Diurnal Variation of the Peripheral Cholinergic Antiinflammatory Function in Mice.

AIMS: Cholinergic antiinflammatory (CAI) pathway functions importantly in inflammation via α7 nicotinic acetylcholine receptors (α7nAChR). The present work tested circadian rhythm in peripheral CAI activity and validities of CAI activity and glucocorticoids in chronotherapy for lipopolysaccharide (LPS)-induced shock. METHODS: Vesicular acetylcholine transporter (VAChT) expressed in liver and kidney was examined every 3 h in C57BL/6 mice. Proinflammatory cytokines in serum and survival time in shock were monitored after LPS injection every 3 h. Mifepristone, antagonist of glucocorticoid receptors, and methyllycaconitine (MLA), antagonist of α7nAChR, were administrated before LPS to block antiinflammatory function of endogenous glucocorticoids and acetylcholine. RESULTS: Both levels of tumor necrosis factor α, interleukin 1ß, and interleukin 6 and mortality exhibited diurnal variations with prominent peaks when LPS was given at 15:00, and the minimum mortality occurred at 00:00. Expression of VAChT increased during resting period. MLA increased serum proinflammatory cytokines slightly, but not affected survival rate. Both differences in cytokines and in survival times between LPS injection at 15:00 and 00:00 were eliminated by mifepristone, but not by MLA. CONCLUSION: Peripheral CAI pathway exerts more powerful antiinflammatory effect during resting period. Glucocorticoids appear to be efficient in chronotherapy for septic shock.

Differential antagonism and tolerance/cross-tolerance among nicotinic acetylcholine receptor agonists: scheduled-controlled responding and hypothermia in C57BL/6J mice.

The tobacco-dependence pharmacotherapies varenicline and cytisine act as partial α4ß2 nAChR agonists. However, the extent to which α4ß2 nicotinic acetylcholine receptors (nAChRs) mediate their in-vivo effects remains unclear. Nicotine, varenicline, cytisine, and epibatidine were studied in male C57BL/6J mice for their effects on rates of fixed ratio responding and rectal temperature alone and in combination with the nonselective nAChR antagonist mecamylamine and the α4ß2 nAChR antagonist dihydro-ß-erythroidine. The effects of nicotine, varenicline, cytisine, epibatidine, and cocaine were assessed before and during chronic nicotine treatment. The rate-decreasing and hypothermic effects of nicotine, varenicline, cytisine, and epibatidine were antagonized by mecamylamine (1 mg/kg), but only the effects of nicotine and epibatidine were antagonized by dihydro-ß-erythroidine (3.2 mg/kg). Chronic nicotine produced 4.7 and 5.1-fold rightward shifts in the nicotine dose-effect functions to decrease response rate and rectal temperature, respectively. Nicotine treatment decreased the potency of epibatidine to decrease response rate and rectal temperature 2.2 and 2.9-fold, respectively, and shifted the varenicline dose-effect functions 2.0 and 1.7-fold rightward, respectively. Cross-tolerance did not develop from nicotine to cytisine. These results suggest that the in-vivo pharmacology of tobacco cessation aids cannot be attributed to a single nAChR subtype; instead, multiple receptor subtypes differentially mediate their effects.

Potential therapeutic uses of mecamylamine and its stereoisomers.

Mecamylamine (3-methylaminoisocamphane hydrochloride) is a nicotinic parasympathetic ganglionic blocker, originally utilized as a therapeutic agent to treat hypertension. Mecamylamine administration produces several deleterious side effects at therapeutically relevant doses. As such, mecamylamine's use as an antihypertensive agent was phased out, except in severe hypertension. Mecamylamine easily traverses the blood-brain barrier to reach the central nervous system (CNS), where it acts as a nicotinic acetylcholine receptor (nAChR) antagonist, inhibiting all known nAChR subtypes. Since nAChRs play a major role in numerous physiological and pathological processes, it is not surprising that mecamylamine has been evaluated for its potential therapeutic effects in a wide variety of CNS disorders, including addiction. Importantly, mecamylamine produces its therapeutic effects on the CNS at doses 3-fold lower than those used to treat hypertension, which diminishes the probability of peripheral side effects. This review focuses on the pharmacological properties of mecamylamine, the differential effects of its stereoisomers, S(+)- and R(-)-mecamylamine, and the potential for effectiveness in treating CNS disorders, including nicotine and alcohol addiction, mood disorders, cognitive impairment and attention deficit hyperactivity disorder.

