KR20050024349A - Epimerized derivatives of k5 polysaccharide with a very high degree of sulfation - Google Patents

Abstract

The present invention provides a novel process for oversulfurizing epiK5-N-sulfate followed by N-sulfurization to obtain epiK5-amine-O-excess sulfate with a very high degree of sulfation. There is provided a novel epiK5-amine-O-supersulfate-derivative, useful in the cosmetic or pharmaceutical art, having a degree of sulphation of at least 4 and essentially inactive on the aggregation parameters. Also described are novel low molecular weight epiK5-sulfates useful as intermediates in the preparation of the corresponding lmw-epiK5-N, O-excess sulfate-derivatives.

Description

EPIMERIZED DERIVATIVES OF K5 POLYSACCHARIDE WITH A VERY HIGH DEGREE OF SULFATION

New derivatives of K5 polysaccharides with a high degree of sulfation, methods for their preparation, novel highly O-sulfated intermediates useful in their synthesis, and the derivatives of K5 polysaccharides as active ingredients which are basically free of activity in coagulation. It relates to a pharmaceutical composition comprising.

In particular, the present invention provides K5 poly, previously N-deacetylated, N-sulfated, and at least 20% C5-epimerized, through O- oversulfuration and subsequent N-sulfurization at appropriate conditions. The present invention relates to a process for preparing epi-K5-N, O- supersulfate obtained by starting with saccharides, and to the epi-K5-N, O-excess sulfate of antiangiogenic and antiviral activity, and epi-K5- A derivative of a novel low molecular weight intermediate polysaccharide of N-sulfate.

Glycosaminoglycans such as heparin, heparan sulfate, dermatane sulfate, chondroitin sulfate and hyaluronic acid are biopolymers extracted industrially from various animal organs.

In particular, heparin, which is mainly obtained by extraction from the gut mucosa of pigs or from bovine lungs, is basically amino linked by uronic acid (glucuronic acid or iduronic acid) and α-1 → 4 or β-1 → 4 bonds. It is polydispersed with a molecular weight distribution of approximately 3,000 to approximately 30,000 D that constitutes a chain mixture consisting of sugar (glucosamine). In heparin, the Euron unit can be O-sulfurized at the 2 position and the glucosamine unit is N-acetylated or N-sulfurized, 6-O-sulfurized and 3- at approximately 0.5% of the glucosamine units present. O-sulfurized.

The character and natural biosynthesis of heparin in mammals is as follows: Lindahl et al., 1986 in Lane, D. and Lindahl, U. (Editor) "Heparin. Chemical and Biological Properties; Clinical Applications", Edward Arnold, London, Pages 159- 190, Lindahl, U. Feingold DS and Roden L, 1986 TIBS, 11, 221-225 and Conrad HE "Heparin Binding Proteins", Chapter 2: Structure of Heparinoids. Academic Press, 1998. Biosynthesis of heparin occurs starting with its N-acetyl-heparoic acid precursor, which constitutes the chain mixture constituting the repetitive glucuronyl-β-1 → 4-N-acetylglucosamine disaccharide unit. The precursors hydrolyze the N-acetyl group partially, enzymatic modifications to displace it with SO ₃ ⁻ groups, and, when industrially extracted, products with approximately twice the number of sulfate groups in terms of carboxyl groups per disaccharide unit. To obtain an O-sulfate group, an enzymatic modification is carried out to epimerize the carboxy at position 5 of the portion of the clocoron unit which is transduced with the iduronic acid unit. These enzymatic modifications bind to antithrombin III (ATIII), called active pentasaccharides, which are structures based on the anticoagulant and antithrombotic activity of heparin itself and essential for the high affinity binding of heparin to ATIII. Leads to the formation of a pentasaccharide region. This pentasaccharide, present inside only a portion of the chain forming heparin, contains glucuronic acid located between disaccharides containing iduronic acid and sulfated glucosamine units at the 3-position.

In fact, the formation of active pentasaccharides is achieved by appropriate sulfateization which also induces the introduction of sulfate groups in the hydroxyl at the 3 position of gulucosamine and by gurucuronyl-C5-epimerase (C5-epimerization). The iduronic acid unit performed is made possible by the epimerization reaction of some of the carboxy of the gurucuron unit. More specifically, the formation of active pentasaccharide actually leads to a widespread induction of products in which C5-epimerization occurs in the cluster, i.e., part of the chain, and contains more iduronic acid units than glucuronic acid units. This is made possible by the fact. In fact, commercial heparin contains approximately 70% of iduronic acid units and 30% of glucuronic acid units.

Along with major anticoagulant and antithrombotic activity, heparin also has antilipaemic, antiproliferative, antiviral, antitumor and antimetastatic activity, but its use as a medicament may cause bleeding. It is hampered by adverse effects due to possible anticoagulant action.

Former technology

Encapsulated K5 polysaccharides isolated from Escherichia coli , described by Vann WF et al., In European Journal of Biochemistry, 1981, 116, 359-364 ("Vann 1981"), are repeating disaccharide units glucuro. It consists of a chain mixture consisting of Nyl-β-1 → 4-N-acetyl glucosamine, thus representing the same repeating sequence (A) of the N-acetyl-heparoic acid precursors of heparin.

Encapsulated K5 polysaccharides, hereafter referred to as "K5 polysaccharides" or more simply "K5", are described by Lormeau et al. And described in Carbohydrate Research, 1994, 263, 271-284, as described in US Pat. No. 5,550,116. Chemically modified by Casu et al. K5-O-sulfates having antitumor, antimetastatic, antiviral, in particular anti-HIV activity are disclosed in EP 333243 and WO 98/34958. K5 was also chemically and enzymatically modified to obtain products with biological activity in vitro in the same form of coagulation as heparin (extracted heparin) extracted from animal organs.

The achievement of a product with activity in the same form of coagulation as that of extractable heparin is in fact a process that mimics development and is expected to be a key step in the overall process of C5-epimerase with D-gulucuroyl C5 epimerase. Caused by

The processes described in IT 1230785, WO 92/17507, WO 96/14425 and WO 97/43317 utilize K5 as starting material. K5 originating from fermentation is subject to N-deacetylation followed by N-sulfurization, and C5-epimerization with C5-epimerase in solution in the K5-N-sulfate thus obtained is carried out, Obtained by either chromatography of a solution of microsomal enzymes from mouse mastocytoma (IT 1230 785) or from bovine liver (WO 92/17507, WO 96/14425 and WO 97/43317).

D-glucuronyl C5 epimerase from bovine liver has been described in Campbell, p. et al. in J. biol. Purified by Chem., 1994, 269/43, 26953-26958 ("Campbell 1994"). The document also provides its composition in amino acids, discloses its use in solution for the transformation of K5-N-sulfate with the corresponding 30% epimerized product, and the disaccharide following the HPLC method. Illustrative formation of iduronic acid by nitrous acid depolymerization.

Document WO 98/48006 was obtained from a recombinant expression vector containing the DNA, followed by Jin-Ping L. et al. in J. Biol Chem. D-glucuronyl C5 epimerase and recombinant D-glucuronyl C5 epi, purified by Campbell et al., As shown by 2001, 276, 20069-20077 (“Jin-Ping 2001”). A DNA sequence encoding for merase is disclosed.

Complete C5-epimerase is described in Crawford B. E. et al. in J. Biol. Chem., 2001, 276 (24), 21538-21543 (Crawford 2001). Document US 2002/0062019 describes a process for preparing epiK5-N, O-sulfate, active in coagulation control, having a degree of sulfation of 2.3 to 2.9 and a molecular weight of 2,000 to 30,000 or a molecular weight of 4,000 to 8,000 or 18,000 to 30,000. do. The process comprises the following steps: (pa) N-deacetylation of K5 polysaccharide and N-sulfurization of the resulting K5-amine, (pb) epimerization of K5-N-sulfate, (pc) epiK5- O-supersulfuration of N-sulfate, (pd) partial O-desulfurization, (pe) selective 6-O-sulfurization, (pf) N-sulfurization of the product thus obtained, and (pb Certain products obtained from the bellows of either) or (pf) may be introduced into the deuterated. The document describes epiK5-N, O-sulfate having a molecular weight of 7,400, obtained by the above steps (pa)-(pf) followed by nitrous acid desaturation at the end of step (pf), with a degree of sulphation of 2.3 to 2.9. do.

The same document also describes moieties of K5 with molecular weights of approximately 5,000, which may face steps (p-a) to (p-f).

In order to standardize the terminology and to make the text easier to understand, in the present specification, a conventional term or expression will be used in the singular or plural, in particular:

"K5" or "K5 polysaccharide" is a capsular polysaccharide from Escherichia coli obtained by fermentation, ie according to the method described in the literature, in particular according to Vann 1981, Manzoni M. et al., Journal of Double in non-reduced initiation, prepared and purified according to Bioactive Compatible Polymers, 1996, 11, 301-311 ("Manzoni 1996") or according to the methods described in WO 01/72848 and WO 02/068447. A chain mixture consisting of disaccharide units (A) optionally containing a bond; It is apparent to those skilled in the art that what is shown here can be applied to N-acetylheparoic acid;

"C5-epimerase" is described in Campbell 1994, WO 98/48006, by Jin-Ping L. et al. in J. Biol Chem. Extracted or recombinant D-glucuronyl C-5 epimerase prepared, isolated and purified as described in 2001, 276, 20069-20077 ("Jin-Ping 2001") or as described in Crawford 2001 it means;

K5-amine means greater than 95% N-deacetylated K5, but the acetyl group is not detectable by current MMR machines;

-"K5-N-sulfate" means, as described hereinafter, N-acetyl groups are not detectable by conventional NMR machines, and therefore generally at least 100%, N-deacetylated and N-sulfate K5 ;

-"Epi K5" refers to K5 and derivatives in which 20-60% of the glucuronic acid units are C5-epimerized with iduronic acid units;

-"Epi K5-N-sulfate" means K5-N-sulfate where 20-60% of the glucuronic acid units are C5-epimerized with iduronic acid units;

"Epi K5-amine-O- supersulfate" means epiK5-amine-O-sulfate having a degree of sulfateization of at least 3.4;

-"Epi K5-N, O- excess sulfate" means a fully N-sulphated epiK5-amine-O-sulfate having a degree of sulphation of at least 4;

Where the general terms and expressions defined above refer to K5 as isolated after fermentation, which generally has a molecular weight distribution of approximately 1,500 to approximately 50,000 with an average molecular weight of 10,000 to 25,000, advantageously 15,000 to 25,000. do;

Where it is preceded by the conventional terms and expressions defined above, the acronym "LMW" (low molecular weight), for example LMW-K5-N-sulfate, LMW-epiK5-N-sulfate Is derived from or consists of K5-N-sulfate having an average molecular weight of approximately 1,500 to approximately 12,000, obtained by depolymerization of K5-N-sulfate or by fractional distillation and calculated from 100 $ N-sulfated products. Low molecular weight products;

Where the general terms and expressions defined above, followed by “-derivatives” are represented as a whole of both natural K5 and derivatives from those of low molecular weight;

The term "approximately" in terms of molecular weight, calculated as 461 for epiK5-N-sulfate-derivatives and 806 for epiK5-N, O- excess sulfate-derivatives having a degree of sulphation 4.26 Mean molecular weight measured by a theoretical mass ± viscosity of disaccharide units;

-The expression "dominant species" is in the form of a grammed representation in a mixture consisting of LMW-epiK5-N-sulfate, LMW-epiK5-amine-O-excess sulfate or LMW-epiK5-N, O-excess sulfate By compound, determined by the peak of the molecular weight curve measured by HPLC;

-Unless specifically stated otherwise, "degree of oxidation" is equally utilized in WO 01/72848 and used in Casu B. et al. means the SO ₃ ⁻ / COO ⁻ ratio, also expressed as the number of sulfate groups per disaccharide unit, measured by conductivity measurement as described by Carbohydrate Research, 1975, 39, 168-176 (Casu 1975). ;

"O-supersulfuration conditions" are described in B. Casu et al. means the maximum O-sulfurization carried out for the examples according to Method C described by Carbohydrate Research, 1994, 263, 271-284 (Casu 1994);

-The term "alkyl" refers to linear or branched alkyl, while the term "tetrabutylammonium" refers to tetra- n -butylammonium.

