New application of Selpatinib

Abstract

The invention discloses a novel application of Selpatinib, and belongs to the field of biological medicines. It is found for the first time that the Selpatinib inhibits retinal pigment epithelial cells from being converted into mesenchymal cells, and the inhibition action mechanism of the Selpatinib is further explored as follows: the TGF beta signal transduction process is stopped by inhibiting the combination of RET and a TGFBR1 receptor I and then blocking RET from entering a nucleus (membrane), and the action mechanism of the Selpatinib RET inhibitor is broken through. The invention discovers the new application of the Selpatinib in treating the ophthalmic diseases related to epithelial-mesenchymal transition of retinal pigment epithelial cells, not only broadens the medicinal application of the Selpatinib and improves the medicinal value of the Selpatinib, but also provides a more targeted new medicine for the ophthalmic diseases related to EMT (epithelial-Mental Transformation), so that the application of the Selpatinib in treating the ophthalmic diseases related to the EMT is developed. The research and development of medicines for inhibiting epithelial-mesenchymal transition related diseases are promoted, and the application prospect is relatively great.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a new use of selpatinib. Background Art

[0002] Selpercatinib (alias: LOXO-292; ARRY-192; Retevmo) is an effective, orally bioavailable, highly selective RET (Receptor Tyrosine Protein kinase) tyrosine kinase inhibitor. It can not only inhibit normal RET signaling, but also combat possible drug-resistant mutations. It is the first highly selective RET inhibitor approved by the FDA. RET, or receptor tyrosine kinase, is a proto-oncogene encoding a transmembrane glycoprotein that interacts with ligands (GFRα1-4) of the glial cell line-derived neurotrophic factor (GDNF) family. If RET becomes abnormal (fusion, rearrangement, or mutation, etc.) and causes it to be overly and continuously activated independent of its ligand, it can trigger abnormal cell proliferation and induce cancer. According to statistics, RET abnormalities can be detected in about 2% of cancers, especially in non-small cell lung cancer, thyroid cancer, etc. Serpatinib binds to the active site of RET kinase, inhibiting the activity of activated point mutants and dimeric RET fusion proteins, thereby blocking downstream signals that promote proliferation and survival. In 2020, Serpatinib was approved for the U.S. market, becoming the first personalized therapeutic drug specifically for cancer patients carrying RET gene mutations.

[0003] Epithelial-mesenchymal transition (EMT) is the process of epithelial cells transforming into mesenchymal cell characteristics under physiological or pathological conditions. In this process, epithelial cells lose polarity and adhesion and acquire mesenchymal characteristics. Retinal pigment epithelial cells (RPE) are a single layer of cells located between the retina and the vascular choroidal vascular layer and are the basic component of the retina. Normal RPE cell layers are tightly connected in a regular manner, and their functions include maintaining selective nutrition and material metabolism, secreting growth factors, participating in the visual cycle, maintaining the blood-retinal barrier, and phagocytosis and antioxidants. However, pathological conditions such as aging, oxidative stress, and inflammation can cause RPE to acquire the characteristics of mesenchymal cells, that is, the RPE-EMT process occurs. Although a variety of signaling molecules can induce this process, the TGFβ signaling pathway is currently known to be the most critical and powerful induction signal for initiating and regulating RPE-EMT. TGFβ directly or indirectly regulates the occurrence and development of EMT by affecting the expression of transcription factors, cell adhesion molecules, and matrix metalloproteinases through its receptor TGFβ receptors (TGFBRs). RPE-EMT can lead to loss of RPE polarity and tight junctions, which in turn may lead to or aggravate major ophthalmic diseases such as age-related macular degeneration (AMD), proliferative vitreoretinopathy (PVR), diabetic retinopathy (DR), central serous chorioretinopathy (CSC), retinal detachment (RD), and secondary cataract with posterior capsule opacity (PCO).

[0004] Over the years, from basic to clinical and various drug research and development institutions, a lot of manpower and material resources have been invested in the development of targeted inhibitors of TGFβ and its receptors, but the progress is slow, and the existing potential inhibitors have significant limitations, which affect their promotion and use in clinical practice. First, the TGFβ signaling pathway plays an important role in a variety of physiological processes, including immune regulation, cell proliferation and differentiation. Therefore, inhibition of TGFβ signaling may lead to a series of adverse reactions and side effects, such as immune system suppression and wound healing disorders. Secondly, the function and regulation of the TGFβ signaling pathway may be different in different types of cells and tissues, so the specificity and selectivity of inhibitors may be low, affecting the function of normal cells and tissues. In addition, there are interactions between the TGFβ signaling pathway and other signaling pathways, and inhibition of TGFβ signaling may affect the function of other signaling pathways. In fact, there is still a lack of specific targeted drugs for the TGFβ signaling pathway in the clinic for the treatment of RPE-EMT-related diseases. Therefore, the discovery of new TGFβ signaling pathway inhibitors has important clinical value.