Nicotinic acetylcholine receptor-based blockade: applications of molecular targets for cancer therapy.

The nicotinic acetylcholine receptor (nAChR) was first characterized in 1970 as a membrane receptor of a neurotransmitter and an ion channel. nAChRs have been shown to be involved in smoking-induced cancer formation in multiple types of human cancer cells. In vitro and in vivo animal studies have shown that homopentameric nAChR inhibitors, such as methyllycaconitine and α-Bgtx, can attenuate nicotine-induced proliferative, angiogenic, and metastatic effects in lung, colon, and bladder cancer cells. Recent publications have shown that α9-nAChR is important for breast cancer formation, and in many in vivo studies, α9-nAChR-specific antagonists (e.g., α-ImI, α-ImI, Vc1.1, RgIA, and It14a) produced an analgesic effect. Vc1.1 functions in a variety of animal pain models and currently has entered phase II clinical trials. For cancer therapy, natural compounds such as garcinol and EGCG have been found to block nicotine- and estrogen-induced breast cancer cell proliferation through inhibition of the α9-nAChR signaling pathway. A detailed investigation of the carcinogenic effects of nAChRs and their specific antagonists would enhance our understanding of their value as targets for clinical translation.

Discovery of non-peptide, small molecule antagonists of α9α10 nicotinic acetylcholine receptors as novel analgesics for the treatment of neuropathic and tonic inflammatory pain.

A series of azaaromatic quaternary ammonium analogs has been discovered as potent and selective α9α10 nicotinic acetylcholine receptor (nAChR) antagonists. The preliminary structure-activity relationships of these analogs suggest that increased rigidity in the linker units results in higher potency in inhibition of α9α10 nAChRs and greater selectivity over α7 nAChRs. These analogs represent a new class of analgesic for the treatment of neuropathic and tonic inflammatory pain.

Investigational medications for treatment of patients with Alzheimer disease.

Development of effective treatments for patients with Alzheimer disease has been challenging. Currently approved treatments include acetylcholinesterase inhibitors and the N-methyl-D-aspartate receptor antagonist memantine hydrochloride. To investigate treatments in development for patients with Alzheimer disease, the author conducted a review of the literature. New approaches for treatment or prevention focus on several general areas, including cholinergic receptor agonists, drugs to decrease ß-amyloid and tau levels, antiinflammatory agents, drugs to increase nitric oxide and cyclic guanosine monophosphate levels, and substances to reduce cell death or promote cellular regeneration. The author focuses on medications currently in clinical trials. Cholinergic agents include orthostatic and allosteric muscarinic M1 agonists and nicotinic receptor agonists. Investigational agents that target ß-amyloid include vaccines, antibodies, and inhibitors of ß-amyloid production. Anti-inflammatory agents, including nonsteroidal anti-inflammatory drugs, the natural product curcumin, and the tumor necrosis factor α inhibitor etanercept, have also been studied. Some drugs currently approved for other uses may also show promise for treatment of patients with Alzheimer disease. Results of clinical trials with many of these investigational drugs have been disappointing, perhaps because of their use with patients in advanced stages of Alzheimer disease. Effective treatment may need to begin earlier-before neurodegeneration becomes severe enough for symptoms to appear.

Potentiated startle as a measure of the negative affective consequences of repeated exposure to nicotine in rats.

RATIONALE: Elevated acoustic startle amplitude has been used to measure anxiety-like effects of drug withdrawal in humans and animals. Withdrawal from a single opiate administration has been shown to produce robust elevations in startle amplitude ("withdrawal-potentiated startle") that escalate in severity with repeated exposure. Although anxiety is a clinical symptom of nicotine dependence, it is currently unknown whether anxiety-like behavior is elicited during the early stages of nicotine dependence in rodents. OBJECTIVE: The objective of this study is to examine whether, as is the case with opiates, single or repeated exposure to nicotine can produce withdrawal-potentiated startle. METHODS: Rats received daily nicotine injections for 14 days, and startle amplitude was tested during spontaneous withdrawal on injection days 1, 7, and 14. RESULTS: Elevated startle responding was observed during nicotine withdrawal on days 7 and 14 but not on day 1, was greater at higher nicotine doses, and was reduced by a nicotine replacement injection given during an additional test session on day 15. Additional experiments demonstrated that nicotine withdrawal-potentiated startle was reduced by the alpha(2)-adrenergic agonist clonidine and that precipitated withdrawal-potentiated startle could not be induced by injection of the nicotinic acetylcholine receptor antagonist mecamylamine. CONCLUSIONS: These results suggest that nicotine withdrawal escalates in severity across days, similar to the previously reported escalation of opiate withdrawal-potentiated startle. Potentiated startle may be a reliable measure of withdrawal from different classes of abused drugs and may be useful in the study of the early stages of drug dependence.

