Pharmaceutical compositions for use in the treatment or prevention of calcium release-activated calcium channel or discoidin domain receptor 2-associated disorders or conditions

Abstract

A pharmaceutical composition is provided for treating or preventing a calcium release-activated calcium (CRAC) channel-related disorder or condition and / or a discoidin domain receptor 2 (DDR2)-related disorder or condition in a subject in need thereof. Also provided is a pharmaceutical composition for inhibiting CRAC channel activation and / or DDR2 activation in a subject's cells. The pharmaceutical composition comprises an effective amount of at least one selected from the group consisting of WRG-28, atovaquone (Av), a WRG-28 precursor, and an atovaquone (Av) precursor; and a pharmaceutically acceptable carrier thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 480,626, filed January 19, 2023, the contents of which are incorporated herein by reference.

[0002] The present disclosure relates to calcium release-activated calcium (CRAC) channels and discoidin domain receptor 2 (DDR2), and in particular to methods for preventing or treating CRAC channel-associated disorders or conditions, and / or DDR2-associated disorders or conditions. [Background technology]

[0003] Calcium release-activated calcium (CRAC) channels are store-operated Ca 2+ Ca influx (SOCE), known as 2+ CRAC channels mediate influx. CRAC channels protect against fibrosis by inhibiting transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition and fibroblast activation. Activation of CRAC channels and discoidin domain receptor 2 (DDR2) is crucial for myofibroblast activation and organ fibrosis. Meanwhile, DDR2, as a downstream molecule of CRAC channels, orchestrates collagen deposition, which is involved in cancer metastasis and organ fibrosis.

[0004] Fibrotic diseases are a global problem, particularly in aging societies. Pulmonary fibrosis induced by infectious diseases, cirrhosis, nephritis, chronic renal fibrosis induced by diabetes or hypertension, and cardiac fibrosis resulting from uremic cardiomyopathy are major causes of death and represent a significant burden on national healthcare expenditures. Myofibroblasts play a pivotal role in organ fibrosis. Resident fibroblasts and pericytes constitute the primary source of myofibroblasts, but other sources, such as macrophages, bone marrow-derived cells, and endothelial cells, have been suggested to contribute to the expansion of myofibroblast populations during disease progression. However, their role remains a subject of debate. Myofibroblasts are recognized for their high capacity in extracellular matrix (ECM) production and remodeling, and α-smooth muscle actin (α-SMA) is a hallmark not only of myofibroblasts but also in the diagnosis of fibrotic diseases.

[0005] Hypercytokinemia, also known as a cytokine storm, is an uncontrolled, excessive inflammatory response resulting from a severe immune response that extends from a local inflammatory response to a viral or bacterial infection. Hyperimmune hyperactivation in hypercytokinemia can occur for a variety of reasons: inappropriate triggering or danger sensing that initiates a response in the absence of a pathogen (e.g., in genetic disorders with inappropriate inflammasome activation); inappropriate or ineffective response amplitude resulting in excessive immune cell activation (e.g., in CAR T cell therapy); uncontrolled infection and prolonged immune activation (e.g., in infection with Epstein-Barr virus, MERS-CoV, or SARS-CoV-2); or an inability to mobilize the immune response and restore homeostasis (e.g., in primary hemophagocytic lymphohistiocytosis).

[0006] Cytokine storm is believed 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 those targeting cytokine function (e.g., tocilizumab, an anti-interleukin-6 receptor antibody), has shown a significant reduction in mortality in COVID-19 patients. Nevertheless, despite its widespread use, two large randomized trials studying tocilizumab failed to demonstrate a survival benefit in hospitalized patients with COVID-19.

[0007] To date, there are no optimal therapeutic agents that specifically target CRAC channel-related disorders, DDR2-related disorders, or both conditions, and therefore there is a need for the development of effective, safe, and tolerable pharmaceutical agents for the treatment or prevention of these conditions. Summary of the Invention

[0008] Methods are provided for treating or preventing a CRAC channel-related disorder or condition and / or a DDR2-related disorder or condition in a subject in need thereof, comprising 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, together with a pharmaceutically acceptable carrier thereof.

[0009] The present disclosure also provides a use of the pharmaceutical composition of the present disclosure for treating or preventing a CRAC channel-related disorder or condition and / or a DDR2-related disorder or condition, comprising administering an effective amount of the pharmaceutical composition of the present disclosure to a subject in need thereof.The present disclosure further provides a pharmaceutical composition for use in treating or preventing a CRAC channel-related disorder or condition and / or a DDR2-related disorder or condition.Furthermore, the present disclosure provides a use of the pharmaceutical composition for manufacturing a medicament for treating or preventing a CRAC channel-related disorder or condition and / or a DDR2-related disorder or condition.

[0010] In some aspects, the present disclosure provides a method for inhibiting CRAC channel and / or DDR2 activation in a subject's cells, comprising administering an effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor, and a pharmaceutically acceptable carrier thereof.

[0011] Also provided herein is the use of the pharmaceutical composition of the present disclosure for inhibiting CRAC channel and / or DDR2 activation in cells of a subject, comprising administering to a subject an effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor, and their pharmaceutically acceptable carriers.The present disclosure further provides a pharmaceutical composition for use in inhibiting CRAC channel and / or DDR2 activation in cells of a subject.Further provided is the use of the pharmaceutical composition for manufacturing a medicament for inhibiting CRAC channel and / or DDR2 activation in cells of a subject.

[0012] In at least one embodiment, an effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor, provides better effects on TGF-β1-induced fibroblast activation, pericyte differentiation to myofibroblasts, and / or inhibition of TGF-β1-induced ECM remodeling.

[0013] In at least one embodiment, an effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor, exhibits a general effect in attenuating TGF-β1-induced myofibroblast activation.

[0014] In at least one embodiment, effective amounts / doses of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor in SOCE exhibit variable abilities to modulate immune responses to different degrees.

[0015] In at least one embodiment, an effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor in SOCE significantly reduces the area stained positive for fibrillar collagen.

[0016] In at least one embodiment, an effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor, exhibits efficacy in inhibiting tubulointerstitial fibrosis and exhibits protective effects against UUO-induced tubular atrophy and apoptosis.

[0017] In some embodiments, an effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor reduces the expansion of fibrotic areas and increases the expression of epithelial markers in mice.

[0018] In some embodiments of the present disclosure, an effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor, reverses fibrosis and promotes tissue repair.

[0019] In some embodiments of the present disclosure, an effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor reduces myofibroblast activation and subsequent ECM remodeling in renal fibrosis.

[0020] In some embodiments of the present disclosure, an effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor enhances tubular differentiation in renal fibrosis.

[0021] In at least one embodiment, an effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor, exhibits significant effects in alleviating failed repair, assisting in tubular regeneration, and treating and protecting renal function.

[0022] In at least one embodiment, an effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor, provides better efficacy in treating, preventing, and protecting the kidney from progressive fibrosis.

[0023] In some embodiments, an effective amount / dose of WRG-28 and / or Av, or WRG-28 precursor and / or Av precursor, shows significant efficacy in reducing pulmonary fibrosis.

[0024] These and other objects of the present invention will no doubt become apparent to those skilled in the art after reading the following detailed description of the preferred embodiment that is illustrated in the various figures and drawings.

