Use of LRRC8 proteins as modulators for prevention and treatment of cardiovascular diseases

Abstract

The present invention relates to various methods for preventing and / or treating cardiovascular thrombosis, hereditary or acquired platelet function deficiency, and hereditary or acquired thrombocythemia, wherein the methods comprise administering to a patient a therapeutically effective amount of an LRRC8 protein modulator. Also described are compounds that are modulators of the LRRC8 protein, including DCPIB (4-[2 [butyl-6, 7-dichloro-2-cyclopentyl-2, 3-dihydro-1-oxo-1H-inden-5-yl) oxy] butyric acid).

Description

Technical Field

[0001] The present invention relates to various methods for inhibiting cardiovascular thrombosis and platelet dysfunction. For example, the present invention relates to various methods for preventing or treating diseases in which cardiovascular thrombosis leads to disease states, such as myocardial ischemia and infarction, stroke, transient ischemic attack, peripheral arterial disease, thromboembolism associated with atrial fibrillation and flutter, and thromboembolism associated with the use of cardiovascular medical devices, as well as diseases associated with platelet dysfunction (including abnormal adhesion, aggregation, activation or thrombosis). Background of the Invention

[0003] The LRRC8 (leucine-rich repeat-containing protein 8) family of protein channels consists of different heterohexameric combinations of one or more of five LRRC8 monomeric proteins (LRRC8 A, E, and F). The genes encoding the five LRRRC8 monomeric proteins are located on three human chromosomes: 1 (LRRC8 B, LRRC8 C, and LRRC8 D), 9 (LRRC8 A), and 19 (LRRC8 E).

[0004] Platelets are megakaryocyte fragments that are an integral part of the in vivo thrombus formation required for hemostasis. A variety of agonist molecules, including thrombin, thromboxane, adenosine diphosphate (ADP), and collagen, stimulate platelet activation, including shape changes and fusion of lysosome-related organelles (LROs) (α-granules, dense granules, and lysosomes) with the platelet plasma membrane. This membrane fusion results in the expression of adhesion molecules on the platelet surface and the release of procoagulant molecules, including von Willebrand factor and fibrinogen (α-granules), as well as ATP, ADP, Ca, and ATP. 2+ and serotonin (dense granules), procoagulant molecules that enable platelets to adhere to the deendothelialized surface of blood vessels and activate coagulation pathways, thereby promoting platelet aggregation and thrombus formation.

[0005] Thrombosis is an integral part of hemostasis after blood vessels are damaged by trauma or surgical procedures. Thrombosis can also cause significant morbidity and mortality when it occurs in ruptured atherosclerotic plaques, the left atrial appendage, or on the surface of medical devices exposed to blood flow. In situ thrombosis on ruptured atherosclerotic plaques can cause myocardial, cerebrovascular, and lower limb ischemia and infarction. Thromboembolism formed in the left atrial appendage of patients with atrial fibrillation and flutter can cause cerebrovascular ischemia and infarction as well as myocardial and lower limb ischemia and infarction. Thrombosis on the surface of in vivo medical devices exposed to blood flow (e.g., prosthetic heart valves, coronary diagnostic and therapeutic catheters; coronary, cerebrovascular, and peripheral stents; intravascular blood flow pumps) or ex vivo medical devices (e.g., extracorporeal membrane oxygenation (ECMO) devices; external ventricular assist devices) can lead to blood conduit occlusion and embolism, causing myocardial, cerebrovascular, and lower limb ischemia and infarction.

[0006] Numerous pharmacologic strategies have been developed to prevent thrombosis, which contributes to morbidity and mortality. These include inhibitors of platelet enzymes involved in thromboxane formation (e.g., acetylsalicylic acid inhibits cyclooxygenase), inhibitors of platelet agonists (e.g., bivalirudin inhibits the proteolytic site on thrombin), and inhibitors of platelet receptors for platelet agonists (e.g., clopidogrel inhibits the P2Y12 purinergic receptor). Despite these therapies, a significant residual risk of morbidity and mortality from cardiovascular thrombosis persists. In the United States, an estimated 605,000 new and 200,000 recurrent myocardial infarctions and 610,000 new and 185,000 recurrent strokes occur annually. In addition, significant morbidity and mortality from thrombosis associated with the use of cardiovascular medical devices, including the use of intravascular diagnostic and therapeutic intravascular catheters, intravascular blood pumps, ventricular assist devices, intra-aortic balloon pumps, extracorporeal membrane oxygenation devices, prosthetic heart valves, and cardiac implantable electronic devices, remains.

[0007] The LRRC8 protein family has five members, encoded by genes on three chromosomes: LRRC8A on human chromosome 9; LRRC8B, LRRC8C, and LRRC8D on human chromosome 1; and LRRC8E on human chromosome 19. These proteins form heterohexamers that function as membrane channels for ions and small molecules. LRRC8A was discovered in a child with agammaglobulinemia associated with a chromosomal translocation that truncates the protein. In 2014, it was shown that the voltage-regulated anion current (VRAC) is mediated by hexameric LRRC8 proteins containing at least LRRC8A. Subsequently, it was shown that LRRC8 proteins are expressed in tissue-specific combinations. In 2020, LRRC8A was shown to be required for normal lysosomal function in multiple cell lines, including HAP1 cells, which are derived from KBM-7 cells taken from a patient with chronic myeloid leukemia. An important step in platelet activation is the fusion of lysosome-related organelles (including α-granules, dense granules, and lysosomes) with the plasma membrane.

[0008] Increased platelet volume has been associated with myocardial infarction and death after myocardial infarction, worse outcomes after acute ischemic stroke, and peripheral arterial disease. Increased platelet volume has also been associated with worse clinical outcomes, including primary percutaneous coronary revascularization, type 2 diabetes, diabetic microvascular complications, and nonalcoholic fatty liver disease.

[0009] The LRRC8 protein has previously been shown to regulate the cell volume of adipocytes and other cells. Recently, it has been shown that small molecules, including but not limited to DCPIB (4-((2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy)butanoic acid), can modulate LRRC8 function in vivo to regulate blood sugar control and other functions.

[0010] Cardiovascular disease (CVD) and type 2 diabetes (T2D) are overlapping global epidemics. An estimated 463 million adults worldwide have T2D, of whom 30-34%, or 149 million, have both T2D and CVD. T2D accelerates the development and severity of CVD, increasing the risk of coronary heart disease death, non-fatal myocardial infarction (MI), and ischemic stroke by 2-4 times, and worsens clinical outcomes in patients who experience a CVD event. CVD is the most common cause of death in patients with T2D—approximately two-thirds of patients with DM die from CVD. Stroke and MI most commonly occur when platelet-rich thrombi form at the site of a ruptured atherosclerotic plaque, partially or completely occluding the vessel lumen and leading to downstream ischemia. In T2D, multiple factors accelerate this process, including a prothrombotic state of platelet hyperreactivity and increased circulating coagulation factors, as well as abnormalities in vessel wall function, including decreased endothelial nitric oxide synthase (eNOS) signaling. Furthermore, multiple factors make platelets less responsive to inhibition by current antiplatelet drugs in patients with T2D. The economic burden of treating stroke and myocardial ischemia in patients with T2D is staggering. In 2018, 1.87 million patients with T2D in the United States were hospitalized for major cardiovascular disease (CVD), including 440,000 for ischemic heart disease, 334,000 for stroke, and 154,000 for lower limb amputations. An estimated 49% of direct care costs for T2D are spent on preventing and treating CVD. Total direct care costs for T2D in the United States increased by 26%, from $188 billion in 2012 to $237 billion in 2017. A meta-analysis of studies from multiple countries suggests that direct costs for CVD care in patients with T2D may account for up to 49% of total direct care costs. At least ten drug classes are approved for the treatment of hyperglycemia associated with T2D. Although newer glycemic control agents (such as SGLT2 inhibitors and GLP1 agonists) can help reduce CVD events in T2D, a significant residual CVD risk remains. Currently, four classes of drugs are commonly used to prevent and / or treat MI or stroke: aspirin, P2Y 12 Receptor inhibitors, thrombin receptor inhibitors and platelet αIIbβ3 inhibitors. They are used alone as monotherapy and in combination (dual antiplatelet therapy; DAPT) to prevent and treat stroke and MI. Although current antiplatelet drugs can reduce CVD events and deaths, their therapeutic potential is limited by the major bleeding associated with their use (e.g., intracranial hemorrhage and hemorrhagic death).

