Pharmaco-EEG analysis of ligands varying in selectivity for α1 subunit-containing GABAA receptors during the active phase in rats.

RATIONALE: Benzodiazepines are known to evoke changes in cortical electrophysiological activity that can be correlated with action at distinct γ-aminobutyric acid type A (GABAA) receptor subtypes. OBJECTIVES: We used electroencephalography (EEG) paired with electromyography (EMG) to evaluate the role of α1 subunit-containing GABAA receptors (α1GABAARs) in benzodiazepine-induced sedation and changes in EEG band frequencies during the active phase of the light/dark cycle. METHODS: Male Sprague-Dawley rats (N = 4/drug) were surgically instrumented with EEG/EMG electrodes. The rats were injected i.p. with zolpidem, an α1GABAAR-preferring compound, or L-838,417, which has selective efficacy for α2/3/5 subunit-containing GABAARs (i.e., α1GABAAR-sparing compound), in comparison with the non-selective benzodiazepine, triazolam. RESULTS: All ligands evaluated induced changes in sleep-wake states during the active phase consistent with an increase in slow-wave sleep (SWS). The degree of SWS increase appeared to be related to the magnitude of delta power band changes induced by the ligands, with the strongest effects engendered by the α1GABAAR-preferring drug zolpidem and the weakest effects by the α1GABAAR-sparing compound, L-838,417. Consistent with other research, a selective increase in beta band power was observed with L-838,417, which may be associated with α2GABAAR-mediated anxiolysis. CONCLUSIONS: Overall, these findings support the establishment of pharmaco-EEG "signatures" for identifying subtype-selective GABAA modulators in vivo.

Behavioral effects of triazolam and pregnanolone combinations: reinforcing and sedative-motor effects in female rhesus monkeys.

Introduction: Benzodiazepines (BZs) are prescribed as anxiolytics, but their use is limited by side effects including abuse liability and daytime drowsiness. Neuroactive steroids are compounds that, like BZs, modulate the effects of GABA at the GABAA receptor. In a previous study, combinations of the BZ triazolam and neuroactive steroid pregnanolone produced supra-additive (i.e., greater than expected effects based on the drugs alone) anxiolytic effects but infra-additive (i.e., lower than expected effects based on the drugs alone) reinforcing effects in male rhesus monkeys, suggestive of an improved therapeutic window. Methods: Female rhesus monkeys (n=4) self-administered triazolam, pregnanolone, and triazolam-pregnanolone combinations intravenously under a progressive-ratio schedule. In order to assess characteristic sedative-motor effects of BZ-neuroactive steroid combinations, female rhesus monkeys (n=4) were administered triazolam, pregnanolone, and triazolam-pregnanolone combinations. Trained observers, blinded to condition, scored the occurrence of species-typical and drug-induced behaviors. Results: In contrast to our previous study with males, triazolam-pregnanolone combinations had primarily supra-additive reinforcing effects in three monkeys but infra-additive reinforcing effects in one monkey. Scores for deep sedation (i.e., defined as atypical loose-limbed posture, eyes closed, does not respond to external stimuli) and observable ataxia (any slip, trip, fall, or loss of balance) were significantly increased by both triazolam and pregnanolone. When combined, triazolam-pregnanolone combinations had supra-additive effects for inducing deep sedation, whereas observable ataxia was attenuated, likely due to the occurrence of robust sedative effects. Discussion: These results suggest that significant sex differences exist in self-administration of BZ-neuroactive steroid combinations, with females likely to show enhanced sensitivity to reinforcing effects compared with males. Moreover, supra-additive sedative effects occurred for females, demonstrating a higher likelihood of this adverse effect when these drug classes are combined.

High-Fat Diet Exacerbates Stress Responsivity in Juvenile Traumatic Brain Injury.

