TWI816381B - Using growth factors to treat pulmonary fibrosis - Google Patents

Abstract

The present invention reveals that epidermal growth factor can be used to treat pulmonary fibrosis.

Description

Using growth factors to treat pulmonary fibrosis

The present invention relates to the use of epidermal growth factor (EGF) to treat pulmonary fibrosis (PF), and in particular to the use of a drug containing epidermal growth factor, polysaccharide and hyaluronic acid (HA). ) composition.

Pulmonary fibrosis (PF) is caused by excessive deposition of extracellular matrix (ECM) [especially collagen] in lung tissue during the wound healing process, and diet, Metabolism, viruses, toxins, genetics, and immune diseases may all be factors that cause pulmonary fibrosis. The main symptoms of pulmonary fibrosis include cough, hypoxemia, shortness of breath, and difficulty breathing. In severe cases, it may cause respiratory failure or even death.

However, there are no clinical drugs that can effectively treat or delay pulmonary fibrosis. Therefore, relevant researchers in this field are committed to developing anti-pulmonary fibrosis drugs.

There are currently studies exploring the correlation between epidermal growth factor receptor (EGFR) signaling and pulmonary fibrosis, for example, in Venkataraman T. et al . (2017), J. Virol. , 91 (12): e00182-17, Venkataraman T. et al. based on many previous studies pointed out that EGFR signaling is a key signaling pathway in the fibrosis process of different organs, and used severe acute respiratory syndrome coronavirus (severe Mice infected with acute respiratory syndrome coronavirus (SARS-CoV) are used as an animal model of virus-induced pulmonary fibrosis to study the role of EGFR. The results showed that mice with sustained EGFR activation after SARS-CoV infection will have increased pulmonary fibrosis and lung lesions, and EGFR signaling is believed to be a key factor in pulmonary fibrosis. As for EGFR ligands, it is thought that they may be released from damaged cells during infection to activate EGFR. Accordingly, preventing the activation of EGFR is considered as a possible way to prevent the progression of pulmonary fibrosis.

In addition, in Vagapova ER et al . (2021), Sci. Rep. , 11:11234, Vagapova ER et al. identified a group of viruses involved in severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) Genes that cause pulmonary fibrosis, including epidermal growth factor (EGF) and its inducible genes, and it has been further found that these genes can be induced by the use of EGFR inhibitors [e.g., dasatinib] and gefitinib] to halt the progression of this viral fibrosis.

Summary of the invention

Although there are the above-mentioned previous studies on epidermal growth factor (EGF) and its receptors, in the present invention, the applicant unexpectedly found that direct use of EGF can effectively treat pulmonary fibrosis (PF) and Improve related tissue lesions, and further combination with polysaccharide (polysaccharide) and hyaluronic acid (HA) can further enhance its effectiveness.

Thus, in a first aspect, the present invention provides the use of an epidermal growth factor supply for the preparation of a composition for treating pulmonary fibrosis. Preferably, the composition further contains polysaccharides and hyaluronic acid.

In a second aspect, the present invention provides a method for treating pulmonary fibrosis, comprising administering to an individual in need thereof a composition as described above.

Detailed description of the invention

It is to be understood that if any prior publication is cited herein, that prior publication does not constitute an admission that the prior publication forms a common practice in the art in Taiwan or any other country. part of knowledge.

For the purposes of this specification, it will be clearly understood that the word "comprising" means "including but not limited to" and the word "comprises" has a corresponding meaning.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used to practice the present invention. Of course, the present invention is in no way limited to the methods and materials described.

The present invention provides an epidermal growth factor (EGF) for use in preparing a composition for treating pulmonary fibrosis (PF).

As used herein, "treating" or "treatment" means preventing, reducing, alleviating, ameliorating, relieving, or controlling. controlling one or more clinical signs of a disease or disorder, and lowering, stopping or reversing a condition being treated or Progression in severity of symptoms.

According to the present invention, the epidermal growth factor can be derived from humans or various animals, plants and microorganisms, and can be a commercially available product, or can be produced by techniques well known and customary to those skilled in the art. The prepared products include, for example, natural products isolated from biological materials or recombinant proteins obtained through genetic engineering or functional fragments thereof. In a preferred embodiment of the present invention, the epidermal growth factor is recombinant human epidermal growth factor (rhEGF).

According to the present invention, the composition further includes polysaccharide and hyaluronic acid (HA).

