Use of LRRC8 protein modulators for preventing and treating cardiovascular disease

Abstract

The present invention relates to various methods for preventing and / or treating cardiovascular thrombosis, inherited or acquired deficiencies in platelet function, and inherited or acquired thrombocytosis, comprising administering to a patient a therapeutically effective amount of an LRRC8 protein modulator. Compounds that are LRRC8 protein modulators are also described, including DCPIB (4-[2[butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic 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 contributes to the disease state, such as myocardial ischemia and infarction, stroke, transient cerebrovascular ischemia, 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 impaired platelet function, including abnormal adhesion, aggregation, activation, or thrombosis. [Background technology]

[0002] The family of LRRC8 (leucine-rich repeat-containing protein 8) protein channels consists of various heterohexameric combinations of one or more of five LRRC8 monomeric proteins (LRRC8A-E). The genes encoding the five LRRC8 monomeric proteins are located on three human chromosomes: 1 (LRRC8B, LRRC8C, and LRRC8D), 9 (LRRC8A), and 19 (LRRC8E).

[0003] Platelets are megakaryocytic cell fragments essential for in vivo thrombosis required for hemostasis. Various agonist molecules, including thrombin, thromboxane, adenosine diphosphate (ADP), and collagen, stimulate platelet activation, which involves a shape change and fusion of lysosome-related organelles (LROs) (alpha granules, dense granules, and lysosomes) with the platelet plasma membrane. This membrane fusion induces the expression of adhesion molecules on the platelet surface, as well as the expression of von Willebrand factor and fibrinogen (alpha granules) as well as ATP, ADP, Ca 2+ and serotonin (dense granules), which allows platelets to adhere to the deendothelialized surface of blood vessels and activate the coagulation pathway, leading to platelet aggregation and thrombus formation.

[0004] Thrombus formation is essential for hemostasis after blood vessels are injured by trauma or surgical manipulation. Thrombosis can also cause significant morbidity and mortality if it occurs on ruptured atherosclerotic plaques, the left atrial appendage, or on the surface of medical devices with surfaces exposed to blood flow. In situ thrombus formation on ruptured arterial atherosclerotic plaques can cause myocardial, cerebrovascular, and lower limb ischemia and infarction. Embolization of thrombi 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. Thrombus formation on the surfaces of medical devices exposed to flowing blood in vivo (e.g., prosthetic heart valves, coronary diagnostic and therapeutic catheters, coronary, cerebrovascular, and peripheral stents, intravascular blood flow pumps) or ex vivo (e.g., extracorporeal membrane oxygenation (ECMO) devices, external ventricular assist devices) can lead to vascular occlusion and embolization causing myocardial, cerebrovascular, and lower limb ischemia and infarction.

[0005] A number of pharmacological strategies have been developed to prevent thrombosis, which causes morbidity and mortality, including inhibitors of platelet enzymes involved in the enzymatic catalysis of platelet formation (e.g., acetylsalicylic acid inhibits cyclooxygenase), inhibitors of platelet agonists (e.g., bivalirudin inhibits proteolytic sites on thrombin), and inhibitors of platelet receptors for platelet agonists (e.g., clopidogrel inhibits the P2Y12 purinergic receptor). Despite these therapies, a very significant residual risk of morbidity and mortality from cardiovascular thrombosis persists. In the United States, it is estimated that there are 605,000 new and 200,000 recurrent myocardial infarctions annually, and 610,000 new and 185,000 recurrent strokes annually. In addition, significant morbidity and mortality persists 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.

[0006] 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 can form heterohexamers that can function as membrane channels for ions and small molecules. LRRC8A was discovered in children with gammopathy associated with a chromosomal translocation that truncates this protein. In 2014, it was shown that voltage-regulated anion current (VRAC) is mediated by hexameric LRRC8 proteins that contain at least LRRC8A. It was subsequently shown that LRRC8 proteins are expressed in tissue-specific combinations. In 2020, it was demonstrated that LRRC8A is required for normal lysosomal function in multiple cell lines, including HAP1 cells derived from KBM-7 cells taken from a patient with chronic myeloid leukemia. An essential step in platelet activation is the fusion of lysosome-related organelles, including alpha granules, dense granules, and lysosomes, with the plasma membrane.

[0007] Increased platelet volume is associated with myocardial infarction and death after myocardial infarction, poor outcomes after acute ischemic stroke, and peripheral arterial disease. Increased platelet volume is also associated with poor clinical outcomes after primary percutaneous coronary revascularization, type 2 diabetes, microvascular complications of diabetes, and nonalcoholic fatty liver disease.

[0008] Previously, it has been shown that the LRRC8 protein regulates cell volume in 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 glycemic control and other functions.

[0009] Cardiovascular disease (CVD) and type 2 diabetes (T2D) are overlapping global pandemics. It is estimated that 463 million adults worldwide have T2D, of which 30-34%, or 149 million, have both T2D and CVD. T2D accelerates the onset and severity of CVD and increases the risk of coronary death, nonfatal myocardial infarction (MI), and ischemic stroke by 2-4 fold, worsening clinical outcomes in patients with CVD events. CVD is the most common cause of death in patients with T2D, and approximately two-thirds of patients with DM die from CVD. Stroke and MI most frequently occur when a platelet-rich thrombus forms at the site of a ruptured atherosclerotic plaque, partially or completely occluding the vascular lumen and resulting in downstream ischemia. In T2D, multiple factors accelerate this process, including a prothrombotic state with hyperreactive platelets and increased circulating coagulation factors, and abnormal vessel wall function, including decreased endothelial nitric oxide synthase (eNOS) signaling. In addition, multiple factors cause platelets in T2D patients to be less responsive to inhibition by current antiplatelet agents. The economic burden of care for stroke and myocardial ischemia in patients with T2D is enormous. In 2018, patients with T2D in the United States had 1.87 million hospitalizations, primarily for CVD, including 440,000 for ischemic heart disease, 334,000 for stroke, and 154,000 for lower limb amputation. It is estimated that 49% of the direct costs of care for T2D are for the prevention and treatment of CVD. The total direct costs of care for T2D in the United States increased from $188 billion in 2012 to $237 billion in 2017, an increase of 26%, and a meta-analysis of studies from multiple countries indicates that the direct costs of CVD care for patients with T2D may account for up to 49% of total direct care costs. There are at least 10 classes of drugs approved to treat hyperglycemia associated with T2D. Newer glycemic control agents such as SGLT2 inhibitors and GLP1 agonists help reduce CVD events in T2D, but significant residual CVD risk remains. Currently, there are four classes of drugs commonly used to prevent and / or treat myocardial infarction or stroke: aspirin, P2Y 12These include 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. Current antiplatelet drugs can reduce CVD events and deaths, but their therapeutic potential is limited by the serious bleeding (e.g., intracranial hemorrhage and hemorrhagic death) associated with their use.

[0010] There is an urgent need for therapies to prevent and treat diseases associated with cardiovascular thrombosis, including myocardial ischemia and infarction, stroke, transient cerebrovascular ischemia, 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 intravascular catheters, intravascular blood pumps, ventricular assist devices, intra-aortic balloon pumps, extracorporeal membrane oxygenation devices, prosthetic heart valves, and cardiac implantable electronic devices. In addition, there is a large unmet clinical need for drugs that improve glycemic control in T2D and also safely prevent cerebral and coronary vascular thrombosis. This unmet clinical need represents a large commercial opportunity. According to current guidelines, it is estimated that there are more than 8 million patients with T2D who are eligible for secondary prevention of MI or stroke with antiplatelet agents. Using the current price of new oral antiplatelet agents (e.g., ticagrelor) as a benchmark, this corresponds to a market of over $20 billion per year just for secondary MI prevention in the US and EU alone. Summary of the Invention

[0011] Various aspects of the invention relate to methods of preventing or treating thrombosis. In various embodiments, a 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 congener thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of

[0012] [ka] JPEG2025513794000003.jpg149169

[0013] and salts and geometric isomers thereof.

