Application of methotrexate or salt thereof in preparation of medicine for preventing and / or treating primary ciliary immobility syndrome

Abstract

The invention provides an application of methotrexate or a salt thereof in preparation of a medicine for preventing and / or treating primary stationary ciliary syndrome, and belongs to the field of medicines. It is found for the first time that methotrexate or the salt thereof can effectively recover the ciliary movement function, and the methotrexate or the salt thereof has a wide application prospect in preparation of the medicine for preventing and / or treating the primary fixed ciliary syndrome.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to the use of methotrexate or a salt thereof in preparing a medicine for preventing and / or treating primary ciliary immobility syndrome. Background Art

[0002] Primary ciliary dyskinesia (PCD), also known as immotile ciliary syndrome or primary immotile ciliary syndrome, is a type of genetic disease that causes a variety of clinical manifestations due to impaired ciliary function, including primary immotile ciliary syndrome, Kartagener syndrome, and ciliary movement direction defects.

[0003] So far, in the process of exploring the treatment of primary ciliary dyskinesia (PCD), researchers have adopted gene therapy, mRNA transcript therapy and read-through therapy for PCD. In the field of gene therapy, researchers such as Chhin, Mclntyre, and Ostrowski used lentivirus or adenovirus as a vector to carry the CRISPR-Cas9 system into a mouse model carrying a ciliary gene mutation to combat PCD. However, the off-target effects that may be caused by the CRISPR-Cas9 system have become a major challenge for gene therapy. In addition, the physiological regulatory changes caused by promoter differences between transgenic and wild-type genes may interfere with the function and interaction of proteins in epithelial cells. The stable integration of transgenes in the host genome may bring serious side effects. For example, in the SCID-X1 trial in France and the United Kingdom, five patients with severe combined immunodeficiency syndrome who received gamma-retroviral gene therapy unfortunately developed leukemia. As an alternative, gene editing technology can directly replace the mutated wild-type gene sequence because it is not limited by gene size. Although gene therapy has shown potential in restoring ciliary activity, many challenges still need to be overcome, such as resistance of airway epithelium to apical transduction of viral vectors, achieving sufficiently high transfection rates to restore ciliary function, inhibitory effects of airway secretions on gene transfer, and maintaining long-term efficacy. At the same time, potential immune responses and off-target effects may cause fatal side effects. Another downstream treatment strategy is transcriptional therapy or RNA therapy, which has the advantage of not directly modifying genomic DNA and the effects are reversible. TRANSLATE-BIO is trying to use lipid nanoparticles as carriers to deliver the DNAI1 gene to the lungs of mice carrying the Dnai1 gene mutation. However, no specific research results on the restoration of ciliary function have been made public so far. In particular, primary ciliary dyskinesia (PCD) involves multiple genetic abnormalities, and some key ciliary-related proteins are large in size, which poses a challenge to the effective delivery of existing vectors, making the potential of RNA therapy in the field of PCD treatment difficult to predict. In addition, more detailed studies are needed to clarify the diffusion efficiency of mRNA encapsulated by lipid nanocarriers in lung tissue and the stability of these mRNA molecules inside cells. In short, although TRANSLATE-BIO has tried to use lipid nanoparticles to carry the DNAI1 gene and inject it into the lungs of Dnai1 mutant mice, it has not yet published data on the recovery of ciliary function. Given that PCD involves multiple genes and some proteins are difficult to transmit, the efficacy of RNA therapy is still uncertain. At the same time, the diffusion of mRNA carried by lipid nanocarriers in the lungs and its stability in cells still need to be explored in depth.

