HK40079918A - Composition for preventing or treating pulmonary diseases comprising hyaluronan and proteoglycan link protein 1 - Google Patents

Description

Composition for preventing or treating pulmonary diseases comprising hyaluronic acid and proteoglycan connexin 1

Technical Field

The present disclosure relates to a recombinant hyaluronic acid and proteoglycan link protein 1 (HAPLN 1) protein and a composition for preventing or treating pulmonary diseases, which includes at least one selected from the group consisting of HAPLN1 (hyaluronic acid and proteoglycan link 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

About two hundred million (five hundred million) (4.8% of the global population) have Chronic Obstructive Pulmonary Disease (COPD), which is the fourth leading cause of death worldwide, 2016, and about 317 million die of COPD, 2015. Furthermore, it is more common in men than women, and the number of patients with chronic obstructive pulmonary disease is increasing year by year.

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 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 aerosols. For these reasons, small airway lung disease and destruction of the lung parenchyma act together to cause airflow limitation, thus causing various clinical symptoms.

According to recent studies, aging (aging) itself may be a significant cause of COPD and has been shown to be a risk factor. In particular, it rapidly increases from the age of 50 years, and then the incidence in the elderly over 60 years is 2 to 3 times higher than in their younger age groups as the age increases. COPD seriously affects the lungs, and in the process, damage to alveoli occurs, and air exchange cannot be smoothly performed due to the damage to the alveoli. It is known that the reason why the disease does not occur in young smokers is because recovery and regeneration are positively performed while being damaged.

With the global trend toward advanced age, chronic inflammation of the lungs occurs as a result of cell aging (cell senescence), telomere shortening, and reduction of certain anti-aging molecules, which may be a direct cause of emphysema known to be caused by narrowing of the small airways and destruction of elastin in lung tissue. The pathogenesis and biochemical changes of these aging emphysema (senile emphysema) are very similar to those of common emphysema. Since emphysema (emphysema) is not reversible but irreversible as asthma or bronchitis, treatment is very difficult, and there is no therapeutic or prophylactic drug that can effectively treat emphysema.

In particular, elastin, which is a constituent of the extracellular matrix (ECM), is a very important protein that provides the gas exchange function of the alveoli with the necessary elasticity and elastic recoil. When the alveolar wall is damaged or injured due to reduction in production or acceleration in decomposition of elastin, the alveolar wall becomes unrecoverable and thus causes a problem of the dilatory action of alveoli, i.e., dyspnea, and finally, COPD progresses.

With COPD and the trend toward higher age-up becoming more important global problems, there is a problem whether COPD caused by aging can be actually treated and prevented. Until recently, it was known that the lungs were an organ that could not be restored or regenerated, and thus the possibility of treating or preventing COPD was recognized to be very small. However, since Butler et al in 2012 demonstrated that adult lung tissue is a re-regenerable organ, recent studies revealed, through cell biological evidence and mechanisms, that the lung epithelial cell group has the ability to self-differentiate and proliferate, and thus can restore or regenerate damaged or degenerated alveoli. With the advancement of regeneration and restoration techniques of degenerated alveoli, it is expected that therapeutic and prophylactic drugs will be more actively studied in the future.

As described above, COPD is a lung disease characterized by irreversible airflow limitation, which occurs and progresses due to damage to airway and lung parenchyma tissues caused by chronic inflammation, but which is known as a disease that can be prevented and treated as described above. Currently, the most commonly used COPD therapeutics worldwide include Inhaled Corticosteroids (ICS) and long-acting muscarinic antagonists (LAMA) as asthma therapeutics, and long-acting beta agonists (LABA) as chronic bronchitis and emphysema therapeutics, but these therapeutics do not completely treat COPD. In particular, since ICS is directly administered into the respiratory tract, it exerts a strong local anti-inflammatory effect. However, systemic side effects may increase when steroids administered in the airways are transported throughout the body from the alveoli to the blood. The side effects of these existing drugs may be anxious to the patient and in severe cases may lead to a refusal of administration, and there is a strong demand in the market for the development of drugs with less side effects.