[0011] There is an urgent need for therapies to prevent and treat conditions associated with cardiovascular thrombosis, including myocardial ischemia and infarction, stroke, transient ischemic attack, peripheral arterial disease, thromboembolism associated with atrial fibrillation and flutter, and thromboembolism associated with the use of cardiovascular medical devices, including intravascular diagnostic and therapeutic catheters, intravascular blood pumps, ventricular assist devices, intra-aortic balloon pumps, extracorporeal membrane oxygenation devices, prosthetic heart valves, and cardiac implantable electronic devices. Furthermore, there is a significant unmet clinical need for a drug that can both improve glycemic control in T2D and safely prevent cerebral and coronary thrombosis. This unmet clinical need represents a significant commercial opportunity. It is estimated that, based on current guidelines, over 8 million patients with T2D are eligible for antiplatelet therapy for secondary prevention of MI or stroke. Using the current price of novel oral antiplatelet agents (e.g., ticagrelor), this equates to a secondary MI prevention market value of over $20 billion annually in the United States and the European Union alone. Summary of the Invention

[0012] Various aspects of the present invention relate to methods for preventing or treating thrombosis. In various embodiments, the method for preventing or treating thrombosis in a subject in need thereof comprises administering to the subject a therapeutically effective amount of DCPIB (4-((2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy)butanoic acid) or a homologue thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:

[0013]

[0014]

[0015] and their salts and geometric isomers.

[0016] In various embodiments, the method comprises administering to the subject a therapeutically effective amount of a compound of formula (I) and its salts and geometric isomers:

[0017]

[0018] in

[0019] R 1 and R 2 are each independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

[0020] R 3 Y-C(O)R 4, Z–N(R 5 )(R 6 ) or –Z–A;

[0021] A is selected from the group consisting of:

[0022]

[0023] R 4 is hydrogen, substituted or unsubstituted alkyl, –OR 7 or –N(R 8 )(R 9 );

[0024] X 1 and X 2 are each independently substituted or unsubstituted alkyl, halo, -OR 10 or –N(R 11 )(R 12 );

[0025] R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 and R 12 are each independently hydrogen or substituted or unsubstituted alkyl;

[0026] Y and Z are each independently a substituted or unsubstituted carbon-containing moiety having at least 2 carbon atoms; and n is 1 or 2.

[0027] In various embodiments, the methods of preventing or treating thrombosis include treating diseases in which thrombosis leads to a disease state. The present invention relates to various methods of preventing or treating diseases in which cardiovascular thrombosis leads to a disease state, such as myocardial ischemia and infarction, stroke, transient ischemic stroke, peripheral arterial disease, thromboembolism associated with atrial fibrillation and flutter, and thromboembolism associated with the use of cardiovascular medical devices, as well as diseases associated with platelet dysfunction (including abnormal adhesion, aggregation, activation or thrombosis).

[0028] Other objects and features will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1We describe how the LRRC8 protein regulates human platelet volume. Single nucleotide polymorphisms (SNPs) assigned to the LRRC8 gene AD were associated with human platelet volume in whole blood in human genome-wide association studies (GWAS), human phenome-wide association studies (PheWAS), and the expression quantitative trait loci (eQTL) database in whole blood. Genome-wide association studies (GWAS) suggest that the LRRC8 protein regulates human platelet volume. A search of the NHGARI-EBI catalog of eligible published human genome-wide association studies (accessed 9 February 2022) identified six SNP variants assigned to three genes on two chromosomes: LRRC8A on 9q34.11 (2 x 10 -10 ) and LRRC8 C on 1p22.2 (5x 10 -23 and 3x 10 -14 ) and LRRC8 D(4x 10 -13 and 6x10 -12 )). A phenome-wide association study (PheWAS) showed that the LRRC8 protein regulates human platelet volume. After identifying six SNPs associated with human platelet volume in the NHGARI-EBI catalog of published human GWAS studies, we searched for PheWAS associations between these six SNP variants and platelet volume in the Common Metabolic Diseases Knowledge Portal (accessed March 3, 2022) and the PheWAS-IEU OpenGWAS Project Catalog (PheWAS Catalog) (8.9x 10 -10 , 2.3x 10 -10 , 5.7x 10 -18 , 7.9x 10 -17 , 1.5x 10 -6 and 1.4x 10 -9 ), as well as four additional SNP variants assigned to LRRC8B, overlapping intervals between LRRC8 B and C, and the association between LRRC8 C and platelet volume, which was initially identified because of its association with blood monocyte count or diastolic blood pressure in the GWAS catalog. In summary, the platelet volume phenotype is associated with 10 SNPs assigned to four LRRC8 genes (LRRC8 AD) on two chromosomes (1p22.2 and 9q34.11). eQTL studies have shown that LRRC8 protein regulates human platelet volume. The GTEXPortal database expression quantitative trait loci (eQTL) database (accessed on March 5, 2022) was then searched and found that LRRC8B in whole blood (P = 2.8x 10 -56 ) and LRRC 8C (P = 4.8 x 10 -63There was a significant association between mRNA transcript levels of β-catenin and SNPs assigned to those genes and associated with platelet volume in the NHGARI-EBI catalog of published human GWAS studies.

[0030] Figure 2 Describe how the LRRC8 protein regulates human platelet count. Single nucleotide polymorphisms (SNPs) assigned to AD in the LRRC8 gene were associated with human platelet count in whole blood in human genome-wide association studies (GWAS), human phenome-wide association studies (PheWAS), and the expression quantitative trait loci (eQTL) database in whole blood. The phenome-wide association study (PheWAS) showed that the LRRC8 protein regulates human platelet count. PheWAS associations between platelet count and 13 SNP variants were searched in the Common Metabolic Diseases Knowledge Portal (accessed on March 3, 2022) with P values ​​< 10 -5 (Accessed February 9, 2022). PheWAS identified associations between three SNP variants and platelet count (4.9 x 10 -11 , 2.6x 10 -10 and 3.8x 10 -7 In conclusion, the platelet count phenotype was associated with three SNPs assigned to two LRRC8 genes (LRRC8 CD) on chromosome 1 (1p22.2). eQTL studies indicate that the LRRC8 protein regulates human platelet count. The GTEX Portal database expression quantitative trait loci (eQTL) database (accessed on March 5, 2022) was searched and LRRC 8C (P = 4.8 x 10 -63 ) mRNA transcript levels were significantly associated with SNPs assigned to LRRC 8C and associated with platelet count in the Common Metabolic Diseases Knowledge Portal (accessed March 3, 2022) or the PheWAS-IEU OpenGWAS Project Catalog (PheWAS Catalog).

[0031] Figure 3 The results depict how LRRC8A-D mRNA transcripts are expressed at high levels in human platelets compared to other mRNA transcripts. The Plateletomics interactive results tool, version 1.0, was used to search the Plateletomics database (accessed on March 5, 2022) and found that mRNA transcripts of LRRC 8A-8D are expressed in human platelets and are in the top 76th, 14th, 43rd, and top 5th percentiles of all mRNA transcripts detected in platelets from 154 patients, respectively. This indicates that mRNA transcripts of LRRC 8A-D are highly expressed in platelets compared to other mRNA transcripts.

[0032] Figure 4 Depicts how human platelets contain mRNA transcripts for LRRC8A, LRRC8B, LRRC8C, and LRRC8D in ratios of 1 to 2.5 to 1.5 to 3.0, respectively. A database search for LRRC8A-E transcripts in Supplementary Table S2A of Simon, LM et al. (Blood 123, e37-45 (2014)) revealed that LRRCA, LRRC8B, LRRC8C, and LRRC8D mRNA transcripts are expressed in human platelets, with LRRC8B, LRRC8C, and LRRC8D expressed at approximately 2.5-fold, 1.5-fold, and 3-fold the level of LRRC8A mRNA, respectively.

[0033] Figure 5 Depicts how the extent of human platelet aggregation stimulated by protease-activated receptor 4 (PAR4) agonists is proportional to LRRC8A expression. The Plateletomics interactive results tool version 1.0 was used to search the Plateletomics database (accessed on March 5, 2022) and found data indicating that the intensity of human platelet aggregation stimulated by protease-activated receptor 4 activating peptide (PAR4 AP), an agonist of the platelet PAR4 thrombin receptor, was significantly correlated with the amount of LRRC8A mRNA expressed in human platelets.

[0034] Figure 6 Describes how the extent of human platelet aggregation stimulated by adenosine diphosphate (ADP) and protease-activated receptor 1 (PAR1) agonists is inversely correlated with LRRC8B expression. The Plateletomics interactive results tool, version 1.0, was used to search the Plateletomics database (accessed March 5, 2022) and found data demonstrating that the magnitude of human platelet aggregation stimulated by adenosine diphosphate (ADP), an agonist of platelet P2Y12 and other ADP receptors, and protease-activated receptor 1 activating peptide (PAR1 AP), an agonist of the platelet PAR1 thrombin receptor, was significantly inversely correlated with the amount of LRRC8B mRNA expressed in human platelets.

[0035] Figure 7Describes how the extent of human platelet aggregation stimulated by protease-activated receptor 4 (PAR4) agonists is inversely correlated with LRRC8C expression. We searched the Plateletomics database (accessed March 5, 2022) using the Plateletomics Interactive Results Tool, version 1.0, and found data demonstrating that the magnitude of human platelet aggregation stimulated by protease-activated receptor 4 activating peptide (PAR4 AP), an agonist of the platelet PAR4 thrombin receptor, is significantly inversely correlated with the amount of LRRC8C mRNA expressed in human platelets.

