Medical use of limaprost

Abstract

Use of limaprost in repairing various acute and chronic lung injuries. These lung injuries include: acute lung injuries caused by bacterial and viral infections, radioactive lung injury, idiopathic and secondary pulmonary fibrosis, chronic obstructive pulmonary disease, interstitial lung disease, pneumoconiosis, and the like. The limaprost has affinity activity to a plurality of pulmonary alveoli regeneration related targets at the same time, and pharmacological tests prove the definite repair effect thereof on acute and chronic lung injuries.

Description

Medical uses of limaprost Technical Field

[0001] The present disclosure relates to the field of medicinal chemistry, and in particular to a new medical use of limaprost. Background Art

[0002] The lungs are the only organ in the human body that is in direct contact with the outside world and exchanges substances, making them extremely susceptible to environmental influences. Various damaging factors, such as smoke, dust, pathogenic microorganisms, and high-energy radiation, can cause lung damage, primarily manifesting as damage to the alveoli. If this damage is not properly repaired, it often progresses to chronic lung diseases such as chronic obstructive pulmonary disease (COPD), interstitial pneumonia, pulmonary fibrosis, and pneumoconiosis.

[0003] There are currently two main categories of drug treatments for chronic lung disease. One category is symptomatic treatment drugs, such as airway dilators and expectorants, which are widely used in the treatment of COPD. These drugs are mainly used to relieve breathing difficulties when the disease worsens and causes breathing difficulties. The other category of drugs alleviates the disease through anti-inflammatory and anti-fibrotic effects, such as pirfenidone and nintedanib for the treatment of pulmonary fibrosis, and some inhaled hormones. However, these drugs can only delay the progression of lung damage at best, and cannot completely prevent or even reverse the disease process.

[0004] The alveoli are the basic functional units of the pulmonary respiratory system and the primary site of lung injury. Alveoli are primarily composed of type I alveolar epithelial cells (AEC I), which mediate gas exchange, and type II alveolar epithelial cells (AEC II), which secrete surfactant. Previously, it was believed that alveoli lacked the ability to repair themselves, relying instead on a vast alveolar reserve to maintain their basic functions. However, recent studies have discovered that a subset of AEC II cells, known as alveolar progenitor cells (AEPs), function as local stem cells within the alveoli. When lung injury leads to the death of a large number of AEC I cells, AEPs proliferate and differentiate to replenish the missing AEC I cells, thereby restoring normal alveolar structure and function. Impaired alveolar regeneration, resulting in reduced or increased proliferation, may be a key mechanism contributing to chronic lung injury or lung cancer.

[0005] Research on lung regeneration based on AEP has become a hot topic in recent years, but currently most studies focus on stem cell therapy, such as the use of induced pluripotent stem cell (iPSC) technology, mesenchymal stem cell (MSC) technology, or the identification and isolation of alveolar epithelial cells with stem cell capacity for autologous stem cell transplantation. A small number of clinical trials of this type of therapy are already underway, but definitive results have not yet been obtained. In addition, the cost of stem cell therapy is high, and the long-term risks are still unclear, so it will take some time for it to be widely used in clinical practice.

[0006] PGI2 analogues can inhibit the migration, proliferation and glial synthesis of fibroblasts in vitro. Therefore, medical workers once believed that PGI2 substances could inhibit interstitial pneumonia and improve pulmonary fibrosis damage. However, because the effects of PGI2 receptors are too broad, when PGI2 substances are applied to the body, the therapeutic effect of improving lung damage is often not reflected, and even serious lung side effects may occur. For example, patent document WO2022203070A1 records that during Ono Pharmaceutical's development of ONO-1301 (a well-known PGI2 receptor agonist used for arteriosclerosis), this PGI2 analogue caused severe lung inflammation and lung abscess side effects. Therefore, medical workers have been looking for more specific targets to improve lung damage.

[0007] In 2023, researchers used transcriptomics to identify drug targets potentially involved in alveolar regeneration in lung tissue from COPD patients. They ultimately identified prostaglandin EP2, EP4, and IP receptors on the surface of AEC II as potential lung regeneration drug targets. Using the nonspecific EP receptor agonist misoprostol and the IP receptor agonist iloprost, they demonstrated in COPD animal models and human lung organoid models that EP2 / 4 and IP receptors mediate alveolar regeneration, providing a potential therapeutic target for alveolar regeneration. However, while prostaglandins are already used to treat respiratory diseases, they act on a variety of cells, such as vascular smooth muscle cells, which cause contraction or dilation, platelets, which cause aggregation or disaggregation, and spinal neurons, which cause pain. Prostaglandins have diverse effects, including but not limited to muscle contraction and inflammation regulation. Their vasodilatory effects are primarily used clinically to treat pulmonary hypertension, such as epoprostenol and iloprost. However, these drugs are rarely used due to their suboptimal efficacy and complex administration. Neither drug is clinically used to treat lung injury. On the other hand, all lung regeneration therapies currently under clinical research are stem cell-based, primarily autologous stem cell transplantation. Stem cell therapy involves extensive pre-processing, screening, and culture induction, and currently can only be applied in a customized manner. This makes it relatively expensive and difficult to meet the treatment needs of the large number of people with lung injury diseases. While promoting alveolar regeneration through drug intervention would undoubtedly be a more economical and convenient solution, there are currently no drugs with alveolar regeneration capabilities.

