Composition for preventing or treating lung diseases comprising hyaluronic acid and proteoglycan linking protein 1

Abstract

The invention relates to a recombinant hyaluronan and proteoglycan link protein 1 (HAPLN1) protein and a composition for preventing or treating lung diseases, wherein the composition comprises at least one selected from the group consisting of HAPLN1 (hyaluronan and proteoglycan link protein 1), a gene encoding HAPLN1 protein, and an effective agent for promoting the expression of HAPLN1 protein or gene or activating its function as an effective ingredient. The recombinant HAPLN1 protein disclosed herein has an excellent effect of improving alveolar damage caused by aging or elastin reduction, thereby effectively preventing or treating lung diseases such as chronic bronchitis, asthma, emphysema, and chronic obstructive pulmonary disease.

Description

Technical Field

[0001] The present disclosure relates to a recombinant hyaluronan and proteoglycan link protein 1 (HAPLN1) protein and a composition for preventing or treating lung diseases, wherein the composition comprises at least one selected from the group consisting of HAPLN1 (hyaluronan and proteoglycan link protein 1) protein, a gene encoding HAPLN1 protein, and an effective agent that promotes the expression of HAPLN1 protein or gene or activates its function as an effective ingredient. Background Art

[0002] As of 2016, approximately 251 million people (4.8% of the world's total population) suffer from chronic obstructive pulmonary disease (COPD), which is the fourth leading cause of death worldwide, and approximately 3.17 million people died of COPD in 2015. In addition, it is more common in men than in women, and the number of patients with COPD is increasing year by year.

[0003] Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease that occurs more frequently than lung cancer and is characterized by irreversible airflow obstruction and the continued development of respiratory symptoms such as cough, sputum, and dyspnea. It is well known that COPD is mainly caused by smoking, occupational exposure, and atmospheric pollution such as particulate matter. For these reasons, small airway lung disease and lung parenchymal destruction work together to cause airflow limitation, thus triggering various clinical symptoms.

[0004] According to recent studies, aging itself may be an important cause of COPD and has been proven to be a risk factor. In particular, it rapidly increases from the age of 50, and then as the age increases, the incidence rate in the elderly over 60 years old is 2 to 3 times higher than that in the younger age group. COPD seriously affects the lungs, and in the process, alveoli are damaged, and air exchange cannot be carried out smoothly due to the damaged alveoli. It is known that the reason why the disease does not occur in young smokers is because recovery and regeneration are actively carried out while being damaged.

[0005] With the global aging trend, cell senescence, telomere shortening and the reduction of certain anti-aging molecules lead to chronic inflammation in the lungs, which may be the direct cause of emphysema, which is known to be caused by narrowing of small airways and destruction of elastin in lung tissue. The pathogenesis and biochemical changes of these senile emphysema are very similar to those of ordinary emphysema. Since emphysema is not reversible like asthma or bronchitis but irreversible, it is very difficult to treat, and there is no therapeutic or preventive drug that can effectively treat emphysema.

[0006] In particular, elastin, a component of the extracellular matrix (ECM), is a very important protein that provides the necessary elasticity and elastic recoil for the gas exchange function of the alveoli. When the alveolar wall is destroyed or damaged due to the reduction of elastin production or the acceleration of its decomposition, the alveolar wall becomes irreparable, thereby causing problems with the expansion and contraction of the alveoli, i.e., dyspnea, and eventually develops into COPD.

[0007] As COPD and the aging trend become more important global issues, there is a question whether COPD caused by aging can actually be treated and prevented. Until recently, it was known that the lungs were organs that could not be restored or regenerated, so it was recognized that the possibility of treating or preventing COPD was very small. However, since the study by Butler et al. in 2012 proved that adult lung tissue is an organ that can regenerate, some recent studies have revealed through cell biological evidence and mechanisms that the lung epithelial cell group has the ability to self-differentiate and proliferate, and can therefore restore or regenerate damaged or degenerated alveoli. As the regeneration and restoration technology of degenerated alveoli advances, it is expected that therapeutic and preventive drugs will be studied more actively in the future.

