KR100568962B1 - Therapeutic Agent for AIDS Which Comprises Orientia tsutsugamushi - Google Patents

Abstract

The present invention relates to acquired immunodeficiency syndrome (AIDS) immunotherapy and vaccine comprising an attenuated cell of Orientia tsutsugamushi as an active ingredient and a pharmaceutically acceptable carrier. According to the present invention, since it is possible to effectively suppress the infection of HIV, the causative agent of AIDS, by using the Orientia Tsutsugamu antigen, it will be widely used for the development of preventive vaccines as well as AIDS immunotherapy.

Orientia tsutsugamushi, AIDS Treatment and Prevention

Description

Therapeutic Agent for AIDS Which Comprises Orientia tsutsugamushi}

FIELD OF THE INVENTION The present invention relates to the use of Orientia Tsutsugashimi cells as acquired immunodeficiency immunotherapy and vaccine. More specifically, the present invention relates to acquired immunodeficiency immunotherapy and vaccine comprising an attenuated cell of Orientia Tsutsugamu as an active ingredient and a pharmaceutically acceptable carrier.

AIDS has emerged as a major threat to human health and social security, as evidenced by the UN report that 40 million people were infected with HIV by the end of 2001 and three million died of AIDS in 2001. In order to treat AIDS, a number of anti-HIV chemotherapeutic agents have been proposed and used for HIV, which is known as its etiology. It is classified as a "reverse transcription inhibitor" and a "pretease inhibitor" that blocks the process of release into the cell after replication. For example, reverse transcriptase inhibitors include zivudine (AZT), lamivudine (3T3), didanoic acid (ddi), and the like, and protease inhibitors include saquinavir, cranciban, and the like.

However, although the drawbacks of electrochemical synthesis agents cause many side effects in the treatment patients due to the toxicity of the drug itself, and drug resistance in long-term use, it has been pointed out. This situation is inevitably used. In order to overcome the problems of electrochemistry, vaccine research using the immune function of the human body has been actively conducted, and efforts have been made to advance the practical use of the vaccine at the national level, but there is no progress yet. to be.

Meanwhile, "AIDSVAX", which has recently been developed as a practically available HIV vaccine, is attracting attention, and is expected to be commercialized in earnest after 2004 when the clinical trial is ended, but the electric vaccine induces only one type of antibody formation. It is known that complete prevention of AIDS, which is expected to require a complex immune response, will be difficult. In addition, International Patent Publication No. WO 00/11035 discloses a technique for extracting an active ingredient from plasma obtained from patients with Orientia Tsutsugamu disease and using it as an AIDS therapeutic. Only a method of treating AIDS by injecting only has been disclosed, and no specific matters have been reported.

Therefore, there is a constant need to develop a method of suppressing HIV infection and treating electric infection by variously inducing the immune function of the human body.

Accordingly, the present inventors have tried to induce a variety of immune functions of the human body to suppress the infection of HIV, and to develop a method for treating the electrical infection, the antiviral immune substance inducing function of Orientia tsutsugamushi , the causative agent of Tsutsugamus disease. In view of the above, the attenuated cells of purified Orientia Tsutsugashimu were immunized to experimental animals to examine their inhibitory function against HIV. As a result, the attenuated cells confirmed that the attenuated cells effectively suppressed the infection of HIV against cells. The invention has been completed.

After all, the main object of the present invention is to provide an AIDS immunotherapeutic agent comprising attenuated cells of Orientia Tsutsugamu as an active ingredient.
Another object of the present invention is to provide an acquired immunodeficiency syndrome (AIDS) vaccine comprising attenuated cells of Orientia Tsutsugamu as an active ingredient.

The AIDS immunotherapeutic agent of the present invention is an attenuated cell of Orientia tsutsugamushi as an active ingredient, and includes a pharmaceutically acceptable carrier, wherein the attenuated cell of Orientia Tstsugamushi Can be obtained through culture and production in a known conventional manner. In addition, the AIDS vaccine of the present invention contains attenuated cells of Orientia tsutsugamushi as an active ingredient and includes a pharmaceutically acceptable carrier.

