Pharmaceutical composition for treating or preventing calcium release-activated calcium channel or discoid domain receptor 2 related diseases or conditions

Abstract

A pharmaceutical composition for use in the treatment or prevention of a disease or condition associated with a calcium release activated calcium (CRAC) channel or a disease or condition associated with discoid domain receptor 2 (DDR2) in an individual in need thereof. A pharmaceutical composition for inhibiting CRAC channel activity and / or DDR2 activity in cells of an individual. The pharmaceutical composition comprises an effective amount of at least one selected from the group consisting of WRG-28, atorvaquone (Av), a WRG-28 precursor, and an atorvaquone (Av) precursor, and a pharmaceutically acceptable carrier thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 480,626, filed on January 19, 2023, the contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to calcium release-activated calcium (CRAC) channels or discoidin domain receptor 2 (DDR2), and more particularly to methods for preventing or treating CRAC channel-related diseases or conditions and / or DDR2-related diseases or conditions. Existing technology

[0004] Calcium release-activated calcium (CRAC) channels regulate Ca 2+ The influx of calcium ions is called store-operated Ca 2+ It inhibits transforming growth factor (TGF)-β1-induced epithelial-to-mesenchymal transition and fibroblast activation, thereby inhibiting fibrosis. Activation of the CRAC pathway and discoidin domain receptor 2 (DDR2) is crucial for myofibroblast activation and organ fibrosis. Furthermore, as a downstream molecule of the CRAC pathway (coordinating collagen deposition), DDR2 is also involved in the mechanisms of cancer metastasis and organ fibrosis.

[0005] Fibrotic diseases are a global problem, particularly pronounced in an aging society. Pulmonary fibrosis induced by infectious diseases, liver cirrhosis, nephritis, chronic renal fibrosis induced by diabetes or hypertension, and cardiac fibrosis caused by uremic cardiomyopathy are leading causes of death and represent a significant burden on public health expenditures. Myofibroblasts play a key role in organ fibrosis. Resident fibroblasts and pericytes constitute the primary source of myofibroblasts, although other sources, such as macrophages, bone marrow-derived cells, and endothelial cells, have been suggested to contribute to myofibroblast numbers during disease progression; however, their roles remain under debate. Myofibroblasts are known for their robust ability to generate and remodel the extracellular matrix (ECM), and α-smooth muscle actin (α-SMA) is not only a marker of myofibroblasts but also a diagnostic tool for fibrotic diseases.

[0006] Hypercytokinemia (also known as cytokine storm) is an uncontrolled, excessive inflammatory response caused by a severe immune response that spreads from a local inflammatory response to a viral or bacterial infection. Immune overactivation in hypercytokinemia can occur for a variety of reasons: inappropriate triggering or danger sensing, initiation of a response in the absence of a pathogen (e.g., in genetic diseases involving inappropriate inflammasome activation); inappropriate or ineffective response amplitude, leading to excessive activation of immune cells (e.g., CAR T-cell therapy); uncontrolled infection and prolonged immune activation (e.g., Epstein-Barr virus, MERS-CoV, or SARS-CoV-2 infection); or failure to resolve the immune response and restore homeostasis (e.g., primary hemophagocytic lymphohistiocytosis).

[0007] Cytokine storms are thought to be a major cause of the high mortality associated with COVID-19. Treatment with anti-inflammatory drugs such as corticosteroids (e.g., dexamethasone) or drugs that target cytokine function (e.g., tocilizumab, an anti-interleukin-6 receptor antibody) has been shown to significantly reduce mortality in patients with COVID-19. However, despite its widespread use, two large randomized trials investigating tocilizumab failed to demonstrate a survival benefit in hospitalized patients with COVID-19.

[0008] To date, there are no optimal therapeutic products specifically targeting CRAC channel-related diseases, DDR2-related diseases, or both conditions. Therefore, there is an unmet need for the development of effective pharmaceutical agents that are safe and tolerable for the treatment or prevention of these conditions. Summary of the Invention

[0009] A method for treating or preventing a CRAC channel-associated disease or condition and / or a DDR2-associated disease or condition in a subject in need thereof is provided. The method comprises administering to the subject an effective amount / dose of WRG-28 and / or atovaquone (Av), or a WRG-28 precursor and / or atovaquone (Av) precursor and a pharmaceutically acceptable carrier thereof.

[0010] The present disclosure also provides a use of a pharmaceutical composition of the present disclosure for treating or preventing a CRAC channel-related disease or condition and / or a DDR2-related disease or condition, comprising administering an effective amount of the pharmaceutical composition of the present disclosure to an individual in need thereof. The present disclosure further provides a pharmaceutical composition for treating or preventing a CRAC channel-related disease or condition and / or a DDR2-related disease or condition. A pharmaceutical composition is further provided for the preparation of a medicament for treating or preventing a CRAC channel-related disease or condition and / or a DDR2-related disease or condition.

[0011] In some aspects, the present disclosure provides a method for inhibiting CARC channel and / or DDR2 activation in cells of an individual, comprising administering an effective amount / dose of WRG-28 and / or Av, or a WRG-28 precursor and / or Av precursor and a pharmaceutically acceptable carrier thereof.

[0012] Also provided is a pharmaceutical composition of the present disclosure for use in inhibiting CRAC channel and / or DDR2 activation in cells of an individual, comprising administering an effective amount / dose of WRG-28 and / or Av, or a WRG-28 precursor and / or an Av precursor and a pharmaceutically acceptable carrier thereof. The present disclosure further provides a pharmaceutical composition for inhibiting CRAC channel and / or DDR2 activation in cells of an individual. Also provided is the use of a pharmaceutical composition in the preparation of a medicament for inhibiting CRAC channel and / or DDR2 activation in cells of an individual.

[0013] In at least one embodiment, the effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor, brings better efficacy in inhibiting TGF-β1-induced fibroblast activation, pericyte-to-myofibroblast differentiation and / or TGF-β1-induced ECM remodeling.

[0014] In at least one embodiment, the effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor exhibits an overall effect of attenuating TGF-β1-induced myofibroblast activation.

[0015] In at least one embodiment, the effective amount / dose of WRG-28 and / or Av, or a WRG-28 precursor and / or an Av precursor in a SOCE exhibits various abilities to modulate immune responses to varying degrees.

[0016] In at least one embodiment, the effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor in SOCE significantly reduces the area positively stained for fibrillar collagen.

[0017] In at least one embodiment, the effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor, shows the efficacy of inhibiting tubulointerstitial fibrosis and has a protective effect on UUO-induced tubular atrophy and cell apoptosis.

[0018] In some embodiments, the effective amount / dose of WRG-28 and / or Av, or a WRG-28 precursor and / or an Av precursor, results in reduced expansion of fibrotic areas and increased expression of epithelial markers in mice.

[0019] In some embodiments, the effective amount / dose of WRG-28 and / or Av, or a WRG-28 precursor and / or an Av precursor reverses fibrosis and promotes tissue repair.

[0020] In some embodiments of the present disclosure, the effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor, reduces the activation of myofibroblasts and the subsequent reorganization of ECMs during renal fibrosis.

[0021] In some embodiments of the present disclosure, the effective amount / dose of WRG-28 and / or Av, or a WRG-28 precursor and / or an Av precursor, increases tubular differentiation during renal fibrosis.

[0022] In at least one embodiment, the effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor, shows profound efficacy in alleviating maladaptive repair, assisting tubular regeneration, and treating and protecting renal function.

[0023] In at least one embodiment, the effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor has better efficacy in treating, preventing and protecting the kidney from progressive fibrosis.

[0024] In some embodiments, the effective amount / dose of WRG-28 and / or Av, or a WRG-28 precursor and / or an Av precursor, exhibits profound efficacy in reducing pulmonary fibrosis.