[0008] As mentioned above, COPD is a pulmonary disease characterized by irreversible airflow limitation, which occurs and develops due to damage to airway and lung parenchymal tissue caused by chronic inflammation, but it is known as a disease that can be prevented and treated as mentioned above. At present, the most commonly used COPD therapeutic agents in the world include inhaled corticosteroids (ICS) and long-acting muscarinic antagonists (LAMA) as asthma therapeutic agents, and long-acting beta agonists (LABA) as chronic bronchitis and emphysema therapeutic agents, but these therapeutic agents cannot completely treat COPD. In particular, since ICS is directly applied to the respiratory tract, it exerts a very strong local anti-inflammatory effect. However, when the steroid applied to the airway is transported from the alveoli to the blood to the whole body, systemic side effects may be increased. The side effects of these existing drugs may make patients feel anxious, and in severe cases, may lead to refusal to apply, so it is strongly required to develop drugs with less side effects on the market. Summary of the invention

[0009] Technical issues

[0010] In order to solve the above problems, the present disclosure provides a recombinant protein and an excellent composition for preventing or treating lung diseases, wherein the composition comprises at least one selected from the group consisting of hyaluronic acid and proteoglycan linking protein 1 (HAPLN1) protein, a gene encoding HAPLN1 protein, and an effective agent that promotes the expression of HAPLN1 protein or gene or activates its function as an effective ingredient.

[0011] Additional aspects will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of presented embodiments of the disclosure.

[0012] Technical Solution

[0013] The recombinant protein according to the present disclosure may be a recombinant hyaluronan and aggrecan linking protein 1 (HAPLN1) protein having the amino acid sequence of SEQ ID NO:1.

[0014] The pharmaceutical composition for preventing or treating lung diseases according to the present disclosure may include at least one selected from the group consisting of HAPLN1 protein, a gene encoding HAPLN1 protein, and an effective agent promoting the expression of HAPLN1 protein or gene or activating the function thereof as an effective ingredient.

[0015] Beneficial Effects

[0016] Compared with existing therapeutic agents for lung diseases, the composition has almost no side effects, and thus can safely and effectively prevent or treat lung diseases that are increasing year by year with the global aging trend.

[0017] It should be understood that the embodiments described herein should be considered to have only descriptive significance, and not for the purpose of limitation. The description of the features or aspects in each embodiment should generally be considered to be applicable to other similar features or aspects in other embodiments. Although one or more embodiments have been described with reference to the accompanying drawings, it will be understood by those of ordinary skill in the art that various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the following claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and other aspects, features and advantages of certain embodiments of the present disclosure will become more apparent from the following description in conjunction with the accompanying drawings, in which:

[0019] Figure 1A , 1B 1C and 1C show the alveolar experimental results of aged mice according to an experimental example of the present disclosure, wherein Figure 1A This is an image observed under a microscope after staining alveolar tissue. Figure 1B and 1C It is a graph quantifying alveolar density. Here, "Young-saline" refers to a group in which phosphate buffered saline (PBS) was administered to a normal control group (young group), "Old-saline" refers to a group in which PBS was administered to an elderly control group (old group), and "Old-rhHAPLN1" refers to a group in which rhHAPLN1 diluted in PBS was administered to an elderly rhHAPLN1-treated group (Old+rhHAPLN1 group).

[0020] Figure 2 It is a schematic diagram of a spray injection (aerosolization) method according to an experimental example of the present disclosure.

[0021] Figure 3A , 3B 3C and 3C show the experimental results of the aerosol inhalation group (AH group) according to an experimental example of the present disclosure, Figure 3A is an image obtained by staining the alveolar tissue of each group and observing it under a microscope, and Figure 3B and 3C The graph is a result of measuring the mean linear intercepts (MLI) values.

