Treatment of venous thrombosis with rilzabrutinib and derivatives thereof

Abstract

Methods related to inhibiting CLEC-2 and treating venous thrombosis with certain BTK inhibitors and / or pharmaceutically acceptable salts thereof are provided.

Description

TREATMENT OF VENOUS THROMBOSIS WITH RILZABRUTINIB AND DERIVATIVES THEREOFTECHNICAL FI ELD

[0001] The present disclosure relates to the use of BTK inhibitors, or pharmaceutically acceptable salts thereof, to inhibit CLEC-2 and to treat venous thrombosis and / or thromboinflammation resulting from venous thrombosis.BACKGROUND

[0002] Deep vein thrombosis (DVT) is a debilitating disease affecting up to 900,000 people annually in the United States. DVT may be classified as an inflammatory disorder where the development of thrombosis is preceded by local inflammation, a phenomenon termed immunothrombosis (Campos et al., Neutrophil extracellular traps and inflammasomes cooperatively promote venous thrombosis in mice, Blood Advances 2021, vol 5(9), 2319-2324). Similarly, hepatic portal vein thrombosis (PVT) is a frequent complication of the continuous hepatic inflammation that occurs in patients with cirrhosis (Villa et al., Enoxaparin prevents portal vein thrombosis and liver decompensation in patients with advanced cirrhosis, Gastroenterology 2012, vol 143(5), 1253-1260). Inflammation and thrombosis are closely related, and uncontrolled immunothrombosis can cause thromboinflammation.

[0003] Novel, safe, and effective oral treatments to reduce the incidence of, or treat, venous thrombosis and thromboinflammation resulting from venous thrombosis, including in DVT and PVT patients, would represent a significant therapeutic advantage over the current standard of care. Accordingly, disclosed herein are novel methods for the treatment and / or prevention of venous thrombosis and thromboinflammation resulting from venous thrombosis, with specific compounds.SUMMARY

[0004] The platelet receptor C-type lectin-like receptor 2 (CLEC-2) has an important role in inflammation driven venous thrombosis. Mice deficient in CLEC-2 exhibit protection from liver thrombosis and PVT following Salmonella typhimurium (STm) infection, as well as in theinferior vena cava (IVC) stenosis model of deep vein thrombosis (DVT) (Hitchcock et al., Inflammation drives thrombosis after Salmonella infection via CLEC-2 on platelets, J. Clin. Invest. 2015, vol 125, 4429-4446; Payne et al., Mice with a deficiency in CLEC-2 are protected against deep vein thrombosis, Blood 2017, vol 129, 2013-2020). In both models, inflammation in the vessel wall causes upregulation of CLEC-2’ s ligand podoplanin, which then triggers platelet activation. In humans, patients with hepatic thrombosis and other inflammatory liver disorders (Chauhan et al., The platelet receptor CLEC-2 blocks neutrophil mediated hepatic recovery in acetaminophen induced acute liver failure, Nature Communications 2020, 1-12), and also in the venous valves of patients with DVT, exhibit upregulation of podoplanin (Nicolson et al., A rationale for blocking thromboinflammation in COVID-19 with Btk inhibitors, Platelets 2020, vol 31, 685-690).

[0005] CLEC-2 is a hem-immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor, signaling through Src, Syk, and Tec family kinases in similar pathway to the other platelet ITAM-containing receptors glycoprotein (GP) VI and FcyRIIA. Inhibition of Syk and the Tec family kinase BTK blocks CLEC-2, GPVI and FcyRIIA signaling (Spalton et al., The novel Syk inhibitor R406 reveals mechanistic differences in the initiation of GPVI and CLEC-2 signaling in platelets, J. Thromb. Haemost., 2009, vol. 7, 1192-1199; Nicolson et al., Inhibition of Btk by Btk-specific concentrations of ibrutinib and acalabrutinib delays but does not block platelet aggregation mediated by glycoprotein VI, Haematologica 2018, vol 103, 2097-2108; Nicolson et al., Low dose Btk inhibitors selectively block platelet activation by CLEC-2, Haematologica 2020, vol 106; Smith et al., Antiplatelet drugs block platelet activation by VITT patient serum, Blood 2021, vol 138, 2733-2740; Smith et al., The Btk inhibitor AB- 95 -LH34 potently inhibits atherosclerotic plaque-induced thrombus formation and platelet procoagulant activity, J. Thromb. Haemost. 2022). CLEC-2 has only a minor or no role in bleeding (Hughes et al., CLEC-2 is not required for platelet aggregation at arteriolar shear, J. Thromb. Haemost.2010, vol 8, 2328-2332; Suzuki-Inoue et al., Essential in Vivo Roles of the C-type Lectin Receptor CLEC-2 Embry onic / Neonatal Lethality of CLEC-2-Deficient Mice by Blood / Lymphatic Misconnections and Impaired Thrombus Formation of CLEC-2 -Deficient Platelets, J. Biol. Chem. 2010, vol 285, 24494-24507). CLEC-2 blockade represents a potentialstrategy for treating venous thrombosis and thromboinflammation resulting from elevated expression levels of podoplanin, with reduced bleeding side effects.

[0006] PRN1008 (also known as rilzabrutinib) is a 3rd generation BTK inhibitor in phase III trials for immune thrombocytopenia (ITP), with no bleeding side effects reported in these patients despite their low platelet counts (Kuter et al., Rilzabrutinib, an Oral BTK Inhibitor, in Immune Thrombocytopenia, New Engl. J. Med. 2022, vol 386, 1421-1431). PRN1008, and another BTK inhibitor PRN473 (also known as atuzabrutinib), have covalent and non-covalent binding regions enabling binding with high potency and long residence time to BTK (Langrish et al., Preclinical Efficacy and Anti-Inflammatory Mechanisms of Action of the Bruton Tyrosine Kinase Inhibitor Rilzabrutinib for Immune-Mediated Disease, J. Immunol. 2021, vol 206(7), 1454-1468; Owens et al, Discovery of Reversible Covalent Bruton’s Tyrosine Kinase Inhibitors PRN473 and PRN1008 (Rilzabrutinib), J. Med. Chem. 2022, vol 65, 5300-5316). These compounds inhibit the related kinases Tec, Txk, and Bmx with lower potency (Xing et al., Preclinical Mechanisms of Topical PRN473, a Bruton Tyrosine Kinase Inhibitor, in Immune- Mediated Skin Disease Models, Immunohorizons 2021, vol 5, 581-589), but have limited binding to off-target kinases such as the Src family kinases (SFK) (Langrish 2021).

[0007] Disclosed herein are studies related to the effects of BTK inhibitors PRN1008 and PRN473 on platelet signaling and function in vitro and ex vivo. Also disclosed are studies on the effects of PRN473 in in vivo models of venous thrombosis.

[0008] Bruton’s agammaglobulinemia tyrosine kinase (BTK) is an essential signaling element downstream of the B-cell receptor (BCR), Fc-gamma receptor (FcyR), and Fc-epsilon receptor (FCER). BTK is a non-receptor tyrosine kinase and a member of the TEC family of kinases. BTK is essential to B cell lineage maturation, and inhibition of BTK activity in cells produces phenotypic changes consistent with blockade of the BCR. Individuals with loss-of-function mutations in the BTK gene develop X-linked agammaglobulinemia (XLA) and lack mature B cells. Illustratively, BTK inhibition results in the down-regulation of various B-cell activities, including cell proliferation, differentiation, maturation, and survival, and the up-regulation of apoptosis.

[0009] Accordingly, a selective BTK inhibitor has the potential to target multiple pathways involved in inflammation. A selective BTK inhibitor may block the initiation and progression of various inflammatory diseases and mitigate tissue damage resulting from these diseases.Although individuals with loss of function mutations in the BTK gene have decreased humoral immunity and are susceptible to pyogenic bacterial and enterovirus infections, requiring treatment with intravenous immunoglobulin, inhibition of BTK in individuals with an intact immune system is not predicted to produce similar susceptibility to infection.

[0010] Some BTK inhibitors can cause bleeding and therefore would not be ideal candidates for use in subjects who are thrombocytopenic, anticoagulated, and / or have intracerebral bleeding (Langrish 2021). However, Compound (I) as disclosed has no impact on normal platelet function in vitro and has not been associated with bleeding in thrombocytopenic patients.

[0011] Compound (I) is a BTK inhibitor of the following structure:wherein *C is a stereochemical center. See PCT Publication No. WO 2014 / 039899, which is incorporated herein by reference, e.g.. Example 31.

[0012] (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l- yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile, having the following structure:is also known as PRN1008 and rilzabrutinib, and is referred to herein also as a compound of Formula (I). This compound has been disclosed in several patent publications, such as, e.g., PCT Publication Nos. WO 2014 / 039899, WO 2015 / 127310, WO 2016 / 100914, WO 2016 / 105531, WO 2018 / 005849, and WO 2021 / 150723, the contents of each of which are incorporated by reference herein.

[0013] Rilzabrutinib is a novel, highly selective, and potent small molecule inhibitor of non-T cell white blood cell signaling via B-cell receptor, FcyR, and / or FCER signaling of the BTK pathway. Rilzabrutinib functions as a reversible covalent BTK inhibitor and is capable of both non-covalently and covalently binding to its target; in particular, its reversible cysteine binding enables high selectivity and precise BTK inhibition without a permanent modification of proteins and peptides (Langrish 2021; Owens 2022; Smith et al, A phase I trial of PRN1008, a novel reversible covalent inhibitor of Bruton’s tyrosine kinase, in healthy volunteers, Br. J. Clin. Pharmacol. 2017, vol 83, 2367-2376). Taken together, these properties allow for enhanced selectivity and extended inhibition with low systemic exposure. In comparison to first and second generation BTKi, rilzabrutinib has shown minimal cross-reactivity with other molecules and is low risk for off-target effects (Smith 2017). Importantly, rilzabrutinib ’s reversible binding minimizes the likelihood of permanently modified peptides (Serafimova et al, Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles, Nat. Chem. Biol. 2012, 8(5), 471-476).

[0014] In addition, rilzabrutinib shows improved kinase selectivity relative to the covalent BTK inhibitor ibrutinib. Preclinical studies in a broad kinase enzyme inhibition panel showed that 1pM rilzabrutinib achieved >90% inhibition of just 6 of 251 kinases sharing a common cysteine in their active site. By contrast, 1 pM ibrutinib inhibited 21 kinases. Rilzabrutinib’ s IC50 values were 1.3 nM for BTK, 0.8 nM for tyrosine protein kinase TEC, 1.0 nM for bone marrow tyrosine kinase on chromosome X (BMX), 1.2 nM for receptor-like kinase (RLK), 6.3 nM for B cell lymphocyte kinase (BLK), and 11 nM for ERBB4. Further preclinical assays with rilzabrutinib showed that binding to BTK persisted while that for other TEC family members decayed rapidly over time.

