[go: up one dir, main page]

WO2002033121A2 - Test diagnostique - Google Patents

Test diagnostique Download PDF

Info

Publication number
WO2002033121A2
WO2002033121A2 PCT/GB2001/004660 GB0104660W WO0233121A2 WO 2002033121 A2 WO2002033121 A2 WO 2002033121A2 GB 0104660 W GB0104660 W GB 0104660W WO 0233121 A2 WO0233121 A2 WO 0233121A2
Authority
WO
WIPO (PCT)
Prior art keywords
alkyl
diabetes
polymoφhism
optionally substituted
phenyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2001/004660
Other languages
English (en)
Other versions
WO2002033121A3 (fr
Inventor
David Hosford
Ian James Purvis
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Glaxo Group Ltd
Original Assignee
Glaxo Group Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Glaxo Group Ltd filed Critical Glaxo Group Ltd
Priority to AU2001295752A priority Critical patent/AU2001295752A1/en
Publication of WO2002033121A2 publication Critical patent/WO2002033121A2/fr
Anticipated expiration legal-status Critical
Publication of WO2002033121A3 publication Critical patent/WO2002033121A3/fr
Ceased legal-status Critical Current

Links

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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • 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 invention relates to the diagnosis of diabetes or susceptibility to diabetes and agents which can be used in the diagnosis.
  • diabetes exhibits a multifactorial inheritance (not a simple Mendelian inheritance). This indicates that many genes contribute to the genetic predisposition to diabetes, making it difficult to identify individual polymorphisms which are involved in causing diabetes.
  • the invention provides a method of diagnosing diabetes or susceptibility to diabetes in an individual, which method comprises typing in a sample from the the individual (i) the insulin receptor gene region or (ii) the insulin receptor protein of the individual, and determining thereby whether the individual has diabetes or is susceptible to diabetes.
  • Figure 1 shows the principle of the Taqman (trade mark) allelic discrimination assay, adapted to detect a polymorphism according to the invention.
  • Two allelic specific primers, G and A differ in their sequence at the polymorphic site (either G or A) and in the fluorescent dye attached to their 5' end (eitlier F or H).
  • G and A differ in their sequence at the polymorphic site (either G or A) and in the fluorescent dye attached to their 5' end (eitlier F or H).
  • the nucleotides containing the fluorescent dye F and quenching agent can be removed from the specific primer by the 5' to 3' endonuclease activity of Taq. Released from the quenching agent, the dye then fluoresces and this can be detected to determine that the allele corresponding to probe G is present in the sample.
  • SEQ ID NO's: 1 to 22 are the sequences of exons 1 to 22 of the insulin receptor gene
  • SEQ ID NO: 23 is the complete coding sequence of the insulin receptor rnRNA
  • SEQ ID NO: 24 is the sequence of the mRNA for the insulin receptor precursor.
  • SEQ ID NO: 25 is the complete sequence from exons 14 to 17 of the insulin receptor gene, including introns.
  • the present invention is concerned with the diagnosis, treatment or prevention of diabetes in an individual.
  • the diabetes is typically type II diabetes.
  • the individual is a human individual.
  • the invention relating to treatment the individual may have diabetes.
  • the insulin receptor gene region or insulin receptor protein of an individual is typed. Wliether the individual has diabetes or is susceptible to diabetes can thus be determined
  • Polymorphisms which are in linkage disequilibrium with each other in a population tend to be found together on the same chromosome. Typically one is found at least 30% of the times, for example at least 40 %, 50%, 70% or 90%, of the time the other is found on a particular chromosome in individuals in the population. Thus polymorphisms which are not functional susceptibility polymorphisms, but are in linkage disequilibrium with the functional polymo ⁇ hisms, may act as a marker indicating the presence of the functional polymorphism.
  • Polymo ⁇ hisms which are in linkage disequilibrium with any of the polymo ⁇ hisms mentioned herein are typically within 500kb, preferably within 400kb, 200kb, 100 kb, 50kb, lOkb, 5kb or 1 kb of the polymorphism.
  • the term "insulin receptor gene region" generally encompasses any of these distances from 5' to the transcription start site and 3' to the transcription termination site.
  • polymo ⁇ hism which is typed may be in the insulin receptor gene region or protein.
  • the polymo ⁇ hism is typically an insertion, deletion or substitution with a length of at least 1, 2, 5 or more base pairs or amino acids.
  • the polymo hism is typically a substitution of 1 base pair, i.e. a single polynucleotide polymo ⁇ hism (SNP).
  • SNP single polynucleotide polymo ⁇ hism
  • the polymo ⁇ hism may be 5' to the coding region, in the coding region, in an intron or 3' to the coding region.
  • the polymo ⁇ hism which is detected is typically the functional mutation which contributes to diabetes, but may be a polymo ⁇ hism which is in linkage disequilibrium with the functional mutation.
  • the polymo ⁇ hism will be associated with diabetes, for example as can be determined in a case/control study (e.g. as mentioned below).
  • the functional polymo ⁇ hism may cause insulin resistance, i.e. reducing the ability of the insulin receptor to activate the insulin signalling pathway in the presence of insulin.
  • the functional polymorphism will cause decreased binding of insulin to the insulin receptor.
  • the polymorphism may cause and/or be associated with migraine.
  • the polymo ⁇ hism may cause a change in any of the characteristics of the receptor discussed herein, such as expression, expression variant, cellular localisation or the pattern of expression in different tissues.
  • the polymo ⁇ hism may modulate (typically decrease) any of the following activities of the insulin receptor: IGF-1 binding, kinase activity (e.g.
  • the polymo ⁇ hism may modulate (typically decrease) the ability of the receptor to cause directly (or indirectly through another component) post-translational modifications, such as serine/threonine phosphorylation, dephosphorylation (via serine /threonine- or tyrosine phosphatases) or glycosylation.
  • the polymo ⁇ hism may be any of the following polymo ⁇ hisms: INSBa, LNSCa, exon ⁇ .poll,- exonl 1. poll, exonl7.pol2, exon ⁇ .poll, exon7.poll, exon7.pol2, exon8.pol2, exon9.pol3, exonl4.poll or INSR-c.4479C>T. These polymo ⁇ hisms are defined in Table 2 below with reference to the sequence flanking the polymorphism . The form of the polymo ⁇ hisms is allele 2 as defined in Table 2 for each of INSBa, LNSCa, exon ⁇ .poll , exonl 1.poll and exonl 7.pol2.
