Treatment and prevention of atherothrombosis by inhibition of Bruton's tyrosine kinase (Btk)

Abstract

The present invention relates to inhibitors of Bruton's tyrosine kinase (Btk) for use in the treatment and / or prevention of atherothrombosis. The present invention further relates to methods for treating and / or preventing atherothrombosis, comprising administering a pharmaceutically effective amount of an inhibitor of Bruton's tyrosine kinase (Btk) to a subject in need thereof.

Description

[Technical field]

[0001] The present invention relates to an inhibitor of Bruton's tyrosine kinase (Btk) 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 pharma- ceutical effective amount of an inhibitor of Bruton's tyrosine kinase (Btk) to a subject in need of such treatment and / or prevention.

[0002] In this specification, a number of documents are cited, for example, patent applications and manufacturer's manuals.The disclosures of these documents are not considered relevant to the patentability of this invention, but are incorporated herein by reference in their entirety.More specifically, all references are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. [Background technology]

[0003] Vascular wall injury leads to platelet activation and the formation of platelet thrombi, followed by further coagulation activity, which leads to the formation of fibrin-containing thrombi that occlude the injured vessel. Thus, hemostasis is an event that begins with the adhesion of platelets to activated components of the subendothelial matrix, such as collagen. Similarly, erosion or rupture of atherosclerotic plaques leads to the formation of platelet- and fibrin-rich thrombi by arresting circulating platelets on exposed materials, such as extracellular matrix components. While normal hemostasis is an important function, thrombus formation resulting from erosion or rupture of atherosclerotic plaques can be harmful, 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 GJ Int Med 2014; 276: 618-632).

[0004] Fibrillar collagens are reactive matrix components that are particularly important in platelet adhesion and activation. Several platelet collagen receptors have been identified so far, among which glycoprotein VI (GPVI) and glycoprotein Ia-IIa (integrin α2β1) are now considered to be of clinical importance. Kuijpers MJE.et al. (FASEB Journal 2003;17(6):685-687) have described the role of these two important collagen receptors GPVI and integrin α2β1 on platelets, as well as the role of Gα in platelet responses induced by TxA2 and ADP via TPα and P2Y1 receptors, respectively. q The role of heterotrimer signaling was investigated. These findings indicate that initial platelet tethering to collagen exposed to flowing blood strongly involves interactions between plasma-deposited GPIb and von Willebrand factor on collagen, and that platelet tethering at high wall shear rates (>500 s -1 showed that stable platelet-collagen adhesion under GPVI-induced IL-1 fusion protein (GPVI) is dependent on both integrin α2β1 and active GPVI receptors. It was hypothesized that platelet binding and activation via GPVI primes cells for integrin-mediated adhesion, and that activated integrin α2β1 stabilizes platelets on collagen. The authors concluded that agents that inhibit integrin α2β1 could potentially be useful in combination with anti-GPVI effects for platelet-directed antithrombotic therapy.

[0005] In Auger et al. (FASEB Journal 2005;19(7):825-827 and full paper published on the World Wide Web under fasebj.org / cgi / doi / 10.1096 / fj.04-1940fje), the authors noted that there is some controversy in the literature about the individual roles of integrin α2β1 and GPVI in platelet adhesion on collagen. The aim of the study was therefore to carry out experiments that address those controversies. For this purpose, both human and mouse platelets were analyzed under identical experimental conditions and using identical or equivalent experimental tools. The authors found that there are two distinct pathways of stable adhesion, one based on the activation of GPVI and Src family kinases and the other dependent on integrin α2β1. Auger et al. further found that integrin α2β1 can mediate adhesion of mouse platelets in the absence of GPVI expression or downstream Src kinase activity and extracellular calcium elevation.

[0006] Since the composition of plaque materials differs from that of healthy blood vessels, one aspect under investigation was and still is the exact nature of their composition and their influence on thrombus formation. A study focusing 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 III positive structures in plaques. The authors of this study found that inhibition of GPVI with antibody 10B12, which specifically binds to the collagen binding site of GPVI, completely blocked platelet thrombus formation on plaques in flowing blood, while no effect was observed with antibody-mediated inhibition of integrin α2β1. This study was supported and expanded by Schulz et al. (Basic Res Cardiol. 2008; 103: 356-367), who further investigated the binding to type I and type III containing structures using a different anti-GPVI antibody (5C4). The authors of these two studies concluded that morphologically diverse collagen type I and type III containing structures in plaques stimulate thrombus formation by activating platelet GPVI, and that inhibition of GPVI may provide a novel antithrombotic strategy to prevent atherothrombosis in patients at cardiovascular risk. Further studies on the inhibition of atherosclerotic plaque-induced platelet activation by using dimeric GPVI-Fc or various anti-GPVI antibodies were described by Jamasbi et al. (J Am Coll Cardiol. 2015; 65: 2404-2415). The authors suggested that the use of GPVI-Fc would be preferred for therapy because there are concerns that antibodies could potentially induce an immune response and increase bleeding, but no such adverse immune responses or increased bleeding effects were noted with the antibodies used in this study.

[0007] An alternative approach is described by Penz et al. (Thromb Haemost. 2007;97:435-443), who focused on the platelet receptor GPIbα, which 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 demonstrated that 1.500s -1 In contrast to static conditions, under flow conditions with a shear rate of 0.01, aspirin could not significantly inhibit plaque-induced thrombus formation, and P2Y1 and P2Y 12 They found that the combination of a receptor antagonist (ADP receptor antagonist) was less effective than GPIbα blockade in reducing plaque-stimulated platelet thrombus formation. Thus, the authors concluded that VWF may play an important role in platelet thrombus formation after plaque rupture and suggested that different antiplatelet drugs, e.g., P2Y1 / P2Y 12 It was suggested that a combination of a receptor antagonist and an inhibitor of GPIbα or GPVI may improve the prevention of human plaque-induced thrombus formation after plaque rupture.

[0008] As shown above, numerous studies have been carried out to elucidate the mechanisms of platelet activation and aggregation (both in healthy blood vessels and in models simulating the rupture of atherosclerotic plaques). However, despite the fact that many attempts are currently being invested in these studies, the most common current treatment remains dual antiplatelet therapy with aspirin and P2Y12 antagonists. Although this treatment reduces ischemic cardiovascular events, its efficacy is limited, and most importantly, this treatment has the drawback of being accompanied by an increased risk of bleeding because these compounds not only target plaque-triggered platelet activation, but also affect physiological hemostatic mechanisms (Bonaca MP.et al.,N Engl J Med.2015;372:1791-1800). Therefore, novel therapies that specifically target plaque-triggered platelet activation with high efficacy but leave physiological hemostatic mechanisms intact would provide great value to the field. Summary of the Invention [Means for solving the problem]

[0009] This need is addressed by the provisions of the embodiments characterized in the claims.

[0010] Thus, the present invention relates to inhibitors of Bruton's tyrosine kinase (Btk) for use in the treatment and / or prevention of atherothrombosis. [Brief description of the drawings]