[0014] In various embodiments, the method comprises administering to the subject a therapeutically effective amount of a compound of formula (I), and salts and geometric isomers thereof:

[0015] [ka]

[0016] During the ceremony, 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, substituted or unsubstituted heteroaryl; R 3 But -YC(O)R 4 , ZN(R 5 )(R 6 ), or -ZA, A is selected from the group consisting of:

[0017] [ka]

[0018] R 4 is hydrogen, substituted or unsubstituted alkyl, -OR 7 , or -N(R 8 )(R 9 ) and X 1 and X 2 each independently represents a substituted or unsubstituted alkyl, halo, -OR 10 , or -N(R 11 )(R 12 ) and 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 two carbon atoms; and n is 1 or 2.

[0019] In various embodiments, methods of preventing or treating thrombosis include treating diseases in which thrombosis contributes to the disease state, and the present invention relates to various methods of preventing or treating diseases in which cardiovascular thrombosis contributes to the disease state, such as myocardial ischemia and infarction, stroke, transient cerebrovascular ischemia, peripheral arterial disease, thromboembolism associated with atrial fibrillation and flutter, and thromboembolism associated with the use of cardiovascular medical devices, and diseases associated with impaired platelet function, including abnormal adhesion, aggregation, activation, or thrombosis.

[0020] Other objects and features will be in part apparent and in part pointed out hereinafter. [Brief description of the drawings]

