Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the present invention relates, in general, to methods for predicting success in cessation of tobacco use in response to a pharmacological treatment with an agonist drug of nicotinic cholinergic receptors, based on the presence of certain polymorphisms and, optionally, in combination, with some clinical variables.
• Tobacco use is one of the biggest public health problems of today because it constitutes one of the main risk factors for various chronic diseases, for example, lung, cardiovascular and cancer diseases.
• tobacco use continues to be widespread throughout the world. In fact, it is estimated that there are about 1 billion smokers in the world. In Spain alone, about 500,000 people a year become ill from passive smoking, 65% of them being young children. Likewise, tobacco smoke is responsible for bronchitis and pneumonia in children under 18 months, between approximately 25,000 and 51,000 cases per year, which require around 1,300 hospitalizations / year for that reason. On the other hand, the average number of non-smoking adults killed by lung cancer is about 400 cases annually.
• Non-nicotinic drug therapy is carried out with the use of drugs that mimic the nicotine molecule and replace it in the fixation in nicotinic receptors, or through other medications with an approved indication to quit smoking; Among the most commonly used medications are bupropion, nortriptyline, varenicline and citisine. By administering these drugs, a success rate is obtained in attempts to quit tobacco consumption that doubles the rate obtained in their absence, so that these drugs contribute substantially to improving health and reducing the risk of premature mortality. (Fant et al. 2009, Handb Exp Pharmacol (192): 487-510).
• TSN nicotine replacement therapy
• chewing gum, patches, pills and inhalers chewing gum, patches, pills and inhalers
• TSN nicotine replacement therapy
• smoked nicotine substitutes can help overcome withdrawal from dependent smokers who quit smoking if used for short periods of time, not exceeding 9 weeks.
• patches are used daily with a constant concentration of nicotine for 3 weeks and then they are exchanged for others with a lower dose of nicotine. Meanwhile the brain adapts to function without nicotine and acquires other healthy habits. The fact that the patches release nicotine steadily does not generate addiction since there is no blood nicotine peak that reinforces the desire to smoke.
• TSN TSN
• psychological support and pharmacological treatment with bupropion, varenicline or nortriptyline have been shown to have a clinically significant effect.
• the efficacy of all treatments can be considered identical at 52 weeks (Aubin et al. 2008, Thorax 63 (8): 717-24).
• Unfortunately, without treatment approximately 3-5% of smokers who wish to quit smoking It achieves one year abstinence rates, but many of those who achieve one year abstinence will fall within the next 2 years (World Health Organization, 2004).
• Smoking represents a complex behavior that includes the beginning of tobacco consumption, its progression to regular use, dependence, abandonment and relapse. Together with environmental factors, there is evidence of the genetic influence on the establishment, maintenance and cessation of tobacco consumption. Association studies report a genotype influence of 40-60% (Uhl et al. 2007, BMC Genet 3: 8-10).
• GWA genome association study
• S Ps single nucleotide polymorphisms
• the change of a single nucleotide can cause: (i) the substitution of an amino acid, if it is in an exonic sequence, varying the function of the protein, (ii) the modification of the amount of the protein, if the SNP is in a regulatory region, or (iii) the change in protein structure, if the change is in a splicing zone.
• nicotine is the main psychoactive compound in tobacco
• most of the candidate genes analyzed in pharmacogenetic studies have focused on nicotinic receptors and genes involved in nicotine metabolism.
• genes involved in nicotine vulnerability are those genes linked to the dopaminergic neurotransmission pathways, especially those of the D2 receptor (DRD2) and the dopamine transporter (DAT or SLC6A3).
• D2 receptor D2 receptor
• DAT dopamine transporter
• the enzymes of the dopaminergic metabolism COMT and MAO-B present interindividual variability that could contribute to the different responses to substances with addictive potential (Barrueco et al. 2005, Med Clin (Barc) 124 (6): 223-8).
• the metabolism of nicotine is subject to genetic variants in the cytochrome P450, responsible for its metabolism to cotinine.
• nicotinic cholinergic receptor subunits may affect the intensity of its effects.
• CHRN nicotinic cholinergic receptor
• CNS central nervous system
• Tobacco use modifies the expression of other subunits such as a7 and a5, thus modifying the structure of the receptor and its coupled functions.
• nicotine metabolism is very sensitive to SNPs in CYP2A6. Certain variants, such as * 2 and * 4 are less active, which means greater sensitivity to the adverse effects of nicotine patches.
• Bupropion is a drug that uses the DAT dopamine transporter and the consequent dopaminergic modulation as a mechanism for smoking cessation. Certain variants of the DAT could represent a resistance to its effects. On the other hand, it has been described, in one of the few methodologically well-designed studies (Berrettini et al. 2007, Biol Psychiatry 61 (1): 111- 118), that bupropion responses may vary depending on the greater or lesser synaptic availability of dopamine controlled by COMT variants. The metabolism of bupropion is also dependent on cytochrome CYP2B6, thus some of its variants, for example, * 4, provides a greater clearance of bupropion and as a consequence, a lower efficacy thereof.
• varenicline In the case of varenicline, given its recent commercial introduction, only a non-independent study of association with genetic variants focused on pharmacogenetics has been published (King et al. 2012, Neuropsychopharmacology 37: 641-650). In this study, continued abstinence is related in patients under treatment with varenicline during weeks 9-12 of treatment with SNPs present in genes related to cholinergic nicotinic receptors (CHRN) and in genes in union imbalance of the Chrl5q25 chromosomal region.
• CHRN cholinergic nicotinic receptors
• the genes related to nicotinic receptors described in this study refer to a pharmacological target of varenicline, which is not extensible to neurobiological dependence processes, since in the state of the art there is no evidence to indicate that the genes described in this study are related to neurobiological dependence processes, they are only described in their relationship with nicotine dependence.
• the interrelation of the CHRNA5-CHRNA3-CHRNB4 cluster with dependence has not been reproduced in the latest meta-analyzes and published studies (Maes HH et al. 2004 Psychol Med 34 (7): 1251-6; Rodr ⁇ guez S et al .
• SNPs single nucleotide polymorphisms
• varenicline a nicotinic cholinergic receptor agonist drug.
• the inventors conducted a study to determine the pharmacogenomic profile associated with the efficacy of a pharmacological treatment for smoking cessation based on varenicline, evaluating the results obtained in terms of withdrawal from smoking at 3 months (short-term efficacy) and 12 months ( long term effectiveness).
• SNPs were genotyped in a total of 807 subjects, of which 479 had received, or were receiving, pharmacological treatment with varenicline, and the association of their genotypes with smoking cessation in response to said pharmacological treatment was found, finding that the alleles of certain SNPs, specifically the SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and / or rs2023239, or any SNP of their corresponding linkage blocks, were significantly associated.
• the SNPs identified in the present application are located in genes that are involved in neurobiological dependency processes, a process that involves the different brain reward systems (mesolimbic and mesocortical areas) and their neurotransmitters and receptors (Lingford-Hughes A & Nutt D 2003 Brit J Psych 182: 97-100; Neuroscience of Psychoactive Substance Use and Dependence 2004 WHO).
• the SNP rs678188 is located in the PARD3 gene (partitioning defective 3)
• the SNP rs9658498 is located in the NOS1 gene (nitric oxide synthase 1)
• the SNP rs4821566 is located in the CSF2RB gene (colony stimulating factor 2 beta receptor)
• the SNP rsl0891510 is located in the NCAM1 gene (neural cell adhesion molecule 1)
• the SNP rsl 1932367 is located in the GRID2 gene (glutamate ionotrophic delta receptor)
• the SNP rs2023239 is located in the CNR1 gene (cannabinoid receiver 1).
• said SNP is selected from the group formed by SNPs rsl0891510, rsl 1932367, rs2023239 and any combination of said SNPs and / or the corresponding SNPs of their linkage blocks; these SNPs (or the corresponding SNPs of their linkage blocks), alone or in combination with each other, prove to be particularly useful for predicting the efficacy of the 12-month treatment response.
• said SNP is SNP rs678188 and / or the corresponding SNPs of its linkage block.
• Figure 1 shows the ROC curves corresponding to the univariate models
• Figure 2 shows the ROC curves corresponding to the univariate (upper graphic) and multivariate (lower graphic) models in relation to the SNP rs9658498.