[0036] Figure 8 Describes how the extent of human platelet aggregation stimulated by protease-activated receptor 1 (PAR1) agonists is inversely correlated with LRRC8D expression. A search of the Plateletomics database (accessed March 5, 2022) using the Plateletomics Interactive Results Tool, version 1.0, revealed data demonstrating that the magnitude of human platelet aggregation stimulated by protease-activated receptor 1 activating peptide (PAR1 AP), an agonist of the platelet PAR1 thrombin receptor, is inversely correlated with the amount of LRRC8D mRNA expressed in human platelets.

[0037] Figure 9 Racial differences in LRRC8A-D expression in human platelets are depicted. The Plateletomics interactive results tool version 1.0 was used to search the Plateletomics database (accessed March 5, 2022) and found data indicating that LRRC8C and LRRC8D mRNA expression was significantly reduced in individuals who identified as Black compared to those who identified as White. LRRC8A and LRRC8B gene expression was also lower in individuals who identified as Black compared to those who identified as White, but did not exceed the significance threshold in this dataset.

[0038] Figure 10Describes how multiple regression analysis demonstrates that LRRC8A, LRRC8B, and LRRC8D play important roles in human platelet aggregation in response to ADP, PAR1 agonist peptides, and PAR4 agonist peptides. The regression coefficients for LRRC 8A-D in the multiple regression analysis of platelet aggregation responses in 5911 commonly expressed mRNAs in human platelets from 154 human subjects in Supplementary Table S3 of Simon, LM et al. (2014) (Blood 123, e37-45) and the corresponding values ​​of significance tests indicate that the extent of ADP-induced platelet aggregation is significantly correlated with the level of LRRC8A mRNA and significantly negatively correlated with the level of LRRC8B mRNA; the extent of PAR1-induced platelet aggregation is significantly negatively correlated with the levels of LRRC8B and LRRC8D mRNA; and the extent of PAR4-induced platelet aggregation is associated with the level of LRRC8D mRNA. The regression set the dependent variable to be the extent of platelet aggregation in response to one of four platelet agonists (ADP, PAR1 AP, PAR 2AP, and arachidonic acid), and the independent variables to be age, race, sex, platelet number, and BMI, as well as the levels of 5,911 commonly expressed mRNAs.

[0039] Figure 11A and 11B Describe how LRRC8A in platelets regulates platelet volume in vivo. Whole blood was collected from the inferior vena cava of 20-week-old WT mice and mice lacking platelet LRRC8A into heparin anticoagulant (15 USP units / mL) and run on an Element HT5 veterinary hematology analyzer (Heska, Loveland). The results showed that the mean platelet volume (MPV) of platelets lacking LRRC8A was significantly greater than that of WT platelets (P < 0.01; unpaired two-sided t-test). Platelet counts were also increased in mice with platelets lacking LRRC8A compared to WT mice, but the difference did not exceed the significance threshold (P = 00.06).

[0040] Figure 12We describe how LRRC8A is required for normal platelet adhesion of human platelets to collagen. Whole blood was collected in heparinized form from the inferior vena cava of 20-week-old WT mice and mice lacking platelet LRRC8A. Heparinized whole blood was fluorescently labeled with DiOC6 (3,39-dihexyloxocarbocyanine iodide). Whole blood was plasma perfused for 5 minutes at a constant upstream pressure through a polydimethylsiloxane (PDMS) microfluidic device coupled to a glass slide patterned with bovine type 1 collagen, followed by 5 minutes of perfusion with PBS. Platelet accumulation was captured using an Olympus IX83 inverted microscope, and the surface area coverage of platelets was quantified using ImageJ. It was found that the absence of LRRC8A significantly reduced platelet adhesion ((P < 0.0001; two-tailed paired t-test).

[0041] Figure 13 We describe how inhibition of LRRC8 with SN-401 (DCPIB) significantly inhibits human platelet adhesion to collagen. Human blood was collected from four donors in heparinized solution, pooled, and incubated for 10 minutes with fluorescently labeled DiOC6 (3,39-dihexyloxocarbocyanine iodide; Invitrogen) and 10 μM SN-401 (DCPIB) or vehicle (DMSO). Whole blood was perfused at a constant upstream pressure for 5 minutes through a polydimethylsiloxane (PDMS) microfluidic device coupled to a glass slide patterned with bovine type 1 collagen, followed by 5 minutes of perfusion with PBS. Platelet aggregation was captured using an Olympus IX83 inverted microscope, and platelet surface area coverage was quantified using ImageJ. SN-401 was found to significantly reduce human platelet adhesion to collagen (vehicle versus SN-401; P < 0.0001; ordinary one-way ANOVA with Turkey's multiple comparison test).

[0042] Figure 14Depicted is how inhibition of LRRC8 with SN-401 (DCPIB) significantly inhibits thrombosis in vivo. To determine whether acute inhibition of LRRC8 channel activity affects platelet function in vivo, 25- to 30-week-old C57BL / 6 mice were administered vehicle or the known LRRC8 channel inhibitor SN-401 (DCPIB) by IP injection, and an assay for tail wound thrombosis was used to quantify thrombosis in vivo. Specifically, 20 minutes after IP injection of vehicle or 10 mg / kg SN-401, the mouse tail was amputated 5 mm from the tip of the tail, placed in 37°C saline for 15 minutes, and the relative blood loss during this period was quantified by estimating the hemoglobin concentration in the saline by measuring the absorbance at 550 nm. It was found that the mean tail blood loss in mice receiving SN-401 was 4 times the blood loss in mice receiving vehicle (P < 0.05; unpaired two-tailed t-test). This demonstrates that pharmacological inhibition of LRRC8 inhibits thrombosis in vivo.

[0043] Figure 15 Depicts how LRRC8 mRNA is highly expressed in human platelets. Shown are the percentile expression of platelet LRRC8A-D relative to all platelet transcripts. Data from the Plateletomics database.

[0044] Figure 16 Depicted are Western blots of LRRC8A, GAPDH of LRRC8A (loading control), β3 integrin, and associated GAPDH in WT and LRRC8A cKO platelets.

[0045] Figures 17A to 17E Depicts how megakaryocyte (MK) patch clamping reveals robust swelling-activated currents. Figure 17A The rat MK perforated patch clamp is described. Figure 17B Depicted are current density over time in response to swelling with hypotonic solution (HYPO, 205 mOsm) and inhibition by 10 μM SN401. Figure 17C The current-voltage relationship in WT MK is depicted. Figure 17D The current-voltage relationship in cKO MK is depicted. Figure 17E Depicted are the average outward and inward currents at +100 mV and -100 mV in WT, WT+SN401, and cKO MK, respectively. *p<0.05; **p<0.01.

[0046] Figure 18 Mean platelet volume of WT, LRRC8A cKO, and PF4-Cre;LRRC8Afl / + controls is depicted. ****p<0.001.

[0047] Figures 19A to 19B Depicted how platelets lacking LRRC8A exhibit impaired aggregation and adhesion. Figure 19A Figure 3. Impaired platelet adhesion to a collagen-coated microfluidic chamber. Two-way ANOVA, p = 0.01. Figure 19B Platelet aggregation in response to thrombin in WT and cKO mice is depicted (n=7 per group). ****p<0.001.

[0048] Figures 20A to 20B Describe how platelet-specific ablation of LRRC8A impairs platelet activation. Figure 20A P-selectin exposure in response to thrombin in WT and cKO mice is depicted. ****p<0.001. Figure 20B Depicted are αllbβ3 activation in response to thrombin in WT and cKO mice. *p<0.05; ****p<0.001.

[0049] Figures 21A to 21B Depicts how LRRC8A cKO mice exhibit impaired in vivo thrombosis in a FeCl3-induced carotid artery injury model. Figure 21A Carotid artery tracings of WT and LRRC8A cKO mice after injury are depicted. Figure 21B Depicted is the time to total occlusion (TTO). ***p<0.001.

[0050] Figures 22A to 22C Describe how LRRC8A is required for platelet thrombus formation but not fibrinogenesis in laser-induced cremaster arteriolar thrombosis. Figures 22A to 22C Depicted are intravital microscopy of WT control and LRRC8A cKO mice. Platelet aggregation and fibrin production were detected by injection of DyLight 649-conjugated anti-CD42c and Alexa 488-conjugated anti-fibrin antibodies, respectively, after laser-induced injury of the cremaster arteriole. Figure 22A Representative images are depicted. Figure 22B Depicted are quantifications of the median integrated fluorescence intensity of the anti-CD42c (Fplatelet) antibody signal after laser injury. Figure 22C Depicted are quantifications of the median integrated fluorescence intensity of the anti-fibrin (F-fibrin) antibody signal after laser injury.

