JPH01207237A - Tumor necrosis factor inducer - Google Patents

Abstract

PURPOSE:To obtain a tumor necrosis factor inducer having tumor necrosis factor inducing activity and low toxicity, comprising trehalose dimycolate as an active ingredient. CONSTITUTION:A tumor necrosis factor inducer containing trehalose dimycolate as an active ingredient. The mycolic acid residue in the active ingredient is a group shown by the formula (R' and R'' are alkyl which may contain unsaturated bond). The active ingredient, for example, is obtained by shaking culture in PGY medium containing 1% Nocadia rubra and extracting from the cell with chloroform methanol. The active ingredient has actions as a first inducer in reference to induction tumor necrosis factor and significantly induces TNF by using a second inducer such as LPS and the first inducer as well.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to the use of tumor necrosis factor (Tumor Necrosis Fac) containing trehalose simicolate as an active ingredient.
tor.

(hereinafter abbreviated as TNF). In particular, the present invention relates to TNF inducers useful as primary inducers for inducing TNF.

[Prior Art/Problems to be Solved by the Invention] In 1975, O1d etc. is a glycoprotein that specifically attacks only cancer cells without causing any damage to normal cells, that is, lI! iTumor necrosis factor (T
N F ) was reported (Proc, Natl.

8cad, Sci, USA, E2. 36
66 (1975)).

According to this, a primary inducer such as BCG live bacteria is administered intravenously to mice, and 1 to 2 weeks later lipopolysaccharide (hereinafter referred to as L
It has been reported that administration of a secondary inducer such as PS) induces TNF in serum. Also B
Pro i killed with formalin instead of CG
It has been reported that when onibacterium acnes is used, TNF can be induced by the same operation as above.

However, live BCG bacteria or dead BCG fallen embryos also contain substances other than those that exert their effects, and their biological activities vary. Since there is heterogeneity and the substance that exhibits the effect does not have a clear structure, it is not possible to produce the substance by means such as synthesis.

On the other hand, compounds whose chemical structures have been determined have uniform physicochemical properties, can be mass-produced, and can be expected to have the same actions and effects when used. The reality is that there is a long-awaited development of a 'r N F inducing agent.

Therefore, the object of the present invention is to provide a TNF inducer that can replace the primary inducer of embryos using live BCG bacteria or killed bacteria, and which uses a compound whose structure has been determined as an active ingredient. An object of the present invention is to provide a TNF-inducing agent.

[Means to solve the problem]

In order to achieve this object, the present inventors conducted extensive research and found that trehalose simicolate has the desired TNF-inducing activity, thereby completing the present invention.

That is, the present invention is a tumor necrosis factor inducer containing trehalose simicolate as an active ingredient.

The mycolic acid residue in trehalose simicolate, which is the active ingredient of the present invention, has the formula It is a concept that also includes those with unsaturated bonds,
It is broader than the usual concept of alkyl group. ).

Regarding formula (1), the alkyl group may be linear or branched, and may have an unsaturated bond (preferably a double bond). The total number of carbon atoms in the mycolic acid residue represented by formula (1) is preferably about 20 to 90.

The alkyl group represented by Ro is preferably as follows. That is, it is a linear or branched chain having 8 to 26 carbon atoms and about 0 to 2 unsaturated bonds (preferably double bonds). In addition, the alkyl group represented by R'' is preferably as follows.That is, it is linear or branched with 20 to 60 carbon atoms, and has 1 to 9 unsaturated bonds (preferably double bonds). It has a certain degree.

Trehalose simicolate, which is the active ingredient of the present invention, is a known compound, and the method for producing it is as follows.

That is, for example, Nocardia rubra (Nocardia rubra).
PGY medium (p) 17.2 containing 1%
．． The resulting bacterial cells were cultured with shaking at 30°C), and trehalose was added to the cells using chloroform/methanol (2: lv/v).
It can be produced by extracting simicolate.

Although the trehalose simicolate can be easily produced by synthesis, a compound produced by synthesis is a preferred embodiment since it contains only a single or active ingredient.

[Action/Effect]

The trehalose simicolate used in the present invention is T
It acts as a primary inducer for inducing NF, and when used in combination with a secondary inducer such as LPS, it significantly induces TNF.

Various reports have been made regarding the toxicity of trehalose simicolate used in the present invention, and it has already been established that its toxicity is extremely low.

Trehalose simicolate, which is the active ingredient of the present invention, can be formulated into any form using a t1 formulation method to prepare the TNFH inducer of the present invention.

Such TNF inducers are prepared by conventional means using any required pharmaceutically acceptable additives (eg, carriers, excipients, etc.). Specific formulations include, for example, oral preparations (e.g. tablets, capsules, powders, liposomes, etc.);
Injections [liposomes, emulsifying injections (e.g. emulsifiers using organic acids, organic bases, surfactants, water-soluble polymers, water-soluble organic solvents, etc.), suspension injections using physiological saline, etc. ] etc. are exemplified.

