JPH06247859A - Bacterium protecting agent - Google Patents

Abstract

PURPOSE:To obtain a bacterium protecting agent useful as a medicine for protecting in vivo transfer of Staphylococcus aureus from intestinal canal in lowering of bacterium protecting function of the intestinal canal, e.g. in carrying out total parenteral nutrition. CONSTITUTION:This bacterium protecting agent consists of a combination of a component composed of specific nucleic acids and/or their pharmacologically permissible salts, e.g. typically the component composed of inosine, cytidine, GMP, uridine and thymidine, especially containing these nucleic acids in molar ratios of 4:4:4:3:1. This bacterium protecting agent protects in vivo transfer of Staphylococcus aureus from intestinal canal in lowering of bacterium protecting function of intestinal canal, e.g. in carrying out total parenteral nutrition.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a bacterial protective agent, more specifically, a bacterial protective agent capable of exerting a pharmacological effect of preventing Staphylococcus aureus from migrating into the body from the intestinal tract when the intestinal tract protective function is deteriorated when a high-calorie infusion is performed. Regarding

[0002]

2. Description of the Related Art Conventionally, for the nutritional management of digestive tract diseases in which oral eating is impossible or difficult, a catheter is inserted up to the ascending or descending vena cava, and a high-concentration amino acid infusion or Nutrition method of injecting glucose solution (Total
parenteral nutrition, TPN: high calorie infusion method) is performed.

However, in the case of artificial nutrition which does not use the intestinal tract, such as the high calorie infusion method, or when only the enteral nutrition such as the elemental diet and the component nutrients is used, the function of the intestinal tract, In particular, the defense function of the intestinal tract against the transfer of intestinal bacteria into the body is impaired, which is presumed to be one of the causes of various infectious diseases, and has been regarded as a serious problem (Alverdy JC, et al., Surgery). ,
1988; 104: 185-190: Jones WG, et al., Surg. Foru
m., 1989; 40: 20-22: Barber AE, et al., JPE
N., 1990; 14: 335-343: Alverdy JC, et al., JP
EN, 1990; 14: 1-6).

However, up to now, there has been no research and development of any drug against the above-mentioned decrease in bacterial defense of the intestinal tract or a drug capable of preventing the transfer of intestinal bacteria from the intestine into the body, and the above problems are left unsolved. It is in the present situation.

The object of the present invention is to prevent or prevent the drug, which has never been researched and developed so far, from reducing the intestinal bacterial defense, that is, the intestinal bacterium, particularly Staphylococcus aureus, from entering the body from the intestinal tract. To provide a possible new bacterial defense agent.

[0006]

Means for Solving the Problems As a result of intensive studies by the present inventors, the above object was achieved by a drug containing a combination of the following specific nucleic acid constituent component and / or a pharmacologically acceptable salt thereof as an active ingredient. Therefore, the present invention has been completed here.

That is, according to the present invention, the following (1) to
Containing any of the combination of the nucleic acid constituent of (3) and / or a pharmacologically acceptable salt thereof as an active ingredient,
Provided is a bacterial protective agent which is characterized by preventing Staphylococcus aureus from entering the body from the intestine when the intestinal defense function is deteriorated.

(1) Inosine, cytidine, GMP, uridine and thymidine or IMP (or inosine), C
MP, GMP, UMP (or uridine) and thymidine (2) CMP, UMP, IMP (or inosine) and thymidine (3) AMP, GMP, CMP, UMP and thymidine However, in the above and the following specification, IMP is Inosine-n'-monophosphate, CMP cytidine-n'-monophosphate, GMP guanine-n'-monophosphate, UM
Let P be uridine-n'-monophosphate and AMP be adenine-n'-monophosphate.

Further, according to the present invention, the above-mentioned bacterial protective agent containing a nucleic acid component consisting of inosine, cytidine, GMP, uridine and thymidine and / or a pharmacologically acceptable salt thereof as an active ingredient, especially 5 above. There is provided the bacterial protective agent containing a nucleic acid constituent of a species and / or a pharmacologically acceptable salt thereof in a specific molar ratio described below.

The nucleic acid constituent used in the present invention is a compound which the applicant of the present invention has been engaged in research for a long time and is already known. For example, JP-A-58-233142 describes specific nucleic acid constituents developed as a nutritional supplement. Further, JP-A-61-277619 describes the same component developed as a therapeutic agent for liver diseases,
Further, JP-A-3-135918 describes the same component as an immunostimulant.