Abstract Traumatic brain injury (TBI) is one of the leading causes of death for children in the United States. Juveniles are more likely to sustain TBIs than most other age groups, and TBI has been shown to result in increased anxiety and stress behaviors. In addition, the hypothalamic-pituitary-adrenal (HPA) axis has previously been shown to become dysregulated after a TBI. Further, many children consume diets high in saturated fats and refined sugars, which are also connected to alterations in HPA axis function and behavior disorders. The goal of the current study was to identify a potential relationship between high-fat diet (HFD) consumption and TBI on HPA axis function in juvenile rats. In the present study, male juvenile Long-Evans rats were fed either a combination of an HFD with a high-fructose corn syrup solution or a standard chow diet. On post-natal Day 30, subjects sustained either a sham TBI or a TBI via the Closed-Head Injury Model of Engineered Rotational Acceleration (CHIMERA). Subjects participated in a trial of the open field test (OFT) following injury. In addition, some rats performed in an acute restraint stress test. All subjects were euthanized 7 days post-injury. Brain and blood plasma samples were collected for use in real-time polymerase chain reaction (RT-PCR), immunohistochemistry, and corticosterone or adrenocorticotropic hormone (ACTH) assays. Immediately following TBI, injured juveniles had increased time to righting and walking, with HFD-fed TBI rats having increased time to walking over Chow-fed TBI rats. HFD-fed TBI rats had a reduced number of entries to the center of the OFT, in addition to reduced time spent in the center compared with HFD Sham controls and Chow TBI rats. During the acute restraint stress test, HFD-fed TBI rats had elevated pre-stress ACTH and corticosterone and post-stress ACTH levels. Pre-stress ACTH levels were significantly elevated in HFD TBI compared with Chow TBI. Further, pre-stress ACTH:corticosterone ratios were elevated in HFD TBI compared with Chow TBI. cFos immunoreactivity in the paraventricular nucleus (PVN) of the hypothalamus following the acute restraint stress test was elevated in HFD-fed TBI rats. HFD TBI rats had greater activation of cFos in the PVN compared with Chow TBI. In addition, RT-PCR showed significantly reduced expression of relevant HPA axis genes, NR3C1, NR3C2, and CRHR2, in the hypothalamus of TBI subjects compared with Sham subjects. Further, AVP and CRHR2 in the hypothalamus were significantly reduced in HFD TBI compared with Chow TBI. These results offer evidence that TBI paired with high-fat diet consumption can cause HPA axis dysfunction, resulting in more anxiety-like behaviors.

Daily, limited access to methamphetamine self-administration during pregnancy leads to increased methamphetamine sensitivity in adult offspring.

Methamphetamine use by women, even throughout pregnancy, is common. But there is limited knowledge about the effects in prenatally methamphetamine-exposed children. This study investigated how prenatal methamphetamine exposure in rats, via maternal i.v. self-administration, affected the sensitivity of adult offspring to methamphetamine in comparison with controls. The offspring were generated from dams either self-administering methamphetamine daily under limited-access conditions prior to and throughout pregnancy, or their respective saline-yoked control dams. Spontaneous and methamphetamine-induced locomotor activity was assessed in male and female offspring of both exposure groups after a range of methamphetamine doses. In a separate group of offspring, acquisition of i.v. methamphetamine self-administration, responding under fixed and progressive ratio schedules of methamphetamine reinforcement, and reinstatement of extinguished drug-seeking behavior were assessed. Methamphetamine dose-dependently increased locomotor activity in both exposure groups. However, methamphetamine-exposed males showed significantly enhanced locomotor activity compared with controls at 1 mg/kg, and methamphetamine-exposed females showed significantly enhanced locomotor activity compared with controls at 3.2 mg/kg. Methamphetamine-exposed offspring of both sexes acquired methamphetamine self-administration faster and showed overall higher levels of methamphetamine-induced reinstatement compared with controls. Taken together, these results indicate that prenatal methamphetamine exposure to relatively low levels alters methamphetamine sensitivity in male and female adult offspring.

8-Substituted Triazolobenzodiazepines: In Vitro and In Vivo Pharmacology in Relation to Structural Docking at the α1 Subunit-Containing GABAA Receptor.

