Composition for preventing or treating Fabry disease

Abstract

The present invention relates to a composition for preventing or treating Fabry's disease, including a composition for preventing or treating Fabry's disease (FD) that contains a selected FDA-approved, preclinical, or clinical compound as an active ingredient, and a composition for preventing or treating Fabry's disease that uses a selected FDA-approved, preclinical, or clinical compound in combination with agalsidase-beta (agalsidase-β, agal).

Description

[Technical Field]

[0001] The present invention relates to a composition for preventing or treating Fabry's disease, including a composition for preventing or treating Fabry's disease (FD) that contains a selected FDA-approved, preclinical, or clinical compound as an active ingredient, and a composition for preventing or treating Fabry's disease that uses a selected FDA-approved, preclinical, or clinical compound in combination with agalsidase-beta (agalsidase-β, agal).

[0002] [Background technology]

[0003] Fabry disease (FD) is an X-chromosome-linked lysosomal storage disorder caused by α-galactosidase (GLA) deficiency. Fabry disease is characterized by the accumulation of globotriaosylceramide (Gb3) in various cell types, particularly vascular endothelial cells. Vascular lesions, including angiokeratomas, typically develop early in life in FD patients and progress to life-threatening vascular disease such as left ventricular hypertrophy (LVH), renal failure, and stroke with age. These complications are thought to be related to abnormalities in microvascular function (Rombach SM, Twickler TB, Aerts JMFG, Linthorst GE, Wijburg FA, Hollak CEM (2010) Vasculopathy in patients with Fabry disease: Current controversies and research directions. Molecular Genetics and Metabolism 99:99-108).

[0004] In recent years, FD patients are generally treated with enzyme replacement therapy (ERT) using recombinant human agalsidase beta (agalsidase-β) or pharmacological chaperone therapy. Patients with Fabry disease are treated with ERT to remove accumulated Gb3.

[0005] However, ERT has limited therapeutic efficacy for patients with late-onset disease or progressive complications. While ERT can temporarily improve the pathophysiological symptoms of FD, it cannot prevent the progression of complications, particularly kidney disease, in FD patients. Furthermore, the therapeutic efficacy of ERT is limited by its short half-life and high immunogenicity in FD patients. Chaperone therapy is also limited in that it is only effective in FD patients with specific GLA mutations. Therefore, new therapeutic strategies for vascular disease in FD are needed.

[0006] Vascular endothelial cells (VECs) derived from dedifferentiated stem cells from FD patients have been reported to have vascular damage due to increased thrombospondin-1 (TSP1) expression and overactivated SMAD2 signaling pathway (Do et al., EBioMedicine, 2020). Furthermore, increased levels of TSP1 and p-SMAD2 proteins in FD vascular endothelial cells (VECs differentiated from FD-iPSCs, FD-VECs) cause overactivation of the TGF-β signaling pathway, resulting in impaired angiogenesis in vitro.

[0007] In this regard, the inventors of the present application have screened for compounds that can down-regulate TSP1 expression and the SMAD2 signaling system (Korean Patent Application Publication No. 10-2022-0170751).

[0008] Meanwhile, TGF-β signaling plays a key role in the endothelial-to-mesenchymal transition (EndMT) of VECs (Cooley et al., 2014), which leads to the development of fibrosis in various tissues. A hallmark of FD is the accumulation of Gb3 in microvessels, which can induce tissue ischemia and induce fibrosis. Although Gb3 accumulation can be partially eliminated by ERT, this therapy remains ineffective for FD patients with progressive tissue fibrosis. Therefore, pharmacological modulation of EndMT may be a useful therapeutic strategy to improve the efficacy of ERT in FD patients.

[0009] Fasudil, a clinically approved ROCK signaling inhibitor, reduced the elevated levels of TSP1 and p-SMAD2 in FD-VECs and increased the expression of angiogenic factors. Fasudil also down-regulated endothelial-mesenchymal transition (EndMT) and ROS generation in FD-VECs. Furthermore, oral administration of fasudil to FD animal model mice alleviated various FD phenotypes, including LVH, renal fibrosis, anhidrosis, and heat insensitivity. Finally, the inventors of this application have demonstrated through these research results that fasudil may be a potential new therapeutic agent for FD patients (Choi JB et al., Molecular Therapy, 2023).

[0010] Under such technical background, the inventors of the present application have confirmed that the selected inhibitor compound is a potent clinical preparation that can be used to restore damaged vascular functionality in FD patients and improve various symptoms in FD patients, and have completed the present invention.

[0011]

[0012]

[0013] Summary of the Invention [Problem to be solved by the invention]

[0014] An object of the present invention is to provide a pharmaceutical composition for preventing or treating Fabry's disease (FD).

[0015] An object of the present invention is to provide a method for preventing or treating Fabry's disease (FD).

[0016] An object of the present invention is to provide a use of a formulation for use in the manufacture of a formulation for the prevention or treatment of Fabry disease.

[0017] An object of the present invention is to provide a composition for preventing or treating Fabry disease in combination with agalsidase-β (agalsidase-β, agal).

[0018] An object of the present invention is to provide a method for preventing or treating Fabry's disease (FD) in combination with agalsidase-β (agalsidase-β, agal).

[0019] An object of the present invention is to provide use of a preparation for use in the manufacture of a preparation for the prevention or treatment of Fabry disease in combination with agalsidase-beta (agalsidase-β, agal).

[0020]

[0021] [Means for solving the problem]

[0022] In order to achieve the above-mentioned object, the present invention provides a pharmaceutical composition for preventing or treating Fabry's disease (FD), which contains, as an active ingredient, a preparation selected from the group consisting of a TGF-β (Transforming Growth Factor-β) receptor activity inhibitor, a PPARγ (peroxisome proliferator-activated receptor) antagonist, a calcium channel blocker, a COX (cyclooxygenase) inhibitor, a piperazine compound, a Rho kinase inhibitor, and a ROCK (Rho-associated protein kinase) inhibitor.

[0023] The present invention also provides a method for preventing or treating Fabry disease, which comprises administering to an individual a formulation selected from the group consisting of a TGF-β (Transforming Growth Factor-β) receptor activity inhibitor, a PPARγ (peroxisome proliferator-activated receptor) antagonist, a calcium channel blocker, a COX (cyclooxygenase) inhibitor, a piperazine compound, a Rho kinase inhibitor, and a ROCK (Rho-associated protein kinase) inhibitor.

[0024] Furthermore, the present invention provides use of a preparation selected from the group consisting of a TGF-β (Transforming Growth Factor-β) receptor activity inhibitor, a PPARγ (peroxisome proliferator-activated receptor) antagonist, a calcium channel blocker, a COX (cyclooxygenase) inhibitor, a piperazine compound, a Rho kinase inhibitor, and a ROCK (Rho-associated protein kinase) inhibitor, for use in the manufacture of a preparation for the prevention or treatment of Fabry disease.

[0025] The present invention also provides a pharmaceutical composition for the prevention or treatment of Fabry's disease (FD), which comprises, as an active ingredient, a preparation selected from the group consisting of a TGF-β (Transforming Growth Factor-β) receptor activity inhibitor, a PPARγ (peroxisome proliferator-activated receptor) antagonist, a calcium channel blocker, a COX (cyclooxygenase) inhibitor, a piperazine compound, a Rho kinase inhibitor, and a ROCK (Rho-associated protein kinase) inhibitor, and is to be used in combination with agalsidase-β (agal).

[0026] The present invention also provides a method for preventing or treating Fabry disease, which comprises administering to an individual a formulation selected from the group consisting of a TGF-β (Transforming Growth Factor-β) receptor activity inhibitor, a PPARγ (peroxisome proliferator-activated receptor) antagonist, a calcium channel blocker, a COX (cyclooxygenase) inhibitor, a piperazine compound, a Rho kinase inhibitor, and a ROCK (Rho-associated protein kinase) inhibitor.

[0027] Furthermore, the present invention provides a use of a formulation selected from the group consisting of TGF-β (Transforming Growth Factor-β) receptor activity inhibitors, PPARγ (peroxisome proliferator-activated receptor) antagonists, calcium channel blockers, COX (cyclooxygenase) inhibitors, piperazine compounds, Rho kinase inhibitors, and ROCK (Rho-associated protein kinase) inhibitors, and agalsidase-beta for use in the manufacture of a formulation for the prevention or treatment of Fabry disease.

[0028]

[0029] [Effects of the Invention]

[0030] The present invention provides a novel use of a formulation selected from the group consisting of TGF-β (Transforming Growth Factor-β) receptor activity inhibitors, PPARγ (peroxisome proliferator-activated receptor) antagonists, calcium channel blockers, COX (cyclooxygenase) inhibitors, piperazine compounds, Rho kinase inhibitors, and ROCK (Rho-associated protein kinase) inhibitors, namely, the preventive or therapeutic effect of Fabry's disease (FD).

[0031] [Brief explanation of the drawings]

[0032] Figure 1. Hit compound screening of clinical chemistry libraries using FD-VEC

[0033] (A) Schematic diagram of the drug screening procedure using FD-VEC differentiated from FD-iPSCs. The effects of 2,107 preclinical or clinical compounds on improving the endothelial cell functionality of FD-VECs were investigated. In the first screening, compounds that induced cytotoxicity and / or abnormal morphological changes in FD-VECs were excluded. The effects of compounds on the ability of FD-VECs to form tube-forming structures were evaluated in the second and third screenings.

[0034] (B) In the second screening stage, scatter plots showing the length of tubes formed by FD-VECs treated with DMSO (negative control group), the TGF-β inhibitor SB431542 (SB; positive control group), or the indicated clinical compounds (5 μM).

