PHARMACEUTICAL COMPOSITION COMPRISING PIBF PROTEIN AS ACTIVE INGREDIENT FOR PREVENTION OR TREATMENT OF INFLAMMATORY DISEASE

Abstract

The present invention relates to a pharmaceutical composition comprising a progesterone-induced blocking factor (PIBF) protein as an active ingredient for prevention or treatment of inflammatory disease. In the present invention, menopausal asthma animal models have been experimentally identified to decrease airway inflammation when treated with PIBF protein. Thus, the composition can be advantageously used in postmenopausal female asthma patients.

Claims

1-9. (canceled)

10. A method of preventing or treating an inflammatory disease, comprising: administering or dosing a composition comprising a progesterone-induced blocking factor (PIBF) protein as an active ingredient to a subject.

11. The method of claim 10, wherein the composition is used to prevent or treat an inflammatory disease in postmenopausal women.

12. The method of claim 10, wherein the PIBF protein has a molecular weight of 25 to 45 kDa.

13. The method of claim 10, wherein the inflammatory disease is asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivity, inflammatory bowel disease, pelvic inflammatory diseases, rheumatoid arthritis, celiac disease, sarcoidosis, interstitial cystitis, or vasculitis.

14. The method of claim 10, wherein the composition further comprises one or more drugs selected from the group consisting of an antiinflammatory agent, a bronchodilator, an antihistamine, a decongestant, and an antitussive.

15. The method of claim 10, wherein the composition is formulated into the form of an oral formulation, a preparation for external use, a suppository, or a sterile injectable solution.

Description

DESCRIPTION OF DRAWINGS

[0035] FIG. 1 is a schematic diagram showing an animal model protocol for identifying the relationship between female hormones and clinical types of asthma.

[0036] FIG. 2 shows the results of analyzing inflammatory cells in a bronchoalveolar lavage fluid in an animal model of postmenopausal female asthma.

[0037] FIG. 3 shows the results of comparing levels of inflammation in lung tissue and changes in tissues in asthma model mice.

[0038] FIG. 4 shows the results of analyzing an effect of airway inflammation in an asthma model using PIBF gene half-deficient transgenic mice.

[0039] FIG. 5 shows the results of comparing levels of inflammation in lung tissue and changes in tissues in the PIBF gene half-deficient transgenic mice.

[0040] FIG. 6 is a schematic diagram showing a protocol for determining an effect of PIBF in an asthma animal model.

[0041] FIG. 7 shows the results of the influx of inflammatory cells into a bronchoalveolar lavage fluid and lung tissue through PIBF treatment.

[0042] FIG. 8A depicts the polynucleotide sequence of a recombinant DNA construct described by SEQ ID NO: 1. The polynucleotide sequences of the sfiI (GGCCACAGCGGC, SEQ ID NO: 6), NheI (TTGGCCGCGTCGGCC, SEQ ID NO: 7) and XhoI (CTCGAG) restriction sites are also depicted. The two stop codons TGATAG are shown at the end of SEQ ID NO: 1 upstream from the XhoI site. SEQ ID NO: 4 corresponds to nucleotides 26 to 919 of SEQ ID NO: 1. This DNA construct encodes PIBF1+FC.

[0043] FIG. 8B depicts the amino acid sequence of SEQ ID NO: 2 which is encoded by the coding sequence of SEQ ID NO: 1.

[0044] FIG. 8C depicts the amino acid sequence of SEQ ID NO: 3.

BEST MODE

[0045] Hereinafter, the present invention will be described in further detail with reference to embodiments thereof. These and other objects, features and advantages of the present invention will be readily understood with reference to the following embodiments. Therefore, it should be understood that the present invention may be embodied in various forms, but is not intended to limit the embodiments disclosed herein. In this case, the embodiments disclosed herein are provided to sufficiently convey the spirit of the present invention to those skilled in the art to which the present invention pertains. Therefore, it will be apparent that the following embodiments are not intended to limit the present invention.

[0046] According to the present invention, an anti-asthmatic inflammation effect of a 35 kDa PIBF protein has been confirmed in a female asthma mouse model in which menopause was induced through the following Examples. To date, focus has been mainly made on the pattern of inflammatory cells in the study on an asthma mechanism, or made on the study of the role of the inflammatory cells, and action mechanisms of several inflammatory cytokines. Based on the clinical diversity of asthma, the development of customized therapeutic agents according to clinical subtypes of asthma was recently proposed as an ideal direction for treatment of asthma. Asthma developing in elderly women after menopause is a unique clinical subtype, and thus elderly women urgently require customized treatment for this subtype. In this case, the present invention may be the most suitable technique. Also, the greatest advantage of the present invention is to avoid the use of steroids having a high risk of side effects in elderly female asthma patients.

EXAMPLES

1. Experimental Methods

[0047] 1-1: Analysis of Inflammatory Cells and Lung Tissues in Bronchoalveolar Lavage Fluid in Postmenopausal Female Asthma Animal Model

[0048] To establish an animal model reflecting postmenopausal female asthma, 6-week-old female C57BL/6 mice (ORIENT BIO Inc.) were subjected to ovariectomy to induce menopause. After stabilization, an acute allergic asthma animal model was induced. To determine an effect of a female hormone on the clinical type of allergic asthma in this model, a female hormone was periodically injected as shown in the protocol of FIG. 1 to compare and evaluate the effects of the female hormone. On day 24 of establishment of the model, the mice were peritoneally anesthetized and thoracically incised. Thereafter, blood was collected from the heart using a 26 gauge syringe so that blood samples were taken simultaneously with euthanasia. Then, a catheter was inserted into the airway, and a bronchoalveolar lavage fluid (BALF) was obtained using 2 mL PBS and a syringe.

