WO2001028539A9

WO2001028539A9 - Agent for treating cephalic pain

Info

Publication number: WO2001028539A9
Application number: PCT/GB2000/004031
Authority: WO
Inventors: Ian James Purvis; Linda Catherine Mccarthy
Original assignee: Glaxo Group Ltd
Current assignee: Glaxo Group Ltd
Priority date: 1999-10-19
Filing date: 2000-10-19
Publication date: 2002-02-07
Anticipated expiration: 2002-04-19
Also published as: WO2001028539A2; WO2001028539A3; AU1036801A

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

AGENT FOR TREATING 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 agonises, 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 genormc DNA or unsphced or spliced mRNA) The component may be an enzyme such as an enzyme m the glucose or lipid metabolic pathways or a kmase The component may be mtracellular or extracellular In one embodiment the component is present in a neuron or a cell in the neurovascular network which is cπtical to the generation of cephalic pain, such as a cell m the tπgeminovascular 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 polysis, increased fatty acid synthesis and incorporation into tπglyceπde. inhibition of gluconeogenesis m hepatocytes

The component may be part of or directly involved in the mtracellular 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 mtracellular 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, duπng 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 mositol 3 k ase, pp60, ppl20, son of sevenless (SOS), MAP kmase, seπne phosphatase, threonme phosphatase, tyrosme kmase, ras, raf, syp, she or a G protein

The agent may modulate components related to the glucose or lipid pathways, l 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, kmase activity (e g tyrosme. threonme or seπne kmase 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 seπne/threonme phosphorylation, dephosphorylation (via seπne /threonme - or tyrosme phosphatases) or glycosylation

The agent may modulate a product which regulates or is part of the expression pathwav 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 m any of the ways descπbed herein in which the agent acts upon the component The product may be the gene trom which any of the components is expressed, an RNA polymerase that can express mRNA. from the gene, the unsphced mRNA which is transcribed from the gene, factors that aid splicing oi the mRNA. the spliced mRNA, nuclear tactors 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 geneb The agent may bind to the component gene either I) 5' to the coding sequence, and/or (π) to the coding sequence, and/or (m) 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 transcπption trom 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 vaπants of the receptor (e g variants produced by different splicing of the mRNA), tissue-specific expression, subcellular localisation or hybπdisation with other receptors (e g the IGF-1 receptor)

The agent typically has an activity which directly or indirectly (e g mediated 1/28539

5 through any of the components discussed above) results in an effect on the msulm receptor or insulin receptor pathway which is generally counter (opposite) to the effect of a polymorphism in the msulm 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 agonist effect, but preferably has an antagonist effect on any of these characteristics of the receptor Generally this will lead to a consequent increase or decrease 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 m another part of the pathway) The polymorphism may be any ot the following polymorphisms INSBa, INSCa, exon8 poll , exonl 1 poll , exonl7 pol2 (the form of these polymorphisms will be allele 2 as defined in table 2) exonό poll , exon7 poll, exon7 po!2, exonδ pol2, exon9 pol3, exonl4 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 (m 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 m the population Polymorphisms which are m 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 ammo 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, m 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 agonist effect on the receptor or pathway As discussed above the agent may act on a component which is downstream of the msulm 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 characteπstics 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 appropπate) 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 increase in the expression or activity of (1) the component which it acts on or (n) on the msulm receptor, for example as measured in any suitable m vitro or in vivo assay mentioned herein and typically at any of the administration doses mentioned herein Agents may cause an increase 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 ug ml^"', lOμg ml ', lOOμg ml ', 500μg ml^"1, lmg ml^"1 l Omg ml ', l OOmg mT Typically the percentage increase represents the percentage increase m expression or activity m a comparison of assays m the presence and absence of the agent Any combination of the above mentioned degrees of percentage increase and 1/28539

7 concentration of agent may be used to define the agent, with a greater percentage increase at a lower concentration being preferred

Typically the agent binds to 1 , 2 or more of the components under physiological (m 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, m contrast with lπeversible 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 mav 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 of the receptor to msulm, l e may increase or decrease any msulm binding-dependent activity of the receptor The agent is typically one which can be used to prevent or treat diabetes, such as non- lnsulin dependent diabetes Typically the agent causes hypoglycemia or antihyperglycemia, stimulates msulm release or reduces the clearance of msulm The agent typically lowers glucose levels by enhancing msulm action action, such as at hepatic sites and/or peripheral sites Thus the agent will typically increase msulin- 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 (PP λR), tvpically PPAR alpha or delta preferably PPAR gamma.