• Figure 3 shows the ROC curves corresponding to the univariate (upper graphic) and multivariate (lower graphic) models in relation to the SNP rs4821566.
• Figure 4 shows the ROC curves corresponding to the univariate (upper graphic) and multivariate (lower graphic) models in relation to SNP rsl0891510.
• Figure 5 shows the ROC curves corresponding to the univariate models
• Figure 6 shows the ROC curves corresponding to the univariate (upper graph) and multivariate (lower graph) models in relation to SNP rs2023239.
• the authors of the present invention have identified alleles of SNPs whose presence is related to a greater or lesser probability of success in abandonment. of tobacco consumption in response to a pharmacological treatment based on varenicline or drugs with a similar mechanism of action such as cytisine and dianicline, among others. Based on that finding, the methods of the present invention have been developed which are described in detail below.
• kits of the invention may include means for extracting the sample from the subject.
• the terms "abandonment” or “cessation” are used interchangeably, in relation to tobacco use, to refer to abstinence, termination or termination of tobacco consumption.
• the abandonment of tobacco consumption can be (i) complete, when there is a total absence of consumption of any type or form of tobacco product from the moment in which it is decided to abandon its consumption, (ii) continued, when the subject who abandons tobacco use has relapses, continuous or punctual, during the 15 days after the date on which he decides to cease or abandon tobacco use, or (iii) punctual, when the subject abandons tobacco consumption for a period variable time although short, in general, a few weeks or months.
• the cessation of tobacco use is a complete cessation.
• the verbal declaration of cessation of tobacco consumption when it is inhaled (smoked), can be validated by determining the levels of carbon monoxide (CO) in exhaled air, being the most common technique cooximetry for such determination.
• CO carbon monoxide
• Levels of 10 or more ppm of CO in exhaled air correspond to smoking subjects.
• tobacco refers to the abuse of tobacco consumption, causing the consumer of tobacco to suffer a physical and psychological dependence that generates a withdrawal syndrome.
• Tobacco has addictive power mainly due to nicotine, which also has Antidepressant effects and symptomatic relief of anxiety.
• the term “smoking” is sometimes used as a synonym for "tobacco addiction”; however, the term “smoking” is also used in relation to chronic intoxication that occurs due to tobacco abuse and groups the effects of repeated tobacco use and developed addiction.
• Tobacco addiction constitutes a risk factor for respiratory and cardiovascular diseases, and is especially harmful during pregnancy.
• Smoking is related to the occurrence of a high number of diseases, including several types of cancer, the most frequent being lung. In addition, tobacco not only harms active smokers, but also those who breathe the same air (passive smokers).
• agonist refers to a compound capable of generating a response in a cell after binding to a cellular receptor.
• the agonist compound binds to a receptor and has an intrinsic effect and, therefore, increases the basal activity of a receptor when it comes into contact with the receptor.
• antagonist refers to a compound that by binding to a cellular receptor blocks the activation of said cellular receptor by the agonist.
• the antagonist compound competes with the agonist compound for binding to a receptor, thereby blocking the action of the agonist on the receptor.
• an antagonist also known as a "neutral” antagonist
• Antagonists mediate their effects by binding to the active site or to allosteric sites in the receptors, or they can interact at unique binding sites that are not normally involved in the biological regulation of receptor activity.
• the antagonist activity can be reversible or irreversible depending on the longevity of the antagonist-receptor complex, which, in turn, depends on the nature of the antagonist-receptor binding.
• the agonists can be partial or complete.
• a "partial agonist" is defined as a compound that has an affinity for a receptor, but unlike a complete agonist, it will cause only a small degree of the peculiar pharmacological response to the nature of the receptor involved, even if a high proportion of receptors are occupied by the compound ..
• allele refers to one, two or more forms of a gene, locus or genetic polymorphism. Sometimes, different alleles can give rise to different phenotypes; however, other times, different alleles will have the same result in the expression of a gene. Most multicellular organisms have two sets of chromosomes, that is, they are diploid. These chromosomes are called homologous chromosomes. Diploid organisms have a copy of each gene (and an allele) on each chromosome. If both alleles are equal, they are homozygous. If the alleles are different, they are heterozygous.
• linkage block or "haplotypic block”, as used herein, refers to the set of SNPs on a particular chromosome that are statistically associated and configure a haplotype (combination of genetic markers).
• cytisine refers to the compound (1R, 5S) -l, 2,3,4,5,6-hexahydro-l, 5-methane-8H-pyrido [l, 2a] [1, 5] diazocine-8-one, of formula:
• Cinsine is a plant alkaloid of relatively rigid conformation, of molecular structure similar to that of nicotine and acetylcholine.
• Citisine is a natural insecticide found in the leaves of Cytisus laburnum and Ulex europaeus, among others.
• the synthesis of citisine and derivatives has been described in Coe JW 2000 Org Lett 2: 4205-4208 and O'Neill BT et al. 2000 Org Lett 2: 4201-4204.
• the term "citisine”, as used herein includes the derivatives mentioned in said scientific publications, among others.
• Cisine is marketed under the brand name Tabex ® and is part of the group of agonist drugs of nicotinic cholinergic receptors and is a compound that has an affinity for the a4B2 subtype of neuronal nicotinic acetylcholine receptors.
• Citisine is a natural insecticide found in the leaves of Cytisus laburnum and Ulex europaeus, among others. The synthesis of citisine and derivatives is described in Coe JW 2000 Org Lett 2: 4205-4208 and O'Neill BT et al. 2000 Org Lett 2: 4201-4204.
• tobacco consumption includes any form of tobacco consumption, constituting the inhalation of the products of the combustion of tobacco its most common form of consumption.
• smoking in the sense used in this description, refers to the action of sucking and firing tobacco smoke.
• the first, or mainstream which is the one that when aspirating a draft passes through the inside of the cigarette until it reaches the lungs of the active smoker
• the second, or secondary current which is the one that is released to the environment from the incandescent end of the cigarette and that can be inhaled by a subject breathing in that contaminated environment, said subject being the passive smoker.
• inhaling tobacco smoke the average smoker consumes between 1 and 2 mg of nicotine per cigarette.
• nicotine quickly reaches its maximum levels in the bloodstream and penetrates the brain.
• cigarette smoking is the most common and popular form of tobacco consumption
• other forms of smokeless tobacco consumption are also contemplated in the present invention, such as, for example, the consumption of tobacco powder or snuff and tobacco consumption to chew, that suppose forms of tobacco consumption by chewing, sucking, snorting, etc.
• These smokeless tobacco products also contain nicotine.
• nicotine is absorbed through the mucous membranes and reaches maximum levels in the blood and in the brain more slowly.
• Electronic cigarettes Another form of tobacco consumption, whose prediction of success or failure of cessation of consumption in subjects undergoing treatment with varenicline also falls within the objectives of the methods provided by the present invention, is given by electronic cigarettes.
• "Electronic cigarettes"("e-Cig") are devices that vaporize the substance contained in the cartridges, causing the expulsion of steam that mimics the smoke in the traditional cigarette and achieving a similar effect on the user.
• the mouthpiece of the device contains a replaceable or refillable cartridge filled with liquid.
• the main substances of said liquid are propylene glycol and / or vegetable glycerin, nicotine in different doses optionally (between 0 and 36 mg / ml in general), optional flavors and aromas.
• dianicline refers to the compound (5aS, 8S, 10aR) -5a, 6.9, 10-tetrahydro, 7H, HH-8, 10a-methanopyrid [2 ', 3': 5.6] pyran [2 , 3-d] azepine and has as formula:
• Dianicline is part of the group of agonist drugs of nicotinic cholinergic receptors and is a compound that has an affinity for the a4B2 subtype of neuronal nicotinic acetylcholine receptors.
• nicotinic cholinergic receptor agonist drug is understood as that drug capable, after interaction with any of the nicotinic cholinergic receptor subunits, to modify cellular response processes and generate a biological response (Florez et al., Pharmacology Humana, 4th edition, Masson, Barcelona, 2003).
• Illustrative, non-limiting examples of such drugs include cytisine, dianicline and varenicline, among others.
• biological sample includes any biological material that can be obtained from a subject, susceptible to conservation and that can house information on the genetic endowment characteristic of the subject.
• the sample of the invention can be of the cellular, tissue or fluid type.
• the sample is of blood and / or saliva.
