Introdução: A grande variabilidade individual em resposta a fármacos antitabagismo sugere que tratamentos específicos podem ser mais efetivos em determinados subgrupos de fumantes. No contexto de medicina personalizada, o principal objetivo do presente estudo foi avaliar se polimorfismos nos genes CHRNA4, CHRNB2, CYP2B6 e ANKK1 estão associados com a resposta às terapias de cessação tabágica em pacientes provenientes de um programa de assistência ao fumante. Métodos: Estudo de coorte com 483 pacientes fumantes que receberam tratamento farmacológico (vareniclina, vareniclina e bupropiona, bupropiona em monoterapia ou coadministrada com terapia de reposição nicotínica). O sucesso na cessação tabágica foi considerado para os pacientes que completaram 6 meses de abstinência contínua. O teste de Fagerström para a dependência à nicotina (FTND) e o escore de consumo situacional Issa foram utilizados para avaliar a dependência à nicotina. Os polimorfismos CHRNA4 (rs1044396 e rs2236196), CHRNB2 (rs2072660 e rs2072661) e ANKK1 (rs1800497) foram genotipados pela análise da curva de melting e os polimorfismos CYP2B6 *9 (rs3745274), *4 (rs2279343), *5 (rs3211371) foram genotipados por restrição enzimática. Resultados: Os pacientes com o genótipo CC para o polimorfismo CHRNA4 (rs10443196) obtiveram menor taxa de sucesso no tratamento com vareniclina (29,5%) em comparação com os portadores dos genótipos CT ou TT (50,9%) (P=0,007; n=167). Os genótipos CT ou TT foram associados com maior odds ratio para o sucesso (OR=1,67; IC 95%=1,10-2,53; P=0,02), em um modelo multivariado. Os pacientes com o genótipo AA para o polimorfismo CYP2B6 (rs2279343) obtiveram maior taxa de sucesso no tratamento com bupropiona (48,0%) em comparação com portadores dos genótipos AG ou GG (35,5%) (P=0,05; n=237). O genótipo AA foi associado com maior odds ratio para o sucesso no tratamento (OR=1,92; IC 95%=1,08-3,42; P=0,03), em um modelo multivariado. Não foram observadas diferenças significativas nos escores FTND e Issa com relação aos polimorfismos estudados. Conclusão: Os polimorfismos CHRNA4 (rs1044396) e CYP2B6 (rs2279343) estão associados com a cessação tabágica em indivíduos tratados com vareniclina e bupropiona, respectivamente. Sugere-se que estes polimorfismos influenciam a resposta farmacológica e podem ser importantes para o desenho de uma farmacoterapia individualizada / Background: The large individual variability in response to drugs for smoking cessation suggests that specific treatments can be more effective in particular subgroups of smokers. In the context of personalized medicine, the main aim of the present study was to evaluate whether the CHRNA4, CHRNB2, CYP2B6 and ANKK1 polymorphisms are associated with response to smoking cessation therapies in patients from a smoker assistance program. Methods: This cohort study enrolled 483 smoking patients patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion in monoterapy or plus nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. The CHRNA4 (rs1044396 and rs2236196), CHRNB2 (rs2072660 and rs2072661) and ANKK1 rs1800497 polymorphisms were genotyped by high resolution melting analysis and the CYP2B6 *9 (rs3745274), *4 (rs2279343) and *5 (rs3211371) were genotyped by restriction fragment lenght polymorphisms. Results: Patients with CHRNA4 rs1044396 CC genotype had lower success rate in treatment with varenicline (29.5%) compared with carriers of CT or TT genotypes (50.9%) (P=0.007, n=167). The CT or TT genotypes were associated with higher odds ratio for success (OR=1.67, 95%CI=1.10-2.53, P=0.02), in a multivariate model. Patients with CYP2B6 rs2279343 AA genotype had higher success rate in treatment with bupropion (48.0%) compared with carriers of AG or GG genotypes (35.5%) (P=0.05, n=237). The AA genotype was associated with higher odds ratio for success (OR=1.92, 95%CI=1.08-3.42, P=0.03), in a multivariate model. We did not observe significant differences in the FTND and Issa scores according to the studied polymorphisms. Conclusion: The CHRNA4 rs1044396 and CYP2B6 rs2279343 are associated with smoking cessation in individuals on varenicline and bupropion terapies, respectively. We suggest that these polymorphisms influence the pharmacological response of these drugs and it might be important in the design of individualized pharmacotherapy