[0025] These and other objects of the present invention will no doubt become tangibly implementable to those skilled in the art after reading the following detailed description of the preferred embodiments illustrated in the figures and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0027] Figure 1A 、 Figure 1B 、 Figure 1C 、 Figure 1D 、 Figure 1E 、 Figure 1F 、 Figure 1G 、 Figure 1H 、 Figure 1I 、 Figure 1J 、 Figure 1K 、 Figure 1L 、 Figure 1M and Figure 1N WRG-28 and atovaquone (Av) were shown to inhibit activation of DDR2 and calcium release-activated calcium (CRAC) channels. Figure 1A HEK293T cells transiently overexpressed with DDR2 were then pretreated for 30 minutes with various compounds (10 μM), including WRG-28, Av, donepezil hydrochloride (Dh), terazosin hydrochloride (Th), verapamil, and flecainide. Verapamil and flecainide were used as negative controls in this disclosure. Cells were then treated with 50 μg / mL type 1 collagen for 12 hours. Cell lysates were obtained and used for Western blotting with antibodies against anti-phosphotyrosine antibody (clone 4G10), DDR2, and β-actin. Figure 1B CRAC channel activation assay was performed in HEK293T cells pretreated with 10 μM BTP2, WRG-28, or Av and treated with Fura 2-AM. 1 μM thapsigargin (Thap.) was used in a calcium-free solution to deplete endoplasmic reticulum calcium stores, followed by reintroduction of 2 mM Ca in the external solution. 2+ To detect CRAC channel activation. As calcium flows through the CRAC channel, the concentration of calcium ions in the cytoplasm increases accordingly, causing a second peak in the cytoplasmic calcium wave. Figure 1C and Figure 1D Shown is a further analysis in which the second peak of each wave and the entry rate are analyzed. Each bar represents the mean value obtained from 47 to 103 cells. Figure 1E Shown is the detection of CRAC channel activation in NRK49F cells treated with 10 μM BTP2, WRG-28, or Av, while the cells were treated with Fura 2-AM. Figure 1F and Figure 1G Shown is a further analysis in which the second peak of each wave and the entry rate are analyzed. Each bar represents the mean value obtained from 83 to 176 cells. Figure 1H Shown are the effects of BTP2, WRG-28, or Av on CRAC channel activity in HK-2 cells. Figure 1I and Figure 1J Further analysis is shown, in which the second peak of each wave and the entry rate are analyzed. Each bar represents the average value obtained from 72 to 108 cells. Orai1 and Stim1 spots were detected in HEK293T cells overexpressing CFP-Orai1 / Stim1-mCherry. Figure 1K )、BTP2( Figure 1L )、WRG-28( Figure 1M ) or Av( Figure 1N ) for 30 minutes and then stimulated with 1 μM thapsigargin for 15 minutes. The cells were then fixed and the spots were observed under a confocal microscope (Olympus, FV-1000). In all figures, each bar represents the mean ± SEM, and *, **, and *** indicate p values ​​< 0.05, 0.01, and 0.001, respectively.

[0028] Figure 2A 、 Figure 2B 、 Figure 2C 、 Figure 2D 、 Figure 2E 、 Figure 2F 、 Figure 2G-1 and Figure 2G-2 We show how WRG-28 and Av inhibit TGF-β1-induced fibroblast activation and myofibroblast differentiation of coat cells. Figure 2A Figure 3 shows TGF-β1-induced renal fibroblast activation detected by upregulation of collagen 1a1 and α-SMA in NRK49F cells treated with 10 μM DMSO, BTP2, WRG-28, or Av for 24 h. Figure 2B The relative protein levels of collagen 1a1 and α-SMA were detected by three independent experiments. Each bar represents the mean ± SEM. Figure 2C Figure 3: TGF-β1-induced myofibroblast differentiation of coat cells in CCL-226 cells treated with 10 μM DMSO, BTP2, WRG-28, or Av for 24 hours. Western blot analysis was used to measure the protein levels of collagen 1a1 and α-SMA under different conditions. Figure 2D NRK49F cells were cultured in collagen gels and co-treated with 10 ng / mL TGF-β1 and / or 10 μM DMSO, BTP2, WRG-28, or Av for 3 days. Figure 2E Gels removed from tissue culture plates are shown, and relative gel areas were measured 8 hours after removal. Each bar represents the mean ± SEM. Figure 2F NRK49F cells cultured in FITC-conjugated collagen gels were co-treated with 10 ng / mL TGF-β1 and / or 10 μM DMSO, BTP2, WRG-28, or Av for 5 days to detect TGF-β1-induced collagen alignment. Images were acquired using a confocal microscope (Olympus, MPE). Figure 2G-1 and Figure 2G-2 The angle between collagen fibers and the cell membrane was measured using Image J. At least five images were analyzed for each condition. Each bar represents the mean ± SEM. In all figures, *, **, and *** indicate p values ​​< 0.05, 0.01, and 0.001, respectively. "ns" indicates a p value > 0.05, indicating no significant difference.

[0029] Figure 3A 、 Figure 3B 、 Figure 3C 、 Figure 3D 、 Figure 3E 、 Figure 3F and Figure 3G WRG-28 and Av inhibited TGF-β1-induced activation of cardiac fibroblasts, lung fibroblasts, and hepatic stellate cells. Figure 3A 、 Figure 3B and Figure 3C As shown, the effects of BTP2, WRG-28, or Av on TGF-β1-induced activation of human cardiac fibroblasts (HCF), human lung fibroblasts (MRC5), and rat hepatic stellate cells (HSC-T6) were examined. Cells were co-treated with various compounds (10 μM) and TGF-β1 for 24 hours. Subsequently, cell lysates were obtained and protein expression was analyzed by Western blot. Figure 3D Immunofluorescence staining was performed to examine TGF-β1-induced fibronectin deposition and α-SMA expression in HCFs treated with 10 μM DMSO, BTP2, WRG-28, or Av. Images were captured using a confocal microscope (Olympus, FV-1000). Figure 3E 、 Figure 3F and Figure 3GFigure 2 shows the activation of CRAC channels in HCF, MRC5, and rat HSC-T6 cells. These cells were treated with 10 μM BTP2, WRG-28, or Av for 45 minutes. The evaluation included measuring the second peak of each calcium wave and the entry rate. Each bar in the figure represents the average of 77 to 91 cells from HCF, 31 to 49 cells from MRC5, and 43 to 94 cells from HSC-T6. In all figures, *, **, and *** indicate p values ​​< 0.05, 0.01, and 0.001, respectively.

[0030] Figure 4A 、 Figure 4B 、 Figure 4C 、 Figure 4D 、 Figure 4E 、 Figure 4F 、 Figure 4G and Figure 4H Figure 3: WRG-28 and Av reduce SOCE in T cells and macrophages, leading to downregulation of cytokine release. Figure 4A ) and macrophages ( Figure 4E ) were used to evaluate the effects of BTP2, WRG-28, Av, and CM-4620 on SOCE. Cells were cultured in 96-well glass-bottomed plates and pretreated with various compounds (10 μM) and Fura 2-AM was added. Subsequently, cytoplasmic calcium signal waves were recorded, and the second peak and entry rate of each wave were evaluated. Each bar represents 43 to 111 cells from T cells ( Figure 4A ) and 52 to 91 cells from macrophages ( Figure 4E ) were averaged. Cytokine induction in T cells and macrophages was performed as described in the Methods section. T cells ( Figure 4B 、 Figure 4C and Figure 4D ) and macrophages ( Figure 4F 、 Figure 4G and Figure 4H ) cytokine levels, e.g., IL-2, TNF-α, and IL-6. Each bar represents mean ± SEM. In all figures, *, **, and *** indicate p values ​​< 0.05, 0.01, and 0.001, respectively.

[0031] Figure 5A 、 Figure 5B 、 Figure 5C 、 Figure 5D 、 Figure 5E 、 Figure 5F 、 Figure 5G 、 Figure 5H 、 Figure 5I 、 Figure 5J and Figure 5KWe show how WRG-28 and Av reduce renal fibrosis induced by unilateral ureteral obstruction (UUO). The left ureter of 7- to 8-week-old male mice was surgically ligated using nylon surgical sutures. Subsequently, the mice were treated with 5 mg / kg / day of DMSO (control), BTP2, WRG-28, or Av for 7 days. Figure 5A Shown is the histological status of kidney tissues of treated mice examined using hematoxylin and eosin (H&E) staining. Figure 5B The expression of fibrillar collagen was assessed by Sirius red staining. Figure 5C The Sirius Red-positive areas measured using Image J are shown. Figure 5D 、 Figure 5E 、 Figure 5F 、 Figure 5G and Figure 5H It was shown that after 5 mg / kg / day of BTP2 ( Figure 5D )、WRG-28( Figure 5F ) or Av( Figure 5H The protein levels of integrin β1, DDR2, collagen 1a1, α-SMA, and E-adhesin were assessed by Western blot in kidney samples of mice treated with BTP2 ( Figure 5E )、WRG-28( Figure 5G ) or Av( Figure 5I )-treated mice. Figure 5J Shown are representative pictures of apoptotic cells assessed by TUNEL assay. Figure 5K Apoptotic cells were evaluated in 5 to 10 images per condition.

[0032] Figure 6A 、 Figure 6B 、 Figure 6C 、 Figure 6D 、 Figure 6E 、 Figure 6F and Figure 6G We show that treatment with WRG-28 and Av during occlusion promotes tissue repair and reduces fibrosis after occlusion is relieved. Figure 6AFigure 3 shows a diagram illustrating the timeline of the operation and drug delivery. The left ureter was surgically ligated (UUO-L) for 7 days, and then the left ureter was reconnected to the bladder (RUUO-L). Various compounds, including DMSO (control group), WRG-28 and Av, were injected intraperitoneally every day during ureteral ligation (UUO-L). An additional 13 days after RUUO-L, the right ureter was ligated (UUO-R). Subsequently, the mice were placed in metabolic cages every other day after surgery (day 21). On day 22, the left kidney was removed for further analysis to assess renal function. Figure 6B Shown are paraffin-embedded tissue sections stained with hematoxylin and eosin (H&E) and Sirius red. Figure 6C The Sirius Red positive area was measured using Image J for 5 to 10 images of each group. Figure 6D ) and Av( Figure 6F )-treated mice, the protein levels of integrin β1, collagen 1a1, DDR2, α-SMA, E-adhesin, SGLT2, NHE1, and β-actin were assessed by Western blot. Figure 6E and Figure 6G ) Quantitative relative protein levels. Each bar represents the mean ± SEM. In all figures, *, **, and *** indicate p values ​​< 0.05, 0.01, and 0.001, respectively.