[0011] [Figure 1-1] Ibrutinib inhibits plaque- and collagen-stimulated static platelet aggregation, but not TRAP- and ADP-stimulated static platelet aggregation. Blood samples were preincubated with vehicle (DMSO, 0.1%) or ibrutinib (1 μM) for 15 min before or without stimulation with plaque (833 μg / ml), collagen (0.1–0.3 μg / ml), TRAP (5 μM), ADP (5 μM), AA (0.6 mM). (A) Representative MEA tracings. Numbers on tracings indicate cumulative aggregation values ​​(AU*min) measured over 10 min. (B) Dose-response curve of ibrutinib for inhibition of plaque-induced platelet aggregation. (C) Dose-response curve of ibrutinib for inhibition of collagen-induced platelet aggregation. (D) Effect of ibrutinib (1 μM) on plaque-, collagen-, TRAP-, ADP-induced platelet aggregation and spontaneous platelet aggregation. Values ​​are mean ± SD (n=5). [Figure 1-2]Ibrutinib inhibits plaque- and collagen-stimulated static platelet aggregation, but not TRAP- and ADP-stimulated static platelet aggregation. Blood samples were preincubated with vehicle (DMSO, 0.1%) or ibrutinib (1 μM) for 15 min before or without stimulation with plaque (833 μg / ml), collagen (0.1–0.3 μg / ml), TRAP (5 μM), ADP (5 μM), AA (0.6 mM). (A) Representative MEA tracings. Numbers on tracings indicate cumulative aggregation values ​​(AU*min) measured over 10 min. (B) Dose-response curve of ibrutinib for inhibition of plaque-induced platelet aggregation. (C) Dose-response curve of ibrutinib for inhibition of collagen-induced platelet aggregation. (D) Effect of ibrutinib (1 μM) on plaque-, collagen-, TRAP-, ADP-induced platelet aggregation and spontaneous platelet aggregation. Values ​​are mean ± SD (n=5). [Figure 1-3] Ibrutinib inhibits plaque- and collagen-stimulated static platelet aggregation, but not TRAP- and ADP-stimulated static platelet aggregation. Blood samples were preincubated with vehicle (DMSO, 0.1%) or ibrutinib (1 μM) for 15 min before or without stimulation with plaque (833 μg / ml), collagen (0.1–0.3 μg / ml), TRAP (5 μM), ADP (5 μM), AA (0.6 mM). (A) Representative MEA tracings. Numbers on tracings indicate cumulative aggregation values ​​(AU*min) measured over 10 min. (B) Dose-response curve of ibrutinib for inhibition of plaque-induced platelet aggregation. (C) Dose-response curve of ibrutinib for inhibition of collagen-induced platelet aggregation. (D) Effect of ibrutinib (1 μM) on plaque-, collagen-, TRAP-, ADP-induced platelet aggregation and spontaneous platelet aggregation. Values ​​are mean ± SD (n=5). [Figure 2-1]Ibrutinib inhibits plaque-induced platelet aggregate formation under arterial flow at low shear rates (600 / s) but not collagen-induced platelet aggregation. Blood samples were preincubated for 15 min with DiOC6 for platelet labeling and with vehicle (0.1% DMSO) or ibrutinib. Representative photomicrographs on the left show the effect of vehicle (0.1% DMSO) or ibrutinib (1 μM) on platelet deposition on plaque homogenates (A), plaque tissue sections (B) and collagen (C) at 0 and 5 min after the start of blood flow. Diagrams on the right show the effect of ibrutinib (0.2, 0.5 and 1 μM) on the kinetics of plaque- and collagen-induced platelet deposition. Inhibition of plaque-induced platelet deposition by ibrutinib (1 μM) is shown in the expanded scale in (A, lower right). Values ​​are mean ± SD (n=5). Significance is shown 5 minutes after initiation of blood flow. [Figure 2-2] Ibrutinib inhibits plaque-induced platelet aggregate formation under arterial flow at low shear rates (600 / s) but not collagen-induced platelet aggregation. Blood samples were preincubated for 15 min with DiOC6 for platelet labeling and with vehicle (0.1% DMSO) or ibrutinib. Representative photomicrographs on the left show the effect of vehicle (0.1% DMSO) or ibrutinib (1 μM) on platelet deposition on plaque homogenates (A), plaque tissue sections (B) and collagen (C) at 0 and 5 min after the start of blood flow. Diagrams on the right show the effect of ibrutinib (0.2, 0.5 and 1 μM) on the kinetics of plaque- and collagen-induced platelet deposition. Inhibition of plaque-induced platelet deposition by ibrutinib (1 μM) is shown in the expanded scale in (A, lower right). Values ​​are mean ± SD (n=5). Significance is shown 5 minutes after initiation of blood flow. [Figure 2-3]Ibrutinib inhibits plaque-induced platelet aggregate formation under arterial flow at low shear rates (600 / s) but not collagen-induced platelet aggregation. Blood samples were preincubated for 15 min with DiOC6 for platelet labeling and with vehicle (0.1% DMSO) or ibrutinib. Representative photomicrographs on the left show the effect of vehicle (0.1% DMSO) or ibrutinib (1 μM) on platelet deposition on plaque homogenates (A), plaque tissue sections (B) and collagen (C) at 0 and 5 min after the start of blood flow. Diagrams on the right show the effect of ibrutinib (0.2, 0.5 and 1 μM) on the kinetics of plaque- and collagen-induced platelet deposition. Inhibition of plaque-induced platelet deposition by ibrutinib (1 μM) is shown in the expanded scale in (A, lower right). Values ​​are mean ± SD (n=5). Significance is shown 5 minutes after initiation of blood flow. [Figure 3-1] Ibrutinib inhibits plaque-induced platelet aggregate formation under arterial flow at high shear rates (1500 / s), but not collagen-induced platelet aggregate formation. Blood samples were preincubated with DiOC6 for platelet labeling, and with vehicle (0.1% DMSO) or ibrutinib. Representative photomicrographs on the left show the effect of vehicle (0.1% DMSO) or ibrutinib (0.5 μM) on platelet deposition onto plaque homogenates (A) and collagen (B) at 0 and 5 min after initiation of blood flow. Diagrams on the right show the effect of ibrutinib (0.2 and 0.5 μM) on the kinetics of plaque- and collagen-induced platelet deposition. Values ​​are mean ± SD (n=5). Significance is indicated 5 min after initiation of blood flow. [Figure 3-2]Ibrutinib inhibits plaque-induced platelet aggregate formation under arterial flow at high shear rates (1500 / s), but not collagen-induced platelet aggregate formation. Blood samples were preincubated with DiOC6 for platelet labeling, and with vehicle (0.1% DMSO) or ibrutinib. Representative photomicrographs on the left show the effect of vehicle (0.1% DMSO) or ibrutinib (0.5 μM) on platelet deposition onto plaque homogenates (A) and collagen (B) at 0 and 5 min after initiation of blood flow. Diagrams on the right show the effect of ibrutinib (0.2 and 0.5 μM) on the kinetics of plaque- and collagen-induced platelet deposition. Values ​​are mean ± SD (n=5). Significance is indicated 5 min after initiation of blood flow. [Figure 4] Oral ibrutinib treatment inhibits plaque- and collagen-induced static platelet aggregation. Blood samples from ibrutinib-treated and control patients were preincubated for 3 min at 37°C before or without stimulation with plaque homogenate (833 μg / ml) or collagen (0.15–0.3 μg / ml). Values ​​are means ± SD (n=5). [Figure 5-1] Oral ibrutinib treatment inhibits platelet thrombus formation on atherosclerotic plaques but not on collagen under arterial flow. Representative photomicrographs on the left show platelet coverage of plaque homogenates (A), plaque tissue sections (B) and collagen (C) at 0 and 6 min after initiation of flow (shear rate 600 / s) with blood from ibrutinib-treated and control patients. Diagrams on the right show the effect of ibrutinib treatment of patients on the kinetics of plaque- and collagen-induced platelet deposition. Inhibition of plaque-induced platelet deposition by ibrutinib is shown in the expanded scale in (A, lower right). Blood samples were preincubated for 10 min with DiOC6 for platelet labeling. Values ​​are mean ± SD (n=5). [Figure 5-2]Oral ibrutinib treatment inhibits platelet thrombus formation on atherosclerotic plaques but not on collagen under arterial flow. Representative photomicrographs on the left show platelet coverage of plaque homogenates (A), plaque tissue sections (B) and collagen (C) at 0 and 6 min after initiation of flow (shear rate 600 / s) with blood from ibrutinib-treated and control patients. Diagrams on the right show the effect of ibrutinib treatment of patients on the kinetics of plaque- and collagen-induced platelet deposition. Inhibition of plaque-induced platelet deposition by ibrutinib is shown in the expanded scale in (A, lower right). Blood samples were preincubated for 10 min with DiOC6 for platelet labeling. Values ​​are mean ± SD (n=5). [Figure 6-1] Effect of oral ibrutinib treatment on in vitro bleeding time (PFA-100). Blood samples from five ibrutinib-treated and five control patients were transferred to (A) collagen / epinephrine or (B) collagen / ADP cartridges and the in vitro closure time was measured by the platelet function analyzer PFA100®. (A, B) Results are shown for each patient. The two horizontal lines indicate the normal range (collagen / EPI 84-160 sec; collagen / ADP 68-121 sec). (C) Mean closure time of control and ibrutinib patients measured by collagen / epinephrine and collagen / ADP cartridges. (D) Mean platelet count (G / l) of control and ibrutinib patients. Values ​​are mean ± SD (n=5). [Figure 6-2]Effect of oral ibrutinib treatment on in vitro bleeding time (PFA-100). Blood samples from five ibrutinib-treated and five control patients were transferred to (A) collagen / epinephrine or (B) collagen / ADP cartridges and the in vitro closure time was measured by the platelet function analyzer PFA100®. (A, B) Results are shown for each patient. The two horizontal lines indicate the normal range (collagen / EPI 84-160 sec; collagen / ADP 68-121 sec). (C) Mean closure time of control and ibrutinib patients measured by collagen / epinephrine and collagen / ADP cartridges. (D) Mean platelet count (G / l) of control and ibrutinib patients. Values ​​are mean ± SD (n=5). [Figure 7] Anti-α2β1 antibody 6F1, but not ibrutinib, inhibits platelet adhesion onto immobilized soluble collagen under arterial flow. Blood was preincubated with DiOC6, abciximab (20 μg / ml), and either DMSO (0.1%; control), ibrutinib (1 μM) or 6F1 (20 μg / ml) for 15 min at 37°C before initiation of flow (shear 600 / s). Values ​​are means + SD (n=4). [Figure 8] Anti-GPIbα antibody 6B4 inhibits plaque and collagen-induced platelet aggregate formation under arterial flow at high shear rates (1500 / s). Blood samples were preincubated with DiOC6 in the absence or presence of 6B4 (5 μg / ml) before perfusion. Effect of 6B4 on the kinetics of plaque and collagen-induced platelet deposition. Values ​​are mean + SD (n = 4-5). [Figure 9]Acalabrutinib (ACP-196) inhibits static platelet aggregation stimulated by plaque and collagen, but not by TRAP, ADP and arachidonic acid (AA). Blood samples were preincubated for 15 min with vehicle (DMSO, 0.1%) or ACP-196 before or without stimulation with plaque (833 μg / ml), collagen (0.1–0.3 μg / ml), TRAP (5 μM), ADP (5 μM), AA. (A) Dose-response curve of ACP-196 for inhibition of plaque-induced platelet aggregation. (B) Effect of ACP-196 (2 μM) on plaque-, collagen-, TRAP-, ADP- and AA-induced platelet aggregation as well as spontaneous platelet aggregation. Values ​​are mean ± SD (n=4). [Figure 10] ONO / GS-4059 inhibits static platelet aggregation stimulated by plaque and collagen, but not by TRAP, ADP and arachidonic acid (AA). Blood samples were preincubated for 15 min with vehicle (DMSO, 0.1%) or ONO / GS-4059 before or without stimulation with plaque (833 μg / ml), collagen (0.1–0.3 μg / ml), TRAP (5 μM), ADP (5 μM), AA (0.6 mM). (A) Dose-response curve of ONO / GS-4059 for inhibition of plaque-induced platelet aggregation. (B) Effect of ONO / GS-4059 (2 μM) on plaque-, collagen-, TRAP-, ADP- and AA-induced platelet aggregation as well as spontaneous platelet aggregation. Values ​​are mean ± SD (n=5). [Figure 11] Acalabrutinib (ACP-196) inhibits plaque-induced platelet thrombus formation but not collagen-induced platelet thrombus formation under arterial flow. Blood samples were preincubated with DiOC6 for platelet labeling and with vehicle (0.1% DMSO) or increasing concentrations of ACP-196 (all in DMSO, final concentration 0.1%) for 15 min before initiation of blood flow at a shear rate of 600 / s. Diagrams show the effect of ACP-196 (0.5, 1 and 2 μM) on the kinetics of plaque- and collagen-induced platelet deposition. Values ​​are mean + SD (n=3). Significance is shown 5 min after initiation of blood flow. [Figure 12] ONO / GS-4059 inhibits plaque-induced platelet thrombus formation but not collagen-induced platelet thrombus formation under arterial flow. Blood samples were preincubated with DiOC6 for platelet labeling and with vehicle (0.1% DMSO) or increasing concentrations of ONO / GS-4059 (all in DMSO, final concentration 0.1%) for 15 min before initiation of blood flow at a shear rate of 600 / s. Diagrams show the effect of ONO / GS-4059 (0.5, 1 and 2 μM) on the kinetics of plaque- and collagen-induced platelet deposition. Values ​​are mean + SD (n=3). Significance is indicated 5 min after initiation of blood flow. [Figure 13] DMSO does not impair plaque and collagen-induced platelet deposition under arterial flow. Blood was preincubated with NaCl or DMSO (0.1%) for 15 min at 37° C. before initiation of flow (shear 600 / s). Values ​​are means ± SD (n=5). [Figure 14] Oral ingestion of a loading dose of Imbruvica® (3×140 mg) inhibits plaque- and collagen-stimulated static platelet aggregation after 3 hours, but not TRAP-, ADP-, and arachidonic acid (AA)-stimulated static platelet aggregation. Blood was collected from two blood donors, A and B, before (control) and 3 hours after ingestion of Imbruvica® (ibrutinib) 3×140 mg, and stimulated with two different pools of plaque homogenates A and B (833 μg / ml), collagen (0.2 μg / ml), TRAP (5 μM), ADP (5 μM), or AA (0.6 mM). Aggregation in blood was measured by MEA. Values ​​are mean + SD of triplicates. [Figure 15]Oral intake of low doses of Imbruvica® for one week inhibits static platelet aggregation stimulated by plaque and collagen, but not by ADP and arachidonic acid (AA). Blood was collected before (control) and one week after intake of Imbruvica® (ibrutinib) (Donor A, 140 mg / day; Donor B, 140 mg every other day) and stimulated with two different pools of plaque homogenates A and B (833 μg / ml), collagen (0.2 μg / ml), TRAP (5 μM), ADP (5 μM), or AA (0.6 mM). Values ​​are the mean + SD of triplicates. [Figure 16] Rapid and sustained inhibition of plaque-induced platelet aggregation by loading and low maintenance doses of Imbruvica®. Blood was drawn before (control), 3 hours after ingestion of Imbruvica® (ibrutinib) 3×140 mg, and on different days after ingestion of Imbruvica® (140 mg / day). Samples were stimulated with plaque homogenate (833 μg / ml), ADP (5 μM), or not stimulated. Values ​​are the mean of duplicates. [Figure 17A] One week of Imbruvica® loading and low maintenance doses rapidly and sustainably inhibit plaque-induced platelet thrombus formation, but not collagen-induced platelet thrombus formation, under arterial flow.Blood was drawn from Donor A before (control), 3 hours after ingestion of Imbruvica® (ibrutinib) 3×140 mg, and after 1 week of ingestion of Imbruvica® (140 mg / day) (A). Blood was collected from donor B before (control), 3 hours after ingestion of Imbruvica® (ibrutinib) 3×140 mg, and 1 week after ingestion of Imbruvica® (140 mg every other day) (B). Samples were preincubated with DiOC6 for platelet labeling for 10 min before initiation of blood flow at a shear rate of 600 / s. Diagrams show the effect of Imbruvica® ingestion on the kinetics of plaque and collagen-induced platelet deposition. Values ​​are mean + SD of triplicates. [Figure 17B] One week of Imbruvica® loading and low maintenance doses rapidly and sustainably inhibit plaque-induced platelet thrombus formation, but not collagen-induced platelet thrombus formation, under arterial flow.Blood was drawn from Donor A before (control), 3 hours after ingestion of Imbruvica® (ibrutinib) 3×140 mg, and after 1 week of ingestion of Imbruvica® (140 mg / day) (A). Blood was collected from donor B before (control), 3 hours after ingestion of Imbruvica® (ibrutinib) 3×140 mg, and 1 week after ingestion of Imbruvica® (140 mg every other day) (B). Samples were preincubated with DiOC6 for platelet labeling for 10 min before initiation of blood flow at a shear rate of 600 / s. Diagrams show the effect of Imbruvica® ingestion on the kinetics of plaque and collagen-induced platelet deposition. Values ​​are mean + SD of triplicates. [Figure 18] Effect of 1 week of Imbruvica® loading and low maintenance doses on in vitro bleeding time (PFA-200). Blood samples from donor A and donor B were taken before (control), 3 hours after, and 1 week after Imbruvica® (ibrutinib) 3×140 mg (140 mg / day: donor A; 140 mg every other day: donor B) and transferred to (A) collagen / epinephrine or (B) collagen / ADP cartridges. In vitro closure times were measured by the platelet function analyzer PFA100®. The two horizontal lines indicate the normal range (collagen / EPI 84-170 sec; collagen / ADP 68-120 sec). Values ​​are single measurements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The term "Bruton's tyrosine kinase", also abbreviated as Btk herein, refers to the tyrosine kinase encoded by the BTK gene in humans. Human Btk protein has NCBI reference sequence: NP_000052.1 (last updated January 5, 2016), and the coding mRNA has NCBI reference sequence: NM_000061.2 (also last updated January 5, 2016). Btk plays a key role in the development of pre-B cells and B lymphocytes by linking the receptor to elevated intracellular free calcium levels. Btk inhibitors, such as ibrutinib, inhibit B cell function and proliferation.

