WO2018002311A1 - Treatment and prevention of atherothrombosis by inhibition of syk kinase - Google Patents

Abstract

The present invention relates to an inhibitor of Syk kinase for use in the treatment and/or prevention of atherothrombosis. The present invention further relates to a method of treating and/or preventing atherothrombosis comprising administering a pharmaceutically effective amount of an inhibitor of Syk kinase to a subject in need thereof.

Description

Treatment and prevention of atherothrombosis by inhibition of Syk kinase

The present invention relates to an inhibitor of Syk kinase for use in the treatment and/or prevention of atherothrombosis. The present invention further relates to a method of treating and/or preventing atherothrombosis comprising administering a pharmaceutically effective amount of an inhibitor of Syk kinase to a subject in need thereof.

In this specification, a number of documents including patent applications and manufacturer's manuals is cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Vessel wall injury results in the activation of platelets and formation of a platelet plug, followed by further coagulant activity, that leads to the formation of fibrin-containing thrombi which occlude the injured vessel. Haemostasis is thus an event that begins with the adherence of platelets to activating components of the subendothelial matrix, such as collagen. Similarly, the erosion or rupture of an atherosclerotic plaque results in the formation of a platelet- and fibrin-rich thrombus, by arresting circulating platelets on the exposed material, such as extracellular matrix components. Whereas normal haemostasis is an important function, the thrombus formation due to erosion or rupture of an atherosclerotic plaque can be detrimental, as it can lead to myocardial infarction and ischemic stroke, two of the leading causes of death worldwide (Fuster V. et al., J Am Coll Cardiol. 2005;46:937-954; Badimon L. and Vilahur G. J Int Med. 2014;276:618-632).

Fibrous collagens are reactive matrix components that are of particular importance in platelet adhesion and activation. Several platelet collagen receptors have been identified so far, of which especially glycoprotein VI (GPVI) and glycoprotein la-lla (integrin α2β1 ) are currently considered to be clinically important. Kuijpers MJE. et al. (FASEB Journal 2003; 17(6):685- 687) investigated the roles of these two important collagen receptors GPVI and integrin α2β1 on platelets, as well as the role of Gocq heterotrimer signaling in platelet responses evoked by TxA₂ and ADP through the TPa and P2Y1 receptors, respectively. Their findings indicated that initial platelet tethering to collagen exposed to flowing blood critically involves the interaction between GPIb and von Willebrand factor deposited from plasma on the collagen, and that stable platelet-collagen adhesion under high wall shear rates (>500 s^"1) depends on active GPVI receptors. It was hypothesised that platelet binding and activation via GPVI primes the cells for integrin-mediated adhesion, and that activated integrin α2β1 stabilises the platelets on collagen. The authors concluded that agents inhibiting integrin α2β1 , potentially in combination with anti-GPVI effects, could be useful for platelet-directed antithrombotic therapies.

In Auger et al. (FASEB Journal 2005; 19(7):825-827 and full article as published in the World Wide Web under fasebj.org/cgi/doi/10.1096/fj.04-1940fje), the authors noted that there are certain controversies in the literature about the individual roles of integrin α2β1 and GPVI in the adhesion of platelets on collagen. The aim of said study was thus to carry out experiments to address these controversies. To this end, both human and mouse platelets were analysed under the same experimental conditions, using identical or equivalent experimental tools. The authors found that there are two distinct pathways of stable adhesion, one based on activation of GPVI and Src family kinases and the other being dependent on integrin 2β1. Auger et al. further found that integrin α2β1 was capable of mediating adhesion of mouse platelets in the absence of GPVI expression or downstream Src kinase activity and intracellular calcium elevation.

Because the composition of plaque material differs from the composition of healthy vessels, one aspect under investigation was, and still is, the precise nature of these compositions and their influence on thrombus formation. Work that focused on thrombus formation using a model simulating the rupture of human lipid-rich atherosclerotic plaques (Penz et al. FASEB Journal 2005; 19(7):898-909) revealed the presence of morphologically altered collagen type I- and type Ill-positive structures in the plaques. The authors of this work found that inhibition of GPVI with the antibody 10B12, which specifically binds the collagen-binding site of GPVI, completely blocked platelet thrombus formation onto plaques in flowing blood, whereas no effect by antibody-mediated inhibition of integrin α2β1 was observed. Moreover, mice platelets lacking GPVI were unable to adhere to and form thrombi onto atheromatous plaques. This work was confirmed and expanded in Schulz et al. (Basic Res Cardiol. 2008;103:356-367), where the binding to type I- and Ill-containing structures was further investigated, using a different anti-GPVI antibody (5C4). The authors of these two studies concluded that the morphologically diverse collagen type I- and type Ill-containing structures in the plaques stimulate the thrombus formation by activating platelet GPVI, and that inhibition of GPVI might provide a novel antithrombotic strategy to prevent atherothrombosis in patients at cardiovascular risk. Further studies into the inhibition of atherosclerotic plaque- induced platelet activation by using dimeric GPVI-Fc or various anti-GPVI antibodies were described in Jamasbi et al. (J Am Coll Cardiol. 2015;65:2404-2415). The authors suggested that the use of GPVI-Fc would be preferable for therapy, because there are concerns that anti-GPVI antibodies could potentially induce immune responses and may increase bleeding, although no such detrimental immune responses or increased bleeding effects were shown for the antibodies employed in this study.

An alternative approach has been described in Penz et al. (Thromb Haemost. 2007; 97:435-443), where the authors focused on GPIboc, the platelet receptor that interacts with von Willebrand factor (VWF) present on plaque collagen structures. This interaction is thought to represent the first step in thrombus formation, inducing initial platelet tethering and transient adhesion. The authors found that under flow conditions with high shear rates of 1.500 s^"1, contrary to static conditions, aspirin failed to significantly inhibit plaque-induced thrombus formation, and a combination of P2Y and P2Yi₂ receptor antagonists (ADP receptor antagonists) was less effective in reducing plaque-stimulated platelet thrombus formation than the blocking of GPIboc. Thus, the authors concluded that VWF might play an important role in platelet thrombus formation after plaque rupture and suggested that a combination of different anti-platelet drugs, such as P2Y₁/P2Y₁₂ receptor antagonists and inhibitors of GPIboc or GPVI might improve the prevention of human plaque-induced thrombus formation after plaque rupture. As shown above, numerous studies have been carried out to elucidate the mechanism of platelet activation and aggregation, both in healthy vessels as well as in a model simulating the rupture of human atherosclerotic plaques. However, despite the fact that a lot of effort is currently being invested into these studies, the most common therapy at present is still a dual anti-platelet therapy with aspirin and a P2Y₁₂-antagonist. Although this therapy reduces ischemic cardiovascular events, its efficacy is limited and, most importantly, this therapy has the drawback of being associated with an increased bleeding risk (Bonaca MP. et al., N Engl J Med. 2015; 372:1791-1800), as these compounds target not only plaque-triggered platelet activation, but also affect physiologic haemostatic mechanisms. Thus, novel therapies aiming specifically at plaque-triggered platelet activation with high efficacy but that leave physiologic haemostatic mechanisms intact would offer tremendous value to the field. This need is addressed by the provision of the embodiments characterised in the claims.

Accordingly, the present invention relates to an inhibitor of Syk kinase for use in the treatment and/or prevention of atherothrombosis.

The term "Syk kinase", also referred to as Syk herein, refers to "spleen tyrosine kinase". Syk kinase is a non-receptor cytoplasmic tyrosine kinases having a characteristic dual SH2 domain separated by a linker domain. Syk kinase is not only expressed in hematopoietic tissues, but in a variety of tissues. Within B and T cells respectively, Syk kinase transmits signals from the B-Cell receptor and T-Cell receptor. It also plays a similar role in transmitting signals from a variety of cell surface receptors including CD74, Fc Receptor, and integrins. Two Syk kinase isoforms are listed in the NCBI data bank for homo sapiens: a short, 612 amino acid isoform Syk(S) (NCBI Accession No: NP_001167639.1 ; entry as of March 15, 2015), and a long, 635 amino acid isoform Syk(L) (NCBI Accession No: NP_001 67638.1 ; entry as of March 15, 2015). There are four known mRNA variants encoding Syk kinase (NCBI Reference Sequences: NM_003177.6; NM_00 35052.3; NM_001 74 67.2 and NM_001174168.2; all entries as of March 15, 2015), as well as two additional, predicted transcript variants X1 to X4 (NCBI Reference Sequences: XM_005252147.3 (as updated on June 6, 2016) and XM_011518946.2 (as updated on June 6, 2016)).

The term "inhibitor" in accordance with the present invention refers to an inhibitor that reduces or abolishes the biological function or activity of a particular target protein, i.e. here Syk kinase. An inhibitor may perform any one or more of the following effects in order to reduce or abolish the biological function or activity of the protein to be inhibited: (i) the transcription of the gene encoding the protein to be inhibited is lowered, i.e. the level of mRNA is lowered, (ii) the translation of the mRNA encoding the protein to be inhibited is lowered, and (iii) the protein performs its biochemical and/or cellular function with lowered efficiency in the presence of the inhibitor. Compounds falling in class (i) include compounds interfering with the transcriptional machinery and/or its interaction with the promoter of said gene and/or with expression control elements remote from the promoter such as enhancers. Compounds of class (ii) comprise antisense constructs and constructs for performing RNA interference (e.g. siRNA) well known in the art (see, e.g. Zamore (2001 ) Nat Struct Biol. 8(9), 746; Tuschl (2001) Chembiochem. 2(4), 239). Compounds of class (iii) interfere with the molecular function of the protein to be inhibited, such as receptor signalling activity and activation of downstream target molecules. Accordingly, active site binding compounds are envisaged. Class (iii) also includes compounds which do not necessarily bind directly to the target, but still interfere with its function or activity, for example by altering the affinity or rate of binding of a known activator to the target, by competing with the binding of a known activator to the target or by displacing a known activator bound to the target. Preferably, the inhibitor binds directly to Syk kinase, thereby directly inhibiting its biological function or activity. Also preferably, the inhibitor binds irreversibly to Syk kinase.

In accordance with the present invention, the inhibitor is an "inhibitor of Syk kinase", i.e. the inhibitor reduces the biological function or activity of Syk kinase. It is particularly preferred that the inhibitor specifically inhibits Syk kinase, i.e. that it only inhibits the biological function or activity of Syk kinase, but not the biological function or activity of other proteins.

