JPH085798B2 - Antibacterial agent - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel antibacterial agent, and more particularly to an antibacterial agent containing a lipoprotein containing apolipoprotein AI as an active ingredient.

2. Description of the Related Art In recent years, individuals with compromised or impaired biological defense (compro
Attention has been paid to the problem of susceptibility to infection in mised hosts, and in particular, so-called opportunistic infections (opportunistic infectious diseases) caused by the pathogenicity of pathogens that were previously harmless to the individual.
infection or terminal infection) has become an important issue in the clinical field. Regarding the pathogens (pathogenic bacteria) of the opportunistic infections, as a result of research in fields such as clinical bacteriology, gram-negative bacilli such as S. epidermidis, Pseudomonas, and Serratia, herpes (Herpes simplex, HSV), Paris cellar sosta (Varicella zoster, VZV), cytomegalovirus (Cytomegalovirus, CMV) and other viruses,
Candida albicans, Aspergillus fumigatus, Nocardia
asteroidea), fungi such as carini (Pneumocystis car
inii), Toxoplasma gondii and other protozoa. However, research on the host side of the infectious disease is insufficient, and, for example, except for the neutrophilic mechanism and the immune mechanism, the bacterial defense ability of serum as a defense mechanism against the infection is almost unknown. However, currently, for the treatment of the opportunistic infections, various known antibiotics are used, but among the currently used antibiotics, there is no one that can exert a sufficient effect on the pathogenic bacterium,
The research and development of a new drug against the opportunistic infection, which takes into account the knowledge of the host defense mechanism, has been earnestly desired.

On the other hand, various studies on plasma lipoproteins have been carried out, and some reports have been made on the relationship between the lipoproteins and immunity and cancer. That is, in 1971, Chan et al. Added that ultra-low density lipoprotein (VLDL)
It has been reported that it has an action of suppressing the production of colony stimulating factor by bone marrow cells. Chisar (Chisar in 1976)
i) et al. reported that lipoproteins inhibit T cell rosette formation, and Bieber et al. reported that lipoproteins suppress PHA testing. See also 197
In 1993, Hagmann et al., In addition to the above VLDL, intermediate-type lipoprotein (IDL), low-density lipoprotein (LD)
L) report that it suppresses the transformation of B lymphocytes by the EV virus. Furthermore, carcinogenesis and the reverse phase of serum cholesterol concentration have been reported at several institutions including the Framingham investigation, and in vitro experiments using lymphocytes, when cholesterol concentration in culture was lowered, lymphocyte membranes were detected. There is also a report that the cholesterol level is lowered and immunological abnormalities are caused. Lipoprotein-induced cytotoxicity to endothelial cells and fibroblasts
There are some reports about toxicity). However, there is no report that any serum component, not to mention lipoprotein, exhibits antibacterial activity against opportunistic infections.

Problems to be Solved by the Invention An object of the present invention is to provide a new antibacterial agent effective for the prevention and treatment of infectious diseases caused by various pathogenic bacteria, particularly opportunistic infectious diseases.

Means for Solving Problems According to the present invention, an antibacterial agent containing a lipoprotein containing apolipoprotein AI (hereinafter referred to as “apo AI”) as an active ingredient is provided.

The antibacterial agent of the present invention exhibits excellent antibacterial activity against pathogens (pathogenic bacteria) of opportunistic infections, and is effective as a preventive and therapeutic agent for the infections. In particular, the antibacterial agent of the present invention, various types of infectious diseases during administration of anticancer agents in which such opportunistic infections are frequently seen, that is, chemotherapy or bone marrow transplantation of acute leukemia, such as gandidiasis, cryptococcosis, aspergillosis, conjugation. Mycosis, black fungus infection,
It is useful as an agent for preventing and treating viral infections, cytomegalovirus pneumonia, and complications such as these.