[0015] Rilzabrutinib has also demonstrated a favorable safety profile in clinical studies. The efficacy of rilzabrutinib has been evaluated in both nonclinical and clinical studies. Rilzabrutinib demonstrated blockade of the rat Arthus reaction, full disease reversal of a rat collagen-induced arthritis model, and reduction in platelet loss in a mouse model of immune thrombocytopenia. Collectively, these and other studies have shown rilzabrutinib to be a safe and effective inhibitor of BTK.

[0016] Compound (II), also known as PRN473 and atuzabrutinib, and is referred to herein as a compound of Formula (II), is a BTK inhibitor of the following structure:where *C is a stereochemical center. This compound has been disclosed in e.g, WO 2012 / 158764 (see, e.g., Compound 125A / 125B in Table 1), which is incorporated herein by reference.

[0017] The effects of selective BTK inhibitors PRN1008 (rilzabrutinib) and PRN473 (atuzabrutinib) on platelet signaling and function mediated by CLEC-2 and the related ITAM-linked receptor GPVI were studied. Platelet CLEC-2 has an important role in venous thrombosis but minimal involvement in hemostasis, and can be blocked by inhibitors of the Tec family kinase BTK. Healthy donor and XLA human platelets were used to determine off-target inhibitor effects. Inferior vena cava (IVC) stenosis and Salmonella infection mouse models were used to assess antithrombotic effects in vivo.

[0018] In human platelets, PRN1008 and PRN473 strongly inhibited CLEC-2-mediated platelet activation to rhodocytin. GPVI-mediated platelet activation to collagen-related peptide (CRP) was also reduced, with only minimal inhibition to collagen. No off-target inhibition of SFKs was seen. PRN1008 treatment of BTK-deficient platelets resulted in minor additional inhibition of aggregation and tyrosine phosphorylation, indicating inhibition of Tec. No effect on GPCR- mediated platelet function was observed. PRN473 significantly reduced the number of thrombi in podoplanin positive vessels following Salmonella infection and the presence of IVC thrombosis following vein stenosis.

[0019] It was surprisingly determined that PRN1008 and PRN473 are potent inhibitors of human platelet CLEC-2, and that PRN473 reduced thrombosis in in vivo models. PRN473 and PRN1008 surprisingly block GPVI-mediated platelet activation. Previous studies looking at PRN1008 in GPVI-mediated platelet function did not show any such blockade (Langrish 2021). It was determined that high concentrations (2 - 5 pM) of drug were required to block CLEC-2 and GPVI-mediated platelet function in whole blood, likely due to a high degree of plasma protein binding not found in washed platelets. PRN1008 and PRN473 are effective inhibitors of CLEC-2 and GPVI mediated platelet function, and have minimal off-target effects on other kinases and platelet signaling pathways. PRN473 reduces thrombosis in two different mouse models of immunothrombosis.

[0020] In accordance herein, the following non-limiting embodiments are encompassed:

[0021] Embodiment 1. A method for inhibiting CLEC-2 in a human subject in need thereof comprising administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from: a compound of Formula (I),a compound of Formula (II),stereochemical center; and pharmaceutically acceptable salts thereof.

[0022] Embodiment 2. A method for inhibiting CLEC-2-mediated platelet activation in a human subject in need thereof comprising administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof.

[0023] Embodiment 3. A method for inhibiting at least one of CLEC-2 mediated platelet activation, thrombus formation, and thrombus propagation in a human subject in need thereof comprising administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof.

[0024] Embodiment 4. The method of Embodiment 3, wherein the thrombi are a result of deep vein thrombosis, venous thrombosis, portal vein thrombosis, or any combination thereof.

[0025] Embodiment 5. A method for reducing GPVLmediated platelet activation in a human subject in need thereof comprising administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof.

[0026] Embodiment 6. A method for inhibiting GPVI-mediated platelet activation in a human subject in need thereof comprising administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof.

[0027] Embodiment 7. A method for reducing the number of thrombi in podoplanin positive blood vessels in a human subject in need thereof comprising administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof.

[0028] Embodiment 8. The method of Embodiment 7, wherein the thrombi are a result of deep vein thrombosis, venous thrombosis, portal vein thrombosis, or any combination thereof.

[0029] Embodiment 9. The method of Embodiment 7, wherein the podoplanin positive blood vessels comprise veins.

[0030] Embodiment 10. A method for treating or preventing venous thrombosis in a human subject having elevated expression levels of podoplanin as compared to normal expression levels, comprising administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof.

[0031] Embodiment 11. The method of Embodiment 10, wherein the venous thrombosis is selected from deep vein thrombosis, portal vein thrombosis, or any combination thereof.

[0032] Embodiment 12. The method of Embodiment 10, wherein the method comprises treating deep vein thrombosis.

[0033] Embodiment 13. The method of Embodiment 10, wherein the method comprises preventing deep vein thrombosis.

[0034] Embodiment 14. The method of Embodiment 10, wherein the at least one BTK inhibitor comprises a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0035] Embodiment 15. The method of Embodiment 10, wherein the at least one BTK inhibitor comprises a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0036] Embodiment 16. A method for treating or preventing thromboinflammation in a human subject having an elevated expression level of podoplanin as compared to a normal expression level, comprising administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, wherein the thromboinflammation results from venous thrombosis.

[0037] Embodiment 17. The method of Embodiment 16, wherein the thromboinflammation results from deep vein thrombosis, portal vein thrombosis, or any combination thereof.

[0038] Embodiment 18. The method of Embodiment 16, wherein the at least one BTK inhibitor comprises a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0039] Embodiment 19. The method of Embodiment 16, wherein the at least one BTK inhibitor comprises a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0040] Embodiment 20. A method for treating or preventing venous thrombosis in a human subject, comprising the steps of:(a) evaluating the subject’s expression levels of podoplanin; and(b) when the subject’s expression levels of podoplanin are elevated as compared to normal levels, administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof.

[0041] Embodiment 21. The method of Embodiment 20, wherein the venous thrombosis is selected from deep vein thrombosis, portal vein thrombosis, or any combination thereof.

[0042] Embodiment 22. The method of Embodiment 20, wherein the method comprises treating deep vein thrombosis.

[0043] Embodiment 23. The method of Embodiment 20, wherein the method comprises preventing deep vein thrombosis.

[0044] Embodiment 24. The method of Embodiment 20, wherein the at least one BTK inhibitor comprises a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0045] Embodiment 25. The method of Embodiment 20, wherein the at least one BTK inhibitor comprises a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0046] Embodiment 26. A method for treating or preventing thromboinflammation in a human subject, comprising the steps of:(a) evaluating the subject’s expression levels of podoplanin; and(b) when the subject’s expression levels of podoplanin are elevated as compared to normal levels, administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, wherein the thromboinflammation results from venous thrombosis.

[0047] Embodiment 27. The method of Embodiment 26, wherein the venous thrombosis is selected from deep vein thrombosis, portal vein thrombosis, or any combination thereof.

[0048] Embodiment 28. The method of Embodiment 26, wherein the method comprises treating thromboinflammation.

[0049] Embodiment 29. The method of Embodiment 26, wherein the method comprises preventing thromboinflammation.

[0050] Embodiment 30. The method of Embodiment 26, wherein the at least one BTK inhibitor comprises a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0051] Embodiment 31. The method of Embodiment 26, wherein the at least one BTK inhibitor comprises a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0052] Embodiment 32. The method of any one of Embodiments 1-14, 16-18, 20-24, and 26-30, wherein the at least one BTK inhibitor is a compound of Formula (I) selected from (R)-2-[3-[4- amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4- methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile, individual (E)- or (Z)- isomers thereof, and pharmaceutically acceptable salts thereof.

[0053] Embodiment 33. The method of Embodiment 32, wherein the at least one BTK inhibitor is a compound of Formula (I) selected from the (E) isomer of (R)-2-[3-[4-amino-3-(2-fluoro-4- phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3- yl)piperazin-l-yl]pent-2-enenitrile and pharmaceutically acceptable salts thereof.

[0054] Embodiment 34. The method of Embodiment 32, wherein the at least one BTK inhibitor is a compound of Formula (I) selected from the (Z) isomer of (R)-2-[3-[4-amino-3-(2-fluoro-4- phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3- yl)piperazin-l-yl]pent-2-enenitrile and pharmaceutically acceptable salts thereof.

[0055] Embodiment 35. The method of Embodiment 32, wherein the at least one BTK inhibitor is a compound of Formula (I) selected from a mixture of (E) and (Z) isomers of (R)-2-[3-[4- amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4- methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile and pharmaceutically acceptable salts thereof.

[0056] Embodiment 36. The method of any one of Embodiments 1-13, 15-17, 19-23, 25-29, and 31, wherein the at least one BTK inhibitor is a compound of Formula (II) selected from (R)-2-(3- (4-amino-3 -(2-fluoro-4-phenoxyphenyl)- lH-pyrazolo[3 ,4-d]pyrimidin- 1 -y l)piperi dine- 1 - carbonyl)-4,4-dimethylpent-2-enenitrile, (S)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH- pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile, a mixture of (R)-2-(3 -(4-amino-3 -(2-fluoro-4-phenoxyphenyl)- lH-pyrazolo[3 ,4-d]pyrimidin- 1 -yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile and (S)-2-(3-(4-amino-3-(2-fluoro-4- phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2- enenitrile, an individual (E)- or (Z)- isomer of any of the above compounds, and pharmaceutically acceptable salts of any of the above compounds.

[0057] Embodiment 37. The method of Embodiment 36, wherein the at least one BTK inhibitor is a compound of Formula (II) selected from the (E) isomer of (R)-2-(3-(4-amino-3-(2-fluoro-4- phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2- enenitrile and pharmaceutically acceptable salts thereof.

[0058] Embodiment 38. The method of Embodiment 36, wherein the at least one BTK inhibitor is a compound of Formula (II) selected from the (Z) isomer of (R)-2-(3-(4-amino-3-(2-fluoro-4- phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2- enenitrile and pharmaceutically acceptable salts thereof.

[0059] Embodiment 39. The method of Embodiment 36, wherein the at least one BTK inhibitor is a compound of Formula (II) selected from a mixture of (E) and (Z) isomers of (R)-2-(3-(4- amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)- 4,4-dimethylpent-2-enenitrile and pharmaceutically acceptable salts thereof.

[0060] Embodiment 40. Use of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, in the manufacture of a medicament for inhibiting CLEC-2 in a human subject.

[0061] Embodiment 41. Use of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, in the manufacture of a medicament for inhibiting at least one of CLEC-2 mediated platelet activation, thrombus formation, and thrombus propagation in a human subject.

[0062] Embodiment 42. Use of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, in the manufacture of a medicament for inhibiting or reducing GPVI-mediated platelet activation in a human subject.

[0063] Embodiment 43. Use of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, in the manufacture of a medicament for reducing the number of thrombi in podoplanin positive blood vessels in a human subject.