[0025] The 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. [Brief explanation of the drawings]

[0026] [Figure 1]Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, and 1N show that WRG-28 and atovaquone (Av) suppress the activation of DDR2 and calcium release-activated calcium (CRAC) channels. Figure 1A shows HEK293T cells transiently overexpressing DDR2 and then pretreated with different compounds (10 μM), including WRG-28, Av, donepezil hydrochloride (Dh), terazosin hydrochloride (Th), verapamil, and flecainide, for 30 min. Verapamil and flecainide were used as negative controls. The cells were then treated with 50 μg / mL type I collagen for 12 h. Cell lysates were collected and subjected to Western blot analysis using antibodies against phosphotyrosine (clone 4G10), DDR2, and β-actin. Figure 1B shows the assessment of CRAC channel activity in HEK293T cells pretreated with 10 μM BTP2, WRG-28, or Av. Cells were loaded with Fura2-AM. CRAC channel activity was assessed by applying 1 μM thapsigargin (Thap.) in a calcium-free solution to deplete endoplasmic reticulum calcium stores, followed by the reintroduction of 2 mM Ca2+ in the external solution. The concentration of cytosolic calcium ions increases as calcium flows through the CRAC channel, resulting in the second peak of the cytosolic calcium wave. Figure 1C and Figure 1D show further analysis of the second peak and influx rate of each wave. Each bar represents the average value obtained from 47–103 cells. Figure 1E shows the evaluation of CRAC channel activity in NRK49F cells treated with 10 μM BTP2, WRG-28, or Av. Cells were loaded with Fura2-AM. Figures 1F and 1G show further analysis, where the second peak and influx rate of each wave were analyzed. Each bar represents the average value obtained from 83 to 176 cells. Figure 1H shows the evaluation of the effect of BTP2, WRG-28, or Av on CRAC channel activity in HK-2 cells. Figures 1I and 1J show further analysis, where the second peak and influx rate of each wave were analyzed. Each bar represents the average value obtained from 72 to 108 cells.Orai1 and Stim1 puncta were assessed in HEK293T cells overexpressing CFP-Orai1 / Stim1-mCherry. Cells were pretreated with 10 μM DMSO (Figure 1K), BTP2 (Figure 1L), WRG-28 (Figure 1M), or Av (Figure 1N) for 30 min, followed by stimulation with 1 μM thapsigargin for 15 min. Cells were then fixed and puncta were observed under a confocal microscope (Olympus FV-1000). In all graphs, each bar represents the mean ± SEM, and *, **, and *** indicate p values ​​<0.05, 0.01, and 0.001, respectively. [Figure 2]Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G-1, and 2G-2 show how WRG-28 and Av suppress TGF-β1-induced fibroblast activation and pericyte-to-myofibroblast differentiation. Figure 2A shows the assessment of TGF-β1-induced renal fibroblast activation, as measured by upregulation of collagen 1a1 and α-SMA, in NRK49F cells treated with 10 μM DMSO, BTP2, WRG-28, or Av for 24 hours. Figure 2B shows the relative protein levels of collagen 1a1 and α-SMA assessed from three independent experiments. Each bar represents the mean ± SEM. Figure 2C shows the evaluation of TGF-β1-induced pericyte-to-myofibroblast activation in CCL-226 cells co-treated with 10 μM DMSO, BTP2, WRG-28, or Av for 24 hours. Collagen 1a1 and α-SMA protein levels were assessed using Western blot analysis under various conditions. Figure 2D shows NRK49F cells 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 shows gels released from tissue culture plates, and relative gel area was assessed 8 hours after release. Each bar represents the mean ± SEM. Figure 2F shows the evaluation of TGF-β1-induced collagen alignment in NRK49F cells cultured in FITC-conjugated collagen gels and co-treated with 10 ng / mL TGF-β1 and / or 10 μM DMSO, BTP2, WRG-28, or Av for 5 days. Images were taken using a confocal microscope (Olympus MPE). Figures 2G-1 and 2G-2 show measurements of the angle between collagen fibers and the cell membrane performed using Image J. At least five different images were analyzed for each condition. Each bar represents the mean ± SEM. In all graphs, *, **, and *** represent p-values ​​<0.05, 0.01, and 0.001, respectively. "ns" indicates a p-value >0.05, indicating no significant difference. [Figure 3]Figures 3A, 3B, 3C, 3D, 3E, 3F, and 3G demonstrate that WRG-28 and Av suppress TGF-β1-induced activation of cardiac fibroblasts, pulmonary fibroblasts, and hepatic stellate cells. As shown in Figures 3A, 3B, and 3C, the effects of BTP2, WRG-28, or Av on TGF-β1-induced activation of human cardiac fibroblasts (HCF), human pulmonary fibroblasts (MRC5), and rat hepatic stellate cells (HSC-T6) were evaluated. Cells were cotreated with various compounds (10 μM) and TGF-β1 for 24 hours. After this time, cell lysates were collected, and protein expression was assessed by Western blot analysis. Figure 3D shows immunofluorescence staining of TGF-β1-induced fibronectin deposition and α-SMA expression in HCFs treated with 10 μM DMSO, BTP2, WRG-28, or Av. Images were taken using an Olympus FV-1000 confocal microscope. Figures 3E, 3F, and 3G show assessment of CRAC channel activity in HCFs, MRC5, and rat HSC-T6 cells. These cells were pretreated with 10 μM BTP2, WRG-28, or Av for 45 min. Assessment included measuring the second peak and influx velocity of each calcium wave. Each bar in the figure represents the mean value obtained from 77–91 cells for HCFs, 31–49 cells for MRC5, and 43–94 cells for HSC-T6. In all graphs, *, **, and *** indicate p-values ​​<0.05, 0.01, and 0.001, respectively. [Figure 4]Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G, and 4H demonstrate how WRG-28 and Av reduce SOCE in T cells and macrophages, resulting in downregulation of cytokine release. The effects of BTP2, WRG-28, Av, and CM-4620 on SOCE were evaluated in T cells (Figure 4A) and macrophages (Figure 4E). Cells were cultured on glass-bottom 96-well plates and pretreated with various compounds (10 μM) while loaded with Fura2-AM. Cytoplasmic calcium signal waves were then recorded, and the second peak and influx rate of each wave were evaluated. Each bar represents the average value obtained from 43–111 cells for T cells (Figure 4A) and 52–91 cells for macrophages (Figure 4E). Cytokine induction in T cells and macrophages was performed as described in the Methods section. The levels of cytokines, including IL-2, TNF-α, and IL-6, in the culture medium were assessed using ELISA kits for T cells (Figure 4B, Figure 4C, and Figure 4D) and macrophages (Figure 4F, Figure 4G, and Figure 4H), respectively. Each bar represents the mean ± SEM. In all graphs, *, **, and *** indicate p values ​​<0.05, 0.01, and 0.001, respectively. [Figure 5]Figure 5A, Figure 5B, Figure 5C, Figure 5D, Figure 5E, Figure 5F, Figure 5G, Figure 5H, Figure 5I, Figure 5J, and Figure 5K demonstrate how WRG-28 and Av reduce renal fibrosis induced by unilateral ureteral obstruction (UUO). Surgical ligation was performed on the left ureter of 7-8 week-old male mice using nylon surgical sutures. Mice were then treated with DMSO (control), BTP2, WRG-28, or Av at 5 mg / kg / day for 7 days. Figure 5A shows kidney histology of treated mice examined by hematoxylin and eosin (H&E) staining. Figure 5B shows the evaluation of fibrillar collagen expression levels by Sirius red staining. Figure 5C shows the measurement of Sirius red-positive areas using Image J. Figures 5D, 5E, 5F, 5G, and 5H show the protein levels of integrin β1, DDR2, collagen 1a1, α-SMA, and E-cadherin in kidney samples from mice treated with 5 mg / kg / day BTP2 (Figure 5D), WRG-28 (Figure 5F), or Av (Figure 5H). These were assessed by Western blot analysis. Relative protein levels in mice treated with BTP2 (Figure 5E), WRG-28 (Figure 5G), or Av (Figure 5I) were quantified using Image J. Figure 5J shows the assessment of apoptotic cells by TUNEL assay; representative photographs are presented. Figure 5K shows the assessment of apoptotic cells from 5 to 10 photographs in each condition. [Figure 6]Figures 6A, 6B, 6C, 6D, 6E, 6F, and 6G demonstrate that treatment with WRG-28 and Av during obstruction promotes tissue repair and reduces fibrosis after release of obstruction. Figure 6A shows the timeline of surgery and drug delivery. The left ureter was surgically ligated for 7 days (UUO-L) and then reconnected to the bladder (RUUO-L). Various compounds, including