  • the polymo ⁇ hism may be a polymo ⁇ hism at the same location as any of these particular polymorphisms (in the case of a SNP, it will be an A, T, C or G at any of the locations).
  • the polymo ⁇ hism may be in linkage disequilibrium with any of these particular polymo ⁇ hisms.
  • the polymo ⁇ hism will have a sequence which is different from or the same as the corresponding region in any one of SEQ ID NOS: 1 to 25.
  • a polymorphism which can be typed to diagnose whether an individual has diabetes or is susceptible to diabetes may be identified by determining whether a candidate polymorphism in the insulin receptor gene region or insulin receptor protein is (i) associated with diabetes or susceptibility to diabetes or (ii) is in linkage disequilibrium with a polymo ⁇ hism which is associated diabetes or susceptibility to diabetes, and thereby determining whether the polymo ⁇ hism can be typed to diagnose diabetes or susceptibility to diabetes.
  • the polymo ⁇ hism is typically detected by directly determining the presence of the polymorphism sequence in a polynucleotide or protein of the individual.
  • a polynucleotide is typically genomic DNA or mRNA, or a polynucleotide derived from these polynucleotides, such as in a library made using polynucleotide from the individual (e.g. a cDNA library).
  • a library made using polynucleotide from the individual e.g. a cDNA library.
  • the presence of the polymo ⁇ hism is determined in a method that comprises contacting a polynucleotide or protein of the individual with a specific binding agent for the polymo ⁇ hism and determining whether the agent binds to a polymorphism in the polynucleotide or protein, the binding of the agent to the polymo ⁇ hism indicating that the individual has diabetes or is susceptible to diabetes.
  • the agent will also bind to flanking nucleotides and amino acids on one or both sides of the polymo ⁇ hism, for example at least 2, 5, 10, 15 or more flanking nucleotide or amino acids in total or on each side.
  • determination of the binding of the agent to the polymo ⁇ hism can be done by determining the binding of the agent to the polynucleotide or protein.
  • the agent is able to bind the corresponding wild-type sequence by binding the nucleotides or amino acids which flank the polymorphism position, although the mamier of binding will be different to the binding of a polynucleotide or protein containing the polymorphism, and this difference will generally be detectable in the method (for example this may occur in sequence specific PCR as discussed below).
  • the presence of the polymo ⁇ hism is being determined in a polynucleotide it may be detected in the double stranded form, but is typically detected in the single stranded form.
  • the 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 polynucleotides.
  • the agent may be molecule which is structurally related to 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, 100 or more amino acids.
  • the agent may be an antibody (including a fragment of such an antibody which is capable of binding the polymo ⁇ hism).
  • a polynucleotide agent which is used in the method will generally bind to the polymo ⁇ hism, and flanking sequence, of the polynucleotide of the individual in a sequence specific manner (e.g. hybridise in accordance with Watso ⁇ -Crick base pairing) and thus typically has a sequence which is fully or partially complementary to the sequence of the polymo ⁇ hism and flanking region.
  • the partially complementary sequence is homologous to the fully complementary sequence.
  • the agent is as a probe. This may be labelled or may be capable of being labelled 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 immobilise 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 immobilised on a solid support and then contacted with the probe.
  • the presence of the probe immobilised to the solid support (via its binding to the polymo ⁇ hism) 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.
  • the solid support is generally made of mtrocellulose or nylon.
  • the method may be based on an ELISA system.
  • the method may be based on an oligonucleotide ligation assay in which two oligonucleotide probes are used. These probes bind to adjacent areas on the polynucleotide which contains the polymo ⁇ hism, allowing (after binding) the two probes to be ligated together by an appropriate ligase enzyme. However the two probes will only bind (in a manner which allows ligation) to a polynucleotide that contains the polymo ⁇ hism, and therefore the detection of the ligated product may be used to determine the presence of the polymo ⁇ hism.
  • the probe is used in a heteroduplex analysis based system to detect polynucleotide polymo ⁇ hisms.
  • a heteroduplex analysis based system to detect polynucleotide polymo ⁇ hisms.
  • the probe when the probe is bound to polynucleotide sequence containing the polymo ⁇ hism it forms a heteroduplex at the site where the polymo ⁇ hism occurs (i.e. it does not form a double strand structure).
  • a heteroduplex structure can be detected by the use of an enzyme which single or double strand specific.
  • the probe is an RNA probe and the enzyme used is RNAse H which cleaves the heteroduplex region, thus allowing the polymo ⁇ hism 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 polymo ⁇ hism (i.e. a sequence- or allele-specific PCR system).
  • a PCR product will only be produced if the polymo ⁇ hism 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 of the primer.
  • the polynucleotide agent will bind to the wild-type sequence but will not act as a primer for a PCR reaction.
  • the method may be an RFLP based system. This can be used if the presence of the polymo ⁇ hism in the polynucleotide creates or destroys a restriction site which is recognised by a restriction enzyme. Thus treatment of a polynucleotide with such a polymo ⁇ hism will lead to different products being produced compared to the corresponding wild-type sequence. Thus the detection of the presence of particular restriction digest products can be used to determine the presence of the polymo ⁇ hism. The presence of the polymo ⁇ liism may be determined based on the change which the presence of the polymo ⁇ hism makes to the mobility of the polynucleotide or protein during gel electrophoresis.
  • SSCP polynucleotide single-stranded conformation polymo ⁇ hism
  • the presence of the polymo ⁇ hism may be determined using a fluorescent dye and quenching agent-based PCR assay such as the Taqman PCR detection system.
