JP2920125B2 - Antimicrobial multiple antigen peptides - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antimicrobial peptide for medical preparations having antimicrobial activity against Escherichia coli, Staphylococcus aureus or fungi, and more particularly to an antimicrobial multiple antigenogen peptide and a positive active ingredient containing the same. It relates to an ionic antibacterial agent.

[0002]

2. Description of the Related Art Lactoferrin has a molecular weight of about 80,000 (70%).
(3 amino acids), which is a protein having antibacterial activity contained in breast milk, tears and the like. Human lactoferrin is expected to be used as a remedy for various symptoms associated with prevention of infectious diseases and immunity, trauma, and eye drops, and is also used as an additive for infant formula.

Incidentally, the journal of the European Biochemical Society (FEB)
S: Federation of European Biochemical Societies)
1996, No. 382, pp. 175-178, describes 46 bases of lactoferrin and its whole loop 20-.
35 bases (HLT1) and short peptide Phe Gln Tr
p Gln Arg Asn Met Arg Lys Val Arg The antibacterial effect of 24-35 bases (HLT2) was tested on Escherichia coli strain (strains) 800.
7 and ML35 using Edwards et al. Are described.

According to the report, lactoferrin itself has almost no antibacterial activity, but the short peptide HL
For T1 or HLT2, 10 ⁷ CFU / ml of control E. coli (CFU indicates colony forming unit) at about 300 μM was added to 300 CFU.
/ Ml, that is, HTL1 or HTL2 shows stronger antibacterial action than whole lactoferrin. In addition, H to which 16 amino acids are bound
It has also been described that LT1 shows stronger antibacterial activity than HLT2, which is 5 residues shorter than this.

Further, Japanese Patent Application Laid-Open No. 5-92994 discloses that
Lactoferrin is converted into a short peptide using an acid or an enzyme, and the results of an antibacterial activity test using 26 Gram-negative strains, 14 Gram-positive strains and two yeasts are described.

[0006]

However, according to the above-mentioned Japanese Patent Application Laid-Open No. 5-92994, JCM 173 of Aspergillus fumigatus is disclosed.
The 9I strain and Rizopus oryzae showed resistance to short peptides derived from lactoferrin, and these short peptides did not exhibit sufficient antibacterial properties. Further, there is no example in which HLT2 having a weaker antibacterial activity than HLT1 is actively used.

[0007] Accordingly, an object of the present invention is to provide a peptide exhibiting superior antibacterial activity as compared with a conventional product by utilizing a specific short peptide having an antibacterial effect in the amino acid sequence constituting lactoferrin. Another object of the present invention is to provide an antibacterial agent having such antibacterial properties.

[0008]

In order to solve the above-mentioned problems, the present invention provides, in the present invention, the C-terminal of a peptide consisting of the following amino acid sequence at the N-terminal of 16 branched peptides to which 15 lysines (Lys) are linked. Are linked to each other to obtain an antimicrobial multiple antigen peptide. Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg or a cationic antibacterial agent containing the above antibacterial multiple antigenogen peptide as an active ingredient, preferably with a concentration (molar concentration) of the active ingredient of 2 μM or more. It was an ionic antibacterial agent.

The antimicrobial multiple antigen peptide having the above structure is obtained by linking a short peptide having the predetermined amino acid sequence to 16 N-terminals of a branched peptide having 15 lysines (Lys) linked thereto. It exhibits excellent antibacterial properties (shown in Examples described later) that could not be exhibited only by short peptides having a predetermined amino acid sequence.

The antimicrobial multiple antigenogen peptide is a cationic peptide, and its antibacterial action is different from the inhibition of bacterial growth, that is, inhibition of DNA synthesis or synthesis enzyme, and causes a change in the membrane potential difference of the bacterial membrane. This is the action of breaking the film. Therefore, the above-mentioned antimicrobial multiple antigenogen peptide hardly provides resistance to bacteria.

[0011] Further, normal antimicrobial peptides are extracted from fungi and the like, and have strong side effects on the human body. However, since lactoferrin is a component derived from a living body, it is considered that it is harmless to the human body and has no side effects.

[0012]

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 known peptide preparation method such as a chemical synthesis method. Specifically, it can be produced by a technique such as chemical synthesis or genetic recombination.

When a peptide is synthesized 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 cross-linked polystyrene, and a commercially available computer controlled by a microcomputer using the amino acid at the C-terminal as a starting point. N-α-t-butoxycarbonylated amino acid (Bo
For c-amino acid) or N-α-9-fluorenylmethoxycarbonylated amino acid (Fmoc-amino acid), the necessary peptide chain is extended in order by removing the protecting group appropriately. Incidentally, as a manufacturing company or a selling company of the peptide synthesizer, there are Pharmacia Biotech, PerkinElmer Japan, Aloka, Shimadzu, and the like.

