JPH08259450A - Enhancer for production of interferon - Google Patents

Abstract

PURPOSE: To obtain a safe enhancer for the production of an interferon containing a microbial cell of a microorganism belonging to the genus Enterococcus or its treated substance as an active ingredient without causing side effects. CONSTITUTION: This enhancer for the production of interferon contains a live or a dead microbial cell obtained by culturing a microorganism belonging to the genus Enterocccus, especially Enterococcus faecalis and Enterococcus casseliflavus or a substance prepared by treating the microbial cell by grinding, extraction with water, etc., an active ingredient. The enhancer can be prepared into a tablet or a granule by blending the active ingredient with a carrier such as starch, lactose or a soybean protein and an additive such as an excipient, a binder, a disintegrating agent, a lubricant, a stabilizer or a flavoring agent therewith according to a conventional method. The daily dose thereof is 0.002-0.1g/kg administered in divided one to several portions. When a large amount of the interferon is administered for a long period in the interferon therapy, side effects such as influenzal symptoms, autoimmune diseases, depressive symptoms, alopecia or an abnormality in thyroid gland functions are caused. This enhancer is capable of avoiding the side effects.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an interferon production enhancer containing, as an active ingredient, cells of a microorganism belonging to the genus Enterococcus or a treated product thereof.

[0002]

BACKGROUND OF THE INVENTION Interferon (IFN) is a general term for proteins having an antiviral activity and a molecular weight of about 20,000. IFN is roughly classified into three types, α, β, and γ, which are mainly produced by immune cells such as lymphocytes. As biological activity, antiviral action, antitumor action, immune response regulating action and the like are known.

Currently, genetically modified IFN and purified I
FN is used as a therapeutic drug for viral infection, viral hepatitis, cancer and the like.

Many substances or compositions that enhance the production of IFN are known, but they do not have sufficient IFN production-enhancing activity, and when used in vivo, there is a problem with safety. There are many. Therefore, a strong IFN production enhancing agent and a safe IFN production enhancing agent are required.

[0005]

IFN is a substance that is produced in the human body, but during IFN therapy, if it is administered from the outside for a long period of time and a large amount of IFN is administered, influenza-like symptoms ( Various side effects such as headache, fever, arthralgia, myalgia, loss of appetite, general malaise, nausea / vomiting), autoimmune diseases, depressive symptoms (such as insomnia and irritability), hair loss, thyroid dysfunction, and blurring. Occurs.

[0006] Therefore, studies have been conducted to prevent viral infection and improve / treat cancer by enhancing IFN production in vivo without administering IFN.

Although many substances and compositions that enhance IFN production are known, many of them are viruses, pathogenic bacteria, etc. and have a safety problem. The present invention is intended to provide a safe IFN production enhancer without side effects.

[0008]

The present inventors have found that microorganisms belonging to the genus Enterococcus, particularly Enterococcus faecalis and Enterococcus caseiflaves bacterial cell samples, enhance IFN production in the body,
The present invention has been completed. Further, the bacterial species used in the agent of the present invention is a kind of lactic acid bacterium isolated from the intestines and silage of healthy subjects, and is therefore a safe bacterial species with no side effects.

Enterococcus faecalis is a kind of indigenous bacteria existing in the intestine (Bergey's Manual of Sys
tematic Bacteriology. 2 (1986)). In the present invention, various strains belonging to this strain can be used,
In particular, NF-10 has a high IFN production enhancing activity.
It is preferable to use 11 strains. The strain has been deposited at the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology as Microorganism Research Institute No. 12564.

Enterococcus casei flavus is one of the bacteria present in silage (Bergey's Manua
l of Systematic Bacteriology. 2 (1986)). In the present invention, various strains belonging to this strain can be used, but N is particularly high in that it has a high IFN production enhancing activity.
It is preferred to use the F-1004 strain. The strain was sent to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Microorganisms Research Institute
Deposited as No. 8.

The following is Enterococcus faecalis NF
-1011 and Enterococcus caseiflaves NF
-1004 shows the means of isolation and the mycological and physiological properties of the strain.

(1) Enterococcus faecalis NF
Separation means of 1011 A 10-fold dilution of feces of a healthy person with heat-sterilized water is smeared on an appropriate selective medium (KMN agar plate and SF agar plate), and cultured at 37 ° C under aerobic conditions for 48 to 72 hours. , A bacterial colony appeared. The bacterial colonies were streaked on another homogeneous plate medium and cultured in the same manner to reappear the bacterial colonies. The same operation was repeated several times to separate a single colony consisting of a single bacterial species. The new isolate was examined for mycological (morphological, biochemical and serological) properties and classified and identified as belonging to Enterococcus faecalis. (2) Enterococcus caseiflaves NF-100
4. Separation means 4 A 10-fold dilution of silage with heat-sterilized water is smeared on an appropriate selective medium (1.5% agar-containing MRS medium), and cultured at 37 ° C under aerobic conditions for 48 to 72 hours to reveal bacterial colonies. Let The bacterial colonies were streaked on another homogeneous plate medium and cultured in the same manner to reappear the bacterial colonies. The same operation was repeated several times to separate a single colony consisting of a single bacterial species. The new isolate was examined for mycological (morphological, biochemical and serological) properties and classified and identified as belonging to Enterococcus casseliflavus.