The agent is typically a compound as descπbed in WO 97/31907, WO 00/08002 or US-A-5,902,726. For example, the agent may be a compound of general formula (I) or a tautomeric form or a pharmaceutically acceptable salt or solvate thereof:

C0₂R '

Alk (0

A — B - o-

wherein A is selected from the group consisting of:

(i) phenyl, wherein said phenyl is optionally substituted by one or more of the following groups; halogen atoms, C,.₆alkyl, C,.₃ alkoxy, C₁ fluoroalkoxy, nitrile, or -NR⁷R⁸ where R⁷ and R^s are independently hydrogen or C alkyl;

(ii) a 5- or 6- membered heterocyclic group containing at least one heteroatom selected from oxygen, nitrogen and sulfur; and

(iii) a fused bicyclic ring wherein C represents a heterocyclic

group as defined in point (ii) above, which bicyclic ring is attached to group B via a ring atom of ring C;

B is selected from the group consisting of:

(iv) C ₆ alkylene;

(v) -MC,.₆ alkylene or C,.₆ alkyleneMC,.₆ alkylene, wherein M is O, S, or -NR² wherein R² represents hydrogen or C,.₃ alkyl;

(vi) a 5- or 6- membered heterocyclic group containing at least one nitrogen heteroatom and optionally at least one further heteroatom selected from oxygen, nitrogen and sulfur and optionally substituted by C,_₃ alkyl; and (vii) Het-C,.₆ alkylene, wherein Het represents a heterocyclic group as defined in point (vi) above; Alk represents C,_₃ alkylene; R¹ represents hydrogen or C,.₃ alkyl; Z is selected from the group consisting of: (viii) -(C,_₃ alkylene) phenyl, which phenyl is optionally substituted by one or more halogen atoms; and

(ix) -NR^JR⁴, wherein R³ represents hydrogen or C,.₃ alkyl, and R⁴ represents -Y-

(C=0)-T-R³ , or -Y-(CH(OH))-T-R⁵, wherein:

(a) Y represents a bond, C,.₆ alkylene, C₂.₆ 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;

(b) T represents a bond, C,_.3 alkyleneoxy, -0- or -N(R⁶)-, wherein R⁶ represents hydrogen or C,.₃ alkyl;

(c) R³ represents C,.₆ alkyl, C₄.₆ cycloalkyl or cycloalkenyl, phenyl (optionally substituted by one or more of the following groups; halogen atoms, C ₃ alkyl, C,.₃ alkoxy groups, C₀.₃ alkyleneNR⁹R'° (where each R⁹ and R'° is independently hydrogen, C,.₃ alkyl, -S0₂C,_₃ alkyl, or -C0₂C,.₃alkyl, -S0₂NHC,.₃alkyl), C₀.₃ alkyleneC0₂H, C₀.₃ alkyleneC0₂C,.₃alkyl, or -OCH₂C(O)NH₂), a 5- or 6- membered heterocyclic group as defined in point (ii) above, a bicylic fused ring

wherein ring D represents a 5- or 6-membered

heterocyclic group containing at least one heteroatom selected from oxygen, nitrogen and sulfur and optionally substituted by (=0), which bicyclic ring is attached to T via a ring atom of ring D: or -C,^ alkyleneMR" wherein M is O, S, or -NR¹² wherein R'² and R" are independently hydrogen or C,.₃ alkyl. /28539

Such compounds are disclosed in WO97/31907.

The terms C₁ 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. The term C₂.₆ alkenyl or aikenylene as used herein contains 2 to 6 carbon atoms and appropriately includes straight chained and branched alkenyl and aikenylene groups, in particular propenylene or the like. The term C,.₃ alkyleneoxy as used herein denotes -O-C,.₃ alkylene-, wherein

C,.₃ alkylene is substantially as defined above, e.g. -O-CH_:- etc.

The terms C _₆ cycloalkyl, C₄.₀ cycloalkylene, C₄.₆ cycloalkenyl and C_4.6 cycloalkenylene include cyclic groups containing 4 to 6 carbon atoms, such as cyclopentane, cyclopentylene, cyclohexane, cyclohexylene, cyclohexene and cyclohexenylene.

The term halogen as used herein includes fluorine, chlorine, bromine and iodine.

The term 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, pφerazinyl, pyrrolidinyl, morpholinyl. pyridyl, pyridazinyl, pyrimidinyl, pyrazmyl, pyrrolyl, pyrazolyl, imidazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, thiadiazolyl. triazolyl or tetrazolyl.