[0035] (C) Relative total tube length formed by FD-VECs treated with DMSO (negative control), the TGF-β inhibitor SB431542 (SB, positive control), or the indicated clinical compounds (0.5 μM, 1 μM, or 5 μM). Data are presented as mean ± SEM (n = 3). *p < 0.05 and ***p < 0.001 (Student's t-test).

[0036] D) Western blot analysis of p-SMAD2, SMAD2, TSP1, KDR, eNOS, and GAPDH in WT-VEC, drug-untreated FD-VEC, and FD-VEC treated with selected compounds. Data are presented as mean ± SEM (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001 (Student's t-test).

[0037]

[0038] Figure 2. Effect of fasudil on angiogenesis in FD-VECs

[0039] (A) Tube formation assay to determine the optimal concentration and EC50 value of fasudil in FD-VEC. Data are presented as mean ± SEM (n = 2).

[0040] (B) Effect of fasudil (5 μM) on the tube-forming ability of VECs derived from FD-iPSC lines harboring distinct GLA mutations. Data are presented as mean ± SEM (n = 3). *p < 0.05 (Student's t-test).

[0041] (C) Analysis of tube formation ability by WT-VEC, untreated (FD) or fasudil-treated FD-VEC, and gene-corrected FD-VEC (FD(c)). Data are shown as mean ± SEM (n = 3). *p < 0.05 (Student's t-test).

[0042] (D) Western blot analysis of p-SMAD2, SMAD2, TSP1, KDR, eNOS, and GAPDH in each cell group described in (A). Data are presented as mean ± SEM (n = 7). *p < 0.05 and ***p < 0.001 (Student's t-test).

[0043]

[0044] Figure 3. Fasudil down-regulates EndMT in FD-VEC.

[0045] (A) Western blot analysis of EndMT-associated factors in WT-VEC, untreated (FD) or fasudil-treated FD-VEC, and gene-corrected FD-VEC (FD(c)). Data are presented as mean ± SEM (n = 4). **p < 0.01 (Student's t-test).

[0046] (B) Immunostaining of EndMT-associated factors in the cells described in (A).

[0047]

[0048] Figure 4. Fasudil alleviates impaired metabolic processes in FD-VEC.

[0049] (A) Representative images and analysis showing ROS fluorescence intensity in WT-VEC, untreated (FD) or fasudil-treated FD-VEC, and gene-corrected FD-VEC (FD(c)). Data are presented as mean ± SEM (n = 4). *p < 0.05 and **p < 0.01 (Student's t-test).

[0050] (B) Extracellular flux analysis of oxygen consumption rate (OCR) in the cells described in (A). Data are presented as mean ± SEM (n=6). O, oligomycin; F, FCCP; R&A, rotenone & antimycin A.

[0051]

[0052] Figure 5. Oral administration of fasudil alleviates the FD phenotype in Gla- / - / TSP1Tg mice.

[0053] (A) Schematic diagram of the experiment in FD mice (Gla- / - / TSP1Tg) orally administered fasudil (10 or 30 mg / kg / day for 6 months). PBS was administered to the control group of FD mice.

[0054] (B) Cardiac ultrasound analysis of WT mice (n = 6) and FD mice treated with PBS (n = 4) or fasudil at 10 or 30 mg / kg (n = 9). Cardiac ejection fraction, fractional shortening, and cardiac output were measured. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (Student's t-test).

[0055] (C) Sweat secretion analysis in WT and FD mice treated with or without fasudil. Data are presented as mean ± SEM (n = 3). *p < 0.05 (Student's t-test).

[0056] (D) Thermal tolerance analysis of WT and FD mice treated with or without fasudil. Increased rate of paw withdrawal indicates hypersensitivity to thermal pain. Data are presented as mean ± SEM (n = 5). *p < 0.05 and **p < 0.01 (Student's t-test).

[0057]

[0058] Figure 6. Oral administration of fasudil alleviates the FD phenotype in Gla- / - / TSP1Tg mice.

[0059] (A) Immunohistochemical analysis of CD31 and ACTA2 in kidney tissues from WT mice (n = 3) and FD mice (Gla- / - / TSP1Tg) treated with PBS (n = 3) or fasudil (n = 4). Data are shown as mean ± SEM. **p < 0.01 (Student's t-test).

[0060] (B) Immunohistochemical analysis of COL1A1 in kidney tissues from WT mice (n = 3) and FD mice (Gla- / - / TSP1Tg) treated with PBS (n = 3) or fasudil (n = 4). Data are shown as mean ± SEM. *p < 0.05 (Student's t-test).

[0061] (C) Immunohistochemical analysis of LCN2 and F4 / 80 in kidney tissues from WT mice (n = 3) and FD mice (Gla- / - / TSP1Tg) treated with PBS (n = 3) or fasudil (n = 4). Data are shown as mean ± SEM. *p < 0.05 and **p < 0.01 (Student's t-test).

[0062] (D) Western blot analysis of fibrosis-related markers (ACTA2 and COL1A1), inflammatory markers (LCN2 and F4 / 80), p-SMAD2, SMAD2, and GAPDH in kidney tissues from fasudil-treated FD mice (Gla- / - / TSP1Tg). Data are presented as mean ± SEM (n = 3). *p < 0.05 and **p < 0.01 (Student's t-test).

[0063]

[0064] Figure 7. Synergistic effect of combined treatment of fasudil and recombinant human agalsidase beta on the function of FD-VEC.

[0065] (A) Experimental schematic for co-treatment of FD-VECs differentiated from FD-iPSCs with fasudil and agA1. FD-VECs were treated with AgA1 every 2 days, followed by a single treatment with fasudil on day 8. The following day, the tube-forming ability of FD-VECs was assessed.

[0066] (B) Tube formation analysis in VECs derived from FD-iPSCs harboring different GLA mutations under combined treatment with fasudil and / or agal (n = 3). Data are presented as mean ± SEM. *p < 0.05 and **p < 0.01 (Student's t-test).

[0067] (C) Western blot of SMAD2, TSP1, KDR, eNOS, and GAPDH in cells described in (B) (n=7). Data are shown as mean ± SEM. *p<0.05 and **p<0.01 (Student's t-test).

[0068] (D) Representative images and analysis of ROS fluorescence intensity in the cells described in (B). (n=5). Data in the graph are shown as mean ± SEM (n=5). *p<0.05 and **p<0.01 (Student's t-test). Scale bar: 50 μm.

[0069] (E) Extracellular flux analysis of oxygen consumption rate (OCR) in cells described in (B). Data are presented as mean ± SEM (n=6). O, oligomycin; F, FCCP; R&A, rotenone & antimycin A.

[0070] Figure 8. Synergistic effect of combined treatment with fasudil and recombinant human agalsidase beta on FD vascular endothelial cell function.

[0071] Figure 9. Synergistic effect of enhancing metabolic function by combined treatment of fasudil and recombinant human agalsidase beta in FD vascular endothelial cells.

[0072] Figure 10. The method for administering fasudil and recombinant human agalsidase beta in Gla- / - model mice.

[0073] Figure 11. Cardiac ultrasound analysis of Fabry disease mice after co-administration of fasudil and recombinant human agalsidase beta.

[0074] Figure 12. Sweat analysis of Fabry mice after co-administration of fasudil and recombinant human agalsidase beta.

[0075] Figure 13. Peripheral nerve analysis of Fabry mice after co-administration of fasudil and recombinant human agalsidase beta.

[0076] Figure 14. Kidney tissue analysis of Fabry disease mice after co-administration of fasudil and recombinant human agalsidase beta.

[0077] Figure 15. Lomerizine improves impaired angiogenesis in FD-VEC.

[0078] (A) Schematic diagram of the drug screening procedure using FD-VECs. FD-VECs were differentiated from FD-iPSCs and secured for phenotypic drug screening. Drugs capable of improving the endothelial cell functionality of FD-VECs were identified based on FDA-approved clinical compounds. In the first screening stage, compounds that induced cytotoxicity or abnormal morphology were eliminated. In the next stage, the remaining compounds were screened for their ability to affect the ability of FD-VECs to form tube-forming structures. Finally, the therapeutic effects of the most effective compounds were tested in FD-mice.

[0079] (B) To investigate the extent to which lomerizine restores the tube-forming ability of FD-VECs, the tube-forming ability was observed at various concentrations, and the EC50 was analyzed based on the data.

[0080] (C) Tube-forming structure formation in WT-VEC, drug-untreated FD-VEC (FD), lomerizine-treated FD-VEC, and gene-corrected FD-VEC (FD(c)). Lomerizine-treated FD-VEC showed a significant increase in tube-forming ability compared with the untreated group. Data are presented as mean ± SEM (n = 3). *** p < 0.05 (Student's t-test); Scale bar: 200 μm; WT, wild-type, normal; FD, Fabry disease; L, lomerizine.

[0081] (D) Results showing that lomerizine treatment can improve the tube-forming ability of FD-VECs differentiated from patient iPSCs with different GLA mutations.

[0082] (E) Western blot of the cells described in (C). Treatment of FD-VEC with lomerizine decreased the levels of p-SMAD2 and TSP1, and increased the levels of KDR and eNOS. Data are shown as mean ± SEM (n = 4). *p < 0.05 and ***p < 0.001 (Student's t-test).

[0083] Figure 16. Lomerizine ameliorates mitochondrial dysfunction in FD-VEC.

[0084] (A) Representative images and analysis of ROS fluorescence intensity in WT-VEC, drug-untreated FD-VEC (FD), lomerizine-treated FD-VEC, and gene-modified FD-VEC (FD(c)). Lomerizine reduced ROS generation in FD-VEC. Data are shown as mean ± SEM (n = 4). *p < 0.05 and **p < 0.01 (Student's t-test).

