Methods for treating drug- and vaccine-induced immune thrombocytopenia by administering certain compounds - Patents.com

Abstract

Methods are provided for treating and / or preventing drug-induced thrombocytopenia (DITP) and vaccine-induced thrombosis and thrombocytopenia syndrome (VITT) with certain BTK inhibitors and / or pharma- ceutically acceptable salts thereof.

Description

[Background technology]

[0001] Drug-induced thrombocytopenia (DITP) and vaccine-induced thrombosis and thrombocytopenia (VITT) have clinical similarities to autoimmune heparin-induced thrombocytopenia (HIT), with the majority of patients testing positive for antiplatelet factor 4 (PF4) antibodies. 1; 2. In HIT, IgG-containing immune complexes bind and crosslink the platelet surface receptor FcγRIIA (CD32a), a low-affinity Fc receptor (FcR) that binds immune complexes with high avidity, initiating platelet activation. 2. Autoimmune HIT, despite its name, occurs rarely and independently of heparin, resulting in persistent severe thrombocytopenia along with DIC and microvascular thrombosis. Id.

[0002] A new, safe and effective oral treatment for maintaining platelet counts in DITP and VITT patients would represent a significant therapeutic advantage over the current standard of care. Accordingly, novel methods for the treatment and / or prevention of DITP and VITT with certain compounds are disclosed herein.

[0003] Bruton's agammaglobulinemia tyrosine kinase (BTK) is an essential signaling element downstream of the B cell receptor (BCR), Fc gamma receptor (FcγR), and Fc epsilon receptor (FcεR). BTK is a non-receptor tyrosine kinase and a member of the TEC family of kinases. BTK is essential for B cell lineage maturation, and inhibition of BTK activity in cells results in phenotypic changes consistent with blocking the BCR. Illustratively, BTK inhibition results in the downregulation of various B cell activities, such as cell proliferation, differentiation, maturation, and survival, as well as the upregulation of apoptosis.

[0004] Rather than acting in an "on / off switch" manner, BTK best appears as an immune function "modulator" (Non-Patent Document 3; Non-Patent Document 4). Important insights into BTK function come from the lack of functional analysis in humans and mice. Individuals lacking functional mutations in the BTK gene develop X-linked agammaglobulinemia (XLA), characterized by a complete absence of circulating B cells and plasma cells and very low levels of all classes of immunoglobulins (Non-Patent Document 5; Non-Patent Document 6). This suggests that BTK inhibition may suppress the production of autoantibodies that are thought to be important in the development of autoimmune diseases.

[0005] While BTK is not expressed in T cells, natural killer cells, and plasma cells and does not have any traceable direct function in T cells and plasma cells (Non-Patent Document 7; Non-Patent Document 8), the enzyme does regulate the activation of other hematopoietic cells, such as basophils, mast cells, macrophages, neutrophils, and platelets. For example, BTK plays a role in the activation of neutrophils, which are key players in the inflammatory response that contributes to wound healing, but can also cause tissue damage (Non-Patent Document 9).

[0006] Thus, selective BTK inhibitors have the potential to target multiple pathways involved in inflammation and autoimmunity, such as, but not limited to, blocking BCR; inhibiting plasma cell differentiation and antibody production; blocking IgG-mediated FcγR activation, phagocytosis, and inflammatory mediators in monocytes or macrophages; blocking IgE-mediated FcεR activation and degranulation in mast cells or basophils; and inhibiting activation, adhesion, recruitment, and oxidative burst in neutrophils. Based on these effects, selective BTK inhibitors may block the initiation and progression of various inflammatory diseases, as well as ameliorate the tissue damage resulting from these diseases. Individuals lacking functional mutations in the BTK gene have reduced humoral immunity and are susceptible to pyogenic bacterial and enteroviral infections, requiring treatment with intravenous immunoglobulin, but inhibition of BTK in individuals with intact immune systems is not expected to result in similar susceptibility to infection.

[0007] For example, several orally administered BTK inhibitors (BTKi), such as ibrutinib (PCI-32765) and spebrutinib (CC-292), are currently on the market or in clinical development for various indications (Non-Patent Document 10). For example, ibrutinib has provided further clinical evaluation of the BTK target and has recently been approved by the US Food and Drug Administration (FDA) for human use in mantle cell lymphoma, Waldenstrom's macroglobulinemia, and chronic lymphocytic leukemia (Non-Patent Document 11). Ibrutinib has also demonstrated activity in other hematological malignancies (Non-Patent Document 12, Non-Patent Document 13), and more recently, use as an antiplatelet agent (Non-Patent Document 14) and offsetting the thrombocytopenia that occurs shortly after vaccination with the COVID-19 vaccine AZD1222 (Vaxzevria) (Non-Patent Document 15). Furthermore, CC-292 has been reported to be well tolerated in healthy volunteer populations at doses that provide 100% occupancy of the BTK enzyme (Non-Patent Document 16). Furthermore, evobrutinib has recently demonstrated efficacy against multiple sclerosis in a Phase 2 trial (Non-Patent Document 17). Other BTKi compounds are in clinical development for various immune-mediated disorders, such as pemphigus (NCT02704429), rheumatoid arthritis (NCT03823378, NCT03682705, NCT03233230), and asthma (NCT03944707) (Non-Patent Document 17; Non-Patent Document 18; Non-Patent Document 19; Non-Patent Document 20; Non-Patent Document 21; Non-Patent Document 22; Non-Patent Document 23).

[0008] Some BTK inhibitors may cause bleeding and therefore would not be ideal candidates for use in thrombocytopenic, anticoagulated, and / or intracerebral hemorrhage subjects. Non-Patent Document 24. PMID: 33674445; PMCID: PMC7980532. However, the disclosed compound (I) has no effect on normal platelet function in vitro and has not been associated with bleeding in thrombocytopenic patients, and has indeed been studied in the treatment of immune thrombocytopenia (ITP). [Prior art documents]

Non-licensed literature

[0009] [Non-licensed document 1] Greinacherら, Thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination, N Engl J Med, doi:10.1056 / NEJMoa2104840(2021) [Non-licensed document 2] Greinacherら, Autoimmune heparin-induced thrombocytopenia, J Thromb Haemost 15(11):2099-114(2017) [Non-licensed document 3] Crofford LJら, 2016

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

[0010] Compound (I) described herein is a BTK inhibitor also known as "rilzabrutinib" and has the following structure: [ka] [In the formula, * C is a stereochemical center. See, e.g., Example 31 of PCT Publication No. WO2014 / 039899, ​​which is incorporated herein by reference.