In order to develop improved anxiolytic drugs, 8-substituted analogs of triazolam were synthesized in an effort to discover compounds with selectivity for α2/α3 subunit-containing GABAA subtypes. Two compounds in this series, XLi-JY-DMH (6-(2-chlorophenyl)-8-ethynyl-1-methyl-4H-benzo [f][1,2,4]triazolo[4,3-a][1,4]diazepine) and SH-TRI-108 [(E)-8-ethynyl-1-methyl-6-(pyridin-2-yl)-4H-benzo [f][1,2,4]triazolo[4,3-a][1,4]diazepine], were evaluated for in vitro and in vivo properties associated with GABAA subtype-selective ligands. In radioligand binding assays conducted in transfected HEK cells containing rat αXß3γ2 subtypes (X = 1,2,3,5), no evidence of selectivity was obtained, although differences in potency relative to triazolam were observed overall (triazolam > XLi-JY-DMH > SH-TRI-108). In studies with rat αXß3γ2 subtypes (X = 1,2,3,5) using patch-clamp electrophysiology, no differences in maximal potentiation of GABA-mediated Cl- current was obtained across subtypes for any compound. However, SH-TRI-108 demonstrated a 25-fold difference in functional potency between α1ß3γ2 vs. α2ß3γ2 subtypes. We evaluated the extent to which this potency difference translated into behavioral pharmacological differences in monkeys. In a rhesus monkey conflict model of anxiolytic-like effects, triazolam, XLi-JY-DMH, and SH-TR-108 increased rates of responding attenuated by shock (anti-conflict effect) but also attenuated non-suppressed responding. In a squirrel monkey observation procedure, both analogs engendered a profile of sedative-motor effects similar to that reported previously for triazolam. In molecular docking studies, we found that the interactions of the 8-ethynyl triazolobenzodiazepines with the C-loop of the α1GABAA site was stronger than that of imidazodiazepines XHe-II-053 and HZ-166, which may account for the non-sedating yet anxiolytic profile of these latter compounds when evaluated in previous studies.

Changes in the elimination and resurgence of alcohol-maintained behavior in rats and the effects of naltrexone.

Resurgence may be a mechanism of relapse in alcohol use disorder patients upon discharge from treatment as part of an abuse-treatment-relapse cycle. Adjunctive pharmacotherapies may be a means to facilitate behavioral treatments and block resurgence. Experiments were conducted using a model of alcohol self-administration to assess the repeatability of the elimination and resurgence of alcohol-maintained behavior and the effects of naltrexone. Experiments had three phases. In Phase 1, behavior was maintained by oral alcohol under a fixed-ratio schedule. In Phase 2, behavior was extinguished via condensed milk delivery under a differential-reinforcement-of-other-behavior (DRO) schedule. In Phase 3, the DRO schedule was eliminated. In Experiment 1, this 3-phase cycle was replicated 4 times. Across replications, response rates and dose of alcohol consumed did not differ in Phase 1, alcohol-maintained behavior was eliminated more rapidly in Phase 2, and the resurgence effect was generally stable in Phase 3. In Experiment 2, naltrexone was administered in Phase 2, Phase 3, or both Phases 2 and 3, to separate groups of rats. Naltrexone facilitated the elimination of alcohol-maintained behavior in Phase 2 and, the resurgence of alcohol-maintained behavior was reduced only for those rats that received naltrexone in both phases. Together, these experiments demonstrate that the resurgence of alcohol-maintained behavior is replicable within-subjects and, further, resurgence of alcohol-maintained behavior may be a useful model to evaluate pharmacological interventions to facilitate behavioral treatments and reduce the likelihood of relapse. Results with naltrexone support the use of medication-assisted therapy approaches to reduce relapse risk in patients. (PsycINFO Database Record (c) 2020 APA, all rights reserved).

Evaluation of the anti-conflict, reinforcing, and sedative effects of YT-III-31, a ligand functionally selective for α3 subunit-containing GABAA receptors.

BACKGROUND: In recent years, pharmacological strategies have implicated α3 subunit-containing GABAA (α3GABAA) receptor subtypes in the anxiety-reducing effects of benzodiazepines, whereas transgenic mouse approaches have implicated α2 or α5 subunit-containing GABAA receptors. AIMS: We investigated the role of α3GABAA subtypes in benzodiazepine-induced behaviors by evaluating the anti-conflict, reinforcing, and sedative-motor effects of the novel compound YT-III-31, which has functional selectivity for α3GABAA receptors. METHODS: Female and male rhesus monkeys were trained under a conflict procedure (n = 3), and a progressive-ratio schedule of reinforcement with midazolam as the training drug (n = 4). Sedative-like behavior was assessed using a quantitative behavioral observation procedure (n = 4). A range of doses of YT-III-31 was administered in all tests using the i.v. route of administration. RESULTS: In the conflict procedure, increasing doses of YT-III-31 resulted only in dose-dependent attenuation of non-suppressed responding. In the progressive-ratio model of self-administration, YT-III-31 maintained average injections/session above vehicle levels at 0.1 and 0.18 mg/kg/injection. In quantitative observation procedures, YT-III-31 engendered mild sedative effects ("rest/sleep posture"), and deep sedation at the highest dose tested (5.6 mg/kg, i.v.), along with a suppression of tactile/oral exploration and increased observable ataxia. In contrast to other benzodiazepine-like ligands, YT-III-31 uniquely engendered a biphasic dose-response function for locomotion and suppressed self-groom. CONCLUSIONS: The finding that YT-III-31 lacked anti-conflict properties is in accordance with transgenic mouse research indicating no role for α3GABAA subtypes in benzodiazepine-mediated anxiety reduction. Instead, our results raise the possibility of a role for α3GABAA receptors in the abuse potential and sedative effects of benzodiazepine-type drugs.