• nicotine refers to an alkaloid compound found in the tobacco plant, responsible for the psychoactive and addictive effects of tobacco. Although about 5,000 chemical compounds have been identified in the different phases (gas, solid or particulate) of tobacco smoke, their nicotine content is what makes tobacco addictive. Nicotine is considered to be responsible for both tobacco addiction and the maintenance of smoking.
• nicotine when inhaling tobacco smoke, nicotine is rapidly absorbed both in the oral mucosa and in the lungs, from where it passes to the circulatory system, distributed throughout the body, and, in 7-10 seconds, nicotine arrives to the brain, where it binds to the nicotinic receptors producing a stimulating and / or sedative, pleasant and rewarding effect, which triggers the appearance of dependence on tobacco (pharmacological or physical dependence).
• the smoker adapts his nicotine inhalation pattern in terms of frequency, depth of inhalations and smoke retention time, depending on his own characteristics and the effect he intends to cause in each circumstance. These variations of the smoking pattern produce different concentrations of nicotine in the blood that yield different levels of nicotine supply to the body and different effects on its action.
• Nicotine produces a series of effects in the body, among which are an increase in blood pressure, an increase in heart rate or tachycardia, an increase in blood glucose and an increase in bowel movement.
• polymorphism refers to substitutions of a single nucleotide for another and that are observed in the general population at a frequency greater than 1%.
• SNP single nucleotide polymorphism
• SNP single nucleotide polymorphism
• SNPs represent one of the most common forms of genetic variation, where each version of the sequence with respect to the polymorphic site is referred to as an allele of the polymorphic site. SNPs occur throughout the entire genome, both in extragenic regions and in coding regions or their regulatory sequences.
• the change of a single nucleotide can cause: 1) the substitution of an amino acid, if it is in an exonic sequence, and vary the effectiveness of the protein's function; 2) modify the amount of the protein, if the SNP is in a regulatory region; or 3) change the structure of the protein, if the change is in a splicing zone.
• Mutations can be used as diagnostic and / or prognostic tools for the identification of subjects with a predisposition to a disease or to a rapid evolution of the disease, genotyping the subject suffering from the disease and facilitating the development of medicines based on the knowledge generated on the role of proteins in the pathogenesis process.
• S Ps can also be used as diagnostic and / or prognostic tools for the identification of subjects that respond well (or do not respond) or that present adverse effects to a specific therapy or to a specific pharmacological treatment since inherited interindividual differences In the pharmacokinetics and pharmacodynamics of drugs they are due to genetic variants that affect gene function or expression.
• These polymorphisms affect drug metabolizing enzymes, transporters and drug targets and can have a great influence on the efficacy and safety of a particular drug therapy or therapy.
• the genetic markers used in the invention are specific alleles at "polymorphic sites" associated with the success or failure of the cessation or abandonment of tobacco use by a subject under treatment with a cholinergic receptor agonist drug. nicotinics to stop using tobacco.
• a nucleotide position in the genome in which more than one sequence is possible in a population is known as a "polymorphic site.”
• a polymorphic site is only one nucleotide in length, the site is commonly called SP, as mentioned above, for example, if at a particular chromosomal location, a member of a population has an adenine (A) and another member of the population has a thymine (T) in the same position, so this position is a polymorphic site and, more specifically, the polymorphic site is an SNP.
• Each version of the sequence with respect to the polymorphic site is known as an allele of the polymorphic site.
• the SNP allows both an A allele and a T allele.
• variants of the nucleotide sequence can lead to changes in the sequence of the encoded polypeptide, which affects their properties (activity alteration, distribution alteration, altered stability , etc.).
• variants of the nucleotide sequence may result in changes that affect the transcription of a gene or the translation of its messenger RNA.
• the alterations can be qualitative, quantitative, or both.
• probability measures the frequency with which a result (or set of results) is obtained when conducting a randomized experiment, of which all possible outcomes are known, under sufficiently stable conditions.
• propensity in combination with the cessation of tobacco use in response to a varenicline-based therapy, refers to the propensity, or actual probability, of a subject to abandon tobacco use in response to said therapy for a certain period of time, for example, for a period of at least 3 months, counted from the start of treatment with varenicline.
• the probability can be "high” or "low.” As those skilled in the art will understand, the probability does not have to be 100% for all the subjects evaluated, although preferably it should be.
• a subject is statistically significant or not, it can be determined without major complications, by a person skilled in the art, using different known tools of statistical evaluation, for example, by determining confidence intervals, determining the value of p, the Student test, Mann-Whitney test, etc. Additional information on these statistical tools can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983.
• Preferred confidence intervals are at least 50%, at least 60%), at least 70%, at least 80%>, at least 90%, or at least 95%. P values are preferably 0, 1, 0.05, 0.02, 0.01 or less.
• subject refers to a human being, female or male, and of any race or age.
• the subject is an active consumer of tobacco, that is, consumes tobacco regularly or sporadically;
• said subject is an active smoker.
• a smoker of more than 20 cigarettes a day could be considered a "severe smoker "
• a smoker of between 10 and 20 cigarettes at day could be considered a "moderate” smoker
• a smoker of less than 10 cigarettes a day could be considered a "mild” smoker.
• tobacco refers to a product processed from the leaves of several plants of the Nicotiana genus, including N. tabacum, N. petunoides, N rustica and N. polidiclia.
• the N. tabacum species can be classified into four varieties, havanensis, brasilensis, virginica and purpurea, which are the origin of the different varieties used in the commercialization of tobacco.
• the tobacco-based products to which the methods of the invention are directed related to the prediction of success or failure of cessation of consumption in subjects undergoing treatment with varenicline include those products that are made totally or partially of tobacco, contain nicotine, and whose consumption occurs, for example, when smoking, sucking, chewing or snorting them.
• tobacco can be presented in the form of cigarettes [(also called cigarettes, cigarettes, etc.) that can be emboquillados or without filter, blond or black, "lights”, “semi-lights” or integers] , cigars, rolling tobacco, pipe smoking tobacco, snuff or snuff, etc.
• treatment refers to the administration of a drug to alleviate or eliminate a pathology or to reduce or eliminate one or more symptoms associated with said pathology.
• the treatment applied to the subject is a pharmacological treatment and is aimed at reducing or eliminating tobacco consumption in said subject and is based on the use of varenicline.
• the subject to whom the treatment is directed is an active consumer of tobacco, for example, an active smoker.
• personalized treatment or “personalized medicine”, as used herein, refers to the design and application of prevention, diagnosis and treatment interventions adapted to the patient's genetic substrate and the molecular profile of the disease.
• varenicline refers to the compound 7,8,9, 10-tetrahydro-6, 10-methane-6H-pyrazino [2,3-h] [3] benzacepin, of the formula: and also includes its pharmaceutically acceptable salts, such as, for example, those mentioned in the international patent application W099 / 35131, as well as its derivatives, for example, N-formyl-varenicline and N-methyl-varenicline. Varenicline is marketed in Spain in the form of its tartrate salt under the brand Chantix ® or Champix ® .
• Varenicline is part of the group of agonist drugs of nicotinic cholinergic receptors and is a compound that acts as a selective partial agonist of nicotinic acetylcholine receptors, which binds with great affinity and selectivity to the a4B2 subtype of neuronal nicotinic receptors of Acetylcholine
• Varenicline is used in the treatment of various diseases, including, by way of illustration, not limitation, chronic inflammatory bowel diseases (including, but not limited to, ulcerative colitis, pyoderma gangrenosum and Crohn's disease), irritable bowel syndrome, dystonia spasmodic, chronic pain, acute pain, celiac disease, reservoritis, vasoconstriction, anxiety, panic disorder, depression, bipolar disorder, autism, sleep disorder, time shift ("jet lag"), amyotrophic lateral sclerosis (ALS), cognitive dysfunction , hypertension, bulimia, anorexia, obesity, cardiac arrhythmia, gastric acid hypersecretion, ulcer, pheochromocytoma, progressive supranuclear paralysis, addiction and chemical dependence (nicotine, tobacco products, alcohol, benzodiazepine, barbiturates, opioids, cocaine), headache, migraine, sudden attack, traumatic brain injury (TBI), obsessive-compulsive disorder, psychosis ,
• the usual treatment for cessation of tobacco use with varenicline includes oral administration, twice a day, of 1 mg of compound, for 12 weeks Absorption is complete and systemic availability high. Maximum plasma concentrations typically occur at 3-4 hours after taking (Faessel HM et al. 2010, Clin Pharmacokinet 49 (12): 799-816).
