WO2001028540A2

WO2001028540A2 - Agent for treating cephalic pain

Info

Publication number: WO2001028540A2
Application number: PCT/GB2000/004051
Authority: WO
Inventors: Ian James Purvis; Linda Catherine Mccarthy
Original assignee: Glaxo Group Limited
Priority date: 1999-10-19
Filing date: 2000-10-19
Publication date: 2001-04-26
Also published as: AU1037301A; WO2001028540A3

Abstract

Use of an agent that modulates directly or indirectly the insulin receptor or insulin receptor signalling pathway in the manufacture of a medicament for use in a method of preventing or treating cephalic pain.

Description

THERAPY OF CEPHALIC PAIN

Field of the invention

The invention relates to a modulator and to a method of screening for the modulator.

Background to the invention

Cephalic pain disorders are generally multifactorial disorder, many of which have an unknown etiology. Both environmental and genetic factors are thought to contribute to cephalic pain disorders. In the case of migraine familial aggregation is observed, although segregation analysis of the pattern of inheritance of migraine within families indicates multifactorial inheritance (not a simple Mendelian inheritance). This implies that many genes contribute to the genetic predisposition to migraine, making it difficult to identify individual genes in linkage studies.

Summary of the invention

The inventors have shown that polymorphisms in the insulin receptor gene contribute to susceptibility to cephalic pain. The insulin receptor is an important component in the regulation of the glucose and lipid metabolism pathways. The present finding allows the treatment of cephalic pain, and in particular migraine, by the manipulation of components of the glucose and lipid metabolism pathways, in particular by manipulation of the insulin receptor.

Accordingly the invention provides use of an agent that modulates directly or indirectly the insulin receptor or insulin receptor signalling pathway in the manufacture of a medicament for use in a method of preventing or treating cephalic pain.

Description of the Figure

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 (either F or H). In the Figure, only the allele corresponding to probe G is present. Probe G can therefore anneal without mismatch to the template and, as Taq DNA polymerase extends from the non- specific primer upstream, 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.

Description of sequences in Sequence Listing 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 mRNA;

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, and

SEQ ID NO: 26 is the amino acid sequence of human PPAR gamma.

Detailed description of the invention

The present invention is concerned with the prevention or treatment of cephalic pain by the use of an agent that modulates, typically antagonises, the insulin receptor or insulin receptor signalling pathway. The cephalic pain may be a cluster headache, chronic paroxysmal hemicrania, headache associated with vascular disorders, headache associated with substances or their withdrawal (for example drug withdrawal), tension headache and in particular migraine with aura or migraine without aura.

The agent may modulate the insulin receptor or the insulin receptor signalling pathway (indirectly) by acting on a component which is able to affect (act on) the receptor or pathway. Such a component is one whose natural activity is generally able to affect the receptor or pathway (i.e. it is operatively linked to the receptor or pathway). Any activity of the receptor or pathway may be affected by the component.

The agent typically modulates the expression or the activity of the component. The component is typically a carbohydrate, lipid, protein or polynucleotide (such as genomic DNA or unspliced or spliced mRNA). The component may be an enzyme such as an enzyme in the glucose or lipid metabolic pathways or a kinase. The component may be intracellular or extracellular. In one embodiment the component is present in a neuron or a cell in the neurovascular network which is critical to the generation of cephalic pain, such as a cell in the trigeminovascular network and all nociceptive connections and afferent modulatory connections to which it is mono- or poly-synaptically linked.

The component may mediate a metabolic or other effect of receptor signalling activity, such as GLUT4 expression at the cell surface, stimulation of glucose or 2- deoxyglucose or 3-O-methyl glucose uptake into cells, increased glycogen synthase phosphorylation, activation and glycogen synthesis, decreased lipolysis, increased fatty acid synthesis and incorporation into triglyceride, inhibition of gluconeogenesis in hepatocytes.

The component may be part of or directly involved in the intracellular signalling pathway of the insulin receptor, i.e. the component may be downstream of the receptor. A downstream component typically mediates or is part of the intracellular changes which occur due to signalling activity. The component may be one which is modified (typically phosphorylated or de-phosphorylated), or whose location in the cell changes, during signalling activity. The component may be one which is capable of binding the insulin receptor. Typically the downstream component is insulin receptor substrate- 1 , -2, -3, or -4, p85, Grb2, Gabl , phosphatidyl inositol 3 kinase, pp60, ppl20, son of sevenless (SOS), MAP kinase, serine phosphatase, threonine phosphatase, tyrosine kinase, ras, raf, syp, she or a G protein.