[0033] Figure 7A 、 Figure 7B 、 Figure 7C 、 Figure 7D 、 Figure 7E 、 Figure 7F and Figure 7G We show how WRG-28 and Av treatment reduce the continued progression of obstruction-induced renal fibrosis after reversal of obstruction. Figure 7A Figure 3 shows a diagram illustrating the timeline of the operation and drug delivery. The left ureter was surgically ligated (UUO-L) for 7 days, and then the left ureter was reconnected to the bladder (RUUO-L). After the ligated left ureter was released (during RUUO-L), various compounds, including DMSO (control group), WRG-28 and Av, were injected into the peritoneal cavity for 13 days. After 13 days of RUUO-L, the right ureter was ligated (UUO-R). Subsequently, the mice were placed in metabolic cages every other day (day 21) after surgery. On day 22, the left kidney was removed for further analysis. Figure 7B Shown are paraffin-embedded tissue sections stained with hematoxylin and eosin (H&E) and Sirius red. Figure 7C The Sirius Red positive area was measured using Image J for 5 to 10 images of each group. Figure 7D ) and Av( Figure 7F)-treated mice, the protein levels of integrin β1, collagen 1a1, DDR2, α-SMA, E-adhesin, SGLT2, NHE1, and β-actin were assessed by Western blot. The relative protein levels were quantified using Image J ( Figure 7E and Figure 7G Each bar represents the mean ± SEM. In all figures, *, **, and *** indicate p values ​​< 0.05, 0.01, and 0.001, respectively. NS indicates p values ​​> 0.05, indicating no significant difference between the two groups.

[0034] Figure 8A 、 Figure 8B 、 Figure 8C 、 Figure 8D 、 Figure 8E 、 Figure 8F 、 Figure 8G and Figure 8H Shown is how WRG-28 and Av restore epithelial differentiation and reduce collagen deposition and myofibroblast expansion in the progression of RUUO-induced renal fibrosis. Frozen tissue blocks were used for immunofluorescence staining. Figure 8A Shown are the expressions of laminin α1 and collagen 1a1 in the kidneys of mice treated with DMSO, WRG-28, or Av during ureteral ligation (7* / 15 days) or after ureteral reocclusion (7 / 15* days). Laminin α1 and collagen 1a1 were detected using primary antibodies followed by secondary antibodies conjugated to Alexa-488 (green) and Alexa-594 (red), respectively. Hoechst 33258 (blue) was used to label cell nuclei. Figure 8A -(ii) shows the fluorescence intensity of collagen 1a1 measured using Image J. Five images were analyzed with 5 to 10 areas per image, and each bar represents the mean ± SEM. Figure 8A -(i) The thickness of laminin α1 was measured in 5 to 10 images analyzed in 5 to 10 areas per image using Image J. The median is marked with a solid line, and the interquartile ranges are indicated with dashed lines. Figure 8B Cy3 (red)-labeled antibody is shown to label α-SMA, while Hoechst 33258 (blue) is used to label cell nuclei. Figure 8B (iii) shows the fluorescence intensity of α-SMA measured using Image J. Five images were quantified using at least five areas per image, and each bar represents the mean ± SEM. Figure 8CPrimary antibodies were used to detect laminin α1 and SGLT2, followed by secondary antibodies conjugated to Alexa-594 (red) and Alexa-647 (cyan), respectively. Hoechst 33258 (blue) was used to label cell nuclei. Figure 8C (iv) Quantification of SGLT2 apical membrane expression in SGLT2-positive cells under each condition. At least five images were analyzed for each condition, and each bar represents the mean ± SEM. Figure 8D DDR2 and AQP1 were detected using primary antibodies, followed by secondary antibodies conjugated to Alexa-488 (green) and Alexa-594 (red), respectively. Hoechst 33258 (blue) was used to label cell nuclei. DDR2 ( Figure 8D -(v)) and AQP1( Figure 8D -(vi)). Each bar in the figure represents the mean ± SEM. In addition, the percentage of cells showing membranes expressing AQP1 was analyzed and quantified ( Figure 8D -(vi)). Figure 8E Apoptotic cells were examined by TUNEL assay in mice treated with different compounds during the blockade period (7* / 15 days). In addition, Ki67-positive cells (representing proliferating cells) were detected by immunohistochemistry using specific antibodies in mice treated with various compounds during the blockade period (7* / 15 days). Figure 8F At least 10 images were displayed for each condition to quantify apoptotic and proliferative tubular cells. Each bar in the figure represents the mean ± SEM. Figure 8G Apoptotic cells were detected by TUNEL assay in mice treated with various compounds after occlusion (7 / 15*d). In addition, Ki67-positive cells (representing proliferating cells) were detected using specific antibodies in mice treated with different compounds after occlusion (7 / 15*d). Figure 8H Quantification of apoptotic and proliferating renal tubular cells is shown. Data were collected from at least 10 images per condition. Each bar represents the mean ± SEM. In all figures, *, **, and *** indicate p values ​​< 0.05, 0.01, and 0.001, respectively.

[0035] Figure 9A and Figure 9B The effects of WRG-28 and Av treatment on the improvement of renal function after obstructive renal injury were shown. Blood samples were collected from mice treated with DMSO, WRG-28, or Av during renal obstruction (7* / 15*d) or after renal obstruction was relieved (7 / 15*d). Blood urea nitrogen (BUN) levels were as follows: Figure 9AAs shown, and the creatinine (CRE) level is as Figure 9B Each bar represents the mean ± SEM. In all figures, *, **, and *** indicate p values ​​< 0.05, 0.01, and 0.001, respectively.

[0036] Figure 10A and Figure 10B Shows how WRG-28 and Av treatment effectively reduce RUUO-induced excessive fibrosis and inflammation. Figure 10A In the study, after the blockage was released (7 / 15*d), ingenuity pathway analysis (IPA) was performed on total RNA extracts from control mice (sham operation group) and mice treated with DMSO, WRG-28, or Av. The resulting genes are listed in Figure 10B . Figure 10A and Figure 10B Large-scale transcriptome analysis further confirmed the strong effects of WRG-28 and Av in preventing and protecting the kidneys from progressive fibrosis.

[0037] Figure 11A 、 Figure 11B 、 Figure 11C and Figure 11D We show how WRG-28 or Av treatment effectively reduces pulmonary fibrosis. Using a Bleomycin (BLM)-induced pulmonary fibrosis mouse model, Figure 11A Showing the timeline of the operation and drug delivery. Figure 11B The evaluation of lung function for various indicators is shown. Hydroxyproline content (as a marker of collagen deposition) was extracted from the right lobe and analyzed; the results are shown in Figure 2. Figure 11C shown. Figure 11D Shown are histological analyses from the left lobe, quantifying the fibrotic area by Masson's Trichrome and Sirius Red staining, respectively. DETAILED DESCRIPTION

[0038] The following examples are provided to illustrate the present disclosure in detail. After reading the disclosure of this specification, those skilled in the art can easily understand the advantages and effects of the present disclosure, and can also be implemented or applied in other different embodiments. Therefore, the following embodiments for performing the present disclosure can be modified and / or changed without violating the scope of its different aspects and applications, and any element or method within the scope of the present disclosure can be combined with any other element or method disclosed in any embodiment of the present disclosure.

[0039] In order to more easily understand the present invention, some terms are first defined. In addition, it should be noted that whenever a value or value range of a parameter is recited, values ​​and ranges intermediate to the recited values ​​are also intended to form part of the present invention.

[0040] As used herein, the singular forms "a," "an," and "the" include plural referents unless specifically and unambiguously limited to one referent. For example, "an element" refers to one element or more than one element, such as a plurality of elements. The term "or" is used interchangeably with the term "and / or," unless expressly stated otherwise.

[0041] As used herein, the terms "comprise," "include," "have," "contain," and any other variations thereof are intended to cover a non-exclusive inclusion. For example, when an object is described as "comprising" an element, unless otherwise stated, it may also include other components, elements, components, structures, regions, parts, devices, systems, steps, or connections, and other elements should not be excluded.

[0042] As used herein, the term "administering" refers to placing an active agent into an individual by a method or route that results in the active agent being at least partially localized at a desired site to produce a desired effect. The active agents described herein can be administered by any suitable route known in the art.

