which THC was administered centrally using solvents that are unsuitable for IT administration in humans because of their toxicity. Our findings suggest that HPßCD may be a useful carrier for IT administration of THC in humans. Drug Dev Res 78 : 411-419, 2017. © 2017 Wiley Periodicals, Inc.

Comparative cytogenetics of six Indo-Pacific moray eels (Anguilliformes: Muraenidae) by chromosomal banding and fluorescence in situ hybridization.

A comparative cytogenetic analysis, using both conventional staining techniques and fluorescence in situ hybridization, of six Indo-Pacific moray eels from three different genera (Gymnothorax fimbriatus, Gymnothorax flavimarginatus, Gymnothorax javanicus, Gymnothorax undulatus, Echidna nebulosa and Gymnomuraena zebra), was carried out to investigate the chromosomal differentiation in the family Muraenidae. Four species displayed a diploid chromosome number 2n = 42, which is common among the Muraenidae. Two other species, G. javanicus and G. flavimarginatus, were characterized by different chromosome numbers (2n = 40 and 2n = 36). For most species, a large amount of constitutive heterochromatin was detected in the chromosomes, with species-specific C-banding patterns that enabled pairing of the homologous chromosomes. In all species, the major ribosomal genes were localized in the guanine-cytosine-rich region of one chromosome pair, but in different chromosomal locations. The (TTAGGG)n telomeric sequences were mapped onto chromosomal ends in all muraenid species studied. The comparison of the results derived from this study with those available in the literature confirms a substantial conservation of the diploid chromosome number in the Muraenidae and supports the hypothesis that rearrangements have occurred that have diversified their karyotypes. Furthermore, the finding of two species with different diploid chromosome numbers suggests that additional chromosomal rearrangements, such as Robertsonian fusions, have occurred in the karyotype evolution of the Muraenidae.

The new compound GET73, N-[(4-trifluoromethyl)benzyl]4-methoxybutyramide, Regulates hippocampal Aminoacidergic transmission possibly via an allosteric modulation of mGlu5 receptor. Behavioural evidence of its "anti-alcohol" and anxiolytic properties.

The present article attempts to provide, on the basis of data emerging from studies carried out in our laboratories, a summary of the chemical and pharmacological properties of the new compound N-[(4-trifluoromethyl)benzyl]4- methoxybutyramide (GET73). Particular emphasis is given to findings obtained in vivo and in vitro suggesting that an allosteric modulation of metabotropic glutamate receptor 5 (mGlu5 receptor) by GET73 may represent the mechanism underlying the effects of the compound produced on rat hippocampal glutamate and GABA transmission. Furthermore, behavioural findings demonstrating how this new compound reduces alcohol intake, displays anxiolytic properties, and influences spatial memory in rats are also summarized. Since mGlu5 receptors play an important role in regulating several central actions of drugs of abuse, and the hippocampus is a crucial brain area involved in addiction, anxiety, and spatial memory, a possible link between mGlu5 receptor allosteric modulation and the profiles of action of GET73 is proposed, although to date no studies have yet explored GET73 binding at the mGlu5 receptor orthosteric and/or allosteric sites. Following a brief overview of glutamatergic neurotransmission, mGlu receptor structures and activation mechanisms, the general properties of mGlu5 receptor and its allosteric modulators are described in the first part of the review.

Procedure of bidirectional selective outbreeding for production of two rat lines differing in sensitivity to the sedative/hypnotic effect of gamma-hydroxybutyric acid.