[0005] At present, clinical use of selpatinib is mainly for specific cancer types (adult patients with RET fusion-positive metastatic non-small cell lung cancer, adults and children aged 12 and above with advanced or metastatic medullary thyroid cancer carrying RET mutations who require systemic treatment, and adults and children aged 12 and above with advanced or metastatic thyroid cancer who are refractory to systemic treatment and radioactive iodine treatment (if applicable), with a small and limited range of therapeutic applications. Therefore, further research and development of new uses for selpatinib has great market value. Summary of the invention

[0006] The purpose of the present invention is to provide a new use of selpatinib to solve the problems existing in the above-mentioned prior art. The present invention has discovered for the first time a new use of selpatinib for treating ophthalmic diseases related to epithelial-mesenchymal transition of retinal pigment epithelial cells, which not only broadens the drug use of selpatinib and improves the drug value of selpatinib, but also provides a more targeted new drug for ophthalmic diseases related to EMT, promotes the research and development of drugs for diseases related to inhibiting epithelial-mesenchymal transition, and has great application prospects.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides the use of selpatinib in preparing a medicine for treating epithelial-mesenchymal transition of retinal pigment epithelial cells.

[0009] The present invention also provides the use of selpatinib in preparing a drug for treating ophthalmic diseases associated with epithelial-mesenchymal transition of retinal pigment epithelial cells.

[0010] Optionally, selpatinib treats the ophthalmic disease by inhibiting epithelial-mesenchymal transition of retinal pigment epithelial cells.

[0011] Optionally, selpatinib inhibits epithelial-mesenchymal transition of retinal pigment epithelial cells by inhibiting TGFβ1 signaling pathway.

[0012] Optionally, the ophthalmic diseases include age-related macular degeneration, proliferative vitreoretinopathy, diabetic retinopathy, central serous chorioretinopathy, retinal detachment and secondary cataract and posterior capsule opacity.

[0013] Optionally, the dosage form of the drug includes injection.

[0014] Optionally, the drug is administered by intravitreal injection.

[0015] The present invention discloses the following technical effects:

[0016] The present invention found through in vitro cell experiments that selpatinib can inhibit the transformation of retinal pigment epithelial cells to mesenchymal cells. Serpatinib blocks the TGFβ signal transduction process by inhibiting the binding of RET to TGFβ receptor I (TGFBR1) and blocking RET from entering the nucleus (membrane). This process breaks through the known mechanism of action of selpatinib as a RET inhibitor and provides a new drug with strong targeting and good therapeutic effect for EMT-related ophthalmic diseases. The present invention lays a foundation for further research and development of more treatment schemes for other diseases related to the TGFβ signaling pathway.

[0017] The present invention discovered for the first time a new use of selpatinib for the treatment of ophthalmic diseases related to epithelial-mesenchymal transition of retinal pigment epithelial cells, which not only broadens the pharmaceutical use of selpatinib and improves the pharmaceutical value of selpatinib, but also provides a more targeted new drug for ophthalmic diseases related to EMT, promotes the research and development of drugs for diseases related to epithelial-mesenchymal transition, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The results of TGFβ1-induced RPE-EMT process; A is the morphology of normal ARPE cells and ARPE cells after 48 hours of TGFβ1 induction; B is the result of EMT marker q-PCR of the two groups of treated cells; C is a representative figure of the results of EMT marker western-blot of the two groups of treated cells; D is a statistical graph of the results of EMT marker western-blot of the two groups of treated cells;

[0020] Figure 2The figure is the result of serpatinib preventing the EMT process induced by TGFβ1; A is the cell survival at different TGFβ1 treatment time and different inhibitor treatment concentrations; B is the normal ARPE cells and ARPE cells after TGFβ1 induction for 48h and cells treated with different concentrations of inhibitors; C is the result of EMT marker q-PCR of the three groups of treated cells; D is the representative figure and data statistical figure of the results of EMT marker western-blot of the three groups of treated cells; G is the representative figure of cell immunofluorescence, which are EMT markers: α-SMA and ZO-1; H is the statistical figure of the fluorescence intensity of α-SMA; I is the statistical figure of the number of intact cells after ZO1 staining;

[0021] Figure 3 The results of the cell migration scratch experiment are shown in Figure 1. A is a representative diagram of three groups of cell scratch experiments; B is a statistical diagram of the scratch experiment data;

