WO1999028351A1

WO1999028351A1 - Persulfated chondroitin sulfates, process for producing the same, and anticoagulants containing the same as the active ingredient

Info

Publication number: WO1999028351A1
Application number: PCT/JP1998/000023
Authority: WO
Inventors: Toshio Imanari; Toshihiko Toida; Robert J. Linhardt
Original assignee: Shin Nippon Yakugyo Co., Ltd.
Priority date: 1997-12-02
Filing date: 1998-01-08
Publication date: 1999-06-10
Also published as: JPH11166001A

Abstract

Novel compounds different from heparin and having a high anticoagulant activity. These compounds are persulfated chondroitin sulfates having at least 3.5, on average, O-sulfate groups per disaccharide repeating unit or pharmacologically acceptable salts thereof. These compounds can be produced by reacting a chondroitin sulfate salt with sulfur trioxide in an aprotic solvent at 30 to 50 °C by regulating the molar ratio of the free hydroxyl groups in the chondroitin sulfate salt to the sulfur trioxide of 1:10 to 1:20.

Description

Specification

Persulfated chondroitin sulfate, method for producing the same, and anticoagulant containing the same as an active ingredient

Technical field

The present invention relates to persulfated chondroitin sulfate, a method for producing the same, and an anticoagulant containing the same as an active ingredient.

Background art

Heparin is a drug widely used in clinical settings as a preoperative and postoperative thrombosis preventive agent. However, serious side effects such as bleeding tendency and thrombocytopenia have been observed in long-term use of heparin, and it is urgently necessary to develop alternative medicines. Recently, the anticoagulant activity of various natural polysaccharides and their chemical derivatives has been investigated, and their potential as pharmaceuticals has been explored.

On the other hand, chondroitin sulfate is a kind of complex carbohydrate called glycosaminoglycan, and N-acetylgalactosamine and glucuronic acid are linear chains that form a repeating unit of 3GalNAc; 81 → 4GlcAβ1 → It is an acidic polysaccharide. On average, there is one sulfate per disaccharide, and usually the 4- or 6-position hydroxyl group of Ν-acetylgalactosamine is sulfated. This polysaccharide is usually found as a proteoglycan as a proteoglycan linked to a serine or threonine residue in a protein via a bridging structure (GlcA-Gato Gato Xy SerVThr). It is assumed to be one of the molecules existing on the extracellular matrix or on the cell surface and responsible for intercellular communication. It is thought that chondroitin sulfate plays a part in the hemostasis mechanism in vivo, but its anticoagulant activity is weaker than that of heparin or the like, so there are few opportunities to use it as a drug.

It has been previously reported that chondroitin sulphate with two to three sulphates per disaccharide unit exhibits anticoagulant activity (Bour in,. Et al, J. Biol. Chem., 265, 15424). -5431 (1990)). It is also true that these chemically sulfated persulfated chondroitin sulfates have structurally different glucuronic acid residues from the iduronic acid contained in heparin and conformations ( Q iu, G., e Pharm. Bull,, 102, 721-726 (1997); Casu, B. Sem inn. Thromb. Haemost., 17S, 9-14 (1991) ₀

However, even conventional persulfated chondroitin sulfate has insufficient anticoagulant activity compared to heparin.

Disclosure of the invention

Therefore, an object of the present invention is to provide a novel compound having high anticoagulant activity, which is different from heparin, and a method for producing the same.

As a result of intensive studies, the present inventor has developed a new method for producing chondroitin sulfate in which the number of sulfate groups per repeating unit is greater than that of conventional persulfated chondroitin sulfate. The present invention has been completed by providing chondroitin sulfate for the first time, and finding that the persulfated chondroitin sulfate has a high anticoagulant activity, which is comparable to that of heparin / phosphorus.

That is, the present invention provides persulfated chondroitin sulfate or pharmacologically acceptable salts thereof having an average of 3.5 or more O-sulfate groups in the disaccharide repeating unit. Further, the present invention relates to a method for forming a salt between chondroitin sulfate and sulfur trioxide in a non-protic solvent at a temperature of 30 ° C. to 50 ° C. Provided is a method for producing persulfated chondroitin sulfate, wherein the reaction is carried out at a molar ratio of 1:10 to 1:20. Furthermore, the present invention provides an anticoagulant comprising the above-mentioned persulfated chondroitin sulfate of the present invention as an active ingredient. Further, the present invention provides a method for preventing blood coagulation, which comprises adding the above-mentioned persulfated chondroitin sulfate of the present invention to blood. Furthermore, the present invention provides the use of the above-mentioned persulfated chondroitin sulfate of the present invention as an anticoagulant.

