JPS62178516A - Remedy for retrovirus infectious disease - Google Patents

Abstract

PURPOSE:The titled remedy comprising sakyomicin A as an active ingredient. CONSTITUTION:A retrovirus infections disease containing sakyomicin A shown by the formula s an active ingredient. Retrovirus infectious diseases include acquired immune deficiency syndrome, adult T-cell leukemia, etc. The compound shown by the formula is readily obtained by cultivating a mold capable of producing sakyomicin A and purifying. Administration is orally or parenterally carried out and powder, granule, injection liquid for internal use, tablet, suppository, ointment, cream, drop, etc., may be cited as a dosage form. 0.01-10mg/kg, preferably 0.1-5mg/kg is orally or parenterally administered. The compound shown by formula inhibits reverse transcriptase of retrovirus, shown inhibitory actions on growth of HTLV-III, a cause virus of AIDS and is effective against retrovirus infectious diseases such as AIDS, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to the field of medicine. More specifically, the present invention relates to a therapeutic agent for retrovirus infections.

Conventional technology Retrovirus is a type of RNA tumor virus.
Among RNA viruses, it is a virus that has a life cycle that converts genetic RNA into DNA, and has reverse transcriptase in each particle, and in addition, the gene for this enzyme is present on the viral genome. be. It is divided into types A, B, C, and D according to its morphology by electron microscopy; mouse mammary tumor virus belongs to type B, Mason-Pfizer virus belongs to type D, and all other sarcoma and leukemia viruses belong to type C. This type C retrovirus has
Acquired Immune Deficiency Syndrome (AIDS)
Immune Deficiency Syndrome
Lymph node enlargement-associated virus (e), which is the causative virus of
V to L: Lylllphadenopatlly
As5ociated Virus), [5cien
ce, vol. 220.

See pages 868-871 (1983), human
T-lymphocyte-tropic retrovirus type 3 (IHLV-1
:11uman T-Cell Lymphotrop
ic Virus tyl) eI[[) [5cien
ce, Vol. 224, pp. 497-500 (1984
) Reference 1 or AIDS-Related (8 RV: AIDS-Re
rated Virus) [5cie11ce, 22nd
5, p. 840 (1984)] and adult hypocellular leukemia (ATL: 8duHT-C).
It also includes adult hypocellular leukemia virus (HTLV-I and ■), which is the causative virus of ELL Leukemia. [See Cell Engineering, Vol. 3, pp. 1053-1063 (1984)].

In recent years, retrovirus infections have become a social problem.

For example, acquired immunodeficiency syndrome (hereinafter referred to as Acquired Immune Deficiency Syndrome).

It is abbreviated as AIDS. ) was named in the United States for cases of cell-mediated immunodeficiency caused by helper T cell dysfunction, which was accompanied by opportunistic infections caused by various microorganisms and Kaposi's sarcoma. In the United States, where the first cases were discovered, by the end of April 1985, there had been 10,000 AIDS patients since 1979, and 49% of them had already died, but the number of cases is still increasing. . Canada again.

AIDS patients are also occurring in European countries, Latin American countries, and Africa [Protein Nucleic Acid Enzymes, Vol. 30.

Pages 1394 to 1407 (1985); Protein nucleic acid enzymes.

Volume 30, pages 1285-1289 (1985)
Reference.

Furthermore, adult hypocellular leukemia is a leukemia that occurs more frequently in southwestern Japan, black people on the Caribbean coast, and Africa, and is an acute leukemia-lymphoma with abnormalities in helper type T cells that develops in adults. This leukemia is extremely malignant and has a high mortality rate, with a 50% survival rate of only 4 months [Pr.
oceedings of National Aca
demy 5science (U, S, 8,) 18th
Volume, page 6476.

(1981); Bfood, Vol. 50, No. 481
(1977)].

As a treatment for these retrovirus infections.

There are methods that are specific to retroviruses, ie, methods that suppress the activity of reverse transcriptase, and methods that are non-specific. As an example of the former.

