RU2333222C2 - Epimerised k5 polysaccharide derivatives with high sulfation degree - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention describes a method of epi-K5-N-sulfate oversulfation for obtaining epi-K5-amine-O-oversulfate with very high sulfation degree, which produces new epi-K5-N,O-oversulfate derivatives with sulfation degree of 4-4.6 on following N-sulfation, the derivatives being almost inactive to fibrillation parametres and applicable in pharmaceutical compositions with antidermatitis and antiviral effect. The invention also describes new low-molecular epi-K5-N-sulfates applicable as transit products in obtaining the respective low-molecular epi-K5-N,O-oversulfate derivatives.

EFFECT: method of epi-K5-N-sulfate oversulfation for obtaining epi-K5-amine-O-oversulfate with extremely high sulfation degree.

55 cl, 4 dwg

Description

FIELD OF THE INVENTION

The present invention relates to new K5 polysaccharide derivatives with a high degree of sulfation, a method for their preparation, new highly O-sulfated intermediates useful in their synthesis, and pharmaceutical compositions containing these K5 polysaccharide derivatives as active ingredients that are practically free from blood coagulation activity.

In particular, the invention relates to a method for producing epi-K5-N, O-hypersulphates, starting from K5 polysaccharide pre-N-deacetylated, N-sulfated and C5-epimerized by at least 20%, by O-hypersulfation under suitable conditions and subsequent N-sulfation, and also relates to data of epi-K5-N, O-hypersulphates having antiangiogenic and antiviral activity, and new low molecular weight intermediates for epi-K5-N-sulfates.

State of the art

Glycosaminoglycans, such as heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate and hyaluronic acid, are biopolymers that are extracted from various animal organs in industry.

In particular, heparin, which is mainly obtained by extraction from the intestinal mucosa of pigs or cattle lungs, is a polydisperse polymer with a molecular weight of approximately from 3,000 to 30,000 D, consisting of mixed chains, mainly consisting of uronic acid (glucuronic or iduronic acid) and amino sugar (glucosamine), interconnected by bonds α-1 → 4- or β-1 → 4. In heparin, the uronic unit can undergo O-sulfation at position 2, and the glucosamine unit can undergo N-acetylation or N-sulfation, 6-O-sulfation and 3-O-sulfation in approximately 0.5% glucosamine units.

The properties and natural biosynthesis of heparin in mammals are described in Lindahl et al., 1986 in Lane D. and Lindahl U. (Editors) "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; Conrad HE "Heparin Binding Proteins", Chapter 2: Structure of Heparinoids. Academic Press, 1998. Heparin biosynthesis begins with its precursor N-acetylheparosan, consisting of mixed chains of repeating units of the glucuronyl-β-1 → 4-N-acetylglucosamine disaccharide. This precursor undergoes enzymatic modifications, which include the partial hydrolysis of N-acetyl, its substitution with the SO ₃ ^- group, the epimerization of carboxyl at position 5 of some part of the glucuronic units with their transformation into iduronic units, and the introduction of O-sulfate groups to form a product that after industrial extraction contains twice as many sulfate groups as compared to carboxyl groups per 1 unit of disaccharide. In addition, these enzymatic modifications lead to the formation of the pentasaccharide binding site of antithrombin III (ATIII), called the active pentasaccharide, which is the structure necessary for the high affinity binding of heparin to ATIII and essential for the anticoagulation and antithrombotic activity of heparin itself. This pentasaccharide, which is present only in some of the chains constituting heparin, contains a sulfated glucosamine unit at position 3 and glucuronic acid located between disaccharides containing iduronic acid.

In nature, the formation of the active pentasaccharide is carried out by the epoxymerization of the carboxyl of a certain part of the glucuronic units into iduronic units under the action of glucuronyl C5 epimerase (C5 epimerization) and corresponding sulfation, which also leads to the introduction of a sulfate group in the hydroxyl at position 3 of glucosamine. In particular, in nature, the formation of active pentasaccharide is carried out in such a way that C5 epimerization occurs in separate "clusters", that is, on part of the chains, and to a large extent, a product is formed that contains more iduronic units than glucuronic ones. In fact, commercial heparin contains approximately 70% of iduronic units and 30% of glucuronic units.

Along with the main anticoagulant and antithrombotic effect, heparin also has antilipemic, antiproliferative, antiviral, anticancer and antimetastatic effects, but its use as a drug is restrained due to side effects due to its anticoagulant effect, which can cause bleeding.

As is known, the capsular polysaccharide K5 isolated from Escherichia coli and described by Vann W.F. et al., European Journal of Biochemistry, 1981, 116, 359-364 ["Vann 1981"], consists of mixed chains consisting of repeating units of glucuronyl-β-1 → 4-N-acetylglucosamine disaccharide, therefore it exhibits the same repeating sequence (A):

as in N-acetylheparosan, the heparin precursor. The K5 capsular polysaccharide, hereinafter referred to as the “K5 polysaccharide” or simply “K5”, was chemically modified by Lormeau et al. As described in US 5550116 and Casu et al. As described in Carbohydrate Research, 1994, 263, 271- 284. K5 O-sulfates having anti-cancer, anti-metastasis, antiviral effects, especially against HIV, are described in EP 333243 and WO 98/34958. K5 was also subjected to chemical and enzymatic modifications in order to obtain products having the same type of biological activity on blood coagulation in vitro as in heparin extracted from animal organs (extractive heparin).

Obtaining products with the same type of blood coagulation activity as extractive heparin is carried out by a method that simulates a process that occurs in nature and involves the entire key stage of C5 epimerization using D-glucuronyl-C5 epimerase.

In the methods described in IT 1230785, WO 92/17507, WO 96/14425 and WO 97/43317, K5 is used as the starting material. The K5 obtained by fermentation is subjected to N-deacetylation and then N-sulfation, and the resulting K5-N-sulfate is subjected to C5 epimerization in solution using C5 epimerase obtained by chromatography of a solution of microsomal enzymes from mouse mastocytoma cells (IT 1230785) or bovine liver (WO 92/17507, WO 96/14425 and WO 97/43317).

Bovine liver D-glucuronyl-C5 epimerase was purified by Campbell P. et al., J. Biol. Chem., 1994, 269/43, 26953-26958 (Campbell 1994), which also obtained its amino acid composition and described its use in solution to convert K5-N-sulfate to the corresponding product, 30% epimerized, attesting to the formation of iduronic acid by HPLC followed by complete depolymerization with nitrous acid to disaccharides.

WO 98/48006 describes a DNA sequence encoding D-glucuronyl-C5 epimerase and recombinant D-glucuronyl-C5 epimerase was prepared using a recombinant expression vector containing this DNA and then purified Campbell et al. As shown in Jin-Ping L. et al., J. Biol. Chem., 2001, 276, 20069-20077 ("Jin-Ping 2001").

The complete sequence of C5 epimerase is described in Crawford B.E. et al., J. Biol. Chem., 2001, 276 (24), 21538-21543 (Crawford 2001).

WO 01/72848 describes a method for producing N-deacetylated N-sulfated derivatives of K5 polysaccharide, epimerized to iduronic acid, by at least 40%, calculated on the sum of uronic acids, with a molecular weight of from 2000 to 30,000, containing from 25% to 50% of the high affinity chains for ATIII and possessing anticoagulation and antithrombotic activity, expressed as the ratio HCII / antiXa, from 1.5 to 4. This document describes the hypersulfation of K5-N-sulfate, epimerized by 40-60%, and it is shown that received product for which cn CTD ¹³ C-NMR, contains 2-3.5 sulfate group on the disaccharide unit 1. By repeating the above hypersulfation under the described conditions and studying ¹³ C-NMR, it was found that the resulting product is actually a free amine containing 80-95% 6-O-sulfate and 30% 3-O-sulfate on amino sugar, but the degree sulfation is 3.2. It also turned out that under the hypersulfation conditions described in WO 01/72848, a degree of sulfation of not more than 3.2 is achieved. US 2002/0062019 describes a method for producing epi-K5-N, O-sulfates that are active in the regulation of blood coagulation, in which the degree of sulfation is from 2.3 to 2.9, with a molecular weight of from 2000 to 30000, or from 4000 up to 8000, or from 18000 to 30000. This method includes the steps of: (p-a) N-deacetylating the K5 polysaccharide and N-sulfating the resulting K5-amine, (pb) epimerizing K5-N-sulfate, (p-c) O- hypersulfation of epi-K5-N-sulfate, (pd) partial O-desulfation, (p-e) selective 6-O-sulfation, (pf) N-sulfation of the resulting product, and any product obtained at the end of one of the stages from (pb) to (pf) can be subjected to depolymerization. This document describes epi-K5-N, O-sulfate with a molecular weight of 7400, obtained by carrying out steps (p-a) to (pf) followed by nitrogen depolymerization at the end of step (pf), in which the degree of sulfation is from 2 , 3 to 2.9.

The same document also describes a separate part of K5 with a molecular weight of about 5000, which can also be subjected to steps (p-a) to (p-f).

In order to standardize terminology and make the text more understandable, conditional terms and expressions, in the singular or plural, will be used in the present description. In particular:

- “K5” or “K5 polysaccharide” means a capsular polysaccharide from Escherichia coli obtained by fermentation, that is, it is a mixed chain consisting of disaccharide units (A) optionally containing a double bond at the non-reducing end, as shown above, and in any case it was obtained and purified by methods described in the literature, in particular by the method of Vann 1981, by the method of Manzoni M. et al., Journal of Bioactive Compatible Polymers, 1996, 11, 301-311 ("Manzoni 1996") or by the method described in WO 01 / 72848 WO 02/068447; the specialist in this field should be clear that everything shown below is applicable to any N-acetylheparosan;

- "C5 epimerase" means B-glucuronyl-C5 epimerase, extractive or recombinant, but in any case, it is obtained, isolated and purified, in particular as described in Campbell 1994; WO 98/48006; Jin-Ping L. et al., J. Biol. Chem. 2001, 276, 20069-20077 ("Jin-Ping 2001") or Crawford 2001;

- "K5-amine" means K5, deacetylated at least 95%, in which N-acetyl groups are not found on a conventional NMR device;

- "K5-N-sulfate" means K5, deacetylated at least 95% and N-sulfated usually 100%, since N-acetyl groups are not detected on a conventional NMR device, as described below;

- “epi-K5” means K5 and its derivatives in which 20-60% of the glucuronic units have undergone C5 epimerization into iduronic units;

- “epi-K5-N-sulfate” means K5-N-sulfate in which 20-60% of the glucuronic units have undergone C5-epimerization to iduronic units;

- "epi-K5-amine-O-hypersulfate" means epi-K5-amine-O-sulfate, in which the degree of sulfation is at least 3.4;

- "epi-K5-N, O-hypersulfate" means epi-K5-amine-O-sulfate, which has undergone complete N-sulfation to a degree of sulfation of at least 4.

