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.

T-maze and food reinforcement: an inexpensive drug discrimination procedure.

The present study provides a detailed description of a novel drug discrimination procedure employing a T-maze and food reinforcement. Three groups of rats (n = 5) were trained to run one of the two arms of the maze after the i.g. administration of either 1.0, 1.5 or 2.0 g/kg ethanol (chosen as training drug) and the opposite arm after administration of water. Only correct trials were rewarded. All the rats learned the discrimination task. Substitution tests with different doses of ethanol in all the three groups and with the non-competitive NMDA antagonist, dizocilpine (MK-801), in the 2.0 g/kg ethanol-trained group provided a pharmacological validation of the paradigm. Advantages and disadvantages of this procedure are discussed.

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.

Cannabinoid modulation of intestinal propulsion in mice.

The effect of cannabinoid receptor activation and blockade on the propulsive activity in the mouse small intestine was assessed in the present study by measuring the transit of an orally administered, non-absorbable marker. The cannabinoid receptor agonist WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3[(morpholinyl)methyl]pyrrolo[1,2,3-de-1, 4benzoxazin-yl]-(1-naphthalenyl)methanone mesylate) inhibited, while the selective cannabinoid CB1 receptor antagonist SR 141716A (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyraz ole-carboxamide) stimulated the marker transit. Furthermore, a per se non-effective dose of SR 141716A reversed WIN 55,212-2-induced reduction of the transit. The results of the present study suggest a role for cannabinoid CB1 receptors in the control of propulsive activity in the mouse small intestine.

Cross-tolerance to ethanol and gamma-hydroxybutyric acid.

In the present study, the development of tolerance to the motor impairing effects of gamma-hydroxybutyric acid (GHBA) and ethanol was compared (Experiment 1). Rats were required to perform a motor coordination task daily shortly after ethanol (3.5 g/kg) and GHBA (1.0 g/kg) administration for 9 consecutive days. Tolerance to the motor impairing effects of ethanol and GHBA developed to a similar extent but with different patterns. On the tenth day, the presence of cross-tolerance to the motor impairing effects of GHBA and ethanol was assessed (Experiment 2). Administration of 1.0 g/kg GHBA produced a significantly lower impairment in ethanol-tolerant rats than in ethanol-naive rats. Similarly, administration of 3.5 g/kg ethanol induced a significantly lower impairment in GHBA-tolerant rats than in GHBA-naive rats. The presence of cross-tolerance between GHBA and ethanol is discussed in terms of common pathways of neuroadaptation to chronic GHBA and ethanol.

Oral self-administration of gamma-hydroxybutyric acid in the rat.

The present study describes the induction of gamma-hydroxybutyric acid (GHB) preference over water in rats. GHB solution (1% w/v in water) was initially offered as the sole fluid available for 14 consecutive days. Subsequently, rats were given a free choice of GHB solution and tap water for 20 consecutive weeks. Under the free-choice regimen, all rats showed periods of preference for GHB solution over water and periods of voluntary abstinence from GHB. On GHB-preference days, GHB was ingested at pharmacologically relevant doses. GHB intake occurred in 2-3 discrete episodes during the nocturnal phase. The development of an animal model of GHB self-administration may constitute a useful tool in the investigation of the neurobiological substrates of GHB-reinforcing properties.

Blockade of ethanol discrimination by isradipine.

The effect of the dihydropyridine Ca2+ channel antagonist, isradipine, on ethanol discrimination was assessed in rats trained to discriminate 1.5 g/kg ethanol from water in a T-maze, food-reinforced drug discrimination procedure. Pretreatment with isradipine (0, 1.0, 3.0 and 5.0 mg/kg i.p.) resulted in a dose-dependent blockade of ethanol discrimination. The results of the present study suggest that L-type Ca2+ channels are involved in the mediation of ethanol discriminative stimulus 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.

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.

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.

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.

Symmetrical generalization between the discriminative stimulus effects of gamma-hydroxybutyric acid and ethanol: occurrence within narrow dose ranges.

Gamma-hydroxybutyric acid (GHB) has been shown to reduce ethanol consumption and suppress ethanol withdrawal syndrome both in laboratory animals and humans. The present study was designed to assess the similarity between the discriminative stimulus effects, or subjective feelings, of GHB and ethanol using a T-maze, food-reinforced drug discrimination procedure. Three groups of rats were trained to discriminate ethanol (1.0 or 2.0 g/kg; p.o.) or GHB (300 mg/kg; p.o.) from water. In the 1.0 g/kg ethanol-trained rats, substitution for ethanol was an inverted U-shape function of GHB dose, with only 300 mg/kg GHB resulting in complete substitution for ethanol. No dose of GHB elicited selection of ethanol-appropriate arm higher than 10% in the 2.0 g/kg ethanol-trained group. In the 300 mg/kg GHB-trained rats, complete substitution for GHB occurred only at the dose of 1.0 g/kg ethanol. Doses of ethanol lower or higher than 1.0 g/kg did not substitute for GHB. The results of the present study indicate that symmetrical generalization between ethanol and GHB occurred within narrow dose ranges. They are discussed in terms of common neurotransmitter systems involved in the mediation of GHB and ethanol effects.

Sardinian alcohol-preferring rats: a genetic animal model of anxiety.