[0008] In summary, there are currently no clinically available therapies or drugs that can promote alveolar regeneration. Current medications commonly used in clinical settings for lung injury, such as pirfenidone and hormones, can actually inhibit alveolar regeneration. Developing novel, orally available drugs that promote alveolar regeneration, thereby offering a new treatment option for patients with lung injury, remains a pressing technical challenge. Summary of the Invention

[0009] Limaprost is an oral prostaglandin E1 analog that has been shown to improve peripheral circulatory failure through vasodilation and antithrombotic effects. It can also improve poor blood flow to nerve tissue in cervical spondylosis and normalize nerve function. It was approved in 1988 for the treatment of ischemic symptoms such as skin ulcers, pain, and coldness associated with thromboangiitis obliterans. The structural formula of limaprost is as follows:

[0010] Limaprost is also used to treat subjective symptoms associated with acquired lumbar spinal stenosis, such as calf pain and numbness and walking difficulties, as additional indications. Limaprost has also been approved for marketing in China as an oral medication for the treatment of spinal stenosis. However, after in-depth research by the inventors, it was found that the compound and its corresponding salts, derivatives and analogs have the efficacy of treating various diseases related to acute and chronic lung damage, and have therapeutic and preventive effects on acute lung injury (ALI), radiation lung injury, pulmonary fibrosis, COPD, interstitial lung disease, pneumoconiosis, etc. caused by various factors. Specifically, the present disclosure provides the following aspects:

[0011] A first aspect of the present disclosure provides use of limaprost, a pharmaceutically acceptable salt thereof, or a derivative thereof in the preparation of a medicament for preventing and / or treating acute and chronic lung injury.

[0012] The second aspect of the present disclosure provides use of limaprost, a pharmaceutically acceptable salt thereof, or a derivative thereof in repairing acute and chronic lung injury.

[0013] In the above-mentioned uses, limaprost, its pharmaceutically acceptable salts, and derivatives play a role in repairing acute and chronic lung injuries by activating alveolar regeneration targets EP2, EP4, and IP.

[0014] In a preferred embodiment, the acute and chronic lung injuries include acute lung injury caused by pathogen infection (e.g., acute lung injury caused by bacteria and / or viruses), acute respiratory distress syndrome, radiation lung injury, idiopathic and / or secondary pulmonary fibrosis, chronic obstructive pulmonary disease, interstitial lung disease, and pneumoconiosis; further preferably, limaprost, its pharmaceutically acceptable salts, and derivatives have therapeutic and preventive effects on radiation lung injury.

[0015] The third aspect of the present disclosure provides the use of limaprost, a pharmaceutically acceptable salt thereof, or a derivative thereof as a co-activator of alveolar regeneration targets EP2EP4 and IP.

[0016] The fourth aspect of the present disclosure provides a medicament for preventing or treating acute or chronic lung injury, which comprises limaprost, a pharmaceutically acceptable salt thereof, or a derivative thereof as an active ingredient.

[0017] A fifth aspect of the present disclosure provides a method for preventing or treating acute or chronic lung injury, comprising administering to a mammal an amount of limaprost, a pharmaceutically acceptable salt thereof, or a derivative thereof that is effective for preventing or treating the symptoms.

[0018] In a preferred embodiment, the acute and chronic lung injury includes acute lung injury caused by pathogen infection (such as acute lung injury caused by bacteria and / or viruses), acute respiratory distress syndrome, radiation lung injury, idiopathic and / or secondary pulmonary fibrosis, chronic obstructive pulmonary disease, interstitial lung disease, and pneumoconiosis.

[0019] The present disclosure offers significant advantages over existing technologies: Currently, no treatments or drugs are available for alveolar regeneration, and only a limited number of stem cell therapies are undergoing clinical research. However, cell therapy is expensive and requires customized production, making it unlikely to benefit a large patient population in the short term. Furthermore, there are risks such as excessive stem cell proliferation leading to tumors. Limaprost, on the other hand, has a 25-year history of human use, with well-defined adverse reactions and dosage methods. As an orally administered prostaglandin derivative, its accessibility and ease of use far surpass those of cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 is a graph showing the effect on the differentiation ability of normal mouse AEC2 cells in 2D culture;

[0021] FIG2 is a graph showing the effect on the stemness of ACE2 cells in normal mice;

[0022] FIG3 is a graph showing the effect of bleomycin on lung hydroxyproline content in a mouse pulmonary fibrosis model;

[0023] FIG4 is a graph showing the survival curve of mice after bleomycin pulmonary fibrosis animal model;

[0024] FIG5 is a graph showing the lung collagen volume fraction (CVF%) after bleomycin-induced pulmonary fibrosis in mice;

[0025] FIG6 is a photograph showing lung tissue of mice with radiation-induced pulmonary fibrosis;

[0026] FIG7 shows photographs of lung tissues of mice with radiation-induced pneumonitis. DETAILED DESCRIPTION

[0027] Each element of the present disclosure will be described in detail below.

[0028] According to the contents disclosed in the examples described below, limaprost can simultaneously activate the alveolar regeneration targets EP2, EP4, and IP, which is superior to other prostaglandin derivatives. In addition, in animal models, it has also been verified that it has a significant efficacy in repairing acute and chronic lung injury, which strongly suggests that limaprost can be used for the treatment of acute and chronic lung injury.

[0029] Limaprost has been used clinically for a very long time, and its safety has not been questioned. Moreover, in the lung inflammation model, its effect is significantly better than existing hormone therapy. Because hormone therapy is generally believed to have obvious side effects, such as femoral head necrosis, treatment is very cautious.