Preferably, the polysaccharide is selected from the group consisting of: chitosan, chitin, glycosaminoglycan, cellulose, starch, peptidoglycan ( peptidoglycan), and their combinations. In a preferred embodiment of the invention, the polysaccharide is chitosan.

According to the present invention, the epidermal growth factor, the polysaccharide and hyaluronic acid may have a weight ratio ranging from 1:0.5:12.5 to 1:250:1250. In a preferred embodiment of the present invention, the weight ratio of the epidermal growth factor, the polysaccharide and hyaluronic acid is 1:5:125.

According to the present invention, the composition may be a food composition, for example, in the form of a food additive, which may be added to an edible material to prepare a food additive. Food products for human or animal consumption. According to the present invention, the types of food products may include, but are not limited to: milk powder, fermented milk, yogurt, butter, beverages (eg, tea, coffee). ), functional beverages, flour products, baked foods, confectionery, candies, fermented foods, animal feeds, health foods (health foods), infant food (infant food), and dietary supplements (dietary supplements).

According to the present invention, the composition may be a pharmaceutical composition.

According to the present invention, the pharmaceutical composition may be in a dosage form suitable for parenteral administration, oral administration, topical administration or respiratory tract administration. .

According to the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier that is widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the group consisting of: solvent, buffer, emulsifier, suspending agent, decomposer ), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent , gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome and the like. The selection and quantities of these reagents are within the professionalism and routine skills of those skilled in the art.

According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for parenteral administration [including injection, for example, sterile aqueous solution] using techniques well known to those skilled in the art. ) or dispersion] and is administered by a route selected from the group consisting of: intraperitoneal injection, intrapleural injection, intramuscular injection ), intravenous injection, intraarterial injection, intraarticular injection, intrasynovial injection, intrathecal injection, intracranial injection ), intraepidermal injection, subcutaneous injection, intradermal injection, intralesional injection, and sublingual administration. Preferably, the pharmaceutical composition is manufactured into a dosage form suitable for intraperitoneal injection, intravenous injection or sublingual administration.

According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for oral administration using techniques well known to those skilled in the art, including, but not limited to: sterile powder, tablet, tablet ( troche), lozenge, pill, capsule, dispersible powder or granule, solution, suspension, emulsion, syrup ), elixirs, slurries and the like.

According to the present invention, the pharmaceutical composition can be manufactured into an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art, including, but not limited to, emulsions. , gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion , serum, paste, foam, drop, suspension, salve and bandage.

According to the present invention, the pharmaceutical composition can also be manufactured into a dosage form suitable for respiratory tract administration [including spray, for example, nasal spray, oral spray] using techniques well known to those skilled in the art. oral spray] and is administered by a route selected from the group consisting of: oral inhalation and nasal inhalation. Preferably, the pharmaceutical composition is manufactured into a dosage form suitable for nasal inhalation and administration.

The present invention also provides a method for treating pulmonary fibrosis, which includes administering (administering) a composition containing epidermal growth factor as described above to an individual in need thereof.

As used herein, the terms "administration" and "administration" may be used interchangeably and mean introducing, providing, or delivering to an individual by any suitable route. delivering) a predetermined active ingredient to perform its intended effect.

As used herein, the term "subject" means any mammal of interest, such as humans, monkeys, cows, sheep, horses, pigs ( pigs, goats, dogs, cats, mice and rats.

According to the present invention, the dosage and frequency of administration of the composition will vary depending on the following factors: the severity of the disease to be treated, the route of administration, and the age, physical condition and response of the individual to be treated. Generally speaking, the compositions according to the invention may be in the form of a single dose or divided into several doses, and may be administered orally, parenterally, topically or respiratory tract. Detailed description of preferred embodiments

The present invention will be further described with reference to the following examples, but it should be understood that these examples are for illustrative purposes only and should not be construed as limitations on the implementation of the present invention. Examples General experimental materials:

The following ingredients were purchased from Leye Biochemical Technology Co., Ltd.: epidermal growth factor (EGF) (Cat. No. 02-108); chitosan (Cat. No. 04-121) ; and hyaluronic acid (HA) (Cat. No. 07-113). General experimental methods: 1. Statistical Analysis:

In the following examples, the experimental data obtained were statistically analyzed using GraphPad Prism software version 9 (v9) (GraphPad Software, San Diego, CA), and based on the standard of "mean ± mean "standard deviation (SD)". All data were analyzed by two-way analysis of variance (ANOVA), followed by Dunnett's test to evaluate the differences between groups. If the obtained analysis result is p <0.05, it represents statistical significance. Example 1. Experimental materials for evaluating the effectiveness of epidermal growth factor in treating pulmonary fibrosis (PF) : 1. Composition containing EGF , chitosan and HA :

The composition containing EGF, chitosan and HA used in this example is obtained by combining the EGF, chitosan and HA in the "general experimental materials" above into a weight ratio of 1:5:125. prepared by mixing and dispensing in sterile water containing 20 µg/g EGF. 2. Experimental animals:

Male C57BL/6 mice (6 weeks old, weighing approximately 16 to 20 g) used in this example were purchased from the National Laboratory Animal Center (ROC). All experimental animals were individually housed in an animal room with 12 hours of light and 12 hours of darkness, room temperature maintained at 21-24°C, and relative humidity maintained at 45-70%, and were fed ad libitum ( ad libitum ). Feed with water and feed. All experimental procedures related to experimental animals were approved by the Institutional Animal Care and Use Committee of National Chung Hsing University and in accordance with the National Institutes of Health (NIH) Conducted according to the Guide for the Care and Use of Laboratory Animals. Experimental methods: A. Induction of pulmonary fibrosis and administration of epidermal growth factor:

First, male C57BL/6 mice were randomly divided into 1 normal control group, 1 pathological control group, and 4 experimental groups (ie, experimental groups 1 to 4) (n=8 for each group). Next, the EGF in the "General Experimental Materials" above and the composition containing EGF, chitosan and HA obtained in item 1 of the "Experimental Materials" above were respectively mixed with phosphate buffered saline. PBS).

EGF (dose 330 µg/kg body weight) was then administered to mice in experimental groups 1 and 2 via nasal inhalation using a nebulizer (PARI, BOY N). and a composition containing EGF, chitosan, and HA (EGF at a dose of 330 µg/kg body weight) for 30 minutes. The mice in experimental groups 3 and 4 were administered EGF (dose 30 μg/kg body weight) and a composition containing EGF, chitosan and HA (including The dose of EGF is 30 µg/kg body weight). Mice in the pathological control group were administered an equal volume of PBS via nasal inhalation. As for the mice in the normal control group, no treatment was performed. Mice in each group were dosed once a day for a total of 19 days.

After administration on day 1, mice in the pathological control group and each experimental group were immediately treated with bleomycin (BLM) (dose 2 mg/kg body weight) via intratracheal instillation. , in order to induce the occurrence of pulmonary fibrosis. As for the mice in the normal control group, no treatment was performed. B. Testing of lung function:

After administration on day 15, methacholine-induced airflow obstruction (methacholine-induced airflow) of mice in each group was measured by Buxco FinePointe whole body plethysmography (DSI Buxco). Airway responsiveness under obstruction. Briefly, mice were allowed to inhale aerosolized PBS to measure Penh and serve as a baseline value, and then the aerosolized PBS was replaced with increasing concentrations (0 mg/mL, 6.25 mg/mL, 12.5 mg/mL). mL, 25 mg/mL and 50 mg/mL) of aerosolized methacholine (Sigma-Aldrich, A2251-25G) and measure the increase in Penh, also known as the Penh index. The lower the measured Penh index, the better the respiratory function.

Afterwards, analyze the experimental data obtained according to the method described in item 1 of the "General Experimental Methods" above. C. Histopathological evaluation:

At the end of the 19th day after the start of drug administration, mice in each group were sacrificed and their lung tissues were removed, followed by treatment with 10% neutral buffered formalin (BiOTnA Biotech, Cat No. TABS06) at room temperature. -4000) for 24 hours, and then the fixed tissue samples were embedded in paraffin and then sliced to obtain tissue sections with a thickness of 5 μm. (tissue sections).

Afterwards, the obtained tissue sections were subjected to hematoxylin-eosin staining and Masson's trichrome staining according to techniques well known and commonly used by those skilled in this art, and then used Olympus CKX41 microscope and observed at a magnification of 400x, followed by Schniering J. et al. (2018), Arthritis Res. Ther. 20:183 and Ruscitti F. et al. (2020), Front. Pharmacol. 11:1117 to score the degree of tissue lesions and fibrosis, and express it from 0 to 5. The higher the value, the more serious the degree of tissue lesions and fibrosis.

