Investigating The Role of AEG-1 in Mouse Models of Pain

Background: Astrocyte Elevated Gene 1 (AEG-1) is a multifunctional protein shown to be a regulator of transcription and multiple intracellular signaling pathways. The role of AEG-1 in cellular inflammation appears to be primarily facilitated by its direct interaction with the transcription factor NFκB, transcriptional regulator of inflammatory cytokines. May be have a potential role in models of pain, particularly chronic inflammatory and chemotherapy induced peripheral neuropathy (CIPN). Methods: C57BL6/J male and female mice, 8-14 weeks old. AEG-1 wild type (WT) and global knockout (KO) male and female mice, 8-14 weeks old. Chronic Inflammatory Pain induced via i.pl. injection of 50% Freund\u27s Complete Adjuvant (CFA) or vehicle into mouse right hind paw. CIPN induced via four 8 mg/kg, i.p. injections of Paclitaxel or vehicle (Toma, et. al). Mechanical hypersensitivity assessed via von frey filaments. Acetone Test was used to assess cold sensitivity. mRNA transcripts collected from tissues were measured via qRT-PCR. Results: AEG-1 KO mice displayed protection from CFA induced mechanical hypersensitivity, thermal sensitivity, and reduces paw edema compare to WT mice. AEG-1 KO mice displayed enhanced recovery from paclitaxel induced mechanical hypersensitivity and cold sensitivity compared to WT mice. AEG-1 expression levels in the periaqueductal grey, spinal cord, and L4-6 corresponding dorsal root ganglia collected from C57BL6/J mice treated with 8mg/Kg paclitaxel or 50% CFA (3 days post injection) showed no difference from control groups. Conclusions: Our data suggest that AEG-1 may be involved in inflammatory and CIPN related nociception in C57BL6/J mice.https://scholarscompass.vcu.edu/gradposters/1093/thumbnail.jp

Function Of Human α3β4α5 Nicotinic Acetylcholine Receptors Is Reduced By The β5(D398N) Variant

Genome-wide studies have strongly associated a non-synonymous polymorphism (rs16969968) that changes the 398th amino acid in the nAChR α5 subunit from aspartic acid to asparagine (D398N), with greater risk for increased nicotine consumption. We have used a pentameric concatemer approach to express defined and consistent populations of α3β4α5 nAChR in Xenopus oocytes. α5(Asn-398; risk) variant incorporation reduces ACh-evoked function compared with inclusion of the common α5(Asp-398) variant without altering agonist or antagonist potencies. Unlinked α3, β4, and α5 subunits assemble to form a uniform nAChR population with pharmacological properties matching those of concatemeric α3β4* nAChRs. α5 subunit incorporation reduces α3β4* nAChR function after coinjection with unlinked α3 and β4 subunits but increases that of α3β4α5 versus α3β4-only concatemers. α5 subunit incorporation into α3β4* nAChR also alters the relative efficacies of competitive agonists and changes the potency of the non-competitive antagonist mecamylamine. Additional observations indicated that in the absence of α5 subunits, free α3 and β4 subunits form at least two further subtypes. The pharmacological profiles of these free subunit α3β4-only subtypes are dissimilar both to each other and to those of α3β4α5 nAChR. The α5 variant-induced change in α3β4α5 nAChR function may underlie some of the phenotypic changes associated with this polymorphism. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc

Astrocyte Elevated Gene-1 (AEG-1) Deletion Selectively Enhances the Antinociceptive Effects of Morphine

Background: Opioids are a class of drugs that are utilized in clinical settings to alleviate acute and chronic pain, but can often lead to development of tolerance, addiction and overdose following prolonged usage. Opioids such as morphine function by activating endogenous µ opioid receptors, which are located in various tissues throughout the body. Astrocyte Elevated Gene-1 (AEG-1) is a multifunctional protein that regulates inflammation, myeloid cell activity and lipid metabolism. Studies have shown interactions and overlaps in cellular signaling between the inflammatory/immune responses and the endogenous opioid system which could suggest a role for AEG-1 in opioids effects. Our goal is to investigate the role of AEG-1 in morphine mediated pharmacological effects including analgesia. Methods: Adult AEG-1 global knockout (KO) and wild-type (WT) male and female mice (C57BL/6J background) were utilized to assess morphine-induced thermal antinociception (The tail immersion assay test), hyperlocomotion, gastrointestinal (GI) transit inhibition, and tolerance. GI transit was assessed via charcoal transit assay. Locomotor boxes were used to assess spontaneous activity in mice. Results: AEG-1 KO mice displayed increased thermal antinociception following acute and repeated morphine administration compared to their WT counterparts. Pretreatment with naloxone blocked the enhancement of morphine thermal antinociception in AEG-1 KO mice. In addition, chronic morphine treated AEG-1 KO mice displayed reduced morphine tolerance development compared to their WT counterparts. No significant differences in morphine-induced hyperlocomotion or GI transit inhibition were observed between AEG-1 KO and WT mice. Conclusions: Our data suggest that AEG-1 deletion enhances the antinociceptive effects of morphine and reduces tolerance to chronic morphine treatment. However, AEG-1 deletion does not impact morphine-induced locomotor activity of GI transit inhibition. Overall, our results suggest that AEG-1 may function as a modulator of the endogenous opioid system.https://scholarscompass.vcu.edu/uresposters/1413/thumbnail.jp