Disclosure of Invention

Technical problem

In order to solve the above problems, the present disclosure provides a recombinant protein and an excellent composition for preventing or treating pulmonary diseases, which includes at least one selected from the group consisting of hyaluronic acid and proteoglycan connexin 1 (HAPLN 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.

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 the presented embodiments of the disclosure.

Technical scheme

The recombinant protein according to the present disclosure may be a recombinant hyaluronic acid and proteoglycan connexin 1 (HAPLN 1) protein having the amino acid sequence of SEQ ID NO: 1.

The pharmaceutical composition for preventing or treating pulmonary 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 that promotes the expression of HAPLN1 protein or gene or activates its function, as an effective ingredient.

The health functional food composition for preventing or improving 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 that promotes the expression of HAPLN1 protein or gene or activates its function, as an effective ingredient.

Advantageous effects

The composition has few side effects compared to existing therapeutic agents for pulmonary diseases, and thus can safely and effectively prevent or treat pulmonary diseases, which have been increasing year by year with the global trend toward aging.

It should be understood that the embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects in each embodiment should generally be considered as available for 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 therein without departing from the spirit and scope of the present invention as defined by the following claims.

Drawings

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

fig. 1A, 1B and 1C illustrate alveolar experimental results of an aged mouse according to an experimental example of the present disclosure, in which fig. 1A is an image observed under a microscope after staining an alveolar tissue, and fig. 1B and 1C are graphs in which alveolar density is quantified. Here, "Young-saline" refers to a group to which Phosphate Buffered Saline (PBS) is administered to a normal control group (Young group), "Old-saline" refers to a group to which PBS is administered to an Old control group (Old group), and "Old-rhHAPLN 1 (Old-rhHAPLN 1)" refers to a group to which rhHAPLN1 diluted in PBS is administered to an Old rhHAPLN1 treatment group (Old + rhHAPLN1 group).

Fig. 2 is a schematic view regarding a spray injection (atomization) manner according to an experimental example of the present disclosure.

Fig. 3A, 3B and 3C show experimental results of aerosol inhalation groups (AH group) according to an experimental example of the present disclosure, fig. 3A is an image obtained by staining alveolar tissues of each group and observing under a microscope, and fig. 3B and 3C are graphs of the results of measuring Mean Linear Intercept (MLI) values thereof.

Fig. 4A, 4B and 4C show experimental results of an endotracheal instillation group (TI group) according to an experimental example of the present disclosure, fig. 4A is an image obtained by staining alveolar tissues of each group and observing under a microscope, and fig. 4B and 4C are graphs of the results of measuring MLI values thereof.

Detailed Description

Reference will now be made in detail to the embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as limited to the description set forth herein. Accordingly, the embodiments are described below only to explain aspects of the present specification by referring to the figures. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. When an expression such as "at least one" precedes a list of elements, the entire list of elements is modified and individual elements of the list are not modified.

Hereinafter, the present disclosure will be described in detail.

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

In the present specification, "prevention" refers to any action of inhibiting or delaying the occurrence of a pulmonary disease or at least one symptom of said disease by administering a pharmaceutical composition or a health functional food composition according to the present disclosure. In addition, treatment of a subject who is in remission in the disease is included to prevent or prevent relapse.

In the present specification, "treatment" refers to any action to ameliorate or beneficially alter (e.g., alleviate, reduce or eliminate) a pulmonary disease or at least one symptom of the pulmonary disease by administering a pharmaceutical composition according to the present disclosure.

In the present specification, "improving" refers to any action that ameliorates or beneficially alters (e.g., alleviates, reduces or eliminates) a lung disease or at least one symptom of the lung disease by ingestion of a nutraceutical composition according to the present disclosure.

In the present specification, "pharmaceutical composition" refers to a composition that is administered for a specific purpose, and for the purposes of the present disclosure, means administered to prevent or treat a pulmonary disease or at least one symptom of the disease.