[0030] The present disclosure provides a method for preventing or treating acute or chronic lung injury, comprising administering to a mammal a prophylactic or therapeutically effective amount of limaprost, a pharmaceutically acceptable salt thereof, or a derivative thereof. Common conditions of acute or chronic lung injury include, but are not limited to, acute lung injury caused by pathogen infection (e.g., acute lung injury caused by bacteria and / or viruses), radiation-induced lung injury, idiopathic and / or secondary pulmonary fibrosis, chronic obstructive pulmonary disease, interstitial lung disease, and pneumoconiosis.

[0031] In one embodiment of the present disclosure, limaprost, its pharmaceutically acceptable salts, and derivatives have therapeutic and preventive effects on radiation-induced lung injury. "Radiation-induced lung injury" generally refers to damage to lung tissue caused by ionizing radiation (such as radiotherapy). All radiotherapy for thoracic tumors will cause radiation-induced lung injury. The radiotherapy includes external beam radiotherapy, internal beam radiotherapy, radionuclide therapy, whole body irradiation, intraoperative radiotherapy, and 4D radiotherapy. More specifically, it includes three-dimensional conformal radiotherapy (3D-CRT), intensity-modulated radiotherapy (IMRT), volumetric modulated radiotherapy (VMAT), image-guided radiotherapy (IGRT), stereotactic body radiation therapy (SBRT), proton therapy, and brachytherapy. "Radiation pneumonitis" is an early manifestation of radiation-induced lung injury. Radiation pneumonitis is different from common inflammatory lung diseases. More specifically, common inflammatory lung diseases such as pneumonia have different causes from radiation pneumonitis. Pneumonia is a lung infection caused by bacteria, viruses, or other microorganisms. This type of infection mainly invades the alveoli, of which bacterial and viral pneumonia are the most common. Clinically, the onset of pneumonia is relatively short, with symptoms primarily consisting of cough and high fever. Treatment primarily focuses on symptomatic treatment and antibiotics to combat bacterial or viral infections. Pneumonia typically resolves on its own after the cause is eliminated. However, radiation-induced lung injury can progress.

[0032] Radiation-induced pulmonary fibrosis (RPF) is a late manifestation of radiation-induced lung injury. It is a type of pulmonary fibrosis with a clear etiology, distinct from idiopathic pulmonary fibrosis (IPF). IPF is a chronic, progressive, fibrosing interstitial lung disease of unknown etiology. Typical symptoms include progressive dyspnea and a dry cough, ultimately leading to respiratory failure and death. IPF is primarily characterized by extracellular fibrotic deposits, with a long and insidious onset. In most cases, lung function is severely compromised by the time of diagnosis.

[0033] The present disclosure provides a pharmaceutical composition for preventing or treating acute or chronic lung injury, comprising limaprost, a pharmaceutically acceptable salt thereof, or a derivative thereof as an active ingredient. Depending on the route of administration, the pharmaceutical composition can be categorized as oral, sublingual or buccal, inhalation, injection, rectal, or transdermal. Oral formulations are preferred.

[0034] The active ingredient of the present disclosure may be present in an amount or dosage of about 0.01 μg to about 10 mg in a unit dose pharmaceutical composition, suitably 0.1 μg-5 mg, 0.1-1000 μg, 0.1-100 μg, 0.5-50 μg, 1.0-40 μg, 1.0-30 μg, 1.0-20, 1.0-10 μg or 1.0-5 μg, for example 0.1 μg, 0.5 μg, 1.0 μg, 5.0 μg, 10 μg, 15 μg, 20 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg. ,90μg,95μg,100μg,125μg,150μg,175μg,200μg,225μg,250μg,275μg,3 00μg, 325μg, 350μg, 375μg, 400μg, 425μg, 450μg, 475μg, 500μg, 525μg, 5 50μg, 575μg, 600μg, 625μg, 650μg, 675μg, 700μg, 725μg, 750μg, 775μg, 8 00μg, 825μg, 850μg, 875μg, 900μg, 925μg, 950μg, 975μg, 1mg, 5mg, 10mg, etc.

[0035] In some embodiments, the pharmaceutical compositions described herein are administered continuously for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 day, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, or at least 80 days.

[0036] In some embodiments, the pharmaceutical compositions of the present disclosure are administered for one or more (e.g., 1-10 courses, 1-8 courses, 1-6 courses, 1-5 courses, 1-4 courses, 1-3 courses, or 1-2 courses, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) courses, wherein each course lasts for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days. , at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days or at least 50 days; and there is an interval of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, two weeks, three weeks or four weeks between each two courses of treatment.

