Cardiovascular safety of dihydroergotamine mesylate delivered by precision olfactory delivery (INP104) for the acute treatment of migraine.

OBJECTIVE: To report the cardiovascular (CV) safety of dihydroergotamine mesylate (DHE) administered by precision olfactory delivery (INP104) from two clinical trials. BACKGROUND: Although the absolute risk is low, migraine is associated with an increased risk of CV events. DHE is a highly effective acute treatment for migraine, but due to its theoretical risk of promoting arterial vasoconstriction, DHE is contraindicated in patients with CV disease or an unfavorable risk factor profile. The INP104 is a novel drug-device combination product approved for acute treatment of migraine that delivers DHE to the upper nasal space using precision olfactory delivery (POD®). METHODS: The STOP 101 was a Phase 1 open-label study that assessed the safety, tolerability, and bioavailability of INP104 1.45 mg, intravenous DHE 1.0 mg, and MIGRANAL (nasal DHE) 2.0 mg in healthy participants. The STOP 301 was a pivotal Phase 3, open-label study that assessed the safety, tolerability, and exploratory efficacy of INP104 1.45 mg over 24 and 52 weeks in patients with migraine. In both studies, active or a history of CV disease, as well as significant CV risk factors, were exclusion criteria. RESULTS: In STOP 101, 36 participants received one or more doses of investigational product. Treatment with intravenous DHE, but not INP104 or nasal DHE, resulted in clinically relevant changes from baseline in systolic blood pressure (BP; 11.4 mmHg, 95% confidence interval [CI] 7.9-15.0) and diastolic BP (13.3 mmHg, 95% CI 9.4-17.1) at 5 min post-dose, persisting up to 30 min post-dose for systolic BP (6.3 mmHg; 95% CI 3.0-9.5) and diastolic BP (7.9 mmHg, 95% CI 3.9-11.9). None of the treatments produced any clinically meaningful electrocardiogram (ECG) changes. In STOP 301, 354 patients received one or more doses of INP104. Over 24 weeks, five patients (1.4%) experienced a non-serious, vascular treatment-emergent adverse event (TEAE). Minimal changes were observed for BP and ECG parameters over 24 or 52 weeks. Off-protocol concomitant use of triptans and other ergot derivatives did not result in any TEAEs. CONCLUSION: In two separate studies, INP104 demonstrated a favorable CV safety profile when used in a study population without CV-related contraindications.

The Gut-brain Connection and Episodic Migraine: an Update.

PURPOSE OF REVIEW: Historical evidence suggests a shared underlying etiology for migraine and gastrointestinal (GI) disorders that involves the gut-brain axis. Here we provide narrative review of recent literature on the gut-brain connection and migraine to emphasize the importance of tailoring treatment plans for patients with episodic migraine who experience GI comorbidities and symptoms. RECENT FINDINGS: Recent population-based studies report the prevalence of migraine and GI disorders as comorbidities as well as overlapping symptomology. American Headache Society (AHS) guidelines have integrated GI symptoms as part of migraine diagnostic criteria and recommend nonoral therapies for patients with GI symptoms or conditions. Nasal delivery is a recommended nonoral alternative; however, it is important to understand potential adverse events that may cause or worsen GI symptoms in some patients due to the site of drug deposition within the nasal cavity with some nasal therapies. Lastly, clinical perspectives emphasize the importance of identifying GI symptoms and comorbidities in patients with episodic migraine to best individualize migraine management. Support for an association between the gut-brain axis and migraine continues to prevail in recent literature; however, the relationship remains complex and not well elucidated. The presence of GI comorbidities and symptoms must be carefully considered when making treatment decisions for patients with episodic migraine.

Δ9-tetrahydrocannabinol and endocannabinoid degradative enzyme inhibitors attenuate intracranial self-stimulation in mice.