By 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,_. alkoxy groups, C_0. alkylene N R⁹R¹⁰ (where each R⁹ and R¹⁰ is idependently hydrogen, C,.₃ alkyl, -S0₂C,.₃ alkyl or C0₂C,.₃ alkyl, -SO₂NHC,.₃ alkyl), C₀.₃ alkylene CO_:H, C₀.₃ alkylene C0₂C,.₃ alkyl, -0CH₂C(0)NH₂, -C,.₃ fluoroalkyl, -CN or SC,.₆ alkyl. In formula (I) above, in the case where Y represents a bond, the nitrogen atom of -NR R⁴ is directly linked to -(C=O) or (CH(OH)) of R\ ie. Z represents -N(R³)- (C=0)-T-R^; or -N(R³)(CH(OH))-T-R⁵. Similarly, m the case where T represents a bond. -(C=0) or (CH(OH)) of R⁴ is directly linked to R le Z represents -N(R^J)-Y- (C=0)-R^J or -N(R^J)-Y-(CH(OH))-R^> It may be the case that both Y and T represent a bond, whereby Z represents -N^XC^-R^{^} or -N(R^J)-(CH(OH))-R

Aptly A represents any of phenyl, heteroaryl (e g pyπdyl) or

wherein fused ring C represents a 5 -membered heteroaryl group contaimng at least one nitrogen heteroatom and optionally a further heteroatom selected from nitrogen and oxygen (e g oxazolyl, lmidazolyl) Particularly A represents any of phenyl, pyπdyl, piperaz yl, or benzoxazol l, any of which can optionally be substituted by one or more C, , alkyl, especially phenyl, piperazmyl, or pyπdyl

B suitably represents any of C, _J alkylene (e g methylene), -N(CH₃)C,. 3alkylene (e g -N(CH₃)(CH₂)₂-) or Het-C, ₀ 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 pyπohdmyl, oxazolyl and thiazolyl) and aptly substituted by C_{t 3} alkyl Particularly B represents -N(CH₃)(CH₂)₂, oxazolyl -C\-6 alkylene, which oxazolyl is optionally substituted by C, alkyl, or thiazolyl which is optionally substituted by C,_.3 alkyl Appropriately Alk represents methylene

Appropriately R¹ represents hydrogen, methyl or ethyl, especially hydrogen Suitably Z may represent -(C, ₃ alkylene) phenyl substituted by one or more halogen atoms, such as optionally substituted benzyl Preferably Z represents -NR³R⁴ substantially as hereinbefore descπbed Generally R^J represents hydrogen As hereinbefore described, R⁴ represents -Y-(C=0)-T-R\ or -Y-(CH(OH))-T-R³ especially -Y(C=0)-T-R⁵, and particular groupings represented by R⁴ include

Y represents phenyl (optionally substituted by one or more halogen atoms, or one or more C, ₃ alkyl e g methyl groups), T represents a bond or an oxygen atom, and R³ 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 descnbed (e g thienyl), T represents a bond and R³ represents phenyl (optionally substituted by one or more halogen atoms or one or more C, ₃ alkyl groups) Y represents C₂.₆ aikenylene- (e.g. propenylene), T represents a bond and R⁵ represents phenyl (optionally substituted by one or more halogen atoms);

Y represents C₄.₆ cycloalkenylene- (e.g. cyclohexenylene), T represents a bond and R^ represents phenyl; Y represents phenyl, T represents a bond and R⁵ represents a heterocyclic group substantially as hereinbefore described (e.g. piperidyl);

Y represents a bond, T represents a bond and R³ represents a bicyclic ring

substantially as hereinbefore described (e.g. D represents a

6-membered heterocyclic ring, in particular pyranyl substituted by (C=0));

Y represents phenyl, T represents C₁ alkyleneoxy (e.g. -O-CH2-) or N(R^ϋ)- (e.g. -NH-) and R^ represents phenyl.

Preferably when Z represents NR R⁴ R^J represents H and R⁴ represents Y- (C=0)-T-R^:ι the said NH and said (C=O) are positioned ortho to each other on Y (which is phenyl), T is a bond or -0-, R³ is C ₆ alkyl, or phenyl (optionally substituted by one or more: halogen atoms, C,.₃ alkyl, C,.₃ alkoxy groups, C_0. ₃alkyleneNR⁹R¹⁰ where each R⁹ and R¹⁰ is independently hydrogen, C,.₃ alkyl. -S0₂C,_. ₃alkyl, or -CO_zC,.₃alkyl, - S0₂NHC,.₃alkyl, C₀.₃ alkyleneCO H, C₀.₃ alkyleneC0₂C,. ₃alkyl, or - OCH₂C(0)NH₂).

Particularly suitably Y represents phenyl, T represents a bond or -0- and R⁵ represents C,.₃ alkyl or phenyl e.g. R⁴ represents

R¹³ wherein R¹³ represents phenyl or OCH₃.

