EP1315837A2 - Polymorphismes du gene du recepteur de la 5-hydroxytryptamine et reponse au traitement - Google Patents

Polymorphismes du gene du recepteur de la 5-hydroxytryptamine et reponse au traitement

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Publication number
EP1315837A2
EP1315837A2 EP01968309A EP01968309A EP1315837A2 EP 1315837 A2 EP1315837 A2 EP 1315837A2 EP 01968309 A EP01968309 A EP 01968309A EP 01968309 A EP01968309 A EP 01968309A EP 1315837 A2 EP1315837 A2 EP 1315837A2
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Prior art keywords
leu
val
ser
pro
arg
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Inventor
Kourtney J GlaxoSmithKline DAVIS
Mary E GlaxoSmithKline FLING
Arlene R GlaxoSmithKline HUGHES
Penelope K GlaxoSmithKline MANASCO
Michael J GlaxoSmithKline STUBBINS
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Glaxo Group Ltd
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Glaxo Group Ltd
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/10—Laxatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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 studies relate to polymorphisms in the 5-hydroxytryptamine 3 receptor (5HT3R) gene, polymorphisms in the UDP-glucuronosyltransferase 1 A4 (UGT1 A4) gene, and phenotypes that are associated or correlated therewith. More particularly, the present studies relate to the correlation of such polymorphisms to the response of subjects with gastrointestinal disorders (such as Irritable Bowel Syndrome (IBS)) to pharmaceutical treatment. The present studies further relate to methods of screening compounds for pharmaceutical activity. The present studies also relate to methods of genotyping subjects for predictive purposes.
  • IBS Irritable Bowel Syndrome
  • IBS Irritable Bowel Syndrome
  • IBS Irritable bowel syndrome
  • IBS may be characterized by altered bowel habit symptoms of either constipation (constipation predominant subtype) or diarrhea (diarrhea predominant subtype), or alternating constipation and diarrhea (alternator subtype).
  • constipation predominant subtype constipation predominant subtype
  • diarrhea diarrhea predominant subtype
  • alternate constipation and diarrhea alternate subtype.
  • IBS there is no single pathophysiological or diagnostic marker of IBS.
  • various diagnostic criteria for IBS are available, e.g.., Thompson et al., Gastroent. Int., 2:92 (1989); Manning et al., Br. Med. J. 2:653 (1978); Thompson et al., Gut 45:1143 (1999).
  • Alosetron hydrochloride (CAS registry number: CAS- 122852-69- 1 ; see US Patent
  • No. 5,360,800 is a 5-HT3 receptor antagonist. Both animal and human studies indicate that 5-HT3 receptor blockade has therapeutic value in the treatment of irritable bowel syndrome, particularly in diarrhea-predominant IBS. (The disclosures of all US patents cited herein are incorporated herein by reference in their entirety.) In double-blind, placebo controlled studies, alosetron hydrochloride has been shown to reduce pain and improve bowel function in female IBS patients whose predominant bowel symptom is diarrhea.
  • 5-hvdroxytryptamine receptors Molecular cloning has revealed the presence of at least fifteen 5- hydroxytryptamine (5-HT) receptor subtypes, which may be divided into various subfamilies based on structure and function.
  • the 5-hydroxytryptamine receptors are not limited to a single tissue type, but are found in the brain, spinal cord, heart and gastrointestinal tract, as well as other cell types.
  • the 5-HT3 receptors are ligand-gated ion channels (Davies et al., Nature 397:359 (1999)); other 5-HT receptors belong to the superfamily of G-protein-coupled receptors. Gerhardt et al., Eur J Pharmacol 1997 Sep 3;334(l):l-23.
  • 5-HT receptors for which little or no pharmacological or functional data exist. Sleight et al., Ann N YAcad Sci 1998 Dec 15;861:91-6. In the gastrointestinal tract, various 5-hydroxytryptamine receptors have been identified and characterized, including 5HT3, 5HT4, and 5HTla receptors; these receptors modulate gut motility and visceral sensory pathways. Various 5HT3 receptor antagonists (e.g., alosetron, granisetron and ondansetron) have been identified for the treatment of IBS. This class of drug appears to reduce visceral sensitivity and has inhibitory effects on motor activity in the distal intestine.
  • 5HT3 receptor antagonists e.g., alosetron, granisetron and ondansetron
  • Full and partial 5HT4 agonists are potential therapeutics to improve constipation-predominant IBS. Preliminary studies suggest that these agents may have therapeutic potential in IBS. Farthing et al., Baillieres Best Pract Res Clin Gastroenterol. 1999 Oct;13(3):461-71. 5HT4 antagonists (piboserod, SB-207266A) have also been suggested for the treatment of IBS.