[0013] Ibrutinib and other compounds that inhibit Btk activity are also known to inhibit platelets. Btk plays a key role in collagen / GPVI and VWF / GPIb triggered platelet signaling pathways (Quek LS.et al.,Current biology:CB.1998;8:1137-1140;Oda A.et al.,Blood.2000;95:1663-1670;Liu J.et al,Blood.2006). Upon platelet GPIb engagement by VWF and GPVI stimulation by collagen, tyrosine kinases Lyn and Syk are first activated, which initiates the assembly of signaling complexes of adaptor proteins and enzymes including Btk and PLCγ2 (Liu J.et al,Blood.2006;108:2596-260;Watson SP.et al.,J Thromb Haemost.2005;3:1752-176). Btk is phosphorylated by Syk and Lyn, and is further autophosphorylated, and is involved in the tyrosine phosphorylation and activation of the effector protein PLCγ2 (Quek LS.et al.,Current biology 1998;8:1137-1140; Watson SP.et al.,J Thromb Haemost.2005;3:1752-176; Liu J.et al,Blood.2006;108:2596-260), which is involved in the cytoplasmic Ca2+ phosphorylation and activation of the effector protein PLCγ2 (PlK LS.et al.,Current biology 1998;8:1137-1140; Watson SP.et al.,J Thromb Haemost.2005;3:1752-176; Liu J.et al.,Blood.2006;108:2596-260), which are two major downstream signaling pathways for platelet activation. 2+This leads to an increase in fibrinogen levels and activation of protein kinase C (Siess W. Physiol Rev. 1989;69:58-178). Furthermore, it has been reported that outside-in signaling of platelet integrin αIIbβ3 during thrombin-induced platelet aggregation stimulates Btk (Laffargue M et al FEBS Letters (1999)443:66-70). Platelet integrin αIIbβ3 is the main fibrinogen receptor and is essential for platelet aggregation.

[0014] Thus, ibrutinib has been found to inhibit collagen- and von VWF-dependent platelet function in static assays and under arterial flow in vitro and ex vivo in patients treated with ibrutinib (Levade M. et al. Blood. 2014; 124: 3991-3995; Kamel S. et al. Leukemia. 2015; 29: 783-787; Kazianka L. et al Leukemia (2016), 1-6). Some of the platelet functions studied after addition of ibrutinib in vitro or ex vivo in ibrutinib-treated patients were collagen- or CRP (GPVI agonist)-stimulated platelet aggregation in PRP, ristocetin-induced platelet aggregation in blood (ristocetin mediates binding of VWF to GPIb), and inhibition of collagen- and vWF-induced platelet thrombus formation in flowing blood (Levade M.et al.Blood.2014;124:3991-3995; Kamel S.et al.Leukemia.2015;29:783-787; Kazianka L.et al Leukemia(2016),1-6; Bye AP et al.Arterioscler.Thromb.Vasc.Biol.(2015),35(11):2326-35). Ibrutinib has also been shown to inhibit outside-in signaling of platelet integrin αIIbβ3 and subsequent platelet adhesion and spreading on immobilized fibrinogen in vitro (Bye AP et al. Arterioscler Thromb Vasc Biol (2015), 35(11): 2326-35). The inhibitory effect of ibrutinib on collagen and VWF / GPIb-mediated platelet responses was accompanied by an increased risk of bleeding in patients taking ibrutinib compared to standard chemotherapy (Levade M. et al. Blood. 2014; 124: 3991-3995; Kamel S. et al. Leukemia. 2015; 29: 783-787; Kazianka L. et al Leukemia (2016), 1-6; Shatzel JJ et al. J Thromb Haemostas (2017), 15: 1-13).Interestingly, treatment of patients with newer, more selective BTK inhibitors (second generation BTK inhibitors, e.g., ACP-196, ONO / GS-4059, and BGB-3111) apparently increases bleeding risk less than treatment of patients with ibrutinib (Wu J et al J Hematol Oncol (2016) 9:80). The new Btk inhibitors BTKI-43607 and BTKI-43761 administered orally for 10 days to non-human primates also showed no increase in template skin bleeding time or effect on coagulation (Rigg RA, et al. Am J Physiol Cell Physiol. 2016;310:C373-380).

[0015] The term "inhibitor" according to the present invention refers to an inhibitor that reduces or stops the biological function or activity of a specific target protein. An inhibitor can perform any one or more of the following effects to reduce or stop the biological function or activity of the protein to be inhibited: (i) reduce the transcription of the gene encoding the protein to be inhibited, i.e., reduce the level of mRNA; (ii) reduce the translation of the mRNA encoding the protein to be inhibited; and (iii) the protein performs its biochemical and / or cellular function with reduced efficiency in the presence of the inhibitor. Compounds that fall into class (i) include compounds that interfere with the transcription machinery and / or its interaction with the promoter of said gene and / or with expression control elements distal to the promoter, such as enhancers. Compounds of class (ii) include antisense constructs and constructs (e.g., siRNAs) for performing RNA interference that are well known in the art (see, for example, Zamore (2001) Nat Struct Biol. 8(9), 746; Tuschl (2001) Chembiochem. 2(4), 239). Class (iii) compounds interfere with the molecular function of the protein to be inhibited, for example, receptor signaling activity and activation of downstream target molecules.Therefore, active site binding compounds are envisaged.Class (iii) also includes compounds that do not necessarily bind directly to the target, but still interfere with its function or activity, for example, by changing the affinity or binding rate 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 directly binds to Btk, thereby directly inhibiting its biological function or activity.

[0016] According to the present invention, the inhibitor is an "inhibitor of Btk", i.e., the inhibitor reduces the biological function or activity of Btk. It is particularly preferred that the inhibitor specifically binds to Btk, i.e., it inhibits only the biological function or activity of Btk, but not the biological function or activity of other proteins.

[0017] The biological function or activity refers in particular to any known biological function or activity of Btk, for example, the one elucidated by the present invention. An example of said biological function or activity is the phosphorylation and activation of PLCγ2 in platelets, which increases cytoplasmic calcium levels and activates the effector molecule protein kinase C (PKC), which then leads to platelet activation. All these functions or activities can be tested using any of a variety of standard methods known in the art, such as tyrosine phosphorylation of Btk or PLCγ2 or increasing cytoplasmic Ca2+ concentration, or based on the teachings of the examples provided below, optionally in conjunction with molecular techniques, such as phosphorylation assays, or the teachings of the literature cited herein.

[0018] In a preferred embodiment, the inhibitor reduces at least one, preferably all, of the above-cited biological functions or activities of Btk by at least 50%, preferably by at least 75%, more preferably by at least 90%, even more preferably by at least 95%, for example, by at least 98% or even 100%. The term "reduction by at least" refers to a decrease in a biological function or activity such that Btk loses one or more, preferably all of the recited amounts of its biological functions or activities. For example, a reduction by at least 75% means that Btk loses 75% of its biological functions or activities, resulting in only 25% of the remaining biological functions or activities compared to uninhibited Btk.

[0019] The function of any of the inhibitors referred to in the present invention can be identified and / or confirmed by using high-throughput screening assays (HTS). High-throughput assays, independent of biochemical, cellular or other assays, can generally be carried out in wells of a microtiter plate, each plate containing, for example, 96, 384 or 1536 wells. Handling of the plate, for example incubation at a temperature other than ambient temperature, and contacting of the test compounds with the assay mixture are preferably carried out by one or more computer-controlled robotic systems, for example pipetting devices. If a large library of test compounds is to be screened and / or if screening is to be performed in a short period of time, a mixture of, for example, 10, 20, 30, 40, 50 or 100 test compounds can be added to each well. If a well shows biological activity, the mixture of test compounds can be deconvoluted to identify one or more test compounds in the mixture that cause the observed biological activity.

[0020] Measurement of binding of a potential inhibitor can be performed, for example, in any binding assay, preferably a biophysical binding assay, which can be used to measure binding of a test molecule prior to performing a functional / activity assay with the inhibitor. Suitable biophysical binding assays are known in the art and include fluorescence polarization (FP) assays, fluorescence resonance energy transfer (FRET) assays and surface plasmon resonance (SPR) assays.

[0021] When the inhibitor acts by reducing the expression level of the target protein, the measurement of the expression level of the protein can be carried out, for example, at the nucleic acid level or at the amino acid level.Methods for measuring the expression of protein at the nucleic acid level include, but are not limited to, Northern blotting, PCR, RT-PCR or real RT-PCR.Methods for measuring the expression of protein at 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.The methods for measuring the expression level of Btk at both the nucleic acid level and the amino acid level are well known in the art.

[0022] A number of Btk inhibitors are currently known and approved for therapy or in clinical trials, and Btk selective irreversible small molecule inhibitors have been discovered (Pan Z.et al.,Chem Med Chem.2007;2:58-61).For example, the small molecule inhibitor ibrutinib (PCI-32765) is a selective Btk inhibitor approved by the FDA for the treatment of mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenstrom's macroglobulinemia, a form of non-Hodgkin's lymphoma (Byrd JC.et al.,N Engl J Med.2013;369:32-42;Wang ML.et al.,N Engl J Med.2013;369:507-516). Further examples of Btk inhibitors currently in clinical trials include acalabrutinib (Phase 3) for relapsed CLL, GS-4059 or ONO / GS-4059 (formerly ONO-4059; Phase 1) for non-Hodgkin's lymphoma and / or CLL, as well as spebrutinib (AVL-292, CC-292; Phase 1) and BGB-3111 (Phase 1) (Wu J et al J Hematol Oncol (2016) 9:80). Additional reversible and irreversible Btk inhibitors have been described in the art, such as CNX-774, imidazoquinoxaline, CGI1746, GDC-0834, RN486, and others, summarized in Whang and Chang, Drug Discovery Today 2014;19:1200-1204 as Btk inhibitors in development for the treatment of rheumatoid arthritis; other irreversible BTK inhibitors are BTKI-43607 and BTKI-43761 (Rigg RA, et al. Am J Physiol Cell Physiol. 2016;310:C373-380).