Antinociceptive activity of coniine in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Hemlock was used as an analgesic in certain ethnopharmacological traditions and there has been no record about the antinociceptive effect of coniine which is the major alkaloid compound of Hemlock. AIM OF THIS STUDY: The present study was undertaken to evaluate the possible antinociceptive activity of coniine. MATERIAL AND METHODS: Antinociceptive activity of coniine was tested dose in Hotplate test (thermal pain model) and in Writhing test (chemical pain model) in different nociception models. RESULTS: Coniine caused a prolongation in reaction time in Hotplate test at 20mg/kg dose. In addition, it was observed that coniine decreased the number of writhes in Writhing test. Both data indicated an antinociceptive effect of coniine. A rotarod test was also conducted in order to clarify, whether this activity was related with a loss of locomotion or with an analgesic activity. None of the chemical agents at those doses used in experiments caused a loss of locomotor activity. It was also shown that antinociceptive effect of morphine was potentialized by coniine which was inhibited by nicotinic receptor blocker mecamylamine (1mg/kg). CONCLUSION: Coniine has antinociceptive effect via the nicotinic receptors. A pharmacological assessment about the painless death of Socrates due to Hemlock (coniine) toxicity has also been presented by using this data.

Targeting alpha7-nicotinic receptor for the treatment of pleural mesothelioma.

Human malignant pleural mesothelioma (MPM) is a dreadful disease and there is still no standard therapy available for a consistent therapeutic approach. This research is aimed at the evaluation of the potential therapeutic effect of a specific nicotinic receptor (nAChR) antagonist, namely alpha-Cobratoxin (alpha-CbT). Its effectiveness was tested in mesothelioma cell lines and in primary mesothelioma cells in vitro, as well as in vivo, in orthotopically xenotransplanted NOD/SCID mice. Cells showed alpha7-nAChR expression and their growth was significantly inhibited by alpha-CbT. Severe induction of apoptosis was observed after exposure to alpha-CbT [IC(80-90)]. Apoptosis was characterised by: change in mitochondrial potential, caspase-3 cleavage, down-regulation of mRNA and protein for survivin, XIAP, IAP1, IAP2 and Bcl-XL, inhibition by caspase-3 inhibitor. In vivo, the alpha-CbT acute LD(50) was 0.15 mg/kg. The LD(100) [0.24 mg/kg] induced fatal respiratory failure and massive kidney necrosis. Phase II experiments with 0.12 ng/kg alpha-CbT (1/1000 of LD(10)) were done in 53 xenotransplanted mice, inhibiting tumour development as confirmed by chest X-ray examinations, autopsy and microscopical findings. The growth of human proliferating T lymphocytes and of mesothelial cells in primary culture was not affected by alpha-CbT. Non-immunogenic derivatives of the alpha-CbT molecule need to be developed for possible human use.

Nicotinic alpha 7 receptors as a new target for treatment of cannabis abuse.

Increasing use of cannabis makes the search for medications to reduce cannabis abuse extremely important. Here, we show that homomeric alpha7 nicotinic receptors are novel molecular entities that could be targeted in the development of new drugs for the treatment of cannabis dependence. In rats, systemic administration of the selective alpha7 nicotinic acetylcholine receptor antagonist methyllycaconitine (MLA), but not the selective heteromeric non-alpha7 nicotinic acetylcholine receptor antagonist dihydrobetaerythroidine, (1) antagonized the discriminative effects of delta-9-tetrahydrocannabinol (THC), the main active ingredient in cannabis, (2) reduced intravenous self-administration of the synthetic cannabinoid CB1 receptor agonist WIN55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone, mesylate salt], and (3) decreased THC-induced dopamine elevations in the shell of the nucleus accumbens. Altogether, our results indicate that blockade of alpha7 nicotinic receptors reverses abuse-related behavioral and neurochemical effects of cannabinoids. Importantly, MLA reversed the effects of cannabinoids at doses that did not produce depressant or toxic effects, further pointing to alpha7 nicotinic antagonists as potentially useful agents in the treatment of cannabis abuse in humans.

