Application of dicafen phenolic acid and derivatives thereof in preparation of medicine for treating pulmonary fibrosis

Abstract

The invention discloses application of dikafen phenolic acid and ester derivatives thereof in preparation of medicines for treating pulmonary fibrosis. On an MRC-5 cell model induced by TGF-beta1, the bikafen phenolic acid and the multiple ester derivatives thereof show the activity of inhibiting alpha-SMA and COL1A1 protein expression; on a bleomycin-induced pulmonary fibrosis mouse model, the dimethyl ester derivative of the dikafen phenolic acid shows a good protective effect on experimental animals. Therefore, the dicafen phenolic acid and the ester derivative thereof have good application in the field of preparation of medicines for treating pulmonary fibrosis diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to new uses of bis(caffeo) acid and its derivatives in the pharmaceutical field, specifically the application of bis(caffeo) acid and its ester derivatives in the preparation of drugs for treating pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis (PF) is a chronic, progressive, and irreversible interstitial lung disease characterized by repeated damage and abnormal repair of alveolar epithelial cells, excessive activation of fibroblasts, and massive deposition of extracellular matrix (ECM). Its pathological endpoint is lung structural destruction and loss of function, ultimately leading to respiratory failure. Among the various types, idiopathic pulmonary fibrosis (IPF) is the most common and has an extremely poor prognosis, with a median survival of only 3-5 years. Globally, the incidence of IPF is increasing year by year. Epidemiological studies indicate that its global prevalence is approximately (2-29) / 100,000 people, and its incidence rate is approximately (0.22-8.8) / 100,000 person-years. With the aging population and the influence of environmental factors (such as smoking and air pollution), the burden of this disease is increasing, posing a serious challenge to public health systems.

[0003] Currently, the clinical selection of drugs for treating pulmonary fibrosis is extremely limited, mainly focusing on antifibrotic drugs and immunosuppressants, but both have significant limitations. The US FDA has approved only nintedanib (a multi-target tyrosine kinase inhibitor) and pirfenidone (a compound with anti-inflammatory and anti-fibrotic effects) for the treatment of IPF. Although these two drugs can slow the rate of decline in lung function (such as forced vital capacity, FVC), they cannot reverse or cure the disease and have significant side effects, such as diarrhea and hepatotoxicity caused by nintedanib, and photosensitivity and gastrointestinal reactions caused by pirfenidone. Furthermore, patient response rates vary, long-term efficacy is limited, and treatment costs are high. Glucocorticoids and immunosuppressants (such as cyclophosphamide and azathioprine) have been used in some non-IPF interstitial lung diseases, but the level of evidence for their efficacy is inconsistent, and long-term use significantly increases the risk of infection and osteoporosis. Therefore, developing new drugs with more definitive efficacy and higher safety is a key scientific problem and clinical need that urgently needs to be addressed in this field.

[0004] Bis-caffeic acid is a neooligan compound obtained by the chemical degradation of tanshinone B, formerly known as SMND-309. Existing literature mainly reports its antioxidant, anti-inflammatory, anti-cerebral ischemia / reperfusion injury, anti-acute myocardial ischemia, and hepatoprotective activities. However, regarding the exact use, effective dosage range, and optimal administration method of bis-caffeic acid and its derivatives (including its esters, amides, ethers, and salts with structural modifications) in the preparation of drugs for the clinical treatment of human pulmonary fibrosis, no disclosure or suggestion has been found in any publicly available academic papers, patents, or clinical reports, both domestically and internationally, to date.

[0005] In summary, given the severe harm of pulmonary fibrosis and the inadequacy of existing treatments, this invention aims to clarify the application of bis(caffeic acid) and its derivatives in the preparation of drugs for treating pulmonary fibrosis, providing a new and more promising treatment strategy for the disease. Summary of the Invention

[0006] This invention provides a technical solution for preparing a drug for treating pulmonary fibrosis, which is achieved by a compound with the structure of Formula I.