[0085] (B) Extracellular flux analysis of oxygen consumption rate (OCR) in the cells described in (A). Lomerizine reduces maximal respiration in FD-VEC. Data are presented as mean ± SEM (n = 6). O, oligomycin; F, FCCP; R&A, rotenone and antimycin A.

[0086] Figure 17. Lomerizine down-regulates EndMT and enhances FD-VEC function.

[0087] (A) Western blot analysis of EndMT-associated factors CD31, COL1A1, ACTA2, SNAI1, and TWIST in WT-VEC, drug-untreated FD-VEC (FD), lomerizine-treated FD-VEC, and gene-corrected FD-VEC (FD(c)). Lomerizine increased the amount of CD31 and decreased the amount of EndMT-associated proteins in FD-VEC. Data are presented as mean ± SEM (n = 4). *p < 0.05 (Student's t-test).

[0088] (B) Immunostaining of EndMT-associated proteins was performed in the cells described in (A). Lomerizine reduced the amount of EndMT-associated proteins in FD-VEC. Scale bar: 50 μm.

[0089] Figure 18. Oral administration of lomerizine reverses the FD phenotype in FD mice.

[0090] (A) Schematic of the protocol for treating FD-mice (Gla- / - / TSP1Tg) with lomerizine (10 or 30 mg / kg / day for 6 months). Each mouse then underwent cardiac ultrasound, sweat secretion testing, and thermal pain testing. The control group of FD-mice received oral DMSO.

[0091] (B) Cardiac ultrasound measurements were performed to measure cardiac function in WT (n = 3) and FD mice treated with DMSO (n = 4) or lomerizine at 10 or 30 mg / kg (n = 9). Left ventricular mass / body weight, ejection fraction, fractional shortening, and cardiac output were measured. Lomerizine improved cardiac function in FD mice. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (Student's t-test).

[0092] (C) Sweat secretion test of WT mice (n = 3) and FD mice treated with or without lomerizine. Data are presented as mean ± SEM (n = 3). *p < 0.05 and **p < 0.01 (Student's t-test).

[0093] (D) Thermal pain testing of WT mice (n = 3) and FD mice (n = 5) treated with or without lomerizine. Increased paw withdrawal latency indicates hypersensitivity to thermal pain. Data are presented as mean ± SEM. *p < 0.05 and **p < 0.01 (Student's t-test).

[0094] Figure 19. Oral administration of lomerizine attenuates fibrosis and inflammation in renal tissue of FD-mice.

[0095] (A) Schematic diagram of the treatment of FD-mice (Gla- / - / TSP1Tg) with lomerizine (10 or 30 mg / kg / day for 6 months) before analyzing kidney tissue. FD-mice (control group) were orally administered DMSO.

[0096] (B) Kidney tissues from WT mice (n = 3) and FD mice (Gla- / - / TSP1Tg) treated with DMSO (n = 3) or lomerizine (n = 4) were analyzed by immunohistochemical labeling of CD31 and ACTA2. Lomerizine reduced ACTA2 fluorescence intensity in CD31+ cells in the kidney tissues of FD mice. Data are shown as mean ± SEM. *p < 0.05 (Student's t-test).

[0097] (C) Renal tissues from WT (n = 3) and FD mice (Gla- / - / TSP1Tg) treated with DMSO (n = 3) or lomerizine (n = 4) were analyzed by immunohistochemical labeling of COL1A1. Lomerizine reduced COL1A1 fluorescence intensity in the kidney tissues of FD mice. Data are shown as mean ± SEM. *p < 0.05 and **p < 0.01 (Student's t-test).

[0098] (D) The levels of inflammation-related markers F4 / 80 and LCN2 were analyzed by immunohistochemical labeling in kidney tissues from WT (n = 3) and FD mice (Gla- / - / TSP1Tg) treated with DMSO (n = 3) or lomerizine (n = 4). Lomerizine reduced F4 / 80 and LCN2 fluorescence intensity in kidney tissues from FD mice. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (Student's t-test).

[0099] (E) Western blot analysis of kidney proteins from FD-mice was performed for fibrosis-related markers ACTA2 and COL1A1, inflammation markers LCN2 and F4 / 80, and GAPDH. Expression of these fibrosis- and inflammation-related markers was reduced in FD-mice treated with lomerizine. Data are presented as mean ± SEM (n = 3). *p < 0.05 (Student's t-test).

[0100] Figure 20. Lomerizine exhibits therapeutic synergy when combined with recombinant human agalsidase beta on FD-VEC function.

[0101] (A) Experimental schematic for co-treatment of FD-VECs differentiated from FD-iPSCs with lomerizine and agA1. FD-VECs were treated with agA1 every 2 days, followed by a single treatment with fasudil on day 8. The following day, the tube-forming ability of FD-VECs was assessed.

[0102] (B) Tube formation analysis in VECs derived from FD-iPSCs harboring different GLA mutations under combined treatment with lomerizine and / or agal (n = 3). Data are presented as mean ± SEM. *p < 0.05 and **p < 0.01 (Student's t-test).

[0103] (C) Western blot of SMAD2, TSP1, KDR, eNOS, and GAPDH in cells described in (B) (n=5). Data are shown as mean ± SEM. *p<0.05 and **p<0.01 (Student's t-test).

[0104] (D) Representative image and analysis of fluorescence intensity in the cells described in (B). (n=5). Data in the graph are shown as mean ± SEM (n=5). *p<0.05 and **p<0.01 (Student's t-test). Scale bar: 50 μm.

[0105] (E) Extracellular flux analysis of oxygen consumption rate (OCR) in cells described in (B). Data are presented as mean ± SEM (n=6). O, oligomycin; F, FCCP; R&A, rotenone & antimycin A.

[0106] Figure 21. Photographs of tube formation in vascular endothelial cells of Fabry disease treated with various ROCK inhibitors.

[0107] Figure 22. The effect of ROCK inhibitor (Y-27632) treatment on the tube formation ability of vascular endothelial cells in Fabry disease patients.

[0108] Figure 23. The effect of ROCK inhibitor (GSK429286A) treatment on the tube formation ability of vascular endothelial cells in Fabry disease patients.

[0109] Figure 24. The effect of ROCK inhibitor (Y-39983) treatment on the tube formation ability of vascular endothelial cells in Fabry disease patients.

[0110] Figure 25. The effect of treatment with a ROCK inhibitor (belmosudil) on enhancing the tube-forming ability of vascular endothelial cells in Fabry disease.

[0111] Figure 26. Photographs showing the tube formation ability of endothelial cells in Fabry disease treated with various ROCK inhibitors. WT, wild type; FD, Fabry disease; SB: SB431542, a SMAD2 signaling inhibitor.

[0112] Figure 27. Shows the effect of ROCK inhibitor (netarsudil) treatment on enhancing the tube-forming ability of vascular endothelial cells in Fabry disease.

[0113] Figure 28. Shows the effect of treatment with a ROCK inhibitor (ripasudil) on enhancing the tube-forming ability of vascular endothelial cells in Fabry disease.

[0114] Figure 29. Shows the effect of ROCK inhibitor (sobesudil) treatment on enhancing the tube-forming ability of vascular endothelial cells in Fabry disease.

[0115] Figure 30. The effect of ROCK inhibitor (AT13148) treatment on the tube formation ability of vascular endothelial cells in Fabry disease.

[0116] Figure 31 shows the effect of ROCK inhibitor (AR13503) treatment on enhancing the tube-forming ability of vascular endothelial cells in Fabry disease.

[0117] Figure 32. The effect of ROCK inhibitor (VX-210) treatment on the enhancement of tube formation in vascular endothelial cells of Fabry disease.

[0118]

[0119] DETAILED DESCRIPTION OF THE INVENTION

[0120] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is that which is well known and commonly used in the art.

[0121] The inventors of the present application screened a library of 2107 FDA-approved, preclinical, and clinical compounds to identify drugs that may be useful therapeutic agents for Fabry's disease (FD).

[0122] Among these, agents selected from the group consisting of TGF-β (Transforming Growth Factor-β) receptor inhibitors, PPARγ (Peroxisome Proliferator-Activated Receptor) antagonists, calcium channel blockers, COX (Cyclooxygenase) inhibitors, piperazine compounds, and Rho kinase inhibitors reduced the levels of p-SMAD2 and TSP1 protein and increased the levels of the angiogenic factors KDR and eNOS in FD-VECs. Furthermore, fasudil inactivated TGF-β-induced EndMT and reduced reactive oxidative stress (ROS) and maximal respiratory volume in FD-VECs. Furthermore, oral administration of fasudil to transgenic mice expressing human TSP1 in vivo (Gla- / - / TSP1Tg; FD mice) improved many FD phenotypes, including LVH, renal fibrosis, anhidrosis, and heat hypersensitivity.

[0123] Among these, calcium channel blockers reduced the protein levels of p-SMAD2 and TSP1 in FD-VECs and increased the levels of the angiogenic factors KDR and eNOS. Furthermore, calcium channel blockers effectively inhibited EndMT in FD-VECs by downregulating ROS production and reducing maximal mitochondrial respiration. Oral administration of calcium channel blockers to FD mice (Gla- / - / TSP1Tg) was confirmed to alleviate various FD phenotypes, including LVH, renal fibrosis, anhidrosis, and heat hypersensitivity.