Summary of the Invention

Unlike what happens with the methods described in IT 1230785, WO 92/17507, WO 96/14425, WO 97/43317, WO 01/72848 and US 2002/0062019, starting with epiK5-N-sulfate, literature, examples For example, a salt of epiK5-N-sulfate is prepared with a tertiary or quaternary organic base, and the reaction mixture is controlled to maintain for 30 to 60 minutes while maintaining the pH at approximately pH 7 with the same organic base, and then O-excess sulfuric acid. To obtain epiK5-amine-O-excess sulfate having a greater degree of sulfation than all other epiK5-amine-O-sulphates described in WO 01/72848, treating the salts obtained with O-sulphurizers under the conditions of oxidation. Surprisingly it has now been found possible.

The resulting epiK5-amine-O-excess sulfate was subjected to N-sulfurization, unlike the products described in IT 1230785, WO 92/17507, WO 96/14425, WO 97/43317, WO 01/72848 and US 2002/0062019. In addition, novel epiK5-N, O-excess sulfates are obtained which are inactive in aggregation and useful for the preparation of pharmaceuticals, in particular for the preparation of pharmaceutical and cosmetic compositions of antiangiogenic and antiviral activity.

By depolymerization with nitrite of epi K5-N, O- excess sulfate, lmw-epi K5-N, O- excess sulfate, which has no activity in aggregation and has anti-angiogenic and antiviral activity, is obtained.

In preparing N, O-sulphate N-deacetylated derivatives of K5 polysaccharides, wherein the iduronic acid is at least 40% epimerized relative to the total uronic acid and has a low molecular weight according to the method described in WO 01/72848, Depolymerization of the high molecular weight product obtained at the end of the final N-sulfurization step of this process generally results in some depolymerized products that exhibit much lower activity on all aggregation parameters compared to the higher molecular weight products that may occur. Since it generates, it has been confirmed that various results can be obtained. This is expected to occur because degradation by nitrous acid is affected by the presence of sulfate groups. In particular, the sulfate at position 3 of glucosamine is described by Nagasawa et al. as described by in Thrombosis Research, 1992, 65, 463-467 (Nagasawa 1992).

By nitrous acid depolymerization of epiK5-N-sulfate with 20% to 60%, preferably 40% to 60%, and preferably about 50% of total uronic acid, high levels of other biological parameters It has now been found that LMW-epiK5-N-excess sulfate is obtained, which constitutes a new effective intermediate for the production of LMW-epiK5-N, O-excess sulfate with and without activity on the aggregation parameters. . In particular, specific LMW-EpiK5-N consisting of a mixture of about 2,000 to about 4,000 new LMW-EpiK5-N-sulfates, more preferably the predominant compound is disaccharide or dodesaccharide or tetrasaccharide. It has been found that it is possible to depolymerize epiK5-N-sulfate to obtain sulfate. These LMW-epiK5-N-excess sulfates also have interesting biological properties that could not be obtained otherwise, epik5-N, O with anti-angiogenic and / or antiviral activity and surprisingly inactive in aggregation. Useful intermediates for the preparation of excess sulfate.

Treat the LMW-epiK5-N-sulfate with the tertiary or quaternary organic bases mentioned above and maintain the reaction mixture for 30-60 minutes while maintaining the pH at approximately pH 7 with the same organic base, and then O By treating the obtained salt with an O-sulphurizing agent under the conditions of oversulfurization, a novel LMW-epiK5-amine-O-excess sulfate is obtained. By subjecting this LMW-epiK5-amine-O-excess sulfate to N-sulfurization, it is surprisingly inactive in aggregation and has anti-angiogenic and / or antiviral activity and is useful in the manufacture of pharmaceutical and cosmetic compositions New N-sulfurized and O-oversulfurized derivatives (LMW-epiK5-N, O-oversulphate) are obtained.

These LMW-epiK5-N-sulfates are recombinant C5-epimerases immobilized on a solid support, isolated and purified K5-N-sulfate, calcium, magnesium, barium and Starting with an epimerization reaction in the presence of one or more divalent ions selected from manganese; It is obtained by nitrous acid depolymerization of the epimerized product obtained subsequently or in reverse order.

Surprisingly, from observations made in the course of epimerization reactions under the above-mentioned conditions, C2-epimerization of every 2 glues of the normal and "strain group" type of substrate, as opposed to what occurs in the biosynthesis of heparin in nature, is surprising. Epi-K5-N-sulfate, characterized by a repeating tetrasaccharide unit consisting of two glycosamine units followed by glucuronic acid units and separated by glucuronic acid units and vice versa. It is possible to predict that it induces a derivative.

details

Therefore, in accordance with one aspect of the present invention, the present invention provides a process for preparing epiK5-N, O-excess sulfate-derivatives and has the following characteristics:

(a) The (epi) K5-N-sulfate-derivative in acidic form is treated with a tertiary or quaternary organic base, the reaction mixture is left for 30-60 minutes and the addition of the same tertiary or quaternary organic base Maintain the pH of the solution at a value of pH 7 and the salt is isolated with the organic base;

(b) the salt of the organic base of the (epi) K5-N-sulfate-derivative is treated with an O-sulfur oxide under conditions of O-supersulfur oxidation;

(c) The epi-K5-amine-O-excess sulfate-derivative thus obtained is treated with N-sulfurizer, and the epiK5-N, O-excess sulfate-derivative is isolated.

In general, N-acyl- (epi) K5-amine-O-excess sulfate-derivatives are isolated in sodium salts and optionally the sodium salts are transformed into another chemically or pharmaceutically acceptable salt.

As used herein, the term "chemically acceptable" refers to the purification of cations, or products, usable for chemical synthesis, such as sodium ions, ammonium, (C ₁ -C ₄ ) tetraalkylammonium, while " Pharmaceutically acceptable "is self-annotating.

Advantageous cations are those derived from alkali metals, alkaline-earth metals, ammonium, (C ₁ -C ₄ ) tetraammonium, aluminum and zinc. Preferred cations are sodium, calcium and tetrabutylammonium ions.

According to an advantageous process, step (a) is K5 polysaccharide, ie, previously N-deacetylated and N-sulfurized, preferably 100%, optionally 20-60% C5-epimerized and optionally nitrous acid depolymerized. Of, passing through a sodium salt solution of epiK5-N-sulfate-derivative, having an average molecular weight from about 1,000 to about 25,000, advantageously from about 1,500 to about 25,000, for example type IR-120 H ⁺ Through an acid ionic exchange resin of elution, the elution also comprises a washing solution of the resin and is neutralized with tertiary or quaternary organic bases, preferably with aqueous tetrabutylammonium hydroxide solution. The solution is left for 1 hour and the pH is maintained at approximately 7 by adding the same base, and the salt thus obtained is recovered by lyophilization.

In step (b), the O-supersulfuration occurs by using an excess of O-sulfurizer and at a temperature of 20 to 70 ° C. for up to 24 hours in an aprotic polar solvent.

Advantageously, ie, having an average molecular weight of approximately 1,000 to approximately 25,000, advantageously approximately 1,500 to approximately 25,000, as isolated in step (a), is previously N-deacetylated and N-sulfurized and preferably Is a 100%, optionally 20-60% C5-epimerized and optionally nitrous acid depolymerized salt of tertiary or quaternary organic base of epiK5-N-sulfate-derivatives dissolved in dimethylformamide. And 2-10 moles of O-sulphurizer for all free hydroxyls at a temperature of 40-60 ° C. for 10-20 hours. Since the O-sulfurizing agent is advantageously used pyridine.SO ₃ adduct at a mass of 2.5-5 moles, preferably 2.5-4 moles per free hydroxyl per disaccharide, the reaction is the addition of 0.1-1 volume of water. And, preferably, neutralized with sodium hydroxide, precipitated with acetone in saturated sodium chloride solution, filtered and possibly ultrafiltration.

The product thus obtained generally has an average molecular weight of about 3,500 to about 40,000, advantageously about 4,500 to about 40,000 and at least 3.4, advantageously at least 3.5, more advantageously at 3.55 to 4, preferably at 3.55 to 3.8 Sodium salt of epiK5-amine-O-supersulfate-derivative having a degree of sulphation with an iduronic acid content of 20-60% of total uronic acid. The sodium salt thus obtained can be converted to another salt. As an example, exchange with calcium can be carried out in operation with a superfiltration membrane.

In step (c), the (epi) K5-amine-O-excess sulfate-derivatives having a high degree of sulfation are N-acylated using methods known in the literature.

In fact, N-sulfurization is epik5-amine-derived in step (b) with sodium carbonate and an N-sulfurization agent, for example (C ₁ -C ₄ ) trialkylamine.SO ₃ or pyridine.SO ₃ adduct. Treatment with an aqueous solution containing O-supersulfate-derivatives, maintaining the mixture at 30-50 ° C. for 8-24 hours and diafiltration of the desired epiK5-N, O-excess sulfate-derivatives, for example diafiltration. Isolating). Optionally, the N-acylation step is repeated until at least 95% of total substitution is obtained.

The novel epiK5-N, O-excess sulfate-derivatives thus obtained are generally in their sodium salt form. The sodium salt can be converted to another chemically or pharmaceutically acceptable salt. In particular, advantageous salts are those of alkali metals, alkaline-earth metals, ammonium, (C ₁ -C ₄ ) tetraalkylammonium, aluminum, and zinc. Salts of sodium, calcium and tetrabutylammonium are preferred.

The starting epiK5-N, O-sulfate-derivatives encountered in step (a) of the process of the invention are derived from K5 polysaccharides, are previously N-deacetylated, virtually 100% N-sulphated, , 20-60%, advantageously 40-60% C5-epimerized, and optionally depolymerized with nitrous acid, having an average molecular weight of about 1,000 to about 25,000, advantageously about 1,500 to about 25,000. Preferably, the material has an average of epi-K5-N-sulfate having an average molecular weight of 10,000 to 25,000 μs or about 1,000 to about 12,000, advantageously about 1,000 to about 10,000, preferably about 1,500 to about 8,000 LMW-epiK5-N-sulfate having a molecular weight.