[0007]

[0008] R1 to R2 are each independently selected from: H; C1 to C5 alkyl groups.

[0009] More preferably, R1 to R2 are each independently selected from H; C1 to C3 alkyl groups.

[0010]

[0011]

[0012] The compound represented by Formula I provided by this invention can be combined with pharmaceutically acceptable excipients or carriers to form a pharmaceutical composition, and can be made into oral preparations such as capsules, soft capsules, pellets, tablets, granules, and aerosols, or injectable preparations such as emulsions for injection, lyophilized powders for injection, microspheres for injection, or nano-preparations for injection.

[0013] The present invention provides the use of a compound as shown in Formula I, or a pharmaceutical composition comprising the compound, in the preparation of a medicament for treating pulmonary fibrosis.

[0014] The dosage of a pharmaceutical composition comprising the compound of Formula I as described in this invention varies depending on the severity of the disease of the patient being treated, individual differences such as the patient's gender, age, weight, and the route of administration and frequency of administration. The effective daily oral dose for adults is determined to be 10–500 mg, preferably 20–200 mg; when administered by injection, the daily injection dose for adults is 5–500 mg, preferably 10–150 mg. Attached Figure Description

[0015] Figure 1 Western blot analysis results (A and B are comparison plots of the effects of different drugs on the expression levels of α-SMA and COL1A1 proteins).

[0016] Figure 2 The curve showing the change in mouse weight during the experiment.

[0017] Figure 3 . Pathological images of lung tissue from each group of experimental mice (A-F are representative HE staining atlases of lung tissue from the control group, model group, pirfenidone group, low-dose dimethyl caffeate group, medium-dose dimethyl caffeate group, and high-dose dimethyl caffeate group, respectively).

[0018] Figure 4 . Pathological images of lung tissue from each group of experimental mice (A-F are representative Sirius red staining atlases of lung tissue from the control group, model group, pirfenidone group, low-dose dimethyl caffeate group, medium-dose dimethyl caffeate group, and high-dose dimethyl caffeate group, respectively).

[0019] Figure 5 Ascrofi score of mouse lung tissue pathological sections. Detailed Implementation

[0020] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions. Unless otherwise stated, percentages and parts are calculated by weight.

[0021] Unless otherwise specified, the compounds and formulations used in the examples were prepared in accordance with the methods provided in the patent applications “A phenolic acid compound, preparation method and pharmaceutical composition and application thereof (application number: 200710015108.8)”, “Bis-caffeophenolic acid derivative, preparation method and application thereof in the preparation of drugs for treating liver injury and liver fibrosis (application number: 202311506629.9)” and “Application of bis-caffeophenolic acid and its derivatives in the preparation of drugs for treating tumors”.

[0022] Example 1: Preparation of Bis(caffeoyl) dimethyl ester aerosol

[0023] prescription:

[0024]

[0025] Procedure: Mix the prescribed amount of dimethyl caffeic acid with cold ethanol (-65℃) and homogenize thoroughly. Then add cold HFA-134a (-65℃), stir and mix. Fill the aerosol container using the cold filling method and cap the valve to obtain the solution-type dimethyl caffeic acid aerosol.

[0026] Example 2: Effects of bis(caffeic acid) and its derivatives on the proliferation of human embryonic lung fibroblasts MRC-5

[0027] 1. Instruments and reagents

[0028] The following equipment was used: Memmert CO2 cell culture incubator (Germany), QuantStudio 5 Applied Biosystems real-time quantitative PCR instrument, DeNovix DS-11 micro-nucleic acid and protein analyzer, Bio-Rad vertical electrophoresis system and transfer system, Beckman Coulter low-temperature ultracentrifuge (USA), Guangzhou Sanrui Technology Co., Ltd. autoclave, Corning 96-well cell culture plate (USA), BD flow cytometer (USA), Thermo Fisher Scientific Molecular devices standard and ultra-low temperature freezer (-80℃), OLYMPUS inverted microscope, Shanghai Lixin Instrument Co., Ltd. ultrapure water system, Eppendorf thermostatic mixing shaker (Germany), and Tianjin De'ant Sensor Technology Co., Ltd. precision electronic balance.