• Method for determining the probability that a subject will quit smoking in response to a therapy based on a nicotinic cholinergic receptor agonist drug (“first method of the invention").
• the invention relates to a method for determining the probability that a subject will quit smoking in response to a therapy based on an agonist drug of nicotinic cholinergic receptors (hereinafter, "first method of the invention” ), which comprises determining in a biological sample of said subject, at least one allele of one or more of the single nucleotide polymorphisms (SNPs) rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, or of any SNP of its corresponding ligation blocks, where
• SNPs single nucleotide polymorphisms
• the invention also relates to a method for determining the probability of a subject quit smoking in response to a therapy based on a nicotinic cholinergic receptor agonist drug, which comprises determining in an biological sample of said subject, at least one allele of one or more of the S Ps rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, or of any SP of its corresponding linkage blocks, where
• At least one allele of one or more of the SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, or any SNP of their subjects is determined in a biological sample of the subject under study corresponding ligation blocks, and the results obtained are correlated with the probability (high or low) that the subject under study quit smoking in response to a therapy based on an agonist drug of nicotinic cholinergic receptors.
• nicotinic cholinergic receptor agonist drugs include cytisine, dianicline and varenicline.
• said nicotinic cholinergic receptor agonist drug is varenicline.
• the subject is a human being of any sex, race or age.
• the subject is an active consumer of tobacco, for example, a smoker of cigars, electronic cigars, cigars or pipes, or a consumer of snuff or snuff.
• said subject is an active consumer of tobacco who voluntarily wishes to stop using tobacco, or who must stop using tobacco, for example, by medical prescription.
• the biological sample from the subject under study is a biological sample that contains a nucleic acid, for example, DNA, genomic DNA (cDNA), complementary DNA (cDNA), RNA, heterogeneous nuclear RNA (mRNA), mRNA, etc., of the subject to evaluate.
• Said biological sample may be isolated or extracted from a tissue, or it may be present in a biological fluid, for example, blood, saliva, etc. Methods for the isolation of biological samples from cells and tissues are well known to those skilled in the art.
• said biological sample is a blood or saliva sample.
• the biological sample when it is a whole blood sample, it can be used directly in the practice of the first method of the invention.
• it is necessary to first extract the nucleic acid from the biological samples for example, cells present in a biological fluid; in that case, the total nucleic acids extracted from said biological samples represents the work material.
• the isolation of nucleic acids from a biological sample that contains can be performed by methods known to those skilled in the art [see for example, Sambrook et al, 2001 "Molecular Cloning: A Laboratory Manual", 3rd edition , Cold Spring Harbor Laboratory Press, New York, vol. 1.3]. Also, sometimes, it may be necessary to amplify the extracted nucleic acid before proceeding with its analysis.
• nucleic acid amplification Numerous methods of nucleic acid amplification are known; many of them are based on an enzymatic reaction, for example, polymerase chain reaction (PCR), ligase chain reaction (LCR), rolling circle amplification assays, "rolling-circle”, etc. . Information on such nucleic acid amplification methods can be found in Sambrook et al, 2001 (cited supra).
• one or more of the SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, or any SNP of their corresponding binding blocks are analyzed, and the nucleotides are determined present in said SNPs, in particular, nucleotides present in the polymorphic site the SNP that allows to identify the corresponding allele of the SNP.
• nucleotides present in one or more of said SNPs in the sample containing nucleic acid of the subject under study can be performed by any method or technique capable of determining nucleotides present in a polymorphic site.
• SNP any method or technique capable of determining nucleotides present in a polymorphic site.
• SNP sequencing, mini-sequencing, hybridization, restriction fragment analysis, oligonucleotide binding assays, allele-specific PCR, HRM (High Resolution Melt) analysis, etc., or by any combination of these methods.
• the person skilled in the art can use any appropriate method or technique to achieve that determination.
• Nucleotides present in SNPs and, in particular, at polymorphic sites can be detected from any strand of nucleic acid or from both strands.
• Illustrative, non-limiting examples of suitable methods, techniques or systems for analyzing nucleotides present at the polymorphic sites of the SNPs identified in the present invention include mini-sequencing methods, nucleic acid sequencing, hybridization, restriction fragment analysis. , oligonucleotide ligation assays, allele-specific PCR, HRM analysis, nucleic acid matrices ("DNA arrays"), for example, the technology available from Aclara BioSciences, Affymetrix, Agilent Technologies, Illumina Inc., Ion Torrent, Fluidigm, Nanopore Tech etc., techniques based on changing the mobility of amplified nucleic acid fragments, single strand conformation polymorphisms (SSCP), denaturing gradient gel electrophoresis (DGGE), CMC ("chemical mismatch cleavage” ), WAVE analysis, etc., or by any combination of such methods. Additional information on the analysis of the nucleotides present in the polymorphic sites of the SNPs can be found in Sambrook
• the first method of the invention comprises genotyping or determining at least one allele of one or more of the SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, and / or the corresponding alleles of the SNPs of their binding blocks, in the biological sample of the subject under study.
• the first method of the invention comprises determining a single allele of one or more of the SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, and / or the corresponding allele of any of the SNPs of its corresponding ligation blocks, that is, determining the nucleotide present in the polymorphic site of said SNP.
• a single allele of SNP rs678188 is determined; In another particular embodiment, a single allele of SNP rs9658498 is determined; in another particular embodiment, a single allele of SNP rs4821566 is determined; in another particular embodiment, a single allele of SNP rsl0891510 is determined; in another particular embodiment, a single allele of SNP rsl 1932367 is determined; in another particular embodiment, a single allele of SNP rs2023239 is determined; while, in another particular embodiment, a single allele of any of the SNPs of the corresponding binding blocks of the SNPs is determined rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 or rs2023239.
• the first method of the invention comprises determining the two alleles of one or more of said SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, and / or of any of the SNPs of their corresponding binding blocks .
• the two alleles of SNP rs678188 are determined; in another particular embodiment, the two alleles of SNP rs9658498 are determined; in another particular embodiment, the two alleles of SNP rs4821566 are determined; in another particular embodiment, the two alleles of SNP rsl0891510 are determined; in another particular embodiment, the two alleles of SNP rsl 1932367 are determined; in another particular embodiment, the two alleles of SNP rs2023239 are determined; while, in another embodiment In particular, the two alleles of any of the S Ps of the corresponding binding blocks of the SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 or rs2023239 are determined.
• the first method of the invention comprises determining one or both alleles of any 1, 2, 3, 4, or 5 SNPs of any of the SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, and / or of the SNPs of their corresponding ligation blocks, while in another particular embodiment, said method comprises determining one or both alleles of the 6 SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, and / or the corresponding SNPs of their corresponding ligation blocks
• the SNPs identified in the present invention associated with the probability (high or low) of a subject giving up tobacco use in response to a therapy based on an agonist drug of nicotinic cholinergic receptors, or with the prediction of the efficacy of a therapy to quit smoking based on an agonist drug of nicotinic cholinergic receptors, or with the selection of a subject for a therapy to quit smoking based on an agonist drug of nicotinic cholinergic receptors, or with the Selection of a therapy to be administered to a subject in need of therapy to abandon consumption, have the following characteristics:
• SNP rs678188 is located in the PARD3 gene (partitioning defective 3), on human chromosome 10 (lOpl 1.21), corresponds to the sequence [ACGCCGCCCATTTCCTATTTCCTACC [C / T] GTGCTGGATTTGCTGA AACCGTCAC] (SEQ ID NO: 1), and is located in the ligation block that extends from nucleotide 34484338 to nucleotide 34509032; SNP rs9658498 is located in the NOS1 (nitric oxide synthase /) gene, on human chromosome 12 (12q24.2-q24.31), corresponds to the sequence [GGTCTCTGATTGTAAAATGAACCAAA [C / T] GTAGCCTCTACCACC TTGCTTAGAA] (SEQ ID NO: 2), and is located in the linkage block that extends from nucleotide 117657521 to nucleotide 117681829;
• SNP rs4821566 is located in the CSF2RB gene (colony stimulating factor 2 beta receptor), on human chromosome 22 (22ql3.1), corresponds to the sequence: [GGGATCATCCTCATATTCTTGCAAGA [C / G] GAAAAGTTT ACCAGTGAGAACTAGG] (SEQ ID NO: 3), and is located in the linkage block that extends from nucleotide 37306899 to nucleotide 37319081;
• - SNP rsl0891510 is located in the NCAM1 gene ⁇ mural cell adhesion molecule /), on human chromosome 11 (1 lq23.1), corresponds to the sequence:
• SNP rsl 1932367 is located in the GRID2 gene (glutamate ionotrophic delta receptor), on human chromosome 4 (4q22), corresponds to the sequence:
• TACACACACA (SEQ ID NO: 5), and is located in the linkage block that extends from nucleotide 94555780 to nucleotide 94579593; Y
• SNP rs2023239 is located in the CNR1 gene ⁇ cannabinoid receptor /), on human chromosome 6 (6ql4-ql5), corresponds to the sequence:
• SNPs rs678188, rs9658, rs96548, rs96548, rs9658, rs9658, rs9658, rs9658, rs9658, rs9658 and rs2023239 correspond to SNPs as they are collected in the NCBI single site polymorphism database (dbSNP, NCBI, http://www.ncbi.nlm.nih.gov/snp; March 2011) and according to HapMap Genome Browser (version 24, Phase 1 & 2).
