Use of PDE4 inhibitor for treating and / or preventing pulmonary diseases

Abstract

Provided is use of a PDE4 inhibitor for treating and / or preventing pulmonary diseases.

Description

Use of PDE4 inhibitors for the treatment and / or prevention of lung diseases

[0001] Cross-reference to related applications

[0002] This application claims priority and benefit to Chinese Patent Application No. 202411597129.5, filed with the China National Intellectual Property Administration on November 8, 2024, and Chinese Patent Application No. 202511604656.9, filed with the China National Intellectual Property Administration on November 4, 2025, the disclosure of which is incorporated herein by reference in its entirety. Invention Field

[0003] This application relates to the use of PDE4 inhibitors for the treatment and / or prevention of lung diseases, particularly the treatment and / or prevention of radiation-induced lung injury, including radiation-induced pneumonia and radiation-induced pulmonary fibrosis.

[0004] Background of the Invention

[0005] Radiation therapy is one of the important treatment methods for thoracic tumors. During the treatment, the lung tissue inevitably receives a certain dose of radiation, which causes radiation-induced lung injury (RILI) of varying degrees. Early manifestation of RILI is radiation pneumonitis (RP), which generally occurs within 6 months after treatment, and most commonly within 1 to 3 months after the start of radiotherapy. Radiation-induced lung fibrosis (RILF) often occurs in the later stage. In reality, RILI is observed as a continuous process, and there is no clear boundary between the two stages.

[0006] According to data released by the National Cancer Center in 2022, there were 4.57 million new cancer cases in China in 2020, including 820,000 cases of lung cancer, 420,000 cases of breast cancer, and 320,000 cases of esophageal cancer. According to statistics, 5%-50% of people undergoing radiation therapy for thoracic tumors develop recurrent pneumonia (RP), and 10%-20% develop severe pneumonia of grade 3 or higher.

[0007] According to the "Chinese Expert Consensus on the Diagnosis and Treatment of Radiation-Associated Pneumonia (2022 Edition)," the current treatment principles for radiation-induced lung injury mainly involve glucocorticoids and / or antibiotics, as well as other adjuvant therapies. However, long-term, high-dose hormone therapy may cause immunosuppression, and the tumor immunosuppressive microenvironment can significantly increase the risk of tumor recurrence and metastasis.

[0008] Currently, the standard treatment for RILI worldwide is limited to glucocorticoid maintenance therapy, and the pipeline of new drugs is still lacking.

[0009] The phosphodiesterase (PDE) family is a class of enzymes that hydrolyze cyclic adenosine monophosphate (cAMP) and / or cyclic guanosine monophosphate (cGMP), playing a dominant role in regulating their intracellular levels. The PDE family comprises 11 enzyme families (PDE1-PDE11), with PDE4 being the earliest discovered and largest family, unique to cAMP. The downstream signaling pathways of PDE4-cAMP mainly include cAMP-dependent protein kinase (PKA) and cAMP-activated exchanger protein (EPAC). These downstream cascades result in the secretion of pro-inflammatory and anti-inflammatory cytokines and inhibit superoxide production. Therefore, inhibiting PDE4 has become a novel strategy for the treatment of inflammatory diseases.

[0010] Furthermore, PDE4 inhibitors can reduce cAMP degradation, thereby enhancing the activity of antifibrotic mediators (PGE2, prostaglandins, and adenosine). These mediators exert their antifibrotic effects by signaling through G protein-coupled receptors. PGE2 exhibits multiple antifibrotic effects, including inhibiting fibroblast activation, enhancing fibroblast susceptibility to apoptosis, and maintaining the integrity of alveolar epithelial cells. PGE2 can stimulate cAMP in lung fibroblasts through prostaglandin 2 receptors, disrupting calcium signaling and inducing antifibrotic effects. PGE2 and treprostacyclin can also inhibit TGF-β-induced myofibroblast differentiation and promote the reversion of differentiated myofibroblasts into fibroblasts.

[0011] Given the limitations of hormone therapy, there is an urgent need for new treatment strategies for radiation-induced lung injury. Summary of the Invention

[0012] This application provides the use of a PDE4 inhibitor for the treatment of lung diseases. Specifically, this application provides the use of compound A (also known as TRD242) or its tautomers for the prevention and / or treatment of lung diseases, including the prevention and / or treatment of lung inflammation and fibrosis, and particularly the prevention and / or treatment of radiation-induced lung injury, including radiation-induced pneumonia and radiation-induced pulmonary fibrosis:

[0013] In vitro cell experiments have shown that PDE4 inhibitors (including compound A) have dual anti-inflammatory and anti-fibrotic effects.

[0014] A radiation-induced lung injury model was established in vivo. Through both prophylactic and therapeutic administration, PDE4 inhibitors (including compound A) were found to have good pharmacodynamic effects.

[0015] This application also provides the use of compounds of formula (I), or their tautomers, solvates, or pharmaceutically acceptable salts, in the preparation of medicaments for the prevention and / or treatment of lung diseases, including use in the preparation of medicaments for the prevention and / or treatment of lung inflammation and / or fibrosis, particularly in the preparation of medicaments for the prevention and / or treatment of radiation-induced lung injury (including radiation-induced pneumonia and / or radiation-induced pulmonary fibrosis):

[0016] in:

[0017] Ring A is a 5-6 member saturated or partially unsaturated heterocycle, which, in addition to the nitrogen atom shown, optionally contains 1-2 additional heteroatoms selected from nitrogen, oxygen and sulfur;

[0018] R1 is selected from hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl groups and -OR;

[0019] R2 is selected from hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl groups and -OR;

[0020] R is selected from hydrogen and C. 1-4 alkyl;

[0021] n is 1, 2, or 3.

[0022] The PDE4 inhibitor / compound of formula (I) may be selected from the following compounds:

[0023] This application also provides a PDE4 inhibitor for the treatment and / or prevention of lung diseases, wherein the PDE4 inhibitor is a compound of formula (I), or a tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0024] This application also provides methods for treating and / or preventing lung diseases, including administering a therapeutically effective amount of a PDE4 inhibitor to a subject, wherein the PDE4 inhibitor is a compound of formula (I), or a tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0025] This application also provides the use of PDE4 inhibitors in the treatment and / or prevention of lung diseases, wherein the PDE4 inhibitor is a compound of formula (I), or a tautomer, solvate, or pharmaceutically acceptable salt thereof. Attached Figure Description

[0026] Figure 1-A shows the TNF-α protein levels in activated PBMC cells after pretreatment with different concentrations of TRD242 compared to the LPS stimulation group.

[0027] Figure 1-B shows the effect of TRD242 on IL-2 production in human PBMCs under PHA-P stimulation compared to the stimulation group. * indicates p < 0.05, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0028] Figure 2A shows the level of FN mRNA after TGF-β1 stimulation; Figure 2B shows the level of PDGF-B mRNA after TGF-β1 stimulation. Compared with TGF-β1, * indicates p < 0.05, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0029] Figure 3 shows the degree of hemocyte exudation in the bronchoalveolar lavage fluid of animals treated with low, medium, and high doses of TRD242. A: Total white blood cell count (WBC); B: Neutrophil count (NEUT); C: Lymphocyte count (LYMPH); D: Platelet count (PLT). All individual data were statistically analyzed using mean ± standard error (SEM). **** P < 0.0001 indicates 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 indicates the statistical analysis results of the TRD242 treatment group or budesonide treatment group compared with the model control group. + P < 0.05 ++ P < 0.01 indicates the statistical analysis results among the low, medium and high dose treatment groups of TRD242.

[0030] Figure 4 shows the H&E staining results, which are typical pathological images of lung tissue from each group of animals. These are lung pathological images under a 200× optical microscope, with an image scale bar of 50 μm (A: normal control group; B: model control group; C: budesonide 0.4 mg / kg; D: TRD242 0.1 mg / kg; E: TRD242 0.5 mg / kg; F: TRD242 1 mg / kg).

[0031] Figure 5 shows the histopathological diagnosis and scoring of lung inflammation in each experimental group based on lung H&E staining results and the lung inflammation scoring criteria. All individual data are expressed as mean ± standard deviation (SD). ****P < 0.0001 indicates the statistical analysis results between the model control group and the normal control group, #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 indicate the statistical analysis results between the TRD242 treatment group or the budesonide treatment group and the model control group.

[0032] Figure 6 shows the BALF detection results. Cell count results are expressed as mean ± standard error (SEM). The differences in efficacy between the blank control group and each drug-treated group and the model control group were analyzed using one-way ANOVA (Dunnett's multiple comparison test). **** represents P < 0.0001.

[0033] Figure 7 shows the results of BALF inflammatory cytokine detection. The results are expressed as mean ± standard error (SEM). P-values ​​were calculated based on the detection results, with data points indicating P < 0.05 marked in parentheses. The differences in efficacy between the treatment groups and the model control group were analyzed using one-way ANOVA (Dunnett's multiple comparison test). ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001.

[0034] Figure 8 shows the H&E staining results.

[0035] Figure 9 shows the histopathological diagnosis and scoring of lung inflammation in each experimental group based on lung H&E staining results and lung inflammation scoring criteria.

[0036] Figure 10 shows the changes in body weight of mice in each experimental group during the experimental period.

[0037] Figure 11 shows the lung function test results of mice in each experimental group.

[0038] Figure 12 shows typical pathological images of lung tissue from mice in each experimental group, with HE staining of lung tissue (100×).

[0039] Figure 13 shows the pathological scores of lung injury in mice of each experimental group. Note: Based on the results of H&E staining of the lungs, the inflammatory response of the lungs in each experimental group was diagnosed and scored according to the lung inflammation scoring criteria. The scoring results are expressed as mean ± standard error (SEM). The differences in efficacy between each drug-treated group and the model control group were analyzed using one-way ANOVA (Dunnett's multiple comparison test). **** represents P < 0.0001.

[0040] Figure 14 shows typical pathological images of lung tissue from mice in each experimental group, with Masson staining of lung tissue (100×).