  • UDP-glucuronosyltransferases catalyze by glucuronidation various endogenous agents, including steroids, bile acids, and bilirubin. The products are rendered more polar, thereby facilitating excretion from the cell.
  • UGTs UDP-glucuronosyltransferases
  • a large family of UGTs exists, allowing for glucuronidation of numerous structurally diverse compounds (Tukey and Strassburg, Ann Rev Pharmacol Toxicol 40:581, 2000). Based upon overall structural similarities, the known UGTs are derived from either the UGT1 or UGT2 gene.
  • the human UGT1 proteins are encoded from a single complex locus on chromosome 2 which contains at least 12 promoters/first exons; each encodes approximately 285 amino acids of the amino-terminal portion of the protein (Ritter et al., J. Biol. Chem 267:3257 (1992); Strassburg et al., Mol. Pharmacol 52:212 (1997)). Exons 2-5 encode the carboxyl terminal half of the UGT1 proteins.
  • the unique UGT1- transcripts are produced by splicing, providing mRNAs that encode different amino- terminal regions but having identical carboxyl-terminal portions. Nomenclature of the UGT gene superfamily is discussed in Mackenzie et al., Pharmacogenetics 7:255 (1997).
  • the present inventors have determined that polymorphisms in the 5- hydroxytryptamine-3 receptor (5HT3R) gene is correlated with the response of subjects with Irritable Bowel Syndrome (IBS) to pharmaceutical therapy. More particularly, they have found that the C178T polymorphism (as defined herein) in the 5HT3R gene is associated with the response of patients with IBS to treatment with a 5HT antagonist; and have identified a genetic subset of IBS patients that displays a higher incidence of adequate relief of IBS symptoms when treated with alosetron (compared to patients with an alternative polymorphism at the same site of the 5HT3R gene).
  • a first aspect of the present invention is a method of screening a patient population to identify those subjects with an increased likelihood of responding favorably to treatment with a 5HT ligand for a gastrointestinal disorder.
  • the subjects may have been previously diagnosed as having IBS, or the screening may be used in conjunction with IBS diagnostic efforts.
  • a further aspect of the present invention is a method of screening a subject suffering from a gastrointestinal disease that is treatable with a 5-hydroxytryptamine (5HT) ligand, as an aid in predicting the subject's response to treatment with a 5HT ligand.
  • the method comprises obtaining a sample of the subject's DNA and determining the genotype of the subject at a polymorphic allelic site in the 5HT3R gene, where different genotypes at that site have been associated with different rates of a phenotypic response to treatment with a 5HT ligand.
  • the genotype that is detected in the sample indicates that the subject is likely to have the phenotypic response associated with that genotype.
  • the genotype of the subject may be determined at a polymorphic site in the UGT1 A4 gene, where different genotypes at that site have been associated with different rates of phenotypic response to treatment with a 5HT ligand.
  • a further aspect of the present invention is a method of screening a subject with irritable bowel syndrome (IBS), as an aid in predicting the subject's response to treatment with a 5HT ligand. The method comprises obtaining a sample of the subject's DNA and determining the genotype of the subject at a polymorphic allelic site in the 5HT3R gene, where different genotypes at that site have been associated with different rates of a phenotypic response to treatment with a 5HT ligand.
  • IBS irritable bowel syndrome
  • a further aspect of the present invention is a method of screening a 5- hydroxytryptamine (5HT) ligand for variations in a measurable phenotypic effects among genetic subpopulations of subjects with a gastrointestinal disorder.
  • the method comprises administering the 5HT ligand to a population of subjects suffering from the gastrointestinal disorder, and obtaining DNA samples from each of the subjects.
  • the DNA samples are genotyped for a polymorphic allele of the 5HT3R gene, and correlations between the polymorphic allele genotype and the occurrence of a phenotypic response in the population of subjects are determined.
  • Detection of a genotype that is correlated with an increased or decreased incidence of a desired therapeutic response or a side effect indicates that the effectiveness of the ligand in treating that gastrointestinal disorder varies among genetic subpopulations.
  • a further aspect of the present invention is a method of treating subjects with Irritable Bowel Syndrome by administering a 5HT3 receptor antagonist, where the patients have a polymorphism in the 5HT3R gene that is predictive of a higher rate of adequate relief of IBS symptoms or a lower incidence of side effects when treated with a 5HT3 receptor antagonist.
  • the rate of relief is increased (and of side effects decreased) compared to subjects who have an alternative polymorphism at the same site of the 5HT3R gene.