A growing body of evidence implicates endogenous cannabinoids as modulators of the mesolimbic dopamine system and motivated behavior. Paradoxically, the reinforcing effects of Δ(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis, have been difficult to detect in preclinical rodent models. In this study, we investigated the impact of THC and inhibitors of the endocannabinoid hydrolytic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) on operant responding for electrical stimulation of the medial forebrain bundle [intracranial self-stimulation (ICSS)], which is known to activate the mesolimbic dopamine system. These drugs were also tested in assays of operant responding for food reinforcement and spontaneous locomotor activity. THC and the MAGL inhibitor JZL184 (4-[bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) attenuated operant responding for ICSS and food, and also reduced spontaneous locomotor activity. In contrast, the FAAH inhibitor PF-3845 (N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide) was largely without effect in these assays. Consistent with previous studies showing that combined inhibition of FAAH and MAGL produces a substantially greater cannabimimetic profile than single enzyme inhibition, the dual FAAH-MAGL inhibitor SA-57 (4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester) produced a similar magnitude of ICSS depression as that produced by THC. ICSS attenuation by JZL184 was associated with increased brain levels of 2-arachidonoylglycerol (2-AG), whereas peak effects of SA-57 were associated with increased levels of both N-arachidonoylethanolamine (anandamide) and 2-AG. The cannabinoid receptor type 1 receptor antagonist rimonabant, but not the cannabinoid receptor type 2 receptor antagonist SR144528, blocked the attenuating effects of THC, JZL184, and SA-57 on ICSS. Thus, THC, MAGL inhibition, and dual FAAH-MAGL inhibition not only reduce ICSS, but also decrease other reinforced and nonreinforced behaviors.

New characterization of dihydroergotamine receptor pharmacology in the context of migraine: utilization of a ß-arrestin recruitment assay.

Introduction: Dihydroergotamine mesylate (DHE) is an established effective acute therapy for migraine and is often characterized by its broad receptor pharmacology. Knowledge of DHE pharmacology largely comes from studies employing older methodologies. Objective: To assess DHE receptor activity using high-throughput methods to screen for functional ß-arrestin activity at G protein-coupled receptors (GPCRs). Methods: Functional receptor activities of DHE and sumatriptan succinate (both 10 µM) were screened against 168 GPCRs using the gpcrMAX assay. Agonist and antagonist effects were considered significant if receptor activity was >30% or inhibited by >50%, respectively. Radiolabeled ligand binding assays were performed for DHE (0.01-300 nM for 5-HT3 and 4E; 0.3-10,000 nM for 5-HT1B, α-adrenergic2B [i.e., α2B-adrenoceptor], D2, and D5) to assess specific binding to select receptors. Results: DHE (10 µM) exhibited agonist activity at α-adrenergic2B, CXC chemokine receptor 7 (CXCR7), dopamine (D)2/5, and 5-hydroxytryptamine (5-HT)1A/1B/2A/2C/5A receptors and antagonist activity at α-adrenergic1B/2A/2C (i.e., α1B/2A/2C-adrenoceptors), calcitonin receptor-receptor activity modifying protein 2 (CTR-RAMP2) or amylin 2 (AMY2), D1/3/4/5, and 5-HT1F receptors. Sumatriptan succinate (10 µM) exhibited agonist activity at the 5-HT1B/1E/1F/5A receptors. DHE demonstrated a half-maximal inhibitory concentration (IC50) of 149 nM at the 5-HT1F receptor and a half-maximal effective concentration (EC50) of 6 µM at the CXCR7 receptor. DHE did not bind to the 5-HT3 receptor at concentrations up to 300 nM and bound poorly to 5-HT4E and D5 receptors (IC50 of 230 and 370 nM, respectively). DHE bound strongly to the D2, 5-HT1B, and α-adrenergic2B receptors (IC50 of 0.47, 0.58, and 2.8 nM, respectively). Conclusion: By using a high-throughput ß-arrestin recruitment assay, this study confirmed the broad receptor profile of DHE and provided an update on DHE receptor pharmacology as it relates to migraine.

Dual blockade of FAAH and MAGL identifies behavioral processes regulated by endocannabinoid crosstalk in vivo.