[0043] As described herein, numerical ranges are inclusive and combinable, and any numerical value falling within the numerical range herein can be regarded as a maximum or minimum value to derive a subrange therefrom. For example, it should be understood that the numerical range "0.1 to 10 μM" includes any subrange between a minimum value of 0.1 μM and a maximum value of 10 μM, such as a subrange from 0.1 μM to 5 μM, from 1.0 μM to 10 μM, and from 0.5 μM to 8 μM, etc. In addition, multiple numerical values ​​used herein can be selectively selected as maximum and minimum values ​​to derive a numerical range. For example, the numerical ranges of 0.1 μM to 5 μM, 0.1 μM to 10 μM, and 5 μM to 10 μM can be derived from the numerical values ​​of 0.1 μM, 5 μM, and 10 μM.

[0044] As used herein, the term "about" generally refers to a value that encompasses a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% of a given value or range. Such variation in values ​​may occur due to, for example, experimental error, typical errors in measurement or handling in manufacturing compounds, compositions, concentrates, or formulations, differences in the source, manufacture, or purity of starting materials or ingredients used in the present disclosure, or similar considerations. Additionally, the term "about" refers to within the standard error range acceptable to one of ordinary skill in the art. Unless expressly stated otherwise, all numerical ranges, amounts, values, and percentages disclosed herein, such as amounts of material, duration of time periods, temperatures, operating conditions, ratios of quantities, and the like, should be understood in all instances to be modified by the term "about."

[0045] As used herein, "subject" refers to any vertebrate, including but not limited to humans or non-human mammals such as deer, mules, elk, and mule deer, for whom amelioration of a condition, disease, or disorder is sought, including CRAC channel and / or DDR2-related diseases or conditions, such as organ fibrosis, hypercytokinemia (i.e., cytokine storm), cancer, and COVID-19. However, the subject is optionally a mammal (e.g., a human) or an animal-like mammal (e.g., a livestock animal such as a dog, cat, horse, rat, mouse, etc.).

[0046] Serum cytokines elevated in individuals with cytokine storm include interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), TNF (tumor necrosis factor), interferon-γ (IFN-γ), macrophage inflammatory protein (MIP) 1α, and MIP 1β. In one embodiment of the present disclosure, the cytokine is selected from the group consisting of any one of interleukins 1 to 36, tumor necrosis factor α, tumor necrosis factor (TNF) α, CD40 ligand, Fas ligand, tumor necrosis factor-related apoptosis-inducing ligand, tumor necrosis factor superfamily member 14, and any combination thereof. In one embodiment of the present disclosure, the cytokine is interleukin-2, interleukin-6, or tumor necrosis factor (TNF) α. Elevated cytokines can lead to endothelial dysfunction, vascular damage and paracrine / metabolic disorders, which in turn cause damage to multiple organ systems. In the early stages of hypercytokinemia, an increase in acute response cytokines (such as TNF and IL-1β) and chemotactic cytokines (such as IL-8 and MCP-1) leads to a sustained increase in IL-6. IL-6 is considered to be one of the more complex cytokines because it is produced by and acts on immune and non-immune cells across multiple organ systems. IL-6 plays a vital role in cytokine storms, contributing to processes such as neutrophil chemotaxis and lymphocyte necrosis. Blocking upstream events associated with cytokine responses, such as inhibiting macrophage signaling to reduce IL-6 production or inhibiting T cell activation to reduce cytokine levels, may represent potential therapeutic targets for managing cytokine storms. In some embodiments of the present disclosure, the cytokine is selected from the group consisting of chemokines, interferons, interleukins, lymphokines and tumor necrosis factors.

[0047] In one embodiment of the present disclosure, the CRAC channel-related disease or condition and / or DDR2-related disease or condition is selected from cytokine storm, fibrotic disease, cancer, arthritis, cardiopulmonary disease, inflammatory disease, autoimmune disease, inflammatory bowel disease (IBD), allergic disease, acute kidney injury (AKI), chronic kidney disease (CKD), uremic cardiomyopathy, nephrogenic systemic fibrosis (NSF), cystic fibrosis, polycystic kidney disease (PKD), pulmonary fibrosis and any combination thereof.

[0048] In one aspect of the present disclosure, the treatment or prevention of the CRAC channel-related disorder or condition and / or DDR2-related disorder or condition comprises inhibiting CRAC channel activation. In one aspect of the present disclosure, the treatment or prevention of the CRAC channel-related disorder or condition and / or DDR2-related disorder or condition comprises inhibiting DDR2 activation. In a further aspect of the present disclosure, the treatment or prevention of the CRAC channel-related disorder or condition and / or DDR2-related disorder or condition comprises inhibiting CRAC channel and DDR2 activation. In some embodiments of the present disclosure, the treatment or prevention of the cytokine storm syndrome comprises reducing SOCE in T cells and macrophages. In at least one embodiment, the WRG-28, the atovaquone, the WRG-28 precursor or the atovaquone precursor inhibits CRAC channel activation, DDR2 activation, SOCE and / or cytokine expression.

[0049] In some embodiments of the present disclosure, the cytokine storm syndrome is an infection-induced cytokine storm syndrome. In other embodiments of the present disclosure, the cytokine storm syndrome is triggered by COVID-19. In other embodiments of the present disclosure, the cytokine storm syndrome is triggered by a pathogen selected from influenza virus, Epstein-Barr virus (EBV), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV2.

[0050] In one embodiment of the present disclosure, the fibrotic disease is tissue fibrosis or organ fibrosis. In another embodiment of the present disclosure, the fibrotic disease is cardiac fibrosis, pulmonary fibrosis, liver fibrosis, nephritis, diabetes, renal fibrosis, or any combination thereof. In some embodiments of the present disclosure, the fibrotic disease is an infection-induced fibrotic disease, an obstruction-induced fibrotic disease, or a drug-induced fibrotic disease. In at least one embodiment of the present disclosure, the infection-induced fibrotic disease is a COVID-19-induced fibrotic disease. In at least one embodiment of the present disclosure, the obstruction-induced fibrotic disease is renal fibrosis caused by ureteral obstruction.

[0051] In at least one embodiment, the treatment or prevention of a fibrotic disease improves renal function, pulmonary function, liver function, or cardiac function; promotes tissue repair and epithelial differentiation; and inhibits collagen deposition, myofibroblast expansion, and / or TGF-β-associated fibroblast activation. In at least one embodiment, the TGF-β-associated fibroblast activation is TGF-β1-associated fibroblast activation. In at least one embodiment, the treatment or prevention of a fibrotic disease includes promoting tissue repair, restoring epithelial differentiation, reducing collagen deposition, inhibiting myofibroblast expansion, and / or inhibiting TGF-β1-associated fibroblast activation in a fibrotic disease or condition.

[0052] In at least one embodiment, the treatment or prevention of a fibrotic disease improves renal function. In some embodiments, the treatment or prevention of a fibrotic disease improves lung function. In other embodiments, the treatment or prevention of a fibrotic disease improves lung function and / or heart function.

[0053] In some embodiments, the cancer is melanoma, head and neck cancer, brain cancer, nervous system cancer, thyroid cancer, thymic cancer, esophageal cancer, stomach cancer, lung cancer, breast cancer, gastrointestinal cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, kidney cancer, adrenocortical cancer, genitourinary system cancer, prostate cancer, bladder cancer, urothelial cancer, uterine cancer, cervical cancer, ovarian cancer, skin cancer, or a hematological tumor. In at least one embodiment, the cancer is small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous cell lung carcinoma, or lung adenocarcinoma. In other embodiments, the cancer is a primary cancer or a secondary cancer. In further embodiments, the cancer is a localized cancer, a regional cancer, an advanced cancer, or a metastatic cancer. In at least one embodiment, the cancer is a solid tumor or a non-solid tumor. In other embodiments, the cancer is a sarcoma, carcinoma, lymphoma, or leukemia.

[0054] Materials and Methods

[0055] Cell culture and treatment Human embryonic kidney cells (HEK293T), rat renal fibroblasts (NRK49F), human cardiac fibroblasts (HCF), human lung fibroblasts (MRC5), and rat hepatic stellate cells (HSC-T6) were purchased from ATCC (through the UK supplier LGC). The mouse pericyte line CCL-226 was a kind gift from Professor SL. Lin (National Taiwan University). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Thermo Scientific) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin according to the manufacturer's instructions. Jurkat T cells and human monocytic THP-1 cells were cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin and 1% streptomycin.

[0056] For TGF-β1-induced cell differentiation, cells were plated on tissue culture dishes coated with type 1 collagen and treated with the various compounds for 4 hours. Cells were then treated with 10 ng / ml TGF-β1 for an additional 24 hours before protein analysis or immunocytochemistry.

[0057] The study used various compounds, including BTP2 (Millipore, CAS 223499-30-7), WRG-28 (MCE#HY-114169), atovaquone (Av) (Cayman#23802), donepezil hydrochloride (Dh) (Millipore, CAS 120011-70-3), and terazosin hydrochloride (Th) (Millipore, CAS 70024-40-7). Compound library screening revealed that Av, donepezil hydrochloride (Dh), and terazosin hydrochloride were structurally comparable to BTP2 and WRG-28.