  • a further aspect of the present invention is a method of treating subjects with Irritable Bowel Syndrome by administering alosetron to the subjects, where they have a polymorphism in the 5HT3R gene that is predictive of a higher rate of relief of IBS symptoms and/or a lower incidence of side effects when treated with alosetron (compared to subjects with an alternative polymorphism at the same site of the 5HT3R gene).
  • a further aspect of the present invention is a method of treating a subject suffering from Irritable Bowel Syndrome (IBS), by identifying the genotype of the subject at a polymorphic allelic site in the 5HT3R gene, where different genotypes at this site are associated with different rates of phenotypic response to treatment of IBS with a 5HT receptor ligand.
  • the subject is administered a 5HT receptor ligand that is associated with an increased rate of a favorable phenotypic response in subjects with the identified genotype.
  • the present invention is concerned with the treatment of gastrointestinal disorders mediated by 5HT receptors, more particularly with the treatment of Irritable Bowel Syndrome (IBS).
  • IBS Irritable Bowel Syndrome
  • the present inventors have determined that polymorphic variations in the 5HT3R and UGT1 A4 genes can be correlated to, or associated with, the response to pharmaceutical treatment, particularly treatment with 5HT receptor ligands and more particularly treatment with 5HT3 receptor antagonists including alosetron.
  • the present inventors have identified that polymorphisms in the 5HT3R and UGT1A4 genes are correlated with the response of subjects with a gastrointestinal disorder to treatment with a 5HT receptor ligand.
  • genetic samples were obtained from 237 subjects enrolled in two clinical trials of alosetron for the treatment of non-constipation predominant IBS.
  • the genetic samples were screened for the presence of the C178T polymorphism (as defined herein) in the 5HT3 Receptor (5HT3R) gene, and for the presence of the C70A polymorphism (as defined herein) in the UGT1 A4 gene (as described below), using polymerase chain reaction (PCR) technology as is known in the art.
  • PCR polymerase chain reaction
  • GenBank accession number AJ005205 (Brass et al., Neuropharmacology 39:308 (2000)).
  • GenBank accession number D49394 GenBank accession number D49394 (Miyake et al., Mol. Pharmacology
  • a polymorphism in the 5' region of the human 5HT3R gene affects position -42
  • this polymorphism is referred to as the C178T polymorphism.
  • Polymorphic changes in the 5' nucleotide sequence of a gene may affect transcriptional efficiency.
  • nucleotide sequence of exon 1 of the human UGT1A4 isozyme is provided at GenBank Accession No. M84128 as:
  • the present inventors determined that, for the C178T polymorphism in the 5HT3R gene, the homozygous genotypes (either homozygous for C at position -42, or homozygous for T at position -42, using the numbering provided above) are associated with an increased rate of adequate relief of IBS symptoms when treated with a 5HT3 receptor antagonist, and therefore an increased incidence of favorable therapeutic response to treatment with a 5HT3 receptor antagonist (compared to subjects with the heterozygous (1,2) genotype treated with the same 5HT3 receptor antagonist, see Table 1).
  • the C178T polymorphism may be either a marker polymorphism or a functional polymorphism.
  • the C70A polymorphism may be either a marker polymorphism or a functional polymorphism.
  • subjects who suffer from a gastrointestinal disease that is treatable with 5HT ligands can be genetically screened, to aid in predicting their response to such treatment.
  • Screening comprises obtaining a sample of DNA from the subject and analyzing the DNA to determine the genotype (presence/absence of polymorphic alleles) at a predetermined polymorphic site in the 5-hydroxytryptamine 3 receptor (5HT3R) gene, where different genotypes at that site have previously been associated with different rates of a phenotypic response to treatment with a 5HT ligand for gastrointestinal disease.
  • 5HT3R 5-hydroxytryptamine 3 receptor
  • the genetic screening may further include determination of the genotype (presence/absence of polymorphic alleles) at a predetermined polymorphic site in the UGT1A4 gene, where different genotypes at that site have previously been associated with different rates of a phenotypic response to treatment with a 5HT ligand for gastrointestinal disease.
  • the method may include stratifying subjects according to both their 5HT3R genotype and their UGT1 A4 genotype, where a particular combination of polymorphic alleles in these genes has been determined to be associated with different rates of a phenotypic response to treatment with a 5HT ligand for gastrointestinal disease. The presence of a particular predetermined genotype therefore indicates an increased likelihood that the individual subject will exhibit the associated phenotype.
  • genotyping a subject as described herein will be an aid in predicting the response a subject will have to treatment with a 5HT ligand, and thus assist in the treatment decision.
  • genotyping a subject (or DNA sample) for a polymorphic allele at a defined genomic locus or "determining the genotype at a polymorphic allelic site” means detecting which forms of the allele are present in a subject (or a sample).