[0021] [Figure 1]We show how LRRC8 protein regulates human platelet volume. Single nucleotide polymorphisms (SNPs) assigned to LRRC8 genes A-D are associated with human platelet volume in human genome-wide association studies (GWAS), human phenome-wide association studies (PheWAS), and databases of expressed quantitative loci (eQTLs) in whole blood. Genome-wide association studies (GWAS) show that LRRC8 protein regulates human platelet volume. We performed a search of the NHGARI-EBI catalog of eligible published human genome-wide association studies (accessed on February 9, 2022) and identified associations between platelet volume and six SNP variants assigned to three genes on two chromosomes: LRRC8A (2 × 10-10) on 9q34.11 and LRRC8C (5 × 10-23 and 3 × 10-14) and LRRC8D (4 × 10-13 and 6 × 10-12) on 1p22.2. Phenome-wide association studies (PheWAS) indicate that 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 the Common Metabolic Diseases Knowledge Portal (accessed March 3, 2022) and the PheWAS-IEU OpenGWAS project catalog (PheWAS catalog) for PheWAS associations between these six SNP variants and platelet volume (8.9x10-10, 2.3x10-10, 5.7x10-18, 7.9x10-17, 1.5x10-6, and 1.4x10-9), as well as four additional SNP variants assigned to LRRC8B, the overlap interval between LRRC8B and C, and LRRC8C that were initially identified in the GWAS catalog for association with blood monocyte count or diastolic blood pressure. Overall, the platelet volume phenotype is associated with 10 SNPs mapped to four LRRC8 genes (LRRC8A-D) on two chromosomes (1p22.2 and 9q34.11). eQTL studies indicate that the LRRC8 protein regulates human platelet volume.We then searched the GTEX Portal Database expression quantitative trait loci (eQTL) database (accessed on March 5, 2022) and found significant associations between levels of LRRC8B (P = 2.8 × 10-56) and LRRC8C (P = 4.8 × 10-63) mRNA transcripts in whole blood and SNPs assigned to those genes associated with platelet volume in the NHGARI-EBI catalog of published human GWAS studies. [Diagram 2] We show how LRRC8 protein regulates human platelet count. Single nucleotide polymorphisms (SNPs) assigned to LRRC8 genes A-D are associated with human platelet count in human genome-wide association studies (GWAS), human phenome-wide association studies (PheWAS), and databases of expressed quantified loci (eQTL) in whole blood. The phenome-wide association studies (PheWAS) show that LRRC8 protein regulates human platelet count. The Common Metabolic Diseases Knowledge Portal (accessed March 3, 2022) was searched for PheWAS associations between platelet count and 13 SNP variants (accessed February 9, 2022) with P values ​​less than 10-5. PheWAS identified associations between three SNP variants and platelet count (4.9 × 10-11, 2.6 × 10-10, and 3.8 × 10-7). Overall, the platelet count phenotype is associated with three SNPs assigned to two LRRC8 genes (LRRC8C-D) on one chromosome (1p22.2). eQTL studies indicate that LRRC8 protein regulates human platelet count. GTEX Portal Database Expression Quantitative Trait Loci (eQTL) database (accessed March 5, 2022) was searched and a significant association was found between the levels of mRNA transcripts of LRRC8C (P=4.8x10-63) in whole blood and SNPs assigned to LRRC8C associated with platelet count in the Common Metabolic Diseases Knowledge Portal (accessed March 3, 2022) or the PheWAS-IEU OpenGWAS Project catalog (PheWAS catalog). [Diagram 3] We show how LRRC8A-D mRNA transcripts are highly expressed in human platelets compared to other mRNA transcripts. Using the Plateletomics Interactive Results Tool version 1.0 to search the Plateletomics database (accessed March 5, 2022), we found that LRRC8A-D mRNA transcripts are expressed in human platelets and that they are in the top 76, 14, 43, and top 5 percentiles of all mRNA transcripts detected in platelets from 154 patients. This indicates that LRRC8A-D mRNA transcripts are highly expressed in platelets compared to other mRNA transcripts. [Figure 4] Figure 1 shows how human platelets contain LRRC8A, LRRC8B, LRRC8C, and LRRC8D mRNA transcripts in a ratio of 1:2.5:1.5:3.0, respectively. LRRC8A-E transcripts were searched in the database in Supplementary Table S2A from Simon, LM et al. (Blood 123, e37-45 (2014)) and found 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. [Diagram 5] We show how the extent of human platelet aggregation stimulated by a protease-activated receptor 4 (PAR4) agonist is proportional to the expression of LRRC8A. Using the Plateletomics Interactive Results Tool version 1.0, we searched the Plateletomics database (accessed March 5, 2022) and found data demonstrating 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, is significantly associated with the amount of LRRC8A mRNA expressed in human platelets. [Figure 6]We show how the degree of human platelet aggregation stimulated by adenosine diphosphate (ADP) and protease-activated receptor 1 (PAR1) agonists is inversely proportional to the expression of LRRC8B. Using the Plateletomics Interactive Results Tool version 1.0 to search the Plateletomics database (accessed March 5, 2022), data were found that demonstrated that the intensity of human platelet aggregation stimulated by adenosine diphosphate (ADP), an agonist of platelet P2Y12 and other ADP receptors, and protease-activated receptor 1-activatin peptide (PAR1 AP), an agonist of the platelet PAR1 thrombin receptor, was significantly inversely related to the amount of LRRC8B mRNA expressed in human platelets. [Figure 7] We show how the degree of human platelet aggregation stimulated by a protease-activated receptor 4 (PAR4) agonist is inversely proportional to the expression of LRRC8C.Using the Plateletomics Interactive Results Tool version 1.0, we searched the Plateletomics database (accessed March 5, 2022) and found data demonstrating 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, is significantly and inversely related to the amount of mRNA for LRRC8C expressed in human platelets. [Figure 8] We show how the degree of human platelet aggregation stimulated by a protease-activated receptor 1 (PAR1) agonist is inversely proportional to the expression of LRRC8D. Using the Plateletomics Interactive Results Tool version 1.0, we searched the Plateletomics database (accessed March 5, 2022) and found data demonstrating 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, is significantly inversely related to the amount of LRRC8D mRNA expressed in human platelets. [Figure 9] Racial differences in expression of LRRC8A-D in human platelets. Using the Plateletomics Interactive Results Tool version 1.0 to search the Plateletomics database (accessed March 5, 2022), data were found demonstrating that mRNA expression of LRRC8C and LRRC8D was significantly lower in people identified as black compared to people identified as white. LRRC8A and LRRC8B gene expression is also lower in people identified as black compared to people identified as white, but did not cross the threshold for significance in this dataset. [Figure 10] Figure 1 shows 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. Regression coefficients of LRRC8A-D in multiple regression analysis of platelet aggregation responses in 5911 commonly expressed mRNAs in human platelets from 154 human subjects from 2014 Supplementary Table S3 from Simon, LM et al. (Blood 123, e37-45 (2014)), and corresponding values ​​for significance tests demonstrate that the degree of ADP-induced platelet aggregation is significantly associated with the levels of LRRC8A mRNA and inversely with the levels of LRRC8B mRNA, that the degree of PAR1-induced platelet aggregation is significantly associated with the levels of LRRC8B and LRRC8D mRNA, and that the degree of PAR4-induced platelet aggregation is associated with the levels of LRRC8D mRNA. The dependent variable in the regression specification was the degree of platelet aggregation in response to one of four platelet agonists (ADP, PAR1 AP, PAR2 AP, and arachidonic acid), and the independent variables were age, race, sex, platelet count, and BMI, as well as the levels of 5,911 commonly expressed mRNAs. [Figure 11A]We show how LRRC8A in platelets regulates platelet volume in vivo. Whole blood was collected into heparin anticoagulant (15 USP units / mL) from the inferior vena cava of 20-week-old WT mice and mice lacking platelet LRRC8A, and whole blood CBC was performed on an Element HT5 veterinary hematology analyzer (Heska, Loveland), which demonstrated that the mean platelet volume (MPV) of platelets lacking LRRC8A was significantly greater than the MPV of WT platelets (P<0.01, unpaired two-tailed t-test). Platelet counts were also increased in mice with platelets lacking LRRC8A compared to WT mice, but did not exceed the threshold for significance (P=0.06). [Figure 11B] We show how LRRC8A in platelets regulates platelet volume in vivo. Whole blood was collected into heparin anticoagulant (15 USP units / mL) from the inferior vena cava of 20-week-old WT mice and mice lacking platelet LRRC8A, and whole blood CBC was performed on an Element HT5 veterinary hematology analyzer (Heska, Loveland), which demonstrated that the mean platelet volume (MPV) of platelets lacking LRRC8A was significantly greater than the MPV of WT platelets (P<0.01, unpaired two-tailed t-test). Platelet counts were also increased in mice with platelets lacking LRRC8A compared to WT mice, but did not exceed the threshold for significance (P=0.06). [Figure 12] We show how LRRC8A is required for normal platelet adhesion to collagen in human platelets. Whole blood was collected into heparin anticoagulant 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-dihexyloxacarbocyanine iodide). Whole blood was perfused for 5 min through a polydimethylsiloxane (PDMS) microfluidic device bound to a glass slide patterned with bovine type 1 collagen at constant upstream pressure. Plasma was then perfused for 5 min with PBS, followed by 5 min of perfusion. Platelet accumulation was captured using an Olympus IX83 inverted microscope, and platelet surface area coverage was quantified using ImageJ. It was found that the absence of LRRC8A significantly reduced platelet adhesion (P<0.0001, paired two-tailed t-test). [Figure 13] We show how inhibition of LRRC8 with SN-401 (DCPIB) significantly inhibited adhesion of human platelets to collagen. Human blood from four donors was collected into heparin anticoagulant, pooled, and incubated with fluorescently labeled DiOC6 (3,39-dihexyloxacarbocyanine iodide, Invitrogen) and either 10 μM SN-401 (DCPIB) or vehicle (DMSO) for 10 min. Whole blood was perfused for 5 min through a polydimethylsiloxane (PDMS) microfluidic device bound to a glass slide patterned with bovine type 1 collagen plasma at constant upstream pressure followed by PBS perfusion for 5 min. Platelet accumulation was captured using an Olympus IX83 inverted microscope and platelet surface area coverage was quantified using ImageJ. It was found that SN-401 significantly reduced adhesion of human platelets to collagen (vehicle vs. SN-401, P<0.0001, ordinary one-way ANOVA with Turkey's multiple comparison test). [Figure 14] We show how inhibition of LRRC8 with SN-401 (DCPIB) significantly inhibited thrombosis in vivo. To determine whether acute inhibition of LRRC8 channel activity affected platelet function in vivo, vehicle or the known LRRC8 channel inhibitor SN-401 (DCPIB) was administered by IP injection to 25-30 week old C57BL / 6 mice, and in vivo thrombosis was quantified using a tail wound thrombosis assay. Specifically, 20 min after IP injection of vehicle or 10 mg / kg SN-401, mouse tails were cut 5 mm from the tail tip and placed in saline at 37°C for 15 min, during which relative blood loss was quantified by estimating hemoglobin concentration in saline by measuring optical absorbance at 550 nm. The average amount of tail blood loss in mice receiving SN-401 was found to be four times that in mice receiving vehicle (P<0.05, unpaired two-tailed t-test), demonstrating that pharmacological inhibition of LRRC8 inhibited thrombosis in vivo. [Figure 15]Figure 1 shows how LRRC8 mRNA is highly expressed in human platelets. Percentile expression of platelet LRRC8A-D relative to all platelet transcripts is shown. Data is from the Plateletomics database. [Figure 16] Western blots of LRRC8A, GAPDH (loading control) for LRRC8A, β3 integrin, and associated GAPDH in WT and LRRC8A cKO platelets are shown. [Figure 17A] Figure 2 shows how megakaryocyte (MK) patch clamp reveals robust swelling-activated currents. Mouse MK perforated patch clamp shown. *p<0.05, **p<0.01. [Figure 17B] FIG. 1 shows how megakaryocyte (MK) patch clamp reveals robust swelling-activated currents. Current density over time in response to swelling with hypotonic solution (HYPO, 205 mOsm) and inhibition with 10 μM SN401. *p<0.05, **p<0.01. [Figure 17C] Figure 1 shows how megakaryocyte (MK) patch clamp reveals robust swelling-activated currents. Current-voltage relationships in WT MK are shown. *p<0.05, **p<0.01. [Figure 17D] Figure 2 shows how megakaryocyte (MK) patch clamp reveals robust swelling-activated currents. Current-voltage relationships in cKO MKs are shown. *p<0.05, **p<0.01. [Figure 17E] FIG. 1 shows how megakaryocyte (MK) patch clamp reveals robust swelling-activated currents. Mean outward and inward currents at +100 mV and -100 mV, respectively, in WT, WT+SN401, and cKO MK are shown. *p<0.05, **p<0.01. [Figure 18] Mean platelet volumes of WT, LRRC8A cKO, and PF4-Cre;LRRC8A fl / + controls are shown. ****p<0.001. [Figure 19A]FIG. 1 shows how platelets lacking LRRC8A show impaired aggregation and adhesion. Impaired adhesion of platelets to collagen-coated microfluidic chambers. Two-way Anova, P=0.01. [Figure 19B] FIG. 19B shows how platelets lacking LRRC8A show impaired aggregation and adhesion. FIG. 19B shows platelet aggregation in WT and cKO mice in response to thrombin (n=7, each group). ****p<0.001. [Figure 20A] Shows how platelet-specific LRRC8A ablation impairs platelet activation. Shows P-selectin exposure in response to thrombin in WT and cKO mice. ****p<0.001. [Figure 20B] Shows how platelet-specific LRRC8A ablation impairs platelet activation. Shows αIIbβ3 activation in response to thrombin in WT and cKO mice. *p<0.05, ****p<0.001. [Figure 21A] FIG. 1 shows how LRRC8A cKO mice exhibit impaired thrombosis in vivo in an FeCl3-induced carotid injury model. Carotid tracings of WT and LRRC8A cKO mice after injury are shown. ***p<0.001. [Figure 21B] FIG. 1 shows how LRRC8A cKO mice exhibit impaired thrombosis in vivo in an FeCl3-induced carotid artery injury model. Time to total occlusion (TTO) is shown. ***p<0.001. [Figure 22A] FIG. 1 shows how LRRC8A is required for platelet thrombus formation, but not fibrin generation, in laser-induced cremaster arteriole thrombosis. Intravital microscopy using WT control and LRRC8A cKO mice is shown. After laser-induced cremaster arteriole injury, platelet accumulation and fibrin generation were detected by injection of DyLight649-conjugated anti-CD42c and Alexa488-conjugated anti-fibrin antibodies, respectively. Representative images are shown. [Figure 22B]Figure 1 shows how LRRC8A is required for platelet thrombus formation, but not fibrin generation, in laser-induced cremaster arteriole thrombosis. Intravital microscopy using WT control and LRRC8A cKO mice is shown. After laser-induced cremaster arteriole injury, platelet accumulation and fibrin generation were detected by injection of DyLight649-conjugated anti-CD42c and Alexa488-conjugated anti-fibrin antibodies, respectively. Quantification of the median integrated fluorescence intensity of the anti-CD42c (F-platelet) antibody signal after laser injury is shown. [Figure 22C] Figure 1 shows how LRRC8A is required for platelet thrombus formation, but not fibrin generation, in laser-induced cremaster arteriole thrombosis. Intravital microscopy with WT control and LRRC8A cKO mice is shown. After laser-induced cremaster arteriole injury, platelet accumulation and fibrin generation were detected by injection of DyLight649-conjugated anti-CD42c and Alexa488-conjugated anti-fibrin antibodies, respectively. Quantification of the median integrated fluorescence intensity of the anti-fibrin (F-fibrin) antibody signal after laser injury is shown. [Figure 23A] FIG. 1 shows how SN-401 impairs human platelet adhesion under physiological shear flow conditions in a collagen-coated microfluidic flow chamber. n=4 blood draws from 4 subjects on 2 separate days. Shown is the percentage of surface area covered by adherent platelets over time in human whole blood treated with vehicle or 10 μM SN-401. p=0.1 for two-way anova. [Figure 23B] Figure 23 shows how SN-401 impairs human platelet adhesion under physiological shear flow conditions in a collagen-coated microfluidic flow chamber. n=4 blood samples are drawn from 4 subjects on 2 separate days. Area under the curve (AUC) calculated from Figure 23A for vehicle and SN-401 are shown. For t-test, p=0.1. [Figure 24]Figure 1 shows how SN-401 treatment increases mouse tail bleeding. Mice were pretreated with vehicle or SN401 (10 mg / kg ip x 1) and tail bleeding was quantified 15 min later by measuring the absorbance of hemoglobin at 550 nm. *p<0.05. [Figure 25A] Figure 2 shows how the congeners SN-418 and SN-418C are potent inhibitors of swelling-activated currents recorded from HEK cells.The chemical structure of SN-418 is shown. [Figure 25B] FIG. 1 shows how the congeners SN-418 and SN-418C are potent inhibitors of swelling-activated currents recorded from HEK cells. The chemical structure of SN-418C is shown. [Figure 25C] Figure 1 shows how congeners SN-418 and SN-418C are potent inhibitors of swelling-activated currents recorded from HEK cells. Currents in the presence of various concentrations of SN-418 and SN-401 normalized to currents activated by hypotonic solution alone are shown. Lines indicate best fits to a logistic equation with IC50s of 420 nM and 4.0 μM for SN-418 and SN-401, respectively. [Figure 25D] Figure 1 shows how congeners SN-418 and SN-418C are potent inhibitors of swelling-activated currents recorded from HEK cells. Currents in the presence of various concentrations of SN-418C and SN-401 normalized to currents activated by hypotonic solution alone are shown. Lines indicate best fits to a logistic equation with IC50s of 265 nM and 4.0 μM for SN-418C and SN-401, respectively. [Figure 26A] Figure 2 shows how SN-418C treatment dose-dependently impairs platelet activation. Flow cytometry mean fluorescence intensity (MFI) of αIIbβ3 in washed platelets upon activation with thrombin or CRP agonists and dose-dependent inhibition by SN-418C. *p<0.05, ***p<0.001. [Figure 26B]Figure 1 shows how SN-418C treatment dose-dependently impairs platelet activation. Flow cytometry mean fluorescence intensity (MFI) of P-selectin in washed platelets upon activation with thrombin or CRP agonists and dose-dependent inhibition by SN-418C is shown. *p<0.05. [Figure 27A] Figure 1 shows how SN-418C treatment inhibits agonist-stimulated platelet aggregation. Washed platelets pretreated with various concentrations of SN418C were shown and aggregation experiments were performed in an aggregometer in the presence of CRP. [Figure 27B] Figure 1 shows how SN-418C treatment inhibits agonist-stimulated platelet aggregation. Washed platelets pretreated with various concentrations of SN418C were shown and aggregation experiments were performed in an aggregometer in the presence of thrombin agonists. [Figure 28] FIG. 1 shows how SN418C inhibits thrombosis in FeCl3-induced carotid artery injury. Time to complete occlusion (TTO) of the carotid artery is shown for vehicle (n=6) and 10 mg / kg ipSN-418C (n=7), p=0.06. [Figure 29A] Figure 1 shows how the human MK cell line MEG01 had robust SN-4XX-inhibited swelling-activated currents. Current density over time in WT MEG01 cells in response to swelling with hypotonic solution (HYPO, 210 mOsm) and inhibition with 10 μM SN401. [Figure 29B] 1 shows how the human MK cell line MEG01 had robust SN-4XX-inhibited swelling-activated currents. 2 shows the current-voltage relationship in response to HYPO and 10 μM SN401 inhibition in WT MEG01 cells. [Figure 29C] Figure 1 shows how the human MK cell line MEG01 had robust SN-4XX-inhibited swelling-activated currents. Figure 1 shows the current-voltage relationship in response to inhibition by HYPO and 3 μM SN418c in WT MEG01 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Various aspects of the present invention relate to methods of preventing and / or treating thrombosis, including methods for preventing and / or treating cardiovascular thrombosis in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an LRRC8 protein modulating agent. The method may comprise administering to the patient a therapeutically effective amount of an LRRC8 protein modulating agent 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 method may comprise administering to the patient a therapeutically effective amount of an LRRC8 protein modulating agent to prevent and / or treat cardiovascular thrombosis and conditions resulting from cardiovascular thrombosis associated with atrial arrhythmias, including atrial flutter and atrial fibrillation. The methods may include administering to the patient a therapeutically effective amount of an LRRC8 protein modulating agent 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.