[0023] According to the present invention, it is particularly preferred that the inhibitor is a platelet-specific inhibitor.In some embodiments, the inhibitor according to the present invention can be provided as a small molecule, a proteinaceous compound, or as a nucleic acid molecule, such as an interference or inhibitory nucleic acid molecule, as described in more detail below.The inhibitor can also be encoded by a nucleic acid molecule that can be incorporated into an expression vector, for example, containing a regulatory element, for example, a megakaryocyte-specific promoter.For example, the silencing of Btk in megakaryocytes and subsequently in platelets can be achieved by using a simian or human immunodeficiency virus type 1-based self-inactivating lentivirus vector carrying a glycoprotein Ibα promoter and an interference or inhibitory nucleic acid molecule, or for example, a nucleic acid molecule that codes for a proteinaceous inhibitor. Methods and suitable vectors for targeted transfection of cells are known in the art, see, for example, Sambrook and Russel ("Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, NY (2001); Ohmori T. et al. Arterioscler Thromb Vasc Biol. 2007 Oct; 27(10): 2266-72; Lavenu-Bombled C. et 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 nucleic acid molecules (siRNA, shRNA) encoding inhibitors or directly affecting Btk transcription into expression vectors allows selective suppression of Btk expression in megakaryocytes and platelets.

[0024] These inhibitors can be administered to a subject by any available and suitable method, for example, orally, intravenously, intradermally, subcutaneously, intramuscularly, intraperitoneally, topically (e.g., by powders, ointments, drops or transdermal patches), buccally, or as an oral or nasal spray. Oral administration is particularly preferred, especially when the inhibitor is ibrutinib.

[0025] The dosage regimen can be determined by the attending physician based on clinical factors. As is well known in the medical arts, the dosage for any one patient depends on 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 co-administered. The effective amount for a given situation is readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician or physician. For example, if the inhibitor is the only compound, the effective amount administered per dose may range from about 70 to 140 mg per person, but as noted above, this is subject to therapeutic discretion. The specific amounts, and corresponding adjustments, in cases where two or more compounds are to be administered can be determined by routine testing well known to those skilled in the art. Administration can be once as a single dose, or repeated administration. The interval time and amount of repetition required can be determined by one skilled in the art without further ado, preferably a dose of 140 mg is administered once every two days. Progress can be monitored by periodic evaluation. The inhibitors of the present invention can 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, such as 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 (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases, and the like.

[0026] The term "arterial thrombosis" refers to thrombosis that occurs in arteries, as opposed to venous thrombosis that occurs in veins.Arterial thrombosis is generally caused by injury to healthy arteries, and the formation of platelet- and fibrin-rich thrombi is essential to seal the wall defect and prevent blood loss.In contrast, atherothrombosis, a special form of arterial thrombosis, is caused by the rupture of atherosclerotic plaques or, less frequently, by the erosion of the endothelium covering atherosclerotic plaques.Thrombosis formation is triggered by atherosclerotic plaque-derived thrombogenic material, which initiates an avalanche of platelet aggregation and fibrin formation.

[0027] Atherosclerosis is a chronic disease of the arterial wall, and atherosclerotic plaques develop during life as a result 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 significantly thickened. The plaque consists of lipids (mostly in the oxidized form), macrophages, smooth muscle cells, their necrotic cellular debris, calcium and extracellular matrix. Virchow coined the term "atheroma" for this material. Various types of collagen accumulate in atherosclerotic plaques and are structurally different from collagen in healthy connective tissues (van Zanten, GH 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 that may alter their platelet reactivity (Monnier VM et al N Engl J Med (1986), 314:403-8; Sell et al Arch Biochem Biophys (2010), 493:192-206). The fundamentally different properties of atherosclerotic plaques compared to the intima of healthy arteries explain their different thrombus formation: atherosclerotic plaques are more thrombus forming than healthy arteries. Platelet deposition is increased on human atherosclerotic coronary arteries compared to normal arteries (van Zanten, GH et al. J Clin Invest. (1994), 93(2):615-32), and in mice, the thrombotic response to injured atherosclerotic carotid arteries is significantly higher than that of injured healthy carotid arteries (Hechler, B. and C. Gachet, Thromb Haemost (2011) 105 Suppl 1:S3-12).

[0028] The term "atherothrombosis" refers to arterial thrombus formation that develops as a result of rupture of atheroma or after erosion of the endothelium covering atherosclerotic plaque. In atherothrombosis, erosion or rupture of vulnerable atherosclerotic plaque in coronary, extracranial or intracranial arteries (carotid or cerebral arteries, respectively) and peripheral (e.g., femoral) arteries arrests circulating platelets and exposes material that triggers thrombus formation. The thrombus may occlude the artery, or the thrombus may detach, embolize, or occlude blood vessels (arteries, arterioles) downstream. Occlusion of the arterial circulation cuts off blood supply to tissues and can cause ischemia and infarction of almost any organ in the body, depending on the localization of the thrombus or thromboembolism, most commonly acute coronary syndrome, myocardial infarction, and ischemic stroke, but also peripheral arterial occlusive disease (PAOD).

[0029] Atherothrombosis can also occur after percutaneous coronary intervention if not prevented by antiplatelet therapy, promoting neointima formation, restenosis and stent thrombosis.

[0030] According to the present invention, it was found that inhibitors of Btk, more specifically, ibrutinib (PCI-32765), an orally applied irreversible Btk inhibitor, inhibit plaque and collagen-triggered platelet aggregation in blood under static conditions, both in vitro and ex vivo.In contrast, platelet aggregation stimulated by TRAP, ADP and arachidonic acid (AA), which activates PAR-1 receptor, was only slightly reduced by ibrutinib in vitro.Importantly, ibrutinib inhibited atherosclerotic plaque-induced platelet thrombus formation in arterial flow in two atherothrombosis models, namely in plaque homogenates exposed to arterial flowing blood and in plaque tissue sections.

[0031] Most importantly, the effects of ibrutinib, whether added to blood in vitro or tested in blood samples from patients on ibrutinib therapy, differed markedly between plaque and collagen stimulation of platelet aggregation under arterial flow conditions: Ibrutinib caused complete and plaque-selective platelet inhibition when blood was perfused over plaque material at shear rates typical of flow in intact coronary arteries (600 / s; see Figures 2 and 5) or over mildly stenosed segments of atherosclerotic coronary arteries (1500 / s; Figure 3).

[0032] This difference between the effects on plaque and collagen-stimulated platelet aggregation under arterial flow conditions can be explained by the observation that the platelet collagen receptor integrin α2β1 plays a key role in platelet adhesion to collagen during arterial flow on isolated collagen fibers similar to the situation in healthy blood vessels, where integrin α2β1 synergizes with GPVI in platelet aggregation to collagen. In contrast, platelet aggregation on plaque under arterial flow depends exclusively 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). Consistent with this, ibrutinib did not inhibit integrin α2β1-mediated platelet adhesion on soluble type I collagen (Figure 7). Thus, Btk does not signal downstream of integrin α2β1, and thus inhibition of Btk by ibrutinib selectively blocks platelet aggregation under flow dependent on GPVI activation, but not on collagen under flow dependent on the combination of GPVI and integrin α2β1 activation.

[0033] In addition to GPVI, Btk has also been reported to play a role in VWF / GPIb signaling. Collagen exposed after arterial wall injury or plaque rupture or erosion can first immobilize circulating VWF, which then binds to the platelet receptor GPIbα. This cross-linking by VWF is crucial to slow down circulating platelets, thereby allowing arterial thrombus growth at shear rates of >1000 / s. In flow experiments shown in the accompanying examples (see, for example, Example 3), at a shear rate of 1500 / s, ibrutinib only had a slight, non-significant effect on collagen-induced platelet deposition, but it effectively inhibited plaque-induced platelet deposition (Figure 3). Moreover, both plaque- and collagen-induced platelet aggregation in high shear rate arterial flow required VWF binding to GPIb. Antibody 6B4 (Penz SM et al. Thromb Haemost. 2007;97:435-443), which inhibits the interaction of GPIbα with VWF, equally inhibited (>95%) both plaque homogenate and collagen-induced platelet thrombus formation at this high shear rate (Figure 8). These results indicate that VWF activation of GPIb is required for both plaque and collagen-induced platelet deposition in high shear arterial flow, but also imply that Btk signaling downstream of GPIb is not functionally relevant.

[0034] Further results in the accompanying Examples show that two new second generation oral irreversible inhibitors of Btk that are more selective than ibrutinib had similar efficacy to ibrutinib.

[0035] Acalabrutinib (ACP-196) and ONO / GS-4059 inhibited plaque- and collagen-triggered platelet aggregation in blood under static conditions in vitro. In contrast, platelet aggregation stimulated by TRAP, ADP or AA, which activate the PAR-1 receptor, was not reduced or only slightly reduced by these inhibitors (Figures 9, 10). The IC50 value of acalabrutinib (ACP-196) for inhibition of plaque-induced platelet aggregation was approximately 0.4 μM, and that of ONO / GS-4059 was approximately 1 μM (Figures 9A, 10A). Importantly, similar to ibrutinib, acalabrutinib (ACP-196) at 2 μM and ONO / GS-4059 at 2 μM caused effective plaque-selective platelet inhibition when blood was perfused over plaque material at arterial shear rates (Figures 11, 12). DMSO (final concentration in blood 0.1%) used to dissolve the Btk inhibitor did not affect platelet responses (Figure 13).

[0036] Preliminary studies in two human volunteers showed that oral ingestion of low doses of ibrutinib (Imbruvica®) was sufficient to inhibit plaque-induced platelet aggregation under static and flow conditions. Plaque- and collagen-induced platelet aggregation was completely inhibited 3 hours after oral ingestion of a loading dose of 3×140 mg Imbruvica® (FIG. 14). Inhibition of plaque-induced aggregation was maintained by oral ingestion of either 140 mg Imbruvica® daily or 140 mg every other day for one week (FIGS. 15, 16). These low ibrutinib doses were also sufficient to inhibit platelet adhesion and aggregation onto atherosclerotic plaques under arterial flow, but not onto collagen (FIGS. 17A, B).

[0037] Ibrutinib, acalabrutinib (ACP-196) and ONO / GS-4059 inhibited plaque-triggered platelet thrombus formation significantly more potently than what was previously observed with the established dual antithrombotic therapy of aspirin and a P2Y12 receptor antagonist (Penz SM et al. Thromb Haemost. 2007;97:435-443; Jamasbi J. et al. J Am Coll Cardiol. 2015;65:2404-2415).

[0038] The results shown in the accompanying examples indicate that targeting Btk can prevent atherothrombosis; in particular, it can prevent occlusive acute atherothrombosis more efficiently and more selectively because it leaves the physiological platelet activation in hemostasis largely intact, as also shown in the accompanying examples.

[0039] This latter finding of intact physiological platelet activation in hemostasis is particularly surprising: Btk-deficient patients, such as those with X-linked agammaglobulinemia (XLA), were previously found to exhibit no spontaneous or severe bleeding (Stewart DM.et al.Current allergy and asthma reports.2001;1:558-565;Futatani T.et al.Br J Haematol 2001;114:141-149), whereas the use of Btk inhibitors, such as ibrutinib, was shown to be associated with perturbation of vWF- and collagen-stimulated platelet activation (Levade M.et al.Blood.2014;124:3991-3995;Kamel S.et al.Leukemia.2015;29:783-787). Low-grade bleeding tendency, e.g., spontaneous discoloration, nosebleeds, or petechiae, is often observed in ibrutinib-treated patients (Wang ML.et al.Blood.2015;126:739-745.2015;Shatzel JJ et al.J Thromb Haemostas(2017)15:1-13). Interestingly, treatment of patients with newer, more selective BTK inhibitors (e.g., ACP-196, ONO / GS-4059, and BGB-3111) apparently causes less bleeding than ibrutinib (Byrd JC et al N Engl J Med(2016)374;4:323-332;Wu J et al J Hematol Oncol(2016)9:80;Walter HS et al Blood(2017)129(20):2808-2810). The new Btk inhibitors BTKI-43607 and BTKI-43761 also showed no increase in template skin bleeding time or effect on coagulation in non-human primates (Rigg RA, et al. Am J Physiol Cell Physiol. 2016;310:C373-380).

[0040] Taken together, the data presented herein indicate that the use of Btk inhibitors remains promising to be more efficient and safer than standard dual platelet therapy with aspirin and a P2Y12 antagonist, while also avoiding the drawbacks of antibody-based interventions, such as those potentially faced when using anti-GPVI antibodies.