[0051] Figures 23A to 23B Depicts how SN-401 impairs human platelet adhesion under physiological shear flow conditions in a collagen-coated microfluidic flow chamber. n = 4 subjects with 4 blood draws on 2 different days. Figure 23ADepicted is the percentage of surface area covered by adhered platelets over time in human whole blood treated with vehicle or 10 μM SN-401. p = 0.1 for two-way ANOVA. Figure 23B Describes the basis Figure 23A Calculated area under the curve (AUC) for vehicle and SN-401. For t-test, p = 0.1.

[0052] Figure 24 Depicts how SN-401 treatment increases tail bleeding in mice. Mice were pretreated with vehicle or SN401 (10 mg / kg pi), and tail bleeding was quantified 15 minutes later by measuring the absorbance of hemoglobin at 550 nm. *p < 0.05.

[0053] Figures 25A to 25D Depicts how the homologues SN-418 and SN-418C are potent inhibitors of swelling-activated currents recorded from HEK cells. Figure 25A The chemical structure of SN-418 is described. Figure 25B The chemical structure of SN-418C is described. Figure 25C Currents in the presence of various concentrations of SN-418 and SN-401 are depicted, normalized to the current activated in hypotonic solution alone. The line shows the best fit to the logistic equation, with IC50 values ​​of 420 nM and 4.0 μM for SN-418 and SN-401, respectively. Figure 25D Currents in the presence of various concentrations of SN-418C and SN-401 are plotted, normalized to the current activated in hypotonic solution alone. The line shows the best fit to the logistic equation, with IC50 values ​​of 265 nM and 4.0 μM for SN-418C and SN-401, respectively.

[0054] Figures 26A to 26B Depicted is how SN-418C treatment dose-dependently attenuates platelet activation. Figure 26A Depicted are flow cytometric mean fluorescence intensity (MFI) of αllbβ3 in washed platelets after activation with thrombin or CRP agonists and dose-dependent inhibition by SN-418C. *p<0.05, ***p<0.001. Figure 26B Depicted are flow cytometric mean fluorescence intensity (MFI) of P-selectin in washed platelets following activation with thrombin or CRP agonists and dose-dependent inhibition by SN-418C. *p<0.05.

[0055] Figures 27A to 27B Delineating how SN-418C treatment inhibits agonist-stimulated platelet aggregation. Figure 27ADepicted are washed platelets pretreated with different concentrations of SN418C and subjected to aggregation experiments in the presence of CRP by aggregometry. Figure 27B Depicted are washed platelets pretreated with different concentrations of SN418C and subjected to aggregation experiments in the presence of a thrombin agonist by aggregometry.

[0056] Figure 28 Figure 3 shows how SN418C inhibits thrombus formation in FeCl3-induced carotid artery injury. Shown are carotid artery total occlusion time (TTO) for vehicle (n=6) and 10 mg / kg ip SN-418C (n=7); p=0.06.

[0057] Figures 29A to 29C Depicts how the human MK cell line MEG01 possesses robust, SN-4XX-inhibited, swelling-activated currents. Figure 29A Depicted are the time course of current density in WT MEG01 cells in response to swelling with hypotonic solution (HYPO, 210 mOsm) and inhibition by 10 μM SN401. Figure 29B Depicted are the current-voltage relationships in WT MEG01 cells in response to HYPO and inhibition by 10 μM SN401. Figure 29C Depicted are the current-voltage relationships in WT MEG01 cells in response to HYPO and inhibition by 3 μM SN418c. DETAILED DESCRIPTION

[0058] Various aspects of the present invention relate to methods for preventing and / or treating thrombosis. The methods include methods for preventing and / or treating cardiovascular thrombosis in a patient in need of such therapy, comprising administering to the patient a therapeutically effective amount of a LRRC8 protein modulator. The methods may include administering to the patient a therapeutically effective amount of a LRRC8 protein modulator to the patient to prevent and / or treat conditions caused or exacerbated by cardiovascular thrombosis, including but not limited to myocardial ischemia, myocardial infarction, cerebrovascular transient ischemic attack, cerebrovascular ischemic stroke, or peripheral arterial occlusion. The methods may include administering to the patient a therapeutically effective amount of a LRRC8 protein modulator to the patient to prevent and / or treat cardiovascular thrombosis and conditions caused by cardiovascular thrombosis, which are associated with atrial arrhythmias, including atrial flutter and atrial fibrillation. The method may comprise administering to a patient a therapeutically effective amount of a LRRC8 protein modulator to the patient to prevent and / or treat cardiovascular thrombosis associated with acute or chronic use of medical devices exposed to intravascular blood, including but not limited to intravascular diagnostic and therapeutic catheters, intravascular blood pumps, ventricular assist devices, intra-aortic balloon pumps, extracorporeal membrane oxygenation devices, prosthetic heart valves, and cardiac implantable electronic devices.

[0059] The present disclosure further relates to a method for preventing and / or treating an inherited or acquired platelet function defect in a patient in need of such therapy, the method comprising administering to the patient a therapeutically effective amount of a LRRC8 protein modulator. The method may comprise administering to the patient a therapeutically effective amount of a LRRC8 protein modulator to the patient so as to normalize platelet function by increasing the contents of platelet alpha granules, dense granules, or other intracellular organelle contents, including but not limited to the release or membrane expression of platelet activators, coagulation factors, and adhesion molecules. The method may comprise administering to the patient a therapeutically effective amount of a LRRC8 protein modulator to the patient so as to normalize platelet function by normalizing platelet volume.

[0060] The present disclosure also relates to methods for preventing and / or treating hereditary or acquired thrombocythaemia in a patient in need of such treatment, comprising administering to the patient a therapeutically effective amount of a modulator of the LRRC8 protein.

[0061] The LRRC8 modulator may be DCPIB.

[0062] Administration of the compound may be sufficient to upregulate expression of LRRC8 or LRRC8-related proteins. Administration of the compound may be sufficient to stabilize the LRRC8 protein complex or LRRC8-related proteins. Administration of the compound may be sufficient to promote membrane trafficking and activity of the LRRC8 protein complex or LRRC8-related proteins. Administration of the compound may be sufficient to enhance LRRC8-mediated signaling or trafficking to the membranes of intracellular organelles, including but not limited to lysosome-related organelles.

[0063] In various embodiments, a method of preventing and / or treating thrombosis in a subject in need thereof comprises administering to the subject a therapeutically effective amount of DCPIB (4-[2[butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid or a homologue thereof.

[0064] In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:

[0065]

[0066]

[0067] and their salts and geometric isomers.

[0068] In various embodiments, the method comprises administering to the subject a therapeutically effective amount of a compound of formula (I) and its salts and geometric isomers:

[0069]

[0070] where R 1 and R 2 are each independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

[0071] R 3 Y-C(O)R 4 、–Z–N(R 5 )(R 6 ) or –Z–A;

[0072] A is selected from the group consisting of:

[0073]

[0074] R 4 is hydrogen, substituted or unsubstituted alkyl, –OR 7 or –N(R 8 )(R 9 );

[0075] X 1 and X 2 are each independently substituted or unsubstituted alkyl, halo, -OR 10 or –N(R 11 )(R 12 );R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 and R 12 are each independently hydrogen or substituted or unsubstituted alkyl;

[0076] Y and Z are each independently a substituted or unsubstituted carbon-containing moiety having at least 2 carbon atoms; and n is 1 or 2.

[0077] In various embodiments, R 1 or R 2 At least one of is a substituted or unsubstituted linear or branched alkyl group having at least 2 carbon atoms. In a further embodiment, R 1 is hydrogen or C1 to C6 alkyl. For example, in some embodiments, R 1 In various embodiments, R 2 is a cycloalkyl group (e.g., a cyclopentyl group). In various embodiments, R 1 or R 2At least one of is selected from the group consisting of:

[0078]

[0079] In various embodiments, R 3 Y-C(O)R 4 In various embodiments, R 3 for –Z–N(R 5 )(R 6 ). In various embodiments, R 3 In certain embodiments, R 3 Select from the group consisting of:

[0080]

[0081] In certain embodiments, R 3 Select from the group consisting of:

[0082]

[0083] In various embodiments, A is selected from the group consisting of:

[0084]

[0085] In various embodiments, R 4 for –OR 7 or –N(R 8 )(R 9 ).

[0086] In various embodiments, X 1 and X 2 Each is independently substituted or unsubstituted C1 to C6 alkyl or halo. In some embodiments, X 1 and X 2 Each is independently C1 to C6 alkyl, fluorine, chlorine, bromine or iodine. In certain embodiments, X 1 and X 2 are each independently methyl, fluoro or chloro.

[0087] In various embodiments, R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 and R 12 Each is independently hydrogen or alkyl. For example, in some embodiments, R 5 、R 6 、R 7 、R8 、R 9 、R 10 、R 11 and R 12 Each is independently hydrogen or C1 to C3 alkyl.