• nucleotide positions indicated for the linkage blocks are defined according to the Haploview software version 4.2 (corresponding to the September 2009 version).
• Polymorphic sites are the following:
• polymorphic site of SNP rs678188 corresponds to position 27 of the sequence SEQ ID NO: 1;
• polymorphic site of SNP rs9658498 corresponds to position 27 of the sequence SEQ ID NO: 2;
• polymorphic site of SNP rs4821566 corresponds to position 27 of the sequence SEQ ID NO: 3;
• polymorphic site of SNP rsl0891510 corresponds to position 27 of the sequence SEQ ID NO: 4;
• polymorphic site of SNP rsl 1932367 corresponds to position 27 of the sequence SEQ ID NO: 5; Y
• polymorphic site of SNP rs2023239 corresponds to position 27 of the sequence SEQ ID NO: 6.
• the specific nucleotide present at the polymorphic site of said SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239 allows to identify the corresponding allele; thus, the C allele of SNP rs678188 corresponds to the allele in which there is a C in the polymorphic site of said SNP; the T allele of SNP rs9658498 corresponds to the allele in which there is a T in the polymorphic site of said SNP; the G allele of SNP rs4821566 corresponds to the allele in which there is a G in the polymorphic site of said SNP; the G allele of SNP rsl0891510 corresponds to the allele in which there is a G in the polymorphic site of said SNP; the G allele of SNP rsl 1932367 corresponds to the allele in which there is a G
• Example 1 Studies carried out by the inventors (Example 1) have allowed the identification of nucleotides present in the polymorphic sites of the SNPs described in this document associated with a high probability that the subject under study will quit smoking in response to a varenicline-based therapy. , sometimes referred to in this description as "alleles associated with a high probability that a subject will quit smoking in response to a therapy based on a agonist drug of nicotinic cholinergic receptors "or simply” alleles of success. "These” alleles of success "are shown in Table 1.
• the success alleles indicated in Table 1 correspond to the TOP strand of the DNA according to the Ilumina nomenclature for the identification of DNA strands.
• the simplest case for the denomination of a strand occurs when one of the possible variations of the SNP is an adenine (A), and the remaining variation is either a cytosine (C) or a guanine (G).
• the sequence of that SNP is called TOP.
• T thymine
• BOT sequence of this SNP is called BOT.
• the SNP is a [A / T] or a [C / G]
• Illumina uses a "sequential walk" technique to name the strand [A / T] and [C / G] of the SNPs.
• the actual SNP is considered to be in the 'n' position.
• the sequences immediately before and after the SNP are ' ⁇ - and' ⁇ +, respectively.
• two base pairs before the SNP are at 'n-2' and two base pairs after the SNP 'n + 2', etc.
• the sequential walk continues until an unambiguous pairing occurs (A / G, A / C, T / C, or T / G).
• nucleotide A or T of the first unambiguous pair when nucleotide A or T of the first unambiguous pair is on the 5 'side of the SNP, then the sequence is called TOP.
• nucleotide A or T of the first unambiguous pair is on the 3 'side of the SNP, then the sequence is called BOT.
• the first method of the invention once at least one of the alleles of one or more of the SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, and / is determined in a biological sample of said subject or of any SNP of its corresponding linkage blocks, it is possible to determine the likelihood that the subject under study will quit smoking in response to a therapy based on an agonist drug of nicotinic cholinergic receptors, for example, varenicline, cytisine, dianicline, or any other drug with a similar mechanism of action.
• an agonist drug of nicotinic cholinergic receptors for example, varenicline, cytisine, dianicline, or any other drug with a similar mechanism of action.
• the establishment of the probability (high or low) for the subject under study to quit smoking in response to a therapy based on an agonist drug of nicotinic cholinergic receptors can be carried out based on the presence or absence of success alleles (that is, based on the presence or absence of nucleotides present at the polymorphic sites of said SNPs associated with a probability (high or low) that the subject under study quit smoking in response to therapy based on a nicotinic cholinergic receptor agonist drug) of said SNPS in the genome of the subject under study. Therefore, as previously mentioned:
• the first method of the invention - the presence of both alleles C of the S P rs678188, or of the corresponding alleles of the S Ps of its linkage block;
• high applied to the probability that a subject will quit smoking in response to a therapy based on an agonist drug of nicotinic cholinergic receptors, as used herein, refers to the frequency with which a result is obtained, in this case, that a subject stops using tobacco in response to a treatment with an agonist drug of nicotinic cholinergic receptors, is greater than 50%.
• the specific nucleotide present at the polymorphic site of said SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239 allows to identify the corresponding allele; thus, the T allele of SNP rs678188 corresponds to the allele in which there is a T in the polymorphic site of said SNP; the C allele of SNP rs9658498 corresponds to the allele in which there is a C in the polymorphic site of said SNP; the C allele of SNP rs4821566 corresponds to the allele in which there is a C in the polymorphic site of said SNP; the T allele of SNP rsl0891510 corresponds to the allele in which there is a T in the polymorphic site of said SNP; the A allele of SNP rsl 1932367 corresponds to the allele in which there is an A at
• the term "low” applied to the likelihood that a subject will quit smoking in response to a therapy based on an agonist drug of nicotinic cholinergic receptors refers to the frequency with which it obtains a result, in this case, that a subject stops using tobacco in response to a treatment with a nicotinic cholinergic receptor agonist drug, is less than 50%.
• SNPs rs678188, rs9658498 and rs4821566 formed by SNPs rs678188, rs9658498 and rs4821566; these SNPs are related to a high probability that a subject in response to a therapy based on an agonist drug of nicotinic cholinergic receptors will quit smoking for a period of at least 3 months from the start of said therapy; Y
• SNPs rsl0891510, rsl 1932367 and rs2023239 are related to a high probability that a subject in response to a therapy based on an agonist drug of nicotinic cholinergic receptors will quit smoking for a period of at least 12 months from the start of said therapy.
• the first method of the invention contemplates the possibility of analyzing the presence or absence in a biological sample of said subject at least one of the alleles of one or more of the SNPs rs678188, rs9658498, rs4821566 or any combination of said SNPs, and / or the corresponding SNPs of their linkage block, wherein
• the first method of the invention contemplates the possibility of analyzing the presence or absence in a biological sample of said subject at least one of the alleles of one or more of the SNPs rs678188, rs9658498, rs4821566 or any combination of said SNPs, and / or the corresponding SNPs of their linkage block, where
• the first method of the invention contemplates the possibility of analyzing the presence or absence in a biological sample of said subject at least one allele of one or more of the SNPs rsl0891510, rsl 1932367 and rs2023239, or any combination of said SNPs, and / or the corresponding SNPs of their linkage block, wherein
• the first method of the invention contemplates the possibility of analyzing the presence or absence in a biological sample of said subject at least one allele of one or more of the SNPs rsl0891510, rsl 1932367 and rs2023239, or any combination of said SNPs, and / or the corresponding SNPs of their linkage block, wherein
• the first method of the invention makes it possible to establish the probability that a subject will quit smoking in response to a therapy based on an agonist drug of nicotinic cholinergic receptors. If said probability is high, for example, due to the presence of a successful allele in at least one of the SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 or rs2023239 (Table 1), or the corresponding alleles of the SNPs of its linkage block, then said subject may be an a priori appropriate candidate to receive a therapy based on a agonist drug of nicotinic cholinergic receptors in order to quit smoking, for example, varenicline, cytisine or dianicline, among others. On the contrary, if said probability is low, then said subject is not an appropriate a priori candidate to receive a therapy based on an agonist drug of nicotinic choline
• the inventors have studied the possible influence of clinical variables, such as subjective stress, reasons for smoking and dependence according to the Fagerstrom scale, on the results of efficacy of said therapy based on an agonist drug of nicotinic cholinergic receptors with the in order to abandon tobacco consumption, as well as its role as confusing or modifying variables of the association studied, through the use of multiple logistic regression models.