Delta(9)-tetrahydrocannabinol (THC), the psychoactive component of marijuana, and other direct cannabinoid receptor (CB1) agonists produce a number of neurobehavioral effects in mammals that range from the beneficial (analgesia) to the untoward (abuse potential). Why, however, this full spectrum of activities is not observed upon pharmacological inhibition or genetic deletion of either fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), enzymes that regulate the two major endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), respectively, has remained unclear. Here, we describe a selective and efficacious dual FAAH/MAGL inhibitor, JZL195, and show that this agent exhibits broad activity in the tetrad test for CB1 agonism, causing analgesia, hypomotilty, and catalepsy. Comparison of JZL195 to specific FAAH and MAGL inhibitors identified behavioral processes that were regulated by a single endocannabinoid pathway (e.g., hypomotility by the 2-AG/MAGL pathway) and, interestingly, those where disruption of both FAAH and MAGL produced additive effects that were reversed by a CB1 antagonist. Falling into this latter category was drug discrimination behavior, where dual FAAH/MAGL blockade, but not disruption of either FAAH or MAGL alone, produced THC-like responses that were reversed by a CB1 antagonist. These data indicate that AEA and 2-AG signaling pathways interact to regulate specific behavioral processes in vivo, including those relevant to drug abuse, thus providing a potential mechanistic basis for the distinct pharmacological profiles of direct CB1 agonists and inhibitors of individual endocannabinoid degradative enzymes.

Dissimilar cannabinoid substitution patterns in mice trained to discriminate Δ(9)-tetrahydrocannabinol or methanandamide from vehicle.

Δ(9)-Tetrahydrocannabinol (THC) discrimination in rodents is a behavioral assay that has been used to probe differences among classes of cannabinoids in rats. The purpose of this study was to determine whether traditional and anandamide-like cannabinoids were distinguishable in cannabinoid discrimination procedures in mice. Male mice were trained to discriminate 30 mg/kg THC or 70 mg/kg methanandamide from vehicle in a two-lever milk-reinforced drug discrimination procedure. After acquisition, agonist tests with THC, methanandamide, CP 55940, and anandamide were conducted, as were antagonism tests with rimonabant. Substitution (agonism) and antagonism tests were also carried out in female mice trained to discriminate THC. THC and CP 55940 fully substituted in THC-trained mice of both sexes. Further, THC substitution was rimonabant reversible. In contrast, mice injected with methanandamide or anandamide failed to respond substantially on the THC lever, even up to doses that decreased overall responding. In methanandamide-trained mice, methanandamide fully generalized to the methanandamide training dose. Rimonabant did not reverse this generalization. Although THC, CP 55940, and anandamide also increased responding on the methanandamide lever, the magnitude of substitution was less than for methanandamide. These results suggest incomplete overlap in the underlying mechanisms mediating endocannabinoid pharmacology and marijuana intoxication. Further, they suggest that methanandamide discrimination may involve a non-CB(1) receptor mechanism that is particularly prominent at higher doses.

Effects of hydroxymetabolites of bupropion on nicotine dependence behavior in mice.

Bupropion is an atypical antidepressant that also has utility as a smoking cessation aid. Hydroxybupropions are major metabolites of bupropion and are believed to contribute to antidepressant and perhaps smoking cessation activities. Because bupropion metabolism is more similar in humans and mice than in humans and rats, the present study investigated effects of hydroxybupropion enantiomers in mouse behavioral models measuring various aspects of nicotine dependence. Bupropion and (2S,3S)-hydroxybupropion, but not (2R,3R)-hydroxybupropion, significantly decreased the development of nicotine reward as measured in the conditioned place preference and withdrawal paradigm in mice. Bupropion and both of its metabolites reversed affective and somatic withdrawal signs in nicotine-dependent mice, but the (2S,3S)-hydroxymetabolite had higher potency. Bupropion and (2S,3S)-, but not (2R,3R)-hydroxybupropion, produced partial substitution for nicotine in drug discrimination tests. Our findings support the hypothesis that the effects of bupropion on measures of nicotine dependence reflect actions of bupropion itself, its hydroxymetabolites, or a combination and suggest that the (2S,3S)-hydroxy isomer is the most active principle, making it a potentially better drug candidate for smoking cessation than bupropion.

Divergent effects of cannabidiol on the discriminative stimulus and place conditioning effects of Delta(9)-tetrahydrocannabinol.

