JPS63190832A - Antimicrobial agent - Google Patents

Abstract

PURPOSE:To obtain an antimicrobial agent comprising lipoprotein containing apolipoprotein A-I as an active ingredient. CONSTITUTION:An antimicrobial agent comprising lipoprotein (e.g. human plasma lipoprotein preferably having 1.006-1.210 specific gravity and 180,000-2,300,000 molecular weight by gel filtration) containing apolipoprotein A-I as an active ingredient. The agent is effective as a preventive and a remedy against various infectious diseases (e.g. candidiasis, cryptococcosis, aspergillosis, zygomycetes disease, black fungus infections disease, virus infectious disease, etc.) in administration of anti-cancer drug, namely in chemotherapy of acute leukemia, transplantation of bone marrow, etc. The amount of the active ingredient is 0.05-50wt.%, preferably 0.1-30wt.% calculated as protein amount in the whole composition and a dose is 0.1-100mg/kg/day calculated as protein amount.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibacterial agent, and more particularly to an antibacterial agent containing a lipoprotein containing apolipoprotein A-I as an active ingredient.

Conventional technology In recent years, individuals with reduced or impaired biological defense ability (comp
Attention has been focused on the problem of susceptibility to infection in romised hosts, and in particular, so-called opportunistic infections (opportunistic infections) caused by pathogens that were previously harmless to the individual becoming pathogenic.
OppOrttlniStiC1nfection or terminal 1infection) has become an important problem in the clinical field. As a result of research in fields such as clinical bacteriology, the pathogen (causing bacteria) of this opportunistic infection has been identified as Staphylococcus epidermidis (3, epidermiformis).
dis), Pseudomonas
, Durham-negative bacilli such as Serratia (3erratia), Herpes simplex, t
-! SV), Variserazosta (Vari)
cell la zoster, VZV), cytomegalovirus (Cytomegalovirus)
, CMV) and other viruses, Candi
da albicans), Aspergillus (As
pcrc+1llus fumigatus), Nocardia (N ocardia asteroidea)
), fungi such as Pneumocystis carinii
carini i), toxopraz? (Toxopi
It has been revealed that the virus is caused by protozoa such as Asma c+ondii). However, research on the host side in this infectious disease is insufficient, and for example, with the exception of the neutrophil mechanism and the immune system, the bacterial defense ability of serum as a means of defense against infection is still largely unknown. Currently, various known antibiotics are used to treat the above-mentioned opportunistic infections, but none of the antibiotics currently in use are sufficiently effective against the above-mentioned pathogenic bacteria. There is a strong need for research and development of new drugs for these opportunistic infections that take into consideration the knowledge of the host's infection defense mechanism.

On the other hand, various studies have been conducted on plasma lipoproteins, and several reports have been made regarding the relationship between these lipoproteins and immunity and cancer.

That is, in 1971, Chan et al. reported that very low density lipoprotein (VLDL) has the effect of suppressing the production of colony-stimulating factor by bone marrow cells.In 1976, Chisari et al. showed that lipoproteins inhibit T cell rosette formation, and Bieber et al.
Each has been reported to inhibit the A test. Also 197
In 9 years:/' + Tsuk Vun (Hagmann et al.) above V
In addition to LDL, intermediate lipoprotein (IDL) and low-density lipoprotein (LDL) are
It has been reported that it suppresses lymphocyte transformation. Furthermore, an inverse relationship between carcinogenesis and serum cholesterol concentrations has been reported at several institutions, including the Framingham study, and in vitro experiments using lymphocytes have shown that lowering cholesterol concentrations in culture results in an increase in lymphocyte membrane formation. There are also reports that cholesterol levels decrease and immune abnormalities occur. Cytotoxicity against endothelial cells and fibroblasts caused by lipoproteins
There are also some reports regarding y). However, there are no reports that lipoproteins or any baijiu ingredients exhibit antibacterial activity against opportunistic infections.

Problems to be Solved by the Invention The purpose of the present invention is to provide a new antibacterial agent that is effective in the prevention and treatment of infections caused by various pathogenic bacteria, particularly opportunistic infections.

Means for Solving the Problems According to the present invention, there is provided an antibacterial agent whose active ingredient is a lipoprotein containing apolipoprotein A-IJ (hereinafter referred to as ApoA-IJ).