The lipoprotein as an active ingredient of the antibacterial agent of the present invention essentially contains apoA-I as an apoprotein. However, the above lipoprotein is not particularly limited as long as it contains apo AI, and any ordinary soluble lipoprotein can be used as an active ingredient of the antibacterial agent of the present invention. In particular, human plasma lipoprotein is preferable as the above-mentioned active ingredient, and among them, usual methods such as specific gravity centrifugation, electrophoresis, precipitation, gel filtration, ion exchange chromatography, immunological methods and those High density lipoprotein (HDL) separated by combination etc.
Is preferred. The lipoprotein that can be preferably used as the active ingredient of the antibacterial agent of the present invention has a specific gravity of 1.006 to 1.2.
10, preferably 1.063 to 1.210, and those having a molecular weight of 180,000 to 23,000, preferably 180,000 to 360,000 by gel filtration.

As the active ingredient of the present invention, the lipoprotein obtained by the above-mentioned immunological method focusing on apoA-I, in particular, affinity chromatography using the apoA-I antibody is most preferable.

The antibacterial agent of the present invention is usually used in the form of a general pharmaceutical preparation. The preparation includes various carriers commonly used in this field, for example, fillers, fillers, binders, moisturizers, disintegrants,
It is prepared using a diluent or excipient such as a surface active agent and a lubricant. Various forms of this pharmaceutical preparation can be selected according to the therapeutic purpose, and typical examples thereof include tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules,
Suppositories, injections (solutions, suspensions, etc.) and the like can be mentioned. When molding into a tablet form, as a carrier, for example, lactose,
Excipients such as sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, 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, dry starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene Sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch,
Disintegrants such as lactose, sucrose, stearin, cocoa butter, disintegration inhibitors such as hydrogenated oil, quaternary ammonium base, absorption promoters such as sodium lauryl sulfate, humectants such as glycerin and starch, starch, lactose, kaolin Adsorbents such as bentonite and colloidal silicic acid, purified talc, stearates, boric acid powders, and lubricants such as polyethylene glycol can be used. Further, the tablets may be tablets coated with a usual coating, for example, sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets or double tablets or multilayer tablets. In the case of molding in the form of pills, as a carrier, for example, glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc sugar excipient, gum arabic powder, tragacanth powder, gelatin, binder such as ethanol, laminaran A disintegrant such as agar can be used. When molded 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. When prepared as an injection,
Liquids, emulsions and suspensions are preferably sterilized and isotonic with blood, and when molded into these forms,
As the diluent, for example, water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, etc. can be used. still,
In this case, a sufficient amount of salt, grape, etc., glycerin, etc. may be contained in the preparation to prepare an isotonic solution, and a usual solubilizing agent, buffer, soothing agent, etc. may be added. Good. In the preparation of the present invention in each of the above-mentioned forms, a coloring agent, a preservative, a flavoring agent, a flavoring agent, a sweetening agent and the like which are commonly used can be further incorporated, if necessary, and other active pharmaceutical ingredient compounds are contained. You can also

The amount of the active ingredient to be contained in the antibacterial agent of the present invention is not particularly limited and is appropriately selected from a wide range, but is generally 0.05 to 50% by weight, preferably 0.1 to 3% by weight in the total composition as the amount of protein.
An amount of 0% by weight is suitable.

The administration method of the antibacterial agent of the present invention is not particularly limited, and can be appropriately determined according to various dosage forms, patient's age, sex and other conditions, degree of disease and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules, capsules and the like are administered orally. The injections are administered intravenously alone or in admixture with usual replenishers such as glucose and amino acids, and if necessary, they are administered intramuscularly, intracutaneously, subcutaneously or intraperitoneally alone. Suppositories are administered rectally.

The dose of the antibacterial agent of the present invention is appropriately selected depending on the usage, the age of the patient, the sex and other conditions, the degree of wetness, etc., but usually the amount of the active ingredient is about 0.1 to about 1 to 1 kg of body weight per day as the protein amount.
The amount should be 100 mg.

Experimental Examples Experimental examples will be given below in order to explain the present invention in more detail.

Experiment I This experiment was conducted for diseases that are prone to opportunistic infections.
This is a study of antibacterial activity in serum.

