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Opioid use disorder reflects a major public health crisis of morbidity and mortality in which opioid withdrawal often contributes to continued use. However, current medications that treat opioid withdrawal symptoms are limited by their abuse liability or lack of efficacy. Although cannabinoid 1 (CB 1 ) receptor agonists, including Δ 9 -tetrahydrocannabinol, ameliorate opioid withdrawal in both clinical and preclinical studies of opioid dependence, this strategy elicits cannabimimetic side effects as well as tolerance and dependence after repeated administration. Alternatively, CB 1 receptor positive allosteric modulators (PAMs) enhance CB 1 receptor signaling and show efficacy in rodent models of pain and cannabinoid dependence but lack cannabimimetic side effects. We hypothesize that the CB 1 receptor PAM ZCZ011 attenuates naloxone-precipitated withdrawal signs in opioid-dependent mice. Accordingly, male and female mice given an escalating dosing regimen of oxycodone, a widely prescribed opioid, and challenged with naloxone displayed withdrawal signs that included diarrhea, weight loss, jumping, paw flutters, and head shakes. ZCZ011 fully attenuated naloxone-precipitated withdrawal-induced diarrhea and weight loss and reduced paw flutters by approximately half, but its effects on head shakes were unreliable, and it did not affect jumping behavior. The antidiarrheal and anti-weight loss effects of ZCZ0111 were reversed by a CB 1 not a cannabinoid receptor type 2 receptor antagonist and were absent in CB 1 (-/-) mice, suggesting a necessary role of CB 1 receptors. Collectively, these results indicate that ZCZ011 completely blocked naloxone-precipitated diarrhea and weight loss in oxycodone-dependent mice and suggest that CB 1 receptor PAMs may offer a novel strategy to treat opioid dependence. SIGNIFICANCE STATEMENT: Opioid use disorder represents a serious public health crisis in which current medications used to treat withdrawal symptoms are limited by abuse liability and side effects. The CB 1 receptor positive allosteric modulator (PAM) ZCZ011, which lacks overt cannabimimetic behavioral effects, ameliorated naloxone-precipitated withdrawal signs through a CB 1 receptor mechanism of action in a mouse model of oxycodone dependence. These results suggest that CB 1 receptor PAMs may represent a viable strategy to treat opioid withdrawal., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Chronic pain and inflammatory diseases can be regulated by complex mechanisms involving α 7 nicotinic acetylcholine receptors (nAChRs), making this subtype a promising drug target for anti-inflammatory therapies. Recent evidence suggests that suchtreatment of inflammatory pain may rely on metabotropic-like rather than ionotropic activation of the α 7 receptor subtype in non-neuronal cells. We previously identified para-trifluoromethyl ( p -CF 3 ) N , N -diethyl- N '-phenylpiperazinium (diEPP) iodide to be among the compounds classified as silent agonists, which are very weak α 7 partial agonists that are able to induce positive allosteric modulator (PAM)-sensitive desensitization. Such drugs have been shown to selectively promote α 7 ionotropic-independent functions. Therefore, we here further investigated the electrophysiological profile of p -CF 3 diEPP and its in vivo antinociceptive activity using Xenopus oocytes expressing α 7, α 4 β 2, or α 3 β 4 nAChRs. The evoked currents confirmed p -CF 3 diEPP to be α 7-selective with a maximal agonism 5% that of acetylcholine (ACh). Coapplication of p -CF 3 diEPP with the type II PAM 4-naphthalene-1-yl-3a,4,5,9b-tetrahydro-3- H -cyclopenta[c]quinoline-8-sulfonic acid amide (TQS) produced desensitization that could be converted to PAM-potentiated currents, which at a negative holding potential were up to 13-fold greater than ACh controls. Voltage-dependence experiments indicated that channel block may limit both control ACh and TQS-potentiated responses. Although no p -CF 3 diEPP agonist activity was detected for the heteromeric nAChRs, it was a noncompetitive antagonist of these receptors. The compound displayed remarkable antihyperalgesic and antiedema effects in in vivo assays. The antinociceptive activity was dose and time dependent. The anti-inflammatory components were sensitive to the α 7-selective antagonist methyllycaconitine, which supports the idea that these effects are mediated by the α 7 nAChR., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Although paclitaxel effectively treats various cancers, its debilitating peripheral neuropathic pain side effects often persist long after treatment has ended. Therefore, a compelling need exists for the identification of novel pharmacologic strategies to mitigate this condition. As inhibitors of monoacylglycerol lipase (MAGL), the primary