[0011] This compound is disclosed in several patent publications, such as PCT Publication Nos. WO2014 / 039899, ​​WO2015 / 127310, WO2016 / 100914, WO2016 / 105531, and WO2018 / 005849, the contents of each of which are incorporated herein by reference.

[0012] Rilzabrutinib is a novel, highly selective, small molecule inhibitor of non-T cell leukocyte signaling via the B cell receptor, FcγR and / or FcεR signaling in the BTK pathway. Rilzabrutinib functions as a reversible covalent BTK inhibitor, as well as forming both non-covalent and covalent bonds with its target, allowing for enhanced selectivity and extended inhibition with low systemic exposure. Compared to first and second generation BTKis, rilzabrutinib has shown minimal cross-reactivity with other molecules and is at low risk for off-target effects (Smith PF et al., 2017). Importantly, the reversible binding of rilzabrutinib minimizes the possibility of permanently altered peptides (Serafimova IM 2012). Additionally, rilzabrutinib exhibits improved kinase selectivity compared to the covalent BTK inhibitor ibrutinib, with rilzabrutinib (1 μM) achieving >90% inhibition against 6 kinases compared to 21 kinases with ibrutinib (1 μM) in a panel of 251 kinases.

[0013] Rilzabrutinib has shown promising results for the treatment of immune-mediated diseases. Rilzabrutinib is the most advanced BTKi in development for autoimmune diseases (Phase 3, NCT03762265) and the first BTKi to be evaluated in the treatment of pemphigus, an autoantibody-driven blistering disease like ITP. In humans, rilzabrutinib is rapidly absorbed after oral administration with a fast half-life (3-4 hours) and variable pharmacokinetics (PK).

[0014] Compound (II) described herein is a BTK inhibitor also known as "azabrutinib" and has the following structure: [ka] [In the formula, * C is a stereochemical center. This compound is disclosed, for example, in WO2012 / 158764 (see, for example, compounds 125A / 125B in Table 1), which is incorporated herein by reference.

[0015] The following non-limiting embodiments are hereby encompassed: Embodiment 1. A method for treating or preventing drug-induced thrombocytopenia (DITP) 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 Compound (I), Compound (II), and pharma- ceutically acceptable salts thereof. Embodiment 2. A method for treating or preventing Vaccine Induced Thrombosis and Thrombocytopenia Syndrome (VITT) 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 Compound (I), Compound (II), and pharma- ceutically acceptable salts thereof. Embodiment 3. A method of increasing platelet count in a human subject having Drug-Induced Thrombocytopenia (DITP) or Vaccine-Induced Thrombosis and Thrombocytopenia Syndrome (VITT), comprising administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from Compound (I), Compound (II), and pharma-ceutically acceptable salts thereof. Embodiment 4. A method of reducing platelet aggregation in a human subject having Drug-Induced Thrombocytopenia (DITP) or Vaccine-Induced Thrombosis and Thrombocytopenia Syndrome (VITT), comprising administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from Compound (I), Compound (II), and pharma-ceutically acceptable salts thereof. Embodiment 5. Prior to administration, the human subject receives one or more of the following: A. High D-dimer levels; b. Thrombosis; and c. Anti-platelet factor 4 (PF4) antibody The method according to any one of the preceding embodiments, having at least one feature selected from the following: Embodiment 6. The method of any one of embodiments 1 and 3-5, wherein drug-induced thrombocytopenia (DITP) is induced by administration of a small molecule, protein, or component, diluent, excipient, or the like found in a therapeutic regimen. Embodiment 7. Drug-induced thrombocytopenia (DITP) is caused by the use of unfractionated heparin, enoxaparin, dalteparin, tinzaparin, acenocoumarol, acetaminophen, acetyldigoxin, alfacalcidol, allopurinol, alteplase, amphotericin B, argatroban, aspirin, atenolol, azathioprine, bivalirudin, bortezomib, capecitabine, captopril, carbamazepine, carboplatin, carfilzomib, ceftriaxone, cephalexin, chlorthalidone, cilastatin / imipenem, clopridine ... Pidogrel, clozapine, cyclocytidine, dactinomycin / actinomycin, deferasirox, deferiprone, diflunisal, digoxin, dipyridamole, drospirenone / ethinyl estradiol, eltrombopag, epoetin alfa, epoprostenol, eptifibatide, famotidine, fluconazole, fluorouracil, furosemide, fusidic acid, ganciclovir, gemcitabine, gentamicin, glycoprotein IIB / IIA inhibitors, gold, hydrochlorothiazide, hydrochlorothiazide / triamterene, Hydroxychloroquine, imatinib, inamrinone, integrilin, ITP drugs, ixazomib, lenalidomide, levetiracetam, linezolid, melperone, menatetrenone, meropenem, methotrexate, metoprolol, molsidomine, nedaplatin, nicotinamide, nitrofurantoin, nonsteroidal anti-inflammatory drugs (NSAIDS) (e.g., ibuprofen, naproxen, celecoxib, diclofenac, and similar), octreotide, pantoprazole, penicillamine, phenytoin, piperacillin, propranolol The method of any one of embodiments 1 and 3-6, wherein the inflammatory bowel syndrome is induced by administration of rifampin, proton pump inhibitors, quinine, rifampin, ruxolitinib, ruxolitinib phosphate, sirolimus, spironolactone, streptokinase, sulfamethoxazole, sulfisoxazole, sunitinib, teicoplanin, temozolomide, temsirolimus, ticlopidine, tirofiban, trimethoprim / sulfamethoxazole, urokinase, valganciclovir, valproic acid, vancomycin, warfarin, or a combination thereof. Embodiment 8. The method of any one of embodiments 1 and 3-6, wherein the drug-induced thrombocytopenia (DITP) is induced by administration of filgrastim (granulocyte colony-stimulating factor; G-CSF), interferon, interferon alpha, pegylated interferon alpha 2B, pegylated interferon alpha 2B / ribavirin, factor VIII, TNF alpha, INF gamma, or a combination thereof. Embodiment 9. The method of any one of embodiments 1 and 3-6, wherein the drug-induced thrombocytopenia (DITP) is induced by administration of abciximab, adalimumab, alemtuzumab, an antibody-drug conjugate, antithymocyte globulin, brentuximab, cixutumumab, efalizumab, natalizumab, rituximab, trastuzumab, or a combination thereof. Embodiment 10. The method of any one of embodiments 2 to 5, wherein Vaccine-Induced Thrombosis and Thrombocytopenia Syndrome (VITT) is induced by administration of a vaccine. Embodiment 11. The method of any one of embodiments 2-5 and 10, wherein Vaccine-Induced Thrombosis and Thrombocytopenia Syndrome (VITT) is induced by administration of a vaccine delivered in an adenoviral vector. Embodiment 12 The method of embodiment 11, wherein the adenoviral vector comprises a therapeutic and / or prophylactic agent. Embodiment 13 The method of embodiment 12, wherein the therapeutic or prophylactic agent is a gene therapy agent. Embodiment 14. The method of any one of embodiments 10 to 13, wherein the vaccine is for preventing coronavirus infection. Embodiment 15. The method of embodiment 14, wherein the coronavirus infection is COVID-19. Embodiment 16. The method of any one of embodiments 10-13, wherein the vaccine is for preventing an infectious disease selected from measles, mumps, rubella, chicken pox, herpes simplex virus 1, herpes simplex virus 2, chicken pox, rotavirus, influenza, yellow fever, smallpox, hepatitis B, human papillomavirus, pneumococcus, hepatitis A, splenic varicella, diphtheria, acellular pertussis, Haemophilus influenzae, such as type B, meningococcus C, meningitis, typhoid fever, rabies, Lyme disease, tetanus, or any combination thereof. Embodiment 17. Compound I is selected from the group consisting of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, (S)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, 17. The method of any one of the preceding claims, wherein the compound is a mixture of (S)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile and (S)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile; or an individual (E) or (Z) isomer of any of the above compounds; and / or a pharma- ceutically acceptable salt of any of the above compounds. Embodiment 18. The method of embodiment 17, wherein compound I is the (E)-isomer of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile or a pharma- ceutically acceptable salt thereof. Embodiment 19. The method of embodiment 17, wherein compound I is the (Z)-isomer of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile or a pharma- ceutically acceptable salt thereof. Embodiment 20. The method of embodiment 17, wherein compound I is a mixture of the (E) and (Z) isomers of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile or a pharma- ceutically acceptable salt thereof. Embodiment 21. Compound II is (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, (S)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, 18. The method of any one of embodiments 1-17, wherein the compound is a mixture of (S)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile and (S)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, or an individual (E) or (Z) isomer of any of the above compounds; and / or a pharma- ceutically acceptable salt of any of the above compounds. Embodiment 22. The method of embodiment 21, wherein compound II is the (E)-isomer of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile or a pharma- ceutically acceptable salt thereof. Embodiment 23. The method of embodiment 21, wherein compound II is the (Z)-isomer of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile or a pharma- ceutically acceptable salt thereof. Embodiment 24. The method of embodiment 21, wherein compound II is a mixture of the (E) and (Z) isomers of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, or a pharma- ceutically acceptable salt thereof.