[0029] MEM culture medium, FBS fetal bovine serum, and P / S double antibiotics (penicillin-streptomycin mixture) were all purchased from Wuhan Pronosei Biotechnology Co., Ltd.; recombinant human TGF-β1 protein and Sirius red staining solution were purchased from Solarbio; Cell counting kit-8 was purchased from Shanghai Yisheng Biotechnology Co., Ltd.; TRIzol... TMReagents, reverse transcription kits, SYBR Green qPCR premix, BCA protein concentration assay kits, rabbit anti-human α-SMA, rabbit anti-human COL1A1, rabbit anti-human p-Smad2 / 3, mouse anti-human GAPDH, and RP-labeled goat anti-rabbit / mouse secondary antibodies were all purchased from Beyotime Biotechnology. Pirfenidone (PFD) capsules were manufactured by Beijing Kangtini Pharmaceutical Co., Ltd., and DMSO was manufactured by Sigma-Aldrich, USA. Bis-caffeic acid (BA), dimethyl bis-caffeic acid (DMB), 9-methyl bis-caffeic acid (9-MMB), and 9'-methyl bis-caffeic acid (9'-MMB) were all prepared in the laboratory. Human embryonic lung fibroblast MRC-5 cell line was purchased from Wuhan Pronosei Life Sciences Co., Ltd., and was thawed, passaged, cryopreserved at -80℃, and cultured according to standard cell handling procedures.

[0030] 2. Experimental Methods

[0031] 2.1 Cell Culture and Grouping: MRC-5 cells were cultured in MEM medium containing 10% FBS and 1% penicillin-dextrose antibiotics at 37°C and 5% CO2 under standard conditions. Cells in the logarithmic growth phase were used for experiments. The experiments were divided into the following groups:

[0032] ① Control group: MEM medium containing 0.5% FBS;

[0033] ② Model group: MEM medium containing 0.5% FBS + 5 ng / mL TGF-β1;

[0034] ③ Positive drug control group: MEM medium containing 0.5% FBS + 5 ng / mL TGF-β1 + 10 mM pirfenidone;

[0035] ④ Three treatment groups of bis(caffeine) at different concentrations: MEM medium containing 0.5% FBS + 5 ng / mL TGF-β1 + different concentrations (5, 10, 20 μM);

[0036] ⑤ Three treatment groups of bis(caffeoyl) methyl ester: MEM medium containing 0.5% FBS + 5 ng / mL TGF-β1 + different concentrations (5, 10, 20 μM);

[0037] ⑥ Three treatment groups of bis(caffeic acid) 9-methyl ester: MEM medium containing 0.5% FBS + 5 ng / mL TGF-β1 + different concentrations (5, 10, 20 μM);

[0038] ⑦ Three treatment groups of bis(caffeic acid) 9'-methyl ester: MEM medium containing 0.5% FBS + 5 ng / mL TGF-β1 + different concentrations (5, 10, 20 μM).

[0039] 2.2 Drug treatment and sample collection: MRC-5 cells were seeded in 6-well plates (2 × 10⁻⁶ cells / well). 5 / hole) and 96-hole plate (5×10) 3 / well). After cell attachment, the medium was replaced with 0.5% FBS MEM medium for starvation and synchronization for 24 hours. The old medium was discarded, and the cells were pretreated for 2 hours with the corresponding drug in the above-mentioned groups. Except for the control group, TGF-β1 was added to the remaining groups to a final concentration of 5 ng / mL. Samples were collected after 48 hours of culture.

[0040] 2.3 Cell viability assay (CCK-8 assay): After 48 hours of drug treatment, 10 μL of CCK-8 solution was added to each well of a 96-well plate and incubated for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability (%) = (OD value) 药物组 -OD 空白 ) / (OD 对照组 -OD 空白 )×100%.