[0064] Embodiment 44. Use of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, in the manufacture of a medicament for treating or preventing venous thrombosis in a human subject having elevated expression levels of podoplanin as compared to normal expression levels.

[0065] Embodiment 45. Use of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, in the manufacture of a medicament for treating or preventing thromboinflammation in a human subject having elevated expression levels of podoplanin as compared to normal expression levels, wherein the thromboinflammation results from venous thrombosis.

[0066] Embodiment 46. At least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in inhibiting CLEC-2 in a human subject.

[0067] Embodiment 47. At least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in inhibiting at least one of CLEC-2 mediated platelet activation, thrombus formation, and thrombus propagation in a human subject.

[0068] Embodiment 48. At least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in inhibiting or reducing GPVI-mediated platelet activation in a human subject.

[0069] Embodiment 49. At least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in reducing the number of thrombi in podoplanin positive blood vessels in a human subject.

[0070] Embodiment 50. At least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in treating or preventing venous thrombosis in a human subject having elevated expression levels of podoplanin as compared to normal expression levels.

[0071] Embodiment 51. At least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in treating or preventing thromboinflammation in a human subject having elevated expression levels of podoplanin as compared to normal expression levels, wherein the thromboinflammation results from venous thrombosis.

[0072] Embodiment 52. At least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in inhibiting CLEC-2 in a human subject in need thereof.

[0073] Embodiment 53. At least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in inhibiting at least one of CLEC-2 mediated platelet activation, thrombus formation, and thrombus propagation in a human subject in need thereof, optionally wherein the thrombi are a result of deep vein thrombosis, venous thrombosis, portal vein thrombosis, or any combination thereof.

[0074] Embodiment 54. At least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in reducing or inhibiting GPVI-mediated platelet activation in a human subject in need thereof.

[0075] Embodiment 55. At least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in reducing the number of thrombi in podoplanin positive blood vessels in a human subject in need thereof, optionally wherein the thrombi are a result of deep vein thrombosis, venous thrombosis, portal vein thrombosis, or any combination thereof, and optionally wherein the podoplanin positive blood vessels comprise veins.

[0076] Embodiment 56. At least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in treating orpreventing venous thrombosis in a human subject having elevated expression levels of podoplanin as compared to normal expression levels, optionally wherein the venous thrombosis is selected from deep vein thrombosis, portal vein thrombosis, or any combination thereof.

[0077] Embodiment 57. At least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in treating or preventing thromboinflammation in a human subject having an elevated expression level of podoplanin as compared to a normal expression level, wherein the thromboinflammation results from venous thrombosis, optionally wherein the thromboinflammation results from deep vein thrombosis, portal vein thrombosis, or any combination thereof.

[0078] Embodiment 58. At least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in treating or preventing venous thrombosis in a human subject, the use comprising the steps of (a) evaluating the subject’s expression levels of podoplanin, and (b) when the subject’s expression levels of podoplanin are elevated as compared to normal levels, administering to the human subject the at least one BTK inhibitor, optionally wherein the venous thrombosis is selected from deep vein thrombosis, portal vein thrombosis, or any combination thereof.

[0079] Embodiment 59. At least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in treating or preventing thromboinflammation in a human subject, the use comprising the steps of (a) evaluating the subject’s expression levels of podoplanin, and (b) when the subject’s expression levels of podoplanin are elevated as compared to normal levels, administering to the human subject the at least one BTK inhibitor, wherein the thromboinflammation results from venous thrombosis, optionally wherein the venous thrombosis is selected from deep vein thrombosis, portal vein thrombosis, or any combination thereof.

[0080] Embodiment 60. The at least one BTK inhibitor for use of any one of Embodiments 52- 59, comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0081] Embodiment 61. The at least one BTK inhibitor for use of any one of Embodiments 52- 59, comprising a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

[0082] Embodiment 62. The at least one BTK inhibitor for use of Embodiment 60, wherein the at least one BTK inhibitor is a compound of Formula (I) selected from (R)-2-[3-[4-amino-3-(2- fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4- (oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile, individual (E)- or (Z)- isomers thereof, a mixture of (E) and (Z) isomers thereof, and pharmaceutically acceptable salts of any of the above compounds.

[0083] Embodiment 63. The at least one BTK inhibitor for use of Embodiment 61, wherein the at least one BTK inhibitor is a compound of Formula (II) selected from (R)-2-(3-(4-amino-3-(2- fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4- dimethylpent-2-enenitrile, (S)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4- d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile, a mixture of (R)-2-(3-(4- amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)- 4,4-dimethylpent-2-enenitrile and (S)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH- pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile, an individual (E)- or (Z)- isomer of any of the above compounds, a mixture of (E) and (Z) isomers of any of the above compounds, and pharmaceutically acceptable salts of any of the above compounds.

[0084] Additional objects and advantages will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0085] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0086] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, serve to explain the principles described herein.BRI EF DESCRIPTION OF THE DRAWINGS

[0087] Figures 1A-1D show that PRN1008 and PRN473 inhibit CLEC-2 and GPVI mediated signalling. Healthy donor washed platelets (4xl08 / ml) were incubated with vehicle (0.02% DMSO) or indicated concentration (50, 200, 500 or 2000 nM) of BTK inhibitors PRN1008 and PRN473 for 1 hour then stimulated with snake venom toxin rhodocytin 300 nM (Figures 1 A and IB) or collagen related peptide (CRP) 10 pg / ml (Figures 1C and ID) for 180 seconds in the presence of eptifibatide (9 pM) and lysed with reducing sample buffer. Whole cell lysates were then separated by SDS-PAGE and western blotted for tyrosine phosphorylation of indicated proteins. Total LAT was used as a loading control. Figures 1A and 1C show representative western blots for stimulation with rhodocytin (Figure 1A) and CRP (Figure 1C). Figures IB and ID show normalized densitometry quantification for stimulation with rhodocytin (Figure IB) and CRP (Figure ID). Mean ± SEM of four identical experiments.

[0088] Figures 2A-2B show that PRN1008 and PRN473 inhibit CLEC-2- and GPVI-mediated platelet aggregation. Healthy donor washed platelets (2xl08 / ml) were incubated with vehicle (0.02% DMSO) or indicated concentration (50, 200, 500 or 2000 nM) of BTK inhibitors PRN1008 and PRN473 for 1 hour before platelet aggregation to snake venom toxin rhodocytin (A) 100 nM and (B) 300 nM; collagen (C) 3 pg / ml and (D) 10 pg / ml; collagen-related peptide (CRP) (E) 1 pg / ml and (F) 3 pg / ml; or (G) thrombin (0.04 U / ml) and thromboxane A2 mimetic U46619 (3 pM), which were measured by lumi-aggregometry. Figure 2A shows (i) representative traces for aggregation to rhodocytin at (A) 100 nM and (B) 300 nM, and to collagen at (C) 3 pg / ml and (D) 10 pg / ml, and (ii) quantification of the aggregation data. Figure 2B shows (i) representative traces for aggregation to collagen-related peptide (CRP) at (E) 1 pg / ml and (F) 3 pg / ml, and to (G) thrombin at (0.04 U / ml) and thromboxane A2 mimetic U46619 at (3 pM); (ii) quantification of the CRP aggregation data; and (iii) quantification of the thrombin and U46619 aggregation data. Mean aggregation ± SEM, n=6.

[0089] Figures 3A-3B show that PRN1008 inhibits CLEC-2 and GPVI-mediated platelet activation and CLEC-2 induced thrombus formation in whole blood. Citrated healthy donor whole blood was incubated with vehicle (0.02% DMSO) or indicated concentration (0.2, 0.5, 2 or 5 pM) of BTK inhibitors PRN1008 and PRN473 for 1 hour. Figure 3 A (i+ii) shows plateletactivation, indicated by activated integrin allb03 (PAC-1) and P-selectin surface expression, that was then assessed by flow cytometry in response to stimulation with (A) snake venom toxin rhodocytin (100 and 300 nM) or (B) collagen related peptide (CRP, 3 and 10 pg / ml) or (C) thrombin receptor activating peptide (TRAP, 30 pM). Mean ± SEM, n=4-8. Figure 3B (Di) shows representative images of citrated healthy donor whole blood that was incubated with vehicle (0.02% DMSO) or indicated concentration (0.5 or 5 pM) of BTK inhibitors PRN1008 or PRN473 for 1 hour. Blood was then labelled with DiOCe dye (10 minutes) and perfused over recombinant podoplanin (10 pg / ml) coated channels at 150 s'1for 8 minutes. Figure 3B (ii+iv) shows quantification of platelet surface area coverage and (iii+v) shows mean aggregate size in images captured every 10 seconds. Mean ± SEM, n=3-4 per condition. Scale bar 100 pm. Statistical analysis by two-way ANOVA with Tukey’s correction for multiple comparisons. *p<0.05, Comparison to vehicle indicated by color.

[0090] Figures 4A-4C show that PRN1008 blocks GPVI-mediated aggregation and PLCy2 phosphorylation in XLA platelets at low agonist concentrations. Healthy donor and XLA washed platelets (2xl08 / ml) were incubated with vehicle (0.02% DMSO) or the indicated concentration (0.5 and 5 pM) of PRN1008 for 1 hour before platelet aggregation to (Figure 4A, Ai and Aii) snake venom toxin rhodocytin 300 nM, collagen at (Figure 4A, Bi and Bii) 3 pg / ml and at (Figure 4A, Ci and Cii) 10 pg / ml, and (Figure 4A, Di and Dii) collagen-related peptide (CRP, 3 pg / ml) was measured by lumi-aggregometry. Figures 4A (i) show representative traces and Figures 4A (ii) show quantification; n=3 for healthy donor, n=2 for XLA. Healthy donor and XLA washed platelets (4xl08 / ml) were incubated with vehicle (0.02% DMSO) or the indicated concentration (0.5 or 5 pM) ofPRN1008 for 1 hour then stimulated with (Figure 4B) collagen related peptide (CRP) 10 pg / ml or (Figure 4C) snake venom toxin rhodocytin 300 nM pg / ml for 180 seconds in the presence of eptifibatide (9 pM) and lysed with reducing sample buffer. Whole cell lysates were then separated by SDS-PAGE and western blotted for tyrosine phosphorylation of indicated proteins. Total LAT was used as a loading control. Figure 4B shows a western blot for stimulation with CRP, and Figure 4C shows a western blot for stimulation with rhodocytin, n=l.

[0091] Figure 5 shows that PRN1008 and PRN473 block platelet activation in mouse whole blood. (A) Mouse washed platelets (2xl08 / ml) were incubated with vehicle (0.02% DMSO) or indicated concentration (0.5, 2, 5 or 10 pM) of BTK inhibitors PRN1008 or PRN473 for 1 hour. Platelet activation, indicated by activated integrin allb[33 (JON / A) and P-selectin surface expression, was then assessed by flow cytometry in response to stimulation with (A) snake venom toxin rhodocytin at (i) 100 nM and (ii) 300 nM; or (B) collagen related peptide (CRP) at (i) 3 pg / ml and (ii) 10 pg / ml; or (C) PAR4 receptor activating peptide (100 or 500 nM). Mean ± SEM, n=3-6.

