JP2000212196A - Physiologically active substance dm9809 and medicine containing the same as active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicine effective as a therapeutic agent for AIDS and hardly having side effect. SOLUTION: This physiologically active substance DM9809 has the following physicochemical properties. (1) High-performance liquid chromatography detects peaks at 1.967 min, 2.400 min, 2.767 min and 3.800 min in (A) 205 nm and detects peak at 2.405 min in (B) 280 nm; (2) nuclear magnetic resonance spectrum has characteristic multiplet signals in 0.6-1.4 ppm; (3) infrared absorption spectrum has characteristic signals in 3336, 2958, 2925, 2854, 1654, 1560 and 1406 cm-1 and (4) mass spectrum has characteristic signals in 537.8, 1074.6, 1610.0 and 2121.2 mHz.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel bioactive substance, and more particularly, to a novel bioactive substance having high anti-HIV activity,
Novel bioactive substance DM9 useful for applications such as medicines and animal drugs
809.

[0002]

2. Description of the Related Art The number of patients with acquired immunodeficiency syndrome (AIDS) has been increasing in recent years, but an effective therapeutic method has not yet been established. AIDS is HIV
(Human Immunodeficiency Virus), it is thought that inhibiting the growth or infectivity of HIV can suppress the development of AIDS or delay the progress of the disease. The AIDS treatments that have been put to practical use include amidothymidine (AZ)
T), but it has not always been sufficiently satisfactory in terms of its efficacy and side effects such as cytotoxicity.

[0003]

Therefore, there is a need for a drug which is effective as a remedy for AIDS and has few side effects on the human body.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that a specific substance derived from a culture of Bacillus subtilis has high anti-HIV activity. The present invention has been completed.

That is, the present invention provides the following physicochemical properties: (1) High-performance liquid chromatography: At 205 nm, 1.967 minutes, 2.400 minutes,
Detected at 2.767 minutes and 3.800 minutes. At 280 nm, it is detected at 2.405 minutes. (2) Nuclear magnetic resonance spectrum: has a characteristic multiplet signal at 0.6 to 1.4 ppm. (3) Infrared absorption spectrum: 3336, 2958, 2
925, 2854, 1654, 1560, 1406cm
^-1 has a characteristic signal. (4) Mass spectrum: 537.8, 1074.6, 16
It has a characteristic signal at 10.0, 2121.2. A biologically active substance DM9809 having the following formula:

[0006] The present invention also provides a medicine containing the above-mentioned physiologically active substance as an active ingredient.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The biologically active substance DM980 of the present invention
9 can be isolated from a culture of Bacillus subtilis by fractionation and purification using a combination of means such as solvent extraction and column chromatography, as exemplified below.

An example of the preferred method is a method comprising the following steps. After culturing Bacillus subtilis, the resulting culture is adjusted to pH 3, extracted with ethyl acetate, the ethyl acetate phase is washed with a sodium carbonate-sodium bicarbonate buffer (pH 9), and the ethyl acetate phase is treated with hydroxide. -Chloride buffer (pH 1
After the extraction in 2), the obtained aqueous phase was adjusted to pH 6, and then extracted with ethyl acetate. This ethyl acetate phase was subjected to column chromatography.
Flow chloroform, ethyl acetate and methanol, and collect the fraction eluted with methanol.

[0009] In order to carry out the above method, it is first necessary to culture Bacillus subtilis (step). This cultivation is carried out by a conventional method, and the Bacillus subtilis preferably used is exemplified by strain DB9011 (Bacillus subtilis DB9011). This bacterium is designated as FERMBP-3418 as 1
It has been deposited on May 21, 991 at the Research Institute of Biotechnology and Industrial Technology (I 1-3, Higashi 1-3-1, Tsukuba, Ibaraki Prefecture).

A preferred culture method for producing the physiologically active substance of the present invention in Bacillus subtilis is, for example, pH 6.
A method of culturing at 37 ° C. for about 4 days using a DB9011 medium adjusted to 95 is used. D used here
The composition of the B9011 medium is as shown below. <Medium composition> (Ingredients) (Mixing amount) glucose 20g polypeptone 10g sodium chloride 5g beef extract 3g Yeast extract 2g phosphate over potassium 1g magnesium 0.5g calcium chloride _sulfate-2H 2 O 0.15 g ferric chloride -6H ₂ O 0.0081 g Manganese chloride-4H ₂ 0.0024 g Ferrous sulfate-7H ₂ O 0.0003 g Ion-exchanged water 1 liter

Next, the culture obtained above is adjusted to pH 3 and extracted with ethyl acetate (step). In this step, the culture (preferably the culture supernatant) is preferably subjected to conventional means such as centrifugation and filtration to remove insoluble substances such as cells. The pH adjustment is performed using, for example, 6N hydrochloric acid, and the extraction with ethyl acetate is performed as follows.
Using a separating funnel, a rotary shaker, etc., for example, 9
It is preferable to carry out at 2 rpm at room temperature for about 1 hour.

