JPH0454125A - Drug for treating and preventing disease infected with retrovirus - Google Patents

Abstract

PURPOSE:To obtain a new drug treating and preventing infectious diseases caused by retrovirus, especially AIDS resulting from human immune deficiency virus, comprising a sulfated polysaccharide derived from 1,3-beta-glucan (paramiron for short) obtained by culturing Euglena, prepared by sulfating paramiron, as an active ingredient. CONSTITUTION:A remedy and preventive for infectious diseases caused by retrovirus, especially AIDS resulting from human immune deficiency virus (HIV), comprising sulfated polysaccharide derived from 1,3-beta-glucan (paramiron), obtained by sulfating a polysaccharide, namely paramiron, prepared by culturing Euglena, as an active ingredient. Since paramiron used exists as waterinsoluble granules in the cells of Euglena, paramiron is readily separated and purified and yield of about 70% based on dry weight of cell can be attained under an optimum culture condition. Since the raw material is 1,3-beta-D-glucan, hardly decomposed with an enzyme in organisms and the effect can be maintained for a long period of time.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a drug for treating or preventing infections caused by retroviruses, particularly AIDS caused by human immunodeficiency virus (HIV). Regarding new drugs that are expected to treat and prevent cancer.

Acquired immunodeficiency syndrome (acquired immunodeficiency syndrome) is attracting attention as the most bizarre disease of this century.
icien-cy syndrome (AIDS) is a human immunodeficiency virus (HIV) that belongs to retroviruses.
) is a viral disease caused by. This disease is 19
In 1981, a weekly report from the US CDC reported that five homosexual men in Los Angeles developed carinii pneumonia (
Centers for disease control
ol:MMVR, 30250°1981). After that, this disease quickly spread all over the world, and according to the tally of the World Health Organization (WHO), as of August 12, 1987, the disease had already been detected in 122 countries, with a total of 60,000 patients. exceeds. In Japan, as of September 4, 1987, 50 patients were confirmed, of which 2
Eight people have already died. Despite the rapid spread of AIDS around the world, there are almost no established methods to prevent AIDS. Although much research is being carried out to develop vaccines for the prevention of H.
Type IV is at least HIV-1t-HIV-2
(7) The existence of both viruses and the fact that this virus mutates at a faster rate than the influenza virus make it difficult to put vaccine development to practical use. The world's leading AIDS scientists do not deny the possibility that new HiV types will be discovered in the future.

As a means of eliminating AIDS, it is considered extremely difficult to use vaccines, so anti-HIV
Treatment with drugs or preventive treatment is expected. AI
The reason behind the rapid increase in the number of DS patients is the rAIDS pre-existing condition.”
There are many people with HIV who have not developed symptoms (carriers). How to prevent these carriers from developing disease is an important issue in countermeasures against AIDS. Anti-HIV agents meet this requirement, and their development is strongly desired.

[Prior Art] Acidothymidine (AZT) is the only anti-HIV agent that has been put into practical use (Nature 326
゜430, 1987). It was synthesized as an anti-cancer drug and is an anti-HIV drug based on the inhibitory effect of HIV reverse transcriptase, but it is highly toxic to the blood-forming tissues of living organisms, causing anemia in many cases. I know. Many other substances are being studied as candidates for anti-HIV agents, but it cannot be said that an effective and safe anti-HIV agent has been developed yet.

Interferon is an example of a substance with anti-HIV effect found in a living body or a substance derived from a living body (Int, J
, Cancer 3B, 433.1986), but there are no clear reports regarding its effectiveness. Furthermore, recently, the anti-HIV effect of a compound containing a sulfate group bound to the internal tank (inositol) contained in the muscle was confirmed by an in vitro reaction;
Since a concentration of mg per ml is required, anti-H
It is thought that the IV activity is low.

Regarding sulfated sugars, for example, dextran sulfate is known to exhibit antiviral effects against certain viruses (Ann, N, Y.

Acad, Sci 13, 365, 1965) H
It has been reported that it also exhibits antiviral activity against IV (THELANCET, JUNE 13.13).
79.1987). However, there are concerns that polysaccharides with α-glucoside bonds, such as starch and dextran, are degraded by enzymes in the body, while the physiological activity of polysaccharides often depends on their molecular weight, so they cannot be used in vivo. has elements that do not necessarily produce the expected effects. Therefore, some of the inventors of the present invention
We believe that a sulfated polysaccharide with a main chain of 1,3-β-D-glucan, which is less susceptible to decomposition, is desirable as a means to solve this problem. 116499) was found to have anti-AIDS activity. In addition, the anti-AIDS effect of sulfated 1.3-β-D-glucan has been demonstrated by the laminarin (La) of kelp.
minarin) and shiitake mushroom lentinan
) etc. are known.