The dose of trehalose simicolate, the active ingredient of the present invention, to be administered to humans varies depending on the dosage form, administration route, patient's severity, tolerance to the drug, etc., but usually per adult:
10rag~2000g, preferably 500~100g
It is recommended that the dose be administered once or in several doses within a range of 0.

1 to 3 days after administration of the TNF inducer of the present invention, TNF is induced by administering a secondary inducer such as LPS, for example, in the same amount as a conventional dose.

[Test Example] Test Example 1 (Cytotoxicity to L-929 cells) (1
) - group of 8 ICR strain 5-week-old male mice (Shizukyo)
．． each Nocardia suspended in a water-in-oil-in-water emulsion containing 2% Freund's incomplete adjuvant.
Trehalose simicolate derived from Nr.rubra (hereinafter abbreviated as Nr-derived TDM) was administered to the tail vein at 30 pg/mouse 10 times every other day.

As a control, a water-in-oil-in-water emulsion containing no Nr-derived TDM was similarly administered at a rate of 0.2 d/mouse.

1 to 3 days after the end of administration, 00 pg/mouse of LPSI derived from Escherichia coli (E. coli) was administered into the tail vein, and 2 hours later, whole blood was collected from the mouse, and serum (T
(including NF) were separated. A group without LPS administration was set as a control.

(2) 3 x 10' cells/d of L-929 cells (fibroblasts) in Eagle MEM medium supplemented with 10% fetal bovine serum
Suspend 100. The cells were dispensed into 96-well microplates and cultured at 37°C in 5% COz. Approximately 24 hours later, 50μ and 1 pg of mouse serum of (1) was serially diluted (26-2!2 times diluted) in Eagle's MEM medium.
/ml actinomycin D50p1 (final concentration 0.2
5 pg/IRE) was dispensed and culture was continued. 18
After an hour, the culture supernatant was removed by suction, and the cells on the plate were stained with crystal violet.The dye incorporated into the living cells was extracted with 33% acetic acid, and the absorbance at 550 nm was measured using a microplate reader. . From the absorbance value, L-929 cytotoxicity (%) was determined using the following formula.
I asked for

(Absorbance of control (no serum added)) The results are shown in Figure 1. In Figure 1, the vertical axis is L-9.
29 cytotoxicity (%), the horizontal axis is the log 2 of the dilution factor.
I took logarithmic values. The value of 50% cytotoxicity was read from this graph and expressed as an approximate decimal value. Table 1 shows the numerical values.

(Left below) Table 1 - LPS Nr-derived T without cytotoxic effect
DM'l □ Nr-derived TDM without cytotoxic effect”
LPS approx. 2.5X105Nr derived T D
M”: Journal of Pharmacob
io-Dynas+1cs10, 113-123 (1
987) [Example] Example 1 A solution of egg yolk phosphatidylcholine (15 μmole) and trehalose (1 mg) dissolved in chloroform was placed in an eggplant-shaped colben, and the chloroform was distilled off under reduced pressure in a water bath at 25 to 30°C. A thin film was formed on the inner wall of the Kolben. Al17.0 phosphate buffered saline (1 ml) was added to this Colchen and shaken until the thin film on the inner wall was peeled off. The exploded liposomes are frozen with dry ice and methanol, and freeze-dried using a freeze dryer.
A lyophilized powder of trehalose simicolate-containing liposomes was obtained.

Example 2 The same procedure as in Example 1 was carried out using soybean phosphatidylcholine instead of egg yolk phosphatidylcholine to obtain a freeze-dried powder of trehalose simicolate-containing liposomes.

Example 3 The same procedure as in Example 1 was carried out using dipalmitoylphosphatidylcholine instead of egg yolk phosphatidylcholine to obtain a lyophilized powder of trehalose simicolate-containing liposomes.

Example 5 Egg yolk phosphatidylcholine and trehalose simycolate? '8? Further, cholesterol In+g was added to &, and the following procedure was carried out in the same manner as in Example 1 to obtain a freeze-dried powder of trehalose dimycolate-containing liposomes.

Example 5 A QQ injection with % and % of a liposome having the following formulation was produced by a conventional method.

Trehalose simicolate 300■Egg yolk phosphatidylcholine 720■Physiological saline
Appropriate amount: 5d Example 6 An injection using a non-aqueous solvent having the following formulation was produced by a conventional method.

Trehalose Simicolate 100■Olive oil
Suitable 1 Total N1 ml Example 7 Take the specified amount of the ingredients other than water from the following ingredients, and stir while heating to 40°C to make it homogeneous. Purified water was added to this mixture and stirred to produce a cloudy, milky injection solution.

Trehalose Simicolate 300■Olive oil
100■d2-α-tocopherol
50 ■ Polysorbate 80 80 ■ Sorbicune sesquioleate 60 ■ Purified water
Appropriate amount 5 rnl

[Brief explanation of the drawing]

Figure 1 shows the Nr-derived TD based on the absorbance value at 550 nm.
This is a graph showing the results of L-929 cytotoxicity (%) of M.

Claims (1)

[Claims] A tumor necrosis factor inducer containing trehalose dimycolate as an active ingredient.