JPH0573760B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for producing antitumor active substances. The present inventor has discovered that although the crude extract from cartilage tissue does not exhibit antitumor activity as it is, it was extracted with an aqueous solvent and then precipitated with a hydrophilic organic solvent.
After separating 300,000 fractions, they found that they exhibited antitumor activity, and based on this discovery, they completed the present invention. That is, this substance contains somatomedin-like growth factor CDF from the above crude extract.
(Cartilage-Derived Factor) is extracted and purified from the residue separated. Fetal cartilage, particularly fetal bovine cartilage, is preferred because animal cartilage used as a starting material in the present invention is available in large quantities. The target substance of the present invention is soluble in water and sparingly soluble in hydrophilic organic solvents, but in order to extract it from the starting material, it is dissolved in an aqueous guanidine salt solution with a pH of about 5 to 7, and then acetone, What is necessary is to add a hydrophilic organic solvent such as ethanol to re-precipitate and fractionate. For example, a membrane fractionation method may be used to separate a fraction with a molecular weight of 20,000 to 300,000. Furthermore, the target substance of the present invention has vascular endothelial cell proliferation inhibitory activity. The vascular endothelial cell proliferation inhibitory activity may be measured using any known measuring means, but in the present invention it was carried out by the method described below. Although it has not been confirmed whether the vascular endothelial cell growth inhibiting active ingredient and the antitumor active ingredient are the same, fractions obtained by the above treatment means and having vascular endothelial cell growth inhibiting activity, such as Samples D, F and Since G has antitumor activity, it is the target substance of the present invention. Specifically, for example, fetal bovine cartilage is sliced, homogenized in an aqueous solvent such as a salt solution, fractionated with acetone (approximately 45 to 65% precipitation), and the resulting precipitate is redissolved in the above-mentioned salt solution. Fraction (molecular weight 2
~300,000), but it is also possible to separate substances that have vascular endothelial cell growth inhibiting activity. Or even diethylaminoethyl (DEAE)
After adsorption on agarose (PH7-9), elution may be performed with saline (0.25-0.6M). If desired, it can be purified by conventional protein purification methods such as dialysis, freeze-dried, and stored. The lyophilized product thus obtained is a pale yellow powder that is soluble in water. The pH value of the aqueous solution is 6-7
It is. When the substance obtained according to the present invention is used as an anticancer agent, it may be administered orally as it is or together with a suitable carrier, or dissolved in physiological saline and administered by injection. The product of the present invention has few side effects because it is derived from a living body, and since it uses animal cartilage, which is available in large quantities, as a starting material, it is expected to be useful as an anticancer agent after further purification if necessary. Hereinafter, the present invention will be explained in detail with reference to Examples. Example: Measurement of vascular endothelial cell proliferation inhibitory activity Vascular endothelial cells, such as bovine pulmonary artery endothelial cells, were cultured using a conventional method, such as 20% of 0.1 ml of 5×10 ³ cells per well in a 96-well multi-culture plate with a diameter of 6 mm. Minimum essential culture medium containing fetal bovine serum
Suspend and seed in 5%
The cells were cultured at 37°C under carbon dioxide aeration. After 3 days of culture, the medium was replaced, and the sample solution (sample dissolved in a balanced salt solution) or the balanced salt solution (control) used to dissolve the sample was added, and 22 hours later,
³ H-thymidine (final concentration 1 μC/ml) was added, and after another 2 hours, ^{the 3} H radioactivity incorporated into the DNA fraction was measured and determined as "DNA synthesis." sample and control
From the DNA synthesis value, determine the inhibition rate of the sample using the following formula. An example is shown in Figure 1. DNA synthesis inhibition rate (%) = (1 - DNA synthesis of sample / DNA synthesis of control) × 100 ○: Sample G, △: Sample F Activate the sample that shows an inhibition rate of 50% when added to 1 ml of medium. It is considered as 1 unit. In addition, the culture plate diameter was 16 mm, and 2×
FIG. 2 shows the inhibitory effect of sample D (described later) on the number of proliferating cells when 10 ⁴ cells were seeded and cultured in the same manner for 1 to 2 weeks with medium exchange performed every other day. ●: Addition of sample D 200 μg/ml (final concentration) ○: Control example (1) Slice 200 g of bovine fetal cartilage, add 1M guanidine hydrochloride - 0.1M 6-amino-n-caproic acid (PH
6.0) homogenized using a polytron. After stirring for 48 hours at 4°C, centrifuge at 8000 rpm for 20 minutes (4°C) and add 45% acetone to the supernatant.
(final concentration) and left at 0°C for 20 minutes. Next, the supernatant was centrifuged at 8000 rpm for 20 minutes (4°C), and acetone was further added to the resulting supernatant to give a final concentration of 65%.
°C) and centrifuged. The obtained precipitate was dissolved in 50 ml of distilled water, and dialyzed against distilled water at 4° C. for 48 hours. After freeze-drying, 4 g of an acetone fraction (sample A) was obtained. Dissolve 3g of sample A in 200ml of 4M guanidine hydrochloric acid, 0.01M ethylenediaminetetraacetic acid (EDTA), and 0.1M 6-amino-n-caproic acid (PH6.5),
Centrifuge at 8000 rpm for 20 minutes (4°C), and ^transfer the resulting supernatant to Amicon's molecular weight 30
The ultrafiltrate was obtained by passing it through a million-cut ultrafiltration membrane "XM-300". Furthermore, under pressure of 2.0Kg/ ^cm2 ,
An internal solution (molecular weight fraction of 300,000 to 20,000) (sample B) and an ultrafiltrate (sample C) were obtained through an ultrafiltration membrane "UP20" with a molecular weight of 20,000 cut manufactured by Toyo Roshi Co., Ltd.
Sample B was similarly dialyzed and freeze-dried to obtain 266 mg of sample B dried product. After subcutaneously transplanting Sarcomer 180 tumor cells into ICR female mice (5 weeks old) to make them tumor-bearing, 2 mg each of sample A and B dried products were dissolved in physiological saline, and as a control group, only physiological saline was used. was subcutaneously administered twice, and the tumor volume was measured from the short and long axes of the tumor 5 weeks after tumor implantation, and the tumor inhibition rate (IR) was calculated using the following formula. The results are shown in Table 1. IR = [Tumor volume of control group] - [Tumor volume of sample administration group] / [Tumor volume of control group] × 100% As shown in Table 1, sample A did not show antitumor activity, whereas sample A did not exhibit antitumor activity. Sample B purified thereby showed antitumor activity. On the other hand, somatomedin activity for samples B and C (Y, Kato, Y. Nomura, M. Tsuji, H.
Ohmae, M. Kinoshita, S. Hamamoto and F.
Suzuki “Cartilage−Derived Factor (CDF). Somato medin−Like Action on Cultured
Chodrocytes”Exp Cell Res.vol.132 P339−347
(1981)), activity was observed only in sample C.