However, the content of 1,3-β-D-glucan contained in these is small, and the productivity is inferior to that derived from microorganisms. In addition, since most of the separation of the raw material 1,3-β-D-glucan relies on extraction methods using hot water or alkali, many impurities that are soluble in the extraction solvent are also extracted, which is used as a drug substance. requires a complicated process to remove these impurities from the target substance. The present inventors believe that the difficulty in obtaining anti-HIV pharmaceutical ingredients and the strong side effects of anti-HIV pharmaceutical ingredients will limit the increase in AIDS patients, in response to the needs of people infected with AIDS, which are expected to continue to increase in the future, and people who are trying to prevent infection. Considering this to be undesirable as a strategy, we have been searching for raw materials for sulfated polysaccharides that have anti-HIV effects that are relatively stable in living organisms, have excellent productivity, and are highly pure.

[Means for explaining the problem] Under the above-mentioned circumstances, the present inventors have conducted repeated research in search of new drugs that are effective for the treatment or preventive treatment of various diseases caused by retroviruses. As a result, we discovered that sulfated paramylon (sulfated paramylon), a polysaccharide (1,3-β-D-glucan) obtained by culturing Euglena, has the ability to suppress the proliferation of retroviruses. This led to the invention. Since paramylon used in the present invention exists as water-insoluble granules within Euglena cells, it is easy to separate and purify, and furthermore, under optimal culture conditions, its content can be as high as approximately 70% of the dry weight of the cells. can get. The efficient cultivation method of Euglena and the separation and purification method of paramylon have already been described by some of the present inventors (Japanese Patent Application Laid-open No. 37297/1983). Euglena mentioned here is a protozoan that belongs to the genus Euglena (genus Euglena) according to the zoological classification, and includes all species, varieties, and variants belonging to this. A typical example is Euglena.
Gracilis strain (Eu 1ena racillis)
, Euglena gracilis bacillis var. (Eu 1
ena racillis var, bacilla
ris), Euglena viridis (Eu 1ena
viridis), Astasia longa (Astasi
a Jon a) etc. Furthermore, Euglena naturally inhabits natural water systems such as ponds and swamps, and these can also be collected and used. Various mutant strains obtained by treating these with known methods such as ultraviolet ray treatment, heat treatment, antibiotic treatment, and chemical mutagen treatment can also be used. In addition, paramylon obtained from these Euglena cells may be degraded with acids or enzymes, secondary modified with functional groups other than sulfate groups, or Although it is possible to add a crosslinking treatment, a compound essentially made from paramylon and subjected to sulfation does not fall within the scope of a drug as defined in the present invention.

As a method for sulfating paramylon, any known method for sulfating polysaccharides may be used; for example, a method using sulfuric acid, a method using chlorosulfonic acid, a method using sulfur trioxide, etc. can be used. . Among these methods, the chlorosulfonic acid method and the method using sulfur trioxide, which are used in combination with a Lewis base, cause less decomposition of polysaccharides than the method using sulfuric acid, so they are less stable as pharmaceutical materials. It is an excellent manufacturing method in terms of ensuring Lewis bases include pyridine, triethylamine, dioxane and bis(2-
(tetraloethyl) ether, etc. are used. Furthermore, a method in which a pyridine sulfuric anhydride complex is reacted with paramylon in pyridine or dimethyl sulfoxide to sulfate it is also used as a simple method. The prepared sulfated paramylon is preferably in the form of an appropriate salt such as an alkali metal salt or an ammonium salt from the viewpoint of storage stability.

As will be clear from the later examples, the sulfated paramylon used in the present invention is a human T cell-derived cell line (Molt-4
In vitro anti-H using C1one N118 cells)
In the IV activity test, the minimum effective concentration to completely suppress the proliferation of HIV is approximately 10 gg/ml, and the minimum effective concentration of dextran sulfate, which is said to be the most effective conventionally, is several μs/ml to 10 μg/ml. It seems that they are almost the same compared to each other.

The drug of the present invention is advantageous in that its raw material, paramylon, is 1,3-β-D-glucan, in addition to its excellent medicinal effect of suppressing virus proliferation. In other words, since sulfated paramylon is a sulfated product of 1,3-β-D-glucan, it is less likely to be degraded by enzymes in the body and maintains its effective concentration for a relatively long time even when administered to patients. It is expected that it will be possible.

Hereinafter, in order to deepen the understanding of the present invention, the present invention will be explained along with Examples showing the anti-HIV activity of the drug of the present invention, but the present invention is not limited to these Examples. For example, in the following examples, HIV (human immunodeficiency virus) is HIV-1 (LAB or HTLV-1).
II [also called 8], the drug of the present invention has similar effects against HIV-2 and other viruses.

[Production Example 1] Preparation of Paramylon Paramylon in the present invention was prepared, for example, by the method described below. Euglena gracilis (Eug-1ena)
gracilis) strain was inoculated into a test tube containing 5 ml of the preculture medium (Table 1) and cultured with shaking at 28° C. for 3 days. Next, the culture solution was inoculated into a Sakaro flask containing 100 ml of preculture medium, and cultured with shaking at 28°C for 60 hours.