In the present specification, "health functional food" means a food including a food produced and processed by using a raw material or ingredient having a useful function for a human body according to the law No. 6727 related to health functional foods, and for the purpose of the present disclosure, means a food having a high medical and medical effect, which is processed so that bioregulatory functions, such as prevention or improvement of lung diseases, biological defense, immunity and recovery, etc., can be effectively exerted in addition to nutrient supply.

The present disclosure provides recombinant hyaluronic acid and proteoglycan linker 1 (HAPLN 1) having the amino acid sequence of SEQ ID NO: 1.

The "HAPLN1" is a protein that stabilizes hyaluronic acid by linking it to proteoglycan, and is a constituent protein in an extracellular matrix that is first found in joints of vertebrates.

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

The present disclosure provides a pharmaceutical composition for preventing or treating a pulmonary disease, the composition comprising 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.

In certain embodiments, the HAPLN1 proteins provided herein can comprise an amino acid sequence having at least 95% sequence identity to SEQ ID No. 1. In some of these embodiments, the HAPLN1 protein can 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 to the amino acid sequence of SEQ ID No. 1 described above. Also, the polypeptides disclosed in the present specification may include the peptide of SEQ ID No. 1, fragments thereof, and peptides in which at least one, two, three, four, five, six, or seven amino acids are modified.

As used herein, the term "identity" is a relationship between two or more polypeptide sequences, and is determined by comparing the sequences as appropriate. In the art, "identity" also means the degree of sequence relatedness between polypeptide sequences, and as the case may be, is determined by the match between strings of such sequences. "identity" can be readily calculated by known methods, for example, using BLAST (Basic Local Alignment Search Tool) version 2.2.1 software with default parameters, national Center for Biological Information: NCBI.

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

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

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

As used herein, the term "effective agent" refers to a HAPLN1 expression promoter that promotes expression of HAPLN1 protein or gene; or a HAPLN1 activator that activates the function of a HAPLN1 protein or gene. As used herein, effective agents include a variety of compounds, proteins or peptides, base sequences, etc., capable of enhancing expression of the HAPLN1 gene or HAPLN1 protein and/or fragments thereof, or activating the function of the HAPLN1 protein or gene. The active agents also include various metabolites, precursors, pharmaceutical equivalents of the compounds, proteins or peptides or base sequences.

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 injury.

The pharmaceutical compositions according to the present disclosure may be prepared according to conventional methods in the pharmaceutical art. The pharmaceutical composition may be mixed with a pharmaceutically acceptable suitable carrier according to the dosage form, in addition to the effective ingredient, and may further include excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, and the like, 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 may be variously selected depending on the administration form and dosage form.

The pharmaceutical composition according to the present disclosure may be applied in any dosage form, and in more detail, may be formulated according to a conventional method into oral dosage forms, external preparations, suppositories, and parenteral dosage forms of sterile injection solutions for use.

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

In the oral dosage form, the liquid dosage form corresponds to a suspension, an internal liquid, an emulsion, a syrup, and the like, and may include various excipients such as a wetting agent, a sweetener, an aromatic agent, a preservative, and the like, in addition to a simple diluent, i.e., water and liquid paraffin, which are generally used.

The parenteral dosage forms may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. As the 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 base of the suppository, witepsol, polyethylene glycol, tween 61, cacao butter, lauric acid glyceride (laurin button), glycerogelatin, and the like can be used. The present disclosure is not so limited and any suitable formulation known in the art may be used.

In addition, the pharmaceutical composition according to the present disclosure may further be added with calcium or vitamin D ₃ Etc. to improve the therapeutic efficacy.

Pharmaceutical compositions according to the present disclosure may be administered in a pharmaceutically effective amount.

In the present specification, a "pharmaceutically effective amount" refers to an amount sufficient to be suitable for the treatment of a disease at a reasonable benefit/risk ratio suitable for medical treatment without causing side effects.