hydrolytic enzyme of the endogenous cannabinoid, 2-arachidonyolglycerol, produces antinociceptive effects in numerous rodent models of pain, we investigated whether inhibitors of this enzyme (i.e., JZL184 and MJN110) would reverse paclitaxel-induced mechanical allodynia in mice. These drugs dose dependently reversed allodynia with respective ED 50 values (95% confidence limit) of 8.4 (5.2-13.6) and 1.8 (1.0-3.3) mg/kg. Complementary genetic and pharmacologic approaches revealed that the antiallodynic effects of each drug require both cannabinoid receptors, CB 1 and CB 2 MJN110 reduced paclitaxel-mediated increased expression of monocyte chemoattractant protein-1 (MCP-1, CCL2) and phospho-p38 MAPK in dorsal root ganglia as well as MCP-1 in spinal dorsal horn. Whereas the antinociceptive effects of high dose JZL184 (40 mg/kg) underwent tolerance following 6 days of repeated dosing, repeated administration of a threshold dose (i.e., 4 mg/kg) completely reversed paclitaxel-induced allodynia. In addition, we found that the administration of MJN110 to control mice lacked intrinsic rewarding effects in the conditioned place preference (CPP) paradigm. However, it produced a CPP in paclitaxel-treated animals, suggesting a reduced paclitaxel-induced aversive state. Importantly, JZL184 did not alter the antiproliferative and apoptotic effects of paclitaxel in A549 and H460 non-small cell lung cancer cells. Taken together, these data indicate that MAGL inhibitors reverse paclitaxel-induced neuropathic pain without interfering with chemotherapeutic efficacy., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Chemotherapy-induced peripheral neuropathy (CIPN), a consequence of peripheral nerve fiber dysfunction or degeneration, continues to be a dose-limiting and debilitating side effect during and/or after cancer chemotherapy. Paclitaxel, a taxane commonly used to treat breast, lung, and ovarian cancers, causes CIPN in 59-78% of cancer patients. Novel interventions are needed due to the current lack of effective CIPN treatments. Our studies were designed to investigate whether nicotine can prevent and/or reverse paclitaxel-induced peripheral neuropathy in a mouse model of CIPN, while ensuring that nicotine will not stimulate lung tumor cell proliferation or interfere with the antitumor properties of paclitaxel. Male C57BL/6J mice received paclitaxel every other day for a total of four injections (8 mg/kg, i.p.). Acute (0.3-0.9 mg/kg, i.p.) and chronic (24 mg/kg per day, s.c.) administration of nicotine respectively reversed and prevented paclitaxel-induced mechanical allodynia. Blockade of the antinociceptive effect of nicotine with mecamylamine and methyllycaconitine suggests that the reversal of paclitaxel-induced mechanical allodynia is primarily mediated by the α 7 nicotinic acetylcholine receptor subtype. Chronic nicotine treatment also prevented paclitaxel-induced intraepidermal nerve fiber loss. Notably, nicotine neither promoted proliferation of A549 and H460 non-small cell lung cancer cells nor interfered with paclitaxel-induced antitumor effects, including apoptosis. Most importantly, chronic nicotine administration did not enhance Lewis lung carcinoma tumor growth in C57BL/6J mice. These data suggest that the nicotinic acetylcholine receptor-mediated pathways may be promising drug targets for the prevention and treatment of CIPN., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Chemotherapy-induced peripheral neuropathy (CIPN) represents a serious complication associated with antineoplastic drugs. Although there are no medications available that effectively prevent CIPN, many classes of drugs have been used to treat this condition, including anticonvulsants, serotonin and noradrenaline reuptake inhibitors, and opioids. However, these therapeutic options yielded inconclusive results in CIPN clinical trials and produced assorted side effects with their prolonged use. Thus, there is an urgent need to develop efficacious and safe treatments for CIPN. In this report, we tested whether the endogenous lipid palmitoylethanolamide (PEA) alone or in combination with the anticonvulsant gabapentin would reduce allodynia in a mouse paclitaxel model of CIPN. Gabapentin and PEA reversed paclitaxel-induced allodynia with respective ED 50 doses (95% confidence interval) of 67.4 (61.52-73.94) and 9.2 (8.39-10.16) mg/kg. Isobolographic analysis of these drugs in combination revealed synergistic antiallodynic effects. The PPAR-α antagonist receptor antagonist GW6471 [N-((2S)-2-(((1Z)-1-methyl-3-oxo-3-(4-(trifluoromethyl)phenyl)prop-1-enyl)amino)-3-(4-(2-(5-methyl-2-phenyl-1,3-oxazol-4-yl)ethoxy)phenyl)propyl)propanamide] completely blocked the antinociceptive effects of PEA. In addition, PEA administered via intraplantar injection into a paw, intrathecal injection, and intracerebroventricular