Epimerised k5 polysaccharide derivatives with high sulfation degree

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

 

The technical field to which the invention relates.

The present invention relates to new derivatives of K5 polysaccharide with a high degree of sulfation, the method of their derivation, much new O-sulfated intermediates useful in their synthesis and pharmaceutical compositions containing data derivatives of K5 polysaccharide as an active ingredient, is almost devoid of activity on the coagulation of blood.

In particular, the invention concerns a method of obtaining an EPI-K5-N,O-hypersulfated, based on the polysaccharide K5, pre-N-deacetylating, N-sulfated and C5-Ameritania at least 20%, by O-hypersalivation in suitable conditions, and subsequent N-sulfation, as well as concerns data EPI-K5-N,O-hypersulfated with antiangiogenic and anti-virus activity, and new low-molecular-weight intermediates for EPI-K5-N-sulfates.

The level of technology

Glycosaminoglycans, such as heparin, heparansulfate, dermatooncology, chondroitin sulfate and hyaluronic acid is a biopolymer, which in industry is extracted from various organs of animals.

In particular, heparin, which is mainly produced by extracting from the intestinal mucosa of pigs or the lungs of cattle, is polidispersen the m polymer with molecular weight approximately in the range from 3000 to 30000 D, consisting of mixed chains, mainly consisting of uronic acid (glucuronic or iduronovoy acid) and amino sugar (glucosamine), interconnected links α-1→4 or β-1→4. The heparin uronic link may be O-sulfation in position 2, and glucosamine link undergoes N-acetylation or N-sulfation, 6-O-sulfation and 3-O-sulfation in approximately 0.5% of the glucosamine units.

Properties and natural biosynthesis of heparin in a mammal is 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 D.S. and L. Roden, 1986 TIBS, 11, 221-225; H.E. Conrad "Heparin Binding Proteins", Chapter 2: Structure of Heparinoids. Academic Press, 1998. Biosynthesis of heparin occurs, beginning with his predecessor N-acetylpiperidine, consisting of mixed chains consisting of repeating units of the disaccharide glucuronyl-β-1→4-N-acetylglucosamine. This precursor is subjected to enzymatic modifications, which include partial hydrolysis of N-acetyl substitution is a group of SO3-the epimerization of carboxyl in position 5 some part glucurone links with turning them into Euronova links and the introduction of O-sulfate groups with the formation of the product, which after the industrial extraction contains twice the sulfate groups with whom avanyu with carboxyl groups per 1 unit of the disaccharide. In addition, these enzymatic modifications lead to the formation of pentasaccharide binding site of anti-thrombin III (ATIII), called active pentasaccharide, which is a structure required for high-affinity binding of heparin to ATIII and essential for the anticoagulant and antithrombotic activity of the heparin. This pentasaccharide, which is present only in some circuits, the components of heparin, contains sulfated link glucosamine at position 3 and glucuronic acid located between disaccharides containing Euronova acid.

In nature, the formation of active pentasaccharide through the epimerization reaction of carboxyl some part glucurone links in Euronova links under the action of glucuronyl-C5-epimerase (C5-epimerization) and the corresponding sulfation, which also leads to the introduction of a sulfate group to the hydroxyl at position 3 of the glucosamine. In particular, in the nature of the formation of the active pentasaccharide is carried out in such a way that C5-epimerization occurs separate "clusters", i.e. on the side chains, and to a large degree, this produces a product containing more euronavy links than glucurone. In fact, commercial heparin contains about 70% euronavy links and 30% glucurone links./p>

Along with the main anticoagulant and antithrombotic activity of heparin also has antilipemics, antiproliferative, antiviral, anticancer and protivoglistotnoe action, but its use as a medicine is constrained because of side effects, due to its anticoagulant effect, which can cause bleeding.

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

as for N-acetylpiperidine the predecessor of heparin. Capsular polysaccharide K5, hereinafter referred to as "polysaccharide K5" or simply "K5", was subjected to chemical modification Lormeau et al., as described in US 5550116, and Casu et al., as described in Carbohydrate Research, 1994, 263, 271-284. O-sulfate K5 with cancer, protivoglistnym, antiviral activity, particularly against HIV, 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 for blood coagulation in vitro, as heparin, is xtraining from animal organs (extractive heparin).

Obtaining products having the same type of activity for blood coagulation as extractive heparin, is a method that mimics the process that occurs in nature and involving all key stage C5-epimerization using the D-glucuronyl-C5-epimerase.

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

D-glucuronyl-C5-epimerase from bovine liver was purified P. Campbell et al., J. Biol. Chem., 1994, 269/43, 26953-26958 (Campbell 1994), which also received its amino acid composition and described its application in the solution for the conversion of K5-N-sulfate to the corresponding product, emerytalny 30%, the evidence education iduronovoy acid HPLC method with subsequent complete depolymerization using nitrous acid disaccharides.

In WO 98/48006 described DNA sequence that encodes a D-glucuronyl-C5-epimerase, and was obtained recombinant D-glucuronyl-C5-epimerase with expressing recombinant vector containing the DNA, and then cleaned Campbell et al., as shown in Jin-Ping L. et al., J. Biol. Chem., 2001, 276, 20069-20077 ("Jin-Ping 2001").

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

In WO 01/72848 describes a method for N-deacetylating N-sulfated derivatives of K5 polysaccharide, emerytalnych to iduronovoy acids, at least 40% in terms of the amount of uronic acids, with a molecular weight of from 2,000 to 30,000, containing from 25% to 50% of the high-affinity circuits for ATIII and having anticoagulant and antithrombotic activity, expressed as the ratio of HCII/Antica, from 1.5 to 4. This document describes hypersalivation K5-N-sulfate, Ameritania by 40-60%, and it is shown that the resulting product, for which the range of13C-NMR, contains 2-3,5 sulfate group 1 link disaccharide. By repeating the above hypersalivation in the described conditions and study13C-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 the amino sugar, but the degree of sulfation is 3.2. It also appeared that in terms of hypersalivation described in WO 01/72848, the degree of sulfation is not more than 3.2. In the US 2002/0062019 describes a method for EPI-K5-N,O-sulfates active in the regulation of sort is of blood, in which the degree of sulfation is from 2.3 to 2.9, with a molecular weight of from 2,000 to 30,000, or from 4000 to 8000, or from 18000 to 30000. This method involves the following stages: (p-a) N-deacetylation of the polysaccharide K5 and N-sulfation of the resulting K5-amine (p-b) epimerization K5-N-sulfate, (p-C) O-hypersalivation EPI-K5-N-sulfate, (p-d) partial O-desulfation, (p-e) selective 6-O-sulfation, (p-f) N-sulfation resulting product, and any product obtained at the end of one stage (p-b) to (p-f), can be subjected to depolymerization. This document describes the EPI-K5-N,O-sulfate with a molecular weight of 7400, obtained during stages from (p) to (p-f) followed nitrous depolymerization at the end of stage (p-f), and whose degree of sulfation is from 2.3 to 2.9.

In the same document are described and a separate part of the K5 with a molecular weight of about 5000, which can also be subjected to the stages of (p-a) to (p-f).

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

- "K5" or "K5 polysaccharide" means a capsular polysaccharide from Escherichia coli obtained by fermentation, that is, mixed chain, consisting of disaccharide units (A), it is certainly containing a double bond at the non end as shown above, and in any case it was obtained and purified by the methods described in the literature, in particular by the method Vann 1981, by the method of M. Manzoni 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; specialist in this area should be clear that all that is shown in further applicable to any N-acetylpiperidine;

- C5-epimerase" means-glucuronyl-C5-epimerase, extractive or recombinant, but in any case it is extracted, 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, deacetylating, at least 95%, at which the N-acetyl groups are not detected on conventional device for NMR;

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

- "EPI-K5" means K5 and its derivatives, in which 20-60% glucurone links underwent C5-epimerization in Euronova links;

- "EPI-K5-N-sulfate" means K5-N-sulfate, which 20-60% glucurone links underwent C5-epimerization in Euronova links;

- "EPI-K5-amine-O-hypersurface" means an EPI-K5-amine-O-sulfate, in which the degree of sulfation is at least 3,4;

- "EPI-K5-N,O-g is the persulfate" means an EPI-K5-amine-O-sulfate, undergone a complete N-sulfation to the degree of sulfation of at least 4.

In addition:

- conditional terms and expressions defined above refer to K5, isolated after fermentation, typically with a molecular weight approximately in the range of 1,500 to 50,000 and an average molecular weight 10000-25000, preferably 15000-25000;

- 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 by depolymerization K5-N-sulfate and consisting of or originating from K5-N-sulfate with an average molecular weight from about 1500 to 12000 in terms of 100% N-sulfated product;

- conditional terms and expressions defined above, if they are worth the word "derived"in General refers to both derivatives of native C5 and low-molecular K5;

- the expression "approximately" in relation to the molecular weight means a molecular weight, measured by means of viscometry, ± theoretical weight disaccharide glycosides management including weight of sodium, which is assumed to be 461 in the case of derivative EPI-K5-N-sulfates and 806 in the case of derivative EPI-K5-N,O-hypersulfated with the degree of sulfation 4.26 deaths;

- the expression "dominant type" means with the Association, which in a mixture consisting of NM EPI-K5-N-sulfate, NM EPI-K5-amine-O-hypersurface or NM EPI-K5-N,O-hypersurface presented in the highest degree that is determined by the peak on the graph of the molecular weight when measured by the method of HPLC;

- unless otherwise agreed, the degree of sulfation" means the ratio of SO3-/COO-also expressed by the number of sulfate groups per 1 unit of the disaccharide in the measurement of conductivity by the method described in Casu C. et al. Carbohydrate Research, 1975, 39, 168-176 (Casu 1975), which was also used in WO 01/72848;

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

- the term "alkyl" means a linear or branched alkyl, then as tetrabutylammonium" means the group of Tetra-p-butylamine.

The invention

It was shown that in contrast to what happens when using 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-hypersurface 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 salts of tertiary or Quaternary organic bases of this EPI-K5-N-sulfate with the obligatory about what followed the reaction mixture for 30-60 minutes while maintaining a pH of about 7 using the same organic base, and then processing the obtained salt of O-sulfation reagent under conditions O-hypersalivation.

Subjecting the acquired EPI-K5-amine-O-hypersurface N-sulfation, get a new EPI-K5-N,O-hypersurface that in contrast to the products described in IT 1230785, WO 92/17507, WO 96/14425, WO 97/43317, WO 01/72848 and US 2002/0062019, devoid of activity on the coagulation of blood and is useful for obtaining drugs, in particular pharmaceutical compositions, possessing antiangiogenic and anti-virus activity or cosmetic compositions.