[0041] Figure 15 shows the lung fibrosis scores of mice in each experimental group. Note: Based on the Masson staining results of the lungs, the degree of lung fibrosis in each experimental group was diagnosed and scored according to the lung fibrosis scoring criteria. The scoring results are expressed as mean ± standard error (SEM). The differences in efficacy between each drug-treated group and the model control group were analyzed using one-way ANOVA (Dunnett's multiple comparison test). **** represents P < 0.0001.

[0042] Figure 16 shows IHC staining of lung tissue from mice in each experimental group, and expression of collagen I in lung tissue (100×).

[0043] Figure 17 shows the percentage of collagen 1-positive cells in the lung tissue of mice in each experimental group. Note: Collagen 1 detection results are expressed as mean ± standard error (SEM). The differences in efficacy between the drug-treated groups and the model control group were analyzed using one-way ANOVA (Dunnett's multiple comparison test). **** represents P < 0.0001.

[0044] Figure 18 shows typical pathological images of lung tissue from mice in each experimental group, with F4 / 80 expression in the lung tissue (100×).

[0045] Figure 19 shows the percentage of F4 / 80 positive cells in the lung tissue of mice in each experimental group.

[0046] Figure 20 shows IHC staining of lung tissue from mice in each experimental group and expression of α-SMA in lung tissue (200×).

[0047] Figure 21 shows the percentage of α-SMA positive cells in the lung tissue of mice in each experimental group.

[0048] Note: α-SMA test results are expressed as mean ± standard error (SEM). The differences in efficacy between each treatment group and the model control group were analyzed using one-way ANOVA (Dunnett's multiple comparison test). ** represents P < 0.01, **** represents P < 0.0001.

[0049] Figure 22 shows the mRNA expression levels of pulmonary fibrosis-related indicators in the lung tissue of mice in each experimental group. Note: qPCR results are expressed as mean ± standard error (SEM). The differences in efficacy between the drug-treated groups and the model control group were analyzed using one-way ANOVA (Dunnett's multiple comparison test). * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001.

[0050] Figure 23 shows the HYP levels in the lung tissue of mice in each experimental group. Note: HYP test results are expressed as mean ± standard error (SEM). P-values ​​were calculated based on the test results, with data points indicating P < 0.05 marked in parentheses. The differences in efficacy between the drug-treated groups and the model control group were analyzed using one-way ANOVA (Dunnett's multiple comparison test). * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001. Detailed Implementation Plan

[0051] definition

[0052] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques that are obvious to one of ordinary skill in the art. While it is believed that the following terms will be well understood by one of ordinary skill in the art, the following definitions are set forth to better interpret this disclosure.

[0053] The terms “including,” “comprising,” “having,” “containing,” or “involving,” as used herein, and their other variations thereof, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0054] “C 1-4 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1 to 4 carbon atoms, including methyl, ethyl, n-propyl, 2-propyl-n-butyl, 2-butyl, 3-methyl-2-propyl, 1,1-dimethylethyl, etc.

[0055] "Halogen" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0056] “C 1-4 "Halogenated alkyl" refers to a C-aryl group that has been replaced by 1-3 halogen atoms. 1-4 Alkyl groups, including fluoromethyl, difluoromethyl, trifluoromethyl, bromomethyl, fluoroethyl, trifluoroethyl, etc.

[0057] "Heterocycle" refers to a 3-7 member saturated or partially unsaturated cyclic group containing 1-3 nitrogen, oxygen and sulfur atoms. Preferably, the heterocycle is a 5-6 member saturated or partially unsaturated heterocycle, which optionally contains 1-2 additional heteroatoms selected from nitrogen, oxygen and sulfur in addition to the nitrogen atom shown.

[0058] "Radiation-induced lung injury" generally refers to damage to lung tissue caused by ionizing radiation (such as radiotherapy). Radiation therapy used for thoracic tumors can lead to radiation-induced lung injury. These radiation therapies include external beam radiation therapy, internal beam radiation therapy, radionuclide therapy, total body irradiation, intraoperative radiation therapy, and 4D radiation therapy. More specifically, they include three-dimensional conformal radiotherapy (3D-CRT), intensity-modulated radiotherapy (IMRT), volumetric modulated arc therapy (VMAT), image-guided radiotherapy (IGRT), stereotactic body radiotherapy (SBRT), proton therapy, and brachytherapy.

[0059] Radiation-induced pneumonia is an early manifestation of radiation-induced lung injury. It differs from common inflammatory lung diseases. More specifically, common inflammatory lung diseases, such as pneumonia, have different causes than radiation-induced pneumonia. Pneumonia is an infection of the lungs caused by bacteria, viruses, or other microorganisms, primarily affecting the alveoli. Bacterial and viral pneumonias are the most common. Clinically, it has a shorter onset time, with symptoms mainly including cough and high fever. Treatment primarily involves symptomatic relief and antibiotics to combat the bacterial or viral infection, and it usually resolves spontaneously once the underlying cause is removed. In contrast, radiation-induced lung injury continues to progress.

[0060] Radiation-induced pulmonary fibrosis (RFP) is a late-stage manifestation of radiation-induced lung injury. It is a type of pulmonary fibrosis with a relatively clear etiology, unlike idiopathic pulmonary fibrosis (IPF). IPF is a chronic, progressive, fibrotic interstitial lung disease of unknown cause, with typical symptoms including progressive dyspnea and dry cough, ultimately leading to death from respiratory failure. IPF is a disease primarily characterized by extracellular fibrosis deposition, with a long and insidious onset; in most cases, lung function is already severely impaired at diagnosis. In contrast, patients with early-stage radiation-induced lung injury will exhibit clinical symptoms, and early anti-inflammatory drug intervention often prevents progression to pulmonary fibrosis. This disclosure describes a PDE4 inhibitor with significant efficacy in the treatment and / or prevention of radiation-induced lung injury.

[0061] The term "treatment" generally refers to achieving the desired pharmacological and / or physiological effect. This effect can be therapeutic, depending on whether it partially or completely stabilizes or cures the disease and / or causes side effects due to the disease. As used herein, "treatment" encompasses any treatment of a patient's disease, including: (a) suppressing the symptoms of the disease, i.e., preventing its progression; or (b) alleviating the symptoms of the disease, i.e., causing the disease or symptoms to regress.

[0062] The term "treatment" means administering the compound or preparation described in this application to improve or eliminate a disease or one or more symptoms related to said disease, and includes:

[0063] (i) Suppress the disease or disease state, that is, curb its development;

[0064] (ii) Relieve the disease or disease state, even if the disease or disease state subsides.

[0065] The term “prevention” means administering the compound or formulation described in this application to prevent a disease or one or more symptoms associated with the disease, and includes: preventing the occurrence of a disease or disease state in mammals, particularly when such mammals are susceptible to the disease state but have not yet been diagnosed with the disease state.

[0066] The terms “effective amount” or “therapeutic effective amount” mean (i) the amount of the disclosed compound used to treat or prevent a particular disease, condition, or disorder; (ii) to reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) to prevent or delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the disclosed compound constituting a “therapeutic effective amount” varies depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal to be treated, but may routinely be determined by a person skilled in the art based on their own knowledge and the content of this disclosure.

[0067] The terms “subject” or “patient” are used interchangeably in this document and refer to an animal, preferably a mammal, and most preferably a human, that has been used as a subject of treatment, observation or experimentation.

[0068] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0069] Specifically, this disclosure relates to the following technical solutions.

[0070] In one embodiment, this disclosure relates to the use of PDE4 inhibitors in the preparation of medicaments for the treatment and / or prevention of lung diseases.

[0071] In a further embodiment, this disclosure relates to the use of a PDE4 inhibitor in the preparation of a medicament for the treatment and / or prevention of lung diseases, wherein the PDE4 inhibitor is a compound of formula (I), or a tautomer, solvate, or pharmaceutically acceptable salt thereof:

[0072] in:

[0073] Ring A is a 5-6 member saturated or partially unsaturated heterocycle, which, in addition to the nitrogen atom shown, optionally contains 1-2 additional heteroatoms selected from nitrogen, oxygen and sulfur;

[0074] R1 is selected from hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl groups and -OR;

[0075] R2 is selected from hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl groups and -OR;

[0076] R is selected from hydrogen and C. 1-4 alkyl;

[0077] n is 1, 2, or 3.

[0078] In a further embodiment, the PDE4 inhibitor is selected from the following compounds, or their tautomers, solvates, or pharmaceutically acceptable salts:

[0079] In a further embodiment, the PDE4 inhibitor is a compound of the following formula, or a tautomer, solvate, or pharmaceutically acceptable salt thereof:

[0080] In some embodiments, this disclosure relates to a PDE4 inhibitor for the treatment and / or prevention of lung diseases, wherein the PDE4 inhibitor is a compound of formula (I), or a tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0081] In some embodiments, this disclosure relates to methods of treating and / or preventing lung diseases, including administering a therapeutically effective amount of a PDE4 inhibitor to a subject, wherein the PDE4 inhibitor is a compound of formula (I), or a tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0082] In some embodiments, this disclosure relates to the use of PDE4 inhibitors in the treatment and / or prevention of lung diseases, wherein the PDE4 inhibitor is a compound of formula (I), or a tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0083] In a further embodiment, the lung disease is lung injury.

[0084] In a further embodiment, the lung disease is lung inflammation.

[0085] In a further embodiment, the lung disease is pulmonary fibrosis.

[0086] In a further embodiment, the lung disease is radiation-induced lung injury.

[0087] In a further embodiment, the radiation-induced lung injury is caused by radiation therapy for a thoracic tumor.

[0088] In a further embodiment, the radiation-induced lung injury is caused by a radiotherapy selected from: external beam radiation therapy, internal beam radiation therapy, radionuclide therapy, whole-body irradiation, intraoperative radiation therapy, and 4D radiation therapy.