According to the present invention, surprisingly, the above-mentioned nucleic acid component has a foreign action which is not related to the previously developed pharmacological action for pharmaceutical use, that is, from the intestinal tract of Staphylococcus aureus when the intestinal protective function is lowered. It has been completed based on the new finding that it has an action of preventing the in vivo transfer of bacterium, and in the following specification, the action of preventing the in vivo transfer of S. aureus from the intestinal tract is referred to as "bacterial protective action". A drug that exerts this action is simply a "bacterial defense agent".
Say.

The nucleic acid constituents used as the active ingredient in the present invention are premised on the above-mentioned specific combination.
The combination ratio thereof is not particularly limited and can be appropriately selected from a wide range. In the case of the above combination (1), for example, inosine: cytidine: GMP: uridine: thymidine = 4: 4: 4:
3: 1 and IMP: CMP: GMP: UMP: thymidine = 4: 4: 4: 3: 1 can be exemplified, and in the case of the combination of the above (2), for example, CMP: UMP: IMP: thymidine =
4: 3: 8: 1 or 7.5: 4: 12.5: 1 can be exemplified, and in the case of the combination of the above (3), for example, AMP:
GMP: CMP: UMP: thymidine = 4: 4: 4: 3:
1 or 2: 2: 2: 1: 1 can be exemplified.

Among the above, the combination suitable for exhibiting the most excellent effect as a bacterial protective agent is the combination of the above (1), particularly the combination of inosine, cytidine, GMP, uridine and thymidine, and the composition thereof. Is 4: 4: 4: 3:
A relative molar ratio of 1 is preferred.

The nucleic acid constituent as the active ingredient used in the present invention may be in the form of a pharmacologically acceptable salt. Various pharmacologically acceptable salts may be used, and especially GMP is a highly soluble disodium salt (GM
P.2Na) and the like).

The dosage unit form of the bacterial protective agent of the present invention can be appropriately selected, for example, in the form of a liquid agent such as an injection, or a tablet,
It can be appropriately used in the form of oral preparations such as granules, powders, solutions, suspensions and emulsions. Of these, the injection form is preferred.

Adjustment to various forms can be carried out in accordance with ordinary methods. For example, the preparation of the present invention in the form of an injection is typically prepared by injecting distilled water for injection in the same manner as in the case of an ordinary injection. Mix and dissolve a prescribed amount of the above, and if necessary, various commonly used additive components such as hydrochloric acid, acetic acid, lactic acid, malic acid, citric acid, sodium hydroxide, potassium hydroxide, etc. It can be adjusted by adding an appropriate amount of an agent or the like and sterilizing the resulting aqueous solution by heat sterilization or sterile filtration.

The thus-prepared liquid agent-form bacterial protective agent of the present invention can have a pH similar to that of an injectable agent such as an ordinary infusion agent. Its preferred pH can range from about 3.0 to 9.0, particularly preferably from about 5.0 to 8.0. The concentration of the nucleic acid component and / or its pharmacologically acceptable salt in the preparation is usually in the range of about 0.5 to 10 w / v%, preferably about 2 to 8 w / v%. Is appropriate.

Further, the preparation of the present invention may be in the form of a solid formulation such as a tablet. For example, when molding the tablet, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate as a carrier, Excipients such as kaolin, crystalline cellulose, silicic acid, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone and other binders, drying Starch, sodium alginate,
Agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose disintegrant, sucrose stearin, cocoa butter, hydrogenated oil, etc. Agents, quaternary ammonium bases, absorption promoters such as sodium lauryl sulfate, humectants such as glycerin and denbun, adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid, purified talc, stearate, boro Lubricants such as acid powder and polyethylene glycol can be used. Further, the tablet is a tablet coated with a usual coating as necessary, for example, a sugar-coated tablet,
It can be a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, or a multi-layer tablet.

In the case of molding in the form of pills, examples of carriers include excipients such as glucose, lactose, starch, cacao butter, hardened vegetable fat, kaolin, talc, gum arabic powder, tragacanth powder, gelatin, ethanol and the like. Binder,
A disintegrating agent such as laminaran or agar can be used. In the case of molding into a suppository, as a carrier, for example, polyethylene glycol, cocoa butter, higher alcohol,
Esters of higher alcohols, gelatin, semi-synthetic glycerides and the like can be used.