Besides:

- conditional terms and expressions defined above refer to K5 isolated after fermentation, usually with a molecular weight of approximately from 1500 to 50,000 and an average molecular weight of 10,000 to 25,000, preferably 15,000 to 25,000;

- conditional terms and expressions defined above, if they are preceded by the abbreviation "NM" (low molecular weight), for example NM K5-N-sulfate, NM epi-K5-N-sulfate, mean low molecular weight products obtained by fractionation or depolymerization K5-N sulphate and consisting of or derived from K5-N-sulphates with an average molecular weight of from about 1,500 to 12,000, based on 100% N-sulfated product;

- conditional terms and expressions defined above, if the word "derivative" is used with them, generally mean both derivatives of native K5 and low molecular weight K5;

- the expression “approximately” with respect to molecular weight means the molecular weight measured by viscometry, ± the theoretical weight of the disaccharide unit, including the weight of sodium, which is assumed to be 461 in the case of derivatives of epi-K5-N-sulfates and 806 in the case of derivatives of epi-K5- N, O-hypersulphates with a sulfation degree of 4.26;

- the expression "predominant variety" means a compound which in the mixture consisting of NM epi-K5-N-sulfate, NM epi-K5-amine-O-hypersulphate or NM epi-K5-N, O-hypersulphate , which is determined by the peak on the molecular weight graph when measured by HPLC;

- unless otherwise specified, "degree of sulfation" means the ratio of SO ₃ ^- / COO ^- , also expressed by the number of sulfate groups per 1 unit of disaccharide, when measured by the conductometric method described in Casu B. et al .. Carbohydrate Research, 1975, 39, 168-176 (Casu 1975), which was also used in WO 01/72848;

- "O-hypersulfation conditions" means the ultimate O-sulfation, which is carried out, for example, according to method C described in B. Casu et al., Carbohydrate Research, 1994, 263, 271-284 (Casu 1994);

- the term “alkyl” means linear or branched alkyl, while “tetrabutylammonium” means a tetra-p-butylammonium group.

SUMMARY OF THE INVENTION

It has been shown that, unlike what happens when applying the methods described in IT 1230785, WO 92/17507, WO 96/14425, WO 97/43317, WO 01/72848 and US 2002/0062019, based on epi-K5 -N-sulfate can be obtained epi-K5-amine-O-hypersulphate with a greater degree of sulfation than any other epi-K5-amine-O-sulfate described in the literature, for example, in WO 01/72848, by obtaining a tertiary salt or a quaternary organic base of this epi-K5-N-sulfate with the obligatory leaving the reaction mixture for 30-60 minutes while maintaining a pH of about 7 using the same organic base Bani, and then treating the resulting salt O-sulfating agent under O-gipersulfatirovaniya.

Subjecting the epi-K5-amine-O-hypersulfates thus obtained to N-sulfation, new epi-K5-N, O-hypersulphates are obtained, which, unlike the products described in IT 1230785, WO 92/17507, WO 96/14425, WO 97/43317, WO 01/72848 and US 2002/0062019, are deprived of blood clotting activity and are useful for the manufacture of drugs, in particular pharmaceutical compositions having antiangiogenic and antiviral activity, or cosmetic compositions.

Upon depolymerization of these epi-K5-N, O-hypersulphates with nitrous acid, new NM epi-K5-N, O-hypersulphates deprived of blood coagulation activity and possessing antiangiogenic and antiviral activity are obtained.

Upon receipt of N, O-sulfate from N-deacetylated derivatives of K5 polysaccharide, epimerized by at least 40% to iduronic acid, calculated on the sum of uronic acids and having a low molecular weight, by the method described in WO 01/72848, it was found that depolymerization of a high molecular weight product obtained at the end of the final stage of the N-sulfation process can give different results, since it usually leads to the production of some depolymerized products with significantly lower activity than those high molecular weight products from which they are formed, for all parameters of blood coagulation. It is assumed that this is because the presence of sulfate groups affects the degradation using nitrous acid. In particular, sulfates at position 3 of glucosamine lead to the formation of heterogeneous products, as described in Nagasawa et al., Thrombosis Research, 1992, 65, 463-467 (Nagasawa 1992).

Then it was found that during nitrogen depolymerization of epi-K5-N-sulfate, in which the content of iduronic acid in terms of the sum of uronic acids is 20-60%, preferably 40-60%, more preferably about 60%, NM epi- K5-N-sulfates, which are new effective intermediate products for the production of NM epi-K5-N, O-hypersulfates, which have high activity on various biological parameters, in particular, with or without activity on blood coagulation parameters. It turned out that by depolymerization of epi-K5-N-sulfate it is possible to obtain new NM epi-K5-N-sulfates with an average molecular weight of about 2000 to 4000, in particular certain NM epi-K5-N-sulfates, consisting of mixtures, in which the predominant compound is a decasaccharide or dodecasaccharide or tetradecasaccharide. In addition, these NM epi-K5-N-sulfates, which cannot be obtained in any other way, have interesting biological properties and are important intermediate products for the production of NM epi-K5-N, O-hypersulphates with antiviral and / or antiangiogenic activity and unexpectedly deprived of blood clotting activity.

Subjecting NM epi-K5-N-sulfate to the above salt production method using a tertiary or quaternary organic base, leaving the reaction mixture for 30-60 minutes while maintaining the pH at about 7 using the same organic base, and then treating the resulting salt with O-sulfating reagent under conditions of O-hypersulfation, get new NM epi-K5-amine-O-hypersulfates. Subjecting NM epi-K5-amine-O-hypersulphate to N-sulfation, new N-sulfated and O-hypersulphated derivatives (NM epi-K5-N, O-hypersulphates) unexpectedly lacking blood coagulation activity and having antiviral and / or antiangiogenic activity, useful for obtaining pharmaceutical or cosmetic compositions.

These NM epi-K5-N-sulfates are prepared from K5-N-sulfate by an epimerization reaction using isolated and purified recombinant C5 epimerase immobilized on a solid support at a temperature of about 30 ° C and a pH of about 7 for 12-24 hours in the presence of a divalent cation selected from calcium, magnesium, barium and manganese, and the subsequent reaction of nitrogen depolymerization of the product obtained in this case, or in reverse order.

Surprisingly, monitoring the progress of the epimerization reaction under the above conditions suggests that, unlike what happens in nature during heparin biosynthesis, it is not “cluster” but normal C5-epimerization of the substrate every 2 units of glucuronic acid that leads to the formation of derivatives epi-K5-N-sulfates, characterized by the presence of a repeating tetrasaccharide unit, consisting of two glucosamine units, separated first by a glucuronic unit and then by an iduronic unit, or vice versa.

DETAILED DESCRIPTION OF THE INVENTION

So, in accordance with one aspect of the present invention provides a method for producing derivatives of epi-K5-N, O-hypersulfates, characterized in that:

(a) an epi-K5-N-sulfate derivative, in acid form, is treated with a tertiary or quaternary organic base, leaving the reaction mixture for 30-60 minutes, and the pH of the solution is maintained at about 7 by adding the same tertiary or quaternary organic base, and isolated a salt of a given organic base;

(b) this salt of an organic base and this epi-K5-N-sulfate derivative is treated with an O-sulfating reagent under O-hypersulfation conditions;

(c) the resulting epi-K5-amine-O-hypersulphate derivative is treated with an N-sulfating reagent and the epi-K5-N, O-hypersulphate derivative is isolated.

In general, an epi-K5-N, O-hypersulfate derivative is isolated in the form of a sodium salt, and optionally, this sodium salt is converted to another chemically or pharmaceutically acceptable salt.

In this context, the term "chemically acceptable" refers to a cation used in chemical synthesis, such as sodium, ammonium, (C ₁ -C ₄ ) tetraalkylammonium ion, or in purification of a product, while "pharmaceutically acceptable" requires no explanation.

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

According to a preferred embodiment of the method, step (a) is carried out by passing a solution of the sodium salt of the epi-K5-N-sulfate derivative, i.e., the K5 polysaccharide pre-N-deacetylated, N-sulfated, usually 100%, C5-epmerized at 20- 60% and optionally depolymerized with nitrous acid, with an average molecular weight of from about 1000 to 25000, preferably from 1500 to 25000, through a column with an acidic anion exchanger, for example, type IR-120 H ⁺ collecting the eluate together with the washing solution and neutralizing the elu ata with a tertiary or quaternary organic base, preferably an aqueous solution of tetrabutylammonium hydroxide. The solution was left for 1 hour, maintaining the pH at about 7 by adding the same base, and the resulting salt was isolated by lyophilization.

In step (b), O-hypersulfation is carried out using an excess of O-sulfating reagent and treatment at a temperature of from 20 to 70 ° C for up to 24 hours in an aprotic polar solvent.

Preferably, the salt of the tertiary or quaternary organic base and the epi-K5-N-sulfate derivative, i.e., the K5 polysaccharide pre-N-deacetylated, N-sulfated, preferably 100%, C5-epimerized 20-60% and optionally depolymerized with nitrous acid, with an average molecular weight of from about 1000 to 25000, preferably from 1500 to 25000, dissolved in dimethylformamide and treated with O-sulfating reagent in an amount of 2-10 mol for each free hydroxyl at temper ature 40-60 ° C for 10-20 hours. The pyridine · SO ₃ compound is preferably used as an O-sulfating reagent in an amount of 2.5-5 mol, preferably 2.5-4 mol per 1 free hydroxyl per 1 disaccharide, and the reaction is carried out preferably at 50-60 ° C., more preferably at 55 ° C during the night.

The product obtained upon completion of the reaction is isolated by adding 0.1-1 volumes of water and neutralizing, preferably with sodium hydroxide, precipitating with a saturated solution of sodium chloride in acetone, filtering and optionally ultrafiltration.

The product thus obtained is usually a sodium salt of an epi-K5-amine-O-hypersulfate derivative with an iduronic acid content of 20-60% of the sum of uronic acids, with an average molecular weight of from about 3500 to 40,000, preferably from 4500 to 40,000, with the degree of sulfation is not less than 3.4, preferably at least 3.5, more preferably from 3.55 to 4, most preferably from 3.55 to 3.8. The salt thus obtained can be converted into another salt. For example, an exchange of ions for calcium ion can be carried out using ultrafiltration membranes.