  • a fluorescent dye and quenching agent-based PCR assay such as the Taqman PCR detection system.
  • This assay uses an allele specific primer comprising the sequence around, and including, the polymo ⁇ hism.
  • the specific primer is labelled 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 hybridising to either allele 5' of the polymorphism.
  • Taq DNA polymerase adds nucleotides to the non-specific primer until it reaches the specific primer. It then releases polynucleotides, the fluorescent dye and 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 is not release from the quenching agent. Therefore by measuring the fluorescence emitted the presence or absence of the polymo ⁇ hism can be determined.
  • a polynucleotide comprising the polymo ⁇ hic region is sequenced across the region which contains the polymo ⁇ hism to determine the presence of the polymo ⁇ hism.
  • the presence of the polymo ⁇ hism may be determined indirectly, for example by measuring an effect which the polymo ⁇ hism causes. This effect may be in the expression or activity of the insulin receptor.
  • the presence of the polymo ⁇ hism may be determined by measuring the activity or level of the expression of the insulin receptor in the individual.
  • the expression of the insulin receptor may be determined by directly measuring the level of the receptor in the cell or indirectly by measuring the level of any other suitable component in the cell, such as measuring mRNA levels (e.g. using quantitative PCR, such as by a Taqman based method).
  • the method is carried out in vivo, however typically it is carried out in vitro on a sample from the individual, typically a blood, saliva or hair root sample.
  • the sample is typically processed before the method is carried out, for example DNA extraction may be carried out.
  • the polynucleotide or protein in the sample may be cleaved either physically or chemically (e.g. using a suitable enzyme).
  • the part of polynucleotide in the sample is copied (or amplified), e.g. by cloning or using a PCR based method. Polynucleotide produced in such a procedure is understood to be covered by the term "polynucleotide of the individual" herein.
  • the invention also provides a diagnostic kit that comprises a polynucleotide, probe, primer, antibody (including an antibody fragment) or agent as defined herein.
  • the kit may additionally comprise one or more other reagents or instruments (such as mentioned herein) which enable any of the embodiments of the method mentioned above to be carried out.
  • Such reagents or instruments include one or more of the following: a means to detect the binding of the agent to the polymo ⁇ hism, an enzyme able to act on a polynucleotide (typically a polymerase or restriction enzyme), suitable buffer(s) (aqueous solutions) for enzyme reagents, PCR primers which bind to regions flanking the polymo ⁇ hism, a positive and/or negative control, a gel electrophoresis apparatus and a means to isolate DNA from sample.
  • a means to detect the binding of the agent to the polymo ⁇ hism typically a polymerase or restriction enzyme
  • suitable buffer(s) aqueous solutions
  • PCR primers which bind to regions flanking the polymo ⁇ hism
  • a positive and/or negative control a gel electrophoresis apparatus and a means to isolate DNA from sample.
  • the invention also provides a non-human animal which has diabetes (typically type II diabetes) or is susceptible to diabetes and which is also transgenic for a polymo ⁇ hism as mentioned above.
  • the animal may be any suitable mammal such as a rodent (e.g. a mouse, rat or hamster) or primate.
  • the animal can be used in a method to assess the efficacy of agents in treating or preventing diabetes.
  • the invention provides a method for treating a patient who has been diagnosed as having or being susceptible to diabetes by a method of the invention, comprising administering an effective amount of an anti-diabetes agent or an agent that prevents the development of diabetes to the patient.
  • the invention also provides: use of an anti-diabetes agent in the manufacture of a medicament for treating . a patient who has been diagnosed as having diabetes by a method of the invention; use of an agent that prevents the development of diabetes in the manufacture of a medicament for treating a patient who has been diagnosed as being susceptible to diabetes by a method of the invention; and a pharmaceutical pack comprising an anti-diabetes pain agent or an agent that prevents the development of diabetes and instructions for administering of the agent to humans diagnosed by the method of the invention.
  • the anti-diabetes agent used is typically an agent reduces blood glucose levels (a hypoglycaemic).
  • the agent may be one which enhances insulin sensitivity (such as in skeletal muscle, liver or adipose tisssue).
  • the agent may be a thiazolidinedione (e.g. as described in U.S. Pat Nos. 5,089,514, 4,342,771, 4,367,234, 4,340,605, 5,306,726).
  • the agent may be a beta 3 agonist.
  • the agent may antagonise atypical beta- adrenoceptors which occur in adipose tissue and the gastrointestinal tract.
  • Such agonists have been found to be particularly useful as thermogenic anti-obesity agents and as anti-diabetic agents. These agonists are described for example in WO 97/21665, WO 97/21666, WO 98/43953, WO 99/65877, WO 95/33724, EP 0455006 and EP 0543662.
  • the agent may affect the sensitivity of the receptor to insulin, i.e. may increase or decrease any insulin binding-dependent activity of the receptor.
  • the agent causes hypoglycemia or antihyperglycemia, stimulates insulin release or reduces the clearance of insulin.
  • the agent typically lowers glucose levels by enhancing insulin action action, such as at hepatic sites and/or peripheral sites.
  • the agent will typically increase insulin-dependent glucose disposal and/or inhbit hepatic glucose output (HGO).
  • the agent which activates the receptor may be an agonist or antagonist of a peroxisome proliferator-activated receptor (PPAR), typically PPAR alpha or delta, preferably PPAR gamma.
  • PPAR peroxisome proliferator-activated receptor
  • the agent is typically a compound as described in WO 97/31907, WO 00/08002 or US-A-5,902,726.
  • the agent may be a compound of general formula (I) or a tautomeric form or a pharmaceutically acceptable salt or solvate thereof:
  • A is selected from the group consisting of:
  • phenyl wherein said phenyl is optionally substituted by one or more of the following groups; halogen atoms, C ⁇ alkyl, C, .3 alkoxy, C w fluoroalkoxy, nitrile, or -NR 7 R 8 where R 7 and R 8 are independently hydrogen or C ⁇ alkyl;
  • B is selected from the group consisting of:
  • Z is selected from the group consisting of:
  • Y represents a bond, C ⁇ alkylene, C 2 . 6 alkenylene, C 4 . 6 cycloalkylene or cycloalkenylene, a heterocyclic group as defined in point (vi) above, or phenyl optionally substituted by one or more C,_ 3 alkyl groups and/or one or more halogen atoms;
  • T represents a bond, C ⁇ _ 3 alkyleneoxy, -0- or -N(R 6 )-, wherein R 6 represents hydrogen or C ⁇ alkyl;
  • R 5 represents C ⁇ alkyl, C 4 . 6 cycloalkyl or cycloalkenyl, phenyl (optionally substituted by one or more of the following groups; halogen atoms, C j . 3 alkyl, C [ . 3 alkoxy groups, C 0 . 3 alkyleneNR 9 R 10 (where each R 9 and R 10 is independently hydrogen, C,_ 3 alkyl, -SO J C J ⁇ alkyl, or -CO ⁇ alkyl, -SO 2 NHC 1 . 3 alkyl), C 0 . 3 alkyleneCO 2 H, C 0.3 alkyleneCO 2 C 1 . 3 alkyl, or -OCH 2 C(O)NH 2 ), a 5- or 6- membered heterocyclic group as defined in point (ii) above, a bicylic fused ring
  • ring D represents a 5- or 6-membered
  • R 12 and R u are independently hydrogen or C N3 alkyl.
  • C, remedy 3 alkyl or alkylene and C,_ 6 alkyl or alkylene as used herein respectively contain 1 to 3 or 1 to 6 carbon atoms and appropriately include straight chained and branched alkyl or alkylene groups, typically methyl, methylene, ethyl and ethylene groups, and straight chained and branched propyl, propylene, butyl and butylene groups.
  • C 2 . 6 alkenyl or alkenylene as used herein contains 2 to 6 carbon atoms and appropriately includes straight chained and branched alkenyl and alkenylene groups, in particular propenylene or the like.
  • C ⁇ alkyleneoxy as used herein denotes -O-C ⁇ alkylene-, wherein
  • C ⁇ _ 3 alkylene is substantially as defined above, e.g. -O-CH 2 - etc.
  • C 4 . 6 cycloalkyl, C 4 . 6 cycloalkylene, cycloalkenyl and C 4 . 6 cyclo alkenylene include cyclic groups containing 4 to 6 carbon atoms, such as cyclopentane, cyclopentylene, cyclohexane, cyclohexylene, cyclohexene and cyclohexenylene.
  • halogen as used herein includes fluorine, chlorine, bromine and iodine.
  • 5- or 6-membered heterocyclic group as used herein includes 5- or
  • 6- membered unsubstituted heterocycloalkyl groups and substituted or unsubstituted heteroaryl groups e.g. substituted or unsubstituted imidazolidinyl, piperidyl, piperazinyl, pyrrolidinyl, mo ⁇ holinyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl, pyrazolyl, imidazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, thiadiazolyl, triazolyl or tetrazolyl.
  • substituted heterocyclic group is meant a 5 or 6 membered heteroaryl group substituted by one or more of the following; halogen atoms, C,. 3 alkyl, C, .3 alkoxy groups, C 0 . 3 alkylene N R 9 R 10 (where each R 9 and R 10 is idependently hydrogen, C,. 3 alkyl, -SO;, ⁇ alkyl or CO 2 C w alkyl -SO 2 NHC,_ 3 alkyl), C 0 _ 3 alkylene CO 2 H, C 0 _ 3 alkylene CO 2 C,. 3 alkyl, -OCH 2 C(O)NH 2 , -C,. 3 fluoroalkyl, -CN or SC M alkyl.
  • Aptly A represents any of phenyl, heteroaryl (e.g. pyridyl) or wherein fused ring C represents a 5-membered heteroaryl group containing at least one nitrogen heteroatom and optionally a further heteroatom selected from nitrogen and oxygen (e.g. oxazolyl, imidazolyl).
  • Particularly A represents any of phenyl, pyridyl, piperazinyl, or benzoxazolyl, any of which can optionally be substituted by one or more C ⁇ virgin 3 alkyl, especially phenyl, piperazinyl, or pyridyl.
  • B suitably represents any of C L3 alkylene (e.g. methylene), -N(CH 3 )C ⁇ _ 3alkylene (e.g. -N(CH 3 )(CH 2 ) 2 -) or alkylene, wherein Het represents a 5- membered heterocyclic group containing at least one nitrogen heteroatom and optionally at least one further heteroatom selected from oxygen and sulfur (e.g. pyrrolidinyl, oxazolyl and thiazolyl) and aptly substituted by Cj. 3 alkyl.
  • C L3 alkylene e.g. methylene
  • -N(CH 3 )C ⁇ _ 3alkylene e.g. -N(CH 3 )(CH 2 ) 2 -
  • Het represents a 5- membered heterocyclic group containing at least one nitrogen heteroatom and optionally at least one further heteroatom selected from oxygen and sulfur (e.g. pyrrolidinyl, oxazolyl and thi
  • B represents -N(CH 3 )(CH 2 ) 2 , oxazolyl -Ci-6 alkylene, which oxazolyl is optionally substituted by C,_ 3 alkyl, or thiazolyl which is optionally substituted by C,_ 3 alkyl.
  • Alk represents methylene
  • R 1 represents hydrogen, methyl or ethyl, especially hydrogen.
  • Z may represent -(C ⁇ _ 3 alkylene) phenyl substituted by one or more halogen atoms, such as optionally substituted benzyl.
  • Z represents -NR 3 R 4 substantially as hereinbefore described.
  • R 3 represents hydrogen.
  • Y represents phenyl (optionally substituted by one or more halogen atoms, or one or more C j _ 3 alkyl e.g. methyl groups), T represents a bond or an oxygen atom, and R 5 represents C 3 alkyl or phenyl (optionally substituted by one or more halogen atoms or one or more C,_ 3 alkyl groups);
  • Y represents a heterocyclic group substantially as hereinbefore described (e.g. thienyl), T represents a bond and R 5 represents phenyl (optionally substituted by one or more halogen atoms or one or more C,. 3 alkyl groups);
  • Y represents C 2 . 6 alkenylene- (e.g. propenylene), T represents a bond and R 5 represents phenyl (optionally substituted by one or more halogen atoms);
  • Y represents C 4 . 6 cycloalkenylene- (e.g. cyclohexenylene), T represents a bond and R 5 represents phenyl; Y represents phenyl, T represents a bond and R 5 represents a heterocyclic group substantially as hereinbefore described (e.g. piperidyl);
  • Y represents a bond
  • T represents a bond
  • R 5 represents a bicyclic ring
  • Y represents phenyl
  • T represents C 3 alkyleneoxy (e.g. -O-CH2-) or N(R 6 )- (e.g. -NH-) and R 5 represents phenyl.
  • alkyleneNR 9 R 10 where each R 9 and R 10 is independently hydrogen, C ⁇ alkyl, -SO ⁇ . ⁇ ⁇ ,al.kyl, or -CO 2 C 1 . 3 alkyl, - SO 2 NHC 1 . 3 alkyl, C 0 . 3 alkyleneCO 2 H, C 0 . 3 al yleneCOjC L 3 alkyl, or - OCH 2 C(O)NH 2 ).
  • Y represents phenyl
  • T represents a bond or -O-
  • R 5 represents C w alkyl or phenyl e.g. R 4 represents
  • R 13 represents phenyl or OCH 3 .
  • a and B are substantially as hereinbefore described, and Ar represents phenyl or a 5- or 6- membered heteroaryl group containing at least one heteroatom selected from oxygen, nitrogen and sulfur; and salts and solvates thereof.
  • A is selected from phenyl, pyridyl and benzoxazoyl.
  • a in Formula (la) represents phenyl or pyridyl.
  • B in Formula (la) represents phenyl or pyridyl.
  • Formula la) is suitably selected from -NR 2 C ⁇ _ 6 alkylene substantially as hereinbefore described and alkylene optionally substituted by C,. 3 alkyl substantially as hereinbefore described.