The ethyl acetate phase obtained by the above extraction is
Then a sodium carbonate-sodium bicarbonate buffer (p
H9) for washing (step). This cleaning is performed, for example, in 9
It is preferable to carry out the reaction at 2 rpm at room temperature for about 1 hour. Since the washing solution contains a trace amount of the physiologically active substance of the present invention or a substance similar thereto, if necessary, wash the washing solution with ethyl acetate. And may be added to the original ethyl acetate phase.

The washed ethyl acetate phase is subjected to extraction after adding a hydroxide-chloride buffer (pH 12), and the aqueous phase is separated (step). This extraction is performed, for example, at 92 rpm.
At room temperature for about 1 hour.

After the obtained aqueous phase is adjusted to pH 6,
Extracted again with ethyl acetate (step). This extraction
Using a separating funnel, a rotary shaker, etc., for example, 9
It is preferable to carry out at 2 rpm at room temperature for about 1 hour.

Since the physiologically active substance DM9809 of the present invention is contained in the ethyl acetate phase obtained as described above,
Next, purification is performed by column chromatography (step). As a carrier used for column chromatography, silica gel or the like can be used. Preferred examples of commercially available products include WAKO C-200 (manufactured by Wako Pure Chemical Industries, Ltd.).

Hexane, chloroform, ethyl acetate and methanol are successively passed through the column chromatography supporting the above ethyl acetate fraction to separate into fractions eluted with each solvent (step).

Since the biologically active substance DM9809 of the present invention is eluted with methanol among the above-mentioned solvents, it is collected and collected as a methanol fraction.
09 (step). In addition, the obtained methanol fraction is further subjected to treatment such as purification, solvent evaporation, powdering, and the like, as necessary, to thereby perform a highly purified physiologically active substance DM9809. FIG. 1 shows the entire process described above.
It is as follows.

The physiologically active substance D of the present invention thus obtained
M9809, high performance liquid chromatography (hereinafter, referred to as M9809)
“HPLC”), nuclear magnetic resonance spectrum (hereinafter “NM”).
R "), infrared absorption spectrum (hereinafter" IR ") and mass spectrum (hereinafter" MS ") are as follows.

(HPLC) Chromatograms under the following conditions are as shown in FIGS. According to this figure, at 205 nm, 1.967 minutes, 2.400 minutes,
It has characteristic peaks at 2.767 minutes and 3.800 minutes, and at 280 nm at 2.405 minutes, respectively.

<HPLC conditions> Model: Beckman HPLC system Column: SHIMAZU Prodigy ODS
5 m 4.6 × 250 m / m Elution solvent: acetonitrile / 10 mM ammonium acetate = 3/4 (v / v) Elution rate: 1.0 ml / min Detection: 205 nm and 280 nm, using Beckman UV detector Temperature: room temperature

(NMR) NMR measured under the following conditions
The spectrum is as shown in FIG. From this spectrum, it is understood that it has a characteristic signal in the range of 0.6 to 1.4 ppm.

<NMR conditions> Model: Gemini 200 manufactured by VARIAN Solvent: deuterated chloroform Temperature: room temperature

(IR) The IR spectrum measured under the following conditions is as shown in FIG. According to this figure, 33
36, 2958, 2925, 2854, 1654, 15
It has a characteristic peak at 60, 1406 cm ^-1 .

<IR condition> Model: 270-50 type infrared spectroscopic altimeter, Hitachi, Ltd. Measurement method: KBr method Temperature: room temperature

(MS) MS spectra measured under the following conditions are as shown in FIGS. From this spectrum, 537.8, 1074.6, 1610.0, 21
It is seen that it has a peak at 21.2.

<MS conditions> Model: API-100G, high performance quadrupole mass spectrometer manufactured by Perkin Elmer Co., Ltd. Measurement mode: Positive Temperature: Room temperature

Although the chemical structure of the physiologically active substance DM9809 is currently being studied from the above-mentioned physicochemical properties, a novel substance having the above-mentioned physicochemical properties has not been found as a compound having high anti-HIV activity. Is assumed.