[0041] 2.4 Western Blot: Total protein was extracted from cells using RIPA lysis buffer, and the concentration was determined by the BCA method. 30 μg of protein was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking with 5% skim milk, the membrane was incubated sequentially with primary antibody (4°C overnight) and the corresponding secondary antibody (room temperature for 1 hour). After ECL development, the band gray values ​​were analyzed using ImageJ software, and normalization was performed using GAPDH as an internal control.

[0042] 2.5 Statistical Analysis: All experiments were independently repeated three times, and data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 10.0 software. One-way ANOVA was used for comparisons among multiple groups, and Tukey's post-hoc test was used for pairwise comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0043] 3. Experimental Results and Analysis

[0044] 3.1 Effects of bis(caffeic acid) and its derivatives on the survival rate of MRC-5 cells:

[0045] See Table 1. Within the concentration range of 10-40 μM, compared with the control group, MRC-5 cells grew normally after 48 hours of drug treatment (survival rate >85% was considered to have no significant effect on cell growth), indicating that the antifibrotic effect within this concentration range was not due to cytotoxicity.

[0046] Table 1. Survival rate of MRC-5 cells after drug treatment (compared to the control group)

[0047]

[0048] 3.2 Effects of bis(caffeic acid) and its derivatives on the expression levels of α-SMA and COL1A1 proteins:

[0049] To facilitate comparison of drug activity, results at the same drug concentration (10 μM) were used for graphical comparison. See appendix. Figure 1 .

[0050] α-SMA is a specific marker of myofibroblast activation. In normal lung tissue, it is mainly found in vascular smooth muscle cells, and is almost not expressed in lung fibroblasts. Under stimulation by profibrotic factors such as TGF-β1, quiescent lung fibroblasts are activated and transform into myofibroblasts. If a drug can reduce α-SMA expression, it indicates that it can reverse or prevent the transformation of fibroblasts into myofibroblasts, directly targeting the "driver cells" of the fibrosis process. This is a key upstream mechanism by which the drug exerts its effect.

[0051] COL1A1, or type I collagen α1 chain, is the main protein component of human scar and fibrous tissue, and a direct product of excessive extracellular matrix deposition. The direct pathological manifestation of pulmonary fibrosis is that activated myofibroblasts synthesize and secrete large amounts of extracellular matrix, primarily composed of type I collagen (COL1A1 being its main component). This excessive collagen fibers accumulate uncontrollably in the alveolar interstitium, ultimately leading to lung hardening, loss of elasticity, and impaired gas exchange. If a drug can reduce COL1A1 expression, it indicates that it can directly inhibit the formation of scar tissue (extracellular matrix). This is the most direct and downstream efficacy indicator for measuring whether a drug can ultimately alleviate the pathological structure of fibrosis and prevent disease progression.

[0052] From the appendix Figure 1 It can be seen that the expression of α-SMA and COL1A1 was significantly increased after the addition of TGF-β1, proving that the in vitro fibrosis model was successfully constructed. Pirfenidone (PFD), bis-caffeic acid (BA), dimethyl bis-caffeic acid (DMB), 9-methyl bis-caffeic acid (9-MMB), and 9'-methyl bis-caffeic acid (9'-MMB) can all reduce the expression levels of α-SMA and COL1A1, indicating that these drugs have anti-pulmonary fibrosis activity.

[0053] Example 3: Study on the protective effect of bis(caffeoyl) dimethyl ester against bleomycin-induced pulmonary fibrosis in mice.

[0054] 1. Materials

[0055] 1.1 Laboratory Animals

[0056] Male C57BL / 6 mice aged 7–8 weeks, weighing 20±2g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., with animal qualification certificate number SCXK(Su)2023-0009.

[0057] 1.2 Instruments and Reagents

[0058] Thermo Fisher Scientific ultra-low temperature freezer (-80℃), Shanghai Lixin Instrument Co., Ltd. ultrapure water system, Eppendorf GmbH (Germany) constant temperature mixing shaker and speed-regulating refrigerated centrifuge, Shanghai Boxun Industrial Co., Ltd. clean bench, Suzhou Sainz Instrument Sartorius CPA225D precision electronic balance, Hubei Aiterui ATR-SQ525 tissue slicer.

