JPH07126172A - Lps-containing anti-mrsa agent and anti-mrsa agent for animal - Google Patents

Abstract

PURPOSE:To obtain an anti-MRSA agent usable for a long period of time, having excellent anti-MRSA effect, containing a lipopolysaccharide (LPS) produced by a microorganism belonging to the genus Panthera. CONSTITUTION:This anti-MRSA agent contains LPS as an active ingredient. The anti-MRSA agent (anti-methicillin-resistant Staphylococcus aureus agent) has excellent anti-MRSA effect including preventive effect, can be medicated for a long period of time by any route of oral administration, percutaneous administration, injection or intradermic injection and exhibits excellent effect especially by intradermic injection. Since the anti-MRSA agent is not an antibiotic, it will not bring about a new resistant bacterium. A dose is 1mug to 100mg per adult (60kg) by oral administration, 1ng to 10mg by intravenous administration and 1ng to 10mg by intradermic injection and is administered once a day.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anti-methicillin-resistant Staphylococcus aureus (hereinafter, "methicillin-resistant Staphylococcus aureus" is referred to as "MRSA") agent containing LPS. More specifically, it relates to an anti-MRSA agent containing LPS produced from Pantoea.

[0002]

MRSA is a multidrug-resistant Staphylococcus aureus that is resistant to methicillin and other antibacterial agents.

[0003] Methicillin was originally a semi-synthetic penicillin that was developed and used around 1960 as an antibacterial agent against multidrug-resistant Staphylococcus aureus that is resistant to broad spectrum antibiotics such as tetracycline. It is a first-generation cepham anti-substance. This first-generation and subsequent second-generation cepham antibiotics were antibacterial against multidrug-resistant Staphylococcus aureus, but in the 1980s, in order to cope with intractable bacterial infections such as Pseudomonas aeruginosa The developed and abused third-generation cepham antibiotics had weak antibacterial activity against staphylococci, so that MRSA selectively proliferated as a bacterium or induced with resistance to mesitillin or the like.

MRSA is not more toxic than ordinary Staphylococcus aureus, and generally has a good course in superficial infections, and has almost the same sensitivity to various disinfectants. However, deep infections cause serious infections and many antibiotics are ineffective. In particular, nosocomial infections have recently attracted attention due to the aging of hospitalized patients due to the extension of life, the increased use of immunosuppressants, the introduction of highly invasive medical care, and the like. However, at present, there is no other effective way to deal with post-operative immunity decline in patients than relying on high doses of antibiotics. (Above, "Ministry of Health and Welfare National Hospital Division / National Sanatorium Division supervision," Guide for nosocomial infection control-Focusing on MRSA "", pages 1-38, 1992, published by Nankodo).
Therefore, at present, MRSA infection is inevitable.

In view of the current situation, various anti-MRSA agents have been developed and used. Oral drugs effective for MRSA include new quinolone type norfloxacin and ofloxacin. In addition, minocycline, rifampicin, arbekacin, vancomycin and the like are used, and vancomycin is said to be most effective. (Ibid, "Guideline for Nosocomial Infection Control-Focusing on MRSA", page 38).

[0006]

However, in recent years, new quinolone type norfloxacin and the like have increased MRSA resistance to it, and also have serious side effects such as hypersensitivity symptoms and renal / liver disorders. (Previously, "Guide for Countermeasures for Nosocomial Infection-MRS
Pay attention to A- ", p. 38)

[0007] In addition, there is no report of resistance to vancomycin at present ("Guide to Nosocomial Infection Control-Focus on MRSA", page 38), but there are serious side effects such as shock symptoms and hypersensitivity symptoms. In addition, there are many restrictions on use, such as the need for careful administration to patients with renal or hepatic disorders. (The Japan Pharmaceutical Information Center, ed., "Medical Drugs Japan Pharmaceuticals Collection, 1993", pages 897-899, 1993, Pharmaceutical Industry Times Co., Ltd.)

More importantly, all of these conventional anti-MRSA agents are antibiotics, and their administration has a very high risk of producing new resistant bacteria, and their continuous use must be avoided. In addition, there are no reports of preventive effects.

In view of the present situation, the present invention has a remarkable anti-MRSA effect including a preventive effect, can be used for a long time, and can be administered by any of oral, transdermal, injection and intradermal routes. In particular, intradermal administration exerts a remarkable effect, and
It is an object of the present invention to provide a novel anti-MRSA agent which is not an antibiotic and therefore does not lead to the development of new resistant bacteria.

[0010]

[Means for Solving the Problems] The anti-MRSA agent of the present invention is a lipopolysaccharide (LPS) produced by Pantoea bacteria.
(Hereinafter, referred to as LPSp) is included.