In step (c), a very high sulfation-derived epi-K5-amine-O-hypersulfate derivative is subjected to N-sulfation using N-sulfation techniques known in the literature.

In practice, N-sulfation is carried out by treating an aqueous solution containing an epi-K5-amine-O-hypersulfate derivative obtained in step (b) with sodium carbonate and an N-sulfating reagent, for example a compound (C ₁ -C ₄ ) -trialkylamine SO ₃ or pyridine · SO ₃ , keeping the mixture at 30-50 ° C for 8-24 hours and isolating the desired derivative of epi-K5-N, O-hypersulfate, for example, by diafiltration. Optionally, the N-sulfation step is repeated until a degree of substitution of more than 95% is obtained, preferably until complete.

The new derivatives of epi-K5-N, O-hypersulphates obtained in this way are usually in the form of the sodium salt. The sodium salt may be converted to another chemically or pharmaceutically acceptable salt. Salts of alkali metals, alkaline earth metals, ammonium, (C ₁ -C ₄ ) -tetraalkylammonium, aluminum and zinc are particularly preferred. Most preferred are sodium, calcium and tetrabutylammonium salts.

The starting epi-K5-N-sulfates sent to step (a) of the process of the present invention are derived from the K5 polysaccharide pre-N-deacetylated, N-sulfated practically 100%, C5-epmerized 20-60%, preferably 40- 60%, and optionally depolymerized with nitrous acid, with an average molecular weight of from about 1000 to 25000, preferably from 1500 to 25000. Preferably, the starting material is epi-K5-N-sulfate with an average molecular weight of from 10000 to 25000 or NM epi-K5 N-Molecular Sulfate m weight of from about 1,000 to 12,000, more preferably 1,000 to 10,000, most preferably from 1500 to 8000.

Epi-K5-N-sulfates obtained by C5-epimerization of K5-N-sulfates are well known in the literature and are widely described, for example, in WO 92/17507, WO 01/72848, WO 98/14425, WO 97/43317 or US 2002/0062019. Their preparation by C5-epimerization of the glucuronic unit of K5-N-sulfate using D-glucuronyl-C5-epimerase is described in the documents cited above.

NM epi-K5-N-sulfate with an iduronic acid content of approximately 20%, obtained by N-deacetylation, N-sulfation and C5-epimerization of a K5 fragment with an average molecular weight of 5000, is described in WO 92/17507. However, such NM K5-N-sulfate contains a significant amount of acetyl groups.

Epi-K5-N-sulfate with an iduronic acid content of 40-60%, particularly preferred as a starting material, is obtained by epimerization of K5-N-sulfate, practically devoid of acetyl groups, which, in turn, is obtained from especially pure K5, in particular not containing lipophilic substances described in WO 02/068477. According to a preferred embodiment of the process, epimerization uses K5-N-sulfate derived from K5 devoid of lipophilic substances, such as that described in WO 02/068477, and C5-epimerization is carried out using D-glucuronyl-C5 epimerase isolated , purified and immobilized on a solid support, at a pH of about 7, at a temperature of about 30 ° C and for 12-24 hours, in the presence of at least one divalent ion selected from calcium, magnesium, barium and manganese.

NM epi-K5-N-sulfates with a higher content of iduronic acid, in particular 40-60%, preferably 50-55%, are new, especially preferred products as starting materials for obtaining NM derivatives of epi-K5-N, O hypersulphates.

The above NM epi-K5-N-sulfates are prepared by a process which is characterized in that K5-N-sulfate is subjected, in any order,

(i) C5 epimerization with O-glucuronyl C5 epimerase isolated, purified and in solution or immobilized on a solid support, at a pH of about 7, at a temperature of about 30 ° C and for 12-24 hours, in the presence of at least one divalent ion selected from among calcium, magnesium, barium and manganese; and

(ii) nitrogen depolymerization, which is optionally followed by reduction, usually with sodium borohydride.

The expression "in any order" means that the method can be performed both in the direction (i) - (ii), that is, in the order above, and in the reverse order, that is, in the direction (ii) - (i), subjecting K5-N-sulfate first to nitrogen depolymerization reactions, and then optionally reducing with sodium borohydride and then C5-epimerization under the above conditions. The preferred order is in the direction (i) → (ii). The sequence (ii) - (i) is preferably used when starting from NM K5-N-sulfates with an average molecular weight of more than 4000, preferably about 6000. For example, you can determine the amount of sodium nitrite, which is based on 1 g of epi-K5- N-sulfate allows you to get NM epi-K5-N-sulfate with a molecular weight of more than 4000, preferably not less than 6000, in order to obtain useful intermediate products for the production of NM epi-K5-N, O-hypersulphates. In fact, in this case, at stage (ii), an optimal degree of epimerization is achieved.

In accordance with a preferred aspect of the invention, C5 epimerase is immobilized on an inert solid support.

C5 epimerase, preferably recombinant, isolated and purified, for example, according to Campbell 1994, WO 98/48006, Jin-Ping 2001 or Crawford 2001, is immobilized on an inert carrier in the presence of a substrate, i.e. in the presence of the starting derivative K5-N- sulfate or in the presence of NM K5-N-sulfate, preferably with an average molecular weight of more than 4000, more preferably not less than 6000. Immobilization is carried out in accordance with standard methods, for example, as described in WO 01/72848.

The C5 epimerization reaction is carried out by passing 20-1000 ml of a 25 mM Hepes solution, pH about 7, containing 0.001-10 g of a substrate (K5-N-sulfate or NM K5-N-sulfate, preferably with a molecular weight of more than 4000, more preferably at least 6000) and a cation selected from calcium, magnesium, barium and manganese in a concentration of from 10 to 60 mm, through a column containing from 1.2 × 10 ⁷ to 3 × 10 ¹¹ cpm of the immobilized enzyme, at a pH of about 7 and a temperature of about 30 ° C, with a transmission rate of 30-220 ml / hour for 12-24 hours, preferably 15-24 hours.

Preferably, this solution is passed at a rate of 200 ml / hour overnight (15-20 hours). The resulting product is purified and separated in accordance with known methods, for example, ultrafiltration and ethanol precipitation. The product thus obtained consists either of epi-K5-N-sulfate (in this case it is dissolved in water and subjected to depolymerization), or of NM epi-K5-N-sulfate (in this case, it is the final product). The degree of epimerization, which in practice means the number of iduronic units relative to glucuronic ones, is calculated using ¹ H-NMR by the method described in WO 96/4425.

The nitrogen depolymerization reaction is carried out in accordance with known methods of heparin depolymerization, for example, in accordance with the methods described in EP 37319, WO 82/03627, or in accordance with the K5-N-sulfate depolymerization method described in EP 544592, but starting from K5- N-sulfate or epi-K5-N-sulfate containing from 0 to 10%, preferably not more than 5%, of acetyl groups. Preferably, the depolymerization is carried out using sodium nitrite and hydrochloric acid on epi-K5-N-sulfate, practically devoid of acetyl groups, followed by in situ reduction with sodium borohydride.

In practice, a cold aqueous solution of epi-K5-N-sulfate is adjusted to an acidic pH (approximately 2) with hydrochloric acid and treated in the cold with sodium nitrite at a constant temperature (approximately 4 ° C) and pH (approximately 2), and upon completion of depolymerization ( about 15-30 minutes) the solution is neutralized with sodium hydroxide and treated, again at a temperature of about 4 ° C, with an aqueous solution of sodium borohydride. Upon completion of the recovery (approximately 4 hours), the excess sodium borohydride is removed with hydrochloric acid, the solution is neutralized with sodium hydroxide and the depolymerized (and reduced) product is isolated in accordance with known methods, for example, by direct precipitation with ethanol or acetone. The product obtained at the end of depolymerization can be either HM epi-K5-N-sulfate (in this case, it is the final product) or NM K5-N-sulfate (in this case, it directly undergoes C5-epimerization, as described above , after isolation or in solution without prior isolation), in particular, if its molecular weight is more than 4000, preferably at least 6000, or it is disposed of to obtain NM K5-N, O-hypersulphate with antiangiogenic and antiviral activity . With proper control of the depolymerization reaction, in particular using various amounts of sodium nitrite / hydrochloric acid, NM K5-N-sulfates or NM epi-K5-N-sulfates are obtained with an average molecular weight over the entire range from about 1500 to 12000, preferably from 1500 to 10000, more preferably from 1500 to 7500, which is calculated from the ¹³ C-NMR spectrum by integrating the signal related to the C2 atom of 2,5-anhydromannitol and the signal from the anomeric carbon of glucosamine on the polysaccharide chain.

In accordance with the general implementation of the method, for example, starting from 1 g of epi-K5-N-sulfate, the starting product is dissolved in 100-200 ml of deionized water and placed in a thermostat at 4 ° C. Then, such an amount of sodium nitrite is added to obtain the desired molecular weight, for example, from 2000 to 4000. Thus, based on epi-K5-N-sulfate with an average molecular weight of 20,000 when measured by HPLC on a BioRad BioSil 250 column using a standard heparin with a known molecular weight, you will need to add from 330 to 480 mg of sodium nitrite in the form of a 0.2% aqueous solution. A solution containing epi-K5-N-sulfate and sodium nitrite at 4 ° C was adjusted to pH 2 by adding 0.1 N HCl, cooled to 4 ° C. The solution is left for 20-40 minutes for the reaction to proceed with slow stirring, and then neutralized with 0.1 N NaOH. The resulting product was brought to room temperature and treated with a reducing reagent, for example sodium borohydride (250-500 mg was dissolved in 50-100 ml of water) and left to react for 4-8 hours. Excess sodium borohydride is removed by adjusting the pH to 5-5.5 with 0.1 N HCl and left for another 2-4 hours. Finally, the solution is neutralized with 0.1 N NaOH and the product is isolated by precipitation with acetone or ethanol after concentration of the product by evaporation under reduced pressure.

Similarly, you can determine the amount of sodium nitrite, which, based on 1 g of K5-N-sulfate or epi-K5-N-sulfate, allows you to get NM K5-N-sulfate or NM epi-K5-N-sulfate with an average molecular weight of 4000 to 12000, preferably from 4000 to 7500, more preferably from 6000 to 7500.

The resulting NM epi-K5-N-sulfate with an iduronic acid content of from 20 to 60%, preferably from 40 to 60%, more preferably from 50 to 55%, and practically devoid of -NH ₂ and N-acetyl groups, with an average molecular weights from about 1,500 to 12,000, preferably from 1,500 to 10,000, more preferably from 1,500 to 7,500, as well as its chemically and pharmaceutically acceptable salts, are new products useful as starting materials of particular interest for the production of epi-K5-N NM , O-hypersulphates, but they themselves are useful in quality The active ingredients of the pharmaceutical or cosmetic compositions constitute an additional aspect of the present invention.

