JPH0684389B2 - Cell growth promoting substance WAS-α391101 - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention promotes proliferation of various cells such as human fetus-derived fibroblasts, human adult skin-derived fibroblasts, mouse fetus-derived normal fibroblasts, mouse lymphocytic ascites tumor-derived cells and the like. WAS-α39110, a novel cell growth promoting substance with action
The present invention relates to 1 and its production method, a cell growth promoter containing the substance, and the like.

The present inventors focused on the fact that a large amount of body hair is mixed in the feces of hairy mammals such as rats and cats, and as a result of earnest research from the viewpoint of its ecological significance, Physiology that has strong growth-promoting activity on various cells such as human fetus-derived fibroblasts, human adult skin-derived fibroblasts, mouse fetus-derived normal fibroblasts, mouse lymphocytic ascites tumor-derived cells in mammalian hair The present invention has been accomplished by finding that an active substance exists and succeeding in its isolation and identification.

That is, the substance WAS-α391101 of the present invention promotes the cell proliferation of normal cells without the transformation thereof, and since the action is reversible, it is used as a wound treatment agent, an ulcer treatment agent, a hair growth stimulating agent, etc. It can be said to be extremely useful.

Hereinafter, the physicochemical properties, production method, physiological properties and usage of the substance WAS-α391101 of the present invention will be explained in detail.

Physicochemical properties of WAS-α391101 i) Elemental analysis values C: 36.02%, H: 7.54%, N: 14.24% and balance oxygen.

ii) Ultraviolet absorption spectrum As shown in Figure 1 attached, iii) Infrared absorption spectrum The spectrum measured by the KBr tablet method is as shown in the attached Fig. 2. Its characteristic absorption is iv) ¹³ C-NMR spectrum The spectrum of the solvent; heavy water, internal standard; tetramethylsilane is as shown in Fig. 3 and its chemical shift values are δ (ppm), 17.391, 20.750, 24.975, 30.177, 42.747,
44.427, 47.352, 51.849, 57.647, 61.440, 61.656, 6
7.129, 72.384, 73.685, 171.699, 173.484 and 176.900
ppm.

v) Melting point 60 ° C (decomposition) vi) Amino acid analysis of acid hydrolyzate Hydrolyzed with 6N hydrochloric acid at 110 ° C for 20 hours and analyzed by an automatic amino acid analyzer. As shown in Fig. 4, glycine, Alanine, serine and threonine were detected.

vii) Solubility Soluble in water. Soluble in acetonitrile and dimethyl sulfoxide. Insoluble in alcohol, ether, chloroform and benzene.

viii) Appearance White candy with a slight yellow tint.

ix) Estimated molecular weight The estimated molecular weight by a high resolution gas chromatography mass spectrometer (JEOL JMS-DX300 type) is 468.

Based on the above analysis data, WAS-α391101 was identified as a single substance having an oligopeptide as a main structural component.

In addition, if the model of the measuring equipment is additionally mentioned, an ultraviolet spectrophotometer (Hitachi 220A type double beam spectrophotometer) infrared spectrophotometer (Shimadzu IR-420 type infrared spectrophotometer), an NMR spectrometer (JEOL JMN -FX90Q type FT-NMR spectrometer),
For example, an automatic amino acid analyzer (Hitachi 655 type high performance liquid chromatograph Hitachi F-1000 type spectrofluorometer).

Production of WAS-α391101 Rat, mouse, sheep, etc. mammalian hair is subjected to water extraction treatment to obtain a water-soluble fraction, and based on the physicochemical properties, dialysis method, gel filtration method, ion exchange method, reverse phase chromatograph It can be isolated and prepared by appropriately using various known fractionation and purification means such as methods.

Physiological properties of WAS-α391101 As shown in the experimental examples below, the substance WAS-α391101 of the present invention shows a strong stimulatory activity on cell proliferation, and its action is reversible without transformation of normal cells. Therefore, it can be extremely usefully used as a wound healing agent, an ulcer healing agent, a hair growth promoting agent, and also as a cosmetic because of its high hygroscopicity.

Acute toxicity As shown in Examples below, LD of the substance WAS-α391101 of the present invention
₅₀ value is subcutaneously 492.5 mg / kg body weight / mouse, orally
1950 mg / kg body weight / mouse.

Use mode of WAS-α391101 The substance WAS-α391101 of the present invention can be used by means such as oral administration, subcutaneous injection or intravenous injection, and its dose is usually about 10 μm.
g to 0.5 g / kg body weight (once), more preferably about 100 μg to about 0.1 g / kg body weight (once) by oral administration, and its dosage form is a solution prepared by dissolving in physiological saline or the like. Injectable solutions, emulsified preparations, powders by lyophilization or suppositories, enteric coatings, sublingual tablets, granules, tablets, capsules, etc., and a usual dosage form is appropriately selected together with a suitable carrier, bulking agent, diluent, etc. obtain.

Further, an ointment form is preferable for repairing damaged skin, and as the base of the ointment, a usual base such as purified lanolin, liquid paraffin, white petrolatum, polyethylene glycols, higher aliphatic alcohols and vegetable oils is used. Moreover, the manufacturing method follows a conventional method.