The agent may modulate components related to the glucose or lipid pathways, i.e. components which are upstream of the insulin receptor. The component which the agent modulates may be the insulin receptor itself. The agent may thus modulate any of the following activities of the receptor: insulin binding, IGF-1 binding, kinase activity (e.g. tyrosine, threonine or serine kinase activity), autophosphorylation, internalisation, re-cycling, interactions with regulatory proteins, or interactions with signalling complexes. The agent may modulate 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 agent may modulate a product which regulates or is part of the expression pathway of the component. The product may be one which is specific to the expression pathway of that component. The agent may act upon the product in any of the ways described herein in which the agent acts upon the component. The product may be the gene from which any of the components is expressed, an RNA polymerase that can express mRNA from the gene, the unspliced mRNA which is transcribed from the gene, factors that aid splicing of the mRNA, the spliced mRNA, nuclear factors that bind to the mRNA and/or transport the mRNA from the nucleus to the cytoplasm, translation factors that contribute to translating the mRNA to protein.

Thus the agent may modulate transcription from the component gene or translation of the component mRNA. Preferably the agent is a specific inhibitor of transcription from the component gene, and does not inhibit transcription from other genes. The agent may bind to the component gene either (i) 5' to the coding sequence, and/or (ii) to the coding sequence, and/or (iii) 3' to the coding sequence. Thus the agent may bind to the promoter, and inhibit the initiation of transcription. As discussed above the agent may bind and inhibit the action of a protein which is required for transcription from the component gene.

The agent may bind to the untranslated or translated regions of the component mRNA. This could modulate the initiation of translation. The agent may modulate, in particular agonise, expression by modulating the rate at which the component is broken down. In particular in the case where the component is the insulin receptor the agent may modulate the expression of different variants of the receptor (e.g. variants produced by different splicing of the mRNA), tissue-specific expression, subcellular localisation or hybridisation with other receptors (e.g. the IGF-1 receptor).

The agent typically has an activity which directly or indirectly (e.g. mediated through any of the components discussed above) results in an effect on the insulin receptor or insulin receptor pathway which is generally counter (opposite) to the effect of a polymorphism in the insulin receptor gene which causes susceptibility to migraine. The polymorphism will generally cause a change in any of the characteristics of the receptor discussed herein, such as expression, activity, expression variant, cellular localisation or the pattern of expression in different tissues. The polymorphism may have an antagonist effect, but preferably has an agonist effect on any of these characteristics of the receptor. Generally this will lead to a consequent decrease or increase in particular parts of the activity of the pathway (particular polymorphisms may cause an increase in activity in one part of the pathway and also cause a decrease in activity in another part of the pathway).

The polymorphism may be any of the following polymorphisms: INSBa, INSCa, exonδ.poll , exonl 1.poll , exonl 7.pol2 (the form of these polymorphisms will be allele 2 as defined in table 2) exonό.poll, exon7.poll , exon7.pol2, exon8.pol2, exon9.pol3, exonl 4.poll or INSR-c.4479C>T (the form of these polymorphisms will be allele 1 or 2 which is in linkage disequilibrium with the associated polymorphism). These polymorphisms are defined in Table 2 below with reference to the sequence flanking the polymorphism. The polymorphism may be a polymorphism 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 polymorphism may be in linkage disequilibrium any of these particular polymorphisms mentioned above. 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. Polymorphisms which are in linkage disequilibrium with any of the polymorphisms mentioned herein are typically within 500kb, preferably within 400kb, 200kb, 100 kb, 50kb, lOkb, 5kb or 1 kb of the polymorphism. The polymorphism is typically an insertion, deletion or substitution with a length of at least 1, 2, 5 or more base pairs or amino acids. In the case of a gene region polymorphism the polymorphism is typically a substitution of 1 base pair, i.e. a single polynucleotide polymorphism (SNP). The polymorphism may be 5' to the coding region, in the coding region, in an intron or 3' to the coding region.