[0023] The disclosure further relates to a method for performing therapy, such as prevention and / or treatment of inherited or acquired defects in platelet function in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an LRRC8 protein modulating agent. The method may comprise administering to the patient a therapeutically effective amount of an LRRC8 protein modulating agent to normalize platelet function by increasing the release or membrane expression of platelet alpha granules, dense granules, or other intracellular organelle contents, including, but not limited to, platelet activators, coagulation factors, and adhesion molecules. The method may comprise administering to the patient a therapeutically effective amount of an LRRC8 protein modulating agent to normalize platelet function by normalizing platelet volume.

[0024] The present disclosure further relates to methods for therapy, such as prevention and / or treatment, of hereditary or acquired thrombocytosis in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an LRRC8 protein modulating agent.

[0025] The LRRC8 modulating agent may be DCPIB.

[0026] The administration of the compound may be sufficient to upregulate the expression of LRRC8 or the expression of LRRC8-associated proteins. The administration of the compound may be sufficient to stabilize the LRRC8 protein complex or the LRRC8-associated proteins. The administration of the compound may be sufficient to promote the membrane trafficking and activity of the LRRC8 protein complex or the LRRC8-associated proteins. The administration of the compound may be sufficient to enhance LRRC8-mediated signaling or trafficking to the membrane of intracellular organelles, including but not limited to lysosome-related organelles.

[0027] In various embodiments, a method for 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 congener thereof.

[0028] In some embodiments, the method comprises administering to the subject a therapeutically effective amount of

[0029] [ka] JPEG2025513794000007.jpg150169

[0030] and salts and geometric isomers thereof.

[0031] In various embodiments, the method comprises administering to the subject a therapeutically effective amount of a compound of formula (I), and salts and geometric isomers thereof:

[0032] [ka]

[0033] In the formula, 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, substituted or unsubstituted heteroaryl; R 3 But -YC(O)R 4 , -ZN(R 5 )(R 6 ), or -ZA, A is selected from the group consisting of:

[0034] [ka]

[0035] R 4 is hydrogen, substituted or unsubstituted alkyl, -OR 7 , or -N(R 8 )(R 9 ) and X 1 and X 2 each independently represents a substituted or unsubstituted alkyl, halo, -OR 10 , or -N(R 11 )(R 12 ) and 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 two carbon atoms; and n is 1 or 2.

