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United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) - R01 DA12610 - R01 DA032246 - T32 DA007027-41 United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Alcohol Abuse & Alcoholism (NIAAA) - P50AA022537 United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission - T32DA007027 - R01DA032246 - R01DA012610 Chronic tobacco use dramatically increases health burdens and financial costs. Limitations of current smoking cessation therapies indicate the need for improved molecular targets. The main addictive component of tobacco, nicotine, exerts its dependency effects via nicotinic acetylcholine receptors (nAChRs). Activation of the homomeric alpha 7 nAChR reduces nicotine's rewarding properties in conditioned place preference (CPP) test and i.v. self-administration models, but the mechanism underlying these effects is unknown. Recently, the nuclear receptor peroxisome proliferator-activated receptor type-alpha (PPAR alpha) has been implicated as a downstream signaling target of the alpha 7 nAChR in ventral tegmental area dopamine cells. The present study investigated PPAR alpha as a possible mediator of the effect of alpha 7 nAChR activation in nicotine dependence. Our results demonstrate the PPAR alpha antagonist GW6471 blocks actions of the alpha 7 nAChR agonist PNU282987 on nicotine reward in an unbiased CPP test in male ICR adult mice. These findings suggests that alpha 7 nAChR activation attenuates nicotine CPP in a PPAR alpha-dependent manner. To evaluate PPAR alpha activation in nicotine dependence we used the selective and potent PPAR alpha agonist, WY-14643 and the clinically used PPAR alpha activator, fenofibrate, in nicotine CPP and we observed attenuation of nicotine preference, but fenofibrate was less potent. We also studied PPAR alpha in nicotine dependence by evaluating its activation in nicotine withdrawal. WY-14643 reversed nicotine withdrawal signs whereas fenofibrate had modest efficacy. This suggests that PPAR alpha plays a role in nicotine reward and withdrawal and that further studies are warranted to elucidate its function in mediating the effects of alpha 7 nAChRs in nicotine dependence.

Pain encompasses both a sensory as well as an affective dimension and these are differentially processed in the brain and periphery. It is therefore important to develop animal models to reflect the non-reflexive assays in pain. In this study, we compared effects of the mu opioid receptor agonist morphine, the nonsteroidal anti-inflammatory drug ketoprofen and the kappa receptor opioid agonist U50,488H and antagonist JDTic on acetic acid-induced stretching and acetic acid-induced aversion in the condition place aversion (CPA) test in male ICR mice. Intraperitoneal administration of acetic acid (0.32-1%) was equipotent in stimulating stretching and CPA. Ketoprofen, morphine and U50,488H all inhibited the acid induced stretching. Ketoprofen and morphine also blocked the acid-induced CPA but U50,488H failed to do so. The reversal ability of ketoprofen and morphine on acid-induced CPA is unique to pain-stimulated place aversion since these drugs failed to reduce non-noxious LiCl-induced CPA. Overall, this study characterized and validated a preclinical mouse model of pain-related aversive behavior that can be used to assess genetic and biological mechanisms of pain as well as improving the predictive validity of preclinical studies on candidate analgesics. United States Department of Health & Human Services National Institutes of Health (NIH) - USA - R01DA-019377 - R01NS070715 - R01DA030404 NIH National Institute of Neurological Disorders & Stroke (NINDS) - R01NS070715 NIH National Institute on Drug Abuse (NIDA) - R01DA030404 European Commission

Recently, alpha 7 nicotinic acetylcholine receptors (nAChRs), primarily activated by binding of orthosteric agonists, represent a target for anti-inflammatory and analgesic drug development. These receptors may also be modulated by positive allosteric modulators (PAMs), ago-allosteric ligands (ago-PAMs), and alpha 7-silent agonists. Activation of 00 nAChRs has been reported to increase the brain levels of endogenous ligands for nuclear peroxisome proliferator-activated receptors type-alpha (PPAR-alpha), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), in a Ca2+-dependent manner. Here, we investigated potential crosstalk between alpha 7 nAChR and PPAR-alpha, using the formalin test, a mouse model of tonic pain. Using pharmacological and genetic approaches, we found that PNU282987, a full alpha 7 agonist, attenuated formalin-induced nociceptive behavior in alpha 7 -dependent manner. Interestingly, the selective PPAR-alpha antagonist GW6471 blocked the antinociceptive effects of PNU282987, but did not alter the antinociceptive responses evoked by the alpha 7 nAChR PAM PNU120596, ago-PAM GAT107, and silent agonist NS6740. Moreover, GW6471 administered systemically or spinally, but not via the intraplantar surface of the formalin-injected paw blocked PNU282987-induced antinociception. Conversely, exogenous administration of the naturally occurring PPAR-alpha agonist PEA potentiated the antinociceptive effects of PNU282987. In contrast, the cannabinoid 031 antagonist rimonabant and the CB2 antagonist SR144528 failed to reverse the antinociceptive effects of PNU282987. These findings suggest that PPAR-alpha plays a key role in a putative antinociceptive alpha 7 nicotinic signaling pathway. United States Department of Health & Human Services National Institutes of Health (NIH) - USA - GM57481 - R01 CA206028 United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Cancer Institute (NCI) - R01CA206028 United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of General Medical Sciences (NIGMS) - R01GM057481 United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission - T32DA007027

