JPH02131433A - Preventive and therapeutic agent for nonspecific infectious diseases - Google Patents

Abstract

PURPOSE:To obtain a preventive and therapeutic agent which is effective for prevention and treatment for infectious diseases caused by bacteria other than Salmonella and viruses by using inactivated Salmonella and antibiotic substances. CONSTITUTION:The subject preventive and therapeutic agent is composed of (A) inactivated Salmonella which is prepared by treating a bacterium in Salmonella such as Salmonella typhimurium (rat typhoid causative bacterium) with heat or formalin, and (B) antibiotics, preferably streptomycin, ampicillin, chloramphenicol, cefazolin at a A/B weight ratio of 1/0.001 to 1/20, preferably 1/0.1 to 1/1. Said preventive and therapeutic agent develops better effect than in single use of an antibiotic, thus the amount of the antibiotic can be reduced whereby the appearance of resistant bacteria due to excessive use of antibiotics, opportunistic infection, adverse effect on living body functions by the disturbance of intestinal bacterial flora can be avoided.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a non-specific infection prevention and treatment agent. In particular, the present invention relates to a prophylactic and therapeutic agent based on inactivated Salmonella that is effective for preventing and/or treating infectious diseases caused by bacteria, fungi, and viruses other than Salmonella.

[Conventional technology]

To prevent and treat infectious diseases caused by bacteria, fungi, and viruses in humans and animals, specific antibiotics that are effective against the pathogens and vaccines that act specifically against the pathogens have been used and administered. However, the heavy use of antibiotics has become a major problem as it often causes a decrease in effectiveness due to the appearance of resistant bacteria, occurrence of opportunistic infections, and adverse effects on biological functions due to disturbance of intestinal flora. In particular, when antibiotics are administered to livestock, there are concerns not only about the effects on the livestock, but also on the human body that consumes the livestock products. Furthermore, in the case of a vaccine, a pathogen that is the same type or closely related to the pathogen to be prevented or treated is inactivated or its activity is weakened; It is used specifically as a pathogen and usually has little effect on foreign or distantly related pathogens.

Even if a vaccine produced from one specific pathogen is effective in preventing or treating an infection caused by a completely different type of pathogen, this is extremely rare.

[Contents of the invention]

The present inventors have continued research on preventive agents against Salmonella infection, and as a result, a combination of a specific antibiotic and inactivated Salmonella is extremely effective as a preventive agent against Salmonella infection. After discovering this, he filed an application as Japanese Patent Application No. 41168/1983 (Japanese Unexamined Patent Publication No. 294623/1982).

The present inventors conducted further research on the efficacy of drugs consisting of antibiotics and inactivated Salmonella, and found that among many fungi, this Salmonella genus belongs to an extremely limited genus. It was discovered that the above-mentioned combination of substances and antibiotics is extremely effective not only for preventing Salmonella infections, but also for preventing and treating infections caused by bacteria, molds, viruses, etc. other than Salmonella. did. This discovery of the present invention is completely unexpected from the common sense that vaccines are only effective against pathogens that are the same or similar to the pathogens that make up the vaccine, and are unique to inactivated Salmonella. . Moreover, it was also unexpectedly discovered that the combination of antibiotics with inactivated Salmonella was more effective than when antibiotics were used alone.

Therefore, the present invention was completed based on this discovery, and is a non-specific infection prevention and treatment agent containing inactivated Salmonella and an antibiotic.

Here, the term "non-specific infection prevention and treatment agent" as used in the present invention means that the drug of the present invention is effective for the prevention and/or treatment of infectious diseases caused by pathogens such as bacteria, molds, and viruses other than Salmonella. means to be used as

Bacteria belonging to the genus Salmonella include Salmonella abor
tvoequina) (Equine Abortus), SalmoneLLa abortus
bovis) (bovine abortion fungus), Salmonella choleraesui
s) (swine fever), Salmonella enteritidis
dis) (Salmonella enteritidis), Salmonella gallinarum (Sal
monella gallinaru+nu) (Salmonella trityphi), Salmonella hirschfeldi (S
Salmonella ichteroides (Falmonella paratyphi C), Salmonella icteroides (Salm
onella icteroides), Salmonella paratihii (Salmonella lapa)
Salmonella typhi A) (Salmonella typhi A), Salmonella typhi (Salmonella typhi), Salmonella teinurium (Salmone
lla typhimurium)
Although many other bacteria are known, the term "inactivated Salmonella" as used in the present invention includes any inactivated bacteria belonging to the genus Salmonella.