EpiK5-N-sulfate prepared by C5-epimerization of K5-N-sulfate is well known in the literature, for example WO 92/17057, WO 01/72848, WO 98/14425, WO 97/43317 or US Widely described in 2002/0062019. Their preparation by C-5 epimerization of glucuronic acid units of K5-N-sulfate with D-glucuronyl C5 epimerase has been described in the literature cited herein.

LMW epiK5-N-sulfuric acid having an iduronic acid unit content of approximately 20%, obtained for C5-epimerization and N-sulfurization, N-deacetylation of a moiety of K5 with an average molecular weight of 5,000 is disclosed in WO 92. Although described in / 17507, this product contains a significant mass of acetyl groups.

EpiK5-N-sulfate having an iduron acid content of 40-60%, which is particularly advantageous as starting material, is obtained by epimerization of K5-N-sulfate which is virtually free of acetyl groups and in turn is prepared from particularly pure K5, in particular WO 02 It does not contain a lipophilic substance described in / 068477. According to a preferred process, K5-N-sulfate by epimerization is used to obtain from K5 free of lipophilic substances such as those described in WO 02/068477, and C5 epimerization is at least one divalent selected from calcium, magnesium, barium and manganese. Immobilized, purified and isolated in a solid support at a pH of about 7 for 12-24 hours and at a temperature of approximately 30 ° C. in the presence of ions.

Especially as a starting material in the preparation of LMW-epi K5-N-Osulfate derivatives having a higher content of iduronic acid units at 40-60%, preferably 50-55% It is a particularly advantageous new material.

As shown herein, LMW-EpiK5-N-sulfate is a K5-N-sulfate in either order;

(Iii) immobilized on a solid support or purified and isolated in solution for about 12-24 hours at a temperature of approximately pH 7, at a temperature of approximately 30 ° C. and in the presence of one or more divalent ions selected from calcium, magnesium, barium unmanganese C5-epimerization with D-glucuronyl C5-epimerase; And

(Ii) generally produced by a process characterized in that reduction to sodium borohydride is subject to optionally subsequent nitrous acid depolymerization.

The expression “in any order” means that the process is followed by reduction in directions (i) to (ii), ie in this turn, and in the reverse direction, ie optionally to sodium borohydride, and subsequently to C5-epimerization under these conditions. It is also shown that in the directions (ii) to (iii), which K5-N-sulphate faces for the first time in the nitrous acid depolymerization reaction, which is confronted with all of them. The preferred order is directions (i) to (ii). Steps (ii) to (iii) are preferably used starting with LMW-K5-N-sulfate having an average molecular weight of at least 4,000, preferably starting at approximately 6,000. For example, starting with 1 g of epiK5-N-sulfate, one determines the amount of sodium nitrite, and in order to obtain useful intermediates for the production of LMW epiK5-N, O- excess sulfate, at least 4,000, in particular 6,000 It is possible to obtain lmw-epi K5-N-sulfate having the above average molecular weight. In fact, in this case, the maximum percentage of epimerization in step (ii) is obtained.

According to a preferred aspect of the present invention, C5-epimerase is immobilized on an inert solid support.

According to Campbell 1994, WO 98/48006, Jin-Ping 2001 or Crawford 2001, for example, preferably purified, isolated, recombinant C5-epimerase is present in the presence of a substrate, ie, at least 4,000, It is preferably immobilized on an inert support in the presence of the initiating K5-N-sulfate-derivative or in the presence of LMW-K5-N-sulfate, advantageously having an average molecular weight of at least 6,000. Immobilization is performed according to conventional methods, for example as described in WO 01/72848.

C-5 epimerization reactions range from 0.001 to 10 g of substrate (preferably at least 4,000, in particular K5-N-sulfate or LMW-K5-N-sulfate, having an average molecular weight of at least 6,000) and from 1.2 × 10 ⁷ to immobilized enzyme. Recycling 20-1,000 ml of 25 mM HEPES solution at approximately pH 7 containing cations selected from calcium, magnesium, barium and manganese at a concentration of 10-60 mM through a column containing 3 × 10 ¹¹ cpm and 12-24 For hours, it is advantageously carried out by maintaining approximately pH 7 at approximately 30 ° C. at a flow rate of 30-220 ml / hour for 15-24 hours.

Preferably the solution is recycled at a flow rate of approximately 200 ml / hour overnight (15-20 hours). The resulting product is separated and purified according to known methods, for example ultrafiltration and precipitation with ethanol. The product thus obtained includes one of epiK5-N-sulfate (and in this case dissolved and depolymerized in water) or LMW-epiK5-N-sulfate (in this case it forms the final product). The amount of iduronic acid units in terms of epimerized fulcent, actually glucuronic acid units, is calculated by ¹ H-NMR according to the method described in WO 96/4425.

The nitrous acid depolymerization reaction is carried out according to the well-known method by depolymerization of heparin, for example in EP 373189, according to the method described in WO 82/03627 or by depolymerization of K5-N-sulfate described in EP 544592. And epi-K5-N-sulfate or K5-N-sulfate containing up to 0-10%, preferably up to 5% acetyl group. Preferably, depolymerization carried out with sodium nitrite and hydrochloric acid in epiK5-N-sulfate free of acetyl groups leads to in situ reduction with sodium borohydride.

Indeed, the cooled aqueous solution of epiK5-N-sulfate is brought to sodium nitrite while keeping the acidic pH (approx. 2) with hydrochloric acid and still cooled and the temperature (approx. 4 ° C.) and pH (approx. 2) constant. At the end of depolymerization (approximately 15-30 minutes), the solution is neutralized with sodium hydroxide and still treated with an aqueous sodium borohydride solution at approximately 4 ° C. At the end of the reduction (approximately 4 hours), the excess sodium borohydride is destroyed with hydrochloric acid, the solution is neutralized with sodium hydroxide, and the depolymerized (and reduced) product is known directly, for example with ethanol or acetone. It is isolated by sedimentation. The product obtained at the end of the depolymerization is in particular in the case of having an average molecular weight of at least 4,000, in particular at least 6,000, or utilized to prepare LMW-K5-N, O- oversulfurized with antiangiogenic and antiviral activity. lmw-epiK5-N-sulfate (in this case it constitutes a termination product) or lmw-K5-N-sulfate (and in this case directly after isolation or in solution without isolation in advance, as shown here) C5-epimerized). By appropriately controlling the depolymerization reaction, in particular utilizing the distinct amounts of sodium nitrite / hydrochloric acid, LMW-K5-N-sulfate or LMW-epiK5-N-sulfate is the anomeric of glucosamine in the polysaccharide chain. from about 1,500 to about 12,000, advantageously from about 1,500 to about 10,000, calculated in ¹³ C-NMR through the integration of the signal contributed to C2 of 2,5-anhydromannitol with an (anomeric) cation Preferably, those having an average molecular weight at an overall difference of about 1,500 to about 7,500 are obtained.

According to a general process starting with 1 g of K5-sulfate or epiK5-N-sulfate, for example, the starting material is dissolved in 100-200 ml of deionized water and temperature controlled at 4 ° C. The amount of sodium nitrite is then added to obtain an average molecular weight of the desired, for example, about 2,000 to about 4,000. Therefore, starting with EpiK5-N-sulfate having a molecular weight of 20,000 as determined by HPLC method equipped with a BioRad BioSil 250 column, and utilizing a known molecular weight heparin standard, it is possible to use from 330 to 330 of sodium nitrite dissolved in 0.2% aqueous solution. Will require the addition of 480 mg. The solution containing sodium nitrite and epiK5-N-sulfate, maintained at 4 ° C., is brought to pH 2 through the addition of 0.1N HCl cooled to 4 ° C. It is reacted under slow stirring for 20-40 minutes and then neutralized with 0.1N NaOH. The product obtained is warmed to room temperature and treated with a reducing agent such as, for example, sodium borohydride (250-500 mg dissolved in 50-100 ml of water) and reacted for 4-8 hours. Excess sodium borohydride is removed with 0.1 N HCl to a pH of 5-5,5 and left for an additional 2-4 hours. Finally, it is neutralized with 0.1 N NaOH and the product is recovered by precipitation with acetone or ethanol after obtaining the concentrated product through evaporation at reduced pressure.

Similarly, the amount of sodium nitrite, starting with 1 g of K5-N-sulfate or epiK5-N-sulfate, has an average molecular weight of about 4,000 to about 12,000, advantageously about 4,000 to 7,500, in particular 6,000 to 7,000 It can be determined to enable the acquisition of lmw-epiK5-N-sulfate or lmw-K5-N-sulfate with.

Have an average molecular weight of about 1,500 to about 12,000, advantageously about 1,500 to about 10,000, preferably about 1,500 to about 7,500, virtually free of NH ₂ and N-acetyl groups and of 20 to 60%, advantageously 40 The thus obtained LMW-epi K5-N-sulfate and their chemically or pharmaceutically acceptable salts having an iduron acid content of from 60% to 60%, preferably 50-55%, are LMW-epi K5-N, O- Not only are new products useful as starting materials of particular interest in the preparation of excess sulfate, but also those useful as active ingredients of pharmaceutical or cosmetic compositions, and which form a further aspect of the present invention.

Advantageously, the starting material in the preparation of the epiK5-N, O- excess sulfate-derivatives of the invention is an epiK5-N-sulfate-derivative consisting of a chain mixture, wherein at least 90% of the chains are represented by Have,

Wherein the uronic acid unit comprises 20-60% of the iduronic acid, n is an integer from 2 to 100, advantageously from 3 to 100, and the corresponding cation is chemically or pharmaceutically acceptable. More advantageously, the starting epiK5-N-sulfate-derivative consists of a chain mixture, the uronic acid unit comprises 40-60% of the iduronic acid, n is from 2 to 100, advantageously from 3 to 100 Is an integer and the corresponding cation is chemically acceptable. Preferred starting materials are such LMW-epiK5-N-sulfates consisting of a chain mixture, wherein at least 90% of the chains have the formula I, wherein the uronic acid unit is 40-60% of the iduronic acid, preferably 50 to 55%, n is an integer from 2 to 20, advantageously from 3 to 15, and the corresponding cation is chemically acceptable.

In fact, the LMW-epiK5-N-sulfate consists of a chain mixture, wherein at least 90% of the chains have the formula I ',

Wherein the uronic acid unit comprises 40-60%, advantageously 40-60%, preferably 50-55%, of iduronic acid, q is an integer from 2-20, and the corresponding cation is chemically or pharmaceutical Academicly acceptable.

As used herein, the term “chemically” refers to sodium, ammonium, (C ₁ -C ₄ ) tetraalkylammonium ions, or cations useful for chemical synthesis or for purification of the product.

Advantageous cations are those derived from alkali metals, alkaline-earth metals, ammonium, (C ₁ -C ₄ ) tetraalkylammonium, aluminum and zinc. Preferred cations are sodium, calcium and tetrabutylammonium ions.