Preferably, the starting materials in the preparation of the epi-K5-N, O-hypersulfate derivatives of the present invention are the epi-K5-N-sulfates derivatives consisting of mixed chains of which at least 90% correspond to formula I:

in which the uronic units are 20-60% composed of iduronic acid, n is an integer from 2 to 100, preferably from 3 to 100, and the corresponding cation is chemically or pharmaceutically acceptable.

More preferably, these starting derivatives of epi-K5-N-sulfates consist of mixed chains of which at least 90% correspond to formula I, in which uronic units are 40-60% composed of iduronic acid, n is an integer from 2 up to 100, preferably from 3 to 100, and the corresponding cation is chemically or pharmaceutically acceptable. Most preferably, the starting materials are NM epi-K5-N-sulfates presented above, consisting of mixed chains, of which at least 90% correspond to formula I, in which the uronic units are 20-60%, preferably 40- 60%, more preferably 50-55%, are composed of iduronic acid, n is an integer from 2 to 20, preferably from 3 to 15, and the corresponding cation is chemically acceptable.

In practice, these preferred NM epi-K5-N-sulfates are composed of mixed chains of which at least 90% correspond to formula I ':

in which the uronic units are 20-60%, preferably 40-60%, more preferably 50-55%, are composed of iduronic acid, q is an integer from 2 to 20, preferably from 3 to 15, and the corresponding cation is chemically or pharmaceutically acceptable.

In this context, the term "chemically" refers to a cation used in chemical synthesis, such as sodium ions, ammonium, (C ₁ -C ₄ ) -tetraalkylammonium, or in the purification of a product.

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

Of particular interest are NM epi-K5-N-sulfates consisting of mixed chains of which at least 90% correspond to the above formula I ', which are obtained by nitrogen depolymerization of the corresponding epi-K5-N-sulfates above, and subsequent optional recovery, for example, sodium borohydride. Of these, NM epi-K5-N-sulfates consisting of mixed chains are preferred, among which chains according to the formula I'a prevail:

in which the uronic units are 60-40% glucuronic acid and 40-60% iduronic acid, p is an integer from 4 to 8. The average molecular weight of these products is approximately 2000 to 4000, and the corresponding cation is chemically or, pharmaceutically acceptable.

The preparation of these epi-K5-M-sulfates, starting from the stage of nitrogen depolymerization, involves the presence at the reducing end of most chains of this mixture of a 2,5-anhydromanose link or, if, for example, sodium borohydride, a 2,5-anhydromannitol link is reduced structure (a):

in which X means formyl or hydroxymethyl. Therefore, the reducing end in most (60-70%) chains is actually represented by structure (b):

in which X is defined above.

The presence of structure (a) does not affect the chemical characteristics of epi-K5-N-sulfates and their derivatives, since sulfation can only lead to the introduction of one or two sulfate groups, but this will not significantly change the degree of sulfation of O-sulfated derivatives. However, it is preferable that nitrogen depolymerization is followed by reduction, for example, with sodium borohydride, since, according to the method of the present invention, NM epi-K5-N-sulfates undergo sulfation and acylation reactions, the effects of which on the formyl group, when X represented by formyl, the 2,5-anhydromanose radical of structure (a) is unknown. In addition, the presence of structure (a) does not affect the biological activity of products, as shown in Østergaard R.V. et al., Thrombosis Research, 1987, 45, 739-749 (Østergaard 1987) for low molecular weight heparins.

Particularly preferred NM epi-K5-N-sulfates of the present invention are composed of mixed chains, among which the compounds of formula I'b predominate:

in which X is formyl or preferably hydroxymethyl, m is 4, 5 or 6, the corresponding cation is chemically or pharmaceutically acceptable, and the glucuronic and iduronic units alternate starting from the glucuronic or iduronic unit, and the ratio between the glucuronic and iduronic units is from 45 / 55 to 55/45, i.e. approximately 50/50.

Therefore, the use of C5 epimerase, preferably recombinant, preferably immobilized on a solid support under the conditions described above, promotes not "cluster" epimerization of derivatives of K5-N-sulfates to derivatives of epi-K5-N-sulfates, as occurs in nature, but epimerization of the usual type.

Thus, in accordance with a further aspect, the present invention provides for the use of isolated and purified C5 epimerase to convert derivatives of K5-N-sulfates to the corresponding derivatives of epi-K5-N-sulfates, characterized by the presence of a repeating tetrasaccharide unit consisting of two glucosamine units, separated first glucuronic link, and then the iduronic link, or separated first by the iduronic link, and then by the glucuronic link.

This epimerization occurs optimally if it is carried out with a derivative of K5-N-sulfate with an average molecular weight of more than 4000, preferably from 6000 to 7500.

In accordance with the present invention, the starting derivatives of epi-K5-N-sulfates, preferably 100% N-sulfated (in particular, derivatives of epi-K5-N-sulfates, consisting of mixed chains of which at least 90% correspond to the formula I or I ', or among which chains according to the formula I'a or I'b prevail, where X is hydroxymethyl) are sent to the above stages (a) and (b), after which the corresponding new epi-K5- derivatives are isolated amine-O-hypersulphates in which the amino group is not substituted and which are usually o have the form of a sodium salt, which can be converted into another chemically or pharmaceutically acceptable salt. Salts of alkali metals, alkaline earth metals, ammonium, (C ₁ -C ₄ ) -tetraalkylammonium, aluminum and zinc are especially preferred, and of these, sodium, calcium and tetrabutylammonium salts are preferred.

Thus, in accordance with the following aspect, the present invention relates to new derivatives of epi-K5-amine-O-hypersulphates and their chemically or pharmaceutically acceptable salts, obtained by a method which is characterized in that:

(a) an epi-K5-N-sulfate derivative, in acid form, is treated with a tertiary or quaternary organic base, leaving the reaction mixture for 30-60 minutes and maintaining the pH of the solution at about 7 by adding this tertiary or quaternary organic base, and its salt is isolated formed with this organic base;

(b) this salt of the organic base and this epi-K5-N-sulfate derivative is treated with an O-sulfating reagent under O-hypersulfation conditions and an epi-K5-amine-O-hypersulfate derivative is isolated.

Using as starting material for stage (a) derivatives of epi-K5-N-sulfates, consisting of mixed chains, of which at least 90% correspond to the above formula I, in which the uronic units are 20-60% composed of iduronic acid, n is an integer from 3 to 100, and the corresponding cation is chemically or pharmaceutically acceptable, at the end of step (b) an epi-K5-amine-O-hypersulfate derivative is obtained consisting of mixed chains of which at least 90% correspond to formula II:

in which the uronic units are 20-60% composed of iduronic acid, n is an integer from 2 to 100, preferably from 3 to 100, R, R 'and R "are hydrogen or SO ₃ ^- , and the degree of sulfation is at least 3.4, preferably at least 3.5, more preferably from 3.55 to 4, most preferably from 3.55 to 3.8, and the corresponding cation is chemically or pharmaceutically acceptable.

These very-sulfated, epi-K5-amine-O-hypersulfates are new products useful as intermediates for their N-sulfated or H- (C ₂ -C ₄ ) acylated derivatives, which are practically devoid of activity on blood coagulation parameters, but with other interesting pharmacological properties.

Preferably, very high sulfation derivatives of epi-K5-amine-O-hypersulphates consist of mixed chains of which at least 90% correspond to formula II, in which uronic acid units are 40-60% composed of iduronic acid, n - an integer from 2 to 100, preferably from 3 to 100, and have an average molecular weight of from about 2000 to 40,000, preferably from 4500 to 40,000, R of at least 40%, preferably 50-80%, is represented by SO ₃ ^- , R 'and R''are both represented by SO ₃ ^- either one of them is represented by hydrogen, and the other is 5-10% represented by SO ₃ ^- in glucuronic acid monosulfate and 10-15% represented by SO ₃ ^- in iduronic acid monosulfate, the degree of sulfation being more than 3.4, preferably at least 3.5, more preferably from 3.55 to 4, most preferably 3.55 to 3.8, and the corresponding cation is chemically or pharmaceutically acceptable.

Preferred derivatives of epi-K5-amine-O-hypersulphates with a very high degree of sulfation are NM epi-K5-amine-O-hypersulphates consisting of mixed chains of which at least 90% correspond to formula II, in which units of 40-60%, preferably 50-55%, are composed of iduronic acid, R, at least 40%, preferably 50-80%, more preferably 65% are SO ₃ ^- , R 'and R''both are represented by SO ₃ ^- either one of them is represented by hydrogen, and the other - by 5-10% is represented by SO ₃ ^- in glucuronic acid and by 10-15% is represented by SO ₃ ^- in iduronic acid, n is an integer from 2 to 20, preferably from 3 to 15, and has an average molecular weight of from about 4000 to 8000, and the corresponding cation is chemically or pharmaceutically acceptable.

In practice, these preferred NM epi-K5-amine-O-hypersulphates are composed of mixed chains of which at least 90% correspond to formula II ′:

in which the uronic units are 20-60% composed of iduronic acid, q is an integer from 2 to 20, preferably from 3 to 15, R, R 'and R''are hydrogen or SO ₃ ^- , the degree of sulfation being at least 3.4, preferably at least 3.5, more preferably from 3.55 to 4, most preferably from 3.55 to 3.8, and the corresponding cation is a chemically or pharmaceutically acceptable ion.

Of these NM epi-K5-amine-O-hypersulphates, those consisting of mixed chains are preferable, among which chains corresponding to formula II'a predominate:

in which the uronic units consist of 20-60% iduronic acid, p is an integer from 4 to 8, R, R 'and R "are already defined above, and the degree of sulfation is at least 3.4, preferably at least 3.5, more preferably from 3.55 to 4, most preferably from 3.55 to 3.8, and the corresponding cation is chemically or pharmaceutically acceptable.

Obtaining new NM epi-K5-amine-O-hypersulphates from NM epi-K5-sulfates obtained by nitrogen depolymerization and subsequent reduction, for example, sodium borohydride, involves the presence on the reducing end of most chains from this mixture of sulfated unit 2.5 anhydromannitol structure (a '):

in which R "means hydrogen or SO ₃ ^- .

Thus, the reducing end in most chains of this mixture is represented by structure (b '):

in which the uronic unit may be glucuronic or iduronic.