  • an individual may be heterozygous or homozygous for a particular allele. More than two forms of an allele may exist, as is the case with microsatellite markers; thus there may be more than three possible genotypes.
  • a "genetic subset" of a population consists of those members of the population having a particular genotype. In the case of a biallelic polymorphism, a population can potentially be divided into three subsets: homozygous for allele 1 (1,1), heterozygous (1,2), and homozygous for allele 2 (2,2).
  • a subject that is "predisposed to" a particular phenotypic response based on genotyping of a polymorphic allele will be more likely to display that phenotype than an individual with a different genotype at that polymorphic allele.
  • the phenotypic response is based on a biallelic polymorphism, the response may differ among the three possible genotypes.
  • a gastrointestinal disease 'treatable with 5HT ligands' is one in which the administration of a 5HT ligand (in an appropriate pharmaceutical formulation, and in a therapeutically effective amount) has been shown to reduce or alleviate symptoms, without causing unacceptable side effects.
  • Regulatory Authority eg FDA, EMEA
  • Therapeutically effective amounts of such compounds can be readily determined by those skilled in the art using, e.g., dose-response studies.
  • the term '5HT ligand' encompasses antagonists and agonists of 5HT receptors, including partial agonists, and drugs that interact with the 5-hydroxytryptamine transporter (5HTT) (e.g., selective serotonin re-uptake inhibitors, SSRIs).
  • 5HT ligands may bind to any subtype of the 5HT receptor, including 5HT3 and 5HT4 receptors; the ligands may be specific for a particular receptor subtype.
  • 5HT-related compounds include 5HT3 receptor antagonists (e.g., alosetron, ondansetron, granisetron, tropisetron, dolasetron, mirtazapine, itasetron, pancopride, zatosetron, azasetron, cliansetron, YM-144 (Yamanouchi) and RS17017 (Roche)).
  • 5HT3 receptor antagonists e.g., alosetron, ondansetron, granisetron, tropisetron, dolasetron, mirtazapine, itasetron, pancopride, zatosetron, azasetron, cliansetron, YM-144 (Yamanouchi) and RS17017 (Roche)).
  • 5HT4 agonists are also known, including tegaserod, prucalopride, norcisapride and the 4-amino-5-chloro-2-methoxy-N-(l -substituted piperidin-4-yl)benzamide known as Y-34959 (Yoshitomi Pharmaceuticals), and buspirone.
  • the use of 5HT4 agonists to treat constipation-predominant IBS has been proposed.
  • 5HT4 antagonists include piboserod (SmithKline Beecham).
  • Dual 5HT3 and 5HT4 receptor agonists include renzapride (SmithKline Beecham) and E3620 (Eisai).
  • a 5HTla receptor agonist is also known, LY315535 (Eli Lilly).
  • Selective serotonin re-uptake inhibitors include fluoxetine, etc.
  • a "side effect" is an undesirable response to the administration of a therapeutic compound, i.e., an effect that is not directed to alleviating the symptoms or cause of the disease being treated. Side effects range from minor inconveniences to more serious events.
  • a "favorable" phenotypic response to treatment is a response in which adequate relief of a symptom (e.g., pain, urgency, altered bowel habit) is achieved with an acceptable level of side effects. A particular phenotypic response may be more favorable (e.g., achieve a more extensive reduction of symptoms) than another.
  • Polymorphisms are variant sequences within the human genome that may or may not have a functional consequence. These variants can be used in all aspects of genetic investigation including the analysis and diagnosis of genetic disease, forensics, evolutionary and population studies. Two types of genetic analyses are typically performed: linkage and association studies.
  • a linkage study provides genetic map information with no prior knowledge or assumption about the function of a gene.
  • a linkage study one uses DNA polymorphisms to identify chromosomal regions that are identical between affected relatives with the expectation that allele sharing frequencies will be higher for a marker (polymorphism) whose chromosomal location is close to that of the disease allele.
  • Physical cloning of a linkage region narrows down the DNA sequence that could harbor the candidate disease gene. While linkage analysis locates the disease locus to a specific chromosome or chromosome region, the region of DNA in which to search for the gene is typically large, on the order of several million base pairs.
  • association shows the coexistence of a polymorphism and a phenotype in a population.
  • Association studies are based upon linkage disequilibrium, a phenomenon that occurs between a marker and a phenotype if the marker polymorphism is situated in close proximity to the functional polymorphism. Since the marker and functional polymorphism are in close proximity, it requires many generations of recombination to separate them in a population. Thus they tend to co-exist together on the same chromosome at a higher than expected frequency.
  • a marker is said to be associated with a specific phenotype when its frequency is significantly higher among one phenotype group compared to its frequency in another. In general, the closer a marker is to the functionally polymorphic site, the stronger the association.
