Use of PDE4 inhibitor for treating and / or preventing chronic obstructive pulmonary disease or related diseases thereof

Abstract

Provided is the use of a PDE4 inhibitor for treating and / or preventing chronic obstructive pulmonary diseases or related diseases thereof.

Description

PDE4 inhibitors are used to treat and / or prevent chronic obstructive pulmonary disease or related conditions.

[0001] Cross-reference to related applications

[0002] This application claims priority and benefit to Chinese Patent Application No. 202411707103.1, filed with the China National Intellectual Property Administration on November 26, 2024, and Chinese Patent Application No. 202511710917.5, filed with the China National Intellectual Property Administration on November 19, 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 chronic obstructive pulmonary disease or related diseases.

[0004] Background of the Invention

[0005] Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease characterized by airflow limitation. It is closely related to the chronic inflammatory response of the airways and lungs to harmful gases or toxic particles and may further develop into pulmonary heart disease and respiratory failure. In 2022, the GOLD (Global Initiative for Chronic Obstructive Lung Disease) global action plan for chronic obstructive pulmonary disease pointed out that COPD ranks fourth among the causes of disease death worldwide. In 2020, a large-scale epidemiological study published in The Lancet showed that there are about 100 million COPD patients in China, which puts great pressure on public health and the national economy. The pathogenesis of COPD is complex. It is generally believed that COPD is the result of the interaction of multiple genetic and environmental factors, and there are currently no safe and effective drugs for the treatment of this disease (Shi Zhe, Wang Shilin, Wang Haiyan, et al., Evaluation and assessment of a screening questionnaire for chronic obstructive pulmonary disease suitable for BMI classification in Chinese people [J]. Chinese Journal of General Practitioners, 2014, 13(3):184-187).

[0006] Currently, first-line drugs for treating COPD mainly consist of various types of bronchodilators, including short-acting and long-acting β2 agonists and short-acting and long-acting anticholinergics. The main function of these drugs is to dilate the bronchi, improve shortness of breath symptoms, and enhance quality of life. During acute COPD episodes, corticosteroids are primarily added to achieve anti-inflammatory effects. Roflumilast is currently the only COPD-specific treatment; however, due to its side effects such as diarrhea and suicidal tendencies, it has not been introduced to the Chinese market.

[0007] In patients with COPD, the inflammatory response shows a significant positive correlation with disease progression; the inflammatory response intensifies as the disease progresses. As a chronic inflammatory disease, COPD has an extremely complex pathological etiology, involving numerous inflammatory responses.

[0008] The phosphodiesterase (PDE) family primarily regulates the level of cyclic adenosine monophosphate (cAMP). CAMP is an important intracellular second messenger; increased intracellular levels can regulate various inflammatory mediators, thereby achieving a broad-spectrum anti-inflammatory effect. Specific mechanisms include: inhibiting inflammatory pathways by upregulating cAMP levels to reduce the proliferation and release of various inflammatory cytokines, proteases, and reactive oxygen species; slowing pulmonary vascular remodeling by reducing the proliferation of pulmonary artery smooth muscle; preventing pulmonary fibrosis by regulating pulmonary fibroblasts and proteolytic enzymes; and relaxing bronchial smooth muscle to dilate the airways.

[0009] The phosphodiesterase family comprises 11 enzyme families (PDE1-PDE11), among which PDE4 is the earliest discovered and largest family, uniquely associated with cAMP. The downstream signaling pathways of PDE4-cAMP mainly include cAMP-dependent protein kinase (PKA) and cAMP-activated exchanger protein (EPAC). These downstream cascades promote 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, including chronic obstructive pulmonary disease (COPD) or related conditions. Summary of the Invention

[0010] This application provides the use of PDE4 inhibitors for the treatment of lung diseases, particularly chronic obstructive pulmonary disease (COPD) or related diseases. Specifically, this application provides the use of compound A (also known as TRD242) or its tautomers for the prevention and / or treatment of COPD or related diseases:

[0011] In vitro cell experiments have shown that PDE4 inhibitors (including compound A) have dual anti-inflammatory and anti-fibrotic effects.