A 16-fold MAP of short peptide (Mu
ltiple antigen peptide) is prepared by using Boc-Lys (Boc) on an insoluble support such as cross-linked polystyrene.
Alternatively, a method of synthesizing a branched peptide using Fmoc-Lys (Fmoc), fixing the C-terminal amino acid of the peptide serving as an antigen thereto, and growing the peptide starting from the amino acid can be adopted. Boc is a tertiary butyloxycarbonyl group, and Fmoc is a fluorenylmethoxycarbonyl group.

That is, as an example of the synthesis of a 16-fold MAP, lysine is dissolved in dioxane water, 1 mol of NaOH and (Boc) ₂ O are added while mixing under ice-cooling, and the mixture is mixed at room temperature for about 30 minutes. Concentrate in vacuo, then 5% KHSO
₄ in acidified (pH 2.3), and extracted with E _t OAc,
After washing all E _t OAc, then dried over Na ₂ SO _4, further concentrated under reduced pressure and crystallized, examples of synthesizing branched peptides 15 lysine (Lys) are linked and the like.

In order to grow the peptide, the Boc method, which is a method for synthesizing the peptide by a conventional solid phase synthesis method, or the Fc method,
The moc method can be adopted. At this time, it should be noted that since the number of reaction points increases, the initial Lys content is set to 0.1 mm.
ol / g, after the amino acid condensation reaction for growing the peptide chain, the unreacted amino group must be acetylated to prevent the subsequent elongation of the peptide chain. Try to prevent generation.

The amount of the active ingredient of the antimicrobial agent comprising the antimicrobial multiple antigen peptide having a predetermined amino acid sequence of the present invention as an active ingredient is preferably set individually under optimum conditions, but is apparent from the experimental results described later. Thus, the concentration of the multiple antigen peptide as an active ingredient was 2%.
It is preferably at least μM.

The antimicrobial agent of the present invention may be used as an external medicine such as a pharmaceutical product, a quasi-drug, or a cosmetic, an oral disinfectant, a food, a sanitizing agent for sanitary use such as a hospital, kitchen, or laundry, and a horticultural plant. It can be used as an antibacterial agent for use (preservative, bactericide), and various antibacterial agents include well-known pharmacologically acceptable excipients and carriers, infiltrant, surfactant, alcohol, fragrance,
Physiological saline may be included.

Examples of the form of the preparation include solid preparations such as tablets and granules, liquid preparations such as solutions and suspensions (particularly, liquid preparations for spraying), and other well-known dosage forms.

[0020]

【Example】

[Example 1] J. Am. Chem., 85, 2149, using an automatic peptide synthesizer (ABI 431A, manufactured by Biosystems).
(1963) (using BOP as a condensing agent in a solid phase synthesis method by the t-Boc method) and Biochemistry Vol. 85, pp. 5409-5413.
(Issued in August 1988)
SP. MAP was produced according to the method of TAM).

That is, a resin obtained by introducing a functional group into a polystyrene resin cross-linked with divinylbenzene (hydroxymethylphenylacetamide resin (also called PAM resin) or 4-methylbenzhydrylamine resin)
An automatic peptide synthesizer using Boc-Lys (Boc) was used.

In an automatic peptide synthesizer, Boc-Ala
0.03 mmol of PAM resin (1/16 of 0.5 mmol) and 0.09 mmol of Boc-Lys (Boc) -OH
1 was coupled with BOP. Next, Boc-Ly
After coupling s (Boc) -OH 0.09 mmol, Boc-Lys (Boc) -OH 0.18 mmol
And Boc-Lys (Boc)
0.36 mmol of -OH and 0.72 mmol of -OH were sequentially coupled. Thus, a branched peptide (MAP16 core) in which 15 lysines (Lys) were linked was synthesized. FIG. 1 shows this synthesis step, and FIG. 2 shows the structure of the core of MAP16 cut out of the resin for reference. Note that K and A in FIG. 1 are Lys and A, respectively.
It is a schematic representation of la.

Next, arginine (Arg) was immobilized by the automatic peptide synthesizer, and the amino acid sequence was added to the 16 N-terminals of the branched peptide by the well-known Boc method using Phe Gln Tr.
A peptide consisting of p Gln Arg Asn Met Arg Lys Val Arg was prepared, and a 16-fold MAP (Multiple antigen peptide) comprising 16 units of a short peptide was produced. The protecting group was removed with a solvent and the peptide was removed. Was.

Solid phase synthesis (Bo) using an automatic peptide synthesizer
In method c, 0.5 mmol scale), a predetermined amino acid is linked to the resin from the C-terminus by repeating the following mechanical operations. The amount of the solution during the operation is about 5
10 ml.

