JP2003040796A - Antichlamydia agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antichlamydia agent surely having antibacterial activity such as washing and sterilization against chlamydiae, especially an antichlamydia agent containing an antibacterial peptide having high antibacterial activity before the infection or directly after the infection of chlamidia, and having excellent infection preventive effect and washing effect, and at the same time free from side effects and evils to a human body, as an active ingredient. SOLUTION: The antichlamydia agent contains one or more kinds of peptides selected from a peptide group consisting of amino acid sequences expressed by the sequence numbers 1-6 of the sequence table (refer to the specification), as active ingredients. Outside a cell, the peptide inhibits the adhesion on and the intrusion into a cell of chlamydia by causing the change in potential difference of the cell membrane in chlamidia (EB) having a cell wall same as in the cell of a gram negative bacterium.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anti-Chlamydia agent that suppresses infection of humans with chlamydia, which is a pathogenic microorganism, and growth after infection.

[0002]

2. Description of the Related Art Chlamy which is a pathogenic microorganism
Since 1974, the genus dia) has been classified as an independent genus, neither a bacterium nor a virus. Chlamydia trachomatis, Chlamydia pneumoniae
(C. pneumoniae), Chlamydia cisttaci (C. psittaci) 3
The species is known. Of these, Chlamydia shittasi is
It is a pathogen common to both humans and animals, and is a pathogen of Aum disease that causes pneumonia by infecting humans from birds, and Chlamydia pneumoniae is known as a pathogen of respiratory infections that infect humans from humans.

[0003] Chlamydia trachomatis is a causative agent of eye infections and urogenital infections, and causes neonatal infections of the eyes and respiratory organs of newborns due to birth canal infection, and in Japan, sexually transmitted diseases: STD)
Is the main pathogenic bacterium.

Unlike the bacterium, the species of the genus Chlamydia, which is such a pathogenic microorganism, has a life cycle that proliferates in a living cell unlike a bacterium, and has a nucleic acid of both DNA and RNA. It is a different pathogen.

By the way, the life cycle of Chlamydia (48 to 72 hours per cycle) will be briefly described as follows.
Chlamydia consists of an elementary body (EB) that propagates from individual to individual or from host cell to cell, and a reticulate that is a non-infectious, mitotic phase form.
There is a time when it becomes body: RB).

[0006] When EB (a sphere having a diameter of about 0.3 µm) is invaded into a host cell and infected, the EB is transformed into an RB (a sphere having a diameter of about 0.5 µm) and further proliferated twice. After that, it matures through an intermediate, is released as EB again, and infects another host or is transferred to another cell of the same host.

Tetracycline antibiotics such as tetracycline, doxycycline, and minocycline are most recommended as conventional anti-chlamydia agents that suppress the infection of human pathogenic microorganisms of the genus Chlamydia and the growth after infection. Next, macrolide antibiotics such as erythromycin and rifampicin have excellent antibacterial activity. In addition, clindamycin, penicillin-G, ampicillin, etc. are known to show some anti-Chlamydia action.

[0008] The mechanism of action of antibiotics such as tetracyclines on Chlamydia utilizes the specificity of tetracycline for ribosomes in host cells and intracellular ribosomes of Chlamydia. That is, the host animal ribosome has a protein precipitation constant of 8
0S, which is composed of 40S and 60S subunits, while the intracellular ribosome of Chlamydia has a protein precipitation constant of 70S, which is 3S.
It is composed of 0S and 50S subunits.

Depending on the difference in the structure of ribosomes, tetracycline antibiotics act on intracellular ribosomes of chlamydia, which is a smaller subunit,
It is said to inhibit the protein synthesis.

[0010]

However, conventional tetracycline antibiotics are therapeutic agents after chlamydia infection, but they are anti-chlamydia agents that positively improve the EB infection preventive property in the life cycle of chlamydia. Without
There is no preventive drug that has a high infection prevention effect or a high cleaning effect immediately after infection, such as a genital cleaning agent or a medical device cleaning agent for preventing STD infection.

In addition, conventional antibiotics have the problems that accidental generation of multidrug-resistant bacteria due to incorrect usage and side effects due to toxin production occur. There is a demand for an anti-Chlamydia agent that does not have side effects or adverse effects on.

