US20050245736A1 - Low molecular weight oversulfated polysaccharide - Google Patents
Low molecular weight oversulfated polysaccharide Download PDFInfo
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- US20050245736A1 US20050245736A1 US10/518,229 US51822905A US2005245736A1 US 20050245736 A1 US20050245736 A1 US 20050245736A1 US 51822905 A US51822905 A US 51822905A US 2005245736 A1 US2005245736 A1 US 2005245736A1
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- Prior art keywords
- lmw
- oversulfate
- chains
- sulfate
- molecular weight
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- GYZVUBZBJRAOOK-UHFFFAOYSA-L O.O.O=C([O-])C1CC(O)C(O)CO1.O=S(=O)([O-])NC1COC(CO)CC1O Chemical compound O.O.O=C([O-])C1CC(O)C(O)CO1.O=S(=O)([O-])NC1COC(CO)CC1O GYZVUBZBJRAOOK-UHFFFAOYSA-L 0.000 description 6
- SCYPVKKKHPCDCV-UHFFFAOYSA-L CC1CC(COS(=O)(=O)[O-])OCC1N.CC1COC(C(=O)[O-])CC1C.O.O Chemical compound CC1CC(COS(=O)(=O)[O-])OCC1N.CC1COC(C(=O)[O-])CC1C.O.O SCYPVKKKHPCDCV-UHFFFAOYSA-L 0.000 description 4
- SVRBRQYDJVJIMS-UHFFFAOYSA-K COC1OC(COS(=O)(=O)[O-])C(OC2OC(C(=O)[O-])C(C)C(C)C2C)C(C)C1NS(=O)(=O)[O-] Chemical compound COC1OC(COS(=O)(=O)[O-])C(OC2OC(C(=O)[O-])C(C)C(C)C2C)C(C)C1NS(=O)(=O)[O-] SVRBRQYDJVJIMS-UHFFFAOYSA-K 0.000 description 4
- QPEVQUKQVCFUHR-UHFFFAOYSA-N CC1C(CO)OC(CO)C1O Chemical compound CC1C(CO)OC(CO)C1O QPEVQUKQVCFUHR-UHFFFAOYSA-N 0.000 description 3
- VOXCERDJIQDLDD-UHFFFAOYSA-L CC1C(COS(=O)(=O)[O-])OC(COS(=O)(=O)[O-])C1C Chemical compound CC1C(COS(=O)(=O)[O-])OC(COS(=O)(=O)[O-])C1C VOXCERDJIQDLDD-UHFFFAOYSA-L 0.000 description 3
- WVUMPGDISSOFSJ-UHFFFAOYSA-N CC1OC(CO)C(C)C1O Chemical compound CC1OC(CO)C(C)C1O WVUMPGDISSOFSJ-UHFFFAOYSA-N 0.000 description 3
- MGTQDUUXGXLPNC-UHFFFAOYSA-K O.O.O.O=C([O-])C1CC(O)C(O)CO1.O=C([O-])C1OCC(O)C(O)C1O.O=S(=O)([O-])NC1COC(CO)CC1O.OCC1CC(O)C(CO)O1 Chemical compound O.O.O.O=C([O-])C1CC(O)C(O)CO1.O=C([O-])C1OCC(O)C(O)C1O.O=S(=O)([O-])NC1COC(CO)CC1O.OCC1CC(O)C(CO)O1 MGTQDUUXGXLPNC-UHFFFAOYSA-K 0.000 description 3
- IKCNSNIHHQZZBR-UHFFFAOYSA-I CC1CC(COS(=O)(=O)[O-])OCC1NS(=O)(=O)[O-].CC1COC(C(=O)[O-])C(O)C1C.CC1COC(C(=O)[O-])CC1C.CC1OC(COS(=O)(=O)[O-])CC1O.O.O.O Chemical compound CC1CC(COS(=O)(=O)[O-])OCC1NS(=O)(=O)[O-].CC1COC(C(=O)[O-])C(O)C1C.CC1COC(C(=O)[O-])CC1C.CC1OC(COS(=O)(=O)[O-])CC1O.O.O.O IKCNSNIHHQZZBR-UHFFFAOYSA-I 0.000 description 2
- 0 C*(C(C(C(CCOCCC(C(C1O)O)OC([*-])C1O)C(CO)O1)O)C1=COCC(C([*-])OC(CCOCCC(C(CO)OC1CO)C1O)C1O)C1O)=C Chemical compound C*(C(C(C(CCOCCC(C(C1O)O)OC([*-])C1O)C(CO)O1)O)C1=COCC(C([*-])OC(CCOCCC(C(CO)OC1CO)C1O)C1O)C1O)=C 0.000 description 1
- YJQKHURICZUYBP-UHFFFAOYSA-M CC(=O)NC1COC(CO)CC1O.O.O.O=C([O-])C1CC(O)C(O)CO1 Chemical compound CC(=O)NC1COC(CO)CC1O.O.O.O=C([O-])C1CC(O)C(O)CO1 YJQKHURICZUYBP-UHFFFAOYSA-M 0.000 description 1
- HJYIBTBWHKLMLP-UHFFFAOYSA-K CC1CC(C)C(N)CO1.CC1CC(COS(=O)(=O)[O-])OC1COS(=O)(=O)[O-].CC1COC(C(=O)[O-])CC1C.O.O.O Chemical compound CC1CC(C)C(N)CO1.CC1CC(COS(=O)(=O)[O-])OC1COS(=O)(=O)[O-].CC1COC(C(=O)[O-])CC1C.O.O.O HJYIBTBWHKLMLP-UHFFFAOYSA-K 0.000 description 1
- OPLOFJIYTPJYRA-UHFFFAOYSA-I CC1CC(COS(=O)(=O)[O-])OC1C.CC1CC(COS(=O)(=O)[O-])OCC1NS(=O)(=O)[O-].CC1COC(C(=O)[O-])C(C)C1C.CC1COC(C(=O)[O-])CC1C.O.O.O Chemical compound CC1CC(COS(=O)(=O)[O-])OC1C.CC1CC(COS(=O)(=O)[O-])OCC1NS(=O)(=O)[O-].CC1COC(C(=O)[O-])C(C)C1C.CC1COC(C(=O)[O-])CC1C.O.O.O OPLOFJIYTPJYRA-UHFFFAOYSA-I 0.000 description 1
- HLQCYTDLHLNTNP-UHFFFAOYSA-H CC1CC(COS(=O)(=O)[O-])OC1COS(=O)(=O)[O-].CC1CC(COS(=O)(=O)[O-])OCC1NS(=O)(=O)[O-].CC1COC(C(=O)[O-])C(C)C1C.CC1COC(C(=O)[O-])CC1C.O.O.O Chemical compound CC1CC(COS(=O)(=O)[O-])OC1COS(=O)(=O)[O-].CC1CC(COS(=O)(=O)[O-])OCC1NS(=O)(=O)[O-].CC1COC(C(=O)[O-])C(C)C1C.CC1COC(C(=O)[O-])CC1C.O.O.O HLQCYTDLHLNTNP-UHFFFAOYSA-H 0.000 description 1
- SKZSQGLSRLWGSE-UHFFFAOYSA-M CC1OC(COS(=O)(=O)[O-])C(C)C1C Chemical compound CC1OC(COS(=O)(=O)[O-])C(C)C1C SKZSQGLSRLWGSE-UHFFFAOYSA-M 0.000 description 1
- JLQHQPDWDPQYJQ-UHFFFAOYSA-L O.O.O.O=C([O-])C1CC(O)C(O)CO1.O=S(=O)([O-])NC1COC(CO)CC1O.OCC1CC(O)C(CO)O1 Chemical compound O.O.O.O=C([O-])C1CC(O)C(O)CO1.O=S(=O)([O-])NC1COC(CO)CC1O.OCC1CC(O)C(CO)O1 JLQHQPDWDPQYJQ-UHFFFAOYSA-L 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- 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
- 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
- A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/16—Emollients or protectives, e.g. against radiation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/06—Antiglaucoma agents or miotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
- C08B37/0078—Degradation products
Definitions
- the present invention refers to new N,O-oversulfated low molecular weight polysaccharides derived from K5 polysaccharide, to a process for their preparation, to new key intermediates in said process and to pharmaceutical compositions containing said oversulfated low molecular weight polysaccharides. More particularly, the present invention refers to a N-deacetylated and N-sulfated K5 having a degree of sulfation between 3.2 and 4 and a mean molecular weight from about 3,000 to about 6,000.