  • B in Formula (la) represents -N(CH 3 )(CH 2 ) 2 - or oxazolyl-C,. ⁇ alkylene, which oxazolyl is optionally substituted by C j . 3 alkyl, e.g. methyl.
  • a particular subgroup of the compounds of formula 1 are compounds of formula (I): wherein;
  • A is selected from the group consisting of:
  • B via a ring atom of ring C;
  • B is selected from the group consisting of: (iv) C j . 6 alkylene; (v) -NR 2 C 1 . 6 alkylene, wherein R 2 represents hydrogen or C ⁇ alkyl;
  • Alk represents C ⁇ alkylene
  • R 1 represents hydrogen or C,_ 3 alkyl
  • T represents a bond, C,_ 3 alkyleneoxy, -0- or -N(R 6 )-, wherein R 6 represents hydrogen or C,. 3 alkyl;
  • R 5 represents C ⁇ alkyl, C 4 . 6 cycloalkyl or cycloalkenyl, phenyl optionally substituted by one or more halogen atoms or one or more C,_ 3 alkyl groups, a 5- or 6- membered heterocyclic group as defined
  • Preferred examples of the compounds of formula (I) include (S)-(2-benzoyl- phenylamino)-3 - ⁇ 4- [2-(5 -methy l-2-phenyl-oxazol-4-yl) ethoxy] -phenyl ⁇ ropionic acid, and 2-(S)-(l-carboxy-2- ⁇ 4- ⁇ 2-(5-methyl-2-phenyl-oxazol-4-yl)-ethoxy]- phenyl ⁇ -ethylamino)-benzoic acid methyl ester.
  • the agent may be a compound of general formula (II) or a tautomeric form, pharmaceutically acceptable salt or solvate thereof:
  • R 2'1 is hydrogen or C ⁇ alkyl
  • R 2"2 is hydrogen, or C ⁇ . 8 alkyl optionally substituted by one or more halogens
  • R 2"3 is C ⁇ an yl, C 4 . 7 cycloalkyl or cycloalkenyl, -OC,. 6 alkyL -NR'R' (where each R' is independently hydrogen or C ⁇ alkyl), a 5 or 6 membered heterocyclic group containing at least one oxygen, nitrogen, or sulfur ring atom (optionally substituted by one or more halogen, C ] .
  • R 2"4 is a 5 or 6 membered heterocyclic group containing at least one oxygen, nitrogen, or sulfur ring atom (optionally substituted by one or more halogen, C ⁇ alkyl optionally substituted by one or more halogens, -O . galkyl optionally substituted by one or more halogens, -CN, or -NO 2 ), or phenyl (optionally substituted by one or more halogen, C ] . 6 alkyl optionally substituted by one or more halogens, -OC ⁇ alkyl optionally substituted by one or more halogens, -NR'R' (as defined above), -CN, or - NO 2 );
  • R 2"5 is hydrogen, halogen, or C ⁇ alkyl optionally substituted by one or more halogens
  • R 2"6 is hydrogen or C ⁇ alkyl
  • X is O or S; and n is 1, 2, or 3.
  • C j . 8 alkyl is preferably C U6 alkyl
  • C,_ 6 is preferably C w alkyl.
  • Typical C ⁇ _ 6 alkyl and C 1-3 alkyl groups are as defined above.
  • Typical C 4.7 • cycloalkyl, C 4 . 7 cycloalkenyl and 5- or 6-membered heterocyclic groups and typical halogen atoms are as defined above.
  • R 2"1 is hydrogen or methyl. Most preferably, R 2"1 is hydrogen.
  • R 2"2 is C,. g alkyl optionally substituted by one or more halogens.
  • said halogen is fluorine.
  • R 2"2 is straight-chain.
  • R 2"3 is pyridine, pyrazine, thiophene, furan, thiazole, or phenyl
  • R 2"3 is phenyl (optionally substituted by one or more halogen, .galkyl optionally substituted by one or more halogens, -OC ⁇ galkyl optionally substituted by one or more halogens, -CN, or -NO 2 ).
  • R 2"4 is phenyl (optionally substituted by one or more halogen, C 6 alkyl optionally substituted by one or more halogens, or -OCj. 6 alkyl optionally substituted by one or more halogens).
  • said halogen is fluorine.
  • R 2"4 is phenyl either unsubstituted or substituted with 1, 2, or 3 fluorine atoms.
  • R 2"5 is hydrogen, halogen, or C ⁇ alkyl optionally substituted by one or more halogens. Most preferably R 2"5 is hydrogen.
  • R 2"6 is methyl or ethyl.
  • n is 2.
  • the carbon atom bonded to CO ⁇ 2"1 is in the S configuration.
  • the absolute configuration around that carbon is:
  • NH Preferred examples of the compounds of general formula (II) include (2S)-2- ⁇ [(Z)-l-methyl-3-oxo-3-phenyl-l-propenyl]amino ⁇ -3- ⁇ 4-(5-methyl-2-phenyl-l,3- oxazol-4-yl) ethoxy]phenyl ⁇ propanoic acid and (2S)-3- ⁇ 4-[2-(5-methyl-2-phenyl- 1 ,3 -oxazol-4-yl)ethoxy]phenyl ⁇ -2- ⁇ [(Z)-3 -oxo-3 -phenyl- 1 -(trifluoromethyl)- 1 - propenyl] amino ⁇ propanoic acid.
  • the agent may be a sulfonylurea (e.g. l-butyl-3-sulfonylurea, tolbutamide, chlo ⁇ ropamide, tolazamide, acetohexamide, glyburide, glipizide or gliclazide), a guanide (guanide or chloroguanide), a biguanide (e.g. phenformin, metformin or buformin) or an ⁇ -glucosidase inliibitor (e.g. acarbose).
  • the agent may be selected from thiazolidinediones, such as the compounds of formula (III)
  • R 3"1 is selected from the group consisting of hydrogen, C ⁇ alkyl, aminoC, . s alkyl, C 1.8 alkylaminoC 1.8 alkyl, heteroarylaminoC,. 6 alkyl, (heteroaryl)(C,. 8 alkyl)aminoC 1 .galkyl, (C 4.8 cycloalkyl)C ].8 alkyl, C ⁇ 8 alkylheteroarylC 1 _ 8 alkyl, 9 or 10 membered heterobicycles which are partially aromatic or substituted 9 or 10 membered heterobicycles which are partially aromatic.
  • a dashed line ( ) is none or one double bond between the two carbon atoms.
  • the dashed line ( ) represents no double bond.
  • Such compounds are described in US-A-5,902,726.
  • C ⁇ alkyl is preferably C ⁇ -allcyl, more preferably C r3 alkyl.
  • Typical alkyl and C w alkyl groups are as defined above.
  • Typical heteroaryl groups are ' 5- or 6-membered heterocyclic groups as defined above.
  • C 4.8 cycloalkyl is preferably C 4 . 7 cycloalkyl such as those defined above.
  • Typical 9 or 10 membered heterobicyles which are partially aromatic include 10-membered rings containing one or more heteroatoms selected from N, O or S.
  • Preferred compounds of formula (III) are those wherein R 3"1 is selected from (i), (ii) or (iii) below: -
  • R 3"2 and R 3"3 are the same or different and each represents a hydrogen atom or a C r C 5 alkyl group;
  • R 3"4 represents a hydrogen atom, a C r Cg aliphatic acyl group, an alicyclic acyl group, an aromatic acyl group, a heterocyclic acyl group, an araliphatic acyl group, a (C,-Cg alkoxy)carbonyl group or an aralkyloxycarbonyl group; and
  • R 3"5 and R 3"6 are the same or different and each represents a hydrogen atom, a C,-C 5 alkyl group or a C]-C 5 alkoxy group, or R 3"5 and R 3"6 together represent a C ] -C 4 alkylenedioxy group.
  • R 3"2 , R 3"3 , R 3"5 and R 3"6 are each methyl and R 3"4 is hydrogen.
  • the agent is troglitazone;
  • heteroarylaminoC ⁇ g alkyl group such as a group
  • R is hydrogen or a C w alkyl group and n is 1, 2, 3 or 4.
  • R is methyl and n is 1, in which case the agent is rosiglitazone.
  • a C ⁇ g alkylheteroaryl C ⁇ _ g alkyl group such as a group
  • R 3"8 is a C ] . 6 alkyl group and n is 1, 2, 3 or 4.
  • R 3"8 is ethyl and n is 1. More preferably the C j _ 8 alkylheteroaryl C,. g alkyl group is
  • the agent in wliich case is pioglitazone.