The physiologically active substance D of the present invention thus obtained
M9809 has high anti-HIV as shown in the Examples below.
Since it is a compound having activity, it is useful as a medicament such as a prophylactic / therapeutic agent for AIDS, an antiviral agent or an animal drug.

When this physiologically active substance DM9809 is used as a drug such as a drug or an animal drug, it can be prepared in any dosage form together with various pharmaceutically acceptable solvents and carriers, and its action is impaired. It is also possible to mix various pharmaceutically acceptable additives to the extent that they are not present.

[0030]

The biologically active substance DM9809 of the present invention has high anti-HIV activity, but its mechanism of action has not yet been completely elucidated. However, it has been confirmed that, instead of inhibiting the reverse transcription process of HIV as in AZT, it inhibits the process of adsorption or fusion of HIV to T cells, thereby reducing its infectivity. It is also suggested that the virus may be lysed by specifically and directly binding to the outer coat protein of the virus particle, and may be a revolutionary antiviral agent or a preventive or therapeutic agent for AIDS.

[0031]

The present invention will be described in more detail with reference to the following Examples and Test Examples, which by no means limit the present invention.

Example 1 Isolation of DM9809: Bacillus subtilis DB9
011 strain; FERM BP-3418) was inoculated into a DB9011 medium and cultured at 37 ° C. for 4 days. After the culture, 13 liters of the culture supernatant was centrifuged (8,000 rpm, three times) to remove insolubles. The obtained culture solution was adjusted to pH 3 with concentrated hydrochloric acid.
Then, the mixture was extracted with 6 liters of ethyl acetate to separate the ethyl acetate phase. Next, 3 liters of a sodium carbonate-sodium bicarbonate buffer solution (pH 9) was added to the ethyl acetate phase, and the mixture was washed by shaking. Further, the ethyl acetate phase was extracted with 3 liters of a hydroxide-chloride buffer (pH 12) to separate the aqueous phase. The aqueous phase was adjusted to pH 6 with hydrochloric acid and extracted with 2 liters of ethyl acetate to separate the ethyl acetate phase.

The obtained ethyl acetate phase was suspended in 0.5 ml of hexane after evaporating the solvent, and silica gel column chromatography [WAKO C-200 (manufactured by Wako Pure Chemical Industries, Ltd.), 10 g. After washing; column diameter 2 cm, length 8 cm]. Hexane 100ml, chloroform 100m as elution solvent
1, 100 ml of ethyl acetate and 100 ml of methanol
And eluted sequentially to obtain corresponding fractions. After evaporating the solvent, each fraction was used as an ethanol solution.

For each of the obtained fractions, anti-HI
When V activity was examined, anti-HIV was added to the fraction eluted with methanol.
Activity was confirmed. The physicochemical properties of this methanol fraction were further measured by high performance liquid chromatography, nuclear magnetic resonance spectrum, infrared absorption spectrum and mass spectrum. The results are as shown in FIGS.

TEST EXAMPLE 1 Screening for anti-HIV activity: Each fraction obtained in Example 1 was used as a sample, and its anti-HIV activity was measured using C816.
Screening was performed by an assay using 6 cells.

That is, C81 which is a human established T cell line
A suspension of 66 cells in RPMI1640 medium (3.0 × 10 ⁵ cells / ml) was inoculated into a 48-well plate in an amount of 100 μl each, and 5 μl (× 2 times) of each of the above samples was added to each well. 0.5%, 0.05%, 0.5%
Added to a final concentration of 005%. 1 at 37 ° C
HIV-1 GU, fresh isolate after incubating for hours, capable of infecting T cell lines and macrophages
The N1WT strain (100 μl) was infected and further incubated at 37 ° C. for 2 days.

The anti-HIV activity was determined by measuring the number of syncytia (Synchia) formed (per well) with an optical microscope. Table 1 shows the results.
Shown in

[0038]

[Table 1] ───────────────────────────────── Number of syncytia ───────── ──────────── Concentration (%) Sample No. 0.5 0.05 0.005 ─────────────────── Ｎ N-hexane eluted fraction (1) 82> 100> 100 (2) 67> 100> 100 Chloroform eluted fraction (1) 70> 100> 100 (2) )> 100> 100> 100 Fraction eluted with ethyl acetate (1) 88> 100> 100 (2) 72> 100> 100 Fraction eluted with methanol (1) 0 42> 100 (2) 0 30> 100 ───────────────────────────── Control (1)> 100 (2)> 100 ───────── ─ ──────────────────────

As is evident from the results shown in Table 1, a strong HIV-1 infection inhibitory activity was observed in the fraction eluted with methanol.