[0041] The present invention further relates to a method for treating and / or preventing atherothrombosis, comprising administering a pharma- ceutical effective amount of an inhibitor of Bruton's tyrosine kinase (Btk) to a subject in need of such treatment and / or prevention.

[0042] All definitions and preferred embodiments provided herein with respect to the inhibitors of the invention apply mutatis mutandis to this method of the invention unless specifically recited otherwise.

[0043] In preferred embodiments of the inhibitors or methods of the invention, the inhibitor is a small molecule, an antibody or antibody mimetic, an aptamer, siRNA, shRNA, miRNA, a ribozyme, or an antisense nucleic acid molecule.

[0044] A "small molecule" as used herein is preferably an organic molecule. An organic molecule refers to or belongs to a class of compounds having a carbon base in which carbon atoms are bonded together by carbon-carbon bonds. The original definition of the term organic refers to the source of the compound, with organic compounds being carbon-containing compounds obtained from plant or animal or microbial sources, while inorganic compounds are obtained from mineral sources. Organic compounds may be natural or synthetic. Organic molecules are preferably aromatic molecules, more preferably heteroaromatic molecules. In organic chemistry, the term aromaticity is used to describe cyclic (ring-shaped), planar (flat) molecules with rings of resonance bonds that exhibit greater stability than other geometries or bonding arrangements with the same set of atoms. Aromatic molecules are extremely stable and do not easily react with other substances without decomposition. In heteroaromatic molecules, at least one of the atoms in the aromatic ring is an atom other than carbon, such as N, S, or O.

[0045] Heteroaromatic molecules are preferably molecules containing an azole motif (i.e. an azole, di-azole, tri-azole or tetra-azole motif) and / or an azine motif (i.e. an azine, di-azine or tri-azine motif). Heteroaromatic molecules are more preferably molecules containing a pyridine (azine), pyrimidine (diazine), triazine, azine, pyrazole or imidazole motif. Heteroaromatic molecules are even more preferably molecules containing a pyrimidine (diazine), pyrazole or imidazole motif. For all the above organic molecules, the molecular weight is preferably in the range of 150 Da to 1500 Da, more preferably in the range of 300 Da to 1000 Da.

[0046] Alternatively, the "small molecule" according to the present invention can be an inorganic compound. Inorganic compounds are derived from mineral resources and include all compounds that do not have 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, for example, less than about 500 amu, and even more preferably, less than about 250 amu. The size of the small molecule can be measured by methods well known in the art, for example, mass spectrometry. Small molecules can be designed, for example, based on the crystal structure of the target molecule, and the sites assumed to be responsible for biological activity can be identified and confirmed in an in vivo assay, for example, an in vivo high throughput screening (HTS) assay. Non-limiting examples of small molecule inhibitors of Btk are ibrutinib (also known as PCI-32765), acalabrutinib (ACP-196), and ONO / GS-4059, which were used in the examples provided herein below. Other examples, such as BTKI-43607 and BTKI-43761, have also been described to irreversibly inhibit Btk in platelets (Rigg RA.et al.Am J Physiol Cell Physiol.2016;310:C373-380). Additional small molecule inhibitors of Btk, such as spebrutinib (AVL-292, CC-292), BGB-3111, CNX-774, imidazoquinoxaline, CGI1746, GDC-0834, RN486, and HM-71224, have been described in the art and are summarized, inter alia, by Wank and Chang, Drug Discovery Today 2014;19:1200-1204, and Wu J et al J Hematol Oncol(2016)9:80). Table 1 below shows the structural formulas of the small molecule inhibitors of Btk: ibrutinib, acalabrutinib (ACP-196), ONO / GS-4059, BGB-3111, CC-292, BMS-986142, CNX-774, imidazoquinoxaline, CGI1746, GDC-0834, RN486, and M-71224.Other Btk inhibitors are BTKI-43607 and BTKI-43761, whose structures have not been disclosed (Rigg RA. et al. Am J Physiol Cell Physiol. 2016;310:C373-380).

[0047] [Table 1] JPEG0007610239000002.jpg177170JPEG0007610239000003.jpg215170JPEG0007610239000004.jpg98170

[0048] The term "antibody" as used according to the present invention includes, for example, polyclonal or monoclonal antibodies. Furthermore, derivatives or fragments thereof that still retain the binding specificity to a target, for example Btk, are also included in the term "antibody". Antibody fragments or derivatives include, inter alia, Fab or Fab' fragments, Fd, F(ab')2, Fv or scFv fragments, single domain VFv fragments, and the like. H or V-like domains, such as VhH or V-NAR-domains, as well as multimeric formats, such as minibodies, diabodies, tri- or triple bodies, 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).

[0049] The term "antibody" also includes such embodiments as chimeric (human constant domain, non-human variable domain), single chain and humanized (human antibody minus the non-human CDRs) antibodies.

[0050] Various techniques for producing antibodies are well known in the art and are described, for example, in Harlow and Lane (1988) and (1999) and Altshuler et al., 2010 (cited above). Thus, polyclonal antibodies can be obtained from the blood of animals after immunization with an antigen in a mixture with additives and adjuvants, and monoclonal antibodies can be produced by any technique that provides antibodies produced by continuous cell line cultures. Examples of such techniques are described, for example, 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, including the hybridoma technique first described by Koehler and Milstein, 1975, the trioma technique, the human B cell hybridoma technique (see, for example, Kozbor D, 1983, Immunology Today, vol. 4, 7; Li J, et al. 2006, PNAS, vol. 103(10), 3557) and the EBV hybridoma technique for producing human monoclonal antibodies (Cole et al., 1985, Alan R. Liss, Inc, 77-96). Furthermore, recombinant antibodies can be obtained from monoclonal antibodies or prepared de novo using various display methods, such as phage, ribosome, mRNA, or cell display. Suitable systems for the expression of recombinant (humanized) antibodies can be selected, for example, from bacteria, yeast, insect, mammalian cell systems or transgenic animals or plants (see, for example, U.S. Pat. No. 6,080,560; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 11265). Furthermore, techniques described for the production of single chain antibodies (see, inter alia, U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies specific for epitopes of Btk.Surface plasmon resonance used in the BIAcore system can be used to increase the efficiency of phage antibodies.

[0051] The term "antibody mimic" as used herein refers to a compound that specifically binds to an antigen, e.g., in this case, Btk, in a manner similar to an antibody, but is not structurally related to an antibody. Antibody mimics are artificial peptides or proteins, usually with a molar mass of about 3-20 kDa. For example, the antibody mimic can be selected from the group consisting of affibodies, adnectins, anticalins, DARPins, avimers, nanophytins, affilins, Kunitz domain peptides, and Fynomer®. These polypeptides are well known in the art and are described in further detail herein below.

[0052] The term "affibody" as used herein refers to a family of antibody mimics derived from the Z domain of Staphylococcus aureus protein A. Structurally, affibody molecules are based on a three-helix bundle domain that can also be incorporated into fusion proteins. Affibody itself has a molecular mass of approximately 6 kDa and is stable at high temperature and under acidic or alkaline conditions. Target specificity, i.e. specificity for Btk, is obtained by randomization of 13 amino acids located in two alpha-helices that are involved in the binding activity of the parent protein domain (Feldwisch J, Tolmachev V.; (2012) Methods Mol Biol. 899: 103-26).

[0053] The term "Adnectin" (also referred to as "monobody") as used herein refers to molecules based on the tenth extracellular domain of human fibronectin III (10Fn3), which adopts a 94-residue Ig-like β-sandwich fold with two to three exposed loops but lacks a central disulfide bridge (Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Adnectins with the desired target specificity, i.e., specificity for Btk, can generally be engineered by introducing modifications in specific loops of the protein.

[0054] The term "anticalins" as used herein refers to engineered proteins derived from lipokines (Beste G, Schmidt FS, Stibora T, Skerra A. (1999) Proc Natl Acad Sci USA. 96(5):1898-903; Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Anticalins have an eight-stranded beta barrel that forms a core unit highly conserved among lipokines and naturally forms a binding site for ligands with four structurally variable loops at the open end. Although anticalins are not homologous to the IgG superfamily, they exhibit features previously considered typical of the binding site of antibodies: (i) high structural plasticity as a result of sequence variation and (ii) increased conformational flexibility that allows induction of fit to targets with different shapes.

[0055] The term "DARPin" as used herein refers to a designed ankyrin repeat domain (166 residues) that provides a rigid interface that typically results from three repeat β-turns.DARPins usually carry three repeats that correspond to an artificial consensus sequence, with six positions per repeat being randomized.As a result, DARPins lack structural flexibility (Gebauer and Skerra, 2009).

[0056] The term "avimer" as used herein refers to a class of antibody mimics consisting of two or more peptide sequences of 30-35 amino acids each derived from the A domain of various membrane receptors and linked by a linker peptide. Binding of the target molecule occurs via the A domain and a domain with the desired binding specificity, i.e., binding specificity for Btk, which can be selected, for example, by phage display technology. The binding specificity of the different A domains contained in the avimer can be identical, but does not have to be identical (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68).

[0057] "Nanophytins" (also known as affitins) are antibody mimetic proteins derived from the DNA-binding protein Sac7d of Sulfolobus acidocaldarius. Nanophytins usually have a molecular weight of approximately 7 kDa and are designed to specifically bind target molecules, such as Btk, by randomizing 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).

[0058] The term "affilin" as used herein refers to an antibody mimic that uses either gamma-B crystallin or ubiquitin as scaffold and develops by modifying the amino acids on the surface of these proteins by random mutagenesis.Selection of affilin with desired target specificity, i.e., specificity for Btk, is performed, for example, by phage display or ribosome display technology.Depending on the scaffold, affilin has a molecular weight of about 10 or 20 kDa.The term affilin as used herein also refers to the dimerization or multimerization form of affilin (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10 (4): 155-68).

[0059] "Kunitz domain peptide" is derived from the Kunitz domain of Kunitz-type protease inhibitors, such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP) or tissue factor pathway inhibitor (TFPI). Kunitz domains have a molecular weight of about 6 kDA, and domains with the required target specificity, i.e., specificity for Btk, can be selected by display techniques, such as phage display (Weidle et al., (2013), Cancer Genomics Proteomics; 10 (4): 155-68).

[0060] The term "Fynomer" as used herein refers to a non-immunoglobulin-derived binding polypeptide derived from human Fyn SH3 domain.Fyn SH3-derived polypeptides are well known in the art and are described, for example, 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).

[0061] Aptamers are nucleic acid or peptide molecules that bind to specific target molecules. 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 molecules. 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, 12, 4:465-483).

[0062] Nucleic acid aptamers are nucleic acid species that are usually composed of (usually short) strands of oligonucleotides. Typically, they are 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.

[0063] Peptide aptamers are peptides or proteins that are usually designed to interfere with other protein interactions inside the cell. 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 peptide aptamers to levels comparable to those of antibodies (nanomolar range). The variable peptide loop typically contains 10-20 amino acids, and the scaffold can be any protein with good solubility properties. Currently, the bacterial protein thioredoxin A is the most commonly used scaffold protein, in which the variable peptide loop is inserted within the redox active site, which is a -Cys-Gly-Pro-Cys-loop in the wild-type protein, where two cysteine ​​side chains can form a disulfide bridge. Peptide aptamer selection can be performed using a variety of systems, but currently the yeast two-hybrid system is the most widely used.

[0064] Aptamers offer utility for biotechnology and therapeutic applications because they offer molecular recognition properties comparable to those of commonly used biomolecules, particularly antibodies. In addition to their differential recognition, aptamers offer advantages over antibodies because they can be fully engineered in a test tube, they are easily produced by chemical synthesis, have desirable storage properties, and induce little or no immunogenicity in therapeutic applications. Unmodified aptamers are rapidly cleared from the bloodstream, with half-lives ranging from minutes to hours due primarily to nuclease degradation and clearance from the body by the kidneys, which are a result of the inherent low molecular weight of the aptamer. Unmodified aptamer applications are currently focused on treating transient conditions, such as blood clotting, or treating organs that allow localized delivery, such as the eye. This rapid clearance can be advantageous in applications such as in vivo diagnostic imaging. Several modifications, such as 2'-fluorine substituted pyrimidines, polyethylene glycol (PEG) conjugation, fusion to albumin or other half-life extending proteins, are available to scientists that can increase the half-life of aptamers by days or even weeks.

