TWI726904B - Medical uses of fungal immunomodulatory proteins in treatment and prophylaxis of alveolar bone loss due to periodontal disease - Google Patents

Abstract

The invention generally related to new medical uses of fungal immunomodulatory proteins. More particularly, the invention relates to using a fungal immunomodulatory protein and compositions containg a fungal immunomodulatory protein for inhibition of osteoclastogenesis, inhibition of bone resorption, and prevention and treatment of bone loss-associated diseases.

Description

Fungal immunomodulatory protein in the treatment and prevention of alveolar caused by periodontal disease Medical uses for bone loss

The present invention generally relates to new medical uses of fungal immunomodulatory proteins. More specifically, the present invention relates to the use of fungal immunomodulatory protein and a composition containing fungal immunomodulatory protein to inhibit the regeneration of osteoclasts, inhibit bone resorption, and prevent and treat bone loss-related diseases.

Ganoderma ( Ganoderma ) is a rare and precious Chinese medicinal material. It has been called "Ganoderma" in China for more than 5000 years. Various species of Ganoderma include: Ganoderma lucidum ( G. lucidum; red), Ganoderma lucidum ( G. applanatum ; brown), pine fir Ganoderma ( G. tsugae ; red-brown), and sweet lucid (G. sinense ; black) And Oregon Ganoderma ( G. oregonense ; dark brown).

Ganoderma has been known to have anti-allergic properties (Chen HY et al. , J. Med. Mycol. 1992; 33: 505-512) and liver protection (Lin JM et al. , Am J Chin Med. 1993; 21(1): 59 -69), anti-cancer (Wasser SP, Crit Rev Immunol 1999.19:65-96) and immunity enhancement (Kino, J Biol.Chem. 1989.264(1):472-8). However, the use of Ganoderma lucidum is still limited to crude extracts (Horner WE et al. , Allergy 1993; 48: 110-116) or small molecule extracts (Kawagishi H., et al. , Phytochemistry 1993; 32: 239-241).

Several proteins derived from edible fungi such as Ganoderma lucidum , Volvariella volvacea and Flammulina velutipes have similar amino acid sequences and immunomodulatory functions. These proteins are named fungal immunomodulatory proteins (FIPs, Ko JL, Eur. J. Biochem. 1995; 228: 224-249).

In 1989, Kino et al. discovered a protein named Ling Zhi-8 (LZ-8) from Ganoderma lucidum (Kino K. et al. , J. Biol. Chem. 1989; 264(1): 472-8). LZ-8 has a positive effect on systemic allergic reactions, and can be used to treat liver cancer and prevent diabetes. LZ-8 and another fungal immunomodulatory protein FIP-fve found from Flammulina velutipes have amino acid sequences and folding structures similar to immunoglobulin heavy chains. In addition, it has been shown that enhancing the expression level of LZ-8 will show immunomodulatory activity, and has a good effect on patients suffering from systemic allergic reactions (Ko JL, Eur. J. Biochem. 1995; 228: 224-249 ). Further research pointed out that FIP can activate human peripheral blood mononuclear cells (HPBMCs, human peripheral blood mononuclear cells), and promote the proliferation of HPBMCs and mouse splenocytes (van der Hem, et al. , Transplantation , 1995; 60,438-443) . Using tritium-calibrated thymidine ( ³ H-thymidine) to analyze the effect of FIP-gts on cell proliferation, it was further found that 5μg/ml FIP-gts or 100μg/ml FIP-fve was compared with phytohemagglutinin (PHA). , phytoagglutinin) is sufficient to allow human lymphocytes to reach the maximum rate of proliferation. For the two populations of non-B and non-T cells, studies have found that FIP- gts can only promote the proliferation of non-B cells.

Similar to lectin and other lectin mitogens, LZ-8 promotes cell division. LZ-8 mainly proliferates T-cells with the help of monocytes. A new family of fungal immunomodulatory proteins (FIPs) has recently been identified (Ko JL et al. Eur J Biochem 1995; 228(2):244-249). From Ganoderma lucidum , Flammulina veltipes , Volvariella volvacea , Ganoderma tsugae , Ganoderma japoncium , Ganoderma microsporum , Ganoderma microsporum, Ganoderma sinense At least 10 FIPs have been identified and isolated from Nectria haematococca (Nectria haematococca), Tremella fuciformis ( Tremella fuciformis ) and Antrodia camphorate (Antrodia camphorate), and they were named LZ-8 (also known as FIP- glu ). , FIP- fve , FIP- vvo , FIP- gts , FIP- gja (GenBank: AY987805), FIP-gmi , FIP- gsi , FIP- nha , FIP- tfu (GenBank: EF152774) and FIP- aca (Hsu HC, et al. , Biochem J 1997; 323(Pt 2): 557-565; Kong et al. , Int. J. Mol. Sci. 2013; 14: 2230-2241; Han et al. , J Appl Microbiol 2010,109 : 1838-44; U.S. Patent No. 7,531,627; and Chinese Patent No. 102241751B). FIPs are mitogenic substances for human peripheral blood lymphocytes (hPBLs) and mouse spleen cells in vitro. The bell-shaped dose-response curve they cause is similar to that of lectin-like cytokines. The activation of hPBLs by FIPs leads to increased production of molecules such as IL-2, IFN-γ, and tumor necrosis factor-α, and is related to the performance of ICAM-1 (Wang PH, et al. , J Agric Food Chem 2004; 52( 9): 2721-2725.). FIPs can also be used as immunosuppressive agents. In vivo, these proteins can prevent allergic reactions throughout the body, and can significantly reduce foot pad edema during the Arthus reaction in rats. These observations show that FIPs can promote health and have curative effects.

Lin et al. purified an immunomodulatory protein from Ganoderma tsugae mycelium and named it FIP- gts (Lin, WH, et al., J Biol. Chem. 1997.272, 20044-20048.). Only purified from the mycelium of Ganoderma tsugae FIP- gts immunomodulatory effect, fruiting body of Ganoderma tsugae and purified FIP- gts had no effect. After the FIP- gts gene was cloned, it was found that its DNA sequence was the same as the LZ-8 found in Ganoderma lucidum. Both molecules have immunomodulatory effects, showing that they are the same protein.

Using Garnier analysis to predict the secondary structure of FIP-gts , it was found that this protein has two α-helices, seven β-sheets and one β-turn. According to SDS-PAGE analysis, the molecular weight of FIP- gts is 13kDa. Amino acid binding analysis with 20μM glutaraldehyde revealed that FIP- gts is a homodimer composed of two identical subunits with a molecular weight of 26kDa.

It is known that bone is a dynamic tissue that is continuously remodeled by osteoblasts and osteoclasts. Osteoblasts produce and secrete matrix proteins, and transport minerals to the matrix, resulting in Osteogenesis, osteoeclasts perform a bone resorption process, disintegrating bone tissue. Therefore, the balance between the bone resorption process caused by osteoclasts and the bone formation process caused by osteoblasts maintains the level of bone. On the contrary, the imbalance between these two processes may lead to many bones. Related diseases, such as osteoporosis, osteopetrosis, osteogenesis imperfect, Paget's disease, rheumatoid arthritis, periodontal Disease (periodontal disease), osteosarcoma (osteosarcoma) and cancer bone metastasis (cancer bone metastasis). Osteoclasts are special multinucleated cells, which are formed by the fusion of preosteoclasts derived from cells belonging to the bone marrow monocyte/macrophage cell line. An abnormal increase in the number of osteoeclasts and/or excessive activation may cause bone loss. Nuclear factor activated B cell κ light chain enhancer (NF-κB) receptor activator (RANK) and its natural ligand (RANKL) are necessary regulators for the differentiation of the precursor cells of osteoclasts into mature osteoclasts. It implies that drugs targeting RANKL-RANK and its signaling pathways may be suitable for the treatment of bone loss-related diseases (Wada T. et al. , Trend Mol Med , 2006; 12(1): 17-25).