[0089] In a further embodiment, the radiation-induced lung injury is caused by a radiotherapy selected from the following: three-dimensional conformal radiotherapy (3D-CRT), intensity-modulated radiotherapy (IMRT), volumetric modulated intensity-modulated radiotherapy (VMAT), image-guided radiotherapy (IGRT), stereotactic body radiotherapy (SBRT), proton therapy, and brachytherapy.

[0090] In a further embodiment, the radiation-induced lung injury is selected from radiation-induced pneumonia and radiation-induced pulmonary fibrosis.

[0091] In a further embodiment, the drug is used to prevent the lung disease.

[0092] In a further embodiment, the drug is used to treat the lung disease.

[0093] In a further embodiment, the drug also includes a package insert indicating its use in combination with an additional therapeutic agent.

[0094] In a further embodiment, the additional therapeutic agent is selected from antibacterial agents, antiviral agents, antifungal agents, antitumor agents, antihistamines, proteins, enzymes, hormones, nonsteroidal anti-inflammatory substances, cytokines, steroids, and insulin.

[0095] The disclosed medicament can be administered orally or parenterally. When administered orally, it can be given in conventional dosage forms. It can be administered parenterally in the following dosage forms: for example, topical formulations (e.g., inhalers, nasal formulations, and topical preparations), injections, transdermal formulations, or nasal formulations. Oral and rectal formulations include, for example, capsules, tablets, pills, powders, sachets, suppositories, and liquid formulations. Injectable formulations include, for example, sterile solutions or suspensions. Topical formulations include, for example, creams, ointments, lotions, and transdermal formulations, such as adhesives.

[0096] In some embodiments, the drug of this disclosure is administered by inhalation, optionally by nebulization.

[0097] The above dosage forms can be formulated using pharmaceutically acceptable excipients or additives in a conventional manner. Excipients or additives include, for example, carriers, binders, flavoring agents, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, and preservatives. Carriers include, for example, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth gum, methylcellulose, sodium carboxymethyl cellulose, low-melting-point waxes, and cocoa butter.

[0098] Liquid formulations for injection include solutions, suspensions, and emulsions. Such formulations include, for example, aqueous solutions and aqueous propylene glycol solutions. Liquid formulations may optionally contain water. They may also be formulated as solutions of polyethylene glycol and / or propylene glycol.

[0099] Topical formulations include the aforementioned liquid formulations, creams, aerosols, sprays, powders, lotions, and ointments. Topical formulations can be prepared by combining a PDE4 inhibitor (including compound A) with conventional pharmaceutically acceptable diluents and carriers. Ointments and creams can be formulated by adding thickeners and / or gelling agents to an aqueous or oily matrix. Matrixes include, for example, water, liquid paraffin, and vegetable oils. Thickeners include, for example, soft paraffin, aluminum stearate, cetyl stearyl alcohol, propylene glycol, lanolin, hydrogenated lanolin, and beeswax. Lotions can be prepared by adding one or more pharmaceutically acceptable stabilizers, suspending agents, emulsifiers, dispersants, thickeners, colorants, or flavoring agents to an aqueous or oily matrix. If desired, topical formulations may optionally contain preservatives or antibacterial growth promoters, such as methylparaben, propylparaben, chlorocresol, or benzalkonium chloride. PDE4 inhibitors (including compound A) can also be administered in dosage forms such as liquid sprays, powders, dry powders, or as drops or inhalers for pulmonary administration, or as nasal or intranasal administration. Furthermore, liquid formulations or suspensions can also be used as eye drops.

[0100] The dosage or frequency of administration of the disclosed compounds depends on the condition, age, weight, dosage form, etc. In oral administration, adults typically administer one or more doses daily at a dose of about 1 to about 1000 mg, preferably about 2 to about 500 mg, particularly about 5 to about 200 mg. In injectable administration, the disclosed compounds can be administered intravenously one or more times at a dose of about 0.1 to about 300 mg, preferably about 1 to about 200 mg. In the case of pulmonary or inhaled formulations, or nasal or intranasal formulations, the disclosed compounds can be administered one or more times at a dose of about 0.1 to about 300 mg, preferably about 1 to about 200 mg. In the case of topical formulations such as ointments or creams, the disclosed compounds can be applied one or more times at a dose of about 0.1 to about 300 mg, preferably about 1 to about 200 mg. In the case of adhesive formulations, the disclosed compounds can be applied one or more times at a dose of about 0.1 to about 300 mg, preferably about 1 to about 200 mg.

[0101] The PDE4 inhibitor disclosed herein has at least one or more of the following beneficial effects: (1) inhibiting the release of inflammatory factors, such as significantly inhibiting the release of TNF-α protein from PBMC cells; significantly inhibiting the expression level of IL-2 in PBMC cells; (2) inhibiting the fibrotic transformation of cells; (3) providing preventive intervention for radiation-induced lung injury; (4) effectively treating radiation-induced lung injury; (5) having good selective inhibition of PDE4; and (6) effectively treating radiation-induced lung fibrosis.

[0102] Example

[0103] Example 1. Pharmacological effects of PDE4 inhibitors in PBMC cells

[0104] 1.1 Experimental Objective

[0105] The aim of this study was to evaluate the effects of compound A (TRD242) on the production of TNF-α and IL-2 in human PBMCs (peripheral blood mononuclear cells) under stimulation by lipopolysaccharide (LPS) and phytohemagglutinin P (PHA-P).

[0106] 1.2 Instruments and Equipment

[0107] 1.3 Test Methods

[0108] Resuscitate PBMC cells and adjust cell density to 0.5-1×10⁻⁶. 6 Cells / mL, incubated overnight in RPMI 1640 medium (containing 10% inactivated FBS). Next:

[0109] 1) Adjust the PBMC density to 5×10 6 Cells / mL, add 100 μL to each well of a 96-well cell culture plate, and add an equal amount of cells to the blank control wells.

[0110] 2) Prepare 4×TRD242 working solution. Add 50μL to each well of the treatment group, and add an equal amount of culture medium to the stimulation group and the blank group. Incubate for half an hour.

[0111] 3) Add 50 μL of 4×LPS (400 ng / mL) or 4×PHA-P (40 μg / mL). Add an equal amount of the corresponding stimulant to the stimulation group and an equal amount of culture medium to the blank group. The incubation time is 4 hours and 20 hours, respectively.

[0112] 4) Collect the supernatant for the detection of TNF-α and IL-2.

[0113] 1.4 Data Statistics

[0114] GraphPad Prism 8 software was used for statistical analysis and graphing. All data are expressed as mean ± SD. One-way ANOVA with Dunnett's test was used to analyze differences between groups. A p < 0.05 was considered statistically significant.

[0115] 1.5 Test Results

[0116] Compared with the LPS stimulation group, TNF-α protein levels in activated PBMC cells were downregulated by 8.4%, 27.0%, 42.7%, 51.7%, and 61.1% after pretreatment with different concentrations (0.1 nM, 1 nM, 10 nM, 100 nM, and 1 μM) of TRD242. TRD242 significantly inhibited TNF-α protein release from PBMC cells at concentrations ≥1 nM (Figure 1-A).

[0117] TRD242 significantly inhibited IL-2 production in human PBMC cells stimulated by PHA-P. The inhibition rates of IL-2 by 0.5 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 100 nM, and 1000 nM TRD242 were 29.2%, 26.0%, 34.3%, 44.9%, 47.2%, 59.2%, 65.0%, and 70.0%, respectively (p < 0.05, p < 0.05, p < 0.01, p < 0.001, p < 0.0001, p < 0.0001, p < 0.0001), as shown in Figure 1-B.

[0118] Conclusion: The results of this experiment show that TRD242 can inhibit the expression levels of inflammatory factors TNF-α and IL-2 in human PBMC cells in vitro.

[0119] Example 2. Pharmacodynamic effects of PDE4 inhibitors in MRC-5 cells

[0120] 2.1 Experimental Objective

[0121] The aim of this study was to explore the effect of the PDE4 inhibitor, TRD242, on the expression of fibroblast-promoting factor mRNA in MRC-5 fibroblasts under the induction of transforming growth factor β1 (TGF-β1).

[0122] 2.2 Instruments and Equipment

[0123] 2.3 Test Methods

[0124] 2.3.1 Cell Culture

[0125] 1) MRC-5 cells were cultured in EMEM medium containing 10% fetal bovine serum.

[0126] 2) When the cell fusion rate reaches 70-85%, digest with 0.05% trypsin, centrifuge, resuspend in serum-free medium, and use for experiments.

[0127] 2.3.2 Seeding Plates and Cell Treatment

[0128] 1) Count the cells and adjust the density to 1×10⁻⁶. 5 Inoculate 0.5 mL per well of a 24-well plate and culture overnight in serum-free starvation.

[0129] 2) Prepare 4×TRD242 working solution, add 0.25mL to each well; set up a blank control group and a TGF-β1 stimulation group, and add an equal volume of FBS-free culture medium.

[0130] 3) After 1 hour of compound pretreatment, 0.25 mL of TGF-β1 (final concentration of 5 ng / mL) was added to all wells except the blank control group, and the cells were cultured for another 48 hours. Total RNA was then extracted from the cells.

[0131] 2.3.3 Total RNA Extraction

[0132] Total RNA was extracted using the TRIzol method, and the specific steps are as follows:

[0133] 1) After culturing cells for 48 hours, aspirate the culture medium and wash once with pre-cooled PBS;

[0134] 2) Discard the PBS and add 0.5 mL of TRIzol to each well;

[0135] 3) Use a pipette to repeatedly pipette the cells to allow them to detach from the bottom of the plate and fully contact the TRIzol for lysis. Combine the two wells and transfer them into a 1.5 mL centrifuge tube. Incubate at room temperature for 5 min.

[0136] 4) Add 200 μL of chloroform to each tube, shake thoroughly on a vortex mixer for 15 seconds, let stand at room temperature for 3 minutes, and centrifuge at 12000g for 3 minutes.

[0137] 5) Carefully aspirate the upper aqueous phase using a pipette and place it into a new 1.5 mL centrifuge tube;

[0138] 6) Add an equal volume of isopropanol to the aqueous phase, gently invert to mix, let stand at room temperature for 3 min, and centrifuge at 12000g for 3 min.