injection reversed paclitaxel-induced allodynia, suggesting that it may act at multiple sites in the neuroaxis and periphery. Finally, repeated administration of PEA (30 mg/kg, 7 days) preserved the antiallodynic effects with no evidence of tolerance. These findings taken together suggest that PEA possesses potential to treat peripheral neuropathy in cancer patients undergoing chemotherapy., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Opioid-induced constipation is a major side effect that persists with long-term opioid use. Previous studies demonstrated that nicotine-induced contractions are enhanced after long-term morphine exposure in guinea pig ileum. In the present study, we examined whether the increased sensitivity to nicotine could be observed in single enteric neurons after long-term morphine exposure, determined the subunits in mouse enteric neurons, and examined the effect of nicotine in reversing opioid-induced constipation. Nicotine (0.03-1 mM) dose-dependently induced inward currents from a holding potential of -60 mV in isolated single enteric neurons from the mouse ileum. The amplitude of the currents, but not the potency to nicotine, was significantly increased in neurons receiving long-term (16-24 h) but not short-term (10 min) exposure to morphine. Quantitative mRNA analysis showed that nicotinic acetylcholine receptor (nAChR) subunit expression in the mouse ileum was α3 ≥ β2 > β4 > α5 > α4 > β3 > α6. Nicotine-induced currents were obtained in neurons from α7, β2, α5, and α6 knockout mice. The currents were, however, inhibited by mecamylamine (10 μM) and the α3β4 blocker α-conotoxin AuIB (3 μM), suggesting that nicotine-induced currents were mediated by the α3β4 subtype of nAChRs on enteric neurons. Conversely, NS3861, a partial agonist at α3β4 nAChR, enhanced fecal pellet expulsion in a dose-dependent manner in mice that received long-term, but not short-term, morphine treatment. Overall, our findings suggest that the efficacy of nAChR agonists on enteric neurons is enhanced after long-term morphine exposure, and activation of the α3β4 subtype of nAChR reverses chronic, but not acute, morphine-induced constipation., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Smoking is a common addiction and a leading cause of disease. Chronic nicotine exposure is known to activate nicotinic acetylcholine receptors (nAChRs) in immune cells. We demonstrate a novel role for α4 nAChRs in the effect of nicotine on T-cell proliferation and immunity. Using cell-based sorting and proteomic analysis we define an α4 nAChR expressing helper T-cell population (α4(+)CD3(+)CD4(+)) and show that this group of cells is responsive to sustained nicotine exposure. In the circulation, spleen, bone marrow, and thymus, we find that nicotine promotes an increase in CD3(+)CD4(+) cells via its activation of the α4 nAChR and regulation of G protein subunit o, G protein regulated-inducer of neurite outgrowth, and CDC42 signaling within T cells. In particular, nicotine is found to promote a helper T cell 2 adaptive immunologic response within T cells that is absent in α4(-/-) mice. We thus present a new mechanism of α4 nAChR signaling and immune regulation in T cells, possibly accounting for the effect of smoking on the immune system.

Studies showed that nicotine has a positive influence on symptoms of ulcerative colitis. In the present study, we explored the effect of nicotine treatment using different routes of administration in the dextran sodium sulfate (DSS) colitis mouse model. We also investigated the effects of cotinine, a major metabolite of nicotine, in the model. C57BL6 adult male mice were given DSS solution freely in the drinking water for seven consecutive days, and tap water was given thereafter. Disease severity, length of the colon, colon tissue histology, and inflammatory markers, including colonic myeloperoxidase activity and colonic tumor necrosis factor-α levels, were evaluated. The effect of nicotine and cotinine treatments via various different routes of administration were examined the DSS model. In addition, we measured the plasma levels of nicotine and cotinine in our treatment protocols. Administration of low, but not high, doses of oral nicotine in DSS-treated mice resulted in a significant decrease in disease severity, histologic damage scores, as well as colonic level of tumor necrosis factor-α. However, the anti-inflammatory effect of nicotine was not seen after chronic s.c. or minipump infusion of the drug. Differences in plasma levels of nicotine and cotinine do not seem to account for this lack of effect. Finally, oral cotinine alone failed to show a significant effect in the DSS model of colitis. These results highlight that dose and route of administration play a critical role in the protective effect of nicotine in the DSS mouse colitis model.