Further, the antibacterial activity of a given peptide was known for pathogenic microorganisms that can grow in media other than living cells, but the experimental method was different for pathogenic microorganisms of the genus Chlamydia that can grow only in living cells, The judgment as to whether or not it becomes an active ingredient cannot be made without positive experiments using living cells.

Therefore, an object of the present invention is to solve the above-mentioned problems of the prior art and surely have an antibacterial action such as washing and sterilization against chlamydia, and especially, the antibacterial property before or immediately after infection with chlamydia. It is an object of the present invention to provide an anti-chlamydia agent containing an antibacterial peptide as an active ingredient, which is highly effective in preventing infection and cleaning and has no side effects or harmful effects on the human body.

[0014]

In order to solve the above problems, in the present invention, one or more peptides selected from the peptide group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 6 in the sequence listing are used as active ingredients. It was the anti-Chlamydia agent contained.

The peptide used in the present invention is an antibacterial peptide derived from human plasma components, and is apoprotain E (apo
-E) is a 30-residue peptide in part (133-162), and there is knowledge of antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella, etc., but antibacterial activity against Chlamydia is unknown. It was

That is, the peptide used in the present invention is
Extracellularly, it is considered that, like Chlamydia (EB), which has a cell wall like the cells of Gram-negative bacteria, is mutated in the potential difference of the cell membrane to suppress attachment and entry into cells.

In addition, anti-Chlamydia trachomatis (Chlamydia trachom) containing peptides comprising the amino acid sequences represented by SEQ ID NOs: 1 to 6 in the sequence listing as an active ingredient
As is clear from the experimental results described below, the atis) agent seems to surely suppress Chlamydia trachomatis from adhering to or entering cells by the above-mentioned action.

The peptides of SEQ ID NOS: 2 to 6 are shortened (deletion) forms of the peptide of SEQ ID NO: 1, and the peptides of SEQ ID NOS: 5 and 6 are partially substituted forms of the peptide of SEQ ID NO: 1. And these are considered to have similar activity to the peptide of SEQ ID NO: 1.

Therefore, the use of the anti-chlamydia agent containing one or more peptides consisting of the predetermined amino acid sequence of the present invention as an active ingredient is, for example, genital washing sterilization such as eye washing solution, vaginal suppository or vaginal washing solution. It can be applied as an agent and other external preparations, an antibacterial agent used by being added to a laundry detergent at the time of washing, an antibacterial agent used by being held in antibacterial textiles and the like.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION A peptide having a predetermined amino acid sequence according to the present invention can be produced by a well-known peptide preparation method such as a chemical synthesis method. Specifically, it can be produced by methods such as chemical synthesis and gene recombination.

In the case of synthesizing a peptide by the solid phase synthesis method, a C-terminal amino acid of the peptide to be synthesized is immobilized on an insoluble support such as crosslinked polystyrene, and a commercially available computer controlled by a microcomputer is used as a starting point. On the synthesizer, the N-α-t-butoxycarbonylated amino acid (Bo
(c-amino acid) or N-α-9-fluorenylmethoxycarbonylated amino acid (Fmoc-amino acid), the protecting group is appropriately removed to sequentially extend the required peptide chain.

The peptide used in the present invention is -SS-
Since it is a simple linear form that does not contain a bond, it can be easily synthesized by any synthetic method as compared with other antibacterial peptides having a complicated structure.

By the way, as a manufacturing company or a sales company of the peptide synthesizer, there are Pharmacia Biotech Co., Perkin Elmer Japan Co., Aloka Co., Shimadzu, etc.

As the formulation of the anti-Chlamydia agent of the present invention, solid agents such as tablets and granules, liquid agents such as solutions and suspensions, liquid agents for spraying and other well-known dosage forms can be adopted.

Other uses and applications of the anti-Chlamydia agent containing a peptide as an active ingredient include cleaning agents for gynecological, urological, ophthalmological and other test instruments, especially washing of medical instruments such as endoscopes. -A bactericide is included. When it is used as a medical cleaning solution such as an esophageal cleaning solution, a bidet cleaning solution, or a vaginal cleaning solution, it is made isotonic with respect to the living body or is used as an active ingredient in a hypotonic physiological saline solution. .2 to 1.6% by volume is compounded.