According to the present invention, there is provided a novel persulfated chondroitin sulfate having a higher sulfation rate of free hydroxyl groups than before. The persulfated chondroitin sulfate of the present invention has excellent anticoagulant activity, particularly excellent Ila factor inhibitory activity, and is useful as a substitute for heparin having strong side effects.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the raw material chondroitin sulfate (a), persulfate reacted with sulfur trioxide at 0 ° C. FIG. 4 shows the results of gradient PAGE of persulfated chondroitin sulfate (b) and persulfated chondroitin sulfate (c) of the present invention reacted with sulfur trioxide at 40 ° C.

FIG. 2 shows IR spectra of the raw material chondroitin sulfate (A) and the completely O-sulfated chondroitin sulfate (B) of the present invention prepared in Example 1.

Figure 3 shows raw chondroitin sulfate (A), persulfated chondroitin sulfate (B) reacted with sulfur trioxide at 0 ° C, and persulfated chondroitin of the present invention reacted with sulfur trioxide at 40 ° C. ¹ shows the results of one-dimensional ¹ HNMR of sulfuric acid (C).

FIG. 4 shows the results of two-dimensional DOF-GOSY (A) and NOESY (B) spectra of fully O-sulfated chondroitin sulfate.

FIG. 5 shows the equilibrium state of the three-dimensional structures of the conventional persulfated chondroitin sulfate (A) and the completely O-persulfated oxidized chondroitin sulfate (B) of the present invention.

FIG. 6 shows the relationship between the number of sulfate groups in the disaccharide unit of chondroitin sulfate and anticoagulant activity.

BEST MODE FOR CARRYING OUT THE INVENTION

As described above, chondroitin sulfate is a linear acidic polysaccharide in which N-acetylgalactosamine and glucuronic acid constitute a repeating unit of —3Ga I NAc ^ l-→ 4G I cA 1—. There are four sulfateable hydroxyl groups in the repeating unit. In chondroitin sulfate found in living organisms, an average of one of these four hydroxyl groups is a sulfate ester. The chondroitin sulfate of the present invention has an average of 3.5 or more, preferably <3.8 or more, and most preferably 4 of the four hydroxyl groups in the disaccharide repeating unit. Has become an ester. When the average number of sulfate groups in the disaccharide repeating unit is 3.5 or more, preferably 3.8 or more, and most preferably 4, the number of sulfate groups is significantly higher than that of conventional persulfated chondroitin sulfate. Increases anticoagulant activity.

The most preferred persulfated chondroitin sulfate of the present invention in which all four hydroxyl groups in the disaccharide repeating unit are sulfate esters is represented by the following formula [I].

The number of repetitions of the disaccharide repeating unit (π in the formula [I]) is not particularly limited, but is preferably about 10 to 50, and more preferably 20 to 30.

The persulfated chondroitin sulfate of the present invention may be in the form of a pharmacologically acceptable salt. Examples of such salts include, but are not limited to, alkali metal salts such as sodium salts and potassium salts, calcium salts, aluminum salts and the like.

The persulfated chondroitin sulfate of the present invention is obtained by reacting a salt of chondroitin sulfate and sulfur trioxide in an aprotic solvent at a temperature of 30 ° C. to 50 ° C. with free hydroxyl groups in chondroitin sulfate. It can be produced by reacting with sulfur oxide at a molar ratio of 1:10 to 1:20.

The chondroitin sulfate used as a starting material is preferably an organic amine salt, particularly preferably a trialkyl (particularly lower alkyl having 1 to 6 carbon atoms) amine salt, and particularly preferably a triptylamine salt. Organic amine salts of chondroitin sulfate include, for example, chondroitin sulfate sodium salt (a commercially available product) prepared from cartilage of sea urchin is applied to a cation exchange resin, and then the organic amine is added and concentrated by freeze-drying. Thus, it can be easily prepared. The cation exchange resin that can be used here is not limited at all. For example, commercially available products such as Dowex 50W X8 (manufactured by Dow Chemical) and SP Sepharose HP (manufactured by Pharmacia) can be used. Wear. The amount of the organic amine added to chondroitin sulfate after cation exchange is preferably about 1 to 1.2 mol per 1 mol of acidic group (carboxyl group or sulfate group) contained in chondroitin sulfate.