The α7 nicotinic acetylcholine receptor (nAChR) subtype is abundantly expressed in the central nervous system and in the periphery. Recent evidence suggests that α7 nAChR subtypes, which can be activated by an endogenous cholinergic tone, comprising acetylcholine and the α7 nAChR agonist choline, play an important role in subchronic pain and inflammation. This study's objective was to test whether α7 nAChR positive allosteric modulators (PAMs) produce antinociception in in vivo mouse models of acute and persistent pain. Testing type I [N-(5-chloro-2-hydroxyphenyl)-N'-[2-chloro-5-(trifluoromethyl)phenyl] (NS1738)] and type II [1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl) (PNU-120596)] α7 nAChR PAMs in acute and persistent pain, we found that, although neither reduced acute thermal pain, only PNU-120596 dose-dependently attenuated paw-licking behavior in the formalin test. The long-acting effect of PNU-120596 in this test was in discordance with its pharmacokinetic profile in mice, which suggests the involvement of postreceptor signaling mechanisms. Our results with selective mitogen-activated protein kinase kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene monoethanolate (U0126) argues for an important role of extracellular signal-regulated kinase-1/2 pathways activation in PNU-120596's antinociceptive effects. The α7 antagonist MLA, administered intrathecally, reversed PNU-120596's effects, confirming PNU-120596's action, in part, through central α7 nAChRs. Importantly, tolerance to PNU-120596 was not developed after subchronic treatment of the drug. Surprisingly, PNU-120596's antinociceptive effects were blocked by NS1738. Our results indicate that type II α7 nAChR PAM PNU-120596, but not type I α7 nAChR PAM NS1738, shows significant antinociception effects in persistent pain models in mice.

Nicotinic agonists display a wide-range profile of antinociceptive activity in acute, tonic, and chronic pain models. However, their effectiveness is limited by their unacceptable side effects. We investigated the antinociceptive effects of two new α4β2* nicotinic partial agonists, varenicline and sazetidine-A, in acute thermal and tonic pain mouse models. Both drugs failed to induce significant effects in the tail-flick and hot-plate tests after subcutaneous administration. However, they blocked nicotine's effects in these tests at very low doses. In contrast to acute pain tests, varenicline and sazetidine-A dose-dependently induced an analgesic effect in the mouse formalin test after systemic administration. Their antinociceptive effects were mediated, however, by different nicotinic acetylcholine receptor (nAChR) subtypes. Sazetidine-A effects were mediated by β2* nAChR subtypes, whereas varenicline actions were attributed to α3β4 nAChRs. Moreover, low inactive doses of varenicline blocked nicotine's actions in phase II of the formalin test. Overall, our results suggest that the antagonistic actions of varenicline at low doses are mediated by β2*-nAChRs and at higher doses as an agonist by α3β4*-nAChRs. In contrast, both actions of sazetidine-A are mediated by β2*-nAChR subtypes. These results suggest that nicotinic partial agonists possess analgesic effects in a rodent tonic pain model and may provide a potential treatment for the treatment of chronic pain disorders.

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.