[0058] Plasmids and transfection

[0059] DDR2 and CFP-Orai1 / Stim1-mCherry were overexpressed in HEK293T cells using Lipofectamine 2000 (Invitrogen) and 200 ng of plasmid DNA. DDR2 plasmids were a kind gift from Professor B. Leitinger (University College London, UK). CFP-Orai1 and Stim1-mCherry were kind gifts from W.T. Chiu (National Cheng Kung University, Taiwan).

[0060] Cytoplasmic Ca 2+ Imaging

[0061] Cells were treated with different compounds (10 μM) and Fura 2-AM (1 μM) and cultured for 48 hours in glass-bottomed 96-well plates while incubating in the dark for 40 minutes in a solution containing 145 mM NaCl, 2.8 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM D-glucose, 10 mM HEPES, pH 7.4. The cells were then washed and incubated for another 15 minutes with the same solution supplemented with Fura 2-AM to completely deesterify. Before imaging, the cells were washed with a solution containing no CaCl2. 2+ A solution (145 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 10 mM D-glucose, 10 mM HEPES, 0.1 mM EGTA, pH 7.4) was applied to cells. 1 μM thapsigargin was added to induce depletion of endoplasmic reticulum (ER) calcium stores, followed by the addition of a 2 mM calcium solution to assess activation of calcium release-activated calcium (CRAC) channels. Cells were excited alternately at 340 and 380 nm, and images were acquired every 2 seconds. The cytoplasmic calcium signal was represented by the 340 nm / 380 nm ratio (R). All images were analyzed using IGOR Pro software.

[0062] Western blot analysis

[0063] Cell lysates were obtained in RIPA buffer (150 mM NaCl, 1 mM EGTA, 50 mM Tris pH 7.4, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and a protease inhibitor cocktail) and collected from the culture dish using a cell scraper. For tissue sample preparation, half of the kidney tissue was cut into approximately 1 mm3 pieces and then placed in a 2 ml tube containing 500 μl of RIPA buffer. The beads were stirred at high speed for 30 seconds using a mechanical homogenizer to extract tissue proteins. Protein samples were collected from the supernatant by centrifugation at 10,000 g for 10 minutes at 4°C. 20 to 30 μg of protein samples were analyzed by 7.5% or 10% SDS-PAGE. Protein expression levels were assessed using specific primary antibodies, including anti-phosphotyrosine antibodies (clone4G10; Millipore), DDR2 (R&D), α-SMA (Sigma), collagen 1a1 (Boster), E-adhesin (BD Biosciences), SGLT2 (Proteintech), NHE1 (Novus), and β-actin (Clone C4, Millipore), followed by incubation with HRP-conjugated secondary antibodies and detection using an ECL kit (Thermo Scientific).

[0064] Gel shrinkage and collagen alignment detection

[0065] The cells were prepared by mixing 3 ml of rat tail collagen (Corning), 1 ml of 5.7× DMEM, 500 μl of 2.5% NaHCO 3 , 1 ml of 0.1 M HEPES, 100 μl of 0.17 M CaCl 2 , 100 μl of 1 N NaOH and 4.3 ml of culture medium containing 2×10 6 NRK49F cell culture medium was used to create collagen gels. 2 ml of culture medium with or without 10 ng / ml TGF-β1 was applied to each well of a 6-well plate, and the cells were incubated for 3 days. Afterwards, the gels were removed from the plates, and the gel area was quantified using Image J.

[0066] Collagen gels were prepared using FITC-conjugated collagen, and cells were grown in these gels for 5 days. After fixing the cells with 4% paraformaldehyde, phalloidin-TRICT was used as a contrast stain to assess cell bodies. The angle between the fibers and the cell membrane was measured using Image J.

[0067] Cytokine induction and evaluation

[0068] THP-1 monocytes were differentiated into M1 macrophages with 0.1 μg / mL phorbol 12-myristate 13-acetate (PMA, Sigma, 8139) for 24 hours and then cultured with 20 ng / ml IFN-γ (MCE, HY-P7025) for another 3 days. Different doses of drugs were co-treated with induction medium containing 20 ng / ml IFN-γ (MCE, HY-P7025) and 1xLPS (Thermo, 00-4976-93). Jurkat T cells were treated with 1 μg / mL PHA and 1 μg / mL PMA for 24 hours and then pre-treated with different doses of the study compound for 30 minutes before cytokine induction.

[0069] Supernatants were collected at designated time points and cytokines, including IL-2, IL-6, and TNF-α, were detected using ELISA kits according to the manufacturer's instructions. The ELISA kits used in this experiment are listed below: ELISA MAX TM DeluxeSet HumanIL-2 (BioLegend, Cat.no.431804), Human IL-6ELISA MAX TM Deluxe (BioLegend, Cat.No.430504) and ELISA MAX TM Deluxe Set Human TNF-α (BioLegend, Cat. no. 430204) was used, and the absorbance was measured at 450 nm using a microplate analyzer (SpectraMax iD3, USA).

[0070] Immunofluorescence staining

[0071] Cells cultured on chamber slides were fixed with 4% paraformaldehyde for 10 minutes, and then the cells were incubated in PBS containing 0.5% Triton X-100 for 5 minutes for the permeabilization step. The cells were then incubated in SuperBlock blocking buffer (Thermo Scientific) for 1 hour at room temperature. The cells were incubated with specific primary antibodies and then with secondary antibodies against mouse or rabbit IgG conjugated to Alexa-488 or Alexa-594nm (Invitrogen) to detect protein expression. Hoechst 33258 (10 μg / ml) was used for nuclear staining. All images were visualized and captured using a confocal microscope (Olympus, FV-1000).

[0072] Immunofluorescence staining was performed on 15-μm-thick tissue slides embedded in Tissue-Tek OCT compound. Tissues were immersed in ice-cold acetone for 10 minutes to fix and permeabilize the tissue, followed by incubation with SuperBlock blocking buffer (ThermoScientific) at room temperature for 1 hour to minimize background signal. Protein expression and localization were detected using specific primary antibodies, including those against collagen 1a1 (Boster), laminin alpha 1 (R&D), SGLT2 (Proteintech), AQP1 (Novus), DDR2 (R&D systems), and α-SMA (Sigma). Fluorescent protein-conjugated secondary antibodies (Invitrogen) were then used for labeling. Images were captured using a confocal microscope (Olympus, FV-1000).

[0073] Unilateral ureteral obstruction (UUO) and reversible unilateral ureteral obstruction (RUUO)

[0074] Procedures involving individual animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the Laboratory Animal Center. Male C57BL / 6 mice aged 7 to 8 weeks were obtained from the animal center. Unilateral ureteral obstruction (UUO) was performed by ligating the left ureter, and mice were treated with 5 mg / kg / day of DMSO, BTP2, WRG-28 or Av for 7 days after ligation. After 7 days, the mice were sacrificed and the kidneys were removed for analysis. One-quarter of the kidneys were fixed with 4% paraformaldehyde and embedded in paraffin, or one-quarter of the kidneys were embedded in Tissue-TekOCT compound. Half of the kidneys were used for protein analysis. In the reversible unilateral ureteral obstruction (RUUO) experiment, the ligated left ureter was reconnected to the bladder 7 days after the UUO surgery. Compounds WRG-28 and Av were injected during ligation (7* / 15d) or after ligation release (7 / 15*d). To assess renal function, mice were ligated on the right ureter one day before placement in metabolic cages. Urine and blood were collected for determination of blood urea nitrogen (BUN) and creatinine (CRE).

[0075] Hematoxylin and eosin (H&E) staining

[0076] Tissue slides were dewaxed and rehydrated and immersed in filtered Harris hematoxylin for 10 seconds. After extensive rinsing with tap water, the specimens were immersed in alcohol-eosin for 2 minutes. The tissues were then dehydrated and mounted with resin medium and glass coverslips.

[0077] Sirius red staining

[0078] Tissue slides were dewaxed and rehydrated, stained with Sirius Red for one hour, and then thoroughly washed with acidified water. After dehydration through three changes of 95% ethanol, two changes of 100% ethanol, and three changes of xylene, tissue slides were mounted with resin mounting medium and glass coverslips. The tissue was then dehydrated and mounted with resin mounting medium and glass coverslips.

[0079] Immunohistochemistry

[0080] Antigen retrieval was performed on dewaxed and rehydrated tissue slides using citrate buffer and microwave heating. Endogenous peroxidase activation was blocked using 3% H2O2 in methanol, and background signal was reduced using SuperBlock blocking buffer (Thermo Scientific). After the blocking step, tissue slides were inked with anti-Ki67 primary antibody and anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP). HRP signal was then detected using diaminobenzidine (DAB). For detection of cell nuclei, hematoxylin was used as a counterstain. The tissue was then dehydrated and mounted with resin medium and a glass coverslip.

[0081] TUNEL (terminal deoxynucleotidyl transferase dUTP nick and labeling) assay

[0082] The TUNEL assay kit (ab206386) was purchased from Abcam. The procedure was performed according to the manufacturer's instructions. Briefly, dewaxed and rehydrated tissue coverslips were incubated in proteinase K solution at 37°C for 20 minutes, in 3% H₂O₂ for 5 minutes, and then incubated in TdT enzyme and reaction mixture for 1.5 hours. Signals were then detected and developed using DAB solution. Methyl green was used for counterstaining to assess healthy cells. Tissue slides were then dehydrated and mounted with resin medium and glass coverslips.