The polymorphism will have a sequence which is different from or the same as the corresponding region in any one of SEQ ID NO's: 1 to 25. Thus the activity of the agent (which is counter to the effect of the polymorphism) will generally lead to an antagonist effect on the receptor or pathway. As discussed above the agent may act on a component which is downstream of the insulin receptor. Such an agent may or may not have an effect on the receptor but will act on a part of the signalling pathway (in a way which is counter to the effect of the mutation on the pathway).

In one embodiment the agent has a mixed antagonist/agonist effect, acting as an antagonist towards some of the characteristics or effects of the receptor, whilst acting as an agonist towards other characteristics or effects of the receptor.

Some of the components which are discussed herein will have an agonist effect on the expression or activity of the receptor or pathway, whilst others will have an antagonist effect. Thus the activity of some of the components will have an effect which is the same as a mutation that causes susceptibility to migraine and the activity of others will have an effect which is counter to the effect of the mutation. Thus the agents which act directly on these components will act as agonists or antagonists (as appropriate) in order to lead to an effect on the receptor or pathway which is counter to the effects of the mutation.

Typically the activity of the agent will cause at least a 2, 5, 10, 20 or 50 fold decrease in the expression or activity of (i) the component which it acts on or (ii) on the insulin receptor, for example as measured in any suitable in vitro or in vivo assay mentioned herein and typically at any of the administration doses mentioned herein. Agents may cause a decrease of at least 10%, at least 25%, at least 50%, at least 100%), at least, 200%, at least 500% or at least 1000% in such expression or activity at a concentration of the agent of l μg ml^"1, 10μg ml^"1, l OOμg ml^"1, 500μg ml^'1, lmg ml^"1' lOmg ml^"1, l OOmg ml ¹. Typically the percentage decrease represents the percentage decrease in expression or activity in a comparison of assays in the presence and absence of the agent. Any combination of the above mentioned degrees of percentage decrease and concentration of agent may be used to define the agent, with a greater percentage decrease at a lower concentration being preferred.

Typically the agent binds to 1, 2 or more of the components under physiological (in vivo) conditions. Generally the binding is specific. The binding is reversible or irreversible. An agent which binds irreversibly dissociates very slowly from the component because it would be very tightly bound, either covalently or non- covalently. Reversible binding, in contrast with irreversible binding, is characterised by a rapid dissociation of the agent/component complex.

Typically the agent will affect the binding of another substance to the component (such as a substance which naturally bind the component). The agent may bind the component at the same site as the substance binds. The agent is typically able to compete for, or inhibit, the binding of the substance to the component. In one embodiment the agent does not bind the component at a site that overlaps with the site at which the substance binds. Typically such an agent does not compete with the substance for binding to component, but may still inhibit the binding of the agent to the component.

The agent may or may not cause a change in the structure of the component. In one embodiment the agent causes the component to change to a less active or nonfunctional form. This change may be reversible or irreversible. Typically the component only adopts such a changed form when bound to the agent. However the change may be irreversible, for example, if the component is chemically modified or is broken down by the agent, for example by the breaking of peptide bonds.

The agent may affect the sensitivity (such as decreasing the sensitivity) of the receptor to insulin, i.e. may increase or decrease any insulin binding-dependent activity of the receptor. The agent typically causes hyperglycemia or antihypoglycemia, inhibits insulin release or increases the clearance of insulin. The agent typically increases glucose levels, for example by countering the effects of insulin action, such as at hepatic sites and/or peripheral sites. Thus the agent will typically decrease insulin-dependent glucose disposal and/or stimulate hepatic glucose output (HGO).

The agent which inhibits/antagonises the receptor may be an agonist or antagonist of a peroxisome proliferator-activated receptor (PPAR), typically PPAR alpha or delta, preferably PPAR gamma.

Other agents include those that modulate, typically inhibit, a RXR receptor. In one embodiment the agent is a protein, polynucleotide, carbohydrate, lipid or small organic molecule. The agent may be a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof,

(I)

where n is 2, 3, or 4, R, is hexyl, heptyl, or C₄.₆alkyl-phenyl, R₂ is butyl or benzyl optionally substituted with 1 or 2 halogens,

R₃ is butyl, benzyl optionally substituted with a trifluoromethyl group or with 1 to 3 halogens, -C₄H₈OH, p-pyridyl, o-pyridyl, ethylpropionate, propyl, ethyl acetate, o- thiophenmethyl, 2,3-methylenedioxobenzyl, 2-thiazolemethyl, 2-furfuryl, R₄ is -COOII, -NHC(0)NH₂, -NHS(CH₃)0₂, -S(NH₂)0₂, hydantoin, -OH, -OCH₂C0₂H, -OCH₂CONH₂, -OCH₃,

R₅ is hydrogen or R₅ and R₄ are bonded together to form a methylenedioxo ring.