[0037] The dosage of the active ingredient of the pharmaceutical composition of the present disclosure depends on the individual being treated, the severity of the disease or condition, the rate of administration, the handling of the compound and the judgment of the prescribing physician. In general, the dosage of the active ingredient is 0.1 μg / to 50 mg / day, for example, 1 μg / to 10 mg / day, 1 μg / to 200 mg / day, 1 μg / to 100 mg / day, 1 μg / to 1000 μg / day, 1 μg / to 900 μg / day, 1 μg / to 800 μg / day, 1 μg / to 700 μg / day, 1 μg / to 600 μg / day, 1 μg / to 500 μg / day, 1 μg / to 400 μg / day, 1 μg / to 300 μg / day, 1 μg / to 200 μg / day, 1 μg / to 150 μg / day, 1 μg / day to 100 μg / day, 1 μg / day to 90 μg / day, 1 μg / day to 80 μg / day, 1 μg / day to 70 μg / day, 1 μg / day to 60 μg / day, 1 μg / day to 50 μg / day, 1 μg / day to 30 μg / day, 1 μg / day to 20 μg / day, 1 μg / day to 10 μg / day, 1 μg / day to 5 μg / day, for example, 1 μg / day, 5 μg / day, 10 μg / day, 15 μg / day, 20 μg / day, 25 μg / day, 30 μg / day, 35 μg / day, 40 μg / day, 45 μg / day, 50 μg / day, 55 μg / day g / day, 60μg / day, 65μg / day, 70μg / day, 75μg / day, 80μg / day, 85μg / day, 90μg / day, 95μg / day, 100μg / day, 125μg / day, 150μg / day, 175μg / day, 200μg / day, 225μg / day, 250μg / day, 275μg / day, 300μg / day, 325μg / day, 350μg / day, 375μg / day, 400μg / day, 425μg / day, 450μg / day, 475μg / day, 500μg / day, 525μg / day, 550μg / day, 575μg / day The dosage is 5 μg / day, 600 μg / day, 625 μg / day, 650 μg / day, 675 μg / day, 700 μg / day, 725 μg / day, 750 μg / day, 775 μg / day, 800 μg / day, 825 μg / day, 850 μg / day, 875 μg / day, 900 μg / day, 925 μg / day, 950 μg / day, 975 μg / day, 1 mg / day, 5 mg / day, 10 mg / day, 15 mg / day, 20 mg / day, 25 mg / day, 30 mg / day, about 35 mg / day, 40 mg / day, 45 mg / day, and 50 mg / day.

[0038] When preparing oral preparations, optional dosage forms include tablets, pills, lozenges, capsules, liquids, gels, syrups, slurries, suspensions, and the like. Tablets and capsules are preferred. Pharmaceutically acceptable carriers include lactose, sucrose, mannitol, starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, agar, alginic acid, sodium alginate, talc, magnesium stearate, micronized silica gel, fatty oils, liquid paraffin, and liquid polyethylene glycol.

[0039] When preparing an inhalation formulation, it can be in the form of a pressurized aerosol or dry powder inhaler. Suitable propellants include dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide. Pharmaceutically acceptable carriers include lactose and starch.

[0040] When preparing injections, the optional dosage forms include oily injections, aqueous injections, or freeze-dried powder injections. Pharmaceutically acceptable carriers include fatty oils, ethyl oleate, triglycerides, liposomes, sodium carboxymethylcellulose, dextran, cross-linked polyvinyl pyrrolidone, agar, alginic acid, and sodium alginate.

[0041] When preparing a formulation for rectal administration, it may be in the form of a suppository or enema. Pharmaceutically acceptable carriers include cocoa butter and glycerides.

[0042] When preparing a transdermal preparation, it can be in the form of a patch. Pharmaceutically acceptable carriers include sodium polyacrylate, polyacrylate, polyvinyl pyrrolidone, and polyethylene glycol.

[0043] Among numerous pharmaceutical agents, oral preparations are particularly preferred. The above-mentioned preparation contains a cyclodextrin inclusion complex of limaprost, wherein the cyclodextrin is selected from one or more of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-α-cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, carboxymethyl-β-cyclodextrin, dimethyl-β-cyclodextrin, and tert-butyl ether-β-cyclodextrin. The sustained-release agent in the sustained-release agent can be selected from one or more of methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, cyanoethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, chitosan, mannan, galactose, polyethylene oxide, polyethylene glycol, polyvinyl alcohol, sodium alginate, agar, pectin, xanthan gum, polyvinyl acetate, polyacrylic acid, polymethyl acrylate, polyethyl acrylate, and carbomer.

[0044] The pharmaceutically acceptable excipients described in the above-mentioned preparation can be selected from one or more of the group consisting of stabilizers, fillers, and lubricants. The stabilizer can be selected from one or more of the group consisting of citric acid, fumaric acid, tartaric acid, ascorbic acid, amino acids, and acetic acid, with amino acids being preferred. The amino acids can be selected from one or more of the group consisting of glutamic acid, glycine, aspartic acid, tryptophan, tyrosine, serine, cysteine, methionine, asparagine, glutamine, threonine, lysine, arginine, histidine, glycine, alanine, valine, leucine, isoleucine, phenylalanine, and proline. The amino acids can also be selected from glutamic acid or aspartic acid. The filler can be selected from one or more of the group consisting of lactose, microcrystalline cellulose, sodium chloride, pre-gelatinized starch, fructose, starch, and galactose. The lubricant can be selected from one or more of the group consisting of magnesium stearate, calcium stearate, and stearic acid. The above-mentioned oral sustained-release preparation can be in the form of tablets, capsules, or granules.

[0045] When used, the pharmaceutical composition should be administered at an effective therapeutic dose, adjusted appropriately based on the nature of the disease, the patient's age, and weight. Typically, the dosage of limaprost is 1 ng / kg to 8 mg / kg body weight, preferably 5 ng / kg to 1000 ng / kg, and more preferably 8 to 800 ng / kg. The dosage and frequency of administration should ultimately be determined by the physician.

[0046] In order to further illustrate the various elements of the present disclosure, the following examples are provided for illustration. However, the scope of protection of the present disclosure is not limited to the following examples.

