JPH0689013B2 - Cell growth inhibitor WAS-Z414101 - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention suppresses 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. Novel cell growth inhibitor WAS-Z414101 with action
And a method for producing the same, a cell growth inhibitor containing the substance, and the like.

The present inventors have 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 ecological significance, In animal hair, there is a physiologically active substance having a strong growth inhibitory activity against various cells such as mouse fetal-derived normal fibroblasts, human adult skin-derived fibroblasts, mouse lymphocytic ascites tumor-derived cells and the like. The present invention has been achieved by the discovery and the successful isolation and identification thereof.

That is, the substance WAS-Z414101 of the present invention is a carcinostatic agent because of its markedly inhibitory effect on the growth of cancer cells such as mouse lymphocytic ascites tumor-derived cells which are markedly proliferative in contrast to the slight suppression of the growth of normal cells, and immunosuppression. It can be said that it is extremely useful as an agent and the like.

Hereinafter, physicochemical properties of the substance WAS-Z414101 of the present invention, a production method,
The physiological properties and the manner of use will be explained in detail.

Physicochemical properties of WAS-Z414101 i) Elemental analysis values C: 57.80%, H: 5.89%, N: 12.48% and balance oxygen.

The experimental values corresponding to _{C 10 H 12 N 2 O 3} · 1 / 5H 2 O.

ii) High resolution gas chromatograph-mass spectrograph As shown in the attached FIG. 5, it is estimated from these that the molecular weight is 208.

iii) Ultraviolet absorption spectrum As shown in Figure 1 attached, iv) Infrared absorption spectrum The spectrum measured by the KBr tablet method is as shown in the attached Figure 2. Its characteristic absorption Is 3500, 3350, 3050, 1662, 1618, 1580, 1488, 1453,
1392, 1350, 1212, 1160, 750 and 628 cm ^-1 .

v) ¹ H-NMR spectrum Solvent; d ₆ -dimethylsulfoxide, internal standard; tetramethylsilane The spectrum is as shown in FIG. 3, and its chemical shift value δ (ppm) is 2.50, 3.08, 3.1.
8, 3.26, 3.37, 3.41, 3.46, 3.57, 3.60, 3.65, 3.6
9, 6.41, 6.43, 6.49, 6.51, 6.52, 6.58, 6.59, 6.6
3, 6.64, 6.77, 6.79, 7.12, 7.14, 7.16, 7.20, 7.2
1, 7.27, 7.29, 7.60, 7.63, 7.71, and 7.72 ppm.

vi) ¹³ C-NMR spectrum Solvent; d ₆ -dimethylsulfoxide, internal standard; spectrum in tetramethylsilane is as shown in FIG.
Its chemical shift value δ (ppm) is 40.12, 49.83, 114.40, 1
16.45, 116.89, 130.80, 134.07, 151.00 and 169.40pp
m.

vii) Melting point 195-200 ° C (decomposition) viii) Solubility Easily soluble in water. Soluble in acetonitrile and dimethyl sulfoxide. Insoluble in alcohol, ether, chloroform and benzene.

viii) Appearance White powder.

According to the above analytical data, WAS-Z414101 has a molecular formula of C ₁₀ H ₁₂ N ₂
It was identified as a single substance that is O ₃ .

In addition, if the model of the measuring equipment is additionally mentioned, a mass spectrometer (JEOL JMS-DX300 type), an ultraviolet spectrophotometer (Hitachi 220A type double beam spectrophotometer), an infrared spectrophotometer (Shimadzu IR-420)
Type), NMR spectrometer (JEOL JMN-FX90Q type F
T-NMR spectrometer).

Production of WAS-Z414101 Rat, mouse, sheep hair, etc. mammalian body 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-Z414101 As shown in the experimental examples described below, the substance WAS-Z414101 of the present invention shows a strong inhibitory activity on the proliferation of cells, and its action is on cancer cells etc. which are more proliferative than those on normal cells. Remarkably, therefore, it can be said that the anticancer agent, immunosuppressive agent, etc. contained in the substance of the present invention are useful as a therapeutic or preventive agent for diseases such as malignant tumors and leukemias, and as a rejection inhibitor after organ transplantation. obtain.

