JPH0789866A - Antiinfectant - Google Patents

Abstract

PURPOSE:To obtain an antiinfectant for opportunistic infection microorganisms, containing a polypeptide having human monocyte chemotactic properties and an activation factor as the active component. CONSTITUTION:A polypeptide having a monocyte mobilization effect or an activation effect for enhancing the tumor cell cytostatic effect of a monocyte is contained as the active component. This medicine is useful for prevention of intrinsic or exogenous infectious diseases caused by immunological hypofunction of a host, abnormality of hormones, administration of an immunosuppressive agent, an operation, a cancer-carrying state, etc., or by an opportunistic infection microorganism such as Pseudomonas aeruginosa, a Salmonella, a Candida or a methicillin-resistant staphylococcus. A life-prolongation effect is exhibited especially on a mouse with an infectious disease caused by an opportunistic infection microorganism and there is hardly side effect. This medicine is administrated to a human in an amount of 0.01 to 200mg per day in one to five times.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an infection protective agent against opportunistic infectious bacteria containing human monocyte chemotactic and activating factor (hereinafter referred to as MCAF) as an active ingredient.

[0002]

2. Description of the Related Art Gentamicin, amphotericin B, sulfa drugs, sulfisoxazole and the like are used as chemotherapy for opportunistic infections, but there are few specific drugs.

[0003]

[Problems to be Solved by the Invention] Endogenous or extrinsic factors which have not been so much a problem in the past due to physical invasion such as deterioration of host immune function, hormonal abnormalities, use of immunosuppressive agents, surgery, and cancer-bearing state. Pseudomonas
seruginosa), Salmonella (Sa-lm)
onella typhimurium), attenuated bacteria such as Candida-Candida-albicans, or methicillin-resistant Staphylococcus (Methicillin-) which has become resistant due to excessive use of antibacterial antibiotics.
resist-ant Staphylococcus
aureus: MRSA) and infections by opportunistic bacteria. Therefore, there is a demand for the development of a drug that enhances the immunopotency of infection and protects against infection.

[0004]

Therefore, as a result of earnest studies, the present inventors have found that BCMGSeno, which incorporates 400 base pairs of the Pst-1 fragment of human MCAF cDNA, is X63A.
Recombinant human MCAF produced by transfecting g8-653 myeloma cells was prepared, and it was found that this product is useful as an infection protective agent against opportunistic infection bacteria, and the present invention was completed. That is, the present invention relates to an agent for protecting against opportunistic infections, which comprises a polypeptide having human monocyte chemotaxis and activator as an active ingredient.

In the present invention, the peptide having human monocyte chemotaxis and activator means a polypeptide having an activating effect of mobilizing monocytes or enhancing the tumor cell growth suppressing effect of monocytes. is there. The amino acid sequence of this polypeptide and the method for producing it are described in JP-A-2-207.
788.

When the compound of the present invention is administered to humans, an appropriate amount may be administered depending on the body weight, age and symptoms, and for example, 0.01 to 200 mg may be administered 1 to 5 times a day. The compound of the present invention may be administered by an appropriate method of administration, for example, an injectable preparation, and a lyophilized product of the compound of the present invention may be appropriately administered as an osmotic pressure adjusting agent, a preservative, a pH adjusting agent, a saccharide (for example, , Sucrose, etc.) and the like may be dissolved in distilled water for injection to which a stabilizer or the like is added. It is also possible to administer this lyophilized product as a liposome preparation by adding additives such as phospholipids.

Further, the compound of the present invention is used as an oily base for suppositories, for example, oils and fats such as olive oil, peanut oil, sesame oil, soybean oil, rapeseed oil, camellia oil, cacao butter, lard, wool fat, beef tallow, etc. or hydrogenation thereof. In addition to oil, acetylated products, and emulsions obtained by emulsifying these with a surfactant, alone or in a mixture of two or more, or in an aqueous base such as glycerogelatin, a base such as propylene glycol, or a mixture of two or more. It may be prepared as a suppository formulation of 1 to 2 g, or may be a percutaneous absorption formulation or nasal formulation by adding and using an appropriate thickening agent, hygroscopic agent, absorption promoter and the like. The proportion of the compound of the present invention in the whole preparation is about 0.01-
It is about 99%, preferably about 0.1 to about 50%, with the balance being additives and excipients.

