JPS63119500A - Sulfated polysaccharide ds 4152 and vascularization inhibitor and antitumor agent containing the same - Google Patents

Abstract

NEW MATERIAL:A sulfated polysaccharide DS 4152 having the following physical and chemical properties. Molecular weight, 29,000+ or -3,000; elemental analysis (%), C 24.42-25.76, H 3.34-3.98, N 0.51-0.89, S 10.6-11.7, P 0.77-1.06; sugar content, 57+ or -3; protein content, 1+ or -0.5; specific rotation, [alpha]D<25>=-37+ or -1 deg. (0.5% aqueous solution); main IR absorption band, 1,240, 840 (shoulder), 810 (cm<-1>; KBr); solubility, easily soluble in water and almost insoluble in organic solvents such as ether, benzene, chloroform, methanol, ethanol, etc.; pH, 6-8 (3% aqueous solution); etc. USE:A vascularization inhibitor and antitumor agent. The activity can be promoted when combined with a steroid drug. PREPARATION:For example, pyrogenic substance, etc., having a molecular weight of >=15X10<4> are removed by a proper molecular weight fractionation method from DF 4639 separated from a cultured product of Arthrobacter sp. AT (FERM P-5255).

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a novel sulfated polysaccharide DS 4152, an angiogenesis inhibitor and an antitumor agent containing this as an active ingredient, and a steroid agent containing this as an active ingredient. The present invention relates to an angiogenesis inhibitor and an antitumor agent.

[Conventional technology and its problems]

It has been known that the sulfated polysaccharide DF 4639, which has fibrinolysis-inducing, infection-protecting, and interferon-inducing effects, exists in the fermentation products of Micrococcus ap and AT-25 (Japanese Patent Laid-Open No. 56-67301). issue,
JP-A-57-42627 and JP-A-59-2532
No. 9).

[ Means for Solving the Problem] Therefore, in order to remove this pyrogenic substance, the present inventors
Further research revealed that DF 4639 is a mixture of several components, of which DS 4
It has been discovered that one component named 152 is non-pyrogenic and has excellent anti-angiogenic and anti-tumor effects.

Furthermore, the present inventors have found that the combination of DS 4152 and a steroid agent synergistically enhances the angiogenesis inhibitory effect and antitumor effect.

The present invention is based on the above findings, and its purpose is to:
A novel sulfated polysaccharide DS 4152 is provided.

In addition, another object of the present invention is to obtain sulfated polysaccharide DS 415
The object of the present invention is to provide an angiogenesis inhibitor and an antitumor agent containing 2 as an active ingredient.

Furthermore, another object of the present invention is the sulfated polysaccharide DS 415
The object of the present invention is to provide an angiogenesis inhibitor and an antitumor agent containing 2 and a steroid agent as active ingredients.

In this specification, "suppression of angiogenesis" is not only extremely important for embryonic development, lutein formation, wound healing, etc., but also for chronic inflammation including rheumatoid arthritis.

It refers to weakening the angiogenic effect of blood vessels, which is deeply involved in the progression of pathological conditions such as immune response and tumor growth. Therefore, angiogenesis inhibitors can be used to treat various diseases involving the above-mentioned angiogenic effects, such as rheumatoid arthritis,
It is useful for the treatment and prevention of proliferative retinitis, retinopathy, diabetic retinitis, retinopathy of prematurity, etc. In particular, it is said that tumors promote strong angiogenesis, and the blood supplied by the newly generated blood vessels further promotes the growth and development of knee tumors, so it is also effective as an antitumor agent.

The sulfated polysaccharide DS 4152 of the present invention is Arthrobacter sp, AT 25 (Mierococeog sp.

As Person T-25, FERM P-5255 and Art
hrobaetor sp, FBRM as AT-25
DF 4639 (deposited under the number BP-1357) isolated from a culture of
), the molecular weight contained therein is approximately L 5
It can be obtained by removing pyrogenic substances of L O' or higher by an appropriate molecular weight fractionation method, such as gel filtration, ultrafiltration, or alcohol precipitation.

That is, in terms of gel filtration properties, DF 4639 can be mixed with a suitable gel filtration carrier, such as Sephacryl [5ephacr
yl S-300 (manufactured by Pharmacia)], and the resulting fraction was subjected to high-speed gel filtration chromatography (Toyo Soda If! G3000).
SW column) was carried out, and a fraction (H fraction) showing a peak at the exclusion limit (moid volume) was eluted with a molecular weight range of approximately 2XLO' to 8X10' without a peak at moid volume. Each fraction (L fraction) is collected and dialyzed.