Afterwards, analyze the experimental data obtained according to the method described in item 1 of the "General Experimental Methods" above. Results: A. Testing of lung function:

Figure 1 shows the Penh index measured under different concentrations of methacholine in each group of mice at the end of the 15th day after the start of drug administration. As can be seen from Figure 1, compared with the normal control group, the Penh index of the pathological control group will increase rapidly as the concentration of methacholine increases, which means that BLM successfully induces pulmonary fibrosis and has a negative impact on lung function. produce negative effects. Compared with the pathological control group, the increase in Penh index of each experimental group was significantly reduced. Among them, experimental group 2 and experimental group 4 showed a more obvious decrease than experimental group 1 and experimental group 3 respectively. . In particular, the most significant reduction was observed in experimental group 2. The results of this experiment show that EGF can effectively improve the lung function of mice regardless of whether it is administered through nasal inhalation or intraperitoneal injection, and this improvement effect can be enhanced when combined with chitosan and HA. B. Histopathological evaluation:

Figures 2 and 3 respectively show the results of evaluating the degree of tissue lesions and fibrosis by hematoxylin-eosin staining and Masson's trichrome staining at the end of the 19th day after the start of drug administration. As can be seen from Figures 2 and 3, compared with the normal control group, the tissue lesions and the degree of pulmonary fibrosis in the pathological control group were significantly increased, which means that BLM successfully induced pulmonary fibrosis and caused related tissue lesions. . Compared with the pathological control group, the degree of tissue lesions and pulmonary fibrosis in each experimental group were significantly reduced. Among them, experimental group 2 and experimental group 4 were more obvious than experimental group 1 and experimental group 3 respectively. of reduction. In particular, the most significant reduction was observed in experimental group 4. The results of this experiment show that EGF can effectively improve pulmonary fibrosis and related tissue lesions whether administered through nasal inhalation or intraperitoneal injection, and this improvement can be enhanced when combined with chitosan and HA. utility.

Based on the above experimental results, the applicant believes that epidermal growth factor can be used to treat pulmonary fibrosis and improve the related decrease in lung function and tissue lesions caused by it, and can be further combined with polysaccharides (especially chitosan) and hyaluronic acid to enhance its effectiveness.

All patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of conflict, the detailed description of the case (including definitions) will prevail.

Although the invention has been described with reference to the specific embodiments above, it will be apparent that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that this invention be limited only as indicated by the appended claims.

The above and other objects, features and advantages of the present invention will become apparent after referring to the following detailed description and preferred embodiments and the accompanying drawings, wherein: Figure 1 shows the 15th step after the start of drug administration. At the end of the day, the Penh index of mice in each group was measured under different concentrations of methacholine, where “**”, “***” and “****” respectively indicate when compared with the pathological control Comparison between groups, p < 0.01, p < 0.001 and p <0.0001; and Figures 2 and 3 respectively show that at the end of the 19th day after the start of drug administration, the lung tissue samples of mice in each group showed significant differences in tissue lesions and fibrosis. The results of scoring the degree of oxidation, where "*", "**" and "****" respectively indicate p <0.05, p <0.01 and p <0.0001 when compared with the pathological control group.

Claims (8)

An epidermal growth factor is provided for use in the preparation of a composition for treating pulmonary fibrosis. The use of claim 1, wherein the composition further comprises polysaccharides and hyaluronic acid, and the polysaccharides are selected from the group consisting of: chitosan, chitin, glycosaminoglycans, cellulose, starch , peptidoglycan, and their combinations. Such as the use of claim 2, wherein the epidermal growth factor, the polysaccharide and hyaluronic acid are in a weight ratio ranging from 1:0.5:12.5 to 1:250:1250. Such as the use of claim 3, wherein the weight ratio of the epidermal growth factor, the polysaccharide and hyaluronic acid is 1:5:125. The use of claim 2, wherein the polysaccharide is chitosan. Such as the use of claim 1, wherein the composition is a food composition or a pharmaceutical composition. The use of claim 6, wherein the pharmaceutical composition further includes a pharmaceutically acceptable carrier. Such as the use of claim 6, wherein the pharmaceutical composition is in a dosage form for parenteral administration, oral administration, topical administration or respiratory administration.