Biological function or activity denotes in particular any known biological function or activity of Syk kinase, including those elucidated in accordance with the present invention. Examples of said biological function or activity are the phosphorylation and activation of Ρί-Ογ2 in thrombocytes, thereby increasing cytosolic calcium levels and activating the effector molecule protein kinase C (PKC), which in turn result in the activation of platelets such as platelet aggregation and degranulation (secretion). For example, SYK-dependent inhibition of platelet secretion after stimulation with collagen or collagen-related peptide, as measured by reduction of ATP release, is a classic readout of GPVI-dependent platelet activation. All these functions or activities can be tested for either using any of a variety of standard methods known in the art, such as tyrosine phosphorylation of Syk or ΡΙΧγ2 or an increase of cytosolic Ca2+ concentration or on the basis of the teachings of the examples provided below, optionally in conjunction with molecular techniques such as phosphorylation assays or with the teachings of the documents cited therein. Preferably, Syk activity, as well as its inhibition, is measured by cell-based or biochemical assays.

In a preferred embodiment, the inhibitor reduces at least one, and preferably all of the above cited biological functions or activities of Syk kinase by at least 50%, preferably by at least 75%, more preferred by at least 90% and even more preferred by at least 95% such as at least 98% or even by 100%. The term "reduction by at least" refers to a decreased biological function or activity such that Syk kinase loses the recited amounts of one or more, preferably of all its biological functions or activities. For example, a reduction by at least 75% means that Syk kinase loses 75% of its biological function or activity and, consequently, has only 25% of the biological function or activity remaining as compared to Syk kinase that is not inhibited.

The function of any of the inhibitors referred to in the present invention may be identified and/or verified by using high throughput screening assays (HTS). High-throughput assays, independently of being biochemical, cellular or other assays, generally may be performed in wells of microtiter plates, wherein each plate may contain, for example 96, 384 or 1536 wells. Handling of the plates, including incubation at temperatures other than ambient temperature, and bringing into contact of test compounds with the assay mixture is preferably effected by one or more computer-controlled robotic systems including pipetting devices. In case large libraries of test compounds are to be screened and/or screening is to be effected within short time, mixtures of, for example 10, 20, 30, 40, 50 or 100 test compounds may be added to each well. In case a well exhibits biological activity, said mixture of test compounds may be de-convoluted to identify the one or more test compounds in said mixture giving rise to the observed biological activity.

The determination of binding of potential inhibitors can be effected in, for example, any binding assay, preferably biophysical binding assay, which may be used to identify binding of test molecules prior to performing the functional/activity assay with the inhibitor. Suitable biophysical binding assays are known in the art and comprise fluorescence polarization (FP) assay, fluorescence resonance energy transfer (FRET) assay and surface plasmon resonance (SPR) assay.

In cases where the inhibitor acts by decreasing the expression level of the target protein, the determination of the expression level of the protein can, for example, be carried out on the nucleic acid level or on the amino acid level. Methods for determining the expression of a protein on the nucleic acid level include, but are not limited to, northern blotting, PCR, RT- PCR or real RT-PCR. Methods for the determination of the expression of a protein on the amino acid level include, but are not limited to, western blotting or polyacrylamide gel electrophoresis in conjunction with protein staining techniques such as Coomassie Brilliant blue or silver-staining. These methods for the determination of the expression level of Syk kinase on both the nucleic acid level as well as the amino acid level are well known in the art.

A number of Syk kinase inhibitors are presently known and have been developed to treat inflammatory disorders, rheumatic and other autoimmune diseases (Deng GM. et al. Front Immunol. 2016 Mar 7;7:78; Lucas MC and Tan SL. Future Med Chem. 2014;6(16):1811-27; Geahlen RL. Trends Pharmacol Sci. 2014;35(8):414-422; Thorarensen A and Kaila N. Pharm Pat Anal. 2014 Sep;3(5):523-41.

For example, R406 is a small molecule Syk inhibitor (Braselmann S et al J Pharmacol Exp Ther (2006) 319(3):998-1008). The compound R788 (renamed fostamatinib), is the water- soluble prodrug of the biologically active R406 (Sheridan C Nat Biotechnol (2008) 26(2): 143- 4.42). These small molecules, R406 as well as R788, have been shown to inhibit the development of experimental arthritis (Coffey G et al J Pharmacol Exp Ther (2012) 340:350- 9). In a randomized clinical Phase II trial, fostamatinib was effective in the treatment of patients with rheumatoid arthritis (Weinblatt ME. et al. Arthritis Rheum (2008) 58:3309- 8; Weinblatt ME et al N Engl J Med 2010;363:1303-12 ; Genovese MC. et al. Arthritis Rheum (2011) 63:337-45). Other examples of autoimmune diseases which were successfully treated by Syk inhibitors are thrombocytopenic purpura (ITP) and heparin-induced thrombocytopenia (HIT) in which autoantibodies against platelet antigens result in platelet activation, and the opsonization and phagocytosis of both platelets and megakaryocytes by macrophages. The Syk inhibitor fostamatinib blocked platelet loss induced by an antibody (Ab) against integrin αΙΙβ in a mouse model of ITP and a phase II clinical trial in patients demonstrated that fostamatinib can restore platelet counts in approximately 50% of patients with ITP (Podolanczuk A et al Blood (2009) 113:3154-60).

Further inhibitors of Syk kinase known in the art include R343, piceatannol, R112 and P505- 15, all of which are reviewed e.g. in Geahlen RL, Trends Pharmacol Sci. 2014; 35(8): 414- 422. R343 is an inhaled Syk inhibitor designed for the treatment of allergic asthma and R112 an intranasal inhibitor tested for the alleviation of seasonal allergies. P505-15 is an orally available, highly selective Syk inhibitor. R788, R406, R343 and R112 were developed by Rigel Pharmaceuticals and P505-15 by Portola Pharmaceuticals. A new highly specific Syk inhibitor is Entospletinib (GS-9973), which has been developed by Gilead Sciences Inc.. Entospletinib is orally active and has entered phase 1 and 2 clinical trials (Ramathan S et al Clin Drug Invest (2017) 37(2): 195-205; Sharman J Blood (2015) 125(15): 2336-2343). Entospletinib is highly Syk-selective (Syk Kd of 7.6 nM, with only 1 other kinase inhibited with a Kd < 100 nM), whereas R406 is nonselective (Syk Kd of 15 nM) and inhibits other 79 kinases with Kd < 100 nM (Sharman J β/oocf (2015) 125(15): 2336-2343

All are competitive inhibitors of the ATP-binding site of Syk. Piceatannol, a natural stilbene, is a relatively low affinity inhibitor of Syk, not very specific, but one that binds competitively with the phospho-acceptor substrate.

Yet further inhibitors, as well as lead compounds for the further development of improved inhibitors, have been reviewed in Geahlen RL., Trends Pharmacol Sci. 2014; 35(8): 414-422 and Lucas MC and Tan SL. Future Med Chem. 2014;6(16):1811-27, and a summary of said inhibitors is provided in Table 1 below. In addition, Thorarensen and Kaila (Thorarensen A and Kaila N. Pharm Pat Anal. 2014 Sep;3(5):523-41) reviewed patent applications published during 2011 to 2013. All of these inhibitors discussed in the art to be Syk inhibitors may be employed in accordance with the present invention.

It is particularly preferred in accordance with the present invention that the inhibitor is a thrombocyte-specific inhibitor. The inhibitor, in accordance with the present invention, may in certain embodiments be provided as a small molecule, a proteinaceous compound or as a nucleic acid molecule, such as e.g. an interfering or inhibiting nucleic acid molecule as described in more detail below. The inhibitor can also be encoded by a nucleic acid molecule, which can, for example, be incorporated into an expression vector comprising regulatory elements, such as megakaryocyte-specific promoters. For example, silencing of Syk kinase in megakaryocytes and, subsequently, platelets can be achieved by using a simian or human immunodeficiency virus type 1 -based, self-inactivating lentiviral vector harbouring a glycoprotein Iba promoter and an interfering or inhibiting nucleic acid molecule or a nucleic acid molecule encoding an e.g. proteinaceous inhibitor. Methods for targeted transfection of cells and suitable vectors are known in the art, see for example Sambrook and Russel ("Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001); Ohmori T. ef al. Arterioscler Thromb Vase Biol. 2007 Oct;27(10):2266-72; Lavenu- Bombled C. ef al. Stem Cells. 2007 Jun;25(6):1571-7; Ohmori T. et al. FASEB J. 2006 Jul;20(9):1522-4; Yasui K. et al. Microbes Infect. 2005 Feb;7(2):240-7). Incorporation of the nucleic acid molecule encoding the inhibitor or directly affecting Syk kinase transcription (siRNA, shRNA) into an expression vector enables the selective suppression of Syk kinase expression in megakaryoyctes and platelets.

These inhibitors can be administered to the subject by any method available and suitable, including e.g. orally, intravenously, intradermally, subcutaneously, intramuscularly, intraperitoneally, topically (such as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray. Oral administration is particularly preferred, especially when the inhibitor is ibrutinib. The dosage regimen can be determined by the attending physician, based on clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The effective amount for a given situation will readily be determined by routine experimentation and is within the skills and judgement of the ordinary clinician or physician. For example, if the inhibitor is the sole compound, the effective daily dose, for example of fostaminib (R788), can be in the range of about 2x 75 mg to 2x 175 mg per person (see e.g. Podolanczuk A. et al. Blood (2009) 113:3154-60), although, as noted above, this will be subject to therapeutic discretion. The particular amounts, as well as the corresponding adjustments in case more than one compound is to be administered, may be determined by conventional tests which are well known to the person skilled in the art. Administration may be once as a single dose or as repeat administrations. The interval time and amount of repeats required can be determined by the skilled person without further ado. Progress can be monitored by periodic assessment. The inhibitor of the invention may be administered locally or systemically. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like

The term "arterial thrombosis" relates to thrombosis that develops in an artery, as opposed to venous thrombosis, which develops in a vein. Arterial thrombosis is generally caused by injury of a healthy artery, and the formation of a platelet- and fibrin-rich thrombus is essential for sealing the wall defect and stopping blood loss. In contrast the specific form of arterial thrombosis atherothrombosis is caused by the rupture of atherosclerotic plaques, or, less frequently, by erosion of the endothelium covering the atherosclerotic plaque. Thrombus formation is triggered by atherosclerotic plaque-derived thrombogenic substances that set in motion an avalanche of aggregating platelets and fibrin formation.

Atherosclerosis is a chronic disease of the arterial wall, and atherosclerotic plaques develop during a life-time as a consequence of continuous lipid deposition, inflammatory and fibrotic processes in the intima of the arterial wall. Whereas in healthy arteries the intima consists only of a thin inner layer of extracellular matrix, the intima of atherosclerotic plaques is drastically thickened. They consist of lipids (largely oxidised), macrophages, smooth muscle cells, their necrotic cell debris, calcium and extracellular matrix. Virchow coined the term "atheroma" for this material. Collagens of various types accumulate in atherosclerotic plaques and they differ structurally from collagens of healthy connective tissue (van Zanten, G.H. et al. J Clin Invest (1994), 93(2):615-32; Katsuda, S. and T. Kaji, J Atheroscler Thromb (2003). 10(5): 267-74; Penz et al. FASEB Journal 2005; 19(7):898-909). They also contain advanced glycation end products (Monnier VM et al N Engl J Med (1986) 314: 403-8; Sell et al Arch Biochem Biophys (2010) 493: 192-206) which may alter their platelet reactivity. The fundamentally different properties of atherosclerotic plaques compared to the intima of healthy arteries explain their different thrombogenicity: Atherosclerotic plaques are more thrombogenic than healthy arteries. Compared to normal arteries, platelet deposition onto human atherosclerotic coronary arteries is increased (van Zanten, G.H. et al. J Clin Invest. (1994), 93(2):615-32), and in mice the thrombotic response to injured carotid atherosclerotic arteries is much higher than to injured healthy carotid arteries (Hechler, B. and C. Gachet, Thromb Haemost (2011 ) 105 Suppl 1 : S3-12).