When depolymerization data EPI-K5-N,O-hypersulfated using nitrous acid get new NM EPI-K5-N,O-hypersurface, devoid of activity on blood coagulation and possessing antiangiogenic and anti-virus activity.

When receiving the N,O-sulfate of N-deacetylating derivatives of K5 polysaccharide, emerytalnych, at least 40% to iduronovoy acid, calculated on the amount of uronic acids and low molecular weight by the method described in WO 01/72848, it was found that the depolymerization of high molecular weight product obtained at the end of the final stage of the process N-sulfation, can give different results, as it usually results in some depolimerizovannogo products with significantly less active than those Vysokomol the molecular products from which they are formed, all the parameters of blood coagulation. It is assumed that this is because the degradation with the use of nitrous acid is affected by the presence of sulfate groups. In particular, the sulfate at position 3 of the glucosamine lead to the formation of heterogeneous products, as described in Nagasawa et al., Thrombosis Research, 1992, 65, 463-467 (Nagasawa 1992).

It was also found that when nitrous depolymerization EPI-K5-N-sulfate, in which the content iduronovoy acid, calculated on the amount of uronic acids is 20-60%, preferably 40-60%, more preferably about 60%, are formed NM EPI-K5-N-sulfates, which is the new effective intermediate products to obtain NM EPI-K5-N,O-hypersulfated that is highly active on various biological parameters, in particular possessing or not possessing activity on the parameters of blood coagulation. It was found that when the depolymerization EPI-K5-N-sulfate, you can get new NM EPI-K5-N-sulfate with an average molecular weight from about 2000 to 4000, in particular, certain NM EPI-K5-N-sulfates, consisting of mixtures in which the predominant connection is decasare or dodecanoate or tetradecanoate. In addition, these NM EPI-K5-N-sulfates, which are impossible to get otherwise, possess interesting biological properties and Aleuts is important intermediates for the receipt NM EPI-K5-N,O-hypersulfated, having anti-virus and/or antiangiogenic activity and unexpectedly deprived of activity on the coagulation of blood.

Exposing NM EPI-K5-N-sulfate of the above method of obtaining salt with a tertiary or Quaternary organic base with mandatory separation of the reaction mixture for 30-60 minutes while maintaining a pH of about 7 using the same organic base, and then processing the obtained salt of O-sulfation reagent under conditions O-hypersalivation, get new NM EPI-K5-amine-O-hypersurface. Exposing NM EPI-K5-amine-O-hypersurface N-sulfation, get new N-sulfated and O-hypersalivation derivatives (NM EPI-K5-N,O-hypersurface), suddenly devoid of activity in the clotting of blood and has anti-virus and/or angiogenic activity, useful for pharmaceutical or cosmetic compositions.

These NM EPI-K5-N-sulfate obtained from K5-N-sulfate by the reaction of epimerization using isolated and purified recombinant C5-epimerase immobilized on a solid medium at a temperature of about 30°and a pH of about 7 in 12-24 hours in the presence of divalent cation selected from among calcium, magnesium, barium and manganese, and the subsequent reaction of nitrous depolymerization resulting product, or obratnoosmoticheskie.

Suddenly oversee the epimerization reaction in the above conditions suggest that in contrast to what occurs in nature in the biosynthesis of heparin, is not "cluster", and the usual C5-epimerization of the substrate after every 2 units of glucuronic acid, which leads to the formation of derivatives EPI-K5-N-sulfates, characterized by the presence of repetitive tetrasaccharides link, consisting of two levels of glucosamine separated first glucuronosyl link, and then euronavy link, or Vice versa.

Detailed description of the invention

Thus, in accordance with one aspect of the present invention provides a method of obtaining derivatives EPI-K5-N,O-hypersulfated, characterized in that:

(a) derived EPI-K5-N-sulfate, in acidic form, is treated with a tertiary or Quaternary organic base, leaving the reaction mixture for 30-60 minutes, and maintain the value of pH of about 7 by the addition of the same tertiary or Quaternary organic base, and excrete salt of this organic base;

(b) this salt of the organic base and this derived EPI-K5-N-sulfate is treated with O-sulfation reagent under conditions O-hypersalivation;

(c) resulting derived EPI-K5-amine-O-hypersurface treated with N-sulf the exporting reagent and produce derived EPI-K5-N,O-hypersurface.

In the General case derived EPI-K5-N,O-hypersurface isolated in the form of sodium salt and optional this sodium salt is converted into another chemically or pharmaceutically acceptable salt.

In this context, the term "chemically reasonable" refers to the cation used in chemical synthesis, the type of sodium, ammonium ion (C1-C4), tetraalkylammonium, or when cleaning the product, whereas a "pharmaceutically acceptable" requires no explanation.

Preferred cations are alkali metals, alkaline earth metals, ammonium, (C1-C4)-tetraalkylammonium, aluminum and zinc. The most preferred cations are sodium, calcium and tetrabutylammonium.

In accordance with a preferred implementation of the method stage (a) is carried out by passing a solution of sodium salt derived EPI-K5-N-sulfate, i.e. polysaccharide K5, pre-N-deacetylating, N-sulfated, usually 100%, C5-Ameritania by 20-60% and optional depolimerizovannogo nitrous acid with an average molecular weight from about 1000 to 25,000, preferably from 1500 to 25000, through a column of acid anionoobmennika, for example, type IR-120 H+collect the eluate with wash solution and neutralization of the eluate tertiary or Quaternary organic base, preferably aqueous rest the rum tetrabutylammonium hydroxide. The solution is kept for 1 hour, maintaining a pH of about 7 by adding the same basis, and any resulting salt allocate by lyophilization.

At the stage (b) O-hypersalivation is carried out using an excess of O-sulfotyrosine reagent and processing at a temperature of from 20 to 70°over time up to 24 hours in an aprotic polar solvent.

Preferably allocated at the stage of (a) salt of the tertiary or Quaternary organic base and derived EPI-K5-N-sulfate, i.e. polysaccharide K5, pre-N-deacetylating, N-sulfated, preferably 100%, C5-Ameritania by 20-60% and optional depolimerizovannogo nitrous acid with an average molecular weight from about 1000 to 25,000, preferably from 1500 to 25000, dissolved in dimethylformamide and treated with O-sulfation reagent in the amount of 2-10 mol for each free hydroxyl at a temperature of 40-60°C for 10-20 hours. As O-sulfotyrosine reagent is preferably used as a compound pyridine·SO3in the amount of 2.5 to 5 mol, preferably 2.5 to 4 mol per 1 free hydroxyl at the 1 disaccharide and the reaction is carried out preferably at 50-60°S, more preferably at 55°With during the night.

Received upon completion of the reaction product produce by adding 0.1 to 1 volume of the water and neutralization, preferably with sodium hydroxide, precipitation with a saturated solution of sodium chloride in acetone, filtering and optional ultrafiltration.

Resulting product is usually a sodium salt derived EPI-K5-amine-O-hypersurface content iduronovoy acid in 20-60% of the amount of uronic acids with an average molecular weight of from about 3500 to 40,000, preferably from 4500 to 40,000, with a degree of sulfation of not less than 3,4, preferably at least a 3.5, more preferably from 3,55 to 4, most preferably from 3,55 to 3.8. Resulting salt may be converted into another salt. For example, there may be an exchange of ions on calcium ion using membranes for ultrafiltration.

At the stage (C) is derived EPI-K5-amine-O-hypersurface with a very high degree of sulfation is subjected to N-sulfation, using known literature methods N-sulfation.

In practice, N-sulfation is carried out by treating an aqueous solution containing derived EPI-K5-amine-O-hypersurface obtained in stage (b), sodium carbonate and N-sulfation reagent, for example compound (C1-C4-trialkylamine·SO3or pyridine·SO3, keeping mixture at 30-50°C for 8-24 hours and highlight the desired derived EPI-K5-N,O-g is the persulfate, for example, by diafiltration. Optional stage N-sulfation is repeated until the degree of substitution of more than 95%, preferably to complete.

New derivatives of EPI-K5-N,O-hypersurface obtained are typically in the form of sodium salt. The sodium salt may be converted into another chemically or pharmaceutically acceptable salt. Particularly preferred alkali metal salts, alkaline earth metal, ammonium, (C1-C4)-tetraalkylammonium, aluminum and zinc. The most preferred salts of sodium, calcium and tetrabutylammonium.

The original EPI-K5-N-sulfates, directed to the step (a) of the method of the present invention, occur from K5 polysaccharide, previously N-deacetylating, N-sulfated almost 100%, C5-Ameritania by 20-60%, preferably 40-60%, and optional depolimerizovannogo nitrous acid with an average molecular weight from about 1000 to 25,000, preferably from 1500 to 25000. Preferably, the source material is an EPI-K5-N-sulfate with an average molecular weight of from 10000 to 25000 NM or EPI-K5-N-sulfate with an average molecular weight from about 1000 to 12,000, more preferably from 1,000 to 10,000, most preferably from 1500 to 8000.

EPI-K5-N-sulfates obtained by C5-epimerization K5-N-sulfates, is all right known in the literature and broadly described, for example, in WO 92/17507, WO 01/72848, WO 98/14425, WO 97/43317 or US 2002/0062019. Receive by C5-epimerization of glucuronic link K5-N-sulfate with D-glucuronyl-C5-epimerase described in the documents listed above.

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

EPI-K5-N-sulfate content iduronovoy acid in 40-60%, particularly preferred as starting material, was prepared by epimerization K5-N-sulfate, almost devoid of acetyl groups, which, in turn, is produced from particularly pure K5, in particular not containing lipophilic substances described in WO 02/068477. In accordance with a preferred implementation of the method, the epimerization is used K5-N-sulfate obtained from K5, devoid of lipophilic substances, like the one described in WO 02/068477, and C5-epimerization is carried out using D-glucuronyl-C5-epimerase, isolated, purified and immobilized on a solid medium at pH of about 7 at a temperature of about 30°and within 12-24 hours, in the presence of at least one divalent ion selected from calcium, magnesium, barium and manganese.

NM EPI-K5-N with higher content iduronovoy acid, in particular 40-60%, preferably 50-55%, is particularly preferred products as raw materials to obtain NM derived EPI-K5-N,O-hypersulfated.

Above NM EPI-K5-N-sulfates receive method, which is characterized by the fact that K5-N-sulfate is subjected, in any order,

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

(ii) nitrous depolymerization, optionally followed by a recovery, usually with sodium borohydride.

