CA2102207A1 - Compositions for the prevention and/or treatment of patholigical processes - Google Patents
Compositions for the prevention and/or treatment of patholigical processesInfo
- Publication number
- CA2102207A1 CA2102207A1 CA002102207A CA2102207A CA2102207A1 CA 2102207 A1 CA2102207 A1 CA 2102207A1 CA 002102207 A CA002102207 A CA 002102207A CA 2102207 A CA2102207 A CA 2102207A CA 2102207 A1 CA2102207 A1 CA 2102207A1
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- Prior art keywords
- pharmaceutical composition
- lmwh
- tnf
- secretion
- treatment
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7016—Disaccharides, e.g. lactose, lactulose
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7024—Esters of saccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/727—Heparin; Heparan
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Abstract
The present invention relates to pharmaceutical compositions for the prevention and/or treatment of pathological processes involving the induction of TNF-.alpha. secretion comprising a pharmaceutically acceptable carrier and a low molecular weight heparin (LMWH). In the pharmaceutical compositions of the present invention, the LMWH is present in a low effective dose and is administered at intervals of about 5-8 days. Furthermore, the LMWH is capable of inhibiting in vitro TNF-.alpha. secretion by resting T cells and/or macrophages in response to T cell-specific antigens, mitogens, macrophage activators, disrupted extracellular matrix (dECM), laminin, fibronectin, and the like.
Description
W~92/1~24~ PCT/US9~/03626 v ~ 3 ~
COMPOSITIONS FOR T~E PR~VENTION AND/OR
TREATMENT OF PATHOLOGICAL PROCESSES
1. Field of the Invention 5The present invention relates to pharmaceutical compositions for the prevention and/or treatment of pathological process~s involving induction of Tumor Necrosis Factor alpha (TNF-~) secretion, which comprise an effective low dosage of a low molecular weight heparin (LMWH) to be administered at intervals of between five to eight days.
The LMWHs to be used according to the invention have an average molecular weight of from about 3,000 to about 6,000 and inhibit TNF-~ secretion by resting T cells and/or macrophages in vitro in response to activation by T cell-specific antigens, ; T cell mitogens, macrophage activators, disrupted extracellular matrix (dECM), fibronectin, laminin or other ECM components.
. Backqround of the Invention TNF-~, a cytokine produced by monocytes (macrophages) and T lymphocytes, is a key element in the cascade of factors that produce the inflammatory response and has many pleiotropic effects as a major orchestrator of disease states (B. Beutler and A.
C~rami, Ann. Rev. Immunol. (1989) 7:625-655).
~ The biologic effects o~ TNF-~ depend on its - concentration and site of production: at low ~- 30 concentrations, TNF-~ may produce desirable ; homeostatic and defense functions, but at high concentrations, systemically or in certain tissues, TNF-~ can synergize with other cytokines, notably interleukin-1 (IL-1) to aggravate many inflam~atory responses.
The following activities have been shown to b~ induced by TNF-~ (together with IL-l); fever, ..
~' :
WO'~ 24~ PCT/US92/~3626 slow-wave sleep, hemodynamic shock, increased production of acute phase proteins, decreased production of albumin, activation of vascular endothelial cells, increased expression of major histocompatibility complex (MHC) molecules, decreased lipoprotein lipase, decreased cytochrome P450, decreased plasma zinc and iron, fibroblast proliferation, increased synovial cell collagenase, increased cyclo-oxygenase activity, activation of T
cells and B cells, and induction of secretion of the cytokines, TNF-~ itself, IL-l and IL-6.
How TNF-~ exerts its effects is not known in detail, but many of the effects are thought to be related to the ability of TNF-~ to stimulate cells to produce prostaglandins and leukotrienes from arachidonic acid of the cell membrane.
TNF-a, as a result of its pleiotropic effects, has been implicated in a variety of pathologic states in many different organs of the bo~y. In blood vessels, TNF--~ promotes hemorrhagic shock, down regulates endothelial cell thrombomodulin and enhances a procoagulant activity. It causes the adhesion of white blood cells and probably of platelet~ to the walls of blood vessels, and so, may promote processes leading to atherosclerosis, as well as to vasculitis.
TNF-~ activates blood cells and causes the adhesion of neutrophils, eosinophils, monocytes/
macrophages and T and B lymphocytes. By inducing IL-6 and IL-8, TNF-~ augments the chemotaxis of inflammatory cells and their penetration into tissues.
Thus, TNF-~ has a role in the tissue damage of autoimmune diseases, allergies and graft rejection.
TNF-~ has also been called cachectin because it modulates the metabolic activities of adipocytes and contributes to the wasting and cachexia accompanying cancer, chronic infections, chronic heart .
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W~'J~ PCTtUS92/03626 failure, and chronic inflammation. TNF-a may also have a role in anorexia nervosa by inhibiting appetite while enhancing wasting of fatty tissue.
TNF-~ has metabolic effects on skeletal and cardiac muscle. It has also marked effects on the liver: it depresses albumin and cytochrome P450 metabolism and increases production of fibrinogen, ~l-acid glycoprotein and other acute phase proteins.
It can also cause necrosis of the bowel.
In the central nervous system, TNF-~ crosses the blood-brain barrier and induces fever, increased sleep and anorexia. Increased TNF-~ concentration is associated with multiple sclerosis. It further causes adrenal hemorrhage and affects production of steroid hormones, enhances collagenase and PGE-2 in the skin, and causes the breakdown of bone and cartilage by activating osteoclasts.
In short, TNF-a is involved in the pathogenesis of many undesirable inflammatory conditions in autoimmune diseases, graft rejection, vasculitis and atherosclerosis. It may have roles in heart failure, in the response to cancer and in anorexia nervosa. For these reasons, ways have been sought to regulate the secretion of TNF-~ as a means to control a variety of diseases.
The prime function of the immune system is to protect the individual against infection by foreign invaders such ~s microorganisms. It may, however, also attack the individual's own tissues leading to pathologic states known as autoimmune diseases. The aggressive reactions of an individual's immune system against tissues from other individuals are the reasons behind the unwanted rejections of transplanted organs.
Hyper~reactivity of the s~stem against foreign substances causes allergy giving symptoms like asthma, rhinitis and eczema.
W0~)2/l~249 PCT/US92/03626 4 -- _~
r ~ , The cells mastering these reactions are the lymphocytes, primarily the activated T lymphocytes, and the pathologic inflammatory response they direct depends on their ability to traffic through blood vessel walls to and from their target tissue. Thus, reducing the ability of lymphocytes to adhere to and penetrate through the walls of blood vessels may prevent autoimmune attack, graft rejection and allergy. This would represent a new therapeutic principle likely to result in better efficacy and reduced adverse reactions compared to the therapies used today.
Atherosclerosis and vasculitis are chronic and acute examples of pathological vessel in~lammation. Atherosclerosis involves thickening and rigidity af the intima of the arteries leading to coronary diseases, myocardial infarction, cerebral infarction and peripheral vascular diseases, and represent~ a major cause of morbidity and mortality in the Western world. Pathologically, atherosclerosis develops slowly and chronically as a lesion caused by fatty and calcareous deposits. The proliferation of fibrous tissues leads ultimately to an acute condition producing sudden occlusion of the lumen of the blood vessel.
TNF-~ has been shown to facilitate and augment human immunodeficiency virus (HIV) replication in vitro (Matsuyama, T. et al., J. Virol. (19~9) 63(6):2504-2509; Michihiko, S. et al., Lancet (1989) 30 1(8648~:1206-1207) and to stimulate HIV-1 gene expression, thus, probably triggering the development of clinical AIDS in individuals latently infected with HIV-l (Okamoto, T. et al., AIDS Res~ Hum. Retroviruses (1989) 5(2):131-138).
Heparin is a glycosaminoglycan, a polyanionic sulfated polysaccharide, which is used ciinically to prevent blood clotting as an W~2/l~2~l~ PCT/US92/03626 - 5 - ;.~
antithrombotic agent. In animal, models heparin has been shown to reduce the ability of autoimmune T
lymphocytes to reach their target organ (Lider, 0. et al., Eur. J. Immunol. (1990) 20:493-499). Heparin was also shown to suppress experimental autoimmune diseases in rats and to prolong the allograft survival in a model of skin transplantation in mice, when used in low doses ~5 ~g for mice and 20 ~g for rats) injected once a day (Lider, 0. et al., J. Clin.
Invest. (1989) 83:752-756).
The mechanisms behind the observed effects are thought to involve inhibition of release by T
lymphocytes of enzyme(s) necessary for penetration of the vessel wall, primarily the enzyme heparanase that specifically attacks the glycosaminoglycan moiety of the sub-endotheli.al extracellular matrix (ECM) that lines blood vessels (Naparstek, Y. et al., Nature ~1984) 310:241-243). Expression of the heparanase enzyme is associated with the ability of autoimmune T
lymphocytes to penetrate blood vessel walls and to attack the brain in the model disease experimental autoimmune encephalomyelitis (EAE).
European Patent Application EP 0114589 (Folkman et al.) describes a composition for - 25 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-~ isomer of hydrocortisone~ According to the disclosure, heparin by itself or cortisone by itself are ineffective; only the ombination of both gives the desired sffects. Although there is no proof in the literature that there is a connection between anyiogenesis and autoimmune diseases, the descxiption on page 5 of the patent application connects angiogenesis with psoriasis and with arthritis, .. , :
~, .
WCl~)2/l~2~l9 PCT/US92/03~26 6 - ~ ~
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indicating the use of high doses of 25,000 units to 47,000 units of heparin per day.
Horvath, J.E. et al., Aust. N.Z.J. Med.
(1975) 5(6):537-539 describe the effect of subanticoagulant doses of subcutaneous heparin on early renal allograft function. The daily dosage is high (5000 U) and the conclusion of the study is that heparin in subanticoagulant doses has no effect on early graft function or graft survival and that it may be associated with increased hemorrhagic complications. `
Toivanen, M.L. et al., Meth. and Find. Exp.
Clin. Pharmacol. (1982) 4(6):3S~-363, examined the effect of heparin in high dosage (lOoO U/rat) in the inhibition of adjuvant arthritis in rats and found that heparin enhanced the severity of the rat adjuvant arthritis.
PCT Patent: Application PCT/AU88/OOQ17 published under No. W088/0530:L (Parish et al.) ;~ 20 describes sulphated polysaccharides that block or inhibit endoglycosylase activ:ity, such as heparanase activity, for use as antimetastatic and anti-inflammatory agents. Heparin and heparin derivatives, such as periodate oxidized, reduced heparins, that had negligible ant.coagulant activity, were shown to have antimetastatic and anti-inflammatory activity when used in dosages within, the range of 1.6-6.6 mg per rat daily, administered by constant infusion (corresponding to 75-308 mg daily for an adult human patient).
3. Summary of the Invention The present invention is related to pharmaceutical compositions for the prevention and/or treatment of pathological processes in~olving the induction of TNF-a secretion comprisiny a pharmaceutically acceptable carrier and a low WO92/1()2~1~ PC~/US92/03626 - t _ ; ~
molecular weight heparin (LMWH) present in a low effective dose for administration at intervals of about 5-~ days and which LMWH is ~apable of inhibiting in vit~o TNF-~ secretion by resting T cells and/or macrophages in response to T cell-specific antigens, mitogens, macrophage activators, disrupted extracellular matrix (dECM), laminin, fibronectin, or other ECM components.
In a particular embodiment of the present invention the LMWH of the pharmaceutical composition has an average molecular weight of from about 3,000 to about 6,000 and, furthermore, may be administered every fifth or seventh day.
It is also an objective of the present invention to provide pharmaceutical compositions to be administered at intervals of about 5-8 days for the p~evention and/or treatment of pathological processes involving the induction of TNF--~ secretion comprising a pharmaceutically acceptable carrier and a low molecular weight heparin (LMWH) present in a low effective dose and which LMWH is capable of inhibiting experimental delayed type hypersensitivity (DTH) reactions to an applied antigen as evidenced by a reduction in the induration observed after the application of the antigen to the skin five to seven days af~er the administration of the pharmaceutical composition relative to the induration observed after the application of the antigen to the skin in the absence of or after recovery from the administration of the pharmaceutical composition. Examples of the applied antigen include, but are not limited to, tetanus, myelin basic protein, purified protein derivative, oxazolone, and the like.
~urthermore, it is an objective of the present invantion to provide pharmaceutical compositions that may be administered in any manner as dictated ky the particular application at hand ., i , W~92/1')~4') PCT/US92/03626 , " ,j ~ ~."j ., including, but not limited to, enteral administration (including oral) or parenteral administration (including topical or inhalation with the aid of aerosols). In preferred embodiments, the pharmaceutical compositions of the present invention are administered subcutaneously or intravenously.
Thus, the present invention is useful, for example, in delaying or preventing allograft rejection and treating or preventing a variety of patholo~ical processes such as those related to autoimmune diseases, allergy, inflammatory diseases (in particular, inflammatory bowel disease), or acquired immunodeficiency syndrome (AIDS). The present invention also finds utility in the treatment of diabetes type I, periodontal disease, uveitis, rheumatic diseases (in particular, rheumatoid arthritis), atherosclerosis, vasculitis, or multiple sclerosis.
The pharmaceutical compositions of the present invention contain low effective doses of the prescribed LMWH and typically contain a single low dose unit of less than 5 mg of hMWH, preferably from about 0.3 to about 3 mg, and most preferably contain a sin~le low dose unit of from 1 to 1.5 mg.
The present invention also contemplates broadly a method of using a low molecular weight heparin (LMWH) which is capable of inhibiting in vitro TNF-~ secretion by resting T cells and/or macrophages in response to T cell-specific antigens, mitogens, macrophage activators, disrupted extracellular matrix (dECM), laminin, fibronectin, or other E~M components for the preparation of a pharmaceutical composition to be administered at intervals of about 5-8 days for the prevention and/or treatment of pathological processes involving induction of TNF-~ secretion which method comprises combining a low effective dose of the LMWH
with a pharmaceutically acceptable carrier.
, W~92/l924~ PCTtUS92/03626 C~
A still further object of the present invention is to provide a method of treating a subject suffering from a pathological process involving induction of TNF-a secretio~ comprising administering to such subject a pharmaceutical composition, as described above, at intervals of about 5-8 days.
Further objects of the present invention will become apparent to those skilled in the art upon further review of the following disclosure, including the detailed descriptions of specific embodiments of the invention.
4. Detailed Descri~tion of the Invention According to the present invention, it was found that treatment with low molecular weight heparins (LMWHs) inhibited the ability of T cells and macrophages to secrete TNF-a. The functional expression of this effect can be seen in the inhibition in mice and humans of the dalayed type 2~ hypersensitivity (DTH) reaction, an inflammatory reaction triggered by cells involving macrophages and other inflammatory cells. Treatment with LMWHs at doses affecting TNF-a produc ion also was able to ` inhibit a model of autoimmune arthritis called adjuvant arthritis (AA). LMWH treatment also prolonged the survival of allogeneic heart transplants in rats and abrogated insulin dependent diabetes mellitus (IDDM) in NOD mice. Moreover, treatment with LMWHs prevented the induction of TNF-a production by T
cells and macrophages in response to the stimulus of damaged subendothe:lial extracellular matrix (ECM).
Since TNF-a at the site of vascular injury has probably a role~in the process of atherosclerosis inhibition of TNF-a at the site o~ damaged subendothelial ECM will ameliorate the pathogenic ~ process of atherosclerosis. A most surprising aspect :`~ of treatment with LMWHs is that such treatment is most . .
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W~')2/l~2~t') PCT/US92/03626 1 0 -- '`
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effective when administered at low doses at weekly intervals. High doses of LMWHs or doses of LMWHs given daily are not effective in inhibiting TNF-~
secretion or immune reactions.
Low molecular weight heparins, produced by fractionation or controlled depolymerization of heparins, show improved antithrombotic performance but also different pharmacokinetic properties as compared to heparin: the half-life is doubled and the bioavailability is higher with respect to their, anticoagulant effect after subcutaneous injection (Bratt, G. et al. Thrombosis and Haemostasis (1985) 53:208; Bone, B. et al., Thrombosis Research (1987) ~6:845).
Accordiny to the present invention it has now been found that LMW~s administered at subanticoagulant doses at several day intervals are effective in the prevention and/or treatment of pathological processes involving induction of TNF-~.
The LMWHs to be used according to the invention are derived from LMWHs with an average molecular weight of 3000-6000, such as, for example the LMWHs disclosed in European Patent EP 0014184.
Some LMWHs are commercially available under different trade names, e.g., Fragmin~, Fraxiparin~, Fraxiparine, Lovenox~/Clexane~.
LMWHs can be produced in several different ways: enrichment by fractionation by ethanol and/or molecular sieving, e.g., gel filtration or membrane filtration of the LMWH present in standard heparin and controlled chemical ~by nitrous acid, ~-elimination or periodate oxidation~ or enzymatic (by heparinases) depolymerizatior.- The conditions for depolymerization can be carefully controlled to yield products of desired molecular weights. Nitrous acid depolymerization is commonly used. Also employed is depolymerization of the benzylic ester of heparin by .
W~ 2/l9249 PCT/US~2/03626 ~-elimination, which yields the same type of fragments as enzymatic depolymerization using heparinas~s. LMWH
with lo~ anticoagulant activity and retaining basic chemical structure can be prepared by depolymerization 5 using periodate oxidation or by removing the antithrombin-binding fraction of LMWH, prepared by other methods, using immobilized antithrombin for adsorption.
Fragmin~ is a low molecular weight heparin with average molecular weight within the range of 4000-6000 dalton, produced by controlled nitrous acid depolymerization of sodium heparin from porcine intestinal mucosa. It is manufactured by Kabi Pharmacia, Sweden, under the name Fragmin~, for use as an antithrombotic agent as saline solutions for injection in single dose syringes of 2500 IU/0.2 ml and 5000 IU/0.2 ml, corresponding to about 16 mg and 32 mg, respectively.
Fraxiparin~, and Fraxiparine~ are LMWHs with average molecular weight of approximately 4500 dalton, produced by fractionation or controlled nitrous acid depolymerization, respectively, of calcium heparin from porcine intestinal mucosa. It is manufactured by Sano~i (Choay Laboratories) for use as an antithrombo-tic agent in single doses comprising ca. 36 mg,corresponding to 3075 IU/0.3 ml of water.
Lovenox~ (Enoxaparin/e), a LMWH fragment produced by depolymerization of sodium heparin from porcine intestinal mucosa, using ~-elimination, is manufactured by Pharmuka SF, France and distributed by Rhone-Poulenc under the names Clexane~ and Lovenox~
for use as antithrombotic agent in single dose syringes comprising 20 mg/0.2 ml and 40 mg/0.4 ml of ~ater .
As shown in the present application, the novel properties of LMWHs described are a common feature o~ all, LMWHs regardless of the manufacturing ' ~2/l(~24~) PCT~US92/03626 1 ~
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process, the structural differences (created by depolymerization or those dependent on variation in the heparin used as raw material) or the anticoagulant activity, provided the LMWH employed is capable of inhibiting TNF-~ secretion in vitro by resting T cells and/or macrophages in response to activation by contact with specific antigens, mitogens, disrupted ECM or its protein components, such as fibronectin or laminin.
Another test useful to identify the LMWHs that are effective for the purpose of the present invention is the inhibition of experimental delayed type hypersensitivity (DTH) skin reactions, a T
lymphocyte dependent reaction, to a variety of antigens tfor example, tetanus antigen, myelin basic protein (MBP), purified protein derivative (PPD), and oxazolone). The LMWHs also inhibit T cell adhesion to ECM and its protein components.
The LMWHs effective according to the invention are incorporated into pharmaceutical compositions, for example, as water solutions, possibly comprising sodium chloride, stabilizers and other suitable non-active ingredients. The preferable way of administration is by injection, subcutaneous or intravenous, but any other suitable mode of administration is encompassed by the invention.
According to the invention, the LMWH is to be administered at intervals of five to eight days, preferably once a week.
The disorders that can be prevented or treated according to the invention are all disorders linked in pathological processes involving induction of TNF-~ secretion, including atherosclerosis and Yasculitis and pathological processes related thereto;
autoimmune diseases, e.g., rheumatoid arthritis, diabetes type I (ID~M); allergy; gra~t rejection;
acute and chronic inflammatory diseases, e.g. uveitis, ' ` .
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bowel inflammation; anorexia nervosa; hemorrha~ic shock caused by septicemia, and opportunistic infections in AIDS - immunocompromised individuals.
In AIDS, the LMWHs will suppress replication of HIV
and/or enhance resistance against the opportunistic infections, thereby preventing the development of AIDS-related complex (ARC).
It was observed according to the present invention that LMWHs can have a narrow dose range of effectiveness, and that different preparations of LMWHs can show different optimal biological effects.
Therefore, it was necessary to establish a Unit Dose for LMWH preparation to be usPd according to the invention, said unit dose being based on a bioassay of inhibition of TNF-a secretion by mouse spleen cells from mice treated with LMWH or by inhibition of DTH
reactivity by the L~WH. It was found that the ability of a particular dose of LMWH to inhibit TNF-~secretion is positively correlated with its ability to inhibit the delayed type hypers~ensitivity (DTH) reaction. This, cell-mediated inElammatory reaction in vivo is important because it is an expression of the processes involved in autoimmune diseases, graft rejection, some types of blood vessel inflammation and allergy.
It was thus established according to the invention that the lowest dose of LMWH per kg causing inhibition of TNF-~, production or inhibition of DTH
reactivity by at least 50% is considered to constitute 12 mouse inhibitory units per kg (12 u/kg). Because of the differences in surface area and metabolism between mice and humans, humans should be treated with a lower dose of LMWH, and 12 u/kg in mouse is established to correspond to 1 u/kg in humans. For example, the dose of Fragmin~ batch 38609 effective in ` inhibiting both TNF-~ secretion and DTH reactivity is 5 ~g per mouse administered weekly. Since each mouse .
~' ' ' - : `
W0~2/l924~ PCT/US92/03626 , 14 - t weighs about 25 g, the dose of Fragmin~ 38609 equivalent to 12 u/kg is 200 ~g/kg of mice. The dose of 1 u/kg suitable for humans is therefore 200 ~g/kg:
12 = 16.67 ~g/kg. A human weighing about 70 kg would then be treated by a dose of about 1.2 mg given in a single dose subcutaneously once every 7 days. Since individual humans vary biologically, the optimal dose may be different from about 1.2 mg and will lie below 5 mg, particularly within the range of 0.3 to 3 mg.
The dose of LMWH that should be effective in rats can be derived ~rom the fact that the dose of LMWH per kg of rats is one-half the dose per kg of mice, i.e. 6 u/kg. For example, if 12 u of Fragmin~
batch 38609 is 200 ~g/kg, then the 6 u dose suitable for rats should be 100 ~g/kg or 20 ~g per 200 g rat, administered once a week.
The invention will now be illustrated by the following non-limiting examples.
5. Exam~les 5.1. Example 1: Bioassay of Inhibition of TNF-a Secretion Supernatants of spleen cells cultured in the presence or absence of LMWH, or spleen cells obtained from mice treated or untreated with LMWH in vivo are analyzed for their ability to secrete TNF-~. The TNF-~ bioassay is based on the cytotoxic effect of TNF-~ on cycloheximide (CHI)-sensitized cells and its quantitation by the neutral red uptake assay as described by Wallach D., J. ImmunoloqY (1984~
132:2464-2469. Briefly, the killing of CHI-sensitized HeLa cells by TNF-~ present in the supernatants of the cells is measured, the concentration of TNF- in said supernatants being determined by comparison to titration curves of TNF-~ exogenously added. Cell viability is determined by incubation with neutral red for two hours, washing away excess dye, extracting the .. .
.
WO !~2/1924~) P~/US92/03~r26 ~ 1 5 ~ v u 7 neutral red that was taken up by the cells with Sorenson's citrate buffer-ethanol mixture, and quantitating it colorimetrically at 570 nm with a Microelisa Autoreader.
Cells from mice treated with LMWH are obtained as follows: female mice of the BALB/c strain t25 grams, 2 months old), at least 5 mice per group, are injected subcutaneously with various doses of LMWH, usually in the range of 0.5 to 20 ~g per mouse.
Five days later the mice are killed by cervical dislocation, the spleens are removed and suspensions of spleen cells, depleted of red blood cells-, are assayed for the production of TNF-~ in response to induction by dECM, Concanavalin A (Con A) or lipopolysaccharide (LPS).
5.2. Example 2: Bioassay of Inhibition of DTH Reactivitv Groups of mice are sensitized on the shaved abdominal skin with 100 ~l of 3% oxazolone (OX) in acetone/olive oil (4:1, v:v). DTH sensitivity is elicited 5 days later as follows: mice are challenged with 20 ~l of 0.5% OX (10 ~l to each side of the ear) in acetone/olive oil. A constant area of the ear is measured immediately before challenge and 24 and 48 h later with a Mitutoyo engineer's micrometer. The ;; individual measuring ear swelling is blinded to the identity o~ the groups of mice. The increment ~) of ear swellinq is expressed as the mean in units of 10-2 mm or 104 inch (+SE) depending on the micrometer that is used. Percent inhibition is calculated as follows:
Po~itive control - experimental group Inhibition = - x 100 Posltive control - negative control Mice are treated with LMWH as in Example 1, injected the day before primary sensitization to OX.
On the fifth day aEter sensitization to OX, the mice .
W0~2/192~9 PCT/US92/036~6 h ~
are challenged to induce a DTH reaction, as described above.
The positive control is the DTH reaction elicited in immunized mice in the absence of treatment with LMW~. The negative control is the background swelling produced by the antigen in immunized mice.
5.3. Example 3: Induction of TNF~a Secretion by T Cells and Macrophaqes In Vitro Microtiter plates were prepared as follows:
fibronectin (FN) or laminin (LN) (Sigma) were added to flat bottom 96-well plates (Costar) at a concentration of 1 ~g/50 ~1 PBS per well and removed after 16 h.
Remaining binding sites were blocked with BSA/PBS (lo mg/ml) which was added to the wells for 2 h and washed out. ECM-coated wells were prepared as follows:
bovine corneal endothelial cells were cultured in flat bottom 96-well plates. Thc confluent layers of endothelial cells were dissolved and the ECM was le~t intact free of cellular debris (Gospodarowicz, D. et al. J. Biol. Chem. (1978) 253::3736). Disrupted ECM
(dECM) was prepared by gently scratching the ECM three times with a 27G syringe needle and the exposed sites were subsequently coated with BSA/PBS. Resting cloned rat CD4+ T cells, designated Kl, which recognize myelin basic protein (MBP), were propagated and maintained in culture and were added to the wells, 105 cells per well with or without 3 x 105 syngeneic splenic macrophages, in 100 ~l per well RPMI 1640 (Gibco) supplemented with 1% BSA and antibiotics. The splenic macrophages were purified by removing the T
and B cells using specific monoclonal antibodies (mAb). Where indicated, anti-murine TNF-a mAb (Genzyme, diluted 1/300) were added to the wells. MBP
(100 ~g~ml), Con A (2.5 ~g/ml), LPS (1 ~g/ml) were added to the wells where indicated. The plates were incubated at 37 ~C in a humidified incubator for 3 h.
WO92/1~2~9 PCT/US92~03626 - 17 - ~ J ~
Subsequently, the contents of the wells (4 wells per experimental group) were collected, centrifuged, and the media were assayed for TNF-~ secretion as in Example 1: supernatants of cultured macrophages and lymphocytes were added to cultures of HeLa cells, which are sensitive to killing by TNF-a, and death of these cells in the presence of the test media was calibrated in comparison to titration curves of exogenous added TNF-a. Cell death is examined by the release of neutral red dye from the preincubated HeLa cells. The results shown here represent data obtained from a total of six experiments that produced essen~ially similar results.
Table 1 shows that T cells and macrophages cultured together can be induced to secrete TNF-a by contact with specific antigen MBP (group 4), the mitogen Con A (group 6) or LPS (group 8). However, in the absence of antigenic or ]nitogenic stimulus, TNF-a was also induced to be secreted by disrupted extracellular matrix (dECM; group 10) or by the ECM
components fibronectin (FN; group 12) or laminin (LN
group 14). Intact ECM was a weak inducer of TNF-a (group 16).
~5 Ta~le 1. TNF-~ secretion by T cells and macrophages is induced by specific antigen MBP, Con A, LPS or dECM
; ` components.
~1 cells cultured together with Secreted TNF-a(yes) or without TNF-~
30 Group inducer(no) macrophages (pg/ml) ._ 1 none no 50 2 yes 65 3 MBP antigen no 30 4 yes 950 Con A no 120 6 yes 1300 .
, , W0~2/l')2tt~ PCT/US92tO3626 7 LPS no 50 8 yes 1500 g dECM no 30 5 10 yes goo 11 FN no 20 12 yes 650 13 LN no 50 14 yes 500 10 15 ECM no 30 16 yes 120 5.4. Example 4: Regulation of TNF-a Secretion b~ LMWHs 15 T cell and accessory cell cultures were prepared as described in Example 3. LMWH was added to the wells at the beginning of the cell culture. The levels of TNF-~ were examined after 3 h of incubation.
Table 2 shows that the presence of LMWH
~Fragmin~ batch 38609) in vitro inhibited the induction of TNF-~ secretion induced by specific antigen (MBP; group 4), mitogensl (Con A and LPS;
groups 6 and 8), dECM or ~CM components (groups 10, 12 and 14). Since TNF-~ secretion induced by dECM is likely to be involved in atherosclerosis, inhibition of TNF-~ by LMWH will be beneficial in atherosclerosis.
Table 2. Induction of TNF-~ secretion induced in vitro is inhibited by L~H ~Fragmin~ batch 38609).
Secretion of TNF-~ by cultures of ; - T cells and TNF-~ LMWHmacrophages 35 Group Inducer(1 ~g/ml)(pg/ml) . . _ - 1 none none 65 ~ yes 30 .
: ,;,: , ~, :`' ijW0~2/1~249 PCTtUS92/036~6 -- 19 ~ ~ J S`~ f 3 MPB antigen none 950 4 yes 60 Con A none 1300 5 6 yes 80 7 LPS none 1500 8 yes 80 9 dECM none goo I 10 yes go ' 10 11 FN none 650 12 yes go 13 LN none 500 14 none 70 ~3 15 3 5.5. Exam~le 5 To examine the ef~ect of LMWH administered ~' to mice in vivo on the secretion of TNF-~ by spleen cells in vitro, the following experiment was conducted. ~ALB/c mice, 5 pe~r group, were treated With various doses of LMWH tE~ragmin~ batch 38609) dilute.d in saline, injected subcutaneously. After one week, the animals were-killed and their spleen cells, devoid of red blood cells, were examined for their ability to secrete TNF-~ in response to control wells ; without dECM (A) or to wells coated with dECM (B).
Measuring the levels of TNF-~ secretion was done as described in Example 1. Table 3 shows the results which indicate that an injection of 5 ~g of LMWH given once, 7 days earlier, inhibited TNF-~ secretion induced by dECM. H ~hsar or lower doses of LMWH were less effective. Th~s, an optimal dose of LMWH
administered in vivo a week earlier was effective.
.
~ 35 .
': '.::. :
,: -W~2/1~2~9 PCT/~92/03626 r TAble 3. Ex vivo inhibition of T cell mediated TNF-a secretion in response to damaged ECM.
In vitro TNF-a secretion (pg/ml) by spleen cells LMWH cultured on:
treatment of BALB/c mice A. None B. Damaged ECM
(weekly) (% Inhibition) 1 None 30 400 2 0.5 ~g 50 380 (5) 3 1 ~g 25 90 (78) 4 5 ~9 25 60 (85) 10 ~g 30 140 (65) 6 20 ~g 40 320 (20) Table 4 shows that a 5 ~g dose in vivo of the LMWH Fragmin~ batch 38609 was also e~fective in inhibiting TNF-a secretion induced by LPS. BALB/c t4 mice per experimental group) mice were treated with the indicated amounts of LMWH diluted in saline and injected subcutaneously. After one week, the mice were injected intraperitoneally with 10 mg LPS, killed 4 hours later and their spleen cells, devoid of red blood cells, were subsequently cultured in dECM coated wells for 3 hours in a humidif:ied incubator. The levels of TNF-a secreted in response to the dECM was measured in the supernatants of the cultures. The results are given in Table 4.
Table 4. Treatment of mice with LMWH inhibits LPS
mediated secretion of TNF-a by macrophages.
.
In vitro TNF-~
LMWH secretion by treatment macrophages (pg/ml~
of mice t~g) in response to LPS % Inhibition 35 690 __ 0.1 500 28 , .
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' ~: ' W~2/l~24~ Pcr/uss2/o3626 2 ~ 7 2 l 5.6. Example 6 To examine the effect of different LMWHs on the inhibition of TNF-~ secretion and on DTH
responses, mice were treated with th~ indicated LMWH
administered subcutaneously in different concentrations. After one week, some of the mice were killed and the induction of TNF-~ secretion in response to Con A activation in vitro was measured.
The remaining mice were examined for their ability to respond to the antigen oxazolone. The results are express~d in Table 5 as percent inhibition compared to the responses of the LMWH untreated mice.
Two conclusions can be made by inspecting the results shown in Table 5:
1. Different batches of LMWH, each 2~ calibrated for by similar antithrombotic effect (factor X assay) have different optimal doses for inhibition of TNF-a secretion. Moreover, there are preparations of LMWH, such as Clexane~ batch 4096, which have no inhibitory effect on TNF-~, secretion at any of the doses tried. Therefore, it may be concluded that the antithrombotic effect of a LMWH
preparation is not related to the potential of the LMWH preparation for inhibition of TNF-~ secretion.
The two different bioassays are mediated by different factors present in the preparations.
2. The ability of a particular dose of LMWH to inhibit TNF-~ secretion is positively correlated with its ability to inhibit DTH reaction, and the dose of a LMWH preparation optimally effective in inhibitin~ TNF-~ secretion is also optimally effective in inhibiting the DTH reaction.
~ ~ .
:
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W0~2/l924~ PCr/US92/03626 Table 5. Treatment of mice with different LMWHs inhibits both ex vivo TNF-a secretion and DTH
responses.
Weekly treatment Ex vivo Con A
5 of mice Conc: induced TNF-~
with LMWH ~g/mouse secretion DTH responses pq/ml % DTH %
Inhibition Inhibition Fragmin~
Batch 38609 None 450 - 25 : 0.5 425 5 21 12 Batch 45389 None 320 - 28 0.1 280 13 26 6 , 5 260 18 24 13 Clexane~
Batch 2088 None 400 - 22 0.1 360 10 17 23 ; Batch 2066 None 350 - 23 0.1 338 6 20 13 : 5 192 57 7 70 : 10 186 55 7 70 25 Batch 4096 None 320 - 24 0 325 o 26 0 5.7. Example 7: Treatment of Adjuvant Arthritis (AA) in Rats with Low Doses of LMWHs Adjuvant arthritis is an experimental disease inducible in some strains of rats by immuni2ing them to antigens of Mycobacterium ` tuberculosis (Pearson, C.M., Proc. ~oc. Exp. Biol.
~ed. (1956) 91:91). This experimental disease is considered to be a model of human rheumatoid arthritis - .
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~2/l~249 PCT/US92/03626 -- 2 3 -- ,i ,~
(Pearson, C.M., Arthritis Rheum. (1964) 7:80). The arthritis appears to be caused by T lymphocytes that recognize an antigen of M. tuberculosis that is cross-reactive with structures in the joint tissues ~Cohen, I.R., et al., Arthritis Rheum. (1985) 28:841).
Lewis rats were immunized with M.
tuber-culosis (1 mg) in oil to induce adjuvant arthritis (Pearson, C.M., Proc. Soc. Exp. Biol. Med.
(1956) 91:91). Five days later the rats were inoculated subcutaneously as indicated with the doses of LMWH and/or heparin and scored for the development of arthritis on a scale of 0-16 as described (Holoshitz, Y., et al., Science (1983) 219:56). All the experiments were performed with Fragmin~, Batch 38609.
In order to study the dose response to Fragmin~ (Fig. 1) rats immunized to induce A~ were injected subcutaneously weekly, starting on the 5th day after injection with 0.5 ~g (o), 1 ~g (~), 2 ~g (-), 10 ~g (0), 15 ~g (~) 20 ~g (~); 30 ~g (-), 40 ~g (x) and PBS control (0). The dose was maximally effective in inhibiting arthrit:is.
The effect of the 20 ~g dose of Fragmin~ on the course of AA is shown in Fig. 2: PBS control (~);
single treatment on 5th day (~); daily (-); every 5th day (o); weekly (~). It is shown that Fragmin administration both at 5 day intervals and at 7 day intervals inhibits arthritis.
Fig. 3 shows the effect of weekly administration of ~ragmin~ (batch 38609) as compared to standard heparin on AA. Lewis rats were immunized to induce AA. Beginning on day 5, the rats were inoculated subcutaneously at weekly intervals with a 20 ~g dose of Fragmin~ (-), heparin (o) or phosphate buffered saline (PBS) control (0). The results show a dramatic difference in potency between Fragmin~ and :`
.
`.'` ' ' , ' ~. ' : ` .
, ~
W0~2/l9~4'~ PCT/US~2/03626 ~- J~ r ~ v? 7 ~ 4 heparin: Fragmin0 completely inhibited arthritis, while heparin had no inhibitory effect.
No inhibitory effect on A~ was found with daily administration of a 20 ~g dose of LMWH, although surprisingly the inhibitory effect of heparin was stronger than that of Fragmin~ in daily administration, as shown in (Fig. 4: Fragmin~ (batch 38609)(~), heparin (o), PBS control (n)).
A similar inhibitory effect was observed with several other LMWHs administered to Lewis rats immunized to induce AA. Fig. 5 shows the results of the injection of a 20 ~g dose of Fraxiparin~ (daily (0); weekly (t)); Fraxiparine~ (daily (~); weekly (-)), Lovenox~/Clexane~ (daily (~); weekly (o)), and PBS
control (x). All the three LMW~ls of different types and sources showed a marked inhibition of arthritis, when administered weekly, but not daily.
5.8. Example 8: Treatment With LMWH Prevents Rejection of Alloqrafts Wistar rats were subjected to alloge~eic BN
heart transplant (Ono, K. and Linsay, E.S., J. Thorac.
Cardiovasc. Sura. (1969) ~5:225-229). From the day - before transplantation, the rats were injec~ed subcutaneously at 7 day intervals with 20 ~g of Fragmin~ or PBS control (Fig. 6, ~ and O, respectively~
and scored for survival. The day of rejection was determined as the day the transplanted heart stopped be.ating, assayed by palpation o~ the abdomen. Fig. 6 shows that the rats treated with the weekly dose of LMWH had a markedly increased survival of the heart allografts.
.:
S.9. Example 9: Biological Effect of LNWH
On Insulin Dependent Diabetes Mellitus (IDDML of NOD Mice ~- Mice of the NOD strain spontaneously develop a form of type I insulin dependent diabetes mellitus ~` .
, , ' ` ' ., , W~')2/l'~2~t~ PCT/~S92/03626 - 25 - ~, ii ,'J ~
(IDDM) that is the accepted model for human IDDM
(Castano, L. and Eisenbarth, G.S., Annu. Rev. Immunol.
(1990) 8:647-679). The disease begins at 4-5 weeks of age by the appearance of inflammation of the pancreatic islets, insulitis. The insulitis progressively damages the insulin-producing beta cells which are sensitive to damage by TNF-~. At about 4-5 months of age, a sufficient number of beta cells are destroyed so that diabetes becomes overt.
To test whether treatment with LMWH could affect the IDDM process, a group of 10 female NOD mice was treated with weekly subcutaneous injections of 5 ~g per mouse of Fragmin~ (batch 38609~, the dose determined to represent 12 mouse units per kg. A
group of 10 control mice were treated with injections of saline. At the age of 5 months all the mice were bled to determine ~he development of IDDM using a standard procedure (Elias, D. et al., Proc. Natl.
Acad. Sci. U.S.A. (1990) 87:1576-1580). Fig. 7 shows that the control mice ("none") had abnormal blood glucose (400 mg/dl). In contrast the mice treated with LMWH had a normal blood glucose ~100 mg/dl).
Thus treatment with LMWH can indeed cure the IDDM
process.
5.1Ø Example 10: LMWH l`reatment of Human D~H
Figure 8 shows an experiment in which a 40 year old male volunteer weighing 85 kg was tested for DTH reactivity to tetanus antigen (Merieux skin test applicator). About 18 mm of induration was measured at 24 and 48 hours. The volunteer was then treated with a subcutaneous injection of Fragmin~ ~batch 38609) 3 mg. Fi.ve days later the volunteer was again tested for his DTH response to tetanus and the induratio~ was inhibited to about 5 mm. The volunteer was tested again for DTH 3 weeks later ("Recovery") and the test showed positive reactivity (23 mm of ~' ' .
W0~)2/1~4') PCT/US92/03~t6 induration at 24 and 48 hours). The volunteer was then treated with Fragmin~ as before and the DTH
reactivity was measured again 7 days later ("7 days post"). Again the DTH was inhibited to about 5 mm of induration. Recovery of DTH again was found 3 weeks later. Thus, LMWH at a dose of less than 5 mg can inhibit DTH in humans at treatment intervals of 5 and 7 days.
It should be apparent to those skilled in the art that other compositions and methods not specifically disclosed in the instant specification are, nevertheless, contemplated thereby. Such other compositions and methods are considered to be within the scope and spirit of the present invention. Hence, :L5 the invention should not be limited by the description of the specific embodiments disclosed herein but only by the following claims.
.
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COMPOSITIONS FOR T~E PR~VENTION AND/OR
TREATMENT OF PATHOLOGICAL PROCESSES
1. Field of the Invention 5The present invention relates to pharmaceutical compositions for the prevention and/or treatment of pathological process~s involving induction of Tumor Necrosis Factor alpha (TNF-~) secretion, which comprise an effective low dosage of a low molecular weight heparin (LMWH) to be administered at intervals of between five to eight days.
The LMWHs to be used according to the invention have an average molecular weight of from about 3,000 to about 6,000 and inhibit TNF-~ secretion by resting T cells and/or macrophages in vitro in response to activation by T cell-specific antigens, ; T cell mitogens, macrophage activators, disrupted extracellular matrix (dECM), fibronectin, laminin or other ECM components.
. Backqround of the Invention TNF-~, a cytokine produced by monocytes (macrophages) and T lymphocytes, is a key element in the cascade of factors that produce the inflammatory response and has many pleiotropic effects as a major orchestrator of disease states (B. Beutler and A.
C~rami, Ann. Rev. Immunol. (1989) 7:625-655).
~ The biologic effects o~ TNF-~ depend on its - concentration and site of production: at low ~- 30 concentrations, TNF-~ may produce desirable ; homeostatic and defense functions, but at high concentrations, systemically or in certain tissues, TNF-~ can synergize with other cytokines, notably interleukin-1 (IL-1) to aggravate many inflam~atory responses.
The following activities have been shown to b~ induced by TNF-~ (together with IL-l); fever, ..
~' :
WO'~ 24~ PCT/US92/~3626 slow-wave sleep, hemodynamic shock, increased production of acute phase proteins, decreased production of albumin, activation of vascular endothelial cells, increased expression of major histocompatibility complex (MHC) molecules, decreased lipoprotein lipase, decreased cytochrome P450, decreased plasma zinc and iron, fibroblast proliferation, increased synovial cell collagenase, increased cyclo-oxygenase activity, activation of T
cells and B cells, and induction of secretion of the cytokines, TNF-~ itself, IL-l and IL-6.
How TNF-~ exerts its effects is not known in detail, but many of the effects are thought to be related to the ability of TNF-~ to stimulate cells to produce prostaglandins and leukotrienes from arachidonic acid of the cell membrane.
TNF-a, as a result of its pleiotropic effects, has been implicated in a variety of pathologic states in many different organs of the bo~y. In blood vessels, TNF--~ promotes hemorrhagic shock, down regulates endothelial cell thrombomodulin and enhances a procoagulant activity. It causes the adhesion of white blood cells and probably of platelet~ to the walls of blood vessels, and so, may promote processes leading to atherosclerosis, as well as to vasculitis.
TNF-~ activates blood cells and causes the adhesion of neutrophils, eosinophils, monocytes/
macrophages and T and B lymphocytes. By inducing IL-6 and IL-8, TNF-~ augments the chemotaxis of inflammatory cells and their penetration into tissues.
Thus, TNF-~ has a role in the tissue damage of autoimmune diseases, allergies and graft rejection.
TNF-~ has also been called cachectin because it modulates the metabolic activities of adipocytes and contributes to the wasting and cachexia accompanying cancer, chronic infections, chronic heart .
,. ~
W~'J~ PCTtUS92/03626 failure, and chronic inflammation. TNF-a may also have a role in anorexia nervosa by inhibiting appetite while enhancing wasting of fatty tissue.
TNF-~ has metabolic effects on skeletal and cardiac muscle. It has also marked effects on the liver: it depresses albumin and cytochrome P450 metabolism and increases production of fibrinogen, ~l-acid glycoprotein and other acute phase proteins.
It can also cause necrosis of the bowel.
In the central nervous system, TNF-~ crosses the blood-brain barrier and induces fever, increased sleep and anorexia. Increased TNF-~ concentration is associated with multiple sclerosis. It further causes adrenal hemorrhage and affects production of steroid hormones, enhances collagenase and PGE-2 in the skin, and causes the breakdown of bone and cartilage by activating osteoclasts.
In short, TNF-a is involved in the pathogenesis of many undesirable inflammatory conditions in autoimmune diseases, graft rejection, vasculitis and atherosclerosis. It may have roles in heart failure, in the response to cancer and in anorexia nervosa. For these reasons, ways have been sought to regulate the secretion of TNF-~ as a means to control a variety of diseases.
The prime function of the immune system is to protect the individual against infection by foreign invaders such ~s microorganisms. It may, however, also attack the individual's own tissues leading to pathologic states known as autoimmune diseases. The aggressive reactions of an individual's immune system against tissues from other individuals are the reasons behind the unwanted rejections of transplanted organs.
Hyper~reactivity of the s~stem against foreign substances causes allergy giving symptoms like asthma, rhinitis and eczema.
W0~)2/l~249 PCT/US92/03626 4 -- _~
r ~ , The cells mastering these reactions are the lymphocytes, primarily the activated T lymphocytes, and the pathologic inflammatory response they direct depends on their ability to traffic through blood vessel walls to and from their target tissue. Thus, reducing the ability of lymphocytes to adhere to and penetrate through the walls of blood vessels may prevent autoimmune attack, graft rejection and allergy. This would represent a new therapeutic principle likely to result in better efficacy and reduced adverse reactions compared to the therapies used today.
Atherosclerosis and vasculitis are chronic and acute examples of pathological vessel in~lammation. Atherosclerosis involves thickening and rigidity af the intima of the arteries leading to coronary diseases, myocardial infarction, cerebral infarction and peripheral vascular diseases, and represent~ a major cause of morbidity and mortality in the Western world. Pathologically, atherosclerosis develops slowly and chronically as a lesion caused by fatty and calcareous deposits. The proliferation of fibrous tissues leads ultimately to an acute condition producing sudden occlusion of the lumen of the blood vessel.
TNF-~ has been shown to facilitate and augment human immunodeficiency virus (HIV) replication in vitro (Matsuyama, T. et al., J. Virol. (19~9) 63(6):2504-2509; Michihiko, S. et al., Lancet (1989) 30 1(8648~:1206-1207) and to stimulate HIV-1 gene expression, thus, probably triggering the development of clinical AIDS in individuals latently infected with HIV-l (Okamoto, T. et al., AIDS Res~ Hum. Retroviruses (1989) 5(2):131-138).
Heparin is a glycosaminoglycan, a polyanionic sulfated polysaccharide, which is used ciinically to prevent blood clotting as an W~2/l~2~l~ PCT/US92/03626 - 5 - ;.~
antithrombotic agent. In animal, models heparin has been shown to reduce the ability of autoimmune T
lymphocytes to reach their target organ (Lider, 0. et al., Eur. J. Immunol. (1990) 20:493-499). Heparin was also shown to suppress experimental autoimmune diseases in rats and to prolong the allograft survival in a model of skin transplantation in mice, when used in low doses ~5 ~g for mice and 20 ~g for rats) injected once a day (Lider, 0. et al., J. Clin.
Invest. (1989) 83:752-756).
The mechanisms behind the observed effects are thought to involve inhibition of release by T
lymphocytes of enzyme(s) necessary for penetration of the vessel wall, primarily the enzyme heparanase that specifically attacks the glycosaminoglycan moiety of the sub-endotheli.al extracellular matrix (ECM) that lines blood vessels (Naparstek, Y. et al., Nature ~1984) 310:241-243). Expression of the heparanase enzyme is associated with the ability of autoimmune T
lymphocytes to penetrate blood vessel walls and to attack the brain in the model disease experimental autoimmune encephalomyelitis (EAE).
European Patent Application EP 0114589 (Folkman et al.) describes a composition for - 25 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-~ isomer of hydrocortisone~ According to the disclosure, heparin by itself or cortisone by itself are ineffective; only the ombination of both gives the desired sffects. Although there is no proof in the literature that there is a connection between anyiogenesis and autoimmune diseases, the descxiption on page 5 of the patent application connects angiogenesis with psoriasis and with arthritis, .. , :
~, .
WCl~)2/l~2~l9 PCT/US92/03~26 6 - ~ ~
L ~ .L U ~ J )~
indicating the use of high doses of 25,000 units to 47,000 units of heparin per day.
Horvath, J.E. et al., Aust. N.Z.J. Med.
(1975) 5(6):537-539 describe the effect of subanticoagulant doses of subcutaneous heparin on early renal allograft function. The daily dosage is high (5000 U) and the conclusion of the study is that heparin in subanticoagulant doses has no effect on early graft function or graft survival and that it may be associated with increased hemorrhagic complications. `
Toivanen, M.L. et al., Meth. and Find. Exp.
Clin. Pharmacol. (1982) 4(6):3S~-363, examined the effect of heparin in high dosage (lOoO U/rat) in the inhibition of adjuvant arthritis in rats and found that heparin enhanced the severity of the rat adjuvant arthritis.
PCT Patent: Application PCT/AU88/OOQ17 published under No. W088/0530:L (Parish et al.) ;~ 20 describes sulphated polysaccharides that block or inhibit endoglycosylase activ:ity, such as heparanase activity, for use as antimetastatic and anti-inflammatory agents. Heparin and heparin derivatives, such as periodate oxidized, reduced heparins, that had negligible ant.coagulant activity, were shown to have antimetastatic and anti-inflammatory activity when used in dosages within, the range of 1.6-6.6 mg per rat daily, administered by constant infusion (corresponding to 75-308 mg daily for an adult human patient).
3. Summary of the Invention The present invention is related to pharmaceutical compositions for the prevention and/or treatment of pathological processes in~olving the induction of TNF-a secretion comprisiny a pharmaceutically acceptable carrier and a low WO92/1()2~1~ PC~/US92/03626 - t _ ; ~
molecular weight heparin (LMWH) present in a low effective dose for administration at intervals of about 5-~ days and which LMWH is ~apable of inhibiting in vit~o TNF-~ secretion by resting T cells and/or macrophages in response to T cell-specific antigens, mitogens, macrophage activators, disrupted extracellular matrix (dECM), laminin, fibronectin, or other ECM components.
In a particular embodiment of the present invention the LMWH of the pharmaceutical composition has an average molecular weight of from about 3,000 to about 6,000 and, furthermore, may be administered every fifth or seventh day.
It is also an objective of the present invention to provide pharmaceutical compositions to be administered at intervals of about 5-8 days for the p~evention and/or treatment of pathological processes involving the induction of TNF--~ secretion comprising a pharmaceutically acceptable carrier and a low molecular weight heparin (LMWH) present in a low effective dose and which LMWH is capable of inhibiting experimental delayed type hypersensitivity (DTH) reactions to an applied antigen as evidenced by a reduction in the induration observed after the application of the antigen to the skin five to seven days af~er the administration of the pharmaceutical composition relative to the induration observed after the application of the antigen to the skin in the absence of or after recovery from the administration of the pharmaceutical composition. Examples of the applied antigen include, but are not limited to, tetanus, myelin basic protein, purified protein derivative, oxazolone, and the like.
~urthermore, it is an objective of the present invantion to provide pharmaceutical compositions that may be administered in any manner as dictated ky the particular application at hand ., i , W~92/1')~4') PCT/US92/03626 , " ,j ~ ~."j ., including, but not limited to, enteral administration (including oral) or parenteral administration (including topical or inhalation with the aid of aerosols). In preferred embodiments, the pharmaceutical compositions of the present invention are administered subcutaneously or intravenously.
Thus, the present invention is useful, for example, in delaying or preventing allograft rejection and treating or preventing a variety of patholo~ical processes such as those related to autoimmune diseases, allergy, inflammatory diseases (in particular, inflammatory bowel disease), or acquired immunodeficiency syndrome (AIDS). The present invention also finds utility in the treatment of diabetes type I, periodontal disease, uveitis, rheumatic diseases (in particular, rheumatoid arthritis), atherosclerosis, vasculitis, or multiple sclerosis.
The pharmaceutical compositions of the present invention contain low effective doses of the prescribed LMWH and typically contain a single low dose unit of less than 5 mg of hMWH, preferably from about 0.3 to about 3 mg, and most preferably contain a sin~le low dose unit of from 1 to 1.5 mg.
The present invention also contemplates broadly a method of using a low molecular weight heparin (LMWH) which is capable of inhibiting in vitro TNF-~ secretion by resting T cells and/or macrophages in response to T cell-specific antigens, mitogens, macrophage activators, disrupted extracellular matrix (dECM), laminin, fibronectin, or other E~M components for the preparation of a pharmaceutical composition to be administered at intervals of about 5-8 days for the prevention and/or treatment of pathological processes involving induction of TNF-~ secretion which method comprises combining a low effective dose of the LMWH
with a pharmaceutically acceptable carrier.
, W~92/l924~ PCTtUS92/03626 C~
A still further object of the present invention is to provide a method of treating a subject suffering from a pathological process involving induction of TNF-a secretio~ comprising administering to such subject a pharmaceutical composition, as described above, at intervals of about 5-8 days.
Further objects of the present invention will become apparent to those skilled in the art upon further review of the following disclosure, including the detailed descriptions of specific embodiments of the invention.
4. Detailed Descri~tion of the Invention According to the present invention, it was found that treatment with low molecular weight heparins (LMWHs) inhibited the ability of T cells and macrophages to secrete TNF-a. The functional expression of this effect can be seen in the inhibition in mice and humans of the dalayed type 2~ hypersensitivity (DTH) reaction, an inflammatory reaction triggered by cells involving macrophages and other inflammatory cells. Treatment with LMWHs at doses affecting TNF-a produc ion also was able to ` inhibit a model of autoimmune arthritis called adjuvant arthritis (AA). LMWH treatment also prolonged the survival of allogeneic heart transplants in rats and abrogated insulin dependent diabetes mellitus (IDDM) in NOD mice. Moreover, treatment with LMWHs prevented the induction of TNF-a production by T
cells and macrophages in response to the stimulus of damaged subendothe:lial extracellular matrix (ECM).
Since TNF-a at the site of vascular injury has probably a role~in the process of atherosclerosis inhibition of TNF-a at the site o~ damaged subendothelial ECM will ameliorate the pathogenic ~ process of atherosclerosis. A most surprising aspect :`~ of treatment with LMWHs is that such treatment is most . .
:
~ .
.
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W~')2/l~2~t') PCT/US92/03626 1 0 -- '`
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effective when administered at low doses at weekly intervals. High doses of LMWHs or doses of LMWHs given daily are not effective in inhibiting TNF-~
secretion or immune reactions.
Low molecular weight heparins, produced by fractionation or controlled depolymerization of heparins, show improved antithrombotic performance but also different pharmacokinetic properties as compared to heparin: the half-life is doubled and the bioavailability is higher with respect to their, anticoagulant effect after subcutaneous injection (Bratt, G. et al. Thrombosis and Haemostasis (1985) 53:208; Bone, B. et al., Thrombosis Research (1987) ~6:845).
Accordiny to the present invention it has now been found that LMW~s administered at subanticoagulant doses at several day intervals are effective in the prevention and/or treatment of pathological processes involving induction of TNF-~.
The LMWHs to be used according to the invention are derived from LMWHs with an average molecular weight of 3000-6000, such as, for example the LMWHs disclosed in European Patent EP 0014184.
Some LMWHs are commercially available under different trade names, e.g., Fragmin~, Fraxiparin~, Fraxiparine, Lovenox~/Clexane~.
LMWHs can be produced in several different ways: enrichment by fractionation by ethanol and/or molecular sieving, e.g., gel filtration or membrane filtration of the LMWH present in standard heparin and controlled chemical ~by nitrous acid, ~-elimination or periodate oxidation~ or enzymatic (by heparinases) depolymerizatior.- The conditions for depolymerization can be carefully controlled to yield products of desired molecular weights. Nitrous acid depolymerization is commonly used. Also employed is depolymerization of the benzylic ester of heparin by .
W~ 2/l9249 PCT/US~2/03626 ~-elimination, which yields the same type of fragments as enzymatic depolymerization using heparinas~s. LMWH
with lo~ anticoagulant activity and retaining basic chemical structure can be prepared by depolymerization 5 using periodate oxidation or by removing the antithrombin-binding fraction of LMWH, prepared by other methods, using immobilized antithrombin for adsorption.
Fragmin~ is a low molecular weight heparin with average molecular weight within the range of 4000-6000 dalton, produced by controlled nitrous acid depolymerization of sodium heparin from porcine intestinal mucosa. It is manufactured by Kabi Pharmacia, Sweden, under the name Fragmin~, for use as an antithrombotic agent as saline solutions for injection in single dose syringes of 2500 IU/0.2 ml and 5000 IU/0.2 ml, corresponding to about 16 mg and 32 mg, respectively.
Fraxiparin~, and Fraxiparine~ are LMWHs with average molecular weight of approximately 4500 dalton, produced by fractionation or controlled nitrous acid depolymerization, respectively, of calcium heparin from porcine intestinal mucosa. It is manufactured by Sano~i (Choay Laboratories) for use as an antithrombo-tic agent in single doses comprising ca. 36 mg,corresponding to 3075 IU/0.3 ml of water.
Lovenox~ (Enoxaparin/e), a LMWH fragment produced by depolymerization of sodium heparin from porcine intestinal mucosa, using ~-elimination, is manufactured by Pharmuka SF, France and distributed by Rhone-Poulenc under the names Clexane~ and Lovenox~
for use as antithrombotic agent in single dose syringes comprising 20 mg/0.2 ml and 40 mg/0.4 ml of ~ater .
As shown in the present application, the novel properties of LMWHs described are a common feature o~ all, LMWHs regardless of the manufacturing ' ~2/l(~24~) PCT~US92/03626 1 ~
!
U ,~
process, the structural differences (created by depolymerization or those dependent on variation in the heparin used as raw material) or the anticoagulant activity, provided the LMWH employed is capable of inhibiting TNF-~ secretion in vitro by resting T cells and/or macrophages in response to activation by contact with specific antigens, mitogens, disrupted ECM or its protein components, such as fibronectin or laminin.
Another test useful to identify the LMWHs that are effective for the purpose of the present invention is the inhibition of experimental delayed type hypersensitivity (DTH) skin reactions, a T
lymphocyte dependent reaction, to a variety of antigens tfor example, tetanus antigen, myelin basic protein (MBP), purified protein derivative (PPD), and oxazolone). The LMWHs also inhibit T cell adhesion to ECM and its protein components.
The LMWHs effective according to the invention are incorporated into pharmaceutical compositions, for example, as water solutions, possibly comprising sodium chloride, stabilizers and other suitable non-active ingredients. The preferable way of administration is by injection, subcutaneous or intravenous, but any other suitable mode of administration is encompassed by the invention.
According to the invention, the LMWH is to be administered at intervals of five to eight days, preferably once a week.
The disorders that can be prevented or treated according to the invention are all disorders linked in pathological processes involving induction of TNF-~ secretion, including atherosclerosis and Yasculitis and pathological processes related thereto;
autoimmune diseases, e.g., rheumatoid arthritis, diabetes type I (ID~M); allergy; gra~t rejection;
acute and chronic inflammatory diseases, e.g. uveitis, ' ` .
<
W~92/19~49 PCT/US92/03626 .~'?~?,~
13 ~ r ~ ,J~
bowel inflammation; anorexia nervosa; hemorrha~ic shock caused by septicemia, and opportunistic infections in AIDS - immunocompromised individuals.
In AIDS, the LMWHs will suppress replication of HIV
and/or enhance resistance against the opportunistic infections, thereby preventing the development of AIDS-related complex (ARC).
It was observed according to the present invention that LMWHs can have a narrow dose range of effectiveness, and that different preparations of LMWHs can show different optimal biological effects.
Therefore, it was necessary to establish a Unit Dose for LMWH preparation to be usPd according to the invention, said unit dose being based on a bioassay of inhibition of TNF-a secretion by mouse spleen cells from mice treated with LMWH or by inhibition of DTH
reactivity by the L~WH. It was found that the ability of a particular dose of LMWH to inhibit TNF-~secretion is positively correlated with its ability to inhibit the delayed type hypers~ensitivity (DTH) reaction. This, cell-mediated inElammatory reaction in vivo is important because it is an expression of the processes involved in autoimmune diseases, graft rejection, some types of blood vessel inflammation and allergy.
It was thus established according to the invention that the lowest dose of LMWH per kg causing inhibition of TNF-~, production or inhibition of DTH
reactivity by at least 50% is considered to constitute 12 mouse inhibitory units per kg (12 u/kg). Because of the differences in surface area and metabolism between mice and humans, humans should be treated with a lower dose of LMWH, and 12 u/kg in mouse is established to correspond to 1 u/kg in humans. For example, the dose of Fragmin~ batch 38609 effective in ` inhibiting both TNF-~ secretion and DTH reactivity is 5 ~g per mouse administered weekly. Since each mouse .
~' ' ' - : `
W0~2/l924~ PCT/US92/03626 , 14 - t weighs about 25 g, the dose of Fragmin~ 38609 equivalent to 12 u/kg is 200 ~g/kg of mice. The dose of 1 u/kg suitable for humans is therefore 200 ~g/kg:
12 = 16.67 ~g/kg. A human weighing about 70 kg would then be treated by a dose of about 1.2 mg given in a single dose subcutaneously once every 7 days. Since individual humans vary biologically, the optimal dose may be different from about 1.2 mg and will lie below 5 mg, particularly within the range of 0.3 to 3 mg.
The dose of LMWH that should be effective in rats can be derived ~rom the fact that the dose of LMWH per kg of rats is one-half the dose per kg of mice, i.e. 6 u/kg. For example, if 12 u of Fragmin~
batch 38609 is 200 ~g/kg, then the 6 u dose suitable for rats should be 100 ~g/kg or 20 ~g per 200 g rat, administered once a week.
The invention will now be illustrated by the following non-limiting examples.
5. Exam~les 5.1. Example 1: Bioassay of Inhibition of TNF-a Secretion Supernatants of spleen cells cultured in the presence or absence of LMWH, or spleen cells obtained from mice treated or untreated with LMWH in vivo are analyzed for their ability to secrete TNF-~. The TNF-~ bioassay is based on the cytotoxic effect of TNF-~ on cycloheximide (CHI)-sensitized cells and its quantitation by the neutral red uptake assay as described by Wallach D., J. ImmunoloqY (1984~
132:2464-2469. Briefly, the killing of CHI-sensitized HeLa cells by TNF-~ present in the supernatants of the cells is measured, the concentration of TNF- in said supernatants being determined by comparison to titration curves of TNF-~ exogenously added. Cell viability is determined by incubation with neutral red for two hours, washing away excess dye, extracting the .. .
.
WO !~2/1924~) P~/US92/03~r26 ~ 1 5 ~ v u 7 neutral red that was taken up by the cells with Sorenson's citrate buffer-ethanol mixture, and quantitating it colorimetrically at 570 nm with a Microelisa Autoreader.
Cells from mice treated with LMWH are obtained as follows: female mice of the BALB/c strain t25 grams, 2 months old), at least 5 mice per group, are injected subcutaneously with various doses of LMWH, usually in the range of 0.5 to 20 ~g per mouse.
Five days later the mice are killed by cervical dislocation, the spleens are removed and suspensions of spleen cells, depleted of red blood cells-, are assayed for the production of TNF-~ in response to induction by dECM, Concanavalin A (Con A) or lipopolysaccharide (LPS).
5.2. Example 2: Bioassay of Inhibition of DTH Reactivitv Groups of mice are sensitized on the shaved abdominal skin with 100 ~l of 3% oxazolone (OX) in acetone/olive oil (4:1, v:v). DTH sensitivity is elicited 5 days later as follows: mice are challenged with 20 ~l of 0.5% OX (10 ~l to each side of the ear) in acetone/olive oil. A constant area of the ear is measured immediately before challenge and 24 and 48 h later with a Mitutoyo engineer's micrometer. The ;; individual measuring ear swelling is blinded to the identity o~ the groups of mice. The increment ~) of ear swellinq is expressed as the mean in units of 10-2 mm or 104 inch (+SE) depending on the micrometer that is used. Percent inhibition is calculated as follows:
Po~itive control - experimental group Inhibition = - x 100 Posltive control - negative control Mice are treated with LMWH as in Example 1, injected the day before primary sensitization to OX.
On the fifth day aEter sensitization to OX, the mice .
W0~2/192~9 PCT/US92/036~6 h ~
are challenged to induce a DTH reaction, as described above.
The positive control is the DTH reaction elicited in immunized mice in the absence of treatment with LMW~. The negative control is the background swelling produced by the antigen in immunized mice.
5.3. Example 3: Induction of TNF~a Secretion by T Cells and Macrophaqes In Vitro Microtiter plates were prepared as follows:
fibronectin (FN) or laminin (LN) (Sigma) were added to flat bottom 96-well plates (Costar) at a concentration of 1 ~g/50 ~1 PBS per well and removed after 16 h.
Remaining binding sites were blocked with BSA/PBS (lo mg/ml) which was added to the wells for 2 h and washed out. ECM-coated wells were prepared as follows:
bovine corneal endothelial cells were cultured in flat bottom 96-well plates. Thc confluent layers of endothelial cells were dissolved and the ECM was le~t intact free of cellular debris (Gospodarowicz, D. et al. J. Biol. Chem. (1978) 253::3736). Disrupted ECM
(dECM) was prepared by gently scratching the ECM three times with a 27G syringe needle and the exposed sites were subsequently coated with BSA/PBS. Resting cloned rat CD4+ T cells, designated Kl, which recognize myelin basic protein (MBP), were propagated and maintained in culture and were added to the wells, 105 cells per well with or without 3 x 105 syngeneic splenic macrophages, in 100 ~l per well RPMI 1640 (Gibco) supplemented with 1% BSA and antibiotics. The splenic macrophages were purified by removing the T
and B cells using specific monoclonal antibodies (mAb). Where indicated, anti-murine TNF-a mAb (Genzyme, diluted 1/300) were added to the wells. MBP
(100 ~g~ml), Con A (2.5 ~g/ml), LPS (1 ~g/ml) were added to the wells where indicated. The plates were incubated at 37 ~C in a humidified incubator for 3 h.
WO92/1~2~9 PCT/US92~03626 - 17 - ~ J ~
Subsequently, the contents of the wells (4 wells per experimental group) were collected, centrifuged, and the media were assayed for TNF-~ secretion as in Example 1: supernatants of cultured macrophages and lymphocytes were added to cultures of HeLa cells, which are sensitive to killing by TNF-a, and death of these cells in the presence of the test media was calibrated in comparison to titration curves of exogenous added TNF-a. Cell death is examined by the release of neutral red dye from the preincubated HeLa cells. The results shown here represent data obtained from a total of six experiments that produced essen~ially similar results.
Table 1 shows that T cells and macrophages cultured together can be induced to secrete TNF-a by contact with specific antigen MBP (group 4), the mitogen Con A (group 6) or LPS (group 8). However, in the absence of antigenic or ]nitogenic stimulus, TNF-a was also induced to be secreted by disrupted extracellular matrix (dECM; group 10) or by the ECM
components fibronectin (FN; group 12) or laminin (LN
group 14). Intact ECM was a weak inducer of TNF-a (group 16).
~5 Ta~le 1. TNF-~ secretion by T cells and macrophages is induced by specific antigen MBP, Con A, LPS or dECM
; ` components.
~1 cells cultured together with Secreted TNF-a(yes) or without TNF-~
30 Group inducer(no) macrophages (pg/ml) ._ 1 none no 50 2 yes 65 3 MBP antigen no 30 4 yes 950 Con A no 120 6 yes 1300 .
, , W0~2/l')2tt~ PCT/US92tO3626 7 LPS no 50 8 yes 1500 g dECM no 30 5 10 yes goo 11 FN no 20 12 yes 650 13 LN no 50 14 yes 500 10 15 ECM no 30 16 yes 120 5.4. Example 4: Regulation of TNF-a Secretion b~ LMWHs 15 T cell and accessory cell cultures were prepared as described in Example 3. LMWH was added to the wells at the beginning of the cell culture. The levels of TNF-~ were examined after 3 h of incubation.
Table 2 shows that the presence of LMWH
~Fragmin~ batch 38609) in vitro inhibited the induction of TNF-~ secretion induced by specific antigen (MBP; group 4), mitogensl (Con A and LPS;
groups 6 and 8), dECM or ~CM components (groups 10, 12 and 14). Since TNF-~ secretion induced by dECM is likely to be involved in atherosclerosis, inhibition of TNF-~ by LMWH will be beneficial in atherosclerosis.
Table 2. Induction of TNF-~ secretion induced in vitro is inhibited by L~H ~Fragmin~ batch 38609).
Secretion of TNF-~ by cultures of ; - T cells and TNF-~ LMWHmacrophages 35 Group Inducer(1 ~g/ml)(pg/ml) . . _ - 1 none none 65 ~ yes 30 .
: ,;,: , ~, :`' ijW0~2/1~249 PCTtUS92/036~6 -- 19 ~ ~ J S`~ f 3 MPB antigen none 950 4 yes 60 Con A none 1300 5 6 yes 80 7 LPS none 1500 8 yes 80 9 dECM none goo I 10 yes go ' 10 11 FN none 650 12 yes go 13 LN none 500 14 none 70 ~3 15 3 5.5. Exam~le 5 To examine the ef~ect of LMWH administered ~' to mice in vivo on the secretion of TNF-~ by spleen cells in vitro, the following experiment was conducted. ~ALB/c mice, 5 pe~r group, were treated With various doses of LMWH tE~ragmin~ batch 38609) dilute.d in saline, injected subcutaneously. After one week, the animals were-killed and their spleen cells, devoid of red blood cells, were examined for their ability to secrete TNF-~ in response to control wells ; without dECM (A) or to wells coated with dECM (B).
Measuring the levels of TNF-~ secretion was done as described in Example 1. Table 3 shows the results which indicate that an injection of 5 ~g of LMWH given once, 7 days earlier, inhibited TNF-~ secretion induced by dECM. H ~hsar or lower doses of LMWH were less effective. Th~s, an optimal dose of LMWH
administered in vivo a week earlier was effective.
.
~ 35 .
': '.::. :
,: -W~2/1~2~9 PCT/~92/03626 r TAble 3. Ex vivo inhibition of T cell mediated TNF-a secretion in response to damaged ECM.
In vitro TNF-a secretion (pg/ml) by spleen cells LMWH cultured on:
treatment of BALB/c mice A. None B. Damaged ECM
(weekly) (% Inhibition) 1 None 30 400 2 0.5 ~g 50 380 (5) 3 1 ~g 25 90 (78) 4 5 ~9 25 60 (85) 10 ~g 30 140 (65) 6 20 ~g 40 320 (20) Table 4 shows that a 5 ~g dose in vivo of the LMWH Fragmin~ batch 38609 was also e~fective in inhibiting TNF-a secretion induced by LPS. BALB/c t4 mice per experimental group) mice were treated with the indicated amounts of LMWH diluted in saline and injected subcutaneously. After one week, the mice were injected intraperitoneally with 10 mg LPS, killed 4 hours later and their spleen cells, devoid of red blood cells, were subsequently cultured in dECM coated wells for 3 hours in a humidif:ied incubator. The levels of TNF-a secreted in response to the dECM was measured in the supernatants of the cultures. The results are given in Table 4.
Table 4. Treatment of mice with LMWH inhibits LPS
mediated secretion of TNF-a by macrophages.
.
In vitro TNF-~
LMWH secretion by treatment macrophages (pg/ml~
of mice t~g) in response to LPS % Inhibition 35 690 __ 0.1 500 28 , .
::`
. .
' ~: ' W~2/l~24~ Pcr/uss2/o3626 2 ~ 7 2 l 5.6. Example 6 To examine the effect of different LMWHs on the inhibition of TNF-~ secretion and on DTH
responses, mice were treated with th~ indicated LMWH
administered subcutaneously in different concentrations. After one week, some of the mice were killed and the induction of TNF-~ secretion in response to Con A activation in vitro was measured.
The remaining mice were examined for their ability to respond to the antigen oxazolone. The results are express~d in Table 5 as percent inhibition compared to the responses of the LMWH untreated mice.
Two conclusions can be made by inspecting the results shown in Table 5:
1. Different batches of LMWH, each 2~ calibrated for by similar antithrombotic effect (factor X assay) have different optimal doses for inhibition of TNF-a secretion. Moreover, there are preparations of LMWH, such as Clexane~ batch 4096, which have no inhibitory effect on TNF-~, secretion at any of the doses tried. Therefore, it may be concluded that the antithrombotic effect of a LMWH
preparation is not related to the potential of the LMWH preparation for inhibition of TNF-~ secretion.
The two different bioassays are mediated by different factors present in the preparations.
2. The ability of a particular dose of LMWH to inhibit TNF-~ secretion is positively correlated with its ability to inhibit DTH reaction, and the dose of a LMWH preparation optimally effective in inhibitin~ TNF-~ secretion is also optimally effective in inhibiting the DTH reaction.
~ ~ .
:
..
,:, ;
W0~2/l924~ PCr/US92/03626 Table 5. Treatment of mice with different LMWHs inhibits both ex vivo TNF-a secretion and DTH
responses.
Weekly treatment Ex vivo Con A
5 of mice Conc: induced TNF-~
with LMWH ~g/mouse secretion DTH responses pq/ml % DTH %
Inhibition Inhibition Fragmin~
Batch 38609 None 450 - 25 : 0.5 425 5 21 12 Batch 45389 None 320 - 28 0.1 280 13 26 6 , 5 260 18 24 13 Clexane~
Batch 2088 None 400 - 22 0.1 360 10 17 23 ; Batch 2066 None 350 - 23 0.1 338 6 20 13 : 5 192 57 7 70 : 10 186 55 7 70 25 Batch 4096 None 320 - 24 0 325 o 26 0 5.7. Example 7: Treatment of Adjuvant Arthritis (AA) in Rats with Low Doses of LMWHs Adjuvant arthritis is an experimental disease inducible in some strains of rats by immuni2ing them to antigens of Mycobacterium ` tuberculosis (Pearson, C.M., Proc. ~oc. Exp. Biol.
~ed. (1956) 91:91). This experimental disease is considered to be a model of human rheumatoid arthritis - .
, ~ .
, .. , ~ .
.
~2/l~249 PCT/US92/03626 -- 2 3 -- ,i ,~
(Pearson, C.M., Arthritis Rheum. (1964) 7:80). The arthritis appears to be caused by T lymphocytes that recognize an antigen of M. tuberculosis that is cross-reactive with structures in the joint tissues ~Cohen, I.R., et al., Arthritis Rheum. (1985) 28:841).
Lewis rats were immunized with M.
tuber-culosis (1 mg) in oil to induce adjuvant arthritis (Pearson, C.M., Proc. Soc. Exp. Biol. Med.
(1956) 91:91). Five days later the rats were inoculated subcutaneously as indicated with the doses of LMWH and/or heparin and scored for the development of arthritis on a scale of 0-16 as described (Holoshitz, Y., et al., Science (1983) 219:56). All the experiments were performed with Fragmin~, Batch 38609.
In order to study the dose response to Fragmin~ (Fig. 1) rats immunized to induce A~ were injected subcutaneously weekly, starting on the 5th day after injection with 0.5 ~g (o), 1 ~g (~), 2 ~g (-), 10 ~g (0), 15 ~g (~) 20 ~g (~); 30 ~g (-), 40 ~g (x) and PBS control (0). The dose was maximally effective in inhibiting arthrit:is.
The effect of the 20 ~g dose of Fragmin~ on the course of AA is shown in Fig. 2: PBS control (~);
single treatment on 5th day (~); daily (-); every 5th day (o); weekly (~). It is shown that Fragmin administration both at 5 day intervals and at 7 day intervals inhibits arthritis.
Fig. 3 shows the effect of weekly administration of ~ragmin~ (batch 38609) as compared to standard heparin on AA. Lewis rats were immunized to induce AA. Beginning on day 5, the rats were inoculated subcutaneously at weekly intervals with a 20 ~g dose of Fragmin~ (-), heparin (o) or phosphate buffered saline (PBS) control (0). The results show a dramatic difference in potency between Fragmin~ and :`
.
`.'` ' ' , ' ~. ' : ` .
, ~
W0~2/l9~4'~ PCT/US~2/03626 ~- J~ r ~ v? 7 ~ 4 heparin: Fragmin0 completely inhibited arthritis, while heparin had no inhibitory effect.
No inhibitory effect on A~ was found with daily administration of a 20 ~g dose of LMWH, although surprisingly the inhibitory effect of heparin was stronger than that of Fragmin~ in daily administration, as shown in (Fig. 4: Fragmin~ (batch 38609)(~), heparin (o), PBS control (n)).
A similar inhibitory effect was observed with several other LMWHs administered to Lewis rats immunized to induce AA. Fig. 5 shows the results of the injection of a 20 ~g dose of Fraxiparin~ (daily (0); weekly (t)); Fraxiparine~ (daily (~); weekly (-)), Lovenox~/Clexane~ (daily (~); weekly (o)), and PBS
control (x). All the three LMW~ls of different types and sources showed a marked inhibition of arthritis, when administered weekly, but not daily.
5.8. Example 8: Treatment With LMWH Prevents Rejection of Alloqrafts Wistar rats were subjected to alloge~eic BN
heart transplant (Ono, K. and Linsay, E.S., J. Thorac.
Cardiovasc. Sura. (1969) ~5:225-229). From the day - before transplantation, the rats were injec~ed subcutaneously at 7 day intervals with 20 ~g of Fragmin~ or PBS control (Fig. 6, ~ and O, respectively~
and scored for survival. The day of rejection was determined as the day the transplanted heart stopped be.ating, assayed by palpation o~ the abdomen. Fig. 6 shows that the rats treated with the weekly dose of LMWH had a markedly increased survival of the heart allografts.
.:
S.9. Example 9: Biological Effect of LNWH
On Insulin Dependent Diabetes Mellitus (IDDML of NOD Mice ~- Mice of the NOD strain spontaneously develop a form of type I insulin dependent diabetes mellitus ~` .
, , ' ` ' ., , W~')2/l'~2~t~ PCT/~S92/03626 - 25 - ~, ii ,'J ~
(IDDM) that is the accepted model for human IDDM
(Castano, L. and Eisenbarth, G.S., Annu. Rev. Immunol.
(1990) 8:647-679). The disease begins at 4-5 weeks of age by the appearance of inflammation of the pancreatic islets, insulitis. The insulitis progressively damages the insulin-producing beta cells which are sensitive to damage by TNF-~. At about 4-5 months of age, a sufficient number of beta cells are destroyed so that diabetes becomes overt.
To test whether treatment with LMWH could affect the IDDM process, a group of 10 female NOD mice was treated with weekly subcutaneous injections of 5 ~g per mouse of Fragmin~ (batch 38609~, the dose determined to represent 12 mouse units per kg. A
group of 10 control mice were treated with injections of saline. At the age of 5 months all the mice were bled to determine ~he development of IDDM using a standard procedure (Elias, D. et al., Proc. Natl.
Acad. Sci. U.S.A. (1990) 87:1576-1580). Fig. 7 shows that the control mice ("none") had abnormal blood glucose (400 mg/dl). In contrast the mice treated with LMWH had a normal blood glucose ~100 mg/dl).
Thus treatment with LMWH can indeed cure the IDDM
process.
5.1Ø Example 10: LMWH l`reatment of Human D~H
Figure 8 shows an experiment in which a 40 year old male volunteer weighing 85 kg was tested for DTH reactivity to tetanus antigen (Merieux skin test applicator). About 18 mm of induration was measured at 24 and 48 hours. The volunteer was then treated with a subcutaneous injection of Fragmin~ ~batch 38609) 3 mg. Fi.ve days later the volunteer was again tested for his DTH response to tetanus and the induratio~ was inhibited to about 5 mm. The volunteer was tested again for DTH 3 weeks later ("Recovery") and the test showed positive reactivity (23 mm of ~' ' .
W0~)2/1~4') PCT/US92/03~t6 induration at 24 and 48 hours). The volunteer was then treated with Fragmin~ as before and the DTH
reactivity was measured again 7 days later ("7 days post"). Again the DTH was inhibited to about 5 mm of induration. Recovery of DTH again was found 3 weeks later. Thus, LMWH at a dose of less than 5 mg can inhibit DTH in humans at treatment intervals of 5 and 7 days.
It should be apparent to those skilled in the art that other compositions and methods not specifically disclosed in the instant specification are, nevertheless, contemplated thereby. Such other compositions and methods are considered to be within the scope and spirit of the present invention. Hence, :L5 the invention should not be limited by the description of the specific embodiments disclosed herein but only by the following claims.
.
':
:
Claims (31)
1. A pharmaceutical composition for the prevention and/or treatment of pathological processes involving the induction of TNF-.alpha. secretion comprising a pharmaceutically acceptable carrier and a low molecular weight heparin (LMWH) present in a low effective dose unit for administration at intervals of about 5-8 days and which LMWH is capable of inhibiting in vitro TNF-.alpha. secretion by resting T cells and/or macrophages in response to T cell-specific antigens, mitogens, macrophage activators, disrupted extracellular matrix (dECM), laminin, fibronectin, or other ECM components.
2. The pharmaceutical composition of claim 1 in which the LMWH has an average molecular weight of from about 3,000 to about 6,000.
3. The pharmaceutical composition of claim 1 or 2 wherein the dose unit is an effective dose unit for administration every fifth day.
4. The pharmaceutical composition of claim 1 or 2 wherein the dose unit is an effective dose unit for administration every seventh day.
5. The pharmaceutical composition of claim 1 or 2 in which said LMWH is further characterized as being capable of inhibiting experimental delayed type hypersensitivity (DTH) reactions.
6. The pharmaceutical composition of claim 1 or 2 for subcutaneous or intravenous administration.
7. The pharmaceutical composition of claim 1 or 2 which is effective to delay or prevent allograft rejection.
8. The pharmaceutical composition of claim 1 or 2 in which said pathological processes include autoimmune diseases, allergy, inflammatory diseases, and other pathological processes related thereto.
9. The pharmaceutical composition of claim 1 or 2 in which said pathological processes are related to AIDS.
10. The pharmaceutical composition of claim 1 or 2 for the treatment of diabetes type I, periodontal disease, inflammatory bowel disease, uveitis, rheumatic diseases or multiple sclerosis.
11. The pharmaceutical composition or claim 10 for the treatment of rheumatoid arthritis.
12. The pharmaceutical composition of claim 8 for the treatment of chronic inflammation.
13. The pharmaceutical composition of claim 8 for the prevention and/or treatment of atherosclerosis, vasculitis, and pathological processes related thereto.
14. The pharmaceutical composition of claim 2 in which said LMWH is present in a single low dose unit of less than 5 mg.
15. The pharmaceutical composition of claim 2 in which said LMWH is present in a single low dosage unit of less than 3 mg.
16. The pharmaceutical composition of claim 2 in which said LMWH is present in a single low dose unit of from about 0.3 to about 3 mg.
17. The pharmaceutical composition of claim 2 in which said LMWH is present in a single low dose unit of from 1 to 1.5 mg.
18. A method of using a low molecular weight heparin (LMWH) which is capable of inhibiting in vitro TNF-.alpha.
secretion by resting T cells and/or macrophages in response to T cell-specific antigens, mitogens, macrophage activators, disrupted extracellular matrix (dECM), laminin, fibronectin, or other ECM components for the preparation of a pharmaceutical composition to be administered at intervals of about 5-8 days far the prevention of a pharmaceutical composition to be administered at intervals or about 5-8 days for the prevention and/or treatment of pathological processes involving induction of TNF-.alpha. secretion which comprises combining a low effective dose of said LMWH with a pharmaceutically acceptable carrier.
secretion by resting T cells and/or macrophages in response to T cell-specific antigens, mitogens, macrophage activators, disrupted extracellular matrix (dECM), laminin, fibronectin, or other ECM components for the preparation of a pharmaceutical composition to be administered at intervals of about 5-8 days far the prevention of a pharmaceutical composition to be administered at intervals or about 5-8 days for the prevention and/or treatment of pathological processes involving induction of TNF-.alpha. secretion which comprises combining a low effective dose of said LMWH with a pharmaceutically acceptable carrier.
19. The method of claim 18 in which said pharmaceutical composition is to be administered every seventh day.
20. The method of claim 18 in which said LMWH has an average molecular weight of from about 2,000 to about 6,000.
21. The method of claim 18, in which said low effective dose is less than 5 mg of said LMWH.
22. The method of claim 18 in which said pathological processes include allograft rejection, allergy, autoimmune and inflammatory diseases.
23. The method of claim 18 in which said pathological processes are related to AIDS.
24. The method of claim 18 in which said pathological processes is related to diabetes type I, periodontal disease, inflammatory bowel disease, uveitis, rheumatic diseases, chronic inflammation, multiple sclerosis, atherosclerosis, and vasculitis.
25. A pharmaceutical composition to be administered at intervals of about 5-8 days for the prevention and/or treatment of pathological processes involving the induction of TNF-.alpha. secretion comprising a pharmaceutically acceptable carrier and a low molecular weight heparin (LMWH) present in a low effective dose unit capable of inhibiting experimental delayed type hypersensitivity (DTH) reactions of a pre-sensitized individual to an applied antigen as evidenced by a reduction in the induration observed after the application of said antigen to the skin five to seven days after the administration of said pharmaceutical composition relative to the induration observed after the application of said antigen to the skin in the absence of or after recovery from the administration of said pharmaceutical composition.
26. The pharmaceutical composition of claim 25 in which said LMWH has an average molecular weight of from about 3,000 to about 6,000.
27. The pharmaceutical composition of claim 25 in which said antigen is selected from the group consisting of tetanus, myelin basic protein, purified protein derivative, and oxazolone.
28. A method of treating a subject suffering from a pathological process involving induction of TNF-.alpha. secretion comprising administering to such subject a pharmaceutical composition of claim 1, 2, 25, or 26 at intervals of about 5-8 days.
29. The method of claim 28 in which said interval between administrations is five days.
30. The method of claim 28 in which said interval between administrations is seven days.
31. The method of claim 28 in which said pharmaceutical composition contains less than about 5 mg of said LMWH.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IL98028 | 1991-05-02 | ||
| IL9802891A IL98028A (en) | 1991-05-02 | 1991-05-02 | Pharmaceutical compositions comprising a low molecular weight heparin (LMWH) |
| IL98298 | 1991-05-28 | ||
| IL98298A IL98298A0 (en) | 1991-05-28 | 1991-05-28 | Pharmaceutical compositions comprising a heparin derivative |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2102207A1 true CA2102207A1 (en) | 1992-11-03 |
Family
ID=26322260
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002102207A Abandoned CA2102207A1 (en) | 1991-05-02 | 1992-05-01 | Compositions for the prevention and/or treatment of patholigical processes |
Country Status (15)
| Country | Link |
|---|---|
| US (3) | US5474987A (en) |
| EP (1) | EP0583360B1 (en) |
| JP (1) | JPH06507635A (en) |
| KR (1) | KR100235782B1 (en) |
| AT (1) | ATE217796T1 (en) |
| AU (1) | AU668865B2 (en) |
| BR (1) | BR9205961A (en) |
| CA (1) | CA2102207A1 (en) |
| CZ (1) | CZ232593A3 (en) |
| DE (1) | DE69232615T2 (en) |
| FI (1) | FI934849A (en) |
| HU (1) | HUT67136A (en) |
| NO (1) | NO933942L (en) |
| SK (1) | SK120193A3 (en) |
| WO (1) | WO1992019249A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5605938A (en) | 1991-05-31 | 1997-02-25 | Gliatech, Inc. | Methods and compositions for inhibition of cell invasion and fibrosis using dextran sulfate |
| US6750207B1 (en) * | 1992-05-01 | 2004-06-15 | Yeda Research And Development Co. Ltd. | Compositions for the regulation of cytokine activity |
| WO1994011006A1 (en) * | 1992-11-10 | 1994-05-26 | Yeda Research And Development Co. Ltd. | Compositions for the regulation of cytokine activity |
| AU6204594A (en) * | 1993-02-22 | 1994-09-14 | Ulrich-Christoph Von Arnim | Use of heparins for the treatment of inflammatory or immunological diseases |
| US5980865A (en) * | 1995-08-18 | 1999-11-09 | Baker Norton Pharmaceuticals, Inc. | Method for treating late phase allergic reactions and inflammatory diseases |
| US6217863B1 (en) | 1995-10-30 | 2001-04-17 | Massachusetts Institute Of Technology | Rationally designed polysaccharide lyases derived from heparinase I |
| WO1998023279A1 (en) * | 1996-11-27 | 1998-06-04 | Rhone-Poulenc Rorer Pharmaceuticals Inc. | PHARMACEUTICAL COMPOSITION COMPRISING A COMPOUND HAVING ANTI-Xa ACTIVITY AND A PLATELET AGGREGATION ANTAGONIST COMPOUND |
| DE69835016T2 (en) * | 1997-07-14 | 2007-06-28 | Muramatsu, Takashi, Nagoya | USE OF MIDKIN OR ANTI-MIDKIN ANTIBODIES |
| WO2000012726A2 (en) | 1998-08-27 | 2000-03-09 | Massachusetts Institute Of Technology | Rationally designed heparinases derived from heparinase i and ii |
| US7056504B1 (en) | 1998-08-27 | 2006-06-06 | Massachusetts Institute Of Technology | Rationally designed heparinases derived from heparinase I and II |
| IL127851A0 (en) * | 1998-12-30 | 1999-10-28 | Applied Research Systems | Inhibition of TNF activity |
| EP1190364A2 (en) * | 1999-04-23 | 2002-03-27 | Massachusetts Institute Of Technology | System and method for polymer notation |
| CA2377734A1 (en) * | 1999-06-30 | 2001-01-11 | Hamilton Civic Hospitals Research Development, Inc. | Heparin compositions that inhibit clot associated coagulation factors |
| EP1266013B1 (en) | 2000-03-08 | 2014-10-15 | Massachusetts Institute Of Technology | Heparinase iii and uses thereof |
| HUP0300939A2 (en) * | 2000-09-08 | 2003-09-29 | Hamilton Civic Hospitals Research Development, Inc. | Antithrombotic compositions |
| WO2002023190A2 (en) | 2000-09-12 | 2002-03-21 | Massachusetts Institute Of Technology | Methods and products related to low molecular weight heparin |
| CA2423469A1 (en) | 2000-10-18 | 2002-04-25 | Massachusetts Institute Of Technology | Methods and products related to pulmonary delivery of polysaccharides |
| WO2002047696A1 (en) * | 2000-12-16 | 2002-06-20 | Aventis Pharma Deutschland Gmbh | Use of low molecular heparin for treating osteoarthritis |
| WO2003000190A2 (en) * | 2001-06-25 | 2003-01-03 | Depuy | Liposomal encapsulation of glycosaminoglycans for the treatment of arthritic joints |
| US20030036103A1 (en) * | 2001-07-31 | 2003-02-20 | Sivaram Pillarisetti | Methods and compositions for diagnosis and treatment of vascular conditions |
| CA2455038A1 (en) * | 2001-07-31 | 2003-02-13 | Board Of Regents, University Of Texas System | Antigens of and antibodies to translocated molecules of microorganisms and uses thereof |
| US7285536B2 (en) * | 2001-12-05 | 2007-10-23 | Yeda Research And Development Co., Ltd. | Anti-cancer therapeutic compounds |
| AU2003230985A1 (en) * | 2002-04-18 | 2003-11-03 | Stephen H. Embury | Method and composition for preventing pain in sickle cell patients |
| US8071569B2 (en) * | 2002-09-20 | 2011-12-06 | Mousa Shaker A | Oxidized heparin fractions and their use in inhibiting angiogenesis |
| CA2514474C (en) * | 2003-01-30 | 2014-05-06 | Avner Yayon | Freeze-dried fibrin matrices and methods for preparation thereof |
| WO2005054493A2 (en) * | 2003-06-12 | 2005-06-16 | Mayo Foundation For Medical Education And Research | Altered activity of toll-like receptors |
| CA2530533C (en) | 2003-06-27 | 2015-02-10 | Ethicon, Incorporated | Postpartum cells derived from umbilical cord tissue, and methods of making and using the same |
| US9572840B2 (en) | 2003-06-27 | 2017-02-21 | DePuy Synthes Products, Inc. | Regeneration and repair of neural tissue using postpartum-derived cells |
| US20060223177A1 (en) | 2003-06-27 | 2006-10-05 | Ethicon Inc. | Postpartum cells derived from umbilical cord tissue, and methods of making and using the same |
| AU2004278013B2 (en) * | 2003-10-01 | 2009-01-15 | Momenta Pharmaceuticals, Inc. | Polysaccharides for pulmonary delivery of active agents |
| CN1819833A (en) * | 2004-03-29 | 2006-08-16 | 克霖固鲁制药股份有限公司 | Hgf production accelerator containing heparin-like oligosaccharide |
| US8450297B2 (en) * | 2004-08-16 | 2013-05-28 | Massachusetts Institute Of Technology | Rapid two-step synthesis of anti-coagulants |
| CA2616166A1 (en) * | 2005-07-22 | 2007-02-01 | The Regents Of The University Of California | Heparin compositions and selectin inhibition |
| US8921109B2 (en) | 2005-09-19 | 2014-12-30 | Histogenics Corporation | Cell-support matrix having narrowly defined uniformly vertically and non-randomly organized porosity and pore density and a method for preparation thereof |
| AU2009250803A1 (en) * | 2008-05-20 | 2009-11-26 | Crystal Clear Partnership | Separation of polysaccharides by charge density gradient |
| US10077420B2 (en) | 2014-12-02 | 2018-09-18 | Histogenics Corporation | Cell and tissue culture container |
Family Cites Families (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL61201A (en) * | 1979-10-05 | 1984-09-30 | Choay Sa | Oligosaccharides having no more than 8 saccharide moieties,their obtention from heparin and pharmaceutical compositions containing them |
| US4281108A (en) * | 1980-01-28 | 1981-07-28 | Hepar Industries, Inc. | Process for obtaining low molecular weight heparins endowed with elevated pharmacological properties, and product so obtained |
| DK168876B1 (en) * | 1982-12-20 | 1994-07-04 | Harvard College | Agent that can be administered orally or subcutaneously to inhibit mammalian angiogenesis and method of preparing such agent |
| US4916219A (en) * | 1985-03-28 | 1990-04-10 | University Of Iowa Research Foundation | Oligosaccharide heparin fragments as inhibitors of complement cascade |
| IT1214609B (en) * | 1985-05-17 | 1990-01-18 | Opocrin Spa | HEXOSAMINOGLICANS DEPOLYMERIZED SULPHATES FOR ANTI-THROMBOTIC, FIBRINOLITHIC, ANTI-INFLAMMATORY ACTIVITIES, THEIR PREPARATION PROCEDURE AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM. |
| US4889808A (en) * | 1985-10-01 | 1989-12-26 | American Home Products | Method of enchancing t-PA and SCU-PA production |
| EP0240098A3 (en) * | 1986-04-04 | 1989-05-10 | Kabushiki Kaisha Ueno Seiyaku Oyo Kenkyujo | Oligo and polysaccharides for the treatment of diseases caused by retroviruses |
| US4788307A (en) * | 1986-04-30 | 1988-11-29 | Choay S.A. | Oligosaccharidic fractions devoid or practically devoid of antithrombotic activity |
| DE3787996T2 (en) * | 1986-05-16 | 1994-03-03 | Italfarmaco Spa | Heparin free from E.D.T.A., fractions and fragments of heparin, processes for their preparation and pharmaceutical compositions containing them. |
| WO1988000017A1 (en) * | 1986-06-27 | 1988-01-14 | Boon Corporation Pty. Ltd. | Mat |
| IL85145A (en) * | 1987-01-23 | 1994-08-26 | Univ Australian | Anti-metastatic pharmaceutical or veterinary compositions containing modified heparin having reduced anticoagulant activity |
| FR2614026B1 (en) * | 1987-04-16 | 1992-04-17 | Sanofi Sa | LOW MOLECULAR WEIGHT HEPARINS WITH REGULAR STRUCTURE, THEIR PREPARATION AND THEIR BIOLOGICAL APPLICATIONS |
| SE8702254D0 (en) * | 1987-05-29 | 1987-05-29 | Kabivitrum Ab | NOVEL HEPARIN DERIVATIVES |
| WO1990003791A1 (en) * | 1988-10-05 | 1990-04-19 | Nicola Diferrante | Methods for interfering with hiv multiplication and composition for the treatment of aids |
| IL88554A0 (en) * | 1988-12-01 | 1989-07-31 | Hadassah Med Org | Compositions containing a compound binding to a heparin receptor |
| PT93847A (en) * | 1989-04-24 | 1990-11-20 | Harvard College | PROCESS FOR THE PREPARATION OF OLIGOSACARIDES OF LOW MOLECULAR WEIGHT DERIVED FROM HEPARIN OR DESPOLYMENED HEPARAN SULFATE AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM |
| AT500533B8 (en) * | 2000-04-13 | 2007-02-15 | Man Nutzfahrzeuge Ag | GAS OR TROLLEY BUS |
-
1992
- 1992-05-01 JP JP4511483A patent/JPH06507635A/en active Pending
- 1992-05-01 BR BR9205961A patent/BR9205961A/en not_active Application Discontinuation
- 1992-05-01 AU AU19131/92A patent/AU668865B2/en not_active Ceased
- 1992-05-01 AT AT92911373T patent/ATE217796T1/en not_active IP Right Cessation
- 1992-05-01 HU HU9303110A patent/HUT67136A/en unknown
- 1992-05-01 KR KR1019930703303A patent/KR100235782B1/en not_active IP Right Cessation
- 1992-05-01 CA CA002102207A patent/CA2102207A1/en not_active Abandoned
- 1992-05-01 WO PCT/US1992/003626 patent/WO1992019249A1/en active IP Right Grant
- 1992-05-01 DE DE69232615T patent/DE69232615T2/en not_active Expired - Lifetime
- 1992-05-01 CZ CS932325A patent/CZ232593A3/en unknown
- 1992-05-01 SK SK1201-93A patent/SK120193A3/en unknown
- 1992-05-01 EP EP92911373A patent/EP0583360B1/en not_active Expired - Lifetime
-
1993
- 1993-11-01 NO NO933942A patent/NO933942L/en unknown
- 1993-11-02 FI FI934849A patent/FI934849A/en not_active Application Discontinuation
-
1995
- 1995-02-03 US US08/384,203 patent/US5474987A/en not_active Expired - Lifetime
- 1995-06-01 US US08/457,655 patent/US5686431A/en not_active Expired - Lifetime
-
1997
- 1997-11-07 US US08/966,315 patent/US5908837A/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| ATE217796T1 (en) | 2002-06-15 |
| JPH06507635A (en) | 1994-09-01 |
| BR9205961A (en) | 1994-07-26 |
| WO1992019249A1 (en) | 1992-11-12 |
| FI934849A0 (en) | 1993-11-02 |
| DE69232615D1 (en) | 2002-06-27 |
| US5474987A (en) | 1995-12-12 |
| EP0583360A1 (en) | 1994-02-23 |
| AU1913192A (en) | 1992-12-21 |
| CZ232593A3 (en) | 1994-07-13 |
| HUT67136A (en) | 1995-02-28 |
| SK120193A3 (en) | 1994-07-06 |
| DE69232615T2 (en) | 2003-01-30 |
| EP0583360B1 (en) | 2002-05-22 |
| NO933942L (en) | 1993-12-14 |
| US5908837A (en) | 1999-06-01 |
| FI934849A (en) | 1993-12-31 |
| KR100235782B1 (en) | 1999-12-15 |
| NO933942D0 (en) | 1993-11-01 |
| HU9303110D0 (en) | 1994-01-28 |
| AU668865B2 (en) | 1996-05-23 |
| US5686431A (en) | 1997-11-11 |
| EP0583360A4 (en) | 1994-04-06 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued | ||
| FZDE | Discontinued |
Effective date: 20010501 |