[0088] In various embodiments, Y and Z are each independently a substituted or unsubstituted alkylene group having 2 to 10 carbon atoms, a substituted or unsubstituted alkenylene group having 2 to 10 carbon atoms, or a substituted or unsubstituted arylene group. In some embodiments, Y and Z are each independently an alkylene group having 2 to 10 carbon atoms, an alkenylene group having 2 to 10 carbon atoms, or a phenylene group. In certain embodiments, Y is an alkylene group or an alkenylene group having 3 to 8 or 3 to 7 carbon atoms. In various embodiments, Y and Z are each independently a cycloalkylene group having 4 to 10 carbon atoms. For example, Y can be an alkylene group or any alkenylene group having 4 carbon atoms. In further embodiments, Z is an alkylene group having 2 to 4 carbon atoms. For example, Z can be an alkylene group having 3 or 4 carbon atoms. In certain embodiments, Y and Z are each independently selected from the group consisting of:

[0089]

[0090] In various embodiments, when Y is an alkylene group having 2 to 3 carbons, then X 1 and X 2 Each is fluorine or each is a substituted or unsubstituted alkyl group (e.g., methyl or ethyl). In some embodiments, Y is not an alkylene group having 3 carbon atoms. In certain embodiments, R 7 is not hydrogen or C1 to C6 alkyl. In some embodiments, X 1 and / or X 2 In certain embodiments, X 1 and / or X 2 In some embodiments, R 1 and / or R 2 According to an embodiment, the compound of formula (I) may be selected from the group consisting of:

[0091]

[0092] and their geometric isomers and salts.

[0093] In certain embodiments described herein, the compound to be administered is selected from the group consisting of:

[0094]

[0095] and their geometric isomers and salts.

[0096] The compound to be administered may be selected from:

[0097]

[0098] The present disclosure further relates to compounds for use in the above methods. These compounds can modulate or inhibit LRRC8 protein. The compounds are more potent than an equivalent amount of DCPIB (4-[2-[butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid) in modulating or inhibiting the SWELL1 channel.

[0099] In various embodiments, the methods of preventing or treating thrombosis include treating diseases in which thrombosis leads to a disease state. The present invention relates to various methods of preventing or treating diseases in which cardiovascular thrombosis leads to a disease state, such as myocardial ischemia and infarction, stroke, transient ischemic stroke, peripheral arterial disease, thromboembolism associated with atrial fibrillation and flutter, and thromboembolism associated with the use of cardiovascular medical devices, as well as diseases associated with platelet dysfunction (including abnormal adhesion, aggregation, activation or thrombosis).

[0100] The synthesis of this compound can be carried out according to the following scheme:

[0101]

[0102] The above synthetic scheme can be modified to synthesize various compounds described herein, and these modifications are indicated by double arrows. Method: i) AlCl3, DCM, 5°C to room temperature, ii) 12N HCl, iii) 1) paraformaldehyde, dimethylamine, acetic acid, 85°C, iv) DMF, 85°C, v) H2SO4, vi) KOtBu, butyl iodide, vii) pyridine-HCl, 195°C, viii) BrCH2CO2Et, K2CO3, DMF, 60°C, ix) 10N NaOH.

[0103] According to the various methods of the present invention, a pharmaceutical composition comprising a compound of formula (I) is administered to a subject in need thereof. The pharmaceutical composition may be administered by, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal administration. In various embodiments, administration is selected from the group consisting of oral, intranasal, intraperitoneal, intravenous, intramuscular, rectal, and transdermal administration.

[0104] It is within the ability of those skilled in the art to determine a therapeutically effective dose of any one or more of the compounds described herein. A therapeutically effective dose refers to the amount of the active ingredient that provides the desired result. The exact dosage will be determined by a licensed physician based on factors relevant to the subject in need of treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that may be considered include the severity of the disease state, the subject's overall health, the subject's age, weight, and sex, diet, time and frequency of administration, drug combination, reaction sensitivity, and tolerance / response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, weekly, or biweekly, depending on the half-life and clearance rate of the particular formulation.

[0105] Typically, depending on route of administration, the normal dose of compound can vary between about 0.05 to about 100mg / kg body weight. Guidance about specific doses and delivery methods is provided in the literature and is generally available to practitioners in the art. Typically, administration will be made so as to administer, for example, a daily oral dose in the range of about 0.1mg to about 75mg, about 0.5mg to about 50mg, or about 1mg to about 25mg / kg body weight. Active ingredient can be administered every day with a single dose or alternatively with divided doses (for example, twice a day, three times a day, four times a day, etc.). In general, when adopting parenteral route, lower dosage can be administered. Therefore, for example, for intravenous administration, the dosage in the range of, for example, about 0.05mg to about 30mg, about 0.1mg to about 25mg, or about 0.1mg to about 20mg / kg body weight can be administered.

[0106] Pharmaceutical compositions for oral administration can use pharmaceutically acceptable carriers known in the art to be suitable for oral administration in dosage formulations. Such carriers enable pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for subject's uptake. In certain embodiments, compositions are formulated for parenteral administration. Further details about preparation and administration techniques can be found in the latest edition of REMINGTON'S PHARMACEUTICALSCIENCES (Mack Publishing Co., Easton, Pa., which is incorporated herein by reference). After preparing the pharmaceutical compositions, they can be placed in suitable containers and labeled for the treatment of specified conditions. Such labels will include amount, frequency, and method of administration.

[0107] In addition to the active ingredient (e.g., a compound of formula (I)), the pharmaceutical composition may also contain a suitable pharmaceutically acceptable carrier comprising excipients and adjuvants that facilitate processing the active compound into a pharmaceutically acceptable preparation. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or any type of formulation adjuvant. Some examples of materials that can be used as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch, potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate; astragalus powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil; and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; artificial cerebrospinal fluid (CSF) and phosphate buffered solutions, and other nontoxic compatible lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as coloring agents, mold release agents, coating agents, sweetening, flavoring and fragrances, preservatives and antioxidants may also be present in the composition (at the judgment of the formulator based on the intended route of administration).

[0108] Unless otherwise indicated, the alkyl, alkenyl, and alkynyl groups described herein preferably contain 1 to 20 carbon atoms in the backbone. They can be straight or branched or cyclic (e.g., cycloalkyl). Alkenyl groups can contain saturated or unsaturated carbon chains, as long as there is at least one carbon-carbon double bond. Alkynyl groups can contain saturated or unsaturated carbon chains, as long as there is at least one carbon-carbon triple bond. Unless otherwise indicated, the alkoxy groups described herein contain saturated or unsaturated, branched, or straight carbon chains with 1 to 20 carbon atoms in the backbone.

[0109] Unless otherwise indicated herein, the term "aryl" refers to a monocyclic, bicyclic, or tricyclic aromatic group containing 6 to 14 ring carbon atoms and includes, for example, phenyl. The term "heteroaryl" refers to a monocyclic, bicyclic, or tricyclic aromatic group having 5 to 14 ring atoms and containing carbon atoms and at least 1, 2, or 3 oxygen, nitrogen, or sulfur heteroatoms.

[0110] Having described the invention in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention as defined in the appended claims.

[0111] Examples

[0112] Example 1: Genome-wide association studies (GWAS) show that LRRC8 protein regulates human platelet volume ( Figure 1 ).

[0113] The NHGARI-EBI catalog of eligible published human genome-wide association studies was searched (accessed on February 9, 2022). This data catalog is currently mapped to the Genome Assembly GRCh38.p13 and dbSNP Build154. The NHGARI-EBI catalog of published human GWAS studies lists 13 SNP variants assigned to five LRRC8 genes that are associated with six metabolic, inflammatory, and CV traits with P values ​​< 10 -8 Each of the five LRRC8 genes contained at least one of these 13 SNPs (accessed February 9, 2022). GWAS in the database identified an association between platelet volume and six SNP variants assigned to three genes on two chromosomes (LRRC8 A on 9q34.11 (2x 10 -10 ) and LRRC8C on 1p22.2 (5x 10 -23 and 3x 10 -14 ) and LRRC8 D(4x 10 -13 and 6x10 -12 )).

[0114] Example 2: PheWAS shows that LRRC8 regulates human platelet volume ( Figure 1 ).

[0115] After identifying six SNPs associated with human platelet volume in the NHGARI-EBI catalog of published human GWAS studies, we searched for PheWAS associations between platelet volume and 13 SNP variants with P values ​​< 10 in the Common Metabolic Diseases Knowledge Portal (accessed March 3, 2022) and the PheWAS-IEU OpenGWAS Project catalog (PheWAS catalog). -5 (Accessed February 9, 2022). PheWAS identified an association between these SNP variants and platelet volume (8.9 x 10 -10 , 2.3x 10 -10 , 5.7x 10 -18 , 7.9x 10 -17 , 1.5x 10 -6 and 1.4x 10 -9), as well as four additional SNP variants assigned to LRRC8 B, an overlapping interval between LRRC8 B and C, and an association between LRRC8 C and platelet volume, initially identified due to its association with blood monocyte count or diastolic blood pressure in a GWAS catalog. In total, the platelet volume phenotype was associated with 10 SNPs assigned to four LRRC8 genes (LRRC8 AD) on two chromosomes (1p22.2 and 9q34.11).

[0116] Example 3: eQTL studies show that LRRC8 protein regulates human platelet volume ( Figure 1 ).