DMSO (control), WRG-28, and Av, were intraperitoneally injected daily during the ureteral ligation (UUO-L) period. After an additional 13 days of RUUO-L, the right ureter was ligated (UUO-R). Mice were then housed in metabolic cages the day after surgery (day 21). On day 22, the left kidney was removed for further analysis to assess renal function. Figure 6B shows paraffin-embedded tissue sections subjected to hematoxylin and eosin (H&E) and Sirius Red staining. Figure 6C shows measurements of Sirius Red-positive areas in each group, performed using Image J from 5–10 images. Protein levels of integrin β1, collagen 1a1, DDR2, α-SMA, E-cadherin, SGLT2, NHE1, and β-actin were assessed in mice treated with WRG-28 (Figure 6D) and Av (Figure 6F) using Western blot analysis. Relative protein levels were quantified using Image J (Figures 6E and 6G). Each bar represents the mean ± SEM. In all graphs, *, **, and *** indicate p-values ​​<0.05, 0.01, and 0.001, respectively. [Figure 7]Figures 7A, 7B, 7C, 7D, 7E, 7F, and 7G demonstrate how treatment with WRG-28 and Av reduces the persistent progression of obstruction-induced renal fibrosis after obstruction release. Figure 7A shows the timeline of surgery and drug delivery. The left ureter was surgically ligated for 7 days (UUO-L) and then reconnected to the bladder (RUUO-L). Various compounds, including DMSO (control), WRG-28, and Av, were intraperitoneally injected for 13 days after release of the left ureter (RUUO-L). Thirteen days after RUUO-L, the right ureter was ligated (UUO-R). Mice were then housed in metabolic cages the day after surgery (day 21). On day 22, the left kidney was removed for further analysis. Figure 7B shows paraffin-embedded tissue sections subjected to hematoxylin and eosin (H&E) and Sirius Red staining. Figure 7C shows measurements of Sirius Red-positive areas in each group, performed using Image J from 5–10 images. Protein levels of integrin β1, collagen 1a1, DDR2, α-SMA, E-cadherin, SGLT2, NHE1, and β-actin were assessed in mice treated with WRG-28 (Figure 7D) and Av (Figure 7F) using Western blot analysis. Relative protein levels were measured and quantified using Image J (Figures 7E and 7G). Each bar represents the mean ± SEM. In all graphs, *, **, 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. [Figure 8]Figures 8A, 8B, 8C, 8D, 8E, 8F, 8G, and 8H show how WRG-28 and Av restore epithelial differentiation and reduce collagen deposition and myofibroblast proliferation during the persistent progression of RUUO-induced renal fibrosis. Frozen tissue blocks were used for immunofluorescence staining. Figure 8A shows the expression of laminin alpha 1 and collagen 1a1 in mouse kidneys treated with DMSO, WRG-28, or Av during ureteral ligation (7 / 15 days) or after release of obstruction (7 / 15 days). Laminin alpha 1 and collagen 1a1 were detected using primary antibodies followed by secondary antibodies conjugated with Alexa-488 (green) and Alexa-594 (red), respectively. Hoechst 33258 (blue) was used to label nuclei. Figure 8A-(ii) shows the fluorescence intensity of collagen 1a1 measured using Image J. Analysis was performed on 5–10 regions from each of five photographs. Each bar represents the mean ± SEM. In Figure 8A-(i), laminin alpha 1 thickness was measured using Image J by analyzing 5–10 regions from each of five photographs. The median is indicated by a solid line, and the quartiles are indicated by dotted lines. Figure 8B shows α-SMA marked with an antibody labeled with Cy3 (red), and Hoechst 33258 (blue) was used to label nuclei. Figure 8B-(iii) shows the fluorescence intensity of α-SMA measured using Image J. Quantification was performed on at least five regions from each of five photographs. Each bar represents the mean ± SEM. Figure 8C shows the detection of laminin alpha 1 and SGLT2 using primary antibodies followed by secondary antibodies conjugated with Alexa-594 (red) and Alexa-647 (cyan), respectively. Hoechst 33258 (blue) was used to label nuclei. Figure 8C-(iv) shows quantification of apical membrane expression of SGLT2 in SGLT2-positive cells under each condition. At least five photographs were analyzed from each condition. Each bar represents the mean ± SEM. Figure 8D shows detection of DDR2 and AQP1 using primary antibodies followed by secondary antibodies conjugated with Alexa-488 (green) and Alexa-594 (red), respectively.Hoechst 33258 (blue) was used to label nuclei. The expression of DDR2 (Figure 8D-(v)) and AQP1 (Figure 8D-(vi)) was analyzed and quantified based on five to eight photographs from each condition. Each bar represents the mean ± SEM. Furthermore, the percentage of cells showing membrane expression of AQP1 was also analyzed and quantified (Figure 8D-(vi)). Figure 8E shows the examination of apoptotic cells by TUNEL assay in mice treated with different compounds during obstruction (7 days / 15 days). Furthermore, Ki67-positive cells, representing proliferating cells, were detected by immunohistochemistry using specific antibodies in mice treated with various compounds during obstruction (7 days / 15 days). Figure 8F shows the quantification of apoptotic and proliferating tubular cells, performed using at least 10 photographs from each condition. Each bar in the figure represents the mean ± SEM. Figure 8G shows the examination of apoptotic cells by TUNEL assay in mice treated with different compounds 7 days after the release of obstruction (7 / 15 days). Furthermore, Ki67-positive cells, indicating proliferating cells, were identified using specific antibodies in mice treated with different compounds 7 / 15 days after the release of obstruction (7 / 15 days). Figure 8H shows the quantification of apoptotic and proliferating tubular cells using data collected from at least 10 photographs from each condition. Each bar in the graph represents the mean ± SEM. In all graphs, *, **, and *** indicate p-values ​​<0.05, 0.01, and 0.001, respectively. [Figure 9] Figures 9A and 9B show the effects of treatment with WRG-28 and Av on improving renal function after obstruction-induced renal injury. Blood samples were collected from mice treated with DMSO, WRG-28, or Av during renal obstruction (7 / 15 d) or after release of obstruction (7 / 15 d). Blood urea nitrogen (BUN) levels are shown in Figure 9A, and creatinine (CRE) levels are shown in Figure 9B. Each bar represents the mean ± SEM. In all graphs, *, **, and *** indicate p-values ​​<0.05, 0.01, and 0.001, respectively. [Figure 10]Figures 10A and 10B show how treatment with WRG-28 and Av effectively reduces RUUO-induced overwhelming fibrosis and inflammation. In Figure 10A, total ribonucleic acid (RNA) extracts from control mice (sham) and mice treated with DMSO, WRG-28, or Av after obstruction release (7 / 15 days) were subjected to ingenuity pathway analysis (IPA). The resulting genes are listed in Figure 10B. Both Figures 10A and 10B show large-scale transcriptome analysis, further confirming the potent effects of WRG-28 and Av in preventing and protecting the kidney from progressive fibrosis. [Figure 11] Figures 11A, 11B, 11C, and 11D show how treatment with WRG-28 or Av effectively reduces pulmonary fibrosis. A bleomycin (BLM)-induced pulmonary fibrosis mouse model was used, and the timeline of surgery and drug delivery is shown in Figure 11A. Figure 11B shows assessment of pulmonary function across various indices. Hydroxyproline content, a marker of collagen deposition, was extracted from the right lobe and analyzed. The results are shown in Figure 11C. Figure 11D shows histological analysis from the left lobe, quantifying the fibrotic area by staining with Masson's trichrome and picrosirius red, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0027] The following embodiments are provided to explain the present disclosure in detail. Those skilled in the art can easily understand the advantages and effects of the present disclosure after reading the disclosure herein, and can also implement or apply it in other different embodiments. Therefore, without contradicting the scope of the present disclosure, the following embodiments can be modified and / or changed to implement the present disclosure in different aspects and applications, and any element or method within the scope of the present disclosure disclosed herein can be combined with any other element or method disclosed in any embodiment of the present disclosure.