The antibacterial agent of the present invention is particularly suitable for pathogenic bacteria (causing bacteria) of opportunistic infections.
It exhibits excellent antibacterial activity against infectious diseases, and is effective as a prophylactic and therapeutic agent for these infectious diseases. In particular, the antibacterial agent of the present invention can be used to treat various infections such as cancercidosis, cryptococcosis, aspergillosis, and conjugation during the administration of anticancer drugs, where such opportunistic infections are frequently observed, such as chemotherapy for acute leukemia and bone marrow transplantation. It is useful as a prophylactic and therapeutic agent for complications such as mycosis, black fungal infection, viral infection, cytomegalovirus pneumonia, and the like.

The lipoprotein that is the active ingredient of the antibacterial agent of the present invention must contain apoA-I as an apoprotein. The above-mentioned lipoprotein is not particularly limited as long as it contains apoA-I, and any ordinary soluble lipoprotein can be used as an active ingredient of the antibacterial agent of the present invention. In particular, human plasma lipoproteins are preferable as the above-mentioned active ingredient, and among them, conventional methods such as specific gravity centrifugation, electrophoresis, precipitation, gel filtration, ion exchange chromatography, immunological methods, etc. High-density lipoprotein (HDL), which is separated by a combination of
) 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 1.00.
6-1.210, preferably 1.063-'1.210
, 180,000 to 2,300 as molecular weight by gel filtration
000, preferably 180,000 to 360,000.

The active ingredient of the present invention is most preferably a lipoprotein that can be purified by the above-mentioned immunological method focusing on apoA-■, particularly by affinity chromatography using an apoA-I antibody.

The antibacterial agent of the present invention is usually used in the form of a common pharmaceutical preparation. The formulation may contain various carriers commonly used in this field,
For example, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and lubricants. Various forms of this pharmaceutical preparation can be selected depending on the therapeutic purpose, and representative examples include tablets, emulsions,
Examples include powders, solutions, suspensions, emulsions, granules, capsules, suppositories, injections (solutions, suspensions, etc.). When forming into a tablet, carriers include excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and silicic acid, water, ethanol, propatool, Simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, binder such as polyvinylpyrrolidone, dry starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyvinylpyrrolidone, etc. Oxyethylene sorbitan fatty acid esters, lauryl FiAM
Sodium, stearic acid monoglyceride, starch,
Disintegrants such as lactose, disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil, etc., absorption enhancers such as quaternary ammonium bases and sodium lauryl sulfate, humectants such as glycerin and starch, starch, lactose, kaolin , bentonite, adsorbents such as colloidal silicic acid, purification, talc,
Lubricants such as stearate, boric acid powder, polyethylene glycol, etc. can be used. Furthermore, the tablets may be coated with a conventional coating, if necessary, such as sugar-coated tablets, L-latin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, or multilayer tablets. When forming into an emulsion, carriers include excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, and talc, binders such as gum arabic powder, tragacanth powder, gelatin, and ethanol, and laminalan agar. Disintegrants such as can be used. When forming into a suppository, carriers such as polyethylene glycol, cacao butter, higher alcohol,
Higher alcohol esters, gelatin, semi-synthetic glycerides, etc. can be used.

When prepared as injections, solutions, emulsions and suspensions are preferably sterilized and isotonic with blood, and when molded into these forms, diluents such as water, water, etc.
Ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. can be used. In this case, sufficient amounts of salt, glucose, glycerin, etc. to prepare an isotonic solution may be included in the preparation, and usual solubilizing agents, buffers, soothing agents, etc. may also be included. It's okay.

The preparations of the present invention in each of the above forms may further contain commonly used coloring agents, preservatives, fragrances, flavoring agents, sweeteners, etc., as necessary, and may also contain other active pharmaceutical ingredient compounds. You can also do that.

The active ingredients to be contained in the antibacterial agent of the present invention are not particularly limited and are appropriately selected from a wide range, but the protein content is usually 0.05 to 50% in the total composition, preferably 0.
．． A suitable amount is 1 to 30% by weight.

The method of administering the antibacterial agent of the present invention is not particularly limited and can be appropriately determined depending on various formulation forms, age, sex and other conditions of the patient, degree of disease, etc. For example, tablets, emulsions, solutions, suspensions, emulsions, granules, capsules, etc. are administered orally. Injections are administered intravenously alone or mixed with conventional replacement fluids such as glucose and amino acids, and if necessary, are administered alone intramuscularly, intradermally, subcutaneously, or intraperitoneally. Suppositories are administered rectally.