The exogenous administration of gamma-hydroxybutyric acid (GHB), a constituent of the mammalian brain where it likely functions as a neurotransmitter or a neuromodulator, exerts a number of pharmacological effects, including sedation and hypnosis. The present paper describes a procedure for selective breeding of two rat lines which markedly differ in sensitivity to the sedative/hypnotic effect of GHB. Selective breeding originated from Wistar rats showing opposite sensitivity to the sedative/hypnotic effect of 1 g/kg GHB (i.p.). 'Sensitive' Wistar rats, defined as those individuals displaying values of r = sleep time/onset greater than the upper 15th percentile, were mated to generate the GHB-sensitive (GHB-S) line; conversely, 'resistant' Wistar rats (r-values lower than the lower 15th percentile) were mated to generate the GHB-resistant (GHB-R) line. Upper and lower 15th percentiles were also used to establish the selection cut-offs and criteria for rats of subsequent generations. Specifically, r-values of GHB-S rats were required to be r > or =8 on two separate tests with GHB; r-values of GHB-R rats were required to be r < or =2 on two separate tests with GHB. In each of the three generations produced to date, GHB-S rats showed significantly shorter onset, longer sleep times and greater r-scores than GHB-R rats. The selective breeding of GHB-S and GHB-R rats: (a) suggests that sensitivity to GHB is under genetic control, and (b) may constitute a unique model for investigation of the physiological function of GHB.

Selective breeding of two rat lines differing in sensitivity to GHB and baclofen.

Two Wistar-derived rat lines, one sensitive (GHB-S) and the other resistant (GHB-R) to the anesthetic effect of gamma-hydroxybutyric acid (GHB), have been selectively bred. GHB-S and GHB-R rats were also sensitive and resistant, respectively, to the anesthetic effect of baclofen, the prototype GABA(B) receptor agonist, suggesting that they may be useful to elucidate not only the role of endogenous GHB but also that of GABA(B) receptors in sleep and anesthesia.

Alcohol stimulates motor activity in selectively bred Sardinian alcohol-preferring (sP), but not in Sardinian alcohol-nonpreferring (sNP), rats.

The present study was conducted to evaluate the effect of low doses of ethanol on motor activity in selectively bred Sardinian alcohol-preferring (sP) and Sardinian alcohol-nonpreferring (sNP) rats. Ethanol was acutely administered at the doses of 0, 0.25, and 0.5 g/kg (i.p.) immediately before rat exposure to an open-field arena for 15 min. The number of square crossings, used as index of motor activity, was significantly lower in saline-treated sP than in saline-treated sNP rats, suggestive of a genetically determined higher emotional state in sP than in sNP rats. Ethanol administration resulted in a dose-dependent, significant increase in the number of square crossings in sP rats, whereas it was completely ineffective in sNP rats. These results suggest to us that a positive relationship exists between ethanol preference and ethanol-induced motor stimulation in sP/sNP rat lines.

Different sensitivity to ethanol in alcohol-preferring sP and -nonpreferring sNP rats.

BACKGROUND AND OBJECTIVES: Clinical research has proposed that initial sensitivity to ethanol may be negatively correlated with levels of subsequent ethanol intake; consistently, alcohol-preferring P rats were found to be less sensitive to the ataxic and sedative/hypnotic effects of ethanol than -nonpreferring NP rats. The present study investigated the initial sensitivity to the ataxic and sedative/hypnotic effects of ethanol and to the sedative/hypnotic effects of pentobarbital and diazepam in selectively bred Sardinian alcohol-preferring sP and -nonpreferring sNP rats. METHODS: In experiment 1, time to lose (onset) and regain (sleep time) the righting reflex after the acute intraperitoneal (ip) administration of 3.0 and 3.5 g/kg ethanol were measured in sP and sNP rats. In experiment 2, sP and sNP rats were required to perform a motor coordination task on a Rota-Rod after the acute intragastric administration of 2.0, 2.5, and 3.0 g/kg ethanol. Experiment 3 assessed onset and sleep time in sP and sNP rats after the acute injection of pentobarbital (40 mg/kg; ip) and diazepam (15 and 20 mg/kg; ip). RESULTS: In experiment 1, sP rats took shorter times to lose the righting reflex and regained this reflex over longer periods of time and at lower blood ethanol levels than sNP rats. In experiment 2, ethanol affected motor coordination to a greater extent in sP than sNP rats. In contrast, results from experiment 3 showed that sP and sNP rats were not differentially sensitive to the sedative/hypnotic effects of pentobarbital and diazepam. CONCLUSIONS: The results of experiments 1 and 2 suggest that sP rats possess a genetically determined, greater sensitivity to the motor impairing and sedative/hypnotic effects of ethanol than sNP rats. Although caution should be adopted before hypothesizing any comparison to humans, these results may feature sP rats as an experimental model of those subsets of human alcoholics with initial high sensitivity to ethanol challenges. Finally, the results of experiment 3 suggest a minimal involvement of the benzodiazepine and barbiturate recognition sites in the differential sensitivity to ethanol of sP and sNP rats.

