Improving the Utility of a Dynorphin Peptide Analogue Using Mannosylated Glycoliposomes.

Peptide therapeutics offer numerous advantages in the treatment of diseases and disorders of the central nervous system (CNS). However, they are not without limitations, especially in terms of their pharmacokinetics where their metabolic lability and low blood-brain barrier penetration hinder their application. Targeted nanoparticle delivery systems are being tapped for their ability to improve the delivery of therapeutics into the brain non-invasively. We have developed a family of mannosylated glycoliposome delivery systems for targeted drug delivery applications. Herein, we demonstrate via in vivo distribution studies the potential of these glycoliposomes to improve the utility of CNS active therapeutics using dynantin, a potent and selective dynorphin peptide analogue antagonist of the kappa opioid receptor (KOR). Glycoliposomal entrapment protected dynantin against known rapid metabolic degradation and ultimately improved brain levels of the peptide by approximately 3-3.5-fold. Moreover, we linked this improved brain delivery with improved KOR antagonist activity by way of an approximately 30-40% positive modulation of striatal dopamine levels 20 min after intranasal administration. Overall, the results clearly highlight the potential of our glycoliposomes as a targeted delivery system for therapeutic agents of the CNS.

Behavioural sensitization to alcohol: Bridging the gap between preclinical research and human models.

Alcohol use disorder (AUD) is an increasingly prevalent disorder that contributes significantly to the global burden of disease. According to the incentive sensitization theory (IST) of addiction, repeated alcohol exposure produces persistent neuroadaptations that promote the craving, relapse, and drug-taking characteristic of addiction. Critical to the IST model is the prediction that stimulant or hedonic drug effects become more pronounced with repeated exposure (i.e., sensitization). While there is an extensive body of preclinical alcohol sensitization research, there have been few studies examining this aspect of the incentive sensitization model in human alcohol research. In particular, developmental studies assessing sensitization over time in humans are lacking, due largely to ethical considerations precluding alcohol administration in alcohol-naïve individuals, and the lack of a reliable protocol for the prospective measurement of sensitization. The lack of translation between preclinical and human models of alcohol sensitization presents a significant barrier to further understanding the relevance of IST to the development and progression of AUD. The present review discusses how the gap between preclinical and clinical alcohol sensitization research can be bridged and how animal studies can inform human alcohol sensitization research.

Brain CYP2B induction can decrease nicotine levels in the brain.

Nicotine can be metabolized by the enzyme CYP2B; brain CYP2B is higher in rats and monkeys treated with nicotine, and in human smokers. A 7-day nicotine treatment increased CYP2B expression in rat brain but not liver, and decreased the behavioral response and brain levels (ex vivo) to the CYP2B substrate propofol. However, the effect of CYP2B induction on the time course and levels of circulating brain nicotine in vivo has not been demonstrated. Using brain microdialysis, nicotine levels following a subcutaneous nicotine injection were measured on day one and after a 7-day nicotine treatment. There was a significant time x treatment interaction (p = 0.01); peak nicotine levels (15-45 minutes post-injection) were lower after treatment (p = 0.04) consistent with CYP2B induction. Following a two-week washout period, brain nicotine levels increased to day one levels (p = 0.02), consistent with brain CYP2B levels returning to baseline. Brain pretreatment of the CYP2B inhibitor, C8-xanthate, increased brain nicotine levels acutely and after 7-day nicotine treatment, indicating the alterations in brain nicotine levels were due to changes in brain CYP2B activity. Plasma nicotine levels were not altered for any time or treatment sampled, confirming no effect on peripheral nicotine metabolism. These results demonstrate that chronic nicotine, by increasing brain CYP2B activity, reduces brain nicotine levels, which could alter nicotine's reinforcing effects. Higher brain CYP2B levels in smokers could lower brain nicotine levels; as this induction would occur following continued nicotine exposure it could increase withdrawal symptoms and contribute to sustaining smoking behavior.

Effect of Brain CYP2B Inhibition on Brain Nicotine Levels and Nicotine Self-Administration.