The present study was designed to assess the anxiety profile of the selectively bred alcohol-preferring sP and alcohol-nonpreferring sNP rats. Rats were offered either water (ethanol-naive rats) or a free choice of 10% (v/v) ethanol and water (ethanol-experienced rats) for 14 consecutive days prior to the test. Spontaneous exploration of an elevated plus maze was used as a behavioral measure of anxiety. Ethanol-naive sP rats spent less time in and made fewer entries into the open arms of the maze than ethanol-naive sNP rats. These results suggest a higher innate degree of anxiety in sP than in sNP rats. Moreover, time spent in and number of entries into the open arms of the maze were higher in ethanol-experienced than in ethanol-naive sP rats. This finding suggests that ethanol consumed voluntarily produces anxiolytic effects in sP rats. The results of the present study are discussed in terms of (a) anxiety as a genetic trait related to ethanol-preference in sP rats and (b) self-medication of anxiety as a possible factor promoting voluntary ethanol consumption in sP rats.

Gamma-hydroxybutyric acid intake in ethanol-preferring sP and -nonpreferring sNP rats.

Gamma-hydroxybutyric acid (GHB) and ethanol share several pharmacological similarities, suggesting that GHB may exert ethanol-like effects in the central nervous system. The present study was designed to test whether selectively bred ethanol-preferring rats would, unlike ethanol-nonpreferring ones, self-administer GHB, consistent with their higher preference for ethanol. Male ethanol-naive Sardinian alcohol-preferring (sP) and Sardinian alcohol-nonpreferring (sNP) rats were used. In Experiment 1, GHB solution (1% (w/v) in water) was initially offered as the sole fluid available for 14 consecutive days and then presented under the two-bottle, free-choice regimen, one bottle containing water and the other the GHB solution, for an additional 14 consecutive days. During the free-choice phase, high preference for GHB and intake of pharmacologically relevant daily doses of GHB developed in both rat lines, presumably because the 14-day no-choice period would unmask the reinforcing properties of GHB and lead to acquisition of GHB preference also in the supposedly less susceptible sNP rats. In Experiment 2, the forced GHB drinking phase was reduced to 3 days. Under the subsequent free-choice regimen, daily GHB preference and intake were initially low in both sP and sNP rats; however, after approximately 10 days, GHB preference and intake in sP rats rose progressively and then stabilized to significantly higher levels than in sNP rats throughout the entire free-choice phase. It is likely that episodic binges of GHB intake occurring during the first 10 days resulted in experiencing the reinforcing properties of GHB by sP but not sNP rats. The results of the present study suggest that a) sP rats are genetically more sensitive to the reinforcing effects of both ethanol and GHB than sNP rats; and b) disclosure of the higher sensitivity of sP rats to the reinforcing effects of GHB is a function of the length of the induction procedure. The results are also discussed in terms of differences in GHB receptors contributing to the predisposition to ethanol preference and avoidance, respectively.

Blockade of the discriminative stimulus effects of gamma-hydroxybutyric acid (GHB) by the GHB receptor antagonist NCS-382.

The present study was designed to assess the ability of the newly synthetized, selective gamma-hydroxybutyric acid (GHB) receptor antagonist, NCS-382, in blocking the discriminative stimulus effects of GHB in a T-maze, food-reinforced drug discrimination procedure. Two groups of rats were trained to run the left arm of the maze 30 min after the i.g. administration of either 300 or 700 mg/kg GHB and the right arm after water. Once discrimination was acquired, combination of different doses of NCS-382 (0, 12.5, 25.0 and 50.0 mg/kg, IP) and GHB training doses were tested for blockade of GHB discrimination. NCS-382 dose-dependently blocked GHB-appropriate responding in both the 300 and 700 mg/kg GHB rat groups. The results of the present study indicate that the discriminative stimulus properties of GHB are mediated via stimulation of GHB receptors.

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.

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.

Sardinian alcohol-preferring rats prefer chocolate and sucrose over ethanol.

The present study was aimed at determining whether the concurrent availability of highly palatable fluids (i.e., a chocolate-flavored drink and a sucrose solution) would alter voluntary ethanol drinking in selectively bred, alcohol-preferring sP and -nonpreferring sNP rats. Ethanol intake occurred under the three-bottle, free choice regimen between 10% (v/v) ethanol solution, tap water, and the palatable fluids for 24 h per day. When rats were given ethanol and water, but no alternative fluids, mean ethanol intake in sP rats ranged between 6 and 7 g/kg per day and mean preference ratio was steadily higher than 80%, whereas mean ethanol intake and preference ratio in sNP rats were constantly lower than 0.3 g/kg and 5%, respectively. In the presence of either the chocolate-flavored drink or sucrose solution, both prepared as isocaloric to the ethanol solution, absolute ethanol intake in sP rats declined by 60-70%; similarly, the preference ratio was reduced by 80-90%. Ethanol intake in sNP rats was unaffected by the simultaneous presentation of either palatable fluids. The results of the present study closely replicate those previously reported in genetically selected, ethanol-preferring HAD rats; however, they differ from those of ethanol-preferring P rats, which were reported to maintain high levels of ethanol intake and preference in the presence of highly palatable fluids. These results are discussed in terms of a) an alternative reinforcement partially substituting for the reinforcing properties of ethanol in sP rats, resulting in a less urgent need of ethanol, and b) genetic animal models of alcoholism diverging in some neurochemical and behavioral traits (e.g., response to the presentation of palatable fluids), which might parallel the different types of alcoholism observed in humans.