[0022] Figure 4A , 4B4C and 4C show the experimental results of the intratracheal instillation group (TI group) according to an experimental example of the present disclosure, Figure 4A is an image obtained by staining the alveolar tissue of each group and observing it under a microscope, and Figure 4B and 4C The following is a graph showing the results of measuring the MLI value. DETAILED DESCRIPTION

[0023] The embodiments shown in the accompanying drawings will now be referred to in detail, examples, wherein the same reference numerals always represent the same elements. In this regard, the present embodiment may have different forms and should not be construed as being limited to the description set forth herein. Therefore, the embodiments are described below only by reference to the accompanying drawings to explain the various aspects of this specification. As used herein, the term "and / or" includes any and all combinations of one or more associated listed items. When an expression such as "at least one" is before an element list, the entire element list is modified and the individual elements of the list are not modified.

[0024] Hereinafter, the present disclosure will be described in detail.

[0025] The present inventors prepared a recombinant HAPLN1 protein and confirmed its effect of improving alveolar damage and its therapeutic effect in an animal model of chronic obstructive pulmonary disease, thereby completing the present disclosure.

[0026] In this specification, "prevention" refers to any action to inhibit or delay the occurrence of a lung disease or at least one symptom of the disease by administering a pharmaceutical composition according to the present disclosure. In addition, it includes treating a subject who has improved in the disease to prevent or prevent recurrence.

[0027] In the present specification, "treatment" refers to any action to improve or beneficially change (eg, alleviate, reduce or eliminate) a lung disease or at least one symptom of the lung disease by administering the pharmaceutical composition according to the present disclosure.

[0028] In this specification, a "pharmaceutical composition" refers to a composition that is administered for a specific purpose, and for the purposes of this disclosure, is meant to be administered to prevent or treat a lung disease or at least one symptom of the disease.

[0029] The present disclosure provides a recombinant hyaluronan and aggrecan linking protein 1 (HAPLN1) having the amino acid sequence of SEQ ID NO:1.

[0030] The "HAPLN1" is a protein that stabilizes hyaluronic acid by linking hyaluronic acid to proteoglycan, and is a constituent protein in the extracellular matrix first discovered in the joints of vertebrates.

[0031] The "recombinant HAPLN1 protein" according to the present disclosure is a recombinant protein prepared by using the HAPLN1 protein.

[0032] The present disclosure provides a pharmaceutical composition for preventing or treating a lung disease, the composition comprising at least one selected from HAPLN1 protein, a gene encoding HAPLN1 protein, and an effective agent promoting the expression of HAPLN1 protein or gene or activating the function thereof as an effective ingredient.

[0033] In certain embodiments, the HAPLN1 protein provided herein may include an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1. In some of these embodiments, the HAPLN1 protein may include an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% sequence identity with the amino acid sequence of SEQ ID NO: 1. Furthermore, the polypeptide disclosed in the present specification may include a peptide of SEQ ID NO: 1, a fragment thereof, and a peptide in which at least one, two, three, four, five, six or seven amino acids are modified.

[0034] As used herein, the term "identity" is a relationship between two or more polypeptide sequences, and is determined by comparing sequences as appropriate. In the art, "identity" also refers to the degree of sequence relatedness between polypeptide sequences, and is determined by the match between strings of such sequences as appropriate. "Identity" can be easily calculated by known methods, such as using BLAST (National Center for Biological Information: NCBI) Basic Local Alignment Search Tool) Version 2.2.1 software with default parameters.

[0035] In certain embodiments, within the scope of the present disclosure, some amino acids in the peptide may be modified to alter the physicochemical properties of the polypeptide of SEQ ID NO: 1. For example, amino acids may be modified to impart enhanced enzymatic, chemical or thermal stability, altered substrate specificity and altered optimal pH to the peptide.

[0036] The recombinant HAPLN1 protein can improve alveolar damage caused by aging or elastin reduction, and thus can be used as a pharmaceutical composition for preventing or treating lung diseases. In addition, according to an experimental example disclosed herein, it can be confirmed that the recombinant HAPLN1 protein shows a significant therapeutic effect in an animal model of chronic obstructive pulmonary disease.