[0012] PDE4 inhibitors (including compound A) are used to treat COPD. COPD is divided into stable COPD and acute exacerbation COPD, also known as acute exacerbation of chronic obstructive pulmonary disease (AECOPD).

[0013] A COPD model was constructed in vivo. Through both prophylactic and therapeutic administration, PDE4 inhibitors (including compound A) were found to have good pharmacodynamic effects.

[0014] This application also provides the use of a compound of formula (I), or a tautomer thereof, solvate thereof, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the prevention and / or treatment of lung diseases, particularly chronic obstructive pulmonary disease or related diseases:

[0015] in:

[0016] 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;

[0017] R1 is selected from hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl groups and -OR;

[0018] R2 is selected from hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl groups and -OR;

[0019] R is selected from hydrogen and C. 1-4 alkyl;

[0020] n is 1, 2, or 3.

[0021] The compound of formula (I) may be selected from the following compounds:

[0022] This application also provides a PDE4 inhibitor for the treatment and / or prevention of chronic obstructive pulmonary disease or related diseases, wherein the PDE4 inhibitor is a compound of formula (I), or a tautomer, solvate or pharmaceutically acceptable salt thereof.

[0023] This application also provides methods for treating and / or preventing chronic obstructive pulmonary disease or related 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.

[0024] This application also provides the use of PDE4 inhibitors in the treatment and / or prevention of chronic obstructive pulmonary disease or related diseases, wherein the PDE4 inhibitor is a compound of formula (I), or a tautomer, solvate or pharmaceutically acceptable salt thereof. Attached Figure Description

[0025] Figure 1A shows the TNF-α protein levels in activated PBMCs after pretreatment with different concentrations of TRD242 compared to the LPS stimulation group.

[0026] Figure 1B 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.

[0027] 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.

[0028] Figure 3A shows the effect of low, medium, and high dose TRD242 on forced vital capacity (FVC) compared with the normal control group, the commercially available control group, and the model control group; Figure 3B shows the effect of low, medium, and high dose TRD242 on partial pressure of oxygen (PO2) (mmHg) compared with the normal control group, the commercially available control group, and the model control group; Figure 3C shows the effect of low, medium, and high dose TRD242 on oxygen saturation (sO2)% compared with the normal control group, the commercially available control group, and the model control group. Note: 1. The normal control group consisted of 4 animals, and the other groups consisted of 6 animals per group; 2. Compared with the model control group, "**" indicates p≤0.01, and "***" indicates p≤0.001.

[0029] Figure 4A shows the effects of low, medium, and high dose TRD242 on the inflammatory cytokine white blood cells (WBCs) compared to the normal control group, the commercially available control group, and the model control group; Figure 4B shows the effects of low, medium, and high dose TRD242 on the inflammatory cytokine neutrophils (Neut) compared to the normal control group, the commercially available control group, and the model control group; Figure 4C shows the effects of low, medium, and high dose TRD242 on the inflammatory cytokine lymphocytes (Lymph) compared to the normal control group, the commercially available control group, and the model control group; Figure 4D shows the effects of low, medium, and high dose TRD242 on the inflammatory cytokine mononuclear cells (Mono) compared to the normal control group, the commercially available control group, and the model control group. Note: 1. The normal control group consisted of 8 mice, and the other groups consisted of 12 mice per group; 2. Compared to the model control group, "*" indicates p≤0.05, "**" indicates p≤0.01, and "***" indicates p≤0.001.

[0030] Figure 5A shows the H&E staining patterns of TRD242 infiltration of inflammatory cells in the low, medium, and high dose groups, compared with the normal control group, the commercially available control group, and the model control group; Figure 5B shows the histopathological diagnosis and scoring of lung inflammatory response in each experimental group based on lung H&E staining results and according to the lung inflammation scoring criteria. Detailed Implementation Plan

[0031] definition

[0032] 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.

[0033] 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.

[0034] “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.

[0035] "Halogen" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0036] “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.

[0037] "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.

[0038] 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.

[0039] 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:

[0040] (i) Suppress the disease or disease state, that is, curb its development;

[0041] (ii) Relieve the disease or disease state, even if the disease or disease state subsides.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] Specifically, this disclosure relates to the following technical solutions.

[0047] In one embodiment, this disclosure relates to the use of PDE4 inhibitors in the preparation of medicaments for the treatment and / or prevention of chronic obstructive pulmonary disease or related diseases.