[0016] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious 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.

[0017] 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 scope of the claims.

[0018] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments and, together with the description, serve to explain the principles described herein. [Brief description of the drawings]

[0019] [Figure 1A]FIG. 1 shows that serum from patients with vaccine-induced thrombosis and thrombocytopenic thrombocytopenic syndrome (VITT) induces platelet aggregation via FcγRIIA. Washed platelets (2×108 / mL) were stimulated with serum (1:15, v / v) from healthy donors (HD) or patients with VITT (P) before and after intravenous immunoglobulin (IVIg) treatment, in the presence of 10 μg / mL IV.3F(ab) or low-concentration heparin (0.2 U / mL), or after heat inactivation (56° C., 45 min). Platelet aggregation was measured in each condition. FIG. 1A shows representative aggregation traces. FIG. 1B and 1C represent the quantification of maximum aggregation by area under the curve (AUC) as measured at 10 min for pre- and post-IVIg samples P2, P3, P4, and P7 (FIG. 1B) and post-IVIg samples P1, P5, and P6 (FIG. 1C) and plasma exchange samples. Mean ± SEM, n = 3. Statistical analysis was performed by two-way ANOVA with Dunnett's multiple comparisons. *p<0.05, ns: non-significant. [Figure 1B] FIG. 1 shows that serum from patients with vaccine-induced thrombosis and thrombocytopenic thrombocytopenic syndrome (VITT) induces platelet aggregation via FcγRIIA. Washed platelets (2×108 / mL) were stimulated with serum (1:15, v / v) from healthy donors (HD) or patients with VITT (P) before and after intravenous immunoglobulin (IVIg) treatment, in the presence of 10 μg / mL IV.3F(ab) or low-concentration heparin (0.2 U / mL), or after heat inactivation (56° C., 45 min). Platelet aggregation was measured in each condition. FIG. 1A shows representative aggregation traces. FIG. 1B and 1C represent the quantification of maximum aggregation by area under the curve (AUC) as measured at 10 min for pre- and post-IVIg samples P2, P3, P4, and P7 (FIG. 1B) and post-IVIg samples P1, P5, and P6 (FIG. 1C) and plasma exchange samples. Mean ± SEM, n = 3. Statistical analysis was performed by two-way ANOVA with Dunnett's multiple comparisons. *p<0.05, ns: non-significant. [Figure 1C]FIG. 1 shows that serum from patients with vaccine-induced thrombosis and thrombocytopenic thrombocytopenic syndrome (VITT) induces platelet aggregation via FcγRIIA. Washed platelets (2×108 / mL) were stimulated with serum (1:15, v / v) from healthy donors (HD) or patients with VITT (P) before and after intravenous immunoglobulin (IVIg) treatment, in the presence of 10 μg / mL IV.3F(ab) or low-concentration heparin (0.2 U / mL), or after heat inactivation (56° C., 45 min). Platelet aggregation was measured in each condition. FIG. 1A shows representative aggregation traces. FIG. 1B and 1C represent the quantification of maximum aggregation by area under the curve (AUC) as measured at 10 min for pre- and post-IVIg samples P2, P3, P4, and P7 (FIG. 1B) and post-IVIg samples P1, P5, and P6 (FIG. 1C) and plasma exchange samples. Mean ± SEM, n = 3. Statistical analysis was performed by two-way ANOVA with Dunnett's multiple comparisons. *p<0.05, ns: non-significant. [Diagram 2] Figure 2 shows the effect of Btk inhibition by rilzabrutinib on blocking platelet aggregation induced by serum from patients with VITT. Washed platelets (2x108 / mL) were incubated with rilzabrutinib (0.5 μM) or vehicle (0.02% DMSO) for 10 min and then stimulated with serum from patients with VITT (1:15, v / v). Figure 2A shows representative aggregation traces. Figure 2B shows quantification of maximum aggregation. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple comparisons. Mean ± SEM, n=7. *p<0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] definition Unless otherwise stated, the following terms used in the specification and claims are defined for purposes of this application and have the following meanings: All undefined technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0021] As used herein, a noun with "a" or "an" means one or more of that thing; for example, a compound means one or more compounds or at least one compound, unless otherwise specified. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0022] 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 relation to a numerical range, it modifies that range by extending the upper and lower limits of the stated numerical values. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 5%. With respect to specific values, the specific values ​​described herein for a subject population (e.g., subjects of a described clinical trial) represent median, average, or statistical numbers unless otherwise indicated. Thus, aspects of the present disclosure that require specific values ​​in subjects are supported herein by population data in which the relevant values ​​are evaluated to be important limits in the subject population.