[0004] Methotrexate disodium salt is an effective folic acid antagonist and an effective inhibitor of dihydrofolate reductase. First, it has anti-metabolic and anti-folate effects, and its main mechanism of action is to inhibit dihydrofolate reductase. This inhibitory effect can effectively prevent folic acid from being converted into tetrahydrofolate, thereby blocking the synthesis of DNA. Since DNA is a substance necessary for cell proliferation and division, this mechanism of action of methotrexate disodium salt makes it an important component of anti-tumor drugs. Including but not limited to acute lymphoblastic leukemia, malignant lymphoma, multiple myeloma, head and neck cancer, etc. By inhibiting the DNA synthesis and proliferation of tumor cells, methotrexate disodium salt can delay the growth and spread of tumors, and gain more treatment time and opportunities for patients. Second, immunosuppressive and anti-tumor effects. Methotrexate disodium salt also has immunosuppressive activity and can affect the body's immune response. This allows it to play a certain therapeutic effect in the treatment of certain autoimmune diseases, such as rheumatoid arthritis. There are no reports of methotrexate disodium salt being used to treat primary ciliary immobility syndrome. Summary of the invention

[0005] The purpose of the invention is to provide the use of methotrexate or its salt in preparing a drug for preventing and / or treating primary ciliary immobility syndrome.

[0006] The present invention provides use of methotrexate or a salt thereof in preparing a medicament for preventing and / or treating primary immotile cilia syndrome.

[0007] Furthermore, the drug is a drug that restores ciliary movement function.

[0008] Furthermore, the methotrexate salt is methotrexate disodium salt.

[0009] Furthermore, the drug is a pharmaceutical preparation prepared with methotrexate or its salt as an active ingredient and pharmaceutically acceptable excipients.

[0010] Furthermore, the pharmaceutical preparation is an oral preparation or an injectable preparation.

[0011] Furthermore, the oral preparation is a tablet, a granule, or a capsule.

[0012] Furthermore, the injection preparation is a powder injection.

[0013] The present invention discovers for the first time that methotrexate or its salt can effectively restore the ciliary movement function, and has broad application prospects in the preparation of drugs for preventing and / or treating primary immotile ciliary syndrome.

[0014] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0015] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the research of the present invention.

[0017] Figure 2 The PCD model validation process: (A) PCR amplification validation results; (B) RT-PCR technology to verify the relative expression results of Dnah9; (C) The morphology of nasal mucosal cilia of wild-type mice and Dnah9 knockout mice under an optical microscope.

[0018] Figure 3 The morphology of cilia in Dnah9 knockout organoids was shown by immunofluorescence staining.

[0019] Figure 4 The therapeutic effect of methotrexate disodium on PCD: (A) morphological changes of cilia after treatment; (B) effect of treatment on the frequency of cilia beat. DETAILED DESCRIPTION

[0020] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0021] Example 1. Effect of methotrexate disodium salt in treating primary ciliary immobility syndrome 1. Experimental methods The present invention refers to the method described in the "Mouse models" section on the left column of page 959 of the prior art (Dynein axonemal heavy chain 10 deficiency causes primary ciliary dyskinesia in humans and mice. Frontiers of medicine, 17(5), 957–971.), and constructs an in vitro model of PCD caused by intrinsic dynein arm deficiency - lung organoids of Dnah9 KO mice. Based on the organoids, an in vitro model for the treatment of PCD is provided, and the effect of methotrexate disodium salt in the treatment of primary ciliary immobility syndrome is studied. Figure 1 Schematic diagram of the research experiment.

[0022] The specific steps are as follows: 1.1 Primary culture and plating process of lung organoids (1) Prepare digestion solution: Prepare two 1 mL digestion mixtures, each containing 0.25 mg / mL Protease E, 400 U / mL Collagenase I, 10 μmol / L Y27632, and 10 U / mL DNase for subsequent use.

[0023] (2) Tissue sampling and cutting: The lung lobes and trachea were removed from the Dnah9 KO mice and placed in PBS solution to keep them moist. Ophthalmic scissors and forceps were used to cut the lung and tracheal tissues into small pieces with a diameter of approximately 2 mm for subsequent digestion.

[0024] (3) Digestion: Transfer the cut tissue blocks to the prepared digestion solution, place them on a 37°C constant temperature shaker, and digest them at 100 rpm for 60 min to ensure that the tissue blocks are fully dissociated.

[0025] (4) Mechanical separation: Use a pipette to mechanically blow the digested tissue blocks until all tissue blocks can pass through the pipette tip smoothly to form suspended cells. When a large number of suspended cells are observed in the digestion solution, stop digestion.

