Alkaloid-Based Isoxazolylureas: Synthesis and Effect in Combination with Anticancer Drugs on C6 Rat Glioma Model Cells.

Alkaloid-based urea derivatives were produced with high yield through the reaction of anabasine and cytisine with isoxazolylphenylcarbamates in boiling benzene. Their antitumor activity, in combination with the commonly used five anticancer drugs, namely cyclophosphane, fluorouracil, etoposide, cisplatin, ribomustine with different mechanisms of action, was investigated. Based on the quantum chemical calculations data and molecular docking, hypotheses have been put forward to explain their mutual influence when affecting C6 rat glioma model cells.

Improving Wastewater-Based Tobacco Use Estimates Using Anabasine.

In wastewater-based epidemiology (WBE), nicotine metabolites have been used as biomarkers for monitoring tobacco use. Recently, the minor tobacco alkaloids anabasine and anatabine have been suggested as more specific biomarkers for tobacco use since nicotine use can be from both tobacco and non-tobacco sources. This study aimed to provide an in-depth evaluation of the suitability of anabasine and anatabine as WBE biomarkers of tobacco and subsequently estimate their excretion factors for WBE applications. Pooled urine (n = 64) and wastewater samples (n = 277), collected between 2009 and 2019 in Queensland, Australia, were analyzed for nicotine and its metabolites (cotinine and hydroxycotinine), as well as anabasine and anatabine. Anabasine performed as the better biomarker, showing a similar per capita load in pooled urine (2.2 ± 0.3 µg/day/person) and wastewater samples (2.3 ± 0.3 µg/day/person), while the per capita load of anatabine in wastewater was 50% higher than its load in urine. It is estimated that 0.9 µg of anabasine was excreted per cigarette smoked. Triangulation of tobacco sales data and tobacco use estimated from either anabasine or cotinine showed that anabasine-based estimates were 5% higher than sales data, while cotinine-based estimates were between 2 and 28% higher. Our results provided concrete evidence to confirm the suitability of anabasine as a specific biomarker for monitoring tobacco use by WBE.

Nicotine Exerts a Stronger Immunosuppressive Effect Than Its Structural Analogs and Regulates Experimental Colitis in Rats.

Ulcerative colitis (UC) is an intractable disease that causes persistent colonic inflammation. Numerous studies have reported that smoking can afford clinical benefits in UC. This study aimed to elucidate whether nicotine, the main component in cigarettes, can exert pharmacological effects against experimental UC. To achieve this objective, we compared the effects of nicotine with those of structural nicotine analogs in a UC rodent model (Slc: Wistar rats, male, 9-week-old, and 220-250 g/rat). Nicotine, or a respective structural analog (nornicotine, cotinine, anabasine, myosmine, and anatabine), was administered intraperitoneally daily to rats (n = 6/group) exhibiting dextran sulfate sodium-induced experimental colitis. Examining the colon tissues of model rats, we compared disease severity, cytokine secretion, and α7 nicotine acetylcholine receptor (nAChR7) expression. We observed that nicotine administration induced weight loss at 2.35% in 10 days. Notably, the reduction in histological severity (score) of UC was more pronounced in rats treated with nicotine (score = 4.83, p = 0.042) than in untreated rats (score = 8.17). Nicotine administration increased nAChR7 expression 6.88-fold (p = 0.022) in inflammatory sites of the colon, mainly by suppressing the production of interleukin (IL)-1ß and IL-6. Moreover, the secretion of these cytokines was suppressed in lipopolysaccharide-stimulated rat macrophages (MΦ) treated with nicotine. In conclusion, nicotine better alleviates experimental UC than the examined structural analogs by activating nAChR7 expression and suppressing proinflammatory cytokines in MΦ.

Discovery of the Nicotinic Receptor Toxin Anabaseine in a Polystiliferan Nemertean.