[0048] 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 chronic obstructive pulmonary disease or related diseases, wherein the PDE4 inhibitor is a compound of formula (I), or a tautomer, solvate, or pharmaceutically acceptable salt thereof:

[0049] in:

[0050] 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;

[0051] R1 is selected from hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl groups and -OR;

[0052] R2 is selected from hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl groups and -OR;

[0053] R is selected from hydrogen and C. 1-4 alkyl;

[0054] n is 1, 2, or 3.

[0055] In a further embodiment, the PDE4 inhibitor is selected from the following compounds, or their tautomers, solvates, or pharmaceutically acceptable salts:

[0056] In a further embodiment, the PDE4 inhibitor is a compound of the following formula, or a tautomer, solvate, or pharmaceutically acceptable salt thereof:

[0057] In some embodiments, this disclosure relates to a PDE4 inhibitor for the treatment and / or prevention of chronic obstructive pulmonary disease or related diseases, wherein the PDE4 inhibitor is a compound of formula (I), or a tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0058] In some embodiments, this disclosure relates to methods of treating and / or preventing chronic obstructive pulmonary disease or related 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.

[0059] In some embodiments, this disclosure relates to the use of PDE4 inhibitors in the treatment and / or prevention of chronic obstructive pulmonary disease or related diseases, wherein the PDE4 inhibitor is a compound of formula (I), or a tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0060] In a further embodiment, the chronic obstructive pulmonary disease is selected from chronic bronchitis-type chronic obstructive pulmonary disease, emphysema-type chronic obstructive pulmonary disease, and mixed-type chronic obstructive pulmonary disease.

[0061] In a further embodiment, the chronic obstructive pulmonary disease includes stable chronic obstructive pulmonary disease and acute exacerbations of chronic obstructive pulmonary disease (AECOPD).

[0062] In a further embodiment, the compound, or its tautomers, solvates, or pharmaceutically acceptable salts, are used to administer the compound in amounts equivalent to about 0.1 mg / day to about 1000 mg / day.

[0063] In a further embodiment, the compound, or its tautomers, solvates, or pharmaceutically acceptable salts, are administered in amounts equivalent to about 0.1 mg / day, about 0.5 mg / day, 1 mg / day, about 5 mg / day, about 10 mg / day, about 20 mg / day, about 30 mg / day, about 40 mg / day, about 50 mg / day, and about 100 mg / day.

[0064] In a further embodiment, the daily dose of said drug is administered once or in two, three, or four divided doses.

[0065] In a further embodiment, the drug is used to administer the drug via a route selected from: injection, transdermal, oral, sublingual, nasal, transmucosal, topical, intraocular, and inhalation (e.g., nebulized inhalation).

[0066] In a further embodiment, the drug is selected from the following dosage forms: tablets, capsules, lozenges, hard candies, powders, sprays, ointments, suppositories, injections, patches, suspensions, solutions, and syrups.

[0067] In a further embodiment, the drug also includes a package insert indicating its use in combination with an additional therapeutic agent.

[0068] In a further embodiment, the additional therapeutic agent is selected from bronchodilators, inhaled corticosteroids, theophylline, mucolytics, cardiovascular drugs, and osteoporosis prevention drugs.

[0069] In a further embodiment, the additional therapeutic agent is selected from short-acting and long-acting β2 agonists, short-acting and long-acting anticholinergic drugs, antibacterial agents, antiviral agents, antifungal agents, antitumor agents, antihistamines, proteins, enzymes, hormones, nonsteroidal anti-inflammatory substances, cytokines, steroids, and insulin.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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) improving lung function and lung gas exchange capacity in patients with chronic obstructive pulmonary disease; and (4) effectively treating chronic obstructive pulmonary disease, especially chronic obstructive pulmonary disease in the acute exacerbation phase.

[0075] Example

[0076] Example 1. Pharmacological effects of PDE4 inhibitors in PBMC cells

[0077] 1.1 Experimental Objective

[0078] 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).

[0079] 1.2 Instruments and Equipment

[0080] 1.3 Test Methods

[0081] 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).

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 4) Collect the supernatant for the detection of TNF-α and IL-2.