Among the above-mentioned bone loss-related diseases, periodontal disease has been recognized as a major public health problem worldwide, and it is the most common cause of tooth loss in adults. Bacterial invasion is decisive for the occurrence of periodontal disease. Bacteria can also cause the host tissues to exhibit an immune inflammatory response, and this process may lead to alveolar bone resorption, loss of connective tissue and the formation of periodontal sacs, and eventually tooth loss. Studies have shown that in the rat periodontitis model induced by threading, the resorption of alveolar bone can be reduced by inhibiting the signal transmission of RANK/RANKL (Jin Q. et al. , J Periodontol 2007; 78( 7): 1300-1308; Crotti T., et al. , J. Periodontal Res. 2003; 38(4): 380-387; and Lu SH et al. , Evid Based Complement Alternat Med , 2013; Article ID 634095) These results suggest that RANKL plays an important role in periodontal bone resorption, and inhibition of RANKL can suppress periodontal bone resorption, thereby preventing alveolar bone loss and curing periodontal disease.

Studies have pointed out that some inhibitors against the maturation and activity of osteoclasts can prevent or treat bone loss-related diseases (for retrospective papers, please refer to Kim SHand Moon, S.-H., Expert Opin. Ther. Patents , 2013; 23(12): 1591-1610). For example, bisphosphonates can bind to bones and prevent osteoeclasts from removing calcium. It has been found to be very effective in treating osteoporosis. Therapeutic antibodies against RANKL, such as denosumab, have been shown to be effective in preventing and treating osteoporosis. Magnolol (magnolol) is a biologically active plant component isolated from the root and bark of Magnolia officinalis . It has been reported to be effective in inhibiting the regeneration of osteoeclasts, and thus is useful in the treatment of periodontal disease. It is also valid (Lu SH et al., the paper ibid.).

Although the immunomodulatory activity of FIPs has been extensively studied, its effect on bone metabolism is still unclear. In the applicant's PCT application PCT/CN2015/074059, it is described that FIP- gts and FIP-fve can be used to reduce the side effects such as bone loss caused by the chemotherapy drug European paclitaxel (Docetaxel). However, other studies have shown that European paclitaxel itself inhibits the production and activity of osteoeclasts, thereby inhibiting bone resorption (Takahashi M. et al. , J Bone Miner Metab. 2009; 27(1): 24-35 ), which indicates that the bone loss caused by European paclitaxel is not caused by the over-production of osteoclasts, and it is impossible for FIP to improve the side effects of the drug by inhibiting the regeneration of osteoclasts but through other mechanisms. Other studies even pointed out that FIP will slightly increase the number of osteoeclasts in the rabbit sinus floor animal model (Hsu et al. , J Oral Maxillofac Surg , 72(9), 2014; 1703e1-e10). These studies have not shown that FIP is suitable for the prevention or treatment of bone loss-related diseases.

Therefore, there is a need for new medical compositions, medical methods, and medical uses for the prevention or treatment of bone loss-related diseases.

People described below, the invention application FIP -gts, FIP- gmi and FIP- vvo is found to be representative FIP, FIP front osteoclasts co-cultivation, will be suppressed in a dose-dependent manner by the etching induced RANKL Osteocyte formation and osteoecotic bone resorption. The inventors further discovered that FIP- gts inhibits alveolar bone resorption in the rat periodontal disease model induced by threading. Based on these findings, this specification proposes new medical applications for diseases and conditions related to bone loss such as periodontal disease.

In the first aspect, the present application provides a method for inhibiting the renewal of osteoclasts in a patient, which comprises adding an effective amount of fungal immunomodulatory protein (FIP) sufficient to inhibit the renewal of osteoclasts. Give to the patient.

In the second aspect, the present application provides a pharmaceutical composition for inhibiting the neonatal action of osteoclasts, which contains an effective amount of fungal immunomodulatory protein (FIP) sufficient to inhibit the neonatal action of osteoclasts, and a pharmaceutical composition Acceptable carrier or diluent.

In the third aspect, the present application provides the use of a fungal immunomodulatory protein (FIP) in the manufacture of a medicine, which is used to inhibit the renewal effect of osteoclasts in a patient.

In the fourth aspect, this application provides a method for treating a patient suffering from a bone loss-related disease, which comprises administering an effective amount of fungal immunomodulatory protein (FIP) sufficient to inhibit bone loss. The patient. In a preferred embodiment, the bone loss-related disease is periodontal disease.

In the fifth aspect, this application provides a method for preventing a bone loss-related disease from occurring in a patient who is prone to the disease, which comprises adding an effective amount of fungal immunomodulatory protein sufficient to inhibit bone loss (FIP) to the patient. In a preferred embodiment, the bone loss-related disease is periodontal disease.

In the sixth aspect, this application provides a pharmaceutical composition for preventing or treating a bone loss-related disease, which contains an effective amount of fungal immunomodulatory protein (FIP) sufficient to inhibit bone loss, and a pharmaceutical composition Acceptable carrier or diluent. In a preferred embodiment, the bone loss-related disease is periodontal disease.

In the seventh aspect, this application provides the use of a fungal immunomodulatory protein (FIP) in the manufacture of a medicine for the treatment of a patient suffering from a bone loss-related disease. disease. In a preferred embodiment, the bone loss-related disease is periodontal disease.

In the eighth aspect, this application provides the use of a fungal immunomodulatory protein (FIP) in the manufacture of a medicine, which is used to prevent a bone loss-related disease from occurring in a person prone to the disease patient. In a preferred embodiment, the bone loss-related disease is periodontal disease.

In a ninth aspect, this application provides a method for inhibiting bone resorption in a patient, which comprises administering to the patient an effective amount of fungal immunomodulatory protein (FIP) sufficient to inhibit bone resorption .

In a tenth aspect, this application provides a pharmaceutical composition for inhibiting bone resorption in a patient, which contains an effective amount of fungal immunomodulatory protein (FIP) sufficient to inhibit bone resorption, and a pharmaceutical composition Acceptable carrier or diluent.

In the eleventh aspect, this application provides the use of a fungal immunomodulatory protein (FIP) in the manufacture of a medicine for inhibiting bone resorption in a patient.