In addition, ultrafiltration can be performed using a suitable membrane (for example, YMIO% YM30, XM50, PM manufactured by Arnlcon).
30 and Pjltron's N0VA100, O
MEGAloo.

N0VA50, OMgGA50 etc. especially yMto)
Pressure with nitrogen gas or slip starch (p
eristallc) Pressurized by e-amp (Q5~
5 kg/22) and collect the permeate as DS 4152. The solvent used was water-ethanol (10
:2-3) or water is suitable, and it is generally carried out at 4°C to room temperature.

Each of the resulting dialyzed fluids was concentrated and filtered, and the filtrate was poured into several times the amount of ethanol under stirring to collect the white precipitate that was generated. After washing with 90% ethanol, ethanol, and acetone in this order, If dried under reduced pressure, the desired DS 4152
(L fraction) and a pyrogen (H fraction) are obtained, respectively.

Da 4152 thus obtained exhibits the physicochemical properties described below. The physical properties below are for its sodium salt.

(1) Molecular weight (according to the past) 29,000±
aoo.

(2) Elemental analysis value (indicating width of 50 pieces) C24,
42~2376% H3, 34~398%WQ51~0
89% 8 106~117%P 077~1.06
% (3) Sugar and protein content Sugar content (%): 57 ± 3 (phenol-sulfuric acid method, galactose standard) Protein content (%): 1 ± 05 (Lori-Foulin method, bovine serum albumin standard) (4) Specific optical rotation [α-37±1 (05% aqueous solution) (5) Main absorption band 124 in infrared absorption spectrum
o, 840 (shoulder), 810 (clL-'; KB
r ) (6) Soluble in water. Virtually insoluble in organic solvents such as ether, benzene, chloroform, methanol, and ethanol.

(7) Color reactions: phenol-sulfuric acid, anthrone-sulfuric acid, Buret reaction, and Lowry-Foulin reaction were positive. The Erlin-Morgan reaction and Ninhirin reaction of the aqueous solution were also positive. Carbazole reaction and Sakaguchi reaction were negative.

(8) Basic, neutral, acidic pH 6-8 (3% concentration aqueous solution) (9) Content of constituent sugars, sulfate groups, and phosphorus D-glucose, D-galactose, 5o3N Hatake, and P (phosphorus) content The molar ratio is approximately 10 each with D-glucose being 10.
:61 near 3:6.

(To) Analysis of the constituent amino acids and amino sugar acid hydrolysates using an amino acid analyzer revealed the presence of alanine, glycine, glutamic acid, cyamopimelic acid, glucosamine, and muramic acid.

As shown in the examples below, the above-mentioned DS 4152 has an angiogenesis inhibitory effect even when used alone, but it exhibits an even more excellent angiogenesis inhibitory effect when combined with a steroid agent.

In addition, in the angiogenesis inhibitor of the present invention, DS 41
Instead of 52, it is also possible to use, Q-phosphorus, low-molecular-weight compound, tilJ-phosphorus, etc.

Previously, it has been reported that steroid hormones such as 12"rednisolone%6α-methylprednisolone and dexamethasone have the effect of suppressing experimentally induced angiogenesis in the chick embryo chorioallantoic membrane, rabbit cornea, and hamster thread. There is (
Cancer, Ram, 39 1305 (1979)
J, Natl.

Cancer In 5t, 57 789 (1978)
and Proe.

Natl, Acad, Sci, US 78 117
6 (1981)).

Among steroid hormones, glucocorticoid F'
(7'rednisolone, sorednisone, betamethasone, etc.)
is used to treat leukemia, malignant lymphoma, breast cancer, and prostate cancer.

Furthermore, male hormones containing anthrostane, such as testosterone propionate and fluoxymesterone, have been used as agents for pilehole gland tumors.
It has been reported that an efficacy rate of % can be obtained (Oncol
ogia L 072 (1984)).

Furthermore, derivatives of progesterone, derivatives of testosterone, and nistrodiene agents have been used in the treatment of prostate cancer.

Steroids that can be used in combination with the above-mentioned DS 4152 include glucocorticoids, progestins, etran and anthrostane, and more specifically, the following are exemplified.