Suramin hexasodium salt, IIPA23 (tun
gusto-antimoniate), Bossphone 1
These include the 1M trisodium salt and the nucleoside analog ribavirin. Ribavirin inhibits hemoglobin synthesis and T
It is toxic to cells, and other substances are known to temporarily suppress the activity of reverse transcriptase in the blood, so a complete cure cannot be expected. Interferon and famycin derivatives have also been experimentally tried. It is known that interferon suppresses the maturation and release of viruses, but there are also reports that high levels of interferon are already present in the blood of AIDS patients, and there are many problems with interferon. In addition, AI
Since the virus that causes DS (hereinafter abbreviated as HTLV-III) destroys helper T cells, interleukin-2 released by helper T cells is also administered. [Protein Nucleic Acid Enzymes, Volume 30, No. 1
See pages 285 to 1289 (1985). In addition, isoprinosine, azidothymidine, (A7T)
, 5-(2-chloroethyl)-2-deoxyuridine and the like are also being investigated.

The problem that the invention aims to solve is to find a substance that is excellent as a therapeutic agent for retrovirus infections.

Means for Solving the Problems The present inventors conducted intensive research in order to find a therapeutic agent for retrovirus infections.
The present invention was completed by discovering that yomicin A) suppresses the growth of retroviruses. That is, the present invention relates to a therapeutic agent for retrovirus infections containing sakyomycin A as an active ingredient.

Function Sakyomycin A, which is the active ingredient of the therapeutic agent of the present invention, is a known substance represented by the following structural formula. [Jour
nal of Antibiotics, Vol. 37, pp. 693-699 (1984)].

Sakyomycin A Sakyomycin A is Journal of Anti
biotics.

It can be easily obtained by culturing and purifying sakyomycin A-producing bacteria as described in Volume 37, pp. 693-699 (1984), but it can also be obtained as shown in the Examples below. It is.

The drug according to the present invention is prepared as a pharmaceutical composition from sakiyomycin A as an active ingredient and a pharmaceutically acceptable carrier selected according to the method and form of administration of the drug. In other words, the drug of the present invention can be administered orally or parenterally depending on the target for treatment of internal virus diseases or external tissue virus diseases, and can be administered as powders, granules, injections or liquids for internal use9 tablets, suppositories, and ointments. The drug can be prepared as a preparation according to the method of administration using a suitable drug carrier such as a cream, an infusion, or the like. For internal diseases in the body, sakyomycin A should be administered at a dose of about 0,000 to 10,000 mg per kg of body weight of a mammal, preferably at a dose of 0.1 m.
Administer orally or parenterally at a dose of g to 5#I9.

For oral administration, diluent 1 Finely divided powder or granules which may contain dispersants and/or surfactants; solutions in water or syrup; or suspending agents which may contain suspending agents; binders and lubricants. Administered in dosage forms such as liquid tablets; dry, non-aqueous solutions or suspensions in capsules or capsules. Flavoring agents, preservatives, suspending agents, diluents, unifying agents, and emulsifying agents are added to these pharmaceutical compositions as necessary.

Excipients can also be included. Particularly for purposes of oral administration, tablets and granules are preferred, and these may be coated with any coating agent.

When administered parenterally or as a drop for eye diseases, sakyomycin A should be administered at a concentration of about 0.01 to 10
(lIl/v)%, good mass approx. 0.1-5 (W/V)
It can be administered as an aqueous solution at a concentration of %.

Preservatives, buffers, etc. may also be included in this solution.

For diseases of the eyes, skin, and other tissues external to the living body, it can be administered locally in the form of an ointment using a water-soluble ointment base or a cream using an oil-in-water cream base.
(W/V)%, preferably 0.1-5 (W/V)%.