[0022] Figure 4 The results of cell migration in Transwell are shown in Figure 1. A is a representative diagram of three groups of cells in Transwell; B is a statistical diagram of the data of the Transwell experiment;

[0023] Figure 5 The figure is the result of Ki67 cell immunofluorescence staining in the cell proliferation experiment; A is the representative figure of Ki67 immunofluorescence in three groups of cell experiments; B is the statistical figure of Ki67 immunofluorescence data in three groups of cell experiments;

[0024] Figure 6 The results of the cell immunofluorescence experiment of the EMT process of mouse RPE layer cells induced by TGFβ1 injection into the mouse vitreous body; A is a schematic diagram of the vitreous injection; B is a representative immunofluorescence image of the mouse retinal pigment epithelial cell layer spread; C is a comparison of the cell area of ​​the three groups of experiments; D is a comparison of the cell perimeter of the three groups of experiments; E is a comparison of the cell aspect ratio of the three groups of experiments; F is a comparison of the cell roundness of the three groups of experiments;

[0025] Figure 7 The results of the cell immunofluorescence experiment are shown in Figure 1. A is a representative immunofluorescence image; B is a statistical graph of the RET fluorescence intensity of the cell membrane, nucleus, and cytoplasm;

[0026] Figure 8 This is a graph showing the results of a protein interaction Co-IP experiment. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The CAS number of Selpercatinib used in the present invention is: 2152628-33-4, and the molecular formula is: C 29 H 31 N 7 O 3 , molecular weight is 525.6015. It can be obtained through conventional purchase channels in the art.

[0033] Serpatinib is currently clinically used to treat some specific RET mutation-related thyroid cancer and non-small cell carcinoma. After in-depth research on serpatinib, the inventor unexpectedly found that serpatinib can inhibit the TGFβ1 signaling pathway, and thereby prevent the pathological epithelial-mesenchymal transformation of retinal pigment epithelial cells induced by the TGFβ1 signaling pathway. Since the epithelial-mesenchymal transformation of retinal pigment epithelial cells is the primary factor leading to major ophthalmic diseases such as age-related macular degeneration (AMD), proliferative vitreoretinopathy (PVR), diabetic retinopathy (DR), central serous chorioretinopathy (CSC), retinal detachment (RD) and secondary cataract posterior capsule opacity (PCO). Therefore, the inventor further confirmed through cell and animal experiments that serpatinib can effectively prevent the pathological epithelial-mesenchymal transformation process of retinal pigment epithelial cells induced by the TGFβ1 signaling pathway. At the same time, the present invention also discovered the relevant mechanism of RET in the RPE-EMT process through experiments, avoiding the influence of general TGFβ signaling pathway blockers on patients or treatment objects, and can more specifically respond to and treat EMT-related ophthalmic diseases.

[0034] Optionally, the effective concentration of selpatinib for inhibiting the migration and proliferation of retinal pigment epithelial cells induced by the TGFβ1 signaling pathway is 1 to 5 μmol / L.

[0035] In a specific embodiment, the inventors confirmed through in vitro experiments on ARPE-19 cells that the TGFβ1 signaling pathway can induce the occurrence of the EMT process of RPE cells, and after the cells were treated with 1μM (μmol / L) concentration of serpatinib, the EMT process induced by the TGFβ1 signaling pathway can be effectively blocked, and the cell viability is not affected. At the same time, the inventors also confirmed through in vitro cell experiments that 1μM concentration of serpatinib can effectively prevent the migration and proliferation of retinal pigment epithelial cells.

[0036] In addition, the inventors conducted an experiment on mice with proliferative vitreoretinopathy induced by vitreous injection of TGFβ1 and confirmed that selpatinib can successfully prevent the EMT process of RPE layer cells in mice, which fully demonstrated that selpatinib has the effect of treating ophthalmic diseases related to epithelial-mesenchymal transition of retinal pigment epithelial cells.

[0037] In a specific embodiment, the injection concentration of selpatinib in mice with proliferative vitreoretinopathy model induced by inhibiting TGFβ1 signaling pathway is 0.5 mmol / L.

[0038] The present invention is described in detail below in conjunction with specific embodiments.

[0039] Example 1 Experiment on the inhibition of TGFβ1-induced EMT process of RPE cells by serpatinib

[0040] 1. Purpose of the experiment

[0041] A TGFβ1-induced RPE cell EMT model was established to verify that selpatinib can inhibit the TGFβ1-induced EMT process.