[0037] As used herein, the term "agent" refers to any substance, including but not limited to a chemical compound, a small molecule, an antibody, a peptidomimetic, a peptide, or a protein.

[0038] As used herein, the term "effective agent" refers to a HAPLN1 expression promoter that promotes the expression of HAPLN1 protein or gene; or a HAPLN1 activator that activates the function of HAPLN1 protein or gene. As used herein, effective agents include various compounds, proteins or peptides, base sequences, etc. that can enhance the expression of HAPLN1 gene or HAPLN1 protein and / or its fragments or activate the function of HAPLN1 protein or gene. The activator also includes various metabolites, precursors, drug equivalents of compounds, proteins or peptides or base sequences.

[0039] In the pharmaceutical composition according to the present disclosure, the lung disease may be one or more selected from the group consisting of chronic bronchitis, asthma, emphysema and chronic obstructive pulmonary disease (COPD), but is not limited thereto, and may include various diseases caused by lung damage.

[0040] The pharmaceutical composition according to the present disclosure can be prepared according to conventional methods in the pharmaceutical field. In addition to the active ingredient, the pharmaceutical composition can be mixed with a pharmaceutically acceptable appropriate carrier according to the dosage form, and can further include excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants and solvents, etc. as needed. The suitable carriers are those that do not hinder the activity and properties of the recombinant HAPLN1 protein according to the present disclosure, and can be selected differently according to the administration form and dosage form.

[0041] The pharmaceutical composition according to the present disclosure may be used in any dosage form, and in more detail, may be formulated into oral dosage forms, external preparations, suppositories, and parenteral dosage forms of sterile injection solutions according to conventional methods.

[0042] In the oral dosage form, the solid dosage form can be in the form of tablets, pills, powders, granules and capsules, and is prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose and gelatin, etc., and in addition to simple excipients, lubricants such as magnesium stearate and talc can also be included. In addition, in the case of a capsule dosage form, in addition to the above substances, a liquid carrier such as a fatty oil can also be included.

[0043] Among the oral dosage forms, liquid dosage forms correspond to suspensions, internal solutions, emulsions and syrups, etc., and in addition to the commonly used simple diluents, namely water and liquid paraffin, may also include various excipients such as wetting agents, sweeteners, aromatics and preservatives, etc.

[0044] The parenteral dosage form may include sterile aqueous solution, non-aqueous solvent, suspension, emulsion, freeze-dried preparation and suppository. As non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil and injectable ester such as ethyl oleate etc. may be used. As the matrix of suppository, Witepsol, polyethylene glycol, Tween 61, cocoa butter, laurin butter and glycerin gelatin etc. may be used. But the disclosure is not limited thereto, and any suitable preparation known in the art may be used.

[0045] In addition, the pharmaceutical composition according to the present disclosure may further be supplemented with calcium or vitamin D3 to enhance the therapeutic efficacy.

[0046] The pharmaceutical composition according to the present disclosure may be administered in a pharmaceutically effective amount.

[0047] In the present specification, "pharmaceutically effective amount" refers to an amount that is sufficient for disease treatment at a reasonable benefit / risk ratio applicable to medical treatment and does not cause side effects.

[0048] The effective dose level of the pharmaceutical composition is determined differently according to factors including the following items and other factors well-known in the medical field: purpose of use, age, sex, weight and health status of the patient, type of disease, severity of disease, drug activity, sensitivity to drugs, method of administration, time of administration, route of administration and excretion rate, treatment time, drugs used in combination or used simultaneously. For example, although not constant, the pharmaceutical composition can usually be administered in an amount of 0.001 mg / kg to 100 mg / kg per day, preferably 0.01 mg / kg to 10 mg / kg once to several times. The dosage does not limit the scope of the present disclosure in any way.

