GB2451495A - Treatment of inflammation by modulating catecholamine metabolism - Google Patents

Abstract

The use of a compound that modulates catecholamine metabolism in the manufacture of a medicament for use in the modulation of an immune response and/or in the treatment or prevention of an inflammatory condition, or condition in which coagulation occurs. The compound may be a monoamine oxidase (MAO) inhibitor, a catechol-0-methyl transferase (COMT) inhibitor, an aldehyde dehydrogenase (ALDH) inhibitor, a compound that stimulates the production or release of a catecholamine or a compound that mimics the biological activity of a catecholamine. MAO inhibitors include 1-hydrazinophthalazine, R-(-)-deprenyl hydrochloride, N-methyl-N-propargyl-3-(2, 4-dichlorophenoxy)propylamine hydrochloride. The COMT inhibitor may be 2-hydroxy-2,4,6-cycloheptatrien-1-one. The ALDH inhibitor may be 4',7-dihydroxy-iso-flavone 7-hydroxy-3-(4-hydroxy-phenyl)-4H-1-benzo-pyran-4-one. The inflammatory condition includes septic shock, arteriosclerosis, atherosclerosis, stroke, thrombosis, embolism, arthritis, psoriasis and ankylosing spondylitis. Pharmaceuticals and kits are claimed as are methods of determining suitability for such treatment by measuring KLF2 expression.

Description

Catecholamine metabolism modulators

Field of Invention

The present invention relates to compounds and compositions containing such compounds for use in therapy and to numerous related applications. More particularly, the invention relates to the modulation of inflammatory and immune responses, coagulation responses and also the treatment of inflammation or coagulation. Particularly, although not exclusively, the invention relates to compounds that alter the metabolism of catecholamines such as dopamine, adrenaline and noradrenaline for the modulation of immune responses, and also for treatment of inflammatory conditions such as arteriosclerosis, atherosclerosis, thrombosis, stroke, embolism, arthritis, psoriasis and ankylosing spondylitis.

Background of the Invention

Immune responses to antigen challenge are an important component of the human or animal body's defences against a range of insults. These insults include infectious agents such as bacteria, viruses, parasites and prions. Antigen dependent immune responses also. play a role in the defence against cancer or the response to allergens. Modulation of such antigen dependent immune responses represents a potential therapeutic avenue in conditions or pathologies associated with each of the above scenarios.

Inflammation is also part of the human or animal body's response to a range of insults. However, inflammation can sometimes be undesirable or harmful.

Inflammatory conditions cause substantial mortality and morbidity in the population at large. Inflammatory conditions can be acute or chronic. Chronic inflammatory conditions include arteriosclerosis, atherosclerosis, arthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis and ankylosing spondylitis. Examples of acute inflammatory conditions include septic shock and anaphylactic shock amongst others.

Kruppel-like transcription factor 2 (KLF2, according to the Hugo Gene Nomenclature Committee (HGNC), also known as Lung Kruppel-Like Factor (LKLF), was originally identified as an immunomodulatory transcription factor based on a hyper-reactive T-cell phenotype observed in RAG2-reconstituted KLF2 knockout mice (1). More recent studies have extended this phenotype (2) to show that KLF2 functions to promote T-cell egress from the thymus during immune challenge. This takes place in part by the ability of KLF2 to activate expression of a cell surface receptor (S1P1) which is required for thymocyte exit from, as well as recirculation back to, the thymus. This trafficking of T-cells is vital to the maintenance of an appropriate immune response to antigen challenge. Thus KLF2 plays a role in the development of antigen-dependent immune responses.

Subsequently, KLF2 has also attracted interest as a more general modulator of inflammation and localised vascular thrombosis. In particular, KLF2 has been considered to be an atheroprotective factor during stress to the vascular endothelium, the key barrier in determining vascular homeostasis. Forced expression of KLF2 drives endothelial transcriptional programs which regulate inflammation, thrombosis haemostasis, vascular tone, and blood vessel development. Without wishing to be bound by theory, the current opinion in the field is that KLF2 is a master regulator of atheroprotective transcriptional programs (3).

Furthermore, KLF2 plays a role in stem cells (4). Therefore molecules that regulate KLF2 can be used in the field of stem cells. Such uses for example include, stem cell therapies such as the therapeutic use of cardiomyocytes for treatment of damage following myocardial infarction.

KLF2 is also known to play a role in the regulation of endothelial thrombotic function (5). Endothelial thrornbotic function is important within the context of blood hemostasis and the balance between pro-coagulative and anti-coagulative pathways. Overexpression of KLF2 has been found to strongly induce the expression of thrombomodulin, endothelial nitric oxide synthase (eNOS), and reduce the expression of plasminogen activator inhibitor-i (PAI-l). This leads to reduced clotting capacity of blood and therefore molecules that regulate KLF2 can be used to protect against local blood clot formation and treatment of cardiovascular disease.

It would be useful if there were further active compounds useful in the modulation of an immune response and/or the treatment or prevention of an inflammatory condition and blood coagulation process, including responses of the vascular endothelium associated with atherosclerosis.

For the purposes of the present invention, endothelial thrombotic functions (e.g. coagulation) are considered to fall within the broader term of inflammation. However, the skilled reader will recognise that within inflammation' there are several distinct subsets of reactions, the effects on blood haemostasis being just one aspect of these.

It is an object of the invention to provide medicaments useful in the treatment of immune conditions or inflammatory conditions or to at least provide the public and/or medical community with useful alternatives to those already known.

Summary of the Invent ion

The present invention is based around the identification of new modulators of KLF2 levels in cells. The present invention is therefore concerned with the provision of new active agents for the modulation of immune responses and/or for the modulation (for example treatment or prevention) of inflammatory conditions or diseases. The compounds, compositions, uses and methods described herein may thus be useful in circumstances under which it is desirable to modulate one or more immune responses alone, modulate inflammation (or coagulation) alone (for example to treat or prevent one or more inflammatory conditions or diseases), or the combination of both modulation of immune responses and inflammation together.

The present inventors have identified compounds that modulate the metabolism of catecholamjnes as new modulators of KLF2 levels in cells. Catecholamjnes are well known chemical compounds and are derived from the amino acid tyrosine. They contain catechol and amine groups. Examples of catecholamines include dopamine, adrenaline and noradrenaline.

Enzymes involved in catecholamine metabolism include monoamine oxidase (MAO), catechol-o-methyl transferase (COMT) and aldehyde dehydrogenase (ALDH). Monoamine oxidase is found in distinct A and B forms which may be modulated specifically or non-specifically by different classes of compounds. The main enzymes responsible for degradation of catecholamines are MAOs. Thus it is envisaged that inhibitors of MAO, and of ALDH and COMT may find use in the present application.

Examples of compounds that may find use in the present invention include methamphetamine and other compounds that inhibit MAOs, and inhibitors of ALDH or COMT. Compounds known to fall into these categories include methamphetamines, and also drugs used for example to treat Parkinsons disease. Also included are isoflavones such as Daicizein.

Methamphetamine and MAO inhibitors bind to MAOs to inhibit the break down of catecholamines. This is primarily the reason why the effects of amphetamines last longer than cocaine and other such substances. Amphetamines not only causes a release of dopamine, epinephrine, and noradrenaline into the blood stream, but also by inhibition of the breakdown of such catecholamines, maintain raised levels for extended periods of time.

Active compounds exemplified herein include: R-(-)-Deprenyl hydrochloride (Deprenyl, also referred to herein as 1N161), N-Methyl-N-propargyl-3 -(2, 4-dichlorophenoxy)propylarnine hydrochloride (clorgyline, also referred to herein as 1N165), 2-I-lydroxy-2,4,6-cycloheptatrjen-l-one (tropolone also referred to herein as 1N170), l-hydrazinophthalazine monohydrochioride (hydralazine, also referred to herein as 1N452), and 4',7 -Dihydroxy-iso-flavone-7-Hydroxy-3-(4-hydroxy-pheny1) -4H-1-benzo-pyran-4one (Daidzein, also referred to herein as 1N167) Clorgyline is an inhibitor of the A class of MAO, whilst Deprenyl acts on the B class. Hydralazine is a non specific MAO A/B inhibitor. Tropolone is a COMT inhibitor and Daidzein is an inhibitor of ALDH.