[0124] In addition, the present inventors confirmed that 10 ROCK signaling inhibitors with similar functionality to fasudil can restore the damaged vascular functionality of FD-VEC, and as a result, improved vascular functionality of FD-VEC. Through this, the inventors of the present application confirmed that ROCK signaling inhibitors can be useful in the treatment of patients with Fabry disease.

[0125] We propose that fasudil is a potent clinical compound that can be used to restore impaired vascular functionality in FD patients and improve various symptoms in FD patients.

[0126] We propose that calcium channel blockers are potent clinical compounds that can be used to restore impaired vascular functionality and improve various symptoms in FD patients.

[0127] In addition, to select drugs that could be useful therapeutic agents for Fabry's disease (FD), we screened ROCK (Rho-associated protein kinase) inhibitors and confirmed that the screened compounds exhibited the effect of enhancing the tube formation ability of vascular endothelial cells in Fabry's disease.

[0128] Based on this, the present invention relates to a pharmaceutical composition for preventing or treating Fabry's disease (FD), which contains, as an active ingredient, a preparation selected from the group consisting of a TGF-β (Transforming Growth Factor-β) receptor activity inhibitor, a PPARγ (peroxisome proliferator-activated receptor) antagonist, a calcium channel blocker, a COX (cyclooxygenase) inhibitor, a piperazine compound, a Rho kinase inhibitor, and a ROCK (Rho-associated protein kinase) inhibitor.

[0129] Fabry disease is an X-linked recessive genetic disorder caused by mutations in the gene encoding α-galactosidase (GLA). The GLA gene is located at xq22.1 in human exon 7 and encodes a glycoprotein consisting of 370 amino acids, which is processed from a precursor protein consisting of a total of 429 amino acids.

[0130] More than 400 mutation sites have been reported as possible mutation sites in the GLA gene (http: / / www.hgmd.cf.ac.uk), and the severity of Fabry disease symptoms varies depending on the mutation site in the GLA gene. Most mutations result in the complete loss of α-galactosidase activity, and some missense mutations, with only 5% to 10% remaining enzyme activity, do not result in clinically significant pathophysiology.

[0131] The major pathophysiological manifestation of Fabry disease is the accumulation of Gb3 in various cell types, including vascular cells, cardiac cells, kidney epithelial cells, and neuronal cells.

[0132] Agalsidase beta, an α-galactosidase, is administered to remove Gb3 accumulated in various cell types. The enzyme, administered intravenously, enters cells via the mannose 6-phosphate (M6P) receptor on the plasma membrane and then transports to lysosomes.

[0133] The TGF-β receptor activity inhibitor may be SB431542, D-4476, or a pharmaceutically acceptable salt thereof, which comprises the following structure:

[0134] [ka]

[0135] The PPARγ antagonist can be T0070907, which comprises the following structure, or a pharmaceutically acceptable salt thereof:

[0136] [ka]

[0137] The calcium channel blocker can be lomerizine, which has the following structure, or a pharmaceutically acceptable salt thereof:

[0138] [ka]

[0139] The COX inhibitor may be tolfenamic acid, which has the following structure:

[0140] [ka]

[0141] The piperazine compound may be eprazinone or a pharmaceutically acceptable salt thereof.

[0142] [ka]

[0143] The Rho kinase inhibitor can be fasudil, which has the following structure, or a pharmaceutically acceptable salt thereof:

[0144] [ka]

[0145] The ROCK inhibitor may include one or more selected from the group consisting of Y27632, GSK429286A, Y39983, Belmosudil, Netarsudil mesylate, Ripasudil hydrochloride hydrate, AR-13503 (Main Netarsudil Metabolite), AMA-0076 (Sovesudil), AT-13148, and VX-210, or a pharmaceutically acceptable salt thereof.

[0146] The "pharmaceutically acceptable salt" may be an acid addition salt formed with a pharmaceutically acceptable free acid, and the free acid may be an organic acid or an inorganic acid.

[0147] The organic acids include, but are not limited to, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid, and aspartic acid, and the inorganic acids include, but are not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

[0148] For example, if a compound has a functional group that can be anionic (e.g., -COOH can be -COO-), a salt can be formed with a suitable cation. Examples of suitable inorganic cations are alkali metal ions, e.g., Na + and K. + , alkaline earth metal cations, e.g., Ca 2+ and Mg 2+ , and other cations, e.g., Al 3+ Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH + ) and substituted ammonium ions (e.g., NHR + , NH2R2 + , NHR3 + , NR4 + ), including but not limited to:

[0149] Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3). 4+ is.

[0150] If the compound has a cationic or cationic functional group (e.g., -NH2 is -NH3 + (which may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,

[0151] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyloxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose, and the like.

[0152] Specifically, the Rho kinase inhibitor can include fasudil hydrochloride.

[0153] The pharmaceutical composition of the present invention can be administered by any route. The composition of the present invention can be provided to an animal by any suitable means, such as directly (e.g., locally by injection, implantation, or local administration to a tissue site) or systemically (e.g., parenterally or orally). When the composition of the present invention is provided parenterally, such as intravenously, subcutaneously, intraocularly, intraperitoneally, intramuscularly, orally, rectally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternally, intracapsularly, intranasally, or by aerosol administration, the pharmaceutical composition can be, for example, aqueous or comprise a portion of a physiologically compatible body fluid suspension or solution. This allows the carrier or vehicle to be physiologically acceptable so that it can be delivered to a patient after addition to the composition. Thus, physiological saline may generally be included as a carrier such as a body fluid for the formulation.

[0154] The frequency of administration may also vary depending on the pharmacokinetic parameters of the formulation used. Typically, a clinician will administer a pharmaceutical composition until a dosage that achieves the desired effect is reached. Thus, the pharmaceutical composition may be administered as a single dose, two or more doses spaced apart in time, or as a continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routine and falls within the scope of their routine practice.

[0155] The unit dosage for humans is 0.01 μg / kg to 100 mg / kg, specifically 1 μg / kg to 10 mg / kg of body weight. While the above-mentioned amount is the optimal amount, it can vary depending on the disease to be treated and the presence or absence of side effects, and the optimal dosage can be determined through routine experimentation. The fusion protein can be administered by periodic bolus injections, or by continuous intravenous, subcutaneous, or intraperitoneal administration from an external reservoir (e.g., an intravenous bag) or an internal source (e.g., a bioerodable implant).

[0156] Suitable routes include oral, parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, inhalation, intradermal, intrathecal, epidural, and infusion techniques), transdermal, rectal, intranasal, topical (including buccal and sublingual), vaginal, intraperitoneal, pulmonary, and intranasal administration. Topical administration can include using transdermal administration such as transdermal patches or iontophoretic devices.

[0157] The preferred route may vary depending, for example, on the condition of the recipient. For oral administration, it may be formulated as a pill, capsule, tablet, etc. together with a pharmaceutically acceptable carrier, lubricant, or excipient. For parenteral administration, it may be formulated in the form of a unit dose injection together with a pharmaceutically acceptable parenteral vehicle or diluent.

[0158] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, which is typically used in drug formulations and may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. The pharmaceutical composition may further comprise one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives typically used in the manufacture of pharmaceutical compositions.

[0159] An effective amount of the pharmaceutical composition can be administered orally or parenterally. Parenteral administration can be performed by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration. Since proteins or peptides are digested during oral administration, oral compositions can be formulated to coat the active agent or protect it from degradation in the stomach. Furthermore, the composition can be administered by any device that can deliver the active agent to target cells.

[0160] The present invention relates to a method for preventing or treating Fabry's disease (FD), which comprises administering to an individual a formulation selected from the group consisting of a TGF-β (Transforming Growth Factor-β) receptor activity inhibitor, a PPARγ (peroxisome proliferator-activated receptor) antagonist, a calcium channel blocker, a COX (cyclooxygenase) inhibitor, a piperazine compound, a Rho kinase inhibitor, and a ROCK (Rho-associated protein kinase) inhibitor. The individual may be a mammal, preferably a human.

[0161] "Treatment" refers to any indication of success in treating or ameliorating an injury, pathology, or condition, including any subjective or objective parameter, such as relief; remission; a reduction in symptoms or the production of damage, pathology, or condition that is more acceptable to the patient; a slowing of the rate of decline or deterioration; the production of a less debilitating end point of regression; or an improvement in the patient's physical or mental well-being. Treatment or amelioration of symptoms can be based on objective or subjective parameters, including a physical examination, neuropsychiatric examination, and / or psychiatric evaluation.

[0162] An "effective amount" is generally an amount sufficient to reduce the severity and frequency of a symptom, eliminate a symptom and its underlying cause, prevent the onset of a symptom or its underlying cause, or ameliorate or correct damage resulting from or associated with a disease state.

[0163] An effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to correct a disease state or condition, particularly a condition or symptom associated with a disease state, or to otherwise prevent, hinder, delay or reverse the progression of the disease state or any other undesirable symptoms associated with the disease in any way. A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, has the intended prophylactic effect, e.g., preventing or delaying the onset of a disease state or reducing the likelihood of the onset (or recurrence) of a disease state or associated symptoms.

[0164] The present invention also relates to a pharmaceutical composition for the prevention or treatment of Fabry's disease (FD), which comprises, as an active ingredient, a preparation selected from the group consisting of TGF-β (Transforming Growth Factor-β) receptor activity inhibitors, PPARγ (peroxisome proliferator-activated receptor) antagonists, calcium channel blockers, COX (cyclooxygenase) inhibitors, piperazine compounds, Rho kinase inhibitors, and ROCK (Rho-associated protein kinase) inhibitors, and which is to be used in combination with agalsidase-β (agal).

