JP2811503B2 - Anti-HIV agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to, for example, Lactobacillus casei.
The present invention relates to an anti-HIV agent having a human immunodeficiency virus (HIV) growth inhibitory activity containing lactobacillus cells known as illus casei) as an active ingredient.

[Background Art] Acquired Immunological Def
HIV (Human Immunodeficiency Virus), which is the causative virus of iciency syndrome (so-called AIDS), has been found to be HTLV-III (Human T-lymphotrop) due to reasons such as the discoverers.
ic virus type III) and LAV (lymphadenopthy-associa)
ted virus) (hereinafter referred to as HIV).

HIV is one of the retroviruses that are RNA-type viruses, and has a unique life cycle that converts gene RNA into DNA in host cells.

Cells serving as HIV host cells are T4 lymphocyte cells that function as inducers and helper cells in the human immune system, and target the CD4 receptor molecule on the surface of the T4 lymphocyte cells to selectively T4 lymphocytes. (Step 1).

The attached HIV is threshed (step 2) to allow its own gene RNA to enter host cells. Invaded gene R
The NA is similarly transcribed into complementary DNA using reverse transcriptase (RT), which was also brought into the host cell (step 3). Transcribed DNA
Is further formed into double-stranded viral DNA by the same enzyme (step 4). The double-stranded viral DNA is integrated into the cell DNA chain in the nucleus using an endonuclease encoded by its own pol gene (step 6).

Next, the viral DNA is transcribed into viral mRNA by RNA polymerase from the host cell and further translated into viral proteins (step 6). The translated viral protein is cleaved by the protease encoded by its own pol gene, undergoes various modifications by host cell-derived enzymes, and is assembled and packaged together with a newly formed new viral RNA genome. (Step 7), budding from the cell membrane (Step 8), and extracellularly migrate to become a new mature virus.

At present, various effective drugs are being developed based on the above-mentioned unique life cycle of HIV. That is, by inhibiting each of the steps 1 to 8 shown in the life cycle, an effective anti-
HIV drugs are being developed.

At present, azidothymidine (3'-azido-2 ', 3'-dideoxythymidine:
AZT) has been approved only, but it has been confirmed that it has an effect of relatively specifically inhibiting the synthesis of viral DNA by RT. However, AZT has the following side effects:
It has a myelosuppressive effect and is considered for co-administration with other anti-HIV agents or immunopotentiators.

Now, acquired immunodeficiency syndrome due to HIV infection (so-called A
The development of IDS) is caused by the lack of sufficient immune effects due to the selective depletion of T4 lymphocytes.

At present, it is natural to develop anti-HIV agents that have a new HIV infection-inhibiting effect without the side effects such as AZT or the effects that impair the human immune system. Anti-HIV agent with inhibitory action, H
There is a demand for the development of anti-HIV agents that also have the function of actively rebuilding the immune system (T4 lymphocytes and the like) lost by IV infection.

That is, the mechanism of T4 lymphocyte destruction due to HIV infection is still largely unknown, but the mechanism of T4 lymphocyte destruction currently considered is as follows.

(1) There is an unidentified T cell destruction factor in infected individuals and serum.

(2) The infected cells are destroyed by the immune mechanism of the infected individual.

(3) Single cell death of infected cells
deth; a phenomenon in which infected cells are killed only by viral infection in the absence of other factors).

(4) Cell death due to syncytium formation (giant cell formation) between infected cells and specific infected cells.

Various anti-HIV agents that inhibit the T4 lymphocyte destruction mechanism as described above or have an immunopotentiating effect to compensate for the T4 lymphocyte reduced by the destruction mechanism, and also have an anti-HIV effect have been variously searched. I have.

In the search for the above-mentioned new anti-HIV agent, various search methods have been used, but as described above, HIV has a specific target, so there is no model animal. At present, clinical trials on the human body are extremely difficult, for example, there are various problems, and it takes 3 to 10 years for the onset of the disease.

Therefore, a cloned cell line expressing the above CD4 on the cell surface has been used for the search.

For example, the MT-4 cell culture system is a HTLV-I (ATL) antigen-positive (endogenous) cell which is a retrovirus similar to HIV, and has been shown to be highly susceptible to HIV infection (Harada, S. ., Koyanagi, Y.and Yamamoto, N.: Science 22
9,563-566 (1985)), it is known that most of them die when infected with HIV in 5 to 10 days.

