WO2004098627A1

WO2004098627A1 - Inhibition of platelet aggregation through acyclovir and two peptide antagonists

Info

Publication number: WO2004098627A1
Application number: PCT/IE2004/000068
Authority: WO
Inventors: Niamh Moran; Kelly Aylward; Deirdre Larkin; Desmond Fitzgerald
Original assignee: Royal College Of Surgeons In Ireland
Priority date: 2003-05-06
Filing date: 2004-05-06
Publication date: 2004-11-18
Also published as: WO2004098627A9

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

1 integrin conveying the intention to activate while
2 the extracellular domains, which constitute greater
3 _than 90% of the molecules.,.actually respond with an
4 increased affinity for ligand and altered display of
5 antibody epitopes suggestive of altered protein
6 folding. A highly conserved amino acid motif has
7 been identified in the cytoplasmic tails of integrin
8 alpha subunits (Stephens et al 1998 J. Biol . Chem.
9 273, 20317) . This motif, KVGFF R, is critical for
10 the intracellular- ediated activation of llb[beta]3.
11 Deletion or mutation of this sequence causes the
12 integrin to assume an active conformation. .13
14
.15 Statements of Invention
.16
.17 A method for inhibiting platelet or neutroph.il
-18 activation :in humans is ..disclosed.

In :one aspect,
-19 r he method -is based -on the finding :that :the .human
-20 platelet-specific rib[beta]3:and. neutrophil-speci ic
121 0L[beta]2,0Cj!;[beta]2-integrin are ssociated with a nu[sigma]leotide
.22 specific chloride channel Icln -and -that prevention
123 :or -disruption of this -association -has the eff ct :of
-24 inhibiting platelet and/or meufcroph.il ctivation.
.25<">IThus, human platelet and -or neutropliil cells .are
126 contacted with an effective lamount of :

an agent which
27 -disrupts or prevents the association between the
.28 platelet-specific or neutrophil-specific integrin
29 and the chloride channel.
30
31 According to the invention, there is provided a
32 method of inhibiting platelet or neutrophil 1 activation in a human subject comprising the step of
2 treating the subject with an agent which has an -3 effect, of preventing or disruptin _an association
4 between a nucleotide sensitive chloride channel ICLn
5 and a platelet-specific [alpha]iIB[beta]3or neutrophil-specific
6 [alpha]L[beta]2,0C[chi][beta]2Integrin. 7
8 In one embodiment, the agent is an inhibitor of the
9 nucleotide sensitive chloride channel ICLn. As such,
10 the agent may be any one of the substituted pu ine
11 compounds disclosed in United States Patent
.12 4,199,574, published 22 April, 1980.

Preferably, the
13 agent is the compound known in the trade as
.14 ACYCLOVIR. Alternatively, the agent may be another
-15 type of nucleotide :analogue. -16
-17 In an alternative embodiment of :the invention,
.18 the agent is a ligand having at -least a mM binding
-19 .affinity for an ramino acid sequence KVGFFKR.
20 Preferably, -the .ligand -is a protein, peptide or
.21 .compound whic -includes -the sequence -AKEEEE or
.22<'>KVGAAKR. Typically, the protein :or peptide -is
.23 rpr[alpha]tected at its :amino and/or carboxy terminus . -In
24 :one preferred -rembodiment of -the invention, the
25 -ligand consists essentially of the ramino acid
26 -sequence AKFEEE :or<">KVGAAKR.

Alternatively, the
27 ligand is a protein, peptide or compound which has
28 at least a mM, preferably a UM, and more preferably
29 a nM, binding affinity for the sequence AKFEEE or 30 KVGAAKR.
31 1 In one embodiment of the invention, the ligand is
2 modified to enhance it's cell permeability.
3 In this regard, the ligand_may be ..modified b.
4 incorporation of a palmitate (Pal) group. 5
6 In a preferred embodiment the agent consists of Pal-
7 AKFEEE or Pal-KVGAAKR. 8
9 The invention also relates to a method of inhibiting
10 platelet or neutrophil activation in a human subject
11 comprising the step of treating the subject with an
12 effective amount of an inhibitor of the nucleotide
13 sensitive chloride channel ICLn.

14
15 The invention also relat.es to a method of -inhibiting
16 platelet or neutrophil activation in a human subject
17 comprising the step of treating the subject with .an
18 effective -amount of a ligand having at least a mM
19 binding affinity for ran ramino acid sequence KVGFFKR.
20 Preferably, the ligand has a [mu]M, or even more
-21 preferably, a .nM binding af inity for the sequence
22 KVGFFKR. In rone embodiment, the liqand -includes, or
.23 consists essentially, of the sequence AKFEEE or
24 KVGAAKR. Alternatively, the ligand is a protein,
.25 peptide or compound which has .at least a mM,
26 preferably a [mu]M, and more preferably a nM, binding
27 affinity for the sequence AKFEEE or KVGAAKR.
28 Suitably, the ligand is modified by incorporation of
29 a palmitate (Pal) group.