[0092] Figures 6A-6C show that PRN473 reduces salmonella-induced liver thrombosis in mice. WT mice were fed control or PRN473 formulated diet for 7 days then (Figure 6A, A) had spleens harvested for BTK occupancy analysis or (Figure 6A, B) citrated whole blood taken for platelet activation analysis by flow cytometry, indicated by activated integrin aflbp3 (JON / A) and P-selectin surface expression, following stimulation with snake venom toxin rhodocytin (100 and 300 nM), collagen related peptide (CRP, 3 and 10 pg / ml) or PAR4 receptor activating peptide (500 nM), as shown in the graphs of Figure 6A. Mean ± SEM. n = 3. Statistical analysis by two-way ANOVA with Sidak’s correction for multiple comparisons. *p<0.05, **p<0.01, ****p<0.0001. WT mice fed control or PRN473 formulated diet were infected with 5 x 105CFU S. Typhimurium (STm) on day 7, with thrombi in portal vein assessed at day 14 (7 days after infection). Figure 6B shows (ii) quantification of thrombus area per unit vessel area, (iii) quantification of number of thrombi, (iv) quantification of number of podoplanin expressing vessels, and (v) number of thrombi in podoplanin positive vessels. n=6. Figure 6C shows peripheral blood counts of STm infected mice at baseline and 7 days after infection for (i) platelets, (ii) MPV, (iii) WBC, and (iv) lymphocytes. Mean ± SEM, n=4.

[0093] Figures 7A-7C show that PRN473 inhibits inferior vena cava thrombosis in mice. Figure 7A: (A) WT mice were gavaged daily with vehicle or PRN473 (80 mg / kg) for 4 days then had (i) platelet activation measured in citrated whole blood by flow cytometry, as indicated by activated integrin allbp3 (JON / A) and P-selectin surface expression, following stimulation with snake venom toxin rhodocytin (100 and 300 nM), collagen related peptide (CRP, 3 and 10 pg / ml) or thrombin (0.03 and 0.1 U / ml). GPRP was added to thrombin stimulation to preventfibrin clot formation. Mean ± SEM, n = 3. Statistical analysis by two-way ANOVA with Sidak’s correction for multiple comparisons. Figure 7B: (B) Inferior vena cava (IVC) stenosis model of deep vein thrombosis (DVT) was performed on WT mice gavaged with vehicle or PRN473 (80 mg / kg) for 4 days. Mice were culled 6 hours post IVC ligation and had (i) spleens harvested for BTK occupancy analysis, (ii) citrated plasma taken for drug concentration measurements, and assessment for thrombus (iii) prevalence, (iv) length and (v) weight. Median. n=16-18 mice / condition for splenic BTK occupancy and thrombus analysis and n=17-21 mice / condition for plasma drug concentration measurements. Statistical analysis by Mann Whitney test. *p< 0.01, ***p=0.0002, ****p<0.0001. Figure 7C: Inferior vena cava (IVC) stenosis model of deep vein thrombosis (DVT) was performed on WT mice gavaged with PRN473 (80 mg / kg) for 4 days. Mice were culled 6 hours post IVC ligation and had citrated plasma taken for drug concentration measurements, and assessment for thrombus (A) length and (B) weight. N=18 mice for plasma drug concentration measurements.

[0094] Figures 8A-8B show PRN1008 and PRN473 inhibit platelet dense granule secretion in response to GPVI and CLEC-2 ligation. Healthy donor washed platelets (2xl08 / ml) were incubated with vehicle (0.02% DMSO) or indicated concentration (50, 200, 500 or 2000 nM) of BTK inhibitors PRN1008 or PRN473 for 1 hour before platelet dense granule secretion to (A) ITAM receptor agonists; snake venom rhodocytin (i) 100 nM and (ii) 300 nM; collagen (iii) 3 pg / ml and (iv) 10 pg / ml; collagen-related-peptide (CRP) (v) 1 pg / ml and (vi) 3 pg / ml (Figure 8A); and to (B) GPCR receptor agonists (i) thrombin 0.04 U / ml, (ii) U46619 3 pM, (iii) thrombin 0.1 U / ml and U46619 10 pM (Figure 8B). Mean ± SEM, n=4-10 per condition.

[0095] Figure 9 shows that PRN1008 inhibits CLEC-2 and GPVI-mediated platelet activation and CLEC-2 induced thrombus formation in whole blood. Citrated healthy donor whole blood was incubated with vehicle (0.02% DMSO) or indicated concentration (0.5 or 5 pM) of BTK inhibitors PRN1008 or PRN473 for 1 hour. Blood was then labelled with DIOCe dye (10 minutes) and perfused over collagen (200 pg / ml) coated channels at 1000 s'1for 8 minutes. Figure 9 shows (A) representative images, and (Bi+iii) quantification of platelet surface area coverage and (Bii+iv) aggregate size in images captured every 30 seconds are shown. Mean ±SEM, n=3-7 per condition. Statistical analysis by two-way ANOVA with Tukey’s correction for multiple comparisons. *p<0.05, Comparison to vehicle indicated by color. Scale bar 100 pm.

[0096] Figures 10A-10B show that PRN473 reduces platelet adhesion and activation when flowed over collagen and fibrinogen. Heparinized mouse blood was treated with 40 pM PPACK and then recalcified before perfusion over (A) collagen (Figure 10A) and (B) fibrinogen (Figure 10B) microspots in the Maastricht flow chamber at arterial shear (1000 s'1) for 4 min. End point images were obtained and analyzed for (i) mean platelet coverage and (ii) multilayered thrombi. Thrombi were perfused with antibodies and imaged to assess platelet activation markers (iii) Phosphatidyl Serine (PS), (iv) P-selectin and (v) activated ullbp3 expression. Mean ± SEM, n=3-4 per condition. Statistical analysis by 2 way ANOVA.DETAILED DESCRIPTIONDefinitions

[0097] Unless otherwise stated, the following terms used in the specification and claims are defined for the purposes of this disclosure and have the following meanings. All undefined technical and scientific terms used in this disclosure have the meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0098] As used herein, “a” or “an” entity refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound unless stated otherwise. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.

[0099] As used herein, the term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 5%. In an embodiment, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10%. With regard to specific values, it should be understood that specific values described herein represent median, mean, or statistical numbers, unless otherwise provided.

[0100] As used herein, the term “active pharmaceutical ingredient” or “therapeutic agent” (“API”) refers to a biologically active compound.

[0101] As used herein, the terms “administer,” “administering,” or “administration” herein refer to providing, giving, dosing, and / or prescribing by either a health practitioner or an authorized agent and / or putting into, taking or consuming by the patient or person herself or himself. For example, “administration” of an API to a patient refers to any route (e.g., oral delivery) of introducing or delivering the API to the patient. Administration includes selfadministration and administration by another.

[0102] As used herein, “BID” and “bid” are used interchangeably to refer to twice a day.

[0103] As used herein, “CLEC-2” refers to C-type lectin-like receptor 2, also known asCLEC-lb, that is expressed on platelets and certain immune cells. CLEC-2 binds to various ligands including the mucin-like protein podoplanin (PDPN) and the snake venom toxin rhodocytin. CLEC-2 is involved in platelet activation, which can lead to thrombus formation and / or thrombus propagation.

[0104] As used herein, a “pharmaceutically acceptable carrier or excipient” means a carrier or an excipient that is useful in preparing a pharmaceutical composition that is generally safe, and neither biologically nor otherwise undesirable, such as, e.g., a carrier or an excipient that is acceptable for mammalian pharmaceutical use.

[0105] As used herein, the term “pharmaceutically acceptable salt” refers to a salt form, e.g., an acid addition salt, of an active pharmaceutical agent that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the API of which the salt is made. Pharmaceutically acceptable salts are well known in the art and include those derived from suitable inorganic and organic acids. Such salts include, but are not limited to, salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like; or formed with organic acids such as formic acid, acetic acid, propionic acid, hexanoic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2- ethanedisulfonic acid, benzenesulfonic acid, 4-toluenesulfonic acid, and the like. S. M. Berge etal., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19.

[0106] As used herein, “podoplanin” refers to the mucin-type transmembrane glycoprotein encoded by the PDPN gene. Podoplanin is an endogenous ligand for CLEC-2 and activates CLEC-2 expressing platelets. Elevated expression levels of podoplanin as compared to normal levels indicate inflammation.

[0107] “Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease, disorder, or condition in a subject that may be predisposed to the disease, disorder, or condition but has not yet been diagnosed with the disease, disorder, or condition. Unless otherwise specified, the terms “prevent,” “prevention,” “reduce,” “inhibit,” or “prevent” do not denote or require complete prevention over all time.

[0108] As used herein, the terms “PRN1008,” “rilzabrutinib,” “(R)-2-[3-[4-amino-3-(2- fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4- (oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile;” “the compound of Formula (I);” and “2-[(3R)-3- [4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]-pyrimidin-l-yl]piperidine-l-carbonyl]- 4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile” are used interchangeably to refer to a compound having the structure:which is also referred to as 2-[(3R)-2-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4- d]pyrimidin-l-yl]piperdine-l-carbonyl]-4-methyl-4[4-(oxetan-3-yl)piperazin-l-yl]-(E and Z)- pent-2-enenitrile; (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine- 1 -carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin- 1 -yl]pent-2-enenitrile; 1 - piperidinepropanenitrile, 3-[4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4- d]pyrimidin-l-yl]-a-[2-methyl-2-[4-(3-oxetanyl)-l-piperazinyl]propylidene]-P-oxo-, (3R)-; (EZ)- 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l- carbonyl]-4-methyl-4[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile; and also by the International Nonproprietary Names for Pharmaceutical Substances (INN) as published by theWorld Health Organization (https: / / cdn.who.int / media / docs / default-source / international- nonproprietary-names-(inn) / pll21.pdf?sfvrsn=69617906_15&download=true) having the following structure:The compound of Formula (I) includes E and Z isomers, as indicated by the wavy bond in the structure shown above. The compound of Formula (I) may be present as a salt form.

[0109] An isomer of rilzabrutinib may contain the corresponding (Z) isomer as an impurity in less than about 1% by weight; a dose of the (Z) isomer of rilzabrutinib may contain the corresponding (E) isomer as an impurity in less than about 1% by weight. When rilzabrutinib is denoted as a mixture of (E) and (Z) isomers of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy- phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3- yl)piperazin-l-yl]pent-2-enenitrile, it means that the amount of (E) or (Z) isomer in the mixture is greater than about 1% by weight. In some embodiments, the molar ratio of (E) to (Z) isomer is 9: 1. Rilzabrutinib or a pharmaceutically acceptable salt thereof may also be referred to herein as a “drug,” “active agent,” “a therapeutically active agent,” or “API.”