Potential use of medicinal plants in the treatment of alcoholism.

The present paper briefly reviews the most relevant experimental data on the reducing effect of some medicinal herbs on voluntary alcohol intake in animal models of alcoholism. Pueraria lobata, Tabernanthe iboga, Panax ginseng, Salvia miltiorrhiza and Hypericum perforatum proved to be effective in decreasing alcohol consumption. Reduction of alcohol absorption from the gastrointestinal system appears to be a common feature among most of the above plants. These data suggest that medicinal plants may constitute novel and effective pharmacotherapies for alcoholism.

Development of short-lasting alcohol deprivation effect in sardinian alcohol-preferring rats.

Alcohol deprivation effect (ADE), defined as a temporary increase in voluntary alcohol intake following a period of alcohol abstinence, was evaluated in selectively bred Sardinian alcohol-preferring (sP) rats. Alcohol was initially offered in free choice with water for 35 consecutive days (predeprivation phase). Subsequently, one group of rats was deprived of alcohol for 1, 3, 7, 15, 30, 90 or 180 consecutive days, while the second group had continuous access to alcohol (deprivation phase). Once alcohol was re-presented, alcohol intake in alcohol-deprived rats was recorded 1 and 24 h after alcohol re-presentation and compared to that monitored in alcohol-nondeprived rats over the same time periods (postdeprivation phase). Alcohol deprivation for 3 to 30 days resulted in a significant increase in voluntary alcohol intake only in the first hour of re-access. These results demonstrate the development of ADE in sP rats. However, the rapid return of alcohol intake to control levels is discussed as evidence in favor of a set-point mechanism capable of regulating alcohol-drinking behavior in sP rats.

Characterization of the discriminative stimulus effects of gamma-hydroxybutyric acid as a means for unraveling the neurochemical basis of gamma-hydroxybutyric acid actions and its similarities to those of ethanol.

The present paper reviews the drug discrimination studies, both from the literature and from this laboratory, conducted to investigate the pharmacological profile of the discriminative stimulus effects of gamma-hydroxybutyric acid. Collectively, the results of these studies suggest that: (1) the discriminative stimulus effects of gamma-hydroxybutyric acid are composed of different cues, each one being the effect of gamma-hydroxybutyric acid on a specific receptor system; (2) the proportion of each component cue varies as the training dose of gamma-hydroxybutyric acid is increased; (3) the gamma-aminobutyric acid B-mediated cue is a major ingredient of the mixed stimulus of gamma-hydroxybutyric acid, but it is more prominent at high training doses than at low training doses of gamma-hydroxybutyric acid; and (4) positive modulation of the gamma-aminobutyric acid A receptor is a relevant part of the discriminative stimulus effects of low gamma-hydroxybutyric acid doses. Finally, data indicating symmetrical generalization between the discriminative stimulus effects of a specific range of doses of gamma-hydroxybutyric acid and those of ethanol are discussed in regard to their further support of the hypothesis that gamma-hydroxybutyric acid may exert its antialcohol effects through a substitution mechanism.

Mechanism of the antialcohol effect of gamma-hydroxybutyric acid.