Accordingly, in a first aspect, the present invention provides use of a compound that modulates catecholamine metabolism in the manufacture of a medicament for use in the modulation of an immune response and/or in the treatment or prevention of an inflammatory condition or condition in which coagulation occurs.

"Modulation of catecholamine metabolism" means alteration of the biochemical pathways leading to the production or breakdown of catecholamines. It may also comprise alterations to the pathways which remove catecholamines from the body. Typically such modulation is inhibition of breakdown and/or removal of catecholamines. Such modulation leads to an increase in the level of catecholamines released by cells, or to delay in the removal of catecholamines from tissues. However, a decrease in catecholamine levels may also occur. The biochemical pathways involved in catecholamine metabolism are complex and interrelated and therefore changes leading to alterations in the levels of a particular catecholamine may not have an equivalent effect on all catecholamines.

The levels of different individual/specific catecholamines may be affected in different direction or to different extents by modulation of catecholamine metabolism. For example such modulation may lead to a eneralised increase, or a generalised decrease in the levels of catecholamines.

Alternatively the levels of some catecholamines may be increased, while others are decreased or unaffected.

Normally, an increase in the levels of catecholamines is preferred, since this is associated with an increase in KLF2 expression.

Compounds which modulate catecholamine metabolism are known to include for example, monoamine oxidase (MAO) inhibitors, catechol-o- methyl transferase (çOMT) inhibitors or aldehyde dehydrogenase inhibitors. In preferred aspects such compounds are used.

Preferred MAO inhibitors include 1-hydrazinophthalazine monohydrochioride, R-(-)-Deprenyl hydrochloride, or N-Methyl-N-propargyl- 3 -(2, 4-dichiorophenoxy) propylamine hydrochloride.

Preferred COMT inhibitors include 2-Hydroxy-2,4,6-cycloheptatrien-l-one.

Preferred ALDH inhibitors include 4',7-Dihydroxy-iso-flavone 7-Hydroxy-3-(4-hydroxy-phenyl) -4H-1-benzo-pyran-4-one.

In addition it is envisaged that compounds which increase catecholamine levels through other means may find use in the present invention. For example, compounds that stimulate the production of catecholamines, or stimulate the release of catecholamines that have already been synthesised. Such compounds include methamphetamine which both stimulates release of catecho].amines and inhibits their breakdown.

Other examples include Pergolide, L-DOPA, Nicotine, Caffeine, bromocriptine, Bypropion (Zyban), all of which increase dopamine levels.

As a further related alternative, compounds that mimic the effect of catecholamines (but which would not normally be classified as catecholamines per se) may be used in the present invention. For example, sympathomimetic drugs are substances that mimic the effects epinephrine and norepinephrine, and may find use in the present invention.

By "modulation of an immune response" it is meant that a response to an antigen is altered relative to the response to that antigen when the compound of the invention is absent. Generally, the "modulation" comprises an elevated or improved immune response to the particular antigen. In a specific embodiment the immune response may be modulated such that an antigen dependent response to a vaccine is enhanced or boosted.

For example, modulation of an immune response may be achieved by the adjuvant activity of compounds of the inventions, i.e. enhancing said immune response, such as boosting antigen-dependent immune responses when co-administered with a vaccine. In preferred embodiments, medicaments of the invention may additionally comprise one or more vaccines.

The term "inflammatory condition" is used herein to refer to unwanted or pathological inflammation. The inflammatory condition may be selected from any disease that involves undesirable or pathological inflammation of any bodily tissue. In preferred embodiments, the inflammatory condition to be treated with the medicament according to the present invention is selected from the group comprising acute inflammation, chronic inflammation, septic shock, arteriosclerosis, atherosclerosis, thrombosis, stroke, embolism, arthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis or ankylosing spondylitis.

As discussed above, inflammation is a complex process involving many different biochemical pathways. Hemostasis is the balance between pro-coagulative and anti-coagulative pathways in the body. In inflammatory responses and conditions the balance between these two coagulative pathways is often tipped in favour of pro-coagulative responses. Thus coagulation is a feature of many inflammatory responses or conditions. For example the development of thrombosis may occur or be enhanced as a result of pro-coagulative pathways involved in inflammation.

Coagulation is particularly important in the context of thrombosis which may be related to, or occur in conjunction with atherosclerosis, arteriosclerosis, embolism or stroke.

Thus it would be clear to the skilled person when an inflammatory response included a pro-coagulative component, or when coagulation was occurring. The skilled person would -10 -also recognise when coagulation was occurring that was not desirable, or was pathological. These scenarios would be encompassed by the term "a condition in which coagulation occurs".

The medicaments, compounds and other aspects of the present invention are particularly suitable for the treatment of arteriosclerosis and atherosclerosis. KLF2 expression in endothelial cells is known to be associated with anti-inflammatory and anti-coagulative effects. Similarly the effect of raised KLF2 expression on reduced coagulation would be expected to have a beneficial effect in conditions in which coagulation plays a role. Accordingly, preferred medicament compounds and other aspects of the invention are for use in the treatment of arteriosclerosis, atherosclerosis, thrombosis, strokes and embolisms.

The medicarnents of the invention may be formulated for any suitable dosage regimen. Particularly preferred embodiments are medicaments formulated for systemic administration or for localised administration. Systemic administration may be by any form of systemic administration known, for example, orally, intravenously or intraperitoneally. Local administration may be by any form of local administration known, for example topically.

The medicaments of the invention may contain any number of pharmaceutically acceptable excipients. Those excipients used in the art are generally well known and may include fillers, lubricants, colours, flavours, wetting agents, solvents, buffering agents, preservatives and the like.

-11 -In some embodiments, the medicaments may contain one or more additional pharmaceutical compounds. In preferred embodiments the medicaments of the invention contain at least one additional anti-inflammatory drug and/or anti-atherosclerotic drug. Any suitable anti-inflammatory and/or anti-atherosclerotic may be utilised.

In preferred embodiments, the at least one additional anti-inflammatory drug comprises a non-steroidal anti-inflammatory drug (NSAID). Preferably the medicaments comprise at least one compound according to the present invention together with ibuprof en and/or aspirin.

The medicament may be in any form suitable for use in therapy.

As discussed in further detail herein, by "treatment" is meant at least improvement, preferably cure of the condition in question. Treatment may also include prophylactic, i.e. preventative treatment aimed at preventing the occurrence or severity of the condition in a subject judged to be at risk of developing a condition to be treated.

The description of terms relating to the first aspect of the present invention (i.e. medicaments) provided herein above applies mutatis mutandis to all aspects of the invention.

In a further aspect the present invention provides a method of modulating an immune response and/or of treating or preventing an inflammatory condition, or condition in which coagulation occurs comprising administering to a subject in need thereof a therapeutically effective quantity of a -12 -compound that modulates catecholamine metabolism, to thereby modulate said immune response or treat or prevent said inflammatory condition.

Preferred classes of compounds and specific compounds that modulate catecholamjne metabolism are as described for the first aspect above.

The method may comprise simultaneous, separate, or sequential administration of one or more compounds of the invention and of a vaccine. In this way an immune response to the vaccine may be modulated. Said modulation may be enhancement or boosting of the immune response.