Therefore the term "atherothrombosis" refers to an arterial thrombosis that develops as a consequence of the rupture of atheroma or after erosion of the endothelium covering the atherosclerotic plaque. In atherothrombosis erosion or rupture of vulnerable atherosclerotic plaques in coronary, extra- or intracranial (carotis or cerebral arteries, respectively) and peripheral (for example femoral) arteries exposes material that arrests circulating platelets and triggers thrombosis. The arterial thrombus can either occlude the artery, or the thrombus can detach, embolise and occlude the vessels (arteries, arterioles) downstream. Occlusion of the arterial circulation cuts blood supply to the tissue, and can cause ischemia and infarction of almost any organ in the body, dependent on the localisation of the thrombi or thrombo- emboli, most commonly acute coronary syndrome, myocardial infarction and ischemic stroke, but also peripheral arterial occlusive disease (PAOD). Atherothrombosis, if not prevented by antiplatelet therapy, occurs also after percutaneous coronary intervention, and can accelerate neointima formation, restenosis and stent thrombosis.

Compounds which inhibit Syk activity are known to inhibit platelets. Syk has been shown to play a key role in the activation of human and mouse platelets by GPVI (collagen receptor), by GPIb (von Willebrand factor receptor), and the outside-in signalling of the integrin α2β1 (collagen receptor) and the integrin allbp3 (fibrinogen receptor) (Spalton JC et al. J Thromb Haemost (2009) 7(7): 1192-1199; Gardiner EE et al. Platelets (2010) 21(4): 244-252; Stegner D et al. Arterioscler Thromb Vase Biol (2014) 34(8): 1615-1620). Accordingly various Syk inhibitors have been found to inhibit collagen-induced human and mouse platelet aggregation under static conditions, platelet deposition onto collagen under flow, and shear stress-induced platelet thrombus formation (Spalton JC et al. J Thromb Haemost (2009) 7(7): 1192-1199; Andre P et al. Blood (2011 ) 118(18): 5000-5010; Speich HE et al. Circulation (2008)118, Suppl.2: S408-S409). In accordance with these findings Syk inhibitors such as PRT060318 provided protection from arterial and venous thrombosis induced by vascular injury in various animal models in vitro and ex vivo (Andre P et al. Blood (2011 ) 118(18): 5000-5010) . Recently the novel oral Syk inhibitor, BI1002494, was shown to protect mice from cerebral arterial thrombosis and brain infarction. The authors used a model in which focal cerebral ischemia was induced by advancing a nylon monofilament through the carotid artery up to the origin of the middle cerebral artery (transient middle cerebral artery occlusion) (van Eeuwijk et al. Arterioscler Thromb Vase Biol (2016) 36:1247-1253). However, in none of the previous studies the effect of Syk inhibitors on atherothombosis has been studied. In accordance with the present invention, it was found that an inhibitor of Syk kinase, more specifically Syk inhibitor II (CAS 227449-73-2), a reversible Syk kinase inhibitor, inhibited plaque and collagen- triggered platelet under static and flow conditions in vitro, whereas TRAP-induced platelet aggregation and ATP-secretion relevant in homeostasis was partially preserved even at high inhibitor concentrations. Surprisingly, the effects of PD173952 (a Src- family kinase inhibitor) and Syk inhibitor II on plaque- and collagen-induced platelet aggregation under arterial flow revealed functionally relevant differences between the two tyrosine kinase inhibitors. In contrast to PD173952, Syk inhibitor II showed a better inhibition of platelet aggregate formation onto plaque homogenate and plaque tissue sections than onto collagen fibers at all concentrations tested. Collagen is a key component of healing wounds. Collagens, located in the matrix underlying vascular endothelial cells, are not exposed to flowing blood. After injury, however, blood will flow directly over subendothelial structures including connective tissue that contains a high percentage of collagen. Thus collagen is ideally situated to initiate hemostasis. There is ample evidence that collagen is one of the major activators of the platelet response after injury. Collagen is the only matrix protein which supports both platelet adhesion and complete activation. When collagen becomes exposed to flowing blood, platelets rapidly adhere, spread, become activated and begin to form an aggregate. It was surprisingly found that the two Syk inhibitors Syk inhibitor II and R406, but not the inhibitor PD173952 of the upstream Src-family kinases were platelet- inhibiting compounds that specifically targets atherosclerotic plaque-triggered platelet activation with high efficacy while leaving collagen-induced platelet activation under arterial flow essentially intact. The atherosclerotic plaque-selective effect was observed at low concentrations of these inhibitors. Thus it is to expect that the doses of Fostamatinib (R788), the oral prodrug of R406, which need to be applied to inhibit atherosclerotic plaque-induced platelet activation under arterial flow are lower than the concentrations which inhibit collagen- induced platelet activation under arterial flow. Based on the pharmacokinetics of fostamatinib in a phase I study (Baluom et al., Br J Clin Pharmacol 76(1): 78-88) and two previous clinical studies (Weinblatt ME et al., N Engl J Med 2010;363:1303-12; Podolanczuk A et al., Blood (2009) 113:3154-60) low doses of 50-75mg twice daily or 100-150 mg once daily are expected to advantageously provide the selective inhibition of atherosclerotic plaque-induced platelet activation. Therefore, in a preferred embodiment the inhibitor or the method of the invention R406 is used/administered at low doses of 50-75mg twice daily or 100-150 mg once daily.

A new Syk kinase inhibitor is Entospletinib (GS-9973), a recently developed selective oral small molecule Syk-inhibitor. Its pharmacokinetics and safety have been tested in a phase I trial (Ramathan S et al Clin Drug Invest (2017) 37(2): 195-205), and a phase 2 trial showed clinical activity in CLL (Sharman J Blood (2015) 125(15): 2336-2343). Entospletinib shows by far greater selectivity for Syk than R406 (Sharman J Blood (2015) 125(15): 2336-2343. In accordance with the present invention, it was found that Entospletinib (GS-9973) dose- dependently inhibited plaque-stimulated platelet aggregation under static conditions in vitro with an IC50 of about 1 Μ, whereas TRAP-, ADP and AA-induced platelet aggregation were much less inhibited. After a loading dose of twice 600mg the first day to achieve maximal protection from plaque-induced platelet activation, low doses of 200 to 400mg twice daily are expected to provide the selective inhibition of atherosclerotic plaque-induced platelet activation. Therefore, in a preferred embodiment the inhibitor or the method of the invention Entospletinib is used/administered at twice 600mg the first day, and 200 to 400mg twice daily subsequently.

Hemostasis is the process which causes bleeding to stop, meaning to keep blood within a damaged blood vessel. It is the first stage of wound healing. Hence, the Syk inhibitor II and R406 advantageously treat atherothrombosis without impairing the stop of bleeding and wound healing. By contrast, the currently used standard dual anti-platelet therapy of atherosclerosis with aspirin and a P2Y12-antagonist affects hemostatic platelet responses and thereby increases bleeding risk and has a limited efficacy to inhibit atherosclerotic plaque-induced platelet thrombus formation.

Src-family and Syk tyrosine kinases are early signaling steps downstream of GPVI activation, and in platelets the Src kinases Fyn and Lyn are upstream of Syk. Under static conditions, platelet aggregation by plaque and collagen fibers is entirely dependent on platelet GPVI activation and the difference in inhibition of plaque and collagen- induced platelet aggregation between the two inhibitors was found to be marginal. However, the difference observed between the effect on plaque- and collagen-stimulated platelet aggregation under arterial flow conditions might be explained with the observation that the platelet collagen receptor integrin α2β1 plays an important role in platelet adhesion to collagen during arterial flow over isolated collagen fibres, which resembles the situation in healthy blood vessels, where integrin α2β1 synergizes with GPVI in platelet aggregation to collagen. In contrast, platelet aggregation onto plaque under arterial flow exclusively depends on platelet GPVI but not on integrin α2β1 (Penz et al. FASEB Journal 2005; 19(7):898-909; Schulz et al. Basic Res Cardiol. 2008;103:356-367). Src kinases signal also downstream of integrin α2β1 and mediate platelet spreading on collagen (Inoue O. et al. J Cell Biol. 2003;160:769-780). This might explain the superior inhibition of collagen-induced aggregate formation under flow by PD173952 as compared to Syk inhibitor II. This interpretation is, however, at variance with the demonstration of PD173952 resistant integrin α2β1 -mediated stable platelet adhesion to collagen under flow (Auger JM. et al. Faseb J. 2005;19:825-827), and also with the findings that immobilized GFOGER peptide binding to the integrin α2β1 binding sites activates not only Src-family, but also Syk kinases (for review see Nieswandt B. and Watson SP. Blood. 2003;102:449-461). Thus the underlying mechanism for the superior inhibition of plaque- induced platelet thrombus formation by Syk inhibitor II over collagen as compared to PD173952 remains intriguing.

The results provided in the appended examples show that Syk inhibitor II expressed full potency and nearly complete plaque selectivity already at 2 μΜ both for plaque homogenate and plaque tissue (see Figs. 3 and 6 for comparison). GS-9973 expressed full potency for plaque-induced platelet aggregation at 5μΜ. Further, Syk inhibitor II inhibited atherosclerotic plaque-induced platelet aggregation in both atherothrombosis models, i.e. in plaque homogenate as well as in plaque tissue sections. R406 at 15μΜ showed plaque- selective inhibition under flow (see Fig. 11). It is expected that GS-9973 at 5μΜ also shows plaque- selective inhibition under flow. Syk inhibition by Syk inhibitor II and R406 suppressed plaque- triggered platelet thrombus formation more potently than previously observed with aspirin plus P2Y12 receptor antagonists (Penz SM. et al. Thromb Haemost. 2007;97:435-443; Jamasbi J. et al. J Am Coll Cardiol. 2015;65:2404-2415), the established dual anti-platelet therapy.

Overall, the data presented herein show that targeting Syk holds promise to be a potent and selective therapy against atherothrombosis, that is also more efficient and safer than standard dual platelet therapy with aspirin and P2Y12 antagonists. Furthermore, such an approach would circumvent the drawbacks of an antibody based intervention, such as potentially encountered when using anti-GPVI-antibodies.