[0083] 5 / 6 nephrectomy

[0084] Experiments were conducted using 7- to 8-week-old male C57BL / 6 mice. The upper and lower poles of the left kidney were ligated, and the right kidney was removed one week later. Four weeks later, mice were injected intraperitoneally with 5 mg / kg / day of DMSO, WRG-28, or Av for the next four weeks. Urine and blood were collected for blood urea nitrogen (BUN) and creatinine (CRE) measurements, and kidneys were used for protein and histological analyses.

[0085] Blood urea nitrogen and creatinine testing

[0086] The levels of BUN and CRE in serum and urine were determined using a fully automated clinical biochemical analyzer (Fuji, Dri-Chem 4000i). All procedures followed the manufacturer's instructions.

[0087] Next-generation sequencing-based RNA sequencing and analysis

[0088] Total RNA was extracted from the kidneys by homogenizing the sample in TRIzol reagent (Invitrogen). Chloroform was added, and the homogenate separated into three layers. The upper, clear aqueous layer containing the RNA was transferred to a new tube, where the RNA was precipitated by adding isopropanol. The precipitate was washed with 70% ethanol and then dissolved in RNase-free water. RNA that passed quality control (including RNA integrity number (RIN) >7 and OD260 / OD280 ~1.8) was sent for sequencing. Transcriptomes were analyzed by ingenuity pathway analysis (IPA).

[0089] Bleomycin-induced pulmonary fibrosis model

[0090] Eight to ten-week-old male WT (C57BL / 6) mice received intratracheal instillation of bleocin (3 mg / kg) diluted in PBS or PBS alone (sham group). To investigate fibrosis, lung samples were collected on day 21 for further analysis.

[0091] Lung function tests

[0092] Pulmonary function was assessed using the flexiVent system (Scireq, Montreal, QC, Canada). Mice were tracheotomized and ventilated at a rate of 150 breaths / min, a tidal volume of 10 ml / kg, and a positive end-expiratory pressure (PEEP) of 2 to 3 cmH2O. Deep inflation perturbations were used to estimate inspiratory capacity (IC). A pressure-volume loop was generated by continuously increasing the pressure and then periodically decreasing it. Other lung function parameters, such as resistance, compliance, and elastance, were measured using the SnapShot-150.

[0093] Hydroxyproline test

[0094] Hydroxyproline content in lung tissue was measured using a Hydroxyproline Colorimetric Detection Kit (BioVision, Milpitas, CA, USA). Briefly, 10 mg of frozen right middle lobe was homogenized in 100 μL of 12N hydrochloric acid (HCl) and hydrolyzed at 120°C for 3 h. Then, 10 μL of each sample was used to quantify absorbance at 560 nm. Hydroxyproline content is expressed in μg / lobe.

[0095] Histology

[0096] The left lung of mice was fixed in 4% paraformaldehyde (PFA) overnight, embedded in paraffin, and cut into 5 μm thick sections for Sirius red staining (Abcam, Cambridge, UK) and Masson's trichrome staining (Sigma Aldrich, MO, USA). The measurement of fibrosis area was quantified using ImageJ software (NIH, http: / / rsbweb.nih.gov / ij / ).

[0097] Statistical analysis

[0098] All results are presented as mean ± SEM. Differences between two groups in all experiments were compared using a two-tailed Student's t-test. Statistical significance was set at a P value < 0.05 using GraphPad Prism. In all figures, *, **, and *** indicate P values ​​< 0.05, 0.01, and 0.001, respectively.

[0099] Example

[0100] Exemplary embodiments of the present disclosure are further described in the following examples, which should not be construed as limiting the scope of the present disclosure.

[0101] Example 1. WRG-28 and Av inhibit the activation of DDR2 and SOCE

[0102] The results of drug 3D structure screening showed that Av, Dh and Th have structural similarities with WRG-28 and BTP2. In order to examine the effects of these compounds on DDR2 activation, HEK293T cells that overexpress DDR2 were pretreated with various compounds. Subsequently, fibrillar collagen was introduced into the culture medium and incubated for 12 hours. The activation of DDR2 was assessed using an anti-phosphotyrosine antibody. WRG-28 has been used as a positive control group. In cells pretreated with Av, Dh and Th, the activation of DDR2 was significantly reduced ( Figure 1A ).

[0103] Next, we investigated the effects of WRG-28, Av, Dh, and Th on SOCE in HEK293T cells. WRG-28 and Av significantly reduced the second peak and rate of SOCE, while Dh and Th showed no significant effect ( Figure 1B 、 Figure 1C and Figure 1D Due to the structural similarity of the above compounds, WRG-28 and Av have great potential to target Orai1 (the pore-forming domain of CRAC channels), which mainly controls SOCE in HEK293T cells. The effects of WRG-28 and Av on SOCE were also tested in rat kidney fibroblasts (NRK49F) cells ( Figure 1E 、 Figure 1F and Figure 1G ) and human proximal tubular cells (HK-2) ( Figure 1H 、 Figure 1I and Figure 1J ).

[0104] To verify the inhibitory effects of WRG-28 and Av on CRAC channel activation, HEK293T cells overexpressing CFP-Orai1 and Stim1-mCherry were pretreated with different compounds. Subsequently, CRAC channel activation was induced by application of thapsigargin, a sarcoendoplasmic reticulum calcium transport ATPase (SERCA) pump inhibitor. The formation of Stim1 or Orai1 puncta induced by store depletion was not affected by BTP2, WRG-28, or Av pretreatment ( Figure 1K 、 Figure 1L 、 Figure 1M and Figure 1N The results showed that the aggregation of STIM1 or the coupling between STIM1 and Orai1 were not affected by these compounds.

[0105] Example 2. WRG-28 and Av inhibit TGF-β1-induced fibroblast activation and pericyte differentiation into myofibroblasts.

[0106] Both DDR2 and CRAC channels are involved in fibroblast activation. Therefore, tests were performed to evaluate the effects of various compounds on rat renal fibroblasts. Pretreatment of NRK49F cells with BTP2, WRG-28, or Av significantly inhibited TGF-β1-induced upregulation of collagen 1a1 and α-SMA ( Figure 2A and Figure 2BTGF-β1 treatment also induced the upregulation of collagen 1a1 and α-SMA in coat cells (CCL-226), which was significantly inhibited by pretreatment with BTP2, WRG-28 or Av ( Figure 2C The results showed that WRG-28 and Av had a potent effect in inhibiting TGF-β1-induced fibroblast activation and pericyte differentiation into myofibroblasts.

[0107] The arrangement of the extracellular matrix (ECM) is a unique characteristic of myofibroblasts, which changes the physical properties of the tissue microenvironment and creates a constant threat to the tissue during organ fibrosis. The effects of different compounds on the ECM arrangement ability of myofibroblasts after TGF-β1 stimulation were then examined. Representative figures show that the gel area of ​​the control group (DMSO) was significantly reduced after release from the culture dish under TGF-β1 stimulation, indicating an increase in the contractility of NRK49F cells. In addition, gel contraction was significantly reduced in cells co-treated with BTP2, WRG-28 or Av ( Figure 2D and Figure 2E After examining the arrangement of collagen fibers using FITC-labeled collagen, vertical arrangement of collagen fibers around cells was observed, especially in the TGF-β1-treated group ( Figure 2F However, treatment with BTP2, WRG-28, or Av reduced TGF-β1-induced collagen alignment ( Figure 2F 、 Figure 2G-1 and Figure 2G-2 Overall, WRG-28 and Av inhibited TGF-β1-induced ECM remodeling.

[0108] Example 3. WRG-28 and Av inhibit TGF-β1-induced activation of cardiac, lung, and liver fibroblasts.

[0109] To evaluate the overall effects of WRG-28 and Av on TGF-β1-induced fibroblast activation, human cardiac fibroblasts (HCF), human pulmonary fibroblasts (MRC5), and rat hepatic stellate cells (HSC-T6) were used. Upregulation of α-SMA induced by TGF-β1 treatment was observed, but this effect was inhibited by co-treatment with multiple compounds, including BTP2, WRG-28, and Av, in all cells tested ( Figure 3A 、 Figure 3B and Figure 3CThe expression of fibronectin and α-SMA induced by TGF-β1 in HCFs was examined, and this effect was again inhibited by treatment with BTP2, WRG-28, or Av ( Figure 3D Furthermore, treatment with TGF-β1 significantly increased the cell expansion area, a phenomenon that was alleviated by co-treatment with BTP2, WRG-28, or Av in HCFs ( Figure 3D ).

[0110] Cytoplasmic calcium measurements were performed to verify the effects of WRG-28 or Av on SOCE in various cell lines. Figure 3E )、MRC5(3F) and HSC-T6( Figure 3G ), WRG-28 or Av treatment significantly reduced SOCE. These results indicate that different compounds have a holistic effect in alleviating TGF-β1-induced myofibroblast activation.

