JPH0892085A - Antiretroviral agent - Google Patents

Abstract

PURPOSE: To obtain an antiretroviral agent capable of effectively inhibiting HIV infection and the spread thereof owing to its excellent cell membrane fusion blocking action, containing, as active ingredient, bryostatin 10 derived from Terebellidae. CONSTITUTION: This antiretroviral agent contains, as active ingredient, a compound of the formula. This compound is obtained, for example, by the following process: an extract obtained by subjecting Terebellidae to extraction with dichloromethane is fractionated with a Sephadex LH-20 column to obtain a fraction and residues; these are screened with sea urchin fertilized egg cleavage inhibitory action as indicator to obtain an active fraction, which is then further fractionated with flush chromatography to obtain a fraction, which is then screened again with sea urchin fertilized egg cleavage inhibitory action as indicator to obtain an active fraction, which is, in turn, fractionated with HPLC. The dose of this agent (in terms of the active ingredient) is pref. 0.01-100mg/kg b.w. a day per adult.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antiretroviral agent containing a compound having an antiretroviral activity as an active ingredient.

[0002]

Various attempts have been made to develop a therapeutic agent for viral diseases, but no useful one has been found so far, and prevention of infection by a vaccine is the only effective response to viral diseases. It is a means. Vaccine development research is also being carried out against the human immunodeficiency virus (HIV), but there are many difficulties that cannot be overcome only by following the conventional methods of virus vaccine development. That is, HIV is a major antigen, the envelope glycoprotein gp120.
It is not easy to develop a vaccine that is effective against many HIV mutant strains, because the gene that defines the structure of E.
Furthermore, infection is often established not only by extracellular HIV particles but also by direct fusion of HIV-infected cells with non-infected cells, and it is not possible to prevent infection with only neutralizing antibodies. Making it difficult. Under these circumstances, since HIV is a retrovirus, drugs that inhibit the activity of retrovirus-specific reverse transcriptase have attracted attention, and azidothymidine [Medical History: Vol. 142, No. 9, 619-622 (1987)] and the like are actually used clinically.
However, such drugs have a problem that they cannot be administered for a long period of time, because they have side effects such as bone marrow suppression, white blood cell decrease, and anemia. Currently, research is being conducted to reduce these side effects, but the current situation is that the problems have not been completely resolved.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have found that intercellular infection from infected cells to non-infected cells plays a major role in the spread of infection, and these intercellular infections are mediated by cell membrane fusion. Therefore, it is intended to provide a clinically applicable drug having a cell membrane fusion inhibitory action. Sulfated polysaccharides (Japanese Patent Laid-Open Nos. 63-45223 and 64-25724) and the like are known as drugs of this type, but their clinical effects are weak and they have not been established as therapeutic drugs. .

[0004]

Means for Solving the Problems In order to obtain a clinically applicable inhibitor, the present inventors mediated cell fusion when HIV-infected lymphocyte cells and non-infected same cells were mixed and cultured. Screening was carried out using the resulting inhibitory effect on the formation of multinucleated giant cells as an index. As a result, bryostatin 10 (bryostatin1) derived from the leaf beetle represented by the chemical formula (1)
The activity was found in 0) and the present invention was completed. That is,
The present invention provides an antiretroviral agent containing a compound represented by the chemical formula (1) as an active ingredient and optionally a pharmaceutically acceptable carrier and the like.

[0005]

[Chemical 2]

The antiretroviral agent of the present invention contains bryostatin 10 as an active ingredient. Bryostatin 10 is a kind of macrolide, and is known to exist in the hemlock bug. The present inventors used bryostatin 10 isolated from Fusamushi worms collected in Asamushi, Aomori Prefecture (Aomori Bay).

The antiretroviral agent of the present invention only needs to contain bryostatin 10 as an active ingredient, and the bryostatin 10 used is not limited to the above-mentioned extract from Physcomitrella patens. Further, the antiretroviral agent of the present invention may be an oral or parenteral preparation. For parenteral preparations, veins, arteries, skin, subcutaneous, muscle, digestive tract (stomach,
It is administered via the intestine). The dose varies depending on the dosage form, dosage form, age of the patient, and condition, and is not uniform, but for adults, 10 μg / kg (body weight) to 100 mg / kg per day
(Body weight) is suitable.