[0110] As used herein, “PRN473,” “the compound of Formula (II)” and “atuzabrutinib,” are used interchangeably to refer to the (E) isomer, (Z) isomer, or a mixture of (E) and (Z) isomers of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile, (S)-2-(3-(4-amino-3-(2-fluoro-4- phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2- enenitrile, or a mixture of (R) and (S) enantiomers of 2-(3-(4-amino-3-(2-fluoro-4- phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2- enenitrile, which has the following structure:where *C is a stereochemical center.

[0111] When the compound of Formula (II) is denoted as (R)-2-(3-(4-amino-3-(2-fluoro- 4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile, it may also contain the corresponding (S) enantiomer as an impurity in less than 5% by weight, such as, e.g., an impurity in less than 1% by weight. Accordingly, when the compound of Formula (II) is denoted as a mixture of (R) and (S) enantiomers of 2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4- dimethylpent-2-enenitrile, the amount of (R) or (S) enantiomer in the mixture is greater than 1% by weight. Similarly, when compound of Formula (II) is denoted as the (E) isomer, it may contain the corresponding (Z) isomer as an impurity in less than 5% by weight, such as less than 1% by weight. Accordingly, when the compound of Formula (II) is denoted as a mixture of (E) and (Z) isomers of 2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin- l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile, the amount of (E) or (Z) isomer in the mixture is greater than 1% by weight.

[0112] In some embodiments, the compound of Formula (II) is a mixture of (R) and (S) enantiomers of 2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l- yl)piperidine- 1 -carbonyl)-4,4-dimethylpent-2-enenitrile.

[0113] In some embodiments, the compound of Formula (II) is substantially (R)-2-(3-(4- amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)- 4,4-dimethylpent-2-enenitrile. In some embodiments, the compound of Formula (II) is at least about 75%, e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, by weight (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l- yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile. In some embodiments, the compound of Formula (II) is at least about 95% by weight (R)-2-(3-(4-amino-3-(2-fluoro-4- phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2- enenitrile.

[0114] As used herein, the term “therapeutically effective amount” refers to that an of a compound that produces the desired effect for which it is administered (e.g., inhibiting CLEC-2 or CLEC-2 mediated platelet activation; or reducing the severity of venous thrombosis or a symptom of venous thrombosis; or reducing the severity of thromboinflammation resulting from elevated expression levels of podoplanin or a symptom of thromboinflammation resulting from elevated expression levels of podoplanin; or lessening the severity of DVT or a symptom of DVT; or lessening the severity of PVT or a symptom of PVT). The exact amount of an effective dose will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0115] As used herein, “thromboinflammation” refers to the coordinated activation of thrombotic and inflammatory response that can develop into thrombosis. In some embodiments, DVT and PVT may result from thromboinflammation.

[0116] As used herein, the term “treat,” “treating,” or “treatment,” when used in connection with a disorder or condition, includes any effect, e.g., lessening, reducing, modulating, ameliorating, or eliminating, that results in the improvement of the disorder orcondition. Improvements in or lessening the severity of any symptom of the disorder or condition can be readily assessed according to standard methods and techniques known in the art.

[0117] As used herein, “venous thrombosis” encompasses deep vein thrombosis (DVT) and portal vein thrombosis (PVT). Venous thrombosis is a disorder characterized by thrombus (blood clot) formation in a vein. When a blood vessel is injured, platelets and fibrin form a blood clot to prevent blood loss. In some embodiments, venous thrombosis may be induced by inflammation, such as thromboinflammation. PVT occurs when a blood clot occurs in the hepatic portal vein that supplies blood to the liver.

[0118] In accordance with the description, provided herein is a method for inhibiting CLEC-2 in a human subject in need thereof, the method including administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from: a compound of Formula (I),a compound of Formula (II),stereochemical center; and pharmaceutically acceptable salts thereof.

[0119] Also provided herein is a method for inhibiting CLEC-2-mediated platelet activation in a human subject in need thereof, the method including administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof.

[0120] In some embodiments, a method for inhibiting at least one of CLEC-2 mediated platelet activation, thrombus formation, and thrombus propagation in a human subject in need thereof is provided, the method including administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof.

[0121] Further provided herein is a method for reducing GPVI-mediated platelet activation in a human subject in need thereof, the method including administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof.

[0122] Additionally provided herein is a method for inhibiting GPVI-mediated platelet activation in a human subject in need thereof, the method including administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from acompound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof.

[0123] In some embodiments, provided herein is a method for reducing the number of thrombi in podoplanin positive blood vessels in a human subject in need thereof, the method including administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof

[0124] Provided herein is additionally a method for treating or preventing venous thrombosis in a human subject having elevated expression levels of podoplanin as compared to normal expression levels, the method including administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof.

[0125] Further provided herein is a method for treating or preventing thromboinflammation in a human subject having an elevated expression level of podoplanin as compared to a normal expression level, the method including administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof. In certain embodiments, the thromboinflammation results from venous thrombosis. In some embodiments, the thromboinflammation results from deep vein thrombosis, portal vein thrombosis, or a combination of deep vein thrombosis and portal vein thrombosis.

[0126] Additionally provided herein is a method for treating or preventing venous thrombosis in a human subject, the method including the steps of (a) evaluating the subject’s expression levels of podoplanin, and (b) when the subject’s expression levels of podoplanin are elevated as compared to normal levels, administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof.

[0127] Further provided herein is a method for treating or preventing thromboinflammation in a human subject, the method including the steps of: (a) evaluating thesubject’s expression levels of podoplanin; and (b) when the subject’s expression levels of podoplanin are elevated as compared to normal levels, administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, wherein the thromboinflammation results from venous thrombosis.

[0128] In certain embodiments, the disclosed method involves thrombi that are a result of deep vein thrombosis, venous thrombosis, portal vein thrombosis, or any combination of deep vein thrombosis, venous thrombosis, and portal vein thrombosis.

[0129] In some embodiments, the disclosed method involves podoplanin positive blood vessels that include veins. In some embodiments, the disclosed method involves podoplanin positive blood vessels that are veins.

[0130] In certain embodiments, the disclosed method involves venous thrombosis that is selected from deep vein thrombosis, portal vein thrombosis, or any combination thereof. In some embodiments, the disclosed method involves venous thrombosis that is selected from deep vein thrombosis and portal vein thrombosis.

[0131] In some embodiments, the disclosed method involves venous thrombosis that is deep vein thrombosis or portal vein thrombosis. In some embodiments, the disclosed method involves venous thrombosis that is a combination of deep vein thrombosis and portal vein thrombosis.

[0132] In some embodiments, the disclosed method involves treating deep vein thrombosis. In certain embodiments, the disclosed method involves preventing deep vein thrombosis. In some embodiments, the disclosed method involves treating and preventing deep vein thrombosis.

[0133] In some embodiments, the disclosed method involves treating thromboinflammation. In certain embodiments, the disclosed method involves preventing thromboinflammation. In some embodiments, the disclosed method involves treating and preventing thromboinflammation.

[0134] Provided herein are methods that involve administration of at least one BTK inhibitor wherein the at least one BTK inhibitor includes a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the methods include administration of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0135] Also provided herein are methods that involve administration of at least one BTK inhibitor wherein the at least one BTK inhibitor includes a compound of Formula (II), or a pharmaceutically acceptable salt thereof. In some embodiments, the methods include administration of a compound of Formula (II) or a pharmaceutically acceptable salt thereof.

[0136] In some embodiments, the disclosed method involves administration of at least one BTK inhibitor, wherein the at least one BTK inhibitor is a compound of Formula (I) selected from (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l- yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile, individual (E)- or (Z)- isomers thereof, and pharmaceutically acceptable salts thereof.

[0137] In certain embodiments, the disclosed method involves administration of at least one BTK inhibitor, wherein the at least one BTK inhibitor is a compound of Formula (I) selected from the (E) isomer of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4- d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2- enenitrile and pharmaceutically acceptable salts thereof.

[0138] In some embodiments, the disclosed method involves administration of at least one BTK inhibitor, wherein the at least one BTK inhibitor is a compound of Formula (I) selected from the (Z) isomer of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4- d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2- enenitrile and pharmaceutically acceptable salts thereof.

[0139] In some embodiments, the disclosed method involves administration of at least one BTK inhibitor, wherein the at least one BTK inhibitor is a compound of Formula (I) selected from a mixture of (E) and (Z) isomers of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy- phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3- yl)piperazin-l-yl]pent-2-enenitrile and pharmaceutically acceptable salts thereof.

[0140] In some embodiments, the disclosed method involves administration of at least one BTK inhibitor, wherein the at least one BTK inhibitor is a compound of Formula (II) selected from (R)-2-(3 -(4-amino-3 -(2-fluoro-4-phenoxyphenyl)- lH-pyrazolo[3 ,4-d]pyrimidin- 1 - yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile, (S)-2-(3-(4-amino-3-(2-fluoro-4- phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2- enenitrile, a mixture of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4- d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile and (S)-2-(3-(4-amino-3- (2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4- dimethylpent-2-enenitrile, an individual (E)- or (Z)- isomer of any of the above compounds, and pharmaceutically acceptable salts of any of the above compounds.

[0141] In certain embodiments, the disclosed method involves administration of at least one BTK inhibitor, wherein the at least one BTK inhibitor is a compound of Formula (II) selected from the (E) isomer of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH- pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile and pharmaceutically acceptable salts thereof

[0142] In some embodiments, the disclosed method involves administration of at least one BTK inhibitor, wherein the at least one BTK inhibitor is a compound of Formula (II) selected from the (Z) isomer of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH- pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile and pharmaceutically acceptable salts thereof

[0143] In some embodiments, the disclosed method involves administration of at least one BTK inhibitor, wherein the at least one BTK inhibitor is selected from a mixture of (E) and (Z) isomers of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l- yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile and pharmaceutically acceptable salts thereof.

[0144] Also provided herein is the use of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, in the manufacture of a medicament for inhibiting CLEC-2 in a human subject.

[0145] Further provided herein is the use of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, in the manufacture of a medicament for inhibiting at least one of CLEC-2 mediated platelet activation, thrombus formation, and thrombus propagation in a human subject. In some embodiments, the use is for inhibiting CLEC-2 mediated platelet activation. In some embodiments, the use is for inhibiting CLEC-2 mediated thrombus formation. In certain embodiments, the use is for inhibiting CLEC-2 mediated thrombus propagation.

[0146] Additionally provided herein is the use of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, in the manufacture of a medicament for inhibiting or reducing GPVI-mediated platelet activation in a human subject. In some embodiments, the use is for inhibiting GPVI- mediated platelet activation. In certain embodiments, the use is for reducing GPVI-mediated platelet activation.

[0147] In some embodiments, provided herein is the use of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, in the manufacture of a medicament for reducing the number of thrombi in podoplanin positive blood vessels in a human subject.