The expression "in any order" means that the method can be performed as in the direction of (i)-(ii), i.e. in the manner described above, and in reverse order, i.e. in the direction of (ii)-(i)in a manner that K5-N-sulfate first reaction of nitrous depolymerization, and then optional recovery with sodium borohydride and then C5-epimerization in the above conditions. Preferred order in the direction (i)→(ii). The sequence (ii)-(i) is preferably used when coming from NM K5-N-sulfate with an average molecular weight more than the 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 to obtain NM EPI-K5-N-sulfate with a molecular weight of more than 4000, preferably not less than 6000 in order to get useful intermediates for obtaining NM EPI-K5-N,O-hypersulfated. In fact, in this case, at the stage (ii), an optimum degree of epimerization.

In accordance with a preferred aspect of the invention, the C5-epimerase subjected to immobilization on inert solid carrier.

C5-epimerase, preferably recombinant, isolated and purified, for example, according to Campbell 1994, WO 98/48006, Jin-Ping 2001 or Crawford 2001, is subjected to immobilization on inert medium in the presence of the substrate, i.e. in the presence of the source derived K5-N-sulfate or NM in the presence of 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 reaction of C5-epimerization is carried out by passing 20-1000 ml of 25 mm Hepes solution, pH 7, containing 0.001 to 10 g of 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 among calcium, magnesium, barium and manganese in a concentration of from 10 to 60 mm, through a column containing from 1.2×107up to 3×1011cpm immobilized enzyme, at pH of about 7 and a temperature of about 30°when speed transmission in 30-220 ml/HR for 12-24 hours, preferably 15 to 24 hours.

Preferably this solution is passed with a speed of 200 ml/hour during the night (15-20 hours). The resulting product is distilled and separated in accordance with known methods, for example, ultrafiltration and precipitation with ethanol. The product consists of any of the EPI-K5-N-sulfate (in this case, it is dissolved in water and subjected to depolymerization)or from NM EPI-K5-N-sulfate (in this case, it is the final product). The degree of epimerization, which in practice means the number euronavy links regarding glucurone, estimated using1H-NMR method described in WO 96/4425.

The reaction of nitrous depolymerization is carried out in accordance with known methods of depolymerization of heparin, for example in accordance with the methods described in EP 37319, WO 82/03627, or in accordance with the method of depolymerization K5-N-sulfate, as described in EP 544592, but on the basis of 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 at EPI-K5-N-sulfate, almost if the hinnon acetyl group, with subsequent restoration in situ with sodium borohydride.

In practice, a cold aqueous solution of EPI-K5-N-sulfate is brought to acidic pH (about 2) with hydrochloric acid and treated in the cold with sodium nitrite at a constant temperature (about 4° (C) and pH (about 2), and at the end depolymerization (approximately 15-30 minutes), the solution is neutralized with sodium hydroxide and treated again at a temperature of about 4°With, an aqueous solution of sodium borohydride. At the end of recovery (approximately 4 hours) the excess of sodium borohydride eliminate hydrochloric acid, the solution is neutralized with sodium hydroxide and produce depolimerizovannogo (and restored) product in accordance with known methods, for example by direct precipitation with ethanol or acetone. The product obtained after 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 is directly exposed C5-epimerization, as described above, after isolation or in solution without pre-allocation), in particular, if its molecular weight is more than 4000, preferably at least 6000, or disposed of to obtain NM K5-N,O-hypersurface with antiangiogenic and anti-virus activity. Owned by monitoring the reaction of depolymerization, in particular, using different amounts of sodium nitrite/hydrochloric acid, receive NM K5-N-sulfates or NM EPI-K5-N-sulfate with an average molecular weight in the whole interval from about 1,500 to 12,000, preferably from 1500 to 10000, more preferably from 1500 to 7500, which is calculated from the spectrum13C-NMR by integration of the signal related to the atom C2 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, based on 1 g of EPI-K5-N-sulfate, the raw product is dissolved in 100-200 ml of deionized water and placed in a thermostat at 4°C. Then add this amount of sodium nitrite to obtain the desired molecular weight, for example, from 2000 to 4000. Thus, on the basis of EPI-K5-N-sulfate with an average molecular weight of 20,000 in the measurement method HPLC on a column of BioRad BioSil 250 using standard heparin with known molecular weights, you will need to add from 330 to 480 mg of sodium nitrite in the form of a 0.2% aqueous solution. The solution containing EPI-K5-N-sulfate and sodium nitrite, 4°adjusted to pH 2 by addition of 0.1 N HCl, cooled to 4°C. the Solution is kept at 20 to 40 minutes for reaction with slow stirring, and then neutralized with a 0.1 N NaOH. The resulting product is brought to room temperature and quenching the see reducing reagent, for example with sodium borohydride (250-500 mg dissolved in 50-100 ml of water) and left for reaction for 4-8 hours. The excess sodium borohydride is removed by bringing the pH to 5-5 .5 with a 0.1 N HCl and left for another 2-4 hours. Finally, the solution is neutralized with a 0.1 N NaOH and produce the product 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 to obtain NM K5-N-sulfate or NM EPI-K5-N-sulfate with an average molecular weight from 4000 to 12000, preferably from 4000 to 7500, more preferably from 6000 to 7500.

Received this NM EPI-K5-N-sulfate content iduronovoy acid from 20 to 60%, preferably from 40 to 60%, more preferably from 50 to 55%, and almost devoid-NH2and N-acetyl groups with an average molecular weight from about 1,500 to 12,000, preferably from 1500 to 10000, more preferably from 1500 to 7500, and it is chemically and pharmaceutically acceptable salts represent new products, applicable as of special interest starting materials to obtain NM EPI-K5-N,O-hypersulfated, but by themselves they are useful as active ingredients of pharmaceutical or cosmetic compositions and are on the additional aspect of the present invention.

Preferably the starting materials for obtaining derivatives EPI-K5-N,O-hypersulfated of the present invention are derived EPI-K5-N-sulfates, consisting of mixed circuits, of which at least 90% conform to the formula I:

in which the uronic units by 20-60% consist of iduronovoy 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, the data source derived EPI-K5-N-sulfates consist of mixed circuits, of which at least 90% conform to the formula I, in which the uronic units 40-60% consist of iduronovoy acid, n is an integer from 2 to 100, preferably from 3 to 100, and the corresponding cation is chemically or pharmaceutically acceptable. The most preferred starting materials are NM EPI-K5-N-sulfates, presented above, consisting of mixed circuits, of which at least 90% conform to the formula I, in which the uronic units by 20-60%, preferably 40-60%, more preferably 50-55% consist of iduronovoy acid, n is an integer from 2 to 20, preferably from 3 to 15, and the corresponding cation is chemically acceptable.

In practice, data is preferable NM EPI-K5-N-sulfates consist of a mixture is the R circuits of which at least 90% conform to the formula I':

in which the uronic units by 20-60%, preferably 40-60%, more preferably 50-55% consist of iduronovoy 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 "chemical" refers to the cation used in chemical synthesis, the type of ions of sodium, ammonium, (C1-C4), tetraalkylammonium, or when cleaning the product.

Preferred cations are alkali metals, alkaline earth metals, ammonium, (C1-C4)-tetraalkylammonium, aluminum and zinc. The most preferred cations are sodium, calcium and tetrabutylammonium.

Of particular interest NM EPI-K5-N-sulfates, consisting of mixed circuits, of which at least 90% conform to the formula I', which is obtained by nitrous depolymerization of the corresponding EPI-K5-N-sulfates, above, and the following optional recovery, for example, with sodium borohydride. Among them, preferable NM EPI-K5-N-sulfates, consisting of mixed circuits, which are dominated by chains according to the formula I:

in which the uronic units at 60-40% consist of glucuronic is islote and 40-60% - from iduronovoy acid, R 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.

These EPI-C5-M-sulfates, starting from the stage of nitrous depolymerization, includes the presence of a pampering the end of most circuits of this mixture level 2.5-anhydromannose or in the case of recovery, for example, with sodium borohydride link 2,5-anhydromannitol patterns:

in which X is formyl or hydroxymethyl. Therefore, regenerating the end the majority (60-70%) of the chains are actually represented by the structure (b):

in which X is defined above.

The presence of the structure (a) does not affect the chemical characteristics of EPI-K5-N-sulfates and their derivatives, as the sulfation can only lead to the introduction of one or two sulfate groups, however, this will not change significantly the degree of sulfation O-sulfated derivatives. However, it is preferable that for nitrous depolymerization followed the restoration, for example, with sodium borohydride as in accordance with the method of the present invention NM EPI-K5-N-sulfates are the reactions of sulfation and acyl is the formation, about the impact on the formyl group, when X is represented by a formyl, radical 2.5-anhydromannose structure (a) is unknown. In addition, the presence structure (a) does not affect the biological activity of the products, as shown in Østergaard W. et al., Thrombosis Research, 1987, 45, 739-749 (Østergaard 1987) in respect of the heparins of low molecular weight.

Especially preferred NM EPI-K5-N-sulfate according to the present invention consist of mixed circuits, which are dominated by the compounds of formula I (b:

in which X is formyl or preferably hydroxymethyl, m is 4, 5 or 6, the corresponding cation is chemically or pharmaceutically acceptable, and glucuronic and Euronova links alternate, starting with a glucuronic or Euronova link, and the ratio between glucuronosyl and iduronate links is from 45/55 to 55/45, that is approximately 50/50.

Consequently, the use of C5-epimerase, preferably recombinant, preferably immobilized on a solid medium under the conditions described above, contributes not a "cluster" of epimerization derivatives of K5-N-sulfate derivatives in EPI-K5-N-sulfates, as it occurs in nature, and the epimerization of the usual type.

Thus, in accordance with the following aspect the present invention provides for the use of the isolated and purified C5-epimerase for making derivatives of K5-N-sulfates corresponding derivatives EPI-K5-N-sulfates, characterized by the presence of repetitive tetrasaccharides link, consisting of two levels of glucosamine separated first glucuronosyl link, and then euronavy link, or divided first euronavy link, and then glucuronosyl link.

This epimerization is optimal if it is derived from K5-N-sulfate with an average molecular weight of 4000, preferably from 6000 to 7500.

In accordance with the present invention the source-derived EPI-K5-N-sulfates, preferably N-sulfated 100% (especially derivatives EPI-K5-N-sulfates, consisting of mixed circuits, of which at least 90% conform to the formula I or I', or dominated by a chain of the formula I a or I b, where X means hydroxymethyl)are directed to the above stage (a) and (b), after which there is a selection of relevant new derivatives EPI-K5-amine-O-hypersulfated, in which the amino group is not substituted, and which usually have the form of sodium salt, which can be transformed into another chemically or pharmaceutically acceptable salt. Particularly preferred alkali metal salts, alkaline earth metal, ammonium, (C1-C4)-tetraalkylammonium, aluminum and zinc, and of these, preferred salts of sodium, calcium and tetrabutylammonium.