Further, the extract obtained is impregnated into a textile product such as a woven or non-woven fabric, and if necessary, it is used in combination with a well-known anti-inflammatory agent, and preferably added to jelly and applied to the affected area of infectious inflammation. Can also be used.

In addition, when washing clothes that may be infected with chlamydia, the anti-chlamydia agent of the present invention is added together with a detergent, that is, it is used as an antibacterial cleaning additive and attached to clothes such as underwear. Chlamydia that are present can also be sterilized and removed.

[0028]

[Examples and Comparative Examples] [Examples 1 to 6] The peptide represented by SEQ ID NO: 1 by a chemical synthesis method using a peptide synthesizer (HATU was used as a condensing agent in the solid-phase synthesis method by the Fmoc method). Was synthesized and purified by high performance liquid chromatography (HPLC) using a reverse phase column. For the HPLC, a column (Kromasil 100C18) with a length of 125 mm packed with C18 silica gel having a particle size of 5 μm was used. The elution conditions were 0.1% tetrafluoroacetic acid aqueous solution (TFA) and 80% acetonitrile aqueous solution. The aqueous solution was supplied to the column at a flow rate of 0.75 ml per minute for 30 minutes with a concentration gradient provided over a time range of 20 to 80%. The peak detection means has a wavelength of 215 nm
Absorbance was detected using the UV detector of.

Similarly, the peptides (A-1, A-2, A-3, B-1, B-2) shown in SEQ ID NOs: 2 to 6 were synthesized.

Next, among the above peptides, the peptide of SEQ ID NO: 1 was used as an active ingredient, and the ratio (μ) shown in Table 1 was added to physiological saline.
(g / ml) blended with anti-chlamydia agent (detergent)
And

Determination of anti-Chlamydia effect (MLC measurement)
Was carried out by the standard method of Japan Chemotherapy Society as follows. That is, the inhibition of growth of chlamydia was evaluated by the inhibition rate (%) determined by the MLC method (presence or absence of destruction of the reticulate body), and the results are also shown in Table 1.

<Chlamydia MLC measurement method (Japan Chemotherapy Association standard method)> A 24-well cell culture plastic plate was used, on which a Heera 22 of 1500-2000 / ml was used.
A culture medium containing 9 cells (Hela cell) was used. The composition of the culture medium was Eagle MEM and heat-inactivated fetal bovine serum,
Add 1 ml to each well of the plate,
After culturing for 24 hours in an incubator at 5 ° C. and 5% carbon dioxide gas (CO ₂ ), after confirming the formation of the conflict monomer of the cells, the culture solution was removed by suction.

4000 / ml chlamydia in this well
0.25 ml of the trachomatis strain (D / UW-3 / CX: distributed by the National Institute of Infectious Diseases) was inoculated, and this was inoculated at 50 at room temperature.
Centrifugal adsorption was carried out for 1 hour by applying a gravity acceleration of 0 to 900G.

The antibacterial liquid (peptide consisting of SEQ ID NO: 1) is diluted by master dilu-
Using Eagle and heat-inactivated fetal bovine serum, the concentration is 0 (control), 25 μg / ml, 50 μg / ml, 100 μ
g / ml, 200 μg / ml, and add 1 ml to the wells in which Chlamydia trachomatis was inoculated into the cells,
After culturing at 37 ° C and 5% CO ₂ for 72 hours, the drug in the wells was removed by suction, and the wells were washed 3 times with serum-free Eagle MEM, and 1 m of heat-inactivated fetal bovine serum supplemented with Eagle MEM was added.
Then, the cells were incubated at 37 ° C. and 5% CO ₂ for 24 hours, and the antibacterial effect was examined as follows.

That is, the culture solution in the cultivated well was aspirated, 1 ml of 100% ethanol was added to the well, the cells were fixed, stained by the DFA method, and converted from the rate at which Chlamydia infected the inclusion body of Helas. The inhibition rate was evaluated, and the results are shown in Table 1.

The rate of Chlamydia infecting the inclusion bodies of Helas was examined by counting the number of inclusion bodies observed in one visual field with a fluorescence microscope at 200 times.