Antibody production and pharmacokinetics of METH in rats following vaccination with the METH vaccine, IXT-v100, adjuvanted with GLA-SE.

BACKGROUND: Methamphetamine use disorder continues to be inadequately treated, but improvements are being made in the field of immunotherapeutics, including vaccines, which could provide new options for treatment. Cocaine and nicotine vaccines have been tested clinically, but have yet to elicit the necessary antibody concentrations required to be effective. Methamphetamine vaccines have been tested in multiple nonclinical models and appear promising. Improved adjuvants have the potential to further stimulate the immune system to reach effective levels of antibodies. Previously, the methamphetamine vaccine IXT-v100 was administered with GLA-SE, a toll-like receptor 4 agonist, in mice to produce higher levels of antibodies than when it was administered with two other widely used adjuvants, Alhydrogel and Sigma Adjuvant System. METHODS: The purpose of this research was to evaluate IXT-v100, given in combination with the adjuvant GLA-SE, to determine its efficacy in antagonizing methamphetamine disposition in a rat pharmacokinetic study. Additional rat studies were conducted to compare the ability of IXT-v100 manufactured with greater hapten densities to elicit higher antibody levels. RESULTS: As expected based on prior studies with anti-methamphetamine monoclonal antibodies, the antibodies resulting from vaccination with IXT-v100 altered methamphetamine pharmacokinetics by increasing serum concentrations and extending the half-life. Furthermore, intentional variations in the ratio of components during manufacturing led to production of vaccines with higher hapten densities. The higher hapten densities resulted in production of antibodies that maintained the ability to bind methamphetamine with high affinity. CONCLUSIONS: The results support continued development of IXT-v100 for the treatment of methamphetamine use disorder.

GABAA Receptor Subtypes and the Abuse-Related Effects of Ethanol in Rhesus Monkeys: Experiments with Selective Positive Allosteric Modulators.

BACKGROUND: Previous studies have investigated α1GABAA and α5GABAA receptor mechanisms in the behavioral effects of ethanol (EtOH) in monkeys. However, genetic studies in humans and preclinical studies with mutant mice suggest a role for α2GABAA and/or α3GABAA receptors in the effects of EtOH. The development of novel positive allosteric modulators (PAMs) with functional selectivity (i.e., selective efficacy) at α2GABAA and α3GABAA receptors allows for probing of these subtypes in preclinical models of the discriminative stimulus and reinforcing effects of EtOH in rhesus macaques. METHODS: In discrimination studies, subjects were trained to discriminate EtOH (2 g/kg, intragastrically) from water under a fixed-ratio (FR) schedule of food delivery. In oral self-administration studies, subjects were trained to self-administer EtOH (2% w/v) or sucrose (0.3 to 1% w/v) under an FR schedule of solution availability. RESULTS: In discrimination studies, functionally selective PAMs at α2GABAA and α3GABAA (HZ-166) or α3GABAA (YT-III-31) receptors substituted fully (maximum percentage of EtOH-lever responding ≥80%) for the discriminative stimulus effects of EtOH without altering response rates. Full substitution for EtOH also was engendered by a nonselective PAM (triazolam), an α5GABAA -preferring PAM (QH-ii-066) and a PAM at α2GABAA , α3GABAA , and α5GABAA receptors (L-838417). A partial (MRK-696) or an α1GABAA -preferring (zolpidem) PAM only engendered partial substitution (i.e., ~50 to 60% EtOH-lever responding). In self-administration studies, pretreatments with the functionally selective PAMs at α2GABAA and α3GABAA (XHe-II-053 and HZ-166) or α3GABAA (YT-III-31 and YT-III-271) receptors increased EtOH, but not sucrose, drinking at doses that had few, or no, observable sedative-motor effects. CONCLUSIONS: Our results confirm prior findings regarding the respective roles of α1GABAA and α5GABAA receptors in the discriminative stimulus effects of EtOH and, further, suggest a key facilitatory role for α3GABAA and potentially α2GABAA receptors in several abuse-related effects of EtOH in monkeys. Moreover, they reveal a potential role for these latter subtypes in EtOH's sedative effects.