Of particular interest is LMW-epiK5-N-sulfate consisting of a chain mixture, in which at least 90% of the chains are subjected to nitrous acid depolymerization of the corresponding epiK5-N-sulfate and subsequent possible reduction to, for example, sodium borohydride. Obtained by the above formula (I '). Of these, preferred are novel LMW- (epi) K5-N-sulfates consisting of a chain mixture, with the predominant species having the general formula (I'a),

Here, the uronic acid unit consists of 60-40% of glucuronic acid and 40% -60% of iduronic acid, and p is an integer of 4-8. The average molecular weight of these products is from about 2,000 to about 4,000 and the corresponding cations are chemically pharmaceutically acceptable.

The origin of these epiK5-N-sulfates from the step of nitrous acid depolymerization is the reduction of 2,5-anhydromannose units at most reducing ends of the chain in the chain mixture to, for example, sodium borohydride. In the presence of 2,5-mannmann anhydride of the following structure (a),

Here, X represents a formyl group or a hydroxymethyl group. Therefore, the reducing end of most (60-70%) of the chain is in fact represented by structure (b),

Where X is as defined above.

Since certain sulfateizations will involve the possible introduction of one or two sulfate groups, which do not significantly change the degree of sulfation of the O-oxidized derivatives, the presence of structure (a) is known as epiK5-N-sulfate and their It does not affect the chemical properties of the derivative. According to the process of the present invention, the LMW-epiK5-N-sulfate is sulfated and acylated, where the influence of 2,5-anhydrous mannose lycal of structure (a) is unknown in the formyl group where X represents formyl. Since the reaction is faced, it is preferred that the nitrous acid depolymerization is followed by a reduction to, for example, sodium borohydride. In addition, the presence of structure (a) is shown by the Øtergaard P.B. et al. As exemplified by Thrombosis Research, 1987, 45, 739-749 (Øtergaard 1987), it does not affect the biological activity of the product.

Particularly advantageous LMW-epiK5-N-sulfate according to the invention consists of a chain mixture, wherein the predominant species is a compound of formula I'b,

Wherein X is formyl or preferably hydroxymethyl, m is 4,5 or 6 and the corresponding cation is one chemically or pharmaceutically acceptable ion, glucuronic acid unit and uronic acid unit Are all of the glucuronic acid or the glucuronic acid unit and the iduronic acid unit are alternatively present, starting with the glucuronic acid unit or the iduronic acid unit. In this case, the glucuron / iduron ratio is 45/55 to 55/45, approximately 50/50.

Therefore, the use of C5-epimerase, preferably recombined, preferably immobilized on a solid support under these conditions, is not a common form, but as Kk-N-sulfate-derived epi-K5-N-sulfate-derived as occurs in nature No “strain group” epimerization of N-sulfate-derivatives is possible.

Therefore, according to another aspect of the present invention, the present invention relates to two glucosamines separated by glucuronic acid units and followed by iduronic acid units or separated by iduronic acid units and by subsequent glucuronic acid units. Provides the use of isolated and purified C5-epimerase for the conversion of K5-N-sulfate-derivatives to the corresponding epiK5-N-sulfate-derivatives, characterized by repetitive tetrasakalite units consisting of units. do.

The epimerization occurs best if performed on a K5-N-sulfate-derivative having an average molecular weight of at least 4,000, preferably of 6,000 to 7,500.

According to the invention, preferably at least 90% of the chains are 100% N-sulphated starting epi-K5-N-sulfate-derivatives (especially epiK5-N-sulfate-derivatives consisting of a chain mixture) Or a species having or predominantly I 'and having formula I'a or I'b, wherein X is hydroxymethyl), resulting in the isolation of the corresponding, novel epiK5-amine-O- excess sulfate-derivative In which steps (a) and (b) are encountered, wherein the amines are generally unsubstituted in the form of sodium salts and can be transformed here with another chemically or pharmaceutically acceptable salt. . Particularly advantageous salts are those of alkali metals, alkaline-earth metals, ammonium, (C ₁ -C ₄ ) tetraalkylammonium, aluminum, and zinc, among which salts of sodium, calcium and tetrabutylammonium are preferred.

Therefore, according to another aspect, the present invention relates to (a) in the acid form, wherein (epi) K5-N-sulfate-derivatives are treated with tertiary or quaternary organic bases, and the reaction mixture is subjected to a period of 30-60 minutes. Left to stand, and the addition of the tertiary or quaternary organic base maintains the pH of the solution at a value of approximately 7 and its salt is isolated with the organic base; (b) the salt of the organic base of the (epi) K5-N-sulfate-derivative is treated with an O-sulphurizing agent under conditions of O-supersulfuration and the (ep) K5-amine-O-excess sulfate-derivative is Novel (epi) K5-amine-O-supersulfate-derivatives and their chemically or pharmaceutically acceptable salts, which can be obtained by a process characterized in that they are isolated.

As an advantageous starting material of step (a), at least 90% of the chains consist of 20 to 60% of uronic acid units consisting of iduronic acid, n is an integer from 3 to 100, and the corresponding cation is chemically or pharmaceutical With an epiK5-N-sulfate-derivative consisting of a chain mixture having the above formula (I), which is acceptable, at the end of step (b), the epiK5-amine-O- excess sulfate-derivative is 90 It is obtained that at least% consists of a chain mixture having the formula (II).

Wherein uronic acid units from 20 to 60% is made of a biceps acid, n is 2 an integer of 1 to 100, preferably an integer from 3 to 100, R, R ', and R "is hydrogen or SO ₃ ^- and The degree of sulfation is at least 3.4, advantageously at least 3.5, more advantageously from 3.55 to 4, preferably from 3.55 to 3.8 and the corresponding cation is chemically or pharmaceutically acceptable.

These epiK5-amine-O- excess sulfate-derivatives with very high degrees of sulfation are basically N-sulfates or N- (C ₂ -C ₄₎ , which have basically no coagulation index activity but have other interesting pharmaceutical properties. It is a new product useful as an intermediate in the preparation of acylated derivatives.

Advantageous epiK5-amine-O-supersulfate-derivatives with very high degrees of sulfation consist of a chain mixture in which at least 90% of the chains have the formula (II) with the following conditions: In formula (II), 40 to 60% of uronic acid units are biceps It is advantageously about 4,500-40,000 with n being 2 to 100, preferably an integer of 3 to 100, an average molecular weight of about 2,000 to 40,000, and R of at least 40%, preferably 50 to 80% of SO ₃ ⁻ , R ′ and R ″ are both SO ₃ ⁻ or one is hydrogen and the other is 5 to 10% of SO ₃ ⁻ in glucuronic acid monosulfate and 10 to 10 in iduronic acid monosulfate 15% SO ₃ ⁻ , the degree of sulfation is greater than 3.4, advantageously at least 3.5, more advantageously from 3.55 to 4, preferably from 3.55 to 3.8, and the corresponding cation is chemically or pharmaceutically acceptable It is possible.

Preferred epiK5-amine-O-supersulfate-derivatives with very high degrees of sulfation are LMW-epiK5-amine-O-supersulfates consisting of a chain mixture in which at least 90% of the chains have the formula II: In formula (II), 40 to 60% of uronic acid units, preferably 50 to 55%, consist of iduronic acid and R is at least 40%, advantageously 50 to 80%, preferably about 65% of SO ₃ ^-, R 'and R "is SO ₃ both a ^- or one is hydrogen and the other is Glu SO ₃ in 5 to 10% in the ku acid ^- and, in the biceps acid 10 to 15% SO ₃ ^-, and n is An integer of 2 to 20, advantageously 3 to 15, an average molecular weight of about 4,000 to 8,000, and the corresponding cation is chemically or pharmaceutically acceptable.

Indeed, the preferred LMW-epiK5-amine-O-excess sulfate-derivatives described above consist of a chain mixture in which at least 90% of the chains have the formula II '.

Wherein uronic acid unit is from 20 to become 60% composed of biceps acid, q is 2 to 20, and particularly 3 to 15 integer glass, R, R ', and R "is hydrogen or SO ₃ ^-, and The degree of sulfation is at least 3.4, advantageously at least 3.5, more advantageously from 3.55 to 4, preferably from 3.55 to 3.8, and the corresponding cation is a chemically or pharmaceutically acceptable ion.

Of these LMW-epiK5-amine-O-excess sulfates, the predominant species preferably consists of a chain mixture having the formula II'a:

Wherein the uronic acid unit consists of 20 to 60% of iduronic acid, p is an integer from 4 to 8, R, R ', and R "are as described above, and the degree of sulfation is at least 3.4, advantageously Is at least 3.5, more preferably 3.55 to 4, preferably 3.55 to 3.8, and the corresponding cation is a chemically or pharmaceutically acceptable ion.

The origin of the new LMW-epi K5-amine-O-excess sulfate from the LMW-epi K5-N-sulfate obtained by depolymerization of nitrogen and continuous reduction reactions, for example with sodium borohydride or the like, reduced the number of parts of the chain in the chain mixture. At the ends includes the presence of sulfated 2,5-mannus anhydride of formula (a ').

Wherein R ″ represents hydrogen or SO ₃ ⁻ .

Thus, the reducing ends of the majority of the chains in the chain mixture are represented by formula (b ').

In the above formula, the uronic acid unit may be glucuron or iduron.

Of the novel LMW-epi K5-amine-O-excess sulfate, the predominant species preferably consists of a mixture which is a compound of formula II'b.

Wherein the uronic acid unit consists of 40 to 60% of iduronic acid, m is 4, 5, or 6, R, R ', and R "are hydrogen or SO ₃ ^- and X" is OH or OSO ₃ ⁻ , the degree of sulfation is at least 3.4, advantageously at least 3.5, more advantageously from 3.55 to 4, preferably from 3.55 to 3.8, and the uronic acid units are present alternately starting with glucuron or iduronic acid units And the corresponding cation is a chemically or pharmaceutically acceptable ion.

All of these epiK5-amine-O- excess sulfate-derivatives with very high degrees of sulfation are new products useful as useful intermediates for the preparation of new N-substituted epiK5-amine-O- excess sulfate-derivatives, Constitute an additional perspective of

In particular, according to another aspect, the present invention relates to the above having a very high degree of sulfidation for the production of new N-substituted, especially N-sulfurized or N-acylated epiK5-amine-O-excess sulfate-derivatives. It relates to the use of one epiK5-amine-O-excess sulfate-derivative.

EpiK5-amine-O- excess sulfate-derivatives obtained at the end of step (b) (in particular, epiK5-amine-O- excess sulfate-derivatives have at least 90% of the chains having the formula II or II ', or At the end of step (c) of the process of the invention, consisting of N-sulfurization of a chain mixture having the formula II'a or II'b, the iduronic acid content is 20 to 60% of the total uronic acid, EpiK5-N, O-excess sulfate-derivatives having a degree of sulfation of 4 or more, preferably 4 to 4.6, are obtained.

Thus, according to another aspect, the present invention relates to iduron acid with at least 20% of the total O-sulfurized and N-sulfurized uronic acid units having an average molecular weight of about 2,000 to 45,000 and having a degree of sulfation of 4 or more. Provided are new N-deacetylated derivatives of C5-epimerized K5 polysaccharides, which are basically inert at the coagulation index.