[0139] 7) Place the centrifuge tube on ice, discard the isopropanol, add 75% ethanol solution prepared with DEPC-treated water, mix gently, and centrifuge at 7500g for 5 minutes.

[0140] 8) Discard the ethanol and let it stand at room temperature for 5-10 minutes to remove residual ethanol. Add 20 μL of DEPC-treated water to each tube and check the RNA concentration. The 260 / 280 nm ratio should be greater than 1.8.

[0141] 2.4 Data Statistics

[0142] Adjust the baseline and threshold to determine the cycle threshold (C) of the amplification curve. T Using relative quantification (2) ΔΔCT The results were analyzed using the following methods. GraphPad Prism 8 software was used for statistical analysis and graphing. All data are expressed as mean ± SD. One-way ANOVA with Dunnett's test was used to analyze differences between groups. A p < 0.05 was considered statistically significant.

[0143] 2.5 Test Results

[0144] TGF-β1 stimulation upregulated FN (fibronectin) mRNA levels by 1.4-fold. Pretreatment with 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, and 1 μM TRD242 reduced FN mRNA levels by 20.8%, 33.9%, 52.7%, 46.1%, 43.0%, 49.0%, and 72.3%, respectively (Figure 2A).

[0145] TGF-β1 stimulation upregulated platelet-derived growth factor (PDGF-B) mRNA levels by 107-fold. Pretreatment with 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, and 1 μM TRD242 reduced PDGF-B mRNA levels by 0.7%, 2.0%, 29.3%, 59.2%, 71.8%, 84.8%, and 87.2%, respectively (Figure 2B).

[0146] Example 3. Pharmacodynamic effects of prophylactic administration of PDE4 inhibitors on radiation-induced lung injury.

[0147] 3.1 Experimental Objective

[0148] By constructing a radiation-induced lung injury model, PDE4 inhibitors were administered prophylactically to observe their pharmacodynamic effects.

[0149] 3.2 Instruments and Equipment

[0150] 3.3 Test Methods

[0151] Preparation before model construction: On the day of grouping, the experimental animals were randomly divided into 6 groups according to the most recent body weight parameters: normal control group, model control group, positive control drug treatment group, and low, medium and high dose treatment groups of test product, with 10 animals per group, for a total of 60 animals. On the day of grouping, the animals were fasted but allowed to drink water for 12-16 hours.

[0152] Model construction procedure: On the day of irradiation (D0), except for the normal control group, all animals in the experimental groups underwent thoracic irradiation using a small animal irradiator. The specific procedure was as follows: After anesthesia, the mice's head, abdomen, and lower urethra were shielded using a lead shielding device, exposing only the thoracic cavity. They were then placed in the irradiation chamber, and the instrument was activated for thoracic irradiation at a dose of 15 Gy, a single irradiation. Model construction was carried out according to group order, with 5 animals per irradiation session; the number of animals per group was 10, for a total of 60 animals.

[0153] Sex ratio: Single sex, all males;

[0154] Grouping method: Based on the most recent animal reordering before grouping, random numbers were assigned, and the random numbers were then ordered in descending order. The groups were designated as groups 1, 2, 3, 4, 5, and 6 from low to high, representing the normal control group, the model control group, the positive control drug treatment group, the low-dose TRD242 treatment group, the medium-dose TRD242 treatment group, and the high-dose TRD242 treatment group, respectively.

[0155] 3.4 Data Statistics

[0156] The measured indicators are expressed as mean ± standard deviation. Data from groups with a sample size of less than 3 are not included in the statistical comparison.

[0157] Animal experimental data in the tables are expressed as mean ± standard deviation (Mean ± SD), while experimental data in the images are described as mean ± standard error (Mean ± SEM). For comparisons among groups with normality and homogeneity of variance, one-way ANOVA was first used, followed by Tukey's HSD test for further multiple comparisons. For groups not conforming to normality or with unequal variances, the Kruskal-Wallis H test (KW method) was used, and Dunn's method was used for multiple comparisons. P < 0.05 was considered statistically significant. All statistical analyses were performed using Stata 15 software.

[0158] 3.5 Test Results

[0159] (1) PDE4 inhibitors can significantly improve the infiltration of inflammatory cells in bronchoalveolar lavage fluid.

[0160] The results of the bronchoalveolar lavage fluid blood cell count at the experimental endpoint showed that, compared with the normal control group, the model control group animals showed obvious pulmonary inflammatory damage after thoracic irradiation. The degree of blood cell exudation in their bronchoalveolar lavage fluid was significantly increased, with the most significant increases in white blood cells, neutrophils, lymphocytes and platelets. The differences were all statistically significant (P<0.05), indicating that the model animals had inflammatory and vascular damage in their lungs.

[0161] PDE4 inhibitors showed significant therapeutic effects on inflammatory lung injury in model animals under the established treatment regimen, and their therapeutic effect was significantly dose-dependent. Under the same detection and analysis conditions, the degree of hemocyte exudation in the bronchoalveolar lavage fluid of animals treated with low, medium and high doses of TRD242 decreased to varying degrees compared with the control group at the same time point. The improvement was most significant in the medium and high dose treatment groups, and the differences were statistically significant (P < 0.05), as shown in Figure 3.

[0162] Note: At the end of the experiment, bronchoalveolar lavage fluid was collected from animals in each experimental group by dissection, and blood cell counts were performed using a fully automated hematology analyzer. All individual data are expressed as mean ± standard deviation (SD). **** P < 0.0001 indicates 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 indicates the statistical analysis results of the TRD242 treatment group or budesonide treatment group compared with the model control group. + P < 0.05 indicates the statistical analysis results among the low, medium and high dose treatment groups of TRD242.

[0163] (2) HE pathological staining showed that PDE4 inhibitors significantly improved the infiltration of inflammatory cells.

[0164] This study collected lung tissue samples from animals in each experimental group at the end of the dissection. The samples underwent routine fixation and H&E staining. The histopathological characteristics of the lung tissue (full field of view) were analyzed under 100× and 200× optical microscopes to analyze the progression of lung injury in the model animals and the effect of the test substance TRD242 on the histological characteristics of the lung tissue. Based on the H&E staining results, lung inflammation in each experimental group was diagnosed, graded, and scored to analyze the degree of inflammatory response in the lungs and the effect of the test substance TRD242 on the inflammatory response in the lungs of the model animals.

[0165] PDE4 inhibitors also showed significant therapeutic effects on radiation-induced lung inflammation and infiltration under the set treatment regimen. Under the same diagnostic analysis conditions, the degree of lung inflammation and alveolar structural damage in the low, medium and high dose TRD242 treatment groups was significantly lower than that in the model control group, as shown in Figure 4.

[0166] Note: Based on the results of H&E staining of the lungs, the lung inflammation response of animals in each experimental group was diagnosed and scored according to the lung inflammation scoring criteria, as shown in Figure 5. All individual data are expressed as mean ± standard deviation (SD). **** P < 00001 indicates 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 indicates the statistical analysis results of the TRD242 treatment group or budesonide treatment group compared with the model control group.

[0167] Conclusion: Compared with hormones, PDE4 is safer for early prophylactic administration in the long run.

[0168] Example 4: Pharmacodynamic effects of PDE4 inhibitors in the treatment of radiation-induced lung injury

[0169] 4.1 Test Plan

[0170] Male SPF-grade C57BL / 6 mice, aged 6–8 weeks, were purchased from Shanghai Lingchang Biotechnology Co., Ltd. They were routinely housed in the SPF-grade laboratory of Shanghai Paisi New Biotechnology Experimental Animal Center under conditions of 40–70% relative humidity and 20–26°C, with 12-hour light-dark cycles. All animals had free access to food and water. Experiments began after one week of acclimatization. The experimental animals were randomly divided into 7 groups based on body weight: a blank control group, a model control group, positive drug group 1 and 2, and low, medium, and high-dose PDE4 inhibitor groups. After anesthesia and fixation, the mice underwent a single whole-chest irradiation with X-rays at a dose of 18 Gy. During irradiation, other areas outside the chest of the mice were shielded using a 1 mm thick specially made lead box. After the mice regained consciousness, they were returned to the barrier and continued to be fed. After irradiation for 2 weeks, the mice were given medication. The low, medium and high dose groups of PDE4 inhibitors were given TRD242 via bronchial nebulization at the corresponding dose once a day until the end of the experiment. The positive control group 1 was given dexamethasone orally at the corresponding dose once a day until the end of the experiment. The positive control group 2 was given budesonide via bronchial nebulization at the corresponding dose once a day until the end of the experiment. The blank control group and the model control group were given the corresponding solvent via bronchial nebulization once a day until the end of the experiment.

[0171] 4.2 Data Statistics

[0172] GraphPad Prism v9.0.0 software was used for statistical analysis. All experimental data are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA (Dunnett's multiple comparison test) was used to evaluate the differences between each treatment group and the model control group. A p-value < 0.05 was considered statistically significant, and a p-value > 0.05 was considered not statistically significant. Specific data are presented in graphs and tables.

[0173] 4.3 Test Results

[0174] (1) BALF white blood cell count

[0175] Figure 6 shows the BALF detection results. The white blood cell counts in the normal group, model group, dexamethasone group, budesonide group, and the low, medium, and high dose TRD242 groups were 9467.36 / mL, 226374.43 / mL, 9262.79 / mL, 19612.52 / mL, 18139.80 / mL, 26794.65 / mL, and 25998.97 / mL, respectively. Compared with the normal control group, the number of sclerotia in the BALF of the model control group was significantly increased after thoracic irradiation induction (P < 0.0001). Compared with the model group, the number of sclerotia in the BALF of each treatment group (dexamethasone group, budesonide group, and low, medium, and high dose TRD242 groups) was significantly decreased (P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001).