Among the bacteria belonging to the genus Salmonella, the present invention uses Salmonella enteritidis.
Enteritidis), Salmonella para
typhi A) (Salmonella typhi A), Salmonella typhi (Salmonella typhiX), Salmonella tinurium (Salmonella
Salmonella typhimurium (Salmonella typhimurium), among others, are particularly effective against Salmonella tinurium
Inactivated Salmonella typhimurium (Salmonella typhimurium) is preferred because it acts effectively against infections caused by various pathogens.

Any method commonly used for inactivating bacteria can be used to inactivate Salmonella, but inactivation by heating or formalin treatment is preferred because it is simple and maintains high medicinal efficacy. When inactivating Salmonella by heating, it is best to heat to a temperature of about 50 to 130°C, and when treating with formalin, Salmonella is reduced to about 0.1 to 0.5
It is recommended that the treatment be carried out by immersing it in an aqueous formalin solution with a concentration of 50%.

The present invention makes it essential to use an antibiotic in combination with inactivated Salmonella. As mentioned above, the combination of antibiotics and inactivated Salmonella provides better prevention and protection against infections caused by pathogens other than Salmonella, such as bacteria, fungi, and viruses, than when antibiotics are used alone. A therapeutic effect can be obtained. Such a non-specific synergistic effect by the inactivated Salmonella of the present invention is completely unexpected.

In the present invention, the antibiotics include aminoglycoside antibiotics such as kanamycin, streptomycin, and fradiomycin; penicillin antibiotics such as benzylpenicillin, phenoxymethylpenicillin, ampicillin, amoxicillin, and bacanbicillin; and polypeptide antibiotics such as colistin and bacitracin. Macrolide antibiotics such as erythromycin, spiramycin, kitasamycin, and ampicillin; Cefem antibiotics such as cephalexin and cefazolin; Chloramphenicol antibiotics such as chloramphenicol and thiamphenicol: oxytetracycline, Tetracycline antibiotics such as doxycycline; antibiotics such as phosphorus-containing polysaccharide antibiotics such as kebemycin and macaropomycin; and any of their pharmaceutically acceptable salts or esters can be used. Among these antibiotics, streptomycin, ampicillin, chloram-7-anycol, and cefazolin are particularly suitable in the present invention.

Depending on the type of infectious disease to be prevented and treated, the type of inactivated Salmofila and the type of antibiotic should be selected and combined.

The non-specific infection prevention and treatment agent of the present invention is effective not only against various infectious diseases other than Salmonella infection in humans, but also in cows, horses, pigs, sheep, goats, chickens, 70,000 birds, ducks, etc. It has high preventive and therapeutic effects on infectious diseases other than Salmonella infections in livestock, poultry, mice, rats, guinea pigs, rabbits, dogs, cats, and other animals, as well as fish. Among them, the drug of the present invention is effective against Pseudomonas aeruginosa (Pseudomonas aeruginosa).
seudomonas aeruginosa AICC
27853) Infectious disease, one of the respiratory diseases of calves, Pasteurella multoshi'l (PasLeurella)
mu l tocida) infections, Salmonella enteritidis (Sa l
lumone l laenteritidis R
B-1 strain) infection, Staphylococcus aureus (Staphylo
coccus aureus ATCC 25723)
Candida, a type of infectious disease and fungus
albicans) infection, murine hepatitis virus infection, Sendai virus infection, etc., and in particular, a combination of inactivated Salmonella consisting of inactivated Salmonella typhimurium and antibiotics is effective against the above-mentioned Effective against infectious diseases.

In the prophylactic and therapeutic agent of the present invention, inactivated Salmonella and antibiotics are usually mixed in a ratio of about 1:0. It is preferably contained in a weight ratio of ool to l:20, particularly 1:o. t-t:
It is preferable to set the ratio to 1.

The dosage of the prophylactic and therapeutic agent of the present invention will naturally vary depending on the species neck, body weight and age of the subject, the type of infectious disease, its symptoms, the number of days of administration, etc. The amount of wet bacteria of inactivated Salmonella per kg of animal body weight is approximately 1 = 100 m9, and the amount of antibiotics is approximately 1
It is preferable to administer at a rate of ~100 mg.

The prophylactic and therapeutic agents of the present invention are preferably administered orally. The dosage form for oral administration may be liquid, semi-solid (e.g. paste) or solid, including conventional oral dosage forms such as liquids, pastes, powders, granules, tablets, coated tablets, capsules and pills. Any of the dosage forms for administration can be employed. Used in liquid and semi-solid form. In this case, inactivated Salmonella and antibiotics may be dispersed or mixed in a medium such as water.