[0117] We searched the GTEX Portal database for expression quantitative trait loci (eQTL) (accessed on March 5, 2022) and found that LRRC8B (P = 2.8 x 10 -56 ) and LRRC 8C (P = 4.8 x 10 -63 There was a significant association between mRNA transcript levels of β-catenin and SNPs assigned to those genes and associated with platelet volume in the NHGARI-EBI catalog of published human GWAS studies.

[0118] Example 4: PheWAS shows that LRRC8 protein regulates human platelet count ( Figure 2 ).

[0119] We searched the Common Metabolic Diseases Knowledge Portal (accessed March 3, 2022) for PheWAS associations between platelet count and 13 SNP variants with P values ​​< 10 -5 (Accessed February 9, 2022). PheWAS identified associations between three SNP variants and platelet count (4.9 x 10 -11 , 2.6x 10 -10 and 3.8x 10 -7 In conclusion, the platelet count phenotype was associated with three SNPs assigned to two LRRC8 genes (LRRC8 CD) on one chromosome (1p22.2).

[0120] Example 5: eQTL studies show that LRRC8 protein regulates human platelet count ( Figure 2 ).

[0121] We searched the GTEX Portal database for expression quantitative trait loci (eQTL) (accessed on March 5, 2022) and found that LRRC 8C (P = 4.8 x 10 -63) mRNA transcript levels were significantly associated with SNPs assigned to LRRC 8C and associated with platelet count in the Common Metabolic Diseases Knowledge Portal (accessed March 3, 2022) or the PheWAS-IEU OpenGWAS Project Catalog (PheWAS Catalog).

[0122] Example 6: LRRC8A-D mRNA transcripts are expressed at high levels in human platelets compared to other mRNA transcripts ( Figure 3 ).

[0123] The Plateletomics interactive results tool, version 1.0, was used to search the Plateletomics database (accessed on March 5, 2022) and found that mRNA transcripts of LRRC 8A-8D were expressed in human platelets and ranked in the top 76th, 14th, 43rd, and top 5th percentiles of all mRNA transcripts detected in platelets from 154 patients, respectively. This suggests that mRNA transcripts of LRRC 8A-D are highly expressed in platelets compared to other mRNA transcripts.

[0124] Example 7: Human platelets contain LRRC8A, LRRC8B, LRRC8C, and LRRC8D mRNA transcripts in ratios of 1 to 2.5 to 1.5 to 3.0, respectively ( Figure 4 ).

[0125] We searched the database for LRRC 8A-E transcripts in Supplementary Table S2A of the following 2014 publication by Simon, LM et al. (Blood 123, e37-45 (2014)) and found that LRRCA, LRRC 8B, LRRC 8C, and LRRC 8D mRNA transcripts are expressed in human platelets, and LRRC 8B, LRRC8C, and LRRC8D are expressed at approximately 2.5-fold, 1.5-fold, and 3-fold the levels of LRRC 8A mRNA, respectively.

[0126] Example 8: The extent of human platelet aggregation stimulated by protease-activated receptor 4 (PAR4) agonists is proportional to LRRC8A expression ( Figure 5 ).

[0127] We searched the Plateletomics database (accessed March 5, 2022) using the Plateletomics Interactive Results Tool, version 1.0, and found data indicating that the intensity of human platelet aggregation stimulated by protease-activated receptor 4-activating peptide (PAR4 AP), an agonist of the platelet PAR4 thrombin receptor, was significantly correlated with the amount of LRRC8A mRNA expressed in human platelets.

[0128] Example 9: The extent of human platelet aggregation stimulated by adenosine diphosphate (ADP) and protease-activated receptor 1 (PAR1) agonists is inversely proportional to LRRC8B expression. Figure 6 ).

[0129] We searched the Plateletomics database (accessed March 5, 2022) using the Plateletomics Interactive Results Tool, version 1.0, and found data indicating that the magnitude of human platelet aggregation stimulated by adenosine diphosphate (ADP), an agonist of platelet P2Y12 and other ADP receptors, and by protease-activated receptor 1-activating peptide (PAR1 AP), an agonist of the platelet PAR1 thrombin receptor, was significantly inversely correlated with the amount of LRRC8B mRNA expressed in human platelets. Example 10: The extent of human platelet aggregation stimulated by protease-activated receptor 4 (PAR4) agonists is inversely correlated with LRRC8C expression ( Figure 7 ).

[0130] We searched the Plateletomics database (accessed March 5, 2022) using the Plateletomics Interactive Results Tool, version 1.0, and found data indicating that the intensity of human platelet aggregation stimulated by protease-activated receptor 4-activating peptide (PAR4 AP), an agonist of the platelet PAR4 thrombin receptor, was significantly inversely correlated with the amount of LRRC8C mRNA expressed in human platelets.

[0131] Example 11: The extent of human platelet aggregation stimulated by a protease-activated receptor 1 (PAR1) agonist is inversely proportional to LRRC8D expression ( Figure 8 ).

[0132] We searched the Plateletomics database (accessed March 5, 2022) using the Plateletomics Interactive Results Tool, version 1.0, and found data indicating that the intensity of human platelet aggregation stimulated by protease-activated receptor 1-activating peptide (PAR1 AP), an agonist of the platelet PAR1 thrombin receptor, was significantly inversely correlated with the amount of LRRC8D mRNA expressed in human platelets.

[0133] Example 12: Racial differences in LRRC8A-D expression in human platelets ( Figure 9 ).

[0134] A search of the Plateletomics database (accessed March 5, 2022) using the Plateletomics Interactive Results Tool, version 1.0, found data indicating that LRRC8C and LRRC8D mRNA expression was significantly reduced in people who identified as Black compared to people who identified as White. LRRC8A and LRRC8B gene expression was also lower in people who identified as Black compared to people who identified as White, but did not exceed the significance threshold in this dataset.

[0135] Example 13: Multiple regression analysis demonstrated that LRRC8A, LRRC8B, and LRRC8D play an important role in human platelet aggregation in response to ADP, PAR1 agonist peptide, and PAR4 agonist peptide ( Figure 10 ).

[0136] The regression coefficients of LRRC 8A-D in the multiple regression analysis of platelet aggregation responses in 5911 commonly expressed mRNAs in human platelets from 154 human subjects in Supplementary Table S3 of Simon, LM et al., 2014 (Blood 123, e37-45 (2014)) and the corresponding values ​​of significance tests showed that the extent of ADP-induced platelet aggregation was significantly correlated with the level of LRRC8A mRNA and significantly negatively correlated with the level of LRRC8B mRNA; the extent of PAR1-induced platelet aggregation was significantly negatively correlated with the levels of LRRC8B and LRRC8D mRNA; and the extent of PAR4-induced platelet aggregation was associated with the level of LRRC8D mRNA. The regression set the dependent variable to be the extent of platelet aggregation in response to one of four platelet agonists (ADP, PAR1AP, PAR2AP, and arachidonic acid), and the independent variables to be age, race, sex, platelet number, and BMI, as well as the levels of 5,911 commonly expressed mRNAs.

[0137] Example 14: LRRC8A in platelets regulates platelet volume in vivo (Figure 11).

[0138] Whole blood was collected from the inferior vena cava of 20-week-old WT mice and mice lacking platelet LRRC8A into heparin anticoagulant (15 USP units / mL) and run on an Element HT5 veterinary hematology analyzer (Heska, Loveland). The results showed that the mean platelet volume (MPV) of platelets lacking LRRC8A was significantly greater than that of WT platelets (P < 0.01; unpaired two-sided t-test). Platelet counts were also increased in mice with platelets lacking LRRC8A compared to WT mice, but the difference did not exceed the significance threshold (P = 0.06).

[0139] Example 15: Normal platelet adhesion of human platelets to collagen requires LRRC8A ( Figure 12 ).

[0140] Whole blood was collected from the inferior vena cava of 20-week-old WT mice and mice lacking platelet LRRC8A in heparinized anticoagulant. Heparinized whole blood was fluorescently labeled with DiOC6 (3,39-dihexyloxocarbocyanine iodide). Whole blood was plasma perfused for 5 minutes under constant upstream pressure through a polydimethylsiloxane (PDMS) microfluidic device coupled to a glass slide patterned with bovine type 1 collagen, followed by PBS perfusion for 5 minutes. Platelet accumulation was captured using an Olympus IX83 inverted microscope, and the surface area coverage of platelets was quantified using ImageJ. It was found that the loss of LRRC8A significantly reduced platelet adhesion ((P < 0.0001; two-tailed paired t-test).

[0141] Example 16: Inhibition of LRRC8 by SN-401 (DCPIB) significantly inhibits human platelet adhesion to collagen ( Figure 13 ).