The methods of the invention are also applicable to the treatment of one or more inflammatory conditions. The term It inflammatory conditiontt is used herein to refer to unwanted or pathological inflammation. The inflammatory condition may be selected from any disease that involves undesirable or pathological inflammation of any bodily tissue. In preferred embodiments, the inflammatory condition to be treated by the method according to the present invention is selected from the group comprising acute inflammation, chronic inflammation, septic shock, arteriosclerosis, atherosclerosis, thrombosis, stroke, embolism, arthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis or ankylosing spondylitis. Atherosclerosis, arteriosclerosis, thrombosis, stroke, embolism, and inflammatory conditions relating particularly to the endothelium are particularly preferred given the connection between KLF2 and endothelial cells/inflammation.

-13 -In preferred embodiments the method may comprise systemic or localised administration or delivery of the compound. This is discussed further in the context of the first aspect.

In a further aspect, the invention relates to a pharmaceutical composition comprising a compound that modulates catecholamjne metabolism and at least one additional pharmaceutical compound which is selected from a vaccine, an anti-inflammatory drug and an anti-athersclerotic drug.

In an alternative aspect, the invention also relates to a product containing a compound that modulates catecholamine metabolism and an additional pharmaceutical compound for simultaneous, separate or sequential use in the modulation of an immune response and/or the treatment or prevention of an inflammatory condition, or condition in which coagulation occurs.

In another aspect the present invention relates to a coating composition for a medical device wherein the coating composition comprises a therapeutically effective amount of a compound that modulates catecholamine metabolism.

In a yet another aspect the present invention relates to a medical device comprising a therapeutically effective amount of a compound that modulates catecholamine metabolism, wherein the compound is comprised in the medical device itself, or the compound is comprised in a coating which has been applied to the medical device.

-14 -In a further aspect the present invention relates to a kit comprising a composition comprising a compound that modulates catecholamine metabolism and a medical device wherein the composition may be used for coating the medical device.

In yet a further aspect the present invention relates to a kit a composition comprising a compound that modulates catecholamine metabolism and a coating composition or a composition that is suitable for use as a coating composition or a coating composition precursor, wherein the former composition may be combined with the latter composition to form a coating composition for a medical device.

In the aspects discussed above, preferred catecholamine modulating compounds are as already described in connection with the first aspect of the invention. As already established the medicarnents, pharmaceutical compositions and products of the present invention may be administered systemically or locally.

In particularly preferred embodiments the pharmaceutical composition includes at least one pharmaceutically acceptable excipient.

The compositions, medicaments and products of the invention for use in the methods of the invention may take the form of a tablet, capsule, injectable solution, implantable slow release matrix or device or any other suitable form.

-15 -The compositions, medicaments and products according to the invention may also take the form of a powder for direct inhalation, a suppository, or a solution which may be suitable for transdermal application. For example, one S medicament according to the invention may simply comprise a solution of the active agents in a suitable solvent, for example dimethyl sulphoxide.

The compositions, medicaments and products according to the invention may be manufactured using any suitable method.

For example, the compositions may be dry milled and mixed prior to tableting and the composition may therefore necessarily contain other pharmaceutically acceptable excipients such as a lubricant selected from the group comprising calcium stearate, magnesium stearate, zinc stearate, stearic acid, talc and combinations thereof, a binding agent selected from the group comprising hydroxypropylmethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose and a polyvinyl pyrrolidone (PVP).

Compositions, medicaments and products according to the invention may contain any pharmaceutically acceptable excipients such as binders, fillers, pigments, disintegrating agents, lubricants, wetting agents, buffers and other excipients conventionally used in the pharmaceutical and chemical fields. Some examples of excipients for use in the compositions, medicaments and products of the present invention are microcrystalline cellulose, lactose, starch, colloidal silica, talc, glycerol esters, sodium stearyl fumarate, and titanium dioxide.

-16 -For oral administration compositions medicaments, or products of the invention may be administered with any inert diluent or with an edible carrier. They may be incorporated directly into food or beverages making up part of the patient's diet. The compositions or medicaments of the invention may be formulated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspension syrups, wafers, and the like.

The tablets, troches, pills, capsules and the like may contain those excipients already mentioned and in some cases may also contain sweetening agents, such as sucrose, glucose, aspartame or saccharin, flavouring agents such as essential oils of mint, peppermint, spearmint or any other suitable flavouring. When the dosage unit is a capsule it may additionally contain a liquid carrier such as an oil or buffered aqueous solution.

Medicaments, products and compositions of the invention may also be formulated with phospholipids or fatty acids or other synthetic nanoparticles as carriers.

Medicaments, products and compositions of the invention may take the form of formulations for parenteral administration and may include sterile aqueous solutions or dispersions, and sterile powders for the preparation of sterile, injectable solutions or dispersions. The solutions or dispersions may also contain buffers, diluents, and other suitable additives that may be designed to promote the cellular uptake of the active agents in the composition, for

example, liposomes.

-17 -Pharmaceutical formulations for topical administration may be especially useful for localized treatment. Formulations for topical treatment include ointments, sprays, gels, suspensions, lotions, creams, and the like. Formulations for topical administration may include known carrier materials such as isopropanol, glycerol, paraffin, stearyl alcohol, polyethylene glycol, and the like.

Medicarnents and compositions of the invention may also include a known chemical absorption promoter. Absorption promoters include, for example, trichioroethanol, ti-if luoroethanol, and certain alcohols and mixtures thereof (according to GB 1,001, 949 to Meyer and GB 1,464, 975 to AstraLakemedel, these references are hereby incorporated in their entirety).

Medicaments and compositions of the invention suitable for rectal or vaginal administration may be presented as a suppository, which may include one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Disclosed herein are medicaments, uses, compositions arid methods for modulating an immune response and/or treating -18 -inflammatory conditions in a subject, preferably a subject in need of such treatment. The subject is most preferably human. As used herein, a subject in need of treatment may be an individual diagnosed with an inflammatory condition, or a patient wanting to prevent or delay the onset of a inflammatory condition, for example, someone with a family history of arteriosclerosis, atherosclerosis, thrombosis, stroke, embolism, arthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis or ankylosing spondylitis.

However, the medicament compositions may be simply taken as a prophylactic.

As mentioned above, the terms "treating" and "treatment" as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediaton of damage.

Thus, for example, the present method of "treating" an inflammatory condition, as the term is used herein, encompasses both prevention of the disorder and treatment of the disorder in a clinically symptomatic individual.

The term "pharmaceutically acceptable carrier" includes a material which is not biologically or otherwise undesirable.

Such a material may be administered to an individual along with the selected active agent without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

In preferred embodiments the pharmaceutical composition of the invention further comprises at least one additional -19 -pharmaceutical compound. The at least one additional pharmaceutical compound may for example be a vaccine or vaccine composition, an anti-inflammatory compound or drug, or an anti-atherosclerotic or anti-arteriosclerotic compound or drug. Such combination of further pharmaceutical compounds with the compounds of the invention may allow for additive or potentially synergistic effects between the compounds of the invention and the further pharmaceutical compounds. Furthermore, the combination of active ingredients may allow for a simpler treatment or dosage regime, for example by reducing the number of individual tablets or injections that are required to be taken by the subject.

In a particularly preferred embodiment of the invention, the pharmaceutical composition comprises at least one compound which alters catecholamine metabolism along with at least one additional pharmaceutical compound. The at least one additional pharmaceutical compounds may be an anti-inflammatory drug and/or anti-atherosclerotic drug. Any suitable anti-inflammatory and/or anti-atherosclerotic may be used.

In preferred embodiments, the at least one additional anti-inflammatory drug comprises a non-steroidal anti-inflammatory drug (NSAID). Preferably the medicaments comprise at least one compound according to the present invention together with ibuprof en and/or aspirin.

Preferably the ibuprof en and/or aspirin is present in an amount of less than 250 mg, more preferably less than 100 mg -20 -daily. Preferably the medicament is formulated so that the daily amounts may be incorporated into a single tablet.