[0023] As used herein, the term "active pharmaceutical ingredient" or "therapeutic agent" ("API") means a biologically active compound.

[0024] As used herein, the terms "administer", "administering", or "administration" refer herein to providing, giving, dispensing, and / or prescribing, either by a healthy actor or authorized agent, and / or injecting, ingesting, or consuming by the patient or human self. For example, "administration" of an API to a patient refers to any route of introduction or delivery of the API to the patient (e.g., oral delivery). Administration encompasses self-administration and administration by another person.

[0025] As used herein, "immune thrombocytopenia" (ITP) encompasses or at least refers to other commonly used terms such as idiopathic thrombocytopenia and idiopathic thrombocytopenic purpura. There are two main types of ITP: short-term (acute) and chronic (long-term). Acute ITP typically lasts for less than 6 months, while chronic ITP can last for 6 months or more. ITP affects multiple age groups and can be seen in children, adolescents, and adults.

[0026] ITP is a disorder that can result in mild or excessive bruising and bleeding. The bleeding is due to abnormally low levels of platelets. ITP can be due to the development of antibodies against structural platelet antigens. In childhood ITP, the antibodies can be caused by viral antigens. In adults, the cause is unknown, but ITP has been associated with Helicobacter pylori infection, and treatment of that infection was followed by remission of ITP. ITP can worsen during pregnancy, increasing the risk of maternal morbidity. In some embodiments, ITP can be induced by drugs (i.e., drug-induced immune thrombocytopenia; DITP), such as small molecules or antibodies.

[0027] As used herein, "drug-induced immune thrombocytopenia" (DITP) refers to acute immune thrombocytopenia that may be suspected when a subject has sudden severe thrombocytopenia. DITP is induced by a drug. Exemplary non-limiting drugs that may induce DITP include small molecules, proteins, antibodies, and compositions and / or compounds used in therapeutic therapy.

[0028] As used herein, "vaccine-induced immune thrombosis and thrombocytopenia" (VITT) refers to blood clotting with low levels of platelets (i.e., thrombosis with thrombocytopenia), disseminated intravascular coagulation (DIC), and hemorrhage with high mortality induced by a vaccine. In some embodiments, VITT is induced by a COVID-19 vaccine. The COVID-19 vaccine may include a whole virus, an attenuated virus, a viral particle, a protein, a nucleic acid, and / or a viral vector. In some embodiments, the COVID-19 vaccine is a viral vector vaccine. In some embodiments, the COVID-19 vaccine is an adenovirus vector vaccine. In some embodiments, the vaccine is AZD1222 (Oxford-AstraZeneca; formerly ChAdOx1 nCoV-19). VITT is sometimes referred to as "vaccine-induced prothrombotic immune thrombocytopenia" (VIPIT), both of which are encompassed herein (i.e., VITT includes VITT and VIPIT).

[0029] As used herein, a "pharmaceutically acceptable carrier or excipient" means a carrier or excipient that is generally safe and not biologically or otherwise undesirable and useful in the preparation of a pharmaceutical composition, such as a carrier or excipient that is acceptable for pharmaceutical use in mammals.

[0030] As used herein, the term "pharmaceutical acceptable salt" refers to a salt form of an active pharmaceutical agent, such as an acid addition salt, that is pharmaceutical acceptable and retains the desired pharmacological activity of the API from which the salt is formed. Pharmaceutically acceptable salts are well known in the art and include those obtained 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 salts 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. SMBerge et al. describe pharma- ceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19.

[0031] As used herein, the terms "Compound (I)", "rilzabrutinib", "(R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile", and "2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile" are used interchangeably to mean a compound having the structure: [ka] [In the formula, * C is a stereochemical center].

[0032] A dose of rilzabrutinib may contain less than about 5% by weight of the corresponding (S) enantiomer as an impurity material, such as less than about 1% by weight of the corresponding (S) enantiomer. Similarly, a dose of the (E) isomer of rilzabrutinib may contain less than about 1% by weight of the corresponding (Z) isomer as an impurity material; a dose of the (Z) isomer of rilzabrutinib may contain less than about 1% by weight of the corresponding (E) isomer as an impurity material. When rilzabrutinib is referred to as a mixture of the (E) and (Z) isomers of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, it means that the amount of the (E) or (Z) isomer in the mixture is greater than about 1% by weight. In some embodiments, the molar ratio of the (E) to (Z) isomer is 9:1.

[0033] As used herein, “Compound (II)” and “azabrutinib” refer to (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, (S)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine- 1-carbonyl)-4,4-dimethylpent-2-enenitrile, or a mixture of the (E)- and (Z)-isomers thereof, or a mixture of the (R)- and (S)-enantiomers of 2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, having the following structure: [ka] [In the formula, * C is a stereochemical center].

[0034] When compound (II) is referred to as (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, it may also contain the corresponding (S) enantiomer, such as less than 5% by weight of impurity material, for example less than 1% by weight of impurity material. Thus, when compound (II) is referred to as a mixture of the (R) and (S) enantiomers of 2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, the amount of the (R) or (S) enantiomer in the mixture is greater than 1% by weight. Similarly, when compound (II) is referred to as an (E) isomer, it may contain less than 5% by weight, for example less than 1% by weight, of the corresponding (Z) isomer as an impurity material. Thus, when compound (II) is referred to as a mixture of the (E) and (Z) isomers of 2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, the amount of the (E) or (Z) isomer in the mixture is greater than 1% by weight.

[0035] In some embodiments, compound (II) is a mixture of the (R) and (S) enantiomers of 2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile.

[0036] In some embodiments, compound (II) is substantially (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile. In some embodiments, compound (II) is at least about 75% by weight, e.g., at least about 80% by weight, at least about 85% by weight, at least about 90% by weight, at least about 95% by weight is (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile. In some embodiments, compound (II) is at least about 95% by weight (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile.

[0037] As used herein, the term "therapeutically effective amount" refers to the amount of compound administered to produce the desired effect for which it is intended (e.g., amelioration of DITP or symptoms of DITP, or reduction in the severity of DITP or symptoms of DITP, or amelioration of VITT or symptoms of VITT, or reduction in the severity of VITT or symptoms of VITT). The exact amount of the effective amount will depend on the purpose of treatment and can be ascertained by the skilled artisan using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0038] As used herein, the terms "treat," "treating," or "treatment," when used in reference to a disorder or condition, encompass any effect, e.g., alleviation, reduction, modulation, amelioration, or elimination, that results in an improvement of the disorder or condition. Improvement or alleviation of the severity of any symptom of the disorder or condition can be readily assessed according to standard methods and techniques known in the art.