The above and other objectives, features and effects of the present invention will become apparent with reference to the description of the following preferred specific examples together with the accompanying drawings. Among them: Figures 1A-1C are bar graphs showing the administration of 2.5 to 10 μg/ml FIP is not toxic to pre-osteocytes; Figures 2A-2C are bar graphs, showing that FIP inhibits the differentiation of RANKL-induced pre-osteocytes into osteoclasts in a dose-dependent manner, where the symbol ^* represents relative to the control containing only medium The group showed P <0.05, and the symbol ^** represents P <0.01 relative to the control group, and the data is expressed as mean±standard deviation; Figure 3A shows the histological image of mature osteoeclasts stained with TRAP, which shows anterior erosion The morphological changes of bone cells after being treated with RANKL(R) and different concentrations of FIP- gts ; Figure 3B is a bar graph showing that in the presence of 0-10μg/ml FIP- gts , RNAKL induced The number of osteoclasts (OC) calculated in the differentiation of osteoclasts is compared with the untreated control group; Figure 4 shows the experimental procedure used in the in vitro model. The cells are pre-treated with RANKL and then treated with RANKL and FIP. -gts co-treatment (left picture), the histogram shows that FIP- gts has an inhibitory effect on RANKL-induced osteoclastogenesis (right picture), where the symbol ^** represents P <compared to the control group containing only medium 0.01, and the symbol ^*** represents P <0.001 relative to the control group, and the data is represented by the mean ± standard deviation; Figure 5A shows the setting of the ties in the rat model, where the root bifurcation area is marked by the symbol ◎ The area around the line is marked with the symbol ☆, the area close to the tie is marked with the symbol ￥, and the area away from the tie is marked with the symbol ※; Figure 5B shows the microcomputer obtained by FIP-gts when the tie is set in the rat mode Tomography (micro-CT) image; Figure 5C shows the CEJ-ABC values measured in four areas near the tie line in the rat model of Example 5; Figure 5D shows the proximal surface of the second molar in the rat model of Example 5 passing by The histological image of hematoxylin and eosin staining. The positions of the enamel junction (CEJ) and the alveolar crest (ABC) are marked by white and black arrows, respectively; Start giving FIP- gts the microcomputer tomography image obtained; Figure 6B shows the CEJ-ABC values measured in the four areas near the tie line of the rat model of Example 6; Figure 6C shows the second molar tooth of the rat model of Example 6 The histological image of the proximal surface stained with hematoxylin and eosin. The positions of the enamel junction (CEJ) and the alveolar crest (ABC) are marked by white and black arrows, respectively; Figure 7 shows the presence or absence of In the presence of FIP- gts , the fluorescent image of the actin ring formed in RANKL-induced osteoclast cell culture; Figure 8A shows the presence or absence of FIP- gts in the bar In this case, the lacuna formation image of the osteoclast cell culture induced by RANKL; and Figures 8B and 8C are histograms showing the bone erosion induced by RANKL in the presence or absence of FIP- gts . The area and number of pits formed in cell culture.

Unless otherwise stated, the following terms used in this specification and each claim have the definitions given below. Please note that the singular term "一" used in the description of this case and each claim is intended to cover one and more than one item, such as at least one, at least two, or at least three, rather than implying only a single item. A contained matter. In addition, the open connectives such as "include" and "have" used in each claim in this case mean that the combination of elements or components described in the claim does not exclude other elements or components that are not specified in the claim. It should also be noted that the term "or" generally also includes "and/or" in its meaning, unless the content clearly indicates otherwise.

The term "fungal immunomodulatory protein" or abbreviated as "FIP" as used in this application refers to the similarity between the amino acid sequence and the immune response effect, which is first defined in Ko et al. , Eur.J.Biochem. 1995;228: A protein in the protein family of 244-249. It has been reported that the immunomodulatory proteins of the FIP family share at least 57% sequence identity. In particular, the primary structures of FIP-gts , FIP-fve , FIP-vvo and LZ-8 exhibit 60-70% sequence identity (Kong et al. , Int.J.Mol.Sci. 2013,14,2230-2241 ; Chinese Patent No. CN102241751B; Wang XF et al. , Curr. Topics Nutraceutical Res. 2012, 10(1): 1-12; and Li OZ et al. , Crit. Rev. Biotech. 2011, 31(4): 365-375). Therefore, the term "fungal immunomodulatory protein" used in this application is intended to cover the amino acid sequence of FIP-gts shown in SEQ ID NO. 1 having at least 57%, preferably at least 60%, at least 70%, at least Any polypeptide that is 80%, at least 90%, or at least 95% amino acid identical and has the ability to initiate, enhance, or extend an individual's immune response. In a preferred embodiment, the fungal immunomodulatory protein has an amino acid sequence selected from the group consisting of: sequence identification number 1 (FIP- gts ), sequence identification number 2 ( FIP-fve ), serial identification number 3 ( FIP-vvo ), serial identification number 4 (LZ-8), serial identification number 5 ( FIP-gja ), serial identification number 6 (FIP- gmi ), Sequence ID No. 7 ( FIP-gsi ) and Sequence ID No. 8 ( FIP-nha ), and their functional variants as immunomodulators. In another preferred embodiment, the FIP is derived from Ganoderma or Straw mushroom, especially having an amino acid sequence selected from the group consisting of: sequence identification number No. 1, sequence identification No. 2, Serial ID No. 3, Serial ID No. 4, Serial ID No. 5, Serial ID No. 6, and Serial ID No. 7. More preferably, the FIP has an amino acid sequence selected from the group consisting of Sequence ID No. 1, Sequence ID No. 2, Sequence ID No. 3, and Sequence ID No. 4. The best one includes the amino acid sequence of SEQ ID No. 1, basically consisting of the amino acid sequence of SEQ ID No. 1, or the amino acid sequence of SEQ ID No. 1.

The FIP used in this application can be obtained from natural sources, fungal cultures, or recombinantly expressed in prokaryotic or eukaryotic microbial hosts such as bacteria or yeast hosts. The FIP thus obtained may be in the form of a crude product, or in the form of a refined formulation separated, fractionated, or partially or substantially purified from the fungal substance by any suitable technique. Preferably, the protein is at least partially purified before use. One method available for preparing FIP is disclosed in WO2005/040375A1, and the method involves culturing a transformed yeast strain carrying an FIP gene expression vector, and collecting recombinant FIP protein from the yeast culture. Other available methods can be found in, for example, Kong et al. (supra) and CN101205553A; and Wang XF et al. , Curr. Topics Nutraceutical Res. 2012; 10(1): 1-12.

When the FIP used is LZ-8 or FIP- gts , the FIP can be directly isolated from Ganoderma lucidum or Pine Fir Ganoderma lucidum, or prepared by recombinant protein technology in a host cell system. The host cell can be a yeast or a bacterial system. Preferably, the host cell system is selected from the group consisting of Saccharomyces cerevisiae , Pichia pastoris , Hansenula polymorpha , Candida utilis , Boyd Candida (Candida boidinii), maltose Candida (Candida maltose), lactose Kluyveromyces (Kluyveromyces lactis), Yarrowia lipolytica yeast (Yarrowia lipolytica), Schwanniomyces occidentalis (Schwanniomyces occidentalis) , Schizosaccharomyces pombe (Schizosaccaromyces pombe), Torulopsis (Torulopsis sp.), yeast terrestrial (Arxula adeninivorans), Aspergillus (Aspergillus sp.) (e.g. A. nidulans (the A. nidulans), Aspergillus niger (A niger ), Aspergillus awamori ( A.awamori ) and Aspergillus oryzae ( A.oryzae ), and Tricoderma sp. (for example, T.reesei ).

The FIPs used in this application are substantially isolated or purified according to the above method, for example, the method disclosed in WO2005/040375A1. The term "substantially isolated" or "substantially purified" as used in this application refers to FIPs that are removed from the natural environment or host system and are at least 60% free, preferably at least 75% free, and more It is preferably at least 90% free, and even more preferably at least 95% free from other components that are naturally bound or accompanied by the host system.