The present invention also provides one or more compounds that modulate catecholamine metabolism and an additional pharmaceutical compound for simultaneous, separate or sequential use in the modulation of an immune response and/or the treatment or prevention of an inflammatory condition. In this way the compound of the invention and the additional pharmaceutical compound may be administered in combination, but in a variety of different forms or routes. The skilled person would be able to determine the optimum dosage, route of administration and relative temporal interval between administration of said compounds of the invention and said additional pharmaceutical compounds.

In preferred embodiments of the invention, the coating composition may find use in providing a coating for a medical device wherein said medical device may or may not itself comprise one or more of the compounds of the invention.

By "therapeutically effective amount" is meant an amount of the compound sufficient to give rise to the desired therapeutic effect. For example, in the context of an inflammatory condition, the therapeutically effective amount is an amount sufficient to modulate an immune response or to treat or prevent an inflammatory condition.

In some preferred embodiments the medical device according to the invention may be manufactured separately and then -21 -have the compound that modulates catecholamine metabolism applied to one or more surfaces.

Alternatively compounds of the invention may be, for example, blended with the ingredients used to make the medical device. It is envisaged that medical devices comprising compounds according to the present invention may comprise said compounds in a slow release formulation, thereby providing continuous delivery of the compound of the invention over time.

The compounds of the invention may be provided in the form of a medicament or pharmaceutical composition as described in detail herein.

The medical device may comprise any medical device, in particular a medical device, which is introduced into a

subject. The introduction of the device may be by

implantation or insertion for example. The device may be inside the subject for any length of time, dependent upon the particular device which is being utilised. This may include permanent introduction or temporary introduction.

In specific embodiments, the device is any one of: a catheter, stent, guidewire, sensor, ventricular assist device (VAD), graft, valve such as an aortic valve, pacemaker, artificial joint, or infusion system/pump. As for other aspects of the invention, the subject is most preferably a human subject.

In one preferred embodiment, the device comprises, consists essentially of or consists of a stent which has had coated -22 -on at least one surface thereof such that in use a sublect will be exposed to the at least one compound of the invention.

In further preferred embodiments the device may be biodegradable, for example a biodegradable stent. In addition, or alternatively, the coating comprising the compound of the invention may be biodegradable.

In one embodiment, the compound of the invention is adsorbed onto a suitable coating, such as a known hydromer coating for example. Any suitable (biocompatible) coating for a medical device may be utilised. The incorporation of into a stable coating facilitates a prolonged, controlled release of the compound or compounds of the invention from the surface on dissolution of the biodegradable polymers.

In another embodiment, a co-polymer blend is utilised.

Examples of suitable polymers into which the compounds of the invention may be (non-covalently) incorporated include, but are not limited to, poly(hexano-6-lactone), poly(ethylene-co-vinyl acetate) (EVA), poly(ethylene oxide) (PEO), polyvinylpyrollidone, poly(tetrafluoroe-thylene) (PTFE), poly(dimethylsilo-xane), polypropylene, poly(ethyleneterephthalate) (PET), polyamides (nylons), poly(ether urethane) (e.g. Pellethane), poly(ether urethane urea) (e.g. Biomer), low density polyethylene (LDPE), high density polyethylene (HDPE), polysulfones, polyvinylchloride (PVC), poly(2-hydroxyethylmethacrylate (PHEMA) and polylactide and blends thereof such as poly(hexano-6-lactone)/polyactjde blends and EVA/PEO blends. Co-polymers such as these offer a range of drug release properties and -23 -are clinically acceptable, and may be readily optimized for the desired release kinetics of the compounds of the invention.

Polymer coatings may be prepared by any suitable method, such as by standard dip-coating methods. Suitable polymer-blends may be dissolved in an appropriate solvent. Examples of a solvent which may be utilised to produce coatings according to the invention include dichloromethane and methylethylketone.

The dosage of the compositions of the invention depends on the individual patients and the disorder to be treated. An exemplary single dose of a composition of the invention is within the range of about 0.001 to about 400 mg. However, smaller dosages may be sufficient, and larger doses may also be required.

Typical daily doses may for example range from 1mg/kg to 5mg/kg. This would equate therefore to 80 to 400mg per day for a 80kg person. Lower or higher doses may be appropriate as the circumstances dictate. In common with many other drugs, higher doses may achieved by multiple dosing if necessary. Alternative routes of delivery may affect the required dose and this would be within the routine abilities of the skilled person to determine. For example, localised delivery of the compounds of the invention (for example in the context of an implanted device, such as a stent comprising the compounds of the invention in a coating or otherwise) may lead to lower doses being required.

Conversely, other routes of administration may require increased doses.

-24 -In one specific embodiment, the polymer which incorporates one or more compounds of the invention also incorporates polyethylene glycol (PEG) molecules/monomers therein. PEG is a well known hydrophilic molecule which has many uses in the coatings of the invention. Various forms of PEG are commercially available. Examples of suitable monomers include polyethylene glycol acrylates, including momo-methoxy triethylene glycol mono(meth)acrylate, mono-methoxy tetraethylene glycol mono(meth)acrylate and polyethylene glycol mono (meth) acrylate.

In a further aspect, the invention relates to use of a coating composition as described herein for coating a medical device.

In a further aspect, the present invention also relates to a method of manufacturing a coating for a medical device comprising combining a compound that modulates catecholamine metabolism with a polymer to form a coating.

In a further aspect, the present invention also relates to a method of manufacturing a medical device comprising coating at least a portion of said medical device with a composition comprising a compound that modulates catecholamine metabolism.

In yet a further aspect, the present invention also relates to a method of assessing the suitability of an individual for treatment with a compound that modulates catecholamine metabolism, the method comprising: -25 -a) in a test sample taken from the individual, determining the level of KLF2 expression, wherein a low level of KLF2 expression is indicative of the suitability of the individual to treatment with the compound, and/or b) in a test sample taken from the individual, determining the level of KLF2 expression before and after treatment with the compound, wherein an increase in KLF2 expression as a result of treatment with the compound is indicative of the suitability of the individual to treatment with the compound; and/or c) in a test sample taken from the individual, determining the DNA sequence upstream of a KLF2 gene present in the test sample and comparing the DNA sequence obtained with the sequence of any one of SEQ ID NOs. 1-5, wherein a level of sequence identity of at least 90% is indicative of the suitability of the individual to treatment with a compound as defined above.

By "upstream" is meant the portion of the nucleic acid molecule to be found in a 5' direction from the proposed transcriptional start site. This portion of the molecule may be 10 000 base pairs in length, or more preferably 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1500, 1000, 500, 482, 400, 300, 200, 150, 111, 100, 63, 50, 25, 10 or 8 base pairs in length. In preferred embodiments according to (C) above the level of sequence identity may be 91%, 92%, 939, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

Methods for determining sequence identity are routine in the art and include use of the BLAST software and EMBOSS software (The European Molecular Biology Open Software Suite (2000), Rice, P. Longden, I. and Bleasby, A. Trends in -26 -Genetics 16, (6) pp276-277) . The term "identity" as used herein refers to the relationship between sequences at the nucleotide level. The expression " identical" is determined by comparing optimally aligned sequences, e.g. two or more, over a comparison window wherein the portion of the sequence in the comparison window may comprise insertions or deletions as compared to the reference sequence for optimal alignment of the sequences. The reference sequence does not comprise insertions or deletions. The reference window is chosen from between at least 10 contiguous nucleotides to about 50, about 100 or to about 150 nucleotides, preferably between about 50 and 150 nucleotides. " identity" is then calculated by determining the number of nucleotides that are identical between the sequences in the window, dividing the number of identical nucleotides by the number of nucleotides in the window and multiplying by 100.

It will also be apparent to the skilled person that the method described above could be considered as a way of assessing whether compounds of the invention are suitable for treating a given individual.