  • the present inventors have determined that polymorphisms in the 5HT3R and the UGT1A4 genes play a role in the response of subjects to pharmaceutical treatment of IBS, thus the genotyping of these genes (either directly or via the gene's expression product) will be useful in identifying therapeutic compounds with measurable effects that vary among subject genotypes.
  • the effect to be measured will depend on the particular gastrointestinal condition, therapeutic compound, and patient population, as will be apparent to one skilled in the art.
  • the measurable effect may be the relief of, or change in, a pathologic sign or symptom or the occurrence of a side effect related to compound administration. Measurement may be objective or subjective (e.g., by patient self- reporting).
  • the association of a 5HT3R and/or UGT1A4 genotype with a therapeutic response will provide a method of determining the probability that an individual subject will respond in a particular way to treatment with 5HT ligands for gastrointestinal disease, hi genotyping, the characteristic that is typically measured is one that can be influenced by a polymorphism in the gene or its expression product.
  • polymorphism includes Single Nucleotide Polymorphisms (SNPs), insertion/deletion polymorphisms; microsatellite polymorphisms; and variable number of tandem repeat (VNTR) polymorphisms.
  • a compound with 5HT ligand activity may be screened for variation in its effects among genetic subpopulations of subjects with a gastrointestinal disorder.
  • Such methods involve administering the compound to a population of subjects suffering from a 5HT-mediated gastrointestinal disorder, obtaining DNA samples from the subjects (which may be done either prior to or after administration of the compound), genotyping polymorphic allelic sites in the 5HT3R gene and/or the UGT1A4 gene, and correlating the genotype of the subjects with their phenotypic responses (both favorable and unfavorable) to the treatment.
  • the methods of the present invention may be used to determine the correlation of a known 5HT3R polymorphic allele, and/or a known UGT1 A4 polymorphic allele, with the response of subjects with gastrointestinal disorders (such as IBS) to treatment with a 5HT ligand.
  • Subjects with the disease of interest are stratified according to genotype for the particular polymorphic allele, and their response to a therapeutic agent is assessed (either prospectively or retrospectively) and compared among the genotypes.
  • the response to the therapeutic agent may include either, or both, desired therapeutic responses (e.g., the alleviation of signs or symptoms) and undesirable side effects.
  • genotypes that are associated with an increased (or decreased) rate of therapeutic efficacy, or an increased (or decreased) incidence of a particular side effect may be identified.
  • the increase or decrease in response is in comparison to the other genotypes, or to a population as a whole.
  • Polymorphic alleles are typically detected by directly determining the presence of the polymorphic sequence in a polynucleotide or protein from the subject, using any suitable technique as is known in the art.
  • a polynucleotide is typically genomic DNA, or a polynucleotide fragment derived from this genomic polynucleotide, such as in a library made using genomic material from the individual (e.g. a cDNA library).
  • genomic DNA or a polynucleotide fragment derived from this genomic polynucleotide, such as in a library made using genomic material from the individual (e.g. a cDNA library).
  • the presence of the polymorphism is determined in a method that comprises contacting a polynucleotide or protein of the individual with a specific binding agent for the polymorphism and determining whether the agent binds to the polynucleotide or protein, where the binding indicates that the polymorphism is present.
  • the binding agent may also bind to flanking nucleotides and amino acids on one or both sides of the polymorphism, for example at least 2, 5, 10, 15 or more flanking nucleotide or amino acids in total or on each side, h one embodiment, the agent is able to bind the corresponding wild-type sequence by binding the nucleotides or amino acids which flank the polymorphism position.
  • the presence of the polymorphism is being determined in a polynucleotide it may he detected in the double stranded form, but is typically detected in the single stranded form.
  • the binding agent may be a polynucleotide (single or double stranded) typically with a length of at least 10 nucleotides, for example at least 15, 20, 30, or more nucleotides.
  • the agent may be a molecule that is structurally similar polynucleotides that comprises units (such as purines or pyrimidines) able to participate in Watson-Crick base pairing.
  • the agent may be a protein, typically with a length of at least 10 amino acids, such as at least 20, 30, 50, or 100 or more amino acids.
  • the agent may be an antibody (including a fragment of such an antibody that is capable of binding the polymorphism).
  • a polynucleotide agent which is used in the method will generally bind to the polymorphism of interest, and the flanking sequence, in a sequence specific manner (e.g. hybridize in accordance with Watson-Crick base pairing) and thus typically has a sequence which is fully or partially complementary to the sequence of the polymorphism and flanking region.
  • a binding agent is used as a probe.
  • the probe may be labeled or may be capable of being labeled indirectly.
  • the detection of the label may be used to detect the presence of the probe on (and hence bound to) the polynucleotide or protein of the individual.