(3) Mycological and Physiological Properties ──────────────────────────── Property NF-1011 NF-1004 ── ────────────────────────── Gram stain ＋ ＋ Bacterial morphology Spherical sphere Catalase − − Hemolytic αα Serogroup D D Proliferation 10 ° C + + 45 ° C + + 50 ° C + + Thermotolerance 60 ° C 30 minutes + + Growth on medium supplemented with bile esculin + + Growth on medium pH 9.6 + + + Methylene blue staining + 6.5% Sex ++ Gelatin liquefaction --- Growth in 0.01% TTC-supplemented medium ++ Growth in tellurite-supplemented medium ++ Presence / absence of acid production Glycerol + + L-arabinose- + D-ribose + + D-xylose- + D -Glucose + + D-galactose + + D-fract Source + + D-mannose + + Maltose + + Mannitol + + Sucrose + + L-sorbose − − D-sorbitol + − L-rhamnose + + Lactose + + amygdalin + + + esculin + + cellobiose in + + − + Merezitose + − ──────────────────────────── +; Positive, −; Negative TTC; 2,3,5-Triphenyl Tetrazolium chloride

The cells obtained by culturing Enterococcus faecalis and Enterococcus casei flavus used in the present invention are dead cells or viable cells, or cells that have been subjected to treatment such as grinding and water extraction are used. You can To formulate these, tablets and tablets are prepared by a known method using additives such as carriers such as starch, lactose and soybean protein, excipients, binders, disintegrating agents, lubricants, stabilizers, and corrigents. It is formulated into granules.

The amount used depends on symptoms, age, etc.,
As an active ingredient, 0.002 to 0.1 g / kg body weight per day can be usually administered to an adult once a day or in several divided doses.

The experimental method for measuring IFN titer is shown below.

(1) Preparation of mouse spleen cells The spleen of a C3H / He N strain mouse (female 6 to 12 weeks old) was aseptically removed and cultured in 5% FCS-RPMI 1640 medium (hereinafter referred to as culture medium). Transfer to a dish, cut into small pieces with scissors, and transfer to a centrifuge tube through a nylon mesh. The cell suspension is centrifuged (1,200 rpm), the precipitated cells are suspended in Tris buffer for red blood cell removal, and the cells are centrifuged again to suspend the precipitated cells in the culture medium. The number of splenocytes in this cell suspension is 5 ×
Adjust so as to be 10 ⁶ pieces / ml to 1 × 10 ⁷ pieces / ml.

(2) Preparation of IFN solution When enhancing IFN production from splenocytes in plate wells: A 96-well flat-bottom plate was used to make a final concentration of the test sample in a mouse splenocyte suspension (100 μl / well). 10 μg /
Add so that the amount becomes ml, and add ₂ in a CO ₂ incubator.
Incubate for 0 hour. As a control, LPS (lipopolysaccharide) or PHA-P (hemagglutinin, which enhances IFN production) is added in the same amount. The centrifugal supernatant obtained after the culturing was
Use FN liquid.

In the case of enhancing IFN production in vivo, cyclophosphamide (200 mg / kg body weight) was intraperitoneally administered to each of two groups of C3H / He N mice (female 8 weeks old), and one of The cell preparation to be tested is orally or intraperitoneally administered to the group for 3 consecutive days from the next day. The other control mouse is similarly administered with physiological saline. Next, a mouse splenocyte suspension was prepared by the method of Experimental method (1), and LPS or PHA-P as an IFN inducer was added thereto,
2 in the CO ₂ incubator as in (2)
Incubate for 0 hour. The centrifugal supernatant obtained after the culture is used as an IFN solution.

(3) Measurement of IFN titer by CPE (cytopathic effect) inhibition method In a 96-well flat bottom plate, 5 × 10 ⁵ cells / m 2 were prepared in advance.
L ₉₂₉ cells prepared in 1 are added and cultured in a CO ₂ incubator for 6 hours to attach the cells to the plate.
The IFN solution prepared in (2) was added to this, the mixture was incubated in a CO ₂ incubator for 16 hours, and the supernatant was discarded.
Add 2,500 PFU / well of sicular Stomatitis Virus (VSV) and incubate for another 48 hours. After culturing, the supernatant is discarded, and the cells denatured and detached by VSV are removed.
The cells that remain attached are stained with crystal violet and the absorbance at 620 nm is measured.

[0021]

EXAMPLES Examples will be shown below, but the present invention is not limited to the description of these Examples.