[0042] Establishment of TGFβ1-induced RPE cell EMT model: This model is currently known to be recognized and widely used RPE-EMT model. The expression levels of cell tight contact markers ZO1, FN1, α-SMA, snail and cell morphology can explain the loss of polarity and tight contact.

[0043] 2. Experimental methods

[0044] (1) Experiment on the occurrence of TGFβ1-induced EMT in RPE cells

[0045] To perform ARPE-19 cell culture experiments, cells need to be seeded into 12-well plates at a density of 1×10 5 cells, using DMEM / F12 medium containing 10% FBS. When the cell fusion rate reached 70% to 80%, the medium was replaced with serum-free DMEM / F12 medium. The purpose of this is to reduce the influence of serum on the experimental results and provide a more accurate basis for subsequent experimental operations. The cells were then pretreated with 20ng / mL TGFβ1 with or without 1μM Selpercatinib for 6 to 8 hours. The morphology of the cells was observed under a microscope, and whether they had a scattered, elongated fibroblast-like morphology was assessed. This morphological change may indicate that the cells have undergone epithelial-mesenchymal transition (EMT), which is the process of cell transformation from epithelial cells to mesenchymal cells. By observing and evaluating the cell morphology, it can be preliminarily determined whether ARPE-19 cells have undergone EMT, providing important reference and guidance for subsequent experiments.

[0046] (2) CCK8 experiment

[0047] 100 mL of cell suspension (about 1×10 4 cells / well) at 37°C / 5% CO 2Cultured in a constant temperature and humidity incubator. When the cells reached 75% to 80% confluence, they were replaced with serum-free medium. After pretreatment with Selpercatinib for 6 to 8 hours, the cells were treated with 20ng / mL TGFβ1. After culturing for 24 hours, 10μL of CCK-8 solution was added to the corresponding wells of the well plate (be careful not to introduce bubbles into the wells, which may interfere with OD value detection), and the 96-well plate was placed in the incubator for 1 hour. After the absorbance at 450nm was measured by the microplate reader, the liquid in the corresponding well was aspirated and the remaining cells were placed in the incubator for continued culture. The absorbance was measured at 450nm at 48h and 72h, respectively.

[0048] Cell viability (%) = [(AS-Ab) / (Ac-Ab)] × 100, AS = absorbance of experimental wells (absorbance of wells containing cell culture medium, CCK-8 and test compounds), Ab = absorbance of blank wells (absorbance of wells containing culture medium and CCK-8), Ac = absorbance of control wells (absorbance of wells containing cells, culture medium and CCK-8).

[0049] (3) Cell protein extraction and western blotting experiments

[0050] ① Extraction of total cell protein:

[0051] Wash the cells with 4°C pre-cooled PBS solution, remove the PBS, add a small amount of pre-cooled PBS, and collect the cells into a 1.5mL EP tube with a suitable cell scraper. Centrifuge at 2500rpm for 10 minutes at 4°C to precipitate the cells, and then remove the supernatant. Next, add RIPA cell lysis buffer containing protease inhibitors and rotate at 4°C for 30 minutes to lyse the cells. After that, centrifuge at 12000rpm for 10 minutes, collect the supernatant and transfer it to a new EP tube.

[0052] ②Determination of protein concentration:

[0053] According to the instructions of the BCA kit, mix solution A and solution B in a ratio of 50:1 to prepare the test solution and add it to each well of the ELISA plate, 200 μL per well. Then, add the standard protein solution to the wells of the ELISA plate according to a certain gradient to establish a standard curve. Take 2 μL of protein sample and add it to the test well. After gently mixing, place the plate in a 37°C environment for 30 minutes. Use an ELISA reader to read the absorbance at a wavelength of 550 nm. According to the standard curve, calculate the protein concentration of the sample and adjust the sample concentration to maintain consistency. Finally, add 6× SDS buffer and boil at 95°C for 5 minutes to prepare the sample.

[0054] ③ Protein blotting:

[0055] A. First, you need to prepare the SDS-PAGE gel: For the stacking gel, a fixed concentration of 3.75% is usually used, while the concentration of the separation gel is set according to the molecular weight of the target protein. Proteins with smaller molecular weights require a higher concentration of separation gel. For example, for an 8% separation gel, the solidification time at 25°C is about 20 to 30 minutes, while it may take 40 to 50 minutes in winter. Once a clear dividing line is formed between the separation gel and the upper liquid, the upper liquid can be removed and the stacking gel can be prepared. The solidification time is about 15 minutes at 25°C.