First, sterilized phosphate-buffered saline (PBS) was dispensed in 20 μm aliquots onto a 60-well microplate, and 0.45
Each test serum sterilized by μm Millipore filter μ
Was added to dilute multiples.

As a bacterial solution, Staphylococcus (S. epidermidis H155 strain)
Was cultivated in tryptic soy broth medium (TSB) for 12 hours, then diluted 100-fold with the same medium and cultivated for 3 hours, and the number of bacteria was adjusted to 1 × 10 ⁸ cfu / ml. The culture medium was diluted 10 ⁴ times with caditon glucose broth, and the number of cells was readjusted to 10 ⁴ cfu / ml.

5 μl of the above-prepared bacterial solution was dispensed into each well (at this time, the number of bacteria was 50 cfu / well) and cultured at 37 ° C. for 12 hours, and the growth rate of bacterial cells was observed.

In the above, the strength of the antibacterial action of the test serum was judged by using as an index the multiplication factor of the test serum up to a multiple dilution.

Table 1 below shows the results obtained by using the sera of patients with various diseases and healthy individuals as test sera.

From Table 1 above, healthy normal human serum shows antibacterial activity up to 8 to 24 times dilution, but in the serum of patients such as leukemia, granulocytopenia, cancer, etc.
It can be seen that the antibacterial activity is exhibited only up to 6-fold dilution, and the biological defense mechanism is clearly reduced.

Experiment 2 In this experiment, the molecular weight fractionation of serum was carried out to determine the molecular weight of the substance exhibiting antibacterial activity in the serum.

3 ml of the test serum was fractionated using Urotrogel AcA54 under the following conditions.

Column size: 2.5 x 100 cm Eluent: 2.5 mM Tris-HCl buffer (pH 7.
8) Fraction: 2 ml / tube As a result, a substance having a strong antibacterial activity (measurement according to Experiment 1) was obtained in fractions Nos. 70 and 71.

The molecular weight of this substance corresponded to human blood high density lipoprotein (HDL).

Experiment 3 Based on the result of Experiment 2, the antibacterial activity of human HDL was examined as follows.

5 ml of test serum was subjected to ion exchange chromatography DE5
2 Cellulose (Whatman) was used for fractionation under the following conditions.

Resin weight used: 10 g (volume 18 ml) Eluent: 2.5 mM Tris-HCl buffer (pH 7.8), 0 → 1M NaC
l Gradient elution Fraction: 1 ml / tube The antibacterial activity of each fraction was measured according to Experiment 1, and as a result, the activity was observed in fraction Nos. 2 to 13, in particular No. 12 to 13 and No. 4 to 6 Had strong antibacterial activity.

The above-obtained fractions having antibacterial activity (fraction Nos. 12 to 13) were gel-filtered by high performance liquid chromatography (HPLC) using TSK G3000SW. The conditions are: flow rate 0.5 ml / min, 300 psi, 0.5 AuFs, 0.5
ml / tube = 10 drops, eluent = 0.2M Na ₂ HPO ₄ buffer (pH
6.8).

As a result, a francion of a substance having a strong public money activity (measurement is the same as in Experiment 1) was obtained at a retention time of 29 minutes to 39 minutes.

The antibacterial active substance obtained above was observed for migration position by SDS gel [Clinical examination, Vol.29, No, 1985, pp1416-142.
1].

As a result, the presence of a substance that migrates to the same position as Apo AI in the above-mentioned antibacterial active substance was confirmed.
The existence of antibacterial action of HD was confirmed.

Experiment 4 This experiment examined the antibacterial activity of lipoproteins.

From the test serum, each lipoprotein fraction was separated by ultracentrifugation, and HDL, LDL and VLDL were collected, respectively.

Each of the above HDL, LDL and VLDL is 50 mM NH ₄ HCO ₃
After adjusting the same concentration with (pH8.0) buffer and passing through a 0.45 μm Millipore filter, 1 ml of each was dispensed into a small test tube. As a control, 1 ml of the same buffer was prepared by the same operation.