Ideally, the ligand
30 consists essentially of Pal-KVGAAKR or Pal-AKFEEE. 1 The methods of the invention may be used to prevent
2 or treat diseases or conditions which are
__3 conventionally treated with_platelet activity
4 modulating agents, and particularly agents which
5 decrease platelet activity. One such condition is
6 stroke.

Other conditions or diseases include
7 Atherosclerosis and Coronary Artery Disease,
8 Ischemic Cerebrovascular Disease, Peripheral
9 Vascular Disease, Diabetes Mellitus, Renal Disease,
10 Inflammation, Antimicrobial Host Defence, Tumor
11 Growth and Metastasis, Alzheimer Disease and
12 Psychiatric Disorders. 13
14 The methods of the invention may be used to prevent
-15 or treat diseases or conditions which are
-16 conventionally treated with neutrophil activity
17 modulating agents, rand particularly agents which
-18 decrease neutrophil activity. -Examples of such
-19 diseases or conditions include -inflammatory diseases
20 such as rheumatoid .inflammation, response to
21 infection, ischemia-reperfusion rinjury -and graft
22 rejection, -and luekocyte adhesion deficiency.

.23
24 The invention also relates to the use of ran agent in
25 the manufacture of a medicament for -the -inhibition
26 of platelet or .neutrophil activation, which agent
27 has an effect of preventing or disrupting an
28 association between a nucleotide sensitive chloride
29 channel ICLn and a platelet-specific ( llb[beta]3) or
30 neutrophil-specific ([alpha]L[beta]2, [alpha]x[beta]2) Integrin. In one
31 embodiment of the invention, the agent is an
32 inhibitor of the nucleotide specific chloride - 1 channel, ICln. In an alternative embodiment, the
2 agent is a ligand having at least a mM binding __3 af-finity. for."the .a ino acid sequences.KVGFFKR_ or
4 AKFEEE.

In one embodiment, the ligand includes, or
5 consists of a peptide having a sequence selected
6 from the group comprising: AKFEEE and KVGAAKR.
7 Typically, the ligand is modified by incorporation
8 of a palmitate (Pal) group. 9
10 The invention also relates to the use of an agent in
11 the manufacture of a medicament for the prevention
12 or treatment of a disease or condition which is
13 conventionally treated with platelet or neutrophil
14 .antagonists, which agent has an effect of preventing -15 or .disrupting an .association between a nucleotide
16 rsensitive chloride channel ICLn :

and a platelet or
17 meutrophil specific .Integrin. iExamples of such .18 ^diseases or conditions .are provided .above. -In one
19 ^embodiment -of the nvention, the agent -is -an
20 -inhibitor of the nucleotide specific .chloride
21 rrchannel, ICln. -In .an .alternative embodiment, the
22 [identical to]agent is a .ligand .having at least a -mM .binding
23 raf+-inity for the amino acid sequences<">KVGFFKR or
24 [Xi]AKEEEE. -In one embodiment, the -ligand -includes, or
25 rconsists of, a peptide :having a ^sequence selected
26 -f oom the group comprising:
27 iAKFEEE and KVGAAKR.

Typically, the ligand is
28 "modified by incorporation of a palmitate group. 29
30 The invention also relates to a peptide, protein or
31 compound which includes the sequence AKFEEE or 1 KVGAAKR, and the use of the peptide, protein or
2 compound as a medicament . - 3 -
4 The invention also relates to an inhibitor of the
5 chloride channel ICLn for use as a medicament . 6
7 The invention also relates to a method of
8 identifying a platelet or neutrophil antagonist
9 comprising:
10 -providing a candidate compound;
11 -contacting a (poly)peptide with the candidate
12 compound, the (poly) peptide including an amino acid
13 sequence selected .from the group comprising:

A-K-F
14 and K-V-G; and
15 -determining whether the candidate compound binds to
16 the (poly) peptide . 17
18 Suitably, the peptide includes, or consists of, a
19 sequence selected from rthe group comprising:
20 KVGFFKR; and AKFEEE. .21
-22 In one embodiment, the (poly) eptide consists of .the
:23 platelet -or neutrophil rspecific -integrins -described
124 .above. In .another ^embodiment, the (poly) eptide
.25 consists .of recombinant nucleotide sensitive
.26 chloride rchannel ZCLn. 27
28 In a preferred embodiment, the (poly) eptide is
29 immobilised to a support. Typically, the support is
30 a 96 or 384 well plate. Suitably, the (polypeptide)
31 will include a biotin moiety or a HIS-tag to
32 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 .af inity 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 rec[alpha]mbinant I.CLn, 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 preincubated 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 fluores.cence .