[0036] In various embodiments, R 1 or R 2 At least one of R is a substituted or unsubstituted linear or branched alkyl having at least two carbon atoms. 1 is hydrogen or C1-C6 alkyl. For example, in some embodiments, R 1 is butyl. In various embodiments, R 2 is cycloalkyl (e.g., cyclopentyl). In various embodiments, R 1 or R 2 At least one of is selected from the group consisting of:

[0037] [ka]

[0038] In various embodiments, R 3 is -YC(O)R 4 In various embodiments, R 3 is -ZN(R 5 )(R 6 In various embodiments, R 3 In certain embodiments, R 3 teeth,

[0039] [ka]

[0040] In certain embodiments, R 3 teeth,

[0041] [ka]

[0042] is selected from the group consisting of:

[0043] In certain embodiments, A is selected from the group consisting of:

[0044] [ka]

[0045] In various embodiments, R 4 -OR 7 Or -N(R 8 )(R 9 ).

[0046] In various embodiments, X 1 and X 2 are each independently substituted or unsubstituted C1-C6 alkyl or halo. 1 and X 2 are each independently C1-C6 alkyl, fluoro, chloro, bromo, or iodo. 1 and X 2 are each independently methyl, fluoro, or chloro.

[0047] In various embodiments, R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are each independently hydrogen or alkyl. For example, in some embodiments, R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are each independently hydrogen or C1 to C3 alkyl.

[0048] In various embodiments, Y and Z are each independently a substituted or unsubstituted alkylene having 2 to 10 carbons, a substituted or unsubstituted alkenylene having 2 to 10 carbons, or a substituted or unsubstituted arylene. In some embodiments, Y and Z are each independently an alkylene having 2 to 10 carbons, an alkenylene having 2 to 10 carbons, or a phenylene. In certain embodiments, Y is an alkylene or alkenylene having 3 to 8 or 3 to 7 carbons. In various embodiments, Y and Z are each independently a cycloalkylene having 4 to 10 carbons. For example, Y can be an alkylene or any alkenylene having 4 carbons. In further embodiments, Z is an alkylene having 2 to 4 carbons. For example, Z can be an alkylene having 3 or 4 carbons. In certain embodiments, Y and Z are each independently selected from the group consisting of:

[0049] [ka]

[0050] In various embodiments, when Y is an alkylene having 2 to 3 carbons, X 1 and X 2 and each are fluoro or each are substituted or unsubstituted alkyl (e.g., methyl or ethyl). In some embodiments, Y is not an alkylene having 3 carbons. In certain embodiments, R 7 is not hydrogen or C1-C6 alkyl. 1 and / or X 2 is not halo. In certain embodiments, X 1 and / or X 2 is not chloro. In some embodiments, R 1 and / or R 2 is not alkyl. According to an embodiment, the compound of formula (I) is

[0051] [ka]

[0052] and geometric isomers and salts thereof.

[0053] In certain embodiments described herein, the compound administered is

[0054] [ka]

[0055] and geometric isomers and salts thereof.

[0056] The compound administered is

[0057] [ka] JPEG2025513794000018.jpg87169

[0058] may be selected from:

[0059] The present disclosure further relates to compounds used in the above methods. These compounds can regulate or inhibit LRRC8 protein. The compounds can have higher potency in regulating or inhibiting SWELL1 channel than equivalent amount of DCPIB (4-[2[butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid).

[0060] In various embodiments, methods of preventing or treating thrombosis include treating diseases in which thrombosis contributes to the disease state, and the present invention relates to various methods of preventing or treating diseases in which cardiovascular thrombosis contributes to the disease state, such as myocardial ischemia and infarction, stroke, transient cerebrovascular ischemia, peripheral arterial disease, thromboembolism associated with atrial fibrillation and flutter, and thromboembolism associated with the use of cardiovascular medical devices, and diseases associated with impaired platelet function, including abnormal adhesion, aggregation, activation, or thrombosis.

[0061] The synthesis of the compounds can proceed according to the following scheme.

[0062] [ka]

[0063] Modifications to the above synthetic scheme that can be made to synthesize various compounds described herein 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.

[0064] According to 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 a route including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. In various embodiments, administration is selected from the group consisting of oral, intranasal, intraperitoneal, intravenous, intramuscular, rectal, and transdermal.

[0065] Determination of a therapeutically effective dose of any one or more of the compounds described herein is within the ability of one of ordinary skill in the art. A therapeutically effective dose refers to that amount of active ingredient that produces a desired result. The exact dosage will be determined by the practitioner in light of factors related to the subject requiring 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 general health, the subject's age, weight, and sex, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerability / response to the therapy. Long-acting pharmaceutical compositions may be administered every 3-4 days, every week, or once every two weeks, depending on the half-life and clearance rate of the particular formulation.

[0066] Typically, the amount of a typical dosage of the compound may vary from about 0.05 to about 100 mg per kg body weight, depending on the route of administration. Guidance regarding specific dosages and delivery methods is provided in the literature and is generally available to those of skill in the art. It will generally be administered to provide a daily oral dose ranging, for example, from about 0.1 mg to about 75 mg, from about 0.5 mg to about 50 mg, or from about 1 mg to about 25 mg per kg body weight. The active ingredient may be administered in a single dose per day, or alternatively, in divided doses (e.g., twice a day, three times a day, four times a day, etc.). Generally, lower doses may be administered when parenteral routes are used. Thus, for example, for intravenous administration, doses ranging, for example, from about 0.05 mg to about 30 mg, from about 0.1 mg to about 25 mg, or from about 0.1 mg to about 20 mg per kg body weight may be used.

[0067] Pharmaceutical compositions for oral administration can be formulated using pharma- ceutically acceptable carriers known in the art in dosages suitable for oral administration. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like for ingestion by a subject. In certain embodiments, the compositions are formulated for parenteral administration. Further details of techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, Pa., incorporated herein by reference). After the pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of the indicated condition. Such labeling includes the amount, frequency, and method of administration.

[0068] In addition to the active ingredient (e.g., a compound of formula (I)), the pharmaceutical composition may contain a suitable pharma- ceutically acceptable carrier, including excipients and adjuvants that facilitate processing of the active compound into a pharma- ceutically usable preparation. As used herein, the term "pharma- ceutically acceptable carrier" refers to any type of non-toxic, inert solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary. Some examples of materials that can function as pharma- ceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; excipients such as powdered tragacanth, malt, gelatin, talc, cocoa butter, and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; ethyl oleate and Esters such as 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, ethyl alcohol, artificial cerebrospinal fluid (CSF), and buffers such as phosphate buffer, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants may also be present in the composition based on the desired route of administration, according to the judgment of the formulator.

[0069] Unless otherwise indicated, the alkyl, alkenyl, and alkynyl groups described herein preferably contain 1 to 20 carbon atoms in the main chain. They may be straight or branched chain or cyclic (e.g., cycloalkyl). Alkenyl groups can contain saturated or unsaturated carbon chains, so long as there is at least one carbon-carbon double bond. Alkynyl groups can contain saturated or unsaturated carbon chains, so 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 unbranched carbon chains having 1 to 20 carbon atoms in the main chain.

[0070] Unless otherwise indicated in the specification, the term "aryl" refers to a monocyclic, bicyclic, or tricyclic aromatic group containing 6 to 14 ring carbon atoms, including, for example, phenyl. The term "heteroaryl" refers to a monocyclic, bicyclic, or tricyclic aromatic group having 5 to 14 ring atoms, containing carbon atoms and at least 1, 2, or 3 oxygen, nitrogen, or sulfur heteroatoms.

