WO2004098627A9

WO2004098627A9 - Inhibition of platelet aggregation through acyclovir and two peptide antagonists

Info

Publication number: WO2004098627A9
Application number: PCT/IE2004/000068
Authority: WO
Inventors: Niamh Moran; Kelly Aylward; Deirdre Larkin; Desmond Fitzgerald
Original assignee: Royal College of Surgeons in Ireland
Current assignee: Royal College of Surgeons in Ireland
Priority date: 2003-05-06
Filing date: 2004-05-06
Publication date: 2005-01-20
Anticipated expiration: 2005-11-06
Also published as: WO2004098627A1

Abstract

A method of inhibiting platelet activation in an human subject comprises the step of treating the subject with an agent which has an effect of preventing or disrupting an association between a nucleotide sensitive chloride channel ICLn and a platelet specific Integrin αIIbβ3. The agent may be an inhibitor of the nucleotide sensitive chloride channel ICLn or a ligand having at least a mM binding affinity for an amino acid sequence KVGFFKR, such as a peptide comprising the sequence AKFEEE or the sequence KVGAAKR.

Description

INHIBITION OF PLATELET AGGREGATION THROUGH ACYCLOVIR AND TWO PEPTIDE ANTAGONISTS

Introduction

The invention relates to a method of, and medicaments for, inhibiting platelet or neutrophil activation in a human subject.

Under normal conditions of haemostasis, circulating blood platelets do not bind to plasma fibrinogen. However, under prothrombotic conditions, the conformation of the platelet-specific integrin αllbβ3 changes radically, allowing high affinity binding of the bivalent fibrinogen. Little is known about this conformational change in αllbβ3 although its occurrence can be tracked using monoclonal antibodies. Many drugs are in development which act to competitively inhibit fibrinogen binding to activated αllbβs and therefore prevent platelet aggregation. However, results from clinical trials are indicating that these drugs are not as effective as predicted in the treatment of ischemic diseases . This is probably because many integrin signalling events are dependent on activation of αllbβ3 and independent of ligand occupancy of the integrin.

The conformational change in αllbβ3 is the composite result of at least two processes: intracellular signals converge on the cytoplasmic tails of the integrin conveying the intention to activate while the extracellular domains, which constitute greater than 90% of the molecules, actually respond with an increased affinity for ligand and altered display of antibody epitopes suggestive of altered protein folding. A highly conserved amino acid motif has been identified in the cytoplasmic tails of integrin alpha subunits (Stephens et al 1998 J. Biol . Chem. 273, 20317) . This motif, KVGFFKR, is critical for the intracellular-mediated activation of αllbβ3. Deletion or mutation of this sequence causes the integrin to assume an active conformation.

Statements of Invention

A method for inhibiting platelet or neutrophil activation in humans is disclosed. In one aspect, the method is based on the finding that the human platelet-specific llbβ₃ and neutrophil-specific _Lβ₂,0Cχβ₂ integrin are associated with a nucleotide specific chloride channel Icln and that prevention or disruption of this association has the effect of inhibiting platelet and/or neutrophil activation. Thus, human platelet and or neutrophil cells are contacted with an effective amount of an agent which disrupts or prevents the association between the platelet-specific or neutrophil-specific integrin and the chloride channel.

According to the invention, there is provided a method of inhibiting platelet or neutrophil activation in a human subject comprising the step of treating the subject with an agent which has an effect of preventing or disrupting an association between a nucleotide sensitive chloride channel ICLn and a platelet-specific αlI_Bβ₃ or neutrophil-specific 06_Lβ₂, 0Cχβ₂ Integrin. In one embodiment, the agent is an inhibitor of the nucleotide sensitive chloride channel ICLn. As such, the agent may be any one of the substituted purine compounds disclosed in United States Patent 4,199,574, published 22 April, 1980. Preferably, the agent is the compound known in the trade as ACYCLOVIR. Alternatively, the agent may be another type of nucleotide analogue.