[0176] (2) Results of BALF inflammatory cytokine detection

[0177] The results of BALF inflammatory cytokine detection are shown in Figure 7. Compared with the normal group, the levels of inflammatory cytokines TNF-α, IL-1β, and IL-6 in the BALF of mice in the model control group were significantly increased (p<0.0001, p<0.01, p<0.0001), with levels of 319.313 pg / mL, 34.370 pg / mL, and 316.868 pg / mL, respectively. Compared with the model control group, the levels of inflammatory cytokines TNF-α, IL-1β, and IL-6 in the BALF of mice in the low, medium, and high dose groups of dexamethasone, budesonide, and TRD242 were significantly decreased.

[0178] (3) Pathological examination results of lung tissue

[0179] The H&E staining results are shown in Figure 8. In the blank control group, the lung tissue structure of mice was intact, with no obvious inflammatory cell infiltration. Compared with the blank control group, the lung tissue morphology and structure of mice in the model control group were disrupted, with significant thickening of the alveolar walls accompanied by hemorrhage and edema, smaller alveolar cavities, and abundant inflammatory cell infiltration, resulting in a significantly higher pathological score (P < 0.0001). Compared with the model control group, the degree of inflammatory cell infiltration in the lung tissue of mice in the low, medium, and high dose groups of dexamethasone, budesonide, and TRD242 was significantly reduced, alveolar wall thickening and hemorrhage were significantly alleviated, and the pathological score was significantly lower (P < 0.0001, P < 0.001, P < 0.001, P < 0.001, P < 0.001, P < 0.001), with no significant differences among the drug administration groups.

[0180] Based on the results of H&E staining of the lungs, the inflammatory response of the lungs in each experimental group was diagnosed and scored according to the lung inflammation scoring criteria. The scoring results are expressed as mean ± standard error (SEM). The differences in efficacy between each drug-treated group and the model control group were analyzed using one-way ANOVA (Dunnett's multiple comparison test). *** represents P < 0.001, **** represents P < 0.0001, see Figure 9.

[0181] Conclusion: For therapeutic administration, PDE4 inhibitors have advantages over the strict control of dosage and gradual reduction of hormones in clinical use.

[0182] Example 5: TRD242 Enzymatic Inhibition Test

[0183] 5.1 Research Objectives

[0184] This experiment used the AMP-Glo ​​method to test the inhibitory effect of the test compound TRD242 on the enzyme activities of PED1C / PDE2A / PDE3A / PDE3B / PDE4A1A / PDE4B2 / PDE4C1 / PDE4D2 / PDE4D3 / PDE7A1 / PDE8A1 / PDE10A1 / PDE10A2 / PDE11A4, the PDE-Glo method to test the inhibitory effect of the test compound TRD242 on the enzyme activities of PDE5A1 / PDE6C, and the IMAP method to test the inhibitory effect of the test compound TRD242 on the enzyme activities of PDE1A / PDE4B1 / PDE9A2.

[0185] 5.2 Reagents and Consumables

[0186] 5.2.1 Materials and Reagents

[0187] 5.2.2 Instruments and Consumables

[0188] 5.3 Experimental Procedure

[0189] 5.3.1 Preparation and Processing of Compounds

[0190] 5.3.1.1 Preparation of Compounds

[0191] 1) Dissolve the positive control drug PDE1-IN-2 in DMSO to prepare a 20 mM stock solution;

[0192] 2) Dissolve the positive control drug PF-05180999 in DMSO to prepare a 50mM stock solution;

[0193] 3) Dissolve the positive control drug Cilostamide in DMSO to prepare a 70 mM stock solution;

[0194] 4) Dissolve the positive control drug Roflumilast in DMSO to prepare a 50 mM stock solution;

[0195] 5) Dissolve the positive control drug Sildenafil (citrate) in DMSO to prepare a 50 mM stock solution;

[0196] 6) Dissolve the positive control drug BRL-50481 in DMSO to prepare a 50 mM stock solution;

[0197] 7) Dissolve the positive control drug PF-04957325 in DMSO to prepare a 20 mM stock solution;

[0198] 8) Dissolve the positive control drug Mardepodect in DMSO to prepare a 50 mM stock solution;

[0199] 9) Dissolve the positive control drug TAK-063 in DMSO to prepare a 50 mM stock solution;

[0200] 10) Dissolve the positive control drug Dipyridamole in DMSO to prepare a 50 mM stock solution;

[0201] 11) Dissolve the positive control drug 3-isobutyl-1-methylxanthine in DMSO to prepare a 100 mM stock solution;

[0202] 12) Dissolve 10 mg of compound TRD242 in DMSO to prepare a 50 mM stock solution;

[0203] 13) Visually inspect the solubility; all concentrations dissolved completely without any visible precipitate.

[0204] Table 1 Information on the tested compounds

[0205] 5.3.1.2 Preservation of Compounds

[0206] Compound TRD242 was dissolved in DMSO and stored at -20°C for later use.

[0207] 5.3.1.3 Preparation of the compound working solution

[0208] 1) The positive control drug PDE1-IN-2 was serially diluted 3-fold with DMSO to 10 concentration points to prepare a 200× compound working solution for later use;

[0209] 2) The positive control drug PF-05180999 was serially diluted 3-fold with DMSO to 10 concentration points to prepare a 200× compound working solution for later use;

[0210] 3) The positive control drug Cilostamide was serially diluted 3-fold with DMSO to 10 concentration points to prepare a 200× compound working solution for later use;

[0211] 4) The positive control drug Roflumilast was serially diluted 3-fold with DMSO to 10 concentration points to prepare a 200× compound working solution for later use;

[0212] 5) The positive control drug Roflumilast was serially diluted 4-fold with DMSO to 10 concentration points to prepare a 200× compound working solution for later use;

[0213] 6) The positive control drug Sildenafil (citrate) was serially diluted 3-fold with DMSO to prepare 10 concentration points to form a 200× compound working solution for later use;

[0214] 7) The positive control agent BRL-50481 was serially diluted 3-fold with DMSO to 10 concentration points to prepare a 200× compound working solution for later use;

[0215] 8) The positive control drug PF-04957325 was serially diluted 3-fold with DMSO to prepare 10 concentration points to form a 200× compound working solution for later use.

[0216] 9) The positive control drug Mardepodect was serially diluted 3-fold with DMSO to 10 concentration points to prepare a 200× compound working solution for later use;

[0217] 10) The positive control drug TAK-063 was serially diluted 3-fold with DMSO to prepare 10 concentration points to form a 200× compound working solution for later use;

[0218] 11) The positive control drug Dipyridamole was serially diluted 3-fold with DMSO to 10 concentration points to prepare a 200× compound working solution for later use;

[0219] 12) The positive control drug 3-isobutyl-1-methylxanthine was serially diluted 3-fold with DMSO to 10 concentration points to prepare a 100× compound working solution for later use;

[0220] 13) Compound TRD242 was serially diluted 5-fold with DMSO to 10 concentration points to prepare a 200× compound working solution for later use;

[0221] 14) Compound TRD242 was serially diluted 5-fold with DMSO to 10 concentration points to prepare a 100× compound working solution for later use.

[0222] Table 2. Compound concentration settings in the PDE enzyme experiment reaction system

[0223] 5.3.2 Determination of the activity inhibition of compounds using different experimental methods

[0224] 5.3.2.1 Activity Inhibition Determination Using the AMP-Glo ​​Experimental Method

[0225] 1) Transfer 20 nL of 200× compound working solution to the experimental plate via Echo for later use. Add the same volume of 200× positive drug working solution to the positive control well PC, and add the same volume of 200× 100% DMSO (1× final concentration: 0.5%) to the negative control well VC.

[0226] 2) Prepare 2×PED1C / PDE2A / PDE3A / PDE3B / PDE4A1A / PDE4B2 / PDE4C1 / PDE4D2 / PDE4D3 / PDE7A1 / PDE8A1 / PDE10A1 / PDE10A2 / PDE11A4 solutions using experimental buffer, and set aside for later use;

[0227] 3) Add 2 μL of 2×PED1C / PDE2A / PDE3A / PDE3B / PDE4A1A / PDE4B2 / PDE4C1 / PDE4D2 / PDE4D3 / PDE7A1 / PDE8A1 / PDE10A1 / PDE10A2 / PDE11A4 solution to the 384-well experimental plate, centrifuge at 1000 rpm for 1 minute, and incubate the experimental plate at room temperature for 10 minutes;

[0228] 4) Prepare a 2×cAMP solution using experimental buffer and set aside for later use;

[0229] 5) Add 2 μL of 2×cAMP solution to the 384-well experimental plate, centrifuge at 1000 rpm for 1 minute, and incubate the experimental plate at room temperature for 60 minutes;

[0230] 6) Add 4 μL of AMP-Glo ​​Reagent to the 384-well plate and centrifuge at 1000 rpm for 1 minute; incubate the plate at room temperature for 60 minutes.

[0231] 7) Add 8 μL of Kinase Detection Reagent to a 384-well experimental plate and centrifuge at 1000 rpm for 1 minute;

[0232] 8) Read the Luminescence signal data through Envision.

[0233] 5.3.2.2 Activity Inhibition Determination Using the PDE-Glo Experimental Method

[0234] 1) Transfer 20 nL of 200× compound working solution to the experimental plate via Echo for later use. Add the same volume of 200× positive compound working solution to the positive control well PC, and add the same volume of 200× 100% DMSO (1× final concentration: 0.5%) to the negative control well VC.

[0235] 2) Prepare a 2×PDE5A1 / PDE6C solution using experimental buffer solution and set aside for later use;

[0236] 3) Add 2 μL of 2×PDE5A1 / PDE6C solution to the 384-well experimental plate, centrifuge at 1000 rpm for 1 minute, and incubate the experimental plate at room temperature for 10 minutes;

[0237] 4) Prepare a 2×cGMP solution using experimental buffer solution and set aside for later use;

[0238] 5) Add 2 μL of 2×cGMP solution to the 384-well experimental plate, centrifuge at 1000 rpm for 1 minute, and incubate the experimental plate at room temperature for 90 minutes;

[0239] 6) Add 4 μL of PDE-Glo Reagent to the 384-well experimental plate, centrifuge at 1000 rpm for 1 minute; incubate the experimental plate at room temperature for 20 minutes;

[0240] 7) Add 8 μL of PDE-Glo Detection Reagent to a 384-well experimental plate and centrifuge at 1000 rpm for 1 minute;

[0241] 8) Read the Luminescence signal data through Envision.