Introduction: Rodent studies suggest that nicotine metabolites and minor tobacco alkaloids such as nornicotine and cotinine may promote cigarette smoking by enhancing nicotine rewarding and reinforcing effects. However, there is little information on the effects of these minor tobacco alkaloids on nicotine withdrawal. The present studies were conducted to determine whether the minor tobacco alkaloids nornicotine and cotinine exhibit nicotine-like behavioral effects in a mouse model of spontaneous nicotine withdrawal. Methods: Mice were infused with nicotine or saline for 14 days. Experiments were conducted on day 15, 18-24 hours after minipump removal. Ten minutes prior to testing, nicotine-dependent ICR male mice received an acute injection of nicotine (0.05 and 0.5 mg/kg), nornicotine (2.5 and 25 mg/kg), or cotinine (5 and 50 mg/kg) to determine effects on somatic signs, anxiety-like behaviors, and hyperalgesia spontaneous signs of withdrawal. Results: Nicotine and the minor tobacco alkaloid nornicotine, but not cotinine, produced dose-dependent reversal of nicotine withdrawal signs in the mouse. Implications: The minor tobacco alkaloid and nicotine metabolite nornicotine at high doses have nicotinic like effects that may contribute to tobacco consumption and dependence. United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission - DA-05274 - DA-032246 - R01DA032246

Metacyclic trypomastigotes are essential for the understanding of the biology of Trypanosoma cruzi, the agent of Chagas disease. However, obtaining these biological stages in axenic medium is difficult. Techniques based on charge and density of the parasite during different stages have been implemented, without showing a high efficiency in the purification of metacyclic trypomastigotes. So far, there is no protocol implemented where sepharose-DEAE is used as a resin. Therefore, herein we tested its ability to purify metacyclic trypomastigotes in Liver Infusion Triptose (LIT) medium cultures. A simple, easy-to-execute and effective protocol based on ion exchange chromatography on Sepharose-DEAE resin for the purification of T. cruzi trypomastigotes is described. T. cruzi strains from the Discrete Typing Units (DTUs) I and II were used. The strains were harvested in LIT medium at a concentration of 1 × 107 epimastigotes/mL. We calculated the time of trypomastigotes increment (TTI). Based on the data obtained, Ion exchange chromatography was performed with DEAE-sepharose resin. To verify the purity and viability of the trypomastigotes, a culture was carried out in LIT medium with subsequent verification with giemsa staining. To evaluate if the technique affected the infectivity of trypomastigotes, in vitro assays were performed in Vero cells and in vivo in ICR-CD1 mice. The technique allowed the purification of metacyclic trypomastigotes of other stages of T. cruzi in a percentage of 100%, a greater recovery was observed in cultures of 12 days. There were differences regarding the recovery of metacyclic trypomastigotes for both DTUs, being DTU TcI the one that recovered a greater amount of these forms. The technique did not affect parasite infectivity in vitro or/and in vivo. © 2017 Elsevier B.V.

Chronic pain remains one of the most challenging of all neurologic diseases and, currently approved drugs have only a modest efficacy in several patient groups and numerous side effects. In recent years, a variety of structurally distinct α7 nicotinic acetylcholine receptor (nAChR) agonists have been developed and profiled for a variety of neurologic diseases. In particular, some of these agonists have been shown to have therapeutic significance in reducing inflammation and nociception, as well as in providing neuroprotection in animal models.1-8 The α7 nAChRs were targeted because they are expressed on both supraspinal and spinal pain transmission pathways.9-14 These receptors are also found on immune and nonimmune cytokine-producing cells, such as macrophages and keratinocytes.15 However, there are still a number of uncertainties in the development of α7 nAChR agonists for the treatment of pain, including receptor selectivity (namely cross-reactivity with 5-HT3 receptors, which have high homology with α7 nAChRs) and possible adverse effects.16,17 In addition, although α7 nAChR agonists have shown beneficial effects in chronic pain models in some studies, this effect was not consistently seen in other studies.18 An alternative therapeutic approach has been the development of positive allosteric modulators (PAMs), which can synergize and augment orthosteric-site–mediated signaling of endogenous neurotransmitters, including acetylcholine and choline, without, in most cases, directly activating or desensitizing the receptor. Several selective α7 nAChR PAMs have been reported and classified as type I and type II. Type I PAMs increase peak agonist-evoked responses, but have little or no effect on the decay rate of macroscopic currents or the equilibrium desensitization of α7 nAChRs, whereas type II PAMs both increase peak currents and slow down the apparent desensitization profile of the agonist response and/or recover nAChRs from the desensitized state.19,20 Both PAM types have been recently tested in vivo for their efficacy in animal models of inflammation and neuropathic pain; however, type II but not type I PAMs were shown to be effective in neuropathic pain models.21,22 Most studies with α7 nAChR PAMs in chronic pain models were conducted with PNU-120596,21-24 and more recently with the newly reported 2,4,20,50-tetrahydroxychalcone.25 Hence, additional studies are needed to fully explore and investigate the analgesic-like properties of α7 nAChR PAMs in animal models of chronic pain. Therefore, in this study, we sought to evaluate the anti-nociceptive and antiinflammatory effects of 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a novel and putative selective α7 nAChR type II PAM,26,27 using different mouse models of chronic pain. PAM-2 has recently been found to enhance α7 nAChR activity in vitro26 and to produce antidepressant activity in mice in vivo.27 The first aim of this study was to evaluate whether PAM-2 produces antiallodynic or antihyperalgesic activities in several mouse inflammatory and neuropathic pain models. We also assessed the effects of PAM-2 on aversion, an important affective component of pain.