[0111] Example 4. WRG-28 and Av reduce cytokine secretion in T cells and macrophages.

[0112] CRAC channel activation is involved in T cell differentiation and cytokine expression. Subsequently, the effects of WRG-28 and Av on SOCE and cytokine secretion in T cells and macrophages were tested. Treatment with WRG-28 and Av showed that blocking T cell ( Figure 4A ) and macrophages ( Figure 4E ) in the multiple capacities of SOCE in T cells. Overall, WRG-28 showed higher efficacy than Av and had similar potential in inhibiting SOCE as CM-4620 (the latest FDA-approved compound for blocking cytokine release). Av showed lower potency but still significantly reduced T cell ( Figure 4A ) and macrophages ( Figure 4E ) in SOCE. The effect of blocking SOCE is reflected in the release of cytokines. WRG-28, BTP2 and CM-4620 significantly reduced the release of IL-2 ( Figure 4B ) and TNF-α( Figure 4D ) secretion and IL-2 ( Figure 4F ), IL-6( Figure 4G ) and TNF-α( Figure 4H In contrast, Av showed relatively lower potency but still significantly reduced IL-2 in T cells ( Figure 4C ) and TNF-α( Figure 4D It showed that IL-2 ( Figure 4F ), IL-6( Figure 4G ) and TNF-α( Figure 4HThese results suggest that WRG-28 and Av in SOCE have diverse abilities to modulate immune responses to varying degrees.

[0113] Example 5. WRG-28 and Av reduce UUO-induced renal fibrosis

[0114] To evaluate the efficacy of WRG-28 and Av in protecting the kidneys from fibrotic damage, conventional unilateral ureteral obstruction (UUO) surgery was performed. Mice underwent UUO surgery and were simultaneously administered the studied compounds for 7 days. Histological results showed that UUO treatment induced tubular dilatation and interstitial dilatation. However, administration of BTP2, WRG-28, or Av seemed to alleviate the effects of UUO ( Figure 5A Sirius red staining results showed that fibrillar collagen was accumulated in the tubulointerstitial space 7 days after UUO surgery. However, administration of BTP2, WRG-28 or Av significantly reduced the area of ​​fibrillar collagen positive staining ( Figure 5B and Figure 5C ).

[0115] Protein analysis showed that in the kidneys treated with UUO, there was upregulation of mesenchymal marker proteins (such as integrin β1, DDR2, collagen 1a1, and α-SMA), while downregulation of epithelial marker proteins (E-adhesin). Figure 5D and Figure 5E )、WRG-28( Figure 5F and Figure 5G ) or Av( Figure 5H and Figure 5I ) inhibited UUO-induced upregulation of integrin β1, DDR2, collagen 1a1, and α-SMA, and also reversed UUO-induced downregulation of E-adhesin. These findings suggest that WRG-28 and Av have the efficacy of inhibiting tubulointerstitial fibrosis.

[0116] The loss of renal tubular epithelial cells leads to tubular atrophy and impairs renal function. In the kidneys treated with UUO, apoptotic tubular cells increased significantly, whereas in the kidneys treated with BTP2, WRG-28, or Av, apoptotic tubular cells decreased significantly ( Figure 5J and Figure 5K These results indicate that BTP2, WRG-28, and Av have protective effects on UUO-induced renal tubular atrophy and cell apoptosis.

[0117] Example 6. WRG-28 and Av protect and promote tissue repair, preventing obstruction-induced tissue damage and fibrosis

[0118] In traditional UUO, the gradual increase in hydrostatic pressure and the toxicity of accumulated excreta can lead to irreversible damage. Another disadvantage of traditional UUO is that it is impossible to assess the remaining renal function on the injured side. Therefore, in order to further understand the effects of the studied compounds on renal protection or post-injury repair, a reversal of unilateral ureteral obstruction (RUUO) surgery was performed. The left ureter was surgically ligated for 7 days and then reconnected to the bladder. Recent results show that surgical reconnection within 3 days after ligation can reverse damage and fibrosis markers. However, when the ligation is continued for 7 days and then reconnected, it will cause irreversible and continued worsening of fibrosis.

[0119] To evaluate whether administration of WRG-28 or atovaquone (Av) promotes renal repair, mice were injected with WRG-28 or atovaquone (Av) for 7 days (7* / 15d) during ureteral obstruction. Thirteen days after the obstruction was relieved, the right ureter was ligated and renal function was assessed by measuring the levels of BUN and CRE in the blood. Figure 6A Histological results showed that the DMSO-treated group had severe fibrotic scars; however, the administration of WRG-28 and Av preserved intact tubular structures ( Figure 6B After the blockage was released in the control group, the collagen-positive area increased from 2% to 22% ( Figure 6C ), but WRG-28 or Av treatment significantly reduced the expansion of fibrotic area ( Figure 6B and Figure 6C ).

[0120] Occlusion for 7 days followed by release for 13 days resulted in irreversible fibrosis and persistently high levels of mesenchymal markers (including integrin β1, DDR2, collagen 1a1, and α-SMA), and low levels of epithelial markers (such as E-adhesin, SGLT2, and NHE1). Figure 6D and Figure 6E ) and Av( Figure 6F and Figure 6G ) significantly reduced the expression of interstitial marker proteins. Importantly, the use of WRG-28 ( Figure 6D and Figure 6E ) and Av( Figure 6F and Figure 6G )-treated mice, the expression of epithelial markers was significantly increased.

[0121] Example 7. WRG-28 and Av treatment reduces the continued progression of obstruction-induced renal fibrosis after obstruction relief.

[0122] Next, we tested whether administration of WRG-28 or Av could promote renal repair and reduce the continued progression of fibrosis after ureteral obstruction was relieved. 13 days after ureteral obstruction was relieved, mice received WRG-28 or Av injection (7 / 15*d) ( Figure 7A Compared with DMSO-treated mice, the WRG-28 and atovaquone (Av)-treated groups showed many intact tubular structures, less inflammation and scar tissue ( Figure 7B The collagen-positive area was also reduced due to WRG-28 or Av treatment ( Figure 7B Below and Figure 7C ). WRG-28( Figure 7D and Figure 7E ) and Av( Figure 7F and Figure 7G ) treatment alleviated the blockage-induced upregulation of integrin β1, DDR2, collagen 1a1, and α-SMA. However, downregulation of epithelial markers E-adhesin, SGLT2, and NHE1 was observed in WRG-28 ( Figure 7D and Figure 7E ) and Av( Figure 7F and Figure 7G These results show that administration of WRG-28 and Av can reverse fibrosis and promote tissue repair.

[0123] Example 8. WRG-28 and Av restore epithelial differentiation and reduce collagen deposition and myofibroblast expansion in occlusion-induced persistent fibrosis.

[0124] Immunofluorescence studies further examined the arrangement and appearance of ECMs in kidneys treated with different compounds at different time points. Laminin α1, a major component of the basement membrane, formed a thin layer beneath renal tubular epithelial cells and between podocytes and endothelial cells in the Bowman's capsule of control kidneys (sham group; e.g., Figure 8A In kidneys blocked for 7 days and then released for 15 days (RUUO-7 / 15*d-DMSO), significant tubular atrophy with thickening of the basement membrane was observed ( Figure 8A and Figure 8A -(i)). Compared with the sham group, the average thickness increased significantly ( Figure 8A -(i)). However, in the WRG-28 and atovaquone (Av)-treated groups, they always showed uniform and unchanged basement membrane width during the blocking period (7* / 15d) or after its release (7 / 15*d) ( Figure 8A and Figure 8A -(i)). Most notably, fewer atrophic tubules were observed in the groups treated with WRG-28 and Av ( Figure 8A and Figure 8A-(i)). In addition, the expression of collagen 1a1 was significantly increased in the DMSO-treated group (RUUO-7 / 15*d-DMSO), while the administration of WRG-28 and Av reduced the accumulation of collagen 1a1 ( Figure 8A and Figure 8A -(ii)). The expression of α-SMA was significantly increased in the DMSO-treated group, but significantly decreased in the WRG-28 and Av-treated groups ( Figure 8B and Figure 8B -(iii)). In the DMSO-treated group (RUUO-7 / 15*d-DMSO), the collagen upstream signal DDR2 was significantly upregulated ( Figure 8D and Figure 8D -(v)); however, administration of WRG-28 and Av decreased the expression of DDR2 compared with the DMSO-treated group ( Figure 8D and Figure 8D -(v)). These results show that administration of WRG-28 and Av reduces the activation of myofibroblasts and the subsequent reassembly of ECMs during renal fibrosis.