[0086] 1.4 Data Statistics

[0087] 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.

[0088] 1.5 Test Results

[0089] 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 1A).

[0090] TRD242 significantly inhibited IL-2 production in human PBMC cells stimulated by PHA-P. The inhibition rates of IL-2 by TRD242 at concentrations of 0.5 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 100 nM, and 1000 nM 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 1B.

[0091] 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.

[0092] Example 2. Pharmacodynamic effects of PDE4 inhibitors in MRC-5 cells

[0093] 2.1 Experimental Objective

[0094] The aim of this study was to investigate the effect of PDE4 inhibitors on the expression of fibroblast-promoting factor mRNA in MRC-5 fibroblasts induced by transforming growth factor β1 (TGF-β1).

[0095] 2.2 Instruments and Equipment

[0096] 2.3 Test Methods

[0097] 2.3.1 Cell Culture

[0098] 1) MRC-5 cells were cultured in EMEM medium containing 10% fetal bovine serum.

[0099] 2) When the cell fusion rate reaches 70-85%, digest with 0.05% trypsin, centrifuge, resuspend in serum-free medium, and use for experiments.

[0100] 2.3.2 Seeding Plates and Cell Treatment

[0101] 1) Count the cells and adjust the density to 1×10⁵ cells / mL. Seed 0.5 mL in each well of a 24-well plate and culture overnight without serum starvation.

[0102] 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.

[0103] 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.

[0104] 2.3.3 Total RNA Extraction

[0105] Total RNA was extracted using the TRIzol method, and the specific steps are as follows:

[0106] 1) After culturing cells for 48 hours, aspirate the culture medium and wash once with pre-cooled PBS;

[0107] 2) Discard the PBS and add 0.5 mL of TRIzol to each well;

[0108] 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.

[0109] 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.

[0110] 5) Carefully aspirate the upper aqueous phase using a pipette and place it into a new 1.5 mL centrifuge tube;

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 2.4 Data Statistics

[0115] 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.

[0116] 2.5 Test Results

[0117] 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, as shown in Figure 2A.

[0118] 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 (as shown in Figure 2B).

[0119] Example 3. Pharmacodynamic effects of PDE4 inhibitors on an animal model of chronic obstructive pulmonary disease.

[0120] 3.1 Experimental Objective

[0121] By constructing an animal model of COPD, the pharmacodynamic effects of PDE4 inhibitors were observed after administration.

[0122] 3.2 Instruments and Equipment

[0123] 3.3 Test Methods

[0124] Model Construction: Smoke Exposure: Animals from D1 to D70 were exposed to smoke generated by a single-channel smoke generator, which mixed the smoke from lit cigarettes with air. The resulting smoke entered the exposure chamber from the top (50cm×40cm×36cm) and exited from the bottom side, creating a dynamic equilibrium. No more than 10 animals were placed in each layer of the chamber. Smoke generation occurred approximately every 10–25 seconds, with a generation volume of 30–60 mL per generation, for 1.5 hours per cycle, 2 cycles per day, and approximately 25 (±5) cigarettes per cycle. Animals were not exposed to smoke on days when given lipopolysaccharide or elastase.

[0125] Lipopolysaccharide (LPS) administered via airway nebulization: D1, D8, D15, D22, D29, D36. The amount of LPS (2 mg / kg, 1 mL / kg) administered to each animal in the model animals was determined based on their body weight. Normal control animals received sodium chloride injection via airway nebulization (1 mL / kg). Sex ratio: Single sex, all males.

[0126] Nebulized proteases in the airway: D42, D45, D48, D55, D62. The amount of porcine pancreatic protease (PPE, 7U / kg, 1mL / kg) administered to each animal in the model animals was determined based on their body weight.

[0127] Normal control group animals received nebulized sodium chloride injection (1 mL / kg) via airway;

[0128] Dosing began on day 42, once daily for a total of 28 days, and samples were collected for observation on day 70.