.An appropπate subgroup of compounds according to the present invention can be represented by formula (la) CO,H

A — B - 0- NH (la)

Ar

O ^ '

wherein 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.

Suitably in formula (la), A is selected from phenyl, pyridyl and benzoxazoyl. in particular, A in Formula (la) represents phenyl or pyridyl. Furthermore, B in Formula la) is suitably selected from -NR²C,.₆ alkylene substantially as hereinbefore described and Het-C,.₆ alkylene optionally substituted by C,.₃ alkyl substantially as hereinbefore described. In particular, B in Formula (la) represents -N(CH₃)(CH_^)₂- or oxazolyl-C,.₆ alkylene, which oxazolyl is optionally substituted by C,.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:

(i) phenyl optionally substituted by one or more halogen atoms; (ii) a 5- or 6- membered heterocyclic group containing at least one heteroatom selected from oxygen, nitrogen and sulfur; and

(iii) a fused bicyclic ring C represents a heterocyclic

group as defined in point (ii) above, which bicyclic ring is attached to group

B via a ring atom of ring C; /28539 l-i

B is selecteα from the group consisting of

(IV) C, ₀ alkylene,

(v) -NR²C, ₆alkylene, wherein R² represents hydrogen or C, ₃ alkyl, a 5- or 6- membered heterocyclic group containing at least one nitrogen heteroatom and optionally at least one further heteroatom selected from oxygen, nitrogen and sulfur and optionally substituted by C, ₃ alkyl, and (vπ) Het-C, ₆alkylene, wherein Het represents a heterocyclic group as defined m point (vi) above,

Alk represents C, ₃alkylene,

R' represents hydrogen or C,.₃ alkyl, Z is selected from the group consisting of (vm) -(C, ₃alkylene) phenyl, which phenyl is optionally substituted by one or more halogen atoms; and (ix) -NR'R⁴, wherein R^J represents hydrogen or C, ₃ alkyl, and R⁴ represents -Y-

(C=0)-T-R⁵ , or -Y-(CH(OH);-T-R⁵, wherein

(a) Y represents a bond, C, ₆ alkylene, C_{2 6} aikenylene, 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,

(b) T represents a bond, C, ₃ alkyleneoxy, -0- or -N(R⁶)-, wherein R⁶ represents hydrogen or C_[_₃ alkyl, (c) R^ represents C, ₆ alkyl, C₄.₆ cycloalkyl or cycioalkenyl, phenyl optionally substituted by one or more halogen atoms or one or moie C, ₃ alkyl groups, a 5- or 6- membered heterocyclic group as defined

in point (n) above, or a bicyclic fused rmg

wherein ring D represents a 5- or 6-membered heterocyclic group containing at least one heteroatom selected from oxygen, nitrogen and sulfur and 1/2

15 optionally substituted by (=0), which bicyclic ring is attached to T via a ring atom of ring D; or a tautomeric form thereof, and/or a pharmaceutically acceptable salt or solvate thereof. Preferred examples of the compounds of formula (I) include (S)-(2-benzoy - phenylamino)-3-{4-[2-(5-methyl-2-phenyl-oxazol-4-yl) ethoxy]-phenyl}propionic acid, and 2-(S)-(l-carboxy-2-{4- {2-(5-methyl-2-phenyl-oxazol-4-yl)-ethoxy]- phenyl}-ethylamino)-benzoic acid methyl ester.

Alternatively the agent may be a compound of general formula (II) or a tautomeric form, pharmaceutically acceptable salt or solvate thereof:

(II) wherein;

R^2'1 is hydrogen or C_halky!;

R^:"2 is hydrogen, or C,.₃alkyl optionally substituted by one or more halogens;

R^2"3 is C,.₆alkyl, C₄.₇cycloalkyl or cycloalkenyl, -OC,.₆alkyl, -NR'R' (where each R' is independently hydrogen or C,.₃alkyl), 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,.₆alkyl optionally substituted by one or more halogens, -OC,.₆alkyl optionally substituted by one or more halogens, -CN, or -N0₂), or phenyl (optionally substituted by one or more halogen, C,_₆alkyl optionally substituted by one or more halogens, -OC,.₆alkyl optionally substituted by one or more halogens, -CN, or -N0₂);

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, -OC^alkyl optionally substituted by one or more halogens, -CN, or -N0₂), or phenyl (optionally substituted by one or more halogen, C₁.₆alkyl optionally substituted by one or more halogens, -OC,_.6alkyl optionally substituted by one or more halogens, -NR'R' (as defined above), -CN, or - NO_:); R²° is hydrogen, halogen, or C,_.3alkyl optionally substituted by one or more halogens;

R^2'6 is hydrogen or C,.₃alkyl; X is O or S; and n is 1 , 2, or 3. Such compounds are disclosed in WO 00/08002.