[0047] Example

[0048] Example 1. Receptor binding verification

[0049] Experimental methods

[0050] Test cells: In this experiment, EP1-HEK293, EP2-HEK293, EP3-HEK293, EP4-HEK293, Rat EP2-CHO, Rat EP4-CHO, IP-HEK293, and FP-HEK293 cells were all constructed by Beijing Aisiyipu Biotechnology Co., Ltd.

[0051] Instrument Information

[0052] Main reagent information

[0053] Determination method

[0054] 1) According to Prepare 1× Stimulation Buffer according to the instructions of the Ultra cAMP Kit (Human EP2, Human EP4, Rat EP2, Rat EP4, Human IP receptor) or IP-One-Gq kit (Human EP1, Human EP3, Human FP).

[0055] 2) Perform a serial dilution of the positive compound through 10 concentrations, and the test compound through 10 concentrations, followed by dilution to 10x with 1× Stimulation Buffer. (Human IP targets are serially diluted through 11 concentrations, and Human EP2, Human EP4, Rat EP2, and Rat EP4 are serially diluted through 10 concentrations.)

[0056] 3) Stably transfected cells were cultured to 80% confluency; cells were collected by trypsin digestion, counted, and inoculated into 384-well plates at 9 μL / well.

[0057] 4) Add 1-2 μL of the diluted 10× compound to the corresponding experimental wells and incubate at 37°C for 30 minutes.

[0058] 5) Dilute Eu-cAMP or d2-IP1 to the working concentration in detection buffer and add 5 μL / well to the corresponding experimental wells.

[0059] 6) ULight TM -Anti-cAMP or Anti-IP1-Cryptate antibody was diluted to the working concentration with detection buffer, and 5 μL / well was added to the corresponding experimental wells; after centrifugation, incubated at room temperature for 1 hour.

[0060] 7) After incubation, use a microplate reader to measure the values ​​at 665 nm and 620 nm under excitation at a wavelength of 330 nm.

[0061] Test results

[0062] Table 1. Receptor binding activity expressed as EC50

[0063] Among all orally available prostaglandins, limaprost has high agonist activity at EP2, EP4, and IP receptors. Meanwhile, misoprost lacks activity at IP receptors, and beraprost lacks activity at EP receptors. Therefore, although limaprost's activity at EP2 / 4 receptors is comparable to or slightly weaker than that of misoprost, and its activity at IP receptors is also weaker than that of beraprost, it is an oral prostaglandin that has sufficient binding activity at all three receptors associated with alveolar regeneration.

[0064] Example 2. Study of limaprost in interstitial lung disease organoids

[0065] A human primary lung bronchial epithelial model was constructed by resuscitating normal human primary lung bronchial epithelial cells and expanding them to a sufficient number of cells before model construction. A human primary lung bronchial epithelial model was constructed on a Transwell plate. After cell inoculation, expansion culture and air-liquid culture were performed separately. After 14 days, an air-liquid co-culture bronchial epithelial model was completed. The drug was then incubated for 5 days. After the incubation, high-content 10x fluorescence photography was performed to record the results. The results showed that the total fluorescence intensity of Vimentin in the limaprost-treated group was significantly reduced compared to the negative control, indicating that this product has the effect of inhibiting lung epithelial-mesenchymal transition.

[0066] Example 3. Effects of limaprost on proliferation and differentiation of mouse alveolar epithelial ACE2 cells

[0067] The purpose of the experiment was to investigate the effects of the test substance on the proliferation and differentiation function of primary mouse AEC2 cells.

[0068] Materials and methods

[0069] experimental animals

[0070] C57BL / 6J male mice (18-20 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (license number: SCXK (Beijing) 2020-0004).

[0071] cell

[0072] MIg2908 mouse lung fibroblasts were purchased from ATCC and cultured in MEM medium containing 10% FBS at 37°C and 5% CO2.

[0073] 2D culture of AEC II cells

[0074] Five normal mice and five C57BL / 6J mice were anesthetized and the thoracotomy was performed to isolate a single lung lobe. Excess tissue and the extrapulmonary trachea were removed. The lung lobes were digested and prepared into a single-cell suspension, which was collected in a 1.5 mL EP tube. To each 1.5 mL centrifuge tube, 5 μL of the following biotinylated antibodies (anti-CD45, anti-CD16 / 32, anti-CD31, anti-Ter119, and anti-integrinβ4, all at 0.5 mg / mL) was added. Nonspecific cells were separated by magnetic bead sorting, and adherent cells were collected and centrifuged at 300 g for 15 min at 4°C to obtain purified AEC II cells.

[0075] Take a 12-well plate, add 5-10 μg / cm2 rat tail collagen to each well, and incubate in the incubator for 2 hours. Add the AEC II cell suspension to the culture plate pretreated with rat tail collagen, and adjust the cell count to 1^10 6 After gently pipetting and evenly dispersing, add 1 mL of cell suspension to each well. Stimulate with different concentrations of the test drug in the corresponding wells and incubate in an incubator for 48 hours. Observe the ratio of AEC2 cells to AEC1 cells under a microscope to examine the effect of different doses of the test drug on the differentiation potential of AEC II cells.