Similar to the foregoing, the new derivatives are collectively referred to by the general term “epi K5-N, O-oversulfate-derivatives” regardless of their molecular weight.

In particular, these derivatives have an average molecular weight of about 2,000 to 45,000 since they are derived from epi-K5-N-sulfate obtained by N-deacetylation and N-sulfurization of K5 by fermentation or by the latter nitrogen depolymerization.

By controlling the nitrogen depolymerization, it is possible to obtain low molecular weight derivatives at substantially all of the above intervals. However, in order to use the derivative of the present invention as a pharmaceutical or cosmetic product, the average molecular weight is about 2,000 to 16,000, advantageously 3,500 to 13,000, the molecular weight distribution is about 1,000 to 15,000, preferably about 4,500 to 9,000 It is advantageous to prepare low molecular weight derivatives, low molecular weight derivatives having a molecular weight distribution of about 2,000 to 10,000 or high molecular weight derivatives derived from unfractionated K5 having an average molecular weight of about 20,000 to 45,000 and a molecular weight distribution of about 2,000 to 70,000. Do.

In the epiK5-N, O-excess sulfate-derivative of the present invention, the degree of sulfation is very high, preferably 4 to 4.6, and the nitrogen of glucosamine is substantially 100% sulfated. Further, considering that the degree of sulfation is 4 or more, the epiK5-N, O-excess sulfate-derivatives are 100% 6-O-sulfated and 50-80% 3-O-sulfate in their glucosamine units. In the glucuronic acid unit, 5 to 10% mono-sulfated, 10 to 15% in the iduronic acid unit, and mono-sulfurized, and the remaining uronic acid unit is 2,3-di-O. Sulfurized.

Advantageous epiK5-N, O-oversulfate-derivatives according to the invention consist of at least 90% of the chains, 20 to 60% of uronic acid units consisting of iduronic acid, n is from 2 to 100, advantageously from 3 to 100 EpiK5-amine-O- excess sulfate-, in turn, prepared from an epiK5-N-sulfate-derivative consisting of a chain mixture having the above formula (I) wherein the corresponding cation is chemically or pharmaceutically acceptable Obtained via a derivative.

In this case, the new epiK5-N, O-excess sulfate-derivative consists of a chain mixture in which at least 90% of the chains have the formula III.

Wherein uronic acid unit is from 20 to 60% is made of a biceps acid, n is 2 an integer of 1 to 100, preferably an integer from 3 to 100, R, R ', and R "is hydrogen or a SO ₃ ^- Z is SO ₃ ⁻ , a degree of sulfation is at least 4, preferably 4 to 4.6, and the corresponding cation is chemically or pharmaceutically acceptable.

The cations are advantageously cations of alkali metals, alkaline-earth metals, ammonium, (C ₁ -C ₄ ) tetraalkylammonium, aluminum and zinc, of which salts of sodium, calcium and tetrabutylammonium are preferred.

Among derivatives, all of which are at least 90% of the chain, R is a chain of 50 to 80%, preferably from about 65% SO ₃ ^{- -} the new epi K5- amine -N, O- excess sulfate, and the degree of sulfuric acid Is 4 or more, advantageously 4 to 4.6, preferably 4 to 4.3, consisting of a chain mixture having the above formula III.

Advantageous epiK5-N, O-extrasulfate-derivatives with very high degrees of sulfation consist of a chain mixture in which at least 90% of the chains have the formula III in which: Z is SO ₃ ^- and uronic acid 40 to 60% of the unit consists of iduronic acid, n is an integer of 2 to 100, preferably 3 to 100, an average molecular weight of about 2,000 to 45,000, advantageously about 4,500 to 45,000, and R is at least 40% , Preferably 50 to 80% SO ₃ ⁻ , R ′ and R ″ are both SO ₃ ^−, or one is hydrogen and the other is 5 to 10% SO ₃ ⁻ in glucuronic acid monosulfate, biceps 10 to 15% SO ₃ ⁻ in lonic acid monosulfate, a degree of sulfation of 4 or more, advantageously 4 to 4.6, and the corresponding cation is chemically or pharmaceutically acceptable.

Preferred N-substituted epiK5-amine-O-excess sulfate-derivatives are LMW-epiK5-amine-O-excess sulfates consisting of a chain mixture in which at least 90% of the chains have the following conditions: In which 40 to 60%, preferably 50 to 55% of uronic acid units comprise iduronic acid and R is at least 40% and advantageously 50 to 80% of SO ₃ ⁻ and , R 'and R "are both SO ₃ ^- or one is hydrogen and the other is 5 to 10% SO ₃ ^- in glucuronic acid, 10 to 15% SO ₃ ^- in iduronic acid, Z is 100% SO ₃ ^- or (C ₂ -C ₄ ) acyl, n is an integer from 2 to 20, advantageously from 3 to 15, an average molecular weight is from about 4,000 to 8,500, and the corresponding cation is chemically or pharmaceutically It is acceptable.

In fact, the preferred epiK5-N, O-sulfate-derivatives with very high degrees of sulfation consist of a chain mixture in which at least 90% of the chains have the formula III ':

Wherein the uronic acid unit consists of 20 to 60% of iduronic acid, q is an integer from 2 to 20, advantageously from 3 to 15, and R, R ', and R "are hydrogen or SO ₃ ^- groups , Z is SO ₃ ⁻ , a degree of sulfation is at least 4, preferably 4 to 4.6, and the corresponding cation is a chemically or pharmaceutically acceptable ion.

Of particular interest are chain mixtures of the formula III 'with the following conditions: In the formula III', the uronic acid unit consists of 40 to 60%, preferably 50 to 55% of iduronic acid and R is at least 40% Preferably 50 to 80%, preferably about 65% SO ₃ ⁻ , R ′ and R ″ are both SO ₃ ^−, or one is hydrogen and the other is 5 to 10% SO ₃ in glucuronic acid. ^- and, in the biceps acid 10 to 15% SO ₃ ^-, and n is 2 to 20, advantageously from 3 to 15 and an average molecular weight of about 2,000 to 16,000, advantageously about 3,500 to about 13,000, preferably about 4,500 And 9,000, and the corresponding cation is one that is chemically or pharmaceutically acceptable.

Among these LMW-epi K5-N, O-excess sulfates, the predominant species preferably consists of a chain mixture having the formula III'a:

Wherein 20 to 60% of the uronic acid unit consists of iduronic acid, p is an integer from 4 to 8, Z is SO ₃ ⁻ , R, R ′, and R ″ are hydrogen or SO ₃ ⁻ , The degree of sulfation is at least 4, preferably from 4 to 4.6, and the corresponding cation is chemically or pharmaceutically acceptable.

The origin of the new LMW-epiK5-N, O-excess sulfate from the LMW-epiK5-sulfate obtained by depolymerization of nitrogen and continuous reduction reactions, for example with sodium borohydride, etc. the illustrated structural formulas (a ') sulfated 2,5-mannitol, anhydrous unit of the above (wherein R "is hydrogen or SO ₃ ^- represents a) a presence of a.

Thus, the reducing ends of the majority of the chains in the chain mixture are represented by the following structural formula (b ").

Wherein Z is SO ₃ ⁻ , and the uronic acid unit is glucuron or iduron.

Of the new LMW-epi K5-N, O-excess sulfate, the predominant species preferably consists of a mixture which is a compound of formula III'b.

Wherein R, R, R ', and R "is hydrogen or SO ₃ ^- and, Z is SO ₃ ^- and, X" is OH or OSO ₃ ^-, and, m is 4, and 5, or 6, sulfuric degree 4 Or more, preferably 4 to 4.6, the uronic acid units are present alternately starting with glucuron or iduronic acid units, and the corresponding cations are chemically or pharmaceutically acceptable ions. The cations are advantageously alkali metals, alkaline-earth metals, ammonium, (C ₁ -C ₄ ) tetraalkylammonium, aluminum, and zinc cations, of which ions of sodium, calcium, and tetrabutylammonium are preferred. .

EpiK5 is pre-N-deacetylated, usually 100% N-sulfated, 20 to 60% C5-epimerized and depolymerized at the beginning of the process according to the invention, ie at the end of step (c) If used as a non-K5 polysaccharide, epiK5-N, O-excess sulfate is separated and depolymerized under nitrogen and subsequently reduced with sodium borohydride or the like to yield the corresponding LMW-epiK5-N, O-excess with the same degree of sulfation Sulphate can be obtained. In particular, at least 90% of the chains, 20-60% of the uronic acid units are made of iduronic acid, q, R, R ', R ", and Z have the meaning as described above, the degree of sulfation is 4 or more, Preferably 4 to 4.6 and the corresponding cation is chemically or pharmaceutically acceptable, an LMW-epiK5-N, O-excess sulfate, consisting of a chain mixture having the formula III 'or III'a In this case, the origin of these LMW-epiK5-N, O-excess sulfates obtained by depolymerization reaction and continuous reduction reaction with, for example, sodium borohydride or the like can be obtained by the following structural formula ( a "), the presence of 2,5-anhydrous manno units.

Wherein X is formyl or hydroxymethyl and R ″ is hydrogen or SO ₃ ⁻ .

The new epiK5-N, O-oversulfate-derivatives, especially their salt forms, are highly anionic products capable of trapping free radicals and are used in the cosmetic industry as a supplement to make "anti-aging" creams or to prevent hair loss. It is also useful in the pharmaceutical industry as a therapeutic agent for dermatitis. In addition, the epi-K5-N, O- oversulfate-derivatives of the present invention, especially LMW-epi K5-N, O- oversulfate, have anti-angiogenic and anti-viral activity, so that the active ingredient for pharmaceutical preparation Configure.

Thus, in one of the further aspects, the present invention, in admixture with a pharmaceutical additive, comprises one of the epiK5-N, O-excess sulfate-derivatives or pharmaceutically acceptable salts thereof as described above as one of the active ingredients. It provides a pharmaceutical composition comprising a pharmaceutically active content of.

In the pharmaceutical compositions of the invention for oral, subcutaneous, intravenous, transdermal, or topical administration, the active ingredient is preferably administered in the form of a dosage unit in admixture with traditional pharmaceutical additives or excipients. Dosages can vary widely depending on the age, weight and health of the patient. This dosage includes administration of 1 to 1000 mg, advantageously 10 to 750 mg, preferably 250 to 500 mg once or three times daily by intravenous, subcutaneous, oral, transdermal, or topical administration.

The pharmaceutical composition of the present invention is formulated with traditional additives suitable for administration of other methods. Particularly advantageous are preparations in the form of creams, ointments, topical drugs, gels, foams, balsams (aromatic ointments, balsam), vaginal suppositories, suppositories, solutions, or suspensions suitable for topical administration.