The present invention further relates to a method of treating and/or preventing atherothrombosis comprising administering a pharmaceutically effective amount of an inhibitor of Syk kinase to a subject in need thereof.

All definitions and preferred embodiments provided herein with regard to the inhibitor of the invention apply mutatis mutandis to this method of the invention, unless specifically detailed otherwise.

In a preferred embodiment of the inhibitor or the method of the invention, the inhibitor is a small molecule, an antibody or antibody mimetic, an aptamer, a siRNA, a shRNA, a miRNA, a ribozyme, or an antisense nucleic acid molecule.

The "small molecule" as used herein is preferably an organic molecule. Organic molecules relate or belong to the class of chemical compounds having a carbon basis, the carbon atoms linked together by carbon-carbon bonds. The original definition of the term organic related to the source of chemical compounds, with organic compounds being those carbon- containing compounds obtained from plant or animal or microbial sources, whereas inorganic compounds were obtained from mineral sources. Organic compounds can be natural or synthetic. The organic molecule is preferably an aromatic molecule and more preferably a heteroaromatic molecule. In organic chemistry, the term aromaticity is used to describe a cyclic (ring-shaped), planar (flat) molecule with a ring of resonance bonds that exhibits more stability than other geometric or connective arrangements with the same set of atoms. Aromatic molecules are very stable, and do not break apart easily to react with other substances. In a heteroaromatic molecule at least one of the atoms in the aromatic ring is an atom other than carbon, e.g. N, S, or O.

The heteroaromatic molecule is preferably a molecule comprising an azole motif (i.e. azole, di-azole, tri-azole or tetra-azole motif) and/or an azine motif (i.e. azine, di-azine, or tri-azine motif). The heteroaromatic molecule is more preferably a molecule comprising a pyridine (azine), pyrimidine (diazine), triazine, azine, pyrazole or imidiazole motif. The heteroaromatic molecule is even more preferably a molecule comprising a pyrimidine (diazine), pyrazole or imidiazole motif. The heteroaromatic molecule is most preferably a molecule comprising a 2- aminopyrimidine, 4-aminopyrimidine, or 2,4-diaminopyrimidine motif, wherein heteroaromatic molecules comprising a 2,4-diaminopyrimidine motif are particularly preferred. For all above- described organic molecules the molecular weight is preferably in the range of 200 Da to 1500 Da and more preferably in the range of 300 Da to 1000 Da.

Alternatively, the "small molecule" in accordance with the present invention may also be an inorganic compound. Inorganic compounds are derived from mineral sources and include all compounds without carbon atoms (except carbon dioxide, carbon monoxide and carbonates). Preferably, the small molecule has a molecular weight of less than about 2000 amu, or less than about 1000 amu such as less than about 500 amu, and even more preferably less than about 250 amu. The size of a small molecule can be determined by methods well-known in the art, e.g., mass spectrometry. The small molecules may be designed, for example, based on the crystal structure of the target molecule, where sites presumably responsible for the biological activity, can be identified and verified in in vivo assays such as in vivo high-throughput screening (HTS) assays.

A non-limiting example of a small molecule inhibitor of Syk is Syk inhibitor II (also referenced under CAS 227449-73-2), which has been employed in the examples provided herein below. Syk inhibitor II is a pyrimidine-5-carboxamide derivative, and selectively inhibits Syk in in vitro kinase assays, with an IC₅₀ of 41 nM, and the passive cutaneous anaphylaxis reaction in mice, with an ID₅₀ of 13 mg/kg following subcutaneous administration (Syk inhibitor II is compound 9a in Hisamichi et al. Bioorganic & Medicinal Chemistry (2005) 13 :4936-4951). Another small molecule inhibitor, PRT-060318 (PRT318, also referred to as P142-76) is a derivative of pyrimidine-5-carboxamide (U.S. patent number 6432963) and, as Syk inhibitor II, and has been shown to prevent heparin-induced thrombocytopenia and thrombosis in a transgenic mouse model after intraperitoneal application (Reilly MP et al. Blood (2011 ) 117: 2241-2246.

Other examples, such as R406 (Braselmann S et al. J Pharmacol Exp Ther (2006) 319(3):998-1008), and R788 (fostamatinib) (Sheridan C Nat Biotechnol (2008) 26(2): 143- 4.42)) the water-soluble prodrug of the biologically active R406, which is applied orally, have also been described. A new compound is the oral Syk inhibitor BI1002494 which is more specific than previous Syk inhibitors such as R406 (van Eeuwijk et al. Arterioscler Thromb Vase Biol (2016) 36:1247-1253). A further very specific oral Syk oral small molecule inhibitor is Entospletinib (GS-9973). Entospletinib shows by far greater selectivity for Syk than R406. Whereas Entospletinib is Syk-selective (Syk Kd of 7.6 nM, with only 1 other kinase inhibited with a Kd < 100 nM), R406 is nonselective (Syk Kd of 15 nM) with 25 kinases with Kd < 15 nM and 54 additional kinases with Kd < 100 nM inhibited) (Sharman J β/oocf (2015) 125(15): 2336-2343. Further compounds such as e.g. R343, piceatannol, R112 and P505-15 have been reviewed in e.g. Geahlen RL, Trends Pharmacol Sci. 2014 August; 35(8): 414-422. Furthermore, Lucas MC and Tan SL. Future Med Chem. 2014;6(16):1811-27 as well as review further inhibitors and provide an overview over relevant patent applications and scientific publications disclosing a diverse range of different small molecule Syk inhibitors. A summary of exemplary (i.e. non-limiting) small molecule inhibitors of Syk, which can be employed in accordance with the present invention, are provided in Table 1 below. In addition, Thorarensen and Kaila (Thorarensen A and Kaila N. Pharm Pat Anal. 2014 Sep; 3(5):523-41) reviewed patent applications published during 2011 to 2013. Also these inhibitors discussed in Thorarensen and Kaila may be employed in accordance with the present invention.

The term "antibody" as used in accordance with the present invention comprises, for example, polyclonal or monoclonal antibodies. Furthermore, also derivatives or fragments thereof, which still retain the binding specificity to the target, e.g. Syk, are comprised in the term "antibody". Antibody fragments or derivatives comprise, inter alia, Fab or Fab' fragments, Fd, F(ab')₂, Fv or scFv fragments, single domain V_H or V-like domains, such as VhH or V-NAR-domains, as well as multimeric formats such as minibodies, diabodies, tribodies or triplebodies, tetrabodies or chemically conjugated Fab'-multimers (see, for example, Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 198; Harlow and Lane "Using Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, 1999; Altshuler EP, Serebryanaya DV, Katrukha AG. 2010, Biochemistry (Mosc)., vol. 75(13), 1584; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 1126).

The term "antibody" also includes embodiments such as chimeric (human constant domain, non-human variable domain), single chain and humanised (human antibody with the exception of non-human CDRs) antibodies.

Various techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane (1988) and (1999) and Altshuler et al., 2010, loc. cit. Thus, polyclonal antibodies can be obtained from the blood of an animal following immunisation with an antigen in mixture with additives and adjuvants and monoclonal antibodies can be produced by any technique which provides antibodies produced by continuous cell line cultures. Examples for such techniques are described, e.g. in Harlow E and Lane D, Cold Spring Harbor Laboratory Press, 1988; Harlow E and Lane D, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999 and include the hybridoma technique originally described by Kohler and Milstein, 1975, the trioma technique, the human B-cell hybridoma technique (see e.g. Kozbor D, 1983, Immunology Today, vol.4, 7; Li J, et al. 2006, PNAS, vol. 103(10), 3557) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, Alan R. Liss, Inc, 77-96). Furthermore, recombinant antibodies may be obtained from monoclonal antibodies or can be prepared de novo using various display methods such as phage, ribosomal, mRNA, or cell display. A suitable system for the expression of the recombinant (humanised) antibodies may be selected from, for example, bacteria, yeast, insects, mammalian cell lines or transgenic animals or plants (see, e.g., US patent 6,080,560; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 11265). Further, techniques described for the production of single chain antibodies (see, inter alia, US Patent 4,946,778) can be adapted to produce single chain antibodies specific for an epitope of Syk. Surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies.

As used herein, the term "antibody mimetics" refers to compounds which, like antibodies, can specifically bind antigens, such as Syk in the present case, but which are not structurally related to antibodies. Antibody mimetics are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa. For example, an antibody mimetic may be selected from the group consisting of affibodies, adnectins, anticalins, DARPins, avimers, nanofitins, affilins, Kunitz domain peptides and Fynomers . These polypeptides are well known in the art and are described in further detail herein below.

The term "affibody", as used herein, refers to a family of antibody mimetics which is derived from the Z-domain of staphylococcal protein A. Structurally, affibody molecules are based on a three-helix bundle domain which can also be incorporated into fusion proteins. In itself, an affibody has a molecular mass of around 6kDa and is stable at high temperatures and under acidic or alkaline conditions. Target specificity, i.e. against Syk, is obtained by randomisation of 13 amino acids located in two alpha-helices involved in the binding activity of the parent protein domain (Feldwisch J, Tolmachev V.; (2012) Methods Mol Biol. 899:103-26).

The term "adnectin" (also referred to as "monobody"), as used herein, relates to a molecule based on the 10th extracellular domain of human fibronectin III (10Fn3), which adopts an Ig- like β-sandwich fold of 94 residues with 2 to 3 exposed loops, but lacks the central disulphide bridge (Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Adnectins with the desired target specificity, i.e. against Syk, can be genetically engineered by introducing modifications in specific loops of the protein.

The term "anticalin", as used herein, refers to an engineered protein derived from a lipocalin (Beste G, Schmidt FS, Stibora T, Skerra A. (1999) Proc Natl Acad Sci U S A. 96(5): 1898- 903; Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Anticalins possess an eight-stranded β-barrel which forms a highly conserved core unit among the lipocalins and naturally forms binding sites for ligands by means of four structurally variable loops at the open end. Anticalins, although not homologous to the IgG superfamily, show features that so far have been considered typical for the binding sites of antibodies: (i) high structural plasticity as a consequence of sequence variation and (ii) elevated conformational flexibility, allowing induced fit to targets with differing shape.

As used herein, the term "DARPin" refers to a designed ankyrin repeat domain (166 residues), which provides a rigid interface arising from typically three repeated β-turns. DARPins usually carry three repeats corresponding to an artificial consensus sequence, wherein six positions per repeat are randomised. Consequently, DARPins lack structural flexibility (Gebauer and Skerra, 2009). The term "avimer", as used herein, refers to a class of antibody mimetics which consist of two or more peptide sequences of 30 to 35 amino acids each, which are derived from A- domains of various membrane receptors and which are connected by linker peptides. Binding of target molecules occurs via the A-domain and domains with the desired binding specificity, i.e. for Syk, can be selected, for example, by phage display techniques. The binding specificity of the different A-domains contained in an avimer may, but does not have to be identical (Weidle UH, et al., (2013), Cancer Genomics Proteomics;10(4): 155-68).