[0129] Intratracheal nebulization: Animals are anesthetized with isoflurane inhalation and then fixed to a restraint device. Using an anesthesia laryngoscope, the glottis is exposed. A blunt needle of a micro-nebulizer containing a measured amount of modeling reagent or sodium chloride injection is gently inserted into the trachea. The piston is then quickly pushed to nebulize the modeling reagent or sodium chloride injection into the lungs. The needle is quickly withdrawn, and the animal is removed from the restraint device with its head facing upward. The animal is rotated left and right to distribute the solution as evenly as possible in each lung lobe. The volume is rounded to the nearest whole number; if the volume falls between two graduations, the upper graduation value is used.

[0130] 3.4 Data Statistics

[0131] The data were processed using IBM SPSS Statistics 28.0 and / or GraphPad Prism 8 statistical software. All statistical analyses employed two-tailed analyses, with a statistical significance level set at P ≤ 0.05. All indicators are expressed as mean ± standard deviation, and the analysis followed these steps: First, Levene's Test was used to test the data for homogeneity. If the data were homogeneous (P > 0.05), a one-way ANOVA was performed. If the ANOVA was significant (P ≤ 0.05), the differences between the model control group and other groups were compared using the LSD test. If the Levene's Test result was significant (P ≤ 0.05), the Kruskal-Wallis nonparametric test was performed. If the Kruskal-Wallis nonparametric test result was significant (P ≤ 0.05), the Mann-Whitney U test was further used for pairwise comparisons.

[0132] 3.5 Test Results

[0133] (1) PDE4 inhibitors can improve lung function and lung gas exchange capacity.

[0134] Compared with the corresponding indicators in the model control group, the mean FVC (mL) of the low, medium, and high dose groups of the test product TRD242 (0.1 mg / kg, 0.5 mg / kg, and 1 mg / kg, respectively) and the commercially available control product (budesonide suspension, 0.5 mg / kg) group were increased, with the maximum increases being approximately 26%, 31%, 15%, and 33%, respectively. Except for the high-dose group, all other groups showed statistically significant differences (P ≤ 0.05). (See Figure 3A).

[0135] Compared with the corresponding indicators in the model control group, the mean PO2 (mmHg) values ​​of animals in the low, medium, and high dose groups of the test product TRD242 (0.1 mg / kg, 0.5 mg / kg, and 1 mg / kg, respectively) and the commercially available control product (budesonide suspension, 0.5 mg / kg) group were increased, with the maximum increases being approximately 25%, 32%, 35%, and 44%, respectively, and all were statistically significant (P≤0.01). (See Figure 3B).

[0136] Compared with the corresponding indicators in the model control group, the mean SO2% values ​​of animals in the low, medium, and high dose groups of the test product TRD242 (0.1 mg / kg, 0.5 mg / kg, and 1 mg / kg, respectively) and the commercially available control product (budesonide suspension, 0.5 mg / kg) group were increased, with the maximum increases being approximately 15%, 18%, 20%, and 22%, respectively, and all were statistically significant (P≤0.001). (See Figure 3C).

[0137] (2) PDE4 inhibitors can improve the infiltration of inflammatory factors in bronchoalveolar lavage fluid.

[0138] As shown in Figures 4A-4D, compared with the corresponding indicators of the model control group, the mean WBC values ​​of animals in the low, medium, and high dose groups of the test product TRD242 (0.1 mg / kg, 0.5 mg / kg, and 1 mg / kg, respectively) and the commercially available control product (budesonide suspension, 0.5 mg / kg) were decreased, with maximum reductions of approximately 40%, 48%, 70%, and 64%, respectively. Except for the low-dose group, all other groups showed statistically significant differences (P ≤ 0.05). The mean Neut values ​​of animals in the low, medium, and high dose groups of the test product TRD242 and the commercially available control product (budesonide suspension) were also decreased, with maximum reductions of approximately 47%, 55%, 78%, and 72%, respectively. The high-dose group showed the highest reduction. Statistical differences were not observed (P≤0.05). The mean Lymph value of animals in the low, medium, and high dose groups of the test product TRD242 and the commercially available control product (budesonide suspension) group decreased, with the maximum decrease being approximately 42%, 47%, 61%, and 55%, respectively. Except for the low dose group, all other groups showed statistical differences (P≤0.05). The mean Mono value of animals in the medium and high dose groups of the test product TRD242 and the commercially available control product (budesonide suspension) group decreased, with the maximum decrease being approximately 23%, 69%, and 69%, respectively. The high dose group and the commercially available control product (budesonide suspension) group showed statistical differences (P≤0.01). The mean Mono value of animals in the low dose group increased slightly, but there was no statistical difference.