Next, a more specific explanation will be given by giving a test example on the pharmacological action of sakyomycin A, which is the active ingredient of the therapeutic agent for retrovirus infection of the present invention. Test Example 1: The solution for measuring the inhibitory effect on reverse transcriptase activity was 100 mHTris-HCI buffer (
state, d), Po1y(γA) (10μQ/ll11)
,01ioo(dT) 12-18(0,04u/d
) and Birth transcributase (3, Ou /d) derived from avian myeloblastosis tyrus.

Dissolve sakyomycin A in dimethyl sulfoxide and get 5 my/mf! A solution having a concentration of 1 was prepared, and then this solution was diluted with distilled water to a predetermined sakyomycin A concentration and used as a test sample solution.

Mix the measurement solution and the test sample solution by 50% each,
After incubating for 60 minutes with shaking at 7°C, the reaction was stopped by cooling on ice. Take 50% of the reaction solution obtained in this way, and add it to a DEAEifl with a diameter of 2.4 cm.
I let it soak into W paper. Next, apply this filter paper to 5% Na2HP.
It was washed three times with O4-12H20 solution, lightly washed with distilled water and ethanol in that order, and then dried. The radioactivity contained in both components captured on this filter paper was measured using a scintillation counter.

The reverse transcriptase inhibition rate (%■) of the test sample sakyomycin A was calculated from the radioactivity value (T) of the test sample group and that of the control group (C) using the following formula.

-T Inhibition rate %I = -X 100 (blank space below) Table 1 Test example 2: Suppressive effect on the growth of HTLV-III Preparation of HTLV-I [For virus preparation, loft 4/HTLV-m cells were % immobilized calf serum (Fe2), penicillin 1 ooru/ml and streptomycin 100 μQ
The cells were prepared in RPHI 1640 medium (hereinafter referred to as complete medium) containing 30×10 4 cells/d and cultured at 37° C. in a 5% CO2 incubator for 4 days.

In the following, unless otherwise specified, the cell culture was carried out at 37°C. From this cell culture solution, the cells were sedimented by centrifugation for 10 minutes using Molt 4/1tTL.
VI [[The culture supernatant was filtered with a Millipore filter (pore size 0.22 μm) to obtain a virus-containing solution. This virus-containing solution is mixed into 1. Prepare one cell line by dispensing 11 oz. and 37 ml in complete medium for VI-I-bearing HT-4 cells until use.
The cells were subcultured at ℃. Two days before the start of the experiment, live cells were counted by live/dead cell differential staining using Trivan blue, and the number of live cells was adjusted to 30xlOcells/ml.

) 0.005. i.e. 60x10 ce
1s/rd HT-4 cells were mixed with a 100-fold dilution of the virus (formerly V-III) solution prepared as described above, and cultured at 37°C for 1 hour to adsorb the virus.

I was infected with the vaping virus)! T-4 cells were washed once and resuspended in complete medium.

Measuring method for growth inhibition of HTLV-III (immunofluorescence method) Sakyomycin A at 1 rItg/ld! was suspended in dimethyl sulfoxide at a concentration of . Using this solution in complete medium, T, 2.0.0.2.0.02 and U O, 002μ
It was diluted to give g/rrdl. HTLV-III
Infection) 4T-4 cells at 60x10 cells/m
1 and mixed with an equal amount of sakyomycin A diluted solution. This results in a cell concentration of 30x10 cells.
/d, the concentration of sakyomycin A is 1.0.0.1.0
．． 01 and 0.001 μg/-.

On the third and fifth day of culture after infection, a smear of HTLV-III-infected HT-4 cells was prepared, dried, and then fixed with cold methanol for 3 minutes. Fixed cells were treated with anti-HTLV-I
I human serum (II'Z italt: 4096) c
7) Reacted with a 1.000-fold diluted solution at 37°C for 30 minutes. After that, the two machines were mixed with phosphate buffered saline (PBS).
Washed for 15 minutes. To this was added an anti-human immunoglobulin G antibody (manufactured by Dakopatts A/S) labeled with fluorescein isothiocyanate.

