JP2002114704A - Antibacterial agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a human-derived antibacterial agent exhibiting an effective antibacterial activity against oral bacteria and the like that cause caries and periodontal disease, and a sterilization method using the antibacterial agent. SOLUTION: An antibacterial agent against a streptococcus group containing an antibacterial peptide as an active ingredient; and a method for sterilizing the streptococcus group, which comprises using such an antibacterial agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an antibacterial agent for a streptococcus group containing an antibacterial peptide as an active ingredient, and a method for killing a streptococcal group using such an antibacterial agent.

[0002]

BACKGROUND OF THE INVENTION Mammalian antimicrobial peptides, collectively referred to as Defensins, play an important role in host defense of mucosal surfaces. These peptides, which are important components of innate immunity to infection, are produced by several types of cells, such as phagocytes and epithelial cells, and have various structural diversity.

[0003] The defensin family is characterized as a cationic peptide in which six cysteine residues form three pairs of intramolecular disulfide bonds. Due to the combination of the disulfide bonds of these cysteine residues, the human defensin family has two α- and β-
Classified into species subfamilies. In 1985, Ganz et al. (J.
Clin. Invest.), There are currently six different molecules reported for human α-defensin (Lehrer, Annu. Rev. Immunol. 1993; Martin,
J. Leukoc. Biol. 1995). Human neutrophil peptide-1-4 (HNP-1-4)
Four molecules, referred to as, are located in the azure granules. Two types of α-, called human defensins-5 and -6 (HD-5 and -6)
Defensins are present in secretory granules of intestinal Paneth cells and epithelial cells of female genital tracts (Quayle, Am. J. Pathol. 1998,
Japanese Patent Publication No. Hei 7-507213.

In 1995, a human defensin structurally different from α-defensin was separated from hemofiltrate (Bensch, FEBS Lett. 1
995), and was named β-defensin. Human β-defensin-1 (hBD-1) is known to be constitutively expressed in kidney, pancreas, ureter, respiratory tract, and several other epithelial tissues (Zhao, FEBS Lett. 1996; McCray, A
m. J. Respir 1997; Valore, Cell Mol. Biol. 1998;
Bensch, J. Clin. Invest. 1995; Goldman, Cell199.
7). Further, human β-defensin, which is a cysteine-rich cationic peptide consisting of 41 amino acid residues,
2 (hBD-2) was found in human skin in 1997 (Harde
r, Nature 1997), and later confirmed to be present in lung, oral mucosa, saliva, and the like. hBD-2 has been reported to be significantly inducibly expressed when organisms are exposed to bacteria and inflammatory cytokines (Harder, Nature 1997;
Singh, Proc. Natl. Acad. Sci. USA 1998; Bals, JC
lin. Invest. 1998; Mathews, Infect Immunol. 199
9).

[0005] hBD-1 mRNA is constitutively expressed in gingival epithelial cells (Krisanaprakornkit, Infect Immun.
1998). In contrast, hBD-2 mRNA is inducibly expressed in cultured cells of the gingival epithelium (Weinberg, Crit. Rev. Oral Biol .. Med. 1998) and salivary glands (Bonass, Oral Microbiol. Immunol. 1999). . It is known that the concentration of hBD-2 in saliva is about 150 ng / ml (Mathews, Infe
ct Immun. 1999). As described above, for hBD-2,
Various reports have been made on the tissue distribution and expression level.

In recent years, there has been an increasing interest in diseases caused by streptococci, such as caries, periodontal disease, and pneumonia, and these streptococci have been used for disinfection using natural antibacterial substances, particularly human-derived antibacterial substances. Much research has been done.

[0007]

However, so far, few effective antibacterial substances have been found.

Accordingly, an object of the present invention is to provide a human-derived antibacterial agent exhibiting an effective antibacterial activity against a streptococcal group, and a method for sterilizing the streptococcal group using such an antibacterial agent.

[0009]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, have found that an antibacterial peptide exhibits an effective antibacterial activity against a streptococcus group.
The present inventors have found that the antibacterial spectrum is broadened by using a protease inhibitor in combination, and completed the present invention.

[0010] That is, the first aspect of the present invention is an antibacterial agent against a streptococcus group containing an antibacterial peptide as an active ingredient. The invention according to claim 2 is characterized in that the streptococci group includes streptococcus pneumoniae, pyogenes streptococci, cariogenic streptococci, Streptococcus gordonii ( Streptococcus gordonii).
ii ), S. sangui
s ), S. cricetu
s ), Streptococcus ratus ( S. rattus ), and at least one member selected from the group consisting of Streptococcus salivarius ( S. salivarius ). The invention according to claim 3, wherein the antimicrobial peptide is
The antibacterial agent according to claim 1 or 2, which is human β-defensin-2. The invention according to claim 4 is the antibacterial agent according to any one of claims 1 to 3, further comprising a protease inhibitor. The invention according to claim 5 is the antibacterial agent according to claim 4, wherein the protease inhibitor is a serine protease inhibitor. The invention according to claim 6 is
A method for sterilizing a group of streptococci, comprising using the antibacterial agent according to any one of claims 1 to 5. Claim 7
The invention according, Streptococcus anginosus, which comprises using the antibacterial agent of claim 4 or 5, wherein (S.anginosus), Streptococcus con Suterata scan (S.constellatus), Streptococcus intermedius (S.intermedius) or a sterilization method of Streptococcus mitis (S.mitis).