The term "osteoclastogenesis" used in this application refers to the process of fusion of monocyte/macrophage cells to form osteoeclasts, and the term "osteoblastogenesis" It refers to the process by which stem cells and precursor cells such as mesenchymal stem cells differentiate into functional osteoblasts. "Bone resorption" refers to the loss of undesired bone due at least in part to abnormally increased osteoclast activity or osteoclast production.

The term "inhibition" used in this application means to reduce the amount, quality or effect of a specific activity. For example, "inhibition" means to reduce the production of osteoeclasts by administering an effective amount of FIP to the patient, thereby Reduce its bone erosion activity.

As disclosed in the present specification, the present invention is primarily based on the inventors have found that, FIP- gts, FIP- gmi and other FIP in newborn FIP- vvo osteoclasts effect capable of exhibiting a strong inhibitory effect. The following examples 2-4 and 7-8 used several established in vitro models, and they showed that FIP inhibited RANKL-induced differentiation of RAW264.7 macrophages into osteoclasts in a dose-dependent manner. Since the new effect of osteoecotic cells is more active than that of osteoblasts, it has been identified as the cause of increased bone resorption, and it is closely related to the beginning or development of various bone loss-related diseases, so this application It shows that FIP is a potential drug in the treatment and prevention of many diseases described below.

The term "bone loss-related diseases" is used in its broadest sense in this application, and it covers diseases, conditions, disorders, and syndromes in which the decrease in bone mass or bone density is a symptom or pathological state. Preferably, the bone loss-related diseases are mainly caused by bone resorption net higher than bone production, especially the new effect of osteoblasts is more active than that of osteoblasts, resulting in metabolic imbalance. These diseases can include, but are not limited to, osteoporosis, juvenile osteoporosis, osteogenesis imperfecta (osteogenesis imperfecta), hypercalcemia (hypercalcemia), hyperparathyroidism, osteomalacia (osteomalacia), osteocalcemia (osteohalisteresis), osteolytic bone disease (osteolytic bone disease), osteonecrosis ( osteonecrosis), Paget's disease, bone loss caused by rheumatoid arthritis, bone loss caused by inflammatory arthritis, bone loss caused by osteomyelitis, and steroid treatment Bone loss, periodontal disease, bone loss caused by cancer, age-related bone loss and other types of osteodeficiencies.

The use of FIP to inhibit the regeneration of osteocytes can also be applied to situations that want to promote bone repair and renewal, such as fractures, bone defects, plastic surgery, dental implants and other transplants.

In a particularly preferred embodiment, the disease is periodontal disease. As mentioned earlier, periodontal disease is believed to occur due to infection by oral bacterial membrane microorganisms. The bacterial infection in turn triggers the host's immune response and activates the new role of osteoclasts, thus causing an imbalance between bone resorption and bone production. , And the degeneration of the alveolar bone used to support the teeth. It is generally believed that alveolar bone resorption can be inhibited by suppressing the new effect of eroded bone cells, thereby preventing or treating various complications related to periodontal disease. The term "periodontal disease" used in this application means a tissue-destructive disease involving resorption of alveolar bone and/or loss of periodontal attachment. In the pre-clinical analysis experiments shown in Examples 5-6, an established rat tying pattern was used, in which a tying was set around the teeth of the experimental animal, which caused plaque to accumulate and cause gingival sulcus epithelial ulcers, thereby triggering the periphery of the teeth Loss of bone and tissue. FIP-gts was chosen to represent the FIP defined above, which was shown to inhibit inflammation and alveolar bone loss in the periodontitis test induced by threading. It is important that FIP is administered before or approximately at the same time as the ligature is set, which can beneficially improve the symptoms that occur in the rat model, which proves that FIP has therapeutic and preventive effects on periodontal disease.

The term "prevention" means that before a bone loss-related disease begins to occur, in a patient who may be exposed to a pathogenic agent or is prone to suffering from the disease, the risk of developing or developing the disease is reduced, that is to say So that it will not develop at least one clinical sign of the disease. The term "treatment" includes the relief of at least one clinical sign of a bone loss-related disease. However, this term should not be understood in the sense that it is always 100% protective against the disease, but is used to mean that a clinical symptom that has occurred or may be about to occur in the patient is substantially reduced or eliminated.

According to the disclosure provided in this application, the term "patient" is intended to cover human or non-human vertebrates, such as non-human mammals, who need to prevent or treat bone loss-related diseases or their conditions. Non-human mammals include livestock animals, companion animals, laboratory animals, and non-human primates. Non-human individuals also include, but are not limited to, horses, cows, pigs, goats, dogs, cats, mice, rats, guinea pigs, gerbils, hamsters, mink, rabbits, and fish. It should be understood that the preferred patients are humans, especially human patients who suffer from a bone loss-related disease or are at risk of suffering from this disease.

A patient with a bone loss-related disease can be identified by conventional diagnostic methods and conditions. A general practitioner in this technical field can identify patients who are susceptible to a bone loss-related disease based on family medical history and/or whether there are genetic markers or genes related to bone loss-related diseases. The term "susceptible to" used in this application means that compared with the general population, there is an increased risk and/or tendency for a bone loss-related disease (usually based on genetic predisposition, environmental factors, or a combination thereof). As the basis). The term "easy to suffer" in its meaning also intends to cover the so-called "easy to suffer" bone loss-related diseases, and may never be diagnosed with this disease.

According to the disclosure provided in this application, the term "administering to a patient" includes allocating, delivering, or administering FIP to a patient in a suitable pharmaceutical formulation by any suitable means for delivering FIP to the desired location of the individual. In order to make FIP contact the target cell, tissue or organ.

FIP can be administered to the individual by any suitable route, such as topical, rectal, enteral or parenteral routes, for example, oral, intravenous, subcutaneous, intratumoral, intramuscular, intraperitoneal, transdermal, spinal Intrathecal, or intracerebral route. Administration can be rapid, such as by injection, or over a period of time, such as by slow infusion or administration of sustained release formulations.

In a preferred embodiment, FIP is intended to be administered orally and prepared in the form of an orally administrable formulation. Such formulations are preferably formulated with suitable carriers, excipients, lubricants, emulsifiers, suspending agents, sweeteners, flavoring agents, and preservatives, and are formulated into tablets or encapsulated into solid capsules or soft capsules. It is also envisaged that such formulations can be designed into the following dosage forms: oral solutions, or oral sachets, or oral pills. Or in addition to oral administration, it is also conceivable that such formulations can be designed as enema, or suppository, or implant, or patch, or cream, or ointment. Some examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, gelatin, syrup, methylcellulose, methyl-propyl-hydroxybenzate, talc, magnesium stearate, water, mineral oil, etc. The formulation may additionally include lubricants, wetting agents, emulsifying and suspending agents, preservatives, sweetening agents or flavoring agents. The FIP composition can be formulated to be able to quickly, continuously or delay the release of active ingredients after being administered to patients using procedures known in the art. The formulation may also contain substances that reduce proteolysis, nucleic acid and other degradation and/or promote absorption, such as, for example, surfactants. The composition can be complexed with polyethylene glycol (ie PEGylation), albumin or the like to help promote stability in the bloodstream.

In some preferred embodiments, FIP is formulated into a dosage form that can be administered orally.

Another preferred FIP formulation is to use a physiological saline solution carrier; other pharmaceutically acceptable carriers are conceivable, for example, non-toxic salts or physiological concentrations of compounds, 5% dextrose aqueous solution, sterile water or the like can also be used. It may also be desirable to have a suitable buffer in the composition. If necessary, such solutions can be lyophilized and stored in sterile ampoules, and can be used for injection after being reconstituted with sterile water. The main solvent can be aqueous or non-aqueous.