For any given well,_ the presence of a platelet or neutrophil antagonist will be indicated by a reduction in luorescence 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 [sigma]onfocal microscopy picture demonstrating localisation of the integrin [alpha]llb[beta]3 and Chloride -Channel XCin;
Fig. 2 is a Wester -blot showing precipitation rfrom platelet lysate .using a bi tinylated<">KVGFFKR peptide, rand a control bi[alpha]tinylated KAAAAAR peptide;
Fig. 3 is a Western blot .of immunoprecipitated platelet lysate using a commercial ICln antibody and a control [alpha]ilb specific rantibody, 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 [alpha]llb[beta]3; 10
1 Figs. 6A-6C are graphs showing the dose response
2 effect of the peptide AKFEEE and a control peptide
3 on Platelet, aggregation; and
4
5 Figs . 7A and 7B are graphs showing the antagonist
6 effect of the peptide Pal-KVGAAKR (Pal-AA) on
7 thro bin-induced platelet aggregation. 8
9 Detailed Description
10
11 The platelet integrin [alpha]llb[beta]3 plays a major role in
12 thrombus development at sites of vascular damage. It
13 is the most abundant receptor. However little is
14 known about the molecular mechanisms involved in its .15 function.

The KVGFFKR sequence within the
16 cytoplasmic tail of the [alpha]ilb subunit, has been .shown
17 to be -highly conserved among rail lintegrin
18 rsubunits, -and play a critical role in the rregulation -19 rof platelet function. In :the -.present work, the
.20 identification of potential regulators of rthe
.21 r+-ntegrin [alpha]llb[beta]3 , through .association with +-he
.22<">KVGFFKR sequence, was investigated. Using a rhuraan
-23 rrfoetal brain expression array (37,200.clones)
24 ^screened with a bi[alpha]tinylated<">KVGFFKR peptide,
25 rproteins demonstrating rhigh affinity ihinding were
26 -identified :and -included the nucleotide sensitive
27 chloride channel ICln, a chloride channel -that
28 regulates cell volume. Referring to Fig. 1, the
29 presence of ICln in platelets has been demonstrated
30 by fluorescent microscopy in which Fig. 1A shows the
31 integrin stain, Fig.

IB shows the ICln stain, Fig.
32 IC is an overlap of the stains of Figs. 1A and IB 11
1 showing co-localisation, and Fig. ID shows the
2 platelets used in the investigation.
3 .
4 It was then demonstrated that this ICln protein has
5 specific binding to the KVGFFKR sequence and not to
6 a control biotinylated KAAAAAR peptide. Referring
7 to Fig. 2, platelet lysate was produced using a mild
8 detergent, and then precipitated using the
9 biotinylated peptides, KVGFFKR and the control
10 KAAAAAR, and run on 7.5% PAGE gels. The Western blot
11 shown was probed with a commercial ICln antibody and
12 shows that ICln is associated with the KVGFFKR
13 motif. 14
15 Fig. 3 shows a Western blot of immunoiprecipitated
16 platelet lysate .using ICln .antibody, probed with a
17 control llb specific antibody, SZ22.

Platelets were .18 lysed using a mild detergent, rand precipitated with .19 the ICln antibody, -and -the control, SZ22- When
20 probed with the SZ22 antibody, .the .blot shows a band -21 at approximately IZSKda (see arrow rmarked 1) wiiere .22<¯>the [alpha]llb subunit was precipitated from platelet
.23 .lysate using this .antibody...Association between -the
-24 TlCln channel and [alpha]LIb[beta]3 is seen at the -12<">5Kda mark
.25 when platelt lysate .is precipitated with ran ICln
.26 rantibody. This is shown by the arrows marked 2.
27 -Bands below this are the heavy and light chains of
28 the antibody, from the precipitation. 29
30 Platelet Activity Tests 31 12
1 Platelet activity can be determined by measuring
2 platelet aggregation.

The formation of platelet "3 - aggregates can be^qua tifi d by_t,he light
4 transmission properties of platelet suspensions .
5 Stirring is required to provide the close cell
6 contacts necessary for aggregation. As platelets
7 clump, the total particle number in the suspension
8 drops, so the light transmission increases. In the
9 following examples, platelet aggregation was assayed
10 in a BioData PAP-4 aggregometer. JNL buffer was used
11 to set 100% aggregation. Platelet suspensions were
12 stirred at llOOrpm unless otherwise stated.
13 Aggregations were performed at 37 degrees C in a
14 final volume of 400[mu].L of WP for 3-4 minutes.
15<">Thrombin, ADP and thrombin receptor activating
16 peptide CTRAP6-SFLLRN) were thawed on ice and
17 -diluted from stock solutions in ice cold water, for -18 ruse r.as platelet agonist controls .