In addition, even if infected with HIV, Mol
There is a t-4 cell culture system. This Molt-4 cell culture system comprises
It is a lymphoblastoid cell derived from LL (acute promyelocytic leukemia) and is highly sensitive to HIV, among which clone No. 8
Is known to be a strain that forms giant cells upon infection (Harada, S. et al .: Virology 146, 272-281 (198
Five)).

One of the immunological differences between the two cloned cell culture systems is that the Molt-4 cell culture is derived from immature T cells compared to the MT-4 cell culture. (The HLA-DR antigen, a marker of activated T cells, is present on the MT-4 cell surface).

On the other hand, it has recently been revealed that a polysaccharide-peptidoglycan complex derived from the cell wall derived from Lactobacillus has an immunopotentiating effect, and clinical applications have been attempted (Japanese Patent Application Laid-Open Nos. 63-1226827 and 63-26827). No. 196521).

[Problems to be Solved by the Invention] However, some immunopotentiators are in vitro.
In some cases, when an immunopotentiator is administered to an infected person in combination with an anti-HIV agent, the administered immunopotentiator is administered in vitro. It was necessary to determine what effect it had on growth.

Therefore, in vit of Lactobacillus cells having an immunopotentiating effect
The inventors of the present invention have attempted to determine what effect it has on HIV proliferation in ro and confirmed that it has a good anti-HIV effect, leading to the present invention.

[Means for Solving the Problems] The anti-HIV agent according to the present invention contains lactobacillus cells as an active ingredient.

[Action] The anti-HIV agent of the present invention contains lactobacillus cells as an active ingredient, but its action mechanism has not yet been sufficiently analyzed. However, without losing guesswork,
The lactobacillus cells of the present invention act on cells by (1) suppressing the binding between cells and HIV or (2) acting on cells due to high mitogen activity for stimulating lymphocytes, and in some way increasing the proliferation of HIV. It is thought that it is related to suppression.

By any of these action mechanisms or multiple actions,
It is presumed to have anti-HIV activity.

In the method of purifying lactobacilli according to the present invention, the lactobacilli as a raw material may be those cultured by a conventional method using an appropriate medium that can be used for culturing lactobacilli,
It does not need to be by a special culture method. After the culture, the cells are washed by a conventional method, and desirably sterilized by heating to obtain the lactobacillus cells (WC) of the present invention.

On the other hand, the fraction used as a comparison is obtained by dissolving and fractionating the lactobacillus cells, specifically, suspended in water in the WC and treated with N-acetylmuramidase, When the cells are treated at a pH of about 5 to 6 and a temperature of about 37 to 50 ° C. for about 12 to 24 hours, the cell wall is first dissolved. When the reaction mixture is centrifuged, CW (Call Wall) as a residue is obtained. After the supernatant of the centrifugation is again treated with an enzyme, the diffusion is decomposed with a diffusion-decomposing enzyme (DNase, RNase), and trypsin is further added to decompose the protein. Next, NA as a nucleic acid component is obtained from the inner solution dialyzed with distilled water. Furthermore, the dialysate is fractionated and purified by gel filtration to obtain c-GP and p-GP. Each fraction was obtained as described above, and
Comparative study of HIV drugs was conducted.

[Examples] Example 1 (In vitro HI by Molt-4 clone No. 8)
Effect on V Proliferation) Test cells used were Molt-4 clone No. 8, and HIV was HTLV / B. The culture solution used was RPMI1640 medium supplemented with kanamycin and 10% fetal calf serum (FCS) as antibiotics. Culture conditions were 37 ° C. and 5% CO ₂ .

The test cells were infected with HIV at a MOI (multiplicity of infection: ratio of virus to host cells) of 0.002, and then the lactobacillus cells (WC) and four of the lactobacillus fractions (c) -GP, p-GP, CW, NA, WC), and cultured for 3 days. The culture solution and the polysaccharide-peptidoglycan complex were exchanged, and the cells were cultured for another 2 days.
It was measured by the blue exclusion method. Uninfected cells were used as a control as a control.

As shown in FIG. 1, the results were as follows: c-GP, p-GP, NA
No growth inhibitory effect was observed in the range of 15.6-62.5 μg / ml.

In addition, the effect of inhibiting the growth of infected and non-infected cells according to the concentration was demonstrated. At concentrations higher than 250 μg / ml, these three substances suppressed cell growth of Molt-4 clone No. 8.

On the other hand, a cell growth inhibitory effect was observed at 15.6 μg / ml or more for CW and WC.