Next, the obtained salt of chondroitin sulfate and sulfur trioxide are mixed in an aprotic solvent. Then, at a temperature of 30 ° C. to 50 ° C., the reaction is carried out at a molar ratio of free hydroxyl groups in chondroitin sulfate to sulfur trioxide of 1:10 to 1:20. Here, preferred aprotic solvents include, but are not limited to, N, N-dimethylformamide, dimethylsulfoxide and the like. The reaction temperature is from 30 to 50 ° C, preferably from 37 to 43 ° C, most preferably about 40 ° C. The molar ratio of free hydroxyl groups to sulfur trioxide in chondroitin sulfate is from 1:10 to 1:20, preferably from 1:13 to 1:17, and most preferably about 1: One is five. The reaction time is about 30 minutes or more, preferably 30 minutes to 2 hours, and more preferably 45 to 90 minutes.

Sulfur trioxide may be reacted alone with chondroitin sulfate.However, a cyclic amine such as pyridine is reacted with sulfur trioxide in advance to form a complex, which is then reacted with chondroitin sulfate. May be. In this case, the reaction conditions between the cyclic amine and sulfur trioxide are, for example, as follows. In other words, the fuming sulfuric acid is distilled and the outflow so ₃ gas is cooled. To so ₃ that exhibited asbestos-like, under cooling, was added dropwise a cyclic amine, after becoming liquid, returning to room temperature, continue dropping until the such no longer observed smoke.

The above method produces the persulfated chondroitin sulfate of the present invention. The reaction can be stopped by adding distilled water. The obtained crude product can be precipitated with cold ethanol saturated with sodium acetate and recovered by centrifugation. Furthermore, the product can be purified by dissolving the recovered product in distilled water, dialyzing against water, and freeze-drying.

The persulfated chondroitin sulfate of the present invention can be used as an anticoagulant similarly to heparin. The dose is appropriately selected according to the symptoms and the mode of use.For example, when used for the prevention of coagulation of perfused blood during extracorporeal blood circulation, 100 to 300 units of heparin international unit may be used. It can be administered intermittently at 500 to 1500 units per hour after administration. As a composition at the time of application, for example, physiological saline or distilled water for injection can be added and dissolved at the time of use so as to be 10000 units per 1 ml. The persulfated chondroitin sulfate of the present invention is derived from chondroitin sulfate, which is a constituent of living organisms, and has no toxicity because the sulfate group is also non-toxic. Can be

Example

Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Example 1 Production of persulfated chondroitin sulfate

100 100 mg of sodium chondroitin sulfate derived from tracheal cartilage was applied to cation exchange resin (Dowex 50W X8). At this time, a column with a column size of 1 cm (inner diameter) x 15 cm (length) was eluted with water at a flow rate of 1. OmIZ. Then, Tribylamine 100 I was added, and the mixture was concentrated by freeze-drying to obtain chondroitin sulfate triptylamine. 1 Omg of the obtained chondroitin sulfate triptylamine salt was dissolved in 0.8 ml of N, N-dimethylformamide (DMF). On the other hand, the fuming sulfuric acid was distilled, the outgoing so ₃ gas was cooled, and cyclic amine was dropped into the asbestos-like so ₃ under cooling, and after cooling to a liquid state, the temperature was returned to room temperature, and the fuming no longer occurred. The pyridine 'sulfur trioxide complex obtained by continuing dropping until no longer observed was added to the chondroitin sulfate triptylamine salt DMF solution in an amount such that sulfur trioxide became 15 times the moles of free hydroxyl groups of chondroitin sulfate. . After reacting at 40 ° C for 1 hour, 1.6 ml of distilled water was added to stop the reaction. The crude product was precipitated with cold ethanol (3 volumes) saturated with sodium acetate and collected by centrifugation. The obtained complete O-sulfated chondroitin sulfate was dissolved in double-distilled water, and the salts were removed by dialysis, and then recovered by freeze-drying.