A "nanofitin" (also known as affitin) is an antibody mimetic protein that is derived from the DNA binding protein Sac7d of Sulfolobus acidocaldarius. Nanofitins usually have a molecular weight of around 7kDa and are designed to specifically bind a target molecule, such as e.g. Syk, by randomising the amino acids on the binding surface (Mouratou B, Behar G, Paillard- Laurance L, Colinet S, Pecorari F., (2012) Methods Mol Biol.; 805:315-31 ).

The term "affilin", as used herein, refers to antibody mimetics that are developed by using either gamma-B crystalline or ubiquitin as a scaffold and modifying amino-acids on the surface of these proteins by random mutagenesis. Selection of affilins with the desired target specificity, i.e. against Syk, is effected, for example, by phage display or ribosome display techniques. Depending on the scaffold, affilins have a molecular weight of approximately 10 or 20kDa. As used herein, the term affilin also refers to di- or multimerised forms of affilins (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68). A "Kunitz domain peptide" is derived from the Kunitz domain of a Kunitz-type protease inhibitor such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP) or tissue factor pathway inhibitor (TFPI). Kunitz domains have a molecular weight of approximately 6kDA and domains with the required target specificity, i.e. against Syk, can be selected by display techniques such as phage display (Weidle et al., (2013), Cancer Genomics Proteomics; 10(4): 55-68).

As used herein, the term "Fynomer^®" refers to a non-immunoglobulin-derived binding polypeptide derived from the human Fyn SH3 domain. Fyn SH3-derived polypeptides are well-known in the art and have been described e.g. in Grabulovski et al. (2007) JBC, 282, p. 3196-3204, WO 2008/022759, Bertschinger et al (2007) Protein Eng Des Sel 20(2):57-68, Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255, or Schlatter et al. (2012), MAbs 4:4, 1-12).

Aptamers are nucleic acid molecules or peptide molecules that bind a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches. Aptamers can be used for both basic research and clinical purposes as macromolecular drugs. Aptamers can be combined with ribozymes to self-cleave in the presence of their target molecule. These compound molecules have additional research, industrial and clinical applications (Osborne et. al. (1997), Current Opinion in Chemical Biology, 1 :5-9; Stull & Szoka (1995), Pharmaceutical Research, 2, 4:465-483).

Nucleic acid aptamers are nucleic acid species that normally consist of (usually short) strands of oligonucleotides. Typically, they have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms.

Peptide aptamers are usually peptides or proteins that are designed to interfere with other protein interactions inside cells. They consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody's (nanomolar range). The variable peptide loop typically comprises 10 to 20 amino acids, and the scaffold may be any protein having good solubility properties. Currently, the bacterial protein Thioredoxin-A is the most commonly used scaffold protein, the variable peptide loop being inserted within the redox- active site, which is a -Cys-Gly-Pro-Cys- loop in the wild protein, the two cysteins lateral chains being able to form a disulfide bridge. Peptide aptamer selection can be made using different systems, but the most widely used is currently the yeast two-hybrid system.

Aptamers offer the utility for biotechnological and therapeutic applications as they offer molecular recognition properties that rival those of the commonly used biomolecules, in particular antibodies. In addition to their discriminatory recognition, aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications. Non-modified aptamers are cleared rapidly from the bloodstream, with a half-life of minutes to hours, mainly due to nuclease degradation and clearance from the body by the kidneys, a result of the aptamers' inherently low molecular weight. Unmodified aptamer applications currently focus on treating transient conditions such as blood clotting, or treating organs such as the eye where local delivery is possible. This rapid clearance can be an advantage in applications such as in vivo diagnostic imaging. Several modifications, such as 2'-fluorine-substituted pyrimidines, polyethylene glycol (PEG) linkage, fusion to albumin or other half life extending proteins etc. are available to scientists such that the half-life of aptamers can be increased for several days or even weeks. The term "peptide" as used herein describes a group of molecules consisting of up to 30 amino acids, whereas the term "polypeptide" (also referred to as "protein") as used herein describes a group of molecules consisting of more than 30 amino acids. The group of peptides and polypeptides are referred to together by using the term "(poly)peptide".

In accordance with the present invention, the term "small interfering RNA (siRNA)", also known as short interfering RNA or silencing RNA, refers to a class of 18 to 30, preferably 19 to 25, most preferred 21 to 23 or even more preferably 21 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g. as an antiviral mechanism or in shaping the chromatin structure of a genome. siRNAs naturally found in nature have a well defined structure: a short double-strand of RNA (dsRNA) with 2-nt 3' overhangs on either end. Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs. siRNAs can also be exogenously (artificially) introduced into cells to bring about the specific knockdown of a gene of interest. Essentially any gene for which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA. The double- stranded RNA molecule or a metabolic processing product thereof is capable of mediating target-specific nucleic acid modifications, particularly RNA interference and/or DNA methylation. Exogenously introduced siRNAs may be devoid of overhangs at their 3' and 5' ends, however, it is preferred that at least one RNA strand has a 5'- and/or 3'-overhang. Preferably, one end of the double-strand has a 3'-overhang from 1 to 5 nucleotides, more preferably from 1 to 3 nucleotides and most preferably 2 nucleotides. The other end may be blunt-ended or has up to 6 nucleotides 3'-overhang. In general, any RNA molecule suitable to act as siRNA is envisioned in the present invention. The most efficient silencing was so far obtained with siRNA duplexes composed of 21 -nt sense and 21 -nt antisense strands, paired in a manner to have a 2-nt 3'- overhang. The sequence of the 2-nt 3' overhang makes a small contribution to the specificity of target recognition restricted to the unpaired nucleotide adjacent to the first base pair (Elbashir et al. 2001 ). 2'-deoxynucleotides in the 3' overhangs are as efficient as ribonucleotides, but are often cheaper to synthesize and probably more nuclease resistant. Delivery of siRNA may be accomplished using any of the methods known in the art, for example by combining the siRNA with saline and administering the combination intravenously or intranasally or by formulating siRNA in glucose (such as for example 5% glucose) or cationic lipids and polymers can be used for siRNA delivery in vivo through systemic routes either intravenously (IV) or intraperitoneally (IP) (Fougerolles et al. (2008), Current Opinion in Pharmacology, 8:280-285; Lu et al. (2008), Methods in Molecular Biology, vol. 437: Drug Delivery Systems - Chapter 3: Delivering Small Interfering RNA for Novel W 201

21

Therapeutics).

A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the siRNA that is bound to it. si/shRNAs to be used in the present invention are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Suppliers of RNA synthesis reagents are Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK). Most conveniently, siRNAs or shRNAs are obtained from commercial RNA oligo synthesis suppliers, which sell RNA-synthesis products of different quality and costs. In general, the RNAs applicable in the present invention are conventionally synthesized and are readily provided in a quality suitable for RNAi. Further molecules effecting RNAi include, for example, microRNAs (miRNA). Said RNA species are single-stranded RNA molecules. Endogenously present miRNA molecules regulate gene expression by binding to a complementary mRNA transcript and triggering of the degradation of said mRNA transcript through a process similar to RNA interference. Accordingly, exogenous miRNA may be employed as an inhibitor of Syk after introduction into the respective cells.

A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyses a chemical reaction. Many natural ribozymes catalyse either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyse the aminotransferase activity of the ribosome. Non-limiting examples of well-characterised small self-cleaving RNAs are the hammerhead, hairpin, hepatitis delta virus, and in vitro- selected lead-dependent ribozymes, whereas the group I intron is an example for larger ribozymes. The principle of catalytic self-cleavage has become well established in recent years. The hammerhead ribozymes are characterised best among the RNA molecules with ribozyme activity. Since it was shown that hammerhead structures can be integrated into heterologous RNA sequences and that ribozyme activity can thereby be transferred to these molecules, it appears that catalytic antisense sequences for almost any target sequence can be created, provided the target sequence contains a potential matching cleavage site. The basic principle of constructing hammerhead ribozymes is as follows: A region of interest of the RNA, which contains the GUC (or CUC) triplet, is selected. Two oligonucleotide strands, each usually with 6 to 8 nucleotides, are taken and the catalytic hammerhead sequence is inserted between them. The best results are usually obtained with short ribozymes and target sequences.

A recent development, also useful in accordance with the present invention, is the combination of an aptamer, recognizing a small compound, with a hammerhead ribozyme. The conformational change induced in the aptamer upon binding the target molecule can regulate the catalytic function of the ribozyme.

The term "antisense nucleic acid molecule", as used herein, refers to a nucleic acid which is complementary to a target nucleic acid. An antisense molecule in accordance with the invention is capable of interacting with the target nucleic acid, more specifically it is capable of hybridizing with the target nucleic acid. Due to the formation of the hybrid, transcription of the target gene(s) and/or translation of the target mRNA is reduced or blocked. Standard methods relating to antisense technology have been described (see, e.g., Melani et al., Cancer Res. (1991 ) 51 :2897-2901 ).

In a more preferred embodiment of the inhibitor or the method of the invention, the inhibitor is syk inhibitor-ll, R406, Entospletinib, or an analogue thereof, preferably R788 (fostaminib).

Syk inhibitor II can be commercially obtained from e.g. Calbiochem under the order number 574712-1 MG and is also known as CAS 227449-73-2. It is a reversible and ATP-competitive inhibitor of Syk with an IC₅o of 41 nM (i.e. compound 9a in Hisamichi et al. Bioorganic & Medicinal Chemistry (2005) 13 :4936-4951).

In accordance with the present invention, the inhibitor can also be an analogue of syk inhibitor-ll. Such analogues include functional as well as structural analogues of syk inhibitor- ll. Non-limiting examples of such analogues of syk inhibitor-ll include R406 (Braselmann S et al J Pharmacol Exp Ther (2006) 319(3):998-1008) well as its water-soluble prodrug R788 (fostaminib) (Sheridan C Nat Biotechnol (2008) 26(2): 143-4.42). Another functional analogue of syk inhibitor-ll includes, without being limiting, PRT-060318 (Reilly MP. Blood 2011 117: 2241-2246). A comprehensive review of patent applications for Syk inhibitors published during 2011-2013 is provided by Thorarensen A and Kaila N (Pharm. Pat. Anal. 2014; 3(5), 523-541 ). In general the structural theme of the compounds in these applications is a traditional type I ATP competitive inhibitor with each organization having a different hinge-binding element. Disclosure of the full length crystal structure of Syk by Merck (Moretto AF et al Recent Pat. Inflamm. Allergy Drug Discov. 2012; 6(2), 97-120) and structure-based drug design (Padilla F et al J. Med. Chem. 2013; 56, 1677).have allowed to identify numerous potent Syk inhibitors with IC50 values of < 10nM in enzymatic assays (Thorarensen A and Kaila N, Pharm. Pat. Anal. 2014; 3(5), 523-541 ) Two key residues, Pro455 and Asn457, have been identified in the ADP ribose binding pocket, which are unique to Syk. The spatial proximity of Pro455 and Asn457 allowed the design of inhibitors that interacted with these two residues. All of these Syk inhibitors have been described in the art and exemplary inhibitors are summarised in Table 1.