Nemerteans (also called Nemertines) are a phylum of predominantly marine worms that use toxins to capture prey and to defend themselves against predators. Hoplonemerteans have a proboscis armed with one or more stylets used in prey capture and are taxonomically divided into Order Monostilifera, whose members possess a single large proboscis stylet, and Order Polystilifera, whose members have multiple small stylets. Many monostiliferans contain alkaloidal toxins, including anabaseine, that stimulate and then desensitize nicotinic acetylcholine receptors that are present in all animals. These compounds also interact with pyridyl chemoreceptors in crustaceans, reducing predation and larval settlement. Anabaseine has been a lead compound in the design of alpha7 nicotinic acetylcholine receptor agonists like GTS-21 (also called DMXBA) to treat disorders of cognition such as Alzheimer's disease and schizophrenia. These drug candidates also display anti-inflammatory activities of potential medical importance. Most polystiliferans live deep in open oceans and are relatively inaccessible. We fortunately obtained two live specimens of a large benthic polystiliferan, Paradrepanophorus crassus (Pc), from the coast of Spain. MS and NMR analyses of the Ehrlich's reagent derivative allowed identification of anabaseine. A spectrophotometric assay for anabaseine, also based on its reaction with Ehrlich's reagent, revealed high concentrations of anabaseine in the body and proboscis. Apparently, the biosynthetic mechanism for producing anabaseine was acquired early in the evolution of the Hoplonemertea, before the monostiliferan-polystiliferan divergence.

Synthesis and Biological Activity of N-acyl Anabasine and Cytisine Derivatives with Adamantane, Pyridine and 1,2-Azole Fragments.

A series of N-acyl derivatives of anabasine and cytisine were prepared, to discover novel, natural product-based medicinal agents. All synthesized compounds were tested for antimicrobial, antifungal, antiviral and analgesic activity. The most pronounced antibacterial activity was shown by the compounds with isoxazole fragments, while the adamantane derivatives showed the greatest antiviral effect. It was found that the majority of anabasine derivatives showed significant analgesic activity, reducing the pain response of animals to the irritating effect of acetic acid. The presence of a high level of antimicrobial and antiviral activity in newly synthesized compounds makes it possible to consider them promising for further study of their pharmacological properties.

Combined Computational and Experimental Studies of Anabasine Encapsulation by Beta-Cyclodextrin.

The encapsulation of the famous alkaloid, anabasine, with ß-CD was studied to obtain a more stable and bioavailable inclusion complex. Various in silico and experimental studies of the obtained ß-CD-anabasine complex are presented. Firstly, molecular docking studies were conducted against the α, ß, and γ cyclodextrins to explore which subclass is the best for encapsulation. The obtained results that pointed at ß-cyclodextrin were further confirmed by five MD simulations and MM-PBSA studies. Experimentally, the spectral properties of the anabasine ß-cyclodextrin complex were determined by FT-IR, 1H, and 13C-NMR spectroscopic methods. Additionally, the surface morphology of the anabasine ß-cyclodextrin was investigated using a scanning electron microscope. Furthermore, the outputs of the thermographic measurements utilizing a differential scanning calorimeter were displayed. The activation energy of the reaction of thermo-oxidative destruction of the clathrate complex was calculated, and the kinetic parameters of the thermal destruction processes were decided using the Freeman-Carroll, Sharpe-Wentworth, Achar, and Coates-Redfern methods. The kinetic parameters of the thermal decomposition of the anabasine ß-cyclodextrin were in agreement and verified the reliability of the obtained results. The obtained computational, spectral, morphological, and thermogravimetric results verified the successful formation of the anabasine ß-cyclodextrin complex.

Anabasine and Anatabine Exposure Attributable to Cigarette Smoking: National Health and Nutrition Examination Survey (NHANES) 2013-2014.