[0165] The TGF-β receptor activity inhibitor may be SB431542, D-4476, or a pharmaceutically acceptable salt thereof, which comprises the following structure:

[0166] [ka]

[0167] The PPARγ antagonist can be T0070907, which comprises the following structure, or a pharmaceutically acceptable salt thereof:

[0168] [ka]

[0169] The calcium channel blocker can be lomerizine, which has the following structure, or a pharmaceutically acceptable salt thereof:

[0170] [ka]

[0171] The COX inhibitor can be tolfenamic acid, which comprises the following structure, or a pharmaceutically acceptable salt thereof:

[0172] [ka]

[0173] The piperazine compound can be eprazinone or a pharmaceutically acceptable salt thereof.

[0174] [ka]

[0175] The Rho kinase inhibitor can be fasudil, which has the following structure, or a pharmaceutically acceptable salt thereof:

[0176] [ka]

[0177] The ROCK inhibitor may include one or more selected from the group consisting of Y27632, GSK429286A, Y39983, belmosudil, netarsudil mesylate, ripasudil hydrochloride hydrate, AR-13503 (Main Netarsudil Metabolite), AMA-0076 (Sovesudil), AT-13148, and VX-210, or a pharmaceutically acceptable salt thereof.

[0178] The preparation selected from the group consisting of TGF-β (Transforming Growth Factor-β) receptor activity inhibitors, PPARγ (peroxisome proliferator-activated receptor) antagonists, calcium channel blockers, COX (cyclooxygenase) inhibitors, piperazine compounds, Rho kinase inhibitors, and ROCK (Rho-associated protein kinase) inhibitors, and agalsidase-β (agal) preferably have complementary activities, so that they do not adversely affect each other.

[0179] The compositions of the present invention can be (1) co-formulated in a combined dosage form and administered or delivered simultaneously; or (2) administered or delivered simultaneously or sequentially as separate dosage forms.

[0180] In the case of the combined dosage form, a formulation selected from the group consisting of a TGF-β (Transforming Growth Factor-β) receptor inhibitor, a PPARγ (Peroxisome Proliferator-Activated Receptor) antagonist, a calcium channel blocker, a COX (Cyclooxygenase) inhibitor, a piperazine compound, a Rho kinase inhibitor, and a ROCK (Rho-associated protein kinase) inhibitor, and agalsidase-β (agal) may be present in the same composition. The dosage form may be, for example, but is not limited to, a dry powder composition, a solution, or a suspension.

[0181] The preparation selected from the group consisting of TGF-β (Transforming Growth Factor-β) receptor activity inhibitors, PPARγ (peroxisome proliferator-activated receptor) antagonists, calcium channel blockers, COX (cyclooxygenase) inhibitors, piperazine compounds, Rho kinase inhibitors, and ROCK (Rho-associated protein kinase) inhibitors, and agalsidase-beta can be administered simultaneously or sequentially.

[0182] The preparation selected from the group consisting of TGF-β (Transforming Growth Factor-β) receptor inhibitors, PPARγ (peroxisome proliferator-activated receptor) antagonists, calcium channel blockers, COX (cyclooxygenase) inhibitors, piperazine compounds, Rho kinase inhibitors, and ROCK (Rho-associated protein kinase) inhibitors, and agalsidase beta are generally administered separately from each other and may be administered simultaneously or sequentially. If administered sequentially, they may be administered two or more times. When administered sequentially, one of a preparation selected from the group consisting of TGF-β (Transforming Growth Factor-β) receptor activity inhibitors, PPARγ (peroxisome proliferator-activated receptor) antagonists, calcium channel blockers, COX (cyclooxygenase) inhibitors, piperazine compounds, Rho kinase inhibitors, and ROCK (Rho-associated protein kinase) inhibitors, and agalsidase-beta, and one of the following may be administered, or two of a preparation selected from the group consisting of TGF-β (Transforming Growth Factor-β) receptor activity inhibitors, PPARγ (peroxisome proliferator-activated receptor) antagonists, calcium channel blockers, COX (cyclooxygenase) inhibitors, piperazine compounds, Rho kinase inhibitors, and ROCK (Rho-associated protein kinase) inhibitors, and agalsidase-beta may be administered alternately with an interval between administrations.

[0183] The dosage of agalsidase-beta may be the amount currently commonly used in the industry, and the dosage can be reduced by using it in combination with a preparation selected from the group consisting of TGF-β (Transforming Growth Factor-β) receptor activity inhibitors, PPARγ (peroxisome proliferator-activated receptor) antagonists, calcium channel blockers, COX (cyclooxygenase) inhibitors, piperazine compounds, Rho kinase inhibitors, and ROCK (Rho-associated protein kinase) inhibitors.

[0184]

[0185] [Example]

[0186] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0187]

[0188] Example 1. Compound screening of a clinical chemistry library using FD-VEC

[0189]

[0190] FD-iPSCs were generated from fibroblasts of three FD patients with GLA mutations. A wild-type (WT) iPSC line derived from foreskin fibroblasts and an FD1 isogenic iPSC line were used as controls. Human iPSCs (hiPSCs) were maintained in mTeSR1 medium (STEMCELL Technologies, Vancouver, Canada) in culture plates pre-coated with Corning Matrigel® hESC-Qualified Matrix. Cells were maintained at 37°C in a 5% CO2 incubator. After 7 days of culture, hiPSC colonies were split at a ratio of 1:50 using Accutase solution (eBioscience, San Diego, CA, USA) and grown under the same culture conditions.

[0191] Human iPSCs were further optimized and differentiated into VECs as described above. Briefly, hiPSCs were transferred to Matrigel® (BD Biosciences, Franklin Lakes, NJ, USA)-coated dishes and cultured in mTESR1 medium (STEMCELL Technologies) at 37°C and 5% CO for 2 days. Subsequently, the cells were cultured in VEC medium (RPMI (HyClone, Logan, MI, USA), 1% B27 (Invitrogen, Carlsbad, CA, USA), and penicillin / streptomycin (Invitrogen)), 50 ng / ml Activin A (PeproTech, Rocky Hill, NJ, USA), 20 ng / ml BMP4 (PeproTech), and 3 μM CHIR990921 (Sigma) at 37°C and 5% CO for an additional 2 days. The cells were then cultured for 3 days at 37°C and 5% CO2 in VEC medium containing 50 ng / ml VEGF-A (PeproTech) and 50 ng / ml bFGF. To differentiate vascular progenitor cells into VECs, CD31+ cells were sorted from the differentiated cells by MACS sorting using CD31+ Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA). The CD31+ cells were transferred to gelatin-coated plates and cultured for 3 days at 37°C and 5% CO2 in EGM-2 medium (Lonza, Basel, Switzerland) supplemented with 100 ng / ml VEGF-A and 50 ng / ml bFGF. The medium was changed daily. Finally, the vascular progenitor cells were cultured for 4 days in growth factor-free EGM-2 medium with daily medium changes.

[0192] To screen for clinical compounds that can treat FD-VEC dysfunction, VECs were plated onto gelatin-coated 96-well plates (SPL Life Sciences, Pocheon, Republic of Korea) at 3 × 10 3 cells / cm 2The cells were plated at a density of 100 μM and maintained in EGM-2 medium in the absence of growth factors for 24 hours. FD-VECs were treated with each compound (5 μM) for 24 hours to select compounds that did not affect cell viability or morphology. The ability of FD-VECs treated with each selected compound to form tubular structures on Matrigel®-coated plates was then confirmed. The tubular structure formation assay was repeated at least three times using different concentrations of each selected compound (0.5 μM, 1 μM, and 5 μM).

[0193] Figure 1A shows a schematic diagram of the clinical compound screening process. FD-VECs were differentiated and isolated from FD-iPSCs as previously described (Do et al., 2020). Briefly, FD-iPSCs were isolated as a CD31+ cell population, then sorted using CD31+ magnetic beads and allowed to mature into FD-VECs in vitro for 7 days. A library of 2107 FDA-approved preclinical and clinical compounds was used in the screening assay. The first round of screening identified 451 compounds that were not cytotoxic to FD-VECs and did not induce morphological changes in vascular endothelial cells. The second round of screening identified 15 compounds capable of promoting tube formation by FD-VECs. The tube lengths formed by FD-VECs cultured with these 15 compounds (treatment concentration: 5 μM) were at least 1.5-fold longer than those of DMSO-treated control FD-VECs and even longer than those of FD-VECs treated with a TGF-β inhibitor (SB431542) (Figure 1B). Next, we repeated the tube formation assay using various concentrations of the 15 compounds (0.5 μM, 1 μM, 5 μM). We found that six compounds induced relatively higher tube formation than the DMSO control (Figure 1C). The selected compounds included two preclinical compounds (a TGF-β receptor inhibitor (D-4476) and a PPARγ antagonist (T0070907)) and four clinical compounds (a calcium channel blocker (lomerizine HCl), a COX inhibitor (tolfenamic acid), a member of the piperazine family (eprazinone 2HCl), and a Rho kinase inhibitor (fasudil)). FD-VECs treated with each of the six selected compounds were able to form tube-like structures. Furthermore, Western blot analysis confirmed that each compound down-regulated the levels of p-SMAD2 and TSP1 in FD-VECs and significantly increased the levels of the angiogenic factors KDR and eNOS (Figure 1D). Thus, we established a new phenotypic screening platform for drug repurposing, including in vitro functional testing of FD-VECs.

[0194]

[0195] Example 2. Fasudil improves defective tube formation in FD-VEC

[0196]

[0197] Of the six compounds selected through the screening analysis, fasudil was the most effective in promoting FD-VEC tube formation and was therefore selected for further analysis.