(1) Steroid hormone whose core is 7° regnan,
That is, glucocorticoids, such as corchicin and its derivatives (acetate, enanthate, randesylate, etc.); hydrocortiscin and its derivatives (acetate, hemisuccinate, caproate, etc.);
Zolednisone and its derivatives:':f Rednisolone and its derivatives (acetate, hemisuccinate, phosphate, butyl acetate, tetrahydrophthalate, trimethyl acetate, etc.); Methylprednisolone and its derivatives (acetate, hemisuccinate, etc.)
; Examples include betamethasone and its derivatives (phosphate, valerate, etc.).

Also included are isomers in which the hydroxyl group at the C-11 position of glucocorticoids is in the α configuration (for example, 11α-epihydrococcicin), and tetrano-1 hydrometabolites of the glucocorticoids (the presence or absence of glucocorticoid activity is (not related) is also included.

Further examples include progesterone proposterone, metroxyprogesterone and its derivatives (acetate, etc.), ditrogstrone and its 17α-acetoxy derivative (duphaston), and the like.

Furthermore, mineralocorticoids such as aldosterone, denxycorticosterone, and derivatives thereof (acetate, trimethyl acetate, enanthate, phenylpropionate, etc.) are also mentioned.

(2) Steroid hormones having anthrostane as their core, that is, male hormones, such as b-androsterone, testosterone, and derivatives thereof (propionate, enanthate, butyrate, caprylate, etc.). Also included are evithiostanol and its derivatives, mypiteostane.

Further included are fluoxymesterone and its derivatives, methyltestrone and its derivatives, and stanolone and its derivatives.

(3) Steroid hormones with estrone as their core, that is, follicular hormones, such as estrone and its derivatives, estradiol and its derivatives (benzoate, zozologionate, valerate, undecenoate, etc.), est IJol and its derivatives ( tripropionate, etc.).

The dosage form of the angiogenesis inhibitor of the present invention includes various forms containing the active ingredient and a medically acceptable carrier and excipient.
Examples include solutions dissolved in water or various infusion preparations, powders, granules, tablets, injections, suppositories, and the like.

When the angiogenesis inhibitor of the present invention contains DS 4152 and a steroid agent, these may be prepared separately into a single drug of the above dosage form to form a combination drug, or a combination drug containing both components may be prepared. It can also be formulated into a drug.

The angiogenesis inhibitor of the present invention can be administered intravenously, intraarterially, orally, subcutaneously, rectally, intramucosally, or locally at the affected area. The dosage is an adult oral daily dose of DS 4
152 is about 1 to 2000 Ing, and steroids are androgen and glucocorticoids are about 10 to 2000 Ing.
10oOWLg, usually 30-607@ is appropriate, and it may be preferable to gradually decrease. For progesterone agents, 100 to 1200+9 is appropriate. For administration by injection, an oral dose of 115 is usually appropriate.

Furthermore, when the angiogenesis inhibitor of the present invention is used as an antitumor agent, the administration method and dosage are essentially the same as above.

〔Effect of the invention〕

DS 4152 of the present invention exhibits an angiogenesis inhibitory effect even when used alone, but when it is further combined with a steroid agent, it exhibits an even more excellent angiogenesis inhibitory effect.

Therefore, DS 4152 alone is useful as an angiogenesis inhibitor, but when combined with a steroid agent, its action is synergistically enhanced, so it can be used, for example, to inhibit tumor angiogenesis and cancer growth. It is particularly useful as an antiangiogenic agent.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 (A) DF 4639 (ao y ) obtained by the method described in JP-A No. 58-67301 was subjected to 15-Q I M
A column (Sephacryl S-300; aox
socIIL) and eluted with the same solvent, 18tif
The eluate was collected. The obtained fraction was subjected to high-speed glue chromatography (Toyo Soda G30).
0o sw column, solvent 0.1 M potassium acetate buffer pH 6,5) to remove voids, l? Does not give a peak at +7 um, molecular weight (dextran standard) is approximately 2X
The 7-lactone eluting in the 10' to 8 x 10' range was collected (approximately 700 yd) and dialyzed against deionized water. The dialyzed fluid was concentrated to about 50 m and then filtered. The filtrate was poured into approximately 400,111 ethanol with stirring, the resulting precipitate was collected, washed with 90% ethanol, ethanol, and acetone in this order, and then dried under reduced pressure (50°C).
, 6 hours) and the target DS 4152 white powder 38
? I got it.