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

The pharmaceutical compositions according to the present disclosure may be administered to any animal in which pulmonary disease may occur, and the animal may include, for example, humans and primates, as well as livestock such as cows, pigs, horses, and dogs.

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

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

Further, the present disclosure provides a health-care functional food composition for preventing or improving lung diseases, comprising 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.

In certain embodiments, HAPLN1 proteins provided herein can comprise an amino acid sequence having at least 95% sequence identity to SEQ ID No. 1. In some of these embodiments, the HAPLN1 protein can 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 to the amino acid sequence of SEQ ID No. 1 described above. Also, the polypeptides disclosed in the present specification may include the peptide of SEQ ID No. 1, fragments thereof, and peptides in which at least one, two, three, four, five, six, or seven amino acids are modified.

The recombinant HAPLN1 protein can have an effect of improving alveolar damage caused by aging or elastin reduction, and thus can be used as a health functional food composition for preventing or improving pulmonary diseases.

In the health functional food 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 injury.

In the health-care functional food composition according to the present disclosure, the health-care functional food may be prepared in the form of powder, granules, tablets, capsules, syrup, or beverage, etc. to prevent or improve pulmonary diseases, and the form of the food is not limited and may include all foods in the conventional sense. For example, the food may include beverages and various drinks, fruits and their processed foods (canned fruits, jams, etc.), fishes, meats and their processed foods (ham, bacon, etc.), breads and noodles, biscuits and snacks, dairy products (butter, cheese, etc.), and may include all functional foods in the general sense. In addition, food products for use as animal feed may also be included.

The health functional food composition according to the present disclosure may be prepared by further comprising a dietetically acceptable food additive and suitable other auxiliary ingredients generally used in the art.

Unless otherwise stated, the applicability of the Food additive may be determined by specifications and standards related to the respective items according to the General Rules and General Test methods (General Rules and General Test of the Korean Food Additives Code) of the Korean Food additive Code approved by the Korean Food and Drug Safety agency (Korean national institute of Food and Drug Safety). Items listed in the "food additive code" are, for example, synthetic additives such as ketones, glycine, potassium citrate, niacin, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sorghum pigment, guar gum, and the like; and mixture additives such as sodium L-glutamate, flour-like alkali additives, preservative agents, tar coloring agents, and the like.

The other auxiliary ingredients may further contain, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, colorants, pectic acids, alginic acids, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, and carbonating agents, and the like. In particular, as the natural carbohydrate, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, erythritol, and the like can be used. Examples of the sweetener may include natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame, and the like.

The effective dose of the recombinant HAPLN1 protein contained in the health functional food according to the present disclosure may be appropriately adjusted according to the purpose of use (such as prevention or improvement of lung diseases). Since the composition comprises food as an ingredient, there are advantages in that: it has no side effects which may occur when ordinary medicines are taken for a long time. Due to its excellent portability, it can be used as a supplement for preventing or improving lung diseases.

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

Further, the present disclosure may provide a method of preventing or treating a pulmonary disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition according to the present disclosure.

The subject is preferably a mammal, including a human and may be a patient in need of treatment for a pulmonary disease. The patients may include patients undergoing treatment for a pulmonary disease, patients in need of treatment for a pulmonary disease, and patients undergoing surgical treatment for a pulmonary disease. Administration of a pharmaceutical composition according to the present disclosure to an individual may result in the alleviation or treatment of pulmonary disease.

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

As used herein, the term "administering" refers to introducing a pharmaceutical composition according to the present disclosure into a subject using any suitable method. Routes of administration may include, but are 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 example, the pharmaceutical composition may be administered in the form of an aerosol or may be administered by instillation.