De-Boc removal 50% TFA / dichloromethane 5 min vortex (stirring) → discharge solution → 50% TFA / dichloromethane 30 min vortex → discharge and wash the solution (dichloromethane 1 min vortex → discharge the solution) × 3 times → (DMF 1 min vortex) → solution discharge) × 3 times condensation Boc- amino acid 1.5mmol / DMF, B
OP 1.5 mmol / DMF, DIEA 2 mmol 40
min → discharge washing (DMF 1 min vortex → discharge solution) × 3 times → (dichloromethane 1 min vortex → discharge solution) × 3 times.

Next, at the same time as the cleavage from the resin, the protecting group of the amino acid side chain was deprotected with hydrogen fluoride (HF). That is, 500 mg of the resin, 1 ml of m-cresol, 0.7 ml of ethanediol, and 0.5 ml of dimethyl sulfide were placed in an HF reaction tube, and mounted on an HF apparatus. The reaction tube was cooled to −78 ° C. under reduced pressure, and 10 ml of HF was introduced into the reaction tube. After stirring at 0 ° C. for 1 hour, HF was distilled off.
Add anhydrous ether, transfer the residue to a glass filter,
Wash well with ether dichloromethane. The peptide was dissolved in 10% aqueous acetic acid, and the filtrate was lyophilized.

The obtained peptide (MAP) was purified by high performance liquid chromatography (HPLC) using a reversed phase column. The HPLC conditions were as follows: an inner diameter of 4.6 mm manufactured by Wako Pure Chemical Industries, which was packed with _C18 silica gel having a particle size of 5 μm,
250mm long column (Wakosil-2, 5C18 A
R), the initial solvent was 0.1%
A mixed solution of 5% acetonitrile (CH ₃ CN) in an aqueous solution of tetrafluoroacetic acid (TFA) was prepared, and a gradient of acetonitrile was formed over time in the range of 5 to 65% at a flow rate of 1.5 ml per minute. Elution was performed for 30 minutes.

As a result of HPLC, a peak of absorbance was detected using an ultraviolet detector having a wavelength of 220 nm, and the result is shown in FIG. The peak report at that time is as follows.

Peak number Detection time (min) Area Height Concentration 1 13.6 46652 3157 1.0 2 15.5 4776076 30762 98.5 3 23.0 22408 1499 0.5 As shown in FIG.

[Comparative Example 1] J. Am. Chem., 85, 2149, using an automatic peptide synthesizer (manufactured by Biosystems: 431).
According to the chemical synthesis method described in (1963) (BOP is used as a condensing agent in the solid phase synthesis method by the t-Boc method), the amino acid sequence is Phe Gln Trp Gln Arg Asn Met Arg Lys
A peptide consisting of Val Arg was prepared and purified by high performance liquid chromatography (HPLC) using a reversed-phase column under exactly the same conditions as in Example 1.

The HPLC conditions were as follows: an ultraviolet detector having a wavelength of 220 nm was used to detect the peak of the absorbance, and the results were shown in FIG.
It was shown to. The peak report is as follows.

Peak number Detection time (minutes) Area Height Concentration 1 11.4 31882 777 1.1 2 11.9 28608899 2007070 98.5 3 12.3 128984 6075 0.4 Implementation obtained as above The following antibacterial test 1 was performed on Example 1 (MAP-16) and Comparative Example 1 (short peptide), and the results are shown in Table 1.

<< Antibacterial Test 1 >> Examples 1 and Comparative Example 1 were diluted with sterile physiological saline to obtain samples having the concentrations shown in Table 1. Then, Escherichia Coli standard strain (ATCC 25) suspended in 1% peptone water was added to each sample.
922), Staphylococcus aureus
ATCC 25923) or fungi (Candida albican)
s: JCM 2900) obtained from RIKEN was mixed and placed in a test tube so that the final bacterial concentration was 1 × 10 ⁷ CFU / ml. Then, after reacting at 37 ° C. for 2 hours, each sample was mixed with a blood agar medium (manufactured by Becton / Deckinson), and further cultured at 37 ° C. for 48 hours, and the state of bacterial growth of each sample was examined. The growth of the bacteria was CFU (1 colony corresponds to 100 cells,
Edward et al., Journal of the European Biochemical Society (FEBS: Fede)
ration of European Biochemical Societies), 199
6 years, No. 382, pages 175 to 178, the same as the evaluation method. ). In addition, 3 for each concentration
The measurement was performed three times using one sample (n = 3), and the values in Table 1 are the average values.