[0026] (5) Digestion termination and filtration: Add 300 μl of FBS to the digestion solution to terminate the digestion process. For tracheal tissue, filter through a 40-μm filter and collect the filtrate; for lung lobe tissue, also filter through a 40-μm filter, but at this time, discard the filtrate and backwash the remaining part on the filter with PBS and collect it.

[0027] (6) Washing and preparation of cell suspension: Wash the filtered and rinsed tissue, remove the supernatant and retain the precipitate. Adjust the amount of liquid according to the number of wells required for plating, and mix it evenly with the precipitate to form a cell suspension.

[0028] (7) Mix Matrigel with cell suspension: Mix the liquefied Matrigel with the cell suspension thoroughly. Depending on the type of well plate (24-well plate, 48-well plate or 96-well plate), add an appropriate amount of Matrigel (30 μl for 24-well plate, 15 μl for 48-well plate, 6 μl for 96-well plate, and the concentration must be kept above 80%). When plating, ensure that the tissue and Matrigel are mixed evenly so that they can be evenly distributed in the Matrigel matrix. After plating, place the well plate in a 37°C incubator to allow the Matrigel to solidify for 15 minutes.

[0029] (8) Add culture medium: After Matrigel is completely solidified, add an appropriate amount of culture medium to each well (500 μL for 24-well plate, 200 μL for 48-well plate, 100 μL for 96-well plate). During the culture process, the culture medium needs to be replaced according to cell growth and color change (yellowening). The culture medium is usually replaced every 4 days.

[0030] 1.2 Organoid passaging steps (1) Collection and initial centrifugation: Use a pipette to gently scrape the organoid and matrix gel mixture from the bottom of the culture dish and transfer it to an EP tube. Subsequently, the mixture in the EP tube is centrifuged at 200G for 3 minutes.

[0031] (2) Preliminary treatment and standing: After centrifugation, remove the supernatant and add 1 ml of cell recovery solution to the EP tube. Mix the solution evenly by blowing and place it on ice for 20 minutes. Then, centrifuge again.

[0032] (3) Washing step: After centrifugation, remove the supernatant, add 1 ml of WASH solution, and mix by pipetting. Then, centrifuge again at 200G for 3 minutes.

[0033] (4) Digestion and termination: After removing the supernatant, add 1 ml of Tryple solution to the EP tube and mix it evenly by blowing. Place the EP tube in an incubator at 37 degrees Celsius for digestion, and the digestion time is 5-10 minutes. During this period, it is necessary to take it out every 5 minutes for blowing and observe the digestion status. If the digestion is incomplete, the digestion time can be appropriately extended until no white precipitate appears. After the digestion is completed, add an appropriate amount of WASH solution to terminate the digestion process, and centrifuge again at 200G for 3 minutes.

[0034] (5) Repeat washing: After centrifugation, pour off the supernatant and add 1 ml of WASH solution again to mix. This step needs to be repeated twice, each time centrifuged at 200G for 3 minutes.

[0035] (6) Filtration (optional): If rapid culture of organoids is required, a 100 μm filter can be used for coarse filtration.

[0036] (7) Glue spreading and standing: After removing the supernatant, add an appropriate amount of matrigel glue (30ul per well) to the EP tube and mix it evenly by blowing. Then, spread the matrigel glue evenly in the wells of the 24-well plate (30ul per well). Place the 24-well plate with glue spread in a 37°C incubator and let it stand for 15 minutes.

[0037] (8) Add culture medium and culture: Take out the 24-well plate from the incubator and add an appropriate amount of culture medium to each well (0.5 ml per well). Finally, put the 24-well plate with culture medium back into the 37-degree Celsius incubator for culture.

[0038] 1.3 Drug intervention steps After digestion, the pulmonary tracheal organoids were converted into single-cell state and then evenly spread in a 48-well culture plate. Next, the drug methotrexate disodium salt was diluted in the culture medium according to a predetermined gradient concentration, and then the culture media containing different concentrations of the drug were added to the corresponding wells one by one. In order to set up a control group, another group of wells were selected to add only PBS solution. To ensure the stability and reliability of the experimental data, each group of experiments was repeated 4 times. The entire drug intervention process lasted for one week.