[0071] After the invention has been described 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. EXAMPLES

[0072] Example 1: Genome-wide association studies (GWAS) indicate that LRRC8 protein regulates human platelet volume (Figure 1). A search of the NHGARI-EBI catalogue of eligible published human genome-wide association studies (accessed February 9, 2022) was performed. The catalogue of data is currently mapped to Genome Assembly GRCh38.p13 and dbSNP Build154. The NHGARI-EBI catalogue of published human GWAS studies included all GWAS studies with a P value <10 -8We listed 13 SNP variants assigned to five LRRC8 genes associated with six metabolic, inflammatory, and CV traits in the 19-year-old male. Each of the five LRRC8 genes contained at least one of these 13 SNPs (accessed February 9, 2022). GWAS in the database identified associations between platelet volume and three genes on two chromosomes: LRRC8A on 9q34.11 (2 × 10 -10 ) and LRRC8C on 1p22.2 (5 × 10 -23 and 3 x 10 -14 ) and LRRC8D(4×10 -13 and 6 x 10 -12 It was found that associations between six SNP variants assigned to

[0073] Example 2: Phenome-wide association study (PheWAS) shows that LRRC8 protein regulates human platelet volume (Figure 1). After identifying six SNPs associated with human platelet volume in the NHGARI-EBI catalog of published human GWAS studies, we compared the associations between platelet volume and P value < 10 in the Common Metabolic Diseases Knowledge Portal (accessed on March 3, 2022) and the PheWAS-IEU OpenGWAS project catalog (PheWAS catalog). -5 We searched for PheWAS associations between 13 SNP variants in (accessed February 9, 2022). PheWAS was able to predict associations between these SNP variants and platelet volume (8.9x10 -10 , 2.3x10 -10 , 5.7x10 -18 , 7.9x10 -17 , 1.5x10 -6 , and 1.4x10 -9), as well as four additional SNP variants assigned to LRRC8B, an overlapping interval between LRRC8B and C, and LRRC8C, which were initially identified in the GWAS catalogue for their association with blood monocyte count or diastolic blood pressure. Overall, the platelet volume phenotype is associated with 10 SNPs assigned to four LRRC8 genes (LRRC8A-D) on two chromosomes (1p22.2 and 9q34.11).

[0074] Example 3: eQTL studies indicate that LRRC8 protein regulates human platelet volume (Figure 1). We searched the GTEX Portal database expression quantitative trait loci (eQTL) database (accessed on March 5, 2022) to find the presence of LRRC8B (P = 2.8 × 10 -56 ) and LRRC8C (P = 4.8 × 10 -63 ) mRNA transcript levels and SNPs assigned to those genes associated with platelet volume in the NHGARI-EBI catalog of published human GWAS studies were found to have significant associations.

[0075] Example 4: Phenome-wide association study (PheWAS) shows that LRRC8 protein regulates human platelet count (Figure 2). The Common Metabolic Diseases Knowledge Portal (accessed March 3, 2022) was used to compare platelet counts and P values ​​of 10 -5 We searched for PheWAS associations between 13 SNP variants (accessed February 9, 2022) and platelet count (4.9 × 10 -11 , 2.6×10 -10 , and 3.8 × 10- 7 Overall, platelet count phenotypes are associated with three SNPs mapped to two LRRC8 genes (LRRC8C-D) on one chromosome (1p22.2).

[0076] Example 5: eQTL studies indicate that LRRC8 protein regulates human platelet count (Figure 2). We searched the GTEX Portal database expression quantitative trait loci (eQTL) database (accessed on March 5, 2022) to find the presence of LRRC8C (P = 4.8x10 -63 Significant associations were found between levels of mRNA transcripts of and SNPs assigned to LRRC8C associated with platelet count in the Common Metabolic Diseases Knowledge Portal (accessed March 3, 2022) or the PheWAS-IEU OpenGWAS Project catalogue (PheWAS catalogue).

[0077] Example 6: LRRC8A-D mRNA transcripts are expressed at high levels in human platelets compared to other mRNA transcripts (Figure 3). Using the Plateletomics Interactive Results Tool version 1.0, we searched the Plateletomics database (accessed on March 5, 2022) and found that LRRC8A-8D mRNA transcripts are expressed in human platelets and that they are in the top 76th, 14th, 43rd, and top 5th percentiles of all mRNA transcripts detected in platelets from 154 patients, indicating that LRRC8A-D mRNA transcripts are highly expressed in platelets compared to other mRNA transcripts.

[0078] Example 7: Human platelets contain mRNA transcripts for LRRC8A, LRRC8B, LRRC8C, and LRRC8D in ratios of 1:2.5:1.5:3.0, respectively (Figure 4). The transcripts of LRRC8A-E were searched in the database in Supplementary Table S2A from the 2014 publication by Simon, LM et al. (Blood 123, e37-45 (2014)) and it was found that mRNA transcripts of LRRCA, LRRC8B, LRRC8C, and LRRC8D are expressed in human platelets, with LRRC8B, LRRC8C, and LRRC8D being expressed at approximately 2.5-fold, 1.5-fold, and 3-fold the levels of LRRC8A mRNA, respectively.

[0079] Example 8: The extent of human platelet aggregation stimulated by protease-activated receptor 4 (PAR4) agonists is proportional to the expression of LRRC8A (Figure 5). The Plateletomics database was searched using the Plateletomics Interactive Results Tool, version 1.0 (accessed March 5, 2022) and data were found demonstrating that the strength of human platelet aggregation stimulated by protease-activated receptor 4-activating peptide (PAR4 AP), an agonist of the platelet PAR4 thrombin receptor, is significantly associated with the amount of LRRC8A mRNA expressed in human platelets.

[0080] 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). A search of the Plateletomics database using the Plateletomics Interactive Results Tool version 1.0 (accessed March 5, 2022) found data demonstrating that the strength of human platelet aggregation stimulated by adenosine diphosphate (ADP), an agonist of platelet P2Y12 and other ADP receptors, and protease-activated receptor 1-activatin peptide (PAR1 AP), an agonist of the platelet PAR1 thrombin receptor, was significantly inversely associated with the amount of LRRC8B mRNA expressed in human platelets.

[0081] Example 10: The extent of human platelet aggregation stimulated by protease-activated receptor 4 (PAR4) agonists is inversely proportional to the expression of LRRC8C (Figure 7). The Plateletomics database was searched using the Plateletomics Interactive Results Tool, version 1.0 (accessed March 5, 2022) and data were found demonstrating that the strength of human platelet aggregation stimulated by protease-activated receptor 4-activating peptide (PAR4 AP), an agonist of the platelet PAR4 thrombin receptor, was significantly and inversely associated with the amount of LRRC8C mRNA expressed in human platelets.

[0082] Example 11: The extent of human platelet aggregation stimulated by protease-activated receptor 1 (PAR1) agonists is inversely proportional to the expression of LRRC8D (Figure 8). The Plateletomics database was searched using the Plateletomics Interactive Results Tool, version 1.0 (accessed March 5, 2022) and data were found demonstrating that the strength of human platelet aggregation stimulated by protease-activated receptor 1-activating peptide (PAR1 AP), an agonist of the platelet PAR1 thrombin receptor, was significantly and inversely associated with the amount of LRRC8D mRNA expressed in human platelets.

[0083] Example 12: There are racial differences in LRRC8A-D expression in human platelets (Figure 9). The Plateletomics database was searched using the Plateletomics Interactive Results Tool version 1.0 (accessed March 5, 2022) and data was found demonstrating that mRNA expression of LRRC8C and LRRC8D was significantly lower in people identified as black compared to people identified as white. LRRC8A and LRRC8B gene expression was also lower in people identified as black compared to people identified as white, but did not cross the threshold for significance in this dataset.