[0039] "Preventing," as used herein, includes providing protection against the onset or recurrence of a disease, disorder, or condition in a subject who may be susceptible 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 imply or require complete prevention throughout the entire treatment period.

[0040] According to the description, there is provided a method for treating or preventing drug-induced thrombocytopenia (DITP) in a human subject in need thereof, comprising administering to a subject a compound (I): [ka] [In the formula, * C is a stereochemical center], Compound (II): [ka] [In the formula, * C is a stereochemical center], and pharma- ceutically acceptable salts thereof Provided herein are such methods comprising administering to a human subject a therapeutically effective amount of at least one BTK inhibitor selected from the group consisting of:

[0041] Also provided herein is a method for treating or preventing Vaccine Induced Thrombosis and Thrombocytopenia Syndrome (VITT) 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 Compound (I), Compound (II), and pharmaceutically acceptable salts thereof.

[0042] In some embodiments, a method of increasing platelet count in a human subject having Drug-Induced Thrombocytopenia (DITP) or Vaccine-Induced Thrombosis and Thrombocytopenia Syndrome (VITT), comprising administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from Compound (I), Compound (II), and pharmaceutically acceptable salts thereof.

[0043] In some embodiments, a method of reducing platelet aggregation in a human subject having Drug-Induced Thrombocytopenia (DITP) or Vaccine-Induced Thrombosis and Thrombocytopenia Syndrome (VITT), comprising administering to the human subject a therapeutically effective amount of at least one BTK inhibitor selected from Compound (I), Compound (II), and pharmaceutically acceptable salts thereof.

[0044] In each case, in some embodiments, prior to administration, the human subject has at least one characteristic selected from the following: high D-dimer levels, thrombosis; and anti-platelet factor 4 (PF4) antibodies.

[0045] In embodiments relating to drug-induced thrombocytopenia (DITP), DITP can be induced by administration of small molecules, proteins, or components, diluents, excipients, or the like found in therapeutic regimens.

[0046] In some embodiments, drug-induced thrombocytopenia (DITP) is treated with unfractionated heparin, enoxaparin, dalteparin, tinzaparin, acenocoumarol, acetaminophen, acetyldigoxin, alfacalcidol, allopurinol, alteplase, amphotericin B, argatroban, aspirin, atenolol, azathioprine, bivalirudin, bortezomib, capecitabine, captopril, carbamazepine, carboplatin, carfilzomib, ceftriaxone, cephalexin, chlorthalidone, silas. statins / imipenem, clopidogrel, clozapine, cyclocytidine, dactinomycin / actinomycin, deferasirox, deferiprone, diflunisal, digoxin, dipyridamole, drospirenone / ethinyl estradiol, eltrombopag, epoetin alfa, epoprostenol, eptifibatide, famotidine, fluconazole, fluorouracil, furosemide, fusidic acid, ganciclovir, gemcitabine, gentamicin, glycoprotein IIB / IIA inhibitors, gold, hydrochlorothiazide, hydrochloride, Lochlorothiazide / triamterene, hydroxychloroquine, imatinib, inamrinone, integrilin, ITP drugs, ixazomib, lenalidomide, levetiracetam, linezolid, melperone, menatetrenone, meropenem, methotrexate, metoprolol, molsidomine, nedaplatin, nicotinamide, nitrofurantoin, nonsteroidal anti-inflammatory drugs (NSAIDS) (e.g., ibuprofen, naproxen, celecoxib, diclofenac, and similar), octreotide, pantoprazole, penicillamine , phenytoin, piperacillin, propranolol, proton pump inhibitors, quinine, rifampin, ruxolitinib, ruxolitinib phosphate, sirolimus, spironolactone, streptokinase, sulfamethoxazole, sulfisoxazole, sunitinib, teicoplanin, temozolomide, temsirolimus, ticlopidine, tirofiban, trimethoprim / sulfamethoxazole, urokinase, valganciclovir, valproic acid, vancomycin, warfarin, or a combination thereof.

[0047] In some embodiments, drug-induced thrombocytopenia (DITP) is induced by administration of filgrastim (granulocyte colony-stimulating factor; G-CSF), interferon, interferon alpha, pegylated interferon alpha 2B, pegylated interferon alpha 2B / ribavirin, factor VIII, TNF alpha, INF gamma, or a combination thereof.

[0048] In some embodiments, drug-induced thrombocytopenia (DITP) is induced by administration of abciximab, adalimumab, alemtuzumab, an antibody-drug conjugate, antithymocyte globulin, brentuximab, cixutumumab, efalizumab, natalizumab, rituximab, trastuzumab, or a combination thereof.

[0049] In embodiments related to Vaccine-Induced Thrombosis and Thrombocytopenia Syndrome (VITT), VITT can be induced by administration of a vaccine. In some embodiments, (VITT) is induced by administration of a vaccine delivered in an adenoviral vector. In certain aspects, the adenoviral vector comprises a therapeutic and / or prophylactic agent. In some embodiments, the therapeutic or prophylactic agent is a gene therapy agent. In some embodiments, the vaccine is for preventing coronavirus infection. In some embodiments, the coronavirus infection is COVID-19. In some embodiments, the vaccine is AZD1222 (Oxford-AstraZeneca COVID-19 vaccine).

[0050] In some embodiments, the vaccine is for preventing an infectious disease selected from measles, mumps, rubella, chicken pox, herpes simplex virus 1, herpes simplex virus 2, chicken pox, rotavirus, influenza, yellow fever, smallpox, hepatitis B, human papillomavirus, pneumococcus, hepatitis A, splenic hepatitis, diphtheria, acellular pertussis, Haemophilus influenzae, such as type B, meningococcus C, meningitis, typhoid fever, rabies, Lyme disease, tetanus, or any combination thereof.

[0051] In some embodiments, compound I is (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, (S)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, (R)-2-[3-[4-amino-3-(2-fluoro-4 a mixture of (S)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile and (S)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile; or an individual (E) or (Z) isomer of any of the above compounds; and / or a pharma- ceutically acceptable salt of any of the above compounds.

[0052] In some embodiments, compound I is the (E)-isomer of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile or a pharma- ceutically acceptable salt thereof.

[0053] In some embodiments, compound I is the (Z)-isomer of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile or a pharma- ceutically acceptable salt thereof.

[0054] In some embodiments, compound I is a mixture of the (E) and (Z) isomers of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, or a pharma- ceutically acceptable salt thereof.