As a bacterial solution, Staphylococcus (S. epidermidis H155 strain)
After culturing in TSB for 12 hours, dilute it 100 times with the same medium and
Incubate at 37 ℃ for 100 hours, and add 100
A one-fold diluted solution was used. This was added to each of the small test tubes prepared above in an amount of 10 μm to give a bacterial count of 5 × 10 ₃ cfu / test tube.

Keep each test tube above at 37 ° C in a constant temperature bath, sample 50μ from each test tube over time, measure the number of cells under a microscope, and examine the antibacterial activity of lipoprotein in each test tube. It was

Table 2 shows the results of calculating the change in the number of bacteria over time in each sample, with the number of bacteria at 0 hours being 1.0 (standard). The test sera were collected from three test subjects and tested.

From Table 2 above, it can be seen that the antibacterial activity of lipoproteins is HDL>LDL> VLDL. For more details,
Each of the above ribbon protein fractions well inhibited the growth of bacterial cells after 3 hours of culture, compared to the control.
After 9 hours of culturing, VLDL rather promotes the growth of bacterial cells. On the other hand, it is clear that HDL satisfactorily inhibits the growth of bacterial cells even after 21 hours of culture.

Experiment 5 Purification of Lipoprotein by Affinity Chromatography of Anti-Apo AI Antibody The antibacterial active substance fractions (retention time 31 minutes, 32 minutes and 33 minutes fractions) obtained in Experiment 3 were used as raw material fractions N31 and N32. And used as N33.

Each raw material fraction was further added to an anti-apo AI monoclonal antibody-binding affinity column (Apo column AI: manufactured by Japan Antibody Research Institute, column volume 1 ml), and 0.15 M NaCl was first added.
Elute with 0.01M phosphate buffer (pH7.2)
An eluate fraction (column non-adsorbed fraction) was obtained. Then, after washing the column with the same buffer, the fraction adsorbed to the column was eluted with 1M acetate buffer containing 0.5M NaCl, and the resulting eluate was diluted with 0.5 volume (v / v) of 3.5M Tris aqueous solution. The mixture was neutralized with to obtain a second eluate fraction (apo AI-containing protein).

Each raw material fraction used above, each elution fraction (first eluate and second eluate) obtained from these, and 0.15M NaCl-containing phosphate buffer (pH 7.2) as a control (Control I)
And 0.5M NaCl-containing 1M acetate buffer + 0.5 times volume (v / v) 3.5M
The antibacterial activity of each of the Tris-hydrochloric acid buffer solutions (Control II) was examined by the microplate method of Experiment 1. However, in this examination, the presence or absence of bacterial growth was determined as (+) and the absence thereof was determined as (-).

 The results obtained are shown in Table 3 below.

The results of determining the protein concentration (mg / ml) contained in each fraction are as shown in Table 4 below.

In addition, for each of the above fractions,
The protein contained in each was analyzed in the same manner as in Example 1 by adding it to SDSS gel.

As a result of analyzing the pattern of the SDS gel electrophoresis, the presence of apo AI was not observed in the first eluate, and apo AI was detected in the second eluate.

From the results of Tables 3 and 4 above, it is clear that the second eluate fraction exhibits a strong antibacterial activity, even though the protein concentration is lower than that of the first eluate fraction.
From this, it was found that the particles (lipoprotein) having apoA-I have a strong activity, and that this lipoprotein is effective as an antibacterial agent.

Hereinafter, formulation examples of the agent of the present invention will be given. In each example, the second eluate fraction (apo AI containing lipoprotein) obtained in Experiment 5 was used as the active ingredient.

Production Example 1 An injection having the same composition as a conventional amino acid preparation and a fat emulsion was prepared, except that the active ingredient was added so that the amount of protein was 5% by weight.

Production Example 2 A hydrophilic ointment was prepared in the same manner as the hydrophilic ointment described in the Japanese Pharmacopoeia, except that an active ingredient of 10% by weight as a protein amount was blended.

Claims (1)

[Claims] 1. An antibacterial agent comprising a lipoprotein containing apolipoprotein AI as an active ingredient.