In an alternative embodiment of the invention, the agent is a ligand having at least a mM binding affinity for an amino acid sequence KVGFFKR. Preferably, the ligand is a protein, peptide or compound which includes the sequence AKFEEE or KVGAAKR. Typically, the protein or peptide is protected at its amino and/or earboϋry terminus. In one preferred embodiment of the invention, the ligand consists essentially of the amino acid sequence AKFEEE or KVGAAKR. Alternatively, the ligand is a protein, peptide or compound which has at least a mM, preferably a μM, and more preferably a nM, binding affinity for the sequence AKFEEE or KVGAAKR . In one embodiment of the invention, the ligand is modified to enhance it's cell permeability. In this regard, the ligand may be modified by incorporation of a palmitate (Pal) group. In a preferred embodiment the agent consists of Pal- AKFEEE or Pal-KVGAAKR.

The invention also relates to a method of inhibiting platelet or neutrophil activation in a human subject comprising the step of treating the subject with an effective amount of an inhibitor of the nucleotide sensitive chloride channel ICLn.

The invention also relates to a method of inhibiting platelet or neutrophil activation in a human subject comprising the step of treating the subject with an effective amount of a ligand having at least a mM binding affinity for an amino acid sequence KVGFFKR. Preferably, the ligand has a μM, or even more preferably, a nM binding affinity for the sequence KVGFFKR. In one embodiment, the ligand includes, or consists essentially, of the sequence AKFEEE or KVGAAKR. Alternatively, the ligand is a protein, peptide or compound which has at lea.st a mM, preferably a μM, and more preferably a nM, binding affinity for the sequence AKFEEE or KVGAAKR. Suitably, the ligand is modified by incorporation of a palmitate (Pal) group. Ideally, the ligand consists essentially of Pal-KVGAAKR or Pal -AKFEEE. The methods of the invention may be used to prevent or treat diseases or conditions which are conventionally treated with platelet activity modulating agents, and particularly agents which decrease platelet activity. One such condition is stroke. Other conditions or diseases include Atherosclerosis and Coronary Artery Disease, Ischemic Cerebrovascular Disease, Peripheral Vascular Disease, Diabetes Mellitus, Renal Disease, Inflammation, Antimicrobial Host Defence, Tumor Growth and Metastasis, Alzheimer Disease and Psychiatric Disorders.

The methods of the invention may be used to prevent or treat diseases or conditions which are conventionally treated with neutrophil activity modulating agents, and particularly agents which decrease neutrophil activity. Examples of such diseases or conditions include inflammatory diseases such as rheumatoid inflammation, response to infection, ischemia-reperfusion injury and graft rejection, and luekocyte adhesion deficiency.

The invention also relates to the use of an agent in the manufacture of a medicament for the inhibition of platelet or neutrophil activation, which agent has an effect of preventing or disrupting an association between a nucleotide sensitive chloride channel ICLn and a platelet-specific ( llbβ3) or neutrophil-specific ( _Lβ₂, o._xβ₂) Integrin. In one embodiment of the invention, the agent is an inhibitor of the nucleotide specific chloride channel, ICln. In an alternative embodiment, the agent is a ligand having at least a mM binding affinity for the amino acid sequences KVGFFKR or AKFEEE. In one embodiment, the ligand includes, or consists of a peptide having a sequence selected from the group comprising: AKFEEE and KVGAAKR. Typically, the ligand is modified by incorporation of a palmitate (Pal) group.

The invention also relates to the use of an agent in the manufacture of a medicament for the prevention or treatment of a disease or condition which is conventionally treated with platelet or neutrophil antagonists, which agent has an effect of preventing or disrupting an association between a nucleotide sensitive chloride channel ICLn and a platelet or neutrophil specific Integrin. Examples of such diseases or conditions are provided above. In one embodiment of the invention, the agent is an inhibitor of the nucleotide specific chloride channel, ICln. In an alternative embodiment, the agent is a ligand having at least a mM binding affinity for the amino acid sequences KVGFFKR or AKFEEE. In one embodiment, the ligand includes, or consists of, a peptide having a sequence selected froom the group comprising: AKFEEE and KVGAAKR. Typically, the ligand is modified by incorporation of a palmitate group.