Many irreversible antagonists are known to bind irreversibly to pharmacological receptors. However, few studies suggest that these irreversible antagonists may also display irreversible non-specific antagonism by binding irreversibly to non-syntopic binding sites on the receptor macromolecule, whereby they modulate the signal transduction of these receptors or reduce the agonist binding affmity. The aim of this study was to investigate whether the classical irreversible antagonists phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible nonspecific antagonism at various G protein-coupled receptor (GPCR) types. In addition, the subcellular mechanism whereby benextramine displays irreversible non-specific antagonism was investigated. Three cell lines were employed to investigate the antagonism by these irreversible antagonists: Chinese hamster ovary (CHO-K1) cells transfected to express the porcine a2A-adrenoceptor (a2A-AR) at higher (a2A-H) or lower (a2A-L) numbers, human neuroblastoma (SH-SY5Y) cells that endogenously express muscarinic acetylcholine receptors (mACh-Rs), and SH-SY5Y cells transfected (5HT2A-SH-SY5Y)o express the human 5HT2A-serotonirne ceptor (5HTZA-R).C ells of the appropriate cell line were pre-treated at the appropriate concentrations and incubation times with an appropriate irreversible antagonist, with or without an appropriate reversible competitive antagonist at a sufficient concentration to protect the specific receptors. This was followed by washing procedures with drug-free media to rinse any unbound or reversibly bound drugs from the cells. When appropriate, cell membranes were prepared. Receptor function was evaluated by measuring whole-cell [3H]-cAMP or [3H]-IPx acumulation, or the binding of [35S]-GTPyS to membraness. Receptor concentrations were determined from radioligand-binding assays. In addition, the constitutive [35S]-GTPyS binding to Go protein before and after pre-treatment with benextramine was investigated. Results suggest that phenoxybenzamine (100 uM, 20 minutes) and benextramine (10 uM, 20 minutes) display irreversible non-specific antagonism at a2A-ARs when measuring Gi-mediated effects in a2A-L cells, but the affinity for a2A-ARs in a2A-H cells was not changed. In addition, it was found that the observed irreversible nonspecific antagonism by benextramine appears to be time- and concentration-dependent. When the mechanism of irreversible antagonism by benextramine was further investigated, benextramine reduced the binding of [35S]-GTPyS to a2A-H membranes with protected a2A-ARs, but did not modulate the constitutive binding of [35S]-GTPyS to Go. In addition, benextramine displays irreversible non-specific antagonism by inhibiting the G,-mediated effects of a2A-ARs in a2A-H cells and the Gq-mediated effects of mACh-Rs or 5HT2A-Rs in SH-SY5Y or 5HT2A-SH-SY5Y cells respectively. 4-DAMP mustard (100 uM, 20 minutes) did not display irreversible non-specific antagonism at mACh-Rs in SH-SY5Y cells, but irreversible non-specific antagonism was observed when the incubation time was increased (100 uM, 60 minutes). In conclusion it was found that phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible non-specific antagonism at typical experimental conditions. These findings confirm concerns in literature and supports the possibility that more irreversible antagonists could display irreversible non-specific antagonism, and that could influence the interpretation of data obtained with such drugs. In addition, benextramine may prove to be a useful experimental drug in studying GPCR signalling. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Many irreversible antagonists are known to bind irreversibly to pharmacological receptors. However, few studies suggest that these irreversible antagonists may also display irreversible non-specific antagonism by binding irreversibly to non-syntopic binding sites on the receptor macromolecule, whereby they modulate the signal transduction of these receptors or reduce the agonist binding affmity. The aim of this study was to investigate whether the classical irreversible antagonists phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible nonspecific antagonism at various G protein-coupled receptor (GPCR) types. In addition, the subcellular mechanism whereby benextramine displays irreversible non-specific antagonism was investigated. Three cell lines were employed to investigate the antagonism by these irreversible antagonists: Chinese hamster ovary (CHO-K1) cells transfected to express the porcine a2A-adrenoceptor (a2A-AR) at higher (a2A-H) or lower (a2A-L) numbers, human neuroblastoma (SH-SY5Y) cells that endogenously express muscarinic acetylcholine receptors (mACh-Rs), and SH-SY5Y cells transfected (5HT2A-SH-SY5Y)o express the human 5HT2A-serotonirne ceptor (5HTZA-R).C ells of the appropriate cell line were pre-treated at the appropriate concentrations and incubation times with an appropriate irreversible antagonist, with or without an appropriate reversible competitive antagonist at a sufficient concentration to protect the specific receptors. This was followed by washing procedures with drug-free media to rinse any unbound or reversibly bound drugs from the cells. When appropriate, cell membranes were prepared. Receptor function was evaluated by measuring whole-cell [3H]-cAMP or [3H]-IPx acumulation, or the binding of [35S]-GTPyS to membraness. Receptor concentrations were determined from radioligand-binding assays. In addition, the constitutive [35S]-GTPyS binding to Go protein before and after pre-treatment with benextramine was investigated. Results suggest that phenoxybenzamine (100 uM, 20 minutes) and benextramine (10 uM, 20 minutes) display irreversible non-specific antagonism at a2A-ARs when measuring Gi-mediated effects in a2A-L cells, but the affinity for a2A-ARs in a2A-H cells was not changed. In addition, it was found that the observed irreversible nonspecific antagonism by benextramine appears to be time- and concentration-dependent. When the mechanism of irreversible antagonism by benextramine was further investigated, benextramine reduced the binding of [35S]-GTPyS to a2A-H membranes with protected a2A-ARs, but did not modulate the constitutive binding of [35S]-GTPyS to Go. In addition, benextramine displays irreversible non-specific antagonism by inhibiting the G,-mediated effects of a2A-ARs in a2A-H cells and the Gq-mediated effects of mACh-Rs or 5HT2A-Rs in SH-SY5Y or 5HT2A-SH-SY5Y cells respectively. 4-DAMP mustard (100 uM, 20 minutes) did not display irreversible non-specific antagonism at mACh-Rs in SH-SY5Y cells, but irreversible non-specific antagonism was observed when the incubation time was increased (100 uM, 60 minutes). In conclusion it was found that phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible non-specific antagonism at typical experimental conditions. These findings confirm concerns in literature and supports the possibility that more irreversible antagonists could display irreversible non-specific antagonism, and that could influence the interpretation of data obtained with such drugs. In addition, benextramine may prove to be a useful experimental drug in studying GPCR signalling. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Introdução: A identificação de variantes genéticas que predispõem a maior susceptibilidade à dependência à nicotina pode ser importante para a prevenção e o tratamento do tabagismo. No contexto de medicina personalizada, os principais objetivos do presente estudo foram avaliar se polimorfismos nos genes CHRNA2, CHRNA3, CHRNA5 e CHRNB3 estão associados com o nível de dependência em indivíduos fumantes e com o resultado do tratamento antitabágico. Métodos: Estudo de coorte com 1049 pacientes fumantes que receberam tratamento farmacológico (vareniclina, vareniclina e bupropiona, bupropiona e/ou terapia de reposição nicotínica). O sucesso na cessação tabágica foi considerado para os pacientes que completaram 6 meses de abstinência contínua. O teste de Fagerström para a dependência à nicotina (FTND) e o escore de consumo situacional Issa foram utilizados para avaliar a dependência à nicotina. A escala de conforto PAF foi utilizada para avaliar o conforto do paciente durante o tratamento. Os polimorfismos CHRNA2 rs2472553, CHRNA3 rs1051730, CHRNA5 rs16969968, CHRNA5 rs2036527 e CHRNB3 rs6474413 foram genotipados pela análise da curva de melting. Resultados: As mulheres portadoras dos genótipos GA e AA para os polimorfismos CHRNA5 rs16969968 e rs2036527 obtiveram maior taxa de sucesso no tratamento antitabagismo: 44,0% e 56,3% (rs16969968), 41,5% e 56,5% (rs2036527), respectivamente; em comparação com as mulheres portadoras do genótipo GG: 35,7% (rs16969968) e 34,8% (rs2036527), (P=0,03; n=389; P=0,01; n=391). Os genótipos GA ou AA para os rs16969968 e rs2036527 foram associados com maior OR para o sucesso em mulheres (OR=1,63; IC 95%=1,04-2,54; P=0,03 e OR=1,59; IC 95%=1,02-2,48; P=0,04; respectivamente), em um modelo multivariado. Não foi encontrada associação dos polimorfismos no gene CHRNA5 com o escore de FTND. Para os polimorfismos CHRNA2 rs2472553, CHRNA3 rs1051730 e CHRNB3 rs6474413 não foram encontradas associações significativas com os fenótipos estudados. Conclusão: Os polimorfismos rs16969968 e rs2036527 no gene CHRNA5 foram associados com maior taxa de sucesso no tratamento antitabagismo em mulheres. Estes resultados podem contribuir com avanços na terapêutica baseada em medicina personalizada / Background: The identification of genetic variants that predispose increased susceptibility to nicotine dependence becomes increasingly important for the prevention and smoking treatment. In the context of personalized medicine, the main aims of this study were to evaluate whether the CHRNA2, CHRNA3, CHRNA5 and CHRNB3 polymorphisms are associated with the level of dependence in smokers and the result of smoking treatment. Methods: This cohort study enrolled 1049 smoking patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion plus/or nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. PAF comfort scale was used to evaluate the comfort of the patient during treatment. The CHRNA2 rs2472553, CHRNA3 rs1051730, CHRNA5 rs16969968 and rs2036527 and CHRNB3 rs6474413 polymorphisms were genotyped by high resolution melting analysis. Results: Females with GA and AA genotypes for CHRNA5 rs16969968 and rs2036527polymorphisms had higher success rate in smoking cessation treatment: 44.0% and 56.3% (rs16969968), 41.5% and 56.5% (rs2036527), respectively; compared with carriers of the GG genotypes: 35.7% (rs16969968), 34.8% (rs2036527), (P=0.03, n=389; P=0.01, n=391). The GA or AA genotypes to the rs16969968 and rs2036527 were associated with higher odds ratio for success in women (OR=1.63; 95%CI=1.04 to 2.54; P=0.03 and OR=1.59, 95%CI=1.02 to 2.48; P=0.04; respectively), in a multivariate model. We found no association of these polymorphisms with FTND score for nicotine dependence. For the CHRNA2 rs2472553, CHRNA3 rs1051730 and CHRNB3 rs6474413 polymorphisms no significant associations were found with phenotypes studied. Conclusion: The CHRNA5 rs16969968 and rs2036527 were associated with higher success rate in the smoking cessation treatment in women. These results can contribute to major advances in personalized medicine based therapy