On the other hand, the gate and
Collect the frac/yon that gives the peak volume (approximately 9
0yLl) and treated in the same manner as in the case of DS4152 described above to obtain als with the H fraction as a slightly yellow powder.

The physicochemical and biological properties of DS 4152 are shown in comparison with DF 4639 and its H fraction.

(,) Sugar, protein, S and P content (Table 1) Table 1 1) Phenol-sulfuric acid method (galactose conversion) 2) Antono? Russ's method (C, A, Antonopoulos
.

Acta Chem, 5cand, 16, 1521
(1962) ) 3) Lowry-7-Olin method (bovine serum albumin conversion) 4
) Cheno et al.'s method (P, S, Chen et aL,...
Anal, Ch@m.

28.1758 (1956)).

(b) Constituent molar ratio of galactose, glucose, sulfate group, and phosphorus The sample was hydrolyzed with LOO''C in 1N sulfuric acid for 5 hours, desalted with an ion exchange resin, and then converted to arzotol acetate using a gas chromatograph using a conventional method. The molar ratio of sulfate groups and phosphorus was calculated from the S and P contents (%).

Table 2 Table 2 shows an example of the molar ratio of each component when glucose is 1.0 mol.

(c) Identification of constituent amino acids and amino sugars DS 415
After hydrolyzing 2 in 3N hydrochloric acid at 100°C for 16 hours,
As a result of analysis using an amino acid analyzer according to a conventional method, peaks of alanine, glycine, glutamic acid, cyamopimelic acid, glucosamine, and muramic acid were observed.

(d) Specific rotation: [α)'', (e = 0.5
, water) Table 3 (,) Glu filtration elution Q-turn Figures 1, 2 and 3 show DS 415, respectively.
2. High-speed gel filtration chromatograms of DF 4639 and 3 fractions are shown (using Toyo Soda G3000 sw column, solvent QIM potassium acetate buffer pH 5, a
9d/min, standards dextran T-10 and T-4
0).

(f) Ultraviolet absorption spectrum 2 m9/d 220-340 nm in aqueous solution
No maximum absorption Fi is observed.

(g) Infrared absorption spectrum (KBr tablet) 1240
．． 840 (shoulder) and 810 cm-' show absorption characteristic of sulfated polysaccharides.

The structure of DS4152 is estimated to be a sulfated polysaccharide in which a pezotidoglycan moiety is bonded to a carbohydrate moiety mainly consisting of D-galactose and D-glucose via muramic acid phosphate.

(h) Pyrogenicity test The results of the pyrogenicity test conducted according to the Japanese Pharmacopoeia (10th revision) are shown in Table 4.

Margin below (i) Acute toxicity of DS 4152 (mouse, intravenous injection)
The LD and o are over 2000m9/kp.

Example 1 (B) DF 4639 (ao t ) was dissolved in 300-water-ethanol/ethanol (10:3) solvent and added to YMIO membrane (4x,
Ultrafiltration was carried out at room temperature under nitrogen pressure ('L51C9/c!IL2). The above solvent was added until the amount of permeate reached about 3 tons. 1 to the concentrated solution of permeate (approx. 50+u/)
After adding and dissolving 001119 in sodium acetate, the supernatant obtained by centrifugation was dropped into approximately 500 m/ml of ethanol under stirring. The generated precipitate was collected, washed sequentially with 90% ethanol, ethanol, and acetone, and then dried under reduced pressure (55°C, 5 hours) to obtain DS 4152 white powder 33F.

The physicochemical properties of this product were the same as DS 4152 of Example 1(A) except for the following sugar, protein, S and P contents.

Sugar content: 58% S content: 11.3% Protein content: 09% P content: 0.92% The high-speed Glue f perchromatogram is shown in Figure 4 (G300
0 SW column, QIM sodium acetate buffer (pa
a5), Q8iE//min).

Example 2 Chicken embryo chorioallantoic angiogenesis inhibition test (direct method): Using chicken embryos,
Tiller and Folkman (Nature 297: 307. (1982)
The following method was used, which is a partial improvement of the method described in ).