Treatment with gamma-hydroxybutyric acid has been reported to effectively decrease alcohol craving and consumption as well as alcohol withdrawal symptoms in alcoholics. We describe the results of animal studies demonstrating the ability of gamma-hydroxybutyric acid to reduce (1) the severity of ethanol withdrawal signs in rats rendered physically dependent on ethanol and (2) voluntary ethanol intake in selectively bred Sardinian alcohol-preferring rats. Furthermore, we review experimental data suggesting that gamma-hydroxybutyric acid and ethanol have several pharmacological effects in common. Relevant similarities are: (1) stimulation of firing rate of dopaminergic neurons and dopamine release in specific rat brain areas; (2) development of cross-tolerance to the motor-impairing effects after repeated administration in rats; 3) abuse potential, as indicated by self-administration of pharmacologically relevant doses of gamma-hydroxybutyric acid in rats and mice; (4) induction of anxiolytic effects in rats; and (5) induction of similar discriminative stimulus effects, as evidenced by symmetrical generalization in a drug discrimination study in rats. These lines of evidence are discussed in relation to gamma-hydroxybutyric acid exerting its antialcohol effects by a substitution mechanism.

Dissociation of ethanol and saccharin preference in sP and sNP rats.

BACKGROUND: It has been proposed that ethanol intake and consumption of sweet tasting solutions are positively correlated in rodents. Experiment 1 of the present study investigated whether selectively bred ethanol-preferring (sP) and -nonpreferring (sNP) rats differed, consistently with the above hypothesis, as to saccharin intake and preference. Experiment 2 evaluated whether saccharin addition to the ethanol solution, likely resulting in a highly palatable fluid, would result in an increase in voluntary ethanol intake in sP rats. METHODS: The saccharin solution was offered, in free choice with water, at a fixed concentration of 1 g/liter for 6 consecutive days in Experiment 1A or at ascending concentrations (0.002 to 16.4 g/liter, doubling the concentration every day) in Experiment 1B. In Experiment 2, 1 g/liter saccharin was added to the standard 10% ethanol solution and offered to sP rats in free choice with water for 7 consecutive days. RESULTS: In both Experiments 1A and 1B, sP and sNP rats showed avidity for the saccharin solution with marginal line difference in saccharin intake and preference. In Experiment 2, daily ethanol intake remained stable at baseline levels (6-7 g/kg), irrespective of the saccharin addition to the ethanol solution. CONCLUSIONS: The results of Experiments 1A and 1B suggest that saccharin drinking behavior in sNP rats deviates from the hypothesis that saccharin and ethanol intakes may co-vary; thus, at least in sNP rats, saccharin and ethanol intakes do not appear to be influenced by the same genetic factors. The results of Experiment 2 provide further support to the existence of a central set-point mechanism that regulates daily ethanol intake in sP rats, likely based on the pharmacological effects of ethanol.

Ability of baclofen in reducing alcohol intake and withdrawal severity: I--Preclinical evidence.

BACKGROUND: The similarities between the pharmacological effects of the gamma-aminobutyric acid receptor agonist, baclofen, and the alcohol-substituting agent, gamma-hydroxybutyric acid, led us to investigate whether baclofen was capable of reducing (a) ethanol withdrawal syndrome in ethanol-dependent rats and (b) voluntary ethanol intake in ethanol-preferring rats. METHODS: In experiment 1, Wistar rats were rendered physically dependent on ethanol by the repeated administration of intoxicating doses of ethanol for 6 consecutive days. Baclofen was acutely administered intraperitoneally at doses of 10, 20, and 40 mg/kg. In experiment 2, baclofen (0, 2.5, 5, and 10 mg/kg, intraperitoneally) was administered once a day for 14 consecutive days to ethanol-preferring sP rats that had continuous access to ethanol (10%, v/v) and water under the two-bottle free choice regimen. RESULTS: In experiment 1, baclofen dose-dependently decreased the intensity of ethanol withdrawal signs; furthermore, 20 mg/kg of baclofen protected from audiogenic seizures in ethanol-withdrawn rats. In experiment 2, baclofen selectively and dose-dependently reduced voluntary ethanol intake; a compensatory increase in water intake left total fluid intake virtually unchanged. CONCLUSIONS: These results are in close agreement with those of a preliminary clinical study and suggest that baclofen may constitute a novel therapeutic agent for alcoholism.

Contribution of GABA(A) and GABA(B) receptors to the discriminative stimulus produced by gamma-hydroxybutyric acid.