Advantageously, the composition according to the invention is mixed with a pharmaceutical additive and, as one of the active ingredients, starts from the epiK5-derivatives according to steps (a), (b) and (c) of the above-described process, EpiK5-N, which can be obtained starting from unpolymerized epiK5 according to steps (a), (b), and (c) of the above process and optionally further proceeding with subsequent nitrogen depolymerization after step (c), O-supersulfate-derivatives or one of its pharmaceutically acceptable salts. Advantageously, the epiK5-N, O-excess sulfate-derivative is the predominant species a compound of formula III'a or III'b, or at least 90% of the chains consist of a chain mixture having formula III or III '. Preferred active ingredients have a degree of sulfation of 4, preferably from 4 to 4.6, advantageously having an average molecular weight of from about 3,500 to 11,000, more advantageously from about 3,500 to 5,200 and basically free of N-acetyl groups LMW-epiK5 -N, O- excess sulfate.

Finally, in another aspect, the present invention provides a cosmetic composition comprising, in admixture with a cosmetic additive, an effective amount of one of the EpiK5-N, O-excess sulfate-derivatives or pharmaceutically acceptable salts thereof. .

Salts selected from the group consisting of salts of sodium, potassium, calcium, magnesium, aluminum, and zinc of the epiK5-N, O-supersulfate-derivatives, in particular the predominant species are compounds of formula III'a or III'b or Salts consisting of a chain mixture of at least 90% of which have the formula III or III 'constitute an active active ingredient of the pharmaceutical or cosmetic composition of the invention.

The following examples are intended to illustrate the invention, but are not intended to limit it.

Preparation Method I

Preparation of K5 Polysaccharides from E. Coli

First fermentation is carried out in Erlenmeyer flasks using the following medium:

Defatted soybean meal 2 g / ℓ

K ₂ HPO ₄ 9.7 g / ℓ

KH ₂ PO ₄ 2 g / ℓ

MgCl ₂ 0.11 g / L

0.5 g / l sodium citrate

Ammonium Sulfate 1 g / L

2 g / l glucose

Hot spring water 1000 ml

pH = 7.3

The medium is sterilized at 120 ° C. for 20 minutes. Glucose is prepared separately in the form of a sterile solution at 120 ° C. for 30 minutes and added to the medium in a sterile manner. A suspension of Escherichia coli cells Bi 8337/41 (O10: K5: H4) obtained from a slope maintained in Tryptic Soy Agar is inoculated into a Erlenmeyer flask and incubated at 37 ° C. for 24 hours under controlled stirring (160 rpm, 6 cm stroke). . Bacterial growth is measured by counting cells using a microscope. In a continuous process, inoculate 0.1% of the culture solution of the Erlenmeyer flask in a 14 l Chemap-Braun fermenter containing the same medium as above, and stir at 400 rpm at 37 ° C. for 18 hours, 1 vvm (vvm = air per liquid volume per minute) Fermentation is carried out by air injection. During fermentation, pH, oxygen, glycosic acid balance, resulting K5 polysaccharides, and bacterial growth are measured. Cells are separated from the media by centrifugation at 10,000 rpm, and the supernatant is ultrafiltered using an SS316 (MST) module immobilized with a PES membrane with nominal cut off of 800 and 10,000 D to reduce its volume to 1/5. . The K5 polysaccharide is then precipitated by adding four times the volume of acetone at 4 ° C. and left overnight at 4 ° C. The polysaccharides are recovered by centrifugation or filtration for 20 minutes at 10,000 rpm. The deproteinization of the obtained solid phase was carried out in Aspergillus duck in a buffer of 0.15 M EDTA and 0.1 M NaCl at pH 8 containing SDS (0.5% sodium dodecyl sulfate) (filtrate 10 mg / L) for 90 minutes at 37 ° C. From type 2 protease. The resulting solution is ultrafiltered on model SS 316 with a membrane of 10,000 D nominal cut off, extracted twice with 1M NaCl and washed with water until the absorbance disappears. K5 polysaccharide is then crystallized with acetone and a yield of 850 mg per liter of fermenter is obtained. The purity of the resulting polysaccharides is determined by determining uronic acid (carbazole method), proton and carbon 13 NMR, UV and protein content. Purity is over 80%.

A Pharmacia 75 HR column with a single moiety with a retention time of about 9 minutes was used using two serial columns of Na ₂ SO ₄ and Bio-Sil SEC 250 (Bio Rad) as mobile phase at a flow rate of 0.5 ml / min. As indicated by HPLC using, the polysaccharide obtained consists of two moieties of different molecular weights of 30,000 and 5,000 D, respectively. The measurement is performed on a standard curve obtained with a heparin moiety having a known molecular weight.

The ¹ H-NMR spectrum of the purified K5 thus obtained shows a different signal due to the methyl of the lipophilic substance.

Preparation II

K5 tablets

100 ml of saturated aqueous sodium chloride solution is maintained at 4 ° C, and 1 g of K5 obtained at the end of Preparation I is dissolved therein, and three times cold isopropanol is added to the obtained solution by volume. The salt concentration of the solution is brought to 3M by addition of the calculated content of saturated sodium chloride solution and the resulting solution is placed in a cold environment (about 4 ° C.) overnight. The precipitate formed is separated by centrifugation at 10,000 rpm for 20 minutes and the product purity is determined by the subsequent ¹ H-NMR spectral test where the purity of the product must be overnight dialysis and no signal in the region below 1.5 ppm. The procedure is dissolved in water, optionally saturated with NaCl, and precipitated with isopropanol. Ultrafiltration to a 10,000D cut-off miniplate millipore membrane until the precipitate is dissolved in water and the salt disappears. As a result, K5 having a purity of 99% or more without traces of lipophilic impurities in the region of 1.5 ppm or less is obtained from the ¹ H-NMR spectrum.

Preparation III

K5-N-sulfate

(i) N-deacetylation

10 g of pure K5 polysaccharide prepared as described in Preparation II is dissolved in 1000 ml of 2N sodium chloride and the resulting solution is left at 60 ° C. for 24 hours. The solution is brought to room temperature and then brought to neutral pH (pH 7) with 6N hydrochloric acid.

(ii) N-sulfurization

Was added to 16g of sodium carbonate to the solution containing the deacetylated K5 is maintained at 40 ℃ four hours after pyridin .SO ₃ ^- is added to 16g. At the end of the reaction after 24 hours, the solution was brought to room temperature and brought to pH 6.5-7 with 5% hydrochloric acid solution. The product is purified from salt by diafiltration using a 1000D spiral wound membrane (prepscale cartridge Millipore). The process is terminated when the permeate conductivity is less than 1000 μS, preferably less than 100 μS. Intradialysis is reduced when the concentration of polysaccharides of 10% is obtained using intradialysis in a concentration dialysis system. The concentrated solution is dried by lyophilization. ¹³ C-NMR spectral analysis revealed no N-acetyl or NH ₂ residues.

Preparation IV

LMW-K5-N-sulfate

The product obtained as disclosed in Example 1, steps (i) and (ii) of WO 02/068477 is depolymerized by decomposition with nitrous acid and continuous reduction of the aldehyde formed. 1 g of K5-N-sulfate is dissolved in 200 ml of distilled water, and it is added to 480 mg of sodium nitrite dissolved in 240 ml of distilled water. The solution is then brought to pH 2 with 0.1 N HCl at 4 ° C. and held for 30 minutes. At the end of the reaction, the solution is brought to pH 7 with 0.1 M NaOH and then brought to room temperature. Then, 450 mg of NaBH ₄ is added to the solution and allowed to react for 4 hours. Excess NaBH ₄ is removed with HCl and the pH is brought to 5-6. The product is neutralized with 0.1 M NaOH, precipitated with 3 times acetone by volume at 4 ° C., filtered with funnel and dried in a 40 ° C. vacuum oven to recover. 900 mg of LMW-K5-N-sulfate having an average molecular weight of about 2,000 consisting of a chain mixture of a dominant paper, a uronic acid unit is a glucuronic acid, and m is a compound of the formula (I'b) of 4 is obtained.

Example 1

LMW-EpiK5-N-Sulfate. Step (i) → (ii)

(i) Epimerization of EpiK5-N-Sulfate

10 g of K5-N-sulfate obtained as disclosed in Example 1, steps (i) and (ii) of WO 02/068477, in which no signal related to acetyl group or NH ₂ appears on the ¹ H-NMR spectrum. It is dissolved in 600 ml of 25 mM HEPES buffer at pH 7, containing CaCl ₂ at a concentration of 50 mM. The resulting solution is recycled through a 50 ml column filled with Sepharose 4B resin containing 5 g of immobilized recombinant C5-epimerase (WO 96/14425) as disclosed in Example 1 of WO 01/72848. . The reaction is carried out at pH 7 with a flow of 200 ml / h at 30 ° C. for 24 hours. The resulting product is purified by ultrafiltration and crystallized with ethanol. EpiK5-N-sulfate is obtained with an iduronic acid content of 54%.

(ii) Depolymerization of EpiK5-N-Sulfate

1 g of the product obtained is dissolved in 25 ml of distilled water and added to 230 mg of sodium nitrite dissolved in 115 ml of distilled water. The solution is then brought to pH 2 with 0.1 N HCl at 4 ° C. and held for 30 minutes. At the end of the reaction, the solution is brought to room temperature to pH 7 with 0.1M NaOH. Then, 450 mg of NaBH ₄ is added to the solution and allowed to react for 4 hours. The product is precipitated with 3 times acetone by volume at 4 ° C., filtered with funnel and recovered by drying in a vacuum oven at 40 ° C. 900 mg of LMW-EpiK5-N-sulfate having 54% iduronic acid content and a molecular weight distribution of 1,000 to 4,000 as determined by HPLC method is obtained.

Example 2

LMW-EpiK5-N-Sulfate. Step (ii) → (i)

(ii) Depolymerization of K5-N-Sulfate

2 g of K5-N-sulfate obtained as disclosed in Example 1, Steps (i) and (ii) of WO 02/068477 were prepared using 100 mg of sodium nitrite and 300 mg of sodium borohydride as described in Preparation I. Depolymerize. 1.8 g of LMW-K5-N-sulfate having an average molecular weight of 5,000 is obtained.

(i) Epimerization of LMW-K5-N-Sulfate

1 g of LMW-K5 N-sulfate obtained in step (ii) was treated as disclosed in step (i) of Example 1. An epimerized product is obtained with an iduronic acid / glucuronic acid ratio of 44/56 relative to a ratio of 0/100 in the starting product with an average molecular weight of 5,000 D and a molecular weight distribution of 2,000 to 10,000. The carbazole method (Bitter and Muir Anal. Biochem. 39, 88-92-1971) measured the content of uronic acid in the standard to yield a yield of 90%.

Example 3

LMW-EpiK5-N-Sulfate. Step (i) → (ii)

(i) Epimerization of K5-N-Sulfate

2 g of K5 N-sulphate obtained as disclosed in Example 1, steps (i) and (ii) of WO 02/068477 are dissolved in 120 ml of 25 mM HEPES buffer at pH 7 containing 50 mM CaCl ₂ . The resulting solution is recycled through a 50 ml column filled with a resin containing immobilized enzyme obtained as disclosed in WO 96/14425. This procedure is carried out at 30 ° C. with a flow of 200 ml / h for 24 hours. The resulting product is purified by ultrafiltration through a 1000D membrane, passed through an ion exchange column IR 120 H ⁺ , and the eluate is neutralized with 1N NaOH. The sample is recovered by crystallization with ethanol and acetone. Epimerized product is obtained with an iduronic acid / glucuronic acid ratio of 55/45 relative to a ratio of 0/100 in the starting product. Epimerization ratio was calculated by ¹ H-NMR according to the method as disclosed in WO96 / 14425. The carbazole method (Bitter and Muir Anal. Biochem. 39, 88-92-1971) measured the content of uronic acid in the standard to yield a yield of 90%.