Therefore clicks the zoom, in accordance with the following aspect of the present invention relates to new derivatives EPI-K5-amine-O-hypersurfaces and their chemically or pharmaceutically acceptable salts obtained by the method, which is characterized by the fact that:

(a) derived EPI-K5-N-sulfate, in acidic form, is treated with a tertiary or Quaternary organic base, leaving the reaction mixture for 30-60 minutes and maintaining the pH value of the solution to about 7 by the addition of this tertiary or Quaternary organic base, and produce its salt formed with this organic base;

(b) this salt of the organic base and this derived EPI-K5-N-sulfate is treated with O-sulfation reagent under conditions O-hypersalivation and produce derived EPI-K5-amine-O-hypersurface.

Using as a starting material for the stage (a) derived EPI-K5-N-sulfates, consisting of mixed circuits, of which at least 90% conform to the formula I, in which the uronic units by 20-60% consist of iduronovoy acid, n is an integer from 3 to 100, and the corresponding cation is chemically or pharmaceutically acceptable at the end of stage (b) receive-derived EPI-K5-amine-O-hypersurface, consisting of mixed circuits, of which at least 90% correspond to the formula II:

in which the uronic units by 20-60% consist of iduronovoy acid, n is an integer from 2 to 100, preferably from 3 to 100, R, R' and R" represent hydrogen or SO3-and the degree of sulfation is at least 3,4, preferably at least a 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 derived EPI-K5-amine-O-hypersurface with a very high degree of sulfation is a new products, applicable as intermediates for N-sulfated or N-(C2-C4)-acylated derivatives, virtually devoid of activity on the parameters of blood coagulation, but with other interesting pharmacological properties.

Preferably derived EPI-K5-amine-O-hypersurface with a very high degree of sulfation consist of mixed circuits, of which at least 90% conform to the formula II, in which the uronic units 40-60% consist of iduronovoy acid, n is an integer from 2 to 100, preferably from 3 to 100, and have an average molecular weight from about 2000 to 40,000, preferably from 4500 to 40000, R, of at least 40%, preferably 50-80%, presents SO3-, R' and R" both the redstavleny SO 3-either one of them is represented by hydrogen, and the other 5-10% of SO3-in monosulfata glucuronic acid and 10-15% of SO3-in monosulfata iduronovoy acid, and the degree of sulfation is more than 3.4, preferably at least a 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.

Preferred derivatives EPI-K5-amine-O-hypersurface with a very high degree of sulfation is NM EPI-K5-amine-O-hypersurface, consisting of mixed circuits, of which at least 90% conform to the formula II, in which the uronic units by 40-60%, preferably 50-55% consist of iduronovoy acid, R is at least 40%, preferably 50-80%, more preferably 65% presents SO3-, R' and R" are both represented SO3-either one of them is represented by hydrogen, and the other 5-10% of SO3-in glucuronic acid and 10-15% of SO3-in iduronovoy acid, n is an integer from 2 to 20, preferably from 3 to 15, and have an average molecular weight of from about 4000 to 8000, and the corresponding cation is chemically or pharmaceutically acceptable.

In practice, d is preferred by NM EPI-K5-amine-O-hypersurface consist of mixed circuits, of which at least 90% conform to the formula II':

in which the uronic units by 20-60% consist of iduronovoy acid, q is an integer from 2 to 20, preferably from 3 to 15, R, R' and R" is represented by hydrogen or SO3-and the degree of sulfation is at least 3,4, preferably at least a 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 ion.

Of these NM EPI-K5-amine-O-hypersulfated preferred those which consist of mixed chains, dominated by circuit corresponding to the formula II a:

in which the uronic units by 20-60% consist of iduronovoy acid, R is an integer from 4 to 8, R, R' and R" has already been defined above, and the degree of sulfation is at least 3,4, preferably at least a 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.

Getting new NM EPI-K5-amine-O-hypersulfated from NM EPI-K5-sulfates obtained by nitrous depolymerization and subsequent recovery, for example, sodium borohydride, includes the presence of a pampering con the e most circuits of this mixture of sulfated 2,5 link-anhydromannitol structure (a'):

in which R ' means hydrogen or SO3-.

Thus, regenerating the end of most circuits of this mixture is represented by the structure (b'):

in which the uronic link can be glucuronosyl or euronavy.

From the above new NM EPI-K5-amine-O-hypersulfated preferred those which consist of mixed chains, among which are compounds according to formula II b:

in which the uronic units 40-60% consist of iduronovoy acid, m is 4, 5 or 6, R, R' and R" is represented by hydrogen or SO3-X" means HE or OSO3-and the degree of sulfation is at least 3,4, preferably at least a 3.5, more preferably from 3,55 to 4, most preferably from 3,55 to 3.8, Euronova links alternate, starting with a glucuronic or Euronova link, and the corresponding cation is chemically or pharmaceutically acceptable.

All these derived EPI-K5-amine-O-hypersurface with a very high degree of sulfation are new products that are useful intermediate products for new N-substituted derivatives EPI-K5-amine-O-hypersulfated, so they are updat the enforcement aspect of the present invention.

In particular, in accordance with the following aspect of the present invention concerns the application of the above derivatives EPI-K5-amine-O-hypersulfated with a very high degree of sulfation to obtain a new N-substituted derivatives EPI-K5-amine-O-hypersulfated, in particular N-sulfated or N-acylated.

Upon completion of stage (C) of the method of the present invention consisting of N-sulfation derived EPI-K5-amine-O-hypersulfated obtained in stage (b) (in particular derivatives EPI-K5-amine-O-hypersulfated, consisting of mixed circuits, of which at least 90% conform to the formula II or II', or dominated by a chain of the formula II a or II b), we obtain the derived EPI-K5-N,O-hypersurface, which Euronova acid is 20-60% from the amount of uronic acids, and the degree of sulfation is at least 4, preferably from 4 to 4.6.

Thus, in accordance with the following aspect the present invention provides new N-diacetylpyridine derivatives of K5 polysaccharide, O-sulfated and N-sulfated, C5-Ameritania to iduronovoy acid, at least 20% of the total uronic units, with an average molecular weight from about 2000 to 45,000, with a degree of sulfation of at least 4, and the data derived have little or no activity is calling on the parameters of blood coagulation.

Similarly to what was said above, the new derivatives in General denote a generic term derived EPI-K5-N,O-hypersurface", regardless of their molecular weight.

In particular, their average molecular weight is from about 2000 to 45,000, as these derivatives occur either from EPI-K5-To-sulfate obtained by N-deacetylation and N-sulfation K5 fermentation, either by nitrous depolymerization. By controlling nitrous depolymerization can be obtained low-molecular derivatives almost all the specified interval. However, to use derivatives of the present invention as a pharmaceutical or cosmetic products preferably obtaining low molecular weight derivatives with an average molecular weight from about 2000 to 16,000, preferably from 3,500 to 13,000 with a distribution of molecular weights between 1000 and 15,000, more preferably from 4500 to 9000 with a distribution of molecular weights between 2,000 and 10,000, or high-molecular derivatives, originating from nefrackzionirovannam K5, with an average molecular weight from about 20000 to 45000, with a distribution of molecular weights between 2000 and 70000.

The derived EPI-K5-N,O-hypersulfated of the present invention, the degree of sulfation is very high, preferably from 4 to 4.6, if e is ω nitrogen atom of sulfated glucosamine almost 100%. In addition, derivatives EPI-K5-N,O-hypersurface 6-O-sulfotyrosine 100% and 3-O-sulfotyrosine 50-80% on glucosamine links, 3-O-monosulfate 5-10% glucuronosyl links, monocultivar 10-15% euronavy links and 2,3-di-O-sulfotyrosine for other euronavy links, given that the degree of sulfation is at least 4.

Preferred derivatives EPI-K5-N,O-hypersurface of the present invention obtained through derivatives EPI-K5-amine-O-hypersurface, which, in turn, is obtained from the derivative EPI-K5-N-sulfates, consisting of mixed circuits, of which at least 90% conform to the formula I, in which the uronic units by 20-60% consist of iduronovoy 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-hypersurface consist of mixed circuits, of which at least 90% correspond to the formula III:

in which the uronic units by 20-60% consist of iduronovoy acid, n is an integer from 2 to 100, preferably from 3 to 100, R, R' and R" is represented by hydrogen or SO3-Z means SO3-and the degree of sulfation is at least 4, preferably from 4 d is 4,6, and the corresponding cation is chemically or pharmaceutically acceptable.

Preferred cations are alkali metals, alkaline earth metals, ammonium, (C1-C4)-tetraalkylammonium, aluminum and zinc, and of these the most preferred salts of sodium, calcium and tetrabutylammonium.

Of the above new derivatives EPI-K5-N,O-hypersulfated those that consist of mixed circuits, of which at least 90% correspond to the formula III in which R is represented SO3-in 50-80% of the chains, preferably approximately 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 derived EPI-K5-N,O-hypersurface with a very high degree of sulfation consist of mixed circuits, of which at least 90% correspond to the formula III in which Z signifies SO3-, uronic units 40-60% consist of iduronovoy acid, n is an integer from 2 to 100, preferably from 3 to 100, and have an average molecular weight from about 2000 to 45,000, preferably from 4500 to 45,000, R, of at least 40%, preferably 50-80%, presents SO3-, R' and R" are both represented SO3-either one of them is represented by hydrogen, and the other 5-10% of SO3-in monocul the veil glucuronic acid and 10-15% of SO 3-in monosulfata iduronovoy acid, and the degree of sulfation is 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-hypersalivation derivatives NM-EPI-5-amino-O-hypersalivation derivative is a mixture of chains in which at least 90% of the chains have the formula III, in which 40-60% of the uronic units preferably contain 50-55% iduronovoy acid, R is at least 40%, preferably 50-80%, particularly preferably 65% SO3-, R' and R" both are SO3-or one is hydrogen and the other is 5-10% SO3-in glucuronic acid and 10-15% SO3-in iduronovoy acid, Z is 100% SO3-or (C2-C4)Atilla, n is an integer from 2 to 20, preferably from 3 to 15, with a molecular weight of from about 4000 to about 8500 and the corresponding cation is acceptable chemically or pharmaceutically.

In practice these preferred derivatives EPI-K5-N,O-sulfates with a very high degree of sulfation consist of mixed circuits, of which at least 90% correspond to the formula III':

in which romovye links on 20-60% consist of iduronovoy acid, q is an integer from 2 to 20, preferably from 3 to 15, R, R' and R" is represented by hydrogen or SO3-Z means SO3-and the degree of sulfation is at least 4, preferably from 4 to 4.6, and the corresponding cation is chemically or pharmaceutically acceptable ion.