In a related aspect, the invention also relates to a method of assessing the effect of treatment with a compound that modulates catecholarnine metabolism, the method comprising: a) in a test sample taken from the individual before treatment of the individual with a compound of the invention determining the level of KLF2 expression, b) in a test sample taken from the individual after treatment of the individual with a compound of the invention determining the level of KLF2 expression, -27 -c) comparing the levels of KLF2 expression obtained in a) and b) wherein an increase in KLF2 expression as a result of treatment of the individual with the compound is indicative of an effect on KLF2 expression by treatment of the individual with a compound of the invention. The effect on KLF2 expression is indicative of a beneficial or positive effect on treatment of the individual.

In preferred embodiments the method further comprises comparing the levels of KLF2 expression in further samples that have been taken from the subject at time points subsequent to the time points where steps a) and b) are carried out.

The differences in the levels of expression must be statistically significant in order to provide a reliable test for assessing the suitability of an individual for treatment with a compound of the invention, or for assessing the effect of treatment with a compound of the invention.

By "a low level of expression" is meant a level of expression that is, statistically speaking, lower than that expected or observed in a normal' sample or individual.

Any method for determining the relative expression level of a gene, or whether the expression level of a gene is significantly increased or decreased may be utilised. Such methods are well known in the art and routinely employed.

For example, statistical analyses may be performed using an analysis of variance test. A typical P value for use in such a method would be P values of < 0.05 when determining whether the relative expression is statistically significant. A difference in, or change in expression may -28 -be deemed significant if there is at least a 10% difference or increase or decrease for example. The test may be made more selective by making the change at least 15%, 20%, 25%, 30%, 35%, 40% or 50%, for example, in order to be considered statistically significant.

In appropriate embodiments, the level of KLF2 expression is determined with reference to a control sample. This control sample is preferably taken from normal tissue in the subject, or from a normal tissue from a healthy subject.

Alternatively, the control sample is taken from the same tissue as that under test at an earlier time point. This is particularly relevant for assessing the effect of treatment, for example in order to ensure that treatment has been effective to modulate an immune response and/or treat or prevent an inflammatory condition, or condition in which coagulation occurs. Thus, suitability of an individual for treatment with a compound that modulates catecholamine metabolism, or the effect of treatment of an individual may be monitored by determining the levels of KLF2 expression at appropriate time points. The skilled person can identify appropriate time points before, during and after treatment through exercise of their ordinary skill.

Suitable additional controls may also be included to ensure that the methods are working properly, such as measuring expression of a suitable reference gene in both test and control samples.

-29 -The compound used in the method of assessing the suitability or effect of treatment may be in the form of a medicament or pharmaceutical composition of the invention.

It will be apparent to one of skill in the art that the methods of assessing suitability of an individual to treatment or assessing the effect of treatment on an individual with a compound that modulates catecholamine metabolism may be used in order to provide a personalised medicine approach to treatment of the individual. According to these methods it is possible to determine if an individual is likely to respond positively to treatment with the compounds of the invention and furthermore, if such treatment is undertaken, to assess the effect of said treatment.

Such methods provide the clear advantage that the most appropriate medicament, pharmaceutical composition or treatment for the individual may be selected from the full range of options available. In addition, the methods of the invention provide the advantage that the effect of the medicament, pharmaceutical composition or treatment on the individual may be monitored, and if necessary changes made, for example in dosage, route of administration, treatment regimen etc. In a further aspect of the invention, there is provided a method of identifying genes whose expression is amenable to modulation by a compound that modulates catecholamine metabolism, wherein the method comprises comparing the nucleotide sequence of a nucleic acid molecule of interest to the nucleotide sequence of any one of SEQ ID NOs. 1-5, -30 -wherein an identity of at least 90% between said any one of SEQ ID NOs.l-5 and a corresponding portion of the nucleotide sequence of the nucleic acid molecule of interest indicates that the nucleic acid molecule is operably linked to, or represents at least a portion of a gene whose expression is amenable to modulation by a compound according to the present invention.

The term "operably linked" as used herein refers to a functional linkage between a "regulatory" sequence and a nucleic acid molecule of interest, such that the "regulatory" sequence is able to initiate transcription of the nucleic acid molecule. The regulatory sequence may comprise any type of sequence known to influence gene expression, such as promoters and enhancers for example.

The regulatory sequence is preferably at least a portion of a promoter.

Methods for determining sequence identity are discussed above in more detail. A method applicable to the present invention may be an in silico method, such as one that can be determined by bioinformatics homology/identity searches, for example by BLAST searches. Software for performing such homology/identity searches is widely available, for example via the internet.

In preferred method of identifying genes as above, the level of sequence identity may be 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

Preferred compounds and classes of compound that modulate catecholamine metabolism have already been discussed above.

-31 -In yet a further aspect the present invention relates to a compound that modulates catecholamine metabolism for use in the modulation of an immune response and/or in the treatment or prevention of an inflammatory condition, or condition in which coagulation occurs.

Preferred embodiments use catecholamine modulating compounds as already described in connection with the previous aspects of the invention. Preferred inflammatory conditions are also as discussed previously. In common with the other aspects of the present invention, atherosclerosis arteriosclerosis, thrombosis, stroke, embolism, and inflammatory conditions relating particularly to the endothelium are particularly preferred given the connection between KLF2 and endothelial cells/inflammation.

As already established the medicaments, pharmaceutical compositions and products of the present invention may be administered systemically or locally.

It is envisaged that the compounds, methods and uses of the invention may find application in an in vivo, ex vivo or in vitro context. It will be appreciated that the teaching of the invention applies to whole organisms and individual cells of said organisms either in situ or when removed ex vivo and maintained in culture for a period of time.

Cultured cells may be returned to the organism after having been removed. Alternatively, the teaching of the present invention may be applied to cell lines grown in culture.

Such cultured cell lines may be of any sort known to one skilled in the art, for example primary cell cultures or -32 -stable cell lines, optionally comprising further modifications.

Brief description of the Figures

The invention will now be described by way of exemplification only and with reference to the accompanying figures, in which: Figure 1 shows the KLF2 expression levels in control cells and cells treated with each of the exemplified compounds described herein at a variety of concentrations.

Stimulation of KLF2 expression was observed in all cases in comparison with control cells.

Figure 2 shows flow cytometric analysis of cultured human umbilical vein endothelial cells (HUVECs) treated with TNFa or TNF in combination with Daidzein or Tropolone in comparison with untreated control cells. Treatment of cells with these compounds reduced the upregulation of VCAM-l expression in HtJVECs in comparison to cells treated with TNFa alone.

Figure 3 shows the results of sandwich ELISA analysis of IL- 6 production by cultured human umbilical vein endothelial cells (HUVECs). Control (untreated cells) are compared with cells exposed to TNFa alone, and cells exposed to TNFc along with compounds of the invention. Inhibition of TNFc mediated IL-6 production was observed in cells exposed to compounds of the invention.

Figure 4 shows the results of sandwich ELISA analysis of MCP-l production by cultured human umbilical vein endothelial cells (HIJVECs). Control (untreated cells) are compared with cells exposed to TNFcx alone, and cells exposed to TNFc along with compounds of the invention. Inhibition -33 -of TNFa mediated MCP-1 production was observed in cells exposed to compounds of the invention.

Figure 5 shows the results of sandwich ELISA analysis of endothelin production by cultured human umbilical vein endothelial cells (HUVECs). Control (untreated cells) are compared with cells exposed to TNFa alone, and cells exposed to TNFc along with compounds of the invention. Inhibition of TNFc mediated endothelin production was observed in cells exposed to compounds of the invention.

Figure 6 shows an alignment between a highly conserved 63 base pair region within the Apal/Saci fragment described above for both mouse and human sequences.

The invention will now be described by way of reference to the examples. The examples discussed in the description are intended in no way to limit the scope of the invention as defined in the accompanying claims.