The dosage of the antibacterial agent of the present invention is appropriately selected depending on the method of use, the patient's age, sex and other conditions, the severity of the disease, etc., but the amount of active ingredient is usually about 0.00% protein per 1 kg of body weight per day.
The amount is preferably 1 to 100 mg.

X-Shi Ichiku In order to explain the present invention in more detail, experimental examples will be given below.

Experimental Engineering This experiment investigated the antibacterial activity in serum for diseases that tend to cause opportunistic infections.

■ First, 20 μQ of sterilized phosphate buffered saline (PBS) was dispensed into a 60-well microplate, and 20 μQ of each test serum that had been sterile-filtered with a 0.45 μm Millipore filter was added for multiple dilution. .

■ Staphylococcus (S, epidermi
disH155 strain) in trypto soy broth medium (T
SB) for 12 hours, then diluted 100 times with the same medium, cultured for 3 hours, and the number of bacteria was reduced to 1X 108 cfu/
m112, further diluted 10' times with casiton glucose medium (casiton glucose broth) of 5 times concentration, and readjusted the number of bacteria to 10' cfu/peng.

■ Dispense 5 μQ of the bacterial solution prepared in step ■ above into each well in ■. At this time, the number of bacteria will be 50 cfu/well), at 37°C.
The cells were cultured for 12 hours and the growth rate of the bacterial cells was observed.

(2) The strength of the antibacterial action of the test serum was determined based on the number of times the test serum was diluted to inhibit the growth of bacterial cells in (2) above.

The results obtained using serum from patients with various diseases and healthy individuals as test serum are shown in Table 1 below.

Table 1 From Table 1 above, serum from healthy individuals exhibits antibacterial activity up to an 8- to 24-fold dilution, but serum from patients with diseases that are prone to opportunistic infections, such as leukemia, granulocytopenia, cancer, etc. ~
It shows antibacterial activity only up to 6-fold dilution, indicating that the biological defense mechanism is clearly reduced.

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

(3) 3 ml of test serum was fractionated using Urotrogel ACA54 under the following conditions.

Column size: 2.5x 100cm Eluent: 0.3
2.5mM Tris-HCl buffer containing NaCQ (pH
7, 8) Fraction: 2m12/tube As a result, substances exhibiting strong antibacterial activity (measured according to Experiment 1) were obtained in fractions No. 0.70 and 71.

The molecular weight of this substance is the human blood high-density lipoprotein (
HDL).

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

(2) 5mf2 of the test serum was fractionated using ion exchange chromatography DE52 cellulose (Whatman) under the following conditions.

Weight of resin used: 10Cl (18 precepts) Eluent: 2.5
mM Tris-HCl buffer (pH 7,8>, O → 1M N
aC2 gradient elution fraction: 1 ml/tube The antibacterial activity of each fraction was measured according to Experiment 1, and activity was observed in fractions No. 2 to 13.
Among them, Nos. 12 to 13 and Nos. 4 to 6 had particularly strong antibacterial activity.

■ Fraction with antibacterial activity obtained in above (■)
Fraction No. 12-13), TSK G30
High performance liquid chromatography (HPL) using 00SW
C) was subjected to gelcin filtration. The condition is that the flow rate is 0.5
m12/min, 30C) psi, 0.5AuFs, 0.
5mG/tube = 10 drops, eluent = 0.2M N
a2HPOt buffer (pH 6,8>).

As a result, a fraction of a substance showing strong antibacterial activity (measurement was the same as in Experiment 1) at a retention time of 29 minutes to 39 minutes was obtained.

■ The antibacterial active substance obtained in the above (■) was run on an SDS gel and the migration position was observed (clinical test, V).

1.29. No. 11. 1985 1) I) 1416~
1421).

As a result, we confirmed the presence of a substance migrating at the same position as Apo A-I among the antibacterial active substances mentioned above, and from this, H
The existence of antibacterial activity of DL was confirmed.

Experiment 4 This experiment investigated the antibacterial activity of lipoproteins.

(2) Each lipoprotein fraction was separated from the test serum by ultracentrifugation, and HDL, LDL, and VLDL were collected.