When used in solid form, inactivated Salmorrha and antibiotics may be made into separate powders or granules and then mixed.
Alternatively, the two may be mixed and then formed into powder, granules, tablets, pills, etc. Furthermore, the prophylactic and therapeutic agents of the present invention include:
Various additives used in this type of drug, such as excipients, fillers, binders, disintegrants, etc., can be added as long as they do not interfere with the effectiveness of the drug.

Further, the prophylactic and therapeutic agent of the present invention may be directly orally administered as it is, or may be administered in addition to feed, food, drinking water, etc.

Preferable results are obtained when the prophylactic and therapeutic agent of the present invention is administered continuously, and it is particularly preferable to administer it for one week or more.

The prophylactic and therapeutic agent of the present invention has almost no toxicity.
Also, no side effects other than those traditionally associated with the antibiotics used are observed.

The present invention will be specifically explained below using Examples, etc.
The present invention is not limited thereto.

Example 1 (A) Salmonella typhimurium
hinurium ATCC 14028) at 37°C in a liquid medium consisting of plain heart infusion.
After culturing for 4 hours, it was centrifuged. After washing with physiological saline, 1 at 1 2 1 'O while suspended in physiological saline.
It was inactivated by heating for 0 minutes.

(B) The inactivated Salmonella Typhimurium obtained in (A) above is expressed as a wet bacteria amount of 0.5 m per 1 mQ of drinking water.
g, and streptomycin sulfate at 0.0 g per 1v2 of drinking water. Drinking water to which 2 mg was added was prepared.

(C) For comparison, drinking water was prepared by adding 0.2 mg of streptomycin sulfate per 1 mQ of drinking water.

(D) BALB/C type? Usu (medium 7., 5 weeks old)
Two groups of 10 animals each were prepared (average weight 19 g/animal),
Each group was given each of the drinking waters prepared above ad libitum for 3 weeks at the same time as regular feed. After completion of administration,
Both groups of mice were fed water containing neither inactivated Salmonella typhimurium nor streptomycin sulfate for one week along with normal chow. Each group of mice was then infected with Pseudomonas aerugi.
nosa AICC 27853) live bacteria were inoculated intraperitoneally at a rate of 107 cells/mouse.

Five days after inoculation, 3 out of 10 mice died when the drinking water (B) above containing both inactivated Salmonella typhimurium and streptomycin sulfate was given (an example of the present invention). In the case where only streptomine sulfate was added and the drinking water of (C) above was given (comparative example), 7 out of 10 mice.

From the above results, the drug of the present invention, which is made by using inactivated Salmonella typhimurium, which is an inactivated Salmonella if, in combination with the antibiotic streptomycin sulfate, is effective against Pseudomonas aeruginosa infection, which belongs to a completely different genus from Salmonella typhimurium. It has a non-specific preventive effect against antibiotics, and the effect is superior to that of using antibiotics alone.

Example 2 Salmonella Typhimurium isolated from the diarrheal stool of a calf reared in the field was cultured and inactivated in the same manner as described in Example 1 (A).

2 Holstein cows (male, 1 week old), 5 cows per group
Groups (average weight 45 kg/head) were prepared.

For the first group of calves, 2Q of milk replacer was added to the milk replacer fed in the morning and evening.
50 mg of the above-mentioned inactivated Salmonella typhimurium per wet bacteria amount
The animals were continuously fed for 2 weeks by adding ampicillin sodium loomg/time.

In addition, for comparison, a milk replacer prepared by adding only ampicillin sodium loo+ng/dose per 2Q of milk replacer to the same type of milk replacer was similarly provided to the second group of calves for 2 weeks.

After the calves in each group were raised until they were 5 weeks old, bacterial tests in the nasal cavities of the calves were conducted. Pasteurella multocida, one of the causative bacteria of calf respiratory disease, was detected from one group of calves (an example of the present invention).
However, the pathogenic bacterium was detected in two calves in the second group (comparative example) that received milk replacer containing only ampicillin sodium; Clinical symptoms such as rhinorrhea and coughing were also observed in calves.

Example 3 (A) After culturing, centrifuging, and washing Salmonella typhimurium in the same manner as in (A) of Example 1, 500 mg of the same was added to 100++ l of a 0.3% formalin aqueous solution as a wet bacterial amount.
Inactivate it by soaking it in water for 24 hours.

(B) 0.2 mg of the inactivated Salmonella Typhimurium obtained in (A) above per ltQ of drinking water as a wet bacteria amount.
In addition, drinking water was prepared in which 0.1 mg of chloram 7-enicol sodium succinate was added per ml of drinking water.

(C) For comparison, only chloramphenicol sodium succinate was added to drinking water at a concentration of O. A sample to which 1mg was added was prepared.