- Glycosamioglycans such as heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate and hyaluronic acid are biopolymers industrially extracted from different animal organs.
- heparin mainly obtained by extraction from pig intestinal mucosa or from beef lung, is a polydisperse copolymer with a molecular weight distribution from about 3,000 to about 30,000 D formed by a mixture of chains essentially consisting of an uronic acid (glucuronic acid or iduronic acid) and of an aminosugar (glucosamine) linked by ⁇ -1 ⁇ 4 or ⁇ -1 ⁇ 4 bonds.
- the uronic unit can be O-sulfated in position 2 and the glucosamine unit is N-acetylated or N-sulfated, 6-O sulfated and 3-O sulfated in about 0.5% of the glucosaminic units present.
- heparin Besides the main anticoagulant and antithrombotic activities, heparin also exerts antilipemic, antiproliferative, antiviral, antitumoral and antimetastatic activity, but its use as a drug for such indications is hampered by the side effects due to its anticoagulant activity that can cause bleeding.
- capsular polysaccharide isolated from strains of Escherichia Coli (herein below also simply referred to as “K5”) described by W. F. Vann et al. (1981) in Eur. J. Biochem 116, 359-364 consists of a mixture of chains formed by a repetitive disaccharide unit formed by D-glucuronic acid and N-acetylglucosamine linked ⁇ 1 ⁇ 4, while the disaccharide units D-glucuronyl-N-acetylglucosamine are linked ⁇ 1 ⁇ 4, thus showing the same repeated sequence (A) as the N-acetylheparosan biosynthetic precursor of heparin and heparan sulfate.
- K5 capsular polysaccharide isolated from strains of Escherichia Coli
- Said process comprises the steps (s-a) a N-deacetylation of K5 polysaccharide and a N-sulfation of the resulting K5-amine, (s-b) an epimerization of the N-sulfate-K5, (s-c) an O-oversulfation of the epiK5-N-sulfate, (s-d) a partial O-desulfation, (s-e) a selective 6-O-sulfation, (s-f) a N-sulfation of the so obtained product, whatever product obtained at the end of steps (s-b)-(s-f) being optionally submitted to a depolymerization.
- N-sulfated-epiK5 having a molecular weight of 7,400 and a degree of sulfation of from 2.3 to 2.9, obtained by the above-mentioned steps (s-a)-(s-f) followed by a nitrous depolymerization at the end of the (s-f) step.
- LMW-K5-N,O-oversulfate having a mean molecular weight of from 2,000 to 5,000 obtained by depolymerization of the K5-N,O-oversulfate or a LMW-K5-N,O-oversulfate of mean molecular weight of about 6,500 directly obtained from the above mentioned fraction of K5 by the steps (a)-(c).
- LMW-K5-N-sulfate it is possible to depolymerize a K5-N-sulfate to obtain new LMW-K5-N-sulfate derivatives which constitute useful starting materials for the preparation of new LMW-K5-N,O-oversulfates.
- new LMW-K5-N-sulfates with very low mean molecular weight, in particular from about 2,000 to about 4,000, more particularly specific LMW-K5-N-sulfates formed by mixtures in which the preponderant compound is a decasaccharide or a dodecasaccharide or a tetradecasaccharide.
- These new LMW-N-sulfates are useful intermediates for the preparation of LMW-K5-N,O-oversulfates with antiviral and/or antiangiogenic activity and devoid of anticoagulant activity.
- a LMW-K5-N,O-oversulfate having a mean molecular weight of from about 3,000 to about 6,000 in particular a LMW-K5-N,O-oversulfate consisting of a mixture in which the preponderant species is a deca-, dodeca- or tetradecasaccharide, with a degree of sulfation of from 3.2 to 4, has an antiangiogenic activity higher than that of the K5-N,O-oversulfate described by Leali 2001.
- LMW-K5-N,O-oversulfates having a mean molecular weight from about 3,000 to about 6,000, in particular LMW-K5-N,O-oversulfates consisting of a mixture in which the preponderant species is a deca-, dodeca- or tetradecasaccharide, avoid of anticoagulant activity, have a good activity on HIV-1 virus.
- LMW-K5-N,O-oversulfate having a mean molecular weight of from about 3,000 to about 6,000 and a degree of sulfation of from 3.2 to 4, advantageously from 3.5 to 4, preferably from 3.7 to 3.9.
- Preferential compounds are LMW-K5-N,O-oversulfates formed by a mixture of chains in which the preponderant species has the formula I in which q is 4, 5, 6, 7 or 8 and R, R′ and R′′ represent hydrogen or a SO3- group, for a degree of sulfation of from 3.2 to 4, advantageously from 3.5 to 4, preferably from 3.5 to 3.9 and the corresponding cation is a chemically or pharmaceutically acceptable one.
- chemically acceptable refers to a cation useful in the chemical syntheses, such as sodium, ammonium, tetra(C 1 -C 4 )alkylammonium, or for the purification of the product, while “pharmaceutically acceptable” is self explanatory.