On the other hand, for comparison, the same operation as above was carried out except that the reaction temperature of chondroitin sulfate triptylamine salt and sulfur trioxide was 0 ° C to obtain partially sulfated chondroitin sulfate in which the sulfation of hydroxyl groups was incomplete. Obtained. In the following examples, chondroitin sulfate (no sulfation treatment) used as a raw material was also examined. Example 2 Properties of persulfated chondroitin sulfate

(1) Measurement of molecular weight The average molecular weight of each chondroitin sulfate was calculated by gradient (concentration gradient) PAGE. The measurement was carried out according to the method of Edens et al. (Edens, RE et al., J. Pharm. Sci., 81, 823-827 (1992)), using 12-22% gradient minigel, and staining with Alcian Blue. Was. Each lane was ablated with 20 μg of chondroitin sulfate. The relative molecular weight of each chondroitin sulfate was confirmed by elution position in GPC-HPLC. 50 mM sodium acetate eluent _(P H 7.4, a flow rate of 1 ml / min) using a detection was conducted at absorbance at 206 nm.

The results are shown in Figure 1. In Fig. 1, lane a shows the results for chondroitin sulfate used as a raw material (no sulfation treatment), and lane b shows the results for partially sulfated chondroitin sulfate reacted with sulfur trioxide at 0 ° C. Lane c shows the results for the persulfated chondroitin sulfate of the present invention reacted with sulfur trioxide at 40 ° C. Lane d shows the oligosaccharide molecular weight marker (4 g). Sulfation of chondroitin sulfate hardly changed the molecular weight, but a slight increase in molecular weight was observed. Gel permeation chromatography (GPC) (Toida, T., Biochem. J., 322, 499-506 (1997)) also showed a slight increase in molecular weight due to sulfation. These results not only suggest the introduction of a new sulfate group into the raw material chondroitin sulfate, but also indicate that the glycosidic bonds in the raw material chondroitin sulfate are stable against this sulfation reaction. The range of molecular weight measured by the above method was 10,000 to 20,000.

(2) Analysis of sulfate group content

Chondroitin sulfate for quantification of sulfate groups was thoroughly dialyzed against distilled water in a dialysis tube having a molecular weight cutoff of 12,000, freeze-dried, and dried in a desiccator in the presence of diphosphorus pentoxide for 2 days. After oxidization, the sulfate group was measured by HPLC using a CM-8 conductivity detector manufactured by Tosouichi Co., Ltd.

As a result, the number of sulfate groups in the disaccharide repeating unit was such that the raw material chondroitin sulfate was sulfated at 1,0 ° C, and that the persulfated chondroitin sulfate was 2.5-3.3, and the sulfated at 40 ° C. The persulfated chondroitin sulfate of the present invention was 4, and it was confirmed that all of the free hydroxyl groups in chondroitin sulfate were sulfated by the method of the present invention described above. Was.

(3) IR spectrum

The infrared absorption spectrum of the solid sample was measured using FT / 1R-230 manufactured by JASCO Corporation. 100 _g of glycosaminoglycan was mixed with 500 g of dried potassium bromide to prepare a tablet having a diameter of 3 mm, which was set in a spectrometer.

Fig. 2 shows the obtained IR spectrum. In FIG. 2, A shows the IR spectrum of the raw material chondroitin sulfate, and B shows the IR spectrum of the complete O-sulfated chondroitin sulfate of the present invention prepared in Example 1. Figure 2 shows that the hydroxyl groups in all chondroitin sulfates have been converted to axial sulfates. The strong absorptions of 1240 and 820-850 Kaiser are attributed to S = 0, C = 0 = S stretching vibration, respectively. Similarly, it is clear that the signals assigned to the C-0-H bending vibrations at 2900, 1440, 1380, and 1100 Kaiser are reduced in the spectrum of completely 0-sulfated chondroitin sulfate. In Fig. 2B, peak 1 is O-H stretching vibration, peak 2 is O-H bending vibration, peak 3 is S = 0 stretching vibration, peak 4 is C-OS bending deformation and axial C-1. shows the stretching vibration of O- S_〇 _3. For the assignment of IR spectra, see Gasu, B. et al., Carbohydr. Res., 63, 13-27 (1978); Orr, SFD, Biochim. Biophys. Acta, 14, 173-181 (1954); Bychkov, SM, Biol. Med., 92, 680-683 (1981); and Sanderson, PN et al, Biochem. J., 243, 175-181 (1987).