The carrier may also contain other pharmaceutically acceptable excipients to adjust or maintain the pH, osmotic pressure, viscosity, clarity, color, sterility, stability, dissolution rate, or odor of the formulation. Similarly, the carrier may additionally contain other pharmaceutically acceptable excipients to adjust or maintain the release or absorption or penetration of the formulation through the blood-brain barrier. Such excipients are substances customarily used to formulate pharmaceuticals for parenteral administration in unit dose or multiple dose forms or for direct infusion by continuous or periodic infusion.

FIP can be suitably formulated into the above-mentioned unit dosage form by using conventional crude drug technology. Such techniques include the step of combining FIP with physiologically acceptable carriers, diluents, adjuvants, and/or excipients. Generally speaking, the formulation is prepared by uniformly and tightly combining FIP and a liquid carrier or a finely divided solid carrier or both, and then forming various products as necessary.

FIP is administered to the patient in a therapeutically or preventively effective amount sufficient to inhibit the neonatal effect of osteoeclasts. Therefore, the term "effective amount" and the term "administered in an effective amount" can be used interchangeably in this application, and it is intended to refer to the effect of inhibiting the regeneration of osteoeclasts or reducing bone quality in diseased tissues or susceptible tissues of patients. The amount of FIP for lost medical effects can be observed when an effective amount of FIP is administered to patients suffering from or prone to suffering from a bone loss-related disease. Although the effective amount is usually determined by the effect of the effective amount compared to the effect observed in a composition that does not include the FIP described in this application (ie the control group) administered to patients with similar conditions, the actual dosage is based on the selected The specific route of administration is calculated. Necessary to determine the appropriate dosage The necessary further detailed calculations are carried out in a conventional manner by persons with ordinary knowledge in the field. Therefore, when administered to a human individual, FIP is preferably administered between 0.01 mg/kg body weight/day to 100 mg/kg body weight/day, more preferably 0.1 mg/kg daily, weekly or twice a week /Day to 10 mg/kg/day. Depending on the pharmacokinetic parameters of the pharmaceutical formulation and the route of administration used, multiple doses can be administered repeatedly.

The following examples are for illustrative purposes only, and are not intended to limit the scope of the present invention.

Example 1: Preparation of FIPs

The FIP-gts with sequence identification number 1 was expressed recombinantly in S. cerevisiae host cells as described in WO2005/040375A1. The cells expressing FIP- gts were broken up, centrifuged, and the supernatant was passed through a filter and molecular sieve to obtain a protein between 10kDa and 100kDa. The filtrate was further purified with Superdex® 75 column (GE Healthcare) using FPLC. The purity is determined by FPLC to exceed 95%.

FIP-vvo (Sequence ID No. 3) and FIP-gmi (Sequence ID No. 6) were prepared using the same method described in this application, and it was found that the protein prepared according to this method had a purity of more than 95%.

Example 2: FIP is not toxic to pre-osteoecotic cells

RAW 264.7 cells are a murine leukemia monocyte/macrophage cell line purchased from the Biological Resources Conservation and Research Center of the Food Industry Development Institute (Hsinchu, Taiwan). RAW 264.7 cells were implanted in a 24-well flat plate culture dish at a density of ^{1×10 5} cells/well, and in the presence of various concentrations of FIP-gts prepared in Example 1 (2.5-20 Μg/ml), cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-deactivated calf serum (FBS; Gibco, New York, USA); Sigma Chemical Co, St. Louis, Missouri, USA .), which lasted 5 days. Remove the supernatant and add 25 μl of 2.5 mg/ml MTT (3-[4,5-dimethylpyridin-2-yl]-2 in phosphate buffered saline (PBS) to each well , 5-Diphenyltetrazolium bromide; Sigma Chemical Co., St. Louis, Missouri, USA). The petri dish was incubated at 37°C for 1 hour to allow MTT to transform into water-insoluble formazan crystals. Subsequently, the supernatant was removed and dimethyl sulfoxide (DMSO) was added to all wells and mixed thoroughly to dissolve the dark blue crystals. After placing it at room temperature for a few minutes to ensure that all crystals are completely dissolved, the readings of each well in the flat petri dish are taken at 570nm.

The results shown in Figure 1A show that the survival rate of cells treated with FIP-gts at a concentration of 2.5 to 10 μg/ml is approximately the same as that of untreated cells. This indicates that FIP- gts is better than RAW 264.7 at the above concentration. Osteocytes are not toxic.

Repeat the previous experiment, but use FIP-gmi and FIP-vvo prepared in Example 1 to replace FIP-gts . The results are shown in Figures 1B and 1C, where the data are representative of 4 reproducibility experiments. Consistent with the results obtained using FIP-gts as shown in Figure 1A , FIP-gmi and FIP-vvo are not toxic to RAW 264.7 pre-osteoclasts at a concentration of 2.5 to 10 μg/ml.

Example 3: The inhibitory effect of FIP on the formation of osteoclasts

RAW 264.7 cells were suspended in DMEM supplemented with 10% heat-deactivated calf serum, and implanted in a 24-well flat plate at a density of ^{1×10 5 cells/well, and then cultured to the septum.} day. On day 1, the medium was removed, and α-minimal basal medium (α-MEM; GIBCO Life Technologies, Grand Island, New York, USA) was added to the cell culture, with or without 2.5-10μg/ml FIP- gts and 100ng/ml RANKL (PeproTech Inc, Rocky Hill, New Jersey, USA). On day 5, the α-MEM was removed, and the cell culture was washed twice with PBS.

Subsequently, relying on the fact that only mature osteoclasts can secrete tartrate-resistant acid phosphatase (TRAP), the TRAP activity analysis was used to evaluate the pre-eclipse induced by RANKL The degree of cell differentiation. In this analysis, 200 microliters of 0.1% Triton X-100 was used to treat the cell culture in each well, and the cell culture was washed once with PBS. Next, add 100 microliters of TRAP solution (2 ml of 0.1 M sodium acetate, pH 5.8; 1 ml of 1 mM ascorbic acid; 1.9 ml of 0.15 M KCl; 50 mM of sodium tartrate; and 0.1 Milliliters of 10 mM p-nitrophenyl phosphate). After incubating at 37°C for 0.5 to 1 hour, the reaction mixture was transferred to a new flat-plate petri dish, and an equal volume of 0.3N NaOH was added to quench the reaction. A microdisk spectrophotometer was used to measure the optical density of each cell culture at 410nm. The results are shown in Figure 2A.

In a separate TRAP staining analysis, RAW264.7 cells were cultured under the aforementioned conditions with or without 2.5-10μg/ml FIP- gts and 100ng/ml RANKL. On the 5th day, the cells were fixed with 3.7% formalin (Sigma Chemical Co., St. Louis, Missouri, USA) for 10 minutes, and treated with 0.1% Triton-X100 for 1 minute, followed by TRAP staining solution (USA Sigma Chemical Co., St. Louis, Missouri, was dyed for 30 minutes. The images showing TRAP-positive multinucleated cells (≧three nuclei) were taken (Figure 3A), and the number of mature osteoeclasts was calculated under an optical microscope (Leica, Vertrieb Deutschland, Germany) (Figure 3B).