  • the binding of the probe to the polynucleotide or protein may be used to immobilize either the probe or the polynucleotide or protein (and thus to separate it from one composition or solution).
  • the polynucleotide or protein of the individual is immobilized on a solid support and then contacted with the probe.
  • the presence of the probe immobilized to the solid support (via its binding to the polymorphism) is then detected, either directly by detecting a label on the probe or indirectly by contacting the probe with a moiety that binds the probe, hi the case of detecting a polynucleotide polymorphism the solid support is generally made of nitrocellulose or nylon.
  • the method may be based on an ELIS A system.
  • the present methods may be based on an oligonucleotide ligation assay in which two oligonucleotide probes are used.
  • probes bind to adjacent areas on the polynucleotide which contains the polymorphism, allowing (after binding) the two probes to be ligated together by an appropriate ligase enzyme.
  • the two probes will only bind (in a manner which allows ligation) to a polynucleotide that contains the polymorphism, and therefore the detection of the ligated product may be used to determine the presence of the polymorphism.
  • the probe is used in a heteroduplex analysis based system to detect polymorphisms, ha such a system when the probe is bound to a polynucleotide sequence containing the polymorphism, it forms a heteroduplex at the site where the polymorphism occurs (i.e. it does not form a double strand structure).
  • a heteroduplex structure can be detected by the use of an enzyme that is single or double strand specific.
  • the probe is an RNA probe and the enzyme used is RNAse H that cleaves the heteroduplex region, thus allowing the polymorphism to be detected by means of the detection of the cleavage products.
  • the method may be based on fluorescent chemical cleavage mismatch analysis which is described for example in PCR Methods and Applications 3:268-71 (1994) and Proc. Natl. Acad. Sci. 85:4397-4401 (1998).
  • the polynucleotide agent is able to act as a primer for a PCR reaction only if it binds a polynucleotide containing the polymorphism (i.e. a sequence- or allele-specific PCR system).
  • a polynucleotide containing the polymorphism i.e. a sequence- or allele-specific PCR system.
  • a PCR product will only be produced if the polymorphism is present in the polynucleotide of the individual.
  • the presence of the polymorphism may be determined by the detection of the PCR product.
  • the region of the primer which is complementary to the polymorphism is at or near the 3' end the primer.
  • the polynucleotide the agent will bind to the wild-type sequence but will not act as a primer for a PCR reaction.
  • the method may be a Restriction Fragment Length Polymorphism (RFLP) based system.
  • RFLP Restriction Fragment Length Polymorphism
  • This can be used if the presence of the polymorphism in the polynucleotide creates or destroys a restriction site that is recognized by a restriction enzyme.
  • treatment of a polynucleotide that has such a polymorphism will lead to different products being produced compared to the corresponding wild-type sequence.
  • the detection of the presence of particular restriction digest products can be used to determine the presence of the polymorphism.
  • the presence of the polymorphism may be determined based on the change that the presence of the polymorphism makes to the mobility of the polynucleotide or protein during gel electrophoresis.
  • SSCP polynucleotide single-stranded conformation polymorphism
  • Denaturing gradient gel electrophoresis DGGE Denaturing gradient gel electrophoresis DGGE is a similar system where the polynucleotide is electrophoresed through a gel with a denaturing gradient, a difference in mobility compared to the corresponding wild-type polynucleotide indicating the presence of the polymorphism.
  • the presence of the polymorphism may be determined using a fluorescent dye and quenching agent-based PCR assay such as the TAQMANTM PCR detection system, hi brief, this assay uses an allele specific primer comprising the sequence around, and including, the polymorphism.
  • the specific primer is labeled with a fluorescent dye at its 5' end, a quenching agent at its 3' end and a 3' phosphate group preventing the addition of nucleotides to it. Normally the fluorescence of the dye is quenched by the quenching agent present in the same primer.
  • the allele specific primer is used in conjunction with a second primer capable of hybridizing to either allele 5' of the polymorphism.
  • Taq DNA polymerase adds nucleotides to the nonspecific primer until it reaches the specific primer. It then releases polynucleotides the fluorescent dye and the quenching agent from the specific primer through its endonuclease activity. The fluorescent dye is therefore no longer in proximity to the quenching agent and fluoresces.
  • the mismatch between the specific primer and template inhibits the endonuclease activity of Taq and the fluorescent dye in not released from the quenching agent. Therefore by measuring the fluorescence emitted the presence or absence of the polymorphism can be determined.
  • a polynucleotide comprising the polymorphic region is sequenced across the region which contains the polymorphism to determine the presence of the polymorphism. Accordingly, any of the following techniques may be utilized in the present methods for genotyping, as is known in the art.