Such compounds are described in WO 00/27832. The disclosure of all WO publications and other publications mentioned herein is incorporated herein by reference. As used herein, C₄.₆alkyl includes straight or branched C₄_₆ alkyl chains such as n-butyl, tert-butyl, iso-butyl, pentyl or hexyl groups; halogen includes fluorine, chlorine, bromine and iodine, especially chlorine and bromine.

Preferably, when any of the R groups in Formula (I) are alkyl, they are straight chain alkyl. Preferably, R_a is butyl, benzyl optionally substituted with 1 or 2 halogen, or p-pyridyl.

The invention may be carried out by administering a substance which provides an agent with any of the above properties in vivo. Such a substance is also included in the term 'agent'. Typically the substance is an inactive or precursor form of the agent which can be processed in vivo to provide the agent. Thus the substance may comprise the agent associated, covalently or non-covalently, with a carrier. The substance can typically be modified or broken down to provide the agent.

The invention provides a method for screening for the agent comprising contacting a candidate substance with a product selected from: (i) one or more components as defined above,

(ii) any part of the expression pathway for a component as defined in (i); or (iii) a function d analogue of (i) or (ii), and determining whether the candidate substance binds or modulates the product, typically in a manner which decreases directly or indirectly the activity or expression of the receptor or pathway.

The method may be carried out in vitro (inside or outside a cell) or in vivo, i.e. the product may be provided in a form which is inside or outside a cell, which cell may be in vitro or in vivo. In one embodiment the method is carried out on a cell, cell culture or cell extract which comprises the component. The cell may be any of the cells mentioned herein, and is preferably the cell is one in which the component or part is naturally expressed.

The method may be carried out in an animal (such as any animal mentioned herein) whose insulin receptor gene comprises a polymorphism which causes susceptibility to cephalic pain, such as any such polymorphism mentioned herein. Typically such an insulin receptor gene is a polynucleotide provided by the invention (as described below) or comprises sequence from such a polynucleotide.

In the case where the product is a functional analogue (iii), this will have some or all of the relevant activity of (i) or (ii) will have surface that mimics the surface of (i) or (ii). Typically the analogue is or comprises a fragment of (i) or (ii). In the case where (i) or (ii) is a polynucleotide or polypeptide the analogue typically has homology with (i) or (ii). The product (i), (ii) or (iii) may be a polynucleotide or protein of the invention as described below.

Any suitable binding assay format can be used to determine whether the product binds the candidate substance, such as the formats discussed below.

The term 'modulate' includes any of the ways mentioned herein in which the agent of the invention is able to modulate a component. Whether or not a candidate substance modulates the activity of (i) or (ii) may be determined by providing the candidate substance to (i) or (ii) under conditions that permit activity of (i) or (ii), and determining whether the candidate substance is able to modulate the activity of the component. The activity which is measured may be any of the activities which is mentioned herein, and may the measurement of a change in a component or an effect on a cell or an effect on an animal in which the method is being carried out. The effect may be one which is associated with cephalic pain, and in the case of an animal may be a symptom of cephalic pain, in particular migraine. The symptom may be a behavioural change, vomiting, photophobia or phonophobia; or a electrophysiological or vasomodulatory change of the substance may be measured.

Typically the assay measure the effect of the candidate substance on the binding between the component and another substance (such as a ligand). Suitable assays in order to measure the changes in such interactions include fluorescence imaging plate reader assays, and radioligand binding assays.

In the case where the activity is transcription from a gene of a component the method may comprise measuring the ability of the candidate substance to modulate transcription, for example in a reporter gene assay. Typically such an assay comprises: (a) providing a test construct comprising a first polynucleotide sequence with the promoter activity of the gene of the component operably linked to a second polynucleotide sequence to be expressed in the form of mRNA;

(b) contacting the candidate substance with the test construct under conditions that would permit the second polynucleotide sequence to be expressed in the form of mRNA in the absence of the substance; and

(c) determining whether the substance modulates expression from the construct.