Acute toxicity LD _{50 of the} substance WAS-Z414101 of the present invention as shown in the experimental examples described below.
The value is 152.5 mg / kg body weight mouse by subcutaneous administration and 522 by oral administration.
5 mg / kg body weight mouse.

Use mode of WAS-Z414101 The substance WAS-Z414101 of the present invention can be applied by such means as oral administration, subcutaneous injection or intravenous injection, and the dose is usually about 100 ng.
To about 100 mg / kg body weight (once), more preferably about 100 μg to about 10 mg / kg body weight (once) by oral administration. The dosage form is a solution in physiological saline or the like, an injection solution. , Powder or suppository by freeze-drying, enteric coating, sublingual tablet,
Ordinary dosage forms such as granules, tablets and capsules can be appropriately selected together with a suitable carrier, filler, diluent and the like.

Experimental Example I WAS-Z414101 Production Method and Purification Wistar germ-free rats (JCL-Wistar GF: CLEAR JAPAN CO., LTD.) 100 g 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 about 200 ml of pure water, and dialyzed twice with 1 day of pure water using a dialysis tube (Union Carbide desalting cellulose tube, pore size 24Å), 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. 6 (1 fraction corresponds to 10 ml in the figure). Then, by the activity assay method shown in Experimental Example II described below, the shaded area in the figure, which is recognized as the active fraction, was collected and subjected to gel filtration column (Toyo Soda TSKgel G1000
PW, φ21.5 mm × 60 cm, eluent: 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-SH34
5, φ2cm x 50cm, eluent: 0.01M formic acid-ammonia buffer (pH3.0) 85:15 mixture with acetonitrile, elution rate
High performance liquid chromatography was performed using 5 ml / min) to obtain 10 mg of a purified standard. 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-Z414101 1. Fibroblast proliferation inhibitory effect (activity assay method) Mouse fetal normal fibroblast SC-1 was seeded on a 96-well multiplate at 3,000 cells per well, and 5 0.2 ml Eagle MEM medium containing% fetal bovine serum (Earl's salt, Gibco)
Incubate at 37 ℃ in the atmosphere of 5% CO ₂ balance air overnight, then change the medium to 0.2 ml MEM medium containing 1% fetal bovine serum and continue culturing for another 5 days. WAS-Z414101 purified sample 10 μg / ml
The number of cells in the (medium) addition hole was measured by a Coulter counter (Model ZM, Coulter Electronics Co.).

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.

2. Human adult skin-derived fibroblasts are seeded in a 96-well multiplate at 3000 cells per well (well) containing 5% fetal bovine serum 0.2m
l Eagle MEM medium (Earl's salt, Gibco) 37 ℃, 5
Cultivated overnight under the condition of% CO ₂ balance air, then culture medium 1
The culture was continued for another 5 days by changing to a 0.2 ml MEM medium containing 10% fetal bovine serum, and the number of cells in the well-added 10 μg / ml (medium) purified WAS-Z414101 sample was measured by a Coulter counter (Model ZM Coulter Electronics Co.). .

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.

3. Mouse lymphocytic ascites tumor-derived cells L5178Y were seeded in a 96-well multiplate at 2000 cells per well (well) at 37 ° C in 0.2 ml Eagle MEM medium containing 1% fetal bovine serum (Earl's salt, Gibco), The cells were cultured for 3 days under the condition of 5% CO ₂ balance air, and the number of cells in the wells containing 10 μg / ml (medium) of the purified WAS-Z414101 sample was measured by 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.

4. High-Breast Cancer Mouse (SHN) Breast cancer primary culture cells were seeded in 96-well multi-plate at 1200 cells per well 37 in 0.2 ml Eagle MEM medium containing 5% fetal bovine serum (Earl's salt, Gibco).
° C., 5% CO ₂ balance with conditions overnight culture of air and then continued for another 3 days of culture instead of medium 1% fetal bovine serum-containing 0.2mlMEM medium, WAS-Z414101 purified sample 10 [mu] g / ml (medium) The number of cells in the addition hole was counted by a Coulter counter (Model ZM, Coulter Electronics Co.).

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.