[0028] In order that the present invention may be more readily understood, certain terms are first defined. Furthermore, it should be noted that whenever a value or range of values ​​for a parameter is listed, values ​​and ranges intermediate to the listed values ​​are also intended to be part of the invention.

[0029] As used herein, the singular forms "a," "an," and "the" include plural referents unless expressly and unambiguously limited to one referent. For example, "an element" means one element or more than one element, e.g., a plurality of elements. The term "or" is used interchangeably with the term "and / or" unless the context clearly dictates otherwise.

[0030] As used herein, the terms "comprising," "comprises," "include," "including," "have," "having," "contain," "containing," and any other variations thereof, are intended to cover non-exclusive inclusions. For example, if the subject matter states that a limitation "comprises," unless otherwise specified, it may further include other components, elements, components, structures, regions, parts, devices, systems, steps, or links, etc., and should not exclude other limitations.

[0031] As used herein, the term "administer" or "administration" refers to the placement of an active agent into a subject by a method or route that results in at least partial localization of the active agent at a desired site to achieve a desired effect. The active agents described herein can be administered by any suitable route known in the art.

[0032] Numerical ranges used herein are inclusive and combinable, and any numerical value within a numerical range herein may be considered the maximum or minimum value for deriving a subrange therefrom. For example, the numerical range "0.1 to 10 μM" is understood to include any subrange between the minimum value of 0.1 μM and the maximum value of 10 μM, such as the subranges 0.1 μM to 5 μM, 1.0 μM to 10 μM, and 0.5 μM to 8 μM. Furthermore, numerical values ​​used herein may be arbitrarily selected as maximum and minimum values ​​for deriving a numerical range. For example, the numerical ranges 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 ​​0.1 μM, 5 μM, and 10 μM.

[0033] As used herein, the term "about" generally refers to a numerical value that is meant to encompass a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% from a given value or range. Such variation in numerical value may result, for example, from experimental error, typical error in measuring or handling procedures for making a compound, composition, concentrate, or formulation, differences in the source, manufacture, or purity of starting materials or components used in the present disclosure, or similar considerations. Alternatively, the term "about" may mean within an acceptable standard error of the mean as considered by one of ordinary skill in the art. Unless otherwise expressly specified, all numerical ranges, amounts, values, and percentages, such as amounts of materials, durations of periods, temperatures, operating conditions, ratios of amounts, etc., disclosed herein should be understood to be modified in all instances by the term "about."

[0034] As used herein, "subject" refers to any vertebrate, including, but not limited to, a human or a non-human mammal such as a deer, mule, elk, or mule deer, in whom a CRAC channel and / or DDR2-related disorder or condition, e.g., a condition, disorder, or disease, including organ fibrosis, hypercytokinemia (i.e., cytokine storm), cancer, and COVID-19, is sought to be ameliorated. Advantageously, however, the subject is a mammal, such as a human, or a domesticated mammal, e.g., a dog, cat, horse, rat, mouse, or other mammal.

[0035] Serum cytokines, including 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, are elevated in individuals with cytokine storm. In one embodiment of the present disclosure, the cytokine is selected from the group consisting of any one of interleukin-1 through interleukin-36, tumor necrosis factor alpha, tumor necrosis factor (TNF) α, CD40 ligand, Fas ligand, tumor necrosis factor-related apoptosis-inducing ligand, and 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 result in endothelial dysfunction, vascular injury, and paracrine / metabolic dysregulation, resulting in damage to multiple organ systems. During the early stages of hypercytokinemia, elevated acute response cytokines such as TNF and IL-1β, as well as chemotactic cytokines such as IL-8 and MCP-1, contribute to the sustained increase in IL-6. IL-6 is considered one of the more complex cytokines due to its production by and action on both immune and non-immune cells across multiple organ systems. IL-6 plays a key role in cytokine storm, contributing to processes such as neutrophil chemotaxis and lymphocyte necrosis. Blocking upstream events associated with the cytokine response, such as inhibiting IL-6 production or macrophage signaling to reduce T cell activity and lower cytokine levels, may represent a potential therapeutic target for managing cytokine storm. In some embodiments of the present disclosure, the cytokine is selected from the group consisting of chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors.

[0036] In one embodiment of the present disclosure, the CRAC channel-related disorder or condition and / or DDR2-related disorder or condition is selected from the group consisting of cytokine storm syndrome, fibrotic disorder, 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.

[0037] In one aspect of the present disclosure, the treatment or prevention of CRAC channel-related disorders or conditions and / or DDR2-related disorders or conditions comprises inhibiting CRAC channel activation. In another aspect of the present disclosure, the treatment or prevention of CRAC channel-related disorders or conditions and / or DDR2-related disorders or conditions comprises inhibiting DDR2 activation. In a further aspect of the present disclosure, the treatment or prevention of CRAC channel-related disorders or conditions and DDR2-related disorders or conditions comprises inhibiting the activation of both CRAC channel and DDR2. In one embodiment of the present disclosure, the treatment or prevention of cytokine storm syndrome comprises reducing SOCE in T cells and macrophages. In at least one embodiment, WRG-28, atovaquone, WRG-28 precursor, or atovaquone precursor inhibits CRAC channel activation, DDR2 activation, SOCE, and / or cytokine expression.

[0038] In some embodiments of the present disclosure, the cytokine storm syndrome is an infectious disease-induced cytokine storm syndrome. In other embodiments of the present disclosure, the cytokine storm syndrome is caused by COVID-19. In some embodiments of the present disclosure, the cytokine storm syndrome is caused 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.

[0039] In one embodiment of the present disclosure, the fibrotic disorder is tissue fibrosis or organ fibrosis. In another embodiment of the present disclosure, the fibrotic disorder is cardiac fibrosis, pulmonary fibrosis, liver fibrosis, nephritis, diabetes, renal fibrosis, kidney fibrosis, or any combination thereof. In some embodiments of the present disclosure, the fibrotic disorder is infection-induced fibrotic disorder, obstruction-induced kidney injury fibrotic disorder, or drug-induced fibrotic disorder. In at least one embodiment of the present disclosure, the infection-induced fibrotic disorder is COVID-19-induced fibrotic disorder. In at least one embodiment of the present disclosure, the obstruction-induced fibrotic disorder is ureteral obstruction-induced kidney fibrosis.

[0040] In at least one embodiment, the treatment or prevention of a fibrotic disorder includes improving renal function, pulmonary function, liver function, or cardiac function; promoting tissue repair and epithelial differentiation; and inhibiting collagen deposition, myofibroblast proliferation, 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 disorder includes promoting tissue repair, restoring epithelial differentiation, reducing collagen deposition, suppressing myofibroblast proliferation, and / or suppressing TGF-β1-associated fibroblast activation in fibrotic disorders or conditions.

[0041] In at least one embodiment, treating or preventing a fibrotic disorder improves kidney function. In some embodiments, treating or preventing a fibrotic disorder improves lung function. In other embodiments, treating or preventing a fibrotic disorder improves liver function and / or cardiac function.

[0042] In some embodiments, the cancer is melanoma, or carcinoma of the head and neck, brain, nervous system, thyroid, thymus, esophagus, stomach, lung, breast, gastrointestinal tract, colon and rectum, liver, pancreas, kidney, adrenal cortex, genitourinary system, prostate, bladder, urothelium, uterus, cervix, ovary, skin, or hematological tumor. In at least one embodiment, the cancer is small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous cell carcinoma of the lung, or adenocarcinoma of the lung. In other embodiments, the cancer is a primary or secondary cancer. In further embodiments, the cancer is localized, regional, advanced, or 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.

[0043] material and method Cell culture and treatments Human embryonic kidney cells (HEK293T), rat kidney 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 cell line, CCL-226, was a gift from Professor S.L. Lin (National Taiwan University). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Thermo Scientific) containing 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) containing 10% fetal bovine serum (Invitrogen), 1% penicillin, and 1% streptomycin.

[0044] For TGF-β1-induced cell differentiation, cells were seeded onto tissue culture dishes coated with type I collagen and then treated with different compounds for 4 hours. The cells were then treated with 10 ng / ml TGF-β1 for an additional 24 hours before protein analysis or immunohistochemical staining studies.

[0045] Different compounds were used in this study, 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). Av, donepezil hydrochloride (Dh), and terazosin hydrochloride were structurally equivalent to BTP2 and WRG-28 based on compound database screening.

[0046] Plasmids and gene transfer DDR2 and CFP-Orai1 / Stim1-mCherry were overexpressed in HEK293T cells using Lipofectamine 2000 (Invitrogen) with 200 ng of plasmid DNA. DDR2 plasmid was a gift from Professor B. Leitinger (University College London, UK). CFP-Orai1 and Stim1-mCherry were gifts from W.T. Chiu (National Cheng Kung University).