[0059] Paraformaldehyde, hematoxylin-eosin staining solution, and Sirius red staining solution were all products of Wuhan Sewell Biotechnology Co., Ltd.; 1,2-propanediol was purchased from Jiangxi Yipusheng Pharmaceutical Co., Ltd.; dimethyl sulfoxide was purchased from Sichuan Jinshan Pharmaceutical Co., Ltd.; pirfenidone capsules were purchased from Beijing Kangtini Pharmaceutical Co., Ltd.; ethanol was produced by Shandong Jiekai Pharmaceutical Co., Ltd.; and bleomycin (BLM) was a product of Nippon Kayaku Co., Ltd. Dimethyl bis(caffeoyl)ate (DMB) was prepared in the laboratory with a purity of 99.2% (detected by HPLC).

[0060] 2. Methods

[0061] 2.1 Animal grouping and modeling:

[0062] After being purchased, the mice were acclimatized for 7 days and then randomly divided into a blank control group, a pulmonary fibrosis model group, a positive control drug pirfenidone group, and three DMB low, medium and high dose groups (20, 40 and 80 mg / kg), with 8 mice in each group.

[0063] The normal control group received saline solution at a dose of 5 mg / kg via endotracheal nebulization; the other groups received a single dose of bleomycin at a dose of 5 mg / kg via endotracheal spray.

[0064] Two weeks after modeling, the drugs were administered by gavage for 14 consecutive days according to the dosage and dosage shown in Table 2. Both pirfenidone and dimethyl caffeic acid were dissolved to the specified concentration using ethanol:1,2-propanediol:DMSO water (1:3:1:5) as the solvent.

[0065] During the experiment, the mice's fur luster, mental state, appetite, and weight changes were observed daily. During the drug administration period, the mice were weighed every 72 hours, and a weight change curve was plotted.

[0066] Table 2 Grouping and administration of experimental animals

[0067]

[0068] 2.2 Sampling and Testing

[0069] Twenty-four hours after the last administration, mice in each group were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg / kg), their positions were fixed, and their lungs were exposed. Fresh lung tissue was harvested, and the left lung was fixed with paraformaldehyde for HE staining and Sirius red staining for pathological analysis. The remaining tissue was washed with physiological saline, labeled, and cryopreserved in liquid nitrogen for later use.

[0070] 2.3 Statistical Analysis

[0071] The severity of pulmonary fibrosis in the lung tissue sections of mice in each group was assessed using the Ascroft scoring system. Data analysis was performed using Graphpad Prism 10.0 and Microsoft Excel 2019.

[0072] 3. Results and Discussion

[0073] 3.1 Changes in mouse body weight during drug administration

[0074] Pulmonary fibrosis can lead to weight loss in mice. This is because lung damage can impair respiratory function, which in turn affects food intake and activity, resulting in weight changes. If a drug can alleviate pulmonary fibrosis, the mice may experience less weight loss or recover more quickly. Therefore, in this experiment, we monitored changes in mouse weight during the experiment to determine the protective effect of DMB on mice with pulmonary fibrosis.

[0075] The results are attached. Figure 2 As shown, the model group mice experienced a decrease in body weight, while the drug-treated group mice showed a slight decrease in body weight at the beginning of modeling, but this was not significant and then recovered. The recovery may be due to the animals' own self-healing mechanism at work. The weight change trend of the mice in the high-dose DMB group was similar to that of the normal control group (Control group), indicating that this dose of DMB had a better protective effect on mice with pulmonary fibrosis.