After reacting at 37°C for 30 minutes, the mixture was washed again with PBS.

Fluorescently labeled cells were observed using a fluorescence microscope, and the percentage of fluorescence-positive cells was calculated. The results are shown in Figure 1.

In addition, on the 3rd and 5th day of sowing, Kyomycin A
The treated non-infected) 1'-4 cells were stained for live and dead cells with 1 to lipan blue, and the cytotoxicity of sakyomycin A was calculated. This was also done for IT-4 cells (infected with IITLV-III) to measure the cytopathic effect of the virus. The results are shown in FIGS. 2 and 3.

Test Example 3: Inhibitory effect on virus growth (plaque method) +1TLV-■ Infected cells, 1.0.0.1.0.
The cells were cultured for 3 days in the presence of zakyomycin A at different concentrations of 01 and ooiμg/-. Each culture solution obtained in this way was centrifuged at 1500 Cj for 10 minutes, and the supernatant was filtered through a Millipore filter (bore length: 0.22
μm) to obtain a +1TLV-III-containing solution.

A plaque formation test was performed on this 11 and v-■ containing solution to measure the number of infectious) fTLV-III particles. That is, in order to make HT-4 cells form a monolayer on a culture dish, 50μO/d was placed in a 35M diameter polystyrene culture dish in advance.
Poly L-lysine (PLL) (manufactured by Sigma) 1d was added dropwise to the plate and then incubated at room temperature for 1 hour to perform surface treatment. The PLL surface-treated culture dish is
Washed three times with BS. 150x104cells/d
! 1.5-mL of MT-4m cell solution was spread on this PLL-treated culture dish and cultured at room temperature for 1 hour. The culture dish was then gently washed twice with PBS to remove non-adherent cells. After forming a monolayer of HT-4 cells, a 100-fold diluted HTLV-nI-containing solution 100'' was gently added to the culture dish, and cultured at room temperature for 1 hour.

After HTLV-m adsorption, 10% Fe2. After antibiotics and 0.6% agar, agarose overlay medium 1- containing neutral red was further overlaid on each culture dish. This culture dish was further cultured for 3 days, and the number of plaques observed was counted. The results are shown in Figure 1. In addition, all experiments were conducted with one group of 3
IITLV-III infected HT-
4$111 cells and Sakyomycin A diluted solution are Test Example 2
Prepared in the same manner.

Test Example 4: Acute Toxicity Regarding sakyomycin A, which is the active ingredient of the present invention,
Acute toxicity was measured by intraperitoneal administration in male ddy mice. As a result, Si05o is approximately 20m1/K
! It was J.

As can be seen from the above test examples, sakyomycin A inhibits retrovirus reverse transcriptase and also inhibits the growth of IITLV-11.
For example, sakyomycin A is extremely useful as a therapeutic agent for diseases caused by retrovirus infection, such as AIDS and adult Tel cell leukemia.

In addition, sakyomycin B, which has the same basic skeleton structure as sakyomycin A, sakyomycin C, sakyomycin D, and aquayamycin
), vineomycin A1 (VineO+tyCini
Benzanthracenone antibiotics such as nA), keriamycin B, and keriamycin C are also expected to have the same retrovirus-inhibiting effect as sakyomycin A.

Reference Example: Sakiyomycin A-producing bacteria of the genus Nocardia isolated and collected from soil in the Asama district of Gunma Prefecture were placed in an aqueous medium 100 containing 2% oatmeal and 0.1% yeast extract. The mixture was inoculated into two 51-liter Sakalo flasks, and cultured with reciprocal shaking at 27°C for 3 days. Next, 2% of the preculture solution was added to each of five 51-sized Erlenmeyer flasks containing the aqueous medium 1.51, and cultured with rotary shaking at 27° C. for 5 days. The obtained culture solution was centrifuged (10
,000 rpm, 10 minutes) to remove bacterial cells.