• the first method of the invention further comprises analyzing at least one clinical variable related to tobacco consumption.
• said clinical variable related to tobacco consumption is selected from the group consisting of subjective stress, reasons for smoking, dependence according to the Fagerstrom scale, and Any of your combinations.
• This probabilistic model based on the determination of at least one of the alleles of one or more of the SNPs identified in the present invention, or of the corresponding alleles of the SNPs of its linkage block, optionally together with a structured clinical evaluation, constitutes a valuable tool for predicting success or failure, based on a high or low probability, of smoking cessation by a subject, in response to treatment with an agonist drug of nicotinic cholinergic receptors, for example, varenicline, cytisine or dianicline, or any other drug with a similar mechanism of action.
• an agonist drug of nicotinic cholinergic receptors for example, varenicline, cytisine or dianicline, or any other drug with a similar mechanism of action.
• the specialist eg, a doctor
• the specialist may optimize the therapeutic care of the subject by choosing, from the outset, the most appropriate therapy based on the probability (high or low) that the subject quit smoking in response to a therapy based on a nicotinic cholinergic receptor agonist drug and closely monitor the subjects under treatment. Therefore, the methods and means provided by the present invention can help physicians select the therapy to quit smoking more appropriate for a subject.
• side effects associated with treatment with nicotinic cholinergic receptor agonists in subjects whose treatment with that drug is ineffective can be avoided.
• Second method of the invention Method for predicting the efficacy of a therapy to quit smoking based on a nicotinic cholinergic receptor agonist drug.
• the invention in another aspect, relates to a method for predicting the efficacy of a therapy for quitting tobacco use based on an agonist drug of nicotinic cholinergic receptors in a subject (hereinafter, "second method of the invention"), comprising determining in an biological sample of said subject at least one allele of one or more of the single nucleotide polymorphisms (S Ps) rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, or any SP of their corresponding blocks ligation
• S Ps single nucleotide polymorphisms
• the invention also relates to a method of predicting the efficacy of a therapy for quitting tobacco use based on an agonist drug of nicotinic cholinergic receptors in a subject, which comprises determining in a biological sample of said subject, at least one allele of one or more of the SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, or of any SNP of its corresponding ligation blocks, wherein
• predict the efficacy of a therapy refers to the determination of the result obtained by a subject in response to a therapy to quit based tobacco use.
• a nicotinic cholinergic receptor agonist drug for example, varenicline, cytisine and dianicline, among others.
• At least one allele of one or more of the SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, or any SNP of their subjects is determined in a biological sample of the subject under study corresponding ligation blocks, and the results obtained are correlated with the prediction of the efficacy of a therapy to quit smoking based on an agonist drug of nicotinic cholinergic receptors in a subject.
• nicotinic cholinergic receptor agonist drugs include cytisine, dianicline and varenicline.
• said nicotinic cholinergic receptor agonist drug is varenicline.
• the prediction that such therapy will be effective or ineffective in a subject can be carried out based on the presence or absence of the success alleles (i.e., based on the presence or absence of nucleotides present at the polymorphic sites of said SNPs associated with a prediction that said therapy based on a nicotinic cholinergic receptor agonist drug will be effective or ineffective) of said SNPS in the genome of the subject under study.
• Such alleles of success in this case, alleles whose presence is associated with a prediction that therapy based on an agonist drug of nicotinic cholinergic receptors will be effective
• Table 1 In a particular embodiment, of the second method of the invention:
• the second method of the invention contemplates the possibility of determining at least one allele of one or more of the SNPs rs678188, rs9658498, rs4821566 or any combination of said SNPs, and / or the corresponding SNPs of their block of ligation where
• said therapy based on a nicotinic cholinergic receptor agonist drug will be effective in a subject for a period of at least 3 months from the start of said therapy.
• the second method of the invention contemplates the possibility of determining at least one allele of one or more of the SNPs rsl0891510, rsl 1932367, rs2023239, or any combination of said SNPs, and / or the corresponding SNPs of its linkage block, where
• said therapy based on a nicotinic cholinergic receptor agonist drug will be effective in a subject for a period of at least 12 months from the start of said therapy.
• the second method of the invention further comprises analyzing at least one clinical variable related to tobacco consumption.
• said clinical variable related to tobacco consumption is selected from the group consisting of subjective stress, reasons for smoking, dependence according to the Fagerstrom scale, and any of your combinations
• the combination of SNPs with clinical variables generally provides better models.
• the second method of the invention makes it possible to predict the efficacy of a therapy for abandoning tobacco consumption based on an agonist drug of nicotinic cholinergic receptors in a subject, so that if the result of said prediction is that the therapy will be effective, then the subject can be treated with a nicotinic cholinergic receptor agonist drug to quit smoking, while, otherwise, if the result of such prediction were that the therapy will be ineffective, then the subject should not be treated with a agonist drug of nicotinic cholinergic receptors.
• nicotinic cholinergic receptor agonist drugs include varenicline, cytisine or dianicline, or any other drug with a similar mechanism of action.
• This predictive model based on the determination of at least one of the alleles of one or more of the SNPs identified in the present invention, or of the corresponding alleles of the SNPs of its linkage block, optionally together with a structured clinical evaluation, constitutes a valuable tool for predicting the efficacy or ineffectiveness of a treatment with an agonist drug of nicotinic cholinergic receptors, for example, varenicline, cytisine or dianicline, or any other drug with a similar mechanism of action.
• an agonist drug of nicotinic cholinergic receptors for example, varenicline, cytisine or dianicline, or any other drug with a similar mechanism of action.
• the specialist eg, a doctor
• the specialist will be able to optimize the therapeutic attention to the subject by choosing, from the outset, the most appropriate therapy based on the prediction of the efficacy or ineffectiveness of the subject quit smoking in response to a therapy based on an agonist drug of nicotinic cholinergic receptors, and closely monitor the subjects under treatment.
• the methods and means provided by the present invention can help physicians select the therapy to quit smoking more appropriate for a subject.
• side effects associated with treatment with nicotinic cholinergic receptor agonists in subjects whose treatment with that drug is ineffective can be avoided.
• said nicotinic cholinergic receptor agonist drug is varenicline.
• the invention relates to a method for selecting a subject for a therapy to quit tobacco use based on a nicotinic cholinergic receptor agonist drug (hereinafter, "third method of the invention"), which comprises determining in a biological sample of said subject at least one allele of one or more of the single nucleotide polymorphisms (S Ps) rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, or any SP of its corresponding ligation blocks, where said subject is selected for said therapy if he has
• S Ps single nucleotide polymorphisms
• a subject is selected for a therapy to quit smoking based on a nicotinic cholinergic receptor agonist drug, when said subject has
• the characteristics of the biological sample, the subject, the SNPs (rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239) and their corresponding ligation blocks, as well as those of their polymorphic sites, have already been mentioned previously in relation to the first method of the invention, as well as the methods for detrminating said SNPs and identifying the nucleotides present at the polymorphic sites of said SNPs; All this is incorporated into this method by reference.
• At least one allele of one or more of the SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, or any SNP of its subjects is determined in a biological sample of the subject under study corresponding ligation blocks, and the results obtained are correlated with the possibility of selecting a subject to receive a therapy to quit smoking based on an agonist drug of nicotinic cholinergic receptors in a subject.
• nicotinic cholinergic receptor agonist drugs include cytisine, dianicline and varenicline.
• said nicotinic cholinergic receptor agonist drug is varenicline.