Of particular interest are mixed circuit according to the formula III', in which the uronic units by 40-60%, preferably 50-55% consist of iduronovoy acid, R is at least 40%, preferably 50-80%, more preferably 65% presents SO3-, R' and R" are both represented SO3-either one of them is represented by hydrogen, and the other 5-10% of SO3-in glucuronic acid and 10-15% of SO3-in iduronovoy acid, n is an integer from 2 to 20, preferably from 3 to 15, and have an average molecular weight from about 2000 to 16,000, preferably from 3,500 to 13,000, more preferably from 4500 to 9000, and the corresponding cation is chemically or pharmaceutically acceptable.

Of these NM EPI-K5-N,O-hypersulfated preferred those which consist of mixed chains, dominated by circuit corresponding to the formula III'as:

in which the uronic units by 20-60% consist of iduronovoy sour is s, p is an integer from 4 to 8, Z is SO3-, R, R' and R" is represented by hydrogen or SO3-and the degree of sulfation is at least 4, preferably from 4 to 4.6, and the corresponding cation is chemically or pharmaceutically acceptable.

Getting new NM EPI-K5-N,O-hypersulfated from NM EPI-K5-sulfates obtained by nitrous depolymerization and subsequent recovery, for example, sodium borohydride, includes the presence of a pampering the end of most circuits of this mixture, sulfated 2,5 link-anhydromannitol structure (a')above, in which R ' means hydrogen or SO3-.

Thus, regenerating the end of most circuits of this mixture is represented by the structure (b):

in which Z signifies SO3-and uronic link can be glucuronosyl or euronavy.

From the above new NM EPI-K5-N,O-hypersulfated preferred those that are mixtures, which are dominated by species which are compounds of formula III'b:

in which R, R' and R" is represented by hydrogen or SO3-Z means SO3-and X" IS HE or OSO3-, m is 4, 5 or 6, and the degree of sulfate the Finance is at least 4, preferably from 4 to 4.6, the uronic units are alternated, starting with a glucuronic or Euronova link, and the corresponding cation is chemically or pharmaceutically acceptable. Preferred cations are alkali metals, alkaline earth metals, ammonium, (C1-C4)-tetraalkylammonium, aluminum and zinc, and of these the most preferred sodium ions, calcium and tetrabutylammonium.

If the method of the present invention as a source derived EPI-K5 is used EPI-K5, that is, the polysaccharide K5, pre-N-deacetylating, N-sulfated usually 100%, C5-emerytalny by 20-60% and not depolimerizovannogo, upon completion of stage (C) is allocated EPI-K5-N,O-hypersurface, which may be subjected to nitrous depolymerization and subsequent recovery, for example, with sodium borohydride to obtain the corresponding NM EPI-K5-N,O-hypersurface with the same degree of sulfation. In particular, we obtain NM EPI-K5-N,O-hypersurface, consisting of mixed circuits, of which at least 90% correspond to the formula III' or III'and in which the uronic units by 20-60% consist of iduronovoy acid, q, R, R', R" and Z are defined above, and the degree of sulfation is at least 4, preferably from 4 to 4.6, and the corresponding cation is Henichesk is or pharmaceutically acceptable. In this case, the receipt of these NM EPI-K5-N,O-hypersulfated by reaction of nitrous depolymerization and possible subsequent recovery, for example, with sodium borohydride includes the presence of a pampering the end of most circuits of this mixture level 2,5-anhydromannitol structure (a"):

in which X is formyl or hydroxymethyl and R" denotes hydrogen or SO3-.

New derivatives of EPI-K5-N,O-hypersurface, especially in the form of salts, are strongly anionic products, capable of capturing free radicals, and they can be used in the cosmetic industry as adjuvants against hair loss or to obtain creams "anti-aging" and in the pharmaceutical industry as a drug for the treatment of dermatitis. In addition, derivatives EPI-K5-N,O-hypersurface of the present invention, in particular NM EPI-K5-N,O-hypersurface have antiangiogenic and anti-virus activity and can therefore serve as active ingredients for obtaining medicines.

Thus, in accordance with one of the additional aspects of the present invention provides pharmaceutical compositions comprising as an active ingredient a pharmacologically active amount derived EPI-K5-N,O-is persulfate, described above, or one of its pharmaceutically acceptable salts, together with a pharmaceutical excipient.

In the pharmaceutical compositions of the present invention for oral, subcutaneous, intravenous, transdermal or topical application of the active ingredients are preferably introduced in the form of dose units, together with classical pharmaceutical excipients or carriers. The dosage may vary markedly depending on the age, weight and health of the patient. It includes the dose from 1 to 1000 mg, preferably from 10 to 750 mg, more preferably from 250 to 500 mg, one to three times a day intravenous, subcutaneous, oral, transdermal or external way. The pharmaceutical compositions of the present invention are prepared together with classic fillings suitable for different applications. Particularly preferred compositions in the form of creams, ointments, liquid, ointments, gels, foams, balms, vaginal rings, suppositories, solutions or suspensions suitable for local application.

Preferably the compositions of the present invention includes as one of its active ingredients derived EPI-K5-N,O-hypersurface obtained on the basis of the derived EPI-K5 in accordance with the stages (a), (b) and (C) of the method described above, whether what about proceeding from neapolitano EPI-K5 in accordance with the stages (a), (b) and (C) of the method described above with possible subsequent nitrous depolymerization after stage (C), or one of its pharmaceutically acceptable salts, together with a pharmaceutical excipient. Preferably this derived EPI-K5-N,O-hypersurface consists of mixed circuits, of which at least 90% correspond to the formula III or III', or dominated by compounds according to the formula III'a or III'b. The preferred active ingredient is NM EPI-K5-N,O-hypersurface 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 almost devoid of N-acetyl groups.

Finally, in accordance with the following aspect, the present invention provides a cosmetic composition comprising an effective amount derived EPI-K5-N,O-hypersurface or its pharmaceutically acceptable salt together with a cosmetic filler.

Salt selected from the group consisting of salts of sodium, potassium, calcium, magnesium, aluminum and zinc derivatives EPI-K5-N,O-hypersulfated, in particular consisting of mixed circuits, of which at least 90% correspond to the formula III or III', or dominated by compounds according to the formula III'a or III'b, is an effective active the m ingredient in pharmaceutical or cosmetic compositions of the present invention.

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

Preparative example I

Getting K5 polysaccharide from Escherichia coli

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

Defatted soybean powder2
To2HPO4the 9.7
KN2PO42
MgCl20,11
Sodium citrate0,5
Ammonium sulfate1
Glucose2
Drinking water, ml1000

pH=7,3

The environment is subjected to sterilization at 120°C for 20 minutes. Glucose is prepared separately in the form of a solution, which was sterilized at 120°C for 30 minutes and added to the medium aseptically. In the conical flask inoculant suspension of E. coli cells Bi 8337/41 (O10:K5:H4), originating from jamb with trypticase soy agar, and incubated at 37°within 24 hours with moderate shaking (160 rpm, wingspan 6 cm). Bacterial growth was measured by counting cells under a microscope. The next stage of the Chemap fermenter-Braun 14 l containing the same environment, inoculant 0.1% culture from the conical flask and carry out fermentation with about what Oskanian air (1 volume of air to 1 volume of liquid for 1 minute) with stirring at 400 rpm at a temperature of 37° C for 18 hours. During fermentation the measurements of pH, oxygen, glucose, production of polysaccharide K5 and bacteria growth. At the end of fermentation the temperature is brought to 80°With 10 minutes. Cells are separated from the medium by centrifugation at 10,000 rpm and the supernatant is subjected to ultrafiltration module 316 SS (MST)with PES membrane with a nominal cut off from 800 to 10000 D, reducing the volume to 1/5. Then the K5 polysaccharide is then precipitated by adding 4 volumes of acetone at 4°and leave overnight at 4°C. After that select by centrifugation at 10,000 rpm for 20 minutes or by filtration. From the obtained substances remove proteins with protease type II 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 of filtrate) at 37°C for 90 minutes the resulting solution is subjected to ultrafiltration module 316 SS with PES membrane with a nominal cut off up to 10,000 D, with two extraction 1 M NaCl, and washed with water until the disappearance of optical absorption in the ultrafiltrate. Then the K5 polysaccharide is then precipitated with acetone to yield 850 mg of 1 l fermentor. The purity of the obtained polysaccharide was measured by determination of uronic acids (carbazolyl method),1The h and13C-NMR, UV and protein content. Purity is more than 80%.

The obtained polysaccharide consists of a jet, the x molecules of different molecular weight of 30,000 and 5,000 D respectively, as follows from the definition of method HPLC on a column of Pharmacia 75 HR, and one molecule with a retention time of about 9 minutes when using two consecutive columns Bio-Sil SEC 250 (BioRad) and Na2SO4as the mobile phase at room temperature at a rate of 0.5 ml/min. and the Determination is performed according to a standard curve obtained with the use of heparin molecules of known molecular weight.

Range1H-NMR obtained with the purified K5 shows various signals characteristic of the metal groups of lipophilic substances.

Preparative example II

Cleaning K5

100 ml of a saturated aqueous solution of sodium chloride at 4°dissolve 1 g of K5 obtained in Preparative example I, and the resulting solution add 3 volumes of cold isopropanol. 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 cool place (about 4° (C) overnight. The formed precipitate was separated by centrifugation at 10,000 rpm for 20 min and check the purity of the product by dialysis during the night and explore the range1H-NMR, which shall not be of the signals in the range of less than 1.5 ppm. Optional repeat the operation of dissolution in water, saturated NaCl, and precipitation with isopropanol. The precipitate is dissolved in water and subjected to ultrafiltration through a membrane Miniplate company Millipore with a cutoff of 10,000 D to the disappearance of salts. You get a K5 with a purity not less than 99%, which in the spectrum of1N-not detected NMR signals lipophilic impurities in the region of less than 1.5 ppm.

Preparative example III

Getting KS-N sulfate

(i) N-Deacetylation

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

(ii) N-Sulfation

In a solution containing deacetylating K5, located at 40°make 16 g of sodium carbonate and 4 hours and 16 g of pyridine-SO3-. Upon completion of the reaction after 24 hours, the solution is brought to room temperature, and then to pH 6.5-7 with 5% hydrochloric acid. The product is purified from salts by diafiltration through the spiral membrane with a cutoff at 1000 D (preparative cartridge company Millipore). The process stops upon reaching the conductivity of the solution is less than 1000 μs, preferably less than 100 µs. The volume of dialysate reduced to achieve a concentration of polysaccharide in the 10°/o, using the same system of dialysis. A concentrated solution is dried by lyophilization. When the spectrum analysis13C-NMR residues of N-acetyl or-NH2not found.