[0049] Next, after reperfusion with physiological saline, lung tissue was obtained. In this case, left lung was fixed in 10% formalin to perform a histopathological test, and the remainder was set aside for extraction of proteins and RNA and stored at −80° C. The total number of cells in the BALF was calculated using a hemocytometer. The cells were immediately cytospun, stained with Diff-Quik, and then differentially counted. The lung tissue was fixed for a day, embedded in paraffin melted at a temperature of 61° C. or less, and then cut into slides having a thickness of 4 μm using a sliding microtome. The observation of inflammation in the lung tissue was confirmed through H&E staining. An acute asthma animal model was induced as follows. On days 0 and 14, 20 μg of ovalbumin and 2 mg of alum were mixed with PBS to prepare 100 μL of the mixture. The mixture was intraperitoneally administered to induce systemic sensitization to OVA. On days 21, 22, and 23, 5% OVA was inhaled into the airway using a nebulizer to establish an animal model.

[0050] 1-2: Analysis of Effect of Airway Inflammation in Asthma Model Using PIBF Gene Half-Deficient Transgenic Mice

[0051] To confirm an effect of PIBF induced by the female hormone on a phenotype of asthma, a postmenopausal female asthma animal model was induced, and normal mice and PIBF half-deficient mice were treated with the female hormone. Thereafter, the influx of cells into a bronchoalveolar lavage fluid was analyzed. This experiment was performed in the same manner as in Example 1-1, except that the PIBF half-deficient mice were used.

[0052] 1-3: Analysis Results of Inflammatory Cell Influx in Bronchoalveolar Lavage Fluid and Lung Tissue Through PIBF Treatment

[0053] An acute asthma animal model was established in the normal mice and the mice in which menopause was induced by ovariectomy, and treated with PIBF (a 35 kDa PIBF1 recombinant protein: SEQ ID NO: 1) as shown in the protocol of FIG. 6 to determine an effect of the PIBF. This experiment was performed in the same manner as in Example 1-1, except that the mice were treated with PIBF as shown in the protocol of FIG. 6. The PIBF recombinant protein has a form in which FC is bound to PIBF and was prepared by commissioning Y-Biologics Inc. The prepared recombinant protein had a polypeptide sequence encoded by the base sequence set forth in SEQ ID NO: 1 (FIG. 8A, PIBF1+FC) or an amino acid sequence set forth in SEQ ID NO: 2 (FIGS. 8A and 8B, PIBF1+FC).

2. Experimental Results

[0054] 2-1: Results of Analysis of Inflammatory Cells and Lung Tissues in Bronchoalveolar Lavage Fluid in Postmenopausal Female Asthma Animal Model

[0055] In the mice in which asthma was induced after the induction of menopause, the infiltration of inflammatory cells in the airway tended to increase compared to the normal mice. In this case, it was confirmed that the airway inflammation decreased again in the group treated with the female hormone (FIG. 2).

[0056] From the results of histological analysis, it was confirmed that the infiltration of inflammatory cells in the lung tissue increased in the mice having undergone the ovariectomy, compared to the normal mice, and then decreased when the mice were again treated with the female hormone (FIG. 3).

[0057] 2-2: Effect of Airway Inflammation in Asthma Model Using PIBF Gene Half-Deficient Transgenic Mice

[0058] As shown in FIG. 4, it was confirmed that the influx of inflammatory cells increased in the PIBF half-deficient mice. From the results of lung tissue analysis, in the mice in which asthma was induced among the PIBF half-deficient mice, it was also confirmed that the airway wall became thicker even when the mice were treated with the female hormone, and the infiltration of inflammatory cells increased, compared to the normal mice (FIG. 5).

[0059] 2-3: Inflammatory Cell Influx Results in Bronchoalveolar Lavage Fluid and Lung Tissue Through PIBF Treatment

[0060] From the results of analysis, it can be seen that airway inflammation worsened when asthma was induced in the mice in which menopause was induced, compared to the normal mice. In this case, it was confirmed that inflammation decreased in a dose-dependent manner when the mice were treated with PIBF. On the other hand, it was confirmed that no effect of decreasing inflammation was observed in an animal model in which asthma was induced in the normal mice, and tissue findings also reflected the same pattern (FIG. 7).

MODE FOR INVENTION

Preparation Examples

Preparation Example 1: Preparation of Pharmaceutical Formulation

[0061] 1-1. Preparation of Powder

TABLE-US-00001 PIBF protein of the present invention 0.1 g Lactose 1.5 g Talc 0.5 g

[0062] The above-described components were mixed, and filled in an airtight bag to prepare a powder.

[0063] 1-2. Preparation of Tablet

TABLE-US-00002 PIBF protein of the present invention 0.1 g Lactose 7.9 g Crystalline cellulose 1.5 g

[0064] The above-described components were mixed, and then subjected to a direct tableting method to prepare a tablet.

[0065] 1-3. Preparation of Capsule

TABLE-US-00003 PIBF protein of the present invention 0.1 g Corn starch 5 g Carboxy cellulose 4.9 g

[0066] The above-described components were mixed to prepare a powder, and the powder was then filled in a hard capsule according to a conventional method of preparing a capsule to prepare a capsule.

Preparation Example 2: Preparation of Health Foods

[0067] 2-1. Preparation of Flour-Based Food

[0068] 0.5 to 5.0 parts by weight of the PIBF protein of the present invention was added to wheat flour, and mixed. Then, the resulting mixture was used to prepare bread, cakes, cookies, crackers, and noodles.

[0069] 2-2. Preparation of Dairy Products

[0070] 5 to 10 parts by weight of the PIBF protein of the present invention was added to milk, and the resulting milk was used to prepare various dairy products such as butter and ice cream.