Impact of menthol on oral nicotine consumption in female and male sprague dawley rats

Introduction: One of the preferable flavors in oral nicotine delivery systems is menthol which masks the harshness of tobacco. However, possible interactions between oral menthol and nicotine on intake and preference remain unclear. Therefore, we aimed to determine the impact of menthol on oral nicotine consumption. Methods: Adult Sprague Dawley female and male rats (n = 8 per group) were given a choice of water or drug solution by using two-bottle free choice paradigm for 2 weeks: vehicle (5% ethanol), nicotine (20 mg/L), menthol (1 g/L) and mentholated nicotine groups. At the end of the study, plasma nicotine levels were determined. Results: When rats were given a choice of nicotine or water, nicotine intake was similar between female and male rats. Menthol addition to nicotine solution significantly increased nicotine intake and preference in male but not female rats without a considerable effect on total fluid intake and body weight change in either sex. The average nicotine intake in male rats was 0.5 +/- 0.05 and 1.4 +/- 0.12 mg/kg/day for nicotine and menthol-nicotine combination (p .05), respectively. Plasma nicotine levels were not significantly different between the groups in either male (nicotine group: 20.8 +/- 4.9, mentholated nicotine group: 31.9 +/- 3.2 ng/mL) or female (nicotine group: 24.0 +/- 3.3, mentholated nicotine group: 17.8 +/- 2.9 ng/mL) rats (p >.05). Conclusions: Menthol increases oral nicotine consumption in male, but not female, rats.Canada Research Chair in PharmacogenomicsTraining and Education Core of the National Institute on Drug Abuse of the National Institutes of Health (NIH) (P50DA036105)Center for Tobacco Products of the US Food and Drug AdministrationCanadian Institutes of Health Research (FDN-154294)Rat für Forschung und Technologieentwicklun

The role of alpha5 nicotinic acetylcholine receptors in mouse models of chronic inflammatory and neuropathic pain

The aim of the present study was to determine the impact of as nicotinic acetylcholine receptor (nAChR) subunit deletion in the mouse on the development and intensity of nociceptive behavior in various chronic pain models. The role of as-containing nAChRs was explored in mouse models of chronic pain, including peripheral neuropathy (chronic constriction nerve injury, CCI), tonic inflammatory pain (the formalin test) and short and long-term inflammatory pain (complete Freund's adjuvant, CFA and carrageenan tests) in alpha(5) knock-out (1(0) and wild-type (WT) mice. The results showed that paw-licking time was decreased in the formalin test, and the hyperalgesic and allodynic responses to carrageenan and CFA injections were also reduced. In addition, paw edema in formalin-, carrageenan- or CFA-treated mice were attenuated in alpha(5)-K-O mice significantly. Furthermore, tumor necrosis factor-alpha (TNF-alpha) levels of carrageenan-treated paws were lower in alpha(5)-K-O mice. The antinociceptive effects of nicotine and sazetidine-A but not varenicline were alpha(5)-dependent in the formalin test. Both hyperalgesia and allodynia observed in the CCI test were reduced in alpha(5)-K-O mice. Nicotine reversal of mechanical allodynia in the CCI test was mediated through alpha(5)-nAChRs at spinal and peripheral sites. In summary, our results highlight the involvement of the et, nAChR subunit in the development of hyperalgesia, allodynia and inflammation associated with chronic neuropathic and inflammatory pain models. They also suggest the importance of alpha(5)-nAChRs as a target for the treatment of chronic pain.United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) (DA-12610)United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (R01DA032246)United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (R01DA012610

C57BL/6 Substrain Differences in Pharmacological Effects after Acute and Repeated Nicotine Administration.