The invention also relates to a peptide, protein or compound which includes the sequence AKFEEE or KVGAAKR, and the use of the peptide, protein or compound as a medicament . The invention also relates to an inhibitor of the chloride channel ICLn for use as a medicament .

The invention also relates to a method of identifying a platelet or neutrophil antagonist comprising: -providing a candidate compound; -contacting a (poly) peptide with the candidate compound, the (poly) peptide including an amino acid sequence selected from the group comprising: A-K-F and K-V-G; and -determining whether the candidate compound binds to the (poly) eptide.

Suitably, the peptide includes, or consists of, a sequence selected from the group comprising: KVGFFKR; and AKFEEE.

In one embodiment, the (poly) peptide consists of the platelet or neutrophil specific integrins described above. In another embodiment, the (poly) eptide consists of recombinant nucleotide sensitive chloride channel ICL_n.

In a preferred embodiment, the (poly) peptide is immobilised to a support. Typically, the support is a 96 or 384 well plate. Suitably, the (polypeptide) will include a biotin moiety or a HIS-tag to facilitate immobilisation. In one embodiment, after incubating the (poly) peptide with the candidate compound, the (poly) peptide is contacted with a moiety which has a binding affinity for the polypeptide, which moiety includes a detectable label. Typically, the detectable level is a fluorescent label such as, for example, FITC, carboxy-fluorescein or Cy-dye. Where the candidate compound has antagonist activity, reduced levels of fluorescence will be detected compared with a control .

When the (poly) peptide contains the sequence KVG, i.e. KVGFFKR or the platelet or neutrophil specific integrin, the moiety which has a binding affinity for the (poly) peptide preferably comprises the sequence AKFEEE, ideally fluorescent AKFEEE. Likewise, when the (poly) peptide contains the sequence AKF, i.e. AKFEEE or recombinant ICL_n, the moiety which has a binding affinity for the (poly) peptide preferably comprises the sequence KVGFFKR, ideally fluorescent KVGFFKR.

Thus, in one embodiment of the invention, the polypeptide (say for example biotin tagged AKFEEE peptide) is immobilised in the wells of a 96 well plate. The AKFEEE coated plate is then pre- incubated with different candidate antagonist compounds (one candidate per well) with some wells being left blank for the purpose of positive controls. Fluorescently labelled KFGFFKR is then added to each well and allowed to incubate for a period of time. Excess unbound peptide is then washed from the wells, and the wells in the plate are then read for fluorescence. For any given well, the presence of a platelet or neutrophil antagonist will be indicated by a reduction in fluorescence compared with a positive control.

Brief Description of the Figures

The invention will be more clearly understood from the following detailed description, given by way of example only, in which:

Fig. 1 is a confocal microscopy picture demonstrating localisation of the integrin llbβ3 and Chloride Channel ICln;

Fig. 2 is a Western blot showing precipitation from platelet lysate using a biotinylated KVGFFKR peptide, and a control biotinylated KAAAAAR peptide;

Fig. 3 is a Western blot of immunoprecipitated platelet lysate using a commercial ICln antibody and a control αilb specific antibody, SZ22;

Figs 4A-4D are graphs showing the dose response effect of Acyclovir on Platelet aggregation;