[0148] Provided herein is also the use of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, in the manufacture of a medicament for treating or preventing venous thrombosis in a human subject having elevated expression levels of podoplanin as compared to normal expression levels. In certain embodiments, the use is for treating venous thrombosis. In some embodiments, the use is for preventing venous thrombosis.

[0149] Further provided is the use of at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, in the manufacture of a medicament for treating or preventing thromboinflammation in a human subject having elevated expression levels of podoplanin as compared to normal expression levels, wherein the thromboinflammation results from venous thrombosis.

[0150] Also provided is at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in inhibiting CLEC-2 in a human subject.

[0151] Provided herein is also at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in inhibiting at least one of CLEC-2 mediated platelet activation, thrombus formation, and thrombus propagation in a human subject. In some embodiments, the at least one BTK inhibitor is for use in inhibiting CLEC-2 mediated platelet activation. In some embodiments, the at least one BTK inhibitor is for use in inhibiting CLEC-2 mediated thrombus formation. In certain embodiments, the at least one BTK inhibitor is for use in inhibiting CLEC-2 mediated thrombus propagation.

[0152] Additionally provided is at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in inhibiting or reducing GPVI-mediated platelet activation in a human subject. In some embodiments, the at least one BTK inhibitor is for use in inhibiting GPVI-mediated platelet activation. In certain embodiments, the at least one BTK inhibitor is for use in reducing GPVI- mediated platelet activation.

[0153] In some embodiments, at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, is provided for use in reducing the number of thrombi in podoplanin positive blood vessels in a human subject. In some embodiments, the thrombi are a result of deep vein thrombosis, venous thrombosis, portal vein thrombosis, or any combination thereof. In some embodiments, the podoplanin positive blood vessels are veins.

[0154] In certain embodiments, at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, is provided for use in treating or preventing venous thrombosis in a human subject having elevated expression levels of podoplanin as compared to normal expression levels. In certain embodiments, the at least one BTK inhibitor is for use in treating venous thrombosis. In some embodiments, the at least one BTK inhibitor is for use in preventing venous thrombosis. In someembodiments, the thrombi are a result of deep vein thrombosis, venous thrombosis, portal vein thrombosis, or any combination thereof.

[0155] Also provided is at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in treating or preventing thromboinflammation in a human subject having elevated expression levels of podoplanin as compared to normal expression levels, wherein the thromboinflammation results from venous thrombosis. In some embodiments, the thromboinflammation is a result of deep vein thrombosis, portal vein thrombosis, or any combination thereof.

[0156] Provided herein is also at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in treating or preventing venous thrombosis in a human subject having elevated expression levels of podoplanin as compared to normal expression levels, the use comprising the steps of (a) evaluating the subject’s expression levels of podoplanin, and (b) when the subject’s expression levels of podoplanin are elevated as compared to normal levels, administering to the human subject the at least one BTK inhibitor. In some embodiments, the venous thrombosis is selected from deep vein thrombosis, portal vein thrombosis, or any combination thereof.

[0157] Further provided herein is at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use in treating or preventing thromboinflammation in a human subject, the use comprising the steps of (a) evaluating the subject’s expression levels of podoplanin, and (b) when the subject’s expression levels of podoplanin are elevated as compared to normal levels, administering to the human subject the at least one BTK inhibitor, wherein the thromboinflammation results from venous thrombosis. In some embodiments, the venous thrombosis is selected from deep vein thrombosis, portal vein thrombosis, or any combination thereof.

[0158] Provided herein is at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use, wherein the at least one BTK inhibitor includes a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the use includes administration of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0159] Also provided herein is at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use, wherein the at least one BTK inhibitor includes a compound of Formula (II) or a pharmaceutically acceptable salt thereof. In some embodiments, the use includes administration of a compound of Formula (II) or a pharmaceutically acceptable salt thereof.

[0160] In some embodiments, the disclosed at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use involves the at least one BTK inhibitor being a compound of Formula (I) from (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l- carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile, individual (E)- or (Z)- isomers thereof, a mixture of (E) and (Z) isomers thereof, and pharmaceutically acceptable salts of any of the above compounds. In some embodiments, the compound for use is selected from (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l- carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile, individual (E)- or (Z)- isomers thereof, and pharmaceutically acceptable salts thereof. In some embodiments, the compound for use is selected from the (E) isomer of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy- phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3- yl)piperazin-l-yl]pent-2-enenitrile and pharmaceutically acceptable salts thereof. In some embodiments, the compound for use is selected from the (Z) isomer of (R)-2-[3-[4-amino-3-(2- fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4- (oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile and pharmaceutically acceptable salts thereof. In some embodiments, the compound for use is selected from a mixture of (E) and (Z) isomers of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l- carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile and pharmaceutically acceptable salts thereof.

[0161] In some embodiments, the disclosed at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use involves the at least one BTK inhibitor being a compound of Formula (II) selected from (R)-2-(3 -(4-amino-3 -(2-fluoro-4-phenoxyphenyl)- lH-pyrazolo[3 ,4-d]pyrimidin- 1 -yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile, (S)-2-(3-(4-amino-3-(2-fluoro-4- phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2- enenitrile, a mixture of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4- d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile and (S)-2-(3-(4-amino-3- (2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4- dimethylpent-2-enenitrile, an individual (E)- or (Z)- isomer of any of the above compounds, a mixture of (E) and (Z) isomers of any of the above compounds, and pharmaceutically acceptable salts of any of the above compounds. In some embodiments, the compound for use is selected from (R)-2-(3 -(4-amino-3 -(2-fluoro-4-phenoxyphenyl)- lH-pyrazolo[3 ,4-d]pyrimidin- 1 - yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile and pharmaceutically acceptable salts thereof. In some embodiments, the compound for use is selected from (S)-2-(3-(4-amino-3-(2- fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4- dimethylpent-2-enenitrile and pharmaceutically acceptable salts thereof. In some embodiments, the compound for use is selected from a mixture of (R)-2-(3-(4-amino-3-(2-fluoro-4- phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2- enenitrile and (S)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l- yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile, and pharmaceutically acceptable salts of any of these compounds. In some embodiments, the compound for use is selected from an individual (E)- or (Z)- isomer of any of the above compounds, and pharmaceutically acceptable salts of any of these compounds.

[0162] In certain embodiments, the disclosed at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use involves the at least one BTK inhibitor being a compound of Formula (II) selected from the (E) isomer of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH- pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile and pharmaceutically acceptable salts thereof. In some embodiments, the disclosed at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use involves the at least one BTK inhibitor being a compound of Formula (II) selected from the (Z) isomer of (R)-2-(3-(4-amino-3-(2-fluoro-4- phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile and pharmaceutically acceptable salts thereof. In some embodiments, the disclosed at least one BTK inhibitor selected from a compound of Formula (I), a compound of Formula (II), and pharmaceutically acceptable salts thereof, for use involves the at least one BTK inhibitor selected from a mixture of (E) and (Z) isomers of (R)-2-(3-(4-amino-3-(2-fluoro-4- phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2- enenitrile and pharmaceutically acceptable salts thereof.EXAMPLESExample 1. Selective Btk inhibition with PRN1008 and PRN473 block human CLEC-2 and GPVI-mediated platelet function and reduce venous thrombosis formation in mice.

[0163] Methods.

[0164] Antibodies and reagents. The a-phospho-tyrosine (4G10) monoclonal antibody(mAb) was from Millipore (Abingdon, UK). The HRP -conjugated Goat a-rat IgG (SC 2032) and a-Syk pAb (SC-1077) were from Santa Cruz Biotechnology (Dallas, USA). Phosphospecific pAb against pY1217 was from Cell Signalling Technology (Hitchin, UK), against LAT pY200 and BTK pY223 were from Abeam (Cambridge, UK), against BTK pY551 was from BD Biosciences (San Jose, CA) and against Src pY418 was from Life Technologies (Carlsbad, CA). Eptifibatide was from GSK (Brentford, UK). The FITC-conjugated rat a-mouse P-selectin mAb and PE-conjugated rat a-mouse integrin al Ib[33 mAb were from Emfret Analytics (Eibelstadt, Germany). The FITC-conjugated rat a-mouse, HRP-conjugated a-mouse and a-rabbit secondary pAbs and Hyperfilm enhanced chemiluminescence (ECL) autoradiography film were from Amersham Biosciences (GE Healthcare, Bucks, UK). The a-hamster podoplanin primary mAb was from Invitrogen (Paisley, UK). The rat a-mouse CD31 primary mAb was from BD Pharmigen (Wokingham, UK). The PE-conjugated rat a-mouse CD41 mAb was from Biolegend (London, UK). The AF647-conjugated goat a-hamster IgG secondary pAb was from Invitrogen (Paisley, UK). The FITC-conjugated goat a-rat IgG secondary pAb was from DAKO (Hamburg, Germany). The AF568-conjugated rabbit a-mouse annexin A5 pAb was from ThermoFisher (Waltham, MA). The AF647-conjugated rat a-mouse CD62P mAb was from BD Pharmigen (Wokingham, UK). The FITC-conjugated rabbit a-mouse fibrinogen pAb was from DAKO (Hamburg, Germany). The Glucose, pH 7 and pH 10 buffer solutions, Bolt running buffer, Boltantioxidant, ECL reagent were from ThermoFisher (Waltham, MA). Rhodocytin was a gift from Johannes Eble (Munster, Germany). Collagen related peptide (CRP) was from CambCol (Cambridge, UK). Collagen (Horm, equine tendon, 95% type-1, 5% type IV) from Takeda (Linz, Austria). Thrombin Receptor Activating Peptide (TRAP) was from Severn Biotech (Kidderminster, UK). PAR4 peptide was from Alta Biosciences (Birmingham, UK). Podoplanin-Fc was made in house in HEK 293T-Cells as described herein. PRN1008 and PRN473 were from Principia Biopharma (Palo Alto, CA). Non-fatty acid free Bovine Serum Albumin (BSA) was from First Link (UK) ltd (Birmingham, UK). ChronoLume® and ATP standard were from ChronoLog Corporation (Havertown, PA). Methanol and ethanol were from VWR Chemicals (Lutterworth, UK). Transblot Turbo western blotting buffer was from Bio-Rad (Kidlington, UK). All other reagents were purchased from Sigma-Aldrich (Poole, UK).

[0165] Podoplanin-Fc generation. Human Podoplanin-Fc plasmid was made by inserting a human podoplanin extracellular domain sequence into the IgFc mammalian vector pFuse-rlgG- Fc (Invitrogen), to yield a construct encoding podoplanin fused at the COOH terminus to the Fc region of rabbit IgGl. For expression and purification of recombinant podoplanin-Fc fusion protein, 293 T cells were transfected with the construct DNA by polyethylenimine (PEI) method. Three days after transfection, the cell culture supernatant was harvested, filtered with a 0.22 pM filter and then purified through a protein A agarose affinity column. The recombinant protein was then eluted by IM Glycine solution at pH 3.0, followed by neutralization and dialysis in phosphate-buffered saline (PBS). The protein solution was aliquoted and stored at -20°C before use. The molecular weight and purity of protein were verified by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).