Tobacco smoking is the major cause of disability and death in the United States and around the world. In addition, tobacco dependence and addiction express themselves as complex behaviors involving an interplay of genetics, environment, and psychological state. Mouse genetic studies could potentially elucidate the novel genes and/or gene networks regulating various aspects of nicotine dependence. Using the closely related C57BL/6 (B6) mice substrains, recent reports have noted phenotypic differences within C57BL/6J (B6J) and C57BL/6N (B6N) mice for some drugs of abuse: alcohol, opiates, and cocaine. However, the differences in nicotine\u27s effects have not yet been described in these substrains. We examined the phenotypic differences in these substrains following the acute and repeated administration of nicotine in several pharmacological measures, including locomotion (after acute and repeated exposure), body temperature, nociception, and anxiety-like behaviors. We report substrain differences in the pharmacological effects of acute and repeated nicotine administration in the B6 substrains. Overall, we show enhanced nicotine sensitivity to locomotion, hypothermia, antinociception, and anxiety-like behaviors in the B6J mouse substrain compared to B6N. In the repeated administration paradigm, both the B6N and B6J substrains showed no sensitized locomotor responses after repeated exposure to nicotine at the two doses tested. This study thus provides evidence that the B6 mouse substrains may be useful for genetic studies to elucidate some of the genetic variants involved in tobacco dependence and addiction

Synthesis And Biological Evaluation Of Bupropion Analogues As Potential Pharmacotherapies For Smoking Cessation

Bupropion (2a) analogues were synthesized and tested for their ability to inhibit monoamine uptake and to antagonize the effects of human α3β4*, α4β2, α4β4, and α1 * nAChRs. The analogues were evaluated for their ability to block nicotine-induced effects in four tests in mice. Nine analogues showed increased monoamine uptake inhibition. Similar to 2a, all but one analogue show inhibition of nAChR function selective for human α3β4*-nAChR. Nine analogues have higher affinity at α3β4*-nAChRs than 2a. Four analogues also had higher affinity for α4β2 nAChR. Analogues 2r, 2m, and 2n with AD 50 values of 0.014,0.015, and 0.028 mg/kg were 87,81, and 43 times more potent than 2a in blocking nicotine-induced antinociception in the tail-flick test. Analogue 2x with IC50 values of 31 and 180 nM for DA and NE, respectively, and with IC50 of 0.62 and 9.8 μm for antagonism of α3β4 and α4β2 nAChRs had the best overall in vitro profile relative to 2a. © 2010 American Chemical Society

Effects of methoxsalen, a CYP2A5/6 inhibitor, on nicotine dependence behaviors in mice

Metabolism of nicotine to inactive cotinine by hepatic enzyme CYP2A6 is the principal pathway by which active nicotine is removed from circulation. We therefore hypothesized that inhibition of mouse CYP2A5, the ortolog of human CYP2A6, by methoxsalen (8-methoxypsoralen) alter dependence-related behaviors of nicotine in the mouse. Conditioned place preference (CPP) test was used to assess the appetitive reward-like properties and precipitated nicotine withdrawal to assess physical (somatic and hyperalgesia) and affective (anxiety-related behaviors) measures. The nicotine plasma levels were also measured with or without methoxsalen pretreatment. Methoxsalen (15 and 30 mg/kg, intraperitoneally) pretreatment enhanced nicotine-induced preference in mice (p < 0.05). However, there was a lack of enhancement of nicotine in the CPP test after the highest dose of the CYP-2A5 inhibitor. Similarly to the CPP results, repeated administration of methoxsalen increased the intensity of mecamylamine-precipitated withdrawal signs. The potentiation of nicotine preference and withdrawal intensity by methoxsalen was accompanied by significant increase in nicotine plasma levels in mice (p < 0.05). Finally, methoxsalen enhanced the ability of a very low dose of nicotine (0.05 mg/kg) to reverse withdrawal signs in mice undergoing spontaneous withdrawal after chronic nicotine infusion (p < 0.05). In conclusion, inhibition of nicotine metabolism by methoxsalen alters the behavioral effects of nicotine in the mouse. Combining CYP2A6 inhibitors with low dose nicotine replacement therapies may have a beneficial role in smoking cessation because it will decrease the drug elimination rate and maintain plasma and brain nicotine levels.United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (DA-05274)United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (R01DA032246) (P30DA033934) (P50DA005274

Nicotinic Acetylcholine Receptor Efficacy And Pharmacological Properties Of 3-(Substituted Phenyl)-2β-Substituted Tropanes

There is a need for different and better aids to tobacco product use cessation. Useful smoking cessation aids, bupropion (2) and varenicline (3), share some chemical features with 3-phenyltropanes (4), which have promise in cocaine dependence therapy. Here we report studies to generate and characterize pharmacodynamic features of 3-phenyltropane analogues. These studies extend our work on the multiple molecular target model for aids to smoking cessation. We identified several new 3-phenyltropane analogues that are superior to 2 in inhibition of dopamine, norepinephrine, and sometimes serotonin reuptake. All of these ligands also act as inhibitors of nicotinic acetylcholine receptor (nAChR) function with a selectivity profile that favors, like 2, inhibition of α3β4*-nAChR. Many of these ligands also block acute effects of nicotine-induced antinociception, locomotor activity, and hypothermia. Importantly, all except one of the analogues tested have better potencies in inhibition of nicotine conditioned place preference than 2. We have identified new compounds that have utility as research tools and possible promise for treatment of nicotine dependence. © 2010 American Chemical Society