[0198] FD-VEC were treated with Accutase (eBioscience) at 37°C for 5 minutes and then dissociated. Dissociated cells (1 × 10 4 The cells were transferred to Matrigel® matrix (BD Biosciences) in EGM-2 medium supplemented with 100 ng / ml VEGF-A and cultured for 24 hours at 37°C and 5% CO2. Images of the vessel-like structures were taken using an inverted microscope (Olympus, Tokyo, Japan). The total length of the tubes was measured using ImageJ software and the Angiogenesis Analyzer plugin (Carpentier, 2012), provided free of charge by the National Institute of Mental Health (NIMH, Bethesda, MD, USA).

[0199] Based on the length of the formed tube structures, the optimal concentration and half-maximal effective concentration (EC50) of fasudil were determined to be 5 μM and 0.4183 μM, respectively (Figure 2A). Therefore, a concentration of 5 μM was used in subsequent experiments. Treatment with 5 μM fasudil significantly increased the tube length of FD-VEC compared to the untreated group, and the tube length of fasudil-treated FD-VEC was increased by the same amount as that of VEC derived from gene-corrected FD-VEC (FD(c)-VEC) cells (a control group with normal genes established through genetic scissors in FD-iPSCs) (Figure 2B). Fasudil also improved the tube-forming ability of VEC derived from FD-iPSC lines harboring other GLA mutations (Figure 2C). Furthermore, compared with untreated FD-VECs, both FD(c)-VECs and fasudil-treated FD-VECs decreased the levels of p-SMAD2 and TSP1, and increased the levels of KDR and eNOS (Figure 2D). Overall, these findings indicate that fasudil is effective in alleviating defective angiogenesis in FD-VECs in vitro.

[0200]

[0201] Example 3. Fasudil down-regulates EndMT and improves FD-VEC function.

[0202]

[0203] Next, we investigated the mechanism by which fasudil improves the angiogenic potential of defective FD-VECs. Hyperresponsive SMAD2 signaling induces EndMT in VECs and reduces angiogenic function. Given its ability to reduce p-SMAD2 expression in FD-VECs, we investigated the effect of fasudil on EndMT in these cells. RNA sequencing of FD-VECs and FD(c)-VECs was performed before drug treatment to investigate the underlying mechanism of defective vascular disease in FD-VECs. Compared to the protein levels of WT-VECs and FD(c)-VECs, the protein levels of CD31 were down-regulated, while the protein levels of mesenchymal-related genes (COL1A1, ACTA2, SNAI1, and TWIST) were up-regulated in FD-VECs (Figure 3A). Fasudil treatment increased the amount of CD31 protein and decreased the levels of mesenchymal-associated markers in FD-VEC (Figure 3A). Immunohistochemistry confirmed that the fluorescence intensity of ACTA2, SNAI1, and COL1A1 decreased after fasudil treatment of FD-VEC (Figure 3B). Collectively, these results demonstrate that impaired angiogenesis in FD-VEC is associated with increased EndMT due to overactive TGF-β signaling and that fasudil can inhibit EndMT induced by TGF-β activation.

[0204]

[0205] Example 4. Fasudil ameliorates impaired metabolic processes in FD-VEC.

[0206]

[0207] Next, we investigated the mechanism by which fasudil inhibits TGF-β-induced EndMT in FD-VEC. ROS are a major contributor to vascular injury due to TGF-β-induced EndMT. As previously reported (Tseng et al., 2017), FD-VEC have higher ROS levels than WT-VEC (Figure 4A). However, fasudil treatment reduced ROS levels to levels similar to those of WT-VEC. Mitochondrial OCR (oxygen consumption rate) was measured in FD(c)-VEC, WT-VEC, and FD-VEC. FD-VEC showed improved basal respiration (I), ATP production (II), maximal respiration (III), and spare capacity (IV). Treatment of FD-VEC with fasudil partially reduced maximal respiration, but did not alter basal respiration, ATP production, or spare capacity (Figure 4B). These results suggest that fasudil promotes ROS removal in FD-VEC, partially restoring impaired metabolic processes by regulating mitochondrial oxygen consumption.

[0208]

[0209] Example 5. Fasudil ameliorates the FD phenotype (LVH, anhidrosis and heat hypersensitivity) in vivo.

[0210]

[0211] Currently available GLA-deficient FD animal models cannot fully mimic the vascular disease of human FD patients. Renal tissue biopsies from FD patients have been found to have higher TSP1 levels than healthy donors. Therefore, we hypothesized that transgenic mice expressing human TSP1 (Gla- / - / TSP1Tg; hereafter referred to as FD mice) could be an effective animal model for studying FD-associated vascular disease. Fasudil (10 or 30 mg / kg / day) was administered orally to FD mice for 6 months, starting at 2 months of age (Figure 5A). LVH is known to occur in one-half of men and one-third of women with FD (Kampmann C, Linhart A, Baehner F, Palecek T, Wiethoff CM, Miebach E, Whybra C, Gal A, Bultas J, Beck M (2008) Onset and progression of the Anderson-Fabry disease-related cardiomyopathy. International journal of cardiology 130:367-73). Therefore, we investigated cardiac function in FD mice via cardiac ultrasound. Compared with WT mice, FD mice exhibited a higher left ventricular (LV) mass / body weight (BW) and lower cardiac ejection fraction, fractional shortening, and cardiac output (Figure 5B). LV mass / BW was significantly reduced in FD mice treated with fasudil (10 or 30 mg / kg) for 6 months (Figure 5B). Furthermore, fasudil administration improved the cardiac ejection fraction, fractional shortening, and cardiac output in FD mice (Figure 5B), indicating that fasudil administration improves the impaired cardiac function of FD mice.

[0212] We also investigated the effects of fasudil on other FD phenotypes, including anhidrosis and heat hypersensitivity. Compared with WT mice, FD mice exhibited reduced sweat secretion and increased paw withdrawal in the heat tolerance test. However, oral administration of fasudil significantly improved anhidrosis and heat hypersensitivity in FD mice (Figures 5C and 5D). Overall, these findings suggest that in vivo administration of fasudil to FD mice alleviates various FD clinical phenotypes, including the progression of renal fibrosis and inflammation.

[0213]

[0214] Example 6. Fasudil alleviates the FD phenotype in vivo.

[0215]

[0216] Another major symptom of FD is renal fibrosis. Fibrosis generally occurs through the EndMT, along with inflammation, in VECs in various tissues. Immunohistochemical analysis confirmed that CD31+ACTA2+ cells were more abundant in the kidney tissue of FD mice than in WT mice, but their abundance decreased after fasudil administration (Figure 6A). Furthermore, fasudil administration downregulated the expression of another EndMT marker, COL1A1, in the kidney tissue of FD mice (Figure 6B). The expression levels of inflammation-related proteins F4 / 80 and LCN2 were also increased in the kidney tissue of FD mice but decreased after fasudil administration (Figure 6C). These findings were also confirmed by Western blot analysis of kidney tissue from FD mice treated with or without fasudil (Figure 6D).

[0217]

[0218] Example 7. Effect of combined treatment with fasudil and recombinant human agalsidase beta

[0219]

[0220] We previously reported that treatment with recombinant human agalsidase beta (agalsidase-β, agal) did not significantly enhance impaired tube formation in FD-VECs (Do HS, Park SW, Im I, Seo D, Yoo HW, Go H, Kim YH, Koh GY, Lee BH, Han YM (2020) Enhanced thrombospondin-1 causes dysfunction of vascular endothelial cells derived from Fabry disease-induced pluripotent stem cells. EBioMedicine 52:102633). Therefore, we analyzed whether coadministration of fasudil and agal could synergistically support the therapeutic effects of fasudil. To this end, FD-VECs were treated with agal and fasudil. FD-VECs were treated with agal every 2 days, followed by a final fasudil treatment, after which their tube formation ability was analyzed (Figure 7A). Surprisingly, the combined treatment restored FD-VEC tube formation functionality more effectively than fasudil treatment alone (Figure 7B). Furthermore, the combined treatment efficiently reduced the levels of TSP1 and p-SMAD2 and increased the expression of angiogenic factors (KDR and eNOS) in FD-VEC compared with either treatment alone (Figure 7C). The combined treatment also efficiently reduced ROS generation in FD-VEC compared with either treatment alone (Figure 7D). Furthermore, agal treatment effectively reduced basal respiration (I), ATP production (II), maximal respiration (III), and spare capacity (IV) in FD-VEC (Figure 7E). Surprisingly, the combined treatment of fasudil and agal effectively reduced maximal respiration (maximal oxygen consumption rate [OCR]) in FD-VEC compared with either treatment alone (Figure 7E). Thus, fasudil is a potential compound that can act as a combination drug to improve the therapeutic effect of agalsidase-β in FD-VEC.

[0221]

[0222] Example 8. Effect of combined treatment with ERT (Enzyme Replacement Therapy) and fasudil

[0223]

[0224] To treat Fabry disease, patients receive intravenous injections of a recombinant enzyme (beta-galactosidase) every two weeks. We investigated whether fasudil, developed in this study, would also have a therapeutic effect when administered together with enzyme therapy.

[0225]

[0226] 8-1. Synergistic effect of combined treatment on FD vascular endothelial cell function

[0227] To investigate whether tube formation occurs in vascular endothelial cells derived from dedifferentiated stem cells of Fabry disease, the cells were treated with the enzyme at 2-day intervals, and finally with fasudil.

[0228] The results are shown in Figure 8. The combined administration of the enzyme and fasudil showed significantly higher tube formation ability than the enzyme alone. This indicates that fasudil has a greater effect on enhancing the functionality of vascular endothelial cells in Fabry disease than the enzyme.