(D) Two groups of C3H/HeJ mice (female, 5 weeks old) with 5 mice in each group were prepared (average weight 169/mouse).
Each group was given the above-prepared drinking water ad libitum for 3 weeks at the same time as regular feed.

After the administration, the animals were allowed to ingest water containing neither inactivated Salmonella Typhimurium nor chloram-7-anycol sodium succinate for one week together with normal feed. Each group of mice was then challenged with Salmonella enteritidis.
Enteritidis RB-1 strain) live bacteria 5X
The mice were inoculated intraperitoneally at a rate of 10' cells/mouse.

Death 28 days after inoculation occurred when drinking water of (B) above containing both inactivated Salmonella Typhimurium and chloram 7-anycol sodium succinate was given (Example of the present invention)
In contrast, when only chloramphenicol sodium succinate was added and the drinking water of (C) above was given (comparative example), 4 out of 5 there were.

Example 4 Five groups of BALB/C mice (female, 5 weeks old) with 5 mice per group (average weight 19 g/mouse) were prepared.

The first group of mice was treated with the same inactivated Salmonella Typhimurium obtained in Example 1 (A) at a moisture content of 0.5 mg/m (
Drinking water containing sodium 2 and se7 azolin at 0.5 my/mQ was given ad libitum for 3 weeks at the same time as regular feed.

A second group of mice was treated with the same drinking water as above for the first group of mice, except that 0.5 mg/mQ of se7azoline sodium was replaced with 0.1 mg/m of tetracycline hydrochloride <1. The animals were given free access for a week.

The same drinking water as for the mice in the first group was given to the mice in the third group, except that cefazolin sodium 0.5 m9/m (2 was replaced with erythromycin ethylsuccinate O.lmg/n+Q.). The animals were given free access for 3 weeks.

Se7azoline sodium 0.5+ng/m0. The mice of the fourth group were given the same drinking water ad libitum for 3 weeks as above for the mice of the first group, except that 400 units of colistin metasulfonate sodium was substituted.

For comparison, mice in Group 5 were given free access to drinking water containing neither inactivated Salmonella typhimurium nor antibiotics for 3 weeks. After the administration, the mice were allowed to ingest water containing neither inactivated Salmonella typhimurium nor antibiotics for one week along with normal feed.

Each group of mice was then injected with live Pseudomonas aeruginosa for 10'
The mice were inoculated intraperitoneally at a ratio of 2 mice per animal.

All mice in groups 1 to 3 died 5 days after inoculation.
The number of deaths was extremely low, being 1 out of 5 mice and 2 out of 5 mice in the 4th group, whereas the number of deaths was high at 4 out of 5 mice in the 5th group, which is a comparative example. Ta.

Example 5 Two groups of C3H/HeN mice (female, 6 weeks old) with 5 mice per group (average weight 19 g/mouse) were prepared.

The first group of mice was treated with 0.0.0. 5mg/mQ,
Furthermore, drinking water containing streptomycin sulfate at a rate of 0.1 mg/mQ was allowed to be freely taken from a water bottle for 3 weeks at the same time as regular feed.

After the administration, the mice were allowed to ingest water containing neither inactivated Salmonella typhimurium nor streptomycin sulfate together with normal feed for one week.

The mice in the second group were given free access to water only for 4 weeks.

Then, peritoneal exudate cells and serum from mice in each group were collected and pooled for each group. Staphylococcus aureus (Staphylococcus aureus) previously incubated with this serum.
us aureus ATCC 25923) live bacteria were added to each peritoneal exudate cell and incubated, and the number of Staphylococcus aureus was measured over time.

When the peritoneal exudate cells collected from the first group of mice (an example of the present invention) were incubated, the increase in Staphylococcus aureus was suppressed to 28 times that of the second group of mice (comparative example).

Example 6 C 3 H / H e N mouse (female, 6 weeks old)
Two groups of 2 animals each (average weight 19 g/mouse) were prepared.

The first group of mice was treated with the inactivated Salmonella typhimurium obtained in Example 1 (A) at a wet bacterial dose of 0. 5mg/m
Q, and also streptomycin sulfate. lmg/l
The drinking water contained in the TIQ was given ad libitum from a water bottle for 3 weeks at the same time as regular feed.

After the administration, the animals were allowed to consume water containing neither inactivated Salmonella typhimurium nor streptomycin sulfate together with normal feed for one week.

The mice in the second group were given free access to water only for 4 weeks.

Then, peritoneal exudate cells and serum from mice in each group were collected and pooled for each group. Candida albicans previously incubated with this serum.
s) to each peritoneal exudate cell and incubate;
The number of Candida was measured in each case over time.