[0047] Cytoplasmic Ca 2+ Imaging Cells cultured for 48 hours on glass-bottom 96-well plates were treated simultaneously with different compounds (10 μM) and Fura2-AM (1 μM) for 40 minutes in the dark in a solution containing 145 mM NaCl, 2.8 mM KCl, 2 mM CaCl, 2 mM MgCl, 10 mM D-glucose, and 10 mM HEPES at pH 7.4. The cells were then washed and incubated for an additional 15 minutes in the same solution supplemented with Fura2-AM for complete deesterification. 2+A calcium-free solution (145 mM NaCl, 2.8 mM KCl, 2 mM MgCl, 10 mM D-glucose, 10 mM HEPES, 0.1 mM EGTA, pH 7.4) was applied to the cells before imaging. 1 μM thapsigargin was added to induce endoplasmic reticulum (ER) calcium store depletion, followed by application of a 2 mM calcium solution to assess calcium release-activated calcium (CRAC) channel activation. Cells were alternately excited at 340 nm and 380 nm and imaged every 2 seconds. The cytosolic calcium signal was expressed by the 340 nm / 380 nm ratio (R). All images were analyzed using IGOR Pro software.

[0048] Western blot analysis Cell lysates were collected 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 protease inhibitor cocktail) and harvested from the culture dish using a cell scraper. For tissue sample preparation, half of the kidney tissue was cut into approximately 1 mm pieces. 3 The tissue was cut into strips and then placed in 500 μl of RIPA buffer in a 2 ml tube. Tissue proteins were extracted by stirring the beads at high speed for 30 seconds using a mechanical homogenizer. The tubes were centrifuged at 10,000 g for 10 minutes at 4°C, and the protein samples in the supernatant were collected. 20–30 μg of protein samples were separated by 7.5% or 10% SDS-PAGE. Protein expression levels were assessed using specific primary antibodies, including antibodies against phosphotyrosine (clone 4G10; Millipore), DDR2 (R&D), α-SMA (Sigma), Collagen1a1 (Boster), E-cadherin (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).

[0049] Gel contraction and collagen alignment assays 3 ml of rat tail collagen (Corning), 1 ml of 5.7× DMEM, 500 μl of 2.5% NaHCO3, 1 ml of 0.1 M HEPES, 100 μl of 0.17 M CaCl2, 100 μl of 1 N NaOH, and 2 × 10 6 Collagen gels were prepared by mixing 4.3 ml of medium with 1000 cells of NRK49F cells. Two ml of medium containing or without 10 ng / ml TGF-β1 was applied onto the gel in each well of a 6-well plate, and the cells were incubated for 3 days. The gels were then released from the culture plate, and the gel area was quantified using Image J.

[0050] Collagen gels were prepared using FITC-conjugated collagen, and cells were grown in the gels for 5 days. Cells were then fixed with 4% paraformaldehyde, and phalloidin-TRICT was used as a counterstain to assess cell soma. The angle between the fibers and the cell membrane was measured using Image J.

[0051] Cytokine induction and assessment THP-1 monocytes were differentiated into M1 macrophages by incubation with 0.1 μg / mL phorbol 12-myristate 13-acetate (PMA, Sigma, P8139) for 24 hours, followed by 20 ng / mL IFN-γ (MCE, HY-P7025) and 1× LPS (Thermo, 00-4976-93) for an additional 3 days. Different doses of drugs were co-treated in induction medium containing 20 ng / mL IFN-γ (MCE, HY-P7025) and 1× LPS (Thermo, 00-4976-93). Jurkat T cells were treated with 1 μg / mL PMA and 1 μg / mL PMA for 24 hours, followed by pretreatment with different doses of test compounds for 30 minutes before cytokine induction.

[0052] The supernatant was collected at the indicated time points, and cytokines including IL-2, IL-6, and TNF-α were analyzed by ELISA kits according to the manufacturer's protocol. The ELISA kits used in the experiment were as follows: ELISA MAX™ Deluxe Set Human IL-2 (BioLegend, Cat. No. 431804), Human IL-6 ELISA MAX™ Deluxe (BioLegend, Cat. No. 430504), and ELISA MAX™ Deluxe Set Human TNF-α (BioLegend, Cat. No. 430204). The absorbance was measured at 450 nm using a microplate reader (SpectraMax iD3, USA).

[0053] Immunofluorescence staining Cells cultured on chamber slides were fixed with 4% paraformaldehyde for 10 minutes, followed by a permeabilization step by incubating the cells in 0.5% Triton X-100 in PBS for 5 minutes. Cells were then incubated in SuperBlock blocking buffer (Thermo Scientific) for 1 hour at room temperature. Protein expression was detected by incubating the cells with specific primary antibodies, followed by secondary antibodies against mouse or rabbit IgG conjugated with Alexa-488 or -594 nm (Invitrogen), respectively. Hoechst 33258 (10 μg / ml) was used for nuclear staining. All images were visualized and captured using a confocal microscope (Olympus FV-1000).

[0054] Fifteen-micrometer-thick tissue slides from Tissue-Tek OCT compound-embedded tissue blocks were used for immunofluorescence staining. Tissues were fixed and permeabilized by immersion in ice-cold acetone for 10 minutes, followed by incubation with SuperBlock blocking buffer (Thermo Scientific) at room temperature for 1 hour to minimize background signal. Protein expression and localization were detected with 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), followed by labeling with secondary antibodies conjugated to fluorescent proteins (Invitrogen). Images were captured using a confocal microscope (Olympus FV-1000).

[0055] Unilateral ureteral obstruction (UUO) and reversible unilateral ureteral obstruction (RUUO) Procedures involving animal subjects were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Laboratory Animal Center. Male C57BL / 6 mice, 7–8 weeks old, were obtained from the animal center. Unilateral ureteral obstruction (UUO) was performed by ligating the left ureter, and the mice were treated with DMSO, BTP2, WRG-28, or Av at 5 mg / kg / day for 7 days after ligation. After 7 days, the mice were sacrificed, and the kidneys were removed and used for analysis. One quarter of the kidney was fixed in 4% paraformaldehyde and embedded in paraffin, or one quarter of the kidney was embedded in Tissue-Tek OCT compound. Half of the kidney was used for protein analysis. In reversible unilateral ureteral obstruction (RUUO) experiments, the ligated left ureter was reconnected to the bladder for 7 days after the UUO surgery. Compound WRG-28 and Av were injected during ligation (7 / 15 days) or after ligation release (7 / 15 days). To evaluate renal function, the right ureter was ligated the day before the mice were placed in metabolic cages. Urine and blood were collected for blood urea nitrogen (BUN) and creatinine (CRE) tests.

[0056] Hematoxylin and eosin (H&E) staining Deparaffinized and rehydrated tissue slides were immersed in filtered Harris hematoxylin for 10 seconds. After thorough rinsing with tap water, the specimens were immersed in alcoholic eosin for 2 minutes. The tissues were then dehydrated and mounted with resin medium and glass coverslips.

[0057] Sirius Red staining Deparaffinized and rehydrated tissue slides were stained with picrosirius red for 1 hour, followed by extensive washing with acidified water. After dehydration through three changes of 95% ethanol, two changes of 100% ethanol, and three changes of xylene, the tissue slides were mounted with resin mounting medium and glass coverslips. The tissues were then dehydrated and mounted with resin mounting medium and glass coverslips.

[0058] Immunohistochemistry Deparaffinized and rehydrated tissue slides were subjected to antigen retrieval using citrate buffer and microwave heating. Endogenous peroxidase activity was blocked using a 3% H2O2 solution in methanol, and background signals were minimized using SuperBlock blocking buffer (Thermo Scientific). After the blocking step, the tissue slides were blotted with anti-Ki67 primary antibody and an antibody against mouse IgG conjugated with horseradish peroxidase (HRP). The HRP signal was then detected using diaminobenzidine (DAB). Hematoxylin was used as a counterstain for nuclear evaluation. The tissues were then dehydrated and mounted with resin medium and glass coverslips.