[0229]

[0230] 8-2. Synergistic effect of combined treatment on metabolic function enhancement in FD vascular endothelial cells

[0231]

[0232] The combined treatment was performed in the same manner as in Example 8-1, and the results are shown in Figure 9. The combined treatment further increased the expression of angiogenesis-related factors (KDR, eNOS) and further reduced TSP1 expression and TFG-beta activity (Figure 9A). The combined treatment also reduced ROS production (Figure 9B) and further effectively reduced maximum OCR(III) (Figure 9C).

[0233]

[0234] 8-3. Combination administration in Gla- / - model mice

[0235] Based on the results of in vitro experiments, Fabry disease mice lacking the α-galactosidase gene (Gla- / -) were orally administered fasudil at a dose of 30 mg / kg daily, and the enzyme (1 mg / kg) was administered intravenously into the tail of the mice once a week for 6 months (Figure 10).

[0236]

[0237] (1) Cardiac ultrasound analysis of Fabry disease mice after co-administration

[0238]

[0239] After the combined administration, the cardiac ultrasound analysis results of the Fabry disease mice are shown in FIG.

[0240] While the experimental group administered the enzyme alone (dark blue) showed a decrease in cardiac function similar to the Fabry disease group (light blue), the fasudil monotherapy and combination therapy groups showed an improvement in cardiac function (Figure 11A), and the left ventricular hypertrophy, a typical phenotype of Fabry disease, was significantly reduced (Figure 11B). These results indirectly suggest that enzyme therapy is currently unable to restore cardiac function and alleviate the symptoms of left ventricular hypertrophy.

[0241]

[0242] (2) Sweating analysis of Fabry disease mice after co-administration

[0243]

[0244] Figure 12 shows the results of sweating analysis of Fabry mice after combined administration. The group treated with enzyme alone (dark blue) maintained the same sweating symptoms as the Fabry mice (light blue), while the group treated with fasudil alone (light purple) and the combined administration group (dark purple) showed improvement in sweating symptoms. This indicates that enzyme therapy is not effective in improving sweating symptoms.

[0245]

[0246] (3) Peripheral nerve analysis of Fabry disease mice after co-administration

[0247]

[0248] Many patients with Fabry disease suffer from peripheral neuralgia, and one way to indirectly observe this in experimental animals is to measure the time it takes for the patient to remove their hind paws from a 55°C hot plate. The results are shown in Figure 13.

[0249] Normal mice (gray) were able to remove their hind paws from the hot plate within 20 seconds, while Fabry disease mice (light blue) were able to withstand the heat for more than 30 seconds. The group treated with enzyme alone (dark blue) showed a high latency similar to that of Fabry disease mice, while the group treated with fasudil alone and the combined treatment group showed some reduction in latency.

[0250] These results indirectly suggest that enzyme therapy is also ineffective in treating peripheral neuralgia in patients with Fabry disease, and that fasudil may be useful in alleviating peripheral neuralgia.

[0251]

[0252] (4) Kidney tissue analysis of Fabry disease mice after co-administration

[0253]

[0254] After the combined administration, the kidney tissue analysis results of the Fabry disease mice are shown in FIG.

[0255] We also confirmed that the expression of fibrotic markers (COL1A1) and inflammatory markers (LCN2, F4 / 80) was reduced in the kidney tissue of mice with Fabry disease when fasudil was administered alone or in combination with other drugs. This suggests that renal failure can be prevented by suppressing the accelerated fibrosis that causes renal failure in patients with Fabry disease.

[0256]

[0257] GLA deficiency causes the continuous accumulation of the intermediate metabolite Gb3 in various tissue cells of patients with Fabry disease, and enzyme therapy is necessary to remove this. However, enzyme therapy cannot be a fundamental treatment for the symptoms of Fabry disease. Therefore, it is expected that a new treatment method using combined enzyme administration will enable a fundamental treatment for patients with Fabry disease.

[0258]

[0259] Example 9. Lomerizine improves defective angiogenesis in FD-VEC.

[0260]

[0261] It has been reported that FD-VECs have impaired vascular endothelial cell function. Using a cell-based screening platform using FD-VECs, we identified compounds that affect angiogenesis. Lomerizine was identified in the FDA-approved clinical compound library provided by the Korea Chemical Bank (www.chembank.org) (Figure 15A). To investigate the extent to which lomerizine restores the tube-forming ability of FD-VECs, we monitored the tube-forming ability at various concentrations and measured the EC50. We concluded that 1 μM was the optimal concentration for maximum effect (Figure 15B). In a tube-forming assay, lomerizine treatment significantly increased the total tube length of FD-VECs (Figure 15C). We also found that lomerizine treatment improved the tube-forming ability of FD-VECs carrying distinct GLA mutations (Figure 15D). Furthermore, lomerizine treatment reduced the expression of p-SMAD2 and TSP1 in FD-VECs and increased the expression of KDR and eNOS (Figure 15E). Therefore, lomerizine is considered to be an effective therapeutic agent for restoring the functionality of FD-VEC in vitro.

[0262]

[0263] Example 10. Lomerizine ameliorates mitochondrial dysfunction in FD-VEC.

[0264]

[0265] We explored how lomerizine improves the angiogenic capacity of FD-VEC. Gb3 accumulation has been reported to cause mitochondrial dysfunction, including dysregulation of ROS production (Stepien et al., 2020; Tseng et al., 2017). An imbalance between ROS generation and the antioxidant defense system can lead to endothelial dysfunction. Lomerizine treatment reduced the overproduction of ROS in FD-VEC by the same amount as in WT- and FD(c)-VEC (Figure 16A). We found that FD-VEC exhibited increased basal respiration (I), ATP production (II), maximal respiration (III), and spare capacity (IV) compared with WT- and FD(c)-VEC (Figure 16B). Furthermore, we found that FD-VEC exhibited increased basal respiration (I), ATP production (II), maximal respiration (III), and spare capacity (IV) when compared with WT- and FD(c)-VEC (Figure 16B). Furthermore, in this data, we observed that when FD-VEC was treated with lomerizine, the maximum respiration rate was slightly decreased.The above results indicate that lomerizine can effectively regulate ROS production in FD-VEC.

[0266]

[0267] Example 11. Lomerizine down-regulates EndMT and improves FD-VEC function.

[0268]

[0269] Next, we investigated how lomerizine overcomes the dysfunction of FD-VEC vascular endothelial cells through ROS scavenging. ROS are known to induce EndMT (Piera-Velazquez & Jimenez, 2019). We found that treatment of FD-VEC with lomerizine reduced the expression of mesenchymal cell-related genes COL1A1, ACTA2, SNAI1, and TWIST compared to WT-VEC or FD(c)-VEC (Figure 17A). Furthermore, immunohistochemistry revealed that treatment of FD-VEC with lomerizine reduced the expression of EndMT-related genes (Figure 17B). These results indicate that lomerizine inhibits EndMT in FD-VEC and improves the functionality of damaged vascular endothelial cells.

[0270]

[0271] Example 12. Oral administration of lomerizine reverses the FD phenotype in FD mice.

[0272]

[0273] Existing FD animal models cannot fully recapitulate the vascular disease observed in human FD patients (Miller et al., 2019; Ohshima et al., 1999; Taguchi et al., 2013). Previous studies have demonstrated that Gla- / - / TSP1Tg mice effectively recapitulate FD-associated vascular disease. To this end, we crossed Gla- / - mice with transgenic mice expressing human TSP1 to generate FD-mice (Gla- / - / TSP1Tg). These mice were then orally administered lomerizine (10 or 30 mg / kg / day) for 6 months (Figure 18A). Cardiac ultrasound revealed that FD mice had a higher left ventricular (LV)-to-body weight (BW) ratio than WT mice, and lomerizine administration reduced this LV / BW ratio (Figure 18B). Furthermore, FD-mice exhibited reduced cardiac function (i.e., cardiac ejection fraction, fractional shortening, and cardiac output) compared with WT mice, but long-term lomerizine administration significantly improved cardiac function. Therefore, these results indicate that lomerizine administration can improve the impaired cardiac function of FD mice. Furthermore, the number of sweat spots and heat sensitivity were reduced in FD-mice compared with WT mice (Figures 18C and 18D), and lomerizine administration reversed this phenotype. Overall, these results indicate that oral administration of lomerizine alleviates various FD-like symptoms in FD-mice.

[0274]

[0275] Example 13. Oral administration of lomerizine attenuates fibrosis and inflammation in renal tissue of FD mice.

[0276]

[0277] Renal fibrosis is another major symptom of FD (Weidemann et al., 2013). Fibrosis and inflammation generally occur when VECs in various tissues (including the kidney) undergo EndMT (Ma et al., 2020). Therefore, we investigated how oral administration of lomerizine affects tissue fibrosis and inflammation in the kidney tissue of FD mice. To this end, we analyzed the expression of EndMT-related genes in Fabry disease mice (Figure 19A). We found significantly more CD31+ / ACTA2+ cells in the kidney tissue of FD mice than in WT mice, and oral administration of lomerizine reduced this increase (Figure 19B).

[0278] Furthermore, FD-mice exhibit EndMT in renal microvessels. Oral administration of lomerizine to FD-mice downregulated the renal expression of the EndMT marker COL1A1 (Figure 19C) and reduced the increased expression of the inflammation-related proteins F4 / 80 and LCN (Figure 19D). Similarly, Western blot analysis of renal tissues from FD-mice and lomerizine-treated FD-mice also demonstrated a reduction in the protein levels of fibrotic and inflammatory markers (Figure 19E). Finally, these results suggest that lomerizine can effectively suppress the progression of renal fibrosis and inflammation in FD-mice and suggest that its use in human FD patients should be considered.