For an application in human beings one preferred Syk kinase inhibitor is Entospletinib (GS- 9973), a recently developed selective oral small molecule Syk-inhibitor. Its pharmacokinetics and safety has been tested in a phase I trial (Ramathan S et al Clin Drug Invest (2017) 37(2): 195-205), and a phase 2 trial showed its clinical activity in CLL (Sharman J Blood (2015) 125( 5): 2336-2343). Entospletinib (GS-9973) shows by far greater selectivity for Syk than R406. Whereas Entospletinib is Syk-selective (Syk Kd of 7.6 nM, with only 1 other kinase inhibited with a Kd < 100 nM), R406 is nonselective (Syk Kd of 15 nM) with 25 kinases with Kd < 15 nM and 54 additional kinases with Kd < 100 nM inhibited) (Sharman J Blood (2015) 125(15): 2336-2343. Preferred oral dosages of Entospletinib to be used are, without being limiting, 600mg twice the first day (loading dose), and 200 to 400mg twice daily subsequently (maintenance dose) to ensure selectivity for inhibition of atherosclerotic plaque-induced platelet activation. In a further preferred embodiment of the inhibitor or the method of the invention, the inhibitor is comprised in a pharmaceutical composition, optionally further comprising a pharmaceutically acceptable carrier, excipient and/or diluent.

The term "pharmaceutical composition", as used herein, relates to a composition for administration to a patient, preferably a human patient. The pharmaceutical composition of the invention comprises at least one, such as at least two, e.g. at least three, in further embodiments at least four such as at last five of the above mentioned inhibitors. In cases where more than one inhibitor is comprised in the pharmaceutical composition it is understood that none of these inhibitors has any essentially inhibitory effect on the other inhibitors also comprised in the composition.

The composition may be in solid, liquid or gaseous form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s).

It is preferred that said pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient and/or diluent. Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, lubricants, binding agents, fillers, sterile solutions etc.

Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. All definitions and preferred embodiments provided herein above with regard to the administration of the inhibitor of the invention, such as routes of administration, preferred dosages as well as means of determining same, apply mutatis mutandis to the administration of the pharmaceutical composition of the present invention.

It is particularly preferred that said pharmaceutical composition comprises further agents known in the art to antagonize atherothrombosis. Since the pharmaceutical preparation of the present invention relies on the inhibitors referred to herein, it is preferred that those mentioned further agents are only used as a supplement, i.e. at a reduced dose as compared to the recommended dose when used as the only drug, so as to e.g. reduce side effects conferred by the further agents.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the patent specification, including definitions, will prevail.

Regarding the embodiments characterised in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise. Similarly, and also in those cases where independent and/or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1 , a dependent claim 2 referring back to claim 1 , and a dependent claim 3 referring back to both claims 2 and 1 , it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1 , of claims 4, 2 and 1 , of claims 4, 3 and , as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

The above considerations apply mutatis mutandis to all appended claims. To give a non- limiting example, the combination of claims 6, 5 and 3 is clearly and unambiguously envisaged in view of the claim structure. The same applies for example to the combination of claims 6, 5 and 4, etc.

The figures show: Figure 1. Effect of the Src-kinase inhibitor PD173952 on plaque-, collagen- and TRAP- induced platelet aggregation and ATP-secretion in blood under static conditions

Samples were pre-incubated for 3 minutes with solvent (DMSO, 0.1 %) or PD173952 before stimulation with (A) plaque (833 pg/ml), (B) collagen (0.5 pg/ml) or (C) TRAP (5 μΜ). Left, representative MEA tracings (PD173952, 10pM); the numbers in the tracings indicate cumulative aggregation values (AU*min) measured at 10 minutes. Right, dose-response curves of PD173952. Values are mean ± SD (n = 5), *: p<0.05; **: p<0.01 , ***: p<0.001 for secondary pair comparison to solvent control. (D) Effect of PD173952 on plaque-, collagen- and TRAP-induced ATP-secretion in blood. Blood samples were pre-incubated with solvent (DMSO, 0.1 %), PD173952 (20 μΜ), abciximab (20 pg/ml) alone or in combination with PD173952 before stimulation with plaque (833 pg/ml), collagen (0.5 pg/ml) and TRAP (5μΜ). The numbers are % of maximal ATP-secretion (control 100 %) measured 3 min after stimulation. Values are mean ± SD, (n = 4-5), *: p<0.05; ***: p<0.001 for secondary pair comparison as indicated by horizontal bars. Figure 2. Inhibition of plaque- and collagen-induced platelet deposition by PD173952 under flow. Left, representative micrographs show platelet coverage of plaque homogenate (A), plaque tissue sections (B) and collagen (C) at 0 min and 4 min after start of blood flow W 201

26

(shear 6007s). Right, the line diagrams show the effect of 10 μΜ PD173952 on plaque- and collagen-induced platelet deposition over time. Top right, inhibition of plaque-induced platelet deposition by PD173952 is shown at blown-up scale. Blood samples were pre-incubated with mepacrine for platelet labeling, and with solvent (0.1 % DMSO) or PD173952 (10 μΜ). Values are mean ± SD (n = 5), ^*: p<0.05; ^**: p<0.01 ; ^***: p<0.001 for comparison to control at full minutes.

Figure 3. Dose-response curves of PD173952 on inhibition of plaque- and collagen- induced platelet deposition under flow. Blood samples were pre-incubated with solvent (DMSO, 0.1%), or PD173952 (2, 5 or 10 μΜ), and platelet coverage was measured 4 min after start of flow (shear 600/s). Other details as in legend of Figure 3. Values are mean ± SD (n = 5). *: p<0.05 for secondary pair comparison of stimuli as indicated by vertical bar.

Figure 4. Effect of syk inhibitor II on plaque-, collagen- and TRAP-induced platelet aggregation and ATP-secretion in blood under static conditions. Samples were pre- incubated for 3 minutes with solvent (DMSO, 0.1 %) or syk inhibitor II before stimulation with (A) plaque (833 pg/ml), (B) collagen (0.5 pg/ml) or (C) TRAP (5 μΜ). Left, representative MEA tracings (syk inhibitor II, 5 μΜ); the numbers in the tracings indicate cumulative aggregation values (AlTmin) measured at 10 minutes. Right, dose-response curves of syk inhibitor II. Values are mean ± SD (n = 5), *^**: p<0.001 for secondary pair comparison to solvent control. (D) Effect of syk inhibitor II on plaque-, collagen- and TRAP- induced ATP- secretion in blood. Blood samples were pre-incubated with solvent (NaCI), syk inhibitor II (20 μΜ), abciximab (20 μg ml) alone or with syk inhibitor II for 3 min at 37°C before stimulation with plaque (A), collagen (B) and TRAP (C). For other details see legend of Fig. 1C. Values are mean ± SD (n = 4-5), ^*: p<0.05; ^**: p<0.01 for secondary pair comparison as indicated by horizontal bars.

Figure 5. Differential inhibition of plaque- and collagen-induced platelet deposition by syk inhibitor II under arterial flow. Left, representative micrographs show platelet deposition on plaque homogenate (A), plaque tissue sections (B) and collagen (C) at 0 min and 4 min after start of blood flow (shear 600/s). Right, the line diagrams show the effect of 2 μΜ syk inhibitor II (dissolved in NaCI) on the kinetic of plaque- and collagen-induced platelet coverage. Top right, almost complete inhibition of plaque-induced platelet coverage by syk inhibitor II is shown at blown-up scale. Other details as in legend of Fig. 2. Values are mean ± SD (n = 5). ^*: p<0.05; ^**: p<0.01 , *^**: p<0.001 for comparison to control at full minutes. Figure 6. Dose-response curves of syk inhibitor II on inhibition of plaque- and collagen-induced platelet coverage under arterial flow. Blood samples were pre- incubated without or with 2, 5 or 10 μΜ syk inhibitor II, and platelet coverage was measured 4 min after start of flow (shear 600/s). Other details are as provided in the legend of Fig. 5. Values are mean ± SD (n = 5). ^**: p<0.01 , **^*: p<0.001 for secondary pair comparison of stimuli as indicated by vertical bar.

Figure 7. Plaque- and collagen-induced platelet deposition in the presence of DMSO under arterial flow. Blood was pre-incubated with NaCI or DMSO (0.1 %) for 5 minutes at 37°C before start of flow (shear 600/s). Values are mean ± SD (n = 5).

Figure 8. Effect of 2μΜ and 5 μΜ PD173952 on the kinetics of plaque- and collagen- induced platelet deposition under arterial flow. For details see legend of Fig.3. Values are mean ± SD (n = 5). ^*: p<0.05; ^**: p<0.01 for secondary pair comparisons at 4 min as indicated by vertical bars.

Figure 9. Effect of 5 and 10 μΜ syk inhibitor II on the kinetics of plaque- and collagen- induced platelet deposition under arterial flow (shear rate 600/s). Inhibition of platelet aggregation on plaque homogenate and plaque tissue by syk inhibitor II is also shown at blown-up scale. For details see legend of Fig.3. Values are mean ± SD (n = 5). *: p<0.05; ***: p<0.001 for secondary pair comparisons at 4 min as indicated by vertical bars.

Figure 10. Effect of the Syk inhibitor R406 on plaque-, collagen-, ADP and TRAP- induced platelet aggregation in blood under static conditions

A. Dose-response curve. Samples were pre-incubated for 3 minutes with solvent (DMSO, 0.1 %; control) or increasing concentrations of R406 (dissolved in DMSO, 0.1%) before stimulation with plaque (833 pg/ml) (mean ± SD, n=6) . (B) Bar diagram. R406 (15μΜ) inhibits plaque- and collagen (0.5 pg/ml) -induced platelet aggregation more than TRAP (5μΜ)- and ADP- 5μΜ) stimulated aggregation. Values are mean ± SD (n= 4-6), **: p<0.01 , ***: p<0.001 for secondary pair comparison to solvent control.