Diaion IIP-2040 was added to the obtained culture filtrate 11.
After stirring for 30 minutes, the mixture was washed with water and eluted with methanol. After concentrating the methanol eluate to about 100 d, it was extracted three times with 200 d of ethyl acetate, the separated ethyl acetate layers were combined, and the solvent was distilled off. The obtained residue was dissolved in chloroform and applied to a silica gel column (Fuko-gel C-200
, column volume l: inner diameter 1. Sc@X length 52.7cm”)
and methanol-chloroborm (1:10 v
/v). Collect active fractions,
It has shrunk by 1. Transfer this concentrated solution to a silica gel column (ie
selgel 60. Column capacity: inner diameter 4cm x length 1
1 cm) and methanol-chloroborum (1:1
The active fractions were collected.

After concentrating this active fraction to dryness, the residue was dissolved in methanol and further fractionated by high performance liquid chromatography under the following conditions. When the active fractions were collected and concentrated to dryness, a red powder with a concentration of 1.38 (J) was obtained, and the physical property data such as nuclear magnetic resonance spectra were consistent with that of sakyomycin A described in the literature. It was identified as

Examples of Conditions for High Performance Liquid Chromatography Examples of the therapeutic agent for retrovirus infections according to the present invention are shown below.

Example 1: Injection (Formulation A) Sakyomycin A 3.0rrrg
Sodium citrate 5.7my Sodium carboxymethyl cellulose (low viscosity) 2. OrItgp
-Methyl hydroxybenzoate 1.5myp-Propyl hydroxybenzoate 0.2Ing Water for injection (add to make the total amount 1.0d) (Formulation B) Sakyomycin A 5.0#Ig Sodium citrate 5. 7Irtg Sodium carboxymethyl cellulose (low viscosity) 2.0 parts gp-Methyl hydroxybenzoate 1.5 mg p-Hydroxybenzoate propyl 0.2 mFJ water for pouring (additionally total amount is 1. (7)) Example 2: Tablet Sakyomycin A 5.0 parts Lactose 91.0 parts Cornstarch (dried) 51.5 parts Gelatin
2.5 parts Magnesium stearate
Pass 1.0 part of Kyomycin A powder through a sieve, mix well with lactose and 30.0 parts of corn starch, and pass both through a sieve. The mixed powder was stirred with gelatin in water and heated to form a 10% (W/W) solution. The granules were dried at 40°C. The dried granules were passed through a sieve to be granulated again, and the remaining starch and magnesium stearate were added and thoroughly mixed.

The granules were compressed to prepare tablets each weighing 150 and 71 g.

Example 3: Capsule Sakyomycin A 10.0 parts Lactose 190.0 parts Pass Kyomycin A and lactose through a sieve.

The resulting powder was thoroughly mixed and then filled into hard gelatin capsules of appropriate size. Each capsule contains 2 mixed powders
Contains 00mg.

Effects of the Invention Sakyomycin A, which is the active ingredient of the therapeutic agent of the present invention, inhibits reverse transcriptase of retrovirus, and also inhibits retrovirus (ITLV-) which is the causative virus of AIDS.
I Cell rate (○) and number of plaque formation (
・) indicates a change in

Figure 2 shows the number of viable uninfected HT-4 cells (b) and the number of viable HTLV-III-infected HT-4 cells (flash) at each concentration of sakyomycin A on the third day of culture.
shows the change in

Figure 3 shows the number of viable uninfected 14T-4 cells (b) and the number of viable HTLV-II [infected] IT-4 cells (b) at each concentration of sakyomycin A on the 5th day of culture.
(mother) changes.

Claims (3)

[Claims] (1) A therapeutic agent for retrovirus infections characterized by containing sakyomycin A as an active ingredient. (2) The therapeutic agent for retrovirus infection according to claim (1), which is used as a therapeutic agent for acquired immunodeficiency syndrome. (3) The therapeutic agent for retrovirus infection according to claim (1), which is used as a therapeutic agent for adult T-cell leukemia.