[0076] 3.2 Pathological analysis of mouse lung tissue

[0077] ① HE staining: HE staining, also known as hematoxylin-eosin staining, utilizes the fact that the base pairs in the cell nucleus face outwards, readily binding with hematoxylin and staining blue; while hemoglobin, collagen fibers, and muscle fibers in the cytoplasm bind with eosin, staining red or pink, thus revealing pathological changes in lung tissue. The HE staining results for this experiment are attached. Figure 3The lung tissue of the normal control group (A) had clear texture and intact alveoli; while the lung tissue morphology of the model group (B, i.e., BLM group) mice was significantly altered, such as alveolar depression and adhesion, diffuse fibrosis, and a large number of inflammatory cell infiltration, indicating successful modeling; the lung tissue of mice in the pirfenidone treatment group and each DMB treatment group showed significantly improved lesion severity compared to the bleomycin group (model group), such as narrowed alveolar septa, reduced inflammatory cells, and clear texture, indicating a reduction in the degree of pulmonary fibrosis.

[0078] ②Sirius red staining: Sirius red staining solution is strongly acidic and readily binds to the basic groups in collagen fibers, staining the abnormally proliferating collagen fibers in pulmonary fibrosis tissue red, thus revealing changes in collagen deposition. The results of Sirius red staining in this experiment are attached. Figure 4 As shown: No obvious collagen deposition was observed in the lung fibrosis tissue of mice in the normal control group; while a large amount of red-stained collagen fibers (B) were observed in the lung tissue of mice in the bleomycin group (i.e., the model group); after treatment with pirfenidone and DMB, the collagen deposition in mice in each treatment group was reduced compared with that in the model group.

[0079] ③ Pathological scoring: Results are attached. Figure 5 The pathological scores of lung fibrosis sections from mice in each group were assessed using the Ascroft scoring system, with the pathological severity of the NC group defined as 0 and the Model group as 7. The severity of pulmonary fibrosis in each group was evaluated and compared. The pathological scores showed that all DMB dose groups and the PFD group were lower than the Model group (but the low-dose group showed no statistically significant difference compared to the Model group), indicating that both DMB and PFD improved the degree of pulmonary fibrosis in mice. Low DMB (20 mg / kg) was less effective than PFD (300 mg / kg), but medium-dose (40 mg / kg) and high-dose (80 mg / kg) showed better protective effects against pulmonary fibrosis than PFD. The high-dose group showed a statistically significant difference (P>0.05) and a dose advantage (pirfenidone dose of 300 mg / kg was higher than DMB dose of 80 mg / kg).

[0080] These results indicate that bis(caffeic acid) and its ester derivatives are promising for applications in the preparation of drugs for treating pulmonary fibrosis. Based on the above experimental results, the dosage for animals and humans was converted to determine that the effective daily oral dose for adults is 10–500 mg, preferably 20–200 mg; and the daily injection dose for adults is 5–500 mg, preferably 10–150 mg.

[0081] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The use of bis(caffeic acid) and its derivatives as shown in Formula I in the preparation of drugs for treating pulmonary fibrosis.

2. The bis(caffeic acid) and its derivatives according to claim 1, characterized in that: In compounds of formula I, R1 and R2 are H or groups with 1 to 5 carbon atoms.

3. The bis(caffeic acid) and its derivatives according to claim 2, characterized in that: In compounds of formula I, R1 and R2 are H or groups with 1 to 3 carbon atoms.

4. The application according to claim 1, characterized in that: When the compound of Formula I is used to prepare a drug for treating pulmonary fibrosis, the effective daily oral dose for adults ranges from 10 to 500 mg, preferably from 20 to 200 mg; when administered by injection, the daily injection dose for adults ranges from 5 to 500 mg, preferably from 10 to 150 mg.

5. The application according to claim 1, characterized in that: When used to prepare drugs for treating pulmonary fibrosis, the compound of formula I can be combined with pharmaceutically acceptable excipients or carriers to form a drug composition, which can be made into oral preparations such as capsules, soft capsules, pellets, tablets, granules, and aerosols, or injectable preparations such as emulsions for injection, lyophilized powders for injection, microspheres for injection, or nano-preparations for injection.

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