[0056] B. Electrophoresis: Carefully add the protein sample that has been stained with bromophenol blue and heat-treated to the electrophoresis tank. Initially, set the voltage to 80V for about 30 minutes until the sample completely enters the separation gel layer from the stacking gel layer. Subsequently, increase the voltage to 100V and continue electrophoresis until the protein sample reaches the bottom of the gel.

[0057] C. Transfer: Remove the concentrated gel layer and soak the separation gel layer in transfer buffer for several minutes. Prepare a nitrocellulose membrane that matches the size of the separation gel, use a "sandwich" structure for transfer, and perform electrophoresis at a constant voltage of 100V for 40 to 60 minutes to transfer the protein from the gel to the membrane. After transfer, remove the membrane, mark the front and back sides, and gently wash with 1×TBS-T solution, then block with 5% skim milk and let stand at room temperature for 1 hour.

[0058] D. Antibody incubation: After blocking, dilute the antibody with 5% skim milk according to the type of antibody and incubate overnight on a shaker at 4°C. The next day, remove the membrane from the milk solution and gently wash it three times with 1×TBS-T solution, 5 minutes each time; then add the secondary antibody, incubate it at room temperature for 1 hour after the same dilution, and then wash it three times with 1×TBS-T solution, 5 minutes each time.

[0059] E. Development: Mix ECL colorimetric solution A and B in a ratio of 1:1, use absorbent paper to absorb the excess liquid on the surface of the membrane, and then evenly apply the mixed colorimetric solution on the surface of the membrane. Place the membrane into a chemiluminescent imaging device for development and save the results.

[0060] 3. Experimental results:

[0061] The experimental results are as follows Figure 1 and Figure 2 As shown. Figure 1 This is the result of TGFβ1 inducing the EMT process of RPE cells. Figure 2 This figure shows the result that selpatinib can effectively prevent the EMT process induced by TGFβ1 in ARPE-19 cell in vitro experiments.

[0062] from Figure 1 and Figure 2 It can be seen that in the in vitro experiment of ARPE-19 cells, after 48 hours of induction of retinal pigment epithelial cells with TGFβ1 at a concentration of 20ng / mL, almost 100% of the cells underwent EMT, that is, they changed from tightly connected regularly arranged polygons to more slender spindle-shaped cells. After pre-treatment with 1μM concentration of selpatinib, more than 75% of the cells were still tightly connected regularly arranged polygons, indicating that they did not transform into mesenchymal cells. This shows that treatment with 1μM concentration of selpatinib can effectively prevent the EMT process induced by TGFβ1, and cell viability is not affected.

[0063] Example 2 Serpatinib inhibits the migration and proliferation of RPE cells

[0064] 1. Purpose of the experiment

[0065] Verify that selpatinib has the effect of inhibiting the migration and proliferation of RPE cells.

[0066] 2. Experimental methods

[0067] 2.1 Scratch test

[0068] (1) Before the experiment begins, use alcohol to disinfect the ruler and marker pen and place them on a clean bench to be sterilized by ultraviolet light for 30 minutes.

[0069] (2) Using a sterilized ruler and marker, draw three straight lines evenly spaced approximately 0.5-1 cm apart on the back of a 12-well culture plate.

[0070] (3) Digest each group of cells, count them, and add 1×10 5 Gently shake the plate to ensure that the cells are evenly distributed, then return the plate to the cell culture incubator for continued culture.

[0071] (4) After the cells have grown to cover the holes, use a sterile 100 μL pipette tip to scratch the plate perpendicular to a straight line on the back of the culture plate. During the scratching process, make sure the pipette tip remains vertical to avoid tilting, and move quickly to maintain the continuity of the scratch.

[0072] (5) Wash the cells three times with PBS buffer to remove unattached cells, and then add serum-free DMEM / F12 medium. Observe and photograph the cells under an inverted microscope, and randomly select three scratch areas for each group to record.

[0073] (6) Place the culture plate back into the cell culture incubator and continue culturing. Observe and photograph the cell status again after 24 and 48 hours.

[0074] (7) Use ImageJ software to analyze the width changes of the scratch area.

[0075] The above steps were repeated three times while keeping the experimental conditions consistent.

[0076] 2.2 Transwell experiment

[0077] (1) Preparation of cell suspension: First, ARPE-19 cells were starved in serum-free medium for 24 hours, and then the cells were collected by trypsin treatment and centrifugation. The cells were resuspended in serum-free DMEM / F12 medium and counted. To terminate the digestion process, serum-containing medium was added, followed by centrifugation at 1000 rpm, and the supernatant was removed. The cells were washed 1 to 2 times with PBS solution until the cell suspension concentration reached 4×10 5 Pieces / mL.