The obtained culture solution was adjusted to a glucose concentration of 2% in a 2L jar fermenter containing main culture medium (Table 2) 1.21.
Culture was carried out at 28° C. for 60 hours while controlling the temperature within 30 minutes. Collect the cells from the culture solution obtained here using a centrifuge, and add 2L of water.
The cells were resuspended in a high-pressure emulsifier at a pressure of 350 kg/am'' to disrupt the cells.Then, the cells were centrifuged to recover the lower white glucan layer.Furthermore, they were suspended in 2 L of a 1% sodium lauryl sulfate aqueous solution and incubated at 100°C. The mixture was stirred for 60 minutes to remove lipids and proteins.

Next, this was repeatedly centrifuged and washed with water, dispersed and dehydrated in acetone, and then dried to obtain 30 g of paramylon white powder with a purity of 9968%. The number average molecular weight of paramylon obtained here was determined by the Manners method (Mannerset aL,
It was measured to be approximately 120,000 using a modified method of (1971).

Table 1 [Production Example 2] Preparation of sulfated paramylon The paramylon obtained by the method of Production Example 1 was prepared into a sulfated product, for example, as follows.

30 ml of anhydrous pyridine was placed in a 100 ml Erlenmeyer flask with a stopper, and under water cooling, a mixed solution of 2.0 ml of chlorsulfuric acid and 1.0 g of paramylon in 30 ml of dimethyl sulfoxide was gradually added. The reaction was stirred at 85° C. for 3 hours, and after cooling to room temperature, the supernatant pyridine was removed by decanting. Add 10ml of distilled water to the lower layer, then add 200ml of methanol
Added l. The resulting precipitate was filtered and added with 5% sodium chloride 1
Add 50ml, then add 6N sodium hydroxide and p
It was set as H9. Three times the volume of acetone was added to this solution, the resulting precipitate was dried, and 0.7 g of sulfated paramylon with a substitution rate of 53% (ratio of sulfated hydroxyl groups to all hydroxyl groups not involved in bonds constituting glucan) was added. Obtained.

In order to evaluate the antiretroviral activity of sulfated paramylon prepared by the method described above, tests described in the following examples were conducted.

[Example 1] Measurement of anti-HIV activity (test of suppressive activity of cytopathic effect by virus) Molt-4 clone No. 8 strains of cultured cells were infected with HIV virus at concentrations of O, Olm, o, and i. Thereafter, the cells were washed with a medium to remove unadsorbed viruses, and the cells were used for testing. In addition, virus-uninfected cells were also used as a control.

Add 1 ml of a cell suspension of 3.5 x 10' cells/ml to each well of a 24-well microplate, and add the sample, ie, sulfated paramylon prepared in Production Example 2, to a final concentration of 10 μs/ml.
1 ml of the medium was added and cultured for 6 days. In addition, as a control, 1 ml of medium was added with dextran sulfate at a final concentration of 10 μg/ml.
, and 1 ml of medium without additives were cultured in the same manner.

As a result of observing the cells after culturing and confirming the presence or absence of the cytopathic effect caused by the virus, it was found that the cytopathic effect caused by the virus was observed in the medium without additives, but the cytopathic effect caused by the virus was observed in the sulfated paramylon and dextran sulfate. No, it was confirmed that these samples had an activity of suppressing the cytopathic effect caused by the virus, that is, an anti-HIV activity.

[Example 2] Measurement of anti-HIV activity (measurement of viral reverse transcription oxygen activity) The viral reverse transcriptase activity of the culture supernatant of Example 1 was measured at 5 ci
ence 228 (1983) p. 336. That is, 1 ml of culture supernatant was added to 12. 9 in OOOXg
The precipitate obtained by centrifugation for 0 minutes was mixed with 10mM Tri at pH 7 and 8 containing 1% NP-40 and 1mM EDTA.
Add 10J of s-HCI buffer, and add 0.0J of s-HCI buffer.
1M MgCl2゜0.045M KCI, 4mM
DTT, 100 μg/ml Po1y(rA)
:oligo (dT) 12-18 (rAdT)
90 μl of 10 mM Tris-HCI buffer containing pH 7 and 8 was added. To this, 2.5 μm (meth
yl-3H) thymidine 5°-trjph
o-phate (30 Ci/mmol) was added and reacted at 37°C for 60 minutes. After the reaction, add 100 μl to 2.
Spotted on 4 cm filter paper (Watmann), 5%
After washing with NaaHPO4 solution and immersing in 99% ethanol, radioactivity was measured using a liquid scintillation counter. As a result, as shown in Table 3, viral reverse transcriptase activity was observed in the medium without any additives, but no viral reverse transcriptase activity was observed in sulfated paramylon and sulfated dextran, indicating that anti-HIV It was confirmed that it is active.

Table 3 Measurement results of viral reverse transcriptase activity Patent applicant Harima Kasei Co., Ltd. Meiji Seika Co., Ltd.

Claims (2)

[Claims] (1) 1,3-β-D derived from Euglena as an active ingredient
- A drug for the treatment and prevention of retrovirus infections, which contains as an active ingredient a sulfated polysaccharide derived from paramylon obtained by sulfating glucan (hereinafter referred to as paramylon). (2) The retrovirus is human immunodeficiency virus (HIV).
) The drug according to claim (1).