[0065] The term "peptide" as used herein describes a group of molecules consisting of up to 30 amino acids, while the term "polypeptide" (also called "protein") as used herein describes a group of molecules consisting of more than 30 amino acids. The groups of peptides and polypeptides are referred to together by using the term "(poly)peptide".

[0066] According to the present invention, the term "small interfering RNA (siRNA)", also known as short interfering RNA or silencing RNA, refers to a class of double-stranded RNA molecules of 18-30, preferably 19-25, most preferably 21-23 or even more preferably 21 nucleotides in length that play various roles in biology. Most notably, siRNAs are involved in the RNA interference (RNAi) pathway, where siRNAs interfere with the expression of specific genes. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, for example, as an antiviral mechanism or in shaping the chromatin structure of the genome.

[0067] siRNAs that are usually found in nature have a well-defined structure: short double strands of RNA (dsRNA) with 2nt 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 the enzyme Dicer, which converts either long dsRNA or small hairpin RNA into siRNA, which can also be exogenously (artificially) introduced into cells to cause specific knockdown of a gene of interest. Thus, essentially any gene whose sequence is known can be targeted based on sequence complementarity with appropriately tailored siRNA. Double-stranded RNA molecules or their metabolic processing products can mediate target-specific nucleic acid modification, particularly RNA interference and / or DNA methylation. Exogenously introduced siRNAs can lack overhangs at their 3' and 5' ends, but it is preferred that at least one RNA strand has a 5'- and / or 3'-overhang. Preferably, one end of the duplex has a 3'-overhang of 1-5 nucleotides, more preferably 1-3 nucleotides, most preferably 2 nucleotides. The other end can be blunt-ended or has a 3'-overhang of up to 6 nucleotides. In general, any RNA molecule suitable for acting as an siRNA is envisaged in the present invention. The most efficient silencing has been obtained so far for siRNA duplexes composed of 21 nt sense and 21 nt antisense strands paired in a manner with a 2 nt 3'-overhang. The sequence of the 2 nt 3'-overhang makes a small contribution to the specificity of target recognition that is restricted to the unpaired nucleotide adjacent to the first base pair (Elbashir et al. 2001). 2'-deoxynucleotides in the 3'-overhang are as efficient as ribonucleotides, but are often cheaper to synthesize and may be more nuclease-resistant.Delivery of siRNA can be accomplished using any method known in the art, for example, by combining the siRNA with saline and administering the combination intravenously or intranasally, or by formulating the siRNA in glucose (e.g., 5% glucose, etc.), or cationic lipids and polymers can be used for in vivo siRNA delivery via either intravenous (IV) or intraperitoneal (IP) systemic routes (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 Therapeutics).

[0068] 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 that is introduced into cells, utilizing a U6 promoter to ensure that the shRNA is always expressed. This vector is usually propagated to daughter cells, allowing 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 mRNA that matches the siRNA bound to it. The si / shRNA that can be used in the present invention is 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 (a division of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK). Most conveniently, siRNA or shRNA is obtained from a commercial RNA oligo synthesis supplier that sells RNA synthesis products of different quality and cost. In general, the RNA applicable in the present invention is conventionally synthesized and easily provided with quality suitable for RNAi.

[0069] Further molecules that perform RNAi include, for example, microRNA (miRNA).The RNA species is a single-stranded RNA molecule.Endogenously present miRNA molecules bind to complementary mRNA transcripts and trigger the degradation of the mRNA transcripts through a process similar to RNA interference, thereby regulating gene expression.Therefore, exogenous miRNA can be used as an inhibitor of Btk after being introduced into respective cells.

[0070] Ribozymes (from ribonucleic acid enzyme, also called RNA enzymes or catalytic RNA) are RNA molecules that catalyze chemical reactions. Many natural ribozymes catalyze either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyze the aminotransferase activity of ribosomes. Non-limiting examples of well-characterized small self-cleaving RNAs are hammerhead, hairpin, hepatitis delta virus, and in vitro selection lead-dependent ribozymes, while group I introns are an example for larger ribozymes. The principle of catalytic self-cleavage has been well established in recent years. Hammerhead ribozymes are the best characterized of the RNA molecules with ribozyme activity. It has been shown that hammerhead structures can be incorporated into heterologous RNA sequences and thereby transfer ribozyme activity to those molecules, so it is conceivable that catalytic antisense sequences can be created for almost any target sequence, provided that the target sequence contains a potential matching cleavage site. The basic principle of constructing a hammerhead ribozyme is as follows: select a region of interest in RNA that contains a GUC (or CUC) triplet. Take two oligonucleotide strands, usually with 6-8 nucleotides each, and insert the catalytic hammerhead sequence between them. Best results are usually obtained with short ribozymes and target sequences.

[0071] A recent development that is also useful in the present invention is the combination of an aptamer that recognizes small molecule compounds with a hammerhead ribozyme. Conformational changes induced in the aptamer upon binding to a target molecule can modulate the catalytic function of the ribozyme.

[0072] The term "antisense nucleic acid molecule" as used herein refers to nucleic acid that is complementary to target nucleic acid.Antisense molecule according to the present invention can interact with target nucleic acid, more specifically, it can hybridize with target nucleic acid.Due to the formation of hybrid, the transcription of (one or more) target gene and / or the translation of target mRNA is reduced or blocked.Standard methods for antisense technology are described (see, for example, Melani et al., Cancer Res. (1991) 51:2897-2901).

[0073] In a further preferred embodiment of the inhibitors or methods of the invention, the inhibitor binds irreversibly to Btk.

[0074] "Irreversible binding" according to the present invention refers to the binding of an inhibitor to Btk in such a manner that the two molecules remain bound until the disappearance of Btk or the cells containing said Btk molecule, for example due to the degradation of Btk or the death of the respective cells. Methods for determining whether a Btk inhibitor irreversibly binds to Btk are well known, including, but not limited to, testing whether the inhibitor covalently binds to cysteine ​​481 (Cys-481) in the active domain of Btk by tagging the inhibitor with a fluorophore, for example Bodipy-FL, incubating it with a cell lysate, and detecting fluorescent Btk by fluorescence scanning after gel electrophoretic protein separation, as described in Honigberg et al, PNAS 2010.

[0075] In a more preferred embodiment of the inhibitor or method of the present invention, the inhibitor is ibrutinib or an analog thereof.

[0076] Ibrutinib is also known as PCI-32765, is commercially available under the name Imbruvica, and has the IUPAC name 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one. It binds covalently to a cysteine ​​residue (Cys-481) in the active site, thereby potently and irreversibly inhibiting Btk (Honigberg LA.et al.,Proc Natl Acad Sci USA.2010;107:13075-13080). Ibrutinib is commercially available from Selleckchem (Houston, USA).

[0077] According to the present invention, the inhibitor may be an analog of ibrutinib. Such analogs include functional and structural analogs of ibrutinib. Further examples of Btk inhibitors include acalabrutinib (ACP-196), ONO / GS-4059, spebrutinib (AVL-292, CC-292), and BGB-3111 (Wu J et al J Hematol Oncol (2016) 9:80). Further reversible and irreversible Btk inhibitors, such as CNX-774, imidazoquinoxaline, CGI1746, GDC-0834, RN486, etc., have been described in the art and are summarized in Whang and Chang, Drug Discovery Today 2014;19:1200-1204) as Btk inhibitors under development for the treatment of rheumatoid arthritis. Other irreversible BTK inhibitors are BTKI-43607 and BTKI-43761, which are analogs of ibrutinib that form a covalent bond with a cysteine ​​residue on Btk (Rigg RA, et al. Am J Physiol Cell Physiol. 2016; 310: C373-380). Functional analogs of ibrutinib include, but are not limited to, Btk inhibitors that covalently bind to a cysteine ​​residue at position 481 in the active site of the kinase domain, such as the irreversible inhibitors CC-292, CNX-774, and imidazoquinoxaline, all of which have been described in the art and summarized in Whang and Chang, Drug Discovery Today 2014; 19: 1200-1204.

[0078] According to a more preferred embodiment of the inhibitor or method of the present invention, the inhibitor (preferably ibrutinib, acalabrutinib (ACP-196), ONO / GS-4059, BGB-3111, or an analogue thereof) is used / administered in a dosage of 10-140 mg per day or 20-280 mg every other day. This dosage is preferably preceded by a single loading dose of 250-560 mg.

[0079] Within the list of ibrutinib, acalabrutinib (ACP-196), ONO / GS-4059, BGB-3111, and their analogs, ibrutinib, acalabrutinib (ACP-196), ONO / GS-4059, and their analogs are preferably used / administered as inhibitors. A preferred dosage of ibrutinib that can be used is a low drug dose of 35-140 mg per day (preferably 70-140 mg per day), or 70-140 mg every other day. A preferred dosage of acalabrutinib that can be used is a low drug dose of 10-50 mg twice per day, resulting in a total daily dose of 20-100 mg. A dosage of ONO / GS-4059 that can be used is preferably a low drug dose of 20-100 mg per day and 50-100 mg every other day. A preferred dosage of BGB-3111 that can be used is a low drug dose of 20-80 mg twice daily for a total daily dose of 40-160 mg.

[0080] By using such low doses of ibrutinib, acalabrutinib, ONO / GS-4059, BGB-3111, or their analogs, selective inhibition of Btk can be achieved primarily, if not exclusively, in platelets. Such low doses ensure that ibrutinib, acalabrutinib, ONO / GS-4059, BGB-3111, or their analogs covalently inactivate Btk in platelets passing through the portal circulation during the absorption phase. This approach takes advantage of the lack of de novo enzyme synthesis in platelets, allowing inhibition of a key mechanism for plaque-initiated thrombus formation in platelets throughout their lifespan, but does not inhibit Btk in other cells.

[0081] Preferably, an initial loading dose is used / administered before the low doses of ibrutinib, acalabrutinib, ONO / GS-4059, BGB-3111, or one of their analogs are used / administered, the loading dose being higher than the low doses described above to ensure an immediate effect on platelets in the patient to be treated.

[0082] According to an even more preferred embodiment of the inhibitor or method of the present invention, the inhibitor is used / administered at a loading dose of 280-560 mg and a maintenance dose of 10-140 mg per day or 40-280 mg every other day.

[0083] Preferred loading doses (taken once) are 280-560 mg for ibrutinib, 200-300 mg for acalabrutinib, 320-480 mg for ONO / GS-4059, and 320-480 mg for BGB-3111.

[0084] In a further preferred embodiment of the inhibitor or method of the invention, the inhibitor is comprised in a pharmaceutical composition, optionally further comprising a pharma- ceutically acceptable carrier, excipient and / or diluent.

[0085] The term "pharmaceutical composition" used herein refers to a composition for administration to a patient, preferably a human patient.The pharmaceutical composition of the present invention comprises at least one of the above inhibitors, for example at least two, for example at least three, in further embodiments at least four, for example at last five.When two or more inhibitors are contained in the pharmaceutical composition, it is understood that none of these inhibitors have essentially any inhibitory effect on other inhibitors that are also contained in the composition.

[0086] The compositions may be in solid, liquid or gas form, inter alia, in the form of powder(s), tablet(s), liquid(s) or aerosol(s).

[0087] The pharmaceutical composition preferably comprises a pharma- ceutically 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, e.g., oil / water emulsions, various types of wetting agents, lubricants, binders, fillers, sterile solutions, and the like.

[0088] Compositions containing such carriers can be formulated by well-known conventional methods.These pharmaceutical compositions can be administered to subjects in suitable doses.Dosage regimens are determined by the attending physician and clinical factors.All definitions and preferred embodiments provided herein above with respect to administration of the inhibitors of the present invention, such as administration route, preferred dosage and means for determining same, apply mutatis mutandis to administration of the pharmaceutical compositions of the present invention.