From the above results, in the case of immature lymphocytes, Molt-4 clone No. 8, Lactobacillus cells (W
C) and four fractions (c-GP, p-GP, CW, NA, WC), c
-GP, p-GP, and NA did not show the effect of suppressing HIV proliferation.
On the other hand, in CW and WC, the cell growth inhibitory effect was observed, particularly in the case of the lactobacillus cells or polysaccharide- according to the present invention.
It is considered that the cytotoxic substance (lymphotoxin-like substance) was secreted or internalized by the peptidoglycan complex.

However, some immunopotentiators induce HIV enhancement even in vitro, so that Lactobacillus cells and polysaccharide-
The absence of an HIV growth-enhancing effect in the active ingredient of the peptidoglycan complex is expected to have good clinical results.

Experimental Example 2 (Influence of MT-4 on HIV proliferation in vitro (WC)) MT-4 was used as a cell to be treated, and the other conditions were the same as those in Experimental Example 1.

After infecting the test cells with HIV so that the MOI becomes 0.002, WC is added and cultured for 3 days, the culture medium and the drug are exchanged, and the cells are further cultured for 2 days. Was measured. Uninfected cells were used as a control as a control.

As shown in FIG. 2, the results were obtained at 100 μg / ml or less.
No effect on cell proliferation was observed in infected and uninfected cells, and favorable HIV growth was inhibited in infected cells in a concentration-dependent manner.

The lactic acid bacterium WC according to the present invention may stimulate MT-4 cells to secrete lymphokines.

That is, the anti-HIV effect was not confirmed in the immature lymphocyte Molt-4 cell culture system of Experimental Example 1, and the anti-HIV effect was confirmed in the mature lymphocyte MT-4 cell culture system. The anti-HIV agent according to the present invention acts as a mitogen and has a high possibility of secreting or internalizing INF-γ produced by immunostimulating mature T cells.

Experimental Example 3 (Effect of MT-4 on HIV proliferation in vitro (c-GP, p-GP, CW, NA)) Using c-GP, p-GP, CW, NA as a test substance, The conditions were the same as in Experimental Example 2.

After infecting the test cells with HIV at a MOI of 0.002, each of c-GP, p-GP, CW, and NA was added and cultured for 3 days.
The culture medium and the drug were exchanged, and the cells were cultured for another 2 days, and the number of viable cells was measured by the trypan blue exclusion method. Uninfected cells were used as a control as a control.

As a result, each drug was up to a concentration of 500 μg / ml,
No significant HIV inhibitory effect was observed.

From the above, to stimulate MT-4 cells to secrete or endogenous lymphokines having an anti-HIV effect,
It was confirmed that the fraction was not induced.

Although there is still no accumulation of data up to a sufficient explanation, it does not escape the guesswork, but as mentioned above, lactobacilli have no pathogenicity and are believed to play some role in host defense. By increasing the antibody production of Peyer's patch cells, Lactobacillus cells themselves act as mitogens, and their fractions are poor in their action. Experimental example 2 has some effect on lymphocyte activation (learning)
In view of the fact that the Lactobacillus cells showed significant anti-HIV activity and that the fractions did not induce the anti-HIV effect, a site that stimulates lymphocyte cells on the surface of the Lactobacillus cell wall was considered. Maybe it exists.

The secreted or endogenous lymphokines do not lose their meaning, but are not limited to one.
As an effective factor of the V action, interferon or an interferon-like substance having an antiviral action is expected.

Experimental Example 4 - Toxicity of Lactobacillus bacteria (polysaccharide acute toxicity test of the peptidoglycan complex) (WC) is clear from the LD ₅₀ value of the following (7 weeks old, the value of the BALA / c male mice weighing about 25 g) In any case, it is not recognized at all.

Oral administration 2000 mg / kg or more Intravenous administration 300 mg / kg or more Intraperitoneal administration 300 mg / kg or more [Effect of the Invention] As described above, the present invention has a good anti-HIV activity against Lactobacillus cells. Is an effective drug to prevent AIDS caused by HIV infection and an antidote that has the potential to be an effective drug to reduce the incidence of ARC and AC patients
It has the effect of obtaining HIV drugs.

[Brief description of the drawings]

FIG. 1 shows HIV in vitro by Molt-4 clone No. 8
FIG. 2 is a diagram showing the results of the effect on the proliferation of HIV in vitro by MT-4 cells.

Claims (1)

(57) [Claims] [1] An anti-HIV agent comprising a lactobacillus cell as an active ingredient.