[0242] 5.3.2.3 Activity Inhibition Determination Using IMAP Experimental Method

[0243] 1) Transfer 200 nL of 100× compound working solution to the experimental plate via Echo for later use. Add 200 nL of 100× 3-isobutyl-1-methylxanthine (1× final concentration: 1000 uM) working solution as positive control. Add 200 nL of 100% DMSO (1× final concentration: 1%) to the positive control well PC and the negative control well VC.

[0244] 2) Prepare 1× experimental buffer: 1× IMAP Reaction Buffer, 1mM DTT, replenished with ddH2O, ready for use;

[0245] 3) Prepare a 2×PDE1A / PDE4B1 / PDE9A2 solution using experimental buffer and set aside for later use;

[0246] 4) Add 10 μL of 2×PDE1A / PDE4B1 / PDE9A2 to the 384-well experimental plate, centrifuge at 1000 rpm for 1 minute, and incubate the experimental plate at room temperature for 30 minutes.

[0247] 5) Prepare a 2×cAMP / cGMP solution using experimental buffer and set aside for later use;

[0248] 6) Add 10 μL of 2×cAMP / cGMP solution to the 384-well experimental plate, centrifuge at 1000 rpm for 1 minute, and incubate the experimental plate at room temperature for 90 minutes;

[0249] 7) Add 60 μL of diluted binding agent to the 384-well experimental plate, centrifuge at 1000 rpm for 1 minute; incubate the experimental plate at room temperature for 60 minutes.

[0250] 8) Read the FP signal data through Envision.

[0251] 5.3.3 Data Analysis

[0252] 1) Z' factor = 1 - 3 × (SD) VC +SD PC ) / (average value VC -average value PC );

[0253] 2) S / B = Signal Ave_VC / Signal Ave_PC ;

[0254] 3) Calculate the compound IC using the Graph Pad nonlinear fitting formula. 50 Y = bottom + (top - bottom) / (1 + 10^(LogIC)) 50 -X)*Hillslope))

[0255] X: Log value of compound concentration; Y: Inhibition %

[0256] 4) Inhibition rate calculation: %inhibition = 100 - (signal) cmpd -Signal Ave_PC ) / (Signal Ave_VC -Signal Ave_PC )×100

[0257] Among them, the AMP-Glo / PDE-Glo experimental method:

[0258] PC: Positive control well value, i.e., the highest concentration reading of the positive control standard added.

[0259] VC: Negative control well value, i.e., DMSO well reading.

[0260] Signal Ave_PC : The average value of all positive control wells on the entire plate.

[0261] Signal Ave_VC : The average value of all negative control wells on the entire plate.

[0262] The IMAP experimental method includes:

[0263] PC: Value of the positive control well, i.e., the value after adding enzyme and substrate.

[0264] VC: Value in the negative control well, i.e., the value after adding buffer and substrate.

[0265] Signal Ave_PC : The average value of all positive control wells on the entire plate.

[0266] Signal Ave_VC : The average value of all negative control wells on the entire plate.

[0267] 5.4 Conclusion

[0268] Table 3 shows the IC50 of the tested compounds against PDE enzyme. 50 and % maximum inhibition rate

[0269] According to the results in Table 3, the IC50 values ​​of the positive control drug 3-isobutyl-1-methylxanthine in the PDE1A, PDE4B1, and PDE9A2 enzyme assays were [data missing]. 50 The IC50 values ​​for the positive control drug PDE1-IN-2 in the PDE1C enzymology assay were 6189.000 nM, 13975.000 nM, and 238641.000 nM, respectively. 50The IC50 value of the positive control PF-05180999 in the PDE2A enzymology assay was 15.280 nM. 50 The IC50 value of the positive control drug Cilostamide in PDE3A and PDE3B enzymatic assays was 0.678 nM. 50 The IC50 values ​​for the positive control drug Roflumilast in enzymatic assays for PDE4A1A, PDE4B2, PDE4C1, PDE4D2, and PDE4D3 were 53.370 nM and 83.700 nM, respectively. 50 The IC50 values ​​of the positive control drug Sildenafil (citrate) in PDE5A1 and PDE6C enzymatic assays were 0.661 nM, 0.249 nM, 4.447 nM, 0.147 nM, and 0.159 nM, respectively. 50 The IC50 values ​​for the positive control drug BRL-50481 in the PDE7A1 enzyme assay were 2.970 nM and 48.790 nM, respectively. 50 The IC50 value of the positive control PF-04957325 in the PDE8A1 enzyme assay was 497.600 nM. 50 The IC50 of the positive control drug Mardepodect in the PDE10A1 enzymology assay was 2.883 nM. 50 The IC50 value of the positive control drug TAK-063 in the PDE10A2 enzyme assay was 0.794 nM. 50 The IC50 value of the positive control drug Dipyridamole was 0.927 nM, and its IC50 value in the PDE11A4 enzymology assay was 0.927 nM. 50 The value was 389.7 nM, which is basically consistent with historical data, indicating that the experimental system is stable and reliable.

[0270] The test compound TRD242 showed no inhibitory activity in enzymatic experiments on PDE1A, PDE1C, PDE2A, PDE3A, PDE3B, PDE5A1, PDE6C, PDE7A1, PDE8A1, PDE9A2, PDE10A1, PDE10A2, and PDE11A4. However, it showed inhibitory activity in several isoforms of PDE4, with PDE4A1A, PDE4B1, PDE4B2, PDE4D2, and PDE4D3 showing the best inhibitory activity and an IC50 value of [missing value]. 50 The inhibitory activity of PDE4C1 enzyme was relatively weak at concentrations of 15.340 nM, 11.030 nM, 6.607 nM, 2.890 nM, and 3.096 nM, respectively, with its IC50 value being [missing information]. 50 The value is 83.920 nM.

[0271] Example 6. Efficacy study of a TRD242 radioactive pulmonary fibrosis model

[0272] Purpose:

[0273] A mouse model of radiation-induced pulmonary fibrosis (RIPF) was established to evaluate the efficacy of the test drug TRD242 in this model.

[0274] method:

[0275] A mouse model of radiation-induced pulmonary fibrosis was established using male C57BL / 6 mice through a single 18 Gy X-ray irradiation of the entire chest cavity. Eight weeks after irradiation, animals were randomly divided into six groups based on body weight: a normal control group, a model control group, a positive control group (nintedanib, 60 mg / kg), and low-dose (0.1 mg / kg), medium-dose (0.5 mg / kg), and high-dose (1 mg / kg) groups of the test drug TRD242, with ten animals in each group. All groups received the corresponding drug or solvent once daily starting eight weeks after X-ray irradiation, followed by a two-week break after four weeks of administration, and then another two weeks of administration until the end of the experiment. The normal control group and model group received 50 μL of physiological saline via bronchial nebulization, while the low-, medium-, and high-dose TRD242 groups received 50 μL of the corresponding dose of TRD242 via bronchial nebulization. The positive control group received nintedanib by gavage. During the experiment, body weight was recorded twice a week. At the end of the experiment, tidal volume (TV), airway resistance (RI), and airway compliance (Cdyn) of mice were measured using an invasive pulmonary function instrument. HE staining was used to observe and score the lung injury of each group of mice. Masson staining was used to observe and score the degree of pulmonary fibrosis of each group of mice. IHC staining was used to observe the expression levels of α-SMA, F4 / 80, and collagen 1 in the lung tissue of each group of mice. qPCR was used to detect the relative expression levels of α-SMA, TGF-β1, collagen 1, collagen 3, MMP12, and TIMP-1 mRNA in the lung tissue of each group of mice. Alkaline hydrolysis was used to detect the content of hydroxyproline (HYP) in the lung tissue of each group of mice.

[0276] Research findings:

[0277] All groups of mice experienced weight loss after irradiation, followed by a gradual increase in weight after 4 days. After the start of nebulized drug administration (8 weeks after irradiation), all groups of mice experienced weight loss as they adapted to the drug administration process. After adaptation, the weight of mice in all drug administration groups and the model control group showed a slow trend of increase.

[0278] Lung function tests in mice showed that, compared with the normal control group, the model control group mice exhibited significantly decreased tidal volume (TV) (P < 0.05), significantly increased airway resistance (RI) (P < 0.001), and significantly decreased airway compliance (CDyn) (P < 0.001), indicating lung dysfunction. Compared with the model group, in all treatment groups—nintedanib group, and low, medium, and high dose TRD242 groups—TV was significantly increased (P < 0.05, P < 0.05, P < 0.05, P < 0.05), RI was significantly decreased (P < 0.05, P < 0.05, P < 0.05, P < 0.05), and CDyn was significantly increased (P < 0.01, P < 0.05, P < 0.01, P < 0.001).

[0279] HE staining results showed that the lung tissue of normal control mice was structurally intact, with no obvious inflammatory cell infiltration. Compared with the normal control group, the alveolar structure of model control mice was disrupted, alveolar septa were thickened, and there was a large amount of inflammatory cell infiltration in the lung tissue, with a significantly increased lung injury score (P < 0.0001). Compared with the model control group, the degree of inflammatory cell infiltration and alveolar septal thickening in the lung tissue of mice in the nintedanib group, and the low, medium, and high dose groups of TRD242 were reduced to varying degrees, with no significant differences among the treatment groups.

[0280] Masson staining results showed that the alveolar wall structure of normal control mice was not significantly abnormal, and the pulmonary septa were not significantly thickened. Compared with the normal control group, the alveolar wall structure of the model control group was disordered, with obvious dense fibrous bands and collagen deposition, and the fibrosis score was significantly increased (P < 0.0001). Compared with the model control group, the alveolar septa thickening and the degree of fibrosis were significantly reduced in the nintedanib group and the low, medium and high dose TRD242 groups (P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001).