  • Hybridization based solid phase hybridization (dot blots, MASDA, reverse dot blots, oligonucleotide arrays (chips)); solution phase hybridization (TAQMANTM, Molecular Beacons);
  • the present invention also provides for a predictive (patient care) test or test kit.
  • a predictive (patient care) test or test kit will aid in disease management of gastrointestinal disease based on predetermined associations between genotype and phenotypic response to 5HT ligands in treating gastrointestinal disease.
  • Such a test may take different formats, including:
  • test kit may include one or more of the following reagents or instruments: a means to detect the binding of the agent to the polymorphism, an enzyme able to act on a polynucleotide (typically a polymerase or restriction enzyme), suitable buffers for enzyme reagents, PCR primers which bind to regions flanking the polymorphism, a positive or negative control (or both), a gel electrophoresis apparatus, and a means to isolate DNA from a sample.
  • the product may utilise one of the chip technologies as described by the current state of the art.
  • the test kit would include printed or machine readable instructions setting forth the correlation between the presence of a specific polymorphism or genotype and the likelihood that a subject with a gastrointestinal disease such as IBS will respond favorably to therapy with a 5HT ligand;
  • test kit (b) a biochemical test which analyses materials derived from the subject's body, including proteins or metabolites, that indicate the presence of a pre-determined polymorphism.
  • An appropriate test kit would comprise a molecule, aptamer, peptide or antibody (including an antibody fragment) that specifically binds to a predetermined polymorphic region (or a specific region flanking the polymorphism), or a binding agent as defined herein.
  • the product may additionally comprise one or more additional reagents or instruments (as are known in the art).
  • the test kit would also include printed or machine-readable instructions setting forth the correlation between the presence of a specific polymorphism or genotype and the likelihood that a subject with IBS will respond favorably to therapy with a 5HT ligand.
  • the present invention provides a method for screening a subject diagnosed with
  • the method comprises screening the subject for a polymorphism in the 5HT3R gene, and/or the UGT1A4 gene, that have previously been associated with a high or low rate of a particular desirable therapeutic outcome (compared to the rate in subjects with other genotypes), or associated with a high or low incidence of an undesired side effect (compared to the incidence in subjects with other genotypes).
  • Subjects are mammalian, and preferably humans.
  • Treatment of a subject with a 5HT ligand comprises administration of an effective amount of the pharmaceutical agent to a subject in need thereof.
  • the dose of agent is determined according to methods known and accepted in the pharmaceutical arts, and can be determined by those skilled in the art.
  • a suitable dosage range and plasma concentration for alosetron are provided in the disclosure of US Patent Number 5,360,800, the entire disclosure of which is hereby incorporated herein by reference.
  • Linkage disequilibrium refers to refers to the tendency of specific alleles at different genomic locations to occur together more frequently than would be expected by chance.
  • Linkage disequilibrium may be quantitated by methods as are known in the art (see, e.g., Devlin and Risch, Genomics 29:311 (1995); BS Weir, Genetic Data Analysis II, Sinauer Associates, Sunderland, MD (1996)). For example, alleles at given loci may be defined as in complete equilibrium if the frequency of any particular set of alleles (or haplotype) is the product of their individual population frequencies A commonly used measure of linkage disequilibrium is r:
  • genotyping methods as are well known in the art, genetic data were obtained from 237 subjects enrolled in clinical trials of alosetron for the treatment of non- constipation predominant IBS. Samples of subjects' DNA were genotyped, including typing for the presence of the C178T polymorphism in the 5HT3R gene, and for the - presence of the C70A polymorphism in the UGT1 A4 gene (as described above).
  • alosetron In the double-blind, placebo controlled clinical trials, female subjects received up to 12 weeks of treatment with either alosetron or a placebo. A favorable response to alosetron was defined as when a subject reported adequate relief of her IBS symptoms of pain and discomfort; the duration of symptom relief was recorded. Each subject's response to alosetron in the clinical trial setting was then analysed in conjunction with genotyping data, to identify genotypes associated with an increased (or decreased) rate of therapeutic efficacy.
  • 'adequate relief from pain and discomfort' (referred to herein as 'adequate relief).
  • Analyses were conducted using three different coding schemes: continuous (ranging from 0 weeks of adequate relief to 12 weeks of adequate relief, 3 levels (relief occurring for 0 weeks, for 1-4 weeks, or for 5-12 weeks and dichotomous (0-4 weeks of adequate relief, versus 5-12 weeks of adequate relief).