Experimental Example 1 Production method and purification of WAS-α391101 Wistar germ-free rats (JCL-Wistar GF: Clear Japan Co., Ltd.) 100g of hair was collected, and sterile distilled water (hereinafter,
After washing with pure water), it was immersed and stirred in 2 l of ice-cooled pure water for 24 hours. Then dip the immersion liquid in a Millipore filter (average pore size
45 μm, HAWP, Millipore), and lyophilized. The freeze-dried product was suspended in approximately 200 ml of pure water, and dialyzed against 1 pure water twice with a dialysis tube (Union Carbide desalting cellulose tube, pore size 24Å) twice each day and night, and the dialyzed external solution was freeze-dried. And roughly 1g
Got Next, using a Toyopearl HW-40F column (molecular weight cutoff 100 to 10,000, φ2 cm × 50 cm, Toyo Soda Co., Ltd.),
Gel filtration was performed with 0.01 M formic acid-ammonia buffer (pH 3.0) as an eluent at an elution rate of 1 ml / min. Its UV absorption 21
The elution pattern at 4 nm is shown in Fig. 5 (in the figure, one fraction corresponds to 10 ml). Then, by the activity assay method shown in Experimental Example II described later, the shaded area in the figure, which is recognized as the active fraction, was collected and subjected to gel filtration column (Toyo Soda TSKgel G1000P
W, φ21.5 mm × 60 cm, after elution: 0.01 M formic acid-ammonia buffer (pH 3.0) elution rate 3 ml / min) was used for high performance liquid chromatography. The elution pattern by UV absorption at 214 nm is shown in FIG. The active fraction, which is the shaded area in the figure, was collected, and the reversed-phase column (Yamamura Chemical YMC-SH345,
φ2 cm x 50 cm, eluent: 0.01 M formic acid-ammonia buffer (p
85:15 mixture of H3.0) and acetonitrile, elution rate 5 ml
/ Min) to give 10 mg of a purified sample. The elution pattern calibrated by the absorbance at 214 nm is shown in FIG. In addition, in the figure, the shaded portion is the active portion, and therefore the active factor can be said to be almost completely isolated at this stage.

The physicochemical properties and the like of the purified preparation thus obtained are as described above.

Experimental Example II Physiological activity of WAS-α391101 1. Fibroblast proliferation promoting effect (activity assay method) Mouse fetal normal fibroblast SC-1 was seeded on 96-well multiplate at 1500 cells per well, and 5% Cultured in 0.2 ml Eagle MEM medium containing fetal bovine serum (Earl's salt, Gibco) overnight at 37 ° C under the condition of 5% CO ₂ remaining air, and then replacing the medium with 0.2 ml MEM medium containing 1% fetal bovine serum. Cultivation was continued for 5 days and purified WAS-α391101 1 μg / ml
The number of cells in the (medium) addition hole was counted by a Coulter counter (Model ZM, Coulter Electronics).

The control is the standard-free group, and the results are summarized in FIG. In the figure, symbol C represents the average number of cells in the control group, and symbol S represents the husband of the standard addition group.

2. Human adult skin-derived fibroblasts are seeded in 96-well multiplate at 1500 cells per well, 0.2 ml Eagle MEM medium containing 5% fetal bovine serum (Earl's salt, Gibco) at 37 ° C, 5% CO ₂ balance air Under the conditions described in 1., the medium was changed to 0.2 ml MEM medium containing 1% fetal bovine serum, and the culture was continued for another 5 days. The number of cells in the 1 μg / ml (medium) purified WAS-α391101 standard was added to the Coulter counter. (Coulter Electronics model ZM).

The control is the standard non-addition group, and the results are summarized in FIG. In the figure, symbol C represents the average number of cells in the control group, and symbol S represents the husband of the standard addition group.

3. Mouse lymphocytic ascites tumor-derived cells L5178Y were seeded in a 96-well multiplate at 2000 cells per well, and in 0.2 ml Eagle MEM medium containing 1% fetal bovine serum (Earl's salt, Gibco).
Incubate for 2 days at 37 ° C with 5% CO ₂ remaining air, then WAS-α
391101 Purified sample 1 µg / ml (medium)
M).

The control is the standard-free group, and the results are summarized in Fig. 10. In the figure, symbol C represents the average number of cells in the control group, and symbol S represents the husband of the standard addition group.

4. Human fetal skin-derived fibroblasts were seeded on a 96-well multiplate at 1300 cells per well, and 0.2% Eagle MEM medium containing 5% fetal bovine serum (Earl's salt, Gibco) at 37 ° C, 5% CO ₂
After culturing for one day under the condition of the rest of the air, and then changing the medium to 0.2 ml MEM medium containing 1% fetal bovine serum and continuing the culturing for another 5 days, the number of cells in the 1 μg / ml (medium) purified hole of WAS-α391101 purified sample was adjusted. It was measured with a Coulter counter (Model ZM, Coulter Electronics Co.).