The results shown in Figures 2A and 3A-3B consistently pointed out that, compared to the control experiment containing only medium, the addition of RANKL induced the differentiation of RAW 264.7 pre-osteoeclasts into osteoeclasts, while FIP- gts would In a dose-dependent manner, RANKL inhibits the maturation of RAW 264.7 cells into osteoblasts.

Repeat the above TRAP activity analysis, but replace FIP-gts with FIP-gmi and FIP-vvo prepared in Example 1 respectively. The results are shown in Figures 2B and 2C, which indicate that these two FIPs exhibit similar inhibitory effects on osteoclast production induced by RANKL.

Example 4: FIP is the main cause of the inhibition of osteoclast production

As described in Example 3, RAW 264.7 cells were treated with 10 μg/ml FIP- gts and 100 ng/ml RANKL, but FIP- gts was added at 0, 1, 2, or 3 days after RANKL was added to the cell culture. As shown in Figure 4, the inhibitory effect of FIP-gts on the formation of osteoclasts in RAW 264.7 cells induced by RANKL decreased as the time interval between adding RANKL and adding FIP-gts increased. This result indicates that FIP- gts is caused by RANKL. The main reason for the inhibition of the induced formation of osteoeclasts.

Example 5: The therapeutic effect of FIP on periodontal disease

In order to test the effect of FIP on the formation of osteoeclasts, this manual uses a rat stringing model according to the experimental procedure described in Cheng et al. , J.Periodont.Res. 2010;45:788-795. To produce the clinical features of periodontal bone loss. Eighteen Sprague-Dawley rats were randomly divided into 3 groups, 6 in each of the control group, the tie group and the tie+FIP- gts group. The rats in the control group were not applied with ligature, while the rats in the ligature group and the ligature+FIP-gts group were wrapped with 3-O silk sutures (diameter 0.2 Mm; Taipei City Jiahe Medical Materials Co., Ltd., Taiwan), and knotted at the proximal buccal side of the second molar of the upper jaw and the first molar of the lower jaw. By tying a silk thread around the neck of the tooth to accumulate bacteria, it induces periodontitis for experimental use in the rat model. These rats were bred under pathogen-free conditions on a 12-hour day/12-hour night cycle, and were given an autoclaved diet, with free access to standard rodent diet (Laboratory Rodent Diet 5001, LabDiet, St. Missouri, USA). Louis City) and drinking water. On the day before tying , rats in the tying+FIP-gts group were fed with FIP-gts at a dose of 1.5 mg per kilogram of body weight, and daily feed and drinking water were continuously supplied throughout the experiment. On the 0th, 3rd, 7th, and 10th days, the morphology of the molar teeth and alveolar bone of each group of rats were examined by micro-computer tomography (micro-CT). All animals were sacrificed _{by CO 2} suffocation on the 14th day. Obtain maxillary bone and maxillary bone samples, fix them with 4% paraformaldehyde, and prepare for micro-computer tomography and histological analysis.

Micro computed tomography is performed using a polymorphic preclinical imaging system with a tomographic sub-system (FLEX Triumph ^TM preclinical PET/CT scanner produced by Gamma Medica-Ideas Inc., Northridge, California, USA). The X-ray tube is operated at an acceleration potential of 75 kilovolt peak (kVp), and the scanning beam current is 120 mA. The field of view of the microcomputer tomography is fixed at 61.44 mm, which magnifies the image by 2 times. In the flight mode, the microcomputer tomography image is captured, 1024 times of projection, one frame each time, to achieve a voxel size of 120 mm × 120 mm × 120 mm. The micro computed tomography data was obtained and reconstructed using Triumph XO software (Gamma Medica-Ideas Inc., Northridge, California, USA), and then visualized and analyzed using VIVID software (Gamma Medica-Ideas Inc., Northridge, California, USA). Using micro-computer tomography data and reconstructed three-dimensional images, in the root bifurcation area (marked by the symbol ◎ in Figure 5A), the area around the tie line (marked by the symbol ☆), the close tie area (marked by the symbol ￥) and Estimate the distance between the cement-enamel junction (CEJ) and the crest of the alveolar bone (ABC) away from the four areas (marked with the symbol ※) and other areas away from the tie line. The resulting microcomputer tomography image and the measured CEJ-ABC value are shown in Figures 5B and 5C, respectively.

The maxillary bone sample obtained on day 14 was fixed in 10% buffered neutral formalin solution for 48 hours and in 10% ethylenediaminetetraacetic acid (EDTA; Sigma, St. Louis, Missouri, USA) Decalcification lasted 4 weeks. Each sample was embedded in paraffin, and several 4 micron thick sections were cut in the mesiodistal direction. The sections were placed on glass slides and stained with hematoxylin-eosin (H&E staining). Measure the second for each rat The distance from CEJ to ABC on the proximal surface of the molar tooth. The results are shown in Figure 5D, where the positions of CEJ and ABC are marked with white and black arrows, respectively.

Repeated measures analysis of variance (ANOVA) was used to evaluate the results of the ligation treatment. The p value<0.05 is considered significant.

The microcomputer tomography data shown in Figures 5B-5C indicate that the root bifurcation area, the area around the ligature and the alveolar bone position close to the ligation area are closer to the root tip than the ligation area, and far away from the ligation area. The location of the alveolar bone is similar in the three animal groups. The results further showed that on days 7, 10 and 14, the crest of the alveolar bone in the ligation group was significantly closer to the root tip than in the control group. This indicated that the ligation group was caused by silk ties. Alveolar bone loss began to appear on the 7th day. However, compared with the ligature group, the ligature + FIP- gts group recovered the alveolar ridge in the root bifurcation area (starting on day 10), the area around the ties and the area close to the ties (starting on the 14th day). The height of the top. Similar results can be seen in the histological data shown in Figure 5D. Compared with the ligature group, the alveolar bone crest position of the ligature+FIP-gts group is closer to the crown, the degree of inflammatory cell infiltration is lower, and the connection is The degree of tissue adhesion is also relatively low. These histological data further confirm the results shown in Figures 5B-5C, showing that the degree of alveolar bone resorption obtained by measuring the distance from CEJ to ABC, in the ligature + FIP- gts group compared to the ligature group Significantly inhibited. All the data disclosed in this example point out that FIP- gts can successfully improve the symptoms that occur in the rat model after setting up the thread or roughly at the same time, which proves that FIP-gts has a therapeutic effect on periodontal disease. .

Example 6: Preventive effect of FIP on periodontal disease

Example 5 was repeated, but the rats in the ligation +FIP-gts group were fed FIP-gts 7 days before the ligation. At 0, 3, 7, 10 and 14 days after the threading, the morphology of the molar teeth and alveolar bones of the rats in each group were examined by micro-computer tomography. The resulting microcomputer tomography image and the measured CEJ-ABC value are shown in Figures 6A and 6B, respectively. These micro computed tomography images showed that the crest of the alveolar bone in the ligation group was significantly closer to the root apex than the control group on days 7, 10, and 14, which indicated that the ligation group was ligated by silk. The induced alveolar bone loss began to appear on the 7th day after the threading. Compared with the ligature group, the ligature+FIP- gts group significantly restored the height of the alveolar crest in the root bifurcation area, the area around the ligature and the area close to the ligature from the 7th day. The histopathological examination of the maxillary bone sample collected on the 14th day (Figure 6C) also confirmed the results obtained by the micro-computed tomography, which showed that the administration of FIP-gts to the rats that received the ligature can significantly inhibit the teeth. The trough bone resorption can be confirmed by the shorter CEJ-ABC distance and lower infiltration of inflammatory cells in the ligature +FIP-gts group than the ligature group. All the data disclosed in this example point out that giving FIP- gts before setting the ligature can successfully improve the symptoms that occur in the rat model, which proves that FIP- gts has a preventive effect on periodontal disease.