[0055] In some embodiments, compound II is (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, (S)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, (R)-2-(3-(4-amino-3-(2-fluoro-4 or an individual (E) or (Z) isomer of any of the above compounds; and / or a pharmaceutically acceptable salt of any of the above compounds.

[0056] In some embodiments, compound II is the (E)-isomer of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile or a pharma- ceutically acceptable salt thereof.

[0057] In some embodiments, compound II is the (Z)-isomer of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile or a pharma- ceutically acceptable salt thereof.

[0058] In some embodiments, compound II is a mixture of the (E) and (Z) isomers of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, or a pharma- ceutically acceptable salt thereof. EXAMPLES

[0059] method A. Eligibility Patients presenting with thrombosis and thrombocytopenia following vaccination with the AstraZeneca vaccine AZD1222 (previously ChAdOx1 nCoV-19) were recruited.

[0060] B. Antibodies and Reagents A mouse monoclonal IgG2b antibody against human CD32 (IV.3) was purified from hybridoma cell supernatant and IV.3 F(ab) fragments were generated using the Pierce Fab Preparation Kit (Thermo Fisher Scientific, catalog #44985).

[0061] Serum preparation: Patient and healthy donor serum was collected after centrifugation of clotted whole blood (2000×g, 10 min, room temperature (RT)). Patient serum was collected before and after treatment with dexamethasone and intravenous immunoglobulin (IVIg; see Table 1 in Example 2).

[0062] C. Preparation of Human Platelets Washed platelets were prepared from citrated whole blood as described in Nicolson et al., Low-dose BTK inhibitors selectively block platelet activation by CLEC-2, Haematologica 106(1):208-19 (2021). Briefly; citrated blood was drawn from healthy drug-free volunteers, mixed with citrate dextrose (1:10, v / v), and centrifuged (200 × g, 20 min, room temperature) to generate platelet-rich plasma. The platelet-rich plasma was then centrifuged (1000 × g, 10 min, room temperature) in the presence of 0.2 μg / mL prostacyclin. The platelet pellet was resuspended in modified Tyrode's-HEPES buffer, citrate dextrose, and 0.2 μg / mL prostacyclin prepared as described in Nicolson et al., and centrifuged (1000 × g, 10 min, room temperature). Platelet pellet: 2 x 10 8 The mixture was resuspended in modified Tyrode's-HEPES buffer to a concentration of 100 µg / mL and allowed to stand for 30 minutes before use.

[0063] D. Light Transmitted Agglutination Assay (LTA) After stimulation with serum (1:15, v / v) for 20 min, washed platelets (2 × 10 8 Aggregation was measured at 100 µg / mL with stirring (1200 rpm) at 37° C. Washed platelets were preincubated with IV.3 F(ab) for 5 min or with inhibitors for 10 min prior to stimulation with serum. Modified Tyrode's-HEPES buffer or dimethyl sulfoxide (DMSO) was used as vehicle.

[0064] E. Statistical analysis All data are presented as mean ± standard error of the mean (SEM) and p < 0.05 was considered statistically significant. Statistical analysis was performed in GraphPad Prism9 (GraphPad Software Inc.) using one-way or two-way ANOVA with Dunnett's correction for multiple comparisons. EXAMPLES

[0065] result A. Patients with Vaccine-Induced Thrombosis and Thrombocytopenia Syndrome (VITT) exhibit thrombosis, thrombocytopenia, high D-dimer levels, and antiplatelet factor 4 (PF4) antibodies. The presentation, findings, treatment, and outcomes of the seven patients with VITT are summarized in Table 1 .

[0066] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0067] All seven patients were Caucasian, under 50 years of age, and had no previous, symptomatic COVID-19. Patients presented with thrombosis (six patients had cerebral venous sinus thrombosis [CVST] and one patient had an ischemic stroke) and thrombocytopenia 9-14 days after first AZD1222 vaccination. All patients presented with headaches, and one also had expressive aphasia 10-14 days after dosing with AZD1222. At presentation, clinical studies showed that all patients were thrombocytopenic (range: 7-113 × 10) with very high D-dimer and low fibrinogen levels. 9Four patients were male and three were female.

[0068] Heparin-induced thrombocytopenia (HIT) screening using the antiplatelet factor 4 (PF4) IgG assay (Immucor catalog #HAT45G) showed strong reactivity in all patients, despite no prior exposure to heparin. By using the Heparin-induced Platelet Activation (HIPA) Assay (HITAlert™ Kit; IQ Products catalog IQP-396), sera from four of the patients tested showed platelet activation that was reduced by low concentrations of heparin and blocked by high concentrations of heparin, compared with the sera from patients whose platelets were activated by low concentrations of heparin. These results are similar to reports of other patients with VITT. See, e.g., Greinacher et al., Thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination, N Engl J Med, doi:10.1056 / NEJMoa2104840 (2021); Schlutz et al., Thrombosis and thrombocytopenia after ChAdOx1 nCoV-19 vaccination, N Engl J Med, doi:10.1056 / NEJMoa2104882 (2021). Brain tomography confirmed cerebral venous sinus thrombosis (CVST) and intracerebral hemorrhage in three patients, and ischemic stroke caused by arterial thrombosis of the internal carotid artery in one patient.

[0069] All patients received intravenous immunoglobulin (IVIg) and the steroid dexamethasone as recommended by the British Society of Haematology guidelines for VITT (British Society of Haematology, Guidance produced from the Expert Haematology Panel (EHP) focused on Covid-19 vaccines inducing thrombosis and thrombocytopenia (VITT) (2021)). Platelet counts improved over 1–4 days in all patients except for one who died 24 hours after presentation. At the time of writing, three patients have recovered and been discharged from hospital with normal platelet counts, one patient remains hospitalized, and two patients have died due to sequelae of CVST and secondary intracerebral hemorrhage. One discharged patient who received dabigatran had recurrent thrombocytopenia and headaches, but no increase in thrombosis or D-dimers 8 weeks after discharge, and required repeated treatment with IVIg and corticosteroids. Two patients underwent plasma exchange. Of note, IVIg has also been shown to rapidly inhibit HIT antibodies that induce platelet activation. Warkentin, High-dose intravenous immunoglobulin for the treatment and prevention of heparin-induced thrombocytopenia: a review, Expert Rev Hematol 12(8):685-98, doi:10.1080 / 17474086.2019.1636645(2019). Patients also received non-heparin anticoagulants, and two patients required intensive care unit support.