The CYP2B enzyme is expressed in human and rat brain, and metabolizes many CNS-acting drugs. The gene that encodes human CYP2B6 is highly polymorphic, where the variation in brain enzyme levels could result in altered brain drug levels. CYP2B can metabolize nicotine, the main psychoactive ingredient in cigarettes; if altered brain CYP2B activity can influence nicotine brain levels, it could influence nicotine-mediated behaviors. To investigate this, a mechanism-based inhibitor selective for CYP2B, C8-xanthate (20 µg), was administered intracerebroventricularly (ICV) into the brain of rats, and 22 h later, nicotine levels were measured by in vivo microdialysis following nicotine (150 µg/kg intravenous). Brain nicotine levels from 15 to 30 min and the AUC0-45 min were both twofold higher (p<0.05) with C8-xanthate vs vehicle pretreatment; there was no difference in peripheral nicotine levels. Rats were then given ICV pretreatment with C8-xanthate/ASCF and underwent intravenous nicotine self-administration with 3.75-30 µg/kg per infusion dose. C8-xanthate pretreatment increased responding in progressive ratio (15 µg/kg per infusion dose, p<0.05). In a separate cohort, C8-xanthate increased the percentage of rats that acquired self-administration (7.5 µg/kg per infusion dose, p<0.05) from 40% after vehicle pretreatment to 100%, with no difference in peripheral nicotine levels measured at the end of behavior. In a third cohort, C8-xanthate increased the number of sessions required to meet extinction criteria (p<0.05). Together these data demonstrate that the brain CYP2B activity can influence nicotine brain levels and subsequent behaviors independent of hepatic metabolism. This suggests that human smokers with variable CYP2B brain levels could have different nicotine levels and reinforcement, which might have a role in smoking behaviors and dependence.

Effect of food training and training dose on nicotine self-administration in rats.

Few studies investigate factors that influence acquisition in nicotine self-administration (NSA), such as food training and training dose. Most have utilized peak doses along nicotine's dose-response curve (15 and 30µg/kg) that establish NSA in the majority of animals. To investigate the specific and combined effects of training and dose on NSA acquisition, separate and head-to-head experiments using food training (FT) or spontaneous acquisition (SP) at multiple doses on the ascending limb of the dose-response curve were tested. First, rats underwent FT or SP under fixed ratio (FR1 and FR2) and progressive ratio (PR) schedules using 7.5-30µg/kg nicotine. More rats acquired NSA with FT vs. SP at 3.75µg/kg (56% vs. 38%) and 7.5µg/kg (88% vs. 40%, p<0.05) and FT rats responded higher under PR. Based on these findings, rats then underwent identical NSA acquisition and PR (with and without nicotine), extinction and reinstatement induced by cue exposure and nicotine in a head-to-head comparison of FT and SP using 7.5µg/kg. Acquisition differences were replicated: 100% FT and 60% SP rats met criteria (p<0.05). Without nicotine (cue only), no FT rats and 8% SP rats met criteria. FR and PR responding, extinction, and cue and nicotine-induced reinstatement did not differ between FT and SP. FT versus SP enhances acquisition at lower nicotine doses but does not alter subsequent behaviours. Lower doses can reinforce NSA and be used, in the absence of FT, to study influences on acquisition more closely modelling the initial phases of human smoking.

Effects of the 5-HT2C receptor agonist Ro60-0175 and the 5-HT2A receptor antagonist M100907 on nicotine self-administration and reinstatement.