Anabasine and anatabine are minor alkaloids in tobacco products and are precursors for tobacco-specific nitrosamines (TSNAs). The levels of these two compounds have been used to differentiate tobacco product sources, monitor compliance with smoking cessation programs, and for biomonitoring in TSNA-related studies. The concentrations of urinary anabasine and anatabine were measured in a representative sample of U.S. adults who smoked cigarettes (N = 770) during the 2013−2014 National Health and Nutrition Examination Survey (NHANES) study cycle, which was the first cycle where urinary anabasine and anatabine data became available. Weighted geometric means (GM) and geometric least squares means (LSM) with 95% confidence intervals were calculated for urinary anabasine and anatabine categorized by tobacco-use status [cigarettes per day (CPD) and smoking frequency] and demographic characteristics. Smoking ≥20 CPD was associated with 3.6× higher anabasine GM and 4.8× higher anatabine GM compared with smoking <10 CPD. Compared with non-daily smoking, daily smoking was associated with higher GMs for urinary anabasine (1.41 ng/mL vs. 6.28 ng/mL) and anatabine (1.62 ng/mL vs. 9.24 ng/mL). Urinary anabasine and anatabine concentrations exceeded the 2 ng/mL cut point in 86% and 91% of urine samples from people who smoke (PWS) daily, respectively; in comparison, 100% of them had serum cotinine concentrations greater than the established 10 ng/mL cut point. We compared these minor tobacco alkaloid levels to those of serum cotinine to assess their suitability as indicators of recent tobacco use at established cut points and found that their optimal cut point values would be lower than the established values. This is the first time that anabasine and anatabine are reported for urine collected from a U.S. population-representative sample of NHANES study participants, providing a snapshot of exposure levels for adults who smoked during 2013−2014. The results of this study serve as an initial reference point for future analysis of NHANES cycles, where changes in the national level of urinary anabasine and anatabine can be monitored among people who smoke to show the effect of changes in tobacco policy.

Heck Reaction of N-Heteroaryl Halides for the Concise Synthesis of Chiral α-Heteroaryl-substituted Heterocycles.

The Heck reaction between N-heteroaryl halides and heterocyclic alkenes provides a convenient approach to biologically relevant α-heteroaryl functionalized heterocycles, yet reactions of this type have been challenging due to strong N-heteroaryl coordination to palladium metal, which causes catalyst poisoning. In this report, an efficient palladium-catalyzed Heck reaction between N-heteroaryl halides and heterocyclic olefins is established, leading to a variety of α-heteroaryl substituted heterocycles. The method features an unprecedented broad substrate scope and excellent functional group compatibility. The employment of a sterically bulky P, P=O ligand containing an anthryl moiety is crucial for this transformation due to the coordinative unsaturation facilitated by its steric bulkiness. The asymmetric variant of the Heck reaction is achieved with (S)-DTBM-SEGPHOS via a cationic palladium pathway, which has enabled an efficient asymmetric synthesis of (S)-nicotine and its analogues.

UPLC-HRESI-MS and GC-MS analysis of the leaves of Nicotiana glauca.

The alkaloid-rich fraction obtained by fractionation of the crude methanolic extract of the leaves of wild tobacco tree Nicotiana glauca Graham (Solanaceae) was analyzed using UPLC-MS and GC-MS. Anabasine, a piperidine alkaloid, was identified as the major constituent with approximately 60 % (m/m) of the alkaloid-rich fraction. In addition to anabasine, six secondary metabolites were identified using high-resolution UPLC-MS. Anabasine was quantified in the leaves to be 1 mg g-1 dry plant material. The GC-MS analysis revealed five compounds with anabasine as the major component, while nicotine was not detected. Moreover, GC-MS was used for the analysis of the volatile oil that was obtained by hydro-distillation from the leaves of N. glauca. The volatile plant oil was found to be rich in oxygenated sesquiterpenes (e.g., ß-bisabolol) and carboxylic acids and esters (e.g., ethyl linoleate and hexadecanoic acid), whereas anabasine was not detected.

DNA methylation differentiates smoking from vaping and non-combustible tobacco use.

Increasing use of non-combusted forms of nicotine such as e-cigarettes poses important public health questions regarding their specific risks relative to combusted tobacco products such as cigarettes. To fully delineate these risks, improved biomarkers that can distinguish between these forms of nicotine use are needed. Prior work has suggested that methylation status at cg05575921 may serve as a specific biomarker of combusted tobacco smoke exposure. We hypothesized combining this epigenetic biomarker with conventional metabolite assays could classify the type of nicotine product consumption. Therefore, we determined DNA methylation and serum cotinine values in samples from 112 smokers, 35 e-cigarette users, 19 smokeless tobacco users, and 269 controls, and performed mass spectroscopy analyses of urine samples from all nicotine users and 22 verified controls to determine urinary levels of putatively nicotine product-specific substances; propylene glycol, 2-cyanoethylmercapturic acid (CEMA), and anabasine. 1) Cigarette smoking was associated with a dose dependent demethylation of cg05575921 and increased urinary CEMA and anabasine levels, 2) e-cigarette use did not demethylate cg05575921, 3) smokeless tobacco use also did not demethylate cg05575921 but was positively associated with anabasine levels 4) CEMA and cg05575921 levels were highly correlated and 5) propylene glycol levels did not reliably distinguish use groups. Cg05575921 assessments distinguish exposure to tobacco smoke from smokeless sources of nicotine including e-cigarettes and smokeless tobacco, neither of which are associated with cg05575921 demethylation. A combination of methylomic and metabolite profiling may allow for accurate classification use status of a variety of nicotine containing products.