[0089] It is particularly preferred that said pharmaceutical composition comprises an additional agent known in the art to have an antagonistic effect on arterial thrombosis.Because the pharmaceutical preparation of the present invention relies on the inhibitor referred to herein, it is preferred that the additional agent mentioned is used only as an adjuvant, i.e., at a reduced dose compared to the recommended dose when used as the sole drug, for example, to reduce the side effects caused by the additional agent.

[0090] 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 present patent specification, including definitions, will control.

[0091] In this specification, particularly with regard to embodiments characterized in the claims, each embodiment recited in a dependent claim is intended to be combined with each embodiment of each claim (independent or dependent) from which said dependent claim depends. For example, in the case of independent claim 1 reciting three alternatives A, B and C, dependent claim 2 reciting three alternatives D, E and F, and claim 3 dependent on claims 1 and 2 and reciting three alternatives G, H and I, it should be understood that the specification expressly discloses embodiments corresponding to the 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.

[0092] Similarly, and even if the independent and / or dependent claims do not cite alternatives, it is understood that when a dependent claim refers back to more than one previous claim, any combination of the subject matter covered thereby is considered to be explicitly disclosed. For example, in the case of independent claim 1, dependent claim 2 which refers back to claim 1, and dependent claim 3 which refers back to both claims 2 and 1, the combination of the subject matter of claims 3 and 1 will be clearly and obviously disclosed as will the combination of the subject matter of claims 3, 2 and 1. If there is a further dependent claim 4 which refers to any one of claims 1-3, then the combination of the subject matter of claims 4 and 1, of claims 4, 2 and 1, of claims 4, 3 and 1, and of claims 4, 3, 2 and 1 will be clearly and obviously disclosed.

[0093] The above considerations apply mutatis mutandis to all appended claims. By way of non-limiting example, the combination of claims 7, 6 and 4 is clearly and obviously envisaged in view of the claim structure. The same applies to, for example, combinations of claims 7, 6 and 5, etc.

[0094] The examples illustrate the invention. EXAMPLES

[0095] Example 1: Materials and Methods reagent Ibrutinib, acalabrutinib (ACP-196) and ONO / GS-4059 were obtained from Selleckchem (Houston, USA). Dimethyl sulfoxide (DMSO) and albumin from human serum (fatty acid free) were from Sigma-Aldrich (Taufkirchen, Germany). Collagen (Horm) and diluent were purchased from Takeda (Linz, Austria). Thrombin receptor activating peptide (TRAP) was obtained from Bachem AG (Bubendorf, Switzerland). 3'-phosphate 5'-phosphate (ADP) was from Biopool (Wicklow, Ireland). DiOC6 was obtained from Life Technologies (Eugene, Oregon, USA). PBS (Dulbecco's phosphate buffered saline) was from Gibco (Grand Island, New York, USA). Recombinant lepirudin (Refludan®) was from Celgene (Windsor, UK). Tissue-Tek® as embedding medium for cryotomy was from Sakura (Alphen aan den Rijn, the Netherlands).

[0096] Stock solution Ibrutinib, acalabrutinib (ACP-196) and ONO / GS-4059 were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mM. Aliquots (30 μl) were stored at −80° C. Before each in vitro experiment, dilutions were made in DMSO (0.1, 0.2, 0.5, 1 and 2 mM) to obtain final blood concentrations of 0.1 μM, 0.2 μM, 0.5 μM, 1 μM and 2 μM, respectively. The final concentration of DMSO in blood was 0.1%. Collagen (1000 μg / ml) was diluted in the dilution solvent at a ratio of 1:10 (100 μg / ml) for aggregometry experiments or at a ratio of 1:50 (20 μg / ml) for flow experiments. TRAP was dissolved in 0.9% NaCl at a concentration of 500 μM. ADP was dissolved in distilled water at a concentration of 500 μM. DiOC6 for platelet labeling was dissolved in DMSO at a concentration of 5 mM. Aliquots (10 μl) were stored at −20° C. Before each flow experiment, the stock solution was dissolved in human serum albumin (HSA, 0.5% in PBS) at a concentration of 100 μM to obtain a final concentration in blood samples of 1 μM.

[0097] Human carotid atherosclerotic plaque Atherosclerotic plaque specimens were donated by patients undergoing endarterectomy for high-grade carotid artery stenosis as previously described (Brandl R. et al., Circulation. 1997; 96: 3360-3368; Reininger AJ. et al., J Am Coll Cardiol. 2010; 55: 1147-11585; Penz S. et al. Faseb J. 2005; 19: 898-909). Patient consent was obtained as approved by the Ethics Committee of the Faculty of Medicine of the University of Munich. Specimens containing lipid-rich plaque were collected. Atherosclerotic plaques were carefully excised under sterile conditions from other areas of atherosclerotic tissue specimens (Brandl R. et al., Circulation. 1997; 96: 3360-3368; Reininger AJ. et al., J Am Coll Cardiol. 2010; 55: 1147-11585; Penz S. et al. Faseb J. 2005; 19: 898-909). Plaques were processed to obtain either homogenates or serial tissue sections.

[0098] For plaque homogenates, plaque specimens were weighed and homogenized in buffer (150 mM NaCl, 1 mM EDTA, pH 7.4), adjusted to a concentration of 100 mg wet weight / ml, and stored at -80°C. Plaque homogenates from five patients were pooled (Dwivedi S. et al., J Transl Med. 2010;8:128). The pooled plaque homogenates were diluted with PBS in a 1:1 ratio for static testing in the Multiplate® analyzer and in a 1:20 ratio for flow experiments.

[0099] For serial plaque tissue sections, atherosclerotic tissue specimens from four different patients were embedded in Tissue-Tek® and cut transversely into 3-micron-thick sections with a cryostat (Leica CM3050S, Wetzlar, Germany) at −20° C. and coated onto glass cover slips. Tissue sections were stored at −80° C.

[0100] Blood collection For the in vitro ibrutinib supplementation experiments, blood was obtained from healthy adults who had not been taking any medications affecting platelet function for at least 2 weeks prior to the experiment.

[0101] For the ex vivo experiments, CLL patients (n=5) on continuous ibrutinib treatment (Imbruvica, 3x140mg capsules once daily, Janssen Pharmaceutica NV, B-2340 Beerse, Belgium) and control patients (n=5) with platelet counts above 150 G / l were selected to allow analysis of platelet function in uncompromised blood due to their platelet concentration. Four of the control patients had CLL and one had MCL and did not require treatment. For each experimental day, one pair of ibrutinib and control patients with similar platelet counts were selected and their platelet function was tested in parallel. Patients' informed consent was obtained according to the Helsinki protocol.

[0102] Blood was collected by venipuncture using a 21-gauge needle and a plastic syringe containing 1 / 10 volume of recombinant hirudin (final concentration in blood approximately 200 U / ml: 13 μg / ml) dissolved in 0.9% NaCl. The first 3 ml of blood drawn into the empty syringe was discarded. Experiments were performed between 30 min and 4 h after venipuncture.

[0103] Platelet aggregation in the blood Platelet aggregation in blood was measured by multiple electrode aggregometry (MEA) using a Multiplate® device from Dynabyte (Munich, Germany) (Toth O. et al. Thromb Haemost. 2006; 96: 781-8). Ibrutinib, acalabrutinib (ACP-196), or ONO / GS-4059 (0.1, 0.2, 0.5, 1 or 2 μM) or DMSO (0.1%, control) were first added to 0.3 ml of saline in an MEA cuvette, followed by 0.3 ml of blood. Samples were preincubated in the test cuvette for 15 min in the absence of agitation. In ex vivo experiments with patient blood, samples were preincubated for 3 min at 37°C in the absence of agitation (Bampalis VG. et al. J Thromb Haemost. 2012; 10: 1710-4). Subsequently, plaque homogenate (833 μg / ml) or collagen (0.2-0.4 μg / ml; tested in individual blood donors to induce aggregation values ​​identical to those of plaque homogenate), TRAP (5 μM) or ADP (5 μM) were added, stirring was initiated, and impedance changes were recorded continuously for 10 min in duplicate samples. Cumulative aggregation values ​​over a 10 min time period were expressed as AU. * Expressed as min(AU, agglomeration units).

[0104] Analysis of platelet aggregation and thrombus formation in flowing whole blood For experiments in flowing blood, glass cover slips (Menzel, 24 x 60 mm, #1.5) coated with collagen (20 μg / ml), plaque homogenates or plaque tissue sections were assembled in a parallel plate flow chamber using adhesive slides (0.1 Luer adhesive slides, ibidi®, Martinsried, Germany) previously blocked with human serum albumin (HSA; 4% in PBS). 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 chamber was perfused with PBS and subsequently blocked for 2 min with PBS containing 4% HSA to prevent non-specific binding of platelets to the glass cover slip.

[0105] Ibrutinib, acalabrutinib (ACP-196), or ONO / GS-4059 (0.1, 0.2, 0.5, 1 or 2 μM) or DMSO (final concentration in blood 0.1%; control) was first added to 100 μl of NaCl (0.9%) placed in a Falcon tube, followed by the addition of the blood sample (2 ml). This procedure avoided hemolysis and ensured that ibrutinib was entirely present in the blood sample. DiOC6 (1 μM) was added to the blood for fluorescent labeling of platelets, and the sample was incubated at 37° C. for 15 min. 7 Control runs were performed with DMSO or NaCl alone. In ex vivo experiments, 3 ml of blood from patients with or without ibrutinib treatment was preincubated with DiOC6 (1 μM) for 10 min at 37°C.

[0106] Blood was perfused through the flow chamber at two shear rates (600 / s; 1500 / s) using an extractable syringe pump (Harvard Apparatus, Holliston, Massachusetts, USA) (Jamasbi J. et al. J Am Coll Cardiol. 2015;65:2404-15). Fluorescence microscopy (excitation: 485 / 25 nm, emission: 528 / 38 nm) was performed for real-time measurements of platelet adhesion and aggregate formation using a 10× air objective (NA 0.4) and a CoolSNAP HQ2 CCD camera (Photometrics, Tucson, USA). Fluorescence images were recorded continuously for 5-7 min (1 frame / s). They were analyzed by quantifying the binary fluorescent area fraction (1.0 = total area) after subtraction of plaque autofluorescence at time 0 min using the NIS-element3.2 (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 μm × 896 μm (Jamasbi J. et al. J Am Coll Cardiol. 2015;65:2404-15). Values ​​are the mean ± SD (measurements per second) of five experiments for each inhibitor using blood from different donors (Jamasbi J. et al. J Am Coll Cardiol. 2015;65:2404-15).

[0107] In vitro closure time (CT) analysis in whole blood The PFA100® and PFA200® devices (Siemens Healthcare, Erlangen, Germany) are further developments of the Thrombostat system used to simulate primary hemostasis and measure bleeding times in vitro (Kratzer MA. et al. Semin Thromb Hemost. 1995; 21 Suppl 2: 25-31; Kundu SK, et al. Semin Thromb Hemost. 1995; 21 Suppl 2: 106-12). The instrument draws citrate-anticoagulated blood (0.8 ml) under constant vacuum from a container through a capillary and a small hole in a membrane filter coated with collagen and ADP (collagen / ADP test cartridge) or collagen and epinephrine (collagen / EPI test cartridge). The time required to obtain complete occlusion of the opening was recorded as the "in vitro closure time". Closure times were measured in two healthy donors and five patients with or without ibrutinib treatment. The five ibrutinib patients were identical to those tested above in static and flow experiments.

[0108] statistics The means of two parallel experimental conditions were compared by paired Student's t test ( * :p<0.05; ** :p<0.01, *** : p<0.001). Three or more simultaneous experimental conditions were tested by ANOVA for repeated measures, followed by pairwise comparisons by Bonferroni tests (§: p<0.05; §§: p<0.01, §§§: p<0.001).