A growing body of evidence suggests that the endogenous cannabinoid system modulates the addictive properties of nicotine, the main component of tobacco that produces rewarding effects. In our study, complementary transgenic and pharmacological approaches were used to test the hypothesis that the endocannabinoid system modulates nicotine reward and dependence. An acute injection of nicotine elicited normal analgesic and hypothermic effects in cannabinoid receptor (CB)(1) knockout (KO) mice and mice treated with the CB(1) antagonist rimonabant. However, disruption of CB(1) receptor signaling blocked nicotine reward, as assessed in the conditioned place preference (CPP) paradigm. In contrast, genetic deletion, or pharmacological inhibition of fatty acid amide hydrolase (FAAH), the enzyme responsible for catabolism of the endocannabinoid anandamide, enhanced the expression of nicotine CPP. Although the expression of spontaneous nicotine withdrawal (14 days, 24 mg/kg/day nicotine) was unaffected in CB(1) KO mice, acute administration of rimonabant (3 mg/kg) ameliorated somatic withdrawal signs in wild-type mice. Increasing endogenous levels of anandamide through genetic or pharmacological approaches exacerbated the physical somatic signs of spontaneous nicotine withdrawal in a milder withdrawal model (7 days, 24 mg/kg/day nicotine). Moreover, FAAH-compromised mice displayed increased conditioned place aversion in a mecamylamine-precipitated model of nicotine withdrawal. These findings indicate that endocannabinoids play a role in the rewarding properties of nicotine as well as nicotine dependence liability. Specifically, increasing endogenous cannabinoid levels magnifies, although disrupting CB(1) receptor signaling, attenuates nicotine reward and withdrawal. Taken together, these results support the hypothesis that cannabinoid receptor antagonists may offer therapeutic advantages to treat tobacco dependence.

A patch-clamp technique in a whole-cell configuration was used to examine the functional activity of recently developed 2-fluoro-3-(substituted phenyl)deschloroepibatidine analogs on two major subtypes of neuronal nicotinic acetylcholine receptors (nAChRs), alpha4beta2 and alpha3beta4, that predominate in the central and peripheral nervous systems, respectively. These epibatidine analogs have been shown previously to possess high binding affinity to alpha4beta2 but not to alpha7 nAChRs and to inhibit nicotine-induced analgesia in behavioral pain tests. The 2-fluoro-3-(4-nitro-phenyl)deschloroepibatidine (4-nitro-PFEB) exhibited the most pronounced antagonist activity among these analogs when tested electrophysiologically on alpha4beta2 nAChRs. It inhibited acetylcholine (ACh)-induced currents in a concentration-dependent manner with an IC(50) value of 0.1 microM and produced complete inhibition at approximately 1 microM concentration. 4-Nitro-PFEB at 0.1 microM concentration produced a 4-fold rightward shift in the ACh concentration-response curve without altering maximum ACh-induced response. This inhibitory effect of 4-nitro-PFEB was voltage- and use-independent and was partially reversible at its 1 microM concentration. The rise and decay kinetics of ACh-induced currents was not altered in the presence of 4-nitro-PFEB. In contrast to alpha4beta2 nAChRs, this compound did not affect alpha3beta4 nAChR-mediated currents at < or =1 microM (IC(50) approximately 63.9 microM). Overall, these functional data agree with previous binding and behavioral findings and suggest collectively that 4-nitro-PFEB is the most effective and selective antagonist of alpha4beta2 versus alpha3beta4 and alpha7 nAChRs among the tested analogs, acting on alpha4beta2 nAChR through a competitive mechanism with a potency 17-fold higher than that of dihydro-beta-erythroidine.

Bupropion is an atypical antidepressant that also has usefulness as a smoking-cessation aid. Because hydroxybupropion, a major metabolite of bupropion, is believed to contribute to its antidepressant activity, this metabolite may also contribute to the smoking-cessation properties of bupropion. This study investigated the effects of hydrobupropion enantiomers on monoamine transporters and nicotinic acetylcholine receptor (nAChR) subtypes. Racemic bupropion and hydroxybupropion inhibit [(3)H]norepinephrine (NE) uptake with similar potency (IC(50) values of 1.9 and 1.7 microM, respectively), but most of the latter activity resides in the (2S,3S)-hydroxy isomer (IC(50) = 520 nM) rather than (2S,3R)-hydroxybupropion (IC(50) > 10,000 nM). Similar results were found with [(3)H]dopamine (DA) uptake. The effects of bupropion and enantiomers of hydroxybupropion on human nAChR subtypes indicate that the (2S,3S) isomer is more potent than the (2S,3R) isomer or racemic bupropion as an antagonist of alpha(4)beta(2) (functional IC(50) = 3.3 microM). In addition, (2S,3S)-hyroxybupropion and bupropion were considerably more potent than (2R, -3R)-hydroxybupropion in a mouse depression model (forced swimming test) and in antagonism of acute nicotine effects in mice. Together, our results suggest that clinical and behavioral effects of bupropion arise from actions at nAChR as well as DA and NE transporters. Furthermore, our data suggest that the (2S,3S)-hydroxybupropion isomer may be a better drug candidate for smoking cessation than bupropion because of its higher potency at the relevant targets.