Cannabis sativa (marijuana plant) contains myriad cannabinoid compounds; yet, investigative attention has focused almost exclusively on Delta(9)-tetrahydrocannabinol (THC), its primary psychoactive substituent. Interest in modulation of THC's effects by these other cannabinoids (e.g., cannabidiol (CBD)) has been stimulated anew by recent approval by Canada of Sativex (a 1:1 dose ratio combination of CBD:THC) for the treatment of multiple sclerosis. The goal of this study was to determine the degree to which THC's abuse-related effects were altered by co-administration of CBD. To this end, CBD and THC were assessed alone and in combination in a two-lever THC discrimination procedure in Long-Evans rats and in a conditioned place preference/aversion (CPP/A) model in ICR mice. CBD did not alter the discriminative stimulus effects of THC at any CBD:THC dose ratio tested. In contrast, CBD, at CBD:THC dose ratios of 1:1 and 1:10, reversed CPA produced by acute injection with 10mg/kg THC. When administered alone, CBD did not produce effects in either procedure. These results suggest that CBD, when administered with THC at therapeutically relevant ratios, may ameliorate aversive effects (e.g., dysphoria) often associated with initial use of THC alone. While this effect may be beneficial for therapeutic usage of a CBD:THC combination medication, our discrimination results showing that CBD did not alter THC's discriminative stimulus effects suggest that CBD:THC combination medications may also produce THC-like subjective effects at these dose ratios.

The effects of acute and repeated nicotine doses on spontaneous activity in male and female Sprague Dawley rats: analysis of brain area epibatidine binding and cotinine levels.

Previous research in this laboratory has shown that nicotine's effects on spontaneous activity are contingent on individual differences, attenuating activity in high active rats and increasing it in low active rats. This study was designed to further evaluate this phenomenon, and to compare it with nicotine's effects on nicotinic acetylcholine receptor (nAChR) expression in several brain regions. Male and female Sprague-Dawley rats selected for differences in baseline activity were administered nicotine twice daily for 14 days, and its effects on spontaneous activity were evaluated following 1, 13 and 27 doses. Furthermore, [(3)H] epibatidine binding and plasma cotinine levels were evaluated 24 h after the 28th dose. Contrary to previous findings, the effects of repeated nicotine on spontaneous activity were minimally contingent on baseline activity levels. Following an initial attenuation, males, but not females, exhibited sensitization to nicotine's effects on spontaneous activity. [(3)H] epibatidine was significantly increased in several brain regions in both male and female nicotine-treated animals, and in females selected for high activity at baseline. However, a clear relationship between these effects and spontaneous activity was not found, due to the lack of consistent effects of nicotine administration and baseline activity on spontaneous activity. Interestingly, significant correlations suggest that rats exhibiting higher spontaneous activity on the final test day were differentially marked by higher [(3)H] epibatidine. Cotinine levels were higher in low activity males than in high activity males, but no differences were observed between high and low activity females. Thus, no clear relationship between this variable and spontaneous activity could be discerned. Based on these data, no simple relationships between the effects of nicotine administration or baseline activity on [(3)H] epibatidine binding, nicotine metabolism, or spontaneous activity were observed. However, a relationship between [(3)H] epibatidine and spontaneous activity on the final test day is suggested.

Acute nicotine reduces and repeated nicotine increases spontaneous activity in male and female Lewis rats.

The Lewis (LEW) strain of rat appears more sensitive to nicotine than other strains in self-administration, conditioned place preference, and drug discrimination behavioral studies. The present study sought to further evaluate the behavioral effects of chronic nicotine treatment in the LEW strain by assessing spontaneous activity, which has consistently revealed sensitization to chronic nicotine administration in Sprague Dawley (SD) rats. High active and low active male and female LEW rats (N=8 per group) were treated twice daily with either nicotine (0.4 mg/kg, sc) or vehicle for 14 consecutive days. Regardless of baseline activity level or sex, spontaneous activity was significantly decreased, compared to saline-treated rats, after a single nicotine injection. However, spontaneous activity increased in both low- and high-activity rats (both sexes) over the two weeks of nicotine administration to levels that were significantly higher than saline-treated rats. Based on these findings, acute and chronic nicotine administration had greater suppressive and enhancing effects on spontaneous activity in LEW rats compared to other strains of rats previously studied. These results further clarify the behavioral sensitivity of the LEW strain of rat to nicotine exposure and lend credence to the role of genetics in the individual susceptibility to nicotine dependence.