[0034]

[Table 1]

As is clear from the results shown in Table 1, the antimicrobial multiple antigen peptide of Example 1 was obtained from Escherichia coli 1
Since 0 ⁷ CFU / ml was antimicrobial at a concentration of 20 μM, and the short peptide of Comparative Example 1 was antimicrobial against the same amount of E. coli at a concentration of 20 mM, Example 1 showed 100 times the antimicrobial activity of Comparative Example 1.

Similarly, Example 1 showed 400 times the antibacterial activity against Staphylococci and fungi as compared with Comparative Example 1. These antibacterial properties show that the antimicrobial multiple antigen peptide of Example 1 not only has an amino acid sequence derived from 16 lactoferrins, but also has the amino acid sequence of lysine (Lys).
This was considered to have been obtained by linking to a branched peptide to which 15 were linked to form a MAP type.

Example 2 Example 2 was changed to 2 μM, 20 μM
The solution was diluted with sterile physiological saline to a concentration of M, 200 nM, or 100 nM to produce a liquid antibacterial agent that can be used for a skin washing solution or the like.

The following Example 2 (antibacterial agent) was subjected to the following antibacterial test 2. The results are shown in Table 2.

<< Antibacterial test 2 >> Escherichia coli
i) A standard strain (ATCC 25922), Staphylococcus aureus ATCC 25923 or a fungus (Candida albicans: JCM 2900 obtained from RIKEN) were cultured on a blood agar medium (manufactured by Becton Dekinson). That is, each of the stock strains was cultured for 48 hours at 37 ° C., and one platinum loop was applied to a separately prepared blood agar medium. Then, the peptide of each concentration of Example 1, which was immediately put into a 25 ml glass container sprayer (spray), was placed about 10 cm above the medium.
Spray vertically once from a distance of
1) After that, the cells were cultured at 37 ° C. for 48 hours, and an antibacterial test 1
The growth status of the bacteria in each sample (CFU
Number). In addition, it measured three times using three samples for each density | concentration, and the numerical value in Table 2 is the average value.

[0040]

[Table 2]

From the results shown in Table 2, it can be seen that the antimicrobial agent containing the antimicrobial multiple antigen peptide of Example 1 as an active ingredient infects Escherichia coli at 10 ⁷ CFU / ml at a concentration of 2 μM. Concentration 2μM
It is understood that the above is preferable.

[0042]

As described above, the invention relating to the antimicrobial multiple antigen peptide of the present invention relates to an antimicrobial multiple antigen in which a short peptide having a predetermined amino acid sequence derived from lactoferrin is linked to 16 N-terminals of a branched peptide. Since it is an antigenic peptide, it has an advantage that it is a peptide exhibiting excellent antibacterial properties as compared with a peptide having a predetermined amino acid sequence constituting lactoferrin alone.

Further, the invention relating to the cationic antibacterial agent has the advantage that it has the above antibacterial properties, hardly imparts resistance to bacteria, is harmless to the human body, has few side effects, and has excellent antibacterial properties. There is.

[0044]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg 1 5 10

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is an explanatory diagram of a synthesis process of a branched peptide (core of MAP16).

FIG. 2 is an explanatory diagram showing the structure of a branched peptide in which 15 lysines (Lys) are linked.

FIG. 3 Example 1 consisting of a branched peptide (MAP)
Illustration showing the amino acid sequence of

FIG. 4 is a chart showing an HPLC measurement chart of Example 1.

FIG. 5 is a chart showing an HPLC measurement chart of Comparative Example 1.

Continuation of the front page (73) Patent holder 596141952 Higashi Narumi 137-1 Shiomi-cho, Showa-ku, Nagoya-shi, Aichi 1 (72) Inventor Carlos Adriel del Carpio 32-2-2202, Hamamichi-cho, Toyohashi-shi, Aichi ) Inventor Yasuki Kojima 1 at Tawara, Tahara-cho, Atsumi-gun, Aichi, 20-family Heights 202 (56) Reference FEBS Lett. , Vol. 382 (1996) p. 175-8 Methods Enzymol. , Vol. 289 (1997) p. 612-37 Proc. Natl. Acad. Sc i. U. S. A. , Vol. 85 (1988) p. 5409-13 Biochemistry, Vol. 27 (1988) p. 5640-5 (58) Fields investigated (Int. Cl. ⁶ , DB name) C07K 14/79 ZNA A01N 37/44 CA (STN) REGISTRY (STN) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. An antimicrobial multiple antigen peptide obtained by linking the C-terminal of a peptide having the following amino acid sequence to 16 N-terminals of a branched peptide to which 15 lysines (Lys) are linked. Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg 2. A cationic antibacterial agent comprising the antimicrobial multiple antigenogen peptide according to claim 1 as an active ingredient. 3. The cationic antibacterial agent according to claim 2, wherein the concentration of the active ingredient is 2 μM or more.