[0279]

[0280] Example 14. Combination therapy with lomerizine and recombinant human α-galactosidase demonstrates effective therapeutic effects.

[0281]

[0282] Previous studies have reported that recombinant human α-galactosidase (agalsidase-β, agal) treatment did not significantly enhance impaired tube formation in FD-VECs (Do et al., 2020). Therefore, we analyzed whether the combined administration of lomerizine and agal could enhance the therapeutic effect of agal. To this end, FD-VECs were treated with agal and lomerizine. FD-VECs were treated with agal every two days, followed by a single lomerizine treatment, and then their tube formation ability was analyzed (Figure 20A). Surprisingly, the combined treatment restored FD-VEC tube formation functionality more effectively than lomerizine treatment alone (Figure 20B). Furthermore, the combined treatment efficiently reduced the levels of TSP1 and p-SMAD2 and increased the expression of angiogenic factors (KDR and eNOS) in FD-VECs compared to either treatment alone (Figure 20C). The combined treatment reduced ROS generation in FD-VEC more efficiently than either treatment (Figure 20D). Furthermore, agal treatment was effective in reducing basal respiration (I), ATP production (II), maximal respiration (III), and spare capacity (IV) in FD-VEC (Figure 20E). Surprisingly, combined treatment with lomerizine and agal effectively reduced maximal respiration (maximal oxygen consumption rate [OCR]) in FD-VEC compared with either treatment alone (Figure 20E). Therefore, lomerizine is a potential compound that can act as a combination drug to improve the therapeutic effect of agalsidase-β in FD-VEC.

[0283]

[0284] Example 15. ROCK inhibitors

[0285] [Types of ROCK inhibitors used in the experiment]

[0286] First, we performed a tube formation experiment to confirm whether the reduced tube formation was restored when vascular endothelial cells differentiated from Fabry disease dedifferentiated stem cells were treated with four ROCK inhibitors (Y27632, GSK429286A, Y39983, and belmosudil) at a concentration of 5 μM (Table 1).

[0287] [Table 1]

[0288] Secondary: Tube formation experiments were performed to confirm whether the reduced tube formation was restored by seven ROCK inhibitors (netarsudil, ripasudil, sobesudil, AT13148, AR13503, VX-210, and latanoprost) in vascular endothelial cells differentiated from a dedifferentiated stem cell line derived from Fabry disease (Table 2).

[0289] [Table 2]

[0290]

[0291] Example 16. Efficacy evaluation

[0292]

[0293] After coating Matrigel on a 96-well plate, the plate was stored in a 5% CO2 incubator at 37°C for 1 day. The next day, ROCK inhibitors were added to each well at the appropriate concentrations (5 μM, 1 μM, 0.5 μM, 0.1 μM, 0.05 μM, 0.01 μM) along with 100 μg / ml VEGF in EGM-2 medium.

[0294] 10,000 endothelial cells were plated onto solidified Matrigel per well, and after one day, each well was photographed under a microscope, and the total length of the tubes was analyzed using Image J to compare the increase or decrease in tube formation.

[0295] As shown in Figures 21 to 25, the results of the first experiment showed that when Fabry disease vascular endothelial cells were treated with Y27632, GSK429286A, Y39983, and belmosudil at a concentration of 5 μM, each compound was observed to have the effect of improving the impaired tube formation ability of Fabry disease vascular endothelial cells.

[0296] In the results of the second additional experiment, as shown in Figures 26 to 32, it was confirmed that Fabry disease-derived vascular endothelial cells had reduced tube formation ability compared to normal vascular endothelial cells, and that tube formation ability was increased in the Fabry disease-derived vascular endothelial cells treated with SB431542 (SMAD2 signaling inhibitor) as a control.In addition, it was confirmed that netarsudil, ripasudil, sobesudil, AT13148, AR13503, and VX-210, all of which were used in the second experiment, increased the tube formation ability of Fabry disease vascular endothelial cells at a concentration of 5 μM.

[0297]

[0298] Although the present invention has been described in detail above, it will be apparent to those skilled in the art that the specific details are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for preventing or treating Fabry's disease (FD), comprising, as an active ingredient, a preparation selected from the group consisting of TGF-β (Transforming Growth Factor-β) receptor activity inhibitors, PPARγ (peroxisome proliferator-activated receptor) antagonists, calcium channel blockers, COX (cyclooxygenase) inhibitors, piperazine compounds, Rho kinase inhibitors, and ROCK (Rho-associated protein kinase) inhibitors.

2. The pharmaceutical composition according to claim 1, wherein the TGF-β receptor activity inhibitor comprises SB431542, D-4476, or a pharmaceutically acceptable salt thereof having the following structure: 【Chemistry 1】

3. 2. The pharmaceutical composition of claim 1, wherein the PPARγ antagonist comprises T0070907 having the following structure or a pharmaceutically acceptable salt thereof: 【Chemistry 2】

4. 2. The pharmaceutical composition of claim 1, wherein the calcium channel blocker comprises lomerizine having the following structure or a pharmaceutically acceptable salt thereof: 【Transformation 3】

5. 2. The pharmaceutical composition of claim 1, wherein the COX inhibitor is tolfenamic acid or a pharmaceutically acceptable salt thereof, comprising the following structure: 【Chemistry 4】

6. 2. The pharmaceutical composition according to claim 1, wherein the piperazine compound is eprazinone or a pharmaceutically acceptable salt thereof. 【Transformation 5】

7. 2. The pharmaceutical composition of claim 1, wherein the Rho kinase inhibitor is fasudil having the following structure or a pharmaceutically acceptable salt thereof: 【Transformation 6】

8. 2. The pharmaceutical composition according to claim 1, wherein the ROCK inhibitor comprises one or more selected from the group consisting of Y27632, GSK429286A, Y39983, Belmosudil, Netarsudil mesylate, Ripasudil hydrochloride hydrate, AR-13503 (Main Netarsudil Metabolite), AMA-0076 (Sovesudil), AT-13148, and VX-210, or a pharmaceutically acceptable salt thereof.

9. 10. The pharmaceutical composition of claim 1, which exhibits the following characteristics: reduction of p-SMAD2 or TSP1 protein; Increased levels of the angiogenic factors KDR or eNOS; Decreased reactive oxidative stress (ROS) and maximal respiration; amelioration of a Fabry disease phenotype selected from the group consisting of left ventricular hypertrophy (LVH), renal fibrosis, anhydrosis, and heat intolerance; or Improved tube-forming ability of damaged vascular endothelial cells.

10. A pharmaceutical composition for the prevention or treatment of Fabry's disease (FD), which comprises, as an active ingredient, a preparation selected from the group consisting of TGF-β (Transforming Growth Factor-β) receptor activity inhibitors, PPARγ (peroxisome proliferator-activated receptor) antagonists, calcium channel blockers, COX (cyclooxygenase) inhibitors, piperazine compounds, Rho kinase inhibitors, and ROCK (Rho-associated protein kinase) inhibitors, and which is to be used in combination with agalsidase-β (agal).

11. The pharmaceutical composition according to claim 10, wherein the TGF-β receptor activity inhibitor comprises SB431542, D-4476, or a pharmaceutically acceptable salt thereof having the following structure: 【Transformation 7】

12. 11. The pharmaceutical composition of claim 10, wherein the PPARγ antagonist comprises T0070907, or a pharmaceutically acceptable salt thereof, comprising the following structure: 【Transformation 8】

13. 11. The pharmaceutical composition of claim 10, wherein the calcium channel blocker comprises lomerizine having the following structure or a pharmaceutically acceptable salt thereof: 【Chemistry 9】

14. 11. The pharmaceutical composition of claim 10, wherein the COX inhibitor is tolfenamic acid or a pharmaceutically acceptable salt thereof, comprising the following structure: 【Chemistry 10】

15. 11. The pharmaceutical composition according to claim 10, wherein the piperazine compound is eprazinone or a pharmaceutically acceptable salt thereof. 【Chemistry 11】

16. 11. The pharmaceutical composition of claim 10, wherein the Rho kinase inhibitor is fasudil having the following structure or a pharmaceutically acceptable salt thereof: 【Chemistry 12】

17. The pharmaceutical composition according to claim 10, wherein the ROCK inhibitor comprises one or more selected from the group consisting of Y27632, GSK429286A, Y39983, Belmosudil, Netarsudil mesylate, Ripasudil hydrochloride hydrate, AR-13503 (Main Netarsudil Metabolite), AMA-0076 (Sovesudil), AT-13148, and VX-210, or a pharmaceutically acceptable salt thereof.

18. 11. The pharmaceutical composition according to claim 10, wherein the preparation selected from the group consisting of a TGF-β (Transforming Growth Factor-β) receptor activity inhibitor, a PPARγ (peroxisome proliferator-activated receptor) antagonist, a calcium channel blocker, a COX (cyclooxygenase) inhibitor, a piperazine compound, a Rho kinase inhibitor, and a ROCK (Rho-associated protein kinase) inhibitor, and agalsidase-beta are contained in a combined dosage form.

19. 11. The pharmaceutical composition according to claim 10, wherein the preparation selected from the group consisting of a TGF-β (Transforming Growth Factor-β) receptor activity inhibitor, a PPARγ (peroxisome proliferator-activated receptor) antagonist, a calcium channel blocker, a COX (cyclooxygenase) inhibitor, a piperazine compound, a Rho kinase inhibitor, and a ROCK (Rho-associated protein kinase) inhibitor, and agalsidase-beta are administered simultaneously or sequentially.

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