[0125] The integrity and structure of the basement membrane influence epithelial differentiation, and the arrangement of specialized membrane microdomains is one of its defining characteristics. Sodium-glucose cotransporter-2 (SGLT-2) is specifically expressed in proximal tubular cells and is located in the apical space of healthy kidneys (sham-operated group, Figure 8C and Figure 8C -(iv)). Blockage for 7 days followed by release for 15 days (RUUO-7 / 15*d-DMSO) induced downregulation of SGLT-2 ( Figure 7D 、 Figure 7F 、 Figure 8C and Figure 8C -(iv)). In addition, in the kidneys of mice treated with DMSO alone, the protein lacked apical membrane distribution in cells expressing SGLT-2 ( Figure 8C and Figure 8C -(iv). However, both WRG-28 and Av treatment reversed the apical membrane expression of SGLT-2. These phenomena were also evident in mice treated during occlusion and then released, showing a distribution very close to normal ( Figure 8C and Figure 8C -(iv)), although SGLT2 protein levels were only slightly altered ( Figure 7D 、 Figure 7E 、 Figure 7F and Figure 7G Similar phenomena were observed in other epithelial marker proteins, including AQP1 ( Figure 8D and Figure 8D -(vi)) and NHE1 (data not shown). In conclusion, administration of WRG-28 and Av enhances tubular differentiation during renal fibrosis.

[0126] A profound imbalance between apoptotic and proliferating renal tubular cells, partly due to cellular aging, leads to maladaptive repair of fibrotic kidneys. During ureteral obstruction, administration of WRG-28 or Av significantly reduced apoptotic cells ( Figure 5J and Figure 5K Even 15 days after the blockage was relieved, a large number of apoptotic (TUNEL-positive) renal tubular cells were still present in DMSO-treated mice ( Figure 8E and Figure 8F However, only a small number of apoptotic cells were observed in the groups treated with WRG-28 and Av during the occlusion period, and their numbers remained low even 15 days after the occlusion was released ( Figure 8E and Figure 8F The number of proliferating tubular cells (Ki67-positive) did not differ significantly between the groups, but more proliferating interstitial cells ( ) were present in DMSO-treated mice compared with the WRG-28 and atovaquone (Av)-treated groups. Figure 8E and Figure 8F ). The administration of WRG-28 and Av after the blockage was released also significantly reduced the number of apoptotic cells (TUNEL-positive cells) ( Figure 8G and Figure 8H ), indicating their preventive and therapeutic effects. Importantly, administration of WRG-28 and Av after occlusion was relieved also increased the number of proliferating renal tubular cells (Ki67-positive cells) ( Figure 8G and Figure 8H ). Therefore, based on the above results, WRG-28 and Av treatment can alleviate maladaptive repair and help renal tubular regeneration.

[0127] Example 9. WRG-28 or atovaquone (Av) treatment improves renal function in obstruction-induced renal injury

[0128] Serum BUN and CRE levels were measured to assess renal function. Obstruction-induced renal injury and continued fibrosis progression resulted in a 2- to 3-fold increase in plasma BUN and CRE levels. However, administration of WRG-28 or Av significantly reduced BUN and CRE levels compared to those in DMSO-treated mice ( Figure 9A and Figure 9B ). These results demonstrate the effectiveness of WRG-28 and Av in treating and protecting renal function.

[0129] Example 10. Transcriptome Analysis of WRG-28 and Av-Treated Mice

[0130] Total ribonucleic acid (RNA) was extracted from the following 2 groups: control mice (sham surgery group) and mice treated with DMSO, WRG-28, or Av after occlusion was relieved (7 / 15*d). Transcriptomes were then analyzed by Ingenuity Pathway Analysis (IPA). The results showed that the inflammatory response, signaling, and fibrosis gene sets were significantly increased in the DMSO-treated group compared with the non-surgical mice. Conversely, administration of WRG-28 and Av reduced the expression of inflammatory response and fibrosis signaling pathways ( Figure 10A Some of these genes are Figure 10B Large-scale transcriptome analysis further confirmed the potent efficacy of WRG-28 and Av in preventing and protecting the kidney from progressive fibrosis.

[0131] Example 11. Administration of WRG-28 or Av effectively reduces pulmonary fibrosis

[0132] The efficacy of WRG-28 and Av in treating pulmonary fibrosis was evaluated in vivo using a BLM-induced pulmonary fibrosis mouse model. On day 0, 50 μL of bleomycin (BLM, 3 mg / kg) or PBS was instilled intratracheally to induce fibrosis or serve as a control group, respectively. Subsequently, various compounds were administered for 14 days (nintedanib: 60 mg / kg / day, WRG-28: 5 mg / kg / day, Av: 5 mg / kg / day). Pulmonary function was assessed on day 21 ( Figure 11A Nintedanib (FDA-approved drug for the treatment of idiopathic pulmonary fibrosis (IPF)) was used as a positive control group. The results of pulmonary function assessment were as follows Figure 11B These results show that BLM administration leads to a decrease in lung capacity and compliance, while increasing lung resistance and elastic resistance. The extent of these changes can be reversed by treatment with nintedanib, WRG-28, or Av. Figure 11C As shown, markers of collagen deposition (hydroxyproline content) were extracted and analyzed to show the extent of fibrosis. BLM can effectively induce pulmonary fibrosis, while the administration of nintedanib, Av or WRG-28 inhibits the progression of fibrosis. Histological analysis was performed by staining techniques to quantify the area of ​​fibrosis. Both Masson's trichrome staining and Sirius red staining showed that nintedanib, Av or WRG-28 treatment effectively reduced the area of ​​fibrosis ( Figure 11D ).

[0133] Those skilled in the art will readily observe that various modifications and variations can be made to the method while maintaining the teachings of the present invention.Accordingly, the above disclosure should be construed as being limited only by the scope and metes and bounds of the appended claims.

Claims

1. A pharmaceutical composition for treating or preventing a CRAC channel-related disease or condition and / or a DDR2-related disease or condition, comprising: an effective amount of at least one selected from the group consisting of WRG-28, atovaquone, a WRG-28 precursor, and an atovaquone precursor; and Pharmaceutically acceptable carrier.

2. The pharmaceutical composition according to claim 1, wherein The CRAC channel-related disease or condition and / or the DDR2-related disease or condition is selected from the group consisting of cytokine storm syndrome, fibrotic disease, cancer, arthritis, cardiopulmonary disease, inflammatory disease, autoimmune disease, inflammatory bowel disease, allergic disease, acute kidney injury, chronic kidney disease, uremic cardiomyopathy, nephrogenic systemic fibrosis, cystic fibrosis, polycystic kidney disease, and any combination thereof.

3. The pharmaceutical composition according to claim 2, wherein The CRAC channel-related disease or condition and / or the DDR2-related disease or condition is cytokine storm syndrome.

4. The pharmaceutical composition according to claim 3, wherein The cytokine storm syndrome is a cytokine storm syndrome induced by infection.

5. The pharmaceutical composition according to claim 4, wherein The cytokine is selected from the group consisting of chemokines, interferons, interleukins, lymphomediators and tumor necrosis factors.

6. The pharmaceutical composition according to claim 5, wherein The cytokine is interleukin or tumor necrosis factor.

7. The pharmaceutical composition according to claim 2, wherein The CRAC channel-related disease or condition and / or the DDR2-related disease or condition is the fibrotic disease.

8. The pharmaceutical composition according to claim 7, wherein The fibrotic disease is selected from the group consisting of cardiac fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, infection-induced fibrotic disease, obstruction-induced fibrotic disease, drug-induced fibrosis, nephritis, diabetes, and any combination thereof.

9. The pharmaceutical composition according to claim 7, wherein The treatment or prevention of fibrotic diseases improves renal function, pulmonary function, liver function and / or cardiac function, promotes tissue repair and / or epithelial cell differentiation, and / or inhibits collagen deposition, myofibroblast expansion and / or TGF-β-related fibroblast activation.

10. The pharmaceutical composition according to claim 9, wherein The treatment or prevention of the fibrotic disease improves the renal function and / or the pulmonary function, promotes the tissue repair and / or the epithelial cell differentiation, and / or inhibits the collagen deposition, the myofibroblast expansion and / or the TGF-β-related fibroblast activation.

11. The pharmaceutical composition according to claim 10, wherein The TGF-β-related fibroblast activation is TGF-β1-related fibroblast activation.

12. The pharmaceutical composition according to claim 1, wherein The WRG-28, the atovaquone, the WRG-28 precursor or the atovaquone precursor inhibits CRAC channel activation, DDR2 activation, store-regulated calcium flux and / or cytokine expression.

13. The pharmaceutical composition according to claim 12, wherein The cytokine is selected from the group consisting of interleukin 1 to interleukin 36, tumor necrosis factor α, tumor necrosis factor β, CD40 ligand, Fas ligand, tumor necrosis factor-related apoptosis-inducing ligand and tumor necrosis factor superfamily member 14 and any combination thereof.

14. The pharmaceutical composition according to claim 13, wherein The cytokine is interleukin-2, interleukin-6 or tumor necrosis factor-α.

15. A pharmaceutical composition for inhibiting CRAC channel activation and / or DDR2 activation in cells of an individual, comprising: An effective amount of at least one selected from the group consisting of WRG-28, atovaquone, a WRG-28 precursor, and an atovaquone precursor; and Pharmaceutically acceptable carrier.

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