Correction to: GABAA Receptor Subtype Mechanisms and the Abuse-Related Effects of Ethanol: Genetic and Pharmacological Evidence.

The chemical name appearing in the first column of Table 1 on the 3rd row from bottom of the table is wrong.

GABAA Receptor Subtype Mechanisms and the Abuse-Related Effects of Ethanol: Genetic and Pharmacological Evidence.

Ethanol's reinforcing and subjective effects, as well as its ability to induce relapse, are powerful factors contributing to its widespread use and abuse. A significant mediator of these behavioral effects is the GABAA receptor system. GABAA receptors are the target for γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the CNS. Structurally, they are pentameric, transmembrane chloride ion channels comprised of subunits from at least eight different families of distinct proteins. The contribution of different GABAA subunits to ethanol's diverse abuse-related effects is not clear and remains an area of research focus. This chapter details the clinical and preclinical findings supporting roles for different α, ß, γ, and δ subunit-containing GABAA receptors in ethanol's reinforcing, subjective/discriminative stimulus, and relapse-inducing effects. The reinforcing properties of ethanol have been studied the most systematically, and convergent preclinical evidence suggests a key role for the α5 subunit in those effects. Regarding ethanol's subjective/discriminative stimulus effects, clinical and genetic findings support a primary role for the α2 subunit, whereas preclinical evidence implicates the α5 subunit. At present, too few studies investigating ethanol relapse exist to make any solid conclusions regarding the role of specific GABAA subunits in this abuse-related effect.

Detrimental effects of self-administered methamphetamine during pregnancy on offspring development in the rat.

BACKGROUND: Methamphetamine (METH) abuse by pregnant women is a commonly observed phenomenon. While the harmful effects of METH are well described for adults, there is only limited knowledge of the effects of METH use during pregnancy on the developing child. In the present study, we investigated how intraveneous (iv) METH self-administration throughout pregnancy affected rat dams and their offspring through weaning, compared to controls. METHODS: Female rats (n=16) were trained to self-administer METH iv; every drug infusion by a dam also resulted in a saline injection to a yoked control (n=16). When stable levels of self-administration were reached, all females were mated. Daily, 2-h self-administration sessions continued until litters were born. General health and weight was assessed daily in dams and pups. In addition, pups were evaluated for achievement of age-appropriate developmental milestones (i.e., righting reflex, negative geotaxis, pinna detachment, fur appearance, incisor eruption and eye opening). RESULTS: Dams self-administered 2-3mg/kg/day METH throughout gestation without consequence to dam health or weight gain during pregnancy. All females produced viable litters, and litter size and composition did not differ between saline and METH dams. Similarly, maternal pup-directed behavior was not affected by prior METH self-administration. However, despite a lack of weight difference in pups, METH-exposed pups were significantly delayed in reaching all assessed developmental milestones compared to controls. CONCLUSION: These results indicate that in utero exposure to moderate METH doses can profoundly and adversely affect offspring development, suggesting that even recreational METH use during pregnancy has potential for harm.

Chronic treatment of (+)-methamphetamine-induced locomotor effects in rats using one or a combination of two high affinity anti-methamphetamine monoclonal antibodies.