As used above, C,.₈ alkyl is preferably C,.₆ alkyl, and C ₆ is preferably C,_.3 alkyl. Typical C,_₆ alkyl and C._₃ alkyl groups are as defined above. Typical C_4.7 cycloalkyl, C₄.₇ cycloalkenyl and 5- or 6-membered heterocyclic groups and typical halogen atoms are as defined above. Preferably, R^2"1 is hydrogen or methyl. Most preferably, R^2"1 is hydrogen.

Preferably, R^2'2 is C,._galkyl optionally substituted by one or more halogens. Preferably, said halogen is fluorine. Most preferably, R^2"2 is straight-chain. Preferably, R^2"3 is pyridine, pyrazine, thiophene, furan, thiazole, or phenyl (any of which may be optionally substituted by one or more halogen, C^alkyl optionally substituted by one or more halogens, -OC,_.6alkyl optionally substimted by one or more halogens, -CN, or -N0₂), or C₄.₇cycloalkyl. Most preferably. R^2"3 is phenyl (optionally substituted by one or more halogen, C,_₆ alkyl optionally substituted by one or more halogens, -OC,.₆alkyl optionally substituted by one or more halogens, -CN, or -NO₂). Preferably R^2"4 is phenyl (optionally substituted by one or more halogen, C,.

₆alkyl optionally substituted by one or more halogens, or -OC,.₆alkyl optionally substituted by one or more halogens). Preferably, said halogen is fluorine. Most preferably R^2"4 is phenyl either unsubstituted or substituted with 1 , 2, or 3 fluorine atoms. Preferably, R^2"5 is hydrogen, halogen, or C,.₃alkyl optionally substituted by one or more halogens. Most preferably R²° is hydrogen. Preferably R^2"6 is methyl or ethyl. 17

Preferably n is 2.

Preferably, the carbon atom bonded to C0₂R^2"' is in the S configuration. In other words, preferably, the absolute configuration around that carbon is:

\ C0₂R^2~' NH

Preferred examples of the compounds of general formula (II) include (2S)-2- ([(Z)-l-methyi-3-oxo-3-phenyl-l -propenyl]amino}-3-{4-(5-methyl-2-phenyl-l ,3- oxazol-4-yl) ethoxyjphenyljpropanoic acid and (2S)-3- (4-[2-(5-methyl-2-phenyl- l ,3-oxazol-4-yl)ethoxy]phenyl}-2-{[(Z)-3-oxo-3-phenyl-l-(trifluoromethyl)-l - propenyl] amino propanoic acid.

The agent may be a sulfonylurea (e.g. l -butyl-3-sulfonylurea, tolbutamide, chlorpropamide, tolazamide, acetohexamide, glyburide, glipizide or gliclazide), a guanide (guanide or chloroguanide), a biguanide (e.g. phenformin, metformin or buformin) or an α-glucosidase inhibitor (e.g. acarbose).

The agent may be selected from thiazolidinediones, such as the compounds of formula (III)

wherein

R^J"' is selected from the group consisting of hydrogen, C,.₈alkyl, aminoC,_. ₈alkyl, C^alkylaminoC^alkyl, heteroarylaminoC,_₆alkyl, (heteroaryl)(C,_. ^lky^aminoC^alkyl, (C₄.₈cycloalkyl)C,_.8alkyl, C,.₈alkylheteroarylC|.₈alkyl, 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. Preferably, the dashed line ( ) represents no double bond. Such compounds are described in US-A-5,902,726.

As used in formula (III), C _i alkyl is preferably C,.₆ alkyl, more preferably C ₃ alkyl. Typical C,.₆ alkyl and C,.₃ alkyl groups are as defined above. Typical heteroaryl groups are 5- or 6-membered heterocyclic groups as defined above. C_4.3 cycloalkyl is preferably C .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, 0 or S.

Prefeπed compounds of formula (III) are those wherein R^3"1 is selected from (i), (ii) or (iii) below:

(i) a substituted 9 or 10 membered heterobicycle which is partially aromatic such as a group

wherein

R^3"2 and R^3"3 are the same or different and each represents a hydrogen atom or a C,-C₅ alkyl group; R^J_4 represents a hydrogen atom, a C,-C₆ aliphatic acyl group, an alicyclic acyl group, an aromatic acyl group, a heterocyclic acyl group, an araliphatic acyl group, a (C,-C₆ alkoxy)carbonyl group or an aralkyloxycarbonyl group; and

R^J and R^3"6 are the same or different and each represents a hydrogen atom, a C_[-C₅ alkyl group or a C,-C₅ alkoxy group, or R³° and R^3"6 together represent a C,-C₄ alkylenedioxy group.