5alpha-cardenolides from Kanahia laniflora inhibit ionotropic acetylcholine receptors.

5alpha-cardenolides isolated from Kanahia laniflora are inhibitors of muscle-type nicotinic acetylcholine receptors expressed in TE671 cells with IC (50) values in the range of 27 - 60 microM, as determined by whole-cell patch-clamp electrophysiological experiments.

Mecamylamine (Targacept).

Targacept Inc, under license from the University of South Florida, is developing a low-dose, liquid gel capsule formulation of the nicotinic acetylcholine antagonist mecamylamine, as a potential treatment for various neuropsychiatric disorders.

Delta9-tetrahydrocannabinol decreases somatic and motivational manifestations of nicotine withdrawal in mice.

The possible interactions between Delta9-tetrahydrocannabinol (Delta9-THC) and nicotine remain unclear in spite of the current association of cannabis and tobacco in humans. The aim of the present study was to explore the interactions between these two drugs of abuse by evaluating the consequences of Delta9-THC administration on the somatic manifestations and the aversive motivational state associated with nicotine withdrawal in mice. Acute Delta9-THC administration significantly decreased the incidence of several nicotine withdrawal signs precipitated by mecamylamine or naloxone, such as wet-dog-shakes, paw tremor and scratches. In both experimental conditions, the global withdrawal score was also significantly attenuated by acute Delta9-THC administration. This effect of Delta9-THC was not due to possible adaptive changes induced by chronic nicotine on CB1 cannabinoid receptors, as the density and functional activity of these receptors were not modified by chronic nicotine administration in the different brain structures investigated. We also evaluated the consequences of Delta9-THC administration on c-Fos expression in several brain structures after chronic nicotine administration and withdrawal. c-Fos was decreased in the caudate putamen and the dentate gyrus after mecamylamine precipitated nicotine withdrawal. However, acute Delta9-THC administration did not modify c-Fos expression under these experimental conditions. Finally, Delta9-THC also reversed conditioned place aversion associated to naloxone precipitated nicotine withdrawal. Taken together, these results indicate that Delta9-THC administration attenuated somatic signs of nicotine withdrawal and this effect was not associated with compensatory changes on CB1 cannabinoid receptors during chronic nicotine administration. In addition, Delta9-THC also ameliorated the aversive motivational consequences of nicotine withdrawal.

Prenylated indole alkaloids from Flustra foliacea with subtype specific binding on NAChRs.

Several brominated indole alkaloids and a diterpene (1-7) were isolated from the dichloromethane extract of the North Sea Bryozoan Flustra foliacea. Alkaloid 4 is a new natural product, whose structure was elucidated by interpretation of spectroscopic data (NMR, mass, UV, and IR). All compounds were tested for their in vitro affinity towards the alpha4beta2* and alpha7* subtype of the neuronal nicotinic acetylcholine receptor (nAChR) using radioligand binding assays. Deformylflustrabromine (3) and deformylflustrabromine B (4) were shown to have affinities in the lower micromolar range for nAChRs, differing in their subtype preference.

Advances in pharmacotherapy for tobacco dependence.

The discovery that bupropion is an effective treatment for tobacco dependence has triggered a rapid increase in development of potential new non-nicotine pharmacotherapies, including bromocriptine, glucose, GTS-21, reboxetine, rimonabant, selegeline and varenicline. Successful new products will need to have excellent side-effect profiles in addition to proven efficacy. New faster delivery nicotine replacement products have the promise of addressing a broader list of indications, including treatment of nicotine withdrawal during temporary abstinence and long-term nicotine maintenance. Nicotine vaccines will need to demonstrate efficacy and also improve certain consumer acceptability characteristics (e.g., frequency of injections required) before they can become widely used and successful therapies. The best hope of improved treatment comes from combining existing and new pharmacotherapies with effective behavioural therapy.

Peripheral and central sites of action for A-85380 in the spinal nerve ligation model of neuropathic pain.