1 in the chorioallantoic membrane of a 4- to 5-day-old fertilized egg of a chicken (Norhynchus)
DS 41152 or He/9 phosphorus dissolved in physiological saline was added and cultured at 37°C.

Two days after the addition of the drug, the development of chorioallantoic blood vessels was compared with a control to which only physiological saline was added, and the probit method was used to evaluate the development of chorioallantoic vessels.
The % angiogenesis inhibition amount (ID5° value) was calculated.

As a result, the ID of the DS 4152 of the present invention. The value is 1
It was 60nf. On the other hand, He, Q-Rin is 100
μ? But it showed no effect.

Example 3 Chicken embryo chorioallantoic angiogenesis inhibition test (direct method): In the same manner as in Example 2, the effect of using steroids and DS 4152 in combination was investigated.

As the steroid, cortiscine acetate was used at 05 μf/chicken embryo IT (an amount that does not affect angiogenesis). For comparison, the activities of DF 4639 and H fractions were also investigated. The results are shown in Table 5.

Table 5 50% angiogenesis inhibition dose (ID, value) Example 4 In the same manner as in Example 2, various steroids and DS 41
ID by combination of 52. Changes in values were examined. As a result, 10n2 of DS 4152 in various steroids
It was revealed that the inhibitory activity of chicken embryo chorioallantoic membrane angiogenesis increases 16 to 100 times by adding
table).

Table 6 Example 5 Angiogenesis inhibitory effect (・XvIvo method): DS 4152
was dissolved in physiological saline and administered subcutaneously or orally to ICR male mice, and blood was collected 6 hours later. 03
Coagulation was inhibited with 13% sodium citrate and added to the chorioallantoic membrane of 5-day-old fertilized chicken eggs in the same manner as in the direct method, and the results were evaluated 2 days later. The results are shown in Table 7.

Table 7 As is clear from the results, a dose-dependent angiogenesis inhibitory effect was observed.

Example 6 Angiogenesis inhibitory effect (ex vivo method): In the same manner as in Example 5, the effect of using steroids and DS 4152 in combination was investigated.

As a steroid, cortiscine acetate was used at a rate of 5 Eg/Yu, and DS 4152 was used at a rate of 9 or 300 Eg/301 Eg/Yu.
■／I adjusted it and added it so that it was clear. Also, for comparison, D
r4639 and H fractions were used. The results are shown in Table 8. The values in the table are the inhibition rates when the degree of development of chorioallantoic blood vessels to which blood collected from control mice to which the same amount of physiological saline was administered is taken as 100%.

Table 8 DS 4152 and DF' 4639 were found to inhibit chorioallantoic neovascularization by both oral and subcutaneous routes.

Example 7 Angiogenesis inhibitory effect (ax vivo method): DS 4152 dissolved in physiological saline was orally administered to ICR male mice. Steroids were administered orally or intramuscularly, either with DS 4152 or alone, suspended in saline.

Blood was collected 6 hours after administration, coagulation was inhibited with 0313% sodium citrate, and this was added to the chorioallantoic membrane of a 5-day-old fertilized egg in the same manner as the direct method, and the effect on angiogenesis was determined 2 days later. The results showed that mice administered only the same amount of saline,
The degree of development of chorioallantoic vessels when blood was added after 6 hours was used as a control, and was expressed as a percentage inhibition.

The results are shown in Table 9.

Below are blank spaces Example 8 Anti-tumor test: Syngeneic ovarian-derived ascites tumor M5 in C57BL/6 male mice
076 was inoculated subcutaneously in t x t o' doses, and on the 5th day,
When S 4152 was subcutaneously administered at 30 Wg/day, 6 times a week, a remarkable antitumor effect and a significant prolongation of survival were observed. That is, as shown in Table 10, the average tumor weight on the 21st day after transplantation was 37% (63% inhibition) of the control group, and the median survival period was 933% longer than that of the control group.

The average tumor weight was determined by measuring the lengths of the major and minor axes of the tumor mass and using the following formula.

Average tumor weight = (long axis) x (short axis) 2 x - Example 9 Antitumor test: ICR male mouse (5 weeks old) K: Sarcoma 180 (8
180) was subcutaneously inoculated in t x t o' pieces, and from the 3rd day, a suspension of cortiscine acetate in physiological saline was injected at 25 Or! tg/
The drug was administered at a rate of 1 w/day for 3 days and at a rate of 100 μ/kIF/day for 1 day.