(4) Optical rotation

A portion of the dried sample was weighed and dissolved in distilled water to a concentration of 5 mg / ml, and the optical rotation was measured. The measurement was performed with sodium D line using DIP-140 manufactured by JASCO Corporation.

As a result, the optical rotation was 130 degrees for the raw material chondroitin sulfate, while it was 18 degrees for the completely O-sulfated chondroitin sulfate. This indicates that the three-dimensional structure of completely O-sulfated chondroitin sulfate has changed compared to that of the raw material chondroitin sulfate.

(5) ¹ H NMR spectrum Persulfated chondroitin sulfate (2 mg) was dissolved in 0.5 ml of heavy water (99.9%), and freeze-drying was repeated to remove exchangeable protons. The sample was placed in a desiccator in the presence of diphosphorus pentoxide under negative pressure, and left at room temperature for 10 minutes. The completely dried sample was dissolved in 0.5 ml of heavy water (99.96%) and passed through a 0.45 fraction syringe filter into an NMR tube (outside diameter 5.0 mm x length 25 cm, pp-528; Wi Imad Glass Co., Buena, NJ). I put it. One-dimensional and two-dimensional NMR measurements were performed using a JSX GSX50 OA spectrum meter equipped with a tunable 5 mm field gradient probe and standard JE0L software. At 303K, other measurements were taken at 333K. The HOD signal was eliminated by pre-saturation for 3 s in the one-dimensional spectrum and 1.5 s in the two-dimensional spectrum. In the two-dimensional spectrum, with an observation width of 2,000 Hz, 512 integrations were performed to obtain 1024 sampling data points, and the time domain data was shifted after zero filling (data matrix size, 1K x 1K). Using sine-bell window function, we amplified for two-dimensional double quantum fi Itered (DQF) -COSY, N0ESY and T0CSY. In the measurement of two-dimensional TOC SY and N0ESY, the mixing time was set to 150, 250, and 500 ms, and as a result, the MLEV-17 mixing sequence of 100 ms was used.

Figure 3 shows the results of the HN MR spectrum. In Figure 3, A is the result for the raw material chondroitin sulfate, B is the persulfated chondroitin sulfate prepared by reacting with sulfur trioxide at 0 ° C (average number of sulfate groups in the disaccharide repeating unit: 3.2) C shows the results for the fully O-sulfated chondroitin sulfate of the present invention. The raw material chondroitin sulfate spectrum shows a slight heterogeneity in the degree of sulfation. In other words, it is clear that this is a mixture of partial structures in which either the 4-position or the 6-position of N-acetylgalactosamine is sulfated. The persulfated chondroitin sulfate prepared at 0 ° C. was as expected (see aarouf i, RM et a, Thromb. Res., 59, 174-758 (1995); Bartolucci, C. et a I., Carbohydr. Res., 276, 401-408 (1995)) The structural heterogeneity was increased compared to the raw material chondroitin sulfate. This increase in structural heterogeneity suggests not only sulfation at positions 4 and 6 of N-acetylgalactosamine but also incomplete sulfonation of glucuronic acid residues at positions 2 and 3. Surprise In addition, the ¹ H NMR spectrum of chondroitin sulfate prepared at 40 ° C was very simple compared to other chemically sulfated samples or raw materials. This clearly implies a reduction in heterogeneity in the sulfation, ie complete 0-sulfation of the sugar chain hydroxyl groups. FIG. 4 shows the two-dimensional ¹ H NMR spectrum, DQF-COSY and N0ESY spectrum of completely 0-sulfated chondroitin sulfate. Each cross peak in the upper panel in FIG. 4 indicates the following. a, Gal pAc H-3 / H-4; b, GlcAp H-1 / H-2; c, GlcAp H-2 / H-3; d, GlcAp H-3 / H-4; e, Gal pAc H -1 / H-2; f, GlcAp H-4 / H-5; g, Gal pAc H-5 / H-6. Each cross peak in the lower panel indicates the next one. a, GalNpAc H-1 / H-3; b, Gal pAc H-1 / GlcAp H—4; c, Gal Ac H-4 / H-5; d, Gal pAc Η-4 / GlcAp H-5; e, Gal pAc H-4 / H-6; f, GlcAp H-3 / H-5. From these spectra, low-field shifts of sugar ring protons due to sulfation are apparent. From the scalar binding and nuclear Overhauser effect, the peak at 4.87 ppm was assigned to H-1 of N-acetylgalactosamine, and the signal at 4.57 ppm was assigned to H-4 of GlcA, indicating that all hydroxyl groups were completely sulfated. It shows that it has been Table 1 shows the chemical shifts and binding constants. According to the Carplus equation, the dihedral angle between adjacent protons of glucuronic acid is 60-70 degrees instead of 180 degrees, and the conformation of glucuronic acid changes from ⁴ C to ¹ C _4. (Figure 5). In FIG. ^{_{5, X 2 = S0 3 -}} , X 3 = H, or ^{^{X 2 = H, X 3 =}} S 0 3 - indicates, S ug denotes a sugar (N- Asechirugaraku Bok the summing).