• the selection of the subject can be carried out based on the presence or absence of the alleles of success (ie, based on the presence or absence of the nucleotides present at the polymorphic sites of said S Ps associated with a prediction that said therapy based on a nicotinic cholinergic receptor agonist drug will be effective) of said SNPS in the genome of the subject under study.
• Such alleles of success in this case, alleles whose presence is associated with a prediction that therapy based on an agonist drug of nicotinic cholinergic receptors will be effective
• Table 1 are mentioned in Table 1.
• the third method of the invention further comprises analyzing at least one clinical variable related to tobacco consumption.
• said clinical variable related to tobacco consumption is selected from the group consisting of subjective stress, reasons for smoking, dependence according to the Fagerstrom scale, and Any of your combinations. The combination of SNPs with clinical variables generally provides better models.
• the third method of the invention allows selecting a subject to receive a therapy to quit smoking based on an agonist drug of the nicotinic cholinergic receptors, so that if the subject is selected then said subject can be treated with an agonist drug. of nicotinic cholinergic receptors to quit smoking, while, otherwise, if the subject is not selected for such therapy then the subject should not be treated with a nicotinic cholinergic receptor agonist drug.
• This selective model based on the determination of at least one of the alleles of one or more of the SNPs identified in the present invention, or of the corresponding alleles of the SNPs of its linkage block, optionally together with a structured clinical evaluation, constitutes A valuable tool for predicting the efficacy or ineffectiveness of a treatment with an agonist drug of nicotinic cholinergic receptors in a subject.
• the specialist eg, a physician
• receptor agonist drugs Nicotinic cholinergics include varenicline, cytisine or dianicline, or any other drug with a similar mechanism of action.
• the invention relates to a method for selecting a therapy for a subject in need of therapy to quit smoking (hereinafter, "fourth method of the invention"), which comprises determining in a biological sample of said subject at least one allele of one or more of the single nucleotide polymorphisms (S Ps) rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, or any SNP of their corresponding linker blocks, wherein a therapy is selected for abandon tobacco use based on a nicotinic cholinergic receptor agonist drug if said subject has
• S Ps single nucleotide polymorphisms
• a therapy is selected to quit smoking based on a nicotinic cholinergic receptor agonist drug, when said subject has
• the characteristics of the biological sample, the subject, the SNPs (rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239) and their corresponding ligation blocks, as well as those of their polymorphic sites, have already been mentioned previously in relation to the first method of the invention, as well as the methods for detrminating said SNPs and identifying the nucleotides present at the polymorphic sites of said SNPs; All this is incorporated into this method by reference.
• At least one allele of one or more of the SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239, or any SNP of its subjects is determined in a biological sample of the subject under study corresponding ligation blocks, and the results obtained are correlated with the possibility of selecting a therapy for a subject in need of receiving therapy to quit smoking.
• the selection of such therapy can be carried out based on the presence or absence of the success alleles (i.e., based on the presence or absence of nucleotides present at the polymorphic sites of said SNPs associated with a prediction that said therapy based on a nicotinic cholinergic receptor agonist drug will be effective) of said SNPS in the genome of the subject under study.
• success alleles i.e., based on the presence or absence of nucleotides present at the polymorphic sites of said SNPs associated with a prediction that said therapy based on a nicotinic cholinergic receptor agonist drug will be effective
• Such alleles of success are mentioned in Table 1.
• the fourth method of the invention further comprises analyzing at least one clinical variable related to tobacco consumption.
• said clinical variable related to tobacco consumption is selected from the group consisting of subjective stress, reasons for smoking, dependence according to the Fagerstrom scale, and Any of your combinations. The combination of SNPs with clinical variables generally provides better models.
• the fourth method of the invention allows to select a therapy to abandon the consumption of tobacco to be administered to a subject based on an agonist drug of the nicotinic cholinergic receptors, so that such therapy is selected, among other therapies to quit smoking , when the genotype of the subject to be subjected to therapy has the previously mentioned characteristics, while, otherwise, if the subject does not have those characteristics, then one would have to select a therapy to quit the consumption of tobacco that was not based on drugs Nicotinic cholinergic receptor agonists.
• This selective model based on the determination of at least one of the alleles of one or more of the SNPs identified in the present invention, or of the corresponding alleles of the SNPs of its linkage block, optionally together with a structured clinical evaluation, constitutes A valuable tool for predicting the efficacy or ineffectiveness of a treatment with an agonist drug of nicotinic cholinergic receptors in a subject.
• the specialist eg, a doctor
• the therapy to be administered to the subject in particular, a therapy based on an agonist drug of nicotinic cholinergic receptors (or other therapy based on others drugs), and closely monitor the efficacy of said selected therapy in subjects undergoing such therapy.
• nicotinic cholinergic receptor agonist drugs include varenicline, cytisine or dianicline, or any other drug with a similar mechanism of action. Kits of the invention
• kits of the invention comprising at least one reagent for determining the presence of at least one allele of one or more of the SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367, rs2023239, and / or the corresponding SNPs of its linkage block, and / or at least one reagent for determining the nucleotide present at the polymorphic site of said SNPs.
• kit of the invention comprising at least one reagent for determining the presence of at least one allele of one or more of the SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367, rs2023239, and / or the corresponding SNPs of its linkage block, and / or at least one reagent for determining the nucleotide present at the polymorphic site of said SNPs.
• the kit of the invention comprises at least one reagent for determining the presence of a single SNP selected from the group consisting of SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367, rs2023239 and the corresponding SNPs of their blocks ligation, and at least one reagent to determine the nucleotide present in the polymorphic site of said SNP.
• the kit of the invention comprises at least one reagent necessary to determine the presence of any 1, 2, 3, 4, 5, or even 6, SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367, rs2023239 , and / or in the corresponding SNPs of their linkage blocks, and / or at least one reagent necessary to determine the nucleotide present in said SNPs.
• the kit of the invention may also include means for extracting the sample from the subject and / or means or reagents for amplifying the nucleic acid present in said sample.
• kits of the invention include one or more reagents for use in accordance with the present invention in suitable containers and packaging materials, including tubes, vials, and packaging techniques such as shrink wrapping and blow molding.
• Materials suitable for inclusion in a kit according to the present invention comprise one or more of the following: pairs of PCR-specific primers (oligonucleotides) that hybridize with domains of the DNA or cDNA sequence flanking the SNPs of interest (SNPs) rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367, rs2023239 and the corresponding SNPs of their linkage blocks); reagents capable of amplifying a domain of a specific sequence, either in the CDNA or cDNA without having to perform the PCR; reagents necessary to discriminate between the different alleles in the sequence of the domains amplified by PCR and PCR-not amplification (for example, restriction endonucleases,
• Kits comprising one or more allele-specific oligonucleotides or polymorphism-specific or pairs of oligonucleotides are specifically contemplated, where each pair of allele-specific oligonucleotides or polymorphism-specific is directed to one of the polymorphism cited herein, i.e. S Ps rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239 and / or the corresponding SNPs of their linkage blocks.
• S Ps rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239 and / or the corresponding SNPs of their linkage blocks i.e. S Ps rs678188, rs9658498, rs4821566, rsl0891510,
• kits comprising a set of probes, comprising a plurality of oligonucleotide probes that interrogate SNPs selected from the group consisting of SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367 and rs2023239 and / or the corresponding SNPs of their linkage blocks, wherein said oligonucleotide probes account for at least 50% of the oligonucleotide probes of the probe set.
• said set of probes comprising a plurality of oligonucleotide probes that interrogate for SNPs selected among said SNPs, wherein said oligonucleotide probes account for at least 50% of the oligonucleotide probes of the probe set, constitutes a further embodiment of this invention.
• the kit comprises one or more pairs of polymorphism-specific or allele-specific oligonucleotides directed to two or more of the SNPs cited above, while another embodiment contemplates one or more oligonucleotides or pairs of polymorphism-specific or allele-specific oligonucleotides targeting all SNPs rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367, rs2023239 and / or the corresponding SNPs of their ligation blocks
• the kit of the invention allows prediction of success in smoking cessation in response to a therapy based on an agonist drug of nicotinic cholinergic receptors. Therefore, in another aspect, the invention relates to the use of the kit of the invention for:
• the invention relates to the use of at least one SNP to: determine the likelihood that a subject will quit smoking in response to therapy based on an agonist drug of nicotinic cholinergic receptors; or for
• S P is selected from the group consisting of S Ps rs678188, rs9658498, rs4821566, rsl0891510, rsl 1932367, rs2023239 and any combination thereof and / or any SNP of their corresponding linkage blocks.