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS It has been known that hBD-2 is present in the oral mucosa and saliva. However, such antibacterial peptides such as hBD-2 have been used in cariogenic streptococci, pneumococcal streptococci, and suppuration. No effective antibacterial activity against streptococci such as streptococci has been known. Furthermore, it was not known at all that the antibacterial spectrum was expanded when a protease inhibitor was used in combination.

The antimicrobial peptide is not particularly limited, but is preferably of human origin, and includes various types of conventionally known human antimicrobial peptides. Among them, human β-defensin-2 (hBD-2)
Is preferred. Such an antimicrobial peptide can be purified from human saliva, oral mucosa and the like according to a conventional method. Further, it can be synthesized by a chemical synthesis method. Furthermore, it is also possible to synthesize and purify using a genetic engineering technique.

The antimicrobial peptides used in the present invention include infectious diseases, skin diseases, and digestive organs caused by a wide variety of microorganisms and viruses such as molds and bacteria (including both gram-positive bacteria and gram-negative bacteria). It is effective for prevention and treatment of systemic inflammation, periodontal disease, etc. Streptococcus
It is particularly excellent in antibacterial activity against Crisetus, Streptococcus ratus, and Streptococcus salivarius.

The antimicrobial peptide can be used in the form of a pharmaceutically acceptable salt. Examples of such salts include inorganic acid salts such as hydrochloride, nitrate and hydrobromide; and organic acid salts such as p-toluenesulfonate, metasulfonate, fumarate, succinate and lactate. Can be

The antibacterial agent of the present invention contains such an antibacterial peptide as an active ingredient. By further containing a protease inhibitor, the antibacterial spectrum is further expanded. The cause of improving the effect by containing the protease inhibitor is not necessarily clear, but the protease inhibitor inhibits the protease activity of the microorganism, making it difficult for the antimicrobial peptide to be decomposed,
As a result, it is speculated that this may be due to an increase in the sensitivity of the microorganism to the antimicrobial peptide, particularly hBD-2. Among the protease inhibitors, serine protease inhibitors are more preferred, and aminoethylbenzenesulfonyl fluoride (AEBSF), tosylphenylalanine chloromethyl ketone (TPCK), phenylmethylsulfonyl fluoride (PMSF) and 3,4-dichloroisocoumarin (3, 4-DCI) is particularly preferred.

The antibacterial agent of the present invention can be used as a pharmaceutical composition or food. The pharmaceutical composition includes both pharmaceuticals and quasi-drugs. These can be made into various dosage forms such as tablets, granules, powders, gels, capsules, suspensions, injections, suppositories, liquids, and external preparations by a conventional method. These pharmaceutical compositions can be produced by blending an antimicrobial peptide or a pharmaceutically acceptable salt thereof with a protease inhibitor and, if necessary, a pharmaceutically acceptable carrier.

Specifically, in the case of a solid preparation, an excipient, a binder, a disintegrant, a bulking agent, a coating agent, a sugar-coating agent and the like are added to an antimicrobial peptide or the like, if necessary, and the mixture is produced by a conventional method. can do. Alternatively, it may be in the form of a preparation in which an antimicrobial peptide or the like is included in a liposome or the like. For liquid formulations, solutions buffered with various salts and buffers,
It can be in the form of a suspension, an emulsion or the like. Salts include alkali or alkaline earth halides, phosphates, or sulfates, such as sodium chloride, potassium chloride, or sodium sulfate. As buffers, for example, citrate, phosphate, HEPES, Tris and the like can be used to the extent that such buffers are to be treated and are physiologically acceptable to the subject to be treated. In the case of an injection, an antimicrobial peptide or the like may be previously dissolved, dispersed, emulsified, or the like in an aqueous carrier such as distilled water for injection to prepare a liquid for injection, or a powder for injection and dissolved when used.

There is no particular limitation on the administration method, and it can be administered in various manners depending on the form of the pharmaceutical composition and the properties of the administration subject. For example, oral, intravenous, subcutaneous, transdermal, intramuscular, intraperitoneal, nasopharyngeal, etc. administration is possible.