Administration of the pharmaceutical compositions according to the present disclosure may be by aerosol, which may be generated by a nebulizer, or by instillation. The pharmaceutical compositions may be administered alone or in combination with a carrier such as a saline solution, dimethylformaldehyde (DMSO), alcohol, or water. It may also be used as a vehicle for intratracheal administration of various agents, such as those that prevent degradation or promote re-synthesis of the elastic fiber. The effective daily amount of the composition is about 0.001mg/kg to 100mg/kg, preferably 0.01mg/kg to 10mg/kg body weight. The dosage administered is not intended to limit the scope of the present disclosure in any way. For example, the amount of the pharmaceutical composition administered intratracheally to a human per day can vary from about 0.001mg/kg to about 100mg/kg of body weight. Preferably, the daily amount is from about 0.01mg/kg to about 10mg/kg body weight of the subject (daily).

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

In addition, the pharmaceutical composition for preventing or treating pulmonary 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, may be administered simultaneously/sequentially in combination with other existing drugs for treating pulmonary diseases or existing treatment methods thereof. Such administration may be a single or multiple administrations. It is important to achieve the maximum effect in the minimum amount without side effects while taking all factors into consideration. The amount can be easily determined by a person skilled in the art.

< example 1> production of recombinant human HAPLN1 (rhHAPLN 1)

1. Amino acid sequence of recombinant human HAPLN1

The amino acid sequence constituting recombinant HAPLN1 is as follows.

2. Expression, purification and preservation of recombinant human HAPLN1 protein (rhHAPLN 1)

To prepare recombinant human HAPLN1 (rhHAPLN 1; gene No. 2678736), a DNA vector encoding the amino acid sequence of rhHAPLN1 was transfected into Expi as a host cell ^TM 293 cells (ThermoFisher Scientific Co., wolterm, mass., USA). For purification, a secretion signal peptide, 10 histidines (H, his), and TEV protease recognition sites were inserted into the amino terminus and expressed. Three days after transfection of the vector, the culture broth was collected and purified by HisTrap column (GE Healthcare, illinois, usa), followed by cleavage of TEV protease-recognition sequence by TEV protease and removal of molecules including histidine using DynaBeads (sequi feishell technology). The solution thus obtained was dialyzed against 40mM Tris-HCl and 1M NaCl at pH 8.0 for 16 hours. The final purified product had a protein concentration of 0.11mg/ml and was aliquoted to single doses using 20mM Tris-HCl, 0.5M NaCl pH 8.0, 50% glycerol as solvent and stored inAnd (4) keeping the mixture in a refrigerator at the temperature of minus 20 ℃ for standby.

< Experimental example 1> examination of alveolar structure improvement effect of aged mice by repeated intraperitoneal administration of recombinant human HAPLN1 protein (rhHAPLN 1)

1. Preparation and raising of laboratory animals

For the experimental animals, 2-month-old male C57BL/6J (Young Bio, korea) mice were set as Young (Young) mice, and 20-month-old male C57BL/6J mice were set as old (old) mice. The mice were allowed free access to water and feed, the temperature of the feeding room was maintained at 21 ℃ to 24 ℃, the humidity was maintained at 40% to 60%, and the day-night cycle was 12 hours, respectively.

The 5 young mice were divided into a normal control group (young group), and 5 old mice were assigned to each of an aged control group (aged group) and an aged rhHAPLN 1-treated group (aged + rhHAPLN1 group). In the aged rhHAPLN 1-treated group (aged + rhHAPLN1 group), rhHAPLN1 diluted in Phosphate Buffered Saline (PBS) was injected intraperitoneally (IP injection) at an administration rate of 0.1mg/kg in an amount of 80. Mu.l at a time, 3 times per week, for 3 weeks. The remaining two control groups were given the same amount of PBS in the same manner.

2. Staining and microscopic visualization of alveolar tissue

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

In addition, for each individual stained tissue section, mean Linear Intercepts (MLI) were calculated using the Image J program to quantify alveolar density. Higher MLI values indicate smaller alveolar surface area and are used as indicators showing 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 arbitrary three sites per mouse to obtain an average value, the values of 5 mice were subjected to statistical analysis using Sigma plot 12.0 and Graph Pad Prism 8. The results are shown in FIG. 1 (B).