Example 7: Actin loop analysis

It is known that during bone resorption, osteoclasts will reorganize their own actin cytoskeleton. Therefore, the formation of actin loops is considered to be a biomarker related to the resorption activity of osteoclasts. The inventor of the present application used the actin ring staining analysis described in this specification to study the effect of FIP on bone resorption regulated by osteoclasts.

In this analysis, RAW264.7 cells were cultured in 100ng/mL RANKL in the presence or absence of FIP- gts for 5 days. The cells were washed, fixed with 3.7% formalin, and incubated in 0.1% Triton-PBS for 15 minutes to permeabilize the cells. The cells were blocked in 1% bovine serum albumin formulated in PBS, and incubated with rhodamine-labeled phalloidin (rhodamine-labeled phalloidin; InvivoGen, San Diego, California, USA) for 30 minutes. After washing, the formation of actin loops was observed under a fluorescent microscope (Axiovert 100; Carl Zeiss GmbH, Oberkochen, Germany). If an actin ring exhibits less than half of the typical actin ring shape, it is considered that its structure has collapsed.

The results are shown in Figure 7. The actin ring was stained with fluorescent phalloidin, showing that its production was inhibited by FIP-gts in a dose-dependent manner. This result confirmed the resorption capacity of FIP for osteoeclasts. Has a suppressive effect.

Example 8: Analysis of functional bone resorption lacuna

Since mature osteoclasts secrete cathepsin K (Cathepsin; CTSK) and tartrate-resistant acid phosphatase (TRAP) and many other enzymes to dissolve the mineral components of bone, this example uses RANKL to induce RAW264.7 cells and Calculate the number and area of the lacunae formed. The cells were cultured on a 24-well Corning® surface petri dish for bone analysis (Corning Life Sciences, New York, USA). The surface of the dish made of styrene is covered with a layer of inorganic calcium phosphate crystals to simulate the bone environment.

RAW264.7 cells were implanted into a culture dish at a density of 1×10 ⁵ cells/well, and then pretreated with FIP-gts or vehicle (0.1% DMSO) for 2 hours, and then 100 ng/Ml RANKL was added. After 5 days of incubation, 1N NaOH was used to decompose the cells for 10 minutes, and the wells were washed twice with PBS. Metamorph image analysis software (Metamorph imaging system; Universal Imaging Corp., Pennsylvania, USA) was used to analyze the number and area of lacunae formed by resorption.

The results are shown in Figures 8A-8C, which indicate that the number and extent of lacunae formed by bone resorption on the petri dish coated with calcium phosphate are reduced in a dependent manner by the dose of FIP- gts. This result again confirms that FIP is effective for erosion. The resorption capacity of bone cells has an inhibitory effect.

Although the present invention has been described with reference to the above preferred specific examples, it should be understood that the preferred specific examples are provided for illustrative purposes only and not intended to limit the scope of the present invention, and various modifications and changes that are extremely obvious to those skilled in the relevant art can be made. Without departing from the spirit and scope of the present invention.

All papers, publications, documents, patents, patent applications, websites, and other printed or electronic documents mentioned in this application-including but not limited to the references listed below-are incorporated by reference in their entirety. In case of conflict, this description, including definitions, will prevail.