DS 4152 was dissolved in physiological saline and administered subcutaneously or orally once a day for 4 days at a dose of Q61 or 6.1 μ/mouse. The animals were sacrificed on the 7th day of transplantation and the tumor weight was compared with the control. As shown in Table 11, there was no difference in tumor weight in the group to which only cortiscine acetate was administered compared to the group to which DS 4152 was administered, but in addition DS 4152 was administered. By doing so, a remarkable growth inhibition effect was obtained, which was 6.9 to 175% of the tumor weight in the control group.

Table 11 * P < Q O5s Hayashi P < QOL Student's test Example 1゜ Granules: DS41326Wt9, lactose 300Rg, corn starch 144rRg, calgeximethyl cellulose calcium 30R9 and hydroxyzorobyl cellulose 2
Using 0Rg, 500 square granules were prepared according to a conventional method. This granule is 500Rg to 5f per day depending on the symptoms.
to take.

Example 11 Injection: 12 mg of DS 4152 and 90 ml of sodium chloride were dissolved in distilled water for injection to make 10 d.

After filtering this solution with a membrane filter, it is filled into ampoules and sterilized at 115°C for 30 minutes to prepare an injection Example 12 Tablets: DS 4152 6m9, Prednisolone 201IIg
, lactose 5o#Ig, tou vo:rsidende 71 a
5'Rg, Cal? oxymethylcellulose calcium 51
1 g, hydroxyzorobyl cellulose 3TRg, and magnesium stearate 05x are mixed and tableted according to a conventional method to make one tablet.

[Brief explanation of the drawing]

Figures 1 to 4 show high-speed Dal r passing Romato Dalam. Figure 1 Figure 2 Elution time (minutes)

Claims (1)

[Claims] 1. Sulfated polysaccharide DS4152 having the following physicochemical properties as a sodium salt. (1) Molecular weight (by gel filtration method) 29,000±3,000 (2) Elemental analysis value C24.42-25.76%H3.34-3.98%N
0.51-0.89%S10.6-11.7%P0.7
7-1.06% (3) Sugar and protein content Sugar content (%): 57 ± 3 (phenol-sulfuric acid method, galactose standard) Protein content (%): 1 ± 0.5 (Lowrie-Follin method,
Bovine serum albumin standard) (4) Specific rotation [α] ^2^5_D-37°±1° (0.5% aqueous solution)
(5) Main absorption band 124 in infrared absorption spectrum
0, 840 (shoulder), 810 (cm^-^1; KBr) (
6) Solubility Easily soluble in water. Virtually insoluble in organic solvents such as ether, benzene, chloroform, methanol, and ethanol. (7) Color reaction phenol-sulfuric acid, anthrone-sulfuric acid, Buret reaction, and Lowry-Foulin reaction were positive. The Elson-Morgan reaction and ninhydrin reaction of the hydrolysis solution were also positive. Carbazole reaction and Sakaguchi reaction were negative. (8) Basic, neutral, acidic pH 6-8 (3% concentration aqueous solution) (9) Content of constituent sugars, sulfate groups, and phosphorus D-glucose, D-galactose, SO_3Na, and P (phosphorus) content molar content The ratio is approximately 10:61:73:6, with D-glucose being 10. (10) Analysis of the constituent amino acids and amino sugar acid hydrolyzate using an amino acid analyzer reveals the presence of alanine, glycine, glutamic acid, diaminopimelic acid, glucosamine, and muramic acid. 2. An angiogenesis inhibitor containing sulfated polysaccharide DS4152 as an active ingredient. 3. The angiogenesis inhibitor according to claim 2, which is effective for rheumatoid arthritis, proliferative retinitis, psoriasis, diabetic retinitis, and retinopathy of prematurity. 4. An antitumor agent containing sulfated polysaccharide DS4152 as an active ingredient. 5. An angiogenesis inhibitor containing sulfated polysaccharide DS4152 and a steroid agent as active ingredients. 6. The angiogenesis inhibitor according to claim 5, wherein the steroid is selected from glucocorticoids, progestins, etran and androstanes. 7. The angiogenesis inhibitor according to claim 5, which is effective for rheumatoid arthritis, proliferative retinitis, psoriasis, diabetic retinitis, and retinopathy of prematurity. 8. An antitumor agent containing sulfated polysaccharide DS4152 and a steroid agent as active ingredients.