Figs. 5A-5F are graphs showing the effect of Acyclovir on the activity of the platelet specific integrin llbβ3; Figs. 6A-6C are graphs showing the dose response effect of the peptide AKFEEE and a control peptide on Platelet aggregation; and Figs. 7A and 7B are graphs showing the antagonist effect of the peptide Pal-KVGAAKR (Pal-AA) on thrombin-induced platelet aggregation. Detailed Description The platelet integrin llbβ3 plays a major role in thrombus development at sites of vascular damage. It is the most abundant receptor. However little is known about the molecular mechanisms involved in its function. The KVGFFKR sequence within the cytoplasmic tail of the αllb subunit, has been shown to be highly conserved among all integrin subunits, and play a critical role in the regulation of platelet function. In the present work, the identification of potential regulators of the integrin llbβ3, through association with the KVGFFKR sequence, was investigated. Using a human foetal brain expression array (37,200 clones) screened with a biotinylated KVGFFKR peptide, proteins demonstrating high affinity binding were identified and included the nucleotide sensitive chloride channel ICln, a chloride channel that regulates cell volume. Referring to Fig. 1, the presence of ICln in platelets has been demonstrated by fluorescent microscopy in which Fig. 1A shows the integrin stain, Fig. IB shows the ICln stain, Fig. 1C is an overlap of the stains of Figs. 1A and IB showing co-localisation, and Fig. ID shows the platelets used in the investigation. It was then demonstrated that this ICln protein has specific binding to the KVGFFKR sequence and not to a control biotinylated KAAAAAR peptide. Referring to Fig. 2, platelet lysate was produced using a mild detergent, and then precipitated using the biotinylated peptides, KVGFFKR and the control KAAAAAR, and run on 7.5% PAGE gels. The Western blot shown was probed with a commercial ICln antibody and shows that ICln is associated with the KVGFFKR motif.

Fig. 3 shows a Western blot of immunoiprecipitated platelet lysate using ICln antibody, probed with a control αllb specific antibody, SZ22. Platelets were lysed using a mild detergent, and precipitated with the ICln antibody, and the control, SZ22. When probed with the SZ22 antibody, the blot shows a band at approximately 125Kda (see arrow marked 1) where the αllb subunit was precipitated from platelet lysate using this antibody. Association between the ICln channel and αllbβ3 is seen at the 125Kda mark when platelt lysate is precipitated with an ICln antibody. This is shown by the arrows marked 2. Bands below this are the heavy and light chains of the antibody, from the precipitation.

Platelet Activity Tests Platelet activity can be determined by measuring platelet aggregation. The formation of platelet aggregates can be quantified by the light transmission properties of platelet suspensions. Stirring is required to provide the close cell contacts necessary for aggregation. As platelets clump, the total particle number in the suspension drops, so the light transmission increases. In the following examples, platelet aggregation was assayed in a BioData PAP-4 aggregometer. JNL buffer was used to set 100% aggregation. Platelet suspensions were stirred at llOOrpm unless otherwise stated. Aggregations were performed at 37 degrees C in a final volume of 400μL of WP for 3-4 minutes. Thrombin, ADP and thrombin receptor activating peptide (TRAP6-SFLLRN) were thawed on ice and diluted from stock solutions in ice cold water, for use as platelet agonist controls. Ppep was used from a 880μM stock solution, and used at concentrations ranging from 0.1 to lOOμM. Inhibitors were pre- incubated for 5min at 37°C in the absence of stirring before addition of agonists unless otherwise stated.

Example 1

Dose Response Effect of Acyclovir on Platelet Aggregation

The effect of the known ICln inhibitor, Acyclovir, on platelet aggregation was determined using a platelet aggregation test. A dose response was set up and the effect observed on thrombin activated platelets. A decrease in aggregation was observed at ImM Acyclovir (Fig. 4C) , ith a greater decrease seen at 5mM Acyclovir (Fig. 4D) . Little or no response is observed at lOOμM (Fig. 4B) . A set of vehicle controls were run, using identical concentrations of DMSO as is used in the Acyclovir concentrations (Fig. 4A) . The DMSO does not have an effect on the aggregation of platelets.