An example of LPSp that can be used in the present invention is Japanese Patent Laid-Open No. 5-155778 (Japanese Patent Application No. 33573/1993), which is a published patent publication published on June 22, 1993.
LPS3 whose production method is specifically described in Production Example 4 of No. 51 application).

[0012] This is a pantoair. Agglomerans inoculum (formerly contributed to the Institute of Microbial Engineering, Institute of Industrial Science and Technology, Ministry of International Trade and Industry from 17th August, 1990 as National Institute of Microbiology 11666, and from 12th August 1991 35th
No. 11, which has been transferred to an international deposit under the Budapest Treaty), has the following physical properties as described in the publication.

Main molecular weight: 6,500 ± 2,500 (S
Phosphorus number: 2 ± 1 / molecular weight 5,000 Hexosamine number: 5 ± 1 / molecular weight 5,000 KDO number: 2 ± 1 / molecular weight 5,000

The SDS-2 method is as described in the above-mentioned patent publication, 10 ml of 10% (w / v) sodium dodecylsulfate (SDS), 17.9 g of tricine and 3.03 g of tris. The electrophoretic buffer prepared by dissolving 1 in 1 liter of distilled water was placed in a slab gel electrophoresis tank manufactured by Marisol, 20% polyacrylamide gel was fixed in the electrophoretic tank, and a sample for molecular weight measurement was placed in the sample groove, and the voltage was set to It is a method of measuring the molecular weight by fixing at 50 v for 1 hour and then at 150 v, and continuing the migration until the dye is eluted from the gel.

As shown in Experimental Example 1, the anti-MRSA effect of the anti-MRSA agent of the present invention will be explained later by administration of cyclophosphamide (hereinafter referred to as CY) to MRSA-immunized mice. Then, MRSA was administered to the mice, and the survival and death of the mice were observed.

The anti-MRSA agent of the present invention is a powder, a granule, a pill, a tablet, a troche, a capsule, a liquid, a patch, an ointment, a liniment, a lotion, and a suppository, according to a conventional formulation technique. , Injectables and the like. However, in particular, it has been confirmed that a remarkable effect can be obtained by intradermal administration in the form of an injection. The site of intradermal administration is not particularly limited.

For animals, it can be further prepared as a feed additive, a premix preparation, and a drinking water additive. When used as a feed additive, it is preferably a powder or granules. The premix formulation refers to a product diluted with a feed component such as starch to facilitate mixing with a feed. The feed to which the anti-MRSA agent of the present invention can be added as a feed additive or a premix preparation may be any commercially available feed. It may also be a feed containing feed additives such as minerals, vitamins and amino acids.

If desired, additives such as excipients, preservatives and buffers may be added to these preparations in order to maintain the storability and homogeneity. Furthermore, flavoring agents,
A flavoring agent and a coloring agent can also be included. As the excipient, for example, lactose or starch can be used. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, paraoxybenzoic acid esters such as propyl paraoxybenzoate, sodium dehydroacetate, and the like.
Phenol, methylparaben, ethylparaben, propylparaben and the like can be used. As the buffer, for example,
Citrate, acetate, phosphate and the like can be used.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. These examples and experimental examples are merely examples and do not limit the present invention. In the following description, the amount of LPS is the E. coli LPS [US Difco (Difco (Difco)
Co) 0128: B8] equivalent amount.

Example 1 (tablet) LPS3 0.04 g 6% HPC lactose 178 g talc stearate 8 g potato starch 14 g The above ingredients were mixed and compressed to give 400 0.5 g tablets containing 0.1 mg LPS3. Prepared.

Example 2 (Liquid for internal use) LPS3 1 mg Purified water 100 ml

Example 3 (Ointment)

Example 4 (Injection) LPS3 0.5 mg Distilled water for injection Total amount 1000 ml

Experimental Example 1 (Anti-MRSA effect) BALB / c male mice aged 6 to 8 weeks (body weight 20 to 25)
g) CY administration group (10 animals) and physiological saline administration group (1)
In the former, 200 mg / kg of CY was dissolved in physiological saline and administered to the former. 5 days later 3 × 10 ⁷
CFU (colony forming unit) MRSA was suspended in physiological saline and intravenously administered, and the survival days of the mice were measured.
As a result, in the physiological saline administration group, no deaths were observed even after 7 days, but in the CY administration group, 5 days later.
The remaining 5 animals died after 2 days, and the survival rate was significantly reduced, and it was confirmed that CY administration resulted in MRSA immunocompromised state.

Next, 10 6-8 week old BALB /
c Male mice (body weight: 20 to 25 g) were administered with 200 mg / kg of CY dissolved in physiological saline, and 5 days later, 3
μg / kg of LPS3 was dissolved in physiological saline and administered intraperitoneally, and 3 hours later, 3 × 10 ⁷ CFU of MRS was administered.
A was suspended in physiological saline and intravenously administered, and the survival days of the mice were measured. As a result, a significant increase in the number of survival days was observed, that is, one animal died one day, two days later, and three days later, while the remaining one survived after seven days. 3μ
The amount of g / kg is the same as L for intradermal administration in allogeneic mice.
It corresponds to 1/4700 of D ₅₀ . Therefore, it is clear that the safety of LPS3 is extremely high and that long-term administration is possible.