We hypothesized that treatment of methamphetamine (METH) effects with a mixture of 2 high affinity anti-METH monoclonal antibodies (mAb) with differing molecular recognition for METH-like structures could increase efficacy compared to treatment with a single mAb. The antibodies studied were mAb7F9 (METH and amphetamine [AMP] KD = 7.7 and 270 nM) and mAb4G9 (16 nM and 110 nM, respectively) in a 50:50 mixture. Adult male Sprague Dawley Rats were treated with iv saline or a loading dose of mAb7F9-mAb4G9 (141 mg/kg of each mAb) followed by 2 weekly doses (70.5 mg/kg total) on days 7 and 14. METH challenge doses (0.56 mg/kg) were administered 4 hrs and 3 days after each mAb7F9-mAb4G9 treatment, and 7 days after the final treatment (day 21). Locomotor activity (0-4 hrs) and serum METH and AMP concentrations (at 5 hrs) were measured after each METH challenge. MAb7F9-mAb4G9 treatment significantly reduced the duration of locomotor activity after 6 of the 7 METH doses (P < 0.05) and significantly increased serum METH and AMP concentrations. Administering three-fold higher METH doses (1.68 mg/kg) on days 24 and 28 showed mAb7F9-mAb4G9 treatment had negligible effects on the duration of METH-induced locomotor activity. These data were then compared to previous monotherapy data. While mAb7F9-mAb4G9 therapy inhibited the effects of multiple METH challenge doses, the inhibition was not as profound or as long lasting as the effects of mAb7F9 treatment alone. These data demonstrate the importance of both mAb affinity and specificity in the production of effective, long-lasting anti-METH mAb therapies.

Combining Active Immunization with Monoclonal Antibody Therapy To Facilitate Early Initiation of a Long-Acting Anti-Methamphetamine Antibody Response.

We hypothesized that an anti-METH mAb could be used in combination with a METH-conjugate vaccine (MCV) to safely improve the overall quality and magnitude of the anti-METH immune response. The benefits would include immediate onset of action (from the mAb), timely increases in the immune responses (from the combined therapy) and duration of antibody response that could last for months (from the MCV). A novel METH-like hapten (METH-SSOO9) was synthesized and then conjugated to immunocyanin monomers of keyhole limpet hemocyanin (IC(KLH)) to create the MCV ICKLH-SOO9. The vaccine, in combination with previously discovered anti-METH mAb7F9, was then tested in rats for safety and potential efficacy. The combination antibody therapy allowed safe achievement of an early high anti-METH antibody response, which persisted throughout the study. Indeed, even after 4 months the METH vaccine antibodies still had the capacity to significantly reduce METH brain concentrations resulting from a 0.56 mg/kg METH dose.

Attenuation of cocaine-induced reinstatement of drug seeking in squirrel monkeys by direct and indirect activation of 5-HT2C receptors.

RATIONALE: 5-Hydroxytryptamine (5-HT) transport inhibitors can attenuate the abuse-related effects of cocaine, and the mechanisms underlying this attenuation may involve activation of 5-HT2C receptors. OBJECTIVES: The objective of this study was to investigate the consequences of direct and indirect pharmacological activation of 5-HT2C receptors on reinstatement of cocaine-seeking behavior induced by cocaine priming and a cocaine-paired stimulus. METHODS: Monkeys were trained to self-administer cocaine under a second-order schedule in which responding was maintained by i.v. cocaine injections and a cocaine-paired stimulus. Drug seeking was extinguished by replacing cocaine with vehicle and eliminating the cocaine-paired stimulus. During reinstatement tests, the animals received a priming injection of cocaine along with restoration of the cocaine-paired stimulus, but only vehicle was available for self administration. RESULTS: Pretreatment with either the 5-HT transport inhibitor fluoxetine (5.6 mg/kg) or the 5-HT2C receptor agonist Ro 60-0175 (1 mg/kg) attenuated reinstatement of drug seeking by cocaine priming. The reinstatement-attenuating effects of both drugs were reversed by the 5-HT2C receptor antagonist SB 242084 (0.03-0.56 mg/kg). Ro 60-0175 (1 mg/kg) attenuated cocaine-induced reinstatement of drug seeking regardless of whether priming injections were or were not accompanied by restoration of the cocaine-paired stimulus. Ro 60-0175 (0.56 mg/kg) was equally effective whether it was administered acutely or chronically. Finally, Ro 60-0175 (0.3-1 mg/kg) had observable behavioral effects suggestive of anxiolytic-like properties. CONCLUSIONS: 5-HT2C receptor mechanisms play a key role in the modulation of cocaine-induced reinstatement by fluoxetine and Ro 60-0175. Direct activation of 5-HT2C receptors may offer a novel, tolerance-free therapeutic strategy for the prevention of cocaine relapse.

Treatment of rats with an anti-(+)-methamphetamine monoclonal antibody shortens the duration of action of repeated (+)-methamphetamine challenges over a one month period.