[0139] (3) Pathological staining showed that PDE4 inhibitors can improve the infiltration of inflammatory factors in bronchoalveolar lavage fluid.

[0140] As shown in Figure 5A, under the experimental conditions, microscopic observation revealed mild to moderate inflammatory cell infiltration around blood vessels / bronchial septa / alveolar septa / alveolar lesions in the model animals; mild to slight fibrosis around blood vessels / alveolar septa / alveolar lesions / alveolar lesions; mild to moderate alveolar dilation; and mild to slight arterial medial hypertrophy. These were the pathological changes expected for model establishment. Compared with the model control group, the severity / incidence of the above lesions showed a decreasing trend in all groups of the test product TRD242 (0.1 mg / kg, 0.5 mg / kg, and 1 mg / kg, respectively) and the commercially available control product (budesonide suspension, 0.5 mg / kg), and a certain degree of dose-relatedness was observed in all groups of the test product TRD242. Compared with the model control group, the fibrosis scores of animals in each group of the test product TRD242 (0.1 mg / kg, 0.5 mg / kg, and 1 mg / kg, respectively) and the commercially available control product (budesonide suspension, 0.5 mg / kg) showed a decreasing trend. The scores of the medium-dose and high-dose groups of the test product TRD242 were close to those of the commercially available control group, as shown in Figure 5B.

[0141] Note: 1. There were 8 normal control group and 12 in each of the other groups; 2. The severity of the lesions was recorded as (-) for no lesions, (+) for mild lesions, (++) for mild lesions, and (+++) for moderate lesions.

Claims

1. Use of PDE4 inhibitors in the preparation of medicaments for the treatment and / or prevention of chronic obstructive pulmonary disease or related 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 chronic obstructive pulmonary disease is selected from chronic bronchitis-type chronic obstructive pulmonary disease, emphysematous-type chronic obstructive pulmonary disease, and mixed-type chronic obstructive pulmonary disease.

6. The use according to any one of claims 1-4, wherein the chronic obstructive pulmonary disease includes stable chronic obstructive pulmonary disease and acute exacerbation chronic obstructive pulmonary disease.

7. The use according to any one of claims 1-4, wherein the compound, or its tautomer, solvate, or pharmaceutically acceptable salt, is administered in an amount equivalent to about 0.1 mg / day to about 1000 mg / day of the compound.

8. The use according to any one of claims 1-4, wherein the compound, or its tautomer, solvate, or pharmaceutically acceptable salt, is administered in amounts equivalent to about 0.1 mg / day, about 0.5 mg / day, 1 mg / day, about 5 mg / day, about 10 mg / day, about 20 mg / day, about 30 mg / day, about 40 mg / day, about 50 mg / day, and about 100 mg / day.

9. The use according to any one of claims 1-4, wherein the daily dose of the drug is for administration in a single dose or in two, three, or four divided doses.

10. The use according to any one of claims 1-4, wherein the drug is for administration via a route selected from: injection, transdermal, oral, sublingual, nasal, transmucosal, topical, intraocular, and inhalation.

11. The use according to any one of claims 1-4, wherein the medicament is a dosage form selected from the following: tablets, capsules, lozenges, hard candies, powders, sprays, ointments, suppositories, injections, patches, suspensions, solutions, and syrups.

12. The use according to any one of claims 1-4, wherein the drug further includes a pharmaceutical instruction leaflet indicating its use in combination with an additional therapeutic agent.

13. The use of claim 12, wherein the additional therapeutic agent is selected from one or more of bronchodilators, inhaled corticosteroids, theophylline, mucolytics, cardiovascular drugs, and osteoporosis prevention drugs.

14. The use of claim 12, wherein the additional therapeutic agent is selected from one or more of short-acting and long-acting β2 agonists, short-acting and long-acting anticholinergic drugs, antibacterial agents, antiviral agents, antifungal agents, antitumor agents, antihistamines, proteins, enzymes, hormones, nonsteroidal anti-inflammatory substances, cytokines, steroids, and insulin.

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