[0166] Approvals and ethics. Ethical approval for collecting blood from healthy volunteers was granted by Birmingham University Internal Ethical Review (ERN_11-0175) in accordance with the Declaration of Helsinki. Ethical approval for collection of blood from patients with XLA was granted by the North West - Haydock NHS Research Ethics Committee (15 / NW / 0079, amendment s).

[0167] Blood collection. Blood was taken by venipuncture from consenting patients or healthy, drug-free volunteers, into 4% sodium citrate.

[0168] Human platelet preparation. Warmed acid citrate dextrose (ACD) 1 : 10 (v / v) was added to citrated whole blood then centrifuged (200 x g;20 minutes, room temperature) and platelet rich plasma (PRP) collected. PRP was then centrifuged (1000 x g, 10 minutes, room temperature) in the presence of 0.2 pg / ml prostacyclin (PGI2), and the supernatant discarded. The platelet pellet was resuspended in 24 ml modified-Tyrode’s-HEPES buffer (134 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM KC1, 12 mM NaHCO3, 20 mM HEPES, 5 mM glucose, 1 mM MgC12; pH 7.3) and 3 ml ACD then centrifuged (1000 x g, 10 minutes, room temperature) in the presence of 0.2 pg / ml PGI2. Supernatant was discarded and platelet pellet resuspended in modified-Tyrode’s-HEPES buffer to the required concentration. Platelets were rested for 30 minutes prior to use in any experiments.

[0169] Light transmission aggregometry (LTA) and granule secretion. Aggregation and adenosine triphosphate (ATP) secretion in washed platelets (2x 108 / ml) or PRP under stirring conditions (1200 rpm) at 37°C were measured in a lumi-aggregometer (Model 700, ChonoLog, Havertown, PA) for 5 minutes. Aggregation was monitored by measuring changes in light transmission, with ChronoLume® (D-luciferin- luciferase mixture) added to enable simultaneous measurement of ATP release by luminescence. Inhibitors (50 nM - 2 pM) or vehicle (dimethyl sulfoxide, DMSO) were incubated with washed platelets or PRP for 1 hour prior to stimulation.

[0170] Protein phosphorylation. Washed platelets at 4xl08 / ml were pre-treated with 9 pM eptifibatide to block integrin al Ib[33 activation. Agonists were added while stirring at 1200 rpm in an aggregometer at 37°C for 180 seconds unless stated otherwise. Inhibitors (50 nM - 2 pM) or vehicle (DMSO) were incubated with washed platelets for 1 hour prior to stimulation. Activation was terminated with 5X SDS reducing sample buffer. Lysates were separated by SDS-PAGE, electro-transferred, and western blotted. Western blots were probed with the stated antibodies and imaged using ECL autoradiography film. For analysis of levels of phosphorylation, western blot films were scanned and band intensity measured using ImageJ 1.5 (National Institutes of Health, USA) with values normalised to those seen in vehicle treated platelets.

[0171] Flow adhesion. Citrated whole blood, stained with 4 pM DiOC6, was perfused over podoplanin (10 pg / ml) or collagen (200 pg / ml) coated channels (Ibidi p-slides VI 0.1) atvenous (150 s'1) or arterial (1000 s'1) shear respectively for 10 minutes. Accumulation of labelled platelets was measured by taking z-stacks (41 images, step size 0.5 pm) every 30 seconds for two separate locations using an Evos FL Auto imaging system (Life Technologies) using a 20x objective. Z-stacks for each condition were then analysed using image J (version 1.52, NIH, USA) and an in-house macro that generates platelet intensity and surface coverage of thrombi for each time point.

[0172] Citrated whole blood was thrombin-inhibited (40 pM PPACK) and recalcified (3.75 mM MgCh and 7.5 mM CaCh) then perfused over fibrinogen and collagen microspots in a Maastricht flow chamber at 1000 s'1. Platelets were labelled for activation markers using antiannexin A5, anti-CD62P and anti-fibrinogen antibodies described above. Endpoint images were obtained on an EVOS AMF4300 microscope (Life Technologies) as described previously (Jooss et al., Anti-GPVI nanobody blocks collagen- and atherosclerotic plaque-induced GPVI clustering, signaling, and thrombus formation. J. Thromb. Haemost. 2022, vol 20, 2617-2631).

[0173] Flow cytometry. Whole blood was diluted 1:9 (v / v) in staining solution (anti-P- selectin and -activated integrin allbp3 antibodies in PBS) then stimulated by indicated agonists 1: 10 (v / v) for 20 minutes in the dark at room temperature. Samples were then fixed with ice- cold 1% paraformaldehyde (PF A) and analyzed on an Accuri C6 flow cytometer (BD Biosciences, Oxford, UK). Platelets were gated using forward and side scatter. Inhibitors (200 nM - 5 pM) or vehicle (dimethyl sulfoxide, DMSO) were incubated in whole blood for 1-hour prior to antibody addition and stimulation.

[0174] Animal models. All animal procedures were undertaken with United Kingdom Home Office approval in accordance with the Animals (Scientific Procedures) Act 1986, license numbers PP9677279, P06779746 and PC427E5DD. All experiments were performed on wild type C57B16 mice.

[0175] PRN473 pre-treatment of mice was by either PRN473 formulated (6.66 g / kg) diet for 7 days or by 80 mg / kg oral gavage once daily for 4 days (Xing 2021). Control diet and oral gavage of vehicle were used in non-treated control animals.

[0176] For ex vivo assessment of platelet function, blood was taken from the IVC of mice into acid citrate dextrose (1 : 10 v:v).

[0177] Salmonella infection thrombosis model was performed as previously described (Hitchcock 2015). PRN473 and control diet treated mice were infected i.p. with 5* 105attenuated Salmonella Typhimurium (STm). Mice continued on assigned diet for 7 days following infection. Tissues were then removed and immediately snap frozen. Frozen tissues were then sectioned and stained by IHC to detect vasculature (CD31), thrombi (CD41) and podoplanin. Sections were imaged and quantified as previously described (Hitchcock 2015).

[0178] IVC stenosis DVT model was performed as previously described (Payne 2017). Briefly, mice were treated with PRN473 or vehicle by oral gavage for 4 days before being anesthetized by isoflurance and a laparotomy performed. IVC side branches were then identified and tied off. A ligature was then placed around the IVC itself to induce stenosis, with a 30- gauge spacer used to maintain a small degree of vessel patency. The incision was closed and mice were allowed to recover. Buprenorphine analgesia was used post-operatively. Mice were culled after 6 hours and the IVC examined for thrombus presence and size.

[0179] BTK occupancy assessment. Frozen spleen samples were thawed on ice and homogenized in Omni tubes pre-loaded with ceramic beads and prepared CelLytic-M + protease / phosphatase lysis buffer using the Omni Bead Ruptor Homogenizer. The homogenate was incubated on ice for complete lysis then clarified by centrifugation. Protein concentration of the cell lysates was determined by BCA Assay (Therm oFi sher-Pierce) according to manufacturer’s instructions.

[0180] Aliquots of vehicle lysate samples were treated with 1 pM PRN473 for 1 hour at room temperature for background reading. Aliquots of all vehicle + dosed spleen lysates and background-reading vehicle lysates were then treated with biotinylated BTK probe PRN299 for 1 hour at room temperature.

[0181] The probe-treated lysates were then transferred to streptavidin coated 96-well plates (Life Technologies) and incubated at room temperature for 1 hour for biotin capture. The captured protein in all assay wells was treated with mouse anti-human BTK Clone 53 primaryantibody (BD Biosciences) overnight at 4°C. After overnight primary antibody incubation, all assay wells were washed and treated with anti-mouse HRP secondary antibody (Jackson Immuno). Peroxidase is detected by prepared SuperSignal ELISA Femto chemiluminescent substrate (ThermoFisher-Pierce) on the Envision2105 Multilabel Reader (Perkin Elmer).

[0182] The average of all background reading wells was calculated, then subtracted from all sample readings in the same plate. The background-subtracted vehicle and dosed readings were then used to calculate % occupancy for each dosed sample by dividing the dosed samples from the vehicle sample, then multiplying by 100 and subtracting from 100. The resulting % occupancy values were then used as a measure of the amount of PRN473 bound to target BTK enzyme in the treated samples at each timepoint.

[0183] Plasma PRN473 levels. Frozen citrated plasma samples were thawed and diluted (1 :5) with 10 ng / mL acetonitrile. Following centrifugation at 4000 rpm for 5 min, the supernatant was diluted (1 : 1) in 1% formic acid before being analyzed by liquid chromatography tandem mass spectrometry.

[0184] Statistical analysis. All data are presented as mean ± standard error of the mean (SEM) with statistical significance taken as p < 0.05 unless otherwise stated. Graphs were plotted using GraphPad Prism 9 (GraphPad Software Inc. La Jolla, Ca). Statistical analysis was performed using one or two-way ANOVA with corrections for multiple comparisons unless otherwise stated. All statistical analyses were performed using GraphPad Prism 9 (GraphPad Software Inc. La Jolla, Ca).

[0185] Results

[0186] PRN 1008 / 473 block CLEC-2- and GPVI-mediated platelet activation.

[0187] The effect of PRN 1008 and PRN473 inhibition on tyrosine phosphorylation downstream of CLEC-2 following activation with rhodocytin was assessed. Both inhibitors blocked BTK Y223 phosphorylation and reduced downstream PLCg2 Y1217 phosphorylation at concentrations >200 nM (Figures 1A and IB). Phosphorylation of LAT Y200 and Src Y418 as well as BTK’ s transphosphorylation site; Y551, all of which are upstream of BTK Y223, was unaffected.

[0188] Blockade of GPVI downstream signaling has also been shown with BTK inhibition, and loss of GPVI-mediated platelet function occurred only at higher concentrations where off-target inhibition of Src phosphorylation was observed (Nicolson 2018). The effect of the inhibitors on GPVI downstream signaling following activation with the specific agonist collagen-related peptide (CRP) was thus assessed (Figures 1C and ID). Phosphorylation of BTK Y223, and PLCg2 Y1217 were again lost at concentrations >200 nM, with BTK Y551 unaffected. No off-target inhibition of Src phosphorylation was observed to either inhibitor, even with high concentrations (2 pM). This shows that platelet CLEC-2 and GPVI signaling can be effectively blocked by PRN1008 and PRN473 BTK inhibition.

[0189] PRN1008 / 473 block (hem)ITAM- but not GPCR-mediated platelet aggregation and secretion.

