CA1226816A - Inhibition of angiogenesis - Google Patents

Inhibition of angiogenesis

Info

Publication number
CA1226816A
CA1226816A CA000443640A CA443640A CA1226816A CA 1226816 A CA1226816 A CA 1226816A CA 000443640 A CA000443640 A CA 000443640A CA 443640 A CA443640 A CA 443640A CA 1226816 A CA1226816 A CA 1226816A
Authority
CA
Canada
Prior art keywords
heparin
cortisone
tumor
day
hydrocortisone
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
CA000443640A
Other languages
French (fr)
Inventor
Moses J. Folkman
Stephanie Taylor
Robert S. Langer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Harvard College
Original Assignee
Harvard College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Harvard College filed Critical Harvard College
Application granted granted Critical
Publication of CA1226816A publication Critical patent/CA1226816A/en
Expired legal-status Critical Current

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters

Abstract

INHIBITION OF ANGIOGENESIS

Abstract of the Disclosure Angiogenesis in mammals is inhibited by administration of heparin or a heparin fragment which is a hexasaccharide or larger together with cortisone, hydrocortisone or the 11-.alpha. isomer of hydrocortisone.
Inhibition of tumor-induced angiogenesis is accompanied by tumor regression, and prevention of metastasis.

Description

12Z6~3~6 AYE

This invention relates to inhibition of angiogenesis and pertains more specifically to treatment of mammals with heparin or heparin fragments and with cortisone, hydrocortisone or the 11-~ isomer of hydrocortisone to inhibit angiogenesis with subsequent regression of large tumor masses and prevention of Tory metastasis in mammals containing such tumors.
Angiogenesis, the growth of new capillary blood vessels, is important in normal processes such as development of the embryo, formation of the corpus luteum and wound healing. It is also a component in pathologic processes such as chronic inflammation, certain immune responses and neoplasia. Furthermore, angiogenesis is a property of most solid tumors and is necessary for their continued growth.
It has previously been reported that heparin enhanced the intensity of angiogenesis induced by tumors in viva, although in the absence of tumor cells or tumor extract neither heparin nor the mast cells which release heparin could induce angiogenesis.
Taylor and Folk man, Nature Vol. 297, 307-312 (1982).
It has also been reported in Tieback et at., J.Natl.Cancer Insight Vol. 57, 769-77~ (1976) that 6 ~-methyl-prednisolone partially suppressed tumor angiogenesis in hamster cheek pouch under certain conditions, but tumor growth was not stopped, and many other publications have reported continued growth of tumors even in the presence of large doses of cortisone. It has also been reported in Gross et at., Proc.Natl.Acad.Sci., USA, Vol. 78, 1176~80 (1981) that medroxyprogesterone, dexamethasone, and to a lesser lZ26~316 extent cortisone, inhibited tumor angiogenesis in rabbit corneas, while eastwardly and testosterone were ineffective.
Heparin, an I, glycosidieally linked highly sulfated eopolymer of Urania acid and glueosamine, has been used clinically as an anticoagulant for half a century. Despite its importance and widespread use;
both the exact structure of heparin and the precise nature by which it acts in blood anti coagulation have not been elucidated. Much of the difficulty in determining the structure of heparin is because it is not a homogeneous substance. Heparin is polydisperse with a molecular weight range from 5,000 to 40,000.
Within a given chain, there are also structural variations such as varying degrees of sulfation, N-acetylation, and C-5 epimerization in the ironic acid residue.
Consequently, the precise composition of commercial heparin varies depending on its source and method of purification. Heparin has been degraded by treatment with heparinase (an enzyme of bacterial origin, Lunger et at. US. Patent 4,341,869) which cleaves the molecule at the ~-glycosidic linkages between N-sulfated-D-glucosamine 6-sulfate and L-iduronic acid 2-sulfate to form fragments including disaccharide, te-trasaccharide, hexasaceharide, and larger oligosaccharides, each being simply a chain-shortened heparin fragment with minor end group modification (the degradation results in a ~-4,5 site of unsaturation in the terminal ironic acid residue).
Lindhardt et at., J.Biol.Chem., Vol. 257, 7310-13 (1982).
It has now been found that angiogenesis in mammals is inhibited and tumor masses in mammals are caused to regress (and metastasis prevented) by administration of heparin or heparin fragments which 1226~16 are hexasaccharides or larger together with cortisone or hydrocortisone or the 11-~ isomer of hydrocortisone.
The full name of the isomer is 11-~, 17, 21-trihydroxypregn-4-ene-3,20-dione. Neither mature non-growing blood vessels nor vascular tissue seems to be affected by the procedure of the present invention.
Inhibition of angiogenesis in accordance with the present invention, in addition to its effect upon tumor regression and metastasis in tumor-bearing mammals, is effective as a contraceptive in females even if first administered after insemination has occurred, is effective to reduce osteoporosis, and is effective in treating diseases involving neovascularization such as neovascular diseases of the eye.
Neither cortisone nor hydrocortisone nor the 11-~ isomer of hydrocortisone effectively inhibits angiogenesis nor causes regression of tumors in the absence of heparin or a heparin fragment. Heparin alone does not inhibit angiogenesis but on the contrary enhances it.
The heparin (or fragment) and cortisone (or hydrocortisone or its 11-~ isomer) may be mixed together prior to administration or may be administered separately. The administration may be oral or parenteral including inter aria topical application, intravenous intraarterial or subcutaneous injection, and including absorption as well as injection and introduction into bodily apertures or orifices. In the case of heparin, which is commercially available in the form of heparin sodium, oral administration leads to degradation in the gastrointestinal tract which results in loss of its anticoagulant activity, but because it has been found that the degradation products include disaccharide and larger fragments, -this mode of ~Z26~316 administration is highly effective for the present invention both for heparin and for heparin fragments.
The heparin and its fragments may be employed in any physiologically acceptable non-toxic form, including their metal salts, preferably as the sodium salts, all of which are embraced in the term "heparin" or "fragment" as used in the present specification and claims. For the best results heparin sold under -the trade name "Panheprin" (Abbott Laboratories) is preferred, but heparin from other sources, such as Hoper, Inc. can also be used although less effective.
Cortisone and its physiologically acceptable non-toxic salts, such as the acetate, are only very slightly soluble in water, hence are preferably administered parenterally, e.g. subcutaneously, not orally. For oral administration, hydrocortisone or its 11-~ isomer (which are relatively water soluble as compared to cortisone) or one of their water-soluble physiologically acceptable non-toxic salts such as the phosphate are preferred.
Water insoluble salts of hydrocortisone or its 11-isomer which are non-toxic and physiologically acceptable, are administered parenterally. The terms "cortisone" and "hydrocortisone" and 11-~ isomer of hydrocortisone as used in the present specification and claims are intended to include both the steroids themselves end their salts as defined above.
Dosages employed are limited only by the well known limits for the administration of -the drugs individually for their usual equates, in the case of cortisone, hydrocortisone, or its 11-~ isomer. Heparin may be administered percutaneously in amounts as large as tolerable without objectionable anticoagulant effects.
Since heparin administered orally has no anticoagulant effect, and since the hexasaccharide fragment has no anticoagulant effect whether given orally or in any lX26~316 other way, large dosages can thus be administered without risk of bleeding. Oral dosages of heparin of the order of 27,000-45,000 units per kg. body weight per day have been found to be effective, but when administered subcutaneously, doses greater than about 600 units per kg. body weight twice daily led to undesirable anticoagulatlon effects. In the case of the hexasaccharide fragment, 7 my per kg. body weight twice daily subcutaneously has been found effective.
Cortisone acetate was effective in subcutaneous dosages of 250 mg/kg/day down to 37 mg/kg/day and hydrocortisone was effective orally in amounts of 0.45 mg/ml drinking water (approximately 75 mg/kg/day). The lo isomer of hydrocortisone is approximately equal to hydrocortisone in activity for the purpose of the present invention.
The dose size required to bring about regression of tumors or to prevent metastasis varies to some extent depending upon the identity of the tumor, as does the length of time required to bring about complete regression of tumors. Tumor size at the beginning of treatment also affects the length of time required for complete regression. Because of the occurrence of angiogenesis in psoriasis and arthritis, it is expected that the present invention may be useful in treating these diseases. Because administration of cortisone, with or without heparin or heparin fragments, may result in pulmonary infection after a number of days, it is desirable to administer a suitable antibiotic as prophylaxis during treatment in accordance with -the present invention.
The heparin (or fragment) and cortisone or hydrocortisone or -the lo isomer of hydrocortisone are best dissolved or suspended in a suitable carrier which itself must be non-toxic and physiologically acceptable, 12X6~316 such as water or normal saline. Compositions containing mixtures of the active agents, either dry or in a suitable carrier, can be employed.
Example 1 Cortisone acetate 0.9 my in 0.9 ml saline was flooded over the chorioallantoic membrane of 8-day chick embryos through a window in the shell made previously.
On day 9, tumor extract (100 go from hepatoma cells (as described in Wetter, Nature Vol. 285, 41-43, 1980) in 5 I H20 was placed on the center of a round 15 mm diameter plastic cover slip and allowed to dry. To the center of each cover slip was then added a 5 I Alcott containing either heparin (6~g, i.e., 1 unit), or water, at least twenty embryos being subjected to each. After drying, the cover slip was placed on the chorioallantoic membrane. Additional control embryos received tumor extract and/or heparin, but were not pretreated with cortisone acetate. The membranes were viewed on day 11 with a-x12 stereoscope. Angiogenesis was present if new capillaries were seen to converge on the spot in the center of the cover slip. All of the embryos treated with water or heparin but no cortisone exhibited angiogenesis, as well as 80% of those treated with cortisone acetate alone. Less -than 2% of those treated with both heparin and cortisone acetate exhibited angiogenesis.
En Porcine mucosal heparin was exhaustively degraded using heparinase by the procedure of Lunger et at., Science Vol. 217, 261-3 (1982) and the products were fractionated using Sephadex* columns equilibrated with lo Nikko. The degraded heparin had no anticoagulant activity as determined by activated partial thromboplastic time or whole blood recalciEication * Registered Trademark `lZX6~316 time. The product or product mixture (250 my) was dissolved in 1 cc of lo Nikko, loaded onto a 75 x 2.5 cm G-15 column, and eluded at 0.5 cc/min. This resulted in several incompletely resolved peaks corresponding to twitter-, hex-, and higher oligosaccharides and a separate peak corresponding to disaccharide product.
The disaccharide peak was freeze-dried, dissolved in 0.2 cc of lo Nikko and rechromatographed on G-15 resulting in the same sharp peak which was freeze-dried. The mixture of twitter-, hex-, and oligosaccharides was freeze-dried, redissolved in 1 cc of lo Nikko, and eluded from a 50 x 1.25 cm G-50 at 2 cumin resulting in an unresolved double peak corresponding to twitter- and hexasaccharide fragments and an additional peak corresponding to oligosaccharides which was freeze-dried. The twitter- and hexasaccharide f fragments were combined, freeze-dried, redissolved in 1 cc of lo Nikko, and loaded onto a G-15 column. The -tetrasaccharide was eluded from a G-15 column in a single peak, the center cut of which was freeze-dried.
The hexasaccharide fraction was f reeze-dried, redissolved in 0.3 cc lo Nikko, eluded from a G-15 column in a single peak, the center cut of which was freeze-dried.
Fragment size was determined by dissolving a weighed amount of each fraction into 0.03M hydrochloric acid and measuring the absorbency of this solution at 232 no. The molecular weight of each fragment was calculated using a molar absorptivity, for the Jo unsaturated carboxylate end group present in each of -these products, of I= 5500. The dip and tetrasaccharides were further characterized by comparing their K avg-values on G-15 with moo-, do-, and trisaccharide standards. Measured molecular - 1226~316 weights were 530, 1210, 1600, and 1870 for the do-, twitter-, hex-, and oligosaccharide fractions respectively.
The various heparin fragments were dissolved into methyl cellulose discs either alone or with cortisone acetate. The discs were then applied to the 4-day yule sac membrane of chick embryos cultured in Putter dishes as described by Taylor and Folk man, Nature, Vol. 297, 307-312 (1982). In the presence of cortisone acetate (100 go as shown in the following table, the hexasaccharide fragment demonstrated the highest antiangiogenesis activity.

lXZ6816 TABLE I
Percent Embryos That Developed Vascular Zones 48 Hours After Implantation of Methyl cellulose Discs On The Davy Old Yolk Sac Membrane COOK. OLIGO- HEX- TWITTER- DIP
( Lug ) SACCHARIDES SACCHARINE SACCHARINE SACCHARINE

Allah died 100~ I 0 ooze 100 us 100~
1 I 50~;
0.1 0 50~

~226~3~6 AYE
Twitter- and disaccharides were inactive.
Oligosaccharides were less active and were toxic at higher concentrations. Therefore, the hexasaccharide fragment was used in subsequent experiments. In the growing 6-day chorioallantoic membrane, discs containing hexasaccharide (12 go and cortisone acetate (100 go produced large vascular zones up to 12.6 0.1 mm diameter by 48 hours. As in the case of heparin (with cortisone acetate), capillaries in the mesodermal layer were absent while the other two tissue layers of the membrane were intact and viable. Hexasaccharide alone did not promote tumor angiogenesis as heparin did.
All discs contained a combination of cortisone acetate (100 go and a heparin fragment. No vascular zones developed in the presence of any heparin fragment alone, or with cortisone or methyl cellulose alone. Ten embryos were used for each group. With hexasaccharide (plus cortisone), the area of the vascular zone was 17%
of the vascular membrane at 12 go and 15~ at 0.1 go For the oligosaccharides, the maximum vascular area was 10%.
Example 3 Fertilized chick embryos were removed from their shell on day 3 (or 4) and incubated in a Putter dish in high humidity and 5% C02 as previously described by Auerbach,et at., J.Devel.Biol., Vol. 41, 391-4 (1974), except that an outer dish and antibiotics were not used. On day 6, a methyl cellulose disc (10 I
containing either heparin (6 go or hexasaccharide heparin fragment (12 go or cortisone acetate (Sigma, powder free of preservatives and suspending agents), or a combination of cortisone acetate + heparin or cortisone acetate + hexasaccharide was implanted on the chorioallantoic membrane. The embryos were examined 48 hours later, and if a clear vascular zone appeared around the methyl cellulose disc, the diameter of the ~226816 zone was measured with a Nixon Profile projector at x20.
Thirty embryos were used in each group. India ink was injected into the heart of some embryos just before formal in fixation so that vessels could be followed to the edge of the vascular zone in histological sections.
Hexasaccharide + cortisone acetate produced vascular zones of 12.6 + 0.1 mm diameter in all embryos. Heparin + cortisone acetate produced vascular zones of 8.9 + 0.7 mm diameter. There were no vascular zones in the presence of any compounds alone, or with methyl cellulose alone.
Histologic cross-sections of the chorioallantoic membranes, revealed that capillaries developed normally in the presence of any compound alone. In contrast, capillaries were completely absent from the mesodermal layer in the face of either hexasaccharide + cortisone acetate, or heparin + cortisone acetate, while the ectodermal and endodermal cell layers remained unaffected. In the mature chorioallan-toic membrane where vessels are no longer growing, the cortisone acetate-heparin or -hexasaccharide fragment combinations were without effect.
Example 4 Polymer pellets of ethylene vinyl acetate copolymer (EVA) of approximately 1 mm diameter were impregnated, using the procedure of Lunger et at., Nature, Vol. 263, 797-800 (1976), with heparin 180 llg(Sigma), or hexasaccharide fragment 300 go or cortisone acetate 1.5 my (Sigma), or a combination of cortisone and heparin.
The pellets were implanted in the cornea of a rabbit eye 1 mm from the limbs and a 1 mm3 piece of V2 carcinoma was implanted distal to the polymer, 2 mm from the limbs. In the opposite eye of each rabbit, control pellets -that were empty were similarly implanted in juxtaposition to the tumor.

1226l~316 Release rates averaged 15 gray for heparin; 21 gray for hexasaccharide fragment; and 5 gray for cortisone. When the compounds were mixed, they released at the same rates. By spectrophotometry, the pellets released heparin for 14 days, hexasaccharide for 11 days, and cortisone for more than 30 days.
As capillary blood vessels grew towards the tumor implant, maximum vessel length was measured every 3 days with a stereoscopic slit lamp at-xlO ( + 0.1 mm). On day 14 the rabbits were killed, and India ink was injected into each carotid artery. The corneas were removed and examined with a stereoscope.
New capillary blood vessels were observed growing towards the tumor and passing over an empty pellet or a pellet containing heparin alone at a mean rate of 0.44 mm/day; and over a pellet containing cortisone alone at 0.22 mm/day. The tumors behind these pellets were vascularized by 6-8 days. When the pellets contained both cortisone and heparin, there was no capillary growth for 13 days. When -the heparin-cortisone pellets were removed or when the pellets were depleted of heparin, capillary growth resumed. Histologic sections showed that tumor cells remained viable and capable of replication even when they were adjacent to the heparin-cortisone pellet.
In -the presence of implanted pellets in which the hexasaccharide fragment of Example 2 replaced the heparin, new capillaries grew toward the tumors a-t a mean rate of 0.30 mm/day in the presence of -the hexasaccharide pellets; 0.14 mm/day when -the pellets contained cortisone; and 0.32 mm/day when the pellets were empty. In the presence of the hexasaccharide-cortisone combination, -there was no capillary growth -throughout the 13-day observation period in 4 rabbits, and in one rabbit a few capillaries grew at 0.07 mm/day.

1~26816 Example 5 (a Ovarian Sarcoma: l mm3 pieces of tumor were implanted with a trucker subcutaneously in the backs of 30 mice; 5 per group. Treatment was begun 10 days later, when mean tumor volume was 1.5 x 102 mm3- Oral heparin was 200 U/ml in drinking water, average daily consumption was 3-5 ml water per 22g mouse. Cortisone acetate was administered subcutaneously once daily in a dose of 250 mg/kg for six days, 125 mg/kg for one day, 75 mg/kg for one day, and thereafter a daily maintenance dose of 37 mg/kg (a "tapered" dosage). Control animals received either saline injections, or heparin alone or cortisone acetate alone. All controls were dead by day 34 with large primary tumors and lung metastasis. All mice treated with oral heparin + cortisone tapered dosage became tumor-free by day 15 and remained so after treatment was discontinued.
An additional group of mice was treated similarly except that heparin was administered twice daily subcutaneously in a dose of 627 units, and cortisone acetate was administered subcutaneously once daily in a uniform dose of 75 mg/kg. Response of the mice was the same as in the first set except that tumors recurred after cessation of treatment; these mice became permanently tumor-free when subjected to the regimen of oral heparin and cortisone acetate tapered dosage described above. One of these mice died on day 31 with no gross primary tumor and no metastasize.
(b) Lewis lung Carcinoma: Treatment began 7 days after implantation of a 1 mm3 piece of tumor in 42 mice: 7 per group. Treatment was with oral heparin and subcutaneous cortisone acetate tapered dosages described above. All controls died by day 33 with large tumors and numerous lung metastasis. In the heparin +
cortisone acetate groups -treatment was discontinued for ~226~3~6 each mouse after tumor had been invisible for approximately 7 days. In the oral heparin + cortisone acetate all mice were off treatment by day 33, and remained tumor-free. In an additional group treated with subcutaneous heparin and subcutaneous cortisone acetate (75 mg/kg), 5 mice were off treatment at day 37 and remained tumor-free. Two mice died of pneumonia on days 30 and 33 respectively with small primary tumors (< 75 mm3). One metastasis was found in one mouse.
To determine if other steroids could substitute for cortisone, heparin was administered with hydrocortisone, dexamethasone, or medroxyprogesterone.
Only hydrocortisone was as effective as cortisone acetate in causing tumor regression when administered with heparin. At the highest tolerable doses neither dexamethasone (3.2 mg/Kg), nor medroxyprogesterone (112 mg/Kg), caused regression of Lewis lung tumors with or without heparin.
(c) B-16 Melanoma: 7.4 x 106 melanoma cells were injected subcutaneously into 40 mice; 5 per group.
Treatment of one group was with oral heparin as described above and oral hydrocortisone, 0.45 mg/ml in drinking water. Another group received oral heparin and subcutaneous cortisone acetate tapered dosage, and a third group received subcutaneous heparin and cortisone acetate 75 m,g/kg. Controls received either water, or heparin alone, or hydrocortisone or cortisone acetate alone. All control animals died by day 31 with large tumors and lung metastasis. In the heparin + cortisone acetate groups, treatment was discontinued after tumor had become invisible for approximately 7 days. In -the oral heparin + cortisone acetate tapered dosage group, 1 mouse died on day 24 with a partially regressed tumor and 2 lung metastasis that were vascular and measured less that 0.lmm. All other mice in the group became tumor-1226~3~6 free and remained so after their treatment was discontinued by day 32. In the subcutaneous heparin +
cortisone acetate group one mouse died on day 18 and another on day 21; neither had lung metastasis.
Treatment was discontinued for the other 3 mice on day 32; tumors 3 weeks later were successfully retreated with oral heparin -I cortisone acetate tapered dosage, and these mice have remained tumor-free. In the oral heparin + oral hydrocortisone group, all mice remained tumor-free after their treatment was discontinued on day 47. The regimen of oral heparin + oral hydrocortisone seemed to be more effective for melanoma than it was for ovarian sarcoma or Lewis lung carcinoma.
- (d) Bladder Carcinoma: 70 mice: 7 per group received a 1 mm3 implant of tumor subcutaneously. All control animals died by day 31, with large primary tumors. No mice bearing bladder carcinoma developed lung metastasis. One group was treated with oral heparin and subcutaneous cortisone acetate tapered dosage, starting on day 9 when mean tumor volume was 140 mm3. Tumors stopped growing, but regressed only partially, and then reached a steady state where tumor volume remained at approximately 70 mm3 for as long as the treatment was continued (i.e., 61 more days). Only one mouse died of pneumonia on day 19. The "dormant"
tumors were viable as evidenced by resumption of tumor growth whenever treatment was discontinued for one mouse at a time, beginning at day 70. For a second group treated with subcutaneous heparin and subcutaneous cortisone acetate 75 mg/kg as described above, the result was similar; i.e., long-term tumor "dormancy".
One mouse died at day 21.
Because of the inability, the standard regimen of oral heparin + cortisone acetate to bring about complete regression, higher concentrations of oral heparin were used with oilier groups. With oral heparin (600 U/ml) -cortisone acetate tapered dosage, there was more significant tumor regression and a steady state ("dormancy") was reached at a smaller tumor volume of approximately 45 mm3. One mouse died. However, with 1000 V/ml heparin, there was complete regression; mice remained tumor-free after discontinuation of treatment on day 39. No mice died in this group.
In summary, all tumors either stopped growing or regressed when the heparin-cortisone acetate combination was administered. In contrast, when either compound was used alone, tumor growth continued at the same rate as in animals receiving only saline injections; all such control animals died with a large tumor burden.
In the majority of animals treated with heparin +
cortisone acetate, it was possible to achieve "complete regression", i.e., tumors did not recur after treatment was discontinued. Thus, with the most effective regimen, oral heparin (200 U/ml) + cortisone (sac.
tapered dosage), it was possible to obtain "complete regression" in 100% of ovarian sarcomas, 100% of Lewis lung carcinomas and 80% of B-16 melanomas. However, when bladder carcinomas were treated with this regimen, there were no "complete regressions" until heparin was increased to 1000 U/ml, following which 100% of tumors regressed without recurrence. With the less effective regimen, heparin (sac.) + cortisone (75 mum), complete regression rate was ovarian, 80%; Lewis lung, 71~; B-16 melanoma 60%; and bladder 0%.
Example 6 The hexasaccharide heparin fragment of Example 2 was dissolved in saline, 1.5. mg/ml. Three mice bearing implanted ovarian sarcoma were treated with subcutaneous injection of the hexasaccharide fragment twice daily at a dosage of 7 mg/kg and subcutaneous injection of 1226~316 cortisone acetate -tapered dosage. Control mice received either cortisone acetate alone or saline. While control tumors grew progressively, the hexasaccharide +
cortisone acetate treated tumors regressed rapidly and were barely visible 4 days later. Their treatment with hexasaccharide was then discontinued, and the tumors reappeared 3-5 days later.
Example 7 To directly observe vascular tumors during systemic therapy, Lewis lung tumors were implanted in the mouse cornea by the procedure of Muthukkaruppan et at., Science, Vol. 205, 1416-18 (1979) and treatment was begun 24 hours later. Heparin oral cortisone acetate tapered dosage significantly inhibited capillary growth (.02 mm/day) compared to cortisone acetate alone (.24 mm/day), heparin alone (.32 mm/day) or saline (.23 mm/day). In the presence of heparin-cortisone acetate, a thin plate of tumor remained vascular. Three-dimensional tumor-growth did not occur. In contrast, in the saline controls or when either heparin or cortisone acetate were administered alone, tumors became vascularized and grew as a three-dimensional mass until they eventually perforated the cornea. These large tumors could be regressed to the flat, thin intracranial phase by the resumption of -the heparin-cor-tisone acetate combination' However, the intracranial tumor cells could not be eradicated; discontinuation of the heparin-cortisone acetate led to recurrence of a vascularized tumor.
Lung metastasize were counted in all animals that died. A x6 stereoscope was used. In all control animals, the lungs were heavily studded with metastasis from the three types of metastasizing -tumors. In contrast, when any combination of heparin + cortisone acetate was used, no metastasis were found in mice bearing ovarian sarcoma; only 1 metastasis was found in a mouse bearing Lewis lung carcinoma; and 2 vascular metastasis less than 0.1 mm diameter were found in one mouse bearing B-16 melanoma. The nearly complete absence of metastasis in heparin + cortisone treated mice was so striking, that the effect can be more readily appreciated by the following expression of data:
Total Number Lung Metastasis Controls = 4553 in 73 animals Heparin + Cortisone = 3 in 39 animals Furthermore, no lung metastasis appeared in any surviving animals that were off treatment.
To exclude the possibility that tumor regression might be caused by direct cytotoxicity, all 4 types of tumor cells were cultured in the presence of 10% serum obtained from mice receiving either heparin, cortisone acetate, heparin-cortisone acetate, or no drug.
Heparin-cortisone acetate did not inhibit cell growth, but in fact stimulated it. Furthermore, histological sections showed no evidence of a cytotoxic effect on bone marrow or intestinal mucus in animals receiving heparin-cortisone acetate.
To exclude the possibility that heparin-cortisone acetate might induce tumor regression by promoting an immune reaction, mice were inoculated with fresh tumor cells at various intervals after they were off treatment. these tumors grew at the same rate as the original implants. Furthermore, if tumor regression was nearly complete and heparin-cortisone acetate was discontinued prematurely, the original tumor resumed its growth. Finally, by stopping and starting treatment, or by using sub optimal doses of heparin-cor-tisone, bladder tumors could be maintained a-t nearly a constant small size (i.e., 45 to 70 mm3) for periods of more than 8 weeks.
Inflammatory angiogenesis induced by implantation of silica particles into the rabbit cornea, immune 1226~316 angiogenesis induced by implantation of lymph node from a different rabbit, were also completely prevented by the cortisone-heparin pellets. Cortisone by itself temporarily delayed the onset of both types of angiogenesis (compared to an empty pellet), and heparin by itself delayed the onset of immune angiogenesis, but neither alone prevented angiogenesis for an extended period of time as did the cor-tisone-heparin combination.
Example 8 A human colon carcinoma was inoculated subcutaneously into nude [athymic] mice, and allowed to grow to a volume of 0.5 cm3. Controls and treated animals received the same compounds, except that heparin in the drinking water was 1000 U/ml. also an additional treatment group included oral hydrocortisone (0.45 mg/ml) and oral heparin. Animals were housed in cages protected by millipore filter. Tumors grew progressively in all control animals, but regressed in animals treated with heparin and cortisone or heparin and hydrocor-tisone. The treated tumors were barely palpable after 6 weeks of therapy.
Example 9 Cal "Swiss" mice were used because this strain breeds easily and the fertilized females almost always conceive and subsequently deliver a full litter.
One maze was left alone in the cage for 24 hours.
His bedding was then changed and two females were added at 5 p.m. The females were then checked at 8 arm. the next morning for the presence of a cervical plug, which indicates insemination. Sufficient male-female cages were set up to obtain at least 20 pregnant females.
Treatment of the inseminated females was started on the day after insemination by offering drinking water to each of four groups, as follows:

~2Z6~316 Group r- Heparin (Hoper) 1,000 U/ml in water II- Hydrocortisone phosphate, 0.45 mg/ml in water III- (L) and (II) together in water IV- Water alone The treatments were continued for only four days, after which female mice were placed one per cage, and followed closely for the presence of offspring. The cages were checked daily for any sign of abortion (fur, fetal remains, etc.).
Groups I, II and IV all produced healthy litters.
In Group III there were no mice born and no evidence of abortion. This supports the conclusion that anti-angiogenesis by heparin-cor-tisone inhibits implantation, <
presumably by inhibiting capillary growth from the uterus.

Claims (5)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. A composition for inhibition of angiogenesis in mammals in which the active agents consist essentially of 1) heparin or a heparin fragment which is a hexasaccharide or larger and 2) cortisone or hydrocortisone or the 11-.alpha. isomer of hydrocortisone.
2. A composition as claimed in claim 1 in which the active agents consist essentially of heparin and hydrocortisone.
3. A composition as claimed in claim 1 in which the active agents consist essentially of a hexasaccharide heparin fragment and cortisone.
4. A composition as claimed in claim 1 in which the active agents consist essentially of a hexasaccharide heparin fragment and hydrocortisone.
5. A composition as claimed in claim 1 in which the active agents consist essentially of a hexasaccharide heparin fragment and hydrocortisone, and including an antibiotic.
CA000443640A 1982-12-20 1983-12-19 Inhibition of angiogenesis Expired CA1226816A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US45143182A 1982-12-20 1982-12-20
US451,431 1982-12-20
US55917583A 1983-12-07 1983-12-07
US559,175 1983-12-07

Publications (1)

Publication Number Publication Date
CA1226816A true CA1226816A (en) 1987-09-15

Family

ID=27036374

Family Applications (1)

Application Number Title Priority Date Filing Date
CA000443640A Expired CA1226816A (en) 1982-12-20 1983-12-19 Inhibition of angiogenesis

Country Status (5)

Country Link
EP (1) EP0114589B1 (en)
AU (1) AU555290B2 (en)
CA (1) CA1226816A (en)
DE (1) DE3373782D1 (en)
DK (1) DK168876B1 (en)

Families Citing this family (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2553287B1 (en) * 1983-10-18 1986-09-12 Choay Sa COMPOSITIONS BASED ON MUCOPOLYSACCHARIDES OR OLIGOSACCHARIDES, ESPECIALLY BASED ON HEPARIN FRACTIONS OR FRAGMENTS, SUITABLE FOR THE TREATMENT OF CELL PROLIFERATION DISORDERS
DE3432661A1 (en) * 1984-09-05 1986-03-06 Albert Prof. Dr. 6907 Nußloch Landsberger CARCINOM THERAPEUTIC
US4771042A (en) * 1985-11-25 1988-09-13 The Upjohn Company Inhibition of angiogenesis involving the coadministration of steroids with heparin or heparin fragments
ATE82126T1 (en) * 1986-04-04 1992-11-15 Angiogenics Ltd PREPARATION TO STOP ANGIOGENESIS AND A CAPILLARY, CELL OR MEMBRANE LEAK.
US4820693A (en) * 1986-05-22 1989-04-11 Angiogenics, Ltd. Method and composition for arresting angiogenesis and capillary, cell or membrane leakage
US4966890A (en) * 1986-04-04 1990-10-30 Angiogenics, Ltd. Method and composition for arresting angiogenesis and capillary, cell or membrane leakage
FR2597484B1 (en) * 1986-04-17 1988-12-23 Sanofi Sa GLYCOSAMINOGLYCANS OF THE HEPARIN OR HEPARANE-SULPHATE TYPE WITH ACTIVITY ON CELL DIVISION AND DIFFERENTIATION, THEIR PREPARATION AND THEIR THERAPEUTIC APPLICATIONS.
IE61758B1 (en) * 1986-05-23 1994-11-30 Daiichi Seiyaku Co Use of a sulfated polysaccharide
IL79255A0 (en) * 1986-06-26 1986-09-30 Hadassah Med Org Composition for metastasis prevention
US5206223A (en) * 1986-06-26 1993-04-27 Yeda Research And Development Co. Ltd. Method for inhibiting heparanase activity
IL85145A (en) * 1987-01-23 1994-08-26 Univ Australian Anti-metastatic pharmaceutical or veterinary compositions containing modified heparin having reduced anticoagulant activity
US5541166A (en) * 1987-01-23 1996-07-30 The Australian National University Sulphated polysaccharides having anti-metastatic and/or anti-inflammatory activity
WO1988007060A1 (en) * 1987-03-19 1988-09-22 Arthropharm Pty. Limited Anti-inflammatory compounds and compositions
US5668116A (en) * 1987-03-19 1997-09-16 Anthropharm Pty. Limited Anti-inflammatory compounds and compositions
WO1990005528A2 (en) * 1988-11-01 1990-05-31 Children's Medical Center Corporation Anti-thrombotic steroids
US6090794A (en) 1990-04-19 2000-07-18 The General Hospital Corporation Inhibition of neurofibrosarcoma growth and angiogenesis
KR0181295B1 (en) * 1990-07-24 1999-04-01 야마타니 와타루 Phospholipid -or lipid-combining glycosaminoglycan production thereof and cancer metastasis inhibitor
US5733892A (en) * 1990-07-24 1998-03-31 Seikagaku Corporation Metastasis inhibitor composition comprising a phospholipid-linked glycosaminoglycan and method for inhibiting metastasis employing the same
US5514667A (en) * 1990-11-05 1996-05-07 Arthropharm Pty. Limited Method for topical treatment of herpes infections
SK120193A3 (en) * 1991-05-02 1994-07-06 Yeda Res & Dev Pharmaceutical composition for the prevention and/or treatment of pathological processes
US6750207B1 (en) 1992-05-01 2004-06-15 Yeda Research And Development Co. Ltd. Compositions for the regulation of cytokine activity
US5861382A (en) * 1992-05-01 1999-01-19 Yeda Research And Development Co. Ltd. Methods for regulation of active TNF-α
WO1998005293A2 (en) * 1996-08-02 1998-02-12 The Children's Medical Center Corporation Method of regulating the female reproductive system through angiogenesis inhibitors
US7056504B1 (en) 1998-08-27 2006-06-06 Massachusetts Institute Of Technology Rationally designed heparinases derived from heparinase I and II
WO2001066772A2 (en) 2000-03-08 2001-09-13 Massachusetts Institute Of Technology Heparinase iii and uses thereof
US6969705B2 (en) 2000-07-21 2005-11-29 Aventis Pharma S.A. Compositions of polysaccharides derived from heparin, their preparation and pharmaceutical compositions containing them
DE10141749A1 (en) * 2000-08-29 2002-03-14 Max Delbrueck Centrum Inhibiting or stimulating angiogenesis by modulating the kinin B1 receptor, useful e.g. for treating solid tumors, macular degeneration, diabetic retinopathy or cardiac infarction
CA2423469A1 (en) 2000-10-18 2002-04-25 Massachusetts Institute Of Technology Methods and products related to pulmonary delivery of polysaccharides
US7285536B2 (en) 2001-12-05 2007-10-23 Yeda Research And Development Co., Ltd. Anti-cancer therapeutic compounds
US20040171819A1 (en) 2002-10-10 2004-09-02 Aventis Pharma S.A. Mixtures of polysaccharides derived from heparin, their preparation and pharmaceutical compositions containing them
US7956046B2 (en) 2003-07-24 2011-06-07 Aventis Pharma S.A. Oligosaccharide mixtures derived from heparin, preparation thereof and pharmaceutical compositions containing them
JP2007523912A (en) * 2004-02-26 2007-08-23 アドバンスト アキュラー システムズ リミテッド Heparin for the treatment of ocular lesions
EP2278978B1 (en) 2008-05-28 2015-09-02 ReveraGen BioPharma, Inc. Non-hormonal steroid modulators of nf-kb for treatment of disease
US9198921B2 (en) 2010-04-05 2015-12-01 Reveragen Biopharma, Inc. Non-hormonal steroid modulators of NF-κB for treatment of disease
WO2014140927A2 (en) 2013-02-13 2014-09-18 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Proteins with modified glycosylation and methods of production thereof
WO2017004205A1 (en) 2015-06-29 2017-01-05 Reveragen Biopharma, Inc. NON-HORMONAL STEROID MODULATORS OF NF-κB FOR TREATMENT OF DISEASE
US11382922B2 (en) 2019-03-07 2022-07-12 Reveragen Biopharma, Inc. Aqueous oral pharmaceutical suspension compositions

Also Published As

Publication number Publication date
DK584483D0 (en) 1983-12-19
DE3373782D1 (en) 1987-10-29
DK584483A (en) 1984-06-21
EP0114589A1 (en) 1984-08-01
DK168876B1 (en) 1994-07-04
AU2258283A (en) 1984-06-28
AU555290B2 (en) 1986-09-18
EP0114589B1 (en) 1987-09-23

Similar Documents

Publication Publication Date Title
CA1226816A (en) Inhibition of angiogenesis
US5001116A (en) Inhibition of angiogenesis
US4994443A (en) Inhibition of angiogenesis
KR0128287B1 (en) Pharmaceutical composition for the inhibition of angiogenesis
Rezkita et al. Curcumin loaded Chitosan nanoparticle for accelerating the post extraction wound healing in diabetes mellitus patient: A review
US4329364A (en) Antiandrogenic agents and methods for the treatment of androgen dependent disease states
JPH06507635A (en) Compositions for the prevention and/or treatment of pathological processes
AU2007303523B2 (en) Radiation sensitizer or anti-cancer chemotherapy sensitizer
US20080214480A1 (en) Method for Treating Sickle Cell Disease and Sickle Cell Disease Sequalae
EP0398925A1 (en) Growth inhibiting agent and the use thereof.
US4966890A (en) Method and composition for arresting angiogenesis and capillary, cell or membrane leakage
US4771056A (en) Method of medical treatment with serotonin antagonists
US4820693A (en) Method and composition for arresting angiogenesis and capillary, cell or membrane leakage
JPS5874608A (en) Pharmaceutical composition having peripheral antagonism for opium medicine
WO2013147689A1 (en) Combination treatment comprising sulphated glycosaminoglycans for inducing labor
CA1289885C (en) Tissue growth regulation
AU2190097A (en) Topical pharmaceutical composition containing heparin and method of treatment
McDONALD et al. Hydrocortisone (compound F) in ophthalmology: Clinical and experimental studies
Clemens et al. Inhibition by ergocornine of initiation and growth of 7, 12-dimethylbenzanthracene-induced mammary tumors in rats: effect of tumor size
AU2013260209A1 (en) Treatment of postpartum haemorrhage with chemically modified heparin or heparan sulphate and a uterotonic agent
JPH0455171B2 (en)
Bloksma et al. Role of histamine in the antitumour activity of endotoxin
Beck et al. Effect of heparin, heparin fragments, and corticosteroids on cerebral endothelial cell growth in vitro and in vivo
CN113842405B (en) Application of broussonetia papyrifera root-bark extract in preparation of anti-allergic and itching-relieving medicine for skin
AU742995B2 (en) Use of low-molecular-weight heparins for preventing and treating cerebral edemas

Legal Events

Date Code Title Description
MKEX Expiry
MKEX Expiry

Effective date: 20040915