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

< example 2> efficacy of recombinant human HAPLN1 protein (rhHAPLN 1) in Chronic Obstructive Pulmonary Disease (COPD) model induced by Porcine Pancreatic Elastase (PPE)

1. Preparation and raising of laboratory animals

Experimental animals female C57BL6/N (Young Bio, korea) mice of 6 to 10 weeks of age, which had body weights of 20 to 25g, were used, and the mice were divided into 5 mice per group. The mice were allowed to freely eat water and feed, the temperature of the feeding room was maintained at 21 to 24 ℃, the humidity was maintained at 40 to 60%, and the day-night periods were 12 hours, respectively.

2. Induction of Chronic Obstructive Pulmonary Disease (COPD)

Mouse models in which COPD is induced were prepared with reference to Suki et al (2017, methods Mol. Biol.1639) and Wright et al (2008, am J Physiol Lung Mol Physiol 295L 1-L15).

To inhale elastase into the lungs, first, porcine pancreatic elastase (EC 134; PPE) and administered once daily to each animal 6IU/30 μ l prior to drug treatment. Administration was divided into two groups according to the inhalation method.

For the aerosol inhalation group (AH), oral administration was carried out using a zonde needle (oral zonde needle) for oral administration of 20G (0.9X 50 mm). Administration can be by carefully stretching the mouth, pulling the tongue, and then dropping the elastase solution on its dorsal side (i.e., the far end of the oropharynx), while inducing inhalation by blocking both nostrils. For the intratracheal instillation group (TI), administration was by nasal drip induction of spontaneous inhalation by using a Ai Bende pipette. Body weights of mice were measured twice weekly, and each group was divided into 4 subgroups to administer the composition agents.

3. Potency of the composition medicament

The aerosol inhalation group (AH group) was subdivided 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 endotracheal instillation group (TI group) was subdivided 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 on COPD, and thus is used as a positive control. The administration and concentration of HA was determined with reference to Cantor et al (2005, experimental Lung Research, 31-430), and thus, hyaluronic acid sodium salt (Hyaluronic acid salt) (Sigma-Aldrich; st. Louis, usa, cat.73641-10 MG) from Streptococcus equi (Streptococcus equi) was purchased and used.

Starting on the first day after elastase administration, samples of each pharmaceutical composition were administered five times a week for nebulization using a Mass Dosing System, in particular an aerosol chamber (Data Science International) (Ball flow meter 3, aerosol 1 hour, work load 30%). A schematic diagram of the atomization is shown in fig. 2.

Using physiological saline as a solvent, each sample was atomized with a nebulizer having concentrations of 0.33% (w/v) rhHAPLN1 and 5.71% (w/v) HA, respectively, by adjusting the volume of a single administration to 6.8ml, the left lung was horizontally divided and the lower part was removed after cardiac perfusion under anesthesia the second day after the last spray administration on day 21, fixed with neutral buffered 10% formalin (NBF), and paraffin tissue sections were prepared so that the section of the upper lung sheet was visible. Tissue sections were stained with hematoxylin & eosin (H & E) method and photographed at 200 magnification using Ni-U (Nikon) microscope and DS-Ri1 (Nikon) digital camera to obtain alveolar tissue photograph data (scale bar =100 μm), and the MLI of alveoli was measured. Higher MLI values indicate smaller alveolar surface area and are used as indicators showing the degree of emphysema or COPD. The formula is Lm (μm) = number of intersections/(grid length (μm) × number of lines). After measuring arbitrary three sites per mouse to obtain an average value, the values of 5 mice were subjected to statistical analysis using Sigma plot 12.0 and Graph Pad Prism 8.

The results of the aerosol inhalation group (AH group) experiment are shown in fig. 3, and the results of the intratracheal instillation group (TI group) experiment are shown in fig. 4.