[0190] The effects of PRN1008 and PRN473 on aggregation and secretion were assessed in washed platelets. Both PRN1008 and PRN473 blocked CLEC-2-mediated platelet aggregation induced by rhodocytin (Figure 2A, Ai and Bi). GPVI-mediated platelet aggregation to low concentrations of CRP was also blocked, however only minor inhibition to high concentrations of CRP and to collagen induced aggregation was observed (Figure 2A, Ci-Di and Figure 2B, Ei-Fi). No effect on G protein-coupled receptor mediated aggregation to thrombin or the thromboxane mimetic U46619 was observed with either inhibitor at high (2 pM) concentrations (Figure 2B, Gi-Ii). Blockade of dense granule secretion was consistently observed at lower concentrations than aggregation to (hem)ITAM receptor agonists (Figures 8A and 8B). No inhibition of ADP-mediated aggregation in platelet rich plasma was observed with either Btk inhibitor, even at 20 pM (Data not shown).

[0191] High concentrations of PRN1008 / 473 inhibit CLEC-2- and GPVI-mediated platelet activation and aggregate size in flow conditions in whole blood.

[0192] Platelet activation in response to CLEC-2 stimulation with rhodocytin was measured using flow cytometry (P-selectin and active integrin allb[33 [PAC-1]) in whole blood following BTK inhibitor incubation. Strong inhibition of platelet activation was observed with 5 pM of both inhibitors (Figure 3A; Ai and Aii). CRP -induced platelet activation was also inhibited at similar concentrations, however not as strongly as CLEC-2 (Figure 3A, Bi and Bii).No inhibition of platelet activation to thrombin receptor activating peptide (TRAP) was observed (Figure 3A, C).

[0193] The effect of PRN1008 and PRN473 in whole blood using flow adhesion assays over recombinant podoplanin at venous shear (150 s'1) and collagen at arterial shear (1000 s'1) was also studied. Significant reductions in platelet adhesion and aggregate size on podoplanin were observed to high (5 pM), but not low (500 nM) concentrations of both inhibitors, whereas high (5 pM) but not low (500 nM) concentrations of both inhibitors reduced thrombus size but not platelet adhesion on collagen (Figure 3B and Figure 9).

[0194] Platelets from patients with XLA do not aggregate in response to CLEC-2 and have reduced responses to GPVI stimulation, which are further inhibited by PRN1008.

[0195] To assess if the observed effects of the inhibitors were due to BTK inhibition or mediated by off-target effects, washed platelets from patients with XLA using aggregometry were studied. Platelets from these patients had no response to CLEC-2 stimulation with rhodocytin and had reduced responses to GPVI stimulation with CRP and collagen (Figure 4A, Ai - Di and Aii - Dii). Addition of low (500 nM) and high (5 pM) concentrations of PRN1008 to XLA platelets had no additional inhibition to high concentrations (10 pg / ml) of collagen but did reduce the aggregation seen to low concentrations (3 pg / ml) of collagen and CRP (Figure 4A, Bi and Di, and Bii and Dii). PLCy2 phosphorylation in healthy donor and XLA platelets was also measured. Low level PLCy2 Y1217 phosphorylation was present in XLA platelets following GPVI stimulation with CRP which was blocked by PRN1008. CLEC-2 stimulation with rhodocytin in the XLA platelets resulted in a very low level of PLCy2 phosphorylation (Figures 4B and 4C).

[0196] Dietary administration of PRN473 to mice blocks CLEC-2 mediated platelet function ex vivo and hepatic thrombosis formation in vivo.

[0197] CLEC-2 plays an important role in mouse models of thromboinflammation (Hitchcock 2015; Payne 2017), therefore the effect of PRN473 in certain in vivo models was studied. Inhibition of mouse platelet activation by PRN473 and PRN1008 in vitro was confirmed. Strong inhibition of mouse platelet activation (P-selectin and active integrin allb[33[JON / A]) was observed at high concentrations (>5 pM) of PRN1008 and PRN473 in whole blood following CLEC-2 and GPVI stimulation (Figure 5, Ai-Bi and Aii-Bii). No effect on PAR4-mediated platelet activation was seen at high (5 pM) concentration (Figure 5, C).

[0198] The effect of dietary administration of PRN473 was studied. Mice fed diet formulated with 6.66 g / kg PRN473 for 7 days had full or near complete BTK occupancy in blood (Figure 6A, A), and exhibited abrogated CLEC-2- and GPVI- but normal PAR4-mediated responses to ex vivo platelet activation (Figure 6A, B). Reduced platelet adhesion, thrombus size, P-selectin and phosphatidyl serine (PS) exposure was also observed when blood from PRN473- treated mice was flowed over Horm collagen at arterial shear (1000 s'1) (Figure 10A). Reduced platelet adhesion to fibrinogen at arterial shear was also seen (Figure 10B).

[0199] To assess the effect of BTK inhibition on Salmonella infection driven thrombosis in the liver, mice were treated with PRN473 or control diet for 7 days before being intraperitoneally (ip) injected with attenuated STm. Livers were then removed at the peak of thrombus formation 7 days after infection and thrombus size assessed (Hitchcock 2015). No difference in relative thrombus area between control and PRN473 treated mice was found (Figure 6B, ii). However, there was a trend towards a reduction in the overall number of thrombi in the PRN473 treated group (Figure 6B, iii-iv), as well as a statistically significant reduction in thrombi forming in podoplanin-expressing vessels (Figure 6B, v). Consistent with this, a trend for reduced platelet consumption and reduced increase in mean platelet volume (MPV) was found in PRN473 -treated mice (Figure 6C, i-ii). As BTK inhibitors also block signaling downstream of the B-cell receptor, white blood cell counts were assessed, with no difference in either total white blood cell count or lymphocyte count between the PRN473 -treated and control mice found (Figure 6C, iii-iv).

[0200] Oral administration of PRN473 blocks deep vein thrombosis formation in vivo.

[0201] Animals with PRN473 or vehicle control administered via oral gavage (80 mg / kg) once daily for 4 days were studied, to determine effects on platelet activation. Blood from these mice had reduced CLEC-2- and GPVI- but normal PAR4-mediated platelet activation (Figure 7A, Ai). After 4 days of dosing by oral gavage, mice underwent IVC stenosis surgery followed by examination for presence of thrombosis 6 hours later. Plasma and spleens were harvested fordrug level analysis. Median splenic occupancy and plasma levels for PRN473 -treated mice were 60.5% and 14.5 ng / mL respectively (Figure 7B, 7Bi-ii). Mice treated with PRN473 had fewer thrombi, and in those mice where thrombi did form, these thrombi were smaller (Figure 7B, Biii- v). All thrombi formed in mice whose plasma concentrations of PRN473 were < 8.5 ng / mL (Figure 7C).EQUIVALENTS

[0202] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0203] As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., + / -5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.

Claims

What is Claimed is:

1. At least one BTK inhibitor selected from: a compound of Formula (I),a compound of Formula (II),stereochemical center; and pharmaceutically acceptable salts thereof; for use in inhibiting CLEC-2 in a human subject in need thereof2. At least one BTK inhibitor selected from: a compound of Formula (I),a compound of Formula (II),stereochemical center; and pharmaceutically acceptable salts thereof; for use in inhibiting at least one of CLEC- 2 mediated platelet activation, thrombus formation, and thrombus propagation in a human subject in need thereof, optionally wherein the thrombi are a result of deep vein thrombosis, venous thrombosis, portal vein thrombosis, or any combination thereof.

3. At least one BTK inhibitor selected from: a compound of Formula (I),a compound of Formula (II),stereochemical center; and pharmaceutically acceptable salts thereof; for use in reducing or inhibiting GPVI- mediated platelet activation in a human subject in need thereof.

4. At least one BTK inhibitor selected from: a compound of Formula (I),a compound of Formula (II),stereochemical center; and pharmaceutically acceptable salts thereof; for use in reducing the number of thrombi in podoplanin positive blood vessels in a human subject in need thereof, optionally wherein the thrombi are a result of deep vein thrombosis, venous thrombosis, portal vein thrombosis, or any combination thereof; and optionally wherein the podoplanin positive blood vessels comprise veins.

5. At least one BTK inhibitor selected from: a compound of Formula (I),a compound of Formula (II),stereochemical center; and pharmaceutically acceptable salts thereof; for use in treating or preventing venous thrombosis in a human subject having elevated expression levels of podoplanin as compared to normal expression levels, optionally wherein the venous thrombosis is selected from deep vein thrombosis, portal vein thrombosis, or any combination thereof.

6. At least one BTK inhibitor selected from: a compound of Formula (I),a compound of Formula (II),stereochemical center; and pharmaceutically acceptable salts thereof; for use in treating or preventing thromboinflammation in a human subject having an elevated expression level of podoplanin as compared to a normal expression level, wherein the thromboinflammation results from venous thrombosis, optionally wherein the thromboinflammation results from deep vein thrombosis, portal vein thrombosis, or any combination thereof.

7. At least one BTK inhibitor selected from: a compound of Formula (I),a compound of Formula (II),stereochemical center; and pharmaceutically acceptable salts thereof; for use in treating or preventing venous thrombosis in a human subject, the use comprising the steps of(a) evaluating the subject’s expression levels of podoplanin; and(b) when the subject’s expression levels of podoplanin are elevated as compared to normal levels, administering to the human subject the at least one BTK inhibitor, optionally wherein the venous thrombosis is selected from deep vein thrombosis, portal vein thrombosis, or any combination thereof.

8. At least one BTK inhibitor selected from: a compound of Formula (I),a compound of Formula (II),stereochemical center; and pharmaceutically acceptable salts thereof; for use in treating or preventing thromboinflammation in a human subject, the use comprising the steps of:(a) evaluating the subject’s expression levels of podoplanin; and(b) when the subject’s expression levels of podoplanin are elevated as compared to normal levels, administering to the human subject the at least one BTK inhibitor, wherein the thromboinflammation results from venous thrombosis, optionally wherein the venous thrombosis is selected from deep vein thrombosis, portal vein thrombosis, or any combination thereof.

9. The at least one BTK inhibitor of any one of claims 1-8, comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

10. The at least one BTK inhibitor of any one of claims 1-8, comprising a compound of Formula (II), or a pharmaceutically acceptable salt thereof.

11. The at least one BTK inhibitor of claim 9, wherein the at least one BTK inhibitor is a compound of Formula (I) selected from (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy- phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3- yl)piperazin-l-yl]pent-2-enenitrile, individual (E)- or (Z)- isomers thereof, a mixture of (E) and (Z) isomers thereof, and pharmaceutically acceptable salts of any of the above compounds.

2. The at least one BTK inhibitor of claim 10, wherein the at least one BTK inhibitor is a compound of Formula (II) selected from (R)-2-(3-(4-amino-3-(2-fluoro-4- phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4- dimethylpent-2-enenitrile, (S)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH- pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile, a mixture of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4- d]pyrimidin-l-yl)piperidine-l-carbonyl)-4,4-dimethylpent-2-enenitrile and (S)-2-(3-(4- amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)piperidine-l- carbonyl)-4,4-dimethylpent-2-enenitrile, an individual (E)- or (Z)- isomer of any of the above compounds, a mixture of (E) and (Z) isomers of any of the above compounds, and pharmaceutically acceptable salts of any of the above compounds.

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