Browsing by Subject "Nicotine"

Now showing 1 - 7 of 7
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    Characterization of b-nicotyrine-mediated inactivation of cytochrome P450 2A6
    (2013-04) Kramlinger, Valerie Marie
    Nicotine, the primary addictive compound in cigarettes, is metabolized in humans by cytochrome P450 2A enzymes. The hepatic enzyme responsible for the metabolism of nicotine in smokers is P450 2A6. P450 2A13, which shares 94% primary sequence homology with P450 2A6, also catalyzes the metabolism of nicotine and is present in the lung. Loss of P450 2A activity is correlated with modified smoking behavior and addiction. Inhibition of these enzymes decreases nicotine metabolism and may be of benefit in smoking cessation. This thesis investigates tobacco-related molecules that may impact P450 2A function and is presented in three parts. In the first, the potential inhibitory potency of (-)-menthol, (R)-(+)-menthofuran, and -nicotyrine of both P450s 2A6 and 2A13 are investigated. All three compounds inhibit P450 2A6 and 2A13 activity. In addition, menthofuran and b-nicotyrine mediate mechanism-based inactivation of P450 2A6 but not 2A13. Second, the P450 2A6 and P450 2A13-mediated metabolism of b-nicotyrine is studied and three metabolites are identified. b-nicotyrine is readily turned over by both P450 2A6 and P450 2A13 as indicated by the calculated Km (4.4µM and 5.0µM, respectively) and Vmax (21 and 37 pmol product/min/pmol, respectively) values. Also in the second section, b-nicotyrine is shown to be a metabolite of P450 2A6-mediated nicotine metabolism. In the last section, attempts to identify a covalent adduct that would result from menthofuran or b-nicotyrine-mediated inactivation are presented, but these are largely unsuccessful.
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    Development of the larval zebrafish as a genetic model for the nicotine response.
    (2010-08) Petzold, Andrew Michael
    Tobacco use is predicted to result in over 1 billion deaths worldwide by the end of the 21st century. How genetic variation contributes to the observed differential predisposition in the human population to drug dependence is unknown. The zebrafish (Danio rerio) is an emerging vertebrate model system for understanding the genetics of behavior. We developed a nicotine behavioral assay in zebrafish and applied it in a forward genetic screen using gene-breaking transposon mutagenesis. We used this method to molecularly characterize bdav/ cct8 and hbog/gabbr1.2 as mutations with altered nicotine response. Each have a single human ortholog, identifying two points for potential scientific, diagnostic, and drug development for nicotine biology and cessation therapeutics. We show this insertional method generates mutant alleles that are reversible through Cre-mediated recombination, representing a conditional mutation system for the zebrafish. Additionally, we developed a conditioned place preference assay for use with larval zebrafish. This assay allows for the perturbation of the differences in genetic function between the physiological and learned response representing one of the first associative learning based assays in the larval zebrafish. The combination of this reporter-tagged insertional mutagen approach and zebrafish provides a powerful platform for a rich array of questions amenable to genetic-based scientific inquiry, including the basis of behavior, epigenetics, plasticity, stress, memory, and learning.
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    Enhancing the efficacy of a nicotine vaccine
    (2013-03) Cornish, Katherine E.
    Tobacco addiction is the leading cause of preventable death worldwide. Many people continue to smoke cigarettes despite clear detrimental health effects. Available smoking cessation therapies are only partially effective, making new treatment approaches necessary to increase smoking cessation rates. Immunization against nicotine features a different mechanism of action than currently available medications. As a pharmacokinetic antagonist, immunization against nicotine alters distribution, metabolism, and clearance of nicotine to attenuate nicotine-induced behavior in animal models. Nicotine vaccines in clinical trials show efficacy but are limited by the modest and highly variable nicotine-specific antibody (NicAb) concentrations produced. This thesis focuses on ways to improve efficacy of a nicotine vaccine by combining it with additional forms of immunotherapy. The first aim of this thesis examined the effects of supplementing vaccination against nicotine with individualized doses of Nic311, a nicotine-specific monoclonal antibody. Compared to either immunotherapy alone, combining active and passive immunization produced greater alterations in nicotine pharmacokinetics and nicotine-induced behavior using a locomotor activity model. Only small doses of Nic311 were necessary to supplement vaccine-generated NicAb concentrations to a previously effective threshold. This decreased cost and use of typically expensive monoclonal antibodies, potentially increasing viability of this approach in a clinical setting. The second aim of this thesis examined the effects of concurrent administration of two immunologically distinct nicotine immunogens in a bivalent vaccine over a range of vaccine formulations and immunization conditions. Immunogens were co-administered in a bivalent vaccine without compromising immunogenicity of either immunogen when delivered subcutaneously in alum, but not when delivered intraperitoneally in Freund's adjuvant. When combined in alum, immune responses elicited by the two immunogens were largely independent of one another. This suggests that subjects who responded poorly to one immunogen may have responded better to the second, immunologically distinct immunogen in the bivalent vaccine. These results indicate that the bivalent vaccine strategy is a feasible way to increase antibody concentrations above what can be achieved using one immunogen alone, but integrity of the response is highly dependent on vaccine formulation and administration conditions.