Direct Evidence That Sunbirds' Gut Microbiota Degrades Floral Nectar's Toxic Alkaloids.

Orange-tufted sunbirds (Cinnyris osea) feed on the nectar of the tobacco tree (Nicotiana glauca) which contains toxic pyridine alkaloids characterized by high concentrations of anabasine and much lower concentrations of nicotine. We aimed at determining whether the gut microbiota of sunbirds harbors bacterial species that enable the birds to cope with these toxic alkaloids. An in vivo experiment that included 12 birds showed that inducing dysbiosis in sunbirds' guts by the addition of sulfamethoxazole and trimethoprim, significantly reduced the birds' ability to degrade anabasine (n = 3) compared to control birds (n = 3) with undisturbed microbiota. Sunbirds whose gut bacterial communities were altered by the antibacterial agents and who were fed with added nicotine, also showed a lower percentage of nicotine degradation (n = 3) in their excreta compared to the sunbirds with undisturbed microbiota (n = 3), though this difference was not significant. In an in vitro experiment, we studied the ability of Lactococcus lactis, Enterobacter hormaechei, Chryseobacterium gleum, Kocuria palustris, and Methylorubrum populi that were isolated from sunbirds' excreta, to degrade anabasine and nicotine. By using gas chromatography-mass spectrometry (GC-MS) analysis, we successfully demonstrated, for the first time, the ability of these species to degrade the focal secondary metabolites. Our findings demonstrate the role of gut bacteria in detoxifying toxic secondary metabolites found in the N. glauca nectar. The degradation products may supply the birds with nitrogen which is scarce in nectar-rich diets. These findings support another role of bacteria in mediating the interactions between plants and their pollinators.

An essay on ecosystem availability of Nicotiana glauca graham alkaloids: the honeybees case study.

BACKGROUND: Invasive plant species pose a significant threat for fragile isolated ecosystems, occupying space, and consuming scarce local resources. Recently though, an additional adverse effect was recognized in the form of its secondary metabolites entering the food chain. The present study is elaborating on this subject with a specific focus on the Nicotiana glauca Graham (Solanaceae) alkaloids and their occurrence and food chain penetrability in Mediterranean ecosystems. For this purpose, a targeted liquid chromatography electrospray tandem mass spectrometric (LC-ESI-MS/MS) analytical method, encompassing six alkaloids and one coumarin derivative, utilizing hydrophilic interaction chromatography (HILIC) was developed and validated. RESULTS: The method exhibited satisfactory recoveries, for all analytes, ranging from 75 to 93%, and acceptable repeatability and reproducibility. Four compounds (anabasine, anatabine, nornicotine, and scopoletin) were identified and quantified in 3 N. glauca flowers extracts, establishing them as potential sources of alien bio-molecules. The most abundant constituent was anabasine, determined at 3900 µg/g in the methanolic extract. These extracts were utilized as feeding treatments on Apis mellifera honeybees, resulting in mild toxicity documented by 16-18% mortality. A slightly increased effect was elicited by the methanolic extract containing anabasine at 20 µg/mL, where mortality approached 25%. Dead bees were screened for residues of the N. glauca flower extracts compounds and a significant mean concentration of anabasine was evidenced in both 10 and 20 µg/mL treatments, ranging from 51 to 92 ng/g per bee body weight. Scopoletin was also detected in trace amounts. CONCLUSIONS: The mild toxicity of the extracts in conjunction with the alkaloid and coumarin residual detection in bees, suggest that these alien bio-molecules are transferred within the food chain, suggesting a chemical invasion phenomenon, never reported before.