TEST EXAMPLE 2 MT-4 / IIIB Assay: Human Established T Cell Line MT-
No. 4 was infected with the HIV-1 IIIB strain, and the virus protein generated after the infection was examined by the indirect fluorescent antibody method (IFA method) to confirm the virus inhibitory activity of the drug.

MT, an HIV-sensitive human established T cell
-4 cells were suspended in an RPMI 1640 medium (1.0 × 10 ⁵ cells / ml), and a 48-well plate was inoculated in an amount of 500 μl into each of the wells.
DM9809 of the present invention (methanol eluted fraction) obtained in
Was added to give concentrations of 0.5%, 0.05% and 0.005%. After incubating at 37 ° C for 1 hour,
The cells were infected with the T cell-tropic HIV-1IIIB strain and incubated at 37 ° C. for 4 days.

After the incubation, the HIV-
One antigen-positive cell was determined by the IFA method (indirect fluorescent antibody method). Table 2 shows the results.

[0043]

Table 2: Ratio of HIV antigen-positive cells (%) ─────────────────── Concentration (%) sample 0.5 0.05 0.005 ───────────── ─────────────────────DM9809 0.150> 90────────────────────── ──────────── Reference> 90 ──────────────────────────────────

From the results shown in Table 2, the physiologically active substance D of the present invention was obtained.
M9809 was also found to show strong anti-HIV-1 activity in tests using MT-4 cells and HIV-1 IIIB strain.

Test Example 3 Examination of Action Mechanism of Anti-HIV Activity (1): Syncyti
um assay showed that DM9809 of the present invention was HIV
Was determined to act on the cell adsorption and fusion processes.

C8166 cells, human established T cells, were
Suspension (5.0 × 10 ⁵⁾ suspended in PMI1640 medium
cells / ml) in a 48-well plate
of DM9809 obtained in Example 1.
(Methanol eluted fraction) was 0.5%, 0.05%, 0.5%
005%. After incubation at 37 ° C. for 1 hour, Molt-4 / IIIB cells (H
(Molt-4 cells persistently infected with IV-1 IIIB strain)
Mix 100 μl of 5.0 × 10 ⁴ cells / ml,
Incubated at 37 ° C for 14 hours.

The action mechanism of the anti-HIV activity was determined by measuring the number of syncytium (Synchium) formed (per well) with an optical microscope. Table 3 shows the results.

[0048]

[Table 3] ────────────────────────────────── Number of syncytia ──────── ────────────── Concentration (%) Sample No. 0.5 0.05 0.005 ───────────────── {DM9809 (1) 055> 100 (2) 039> 100} ──────────────── Control (1)> 100 (2)> 100 ────────────────────── ────────────

The results in Table 3 show that the already known anti-HIV drugs dextran sulfate (a fusion inhibitor of HIV) and AZ
From the results of a similar test for T (reverse transcription inhibitor), DM98
09 was found to inhibit the process of adsorption or fusion of HIV-1 to cells.

Test Example 4 Examination of the Mechanism of Action of Anti-HIV Activity (2) By treating HIV or cells in advance with DM9809, the inhibition of the anti-HIV activity of DM9809 was reduced by HI.
It was examined whether it occurs by acting on V or by acting on cells.

(1) Virus treatment: Test virus (HI
V-1GUN1WT) in 100 μl, 5 μl of DM9809 (methanol eluted fraction) of the present invention obtained in Example 1 above
Was added and incubated at 37 ° C. for 1 hour, followed by centrifugation (15,000 rpm, 90 minutes). The obtained precipitate (HIV-1GUN1WT) was inoculated to C8166 cells (human established T cells) in RPMI1640 medium (200 μl) to infect. After inoculation, the plate was incubated for 2 days, and the number of syncytia (Synchia) formed (per well) was measured with a light microscope.

(2) Cell treatment RPMI1640 of C8166 cells (human established T cells)
The DM9 of the present invention obtained in Example 1 was added to 100 μl of the medium.
809 (methanol eluted fraction) 5 μl of DM9809 was added, and the mixture was incubated at 37 ° C. for 1 hour, followed by centrifugation (5,000 rpm, 5 minutes). The obtained sediment (C8166 cells) was previously kept at 37 ° C. for 1 hour, and then centrifuged (15,000 rpm, 90 minutes).
HIV-1 GUN1 suspended in RPMI 1640 medium
Infected with WT. Incubate for 2 days after infection,
The number of syncytia (Synchia) formed (per well) was measured with a light microscope. Table 4 also shows the results.