Dosage amount, administration interval, toxicity value The amount and administration interval of the anti-MRSA agent or the animal anti-MRSA agent of the present invention are, of course, subject to administration under the strict control of the attending physician or veterinarian. It is individually determined in consideration of age, symptoms, body weight, and administration effect, but is 1 μg to 100 m by oral administration in human adult (60 kg) in terms of Escherichia coli LPS [0128: B8 manufactured by Difco, USA].
g, 10 ng to 10 mg by intravenous administration, 100 by transdermal administration
ng to 10 mg, 1 ng to 10 mg by intradermal administration per day
It serves as a rough guideline for the dose.

With regard to animals, for large animals such as cows and horses, 1/60 of the above amount is used as a standard for the amount per 1 kg of body weight, and pigs,
For medium and small-sized animals such as dogs and cats, double that amount and weigh 1k.
As a guideline for the amount per gram, and for birds such as chickens, a double dose thereof can be administered as a guideline for the amount per 1 kg of body weight.

[0028] The LD ₅₀ of LPS3 measured in the C3H / He male mouse of 7-week-old with an average body weight of 22 g was 180 μg / mouse, as measured by the Behrens-Kelber method.
E. coli LPS [012 manufactured by Difco, USA
8: B8] was 300% / animal 60%.

As described below, LPS3 was also confirmed to have "postoperative pain suppressing effect" and "cancer metastasis suppressing effect". The dose for which these effects are expected is the same as in the case of the anti-MRSA agent.

Experimental Example 2 (Postoperative pain suppressing effect) General anesthesia (oxygen-smile-isoflurane) for gallstone disease.
19 patients underwent endoscopic cholecystectomy under the LPS [LPS
The post-operative pain suppressing effect of LPS3 was confirmed in 9 cases (ages 32 to 63 years old) in the 3 administration group and in 10 cases (ages 34 to 58 years old) in the non-administration group.

For the LPS3 administration group, 30 μg of LPS3 [prepared as 3 ml of 50% (w / v) glycerol solution] at the time of anesthesia immediately before surgery, 8 pm before bed on the day of surgery
10 μg [50% of 1 ml (w
/ V) Prepared as glycerol solution], a total of 3 times. The clinical evaluation of the results was carried out by evaluating the pain and sleep condition on the next morning after the surgery in 5 grades, respectively, and comparing the amount of analgesic used for suppressing postoperative pain.

1) Evaluation of Pain Five-level evaluation was carried out, with "0" indicating no pain and "4" indicating extremely pain. Evaluation of the LPS3 administration group was “0” in 3 cases,
“1” was 4 cases, “3” was 2 cases, and the average was 2.0 ± 2.8, showing a significant (p <0.05) analgesic effect.

On the other hand, in the non-administration group, "1" was evaluated in 5 cases,
“2” was 2 cases, “3” was 2 cases, and “4” was 1 case, and the average was 3.8 ± 2.3.

2) Evaluation of Sleep Situation A five-step evaluation was performed with deep sleep being “0” and sleep inability being “4”. Evaluation of LPS3 administration group was “0” in 5 cases, “1”
Was 1 and “2” was 3 in average of 1.4 ± 2.6, showing a significant (p <0.04) effect.

On the other hand, the non-administration group was evaluated as "0" in 1 case,
“1” was 4 cases, “2” was 1 case, and “3” was 4 cases, and the average was 3.6 ± 5.0.

3) Analgesic Usage The usage from the postoperative period to the clinical evaluation on the next morning was as follows. In the LPS3 administration group, pentazocine 15 was observed in 3 cases.
Simultaneous intramuscular injection of mg and hydroxyzine 25 mg was required once, and administration of analgesics was not required in the other 6 cases (67%).

On the other hand, in the non-administration group, 2 cases were given the intramuscular injection 1 times.
3 times required 2 times or more, and 2 cases required administration of 25 mg of indomethacin suppository once. It was 3 cases (30%) that the administration of the analgesic was not necessary.

4) Changes in Body Temperature There was no significant difference between the two groups before and after surgery.

5) Biochemically Measured Values The measured values immediately before the first LPS3 administration and after 2 hours from the administration are compared as follows.

Serum M-CSF (macrophage-colony stimulating factor) was not significantly different between the two groups.

Serum TNF in the placebo group was
6.29 ± 1.55 pg / ml to 4.76 ± 1.87
It was significantly (p <0.04) decreased to pg / ml, and the average reduction rate was 24.3%.