To further form a powder, the crystals of the active ingredient of the present invention, which have been sieved with a sieve, can be used as carriers such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, and crystals. It may be uniformly mixed with an excipient such as cellulose or silicic acid.

The applied amount (dose) of the bacterial protective agent of the present invention to a patient is appropriately selected according to the pathological condition, age, weight, sex, degree of disease of the patient to which it is to be administered, and is not particularly limited. Usually, about 0.5 to 5.0 g per adult per day, preferably about 1.5 to 2.
An amount of 5 g can be used as a guide, and this amount can be appropriately increased or decreased, and the above dose can be administered in 1 to 4 divided doses per day.

[0022]

EXAMPLES In order to further clarify the present invention, production examples of the bacterial protective agent of the present invention will be given below, followed by test examples. In addition, the molar ratio in the production example shows an approximate value.

[0023]

[Production Example 1] Pure crystals of the respective components having the following compositions were added to distilled water for injection, dissolved by stirring, and the pH was adjusted to about 7.4 using hydrochloric acid as a pH adjuster. Then, the obtained aqueous solution is aseptically filtered and filled in a container for injection, and after purging with nitrogen, the container is closed and placed in an autoclave at 105 ° C. under 4 ° C.
After sterilization for 0 minutes, the bacterial protective agent of the present invention (total free nucleic acid concentration 8 w / v%) was prepared as an injection.

[0024]

[0025]

[Production Example 2] Pure crystals of the respective components in the amounts shown below were added to distilled water for injection, dissolved by stirring, and the pH was adjusted to about 7.3 using acetic acid as a pH adjuster. Then, the obtained aqueous solution is aseptically filtered and filled in a container for injection, and after purging with nitrogen, the container is closed and placed in an autoclave at 110 ° C. for 4 hours.
After sterilization for 0 minutes, the bacterial protective agent of the present invention (total free nucleic acid concentration 8 w / v%) was prepared as an injection.

[0026]

[0027]

[Production Example 3] Pure crystals of the respective components in the amounts shown below were added to distilled water for injection, dissolved by stirring, and the pH was adjusted to about 6.4 using hydrochloric acid as a pH adjuster. Then, the obtained aqueous solution is aseptically filtered and filled in a container for injection, and after purging with nitrogen, the container is closed and placed in an autoclave at 105 ° C. under 4 ° C.
After sterilization for 0 minutes, the bacterial protective agent of the present invention (total free nucleic acid concentration 4 w / v%) was prepared as an injection.

[0028]

[0029]

[Production Example 4] The same as in Production Example 1, the bacterial protective agent of the present invention as an injectable composition having the following composition (total free nucleic acid concentration 8 w / v%)
Was adjusted.

[0030]

[0031]

[Production Example 5] In the same manner as in Production Example 1, the bacterial protective agent of the present invention as an injection having the following composition (total free nucleic acid concentration 8 w / v%)
Was adjusted.

[0032]

[0033]

[Production Example 6] In the same manner as in Production Example 1, the bacterial protective agent of the present invention as an injection having the following composition (total free nucleic acid concentration: 5 w / v%)
Was adjusted.

[0034]

[0035]

[Production Example 7] The bacterial protective agent of the present invention (total free nucleic acid concentration) as an injection having the following composition was produced in the same manner as in Production Example 1 except that sodium hydroxide was used as a pH adjuster to adjust the pH to about 8.0. 7 w / v%).

[0036]

[0037]

[Production Example 8] In the same manner as in Production Example 7, the bacterial protective agent of the present invention as an injectable composition having the following composition (total free nucleic acid concentration: 8.2 w / v)
%) Was adjusted.

[0038]

[0039]

[Production Example 9] 2.34 g of AMP / 2Na, CMP / 2N
a 2.20g, GMP ・ 2Na2.44g, UMP ・ 2
After filtering each pure crystal of 1.65 g of Na and 0.36 g of thymidine with a 60 mesh sieve, 91.01 g of starch as an excipient was added to them and mixed in the Near East,
The bacterial protective agent of the present invention in powder form was prepared.