The reinforcing effects of nicotine are mediated in part by brain dopamine systems. Serotonin, acting via 5-HT(2A) and 5-HT(2C) receptors, modulates dopamine function. In these experiments we examined the effects of the 5-HT(2C) receptor agonist Ro60-0175 and the 5-HT(2A) receptor antagonist (M100907, volinanserin) on nicotine self-administration and reinstatement of nicotine-seeking. Male Long-Evans rats self-administered nicotine (0.03 mg/kg/infusion, IV) on either a FR5 or a progressive ratio schedule of reinforcement. Ro60-0175 reduced responding for nicotine on both schedules. While Ro60-0175 also reduced responding for food reinforcement, response rates under drug treatment were several-fold higher than in animals responding for nicotine. M100907 did not alter responding for nicotine, or food, on either schedule. In tests of reinstatement of nicotine-seeking, rats were first trained to lever press for IV infusions of nicotine; each infusion was also accompanied by a compound cue consisting of a light and tone. This response was then extinguished over multiple sessions. Injecting rats with a nicotine prime (0.15 mg/kg) reinstated responding; reinstatement was also observed when responses were accompanied by the nicotine associated cue. Ro60-0175 attenuated reinstatement of responding induced by nicotine and by the cue. The effects of Ro60-0175 on both forms of reinstatement were blocked by the 5-HT(2C) receptor antagonist SB242084. M100907 also reduced reinstatement induced by either the nicotine prime or by the nicotine associated cue. The results indicate that 5-HT(2C) and 5-HT(2A) receptors may be potential targets for therapies to treat some aspects of nicotine dependence.

Serotonin receptors as potential targets for modulation of nicotine use and dependence.

Nicotine use carries considerable health risks and plays a major role in a variety of diseases. Current pharmacological treatments to aid in smoking cessation include nicotine-replacement therapy and non-nicotinic strategies such as bupropion and varenicline. While these treatments benefit some individuals there is still a need for better and more effective treatment strategies. Nicotine is the major psychoactive substance in tobacco. Some behavioural effects of nicotine, including its reinforcing efficacy result in part from activation of mesolimbic dopamine neurons. Modulation of dopamine function is one potential treatment strategy that could treat nicotine dependence. Serotonergic neurons modulate the functioning of dopamine neurons in a complex fashion. Much of this complexity arises from the fact that serotonin (5-HT) exerts its effects through multiple receptor subtypes, some of which even act in apparent functional opposition to each other. This article reviews evidence, primarily from animal experiments, using behavioural procedures relevant to nicotine use on the potential for 5-HT receptors as targets for treating nicotine dependence. The 5-HT(1A, 2A, 2C, 3, 4, 6) receptor subtypes have received most experimental attention, with the 5-HT(1A) and 5-HT(2C) receptors being the best studied. Several studies have now shown that 5-HT(1A) receptor antagonists alleviate some of the behavioural signs induced by nicotine withdrawal. Electrophysiological and neurochemical studies show that stimulation of 5-HT(2C) receptors reduces the function of the mesolimbic dopamine pathway. 5-HT(2C) receptor agonists block the stimulatory action of nicotine on midbrain dopamine function. They also reduce several behavioural effects of nicotine including its discriminative stimulus properties and reinforcing effects. Although more work remains to be done, 5-HT(2C) receptor agonists perhaps hold the most promise as potential therapies for smoking cessation.

Effects of pharmacological stressors on c-fos and CRF mRNA in mouse brain: relationship to alcohol seeking.

A marked heterogeneity exists among stressors in their ability to reinstate alcohol seeking in rats. We have reported that the pharmacological stressor yohimbine, an alpha-2 adrenoceptor antagonist, potently reinstated alcohol seeking, but FG-7142, a benzodiazepine inverse agonist was ineffective. In rats, we determined that yohimbine elicits patterns of brain expression of the mRNAs for c-fos, a marker of neuronal activation, and corticotropin-releasing factor (CRF) a stress-related peptide, distinct from that produced by FG-7142. The purpose of the present experiment is to determine if these differential effects of yohimbine and FG-7142 on regional c-fos and CRF mRNA expression generalize to another animal commonly used in alcohol research, the C57 BL/6J mouse. In comparing the results of the present study to those of our previous one, we found a number of commonalities in the patterns of activation elicited by yohimbine and FG-7142 between the two species, and some notable differences. As we found in the rat, yohimbine selectively increased c-fos mRNA in the mouse NACs, BLA and CeA. Yohimbine increased CRF mRNA only in the mouse PVN, but was without effect on CRF mRNA in extrahypothalamic sites, the BNST and CeA. This differs from what we saw in the rat, where yohimbine increased CRF mRNA in these extrahypothalamic regions, but not the PVN. The selective induction of c-fos in the NACs, BLA and CeA of mice and rats by yohimbine offers further support for the idea that activation of these structures participates in reinstatement induced by such stressors.