Cellular nicotinic receptor desensitization correlates with nicotine-induced acute behavioral tolerance in rats.

RATIONALE: Individuals vary in their susceptibility to nicotine addiction. However, there is little evidence that behavioral sensitivity to nicotine is dependent upon the functional state of nicotinic cholinergic receptors (nAChRs). OBJECTIVE: This study aims to determine the relationship between in vivo behavioral desensitization and in vitro desensitization of nAChR function. METHODS: Male Sprague-Dawley rats trained to discriminate nicotine were tested for development of acute behavioral tolerance. The rats were injected with nicotine (0.4 mg/kg free base, s.c.), tested for nicotine discrimination for 2 min, then injected with the same dose of nicotine 90, 180, and 270 min after the first injection and tested for nicotine discrimination after each injection. Susceptibility of nAChRs of individual rats to desensitization was assessed by use of the (86)Rb(+) efflux assay using synaptosomes prepared from the "thalamus," which included the hypothalamus and midbrain as well as the thalamic nuclei. To desensitize nAChRs, synaptsosomes were superfused with low concentrations of nicotine (5, 10, 20, and 30 nM) before stimulation of (86)Rb(+) efflux with nicotine (10 muM). RESULTS: The slopes of the behavioral desensitization were plotted as a function of the decline of nicotine-stimulated (86)Rb(+) efflux after in vitro desensitization. A significant correlation was observed between the in vitro desensitization of thalamic (86)Rb(+) efflux and the extent of behavioral desensitization of individual rats. CONCLUSIONS: These findings are consistent with the idea that production of acute behavioral tolerance by nicotine is related to its ability to induce nAChR desensitization at the cellular level.

Dose, duration, and pattern of nicotine administration as determinants of behavioral dependence in rats.

RATIONALE: Relatively little is known about the role of dose, duration, and pattern of nicotine exposure in the development of dependence. Disruption of learned behavior during antagonist-precipitated withdrawal can be a sensitive, quantitative measure of behavioral dependence. OBJECTIVES: The present study sought to determine whether behavioral dependence upon nicotine could be induced in rats and, if so, what exposure conditions were essential for inducing it. Our primary focus was on whether continuous exposure over several days was necessary to produce dependence. METHODS: Male Sprague-Dawley rats were trained to lever press under fixed-ratio 10 schedules of food reinforcement during daily, 15-min experimental sessions. Nicotine was then administered s.c. via osmotic minipumps that delivered various nicotine dosage regimens, some including 24-h nicotine-free periods, to manipulate pattern of exposure. The presence of dependence was tested with challenges with the nicotinic acetylcholine receptor antagonist, mecamylamine, or during spontaneous withdrawal. RESULTS: After 7 days of 3, 6, and 12 mg kg(-1) day(-1) nicotine administration, response rates were significantly reduced in nicotinized, but not in saline-treated rats following mecamylamine challenges. Subsequent studies demonstrated that 4 days, but not 3 days, of cumulative 3 mg kg(-1) day(-1) nicotine administration was sufficient to induce dependence. The induction of dependence could be prevented by imposing a nicotine-free period between the first and second days during these 4-day regimens but not at other times. CONCLUSION: Behavioral dependence upon nicotine can be induced in the rat, and its induction is dependent upon its cumulative duration and pattern of exposure suggesting that tobacco dependencies could be controlled by similar determinants.

Evidence of cellular nicotinic receptor desensitization in rats exhibiting nicotine-induced acute tolerance.