Long-term trends in tobacco use assessed by wastewater-based epidemiology and its relationship with consumption of nicotine containing products.

Measurement of population tobacco use via wastewater-based epidemiology (WBE) provides objective data to evaluate the efficacy of tobacco control strategies. However, current WBE tobacco-use estimates based on nicotine metabolites (cotinine and hydroxycotinine) can be masked by use of non-tobacco nicotine-containing products. To better understand nicotine and tobacco use, we analysed tobacco-specific biomarkers, anabasine and anatabine, as well as nicotine metabolites, cotinine and hydroxycotinine, in wastewater samples collected for 6 weeks per year over 6 years (2012-2017) from an Australian wastewater treatment plant serving approximately 100,000 people. Population-normalised mass loads were used to estimate tobacco and nicotine use trends and were compared with surveys and taxation statistics. Significant annual declines were observed for anabasine, anatabine, cotinine and hydroxycotinine of -3.0%, -2.7%, -2.4%, and -2.1%, respectively. The results corresponded with the annual declining trends reported from surveys (-5%) and taxation statistics (-4%). Significant annual decreases in the ratios of anabasine to cotinine (-1.2%) and anatabine to cotinine (-1.0%) suggested a relative increase in the use of non-tobacco nicotine products at the same time that tobacco use was declining. Monitoring tobacco use with anabasine and anatabine removed influence from nicotine-containing products, showing larger reductions in this Australian city than via nicotine biomarkers, whilst also demonstrating their suitability for monitoring long-term trends.

Determination of anabasine, anatabine, and nicotine biomarkers in wastewater by enhanced direct injection LC-MS/MS and evaluation of their in-sewer stability.

Wastewater-based epidemiology (WBE) has been used to estimate tobacco use in the population. However, the increased use of nicotine replacement therapies and e-cigarettes contributes to the load of nicotine metabolites in wastewater, causing over-estimation of tobacco use if nicotine metabolites were used in WBE back-estimation. This study aims to develop a rapid method for determining the tobacco-specific biomarkers, anabasine and anatabine, in wastewater and to evaluate their in-sewer stability for better estimation of tobacco use by WBE. An enhanced direct injection LC-MS/MS was developed to quantify anabasine and anatabine as well as nicotine biomarkers (nicotine, cotinine and hydroxycotinine). The method was optimal when wastewater was filtered through 0.2 µm RC syringe filters and a pre-conditioned SPE cartridge (Oasis HLB 1 cc, 30 mg) before 50 µL was injected into the LC-MS/MS system. Limits of quantification varied between 2.7 and 54.9 ng/L with recoveries from 76% to 103% for all five compounds. In sewer reactors, anabasine and anatabine were less stable than cotinine and hydroxycotinine. They were more stable in the gravity sewer reactor with <20% loss in 12 h than in the rising main sewer reactor with ~30% loss in the same period. We then applied the new method to 42 daily wastewater influent samples collected from an Australian wastewater treatment plant. The five biomarkers were detected in all samples with concentrations ranging from 9.2 to 7430 ng/L. All five compounds were positively correlated with one another. Our results suggested a high throughput analytical method for feasible application in anabasine and anatabine as biomarkers of tobacco use in routine wastewater monitoring.

Alkaloid chemophenetics and transcriptomics of the Nicotiana genus.

In this study, we determined the pyridine alkaloid content (nicotine, nornicotine, anabasine, anatabine, cotinine, and myosmine) of 58 species and 2 subspecies of the Nicotiana genus by ultra-high-performance liquid chromatography coupled with mass spectrometry. We observed clear correlation between Noctiflorae and Suaveolentes sections and their above average accumulation of anabasine in the genus. In addition, the results demonstrated the presence of not only trace amounts but quantifiable levels of myosmine, an alkaloid previously detected in only minute quantities, in the leaves and roots of 16 species. In this study, analysis of gene expression of 58 species and 2 subspecies from the Nicotiana genus by mRNA sequencing was performed for the first time. Sequencing reads were mapped against annotated genes of a Nicotiana tabacum reference genome and expression values were subsequently calculated. Hierarchical clustering of alkaloid biosynthesis pathway genes and alkaloid content composition revealed patterns clearly segregating Nicotiana sections. Correlation of gene expression with alkaloid accumulation phenotypes was evident, including low putrescine methyltransferase expression for all species in the Suaveolentes section or clear correlation of nicotine demethylase with conversion rates of nicotine to nornicotine in the majority of species. Multiple additional correlations between alkaloid accumulation and gene expression values were identified, which makes this study an important fundament toward future scientific exploration of the Nicotiana genus.