[Table] (2) 150g of bovine fetal cartilage and 0.1M6 of 1M guanidine hydrochloride
-Amino-n-caproic acid (PH6.0) 1.5 and homogenized in a mixer. After stirring for 48 hours at 4°C, centrifuge at 8000 rpm for 20 minutes (4°C), add acetone to the resulting supernatant at a final concentration of 45%, and centrifuge at 0°C.
I left it for 20 minutes. The mixture was then centrifuged at 8000 rpm for 20 minutes (4°C), and acetone was further added to the resulting supernatant to give a final concentration of 65%, and the mixture was left at 0°C for 20 minutes. 8000rpm20
After centrifugation for 4°C, the resulting precipitate was dissolved in 50 ml of distilled water, dialyzed against distilled water at 4°C for 48 hours, and then freeze-dried. This was mixed with 200ml of 4M guanidine hydrochloride, 0.01M EDTA, 0.1M 6-amino-n-
Dissolved in caproic acid (PH6.5), passed through ultrafiltration membrane "XM-300" under pressure of 1.0Kg/ ^cm2 , and ultrafiltrated the obtained ultrafiltrate under pressure of 1.5Kg/ ^cm2 . The internal solution was collected through the membrane "UP20", dialyzed against distilled water in the same manner, and then freeze-dried to obtain 172 mg of powder (sample D). Contains 64.5% protein (by phorin method) by weight. After subcutaneously transplanting Sarcomer 180 tumor cells into ICR female mice (5 weeks old) and making them tumor-bearing,
D2mg dissolved in physiological saline and as a control group, physiological saline alone was subcutaneously administered 4 times,
The tumor volume was measured 5 weeks after tumor implantation to determine the tumor inhibition rate, and the results are shown in Table 2.