[0078] (2) Inoculation of cells: Add 600 μL of DMEM / F12 medium containing 10% FBS to the lower chamber of a 24-well plate. For the TGFβ1-treated group, add 20 ng / mL of TGFβ1. Place a Transwell chamber with a pore size of 8 μm in a 24-well plate and add 200 μL of the cell suspension to the upper chamber of the Transwell chamber. Ensure that there are no bubbles between the Transwell chamber and the culture medium in the lower chamber of the 24-well plate. If there are bubbles, re-cover the chamber until the bubbles disappear.

[0079] (3) Cultivating cells: Place the cells in a biosafety cabinet for 1 minute, then place the 24-well culture plate containing the Transwell chamber in a constant temperature cell culture incubator and continue culturing for 18 hours.

[0080] (4) Cell staining: Remove the Transwell chamber, remove the culture medium, and wash twice with PBS. Then immerse the chamber in 4% paraformaldehyde solution and fix it at room temperature for 20 minutes. Gently wipe the RPE cells that have not migrated in the upper chamber with a cotton swab, and then wash twice with PBS. Next, use crystal violet staining solution to stain the RPE cells that have migrated to the lower chamber for 15 minutes, and then wash twice with PBS.

[0081] (5) Statistical results: The migration of ARPE-19 cells to the lower chamber was observed and recorded using an inverted phase contrast microscope. Three fields of view were randomly selected from each group at a magnification of 20×, and the number of RPE cells that migrated to the lower chamber in each field of view was counted and analyzed.

[0082] 2.3. Ki67 immunofluorescence experiment:

[0083] (1) Cell culture: ARPE-19 cells were seeded at an appropriate density into a 24-well plate containing a cell slide and cultured to an appropriate density.

[0084] (2) Cell fixation: After cell treatment, the culture medium was removed, the cells were washed with 1× PBS buffer and then discarded, 500 mL of 4% paraformaldehyde solution was added to fix the cells, and the solution was removed after standing at room temperature for 15 minutes.

[0085] (3) Wash again: Add 500 μL of room temperature 1× PBS buffer, place the well plate on a shaker, gently wash at 100 rpm for 5 minutes, and then remove the solution.

[0086] (4) Cell membrane permeabilization: Add 500 L of 0.3% TritonX-100 solution to permeabilize the cell membrane, let stand at room temperature for 5 minutes, and then remove the solution.

[0087] (5) Repeat the washing step: add 500 mL of room temperature 1× PBS buffer again and wash the cells in the same way.

[0088] (6) Blocking nonspecific binding sites: Add sterile water to a wet box and place the plate in the well, add 150 μL of 1% BSA solution for blocking, and incubate at 37°C for 1 hour.

[0089] (7) PBST washing: Add 500 μL of room temperature 1× PBST buffer, wash the cells at 100 rpm for 5 minutes, and remove the solution.

[0090] (8) Primary antibody incubation: Remove the glass slide, absorb excess liquid, and then drop 50 μL of diluted primary antibody on the plastic plate. Place the glass slide with the cell side facing down on the antibody and incubate at 37°C for 1 hour.

[0091] (9) Repeat PBST washing: Add 500 μL of room temperature 1× PBST buffer again, wash the cells for 5 minutes, and repeat four times.

[0092] (10) Secondary antibody incubation: Place the glass slide back into the well plate with the cell side facing up, add 150 mL of secondary antibody dilution, seal with tin foil to avoid light, and incubate at 37°C for 1 hour.

[0093] (11) Washing: Add 500 mL of room temperature 1× PBST buffer and wash the cells at 100 rpm for 5 minutes. Repeat four times. This step must be performed in a dark room and always kept away from light.

[0094] (12) Sealing: In a dark room, add a sealing solution containing DAPI to the glass slide, remove the glass slide, absorb the excess liquid, place the cells face down on the sealing solution, fix with nail polish, and store at 4°C.

[0095] (13) Imaging: Image acquisition was performed using a fluorescence microscope.

[0096] 3. Experimental results:

[0097] The experimental results are as follows Figure 3 to Figure 5 As shown. Among them: Figure 3 The results of the cell migration scratch experiment are shown in Figure 2. Figure 4 This is the result of cell migration Transwell. Figure 5 This is the result of cell proliferation experiment - Ki67 cell immunofluorescence staining.