[0049] The pharmaceutical composition according to the present disclosure can be administered to any animal that may develop a lung disease, and the animal may include, for example, humans and primates, and livestock such as cattle, pigs, horses, and dogs.

[0050] The pharmaceutical composition according to the present disclosure can be administered by an appropriate route of administration according to the form of the preparation, or can be administered by various routes of oral or parenteral routes, as long as the target tissue can be reached. The method of administration is not particularly limited, and can be administered by conventional methods such as oral, rectal, intravenous, intramuscular, topical, subcutaneous, respiratory inhalation, endometrial or intraventricular (intracerebroventricular) injection, etc.

[0051] The pharmaceutical composition according to the present disclosure may be used alone or in combination with surgery or other chemotherapy for the prevention or treatment of lung diseases.

[0052] In certain embodiments, the HAPLN1 protein provided herein may include an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1. In some of these embodiments, the HAPLN1 protein may include an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% sequence identity with the amino acid sequence of SEQ ID NO: 1. Furthermore, the polypeptide disclosed in the present specification may include a peptide of SEQ ID NO: 1, a fragment thereof, and a peptide in which at least one, two, three, four, five, six or seven amino acids are modified.

[0053] Hereinafter, for better understanding, the present disclosure will be described in detail with reference to exemplary embodiments. However, the following embodiments are only for illustrative purposes, and the scope of the present disclosure is not limited to the following embodiments. The embodiments of the present disclosure are provided to explain the present disclosure more completely to those of ordinary skill in the art.

[0054] Furthermore, the present disclosure may provide a method of preventing or treating a lung disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition according to the present disclosure.

[0055] The subject is preferably a mammal, including a human, and may be a patient in need of treatment for a lung disease. The patient may include a patient who is receiving treatment for a lung disease, a patient who has received treatment for a lung disease, a patient in need of treatment for a lung disease, and a patient who has received surgery to treat a lung disease. Administering a pharmaceutical composition according to the present disclosure to an individual may alleviate or treat a lung disease.

[0056] As used herein, the term "alleviate" refers to any effect that reduces or benefits lung disease by administering the pharmaceutical composition according to the present disclosure. The pharmaceutical composition according to the present disclosure is administered in a pharmaceutically effective amount.

[0057] As used herein, the term "administering" refers to introducing a pharmaceutical composition according to the present disclosure into a subject using any suitable method. The route of administration may include, but is not limited to, intratracheal or intrabronchial instillation or by inhalation, or any type of injection, or any type of oral medication or any type of transdermal medication. As one of the examples, the pharmaceutical composition may be administered in the form of an aerosol or may be administered by instillation.

[0058] The administration of the pharmaceutical composition according to the present disclosure can be carried out by aerosol or by instillation, and the aerosol can be produced by a nebulizer. The pharmaceutical composition can be administered alone or with a carrier such as a saline solution, dimethylformaldehyde (DMSO), alcohol or water. It can also be used as a medium for administering various reagents (such as those that prevent elastic fiber degradation or promote its resynthesis) in the trachea. The daily effective amount of the composition is about 0.001mg / kg to 100mg / kg, preferably 0.01mg / kg to 10mg / kg body weight. The dosage does not limit the scope of the present disclosure in any aspect. For example, the amount of the pharmaceutical composition administered to the human trachea every day can vary within about 0.001mg / kg to about 100mg / kg body weight. Preferably, the daily amount is about 0.01mg / kg to about 10mg / kg body weight (daily) of the subject.

[0059] In addition, as is well known in the art, the time of administering the pharmaceutical composition can be varied to achieve the desired result. For example, the pharmaceutical composition can be administered in the form of an aerosol for about 30 seconds to about 1 hour, 1-5 times per day, or until the desired daily dose is fully administered in each treatment regimen.