Introdução: A identificação de variantes genéticas que predispõem a maior susceptibilidade à dependência à nicotina pode ser importante para a prevenção e o tratamento do tabagismo. No contexto de medicina personalizada, os principais objetivos do presente estudo foram avaliar se polimorfismos nos genes CHRNA2, CHRNA3, CHRNA5 e CHRNB3 estão associados com o nível de dependência em indivíduos fumantes e com o resultado do tratamento antitabágico. Métodos: Estudo de coorte com 1049 pacientes fumantes que receberam tratamento farmacológico (vareniclina, vareniclina e bupropiona, bupropiona e/ou terapia de reposição nicotínica). O sucesso na cessação tabágica foi considerado para os pacientes que completaram 6 meses de abstinência contínua. O teste de Fagerström para a dependência à nicotina (FTND) e o escore de consumo situacional Issa foram utilizados para avaliar a dependência à nicotina. A escala de conforto PAF foi utilizada para avaliar o conforto do paciente durante o tratamento. Os polimorfismos CHRNA2 rs2472553, CHRNA3 rs1051730, CHRNA5 rs16969968, CHRNA5 rs2036527 e CHRNB3 rs6474413 foram genotipados pela análise da curva de melting. Resultados: As mulheres portadoras dos genótipos GA e AA para os polimorfismos CHRNA5 rs16969968 e rs2036527 obtiveram maior taxa de sucesso no tratamento antitabagismo: 44,0% e 56,3% (rs16969968), 41,5% e 56,5% (rs2036527), respectivamente; em comparação com as mulheres portadoras do genótipo GG: 35,7% (rs16969968) e 34,8% (rs2036527), (P=0,03; n=389; P=0,01; n=391). Os genótipos GA ou AA para os rs16969968 e rs2036527 foram associados com maior OR para o sucesso em mulheres (OR=1,63; IC 95%=1,04-2,54; P=0,03 e OR=1,59; IC 95%=1,02-2,48; P=0,04; respectivamente), em um modelo multivariado. Não foi encontrada associação dos polimorfismos no gene CHRNA5 com o escore de FTND. Para os polimorfismos CHRNA2 rs2472553, CHRNA3 rs1051730 e CHRNB3 rs6474413 não foram encontradas associações significativas com os fenótipos estudados. Conclusão: Os polimorfismos rs16969968 e rs2036527 no gene CHRNA5 foram associados com maior taxa de sucesso no tratamento antitabagismo em mulheres. Estes resultados podem contribuir com avanços na terapêutica baseada em medicina personalizada / Background: The identification of genetic variants that predispose increased susceptibility to nicotine dependence becomes increasingly important for the prevention and smoking treatment. In the context of personalized medicine, the main aims of this study were to evaluate whether the CHRNA2, CHRNA3, CHRNA5 and CHRNB3 polymorphisms are associated with the level of dependence in smokers and the result of smoking treatment. Methods: This cohort study enrolled 1049 smoking patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion plus/or nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. PAF comfort scale was used to evaluate the comfort of the patient during treatment. The CHRNA2 rs2472553, CHRNA3 rs1051730, CHRNA5 rs16969968 and rs2036527 and CHRNB3 rs6474413 polymorphisms were genotyped by high resolution melting analysis. Results: Females with GA and AA genotypes for CHRNA5 rs16969968 and rs2036527polymorphisms had higher success rate in smoking cessation treatment: 44.0% and 56.3% (rs16969968), 41.5% and 56.5% (rs2036527), respectively; compared with carriers of the GG genotypes: 35.7% (rs16969968), 34.8% (rs2036527), (P=0.03, n=389; P=0.01, n=391). The GA or AA genotypes to the rs16969968 and rs2036527 were associated with higher odds ratio for success in women (OR=1.63; 95%CI=1.04 to 2.54; P=0.03 and OR=1.59, 95%CI=1.02 to 2.48; P=0.04; respectively), in a multivariate model. We found no association of these polymorphisms with FTND score for nicotine dependence. For the CHRNA2 rs2472553, CHRNA3 rs1051730 and CHRNB3 rs6474413 polymorphisms no significant associations were found with phenotypes studied. Conclusion: The CHRNA5 rs16969968 and rs2036527 were associated with higher success rate in the smoking cessation treatment in women. These results can contribute to major advances in personalized medicine based therapy