In addition, pharmaceutically acceptable carriers are not particularly limited, but include adjuvant (eg, aluminum hydroxide gel), binder (eg, polyvinylpyrrolidone, hydroxypropylcellulose), disintegrant (eg, carboxymethyl Cellulose calcium, sodium starch glycolate), diluents (e.g. corn starch, lactose, soybean oil, crystalline cellulose, mannitol), glidants (e.g. magnesium stearate, talc), sweeteners (e.g. white sugar, fructose, sorbitol, aspartame), Stabilizers (e.g. sodium carboxymethylcellulose, alpha or beta cyclodextrin, vitamin C, citric acid, lead), preservatives (e.g. methyl paraoxybenzoate, propyl paraoxybenzoate, sodium benzoate), fragrances (e.g. ethyl vanillin, after masking Leva, menthol flavono, herbal flavors) or mixtures thereof.

The treatment for acquired immunodeficiency syndrome of the present invention may be prepared in pharmaceutical preparations such as tablets, capsules, soft capsules, solutions, ointments or injections.

Hereinafter, the present invention will be described in detail.

The present inventors have been informed that a relatively small number of HIV exists in patients with both acquired immunodeficiency syndrome and Tsutsugamushi disease, and have conducted various studies on the association between HIV and Orientia Tsutsugamushi strain, the cause of Tsutsugamushi disease. As a result, it was confirmed that HIV infection by inoculation of the purified Orientia Tsutsugamu antigen can be suppressed.

First, the cells of Orientia Tsutsugamu (Giliam, Karp, Kato) are infected with L-929 cells in a conventional manner, followed by culturing the infected cells, and obtaining the cells at the time when the infection is maximized due to bacterial growth. Inactivated and purified by a known method to obtain the orientia Tsutsugamu attenuated cells. Subsequently, the attenuated cells obtained were administered to the experimental animals, and the HIV-sensitive cell line after mixing the OT immune plasma obtained from the blood of the experimental animals with HIV to determine whether an immune substance having HIV inhibitory ability was formed. HIV was inoculated to determine whether to suppress the proliferation of HIV, and compared with plasma obtained from blood of normal animals not vaccinated with the Orientia Tsutsugamu antigen. However, the animals inoculated with the glycocells were able to confirm the formation of immune substances that significantly inhibit HIV infection.

According to the present invention, the attenuated cells of Orientia Tsutsugamu form an immune substance that can inhibit the growth of HIV by 80% or more in experimental animals, and thus can be widely used in the development of immunotherapy and preventive vaccine against AIDS caused by HIV. There will be.
In fact, comparing the present invention with the above-described International Patent Publication No. WO 00/11035 using Orientia Tsutsugamushi, the biggest difference is that in WO 00/11035, one thing obtained from an animal immunized with Orientia Tsutsugamu Although a method of treating AIDS is provided by administering an immune component to a patient, in the present invention, AIDS is treated by directly administering an attenuated Orientia Tsutsugamus cell component to a patient, thereby stimulating a complex immune component to be produced in the patient's body. Provide a way to. The problem of WO 00/11035, which provides a conventional passive immunization for obtaining one type of antibody and administering the same, is described in the present invention by administering the attenuated Orientia Tsutsugamus cell, which is an antigenic component, of human body against HIV. Since the solution has been solved by active immunization which induces defense mechanism, it is obvious that the present invention is an advanced invention in terms of the technical spirit of the present invention as compared to WO 00/11035, and it is obvious that the present invention is clearly distinguished in terms of construction and effect. It is.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1: Oriental thiazol tsutsugamushi (O. tsutsugamushi) culture of the strain

Mouse fibroblast line L-929 (ATCC CCL-1) cell cells were used with Eagle's Minimun Essential Media (EMEM, Sigma Chem. Co., USA) containing 10% (v / v) fetal calf serum. Incubated at 5% (v / v) CO ₂ , 37 ° C. After incubation, the medium was removed, an O. tsutsugamushi ( OT) standard strain suspension was added, and the L-929 cells were infected by standing in a carbon dioxide incubator at 37 ° C for 90 minutes. After infection, medium was changed every 3 days with EMEM containing 0.4ug / ml of Daunomycin and 5% (v / v) fetal bovine serum, at 5% (v / v) CO ₂ , 34 ° C. Cells were incubated for 12 days and cells were collected.