[0084] Example 13: 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 (Figure 10). Regression coefficients for LRRC8A-D in a multiple regression analysis of platelet aggregation responses at 5911 commonly expressed mRNAs in human platelets from 154 human subjects from Simon, LM et al. (Blood 123, e37-45 (2014)) in 2014 Supplementary Table S3, and corresponding values ​​for significance tests, demonstrate that the degree of ADP-induced platelet aggregation is significantly associated with the levels of LRRC8A mRNA and inversely with the levels of LRRC8B mRNA, that the degree of PAR1-induced platelet aggregation is significantly associated with the levels of LRRC8B and LRRC8D mRNA, and that the degree of PAR4-induced platelet aggregation is associated with the levels of LRRC8D mRNA. The dependent variable in the regression specification was the degree of platelet aggregation in response to one of four platelet agonists (ADP, PAR1 AP, PAR2 AP, and arachidonic acid), and the independent variables were age, race, sex, platelet count, and BMI, as well as the levels of 5,911 commonly expressed mRNAs.

[0085] Example 14: LRRC8A in platelets regulates platelet volume in vivo (Figure 11). Whole blood was collected into heparin anticoagulant (15 USP units / mL) from the inferior vena cava of 20-week-old WT mice and mice lacking platelet LRRC8A, and whole blood CBCs were performed on an Element HT5 veterinary hematology analyzer (Heska, Loveland), which demonstrated that the mean platelet volume (MPV) of platelets lacking LRRC8A was significantly greater than the MPV of WT platelets (P<0.01, unpaired two-tailed t-test). Platelet counts were also increased in mice with platelets lacking LRRC8A compared to WT mice, but did not exceed the threshold for significance (P=0.06).

[0086] Example 15: LRRC8A is required for normal platelet adhesion to collagen of human platelets (Figure 12). Whole blood was collected into heparin anticoagulant 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-dihexyloxacarbocyanine iodide). Whole blood was perfused for 5 min through a polydimethylsiloxane (PDMS) microfluidic device bound to a glass slide patterned with bovine type 1 collagen at constant upstream pressure. Plasma was then perfused for 5 min with PBS, followed by 5 min of perfusion. Platelet accumulation was captured using an Olympus IX83 inverted microscope, and platelet surface area coverage was quantified using ImageJ. It was found that the absence of LRRC8A significantly reduced platelet adhesion (P<0.0001, paired two-tailed t-test).

[0087] Example 16: Inhibition of LRRC8 with SN-401 (DCPIB) significantly inhibited adhesion of human platelets to collagen (Figure 13). Human blood from four donors was collected into heparin anticoagulant, pooled, and incubated with fluorescently labeled DiOC6 (3,39-dihexyloxacarbocyanine iodide, Invitrogen) and either 10 μM SN-401 (DCPIB) or vehicle (DMSO) for 10 min. Whole blood was perfused for 5 min through a polydimethylsiloxane (PDMS) microfluidic device bound to a glass slide patterned with bovine type 1 collagen plasma at constant upstream pressure, followed by 5 min of PBS perfusion. Platelet accumulation was captured using an Olympus IX83 inverted microscope, and platelet surface area coverage was quantified using ImageJ. It was found that SN-401 significantly reduced adhesion of human platelets to collagen (vehicle vs. SN-401, P < 0.0001, ordinary one-way ANOVA with Turkey's multiple comparison test).

[0088] Example 17: Inhibition of LRRC8 with SN-401 (DCPIB) significantly inhibited thrombosis in vivo (Figure 14). To determine whether acute inhibition of LRRC8 channel activity affected platelet function in vivo, vehicle or the known LRRC8 channel inhibitor SN-401 (DCPIB) was administered by IP injection to 25-30 week old C57BL / 6 mice, and in vivo thrombosis was quantified using a tail wound thrombosis assay. Specifically, 20 min after IP injection of vehicle or 10 mg / kg SN-401, mouse tails were cut 5 mm from the tail tip and placed in saline at 37°C for 15 min, during which time relative blood loss was quantified by estimating hemoglobin concentration in saline by measuring optical absorbance at 550 nm. The average amount of tail blood loss in mice receiving SN-401 was found to be four times that of blood loss in mice receiving vehicle (P<0.05, unpaired two-tailed t-test). This demonstrated that pharmacological inhibition of LRRC8 inhibited thrombosis in vivo.

[0089] Example 18: Functional studies (Figures 15-29C) As mentioned above, four of the five LRRC8 subunit genes, LRRC8A-D, were shown to be associated with mean platelet volume (MPV) in genome-wide association studies (GWAS) and phenome-wide association studies (PheWAS), implicating LRRC8 channels in human platelet function and thrombosis. These findings are supported by human platelet mRNA expression and platelet activation data in the Plateletomics database, which reveal that 1) LRRC8A-D are expressed in platelets (Figure 15), and 2) transcript levels of LRRC8A, 8B, and 8D are significantly correlated (P<0.007-P<0.03) with platelet activation by known soluble platelet agonists. Notably, LRRC8E was not detected in human platelets and was not associated with platelet traits in GWAS / PheWAS, which is consistent with LRRC8A, B, C, and D being true associations.

[0090] To validate the biology suggested by human genetic studies, we fl / fl Mouse (SWELL1fl / fl ) 35~38 were crossed with platelet factor 4-driven Cre recombinase mice (Pf4-Cre) to fl / fl;Pf4-cre+ / - (cKO) and littermate control LRRC8A fl / fl We generated megakaryocyte (MK)-specific LRRC8A conditional knockout (cKO) mice by generating wild-type mice (WT). We confirmed the complete deletion of LRRC8A protein from cKO mouse platelets while maintaining the expression of the platelet marker β3 integrin (Figure 16). We demonstrated that LRRC8 protein regulates functional VRAC / I expression in platelets. Cl,SWELL To confirm that a channel is formed, Cl,SWELL was measured in freshly isolated mouse MKs using the perforated patch configuration of the patch clamp technique (Figure 17A). Cl,SWELL Currents observed in response to hypotonic swelling of WT MKs were completely inhibited upon application of 10 μM of the tool compound SN-401, with virtually no appreciable background current (Figures 17B-17D). This hypotonically activated swelling-activated current was completely abolished in cKO MKs (Figures 17C-17D), confirming that the SN-401-inhibited current in mouse MKs is entirely LRRC8A-mediated.

[0091] After confirming the ablation of LRRC8A protein and LRRC8A-mediated function in cKO platelets, platelet volume and platelet count were then measured. Consistent with the increased MPV associated with the SNP assigned to LRRC8A in the GWAS / PheWAS study, platelet-targeted deletion of LRRC8A also increases MPV compared to both WT and platelet-targeted heterozygous controls without increasing platelet count (Figure 18). Thus, deletion of LRRC8A in mouse platelets causes an increase in MPV, consistent with the variant human SNP allele located in the 5' untranslated exon region of LRRC8A identified in the GWAS / PheWAS study.

[0092] We then examined the functional consequences of LRRC8A deletion in platelets. Platelet adhesion, mediated primarily by the collagen receptor GPVI, alpha and dense granule secretion, and aggregation, mediated by activated αIIbβ3 integrin fibrinogen receptor, are three platelet functions essential for normal thrombus formation. Depletion of LRRC8A significantly impairs platelet adhesion to fibrillar collagen-coated surfaces under shear conditions (Figure 19A) and thrombin-stimulated aggregation (Figure 19B). Furthermore, LRRC8A deletion reduced thrombin-stimulated platelet activation as assessed by P-selectin exposure, a measure of alpha granule release (Figure 20A) and αIIbβ3 integrin activation (Figure 20B). However, LRRC8A-null platelets still exhibited poor Ca uptake. 2+ In response to the ionophore A23187, LRRC8 upregulates intracellular Ca 2+ These results suggest that LRRC8 regulates platelet signaling pathways upstream of LRRC8. In summary, these data indicate that LRRC8 channel signaling is required for normal platelet volume, adhesion, granule release, and aggregation.