[0008]

Next, the action of MCAF to prevent infection will be specifically described with reference to test examples. Test Example 1 MCAF P. Protective effect against aeruginosa infection Test method BALB / c mice (8-12 weeks old, 10 mice per group) were intraperitoneally supplemented with 25 ng, 250 ng or 2.5 ng of MCAF.
A dose of μg / mouse was administered, and an untreated group was set as a control. 6 hours later, 1 × 10, which is 25 times the LD ₅₀
⁷ CFU (colony forming unit; viable cell count) P. aeru
Ginosa was inoculated intraperitoneally. The results are shown in Table 1. According to the comparison of the survival rate 48 hours after the bacterial inoculation, the control group died within 48 hours, but the MCAF 2.5 μg administration group completely survived the mice. In the 250 ng administration group, 4 out of 10 survived, and in the 25 ng administration group, 3 survived.

Table 1 ─────────────────────────────────── Group survival comparison (48 hr) ─── ──────────────────────────────── Untreated group 0/10 MCAF 25 ng administration group 3/10 250 ng administration group 4 / 10 2.5 μg administration group 10/10 ────────────────────────────────────

Test Example 2 P. of MCAF in leukopenic mice. aerugi
Protective effect against nosa infection Test method As for leukopenic mice, BALB / c mice (8 to 12 weeks old, 10 mice per group) were intraperitoneally injected with cyclophosphamide at a dose of 200 mg / kg 4 days before infection. Created. The infection was 1.5 × 10 ⁴ CFU of P. aerugin
It was induced by inoculating intraperitoneally with osa. MCAF was intraperitoneally 25 ng, 250 ng, or 2. 6 hours before infection.
It was administered at a dose of 5 μg / mouse. The results are shown in Table 2. The control group died due to infection in all cases within 3 days after the bacterial inoculation, but a protective effect was observed in the MCAF administration group.

Table 2 ─────────────────────────────────── Group survival rate comparison (3day) ─── ──────────────────────────────── Untreated group 0/10 MCAF 25 ng administration group 6/10 250 ng administration group 6 / 10 2.5 μg administration group 9/10 ────────────────────────────────────

Test Example 3 MCAF S. Protective effect against typhimurim infection Test method C3H / HeJ mice (8 to 12 weeks old, 10 mice per group) were intraperitoneally administered with 25 or 250 ng of MCAF or 2.
It was administered at a dose of 5 μg / mouse, and an untreated group was set as a control. After 6 hours, 1 × 10 ⁶ CFU of S. type
Himuri-um was inoculated intraperitoneally. The results are shown in Table 3. According to the comparison of the survival rate on the 6th day after the bacterial inoculation, the control group died within 6 days, but the mice completely survived in the MCAF2.5 μg administration group. LD ₅₀ is 0.5 μg / m
I was able to estimate it as "use". 6/10 mice survived in the 250 ng group, and 3/10 in the 25 ng group.
10 were able to survive.

Table 3 ─────────────────────────────────── Group survival rate comparison (6day) ─── ──────────────────────────────── Untreated group 0/10 MCAF 25 ng administration group 3/10 250 ng administration group 6 / 10 2.5 μg administration group 10/10 ────────────────────────────────────

Test Example 4 M in vitro for phagocytosis and sterilization of bacteria
Effect of CAF Test method Mice were intraperitoneally pre-administered 2 ml of thioglycollate, and 4 days later, induced peritoneal cells were collected. The cells in the peritoneal cavity were suspended in RPMI culture medium containing 5% bovine serum, and 2
1 x 10 ⁶ cells / well on a 24-well plate
It was sprinkled at the concentration of. After removing non-adherent cells, adherent macrophages were treated with 0.1, 1.0, 10 and 100 ng of M.
Precultured with CAF for 12 hours. And 1 x 10
⁶ CFU P. aeruginosa was infected. Three
After culturing for 0 minutes, the wells were washed to remove non-phagocytic bacterial cells, and the wells of one plate were used for the experiment of phagocytosis to 0.
It was dissolved in a 05% Triton X-100 solution. The well of the other plate was incubated with a new culture solution for another 150 minutes for the sterilization experiment, and then 0.05% Triton X-1.
It was dissolved in the 00 solution. Then, CFU in the cell disruption solution was measured by quantitative culture. Data are mean ± SE of 4 experiments. The phagocytosis rate was expressed by the number of intracellular bacteria. The sterilization rate was calculated according to the following formula.