Example 2 Obtaining OT Immune Plasma Against OT Antigen

Infected cells collected in Example 1 were suspended in TS buffer, sufficiently disrupted for 2 minutes using a grinder, and then centrifuged at 1,000 rpm for 5 minutes to obtain a supernatant. Percoll was added to the supernatant thus obtained to give a final concentration of 40% (v / v), and isothermal centrifugation was performed at 25,000 rpm for 60 minutes to obtain an OT band of the lower layer. Then, the obtained OT band was washed six times with PBS to obtain an OT antigen.

The obtained OT antigen was intramuscularly injected to experimental animals (Balb / c mouse) three times at five-day intervals in a conventional manner, and then collected one month after the final inoculation, and blood cells were collected from the blood collected by centrifugal sedimentation. Components were removed to obtain OT immune plasma.

Example 3 Measurement of Anti-HIV Effect of OT Immune Plasma

The OT immune plasma obtained in Example 2 was diluted 5-fold stepwise in RPMI cell culture medium (Sigam Chem. Co., USA) and diluted to 1/5, 1/25, 1/125 or 1/625. After dilutions were prepared, they were mixed with the medium containing HIV and left to stand at room temperature for 1 hour. Then, mixed with MT-2 cells (immune system T cells extracted from human blood, HIV sensitive cells) prepared in advance, electrostatically infected HIV was infected with MT-2 cells. Infected MT-2 cells were aliquoted into 24-well plates, incubated at 37 ° C. for 4 days, and then microscopically observed to count wells in which multinucleated giant cells (syncytia) formed by HIV were counted, By measuring, the degree of inhibition of multinuclear giant cell formation was determined (see Table 1). At this time, as a control, HIV treated with normal plasma obtained from normal experimental animals not inoculated with OT antigen was used.

Multinuclear Giant Cell Incidence (%) Dilution Ratio Control Experimental group 1/5 100 17 1/25 100 50 1/125 100 75 1/625 100 92

As shown in Table 1, it was found that the immune plasma obtained from the OT antigen of the present invention inhibits HIV infection by up to 83%. Since the OT antigen of the present invention acts as an effective HIV inhibitor, It was confirmed that it could be used for treatment.

Acute Toxicity Test

Non-rodent beagle dogs 6 to 7 weeks old were administered parenterally to the OT antigen of the present invention and examined for mortality within 24 hours, wherein females were 6 to 8 kg and males were 7 to 9 kg. 8 dogs each were used. As a result, it was confirmed that even when the OT antigen was administered up to 1 mg / kg corresponding to 1000 times the daily clinical dose of the expected person, no dead individuals occurred. Therefore, the OT antigen of the present invention is safe enough to observe acute toxicity even up to 1mg per kg, it can be seen that it can be safely administered in vivo.

Dosage

The dosage of OT immune plasma, which is an active ingredient in the pharmaceutical composition of the present invention, may be administered from 0.1 to 0.5 mg / kg body weight once or three times per day depending on the patient's age, sex, symptoms, administration method or prevention purpose. have.

As described and demonstrated in detail above, the present invention provides an acquired immunodeficiency syndrome (AIDS) immunotherapy and vaccine comprising an attenuated cell of Orientia tsutsugamushi as an active ingredient and a pharmaceutically acceptable carrier. do. According to the present invention, since the immune substance induced by the Orientia Tsutsugamu antigen can effectively suppress the infection of HIV, the causative agent of AIDS, the AIDS immunotherapy agent as well as the development of a prophylactic vaccine will be widely used.

Claims (3)

A therapeutic agent for acquired immunodeficiency syndrome (AIDS) comprising an attenuated cell of Orientia tsutsugamushi as an active ingredient and a pharmaceutically acceptable carrier. delete delete