Supramolecular Nano-Encapsulation of Anabasine Reduced Its Developmental Toxicity in Zebrafish.

Anabasine (ANA), a major piperidine alkaloid originally isolated from wild tobacco trees (Nicotiana glauca), has been known to induce serious developmental toxicities such as skeletal deformities in livestock and humans. In this study, we thoroughly investigated the supramolecular nano-encapsulations of ANA by an artificial nanocontainer, cucurbit[7] uril (CB[7]), and examined the influences of the nano-encapsulation on ANA's inherent developmental toxicities on a zebrafish model. We have shown that CB[7] formed 1:1 host-guest inclusion complexes with ANA via a relatively high binding strength [K a of (7.45 ± 0.31) × 104 M-1] in an aqueous solution, via UV-vis and 1H nuclear magnetic resonance spectroscopic titrations, as well as isothermal titration calorimetry titration. As a consequence, CB[7] significantly attenuated the developmental toxicity of ANA on zebrafish in vivo. In contrast, for a comparative purpose, ß-CD didn't exert any influence on the toxicity of ANA due to its weak binding with ANA, which was not even measurable via either spectroscopic methods or ITC titration. This is the first head-to-head comparison of this pair of nanocontainers, CB[7] and ß-CD, on their potential roles in influencing the toxicity of guest molecules and the results suggested that CB[7] could become a more promising functional excipient for reducing the inherent toxicities of active pharmaceutical ingredients, particularly alkaloids that may form relatively strong host-guest binding species with the host.

A Pharmacological Comparison of Two Isomeric Nicotinic Receptor Agonists: The Marine Toxin Isoanatabine and the Tobacco Alkaloid Anatabine.

Many organisms possess "secondary" compounds to avoid consumption or to immobilize prey. While the most abundant or active compounds are initially investigated, more extensive analyses reveal other "minor" compounds with distinctive properties that may also be of biomedical and pharmaceutical significance. Here, we present an initial in vitro investigation of the actions of two isomeric tetrahydropyridyl ring-containing anabasine analogs: isoanatabine, an alkaloid isolated from a marine worm, and anatabine, a relatively abundant minor alkaloid in commercial tobacco plants. Both compounds have a double bond that is distal to the piperidine ring nitrogen of anabasine. Racemic isoanatabine and anatabine were synthesized and their S- and R-enantiomers were isolated by chiral high pressure liquid chromatography (HPLC). Both isoanatabines displayed higher efficacies at α4ß2 nicotinic acetylcholine receptors (nAChRs) relative to the anatabines; R-isoanatabine was most potent. Radioligand binding experiments revealed similar α4ß2 nAChR binding affinities for the isoanatabines, but R-anatabine affinity was twice that of S-anatabine. While the two anatabines and S-isoanatabine were highly efficacious agonists at α7 nAChRs, R-isoanatabine was only a weak partial agonist. The four compounds share an ability to stimulate both α4ß2 and α7 nAChRs, a property that may be useful in developing more efficacious drugs to treat neurodegenerative and other medical disorders.