■ Adjust the above HDL, LDL and VLDL (7) 1L to the same concentration with 50mM NH4HCO3 (pH 8, 0>1 buffer), pass through a 0.45μm Millipore filter, and then add 1mQ of each to a small test tube. As a control, 1 scoop of the same buffer was prepared by the same procedure.

■ Staphylococcus (3, epidermid
isH155 strain) was cultured in TSB for 12 hours, diluted 100 times with the same medium and cultured at 37°C for 3 hours, and the resulting culture solution was further diluted 100 times with the same medium and used. 10 μQ of this was added to each small test tube prepared in step ① above to obtain a bacterial count of 5×10 3 cfu/test tube.

■ Keep each test tube in ■ above at 37°C in a constant temperature bath.
50 μQ was taken from each test tube over time, the number of bacterial cells was measured under a microscope, and the antibacterial activity of the lipoprotein in each test tube was investigated.

Table 2 below shows the results of calculating the change in the number of bacteria over time in each sample, assuming that the number of bacteria at 0 hours is 1.0 (standard). Incidentally, the test serum samples collected from three subjects were tested.

Table 2 From Table 2 above, the strength of the antibacterial activity of lipoproteins is:
It can be seen that HDL>LDL>VLDL. More specifically, each of the above lipoprotein fractions inhibited the growth of bacterial cells better than the control after 3 hours of culture, but VLDL inhibited the growth of bacterial cells after 9 hours of culture. In fact, it has resulted in promotion. On the other hand, it is clear that HDL effectively inhibits the growth of bacterial cells even after 21 hours of culture.

Experiment 5 Purification of lipoproteins by affinity chromatography of anti-apo A-I antibody ■ The antibacterial active substance fractions obtained in Experiment 3 (retention time fractions of 31 minutes, 32 minutes, and 33 minutes) were used as raw material fraction N3.
1, N32 and N33.

Each raw material fraction was further added to an anti-apoA-I monoclonal antibody binding affinity column (Apocolumn A-I: manufactured by Nippon Antibody Research Institute, column volume 1-), and first 0.15M
0.01M phosphate buffer containing NaCQ (pH 7,
2> to obtain a first eluate fraction (column non-adsorbed fraction). Next, after washing the column with the same buffer, 0.
Elute the fraction adsorbed on the column with 1M acetate buffer containing 5M NaCQ, and divide the resulting eluate by 0.5xff1.
Neutralize with 3.5M Tris aqueous solution (v/v), and
ApoA-I-containing protein) was obtained by fractionating the eluate.

■Each raw material fraction used in above (■), each elution fraction obtained from these (first eluate and second eluate), and a phosphate buffer containing 0.15M NaCQ (117
, 2) (control ■) and 0.5M NaCQ-containing 1-containing 1-mild acetic acid buffer (v/v) 3.5M Tris-HCl buffer (control ■) using the microplate method in Experiment 1. Antibacterial activity was investigated. However, in this study, the presence of bacterial growth was determined as (+) and the absence of bacterial growth was determined as (-).

The results obtained are shown in Table 3 below.

Table 3 Concentration of protein contained in each portion (mg/m2)
The results obtained are as shown in Table 4 below.

■ Also, for each of the fractions in ■ above,
They were added to an SDS gel in the same manner as in (2) above, and the composition of the proteins contained in each was analyzed.

As a result of analyzing the above SDS gel electrophoresis pattern, the presence of apoA-I was not observed in the first eluate, but apoA-I was detected in the second eluate.

From the results in Tables 3 and 4 above, the second eluate fraction is:
Although the protein concentration is lower than that of the first eluate fraction, it is clear that it exhibits strong antibacterial activity, and from this, particles containing apoA-I (lipoproteins)
This lipoprotein was found to be effective as an antibacterial agent.

Examples of formulations of the antibacterial agent of the present invention are listed below. In addition, on each side, the second eluate fraction (apoA-I-containing protein) obtained in Section 2 of Experiment 5 above was used as the active ingredient.

Formulation Example 1 Injections having approximately the same composition as a normal amino acid preparation and fat emulsion were prepared, except that the active ingredients were blended so that the protein content was 5% by weight.

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

(that's all)

Claims (1)

[Claims] (1) An antibacterial agent containing a lipoprotein containing apolipoprotein A-I as an active ingredient.