On the other hand, in the LPS3 administration group, 6.00 ±
1.48 pg / ml to 6.92 ± 2.56 pg / ml
, And the average rate of increase was 15%, showing a significant (p <0.05) increase after surgery in the non-administration group.

The TNF production amount of mononuclear cells decreased in both groups, and there was no significant difference between the groups.

The amount of β-endorphin in serum was determined by LPS.
In the 3 administration groups, 23.8 ± 9.8 pg / ml to 17
It increased to 1.5 ± 78.9 pg / ml. Even in the non-administered group, 39.7 ± 17.6 pg / ml to 151.3 ± 1
It increased to 29.6 pg / ml, but the average rate of increase was LPS
Only 53% of the 3 dose groups.

Experimental Example 3 (Cancer Metastasis Inhibitory Effect) Cancer cells were intravenously injected into mice, and the number of cancer nodules in the lungs was visually confirmed and confirmed. This depends on the difference in the primary tumor, but in general, hematogenous metastasis and lymphatic metastasis are one of the cases of hematogenous metastasis, lymphatic metastasis, destructive metastasis, and contact metastasis, which are cancer metastasis modes. In most cases, even in the case of lymphatic metastasis, cancer cells flow through the lymphatic vessels and eventually enter the blood, and metastasis to the lung is most often observed (Shigeru Toyoshima, "Recurrent and metastatic cancer"). , Page 6, 1981
Jiyusha Co., Ltd .; Hiroshi Kobayashi, Oncology, p. 40, 198
(4th year, Nanzandou), it is considered most appropriate to observe lung metastases due to hematopoiesis. Specifically, it is as follows.

Mouse (BALB / c, male, average 8 weeks old,
Meth solid cancer Meth with high lung metastases in an average body weight of 25 g)
A sarcoma cells (1 × 10 ⁵ cells / mouse) were injected through the tail vein,
On the 20th day after the injection, the lung was taken out by incision and the number of cancer nodules in the lung was visually observed. The lung metastasizing strain was prepared by the following method.

1) 5 × 10 ⁶ of ascites-type MethA sarcoma cells
The cells / ml suspension was prepared with PBS (−) (manufactured by Nissui Pharmaceutical Co., Ltd.), and 200 μl thereof was injected from the tail vein of BALB / c male mice (1 × 10 ⁶ cells / mouse), and 20 after injection.
On day one, cancer nodules in the lungs were excised.

2) The above cancer nodules were treated with 500 units / ml of dispase (manufactured by Sanko Junyaku Co., Ltd.) solution prepared by Hank's solution (manufactured by Nissui Pharmaceutical Co., Ltd.) at 37 ° C. for 30 minutes to prepare a cell suspension. Was prepared. Add this cell suspension to another BA
LB / c male mice were intraperitoneally transplanted and ascites.

3) The cells obtained by repeating the above 1) to 2) a total of 5 times were used as the above lung metastases.

In the LPS3 administration group, 12 after injection of sarcoma cells
4 days from the first day, 0.1μg, 1μg, 10μ respectively
LPS3 (5 animals in each group) was dissolved in 0.05 ml of physiological saline and intradermally administered to the side of the abdomen. On the other hand, the control group (5
0.05 ml of physiological saline alone was administered to each mouse. As a result, the average number of cancer nodules was 56.2 ± 4.
4 and 0.1 μg administration group 48.0 ± 5.6, 1 μg administration group 30.8 ± 9.7, 10 μg administration group 18.4 ±
It was confirmed that LPS3 suppressed cancer metastasis in a dose-dependent manner. Particularly, in the 10 μg administration group, a significant (p <0.001) suppression of 74% was observed as compared with the control group.

[0051]

INDUSTRIAL APPLICABILITY According to the present invention, it has an excellent anti-MRSA effect, can be used for a long period of time, and can be administered by any of the oral, transdermal, injection and intradermal routes. Provided are a novel anti-MRSA agent and an animal anti-MRSA agent that exert an effect and do not cause the generation of new resistant bacteria.

 ─────────────────────────────────────────────────── --- Continuation of the front page (72) Inventor Denichi Mizuno 255-4 Kokuki, Shiraoka-cho, Minami-Saitama-gun, Saitama Prefectural University of Technology, Biotechnology Research Center, Teikyo University (72) Takafumi Okutomi Kokuki, Shiraoka-cho, Minami-Saitama-gun, Saitama 255-4 Patent Department, Biotechnology Research Center, Teikyo University

Claims (4)

[Claims] 1. An anti-MRS comprising an LPS.
Agent A. 2. LPS is L produced by Pantoea spp.
Anti-MRS according to claim 1, characterized in that it is PS.
Agent A. 3. A Pantoea spp. Is Pantoea. The anti-MRSA agent according to claim 2, which is an agglomerans inoculum. 4. An anti-MRSA agent for animals, which comprises LPS.