- Advantageous cations are those derived from alkaline metals, earth-alkaline metals, ammonium, tetra(C 1 -C 4 )alkylammonium, aluminum and zinc.
- the LMW-K5-N,O-oversulfate of the present invention can be prepared by depolymerization of K5-N,O-oversulfates of the type described in Leali 2001 and prepared with the method described in it.
- Said depolymerization is performed according to the known methods for the depolymerization of heparin, for instance according to the method described in EP 37319, in WO 82/03627 or the method for the depolymerization of a N,O-sulfated-K5 described in EP 544592.
- the depolymerization performed with sodium nitrite and hydrochloric acid (“nitrous depolymerization”), is followed by an in situ reduction with sodium borohydride.
- LMW-K5-N,O-oversulfates are obtained having the desired molecular weight.
- the starting product is dissolved in 100-200 ml of deionized water and thermostated at 4° C. Then an amount of sodium nitrite to obtain the desired mean molecular weight from about 3,000 to about 6,000 is added. Consequently, starting from a K5-N,O-oversulfate having a molecular weight of 20,000 measured by HPLC equipped with a BioSil 250 Biorad column and using standards of heparin with known molecular weight, from 160 to 230 mg of sodium nitrite dissolved in a water solution at 0.2% shall be added.
- a reducing agent such as sodium borohydride (250-500 mg dissolved in 50-100 ml of water)
- LMW-K5-N,O-oversulfates from the nitrous depolymerization of a K5-N,O-oversulfate and subsequent possible reduction with for instance sodium borohydride implies, at the reducing end of the majority of the chains in said mixture of chains, the presence of a sulfated 2,5-anhydromanno unit having the structure (a°) in which X′ represents formyl or hydroxymethyl and R′′ represents hydrogen or SO ⁇ .
- preferred products are the LMW-K5-N,O-oversulfate, obtainable by nitrous depolymerization of a K5-N,O-oversulfate and optional subsequent reduction with, for instance, sodium borohydride, consisting of mixtures of chains in which the preponderant species is a compound of formula I° wherein m is 4, 5 or 6, R,R′ and R′′ are hydrogen or SO ⁇ , X′ is formyl or hydroxymethyl, for a degree of sulfation of from 3.2 to 4, advantageously from 3.5 to 4, preferably from 3.5 to 3.9 and the corresponding cation is a chemically or pharmaceutically acceptable one.
- the mean molecular weight of each mixture advantageously is 3,750-4,250, 4,750-5,250, 5,750-6,250.
- LMW-K5-N,O-oversulfates have high antiangiogenic activity with a favourable ratio to the global anticoagulant activity and can be used in the preparation of pharmaceutical compositions for the treatment of angiogenesis-dependent pathologies in doses in which the risk of hemorragic side effects is extremely reduced.
- Angiogenesis dependent pathologies that can be treated with the LMW-K5-N,O-oversulfates are for example, among those found in the human beings, diabetic retinopathy, neovascularization of the transplanted cornea, neovascular glaucoma, trachoma, retrolental fibrodisplasia, psoriasis, pyogenic glaucoma, development of the ateroschlerotic plaque, hemangioma and angiofibroma, artero-venous malformations, arthritis, and in the combinatorial therapy of solid tumors.
- the new LMW-K5-N,O-oversulfates of the present invention were active in the in vivo test of the inhibition of the angiogenesis on chicken embryo choroallantioic membrane (CAM) according to Ribatti D. et al., J. Vasc. Res. 1997, 34, 455-463 (Ribatti 1997).
- CAM chicken embryo choroallantioic membrane
- sponge implants of Gelfoam are applied on the CAM of chicken embryos at the 8th day of development and, immediately after the application, 3 ⁇ l of a solution of physiologic saline containing 50 ⁇ g of LMW-K5-N,O-oversulfate are applied or, as reference compound, of K5-N,O-oversulfate with 15,000 molecular weight and degree of sulfation of 3.84 described in Leali 2001.
- the sponges are examined every day till the 12th day of incubation.
- the score of angiogenesis is obtained by counting the number of macroscopic vessels observable around the sponge at the different days of development and at the number of embryos (eggs) on which the compound is active.
- LMW-K5-N,O-oversulfates having a mean molecular weight of from about 3,000 to about 6,000 can be also prepared according to a process that is not described in literature and that is generally applicable to the preparation of new LMW-K5-N,O-oversulfates having a mean molecular weight from about 3,000 to about 12,000. Said new process and said new LMW-K5-N,O-oversulfates represent further aspects of the present invention.
- the present invention also provides a process for the preparation of new LMW-K5-N,O-oversulfates having a degree of sulfation of from 3.2 to 4, advantageously from 3.5 to 4, preferably from 3.5 to 3.9, which comprises
- a LMW-K5-N-sulfate obtained from a K5 by a practically complete N-deacetylation, subsequent N-sulfation, and final nitrous depolymerization followed by reduction of the LMW-K5-N-sulfate, as above illustrated, is used as starting material for the process of the present invention. Said reduction is necessary since the LMW-K5-N-sulfate subsequently undertakes reactions wherein the influence on the formyl group of the 2,5 anhydromannose radical is unknown.
- LMW-K5-N-sulfate can be obtained, having a mean molecular weight in the whole range from about 1,500 to about 10,000, preferably from about 1,500 to about 7,500, as calculated from the 13C-NMR spectrum by the integration of the signal attributable to the C-2 of 2,5 anhydromannitol with that of the anomeric carbon of the glucosamine inside the polysaccharide chain.
- LMW-K5-N-sulfates having a mean molecular weight from about 1,500 to about 7,500 and their chemically or pharmaceutically acceptable salts are new products useful as intermediates and also pharmaceutically active.
- the molecular weight distribution of said LMW-K5-N-sulfates can be from about 1,000 to about 10,000.
- the starting product is dissolved in 100-200 ml of deionized water and thermostated at 4° C. Then an amount of sodium nitrite to obtain the desired mean molecular weight of from about 2,000 to about 4,000 is added. Consequently, starting from a K5-N-sulfate having a molecular weight of 20,000 measured by HPLC equipped with a BioSil 250 Biorad column and using standards of heparin with known molecular weight, from 330 to 480 mg of sodium nitrite dissolved in a 0.2% water solution shall be added.
- the solution containing the K5-N-sulfate and sodium nitrite, kept at 4° C. is brought to pH 2 by adding 0.1N HCl cooled at 4° C.
- the reaction is left to react for 20-40 minutes, then neutralized with 0.1 N NaOH.
- the obtained product is warmed up to room temperature and treated with a reducing agent such as sodium borohydride (250-500 mg dissolved in 50-100 ml of water) and left to react for 4-8 hours.