[0098] from Figure 3 to Figure 5 It can be seen that:

[0099] (1) Through the cell migration experiment-scratch experiment, it was found that the migration rate of normal retinal pigment epithelial cells at 24h was 38%, while the migration rate of retinal pigment epithelial cells induced by 20ng / mL TGFβ1 was 58%, but the migration rate of retinal pigment epithelial cells pretreated with 1μM concentration of selpatinib and induced by 20ng / ml TGFβ1 was 36%. Similarly, the migration rate of normal retinal pigment epithelial cells at 48h was 57%, while the migration rate of retinal pigment epithelial cells induced by 20ng / mL TGFβ1 was 88%, but the migration rate of retinal pigment epithelial cells pretreated with 1μM concentration of selpatinib and induced by 20ng / mL TGFβ1 was 54%. The above data show that selpatinib can effectively prevent the migration of retinal pigment epithelial cells.

[0100] (2) Through the cell migration experiment-Transwell experiment, it was found that the average number of cells in the normal treatment group that passed through was 89 / standard visual field, the average number of cells in the treatment group that was induced by 20ng / mL TGFβ1 was 275 / standard visual field, and the average number of cells in the treatment group that was pretreated with 1μM concentration of selpatinib and induced by 20ng / mL TGFβ1 was 132 / standard visual field. The above data show that selpatinib can effectively prevent the migration of retinal pigment epithelial cells.

[0101] Through the cell proliferation experiment-ki67 cell immunofluorescence staining, it was found that the higher the percentage of positive cells, the higher the cell proliferation. The average percentage of positive cells in the normal treatment group was 7.5%, the average percentage of positive cells in the treatment group induced by 20ng / mL TGFβ1 was 18.5%, and the average percentage of positive cells in the treatment group induced by 20ng / mL TGFβ1 and pretreated with 1μM concentration of selpatinib was 6.8%. The above data show that selpatinib can effectively prevent the proliferation of retinal pigment epithelial cells.

[0102] Example 3 Experimental study on the process of serpatinib preventing EMT of RPE layer cells in mouse eyes

[0103] 1. Purpose of the experiment

[0104] It was verified that selpercatinib can effectively prevent the EMT process of RPE layer cells in the eyes of mice with proliferative vitreoretinopathy model induced by injection of TGFβ1.

[0105] 2. Experimental methods

[0106] 2.1 In vivo experiments in mice

[0107] Mice were injected intravitreally, and three days later, samples were taken for perfusion and immunofluorescence analysis:

[0108] The proliferative vitreoretinopathy animal model was induced by intravitreal injection of TGFβ1 in mice, with two mice in each group (four eyes, n=4). The treatments were as follows: the control group mice were injected with 2.5 μL of PBS solution, the experimental group 1 mice were injected with 2 μL of 20 ng / mL TGFβ1+0.5 μL PBS, and the experimental group 2 mice were injected with 2 μL PBS+0.5 μL 0.5 mM selpercatinib.

[0109] (1) Mice were anesthetized with sodium pentobarbital and then perfused.

[0110] (2) Cut the eyeball open along the limbus of the cornea and sclera, cut off the optic nerve, and separate the neural retina. Use one forceps to hold the RPE at the back, and use the other forceps to pull out the white neural retina, and try to shake the remaining RPE-choroid-sclera complex into a bowl shape.

[0111] (3) Place the RPE in a sterile 1.5 mL centrifuge tube containing 1 mL of 4% PFA, fix at room temperature for 15 min, discard the solution, and wash with PBS three times, 5 min each time.

[0112] (3) Permeabilize with 0.3% triton X100 for 15-30 minutes, discard the solution, and wash with PBS three times, 10 minutes each time.

[0113] (4) Block with 2.5% BSA for 30 minutes.

[0114] (5) Primary antibody incubation: Add the diluted primary antibody mixture and incubate at 4°C overnight.

[0115] (6) Secondary antibody incubation: Wash with PBS three times, 10 min each time, add appropriate amount of fluorescent secondary antibody dilution solution and incubate at room temperature in the dark for 1 h.

[0116] (7) Nuclear staining: Wash with PBS three times, 10 min each time, add appropriate amount of DAPI dilution, and incubate at room temperature in the dark for 10 min.

[0117] (8) Sealing: Take out the RPE-choroidal sclera complex from the centrifuge tube and place it on a glass slide. Cut it into a petal-like shape along the lower edge of the cornea and sclera. Add anti-fluorescence attenuation sealing agent to seal the slide. Observe and photograph under an inverted fluorescence microscope.

[0118] 2.2. Cell immunofluorescence experiment is as shown in 2.3 of Example 2.

[0119] 3. Experimental results:

[0120] The experimental results are as follows Figure 6 As shown. Among them: Figure 6 This is the result of a cell immunofluorescence experiment to show the EMT process of RPE cells induced by intravitreal injection of TGFβ1.