In a preferred embodiment the method for screening for the agent determines whether the agent acts as an agonist or antagonist of a PPAR, preferably gamma (e.g. a PPAR which is the same or homologous to SEQ ID NO:26), in a manner that leads to antagonising of insulin receptor activity. Such a method may be based on the methods described Willson et al (2000) J. Medicinal Chemistry 43,527-550. In one embodiment the method determines whether the agent decreases the expression or activity of an RXR ligand which has the desired effect on PPAR, i.e. an effect which leads to the antagonising of the insulin receptor.

Suitable candidate substances which tested in the above screening methods include antibody agents (for example, monoclonal and polvclonal antibodies, single chain antibodies, chimeric antibodies and CDR-grafted antibodies). Furthermore, combinatorial libraries, defined chemical identities, peptide and peptide mimetics, oligonucleotides and natural agent libraries, such as display libraries (e.g. phage display libraries) may also be tested. The candidate substances may be chemical compounds, which are typically derived from synthesis around small molecules which may have any of the properties of the agent mentioned herein. Batches of the candidate substances may be used in an initial screen of. for example, ten substances per reaction, and the substances of batches which show inhibition tested individually.

The invention also provides an isolated polynucleotide or protein that comprises (i) a polymorphism that causes susceptibility to cephalic pain, or (ii) a naturally occurring polymorphism that is in linkage disequilibrium with (i). Such polymorphisms may be any of the polymorphisms mentioned herein. The polymorphism that causes susceptibility may be one which is or which is not found in nature. The polynucleotide or protein may comprise human or animal sequence (or be homologous to such sequence). Such an animal is typically a mammal, such as a rodent (e.g a mouse, rat or hamster) or a primate. Such a polynucleotide or protein may comprise any of the human polymorphisms mentioned herein at the equivalent positions in the animal polynucleotide or protein sequence. The polynucleotide or protein typically comprises the insulin receptor gene region sequence or the insulin receptor protein sequence, or is homologous to such sequences; or is part of (a fragment of) such sequences (as discussed below such sequences may be of a human or animal). In particular the part of the sequence may correspond to any of the sequences given herein in or parts of such sequences. The polynucleotide is typically at least 5, 10, 15, 20. 30. 50. 100, 200, 500. bases long, such as at least lkb. lOkb, lOOkb, 1000 kb or more in length.

The polynucleotide of the invention is generally capable of hybridising selectively with a polynucleotide comprising all or part of the insulin receptor gene region sequence, including sequence 5' to the coding sequence, coding sequence, intron sequence or sequence 3' to the coding sequence. Thus it may be capable of selectively hybridising with all or part of the sequence shown in any one of SEQ ID NOS:l to 25 (including sequence complementary to that sequence).

Selective hybridisation means that generally the polynucleotide can hybridize to the gene region sequence at a level significantly above background. The signal level generated by the interaction between a polynucleotide of the invention and the gene region sequence is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the gene region sequence. The intensity of interaction may be measured, for example, by radiolabelling the polynucleotide, e.g. with ³²P. Selective hybridisation is typically achieved using conditions of medium to high stringency (for example 0.03M sodium chloride and either 0.003 or 0.03M sodium citrate at from about 50°C to about 60°C). Polynucleotides of the invention may comprise DN A or RNA. The polynucleotides may be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to polynucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art.

The protein of the invention can be encoded by a polynucleotide of the invention. The protein may comprise all or part of a polypeptide sequence encoded by any of the polynucleotides represented by SEQ ID NO's: 1 to 25, or be a homologue of all or part of such a sequence. The protein may have one or more of the activities of the insulin receptor, such as being able to bind insulin and/or signalling activity. The protein is typically at least 10 amino acids long, such as at least 20, 50, 100, 300 or 500 amino acids long. The protein may be used to produce antibodies specific to the polymorphism, such as those mentioned herein. This may be done for example by using the protein as an immunogen which is administered to a mammal (such as any of those mentioned herein), extracting B cells from the animal, selecting a B cell from the extracted cells based on the ability of the B cell to produce the antibody mentioned above, optionally immortalising the B cell and then obtaining the antibody from the selected B cell. Polynucleotides or proteins of the invention may carry a revealing label.