Ppep was used rfrom -19 -a 880[mu]M stock solution, rand -used at concentrations -20 rranging from 0.1 to 100[mu]M. .Inhibitors were pre-
.21 -incubated 'for 5min at 37[deg.]C -in tne -absence .of
-22 rstirring before .addition of agonists unless
.23 otherwise<">Stated. .24 -25 26 -Example 1
27
28 Dose Response Effect of Acyclovir on Platelet
29 Aggregation 30
31 The effect of the known ICln inhibitor, Acyclovir,
32 on platelet aggregation was determined using a 13
platelet aggregation test. A dose response was set up and the effect observed on thrombin activated platelets. A decrease in aggregation^as observed at ImM Acyclovir (Fig. 4C) , with a greater decrease seen at 5mM Acyclovir (Fig. 4D) . Little or no response is observed at lOO[mu]M (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 tne Peptide AKFEEE on Plateie[tau]r Aggregation
The peptide AKFEEE is a ligand having a proposed -high affinity binding to the integrin motif KVGFFKR.<¯>The effect of the peptide AKEE[Xi]E on platelet aggregation was determined using a platelet aggregation test . A dose response was set up and tne -effect observed on -thrombin activated platelets. A -decrease rin aggregation was observed at 10[mu]M -peptide, with greater decreases seen at 50[mu]M and 80[mu]M peptide (Fig. 6A) . A set of vehicle controls were run, using DMSO and different concentrations of a control peptide, L[Xi]FEEE. 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 14
1 aggregation by the peptide AKFEEE is shown in Fig.
2 6C.
4 Example 3
5
6 PAC-1 and CD62 binding in the Presence of Dose
7 Response Acyclovir 8
9 The effect of Acyclovir on the activity of [alpha]llb[beta]3
10 was examined through activation of the integrin and
11 platelet secretion. PAC-1, measuring the activation
12 of the integrin llb[beta]3 , shows a dose dependant
13 decrease in the activation of [alpha]llb[beta]3 in the presence
14 of lOO[mu]M, lO M and 50mM Acyclovir (Figs 5A-5C) .

It
15 would appear that Acyclovir has no effect on
16 granular secretion -initiated by -thrombin, .as little
17 or no shift is seen in the binding of<">CD62-PE (Figs
18 5D-5F) . -19
.20 -Example- 4
.21
-22 Figs. 7A and 7B show the agonist .antagonist effect
.23 of three peptides (50[mu]M) on platelet aggregation.
24 The peptides are KVGAAKR (Pal-AA) , KVGFFKR (Pal-<'>FF)
25 and KVGAFKR (Eal-AF) . Peptide (50[mu]M) was added rfco
26 platelets and -allowed to aggregate for 3.minutes.
27 It can be seen that Pal-AA has not agonist effect on
28 platelet aggregation (Fig. 7A) . To test the
29 antagonist effect of the peptides, platelets were
30 pre-incubated with peptide for 3 minutes prior to
31 addition of thrombin (0.2[mu]/ml) and allowed to
32 aggregate for a further 3 minutes.

It can be seen 15
in Fig. 7B that Pal-AA completely inhibits thrombininduced 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 control . Fluorescent KVGFFKR is then added to each well, and allowed to incubate in .the well for a suitable period of time. After rrincubation, 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
Recomibmant ICLn protein is immobilised ron 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 16
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 ramount of the active ingredient. .Examples of such dosage units are capsules, tablets, powders, rgranules 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 17
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 rmay be present in unit or multi-dose containers such .as seated rampoules or vials. To facilitate cell penetration, rthe active peptide may be .lipidated, preferably .using palmitate.
-If the disease or condition is localized -in the G.I. rtract, the compound may be formulated with acidrstable, base-liable coatings known in the art which began to dissolve in the high pH intestine.

Formulations to enhance local pharmacologi[sigma] effects and reduce systemic uptake are preferred.
Formulations suitable for administration conveniently comprise a sterile aqueous preparation 18
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 stearate.
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 19
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 rabout 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 20 may likewise be applied to the treatment of nonhuman mammals.

Claims

21 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/ 0Cχβ₂.

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. 22

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 mM 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 23

nucleotide sensitive chloride channel ICLn and a platelet specific (αllbβ3) or neutrophil specific (α_Lβ₂, χβ₂) 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 (αllbβ3) or neutrophil specific (α_Lβ₂, θχβ₂) 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. 24

18. Use as claimed in any of Claims 13 to 15 in which the agent is a ligand having at least a M 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. 25

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) eptide with the candidate compound, the (poly) eptide 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. 26

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 .