Figure 11. Differential inhibition of plaque- and collagen-induced platelet deposition by R406 under arterial flow, (shear rate 600/s). Blood samples were pre-incubated for 10 min with DiOC6 for platelet labeling, and with solvent (0.1 % DMSO; control) or increasing concentrations of R406. Values are mean ± SD (n = 4). *: p<0.05; *^**: p<0.001 for secondary pair comparisons at 4 min as indicated by vertical bars. W

28

Figure 12. Effect of Entospletinib (GS-9973) on plaque-, collagen-, TRAP-, ADP, and arachidonic acid (AA) induced platelet aggregation in blood under static conditions A. Dose-response curve. Samples were pre-incubated for 15 minutes with solvent (DMSO, 0.1 %; control) or increasing concentrations of GS-9973 (dissolved in DMSO, 0.1%) before stimulation with plaque (833 pg/ml) (mean ± SD, n=3) (B) Bar diagram. GS-9973 (10μΜ) inhibits plaque- and collagen (0.5 pg/ml) -induced platelet aggregation more than TRAP (5μΜ) -, ADP (5μΜ)- or AA (0.6mM)- stimulated aggregation. Values are mean ± SD (n= 3).

The examples illustrate the invention.

Example 1 : Materials and methods

Reagents

Syk inhibitor II (catalog #574712) was obtained from Calbiochem (San Diego, USA). PD173952 (catalog #PZ0113), dimethyl sulfoxide (DMSO), albumin from human serum (fatty acid free), mepacrine (quinacrine dihydrochloride) came from Sigma-Aldrich (Taufkirchen, Germany). Abciximab (ReoPro^®, catalog #402716) was obtained from Janssen Biologies B.V. (Leiden, the Netherlands). R406 and GS-9973 were from Selleckchem (Houston, TX, USA). (Horm, catalog #4047562) and diluting solvent were purchased from Takeda (Linz, Austria). Thrombin receptor activating peptide (TRAP, catalog #1045040) was obtained from Bachem AG (Bubendorf, Switzerland). PBS (Dulbecco's Phosphate Buffered Saline) was from Gibco (Grand Island, New York, USA). Recombinant lepirudin (Refludan^®) came from Celgene (Windsor, UK). Luciferin-Luciferase reagent, ATP standard was from Chrono-Log Corporation (Havertown, USA). Tissue-Tek® as embedding medium for cryotomy came from Sakura (Alphen aan den Rijn, the Netherlands).

Stock solutions

Syk inhibitor II was first dissolved in distilled water at a concentration of 4 mM. Physiological saline was subsequently added in a 1 :1 ratio. Using this procedure precipitate formation is avoided. Aliquots of the stock solutions of syk inhibitor II (2 mM) were stored at -20°C. PD173952, GS-9973 and R406 were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mM. Aliquots (50 pi) were stored at -20°C. Before each experiment, dilutions were made in DMSO (1 , 2, 5, 10, 20 mM) to obtain final concentrations in blood of 1 μΜ, 2 μΜ, 5 μΜ, 10 μΜ for PD173952 and GS-9973, 2.5 μΜ, 5 μΜ, 10 μΜ, 15μΜ and 20μΜ for R406, and respectively. The final concentration of DMSO in blood was 0.1 %. Collagen (1000 pg/ml) suspension was diluted 1 :10 with diluting solvent (100 pg/ml). TRAP was dissolved in 0.9% NaCI at a concentration of 500 μΜ. Human carotid atherosclerotic plaques

Atherosclerotic tissue specimens were donated by patients who underwent endarterectomy for high-grade carotid artery stenosis as described previously (Reininger AJ. et al. J Am Coll Cardiol 2010; 55: 1147-58; Penz S. ef al. Faseb J 2005; 19: 898-909; Brandl R. ef al. Circulation 1997; 96: 3360-8). Patient consent was obtained as approved by the Ethics Committee of the Faculty of Medicine of the University of Munich. The atheromatous plaques were carefully dissected under sterile conditions from other regions of the atherosclerotic tissue specimens (Reininger AJ. et al. J Am Coll Cardiol 2010; 55: 1147-58; Penz S. ef al. Faseb J 2005; 19: 898-909; Brandl R. er a/. Circulation 1997; 96: 3360-8). The plaques were further processed to obtain homogenates and tissue sections. For plaque homogenates, the plaque specimens were weighed, homogenized in buffer (150 mM NaCI, 1 mM EDTA, pH 7.4) adjusted to a concentration of 100 mg wet weight/ml and stored at -80°C. Plaque homogenates from 5 patients were pooled. Aliquots of pooled plaque homogenates were diluted with PBS for the static tests in the Multiplate® analyzer and in the lumi-aggregometer in a 1 :1 ratio and for the flow experiments in a 1 :20 ratio.

For sequential plaque tissue sections, atherosclerotic tissue specimens from 4 different patients were embedded in Tissue-Tek®, transversely cut into 3 microns thin sections at - 20°C by cryostat (Leica CM 3050S, Wetzlar, Germany) and coated onto glass cover-slips. The tissue sections were stored at -80°C.

Blood collection

Blood was obtained from healthy adults who had not taken any medication affecting platelet function for at least 2 weeks preceding the experiment. Informed consent was obtained in accordance with the Helsinki protocol. After venipuncture using a 21 -gauge needle, the first 3 ml blood were discarded and then collected into a plastic syringe containing 1/10 volume recombinant hirudin dissolved in 0.9% NaCI (final concentration in blood ~ 200 U/ml; 13 pg/ml). Experiments were performed between 30 min and 4 h after venipuncture.

Platelet aggregation in blood

Blood platelet aggregation was measured by multiple electrode aggregometry (MEA) using the Multiplate® device from Dynabyte (Munich, Germany) (Toth O. et al. Thromb Haemost

2006; 96: 781-8). PD173952, R406, GS-9973 or DMSO (control) was first added to 0.3ml of physiological saline in the MEA cuvettes before addition of 0.3ml of blood to avoid hemolysis.

Syk inhibitor II or saline (control) were added to the blood/saline mixture. The samples were pre-incubated in the absence of stirring for 3 minutes (PD173952, Syk inhibitor II, R406,

DMSO) or 15 min (GS-9973, DMSO) at 37°C in the test cuvettes (Bampalis VG ef al. J

Thromb Haemost 2012; 10: 1710-4). Subsequently, buffer (control), collagen (0.5 Mg/ml; diluted with SKF buffer), plaque homogenate (833 pg/ml), TRAP (5 μΜ), arachidonic acid (AA) or ADP (5μΜ) was added, and stirring was started. The impedance change was recorded continuously for 10 minutes in duplicate samples by two independent electrode pairs. The mean values of the two independent determinations were expressed in arbitrary "aggregation units" (AU), and cumulative aggregation values over the 10 min time period are expressed as AU*min.

ATP-secretion in blood

ATP-secretion from stimulated platelets in blood was measured according to a method described previously (Ingerman CM. et a/. Thromb Res 1979; 16: 335-44). Similar experimental conditions as for the MEA measurements were used. Saline (150 pi) and hirudin-anticoagulated blood (150 μΙ) were incubated with the inhibitors for 3 minutes at 37°C without stirring as described above. In some samples, abciximab (20 μg/ml) (an anti-integrin αικ,β₃ antibody) was used alone or in combination with the tyrosine kinase inhibitors to block aggregation. After pre-incubation, luciferase-luciferin reagent (50 μΙ of 17.6 U/ml) was added to the samples, stirring (1000 rpm) was started, and the increase of luminescence after stimulation with collagen (0.5 pg/ml), plaque homogenate (833 pg/ml) or TRAP (5 μΜ) was measured in the lumi-aggregometer (Chronolog, Havertown, PA, USA) (Dwivedi S. et al. J Transl Med 2010; 8: 128).

Analysis of platelet aggregation and thrombus formation in flowing blood

For experiments in flowing blood, glass cover slips (Menzel, 24x60mm, # 1.5) coated with collagen (100 pg/ml), plaque homogenates or plaque tissue sections were assembled into parallel plate flow chambers using sticky slides (0.1 Luer sticky slides, ibidi^®, Martinsried, Germany) which had been previously blocked with human serum albumin (HSA; 4% in PBS) [8]. The flow chamber was then mounted on the stage of a fluorescence microscope (TE2000-E, Nikon, Tokyo, Japan) equipped with an incubation chamber (37°C). The flow chambers were perfused with PBS and subsequently blocked with PBS containing 4% HSA for 2 min to prevent non-specific binding of platelets to the glass cover slips.

Syk inhibitor II (2 μΜ, 5 μΜ, or 10 μΜ) was directly added to blood (2ml) in various concentrations. PD173952 (2 μΜ, 5 μΜ, or 10 μΜ), R406 ( 5 μΜ, 10μΜ, or 15μΜ) and DMSO (final concentration in blood 0.1%; control) was first added to 100 μΙ of NaCI (0.9 %) which was then transferred to the 2 ml of blood in order to avoid hemolysis. Mepacrine (10 μΜ) or DiOC6 (1μΜ) was added to blood for fluorescence labelling of platelets, and the samples were then incubated for 5 min at 37°C. Control runs were performed without platelet inhibitors or DMSO. Blood was perfused through the flow chamber at a shear rate of 600/s by a withdrawal syringe pump (Harvard Apparatus, Holliston, Massachusetts, USA). Fluorescence microscopy (excitation: 485/25nm, emission: 528/38 nm) was performed for real time monitoring of platelet adhesion and aggregate formation using a 10x air objective (NA 0.4) and a CoolSNAP HQ2 CCD camera (Photometries, Tucson, USA). Fluorescence images were continuously recorded (1 frame/sec) for 5 min. They were analyzed by quantifying the binary fluorescent area fraction (1.0=total area) after subtraction of the plaque autofluorescence at time 0 min using the NIS-element 3.2 (Nikon, Tokyo, Japan) software package (Jamasbi J. et al. J Am Coll Cardiol 2015; 65: 2404-15). The area visualized in this setting was 669 pm x 896 pm. Values are the mean ± SD (measured each sec) of 5 experiments for each inhibitor with blood from different donors.

Statistics

Results are shown as mean + SD from experiments conducted with blood from every donor at all concentrations of dose response curves or all time points of kinetics. Means of two parallel experimental conditions were compared by Student^'s t-test, if appropriate, or by Mann-Whitney^'s U-test (^*: p<0.05; *^*: p<0.01 , *^**: p<0.001). More than two concurrent experimental conditions were compared by repeated measures ANOVA, if appropriate, or by Friedman repeated measures ANOVA on ranks followed by secondary pair comparisons by Bonferroni's method or Tukey's test, respectively. Significance of secondary pair comparisons is indicated by bars and asterisks (*: p<0.05; ^**: p<0.01 , ***: p<0.001).

Example 2: The Src-family kinase inhibitor PD173952 inhibits plaque-, collagen- and TRAP- induced platelet aggregation in blood under static conditions

The Src-family kinase inhibitor PD173952 (>5 μΜ) completely inhibited plaque- and collagen- induced platelet aggregation in blood as measured by impedance aggregometry (Fig.1 A, B). Inhibition of platelet aggregation stimulated by plaque and collagen by PD173952 showed similar dose-response curves with IC₅₀ values of 1.72 ± 0.08 μΜ, and 2 ± 0.3 μΜ, respectively.