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Example 3 Anticoagulant effect

Normal human plasma (NHP) was collected from healthy volunteers. Anti-factor Xa activity was measured using Coatest and hepar in / hepar in kit (Chromogen ix, olndal, Sweden; Chondroitin sulfate, persulfated chondroitin sulfate derivative and low molecular weight heparin standard were diluted) Dissolved in normal human plasma 2.9 mM Chromogen ic Xa substrate S-2732 (50 mM Tr is, containing Sue-1 IeG Iu (~ iPeridyI) -GI y-Ar -pNA) Ί.5 μΜ EDTA buffer (pH 8.4) 200 μl of Bacillus factor Xa (1.25 / ml) was added, incubated at 37 ° C for 8 minutes, and then 200 μl of 20% acetic acid was added. Factor Xa activity was measured by absorbance at 405 nm.

The anti-Ila factor activity was determined by mixing 50 μl of an aqueous solution of chondroitin sulfate or a persulfated derivative, 850 μl of Tris buffer (ρΗ8.3), 30 μl of normal human plasma, and human thrombin (1.2 NIH units / ml). 1.9mM after incubating at 37 ° C for 30 seconds

Chromogenic TH ^ ethyImaIonyI-Pro-Arg-p-nitroaniide hydrochloride) was caloried, and Ila factor activity was measured by absorbance at 405 nm. AGL 300 plus purchased from instrument at ion (Lexington, MA) was used for measurement, and USP Heparin reference Standard of U.S. Ph armacopeial Convention (ROCK I Ile, MD) was used as a control.

(K-3) was used.

Fig. 6 shows the results. In FIG. 6, squares indicate anti-Ila factor activity, and circles indicate anti-factor Xa activity. As shown in Figure 6, complete 0-sulfation dramatically increased the Ila factor inhibitory activity. On the other hand, the anti-factor Xa activity increased slightly with sulfation, but was not as high as that of the Ila factor. This result suggests that the increase in the inhibitory activity against factor Xa is simply due to the nonspecific increase in the negative charge due to the introduction of the sulfate group.

Claims

The scope of the claims

1. A persulfated chond having an average of 3.5 or more O-sulfate groups in a disaccharide repeating unit. Itine sulfate or a pharmacologically acceptable salt thereof.

2. The persulfated chondroitin sulfate or a pharmacologically acceptable salt thereof according to claim 1, which has an average of 3.8 or more 0-sulfate groups in the disaccharide repeating unit.

3. The persulfated chondroitin sulfate or pharmacologically acceptable salt thereof according to claim 2, wherein the disaccharide repeating unit has an average of four O-sulfate groups.

4. Salt of chondroitin sulfate and sulfur trioxide in an aprotic solvent at 30 ° C

A method for producing persulfated chondroitin sulfate, wherein the reaction is carried out at a temperature of 50 ° C with a molar ratio of free hydroxyl groups in chondroitin sulfate to sulfur trioxide of 1:10 to 1:20.

5. The method according to claim 4, wherein the reaction temperature is 37 to 43 ° C, and the molar ratio is 1:13 to 1:17.

6. The reaction temperature is 40. It is C, wherein the molar ratio of _1: 1 5 The method of claim 5, wherein.

7. The method according to any one of claims 4 to 6, wherein the salt is an organic amine salt.

8. An anticoagulant comprising the persulfated chondroitin sulfate according to any one of claims 1 to 3 as an active ingredient.