(ii) Depolymerization of Epi-K5-N-Sulfate

1 g of the product obtained in step (i) is depolymerized by the process of decomposition with nitrous acid and the continuous reduction of the aldehyde formed. In particular, the product is dissolved in 25 ml of distilled water and added to 230 mg of sodium nitrite dissolved in 115 ml of distilled water. The solution is then brought to pH 2 with 0.1 N HCl at 4 ° C. and held for 30 minutes. At the end of the reaction, the solution is brought to room temperature to pH 7 with 0.1M NaOH. Then, 450 mg of NaBH ₄ is added to the solution and allowed to react for 4 hours. The product is precipitated with 3 times acetone by volume at 4 ° C., filtered with funnel and recovered by drying in a vacuum oven at 40 ° C. 900 mg of LMW-epiK5-N-sulfate having a glucuronic acid unit content of 45% and an iduronic acid unit content of 55% and having a molecular weight distribution in the range of 1,000 to 4,000 measured by HPLC method is obtained.

Example 4

Epi K5-N, O-Excess Sulfate

(a) Tetrabutylammonium Salt of EpiK5-N-Sulfate

A solution containing 400 mg of EpiK5-N-sulphate obtained at the end of step (i) of Example 1 in 40 ml of water was kept at 4 ° C. with a thermostat and then preconditioned with water at 4 ° C. IR Passed 120 H ⁺ . The resulting eluate, consisting of 100 ml of a solution of pH 1.94, is neutralized with 15% tetrabutylammonium hydroxide solution, left at room temperature for 1 hour, and then maintained at pH 7 by addition of 15% tetrabutylammonium hydroxide, and finally lyophilized. . As a result, 805 mg of tetrabutylammonium salt of epiK5-N-sulfate was obtained.

(b) EpiK5-amine-O- excess sulfate

The solution containing 805 mg of the obtained salt in 30 ml of dimethylformamide was fixed at 55 ° C. and treated with 30 ml of dimethylformamide containing 2.26 g of pyridine.SO ₃ adduct. The reaction is continued overnight at 55 ° C. and then 60 ml of water are added to the mixture, neutralized with 1N NaOH, then the product precipitates with acetone tripled in volume saturated with NaCl and left at 4 ° C. overnight. The precipitate is filtered on guch G4, ultrafiltered with a 1000D TFF Millipore system, dried under reduced pressure and recovered. It has 54% of iduronic acid content, 100% of glucosamine-6-O-sulfate content, 60% of glucosamine 3-O-sulfate content, 10% of glucuronic acid monosulfate, 15% of iduronic acid monosulfate, and the rest of the uronic acid unit. Is bisulfated, and 550 mg of epi-K5-amine-O- excess sulfate having a degree of sulfation of 3.55 measured according to the conductivity measurement method of Casu et al. (1975) is obtained.

(c) EpiK5-amine-O-supersulfate-N-sulfate

400 mg of sodium carbonate was added to a solution containing 250 mg of epi-K5-amine-O-excess sulfate obtained in step (b) in 15 ml of water, followed by 400 mg of slightly solid pyridine.SO ₃ adduct after 4 hours. Add. The reaction mixture is kept at 55 ° C. overnight and then stopped by bringing the pH to 7 with 0.1N hydrochloric acid solution. Ultrafiltration using a 1,000D membrane was followed by addition of acetone in a volume ratio three times saturated with sodium chloride and the precipitate was recovered by centrifugation at 5000 rpm for 5 seconds. 244 mg of epi K5-N, O-excess sulfate was obtained having a degree of sulfation of 4.25 as measured by Casu et al. (1975). ^{From the 1} H-NMR spectral analysis, the epiK5-N, O-excess sulfate was obtained with iduron acid content 54%, 6-O-sulfate content 100%, N-sulfate content 100%, glucosamine 3-O-sulfate content 60% , Glucuronic acid monosulfate content 10%, iduronic acid monosulfate content 15%, the rest of the uronic acid unit appears to be bisulfated. Thus, taking into account the error limits of the process, the degree of sulfation corresponding to the degree of sulfation of 100% N-sulfated epiK5-amine-O-excess sulfate, calculated at the end of step (b), is calculated to be 4.35. Therefore, it can be predicted that if a certain ratio of sulfuric acid groups is exceeded, the degree of sulfation determined by the conductivity measuring method may be underestimated due to the strong anionic property of the product.

Claims (70)