[Table] From Table 2, sample D showed remarkable antitumor activity.
As a result, it was understood that the tumors had completely regressed in 3 out of 7 animals. (3) Bovine fetal cartilage 350g 1M guanidine hydrochloride 0.1M,
It was homogenized in a mixer in 6-amino-n-caproic acid (PH6.0) 3.5. 4℃ below 48
After stirring for an hour, centrifuge at 8000 rpm for 20 minutes (4°C) and add acetone to the resulting supernatant to a final concentration of 65.
% and left at 0°C for 20 minutes. After centrifugation at 8000 rpm for 20 minutes (4°C), the resulting precipitate was dissolved in 50 ml of distilled water, dialyzed against distilled water at 4°C for 48 hours, and then freeze-dried. This was dissolved in 200ml of 4M guanidine hydrochloric acid, 0.01M EDTA, 0.1M 6-amino-n-caproic acid (PH6.5) under a pressure of 1.0Kg/ ^cm2 .
The obtained ultrafiltrate is passed through the ultrafiltration membrane "UP20" under pressure of 1.5Kg/cm ² to collect the internal fluid, which is then filtered to 1Kg/cm2. ² Under pressure, ultrafiltration membrane “Spectrofilter UF(A) (manufactured by Spectropore; molecular weight
100,000 cuts) to collect the internal fluid, then similarly dialyzed against distilled water and freeze-dried.
270 mg of sample E was obtained, dissolved in 30 ml of 10 mM sodium phosphate buffer (PH8.0), and insoluble matter was removed by centrifugation to obtain sample F (138 mg of protein). This was applied to a “DEAE Sepharose CL-6B” (DEAE agarose; manufactured by Pharmacia) column (diameter 1.5
cm Fractions were collected (1 fraction 13 ml). The elution pattern (absorbance at 280 nm, vascular endothelial cell proliferation inhibitory activity) is shown in FIG. Fraction number 19
~21 (sodium chloride 0.26-0.33M elution fraction)
A fraction with vascular endothelial cell proliferation inhibitory activity was collected (activity recovery rate 103%, specific activity 6400 units/
mg, protein, ×70.4 times purified). This was designated as sample G. After transplanting B16 melanoma tumor cells (3 × 10 ⁵ cells) into the soles of C57BL female mice and making them tumor-bearing,
Sample E (0.5 mg) dissolved in physiological saline,
As a control group, only physiological saline was subcutaneously administered 6 times, and the tumor volume was measured 6 weeks after tumor implantation to determine the tumor inhibition rate. The results are shown in Table 3.

[Table] It is understood from Table 3 that Sample E shows significant antitumor activity. B16 melanoma tumor cells (3 × 10 ⁵ cells) were transplanted into the soles of C57BL female mice to make them tumor-bearing.
F2 30 μg protein dissolved in physiological saline, sample G 10 μg protein dissolved in physiological saline,
As a control group, physiological saline alone was subcutaneously administered six times, and the tumor volume was measured 2.5 weeks after tumor implantation to determine the tumor inhibition rate. The results are shown in Table 4.

[Table] From Table 4, it is understood that Samples E and G exhibit remarkable antitumor activity. The vascular endothelial cell proliferation inhibitory activity in a single dose of Samples F and G was 21 units and 65 units, respectively.

[Brief explanation of drawings]

Figure 1 shows an example of DNA synthesis inhibition rate measurement, Figure 2 shows the vascular endothelial cell proliferation inhibitory activity of sample D, and Figure 3 shows the measurement of the inhibition rate of DNA synthesis.
The figure shows the DEAE agarose column elution pattern (absorbance at 280 nm, vascular endothelial cell proliferation inhibitory activity) and the sodium chloride concentration of the sodium chloride eluate.

Claims (1)

[Claims] 1. Animal cartilage is extracted with an aqueous solvent, a hydrophilic solvent is added to this extract to precipitate a precipitate, and a fraction with a molecular weight of 20,000 to 300,000 is separated from the precipitate using an ultrafiltration method. A method for producing a characteristic antitumor substance.