The present study examined the involvement of GABA(A) and GABA(B) receptors in the discriminative stimulus effects of gamma-hydroxybutyric acid (GHB). Rats were trained to discriminate either 300 or 700 mg/kg GHB IG from water using a T-maze, food-reinforced drug-discrimination procedure. The direct GABA(B) agonist, baclofen, substituted completely for both training doses of GHB; its potency to substitute for GHB increased moderately as the training dose of GHB was increased. The positive GABA(A) modulator, diazepam, substituted partially for 300 mg/kg GHB, but failed to elicit GHB-appropriate responding in rats trained with the higher GHB dose. Finally, the GABA(B) antagonist, CGP 35348, completely blocked the discriminative stimulus effects of the high training dose of GHB, but only partially antagonized the effects of the low training dose. These results suggest that (a) GHB produces a compound stimulus, and (b) both GABA(B)- and GABA(A)-mediated cues are prominent components of this compound stimulus; the contribution of each component, however, appears to vary as the training dose of GHB is increased.

Salvia miltiorrhiza extract inhibits alcohol absorption, preference, and discrimination in sP rats.

Experiment 1 of the present study investigated the ability of a standardized extract of Salvia miltiorrhiza in reducing voluntary ethanol intake in ethanol-preferring rats of the sP line. Ethanol intake occurred under the two-bottle free-choice regimen between 10% (v/v) ethanol and water in daily 4-h scheduled access periods; water was present 24 h/day. Intragastric administration of 200 mg/kg Salvia miltiorrhiza extract resulted in approximately 40% reduction in ethanol intake and preference throughout the 4-day treatment. This effect of Salvia miltiorrhiza extract was likely due to its ability of altering ethanol absorption from the gastrointestinal tract. Indeed, Experiments 2 and 3 of this study demonstrated that 200 mg/kg Salvia miltiorrhiza extract reduced blood ethanol levels (BELs) up to 60% in comparison to control rats, when ethanol was given IG, whereas it failed to modify BELs when ethanol was injected IP. The reducing effect of Salvia miltiorrhiza extract on ethanol absorption may have therefore resulted in an attenuated perception of the psychoactive effects of ethanol sought by ethanol-drinking rats. Consistently, the results of Experiment 4 of the present study demonstrated that a combination of 200 mg/kg Salvia miltiorrhiza extract IG and 1 or 2 g/kg ethanol IG resulted in a partial blockade of the discriminative stimulus effects of ethanol in sP rats trained to discriminate these doses of ethanol from water in a drug discrimination procedure. Collectively, the results are discussed as being suggestive that drugs curbing ethanol absorption from the gastrointestinal tract may constitute a novel strategy for controlling excessive alcohol consumption in human alcoholics.

Reduction of blood ethanol levels by the gamma-hydroxybutyric acid receptor antagonist, NCS-382.

The present study demonstrates that the gamma-hydroxybutyric acid receptor antagonist, NCS-382, markedly reduces blood ethanol levels (BELs) in rats when ethanol is administered via the intragastric route, whereas it is completely ineffective when ethanol is injected IP. The reducing effect of NCS-382 on BELs is likely due to a lessened absorption of ethanol from the gastrointestinal tract.

Gamma-hydroxybutyric acid reducing effect on ethanol intake: evidence in favour of a substitution mechanism.

Experiment 1 in the present study investigated the time course and dose range of gamma-hydroxybutyric acid (GHB) to reduce voluntary ethanol intake in selectively bred Sardinian ethanol-preferring (sP) rats. Ethanol (10%, v/v) and tap water were offered under the two-bottle free choice regimen with unlimited access. GHB (200, 300, and 400 mg/kg, i.p.) was administered 15 20 min prior to the start of the dark phase of the light-dark cycle. Ethanol and water intakes were recorded at different time intervals during the dark phase. GHB significantly reduced ethanol intake at doses of 300 and 400 mg/kg; statistical significance occurred only at the 15-min and 30-min observation times. The GHB dose of 300 mg/kg was devoid of any sedative effect, as demonstrated in Experiment 2 by the lack of any impairment of spontaneous locomotor activity. Finally, this dose of GHB was also found to exert a robust anxiolytic effect in sP rats tested on the elevated plus maze (Experiment 3). Collectively, the results of the present study demonstrate that a non-sedative and anxiolytic dose of GHB effectively reduced voluntary ethanol intake in sP rats. The rapid onset of the reducing effect of GHB on ethanol intake, as well as its anxiolytic effect, are discussed in terms of adding further support to the hypothesis that GHB may control alcohol craving and consumption in humans by substituting for ethanol's reinforcing effects.