[0142] Human blood was collected from four donors in heparinized anticoagulant, pooled, and incubated for 10 minutes with fluorescently labeled DiOC6 (3,39-dihexyloxocarbocyanine iodide; Invitrogen) and 10 μM SN-401 (DCPIB) or vehicle (DMSO). Whole blood was perfused at a constant upstream pressure through a polydimethylsiloxane (PDMS) microfluidic device coupled to a glass slide patterned with bovine type 1 collagen for 5 minutes, followed by 5 minutes of perfusion with PBS. Platelet accumulation was captured using an Olympus IX83 inverted microscope, and platelet surface area coverage was quantified using ImageJ. SN-401 was found to significantly reduce human platelet adhesion to collagen (vehicle versus SN-401; P < 0.0001; ordinary one-way ANOVA with Turkey's multiple comparison test).

[0143] Example 17: Inhibition of LRRC8 with SN-401 (DCPIB) significantly inhibits thrombus formation in vivo ( Figure 14 ).

[0144] To determine whether acute inhibition of LRRC8 channel activity affects platelet function in vivo, 25- to 30-week-old C57BL / 6 mice were administered vehicle or a known LRRC8 channel inhibitor, SN-401 (DCPIB), by IP injection, and in vivo thrombosis formation was quantified using an assay for tail wound thrombosis. Specifically, 20 minutes after IP injection of vehicle or 10 mg / kg SN-401, the mouse tail was amputated 5 mm from the tip of the tail, placed in 37°C saline for 15 minutes, and the relative blood loss during this period was quantified by estimating the hemoglobin concentration in the saline by measuring absorbance at 550 nm. It was found that the mean tail blood loss in mice receiving SN-401 was 4 times the blood loss in mice receiving vehicle (P < 0.05; unpaired two-tailed t-test). This demonstrates that pharmacological inhibition of LRRC8 inhibits thrombosis in vivo.

[0145] Example 18: Functional study ( Figures 15 to 29C )

[0146] As mentioned above, in genome-wide association studies (GWAS) and phenome-wide association studies (PheWAS), four of the five LRRC8 subunit genes (LRRC8A-D) were shown to be associated with mean platelet volume (MPV), suggesting that the LRRC8 pathway is involved in human platelet function and thrombosis. These findings are supported by human platelet mRNA expression and platelet activation data from the Plateletomics database, which revealed that 1) LRRC8A-D is expressed in platelets ( Figure 15 ), and 2) transcript levels of LRRC8A, 8B, and 8D were significantly associated with platelet activation by known soluble platelet agonists (P < 0.007 to P < 0.03). Notably, LRRC8E was not detected in human platelets in GWAS / PheWAS and was not associated with platelet traits, consistent with a true association of LRRC8A, B, C, and D.

[0147] To verify the biological features suggested by human genetic studies, we fl / fl Mouse (SWELL1 fl / fl ) 35-38 Crossing with platelet factor 4-driven Cre recombinase mice (Pf4-Cre) to generate LRRC8A fl / fl；Pf4-cre+ / - (cKO) and littermate controls LRRC8A fl / fl We generated megakaryocyte (MK)-specific LRRC8A conditional knockout (cKO) mice by culturing WT mice. We confirmed the complete absence of LRRC8A protein in platelets of cKO mice, while maintaining the expression of the platelet marker β3 integrin ( Figure 16 To confirm that LRRC8 protein forms functional VRAC / I in platelets Cl,SWELL Channels, I was measured in freshly isolated rat MK using the perforated patch configuration of the patch clamp technique. Cl,SWELL ( Figure 17A A robust I was observed in response to hypotonic swelling in WT MKs. Cl,SWELL The current was completely suppressed after application of 10 μM tool compound SN-401, with almost no obvious background current ( Figures 17B to 17D This hypotonicity-activated, swelling-activated current was completely abolished in cKO MK ( Figures 17C to 17D ), confirming that the SN-401 inhibitory current in mouse MK was entirely LRRC8A-mediated.

[0148] Having confirmed the ablation of LRRC8A protein and LRRC8A-mediated functions in cKO platelets, we next measured platelet volume and number. Consistent with the increased MPV associated with SNPs assigned to LRRC8A in GWAS / PheWAS studies, platelet-targeted deletion of LRRC8A also increased MPV compared to both WT and platelet-targeted heterozygous controls. Figure 18 ), without increasing platelet counts. Thus, deletion of LRRC8A in mouse platelets results in increased MPV, consistent with a variant human SNP allele located in the 5'-untranslated exonic region of LRRC8A identified in GWAS / PheWAS studies.

[0149] Next, the functional consequences of LRRC8A deletion in platelets were examined. Platelet adhesion, α-granule and dense granule secretion, and aggregation mediated by the activated αIIbβ3 integrin fibrinogen receptor are three platelet functions required for normal thrombus formation. LRRC8A depletion significantly impaired platelet function under shear conditions ( Figure 19A ) and thrombin-stimulated aggregation ( Figure 19B ) to fibrillar collagen-coated surfaces. Furthermore, LRRC8A deletion reduced thrombin-stimulated platelet activation, as measured by P-selectin exposure, a measure of α-granule release ( Figure 20A ) and αIIbβ3 integrin activation ( Figure 20B However, LRRC8A-deficient platelets still respond to Ca 2+ ionophore A23187, indicating that LRRC8 regulates intracellular Ca 2+ Upstream platelet signaling pathways. Taken together, these data indicate that LRRC8 channel signaling is required for normal platelet size, adhesion, granule release, and aggregation.

[0150] Because in vivo thrombosis also integrates many other factors observed in vitro in addition to platelet aggregation and adhesion, we examined arterial thrombosis in WT and cKO mice using two different in vivo thrombosis models: (1) FeCl3-induced carotid artery injury ( Figures 21A to 21B ); and (2) laser-induced cremaster arteriolar injury ( Figure 22A 、 22B and 22C). Notably, a significantly prolonged time to occlusion (TTO) of the FeCl3-injured carotid artery was observed in cKO mice compared with WT mice ( Figures 21A to 21B ). It is worth noting that, if Figure 21B As shown, the TTO of 5 / 7 cKO mice was greater than the 30-min (1800-s) time limit of the experiment, and thus the average TTO underestimates the true TTO because the values ​​were censored at 30 min. In the laser-induced cremaster arteriolar injury model, MK-specific deletion of LRRC8A also significantly reduced platelet thrombus formation compared with WT mice ( Figure 22A 、 22B and 22C), whereas fibrin production was unaffected in cKO mice ( Figure 22A 、 22B and 22C), consistent with a platelet-specific defect in LRRC8 signaling.

[0151] Furthermore, preserved fibrin production in cKO mice suggests that pharmacological inhibition may have the potential to selectively impair platelet thrombus formation while preserving hemostasis associated with intact fibrin production, suggesting that the LRRC8 channel may be a drug target for platelet-prone individuals who are less susceptible to major bleeding while still serving as effective antithrombotic agents. These data, spanning human genetics to mouse platelet-targeted loss-of-function models, rigorously validate the platelet LRRC8 channel as an innovative drug target for antiplatelet drugs with previously unknown mechanisms of action. Furthermore, platelet LRRC8 inhibitors are being developed as antithrombotic agents for both non-diabetic and diabetic patients.

[0152] Furthermore, SN-4XX compounds have been shown to inhibit platelet activity.

[0153] Patch clamp studies using the tool compound SN-401 revealed a strong inhibition of LRRC8 channel activity in mouse MK ( Figures 17A to 17E ), suggesting that if platelet activation requires LRRC8 channel activity, then SN-401 may also inhibit platelet activity. Preliminary platelet adhesion studies using microfluidic devices on human blood (4 different donors, performed on 4 different days) showed that SN-401 significantly inhibited platelet adhesion ( Figures 23A to 23B Consistent with this, ip injection of 10 mg / kg SN-401 in mice showed increased tail bleeding in vivo ( Figure 24 These data provide proof of concept that inhibition of LRRC8 channels with SN-4XX compounds has antiplatelet and antithrombotic activities.

[0154] SAR-based approaches have been successfully applied to develop more potent LRRC8 channel inhibitors. Examples of these are SN-418 and SN-418C ( Figure 25A and 25B ), which includes modifications in the alkyl chain + / - cyclopropyl groups to increase LRRC8 channel inhibition to approximately 10-fold that of SN-401 ( Figure 25C and 25D SN-418C was then tested for its in vitro antiplatelet activity. Significant inhibition of thrombin-induced platelet activation, such as activation of αIIbβ3 integrin ( Figure 26A ) and P-selectin exposure ( Figure 26B Consistent with these findings, SN-418C also strongly inhibited platelet aggregation in response to collagen-related peptide (CRP) and thrombin in a dose-dependent manner ( Figures 27A to 27B In fact, this level of platelet aggregation inhibition was comparable to that observed in LRRC8A-deficient platelets ( Figure 19B Finally, SN-418C was injected ip 4 days before FeCl3 carotid artery injury and then 20 minutes later, and the results showed that SN-418C significantly increased the occlusion time (TTO, Figure 28 ), which was 3.7-fold higher than that in vehicle-treated mice. Similar to what was observed with LRRC8AcKO mice ( Figures 21A to 21B ), SN-418C treatment caused TTO in 4 / 7 mice to be greater than the experimental standard time limit of 30 minutes, indicating that the increase in TTO observed at this 10 mg / kg dose of SN-418 was underestimated. Figure 29A 、 Figure 29B and Figure 29C As shown, MEG01 cells also have a robust I response to hypotonic swelling Cl,SWELL currents, and these were completely inhibited by SN-401 and SN-418C.