Sterilization rate = [1- (CFU / 3 after 180 minutes
CFU after 0 minutes] × 100% The untreated control group after 30 minutes of culture is indicated in parentheses on the right side of the phagocytosis rate of 1%.
The ratio is shown. The results are shown in Table 4. Based on this data, MCAF is a macrophage P. aer-ug
It was shown that the phagocytosis against inosa was clearly enhanced in a concentration-dependent manner. MCAF 100 ng / ml treatment induced 5 times more phagocytosis than the untreated control group. Further MC
AF treatment significantly increased the bactericidal rate of the bacteria. Similarly, the enhancement of phagocytic ability and the increase of bactericidal rate are S. typhimuri
um, methicillin resistant S. aure-us and C.I. alb
It was similarly observed in icans.

Table 4 Group culture time Phagocytosis rate Germicidal rate (min) CFU (1 × 10 ³ ) (%) ───────────────────────── ─────── Untreated group 30 2.0 ± 0.4 (1.0) 180 1.4 ± 0.3 30.0 MCAF 1.0 ng / ml 30 4.1 ± 0.6 (2. 1) 180 2.6 ± 0.5 36.6 MCAF 0.1 ng / ml 30 5.9 ± 0.2 (2.9) 180 3.5 ± 0.4 40.7 MCAF 10 ng / ml 30 9.2 ± 0.5 (4.6) 180 2.3 ± 0.2 75.0 MCAF100ng / ml 30 10.1 ± 0.9 (5.0) 180 2.8 ± 0.1 72.3

[0017]

INDUSTRIAL APPLICABILITY MCAF has a significant life-prolonging effect in mice infected with opportunistic infections, and has almost no side effects, and it has been revealed that an infection preventive agent containing the peptide as an active ingredient is extremely effective.

[0018]

EXAMPLES Hereinafter, formulation examples will be shown as examples of the present invention. Example 1 20 mg of high-purity MCAF and 30 mg of sucrose were added to 5 m of salt.
The solution was dissolved in 10 ml of 5 mMol trisphosphate 0.5M phosphate buffer containing g, and this solution was freeze-dried to obtain an MCAF preparation for the present invention having the following composition. MCAF 20 mg Sodium phosphate 5 mmol Mol salt 5 mg Sucrose 30 mg Example 2 Dipalmitoylphosphatidylcholine (DPPC) 58.7 mg, Dioleoylphosphatidylcholine (D
OPC) 15.7 mg, cholesterol (chol)
9.6 mg was dissolved in 7.5 ml of diethyl ether.
High-purity MCAF in phosphate buffered saline (pho
sphate-buffered saline, PB
S) 2.5 ml of a solution (50 mg / ml) was added and dispersed with a tank type ultrasonic disperser, and diethyl ether was distilled off with an evaporator to form a gel. PBS (6 ml) was added to this gel and dispersed while rotating with an evaporator to obtain liposomes. The liposomes were washed by centrifugation. Then, the cells were sterilized by a sequential filtration method through a membrane having a pore size of 0.2 μm. This is diluted with sterile PBS and MC
An AF concentration of 4.8 mg / ml was filled in an ampoule to prepare a preparation.

Claims (2)

[Claims] 1. A protective agent against opportunistic infectious bacteria, which comprises a polypeptide having human monocyte chemotaxis and activator as an active ingredient. 2. The infection protective agent according to claim 1, wherein the infectious bacteria are Pseudomonas aeruginosa, Salmonella, Candida, and methicillin-resistant Staphylococcus.