[0076] 3D culture of AEC II cells

[0077] After isolating primary mouse AEC II cells, cultured normal mouse lung fibroblasts (Mlg2908) were immediately trypsinized, centrifuged at 1000g at room temperature, and resuspended in 200μL of complete culture medium. After a 10-fold dilution, the cells were counted. AEC2 cell to Mlg2908 cell ratio was 6,000:90,000. 100μL of cells was mixed with 100μL of Matrigel at a 1:1 ratio and then slowly added to a Transwell chamber for sphering. 500μL of complete culture medium / drug-containing medium was added to the outer chamber, and the cells were cultured in an incubator. The culture medium in the outer chamber was replaced every two days. After approximately two weeks of cell culture, the AEC2 cells were observed under an inverted microscope for sphering, and the sphering rate and size of the spheres were calculated.

[0078] Experimental results

[0079] In the AEC II primary cell 2D culture model, the data on the differentiation ratio of AEC2 cells to AEC1 cells after stimulation with different doses of limaprost are shown in Figure 1 below. Limaprost administration at 0.5, 1, and 1.25 ng / mL significantly increased the differentiation ratio of AEC2 cells to AEC1 cells, demonstrating that limaprost has a significant pro-differentiation effect on ACE2 cells.

[0080] In a 3D culture model of primary AEC II cells, the clone formation efficiency (CFE%) and sphere diameter data of AEC II cells after stimulation with different doses of limaprost are shown in Figure 2 below. Limaprost can increase the clone sphere formation rate of AEC II cells, with the 1 ng / mL and 1.25 ng / mL groups showing statistically significant differences compared to the control group. Clone diameter is another important indicator for assessing the stemness of AEC II cells. All doses of limaprost significantly increased the diameter of AEC II cell clone spheres, and the effect of limaprost on clone diameter was more significant than its effect on CFE%, indicating that limaprost has a higher differentiation-promoting effect.

[0081] Example 4. Validation of limaprost in a pulmonary fibrosis model

[0082] Purpose of the experiment

[0083] The pharmacodynamic activities of compounds G1 and G2 against bleomycin (BLM)-induced pulmonary fibrosis (PF) model mice were evaluated.

[0084] Materials and methods

[0085] experimental animals

[0086] C57BL / 6J male mice (18–20 g); 8 mice per group. Purchased from Beijing Huafukang Biotechnology Co., Ltd.; License No.: SCXK(Beijing)2019-0008.

[0087] Experimental groups

[0088] PF model group (model group): DDW was administered orally with a volume of 0.2 ml / 20 g.

[0089] Limaprost group (LM, 100 μg / kg): prepared with 0.5% CMC-Na, stored at 4°C, and administered orally once a day with a volume of 0.2 ml / 20 g.

[0090] Beraprost group (BQ, 100 μg / kg): prepared with 0.5% CMC-Na, stored at 4°C, and administered orally once a day with a volume of 0.2 ml / 20 g.

[0091] Experimental methods

[0092] After seven days of acclimatization in an SPF animal facility, mice were randomly divided into three groups, each consisting of eight mice. PF was established in the model group and each treatment group by endotracheal intubation and administration of 50 μl of BLM (3 mg / kg). The model group was gavaged with DDW once daily (0.2 ml / 20 g). Each treatment group was gavaged once daily (0.2 ml / 20 g) starting from the second day of modeling. One to seven days after modeling, the model group animals showed obvious mental depression, decreased activity, and crackles during breathing, which are typical PF lesions. The experiment was terminated on the 21st day after modeling, and the animals in each group were sacrificed. Pulmonary fibrosis-related evaluation indicators (weight change rate, survival index, lung coefficient, hydroxyproline (HYP) content per milligram of lung tissue and HYP content per whole lung weight) were measured. Lung tissues were stained with HE and Masson staining to comprehensively evaluate the anti-PF pharmacodynamic activity of the compound.

[0093] Experimental results

[0094] By the end of the experiment, 2 animals in the model group (Model), 1 in the limaprost group (LM), and 1 in the beraprost sodium group (BQ) died of respiratory distress. Death occurred later in the limaprost group, as shown in Figure 3.

[0095] The results of the determination of hydroxyproline content in the lungs showed that the hydroxyproline content in the lungs of the animals in the limaprost group was significantly lower than that in the model group, indicating that the degree of collagen deposition in the lungs of the animals in this group was relatively mild, as shown in Figure 4.

[0096] HE and Masson staining revealed significant pathological changes in the lungs of mice in all groups, including fibrosis, collagen deposition, and inflammatory cell infiltration. However, compared with the control group, the limaprost group showed significantly less lesions, a significantly lower pathological score, and a significant decrease in the collagen volume fraction (CVF%) in lung tissue (see Figure 3).

[0097] Example 5. Validation of Limaprost in a Radiation-Induced Pulmonary Fibrosis Model

[0098] A radiation-induced pulmonary fibrosis model was established by irradiating the chest of C57BL / 6J male mice with a single 15 Gy dose using a Gammacell 40Exactor biological irradiator at a dose rate of 0.88 Gy / min, using a specialized shielding device for localized irradiation. Drug treatment began the day before modeling, with oral administration twice per day per mouse for a tentative 21-day duration. After treatment, lung tissue from each group was obtained for pathological examination. The results are shown in Figure 4.

[0099] Results showed that limaprost at doses of 0.05, 0.1, and 0.2 mg / kg effectively inhibited collagen deposition, significantly outperforming the active agent methylprednisolone. The 0.3 and 0.4 mg / kg groups showed similar effects to methylprednisolone. These results suggest that limaprost, within a certain dose range, has the potential to prevent radiation-induced pulmonary fibrosis.

[0100] Example 6. Validation of Limaprost in Radiation Pneumonia Model

[0101] The purpose of the experiment was to establish a mouse radiation lung disease / pneumonia model that conforms to the clinical characteristics of the disease by chest irradiation, and on this basis, to evaluate the therapeutic effect of limaprost on the mouse radiation lung disease model.