Description of the Invention

It has surprisingly been found that the expression of KLF2 may be modulated by compounds that modulate catecholamine metabolism.

Treatment of cultured cells, for example osteoblast cells or mesenchymal stem cells, with compounds according to the invention leads to enhanced KLF2 expression. Studies of endothelial cells in culture have also shown that treatment with compounds of the invention inhibits the expression of the acute-phase inflammatory cytokirie, IL-6, the chemokine (MCP-l) which promotes monocyte recruitment to the endothelium, and the vasoconstricting peptide endothelin.

These observations indicate that compounds according to the invention may be used to modulate KLF2 expression, thereby -34 -providing an opportunity to modulate stem cell behaviour, immune responses and inflammatory responses.

Stem cell behaviour may be modulated in an appropriate manner for therapeutic applications such as the therapeutic use of cardiomyocytes for treatment of damage following myocardial infarction. Immune responses may be modulated for example such that antigen dependent responses are enhanced. Inflammatory responses may for example be modulated at the level of endothelial cells.

Analysis of the responsiveness of KLF2 expression to modulation by compounds of the invention was carried out. A reporter construct comprising the promoter region from KLF2, cloned upstream of the firefly luciferase gene was transfected into mouse C2C12 cells. The effect of the compounds of the invention on the expression of the reporter gene, as driven by the KLF2 promoter, was then observed. It was clear that the compounds that modulate catecholamine metabolism were giving rise to an increase in KLF2 expression (see Figure 1).

The sequence of the KIJF2 promoter region is provided in SEQ ID NO. 1. Deletion analysis of this promoter region defines a 111 base-pair Apal/Saci fragment within the KLF2 promoter as the specific element which is responsive to the compounds of the invention.

Within this fragment there are several putative transcription factor binding sites. As such, and of particular interest is the 63 base pair sequence shown in SEQ ID NO. 3. There is very high homology between the mouse -35 -and human (SEQ ID NO. 4) sequences in this region. See Figure 6 which shows an alignment of the human and mouse sequences illustrating identity between 62 of the 63 nucleotides at this highly conserved locus.

The TAAATTTA palindrome (SEQ ID NO. 5) which is present within the highly conserved 63 base pair region identified above, and identical between the human and mouse sequences, is a good match for the Mef 2 family of transcription factors. Without wishing to be bound by theory, it seems plausible that the compounds of the invention act on KLF2 via a member of the Mef2 transcription factor family.

Studies on KLF2 expression were carried out in primary cultures of human umbilical vein endothelial cells (HUVECs).

These cells are the in vitro tool of choice for modelling the cell biology of the endothelium, including the vascular endothelium lining blood vessels. The surprising finding that the compounds of the invention enhanced the expression of KLF2 in C2C12 cells was thus extended to HUVECs, i.e. compounds of the invention also enhance the expression of KLF2 in HUVECs.

It was also noted that whereas inflammatory abuse of HUVECs with the cytokine TNFc decreases expression of KLF2, coincident treatment with compounds of the invention opposes this effect and maintains KLF2 expression at basal levels.

Thus, these observations strongly suggest that compounds of the invention can oppose pro-inflammatory insults to cells.

Inflammatory activation of cells, such as endothelial cells, also leads to the production of pro-inflammatory cytokines -36 -which are important in the inflammatory process. For example, endothelial cell activation and the production of pro-inflammatory cytokines are believed to be important in the atherosclerotic process.

The effect of the compounds of the invention on expression of the endothelial activation marker, VCAI'1-l, was analysed.

VCAM-1 is an adhesion molecule which when expressed on the endothelial surface, adheres to and encourages the extravasation of inflammatory cells (such as monocytes) from the blood. VCAIvI-l is therefore a key regulator of the inflammatory state.

VCAM-]. has also been shown to be repressed by ectopic KLF2 expression (5) and the present inventors have demonstrated that the compounds of the invention also regulate VCAM-l expression, presumably through its effects on KLF2 expression. Flow cytometry shows that the compounds of the invention do indeed block VCAM-1 expression induced by TNFa, significantly reducing the population of VCAN-1 positive cells (Figure 2) In a similar manner, 11-6, MCP-1 and endothelin, all molecules which are involved in inflammation were investigated. As demonstrated in Figures 2, 3, 4 and 5, the compounds of the invention antagonise the ability of TNF to enhance the production of VCAN-1, IL-6, MCP-l and endothelin respectively.

These findings have several consequences. Firstly, by reducing IL-6 production, the compounds of the invention appear to function as anti-inflammatory signals, blocking -37 -the production of an acute-phase inflammatory response.

Secondly, in opposing vCAM-l and MCP-1 production (for example in endothelial cells) the compounds of the invention would prevent the chemotaxis of monocytes from the circulation towards the endothelial cell layer and their subsequent extravasation. In addition, endothelin production is also opposed, reducing vasoconstriction and other pro-inflammatory effects mediated by this molecule.

These processes are important in the development of an inflammatory response, in particular in the development of atherosclerotic lesions.

The compounds of the invention enhance expression of KLF2, thereby regulating expression of proposed KLF2 target genes, an example of which is. VCAM-1, a key cell surface adhesion molecule involved in the extravasation of white blood cells and the regulation of the inflammatory state. Given that absence of KLF2 leads to inappropriate immune responses, drugs capable of increasing KLF2 levels may also have the effect of enhancing immune responses. The compounds of the invention therefore constitute a new class of adjuvants for boosting antigen-dependent immune responses in vaccines.

Examples

Example 1 Luciferase Reporter gene assay 482 base-pairs immediately 5' of the proposed transcriptional start of the murine KLF2 promoter (SEQ ID NO. 1) were PCR-amplified and cloned upstream of the firefly luciferase reporter gene. This construct therefore comprises the upstream regulatory element for KLF2. A putative 8 nucleotide transcription factor binding site for has been -38 -identified in this conserved region for the Mef2 family of transcription factors.

The construct described above was transfected into C2C12 cells along with a Renilla luciferase construct for riormalisation, and cells were stimulated for 24 hours with varying concentrations of compounds of the invention.

Firefly luciferase activity was determined by a luminescence assay and normalised to Renilla lucif erase activity. The results are shown in table 1 below and Figure 1.

KLF2 reporter expression Table 1 Arbitrary Units control 1.2 1N161 Deprenyl (500nM) 2.5 IN161 Deprenyl(5OnM) 1.85 IN 165 Clorgyline (500nM) 3.1 IN 165 Clorgyline (5OnM) 2.9 IN 165 Clorgyline (5nM) 2 1N167 Daidzein (5OpM) 3.2 IN167 Daidzein (5pM) 2.5 1N167 Daidzein (500nM) 1.5 IN 170 Tropolone (5OpM) 3 1N452 Hydrazaline (5pM) 2.5 1N452 Hydrazaline (500nM) 1.5 Example 2 anti-inflammatory action in HUVECs (1) Inhibition of TNFcx-induced induction of VCAN-1 by inhibitors of catecholamine metabolism was observed. 1-IUVECs were cultured for 24 hours in media alone (control), media supplemented with long/mi recombinant human TNFa, or supplemented with lOng/ml TNFa plus Daidzein or Tropolone at 5OpM. Cells were dissociated with PBS/0.5mI1 EDTA and stained for VCAN-1 expression using a FITC-labelled anti-human VCAN- 1 monoclonal antibody. Staining was detected using a Beckman-Coulter FC500 Flow Cytometer and analysed with CXP software. The results are shown in Figure 2.

-39 -The bars represent regions set against the untreated control to show the proportion of VCAM-l-positive cells under different experimental conditions. The control curve is reproduced in the graph shown for each of the other experimental conditions (TNFcx alone, TNFa + Daidzein, TNFcx + Tropolone) for ease of reference.