An electrophysiological characterization of naturally occurring tobacco alkaloids and their action on human α4ß2 and α7 nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptor (nAChR) subtype-selective pharmacological profiles of tobacco alkaloids are essential for understanding the physiological effects of tobacco products. In this study, automated electrophysiology was used to functionally characterize the effects of distinct groups of tobacco alkaloids on human α4ß2 and α7 nAChRs. We found that, in tobacco alkaloids, pyridine as a hydrogen bond acceptor and a basic nitrogen atom at a distance of 4-7 Šare pharmacophoric elements necessary for molecular recognition by α4ß2 and α7 nAChRs with various degrees of selectivity, potency, and efficacy. While four alkaloids-nicotine, nornicotine, anabasine and R-anatabine-potently activated α4ß2, they were also weak agonists of α7 nAChRs. Nicotine was the most potent agonist of α4ß2, while anabasine elicited the highest activation of α7. None of the tobacco alkaloids enhanced nAChR activity elicited by the endogenous ligand acetylcholine; therefore, none was considered to be a positive allosteric modulator (PAM) of either α4ß2 or α7 nAChRs. In contrast, we identified tobacco alkaloids, such as the tryptophan metabolite 6-hydroxykynurenic acid, that decreased the activity of both α4ß2 and α7 nAChRs. Our study identified a class of alkaloids with positive and negative effects against human α4ß2 and α7 nAChRs. It also revealed human α4ß2 to be the principal receptor for sensing the most abundant alkaloids in tobacco leaves.

Discriminative Stimulus Properties of S(-)-Nicotine: "A Drug for All Seasons".

S(-)-Nicotine is the major pharmacologically active substance in tobacco and can function as an effective discriminative stimulus in both experimental animals and humans. In this model, subjects must detect and communicate the nicotine drug state versus the non-drug state. This review describes the usefulness of the procedure to study nicotine, presents a general overview of the model, and provides some relevant methodological details for the establishment of this drug as a stimulus. Once established, the (-)-nicotine stimulus can be characterized for dose response and time course effects. Moreover, tests can be conducted to determine the similarity of effects produced by test drugs to those produced by the training dose of nicotine. Such tests have shown that the stimulus effects of nicotine are stereoselective [S(-)-nicotine >R(+)-nicotine] and that other "natural" tobacco alkaloids and (-)-nicotine metabolites can produce (-)-nicotine-like effects, but these drugs are much less potent than (-)-nicotine. Stimulus antagonism tests with mecamylamine and DHßE (dihydro-ß-erythroidine) indicate that the (-)-nicotine stimulus is mediated via α4ß2 nicotinic acetylcholine receptors (nAChRs) in brain; dopamine systems also are likely involved. Individuals who try to cease their use of nicotine-based products are often unsuccessful. Bupropion (Zyban®) and varenicline (Chantix®) may be somewhat effective as anti-smoking medications because they probably produce stimulus effects that serve as suitable substitutes for (-)-nicotine in the individual who is motivated to quit smoking. Finally, it is proposed that future drug discrimination studies should apply the model to the issue of maintenance of abstinence from (-)-nicotine-based products.

Lysine-derived Alkaloids: Overview and Update on Biosynthesis and Medicinal Applications with Emphasis on Quinolizidine Alkaloids.

BACKGROUND: Plants produce a vast variety of specialized metabolites which can be a rich source for lead compounds for the development of new drugs. Alkaloids are one the largest groups of plant specialized metabolites important for natural product based pharmaceuticals. Of these, lysine (Lys)-derived alkaloids exhibit a wide range of pharmacological properties which are beneficial for humans. For instance they have anticancer, anti-Alzheimer's disease, anti-inflammatory, hypocholesterolemic and antiarrhtymic effects. Lys-derived alkaloids are widely distributed throughout the plant kingdom: they can be found in various species from clubmosses to flowering plants. Lys is one of the most essential amino acids for humans and livestock and is synthesized in the plastids of land plants. Lys-derived alkaloids can be divided into four major groups including quinolizidine, lycopodium, piperidine, and indolizidine alkaloids. Despite the importance of these compounds, the biosynthetic pathways of Lys-derived alkaloids are not well understood. With the exception of indolizidine alkaloids, Lys decarboxylase (LDC) is the enzyme involved in the first committed step of the biosynthesis by catalyzing the transformation of L-Lys into cadaverine. Cadaverine is then oxidized by copper amine oxidase (CuAO) and spontaneously cyclized to Δ1-piperideine Schiff base which is a universal intermediate for the production of various Lys-derived alkaloids. CONCLUSION: In this review, we briefly summarize the recent understanding about the structures, occurrences, analytical procedures, biosyntheses, and potential health effects and medical applications of Lys-derived alkaloids with emphasis on quinolizidine alkaloids (QAs).