- the excess of sodium borohydride is eliminated by bringing the pH to 5-5.5 with 0.1N HCl and left to react for further 2-4 hours.
- the solution is neutralized with 0.1N NaOH and the product is recovered by precipitation with acetone or ethanol after concentration of the product by reduced pressure evaporation.
- the amount of sodium nitrite that, starting from 1 g of K5-N-sulfate, allows to obtain a LMW-K5-N-sulfate with a molecular weight of from about 4,000 to about 7,500, in particular of at least 6,000 (6,000-7,500), can be established.
- the K5-N-sulfate is very well known in the literature and it is described in the documents herein above cited to illustrate the state of the art.
- the above cited starting material is invariably obtained by N-deacetylation and subsequent N-sulfation of the thus obtained K5-amine.
- the preparation of a K5-N-sulfate practically devoid of acetyl groups or NH2 is facilitated if the K5 from which it is prepared is particularly pure, in particular if it does not contain lipophilic substances.
- a K5-N-sulfate prepared from a K5 free from lipophilic substances is easier oversulfated, as described in the pending patent application IT MI2001A00397 (WO 02/068477). It is then preferable that the depolymerization is performed starting from a K5-N-sulfate prepared from a K5 purified as described in PREPARATION I herein below. Said K5-N-sulfate, whose 13 C-NMR spectrum does not show traces of acetyl groups or NH2, is described in PREPARATION II herein below.
- Advantageous starting materials of the process of the present invention are new LMW-K5-N-sulfates obtained by nitrous depolymerization of a K5 and subsequent reduction, for instance with sodium borohydride, consisting of mixtures of chains in which at least 90% of said chains have the formula II wherein n represents a number from 2 to 20 and the corresponding cation is a chemically or pharmaceutically acceptable one.
- the starting materials are new LMW-K5-N-sulfates consisting of a mixture of chains in which the preponderant species has the formula II′ wherein q is 4, 5, 6, 7, or 8 and the corresponding cation is a chemically or pharmaceutically acceptable one.
- LMW-K5-N-sulfates that constitute an advantageous aspect of the invention, are obtained from a K5-N-sulfate by nitrous depolymerization and subsequent reduction for instance with sodium borohydride as illustrated above. Their mean molecular weight is from about 2,000 to about 4,000.
- the presence of the structure (a′) has no influence on the properties of the LMW-K5-N-sulfates and of their derivatives because possible sulfations would only involve a possible introduction of one or more sulfate groups that would not significantly influence the-degree of sulfation of the O-sulfated derivatives.
- the preferred LMW-K5-N-sulfates are practically devoid of acetyl groups.
- LMW-K5-N-sulfates are formed by mixtures of chains in which the preponderant species is a compound of formula II′ in which m represents 4, 5 or 6 and the corresponding cation is a chemically or pharmaceutically acceptable one.
- the invention relates to a process for the preparation of new LMW-K5-N-sulfates and of their chemically or pharmaceutically acceptable salts, which comprises submitting a K5-N-sulfate to a controlled nitrous depolymerization optionally followed by a reduction and isolating the product so obtained.
- Said products usually are in form of their sodium salt, that can be transformed into another chemically or pharmaceutically acceptable salt.
- said LMW-K5-N-sulfates have a 2,5-anhydromanno unit having the structure (a) in which X represents a formyl or hydroxymethyl group and, preferably, they are formed by mixtures of chains in which at least 90% of said chains has the formula II or by mixtures of chains in which the preponderant species has the formula II′ or II′′.
- X represents a formyl or hydroxymethyl group
- These new LMW-K5-N-sulfates can be used as active ingredients of pharmaceutical compositions.
- the new LMW-K5-N-sulfates are the starting material of the process for the preparation of the LMW-K5-N,O-oversulfates of the present invention.
- the LMW-K5-N-sulfate has a molecular weight distribution from about 1,000 to about 10,000.
- the LMW-K5-N-sulfates starting materials of the process of the present invention are preferably used as sodium salt, unless a tertiary amine or quaternary ammonium salt prepared according to step (a) above illustrated, preferably the tetrabutylammonium salt, is already available.
- step (a) is performed by passing a solution of the sodium salt of the starting LMW-K5-N-sulfate through an acid ionic exchange resin, for example of the type IR-120 H+, by collecting the eluate comprising also the waste of the resin and by neutralizing the eluate with a tertiary amine or quaternary ammonium hydroxide, preferably with an aqueous solution of tetrabutylammonium hydroxide.
- the solution is let to stand for one hour, by concurrently maintaining the pH at 7 by adding the same base and the so obtained salt is isolated by freeze drying.
- step (b) the O-oversulfation is performed using an excess of O-sulfating agent at a temperature from 20 to 70° C. for a period of time up to 24 hours in a polar aprotic solvent.
- the tertiary amine or quaternary ammonium salt of the LMW-K5-N-sulfate so obtained in step (a) is dissolved in dimethylformamide and treated with 2-10 moles of an O-sulfating agent for each free hydroxy group at a temperature of 40-60° C. for 10-20 hours.
- the adduct pyridine.SO 3 is advantageously used, in amounts of 2.5-5 moles, preferably 2.5-4 moles per free hydroxy group and the reaction is advantageously performed at 50-60° C., preferably at 55° C. overnight.
- the product obtained at the end of the reaction is isolated by addition of 0.1-1 volume of water and neutralization, preferably with sodium hydroxide, by precipitation with a saturated solution of sodium chloride in acetone and filtration optionally followed by an ultrafiltration.
- the so obtained product is preferably the sodium salt of a LMW-K5-amine-O-oversulfate having a degree of sulfation of from 2.3 to 3.
- the sodium salt so obtained can be converted into another salt.
- the mean molecular weight of such a product can be from about 3,500 to about 11,000.
- step (c) the LMW-K5-amine-O-oversulfates are submitted to a N-sulfation, performed by treating their aqueous solution with sodium carbonate and a N-sulfating agent, for example a (C 1 -C 4 )trialkylamine.SO 3 or pyridine.SO 3 , by maintaining the mixture at 30-50° C. for 8-24 hours and by isolating the LMW-K5-N,O-oversulfate for example by diafiltration.
- a N-sulfating agent for example a (C 1 -C 4 )trialkylamine.SO 3 or pyridine.
- LMW-K5-amine-O-oversulfates and their chemically or pharmaceutically acceptable salts obtainable according to the above said process, limited to steps (a) and (b), are new products which constitute a further aspect of the present invention.
- the present invention also provides new LMW-K5-amine-O-oversulfates having a degree of sulfation of from 2.3 to 3 and their chemically or pharmaceutically acceptable salts.