[0281] IHC staining results showed that collagen I expression was slightly positive in the lung septa after immunohistochemical staining, indicating type I collagen fiber deposition. The expression level of collagen I in each treatment group showed a decreasing trend compared to the model group, but no significant difference was observed. F4 / 80 immunohistochemical staining showed higher macrophage expression in the lung tissue of the model control group. Macrophage expression in the nintedanib positive control group and the low, medium, and high dose TRD242 groups was significantly lower than that in the model group. The differences in the nintedanib group and the medium and high dose TRD242 groups were statistically significant compared to the model group (P < 0.05, P < 0.05, P < 0.05). α-SMA expression was observed in the bronchial and vascular walls after immunohistochemical staining, and fibroblasts were expressed in the lesion areas of the lung tissue. The expression level of α-SMA in the lung tissue of mice in the nintedanib group was significantly lower than that in the model group (P < 0.01). The expression level of α-SMA in the low, medium, and high dose TRD242 groups showed a decreasing trend compared to the model group, but no significant difference was observed.

[0282] qPCR results showed that, compared with the normal control group, the relative expression levels of α-SMA, TGF-β1, and collagen 3 mRNA in the lung tissue of mice in the model group were significantly increased (P<0.001, P<0.001, P<0.01), while the relative expression levels of collagen 1, MMP12, and TIMP-1 mRNA showed no significant change. Compared with the model group, the relative expression levels of α-SMA, TGF-β1, and collagen 3 mRNA in the lung tissue of mice in the nintedanib group, and the low, medium, and high dose TRD242 groups were decreased to varying degrees, while the relative expression levels of collagen 1, MMP12, and TIMP-1 mRNA showed no significant change.

[0283] Hydroxyproline (HYP) levels were significantly higher in the lung tissue of the model group mice compared with the normal control group (P < 0.01); and significantly lower in the lung tissue of each treatment group compared with the model group (P < 0.05, P < 0.01, P < 0.05, P < 0.05).

[0284] in conclusion:

[0285] Under the conditions of this experiment, a single 18 Gy X-ray irradiation of the entire thoracic cavity successfully established a mouse model of radiation-induced pulmonary fibrosis. Eight weeks after X-ray irradiation, treatment with the test drug TRD242 and the positive control drug nintedanib, once daily for six weeks, significantly improved lung function in fibrotic mice, reduced the expression levels of inflammatory and fibrotic markers such as F4 / 80, α-SMA, TGF-β1, and collagen 3 in the lung tissue to varying degrees, significantly reduced HYP content in the lung tissue, and decreased the degree of inflammatory cell infiltration and fibrosis in the lung tissue. The efficacy of the test drug TRD242 was comparable to that of the positive control drug.

[0286] 6.1 List of Abbreviations

[0287] 6.2 Experimental Objective

[0288] A mouse model of radiation-induced pulmonary fibrosis (RIPF) was established to evaluate the efficacy of the test substance TRD242.

[0289] 6.3 Experimental Materials

[0290] 6.3.1 Test substance

[0291] 6.3.2 Positive Drug

[0292] 6.3.3 Laboratory Animals

[0293] Male SPF-grade C57BL / 6 mice, aged 6–8 weeks, were purchased from Shanghai Lingchang Biotechnology Co., Ltd. They were routinely housed in the SPF-grade laboratory of Shanghai Paisi New Biotechnology Experimental Animal Center under conditions of 40–70% relative humidity and 20–26°C, with 12-hour light-dark cycles. All animals had free access to food and water. Experiments began after one week of acclimatization. This experimental procedure strictly adhered to animal welfare regulations and was approved by the Laboratory Animal Management and Use Committee (IACUC) of Shanghai Paisi New Biotechnology Co., Ltd., ethics approval number: P08240407AN1.

[0294] 6.4 Experiment Content

[0295] 6.4.1 Preparation of the test drug

[0296] TRD242 solution preparation: Before use, sonicate in an ice bath to ensure thorough and uniform dispersion, then prepare with physiological saline to the required concentration before use. Prepare fresh solution daily.

[0297] 6.4.2 Preparation of positive control drug

[0298] Preparation of 0.5% sodium carboxymethyl cellulose (CMC-Na) solution: Accurately weigh 0.5g of sodium carboxymethyl cellulose powder, dissolve it in 100mL of deionized water to prepare a clear and transparent solution with a concentration of 0.5%, and set aside for later use.

[0299] Preparation of nintedanib solution: Weigh nintedanib powder, add 0.5% CMC-Na solution, and prepare a solution with a drug concentration of 6 mg / mL. Prepare fresh solution daily.

[0300] 6.4.3 Animal Grouping

[0301] The experimental animals were randomly divided into 6 groups according to their body weight: normal control group, model control group, positive drug group, and low, medium and high dose groups of TRD242. The specific grouping is shown in Table 4.

[0302] Table 4. Animal grouping and administration

[0303] Note: * indicates that the dosage concentration is adjusted according to the animal's body weight (dosage volume: 50 μL)

[0304] 6.4.4 Construction of a mouse model of radiation-induced pulmonary fibrosis

[0305] After anesthesia and fixation, experimental mice were subjected to a single whole-chest irradiation with X-rays at a dose of 18 Gy. During the irradiation, other parts of the mice outside the chest were shielded using a specially made 1 mm thick lead box. After the mice regained consciousness, they were returned to the shielded area for continued rearing.

[0306] 6.4.5 Dosing regimen

[0307] Eight weeks after irradiation, mice were given TRD242 via bronchial nebulization in the low, medium, and high dose groups. The positive control group received nintedanib orally at the corresponding dose. The normal control and model control groups received the corresponding solvent via bronchial nebulization. The drugs were administered once daily for four weeks, followed by a two-week break, and then another two weeks of administration until the end of the experiment, for a total of six weeks. The specific administration regimen is detailed in Table 4.

[0308] 6.4.6 General condition observation and weight recording of mice

[0309] During the experiment, the mice were observed daily for their general condition, and their weight was recorded twice a week.

[0310] 6.4.7 Lung Function Test

[0311] At the end of the experiment, the mice were anesthetized and fixed in a supine position. They were then intubated and connected to a small animal invasive pulmonary function instrument to measure their invasive pulmonary function indicators.

[0312] 6.4.8 Sample Collection and Detection Analysis

[0313] 6.4.8.1 Sample Collection

[0314] After lung function tests, lung tissue was separated. The left lung was used for histopathological examination, and the right lung was used for the determination of hydroxyprolinease (HYP) content and the detection of mRNA levels of fibrosis-related indicators.

[0315] 6.4.8.2 Lung tissue pathological examination

[0316] Left lung tissue from mice in each group was fixed with 10% formalin, then dehydrated, cleared, embedded in paraffin, and sectioned. The tissue was then stained with hematoxylin-eosin (H&E) and Masson's trichrome stain. Pathological changes in the lung tissue were observed under a light microscope, and pathological scores were calculated. The scoring criteria for the degree of lung inflammation and fibrosis are shown in Tables 5 and 6.

[0317] Table 5. Lung Injury Scoring Criteria

[0318] Table 6. Scoring criteria for pulmonary fibrosis

[0319] 6.4.8.3 Expression of α-SMA, collagen 1, and F4 / 80 in lung tissue

[0320] Immunohistochemical staining was performed to detect the expression of α-SMA, collagen 1, and F4 / 80 in lung tissue.

[0321] 6.4.8.4 Detection of mRNA levels as a fibrosis marker

[0322] The changes in the mRNA levels of fibrosis markers TGF-β1, α-SMA, collagen 1, collagen 3, MMP-12, and TIMP-1 were detected by qPCR.

[0323] 6.4.8.5 Determination of HYP content

[0324] HYP content in lung tissue was detected using an alkaline hydrolysis method, strictly following the instructions for the HYP content assay kit.

[0325] 6.5 Statistical Analysis

[0326] GraphPad Prism v9.0.0 software was used for statistical analysis. All experimental data are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA (Dunnett's multiple comparison test) was used to evaluate the differences between each treatment group and the model control group. A p-value < 0.05 was considered statistically significant, and a p-value > 0.05 was considered not statistically significant. Specific data are presented in graphs and tables.

[0327] 6.6 Experimental Results

[0328] 6.6.1 General condition and weight changes of mice

[0329] Mouse body weight was recorded twice a week during the experiment. The changes in body weight of each group are shown in Figure 10 and Table 7. After irradiation, all groups of mice experienced a decrease in body weight, which gradually increased after 4 days. After the start of nebulized drug administration (8 weeks after irradiation), the weight of mice in each group decreased as they adapted to the drug administration process. After adaptation, the body weight of mice in each drug administration group and the model control group showed a slow growth trend.

[0330] Table 7. Changes in body weight of mice in each experimental group during the experimental period.

[0331] Note: D0 is the body weight of the animals on the day of irradiation, D57 is the body weight of the model group on the day of irradiation, and D116 is the body weight at the end of the experiment. All animals were not fasted. The range of body weight change from D57 to D116 was calculated. The body weight data of each group of mice are expressed as mean ± standard error (SEM).

[0332] 6.6.2 Lung function test results

[0333] The results of pulmonary function tests (Figure 11, Table 8) showed that, compared with the normal control group, the tidal volume (TV) of the model control group mice was significantly decreased (P < 0.05), the airway resistance (RI) was significantly increased (P < 0.001), and the airway compliance (CDyn) was significantly decreased (P < 0.001), indicating pulmonary dysfunction in the mice. Compared with the model group, the TV of mice in each treatment group (nintedanib group, low-, medium-, and high-dose TRD242 groups) was significantly increased (P < 0.05, P < 0.05, P < 0.05, P < 0.05), the RI was significantly decreased (P < 0.05, P < 0.05, P < 0.05, P < 0.05), and the CDyn was significantly increased (P < 0.01, P < 0.05, P < 0.01, P < 0.001).