Preferably R^3"2, R^3"3, R^3"5 and R^3"6 are each methyl and R^3"4 is hydrogen. In this case the agent is troglitazone;

(ii) heteroarylaminoC,.₆ alkyl group such as a group

wherein:

R^{J~ '} is hydrogen or a C _₆ alkyl group and n is 1, 2, 3 or 4. Preferably R^3"7 is methyl and n is 1 , in which case the agent is rosiglitazone.

(iii) a C_[.₈ alkylheteroaryl C,._g alkyl group such as a group

wherein:

R^3"8 is a C,.₆ alkyl group and n is 1, 2, 3 or 4. Preferably R^3"8 is ethyl and n is 1. More preferably the C,_₈ alkylheteroaryl C^ alkyl group is

in which case the agent is pioglitazone.

In the above groups (i), (ii) and (iii), where 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.

Where 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. W ere R^3"4 represents an alicyclic acyl group, it is preferably a cyclopentanecarbonyl, cyclohexanecarbonyl or cycloheptanecarbonyl group. Where R^3"4 represents an aromatic acyl group, the aromatic moiety thereof may optionally have one or more substituents (for example nitro, ammo, alkylamino, diaikylamino, alkoxy, halo, alkyl or hydroxy substituents); examples of such aromatic acyl groups include benzoyl, p-nitrobenzoyl, m- fluorobenzoyl, o-chlorobenzoyl, p-aminobenzoyl, m-(dimethylamino)benzoyl, o- methoxybenzoyl, 3,4-dichlorobenzoyl, 3,5-di-t-butyl-4-hydroxybenzoyl and 1- naphthoyl groups. Where 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. Where 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, alkylam o, dialkylamino, alkoxy. halo, alkyl or hydroxy substituents): examples of such araliphatic acyl groups include the phenylacetyl, p-chlorophenylacetyl, phenylpropionyl and cinnamoyl groups. Where R^3"4 represents a (C,-C₆ 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. Where 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.

Where R^3"3 and R ^"δ 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. Alternatively, R^J° and R^J"δ may together represent a C,-C₄ alky lenedioxy group, more preferably a methylenedioxy 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 a thiazolidinedione as described in U.S. Pat Nos. 5,089,514, 4,342,771, 4,367,234, 4,340,605 or 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 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-phenylalcanoιc acid drivadves, such as those descπbed m WO 97/31907, hypoglycemic alkaloids, such as qumdolme and cryptolepme which may be obtained from extracts from Cryptolepsis sp as disclosed in US-Λ-5.629,319, as well as tπterpenoid substances, such as tnose disclosed US-A-5,691 ,386, and eremophilanohde sesquiterpenes, such as described in US-A-5,747,527

Other suitable agents include polymorphic iorms of troglπazone, terpenoid- type qumones 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 incorporated herein by reference

Other 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 angiotensm II antagonist or angiotensin converting enzyme inhibitor In one embodiment the agent is a protein, polynucleotide, carbohydrate, lipid or small organic molecule

The invention may be carried out by admimsteπng a substance which provides an agent with any of the above properties in vivo Such a substance is also included m the term 'agent' Typically the substance is an inactive or precursor form of the agent which can be processed in vι\o 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 (l) one or more components as defined above, (n) any part of the expression pathway for a component as defined in (I), or (in) a functional analogue of (l) or (n), and determining whether the candidate substance binds or modulates the product, typically in a manner which increases 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, l e the product may be provided m a form which is inside or outside a cell, which cell may be in vitro or in vivo In one embodiment the method is cameα out on a 99 cell, cell culture or cell extract which comprises the component The cell mav 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 earned out in an animal (such as any animal mentioned herein) whose msulm receptor gene comprises a polymorphism which causes susceptibility to cephalic pain, such as any such polymorphism mentioned herein Typically such an msulm receptor gene is a polynucleotide provided by the invention (as descπbed below) or comprises sequence from such a polynucleotide

In the case where the product is a functional analogue (m), this will have some or all of the relevant activity of (I) or (a) will have surface that mimics the surface of (l) or (a) Typically the analogue is or comprises a fragment of (1) or (n) In the case where (l) or (a) is a polynucleotide or polypeptide the analogue typically has homology with (1) or (a) The product (l), (a) or (in) 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 (l) or (a) may be determined by providing the candidate substance to (l) or (a) under conditions that permit activity of (I) or (a), 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 ammal 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, m 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 assavs in order to measure the changes in such interactions include fluorescence imaging plate reader assays, and radiohgand 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 activation/agonising of msulm receptor activity Such a method may be based on the methods described Willson et al (2000) J Medicinal Chemistry 43,527-550 In the embodiment the method determines whether the agent increases the expression or activity of an RXR ligand which has the desired effect on PPAR, l e an effect which leads to the activation of the msulm receptor