- their mean molecular weight is from about 3,500 to about 10,000, more advantageously from about 3,500 to about 5,200.
- a LMW-K5-N-sulfate consisting of a mixture of chains in which at least 90% of said chains has the above mentioned formula II is used as starting material
- LMW-K5-amine-O-oversulfates are new products useful as intermediates in the preparation of their N-sulfated derivatives, but they have per se interesting pharmacological properties, in particular against free radicals.
- the origin of the new LMW-K5-amine-O-oversulfates from LMW-K5-N-sulfates obtained by nitrous depolymerization and subsequent reduction with, for instance, sodium borohydride involves the presence of a 2,5 anhydromannitol sulfated unit of structure (a′′) in which R′′ represents hydrogen or SO ⁇ , at the reducing end of the majority of the chains in said mixture of chains.
- LMW-K5-amine-O-oversulfates those consisting of mixtures in which the preponderant species is a compound of formula (III′′) wherein R, R′, R′′ are hydrogen or SO ⁇ , X′′ is OH or OSO ⁇ , for a degree of sulfation of from 2.2 to 3, m is 4, 5, or 6 and the corresponding cation is a chemically or pharmaceutically acceptable one, are preferred.
- the new LMW-K5-N,O-oversulfates obtained at the end of the process of the present invention are generally present as sodium salt.
- Said sodium salt can be converted into another chemically or pharmaceutically acceptable salt.
- an exchange with the calcium ion can be performed using ultrafiltration membranes.
- Particularly advantageous salts are those of alkaline metals, earth-alkaline metals, ammonium, tetra(C 1 -C 4 )alkylammonium, aluminum, and zinc.
- the sodium, calcium and tetrabutylammonium are preferred salts.
- the LMW-K5-N,O-oversulfates obtained according to the process of the present invention consist of mixtures of chains in which at least 90% of said chains has the structure I′ in which n is an integer from 2 to 20, R, R′ and R′′ represent hydrogen or a SO ⁇ - group, in which the reducing end of the majority of said chains has the structure (a′′), for a degree of sulfation of from 3.2 to 4, advantageously from 3.5 to 4, preferably from 3.5 to 3.9, and the corresponding cation is a chemically or pharmaceutically acceptable one.
- Preferred LMW-K5-N,O-oversulfates among those obtained according to the process of the present invention consist of mixtures of chains in which the preponderant species is a compound of formula I′′ wherein R, R′ and R′′ are hydrogen or SO3-, X′′ is OH or OSO3-, for a degree of sulfation of from 3.2 to 4, advantageously from 3.5 to 4, preferably from 3.5 to 3.9, m is 4, 5 or 6 and the corresponding cation is a chemically or pharmaceutically acceptable one.
- the administration of the active principle can be performed by oral, transdermic or preferably parenteral, in particular subcutaneous, intramuscular or intravenous, or topic route.
- the intended daily dosage for the parenteral administration is of 0.5-500 mg/Kg/die, advantageously of 5-250 mg/kg/die, preferably of 10-150 mg/kg/die, while the dosage intended for the topical route is of 1-1,000 mg/Kg/die, advantageously 10-500 mg/Kg/die, preferably 20-100 mg/Kg/die.
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising, as an active ingredient, a pharmacologically effective amount of a LMW-K5-N,O-oversulfate advantageously consisting of mixture of chains in which the preponderant species is a compound of formula I, I°, I′′ or by mixtures of chains in which at least 90% of said chains has the formula I′, for a degree of sulfation of from 3.2 to 4, advantageoulsy from 3.5 to 4, preferably from 3.5 to 3.9, or of its pharmaceutically acceptable salts, in admixture with a pharmaceutical vehicle.
- Advantageous pharmaceutically acceptable salts are sodium, potassium, calcium, magnesium, aluminum and zinc salts.
- the active ingredients are preferably administered as dosage units, in admixture with the classic excipients or pharmaceutical vehicles.
- the dose can amply change in function of age, weight, and health conditions of the patient, as much as of severity of the infection and of route of administration.
- This dose comprises the administration of a dosage unit of from 1 to 1,000 mg, advantageously from 10 to 750 mg, preferably from 250 to 500 mg, once to three times per day, by intravenous, intramuscular, subcutaneous, oral, transdermic, transmucosal or topical route.
- compositions of the present invention are formulated with the pharmaceutical carriers suitable for the various administration routes.
- the present invention provides a pharmaceutical composition for the treatment of angiogenesis dependent pathologies or for the treatment of HIV infection which comprises, as an active ingredient, a pharmacologically effective amount of a LMW-K5-N,O-oversulfate advantageously consisting of mixtures of chains in which the preponderant species is a compound of formula I, I°, I′′ or by mixtures of chains in which at least 90% of said chains has the formula I′, for a degree of sulfation of from 3.2 to 4, advantageously from 3.5 to 4, preferably from 3.5 to 3.9, or of one of its pharmaceutically acceptable salts, in admixture with a pharmaceutical vehicle or carrier.
- a LMW-K5-N,O-oversulfate advantageously consisting of mixtures of chains in which the preponderant species is a compound of formula I, I°, I′′ or by mixtures of chains in which at least 90% of said chains has the formula I′, for a degree of sulfation of from 3.2 to 4, advantageously
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising, as one of its active ingredients, a new LMW-K5-amine-O-oversulfate obtainable according to steps (a) and (b) of the process above described, especially a LMW-K5-amine-O-oversulfate having a degree of sulfation of from 2.2 to 3, advantageously having a mean molecular weight from about 3,500 to about 11,000, more advantageously from about 3,500 to about 5,200, preferably consisting of a mixture of chains in which at least 90% of said chains has the formula III or in which the preponderant species is a compound of formula III′ or III′′, or one of its pharmaceutically acceptable salts, in admixture with a pharmaceutical carrier.
- said LMW-O-oversulfated-K5 amine with a degree of sulfation of from 2.2 to 3 is substantially free of N-acetyl groups.
- the precipitate formed is separated by centrifugation at 10,000 rpm for 20 minutes and the purity of the product is controlled by overnight dialysis and subsequent 1 H-NMR analysis in order to ascertain that signals in the region below 1.5 ppm are absent. If necessary, the procedure of dissolution in water containing 4M NaCl and precipitation with isopropanol is repeated. The precitate is dissolved in water and ultrafiltrated on a Miniplate membrane Millipore with a 10,000 D cut off till disappearance of the salts. A K5 is obtained having a purity of at least 99% and giving a 1 H-NMR spectrum in which traces of lipophilic impurities in the region below 1.5 ppm are undetectable.