<110> Probiotics Technology Development Co., Ltd. <120> The medical use of fungal immunomodulatory protein in the treatment and prevention of alveolar bone loss caused by periodontal disease <160> Number of serial identification numbers: 8 <210> Sequence Identification number: No. 1 <211> Sequence length: 111 <212> Sequence type: PRT <213> Organism: Ganoderma tsugae (Ganoderma tsugae) <221> Name/Keyword: Peptide <222> Position: (1 )…(111) ＜400＞ Sequence: 1 Met Ser Asp Thr Ala Leu Ile Phe Arg Leu Ala Trp Asp Val Lys Lys 1 5 10 15 Leu Ser Phe Asp Tyr Thr Pro Asn Trp Gly Arg Gly Asn Pro Asn Asn 20 25 30 Phe Ile Asp Thr Val Thr Phe Pro Lys Val Leu Thr Asp Lys Ala Tyr 35 40 45 Thr Tyr Arg Val Ala Val Ser Gly Arg Asn Leu Gly Val Lys Pro Ser 50 55 60 Tyr Ala Val Glu Ser Asp Gly Ser Gln Lys Val Asn Phe Leu Glu Tyr 65 70 75 80 Asn Ser Gly Tyr Gly Ile Ala Asp Thr Asn Thr Ile Gln Val Phe Val 85 90 95 Val Asp Pro Asp Thr Asn Asn Asp Phe Ile Ile Ala Gln Trp Asn 100 105 110 ＜210＞ Serial identification number: No. 2 ＜211＞ Sequence length: 114 ＜212＞ Sequence type: PRT ＜213＞ Organism: Flammulina Velutipes ＜221＞ Name/Keyword: Peptide <222> Position: (1)…(114) <400> Sequence: 2 Ser Ala Thr Ser Leu Thr Phe Gln Leu Ala Tyr Leu Val Lys Lys Ile 1 5 10 15 Asp Phe Asp Tyr Thr Pro Asn Trp Gly Arg Gly Thr Pro Ser Ser Tyr 20 25 30 Ile Asp Asn Leu Thr Phe Pro Lys Val Leu Thr Asp Lys Lys Tyr Ser 35 40 45 Tyr Arg Val Val Val Asn Gly Ser Asp Leu Gly Val Glu Ser Asn Phe 50 55 60 Ala Val Thr Pro Ser Gly Gly Gln Thr Ile Asn Phe Leu Gln Tyr Asn 65 70 75 80 Lys Gly Tyr Gly Val Ala Asp Thr Lys Thr Ile Gln Val Phe Val Val 85 90 95 Ile Pro Asp Thr Gly Asn Ser Glu Glu Tyr Ile Ile Ala Glu Trp Lys 100 105 110 Lys Thr ＜210＞ Serial identification number: No. 3 ＜211＞ Sequence length: 112 ＜212＞ Sequence type: PRT ＜213＞ Organism: Straw mushroom (Volvariella volvacea ) ＜221＞ Name /Keyword: peptide <222> Position: (1)…(112) <400> Sequence: 3 Ser Thr Asp Leu Thr Gln Leu Leu Phe Phe Ile Ala Tyr Asn Leu Gln 1 5 10 15 Lys Val Asn Phe Asp Tyr Thr Pro Gln Trp Gln Arg Gly Asn Pro Ser 20 25 30 Ser Tyr Ile Asp Ala Val Val Phe Pro Arg Val Leu Thr Asn Lys Ala 35 40 45 Tyr Gln Tyr Arg Val Val Thr Gly Asp Lys Asp Leu Gly Ile Lys Pro 50 55 60 Ser Tyr Ser Val Gln Ala Asp Gly Ser Gln Lys Val Asn Leu Leu Glu 65 70 75 80 Tyr Asn Gly Gly Tyr Gly Val Ala Asp Thr Thr Thr Ile Lys Ile Tyr 85 90 95 Val Val Val Asp Pro Ser Asn Gly Asn Gln Tyr Leu Ile Ala Gln Trp Lys 100 105 110 ＜210＞ Serial identification number: No. 4 ＜211＞ Sequence length: 111 ＜212＞ Sequence type: PRT ＜213＞ Organism: Ganoderma lucidum ＜221＞ Name /Keyword: peptide <222> Position: (1)…(111) <400> Sequence: 4 Met Ser Asp Thr Ala Leu Ile Phe Arg Leu Ala Trp Asp Val Lys Lys 1 5 10 15 Leu Ser Phe Asp Tyr Thr Pro Asn Trp Gly Arg Gly Asn Pro Asn Asn 20 25 30 Phe Ile Asp Thr Val Thr Phe Pro Lys Val Leu Thr Asp Lys Ala Tyr 35 40 45 Thr Tyr Arg Val Ala Val Ser Gly Arg Asn Leu Gly Val Lys Pro Ser 50 55 60 Tyr Ala Val Glu Ser Asp Gly Ser Gln Lys Val Asn Phe Leu Glu Tyr 65 70 75 80 Asn Ser Gly Tyr Gly Ile Ala Asp Thr Asn Thr Ile Gln Val Phe Val 85 90 95 Val Asp Pro Asp Thr Asn Asn As p Phe Ile Ile Ala Gln Trp Asn 100 105 110 ＜210＞ Serial identification number: No. 5 ＜211＞ Sequence length: 111 ＜212＞ Sequence type: PRT ＜213＞ Organism: Ganoderma japoncium (Ganoderma japoncium) ＜221＞ Name /Keyword: peptide <222> Position: (1)…(111) <400> Sequence: 5 Met Ser Asp Thr Ala Leu Ile Phe Arg Leu Ala Trp Asp Val Lys Lys 1 5 10 15 Leu Ser Phe Asp Tyr Thr Pro Thr Trp Gly Arg Gly Asn Pro Ser Arg 20 25 30 Phe Val Asp Asn Val Thr Phe Pro Gln Val Leu Ala Asp Lys Ala Tyr 35 40 45 Thr Tyr Arg Val Val Val Ser Gly Arg Asp Leu Gly Val Arg Pro Ser 50 55 60 Tyr Ala Val Gly Ser Asp Gly Ser Gln Lys Val Asn Phe Leu Glu Tyr 65 70 75 80 Asn Gln Gly Tyr Gly Ile Ala Asp Thr Asn Thr Ile Gln Val Phe Val 85 90 95 Ile Asp Pro Asp Thr Asp Ala Asp Phe Ile Ile Ala Gln Trp Asn 100 105 110 ＜210＞ Sequence identification number: No. 6 ＜211＞ Sequence length: 111 ＜212＞ Sequence type: PRT ＜213＞ Organism: Ganoderma microsporum (Ganoderma microsporum) ＜221＞ Name/Keyword: Peptide <222> Position: (1)…(111) <400> Sequence: 6 Met Ser Asp Thr Ala Leu Ile Phe Thr Leu Ala Trp Asn Val Lys Gln 1 5 10 15 Leu Ala Phe Asp Tyr Thr Pro Asn Trp Gly Arg Gly Arg Pro Ser Ser 20 25 30 Phe Ile Asp Thr Val Thr Phe Pro Thr Val Leu Thr Asp Lys Ala Tyr 35 40 45 Thr Tyr Arg Val Val Val Ser Gly Lys Asp Leu Gly Val Arg Pro Ser 50 55 60 Tyr Ala Val Glu Ser Asp Gly Ser Gln Lys Ile Asn Phe Leu Glu Tyr 65 70 75 80 Asn Ser Gly Tyr Gly Ile Ala Asp Thr Asn Thr Ile Gln Val Tyr Val 85 90 95 Ile Asp Pro Asp Thr Gly As n Asn Phe Ile Val Ala Gln Trp Asn 100 105 110 ＜210＞ Serial identification number: No. 7 ＜211＞ Sequence length: 111 ＜212＞ Sequence type: PRT ＜213＞ Organism: Ganoderma sinense ＜221 ＞ Name/Keyword: Peptide <222> Position: (1)…(111) <400> Sequence: 7 Met Ser Asp Thr Ala Leu Ile Phe Arg Leu Ala Trp Asp Val Lys Lys 1 5 10 15 Leu Ser Phe Asp Tyr Thr Pro Thr Trp Gly Arg Gly Asn Pro Ser Arg 20 25 30 Phe Val Asp Asn Val Thr Phe Pro Gln Val Leu Ala Asp Lys Ala Tyr 35 40 45 Thr Tyr Arg Val Val Val Ser Gly Arg Asp Leu Gly Val Arg Pro Ser 50 55 60 Tyr Ala Val Gly Ser Asp Gly Ser Gln Lys Val Asn Phe Leu Glu Tyr 65 70 75 80 Asn Gln Gly Tyr Gly Ile Ala Asp Thr Asn Thr Ile Gln Val Phe Val 85 90 95 Ile Asp Pro Asp Thr Gly Ala A sp Phe Ile Ile Ala Gln Trp Asn 100 105 110 ＜210＞ Sequence identification number: No. 8 ＜211＞ Sequence length: 114 ＜212＞ Sequence type: PRT ＜213＞ Organism: Nectria haematococca ) <221> Name/Keyword: Peptide <222> Position: (1)…(114) <400> Sequence: 8 Met Ala Thr Thr Asn Asp Ser Ala Val Leu Phe Tyr Ile Val Ala Ser 1 5 10 15 Gln Lys Lys Leu Ser Phe Asp Tyr Thr Pro Asn Trp Gly Arg Gly Ser 20 25 30 Pro Asn Ser Tyr Ile Asp Asn Leu Thr Phe Pro Arg Val Leu Thr Asn 35 40 45 Lys Pro Tyr Lys Tyr Arg Val Val Lys Ala Gly Gly Gln Asp Leu Gly Val 50 55 60 Arg Asp Ser Tyr Ser Val Gln Ser Asp Gly Ser Gln Lys Val Asn Phe 65 70 75 80 Leu Glu Tyr Asn Ala Gly Arg Gly Ile Ala Asp Thr Gln Thr Ile Gln 85 90 95 Val Tyr Val Val Asp Pro Asp Asn Gly Asn Gln Tyr Leu Val Ala Gln 100 105 110 Trp Lys

Claims (7)

The use of a fungal immunomodulatory protein (FIP) in the manufacture of a medicine for the treatment of alveolar bone loss caused by periodontal disease in a patient suffering from periodontal disease. The use of a fungal immunomodulatory protein (FIP) in the manufacture of a medicine for preventing alveolar bone loss caused by periodontal disease in a patient prone to periodontal disease. The use according to any one of claims 1 to 2, wherein the FIP has at least 57% amino acid identity with the amino acid sequence of SEQ ID NO. 1. The use of claim 3, wherein the FIP has an amino acid sequence selected from the group consisting of: serial identification number 1, serial identification number 2, and serial identification number 3. , Serial ID No. 4, Serial ID No. 5, Serial ID No. 6, Serial ID No. 7 and Serial ID No. 8. The use according to claim 4, wherein the FIP has an amino acid sequence of sequence identification number 1. The use according to any one of claims 1 to 2, wherein the FIP is in the form of oral administration. The use according to any one of claims 1 to 2, wherein the patient is selected from the group consisting of humans and non-human vertebrates.

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