[0060] In addition, the pharmaceutical composition for preventing or treating lung diseases according to the present disclosure, which includes at least one selected from the group consisting of HAPLN1 protein, a gene encoding HAPLN1 protein, and an effective agent that promotes the expression of HAPLN1 protein or gene or activates its function as an effective ingredient, can be combined with other existing drugs for treating lung diseases or their existing treatment methods for simultaneous / sequential administration. Such administration can be single or multiple administrations. It is important to achieve the maximum effect with the minimum amount without side effects while considering all factors. The amount can be easily determined by those skilled in the art.

[0061] <Example 1> Production of recombinant human HAPLN1 (rhHAPLN1)

[0062] 1. Amino acid sequence of recombinant human HAPLN1

[0063] The amino acid sequence constituting the recombinant HAPLN1 is as follows.

[0064]

[0065] 2. Expression, purification and storage of recombinant human HAPLN1 protein (rhHAPLN1)

[0066] To prepare recombinant human HAPLN1 (rhHAPLN1; gene number 2678736), a DNA vector encoding the amino acid sequence of rhHAPLN1 was transfected into Expi TM293 cells (Thermo Fisher Scientific Co., Waltham, Massachusetts, USA). For purification, a secretion signal peptide, 10 histidines (H, His) and a TEV protease recognition site were inserted into the amino terminus and expressed. Three days after vector transfection, the culture fluid was collected and purified by a HisTrap column (GE Healthcare, Illinois, USA), and then the TEV protease-recognition sequence was cleaved by TEV protease, and the molecules including histidine were removed using DynaBeads (Thermo Fisher Scientific). The solution thus obtained was dialyzed for 16 hours at pH 8.0 using 40mM Tris-HCl and 1M NaCl. The final purified product had a protein concentration of 0.11 mg / ml and was aliquoted into single doses using 20mM Tris-HCl, 0.5M NaCl pH 8.0, and 50% glycerol as solvents, and then stored in a refrigerator at -20°C for use.

[0067] <Experimental Example 1> Examination of the effect of improving alveolar structure in aged mice by repeated intraperitoneal administration of recombinant human HAPLN1 protein (rhHAPLN1)

[0068] 1. Preparation and Housing of Experimental Animals

[0069] For the experimental animals, 2-month-old male C57BL / 6J (Young Bio, Korea) mice were set as young mice, and 20-month-old male C57BL / 6J mice were set as old mice. The mice were allowed to freely access water and feed, and the temperature of the breeding room was maintained at 21°C to 24°C, the humidity was maintained at 40% to 60%, and the day and night cycle was 12 hours.

[0070] Five young mice were divided into a normal control group (young group), and five old mice were assigned to each of the aged control group (old group) and the aged rhHAPLN1-treated group (aged+rhHAPLN1 group). In the aged rhHAPLN1-treated group (aged+rhHAPLN1 group), rhHAPLN1 diluted in phosphate buffered saline (PBS) was intraperitoneally injected (IP injection) at a dosage of 0.1 mg / kg in a single dose of 80 μl, three times a week for three weeks. The other two control groups were given the same amount of PBS in the same manner.

[0071] 2. Staining and Microscopic Observation of Alveolar Tissue

[0072] At the end of the experiment, after cardiac perfusion, the left lung was divided into upper and lower parts, fixed with neutral buffered 10% formalin (NBF), and paraffin tissue sections were prepared so that the cross-section of the upper lung slice could be seen. The tissue sections were stained with hematoxylin-eosin (H&E) and then photographed using a Ni-U (Nikon) microscope and a DS-Ri1 (Nikon) digital camera. The results are shown in Figure 1 (A) (scale bar = 100 μm).

[0073] In addition, for each individual stained tissue section, the mean linear intercept (MLI) was calculated using the Image J program to quantify the alveolar density. The higher the MLI value, the smaller the alveolar surface area, and is used as an indicator to show the degree of emphysema or chronic obstructive pulmonary disease (COPD). The formula is Lm (μm) = number of intersections / (grid length (μm) × number of lines). After measuring any three points of each mouse to obtain the average value, the values ​​of 5 mice were statistically analyzed using Sigma plot 12.0 and Graph PadPrism 8. The results are shown in Figure 1 (B).