• said SNP is selected from the group consisting of SNPs rs678188, rs9658498, rs4821566, or any combination of said SNPs, and / or the corresponding SNPs of its linkage block.
• said SNP is it selects from the group formed by the SNPs rsl0891510, rsl 1932367, rs2023239, or any combination of said SNPs, and / or the corresponding SNPs of its linkage block.
• the invention relates to a therapy for abandoning tobacco use based on an agonist drug of nicotinic cholinergic receptors for use in the treatment of a subject in need of therapy to abandon tobacco use, wherein said subject is selected by the third method of the invention.
• said nictoinic cholinergic receptor agonist drug is selected from varenicline, cytisine and dianicline, preferably carenicline.
• Nucleic acid samples can be genotyped to determine the alleles that are present in a particular gene region of interest, for example, the position of a particular SNP, by methods well known in the state of the art.
• Candidate SNPs were identified by 3 different strategies, mainly aimed at including SNPs with a potential functional impact on protein structure and / or gene expression, as well as from the information of common variations throughout a given gene.
• nsSNPs SNPs located in potential regions (5 'upstream, 5'-UTR, coding region, splicing sites, first intron, 3'-UTR and downstream 3').
• SNPs were searched from 20 kilobases (kb) upstream 5 'from the start codon to 10 kb downstream 3' from the stop codon for a given gene.
• the SNPs included in this group were those with a possible functional effect, specifically those that alter potential binding sites to transcription factors such as those that can be predicted through the use of Pupas View software (Conde et al. 2005, Nucleic Acid Res 33: W501-5) and SNPs located in conserved sequences between species, as determined by the use of UCSC Genome Browser Datábase (http://genome.ucsc.edu/index.html).
• target SNPs were selected.
• the main objective of this selection was to identify a set of SNPs targeting almost all known SNPs in a gene, including those located in potential regulatory regions that showed no evidence of functionality.
• the target SNPs were defined as common SNPs with an estimated R2 greater than 0.8 (r2> 0.8) with other SNPs in subjects with European ancestry (CEU) in the HapMap database (Tagger, Haploview 3.2 ) (Barrett et al, 2005, Bioinformatics 21 (2): 263-265).
• CEU European ancestry
• SNPs with a described frequency of minor allele greater than 5% were selected in Caucasian populations of HapMap-CEU and PERLEGEN-EUR-Panel North America (ENSEMBL genome browser v. 3.3 -3.6; www.ensembl.org), with the objective of guaranteeing informative SNPs. All selected SNPs were filtered using the Illumina technology criteria (score> 0.6 or GoldengateValidated Status, Illumina Inc., San Diego, USA). The genotyping of SNPs was carried out at the National Genotyping Center (CeGen), Spain, using the Illumina Bead Array system (Illumina Inc., San Diego; USA).
• Illumina Bead Array technology is based on particles ("beads") of 3 ⁇ that self-aggregate in microwells on one of the substrates: fiber optic beams or flat silica surfaces. When the particles are randomly assembled on one of these 2 substrates, they are arranged with a uniform separation of approximately 5.7 mm. Each particle is coated by hundreds of thousands of copies of a specific oligonucleotide that acts by capturing the sequences in one of Illumina's assays. On average, each type of particle is represented 30 times per sample, allowing a high degree of accuracy. The GoldenGate Genotyping assay genotyping assay was selected.
• allele-specific oligonucleotides hybridize to gDNA, extend and bind to a locus-specific oligonucleotide (LSO).
• PCR was carried out using universal primers.
• the products of the multiplex reaction were hybridized for detection and analysis to a universal Sentrix Array.
• the results obtained in DNA samples of blood duplicates were consistent with all SNPs genotyped by this method.
• the DNA was quantified at the National Genotyping Center (CeGen) using the PicoGreen technique and diluted to a final concentration of 50 ng / ml. With this technique, the DNA concentration was determined by a fluorescent marker (PicoGreen ® , Molecular Probes) that binds to double stranded DNA and subsequent quantification in a fluorimeter.
• PicoGreen ® fluorescent marker that binds to double stranded DNA and subsequent quantification in a fluorimeter.
• TSN nicotine replacement therapy
• the clinical results were related to the efficacy of pharmacological treatments.
• the efficacy was evaluated 3 and 12 months after the start of treatment, according to previously established withdrawal criteria such as carbon monoxide measurement, clinical interviews and patient information.
• candidate genes 80 candidate genes were selected based on their biological relevance and literature search as well as based on the bets of the candidate candidate genes.
• 384 SNPs, insertion / deletion and tandem repeats were analyzed. For each gene the selection of the SNP was based on the following criteria:
• SNPs located in regions that may influence the function of the gene upstream, 5'UTR, coding region, splicing sites, intron 1, 3'UTR and downstream).
• the results shown below present the adjustment of two prediction models.
• the first is a univariate model that only includes the genotype as an explanatory variable, while the second also includes clinical variables (subjective stress, reasons for smoking, and dependence according to the Fagerstrom scale).
• the explanatory power of both models has been checked using the likelihood ratio test for nested models. A significant value indicates the need to explain the result through the multivariate model, while a non-significant p-value implies that the univariate model (genotype) would be sufficient to explain the result.
• the C / T and C / C genotypes are associated with greater withdrawal at 3 months, with the C allele being the most important factor in explaining the success of withdrawal.
• the prediction is better for the multivariate model (66% based on the area under the curve) than for the univariate.
• the significance of «-value for nested models indicates that the multivariate model is significantly better than the univariate in predicting the outcome.
• Figure 1 shows the ROC curves corresponding to the univariate (upper graphic) and multivariate (lower graphic) models in relation to the SNP rs678188. rs9658498 (Chr 12; US 1)
• Figure 2 shows the ROC curves corresponding to the univariate (upper graphic) and multivariate (lower graphic) models in relation to the SNP rs9658498.
• rs4821566 Chor 22; CSF2RB
• SNPs rs678188, rs9658498 and rs4821566 have a predictive value of efficacy at three months of a pharmacological treatment for smoking cessation based on varenicline when analyzed together.
• T the most important factor in the explanation of failure in withdrawal.
• the prediction is better for the multivariate model (64% based on the area under the curve) than for the univariate.
• the significance of «-value for nested models indicates that the multivariate model is significantly better than the univariate in predicting the outcome.
• Figure 4 shows the ROC curves corresponding to the univariate (upper graphic) and multivariate (lower graphic) models in relation to SNP rsl0891510. rsl 1932367 (Chr 4; GRID2)
• Figure 5 shows the ROC curves corresponding to the univariate (upper graph) and multivariate (lower graph) models in relation to SNP rsl 1932367. rs2023239 (Chr 6; CNR1)
• the C / T and C / C genotypes are associated with greater abstinence at 12 months, with the C allele being the most important factor in explaining success in withdrawal.
• the prediction is better for the multivariate model (66% based on the area under the curve) than for the univariate.
• the significance of «-value for nested models indicates that the multivariate model is significantly better than the univariate in predicting the outcome.
• Figure 6 shows the ROC curves corresponding to the univariate (upper graph) and multivariate (lower graph) models in relation to SNP rs2023239.
• AAIGRT Upstream intergenic transcriptional regulator
• AANF Upstream non-functional
• AARR Upstream regulatory region
• AARRP Upstream promoter region regulator
• AART Upstream transcriptional regulator
• ABNF Downstream non-functional
• ABRT Downstream transcriptional regulator
• CG CpG Site
• IGNF Non-functional intergenic
• IGRT Intergenic transcriptional regulator
• IP Intronic enhancer
• IRT Intronic transcriptional regulator
• ISI Intronic without information
• ND Data not available.
• AAIGRT Upstream intergenic transcriptional regulator
• AANI Upstream no information
• AARR Upstream regulatory region
• AARRP Upstream promoter region regulator
• AART Upstream transcriptional regulator
• ABNF Downstream non-functional
• ABRT Downstream transcriptional regulator
• CG CpG Site
• IGNF Non-functional intergenic
• IGRT Intergenic transcriptional regulator
• IP Intronic enhancer
• IRT Intronic transcriptional regulator
• ISI Intronic without information
• ND Data not available.
• SEQUENCE LISTING means "LIST OF SEQUENCES”.

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