[0155] Overall, these data demonstrate that the SN-40X compound possesses antiplatelet activity and may be used as an antithrombotic agent. This provides proof of concept for an antiplatelet agent, acting via a previously unknown mechanism of action, for the treatment of myocardial infarction, stroke, coronary artery disease, peripheral artery disease, and other thrombotic diseases and complications.

[0156] All documents cited herein are incorporated by reference. Although certain embodiments of the present invention have been described and many details have been set forth for the purpose of illustration, certain details may be changed without departing from the basic principles of the present invention. In the context of describing embodiments of the present invention, the terms "a / an" and "the" and similar terms should be interpreted as encompassing both the singular and the plural, unless otherwise specified herein or clearly contradictory to the context. Unless otherwise specified, the terms "comprise," "have," "include," and "contain" should be interpreted as open-ended terms (i.e., meaning "including but not limited to"). Unless otherwise indicated herein, the description of the range of values ​​herein is intended only to serve as a shorthand method for individually referring to each individual value within the range, and each individual value is incorporated into this specification as if individually narrated herein. In addition to the order described in detail herein, all methods described herein can be performed in any suitable order, unless otherwise specified herein or clearly contradictory to the context. Any and all examples or exemplary language (e.g., "such as") provided herein are used only to better illustrate embodiments of the present invention and are not necessarily intended to limit the scope of the present invention unless specifically stated in the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Claims

1. A method for preventing and / or treating cardiovascular thrombosis in a patient in need of such treatment, the method comprising administering to the patient a therapeutically effective amount of a LRRC8 protein modulator.

2. The method of claim 1, wherein the method comprises administering to the patient a therapeutically effective amount of a LRRC8 protein modulator to the patient in order to prevent and / or treat a condition caused or exacerbated by cardiovascular thrombosis, including but not limited to myocardial ischemia, myocardial infarction, cerebrovascular transient ischemic attack, cerebrovascular ischemic stroke or peripheral arterial occlusion.

3. The method of claim 1, wherein the method comprises administering to the patient a therapeutically effective amount of a LRRC8 protein modulator to the patient in order to prevent and / or treat cardiovascular thrombosis and conditions resulting from cardiovascular thrombosis, which are associated with atrial arrhythmias, including atrial flutter and atrial fibrillation.

4. The method of claim 1, wherein the method comprises administering to the patient a therapeutically effective amount of a LRRC8 protein modulator to the patient in order to prevent and / or treat cardiovascular thrombosis associated with acute or chronic use of medical devices exposed to intravascular blood, including but not limited to intravascular diagnostic and therapeutic catheters, intravascular blood pumps, ventricular assist devices, intra-aortic balloon pumps, extracorporeal membrane oxygenation devices, artificial heart valves, and cardiac implantable electronic devices.

5. A method for preventing and / or treating an inherited or acquired platelet function defect in a patient in need of such treatment, the method comprising administering to the patient a therapeutically effective amount of a LRRC8 protein modulator.

6. The method of claim 5, wherein the method comprises administering to the patient a therapeutically effective amount of a LRRC8 protein modulator to the patient so as to normalize platelet function by increasing the contents of platelet alpha granules, dense granules, or other intracellular organelles, including but not limited to the release or membrane expression of platelet activators, coagulation factors, and adhesion molecules.

7. The method of claim 5, wherein the method comprises administering to the patient a therapeutically effective amount of a LRRC8 protein modulator to the patient so as to normalize platelet function by normalizing platelet volume.

8. A method for preventing and / or treating hereditary or acquired thrombocythemia in a patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a LRRC8 protein modulator.

9. The method according to any one of claims 1 to 8, wherein the LRRC8 modulator is DCPIB.

10. The method according to any one of claims 1 to 9, wherein the LRRC8 modulator is a compound of formula (I) or a geometric isomer or salt thereof: in: R 1 and R 2 are each independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 3 Y-C(O)R 4 ,–Z–N(R 5 )(R 6 ) or –Z–A; A is selected from the group consisting of: R 4 is hydrogen, substituted or unsubstituted alkyl, –OR 7 or –N(R 8 )(R 9 ); X 1 and X 2 are independently substituted or unsubstituted alkyl, halo, –OR 10 or –N(R 11 )(R 12 ); R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are each independently hydrogen or substituted or unsubstituted alkyl; Y and Z are each independently a substituted or unsubstituted carbon-containing moiety having at least 2 carbon atoms; and n is 1 or 2.

11. The method according to claim 10, wherein R 1 or R 2 At least one of the is a substituted or unsubstituted straight or branched chain alkyl group having at least 2 carbon atoms.

12. The method according to claim 10 or 11, wherein R 1 or R 2 At least one of is selected from the group consisting of:

13. The method according to claims 10 to 12, wherein R 1 is hydrogen or C1 to C6 alkyl.

14. The method according to any one of claims 10 to 13, wherein R 1 It is butyl.

15. The method according to any one of claims 10 to 14, wherein R 2 It is a cycloalkyl group.

16. The method according to any one of claims 10 to 15, wherein R 2 It is cyclopentyl.

17. The method according to any one of claims 10 to 16, wherein R 3 Y-C(O)R 4 .

18. The method according to any one of claims 10 to 17, wherein R 4 For –OR 7 or –N(R 8 )(R 9 ).

19. The method according to any one of claims 10 to 18, wherein R 3 =–Z–N(R 5 )(R 6 ).

20. The method according to any one of claims 10 to 19, wherein R 3 It is –Z–A.

21. The method according to any one of claims 10 to 20, wherein A is selected from the group consisting of:

22. The method according to any one of claims 10 to 21, wherein Y and Z are each independently a substituted or unsubstituted alkylene group having 2 to 10 carbons, a substituted or unsubstituted alkenylene group having 2 to 10 carbons, or a substituted or unsubstituted arylene group.

23. The method of any one of claims 10 to 22, wherein Y and Z are each independently an alkylene group having 2 to 10 carbons, an alkenylene group having 2 to 10 carbons, or a phenylene group.

24. The method of any one of claims 10 to 23, wherein Y and Z are each independently a cycloalkylene group having 4 to 10 carbons.

25. The method of any one of claims 10 to 24, wherein Y is an alkylene or alkenylene group having 3 to 8 carbons or 3 to 7 carbons.

26. The method according to any one of claims 10 to 25, wherein Y is an alkylene group or any alkenylene group having 4 carbons.

27. The method of any one of claims 10 to 26, wherein Z is an alkylene group having 2 to 4 carbons.

28. The method of any one of claims 10 to 27, wherein Z is an alkylene group having 3 or 4 carbons.

29. The method according to any one of claims 10 to 28, wherein Y and Z are each independently selected from the group consisting of:

30. The method of any one of claims 10 to 29, wherein when Y is an alkyl group having 2 to 3 carbons, then X 1 and X 2 Each is fluorine or each is substituted or unsubstituted alkyl.

31. The method according to any one of claims 10 to 30, wherein R 3 Select from the group consisting of:

32. The method according to any one of claims 10 to 31, wherein X 1 and X 2 Each is independently substituted or unsubstituted C1 to C6 alkyl or halogen.

33. The method according to any one of claims 10 to 32, wherein X 1 and X 2 Each is independently C1 to C6 alkyl, fluorine, chlorine, bromine or iodine.

34. The method according to any one of claims 10 to 33, wherein X 1 and X 2 are each independently methyl, fluoro or chloro.

35. The method according to any one of claims 10 to 34, wherein R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 Each is independently hydrogen or alkyl.

36. The method according to any one of claims 10 to 35, wherein R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 Each is independently hydrogen or C1 to C3 alkyl.

37. The method according to any one of claims 10 to 36, selected from the group consisting of:

38. The method of any one of claims 10 to 37, wherein the compound modulates or inhibits LRRC8 protein.

39. The method of claim 38, wherein the compound has a greater potency in modulating or inhibiting SWELL1 channels than an equivalent amount of DCPIB (4-[2[butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid).

40. The method of any one of claims 1 to 39, wherein administration of the compound is sufficient to upregulate expression of LRRC8 or expression of a LRRC8-related protein.

41. The method of any one of claims 1 to 40, wherein administration of the compound is sufficient to stabilize the LRRC8 protein complex or LRRC8-associated protein.

42. The method of any one of claims 1 to 41, wherein administration of the compound is sufficient to promote membrane trafficking and activity of the LRRC8 protein complex or LRRC8-associated protein.

43. The method of any one of claims 1 to 42, wherein administration of the compound is sufficient to enhance LRRC8-mediated signaling or trafficking to the membrane of an intracellular organelle, including but not limited to lysosome-related organelles.

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