[0109] Example 2: Ibrutinib inhibits plaque- and collagen-induced platelet aggregation in blood under static conditions Preincubation of blood with ibrutinib (1 μM) almost completely inhibited platelet aggregation upon stimulation with plaque and collagen, as measured by impedance aggregometry (Fig. 1A). Inhibition of plaque- and collagen-stimulated platelet aggregation by ibrutinib showed similar dose-response curves. The IC50 values ​​of ibrutinib for inhibition of plaque- and collagen-stimulated platelet aggregation were 0.18 ± 0.05 μM and 0.12 ± 0.04 μM, respectively, with maximum inhibition reached at 0.5 μM (Fig. 1B, C). In contrast, ibrutinib only modestly reduced platelet aggregation stimulated by TRAP and ADP, which activate the PAR-1 receptor (by −31% and −13%, respectively; Fig. 1A, D). Spontaneous minimal platelet aggregation in the absence of stimulation was also abolished by ibrutinib (Fig. 1A, lower row, D).

[0110] Example 3: Ibrutinib inhibits platelet thrombus formation stimulated by plaque homogenate and plaque tissue, but not by collagen, under arterial flow conditions To simulate platelet activation due to plaque rupture, human blood was perfused in a parallel-plate flow chamber over coverslips coated with human plaque homogenates or plaque tissue sections at physiological wall shear rates for healthy carotid and coronary arteries (600 / s) and mildly stenosed coronary lesions (1500 / s).

[0111] At 600 / s, ibrutinib (1 μM) completely abolished platelet aggregation on plaque homogenates (-98 ± 1%) while preserving platelet adhesion (Figure 2A). Ibrutinib (1 μM) also potently inhibited platelet thrombus formation on plaque tissue (by -76 ± 16%; Figure 2B), but only delayed and did not attenuate final platelet aggregate formation on collagen fibers (+8 ± 2%) (Figure 2C). Lower concentrations of ibrutinib (0.2 and 0.5 μM) still delayed and reduced platelet aggregation on plaque homogenates (-43 ± 27 and -84 ± 8%, respectively) and plaque tissue (-33 ± 23 and -44 ± 30%, respectively), but did not impair collagen-induced platelet aggregation (+1 ± 6 and +3 ± 9%, respectively). DMSO solvent did not affect platelet aggregation (data not shown).

[0112] Even at high shear rates, ibrutinib (0.2 μM and 0.5 μM) did not reduce platelet aggregation onto collagen but still inhibited platelet thrombus formation onto plaque homogenates (by −69 ± 16% and −93 ± 5%, respectively) (Figure 3).

[0113] Example 4: Plaque and collagen-induced platelet aggregation under static and arterial flow conditions in patients undergoing ibrutinib therapy We next analyzed the effect of ongoing ibrutinib therapy (420 mg / day) in five CLL patients. Five matched patients not taking ibrutinib served as controls. Blood platelet counts were similar in ibrutinib patients (174 ± 19 G / l) and controls (186 ± 37 G / l). In blood from patients on ibrutinib, platelet aggregation was inhibited when stimulated in vitro by plaque (-89 ± 7%) or collagen (-84 ± 8%) under static conditions, and unstimulated spontaneous platelet aggregation was also blocked (Figure 4). Remarkably, ibrutinib treatment stopped platelet thrombus formation on plaque homogenates under arterial flow conditions, while preserving platelet adhesion (Figure 5A). Platelet thrombus formation on plaque tissue was also largely prevented (by -83 ± 24%; Figure 5B). In contrast, ibrutinib only minimally slowed early platelet aggregation from flowing blood onto collagen fibers, but reached a maximal response identical to that of controls (FIG. 5C).

[0114] Example 5: Effect of ibrutinib ex vivo on in vitro closure time (CT) To test whether ibrutinib intake could increase bleeding time, measurements were performed with the platelet function analyzer PFA-100 in five patients taking ibrutinib identical to those tested above. This device simulates primary hemostasis in vitro. However, whether abnormal values ​​found with the PFA-100 or PFA-200 devices reflect potential bleeding risk in vivo remains a subject of debate (Kundu SK.et al.,Semin Thromb Hemost.1995;21 Suppl 2:106-112; Paniccia R.et al.,Vascular health and risk management.2015;11:133-148). The mean closure time in patients on ibrutinib therapy was not significantly increased, but two of the five patients showed prolonged closure time for the collagen / epinephrine test cartridge (Figure 6A, C). For collagen / ADP cartridges, closure times were in the normal range in both patient groups (Figure 6B, C). Blood platelet counts were similar in both patient groups (Figure 6D).

[0115] Example 6: Ibrutinib inhibits platelet aggregation onto plaque under arterial flow via a mechanism independent of platelet integrin α2β1 and GPIb binding to VWF Under arterial flow, the integrin α2β1-blocking antibody 6F1, but not ibrutinib, blocked platelet adhesion onto soluble type I collagen (Figure 7), confirming that integrin α2β1 is important for platelet adhesion to soluble collagen (Jung and Moroi J Biol Chem (1998) 273(24):14827-37) and demonstrating that ibrutinib does not interfere with integrin α2β1-mediated platelet adhesion to native collagen in physiological hemostasis.

[0116] To further clarify whether the plaque-specific inhibition of platelet aggregation by ibrutinib at high shear flow was due only to inhibition of GPVI signaling or also to inhibition of GPIb signaling, blood was preincubated with monoclonal antibody 6B4 (Penz SM et al Thromb Haemost (2007);97(3):435-43), which inhibits the interaction of GPIbα with VWF. When perfused at high shear over plaque homogenate and collagen, both plaque homogenate- and collagen-induced platelet thrombus formation were equally well inhibited (>95%) (Figure 8). This indicates that VWF activation of GPIb is required for platelet adhesion and aggregation to both plaque and collagen surfaces at high shear rates, but also implies that Btk signaling downstream of GPIb is not functionally relevant here.

[0117] Example 7: The novel second generation Btk inhibitors acalabrutinib (ACP-196) and ONO / GS-4059 inhibit plaque and collagen-induced platelet aggregation in blood under static conditions Preincubation of blood with acalabrutinib (ACP-196) and ONO / GS-4059 dose-dependently inhibited plaque-induced platelet aggregation in blood as measured by impedance aggregometry (Figure 9A; Figure 10A). The IC50 values ​​of ACP-196 and ONO / GS-4059 were 0.4 μM and 1 μM, respectively, with maximum inhibition (80% and 82%, respectively) reached at 1 and 2 μM, respectively. Collagen-induced platelet aggregation was equally well inhibited (Figure 9B, Figure 10B). In contrast, ACP-196 (2 μM) and ONO / GS-4059 (2 μM) only slightly reduced platelet aggregation stimulated by TRAP, ADP or AA (Figure 9B, Figure 10B). Spontaneous minimal platelet aggregation in the absence of stimulation was abolished by ibrutinib (Figure 1A, bottom, D).

[0118] Example 8: Acalabrutinib (ACP-196) and ONO / GS-4059 inhibit platelet thrombus formation stimulated by plaque homogenate but not collagen under arterial flow conditions. ACP-196 dose-dependently inhibited platelet aggregation on plaque homogenates in arterial flow with a shear rate of 600 / s, with 2 μM inhibiting platelet aggregation by 96% (Figure 11). ONO / GS-4059 also showed dose-dependent inhibition, potently inhibiting platelet thrombus formation on plaque homogenates by -76% at 2 μM; Figure 12). Both Btk inhibitors slightly delayed but did not attenuate final platelet aggregate formation on collagen fibers at the highest concentrations tested (Figures 11, 12). DMSO solvent did not affect platelet aggregation (Figure 13).

[0119] Example 9 Oral administration of low doses of ibrutinib inhibits plaque-induced platelet aggregation under static conditions and arterial flow. To test the potential of ibrutinib as an antiplatelet therapy, two healthy donors (male, 61 and 66 years old) received a loading dose of Imbruvica (3 capsules x 140 mg = 420 mg) followed by a low dose of 140 mg / day for one week (Donor A) or a very low dose of 140 mg every other day for one week (Donor B). This regimen did not have any adverse side effects in the two donors.

[0120] A loading dose of 3 x 140 mg Imbruvica almost completely inhibited platelet aggregation induced by two plaque homogenate pools by 94% and 93%, collagen-induced platelet aggregation by 91% in donor A, and reduced platelet aggregation by 68% and 77% after plaque stimulation and by 58% after collagen stimulation in donor B (Figure 14). Platelet aggregation induced by TRAP, ADP or AA was slightly inhibited in donors A and B (Figure 14).

[0121] In donor A, after one week of Imbruvica (140 mg / day), platelet aggregation stimulated by the two plaque homogenate pools was still inhibited by 94% and 91%, and after collagen stimulation by 91% (Figure 15). In donor B, after one week of very low doses of Imbruvica (140 mg every other day), platelet aggregation stimulated by the two plaque homogenate pools was similarly stimulated by 93% and 91%, and after collagen stimulation was inhibited by 82% (Figure 15). Platelet aggregation induced by ADP or AA was slightly inhibited in donors A and B (Figure 15). Platelet aggregation stimulated by TRAP was reduced by 70% in donor A and 45% in donor B.

[0122] Plaque-induced platelet aggregation was measured on several days during a one-week intake of Imbruvica (140 mg / day) by Donor A. It was found to be >90% inhibited on each test day regardless of whether blood samples were drawn 23 or 3 hours after dosing (FIG. 16).

[0123] Oral ingestion of ibrutinib inhibited platelet thrombus formation stimulated by plaque homogenate under arterial flow conditions, but not by collagen (Figure 17A, B). A loading dose of Imbruvica (3 x 140 mg) inhibited plaque-induced thrombus formation equally well in both donors after 3 hours. After 1 week, both the low dose of Imbruvica 140 mg / day and the very low dose of Imbruvica 140 mg every other day inhibited plaque-induced thrombus formation, with slightly less inhibition observed after the very low dose of Imbruvica 140 mg (compare Figures 17A and B).

[0124] To test whether ibrutinib intake could increase bleeding time, PFA-200 device and closure time were measured in both blood donors. Donor B showed prolonged closure time for collagen / epinephrine test cartridge 3 hours after ibrutinib challenge and 1 week after low dose ibrutinib intake, whereas donor A showed less (Figure 18). It was noted that donor B already had closure time values ​​above the normal range before ibrutinib intake. For collagen / ADP cartridge, closure time was in the normal range after ibrutinib intake in both donors (Figure 18).

[0125] Imbruvica supplementation (either a loading dose or a low dose for 1 week) had no effect on red blood cell, platelet, or white blood cell (lymphocyte, neutrophil, monocyte) counts. B lymphocytes (measured by FACS) increased rather than decreased in both donors (Table 2). Liver enzymes were not altered by Imbruvica supplementation.

[0126] In summary, in two healthy subjects, an oral Imbruvica loading dose (3 × 140 mg) followed by a lower maintenance dose (140 mg / day or 140 mg every other day) provided rapid and sustained protection from plaque-induced platelet aggregation and platelet thrombus formation under arterial flow without adverse side effects.

[0127] [Table 2]

Claims

1. A medicament for treating and / or preventing atherothrombosis comprising a medicament comprising a medicament for treating and / or preventing atherothrombosis ...

2. The pharmaceutical of claim 1, wherein the molecular weight of the Btk inhibitor is in the range of 150 Da to 1500 Da.

3. The pharmaceutical of claim 1 or 2, wherein the inhibitor irreversibly binds to Btk.

4. The pharmaceutical agent according to any one of claims 1 to 3, wherein the inhibitor is ibrutinib, acalabrutinib (ACP-196), ONO / GS-4059, or BGB-3111.

5. The method of any one of claims 1 to 4, wherein the inhibitor is comprised in a pharmaceutical composition optionally further comprising a pharma- ceutically acceptable carrier, excipient and / or diluent.

6. The medicament according to any one of claims 1 to 5, wherein the inhibitor is used / administered at a loading dose of 280-560 mg and a maintenance dose of 10-140 mg per day or 40-280 mg every other day.

Images