RATIONALE: Individuals vary in their susceptibility to nicotine addiction. However, there is little evidence that behavioral sensitivity to nicotine is dependent upon the functional state of nicotinic cholinergic receptors (nAChRs). OBJECTIVE: To determine the relationship between in vivo pharmacological desensitization (in other words, acute tolerance) and brain regional nAChR function. METHODS: Male Sprague-Dawley rats, trained to discriminate nicotine (0.4 mg/kg free base) from saline in a two-lever drug discrimination task, were tested for the development of acute tolerance. Rats were injected with 0.4 mg/kg nicotine, tested for nicotine discrimination for 2 min, then injected with the same dose of nicotine 90 min, 180 min, and 270 min after the first injection and tested for nicotine discrimination after each injection. These subjects were separated into two groups, desensitizers (DZ) and nondesensitizers (NDZ), based upon performance in the repetitive dosing drug discrimination paradigm. The sensitivity of nAChRs in specific brain regions of these two groups was assessed by the use of an 86Rb+ efflux assay using synaptosomes prepared from the frontal cortex, hippocampus, striatum, and "thalamus," which included the midbrain and hypothalamus as well as the thalamus. RESULTS: The nicotine-induced increase in 86Rb+ efflux was significantly greater in NDZ as compared to DZ in the "thalamus." There was no statistically significant difference in the effects of nicotine in the frontal cortex, hippocampus, and striatum of these two groups. A significant correlation was observed between thalamic 86Rb+ efflux and the rate of behavioral desensitization of individual rats. CONCLUSION: These findings are consistent with the concept that the production of acute tolerance by nicotine in vivo correlates directly with its ability to induce nAChR desensitization at the cellular level.

Nicotinic receptor inactivation after acute and repeated in vivo nicotine exposures in rats.

Nicotine tolerance is often accompanied by an upregulation of brain area nicotinic acetylcholine receptors (nAChRs) in both animal and human subjects. This upregulation has been hypothesized to result from repeated or prolonged exposures of these receptors to nicotine. To explore this further, this study examined the level of nAChR desensitization following acute and repeated nicotine administration in the male Lewis rat. Nicotine-stimulated (86)Rb(+) efflux was measured in synaptosomes prepared from the frontal cortex, hippocampus, striatum, and thalamus. Analysis of receptor functionality was achieved by calculating area-under-the-curve (AUC) for nicotine-induced fractional (86)Rb(+) efflux. Nicotine-stimulated (86)Rb(+) efflux from all brain regions was significantly less in rats that received an acute injection of 0.4 mg/kg nicotine (s.c.) 15 min prior to dissection compared to control rats. This decrease in nAChR functional status was also observed in rats treated with 1 day or 14 days of twice-daily nicotine administration. These results are consistent with the concept that acute exposure to nicotine induces rapid desensitization of nAChRs. In addition, following repeated exposure to nicotine, nAChRs did not become tolerant to the loss in receptor function that occurs after an initial nicotine administration. Overall, these data suggest that neuronal adaptations underlying nicotine tolerance may begin upon initial exposure then persist following repeated exposures.

Neurochemical and behavioral effects of bupropion and mecamylamine in the presence of nicotine.

The primary mechanism of action of bupropion, a smoking cessation drug, is commonly believed to involve the dopaminergic system although evidence exists that bupropion also has effects at nicotinic acetylcholine receptors (nAChRs). This study evaluated the disruptive effects of nicotine on response rates in the presence of bupropion and the nAChR antagonist, mecamylamine, as well as the ability of these drugs to alter nicotine-stimulated nAChR function in various brain areas. Rats were trained to respond on a single lever under a variable interval 15 (VI15) schedule for food reinforcement. Initially, dose effect curves were generated for nicotine, bupropion and mecamylamine. Upon determining the dose of nicotine (1.2 mg/kg) effective in completely disrupting rates of responding, it was established that both mecamylamine and bupropion block nicotine's rate-reducing effects. This result suggests that bupropion shares behavioral effects with mecamylamine when administered in the presence of nicotine. To explore this relationship further, the effect of in vivo administration of bupropion or mecamylamine on nicotine-stimulated (86)Rb(+) efflux was studied in synaptosomes prepared from the frontal cortex, hippocampus, striatum and thalamus. Nicotine-stimulated (86)Rb(+) efflux from all brain regions was significantly reduced in rats administered 3.0 mg/kg mecamylamine (s.c.) 15 min prior to dissection compared to control rats. In contrast, a significant increase in nicotine-stimulated (86)Rb(+) efflux was observed in all brain regions from rats administered 30.0 mg/kg bupropion (s.c.) 15 min prior to dissection compared to control rats. Taken together these results demonstrate that when administered in the presence of nicotine, bupropion elicits unique pharmacological differences such that it exhibits both nAChR agonist- and antagonistic-like effects.