When the antiretroviral agent of the present invention is to be prepared as a parenteral preparation, it may be considered to be a sterile water-soluble solution, a non-water-soluble solution or an emulsion. As a base for making a non-water-soluble liquid or an emulsion, propylene glycol, polyethylene glycol, glycerin, ethyl oleate and the like can be considered. As the oral preparation, capsules, tablets, granules, fine granules, powders, syrups and the like can be considered. Various preparations of the antiretroviral agent of the present invention can be carried out by a conventional method in the technical field of pharmaceutical preparation according to a conventional method.

Next, the antiretroviral agent of the present invention will be specifically described.

[Isolation of bryostatin 10] 1.5 kg of the hemlock bug was extracted with CH ₂ Cl ₂ to obtain 36.6 g of an extract. This was loaded on a Sephadex LH-20 column to n-hexane: CH ₂ Cl ₂ : C.
Fractionation was carried out with H ₃ OH = 4: 5: 1 to obtain fractions 1 to 7 and a residue. When these were screened using the fertilized egg cleavage inhibition effect as an index, activity was observed in fractions 2 and 3. These are further purified by flash chromatography _{(ODS; CH 3 CN:.} H 2 O = 50: fractionated in 50 min to obtain a fraction 8 fractions 11 to them to index the sea urchin embryo cleavage inhibitory activity was further screened, the fraction 10 showed activity in fractions _{10 HPLC (ODS; CH 3 OH} :. H 2 O = 8
Fractionation was performed at 5:15) to obtain 15 mg of bryostatin.

[Inhibition of multinucleated giant cell formation] Molt-4 / HIV, which is a lymphocyte in which HIV isolated is persistently infected
When cells and non-infected Molt-4 cells are mixed and cultured, Molt-4
/ HIV envelope glycoproteins gp120 and M expressed on the surface of HIV / HIV cell membranes
By binding to the CD4 receptor on the olt-4 cell membrane surface, both cell membranes fuse with each other to form multinucleated giant cells. By looking at the inhibition of the formation of multinucleated giant cells by the addition of a drug, it is possible to examine the effect on the stage of HIV invasion and invasion and intercellular infection.

1. Method Flat-bottom 96-well microplate (Falcon, Part No. 3072)
100 μl / well of a sample solution diluted to a predetermined concentration with a medium (RPMI1640 [containing 10% Fetal calf serum]) was added thereto.
Next, persistently infected Molt-4 / HIV cell suspension (1
× 10 ⁶ cells / ml) was added at 50 μl / well, and non-infected Molt-
50 μl / well of 4 cell suspension (1 × 10 ⁶ cells / ml) was added and stirred, and mixed culture was carried out at 37 ° C. in the presence of 5% CO ₂ to obtain a test section. For control, the persistent infection Molt-4 / HIV cell suspension was changed to 50 μl / well, and non-infected Molt-4 cell suspension (1 x
10 ⁶ cells / ml) 50 μl / well was added. The formation of multinucleated giant cells was observed within a few hours after the mixed culture, and became remarkable after 20 hours. After 24 hours of culture, the presence or absence of multinucleated giant cells was examined by observation with a microscope, and the number of viable cells was measured by the trypan blue staining method to obtain the Fusion Index (Virology 164,542-54).
6,1988). The method of calculating the Fusion Index is as follows, and the value is 0 if there is a suppressing effect.

Fusion Index = (number of control cells per well / number of test cells per well)-
1 2. Results The results are shown in Figure 1. The dextran sulfate as a control drug has a Fusion Index of 0 in the dose range of 10 to 300 μg / ml.
Bryostatin 10 showed a value around 0 in the dose range of 0.01 to 3 μg / ml, which is lower than this value. Microscopic observations were consistent with this result, with dextran sulfate blocking the formation of multinucleated giant cells at doses of 10-100 μg / ml, whereas bryostatin 10 was 0.01-3.
The dose of μg / ml prevented the formation of multinucleated giant cells.

[0014]

The antiretroviral agent of the present invention contains bryostatin 10 as an active ingredient. Due to the excellent cell membrane fusion inhibitory effect of bryostatin 10, HI
Effectively suppresses V infection and spread.

[Brief description of drawings]

FIG. 1 is a graph showing the inhibitory effect of bryostatin 10 on cell membrane fusion.

Claims (1)

[Claims] 1. An antiretroviral agent comprising a compound represented by the chemical formula (1) as an active ingredient. [Chemical 1]