Example 2

Dose Response Effect of the Peptide AKFEEE on Platelet Aggregation

The peptide AKFEEE is a ligand having a proposed high affinity binding to the integrin motif KVGFFKR. The effect of the peptide AKFEEE on platelet aggregation was determined using a platelet aggregation test. A dose response was set up and the effect observed on thrombin activated platelets. A decrease in aggregatio was observed at 10μM peptide, with greater decreases seen at 50μM and 80μM peptide (Fig. 6A) . A set of vehicle controls were run, using DMSO and different concentrations of a control peptide, LEFEEE . The DMSO and control peptide did not have an effect on the aggregation of platelets (Fig. 6B) . The potent dose dependant inhibition of Pal-KVGFFKR-induced platelet aggregation by the peptide AKFEEE is shown in Fig. 6C. Example 3 PAC-1 and CD62 binding in the Presence of Dose Response Acyclovir The effect of Acyclovir on the activity of αllbβ3 was examined through activation of the integrin and platelet secretion. PAC-1, measuring the activation of the integrin αllbβ3, shows a dose dependant decrease in the activation of αllbβs in the presence of 100μM, lOmM and 50mM Acyclovir (Figs 5A-5C) . It would appear that Acyclovir has no effect on granular secretion initiated by thrombin, as little or no shift is seen in the binding of CD62-PE (Figs 5D-5F) . Example 4

Figs. 7A and 7B show the agonist/antagonist effect of three peptides (50μM) on platelet aggregation. The peptides are KVGAAKR (Pal-AA) , KVGFFKR (Pal-FF) and KVGAFKR (Pal-AF) . Peptide (50μM) was added to platelets and allowed to aggregate for 3 minutes. It can be seen that Pal-AA has not agonist effect on platelet aggregation (Fig. 7A) . To test the antagonist effect of the peptides, platelets were pre-incubated with peptide for 3 minutes prior to addition of thrombin (0.2μ/ml) and allowed to aggregate for a further 3 minutes. It can be seen in Fig. 7B that Pal-AA completely inhibits thrombin- induced aggregation. Screening Assays

Assay I

Biotin-tagged AKFEEE peptide is immobolised on a 96 well plate. Using a library of candidate platelet antagonists, pre-incubate the AKFEEE coated plate with the putative antagonists. Each well of the plate should include only one putative antagonist. A number of the wells are left blank as positive controls. Fluorescent KVGFFKR is then added to each well, and allowed to incubate in the well for a suitable period of time. After incubation, excess unbound fluorescent KVGFFKR is washed from the wells, and the fluorescence of each well is read using a Wallac Fluorescent Plate Reader.

Assay II

Recomibinant ICLn protein, is immobilised on a 96 well plate. Using a library of candidate platelet antagonists, pre-incubate the ICLn coated plate with the putative antagonists. Each well of the plate should include only one putative antagonist. A number of the wells are left blank as positive controls. Fluorescent KVGFFKR is then added to each well, and allowed to incubate in the well for a suitable period of time. After incubation, excess unbound fluorescent KVGFFKR is washed from the wells, and the fluorescence of each well is read using a Wallac Fluorescent Plate Reader. Assay III Immobilise recombinant ICLn protein on a 96 well plate. Prepare a library of fluorescent candidate platelet antagonists. Incubate the immobilised protein with the candidate platelet antagonists (one putative antagonists per well) and probe for bound fluorescent antagonist.

For oral administration, the medicament according to the invention may be in the form of, for example, a tablet, capsule suspension or liquid. The medicament is preferably made in the form of a dosage unit containing a particular amount of the active ingredient. Examples of such dosage units are capsules, tablets, powders, granules or a suspension, with conventional additives such as lactose, mannitol, corn starch or potatoes starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators such as corn starch, potaote starch or sodium carboxymethyl-cellulose; and. with lubricants such as talc or magnesium stearate. The active ingredient may also be administered by injection as a composition wherein, for example, saline, dextrose or water may be used as a suitable carrier. When the active is a peptide, it will be modified to enable it survive conditions in the gut. One means of modification is cyclisation where a cysteine residue is attached at each end of the peptide and the peptide is cyclised by means of disulphide bonds between the two cysteines. Alternatively, the peptide bonds in the peptide may be modified to render them resistant to digestive enzymes in the gut .