[0074] As shown in Figures 1(A) and 1(B), the alveolar MLI value (μm) of aged mice increased by about 141% compared with that of young mice (p<0.001). On the other hand, it was found that the increase caused by aging itself was reduced by about 56% (p<0.0435) by rhHAPLN1 treatment.

[0075] <Example 2> Efficacy of recombinant human HAPLN1 protein (rhHAPLN1) in a chronic obstructive pulmonary disease (COPD) model induced by porcine pancreatic elastase (PPE)

[0076] 1. Preparation and Housing of Experimental Animals

[0077] Female C57BL6 / N (Young Bio, Korea) mice aged 6 to 10 weeks with a body weight of 20 to 25 g were used as experimental animals, and the mice were divided into groups of 5. The mice were allowed to freely access water and feed, and the temperature of the breeding room was maintained at 21°C to 24°C, the humidity was maintained at 40% to 60%, and the day and night cycle was 12 hours.

[0078] 2. Causes of chronic obstructive pulmonary disease (COPD)

[0079] With reference to Suki et al. (2017, Methods Mol. Biol. 1639: 67-75) and Wright et al. (2008, Am J Physiol Lung Mol Physiol 295: L1-L15), a mouse model in which COPD was induced was prepared.

[0080] To inhale elastase into the lung, first, porcine pancreatic elastase (EC134; PPE) was purchased from Elastin Products Company Inc. (Owensville, MO, USA) and 6 IU / 30 μl was administered once a day to each animal before drug treatment. The animals were divided into two groups according to the inhalation method.

[0081] For the aerosol inhalation group (AH), a 20G (0.9×50mm) oral zonde needle was used for oral administration. It can be administered by carefully opening the mouth, pulling the tongue, and then dripping the elastase solution on its dorsal side (i.e., distal to the oropharynx), while inducing inhalation by blocking both nostrils. For the intratracheal instillation group (TI), it was administered by inducing spontaneous inhalation of mice by dripping into the nasal cavity using an Eppendorf pipette. The body weight of the mice was measured twice a week, and each group was divided into 4 subgroups to administer the composition.

[0082] 3. Efficacy of the combined drug

[0083] The aerosol inhalation group (AH group) was further divided into AH-1: no PPE treatment (normal) group, AH-2: PPE + saline group, AH-3: PPE + rhHAPLN1 group and AH-4: PPE + hyaluronic acid (HA) group, and the intratracheal instillation group (TI group) was further divided into TI-1: no PPE treatment (normal) group, TI-2: PPE + saline group, TI-3: PPE + rhHAPLN1 group and TI-4: PPE + HA group. Here, HA is hyaluronic acid, which is known to have efficacy against COPD, and is therefore used as a positive control. The administration and concentration of HA were determined with reference to Cantor et al. (2005, Experimental Lung Research, 31: 417-430), and therefore, Hyaluronic acid sodium salt from Streptococcus equi (Sigma-Aldrich; St. Louis, MO, USA, Cat. 73641-10MG) was purchased and used.

[0084] Starting from the first day after the elastase administration, each pharmaceutical composition sample was administered five times a week using a Mass Dosing System, specifically an aerosol chamber (Data Science International) for nebulization (Ball flow meter 3, aerosol 1 hour, working volume 30%). Figure 2 A schematic diagram of atomization is shown in FIG.