In the case of foods, beverages such as juices and teas;
Foods of any form, such as gel foods such as udon, buckwheat, tofu, kamaboko, and jelly, can be used. They are,
An antimicrobial peptide or the like is added to the raw material of each food, and the food can be produced according to a conventional method.

The dose of the antibacterial agent of the present invention depends on the form of administration, the age, sex, weight, and medical condition of the subject, but is generally preferably 100 μg to 100 g per day for an adult.
mg to 10 g are particularly preferred. The frequency of administration is preferably once to several times per day. When a protease inhibitor is used in combination, it is generally preferable to administer the protease inhibitor per adult day at 100 μg to 100 g, particularly 1 mg to 10 g.

[0021]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

Example 1 Measurement of Antibacterial Activity Antibacterial test of hBD-2 against streptococci 0.02-0.06 OD ₆₂₀ units of streptococci were suspended in phosphate buffer agarose, spread to a thickness of about 1 mm, and cooled. Then, a hole having a diameter of about 3 mm was made, 4.5 μl of 0-250 μg / ml hBD-2 was added, and the cells were cultured at 37 ° C. for 3 hours. Then, 2 × TSB agarose was overlaid. After that, the size of the stop circle on the plate was measured. As a comparison method, the relationship between the radius of the inhibition circle and the concentration was plotted, and the concentration at which the diameter of the inhibition circle was 4.0 mm was calculated from the obtained approximate expression. Table 1 shows the antibacterial effect of human hBD-2 on streptococci.

[0023]

[Table 1]

It was revealed that three strains of the Angionas group and two strains of Streptococcus mitis had resistance to hBD-2. Further, Streptococcus mutans is cariogenic streptococci (S. mutans) and Streptococcus sobrinus (S.sobrinus) and Streptococcus pyogenes (S. pyogenes), Streptococcus pneumoniae (S. pneumoniae) is
It was found to be sensitive to hBD-2. further,
Streptococcus gordonii, Streptococcus sanguis, Streptococcus christetus,
Streptococcus ratus, Streptococcus
Salivaryus was found to be sensitive to hBD-2.

Example 2 Streptococcus angino
Microorganisms for Haussus and Streptococcus mytis
Protease Inhibition Test Derived from 15 kinds of protease inhibitors (antipain, amastatin, aprotinin, bestatin, chymostatin) dissolved in dimethyl sulfoxide (DMSO) in phosphate buffer agarose in which bacteria were suspended with respect to the experimental system of Example 1. , 3,4-dichloroisocoumarin (3,4, -DC
I), E-64, EDTA, leupistin, leupeptin, AEBSF, pepstatin, phosphoramidone, trypsin inhibitor, TPCK) were added at a concentration of 10 nM-10 mM to inhibit the protease activity derived from microorganisms. Otherwise, the experiment was performed under the same conditions as in Example 1. Controls received only DMSO. Bacteria used were Streptococcus anginosus and Streptococcus mitis. The results are shown in FIGS. Here, unit is 10 times the size (mm) of the diameter of the blocking circle minus 3 mm.

When 15 kinds of protease inhibitors were used at the respective optimal concentrations, the resistance of Streptococcus anginosas and Streptococcus mitis was remarkably reduced when the serine protease inhibitors 3,4-DCI and AEBSF were used. . In addition, when TPCK was used, the resistance of only Streptococcus anginosus significantly decreased. The resistance of Streptococcus anginosas and Streptococcus mitis bacteria to hBD-2 was found to be proteolytic activity by proteases. HBD-2 by bacterial protease activity
Although the antibacterial activity of the compound was suppressed, the antibacterial effect could be enhanced by a protease inhibitor.

[0027]

The antimicrobial agent of the present invention contains a human-derived antimicrobial peptide as an active ingredient and is excellent against streptococci, in particular, streptococcus pneumoniae, pyogenes streptococci, cariogenic streptococci, and the like. Shows antibacterial effect. In addition, by using a protease inhibitor in combination, the antibacterial spectrum can be expanded. The antibacterial agent of the present invention is effective for enhancing biological defense and treating various diseases.

[Brief description of the drawings]

FIG. 1 is a view showing the effect of hBD-2 on Streptococcus anginosus.

FIG. 2 is a view showing the combined effect of hBD-2 and antipaine on Streptococcus anginosas.

FIG. 3 is a view showing the combined effect of hBD-2 and amastine on Streptococcus anginosas.

FIG. 4 is a view showing the combined effect of hBD-2 and aprotinin on Streptococcus angiosus.

FIG. 5 is a view showing the combined effect of hBD-2 and bestatin on Streptococcus angiosus.

FIG. 6 is a diagram showing the combined effect of hBD-2 and chymostatin on Streptococcus anginosus.

FIG. 7 is a graph showing the effect of using hBD-2 and 3,4-DCI in combination on Streptococcus angiosus.

