JPS59176213A - Control for intervascular formation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the inhibition of angiogenesis, and more particularly to the inhibition of angiogenesis in which mammals are treated with heparin or heparin fragments and 11-alpha of corchicin, hydrocortiscin or hydrocortiscin.
Treatment with isomer inhibits angiogenesis and anti-tumor animals (7)
Concerning achieving regression of tumor masses and prevention of tumor metastasis.

Angiogenesis, the growth of new capillaries, is integral to normal processes such as embryonic development, corpus luteum formation, and wound healing.

However, it is also involved in pathological processes such as chronic inflammation, certain comb immune responses, and tumor growth. Moreover, angiogenesis is a characteristic of most solid tumors and is essential for their continued growth; heparin in ViVO enhances tumor-induced angiogenesis, but tumor cells or tumor extracts It is known that in its absence, neither heparin nor the mast cells that release heparin induce angiogenesis (Taytor & Folkllan).

Nature, 297: 307-612, 19
82).

In addition, 6α-methylprednisolone partially suppresses tumor angiogenesis in hamster cheeks under persimmon conditions;
Shu't)ik et al. (
J, Natl, cancer, l'net,, 5
7:769-774.1976).

There are many other reports that tumor growth continues even in the presence of large amounts of corchicin. GroBe et al.
oc.

Natl, Acad, Sci, (JSA, 7
8: 1176-1180#1981) Medroxyzorodesterone, dexamethasone, and, to a lesser extent, corchicin have the effect of inhibiting tumor angiogenesis in the rabbit cornea, while estradiol and testosterone have the effect of inhibiting angiogenesis in the rabbit cornea. −j: Reports that there is no such thing.

Heparin, i.e., α, β-glycosidic, heavily tm-oxidized uronic acid and glucosamine covalent,
It has been used clinically as an anticoagulant for half a century. Despite its importance and widespread use, neither the exact structure of heparin nor the mechanism by which it acts as a blood barrier has been elucidated. Much of the inconsistency in determining the structure of heparin is due to the fact that it is not a homogeneous substance. Heparin is a polydisperse substance with molecules i 5,000 to 40.0 [ ] 0.

Within the chain σ,), there are variations in the degree of sulfate, N-acylation, and c-5 uronic acid residue, resulting in structural differences.

Therefore, the exact composition of commercially available heparin varies depending on its source and purification method. For heparin, its molecules are N-sulfated-D-glucosamine = 6-sulfate and L
α-glycosylation between -iduronic acid 2-sulfate
The bond cleavage simply shortened the chain, resulting in a slight modification of the terminal group (Δ-4,
Decomposition occurs at the 5-position unsaturation) to form heparin fragments, trisaccharides, tetrasaccharides, hexasaccharides and higher oligosaccharides (:[,1nhardt et al.: J, B1
11. ahem.

257:7310-7313.1982) Heparin fragment of bacterial origin (I, Anger et al.: U.S. Patent No. 4.
No. 341.869).

The present invention also relates to heparin or heparin fragments, which are polysaccharides of hexasaccharide or higher, and corchicin-1 or hydrocortiscin.
<U Hydrocortezono> 11-α) (Administration of the substance suppressed angiogenesis in mammals! 411.
, discovered that regression of tumor masses in mammals (and prevention of metastasis υ) occurred and was completed with σ〕)0 The basic name of this isomer is 11α, 17゜21-) '
IJ Hydroxyzoregna-4-ene-6°2-1-dione. The inventive operation does not appear to affect mature and ungrown lining or vascular tissue. The angiogenesis inhibitor IL1 according to the present invention can be used not only to prevent regression and metastasis of J1 tumors in 11-year-old animals, but also to act as a deterrent for women even when administered for the first time after insemination, and to prevent bone loss. It is also effective in alleviating symptoms and treating diseases involving pulsation neogenesis, such as ocular angiogenesis diseases.

Neither cortiscin nor hydrocortiscin, nor the 11-α isomer of violet hydrocortiscin, nor heparin 7j, effectively inhibit angiogenesis or cause tumor regression in the absence of heparin fragments. Heparin alone does not inhibit angiogenesis; on the contrary, it promotes it.

Heparin (or a fragment thereof) and cortiscin (also hydrocortiscin or its 11-α isomer) may be mixed prior to administration or may be administered separately. Administration is carried out orally or parenterally, including in particular topical, intravenous, intraarterial, subcutaneous administration, and includes absorption as well as injection, introduction into body cavities or mouth. Heparin, which is commercially available in the form of heparin sodium, is degraded in the gastrointestinal tract when administered orally, reducing its anticoagulant activity; however, the degradation products include trisaccharides and larger fragments; Even in fragments, this route of administration is highly effective in achieving the effects of the present invention. Heparin and its fragments can be used in any physiologically acceptable non-toxic form, including metal salts, preferably sodium salts (all these forms are included in the term heparin or fragments thereof as used herein). included). Product name of “Panheparin” (
Heparin, commercially available from Abbott Laboratories, is preferred for best results;
Other raw materials such as heparin such as Hepar, NC
rrP made by , can also be used in the same way, although it is slightly less effective. Since cortissin and its physiologically acceptable non-toxic salts, such as vinegar salts, are very sparingly soluble in water, they are preferably administered parenterally, eg subcutaneously, and oral administration is not appropriate. For oral administration, use % hydrocortisone or its 11-alpha isomer (which is more soluble in water than corticosteroids) or its physiologically tolerable, non-toxic, bath salts, such as phosphorus. Preferably, the non-tolerable water-insoluble salt of the 11-alpha isomer is administered parenterally. The term cortiscine used in this specification, 〕-idrocortedene 2 and no-idrocortidene 2 to the 11-α isomer F1, refers to the steroid P
Does it include both itself and the salt of the mystery defined above? ) of Oconacin, hydrocortiscin or its 11-alpha isomer, it is well known that when administering the drug at the same time, the cv normal effect can be obtained by the alpha dose When administering heparin subcutaneously, doses can be used as long as undesirable anticoagulant effects are tolerated. Since heparin does not produce an anticoagulant effect even when administered orally, and the Rokutang fragments do not exhibit an anticoagulant effect, large doses can be administered orally or by other routes without the risk of bleeding.
Heparin has been shown to be effective when administered orally on the order of 27,000 to 45,000 units per body weight per day. However, in the case of subcutaneous administration, twice a day at a dose of about 600 units/kg body weight or more, a desirable anticoagulant effect is developed. In the case of hexasaccharide fragments, the effect is observed when administered subcutaneously twice a day at 7 μ/kP body weight. For subcutaneous administration of Acetate Cochin, the dose is 2507/kg/day to 7~/~/day of Kawaishi for oral administration of hydrocortiscin, and for oral administration of hydrocortiscin, the dose is 0.457ng/- (approximately 75 kg/day) in drinking water. (Y/day) was valid. 11-alpha of hydrocortiscin
The isomer exhibits approximately comparable activity to hydrocortiscin with respect to the objects of the present invention.

The dose required to prevent tumor regression or metastasis varies to some extent with the type of tumor and also with the time required for complete tumor regression. r+Ji J) A at the beginning of treatment
The time required for complete regression is also determined by α. Since angiogenesis also occurs in psoriasis and arthritis, it is expected that the present invention will be useful for diseases such as stiffness. Repeated administration of Coach Ann for many days may result in lung infections, whether or not heparin casings or heparin fragments are administered at the same time, and there is no prophylaxis during treatment with the present invention. It is desirable to administer an excessive amount of antibiotics.

Heparin and corchicin, hydrocortiscin or the 11-alpha isomer of hydrocortiscin are themselves non-specific, physiologically acceptable and filterable carriers;
It is best to use a heavy phase or suspension in water or saline, for example. Compositions may be used in which the active ingredient mixture is present in the dry form or in a suitable carrier.

Example 1 Add 0.9cm of open plate coach to 0.9ml of saline solution.9.
7J11 cells were injected onto the chorioallantoic membrane of an 8-day-old chick embryo through a hole previously made in the shell. On day 9, tumor extract from hepatoma cells (100 μg, zetter: Nature
.

285: 41-43.1980) was placed on a plastic overslip with a circular diameter of 15 ruL and allowed to dry. Add 5 μl of a standard solution of either heparin (6 μg or 1 unit) or water to the center of each overslip and treat at least 20 embryos with each. After drying, the overslip was placed on the chorioallantoic membrane. Control embryos were also provided in which tumor extracts and/or fc were treated with heparin, but without cortiscine acetate pretreatment. The membrane was examined with a 12x stereoscope on the 11th day. When new capillaries gathered on the central spot of the overslip were observed, angiogenesis was considered to be present. All embryos treated with water or heparin but not cortiscin showed vasculogenesis in 80% of embryos treated with cortiscin alone. Less than 2% of embryos treated with both heparin and cortiscine acetate showed vasculogenesis.

Example 2 Using porcine mucosal heparin without heparin treatment, Langer
(5science, 217: 261-263
.

1982) l/) method and the product was equilibrated with I M NH40AC.
It was fractionated by ex column. Decomposed heparin is
No anticoagulant activity was demonstrated by activity ratio partial thromboplastin time or whole blood calcium recalcification time. Product-1: or the product mixture (250) was mixed with I M NH,0
Dissolved in A01 cc, 75 x 2-5 cma-15
The column was loaded and eluted at 1.5 cc/min. As a result, several peaks corresponding to tetrasaccharides, hexasaccharides, and higher oligosaccharides, which were not completely separated, and a separated peak corresponding to trisaccharides were obtained. The trisaccharide σ peaks were lyophilized and I M NH40ACO,2CC
The same sharp peak as 4 was obtained by rechromatography on G-15. Redissolve in 1 cc of I M NH40Ac,
Elution from 50X1.25CTLG-50 at 2 CCZ minutes yielded 1% llr corresponding to tetrasaccharide and hexasaccharide fragments.
A double peak corresponding to oligosaccharide V was obtained, and the latter was lyophilized. The tetrasaccharide and hexasaccharide fragments were combined, lyophilized, and IM
Redissolved in NH40AC and applied to G-15 column.

The tetrasaccharide was extracted from the G-15 column as a single peak, and the central portion was lyophilized.

The hexasaccharide fraction was lyophilized and I M NH40AcQ,3
When redissolved in cc and eluted from the G-15 column, a single peak was obtained, and the central portion was lyophilized.

The size of the fragments was determined by dissolving an aliquot of each fraction in 0.03 M hydrochloric acid and measuring the absorbance of this solution at 232 nm. The molecular weight of each fragment was calculated using a molecular absorption rate ξ=5500 due to the presence of α,β unsaturated carboxylate end groups in each product. Trisaccharides and tetrasaccharides were further characterized by comparing the average values on G-15 with monosaccharide, trisaccharide, and trisaccharide standards. The measured molecular weights of the trisaccharide, tetrasaccharide, hexasaccharide and oligosaccharide fractions are 5° and 0°12, respectively.
10.1600 and 187D.

Various heparin fragments were individually dissolved in a methyl cellulose solution and dissolved in a methylcellulose solution.

Next is the disc, Taylor (l:, Folki
an) one method (nature, 297: , 607
-312, 1982) by -(Betl), l11
Applied to the yolk fungal membrane (4th day) of the chick embryo cultured in 1.
Ta. As shown in the following table, acid = 1-thione (I
In the presence of D Oμg) σ, the hexasaccharide fragments showed the most angiogenic activity; Death White 100% 0 Arrogance
D Section 8 100% 100φ 4 25% 100 Section 1 25 Power 50% Tetrasaccharide jiao-yohhi trisaccharide did not show any activity.

Oligosaccharides had low activity and were toxic at high concentrations. Therefore, Rokushi, Class 1 fragments were used in the subsequent experiments. The chorioallantoic membrane on the 6th day of growth contains hexasaccharides (12 μg
) and cortiscine acetate (100 μg) produced a 12.6 ± 0.1” diameter area of aneurysm by 48 hours. In the case of valine (and cortisone acetate) (1), the capillaries of the mesodorum layer were absent, while the other two tissue layers of the membrane remained immobile and viable. The hexasaccharide alone did not promote the formation of ll'JL and θ formation like that of hevarin. All discs contained a combination of conacin I\1 acid (100 μg) and a fragment of IJL. . Both heparin fragments alone, and cortiscin, metelcellulo-
When used alone, no avascular region was observed. Embryos of 10 ftω were used in each group. Six Sugar Chi (+Coach Anne)
In case of 1, the blood vessel area was 17% in the case of 12 µg, and 15 in the case of 0.1 µg.

In the case of oligozatucaride, the highest dog blood pressure range score was 10.

The chick embryos were taken out on the 6th or 4th day from the 5th stage, and externally washed in a dish and antibiotics were not used.
uer't) ach et al. (J, nevel, 13j,
The cells were incubated in a Petri dish at high humidity and 5% CO2 according to the method of OL, A1: RO 91-394.1974). On day 6, heparin (6 μg), or hexate heparin fragments (12 μg), or cortiscine acetate (S
Methylcellulose discs (10 μl) containing either a powder free of igma, preservatives, or clouding agents, or a combination of cortiscine acetate and heparin, or a combination of cortiscine acetate and hexasaccharides). It was transplanted into the chorioallantoic membrane. Examine the embryos on the 4th day and if there is a 1 round avascular zone around the methylcellulose disk, N1k
The diameter of the aneurysm zone was measured using an on-broil projector (20x magnification). 60 embryos were fecalized in each group]-. Some embryonic hearts were injected with InV ink just before the first injection of formalin to enable tracing of the vessels in the tissue section to the first CV region.

In the Rokutate frequent ten'ei cochinisin, all embryos have a diameter of 12,
A hemostasis region of 6±0.1iπ was produced. For heparin + cortidine acetate, the diameter is 8.9±0.7. A haemorrhagic area occurred. In the presence of any of the 16 compounds alone or in the presence of methylcellulose alone, no avascular zone was produced. The statement was accepted.

On the other hand, hexasaccharide + acid cortiscine or heba-1 non+
In the mesoderm embryos to which cortiscine α was applied, the capillaries disappeared to the original state, whereas no effect on the ectoderm and endoderm cell layers was observed. In the mature chorioallantoic membrane, in which the blood vessels have not yet developed, no effect was observed by the hexacarboxylic acid, an-heparin, or the monohexasaccharide combination.

Langer r:
)0) Method (Nature, 263: 797-7
80 jl 976), heparin (Sigma
) 18011g, 'E or hexasaccharide fragment 300μg
1. Taketake vinegar modified hydrocortisone (Sigma) 1
．． 5■ or a combination of cortiscin and heparin. This pellet was implanted into the cornea of a rabbit eye, located one patch from the limbus, and the cancer tissue was implanted at a position two from the limbus distal to the polymer. The contralateral eye of each rabbit was similarly implanted with a control pellet without examples, proximal to the tumor.

Release rates averaged 15 μg/day for heparin, 21 μg/day for hexasaccharide kd cloves, and 5 μg/day for cortiscin.

Even if the compounds are mixed, the release rate of each does not change. According to the colorimetric method, the pellets were treated with heparin for 14 days.
Hexasaccharides were released for 11 days and cortiscin was released for over 30 days.

When the capillary grows in the direction of the transplanted tumor, the maximum surface tube length is measured every day for 6 days by stereoscopic slit ramp 0 (10 times, ±0.
11 ca). On the 14th day, the rabbits were sacrificed and each carotid artery was injected with Indian ink. The cornea was removed and examined using a stereoscope.

New capillaries grow in the direction of the ulcer and become empty)
t or pellets containing heparin alone lf - average speed 0.44
B/day was observed to pass, which was 0.22111 M for pellets containing cortiscin alone. Tumors behind these pellets became vascularized by 6-8 days. When the pellet contained both cortiscin and heparin, there was no capillary growth for 13 days. Upon removal of the heparin-cortiscin pellet or depletion of heparin in the pellet, capillary growth resumed. Histological observations showed that the tumor cells remained viable and cleavable even though they were adjacent to the heparin-cortiscin pellet.

Example 2 When a pellet in which Q heparin was replaced with a hexasaccharide fragment was implanted, new capillaries were directed toward the tumor on average at 0.30 M/day in the presence of the hexasaccharide pellet and on average 0.3 M/day when the pellet contained cortiscine. The growth rate was 14 mm/day, and an average rate of 0.32 B/day when no pellets were included. In the presence of the combination of Rokutachi and cortiscin, 1
No capillary growth was observed in the four rabbits throughout the 3-day observation period, and only capillary growth was observed at a rate of 0.07 on day 14 and at rate of 2.6 on day 7.

Σ (a) Ovarian 1 inhibition: 3 to tumor fragment 1 1N-5 mice,
It was transplanted into the back of 30 mice using a trocar. After 10 days, the tumor CV x1z average volume was 1.5 x 102
It started when it was time 3. Heparin 2 [10 units/tnl was added to the drinking water. Average daily consumption was 6 to 5 -/22 g mouse. Acetic acid Coach 1 Ann is 25 Q nI9/kg17. once a day. ) dose 6
125mg/ki9 for 1 day, 75mg/ki9 for 1 day,
Thereafter, a maintenance dose (attenuated administration) of 37 W/kllJ was administered subcutaneously. Control animals received either saline injections or heparin or Corchione acetate alone. All control animals had primary tumor or fcl by day 64.
``j: The tumor metastasized to the lungs became larger and died. The tumor disappeared by day 15 in the mice that were given oral Hevalin and Coach Ann's tumor reduction device, and it remained in a state of disappearance even after the treatment was discontinued.

Heparin was subcutaneously administered twice a day to a group of 0 mice at a dose of 627, and cortiscine acetate was administered subcutaneously once a day at a constant dose of 75 μ/day, but the same treatment was performed. The mice initially responded similarly, but tumors recurred after treatment was discontinued. These mice were then treated with oral heparin and decreasing doses of Corchione acetate as described above, and the tumors permanently disappeared. These 79
2631 animals died on day 31, but the primary tumor was not visible to the naked eye and there was no metastasis.

(b) Lewis's lung cancer: 7 mice per group with tumor fragment I B3;
Forty-two mice were transplanted and treatment started 7 days later. Treatment consisted of oral heparin as previously described and subcutaneous administration of decreasing doses of cortiscin. All control animals died by day 66 with large tumors and numerous lung metastases. Treatment in the heparinistoric acid acetate group was discontinued for approximately 7 days after tumors were no longer visible to the naked eye in each mouse. In the group receiving oral heparin + cortisone acetate, treatment was discontinued in all mice by day 66, and the tumor was maintained in a state where it disappeared.

Subcutaneous heparin and cortiscine acetate (75 Da/kl
In the subcutaneous administration group, 5 mice stopped treatment on day 37, 3 mice remained free of tumors, and 2 mice died of pneumonia on day 60 and 36, respectively. , 1 child was found to have a small σ primary rhinorrhea!・Metastasis was observed in one place in 7 mice of -01 mice.

To test whether other steroids could be used in Coach Ann, heparin was administered along with hydrocortiscine, dexamethasone, or medroxif steroids. 1 itt administered with heparin
The only drug that was as effective as conacin acetate in causing tumor regression was Hydrocochian (3-2 nt!).
/kg), MeV Roxyprogasterone (112ry
g/i<g), no regression of Lewis lung tumor occurred whether or not Hebalinke was administered simultaneously.

(C) B-16 melanoma: 84 x 105 melanocytes 1
Groups of 5 and 40 mice were injected subcutaneously.

One case was treated with heparin υ line administration and hydrocortisone vv [], a 5 my/ml oral administration of drinking water, as described in -. Oral administration and subcutaneous administration of the last revised coach anthology, Phase 3: Nigeheparin and cortiscine acetate 75■/
9 subcutaneous administrations were performed. Controls received water, heparin alone, hydrocortiscine or cortisone alone. All control animals developed canine tumors and metastases to the lungs and died by day 31. In the heparinist and cortiscine acetate group, treatment was discontinued for approximately 7 days after tumors were no longer observed. In the oral heparinist and cortidine acetate attenuated dose group, one mouse died on day 24, but a partially regressed tumor and two lung metastases were observed. These had a skin texture of 0.1" or less. One remaining tumor in this group disappeared, and even after discontinuing treatment, 62
No recurrence of Katsura's tumor was observed until day 1. Subcutaneous heparin+
In the cortiscine acetate group, one mouse died on the 18th day and the other mouse died on the 21st day.

No metastasis to the lungs was observed in either case. The other 6 are 3
Treatment was discontinued on the second day, and treatment was started 6 weeks later with attenuated doses of parked lohevarin and cortiscine acetate, which was successful.
After that, the +i ulcer disappeared. With oral lohevarin + oral lohydrocortisone cup, tumor recurrence 't' occurred even after 47 days when all mice stopped treatment: uF! I didn't meet it. 7. With oral lohevarin + oral lohi V locortezone. ) treatment is
It appeared to be more effective against ovarian tumors and Lewis lung cancer.

(d,) Bladder cancer: 7 mice in each group, 70 mice were implanted subcutaneously with tumor 1u3 (~. Control animals had large primary tl
All of the patients died in 61 days due to ulcers. Bladder cancer? Lung metastasis occurred in 7t mice with f? :, there were no examples. Group 1 was treated with translohevarin + subcutaneous f4 ili c'r treatment, and the tumor volume was 140.
It started on the 9th [-] when it became urn3. Tumor growth stopped, but regression was only partial, with tumor volume of approximately 706
``It became a constant condition, and it remained constant after one treatment, and I had no sleep (i.e. for more than 61 days).''
.

One animal died of pneumonia on day 19 with 1ζ thrush. Survival of dormant 1l111 tumors was evidenced by the resumption of tumor growth after treatment was discontinued; one animal began to grow 70 days after treatment was discontinued. Subcutaneous heparinist subcutaneous intoxication cortiscine (75my/
The results were similar in the second group treated with kj7) as described above, with prolonged dormancy. One mouse died on day 21.

Since complete regression could not be achieved with the standard therapy of the oral heparinist, a highly concentrated dose of heparin was administered orally to the other groups. Oral hebalin (600 units/r
Attenuating doses of nI and monoacetate resulted in more significant tumor regression and steady state was reached at a smaller volume, ie, approximately 45B3. One mouse died. However, 1000 units of heparin/i'''C pups underwent total regression, and no mice were found to have rhinoplasty on day 69 after treatment was discontinued. There were no deaths in this group.

In summary, combined administration of heparin and cortiscine acetate 2
When this happens, all tumors either stop growing or regress. Conversely, when either compound is used alone, tumors grow at the same rate as those receiving injections of saline alone. Control animals of the small-breasted species died from enlarged tumors.

Complete regression was possible in the majority of motor WVs treated with heparin plus cortiscine acetate. In other words, the ulcer did not recur even after it healed. For example, the most effective μ: quasi-oral hebalin (20 mouth units/1d) +
According to cortiscin (subcutaneous, reduced dose), vl] fruit 11
Medium+g T I Q 3%, Lewis f1j cancer floO%
, B-16 melanoma and 80% zero child was allowed to retire completely. However, for bladder cancer treated with this method, there is no complete photorejection.
The heparin dose was increased to 1000 units/continuation, and complete regression of 100 units without recurrence occurred in 14 cases. A slightly less effective treatment standard, heparinist coach Anne (757n?), resulted in regression of 80% for Ukou liver cancer, 71% for Lewis lung cancer, and 60% for B-16 melanoma.
% in bladder cancer.

6. Add 2Q hexasaccharide buccal heparin fragments to 1.57Q saline solution.
/ To d? Tani j'141~ta. Transfer) 6 mice with 11α oval tumor, 7 ntg/kg, 1
It is treated with subcutaneous injections twice a day, and subcutaneous injections for reducing cortisone. Control mice were given one dose of corticcin alone or saline. In contrast to the control tumors (which grew progressively), the hexasaccharide-treated tumors rapidly regressed and were no longer visible to the naked eye after 4 days. When the treatment with this hexasaccharide was then discontinued, the II tumor reappeared 3 to 5 days later.
ukkaruppan et al. (5science, 2Q 5
A Lewis lung tumor was implanted into the corner of each mouse according to the method of 1979), and treatment was started 24 hours later. Heparin (oral) - acetic acid coach
An attenuated dose inhibited capillary growth by cortiscine acetate alone (0,2411 n/day), heparin alone (0,32 rL
mZ day) and saline solution (0.23r;, ya 7 days) significantly inhibited (0.02F, ln/day). In the presence of heparin-cochrane acetate, the thin plates of the tumor exhibit a sectional appearance. Three-dimensional tumor growth does not occur. In contrast, in the saline control or when heparin or cortiscine acetate was administered, one locus underwent angiogenesis;
It continues to grow in three dimensions until it perforates the cornea. These large tumors could be regressed to a planar, ocular corneal phase by the combined use of heparin-cortiscine acetate. However, the corneal tumor cells could not be eradicated, and when the acid therapy was discontinued, the angiogenic tumor regenerated.

The number of lung metastases was counted for all animals that died.

A 6x stereoscope was used. In control animals, the lungs contained 41
1! Metastasis consisting of a metastatic tumor in the cheek was observed all over. In contrast, when heparin and cortiscine acetate were used in combination, no metastasis was observed in mice with ovarian tumors, and in addition to one case in a mouse with Lewis lung cancer, metastasis and melanoma in mice with melanoma were observed. 7. There was almost complete metastasis in mice treated with phosphocortiscin, which only showed Wm metastases with a diameter of 0.1+ m or less. % 'Km 〃One thing is absolutely serious. This effect will be roughly clear from the following data: Number of transfers: 4,553 out of 76 animals; 6 out of 39 animals; No lung metastasis was observed.

To rule out the possibility that regression of this single locus was due to direct cytotoxicity, all four tumor cells were treated with either heparin, cortiscine acetate, heparin-cortiscine acetate, or no drug. Cultured in the presence of 10% mouse serum. Heparin-cortiscine acetate did not inhibit cell growth and in fact stimulated it. Furthermore, histological examination revealed no signs of cytotoxicity in the bone marrow or small intestinal mucosa of animals receiving heparin-cortisone acetate administration.

To exclude the possibility that heparin-cortiscine acetate induces tumor regression by promoting an immune response, mice were inoculated with new tumor cells at various intervals after treatment discontinuation. They grew at the same rate. Moreover, if heparin-cortiscine acetate is discontinued before tumor regression is almost complete, the initial tumor growth will resume.In addition, discontinuing or starting treatment may result in the use of heparin-cortiscine at a lower than optimal dose. To do that,
ll:1. ), bladder ulcers were maintained at an almost constant small volume (i.e., 45-70η method 1') for as long as 8 weeks1, induced by implantation of silica particles into the cornea of rabbits. ~)? Immune angiogenesis due to the implantation of O-lymph nodes was also completely prevented by cortisone-valine pellets. Heparin itself temporarily delayed the onset of both types of angiogenesis (compared to Beret), and heparin itself also delayed the onset of immune angiogenesis, but not both types of angiogenesis. Nagisa alone (1, cortesone-hebarin combination α tsu yang it α-) did not cause vasculature formation over a long period of time. Adults, valley ft. Neudrocortezone (0,4b+...y/ml) Go 9 and U company lohevarin were administered to C1, which was administered at the same ratio. Reared in cages [7]. In control animals, tumors grew progressively, whereas in animals treated with heparin and cortiscin or heparin and high-IS loccortisone, regression was observed and f-phrase apparatus tumors were palpable [-2] at 6 weeks of treatment. Became.

Example 9 CD-1 Swiss mice were used. This is because this type of mouse is easily fertilized and almost always becomes pregnant and gives birth to a mass production of two litters. One male was left in the cage for 24 hours. I changed the bedding and added two females at 5pm. The vaginal plug was checked at 8 a.m. the next morning, and its presence was determined to indicate fertilization. Enough male-female cages were prepared to accommodate at least 20 pregnant animals.

On the day after fertilization, treatment for fertilized female animals was initiated with the following drinking water administration.

Group 2 - Hevarin (piepar) LOOO units/ml of water.

■Group - Rin-ki Hydrocoach Anne 0.45my/l water If liver-(I) and (n) are added to water I~・Kan-Water alone treatment UA! The mice were harvested for 1 day, and then placed one by one in a separate cage, and parturition was carefully observed. The cage was checked for signs of miscarriage (hair, retained fetus).

[,If:'b- and 111 groups both had healthy pups.I-, 0th 11th'1, there were no close mice, and there were no miscarriages))) Symptoms Ta. This is due to the 771 pressure of hebaline cortisol, probably due to the growth inhibition of capillaries α- from the uterus.
Block it with 5. This effect is thought to be due to its angiogenesis-inhibiting effect (4).

Agent Asamura Continued from page 1 Priority claim @ December 7, 1983
)■5591.75 ■Applicant: Stephanie Tiller 400 Plutzklin Avenue, Boston, Massachusetts, United States of America ■Applicant: Ropert Samuel Langer 46 Greenville Street, Zumerville, Massachusetts, United States of America Procedural Amendment (Mutsu) February 17, 1980 Mr. Commissioner of the Japan Patent Office1, Indication of the case, Patent Application No. 240768 of 19882, Name of the invention, Method for inhibiting angiogenesis3, Relationship with the case by the person making the amendment Address of the patent applicant Name Moses Judah Folkman (first name)
4. Agent Showa Month, Day 6, Copywriting of the specification of the number of inventions increased by supplementation (no change in content)

Claims (1)

[Scope of Claims] (1) Heparin - or one piece of heparin, which is a polysaccharide of six or more polysaccharides, and the 11-α isomer of cortisol, hydrocorchian, or hydrocorchicin. The active ingredients are heparin and hydrocortisone, and the active ingredients are heparin and hydrocortisone. The method according to claim 1 (:3) administration V Yoko ['' is administered as the main ingredient, and the active ingredients are a 1υr piece of Rokuhozuke heparin and a mulch containing hydrocortiscin as the main ingredient. 1id eight request) Tsuko ^1 noj' body le 1ji fjδ self-loading method Otori San~・4) 1 に た う Ｇｕｒｅｎｔ Cυ yoke including the step of administering an antibiotic to the mammalian animal.'' 1st burial mound self-fighting method te
l The administration is parenteral, and the heptapolytic components are hexasaccharide heparin capsules and coch-an, hydrocorti-an or hydrocortidene Q] 11-α isomer as the main ingredients. The method according to claim 1 (6) The method according to claim 5, which comprises the step of administering an antibiotic to the mammal (7) The active ingredient Vi1. ) heparin fragments, which are polysaccharides of heparin size or hexasaccharide or higher, and 2) 11-α isomer of coch-an or hydro-coch-an or high-10 cortiscine, in sentinel mammals. Pharmaceutical composition for inhibiting angiogenesis (8) Pharmaceutical composition according to claim 7, in which the active ingredients are mainly heparin and hydrocortiscin (9) The active ingredients are hexasaccharide heparin fragments and cortiscin. Pharmaceutical composition (1O) according to claim 7, in which the active ingredients are hexasaccharide heparin fragments and hydrocortisin as main ingredients dυ activity The pharmaceutical composition according to claim 7, which contains hexasaccharide heparin fragments and hydrocochycin as the main ingredients, and contains an antibiotic (12+ heparin or a heparin fragment that is a polysaccharide of hexasaccharide or more) and hydrocochycin. Treatment method of administering to a tumor-bearing mammal an effective amount of an active ingredient mainly composed of 11-alpha or hydrocortiscin or the 11-alpha isomer of hydrocortiscin. Claim 12, which contains Cortedene as the main component
Therapeutic method (I4) according to Claim 14) The administration is oral administration, and the active ingredients are hexasaccharide heparin fragments and hydrocortisin as main ingredients. A special feature that controls the process of administering a substance. 'l-Therapeutic method u6) according to claim 12) The administration is parenteral administration, and the active ingredients are the hexasaccharide heharin fragment and corchicin or hydrocorchicin as the main ingredients. 1171. The treatment method according to claim 16, which includes the step of administering an antibiotic to a moaning dwarf animal.