AMP ・ 2Na, CMP ・ 2N of this product
The compounding molar ratio of a, GMP · 2Na, UMP · 2Na and thymidine was about 4: 4: 4: 3: 1, and the total free nucleic acid concentration was 8 w / v%.

[0041]

[Production Example 10] 1.06 w / v% inosine, 1.46 w / v% cytidine, 2.44 w / v% GMP · 2Na,
Uridine 1.10 w / v%, thymidine 0.36 w / v
%, Purified sucrose 20.00 w / v%, ethyl paraoxybenzoate 0.009 W / V% and butyl paraoxybenzoate 0.006 W / V%, purified water is first heated, and a sweetener is added to this. Was added and dissolved with stirring, and after cooling, each nucleic acid component, and ethyl paraoxybenzoate and butyl paraoxybenzoate as preservatives dissolved in a small amount of ethanol were added and dissolved with stirring. Then, after adjusting the liquid volume with purified water, it was filtered, filled in a glass container, and after nitrogen substitution, the container was closed, and the container was heat-sterilized to prepare a bacterial protective agent of the present invention in a liquid drug form.

Inosine: cytidine: GMP
The molar ratio of 2Na: uridine: thymidine is about 4: 4: 4 :.
3: 1 and total free nucleic acid concentration was 6.8 w / v%.

[0043]

[Production Example 11] In the same manner as in Production Example 10, the bacterial protective agent of the present invention as a liquid preparation having the following composition (total free nucleic acid concentration: 4 w / v)
%) Was adjusted.

[0044]

[0045]

[Production Example 12] Pure crystals of the respective components in the following composition were prepared,
It was added to distilled water for injection and dissolved by stirring to make the total amount 1 l. Next, the obtained aqueous solution is aseptically filtered and filled in an injection container, which is closed in an autoclave for 10 minutes.
Sterilize at 5 ° C for 40 minutes, and then inject (2 x 5 ml x 2
00) as the bacterial protective agent of the present invention (total free nucleic acid concentration: 3.
35 w / v%).

[0046]

[0047]

[Production Example 13] Inosine 2.
4 g, cytidine 2.2 g, 5'-GMP.2Na 3.7
g, 1.7 g of uridine and 0.5 g of thymidine were each passed through a 60-mesh sieve and mixed, and then 89.5 g of starch as an excipient was added and mixed evenly to obtain a powder. The bacterial protective agent of the present invention was prepared.

The ratio of each nucleic acid component in the obtained preparation is
Molar ratio of inosine: cytidine: 5'-GMP.2Na:
Uridine: thymidine = 4: 4: 4: 3: 1.

[0049]

MANUFACTURING EXAMPLE 14 The bacterial protective agent of the present invention as a powder was prepared in the same manner as in Manufacturing Example 13 except that the components were adjusted as follows.

[0050]

[0051]

[Production Example 15] The bacterial protective agent of the present invention as a powder was prepared in the same manner as in Production Example 13 except that the components were adjusted as follows.

[0052]

[0053]

[Production Example 16] The bacterial protective agent of the present invention as a powder was prepared in the same manner as in Production Example 13 except that the components were adjusted as follows.

[0054]

[0055]

[Pharmacological Test Example 1] SD male rats (7 weeks old) were divided into 3 groups, each of which was fasted overnight, and then high calorie infusion (T
PN) jugular cannula indwelling was performed to initiate TPN. A predetermined dose of 50% was administered on the first day of infusion and thereafter 100%, and TPN and its management were performed for a total period of 9 days.

Of the 3 groups of the high calorie infusion administration group, 2 groups (the 1st group and the 2nd group) were infusions of usual sugar, amino acid and electrolyte composition [GE-3 (pharmacology and treatment, vol. 20, suppl. 2,199
2, p645-655, Otsuka Pharmaceutical Factory Co., Ltd., Amiparene (made by the same company), and OMV (JJPEN, vol.11,
No. 4, 1989, p405-408, manufactured by Doujinsha Co., Ltd.), which was appropriately mixed so as to have a glucose content of 20.2% and an amino acid content of 4.4%. Was administered to the infusion solution, which was further mixed with a composition liquid containing the bacterial protective agent of the present invention obtained in Production Example 1 in an amount of 0.5 mmol / kg / day.