[0070] B. Serum from patients with VITT induces platelet aggregation Serum was collected from healthy donors and patients with VITT. Patient 1 had serum collected after IVIg was administered. Patients 2, 3, and 4 had serum collected both before and after IVIg administration. Patient 2 received dexamethasone before the first serum collection. To examine the effect on platelet activation, these sera were added to washed platelets and platelet aggregation was measured (Figure 1A-1C). Serum from patients with VITT caused platelet aggregation to a variable extent depending on the platelet donor, which was abolished in the post-IVIg treatment serum. Low titer anti-PF4 antibodies have been found to develop after vaccination in a small proportion of healthy individuals; however, they did not cause platelet aggregation. Aggregation was inhibited after IVIg treatment, except in two patients who did not respond clinically to IVIg and required plasma exchange (Figure 1A). In these two patients, the aggregation response was blocked after plasma exchange. Eptifibatide treatment confirmed that the response was aggregation and not agglutination. Three replicates were performed for each serum sample in platelets from healthy donors known to respond.

[0071] Addition of the integrin αIIbβ3 inhibitor eptifibatide (9 μM) inhibited the response to the patient's serum, confirming that this was aggregation rather than agglutination (data not shown).

[0072] C. Platelet aggregation in response to serum from patients with VITT is abolished by FcγRIIA blockade and heat inactivation Platelet activation in HIT occurs through antibody-mediated clustering of FcγRIIA. Greinacher et al., Autoimmune heparin-induced thrombocytopenia, J Thromb Haemost 15(11):2099-114 (2017). To determine whether a similar mechanism is involved in VITT, we used anti-FcγRIIA blocking IV.3 F(ab). Platelet activation by patient serum was abolished in the presence of IV.3 F(ab), demonstrating that platelet activation in VITT is mediated by FcγRIIA (Figure 1A).

[0073] The effects of PF4 and heparin in HIT in combination with patient serum were evaluated. Both PF4 and low concentrations of heparin were found to enhance platelet responses in the HIT assay, whereas high concentrations of heparin inhibited all responses. Rubino et al., A comparative study of platelet factor 4-enhanced platelet activation assays for the diagnosis of heparin-induced thrombocytopenia, H Thrombo Haemost 19(4):1096-102(2021);Vayne et al., Beneficial effect of exogenous platelet factor 4 for detecting pathogenic heparin-induced thrombocytopenia antibodies, Br J Haematol 179(5):811-9(2017);Padmanabhan et al., A novel PF4-dependent platelet activation assay identifies patients likely to have heparin-induced thrombocytopenia / thrombosis, Chest150(3):506-15(2016). The partial agglutination enhancement observed for the serum of patient 2 was not observed in the presence of 10 μg / mL PF4 (data not shown). Low concentrations of heparin are known to enhance platelet responses in the HIT assay, whereas high concentrations are inhibitory. In contrast, low concentrations (0.2 U / mL) of heparin either prevent (5 of 7 patients) or delay (2 of 7 patients) aggregation. High heparin concentrations (100 U / mL) inhibit aggregation (Figures 1B-1C).

[0074] Immune complexes activating platelets via FcγRIIA have been reported in patients critically ill with COVID-19. In these patients exposed to heparin and exhibiting thrombocytopenia and thrombosis, HIT was excluded by the absence of anti-PF4 antibodies and heparin-independent platelet activity. Platelet activation by these immune complexes was blocked by both low and high concentrations of heparin. We observed that heparin blocked platelet aggregation, indicating that the decision to withhold heparin use in patients with VITT should probably be reconsidered. Unfractionated heparin treatment has been reported in one patient with VITT without adverse effects.

[0075] Anti-SARS-CoV-2 spike protein IgG antibodies from patients with severe COVID-19 have been found to induce apoptosis and increase phosphatidylserine externalization in platelets mediated by FcγRIIA, but IgG aggregates or immune complexes could not be isolated from the patient's serum. A similar mechanism may occur in patients with VITT. FcγRIIA activation can result in phosphatidylserine exposure and procoagulant platelets, which may lead to the extensive thrombosis and thrombocytopenia observed in patients with VITT. To exclude platelet activation from other sources in serum (e.g., thrombin and complement), we used heat inactivation (56°C, 45 min) of the serum of three patients that caused activation. Warkentin et al., The platelet serotonin-release assay, Am J Hematol 90(6):564-72, doi:10.1002 / ajh.24006 (2015). Heat inactivation of the patient's serum prevented aggregation in three of seven patients (Figure 1A), whereas a mild effect on aggregation was observed with compstatin (a C3a inhibitor) and FUT-175 (a C3, C4, and C5 inhibitor; data not shown). These findings indicate that complement is not critical but may reinforce platelet activation. Eculizumab (an anti-C5 monoclonal antibody) treatment has been reported in two patients with VITT in whom anticoagulants and IVIg or plasma exchange had failed. Both patients improved rapidly. In VITT pathology, the involvement of complement, which mediates a widespread thromboinflammatory response involving endothelium, mononuclear cells, and neutrophils, as well as platelets, should be considered. Normal serum complement levels in patients with VITT have been reported.

[0076] D. Inhibition of Bruton's tyrosine kinase (Btk) by rilzabrutinib prevents platelet aggregation induced by serum from VITT patients We tested whether the Btk inhibitor rilzabrutinib (rilzabrutinib powder prepared in DMSO to achieve a concentration of 0.5 μM (final 0.02% DMSO)) could prevent platelet aggregation in patient serum. As shown in Figures 2A-2B, rilzabrutinib prevented platelet aggregation in patient serum (Figures 2A-B). Rilzabrutinib completely inhibited aggregation up to 3 μg / mL collagen (results not shown).

[0077] equivalent The above specification is believed to be sufficient to enable those skilled in the art to practice the embodiments. The above description and examples detail certain embodiments and explain the best mode contemplated by the inventors. However, no matter how detailed the above description may be in the text, the embodiments may be practiced in many ways and should be interpreted according to the appended claims and any equivalents thereof.

[0078] As used herein, the term "about" refers to numerical values, such as integers, fractions, and percentages, whether or not expressly indicated. The term "about" generally refers to a range of numerical values ​​(e.g., + / - 5 to 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). For example, when a term such as "at least" and "about" precedes a list of numerical values ​​or ranges, the term modifies all of the values ​​or ranges provided in the list. In some cases, the term "about" may include numerical values ​​that are rounded to the nearest significant figure.

Claims

1. 1. A pharmaceutical composition for treating or preventing drug-induced thrombocytopenia (DITP) in a human subject in need thereof, comprising: Compound (I): 【Chemistry 1】 [In the formula, ＊ C is a stereochemical center; Compound (II): 【Chemistry 2】 [In the formula, ＊ C is a stereochemical center; and a pharma- ceutically acceptable salt thereof.