  • R 3'2 , R 3"3 , R 3"5 , R 3"6 , R 3"7 or R 3"8 represents an alkyl group
  • this may be a straight or branched chain alkyl group for example methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or isopentyl.
  • R 3"4 represents an aliphatic acyl group, this preferably has from 1 to 6 carbon atoms and may include one or more carbon-carbon double or triple bonds. Examples of such groups include formyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, hexanoyl, acryloyl, methacryloyl and crotonoyl.
  • R 3'4 represents an alicyclic acyl group, it is preferably a cyclopentanecarbonyl, cyclohexanecarbonyl or cycloheptanecarbonyl group.
  • R 3"4 represents an aromatic acyl group
  • the aromatic moiety thereof may optionally have one or more substituents (for example nitro, amino, alkylamino, dialkylamino, alkoxy, halo, alkyl or hydroxy substituents);
  • substituents for example nitro, amino, alkylamino, dialkylamino, alkoxy, halo, alkyl or hydroxy substituents
  • examples of such aromatic acyl groups include benzoyl, p-nitrobenzoyl, m- fluorobenzoyi, o-chlorobenzoyl, p-aminobenzoyl, m-(dimethylamino)benzoyl, o- methoxybenzoyl, 3,4-dichlorobenzoyl, 3,5-di-t-butyl-4-hydroxybenzoyl and 1- naphthoyl groups.
  • R 3"4 represents a heterocyclic acyl group
  • the heterocyclic moiety thereof preferably has one or more, preferably one, oxygen, sulphur or nitrogen hetero atoms and has from 4 to 7 ring atoms.
  • R 3"4 represents an araliphatic acyl group
  • the aliphatic moiety thereof may optionally have one or more carbon-carbon double or triple bonds and the aryl moiety thereof may optionally have one or more substituents (for example nitro, amino, alkylamino, dialkylamino, alkoxy, halo, alkyl or hydroxy substituents); examples of such araliphatic acyl groups include the phenylacetyl, p-chlorophenylacetyL phenylpropionyl and cinnamoyl groups.
  • R 3"4 represents a ( -Cg alkoxy) carbonyl group
  • the alkyl moiety thereof may be any one of those alkyl groups as defined above and the alkoxycarbonyl group represented by R 3"4 .
  • R 3"4 represents an aralkyloxycarbonyl group
  • the aralkyl moiety thereof may be any one of those included within the araliphatic acyl group represented by R 3"4 .
  • R 3"5 and R 3"6 represent alkoxy groups, these may be the same or different and may be straight or branched chain groups, preferably having from 1 to 4 carbon atoms. Examples include methoxy, ethoxy, propoxy, isopropoxy and butoxy.
  • R 3"5 and R 3"6 may together represent a C r C 4 alkylenedioxy group, more preferably a methy lenedioxy or ethylenedioxy group.
  • the agent may also be ciglitazone, darglitazone or englitazone or derivatives of any of the thiozoladinediones (e.g. derivatives referred to in WO 00/35437).
  • Other agents include oxyzolidinediones, such as JTT 501, and non-chiral acyclic agents, such as GW 262570, as well as substituted 4-hydroxyphenylalcanoic acid derivatives with agonist activity to PPAR gamma.
  • the agent may be selected from non-thiazolidinedione insulin sensitizers such as those dislcosed in Buckle et al (1996) Bioorganic and Medicinal Chemistry Letters 6, 2121-6 and substituted 4-hydroxy-phenylalcanoic acid drivatives, such as those described in WO 97/31907, hypoglycemic alkaloids, such as quindoline and cryptolepine, which may be obtained from extracts from Cryptolepsis sp., as disclosed in US-A-5,629,319, as well as triterpenoid substances, such as those disclosed in US-A-5,691,386, and eremophilanolide sesquiterpenes, such as described in US-A-5,747,527.
  • non-thiazolidinedione insulin sensitizers such as those dislcosed in Buckle et al (1996) Bioorganic and Medicinal Chemistry Letters 6, 2121-6 and substituted 4-hydroxy-phenylalcanoic acid drivatives
  • Suitable agents include polymo ⁇ hic fornis of troglitazone, tei enoid- type quinones and C-substituted pentacycloazoles and N-alkyl substituted pentacycloazoles, for example as disclosed in US-A-5,700,820, US-A-5,674,900, US-A-5,641,796.
  • the disclosure of all the US patents, WO publications and other publications mentioned herein is inco ⁇ orated herein by reference.
  • agents include those that activate a RXR receptor that forms a heterodimer with PPAR, for example, ligand 100268, which is an RXR receptor ligand.
  • the agent may be an angiotensin II antagonist or angiotensin converting enzyme inhibitor.
  • the agent is a protein, polynucleotide, carbohydrate, lipid or small organic molecule.
  • the invention also provides a method of treating diabetes (preferably type II) comprising administering an effective amount of an anti-diabetes agent to an individual who has a polymorphism in the insulin gene region and/or insulin receptor protein.
  • the invention additionally provides use of an anti-diabetes agent in the manufacture of a medicament for use in treating an individual with a polymo ⁇ hism in the insulin gene region and/or insulin receptor protein.
  • the invention also provides a method of preventing diabetes comprising administering an effective amount of an agent which prevents diabetes to an individual who has a polymo ⁇ hism in the insulin gene region and/or insulin receptor protein.
  • the invention additionally provides use of an agent which prevents diabetes agent in the manufacture of a medicament for use in treating an individual with a polymorphism in the insulin gene region and/or insulin receptor protein.
  • the polymo ⁇ hism present in the individual is preferably any of the polymorphisms mentioned herein, such as a polymorphism which causes (i) insulin resistance, (ii) reduced binding of insulin to the insulin receptor, or (iii) migraine.
  • the polymo ⁇ hism may be any of the following polymo ⁇ hisms: INSBa, LNSCa, exon ⁇ .poll, exonl 1. oll, exonl7.pol2, exon ⁇ .poll, exon7.poll, exon7.pol2, exon8.pol2, exon9.pol3, exonH.poll or LNSR-c.4479C>T.
  • the agent which is administered to treat or prevent diabetes may be any suitable agent mentioned herein or in any of the US patents, WO publications or other publications mentioned herein.
  • the agent is a compound of formula (I), (II) or (III) as described herein.
  • the agent may be: (S)-(2-benzoyl-phenylamino)-3- ⁇ 4-[2-(5-methyl-2-phenyl-oxazol-4-yl) ethoxy]- phenyl ⁇ propionic acid, 2-(S)-(l-carboxy-2- ⁇ 4- ⁇ 2-(5-methyl-2-phenyl-oxazol-4-yl)- ethoxy]-phenyl ⁇ -ethylamino)-benzoic acid methyl ester, (2S)-2 ⁇ [(Z)-l-rnethyl-3- oxo-3 -phenyl- 1 -propenyl]amino ⁇ -3 - ⁇ 4-(5-methyl-2-phenyl- 1 ,3 -oxazol-4-yl) ethoxy]phenyl ⁇ propanoic acid or (2S)-3- ⁇ 4-[2-(5-methyl-2-phenyl-l,3-oxazol-4- yl
  • an effective amount of such an agent may be given to a human patient in need thereof.
  • the dose of agent may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen.