[0085] Using physiological saline as a solvent, each sample was nebulized with a concentration of 0.33% (w / v) rhHAPLN1 and 5.71% (w / v) HA by adjusting the volume of a single administration to 6.8 ml. The day after the last nebulization on day 21, after cardiac perfusion under anesthesia, the left lung was bisected horizontally, and the lower part was removed, fixed with neutral buffered 10% formalin (NBF), and paraffin tissue sections were prepared so that the cut surface of the upper lung slice could be seen. The tissue sections were stained with hematoxylin & eosin (H&E) staining, and alveolar tissue photograph data (scale bar = 100 μm) was obtained using a Ni-U (Nikon) microscope and a DS-Ri1 (Nikon) digital camera at 200 magnifications, and the MLI of the alveoli was measured. The higher the MLI value, the smaller the alveolar surface area, and it is used as an indicator to show the degree of emphysema or COPD. The formula is Lm (μm) = number of intersections / (grid length (μm) × number of lines). After measuring three random locations of each mouse to obtain the average value, the values ​​of 5 mice were statistically analyzed using Sigma plot 12.0 and Graph Pad Prism 8.

[0086] The experimental results of the aerosol inhalation group (AH group) are shown in FIG3 , and the experimental results of the intratracheal instillation group (TI group) are shown in FIG4 .

[0087] Figure 3 shows the results of the aerosol inhalation group (AH), referring to Figures 3A to 3C , the PPE-treated group (AH-2) 21.2μm increased by about 1.5 times compared with the untreated normal group (AH-1) 13.8μm (p<0.001). Compared with the PPE-treated group 21.2μm, the rhHAPLN1-treated group (AH-3) 14.5μm decreased by about 95% (p<0.00002), indicating that it almost returned to normal.

[0088] In contrast, from the perspective of concentration, the HA-treated group (AH-4) was 5.71% (w / v), which was approximately 17 times higher than 0.33% (w / v). The HA-treated group showed an approximately 73% reduction (p<0.002), indicating that the efficacy of the positive control HA treatment was slightly lower than that of the rhHAPLN1 treatment (approximately 95% reduction).

[0089] FIG4 is the experimental results of the intratracheal instillation group (TI group), and reference FIG. 4A to FIG. 4C The PPE-treated group (TI-2) was 22.1 μm, which was about 1.6 times higher than the untreated normal group (TI-1) of 13.8 μm (p<0.001). Compared with the PPE-treated group (TI-2) of 22.1 μm, the rhHAPLN1-treated group (TI-3) was 16.1 μm, which was about 72% lower (p<0.0009).

[0090] In contrast, from the perspective of concentration, the HA-treated group (TI-4) was 5.71% (w / v), which was approximately 17 times higher than 0.33% (w / v), and the HA-treated group showed approximately 64% reduction (p<0.00123), indicating that the efficacy of the positive control HA treatment was slightly lower than that of the rhHAPLN1 treatment, which showed approximately 72% reduction.

[0091] The recombinant HAPLN1 according to the present disclosure may have an excellent effect of improving alveolar damage caused by aging or elastin reduction, and thus it may be used as a pharmaceutical composition for preventing or treating lung diseases caused by aging or elastin reduction, such as chronic bronchitis, asthma, emphysema, chronic obstructive pulmonary disease, and the like.

[0092] Compared with existing therapeutic agents for lung diseases, the composition has almost no side effects, so it can safely and effectively prevent or treat lung diseases that are increasing year by year with the global aging trend.

[0093] It should be understood that the embodiments described herein should be considered in a descriptive sense only and not for the purpose of limitation. The description of the features or aspects in each embodiment should generally be considered to be applicable to other similar features or aspects in other embodiments. Although one or more embodiments have been described with reference to the accompanying drawings, it will be understood by those of ordinary skill in the art that various changes in form and details may be made without departing from the spirit and scope of the present disclosure as defined by the following claims.

Claims

1. Use of a pharmaceutical composition in preparing a drug for preventing or treating a lung disease, the pharmaceutical composition comprising at least one selected from the group consisting of hyaluronic acid and proteoglycan linking protein 1 HAPLN1 and a gene encoding HAPLN1 protein, wherein the amino acid sequence of the HAPLN1 protein is as shown in SEQ ID NO: 1, and the lung disease is one or more selected from the group consisting of emphysema and chronic obstructive pulmonary disease.

2. The use according to claim 1, wherein the HAPLN1 protein improves alveolar damage caused by aging or elastin reduction.

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