[0121] from Figure 6 It can be seen that by observing the marker of the EMT process: ZO-1, it was found that serpatinib successfully blocked the EMT process of RPE layer cells. The area, perimeter, aspect ratio and roundness of the cells have all changed significantly. In the experimental group 1 mice, the cell area increased significantly (the increase in cell area indicates that the cell structure is destroyed and the cells are unevenly arranged); the cell perimeter increased significantly (the increase in cell perimeter also indicates that the cell structure is destroyed, and the destruction indicates that the cells have a tendency to transform into mesenchyme); the cell aspect ratio increased significantly (indicating that the cells are elongated and the structure is destroyed); the roundness of the cells decreased (the normal cell roundness is close to 1, which indicates that the cells have changed from tightly arranged polygonal cells to irregular cells with unclear structure and reduced roundness, indicating that the cell structure is destroyed), while the cell area of ​​the experimental group 2 mice did not increase significantly, indicating that the inhibitor effectively inhibited the structural destruction of the cells; while the cell perimeter of the experimental group 2 mice did not increase significantly, indicating that the inhibitor effectively inhibited the structural destruction of the cells; the roundness of the cells was close to 1, indicating that the degree of structural destruction of the cells was not great.

[0122] Example 4 Experiment on the drug mechanism of serpatinib in preventing EMT

[0123] 1. Purpose of the experiment

[0124] To explore the drug mechanism of selpatinib in preventing EMT.

[0125] 2. Experimental methods

[0126] 2.1. Cell immunofluorescence experiment is as shown in 2.3 of Example 2.

[0127] 2.2 Protein interaction co-IP experiment:

[0128] (1) Lyse a 10 cm cell culture dish with 1 mL of cell lysis buffer.

[0129] (2) After lysis, incubate at 4°C, 12,000 rpm, for 30 min.

[0130] (3) Add primary antibody and incubate overnight.

[0131] (4) After overnight, add 40uL of beads and incubate at 4℃ for 3-4h.

[0132] (5) Wash beads by adding IP buffer in suspension at 4500 rpm for 3 min and 5 times.

[0133] (6) Add 2× loading, 40 μL, stir with a small pipette tip, 100°C, 5 min.

[0134] (7) Follow up with western blot experiment.

[0135] (8) The presence of bands indicates that there is an interaction between the two proteins.

[0136] 3. Experimental results

[0137] The experimental results are as follows Figure 7 and Figure 8 As shown. Among them: Figure 7 The results of cell immunofluorescence experiment are shown in Figure 2. Figure 8 This is a graph showing the results of a protein interaction co-IP experiment.

[0138] from Figure 7 and Figure 8 It can be seen that:

[0139] Through cell immunofluorescence experiments, it was found that in cells induced by 20ng / mL TGFβ1, RET expression moved to the nucleus, and the expression of RET around the nucleus increased significantly, while the expression of RET around the cell membrane decreased significantly; while in cells pretreated with 1μM concentration of selpatinib, RET expression did not move to the nucleus, and the expression of RET in the nucleus did not increase significantly, while the expression of RET around the cell membrane increased significantly. This indicates that RET has the performance of entering the nucleus during the induction of EMT, but selpatinib effectively inhibits this process.

[0140] Through protein interaction Co-IP experiments, it was found that RET binds to TGFBR1 during TGFβ1-induced EMT and RPE-EMT. Although serpatinib does not affect (or promotes) the binding of the two, it can inhibit RET kinase activity and prevent RET from entering the nucleus when binding to TGFBR1 ( Figure 8 ) to prevent EMT.

[0141] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of selpatinib in the preparation of drugs for treating epithelial-mesenchymal transition of retinal pigment epithelial cells.

2. The use of selpatinib in the preparation of drugs for treating ophthalmic diseases related to epithelial-mesenchymal transition of retinal pigment epithelial cells.

3. The use according to claim 2, characterized in that: The selpatinib treats the ophthalmic disease by inhibiting the epithelial-mesenchymal transition of retinal pigment epithelial cells.

4. The use according to claim 3, characterized in that: Serpatinib inhibits the epithelial-mesenchymal transition of retinal pigment epithelial cells by inhibiting the TGFβ1 signaling pathway.

5. The use according to claim 2, characterized in that: The ophthalmic diseases include age-related macular degeneration, proliferative vitreoretinopathy, diabetic retinopathy, central serous chorioretinopathy, retinal detachment and posterior capsule opacity of secondary cataract.

6. The use according to claim 2, characterized in that: The dosage form of the drug includes injection.

7. The use according to claim 6, characterized in that: The drug is administered by intravitreal injection.

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