FIG. 3 is the result of the aerosol inhalation group (AH), and referring to FIGS. 3A to 3C, the 21.2 μm increase of PPE-treated group (AH-2) was about 1.5 times (p < 0.001) compared to the 13.8 μm increase of untreated normal group (AH-1). The decrease in rhHAPLN 1-treated group (AH-3) 14.5 μm by about 95% (p < 0.00002) compared to PPE-treated group 21.2 μm indicates that it was almost restored to a normal state.

In contrast, from a concentration point of view, the HA-treated group (AH-4) was 5.71% (w/v), about 17-fold higher than 0.33% (w/v), showing about a 73% reduction (p < 0.002), indicating that the positive control HA treatment was slightly less effective than rhHAPLN1 treatment (about a 95% reduction).

FIG. 4 is the experimental result of the intratracheal instillation group (TI group), and referring to FIGS. 4A to 4C, the PPE-treated group (TI-2) had an increase of 22.1 μm by about 1.6 times (p < 0.001) compared with the untreated normal group (TI-1) of 13.8 μm. The rhHAPLN 1-treated group (TI-3) was 16.1 μm, a reduction of about 72% (p < 0.0009) compared to the PPE-treated group (TI-2) 22.1 μm.

In contrast, from a concentration point of view, the HA-treated group (TI-4) was 5.71% (w/v), which was about 17 times higher than 0.33% (w/v), and showed about 64% reduction (p < 0.00123), indicating that the positive control HA treatment was slightly less effective than rhHAPLN1 treatment, which showed about 72% reduction.

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 or health functional food composition for preventing or treating pulmonary diseases such as chronic bronchitis, asthma, emphysema, chronic obstructive pulmonary disease, etc. caused by aging or elastin reduction.

The composition has few side effects compared to existing therapeutic agents for pulmonary diseases, so it can safely and effectively prevent or treat pulmonary diseases that increase year by year with the global aging trend.

It is to be understood that the embodiments described herein are to be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects in each embodiment should generally be considered as available for 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 therein without departing from the spirit and scope of the present disclosure as defined by the following claims.

Claims (10)

1. A pharmaceutical composition for preventing or treating pulmonary diseases, comprising at least one selected from the group consisting of a HAPLN1 (hyaluronic acid and proteoglycan connexin 1) protein, a gene encoding the HAPLN1 protein, and an effective agent that promotes the expression of or activates the function of the HAPLN1 protein or gene as an effective ingredient.

2. The pharmaceutical composition for preventing or treating pulmonary diseases according to claim 1,

the HAPLN1 protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID No. 1.

3. The pharmaceutical composition for preventing or treating pulmonary disease of claim 1, wherein

The HAPLN1 protein ameliorates alveolar damage caused by aging or elastin reduction.

4. The pharmaceutical composition for the prevention or treatment of a pulmonary disease according to claim 1,

the lung disease is one or more selected from the group consisting of chronic bronchitis, asthma, emphysema and Chronic Obstructive Pulmonary Disease (COPD).

5. A health functional food composition for improving or preventing pulmonary diseases, comprising at least one selected from the group consisting of HAPLN1 (hyaluronic acid and proteoglycan connexin 1) protein, a gene encoding HAPLN1 protein, and an effective agent that promotes the expression of or activates the function of HAPLN1 protein or gene as an effective ingredient.

6. The health functional food composition for ameliorating or preventing pulmonary diseases according to claim 5, wherein the HAPLN1 protein ameliorates alveolar damage caused by aging or elastin reduction.

7. The health functional food composition for ameliorating or preventing pulmonary diseases according to claim 5, wherein,

the lung disease is one or more selected from the group consisting of chronic bronchitis, asthma, emphysema and Chronic Obstructive Pulmonary Disease (COPD).

8. A method of preventing or treating a pulmonary disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 1.

9. The method of claim 8, wherein,

the HAPLN1 protein ameliorates alveolar damage caused by aging or elastin reduction.

10. The method of claim 8, wherein,

the lung disease is one or more selected from the group consisting of chronic bronchitis, asthma, emphysema and Chronic Obstructive Pulmonary Disease (COPD).