  • the recursive partitioning analyses (see Tables 1 & 2) identified polymorphisms in two genes that were associated with an increased rate of response to alosetron in the clinical trials. These were the C178T polymorphism in the 5HT3R gene (as defined herem , and the C70A polymorphism in the UGT1 A4 gene (as defined). Subjects' DNA was coded according to the presence of various polymorphisms, including polymorphisms in the 5HT3R and UGT1A4 genes.
  • a subject homozygous for "C” at the C178T polymorphic site in the 5HT3R gene was coded 1,1; a subject homozygous for "T” at the C178T polymorphic site was coded 2,2; and the heterozygous condition was coded 1,2.
  • a subject homozygous for "C” at the C70A polymorphic site in the UGT1A4 gene was coded 1,1; a subject homozygous for "A" at the C70A polymorphic site was coded 2,2; and the heterozygous condition was coded 1,2.
  • the model for alosetron treated subjects only suggested the same gene-gene interaction as the decision tree, where subjects having a 1,1 UGT1A4 genotype and a
  • 5HT3R genotype of either 1,1 or 2,2, were more likely to have good response when compared to other genotypes (p 0.057).
  • DNA samples are obtained from a population of subjects with gastrointestinal disease, and genomic DNA is extracted using standard procedures (automated extraction or using kit formats). The genotypes of the subjects, and any control individuals utilized, are determined for polymorphisms within the 5HT3R and UGT1A4 gene sequences, using either PCR, PCR-RFLP, TAQMANTM allelic discrimination assays, or any other suitable technique as is known in the art.
  • a simple size discrimination assay can be employed to determine the genotype of an individual, hi this case, two primers are employed to specifically amplify the gene of interest in a region surrounding the site of the polymorphism. PCR amplification is carried out, generating products that differ in length, dependent on the genotype (insertion or deletion) they possess. When subjected to gel electrophoresis, the differently sized products are separated, visualized, and the specific genotypes interpreted directly.
  • PCR-RFLP polymerase chain reaction - restriction fragment length polymorphism
  • a PCR-RFLP assay employs two gene- specific primers to anneal to, and specifically amplify a segment of genomic DNA surrounding the polymorphic site of interest.
  • specific restriction endonuclease enzymes are employed to digest the PCR products produced.
  • the enzyme utilized for an assay is selected due to its specific recognition sequence which it requires to bind to, and cleave the PCR product in the presence/absence of the polymorphism, yielding fragments diagnostic of the specific base present at the polymorphic site.
  • gel electrophoresis is employed to separate and visualize the fragments produced.
  • TAQMANTM PE Applied Biosystems, Foster City, CA assays, as are known in the art, may also be utilized to identify polymorphisms.
  • the allelic discrimination assay uses two allele specific probes labeled with a different fluorescent dye at their 5' ends but with a common quenching agent at their 3' ends. Both probes have a 3' phosphate group so that Taq polymerase cannot add nucleotides to them.
  • the allele specific probes comprise the sequence encompassing the polymorphic site and differ in the sequence at this site. The allele specific probes are capable of hybridizing without mismatches to the appropriate site.
  • the allele specific probes are used in conjunction with two primers, one of which hybridizes to the template 5' of the two specific probes, while the other hybridizes to the template 3' of the two probes. If the allele corresponding to one of the specific probes is present, the specific probe will hybridize perfectly to the template.
  • the Taq polymerase extending the 5' primer, will then remove the nucleotides from the specific probe, releasing both the fluorescent dye and the quenching agent. This will result in an increase in the fluorescence from the dye no longer in close proximity to the quenching agent.
  • the mismatch at the polymorphic site will inhibit the 5' to 3' endonuclease activity of Taq and hence prevent release of the fluorescent dye.
  • the ABI7700 sequence detection system is used to measure the increase in the fluorescence from each specific dye at the end of the thermal cycling PCR directly in PCR reaction tubes. The information from the reactions is then analyzed. If an individual is homozygous for a particular allele only fluorescence corresponding to the dye from that specific probe will be released, but if the individual is heterozygous, then both dyes will fluoresce. The genotypes of the individuals are then correlated with their phenotypic response to treatment with a 5HT ligand. Responses that vary among the genetic subpopulations are identified.

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Abstract

L'invention concerne des corrélations entre polymorphismes dans le gène du récepteur de la 5-hydroxytryptamine 3, et/ou de polymorphismes dans le gène UGT1A4, et une réponse phénotypique du sujet au traitement au moyen de ligands de la 5-hydroxytryptamine. Cette invention concerne également des méthodes de dépistage chez des sujets pour l'aide au traitement, des méthodes de criblage de ligands 5HT et des méthodes de traitement.
EP01968309A 2000-09-06 2001-08-31 Polymorphismes du gene du recepteur de la 5-hydroxytryptamine et reponse au traitement Withdrawn EP1315837A2 (fr)

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