Suitable candidate substances which tested in the above screening methods include antibody agents (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimeπc antibodies and CDR-grafted antibodies) Furthermore, combinatorial hbraπes, defined chemical identities, peptide and peptide mimetics, ohgonucleotides and natural agent libraπes, 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 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^ϋC to about 60°C).

Polynucleotides of the invention may comprise DNA 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 ³²P 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 oi tne invention into a rephcable 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 descπbed below m connection with expression vectors

The vector may be an expression vector In such a vector the polynucleotide of the invention m 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 descπbed 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 descnbed 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 transgemc 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 ammal 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%o 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%o 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 refeπed 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 may 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 earner 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 pyπolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurvlsulphates; 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 earner, 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 earner, 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 jor identifying individuals with migraine

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

Viigraine without aura

FIA (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

Genotyping of individuals for SNPs

Samples were obtained from the study group and genomic DNA extracted using a standard kit and a slating out technique (Cambπdge Molecular) The genotypes of the migrameurs with aura and control individuals for individual SNPs within the msulm 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

ϊ u - I _

TGTCTTGAAAGCCCTTATCAGAATGTGACG TGTGGTTCCAACAGTTGGACGGTGGTAGACATTGA

INSR GAGTTCGA1 GGGCAGGATGC CCCACCCC ΓGAGG I C

TTGTAAGCCTCTATAAm ATAT I I I I I GTTC AΛ AATCTTTAATGATGCTTG IATTTAACAACTGG

SI l ATΓI GGAAGGCAΓI GTA C ΓCACTAG Γ I I CC

CGACTGGGACTCAG

90 c. 11 J . GCAGGCAG I ACAGGAACCATGA1 GC . GGLA

CCGAGGAGACCCCGCGCTCCCGCAGCCAT GCGGCGGCCGCGCCGCTGCTGGTGGCGGTGGCC 2M exon 1 poll 1862 GGGCACCGGGGGCCGGCGGGG GCGCTGC1ACTGGGCGC

CAGCCCGACGACCCCACCAAGTGCGTGGC

J-L άxon- pol l 244 c ΓGCCGCAAC . I C I ACCI GGA

TGAGTTCTGGATGTGGGTCTGGGGGGCAG 2b I axon j μo!2 407 CCGAGAGGAGAΛGGAALG I G GCAAACCCAGGGT

CAATGGGGACCAGGCATCCTGTAAGTCACT GAGGGACCGGTTTAGTGGAAGATGGTTTTTCCATG 2b. exon6 poll 43 GGTCCCCAACC I ITTTGGCA GACTGGTGGTGGG1 G c TTCATAGTCAGCCAGGCACA TGGGGAGGA TCACCTCTGCCTTCTCACGGTCCCTCCAGGAAGTG

CD 2b3 exoιι7 poll 169 GACCCTΪAAGGGAATAGCAGC I GGGGGTCCCAbGCT 01 TTCATGCTGTTCTACAAAGAGGCGTAAGTA GGAGGCGAGGGC1 GGCTGGCTCTGTGCTTGCTAC

H ^64 _ fcxu<)7 po!2 431 GAAGAG 11 GAGAGACGC1 G G l I I G TGC I CCAATCT

H CTTCTGCfGTTTTGTCTTGAAAGCCCTTATC GGGCAGGATGCGTGTGGTTCCAACAGTTGGACGG^" 26b C exoiifl poll 151 AGAA I G I GACGGAGTTCGA IGG ΓAGACA I ΓGACCC -1 TTGTCTTGAAAGCCCTTATCAGAATGTGAC TGTGGTTCCAACAGTTGGACGG . GGTAGACATTGA m JJ exon- po!2 i_L_ GGAGTTCGA1 GGCAGGA I GC CCC

CGCCATCCTCCCACCAGCTTTCTTTGCACA TTTCCTTTCTCCCTGGCAGACCCCTCTGTGCCCCT

W 266 exon9 poll 66 c r GΠTCTCA i GATGGACCC GGAICCAATCΓCAGT

OC m GCTTΓCTTTGCACACTGTTTCTCATGATGGA CAGACCCCTC . GTGCCCCTGGATCCAATCTCAGTG m 267 exon9 pϋ!2 103 CCCGTΠ ccrrrcTccciG rc ΓAAC ΓCATCATCC