[0037]

[Table 1]

In addition, as a comparative example, in place of the above-mentioned peptide, minomycin, which is a typical example of conventional antibacterial agents, was added to 0.0
25 μg / ml, 0.5 μg / ml, 1 μg / ml, 2
The inhibition rate was evaluated from the rate of infection of Chlamydia with the inclusion body of Helas in the same manner as above except that the addition was performed at a concentration of μg / ml, and the results are also shown in Table 1.

As is clear from the results shown in Table 1, the anti-Chlamydia agents of the Examples contained 2 peptides each having a predetermined amino acid sequence.
By containing it at a concentration of 5 μg / ml or more, RB corresponding to the period of Chlamydia infection was destroyed and the antibacterial property was clearly shown.

Further, peptides (A-1, A-2, A-3, B- which consist of the amino acid sequences represented by SEQ ID NOs: 2 to 6)
1, B-2) was also evaluated by the above-mentioned MLC method in exactly the same manner, and almost the same result as the above-mentioned result was obtained. From this, it was confirmed that the peptides of SEQ ID NOS: 2 to 6 can also be an active ingredient of the anti-Chlamydia agent.

[0041]

INDUSTRIAL APPLICABILITY As described above, the present invention provides an anti-Chlamydia agent containing a peptide having a predetermined amino acid sequence as an active ingredient. It is an anti-Chlamydia agent that has an antibacterial action such as washing and sterilization against Chlamydia, because it cannot adhere to or enter into.

Therefore, the anti-Chlamydia agent of the present invention has a high antibacterial property before or immediately after infection with Chlamydia, and has an advantage of becoming an anti-Chlamydia agent having excellent infection preventive effect and cleaning effect.

Further, the anti-Chlamydia agent of the present invention is, in particular, Chlamydia trachomatis.
It becomes an anti-Chlamydia agent with a certain effect of preventing infection against.

[0044]

[Sequence list]                               SEQUENCE LISTING <110> Kabushikigaisha enkaku-iryou kenkyusho <120> Anti-chlamidia agent <130> KPO5470-04 <160> 6 <210> 1 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: coji4 <400> 1 Leu Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu Arg Lys Arg Leu   1 5 10 15 Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala Val Tyr              20 25 30 <110> Kabushikigaisha enkaku-iryou kenkyusho <120> Anti-chlamidia agent <130> KPO5470-04 <160> 6 <210> 2 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: A-1 <400> 2 Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu Arg Lys Arg Leu Leu   1 5 10 15 Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu              20 25 <110> Kabushikigaisha enkaku-iryou kenkyusho <120> Anti-chlamidia agent <130> KPO5470-04 <160> 6 <210> 3 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: A-2 <400> 3 Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu Arg Lys Arg Leu Leu   1 5 10 15 Arg Asp Ala Asp Asp Leu              20 <110> Kabushikigaisha enkaku-iryou kenkyusho <120> Anti-chlamidia agent <130> KPO5470-04 <160> 6 <210> 4 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: A-3 <400> 4 Arg Val Arg Leu Ala Ser His Leu Arg Lys Leu Arg Lys Arg Leu Leu   1 5 10 15 Arg Asp <110> Kabushikigaisha enkaku-iryou kenkyusho <120> Anti-chlamidia agent <130> KPO5470-04 <160> 6 <210> 5 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: B-1 <400> 5 Leu Arg Val Arg Leu Ala Ser His Leu Ala Lys Leu Ala Lys Ala Leu   1 5 10 15 Leu Ala Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala Val Tyr              20 25 30 <110> Kabushikigaisha enkaku-iryou kenkyusho <120> Anti-chlamidia agent <130> KPO5470-04 <160> 6 <210> 6 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: B-2 <400> 6 Leu Arg Val Arg Leu Ala Ser His Ala Arg Lys Ala Arg Lys Arg Ala   1 5 10 15 Ala Arg Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala Val Tyr              20 25 30

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 4C084 AA02 BA01 BA08 BA18 BA19                       BA23 MA01 NA14 ZB322                       ZB352                 4H045 AA30 BA18 CA40 EA29 FA34                       FA72 FA74

Claims (2)

[Claims] 1. An anti-Chlamydia agent containing, as an active ingredient, one or more peptides selected from the peptide group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 6 in the sequence listing. 2. An anti-Chlamydia trachomatis containing a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing as an active ingredient.
Agent.