For intravenous, intramuscular, subcutaneous, or intraperitioneal administration, the compound may be combined with a sterile aqueous solution which is preferably isotonic with the blood of the recipient. Such formulations may be prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride, glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. The formulations may be present in unit or multi-dose containers such as seated ampoules or vials. To facilitate cell penetration, the active peptide may be lipidated, preferably using palmitate.

If the disease or condition is localized in the G.I. tract, the compound, may be formulated with acid- stable, base-liable coatings known in the art which began to dissolve in the high pH intestine. Formulations to enhance local pharmacologic effects and reduce systemic uptake are preferred.

Formulations suitable for administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably made isotonic. Preparations for injections may also be formulated by suspending or emulsifying the compounds in non-aqueous solvent, such as vegetable oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol .

For aerosol delivery, the compounds may be formulated with known aerosol exipients, such as saline and administered using commercially available nebulizers. Formulation in a fatty acid source may be used to enhance biocompatibility. Aerosol delivery is the preferred method of delivery for epithelial airway inflammation. When the active is a peptide, it is preferably lipidated with, for example, palmitate.

For rectal administration, the active ingredient may be formulated into suppositories using bases which are solid at room temperature and melt and dissolve at body temperature. Commonly used bases include cocoa butter, glycerinated gelatin, hydrogenated vegetable oil, polyethylene glycols of various molecular weights, and fatty esters of polyethylene tearate.

The dosage form and amount can be readily established by reference to known disease or condition treatments or prophylactic regiments. The amount of therapeutically active compound that is administered and the dosage regimen for treating a disease condition with the compounds and /or compositions of this invention depends on a variety of factors, including the age, weight, sex and medical condition of the subject, the severity of the disease, the route and frequency of administration, and the particular compound employed, the location of the disease or condition, as well as the pharmacokinetic properties of the individual treated, and thus may vary widely. The dosage will generally be lower if the compounds are administered locally rather than systemically, and for prevention rather than for treatment. Such treatments may be administered as often as necessary and for the period of time judged necessary by the treating physician. One of skill in the art will appreciate that the dosage regime or therapeutically effective amount of the inhibitor to be administrated may need to be optimized for each individual. The pharmaceutical compositions may contain active ingredient in the range of about 0.1 to 2000mg, preferably in the range of about 0.5 to 500mg and most preferably between about 1 and 200 mg. A daily dose of about 0.01 to lOOmg/kg body weight, preferably between about 0.1 and about 50mg/kg body weight, may be appropriate. The daily dose can be administered in one to four doses per day.

The invention is not limited to the embodiments hereinbefore described which may be varied in detail without departing from the invention. Thus, while the invention has been described as a treatment for human subjects, the methods and agents of invention may likewise be applied to the treatment of non- human mammals .

Claims

Claims 1. A method of inhibiting platelet or neutrophil activation in a human subject comprising the step of treating the subject with an agent which has an effect of preventing or disrupting an association between a nucleotide sensitive chloride channel ICLn and a platelet specific Integrin αllbβ3 or a neutrophil specific Integrin α_Lβ₂, θχβ₂.

2. A method as claimed in Claim 1 in which the agent is an inhibitor of the nucleotide sensitive chloride channel ICLn.

3. A method as claimed in Claim 2 in which the inhibitor is ACYCLOVIR.

4. A method as claimed in Claim 1 in which the agent is an isolated ligand having at least a mM binding affinity for an amino acid sequence KVGFFKR.

5. A method as claimed in Claim 4 in which the ligand is an isolated, protein, peptide or compound comprising the sequence AKFEEE.

6. A method as claimed in Claim 4 in which the ligand consists essentially of Pal-AKFEEE.

7. A method as claimed in Claim 1 or 2 in which the ligand is a protein, peptide or compound comprising the sequence KVGAAKR.