Stimulation of locomotor activity by voluntarily consumed ethanol in Sardinian alcohol-preferring rats.

Stimulation of motor activity induced by ethanol has been proposed to reflect the positive reinforcing properties of the drug. The present study was designed to assess whether voluntary ethanol intake would stimulate locomotor activity in Sardinian alcohol-preferring (sP) rats, selectively bred for high ethanol preference and consumption. Rats were habituated to a) consume either water alone (water-consuming rats) or ethanol (10%, v/v) as free choice together with water (ethanol-consuming rats) according to a 15-min limited access protocol for 10 consecutive days prior to the test, and b) explore an open field for 10 min immediately after the drinking session in a trial on 3 consecutive days before the test. On the test day, voluntary ethanol consumption in ethanol-consuming rats averaged 1.2 g/kg. Values for activity measures (time spent moving, number of square crossings and number of rearings) were significantly higher in ethanol- than in water-consuming rats at both 5- and 10-min intervals. These results suggest that the euphorigenic effects of ethanol, supposedly represented by the stimulation of locomotor activity, are part of the reinforcing properties of ethanol in sP rats.

Involvement of GABA(A) and GABA(B) receptors in the mediation of discriminative stimulus effects of gamma-hydroxybutyric acid.

The present study was designed to further investigate the pharmacological profile of the discriminative stimulus effects of gamma-hydroxybutyric acid (GHB). Drugs acting at the gamma-aminobutyric acid (GABA)B receptor (baclofen and CGP 35348), GABA(A)/benzodiazepine receptor complex (diazepam), N-methyl-D-aspartate (NMDA) receptor complex (dizocilpine), and cannabinoid receptor (WIN 55,212-2) were tested for substitution or blockade of the GHB interoceptive cue in rats trained to discriminate either 300 or 700 mg/kg of GHB i.g. from water in a T-maze, food-reinforced drug discrimination paradigm. Baclofen completely substituted for both training doses of GHB; however, its potency in substituting for GHB increased as the training dose of GHB was increased. CGP 35348 partially and completely blocked the cue elicited by 300 and 700 mg/kg of GHB, respectively. In contrast, diazepam partially substituted for 300 mg/kg of GHB, while failing to produce a GHB-appropriate response in the rat group trained to the higher GHB dose. Neither dizocilpine nor WIN 55,212-2 substituted for GHB. Collectively, these data suggest that: a) GHB produces a compound stimulus; and b) GABA(B)- and GABA(A)-mediated cues are prominent components of the mixed stimulus of GHB. However, the quality (i.e., the proportion of the component cues) of the stimulus varies as the training dose of GHB is increased; indeed, the contribution of the GABA(A)- and GABA(B)-mediated cues were smaller and greater, respectively, at 700 and 300 mg/kg of GHB training doses.

Appetite suppression and weight loss after the cannabinoid antagonist SR 141716.

The effect of the cannabinoid CB1 receptor antagonist, SR 141716, on food intake and body weight was assessed in adult, non-obese Wistar rats. The daily administration of SR 141716 (2.5 and 10 mg/kg; i.p.) reduced dose-dependently both food intake and body weight. Tolerance to the anorectic effect developed within 5 days; in contrast, body weight in SR 141716-treated rats remained markedly below that of vehicle-treated rats throughout the entire treatment period (14 days). The results suggest that brain cannabinoid receptors are involved in the regulation of appetite and body weight.