This study assessed clinical scenarios of continuing monoclonal antibody (mAb) treatment for (+)-methamphetamine (METH) addiction, and the implications of missing or discontinuing this therapy. We hypothesized that chronic anti-METH mAb7F9 (METH KD=9 nM) treatment of rats could significantly decrease METH-induced behaviors; even with repeated METH challenges, use of METH doses in excess of mAb binding sites, and after discontinuing mAb treatment which results in a 10-fold reduction in mAb7F9 serum concentrations. Male Sprague Dawley rats (n=6/group) were treated with i.v. saline or a loading dose of mAb7F9 to achieve instant steady-state conditions followed by two weekly (141 mg/kg) doses ending on day 14. METH (0.56 mg/kg) was administered 4h and three days after each saline or mAb7F9 treatment, and on day 21. This produced locomotion and rearing behavior that lasted about 120 min in control rats. In mAb7F9 treated rats, METH-induced distance traveled was significantly reduced from 60 to 120 min (P<0.05) on days 0-21 and rearing was significantly reduced from 60 to 120 min on days 0-17. METH serum concentrations determined 5h after METH dosing was significantly increased in mAb7F9-treated rats after all METH challenges. On days 24 and 28 (the final day), the rats were administered a 3-fold higher METH dose (1.68 mg/kg). MAb7F9 treated rats showed a substantially earlier termination of the METH-induced locomotion on both days, even though the METH dose exceeded mAb7F9s binding capacity. METH brain concentrations determined 5h after METH on day 28 were also significantly decreased in mAb7F9-treated rats. In conclusion, over one month, mAb7F9 significantly and continuously bound METH and reduced METH-induced locomotor effects even after discontinuation of mAb treatment and challenge with higher METH doses.

Vaccination protects rats from methamphetamine-induced impairment of behavioral responding for food.

(+)-Methamphetamine (METH) addiction is a chronic disease that interferes with fundamental brain-mediated behaviors and biological functions like eating. These studies present preclinical efficacy and safety profiles for a METH conjugate vaccine (IC(KLH)-SMO9) designed to treat METH abuse. ICKLH-SMO9 efficacy and safety were assessed over a 16-week period by monitoring general health and stability of responding in a food maintained behavioral paradigm. Male Sprague-Dawley rats were trained to lever press for food reinforcers until stable behavior was established. Rats (n=9/group) were then immunized with 100 µg of a control antigenic carrier protein (IC(KLH)-Cys) or IC(KLH)-SMO9 in Alhydrogel adjuvant, with booster immunizations at 4, 8 and 12 weeks. Health, immunization site and behavior were assessed daily. No adverse effects were found. During weeks 14-16, when antibody titers and METH affinity (K(d)=13.9 ± 1.7 nM) were maximal, all rats received progressively higher METH doses (0.3-3.0 mg/kg) every 3-4 days, followed by behavioral testing. Even though the lower METH doses from 0.3 to 1.0 mg/kg produced no impairment in food maintained behavior, 3.0-mg/kg in control rats showed significantly (p<0.05) reduced response rates and number of reinforcers earned, as well as reduced food intake. In sharp contrast, the IC(KLH)-SMO9 group showed no changes in food maintained behavior at any METH dose, even though METH serum concentrations showed profound increases due to anti-METH antibody binding. These findings suggest the IC(KLH)-SMO9 vaccine is effective and safe at reducing adverse METH-induced effects, even at high METH doses.

Modulation of α5 subunit-containing GABAA receptors alters alcohol drinking by rhesus monkeys.

BACKGROUND: Alcohol's ability to potentiate the activity of γ-aminobutyric acid (GABA) at GABAA receptors has been implicated as a key mechanism underlying the behavioral effects of alcohol. The complex molecular biology of these receptors raises the possibility that particular receptor subtypes may play unique roles in alcohol's abuse-related effects and that subtype-selective ligands with therapeutic specificity against alcohol might be developed. This study evaluated the capacity of α5GABAA receptor ligands to alter selectively the reinforcing effects of alcohol. METHODS: Two groups of rhesus monkeys were trained to orally self-administer alcohol or sucrose under fixed-ratio schedules and limited daily access conditions. In addition, following daily self-administration sessions, the behavior of each monkey was scored for both species-typical and drug-induced behaviors. RESULTS: Concentrations of 1 to 6% alcohol maintained self-administration above water levels, engendered pharmacologically relevant blood alcohol levels ranging from 90 to 160 mg/dl, and produced changes in behavior typical of alcohol intoxication. Concentrations of 0.3 to 3% sucrose also reliably maintained self-administration. The α5GABAA receptor agonist QH-ii-066 enhanced and the α5GABAA receptor inverse agonist L-655,708 inhibited alcohol, but not sucrose drinking. The changes in alcohol drinking could be reversed with the α5GABAA receptor antagonist XLi-093. However, L-655,708 increased yawning in both alcohol and sucrose drinkers, possibly indicative of an anxiogenic effect. CONCLUSIONS: These findings suggest a prominent and specific role for α5GABAA receptor mechanisms in the reinforcing effects of alcohol. Moreover, these results suggest that α5GABAA receptors may represent a novel pharmacological target for the development of medications to reduce drinking. Of ligands modulating this receptor, α5GABAA receptor inverse agonists may hold the most promise as alcohol pharmacotherapies.