Neuronal nicotinic receptor (NNR) agonists such as ABT 594 have been shown to be effective in a wide range of preclinical models of acute and neuropathic pain. The present study, using the NNR agonist A-85380, sought to determine if NNR agonists are acting via similar or differing mechanisms to induce anti-nociception and anti-allodynia. A systemic administration of the quaternary NNR antagonist chlorisondamine (0.4 micromol/kg, intraperitoneal (i.p.)) did not alter A-85380-induced (0.75 micromol/kg, i.p.) anti-nociception in the rat paw withdrawal model of acute thermal pain. In contrast, previous studies have demonstrated that blockade of central NNRs by prior administration of chlorisondamine (10 microg i.c.v.) prevents A-85380 induced anti-nociception indicating a predominantly central site of action of NNR agonists in relieving acute pain. In the rat spinal nerve ligation model of neuropathic pain, A-85380 induced a dose-dependent anti-allodynia (0.5-1.0 micromol/kg) that was blocked by pretreatment with mecamylamine (1 micromol/kg). Interestingly, unlike acute pain, both systemic and central administration of chlorisondamine blocked A-85380-induced anti-allodynia, an effect that was determined not to be due to a non-specific effect of chlorisondamine or to chlorisondamine crossing the blood-brain barrier. The peripheral site of action was shown not to be the primary receptive field, since A-85380 had equally potent anti-allodynic effects when it was injected into either the affected or unaffected paw. In contrast, infusion of A-85380 directly onto the L5 dorsal root ganglion on the affected side resulted in a dose-dependent and marked anti-allodynia (10-20 microg) at doses that had no effect when injected systemically. This effect was blocked by pretreatment with chlorisondamine. Together these data further support the idea that different mechanisms underlie different pain states and suggest that the effects of NNR agonists in neuropathic pain may be due in part to peripheral actions of the compounds.

New directions in pain management.

Pain medicine is one of the most rapidly developing medical specialties of today. While there are many modalities that can be used in managing the patient in pain, drug treatment remains, for the most part, the cornerstone of treatment. Opioids retain their position as the foundation of most analgesic strategies, although they tend to be used nowadays in combination with adjuvant analgesics such as paracetamol and nonsteroidal antiinflammatory drugs. The range of available opioids has also been expanded with drugs such as hydromorphone and oxycodone, originally developed almost a century ago. This expanded choice has resulted in the concept of opioid rotation in chronic pain states, an approach that is aimed at maintaining pain control while minimizing adverse effects. Nonsteroidal antiinflammatory drugs continue to play an important role, especially as adjuvants, and the development of drugs such as celecoxib and refecoxib, highly specific for the inhibition of cyclooxygenase 2 pathway has been a further advance. The treatment of neuropathic pain continues to be a challenge to the clinician. While this has traditionally been treated with drugs from the anticonvulsant, antiarrhythmic and anti-depressant groups, results from these treatments have often been less than satisfactory. This has led to the development of completely new drug classes that modulate neuronal transmission in pain pathways, some of which are derived from exotic animal sources, such as the conotoxins from the marine snail family and epibatidine from a species of frog. The role of cannabinoids remains controversial.

Ethnopharmacology in drug discovery: an analysis of its role and potential contribution.

In this paper we discuss some examples of ethnopharmacological research as it has been conducted during the last two centuries and look at the current role of this discipline in drug discovery (especially with respect to the American and European markets) and the further development of these phytotherapeutical resources for local use in the countries of origin (ethnopharmacology). Examples from 19th century research on curare (Humboldt and Bernard), from the 20th century on hallucinogenic mushrooms (Wasson), on Mexican Indian indigenous plants (our own research) and of current industry based research are used to illustrate the development of this discipline and to highlight the challenges for the future.

[Assessment of anti-tremorogenic drugs--nicotine-induced tail-tremor model].

The repeated administration of nicotine at small doses, which do not produce whole body tremor or convulsion, causes tremor only in the tail (tail-tremor) of rats. The tremor is accompanied by locomotor hyperactivity without rigidity and immobility of the whole body, suggesting that the nicotine-induced tail-tremor model is useful for studying the mechanism underlying tremor associated with movement. The tail-tremor induced by nicotine was suppressed by mecamylamine, a nicotinic antagonist, but not by atropine or scopolamine, muscalinic antagonists. Moreover, the tail-tremor was suppressed by the beta-blockers propranolol and pindolol, as well as the benzodiazepines diazepam and clonazepam. Tremor at rest is observed only in Parkinson's disease, which is improved with anti-muscalinic drugs. Essential tremor is one of the typical tremors connected with movement (postural and kinetic tremor) and is improved with beta-blocker. These findings and results suggest that nicotine-induced tail-tremor is useful for the study of essential tremor in animal models.