[0059] TUNEL (terminal deoxynucleotidyl transferase dUTP nick and label) assay The TUNEL assay kit was purchased from Abcam (ab206386). The procedure was performed according to the manufacturer's instructions. Briefly, deparaffinized and rehydrated tissue slides were incubated in proteinase K solution for 20 minutes and in 3% H2O2 for 5 minutes, followed by incubation with TdT enzyme and reaction mixture at 37 °C for 1.5 hours. The signal was then detected and developed with DAB solution. Methyl green was used for counterstaining to assess healthy cells. The tissue slides were then dehydrated and mounted with resin medium and glass coverslips.

[0060] 5 / 6 nephrectomy Male C57BL / 6 mice aged 7–8 weeks were used in the experiment. 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 intraperitoneally injected with DMSO, WRG-28, or Av at 5 mg / kg / day for the next four weeks. Urine and blood were collected for blood urea nitrogen (BUN) and creatinine (CRE) testing, and kidneys were subjected to protein and histological analysis.

[0061] Blood urea nitrogen and creatinine detection The amounts of BUN and CRE in serum and urine were detected using an automated clinical chemistry analyzer (Fujifilm Corporation, DryChem 4000i). All procedures were performed according to the manufacturer's instructions.

[0062] Next-generation sequencing-based RNA sequencing and analysis Total RNA was extracted from kidneys by homogenizing samples in TRIzol Reagent (Invitrogen). Chloroform was added to separate the homogenate into three layers. The upper, clear aqueous layer containing the RNA was transferred to a new tube, and the RNA was precipitated by adding isopropanol. The pellet was washed with 70% ethanol and then dissolved in RNase-free water. RNA that passed quality controls, including an RNA integrity number (RIN) of >7 and an OD260 / OD280 of approximately 1.8, was sent for sequencing. Transcriptomes were analyzed by Ingenuity Pathway Analysis (IPA).

[0063] Bleomycin-induced pulmonary fibrosis model Eight to ten week-old male wild-type (C57BL / 6) mice were intratracheally instilled with 3 mg / kg bleomycin (Nippon Kayaku Co., Ltd.) diluted in PBS or PBS alone (sham). For fibrosis studies, lung samples were collected on day 21 for further analysis.

[0064] Pulmonary function tests Pulmonary function was assessed using the flexiVent system (Scireq, Montreal, Quebec, Canada). Mice were tracheotomized and mechanically ventilated at a rate of 150 breaths / min, a tidal volume of 10 ml / kg, and a positive end-expiratory pressure (PEEP) of 2-3 cmH2O. Deep inflation perturbations were used to estimate inspiratory volume (IC). A pressure-volume loop was generated by a constant increase in pressure followed by a rhythmic decrease in pressure. Other pulmonary function parameters, such as resistance, compliance, and elastance, were measured using the SnapShot-150.

[0065] Hydroxyproline assay Hydroxyproline levels in lung tissue were measured using a hydroxyproline colorimetric assay kit (BioVision, Milpitas, CA, USA). Briefly, 10 mg of frozen right middle lung lobes were homogenized in 100 μL of hydrochloric acid (HCl, 12N) and hydrolyzed at 120°C for 3 hours. Absorbance at 560 nm was then quantified using 10 μL of each sample. Hydroxyproline content was expressed in μg per lobe.

[0066] Histological examination Left lungs from mice were fixed overnight in 4% paraformaldehyde (PFA), embedded in paraffin, and sectioned at 5 μm thickness for Picrosirius Red (Abcam, Cambridge, UK) and Masson's Trichrome staining (Sigma-Aldrich, MO, USA). Fibrosis area was quantified using ImageJ software (NIH, http: / / rsbweb.nih.gov / ij / ).

[0067] statistical analysis All results were expressed as mean ± SEM. A two-tailed Student's t-test was used to compare differences between two groups in all experiments. Statistical significance was set at a P value of <0.05 using GraphPad Prism. In all graphs, *, **, and *** indicate P values ​​of <0.05, 0.01, and 0.001, respectively. [Example]

[0068] The following examples further describe exemplary embodiments of the present disclosure, but should not be construed as limiting the scope of the disclosure.

[0069] Example 1 WRG-28 and Av inhibit the activation of DDR2 and SOCE The results of 3D structural drug screening suggest that Av, Dh, and Th share structural similarities with WRG-28 and BTP2. To examine the effects of these compounds on DDR2 activation, HEK293T cells overexpressing DDR2 were pretreated with various compounds. Subsequently, fibrillar collagen was introduced into the culture medium and incubated for 12 hours. DDR2 activation was assessed using an anti-phosphotyrosine antibody. WRG-28 was used as a positive control. DDR2 activation was significantly reduced in cells pretreated with Av, Dh, and Th (Figure 1A).

[0070] Next, we investigated the effects of WRG-28, Av, Dh, and Th on SOCE in HEK293T cells. Both WRG-28 and Av significantly reduced the second peak and velocity of SOCE, whereas Dh and Th had no significant effect (Figures 1B, 1C, and 1D). Because of their structural similarity, WRG-28 and Av have great potential to target Orai1, the pore-forming domain of the CRAC channel that primarily governs SOCE in HEK293T cells. The effects of WRG-28 and Av on SOCE were also examined in rat renal fibroblast (NRK49F) cells (Figures 1E, 1F, and 1G) and human proximal tubule cells (HK-2) (Figures 1H, 1I, and 1J).

[0071] 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. CRAC channel activation was then induced by applying thapsigargin, a sarcoplasmic reticulum calcium-transporting ATPase (SERCA) pump inhibitor. Store depletion-induced Stim1 or Orai1 puncta formation was not affected by pretreatment with BTP2, WRG-28, or Av (Figure 1K, 1L, 1M, and 1N). This suggests that Stim1 aggregation or the coupling between Stim1 and Orai1 is not affected by these compounds.

[0072] Example 2 WRG-28 and Av suppress TGF-β1-induced fibroblast activation and pericyte to myofibroblast differentiation. Both DDR2 and CRAC channels are involved in fibroblast activation. Therefore, we performed a study to evaluate the effects of various compounds on rat kidney fibroblasts. TGF-β1-induced upregulation of collagen 1a1 and α-SMA was significantly suppressed by pretreatment of NRK49F cells with BTP2, WRG-28, or Av (Figures 2A and 2B). TGF-β1 treatment also induced upregulation of collagen 1a1 and α-SMA in pericytes (CCL-226), which was significantly suppressed by pretreatment with BTP2, WRG-28, or Av (Figure 2C). The results suggest the potent effects of WRG-28 and Av in inhibiting TGF-β1-induced fibroblast activation and pericyte-to-myofibroblast differentiation.

[0073] Extracellular matrix (ECM) alignment is a unique feature of myofibroblasts, altering the physical properties of the tissue microenvironment and posing a threat to tissue homeostasis in organ fibrosis. We then tested the effects of different compounds on the ECM alignment ability of myofibroblasts after TGF-β1 stimulation. Representative images show that upon TGF-β1 stimulation, the gel area significantly decreased in the control group (DMSO) after release from the culture dish, suggesting an increase in contractile force in NRK49F cells. Furthermore, gel contraction was significantly reduced in cells co-treated with BTP2, WRG-28, or Av (Figures 2D and 2E). Using FITC-labeled collagen to examine collagen fiber alignment, we observed vertical alignment of collagen fibers around the cells, especially in the TGF-β1-treated group (Figure 2F). Nevertheless, treatment with BTP2, WRG-28, or Av reduced TGF-β1-induced collagen alignment (Figures 2F, 2G-1, and 2G-2). Collectively, WRG-28 and Av inhibit TGF-β1-induced ECM remodeling.

[0074] Example 3 WRG-28 and Av inhibit TGF-β1-induced activation of cardiac, pulmonary, and hepatic fibroblasts. To evaluate the overall effects of WRG-28 and Av on TGF-β1-induced fibroblast activation, we used human cardiac fibroblasts (HCFs), human lung fibroblasts (MRC5), and rat hepatic stellate cells (HSC-T6). Although TGF-β1 treatment induced upregulation of α-SMA, this effect was suppressed across all cell types tested by cotreatment with various compounds, including BTP2, WRG-28, and Av (Figures 3A, 3B, and 3C). In HCFs, we examined TGF-β1-induced fibronectin and α-SMA expression. Treatment with BTP2, WRG-28, or Av again suppressed this effect (Figure 3D). Furthermore, treatment with TGF-β1 significantly increased the cell spreading area in HCFs, a phenomenon that was attenuated by cotreatment with BTP2, WRG-28, or Av (Figure 3D).