H CTGTGCCCCTGGATCCAATCTCAGTGTCTA TGAAGTGGAAACCACCCTCCGACCCCAATGΩCAAC 26B exon9 μolJ 1b2 ACTCATCAL CCCAGATTATT ATCACCCAC1ACC1G

CCAATCTCAGTGTCTAACTCATCATCCCAG CCCTCCGACCCCAATGGCAACATCACCCACTACCT c 2§9 exon9 put 176 _ ATT I GGTΠ C I GGGAGAG r- CAGGGCCGGCGGCGTGCCAGGCAGA ΓGC TGGCTΠTTGCATGCGGCGGGCAGCTGTGCTGGA m 270 exonl 1 poll 4B CTCGGAGAACCCAGGGGT TC I GAGCAGATGCI ΓCACC t AAGGCTGATGACATTG ITGGCCC I^" G TGACG GTCGTCCACTTGATG RGGCAGGAGCCGAAGGAGC σ. --71 exon !3 poll 145 CATGAAATC r π GAGAAC AA CCAA TGGTC I faATCGT

GCTTAGCACTGAGCATGGTGGGACATTGCA] CAGTGCTGGCCCTGGCCTTGACTCTCAGGCCTATC 272 exon 14 poll 332 GGGGA1 GACTTGGAGAGGCC AGC1 GC1 GCGGTGCT

CAAGGATGCTGTGTAGATAAGTAAGAAGTA G1GCCGGACGAGTGGGAGGTGTCTCGAGAGAAGA

-IL exon!7 poll 155 GTG1TTCCATGC I CTGTGTA TCACCCTCC1T CGAOA

ATTGAGTTCCTCAATGAGGCCTCGGTCATG GTGGTGAGTCCAGfGGGGGTGGGACATGGGCTGG

-Ii exuπ 17 pol2 11- AAGGGCITCACC (GCCATCA c ι r rccTGACcc i rcc

GTTTGAAAGCCTCTGGAAMCTCAGGATTC TGGAG TTCAGAGATCGTTCCTATACA RTTCTGTTCA

INSR - 4359G-A 541 ΓCACGACTCTACCATGTCCA I C ΠAAGGTGGACT

CTAGTCCTGCAGAGGATTTAACTGTGAACC AGTTGCTGCTCCTTTGGGGCAACGACGGTTTCAAA

--L INSR c 4479C=T 661 TGGAGGGCAAGGGGTTTCCA ccA GAi r . ΓG \ G IT

TGTTTCGGGACTGCTGGAAAATCAGGATGT ATGGACAAGGGAGGGAAGGAACAGGGTGGCCCAC

294 INSR IVS14 121G-1 166 GGAAGAGCAGCAGAGAGG Π CCAUCCAGGAGTGGA

CCCGGGCGCAGAGTCCCTTCCTAGGCCAG GCCCGCACGGGCCCAGCTGACGGGCCGCGTTGTT

2_a INSR g 496G>A 1323 A ΓCCGCGCCGCCTTT ICCCGC TACGGGCCCGAGCAGC

TTCCCGCGGCCCGCACGGGCCCAGCTGAC G^'AGCAGCCCTCTCTCCCGCCGCCCGCCCGCCACC

300 INSR-g 453C-G 1366 GGGCCGCGΠ GTTTACGGGCC CGCCAGCCCAGGTGCC

GGCTGTCCCCAGGGGCCTGGCTTGGG . CT CGCGGGCGGGACATCTGGGGGCG CCACGCGCT joa INSR 9 -1029G'! 795 CGCCL-CTGGGCCGGGGCGCAC C ΓGGGACGAGTG ΓCGCT

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 agonist.

3. Use according to claim 1 wherein the agent directly agonises 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 agonises insulin sensitivity.

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

7. Use according to claim 6 wherein the agent agonises the effect of a polymorphism which is selected from INSBa, INSCa, exonδ.pol l, exonl l .pol 1, exonl7.pol2, exonό.pol l , exon7.pol l, exon7.pol2, exon8.pol2, exon9.pol3, exonl4.pol l 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, exonδ.pol l, exonl l .pol 1, exonl7.pol2, exonό.pol l , exon7.pol 1, exon7.pol2, exon8.pol2, exon9.pol3, exonl4.pol l and INSR-C.4479C.T 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 7.

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 polymoφhism which is selected from INSBa, INSCa, exonδ.pol 1, exonl l .pol l, exonl7.pol2, exonό.pol l, exon7.pol l , exon7.pol2, exon8.pol2, exon9.pol3, exonl4.pol l and INSR- 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.