  • a suitable dose may however be from 0.1 to 100 mg/kg body weight such as 1 to 40 mg/kg body weight. Again, a physician will be able to detennine the required route of administration and dosage for any particular patient.
  • the formulation of the agent will depend upon factors such as the nature of the substance and the condition to be treated.
  • the agent is formulated for use with a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutically acceptable carrier or diluent For example it may be formulated for oral, parenteral, intravenous, intramuscular or subcutaneous administration. A physician will be able to determine the required route of administration for each particular patient.
  • the pharmaceutical carrier or diluent may be, for example, an isotonic solution.
  • the effectiveness of particular agents may be affected by or dependent on whether the individual has particular polymorphisms in the insulin receptor gene region or insulin receptor.
  • the invention can allow the determination of whether an individual will respond to a particular agent by determining whether the individual has a polymorphism which affects the effectiveness of that agent.
  • the invention includes a method of treating a " patient who has been identified as being able to respond to the agent comprising administering the agent to the patient.
  • certain agents may produce side effects in individuals with particular polymo ⁇ hisms in the insulin gene region or protein.
  • the invention allows the identification of a patient who is at increased risk of suffering side effects due to such an agent by identifying whether an individual has such a polymo ⁇ hism.
  • polymo ⁇ hism Individuals who carry a particular polymo ⁇ hism in the insulin receptor gene may exhibit differences in their ability to regulate metabolic pathways under different physiological conditions and will display altered reactions to different diseases. In addition, differences in metabolic regulation arising as a result of the polymorphism may have a direct effect on the response of an individual to gene therapy.
  • the polymo ⁇ hism may therefore have the greatest effect on the efficacy of drugs designed to modulate the activity of the insulin receptor or other components in its signalling pathway. However, the polymo ⁇ hisms may also affect the response to agents acting on other biochemical pathways regulated by the insulin receptor. The invention may therefore be useful both to predict the clinical response to such agents and to determine therapeutic dose.
  • the invention may further be used in the development of new drug therapies which selectively target one or more allelic variants of the insulin receptor gene (i.e. wliich have different polymo ⁇ hisms). Identification of a link between a particular allelic variant and predisposition to disease development or response to drug therapy may have a significant impact on the design of new drugs. Drugs may be designed to regulate the biological activity of the variants implicated in the disease process while minimising effects on other variants.
  • HA head ache lasting 4-72 hrs if unsuccessfully treated; HA with at least 2 of the following: unilateral pain; pulsating quality; moderate to severe intensity; aggravation by physical activity; 5 - HA with nausea, or vomiting, or photophobia, or phonophobia (at least 1).
  • HA defined as above, with onset accompanying or following aura within 60 10 minutes.
  • HA fulfills migraine with aura characteristics; aura includes hemiparesis that may be prolonged (> 60 minutes): 15 at least 1 first-degree relative with similar HAs.
  • Samples were obtained from the study group and genomic DNA extracted using a standard kit and a slating out technique (Cambridge Molecular). The 20. genotypes of the migraineurs with aura and control individuals for individual SNPs within the insulin receptor gene were then determined from the DNA samples obtained using the Taqman allelic discrimination assay.
  • the allelic discrimination assay used two allele specific primers labeled with a different fluorescent dye at their 5' ends but with a 25 common quenching agent at their 3' ends. Both primers had a 3' phosphate group so that Taq polymerase could not add nucleotides to them.
  • the allele specific primers comprised the sequence encompassing the polymo ⁇ hic site and differed only in the sequence at this site. The allele specific primers were only capable of hybridizing without mismatches to the appropriate allele. 30 The allele specific primers were used in typing PCRs in conjunction with a third primer, which hybridized to the template 5' of the two specific primers.
  • the specific primer would hybridize perfectly to the template.
  • the Taq polymerase extending the 5' primer, would then remove the nucleotides from the specific probe releasing both the fluorescent dye and the quenching agent. This resulted in an increase in the fluorescence from the dye no longer in close proximity to the quenching agent.
  • the allele specific primer hybridized to the other allele would inhibit the 5' to 3' endonuclease activity of Taq and hence prevent release of the fluorescent dye.
  • the ABI7700 sequence detection system was used to measure the increase in fluorescence from each specific dye during the thermal cycling PCR directly in PCR reaction tubes. The information from the reactions was then analyzed. If an individual was homozygous for a particular allele only fluorescence conesponding to the dye from that specific primer would be released, if the individual was heterozygous both dyes would fluoresce.
  • Table 1 shows the P values for the co-inheritance of the associated SNPs with migraine.
  • Table 2 shows the SNPs typed in the sample group to determine association of the SNP with migraine.
  • the polymorphic site typed is given together with the flanking sequence 5' and 3'.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de diagnostic du diabète ou de l'éventualité d'un diabète chez un individu. Ce procédé consiste à typer dans un échantillon provenant de l'individu (i) la zone de gène de récepteur d'insuline ou (ii) la protéine de récepteur d'insuline de l'individu et à déterminer ainsi si l'individu est atteint de diabète ou susceptible de l'être.
PCT/GB2001/004660 2000-10-19 2001-10-19 Test diagnostique Ceased WO2002033121A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001295752A AU2001295752A1 (en) 2000-10-19 2001-10-19 Test for the diagnosis of diabetes and compounds for the treatment thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB0025678.4A GB0025678D0 (en) 2000-10-19 2000-10-19 Diagnostic test
GB0025678.4 2000-10-19