After maintaining the above TPN for 6 days, the test animals in each group were treated with physiological saline for the first group, and for the second and third groups with distilled water to a concentration of 25 mg / ml of MTX (methotrexate). ) By Fox et al. (ADFox, et al.,
Journal of Parenteral and Enteral Nutrition, 12, 3
According to 25-331 (1988) iv.bolus 0.8 ml / each
kg was administered, and TPN was continued even after the administration.

72 hours after the administration of the physiological saline and MTX, the mesenteric lymph nodes (MLNs) of each test animal were collected, the tissue weight was measured, and 10 times the volume of the heart infusion medium was then added. Was added to homogenate the collected MLNs, and then serially diluted with sterile physiological saline to prepare a sample solution for viable cell count.

With respect to the above sample solution, the total viable count of the enterococci was measured using a heart infusion agar medium, and the viable bacteria (Enterobacteriaceae) count of the Enterobacteriaceae family was measured using a MacConkey agar medium. Number M-EN
Using T medium, Staphylococcus (Staphylococcus
aureus) The viable cell count was quantified by the surface smearing method using an egg yolk-added Mannit salt agar medium. As the culture conditions, 35 ° C. was adopted, and the total viable count, enterococcus count, and Staphylococcus aureus count were measured after 48 hours, and the viable count belonging to Enterobacteriaceae was measured after 24 hours.

According to the above, the total number of viable bacteria per 1 g of MLNs was determined by total colony forming unit (CFU).
The results obtained in step 1 are shown in FIG. Further, the ratio of each bacterium detected in MLNs is shown in Table 1 below (denominator is the number of experiments, numerator is the number of bacteria positives).

[0061]

[Table 1]

The following can be seen from FIG. That is, in the administration group (third group) of the bacterial protective agent of the present invention, M
The total viable cell count in LNs was significantly lower than that in the non-administered group (second group) of the bacterial protective agent of the present invention, which indicates that the transfer of bacteria into the body is significantly suppressed.

Furthermore, from Table 1 above, the detection frequency of bacteria in the mesenteric lymph nodes of TPN rats was as follows: total viable counts, enterobacteriaceae counts and Staphylococcus aureus counts by MTX administration. However, the detection rate of Staphylococcus aureus under MTX administration was 0% in the administration group (the third group) of the bacterial defense agent-containing infusion solution of the present invention, which is higher than that of the second group. It was significantly low, and it was revealed from this that the use of the bacterial protective agent of the present invention can selectively and significantly reduce the number of Staphylococcus aureus.

It was also found that the bacterial protective agent of the present invention can exert the same effect on enterococci other than Staphylococcus aureus.

From the above, it is apparent that the bacterial protective agent of the present invention can completely prevent the transfer of Staphylococcus aureus into the body due to permeation of the intestinal tract membrane during administration of a high-calorie infusion.

[Brief description of drawings]

FIG. 1 is a graph showing the total number of bacteria in the group administered with the bacterial protective agent of the present invention and the comparative control group in the test conducted according to Pharmacological Test Example 1.

Claims (3)

[Claims] 1. A nucleic acid component of the following (1) to (3) and /
Alternatively, a bacterial protective agent containing any of a combination of pharmacologically acceptable salts thereof as an active ingredient and protecting Staphylococcus aureus from entering the body from the intestine when the intestinal defense function is lowered. (1) Inosine, cytidine, GMP, uridine and thymidine or IMP (or inosine), CMP, GM
P, UMP (or uridine) and thymidine (2) CMP, UMP, IMP (or inosine) and thymidine (3) AMP, GMP, CMP, UMP and thymidine [wherein IMP is inosine-n'-monophosphate, CMP Is cytidine-n'-monophosphate, GMP is guanine-n '
-Monophosphate, UMP is uridine-n'-monophosphate,
AMP is adenine-n'-monophosphate. ] 2. A nucleic acid component comprising inosine, cytidine, GMP, uridine and thymidine and / or a pharmacologically acceptable salt thereof as an active ingredient.
Bacterial protective agent according to. 3. The bacterial protective agent according to claim 1, wherein the relative molar ratio of the nucleic acid constituents is the following molar ratio. Nucleic acid components Relative molar ratio Inosine 4 Cytidine 4 GMP 4 Uridine 3 Thymidine 1