- the K5-N-sulfate thus obtained is purified from salts by diafiltration using a spiral membrane of 1,000 D (Prepscale Cartridge-Millipore). The process is ended when the conductivity of the permeate is below 1,000 ⁇ S, preferably below 100 ⁇ S. The intradialysis is reduced till a polysaccharide concentration of 10% using the same in concentration dialysis system. The concentrated solution is freeze dried.
- the 13 C-NMR spectrum of the K5-N-sulfate does not show any signal of residual N-acetyl or NH 2 groups.
- the product obtained in PREPARATION II is depolymerized by the degradation method with nitrous acid and subsequent reduction of the formed aldehyde.
- One gram of K5-N-sulfate is dissolved in 200 ml of distilled water and 480 mg of sodium nitrite dissolved in 240 ml of distilled water are added thereinto.
- the solution is then cooled to 4° C. and the pH is brought to 2 with 0.1N HCl and maintained for 30 minutes. At the end of the reaction the solution is brought to pH 7 with 0.1N NaOH and then to room temperature.
- the solution is then added with 450 mg of NaBH 4 and left to react for 4 hours.
- the excess NaBH4 is eliminated with HCl by bringing the pH to 5-6.
- the product neutralized with 0.1 M NaOH, is recovered by precipitation with 3 volumes of acetone at 4° C., filtration with a glass funnel and dried at 40° C. in vacuum oven.
- 900 mg of a LMW-K5-N-sulfate having a mean molecular weight of about 2,000 are obtained.
- K5-N-sulfate obtained as described in PREPARATION II is dissolved in 100 ml of deionized water and the solution is cooled to 10° C. with a cooling bath and then passed through a cationic exchange resin IR 120 H + or equivalent (50-200 ml). Both the column and the reservoir of the eluate are maintained at 10° C. After the passage of the solution containing the sample, the resin is washed with deionized water till the pH of the permeate is higher than 6 (about 3 volumes of deionized water). The acidic solution is brought to neutrality with tetrabutylammonium hydroxide (15% aqueous solution), then reduced to the minimum volume and freeze dried.
- the tetrabutylammonium salt is dissolved in 40 ml of DMF and added with 3.5 g of adduct pyridine.SO 3 in solid form.
- the solution is kept at 50° C. for 24 hours.
- the solution is cooled to room temperature and added with 3 volumes of a saturated sodium chloride solution in acetone till complete precipitation.
- the precipitate is separated from the solvent by filtration, solubilized with the minimum amount of deionized water (for example 100 ml) and added with sodium chloride to obtain a 0.2M solution.
- the solution is brought to pH 7.5-8 with 2N sodium hydroxide and added with 2 volumes of acetone till complete precipitation.
- the precipitate is separated from the solvent by filtration.
- the solid obtained is solubilized with 100 ml of deionized water and purified from residual salts by ultrafiltration using a spiral membrane of 1,000 D cut-off (prepscale cartridge-Millipore).
- the solution containing the O-sulfated product is treated for the N-sulfation as previously described in step (ii) of PREPARATION II.
- the product has a sulfate to carboxyl ratio of 3.87 measured by conductimetry according to Casu et al. and a mean molecular weight of 20,000 measured by molecular exclusion HPLC.
- K5-N,O-oversulfate obtained as described in PREPARATION IV is dissolved in 200 ml of deionized water and thermostated to 4° C. Then 230 mg of sodium nitrite dissolved in an aqueous solution at the concentration of 0.2% are added.
- the solution containing the K5-N,O-oversulfate and sodium nitrite, kept at 4° C., is brought to pH 2 by addition of 0.1N HCl cooled to 4° C. The solution is left to react under slow stirring for 30 minutes, then neutralized with 0.1N NaOH.
- the solution containing the LMW-K5-N,O-oversulfate so obtained, consisting of a mixture of chains in which the preponderant species is a decasaccharide of formula I° in which m is 4 and X′ is formyl, is brought to room temperature and treated with 250 mg of sodium borohydride dissolved in 50 ml of water and left to react for 4 hours.
- the excess sodium borohydride is eliminated by bringing the pH to about 5 with 0.1N HCl and left for further 2 hours.
- the solution is neutralized with 0.1N NaOH and the product is recovered by precipitation with acetone after concentration of the product by evaporation under reduced pressure.
- the LMW-K5-N,O-oversulfate so obtained shows characteristics of sulfation similar to those of the K5-N,O-oversulfate starting material, a mean molecular weight of about 4,250 measured by viscosimetry and consists of a mixture of chains in which the preponderant species is a decasaccharide of formula I′ in which m is 4 and X′ is CH2OH.
- K5-N,O-oversulfate obtained as described in PREPARATION IV is treated as described in EXAMPLE 1 using 200 mg of sodium nitrite.
- the product obtained shows characteristics of sulfation similar to those of the K5-N,O-oversulfate starting material, a mean molecular weight of about 5,000 measured by viscosimetry and consists of a mixture of chains in which the preponderant species is a dodecasaccharide of formula I′ in which m is 5 and X′ is CH 2 OH.
- K5-N,O-oversulfate obtained as described in PREPARATION IV is treated as described in Example 1 using 160 mg of sodium nitrite.
- the LMW-K5-N,O-oversulfate thus obtained shows characteristics of sulfation similar to those of the K5-N,O-oversulfate starting material, a mean molecular weight of about 6,000 measured by viscosimetry and consists of a mixture of chains in which the preponderant species is a tetradecasaccharide of formula I′ in which m is 6 and X′ is CH 2 OH.
- a solution of 500 mg of LMW-K5-N-sulfate obtained as described in PREPARATION III in 50 ml of water is thermostated to 4° C., then passed through a IR 120 H + ionic exchange resin preconditioned with water at 4° C.
- the eluate obtained consisting of 125 ml of a solution at a pH of about 2, is neutralized with a 15% solution of tetrabutylammonium hydroxide and left at room temperature for one hour, by maintaining the pH at 7 by addition of a 15% tetrabutylammonium hydroxide solution and finally is freeze dried.
- One gram of the tetrabutylammonium salt of LMW-K5-N-sulfate is obtained.
- a solution containing one gram of the so obtained salt in 20 ml of dimethylformamide is kept at 55° C. and treated with 20 ml of dimethylformamide containing 1.7 g of pyridine.SO 3 adduct.
- the reaction at 55° C. is performed overnight, then 40 ml of water are added to the mixture.
- the product is precipitated with 3 volumes of saturated NaCl solution in acetone and kept at 4° C. overnight.
- the precipitate is recovered by filtration on a G4 glass funnel and then ultrafiltrated with a TFF Millipore system with 1,000 D cut off and dried at reduced pressure.
- LMW-K5-amine-O-oversulfate consisting of a mixture of chains in which the preponderant species is a decasaccharide of formula II′′ wherein m is 4, for a degree of sulfation of about 2.9.