Example 1. (Cultivation of Enterococcus faecalis)
s) NF-1011 and Enterococcus casseliflavus NF-1004
Was inoculated into a Rogosa liquid medium having a composition shown below as a typical medium (the number of bacteria: 10 ⁶ cells / ml), and 10-1 at 37 ° C
After culturing for 6 hours, a culture solution containing about 10 ⁹ viable cells / ml was obtained. The obtained culture solution was centrifuged at 12,000 rpm for 20 minutes to collect the cells, and the cells were washed twice with distilled water to obtain cells. The cells were suspended in distilled water in an amount 1/100 of the amount of the liquid medium used, and heated at 110 ° C. for 10 minutes to obtain a suspension of dead cells. Next, it was dried by an appropriate method such as a hot air drying method or a freeze drying method to obtain dead bacterial cell powder (fungal cell preparation).

The composition of Rogosa liquid medium is shown below. Trypticase 10 g Yeast extract 5 g Tryptose 3 g Monopotassium phosphate 3 g Dipotassium phosphate 3 g Triammonium citrate 2 g Tween 80 (surfactant) 1 g Glucose 20 g Cysteine hydrochloride 0.2 g Salt solution (as in 1) 5 ml Distilled water 1000 ml (adjusted to pH 7.0, heat sterilized at 121 ° C. for 15 minutes) (1) Salt solution: MgSO ₄ .7H ₂ O 11.5
_{g FeSO 4 · 7H 2 O 0.68g} MnSO 4 · 2H 2 O 2.4g distilled water 100ml

Embodiment 2 FIG. Enhancement of IFN Production from Splenocytes in Plate Well Using mouse splenocytes prepared according to the experimental method (1), an IFN solution was prepared by the method shown in (2). As test specimens, the NF-1004 bacterial cell preparation and the NF-1011 bacterial cell preparation prepared in Example 1 were used.

According to the experimental method (3), the titer of IFN was measured and used as the activity of enhancing IFN production from splenocytes of the test sample. As the results are shown in Table 1, the test sample showed a strong enhancing activity equivalent to or higher than that of LPS or PHA-P as a control.

[0026]

[Table 1] ─────────────────────────── IFN titer (IU / ml) ─────────── ───────────────── Bacterial preparation NF-1011 216 NF-1004 176 LPS 163 PHA-P 21.6 ───────────── ──────────────

Embodiment 3 FIG. Enhancement of IFN production in vivo (1) Administration of bacterial cell preparation and preparation of splenocytes Cyclophosphamide (200 mg / kg body weight) was intraperitoneally administered to C3H / He N strain mice (female 8 weeks old) After administration, the NF-1011 bacterial cell preparation prepared in Example 1 was administered for 3 consecutive days from the next day. The oral preparation was 2.4 mg / g body weight, and the intraperitoneal administration was 3 days.
Each dose was adjusted to 4.0 μg / g body weight. 2 of the final administration
After a day, mouse splenocytes were prepared according to the experimental method (1).

(2) Preparation of IFN Solution Using the obtained mouse splenocytes, an IFN solution was prepared according to the experimental method (2). That is, 1 μg / ml of LPS was added to a splenocyte suspension of a mouse to which the test bacterial cell preparation was administered,
Alternatively, PHA-P was added to 10 μg / ml and cultured in a CO ₂ incubator for 20 hours in the same manner as in the experimental method (2). The centrifugal supernatant obtained after culturing is IFN
It was a liquid.

(3) Measurement of IFN titer This was based on the experimental method (3). The results are shown in Table 2. NF-
IFN production was markedly enhanced by oral administration or intraperitoneal administration of 1011 bacterial cell preparation.

[0030]

[Table 2] ────────────────────────────── IFN titer (IU / ml) LPS PHA-P ─── ─────────────────────────── Physiological saline 253.0 ± 58.4 65.1 ± 52.7 (control) ────────── ──────────────────── NF-1011 cell preparation 419.9 ± 98.7 ^* 153.4 ± 79.9 ^* Oral administration ────────────── ───────────────── NF-1011 microbial preparation 281.5 ± 143.2 251.3 ± 91.7 ^* Intraperitoneal administration ──────────────── ────────────── ^* : p <0.05 (significantly different from the control value)

[0031]

INDUSTRIAL APPLICABILITY By using cells belonging to the genus Enterococcus, a safe IFN production enhancer without side effects can be provided. Further, since these microorganisms can be orally effective, they can be used as an oral preparation.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:01) (72) Inventor Tetsuro Yamamoto 1406 Entoku-in, Ayama-cho, Ayama-cho, Mie Prefecture 69 issue

Claims (3)

[Claims] 1. An interferon production enhancer containing, as an active ingredient, cells of a microorganism belonging to the genus Enterococcus or a processed product thereof. 2. The interferon production enhancer according to claim 1, wherein the microorganism belonging to the genus Enterococcus is Enterococcus faecalis or Enterococcus caseiflaves. 3. The interferon production enhancer according to claim 1, wherein the microorganism belonging to the genus Enterococcus is Enterococcus faecalis NF-1011 or Enterococcus caseiflaves NF-1004.