Suitable labels include radioisotopes such as ²P or "S, fluorescent labels, enzyme labels or other protein labels such as biotin.

Polynucleotides of the invention can be incorporated into a vector. Typically such a vector is a polynucleotide in which the sequence of the polynucleotide of the invention is present. The vector may be recombinant replicable vector, which may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below in connection with expression vectors.

The vector may be an expression vector. In such a vector the polynucleotide of the invention in the vector is typically operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

Such vectors may be transformed into a suitable host cell as described above to provide for expression of the protein of the invention. Thus, in a further aspect the invention provides a process for preparing the protein of the invention, which process comprises cultivating a host cell transformed or transfected with an expression vector as described above under conditions to provide for expression of the protein, and optionally recovering the expressed protein.

The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes. Promoters and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed.

The invention also provides an animal which is transgenic for a polymorphism as mentioned above. The animal may be any of the animals mentioned herein. Typically the genome of all or some of the cells of the animal comprises a polynucleotide of the invention. Generally the animal expresses a protein of the invention. Typically the animal suffers from cephalic pain, such as migraine.

The binding assay generally comprises contacting the candidate substance with the product and determining whether the binding occurs between the candidate substance and the product. The binding may be determined by measuring a characteristic of the product which changes upon binding, such as spectroscopic changes.

The assay format may be a 'band shift' system, for example based on determining whether the candidate substance advances or retards the product during gel electrophoresis. The assay may be a competitive binding assay . This determines whether the candidate substance is able to inhibit the binding of the product to an agent which is known to bind to the product, such as an antibody specific for the product.

The agent, polynucleotide, protein of the invention or any of the cells mentioned herein may be present in a substantially isolated form. They may be mixed with carriers or diluents and still be regarded as substantially isolated. They may also be in a substantially purified form, in which case it will generally comprise at least 90%, e.g. at least 95%, 98% or 99% of the dry mass of the preparation.

Homologues of polynucleotide or protein sequences are referred to herein. Such homologues typically have at least 70% homology, preferably at least 80, 90%), 95%, 97% or 99% homology, for example over a region of at least 15, 20, 30, 100 more contiguous nucleotides or amino acids. The homology may calculated on the basis of amino acid identity (sometimes referred to as "hard homology"). For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value: the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 1 1. the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1. more preferably less than about 0.01. and most preferably less than about 0.001.

The homologous sequence typically differ by at least 1, 2. 5, 10, 20 or more mutations (which may be substitutions, deletions or insertions of nucleotide or amino acids). These mutation may be measured across any of the regions mentioned above in relation to calculating homology. In the case of proteins the substitutions are preferably conservative substitutions. These are defined according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column mav be substituted for each other:

The formulation of the agent for use in preventing or treating cephalic pain will depend upon factors such as the nature of the substance and the condition to be treated. The agent may be administered in a variety of dosage forms. Thus, they can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. The inhibitors may also be administered parenterally, either subcutaneously. intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The modulators may also be administered as suppositories. A physician will be able to determine the required route of administration for each particular patient.

Typically the agent is formulated for use with a pharmaceutically acceptable carrier or diluent. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose. carboxymethylcellulose or polyvinyl pyrrolidone: disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.

Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum. agar, sodium alginate. pectin, methylcellulose, carboxymethylcellulose. or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

A therapeutically effective amount of agent is administered to a patient. The dose of modulator 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. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg, preferably from about O.lmg/kg to lOmg/kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g.

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 determine the required route of administration and dosage for any particular patient.

The following Example illustrates the invention:

EXAMPLE

Clinical criteria for identifying individuals with migraine

The following criteria were used to identify individuals with specific types of

Migraine without aura: - 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;

HA with nausea, or vomiting, or photophobia, or phonophobia (at least 1).

Migraine with aura:

Aura lasting 4-60 minutes;

HA defined as above, with onset accompanying or following aura within 60 minutes.

Familial hemiplegic migraine:

HA fulfills migraine with aura characteristics; aura includes hemiparesis that may be prolonged (> 60 minutes): at least 1 first-degree relative with similar HAs.