- step (b) To a solution of 500 mg of the LMW-K5-amine-O-oversulfate obtained in step (b) in 30 ml of water, 800 mg of sodium carbonate are added, then 800 mg of pyridine.SO 3 adduct in solid form are added stepwise in 4 hours to the so obtained solution. The reaction mixture is kept at 55° C. overnight, then the reaction is stopped by bringing its pH to 7 by addition of 0.1 N HCl. After ultrafiltration on a 1,000 D membrane, 3 volumes of a saturated solution of NaCl in acetone are added and the precipitate is recovered by centrifugation at 5,000 rpm for 5 minutes.
- LMW-K5-N-sulfate obtained as described in PREPARATION III is submitted to steps (a) and (b) as described in Example 4.
- the LMW-K5-amine-O-oversulfate is recovered by precipitation with 3 volumes of a saturated solution of NaCl in acetone, dissolution of the precipitate obtained in water, ultrafiltration on a 1,000 D cut-off membrane and freeze drying.
- the so obtained product is a LMW-K5-amine-O-oversulfate having a mean molecular weight of about 3,600 measured by viscosimetry and consists of a mixture of chains in which the preponderant species is a decasaccharide of formula III wherein m is 4.
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Application Number | Priority Date | Filing Date | Title |
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ITMI2002A001346 | 2002-06-18 | ||
ITMI2002A001345 | 2002-06-18 | ||
IT2002MI001346A ITMI20021346A1 (it) | 2002-06-18 | 2002-06-18 | Polisaccaridi a basso peso molecolare e procedimento per la loro preparazione |
IT2002MI001345A ITMI20021345A1 (it) | 2002-06-18 | 2002-06-18 | Derivati epimerizzati del polissacaride k5 a elevatissimo grado di solfatazione |
ITMI2002A001854 | 2002-08-27 | ||
ITMI20021854 ITMI20021854A1 (it) | 2002-08-27 | 2002-08-27 | Polisaccaride supersolfatato a basso peso molecolare. |
PCT/IB2003/002347 WO2003106506A1 (en) | 2002-06-18 | 2003-06-17 | Low molecular weight oversulfated polysaccharide |
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US10/518,229 Abandoned US20050245736A1 (en) | 2002-06-18 | 2003-06-17 | Low molecular weight oversulfated polysaccharide |
US10/518,303 Abandoned US20050256079A1 (en) | 2002-06-18 | 2003-06-17 | Process for the manufacture of n-acyl-(epi)k5-amine-o-sulfate-derivatives and products thus obtained |
US12/895,894 Expired - Fee Related US8193166B2 (en) | 2002-06-18 | 2010-10-01 | Epimerized derivatives of K5 polysaccharide with a very high degree of sulfation |
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US12/895,894 Expired - Fee Related US8193166B2 (en) | 2002-06-18 | 2010-10-01 | Epimerized derivatives of K5 polysaccharide with a very high degree of sulfation |
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Cited By (6)
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US20050256079A1 (en) * | 2002-06-18 | 2005-11-17 | Glycores 2000 S.R.1. | Process for the manufacture of n-acyl-(epi)k5-amine-o-sulfate-derivatives and products thus obtained |
US20070259833A1 (en) * | 2004-06-14 | 2007-11-08 | Institut Francais De Recherche Pour L'exploitation De La Mer (Fremer) | Use of Low-Molecular-Weight Highly Sulfated Polysaccharide Derivatives for Modulating Angiogenesis |
US20080146793A1 (en) * | 2001-02-27 | 2008-06-19 | Pasqua Oreste | Highly sulfated derivatives of K5 polysaccharide and their preparation |
US20090005341A1 (en) * | 2002-06-18 | 2009-01-01 | Glycores 2000 S.R.L. | Process for the preparation of n-acyl-(epi)k5-amine-o-sulfate-derivatives and products thus obtained |
US20090170927A1 (en) * | 2005-10-21 | 2009-07-02 | Bezwada Biomedical, Llc | Functionalized phenolic compounds and polymers therefrom |
US7812151B2 (en) | 2003-12-17 | 2010-10-12 | Glycores 2000 S.R.L. | Low molecular weight polysaccharides having antithrombotic activity |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120253029A1 (en) | 2002-06-18 | 2012-10-04 | Pasqua Anna Oreste | Process for the preparation of highly o-sulfated epimerized derivatives of k5 polysaccharide and intermediates therein |
ITMI20031618A1 (it) * | 2003-08-06 | 2005-02-07 | Inalco Spa | Derivati polisaccaridici dotati di alta attivita' |
FR2871476B1 (fr) * | 2004-06-14 | 2011-04-01 | Ifremer | Derives depolymerises sulfates d'exopolysaccharides (eps) provenant de bacteries marines mesophiles, leur procede de preparation et leurs utilisations en regeneration tissulaire |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020062019A1 (en) * | 2000-03-30 | 2002-05-23 | Pasqua Oreste | Glycosaminoglycans derived from K5 polysaccharide having high anticoagulant and antithrombotic activities and process for their preparation |
Family Cites Families (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IT1141483B (it) * | 1980-04-18 | 1986-10-01 | Crinos Industria Farmaco | Resina poliamminica per la separazione selettiva di eparina da altri glucosamminoglicani e procedimento per la sua preparazione |
FR2503714B1 (fr) * | 1981-04-10 | 1986-11-21 | Choay Sa | Procede d'obtention de mucopolysaccharides biologiquement actifs, de purete elevee, par depolymerisation de l'heparine |
DE3787996T2 (de) * | 1986-05-16 | 1994-03-03 | Italfarmaco Spa | Heparine, frei von E.D.T.A., Fraktionen und Fragmente von Heparin, Verfahren zu deren Herstellung und pharmazeutische Zusammensetzungen, welche diese enthalten. |
EP0333243A3 (en) * | 1988-03-10 | 1989-09-27 | Akzo N.V. | Sulphated k5 antigen and sulphated k5 antigen fragments |
FR2669932B1 (fr) * | 1990-12-03 | 1994-07-01 | Sanofi Sa | Nouvel heparosane-n,o-sulfate, son procede de preparation et les compositions pharmaceutiques qui le contiennent. |
GB2286193A (en) | 1991-03-28 | 1995-08-09 | Italfarmaco Spa | Anticoagulants and processes for preparing such |