5. In vivo inhibitory effect of WAS-Z414101 on high breast cancer mouse (SHN) in vivo, high breast cancer mouse (SHN, ♀, ref: Hiroshi Nagasawa) with an average body weight of 25 g and no postnatal reproductive reproductive cancer. Reiko Yauchi et al., European Journal of Cancer (Europ.J.Cance
r,) 12: 1017-1019, 1976. Approximately 4 months old) Aseptic water was freely supplied to the control group and WAS to the treatment group for 50 animals per group.
-Group of Z414101 dissolved in sterile water and ingested 2 mg / kg body weight per day by free water supply, and WAS-Z414101 dissolved in physiological saline and administered by intravenous injection of 0.2 mg / kg body weight per day Three groups in total were raised for 2 months after the start of the experiment, and the spontaneous carcinogenic rate was calculated, and the results are shown in Table 1.

6. Effect of WAS-Z414101 on carcinogenic SHN Highly-developed breast cancer mouse (SHN♀, ref: Hiroshi Nagasawa, Reiko Yanai et al. European Journal of Cancer (Europ.J.Cancer)) whose carcinogenesis (mean diameter 5 mm) was confirmed by palpation 1
2: 1017-1019,1976. Approximately 5 months old) Average weight 30g, 20 per group
The control group was given free access to sterile water, and the treatment group was WA.
A group in which S-Z414101 was dissolved in sterile water and ingested 5 mg / kg body weight per day by free water and WAS-Z414101 was dissolved in physiological saline, and 0.5 mg / kg body weight per day was administered by intravenous injection. Three groups in total were raised for 6 months from the start of the experiment, and the diameter and survival rate of the cancer were calculated and the results are shown in Table 2. The numerical values in the upper row of the table show the mean diameter of cancer ± standard error, and the lower row () shows the survival rate (%).

7. ICR mice (male, 6 weeks old, average body weight 30.0 ± 0.3 g, 10 mice in each group) were used, and 1.5 mg dissolved in 0.5 ml of physiological saline was used.
3.0 mg, 4.5 mg, 6.0 mg, 7.5 mg of the present invention WAS in 5 stages
-Z414101 was subcutaneously administered, and its life and death was observed for 7 days.
LD ₅₀ value calculated according to rens-krber method is 152.5 mg / kg
Was a weight mouse.

8. ICR mice (male, 6 weeks old, average body weight 30.0 ± 0.3 g, 10 mice in each group) were used, 6.0 mg dissolved in 0.5 ml of physiological saline,
10.5 mg, 15.0 mg, 19.5 mg, 24.0 mg of the substance of the present invention in 5 stages
WAS-Z414101 was orally administered, and its life and death was observed for 7 days.
LD ₅₀ values were calculated according to hrens-krber method is 522.5mg / k
It was a g-weight mouse.

Formulation Example 1. 100 μg of WAS-Z414101 was dissolved in 1 ml of sterilized physiological saline to prepare an injection.

2. 20 mg of WAS-Z414101 was thoroughly mixed with 980 mg of lactose, and granules were prepared by a wet method using 5% starch paste solution as a binder and tableted into tablets.

As described above, the substance WAS-Z414101 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 5 are explanatory views showing the physicochemical properties of the substance of the present invention. 6 to 8 are explanatory views of Experimental Example I of the present invention. 9 to 12 show Experimental Examples II-1, II-2, II-3 of the present invention.
FIG. 11 is an explanatory diagram of II-4. The length of the vertical line at the top of the graph shows the standard error.

Claims (1)

[Claims] (A) Molecular formula: C ₁₀ H ₁₂ N ₂ O ₃ (b) Melting point: 195 to 200 ° C. (decomposition) (c) Solubility: readily soluble in water. Soluble in acetonitrile and dimethyl sulfoxide. Insoluble in ethyl alcohol, ether, chloroform and benzene. (D) (E) Infrared absorption spectrum (KBr method): As shown in the attached FIG. (F) Nuclear magnetic resonance spectrum of hydrogen (solvent: d ₆ -dimethylsulfoxide, internal standard: tetramethylsilane): as shown in FIG. 3 attached. (G) Nuclear magnetic resonance spectrum of carbon 13 (solvent: d ₆ -dimethylsulfoxide, internal standard: tetramethylsilane):
It is as shown in the attached FIG. WAS-Z414101, which is a cell growth inhibitor.