Preparative the first example IV

Low-molecular KS-N sulfate

The product obtained as described in Example 1, stage (i) and (ii) of the patent WO 02/068477, is subjected to a depolymerization method of splitting nitrous acid and subsequent recovery of the resulting aldehyde. To do this, dissolve 1 g of K5-N-sulfate in 200 ml of distilled water and added 480 mg of sodium nitrite dissolved in 240 ml of distilled water. Then the solution was adjusted to 4°and to pH 2 using a 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 to the solution was added 450 mg of NaBH4and left to react for 4 hours. Excess NaBH4removed using HCl, bringing the pH to 5-6. After neutralization with 0.1 M NaOH product produce by precipitation with 3 volumes of acetone at 4°With filtering on a funnel and dried at 40°With vacuum drying. Receive 900 mg NM K5-N-sulfate with an average molecular weight of about 2000, consisting of mixed circuits, which are dominated by the compounds of formula I b, in which m is 4, and the uronic units are presented glucuronic acid.

Example 1

NM EPI-K5-N-sulfate. Sequence (i)→(ii)

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

10 g of K5-N-sulfate obtained as described in Example 1, stage (i) and (ii) of the patent WO 02/068477, which in the spectrum of1H-NMR naobnarodovanie signals, the corresponding residues of N-acetyl or-NH2, is dissolved in 600 ml of 25 mm Hepes buffer, pH 7, containing CaCl2at a concentration of 50 mm, and the resulting solution was passed through a column of 50 ml volume, filled with Separate 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°at pH 7 at a throughput rate of 200 ml/h for 24 hours. The resulting product was then purified by ultrafiltration and precipitation with ethanol. You get an EPI-K5-N-sulfate content iduronovoy acid in 54%.

(ii) the Depolymerization EPI-K5-N-sulfate

1 g of the obtained product is dissolved in 25 ml of distilled water and this solution make 230 mg of sodium nitrite dissolved in 115 ml of distilled water. Then the solution was adjusted to 4°and to pH 2 using a 0.1 N HCl and left for 30 minutes At the end of the reaction the solution is brought to room temperature and up to pH 7 with 0.1 M NaOH. Then to the solution was added 450 mg of NaBH4and left to react for 4 hours. The product distinguish by precipitation with 3 volumes of acetone at 4°With filtering on a funnel and dried at 40°With vacuum drying. Receive 900 mg NM EPI-K5-N-sulfate content iduronovoy acid in 54% and a distribution of molecular weights from 1000 to 4000 when measured by HPLC method.

Example 2

NM EPI-K5-N-sulfate. The placenta is the sequence (ii)→ (i)

(ii) the Depolymerization KS-N sulfate

2 g of K5-N-sulfate obtained as described in Example 1, stage (i) and (ii) of the patent WO 02/068477, subjected to depolymerization as described in Preparative example I, using 100 mg of sodium nitrite and 300 mg of sodium borohydride. Get 1.8 g NM K5-N-sulfate with an average molecular weight of 5000.

(i) the Epimerization NM KS-N sulfate

1 g NM K5-N-sulfate obtained at the previous stage (ii), is treated as described in stage (i) of Example 1. Get emerytalny product ratio Euronova/glucuronic acid 44/56 compared with a ratio of 0/100 from the original product, with a distribution of molecular weights from 2,000 to 10,000 and an average molecular weight of 5000 D. the Output is calculated by measuring the content of uronic acids compared with the standard in accordance with carbazole method (Bitter and Muir, Anal. Biochem. 1971, 39, 88-92)is 90%.

Example 3

NM EPI-K5-N-sulfate. Sequence (i)→(ii)

(i) the Epimerization of KS-N sulfate

Portion 2 g of K5-N-sulfate obtained as described in Example 1, stage (i) and (ii) of the patent WO 02/068477, dissolved in 120 ml of 25 mm Hepes buffer, pH 7, containing 50 mm CaCl2. The resulting solution was passed through a column of 50 ml volume, filled with media containing immobilized enzyme obtained as described in WO 96/14425. This operation is carried out at 30°at the speed of transport is Denmark 200 ml/h for 24 hours. The resulting product was then purified by ultrafiltration through a membrane with a cutoff at 1000 D and passing through a column of an ion exchanger IR 120 H+, neutralizer the eluate with 1N NaOH. The sample emit by precipitation with ethanol or acetone. Get emerytalny product ratio Euronova/glucuronic acid 55/45 compared with a ratio of 0/100 the original product. The degree of epimerization calculated by 1H-NMR in accordance with the method described in WO 96/14425. The output is calculated by measuring the content of uronic acids compared with the standard in accordance with carbazole method (Bitter and Muir, Anal. Biochem. 1971, 39, 88-92)is 90%.

(ii) the Depolymerization EPI-K5-N-sulfate

1 g of the product obtained in stage (a), is subjected to a depolymerization method of splitting nitrous acid and subsequent recovery of the resulting aldehyde. This product is dissolved in 25 ml of distilled water and bring back 230 mg of sodium nitrite dissolved in 115 ml of distilled water. Then the solution was adjusted to 4°and to pH 2 with 0,1 N model HC1 and leave for 30 minutes At the end of the reaction the solution is brought to room temperature and up to pH 7 with 0.1 M NaOH. Then to the solution was added 450 mg of NaBH4and left to react for 4 hours. The product distinguish by precipitation with 3 volumes of acetone at 4°With filtered with the eating funnel and dried at 40° With vacuum drying. Receive 900 mg NM EPI-K5-N-sulfate with a distribution of molecular weights in the measurement by the method of HPLC in the range from 1000 to 4000, with the content of glucuronic acid in 45% and the content iduronovoy acid in 55%.

Example 4

EPI-K5-N,O-hypersurface

(a) Tetrabutylammonium salt EPI-K5-N-sulfate

In 40 ml of water was dissolved 400 mg of EPI-K5-N-sulfate obtained at the end of stage (i) of Example 1, the solution is placed in a thermostat at 4°and then passed through the ion exchanger IR 120+pre-balanced with water at 4°C. the Obtained eluate consisting of 100 ml of a solution with pH value of 1.94, neutralized with 15% solution of tetrabutylammonium hydroxide and left at room temperature for 1 hour, maintaining a pH of about 7 by the addition of 15% tetrabutylammonium hydroxide, after which lyophilizer. While getting 805 mg tetrabutylammonium salt EPI-K5-N-sulfate.

(b) EPI-K5-amine-O-hypersurface

The solution containing 805 mg salt obtained above in 30 ml of dimethylformamide, lead up to 55°and treated With 30 ml of dimethylformamide containing of 2.26 g of compound pyridine·SO3. The reaction at 55°To continue throughout the night, and then this mixture was added 60 ml of water. After neutralization with 1N NaOH, the product precipitated with 3 volumes of acetone saturated with NaCl, and leave at 4°With all night. The precipitate is extracted f is trovanjem on guch G4, and then subjected to ultrafiltration using a TFF system company Millipore with a cutoff at 1000 D and dried under reduced pressure. Get 550 mg EPI-K5-amine-O-hypersurface content iduronovoy acid 54%, glucosamine-6-O-sulfate 100%, glucosamine-3-O-sulfate 60%, monosulfata glucuronic acid 10%, monosulfata iduronovoy acid 15%, with the rest of Euronova links desulfation, and the degree of sulfation is 3,55 when measuring conductometric method according to Casu et al., 1975.

(c) N-sulfation EPI-K5-amine-O-hypersurface

250 mg EPI-K5-amine-O-hypersurface obtained in stage (b), was dissolved in 15 ml of water was added 400 mg of sodium carbonate, and then the mixture was made 400 mg pyridine·SO3as a solid in small portions over 4 hours. The reaction mixture was kept at 55°during the night, and then stopped the reaction by bringing to pH 7 with 0.1 N HCl. After ultrafiltration through a membrane with a cutoff of 1000 L) was added to 3 volumes of acetone saturated with sodium chloride and extracted precipitate by centrifugation at 5000 rpm for 5 minutes this was 244 mg EPI-K5-N,O-hypersurface, in which the degree of sulfation was 4.25, when measured by conductometric method according to Casu et al., 1975. From the spectrum analysis1H-NMR follows that received EPI-K5-N,O-hypersurface there is tons of content iduronovoy acid 54%, 6-O-sulfate 100%, N-sulfate 100%, glucosamine-3-O-sulfate 60%, monosulfata glucuronic acid 10%, monosulfata iduronovoy acid 15%, with the rest of Euronova links desulfation. From the spectrum1H-NMR was calculated degree of sulfation in 4,35, which, given the limits of error of the methods corresponds to the degree of sulfation EPI-K5-amine-O-hypersurface achieved at the end of stage (b) and N-sulfated at 100%. Therefore, it is assumed that when exceeding a certain percentage of sulfate groups strongly anionic nature of the product may lead to an underestimation of the degree of sulfation when determining the conductometric method.

1. The method of deriving the N-deacetylating,N,O-hypersalivation, 20-60%C5-Ameritania K5 polysaccharide(EPI-K5-N,O-hypersurface-derived) with the degree of sulfation is equal to 4-4,6, characterized in that:

(a) N-deacetylating, N-hypersalivation, 20-60% C5-emerytalny K5 polysaccharide (EPI-K5-N,O-hypersurface-derived) with an average molecular weight from about 1500 to 25000, in acidic form, is treated with a tertiary or Quaternary organic base, leaving the reaction mixture for 30-60 min at pH about 7, and allocate its salt formed with this organic base;

(b) this salt organic is one reason and this EPI-K5-N-sulfate-derivative is treated with O-sulfation reagent under conditions O-hypersalivation;

(c) thus obtained N-deacetylating,N,O-hypersalivation, 20-60% C5-emerytalny K5 polysaccharide (EPI-K5-N,O-hypersurface-derived) with the degree of sulfation is equal 3,55-3,8, N-sulfation conventional methods and produce received EPI-K5-amine-O-hypersurface-derived.

2. The method according to claim 1, characterized in that EPI-K5-N,O-hypersurface-derived isolated in the form of sodium salt, and optionally converted into another chemically or pharmaceutically acceptable salt.

3. The method according to claim 1, characterized in that in stage (a) as an organic base is tetrabutylammonium hydroxide.

4. The method according to claim 1, characterized in that in stage (b) O-hypersalivation carried out in dimethylformamide, using 2-4 moles of O-sulfotyrosine reagent 1 available group HE 1 disaccharide at a temperature of 40-60°C for 15-20 hours

5. The method according to claim 1, characterized in that as starting material used EPI-K5-N-sulfate-derivative 100% N-sulfated and 40-60% C5-epimerization.