Comparative effects of dextromethorphan and dextrorphan on nicotine discrimination in rats.

While the role of dextrorphan and dextromethorphan as N-methyl-d-aspartate (NMDA) receptor antagonists has received considerable research attention, their effects on nicotinic acetylcholine receptors (nAChR) has been less well characterized. Recent in vitro and in vivo research has suggested that these drugs noncompetitively block alpha3beta4*, alpha4beta2, and alpha7 nAChR subtypes and antagonize nicotine's antinociceptive and reinforcing effects. Both drugs were most potent at blocking alpha3beta4* AChR. This study investigated the effects of dextrorphan and dextromethorphan on nicotine's discriminative stimulus effects. Three groups of rats were trained in a two-lever drug discrimination procedure to discriminate 0.4 mg/kg s.c. nicotine from saline. Nicotine dose-dependently substituted for itself in all three groups. In contrast, when dextrorphan (group 1) or dextromethorphan (group 2) were injected i.p., neither substitution for nor antagonism of nicotine was observed for either drug. Since i.p. administration allows substantial metabolism of dextromethorphan to its parent compound dextrorphan, the two drugs were also tested following s.c. administration (group 3). Discrimination results were similar across both routes of administration, in that neither substitution nor antagonism occurred, however, s.c. administration reduced response rates to a much greater extent than did i.p. administration. Previous work suggests that beta2 subunits are crucial for mediation of nicotine's discriminative stimulus effects and may play a role in its reinforcing effects, albeit other research suggests a role for alpha3beta4* nicotinic receptors in the latter. Our results suggest that alpha3beta4* nicotinic receptors do not play a major role in nicotine's discriminative stimulus effects. Further, they suggest that the role of cholinergic mediation of the behavioral effects of dextrorphan and dextromethorphan related to the abuse properties of nicotine may be minimal.

Discriminative stimulus properties of the atypical antipsychotic clozapine and the typical antipsychotic chlorpromazine in a three-choice drug discrimination procedure in rats.

RATIONALE: The atypical antipsychotic drug (APD) clozapine elicits a robust discriminative cue that is generally selective for other atypical APDS in two-choice drug discrimination (DD) procedures. OBJECTIVES: The present study determined whether a three-choice DD procedure with the atypical APD clozapine (CLZ) versus the typical APD chlorpromazine (CPZ) versus vehicle (VEH) could provide greater selectivity between atypical and typical APDs. METHODS: Sprague-Dawley rats were trained to discriminate 5.0 mg/kg CLZ from 1.0 mg/kg CPZ from VEH in a three-lever DD task with an FR30 food reinforcement schedule. RESULTS: Generalization testing with CLZ produced CPZ-appropriate responding at lower doses (ED50=0.103 mg/kg) and CLZ-appropriate responding at higher doses (ED50=1.69 mg/kg). Generalization testing with the atypical APD olanzapine produced similar results. In contrast, the atypical APD risperidone and the typical APDs CPZ and haloperidol produced only CPZ-appropriate responding. The muscarinic antagonist scopolamine produced CPZ-appropriate responding at lower doses and CLZ-appropriate responding at higher doses in a manner similar to CLZ and olanzapine. The co-administration of haloperidol (0.00625 mg/kg) with scopolamine shifted the dose-response curve for CLZ-appropriate responding to the left. The 5-HT(2A/2C) antagonist ritanserin and the H1 histamine antagonist pyrilamine did not substitute for either CLZ or CPZ. The alpha1 adrenergic antagonist prazosin did not substitute for CLZ, but produced full substitution for CPZ. CONCLUSIONS: The three-choice DD procedure clearly distinguished the atypical APDs CLZ and olanzapine from the typical APDs CPZ and haloperidol; however, the stimulus properties of the atypical APD risperidone were similar to CPZ, but not to CLZ. These findings further suggest that CLZ, as well as CPZ, elicits a compound cue.