[0102] Materials and methods

[0103] experimental animals

[0104] Male C57BL / 6 mice, SPF grade, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.; production license number: SCXK (Beijing) 2019-0010.

[0105] Experimental groups

[0106] Normal control group: normal saline, gavage

[0107] Model group: DDW was administered orally with a volume of 0.2 ml / 20 g.

[0108] Limaprost group (0.1, 0.2 and 0.3 mg / kg): prepared with 0.5% CMC-Na, stored at 4°C, and administered orally twice daily with a volume of 0.2 ml / 20 g.

[0109] Methylprednisolone group (0.6 mg / kg): prepared with 0.5% CMC-Na, stored at 4°C, and administered orally twice daily with a volume of 0.2 ml / 20 g.

[0110] Combined medication group: limaprost 0.2 mg / kg combined with methylprednisolone 0.6 mg / kg

[0111] Experimental methods

[0112] On the day of irradiation (D0), all animals in the experimental groups, except the normal control group, underwent thoracic irradiation using a small animal irradiator. The specific procedure was as follows: after confirming anesthesia, the head, abdomen, and lower urinary tract of the experimental animals were shielded with a lead shield, exposing only the thoracic cavity. The animals were then placed in the irradiation chamber and the instrument was activated for thoracic irradiation. The irradiation dose was 15 Gy, a single irradiation. Models were established sequentially according to group placement, with 6 animals per treatment. Dosing began the day after irradiation (D1) and continued for 42 consecutive days.

[0113] On the day after the end of administration (D43), alveolar lavage fluid samples were collected from the animals in each experimental group, and blood cell counts were performed using an automatic blood cell counter to analyze the lung injury process of the model animals; the collected alveolar lavage fluid was centrifuged at low temperature, and the supernatant was collected. The ELISA method was used to detect the levels of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) in the alveolar lavage fluid of the animals in each experimental group to analyze the inflammatory response process in the lungs of the model animals; at the end of the experimental dissection, the lungs of the animals in each experimental group were grossly dissected and observed, the lung index was calculated after weighing, and HE staining was performed after routine fixation. The degree of lung inflammatory infiltration in the animals in each experimental group was histopathologically diagnosed and scored.

[0114] Experimental results

[0115] Limaprost has a certain degree of improvement effect on the weight loss response of model animals. Compared with the model control group during the same period, the weight loss of animals in the low- and medium-dose limaprost treatment groups was alleviated.

[0116] The results of alveolar lavage fluid blood cell count test showed that limaprost had a significant inhibitory effect on the process of increased inflammatory exudation in the lungs caused by radiation-induced lung injury.

[0117] After 6 weeks of treatment with low, medium, and high doses of limaprost, the levels of leukocytes, neutrophils, and lymphocytes in the bronchoalveolar lavage fluid of the model animals were significantly reduced compared to the control group during the same period, with the differences being statistically significant (P<0.05). However, after 4 and 6 weeks of combined treatment with the medium dose of the test article and methylprednisolone, no significant advantage was shown in improving pulmonary hematocrit in the model animals. See Table 2 below.

[0118] Table 2. Effects of limaprost on the degree of hemocytosis in bronchoalveolar lavage fluid of model animals

[0119] Note: ****P<0.001***P<0.001 represents the statistical analysis results between the model control group and the normal control group, #P<0.05, ##P<0.01, ###P<0.001 represents the statistical analysis results between the treatment group and the model control group.

[0120] Table 3 shows the results of bronchoalveolar lavage fluid inflammatory factor testing. Limaprost significantly inhibited the elevated levels of inflammatory factors (IL-1β, IL-6, and TNF-α) in the lungs of the model animals. The combination therapy was significantly more effective than methylprednisolone in improving TNF-α levels in the lungs.

[0121] Table 3. Effects of the test article TRD401 on the levels of inflammatory factors in the bronchoalveolar lavage fluid of model animals after 6 weeks of intervention

[0122] Note: **P<0.01, ***P<0.001 represent the statistical analysis results of the model control group and the normal control group, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001 represent the statistical analysis results of the treatment group and the model control group, &P<0.05 represents the statistical analysis results of the medium-dose group, the methylprednisolone group, and the medium-dose TRD401 combined with methylprednisolone treatment group.

[0123] Limaprost demonstrated a significant therapeutic effect on macroscopic lung injury symptoms in the model animals, including swelling, hemorrhage, and congestion, as well as elevated lung index. Compared with the model control group, the lung index and degree of swelling in the low-, medium-, and high-dose limaprost groups were significantly improved, and the lung index was significantly lower than that in the model control group (P < 0.05). Combination therapy of the medium-dose test article and methylprednisolone for 4 and 6 weeks showed no significant improvement in macroscopic lung injury in the model animals.

[0124] Lung HE staining and histopathological analysis revealed that limaprost demonstrated a significant therapeutic effect against radiation-induced lung inflammation. Inflammatory infiltration around the alveolar walls, bronchi, and blood vessels, as well as vascular congestion, was significantly reduced in the low-, medium-, and high-dose groups. The inflammatory scores were significantly lower than those in the control group (P < 0.05). Combination therapy with methylprednisolone at a medium dose for 4 and 6 weeks showed no significant improvement in lung histopathological changes in the model animals. Representative pathological photographs are shown in Figure 7, which are pictures showing the pathological state of the lungs under a 400× optical microscope, with an image scale of 50 μm (A, normal control group; B, model control group; C, methylprednisolone 0.6 mg / kg; D, limaprost 0.1 mg / kg; E, limaprost 0.2 mg / kg; F, limaprost 0.3 mg / kg; G, limaprost 0.2 mg / kg + methylprednisolone 0.6 mg / kg).