Of the above-described new NM epi-K5-amine-O-hypersulphates, those consisting of mixed chains are preferred, among which the compounds according to formula II'b predominate:

in which the uronic units are 40-60% composed of iduronic acid, m is 4, 5 or 6, R, R 'and R''are represented by hydrogen or SO ₃ ^- , X''means OH or OSO ₃ ^- , and the degree of sulfation is at least 3.4, preferably at least 3.5, more preferably from 3.55 to 4, most preferably from 3.55 to 3.8, the iduronic units alternate starting from the glucuronic or iduronic unit and the corresponding cation is chemically or pharmaceutically acceptable.

All of these highly sulfated epi-K5-amine-O-hypersulphate derivatives are novel products that are useful intermediates for the preparation of new N-substituted epi-K5-amin-O-hypersulphate derivatives, therefore they constitute an additional aspect of the present inventions.

In particular, in accordance with a further aspect, the present invention relates to the use of the very high sulfation derivatives of epi-K5-amine-O-sulfates described above for the preparation of new N-substituted derivatives of epi-K5-amine-O-hypersulfates, in particular N-sulfated or N-acylated.

Upon completion of step (c) of the method of the present invention, consisting of N-sulfation of derivatives of epi-K5-amine-O-hypersulphates obtained in stage (b) (especially derivatives of epi-K5-amine-O-hypersulphates consisting of mixed chains of which at least 90% correspond to formula II or II ', or among which chains according to formula II'a or II'b predominate), derivatives of epi-K5-N, O-hypersulphates in which iduronic acid is 20-60% of the sum of uronic acids, and the degree of sulfation is at least 4, preferably from 4 to 4 , 6.

Thus, in accordance with a further aspect, the present invention provides new N-deacetylated K5 polysaccharide derivatives, O-sulfated and N-sulfated, C5-epimerized to iduronic acid, by at least 20% of the sum of the uronic units, with an average molecular weight of approximately from 2000 to 45000, with a degree of sulfation of at least 4, and these derivatives have practically no activity on the parameters of blood coagulation.

Similarly to what was said above, these new derivatives are generally referred to by the general term "derivatives of epi-K5-N, O-hypersulphates", regardless of their molecular weight.

In particular, their average molecular weight is from about 2000 to 45000, since these derivatives come either from epi-K5-K-sulfate obtained by N-deacetylation and N-sulfation of K5 during fermentation, or by its nitrogen depolymerization. By controlling nitrogen depolymerization, low molecular weight derivatives can be obtained over virtually the entire specified range. However, for the use of the derivatives of the present invention as pharmaceutical or cosmetic products, it is preferable to obtain low molecular weight derivatives with an average molecular weight of from about 2000 to 16000, preferably from 3500 to 13000 with a molecular weight distribution between 1000 and 15000, more preferably from 4500 to 9000 with a molecular distribution weights between 2000 and 10000, or high molecular weight derivatives derived from unfractionated K5, with an average molecular weight of approximately 20,000 to 45,000, with definiteness of molecular weights between 2000 and 70,000.

In the epi-K5-N, O-hypersulfate derivatives of the present invention, the degree of sulfation is very high, preferably from 4 to 4.6, while the nitrogen atom of glucosamine is almost 100% sulfated. In addition, the derivatives of epi-K5-N, O-hypersulphates 6-O-sulfated at 100% and 3-O-sulfated at 50-80% in glucosamine units, 3-O-monosulfated in 5-10% in glucuronic units, O-monosulfated by 10-15% on the iduronic units and 2,3-di-O-sulfated on the remaining iduronic units, given that the degree of sulfation is at least 4.

Preferred derivatives of epi-K5-N, O-hypersulphates of the present invention are obtained through derivatives of epi-K5-amine-O-hypersulphates, which, in turn, are derived from derivatives of epi-K5-N-sulfates consisting of mixed chains of of which at least 90% correspond to formula I, in which the uronic units are 20-60% composed of iduronic acid, n is an integer from 2 to 100, preferably from 3 to 100, and the corresponding cation is chemically or pharmaceutically acceptable.

In this case, the new derivatives of epi-K5-N, O-hypersulphates consist of mixed chains, of which at least 90% correspond to formula III:

in which the uronic units are 20-60% composed of iduronic acid, n is an integer from 2 to 100, preferably from 3 to 100, R, R 'and R''are hydrogen or SO ₃ ^- , Z is SO ₃ ^- , moreover, the degree of sulfation is at least 4, preferably from 4 to 4.6, and the corresponding cation is chemically or pharmaceutically acceptable.

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

Of the above new derivatives epi-K5-N, O-gipersulfatov those composed of mixed circuits, of which at least 90% conform to the formula III, wherein R is represented by SO ₃ ^- in 50-80% of the chains, preferably 65% of the chains, and the degree of sulfation is at least 4, preferably from 4 to 4.6, more preferably from 4 to 4.3.

Preferably, very high sulfation derivatives of epi-K5-N, O-hypersulphates are composed of mixed chains of which at least 90% correspond to formula III, in which Z is SO ₃ ^- , the uronic units are 40-60% from iduronic acid, n is an integer from 2 to 100, preferably from 3 to 100, and have an average molecular weight of from about 2000 to 45000, preferably from 4500 to 45000, R, at least 40%, preferably 50- 80%, represented by SO ₃ ^-, R 'and R''are both represented SO ₃ ^- or one of them is hydrogen and the other is - 5-10% n edstavlen SO ₃ ^- in monosulfate glucuronic acid and 10-15% is represented by SO ₃ ^- in monosulfate iduronic acid, the degree of sulfation of at least 4, preferably from 4 to 4.6 and the corresponding cation is chemically or pharmaceutically acceptable .

Preferred N-substituted epi-5-amino-O-hypersulphated derivatives of HM-epi-5-amino-O-hypersulphated derivatives are chain mixtures in which at least 90% of the chains have the formula III, in which 40-60% the uronic units preferably contain 50-55% of iduronic acid, R is at least 40%, preferably 50-80%, particularly preferably 65% SO ₃ ^- , R 'and R''are both SO ₃ ^- or one of them is hydrogen and the other is 5-10% SO ₃ ^- in glucuronic acid and 10-15% SO ₃ ^- in iduronic acid, Z is a 100% SO ₃ ^- or (C ₂ - ₄₎ atsillom, n is an integer from 2 to 20, preferably from 3 to 15, a molecular weight of from about 4000 to about 8500 and the corresponding cation is chemically or pharmaceutically acceptable.

In practice, these preferred derivatives of epi-K5-N, O-sulfates with a very high degree of sulfation are composed of mixed chains of which at least 90% correspond to formula III ':

in which the uronic units are 20-60% composed of iduronic acid, q is an integer from 2 to 20, preferably from 3 to 15, R, R 'and R''are hydrogen or SO ₃ ^- , Z is SO ₃ ^- , moreover, the degree of sulfation is at least 4, preferably from 4 to 4.6, and the corresponding cation is a chemically or pharmaceutically acceptable ion.

Of particular interest are the mixed chains according to formula III ', in which the uronic units are 40-60%, preferably 50-55%, of iduronic acid, R is at least 40%, preferably 50-80%, more preferably 65% represented by SO ₃ ^- , R 'and R''both represented by SO ₃ ^- either one of them is represented by hydrogen, and the other - by 5-10% is represented by SO ₃ ^- in glucuronic acid and by 10-15% is represented by SO ₃ ^- in iduronic acid, n is an integer from 2 to 20, preferably from 3 to 15, and have an average molecular weight of from about 2000 to 16000, preferably from 3500 up to 13000, more preferably from 4500 to 9000, and the corresponding cation is chemically or pharmaceutically acceptable.

Of these NM epi-K5-N, O-hypersulphates, those consisting of mixed chains are preferable, among which chains corresponding to formula III'a predominate:

in which the uronic units are 20-60% composed of iduronic acid, p is an integer from 4 to 8, Z is SO ₃ ^- , R, R 'and R''are hydrogen or SO ₃ ^- , and the degree of sulfation is at least 4, preferably from 4 to 4.6, and the corresponding cation is chemically or pharmaceutically acceptable.

Obtaining new NM epi-K5-N, O-hypersulphates from NM epi-K5-sulfates obtained by nitrogen depolymerization and subsequent reduction, for example, sodium borohydride, includes the presence, at the reducing end, on most of the chains of this mixture, sulfated link 2 , 5-anhydromannitol of structure (a ') above, wherein R "is hydrogen or SO ₃ ^- .

Thus, the reducing end in most chains of this mixture is represented by structure (b ''):

in which Z is SO ₃ ^- , and the uronic unit may be glucuronic or iduronic.

Of the above-described new NM epi-K5-N, O-hypersulphates, those which are mixtures are preferred, among which species which are compounds of the formula III'b predominate:

in which R, R 'and R "are hydrogen or SO ₃ ^- , Z is SO ₃ ^- , and X" is OH or OSO ₃ ^- , m is 4, 5 or 6, wherein the degree of sulfation is at least , 4, preferably from 4 to 4.6, the uronic units alternate starting from the glucuronic or iduronic unit, and the corresponding cation is chemically or pharmaceutically acceptable. The cations of alkali metals, alkaline earth metals, ammonium, (C ₁ -C ₄ ) -tetraalkylammonium, aluminum and zinc are preferred, and of these, sodium, calcium and tetrabutylammonium ions are most preferred.

If epi-K5 is used as the starting epi-K5 derivative in the method of the present invention, that is, K5 polysaccharide pre-N-deacetylated, N-sulfated usually 100%, C5-epmerized 20-60% and not depolymerized, then upon completion of step (c) epi-K5-N, O-hypersulfate is released, which can be subjected to nitrogen depolymerization and subsequent reduction, for example, with sodium borohydride, to obtain the corresponding NM epi-K5-N, O-hypersulfate having the same degree of sulfation . In particular, NM epi-K5-N, O-hypersulfates are obtained, consisting of mixed chains, of which at least 90% correspond to formula III 'or III'a, in which the uronic units are 20-60% composed of iduronic acids, q, R, R ′, R ″ and Z are already defined above, the degree of sulfation being at least 4, preferably from 4 to 4.6, and the corresponding cation is chemically or pharmaceutically acceptable. In this case, the preparation of these NM epi-K5-N, O-hypersulphates by means of a nitrogen depolymerization reaction and possible subsequent reduction, for example, with sodium borohydride, involves the presence of a 2,5-anhydromannitol structure (a " ):

in which X is formyl or hydroxymethyl, and R ″ is hydrogen or SO ₃ ^- .

The new derivatives of epi-K5-N, O-hypersulphates, especially in the form of salts, are highly anionic products capable of trapping free radicals, and they can be used in the cosmetics industry as adjuvants against hair loss or to obtain anti-aging creams and in the pharmaceutical industry as a treatment for dermatitis. In addition, the epi-K5-N, O-hypersulphate derivatives of the present invention, in particular HM epi-K5-N, O-hypersulphates, have antiangiogenic and antiviral activity and therefore can serve as active ingredients for the preparation of drugs.