The control is the standard-free group, and the results are summarized in Fig. 11. In the figure, symbol C represents the average number of cells in the control group, and symbol S represents the husband of the standard addition group.

5. Wistar rats (male, 10 weeks old, normal weight 250g-5 groups) were fasted for 16 hours, placed in a stress cage and placed at 23 ° C.
It was immersed in a constant temperature water bath at 16 hours, and after 16 hours, the rat was taken out to cause a stress ulcer. WAS-α391101 10 μg / mouse dissolved in 0.5 ml of physiological saline was orally administered once a day for 3 days, the rat was killed and blood was removed, the stomach was taken out and fixed with formalin, and the length of the linear ulcer was measured. The total sum is expressed as a stress ulcer index (mm) and is shown in Table 1. The control is a saline administration group.

6. Wistar rats (male, 10-week-old, normal weight 250 g-group of 5) were fasted for 16 hours, and then WAS-α391101 10 μg / mouse dissolved in 0.5 ml of physiological saline was orally administered to the strain cage. It was immersed in a constant temperature water bath at 23 ° C. and left for 16 hours. The taken out rat was immediately killed and the abdomen was opened, and the stomach was taken out and fixed with formalin. The length of the linear ulcer was measured and the total sum thereof was expressed as a stress ulcer index (mm), which is shown in Table 2. The control is a saline administration group.

7. Wistar rats (male, 8 weeks old, normal weight 200 g-group of 5) are anesthetized with ether, and after removing hair from the back with a hair remover (manufactured by Shiseido Co., Ltd.), pick up the skin and punch with 10 mm diameter chisel. It was 100 mg of WAS-α391101 was added to 1 kg of white petrolatum ointment and formulated, 100 mg of ointment was applied to the wound surface twice a day, and the area of the wound surface was measured every day. I asked. The control was white petrolatum ointment and the results are shown in Table 3.

8. ICR mice (male, 6 weeks old, average weight 30.0 ± 0.3 g, 10 mice in each group) were used, and 6 mg, 1 dissolved in 0.5 ml of physiological saline.
0.5 mg, 15 mg, 19.5 mg, 24 mg of the present invention substance WAS in 5 steps
Administered α391101 subcutaneously and observed its life and death for 7 days.
LD ₅₀ value calculated according to the ens-krber method is 492.5 mg / kg
Was a weight mouse.

9. ICR mice (male, 6 weeks old, average body weight 30.0 ± 0.3 g, 10 mice in each group) were used, and dissolved in 0.5 ml of physiological saline, 30 mg, 4
Inventive substance WAS-α39 in 5 steps of 5 mg, 60 mg, 75 mg and 90 mg
1101 was subcutaneously administered, and life and death were observed for 7 days.
The LD ₅₀ value calculated according to the Krber method was 1950 mg / kg body weight mouse.

Formulation Example 1. WAS-α391101 10 μg was dissolved in sterilized physiological saline 1 ml to prepare an injection.

2. To 100 mg of WAS-α391101, a white ointment of the pharmacopoeia was gradually added and kneaded to give a total amount of 1 kg to prepare an ointment.

3. WAS-α391101 (10 mg) was dissolved in sterile distilled water (50 ml) and mixed with cholesterol (0.05 g) dissolved in ethanol (50 ml), and an appropriate amount of perfume was added to the solution to prepare a topical liquid for rubbing.

4.WAS-α391101 50mg, menthol 0.6mg, thymol 0.6
mg, eucalyptus oil 0.002 ml and lemon oil 0.02 ml lactose 950 mg
The mixture was thoroughly mixed with, and 5% starch paste solution was used as a binder to prepare granules by a wet method and tabletted to prepare a lozenge of about 1 g per tablet.

5. WAS-α391101 (50 mg) was thoroughly mixed with lactose (950 mg), granules were prepared by a wet method using 5% starch paste solution as a binder, and compressed into tablets.

As described above, the substance WAS-α391101 of the present invention can be made into a desired dosage form having a predetermined activity by dissolving and mixing it in a pharmaceutically acceptable carrier based on the standard dose and the like.

[Brief description of drawings]

1 to 4 are explanatory views showing the physicochemical properties of the substance of the present invention. 5 to 7 are explanatory views of Experimental Example I of the present invention. 8 to 11 show Experimental Examples II-1, II-2, II-3 of the present invention.
FIG. 11 is an explanatory diagram of II-4. The vertical line length at the top of the graph shows the standard error.

Claims (1)

[Claims] (A) Estimated molecular weight: 468 (mass spectrum method) (b) Melting point: 60 ° C (decomposition) (c) Solubility: readily soluble in water. Soluble in acetonitrile and dimethyl sulfoxide. Insoluble in ethyl alcohol, ether, chloroform and benzene. (D) UV absorption maximum wavelength: (E) Infrared absorption spectrum (KBr method): As shown in the attached FIG. (F) Nuclear magnetic resonance spectrum of carbon 13 (solvent; heavy water internal standard; tetramethylsilane): As shown in FIG. WAS-α391101, which is a cell growth promoter.