FIG. 8 is a view showing the combined effect of hBD-2 and E-64 on Streptococcus angionosus.

FIG. 9 is a view showing the combined effect of hBD-2 and EDTA on Streptococcus anginosas.

FIG. 10 is a view showing the combined effect of hBD-2 and leuhistin on Streptococcus angiosus.

FIG. 11 is a view showing the combined effect of hBD-2 and leupeptin on Streptococcus angiosus.

FIG. 12 is a view showing the effect of using hBD-2 and AEBSF in combination on Streptococcus angionosus.

FIG. 13 is a view showing the combined effect of hBD-2 and pepstatin on Streptococcus anginosus.

FIG. 14 is a view showing the combined effect of hBD-2 and phosphoramidon on Streptococcus angiosus.

FIG. 15 is a view showing the combined effect of hBD-2 and trypsin inhibitor on Streptococcus angiosus.

FIG. 16 is a view showing the combined effect of hBD-2 and TPCK on Streptococcus angiosus.

FIG. 17. hB against Streptococcus mitis
It is a figure showing the effect of D-2.

FIG. 18. hB against Streptococcus mitis
It is a figure which shows the combined effect of D-2 and anti-pain.

FIG. 19. hB against Streptococcus mitis
It is a figure which shows the combined effect of D-2 and amasutine.

FIG. 20: hB against Streptococcus mitis
It is a figure which shows the combined effect of D-2 and aprotinin.

FIG. 21. hB against Streptococcus mitis
It is a figure which shows the combined effect of D-2 and bestatin.

FIG. 22: hB against Streptococcus mitis
It is a figure which shows the combined effect of D-2 and chymostatin.

FIG. 23. hB against Streptococcus mitis
It is a figure which shows the combined effect of D-2 and 3,4-DCI.

FIG. 24. hB against Streptococcus mitis
It is a figure which shows the combined effect of D-2 and E-64.

FIG. 25. hB against Streptococcus mitis
It is a figure which shows the combined effect of D-2 and EDTA.

FIG. 26. hB against Streptococcus mitis
It is a figure which shows the combined effect of D-2 and leuhistin.

FIG. 27. hB against Streptococcus mitis
It is a figure which shows the combined effect of D-2 and leupeptin.

FIG. 28. hB against Streptococcus mitis
It is a figure which shows the combined effect of D-2 and AEBSF.

FIG. 29. hB against Streptococcus mitis
It is a figure which shows the combined effect of D-2 and pepstatin.

FIG. 30. hB against Streptococcus mitis
It is a figure which shows the combined effect of D-2 and phosphoramidone.

FIG. 31. hB against Streptococcus mitis
It is a figure which shows the combined effect of D-2 and a trypsin inhibitor.

FIG. 32. hB against Streptococcus mitis
It is a figure which shows the combined effect of D-2 and TPCK.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 31/04 A61P 43/00 111 43/00 111 A61K 37/02 (72) Inventor Akiko Eto Shinjuku, Tokyo 1-23-1-1, Toyama-ku, National Institute for Infectious Diseases (72) Inventor Sho Imai 1-23-1-1, Toyama, Shinjuku-ku, Tokyo National Institute for Infectious Diseases (72) Inventor, Toshiki Nishizawa Tokyo 1-23-1-1 Toyama, Shinjuku-ku, Tokyo National Institute for Infectious Diseases (72) Inventor Nobuhiro Hanada F-term (Reference) 41-208-1, Toyama, Shinjuku-ku, Tokyo National Institute for Infectious Diseases 4C084 AA02 AA17 CA18 DC32 NA14 ZA661 ZA671 ZA891 ZB351 ZC202

Claims (7)

[Claims] 1. An antibacterial agent against a streptococcus group comprising an antibacterial peptide as an active ingredient. 2. The group of streptococci is Streptococcus pneumonia, Streptococcus pyogenes, Streptococcus cariogen , Streptococcus gordonii , Streptococcus sanguis ( S. sanguis ), S. cricetus . Streptococcus Ratusu (S. rattus), antibacterial agent according to claim 1, wherein at least one selected from the group consisting of Streptococcus salivarius (S.salivarius). 3. The antibacterial agent according to claim 1, wherein the antibacterial peptide is human β-defensin-2. 4. The antibacterial agent according to claim 1, further comprising a protease inhibitor. 5. The antibacterial agent according to claim 4, wherein the protease inhibitor is a serine protease inhibitor. 6. A method for sterilizing a group of streptococci, comprising using the antibacterial agent according to any one of claims 1 to 5. 7. A Streptococcus anginosus, which comprises using the antibacterial agent of claim 4 or 5, wherein (S.anginosus), Streptococcus con Suterata scan (S.constellatus), Streptococcus intermedius (S.intermedius) or sterilization method of Streptococcus mitis (S.mitis).