8. A method as claimed in Claim 7 in which the ligand consists essentially of Pal-KVGAAKR.

9. A method as claimed in Claims 5 or 7 in which the ligand is modified to enhance it's cell permeability.

10. A method as claimed in Claim 9 in which the ligand is modified by palmitoylation.

11. A method of inhibiting platelet or neutrophil activation in a human subject comprising the step of treating the subject with an effective amount of an inhibitor of the nucleotide sensitive chloride channel ICLn.

12. A method of inhibiting platelet or neutrophil activation in a human subject comprising the step of treating the subject with an effective amount of a ligand having at least a M binding affinity for an amino acid sequence KVGFFKR.

13. Use of an agent in the manufacture of a medicament for the inhibition of platelet or neutrophil activation, which agent has an effect of preventing or disrupting an association between a nucleotide sensitive chloride channel ICLn and a platelet specific (αllbβ3) or neutrophil specific (α_Lβ₂, θχβ₂) integrin.

14. Use of an agent in the manufacture of a medicament for the prophylaxis or treatment of a disease or condition which is conventionally treated with a platelet or neutrophil activity modulating agent, wherein the agent is characterised by having an effect of preventing or disrupting an association between a nucleotide sensitive chloride channel ICLn and a platelet specific (αllbβs) or neutrophil specific (α_Lβ₂, 0χβ₂) integrin.

15. Use as claimed in Claim 14 in which the disease or condition is selected from the group consisting of: Stroke; Atherosclerosis and Coronary Artery Disease, Ischemic Cerebrovascular Disease, Peripheral Vascular Disease, Diabetes Mellitus, Renal Disease, Inflammation, Antimicrobial Host Defence, Tumor Growth and Metastasis, Alzheimer Disease and Psychiatric Disorders.

16. Use as claimed in any of Claims 13 to 15 in which the agent is an inhibitor of the nucleotide sensitive chloride channel ICLn.

17. Use as claimed in Claim 16 in which the agent is ACYCLOVIR.

18. Use as claimed in any of Claims 13 to 15 in which the agent is a ligand having at least a mM binding affinity for an amino acid sequence KVGFFKR.

19. Use as claimed in Claim 18 in which the ligand is a protein, peptide or compound comprising an amino acid sequence selected from the group: AKFEEE and KVGAAKR.

20. Use as claimed in Claims 18 to 19 in which the ligand is modified to enhance it's cell permeability.

21. Use as claimed in Claim 20 in which the ligand is modified by palmitoylation.

22. An isolated peptide, protein or compound which includes an amino acid sequence AKFEEE.

23. An isolated peptide which consists essentially of the sequence AKFEEE.

24. An isolated peptide which consists essentially of the modified sequence Pal-AKFEEE.

25. An isolated peptide, protein or compound which includes an amino acid sequence KVGAAKR.

26. An isolated peptide which consists essentially of the sequence KVGAAKR.

27. An isolated peptide which consists essentially of the modified sequence Pal-KVGAAKR.

28. A peptide, protein or compound of any of Claims 22 and 27 for use as a medicament.

29. A method of identifying a platelet or neutrophil antagonist comprising: -providing a candidate compound,- -contacting a (poly) peptide with the candidate compound, the (poly) peptide including an amino acid sequence selected from the group comprising: A-K-F and K-V-G; and -determining whether the candidate compound binds to the (poly) peptide.

30. A method as claimed in Claim 29 in which the (poly) peptide includes, or consists of, a sequence selected from the group comprising: KVGFFKR; AKFEEE; and KVGAAKR.

31. A method as claimed in Claims 29 and 30 in which the (poly) peptide is immobilised to a support.

32. A method as claimed in Claim 31 in which the (poly) peptide comprises a biotin moiety or a HIS-tag to facilitate immobilisation.

33. A method as claimed in any of Claims 29 to 32 in which, after incubating the (poly) peptide with the candidate compound, the (poly) peptide is contacted with a moiety which has a high binding affinity for the (poly) peptide, which moiety includes a detectable label .