Publications (2)

Publication Number Publication Date
WO2002033121A2 true WO2002033121A2 (fr) 2002-04-25
WO2002033121A3 WO2002033121A3 (fr) 2003-10-16

Family

ID=9901640

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2001/004660 Ceased WO2002033121A2 (fr) 2000-10-19 2001-10-19 Test diagnostique

Country Status (3)

Country Link
AU (1) AU2001295752A1 (fr)
GB (1) GB0025678D0 (fr)
WO (1) WO2002033121A2 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001028540A2 (fr) * 1999-10-19 2001-04-26 Glaxo Group Limited Therapie de la douleur cephalique
AU1036801A (en) * 1999-10-19 2001-04-30 Glaxo Group Limited Agent for treating cephalic pain

Also Published As

Publication number Publication date
AU2001295752A1 (en) 2002-04-29
WO2002033121A3 (fr) 2003-10-16
GB0025678D0 (en) 2000-12-06

Similar Documents

Publication Publication Date Title
AU2003302472B2 (en) Target for therapy of cognitive impairment
US10272076B2 (en) Methods for the administration of iloperidone
US20050042614A1 (en) Methods to treat diabetes and related conditions based on polymorphisms in the tcf-1 gene
US20100144781A1 (en) Methods of treating psychosis and schizophrenia based on polymorphisms in the erbb4 gene
WO2006081555A2 (fr) Methodes de diagnostique et de traitement de l'asthme, de la rhinite allergique et d'autres maladies respiratoires mettant en oeuvre une association d'haplotypes
AU2004257748A1 (en) Method of diagnosis and treatment for asthma based on haplotype association
US20220259660A1 (en) Methods for the administration of iloperidone
US20050256649A1 (en) High throughput correlation of polymorphic forms with multiple phenotypes within clinical populations
WO2002033121A2 (fr) Test diagnostique
WO2001028539A9 (fr) Agent pour le traitement de la douleur cephalique
CA2540760C (fr) Utilisation de polymorphismes genetiques pour la prediction d'hepatotoxicite induite par medicament
WO2001029256A2 (fr) Marqueurs de susceptibilite aux maux de tete
US20040023225A1 (en) Methods and compositions for identifying risk factors for abnormal lipid levels and the diseases and disorders associated therewith
WO2001028540A2 (fr) Therapie de la douleur cephalique
WO2003056328A1 (fr) Correlation a haut rendement de formes polymorphiques avec plusieurs phenotypes dans des populations cliniques
WO2011146458A1 (fr) Méthode d'administration et de traitement
Dugaich et al. Research Article Role of the Rostral Ventrolateral Medulla in the Arterial Hypertension in Chronic Renal Failure
WO2004019877A2 (fr) Nos-2 et glaucome

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP