WO2001035967A1 - Heparanase inhibitors for the treatment of heart failure - Google Patents

Heparanase inhibitors for the treatment of heart failure Download PDF

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Publication number
WO2001035967A1
WO2001035967A1 PCT/EP2000/011441 EP0011441W WO0135967A1 WO 2001035967 A1 WO2001035967 A1 WO 2001035967A1 EP 0011441 W EP0011441 W EP 0011441W WO 0135967 A1 WO0135967 A1 WO 0135967A1
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Prior art keywords
heparanase
treatment
heart failure
inhibitors
use according
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PCT/EP2000/011441
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German (de)
French (fr)
Inventor
Dieter Herr
Alfred Hahn
Volker Laux
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Abbott Gmbh & Co. Kg
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Priority to AU15221/01A priority Critical patent/AU1522101A/en
Priority to EP00977548A priority patent/EP1229921A1/en
Publication of WO2001035967A1 publication Critical patent/WO2001035967A1/en

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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
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • 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
    • 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
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • 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

Definitions

  • Heparanase inhibitors for the treatment of heart failure are highly paranase inhibitors for the treatment of heart failure.
  • the present invention relates to the use of at least one heparanase inhibitor for the treatment of heart failure and related signs, symptoms and / or malfunctions and methods for the production of pharmaceutical agents for the treatment of heart failure.
  • Proteoglycans are polyanionic substances of high molecular weight in which different types of heteropolysaccharide chains are covalently bound to a polypeptide backbone.
  • the polysaccharide groups of the proteoglycans formerly known as mucopolysaccharides, are now known as glycosaminoglycans.
  • a large number of enzymes are involved in the construction, conversion and degradation of these proteoglycans.
  • Proteolysis can release glycosaminoglycans, which in turn are broken down into smaller fragments under the action of glycosaminoglycan endoglycosidases, while corresponding exoglycosidases release monosaccharides from the non-reducing ends of the glycosaminoglycans.
  • Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPG) occur on the extracellular surface and in the extracellular matrix.
  • the HS chains are generally formed from clusters of sulfated disaccharide units, primarily 1-4 N-sulfated glucosamines bonded to ⁇ -iduronic acid residues, which pass through regions with little or no sulfate, primarily 1-4 at ⁇ -D -Glucuronic acid bound N-acetylated glucosamines, separated from each other. They are believed to play a major role in cell-cell and cell-matrix interactions that are involved in various physiological and pathophysiological processes. Examples include the adhesion, migration, differentiation and proliferation of cells.
  • HS and / or HSPG Various molecules are reported to interact with HS and / or HSPG. These are either growth factors (eg FGF, PDGF, VEGF), cytokines (IL-2), extracellular matrix proteins (fibronectin, collagen), factors involved in hemostasis (heparin factor II), or molecules of a different nature, e.g. lipoproteins, DNA topoisomerases and ⁇ -amyloid proteins (cf.Hileman et al. (1998) BioEssays 20, 156-167; Stringer and Gallagher (1997) Int. J. Biochem Cell Biol 29, 709-714 ; Rapraeger (1993) Curr. Opin Cell. Biol. 5, 844-853; Bernfield et al. (1993) Development 1993 Suppl.
  • growth factors eg FGF, PDGF, VEGF
  • IL-2 cytokines
  • extracellular matrix proteins fibronectin, collagen
  • factors involved in hemostasis heparin factor II
  • heparanase received attention because they were associated with the metastasis of tumors, inflammatory processes and leukocyte migration (WO 95/24907; US-A-5,817,800; US-A-5,262, 403; Vlodavsky et al . (1999) Genbank Accession No. AF 144325; Hulett et al. (1999) Nature Medicine 5,
  • heparanase was originally discovered in murine metastatic melanoma cells. Heparanase cleaves HS into characteristic high molecular weight fragments and this activity has been associated with the metastatic potential of melanoma cells.
  • the object of the present invention is to provide new therapeutic applications for modulating the heparanase activity.
  • the present invention relates to the use of at least one heparanase inhibitor for the treatment of heart failure.
  • cardiac insufficiency means an inability of the heart to provide the necessary support.
  • heart failure describes the state of a heart in which compensation mechanisms such as heart rate, contractility, stroke volume, hypertension are not sufficient to maintain a normal cardiac output. It is a weakness of the pump function.
  • Heart failure can affect the entire heart (global heart failure) or parts of it, for example left or right heart failure.
  • Preferred according to the invention is the treatment of myocardial insufficiency, i.e. Heart failure due to a change in the myocardium.
  • myocardial insufficiency i.e. Heart failure due to a change in the myocardium.
  • cardiomyopathies and preferably primary cardiomyopathies for example hypertrophic obstructive or non-obstructive cardiomyopathies characterized by hypertrophy of the heart, especially the chamber septum and left ventricle, and congestive cardiomyopathies characterized by hypertrophy and dilatation of the heart (also as dilated congestive cardiomyopathies).
  • the forms of heart failure, in particular congestive heart failure, which are preferably treated according to the invention are based on one or more of the changes in the myocardium listed below: hypertrophy of individual or all wall layers, decrease in muscle extensibility, heart enlargement, in particular enlargement of the ventricle, in particular without increasing the thickness of the ventricular muscles, decrease in the thickness of the ventricular muscles and fibrous changes in the ventricular muscles.
  • the indication heart failure to be treated according to the invention is generally characterized by a progressive development, ie the conditions described above change over time, the degree of severity generally increases and, if appropriate, conditions can merge with one another or other conditions can change to existing conditions draw near.
  • heart failures are treated which are preceded or accompanied by a lowering of the pH value of the affected heart parts.
  • Values in the acidic pH range usually around 2 to 6.5, around 3 to 6 and especially around 4.5 to 5.5 are important here.
  • the treatment of heart failure according to the invention or the conditions on which it is based enable a number of other signs, symptoms and / or malfunctions to be treated which are related to heart failure, i.e. especially accompany the disease states described above.
  • These include, for example, changes in the peripheral circulation, in particular congestion in the large and / or in the small circulation, e.g. Lung and liver congestion, a reduction in the blood supply to the peripheral circulation, respiratory disorders (dyspnea), kidney function, e.g. Nocturia, and the electrolyte, e.g. peripheral edema, breast and abdominal addiction, etc.
  • These signs, symptoms and / or malfunctions often form patterns or groups which are referred to as syndromes, so that, according to the invention, the treatment of the heart failure syndrome results.
  • Treatment in the sense of the invention includes not only the treatment of acute or chronic signs, symptoms and / or malfunctions, but also preventive treatment (prevention).
  • One purpose of acute or chronic treatment is to remedy the disorders, regulate the conditions, or alleviate the signs, symptoms and / or malfunctions.
  • the purpose of the treatment is to reduce the activity of the heparanase.
  • One purpose of preventive (preventive) treatment is to prevent the occurrence of the disorders, conditions, signs, symptoms and / or malfunctions, which includes a time delay in the occurrence.
  • Treatment can be symptomatic, for example as symptom suppression be aligned. It can be short-term, medium-term, or it can also be long-term treatment, for example in the context of maintenance therapy.
  • heparanase inhibitor describes substances which inhibit the enzymatic activity of heparanase or its expression.
  • inhibition is understood to mean a reduction in the enzyme activity, especially the activity as endogenous glycosidase, endoglucuronidase, ⁇ -glucuronidase and in particular endo- ⁇ -glucuronidase, or the expression of heparanase.
  • the enzyme activity of heparanase leads, for example, to the cleavage of glycosaminoglycans, optionally as part of proteoglycans, in particular heparan sulfate, or the corresponding proteoglycans.
  • Heparase inhibitors according to the invention preferably bring about a reduction in the HS and HSPG degradation by heparanase.
  • inhibitors of mammalian heparanase EC 3.2.1
  • human heparanase and especially the heparanase with the amino acid sequence SEQ ID NO: 2 encoded by the cDNA with SEQ ID NO: 1 are preferred.
  • Inhibitors according to the invention usually bind to heparanase or to nucleic acids encoding heparanase, e.g. DNA or mRNA. Binding is understood to mean any molecular interaction between inhibitor and enzyme or nucleic acid, in particular under physiological conditions. These are usually classic interactions, which include electrostatic forces, van der Waals forces, hydrogen bonds, hydrophobic bonds, or metal complex-like coordinative bonds. In addition to the reversible molecular interactions mentioned above, irreversible interactions between inhibitor and enzyme can also be considered, e.g. covalent bonds.
  • competitive enzyme inhibitors bind in the region of or one of the active domains of heparanase and compete with other substrates for their binding site (s) (competition).
  • competitive enzyme inhibitors are understood to mean those which compete with a comparison substrate, in the present case preferably heparan sulfate, for binding to heparanase, ie the binding of one hinders the binding of the other. Because of this binding to heparanase, competitive enzyme inhibitors can also be referred to as heparanase substrates.
  • inhibitors are preferably substrates which, in comparison to the natural substrate or substrates, are not or at least less accessible to the catalytic activity of heparanase, ie they are not or only comparatively accessible to a small extent by heparanase, in particular cleaved. It is also possible to use non-competitive inhibitors which, for example, bind essentially irreversibly to active domains or to the heparanase elsewhere and, for example via allosteric effects, have an influence on the enzyme activity.
  • the principle applies that the displacement of a substrate by an inhibitor increases with decreasing binding affinity of the substrate or increasing binding affinity of the inhibitor. It is therefore expedient for inhibitors which can be used according to the invention to have a high binding affinity for heparanase. Such a favorable binding affinity allows an effective displacement of naturally occurring enzyme substrates, for example heparan sulfates and heparan sulfate proteoglycans, the concentration of inhibitor required for binding a certain amount of this inhibitor to the enzyme or for displacing a certain amount of a substrate with increasing Binding affinity of the inhibitor decreases.
  • inhibitors are therefore preferred whose binding affinity is so great that they can be administered as an active ingredient in an effective medical treatment in acceptable amounts.
  • Inhibitors according to the invention are therefore preferably administered in daily doses of approximately 0.01 to 30 mg / kg body weight and in particular approximately 0.1 to 15 mg / kg body weight.
  • the competitive inhibition of the binding of heparanase inhibitors can also be evaluated to the extent that inhibitors preferred according to the invention have half-maximum inhibitory constants IC 50 in vitro of less than 10 -3 M, preferably less than 10 -4 M and in particular less than 10 -5 M and with non-competitive inhibition of less than 10 -4 M, preferably less than 10 -5 M and in particular less than 10 ⁇ 6 M.
  • the expression inhibitors are, in particular, oligonucleotides which act, for example, in the sense of an antisense RNA or DNA, or in the sense of the triple helix technique.
  • heparanase inhibitors are glycosaminoglycans with structural similarity to the natural substrates of heparanase, in particular heparan sulfates. These include heparins, heparin fracture Nen and heparin fragments, for example heparins of a certain molecular weight, heparin derivatives, for example heparins with at least partially reduced carboxyl groups, at least partially N-desulfated, N-acetylated heparins, for example in EP 0 254 067 A2, WO 92/01003 and US Pat. No.
  • N-desulfated, N-acetylated heparin described at least partially N, 0-desulfated, N-resulfated heparins, for example the compounds described in WO 92/01003 and US Pat. No. 5, 206,223, and O-acylated heparins, for example those in the compounds described in EP 0 356 275 AI.
  • Heparin is preferably obtained from natural sources, for example the intestinal mucosa of cattle or pigs. Fragmentation and / or fractionation can be carried out in the usual way.
  • Carboxyl groups can be reduced with NaBH, for example.
  • Sulfate groups can be removed, for example, by treatment with water- or methanol-containing DMSO, the degree of desulfation depending on the reaction time, the reaction temperature and the addition of water or methanol.
  • N-acetylation can be accomplished, for example, with acetic anhydride under alkaline conditions and the resulfation can be accomplished, for example, with a triethylamine-sulfur trioxide complex.
  • glycosaminoglycans can also be derivatized, for example hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate, tertiary sulfate, keratin sulfate and heparan sulfate and their proteoglycans, as described using the example of O-acylation in EP 0 356 275 Al is.
  • sulfated oligosaccharides for example those described in WO 96/33726, ie in particular sulfated mannopentaose phosphates, maltohexaose sulfates and the like, and sulfated polysaccharides, for example those described in WO 88/05301, that is to say in particular heparin, fucoidan, pentosan sulfate, dextran sulfate and carrageenan -Lambda.
  • the oligosaccharides and polysaccharides containing phosphosugars mentioned in WO 90/01938 can also be used.
  • Glycomimetic saccharopeptides for example those of the formula described in WO 96/35700, are also suitable
  • radicals W independently of one another for fucose
  • 3-amino-3-deoxyglucose 4-amino-4-deoxyglucose, glucose, galactose, glucosamine, galactosamine, glucuronic acid, galacturonic acid, glucosaminuronic acid, neuraminic acid, maltose, maltotriose, iduronic acid, 2,5-anhydromannitol, mannose, mannuronic acid, and cellobiose stand;
  • radicals Y independently of one another represent -NR 3 -C (0) - and -C (0) -NR 3 -;
  • radicals X independently of one another represent a difunctional or polyfunctional group, in particular ethylene glycol, ethylene glycol oligomers, lower alkyl, optionally substituted alkyl, amino acids and peptides;
  • R 3 represents -H, alkyl of 1 to 8 carbon atoms and aralkyl of 5 to 8 carbon atoms.
  • Laminarin sulfates also called laminaran sulfates, are linear polymers made from ⁇ -1,3-linked glucose residues with possibly small proportions of ⁇ - (1,6) linkages and 2 to 3% D-mannitol end groups, in particular the sodium salt with a molar ratio of at least 1: 1 sulfate groups to monosaccharide units can also be used as a heparanase inhibitor (cf. WO 95/24907).
  • heparanase inhibitors are suramin and trachyspinic acid.
  • Y stands for -COOH, -P0 3 H 2 -P (0) (OR 6 ) (OH), -P (0) R 6 (OH), tetrazole and -S0 3 H, in which
  • X represents NH, 0 or S
  • R represents a hydrogen atom or -C (0) NHC 6 (R 7 ) 5 , in which C ⁇ (R 7 ) 5 preferably represents optionally mono- to pentasubstituted phenyl and the substituents R 7 are selected from OH, halogen, -COOH , -P0 3 H 2 or -S0 3 H.
  • the salts of these compounds are also included.
  • Illustrative examples of these compounds are (Z) -O- (D-glucopyranuronosylidene) amino-N-phenylcarbamate and (5R, Z) -0- (5-C-phosphonato-D-xylopyranosylidene) amino-N-phenylcarbamate and their sodium salts.
  • These compounds can be prepared in a manner known per se, for example using the processes described in EP 0 642 799.
  • Low molecular weight heparanase inhibitors mostly synthetic compounds, can be used advantageously in many ways.
  • Aptamers which are nucleic acids, usually oligonucleotides, with sufficient affinity for heparanase, can also be used as inhibitors.
  • Heparanase-specific antibodies can also be useful as heparanase inhibitors. It can be polyclonal antisera, monoclonal antibodies, antibody fragments such as F (ab), Fc, etc., chimeric, humanized and recombinant antibodies. Such antibodies can be produced in a manner known per se. Heparanase as such or antigenic fragments thereof, which as a rule are coupled to customary carrier proteins, can be used as the immunogen.
  • Example 8 of WO 99/43830 describes, for example, the production of a polyclonal antiserum against selected peptides of human heparanase.
  • WO 95/04158 describes the production of selected heparinase peptides, in particular a C-terminal sequence, there called SEQ ID NO: 42, and the production of antisera directed against them and their usefulness as heparanase inhibitors.
  • WO 96/08559 describes phosphorothioate or phosphorodithioate antisense oligonucleotides with preferably 7 to 30 nucleotides, which are essentially formed from dG and / or dT nucleotides. Specifically, are suitable for the inhibition of endoglycosidases, in particular heparanases, for example the oligonucleotides of the sequences SEQ ID NO: 2,4,6,10 described there.
  • the application according to the invention is not limited to the inhibitors mentioned above. Rather, any substance in the presence of which the heparanase activity is lower than in the absence thereof can be used according to the invention as a heparanase inhibitor.
  • Test systems are known for measuring the heparanase activity. These are generally based on the use of labeled heparan sulfates or heparan sulfate proteoglycans as a substrate, the conversion, ie the cleavage of this substrate and the associated release of certain fragments, being able to be followed on the basis of the label.
  • the substrates can be obtained biologically, for example by cultivating endothelial cells in radiolabeled 35 SO 4 or injecting radiolabelled sulfate into tumorous experimental animals, and recovering appropriately labeled heparan sulfate proteoglycans from the endothelial or tumor cells.
  • the substrates can also be synthesized chemically, for example by first partially deacetylating heparan sulfate and then reacetylating. By reductive amination of the free ends of heparan sulfate and subsequent addition of suitable labels, for example, fluorescence-labeled heparan sulfates can be produced.
  • the coupling chemistry customary in this area can be used, for example first aminating the reducing ends and then coupling glycosaminoglycans modified in this way to suitable matrices, for example agarose, sepharose and the like.
  • suitable matrices for example agarose, sepharose and the like.
  • Heparan sulfate proteoglycans or heparan sulfate peptides thereof can be coupled, for example, to CNBr-activated Sepharose.
  • Another possibility is to separate the degradation products by gel filtration, precipitation reactions, for example as described in Example J of WO 96/35700, and the like.
  • Fluorescence labels can advantageously be detected by means of HPLC, preferably exclusion HPLC.
  • HPLC preferably exclusion HPLC.
  • HS-binding proteins for example histidine-rich glycoproteins, Use wisely in immobilized form in order to separate non-degraded or only partially degraded heparanase substrate from degraded substrate and thereby enable its detection.
  • the heparanase used in these tests can be of natural or recombinant origin, so heparanase can be purified from a large number of tissues and body fluids, including serum.
  • the expression of human heparanase can be accomplished with the expression systems mentioned in WO 95/04158 and WO 99/43830.
  • the present invention therefore also relates to a method for identifying heparanase inhibitors, the activity of heparanase being determined in the presence and in the absence of at least one test substance.
  • test systems described above and other similarly suitable test systems can form the basis for in vitro screening methods, preferably for primary screening, with which one can read out from a large number of different substances those which are useful with regard to the use according to the invention
  • the present invention thus also relates to corresponding methods for identifying active substances for the treatment of heart failure and, based on this, the production of pharmaceutical agents for the treatment of heart failure.
  • Such a process for the development of active substances for the treatment of heart failure is characterized in that heparanase inhibitors are first selected from a large number of substances and used to produce the agent. For example, using combinatorial chemistry, extensive substance banks can be created that include myriads of potential active substances.
  • the screening of combinatorial substance libraries for substances with the desired activity can be automated. Screening robots are used for the efficient evaluation of the individual assays, which are preferably arranged on microtiter plates.
  • Kits and components for performing this assay can be obtained commercially, for example from Amersham Pharmacia Biotech.
  • solubilized or membrane-bound substrates are immobilized on small fluomicrospheres containing scintillation substance.
  • the substrate is radioactively marked and the scintillation substance is stimulated to emit light as long as the spatial proximity between scintillation Substance and radiolabeling is given, or the radioactive label is inserted into the immobilized substrate by the enzyme activity to be measured and, as a result, the scintillation substance is excited to emit light.
  • FlashPlate® technology known in the field of active substance screening. Kits and components for carrying out this assay can be obtained commercially, for example from NEN ® Life Science Products. This principle is also based on microtiter plates (96 or 384), which are coated with scintillation substance.
  • heparanase inhibitors includes a method as part of the treatment.
  • An effective amount of one or more heparanase inhibitors is administered to the individual to be treated, preferably a mammal, in particular a human, useful or domestic animal.
  • the treatment is carried out by adding it once or several times a day, optionally together or alternating with other active substances or preparations containing the active substance. Whether such treatment is indicated and in what form it must be carried out depends on the individual case and is subject to a medical assessment (diagnosis) to develop the existing signs, symptoms and / or malfunctions, risks, certain signs, symptoms and / or malfunctions , and other factors.
  • the teaching according to the invention is primarily aimed at the production of pharmaceutical agents for the treatment of an individual, preferably a mammal, in particular a human, useful or domestic animal.
  • the inhibitors are usually administered in the form of pharmaceutical compositions which comprise a pharmaceutically acceptable excipient with at least one inhibitor according to the invention, optionally also a mixture of several inhibitors according to the invention, and optionally further active ingredients.
  • These compositions can be administered, for example, by the oral, rectal, transdermal, subcutaneous, intravenous, intramuscular or intranasal routes.
  • suitable pharmaceutical formulations are solid pharmaceutical forms, such as powders, powders, granules, tablets, lozenges, sachets, cachets, coated tablets, capsules such as hard and soft gelatin capsules, suppositories or vaginal pharmaceutical forms, semi-solid medicinal forms.
  • neiforms such as ointments, creams, hydrogels, pastes or plasters
  • liquid pharmaceutical forms such as solutions, emulsions, in particular oil-in-water emulsions, suspensions, for example lotions, injection and infusion preparations, eye and ear drops.
  • Implanted delivery devices can also be used for the administration of inhibitors according to the invention. Liposomes, microspheres or polymer matrices can also be used.
  • inhibitors according to the invention are usually mixed or diluted with an excipient.
  • Excipients can be solid, semi-solid or liquid materials that serve as vehicles, carriers or media for the active ingredient.
  • Suitable excipients include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methyl cellulose.
  • the formulations can be pharmaceutically acceptable carriers or customary auxiliaries, such as lubricants, for example talc, magnesium stearate and mineral oil; Wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; Antioxidants; Antiirritatives; chelating agents; coating aids; Emulsion stabilizers; film formers; gelling agents; Odor masking agents; masking flavors; resins; Hydrocolloids; Solvents; Solubilizing agents; Neutralizing agents; Permeation accelerator; pigments; quaternary ammonium compounds; Refatting and overfatting agents; Ointment, cream or oil base materials; Silicone derivatives; spreading aids; stabilizers; Sterilanzien; Fundamentals of the suppository; Tablet excipients such as binders, fillers, lubricants, disintegrants or coatings; Propellant; Desiccant; Opacifiers; Thickener; waxes; plasticizer
  • a design in this regard is based on expert knowledge, as is shown, for example, in Fiedler, H.P., Lexicon of auxiliaries for pharmacy, cosmetics and related areas, 4th edition, Aulendorf: ECV-Editio-Cantor-Verlag, 1996.
  • FIG. 1 shows the gel electrophoretic separation of the amplificates obtained after RT-PCR according to Example 1 in parallel with molecular weight standards (MWM) and the negative control (negative).
  • MLM molecular weight standards
  • the animal model used was by Wiesener et al. in Circulation 95, 1253-1259 (1997).
  • Five rats treated with the aortic clamp technique (No. 3, 15, 24, 25, 112) developed congestive heart failure (cardiac hypertrophy).
  • the hearts were removed and the mRNA was isolated in the usual way.
  • the amount of heparanase mRNA expressed could be determined by RT-PCR by using the oligonucleotide with the sequence SEQ ID NO: 3 as sense primer and the oligonucleotide with the sequence SEQ ID NO: 4 as antisense primer.
  • the GADPH expression was measured as a housekeeping gene.
  • the same study was carried out on control animals (rats No. 11, 17, 19, 32, 33).
  • the respective amplificates obtained by RT-PCR were separated electrophoretically and their amount was quantified.
  • 1 shows the amplicons obtained after electrophoretic separation for animals 11, 17, 19, 32 and 33 of the control group and animals 3, 15, 24, 25 and 112 of the test group.
  • the quantification gave the following heparanase mRNA levels, each based on GAPDH expression:
  • the mean values are 4.289 ⁇ 0.75 for the control group and 4.153 ⁇ 0.06 for the test group. These values show that the pathological state induced in the model is not associated with regulation of the expression of heparanase mRNA, but 5 the intra- and extracellular acidification occurring in connection with cardiac hypertrophy and heart failure modulates the heparanase activity (Schini-Kerth et al (1997) Circulation 96, 3888-3896; Shrode et al. (1997) J Bioenerg Biomembr 29, 393-399; Kraus and Wolf (1996) Tumor Biol 17, 133-154; Tamagaki et al.

Abstract

The invention relates to the use of heparanase inhibitors for the treatment of heart failure, especially congestive heart failure, and related indications, symptoms and/or dysfunctions such as peripheral edema, pulmonary and liver congestion, dyspnea, hydrothorax and abdominal dropsy. The invention also relates to a method for producing pharmaceutical agents used to treat heart failure.

Description

Heparanase-Inhibitoren zur Behandlung von Herzinsuffizienz.Heparanase inhibitors for the treatment of heart failure.
Die vorliegende Erfindung betrifft die Verwendung wenigstens ei- nes Heparanase-Inhibitors zur Behandlung von Herzinsuffizienz und damit zusammenhängenden Anzeichen, Symptomen und/oder Fehlfunktionen und Verfahren zur Herstellung pharmazeutischer Mittel zur Behandlung von Herzinsuffizienz.The present invention relates to the use of at least one heparanase inhibitor for the treatment of heart failure and related signs, symptoms and / or malfunctions and methods for the production of pharmaceutical agents for the treatment of heart failure.
Proteoglykane sind polyanionische Substanzen hohen Molekulargewichts, in denen verschiedene Arten von Heteropolysaccharid-Ket- ten kovalent an ein Polypeptidrückgrat gebunden sind. Die ehemals als Mucopolysaccharide bezeichneten Polysaccharidgruppen der Proteoglykane werden neuerdings als Glycosaminoglykane bezeichnet. Eine Vielzahl von Enzymen ist an dem Auf-, Um- und Abbau dieser Proteoglykane beteiligt. Durch Proteolyse können Glycosaminoglykane freigesetzt werden, die wiederum unter der Einwirkung von Glycosaminoglykan-Endoglycosidasen in kleinere Fragmente zerlegt werden, während entsprechende Exoglycosidasen Monosaccharide von den nicht-reduzierenden Enden der Glycosaminoglykane freisetzen.Proteoglycans are polyanionic substances of high molecular weight in which different types of heteropolysaccharide chains are covalently bound to a polypeptide backbone. The polysaccharide groups of the proteoglycans, formerly known as mucopolysaccharides, are now known as glycosaminoglycans. A large number of enzymes are involved in the construction, conversion and degradation of these proteoglycans. Proteolysis can release glycosaminoglycans, which in turn are broken down into smaller fragments under the action of glycosaminoglycan endoglycosidases, while corresponding exoglycosidases release monosaccharides from the non-reducing ends of the glycosaminoglycans.
Heparansulfat (HS) und Heparansulfat-Proteoglykane (HSPG) kommen auf der extrazellulären Oberfläche und in der extrazellulären Matrix vor. Die HS-Ketten werden im allgemeinen aus Clustern sulfa- tierter Disaccharid-Einheiten, vornehmlich 1-4 an α-Iduronsäure- Reste gebundene N-sulfatierte Glucosamine, gebildet, die durch wenig oder nicht sulfatierte Regionen, vornehmlich 1-4 an ß-D-Glucuronsäure gebundene N-acetylierte Glucosamine, voneinander getrennt sind. Ihnen wird eine Hauptrolle in Zeil-Zeil- und Zell-Matrix- echselwirkungen zugeschrieben, die an verschiedenen physiologischen und pathophysiologischen Prozessen beteiligt sind. Zu nennen sind hier beispielsweise die Adhäsion, Migration, Differenzierung und Proliferierung von Zellen. Berichten zufolge interagieren verschiedene Moleküle mit HS und/oder HSPG. Es han- delt sich dabei entweder um Wachtstumsfaktoren (z.B. FGF, PDGF, VEGF), Cytokine (IL-2), extrazelluläre Matrixproteine (Fibronek- tin, Collagen), an der Hämostase beteiligte Faktoren (Heparin-Co- faktor II), oder um Moleküle anderer Natur, z.B. Lipoproteine, DNA-Topoisomerasen und ß-Amyloidproteine (vgl. Hileman et al. (1998) BioEssays 20, 156-167; Stringer and Gallagher (1997) Int. J. Biochem Cell Biol 29, 709-714; Rapraeger (1993) Curr. Opin Cell. Biol. 5, 844-853; Bernfield et al. (1993) Development 1993 Suppl. 205-212; Kjellen and Lindahl. (1991) Annu. Rev. Biochem. 60, 443-475; Schlessinger et al. (1995), Cell 83, 367-360; Turnbull und Gallagher (1993) Biochem. Soc. Trans. 21, 477-482; Najjam et al (1997) Cytokine 9, 1013-1022; Ho et al. (1997) J. Biol. Chem. 272, 16838-16844; Pillarisetti et al (1997) J. Biol. Chem. 272, 15753 15759; Kovalszky et al. (1998) Mol. Cell. Biol. 183, 11-23; Schulz et al. (1998) Eur. J. Neuroscience 10, 2085-2093) .Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPG) occur on the extracellular surface and in the extracellular matrix. The HS chains are generally formed from clusters of sulfated disaccharide units, primarily 1-4 N-sulfated glucosamines bonded to α-iduronic acid residues, which pass through regions with little or no sulfate, primarily 1-4 at β-D -Glucuronic acid bound N-acetylated glucosamines, separated from each other. They are believed to play a major role in cell-cell and cell-matrix interactions that are involved in various physiological and pathophysiological processes. Examples include the adhesion, migration, differentiation and proliferation of cells. Various molecules are reported to interact with HS and / or HSPG. These are either growth factors (eg FGF, PDGF, VEGF), cytokines (IL-2), extracellular matrix proteins (fibronectin, collagen), factors involved in hemostasis (heparin factor II), or molecules of a different nature, e.g. lipoproteins, DNA topoisomerases and β-amyloid proteins (cf.Hileman et al. (1998) BioEssays 20, 156-167; Stringer and Gallagher (1997) Int. J. Biochem Cell Biol 29, 709-714 ; Rapraeger (1993) Curr. Opin Cell. Biol. 5, 844-853; Bernfield et al. (1993) Development 1993 Suppl. 205-212; Kjellen and Lindahl. (1991) Annu. Rev. Biochem. 60, 443- 475; Schlessinger et al. (1995), Cell 83, 367-360; Turnbull and Gallagher (1993) Biochem. Soc. Trans. 21, 477-482; Najjam et al (1997) Cytokine 9, 1013-1022; Ho et al. (1997) J. Biol. Chem. 272, 16838-16844; Pillarisetti et al (1997) J. Biol. Chem. 272, 15753, 15759; Kovalszky et al. (1998) Mol. Cell. Biol. 183, 11-23; Schulz et al. (1998) Eur. J. Neuroscience 10, 2085-2093).
5 Angesichts dieser vielfältigen Beteiligung ist HS/HSPG-modulie- renden Enzymen besonderes Interesse entgegengebracht worden (Ernst et al. (1995) Crit. Rev. Biochem. Mol. Biol. 30, 387-444).5 In view of this diverse participation, particular interest has been shown in HS / HSPG-modulating enzymes (Ernst et al. (1995) Crit. Rev. Biochem. Mol. Biol. 30, 387-444).
Endoglycosidasen und insbesondere endo-ß-Glucuronidase (im folgen-Endoglycosidases and especially endo-ß-glucuronidase (hereinafter
10 den Heparanase genannt) fanden Beachtung, da sie mit der Metasta- sierung von Tumoren, endzündlichen Prozessen und der Leukozytenwanderung in Verbindung gebracht wurden (WO 95/24907; US-A-5,817,800; US-A-5,262, 403; Vlodavsky et al. (1999) Genbank Accession Nr. AF 144325; Hulett et al. (1999) Nature Medicine 5,10 called the heparanase) received attention because they were associated with the metastasis of tumors, inflammatory processes and leukocyte migration (WO 95/24907; US-A-5,817,800; US-A-5,262, 403; Vlodavsky et al . (1999) Genbank Accession No. AF 144325; Hulett et al. (1999) Nature Medicine 5,
15 803-809; Toyoshima and Nakajima (1999) J. Bio. Chem 274,15 803-809; Toyoshima and Nakajima (1999) J. Bio. Chem 274,
24153-24160). Tatsächlich wurde Heparanase ursprünglich in muri- nen metastatischen Melanomzellen entdeckt. Heparanase spaltet HS in charakteristische Fragmente hohen Molekulargewichts und diese Aktivität wurde mit dem metastatischen Potential der Melanomzel-24153-24160). In fact, heparanase was originally discovered in murine metastatic melanoma cells. Heparanase cleaves HS into characteristic high molecular weight fragments and this activity has been associated with the metastatic potential of melanoma cells.
20 len korreliert (Nakajima et al. (1983) Science 220, 601-613; Nakajima et al. (1984) J. Biol. Chem.259, 2283-2290). Infolge wurde eine erhöhte Heparanase-Aktivität in weiteren mobilen, in- vasiven Zellen aufgezeigt, beispielsweise in Zusammenhang mit Lymphomen, Mastocytomen, Adenocarzinomen, Leukämien und rheuma-20 len correlated (Nakajima et al. (1983) Science 220, 601-613; Nakajima et al. (1984) J. Biol. Chem. 259, 2283-2290). As a result, increased heparanase activity in other mobile, invasive cells has been demonstrated, for example in connection with lymphomas, mastocytomas, adenocarcinomas, leukaemias and rheumatic
25 toiden Fibroblasten.25 roaring fibroblasts.
Gestützt auf diese Beobachtungen schlug man vor, Heparanase-Inhibitoren zu verwenden, um das invasive Potential von Zellen in Zusammemhang mit pathologischen Zuständen günstig zu beeinflus-Based on these observations, it was proposed to use heparanase inhibitors to favorably influence the invasive potential of cells related to pathological conditions.
30 sen. In diesem Sinne wird in der WO 99/43830 vermutet, daß Inhibitoren der Heparanase-Aktivität auch bei der Behandlung von Arthritis, Asthma und anderen entzündlichen Erkrankungen, vaskulä- rer Restenose, Atherosklerose, Tumorwachstum und -progression und fibroproliferativen Erkrankungen von Nutzen sein könnten, denn30 sen. In this sense it is assumed in WO 99/43830 that inhibitors of heparanase activity could also be useful in the treatment of arthritis, asthma and other inflammatory diseases, vascular restenosis, atherosclerosis, tumor growth and progression and fibroproliferative diseases, because
35 all diesen Zuständen liegt eine Einwanderung von Fremdzellen in das betroffene Gewebe bzw. Organ zugrunde.35 All of these conditions are based on immigration of foreign cells into the affected tissue or organ.
Aufgabe der vorliegenden Erfindung ist es, neue therapeutische Anwendungen für eine Modulation der Heparanase-Aktivität bereit- 40 zustellen.The object of the present invention is to provide new therapeutic applications for modulating the heparanase activity.
Überraschenderweise wurde nun gefunden, daß die Inhibition der Heparanase-Aktivität eine Behandlung von Herzsuffizienz ermöglicht. 45 Gegenstand der vorliegenden Erfindung ist die Verwendung wenigstens eines Heparanase-Inhibitors zur Behandlung von Herzinsuffizienz.Surprisingly, it has now been found that the inhibition of heparanase activity enables the treatment of heart failure. 45 The present invention relates to the use of at least one heparanase inhibitor for the treatment of heart failure.
Unter Herzinsuffizienz (synonym: Myocardinsuffizienz , Herzmuskelschwäche, Insuffizienzia cordis) versteht man erfindungsgemäß ein Unvermögen des Herzens, die erforderliche Förderleistung zu erbringen. Der Begriff Herzinsuffizienz beschreibt den Zustand eines Herzes, in dem Kompensationsmechanismen wie Herzfrequenz, Kontraktivität, Schlagvolumen, Hypertonie, nicht zur Aufrechterhaltung eines normalen Herzzeitvolumens ausreichen. Es handelt sich um eine Schwäche der Pumpenfunktion.According to the invention, cardiac insufficiency (synonym: myocardial insufficiency, cardiac muscle weakness, insufficiency cordis) means an inability of the heart to provide the necessary support. The term heart failure describes the state of a heart in which compensation mechanisms such as heart rate, contractility, stroke volume, hypertension are not sufficient to maintain a normal cardiac output. It is a weakness of the pump function.
Dieser Zustand kann bei Belastung (Belastungsinsuffizienz) oder schon in Ruhe (Ruheinsuffizienz) auftreten. Je nach Schweregrad wird gemäß der New York Heart Association (NYHA) unterschieden zwischen Funktionsklassen I bis IV, d.h. einer völligen Beschwerdefreiheit bei normaler körperlicher Belastung bis hin zu Insuffizienzzeichen bei nahezu jeder körperlichen Tätigkeit, die häu- fig auch in Ruhe bestehen.This condition can occur during exercise (insufficiency) or at rest (insufficiency). Depending on the severity, according to the New York Heart Association (NYHA) a distinction is made between functional classes I to IV, i.e. complete freedom from symptoms with normal physical exertion up to signs of insufficiency in almost every physical activity, which often also exist at rest.
Die Herzinsuffizienz kann das gesamte Herz (globale Herzinsuffizienz) oder Teile davon betreffen, beispielsweise Links- oder Rechtsherzinsuffizienz .Heart failure can affect the entire heart (global heart failure) or parts of it, for example left or right heart failure.
Erfindungsgemäß bevorzugt ist die Behandlung von Myokardinsuffi- zienzen, d.h. Herzinsuffizienzen, die auf eine Veränderung des Myokards zurückzuführen sind. Zu nennen sind in diesem Zusammenhang insbesondere Kardiomyopathien und vorzugsweise primäre Kar- diomyopathien, beispielsweise durch Hypertrophie des Herzens, vor allem des Kammerseptums und des linken Ventrikels gekennzeichnete hypertrophe obstruktive oder nicht-obstruktive Kardiomyopathien und durch Hypertrophie und Dilatation des Herzens gekennzeichnete kongestive Kardiomyopathien (auch als dilatative kongestive Kar- diomyopathien bezeichnet) .Preferred according to the invention is the treatment of myocardial insufficiency, i.e. Heart failure due to a change in the myocardium. In this context, particular mention should be made of cardiomyopathies and preferably primary cardiomyopathies, for example hypertrophic obstructive or non-obstructive cardiomyopathies characterized by hypertrophy of the heart, especially the chamber septum and left ventricle, and congestive cardiomyopathies characterized by hypertrophy and dilatation of the heart (also as dilated congestive cardiomyopathies).
Den erfindungsgemäß bevorzugt behandelten Formen von Herzinsuffizienz, insbesondere kongestiver Herzinsuffizienz, liegen eine oder mehrere der nachfolgend aufgezählten Veränderungen des Myo- kards zugrunde: Hypertrophie einzelner oder aller Wandschichten, Abnahme der Muskeldehnbarkeit, Herzvergrößerung, insbesondere Ventrikelvergrößerung, insbesondere ohne Dickenzunahme der Ventrikelmuskulatur, Dickenabnahme der Ventrikelmuskulatur und fi- brotische Veränderungen der Ventrikelmuskulatur. Die erfindungsgemäß zu behandelnde Indikation Herzinsuffizienz ist in der Regel gekennzeichnet durch eine progressive Entwicklung, d.h. die vorstehend beschriebenen Zustände verändern sich im Laufe der Zeit, in der Regel nimmt der Schweregrad zu und ge- gebenenfalls können Zustände ineinander übergehen oder weitere Zustände zu bereits bestehenden Zuständen hinzutreten.The forms of heart failure, in particular congestive heart failure, which are preferably treated according to the invention are based on one or more of the changes in the myocardium listed below: hypertrophy of individual or all wall layers, decrease in muscle extensibility, heart enlargement, in particular enlargement of the ventricle, in particular without increasing the thickness of the ventricular muscles, decrease in the thickness of the ventricular muscles and fibrous changes in the ventricular muscles. The indication heart failure to be treated according to the invention is generally characterized by a progressive development, ie the conditions described above change over time, the degree of severity generally increases and, if appropriate, conditions can merge with one another or other conditions can change to existing conditions draw near.
In diesem Sinne werden erfindungsgemäß insbesondere Veränderungen des Myokards behandelt, die unter dem Begriff "remodelling" zu- sammengefaßt werden; das sind Vorgänge, die Veränderungen in der Myokardiocyt-Struktur und/oder Veränderungen umgebenden Bindegewebes mit sich bringen.In this sense, changes in the myocardium, which are summarized under the term "remodeling", are treated according to the invention; these are processes that involve changes in the structure of the myocardiocyte and / or changes in the connective tissue surrounding it.
Einem besonderen Aspekt zufolge werden Herzinsuffizienzen behan- delt, denen eine Absenkung des pH-Wertes betroffener Herzteile vorausgeht oder diese begleitet. Werte im sauren pH-Bereich, in der Regel bei etwa 2 bis 6,5, bei etwa 3 bis 6 und vor allem bei etwa 4,5 bis 5,5 sind hier von Bedeutung.According to a special aspect, heart failures are treated which are preceded or accompanied by a lowering of the pH value of the affected heart parts. Values in the acidic pH range, usually around 2 to 6.5, around 3 to 6 and especially around 4.5 to 5.5 are important here.
Durch die erfindungsgemäße Behandlung von Herzinsuffizienz bzw. den ihr zugrundeliegenden Zuständen lassen sich eine Reihe weiterer Anzeichen, Symptome und/oder Fehlfunktionen behandeln, die mit Herzinsuffizienz zusammenhängen, d.h. insbesondere die oben beschriebenen Erkrankungszustände begleiten. Hierzu gehören bei- spielsweise Veränderungen des peripheren Kreislaufs, insbesondere Stauungserscheinungen im großen und/oder im kleinen Kreislauf, z.B. Lungen- und Leberkongestion, eine Verminderung der Blutversorgung der Kreislaufperipherie, Störungen der Atmung (Dyspnoe), der Nierenfunktion, z.B. Nykturie, und des Elektrolytstoff ech- sels, z.B. periphere Ödeme, Brust- und Bauchwassersucht, etc. Diese Anzeichen, Symptome und/oder Fehlfunktionen bilden häufig Muster oder Gruppen, die als Syndrome bezeichnet werden, so daß sich erfindungsgemäß die Behandlung des Syndroms Herzinsuffienz ergibt .The treatment of heart failure according to the invention or the conditions on which it is based enable a number of other signs, symptoms and / or malfunctions to be treated which are related to heart failure, i.e. especially accompany the disease states described above. These include, for example, changes in the peripheral circulation, in particular congestion in the large and / or in the small circulation, e.g. Lung and liver congestion, a reduction in the blood supply to the peripheral circulation, respiratory disorders (dyspnea), kidney function, e.g. Nocturia, and the electrolyte, e.g. peripheral edema, breast and abdominal addiction, etc. These signs, symptoms and / or malfunctions often form patterns or groups which are referred to as syndromes, so that, according to the invention, the treatment of the heart failure syndrome results.
Eine Behandlung im erfindungsgemäßen Sinne umfaßt nicht nur die Behandlung akuter oder chronischer Anzeichen, Symptomen und/oder Fehlfunktionen, sondern auch eine vorbeugende Behandlung (Prävention) . Ein Zweck der akuten oder chronischen Behandlung ist eine Behebung der Störungen, Regulation der Zustände, bzw. Linderung der Anzeichen, Symptome und/oder Fehlfunktionen. Einem besonderen Aspekt zufolge ist es Zweck der Behandlung, die Aktivität der Heparanase zu verringern. Ein Zweck der vorbeugenden (präventiven) Behandlung ist es, das Auftreten der Störungen, Zustände, Anzeichen, Symptome und/oder Fehlfunktionen zu vermeiden, wozu auch eine zeitliche Verzögerung des Auftretens zählt. Die Behandlung kann symptomatisch, beispielsweise als Symptomsuppression ausgerichtet sein. Sie kann kurzzeitig erfolgen, mittelfristig ausgerichtet sein, oder es kann sich auch um eine Langzeitbehandlung, beispielsweise im Rahmen einer Erhaltungstherapie, handeln.Treatment in the sense of the invention includes not only the treatment of acute or chronic signs, symptoms and / or malfunctions, but also preventive treatment (prevention). One purpose of acute or chronic treatment is to remedy the disorders, regulate the conditions, or alleviate the signs, symptoms and / or malfunctions. According to a particular aspect, the purpose of the treatment is to reduce the activity of the heparanase. One purpose of preventive (preventive) treatment is to prevent the occurrence of the disorders, conditions, signs, symptoms and / or malfunctions, which includes a time delay in the occurrence. Treatment can be symptomatic, for example as symptom suppression be aligned. It can be short-term, medium-term, or it can also be long-term treatment, for example in the context of maintenance therapy.
Der Begriff "Heparanase-Inhibitor" beschreibt Substanzen, welche die enzymatische Aktivität von Heparanase oder deren Expression inhibieren. Unter Inhibition wird in diesem Zusammenhang eine Verminderung der Enzymaktivität, vor allem der Aktivität als En- doglycosidase, Endoglucuronidase, ß-Glucuronidase und insbesondere Endo-ß-Glucuronidase, bzw. der Expression von Heparanase verstanden. Die Enzymaktivität von Heparanase führt beispielsweise zur Spaltung von Glycosaminoglykanen, gegebenenfalls als Teil von Proteoglykanen, insbesondere von Heparansulfat, bzw. den entsprechenden Proteoglykane. Vorzugsweise bewirken erfindungsgemäße He- paranase-Inhibtioren eine Verringerung des HS- und HSPG-Abbaus durch Heparanase.The term "heparanase inhibitor" describes substances which inhibit the enzymatic activity of heparanase or its expression. In this context, inhibition is understood to mean a reduction in the enzyme activity, especially the activity as endogenous glycosidase, endoglucuronidase, β-glucuronidase and in particular endo-β-glucuronidase, or the expression of heparanase. The enzyme activity of heparanase leads, for example, to the cleavage of glycosaminoglycans, optionally as part of proteoglycans, in particular heparan sulfate, or the corresponding proteoglycans. Heparase inhibitors according to the invention preferably bring about a reduction in the HS and HSPG degradation by heparanase.
Erfindungsgemäß bevorzugt sind Inhibitoren von Säuger-Heparanase (EC 3.2.1) und insbesondere von humaner Heparanase und vor allem der durch die cDNA mit der SEQ ID NO: 1 kodierten Heparanase mit der Aminosäuresequenz SEQ ID NO: 2.According to the invention, inhibitors of mammalian heparanase (EC 3.2.1) and in particular of human heparanase and especially the heparanase with the amino acid sequence SEQ ID NO: 2 encoded by the cDNA with SEQ ID NO: 1 are preferred.
Erfindungsgemäße Inhibitoren binden in der Regel an Heparanase oder an Heparanase kodierende Nukleinsäuren, z.B. DNA oder mRNA. Unter Bindung versteht man jede molekulare Wechselwirkung zwischen Inhibitor und Enzym bzw. Nukleinsäure, insbesondere unter physiologischen Bedingungen. Dies sind in der Regel klassische Wechselwirkungen, zu denen elektrostatische Kräfte, van-der- Waals-Kräfte, Wasserstoffbrücken-Bindungen, hydrophobe Bindungen, oder metallkomplexartige koordinative Bindungen gehören. Zusätzlich zu den vorstehend genannten, reversiblen molekularen Wechselwirkungen können auch irreversible Wechselwirkungen zwischen Inhibitor und Enzym in Betracht kommen, z.B. kovalente Bindungen.Inhibitors according to the invention usually bind to heparanase or to nucleic acids encoding heparanase, e.g. DNA or mRNA. Binding is understood to mean any molecular interaction between inhibitor and enzyme or nucleic acid, in particular under physiological conditions. These are usually classic interactions, which include electrostatic forces, van der Waals forces, hydrogen bonds, hydrophobic bonds, or metal complex-like coordinative bonds. In addition to the reversible molecular interactions mentioned above, irreversible interactions between inhibitor and enzyme can also be considered, e.g. covalent bonds.
In der Regel binden Enzym-Inhibitoren im Bereich der oder einer der aktiven Domänen von Heparanase und konkurrieren mit anderen Substraten um deren Bindungsstelle (n) (Kompetition) . Dementsprechend versteht man unter kompetitiven Enzym-Inhibitoren diejenigen, die mit einem VergleichsSubstrat, im vorliegenden Fall vor- zugsweise Heparansulfat, um die Bindung an Heparanase konkurrieren, d.h. die Bindung des einen behindert die Bindung des anderen. Wegen dieser Bindung an Heparanase können kompetitive Enzym- Inhibitoren auch als Heparanase-Substrat bezeichnet werden. Vorzugsweise handelt es sich bei diesen Inhibitoren um Substrate, die im Vergleich zu dem oder den natürlichen Substraten der kata- lytischen Aktivität von Heparanase nicht oder zumindest weniger zugänglich sind, d.h. sie werden nicht oder in vergleichsweise geringem Ausmaß durch Heparanase umgesetzt, insbesondere gespalten. Ebenfalls brauchbar sind nicht-kompetitive Inhibitoren, die beispielsweise im wesentlichen irreversibel an aktive Domänen, oder an anderer Stelle an die Heparanase binden und, beispiels- weise über allosterische Effekte, Einfluß auf die Enzymaktivität nehmen.As a rule, enzyme inhibitors bind in the region of or one of the active domains of heparanase and compete with other substrates for their binding site (s) (competition). Accordingly, competitive enzyme inhibitors are understood to mean those which compete with a comparison substrate, in the present case preferably heparan sulfate, for binding to heparanase, ie the binding of one hinders the binding of the other. Because of this binding to heparanase, competitive enzyme inhibitors can also be referred to as heparanase substrates. These inhibitors are preferably substrates which, in comparison to the natural substrate or substrates, are not or at least less accessible to the catalytic activity of heparanase, ie they are not or only comparatively accessible to a small extent by heparanase, in particular cleaved. It is also possible to use non-competitive inhibitors which, for example, bind essentially irreversibly to active domains or to the heparanase elsewhere and, for example via allosteric effects, have an influence on the enzyme activity.
Zumindest für den Fall der kompetitiven Inhibition gilt der Grundsatz, daß die Verdrängung eines Substrats durch einen Inhi- bitor mit abnehmender Bindungsaffinität des Substrats bzw. zunehmender Bindungsaffinität des Inhibitors zunimmt. Zweckmäßigerweise besitzen daher erfindungsgemäß brauchbare Inhibitoren eine hohe Bindungsaffinität für Heparanase. Eine derartig günstig ausfallende Bindungsaffinität gestattet eine wirksame Verdrängung natürlich vorkommender Enzymsubstrate, beispielsweise von Hepa- ransulfaten und Heparansulfat-Proteoglykanen, wobei die erforderliche Konzentration an Inhibitor zur Bindung einer bestimmten Menge dieses Inhibitors an das Enzym bzw. zur Verdrängung einer bestimmten Menge eines Substrats mit zunehmender Bindungsaffini- tat des Inhibitors abnimmt. Im Hinblick auf die medizinische Anwendung werden daher Inhibitoren bevorzugt, deren Bindungsaffinität so groß ist, daß diese als Wirkstoff im Rahmen einer wirksamen medizinischen Behandlung in vertretbaren Mengen verabreicht werden können. Erfindungsgemäße Inhibitoren werden daher vorzugs- weise in Tagesdosen von etwa 0,01 bis 30 mg/kg Körpergewicht und insbesondere von etwa 0,1 bis 15 mg/kg Körpergewicht verabreicht.At least in the case of competitive inhibition, the principle applies that the displacement of a substrate by an inhibitor increases with decreasing binding affinity of the substrate or increasing binding affinity of the inhibitor. It is therefore expedient for inhibitors which can be used according to the invention to have a high binding affinity for heparanase. Such a favorable binding affinity allows an effective displacement of naturally occurring enzyme substrates, for example heparan sulfates and heparan sulfate proteoglycans, the concentration of inhibitor required for binding a certain amount of this inhibitor to the enzyme or for displacing a certain amount of a substrate with increasing Binding affinity of the inhibitor decreases. With regard to medical use, inhibitors are therefore preferred whose binding affinity is so great that they can be administered as an active ingredient in an effective medical treatment in acceptable amounts. Inhibitors according to the invention are therefore preferably administered in daily doses of approximately 0.01 to 30 mg / kg body weight and in particular approximately 0.1 to 15 mg / kg body weight.
Eine Möglichkeit, die Bindungsaffinität auszudrücken, bieten die oben angesprochenen Kompetitionsexperimente, mit denen man dieje- nige Konzentration an Inhibitor ermittelt, die das Enzym im Hinblick auf die Umsetzung eines anderen Substrats zu 50% hemmt (ICso-Werte) . So läßt sich auch die kompetitive Hemmung der Bindung von Heparanase-Inhibitoren dahingehend auswerten, daß erfindungsgemäß bevorzugte Inhibitoren halbmaximale Hemmkonstanten IC50 in vitro von weniger als 10-3 M, vorzugsweise von weniger als 10-4 M und insbesondere von weniger als 10-5 M und bei nicht-kompetiver Hemmung von weniger als 10-4 M, vorzugsweise von weniger als 10-5 M und insbesondere von weniger als 10~6 M aufweisen.One way of expressing the binding affinity is provided by the competition experiments mentioned above, with which one determines the concentration of inhibitor which the enzyme inhibits by 50% with regard to the conversion of another substrate (IC 50 values). Thus, the competitive inhibition of the binding of heparanase inhibitors can also be evaluated to the extent that inhibitors preferred according to the invention have half-maximum inhibitory constants IC 50 in vitro of less than 10 -3 M, preferably less than 10 -4 M and in particular less than 10 -5 M and with non-competitive inhibition of less than 10 -4 M, preferably less than 10 -5 M and in particular less than 10 ~ 6 M.
Bei den Expressionsinhibitoren handelt es sich insbesondere um Oligonukleotide, die beispielsweise im Sinne einer antisense-RNA oder -DNA, oder im Sinne der Triple-Helix-Technik wirken.The expression inhibitors are, in particular, oligonucleotides which act, for example, in the sense of an antisense RNA or DNA, or in the sense of the triple helix technique.
Eine Reihe von Heparanase-Inhibitoren sind bereits bekannt. Es handelt sich vielfach um Glycosaminoglykane mit struktureller Ähnlichkeit zu den natürlichen Substraten der Heparanase, insbesondere Heparansulfate. Hierzu gehören Heparine, Heparinfraktio- nen und Heparinfragmente, z.B. Heparine bestimmten Molekulargewichts, Heparinderivate, beispielsweise Heparine mit zumindest teilweise reduzierten Carboxylgruppen, zumindest partiell N-de- sulfatierte, N-acetylierte Heparine, z.B. in EP 0 254 067 A2, WO 92/01003 und US-A-5,206,223 beschriebenes N-desulfatiertes, N-acetyliertes Heparin, zumindest partiell N,0-desulfatierte, N-resulfatierte Heparine, z.B. die in WO 92/01003 und US-A-5, 206,223 beschriebenen Verbindungen, und O-acylierte Heparine, beispielsweise die in der EP 0 356 275 AI beschriebenen Verbindungen.A number of heparanase inhibitors are already known. Many of them are glycosaminoglycans with structural similarity to the natural substrates of heparanase, in particular heparan sulfates. These include heparins, heparin fracture Nen and heparin fragments, for example heparins of a certain molecular weight, heparin derivatives, for example heparins with at least partially reduced carboxyl groups, at least partially N-desulfated, N-acetylated heparins, for example in EP 0 254 067 A2, WO 92/01003 and US Pat. No. 5,206,223 N-desulfated, N-acetylated heparin described, at least partially N, 0-desulfated, N-resulfated heparins, for example the compounds described in WO 92/01003 and US Pat. No. 5, 206,223, and O-acylated heparins, for example those in the compounds described in EP 0 356 275 AI.
Heparin wird vorzugsweise aus natürlichen Quellen, beispielsweise der intestinalen Mukosa von Rindern oder Schweinen, gewonnen. Eine Fragmentierung und/oder Fraktionierung kann auf die übliche Art und Weise erfolgen. Carboxylgruppen lassen sich beispielsweise mit NaBH reduzieren. Sulfatgruppen können beispielsweise durch eine Behandlung mit wasser- oder methanolhaltigem DMSO entfernt werden, wobei sich der Grad der Desulfatierung nach der Reaktionsdauer, der Reaktionstemperatur und dem Zusatz von Wasser oder Methanol richtet. Eine N-Acetylierung kann beispielsweise mit Essigsäureanhydrid unter alkalischen Bedingungen bewerkstelligt werden und die Resulfatierung gelingt beispielsweise mit einem Triethylamin-Schwefeltrioxid-Komplex.Heparin is preferably obtained from natural sources, for example the intestinal mucosa of cattle or pigs. Fragmentation and / or fractionation can be carried out in the usual way. Carboxyl groups can be reduced with NaBH, for example. Sulfate groups can be removed, for example, by treatment with water- or methanol-containing DMSO, the degree of desulfation depending on the reaction time, the reaction temperature and the addition of water or methanol. N-acetylation can be accomplished, for example, with acetic anhydride under alkaline conditions and the resulfation can be accomplished, for example, with a triethylamine-sulfur trioxide complex.
Anstatt Heparin können auch andere Glycosaminoglykane derivati- siert werden, beispielsweise Hyaluronsäure, Chondroitin-4-sulfat, Chondroitin-6-sulfat, Deπrtatansulfat, Keratansulfat und Heparansulfat und deren Proteoglykane, wie am Beispiel der O-Acylierung in der EP 0 356 275 AI beschrieben ist.Instead of heparin, other glycosaminoglycans can also be derivatized, for example hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate, tertiary sulfate, keratin sulfate and heparan sulfate and their proteoglycans, as described using the example of O-acylation in EP 0 356 275 Al is.
Geeignet sind auch sulfatierte Oligosaccharide, beispielsweise die in WO 96/33726 beschriebenen, also insbesondere sulfatierte Mannopentaosephosphate, Maltohexaosesulfate und dergleichen, und sulfatierte Polysaccharide, beispielsweise die in WO 88/05301 be- schriebenen, also insbesondere Heparin, Fucoidan, Pentosansulfat, Dextransulfat und Carrageenan-Lambda. Auch die in WO 90/01938 genannten Phosphozucker enthaltenden Oligo- und Polysaccharide sind brauchbar.Also suitable are sulfated oligosaccharides, for example those described in WO 96/33726, ie in particular sulfated mannopentaose phosphates, maltohexaose sulfates and the like, and sulfated polysaccharides, for example those described in WO 88/05301, that is to say in particular heparin, fucoidan, pentosan sulfate, dextran sulfate and carrageenan -Lambda. The oligosaccharides and polysaccharides containing phosphosugars mentioned in WO 90/01938 can also be used.
Ebenfalls geeignet sind glycomimetische Saccharopeptide, beispielsweise die in WO 96/35700 beschriebenen der FormelGlycomimetic saccharopeptides, for example those of the formula described in WO 96/35700, are also suitable
W (X)n Y [(X)n W (X)n Y]m (X)n WW (X) n Y [(X) n W (X) n Y] m (X) n W
worin die Reste W unabhängig voneinander für Fucose,wherein the radicals W independently of one another for fucose,
3-Amino-3-deoxyglucose, 4-Amino-4-deoxyglucose, Glucose, Galactose, Glucosamin, Galactosamin, Glucuronsäure, Galacturonsäure, Glucosaminuronsäure, Neuraminsäure, Maltose, Maltotriose, Iduronsäure, 2,5-Anhydromannitol, Mannose, Mannuronsäure, und Cellobiose stehen;3-amino-3-deoxyglucose, 4-amino-4-deoxyglucose, glucose, galactose, glucosamine, galactosamine, glucuronic acid, galacturonic acid, glucosaminuronic acid, neuraminic acid, maltose, maltotriose, iduronic acid, 2,5-anhydromannitol, mannose, mannuronic acid, and cellobiose stand;
die Reste Y unabhängig voneinander für -NR3-C(0)- und -C(0)-NR3- stehen;the radicals Y independently of one another represent -NR 3 -C (0) - and -C (0) -NR 3 -;
die Reste X unabhängig voneinander für eine difunktionelle or polyfunktioneile Gruppe, insbesondere Ethylenglycol, Ethylenglycol-Oligomere, Niedrigalkyl, gegebenefalls substituiertes Alkyl, Aminosäuren und Peptide stehen;the radicals X independently of one another represent a difunctional or polyfunctional group, in particular ethylene glycol, ethylene glycol oligomers, lower alkyl, optionally substituted alkyl, amino acids and peptides;
n jeweils 0 oder 1 ist; m jeweils 0 oder eine ganze Zahl von 1 bis 99 ist;n is 0 or 1; m is each 0 or an integer from 1 to 99;
mit der Maßgabe, daß die Gesamtanzahl von Resten W 2 bis 100 be- trägt;with the proviso that the total number of residues W is 2 to 100;
und R3 für -H, Alkyl mit 1 bis 8 Kohlenstoffatomen und Aralkyl mit 5 bis 8 Kohlenstoffatomen steht.and R 3 represents -H, alkyl of 1 to 8 carbon atoms and aralkyl of 5 to 8 carbon atoms.
Laminarin-Sulfate, auch Laminaran-Sulfate genannt, das sind lineare Polymere aus ß-1,3-verknüpften Glucose-Resten mit gegebenenfalls geringen Anteilen an ß- ( 1 , 6 ) -Verknüpfungen und 2 bis 3% D-Mannitol-Endgruppen, insbesondere das Natriumsalz mit einem molaren Verhältnis von wenigstens 1 : 1 Sulfatgruppen zu Monosaccha- rid-Einheiten ist ebenfalls als Heparanase-Inhibitor brauchbar (vgl. WO 95/24907) .Laminarin sulfates, also called laminaran sulfates, are linear polymers made from β-1,3-linked glucose residues with possibly small proportions of β- (1,6) linkages and 2 to 3% D-mannitol end groups, in particular the sodium salt with a molar ratio of at least 1: 1 sulfate groups to monosaccharide units can also be used as a heparanase inhibitor (cf. WO 95/24907).
Weitere Heparanase-Inhibitoren sind Suramin und Trachyspinsäure.Other heparanase inhibitors are suramin and trachyspinic acid.
Geeignet sind auch die in dem US-Patent 5,817,800 beschriebenen Verbindungen der Formel IThe compounds of the formula I described in US Pat. No. 5,817,800 are also suitable
Figure imgf000009_0001
Figure imgf000009_0001
worin Y für -COOH, -P03H2-P(0) (OR6) (OH), -P(0)R6(OH), Tetrazol und -S03H steht, worinwherein Y stands for -COOH, -P0 3 H 2 -P (0) (OR 6 ) (OH), -P (0) R 6 (OH), tetrazole and -S0 3 H, in which
R6 C!-C4-Alkyl ist,R 6 C ! Is -C 4 alkyl,
X für NH, 0 oder S steht, undX represents NH, 0 or S, and
R für ein Wasserstoffatom oder für -C(0)NHC6(R7)5 steht, worin Cβ(R7)5 vorzugsweise für gegegenenfalls einfach bis fünffach substituiertes Phenyl steht und die Substituenten R7 ausgewählt sind unter OH, Halogen, -COOH, -P03H2 oder -S03H.R represents a hydrogen atom or -C (0) NHC 6 (R 7 ) 5 , in which Cβ (R 7 ) 5 preferably represents optionally mono- to pentasubstituted phenyl and the substituents R 7 are selected from OH, halogen, -COOH , -P0 3 H 2 or -S0 3 H.
Auch die Salze dieser Verbindungen gehören dazu. Veranschaulichende Beispiele dieser Verbindungen sind ( Z ) -O- (D-Glukopyranuro- nosyliden)amino-N-phenylcarbamat und (5R,Z)-0-(5-C-Phosphonato- D-xylopyranosyliden) amino-N-phenylcarbamat bzw. deren Natriumsalze. Diese Verbindungen können in an sich bekannter Weise hergestellt werden, beispielsweise mit den in der EP 0 642 799 beschriebenen Verfahren.The salts of these compounds are also included. Illustrative examples of these compounds are (Z) -O- (D-glucopyranuronosylidene) amino-N-phenylcarbamate and (5R, Z) -0- (5-C-phosphonato-D-xylopyranosylidene) amino-N-phenylcarbamate and their sodium salts. These compounds can be prepared in a manner known per se, for example using the processes described in EP 0 642 799.
Niedermolekulare Heparanase-Inhibitoren, meist synthetische Verbindungen, sind in vielerlei Hinsicht vorteilhaft brauchbar.Low molecular weight heparanase inhibitors, mostly synthetic compounds, can be used advantageously in many ways.
Auch Aptamere, das sind Nukleinsäuren, in der Regel Oligonukleo- tide, mit ausreichender Affinität zu Heparanase, können als Inhibitoren Anwendung finden.Aptamers, which are nucleic acids, usually oligonucleotides, with sufficient affinity for heparanase, can also be used as inhibitors.
Auch Heparanase-spezifische Antikörper können als Heparanase-Inhibitoren brauchbar sein. Es kann sich um polyklonale Antiseren, monoklonale Antikörper, Antikörperfragmente, wie F(ab), Fc, etc., chimäre, humanisierte und rekombinante Antikörper handeln. Die Herstellung solcher Antikörper kann in an sich bekannter Weise erfolgen. Als Immunogen kann man Heparanase als solche oder anti- gene Fragmente davon, die in der Regel an übliche Trägerproteine gekoppelt werden, verwenden. In Beispiel 8 der WO 99/43830 wird beispielsweise die Herstellung eines polyklonalen Antiserums gegen ausgesuchte Peptide humaner Heparanase beschrieben. In ähnlicher Weise wird in WO 95/04158 die Herstellung ausgesuchter Hepa- ranase-Peptide, insbesondere einer C-terminalen Sequenz, dort SEQ ID NO: 42 genannt, und die Erzeugung hiergegen gerichteter Anti- sera sowie deren Brauchbarkeit als Heparanase-Inhibitoren beschrieben.Heparanase-specific antibodies can also be useful as heparanase inhibitors. It can be polyclonal antisera, monoclonal antibodies, antibody fragments such as F (ab), Fc, etc., chimeric, humanized and recombinant antibodies. Such antibodies can be produced in a manner known per se. Heparanase as such or antigenic fragments thereof, which as a rule are coupled to customary carrier proteins, can be used as the immunogen. Example 8 of WO 99/43830 describes, for example, the production of a polyclonal antiserum against selected peptides of human heparanase. Similarly, WO 95/04158 describes the production of selected heparinase peptides, in particular a C-terminal sequence, there called SEQ ID NO: 42, and the production of antisera directed against them and their usefulness as heparanase inhibitors.
Die WO 96/08559 beschreibt Phosphorthioat- oder Phosphordithioat- Antisense-Oligonukleotide mit vorzugsweise 7 bis 30 Nukleotiden, die im wesentlichen aus dG und/oder dT-Nukleotiden gebildet werden. Konkret eignen sich zur Inhibition von Endoglycosidasen, insbesondere Heparanasen, beispielsweise die Oligonukleotide der dort beschriebenen Sequenzen SEQ ID NO:2,4,6,10.WO 96/08559 describes phosphorothioate or phosphorodithioate antisense oligonucleotides with preferably 7 to 30 nucleotides, which are essentially formed from dG and / or dT nucleotides. Specifically, are suitable for the inhibition of endoglycosidases, in particular heparanases, for example the oligonucleotides of the sequences SEQ ID NO: 2,4,6,10 described there.
Die erfindungsgemäße Anwendung ist nicht auf die vorstehend ge- nannten Inhibitoren beschränkt. Vielmehr kann jede Substanz, in deren Gegenwart die Heparanase-Aktivität geringer ist als in deren Abwesenheit, erfindungsgemäß als Heparanase-Inhibitor Anwendung finden.The application according to the invention is not limited to the inhibitors mentioned above. Rather, any substance in the presence of which the heparanase activity is lower than in the absence thereof can be used according to the invention as a heparanase inhibitor.
Zur Messung der Heparanase-Aktivität sind Testsysteme bekannt. Diese beruhen in der Regel auf dem Einsatz von markierten Hepa- ransulfaten oder Heparansulfat-Proteoglykanen als Substrat, wobei die Umsetzung, d.h. die Spaltung dieses Substrats und die damit verbundene Freisetzung bestimmter Fragmente anhand der Markierung verfolgt werden kann. Beispielsweise kann man fluoreszenz-, z.B. FITC-, oder radio-, z.B. 35S04-, 14C- oder 3H-markierte Heparansul- fate oder Heparansulfat-Proteoglykane, insbesondere 35S04-markierte Heparansulfat-Proteoglykane, 1C- oder 3H-acetylierte Hepa- ransulfate, oder fluoreszenzmarkierte Substrate, beispielsweise 4-Methylumbelliferyl-ß-D-glucuronid verwenden.Test systems are known for measuring the heparanase activity. These are generally based on the use of labeled heparan sulfates or heparan sulfate proteoglycans as a substrate, the conversion, ie the cleavage of this substrate and the associated release of certain fragments, being able to be followed on the basis of the label. For example, one can fluoresce-, eg FITC-, or radio-, eg 35 S0 4 -, 14 C- or 3 H-labeled heparan sulfates or heparan sulfate proteoglycans, especially 35 S0 4- labeled heparan sulfate proteoglycans, 1 C- or Use 3 H-acetylated heparan sulfates or fluorescence-labeled substrates, for example 4-methylumbelliferyl-ß-D-glucuronide.
Die Substrate können auf biologischem Wege erhalten werden, beispielsweise indem man Endothelialzellen in radiomarkiertem 35S04 kultiviert oder tumorösen Versuchstieren radiomarkiertes Sulfat injiziert, und aus den Endothelial- bzw. Tumorzellen entsprechend markierte Heparansulfat-Proteoglykane gewinnt. Die Substrate können aber auch auf chemischem Wege synthetisiert werden, beispielsweise indem man Heparansulfat zunächst partiell deacety- liert und anschließend reacetyliert . Durch reduktive Aminierung der freien Enden von Heparansulfat und anschließender Anfügung geeigneter Markierungen können beispielsweise fluoreszenzmarkierte Heparansulfate hergestellt werden. Es kann von Vorteil sein, solche Substrate an einen festen Träger zu koppeln, was den Nachweis freigesetzter Fragmente erleichtert. Zu diesem Zweck kann man die in diesem Bereich übliche Kopplungschemie anwenden, beispielsweise zunächst die reduzierenden Enden aminieren und dann derart modifizierte Glycosaminoglykane an geeignete Matri- ces, beispielsweise Agarose, Sepharose und ähnliches koppeln. Heparansulfat-Proteoglykane oder auch Heparansulfat-Peptide davon können beispielsweise an CNBr-aktivierter Sepharose gekoppelt werden. Eine weitere Möglichkeit besteht in der Abtrennung der Degradationsprodukte durch Gelfiltration, Fällungsreaktionen, z.B. wie in Beispiel J der WO 96/35700 beschrieben ist, und ähnlichem. Mittels HPLC, vorzugsweise der Auschluß-HPLC , lassen sich vorteilhafterweise Fluoreszenzmarkierungen detektieren. Gemäß dem in der WO 98/03638 beschriebenen Testverfahren kann man auch HS- bindende Proteine, z.B. histidinreiche Glycoproteine , Vorzugs- weise in immobilisierter Form verwenden, um nicht oder nur partiell degradiertes Heparanase-Substrat von degradiertem Substrat zu trennen und dadurch dessen Nachweis zu ermöglichen.The substrates can be obtained biologically, for example by cultivating endothelial cells in radiolabeled 35 SO 4 or injecting radiolabelled sulfate into tumorous experimental animals, and recovering appropriately labeled heparan sulfate proteoglycans from the endothelial or tumor cells. However, the substrates can also be synthesized chemically, for example by first partially deacetylating heparan sulfate and then reacetylating. By reductive amination of the free ends of heparan sulfate and subsequent addition of suitable labels, for example, fluorescence-labeled heparan sulfates can be produced. It may be advantageous to couple such substrates to a solid support, which facilitates the detection of released fragments. For this purpose, the coupling chemistry customary in this area can be used, for example first aminating the reducing ends and then coupling glycosaminoglycans modified in this way to suitable matrices, for example agarose, sepharose and the like. Heparan sulfate proteoglycans or heparan sulfate peptides thereof can be coupled, for example, to CNBr-activated Sepharose. Another possibility is to separate the degradation products by gel filtration, precipitation reactions, for example as described in Example J of WO 96/35700, and the like. Fluorescence labels can advantageously be detected by means of HPLC, preferably exclusion HPLC. According to the test method described in WO 98/03638, HS-binding proteins, for example histidine-rich glycoproteins, Use wisely in immobilized form in order to separate non-degraded or only partially degraded heparanase substrate from degraded substrate and thereby enable its detection.
Die in diesen Tests eingesetzte Heparanase kann natürlichen oder rekombinanten Ursprungs sein, so kann Heparanase aus einer Vielzahl von Geweben und Körperflüssigkeiten, Serum eingeschlossen, aufgereinigt werden. Die Expression menschlicher Heparanase kann mit den in WO 95/04158 und WO 99/43830 erwähnten Expressionssy- stemen bewerkstelligt werden.The heparanase used in these tests can be of natural or recombinant origin, so heparanase can be purified from a large number of tissues and body fluids, including serum. The expression of human heparanase can be accomplished with the expression systems mentioned in WO 95/04158 and WO 99/43830.
Gegenstand der vorliegenden Erfindung ist daher auch ein Verfahren zur Identifizierung von Heparanase-Inhibitoren, wobei man die Aktivität von Heparanase in Gegenwart und in Abwesenheit wenig- stens einer Testsubstanz bestimmt.The present invention therefore also relates to a method for identifying heparanase inhibitors, the activity of heparanase being determined in the presence and in the absence of at least one test substance.
Die vorstehend beschriebenen und weitere in ähnlicher Weise geeignete Testsysteme können die Grundlage bilden für in vitro-Scree- ning-Verfahren, vorzugsweise zum primären Screening, mit denen man aus einer Vielzahl verschiedener Substanzen diejenigen auslesen kann, die im Hinblick auf die erfindungsgemäße Anwendung brauchbar sind. So betrifft die vorliegende Erfindung auch entsprechende Verfahren zur Identifizierung von Wirkstoffen zur Behandlung von Herzinsuffizienz und darauf aufbauend die Herstel- lung pharmazeutischer Mittel zur Behandlung von Herzinsuffizienz. Ein solches Verfahren zur Entwicklung von Wirkstoffen zur Behandlung von Herzinsuffizienz ist dadurch gekennzeichnet, daß man zunächst aus einer Vielzahl von Substanzen Heparanase-Inhibitoren auswählt und diese zur Herstellung des Mittels verwendet. Bei- spielsweise können mittels kombinatorischer Chemie umfangreiche Stoffbanken angelegt werden, die Myriaden potentieller Wirkstoffe umfassen. Das Durchmustern kombinatorischer Substanzbibliotheken nach Stoffen mit gewünschter Aktivität ist automatisierbar. Screening-Roboter dienen der effizienten Auswertung der vorzugs- weise auf Mikrotiterplatten angeordneten Einzelassays.The test systems described above and other similarly suitable test systems can form the basis for in vitro screening methods, preferably for primary screening, with which one can read out from a large number of different substances those which are useful with regard to the use according to the invention , The present invention thus also relates to corresponding methods for identifying active substances for the treatment of heart failure and, based on this, the production of pharmaceutical agents for the treatment of heart failure. Such a process for the development of active substances for the treatment of heart failure is characterized in that heparanase inhibitors are first selected from a large number of substances and used to produce the agent. For example, using combinatorial chemistry, extensive substance banks can be created that include myriads of potential active substances. The screening of combinatorial substance libraries for substances with the desired activity can be automated. Screening robots are used for the efficient evaluation of the individual assays, which are preferably arranged on microtiter plates.
Eine besonders effektive Technologie zur Durchführung derartiger Verfahren ist der im Bereich des Wirkstoffscreenings bekannte Scintillation Proximity Assay, kurz SPA genannt. Kits und Kompo- nenten zur Durchführung dieses Assays können kommerziell bezogen werden, beispielweise bei Amersham Pharmacia Biotech. Für enzyma- tische Testanwendungen werden im Prinzip solubilisierte oder membrangebundene Substrate auf Scintillationssubstanz enthaltenden, kleinen Fluomikrosphären immobilisert. Je nach Art der zu testenden enzymatischen Aktivität ist das Substrat radioaktiv markiert und die Scintillationssubstanz wird solange zur Lichtemission angeregt, wie die räumliche Nähe zwischen Scintillations- Substanz und Radiomarkierung gegeben ist, oder es wird die radioaktive Markierung in das immobiliserte Substrat eben durch die zu messende Enzymaktivität eingefügt und als Folge die Scintillationssubstanz zur Lichtemission angeregt. So ergeben sich Testfor- mate, bei denen eine abnehmende bzw. zunehmende Sig-nalintensität gemessen wird.A particularly effective technology for carrying out such methods is the scintillation proximity assay known in the field of active substance screening, or SPA for short. Kits and components for performing this assay can be obtained commercially, for example from Amersham Pharmacia Biotech. In principle, for enzymatic test applications, solubilized or membrane-bound substrates are immobilized on small fluomicrospheres containing scintillation substance. Depending on the type of enzymatic activity to be tested, the substrate is radioactively marked and the scintillation substance is stimulated to emit light as long as the spatial proximity between scintillation Substance and radiolabeling is given, or the radioactive label is inserted into the immobilized substrate by the enzyme activity to be measured and, as a result, the scintillation substance is excited to emit light. This results in test formats in which a decreasing or increasing signal intensity is measured.
Eine weitere besonders effektive Technologie zur Durchführung derartiger Verfahren ist die im Bereich des Wirkstoffscreenings bekannte FlashPlate®-Technologie. Kits und Komponenten zur Durchführung dieses Assays können kommerziell bezogen werden, beispielweise bei NEN® Life Science Products. Dieses Prinzip basiert ebenfalls auf Mikrotiterplatten (96er oder 384er), die mit Scintillationssubstanz beschichtet sind.Another particularly effective technology for carrying out such processes is the FlashPlate® technology known in the field of active substance screening. Kits and components for carrying out this assay can be obtained commercially, for example from NEN ® Life Science Products. This principle is also based on microtiter plates (96 or 384), which are coated with scintillation substance.
Die erfindungsgemäße Verwendung von Heparanase-Inhibitoren beinhaltet im Rahmen der Behandlung ein Verfahren. Dabei wird dem zu behandelnden Individuum, vorzugsweise einem Säuger, insbesondere einem Menschen, Nutz- oder Haustier, eine wirksame Menge eines oder mehrerer Heparanase-Inhibitoren, in der Regel der pharmazeutischen und tierarzneilichen Praxis entsprechend formuliert, verabreicht. Die Behandlung erfolgt in der Regel durch einmaliges oder mehrmaliges tägliches Zuführen gegebenenfalls zusammen oder im Wechsel mit anderen Wirkstoffen oder wirkstoffhaltigen Präpa- raten. Ob eine solche Behandlung angezeigt ist und in welcher Form sie zu erfolgen hat, hängt vom Einzelfall ab und unterliegt einer medizinischen Beurteilung (Diagnose), die vorhandene Anzeichen, Symptome und/oder Fehlfunktionen, Risiken, bestimmte Anzeichen, Symptome und/oder Fehlfunktionen zu entwickeln, und weitere Faktoren miteinbezieht .The use of heparanase inhibitors according to the invention includes a method as part of the treatment. An effective amount of one or more heparanase inhibitors, usually formulated in accordance with pharmaceutical and veterinary practice, is administered to the individual to be treated, preferably a mammal, in particular a human, useful or domestic animal. As a rule, the treatment is carried out by adding it once or several times a day, optionally together or alternating with other active substances or preparations containing the active substance. Whether such treatment is indicated and in what form it must be carried out depends on the individual case and is subject to a medical assessment (diagnosis) to develop the existing signs, symptoms and / or malfunctions, risks, certain signs, symptoms and / or malfunctions , and other factors.
Die erfindungsgemäße Lehre richtet sich vor allem auf die Herstellung pharmazeutischer Mittel zur Behandlung eines Individuums, vorzugsweise eines Säugers, insbesondere eines Menschen, Nutz- oder Haustieres. So werden die Inhibitoren gewöhnlich in Form von pharmazeutischen Zusammensetzungen verabreicht, die einen pharmazeutisch verträglichen Exzipienten mit wenigstens einem erfindungsgemäßen Inhibitor, gegebenfalls auch einem Gemisch mehrerer erfindungsgemäßer Inhibitoren, und gegebenenfalls weiteren Wirkstoffen umfassen. Diese Zusammensetzungen können beispielsweise auf oralem, rektalem, transdermalem, subkutanem, intravenösem, intramuskulärem oder intranasalem Weg verabreicht werden.The teaching according to the invention is primarily aimed at the production of pharmaceutical agents for the treatment of an individual, preferably a mammal, in particular a human, useful or domestic animal. Thus, the inhibitors are usually administered in the form of pharmaceutical compositions which comprise a pharmaceutically acceptable excipient with at least one inhibitor according to the invention, optionally also a mixture of several inhibitors according to the invention, and optionally further active ingredients. These compositions can be administered, for example, by the oral, rectal, transdermal, subcutaneous, intravenous, intramuscular or intranasal routes.
Beispiele geeigneter pharmazeutischer Formulierungen sind feste Arzneiformen, wie Pulver, Puder, Granulate, Tabletten, Pastillen, Sachets, Cachets, Dragees, Kapseln wie Hart- und Weichgelatinekapseln, Suppositorien oder vaginale Arzneiformen, halbfeste Arz- neiformen, wie Salben, Cremes, Hydrogele, Pasten oder Pflaster, sowie flüssige Arzneiformen, wie Lösungen, Emulsionen, insbesondere Öl-in-Wasser-Emulsionen, Suspensionen, beispielsweise Lotionen, Injektions- und Infusionszubereitungen, Augen- und Ohren- tropfen. Auch implantierte Abgabevorrichtungen können zur Verabreichung erfindungsgemäßer Inhibitoren verwendet werden. Ferner können auch Liposomen, Mikrosphären oder Polymermatrizes zur Anwendung kommen.Examples of suitable pharmaceutical formulations are solid pharmaceutical forms, such as powders, powders, granules, tablets, lozenges, sachets, cachets, coated tablets, capsules such as hard and soft gelatin capsules, suppositories or vaginal pharmaceutical forms, semi-solid medicinal forms. neiforms, such as ointments, creams, hydrogels, pastes or plasters, and liquid pharmaceutical forms, such as solutions, emulsions, in particular oil-in-water emulsions, suspensions, for example lotions, injection and infusion preparations, eye and ear drops. Implanted delivery devices can also be used for the administration of inhibitors according to the invention. Liposomes, microspheres or polymer matrices can also be used.
Bei der Herstellung der Zusammensetzungen werden erfindungsgemäße Inhibitoren gewöhnlich mit einem Exzipienten vermischt oder verdünnt. Exzipienten können feste, halbfeste oder flüssige Materialien sein, die als Vehikel, Träger oder Medium für den Wirkstoff dienen.In the preparation of the compositions, inhibitors according to the invention are usually mixed or diluted with an excipient. Excipients can be solid, semi-solid or liquid materials that serve as vehicles, carriers or media for the active ingredient.
Zu geeigneten Exzipienten gehören beispielsweise Lactose, Dextrose, Sucrose, Sorbitol, Mannitol, Stärken, Akaziengummi, Calci- umphosphat, Alginate, Traganth, Gelatine, Calciumsilikat, mikrokristalline Cellulose, Polyvinylpyrrolidon, Cellulose, Wasser, Sirup und Methylcellulose. Ferner können die Formulierungen pharmazeutisch akzeptable Träger oder übliche Hilfsstoffe, wie Gleitmittel, beispielsweise Talk, Magnesiumstearat und Mineralöl; Netzmittel; emulgierende und suspendierende Mittel; konservierende Mittel, wie Methyl- und Propylhydroxybenzoate; Antioxidan- tien; Antireizstoffe; Chelatbildner; Dragierhilfsmittel; Emulsionsstabilisatoren; Filmbildner; Gelbildner; Geruchsmaskierungsmittel; Geschmackskorrigentien; Harze; Hydrokolloide; Lösemittel; Lösungsvermittler; Neutralisierungsmittel; Permeationsbeschleuni- ger; Pigmente; quaternäre Ammoniumverbindungen; Rückfettungs- und Überfettungsmittel; Salben-, Creme- oder Öl-Grundstoffe; Silikon- Derivate; Spreithilfsmittel; Stabilisatoren; Sterilanzien; Suppo- sitoriengrundlagen; Tabletten-Hilfsstoffe, wie Bindemittel, Füllstoffe, Gleitmittel, Sprengmittel oder Überzüge; Treibmittel; Trocknungsmittel; Trübungsmittel; Verdickungsmittel; Wachse; Weichmacher; Weißöle umfassen. Eine diesbezügliche Ausgestaltung beruht auf fachmännischem Wissen, wie beispielsweise in Fiedler, H.P., Lexikon der Hilfsstoffe für Pharmazie, Kosmetik und angrenzende Gebiete, 4. Auflage, Aulendorf: ECV-Editio-Cantor-Verlag, 1996, dargestellt ist.Suitable excipients include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methyl cellulose. Furthermore, the formulations can be pharmaceutically acceptable carriers or customary auxiliaries, such as lubricants, for example talc, magnesium stearate and mineral oil; Wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; Antioxidants; Antiirritatives; chelating agents; coating aids; Emulsion stabilizers; film formers; gelling agents; Odor masking agents; masking flavors; resins; Hydrocolloids; Solvents; Solubilizing agents; Neutralizing agents; Permeation accelerator; pigments; quaternary ammonium compounds; Refatting and overfatting agents; Ointment, cream or oil base materials; Silicone derivatives; spreading aids; stabilizers; Sterilanzien; Fundamentals of the suppository; Tablet excipients such as binders, fillers, lubricants, disintegrants or coatings; Propellant; Desiccant; Opacifiers; Thickener; waxes; plasticizers; Include white oils. A design in this regard is based on expert knowledge, as is shown, for example, in Fiedler, H.P., Lexicon of auxiliaries for pharmacy, cosmetics and related areas, 4th edition, Aulendorf: ECV-Editio-Cantor-Verlag, 1996.
Die vorliegende Erfindung wird anhand der nachfolgenden Beispiele näher erläutert, ohne darauf beschränkt zu sein.The present invention is explained in more detail with reference to the following examples, without being restricted thereto.
Figur 1 zeigt die gelelektrophoretische Auftrennung der nach RT- PCR gemäß Beispiel 1 erhaltenen Amplifikate parallel zu Molekulargewichtsstandards (MWM) und der Negativ-Kontrolle (negativ). Beispiel 1FIG. 1 shows the gel electrophoretic separation of the amplificates obtained after RT-PCR according to Example 1 in parallel with molecular weight standards (MWM) and the negative control (negative). example 1
Heparanase-Expression in einem Ratten-Modell für kongestive HerzinsuffizienzHeparanase expression in a rat model for congestive heart failure
Das verwendete Tier-Modell wurde von Wiesener et al. in Circula- tion 95, 1253-1259 (1997) beschrieben. So entwickelten fünf mit der Aortenklemmtechnik behandelte Ratten (Nr. 3, 15, 24, 25, 112) eine kongestive Herzinsuffizienz (Herzhypertrophie). Die Herzen wurden entnommen, und die mRNA wurde in üblicher Weise isoliert. Mittels RT-PCR konnte die Menge an exprimierter Heparanase-mRNA bestimmt werden, indem man als Sense-Primer das Oligonukleotid mit der Sequenz SEQ ID NO: 3 und als Antisense-Primer das Oligonukleotid mit der Sequenz SEQ ID NO: 4 verwendete. Parallel wurde die GADPH-Expression als Housekeeping-Gen gemessen. Die gleiche Untersuchung wurde an Kontrolltieren (Ratten Nr. 11, 17, 19, 32, 33 ) vorgenommen.The animal model used was by Wiesener et al. in Circulation 95, 1253-1259 (1997). Five rats treated with the aortic clamp technique (No. 3, 15, 24, 25, 112) developed congestive heart failure (cardiac hypertrophy). The hearts were removed and the mRNA was isolated in the usual way. The amount of heparanase mRNA expressed could be determined by RT-PCR by using the oligonucleotide with the sequence SEQ ID NO: 3 as sense primer and the oligonucleotide with the sequence SEQ ID NO: 4 as antisense primer. In parallel, the GADPH expression was measured as a housekeeping gene. The same study was carried out on control animals (rats No. 11, 17, 19, 32, 33).
Die jeweiligen mittels RT-PCR erhaltenen Amplifikate wurden elek- trophoretisch aufgetrennt, und ihre Menge wurde quantifiziert. Fig. 1 zeigt für die Tiere 11, 17, 19, 32 und 33 der Kontrollgruppe sowie die Tiere 3, 15, 24, 25 und 112 der Testgruppe die erhaltenen Amplifikate nach elektrophoretischer Auftrennung. Die Quantifizierung ergab folgende, jeweils auf die GAPDH-Expression bezogene Heparanase-mRNA-Spiegel:The respective amplificates obtained by RT-PCR were separated electrophoretically and their amount was quantified. 1 shows the amplicons obtained after electrophoretic separation for animals 11, 17, 19, 32 and 33 of the control group and animals 3, 15, 24, 25 and 112 of the test group. The quantification gave the following heparanase mRNA levels, each based on GAPDH expression:
Tabelle 1 :Table 1 :
Figure imgf000015_0001
Als Mittelwerte ergeben sich für die Kontrollgruppe 4,289 ± 0.75 und für die Testgruppe 4,153 ± 0,06. Diese Werte zeigen, daß der im Modell induzierte pathologische Zustand nicht mit einer Regulation der Expression von Heparanase-mRNA verbunden ist, sondern 5 die im Zusammenhang mit Herzhypertrophie und Herzinsuffizienz auftretende intra- und extrazelluläre Ansäuerung die Heparanase- Aktivität moduliert (Schini-Kerth et al (1997) Circulation 96, 3888-3896; Shrode et al. (1997) J Bioenerg Biomembr 29, 393-399; Kraus and Wolf (1996) Tumor Biol 17, 133-154; Tamagaki et al.
Figure imgf000015_0001
The mean values are 4.289 ± 0.75 for the control group and 4.153 ± 0.06 for the test group. These values show that the pathological state induced in the model is not associated with regulation of the expression of heparanase mRNA, but 5 the intra- and extracellular acidification occurring in connection with cardiac hypertrophy and heart failure modulates the heparanase activity (Schini-Kerth et al (1997) Circulation 96, 3888-3896; Shrode et al. (1997) J Bioenerg Biomembr 29, 393-399; Kraus and Wolf (1996) Tumor Biol 17, 133-154; Tamagaki et al.
10 (1996) Atherosclerosis 123, 73-82; Brown and Breton (1996) J Exp Biol. 199, 2345-2358; Apkon et al. (1997) Am J Physiol 273, H434-445; Ito et al (1997) J. Clin Invest 99, 125-135; Tajima et al. (1998) Circulation 98, 2760-2764; Flores et al (1996) Kidney Int. Suppl 55, S122 125; Hisatome et al. (1997) Gen Pharmacol 29,10 (1996) Atherosclerosis 123, 73-82; Brown and Breton (1996) J Exp Biol. 199, 2345-2358; Apkon et al. (1997) Am J Physiol 273, H434-445; Ito et al (1997) J. Clin Invest 99, 125-135; Tajima et al. (1998) Circulation 98, 2760-2764; Flores et al (1996) Kidney Int. Suppl 55, S122 125; Hisatome et al. (1997) Gene Pharmacol 29,
15 557-560; Russ et al. (1996) Pflugers Arch 433, 26-34).15 557-560; Russ et al. (1996) Pflugers Arch 433, 26-34).
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Claims

Patentansprüche claims
1. Verwendung wenigstens eines Heparanase-Inhibitors zur Her- Stellung eines pharmazeutischen Mittels zur Behandlung von1. Use of at least one heparanase inhibitor for the manufacture of a pharmaceutical composition for the treatment of
Herzinsuffizienz und damit zusammenhängenden Anzeichen, Symptomen und/oder Fehlfunktionen.Heart failure and related signs, symptoms and / or malfunctions.
2. Verwendung nach Anspruch 1 zur Behandlung von kongestiver Herzinsuffizienz und damit zusammenhängenden Anzeichen,2. Use according to claim 1 for the treatment of congestive heart failure and related signs,
Symptomen und/oder Fehlfunktionen.Symptoms and / or malfunctions.
3. Verwendung nach Anspruch 1 oder 2 zur Behandlung von peripheren Ödemen, Lungen- und Leberkongestion, Dyspnoe, Brust- und Bauchwassersucht.3. Use according to claim 1 or 2 for the treatment of peripheral edema, lung and liver congestion, dyspnea, breast and abdominal addiction.
4. Verwendung nach einem der vorhergenden Ansprüche, wobei wenigstens ein Heparanase-Inhibitor ein Glycosaminoglykan ist.4. Use according to one of the preceding claims, wherein at least one heparanase inhibitor is a glycosaminoglycan.
5. Verwendung nach Anspruch 4 , wobei das Glycosaminoglykan ausgewählt ist unter Heparinen mit zumindest teilweise reduzierten Carboxylgruppen, zumindest partiell N-desulfatierten, N-acetylierten Heparinen, zumindest partiell N,0-desulfatier- ten, N-resulfatierten Heparinen, O-acylierten Heparinen, sul- fatierten und/oder phosphorylierten Oligosacchariden, glyco- mimetischen Saccharopeptiden, Laminarin-Sulfaten, Suramin und Trachyspinsäure .5. Use according to claim 4, wherein the glycosaminoglycan is selected from heparins with at least partially reduced carboxyl groups, at least partially N-desulfated, N-acetylated heparins, at least partially N, 0-desulfated, N-resulfated heparins, O-acylated heparins , sulfated and / or phosphorylated oligosaccharides, glycomimetic saccharopeptides, laminarin sulfates, suramin and trachyspinic acid.
6. Verwendung nach einem der Ansprüche 1 bis 3 , wobei wenigstens ein Heparanase-Inhibitor eine niedermolekulare Verbindung ist.6. Use according to any one of claims 1 to 3, wherein at least one heparanase inhibitor is a low molecular weight compound.
7. Verfahren zur Herstellung eines pharmazeutischen Mittels zur Behandlung von Herzinsuffizienz, dadurch gekennzeichnet, daß man zunächst aus einer Vielzahl von Substanzen Heparanase-Inhibitoren auswählt und diese zur Herstellung des Mittels verwendet . 7. A process for the preparation of a pharmaceutical agent for the treatment of heart failure, characterized in that heparanase inhibitors are first selected from a large number of substances and these are used to prepare the agent.
PCT/EP2000/011441 1999-11-19 2000-11-17 Heparanase inhibitors for the treatment of heart failure WO2001035967A1 (en)

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EP1417304A2 (en) * 2001-07-13 2004-05-12 Imclone Systems, Inc. Method and composition for inhibiting heparanase activity
US7326727B2 (en) 2002-08-05 2008-02-05 Oxford Glycosciences (Uk) Ltd. Furanthiazole derivatives as heparanase inhibitors
EP2343077A1 (en) 2001-09-12 2011-07-13 SIGMA-TAU Research Switzerland S.A. Derivatives of totally N-desulphated glycosaminoglycans as heparanase inhibitors, endowed with antiangiogenic activity and devoid of anticoagulating effect

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CH696701A5 (en) 2001-12-18 2007-10-15 Hoffmann La Roche A method for testing an agent for its ability to inhibit heparanase activity.
DE10258770B4 (en) * 2001-12-18 2005-02-10 F. Hoffmann-La Roche Ag Test for inhibitors of heparanase, useful for treatment of e.g. cancer and inflammatory disease, using immobilized, labeled heparanase substrate
EP1479764A1 (en) 2003-05-19 2004-11-24 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Heparanase-derived peptides for vaccination of tumor patients

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WO1996008559A1 (en) * 1994-09-16 1996-03-21 Cardiac Crc Nominees Pty. Ltd. Glycosaminoglycan-degrading enzyme inhibition and resultant disease therapies
WO1999023214A1 (en) * 1997-10-30 1999-05-14 The Trustees Of The University Of Pennsylvania Compositions and methods for enhancing cardiac contractility mediated by myocyte p2 purinoceptors and models thereof
WO1999043830A2 (en) * 1998-02-24 1999-09-02 Pharmacia & Upjohn Company Human platelet heparanase polypeptides, polynucleotide molecules that encode them, and methods for the identification of compounds that alter heparanase activity

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Publication number Priority date Publication date Assignee Title
EP1417304A2 (en) * 2001-07-13 2004-05-12 Imclone Systems, Inc. Method and composition for inhibiting heparanase activity
EP1417304A4 (en) * 2001-07-13 2005-11-23 Imclone Systems Inc Method and composition for inhibiting heparanase activity
EP2343077A1 (en) 2001-09-12 2011-07-13 SIGMA-TAU Research Switzerland S.A. Derivatives of totally N-desulphated glycosaminoglycans as heparanase inhibitors, endowed with antiangiogenic activity and devoid of anticoagulating effect
US7326727B2 (en) 2002-08-05 2008-02-05 Oxford Glycosciences (Uk) Ltd. Furanthiazole derivatives as heparanase inhibitors

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