[0053]

[Table 4] 数 Number of Syncyria Sample No. Virus treatment Cell treatment ─── ＤＭ DM9809 (1) 0> 100 (2) 0> 100 ＤＭ ────────────────────────

From the results in Table 4, it is clear that DM9809 was dissolved by binding to the coat protein of the virus, for example.
It was suggested that it acts directly and specifically on virus particles and reduces virus infectivity.

[0055]

The novel physiologically active substance DM980 of the present invention
No. 9 has specific and high anti-HIV activity, and thus is useful as a drug such as an antiviral agent or a prophylactic / therapeutic agent for AIDS. It can also be used as an animal drug such as an antiviral agent for immunodeficiency diseases of mammals other than humans such as monkeys and cats.

[Brief description of the drawings]

FIG. 1 is a drawing showing an outline of a fractionation operation of a Bacillus subtilis culture.

FIG. 2 shows the results of high performance liquid chromatography of a methanol eluted fraction containing DM9809 (205n).
m).

FIG. 3 shows the results of high performance liquid chromatography of a methanol eluted fraction containing DM9809 (280n).
m).

FIG. 4 is a drawing showing the result of a nuclear magnetic resonance spectrum of a methanol eluted fraction containing DM9809.

FIG. 5 is a drawing showing the results of an infrared absorption spectrum of a methanol eluted fraction containing DM9809.

FIG. 6 is a view showing the result of mass spectrum of a methanol eluted fraction containing DM9809.

FIG. 7 is an enlarged view of a part of the spectrum of FIG.
20-570 m / z).

FIG. 8 is an enlarged view of a part of the spectrum of FIG.
020-1110 m / z).

9 is an enlarged view of a part of the spectrum shown in FIG.
580-1640 m / z).

FIG. 10 is an enlarged view (2080 to 2200 m / z) of a part of the spectrum in FIG. that's all

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12P 1/04 C12P 1/04 Z // C12N 1/20 C12N 1/20 A (C12P 1/04 C12R 1 : 125) (C12N 1/20 C12R 1: 125) F term (reference) 4B064 AH19 BH02 BH03 BH07 CA02 CE03 CE07 CE08 DA01 4B065 AA19X BA22 BC02 BD15 BD16 BD18 CA44 4C087 AA01 AA02 BC65 CA10 MA01 MA11 NA01 NA14 ZC55 4H0A AB29 AC62 AD20 BA31 CA11

Claims (8)

[Claims] 1. The following physicochemical properties: (1) High performance liquid chromatography: At 205 nm, 1.967 minutes, 2.400 minutes,
Detected at 2.767 minutes and 3.800 minutes. At 280 nm, it is detected at 2.405 minutes. (2) Nuclear magnetic resonance spectrum: has a characteristic multiplet signal at 0.6 to 1.4 ppm. (3) Infrared absorption spectrum: 3336, 2958, 2
925, 2854, 1654, 1560, 1406cm
^-1 has a characteristic signal. (4) Mass spectrum: 537.8, 1074.6, 16
It has a characteristic signal at 10.0, 2121.2. A physiologically active substance DM9809 having the following formula: 2. The biologically active substance DM9809 according to claim 1, which is obtained by the following production method: culturing Bacillus subtilis, adjusting the obtained culture to pH 3, and extracting with ethyl acetate. The ethyl acetate phase was washed with a sodium carbonate-sodium bicarbonate buffer (pH 9), and the ethyl acetate phase was washed with a hydroxide-chloride buffer (pH 1).
After the extraction in 2), the obtained aqueous phase was adjusted to pH 6, and then extracted with ethyl acetate. This ethyl acetate phase was subjected to column chromatography.
Flow chloroform, ethyl acetate and methanol, and collect the fraction eluted with methanol. 3. Bacillus subtilis strain DB9011 (Bacillus s.
The biologically active substance DM9809 according to claim 2, which is ubtilis DB9011). 4. A medicament comprising the physiologically active substance according to claim 1 as an active ingredient. 5. The medicament according to claim 4, which is an antiviral agent. 6. The medicament according to claim 5, wherein the virus is HIV. 7. The medicament according to claim 4, which is a prophylactic or therapeutic agent for AIDS. 8. A preventive or therapeutic agent for a viral immunodeficiency disease in mammals, comprising a culture extract of Bacillus subtilis as an active ingredient.