FR2684385B1 (fr) * | 1991-11-28 | 1997-08-01 | Sanofi Elf | Heparosanes-n,o-sulfates de haute masse moleculaire, leur procede de preparation et les compositions pharmaceutiques qui les contiennent. |
IT1271057B (it) * | 1994-11-04 | 1997-05-26 | Inalco Spa | Polisaccaridi aventi un elevato contenuto di acido iduronico |
US5798356A (en) * | 1995-08-07 | 1998-08-25 | Alcon Laboratories, Inc. | Angiostatic compounds |
IT1282994B1 (it) | 1996-05-10 | 1998-04-03 | Inalco Spa | Derivati del polisaccaride k5 aventi elevata attivita' anticoagulante |
IT1284143B1 (it) * | 1996-09-06 | 1998-05-08 | Istituto Scient Di Chimica E B | Eparansolfati semi-sintetici ad elevata attivita' antimetastica e ridotto rischio emorragico |
GB9620459D0 (en) | 1996-10-01 | 1996-11-20 | Mclaughlin Andrew | Improvements in or relating to blocked isocyanates and their production |
IT1289613B1 (it) * | 1997-02-07 | 1998-10-15 | Inalco Spa | Polisaccaridi batterici o-solfatati |
IT1290814B1 (it) * | 1997-03-24 | 1998-12-11 | Istituto Scient Di Chimica E B | Glicosaminoglicani aventi elevata attivita' antitrombotica |
US6329351B1 (en) * | 1997-08-28 | 2001-12-11 | Istituto Scientifico Di Chimica E Biochimica “G. Ronzoni” | Semi-synthetic sulphaminoheparosansulphates having high anti-metastatic activity and reduced haemorrhagic risk |
US6388060B1 (en) * | 1998-11-06 | 2002-05-14 | Vascular Therapeutics Inc. | Process for the sulfation of uronic acid-containing polysaccharides |
IT1318432B1 (it) * | 2000-03-30 | 2003-08-25 | Inalco Spa | Glicosaminoglicani derivati dal polisaccaride k5 aventi elevataattivita' anticoagulante ed antitrombotica e processo per la loro |
ITMI20032498A1 (it) * | 2003-12-17 | 2005-06-18 | Pasqua Anna Oreste | Polisaccaridi antitrombotici a basso peso molecolare e |
US20050027117A1 (en) * | 2000-12-18 | 2005-02-03 | Pasqua Oreste | Anticoagulant and antithrombotic LMW-glycosaminoglycans derived from K5 polysaccharide and process for their preparation |
ITMI20010397A1 (it) * | 2001-02-27 | 2002-08-27 | Giorgio Zoppetti | Derivati altamente n,o-solfatati del polisaccaride k5 e loro preparazione |
ITMI20010779A1 (it) * | 2001-04-12 | 2002-10-12 | Giorgio Zoppetti | Uso di polisaccaridi batterici solfati atti all'inibizione dell'angiogenesi |
ITMI20011633A1 (it) * | 2001-07-27 | 2003-01-27 | San Raffaele Centro Fond | Uso di polisaccaridi batterici supersolfatati inibitori dell'hiv |
ITMI20012200A1 (it) * | 2001-10-22 | 2003-04-22 | Ibsa Inst Biochimique Sa | Processo per la preparazione di condroitin solfati dal polisaccaride k4 e prodotti ottenuti |
ITMI20021294A1 (it) * | 2002-06-12 | 2003-12-12 | Inalco Spa | Polisaccaridi batterici o-solfatati e loro uso |
MXPA04012714A (es) * | 2002-06-18 | 2005-08-15 | Glycores 2000 Srl | Derivados epimerizados del polisacarido k5 con un grado muy alto de sulfatacion. |
ITMI20032614A1 (it) * | 2003-12-30 | 2005-06-30 | Altergon Sa | Nuova composizione comprendente cs |
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020062019A1 (en) * | 2000-03-30 | 2002-05-23 | Pasqua Oreste | Glycosaminoglycans derived from K5 polysaccharide having high anticoagulant and antithrombotic activities and process for their preparation |
Cited By (13)
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US20080146793A1 (en) * | 2001-02-27 | 2008-06-19 | Pasqua Oreste | Highly sulfated derivatives of K5 polysaccharide and their preparation |
US8513407B2 (en) | 2002-06-18 | 2013-08-20 | Glycores 2000 S.R.L. | Process for the preparation of N-acyl-(epi)K5-amine-O-sulfate-derivatives and products thus obtained |
US20090005341A1 (en) * | 2002-06-18 | 2009-01-01 | Glycores 2000 S.R.L. | Process for the preparation of n-acyl-(epi)k5-amine-o-sulfate-derivatives and products thus obtained |
US20050256079A1 (en) * | 2002-06-18 | 2005-11-17 | Glycores 2000 S.R.1. | Process for the manufacture of n-acyl-(epi)k5-amine-o-sulfate-derivatives and products thus obtained |
US7812151B2 (en) | 2003-12-17 | 2010-10-12 | Glycores 2000 S.R.L. | Low molecular weight polysaccharides having antithrombotic activity |
US20080131472A1 (en) * | 2004-06-14 | 2008-06-05 | Universite Rene Descarties Paris 5 | Sulfated Depolymerized Derivatives of Exopolysaccharides (Eps) from Mesophilic Marine Bacteria, Method for Preparing Same, and Uses Thereof in Tissue Regeneration |
US20070259833A1 (en) * | 2004-06-14 | 2007-11-08 | Institut Francais De Recherche Pour L'exploitation De La Mer (Fremer) | Use of Low-Molecular-Weight Highly Sulfated Polysaccharide Derivatives for Modulating Angiogenesis |
US7833990B2 (en) | 2004-06-14 | 2010-11-16 | Institut Francais De Recherche Pour L'exploitation De La Mer (Ifremer) | Use of low-molecular-weight highly sulfated polysaccharide derivatives for modulating angiogenesis |
US8598142B2 (en) | 2004-06-14 | 2013-12-03 | Institut Francais De Recherche Pour L'exploitation De La Mer (Ifremer) | Sulfated depolymerized derivatives of expolysaccharides (EPS) from mesophilic marine bacteria, method for preparing the same, and uses thereof in tissue regeneration |
US9125883B2 (en) | 2004-06-14 | 2015-09-08 | Institut Francais De Recherche Pour L'exploitation De La Mer (Ifremer) | Sulfated depolymerized derivatives of exopolysaccharides (EPS) from mesophilic marine bacteria, method for preparing same, and uses thereof in tissue regeneration |
US20090170927A1 (en) * | 2005-10-21 | 2009-07-02 | Bezwada Biomedical, Llc | Functionalized phenolic compounds and polymers therefrom |
US8318973B2 (en) * | 2005-10-21 | 2012-11-27 | Bezwada Biomedical, Llc | Functionalized sinapic acid and methyl sinapate |
US8802892B2 (en) | 2005-10-21 | 2014-08-12 | Bezwada Biomedical, Llc | Functionalized phenolic compounds and polymers therefrom |
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