6. The method according to claim 5, characterized by the fact that this source EPI-K5-N-sulfate-derivative has an average molecular weight of from 10000 to 25000.

7. The method according to claim 1, characterized by the fact that the original EPI-K5-N-sulfate-derivative has an average molecular weight of from about to 8000.

8. The method according to claim 1, characterized in that as starting material used EPI-K5-N-sulfate-derivative, consisting of mixed circuits, of which at least 90% conform to the formula I:

in which the uronic units by 20-60% consist of iduronovoy 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 source material consists of mixed circuits, of which at least 90% conform to the formula I, in which the uronic units 40-60% consist of iduronovoy acid.

10. The method according to claim 8, characterized in that in 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 circuits, of which at least 90% conform to the formula I':

in which the uronic units by 20-60% consist of iduronovoy 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 p is obladaet circuit according to the formula I:

in which the uronic units at 60-40% composed of glucuronic acid and 40-60% iduronovoy 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 the source material is from about 2000 to 4000.

15. The method according to 14, characterized in that the source material consists of mixed circuits, which are dominated by chains according to the formula I′b:

in which X is hydroxymethyl, m is 4, 5 or 6, and glucuronic and Euronova links alternate, starting with a glucuronic or Euronova link.

16. The method according to any one of claims 1 to 15, characterized in that the starting material is obtained by N-deacetylation and N-sulfation K5, almost devoid of lipophilic substances.

17. EPI-K5-N,O-hypersurface-derived, obtained in accordance with the method according to claims 1 to 16.

18. EPI-K5-N,O-hypersurface is derived by 17 having content iduronovoy acid 20-60%, the average molecular weight of from about 2000 to 45,000 and the degree of sulfation, at least, 4-4,6, or one of its chemically or pharmaceutically acceptable salts, and this derivative is virtually the key has no effect on the parameters of blood coagulation.

19. EPI-K5-N,O-hypersurface is derived by p, whose average molecular weight is from about 15,000 to 45,000.

20. EPI-K5-N,O-hypersurface is derived by p, whose average molecular weight is from about 4500 to 8500.

21. EPI-K5-N,O-hypersurface is derived by p, characterized by the fact that it is 100% 6-O-sulfated and 50-80% of 3-O-sulfated on glucosamine links, 5-10% O-monosulfate on glucuronosyl links, 10-15% of 3-O-monosulfate on euronavy links and 2,3-desulfation for the rest of uronic links.

22. EPI-K5-N,O-hypersurface is derived by p, characterized in that it consists of mixed circuits, of which at least 90% correspond to the formula III:

in which the uronic units by 20-60% consist of iduronovoy acid, R, R' and R" is represented by hydrogen or SO3-and R presents SO3-at least 40% of the data mixed circuits, Z means SO3-, n is an integer from 2 to 100, and the degree of sulfation is at least 4, and the corresponding cation is chemically or pharmaceutically acceptable.

23. EPI-K5-N,O-hypersurface is derived by p, characterized in that it consists of mixed circuits, of which at least 90% soo which correspond to the formula III, in which the uronic units 40-60% presents iduronovoy acid.

24. EPI-K5-N,O-hypersurface is derived according to article 22, characterized in that it consists of mixed circuits, of which at least 90% correspond to the formula III in which n is an integer from 3 to 100.

25. EPI-K5-N,O-hypersurface is derived by p, characterized in that it is a low-molecular (NM) EPI-K5-N,O-hypersulfated, consisting of mixed circuits, of which at least 90% correspond to the formula III':

in which the uronic units by 20-60% consist of iduronovoy acid, q is an integer from 2 to 20, R, R' and R" is represented by hydrogen or SO3-Z means SO3-and the corresponding cation is chemically or pharmaceutically acceptable.

26. NM EPI-K5-N,O-hypersurface on A.25, characterized in that it consists of mixed circuits, of which at least 90% correspond to the formula III', in which q is an integer from 3 to 15.

27. NM EPI-K5-N,O-hypersurface on A.25, characterized in that it consists of mixed circuits, of which at least 90% correspond to the formula III', in which the uronic units 40-60% consist of iduronovoy acid.

28. NM EPI-K5-N,O-hypersurface on A.25, characterized in that the content iduronovoy acid in it is 50-55%.

p> 29. NM EPI-K5-N,O-hypersurface on p, characterized in that it consists of mixed circuits, of which at least 90% correspond to the formula III', in which R is at least 40% of SO3-, R' and R" are both represented SO3-either one of them is represented by hydrogen, and the other 5-10% of SO3-in glucuronic acid and 10-15% of SO3-in iduronovoy acid.

30. NM EPI-K5-N,O-hypersurface on p, characterized in that the average molecular weight of approximately from 4000 to 8500.

31. NM EPI-K5-N,O-hypersurface on p, characterized in that it consists of mixed circuits, of which at least 90% correspond to the formula III', in which R 50-80% of SO3-.

32. NM EPI-K5-N,O-hypersurface on p, characterized in that it consists of mixed circuits, which are dominated by chains according to the formula III'a:

in which the uronic units by 20-60% consist of iduronovoy acid, p is an integer from 4 to 8, Z is SO3-, R, R' and R" is represented by hydrogen or SO3-and the corresponding cation is chemically or pharmaceutically acceptable.

33. NM EPI-K5-N,O-hypersurface on p, characterized in that it consists of cm is Shannah circuits, dominated by chains according to the formula III'b:

in which R, R' and R" is represented by hydrogen or SO3-Z means SO3-and X" IS HE or OSO3-, m is 4, 5 or 6, Euronova links alternate, starting with a glucuronic or Euronova link, and the corresponding cation is chemically or pharmaceutically acceptable.

34. EPI-K5-N,O-hypersurface-derivative according to any one of p-33, characterized by the fact that this is chemically or pharmaceutically acceptable salt or a salt or cation is a cation of alkali metal, alkaline earth metal, ammonium, (C1-C4)-tetraalkylammonium, aluminum or zinc.

35. EPI-K5-N,O-hypersurface-derivative according to any one of p-33, characterized by the fact that this is chemically or pharmaceutically acceptable salt or a salt or cation is a cation of sodium, calcium, or tetrabutylammonium.

36. EPI-K5-amine-O-hypersurface-derivative having a degree of sulfation 3,55-3,8 one of its chemically or pharmaceutically acceptable salts produced in accordance with the stages (a) and (b) according to claim 1, isolated as sodium salt and, optionally, converted into another chemically or pharmaceutically acceptable salt.

37. EPI-K5-amine-O-hypersurface is derived by p, in which the soda is the content iduronovoy acid is 20-60% of the amount of uronic acids, with an average molecular weight from about 2000 to 40000.

38. EPI-K5-amine-O-hypersurface is derived by p, characterized in that it consists of mixed circuits, of which at least 90% conform to the formula II:

in which the uronic units by 20-60% consist of iduronovoy acid, n is an integer from 2 to 100, R, R' and R" is represented by hydrogen or SO3-and the corresponding cation is chemically or pharmaceutically acceptable.

39. EPI-K5-amine-O-hypersurface-derived in § 38, characterized in that in formula II, n means an integer from 3 to 100.

40. EPI-K5-amine-O-hypersurface-derived in § 38, characterized in that it consists of mixed circuits, of which at least 90% conform to the formula II, in which the uronic units 40-60% consist of iduronovoy acid with an average molecular weight of approximately from 2,000 to 40,000, R, of at least 40% of SO3-, R' and R" are both represented SO3-either one of them is represented by hydrogen, and the other 5-10% of SO3-in monosulfata glucuronic acid and 10-15% of SO3-in monosulfata iduronovoy acid.

41. EPI-K5-amine-O-hypersurface-derived in § 38, characterized in that navsats NM EPI-K5-amine-O-hypersulfated, consisting of mixed circuits, of which at least 90% conform to the formula II, in which the uronic units 40-60% consist of iduronovoy acid, R is at least 40% of SO3-, R' and R" are both represented SO3-either one of them is represented by hydrogen, and the other 5-10% of SO3-in glucuronic acid and 10-15% of SO3-in iduronovoy acid, n is an integer from 3 to 15, has an average molecular weight of from about 4000 to 8000, and the corresponding cation is chemically or pharmaceutically acceptable.

42. EPI-K5-amine-O-hypersurface is derived by p, characterized in that it is NM EPI-K5-amine-O-hypersulfated, consisting of mixed circuits, which are dominated by chains according to the formula II a:

in which the uronic units by 20-60% consist of iduronovoy acid, p is an integer from 4 to 8, R, R' and R" is represented by hydrogen or SO3-.

43. NM EPI-K5-amine-O-hypersurface in § 42, characterized in that it consists of mixed chains, among which are compounds according to formula II b:

in which the uronic units 40-60% consist of iduronovoy acid, m is 4, 5 or 6, R, R' and R" is represented by hydrogen and is and SO 3-X" means HE or OSO3and Euronova links alternate, starting with a glucuronic or Euronova link.

44. NM EPI-K5-N-sulfate, almost devoid-NH2and N-acetyl groups content iduronovoy acid from 20 to 60% and an average molecular weight from about 1,500 to 12,000, or one of its chemically or pharmaceutically acceptable salts.

45. NM EPI-K5-N-sulfate according to item 44, characterized in that the content iduronovoy acid ranges from 40 to 60%, and the average molecular weight of from about 1500 to 10000.

46. NM EPI-K5-N-sulfate according to item 45, characterized in that the average molecular weight is about 1500 to 7500.

47. NM EPI-K5-N-sulfate according to item 46, obtained by the method, characterized in that the K5-N-sulfate is subjected, in any order,

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

(ii) nitrous depolymerization, optionally followed by a recovery.

48. NM EPI-K5-N-sulfate according p, characterized in that it consists of mixed chains, of which less is th 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 p, characterized in that it has a link with the structure (a")

on the restored end of most mixed circuits, where X is a formyl or hydroxymethyl.

50. NM EPI-K5-N-sulfate according p, characterized in that it consists of mixed circuits, of which at least 90% conform to the formula I′where g is an integer from 3 to 15.

51. NM EPI-K5-N-sulfate according p, characterized in that it consists of mixed circuits, which are dominated by chains according to the formula I:

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 § 51, characterized in that it consists of mixed circuits, which are dominated by chains according to the formula I (b:

in which X is hydroxymethyl, m is 4, 5 or 6, the corresponding cation is chemically or pharmaceutically acceptable ion, and glucuronic and Euronova links alternate commenced the traveler with glucuronic or Euronova link.

53. NM EPI-K5-N-sulfate according to any one of paragraphs 44 to 52, characterized in that the salt or cation chosen from among alkali metals, alkaline earth metals, ammonium, (C1-C4)-tetraalkylammonium, aluminum and zinc.