Thus, in accordance with one additional aspect, the present invention provides pharmaceutical compositions comprising, as one of the active ingredients, a pharmacologically active amount of an epi-K5-N, O-hypersulfate derivative described above, or one of its pharmaceutically acceptable salts, together with a pharmaceutical filler.

In the pharmaceutical compositions of the present invention for oral, subcutaneous, intravenous, transdermal or external use, the active ingredients are preferably administered in unit dosage form, together with classical pharmaceutical excipients or carriers. Dosage can vary greatly depending on the age, weight and health status of the patient. It includes administering a dose of 1 to 1000 mg, preferably 10 to 750 mg, more preferably 250 to 500 mg, one to three times a day, by the intravenous, subcutaneous, oral, transdermal or external route. The pharmaceutical compositions of the present invention are formulated with classic excipients suitable for various uses. Formulations in the form of creams, ointments, liquid ointments, gels, foams, balsams, vaginal rings, suppositories, solutions or suspensions suitable for topical application are particularly preferred.

Preferably, the compositions of the present invention comprise, as one of the active ingredients, an epi-K5-N, O-hypersulfate derivative prepared from an epi-K5 derivative in accordance with steps (a), (b) and (c) of the above method, or starting from the undepolymerized epi-K5 in accordance with steps (a), (b) and (c) of the above method with possible subsequent nitrogen depolymerization after step (c), or one of its pharmaceutically acceptable salts, together with a pharmaceutical excipient. Preferably, this epi-K5-N, O-hypersulfate derivative consists of mixed chains of which at least 90% correspond to formula III or III ', or among which compounds of formula III'a or III'b predominate. A preferred active ingredient is NM epi-K5-N, O-hypersulphate with a degree of sulfation of at least 4, preferably from 4 to 4.6, preferably with an average molecular weight of from about 3500 to 11000, more preferably from 3500 to 5200, and virtually devoid of N-acetyl groups.

Finally, in accordance with a further aspect, the present invention provides a cosmetic composition comprising an effective amount of an epi-K5-N derivative, O-hypersulphate or a pharmaceutically acceptable salt thereof, together with a cosmetic excipient.

A salt selected from the group consisting of salts of sodium, potassium, calcium, magnesium, aluminum and zinc derivatives of epi-K5-N, O-hypersulphates, in particular consisting of mixed chains, of which at least 90% correspond to formula III or III ', or among which the compounds of formula III'a or III'b predominate, is an effective active ingredient in the pharmaceutical or cosmetic composition of the present invention.

The following examples disclose the invention but do not limit it.

Preparative example I

Obtaining polysaccharide K5 from Escherichia coli

First, fermentation is carried out in a conical flask using the following medium, g / l:

Low Fat Soya Powder 2 K ₂ HPO ₄ 9.7 KN ₂ PO ₄ 2 MgCl ₂ 0.11 Sodium citrate 0.5 Ammonium sulfate one Glucose 2 Drinking water, ml 1000

pH = 7.3

The medium is sterilized at 120 ° C. for 20 minutes. Glucose is prepared separately in the form of a solution, which is sterilized at 120 ° C for 30 minutes and added to the medium aseptically. A suspension of E. coli Bi 8337/41 cells (O10: K5: H4) originating from a jamb with tryptic soy agar was inoculated into a conical flask and incubated at 37 ° C for 24 hours with moderate shaking (160 rpm, range 6 cm). Bacterial growth is measured by counting cells under a microscope. In the next step, 0.1% culture from a conical flask was inoculated into a 14 L Chemap-Braun fermenter containing the same medium and fermented with air transmission (1 volume of air per 1 volume of liquid in 1 minute) with stirring at 400 rpm min at a temperature of 37 ° C for 18 hours. During fermentation, pH, oxygen, glucose, K5 polysaccharide production and bacterial growth are measured. After fermentation, the temperature was adjusted to 80 ° C for 10 minutes. Cells are separated from the medium by centrifugation at 10,000 rpm and the supernatant is subjected to ultrafiltration in an SS 316 (MST) module equipped with a PES membrane with a nominal cut-off of 800 to 10,000 D, reducing the volume to 1/5. Then the K5 polysaccharide is precipitated by adding 4 volumes of acetone at 4 ° C and left overnight at 4 ° C. After that, it is isolated by centrifugation at 10,000 rpm for 20 minutes or by filtration. Proteins were removed from the resulting material using type II protease from Aspergillus orizae in a buffer of 0.1 M NaCl and 0.15 M EDTA, pH 8, containing SDS (0.5% sodium dodecyl sulfate) (10 mg / L filtrate) at 37 ° C for 90 minutes The resulting solution was ultrafiltered in an SS 316 module equipped with a PES membrane with a nominal cut-off of up to 10,000 D, with two extractions of 1 M NaCl, and washed with water until the optical absorption in the ultrafiltrate disappeared. Then the K5 polysaccharide is precipitated with acetone in a yield of 850 mg per 1 liter of fermenter. The purity of the polysaccharide obtained is measured by determining uronic acids (carbazole method), ¹ H- and ¹³ C-NMR, UV and protein content. Purity is more than 80%.

The resulting polysaccharide consists of two molecules of different molecular weights of 30,000 and 5,000 D, respectively, as follows from the HPLC determination on a Pharmacia 75 HR column, and one molecule with a retention time of about 9 minutes using two sequential Bio-Sil SEC 250 columns (BioRad) and Na ₂ SO ₄ as the mobile phase at room temperature at a rate of 0.5 ml / min. The determination is carried out according to a standard curve obtained using heparin molecules of known molecular weight.

The ¹ H-NMR spectrum of the purified K5 thus obtained displays various signals characteristic of the methyl groups of lipophilic substances.

Preparative example II

Cleaning K5

In 100 ml of a saturated aqueous solution of sodium chloride at 4 ° C, 1 g of K5 obtained in Preparation Example I is dissolved, and 3 volumes of cold isopropanol are added to the resulting solution. The salt concentration in the solution was adjusted to 3 M by adding the calculated volume of a saturated solution of sodium chloride and the resulting solution was left in a cold place (about 4 ° C) overnight. The precipitate formed is separated by centrifugation at 10,000 rpm for 20 minutes and the purity of the product is checked by dialysis overnight and examination of the ¹ H-NMR spectrum, in which there should be no signals in the region of less than 1.5 ppm. Optionally, the operation of dissolving in water saturated with NaCl and precipitating with isopropanol is repeated. The precipitate was dissolved in water and ultrafiltered through a Millipore Miniplate membrane with a cut-off of 10,000 D until salts disappeared. In this case, K5 is obtained with a purity of at least 99%, for which lipophilic impurity signals in the region of less than 1.5 ppm are not detected in the ¹ H-NMR spectrum.

Preparative Example III

Getting KS-N-sulfate

(i) N-Deacetylation

10 g of purified K5 polysaccharide obtained as described in Preparation Example II was dissolved in 1000 ml of 2N NaOH and the resulting solution was left at 60 ° C for 24 hours. The solution was brought to room temperature and then to a neutral pH (pH 7) with 6N hydrochloric acid.

(ii) N-Sulfation

16 g of sodium carbonate are added to a solution containing deacetylated K5 at 40 ° C and after 4 hours - 16 g of pyridine-SO ₃ ^- . At the end of the reaction, after 24 hours, the solution was brought to room temperature, and then to a pH of 6.5-7 using a 5% hydrochloric acid solution. The product is purified from salts by diafiltration through a spiral membrane with a cut-off of 1000 D (preparative cartridge from Millipore). The process is stopped when the conductivity of the solution is less than 1000 μS, preferably less than 100 μS. The dialysate volume is reduced to achieve a polysaccharide concentration of 10 ° / o using the same dialysis system. The concentrated solution is dried by lyophilization. When analyzing the ¹³ C-NMR spectrum, N-acetyl or -NH ₂ residues were not detected.

Preparative Example IV

Low molecular weight KS-N-sulfate

The product obtained as described in Example 1, stage (i) and (ii) of patent WO 02/068477, is subjected to depolymerization by the method of splitting with nitrous acid and subsequent recovery of the resulting aldehyde. For this, 1 g 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 is added. Then the solution was adjusted to 4 ° C and to pH 2 with 0.1 N HCl and left for 30 minutes. At the end of the reaction, the solution was adjusted to pH 7 with 0.1 M NaOH, and then to room temperature. Then, 450 mg of NaBH _{4 was} added to the solution and allowed to react for 4 hours. Excess NaBH _{4 is} removed with HCl, adjusting the pH to 5-6. After neutralization with 0.1 M NaOH, the product is isolated by precipitation with 3 volumes of acetone at 4 ° C with filtering on a funnel and dried at 40 ° C in vacuum drying. Receive 900 mg NM K5-N-sulfate with an average molecular weight of about 2000, consisting of mixed chains, among which the compounds of the formula I'b prevail, in which m is 4, and the uronic units are glucuronic acid.

Example 1

NM epi-K5-N-sulfate. The sequence (i) → (ii)

(i) Epimerization to epi-K5-N-sulfate

10 g of K5-N-sulfate obtained as described in Example 1, steps (i) and (ii) of patent WO 02/068477, in which signals corresponding to N-acetyl or -NH residues are not detected in the ¹ H-NMR spectrum ₂ , dissolved in 600 ml of 25 mM Hepes buffer, pH 7, containing CaCl ₂ at a concentration of 50 mM, and the resulting solution was passed through a 50 ml column filled with Sepharose 4B containing 5 g of recombinant C5 epimerase (WO 96/14425), immobilized as described in Example 1 of WO 01/72848. The reaction is carried out at 30 ° C at pH 7 with a transmission rate of 200 ml / h for 24 hours. The resulting product is purified by ultrafiltration and ethanol precipitation. An epi-K5-N-sulfate is obtained with an iduronic acid content of 54%.

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

1 g of the obtained product is dissolved in 25 ml of distilled water, and 230 mg of sodium nitrite dissolved in 115 ml of distilled water is added to this solution. Then the solution was adjusted to 4 ° C and to pH 2 with 0.1 N HCl and left for 30 minutes. At the end of the reaction, the solution was brought to room temperature and to pH 7 with 0.1 M NaOH. Then, 450 mg of NaBH _{4 was} added to the solution and allowed to react for 4 hours. The product is isolated by precipitation with 3 volumes of acetone at 4 ° C with filtering on a funnel and dried at 40 ° C in vacuum drying. Receive 900 mg of NM epi-K5-N-sulfate with an iduronic acid content of 54% and a molecular weight distribution of from 1000 to 4000 when measured by HPLC.