[0093] Because in vivo thrombosis integrates several additional factors beyond platelet aggregation and adhesion observed in vitro, we investigated arterial thrombosis in WT and cKO mice using two different in vivo thrombosis models: (1) FeCl3-induced carotid artery injury (Figures 21A-21B), and (2) laser-induced cremaster arteriolar injury (Figures 22A, 22B, and 22C). Notably, we observed a marked prolongation of the time to occlusion (TTO) of FeCl3-injured carotid arteries in cKO mice compared to WT mice (Figures 21A-21B). Notably, as shown in Figure 21B, 5 / 7 cKO mice had TTOs greater than the 30 min (1800 s) time limit of the experiment, and thus the average TTOs underestimate the true TTOs because their values ​​are censored at 30 min. In a laser-induced cremaster arteriolar injury model, MK-specific deletion of LRRC8A also significantly reduced platelet thrombus formation compared to WT mice (Figures 22A, 22B, and 22C), whereas fibrin generation was unaffected in cKO mice, consistent with a platelet-specific defect in LRRC8 signaling (Figures 22A, 22B, and 22C).

[0094] Furthermore, preserved fibrin generation in cKO mice suggests that pharmacological inhibition may have the potential to selectively impair platelet thrombus formation but maintain hemostasis associated with intact fibrin generation, suggesting that the LRRC8 channel may be a drug target that is less likely to cause significant bleeding while still being an effective antithrombotic agent. Collectively, these data, which span from human genetics to mouse platelet-targeted loss-of-function models, rigorously validate the platelet LRRC8 channel as an innovative drug target for antiplatelet agents with previously unknown mechanisms of action. Furthermore, platelet LRRC8 inhibitors are being developed as antithrombotic agents for use in both nondiabetic and diabetic patients.

[0095] Additionally, the SN-4XX compounds have been shown to inhibit platelet activity.

[0096] Patch clamp studies with the tool compound SN-401 revealed robust inhibition of LRRC8 channel activity in mouse MKs (Figures 17A-17E), suggesting that SN-401 may also inhibit platelet activity if LRRC8 channel activity is required for platelet activation. Preliminary platelet adhesion studies performed on human blood (four different donors, performed on four separate days) using a microfluidic device demonstrated that SN-401 significantly inhibited platelet adhesion (Figures 23A-23B). Consistent with this, ip injection of 10 mg / kg SN-401 into mice revealed increased tail bleeding in vivo (Figure 24). These data provide proof of concept that LRRC8 channel inhibition by SN-4XX compounds has antiplatelet and antithrombotic activity.

[0097] SAR-based approaches have been successfully applied to develop more potent LRRC8 channel inhibitors. Examples of these are SN-418 and SN-418C (Figures 25A and 25B), which contain modifications of the alkyl chain + / - cyclopropyl group to increase LRRC8 channel inhibition by approximately 10-fold over SN-401 (Figures 25C and 25D). SN-418C was then tested for antiplatelet activity in vitro. Significant inhibition of thrombin-induced platelet activation, assessed by αIIbβ3 integrin activation (Figure 26A) and P-selectin exposure (Figure 26B), was observed. Consistent with these findings, SN-418C also robustly inhibited platelet aggregation in response to collagen-related peptide (CRP) and thrombin in a dose-dependent manner (Figures 27A-27B). Indeed, this level of platelet aggregation inhibition was comparable to that observed in LRRC8A null platelets (Figure 19B). Finally, ip injection of 10 mg / kg SN-418C for 4 days followed by 20 min prior to FeCl3 carotid injury demonstrated that SN-418C significantly increased time to occlusion (TTO, Figure 28) by 3.7-fold over vehicle-treated mice. Similar to what was observed in LRRC8A cKO mice (Figures 21A-21B), SN-418C treatment resulted in 4 / 7 mice with TTO exceeding the standard 30 min time limit of the experiment, indicating that the increase in TTO observed with this 10 mg / kg dose of SN-418 is an underestimate. As shown in Figures 29A, 29B, and 29C, MEG01 cells also showed robust I responses to hypotonic swelling. Cl,SWELL currents, which are completely inhibited by SN-401 and SN-418C.

[0098] Overall, these data indicate that the SN-40X compound has antiplatelet activity and can be used as an antithrombotic agent, providing proof of concept for an antiplatelet agent that functions 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.

[0099] All documents cited herein are incorporated by reference. Although specific embodiments of the invention have been described and many details have been set forth for purposes of illustration, the specific details may be changed without departing from the underlying principles of the invention. The use of the terms "a," "an," "the," and similar terms in the context of describing embodiments of the invention should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms20 (i.e., meaning "including, but not limited to"), unless otherwise noted. References to ranges of values ​​herein are merely intended to serve as a shorthand way of individually referring to each individual value falling within the range, unless otherwise indicated herein, and each individual value is incorporated into the specification as if it were set forth herein individually. In addition to the sequence detailed herein, the methods described herein may be performed in any suitable sequence25, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate embodiments of the invention and does not necessarily limit the scope of the invention unless specifically recited otherwise 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 carrying out therapy, such as the prevention and / or treatment of cardiovascular thrombosis, in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of an LRRC8 protein modulating agent.

2. 2. The method of claim 1, wherein the method comprises administering to the patient a therapeutically effective amount of an LRRC8 protein modulating agent 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. 2. The method of claim 1, wherein the method comprises administering to the patient a therapeutically effective amount of an LRRC8 protein modulating agent to prevent and / or treat cardiovascular thrombosis and conditions resulting from cardiovascular thrombosis associated with atrial arrhythmias, including atrial flutter and atrial fibrillation.

4. 2. The method of claim 1, wherein the method comprises administering to the patient a therapeutically effective amount of an LRRC8 protein modulating agent 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 carrying out therapy such as prevention and / or treatment of an inherited or acquired defect in platelet function in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of an LRRC8 protein modulating agent.

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

7. 6. The method of claim 5, wherein the method comprises administering to the patient a therapeutically effective amount of an LRRC8 protein modulating agent to normalize platelet function by normalizing platelet volume.

8. A method for carrying out therapy, such as prevention and / or treatment, of hereditary or acquired thrombocytosis in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of an LRRC8 protein modulating agent.

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

10. The LRRC8 modulator is a compound of the following formula (I), or a geometric isomer or salt thereof: 【Chemistry 1】 During the ceremony, 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, substituted or unsubstituted heteroaryl; R ３ But -Y-C(O)R ４ , -Z-N(R ５ ) (R ６ ), or -Z-A, A is selected from the group consisting of: 【Chemistry 2】 R ４ is hydrogen, substituted or unsubstituted alkyl, -OR ７ , or -N(R ８ ) (R ９ ) and X １ and X ２ each independently represents a substituted or unsubstituted alkyl, halo, -OR １０ or -N(R １１ ) (R １２ ) and 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 two carbon atoms; The method according to any one of claims 1 to 9, wherein n is 1 or 2.

11. R １ Or R ２ The method of claim 10, wherein at least one of is a substituted or unsubstituted linear or branched alkyl having at least two carbon atoms.

12. R １ Or R ２ 12. The method of claim 10 or 11, wherein at least one of is selected from the group consisting of: 【Chemistry 3】

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

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

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

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

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

18. R ４ But, -OR ７ Or -N(R ８ ) (R ９ The method according to any one of claims 10 to 17, wherein

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

20. R ３ The method of any one of claims 10 to 19, wherein is -ZA.

21. The method of any one of claims 10 to 20, wherein A is selected from the group consisting of: 【Chemistry 4】

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

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

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

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

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

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

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

29. Y and Z are each independently selected from the group consisting of: 【Chemistry 5】 The method according to any one of claims 10 to 28, selected from the group consisting of:

30. When Y is an alkyl group having 2 to 3 carbon atoms, X １ and X ２ The method of any one of claims 10 to 29, wherein both of are each fluoro or each substituted or unsubstituted alkyl.

31. R ３ but, 【Chemistry 6】 The method according to any one of claims 10 to 30, wherein the compound is selected from the group consisting of:

32. X １ and X ２ The method of any one of claims 10-31, wherein each is independently substituted or unsubstituted C1-C6 alkyl or halo.

33. X １ and X ２ The method of any one of claims 10 to 32, wherein each is independently C1 to C6 alkyl, fluoro, chloro, bromo, or iodo.

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

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

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

37. The method of any one of claims 10 to 36, selected from the group consisting of: 【Chemistry 7】 【change】

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

39. 39. The method of claim 38, wherein the compound has 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. 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 an LRRC8-associated protein.

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

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

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

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