When the peritoneal exudate cells collected from the first group of mice (Example) were incubated, the increase in Candida was suppressed to about 37% compared to that of the second group of mice (Comparative Example).

Example 7 Two groups of BALB/C nude mice (female, 6 weeks old) with 10 mice per group were prepared.

The first group of mice was treated with water containing the inactivated Salmonella Typhimurium obtained in Example 1 (A) at a wet bacterial level of 0.5*+g/mQ, and streptomycin sulfate at a rate of 0.1 mg/mI2. The mice were given ad libitum for 8 weeks from 6 to 10 weeks of age and from 30 to 34 weeks of age. During the rest of the period, they were allowed to ingest water that did not contain inactivated S. typhimurium or antibiotics.

A second group of mice received only streptomycin sulfate.
Water containing lm9/mQ was given ad libitum for 8 weeks from 6 weeks of age to 10 weeks of age and from 30 weeks of age to 34 weeks of age, and no inactivated Salmonella typhimurium and antibiotics were not included during the rest of the period. I was given water.

By the age of 85 weeks, all mice in the second group (comparative example) had died of wasting disease caused by the virus, but five mice in the first group were alive and healthy.

〔Effect of the invention〕

In the present invention, by using inactivated Salmonella in combination with antibiotics, extremely excellent effects can be achieved for the prevention and/or treatment of infectious diseases caused by pathogens such as bacteria, fungi, and viruses other than Salmonella. and the effect is better than when antibiotics are used alone.

Therefore, in the present invention, the amount of antibiotics used can be much smaller than conventionally used alone for prevention or treatment. Therefore, it is possible to prevent the emergence of resistant bacteria, occurrence of opportunistic infections, and adverse effects on biological functions due to disturbance of intestinal flora caused by heavy use of antibiotics, which is economical.

In addition, since the present invention uses killed and inactivated Salmonella, it is safe without the risk of environmental contamination or reversion of the attenuated live bacteria to pathogenic bacteria, as seen in vaccines using attenuated live bacteria.

The non-specific infection prevention and treatment agent of the present invention can be effectively used against various infectious diseases in humans, livestock, mice, and other animals, and can be used in a wide range of applications.

Furthermore, since the prophylactic and therapeutic agent of the present invention is administered orally, unlike administration by injection, it is less complicated and dangerous, is safe, and saves labor.

Procedural Amendment September 18, 1999 Director General of the Patent Office Fumi Takeshi Yoshida 1. Display of the case 1988 Special Fraud WJI No. 283743 2. Name of the invention Non-specific infection prevention/treatment agent 3. Relationship with the case of the person making the amendment Patent applicant address: 19-12 Nihonbashi Koami-cho, Chuo-ku, Tokyo Name: Nisshin Seifun Co., Ltd. 4. Agent 7. Correction details l) Correct rAIccJ on page 9, line 7 to rATC.

2) rsallumonellaJ on page 9, line 9
．． Corrected to almonellaJ.

3) rtyphinuriumJ on page 12, line 7
Correct to typhimuriumJ.

4) Correct rAlccJ on page 13, line 10 to rATCCJ.

5) Correct the "moisture content" in the bottom three lines of page 17 to "moist bacteria content".

6) Correct "kono" in line 9 of page 20 to "kono".

7) Correct “kono” in the 4th line from the bottom of page 21 to “kono”.

8) rc CandedaJ in the 2nd line from the bottom of page 21
Corrected to andidaJ.

9) Add the following text between lines 7 and 8 on page 23.

“Example 8 C 3 H / H e N mouse (female, 6 weeks old)
Two groups of 5 animals each (average weight 19 g/mouse) were prepared.

The mice of the first group were treated with l. ml of the inactivated Salmonella typhimurium obtained in (^) of Example 1 as a wet bacterial dose. Q-e/mQ, and drinking water containing streptomycin sulfate at a rate of 0.1 my/m (lc') in a water bottle at the same time as regular feed.
The animals were given free access for a week.

After the administration, the mice were allowed to ingest water containing neither inactivated Salmonella typhimurium nor streptomine sulfate together with normal feed for one week.

The mice in the second group were given free access to water only for 4 weeks.

Each group of mice was then challenged with Klebsiella pneumoniae (Klebs).
iella pneumoniae ATCC 138
83) Bacteria were injected intraperitoneally at a rate of 7 lO/mouse.

Two days after harvesting, 3 out of 5 mice in the first group died, whereas all of them died. ” 1 mouse in the second group

Claims (1)

[Claims] Non-specific infection prevention and treatment agent containing inactivated Salmonella and antibiotics.