Platelet aggregation stimulated by TRAP activating the PAR-1 receptor could maximally be reduced by 74 % by PD173952 (Fig. 1C). The IC₅₀ for inhibition of TRAP-stimulated platelet aggregation was 1.7 ± 0.9 μΜ.

To analyze whether inhibition of platelet aggregation by PD173952 might be due to an effect on platelet secretion, stimulus-induced ATP-secretion from dense granules was measured under the same experimental conditions (stirring of diluted blood samples, see methods). Platelet ATP-secretion stimulated by plaque or collagen was completely abolished by 20 μΜ PD173952 (Fig. 1D). It was concluded that PD173952 might inhibit plaque-and collagen stimulated platelet aggregation in part, by blocking secretion. Abciximab reduced ATP- secretion stimulated by plaque and collagen by 53 ± 28 % and 38 ± 31 %, respectively (Fig. 1D), indicating that fibrinogen-binding to the an_b 3 integrin amplifies secretion confirming previous observations (Dwivedi S. et al.. J Transl Med. 2010;8:128).

In contrast, to the complete inhibition of platelet secretion upon stimulation by plaque and collagen, PD 73952 (20 μΜ) only modestly and non-significantly reduced ATP-secretion stimulated by TRAP (by 28 ± 18 %). Secretion was not further inhibited by the presence of abciximab (35 ± 29 %; Fig. 1D). This indicates that in the case of TRAP stimulation PD173952 does not directly inhibit secretion. Here, the effects of PD173952 and abciximab were functionally similar, suggesting that inhibition of secretion might be rather secondary to inhibition of aggregation by PD173952 interference with Src-kinase mediated

integrin inside-out or outside-in signaling (Senis YA. et al. Blood. 2014;124:2013-2024; Wu Y. et al. J Biol Chem. 2015;290:15825-15834).

Example 3: PD173952 inhibited platelet thrombus formation under arterial flow conditions stimulated by plaque homogenate, plaque tissue and collagen

Hirudin-anticoagulated blood perfused in flow chambers under arterial flow conditions (shear rate 600 s^"1 as in medium sized arteries) showed similar kinetics and extent of platelet adhesion and aggregate formation onto atherosclerotic plaque homogenate, plaque tissue slices or collagen (Fig. 2). PD173952 (10 μΜ) completely inhibited platelet aggregation onto plaque homogenate, however platelet adhesion was preserved (Fig. 2A). PD173952 inhibited platelet aggregate formation onto plaque tissue somewhat less efficiently by 81 % (Fig. 2B), and it reduced platelet aggregate onto collagen only by 50 ± 24 % (Fig. 2C). DMSO used as solvent of PD173952 did not affect platelet aggregation (Fig. 7). Lower concentrations of PD173952 (2 and 5 pM) delayed and reduced platelet aggregation on plaque-homogenate and -tissue as well as on collagen (Fig. 3, 8). The superior inhibition of plaque-homogenate induced platelet aggregate formation compared to collagen stimulation was significant only at 10 μΜ PD173952 (Fig. 3). Example 4: Syk inhibitor II can suppress plaque- and collagen-, but not TRAP-induced platelet aggregation and ATP-secretion in blood under static conditions

Plaque-induced platelet aggregation was completely abolished by 5 μΜ Syk inhibitor II, whereas inhibition of platelet aggregation stimulated by collagen was only reduced by 85 %. The IC₅₀ values of Syk inhibitor II for inhibition of plaque and collagen-induced platelet aggregation were 1.78 ± 0.8 μΜ, and 2.04 ± 0.6 μΜ, respectively (Fig. 4A, B). In contrast, TRAP-induced platelet aggregation was reduced only by - 46 ± 28 % by 10 μΜ Syk inhibitor II (Fig. 4C, right panel).

In parallel, platelet ATP-secretion induced by plaque or collagen was almost completely inhibited (by more than 95 %; Fig. 4D). In the presence of abciximab preventing aggregation, Syk inhibitor II still inhibited the plaque- and collagen- induced ATP secretion completely. Thus, Syk inhibitor II directly (independent of aggregation) inhibits plaque and collagen- induced dense granule secretion and may partly inhibit aggregation by inhibiting secretion.

In contrast, TRAP-induced platelet secretion was only slightly attenuated by Syk inhibitor II by 17 ± 15 % (n.s.), abciximab or both (Fig. 4D). This indicates that after TRAP stimulation, Syk inhibitor II does not prevent dense granule secretion, and reduces platelet aggregation independent of an effect on secretion.

Example 5: Superior action of Syk inhibitor II on plaque- compared to collagen- induced platelet thrombus formation under arterial flow conditions Platelet aggregation stimulated by plaque homogenate was completely abolished by low concentrations (2 μΜ) of the Syk inhibitor II under arterial flow conditions, while platelet adhesion persisted (Fig. 5A). Syk inhibitor II (2 μΜ) also strongly inhibited platelet thrombus formation onto plaque tissue (by 84 ± 16 %, Fig. 5B), but not onto collagen (Fig. 5C). With increasing concentrations of the Syk inhibitor II, plaque tissue induced-platelet aggregate formation was almost completely inhibited, and platelet adhesion to plaque homogenate was further reduced (from 4.4 % at 2 μΜ to 0.5 % at 5 μΜ and 0.2 % 10 μΜ syk inhibitor II) (Fig. 8). Syk inhibitor II suppressed plaque- significantly better than collagen-induced platelet aggregate formation at all concentrations (2μΜ, 5μΜ, 10μΜ) of Syk inhibitor II (Fig. 6). Example 6: R406 suppresses preferably plaque- and collagen-induced platelet aggregation in blood under static conditions.

Inhibition of platelet aggregation stimulated by plaque by R406 was dose-dependent with an IC₅₀ value of 6.05 μ (Fig. 10A). Plaque- and collagen-induced platelet aggregation was completely suppressed by 15 μΜ R406, whereas inhibition of platelet aggregation stimulated by TRAP- or ADP was reduced only by 56% and 43%,respectively with 15 μΜ R406 (Fig. 10B).

Example 7: Superior action of R406 on plaque- compared to collagen-induced platelet thrombus formation under arterial flow conditions

Platelet aggregation stimulated by plaque homogenate under arterial flow was dose- dependently reduced by R406, and significantly inhibited by 15μΜ R406 (Fig. 11 ). No inhibition of collagen-induced platelet aggregate formation was observed at all concentrations tested (5μΜ, 10μΜ, 15μΜ) of R406 (Fig. 11).

Example 8: GS-9973 (Entospletinib) suppresses preferably plaque- and collagen- induced platelet aggregation in blood under static conditions.

Inhibition of platelet aggregation stimulated by plaque by GS-9973 was dose-dependent with an IC₅₀ value of 1 μΜ (Fig. 12A). Plaque- and collagen-induced platelet aggregation was completely suppressed by 10μΜ GS-9973, whereas platelet aggregation stimulated by TRAP- or ADP was much less inhibited. AA-induced platelet aggregation was not affected (Fig. 12B).

Table 1 : Exemplary Syk kinase inhibitors and lead compounds for the development of improved Syk inhibitors described in the art.



40

[2] Gilead; Compound 36

[2] Gilead; Compound 37

[2] Gilead; Compound 38

[2] Pyrrolopyrazine

Compound 39

[2] Pyrrolopyrazine

Compound 40

1 [2] Aminopyridine

Compound 41

[2] Aminopyridine ^IK3

Compound 42

[2] Aminopyridine

Compound 43

[2] Aminopyridine

Compound 44

[6] Type II inhibitor: binds

to the inactive

(DFG-out)

conformation of a

kinase.

Compound 2

[6] Type II inhibitor: binds

to the inactive

(DFG-out)

conformation of a

kinase.

Compound 3 [6] Carboxamide analog

Compound 5

(Alcon)

[6] Carboxamide analog

Compound 6

(Alcon)

[6] Carboxamide analog

Compound 7

(Alcon)

[6] Tricyclic core

Compound 8

(Abbott)

e

R and R1 : hydrogen

R2: 3-indole derivative with various amide substituents

[6] Tricyclic core

Compound 9 ψ

(Abbott)

*

R3: hydrogen,

R4 :small alkyl or saturated heterocycles

R5: phenyls, pyridyls and indoles substituted with polar groups. [6] Indazole- 3-amine analog

Compound 12

(Almiral)

[6] Indazole- 3-amine analog

Compound 13

(Almiral) I [ J

[6] Pyridine-2-amino core

Compound 14a and

14b

(Almiral)

14a H CCH3

14b ϊ N

Pyridopyrazine

derivatives with

generic structure 47 of

233 compounds;

examples are

compounds 47a-d;

(Hutchison)

Nicotinamide as hinge

binding recognition

element in Syk

inhibitors with generic

structure 48; examples

are compounds 48a-d;

(JP Tobacco) 48b

o

[6] Furopyridine series

with generic core

structure 57 and

compounds 58-60

(Merck GmbH) » M

Si

[6] Triazole series with

generic core structure

61 and compounds

64,65

(Merck GmbH)

M

W

52

16] Compounds 113a and

113b belonging to the

pyridazine

carboxamide (left) and

Compounds 1 14a and

1 14b thieno

pyrimidine (right) Syk

inhibitors;

Probably the

heterocyclic core is

the hinge

binder and cyclohexyl

diamine interacts with

the two key residues in

the Syk ribose pocket.

(Roche)

[6] Triazine

core derivatives.

Compounds 117, 18,

1 19 are examples

(Taiho)

[6] Fused heteroaromatic

pyrrolidinones with

core 120 and

derivatives 121-125

(Takeda)

111

123

Claims

Claims

1. An inhibitor of Syk kinase for use in the treatment and/or prevention of atherothrombosis.

2. A method of treating and/or preventing atherothrombosis comprising administering a pharmaceutically effective amount of an inhibitor of Syk kinase to a subject in need thereof.

3. The inhibitor of claim 1 or the method of claim 2, wherein the inhibitor is a small molecule, an antibody or antibody mimetic, an aptamer, an siRNA, an shRNA, a miRNA, a ribozyme, or an antisense nucleic acid molecule.

4. The inhibitor or the method of claim 3, wherein the inhibitor is a small molecule comprising an azole motif and/or an azine motif.

5. The inhibitor or the method of claim 4, wherein the inhibitor comprises a 2- aminopyrimidine, 4-aminopyrimidine, or 2,4-diaminopyrimidine motif.

6. The inhibitor or the method of any one of claims 1 to 5, wherein the inhibitor is Syk inhibitor-ll, R406, Entospletinib or an analogue thereof.

7. The inhibitor or the method of any one of claims 4 to 6, wherein the inhibitor has a molecular weight in the range of 200 Da to 1500 Da, and preferably in the range of 300 Da to 1000 Da.

8. The inhibitor or the method of any one of claims 1 to 7, wherein the inhibitor is comprised in a pharmaceutical composition, optionally further comprising a pharmaceutically acceptable carrier, excipient and/or diluent.