The clastogenic effect of total dichloromethane, methanol and water extracts, four bioactive fractions and three individual constituents from Carthamus lanatus aerial parts were evaluated in mice by bone marrow chromosome aberration assay with mitomycin C as positive control. Significant differences in the percentage of aberrant mitosis of the extracts were observed. The dichloromethane extract exhibited a considerable clastogenic effect and the water extract a negligible one. Different types of chromosome aberrations and time-dependant effects for the active fractions and individual compounds were found.

Whole embryo culture (WEC) of postimplantation rodent embryos is widely used for the study of mammalian embryogenesis and developmental toxicity testing. Its major advantage is that it allows direct access to embryos for experimental manipulations and the monitoring of their consequences that would otherwise not be possible or technically difficult to perform in utero. However, a major drawback of mammalian WEC is that the culture media currently in use display batch variations and are undefined, as they contain serum or serum replacements of unknown composition. Moreover, these media possess cell-signalling activities important for embryogenesis. Therefore, reproducibility of mammalian postimplantation WEC results may be affected by batch variation and their interpretation is complicated because the experimenter is unsure whether the embryo response to experimental perturbations is solely due to their action, or modified as a result of influences from undefined substances/signaling activities present in culture media. To alleviate these problems we investigated whether N2B27, a serum-free and defined medium, can support the in vitro development of postimplantation mammalian embryos. We show that N2B27 allows pre-gastrulation mouse embryos isolated at embryonic day 5.5 to develop to advanced gastrulation, reaching the mid- and late primitive streak stages. This is the first demonstration that postimplantation mammalian embryos can develop in vitro in a defined medium in the absence of serum and provides a novel WEC system for studying developmental mechanisms and testing for developmental toxicity during the early postimplantation period. © 2015 Elsevier Inc. All rights reserved. 468 813 819

The alpha 7 nicotinic acetylcholine receptor (nAChR) is a promising drug target for a number of neurological disorders including chronic pain and inflammatory diseases. Since alpha 7 can function as a ligand-gated ion channel, drug development initially focused on ligands that were selective activators of the alpha 7 ion channel. However, the best alpha 7 drugs for chronic pain and inflammation indications may not be ion channel activators but rather "silent agonists", which bind to the receptor but preferentially induce non-conducting states that modulate signal transduction in non-neuronal cells. One such compound is NS6740. We show that NS6740 selectively induces prolonged desensitization of alpha 7 nAChRs. There are two forms of alpha 7 desensitization that can be distinguished by their sensitivity to the positive allosteric modulators (PAMs). At high concentrations, NS6740 preferentially induces PAM-insensitive desensitization, which over the course of several minutes reverts to the sensitive form. NS6740 was tested in several pain models after in vivo administration in the mouse. Although it had no effects in acute thermal pain, NS6740 induced significant dose- and time-dependent antinociceptive activity in formalin- and acetic acid-induced nociceptive behaviors as well as in the chronic constrictive nerve injury (CCI) model for neuropathic pain. The antinociceptive activity of NS6740 in these models was alpha 7-dependent. In addition, NS6740 administration reversed pain-induced aversion, an important affective component of pain. The time and concentration dependence of the effects were consistent with NS6740 induction of PAM-insensitive non-conducting states, suggesting that signal transduction required for analgesia is accomplished by alpha 7 receptors in that conformation. VCU Massey Cancer Center (A-35337) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (DA032246) United States Department of Health & Human Services National Institutes of Health (NIH) - USA (GM57481) United States Department of Health & Human Services National Institutes of Health (NIH) - USA (DA027113) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of General Medical Sciences (NIGMS) (R01GM057481) 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) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (R03DA027113)

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)