In the method for producing epi K5-N, O- excess sulfate-derivative, (a) treating an epiK5-N-sulfate-derivative having an average molecular weight of about 1,500 to 25,000 in acidic form with a tertiary or quaternary organic base, leaving the reaction mixture at pH about 7 for 30-60 minutes And isolating its salt with said organic base; (b) treating the salt of the organic base of the epiK5-N-sulfate-derivative with an O-sulphurizer under conditions of O-oversulfuration; (c) treating the salt of the tertiary or quaternary organic base of the obtained epiK5-amine-O- supersulfate-derivative with an N-sulfurizing agent and isolating the obtained epiK5-N, O-excess sulfate-derivative The method consists of. The method of claim 1, wherein the epiK5-N, O-excess sulfate-derivative is isolated in the form of a sodium salt and optionally converted to another chemically or pharmaceutically acceptable salt. The process according to claim 1 or 2, wherein in step (a) tetrabutylammonium hydroxide is used as the organic base. The process according to any one of claims 1 to 3, wherein in step (b), the O-supersulfuration is carried out in dimethylformamide using 2-4 moles of O-sulphurizer per OH available per disaccharide. , 15 to 20 hours at a temperature of 40 ~ 60 ℃. 5. The method of claim 1, wherein the epiK5-N-sulfate-derivative used as starting material has an average molecular weight of about 1,000 to about 25,000. 6. 6. The method of claim 5, wherein said starting epiK5-N-sulfate-derivative is 40-60% C5- epimerized. The method of any one of claims 1 to 6, wherein the starting epiK5-N-sulfate-derivative has an average molecular weight of about 1,500 to about 25,000. 8. The method of claim 7, wherein said starting epiK5-N-sulfate-derivative has an average molecular weight of 10,000 to 25,000. 7. The method of any one of claims 1 to 6, wherein the starting material has an average molecular weight of about 1,000 to about 12,000. 10. The method of claim 9, wherein the starting material has an average molecular weight of about 1,500 to about 8,000. The epiK5-N-sulfate-derivative according to claim 5, wherein the epiK5-N-sulfate-derivative used as starting material consists of a chain mixture wherein at least 90% of the chains have the formula Wherein the uronic acid unit consists of 20-60% of iduronic acid, n is an integer from 2 to 100 and the corresponding cation is chemically or pharmaceutically acceptable. 12. The process of claim 11, wherein the starting material consists of a chain mixture in which at least 90% of the chains have the formula I, wherein the uronic acid unit consists of 40-60% of iduronic acid. 13. The method of claim 11 or 12, wherein in formula I, n represents an integer from 3 to 100. The composition of claim 5, wherein the starting material consists of a chain mixture wherein at least 90% of the chains have the formula I ′: Wherein the uronic acid unit consists of 20-60% of iduronic acid, q is an integer from 2 to 20 and the corresponding cation is chemically or pharmaceutically acceptable. 15. The method of claim 14, wherein the starting material consists of a chain composition wherein at least 90% of the chains have the formula I 'wherein n is an integer from 3 to 15. The process of claim 5 wherein the starting material consists of a chain mixture having the predominant species having the formula I'a: Wherein the uronic acid unit is comprised of 60-40% glucuronic acid and 40-60% iduron acid, p is an integer from 4 to 8, and the corresponding cation is chemically or pharmaceutically acceptable. Way. 17. The method of claim 16, wherein the average molecular weight of the starting material is about 2000 to about 4000. 18. The process of claim 16 or 17, wherein the starting material consists of a chain mixture having the predominant species having the formula I'b: Wherein X is hydroxymethyl and m is 4, 5, or 6, starting with glucuronic acid or iduronic acid units, wherein the glucuronic acid and iduronic acid units are alternately present. 19. The method according to any one of claims 5 to 18, wherein the starting material is derived from N-deacetylation and N-sulfurization of K5 which is basically free of lipophilic substances. EpiK5-N, O-excess sulfate-derivatives obtainable by the method according to any one of claims 1 to 19.  EpiK5-N, O-supersulfate-derivatives, or chemically or pharmaceutically acceptable salts thereof, having an iduron acid content of 20-60%, an average molecular weight of about 2,000 to about 45,000, and a degree of sulfation of at least 4, Wherein said derivative is basically inactive on the aggregation parameters. EpiK5-N, O- excess sulfate-derivatives. 22. The epiK5-N, O-supersulfate-derivative of claim 21 wherein the average molecular weight is from about 15,000 to about 45,000.  22. The epiK5-N, O-supersulfate-derivative of claim 21 wherein the average molecular weight is from about 4,500 to about 8,500. The epiK5-N, O-excess sulfate-derivative according to any one of claims 21 to 23, wherein the degree of sulfation is 4 to 4.6. 25. The method of any of claims 21 to 24, wherein 100% 6-O-sulfated and 50-80% 3-O-sulfated in the glycosamine unit and 5-10% in the glucuronic acid unit. EpiK5-N, O characterized by O-monosulfated, 10-15% 3-O-monosulfated in the iduronic acid unit and 2,3-di-O-sulfated in the remaining uronic acid units Excess sulfate-derivatives. 26. The epiK5-N, O-supersulfate-derivative according to any one of claims 21 to 25, wherein at least 90% of the chains consist of a chain mixture having the formula III: Here, the uronic acid unit is composed of a biceps acid of 20 ~ 60%, R, R ' and R "are hydrogen or SO ₃ ^-, and, R is at least 40% SO ₃ in the chain ^mixture present in, and Z is And a SO ₃ ⁻ group, n is an integer from 2 to 100, the degree of sulfation is at least 4, and the corresponding cation is chemically or pharmaceutically acceptable. 27. The epi K5-N, O-excess according to claim 26, wherein at least 90% of the chains consist of a chain mixture having the formula III, wherein the uronic acid unit consists of 40-60% of iduronic acid. Sulfate-derivatives. 28. The epiK5-N, O-supersulfate-derivative according to claim 26 or 27, wherein at least 90% of the chains consist of a chain mixture having the formula III, wherein n is an integer from 3 to 100. . 29. The method of any one of claims 26-28, wherein at least 90% of the chains are lmw-epi K5-N, O-excess sulfate, consisting of a chain mixture having the formula III ': Wherein the uronic acid unit consists of 20-60% of iduronic acid, q is an integer from 2 to 20, and R, R 'and R "are hydrogen or SO ₃ ^- groups for the degree of sulfation of at least 4, 4 to 4.6. And Z is S0 ₃ ^- and the corresponding cation is a chemically or pharmaceutically acceptable ion. EpiK5-N, O- excess sulfate-derivative. 30. The lmw-epiK5-N, O-excess sulfate according to claim 29, wherein at least 90% of the chains consist of a chain mixture having the formula III ', wherein q is an integer from 3 to 15. 31. The lmw-epiK5-N, O-supersulfate salt according to claim 30, wherein at least 90% of the chains consist of a chain mixture having the formula III 'wherein the uronic acid unit consists of 40-60% of iduronic acid. . 32. The lmw-epi K5-N, O-excess sulfate according to claim 31, wherein the content of iduron acid is 50-55%. 33. The composition of any one of claims 29-32, wherein at least 90% of the chains are composed of a chain mixture having the formula III ', wherein R is at least 40% SO ₃ ^- and both R' and R "are SO. Lmw-epiK5-N, O- supersulfate, characterized in that it is ₃ ^- or one is hydrogen and the other is 5 to 10% SO ₃ ^- in glucuronic acid and 10 to 15% SO ₃ ^- in iduronic acid . 34. The lmw-epiK5-N, O-excess sulfate according to claim 33, having an average molecular weight of about 2,000 to about 16,000. 35. The lmw-epiK5-N, O-excess sulfate according to claim 34, having an average molecular weight of about 4,500 to about 9,000. Of claim 33 to claim 35. Compounds according to any one of the preceding, a chain of at least 90% consists of a mixture of chains having the formula Ⅲ ', R is 50 ~ 80% SO ₃ ^- lmw- epi K5-, characterized in that N, O-excess sulfate. 37. The method of claim 32, wherein the predominant species consists of a chain mixture having the formula III'a: Wherein the uronic acid unit consists of 20-60% diuronic acid, p is an integer from 4 to 8, and R, R 'and R "are hydrogen or SO ₃ ^- groups for the degree of sulfateization of 4 to 4.6, The corresponding cation is chemically or pharmaceutically acceptable lmw-epi K5-N, O- excess sulfate. 38. The method of any of claims 33 to 37, wherein the predominant species consists of a chain mixture having the formula III'b: Wherein for the 4 to 4.6 sulphate degree, R, R 'and R "are hydrogen or SO ₃ ^- is a group, Z is SO ₃ ^- group, and, X" is OH or OSO ₃ ^-, and, m is 4, 5, Or 6, starting with glucuronic acid or iduronic acid units alternately with glucuronic acid and iduronic acid units, the corresponding cation being lmw-epiK5-N, O-excess, which is a chemically or pharmaceutically acceptable ion sulfate. The chemically or pharmaceutically acceptable salt or cation according to claim 20, wherein the chemically or pharmaceutically acceptable salt or cation is an alkali metal or alkaline-earth metal, ammonium, (C ₁ -C ₄ ) tetraalkylammonium, aluminum or zinc. EpiK5-N, O- excess sulfate-derivative, characterized in that the salt or cation of. 40. The epiK5-N, O-supersulfate-derivative of claim 39 wherein the chemically or pharmaceutically acceptable salt or cation is a salt or cation of sodium, calcium or tetrabutylammonium. Isolated in the form of epiK5-amine-O-supersulfate-derivatives or sodium salts, obtainable by the process according to steps (a) and (b) of claim 1, and optionally other chemically or pharmaceutically acceptable Chemically or pharmaceutically acceptable salts thereof, converted to salts. EpiK5-amine-O- excess sulfate-derivatives or chemically or pharmaceutically acceptable salts thereof, wherein the iduronic acid content is 20-60% of the total uronic acid, an average molecular weight of about 2,000 to about 40,000 and at least EpiK5-amine-O- excess sulfate-derivatives with a degree of sulfation of 3.4. 43. The process of claim 42, wherein the epiK5-amine-O-excess sulfate-derivative consists of a chain mixture wherein at least 90% of the chains have the formula II: Here, the uronic acid unit is composed of a biceps acid of 20 ~ 60%, n is from 2 to 100, and the integer, R, R 'and R "are hydrogen or SO ₃ ^-, and the sulfuric acid affinity is at least 3.4, the corresponding And the cation is chemically or pharmaceutically acceptable. EpiK5-amine-O- excess sulfate-derivative. 44. The epiK5-amine-O- excess sulfate-derivative according to claim 43, wherein n represents an integer from 3 to 10. 45. The composition of any one of claims 42-44, wherein at least 90% of the chains consist of a chain mixture having the formula II above, wherein the uronic acid unit consists of 40-60% of iduronic acid, and from about 2,000 to about Having an average molecular weight of 40,000, R is at least 40% SO ₃ ⁻ , R ′ and R ″ are both SO ₃ ⁻ or one is hydrogen and the other is 5-10% SO ₃ ⁻ in monosulfate glucuronic acid And 10-15% SO ₃ ⁻ in monosulfate iduronic acid. 46. The compound of any one of claims 42-45, wherein at least 90% of the chains are lmw-epiK5-amine-O-supersulfate consisting of a chain mixture having the formula II above, wherein the uronic acid unit is 40-60. Consisting of% iduronic acid, R is at least 40% SO ₃ ⁻ , R ′ and R ″ are both SO ₃ ⁻ or one is hydrogen and the other is 5-10% SO ₃ ⁻ in glucuronic acid Edu acid in 10 ~ 15% SO ₃ ^-, n is a 3 to 15 integer, having an average molecular weight of from about 4,000 to about 8,000, the corresponding cation is epitaxially, characterized in that acceptable chemically or pharmaceutically K5 -Amine-O-excess sulfate-derivatives. 47. The method of any of claims 42-46, wherein the predominant species is lmw-epiK5-amine-O-excess sulfate, consisting of a chain mixture having the formula II'a: Characterized in that a sulfate degree of at least ^3.4, where, w and acid unit is composed of biceps acid of 20 ~ 60%, p is from 4 to 8 integers, R, R 'and R "are hydrogen or SO ₃ EpiK5-amine-O-excess sulfate-derivative. 48. The composition of claim 47, wherein the predominant species consists of a chain mixture which is a compound having the formula II'b: Wherein the uronic acid unit consists of 40-60% of iduronic acid, m is 4, 5, or 6, for a degree of sulfation of at least 3.4, R, R 'and R "are hydrogen or SO ₃ ^- groups, X ″ is OH or OSO ₃ ⁻ , and lmw epiK5-amine-O-excess sulfate, wherein the iduronic acid unit is present alternately starting with glucuronic acid or iduronic acid unit. Lmw-epiK5-N-sulfate, or its chemically or pharmaceutically acceptable, which is substantially free of NH ₂ and N-acetyl groups and has an iduronic acid content of 20-60%, an average molecular weight of about 1,500 to about 12,000 salt. 50. The lmw-epiK5-N-sulfate according to claim 49, having an iduronic acid content of 40-60% and an average molecular weight of about 1,500 to about 10,000. 50. The lmw-epiK5-N-sulfate according to claim 49, wherein the content of iduron acid is from 50 to 55% and the average molecular weight is from about 1,500 to about 7,500. 52. The lmw-epiK5-N-sulfate according to any one of claims 49-51, obtained by the process of performing the following steps (in any order) to K5-N-sulfate: (i) Calcium, Magnesium, Barium with D-glucuronyl C5-epimerase isolated, purified and immobilized in solution or on a solid support for a temperature of about 7, about 30 ° C. for 12-24 hours. And C5-epimerization in the presence of one or more divalent ions selected from manganese; And (ii) nitrite depolymerization and subsequent reduction. 53. The lmw-epiK5-N-sulfate according to claim 52, wherein in step (ii), depolymerization is followed by reduction with sodium borohydride. The compound of claim 53, wherein at least 90% of the chains consist of a chain mixture having the formula I ′: Wherein the uronic acid unit consists of 20-60% of iduronic acid, q is an integer from 2 to 20, and the corresponding cation is chemically or pharmaceutically acceptable lmw-epiK5-N-sulfate. 55. The lmw-epiK5-N-, wherein the at least 90% of the chains consist of a chain mixture having the formula I 'wherein the uronic acid unit consists of 40-60% of iduronic acid. sulfate. The lmw-epiK5-N-sulfate according to claim 54 or 55, wherein at least 90% of the chains consist of a chain mixture having the formula I ', wherein n is an integer from 3 to 5. 59. The method of claim 54 or 55, wherein the predominant species consists of a chain mixture having the formula I'a: Wherein the uronic acid unit is comprised of 60-40% glucuronic acid and 40-60% iduron acid, p is an integer from 4 to 8, and the corresponding cation is chemically or pharmaceutically acceptable. lmw-epi K5-N-sulfate. 58. The process of any of claims 55-57, wherein the predominant species consists of a chain mixture having the formula I'b: Wherein X is hydroxymethyl, m is 4, 5, or 6, and the corresponding cation is a chemically or pharmaceutically acceptable ion of Ana, starting with glucuronic acid or iduronic acid unit, glucuronic acid and Lmw-epi K5-N-sulfate, characterized by alternating iduronic acid units. 59. The method of any of claims 49-58, wherein the salt or cation is selected from alkali metal or alkali-earth metals, ammonium, (C ₁ -C ₄ ) tetraalkylammonium, aluminum or zinc. Lmw-epiK5-N-sulfate. 60. The lmw-epiK5-N-sulfate according to claim 59, wherein the salt or cation is selected from those of sodium, calcium and tetrabutylammonium. Process for preparing lmw-epi K5-N-sulfate, consisting of performing K5-N-sulfate in the following steps (in any order): (i) Calcium, Magnesium, Barium with D-glucuronyl C5-epimerase isolated, purified and immobilized in solution or on a solid support for a temperature of about 7, about 30 ° C. for 12-24 hours. And C5-epimerization in the presence of one or more divalent ions selected from manganese; And (ii) nitrous acid depolymerization and optionally subsequent reduction. 63. The method of claim 61, wherein the method is carried out in the order (i) to (ii). 63. The method of claim 61, wherein the method is carried out in the order (ii) to (i). 64. The process according to claim 63, wherein the product obtained at the end of depolymerization is lmw-K5-N-sulfate which is subjected to C5-epimerization directly. 65. The method of claim 64, wherein said lmw-K5-N-sulfate has an average molecular weight of at least 4,000. A pharmaceutical composition comprising, as an active ingredient, a pharmacologically active amount of EpiK5-N, O- excess sulfate-derivative according to any one of claims 20 to 40 in admixture with a pharmaceutical excipient. A cosmetic composition comprising an effective amount of the epiK5-N, O-excess sulfate-derivative according to any one of claims 20 to 40 in admixture with a cosmetic excipient. For the use of isolated and purified C5-epimerase for the conversion of K5-N-sulfate-derivatives to the corresponding epiK5-N-sulfate-derivatives, the epiK5-N-sulfate-derivatives are glucuronic acid units. Or characterized by repeated tetrasaccharide units consisting of two glycosamines separated by and followed by an iduronic acid unit or followed by a glucuronic acid unit. 69. The use of claim 68, wherein the K5-N-sulfate-derivative has an average molecular weight of at least 4,000. 70. The use of claim 69, wherein the average molecular weight is between 6,000 and 7,000.