[0334] Table 8. Results of lung function tests in mice of each experimental group

[0335] Note: Lung function test results are expressed as mean ± standard error (SEM). The differences in efficacy between the treatment groups and the model control group were analyzed using one-way ANOVA (Dunnett's multiple comparison test). * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0336] 6.6.3 Pathological examination results of lung tissue

[0337] 6.6.3.1 H&E staining

[0338] The HE staining results are shown in Figure 12. In the normal control group, the lung tissue structure of the mice was intact, with no obvious inflammatory cell infiltration. Compared with the normal control group, the alveolar structure of the model control group mice was disrupted, the alveolar septa were thickened, and there was a large amount of inflammatory cell infiltration in the lung tissue, resulting in a significantly increased lung injury score (Figure 13) (P < 0.0001). Compared with the model control group, the degree of inflammatory cell infiltration and alveolar septal thickening in the lung tissue of mice in the nintedanib group and the low, medium, and high dose TRD242 groups were reduced to varying degrees, with no significant differences among the treatment groups.

[0339] 6.6.3.2 Masson staining

[0340] The Masson staining results are shown in Figure 14. In the normal control group, the alveolar wall structure of the mice showed no obvious abnormalities, and the pulmonary septa did not show significant thickening. Compared with the normal control group, the alveolar wall structure in the lung tissue of the model control group was disordered, with obvious dense fibrous bands and collagen deposition, and the fibrosis score (Figure 15) was significantly increased (P < 0.0001). Compared with the model control group, the alveolar septal thickening and the degree of fibrosis were significantly reduced in the nintedanib group and the low, medium, and high dose TRD242 groups (P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001).

[0341] 6.6.4 Expression of collagen 1, F4 / 80, and α-SMA in lung tissue

[0342] 6.6.4.1 Collagen 1 expression in lung tissue

[0343] The expression levels of collagen 1 in the lung tissue of mice in each group are shown in Figures 16 and 17. After immunohistochemical staining, slight positive expression of collagen 1 was observed in the lung tissue septa, showing type I collagen fiber deposition. The expression level of collagen 1 in each treatment group showed a decreasing trend compared with the model group, but there was no significant difference.

[0344] 6.6.4.2 F4 / 80 expression in lung tissue

[0345] The expression levels of F4 / 80 in the lung tissue of mice in each group are shown in Figures 18 and 19. After immunohistochemical staining, it was observed that the expression level of macrophages in the lung tissue of the model control group was higher. The expression level of macrophages in the positive drug nintedanib group and the low, medium and high dose groups of TRD242 was significantly lower than that in the model group. Among them, the differences in the nintedanib group and the medium and high dose groups of TRD242 were statistically significant compared with the model group (P<0.05, P<0.05, P<0.05).

[0346] Note: F4 / 80 test results are expressed as mean ± standard error (SEM). The differences in efficacy between each treatment group and the model control group were analyzed using one-way ANOVA (Dunnett's multiple comparison test). * indicates P < 0.05, ** indicates P < 0.01.

[0347] 6.6.4.3 α-SMA expression in lung tissue

[0348] The expression levels of α-SMA in the lung tissues of mice in each group are shown in Figures 20 and 21. Immunohistochemical staining revealed α-SMA expression in the bronchial and vascular walls, and fibroblast expression in the lesion areas of the lung tissue. The expression level of α-SMA in the lung tissue of mice in the nintedanib group was significantly lower than that in the model group (P < 0.01). The expression levels of α-SMA in low, medium, and high doses of TRD242 showed a decreasing trend compared to the model group, but there was no significant difference.

[0349] 6.6.5 Lung tissue qPCR detection results

[0350] The expression levels of α-SMA, TGF-β1, collagen 1, collagen 3, MMP12, and TIMP-1 mRNA in the lung tissues of mice in each group are shown in Figure 22 and Table 9. Compared with the normal control group, the relative expression levels of α-SMA, TGF-β1, and collagen 3 mRNA in the lung tissues of mice in the model group were significantly increased (P < 0.001, P < 0.001, P < 0.01), while the relative expression levels of collagen 1, MMP12, and TIMP-1 mRNA showed no significant change compared with the model group. Compared with the control group, the relative expression levels of α-SMA, TGF-β1, and collagen 3 mRNA in the lung tissue of mice in the nintedanib group, and the low, medium, and high dose TRD242 groups were decreased to varying degrees. The relative expression levels of α-SMA mRNA in the model group, nintedanib group, and the low, medium, and high dose TRD242 groups were 2.52, 0.80, 1.88, 1.22, and 1.92, respectively (P < 0.0001, P > 0.05, P < 0.01, P > 0.05). TGF-β1 mRNA expression levels were also significantly lower. The relative expression levels of collagen 3 mRNA were 2.36, 0.65, 1.25, 1.11, and 0.85, respectively (P < 0.0001, P < 0.01, P < 0.001, P < 0.0001), and the relative expression levels of collagen 3 mRNA were 3.45, 0.47, 2.20, 1.83, and 2.55, respectively (P < 0.0001, P > 0.05, P < 0.05, P > 0.05). The relative expression levels of collagen 1, MMP12, and TIMP-1 mRNA showed no significant changes.

[0351] Table 9. mRNA expression levels of pulmonary fibrosis-related indicators in lung tissue of mice in each experimental group.

[0352] Note: qPCR results are expressed as mean ± standard error (SEM). The differences in efficacy between the treatment groups and the model control group were analyzed using one-way ANOVA (Dunnett's multiple comparison test). * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001.

[0353] 6.6.6 HYP Test Results

[0354] The HYP content in the lung tissue of mice in each group was measured by alkaline hydrolysis. The results are shown in Figure 23 and Table 10. Compared with the normal control group, the HYP content in the lung tissue of mice in the model group was significantly increased (P < 0.01). The HYP levels in the normal control group and the model group were 0.79 μg / mg and 1.03 μg / mg, respectively. Compared with the model group, the HYP content in the lung tissue of mice in each treatment group was significantly decreased (P < 0.05, P < 0.01, P < 0.05, P < 0.05). The HYP levels in the lung tissue of mice in the nintedanib group, and the low, medium, and high dose TRD242 groups were 0.84 μg / mg, 0.82 μg / mg, 0.81 μg / mg, and 0.84 μg / mg, respectively.

[0355] Table 10. HYP levels in lung tissue of mice in each experimental group

[0356] Note: HYP test results are expressed as mean ± standard error (SEM). P-values ​​are calculated based on the test results, with data points P < 0.05 marked in parentheses. The differences in efficacy between each treatment group and the model control group were analyzed using one-way ANOVA (Dunnett's multiple comparison test). * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001.

[0357] 6.7 Conclusion

[0358] Under the conditions of this experiment, a single 18 Gy X-ray irradiation of the entire thoracic cavity successfully established a mouse model of radiation-induced pulmonary fibrosis. Eight weeks after X-ray irradiation, treatment with the test drug TRD242 and the positive control drug nintedanib, once daily for six weeks, significantly improved lung function in fibrotic mice, reduced the expression levels of inflammatory and fibrotic markers such as F4 / 80, α-SMA, TGF-β1, and collagen 3 in the lung tissue to varying degrees, significantly reduced HYP content in the lung tissue, and decreased the degree of inflammatory cell infiltration and fibrosis in the lung tissue. The efficacy of the test drug TRD242 was comparable to that of the positive control drug.

Claims

1. Use of PDE4 inhibitors in the preparation of medicaments for the treatment and / or prevention of lung diseases.

2. The use of claim 1, wherein the PDE4 inhibitor is a compound of formula (I), or a tautomer, solvate, or pharmaceutically acceptable salt thereof: in: Ring A is a 5-6 member saturated or partially unsaturated heterocycle, which, in addition to the nitrogen atom shown, optionally contains 1-2 additional heteroatoms selected from nitrogen, oxygen and sulfur; R1 is selected from hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl groups and -OR; R2 is selected from hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl groups and -OR; R is selected from hydrogen and C. 1-4 alkyl; n is 1, 2, or 3.

3. The use of claim 1, wherein the PDE4 inhibitor is selected from the group consisting of compounds, or their tautomers, solvates, or pharmaceutically acceptable salts:

4. The use of claim 1, wherein the PDE4 inhibitor is a compound of the following formula, or a tautomer, solvate, or pharmaceutically acceptable salt thereof:

5. The use according to any one of claims 1-4, wherein the lung disease is lung injury.

6. The use according to any one of claims 1-4, wherein the lung disease is lung inflammation.

7. The use according to any one of claims 1-4, wherein the lung disease is pulmonary fibrosis.

8. The use according to any one of claims 1-4, wherein the lung disease is radiation-induced lung injury.

9. The use of claim 8, wherein the radiation-induced lung injury is caused by radiation therapy for a thoracic tumor.

10. The use of claim 8 or 9, wherein the radiation-induced lung injury is caused by a radiotherapy selected from: external beam radiation therapy, internal beam radiation therapy, radionuclide therapy, total body irradiation, intraoperative radiation therapy, and 4D radiation therapy.

11. The use of claim 8 or 9, wherein the radiation-induced lung injury is caused by a radiotherapy selected from: three-dimensional conformal radiotherapy (3D-CRT), intensity-modulated radiotherapy (IMRT), volumetric modulated intensity-modulated radiotherapy (VMAT), image-guided radiotherapy (IGRT), stereotactic body radiotherapy (SBRT), proton therapy, and brachytherapy.

12. The use of claim 8 or 9, wherein the radiation-induced lung injury is selected from radiation-induced pneumonia and radiation-induced pulmonary fibrosis.

13. The use according to any one of claims 1-12, wherein the drug is used to prevent the lung disease.

14. The use according to any one of claims 1-12, wherein the drug is used to treat the lung disease.

15. The use according to any one of claims 1-14, wherein the medicament further includes a pharmaceutical instruction leaflet indicating its use in combination with an additional therapeutic agent.

16. The use of claim 15, wherein the additional therapeutic agent is selected from one or more of antibacterial agents, antiviral agents, antifungal agents, antitumor agents, antihistamines, proteins, enzymes, hormones, nonsteroidal anti-inflammatory substances, cytokines, steroids, and insulin.

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