A pharmacogenetic model of naltrexone-induced attenuation of alcohol consumption in rhesus monkeys.

BACKGROUND: Variation at the human mu-opioid receptor has been associated with alcohol abuse. The A118G (N40D) polymorphism in humans is functionally mimicked by the C77G (P26R) polymorphism in rhesus monkeys; both show similar in vitro influences on ligand binding and in vivo correlations with physiological measures as well as behavioral measures including predilection towards alcohol consumption. Naltrexone, an antagonist at the receptor, has been used to treat alcoholism in humans and has been reported to show differences in effectiveness depending on genotype. METHODS: Here we describe a study in which we a priori selected rhesus monkeys based on genotype at the OPRM1 C77G single nucleotide polymorphism, trained them to self-administer alcohol, and assessed naltrexone responsiveness. RESULTS: Alcohol intake in rhesus monkeys varied with genotype across a range of alcohol concentrations (0.5-4%, w/v) such that animals with the G/G genotype drank consistently more alcohol than those animals with the C/C genotype. Additionally, naltrexone attenuated alcohol drinking in a dose- and genotype-dependent manner. Animals harboring the G/G genotype were more sensitive to the effects of naltrexone and showed greater reductions in alcohol consumption at lower naltrexone doses compared to animals with a C/G or C/C genotype. CONCLUSIONS: This preliminary study demonstrates a pharmacogenomic response to naltrexone in rhesus monkeys that parallels that seen in humans. This finding provides a basis for developing a pharmacogenetic animal model for naltrexone effect that can expand further our understanding of the causes and treatments of alcohol use disorders.

Attenuation of cocaine-induced reinstatement of drug seeking in squirrel monkeys: kappa opioid and serotonergic mechanisms.

RATIONALE: Kappa agonists can attenuate reinstatement of cocaine-seeking behavior induced by cocaine priming. The mechanisms underlying this effect have not been characterized fully but may have a serotonergic component as kappa agonists also increase the release of serotonin (5-hydroxytryptamine, 5-HT). OBJECTIVES: This study investigated the role of kappa opioid receptor and 5-HT mechanisms in kappa agonist-induced attenuation of cocaine priming in monkeys. METHODS: Squirrel monkeys were trained to self-administer cocaine (0.18-0.3 mg/kg/injection) under a second-order schedule in which drug seeking was maintained jointly by cocaine injections and a cocaine-paired visual stimulus. In extinction sessions, saline was substituted for cocaine, and the cocaine-paired stimulus was omitted. During test sessions, only saline was available for self-administration, and response-contingent presentations of the cocaine-paired stimulus were restored. RESULTS: Priming injections of cocaine (0.1-1.0 mg/kg) induced reinstatement of drug seeking. Maximal levels of responding were similar to those maintained by active cocaine self-administration. Pretreatment with the kappa agonists enadoline (0.01 mg/kg) and spiradoline (0.3 mg/kg) or the 5-HT transport inhibitors fluoxetine (5.6 mg/kg) and citalopram (10.0 mg/kg) attenuated the priming effects of cocaine, shifting the cocaine dose-response function rightward and downward. Inhibition of cocaine-induced reinstatement of drug seeking by spiradoline and fluoxetine was reversed by R(+)8-hydroxy-2-(di-n-propylamino)tetralin (0.03 mg/kg), a 5HT(1A) agonist that inhibits 5-HT release. The effects of spiradoline also were reversed by the kappa antagonist nor-binaltorphimine (10.0 mg/kg). CONCLUSIONS: Results suggest that the capacity of kappa opioid agonists to increase extracellular 5-HT levels may at least partially underlie kappa agonist-induced modulation of cocaine seeking.