Geno typing of individuals for SNPs

Samples were obtained from the study group and genomic DNA extracted using a standard kit and a slating out technique (Cambridge Molecular). The 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.

For each polymorphic site the allelic discrimination assay used two allele specific primers labeled with a different fluorescent dye at their 5^" ends but with a 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 polymorphic 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.

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. If the allele corresponding to one of the specific primers was present 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. If the allele specific primer hybridized to the other allele the mismatch at the polymorphic site 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 corresponding 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'. Table 1

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Claims

1. Use of an agent that modulates directly or indirectly the insulin receptor or insulin receptor signalling pathway in the manufacture of a medicament for use in a method of preventing or treating cephalic pain.

2. Use according to claim 1 wherein the agent is an antagonist.

3. Use according to claim 1 wherein the agent directly antagonises any one of the following components: the insulin receptor, insulin receptor substrate-1 , -2, -3, or -4, p85, Grb2. Gabl. phosphatidyl inositol 3 kinase, pp60, ppl20, son of sevenless

(SOS), MAP kinase, serine phosphatase, threonine phosphatase, tyrosine kinase, ras, raf, syp, she or a G protein.

4. Use according to claim 3, wherein the component is the insulin receptor.

5. Use according to any one of the preceding claims wherein the agent antagonises insulin sensitivity.

6. Use according to any one of the preceding claims wherein the agent antagonises the effect of a cephalic pain susceptibility polymorphism in the insulin receptor gene.

7. Use according to any one of the preceding claims wherein the agent antagonises the effect of a polymorphism which is selected from INSBa, INSCa, exonδ.pol 1, exonl 1. pol l, exonl7.pol2, exonό.pol l, exon7.pol l, exon7.pol2, exon8.pol2, exon9.pol3, exonl4.pol 1 and INSR-C.4479C.T or which is in linkage disequilibrium therewith.

8. A method of screening for an agent as defined in claim 1 comprising contacting a candidate substance with (i) one or more components which affect the insulin receptor or the insulin signalling pathway, (ii) any part of the expression pathway for a component as defined in (i); or (iii) a functional analogue of (i) or (ii), and determining whether the candidate substance binds or modulates (i) ,(ii) or (iii).

9. A method according to claim 8 wherein at least one of the components is the insulin receptor protein, a peroxisome proliferator-activated receptor or an RXR ligand.

10. A method according to claim 8 comprising administering the candidate substance to a mammal whose insulin receptor gene comprises a polymorphism which is selected from INSBa, INSCa, exon8.pol 1, exonl l .pol 1 , exonl7.pol2, exonό.pol 1, exon7.pol 1. exon7.pol2, exon8.pol2. exon9.pol3, exonH.pol 1 and ΓNSR-C.4479CT or which is in linkage disequilibrium therewith; and determining whether the substance counteracts the effect of the polymorphism.

1 1. A method according to claim 10 wherein the effect is a symptom of migraine.

12. An isolated polynucleotide or protein that comprises (i) a polymorphism that causes susceptibility to cephalic pain or (ii) a naturally occurring polymorphism that is in linkage disequilibrium with (i).

13. A polynuclotide or protein according to claim 12 which comprises insulin receptor gene region sequence or insulin receptor protein sequence.

14. A polynucleotide or protein according to claim 13 wherein the polymorphism is as defined in claim 4.

15. A polynucleotide or protein according to any one of claims 12 to 14 which has a length of at least 15 nucleotides or at least 15 amino acids.

16. A vector incorporating a polynucleotide as defined in any one of claims 12 to 15.

17. A process for the preparation of a protein that modulates directly or indirectly the insulin receptor or insulin receptor signalling pathway, which process comprises: (a) culturing a host cell transformed or transfected with a vector as defined in claim 16 under conditions to provide for expression of the protein, and optionally (b) recovering the expressed protein.

18. A non-human animal which is transgenic for a polymorphism which is selected from INSBa, INSCa, exonδ.pol l, exonl 1. pol l, exonl7.pol2, exonό.pol l. exon7.pol l, exon7.pol2. exon8.pol2, exon9.pol3, exonl4.pol l and INS R- C.4479C.T or which is in linkage disequilibrium therewith.

19. A method of treating cephalic pain, which comprise adminstering to a subject in need thereof an agent that modulates directly or indirectly the insulin receptor or insulin receptor signalling pathway.