Example 3 Anti-inflammatory action in HUVECs (2) Control HtJVECs were cultured for 24 hours in media alone, or with long/ml recombinant human TNFa. Test cells were incubated under the same conditions with the addition of Deprenyl (500nN), Clorgyline (500nM), Daidzein (5OpM) or Tropolone (50i.IM). IL-6 production was measured by sandwich ELISA using capture and detection antibodies from R and D Systems, and quantified by comparison to a serially-diluted recombinant IL-6 standard. The results are shown in table 2 below and Figure 3.

Table 2 I L-6

______________________ pg/mi CNTRL 13 TNFa (lOng/mi) 400 TNFa +1N161 Deprenyl (500riM) 400 TNFa +IN 165 Clorgyllne (500nM) 312 INFo +IN 167 Daidzein (50i.iM) 120 TNFa +1N170 Tropolone (5OpM) 250 Example 4 Anti-inflammatory action in HUVECs (3) HUVECs were cultured for 24 hours in media alone, or with lOng/mi recombinant human TNFc. Test cells were incubated under the same conditions with the addition of Deprenyl (500nM), Clorgyline (500nM), Daidzein (5OpM) or Tropolone -40 - (5OpM). MCP-l production was measured by sandwich ELISA using capture and detection antibodies from R and D Systems and quantified by comparison to a serially-diluted recombinant MCP-l standard. The results are shown in table 3 below and Figure 4.

Table 3 MCP-l

___________________________________ ng/mI CONTROL 0.02 TNFa (lOng/mi) 5.6 TNFa +IN 161 Deprenyt (500nM) 5.4 TNFci +IN 165 CIorgyline (500nM) 4.4 TNFa +IN 167 Daidzein (5OpM) 4 TNFa +IN 170 Tropolone (5OpM) 4 6 Example 5 Anti-inflammatory action in}WVECs (4) HtJVECs were cultured for 24 hours in media alone, or with long/mi recombinant human TNFx. Test cells were incubated under the same conditions with the addition of Deprenyl (500nN), Clorgyline (SOOnrvl), Daidzein (5OpM) or Tropolone (5OpM). Endothelin production was measured by sandwich ELISA using capture and detection antibodies from R and D Systems and quantified by comparison to a serially-diluted recombinant endothelin standard. The results are shown in table 4 below and Figure 5.

Table 4 ED-i

______________________________ pglmI CONTROL 400 TNFO (long/mi) 420 TNFa +1N161 Deprenyi (500nM) 380 TNFa +IN 165 Ciorgyline (500nM) 380 TNFa +1N167 Daidzein (5OpM) 280 TNEa +IN 170 Tropoione (5OpM) 380 -41 -Various aspects of the present invention have been exemplified in the preceding section of the specification.

The discussion of these examples is intended for exemplification purposes only and is not intended to restrict in any way the scope of the invention as claimed in the appended claims.

References All of these references are hereby incorporated into the

present disclosure in their entirety.

(1) Kuo CT et al., Science 1997; 278 (5339): 788-9 (2) Carlson CM et al., Nature, 2006; 442 (7100): 299-302 (3) Parmar et al., J. Clin. Invest. 2006, 116, 49-58 (4) Glover et al., PL0S Comput Biol. 2006 Nov 24;2(ll):el58 (5) Lin et al Circulation research 2005 96, e48-e57.

SEQUENCE LISTING

<110> Inion Ltd <120> Catecholamine metabolism inhibitors <130> P89861GB00 <160> 5 <170> Patentln version 3.3 <210> 1 <211> 482 <212> DNA <213> Mus musculus <400> 1 ggccccaaac ttcatccttc ttgtcttagc ttccaaggta gcttaaacaa agatttcaca 60 gagcgttccc tgggtgagca ctgtgacccc agactcccca tgcatcggtc caggtttcca 120 ggggcttgag gctggggaga taccgggaaa gcctcgtcac ccgtgttccc cgcggagtcc 180 caggtgccac cccctccccc gccccccgcg cgcgctaact atgctgttgt gcctggtttg 240 ttgacgccgt cgccataggg acggtcgggg cggggcccg gccgcggggg cggggcttga 300 ggagcgcagt ccgggctccc gcagtagccg ccacagccac caccgccagg cttatatacc 360 gcggctaaat ttaggctgag cccggagctc gtccccatcc gggacgcgtt tccgtcgacg 420 cctcctagcc cggccctctg tgcctgccgc gcctataagg cctgggtggg ccgggccaca 480 gc 482 <210> 2 <211> 111 <212> DNA <213> Mus musculus <400> 2 cggccgcggg ggcggggctt gaggagcgca gtccgggctc ccgcagtagc cgccacagcc 60 accaccgcca ggcttatata ccgcggctaa atttaggctg agcccggagc t 111 <210> 3 <211> 63 <212> DNA <213> Mus musculus <400> 3 ggcttatata ccgcggctaa atttaggctg agcccggagc tcgtccccat ccgggacgcg 60 ttt 63 <210> 4 <211> 63

I

<212> DNA <213> Horno sapiens <400> 4 ggcttatata ccgcggctaa atttagctg cgcccggagc tcgtccccat ccgggacgcg 60 ttt 63 <210> 5 <211> 8 <212> DNA <213> Mus musculus <400> 5 taaattta 8

Claims (47)

1. Use of a compound that modulates catecholamine metabolism in the manufacture of a medicament for use in the modulation of an immune response and/or in the treatment or prevention of an inflammatory condition, or condition in which coagulation occurs.

2. Use according to claim 1 wherein the compound is a monoamine oxidase (MAO) inhibitor, a catechol-o-methyl transferase (COMT) inhibitor, an aldehyde dehydrogenase (ALDH) inhibitor, a compound that stimulates the production of a catecholamine, a compound that stimulates the release of a catecholamine, or a compound that mimics the biological activity of a catecholamine.

3. Use according to claim 2 wherein the MAO inhibitor is l-hydrazinophthalazine monohydrochloride, R-(-) -Deprenyl hydrochloride, or N-Methyl-N-propargyl-3-(2,4-dichlorophenoxy) propylamine hydrochloride.

4. Use according to claim 2 wherein the CONT inhibitor is 2-Hydroxy-2, 4, 6-cycloheptatrien-l-one.

5. Use according to claim 2 wherein ALDH inhibitor is 4,7-Dihydroxy-iso-flavone 7-Hydroxy-3-(4-hydroxy-phenyl) -4H-l-benzo-pyran-4 -one.

6. Use according to any one of the preceding claims wherein the inflammatory condition or condition in which coagulation occurs is selected from acute inflammation, chronic inflammation, septic shock, arteriosclerosis, Lts atherosclerosis, thrombosis, stroke, embolism, arthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis or ankylosing spondylitis.

7. Use according to any one of claims 1 to 6 wherein the medicament is for systemic administration.

8. Use according to any one of claims 1 to 6 wherein the medicament is for localised administration.

9. A method of modulating an immune response and/or of treating or preventing an inflammatory condition, or condition in which coagulation occurs comprising administering to a subject in need thereof a therapeutically effective quantity of a compound that modulates catecholamine metabolism, to thereby modulate said immune response or treat or prevent said inflammatory condition, or condition in which coagulation occurs.

10. The method according to claim 9 wherein the compound is a monoamine oxidase (MAO) inhibitor, a catechol-o-methyl transferase (COMT) inhibitor, an aldehyde dehydrogenase (ALDH) inhibitor, a compound that stimulates the production of a catecholamine, a compound that stimulates the release of a catecholamine, or a compound that mimics the biological activity of a catecholamine.

11. The method according to claim 10 wherein the MAO inhibitor is l-hydrazinophthalazine monohydrochioride, R- (-) -Deprenyl hydrochloride, or N-Methyl-N-propargyl-3-(2,4-dichlorophenoxy) propylamine hydrochloride.

12. The method according to claim 10 wherein the COMT inhibitor is 2-Hydroxy-2,4,6-cycloheptatrien-l-one.

13. The method according to claim 10 wherein ALDH inhibitor is 4' ,7-Dihydroxy-iso-flavone 7-Hydroxy-3-(4-hydroxy-phenyl) -4H-1 -benzo-pyran-4 -one.

14. The method according to any one claims 9 to 13 wherein the inflammatory condition or condition in which coagulation occurs is selected from acute inflammation, chronic inflammation, septic shock, arteriosclerosis, atherosclerosis, thrombosis, stroke, embolism, arthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis or ankylosing spondylitis.

15. The method according to any one claims 9 to 14 wherein the compound is administered systemically.

16. The method according to any one claims 9 to 14 wherein the compound is administered locally.

17. The method of any one of claims 9 to 16 wherein the compound is administered simultaneously, separately or sequentially with a vaccine.

18. A pharmaceutical composition comprising a compound that modulates catecholamine metabolism, and at least one additional pharmaceutical compound which is selected from a vaccine, an anti-inflammatory drug and an anti-atherosclerotic drug.

19. A product containing a compound that modulates catecholamine metabolism and an additional pharmaceutical compound for simultaneous, separate or sequential use in the modulation of an immune response and/or the treatment or prevention of an inflammatory condition, or condition in which coagulation occurs.

20. A product according to claim 19 wherein the additional pharmaceutical compound is a vaccine, an anti-inflammatory drug or an anti-atherosclerotic drug.

21. A coating composition for a medical device wherein the coating composition comprises a therapeutically effective amount of a compound that modulates catecholamine metabolism.

22. A medical device comprising a therapeutically effective amount of a compound that modulates catecholamine metabolism, and wherein; a) the compound is comprised in the medical device itself, or b) the compound is comprised in a coating which has been applied to the medical device.

23. A kit comprising, a) a composition comprising a compound that modulates catecholamine metabolism, and b) a medical device, wherein the composition of a) may be used for coating the medical device of b)

24. A kit comprising, a) a composition comprising a compound that modulates catecholamine metabolism, and, and b) a coating composition or composition that is suitable for use as a coating composition or coating composition pre-cursor, wherein the composition of a) may be combined with the composition of b) to form a coating composition for a medical device.

25. The pharmaceutical composition, product, coating composition, medical device, or kit of any one of claims 15 to 24 wherein the compound that modulated catecholamine metabolism is a monoamine oxidase (MAO) inhibitor, a catechol-o-methyl transferase (COMT) inhibitor an aldehyde dehydrogenase (ALDH) inhibitor, a compound that stimulates the production of a catecholatnine, a compound that stimulates the release of a catecholamine, or a compound that mimics the biological activity of a catecholamine.

26. The pharmaceutical composition, product, coating composition, medical device, or kit of claim 25 wherein the MAO inhibitor is l-hydrazinophthalazine monohydrochioride, R-(-) -Deprenyl hydrochloride, or N-Methyl-N-propargyl-3- (2,4 -dichiorophenoxy) propylamine hydrochloride.

27. The pharmaceutical composition, product, coating composition, medical device, or kit of claim 25 wherein the COMT inhibitor is 2-Hydroxy-2, 4, 6-cycloheptatrien-1-one.

28. The pharmaceutical composition, product, coating composition, medical device, or kit of claim 26 wherein ALDI-i inhibitor is 4', 7 -Dihydroxy-iso-flavone 7-Hydroxy-3 -(4-hydroxy-phenyl) -4H-l-benzo-pyran-4-one.

29. Use of a coating composition as defined in any one of claims 21 or 25 to 28 for coating a medical device.

30. A method of manufacturing a coating for a medical device comprising combining a compound that modulates catecholamine metabolism with a polymer to form a coating.

31. A method of manufacturing a medical device comprising coating at least a portion of said medical device with a therapeutically effective amount of a coating comprising a compound that modulates catecholamine metabolism.

32. A method of increasing the expression of KLF2 in a cell, wherein the method comprises contacting said cell with a compound that modulates catecholamine metabolism.

33. A method of assessing the suitability of an individual for treatment with a compound that modulates catecholamine metabolism, the method comprising, a) in a test sample taken from the individual, determining the level of KLF2 expression, wherein a low level of KL,F2 expression is indicative of the suitability of the individual to treatment with the compound, and/or b) in a test sample taken from the individual, determining the level of KLF2 expression before and after treatment with the compound, wherein an S0 increase in KLF2 expression following treatment with the compound is indicative of the suitability of the individual to treatment with the compound.

34. A method of assessing the effect of treatment of an individual with a compound that modulates catecholamine metabolism the method comprising, a) in a test sample taken from the individual before treatment of the individual with a compound as defined above determining the level of KLF2 expression, b) in a test sample taken from the individual after treatment of the individual with a compound as defined above determining the level of KLF2 expression, c) comparing the levels of KLF2 expression obtained in a) and b) wherein an increase in KLF2 expression following treatment of the individual with the compound is indicative of a positive effect of treatment of the individual with a compound as defined above.

35. The method according to claim 34, further comprising comparing the levels of KLF2 expression in further samples that have been taken from the subject at time points subsequent to time points a) and b).

36. The method according to any one of claims 29 to 35 wherein the compound is a monoamine oxidase (MAO) inhibitor, 5-I a catechol-o-methyl transferase (COMT) inhibitor, an aldehyde dehydrogenase (ALDH) inhibitor, a compound that stimulates the production of a catecholamine, a compound that stimulates the release of a catecholamine, or a compound that mimics the biological activity of a catecholamine.

37. The method according to claim 36 wherein the MAO inhibitor is l-hydrazinophthalazine monohydrochioride, R- (-) -Deprenyl hydrochloride, or N-Methyl-N--propargyl-3-(2,4-dichlorophenoxy) propylamine hydrochloride.

38. The method according to claim 37 wherein the COMT inhibitor is 2-Hydroxy-2, 4, 6-cycloheptatrien-l-one.

39. The method according to claim 37 wherein ALDH inhibitor is 4' ,7-Dihydroxy-iso-'flavone 7-Hydroxy-3-(4-hydroxy-phenyl) -4H-l-benzo-pyran-4-one.

40. A compound that modulates catecholamine metabolism for use in the modulation of an immune response and/or in the treatment or prevention of an inflammatory condition, or condition in which coagulation occurs.

41. A compound according to claim 40 wherein the compound is a monoamine oxidase (MAO) inhibitor, a catechol-o-methyl transferase (COMT) inhibitor, an aldehyde dehydrogenase (ALDH) inhibitor, a compound that stimulates the production of a catecholamine, a compound that stimulates the release of a catecholamine, or a compound that mimics the biological activity of a catecholamine. Sc

42. A compound according to claim 41 wherein the MAO inhibitor is 1-hydrazinophthalazine monohydrochioride, R- (-) -Deprenyl hydrochloride, or N-Methyl-N-propargyl-3-(2,4-dichiorophenoxy) propylamine hydrochloride.

43. A compound according to claim 41 wherein the COMT inhibitor is 2-Hydroxy-2,4,6-cycloheptatrien-l-Ofle.

44. A compound according to claim 4]. wherein ALDH inhibitor is 4' , 7 -Dihydroxy-iso-flavone 7 -Hydroxy-3 -(4-hydroxy-iphenyl) -4H-1-benzo-'pyran-4-one.

45. A compound according to any one claims 40 to 44 wherein the inflammatory condition or condition in which coagulation occurs is selected from acute inflammation, chronic inflammation, septic shock, arteriosclerosis, atherosclerosis, thrombosis, stroke, embolism, arthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis or ankylosing spondylitis.

46. A compound according to any one of claims 40 to 45 wherein the compound is for systemic administration.

47. A compound according to any one of claims 40 to 45 wherein the compound is for localised administration.

840996 MJG, MJG