[0125] Based on the above experimental results, limaprost demonstrated significant alveolar damage repair in multiple lung injury models. This effect is related to limaprost's promotion of the proliferation and differentiation of AEC II, particularly its promotion of AEC II to AEC I differentiation. Limaprost also demonstrated a clear therapeutic effect in improving radiation-induced lung injury.

[0126] Although oral prostaglandin E1 derivatives and prostacyclin derivatives may also have the effect of promoting alveolar regeneration. However, limaprost is a prostaglandin derivative that has obvious affinity activity for three alveolar regeneration-related targets, EP2, EP4 and IP, while other drugs are only active on some of the receptors, which may cause the overall efficacy of these drugs to be weaker than limaprost. In fact, it has been demonstrated in Example 4 that BQ, which only has affinity activity for IP receptors, did not reduce the degree of pulmonary fibrosis in model animals. Limaprost does not produce lung damage repair effects based on the effects of PGI2 or PEG2, but rather through the more downstream EP2, EP4 and IP. Compared with PGI2 or PEG2 substances, the possibility of side effects such as pneumonia and lung abscess caused by the body's compensatory effect is low.

[0127] The pharmacological activity of limaprost in repairing lung injury is not based on the common biological regulatory pathway of prostaglandin analogues, but on the activation of three alveolar regeneration-related targets of limaprost, namely EP2, EP4 and IP. Its effect is excellent and unique.

[0128] While the present disclosure uses the above-described embodiments to illustrate the detailed methods of the present disclosure, the present disclosure is not limited to the above-described detailed methods, and does not necessarily rely on the above-described detailed methods for implementation. Those skilled in the art should understand that any improvements to the present disclosure, equivalent replacements for raw materials in the products of the present disclosure, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present disclosure.

Claims

1. Use of limaprost, its pharmaceutically acceptable salts, or its derivatives in the preparation of drugs for preventing and / or treating acute and chronic lung injury.

2. Use of limaprost, its pharmaceutically acceptable salts, or its derivatives in repairing acute and chronic lung injury.

3. The use according to claim 1 or 2, characterized in that Limaprost, its pharmaceutically acceptable salts, and derivatives play a role in repairing acute and chronic lung injuries by activating alveolar regeneration targets EP2, EP4, and IP.

4. The use according to claim 1 or 2, wherein the acute and chronic lung injuries include acute lung injury caused by pathogen infection, acute respiratory distress syndrome, radiation lung injury, idiopathic and secondary pulmonary fibrosis, chronic obstructive pulmonary disease, interstitial lung disease, and pneumoconiosis.

5. Use of limaprost, its pharmaceutically acceptable salts, or its derivatives as co-activators of alveolar regeneration targets EP2, EP4, and IP.

6. A pharmaceutical composition for preventing or treating acute or chronic lung injury, comprising limaprost, its pharmaceutically acceptable salts, or its derivatives as active ingredients.

7. The pharmaceutical composition according to claim 6, wherein the acute and chronic lung injury includes acute lung injury caused by pathogen infection, acute respiratory distress syndrome, radiation lung injury, idiopathic and secondary pulmonary fibrosis, chronic obstructive pulmonary disease, interstitial lung disease, and pneumoconiosis; the acute and chronic lung injury is preferably radiation lung injury.

8. The pharmaceutical composition according to claim 6 or 7, wherein the content or dosage of the active ingredient in a unit dose of the pharmaceutical composition can be 0.01 μg to about 10 mg, preferably 0.1-1000 μg, further preferably 0.1-100 μg, and more preferably 1-50 μg.

9. The pharmaceutical composition of claim 6 or 7, wherein the pharmaceutical composition is administered continuously for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 day, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days or at least 80 days.

10. The pharmaceutical composition according to claim 6 or 7, characterized in that The pharmaceutical composition is for one or more courses of treatment.

11. The pharmaceutical composition according to claim 6 or 7, characterized in that The total daily dosage of the active ingredient of the pharmaceutical composition is 0.1 μg / day to 50 mg / day, preferably 1 μg / day to 1000 μg / day, more preferably 1 μg / day to 500 μg / day, and more preferably 1 μg / day to 150 μg / day.

12. The pharmaceutical composition according to claim 6 or 7, wherein the dosage of limaprost is 1 ng / kg-8 mg / kg body weight, preferably 5 ng / kg-1000 ng / kg body weight, more preferably 8-800 ng / kg body weight.

13. A method for preventing or treating acute or chronic lung injury, comprising administering to a mammal an amount of limaprost, a pharmaceutically acceptable salt thereof, or a derivative thereof that is effective for preventing or treating the symptoms.

14. Use of limaprost, its pharmaceutically acceptable salts, or its derivatives in the preparation of drugs for preventing or treating acute or chronic lung injury.

15. The use according to claim 14, wherein the acute and chronic lung injuries include acute lung injury caused by pathogen infection, acute respiratory distress syndrome, radiation-induced lung injury, idiopathic and secondary pulmonary fibrosis, chronic obstructive pulmonary disease, interstitial lung disease, and pneumoconiosis.

Images