Intra-median raphe nucleus (MRN) infusions of muscimol, a GABA-A receptor agonist, reinstate alcohol seeking in rats: role of impulsivity and reward.

RATIONALE AND OBJECTIVES: We previously found that the inhibition of median raphe nucleus (MRN) 5-HT transmission by local injections of a 5-HT1A agonist 8-OH-DPAT or corticotrophin-releasing factor (CRF) mimic the effect of foot shock stress to reinstate alcohol seeking. In this study, we further explored the role of the MRN by examining the effect of inhibition of MRN neurons, by injecting the GABA-A receptor agonist muscimol, on the reinstatement of alcohol seeking. MATERIALS AND METHODS: Male rats were trained to lever press for 12% alcohol. Cannulae were implanted aimed at the MRN; some rats were also given intra-MRN injections of 5,7-DHT to destroy ascending 5-HT neurons. After retraining, alcohol responding was extinguished for 14 days. Subsequently, we tested the effect of muscimol injections into the MRN (0, 12.5, 25, 50 ng) on reinstatement. We also tested the effect of MRN muscimol injections on a measure of reward, conditioned place preference (CPP) and performance in the five-choice serial reaction time task (5-CSRTT), which tests a variety of psychological processes including sustained attention and impulsivity. RESULTS: MRN muscimol injections strongly reinstated alcohol seeking and this effect was not reversed by the depletion of 5-HT with 5,7-DHT. MRN muscimol injections did not induce a CPP, but did significantly impair multiple aspects of performance on the 5-CSRTT, including a marked increase in premature, or impulsive, responding. CONCLUSIONS: Together with our previous findings, these results suggest that the inhibition of MRN projection neurons provokes alcohol seeking. Results from the 5-CSRTT suggest that increased impulsivity may contribute to these effects.

3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo[1,2-b]pyridazine: a novel brain-penetrant, orally available corticotropin-releasing factor receptor 1 antagonist with efficacy in animal models of alcoholism.

We describe a novel corticotropin-releasing factor receptor 1 (CRF1) antagonist with advantageous properties for clinical development, and its in vivo activity in preclinical alcoholism models. 3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo[1,2-b]pyridazine (MTIP) inhibited 125I-sauvagine binding to rat pituitary membranes and cloned human CRF1 with subnanomolar affinities, with no detectable activity at the CRF2 receptor or other common drug targets. After oral administration to rats, MTIP inhibited 125I-sauvagine binding to rat cerebellar membranes ex vivo with an ED50 of approximately 1.3 mg/kg and an oral bioavailability of 91.1%. Compared with R121919 (2,5-dimethyl-3-(6-dimethyl-4-methylpyridin-3-yl)-7-dipropylamino-pyrazolo[1,5-a]pyrimidine) and CP154526 (N-butyl-N-ethyl-4,9-dimethyl-7-(2,4,6-trimethylphenyl)-3,5,7-triazabicyclo[4.3.0]nona-2,4,8,10-tetraen-2-amine), MTIP had a markedly reduced volume of distribution and clearance. Neither open-field activity nor baseline exploration of an elevated plus-maze was affected by MTIP (1-10 mg/kg). In contrast, MTIP dose-dependently reversed anxiogenic effects of withdrawal from a 3 g/kg alcohol dose. Similarly, MTIP blocked excessive alcohol self-administration in Wistar rats with a history of dependence, and in a genetic model of high alcohol preference, the msP rat, at doses that had no effect in nondependent Wistar rats. Also, MTIP blocked reinstatement of stress-induced alcohol seeking both in postdependent and in genetically selected msP animals, again at doses that were ineffective in nondependent Wistar rats. Based on these findings, MTIP is a promising candidate for treatment of alcohol dependence.