[0075] Cytosolic calcium measurements were performed to examine the effects of WRG-28 or Av on SOCE in various cell lines. Treatment with WRG-28 or Av significantly reduced SOCE in HCF (Figure 3E), MRC5 (Figure 3F), and HSC-T6 (Figure 3G). These results suggested a common effect of different compounds in attenuating TGF-β1-induced myofibroblast activation.

[0076] Example 4 WRG-28 and Av reduce cytokine secretion in T cells and macrophages CRAC channel activation is involved in T cell differentiation and cytokine expression. We then tested the effects of WRG-28 and Av on SOCE and cytokine secretion in both T cells and macrophages. Treatment with WRG-28 and Av showed variable ability to block SOCE in T cells (Figure 4A) and macrophages (Figure 4E). Overall, WRG-28 showed greater efficacy in inhibiting SOCE compared with Av, demonstrating similar potency to CM-4620, the most recent FDA-approved compound used to block cytokine release. Av showed lower potency but still significantly reduced SOCE in both T cells (Figure 4A) and macrophages (Figure 4E). The effectiveness of blocking SOCE was reflected in cytokine release. WRG-28, together with BTP2 and CM-4620, significantly reduced the secretion of IL-2 (Figure 4B) and TNF-α (Figure 4D) in T cells and IL-2 (Figure 4F), IL-6 (Figure 4G), and TNF-α (Figure 4H) in macrophages. Conversely, Av, although showing a relatively low potency, still significantly reduced the secretion of IL-2 (Figure 4C) and TNF-α (Figure 4D) in T cells. It also showed a slight reduction in the secretion of IL-2 (Figure 4F), IL-6 (Figure 4G), and TNF-α (Figure 4H) in macrophages. These results suggest that the variable capacities of WRG-28 and Av in SOCE may modulate immune responses to different degrees.

[0077] Example 5 WRG-28 and Av attenuate UUO-induced renal fibrosis To evaluate the efficacy of WRG-28 and Av in protecting the kidney from fibrotic injury, we performed conventional unilateral ureteral obstruction (UUO) surgery. Mice underwent UUO surgery and were simultaneously administered the test compounds for 7 days. Histological results showed that UUO treatment induced tubular and interstitial dilation. Nevertheless, administration of BTP2, WRG-28, or Av seemed to alleviate the effects caused by UUO (Figure 5A). Results from Sirius Red staining revealed the accumulation of fibrillar collagen in the tubulointerstitial space 7 days after UUO surgery. However, administration of BTP2, WRG-28, or Av significantly reduced the area positively stained for fibrillar collagen (Figures 5B and 5C).

[0078] Protein analysis revealed upregulation of mesenchymal marker proteins, such as integrin β1, DDR2, collagen 1a1, and α-SMA, along with downregulation of the epithelial marker protein, E-cadherin, in kidneys subjected to UUO treatment. Administration of BTP2 (Figures 5D and 5E), WRG-28 (Figures 5F and 5G), or Av (Figures 5H and 5I) suppressed the UUO-induced upregulation of integrin β1, DDR2, collagen 1a1, and α-SMA, but also reversed the UUO-induced downregulation of E-cadherin. These findings suggest the efficacy of WRG-28 and Av in inhibiting tubulointerstitial fibrosis.

[0079] Loss of tubular epithelial cells leads to tubular atrophy and impaired renal function. In kidneys treated with UUO, there was a significant increase in apoptotic tubular cells, which was significantly reduced in mice treated with BTP2, WRG-28, or Av (Figures 5J and 5K). These results suggest a protective effect of BTP2, WRG-28, and Av against UUO-induced tubular atrophy and apoptosis.

[0080] Example 6 WRG-28 and Av protect and promote tissue repair from obstruction-induced tissue damage and fibrosis The gradual increase in hydrostatic pressure and toxicity from accumulated waste products leads to irreversible damage in conventional UUO. Another drawback of conventional UUO is the inability to assess the remaining renal function of the injured side. Therefore, to gain further insight into the effects of investigational compounds on kidney protection or repair after injury, we performed a reverse unilateral ureteral obstruction (RUUO) procedure. The left ureter was surgically ligated for 7 days and then reconnected to the bladder. Our recent results show that injury and fibrosis markers are reversible when surgical reconnection is performed within 3 days of ligation. However, when reconnection is performed after 7 days of ligation, irreversible and persistent progression of fibrosis was observed.

[0081] 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 during ureteral obstruction (7* / 15 days). After 13 days of relief of obstruction, the right ureter was ligated, allowing assessment of renal function by measuring blood BUN and CRE levels.

[0082] The experimental procedure is shown in Figure 6A. Histological examination revealed severe fibrotic scarring in the DMSO-treated group. However, administration of WRG-28 and Av preserved intact tubular structures (Figure 6B). The collagen-positive area increased from 2% to 22% after removal of the obstruction in the control group (Figure 6C). Treatment with WRG-28 or Av significantly reduced the expansion of the fibrotic area (Figures 6B and 6C).

[0083] Relief of the occlusion for 13 days after 7 days of treatment resulted in irreversible fibrosis, maintaining high levels of mesenchymal marker proteins, including integrin β1, DDR2, collagen 1a1, and α-SMA, while maintaining low levels of epithelial marker proteins, such as E-cadherin, SGLT2, and NHE1. Administration of WRG-28 (Figures 6D and 6E) and Av (Figures 6F and 6G) ​​during occlusion significantly reduced the expression of mesenchymal marker proteins. Importantly, there was a significant increase in the expression of epithelial markers in mice treated with WRG-28 (Figures 6D and 6E) and Av (Figures 6F and 6G).

[0084] Example 7 WRG-28 or Av treatment reduces the persistent progression of obstruction-induced renal fibrosis after relief of obstruction. Next, we tested whether administration of WRG-28 or Av could promote kidney repair and reduce the persistent progression of fibrosis after relief of obstruction. Mice were injected with WRG-28 or Av for 13 days after relief of ureteral obstruction (7 / 15 days) (Figure 7A). WRG-28 and atovaquone (Av)-treated mice showed a greater number of intact tubular structures and less inflammation and scar tissue compared with DMSO-treated mice (Figure 7B, upper panel). Collagen-positive areas were also reduced by WRG-28 or Av treatment (Figure 7B, lower panel, and Figure 7C). Obstruction-induced upregulation of integrin β1, DDR2, collagen 1a1, and α-SMA was attenuated by WRG-28 (Figures 7D and 7E) and Av (Figures 7F and 7G) treatment. However, downregulation of epithelial marker proteins, E-cadherin, SGLT2, and NHE1, was only partially reversed in the WRG-28 (Figures 7D and 7E) and Av (Figures 7F and 7G)-treated groups. These results suggest that administration of WRG-28 and Av can reverse fibrosis and promote tissue repair.

[0085] Example 8 WRG-28 and Av restore epithelial differentiation and reduce collagen deposition and myofibroblast proliferation in the persistent progression of obstruction-induced fibrosis. Immunofluorescence studies further characterized the alignment and expression of the ECM in kidneys treated with different compounds at various time points. Laminin alpha 1, a major component of the basement membrane, forms a thin layer beneath tubular epithelial cells and between podocytes and endothelial cells in Bowman's capsule in control kidneys (sham; shown in Figure 8A). After 7 days of obstruction, kidneys with 15 days of relief (RUUO-7 / 15*d-DMSO) showed significant tubular atrophy accompanied by thickening of the basement membrane (Figure 8A and Figure 8A-(i)). The mean thickness was significantly increased compared to the sham group (Figure 8A-(i)). However, the WRG-28 and atovaquone (Av)-treated groups consistently showed uniform and unchanged basement membrane width, regardless of whether the obstruction was present (7* / 15d) or after its release (7 / 15*d) (Figure 8A and Figure 8A-(i)). Most notably, fewer atrophied tubules were observed in the WRG-28 and Av-treated group (Figures 8A and 8A-(i)). Furthermore, collagen 1a1 expression was significantly elevated in the DMSO-treated group (RUUO-7 / 15*d-DMSO), but administration of WRG-28 and Av reduced collagen 1a1 accumulation (Figures 8A and 8A-(ii)). α-SMA expression was significantly elevated in the DMSO-treated group but significantly decreased in the WRG-28 and Av-treated group (Figures 8B and 8B-(iii)). DDR2, a collagen upstream signaling protein, was significantly upregulated in the DMSO-treated group (RUUO 7 / 15*d-DMSO) (Figures 8D and 8D-(v)). However, administration of WRG-28 and Av reduced DDR2 expression compared with the DMSO-treated group (Figures 8D and 8D-(v)). These results suggest that administration of WRG-28 and Av reduces myofibroblast activation and subsequent ECM remodeling in renal fibrosis.