2. 1. A pharmaceutical composition for treating or preventing Vaccine Induced Thrombosis and Thrombocytopenia Syndrome (VITT) in a human subject in need thereof, comprising: Compound (I): 【Chemistry 3】 [In the formula, ＊ C is a stereochemical center; Compound (II): 【Chemistry 4】 [In the formula, ＊ C is a stereochemical center; and a pharma- ceutically acceptable salt thereof.

3. 1. A pharmaceutical composition for increasing platelet count in a human subject with Drug-Induced Thrombocytopenia (DITP) or Vaccine-Induced Thrombosis and Thrombocytopenia Syndrome (VITT), comprising: Compound (I): 【Chemistry 5】 [In the formula, ＊ C is a stereochemical center; Compound (II): 【Chemistry 6】 [In the formula, ＊ C is a stereochemical center; and a pharma- ceutically acceptable salt thereof.

4. 1. A pharmaceutical composition for reducing platelet aggregation in a human subject having drug-induced thrombocytopenia (DITP) or vaccine-induced thrombosis and thrombocytopenia syndrome (VITT), comprising: Compound (I): 【Chemistry 7】 [In the formula, ＊ C is a stereochemical center; Compound (II): 【Chemistry 8】 [In the formula, ＊ C is a stereochemical center; and a pharma- ceutically acceptable salt thereof.

5. The human subject may be: a. High D-dimer levels; b. Thrombosis; and c. Anti-platelet factor 4 (PF4) antibody The pharmaceutical composition according to any one of claims 1 to 4, having at least one characteristic selected from the group consisting of:

6. Drug-induced thrombocytopenia (DITP) is (a) a small molecule, protein, or component, diluent, excipient, or the like found in a therapeutic regimen; or (b) Unfractionated heparin, enoxaparin, dalteparin, tinzaparin, acenocoumarol, acetaminophen, acetyldigoxin, alfacalcidol, allopurinol, alteplase, amphotericin B, argatroban, aspirin, atenolol, azathioprine, bivalirudin, bortezomib, capecitabine, captopril, carbamazepine, carboplatin, carfilzomib, ceftriaxone, cephalexin, chlorthalidone, cilastatin / imipenem, clopidogrel, clo Zapine, cyclocytidine, dactinomycin / actinomycin, deferasirox, deferiprone, diflunisal, digoxin, dipyridamole, drospirenone / ethinyl estradiol, eltrombopag, epoetin alfa, epoprostenol, eptifibatide, famotidine, fluconazole, fluorouracil, furosemide, fusidic acid, ganciclovir, gemcitabine, gentamicin, glycoprotein IIB / IIA inhibitors, gold, hydrochlorothiazide, hydrochlorothiazide / triazolamide. Amterene, hydroxychloroquine, imatinib, inamrinone, integrilin, ITP drugs, ixazomib, lenalidomide, levetiracetam, linezolid, melperone, menatetrenone, meropenem, methotrexate, metoprolol, molsidomine, nedaplatin, nicotinamide, nitrofurantoin, nonsteroidal anti-inflammatory drugs (NSAIDS) (e.g., ibuprofen, naproxen, celecoxib, diclofenac, and the like), octreotide, pantoprazole, penicillamine, phenytoin, piperacillin, propranolol, proton pump inhibitors, quinine, rifampin, ruxolitinib, ruxolitinib phosphate, sirolimus, spironolactone, streptokinase, sulfamethoxazole, sulfisoxazole, sunitinib, teicoplanin, temozolomide, temsirolimus, ticlopidine, tirofiban, trimethoprim / sulfamethoxazole, urokinase, valganciclovir, valproic acid, vancomycin, warfarin, or combinations thereof; or (c) filgrastim (granulocyte colony stimulating factor; G-CSF), interferon, interferon alpha, pegylated interferon alpha 2B, pegylated interferon alpha 2B / ribavirin, factor VIII, TNF alpha, INF gamma, or a combination thereof; or (d) abciximab, adalimumab, alemtuzumab, antibody-drug conjugates, antithymocyte globulin, brentuximab, cixutumumab, efalizumab, natalizumab, rituximab, trastuzumab, or combinations thereof. The pharmaceutical composition according to any one of claims 1, 3 and 4, wherein the administration of

7. The pharmaceutical composition according to any one of claims 2 to 4, wherein the Vaccine-Induced Thrombosis and Thrombocytopenia Syndrome (VITT) is induced by administration of a vaccine.

8. Vaccine-induced thrombosis and thrombocytopenia syndrome (VITT) is (a) administration of a vaccine delivered in an adenoviral vector; (b) administration of a vaccine delivered in an adenoviral vector that contains a therapeutic and / or prophylactic agent; (c) administration of a vaccine delivered in an adenoviral vector that contains a gene therapy agent; (d) the administration of a vaccine to prevent coronavirus infection; or (e) Administration of vaccines to prevent COVID-19 infection. The pharmaceutical composition according to claim 7, which is induced by

9. 8. The pharmaceutical composition of claim 7, wherein the vaccine is for preventing an infectious disease selected from measles, mumps, rubella, chicken pox, herpes simplex virus 1, herpes simplex virus 2, chicken pox, rotavirus, influenza, yellow fever, smallpox, hepatitis B, human papillomavirus, pneumococcus, hepatitis A, splenic hepatitis, diphtheria, acellular pertussis, Haemophilus influenzae, such as type B, meningococcus C, meningitis, typhoid fever, rabies, Lyme disease, tetanus, or any combination thereof.

10. Compound I is (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, (S)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[ 5. The pharmaceutical composition of any one of claims 1 to 4, which is a mixture of (S)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile and (S)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile; or an individual (E) or (Z) isomer of any of said compounds; and / or a pharma- ceutically acceptable salt of any of said compounds.

11. Compound I is (a) the (E)-isomer of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, or a pharma- ceutically acceptable salt thereof; (b) the (Z)-isomer of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, or a pharma- ceutically acceptable salt thereof; or (c) a mixture of the (E) and (Z) isomers of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, or a pharma- ceutically acceptable salt thereof. The pharmaceutical composition according to claim 10,

12. Compound II is (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, (S)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)- carbonyl)-4,4-dimethylpent-2-enenitrile, (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile and (S)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d ]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, or an individual (E) or (Z) isomer of any of said compounds; and / or a pharma- ceutically acceptable salt of any of said compounds.

13. Compound II is (a) the (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile (E) isomer, or a pharma- ceutically acceptable salt thereof; (b) the (Z)-isomer of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, or a pharma- ceutically acceptable salt thereof; or (c) A mixture of the (E) and (Z) isomers of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-4,4-dimethylpent-2-enenitrile, or a pharma- ceutically acceptable salt thereof. The pharmaceutical composition of claim 12,

Images