54. NM EPI-K5-N-sulfate according to any one of p-52, characterized in that the salt or cation chosen from among sodium, calcium and tetrabutylammonium.

55. Pharmaceutical composition having anticommuting and antiviral activity comprising as an active ingredient a pharmacologically active amount of an EPI-K5-N,O-hypersurface-derivative according to any one of p-40 mixed with a pharmaceutical excipient.

Priority items:

18.06.2002 according to claims 1-7, 10, 12, 16-20, 21, 24, 29, 30, 31, 34-37, 39-41, 46-49, 50, 53-55;

27.08.2002 on PP-15, 51, 52;

17.06.2003 on PP, 9, 11, 22, 23, 25-28, 32, 33, 38, 42-45.



 

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8 ex

FIELD: bioorganic chemistry, chemical technology.

SUBSTANCE: invention describes N-deacylated N-sulfatized derivative of polysaccharide K5 epimerized up to at least to 40% of the content of L-iduronic acid with respect to the total content of uronic acids and with molecular mass 2000-30000 Da and comprising 25-50% by the chain weight showing the high affinity to ATIII and possessing anti-coagulant and anti-thrombosis activity with the ratio of abovementioned activities of HCII/antiXa from 1.5 to 4. Also, invention describes a method for preparing a derivative of polysaccharide K5 comprising isolation of polysaccharide K5 from Escherichia coli cells, N-deacylation and N-sulfatization, C-5-epimerization of D-glucuronic acid to L-induronic acid, supersulfatization, selective O-desulfatization, selective 6-O-sulfatization and N-sulfatization. The reaction of C-5-epimerization is carried out by using the enzyme glucuronosyl-C-5 epimerase in the soluble or immobilized form in the presence of bivalent cations chosen from the group comprising Ba, Ca, Mg and Mn.

EFFECT: improved preparing method, valuable medicinal properties of substances.

12 cl, 3 tbl, 12 dwg, 12 ex

The invention relates to a method for anticoagulant compositions, and compositions obtained thereby

The invention relates to fototermin derived fractions, each of which receive chemical binding photoreactive connection with glycosaminoglycans /hereinafter sometimes referred to for brevity as a "GAG"/ and stitched glycosaminoglycans having a three-dimensional netted structure which is obtained by exposing the above derived expression for the dimerization photoreactive compounds, to processes for their preparation, and, further, to a satisfactory biocompatible materials for medical use, which contain them

FIELD: medicine; pharmacology.

SUBSTANCE: offered invention concerns medicine and pharmacology, exactly to method of Chitosan production. Method includes milling of natural chitin-containing raw material, alternating by turn three stages of raw material deproteinisation, and three stages of decalcification, deproteinisation stages are carried out by sodium hydroxide solution of concentration 3-5% produced by diaphragm electrolysis method within 3-4 hours at temperature 60-65°C; decalcification stages are performed by 3-5% hydrochloric acid solution at temperature 20-25°C within 1-3 hours; deacetylation is performed by 49-55% sodium hydroxide produced by diaphragm electrolysis method; deproteinisation and decalcification stages are performed in series connected six steel devices with internal glass-enamel coating, equipped with stirrers, thermopools, connections for reagents feeding and disposal, jackets for reactionary mass cooling and heating. Afterwards deacetylation follows with 49-55% sodium hydroxide produced by diaphragm electrolysis method. Process is conducted at temperature 95-105°C within 7-9 hours and followed by washing, spinning and drying of finished Chitosan.

EFFECT: production of high-clean Chitosan.

FIELD: medicine.

SUBSTANCE: invention refers to medicine and describes method of glycosaminoglycan release from mineralised connective tissue, containing enzymatic hydrolysis, deproteinisation, deposition, and characterised by the fact that demineralisation is performed within 20 h using 0.5 H HC1, enzymatic hydrolysis is performed within 18 h using pepsin enzyme, deproteinisation is performed by adding of ammonium sulphate to 75% saturation, glycosaminoglycans are deposited with 4% potassium acetate in 96% ethanol, re-deposited with ethanol and lyophilised. Glycosaminoglycans are exposed to chromatographic separation using "ДЕАЕ"-sephadex A-25, and lyophilised. This method is easy-to-use, realised in laboratory environment of patient care institutions, and time-saving.

EFFECT: easy and saving method of glycosaminoglycan release from mineralised connective tissue.

1 ex, 2 dwg

FIELD: fish processing industry.

SUBSTANCE: invention relates to method for production of chitin from raw materials belonging to class Crustacea. Claimed method includes maxillopod grinding, enzymatic hydrolysis of proteins, hydrolyzate separation followed by drying, demineralization deproteinization. Enzymatic hydrolysis is carried out by using own maxillopod enzymes. Autoenzymolisis is carried out in aqueous medium with addition of bioconserving agents in raw/bioconserving agent solution of 1:(3-4) for 21 h at 35°C. As bioconserving agent milky whey or aloe juice is used. After protein hydrolyzate separation chitin semi-product is demineralized.

EFFECT: decreased product cost, product of improved quality, environmentally friendly method.

3 cl, 7 ex, 2 tbl

FIELD: polymers, chemical and biochemical technology.

SUBSTANCE: method for preparing oligomers of chitosan is carried out by enzymatic hydrolysis of chitosan in an aqueous solution at pH 3.0-4.4. Hydrolysis is carried out in the presence of chitosanase and papain taken in the ratio = 1:(1-1.2) by mass. Hydrolyzate is treated with anion-exchange resin, filtered, concentrated to the content of water 62-80% and dried. Method provides decreasing losses by 50-70% and enhancing quality of the end product. Invention can be used in food industry and medicine.

EFFECT: improved preparing method.

2 cl, 4 ex

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention describes chitosanium perchlorate of the following formula: C6O4H9NH3ClO4. This compound shows such properties as explosion-proof, absence of toxicity, resistance to moisture and heating, mechanical effects and possesses high oxidizing and binding properties. This allows its using in power-consuming compositions in mixture with chitosanium dodecahydro-clozododecaborate wherein it functions as a binding agent. The quantitative ratio between chitosanium dodecahydro-clozododecaborate in the composition is determined by required regimen of combustion: the more content of chitosanium perchlorate the higher activity of the composition.

EFFECT: improved preparing method, valuable properties of composition.

3 cl, 1 dwg, 4 ex

FIELD: derivatives of chitosan.

SUBSTANCE: invention relates to preparing biologically active chitosan substances and their derivatives. Invention describes a modified chitosan substance showing pH-neutral reaction and plastic structure of chitosan particles as fractal chitosan particles of size of nanofractals from 1 nm, not less, and to 5000 nm, not above, or as cross-linked net-shaped polymer having multiple cavities of size from 1 nm, not less, to 50 nm, not above. Invention describes methods for their preparing. Invention provides high transdermal penetration of chitosan substance and enhanced capacity for administration of medicinal or biologically active substances into chitosan substance. Invention can be used in manufacturing cosmetic, curative-cosmetic, pharmacological preparations, biologically active food supplements and foodstuffs.

EFFECT: improved and valuable properties of chitosan substances.

14 cl, 4 tbl, 7 dwg, 9 ex

FIELD: chemical engineering.

SUBSTANCE: method involves bringing chitosan and aqueous solution of acrylic acid in interaction under heating in 1:1-2:1 mole proportion, treating with potassium hydroxide, precipitating with acetone and drying the product. Acrylic acid is used as 10-20% aqueous solution. Source reagents are preliminarily kept at room temperature until gel is formed. Heating to 50-70°C is applied for 12-24 h.

EFFECT: high joining degree of acrylic acid to chitosan.

1 tbl

FIELD: polymers, chemical technology.

SUBSTANCE: invention describes a method for synthesis of grafted copolymers of chitin and chitosan with synthetic polymers. Method involves chemical interaction of chitin or chitosan with a synthetic polymer chosen from the group comprising polyacrylamide, polylactic acid, polyvinyl acetate, polyvinyl alcohol, maleic anhydride-modified polyethylene or polypropylene or with a monomer - acrylamide or lactide (cyclic dimer of lactic acid). The reaction is carried in solid aggregate state of reagents by the reaction extrusion at temperature below melting point of synthetic reagent. Method provides excluding the used of catalysts or solvents.

EFFECT: improved method of synthesis.

18 cl, 10 ex

FIELD: natural compounds, chemical technology and biotechnology.

SUBSTANCE: invention describes a method for preparing chitosan oligosaccharides. Method involves enzymatic depolymerization of chitosan with chitosanase in an aqueous solution in the presence of acetic, ascorbic, succinic acid or their mixtures followed by filtration and drying. After drying prepared chitosan oligosaccharides are subjected for low-temperature treatment with liquid nitrogen. Method provides preparing chitosan oligosaccharides without unfavorable after tasting.

EFFECT: improved preparing method.

4 ex

FIELD: natural substances, chemical technology.

SUBSTANCE: invention relates to a method for preparing chitosan and purification from components of the reaction mixture - low-molecular products of deacetylation and alkali excess. Invention relates to a method for purifying chitosan prepared by solid-state method involving treatment of reaction mass with extractant consisting of 3.3-20.0% of water, 32.2-57.1% of ethyl acetate and 24.6-64.5% of ethanol at the extractant boiling point. Also, invention relates to a method for purifying chitosan prepared by suspension method and involving treatment of the reaction mass with ethyl acetate and the following treatment with extractant consisting of 6.2-25.0% of water, 12.5-62.5% of ethyl acetate and 31.3-62.5% of ethanol at the extractant boiling point.

EFFECT: improved isolating and preparing method.

3 cl, 2 tbl, 1 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to cyclodextrin-containing polymeric compounds, which are carriers for delivery of therapeutics, and pharmaceutical preparations based on them. Invention also relates to method of treating subjects with therapeutically effective quantity of said cyclodextrin-containing polymeric compound. Claimed cyclodextrin-containing polymers improve medication stability, increase its solubility and reduce toxicity of therapeutics when used in vivo. Furthermore, by selecting from a variety of linker groups and targeting ligands of said polymers it is possible to realise controlled delivery of therapeutic agents.

EFFECT: obtaining cyclodextrin-containing polymer compounds, improving medication stability, increasing its solubility and reducing toxicity of therapeutics when used in vivo.

56 cl, 13 dwg, 7 tbl, 46 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the mixture of sulfated oligosaccharide having common structure of polyose that is included in heparin composition with average molecular mass ranging from 1500 to 3000 Da and proportion of anti-Xa/anti-IIa more than 30, to the method of their production and antithrombotic pharmaceutical compositions containing them.

EFFECT: production of the pharmaceutical compositions containing sulfated oligosaccharide that has antithrombotic activity.

31 cl, 12 ex

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