[0039] 1.4 Cilia beat assessment method Organoids from wild-type mice, organoids from Dnah9 knockout (KO) mice, and organoid samples treated with specific drugs were placed under an Olympus CKX41 upright optical microscope for observation. The microscope system was equipped with a Sprinter high-speed camera produced by Optronis GmbH and an oil-immersion objective with a 100x magnification to ensure high-definition shooting. During the shooting, a high-speed continuous shooting mode of 200 frames per second was used for 10 seconds to capture the dynamic details of the cilia beating. After the shooting was completed, the video playback speed was reduced to 0.1 times the original speed so that the cilia beating could be analyzed and evaluated in more detail.

[0040] 2. Experimental results (1) PCD model verification process The present invention successfully constructed a Dnah9 gene knockout organoid system. In order to verify the genotype of this system, a method of PCR amplification combined with agarose gel electrophoresis was used. The results showed that the present invention has successfully obtained Dnah9 homozygous knockout organoids (such as Figure 2 In addition, further detection by RT-PCR technology confirmed that the expression of Dnah9 gene in organoids had been effectively knocked out (as shown in Figure 2 B). The morphology of nasal mucosal cilia of wild-type mice and Dnah9 knockout mice is shown under an optical microscope. The nasal mucosal cilia of Dnah9 knockout mice are shorter and more curly ( Figure 2 C).

[0041] (2) Establishment of Dnah9 KD lung tracheal organoids The present invention successfully constructed a Dnah9 gene knockout organoid, and further used immunofluorescence staining to specifically stain the ciliated cells. It was observed that in the Dnah9 knockout organoid model, the number of cilia decreased, the length shortened, and the morphology was curled (see Figure 3 ).

[0042] (3) To explore the therapeutic effect of methotrexate disodium on PCD The present invention uses Dnah9 knockout organoids as a drug screening model, selects diphenyl iodonium chloride as a negative control group, and sets up ATP as a positive control group (this small molecule can increase the frequency of ciliary beating at a concentration of 10µM). On this basis, the present invention sets three concentration gradients of methotrexate disodium salt (10µM, 50µM, and 100µM, respectively), and adds these different concentrations of drugs to the Dnah9 knockout organoids. Four replicate wells are set for each concentration gradient to ensure the reliability of the experimental results. After 16 consecutive days of administration, the beating of the cilia was measured. Figure 4 A shows the morphological changes of organoids after drug treatment. Figure 4 As can be observed in B, the addition of diphenyl iodonium chloride caused the cilia to be completely immobile, while compared with the Dnah9 knockout organoid group without drug addition, the addition of both methotrexate disodium salt and ATP helped to restore the cilia beating function. Further analysis results showed that 50µM and 100µM concentrations of methotrexate disodium salt performed better than 10µM concentration of ATP in restoring the cilia beating function, especially 100µM concentration of methotrexate disodium salt, which significantly increased the cilia beating frequency and was close to the cilia beating frequency of normal wild-type mice (see Figure 4 B).

[0043] Therefore, the results of this study indicate that methotrexate disodium salt has a significant effect in restoring ciliary motility, providing a new potential drug candidate for the treatment of PCD.

[0044] The above results show that methotrexate disodium salt has a significant effect on restoring ciliary motility and can be used to effectively treat primary ciliary immobility syndrome.

[0045] In summary, the present invention discovers for the first time that methotrexate or its salts can effectively restore ciliary motility, and has broad application prospects in the preparation of drugs for preventing and / or treating primary immotile ciliary syndrome.

Claims

1. Use of methotrexate or its salt in the preparation of a medicament for preventing and / or treating primary immotile ciliary syndrome.

2. The use according to claim 1, characterized in that The drug is a drug that restores the ciliary movement function.

3. The use according to claim 1, characterized in that The methotrexate salt is methotrexate disodium salt.

4. The use according to claim 1, characterized in that The medicine is a pharmaceutical preparation prepared by taking methotrexate or its salt as active ingredient and adding pharmaceutically acceptable auxiliary materials.

5. The use according to claim 4, characterized in that The pharmaceutical preparation is an oral preparation or an injection preparation.

6. The use according to claim 5, characterized in that The oral preparations are tablets, granules and capsules.

7. The use according to claim 6, characterized in that The injection preparation is a powder injection.

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