Example 2

NM epi-K5-N-sulfate. The sequence (ii) → (i)

(ii) Depolymerization of KS-N-sulfate

2 g of K5-N-sulfate obtained as described in Example 1, steps (i) and (ii) of patent WO 02/068477 are depolymerized as described in Preparation Example I using 100 mg of sodium nitrite and 300 mg of sodium borohydride . Obtain 1.8 g of NM K5-N-sulfate with an average molecular weight of 5000.

(i) Epimerization of NM KS-N-sulfate

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

Example 3

NM epi-K5-N-sulfate. The sequence (i) → (ii)

(i) Epimerization of KS-N-sulfate

A portion in 2 g of K5-N-sulfate obtained as described in Example 1, steps (i) and (ii) of WO 02/068477 is dissolved in 120 ml of 25 mM Hepes buffer, pH 7, containing 50 mM CaCl ₂ . The resulting solution was passed through a 50 ml column filled with a vehicle containing an immobilized enzyme prepared as described in WO 96/14425. This operation is carried out at 30 ° C. at a flow rate of 200 ml / h for 24 hours. The resulting product was purified by ultrafiltration through a 1000 D cut-off membrane and passing through an IR 120 H ⁺ ion exchanger column, neutralizing the eluate with 1N NaOH. A sample is isolated by precipitation with ethanol or acetone. An epimerized product with an iduronic / glucuronic acid ratio of 55/45 is obtained compared to a 0/100 ratio of the starting material. The degree of epimerization is calculated by 1 H-NMR in accordance with the method described in WO 96/14425. The yield calculated by measuring the uronic acid content compared to the standard according to the carbazole method (Bitter and Muir, Anal. Biochem. 1971, 39, 88-92) is 90%.

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

1 g of the product obtained in stage (a) is subjected to depolymerization by splitting with nitrous acid and subsequent reduction of the resulting aldehyde. For this, the product is dissolved in 25 ml of distilled water and 230 mg of sodium nitrite dissolved in 115 ml of distilled water are added thereto. Then the solution was adjusted to 4 ° C and to pH 2 with 0.1 N HCl and left for 30 minutes. At the end of the reaction, the solution was brought to room temperature and to pH 7 with 0.1 M NaOH. Then, 450 mg of NaBH _{4 was} added to the solution and allowed to react for 4 hours. The product is isolated by precipitation with 3 volumes of acetone at 4 ° C with filtering on a funnel and dried at 40 ° C in vacuum drying. Receive 900 mg of NM epi-K5-N-sulfate with a molecular weight distribution when measured by HPLC in the range from 1000 to 4000, with a glucuronic acid content of 45% and an iduronic acid content of 55%.

Example 4

Epi-K5-N, O-Hypersulfate

(a) Tetrabutylammonium salt of epi-K5-N-sulfate

400 mg of epi-K5-N-sulfate obtained at the end of step (i) of Example 1 is dissolved in 40 ml of water, the solution is placed in a thermostat at 4 ° C, and then passed through an IR 120 ⁺ ion exchanger, previously balanced with water at 4 ° FROM. The resulting eluate, consisting of 100 ml of a solution with a pH of 1.94, is neutralized with a 15% tetrabutylammonium hydroxide solution and left at room temperature for 1 hour, maintaining a pH of about 7 by adding 15% tetrabutylammonium hydroxide, and then lyophilized. This gives 805 mg of the tetrabutylammonium salt of epi-K5-N-sulfate.

(b) Epi-K5-amine-O-hypersulfate

A solution containing 805 mg of the salt obtained above in 30 ml of dimethylformamide was brought to 55 ° C and treated with 30 ml of dimethylformamide containing 2.26 g of the compound pyridine · SO ₃ . The reaction at 55 ° C. was continued overnight, and then 60 ml of water was added to this mixture. After neutralization with 1N NaOH, the product is precipitated with 3 volumes of acetone saturated with NaCl and left at 4 ° C overnight. The precipitate was recovered by filtration on a guch G4 and then ultrafiltered using a Millipore TFF system with 1000 D cut-off and dried under reduced pressure. 550 mg of epi-K5-amine-O-hypersulphate are obtained with an iduronic acid content of 54%, glucosamine-6-O-sulfate 100%, glucosamine-3-O-sulfate 60%, glucuronic acid monosulfate 10%, iduronic acid monosulfate 15% moreover, the remaining iduronic units are disulfated, and the degree of sulfation is 3.55 when measured by the conductometric method according to Casu et al., 1975.

(c) N-sulfation of epi-K5-amine-O-hypersulfate

250 mg of the epi-K5-amine-O-hypersulfate obtained in step (b) was dissolved in 15 ml of water and 400 mg of sodium carbonate was added, and then 400 mg of pyridine · SO ₃ in solid form was added in small portions to the resulting mixture. 4 hours. The reaction mixture was kept at 55 ° C. overnight, and then the reaction was stopped by adjusting to pH 7 with 0.1 N HCl. After ultrafiltration through a membrane with a cut-off of up to 1000 D, 3 volumes of acetone saturated with sodium chloride were added, and the precipitate was recovered by centrifugation at 5000 rpm for 5 min. In this case, 244 mg of epi-K5-N, O-hypersulfate was obtained, in which the degree of sulfation was 4.25 when measured by the conductometric method according to Casu et al., 1975. From an analysis of the ¹ H-NMR spectrum, it follows that the obtained epi-K5 N, O-hypersulfate has an iduronic acid content of 54%, 6-O-sulfate 100%, N-sulfate 100%, glucosamine-3-O-sulfate 60%, glucuronic acid monosulfate 10%, iduronic acid monosulfate 15%, the rest iduronic units are disulfated. From the ¹ H-NMR spectrum, a sulfation degree of 4.35 was calculated, which, taking into account the error limits of the methods, corresponds to the degree of sulfation of epi-K5-amine-O-hypersulfate obtained at the end of step (b) and 100% N-sulfated. Therefore, it is assumed that when a certain percentage of sulfate groups is exceeded, the strongly anionic nature of the product can lead to an underestimation of the degree of sulfation when determined by the conductometric method.

Claims (58)

1. A method of obtaining a derivative of N-deacetylated, N, O-hypersulfated, 20-60% C5-epimerized K5 polysaccharide (epi-K5-N, O-hypersulfate derivative) with a degree of sulfation equal to 4-4.6, characterized in , what: (a) N-deacetylated, N-hypersulfated, 20-60% C5-epimerized K5 polysaccharide (epi-K5-N, O-hypersulfate derivative) with an average molecular weight of about 1500 to 25000, in acid form, is treated with tertiary or a quaternary organic base, leave the reaction mixture for 30-60 minutes at a pH of about 7, and its salt formed with this organic base is isolated; (b) this salt of the organic base and this epi-K5-N-sulfate derivative is treated with an O-sulfating reagent under conditions of O-hypersulfation; (c) the thus obtained N-deacetylated, N, O-hypersulfated, 20-60% C5-epimerized K5 polysaccharide (epi-K5-N, O-hypersulfate derivative) with a degree of sulfation of 3.55-3.8 , N-sulfate using conventional methods and isolate the resulting epi-K5-amine-O-hypersulfate derivative. 2. The method according to claim 1, characterized in that the epi-K5-N, O-hypersulfate derivative is isolated in the form of a sodium salt and optionally converted into another chemically or pharmaceutically acceptable salt. 3. The method according to claim 1, characterized in that at stage (a), tetrabutylammonium hydroxide is used as the organic base. 4. The method according to claim 1, characterized in that in step (b) O-hypersulfation is carried out in dimethylformamide using 2-4 moles of O-sulfating reagent per 1 available OH group per 1 disaccharide at a temperature of 40-60 ° C for 15-20 hours 5. The method according to claim 1, characterized in that the starting material is an epi-K5-N-sulfate derivative 100% N-sulfated and 40-60% C5-epimerized. 6. The method according to claim 5, characterized in that the initial epi-K5-N-sulfate derivative has an average molecular weight of from 10,000 to 25,000. 7. The method according to claim 1, characterized in that the original epi-K5-N-sulfate derivative has an average molecular weight of from about 1500 to 8000. 8. The method according to claim 1, characterized in that the starting material is an epi-K5-N-sulfate derivative consisting of mixed chains, of which at least 90% correspond to formula I:

in which the uronic units are 20-60% composed of iduronic acid, n is an integer from 2 to 100, and the corresponding cation is chemically or pharmaceutically acceptable. 9. The method according to claim 8, characterized in that the starting material consists of mixed chains, of which at least 90% correspond to formula I, in which the uronic units are 40-60% composed of iduronic acid. 10. The method according to claim 8, characterized in that in the formula I n means an integer from 3 to 100. 11. The method according to claim 8, characterized in that the source material consists of mixed chains, of which at least 90% correspond to the formula I ':

in which the uronic units are 20-60% composed of iduronic acid, q is an integer from 2 to 20, and the corresponding cation is chemically or pharmaceutically acceptable. 12. The method according to claim 9, characterized in that in formula I, in which n means an integer from 3 to 15. 13. The method according to claim 5, characterized in that the source material consists of mixed chains, among which the chains according to the formula I'a prevail:

in which the uronic units are 60–40% glucuronic acid and 40–60% iduronic acid, p is an integer from 4 to 8, and the corresponding cation is chemically or pharmaceutically acceptable. 14. The method according to item 13, characterized in that the average molecular weight of this source material is approximately from 2000 to 4000. 15. The method according to 14, characterized in that the source material consists of mixed chains, among which the chains according to the formula I′b prevail:

in which X is hydroxymethyl, m is 4, 5 or 6, and the glucuronic and iduronic units alternate starting from the glucuronic or iduronic unit. 16. The method according to any one of claims 1 to 15, characterized in that the source material is obtained by N-deacetylation and N-sulfation of K5, practically devoid of lipophilic substances. 17. Epi-K5-N, O-hypersulfate derivative obtained in accordance with the method according to claims 1-16. 18. Epi-K5-N, O-hypersulfate derivative according to claim 17, having an iduronic acid content of 20-60%, an average molecular weight of from about 2000 to 45000, and a degree of sulfation of at least 4-4.6, or one of its chemically or pharmaceutically acceptable salts, and this derivative has practically no activity on blood coagulation parameters. 19. Epi-K5-N, the O-hypersulfate derivative of claim 18, wherein the average molecular weight is from about 15,000 to about 45,000. 20. Epi-K5-N, O-hypersulfate derivative according to claim 18, wherein the average molecular weight is from about 4,500 to 8,500. 21. Epi-K5-N, O-hypersulfate derivative according to claim 18, characterized in that it is 100% 6-O-sulfated and 50-80% 3-O-sulfated at glucosamine units, 5-10 % O-monosulfated at glucuronic units, 10-15% 3-O-monosulfated at iduronic units and 2,3-disulfated at other uronic units. 22. Epi-K5-N, O-hypersulfate derivative according to claim 18, characterized in that it consists of mixed chains, of which at least 90% correspond to formula III:

in which the uronic units are 20-60% composed of iduronic acid, R, R 'and R''are hydrogen or SO ₃ ^- , with R being SO ₃ ^- in at least 40% of these mixed chains, Z is SO ₃ ^- , n is an integer from 2 to 100, wherein the degree of sulfation is at least 4, and the corresponding cation is chemically or pharmaceutically acceptable. 23. Epi-K5-N, O-hypersulfate derivative according to claim 18, characterized in that it consists of mixed chains of which at least 90% correspond to formula III, in which the damage units are 40-60% represented by iduronic acid. 24. Epi-K5-N, O-hypersulfate derivative according to claim 22, characterized in that it consists of mixed chains of which at least 90% correspond to formula III, in which n is an integer from 3 to one hundred. 25. Epi-K5-N, O-hypersulphate derivative according to claim 18, characterized in that it is a low molecular weight (HM) epi-K5-N, O-hypersulphate consisting of mixed chains, of which at least 90% correspond to the formula III ':

in which the uronic units are 20-60% composed of iduronic acid, q is an integer from 2 to 20, R, R 'and R''are hydrogen or SO ₃ ^- , Z is SO ₃ ^- , and the corresponding cation is chemically or pharmaceutically acceptable. 26. NM epi-K5-N, O-hypersulfate according to claim 25, characterized in that it consists of mixed chains of which at least 90% correspond to formula III ', in which q is an integer from 3 to fifteen. 27. NM epi-K5-N, O-hypersulfate according to claim 25, characterized in that it consists of mixed chains of which at least 90% correspond to formula III ', in which the damage units are 40-60% composed of iduronic acid. 28. NM epi-K5-N, O-hypersulfate according to claim 25, characterized in that the content of iduronic acid in it is 50-55%. 29. NM epi-K5-N, O-hypersulfate according to claim 26, characterized in that it consists of mixed chains of which at least 90% correspond to formula III ', in which R is at least 40% is represented by SO ₃ ^- , R 'and R''both are represented by SO ₃ ^- either one of them is represented by hydrogen, and the other - by 5-10% is represented by SO ₃ ^- in glucuronic acid and by 10-15% is represented by SO ₃ ^- in iduronic acid. 30. NM epi-K5-N, O-hypersulfate according to claim 26, characterized in that its average molecular weight is approximately from 4000 to 8500. 31. NM epi-K5-N, O-hypersulfate according to claim 26, characterized in that it consists of mixed chains of which at least 90% correspond to formula III ', in which R is 50-80% represented SO ₃ ^- . 32. NM epi-K5-N, O-hypersulfate according to claim 26, characterized in that it consists of mixed chains, among which chains according to formula III'a prevail:

in which the uronic units are 20-60% composed of iduronic acid, p is an integer from 4 to 8, Z is SO ₃ ^- , R, R 'and R''are hydrogen or SO ₃ ^- , and the corresponding cation is chemically or pharmaceutically acceptable. 33. NM epi-K5-N, O-hypersulfate according to claim 32, characterized in that it consists of mixed chains, among which chains according to formula III'b prevail:

in which R, R 'and R''are hydrogen or SO ₃ ^- , Z is SO ₃ ^- , and X' is OH or OSO ₃ ^- , m is 4, 5 or 6, and the iduronic units alternate starting with glucuronic or an iduronic unit, and the corresponding cation is chemically or pharmaceutically acceptable. 34. Epi-K5-N, O-hypersulphate derivative according to any one of claims 18-33, characterized in that the chemically or pharmaceutically acceptable salt or cation is a salt or cation of an alkali metal, alkaline earth metal, ammonium, (C ₁ - C ₄ ) -tetraalkylammonium, aluminum or zinc. 35. Epi-K5-N, O-hypersulfate derivative according to any one of claims 18 to 33, characterized in that the chemically or pharmaceutically acceptable salt or cation is a salt, cation of sodium, calcium or tetrabutylammonium. 36. An Epi-K5-amine-O-hypersulfate derivative having a sulfation degree of 3.55-3.8 is one of its chemically or pharmaceutically acceptable salts, obtained in accordance with stages (a) and (b) according to claim 1, isolated in the form of a sodium salt and, optionally, converted into another chemically or pharmaceutically acceptable salt. 37. The Epi-K5-amine-O-hypersulphate derivative of claim 36, wherein the content of iduronic acid is 20-60% of the sum of uronic acids, with an average molecular weight of about 2000 to 40,000. 38. Epi-K5-amine-O-hypersulfate derivative according to claim 36, characterized in that it consists of mixed chains, of which at least 90% correspond to formula II:

in which the uronic units are 20-60% composed of iduronic acid, n is an integer from 2 to 100, R, R 'and R "are hydrogen or SO ₃ ^- , and the corresponding cation is chemically or pharmaceutically acceptable. 39. Epi-K5-amine-O-hypersulfate derivative according to claim 38, characterized in that in formula II n means an integer from 3 to 100. 40. Epi-K5-amine-O-hypersulphate derivative according to claim 38, characterized in that it consists of mixed chains of which at least 90% correspond to formula II, in which the damage units are 40-60% consist of iduronic acid, with an average molecular weight of about 2000 to 40,000, R is at least 40% represented by SO ₃ ^- , R 'and R''are both represented by SO ₃ ^- either one of them is hydrogen and the other - 5-10% is represented by SO ₃ ^- in monosulfate glucuronic acid and 10-15% is represented by SO ₃ ^- in monosulfate iduronic acid. 41. The Epi-K5-amine-O-hypersulphate derivative of claim 38, characterized in that it is a NM epi-K5-amine-O-hypersulphate consisting of mixed chains of which at least 90% correspond formula II, in which the uronic units are 40-60% composed of iduronic acid, R is at least 40% represented by SO ₃ ^- , R 'and R''are both represented by SO ₃ ^- or one of them is hydrogen, and other - is represented by 5-10% SO ₃ ^- in glucuronic acid and 10-15% is represented by SO ₃ ^- in iduronic acid, n - an integer from 3 to 15, has an average molecular weight of about from 4000 to 8000, and the corresponding cation is chemically or pharmaceutically acceptable. 42. Epi-K5-amine-O-hypersulfate derivative according to clause 36, characterized in that it is HM epi-K5-amine-O-hypersulfate, consisting of mixed chains, among which the chains according to formula II'a prevail:

in which the uronic units are 20-60% composed of iduronic acid, p is an integer from 4 to 8, R, R 'and R''are hydrogen or SO ₃ ^- . 43. NM epi-K5-amine-O-hypersulfate according to claim 42, characterized in that it consists of mixed chains, among which the compounds according to formula II'b predominate:

in which the uronic units are 40-60% composed of iduronic acid, m is 4, 5 or 6, R, R 'and R''are represented by hydrogen or SO ₃ ^- , X''means OH or OSO ₃ , and the iduronic units alternate starting from the glucuronic or iduronic unit. 44. NM epi-K5-N-sulfate, practically devoid of -NH ₂ and N-acetyl groups, with an iduronic acid content of from 20 to 60% and an average molecular weight of from about 1500 to 12000, or one of its chemically or pharmaceutically acceptable salts . 45. NM epi-K5-N-sulfate according to claim 44, characterized in that the content of iduronic acid is from 40 to 60%, and the average molecular weight is from about 1500 to 10000. 46. NM epi-K5-N-sulfate according to item 45, characterized in that the average molecular weight is approximately from 1500 to 7500. 47. NM epi-K5-N-sulfate according to item 46, obtained by the method, characterized in that K5-N-sulfate is subjected, in any order, (i) C5 epimerization using O-glucuronyl-C5 epimerase isolated, purified and in solution or immobilized on a solid support, at a pH of about 7, at a temperature of about 30 ° C and for 12-24 hours, in the presence of at least one divalent ion selected from among calcium, magnesium, barium and manganese; and (ii) nitrogen depolymerization, which is optionally followed by reduction. 48. NM epi-K5-N-sulfate according to clause 47, characterized in that it consists of mixed chains, of which at least 90% correspond to the formula I ':

in which q is an integer from 2 to 20, and the corresponding cation is chemically or pharmaceutically acceptable. 49. NM epi-K5-N-sulfate according to claim 48, characterized in that it contains a unit with a structure (a '')

at the reduced end of most mixed chains, where X is formyl or hydroxymethyl. 50. NM epi-K5-N-sulfate according to claim 48, characterized in that it consists of mixed chains, of which at least 90% correspond to the formula I ′, in which g is an integer from 3 to 15. 51. NM epi-K5-N-sulfate according to claim 48, characterized in that it consists of mixed chains, among which chains according to the formula I'a prevail:

in which p is an integer from 4 to 8, and the corresponding cation is chemically or pharmaceutically acceptable. 52. NM epi-K5-N-sulfate according to paragraph 51, characterized in that it consists of mixed chains, among which chains according to the formula I'b prevail:

in which X is hydroxymethyl, m is 4, 5 or 6, the corresponding cation is a chemically or pharmaceutically acceptable ion, and the glucuronic and iduronic units alternate starting from the glucuronic or iduronic unit. 53. NM epi-K5-N-sulfate according to any one of paragraphs.44-52, characterized in that the salt or cation is selected from among alkali metals, alkaline earth metals, ammonium, (C ₁ -C ₄ ) -tetraalkylammonium, aluminum and zinc . 54. NM epi-K5-N-sulfate according to any one of claims 45-52, characterized in that the salt or cation is selected from sodium, calcium and tetrabutylammonium. 55. A pharmaceutical composition having antidermatitis and antiviral activity, comprising as one of the active ingredients a pharmacologically active amount of epi-K5-N, O-hypersulfate derivative according to any one of claims 20-40 in a mixture with a pharmaceutical excipient. Priority on points: 06/18/2002 according to claims 1-7, 10, 12, 16-20, 21, 24, 29, 30, 31, 34-37, 39-41, 46-49, 50, 53-55; 08/27/2002 according to claims 13-15, 51, 52; 06/17/2003 according to claims 8, 9, 11, 22, 23, 25-28, 32, 33, 38, 42-45.