[0086] 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 tubule cells and localized to the apical lumen of healthy kidneys (sham, Figure 8C and Figure 8C-(iv)). Seven days of obstruction followed by 15 days of relief (RUUO-7 / 15*d-DMSO) induced downregulation of SGLT-2 (Figure 7D, Figure 7F, Figure 8C, and Figure 8C-(iv)). Furthermore, in SGLT-2-expressing cells, the protein lacked apical distribution in kidneys from mice treated with DMSO alone (Figure 8C and Figure 8C-(iv)). However, treatment with both WRG-28 and Av reversed apical membrane expression of SGLT-2. These phenomena were also evident in mice treated during obstruction that subsequently relieved SGLT2 protein levels, although only slight changes were observed in SGLT2 protein levels (Figures 7D, 7E, 7F, and 7G), which showed a distribution very close to normal (Figures 8C and 8C-(iv)). Similar phenomena were observed in other epithelial marker proteins, including AQP1 (Figures 8D and 8D-(vi)) and NHE1 (data not shown). Collectively, administration of WRG-28 and Av enhanced tubular differentiation in renal fibrosis.

[0087] The extreme imbalance between apoptotic and proliferative tubular cells, due in part to cellular senescence, leads to impaired repair in fibrotic kidneys. Administration of WRG-28 or Av during ureteral obstruction significantly reduced apoptotic cells (Figures 5J and 5K). Even after 15 days of relief of obstruction, a large number of apoptotic (TUNEL-positive) tubular cells remained present in DMSO-treated mice (Figures 8E and 8F). However, in the group treated with WRG-28 and Av during obstruction, only a small number of apoptotic cells were observed, and their number remained low even after 15 days of relief (Figures 8E and 8F). Although the number of proliferative tubular cells (Ki67-positive) did not differ significantly between groups, there were more proliferative interstitial cells in DMSO-treated mice compared with the WRG-28 and atovaquone (Av)-treated group (Figures 8E and 8F). Administration of WRG-28 and Av after relief of obstruction also significantly reduced the number of apoptotic cells (TUNEL-positive cells) (Figures 8G and 8H), suggesting both preventive and therapeutic effects of WRG-28 and Av. Importantly, administration of WRG-28 and Av after relief of obstruction also increased the number of proliferating tubular cells (Ki67-positive cells) (Figures 8G and 8H). Therefore, based on the above results, WRG-28 and Av treatment can alleviate failed repair and support tubular regeneration.

[0088] Example 9 Treatment with WRG-28 or Atovaquone (Av) Improves Renal Function in Obstruction-Induced Renal Injury Renal function was assessed by measuring serum BUN and CRE levels. The persistent progression of renal injury and fibrosis induced by obstruction resulted in 2-3 times higher plasma BUN and CRE levels. However, administration of WRG-28 or Av significantly reduced BUN and CRE levels compared with DMSO-treated mice (Figure 9A and Figure 9B). These results demonstrate the effectiveness of WRG-28 and Av in treating and protecting renal function.

[0089] Example 10 Transcriptome analysis in WRG-28 and Av-treated mice Total ribonucleic acid (RNA) was extracted from two groups: control mice (sham) and mice treated with DMSO, WRG-28, or Av after removal of the obstruction (7 / 15 d). The transcriptomes were then analyzed by ingenuity pathway analysis (IPA). Results showed a significant increase in inflammatory response, signaling, and fibrosis gene cohorts in the DMSO-treated group compared with mice without surgical treatment. Conversely, administration of WRG-28 and Av reduced inflammatory response and fibrosis signaling pathways (Figure 10A). Some of these genes are listed in Figure 10B. Large-scale transcriptome analysis further confirms the potent effects of WRG-28 and Av in preventing and protecting the kidney from progressive fibrosis.

[0090] Example 11 Treatment with WRG-28 or Av Effectively Reduces Pulmonary Fibrosis The efficacy of WRG-28 and Av in treating pulmonary fibrosis was investigated in vivo using a BLM-induced pulmonary fibrosis mouse model. 50 μL of bleomycin (BLM, 3 mg / kg) or PBS was administered intratracheally on day 0 to induce fibrosis or serve as a control, respectively. Various compounds were then administered for 14 days (nintedanib: 60 mg / kg / day, WRG-28: 5 mg / kg / day, Av: 5 mg / kg / day). Pulmonary function assessment was performed on day 21 (Figure 11A). Nintedanib, an FDA-approved drug for the treatment of idiopathic pulmonary fibrosis (IPF), was used as a positive control. The results of the pulmonary function assessment are shown in Figure 11B. These results indicate that administration of BLM leads to a decrease in lung volume and compliance while increasing lung resistance and elastance. The extent of these changes can be reversed by treatment with nintedanib, WRG-28, or Av. Hydroxyproline content, a marker of collagen deposition, was extracted and analyzed to demonstrate the extent of fibrosis, as shown in Figure 11C. While BLM can effectively induce pulmonary fibrosis, administration of nintedanib, Av, or WRG-28 inhibits the progression of fibrosis. Histological analysis was performed using staining techniques to quantify the fibrotic area. Both Masson's trichrome staining and picrosirius red staining demonstrate that treatment with nintedanib, Av, or WRG-28 effectively reduces the fibrotic area (Figure 11D).

[0091] Those skilled in the art will readily appreciate that numerous modifications and variations of the present method may be made while retaining the teachings of the present invention. Accordingly, the above disclosure should be construed as limited only by the metes and bounds of the appended claims.

Claims

1. 1. A pharmaceutical composition for use in the treatment or prevention of a CRAC channel-associated disorder or condition, and / or a DDR2-associated disorder 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 and pharmaceutical compositions comprising such pharmaceutically acceptable carriers.

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

3. The pharmaceutical composition of claim 2, wherein the CRAC channel-associated disorder or condition and / or the DDR2-associated disorder or condition is the cytokine storm syndrome.

4. The pharmaceutical composition according to claim 3, wherein the cytokine storm syndrome is an infection-induced cytokine storm syndrome.

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

6. The pharmaceutical composition of claim 5 , wherein the cytokine is an interleukin or a tumor necrosis factor.

7. The pharmaceutical composition of claim 2, wherein the CRAC channel-associated disorder or condition and / or the DDR2-associated disorder or condition is a fibrotic disorder.

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

9. 8. The pharmaceutical composition of claim 7, wherein the treatment or prevention of fibrotic disorders improves renal, pulmonary, hepatic, and / or cardiac function; promotes tissue repair and / or epithelial differentiation; and / or inhibits collagen deposition, myofibroblast proliferation, and / or TGF-β-associated fibroblast activation.

10. 10. The pharmaceutical composition of claim 9, wherein the treatment or prevention of a fibrotic disorder improves the renal and / or pulmonary function; promotes the tissue repair and / or the epithelial differentiation; and / or inhibits the collagen deposition, the myofibroblast proliferation, and / or the TGF-β-associated fibroblast activation.

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

12. The WRG-28, the atovaquone, the WRG-28 precursor, or the atovaquone precursor activates CRAC channels, DDR2, store-operated Ca ２＋ The pharmaceutical composition of claim 1 , which inhibits influx and / or cytokine expression.

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

14. 14. The pharmaceutical composition of claim 13, wherein the cytokine is interleukin 2, interleukin 6, or tumor necrosis factor alpha.

15. 1. A pharmaceutical composition for use in inhibiting CRAC channel activation and / or DDR2 activation in a cell of a subject, 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 and pharmaceutical compositions comprising such pharmaceutically acceptable carriers.

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