Differential behavioral responses to nicotine in Lewis and Fischer-344 rats.

Individual and strain variability in the effects of nicotine suggests the involvement of a genetic component in nicotinic cholinergic receptor (nAChR) function, which may help explain nicotine's variable behavioral and pharmacological effects in different individuals. The present study evaluated differential responses to the discriminative stimulus (DS) and rewarding properties of nicotine in Lewis (LEW) and Fischer-344 (F-344) rats. Drug discrimination (DD) data suggest that the LEW rat is more sensitive to nicotine as LEW rats acquired the nicotine discrimination at a dose of 0.4 mg/kg, whereas F-344 rats acquired the dose of 0.9 mg/kg (all nicotine doses expressed as free base). Similarly, LEW rats exhibited nicotine-conditioned place preference (CPP) at 0.6 mg/kg, whereas the F-344 rats did not. Subsequent testing with a higher dose (0.9 mg/kg) failed to maintain the nicotine-CPP in the LEW rats. Conversely, nicotine-place preference in the F-344 rats was not changed at the higher dose. Taken together, these results suggest potential differences of sensitivities in LEW and F-344 rats to the rewarding and discriminative stimulus (DS) properties of nicotine. These findings support previous research by demonstrating that the F-344 rat is less sensitive to nicotine compared to the LEW rat.

Phenotypic assessment of THC discriminative stimulus properties in fatty acid amide hydrolase knockout and wildtype mice.

A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ(9)-tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with similar THC dose-response curves between groups. Anandamide fully substituted for THC in FAAH knockout, but not wildtype, mice. Conversely, the metabolically stable anandamide analog O-1812 fully substituted in both groups, but was more potent in knockouts. The CB1 receptor antagonist rimonabant dose-dependently attenuated THC generalization in both groups and anandamide substitution in FAAH knockouts. Pharmacological inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), with JZL184 resulted in full substitution for THC in FAAH knockout mice and nearly full substitution in wildtypes. Quantification of brain endocannabinoid levels revealed expected elevations in anandamide in FAAH knockout mice compared to wildtypes and equipotent dose-dependent elevations in 2-AG following JZL184 administration. Dual inhibition of FAAH and MAGL with JZL195 resulted in roughly equipotent increases in THC-appropriate responding in both groups. While the notable similarity in THC's discriminative stimulus effects across genotype suggests that the increased baseline brain anandamide levels (as seen in FAAH knockout mice) do not alter THC's subjective effects, FAAH knockout mice are more sensitive to the THC-like effects of pharmacologically induced increases in anandamide and MAGL inhibition (e.g., JZL184).

Neurochemical Metabolomics Reveals Disruption to Sphingolipid Metabolism Following Chronic Haloperidol Administration.

Haloperidol is an effective antipsychotic drug for treatment of schizophrenia, but prolonged use can lead to debilitating side effects. To better understand the effects of long-term administration, we measured global metabolic changes in mouse brain following 3 mg/kg/day haloperidol for 28 days. These conditions lead to movement-related side effects in mice akin to those observed in patients after prolonged use. Brain tissue was collected following microwave tissue fixation to arrest metabolism and extracted metabolites were assessed using both liquid and gas chromatography mass spectrometry (MS). Over 300 unique compounds were identified across MS platforms. Haloperidol was found to be present in all test samples and not in controls, indicating experimental validity. Twenty-one compounds differed significantly between test and control groups at the p < 0.05 level. Top compounds were robust to analytical method, also being identified via partial least squares discriminant analysis. Four compounds (sphinganine, N-acetylornithine, leucine and adenosine diphosphate) survived correction for multiple testing in a non-parametric analysis using false discovery rate threshold < 0.1. Pathway analysis of nominally significant compounds (p < 0.05) revealed significant findings for sphingolipid metabolism (p = 0.015) and protein biosynthesis (p = 0.024). Altered sphingolipid metabolism is suggestive of disruptions to myelin. This interpretation is supported by our observation of elevated N-acetyl-aspartyl-glutamate in the haloperidol-treated mice (p = 0.004), a marker previously associated with demyelination. This study further demonstrates the utility of murine neurochemical metabolomics as a method to advance understanding of CNS drug effects.