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    Medications to help quit smoking
    (2012-04-09) Bloomberg, Elisabeth
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    Metabolism of nicotine and the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): genetic and phenotypic variation.
    (2009-11) Berg, Jeannette Zinggeler
    Nicotine is the addictive agent in tobacco and differences in nicotine metabolism may affect tobacco use, and consequently exposure to tobacco carcinogens. A lung procarcinogen in tobacco is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its carcinogenic effect is dependent on metabolic activation and is counter-balanced by metabolic detoxification. Nicotine and NNK are structurally related and both are metabolized by cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and flavin-monoxygenase (FMO) enzymes. The goal of this thesis research was to explore variation in nicotine metabolism in vivo and to probe specific enzyme-catalyzed reactions of NNK in vitro. Low nicotine and cotinine glucuronidation were observed among African Americans compared to Europeans, and among individuals with a variant UGT2B10 genotype. In a controlled dose study of ethnic differences in nicotine metabolism (n= 93 smokers), African Americans excreted 30-40 % less nicotine and cotinine as their glucuronide conjugates than European Americans. This difference in glucuronidation explained the higher free cotinine concentrations observed in African Americans compared to European Americans. The most efficient in vitro catalyst of nicotine and cotinine glucuronidation is UGT2B10. We demonstrated that UGT2B10 contributes to in vivo nicotine metabolism in a genotype-phenotype analysis of 325 smokers. Individuals who were heterozygous for the UGT2B10 Asp67Tyr allele excreted less nicotine or cotinine as their glucuronide conjugates than wild-type; the ratio of cotinine glucuronide:cotinine was decreased by 60 %, while increases in urinary and plasma cotinine and trans-3'-hydroxycotinine were observed. Strikingly, a robust biomarker of nicotine intake, nicotine equivalents, were lower among Asp67Tyr heterozygotes compared to individuals without this allele; 58.2 nmol/ml (95 % CI, 48.9 - 68.2) versus 69.2 nmol/ml (95 % CI, 64.3 - 74.5). Individuals with low activity UGT2B10 may smoke less intensely, as reported for individuals with CYP2A6 polymorphisms that cause decreased nicotine C-oxidation. In contrast to nicotine, NNK is a carcinogen. It is metabolized to reactive intermediates that can form DNA and protein adducts, or it is detoxified by glucuronidation. P450 2A13 is the most efficient catalyst of NNK oxidation. We explored the effect of an active site mutant, Asn297Ala, on enzyme function and found that loss of hydrogen bonding to substrate in the active site affected substrate orientation and product formation. The orphan P450 2A7 was considered as a potential catalyst for NNK oxidation, but expression of wild-type or two naturally-occurring variants failed to yield protein with a P450 spectra and no appreciable activity towards P450 2A substrates was observed. Preliminary experiments were conducted to search for the glucuronide conjugate formed from the unstable oxidation product alpha-hydroxymethyl NNK, which has not been identified in any human system. The extent to which variation in metabolism mediates smoking behavior and cancer risk warrants consideration. The enzymes involved are potential drug targets for smoking cessation pharmacotherapy and cancer chemoprevention.
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    Passive Immunization to treat nicotine dependence in rats.
    (2009-01) Roiko, Samuel A.
    Cigarette smoking is the leading preventable cause of death in the United States, primarily due to nicotine addiction. Currently available medications are only partially effective in increasing smoking cessation, and additional therapies are needed. Immunotherapy is an alternative strategy for attenuating nicotine's addictive effects. Vaccination against nicotine alters nicotine pharmacokinetics and nicotine's behavioral effects in rats. In clinical trials, nicotine vaccines reduce smoking, but efficacy is limited by high variability and low mean serum nicotine-specific antibody (NicAb) levels. Passive immunization is an alternative method of immunization, and can potentially circumvent the limitations of vaccination by allowing control over the dose and timing of NicAb administration. The overall goal of this thesis was to examine the use of passive immunization to treat tobacco addiction. The specific aims were to characterize the efficacy of passive immunization on nicotine pharmacokinetics and nicotine-induced behavior, explore the effects of combining vaccination with passive immunization, and examine a potential adverse effect of passive immunization, the precipitation of withdrawal. Passive immunization with the nicotine-specific monoclonal antibody Nic311 reduced nicotine clearance and steady-state volume of distribution, and prolonged nicotine's elimination half-life, demonstrating the underlying mechanism of immunotherapy efficacy. Nic311 attenuated nicotine-induced locomotor sensitization, and combining Nic311 with vaccination resulted in greater reductions in brain nicotine levels and nicotine-induced locomotor sensitization compared to either immunotherapy alone, demonstrating the behavioral efficacy of Nic311 and suggesting a possible clinical role as a supplement for vaccination. Administration of Nic311 to nicotine-dependent rats substantially reduced brain nicotine levels but did not elicit a nicotine withdrawal syndrome. These findings demonstrate the benefits and safety of passive immunization with Nic311, and point to its potential in treating nicotine addiction.
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